A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

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Loo Keat

2012-05-01

Full Text Available Abstract Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele and 0.11 (T allele; for eNOS +894 G > T, the allele frequencies were 0.58 (G allele and 0.43 (T allele; and for eNOS −786 T > C, the allele

¿Cuán liviana puede ser la risa? Nietzsche y levinas frente al “fin del hombre”

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Aïcha Liviana Messina

2012-10-01

Full Text Available El artículo parte de la siguiente lectura: en distintas ocasiones, Levinas se refiere al pensamiento de Nietzsche, a su risa, que “rechaza el lenguaje”; sorprendentemente, Levinas no sitúa a Nietzsche, a diferencia de Heidegger, como el último metafísico, sino que lo descoloca en esta historia, subrayando más bien la “extrema juventud” de la risa nietzscheana. Este artículo propone mostrar cómo la risa nietzscheana se articula con lo que Levinas llamaba una “subjetividad del sujeto inocente de conjunciones ontológicas”. Se concluye con una reflexión sobre la vergüenza y los límites del hombre.

[Morphologic and AFLP analysis of relationships between tulip species Tulipa biebersteiniana (Liliaceae)].

Science.gov (United States)

Kutlunina, N A; Polezhaeva, M A; Permiakova, M V

2013-04-01

In populations of four species of tulips, (Tulipa biebersteiniana, T. patens, T. scytica and T. riparia) from the Volgograd, Kurgansk, Orenburg, and Chelyabinsk regions and the Republic of Bashkortostan, genetic diversity was studied by means of morphological and AFLP analysis. A morphological analysis of seven quantitative and two qualitative criteria was carried out. Three selective EcoRI/MseI primer pairs allowed one to genotype 81 individuals from 13 tulip populations with 87 loci. The low level of variability by AFLP loci were revealed in all species, including T. biebersteiniana (P = 20.41%, UH(e) = 0.075), T. patens (26.97%, 0.082), T. scytica (27.53%, 0.086), and T. riparia (27.72%, 0.096). According to the AMOVA results, the variability proportion that characterizes the differences between the four Tulip species was lower (F(CT) = 0.235) than between populations within species (F(ST) = 0.439). Tulipa patens is well differentiated by means of Nei's distances, coordination, and analysis in the STRUCTURE program. An analysis in the STRUCTURE revealed four genetic groups of tulips that are not completely in accordance with the analyzed species. This acknowledges the presence of complicated genetic process in the tulip population.

AFLP analysis among Ethiopian arabica coffee genotypes

African Journals Online (AJOL)

STORAGESEVER

2008-09-17

Sep 17, 2008 ... AFLP analysis. AFLP analysis was performed following the protocol of Vos et al. ... 72oC. All polymerase chain reactions (PCR) were perform- ... ACT/MseI-CAG showed the highest value for MI ..... WL, Lee M, Porter K (2000).

Quantitative analysis of Terminal Restriction Fragment Length Polymorphism (T-RFLP microbial community profiles: peak height data showed to be more reproducible than peak area Análise quantitativa de perfis de T-RFLP de comunidades microbianas: dados de altura de picos mostraram-se mais reprodutíveis do que os de área

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Roberto A. Caffaro-Filho

2007-12-01

Full Text Available Terminal Restriction Fragment Length Polymorphism (T-RFLP is a culture-independent fingerprinting method for microbial community analysis. Profiles generated by an automated electrophoresis system can be analysed quantitatively using either peak height or peak area data. Statistical testing demontrated that peak height data showed to be more reproducible than peak area data.Terminal Restriction Fragment Length Polymorphism (T-RFLP é um método molecular, independente de cultivo, para análise de comunidades microbianas. Perfis gerados por um sistema automatizado de eletroforese podem ser analisados quantitativamente usando dados de altura ou área dos picos. Os dados de altura mostraram-se mais reprodutíveis do que os de área.

Molecular analysis of bacterial populations in water samples from two Uranium mill tailings by using RISA retrieval

International Nuclear Information System (INIS)

Selenska-Pobell, S.; Radeva, G.

2004-01-01

Ribosomal intergenic spacer amplification (RISA) retrieval was applied to analyse the natural bacterial communities in drain waters of two uranium mill tailings - Gittersee/Coschuetz in Germany and Shiprock in the USA. About 35% of the clones from RISA library constructed for the samples of the German tailings represented a microdiverse population of Planctomycetales. The rest of the clones were affiliated with rather diverse bacterial groups including γ- and δ-Proteobacteria, Cytophaga/Flavobacterium/Bacteroides (CFB), Nitrospira, Verrucomicrobia and Actinobacteria. 8% of the cloned sequences represented a novel bacterial lineage from the recently described division NC3. Bacterial diversity in the Shiprock mill tailings was found to be significantly lower. RISA library constructed for those samples contained only two larger groups of clones representing β-proteobacterial species and one small group which was affiliated with δ-Proteobacteria. (authors)

Molecular analysis of bacterial populations in water samples from two Uranium mill tailings by using RISA retrieval

Energy Technology Data Exchange (ETDEWEB)

Selenska-Pobell, S [Forschungszentrum Rossendorf, Institute of Radiochemistry, Dresden (Germany); Radeva, G [Bulgarian Academy of Sciences, Institute of Molecular Biology, Sofia (Bulgaria)

2004-07-01

Ribosomal intergenic spacer amplification (RISA) retrieval was applied to analyse the natural bacterial communities in drain waters of two uranium mill tailings - Gittersee/Coschuetz in Germany and Shiprock in the USA. About 35% of the clones from RISA library constructed for the samples of the German tailings represented a microdiverse population of Planctomycetales. The rest of the clones were affiliated with rather diverse bacterial groups including {gamma}- and {delta}-Proteobacteria, Cytophaga/Flavobacterium/Bacteroides (CFB), Nitrospira, Verrucomicrobia and Actinobacteria. 8% of the cloned sequences represented a novel bacterial lineage from the recently described division NC3. Bacterial diversity in the Shiprock mill tailings was found to be significantly lower. RISA library constructed for those samples contained only two larger groups of clones representing {beta}-proteobacterial species and one small group which was affiliated with {delta}-Proteobacteria. (authors)

Observation of high seasonal variation in community structure of denitrifying bacteria in arable soil receiving artificial fertilizer and cattle manure by determining T-RFLP of nir gene fragments

DEFF Research Database (Denmark)

Priemé, Anders; Wolsing, Martin

2004-01-01

Temporal and spatial variation of communities of soil denitrifying bacteria at sites receiving mineral fertilizer (60 and 120 kg N ha-1 year-1) and cattle manure (75 and 150 kg N ha-1 year-1) were explored using terminal restriction fragment length polymorphism (T-RFLP) analyses of PCR amplified...... nitrite reductase (nirK and nirS) gene fragments. The analyses were done three times during the year: in March, July and October. nirK gene fragments could be amplified in all three months, whereas nirS gene fragments could be amplified only in March. Analysis of similarities in T-RFLP patterns revealed...... a significant seasonal shift in the community structure of nirK-containing bacteria. Also, sites treated with mineral fertilizer or cattle manure showed different communities of nirK-containing denitrifying bacteria, since the T-RFLP patterns of soils treated with these fertilizers were significantly different...

Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis

Science.gov (United States)

Grant T. Kirker; M. Lynn Prewitt; Walter J. Diehl; Susan V. Diehl

2012-01-01

The effects of wood preservatives on the bacterial community in southern yellow pine were assessed by the molecular method ‘terminal restriction fragment length polymorphism’ (T-RFLP). Stakes, treated with 0.25 % and 0.37 % ammoniacal copper quat (ACQ-C), 0.1 % and 0.25 % chlorothalonil (CTN), 0.1 % and 0.25 % CTN with 2 % butylated hydroxytoluene (BHT), and 2 % BHT...

La risa en Francisco Suárez. El paso de la Edad Media al Barroco

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Manuel Lázaro Pulido

2012-07-01

Full Text Available El estudio muestra la relación entre la tradición medieval presente en la segunda escolástica y las consecuencias en el Barroco del humanismo renancentista, a partir de la referencia que hace Suárez a la risa en el Comentario al De anima. Aparece un desarrollo de la temática clásica y medieval sobre la risa, donde se tiene en cuenta la esfera intelectual del Humanismo científico y se señalan algunas temáticas que se tendrán presente en el Barroco moderno.

Genetic relationships among Arachis species based on AFLP

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Gimenes Marcos A.

2002-01-01

Full Text Available Amplified Fragment Length Polymorphism (AFLP was used to establish the genetic relationships among 20 species from seven of the nine sections of genus Arachis. The level of polymorphism among nine accessions of the cultivated peanut, A. hypogaea L., was also evaluated. Three combinations of primers were used to amplify the AFLPs. The fragments were separated in 6% denaturing acrylamide gels. A total of 408 fragments were analyzed. An average of 135.3 fragments per primer combination were scored, and the largest number of fragments was 169 using primer combination Eco RI - ACC / Mse I - CTG, while the lowest was 108, with Eco RI - ACT / Mse I - CTT. In general, the genetic relationships established using AFLPs agreed with the classification established using morphology and crossability data. The results indicated that AFLPs are good markers for establishing the relationships among Arachis species. The polymorphism detected in A. hypogaea by this method was higher than the one found with other markers, like RAPDs and RFLPs. However, our data suggest that the polymorphism detected be using AFLP with only three primer combinations is still too low to be used for any kind of genetic study in this species.

The Regional Integrated Sciences and Assessments (RISA) Program, Climate Services, and Meeting the National Climate Change Adaptation Challenge

Science.gov (United States)

Overpeck, J. T.; Udall, B.; Miles, E.; Dow, K.; Anderson, C.; Cayan, D.; Dettinger, M.; Hartmann, H.; Jones, J.; Mote, P.; Ray, A.; Shafer, M.; White, D.

2008-12-01

The NOAA-led RISA Program has grown steadily to nine regions and a focus that includes both natural climate variability and human-driven climate change. The RISAs are, at their core, university-based and heavily invested in partnerships, particularly with stakeholders, NOAA, and other federal agencies. RISA research, assessment and partnerships have led to new operational climate services within NOAA and other agencies, and have become important foundations in the development of local, state and regional climate change adaptation initiatives. The RISA experience indicates that a national climate service is needed, and must include: (1) services prioritized based on stakeholder needs; (2) sustained, ongoing regional interactions with users, (3) a commitment to improve climate literacy; (4) support for assessment as an ongoing, iterative process; (5) full recognition that stakeholder decisions are seldom made using climate information alone; (6) strong interagency partnership; (7) national implementation and regional in focus; (8) capability spanning local, state, tribal, regional, national and international space scales, and weeks to millennia time scales; and (9) institutional design and scientific support flexible enough to assure the effort is nimble enough to respond to rapidly-changing stakeholder needs. The RISA experience also highlights the central role that universities must play in national climate change adaptation programs. Universities have a tradition of trusted regional stakeholder partnerships, as well as the interdisciplinary expertise - including social science, ecosystem science, law, and economics - required to meet stakeholder climate-related needs; project workforce can also shift rapidly in universities. Universities have a proven ability to build and sustain interagency partnerships. Universities excel in most forms of education and training. And universities often have proven entrepreneurship, technology transfer and private sector

Innovative nuclear technologies based on radiation induced surface activation (RISA). 1. The project overview

International Nuclear Information System (INIS)

Fujisawa, Kyosuke; Morooka, Shinichi; Hishida, Mamoru

2004-01-01

This research of the Innovative nuclear technologies based on Radiation Induced Surface Activation (RISA) is due to start from 2003 and to be ended to 2006, and performed fund by Ministry of Economy, Trade and Industry (METI) Japan. One of the innovative technologies is to develop a high performance corrosion-proof film to prevent the surface of reactor internals from stress corrosion cracking (SCC), the other one is to develop the film for improving the heat transfer performance a high performance of the nuclear fuel rod. Both of these properties are derived under gamma ray irradiation by the RISA effect. This paper reports about the summary of this subsidy enterprise by METI. (author)

RiSA: A Science Festival for the Bilingual and Bicultural Rio Grande Valley

Science.gov (United States)

Key, Joey Shapiro; Torres, Cristina; Stone, Robert

2014-03-01

The Rio Grande Science and Arts (RiSA) Festival organized by the Center for Gravitational Wave Astronomy (CGWA) at the University of Texas at Brownsville (UTB) will use a wide variety of artforms to bring physics and science topics to the bilingual and bicultural population of the Rio Grande Valley of South Texas. The science and art faculty at UTB will partner with art and education professionals to create an annual community event celebrating science though art. Music, dance, poetry, and visual arts will headline the festival activities. Festival events and products will be produced in both English and Spanish to attract and inform the bilingual local community. The RiSA Festival is supported by the Science Festival Alliance and the Sloan Foundation. Supported by the Science Festival Alliance and the Sloan Foundation.

Expression of bvg-repressed genes in Bordetella pertussis is controlled by RisA through a novel c-di-GMP signaling pathway

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The BvgAS two component system of Bordetella pertussis controls virulence factor expression. In addition, BvgAS controls expression of the bvg-repressed genes through the action of the repressor, BvgR. The transcription factor RisA is inhibited by BvgR, and when BvgR is not expressed RisA induces th...

Association of AFLP and SSR markers with agronomic and fibre ...

Indian Academy of Sciences (India)

We have attempted to tag yield and fibre quality traits with AFLP and SSR markers using F2 and F3 populations of a cross between two Gossypium hirsutum varieties, PS56-4 and RS2013. Out of 50 AFLP primer combinations and 177 SSR primer pairs tested, 32 AFLP and four SSR primers were chosen for genotyping F2 ...

Molecular phylogeny of Fusarium species by AFLP fingerprint ...

African Journals Online (AJOL)

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships within and between natural populations of five Fusarium spp. AFLP templates were prepared by the digestion of Fusarium DNA with EcoRI and MseI restriction endonucleases and ...

AFLP and MS-AFLP analysis of the variation within saffron crocus (Crocus sativus L. germplasm.

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Matteo Busconi

Full Text Available The presence and extent of genetic variation in saffron crocus are still debated, as testified by several contradictory articles providing contrasting results about the monomorphism or less of the species. Remarkably, phenotypic variations have been frequently observed in the field, such variations are usually unstable and can change from one growing season to another. Considering that gene expression can be influenced both by genetic and epigenetic changes, epigenetics could be a plausible cause of the alternative phenotypes. In order to obtain new insights into this issue, we carried out a molecular marker analysis of 112 accessions from the World Saffron and Crocus Collection. The accessions were grown for at least three years in the same open field conditions. The same samples were analysed using Amplified Fragment Length Polymorphism (AFLP and Methyl Sensitive AFLP in order to search for variation at the genetic (DNA sequence and epigenetic (cytosine methylation level. While the genetic variability was low (4.23% polymorphic peaks and twelve (12 effective different genotypes, the methyl sensitive analysis showed the presence of high epigenetic variability (33.57% polymorphic peaks and twenty eight (28 different effective epigenotypes. The pattern obtained by Factorial Correspondence Analysis of AFLP and, in particular, of MS-AFLP data was consistent with the geographical provenance of the accessions. Very interestingly, by focusing on Spanish accessions, it was observed that the distribution of the accessions in the Factorial Correspondence Analysis is not random but tends to reflect the geographical origin. Two clearly defined clusters grouping accessions from the West (Toledo and Ciudad Real and accessions from the East (Cuenca and Teruel were clearly recognised.

Genetic diversity and identification of variety-specific AFLP markers ...

African Journals Online (AJOL)

In all the fenugreek varieties, a total of 25 variety-specific AFLP markers were found. Phylogenetic trees among 5 plant varieties were constructed based on Nei's coefficient standard genetic distances using unweighted pair group method with arithmetic mean (UPGMA) method. For RAPD and AFLP analysis, Gujarat Methi-1 ...

Diversity analysis of bacterial community compositions in sediments of urban lakes by terminal restriction fragment length polymorphism (T-RFLP).

Science.gov (United States)

Zhao, Dayong; Huang, Rui; Zeng, Jin; Yan, Wenming; Wang, Jianqun; Ma, Ting; Wang, Meng; Wu, Qinglong L

2012-11-01

Bacteria are crucial components in lake sediments and play important role in various environmental processes. Urban lakes in the densely populated cities are often small, shallow, highly artificial and hypereutrophic compared to rural and natural lakes and have been overlooked for a long time. In the present study, bacterial community compositions in surface sediments of three urban lakes (Lake Mochou, Lake Qianhu and Lake Zixia) in Nanjing City, China, were investigated using the terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene and clone libraries. Remarkable differences in the T-RFLP patterns were observed in different lakes or different sampling stations of the same lake. Canonical correspondence analysis indicated that total nitrogen (TN) had significant effects on bacterial community structure in the lake sediments. Chloroflexi were the most dominant bacterial group in the clone library from Lake Mochou (21.7 % of the total clones) which was partly associated with its higher TN and organic matters concentrations. However, Bacteroidetes appeared to be dominated colonizers in the sediments of Lake Zixia (20.4 % of the total clones). Our study gives a comprehensive insight into the structure of bacterial community of urban lake sediments, indicating that the environmental factors played a key role in influencing the bacterial community composition in the freshwater ecosystems.

Evaluating the relationship between evolutionary divergence and phylogenetic accuracy in AFLP data sets.

Science.gov (United States)

García-Pereira, María Jesús; Caballero, Armando; Quesada, Humberto

2010-05-01

Using in silico amplified fragment length polymorphism (AFLP) fingerprints, we explore the relationship between sequence similarity and phylogeny accuracy to test when, in terms of genetic divergence, the quality of AFLP data becomes too low to be informative for a reliable phylogenetic reconstruction. We generated DNA sequences with known phylogenies using balanced and unbalanced trees with recent, uniform and ancient radiations, and average branch lengths (from the most internal node to the tip) ranging from 0.02 to 0.4 substitutions per site. The resulting sequences were used to emulate the AFLP procedure. Trees were estimated by maximum parsimony (MP), neighbor-joining (NJ), and minimum evolution (ME) methods from both DNA sequences and virtual AFLP fingerprints. The estimated trees were compared with the reference trees using a score that measures overall differences in both topology and relative branch length. As expected, the accuracy of AFLP-based phylogenies decreased dramatically in the more divergent data sets. Above a divergence of approximately 0.05, AFLP-based phylogenies were largely inaccurate irrespective of the distinct topology, radiation model, or phylogenetic method used. This value represents an upper bound of expected tree accuracy for data sets with a simple divergence history; AFLP data sets with a similar divergence but with unbalanced topologies and short ancestral branches produced much less accurate trees. The lack of homology of AFLP bands quickly increases with divergence and reaches its maximum value (100%) at a divergence of only 0.4. Low guanine-cytosine (GC) contents increase the number of nonhomologous bands in AFLP data sets and lead to less reliable trees. However, the effect of the lack of band homology on tree accuracy is surprisingly small relative to the negative impact due to the low information content of AFLP characters. Tree-building methods based on genetic distance displayed similar trends and outperformed parsimony

Differentiation of species of the family Acetobacteraceae by AFLP DNA fingerprinting: Gluconacetobacter kombuchae is a later heterotypic synonym of Gluconacetobacter hansenii.

Science.gov (United States)

Cleenwerck, Ilse; De Wachter, Marjan; González, Angel; De Vuyst, Luc; De Vos, Paul

2009-07-01

Amplified fragment length polymorphism (AFLP) DNA fingerprinting was investigated as a tool for fast and accurate identification of acetic acid bacteria (AAB) to the species level. One hundred and thirty five reference strains and 15 additional strains, representing 50 recognized species of the family Acetobacteraceae, were subjected to AFLP analysis using the restriction enzyme combination ApaI/TaqI and the primer combination A03/T03. The reference strains had been previously subjected to either DNA-DNA hybridization or 16S-23S rRNA spacer region gene sequence analysis and were regarded as being accurately classified at the species level. The present study revealed that six of these strains should be reclassified, namely Gluconacetobacter europaeus LMG 1518 and Gluconacetobacter xylinus LMG 1510 as Gluconacetobacter xylinus and Gluconacetobacter europaeus, respectively; Gluconacetobacter kombuchae LMG 23726(T) as Gluconacetobacter hansenii; and Acetobacter orleanensis strains LMG 1545, LMG 1592 and LMG 1608 as Acetobacter cerevisiae. Cluster analysis of the AFLP DNA fingerprints of the reference strains revealed one cluster for each species, showing a linkage level below 50 % with other clusters, except for Acetobacter pasteurianus, Acetobacter indonesiensis and Acetobacter cerevisiae. These three species were separated into two, two, and three clusters, respectively. At present, confusion exists regarding the taxonomic status of Gluconacetobacter oboediens and Gluconacetobacter intermedius; the AFLP data from this study supported their classification as separate taxa. The 15 additional strains could all be identified at the species level. AFLP analysis further revealed that some species harboured genetically diverse strains, whereas other species consisted of strains showing similar banding patterns, indicating a more limited genetic diversity. It can be concluded that AFLP DNA fingerprinting is suitable for accurate identification and classification of a broad

The relative contribution of band number to phylogenetic accuracy in AFLP data sets.

Science.gov (United States)

García-Pereira, M J; Caballero, A; Quesada, H

2011-11-01

We examined the effect of increasing the number of sampled amplified fragment length polymorphism (AFLP) bands to reconstruct an accurate and well-supported AFLP-based phylogeny. In silico AFLP was performed using simulated DNA sequences evolving along balanced and unbalanced model trees with recent, uniform and ancient radiations and average branch lengths (from the most internal node to the tip) ranging from 0.02 to 0.05 substitutions per site. Trees were estimated by minimum evolution (ME) and maximum parsimony (MP) methods from both DNA sequences and virtual AFLP fingerprints. The comparison of the true tree with the estimated AFLP trees suggests that moderate numbers of AFLP bands are necessary to recover the correct topology with high bootstrap support values (i.e. >70%). Fewer numbers of bands are necessary for shorter tree lengths and for balanced than for unbalanced tree topologies. However, branch length estimation was rather unreliable and did not improve substantially after a certain number of bands were sampled. These results hold for different levels of genome coverage and number of taxa analysed. In silico AFLP using bacterial genomic DNA sequences recovered a well-supported tree topology that mirrored an empirical phylogeny based on a set of 31 orthologous gene sequences when as few as 263 AFLP bands were scored. These results suggest that AFLPs may be an efficient alternative to traditional DNA sequencing for accurate topology reconstruction of shallow trees when not very short ancestral branches exist. © 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.

Evaluation of interspecific DNA variability in poplars using AFLP and ...

African Journals Online (AJOL)

The objective of this paper was to examine interspecific DNA variation in poplars using AFLP and SSR markers. The AFLP and SSR markers polymorphism and its power of discrimination were determined within 13 genotypes of different genetic background (clones, cultivars, hybrids) of two sections (Aigeiros and ...

Puelles romero, Luis: Honoré Daumier: la risa republicana

Directory of Open Access Journals (Sweden)

Óscar Ortega Ruiz

2015-01-01

Full Text Available Recientemente, Abada Editores ha puesto a disposición de sus lectores el nuevo ensayo de Luis Puelles Romero, titular de Estética y Teoría de las Artes de la Universidad de Málaga. Honoré Daumier: la risa republicana se interna en la vida y la obra de este marsellés nacido en 1808 y muerto en París en 1879, cuyo trabajo se desarrolló durante cincuenta años decisivos de la historia de Francia, desde el final del periodo legitimista hasta los inicios de la III República.

Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting

NARCIS (Netherlands)

Agbo, E.E.C.; Majiwa, P.A.O.; Claassen, H.J.H.M.; Pas, te M.F.W.

2002-01-01

Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple

Genetics and phylogeny of genus Coilia in China based on AFLP markers

Science.gov (United States)

Yang, Qiaoli; Han, Zhiqiang; Sun, Dianrong; Xie, Songguang; Lin, Longshan; Gao, Tianxiang

2010-07-01

The taxonomy of Coilia has been extensively studied in China, and yet phylogenetic relationships among component taxa remain controversial. We used a PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP) to characterize and identify all four species of Coilia in China. We examined the genetic relationships of the four species of Coilia and a subspecies of Coilia nasus with AFLP. A total of 180 AFLP loci were generated from six primer combinations, of which 76.11% were polymorphic. The mean genetic distance between pairs of taxa ranged from 0.047 to 0.596. The neighbor-joining tree and UPGMA dendrogram resolved the investigated species into three separate lineages: (1) C. mystus, (2) C. grayii and (3) C. brachygnathus, C. nasus, and C. nasus taihuensis. Phylogenetic analysis of the AFLP data is inconsistent with current morphological taxonomic systems. The AFLP data indicated a close relationship among C. brachygnathus, C. nasus taihuensis, and C. nasus. Therefore, the two species described under Coilia ( C. brachygnathus and C. nasus taihuensis) are treated as synonyms of C. nasus.

Differentiation of Toxocara canis and Toxocara cati based on PCR-RFLP analyses of rDNA-ITS and mitochondrial cox1 and nad1 regions.

Science.gov (United States)

Mikaeili, Fattaneh; Mathis, Alexander; Deplazes, Peter; Mirhendi, Hossein; Barazesh, Afshin; Ebrahimi, Sepideh; Kia, Eshrat Beigom

2017-09-26

The definitive genetic identification of Toxocara species is currently based on PCR/sequencing. The objectives of the present study were to design and conduct an in silico polymerase chain reaction-restriction fragment length polymorphism method for identification of Toxocara species. In silico analyses using the DNASIS and NEBcutter softwares were performed with rDNA internal transcribed spacers, and mitochondrial cox1 and nad1 sequences obtained in our previous studies along with relevant sequences deposited in GenBank. Consequently, RFLP profiles were designed and all isolates of T. canis and T. cati collected from dogs and cats in different geographical areas of Iran were investigated with the RFLP method using some of the identified suitable enzymes. The findings of in silico analyses predicted that on the cox1 gene only the MboII enzyme is appropriate for PCR-RFLP to reliably distinguish the two species. No suitable enzyme for PCR-RFLP on the nad1 gene was identified that yields the same pattern for all isolates of a species. DNASIS software showed that there are 241 suitable restriction enzymes for the differentiation of T. canis from T. cati based on ITS sequences. RsaI, MvaI and SalI enzymes were selected to evaluate the reliability of the in silico PCR-RFLP. The sizes of restriction fragments obtained by PCR-RFLP of all samples consistently matched the expected RFLP patterns. The ITS sequences are usually conserved and the PCR-RFLP approach targeting the ITS sequence is recommended for the molecular differentiation of Toxocara species and can provide a reliable tool for identification purposes particularly at the larval and egg stages.

AFLP marker linked to water-stress-tolerant bulks in barley (Hordeum vulgare L.

Directory of Open Access Journals (Sweden)

A. Altinkut

2003-01-01

Full Text Available The amplified fragment length polymorphism (AFLP assay is an efficient method for the identification of molecular markers, useful in the improvement of numerous crop species. Bulked Segregant Analysis (BSA was used to identify AFLP markers associated with water-stress tolerance in barley, as this would permit rapid selection of water-stress tolerant genotypes in breeding programs. AFLP markers linked to water-stress tolerance was identified in two DNA pools (tolerant and sensitive, which were established using selected F2 individuals resulting from a cross between water-stress-tolerant and sensitive barley parental genotypes, based on their paraquat (PQ tolerance, leaf size, and relative water content (RWC. All these three traits were previously shown to be associated with water-stress tolerance in segregating F2 progeny of the barley cross used in a previous study. AFLP analysis was then performed on these DNA pools, using 40 primer pairs to detect AFLP fragments that are present/absent, respectively, in the two pools and their parental lines. One separate AFLP fragment, which was present in the tolerant parent and in the tolerant bulk, but absent in the sensitive parent and in the sensitive bulk, was identified. Polymorphism of the AFLP marker was tested among tolerant and sensitive F2 individuals. The presence of this marker that is associated with water-stress tolerance will greatly enhance selection for paraquat and water-stress tolerant genotypes in future breeding programs.

A first linkage map of globe artichoke (Cynara cardunculus var. scolymus L.) based on AFLP, S-SAP, M-AFLP and microsatellite markers.

Science.gov (United States)

Lanteri, S; Acquadro, A; Comino, C; Mauro, R; Mauromicale, G; Portis, E

2006-05-01

We present the first genetic maps of globe artichoke (Cynara cardunculus var. scolymus L. 2n=2x=34), constructed with a two-way pseudo-testcross strategy. A F1 mapping population of 94 individuals was generated between a late-maturing, non-spiny type and an early-maturing spiny type. The 30 AFLP, 13 M-AFLP and 9 S-SAP primer combinations chosen identified, respectively, 352, 38 and 41 polymorphic markers. Of 32 microsatellite primer pairs tested, 12 identified heterozygous loci in one or other parent, and 7 were fully informative as they segregated in both parents. The female parent map comprised 204 loci, spread over 18 linkage groups and spanned 1330.5 cM with a mean marker density of 6.5 cM. The equivalent figures for the male parent map were 180 loci, 17 linkage groups, 1239.4 and 6.9 cM. About 3% of the AFLP and AFLP-derived markers displayed segregation distortion with a P value below 0.01, and were not used for map construction. All the SSR loci were included in the linkage analysis, although one locus did show some segregation distortion. The presence of 78 markers in common to both maps allowed the alignment of 16 linkage groups. The maps generated provide a firm basis for the mapping of agriculturally relevant traits, which will then open the way for the application of a marker-assisted selection breeding strategy in this species.

Verecundia, risa y decoro: Cicerón y el arte de insultar

Directory of Open Access Journals (Sweden)

Mas, Salvador

2015-12-01

estrategia, a despecho de su efectividad y por la misma, puede sobrepasar los límites de lo aceptable o de lo provechoso. Cuestión tanto más grave cuanto que el orador puede y debe reírse de asuntos que, considerados en sí mismos, carecen de gracia. La retórica ciceroniana de la risa plantea y desemboca en una ética y una táctica del humor atenta al estudio y posible codificación de los principios rectores de tan poderoso recurso, que debe utilizarse de manera conveniente, adecuada y decorosa (pues de todas estas formas puede traducirse la palabra griega prépon y la latina decorum, lo cual presupone que no siempre es así y, por lo que interesa en estas páginas, que hay risas indebidas, próximas al vituperio, que pueden acarrear dificultades a quien las provoca, pues siempre es peligroso reírse de los poderosos. Cicerón, sin duda, era un individuo que insultaba mucho, con ingenio, gracia y en ocasiones con extraordinaria agresividad.

The Consortium for Climate Risk in the Urban Northeast: A NOAA RISA Project

Science.gov (United States)

Rosenzweig, C.

2011-12-01

The Consortium for Climate Risk in the Urban Northeast, or CCRUN, was funded in October 2010 under NOAA's Regional Integrated Sciences and Assessments (RISA) program to serve stakeholder needs in assessing and managing risks from climate variability and change. It is currently also the only RISA team with a principal focus on climate change adaptation in urban settings. While CCRUN's initial focus is on the major cities of the urban Northeast corridor (Philadelphia, New York and Boston), its work will ultimately expand to cover small and medium-sized cities in the relevant portions of Massachusetts, Rhode Island, Connecticut, New York, New Jersey and Pennsylvania as well, so that local needs for targeted climate-risk information can be served in a coordinated way. CCRUN is designed to address the complex challenges that are associated with densely populated, highly interconnected urban areas, including such as urban heat island effects; poor air quality; intense coastal development, and multifunctional settlement along inland waterways; complex overlapping institutional jurisdictions; integrated infrastructure systems; and highly diverse, and in some cases, fragile socio-economic communities. These challenges can best be addressed by the stakeholder-driven interdisciplinary approach taken by the CCRUN RISA team. As an important added benefit, the research accomplishments and lessons learned through stakeholder engagement will provide a foundation for managing climate risks in other urban areas in the United States. CCRUN's initial projects are focused in three broad sectors: Water, Coasts, and Health. Research in each of these sectors is linked through the cross-cutting themes of climate change and community vulnerability, the latter of which is especially important in considerations of environmental justice and equity. CCRUN's stakeholder-driven approach to research can therefore support investigations of the impacts of a changing climate, population growth, and

An AFLP marker linked to turnip mosaic virus resistance gene in pak ...

African Journals Online (AJOL)

STORAGESEVER

2009-06-03

Jun 3, 2009 ... polymorphism (AFLP) technique and bulked segregant analysis (BSA) method were used to study the. F2 population. An AFLP marker ... TuRB03 (Hughes et al., 2003), TuRB04-05 (Jenner et al.,. 2002), ConTR01 ... The resistance evaluation was done by visual observation and direct enzyme-linked ...

A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses.

Science.gov (United States)

Yamamoto, F; Yamamoto, M

2004-07-01

We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme NotI in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15 x 2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.

Characterization of four salsola species and their genetic relationship by aflp

International Nuclear Information System (INIS)

Amal, M.E.; Hamid, A.

2016-01-01

Amplified length polymorphism (AFLP) technique was used to characterize and detect molecular genetic markers for four Salsola species collected from Al Jouf region in the northern of Saudi Arabia and to shed light on their genetic relationships. Three primer combinations were used for AFLP analysis of the four Salsola species, they generated a total of 181 fragments of which 133 were species specific markers scored across Salsola species. The dendogram produced by Jaccard's coefficient and the UPGMA clustering method showed one main cluster, subdivided into two subclusters. The first sub cluster included Salsola schweinfurthii and Salsola tetrandra. The second sub cluster included Salsola villosa and Salsola cyclophylla. It is worth mentioning that this is the first study to use AFLP markers to characterize and detect molecular genetic markers for the four Salsola species and their genetic relationships. (author)

(L.) Dunal using RAPD and AFLP markers

African Journals Online (AJOL)

ajl yemi

2011-10-26

Oct 26, 2011 ... Fragment Length Polymorphism (AFLP) markers. Eighteen ... importance due to its simplicity, efficiency, relative ease .... nation, number of polymorphic bands, percentage polymorphism .... roots, stems, leaves, flowers, pollen grains, mature fruits ... genetic changes that isolated it from the wild species.

Biochemical and genetic variation of some Syrian wheat varieties using NIR, RAPD and AFLPs techniques

International Nuclear Information System (INIS)

Saleh, B.

2012-01-01

This study was performed to assess chemical components and genetic variability of five Syrian wheat varieties using NIR, RAPD and AFLP techniques. NIR technique showed that Cham6 was the best variety in term of wheat grain quality due to their lowest protein (%), hardness, water uptake and baking volume and the highest starch (%) compared to the other tested varieties. PCR amplifications with 21 RAPD primers and 13 AFLP PCs primer combinations gave 104 and 466 discernible loci of which 24 (18.823%) and 199 (45.527%) were polymorphic for the both techniques respectively. Our data indicated that the three techniques gave similar results regarding the degree of relatedness among the tested varieties. In the present investigation, AFLP fingerprinting was more efficient than the RAPD assay. Where the letter exhibited lower Marker Index (MI) average (0.219) compared to AFLP one (3.203). The pattern generated by RAPD, AFLPs markers or by NIR separated the five wheat varieties into two groups. The first group consists of two subclusters. The first subcluster involved Cham8 and Bohous6, while the second one includes Cham6 that is very closed to precedent varieties. The second group consists of Bohous9 and Cham7 that were also closely related. Based on this study, the use of NIR, RAPD and AFLP techniques could be a powerful tool to detect the effectiveness relationships of these technologies. (author)

AFLP studies on downy-mildew-resistant and downy-mildew-susceptible genotypes of opium poppy.

Science.gov (United States)

Dubey, Mukesh K; Shasany, Ajit K; Dhawan, Om P; Shukla, Ashutosh K; Khanuja, Suman P S

2010-04-01

Downy mildew (DM) caused by Peronospora arborescens, is a serious disease in opium poppy (Papaver somniferum), which has a world-wide spread. The establishment of DM-resistant cultivars appears to be a sustainable way to control the In this paper, we present the results of a study aimed at the identification of amplified fragment length polymorphism (AFLP) markers for DM-resistance in opium poppy. Three opium poppy genotypes (inbred over about 10 years): Pps-1 (DM-resistant), Jawahar-16 (DM-susceptible) and H-9 (DM-susceptible) were crossed in a diallel manner and the F(1) progeny along with the parents were subjected to AFLP analysis of chloroplast (cp) and nuclear DNA with seven and nine EcoRI / MseI primer combinations, respectively. cpDNA AFLP analysis identified 24 Pps-1 (DM-resistant)-specific unique fragments that were found to be maternally inherited in both the crosses, Pps-1 x Jawahar-16 and Pps-1 x H-9. In the case of nuclear DNA AFLP analysis, it was found that 17 fragments inherited from Pps-1 were common to the reciprocal crosses of both (i) Pps-1 and Jawahar-16 as well as (ii) Pps-1 and H-9. This is the first molecular investigation on the identification of polymorphism between DM-resistant and DM-susceptible opium poppy genotypes and development of DM-resistant opium poppy genotypespecific AFLP markers. These AFLP markers could be used in future genetic studies for analysis of linkage to the downy mildew resistance trait.

Distribution of Penicillium commune isolates in cheese dairies mapped using secondary metabolite profiles, morphotypes, RAPD and AFLP fingerprinting

DEFF Research Database (Denmark)

Lund, Flemming; Nielsen, A.B.; Skouboe, P.

2003-01-01

) and amplified fragment length polymorphism, (AFLP). For a sub-set of 272 P. commune isolates RAPD analysis generated 33 RAPD groups whereas AFLP profiling revealed 55 AFLP groups. This study conclusively showed that the discriminatory power of AFLP was high compared to RAPD and that AFLP fingerprinting matched...... morphotyping, P. commune isolates with identical profiles using all four typing techniques were interpreted as closely related isolates with a common origin and the distribution of these isolates in the processing environment indicated possible contamination points in the cheese dairies. The coating process...... and unpacking of cheeses with growth of P. commune seemed to cause the contamination problems. Several identical P. commune isolates remained present in the processing environment for more than 7 years in both dairies....

Detecting the influence of ornamental Berberis thunbergii var. atropurpurea in invasive populations of Berberis thunbergii (Berberidaceae) using AFLP1.

Science.gov (United States)

Lubell, Jessica D; Brand, Mark H; Lehrer, Jonathan M; Holsinger, Kent E

2008-06-01

Japanese barberry (Berberis thunbergii DC.) is a widespread invasive plant that remains an important landscape shrub represented by ornamental, purple-leaved forms of the botanical variety atropurpurea. These forms differ greatly in appearance from feral plants, bringing into question whether they contribute to invasive populations or whether the invasions represent self-sustaining populations derived from the initial introduction of the species in the late 19th century. In this study we used amplified fragment length polymorphism (AFLP) markers to determine whether genetic contributions from B. t. var. atropurpurea are found within naturalized Japanese barberry populations in southern New England. Bayesian clustering of AFLP genotypes and principal coordinate analysis distinguished B. t. var. atropurpurea genotypes from 85 plants representing five invasive populations. While a single feral plant resembled B. t. var. atropurpurea phenotypically and fell within the same genetic cluster, all other naturalized plants sampled were genetically distinct from the purple-leaved genotypes. Seven plants from two different sites possessed morphology consistent with Berberis vulgaris (common barberry) or B. ×ottawensis (B. thunbergii × B. vulgaris). Genetic analysis placed these plants in two clusters separate from B. thunbergii. Although the Bayesian analysis indicated some introgression of B. t. var. atropurpurea and B. vulgaris, these genotypes have had limited influence on extant feral populations of B. thunbergii.

Divergência genética entre genótipos de alface por meio de marcadores AFLP Genetics divergence among lettuce genotypes by AFLP markers

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Cristina Soares de Sousa

2007-01-01

Full Text Available Considerando a restrita diversidade de espécies disponíveis para nutrir a carência de vitaminas no Brasil, Kerr e colaboradores, desde 1981, vêm desenvolvendo pesquisas para melhoramento genético de hortaliças ricas em vitamina A. Dentre elas, obtiveram uma cultivar de alface, denominada Uberlândia 10.000 com 10.200 UI de vitamina A em 100 gramas de folha fresca. Este trabalho objetivou comparar o grau de divergência genética entre a cultivar Uberlândia 10.000 e seus parentais para avaliar a eficiência da seleção utilizada, por meio da técnica AFLP. Foram utilizados os seguintes genótipos de alface: Maioba, Salad Bowl-Mimosa, Moreninha-de-Uberlândia, Vitória de Santo Antão, Uberlândia 10.000 lisa 8.ª e 9.ª geração e Uberlândia 10.000 crespa 8.ª e 9.ª geração. A técnica AFLP foi eficiente para identificar genótipos muito próximos e para estudos de progênies em alface. O primer PR15 permitiu a separação da forma lisa e crespa com 1,8% de divergência genética e a oitava da nona geração com apenas 0,71%. Com o estudo da filogenia da cultivar pode-se observar que o programa de melhoramento foi desenvolvido com sucesso, pois a cultivar obtida Uberlândia 10.000 possui alto teor de vitamina A e 92% de similaridade com o parental Vitória de Santo Antão. O primer PR11 conseguiu identificar polimorfismo entre cultivares de alta e baixa resistência à septoriose, sugerindo a possibilidade destas bandas estarem relacionadas à resistência.Considering the restricted diversity of species available to counteract vitamin deficiencies in Brazil, Kerr and coworkers have been engaged since 1981, in developing genetic improved garden vegetables rich in vitamin A. One of these vegetables is the lettuce cultivar Uberlândia 10,000, which contains 10,200 UI of vitamin A per 100 grams of fresh leaves. This study compares the genetic diversity between Uberlândia 10,000 and its parental, evaluating selection efficiency through

Genomic variations of Mycoplasma capricolum subsp capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

DEFF Research Database (Denmark)

Kokotovic, Branko; Bolske, G.; Ahrens, Peter

2000-01-01

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size...

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

Science.gov (United States)

Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

2017-03-09

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

Marcadores fAFLP na caracterização de três genótipos de umezeiro selecionados como porta-enxertos para pessegueiro fAFLP markers to characterize three mume genotypes selected as rootstocks for peach tree

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Ester Wickert

2007-12-01

Full Text Available O objetivo deste trabalho foi caracterizar a diversidade genética existente em três genótipos de umezeiro (Clone 05, cv. Rigitano e Clone 15 e identificar marcadores moleculares fAFLP (fluorescent Amplified Fragment Lenght Polymorphism passíveis de serem utilizados na discriminação dos três genótipos de umezeiro selecionados como porta-enxertos para pessegueiro. Foram utilizadas 24 diferentes combinações de primers seletivos fAFLP que geraram 648 marcas, das quais 272 foram diferenciadoras dos três genótipos entre si. As marcas diferenciadoras permitiram o agrupamento dos clones de umezeiro de acordo com sua similaridade através do Método da Distância e algorítmo Neighbour Joining. As mesmas marcas foram utilizadas para calcular a distância genética entre os clones. Com o uso de marcadores fAFLP foi possível discriminar os três genótipos de umezeiro entre si, destacando-se as combinações Fam ACT/CAT, Joe AGG/CTT e Ned AGC/CAA, que permitiram a diferenciação individual de cada um dos clones. A maior distância genética foi encontrada entre a cv. Rigitano e o Clone 15. Os marcadores fAFLP revelaram maior proximidade genética entre o Clone 05 e a cv. Rigitano.The objective of this work was the identification of fAFLP markers to be used in molecular characterization of three mume genotypes selected as rootstocks for peach tree. Twenty-four different fAFLP primer combinations were used and allowed the recognition of 648 markers, comprising 272 markers which were able to discriminate the three clones one from the other. These markers were used to calculate the groupment of the clones according to their similarities with the distance method and neighbour joining algorithm. The same markers were also used to calculate the genetic distance among the clones. The fAFLP markers were efficient to identify the clones, mainly by the combinations of selective primers Fam ACT/CAT, Joe AGG/CTT and Ned AGC/CAA. fAFLP markers allowed the

Comparison of PCR-RFLP pattern with sequencing analysis of the ITS region of Hyrcanain\\'s Tilia

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Hamed Yousefzadeh

2014-01-01

T. hyrcana and T. rubra from Hyrcanian's origin, but it could not separate T. begonifloia from the other hyrcanian species. In this respect, derived results were similar to sequencing one. In conclusion, with regard to less expensive and less time consuming PCR-RFLP technique and high similarity between its result with sequencing, we recommend this method as a simple and economical method with relatively high efficiency studding plant phylogeny.

Cyclic di-GMP regulation of the bvg-repressed genes and the orphan response regulator RisA in Bordetella pertussis

Science.gov (United States)

Expression of Bordetella pertussis virulence factors is activated by the BvgAS two-component system. Under modulating growth conditions BvgAS indirectly represses another set of genes through the action of BvgR, a bvg-activated protein. BvgR blocks activation of the response regulator RisA which is ...

Use of AFLPs to differentiate between Fusarium species causing ...

African Journals Online (AJOL)

STORAGESEVER

2009-09-15

Fusarium spp. and. Helmintosporium sativum) diseases are common. The aim of this study was to use the AFLP technique to determine variation and genetic relationships between Syrian Fusarium isolates; and compare them.

A Three-Legged Stool or Race? Governance Models for NOAA RISAs, DOI CSCs, and USDA Climate Hubs

Science.gov (United States)

Foster, J. G.

2014-12-01

NOAAs Regional Integrated Sciences and Assessments (RISA) Teams, DOIs Climate Science Centers (CSCs), and USDAs Regional Climate Hubs (RCHs) have common missions of integrating climate and related knowledge across scientific disciplines and regions to create "actionable" information that decision-makes can use to manage climate risks and impacts at state and local scales. However, the sponsoring agency programs, university investigators, and local federal officials govern each differently. The three models of program and center governance are 1) exclusively university (RISAs), 2) a hybrid of Federal government and (host) university (CSCs,), and 3) exclusively Federal (Hubs). Each model has it's advantages and disadvantages in terms of legal definition and authority, scientific mission requirements and strategies, flexibility and legitimacy to conduct research and to collaborate regionally with constituencies, leadership and governance approach and "friction points,", staff capacity and ability to engage stakeholders, necessity to deliver products and services, bureaucratic oversight, performance evaluation, and political support at Congressional, state, and local levels. Using available sources of information and data, this paper will compare and contrast the strengths and weakness of these three regional applied climate science center governance models.

A Three-Legged Stool or Race? Governance Models for NOAA RISAs, DOI CSCs, and USDA Climate Hub

Science.gov (United States)

Foster, J. G.

2014-12-01

NOAAs Regional Integrated Sciences and Assessments (RISA) Teams, DOIs Climate Science Centers (CSCs), and USDAs Regional Climate Hubs (RCHs) have common missions of integrating climate and related knowledge across scientific disciplines and regions to create "actionable" information that decision-makes can use to manage climate risks and impacts at state and local scales. However, the sponsoring agency programs, university investigators, and local federal officials govern each differently. The three models of program and center governance are 1) exclusively university (RISAs), 2) a hybrid of Federal government and (host) university (CSCs,), and 3) exclusively Federal (Hubs). Each model has it's advantages and disadvantages in terms of legal definition and authority, scientific mission requirements and strategies, flexibility and legitimacy to conduct research and to collaborate regionally with constituencies, leadership and governance approach and "friction points,", staff capacity and ability to engage stakeholders, necessity to deliver products and services, bureaucratic oversight, performance evaluation, and political support at Congressional, state, and local levels. Using available sources of information and data, this paper will compare and contrast the strengths and weakness of these three regional applied climate science center governance models.

Phylogenic analysis in Acacia senegal using AFLP molecular ...

African Journals Online (AJOL)

Amplified fragment length polymorphism (AFLP) DNA markers were used to characterize the genetic diversity and relationships in gum Arabic tree (Acacia senegal). Twenty eight samples of Acacia senegal collected from populations distributed throughout the Gum Arabic belt were tested in comparison with samples of ...

Risa y aprendizaje: el papel del humor en la labor docente

Directory of Open Access Journals (Sweden)

Eduardo Jáuregui Narváez

2009-01-01

Full Text Available Numerosas corrientes pedagógicas en las últimas décadas han fomentado el humor, la diversión y la risa en la labor docente, citando numerosos beneficios, establecer una mejor relación con los estudiantes, reducir el estrés y la ansiedad, gestionar el conflicto, proporcionar una recompensa emocional que motive la participación y el estudio, y comunicar la materia más eficazmente, estimulando la atención, la creatividad y la memoria. En este artículo se evalúa la justificación teórica y empírica de este modelo más "lúdico" del aprendizaje, y se matizan algunas de las ideas que suelen proponerse en este sentido.

Amplified fragment length polymorphism (AFLP) and genealogy ...

African Journals Online (AJOL)

STORAGESEVER

2010-06-07

Jun 7, 2010 ... Pang CY, Du XM, Ma ZY (2006). Evaluation of the introgressed lines and screening for elite germplasm in Gossypium, Chin. Sci. Bull. 51(1):. 304-312. Pieter V, Rene H, Marjo B (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23(21): 4407-4414. Qian SY, Huang JQ, Zhou BL, Peng ...

Genetic structure in cultivated and wild carrots (¤Daucus carota¤ L.) revealed by AFLP analysis

DEFF Research Database (Denmark)

Shim, S.I.; Bagger Jørgensen, Rikke

2000-01-01

Genetic variation within and among five Danish populations of wild carrot and five cultivated varieties was investigated using amplified fragment length polymorphism (AFLP). Ten AFLP primer combinations produced 116 polymorphic bands. Based on the marker data an UPGMA-cluster analysis and principal...

Application of fluorescence-based semi-automated AFLP analysis in barley and wheat

DEFF Research Database (Denmark)

Schwarz, G.; Herz, M.; Huang, X.Q.

2000-01-01

of semi-automated codominant analysis for hemizygous AFLP markers in an F-2 population was too low, proposing the use of dominant allele-typing defaults. Nevertheless, the efficiency of genetic mapping, especially of complex plant genomes, will be accelerated by combining the presented genotyping......Genetic mapping and the selection of closely linked molecular markers for important agronomic traits require efficient, large-scale genotyping methods. A semi-automated multifluorophore technique was applied for genotyping AFLP marker loci in barley and wheat. In comparison to conventional P-33...

Identification of molecular markers associated with fruit traits in olive and assessment of olive core collection with AFLP markers and fruit traits.

Science.gov (United States)

Ipek, M; Seker, M; Ipek, A; Gul, M K

2015-03-31

The purpose of this study was to characterize olive core collection with amplified fragment length polymorphism (AFLP) markers and fruit traits and to determine AFLP markers significantly associated with these fruit characters in olive. A total of 168 polymorphic AFLP markers generated by five primer combinations and nine fruit traits were used to characterize relationships between 18 olive cultivars. Although all olive cultivars were discriminated from each other by either AFLP markers (markers and fruit traits was not significantly correlated (r = 0.13). Partial clustering of olive cultivars by AFLP markers according to their geographical origin was observed. Associations of AFLP markers with fruits were determined using a multiple-regression analysis with stepwise addition of AFLP markers. Significant associations between eight AFLP markers and fruit traits were identified. While five AFLP markers demonstrated significant negative correlation with fruit and stone weight, width and length and total polyphenols (P markers displayed significant positive correlation with α-tocopherol and γ-tocopherol (P molecular markers with fruit traits in olive. Molecular markers associated with morphological and agronomic traits could be utilized for the breeding of olive cultivars. However, the association power of these markers needs to be confirmed in larger populations, and highly correlated markers should then be converted to PCR-based DNA markers such as sequence-characterized amplified region markers for better utilization.

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

Science.gov (United States)

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Molecular characterization of native potato (Solanum spp. Chungui, Ayacucho, using AFLP

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Juan C. Gonzales Mamani

2014-12-01

Full Text Available Genetic diversity of 25 morphotypes of native potatoes Solanum spp. from Chungui (La Mar, Ayacucho were assess. Morphotypes collected were micropropagated in Murashigue Skoog medium (1962. DNA extraction proceeded using the CTAB method modified from 3 weeks leaves crop, good quality and quantity of DNA was able to use the AFLP. Enzymatic digestion of the DNA was performed using EcoRI and MseI. Three combinations of AFLP primers with three selective nucleotides were used, resulting in a total of 85 clearly discernable bands, of which 63 were polymorphic. The combination E37 – M50 showed the most informative polymorphic index content of 0.43. The presence/absence of polymorphic bands was evaluated using the Simple Matching coefficient similarity and clustering analysis using the UPGMA. The dendrogram produced had a cophenetic correlation coefficient r= 0.7. At the level 0.64 of Simple Matching coefficient similarity, the dendrogram grouped the morphotypes of native potatoes in 4 genetic groups, it not found duplicated morphotypes, despite having some morphotypes very similar. Our results would be showing the highly informative power of AFLP markers for the analysis of genetic diversity of native potatoes.

Using aflp to identify genetic relationships in cassia species from Thailand

International Nuclear Information System (INIS)

Sihanat, A.; Rungsihirunrat, K.; Chareonsap, P. P.; Ruangrungsi, N.

2017-01-01

Several species of Cassia are used in Thai folk medicine as a laxative and a treatment for skin infections. However, the taxonomy of the GenusCassia is quite complex and intriguing. Thus, the correct identification of the species of this genus is necessary for efficacy and safety. The phylogenetic relationships among the 16 species of Cassiagenus existing in Thailand were evaluated using Amplified Fragment Length Polymorphism (AFLP) technique. Combinations of 70 primers were screened and eleven primer combinations produced a total of 849 distinct and reproducible bands ranging from 60 to 100 bands with an average of 77.18 bands per primer combination. The genetic distances were calculated based on the AFLP bands that had been amplified using the eleven primer combinations. The similarity indices (SI) ranged from 0.25 to 0.78. The dendrogram was created using the Unweighted Pair Group Method of the Arithmetic Average (UPGMA) and the genotypes were divided into two major groups. The results indicate that the phylogenetic relationships are associated with the morphological characterization. In conclusion, an AFLP marker could be an efficient and reliable tool for the identification of a Cassia species. (author)

Assessment of genetic diversity in Chinese eared pheasant using fluorescent-AFLP markers

DEFF Research Database (Denmark)

Li, Xiujuan; Zhu, Yaohong; Liu, Panqi

2010-01-01

on the list of the world’s threatened species. In this paper, 74 individuals from the four eared pheasant species were assessed for population genetic diversity by means of fluorescent-AFLP markers. A total of 429 AFLP peaks were amplified by 11 pairs of fluorescent EcoRI/TaqI primer combinations. Out of all...... using Jaccard’s similarity coefficients (SC) and the corresponding dendrogram. It was found that there was a moderate genetic distance between the four species (SC = 0.674–0.832). Brown eared pheasant was genetically closely related to blue eared pheasant (SC = 0.832), while white eared pheasant...

Terapia de la risa en un grupo de mujeres adultas/ Laughter therapy in a group of elderly women

Directory of Open Access Journals (Sweden)

María M. Villam

2013-05-01

Full Text Available Objetivo: explorar los beneficios de la terapia de la risa en un grupo de mujeres adultas pertenecientes a un centro gerontológico del Municipio de Envigado (Colombia. Metodología: estudio con enfoque cualitativo mediante entrevista semiestructurada a 10 adultas entre 59 y 97 años de edad, antes y después de la aplicación de 5 sesiones, una semanal, de terapia de la risa por parte del grupo Mediclaun “payasos hospitalarios”. Resultados: muchos de los cambios percibidos en las adultas coinciden con la literatura en lo que respecta a una actitud más positiva, incremento de la confianza en el otro y la aceptación; expresan el pasado con menos dolor y un mayor agrado frente al acompañamiento familiar. Conclusiones: La terapia de la risa permite al adulto mayor encontrar fortalezas que puedan ayudarle a mejorar su presente, a su vez, lograr cambios en el individuo que se le revierten positivamente y a su relación con el entorno Objective: to explore the benefits of laughter therapy in a group of elderly women belonging to a gerontology center (Envigado, Colombia. Methodology: a qualitative study using a semi-structured interview with 10 adult women aged 59 to 97. The participants were interviewed before and after applying 5 weekly sessions of laughter therapy. Said therapy was administered by the Mediclaun "hospital clowns" group. Results: many of the changes observed in adult women are consistent with the results described in the literature in relation to a more positive attitude, increased confidence in people and acceptance; the participants expressed the past with less pain and had a greater liking towards family support. Conclusions: laughter therapy allows seniors to find the strength to improve their present situation, thus causing positive changes in their own self and in their relationship with their surroundings

Estudo da diversidade genética de Podosphaera xanthii através de marcadores AFLP e seqüências ITS

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Erika Sayuri Naruzawa

2011-06-01

Full Text Available O meloeiro (Cucumis melo L. é uma frutífera largamente cultivada no Brasil, principalmente no nordeste brasileiro, onde é produzida principalmente para a exportação. Plantas da família do meloeiro, como pepino e abóbora, podem ser severamente afetadas pelo oídio, causado por Podosphaera xanthii.. Este fungo apresenta diversas raças fisiológicas cuja correta identificação é importante para o manejo da doença, já que o uso de variedades resistentes é o método mais eficaz de seu controle. No entanto, a identificação destas raças por meio da prática tradicional de inoculações em uma série diferenciadora de variedades de meloeiro é laboriosa e passível de erros. Devido a isso, um método alternativo seria o uso de marcadores moleculares para determinar de forma rápida a identidade das raças. O objetivo deste estudo foi o de analisar a variabilidade entre isolados de P. xanthii previamente classificados em raças através da técnica de AFLP e do seqüenciamento da região ITS 5.8S do rDNA. A partir dos marcadores AFLP obteve-se um dendrograma no qual não houve separação dos isolados quanto às suas raças, origem geográfica ou hospedeiro de origem. Com esta técnica verificou-se alta variabilidade entre isolados, com similaridade genética máxima de 69% e similaridade mínima de 23%. Ao contrário da informação gerada por AFLP, não foi observada variação na sequência da região ITS 5.8S entre isolados. Desta forma, a análise por AFLP indicou que os isolados tem composição genética heterogênea muito embora este fato não tenha sido evidenciado pelo sequenciamento da região ITS.

First isolation and RFLP genotyping of Toxoplasma gondii from crab-eating fox (Cerdocyon thous-Linnaeus, 1766).

Science.gov (United States)

de Almeida, Jonatas Campos; de Melo, Renata Pimentel Bandeira; de Morais Pedrosa, Camila; da Silva Santos, Marcelo; de Barros, Luiz Daniel; Garcia, João Luis; Porto, Wagnner José Nascimento; Mota, Rinaldo Aparecido

2017-05-01

Wild animals may play an important role in the transmission and maintenance of Toxoplasma gondii in the environment. The purpose of the present study was to isolate and genotype T. gondii from a free-ranging crab-eating fox (Cerdocyon thous-Linnaeus, 1766). A crab-eating fox in critical health condition was attended in a veterinary hospital in Recife, Pernambuco State, Brazil. The animal died despite emergency treatment. The brain was collected aseptically and destined for mouse bioassay. One isolate of T. gondii was obtained, and Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) was used to assess genetic variability at 11 markers (SAG1, SAG2, altSAG2, SAG3, BTUB, GRA6, c228, c292, L358, PK1 and APICO). A murine model was used to assess the virulence of the isolate. Using the PCR-RFLP, genotype ToxoDB #13 was identified, which is considered an atypical strain. The isolate was classified as avirulent in the murine model. This is the first study to report T. gondii infection in the crab-eating fox. Copyright © 2017 Elsevier B.V. All rights reserved.

Eukaryotic transcriptomics in silico: Optimizing cDNA-AFLP efficiency

NARCIS (Netherlands)

Stölting, K.N.; Gort, G.; Wüst, C.; Wilson, A.B.

2009-01-01

Background - Complementary-DNA based amplified fragment length polymorphism (cDNA-AFLP) is a commonly used tool for assessing the genetic regulation of traits through the correlation of trait expression with cDNA expression profiles. In spite of the frequent application of this method, studies on

Genetic diversity in some Turkish pepper (Capsicum annuum L ...

African Journals Online (AJOL)

STORAGESEVER

2009-09-15

Sep 15, 2009 ... RFLP, RAPD, AFLP, and micro-satellites have been beneficial by being ... List of pepper genotypes used in the experiment. Genotype. Type. Source. Classification of ..... Artificial selection might decrease the genetic diversity ...

Evaluation of interspecific DNA variability in poplars using AFLP and ...

African Journals Online (AJOL)

STORAGESEVER

2009-10-19

Oct 19, 2009 ... Both markers crearly separated two distinct clusters, one included Populus nigra and the other ... Species of Populus used to test SSR and AFLP primer pair utility. ..... cluster NS001 and NS002 were closely related to 0.273.

First genetic linkage map of Taraxacum koksaghyz Rodin based on AFLP, SSR, COS and EST-SSR markers.

Science.gov (United States)

Arias, Marina; Hernandez, Monica; Remondegui, Naroa; Huvenaars, Koen; van Dijk, Peter; Ritter, Enrique

2016-08-04

Taraxacum koksaghyz Rodin (TKS) has been studied in many occasions as a possible alternative source for natural rubber production of good quality and for inulin production. Some tire companies are already testing TKS tire prototypes. There are also many investigations on the production of bio-fuels from inulin and inulin applications for health improvement and in the food industry. A limited amount of genomic resources exist for TKS and particularly no genetic linkage map is available in this species. We have constructed the first TKS genetic linkage map based on AFLP, COS, SSR and EST-SSR markers. The integrated linkage map with eight linkage groups (LG), representing the eight chromosomes of Russian dandelion, has 185 individual AFLP markers from parent 1, 188 individual AFLP markers from parent 2, 75 common AFLP markers and 6 COS, 1 SSR and 63 EST-SSR loci. Blasting the EST-SSR sequences against known sequences from lettuce allowed a partial alignment of our TKS map with a lettuce map. Blast searches against plant gene databases revealed some homologies with useful genes for downstream applications in the future.

Insight into the genomic diversity and relationship of Astragalus glycyphyllos symbionts by RAPD, ERIC-PCR, and AFLP fingerprinting.

Science.gov (United States)

Gnat, Sebastian; Małek, Wanda; Oleńska, Ewa; Trościańczyk, Aleksandra; Wdowiak-Wróbel, Sylwia; Kalita, Michał; Wójcik, Magdalena

2015-11-01

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR one and these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8-100, 18-100, and 23-100%, respectively.

AFLP genome scanning reveals divergent selection in natural populations of Liriodendron chinense (Magnoliaceae along a latitudinal transect

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Aihong eYang

2016-05-01

Full Text Available Understanding adaptive genetic variation and its relation to environmental factors are important for understanding how plants adapt to climate change and for managing genetic resources. Genome scans for the loci exhibiting either notably high or low levels of population differentiation (outlier loci provide one means of identifying genomic regions possibly associated with convergent or divergent selection. In this study, we combined AFLP genome scan and environmental association analysis to test for signals of natural selection in natural populations of Liriodendron chinense (Chinese Tulip Tree; Magnoliaceae along a latitudinal transect. We genotyped 276 individuals from 11 populations of L. chinense using 987 AFLP markers. Two complementary methods (Dfdist and BayeScan and association analysis between AFLP loci and climate factors were applied to detect outlier loci. Our analyses recovered both neutral and potentially adaptive genetic differentiation among populations of L. chinense. We found moderate genetic diversity within populations and high genetic differentiation among populations with reduced genetic diversity towards the periphery of the species ranges. Nine AFLP marker loci showed evidence of being outliers for population differentiation for both detection methods. Of these, six were strongly associated with at least one climate factor. Temperature, precipitation and radiation were found to be three important factors influencing local adaptation of L. chinense. The outlier AFLP loci are likely not the target of natural selection, but the neighboring genes of these loci might be involved in local adaptation. Hence, these candidates should be validated by further studies.

Development of a simple and powerful method, cDNA AFLP-SSPAG ...

African Journals Online (AJOL)

AJB SERVER

2006-12-18

SSPAG was descry- bed. Using this method, Differential mRNA expression was found between high and low heterosis groups of maize. Differential cDNAs were easily obtained from silver stained cDNA-AFLP separated on.

Human papillomavirus detection and typing using a nested-PCR-RFLP assay.

Science.gov (United States)

Coser, Janaina; Boeira, Thaís da Rocha; Fonseca, André Salvador Kazantzi; Ikuta, Nilo; Lunge, Vagner Ricardo

2011-01-01

It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing.

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Directory of Open Access Journals (Sweden)

Bianca W. Bertoni

2010-01-01

Full Text Available Jacaranda decurrens (Bignoniaceae is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD and amplified fragment length polymorphism (AFLP. Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2% polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA and Pearson's correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006 between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.

PCR-RFLP diagnostic method for identifying Globodera species in Slovenia

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Sasa ŠIRCA

2011-01-01

Full Text Available Species identification within the genus Globodera is based on the morphological and morphometrical characters of the cysts and second stage juveniles, and these are included in the majority of identification keys. Morphometrical methods are fast and can be applied to most of samples but they demand a trained and experienced specialist. Furthermore, some morphometrical characters may overlap between populations and beetwen species, leading to inaccurate identification. To confirm and complement the morphometrical identification of Globodera species molecular methods have been developed. Sequences of the internal transcribed spacer regions ITS1 and ITS2 of the rDNA gene cluster proved to be useful for identifying nematode species identification. A PCR-RFLP molecular method was used to identify Globodera rostochiensis, G. pallida, G. tabacum and G. achilleae. Globodera rostochiensis, G. pallida, G. tabacum and G. achilleae can be distinguished with PCR-RFLP analysis of the rDNA ITS fragment using five restriction enzymes. The RFLP patterns of G. rostochiensis, G. tabacum and G. achilleae were species-specific, while those of G. pallida varied. South American populations of G. pallida differed from other populations as their RFLP patterns were demonstrated to be distinct by in silico restriction of the ITS sequences deposited at NCBI.

Genotyping of friesian horses to detect a hydrocephalus-associated c.1423C>T mutation in B3GALNT2 using PCR-RFLP and PCR-PIRA methods: Frequency in stallion horses in México.

Science.gov (United States)

Ayala-Valdovinos, Miguel Angel; Galindo-García, Jorge; Sánchez-Chiprés, David; Duifhuis-Rivera, Theodor

2017-04-01

Hydrocephalus in Friesian horses is an autosomal recessive hereditary disease that can result in an abortion, a stillbirth, or euthanization of a newborn foal. Here, the hydrocephalus-associated c.1423C > T mutation in B3GALNT2 gene was detected with PCR-RFLP and PCR-PIRA methods for horse genotyping. A preliminary genotyping survey was performed on 83 randomly selected Friesian stallion horses to determine the current allele frequency in Mexico. The frequency of the mutant T allele was 9.6%. Copyright © 2016 Elsevier Ltd. All rights reserved.

Some AFLP amplicons are highly conserved DNA sequences mapping to the same linkage groups in two F2 populations of carrot

Directory of Open Access Journals (Sweden)

Santos Carlos A.F.

2002-01-01

Full Text Available Amplified fragment length polymorphism (AFLP is a fast and reliable tool to generate a large number of DNA markers. In two unrelated F2 populations of carrot (Daucus carota L., Brasilia x HCM and B493 x QAL (wild carrot, it was hypothesized that DNA 1 digested with the same restriction endonuclease enzymes and amplified with the same primer combination and 2 sharing the same position in polyacrylamide gels should be conserved sequences. To test this hypothesis AFLP fragments from polyacrylamide gels were eluted, reamplified, separated in agarose gels, purified, cloned and sequenced. Among thirty-one paired fragments from each F2 population, twenty-six had identity greater than 91% and five presented identity of 24% to 44%. Among the twenty-six conserved AFLPs only one mapped to different linkage groups in the two populations while four of the five less-conserved bands mapped to different linkage groups. Of eight SCAR (sequence characterized amplified regions primers tested, one conserved AFLP resulted in co-dominant markers in both populations. Screening among 14 carrot inbreds or cultivars with three AFLP-SCAR primers revealed clear and polymorphic PCR products, with similar molecular sizes on agarose gels. The development of co-dominant markers based on conserved AFLP fragments will be useful to detect seed mixtures among hybrids, to improve and to merge linkage maps and to study diversity and phylogenetic relationships.

Identificación y mapeo de AFLPs ligados al gen de resistencia al PVX en Solanum commersonii

Directory of Open Access Journals (Sweden)

Mónica Blanco

2005-01-01

Full Text Available Solanum commersonii es una especie silvestre de papa considerada como una fuente de genes de resistencia al PVX. Para identificar marcadores moleculares relacionados con los genes de resistencia a este virus, se realizó un análisis en el que se combinó la técnica de BSA con el uso de AFLPs. Del cruce de 2 padres heterocigotos y resistentes al PVX, provenientes de una F1, se obtuvo una F2. La población fue inoculada con el PVXMS y 30 días después mediante un ELISA, la progenie fue dividida en individuos infectados y no infectados con el PVXMS; a estos 2 grupos se les realizó un BSA. El ADN de los individuos resistentes fue mezclado aparte del ADN de los individuos susceptibles y con la ayuda de AFLPs se logró identificar 22 combinaciones de imprimadores que produjeron bandas específicas relacionadas con el carácter de resistencia al PVX. Las combinaciones de imprimadores seleccionadas fueron utilizadas para evaluar cada uno de los individuos de la F2 en forma independiente. Producto de este análisis se obtuvo 63 bandas polimórficas relacionadas al carácter de resistencia, cuya información fue introducida en el programa MAPRF6. Como resultado se obtuvo 4 grupos de ligamiento. Se encontró un RGA, obtenido en otro estudio que co-segrega (0 cM con el locus del gen de resistencia extrema (Rx y los AFLPs 42 y 39 que están rodeando el mismo locus a 22,6 cM o más. La información obtenida será básica para implementar programas de selección asistida por marcadores moleculares en el mejoramiento genético.

AFLP genetic polymorphism in wild barley (Hordeum spontaneum) populations in Israel

NARCIS (Netherlands)

Turpeinen, T.; Vanhala, T.; Nevo, E.; Nissila, E.

2003-01-01

The genetic diversity produced by the amplified fragment length polymorphism (AFLP) method was studied in 94 genotypes of wild barley, Hordeum spontaneum (C. Koch) Thell., originating from ten ecologically and geographically different locations in Israel. Eight primer pairs produced 204 discernible

Cambios en la depresión y el sentimiento de soledad después de la terapia de la risa en adultos mayores internados

Directory of Open Access Journals (Sweden)

Ángela Quintero

2015-03-01

Conclusión. Se resalta la importancia que tiene la terapia de la risa en la disminución del grado de depresión en adultos mayores. Los resultados concuerdan con la afirmación de que el constructo de soledad es independiente del de depresión.

An integrated interspecific AFLP map of lettuce (Lactuca) based on two L. Saligna x L. sativa F2-populations

NARCIS (Netherlands)

Jeuken, M.; Wijk, van R.; Peleman, J.; Lindhout, P.

2001-01-01

AFLP markers were obtained with 12 EcoRI/ MseI primer combinations on two independent F2 populations of Lactuca sativa 2 Lactuca saligna. The polymorphism rates of the AFLP products between the two different L. saligna lines was 39°between the two different L. sativa cultivars 13 nd between the L.

Identification and characterization of some aromatic rice mutants using amplified fragment length polymorphism (AFLP) technique

International Nuclear Information System (INIS)

Fahmy, E.M.; Sobieh, S. E. S.; Ayaad, M. H.; El-Gohary, A. A.; Rownak, A.

2012-12-01

Accurate identifying of the genotypes is considered one of the most important mechanisms used in the recording or the protection of plant varieties. The investigation was conducted at the experimental form belonging to the egyptian Atomic Energy Authority, Inshas. The aim was to evaluate grain quality characteristics and molecular genetic variation using Amplified Fragment Length Polymorphism (AFLP) technique among six rice genotypes, Egyptian Jasmine aromatic rice cultivar and five aromatic rice mutants in (M3 mutagenic generation). Two mutation (Egy22 and Egy24) were selected from irradiated Sakha 102 population with 200 and 400Gy of gamma rays in the M2 generation, respectively, and three mutations ( Egy32, Egy33, and Egy34) were selected from irradiated Sakha 103 population with 200, 300, 400Gy of gamma rays in the M2 generation, respectively. The obtained results showed that the strong aroma was obtained for mutant Egy22 as compared with Egyptian Jasmine rice cultivar (moderate aroma). Seven primer combinations were used through six rice genotypes on the molecular level using AFLP marker. The size of AFLP Fragments Were Ranged from 51- 494bp. The total number of amplified bands was 997 band among them 919 polymorphic bans representing 92.2%. The highest similarity index (89%) was observed between Egyptian Jasmine and Egy32 followed by (82%) observed between Egyptian Jasmine and Egy34. On the other hand, the lowest similarity index was (48%) between Egyptian Jasmine and Egy24. In six rice genotypes, Egy24 produced the highest number of the AFLP makers giving 49 unique markers (23 positive and 26 negative), then Egy22 showed 23 unique markers (27 positive and 6 negative) while Egy33 was characterized by 17 unique markers (12 positive and 5 negative). At last Egyptian Jasmine was discriminated by the lowest number of markets, 10 (6 positive and 4 negative). The study further confirmed that AFLP technique was able to differentiate rice genotypes by a higher number

Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

DEFF Research Database (Denmark)

Høgdall, Estrid; Vuust, Jens; Lind, Peter

2000-01-01

of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T...

Caracterización de accesiones de papaya (Carica papaya L.) a través de marcadores AFLP en Cuba

OpenAIRE

Maruchi Alonso Esquivel; Martín Bautista Alor; Matilde Ortiz García; Adriana Quiroz Moreno; Wolfgang Rohde; Lorenzo Felipe Sánchez Teyer

2010-01-01

Los marcadores moleculares son herramientas valiosas en los estudios genéticos en plantas, y están siendo empleados exitosamente en programas de mejoramiento principalmente en la elección de progenitores y en la selección. El polimorfismo observado mediante la técnica molecular AFLP (Amplified Fragment Length Polymorphism) ha sido de utilidad para estudios de diversidad genética en frutales. En el presente trabajo se realizó la caracterización molecular de 12 accesiones de papaya (Carica papa...

Caracterización de accesiones de papaya (Carica papaya L.) a través de marcadores AFLP en Cuba

OpenAIRE

Esquivel, Maruchi Alonso; Bautista Alor, Martín; Ortiz García, Matilde; Quiroz Moreno, Adriana; Rohde, Wolfgang; Sánchez Teyer, Lorenzo Felipe

2009-01-01

Los marcadores moleculares son herramientas valiosas en los estudios genéticos en plantas, y están siendo empleados exitosamente en programas de mejoramiento principalmente en la elección de progenitores y en la selección. El polimorfismo observado mediante la técnica molecular AFLP (Amplified Fragment Length Polymorphism) ha sido de utilidad para estudios de diversidad genética en frutales. En el presente trabajo se realizó la caracterización molecular de 12 accesiones de papaya (Carica papa...

Sensitive identification of mycobacterial species using PCR-RFLP on bronchial washings.

Science.gov (United States)

Hidaka, E; Honda, T; Ueno, I; Yamasaki, Y; Kubo, K; Katsuyama, T

2000-03-01

In 98 patients (24 with active pulmonary tuberculosis [TB] lesions, 28 with cured TB lesions, and 46 with nontuberculous opacities [control group] in chest CT scans), we examined whether washing the bronchus after brushing the lesion, then applying polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to the bronchial washings might be useful for diagnosing TB and nontuberculous mycobacteriosis (NTMosis). After biopsy and brushing with a bronchoscope, the bronchus connecting to the lesion was washed with 20 ml saline. The saline used for washing the brushes (5 ml; brushing sample), and 3 to 10 ml saline aspirated through the forceps channel (washing sample) were examined by PCR-RFLP, which proved able to identify Mycobacterium tuberculosis and seven species of nontuberculous mycobacteria (NTM). The values obtained for the sensitivity of the PCR-RFLP with respect to the brushing sample, the washing sample, and both samples mixed together were 70, 76, and 91%, respectively, when only patients who were culture-positive or radiologically improved after antituberculous therapy were considered as showing true infection. A mixture of brushing and washing samples provides useful material for PCR and culture, and the PCR-RFLP used here is a good method for the simultaneous identification of several species of mycobacterium (including M. tuberculosis).

Assessment of Genetic Variation Among East African Cercospora ...

African Journals Online (AJOL)

Rapid flagement length polymorphism (RFLP) and amplified flagement length polymorphism (AFLP) analyses were used to study genetic diversity of Cercospora zeae-maydis isolates collected from Uganda, Kenya and Rwanda. For comparative purposes, isolates from Zimbabwe and the United States of America (USA) ...

AFLP analysis of rice transformed with maize DNA by particle beam

International Nuclear Information System (INIS)

Ji Shengdong; Chen Peng; Wang Jiachuan; Yuan Zhao; Yue Chunhui; Wang Zhifeng

2009-01-01

Many stable heritable rice lines were obtained via five years agricultural selection, which were derived from rice (oryza stative Japonica) Yujing-6 transgened with large fraction DNA of Zhengdan-14 (zea mays L.) by particle beam method. 18 pairs optimum selective primers were got by screening from 64 pairs AFLP selective primers via experiment on two mutant lines, which could amplify many DNA fingerprints and also could amplify polymorphic bands and target bands, both in this two mutant lines. Then the two mutant lines and two controls were analyzed with AFLP, the results showed that many polymorphic bands (such as novel bands, target bands, missing bands) were found in mutant lines. The discrepancy in DNA level indicated that rice, transgened with large fraction DNA of Zhengdan-14 by particle beam, might be inserted maize DNA and inherited steadily in some degree. It also indicated that it was possible to cultivate novel rice variety transformed with wide DNA by particle beam. (authors)

Characterization of Streptococcus suis through serotyping, SE-AFLP and virulence profile

Directory of Open Access Journals (Sweden)

Franco F. Calderaro

Full Text Available Abstract: Streptococcus suis is one of most important pathogens in the swine industry worldwide. Despite its importance, studies of S. suis characterization in South America are still rare. This study evaluates S. suis isolates from distinct Brazilian states, from 1999 to 2004, and its molecular and serological characterization. A total of 174 isolates were studied. S. suis identification was confirmed by PCR and isolates were further serotyped and genotyped by SE-AFLP and amplification of virulence markers. Serotype 1, 2, 3, 4, 7, 18, 22 and 32 were identified among the studied isolates, and only 4% were characterized as non-typeable. The mrp+/epf+/sly+ genotype was the most frequent. The SE-AFLP analysis resulted in 29 patterns distributed in three main clusters with over 65% of genetic similarity. Isolates presented a slight tendency to cluster according to serotype and origin; however, no further correlation with virulence genotypes was observed.

Diversidade genética entre híbridos de laranja-doce e tangor 'Murcott' avaliada por fAFLP e RAPD Genetic diversity among hybrids of sweet orange and 'Murcott' tangor evaluated by fAFLP and RAPD markers

Directory of Open Access Journals (Sweden)

Marinês Bastianel

2006-05-01

Full Text Available O objetivo deste trabalho foi avaliar a diversidade genética em uma população de 148 híbridos de tangor 'Murcott' (Citrus reticulata Blanco x C. sinensis L. Osbeck e laranja 'Pêra' (C. sinensis L. Osbeck obtidos por polinização controlada, pelo uso de marcadores fAFLP e RAPD. Marcadores polimórficos (416 marcadores fAFLP e 33 RAPD foram utilizados para avaliar a similaridade genética entre os híbridos, calculada com o coeficiente Jaccard pelo método UPGMA. A consistência de cada agrupamento foi determinada pelo programa BOOD. Houve alta similaridade genética entre os parentais. A laranja 'Pêra' apresentou maior número (132 de loci em heterozigose em relação ao tangor 'Murcott' (105, corroborando a teoria de origem híbrida para a laranja-doce. Observaram-se dois grupos distintos de plantas, e um deles abrangeu 80% dos híbridos com maior similaridade com a laranja 'Pêra'. A análise bootstrap não revelou consistência estatística entre esses grupos. Marcadores fAFLP são mais eficientes na avaliação do polimorfismo, sendo indicados para seleção de indivíduos híbridos mais próximos a um dos parentais.The objective of this work was to evaluate the genetic diversity in a population of 148 hybrids of 'Murcott' tangor (Citrus reticulata Blanco x C. sinensis L. Osbeck and 'Pêra' sweet orange (C. sinensis L. Osbeck, obtained by controlled polination, using fAFLP and RAPD markers. Polymorphic markers (416 fAFLP and 33 RAPD markers were used to evaluate genetic similarity among the hybrids, calculated by the coefficient of Jaccard, using the UPGMA method. The consistency of each group was determined by software BOOD. There was high genetic similarity within the parents. 'Pêra' sweet orange had a higher number of loci in heterozygosis (132 compared to 'Murcott' tangor (105, supporting the theory of hybrid origin for sweet oranges. Two distinct groups of plants were observed: one group had 80% of the hybrids that displayed

Alkaline Extraction of DNA from Pathogenic Fungi for PCR-RFLP Analysis

OpenAIRE

Matsumoto, Masaru; Mishima, Shinobu; Matsuyama, Nobuaki; 松元, 賢; 松山, 宣明

1997-01-01

For the preparation of DNA samples from fungal mycelia alkaline extraction method was applied and assessed its usefulness for PCR-RFLP analysis. Using alkaline treatment protocols, 18S ribosomal DNAs (rDNA) derived from fungal genomic DNA of Pyricularia oryzae, P. zingiberi, Rhizoctonia solani and R. oryzae were PCR-amplified and digested with Hha I, Msp I and Hae ill. RFLP analysis with HhaI showed the divergent polymorphism between genus Pyricularia and Rhizoctonia. The alkaline DNA extract...

Identification of five sea cucumber species through PCR-RFLP analysis

Science.gov (United States)

Lv, Yingchun; Zheng, Rong; Zuo, Tao; Wang, Yuming; Li, Zhaojie; Xue, Yong; Xue, Changhu; Tang, Qingjuan

2014-10-01

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene ( COI) was used to identifing five sea cucumber species ( Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

PCR-RFLP Method to Identify Salmonid Species of Economic Importance

Directory of Open Access Journals (Sweden)

Andreea Dudu

2011-05-01

Full Text Available The identification of different fish species by molecular methods has become necessary to avoid both the incorrect labelling of individuals involved in repopulation programmes and the commercial frauds on the fish market. Different fish species of great economical importance, like the salmonids, which are very much requested for their meat, can be identified using molecular techniques such as PCR-RFLP. The method is based on the amplification of a target region from the genome by PCR reaction followed by endonucleases digestion to detect the polymorphism of restriction fragments. In our study we analysed the following salmonid species from Romania: Salmo trutta fario, Salmo labrax, Salvelinus fontinalis, Onchorhynchus mykiss, Thymallus thymallus and Hucho hucho. In order to discriminate between the analysed species we amplified a fragment of mitochondrial genome comprising tRNAGlu/ cytochrome b/ tRNAThr/ tRNAPro/ D-loop/ tRNAPhe, followed by digestion with a specific restriction enzyme. The direct digestion of unpurified PCR products generated species-specific restriction patterns and proved to be a simple, reliable, inexpensive and fast method. Thus, it may be successfully utilized in specialized laboratories for the correct identification of the fish species for multiple purposes, including the traceability of fish food products.

A high-density consensus map of barley to compare the distribution of QTLs for partial resistance to Puccinia hordei and of defence gene homologues

NARCIS (Netherlands)

Marcel, T.C.; Varshney, R.K.; Barbieri, M.; Jafary, H.; Kock, de M.J.D.; Graner, A.; Niks, R.E.

2007-01-01

A consensus map of barley was constructed based on three reference doubled haploid (DH) populations and three recombinant inbred line (RIL) populations. Several sets of microsatellites were used as bridge markers in the integration of those populations previously genotyped with RFLP or with AFLP

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping

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Chang Hsueh-Wei

2010-04-01

Full Text Available Abstract Background PCR-restriction fragment length polymorphism (RFLP assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2. Results The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels, gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system. Conclusions The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping.

Science.gov (United States)

Chang, Hsueh-Wei; Cheng, Yu-Huei; Chuang, Li-Yeh; Yang, Cheng-Hong

2010-04-08

PCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2. The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system. The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.

Molecular identification of dermatophytosis by polymerase chain reaction (PCR and detection of source of infection by restricted fragment length polymorphism (RFLP

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BK Jha

2013-09-01

Full Text Available Introduction Dermatophytes are responsible for most superficial fungal infections and the estimated lifetime risk of acquiring a dermatophyte infection is between 10-20%. These fungi are mainly classified in three major genera Microsporum, Trichophyton and Epidermophyton. Materials and Methods Clinically suspected 200 cases of dermatophyte infected patients from K. R. Hospital Mysore and Mission Hospital Mysore were included in this descriptive study from January 2011 to June 2012. All the culture positive smear- 10% Potassium Hydroxide (KOH and culture in different dermatophytic medium patients were confirmed by PCR and source of infection was detected (n=10 from PCR positive patients and (n=10 from their domestic animals by PCR-RFLP methods targeting 18S rDNA regions of fungi. Results Out of 200 clinically suspected cases KOH mount was positive in 143 (71.5% cases and culture was positive in 132(66% cases. The isolates belonged to three genera and eight species as T.mentagrophytes 52(39.4%, T.rubrum 30(22.7%, T.violacium 18(13.6%, T.verrucosum 11(8.3%, E.floccosum 10(7.6%, M.canis 6(4.5%, T.tonsurans 03(2.3% and T.schollenii 2(1.5%. To identify the source of infection 10 animals ,one each from the houses of 10 patients who were PCR positive were also subjected to PCR and RFLP. The animals and the patients were found to be infected by same organisms T.verrucosum .This indicates that T.verrucosum infection is from animal source. Conclusion Dermatophytic infections are more common infectious disease. Preliminary diagnosis of dermatophytosis can be done by KOH mount and culture, which takes longer time to report and cannot differentiate at the genus and species level. Results indicate that PCR-RFLP may be considered as gold standard for the diagnosis and confirmation of source of infection of dermatophytosis and can aid the clinician in initiating prompt and appropriate antifungal therapy. Journal of College of Medical Sciences-Nepal, 2012, Vol-8

Genetic diversity of Chilean and Brazilian Alstroemeria species assessed by AFLP analysis

NARCIS (Netherlands)

Han, T.H.; Jeu, de M.J.; Eck, van H.J.; Jacobsen, E.

2000-01-01

One to three accessions of 22 Alstroemeria species, an interspecific hybrid (A. aurea ́ A. inodora), and single accessions of Bomarea salsilla and Leontochir ovallei were evaluated using the AFLP-marker technique to estimate the genetic diversity within the genus Alstroemeria. Three primer

DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

Science.gov (United States)

Rathore, Mangal S; Jha, Bhavanath

2016-03-01

The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.

Genetic markers and their application in livestock breeding in South ...

African Journals Online (AJOL)

The ultimate use of DNA markers would be to identify quantitative trait loci (QTL) in order to practice genotypic selection. This paper reviews DNA markers (RAPD, DFP, RFLP AFLP, minisatellites, microsatellites, SNP) and provides a brief overview of the current application of these markers in animal breeding.

Hemi-nested PCR and RFLP methodologies for identifying blood meals of the Chagas disease vector, Triatoma infestans.

Science.gov (United States)

Roellig, Dawn M; Gomez-Puerta, Luis A; Mead, Daniel G; Pinto, Jesus; Ancca-Juarez, Jenny; Calderon, Maritza; Bern, Caryn; Gilman, Robert H; Cama, Vitaliano A

2013-01-01

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by hematophagous reduviid bugs within the subfamily Triatominae. These vectors take blood meals from a wide range of hosts, and their feeding behaviors have been used to investigate the ecology and epidemiology of T. cruzi. In this study we describe two PCR-based methodologies that amplify a fragment of the 16S mitochondrial rDNA, aimed to improve the identification of blood meal sources for Triatoma infestans: a.--Sequence analyses of two heminested PCRs that allow the identification of mammalian and avian species, and b.--restriction fragment length polymorphism (RFLP) analysis from the mammalian PCR to identify and differentiate multi-host blood meals. Findings from both methodologies indicate that host DNA could be detected and the host species identified in samples from laboratory reared and field collected triatomines. The implications of this study are two-fold. First, these methods can be used in areas where the fauna diversity and feeding behavior of the triatomines are unknown. Secondly, the RFLP method led to the identification of multi-host DNA from T. infestans gut contents, enhancing the information provided by this assay. These tools are important contributions for ecological and epidemiological studies of vector-borne diseases.

AFLP markers for the assessment of genetic diversity in european and North American potato varieties cultivated in Iran

Directory of Open Access Journals (Sweden)

Saeed Tarkesh Esfahani

2009-01-01

Full Text Available Information about the genetic diversity of potato germplasm in Iran is important for variety identification andto enhance the classification of germplasm collections and exploit them in breeding programs and for the development andintroduction of new varieties. AFLP fingerprinting was applied to a group of cultivated potato varieties to find if there is anygeographical differentiation in potato diversity from Europe and North America. The high level of polymorphism within potatovarieties and the high number of variety-specific bands suggest that AFLPs are powerful markers for diversity analysis inpotato varieties. No region-specific AFLP markers were found (present in varieties from the same origin and absent inothers. The UPGMA dendrogram revealed four distinct clusters corresponding almost to the geographical origin of thevarieties. However, the bootstrap support for branches was rather weak. No clusters clearly distinguished varieties fromEurope and North America. Varieties from the same geographical origins however tended to group together within eachcluster. The mean similarity and the UPGMA dendrogram both suggest that North American varieties have nearly identicalgenetic diversity to European varieties. The results of AMOVA revealed large within-region variations which accounted for94.5% of the total molecular variance. The between-region variation, although accounting for only 5.5% of the total variation,was statistically significant. AFLP technology was successfully used to evaluate diversity between different geographicalgroups of potatoes and is recommended for potato genetic studies.

On some surprising statistical properties of a DNA fingerprinting technique called AFLP

NARCIS (Netherlands)

Gort, G.

2010-01-01

AFLP is a widely used DNA fingerprinting technique, resulting in band absence - presence profiles, like a bar code. Bands represent DNA fragments, sampled from the genome of an individual plant or other organism. The DNA fragments travel through a lane of an electrophoretic gel or microcapillary

Genetic Diversity of Hibiscus tiliaceus (Malvaceae) in China Assessed using AFLP Markers

Science.gov (United States)

TANG, TIAN; ZHONG, YANG; JIAN, SHUGUANG; SHI, SUHUA

2003-01-01

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variations within and among nine natural populations of Hibiscus tiliaceus in China. DNA from 145 individuals was amplified with eight primer pairs. No polymorphisms were found among the 20 samples of a marginal population of recent origin probably due to a founder effect. Across the other 125 individuals, 501 of 566 bands (88·5 %) were polymorphic, and 125 unique AFLP phenotypes were observed. Estimates of genetic diversity agreed with life history traits of H. tiliaceus and geographical distribution. AMOVA analysis revealed that most genetic diversity resided within populations (84·8 %), which corresponded to results reported for outcrossing plants. The indirect estimate of gene flow based on ϕST was moderate (Nm = 1·395). Long-distance dispersal of floating seeds and local environments may play an important role in shaping the genetic diversity of the population and the genetic structure of this species. PMID:12930729

AFLP/SSR mapping of resistance genes to Alectra vogelii in cowpea ...

African Journals Online (AJOL)

To find and map the resistance gene to A. vogelii in cowpea, a F2 population from a cross involving a resistant parent IT81D-994 and a susceptible TVX3236 was screened. Amplified fragment length polymorphism (AFLP) in combination with Single Sequence Repeat (SSR) analysis was used to identify markers that may be ...

Advance of molecular marker application in the tobacco research ...

African Journals Online (AJOL)

Tobacco (Nicotiana spp.) is one of the most important commercial crops in the world. During the last two decades, molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The principles and characteristics of several molecular markers such as RFLP, RAPD, AFLP, ...

Frequencies of VNTR and RFLP polymorphisms associated with factor VIII gene in Singapore

Energy Technology Data Exchange (ETDEWEB)

Fong, I.; Lai, P.S.; Ouah, T.C. [National Univ. of Singapore (Malaysia)] [and others

1994-09-01

The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1 = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.

Molecular characterization and identification of markers for toxic and non-toxic varieties of Jatropha curcas L. using RAPD, AFLP and SSR markers.

Science.gov (United States)

Sudheer Pamidimarri, D V N; Singh, Sweta; Mastan, Shaik G; Patel, Jalpa; Reddy, Muppala P

2009-07-01

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).

Variabilidade genética em populações de pitangueira oriundas de autopolinização e polinização livre, acessada por AFLP Genetic variability in surinam cherry populations originated from self-pollination and cross pollination, estimated by AFLP

Directory of Open Access Journals (Sweden)

Rodrigo Cezar Franzon

2010-03-01

Full Text Available Foram utilizados marcadores AFLP para a avaliação de populações de plantas de pitangueira (Eugenia uniflora oriundas de autopolinização e de polinização livre, com o objetivo de verificar a variabilidade existente entre e dentro dessas populações, visando a fornecer mais informações que ajudem no entendimento do modo de reprodução dessa espécie. O material vegetal utilizado foi oriundo de duas seleções de pitangueira ("Pit 15" e "Pit 52", mantidas na Embrapa Clima Temperado. De cada seleção, foram obtidas duas populações F1, por meio de autopolinização e de polinização livre, totalizando quatro populações. Foram analisados 18 indivíduos de cada população e as duas plantas-mãe, totalizando 74 indivíduos. Foram utilizadas três combinações de primers AFLP e calculada a similaridade genética entre plantas pelo coeficiente de Jaccard. Uma estimativa da variabilidade genética entre e dentro das populações foi estimada pela AMOVA. As três combinações de primers AFLP utilizadas amplificaram um total de 178 locos AFLP, dos quais 114 (64,0% foram polimórficos entre todos os indivíduos. Não houve separação clara entre populações descendentes da mesma planta-mãe. Foi observado maior polimorfismo de marcadores AFLP em populações de polinização livre. A proporção da variabilidade genética total entre populações foi significativa, embora tenha sido menor do que aquela observada dentro das populações. A reprodução da pitangueira é decorrente tanto da autofertilização quanto da polinização cruzada, sendo necessário, no entanto, novos estudos para determinar qual a estratégia de reprodução mais eficiente.AFLP molecular markers were used aiming to study the genetic variability within and between Surinam cherry (Eugenia uniflora populations, originated from self-pollination and open pollination of two selections (Pit 15 and Pit 52 of the Embrapa Clima Temperado collection. The objective was to

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP.

Science.gov (United States)

Shan, X H; Li, Y D; Liu, X M; Wu, Y; Zhang, M Z; Guo, W L; Liu, B; Yuan, Y P

2012-08-17

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.

Genetic diversity of sweet sorghum germplasm in Mexico using AFLP and SSR markers

Science.gov (United States)

The objective of this work was to evaluate the diversity and genetic relationships between lines and varieties of the sweet sorghum (Sorghum bicolor) germplasm bank of the National Institute for Forestry, Agriculture and Livestock Research, Mexico, using AFLP and SSR markers. The molecular markers ...

Species relationships in Lactuca S.L. (Lactuceae, Asteraceae) inferred from AFLP fingerprints

NARCIS (Netherlands)

Koopman, W.J.M.; Zevenbergen, M.J.; Berg, van den R.G.

2001-01-01

An AFLP data set comprising 95 accessions from 20 species of Lactuca s.l. (sensu lato) and related genera was generated using the primer combinations E35/M48 and E35/M49. In phenetic analyses of a data subset, clustering with UPGMA based on Jaccard's similarity coefficient resulted in the highest

Comparison between RFLP and MIRU-VNTR genotyping of Mycobacterium tuberculosis strains isolated in Stockholm 2009 to 2011.

Science.gov (United States)

Jonsson, Jerker; Hoffner, Sven; Berggren, Ingela; Bruchfeld, Judith; Ghebremichael, Solomon; Pennhag, Alexandra; Groenheit, Ramona

2014-01-01

Our aim was to analyze the difference between methods for genotyping of Mycobacterium tuberculosis complex isolates. We collected genotyping results from Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units-Variable Numbers of Tandem Repeat (MIRU-VNTR) in a geographically limited area (Stockholm) during a period of three years. The number and proportion of isolates belonging to clusters was reduced by 45 and 35% respectively when combining the two methods compared with using RFLP or MIRU-VNTR only. The mean size of the clusters was smaller when combining methods and smaller with RFLP compared to MIRU-VNTR. In clusters with confirmed epidemiological links RFLP coincided slightly better than MIRU-VNTR but where there was a difference, the variation in MIRU-VNTR pattern was only in a single locus. In isolates with few IS6110 bands in RFLP, MIRU-VNTR differentiated the isolates more, dividing the RFLP clusters. Since MIRU-VNTR is faster and less labour-intensive it is the method of choice for routine genotyping. In most cases it will be sufficient for epidemiological purposes but true clustering might still be considered if there are epidemiological links and the MIRU-VNTR results differ in only one of its 24 loci.

A detailed linkage map of lettuce based on SSAP, AFLP and NBS markers

NARCIS (Netherlands)

Syed, H.; Sorensen, A.P.; Antonise, R.; van de Wiel, C.; van der Linden, C.G.; van 't Westende, W.; Hooftman, D.A.P.; den Nijs, J.C.M.; Flavell, A.J.

2006-01-01

Abstract Molecular markers based upon a novel lettuce LTR retrotransposon and the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of disease resistance-associated genes have been combined with AFLP markers to generate a 458 locus genetic linkage map for lettuce. A total of 187

Caracterización de accesiones de papaya (Carica papaya L. a través de marcadores AFLP en Cuba

Directory of Open Access Journals (Sweden)

Maruchi Alonso Esquivel

2009-07-01

Full Text Available Los marcadores moleculares son herramientas valiosas en los estudios genéticos en plantas, y están siendo empleados exitosamente en programas de mejoramiento principalmente en la elección de progenitores y en la selección. El polimorfismo observado mediante la técnica molecular AFLP (Amplified Fragment Length Polymorphism ha sido de utilidad para estudios de diversidad genética en frutales. En el presente trabajo se realizó la caracterización molecular de 12 accesiones de papaya (Carica papaya L. del banco de germoplasma del Instituto de Investigaciones en Fruticultura Tropical (IIFT, empleando la técnica AFLP. Se evaluaron seis combinaciones de iniciadores para la amplificación selectiva, las cuales amplificaron un total de 431 bandas con 73,3% de polimorfismo. El número total de patrones de bandas identificados fue igual en todas las combinaciones utilizadas, con un porcentaje de identificación alto, lo que sugiere que dichas combinaciones pudieran ser empleadas para estudios de variabilidad genética en papaya. En general, los resultados presentados demuestran que existe diversidad genética entre las accesiones evaluadas, lo cual constituye un reflejo del origen que presentan los genotipos analizados a partir de la introducción de materiales foráneos y la polinización abierta de un grupo de materiales selectos. Por tanto, se recomienda retomar la prospección y selección de accesiones locales, así como la introducción de nuevos genotipos foráneos, como dos vías fundamentales para aumentar la diversidad genética presente en el banco de germoplasma de papaya de Cuba.

Genetic analysis of floating Enteromorpha prolifera in the Yellow Sea with AFLP marker

Science.gov (United States)

Liu, Cui; Zhang, Jing; Sun, Xiaoyu; Li, Jian; Zhang, Xi; Liu, Tao

2011-09-01

Extremely large accumulation of green algae Enteromorpha prolifera floated along China' coastal region of the Yellow Sea ever since the summer of 2008. Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea. Two hundred reproducible fragments were generated with 8 AFLP primer combinations, of which 194 (97%) were polymorphic. The average Nei's genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations. The E. prolifera originated from different areas and were undergoing a course of mixing.

An AFLP marker linked to turnip mosaic virus resistance gene in pak ...

African Journals Online (AJOL)

An AFLP marker linked to turnip mosaic virus resistance gene in pak-choi. W Xinhua, C Huoying, Z Yuying, H Ruixian. Abstract. Pak-choi is one of the most important vegetable crops in China. Turnip mosaic virus (TuMV) is one of its main pathogen. Screening the molecular marker linked to the TuMV resistance gene is an ...

Highly polymorphic RFLP probes as diagnostic tools

International Nuclear Information System (INIS)

Donis-Keller, H.; Barker, D.F.; Knowlton, R.G.; Schumm, J.W.; Braman, J.C.; Green, P.

1986-01-01

In this paper, we describe the identification of highly polymorphic RFLP loci and their application to genotyping in humans and to mapping the CF gene to chromosome 7. We also report the construction of a high-resolution genetic map of chromosome 7 and summarize progress toward the development of a presymptomatic diagnostic test for CF that should be useful in virtually every case. 25 references, 7 figures, 5 tables

Identification of QTLs for grain yield and grain-related traits of maize (Zea mays L.) using an AFLP-map, different testers, and cofactor analysis

NARCIS (Netherlands)

Ajimone Marsan, P.; Gorni, C.; Chitto, A.; Redaelli, R.; Vijk, van R.; Stam, P.; Motto, M.

2001-01-01

Abstract We exploited the AFLP?1(AFLP? is a registered trademark of Keygene, N.V.) technique to map and characterise quantitative trait loci (QTLs) for grain yield and two grain-related traits of a maize segregating population. Two maize elite inbred lines were crossed to produce 229 F2 individuals

AFLP analysis of genetic diversity and phylogenetic relationships of Brassica oleracea in Ireland.

Science.gov (United States)

El-Esawi, Mohamed A; Germaine, Kieran; Bourke, Paula; Malone, Renee

2016-01-01

Brassica oleracea L. is one of the most economically important vegetable crop species of the genus Brassica L. This species is threatened in Ireland, without any prior reported genetic studies. The use of this species is being very limited due to its imprecise phylogeny and uncompleted genetic characterisation. The main objective of this study was to assess the genetic diversity and phylogenetic relationships of a set of 25 Irish B. oleracea accessions using the powerful amplified fragment length polymorphism (AFLP) technique. A total of 471 fragments were scored across all the 11 AFLP primer sets used, out of which 423 (89.8%) were polymorphic and could differentiate the accessions analysed. The dendrogram showed that cauliflowers were more closely related to cabbages than kales were, and accessions of some cabbage types were distributed among different clusters within cabbage subgroups. Approximately 33.7% of the total genetic variation was found among accessions, and 66.3% of the variation resided within accessions. The total genetic diversity (HT) and the intra-accessional genetic diversity (HS) were 0.251 and 0.156, respectively. This high level of variation demonstrates that the Irish B. oleracea accessions studied should be managed and conserved for future utilisation and exploitation in food and agriculture. In conclusion, this study addressed important phylogenetic questions within this species, and provided a new insight into the inclusion of four accessions of cabbages and kales in future breeding programs for improving varieties. AFLP markers were efficient for assessing genetic diversity and phylogenetic relationships in Irish B. oleracea species. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Toxigenic profile and AFLP variability of Alternaria alternata and Alternaria infectoria occurring on wheat

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María Silvina Oviedo

2013-01-01

Full Text Available The objectives of this study were to evaluate the ability to produce alternariol (AOH, alternariol monomethyl ether (AME and tenuazonic acid (TA by A. alternata and A. infectoria strains recovered from wheat kernels obtained from one of the main production area in Argentina; to confirm using AFLPs molecular markers the identify of the isolates up to species level, and to evaluate the intra and inter-specific genetic diversity of these two Alternaria species. Among all the Alternaria strains tested (254, 84% of them were able to produce mycotoxins. The most frequent profile of toxin production found was the co-production of AOH and AME in both species tested. TA was only produced by strains of A. alternata. Amplified fragment polymorphism (AFLPs analysis was applied to a set of 89 isolates of Alternaria spp (40 were A. infectoria and 49 were A. alternata in order to confirm the morphological identification. The results showed that AFLPs are powerful diagnostic tool for differentiating between A. alternata and A. infectoria. Indeed, in the current study the outgroup strains, A. tenuissima was consistently classified. Characteristic polymorphic bands separated these two species regardless of the primer combination used. Related to intraspecific variability, A. alternata and A. infectoria isolates evaluated seemed to form and homogeneous group with a high degree of similarity among the isolates within each species. However, there was more scoreable polymorphism within A. alternata than within A. infectoria isolates. There was a concordance between morphological identification and separation up to species level using molecular markers. Clear polymorphism both within and between species showed that AFLP can be used to asses genetic variation in A. alternata and A. infectoria. The most important finding of the present study was the report on AOH and AME production by A. infectoria strains isolated from wheat kernels in Argentina on a semisynthetic

New chicken Rfp-Y haplotypes on the basis of MHC class II RFLP and MLC analyses

DEFF Research Database (Denmark)

Juul-Madsen, H R; Zoorob, R; Auffray, C

1997-01-01

New chicken Rfp-Y haplotypes were determined by the use of restriction fragment length polymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B15, B19, B21, B201. The RFLP polymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps...... near a polymorphic lectin gene located in the Rfp-Y system and DNA from families with known segregation of the implicated RFLP polymorphism. For the first time it is shown that major histocompatibility complex class II genes in the Rfp-Y system have functional implications. Sequence information...

Relationship of European Persimmon (Diospyros kaki Thunb.) Cultivars to Asian Cultivars, Characterized Using AFLPs.

Science.gov (United States)

Sixty one persimmon (Diospyros kaki Thunb.) selections, including 17 Italian, 11 Spanish, 13 Japanese, six Korean, five Chinese, one Israeli, and eight of unknown origin, were evaluated for genetic differences by AFLP analysis. Relationships among cultivars were evaluated by UPGMA clustering, Neigh...

Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis

DEFF Research Database (Denmark)

On, Stephen L.W.; Atabay, H.I.; Amisu, K.O.

2004-01-01

Aims: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. Methods and Results: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and Nor...

Isolation and RFLP Genotyping of Toxoplasma gondii in Free-Range Chickens (Gallus domesticus) in Grenada, West Indies, Revealed Widespread and Dominance of Clonal Type III Parasites.

Science.gov (United States)

Chikweto, Alfred; Sharma, Ravindra N; Tiwari, Keshaw P; Verma, Shiv K; Calero-Bernal, Rafael; Jiang, Tiantian; Su, Chunlei; Kwok, Oliver C; Dubey, Jitender P

2017-02-01

The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondii in free-range chickens from Grenada, West Indies. Using the modified agglutination test, antibodies to T. gondii were found in 39 (26.9%) of 145 free-range chickens with titers of 25 in 7 chickens, 50 in 6 chickens, 100 in 2 chickens, and 200 or higher in 24 chickens. The hearts of the 39 seropositive chickens were bioassayed in mice; viable T. gondii was isolated from 20 and further propagated in cell culture. Genotyping of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed 4 genotypes, including ToxoDB PCR-RFLP no. 2 (Type III), no. 7, no. 13, and no. 259 (new). These results indicated that T. gondii population genetics in free-range chickens seems to be moderately diverse with ToxoDB no. 2 (Type III) as the most frequent (15/20 = 75%) compared to other genotypes in Grenada.

Differentiation between a pathogenic and a non-pathogenic form of Gyrodactylus salaris using PCR-RFLP

DEFF Research Database (Denmark)

Kania, Per Walther; Jørgensen, Thomas Rohde; Buchmann, Kurt

2007-01-01

A new method based on PCR-RFLP is presented. It is able to differentiate between the Danish non-pathogenic form of Gyrodactylus salaris and the Norwegian pathogenic form.......A new method based on PCR-RFLP is presented. It is able to differentiate between the Danish non-pathogenic form of Gyrodactylus salaris and the Norwegian pathogenic form....

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

Science.gov (United States)

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

IDENTIFIKASI DAGING BABI MENGGUNAKAN METODE PCR-RFLP GEN Cytochrome b DAN PCR PRIMER SPESIFIK GEN AMELOGENIN (Pork Identification Using PCR-RFLP of Cytochrome b Gene and Species Specific PCR of Amelogenin Gene

Directory of Open Access Journals (Sweden)

Yuny Erwanto

2013-03-01

Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP and species specific PCR methods had been applied for identifying pork in mixture of meat. Pork sample in various levels (1, 3, 5 and 10% was prepared in mixture with beef, chicken and mutton. The primary CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b b (cytochrome b gene and PCR successfully amplified fragments of 359 bp. To distinguish pig species existence, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed that pig mitochondrial DNA was cut into 131 and 228 bp fragments. A polymerase chain reaction (PCR method based on the nucleotide sequence variation in the amelogenin gene has been chosen for the specific identification of pork DNAs in mixture meat. The primers designed generated specific fragments of 353 and 312 bp length for pork. The specificity of the primary designed was tested on 4 animal species including pig, cattle, chicken and goat species. Analysis of experimental mixture meat demonstrated that 1% of raw pork tissues could be detected using PCR-RFLP with BseDI restriction enzyme but detection using species-specific PCR showed the cross reactivity to beef, chicken and mutton. The cytochrome b PCR-RFLP species identification assay yielded excellent results for identification of pig species. PCR-RFLP is a potentially reliable technique for detection of the existence of pork in animal food product for Halal authentication. Keywords: Pork identification, cytochrome b, amelogenin, polymerase chain reaction   ABSTRAK   Penelitian ini dilakukan untuk mengaplikasikan metode deteksi daging babi dalam campuan daging dengan sapi, kambing dan ayam melalui PCR-RFLP dan PCR dengan primer spesifik untuk babi. Level kontaminasi daging babi dibuat sebesar 1, 3, 5 dan 10% dari total daging dalam campuran. Metode PCR-RFLP menggunakan sepasang primer yaitu gen cytochrome b dari mitokondria yang

Genetic differentiation in Pyrenophora teres f. teres populations from Syria and Tunisia as assessed by AFLP markers.

Science.gov (United States)

Bouajila, A; Zoghlami, N; Murad, S; Baum, M; Ghorbel, A; Nazari, K

2013-06-01

To investigate the level of genetic differentiation and diversity among Pyrenophora teres isolate populations originating from different agro-ecological areas of Syria and Tunisia and to determine the potential of AFLP profiling in genotyping Pyrenophora teres f. teres. In this study, AFLP markers have been employed to identify patterns of population structure in 20 Pyrenophora teres f. teres populations from Syria and Tunisia. Ninety-four isolates were studied by the use of a protocol that involved stringent PCR amplification of fragments derived from digestion of genomic DNA with restriction enzymes EcoRI and MesI. Based on 401 amplified polymorphic DNA markers (AFLP), variance analyses indicated that most of the variation was partitioned within rather than between populations. Genotypic diversity (GD) was high for populations from Rihane, local landraces and different agro-ecological zones (GD = 0·75-0·86). There was high genetic differentiation among pathogen populations from different host populations in Syria (Gst  = 0·31, ht = 0·190) and Tunisia (Gst  = 0·39, ht = 0·263), which may be partly explained by the low gene flow around the areas sampled. A phenetic tree revealed three groups with high bootstrap values (55, 68, 76) and reflected the grouping of isolates based on host, or agro-ecological areas. AFLP profiling is an effective method for typing the genetically diverse pathogen Pyrenophora teres f. teres. The study represents a comparative analysis of the genetic diversity in P. teres isolates from two countries spanning two continents and also shows that several distinct P. teres genotypes may be found in a given environment. The implications of these findings for Pyrenophora teres f. teres evolutionary potential and net blotch-resistance breeding in Syria and Tunisia were also discussed. © 2012 The Society for Applied Microbiology.

AFLPs reveal genomic regions not detected by RFLPs: a case study in tomato

NARCIS (Netherlands)

Bonnema, G.; Berg, van den P.; Lindhout, P.

2002-01-01

A set of three tomato chromosome 7 introgression lines (ILs) containing overlapping segments of Lycopersicon pennellii DNA was screened with a set of 10 EcoRI–MseI and 10 PstI–MseI AFLP primer combinations. A large number of markers were identified that mapped to one of the four regions of

Species-specific AFLP markers for identification of Zingiber officinale, Z. montanum and Z. zerumbet (Zingiberaceae).

Science.gov (United States)

Ghosh, S; Majumder, P B; Sen Mandi, S

2011-02-08

The Zingiber genus, which includes the herbs known as gingers, commonly used in cooking, is well known for its medicinal properties, as described in the Indian pharmacopoeia. Different members of this genus, although somewhat similar in morphology, differ widely in their pharmacological and therapeutic properties. The most important species of this genus, with maximal therapeutic properties, is Zingiber officinale (garden ginger), which is often adulterated with other less-potent Zingiber sp. There is an existing demand in the herbal drug industry for an authentication system for the Zingiber sp in order to facilitate their commercial use as genuine phytoceuticals. To this end, we used amplified fragment length polymorphism (AFLP) to produce DNA fingerprints for three Zingiber species. Sixteen collections (six of Z. officinale, five of Z. montanum, and five of Z. zerumbet) were used in the study. Seven selective primer pairs were found to be useful for all the accessions. A total of 837 fragments were produced by these primer pairs. Species-specific markers were identified for all three Zingiber species (91 for Z. officinale, 82 for Z. montanum, and 55 for Z. zerumbet). The dendogram analysis generated from AFLP patterns showed that Z. montanum and Z. zerumbet are phylogenetically closer to each other than to Z. officinale. The AFLP fingerprints of the Zingiber species could be used to authenticate Zingiber sp-derived drugs and to resolve adulteration-related problems faced by the commercial users of these herbs.

AFLP Markers as a tool to reconstruct complex relationships: a case study in Rosa (Rosaceae)

NARCIS (Netherlands)

Koopman, W.J.M.; Wissemann, V.; Cock, de K.; Huylenbroeck, van J.; Riek, de J.; Sabatino, G.J.H.; Visser, D.L.; Vosman, B.; Ritz, K.; Maes, B.; Werlemark, G.; Nybom, H.; Debener, T.; Linde, M.; Smulders, M.J.M.

2008-01-01

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in

Contribución del análisis RFLP del IS6110 de Mycobacterium tuberculosis al diseño y refinamiento de estrategias para el control de la tuberculosis en Colombia Contribution of M. tuberculosis IS6110 based RFLP assay to the design and refinement of tuberculosis control strategies in Colombia

Directory of Open Access Journals (Sweden)

Doris Amanda Rosero

2008-09-01

Full Text Available Una alternativa de mejoramiento de las estrategias para el control de la tuberculosis, la ofrecen los métodos para la diferenciación de cepas de M. tuberculosis. La literatura evidencia que la técnica RFLP del segmento de inserción IS6110 está ampliamente estandarizada a nivel internacional y ha demostrado ser un buen instrumento para orientar estrategias locales de control. Mediante una revisión exhaustiva de la literatura, el presente estudio pretende establecer si esta técnica molecular puede contribuir al diseño y refinamiento de estrategias para el control de la tuberculosis en Colombia. En esta revisión se analizaron los resultados de los estudios publicados entre 1993 y 2008, realizados con la técnica en países en desarrollo, incluida Colombia. Los resultados sugieren que en el contexto colombiano esta técnica puede ofrecer información útil para los directores del programa de control de tuberculosis y, por tanto, debe seguir siendo realizada. Para establecer la periodicidad, las poblaciones blanco y otras condiciones óptimas para la realización de la técnica, se necesitan estudios de investigación operativa que incluyan análisis de costo-efectividad y costo-utilidad.Molecular biology methods offer an alternative for improving tuberculosis control strategies through M. tuberculosis strain typing techniques. The international literature shows that RFLP of the insertion element IS6110 is widely standardized internationally and has proved to be a useful tool to guide local tuberculosis control strategies. By means of a thorough literature review, this study aimed to determine if this molecular based method could be useful for the design and refinement of tuberculosis control strategies in Colombia. Results from epidemiologic studies published between 1995 and 2008 which used this technique in developing countries, including Colombia, were analyzed. Our results suggest that in the Colombian context this molecular technique

Genetic diversity among wild and cultivated barley as revealed by RFLP

DEFF Research Database (Denmark)

Petersen, L.; Østergård, H.; Giese, H.

1994-01-01

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. sponta...... an intermediate level. The proportion of gene diversity residing among,geographical groups (F-ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups....... was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed...

Molecular mapping of the Pinus monticola Cr2 gene using AFLP and SCAR markers

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A.K.M. Ekramoddoullah

2013-12-01

Full Text Available White pine blister rust (WPBR, caused by Cronartium ribicola, is a devastating disease in five-needle pines. Genetic resistance is an important component of integrated strategies to control WPBR. The major resistance gene Cr2, discovered by Kinloch etal.(1999, is also effective against British Columbia (BC isolates of WPBR (Hunt et al. 2004. Pyramiding Cr2 gene with other resistancegenes is being pursued as a strategy in BC white pine breeding. To facilitate this strategy, we have recently identified a few RAPD markerslinked to Cr2 at one side (Liu et al. 2006. The objective of the present study was to identify amplified fragment length polymorphism(AFLP markers linked to both sides of Cr2 for its more precise apping. Use of the AFLP technique combined with bulked segregant analysis (BSA and haploid segregation analysis allowed the identification of five AFLP markers. Of these five AFLP markers in the Cr2 linkage, markers EacccMccgat-365, EactgMcccac- 290, and EacagEacag-750 werelinked in coupling and EacagMcccag-160r and EacccMccgat-180r in repulsion. Following cloning and sequencing of the AFLP andRAPD markers, specific PCR primers were designed and used in the amplification of sequence characterized amplified region(SCAR markers at both sides of Cr2. EacccMccgat- 365 and RAPD marker U570-843 reported previously were converted into SCARmarkers. These two SCARs segregated in a 1:1 (presence:absence ratio and the scoring cosegregated with their respective AFLP orRAPD marker. The SCAR marker EacccMccgat- 365-scar was positioned at 3.1 Kosambi cM from one side of Cr2 and U570-843-scarlocalized at 1.4 Kosambi cM from other side. Both SCAR markers can be useful in breeding programs with marker-assisted selection procedureto screen for resistance. This study represents the first report of the development of PCR-based sequence-specific markers linkedto blister rust resistance in five-needle pines. These findings may

[Comparison of usefulness between variable numbers of tandem repeats (VNTR) analysis and restriction fragment length polymorphism (RFLP) in the genotyping of Mycobacterium avium].

Science.gov (United States)

Kazumi, Yuko; Udagawa, Tadashi; Maeda, Shinji; Murase, Yoshirou; Sugawara, Isamu; Okumura, Masao; Azuma, Yuka; Goto, Mieko; Tsunematsu, Noriko

2007-10-01

Comparison of usefulness of IS1245 RFLP and VNTR in M. avium genotyping. Thirty-six cases (55 strains) from sputum and BALF and twelve cases (29 strains) isolated from blood of HIV-infected patients were used. VNTR and RFLP using IS1245 were performed. Multiple samples were taken from 16 patients and 52 clinical isolates were used for VNTR and RFLP for comparison. (1) VNTR and RFLP results were identical in 12 out of 16 cases whose samples were collected several times. (2) Eight isolates were obtained from one patient. In this eight isolates, there were the cases of M. avium polyclonal infection and of mixed infection with M. intracellulare. VNTR patterns were two types and RFLP were 5 kinds of different in this case. (3) VNTR patterns of six isolates from one HIV-infected patient were identical, but there were three variations in RFLP patterns. There were three cases of mixed infections with M. tuberculosis or M. intracellulare, and six strains polyclonal infection of M. avium (7.1 %) in 84 isolates. These 6 clinical isolates were derived from sputum or BALF (5 strains) and HIV-infected blood (one strain). VNTR patterns were similar in four pairs (9 strains) who did not contact closely, but they were distinguished clearly by RFLP. Seventeen strains had three or less IS1245-related bands in RFLP analyses of 89 strains. As there is a possibility of polyclonal infection with M. avium and mixed infection with other species, the single clonal infection should be confirmed first by VNTR. When single colony was obtained, VNTR and RFLP were performed for genotyping of M. avium. Furthermore, strains with less bands by RFLP should be carefully judged in terms of both VNTR and RFLP. It is recommended that the specimens should be collected from each patient several times.

AFLP polymorphisms allow high resolution genetic analysis of American Tegumentary Leishmaniasis agents circulating in Panama and other members of the Leishmania genus.

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Carlos M Restrepo

Full Text Available American Tegumentary Leishmaniasis is caused by parasites of the genus Leishmania, and causes significant health problems throughout the Americas. In Panama, Leishmania parasites are endemic, causing thousands of new cases every year, mostly of the cutaneous form. In the last years, the burden of the disease has increased, coincident with increasing disturbances in its natural sylvatic environments. The study of genetic variation in parasites is important for a better understanding of the biology, population genetics, and ultimately the evolution and epidemiology of these organisms. Very few attempts have been made to characterize genetic polymorphisms of parasites isolated from Panamanian patients of cutaneous leishmaniasis. Here we present data on the genetic variability of local isolates of Leishmania, as well as specimens from several other species, by means of Amplified Fragment Length Polymorphisms (AFLP, a technique seldom used to study genetic makeup of parasites. We demonstrate that this technique allows detection of very high levels of genetic variability in local isolates of Leishmania panamensis in a highly reproducible manner. The analysis of AFLP fingerprints generated by unique selective primer combinations in L. panamensis suggests a predominant clonal mode of reproduction. Using fluorescently labeled primers, many taxon-specific fragments were identified which may show potential as species diagnostic fragments. The AFLP permitted a high resolution genetic analysis of the Leishmania genus, clearly separating certain groups among L. panamensis specimens and highly related species such as L. panamensis and L. guyanensis. The phylogenetic networks reconstructed from our AFLP data are congruent with established taxonomy for the genus Leishmania, even when using single selective primer combinations. Results of this study demonstrate that AFLP polymorphisms can be informative for genetic characterization in Leishmania parasites, at

A genetic linkage map of willow (Salix viminalis) based on AFLP and microsatelite markers

NARCIS (Netherlands)

Hanley, S.; Barker, J.H.A.; Ooijen, van J.W.; Aldam, C.; Harris, S.L.; Ahman, I.; Larsson, S.; Karp, A.

2002-01-01

The genus Salix (willow) contains a number of species of great value as biomass crops. Efforts to breed varieties with improved biomass yields and resistances to pests and diseases are limited by the lack of knowledge on the genetic basis of the traits. We have used AFLP and microsatellite markers

Assessment of genetic stability in micropropagules of Jatropha curcas genotypes by RAPD and AFLP analysis

KAUST Repository

Sharma, Sweta K.

2011-07-01

Jatropha curcas (Euphorbiaceae), a drought resistant non edible oil yielding plant, has acquired significant importance as an alternative renewable energy source. Low and inconsistent yields found in field plantations prompted for identification of high yielding clones and their large scale multiplication by vegetative propagation to obtain true to type plants. In the current investigation plantlets of J. curcas generated by axillary bud proliferation (micropropagation) using nodal segments obtained from selected high yielding genotypes were assessed for their genetic stability using Randomly Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) analyses. For RAPD analysis, 21 out of 52 arbitrary decamer primers screened gave clear reproducible bands. In the micropropagated plantlets obtained from the 2nd sub-culture, 4 out of a total of 177 bands scored were polymorphic, but in the 8th and 16th sub-cultures (culture cycle) no polymorphisms were detected. AFLP analysis revealed 0.63%, 0% and 0% polymorphism in the 2nd, 8th and 16th generations, respectively. When different genotypes, viz. IC 56557 16, IC 56557 34 and IC 56557 13, were assessed by AFLP, 0%, 0.31% and 0.47% polymorphisms were found, respectively, indicating a difference in genetic stability among the different genotypes. To the best of our knowledge this is the first report on assessment of genetic stability of micropropagated plantlets in J. curcas and suggests that axillary shoot proliferation can safely be used as an efficient micropropagation method for mass propagation of J. curcas. © 2011 Elsevier B.V.

Huyendo del miedo, desterrando el enojo, escapando de la tristeza y cayendo en brazos de la risa: los chistes en México en tiempos de la influenza 2009

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Anna María Fernandez Poncela

2012-03-01

Full Text Available Se trata de revisar el humor, los chistes y la risa en tiempos de la influenza en México en el año 2009. La relación entre narrativas sociales populares (chistes y reacciones emocionales de la sociedad en un momento de incertidumbre y temor social. El por qué y para qué de los chistes, su función social y psicológica. El objetivo es que el chiste nos acompañe a develar la realidad social, política, emocional y cultural. Para ello se aborda un poco el humor, lo cómico, la risa y los chistes. Presentamos algunas definiciones de estos fenómenos y mostramos su funcionalidad psicológica y social, entre otras cosas, como parte de los sentimientos humanos y la cultura en general. También reflexionamos algo en torno al mundo de las emociones y su evolución o desarrollo, y en determinadas situaciones sociales, en concreto en el contexto de los días de la contingencia sanitaria por el virus A H1N1 en México, entre abril y mayo del año 2009.

Genetic diversity of wild and cultivated Rubus species in Colombia using AFLP and SSR markers

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Sandra Bibiana Aguilar

2007-01-01

Full Text Available The Andean blackberry belongs to the genus Rubus, the largest of the Rosaceae family and one of the mostdiverse of the plant kingdom. In Colombia Rubus glaucus Benth, known as the Andean raspberry or blackberry, is one of thenine edible of the genus out of forty-four reported species. In this study wild and cultivated genotypes, collected in the CentralAndes of Colombia were analyzed by AFLP and SSR markers. Sexual reproduction seems to play an important role inmaintaining the genetic variability in R. glaucus, and the viability of using the SSR of Rubus alceifolius to characterizeColombian Rubus species was clearly demonstrated. All species evaluated produced very specific banding patterns,differentiating them from the others. Both AFLP and SSR produced bands exclusive to each of the following species: R.robustus, R. urticifolius, R. glaucus, and R. rosifolius. The SSR markers differentiated diploid and tetraploid genotypes of R.glaucus.

Identification of genes related to Paulownia witches' broom by AFLP and MSAP.

Science.gov (United States)

Cao, Xibing; Fan, Guoqiang; Deng, Minjie; Zhao, Zhenli; Dong, Yanpeng

2014-08-21

DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches' broom (PaWB) infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS) using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L(-1) MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR) showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB.

Identification of Genes Related to Paulownia Witches’ Broom by AFLP and MSAP

Science.gov (United States)

Cao, Xibing; Fan, Guoqiang; Deng, Minjie; Zhao, Zhenli; Dong, Yanpeng

2014-01-01

DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches’ broom (PaWB) infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS) using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L−1 MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR) showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB. PMID:25196603

Risk factors associated with cluster size of Mycobacterium tuberculosis (Mtb) of different RFLP lineages in Brazil.

Science.gov (United States)

Peres, Renata Lyrio; Vinhas, Solange Alves; Ribeiro, Fabíola Karla Correa; Palaci, Moisés; do Prado, Thiago Nascimento; Reis-Santos, Bárbara; Zandonade, Eliana; Suffys, Philip Noel; Golub, Jonathan E; Riley, Lee W; Maciel, Ethel Leonor

2018-02-08

Tuberculosis (TB) transmission is influenced by patient-related risk, environment and bacteriological factors. We determined the risk factors associated with cluster size of IS6110 RFLP based genotypes of Mycobacterium tuberculosis (Mtb) isolates from Vitoria, Espirito Santo, Brazil. Cross-sectional study of new TB cases identified in the metropolitan area of Vitoria, Brazil between 2000 and 2010. Mtb isolates were genotyped by the IS6110 RFLP, spoligotyping and RD Rio . The isolates were classified according to genotype cluster sizes by three genotyping methods and associated patient epidemiologic characteristics. Regression Model was performed to identify factors associated with cluster size. Among 959 Mtb isolates, 461 (48%) cases had an isolate that belonged to an RFLP cluster, and six clusters with ten or more isolates were identified. Of the isolates spoligotyped, 448 (52%) were classified as LAM and 412 (48%) as non-LAM. Our regression model found that 6-9 isolates/RFLP cluster were more likely belong to the LAM family, having the RD Rio genotype and to be smear-positive (adjusted OR = 1.17, 95% CI 1.08-1.26; adjusted OR = 1.25, 95% CI 1.14-1.37; crude OR = 2.68, 95% IC 1.13-6.34; respectively) and living in a Serra city neighborhood decrease the risk of being in the 6-9 isolates/RFLP cluster (adjusted OR = 0.29, 95% CI, 0.10-0.84), than in the others groups. Individuals aged 21 to 30, 31 to 40 and > 50 years were less likely of belonging the 2-5 isolates/RFLP cluster than unique patterns compared to individuals cluster group (adjustment OR = 0.45, 95% CI 0.24-0.85) than unique patterns. We found that a large proportion of new TB infections in Vitoria is caused by prevalent Mtb genotypes belonging to the LAM family and RD Rio genotypes. Such information demonstrates that some genotypes are more likely to cause recent transmission. Targeting interventions such as screening in specific areas and social risk groups, should be a priority

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts.

Science.gov (United States)

Flores Berrios, E P; Alba González, J F; Arrizon Gaviño, J P; Romano, P; Capece, A; Gschaedler Mathis, A

2005-01-01

The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.

PCR and RFLP analyses based on the ribosomal protein operon

Science.gov (United States)

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16Sr RNA gene. RFLP analysis of 16Sr RNA gene sequences has identified 31 16Sr RNA (16Sr) groups and more than 100 16Sr subgroups. Classification of phytoplasma strains can however, become more refin...

Identification, genetic localization, and allelic diversity of selectively amplified microsatellite polymorphic loci in lettuce and wild relatives (Lactuca spp.).

Science.gov (United States)

Witsenboer, H; Michelmore, R W; Vogel, J

1997-12-01

Selectively amplified microsatellite polymorphic locus (SAMPL) analysis is a method of amplifying microsatellite loci using generic PCR primers. SAMPL analysis uses one AFLP primer in combination with a primer complementary to microsatellite sequences. SAMPL primers based on compound microsatellite sequences provided the clearest amplification patterns. We explored the potential of SAMPL analysis in lettuce to detect PCR-based codominant microsatellite markers. Fifty-eight SAMPLs were identified and placed on the genetic map. Seventeen were codominant. SAMPLs were dispersed with RFLP markers on 11 of the 12 main linkage groups in lettuce, indicating that they have a similar genomic distribution. Some but not all fragments amplified by SAMPL analysis were confirmed to contain microsatellite sequences by Southern hybridization. Forty-five cultivars of lettuce and five wild species of Lactuca were analyzed to determine the allelic diversity for codominant SAMPLs. From 3 to 11 putative alleles were found for each SAMPL; 2-6 alleles were found within Lactuca sativa and 1-3 alleles were found among the crisphead genotypes, the most genetically homogeneous plant type of L. sativa. This allelic diversity is greater than that found for RFLP markers. Numerous new alleles were observed in the wild species; however, there were frequent null alleles. Therefore, SAMPL analysis is more applicable to intraspecific than to interspecific comparisons. A phenetic analysis based on SAMPLs resulted in a dendrogram similar to those based on RFLP and AFLP markers.

KARAKTERISTIK GENETIK Kappaphycus alvarezii SEHAT DAN TERINFEKSI PENYAKIT ICE-ICE DENGAN METODE Amplified Fragment Length Polymorphism (AFLP

Directory of Open Access Journals (Sweden)

Emma Suryati

2013-03-01

Full Text Available Infeksi penyakit ice-ice pada Kappaphycus alvarezii seringkali menyebabkan penurunan produksi yang sangat signifikan. K. alvarezii merupakan alga merah penghasil karaginan yang memiliki nilai ekonomi tinggi dan banyak dimanfaatkan dalam berbagai industri, seperti farmasi, makanan, stabilizer, dan kosmetik. Perbaikan genetik sangat diperlukan untuk meningkatkan produksi. Penelitian ini bertujuan untuk mengetahui karakteristik kemiripan genetik K. alvarezii sehat dan terinfeksi penyakit dari Balai Penelitian dan Pengembangan Budidaya Air Payau (BPPBAP, Maros dengan metode Amplified Fragment Length Polymorphism (AFLP. Pada penelitian ini juga dianalisis K. alvarezii asal Bone (BNE, Gorontalo (GRL, Tambalang (TMB, dan Kendari (KND sebagai kontrol rumput laut sehat. Metode AFLP menggunakan enzim restriksi Psti dan Mset, preamplifikasi dan amplifikasi selektif diawali dengan isolsi DNA, uji genimoc DNA, restriksi dan ligasi. Hasil yang diperoleh menunjukkan penggunaan marker AFLP dengan primer forward P11 dan primer reverse M48, M49 dan M50 terhadap K. alvarezii yang berasal dari Takalar (TKL, dan Mataram (MTR, tanpa infeksi (sehat dan terinfeksi penyakit Takalar ice (TKL+, Mataram ice (MTR+, serta K. alvarezii kontrol (BNE, (GRL, (TMB, dan (KND menghasilkan 519 fragmen dalam 122 lokus dengan ukuran 50 - ~370 pb. Kemiripan genetik K. alvarezii yang terinfeksi penyakit ice-ice lebih rendah jika dibandingkan dengan yang sehat. Kemiripan genetik K. alvarezii dari Takalar sehat (TKL dan terinfeksi ice-ice (TKL+ adalah 0,8176 dan MTR-MTR+ adalah 0,8033.

AFLP analysis of Cynodon dactylon (L.) Pers. var. dactylon genetic variation.

Science.gov (United States)

Wu, Y Q; Taliaferro, C M; Bai, G H; Anderson, M P

2004-08-01

Cynodon dactylon (L.) Pers. var. dactylon (common bermudagrass) is geographically widely distributed between about lat 45 degrees N and lat 45 degrees S, penetrating to about lat 53 degrees N in Europe. The extensive variation of morphological and adaptive characteristics of the taxon is substantially documented, but information is lacking on DNA molecular variation in geographically disparate forms. Accordingly, this study was conducted to assess molecular genetic variation and genetic relatedness among 28 C. dactylon var. dactylon accessions originating from 11 countries on 4 continents (Africa, Asia, Australia, and Europe). A fluorescence-labeled amplified fragment length polymorphism (AFLP) DNA profiling method was used to detect the genetic diversity and relatedness. On the basis of 443 polymorphic AFLP fragments from 8 primer combinations, the accessions were grouped into clusters and subclusters associating with their geographic origins. Genetic similarity coefficients (SC) for the 28 accessions ranged from 0.53 to 0.98. Accessions originating from Africa, Australia, Asia, and Europe formed major groupings as indicated by cluster and principal coordinate analysis. Accessions from Australia and Asia, though separately clustered, were relatively closely related and most distantly related to accessions of European origin. African accessions formed two distant clusters and had the greatest variation in genetic relatedness relative to accessions from other geographic regions. Sampling the full extent of genetic variation in C. dactylon var. dactylon would require extensive germplasm collection in the major geographic regions of its distributional range.

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

Science.gov (United States)

Fong, Wai-Ying; Ho, Chi-Chun; Poon, Wing-Tat

2017-05-12

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

Identification of Genes Related to Paulownia Witches’ Broom by AFLP and MSAP

Directory of Open Access Journals (Sweden)

Xibing Cao

2014-08-01

Full Text Available DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches’ broom (PaWB infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS using amplified fragment length polymorphism (AFLP and methylation-sensitive amplification polymorphism (MSAP techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L−1 MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB.

Genetic structure of Pilosocereus gounellei (Cactaceae) as revealed by AFLP marker to guide proposals for improvement and restoration of degraded areas in Caatinga biome.

Science.gov (United States)

Monteiro, E R; Strioto, D K; Meirelles, A C S; Mangolin, C A; Machado, M F P S

2015-12-15

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate DNA polymorphism in Pilosocereus gounellei with the aim of differentiating samples grown in different Brazilian semiarid regions. Seven primer pairs were used to amplify 703 AFLP markers, of which 700 (99.21%) markers were polymorphic. The percentage of polymorphic markers ranged from 95.3% for the primer combination E-AAG/M-CTT to 100% for E-ACC/M-CAT, E-ACC/M-CAA, E-AGC/M-CAG, E-ACT/M-CTA, and E-AGG/M-CTG. The largest number of informative markers (126) was detected using the primer combination E-AAC/M-CTA. Polymorphism of the amplified DNA fragments ranged from 72.55% (in sample from Piauí State) to 82.79% (in samples from Rio Grande Norte State), with an average of 75.39%. Despite the high genetic diversity of AFLP markers in xiquexique, analysis using the STRUCTURE software identified relatively homogeneous clusters of xiquexique from the same location, indicating a differentiation at the molecular level, among the plant samples from different regions of the Caatinga biome. The AFLP methodology identified genetically homogeneous and contrasting plants, as well as plants from different regions with common DNA markers. Seeds from such plants can be used for further propagation of plants for establishment of biodiversity conservation units and restoration of degraded areas of the Caatinga biome.

Proyecto de intervención sobre la implementación de un taller de dinámica de la risa en un grupo de personas mayores

OpenAIRE

Santos Marcos, Pablo

2012-01-01

Las políticas y programas de envejecimiento activo reconocen la necesidad de fomentar y equilibrar la responsabilidad personal. Las personas y familias necesitan planificar y prepararse para la vejez adoptando prácticas de salud positivas. Estos programas son tan importantes como los que mejoran las salud física. Se explican los efectos terapéuticos de la risa a nivel físico, psicológico y social. Trabajo de Fin de Grado en Trabajo Social. Curso 2011-2012

Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers

DEFF Research Database (Denmark)

Giese, H.; Holm-Jensen, A.G.; Jensen, H.P.

1993-01-01

The powdery mildew disease resistance gene Ml(La) was found to belong to a locus on barely chromosome 2. We suggest that this locus be designated MlLa. Linkage analysis was carried out on 72 chromosome-doubled, spring-type progeny lines from a cross between the winter var 'Vogelsanger Gold' and t......' and the spring var 'Alf'. A map of chromosome 2 spanning 119 cM and flanked by two peroxidase gene loci was constructed. In addition to the Laevigatum resistance locus the map includes nine RFLP markers, the two peroxidase gene loci and the six-row locus in barley....

Genetic characterization of rhizobia isolated from Amazonas soils based on the technique of PCR-RFLP / Caracterização genética de rizóbio isolados de solos no Amazonas baseada na técnica de PCR-RFLP

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Arlem Nascimento de Oliveira

2009-12-01

Full Text Available Few are the details concerning the molecular characterization of native rhizobia isolates from Amazonas soils. The aim of the present study was the genetic characterization of rhizobia isolated from nodules of cowpea using the method PCR-RFLP of the ribosome genes 16S rRNA and the restriction enzymes HinfI, MspI, and MboI. The analysis included 20 native isolates and ten reference strains from different origins. The results indicated a great genetic variability with the formation of eight groups in one dendrogram with a varied level of similarity.Poucas são as informações referentes à caracterização molecular de rizóbios nativos isolados de solos no Amazonas. Assim, o objetivo deste trabalho foi a caracterização genética de rizóbios isolados de nódulos de caupi usando o método PCR-RFLP dos genes ribossomais 16S rRNA e as enzimas de restrição HinfI, MspI, e MboI. A análise incluiu 20 isolados nativos e dez estirpes-referência de diferentes origens. Os resultados indicaram uma grande variabilidade genética com a formação de oito grupos em um dendrograma com um nível de similaridade variado.

Variabilidade genética do umbuzeiro no Semi-Árido brasileiro, por meio de marcadores AFLP Genetic variability of umbu trees in Brazilian Semi-Arid region, based on AFLP markers

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Carlos Antonio Fernandes Santos

2008-08-01

Full Text Available O objetivo deste trabalho foi avaliar a distribuição da variabilidade genética do umbuzeiro (Spondias tuberosa, no Semi-Árido brasileiro, por meio de marcadores AFLP, para subsidiar estratégias de prospecção e conservação da espécie. Foram analisados 68 indivíduos de umbuzeiro de 15 ecorregiões, pelo dendrograma UPGMA e pela dispersão em escala multidimensional (MDS, com o coeficiente de Jaccard de 141 bandas polimórficas de AFLP. A análise da variância molecular foi realizada pela decomposição total entre e dentro das regiões ecogeográficas. O dendrograma apresentou valor cofenético de 0,96, e o gráfico MDS apresentou 0,25 para a falta de ajustamento. A variabilidade genética do umbuzeiro foi estimada em 0,3138, o que indica grande variação entre os grupos de indivíduos. Agrupamentos específicos foram observados em seis regiões ecogeográficas, enquanto nas demais regiões observaram-se pares entre alguns indivíduos, sem formação de agrupamentos específicos por local de amostragem, o que indica que a variabilidade genética do umbuzeironão está uniformemente distribuída no Semi-Árido. Sugerem-se estratégias para o estabelecimento de maior número de áreas para conservação in situ ou amostragens de menor número de indivíduos, em várias unidades de paisagens, para conservação ex situ da variabilidade genética do umbuzeiro.The objective of this work was to evaluate the genetic variability distribution of umbu tree (Spondias tuberosa, within Brazilian Semi-Arid region, based on AFLP markers, in order to suggest prospecting and preservation strategies for this species. Sixty-eight umbu trees of 15 ecogeographic regions were analyzed for 141 polymorphic AFLP bands, through the UPGMA dendrogram and the multidimensional scaling (MDS, based on Jaccard's coefficient . Analysis of molecular variance was accomplished by total decomposition among and within ecogeographic regions. The dendrogram presented co

Molecular characterisation of Aspergillus flavus isolates from peanut fields in India using AFLP

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Diwakar Singh

2015-09-01

Full Text Available Aflatoxin contamination of peanut, due to infection by Aspergillus flavus, is a major problem of rain-fed agriculture in India. In the present study, molecular characterisation of 187 Aspergillus flavus isolates, which were sampled from the peanut fields of Gujarat state in India, was performed using AFLP markers. On a pooled cluster analysis, the markers could successfully discriminate among the ‘A’, ‘B’ and ‘G’ group A. flavus isolates. PCoA analysis also showed equivalent results to the cluster analysis. Most of the isolates from one district could be clustered together, which indicated genetic similarity among the isolates. Further, a lot of genetic variability was observed within a district and within a group. The results of AMOVA test revealed that the variance within a population (84% was more than that between two populations (16%. The isolates, when tested by indirect competitive ELISA, showed about 68.5% of them to be atoxigenic. Composite analysis between the aflatoxin production and AFLP data was found to be ineffective in separating the isolate types by aflatoxigenicity. Certain unique fragments, with respect to individual isolates, were also identified that may be used for development of SCAR marker to aid in rapid and precise identification of isolates.

A TaqI RFLP in the locus D9S29 on human chromosome 9

Energy Technology Data Exchange (ETDEWEB)

Weitnauer, L.; Antonelli, A.; Pandolfo, M. (Istituto Neurologico, Milan (Italy))

1989-12-25

Phage LAMP92 was isolated from the Los Alamos chromosome 9 library and its 3.8 Kbp insert was subcloned into the Eco RI site of pUC19. Taq I identifies a three allele RFLP (B1: 7.6 kb + 3 kb, B2: 14 kb + 3 kb, B3: 17 kb). A two allele Pvu II RFLP detected by the same probe was described previously. It was localized on 9q22-q31 by in situ hybridization and linkage analysis. Co-dominant segregation was shown in 14 informative families.

AcuI identifies water buffalo CSN3 genotypes by RFLP analysis

Indian Academy of Sciences (India)

Home; Journals; Journal of Genetics; Volume 93; Online resources. AcuI identifies water buffalo CSN3 genotypes by RFLP analysis. Soheir M. El Nahas Ahlam A. Abou Mossallam. Volume 93 Online resources 2014 pp e94-e96. Fulltext. Click here to view fulltext PDF. Permanent link:

Análisis de segregantes agrupados (BSA para la detección de AFLPs ligados al gen de resistencia a PVX en Solanum commersonii

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Mónica Blanco

2005-01-01

Full Text Available Para identificar polimorfismos asociados al gen de resistencia al PVX en la papa silvestre Solanum commersonii, se realizó un análisis de segregantes agrupados (BSA asistido con AFLPs. Estos polimorfismos están basados en la localización de un locus relacionado con la resistencia al virus X de la papa (PVX. Inicialmente, mediante un análisis de ELISA, los individuos de una progenie F2 previamente inoculados con el PVX, fueron ubicados en 2 grupos, uno con los individuos resistentes y otro con los susceptibles. Posteriormente, para el BSA el ADN de todos los individuos resistentes fue mezclado, lo mismo el ADN de todos los individuos susceptibles. Ambos grupos de ADN fueron analizados independientemente, utilizando 64 diferentes combinaciones de AFLPs. El análisis de los geles resultó en la identificación de 22 combinaciones diferentes de AFLPs que generaron bandas relacionadas exclusivamente con el carácter de resistencia al PVX.

cDNA-AFLP analysis reveals differential gene expression in compatible interaction of wheat challenged with Puccinia striiformis f. sp. tritici

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Huang Lili

2009-06-01

Full Text Available Abstract Background Puccinia striiformis f. sp. tritici is a fungal pathogen causing stripe rust, one of the most important wheat diseases worldwide. The fungus is strictly biotrophic and thus, completely dependent on living host cells for its reproduction, which makes it difficult to study genes of the pathogen. In spite of its economic importance, little is known about the molecular basis of compatible interaction between the pathogen and wheat host. In this study, we identified wheat and P. striiformis genes associated with the infection process by conducting a large-scale transcriptomic analysis using cDNA-AFLP. Results Of the total 54,912 transcript derived fragments (TDFs obtained using cDNA-AFLP with 64 primer pairs, 2,306 (4.2% displayed altered expression patterns after inoculation, of which 966 showed up-regulated and 1,340 down-regulated. 186 TDFs produced reliable sequences after sequencing of 208 TDFs selected, of which 74 (40% had known functions through BLAST searching the GenBank database. Majority of the latter group had predicted gene products involved in energy (13%, signal transduction (5.4%, disease/defence (5.9% and metabolism (5% of the sequenced TDFs. BLAST searching of the wheat stem rust fungus genome database identified 18 TDFs possibly from the stripe rust pathogen, of which 9 were validated of the pathogen origin using PCR-based assays followed by sequencing confirmation. Of the 186 reliable TDFs, 29 homologous to genes known to play a role in disease/defense, signal transduction or uncharacterized genes were further selected for validation of cDNA-AFLP expression patterns using qRT-PCR analyses. Results confirmed the altered expression patterns of 28 (96.5% genes revealed by the cDNA-AFLP technique. Conclusion The results show that cDNA-AFLP is a reliable technique for studying expression patterns of genes involved in the wheat-stripe rust interactions. Genes involved in compatible interactions between wheat and the

Genetic variation in Pythium myriotylum based on SNP typing and development of a PCR-RFLP detection of isolates recovered from Pythium soft rot ginger.

Science.gov (United States)

Le, D P; Smith, M K; Aitken, E A B

2017-10-01

Pythium myriotylum is responsible for severe losses in both capsicum and ginger crops in Australia under different regimes. Intraspecific genomic variation within the pathogen might explain the differences in aggressiveness and pathogenicity on diverse hosts. In this study, whole genome data of four P. myriotylum isolates recovered from three hosts and one Pythium zingiberis isolate were derived and analysed for sequence diversity based on single nucleotide polymorphisms (SNPs). A higher number of true and unique SNPs occurred in P. myriotylum isolates obtained from ginger with symptoms of Pythium soft rot (PSR) in Australia compared to other P. myriotylum isolates. Overall, SNPs were discovered more in the mitochondrial genome than those in the nuclear genome. Among the SNPs, a single substitution from the cytosine (C) to the thymine (T) in the partially sequenced CoxII gene of 14 representatives of PSR P. myriotylum isolates was within a restriction site of HinP1I enzyme which was used in the PCR-RFLP for detection and identification of the isolates without sequencing. The PCR-RFLP was also sensitive to detect PSR P. myriotylum strains from artificially infected ginger without the need for isolation for pure cultures. This is the first study of intraspecific variants of Pythium myriotylum isolates recovered from different hosts and origins based on single nucleotide polymorphism (SNP) genotyping of multiple genes. The SNPs discovered provide valuable makers for detection and identification of P. myriotylum strains initially isolated from Pythium soft rot (PSR) ginger by using PCR-RFLP of the CoxII locus. The PCR-RFLP was also sensitive to detect P. myriotylum directly from PSR ginger sampled from pot trials without the need of isolation for pure cultures. © 2017 The Society for Applied Microbiology.

Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from 18S ribosomal RNA gene of Trypanosoma congolense

International Nuclear Information System (INIS)

Osanyo, A.; Majiwa, P.W.

2006-01-01

Oligonucleotide primers were designed from the conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of protozoans: Trypanosoma brucei, Leishmania donovani, Triponema aequale and Lagenidium gigantum. The primers were used in polymerace chain reaction (PCR) to generate PCR products of approximately 1 Kb using genomic DNA from T. brucei and the four genotypic groups of T. congolense as template. The five PCR products so produced were digested with several restriction enzymes and hybridized to a DNA probe made from T. brucei PCR product of the same 18S rRNA gene region. Most restriction enzyme digests revealed polymorphism with respect to the location of their recognition sites on the five PCR products. The restriction fragment length polymorphism (RFLP) pattern observed indicate that the 18S rRNA gene sequences of trypanosomes: T. brucei and the four genotypes of T.congolence group are heterogeneous. The results further demonstrate that the region that was amplified can be used in specific identification of trypanosomes species and subspecies.(author)

Molecular characterization of enteroinvasive Escherichia coli ipa genes by PCR-RFLP analysis Caracterização molecular do gene ipa de Escherichia coli enteroinvasora pela análise de PCR- RFLP

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Adriana Gibotti

2004-06-01

Full Text Available In this study, polymorphism in ipa genes was found in five out of nine EIEC serotypes studied. When SalI and HindII were used in RFLP-PCR assays many EIEC serotypes showed polymorphism in ipaB and ipaD. On the other hand, no polymorphism was observed in ipaA and ipaC in these strains. The polymorphism present in EIEC strains is serotype-dependent, since restriction patterns were conserved amongst strains belonging to the same serotype. When IpaB deduced amino acid sequences of S. flexneri M90T and FBC124-13 were compared, ten amino acids changes could be observed mainly in the amino-terminal region. The deduced EIEC IpaD amino-acid sequence presented 91% similarity with the Shigella strain. In this case, amino acid changes were spread out through the whole structure, except in the carboxyl-terminal region.No presente estudo, foi encontrado polimorfismo no gene ipa em cinco sorotipos de EIEC, de nove estudados. Quando enzimas de restrição SalI e HindII foram utilizadas no ensaio de PCR-RFLP, amostras de EIEC apresentaram polimorfismo em ipaB e ipaD. Por outro lado, não foram observados polimorfismos nos genes ipaA e ipaC nestas cepas, quando diversas enzimas de restrição foram utilizadas. O polimorfismo presente em cepas de EIEC é sorotipo-dependente, uma vez que os padrões de restrição foram conservados entre as cepas pertencentes ao mesmo sorotipo. Quando a seqüência deduzida de aminoácidos de IpaB de S. flexneri M90T e FBC124-13 foram comparadas, mudanças foram observadas em dez aminoácidos na região amino-terminal. A seqüência deduzida de aminoácidos de IpaD de EIEC apresentou similaridade de 91% com a cepa de Shigella. Neste caso, mudanças de aminoácidos ocorreram em toda a estrutura da molécula de IpaD, exceto na região carboxi-terminal.

Phylogeny and infrageneric delimitation in the genus Spiraea (Rosaceae) inferred from AFLP markers and compared with morphology

Czech Academy of Sciences Publication Activity Database

Záveská Drábková, Lenka; Pospíšková, M.; Businský, R.

2017-01-01

Roč. 185, č. 4 (2017), s. 525-541 ISSN 0024-4074 R&D Projects: GA ČR(CZ) GA16-14649S Institutional support: RVO:67985939 Keywords : morphological analysis * Spiraea * AFLP Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 2.277, year: 2016

ISSR and AFLP analysis of the temporal and spatial population structure of the post-fire annual, Nicotiana attenuata, in SW Utah

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Preston Catherine A

2004-09-01

Full Text Available Abstract Background The native annual tobacco, Nicotiana attenuata, is found primarily in large ephemeral populations (typically for less than 3 growing seasons after fires in sagebrush and pinyon-juniper ecosystems and in small persistent populations (for many growing seasons in isolated washes typically along roadsides throughout the Great Basin Desert of the SW USA. This distribution pattern is due to its unusual germination behavior. Ephemeral populations are produced by the germination of dormant seeds from long-lived seed banks which are stimulated to germinate by a combination of unidentified positive cues found in wood smoke and the removal of inhibitors leached from the unburned litter of the dominant vegetation. Persistent populations may result where these inhibitors do not exist, as in washes or along disturbed roadsides. To determine if this germination behavior has influenced population structure, we conducted an AFLP (244 individuals, ISSR (175 individuals and ISSR+ AFLP (175 individuals analysis on plants originating from seed collected from populations growing in 11 wash and burns over 11 years from the SW USA. Results Genetic variance as measured by both ISSR and AFLP markers was low among sites and comparatively higher within populations. Cluster analysis of the Utah samples with samples collected from Arizona, California, and Oregon as out-groups also did not reveal patterns. AMOVA analysis of the combined AFLP and ISSR data sets yielded significantly low genetic differentiation among sites (Φct, moderate among populations within sites (Φsc and higher genetic differentiation within populations (Φst. Conclusions We conclude that the seed dormancy of this post-fire annual and its resulting age structure in conjunction with natural selection processes are responsible for significantly low among sites and comparatively high within-population genetic variation observed in this species.

Genetic diversity in populations of Isatis glauca Aucher ex Boiss. ssp. from Central Anatolia in Turkey, as revealed by AFLP analysis.

Science.gov (United States)

Özbek, Özlem; Görgülü, Elçin; Yıldırımlı, Şinasi

2013-12-01

Isatidae L. is a complex and systematically difficult genus in Brassicaceae. The genus displays great morphological polymorphism, which makes the classification of species and subspecies difficult as it is observed in Isatis glauca Aucher ex Boiss. The aim of this study is characterization of the genetic diversity in subspecies of Isatis glauca Aucher ex Boiss. distributed widely in Central Anatolia, in Turkey by using Amplified Fragment Length Polymorphism (AFLP) technique. Eight different Eco RI-Mse I primer combinations produced 805 AFLP loci, 793 (98.5%) of which were polymorphic in 67 accessions representing nine different populations. The data obtained by AFLP was computed with using GDA (Genetic Data Analysis) and STRUCTURE (version 2.3.3) software programs for population genetics. The mean proportion of the polymorphic locus (P), the mean number of alleles (A), the number of unique alleles (U) and the mean value of gene diversity (He) were 0.59, 1.59, 20, and 0.23 respectively. The coancestry coefficient (ϴ) was 0.24. The optimal number of K was identified as seven. The principal component analysis (PCA) explained 85.61% of the total genetic variation. Isatis glauca ssp. populations showed a high level of genetic diversity, and the AFLP analysis revealed that high polymorphism and differentiated subspecies could be used conveniently for population genetic studies. The principal coordinate analysis (PCoA) based on the dissimilarity matrix, the dendrogram drawn with UPGMA method and STRUCTURE cluster analysis distinguished the accessions successfully. The accessions formed distinctive population structures for populations AA, AB, E, K, and S. Populations AG1 and AG2 seemed to have similar genetic content, in addition, in both populations several hybrid individuals were observed. The accessions did not formed distinctive population structures for both populations AI and ANP. Consequently, Ankara province might be the area, where species Isatis glauca Aucher

Molecular typing of Trichomonas vaginalis isolates by actin gene sequence analysis and carriage of T. vaginalis viruses.

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Masha, Simon C; Cools, Piet; Crucitti, Tania; Sanders, Eduard J; Vaneechoutte, Mario

2017-10-30

The protozoan parasite Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen. Although T. vaginalis is highly prevalent among women in Kenya, there is lack of data regarding genetic diversity of isolates currently in circulation in Kenya. Typing was performed on 22 clinical isolates of T. vaginalis collected from women attending the antenatal care clinic at Kilifi County Hospital, Kenya, in 2015. Genotyping followed a previously proposed restriction fragment length polymorphism (RFLP) scheme, which involved in silico cleavage of the amplified actin gene by HindII, MseI and RsaI restriction enzymes. Phylogenetic analysis of all the sequences was performed to confirm the results obtained by RFLP-analysis and to assess the diversity within the RFLP genotypes. Additionally, we determined carriage of the four different types of Trichomonas vaginalis viruses (TVVs) by polymerase chain reaction. In silico RFLP-analysis revealed five actin genotypes; 50.0% of the isolates were of actin genotype E, 27.3% of actin genotype N, 13.6% of actin genotype G and 4.5% of actin genotypes I and P. Phylogenetic analysis was in agreement with the RFLP-analysis, with the different actin genotypes clustering together. Prevalence of TVVs was 43.5% (95% confidence interval, CI: 23.2-65.5). TVV1 was the most prevalent, present in 39.1% of the strains and 90% of the T. vaginalis isolates which harbored TVVs had more than one type of TVV. None of the isolates of actin genotype E harbored any TVV. The presence of five actin genotypes in our study suggests notable diversity among T. vaginalis isolates occurring among pregnant women in Kilifi, Kenya. Isolates of the most prevalent actin genotype E lacked TVVs. We found no association between T. vaginalis genotype, carriage of TVVs and symptoms. Further studies with higher number of strains should be conducted in order to corroborate these results.

Inheritance of RFLP loci in a loblolly pine three-generation pedigree

Science.gov (United States)

M.D. Devey; K.D. Jermstad; C.G. Tauer; D.B. Neale

1991-01-01

A high-density restriction fragment length polymorphism (RFLP) linkage map is being constructed for loblolly pine (Pinus taeda L.). Loblolly pine cDNA and genomic DNA clones were used as probes in hybridizations to genomic DNAs prepared from grandparents, parents, and progeny of a three-generation outbred pedigree. Approximately 200 probes were...

Phylogeny and infrageneric delimitation in Spiraea (Rosaceae) inferred from AFLP markers and a comparison with morphology

Czech Academy of Sciences Publication Activity Database

Záveská Drábková, Lenka; Pospíšková, M.; Businský, R.

2017-01-01

Roč. 185, č. 4 (2017), s. 525-541 ISSN 0024-4074 R&D Projects: GA ČR(CZ) GA16-14649S Institutional support: RVO:61389030 Keywords : classification * spiraeoideae * networks * aflp * Bayesian analysis * maximum parsimony * neighbour network * taxonomy Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 2.277, year: 2016

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR-RFLP in Iran.

Science.gov (United States)

Momeni, Zohreh; Sadraei, Javid; Kazemi, Bahram; Dalimi, Abdolhossein

2015-12-01

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral, parasitic sexually transmitted infection in the world. At present, little is known regarding the degree of strain variability of T. vaginalis. A classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, drug resistance, pathogenesis and transmission of T. vaginalis. Eight different types of actin genes have been identified by PCR-RFLP in T. vaginalis; the purpose of this study is to determine the genotypes of this parasite in Karaj city, Iran. Forty-five clinical T. vaginalis isolates from vaginal secretions and urine sediment were collected from Karaj city from 2012 through 2014. DNA was extracted and the actin gene was amplified by nested-PCR; all samples were positive. To determine the genetic differences, sequencing on seven samples was conducted. Then, all PCR products were digested with HindII, MseI, and RsaI restriction enzymes. Of 45 isolates, 23 samples (51.1%) were of actin genotype G, 11 samples (24.4%) of genotype E, six samples (13.3%) of genotype H, three samples (6.6%) of genotype I, and two samples (4.4%) were mixed genotypes of G and E. Genetic diversity of T. vaginalis isolates is notable. The actin genotype G may be the dominant genotype in Karaj city, Iran. Copyright © 2015 Elsevier Inc. All rights reserved.

Clinical application of RT-nested PCR integrated with RFLP in Hantavirus detection and genotyping: a prospective study in Shandong Province, PR China.

Science.gov (United States)

Liu, Yun-Xi; Zhao, Zhong-Tang; Cao, Wu-Chun; Xu, Xiao-Qun; Suo, Ji-Jiang; Xing, Yu-Bin; Jia, Ning; Du, Ming-Mei; Liu, Bo-Wei; Yao, Yuan

2013-01-01

The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.

Characterization of Macrophomina phaseolina isolates by their response to different osmotic potentials and AFLP

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Bárbara J. Gutiérrez Cedeño

2014-01-01

Full Text Available Charcoal rot of Phaseolus vulgaris is caused by the fungus Macrophomina phaseolina, the disease is associated with high temperature and water stress. The objective of this study was to characterize isolates of M. phaseolina by their response to different osmotic potentials and AFLP. The growth of 11 isolates was determined on potato dextrose agar at 48 and 72 h in a gradient of osmotic potential induced using NaCl as well as their effects on germination of sclerotia. Three water groups were statistically different indicating differential response to osmotic potential and all sclerotia grown under these conditions, germinated between 24 and 48h. There were groups of isolates that were tolerant to water stress induced. The AFLP genotyping allowed the formation of five genetic groups, showing a wide genetic variability. Of the nine starters CTA-AT showed a high degree of confidence in the identification of genotypes of M. phaseolina and CAA-AC had the lowest discriminatory power. These results show that M. phaseolina isolates responded differently to osmotic potential and are genetically different between them. Although there was a clear correspondence of genetic groups to water groups; these responses are important features in the search for alternative management in black bean pathosystem. Keywords: molecular marker, M. phaseolina, water deficit

PHYLOGENETIC RELATIONSHIPS AMONGST 10 Durio SPECIES BASED ON PCR-RFLP ANALYSIS OF TWO CHLOROPLAST GENES

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Panca J. Santoso

2013-07-01

Full Text Available Twenty seven species of Durio have been identified in Sabah and Sarawak, Malaysia, but their relationships have not been studied. This study was conducted to analyse phylogenetic relationships amongst 10 Durio species in Malaysia using PCR-RFLP on two chloroplast DNA genes, i.e. ndhC-trnV and rbcL. DNAs were extracted from young leaves of 11 accessions from 10 Durio species collected from the Tenom Agriculture Research Station, Sabah, and University Agriculture Park, Universiti Putra Malaysia. Two pairs of oligonucleotide primers, N1-N2 and rbcL1-rbcL2, were used to flank the target regions ndhC-trnV and rbcL. Eight restriction enzymes, HindIII, BsuRI, PstI, TaqI, MspI, SmaI, BshNI, and EcoR130I, were used to digest the amplicons. Based on the results of PCR-RFLP on ndhC-trnV gene, the 10 Durio species were grouped into five distinct clusters, and the accessions generally showed high variations. However, based on the results of PCR-RFLP on the rbcL gene, the species were grouped into three distinct clusters, and generally showed low variations. This means that ndhC-trnV gene is more reliable for phylogenetic analysis in lower taxonomic level of Durio species or for diversity analysis, while rbcL gene is reliable marker for phylogenetic analysis at higher taxonomic level. PCR-RFLP on the ndhC-trnV and rbcL genes could therefore be considered as useful markers to phylogenetic analysis amongst Durio species. These finding might be used for further molecular marker assisted in Durio breeding program.

Detection and RFLP Analysis of Canine Parvovirus (CPV) DNA by Polymerase Chain Reaction (PCR) in a Dog

OpenAIRE

ÖZKUL, Aykut

2002-01-01

In this study, the detection of canine parvovirus (CPV) in a fecal sample from a dog with enteritis was performed for the first time using the polymerase chain reaction (PCR) in Turkey. The final PCR product was analyzed using the restriction fragment length polymorphysm (RFLP) technique. RFLP analysis using Apa LI and Eco RV restriction endonucleases revealed homology in the nucleotide sequence in at least the VP2 coding region of the virus DNAs detected in the fecal specimen and prepared fr...

DIVERSIDAD GENÉTICA DE MARACUYÁ EN GUATEMALA REVELADA POR MARCADORES AFLP

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Karla Melina Ponciano-Samayoa

2012-01-01

Full Text Available Diversidad genética de maracuyá en Guatemala revelada por marcadores AFLP. Los objetivos de este estudio fueron caracterizar con AFLP nueve genotipos colectados en Guatemala y determinar la diversidad genética existente. En el Instituto de Ciencia y Tecnología Agrícolas (ICTA, en el período julio 2010/mayo 2011, se realizó este análisis preliminar amplifi cando diez combinaciones selectivas con las que se detectaron 106 polimorfi smos. Las combinaciones selectivas E+ACG/M+CAG, E+ACA/M+CTA, E+ACT/M+CTG y E+AAC/M+CTT dieron el mayor grado de información. En promedio se visualizaron diez loci por amplifi cación selectiva. El análisis de similaridad reveló que los genotipos no están duplicados. Los análisis de correspondencia y conglomerados identifi caron dos grupos bien defi nidos. El primero incluyó a los materiales de P. edulis f. edulis Sims y el segundo a los materiales de P. edulis f. fl avicarpa Degener. La diversidad genética de Nei para la colección fue 0,3160. La diferenciación genética (Gst fue 0,2542. El 25,42% de la diversidad se expresó entre grupos mientras que el 74,58% dentro de estos. Los resultados dan evidencia de la cercanía evolutiva de los tipos amarilla y morada de P. edulis Sims. El fl ujo genético fue alto (Nm=1,4670 como se esperaba en una especie alógama en la que se favorece el intercambio inter e intraespecífi co.

BcII RFLP for the human vimentin gene

Energy Technology Data Exchange (ETDEWEB)

Marcus, E M; Smith, B A; Telenius, H; Ponder, B A.J.; Mathew, C G.P. [Haddow Laboratories, Surrey (England); Landsvater, R M; Buys, C H.C.M. [State Univ. of Groningen (Netherlands); Ferrari, S [Temple University Medical School, Philadelphia, PA (USA)

1988-09-26

A 1.1 kb cDNA clone (hp4F1) encoding the human vimentin gene was identified in a human library by screening with 4F1, a hamster vimentin cDNA. BcII (TGATCA) recognizes a two allele polymorphism: bands A1 at 8.1 kb, and A2 at 3.6 kb. The allele frequency was determined in 47 unrelated Caucasian individuals. The RFLP was mapped to chromosome 10pter-10q23 using somatic cell hybrids and to 10p13 by in situ hybridization. Co-dominant segregation was observed in 2 informative families.

SSU rDNA sequence diversity and seasonally differentiated distribution of nanoplanktonic ciliates in neritic Bohai and Yellow Seas as revealed by T-RFLP.

Science.gov (United States)

Dong, Jun; Shi, Fei; Li, Han; Zhang, Xiaoming; Hu, Xiaozhong; Gong, Jun

2014-01-01

Nanociliates have been frequently found to be important players in the marine microbial loop, however, little is known about their diversity and distribution in coastal ecosystems. We investigated the molecular diversity and distribution patterns of nanoplanktonic oligotrich and choreotrich (OC) ciliates in surface water of three neritic basins of northern China, the South Yellow Sea (SYS), North Yellow Sea (NYS), and Bohai Sea (BS) in June and November 2011. SSU rRNA gene clone libraries generated from three summertime samples (sites B38, B4 and H8) were analyzed and revealed a large novel ribotype diversity, of which many were low-abundant phylotypes belonging to the subclass Oligotrichia, but divergent from described morphospecies. Based on the data of terminal-restriction fragment length polymorphism (T-RFLP) analysis of all 35 samples, we found that the T-RF richness was generally higher in the SYS than in the BS, and negatively correlated with the molar ratio of P to Si. Overall, multidimensional scaling and permutational multivariate analysis of variance of the community turnover demonstrated a distinct seasonal pattern but no basin-to-basin differentiation across all samples. Nevertheless, significant community differences among basins were recognized in the winter dataset. Mantel tests showed that the environmental factors, P:Si ratio, water temperature and concentration of dissolved oxygen (DO), determined the community across all samples. However, both biogeographic distance and environment shaped the community in winter, with DO being the most important physicochemical factor. Our results indicate that the stoichiometric ratio of P:Si is a key factor, through which the phytoplankton community may be shaped, resulting in a cascade effect on the diversity and community composition of OC nanociliates in the N-rich, Si-limited coastal surface waters, and that the Yellow Sea Warm Current drives the nanociliate community, and possibly the microbial food webs

Cytogenetic characterization and AFLP-based genetic linkage mapping for the butterfly Bicyclus anynana, covering all 28 karyotyped chromosomes

Czech Academy of Sciences Publication Activity Database

Van´t Hof, A. E.; Marec, František; Saccheri, I. J.; Brakefield, P. M.; Zwaan, B. J.

2008-01-01

Roč. 3, č. 12 (2008), e3882 E-ISSN 1932-6203 R&D Projects: GA ČR GA206/06/1860 Institutional research plan: CEZ:AV0Z50070508 Keywords : Bicyclus anynana * cytogenetic characterization * AFLP-based genetic linkage mapping Subject RIV: EB - Genetics ; Molecular Biology

Huyendo del miedo, desterrando el enojo, escapando de la tristeza y   cayendo en brazos de la risa: los chistes en México en tiempos de la influenza 2009

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Anna María Fernández Poncela

2012-07-01

Full Text Available Se trata de revisar el humor, los chistes y la risa en tiempos de la influenza en México en el año 2009. La relación entre narrativas sociales populares (chistes y reacciones emocionales de la sociedad en un momento de incertidumbre y temor social. El por qué y para qué de los chistes, su función social y psicológica. El objetivo es que el chiste nos acompañe a develar la realidad social, política, emocional y cultural. Para ello se aborda un poco el humor, lo cómico, la risa y los chistes. Presentamos algunas definiciones de estos fenómenos y mostramos su funcionalidad psicológica y social, entre otras co‐ sas, como parte de los sentimientos humanos y la cultura en general. También reflexionamos algo en torno al mundo de las emociones y su evolución o desarrollo, y en determinadas situaciones sociales, en concreto en el contexto de los días de la contingencia sanitaria por el virus A H1N1 en México, en‐ tre abril y mayo del año 2009. 

Key processes for Cheirolophus (Asteraceae diversification on oceanic islands inferred from AFLP data.

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Daniel Vitales

Full Text Available The radiation of the genus Cheirolophus (Asteraceae in Macaronesia constitutes a spectacular case of rapid diversification on oceanic islands. Twenty species - nine of them included in the IUCN Red List of Threatened Species - have been described to date inhabiting the Madeiran and Canarian archipelagos. A previous phylogenetic study revealed that the diversification of Cheirolophus in Macaronesia started less than 2 Ma. As a result of such an explosive speciation process, limited phylogenetic resolution was reported, mainly due to the low variability of the employed molecular markers. In the present study, we used highly polymorphic AFLP markers to i evaluate species' boundaries, ii infer their evolutionary relationships and iii investigate the patterns of genetic diversity in relation to the potential processes likely involved in the radiation of Cheirolophus. One hundred and seventy-two individuals representing all Macaronesian Cheirolophus species were analysed using 249 AFLP loci. Our results suggest that geographic isolation played an important role in this radiation process. This was likely driven by the combination of poor gene flow capacity and a good ability for sporadic long-distance colonisations. In addition, we also found some traces of introgression and incipient ecological adaptation, which could have further enhanced the extraordinary diversification of Cheirolophus in Macaronesia. Last, we hypothesize that current threat categories assigned to Macaronesian Cheirolophus species do not reflect their respective evolutionary relevance, so future evaluations of their conservation status should take into account the results presented here.

Key processes for Cheirolophus (Asteraceae) diversification on oceanic islands inferred from AFLP data.

Science.gov (United States)

Vitales, Daniel; García-Fernández, Alfredo; Pellicer, Jaume; Vallès, Joan; Santos-Guerra, Arnoldo; Cowan, Robyn S; Fay, Michael F; Hidalgo, Oriane; Garnatje, Teresa

2014-01-01

The radiation of the genus Cheirolophus (Asteraceae) in Macaronesia constitutes a spectacular case of rapid diversification on oceanic islands. Twenty species - nine of them included in the IUCN Red List of Threatened Species - have been described to date inhabiting the Madeiran and Canarian archipelagos. A previous phylogenetic study revealed that the diversification of Cheirolophus in Macaronesia started less than 2 Ma. As a result of such an explosive speciation process, limited phylogenetic resolution was reported, mainly due to the low variability of the employed molecular markers. In the present study, we used highly polymorphic AFLP markers to i) evaluate species' boundaries, ii) infer their evolutionary relationships and iii) investigate the patterns of genetic diversity in relation to the potential processes likely involved in the radiation of Cheirolophus. One hundred and seventy-two individuals representing all Macaronesian Cheirolophus species were analysed using 249 AFLP loci. Our results suggest that geographic isolation played an important role in this radiation process. This was likely driven by the combination of poor gene flow capacity and a good ability for sporadic long-distance colonisations. In addition, we also found some traces of introgression and incipient ecological adaptation, which could have further enhanced the extraordinary diversification of Cheirolophus in Macaronesia. Last, we hypothesize that current threat categories assigned to Macaronesian Cheirolophus species do not reflect their respective evolutionary relevance, so future evaluations of their conservation status should take into account the results presented here.

Genotyping of Pasteurella multocida ovine and bovine isolates from Iran based on PCR-RFLP of ompH gene

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A. Ghanizadeh

2015-10-01

Full Text Available Pasteurella multocida (P. multocida, A Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. The outer membrane of Gram-negative bacteria contains of many different protein in very high copy numbers. One of the major outer membrane, the H proteins have functional as high immunogenicity and antigenicity. In this study to increase information about epidemiology of ovine and bovine P. multocida, the 24 isolates from sheep and nine isolates from cattle were investigated by PCR-RFLP analysis of the ompH gene. In all 33 isolates, digestion of the amplified fragment of ompH gene by using EcoRI, cfoI and HindIII produced 3, 5 and 3 different restriction patterns respectively. Sixteen RFLP patterns were found among 33 investigated P.multocida isolates. This study showed that, the PCR RFLP based on ompH gene is potentially a useful method for typing of P. multocida isolates from sheep and cattle. The RFLP patterns of this gene exhibited extensive restriction site heterogeneity, which may be particularly suitable for fingerprinting of P. multocida isolates.Considering ompH protein as a protective immunogenic moiety of P.ultocida, the results of this study showed a heterogenic bacteria and this means the possibility to produce a multivalent vaccine to be protective against diseases caused by this organism in sheep and cattle in Iran.

Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

Science.gov (United States)

Montalvo, Ana M; Fraga, Jorge; Tirado, Dídier; Blandón, Gustavo; Alba, Annia; Van der Auwera, Gert; Vélez, Iván Darío; Muskus, Carlos

2017-07-01

Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.

A note on the measurement of genetic diversity within genebank accessions of lettuce (Lactuca sativa L.) using AFLP markers

NARCIS (Netherlands)

Jansen, J.; Verbakel, H.; Peleman, J.; Hintum, van T.J.L.

2006-01-01

This paper discusses a statistical approach for measuring genetic diversity within genebank accessions of a self-fertilising species. This approach is applied to lettuce (Lactuca sativa L.), using AFLP marker data on a set of 1,390 accessions, representing six different lettuce types. Knowledge of

Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

Science.gov (United States)

Alonso, Sergio; Suzuki, Koichi; Yamamoto, Fumiichiro; Perucho, Manuel

2018-01-01

Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

PCR-RFLP Analysis of a fliC Gene Fragment in Avian Salmonella Isolates

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Zohreh Ebrahimvandi

2014-07-01

Full Text Available Background: Salmonella are a genus of zoonotic bacteria of worldwide economic and health importance. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or watercontaminated by infected feces. Objectives: The aim of this study was to apply a PCR-RFLP method based on the fliC gene to identify the serotypes of Salmonella isolates from Karaj, Iran. Materials and Methods: A total of 30 Salmonella isolates were serotyped by specific antisera. For the PCR-RFLP method based on the fliC gene, extracted DNA was used as the template for amplifying the fliC gene (1500 bp using specific primers. PCR products were subjected to digestion using HhaI restriction endonuclease. Results: This study determined 30 serotypes as Salmonella durban (56.6%, Salmonella uno (23.3%, Salmonella enteritidis (3.3%, Salmonella tinda (3.3%, Salmonella mjimweme (3.3%, Salmonella Thompson (3.3%, Salmonella sIIO8 (3.3 % and Salmonella sIIO7 (3.3%. Observations indicated that HhaI is able to discriminate Salmonella tinda and Salmonella thompson, yet Salmonella enteritidis, Salmonella durban and Salmonella mjimweme had the same pattern with this enzyme. Also Salmonella sIIO8, Salmonella sIIO7 and Salmonella uno showed the same pattern. Thus, regarding the size and the number of resulting fragments from this enzyme, four patterns were obtained for HhaI. Conclusion: A large number of Salmonella serotypes need to be analyzed by the PCR-RFLP method and different enzymes must be used to give reliable results.

Molecular characterization of UV-treated sugar beet somaclones using RFLP markers

International Nuclear Information System (INIS)

Levall, M.W.; Bengtsson, K.; Nilsson, N.-O.; Hjerdin, A.; Hallden, C.

1994-01-01

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment length polymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants. (author)

Molecular typing of Trichomonas vaginalis isolates by actin gene sequence analysis and carriage of T. vaginalis viruses

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Simon C. Masha

2017-10-01

Full Text Available Abstract Background The protozoan parasite Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen. Although T. vaginalis is highly prevalent among women in Kenya, there is lack of data regarding genetic diversity of isolates currently in circulation in Kenya. Methods Typing was performed on 22 clinical isolates of T. vaginalis collected from women attending the antenatal care clinic at Kilifi County Hospital, Kenya, in 2015. Genotyping followed a previously proposed restriction fragment length polymorphism (RFLP scheme, which involved in silico cleavage of the amplified actin gene by HindII, MseI and RsaI restriction enzymes. Phylogenetic analysis of all the sequences was performed to confirm the results obtained by RFLP-analysis and to assess the diversity within the RFLP genotypes. Additionally, we determined carriage of the four different types of Trichomonas vaginalis viruses (TVVs by polymerase chain reaction. Results In silico RFLP-analysis revealed five actin genotypes; 50.0% of the isolates were of actin genotype E, 27.3% of actin genotype N, 13.6% of actin genotype G and 4.5% of actin genotypes I and P. Phylogenetic analysis was in agreement with the RFLP-analysis, with the different actin genotypes clustering together. Prevalence of TVVs was 43.5% (95% confidence interval, CI: 23.2–65.5. TVV1 was the most prevalent, present in 39.1% of the strains and 90% of the T. vaginalis isolates which harbored TVVs had more than one type of TVV. None of the isolates of actin genotype E harbored any TVV. Conclusion The presence of five actin genotypes in our study suggests notable diversity among T. vaginalis isolates occurring among pregnant women in Kilifi, Kenya. Isolates of the most prevalent actin genotype E lacked TVVs. We found no association between T. vaginalis genotype, carriage of TVVs and symptoms. Further studies with higher number of strains should be conducted in order to corroborate these results.

RFLP for the human pepsinogen C gene (PGC)

Energy Technology Data Exchange (ETDEWEB)

Azuma, T; Pals, G; Taggart, R T

1988-10-11

PGC 301 is a 1224 bp cDNA clone containing exons 2-9 of the human pepsinogen C (progastericsin) coding sequence. 100 bp deletion/insertion polymorphism is observed with five restriction endonucleases; BamHI, EcoRI, MstII, PstI, and SacI. The same RFLP is observed with these enzymes. The polymorphic region is located between exons 7 and 8. The frequency was estimated from 40 unrelated Caucasians, with the large fragment allele 82.5% and the small fragment allele 17.5%. PGC gene has been assigned to 6p21.1-pter by somatic cell hybrids. Mendelian inheritance was demonstrated in two families.

Analysis of ELA-DQB exon 2 polymorphism in Argentine Creole horses by PCR-RFLP and PCR-SSCP.

Science.gov (United States)

Villegas-Castagnasso, E E; Díaz, S; Giovambattista, G; Dulout, F N; Peral-García, P

2003-08-01

The second exon of equine leucocyte antigen (ELA)-DQB genes was amplified from genomic DNA of 32 Argentine Creole horses by PCR. Amplified DNA was analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformation polymorphism (SSCP). The PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns, five MspI patterns and two HinfI patterns. EcoRI showed no variation in the analysed sample. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel ELA-DQB alleles. PCR-SSCP analysis exhibited seven different band patterns, and the number of bands per animal ranged from four to nine. Both methods indicated that at least two DQB genes are present. The presence of more than two alleles in each animal showed that the primers employed in this work are not specific for a unique DQB locus. The improvement of this PCR-RFLP method should provide a simple and rapid technique for an accurate definition of ELA-DQB typing in horses.

Use of AFLP marker system on sugarcane somaclones to study their resistance to rust

OpenAIRE

María Ileana Oloriz; Luis Rojas; Víctor Gil; Milady Mendoza; Ariel Arencibia; Elva Rosa Carmona; Elio Jiménez

2002-01-01

AFLPs (amplified fragment length polymorphism) was carried out from genomic DNA of five rust resistant sugar cane somaclons and their susceptible donor, Saccharum officinarum var B 4362, using three combinations of primers (EcoRI/aca: MseI/acc; EcoRI/aca: MseI/atg and EcoRI/aca: MseI/agg). Six polymorphic bands were obtained, two of these only appeared in the resistant genotypes, which are probably DNA sequences, related to rust resistance locus. These fragments have been cloned to study thei...

Differentially expressed genes in Populus simonii x P. nigra in respnse to NaCl stress using cDNA-AFLP

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Salinity is an important environmental factor limiting growth and productivity of plants, and affects almost every aspect of the plant physiology and biochemistry. The objective of this study was to apply cDNA-AFLP and to identify differentially expressed genes in response to NaCl stress vs. no-stre...

Morphological changes of Paulownia seedlings infected phytoplasmas reveal the genes associated with witches' broom through AFLP and MSAP.

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Xibing Cao

Full Text Available Paulownia witches' broom (PaWB caused by phytoplasma might result in devastating damage to the growth and wood production of Paulownia. To study the effect of phytoplasma on DNA sequence and to discover the genes related to PaWB occurrence, DNA polymorphisms and DNA methylation levels and patterns in PaWB seedlings, the ones treated with various concentration of methyl methane sulfonate (MMS and healthy seedlings were investigated with amplified fragment length polymorphism (AFLP and methylation-sensitive amplification polymorphism (MSAP. Our results indicated that PaWB seedlings recovered a normal morphology, similar to healthy seedlings, after treatment with more than 20 mg · L-1 MMS; Phytoplasma infection did not change the Paulownia genomic DNA sequence at AFLP level, but changed the global DNA methylation levels and patterns; Genes related to PaWB were discovered through MSAP and validated using quantitative real-time PCR (qRT-PCR. These results implied that changes of DNA methylation levels and patterns were closely related to the morphological changes of seedlings infected with phytoplasmas.

Non PCR-amplified Transcripts and AFLP fragments as reduced representations of the quail genome for 454 Titanium sequencing

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Leterrier Christine

2010-07-01

Full Text Available Abstract Background SNP (Single Nucleotide Polymorphism discovery is now routinely performed using high-throughput sequencing of reduced representation libraries. Our objective was to adapt 454 GS FLX based sequencing methodologies in order to obtain the largest possible dataset from two reduced representations libraries, produced by AFLP (Amplified Fragment Length Polymorphism for genomic DNA, and EST (Expressed Sequence Tag for the transcribed fraction of the genome. Findings The expressed fraction was obtained by preparing cDNA libraries without PCR amplification from quail embryo and brain. To optimize the information content for SNP analyses, libraries were prepared from individuals selected in three quail lines and each individual in the AFLP library was tagged. Sequencing runs produced 399,189 sequence reads from cDNA and 373,484 from genomic fragments, covering close to 250 Mb of sequence in total. Conclusions Both methods used to obtain reduced representations for high-throughput sequencing were successful after several improvements. The protocols may be used for several sequencing applications, such as de novo sequencing, tagged PCR fragments or long fragment sequencing of cDNA.

AFLP diversity between the Novosibirsk and Tomsk chromosome races of the common shrew (Sorex araneus

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Andrey Polyakov

2009-12-01

Full Text Available Genetic diversity between of the Novosibirsk and Tomsk chromosome races of the common shrew (Sorex araneus was analyzed using 39 polymorphic AFLP (amplified fragments length polymorphism markers. Exact and F-statistics tests for population differentiation demonstrated significant interracial difference in allele frequencies and significant subdivision between the races. The value of the genetic distance between the chromosome races observed in this study corresponds to that found between subspecies of mammals studied so far.

Molecular typing of phlebotomine sand flies in al-madinah and asir regions, Saudi Arabia using PCR–RFLP of 18S

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Abeer A. Al-Dakhil

2017-11-01

Full Text Available Studies on the distribution of sand flies are important for the control of leishmaniasis in endemic and neighboring areas. In the present study polymerase chain reaction (PCR–restriction fragment length polymorphism (RFLP was used to identify the distribution of sand flies in Al-Madinah and Asir Regions of Saudi Arabia using PCR–RFLP of 18S ribosomal RNA gene. Based on the morphological characteristics, the sand flies were differentiated into seven species viz., Phlebotomus papatasi, Phlebotomus sergenti, Phlebotomus bergeroti, Sergentomyia clydei, Sergentomyia antennata, Sergentomyia fallax and Sergentomyia schwetzi. PCR–RFLP of 18S ribosomal RNA (rRNA genes with eight different restriction enzymes resulted in species-specific agarose gel electrophoresis banding patterns. Of the eight restriction enzymes used, not a single restriction enzyme by itself could separate species belonging to the same genera (like P. papatasi and P. sergenti by AseI as well as those belonging to different genera (like P. papatasi and S. clydei by AseI. We therefore conclude that the genetic diversity within sand fly species based on PCR–RFLP technique was nonspecific. Studies are in progress to study the viability of alternate techniques like low-stringency single specific primer polymerase chain reaction which can be used for molecular typing.

Gm typing by immunoglobulin heavy-chain gene RFLP analysis.

OpenAIRE

Jazwinska, E C; Dunckley, H; Propert, D N; Gatenby, P A; Serjeantson, S W

1988-01-01

This study was undertaken to investigate a means of assigning Gm allotypes to Caucasians by RFLP analysis. A single immunoglobulin heavy-chain gamma-4 cDNA probe (HU gamma 4) was hybridized with genomic DNA digested separately with two restriction enzymes, TaqI and PvuII. Results showed excellent correlation (P less than .001) between serologically defined Gm allotypes G1m(1), G1m(2), G2m(23), and G1m;G3m (3;5,10) and RFLPs identified with the (HU gamma 4) probe. We conclude that it is now po...

Software for optimization of SNP and PCR-RFLP genotyping to discriminate many genomes with the fewest assays

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Wagner Mark C

2005-05-01

Full Text Available Abstract Background Microbial forensics is important in tracking the source of a pathogen, whether the disease is a naturally occurring outbreak or part of a criminal investigation. Results A method and SPR Opt (SNP and PCR-RFLP Optimization software to perform a comprehensive, whole-genome analysis to forensically discriminate multiple sequences is presented. Tools for the optimization of forensic typing using Single Nucleotide Polymorphism (SNP and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP analyses across multiple isolate sequences of a species are described. The PCR-RFLP analysis includes prediction and selection of optimal primers and restriction enzymes to enable maximum isolate discrimination based on sequence information. SPR Opt calculates all SNP or PCR-RFLP variations present in the sequences, groups them into haplotypes according to their co-segregation across those sequences, and performs combinatoric analyses to determine which sets of haplotypes provide maximal discrimination among all the input sequences. Those set combinations requiring that membership in the fewest haplotypes be queried (i.e. the fewest assays be performed are found. These analyses highlight variable regions based on existing sequence data. These markers may be heterogeneous among unsequenced isolates as well, and thus may be useful for characterizing the relationships among unsequenced as well as sequenced isolates. The predictions are multi-locus. Analyses of mumps and SARS viruses are summarized. Phylogenetic trees created based on SNPs, PCR-RFLPs, and full genomes are compared for SARS virus, illustrating that purported phylogenies based only on SNP or PCR-RFLP variations do not match those based on multiple sequence alignment of the full genomes. Conclusion This is the first software to optimize the selection of forensic markers to maximize information gained from the fewest assays, accepting whole or partial genome sequence data as input. As

Identification of Spanish isolates of Rhizoctonia solani from potato by anastomosis grouping, ITS-RFLP and RAMS-fingerprinting

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A.M. Elbakali

2003-08-01

Full Text Available Anastomosis grouping, restriction fragment length polymorphism (RFLP of the ITS regions including the 5.85 rDNA, and random amplified microsatellites (RAMS were used to characterize isolates of Rhizoctonia solani collected from Spain and Finland. There was a high similarity between the results obtained with the three techniques. RAMS markers revealed more genetic variation among isolates of R. solani than RFLP. The anastomosis group (AG–3 isolates were clearly separated from isolates belonging to other AGs by RAMS, RFLPs and anastomosis grouping. Almost all the isolates sampled from potato belonged to AG–3. No differences were observed between Spanish and Finnish AG–3 isolates.

Using AFLP markers and the Geneland program for the inference of population genetic structure

DEFF Research Database (Denmark)

Guillot, Gilles; Santos, Filipe

2010-01-01

the computer program Geneland designed to infer population structure has been adapted to deal with dominant markers; and (ii) we use Geneland for numerical comparison of dominant and codominant markers to perform clustering. AFLP markers lead to less accurate results than bi-allelic codominant markers...... such as single nucleotide polymorphisms (SNP) markers but this difference becomes negligible for data sets of common size (number of individuals n≥100, number of markers L≥200). The latest Geneland version (3.2.1) handling dominant markers is freely available as an R package with a fully clickable graphical...

SSU rDNA sequence diversity and seasonally differentiated distribution of nanoplanktonic ciliates in neritic Bohai and Yellow Seas as revealed by T-RFLP.

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Jun Dong

Full Text Available Nanociliates have been frequently found to be important players in the marine microbial loop, however, little is known about their diversity and distribution in coastal ecosystems. We investigated the molecular diversity and distribution patterns of nanoplanktonic oligotrich and choreotrich (OC ciliates in surface water of three neritic basins of northern China, the South Yellow Sea (SYS, North Yellow Sea (NYS, and Bohai Sea (BS in June and November 2011. SSU rRNA gene clone libraries generated from three summertime samples (sites B38, B4 and H8 were analyzed and revealed a large novel ribotype diversity, of which many were low-abundant phylotypes belonging to the subclass Oligotrichia, but divergent from described morphospecies. Based on the data of terminal-restriction fragment length polymorphism (T-RFLP analysis of all 35 samples, we found that the T-RF richness was generally higher in the SYS than in the BS, and negatively correlated with the molar ratio of P to Si. Overall, multidimensional scaling and permutational multivariate analysis of variance of the community turnover demonstrated a distinct seasonal pattern but no basin-to-basin differentiation across all samples. Nevertheless, significant community differences among basins were recognized in the winter dataset. Mantel tests showed that the environmental factors, P:Si ratio, water temperature and concentration of dissolved oxygen (DO, determined the community across all samples. However, both biogeographic distance and environment shaped the community in winter, with DO being the most important physicochemical factor. Our results indicate that the stoichiometric ratio of P:Si is a key factor, through which the phytoplankton community may be shaped, resulting in a cascade effect on the diversity and community composition of OC nanociliates in the N-rich, Si-limited coastal surface waters, and that the Yellow Sea Warm Current drives the nanociliate community, and possibly the

Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of simple PCR-RFLP for preliminary differentiation.

Science.gov (United States)

Mirhendi, H; Zarei, F; Motamedi, M; Nouripour-Sisakht, S

2016-03-01

This work aimed to identify the species distribution of common clinical and environmental isolates of black Aspergilli based on simple restriction fragment length polymorphism (RFLP) analysis of the β-tubulin gene. A total of 149 clinical and environmental strains of black Aspergilli were collected and subjected to preliminary morphological examination. Total genomic DNAs were extracted, and PCR was performed to amplify part of the β-tubulin gene. At first, 52 randomly selected samples were species-delineated by sequence analysis. In order to distinguish the most common species, PCR amplicons of 117 black Aspergillus strains were identified by simple PCR-RFLP analysis using the enzyme TasI. Among 52 sequenced isolates, 28 were Aspergillus tubingensis, 21 Aspergillus niger, and the three remaining isolates included Aspergillus uvarum, Aspergillus awamori, and Aspergillus acidus. All 100 environmental and 17 BAL samples subjected to TasI-RFLP analysis of the β-tubulin gene, fell into two groups, consisting of about 59% (n=69) A. tubingensis and 41% (n=48) A. niger. Therefore, the method successfully and rapidly distinguished A. tubingensis and A. niger as the most common species among the clinical and environmental isolates. Although tardy, the Ehrlich test was also able to differentiate A. tubingensis and A. niger according to the yellow color reaction specific to A. niger. A. tubingensis and A. niger are the most common black Aspergillus in both clinical and environmental isolates in Iran. PCR-RFLP using TasI digestion of β-tubulin DNA enables rapid screening for these common species. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species.

Science.gov (United States)

Şakalar, Çağrı; Kuk, Salih; Erensoy, Ahmet; Dağli, Adile Ferda; Özercan, İbrahim Hanifi; Çetınkaya, Ülfet; Yazar, Süleyman

2014-01-01

To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

PCR-RFLP de las regiones ITS-5.8S como herramienta de identificación de levaduras: ventajas y desventajas

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Luis E. Segura G.

2010-01-01

Full Text Available Las levaduras juegan un papel muy importante en los procesos de fermentación de bebidas yalimentos, por lo que es necesario identificarlas. A finales de los años 90 se propuso la técnica de análisis delpolimorfismo en la longitud de los fragmentos de restricción (RFLP de las regiones ITS-5.8S como una herramienta rápida y confiable para identificarlas. En este trabajo se analizaron 104 cepas de Saccharomyces cerevisiae de la colección de CIATEJ. Además se analizaron los patrones de bandas publicados hasta el momento para levaduras, lo que nos permitió establecer el parámetro de error de dicha técnica. A partir de este análisis se comprobó que esta técnica es rápida y confiable en la gran mayoría de los casos, sin embargo para algunas especies de levaduras existen ambigüedades en los patrones publicados o no permite diferenciar especies cercanas.

Using AFLP-RGA markers to assess genetic diversity among pigeon pea (Cajanus cajan genotypes in relation to major diseases

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Prakash G Pati

2014-06-01

Full Text Available Resistance gene analog (RGA-anchored amplified fragment length polymorphism (AFLP-RGA marker system was used in order to evaluate genetic relationships among 22 pigeon pea genotypes with varied responses to Fusarium wilt and sterility mosaic disease. Five AFLP-RGA primer combinations (E-CAG/wlrk-S, M-GTG/wlrk-S, M-GTG/wlrk-AS, E-CAT/S1-INV and E-CAG/wlrk-AS produced 173 scorable fragments, of which 157 (90.7% were polymorphic, with an average of 31.4 fragments per primer combination. The polymorphism rates obtained with the five primers were 83.3%, 92.0%, 92.3%, 93.0% and 93.1%, respectively. Mean polymorphic information content (PIC values ranged from 0.24 (with E-CAT/S1-INV to 0.30 (with E-CAG/wlrk-AS, whereas resolving power (RP values varied from 11.06 (with M-GTG/wlrk-S to 25.51 (with E-CAG/wlrk-AS and marker index (MI values ranged from 5.98 (with M-GTG/wlrk-S to 12.30 (with E-CAG/wlrk-AS. We identified a positive correlation between MI and RP (r²=0.98, p<0.05, stronger that that observed for the comparison between PIC and RP (r²=0.88, p<0.05. That implies that either MI or RP is the best parameter for selecting more informative AFLP-RGA primer combinations. The Jaccard coefficient ranged from 0.07 to 0.72, suggesting a broad genetic base in the genotypes studied. A neighbor-joining tree, based on the unweighted pair group method with arithmetic mean, distinguished cultivated species from wild species. The grouping of resistant genotypes in different clusters would help in the selection of suitable donors for resistance breeding in pigeon pea.

Identificação espécie-específica de carnes e produtos cárneos de origem bubalina e bovina pela técnica de PCR-RFLP

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L.V. Teixeira

2015-02-01

Full Text Available Para avaliar a viabilidade da metodologia da Reação em Cadeia da Polimerase associada com o Polimorfismo de Fragmentos de DNA (PCR-RFLP na identificação de fraude intencional e contaminação acidental em produtos cárneos de origem bubalina, in natura e processados, foram testadas amostras puras e amostras de carnes com misturas controladas, produzidas em laboratório, com adição de 1%, 5%, 10% e 50% de carne bovina em carne de búfalo, homogeneizada crua e em amostras autoclavada. Foram comparados, ainda, diferentes métodos de extração, usando um kit comercial e a técnica clássica, utilizando fenol/clorofórmio. O resultado estatístico foi obtido por tabela de contingência, analisada pelo teste do qui-quadrado (χ2 e do exato de Fisher. A especificidade encontrada foi altamente significativa (P<0,0001. Observou-se também sensibilidade altamente significativa nas diluições a partir de 10% (P<0,0001. A técnica tem alta especificidade e sensibilidade para detectar até mesmo contaminação de 1%, mas a repetibilidade desse resultado impede a aplicação oficial desse método para a inspeção de contaminação acidental, sendo recomendada somente para inspeção de fraude a partir de 10% de substituição. Em carnes autoclavadas, a eficácia do teste é menor. A técnica pode ser empregada para certificação de produto específico (selo de identidade de espécie.

Application of DNA RFLP procedures in interspecific gene transfer: The Lr19 translocation of wheat

International Nuclear Information System (INIS)

Prins, R.; Marais, G.F.; Marais, A.S.; Pretorius, Z.A.; Janse, B.J.H.

1998-01-01

Twenty-nine lines with deletions in the Lr19 ('Indis') translocated chromosome segment were used to physically map Thinopyrum Restriction Fragment Length Polymorphism (RFLP) loci as well as the Sr25 and Sdl loci. The relative distances between marker loci on the translocation were then calculated. The information was then used as an aid to characterize several recombined forms of the translocation. The data confirmed the reported homoeology between the Lr19 segment and chromosome arm 7DL of wheat. Also, it seems that the Lr19 translocation in 'Indis' is very similar to the Lr19 segment in the T4 source and that the former may not derive from Thinopyrum distichum. Near-isogenic lines of the recombined segments were derived and used to study their expression of leaf rust resistance. It became evident that only one potentially useful recombinant was obtained in an earlier attempt to induce allosyndetic pairing between the Lr19 translocation and 7DL of wheat. (author)

PCR-RFLP Using BseDI Enzyme for Pork Authentication in Sausage and Nugget Products

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Y. Erwanto

2011-04-01

Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP using BseDI restriction enzyme had been applied for identifying the presence of pork in processed meat (beef sausage and chicken nugget including before and after frying. Pork sample in various levels (1%, 3%, 5%, 10%, and 25 % was prepared in a mixture with beef and chicken meats and processed for sausage and nugget. The primers CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b (cyt b gene and PCR successfully amplified fragments of 359 bp. To distinguish existence of porcine species, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed pig mitochondrial DNA was cut into 131 and 228 bp fragments. The PCR-RFLP species identification assay yielded excellent results for identification of porcine species. It is a potentially reliable technique for pork detection in animal food processed products for Halal authentication.

Typing clinical and animal environment Aspergillus fumigatus gliotoxin producer strains isolated from Brazil by PCR-RFLP markers.

Science.gov (United States)

Soleiro, C A; Pena, G A; Cavaglieri, L R; Coelho, I; Keller, L M; Dalcero, A M; Rosa, C A R

2013-12-01

Aspergillus fumigatus, a well-known human and animal pathogen causing aspergillosis, has been historically identified by morphological and microscopic features. However, recent studies have shown that species identification on the basis of morphology alone is problematic. The aim of this work was to confirm the taxonomic state at specie level of a set of clinical (human and animal) and animal environment A. fumigatus strains identified by morphological criteria applying a PCR-RFLP assay by an in silico and in situ analysis with three restriction enzymes. The A. fumigatus gliotoxin-producing ability was also determined. Previous to the in situ PCR-RFLP analysis, an in silico assay with BccI, MspI and Sau3AI restriction enzymes was carried out. After that, these enzymes were used for in situ assay. All A. fumigatus strains isolated from corn silage, human aspergillosis and bovine mastitis and high per cent of the strains isolated from cereals, animal feedstuff and sorghum silage were able to produce high gliotoxin levels. Also, all these strains identified by morphological criteria as A. fumigatus, regardless of its isolation source, had band patterns according to A. fumigatus sensu stricto by PCR-RFLP markers. Aspergillus fumigatus is a well-known human and animal pathogen causing aspergillosis. In this study, clinical (human and animal) and animal environment strains were able to produce high gliotoxin levels and had band profiles according to A. fumigatus sensu stricto by PCR-RFLP markers. The results obtained here suggest that strains involved in human and animal aspergillosis could come from the animal environment in which A. fumigatus is frequently found. Its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs. © 2013 The Society for Applied Microbiology.

Identification of AFLP molecular linked to row- type gene in barley

International Nuclear Information System (INIS)

Sayed- Tabatabaei, B.E.

2005-01-01

Formation of the two-and six-rowed types in barley is predominantly controlled by alleles at a single locus (vrzl) which is located in long armn of chromosome 2H. This gene is a key character on the study of barley domestication and yield. Near-isogenic lines of barley were produced from crosses between Kanto Nakate Gold (tow-rowed) and Azumamugi (six-rowed). The selected lines were used for screening of AFLP polymorphic bands which are linked to vrs1 locus. After screening of a total of 1792 primer combination, five polymorphic bands were identified. A construction of high resolution map around the vrs1 locus was made using recombinant inbred lines. These markers can be used for a map-based cloning of the genes at the vrsl locus

Molecular screening for Candida orthopsilosis and Candida metapsilosis among Danish Candida parapsilosis group blood culture isolates: proposal of a new RFLP profile for differentiation

DEFF Research Database (Denmark)

Mirhendi, Hossein; Bruun, Brita; Schønheyder, Henrik Carl

2010-01-01

. Isolates with RFLP patterns distinct from C. parapsilosis were characterized by sequence analysis of the ITS1-ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610 restriction enzymes were predicted in silico using 12 available sequences. By PCR-RFLP of the SADH......, respectively. This was confirmed using the reference strains and 79 clinical isolates. In conclusion, reliable discrimination was obtained by PCR-RFLP profile analysis of the SADH gene after digestion with NlaIII but not with BanI. C. metapsilosis and C. orthopsilosis are involved in a small but significant...

Diversidad genética intra e inter-específica de ñame (Dioscorea spp. de la región Caribe de Colombia mediante marcadores AFLP Genetic diversity intra and inter-specific yam (Dioscorea spp. from the colombian caribbean region by AFLP markers

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Hernando Javier Rivera-Jiménez

2011-12-01

Full Text Available Conocer la variabilidad genética del ñame, Dioscorea spp., permite apoyar estrategias de mejoramiento y conservación de este recurso fitogenético. El objetivo de este estudio fue la caracterización molecular de 20 accesiones de Dioscorea spp. mediante la técnica molecular de AFLP para determinar cómo se distribuye la variabilidad genética de manera intra e inter-específica. Los datos fueron analizados mediante los métodos de agrupación de correspondencia múltiple y análisis de similaridad de Dice, estableciendo los niveles de confiabilidad de los grupos genéticos mediante remuestreos. En términos de diversidad interespecífica, los valores promedios de similitud variaron entre 41.81% entre D. alata L. y D. rotundata Poir., y 33.51% entre D. trifida L.f. y D. esculenta (Lour. Burkill, lo que sugiere alta diversidad genética entre las accesiones estudiadas, que formaron cuatro grupos genéticos: D. alata, D. rotundata, D. esculenta y D. trifida, confirmando correspondencia entre la caracterización morfológica, clasificación botánica y la caracterización molecular. En términos de diversidad intraespecífica para la especie D. alata, el análisis también reveló una composición heterogénea en la región Caribe colombiana. Estos estudios ayudarán a definir una estrategia adecuada para fines de conservación y apoyar los esfuerzos futuros en los programas de mejoramiento genético.Knowing the genetic variability of yams, Dioscorea spp., is a good tool to support development and conservation strategies of this plant as genetic resource. The aim of this study was to carried out the molecular characterization of 20 accessions of Dioscorea spp. using the AFLP molecular technique to determine how genetic variation is distributed intra-and inter-specifically. Using multiple correspondence analysis and level of reliability of the genetic groups by resampling, the results showed high genetic variability among the accessions studied

Levels of Intra-specific AFLP Diversity in Tuber-Bearing Potato Species with Different Breeding Systems and Ploidy Levels

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Glenn J. Bryan

2017-09-01

Full Text Available DNA-based marker analysis of plant genebank material has become a useful tool in the evaluation of levels of genetic diversity and for the informed use and maintenance of germplasm. In this study, we quantify levels of amplified fragment length polymorphism (AFLP in representative accessions of wild and cultivated potato species of differing geographic origin, ploidy, and breeding system. We generated 449 polymorphic AFLP fragments in 619 plants, representing multiple plants (16–23 from 17 accessions of 14 potato taxa as well as single plants sampled from available accessions (from 3 to 56 of the same 14 taxa. Intra-accession diversities were compared to those of a synthetic ‘taxon-wide’ population comprising a single individual from a variable number of available accessions of each sampled taxon. Results confirm the expected considerably lower levels of polymorphism within accessions of self-compatible as compared to self-incompatible taxa. We observed broadly similar levels of ‘taxon-wide’ polymorphism among self-compatible and self-incompatible species, with self-compatible taxa showing only slightly lower rates of polymorphism. The most diverse accessions were the two cultivated potato accessions examined, the least diverse being the Mexican allohexaploids Solanum demissum and S. iopetalum. Generally allopolyploid self-compatible accessions exhibited lower levels of diversity. Some purported self-incompatible accessions showed relatively low levels of marker diversity, similar to the more diverse self-compatible material surveyed. Our data indicate that for self-compatible species a single plant is highly representative of a genebank accession. The situation for self-incompatible taxa is less clear, and sampling strategies used will depend on the type of investigation. These results have important implications for those seeking novel trait variation (e.g., disease resistance in gene banks as well as for the selection of individuals

Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

Science.gov (United States)

Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

2016-09-01

We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05). © 2016 The Author(s).

Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease.

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Ying Wang

Full Text Available High resolution melting (HRM is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD. We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population.A total of 315 blood samples from 147 CHD patients (male72, female 75 and 168 healthy controls (male 92, female 76 were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs from unconditional logistic regression.All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%.MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping.

Genetic diversity revealed by AFLP markers in Albanian goat breeds

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Hoda Anila

2012-01-01

Full Text Available The amplified fragment length polymorphism (AFLP technique with three EcoRI/TaqI primer combinations was used in 185 unrelated individuals, representative of 6 local goat breeds of Albania, and 107 markers were generated. The mean Nei’s expected heterozygosity value for the whole population was 0.199 and the mean Shannon index was 0.249, indicating a high level of within-breed diversity. Wright’s FST index, Nei’s unbiased genetic distance and Reynolds’ genetic distance were calculated. Pairwise Fst values among the populations ranged from 0.019 to 0.047. A highly significant average FST of 0.031 was estimated, showing a low level of breed subdivision. Most of the variation is accounted for by differences among individuals. Cluster analysis based on Reynolds’ genetic distance between breeds and PCA were performed. An individual UPGMA tree based on Jaccard’s similarity index showed clusters with individuals from all goat breeds. Analysis of population structure points to a high level of admixture among breeds.

RFLP analysis of rice semi-dwarf mutation induced by high energy argon ion radiation

International Nuclear Information System (INIS)

Zhuang Chuxiong; Hu Weimin; Mei Mantong

1997-01-01

Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi-dwarf mutants induced by high energy argon ion radiation, Ar-10, and Xiang-Ar-1, were examined with restriction fragment length polymorphism (RFLP) analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes. Among the markers screened, 9 detected polymorphism were between Bianpizhen and Ar-10, and 11 detected polymorphism were between Xiangzhan and Xiang-Ar-1. Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5.15% and 6.39% for Ar-10 and Xiang-Ar-1 respectively. These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation. Genetic analysis and gene tagging of semi-dwarf mutation in one of the mutant line, Ar-10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4

RFLP Analysis of rice semi dwarf mutation induced by high energy argon ion radiation

International Nuclear Information System (INIS)

Zhuang Chuxiong; Hu Weimin; Mei Mantong

1997-01-01

Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi dwarf mutants induced by high energy argon ion radiation, Ar 10, and Xiang Ar 1, were examined with restriction fragment length polymorphism(RFLP)analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes.Among the markers screened, 9 detected polymorphism were between Bianpizhan and Ar 10, and 11 detected polymorphism were between Xiangzhan and Xiang Ar 1.Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5 15% and 6 39% for Ar 10 and Xiang Ar 1 respectively.These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation.Genetic analysis and gene tagging of semi dwarf mutation in one of the mutant line, Ar 10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4. (author)

Application of RFLP-PCR-Based Identification for Sand Fly Surveillance in an Area Endemic for Kala-Azar in Mymensingh, Bangladesh

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Mohammad Shafiul Alam

2012-01-01

Full Text Available Mymensingh is the most endemic district for kala-azar in Bangladesh. Phlebotomus argentipes remains the only known vector although a number of sand fly species are prevalent in this area. Genotyping of sand flies distributed in a VL endemic area was developed by a PCR and restriction-fragment-length polymorphism (RFLP of 18S rRNA gene of sand fly species. Using the RFLP-PCR analysis with AfaI and HinfI restriction enzymes, P. argentipes, P. papatasi, and Sergentomyia species could be identified. Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found. Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection. The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh.

Primer reporte de empleo de marcadores AFLP en Asteraceae en Cuba

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Grecia Montalvo Fernández

2012-07-01

Full Text Available Título en ingles: First report of the employment of AFLP markers in Asteraceae in Cuba. Resumen: Rhodogeron coronopifolius Griseb., es una especie vegetal de la familia Asteraceae, que se encuentra en peligro crítico de extinción. Es endémico de la provincia Villa Clara en la región central de Cuba. Habita en el matorral xeromorfo sub espinoso sobre serpentina. Existen solo cinco poblaciones naturales dentro de un área protegida, la principal causa de amenaza es la fragmentación de su hábitat por acciones antrópicas. Debido a su situación de conservación, se hace necesario realizar estudios de la diversidad genética de las poblaciones naturales para así generar información básica y diseñar una estrategia de conservación. El objetivo de este trabajo fue analizar de manera preliminar la diversidad genética de cuatro poblaciones de esta especie utilizando marcadores AFLP (Polimorfismo de Longitud de Fragmentos Amplificados. Se emplearon dos combinaciones de iniciadores y se evaluó el porcentaje de polimorfismo así como la similitud entre los individuos.  Se obtuvieron 165 loci de los cuales el 78,7 % fueron polimórficos. La población de mayor polimorfismo fue Corojito con 85,2%, de manera general el polimorfismo fue alto con valores entre 75 y  87%. La similitud entre los individuos también fue alta con un promedio de 0,74. El agrupamiento genético fue independiente a la población de procedencia, lo que sugiere que existe intercambio genético entre las poblaciones y que estas comparten más del 80 % de los alelos que fueron analizados. Los resultados obtenidos son importantes para el mantenimiento in situ de la especie y para tomar decisiones en aras de su conservación. Palabras clave: Rhodogeron coronopifolius, peligro de extinción, polimorfismo, conservación. Abstract: Rhodogeron coronopifolius Griseb., is a specie of Asteraceae family, in critical danger of extinction. It is an endemic of Villa Clara city in the

Glacial survival east and west of the 'Mekong-Salween Divide' in the Himalaya-Hengduan Mountains region as revealed by AFLPs and cpDNA sequence variation in Sinopodophyllum hexandrum (Berberidaceae).

Science.gov (United States)

Li, Yong; Zhai, Sheng-Nan; Qiu, Ying-Xiong; Guo, Yan-Ping; Ge, Xue-Jun; Comes, Hans Peter

2011-05-01

Molecular phylogeographic studies have recently begun to elucidate how plant species from the Qinghai-Tibetan Plateau (QTP) and adjacent regions responded to the Quaternary climatic oscillations. In this regard, however, far less attention has been paid to the southern and south-eastern declivities of the QTP, i.e. the Himalaya-Hengduan Mountains (HHM) region. Here, we report a survey of amplified fragment length polymorphisms (AFLPs) and chloroplast DNA (cpDNA) sequence variation in the HHM endemic Sinopodophyllum hexandrum, a highly selfing alpine perennial herb with mainly gravity-dispersed berries (105 individuals, 19 localities). We specifically aimed to test a vicariant evolutionary hypothesis across the 'Mekong-Salween Divide', a known biogeographic and phytogeographic boundary of north-to-south trending river valleys separating the East Himalayas and Hengduan Mts. Both cpDNA and AFLPs identified two divergent phylogroups largely congruent with these mountain ranges. There was no genetic depauperation in the more strongly glaciated East Himalayas (AFLPs: H(E)=0.031; cpDNA: h(S)=0.133) compared to the mainly ice-free Hengduan Mts. (AFLPs: H(E)=0.037; cpDNA: h(S)=0.082), while population differentiation was consistently higher in the former region (AFLPs: Φ(ST)=0.522 vs. 0.312; cpDNA: Φ(ST)=0.785 vs. 0.417). Our results suggest that East Himalayan and Hengduan populations of S. hexandrum were once fragmented, persisted in situ during glacials in both areas, and have not merged again, except for a major instance of inter-lineage chloroplast capture identified at the MSD boundary. Our coalescent time estimate for all cpDNA haplotypes (c. 0.37-0.48 mya), together with paleogeological evidence, strongly rejects paleo-drainage formation as a mechanism underlying allopatric fragmentation, whereas mountain glaciers following the ridges of the MSD during glacials (and possible interglacials) could have been responsible. This study thus indicates an important role

Highly sensitive detection of the PIK3CAH1047R mutation in colorectal cancer using a novel PCR-RFLP method

International Nuclear Information System (INIS)

Li, Wan-Ming; Hu, Ting-Ting; Zhou, Lin-Lin; Feng, Yi-Ming; Wang, Yun-Yi; Fang, Jin

2016-01-01

The PIK3CA H1047R mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CA H1047R mutation in high effectiveness. A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CA H1047R was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method. The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA H1047R mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CA H1047R mutation and the patients’ age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods. We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CA H1047R mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy

Use of AFLP marker system on sugarcane somaclones to study their resistance to rust

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María Ileana Oloriz

2002-10-01

Full Text Available AFLPs (amplified fragment length polymorphism was carried out from genomic DNA of five rust resistant sugar cane somaclons and their susceptible donor, Saccharum officinarum var B 4362, using three combinations of primers (EcoRI/aca: MseI/acc; EcoRI/aca: MseI/atg and EcoRI/aca: MseI/agg. Six polymorphic bands were obtained, two of these only appeared in the resistant genotypes, which are probably DNA sequences, related to rust resistance locus. These fragments have been cloned to study their nucleotide sequence and to investigate their roll in resistance mechanism develop by these mutants during Puccinia melanocephala infection. Key words: molecular markers, Puccinia. melanocephala, Saccharum

Development of simple and rapid PCR-fingerprinting methods for Vibrio cholerae on the basis of genetic diversity of the superintegron.

Science.gov (United States)

Chowdhury, N; Asakura, M; Neogi, S B; Hinenoya, A; Haldar, S; Ramamurthy, T; Sarkar, B L; Faruque, S M; Yamasaki, S

2010-07-01

To develop simple and rapid PCR-fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains. PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed targeting region between integrase gene in the SI and its nearby ORF, followed by BglI digestion. Besides, a V. cholerae repeat-amplified fragment length polymorphism (VCR-AFLP) assay was also developed. In the PCR-RFLP, 94 El Tor, 29 classical and 54 O139 strains produced nine, three and six different DNA fingerprints, respectively. On the other hand, VCR-AFLP distinguished these El Tor, classical and O139 strains into five, nine and two DNA fingerprints, respectively. Combining both assays the El Tor, classical and O139 strains could be differentiated into 11, 10 and seven different types, respectively. In a comparative study, pulsed-field gel electrophoresis (PFGE) showed similar differentiation for El Tor (11 types), but lower discrimination for O139 (two types) and classical strains (five types). The PCR assays based on SI diversity can be used as a useful typing tool for epidemiological studies of V. cholerae. This newly developed method is more discriminatory, simple, rapid and cost-effective in comparison with PFGE, and thus can be widely applicable. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

RFLP for Duchenne muscular dystrophy cDNA clone 44-1

Energy Technology Data Exchange (ETDEWEB)

Laing, N G; Siddique, T; Bartlett, R J; Yamaoka, L H; Chen, J C; Walker, A P; Hung, W Y; Roses, A D [Duke Univ. Medical Center, Durham, NC (USA)

1988-07-25

Clone 44-1 is one of six cDNA clones which comprise the cDNA for the Duchenne muscular dystrophy gene. It is a 0.9kb fragment in the EcoR1 site of Bluescript. Taq1 (TlCGA) identifies two alleles with bands at 6.8 and 5.7kb, as well as four constant bands at 4.8, 3.9, 3.5 and 2.5kb. Its frequency was studied in 62 unrelated individuals. Mendelian inheritance was demonstrated in one three generation and three two generation informative families, 26 individuals. There were no problems on RFLP analysis under normal stringency conditions.

RFLP for TaqI at the human tyrosinase locus

Energy Technology Data Exchange (ETDEWEB)

Spritz, R; Strunk, K; Oetting, W; King, R

1988-10-25

A 1.4-kb EcoRI-PstI fragment from the mouse tyrosinase cDNA plasmid pTyrs-33 containing virtually the complete coding sequences. TaqI identifies a two-allele polymorphism with fragments of either 2.8 kb or 2.4 kb that contain most of the tyrosinase coding region. Three weak (1.4 kb, 0.9 kb, and 0.6 kb) and two very weak (5.0 and 3.2 kb) constant bands are also seen. The human tyrosinase gene has been regionally mapped to 11q14->21, and a wealy cross-hybridizing tyrosinase-related sequence mapped to 11p11.2->cen. Co-dominant segregation has been shown in two families. The RFLP was observed under normal hybridization and wash conditions.

Molecular characterization of papaya genotypes using AFLP markers

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Eder Jorge de Oliveira

2011-09-01

Full Text Available Due to the low genetic variability reported in the commercial plantations of papaya (Carica papaya L., the objective of this study was analyze the genetic diversity of 32 genotypes including cultivars, landraces, inbred lines, and improved germplasm using the AFLP technique (Amplified Fragment Length Polymorphism. The genetic distance matrix was obtained using the Nei and Li genetic distance and clustering was performed using the unweighted pair-method with arithmetic mean (UPGMA. Using 11 combinations of EcoRI/MseI primers, 383 polymorphic bands were obtained. On average, 34.8 polymorphic bands were obtained per primer combination. Five clusters were formed. The traditional cultivar 'Sunrise' and the inbred line CMF-L30-08 were the closest genotypes, and the improved germplasm (CMF041 and landrace (CMF233 the most distant. The main papaya cultivars commercially grown in Brazil, as well as four inbred lines and three improved germplasm, were clustered together, however, were not grouped in the same branch. The genetic distance between the Sunrise and Golden cultivars was 0.329, and even arising from mutation and selection within the Sunrise variety, the Golden stores considerable genetic variability. Additional variability was observed in the inbred lines derived from papaya breeding program at Embrapa Cassava and Fruits.

Una risa en el desierto: el arte de la resurrección y sermones y prédicas del Cristo de Elqui

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Jorge Ladino Gaitán Bayona

2015-01-01

Full Text Available En este artículo de reflexión se analiza la propuesta carnavalesca de la novela El arte de la resurrección (2010, de Hernán Rivera Letelier, y los poemarios Sermones y prédicas del Cristo de Elqui (1977 y Nuevos sermones y prédicas del Cristo de Elqui (1979, de Nicanor Parra. En estas obras interviene como protagonista un excéntrico predicador de existencia real en la historia de Chile: Domingo Zárate Vega, mejor conocido como el Cristo de Elqui. Se retoman planteamientos teóricos de Mijail Bajtín para explorar la risa y la locura como recursos estéticos que generan una visión crítica sobre la miseria de los mineros en las salitreras del desierto chileno, la dictadura militar de Augusto Pinochet y La matanza de la Escuela Santa María de Iquique (21 de diciembre de 1907.

Genetic relationship and diversity in a sesame (Sesamum indicum L. germplasm collection using amplified fragment length polymorphism (AFLP

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Karlovsky Petr

2006-02-01

Full Text Available Abstract Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia, and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP. Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA. This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

Science.gov (United States)

Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

2017-08-01

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Detection of the mosquitocidal toxin genes encoding Cry11 proteins from Bacillus thuringiensis using a novel PCR-RFLP method Detección de genes que codifican proteínas mosquitocidas Cry11 de Bacillus thuringiensis mediante un método de PCR-RFLP novedoso

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D. H. Sauka

2010-02-01

Full Text Available A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method for detection of cry11 genes from Bacillus thuringiensis was established. Based on the analysis of conserved regions of the cry11 genes, 2 oligonucleotide primers were designed to amplify a 1459-bp fragment of the cry11Aa gene, and a 1471-bp of the cry11Ba and cry11Bb genes. The amplification products were digested with restriction endonuclease HinfI. Exotic B. thuringiensis strains and native isolates collected from soils, leaves and stored product dust of Argentina were analyzed to study the distribution of cry11 genes. The PCR-RFLP patterns revealed the detection of cry11 genes in 3 of 64 exotic strains and in 10 of 107 native B. thuringiensis isolates tested. Just the cry11Aa gene subclass was detected among these bacteria. Since the methodology was also developed to detect cry11Ba and cry11Bb genes, an experimental future confirmation will be required. Based on the results obtained, the PCR-RFLP method presented may be a valuable tool for specific detection of the mosquitocidal toxin genes encoding Cry11 proteins from B. thuringiensis.En el presente estudio se estableció una estrategia basada en la amplificación génica (PCR y el posterior análisis de restricción (RFLP para detectar todos los genes cry11 de Bacillus thuringiensis informados hasta ahora. De acuerdo con el análisis de las regiones conservadas en los genes cry11, se diseñaron dos cebadores para amplificar un fragmento de 1459 pb de los genes cry11Aa y un fragmento de 1471 pb de los genes cry11Ba y cry11Bb. Los productos de la amplificación fueron digeridos con la enzima de restricción HinfI. Se analizaron cepas exóticas de B. thuringiensis y aislamientos nativos de Argentina obtenidos a partir de muestras de suelos, hojas y polvillo de silos, para estudiar la distribución de los genes cry11. Los patrones de PCR-RFLP revelaron la presencia de genes cry11 en 3 de las 64 cepas ex

Comparative Study of Campylobacter spp. Isolated from Children With Gastroenteritis in Bahonar Hospital, Karaj, Using PCR and RFLP

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Arefeh Abdi

2016-05-01

Full Text Available Background: Campylobacter species are responsible for the majority of cases of food-borne gastroenteritis. The sources of the disease outbreaks are often contaminated water or milk, and consumption of undercooked poultry product is the main cause of sporadic campylobacteriosis cases. Objectives: The aims of this study were to determine the prevalence of Campylobacter gastroenteritis in children and to differentiate the interfering species using polymerase chain reaction (PCR and restriction fragment length polymorphism (RFLP methods at the Bahonar hospital in Karaj, Iran. Patients and Methods: A total of 150 stool samples were collected from children under 10 years old during the summer of 2014. PCR was performed using genus- and species-specific primers and RFLP was done using AluI and TasI enzymes. Results: The results showed the amplification of 400 and 491 bp segments and Campylobacter contamination in 30 (20% samples; 5 out of 30 Campylobacter positive samples (16.66% were identified as C. jejuni, 20 (66.66% as C. coli, 3 (10% as C. jejuni and C. coli (mixed infection, and 2 (6.66% were identified as non-jejuni, non-coli Campylobacter using the PCR method. Following the evaluation of RFLP results, 7 positive samples (23.33% showed the electrophoretic pattern of C. jejuni, 21 (70% showed the electrophoretic pattern of C. coli, and 2 (6.6% showed both of the patterns and mixed contamination with jejuni and coli species. The results of digestion with TasI did not show any C. lari or C. upsaliensis patterns. Conclusions: The results of this study showed high percentage of Campylobacter contamination in the tested stool samples. The other surprising finding was the high rate of Campylobacter coli positive samples; the difference between the results of PCR using species-specific primers (hipo and asp and the RFLP method (electrophoretic patterns in some of the positive samples confirms the hypothesis of variations in nucleotide sequences of the

Molecular mapping of powdery mildew resistance gene Eg-3 in cultivated oat (Avena sativa L. cv. Rollo).

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Mohler, Volker; Zeller, Friedrich J; Hsam, Sai L K

2012-05-01

Powdery mildew is a prevalent fungal disease affecting oat (Avena sativa L.) production in Europe. Common oat cultivar Rollo was previously shown to carry the powdery mildew resistance gene Eg-3 in common with cultivar Mostyn. The resistance gene was mapped with restriction fragment length polymorphism (RFLP) markers from Triticeae group-1 chromosomes using a population of F(3) lines from a cross between A. byzantina cv. Kanota and A. sativa cv. Rollo. This comparative mapping approach positioned Eg-3 between cDNA-RFLP marker loci cmwg706 and cmwg733. Since both marker loci were derived from the long arm of barley chromosome 1H, the subchromosomal location of Eg-3 was assumed to be on the long arm of oat chromosome 17. Amplified fragment length polymorphism (AFLP) marker technology featured as an efficient means for obtaining markers closely linked to Eg-3.

GISH and AFLP analyses of novel Brassica napus lines derived from one hybrid between B. napus and Orychophragmus violaceus.

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Ma, Ni; Li, Zai-Yun; Cartagena, J A; Fukui, K

2006-10-01

New Brassica napus inbred lines with different petal colors and with canola quality and increased levels of oleic (approximately 70%, 10% higher than that of B. napus parent) and linoleic (28%) acids have been developed in the progenies of one B. napus cv. Oro x Orychophragmus violaceus F5 hybrid plant (2n = 31). Their genetic constituents were analyzed by using the methods of genomic in situ hybridization (GISH) and amplified fragments length polymorphism (AFLP). No intact chromosomes of O. violaceus origin were detected by GISH in their somatic cells of ovaries and root tips (2n = 38) and pollen mother cells (PMCs) with normal chromosome pairing (19 bivalents) and segregation (19:19), though signals of variable sizes and intensities were located mainly at terminal and centromeric parts of some mitotic chromosomes and meiotic bivalents at diakinesis or chromosomes in anaphase I groups and one large patch of chromatin was intensively labeled and separated spatially in some telophase I nuclei and metaphase II PMCs. AFLP analysis revealed that substantial genomic changes have occurred in these lines and O. violaceus-specific bands, deleted bands in 'Oro' and novel bands for two parents were detected. The possible mechanisms for these results were discussed.

Experimental and clinical studies on simultaneous fat and protein tolerance digestion-absorption test using 131I-triolein and 125I-RISA

International Nuclear Information System (INIS)

Tokura, Yasunobu

1979-01-01

Simultaneous fat and protein tolerance digestion-absorption test using 131 I-triolein and 125 I-RISA was investigated experimentally in rats and clinically in 148 inpatients with various diseases. The results were as follows: Animal experiments. Fat absorption was markedly impaired in the exclusion of bile from the intestine. Each function of fat and protein absorption was independently detected. These animal experiments gave the clinical evaluation of this test for fat and protein digestion-absorption function. Clinical investigations. The test meal was prepared without using commercial Tween 80 as an emulgent. Either fat or protein absorption was markedly impaired in some patients with various diseases. Inpatients with various diseases investigated were divided into 3 groups by correlation coefficient between fecal excretion ratios of fat and protein. Each function of fat and protein digestion-absorption was able to be detected independently by this test simultaneously. This test can further be used as a labolatory aid in evaluating fat and protein digestion-absorption function in patients with malabsorption syndrome. (author)

Benzimidazole -Resistance in Haemonchus Contortus: New PCR-RFLP Method for the Detection of Point Mutation at Codon 167 of Isotype 1 Β-Tubulin Gene

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A Eslami

2012-12-01

Full Text Available Background: Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT-HC167 MF-primer, to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubu­lin gene of Haemonchus contortus.Methods: There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleo­tide polymorphism (SNP at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer, in which the nucleotide T at the posi­tion 443 was substituted through a nucleotide A creating a restriction site for restriction endonuc­lease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achiev­ing a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, respec­tively.Results: All worms had two alleles encoding for phenylalanine (BZss homozygote for both codons.Conclusion: Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP.

Dense genetic linkage maps of three Populus species (Populus deltoides, P. nigra and P. trichocarpa) based on AFLP and microsatellite markers.

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Cervera, M T; Storme, V; Ivens, B; Gusmão, J; Liu, B H; Hostyn, V; Van Slycken, J; Van Montagu, M; Boerjan, W

2001-06-01

Populus deltoides, P. nigra, and P. trichocarpa are the most important species for poplar breeding programs worldwide. In addition, Populus has become a model for fundamental research on trees. Linkage maps were constructed for these three species by analyzing progeny of two controlled crosses sharing the same female parent, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24. The two-way pseudotestcross mapping strategy was used to construct the maps. Amplified fragment length polymorphism (AFLP) markers that segregated 1:1 were used to form the four parental maps. Microsatellites and sequence-tagged sites were used to align homoeologous groups between the maps and to merge linkage groups within the individual maps. Linkage analysis and alignment of the homoeologous groups resulted in 566 markers distributed over 19 groups for P. deltoides covering 86% of the genome, 339 markers distributed over 19 groups for P. trichocarpa covering 73%, and 369 markers distributed over 28 groups for P. nigra covering 61%. Several tests for randomness showed that the AFLP markers were randomly distributed over the genome.

Characterization of South American Snails of the Genus Biomphalaria (Basommatophora: Planorbidae and Schistosoma mansoni (Platyhelminthes: Trematoda in Molluscs by PCR-RFLP

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Roberta Lima Caldeira

2016-01-01

Full Text Available The identification of snails of the genus Biomphalaria can be done using morphological characteristics which depends on the size of the snails and skill and knowledge of researcher. These methods sometimes are not adequate for identification of species. The PCR-RFLP, using the ITS region of the rDNA, has been used to identify Brazilian species of the genus Biomphalaria. Nevertheless, there is a lack of information about snails from other Latin American countries. In addition, some snails may be infected by Schistosoma mansoni and when submitted to PCR-RFLP they show molecular profiles different from those previously standardized for the other mollusc species. In this work the molecular profiles of 15 species and the subspecies were established by PCR-RFLP of ITS-rDNA with the enzyme DdeI. Moreover, the molecular profiles of host species, B. glabrata, B. straminea, B. tenagophila, and B. prona, infected by S. mansoni were also established. The molluscs were dissected to permit morphological identification. These results contribute to a correct identification of snails of the genus Biomphalaria and detection of these snails infected by S. mansoni.

Incidence of Giardia lamblia Subspecies by PCR-RFLP in Stool Specimens of Hospitalized Children at Urmia Mutahhari Hospital, West Azerbaijan Province, Iran.

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Khosro Hazrati Tappeh

2014-12-01

Full Text Available Giardia lamblia is one of the most prevalent intestinal flagellate protozoa that infects a wide range of vertebrate hosts causing severe intestinal disorder in children.This study was performed to determine subspecies of G.lamblia by the PCR-RFLP method, targeting the glutamate dehydrogenase(gdhlocus, in hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province,Iran and determining the infection transformational storages in this area.Overall, 720 stool specimens were collected from the hospitalized children, 34 samples were positive and Giardia cysts were detected under the microscope. Cysts were partially purified by the sucrose density gradient method and then washed with sterile distilled water to remove effectively the PCR inhibitors. Genomic DNA of G. lamblia isolates was extracted by freeze-thaw cycles followed by phenol/ chloroform/isoamyl alcohol method. The single step PCR-RFLP assay was used to differentiate the assemblages between A and B, which were found in humans. In this method, 432 bp expected size was amplified, and then for detection of subspecies, specific restriction RsaI and BspLI enzymes were used.Totally 34 samples were positive in terms of Giardia cyst out of 720 examined samples microscopically, so the parasite spread rate is reported 4.72%. Analysis PCR-RFLP on these samples revealed that 28 samples (93.3% have the genotype BIII and 2 samples (6.7% belong to the subgroup BIV.PCR-RFLP is a proper analytical method for determining the genotype among parasite types, using the glutamate dehydrogenizes zone's genes. Based on the results, an animal origin of infection cycle is suggested.

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene.

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Kumar, Deepak; Singh, S P; Karabasanavar, Nagappa S; Singh, Rashmi; Umapathi, V

2014-11-01

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

BAC-derived markers converted from RFLP linked to Phytophthora capsici resistance in pepper (Capsicum annuum L.).

Science.gov (United States)

Kim, Hyoun-Joung; Nahm, Seok-Hyeon; Lee, Heung-Ryul; Yoon, Gi-Bo; Kim, Ki-Taek; Kang, Byoung-Cheorl; Choi, Doil; Kweon, Oh Yeol; Cho, Myeong-Cheoul; Kwon, Jin-Kyung; Han, Jung-Heon; Kim, Jeong-Ho; Park, Minkyu; Ahn, Jong Hwa; Choi, Soon Ho; Her, Nam Han; Sung, Joo-Hee; Kim, Byung-Dong

2008-12-01

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.

Overexpression of Aldo-Keto-Reductase in Azole-resistant Clinical Isolates of Candida Glabrata Determined by cDNA-AFLP

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Mansour Heidari

2013-01-01

Full Text Available Background: Candida glabrata causes significant medical problems in immunocompromised patients. Many strains of this yeast are intrinsically resistant to azole antifungal agents, and treatment is problematic, leading to high morbidity and mortality rates in immunosuppressed individuals. The primary goal of this study was to investigate the genes involved in the drug resistance of clinical isolates of C. glabrata.Methods: The clinical isolates of C. glabrata were collected in an epidemiological survey of candidal infection inimmunocompromised patients and consisted of four fluconazole and itraconazole resistant isolates, two fluconazoleand itraconazole sensitive isolates, and C. glabrata CBS 138 as reference strain. Antifungal susceptibility patterns ofthe organisms were determined beforehand by the Clinical and Laboratory Standards Institute (CLSI. The potentialgene(s implicated in antifungal resistance were investigated using complementary DNA- Amplified Fragment Length Polymorphism (cDNA-AFLP. Semi-quantitative RT-PCR was carried out to evaluate the expression of gene(s in resistant isolates as compared to sensitive and reference strains.Results and conclusions: The aldo-keto-reductase superfamily (AKR gene was upregulated in the resistant clinicalisolates as assessed by cDNA-AFLP. Semi-quantitative RT-PCR revealed AKR mRNA expression approximately twice that seen in the sensitive isolates. Overexpression of the AKR gene was associated with increased fluconazole and itraconazole resistance in C. glabrata. The data suggest that upregulation of the AKR gene might give a new insight into the mechanism of azole resistance.

La risa natal. Fundamentos antropológicos y religiosos de la comicidad clerical en Navidad

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del Campo Tejedor, Alberto

2014-12-01

Full Text Available The comicalness of many of the popular and clerical festivals in the winter cycle has been mostly interpreted as a pagan and profane survival, objected to Christian faith. This paper discusses this simplistic thesis and adduces evidences that allows to understand the struggles between detractors and sympathizers of the polemic burlesque festivals on Christmas. We study the type of paschal joy that certain theology has justified in consideration of reasons such as the eutrapelia or the exemplary character of the humiliation and the humility in rituals based on investment of roles. Christmas laugh appears then as a fundamental ingredient of a mytho-logic in which turbulence, joviality, even obscenity, constitute liminal forms in collaborating with the eternal cycle of birth, death and resurrection.La comicidad de muchas de las fiestas populares y clericales del ciclo invernal ha sido interpretada mayoritariamente como supervivencia pagana y profana, contraria a la fe cristiana. Este artículo discute esta tesis reduccionista y aporta evidencias que permiten comprender las pugnas entre detractores y simpatizantes de las polémicas fiestas burlescas en torno a la Navidad. Para ello se indaga el tipo de alegría pascual que cierta teología ha justificado en atención a razones como la eutrapelia o el carácter ejemplar de la humillación y la humildad en los rituales de inversión de roles. La risa navideña se muestra como un ingrediente fundamental de una mito-lógica en la que la turbulencia, la jovialidad, incluso la obscenidad, constituyen formas liminales de colaborar en el eterno ciclo de nacimiento, muerte y resurrección.

AFLP genome scans suggest divergent selection on colour patterning in allopatric colour morphs of a cichlid fish.

Science.gov (United States)

Mattersdorfer, Karin; Koblmüller, Stephan; Sefc, Kristina M

2012-07-01

Genome scan-based tests for selection are directly applicable to natural populations to study the genetic and evolutionary mechanisms behind phenotypic differentiation. We conducted AFLP genome scans in three distinct geographic colour morphs of the cichlid fish Tropheus moorii to assess whether the extant, allopatric colour pattern differentiation can be explained by drift and to identify markers mapping to genomic regions possibly involved in colour patterning. The tested morphs occupy adjacent shore sections in southern Lake Tanganyika and are separated from each other by major habitat barriers. The genome scans revealed significant genetic structure between morphs, but a very low proportion of loci fixed for alternative AFLP alleles in different morphs. This high level of polymorphism within morphs suggested that colour pattern differentiation did not result exclusively from neutral processes. Outlier detection methods identified six loci with excess differentiation in the comparison between a bluish and a yellow-blotch morph and five different outlier loci in comparisons of each of these morphs with a red morph. As population expansions and the genetic structure of Tropheus make the outlier approach prone to false-positive signals of selection, we examined the correlation between outlier locus alleles and colour phenotypes in a genetic and phenotypic cline between two morphs. Distributions of allele frequencies at one outlier locus were indeed consistent with linkage to a colour locus. Despite the challenges posed by population structure and demography, our results encourage the cautious application of genome scans to studies of divergent selection in subdivided and recently expanded populations. © 2012 Blackwell Publishing Ltd.

Rice genome mapping and its application in rice genetics and breeding

International Nuclear Information System (INIS)

Eun, M.Y.; Cho, Y.G.; Hahn, J.H.; Yoon, U.H.; Yi, B.Y.; Chung, T.Y.

1998-01-01

An 'MG' recombinant inbred population which consists of 164 F 13 lines has been developed from a cross between a Tongil type variety Milyang 23 and a Japonica type Gihobyeo by single seed descent. A Restriction Fragment Length Polymorphism (RFLP) framework map using this population has been constructed. Morphological markers, isozyme loci, microsatellites, Amplified Fragment Length Polymorphisms (AFLP), and new complementary DNA (cDNA) markers are being integrated in the framework map for a highly saturated comprehensive map. So far, 207 RFLPs, 89 microsatellites, 5 isozymes, 232 AFLPs, and 2 morphological markers have been mapped through international collaboration. The map contains 1,826 cM with an average interval size of 4.5 cM on the framework map and 3.4 cM overall (as of 29 October 1996). The framework map is being used for analyzing, quantitative trait loci (QTL) of agronomic characters and some physico-chemical properties relating to rice quality. The number of significant QTLs affecting each trait ranged from one to five, and 38 QTLs were detected for 17 traits. The percentage of variance explained by each QTL ranged from 5.6 to 66.9%. The isozyme marker, EstI-2, and two RFLP markers, RG109 and RG220, were linked most tightly at a distance less than 1 cM with the semidwarf (sd-1) gene on chromosome 1. These markers could be used for precise in vitro selection of individuals carrying the semidwarf gene using single seeds or very young leaf tissue, before this character is fully expressed. Appropriate application of marker-assisted selection, using EstI-2 and RFLP markers for the semidwarf character, in combination with other markers linked to genes of agronomic importance in rice, holds promise for improving, the efficiency of breeding, and the high-resolution genetic and physical mapping near sd-1, aimed at ultimately cloning this valuable gene

Mapping quantitative trait loci (QTLs for fatty acid composition in an interspecific cross of oil palm

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Sharma Mukesh

2009-08-01

Full Text Available Abstract Background Marker Assisted Selection (MAS is well suited to a perennial crop like oil palm, in which the economic products are not produced until several years after planting. The use of DNA markers for selection in such crops can greatly reduce the number of breeding cycles needed. With the use of DNA markers, informed decisions can be made at the nursery stage, regarding which individuals should be retained as breeding stock, which are satisfactory for agricultural production, and which should be culled. The trait associated with oil quality, measured in terms of its fatty acid composition, is an important agronomic trait that can eventually be tracked using molecular markers. This will speed up the production of new and improved oil palm planting materials. Results A map was constructed using AFLP, RFLP and SSR markers for an interspecific cross involving a Colombian Elaeis oleifera (UP1026 and a Nigerian E. guinneensis (T128. A framework map was generated for the male parent, T128, using Joinmap ver. 4.0. In the paternal (E. guineensis map, 252 markers (199 AFLP, 38 RFLP and 15 SSR could be ordered in 21 linkage groups (1815 cM. Interval mapping and multiple-QTL model (MQM mapping (also known as composite interval mapping, CIM were used to detect quantitative trait loci (QTLs controlling oil quality (measured in terms of iodine value and fatty acid composition. At a 5% genome-wide significance threshold level, QTLs associated with iodine value (IV, myristic acid (C14:0, palmitic acid (C16:0, palmitoleic acid (C16:1, stearic acid (C18:0, oleic acid (C18:1 and linoleic acid (C18:2 content were detected. One genomic region on Group 1 appears to be influencing IV, C14:0, C16:0, C18:0 and C18:1 content. Significant QTL for C14:0, C16:1, C18:0 and C18:1 content was detected around the same locus on Group 15, thus revealing another major locus influencing fatty acid composition in oil palm. Additional QTL for C18:0 was detected on Group 3

MORPHOLOGY AND GENETIC DIVERSITY OF MITOCHONDRIAL DNA D-LOOP REGION USING PCR-RFLP ANALYSIS IN MAGELANG DUCK AND OTHER NATIVE DUCK

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D. Purwantini

2014-10-01

Full Text Available The aim of this study was to investigate the different of plumage colors on morphological diversityof Magelang duck and genetic diversity using PCR-RFLP mtDNA D-loop region analysis of Magelangduck and four others native duck population (Tegal, Mojosari, Bali and Alabio duck in Indonesia. Bloodsample was taken from 50 Magelang ducks and 20 of each native ducks. Morphological characteristicsof body measurement, production ability and egg quality of Magelang duck were analyzed usingCompletely Randomized Design with 11 plumage colors as treatments. PCR technique was administeredto amplify fragments in mtDNA D-loop region and PCR products were digested with endonucleaserestriction enzyme AluI and HaeIII. The result showed that morphology diversity of Magelang duck wasstatistically affected by different plumage colors. PCR-RFLP analysis using AluI and HaeIII restrictionenzyme resulted in six combinations of restriction fragment pattern shown in six haplotypes (A, B, C, D,E and F. Haplotype difference showed genetic diversity in the population of Magelang duck and theother native ducks. In conclusion, the different plumage colors affected morphology diversity ofMagelang duck. Genetic diversity of Indonesian native duck population could be identified by usingPCR-RFLP analysis on mtDNA D-loop region.

Risa y salud: abordajes terapéuticos. ; Laughter and health: therapeutic approaches

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Eder Ariel Lancheros García

2011-01-01

Full Text Available La terapia de la risa hoy en día es utilizada en diversas clínicas y hospitales del mundo como medida coadyuvante en pro del bienestar físico, psicológico, social y emocional de los pacientes. En nuestro país es un tema que poco se conoce y por lo cual es de suma importancia dar a conocer en el ámbito nacional los múltiples beneficios que genera en la salud de las personas. El payaso hospitalario, como protagonista principal, ejerce una importante labor y a través de él se busca lograr una relación armoniosa con el paciente, con el objetivo de mejorar su percepción de la enfermedad, lograr una adecuada adaptación hospitalaria, disminuir los niveles de ansiedad y generar un bienestar global. En Bucaramanga existe la Fundación Corazón en Parches, que se ha encargado de llevar un mensaje de amor y compasión a los pacientes hospitalizados, en especial en edad pediátrica, aplicando la risoterapia a cabalidad, recordando que lo más importante no es curar una enfermedad sino tratar a un paciente que tiene una enfermedad, implementando una visión holística.______________________________________________________________________Laughter therapy is now used in many hospitals and clinics worldwide as adjunctive measure in favor of physical, psychological, social and emotional development of patients. Our country is a topic that little is known and therefore it is of utmost importance to share nationalgenerates multiple benefits in human health. The hospital clown as the main character has an important work and through it seeks to achieve a harmonious relationship with the patient, with the aim of improving their perception of the disease, hospital achieve adequate adaptation, lower levels of anxiety and generate as such a global welfare. InBucaramanga exists Fundación Corazón en Parches, who have managed to bring a message of love and compassion to hospitalized patients, especially pediatric patients, using the fully laughter therapy

A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries.

Science.gov (United States)

Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Amani, Hizem; Mezhoud, Habib; Babba, Hamouda

2016-10-01

Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1-G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6-G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.

Genetic recombination within the human T-cell receptor α-chain gene complex

International Nuclear Information System (INIS)

Robinson, M.A.; Kindt, T.J.

1987-01-01

Genetic analyses of the human T-cell receptor (TCR) α-chain genes indicate that recombination events may occur frequently within this gene complex. Examination of the inheritance of restriction fragment length polymorphisms (RFLP) detected by using probes for constant or variable region gene segments made it possible to assign TCRα haplotypes to the 16 parents and 43 offspring of eight families studied. A total of six RFLP, three for the constant region and three for variable region segments, were examined in the present studies. Most enzyme and probe combinations tested revealed no polymorphism and those finally selected for the study showed limited polymorphism in that only two or, in one case, three allelic forms of the gene were seen. In spite of limited variability at this level, extensive heterogeneity was observed for the combinations of markers present in haplotypes, suggesting that frequent recombination events have occurred. Most strikingly, multiple combinations of RFLP occurring in close proximity of the TCRα constant region gene were observed in this study. A high recombination frequency for the TCRα gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCRα haplotypes

Origins of domestication and polyploidy in oca (Oxalis tuberosa; Oxalidaceae). 3. AFLP data of oca and four wild, tuber-bearing taxa.

Science.gov (United States)

Emshwiller, Eve; Theim, Terra; Grau, Alfredo; Nina, Victor; Terrazas, Franz

2009-10-01

Many crops are polyploids, and it can be challenging to untangle the often complicated history of their origins of domestication and origins of polyploidy. To complement other studies of the origins of polyploidy of the octoploid tuber crop oca (Oxalis tuberosa) that used DNA sequence data and phylogenetic methods, we here compared AFLP data for oca with four wild, tuber-bearing Oxalis taxa found in different regions of the central Andes. Results confirmed the divergence of two use-categories of cultivated oca that indigenous farmers use for different purposes, suggesting the possibility that they might have had separate origins of domestication. Despite previous results with nuclear-encoded, chloroplast-expressed glutamine synthetase suggesting that O. picchensis might be a progenitor of oca, AFLP data of this species, as well as different populations of wild, tuber-bearing Oxalis found in Lima Department, Peru, were relatively divergent from O. tuberosa. Results from all analytical methods suggested that the unnamed wild, tuber-bearing Oxalis found in Bolivia and O. chicligastensis in NW Argentina are the best candidates as the genome donors for polyploid O. tuberosa, but the results were somewhat equivocal about which of these two taxa is the more strongly supported as oca's progenitor.

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Directory of Open Access Journals (Sweden)

Diego Rayan Teixeira de Sousa

2015-05-01

Conclusion: The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

c-Ha-ras BamHI RFLP in human urothelial tumors and point mutations in hot codons

International Nuclear Information System (INIS)

Weismanova, E; Skovraga, M.; Kaluz, S.

1993-01-01

High-molecular weights DNAs from 30 bladder and renal cell carcinomas (RCC) were isolated and the c-Ha-ras the c-Ha-ras gene BamHI RFLP was examined. Amplification of c-Ha-ras with normal localization with regard to the size of alleles was found only in the case. One of the normally localized c-Ha-ras allele termed RCC c-H-ras of a length of about 6.6 kbp was cloned and an oncogene-activating point mutation was identified using two restriction enzymes. After comparison of CfrI and Cfr10I cleavage maps of RCC c-Ha-ras to complete nucleotide sequences of EJ/T24 c-Ha-ras oncogene and its normal counterpart, a point mutation was identified within codon 11 or 12. The use of CfrI and Cfr10I is of value for clinical practice in identification of point mutations in c-Ha-ras PCR product in neoplasia accompanied by somatic mutation of c-Ha-ras. The correlation among c-Ha-ras allele, amplification/loss, presence of point mutation and progression of neoplasia is discussed. (author)

AFLP analysis and improved phytoextraction capacity of transgenic gshI-poplar clones (Populus x canescens L.) for copper in vitro

Energy Technology Data Exchange (ETDEWEB)

Gyulai, G. [St. Stephanus Univ., Dept. of Genetics and PB (Hungary); HAS-SIU Research Group for Molecular Plant Breeding (Hungary); IGER, Plas Gogerddan, Aberystwyth (United Kingdom); Humphreys, M.; Skoet, K.; Skoet, L.; Heywood, S.; Lovatt, A.; Roderick, H.; Abberton, M. [IGER, Plas Gogerddan, Aberystwyth (United Kingdom); Bittsanszky, A.; Kiss, J.; Szabo, Z. [St. Stephanus Univ., Dept. of Genetics and PB (Hungary); Gullner, G.; Koemives, T. [Plant Protection Inst., Hungarian Academy of Sciences, Budapest (Hungary); Radimszky, L. [Soil Science Inst., Hungarian Academy of Sciences, Budapest (Hungary); Rennenberg, H. [Albert-Ludwigs-Univ., Inst. fuer Forstbotanik und Baumphysiologie, Freiburg (Germany); Heszky, L. [St. Stephanus Univ., Dept. of Genetics and PB (Hungary); HAS-SIU Research Group for Molecular Plant Breeding (Hungary)

2005-04-01

Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRI-MseI digestion. Four fragments generated by EcoAGT-MseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. x canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO{sub 4} (10{sup -1} to 10{sup -5} M) incubated for 21 days on aseptic tissue culture media WPM containing 1 {mu}M Cu. ZN{sup 2+} caused phytotoxicity only at high concentrations (10{sup -1} to 10{sup -2} M). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper. (orig.)

Low level of genetic variation within Melica transsilvanica populations from the Kraków-Częstochowa Upland and the Pieniny Mts revealed by AFLPs analysis

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Magdalena Szczepaniak

2011-01-01

Full Text Available Fragmented distribution, the breeding system and effects of genetic drift in small-size populations occurring at edge of the species range play an important role in shaping genetic diversity of such a species. Melica transsilvanica is a plant rare in the flora of Poland, where it reaches the northern limit of its continuous range. Amplified Fragment Length Polymorphism (AFLP DNA profiling method was applied to measure genetic diversity among and within populations of M. transsilvanica. Additionally, genetic relationships between M. transsilvanica and Melica ciliata, two closely related species, were explored. A total of 68 plants from 7 populations of M. transsilvanica and 24 plants from 2 populations of M. ciliata, collected in Poland and outside it, were analyzed. Using 294 AFLP fragments from 3 primer combinations, accessions were grouped into two major clusters associating with M. ciliata and M. transsilvanica, respectively. Further, two subclusters, corresponding to the samples collected from the Pieniny Mts and from the Kraków - Częstochowa Upland were clearly distinguished within the M. transsilvanica group. The hierarchical AMOVA exhibited significant genetic distinction between these geographic regions (60.89%, p < 0.001. The obtained results showed that the most genetic diversity resided between the populations of M. transsilvanica (86.03% while considerably lower genetic variation was found within the populations (13.97%, which is consistent with the results reported for self-plants. The low level of AFLP genetic variation of M. transsilvanica can be caused by the geographic isolation of populations, which preserves the dominant self-mating breeding system of the species. Individual populations of M. transsilvanica are characterized by isolated gene pools differing by a small number of loci.

Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays

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Marize Quinhones Pires

2014-04-01

Full Text Available Introduction During a diagnostic evaluation of canine visceral leishmaniasis (VL, two of seventeen dogs were found to be co-infected by Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi. Methods Specific polymerase chain reaction (PCR and restriction fragment length polymorphism-PCR (RFLP-PCR assays were performed. Results PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi in those fragments. Conclusions This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

Eco RV RFLP at the insulin-like growth factor I (IGF I) locus on chromosome 12

Energy Technology Data Exchange (ETDEWEB)

Schneid, H; Noguiez, P; Girard, F; Binoux, M; Le Bouc, Y [INSERM U 142, Paris (France)

1988-09-26

The human liver IFG I cDNA insert from the {lambda}TG03 (1) was subcloned in PGEM4 (pTG 3906). The 660 bp Eco RI-Bam HI fragment containing the 5{prime} untranslated, the coding and the 3{prime} untranslated region of the IGF IA gene (exons 1, 2, 3 and 5) was used. Eco RV digestion of genomic DNA and hybridization with the IGF I probe reveals invariant fragments of 20; 8, 7 and 5 kb and polymorphic fragments of 13 kb (allele 1) and 11, 5 kb (allele 2). The frequency of alleles was studied in 51 unrelated European Caucasians. Co-dominant segregation was observed in 2 European families (9 individuals). Hind III and Pvu II digestions show previous described RFLP. Allelic frequencies however were: Hind III (5, 2 kb allele = 0, 14 and 4, 9 kb allele = 0, 86) and Pvu II (5 kb allele = 0, 14 and 4, 7 kb allele = 0, 86). Hind III and Pvu II RFLPs are linked. Among the DNA of the 14 individuals, where Eco RV RFLP was found, only one exhibited Hind III and Pvu II RFLPs.

Molecular characterization of cultivated species of the genus Pachyrhizus Rich. ex DC. by AFLP markers

DEFF Research Database (Denmark)

Santayana, Monica; Rossel, Genoveva; Núñez, Jorge

2014-01-01

) molecular markers in order to estimate genetic diversity and interspecific relationships. To complement molecular marker information, individuals from each accession were analyzed in order to confirmploidy levels. Eight AFLP primer combinations detected 136 (68.7 %) polymorphic bands. Shannon’s diversity...... indices (Hs) for each species were 1.04 (P. ahipa), 1.07 (P. tuberosus), and 2.42 (P. erosus), while the total diversity index was 2.45. Phylogenetic analysis, principal coordinate analysis and analysis of molecular variance (FST=0.796) all showed significant species differentiation. All accessions were...... diploid (2n=2x=22), which is characteristic of the tribe Phaseoleae. Finally, a misclassified accession of P. tuberosus was identified. Molecular characterization of accessions is necessary for efficient management of germplasm collections....

A high degree of genetic diversity is revealed in Isatis spp. (dyer's woad) by amplified fragment length polymorphism (AFLP).

Science.gov (United States)

Gilbert (nee Stoker), G.; Garton, S.; Karam, A.; Arnold, M.; Karp, A.; Edwards, J.; Cooke, T.; Barker, A.

2002-05-01

Genetic diversity in 38 genotypes, representing 28 individual genotypes from five landraces of Isatis tinctoria (three German: Tubingen, Potsdam and Erfurt, one Swiss and one English), five genotypes of Isatis indigotica (Chinese woad) and five genotypes of Isatis glauca, were investigated using AFLP analysis. Five primer combinations detected a total of 502 fragments of which 436 (86.9%) were polymorphic. The level of polymorphism recorded within each species was 29.8, 86.9 and 35.8% for I. indigotica, I. tinctoria and I. glauca, respectively. Clearly, genetic diversity within I. tinctoria was greater than that observed in I. indigotica or I. glauca. Cluster analyses of the AFLP data using UPGMA and PCO revealed the complete separation of the genotypes of each species into distinct groups. I. indigotica separated as an entirely independent group, whereas I. glauca formed a separate cluster within the I. tinctoria group. Indeed, I. tinctoria and I. glauca are more closely related to each other than either is to I. indigotica. In addition, the genotypes of each landrace, apart from one from the English group, were clearly discriminated. However, the anomalous genotype did associate with the rest of its group when it was linked with the Erfurt group. These results provide new and useful information about the make-up of the Isatis genome, which has not previously been evaluated. They will be useful in the selection of plant material for variety development and conservation of the gene-pool.

Association of IL-10 gene (âˆ'1082A >G, âˆ'819C >T and âˆ'592C >A ...

Indian Academy of Sciences (India)

AMIT KUMAR MADESHIYA

assessment-insulin resistance in north Indian MetS subjects. [Madeshiya A. K. ... Type 2 diabetes is considered as an inflammatory .... fragment length polymorphism (RFLP) are listed in table 1. ...... γ +874T/A polymorphisms with atopic asthma children from .... 10 –819C/T polymorphism and cancer risk: a HuGE review and.

Genetic Background and Population Genetics of Hungarian Brown Trout Populations Using PCR-RFLP and Microsatellite Markers

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Ágnes Ősz

2015-12-01

4 University of West Hungary, Mosonmagyaróvár Vár 2., 9200 Mosonmagyaróvár, Hungary Based on the analyses of the mitochondrial DNA of several European brown trout populations, five evolutionary lineages of brown trout were indentified (Atlantic, Danubian, Mediterranean, Adriatic, Marble. The species is bred primarily for stock enhancement of natural waters, however the most hatchery-maintained broodstocks originate from the Atlantic lineage. Due to the hydrogeography of Hungary our stocks should theoretically belong to the Danubian lineage; however, this has not been investigated earlier by genetic studies. For our genetic analysis, 702 fin clips were collected from two brown trout broodstocks (Lillafüred and Szilvásvárad as well as populations of natural streams (Bán, Jósva, Kemence, Apátkút, Bittva and Kölöntés in Hungary. Sequencing of the control region in mitochondrial DNA, three PCR-RFLP (mitochondrial DNA control region, lactate dehydrogenase and somatolactin genes and five microsatellite markers were used to distinguish between Danubian and Atlantic lineages of brown trout. The proportion of the mitochondrial haplotype of the Danubian lineage was low, with the exception of the Apátkúti, Kölöntés streams and Szilvásvárad broodstock. Analyses of nuclear PCR-RFLP and microsatellites markers showed various distributions of alleles characteristic of the Atlantic or Danubian lineages, although the Atlantic genotype has dominated in all population. In case of the analyses of microsatellites the polymorphism varied greatly at all locations. In addition we found several alleles that were not described earlier in other populations. Those alleles probably would be typical of Hungarian brown trout populations. Overall the populations were effectively in Hardy-Weinberg equilibrium for both PCR-RFLP and microsatellite markers. The remarkably high proportion of allochthonous Atlantic alleles in the analyzed sites is a clear indicator of the import

Identification of Echinococcus Granulosus Strains in Isolated Hydatid Cyst Specimens from Animals by PCR-RFLP Method in West Azerbaijan – Iran

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Haleh Hanifian

2013-09-01

Full Text Available Background: The aim of this study was DNA extraction from protosco­lecses of Echinococcus granulosus and identification of these strains in West-Azerbai­jan Province, north western Iran.Methods: Thirty one livestock isolates from sheep and cattle were collected from abattoirs of the province. To investigate the genetic variation of the isolates, after DNA extraction by Glass beads-phenol chloroform method; PCR-RLFP analysis of rDNA-ITS1 was performed using three different restric­tion enzymes of Taq 1, Rsa 1 and Alu 1.Result: Amplified PCR products for all isolates were 1000bp band which is expected band in sheep strains (G1-G3 complex. The results of RFLP analy­sis also were the same for all isolates. PCR-RFLP patterns restriction en­zymes were identical as follows, Rsa1 bands under UV showed two bands approximately 655bp and 345bp. Alu1 bands were as follows: two approx­imately 800bp and 200bp and Taq1 did not cut any region and bands were approximately 1000 bp in all samples.Conclusions: Based on PCR-RFLP patterns of ITS1 fragment produced with endonucleases enzyme digestion in animal isolates, it can be concluded that a single strain of E. granulosus (sheep strain or G1-G3 complex is domi­nant genotype in this province

Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C-coli: the CAMPYNET experience

DEFF Research Database (Denmark)

Harrington, Clare S.; Moran, L.; Ridley, A.M.

2003-01-01

Aims: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP(fla typing) methods previously described for Campylobacter. Methods and Results: The sample set(n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two ...

A BanI RFLP at a deletion hotspot in the human dystrophin gene

Energy Technology Data Exchange (ETDEWEB)

Read, A P; Mountford, R [St. Mary' s Hospital, Manchester (England)

1990-01-25

Cf56a is a 0.9 kb EcoRI fragment of dystrophin cDNA in pUC13. Cf56a is identical to Kunkel's cDNA probe 8. Constant bands of 14.4, 11.0, 8.1, 6.2 and 1.3 kb correspond to exons I, N, L, N and K respectively. The polymorphic band is exon J (exon 48, 1.2+3.9 kb HindIII bands). This exon is deleted in 25% of all Duchenne/Becker dystrophy boys. Therefore this RFLP is useful for determining carrier status of at-risk females by showing heterozygosity or apparent non-maternity.

DArT whole genome profiling provides insights on the evolution and taxonomy of edible Banana (Musa spp.)

Czech Academy of Sciences Publication Activity Database

Sardos, J.; Perrier, X.; Doležel, Jaroslav; Hřibová, Eva; Christelová, Pavla; Van den Houwe, I.; Kilian, A.; Roux, N.

2016-01-01

Roč. 118, č. 7 (2016), s. 1269-1278 ISSN 0305-7364 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk LG15017 Institutional support: RVO:61389030 Keywords : multilocus genotype data * arrays technology dart * genetic diversity * population-structure * balbisiana colla * acuminata colla * markers * identification * aflp * domestication * Musa acuminata * Musa balbisiana * Musa spp. * banana * DArT * domestication * taxonomy * classification * domestication Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 4.041, year: 2016

Real-imaging cDNA-AFLP transcript profiling of pancreatic cancer patients: Egr-1 as a potential key regulator of muscle cachexia

Energy Technology Data Exchange (ETDEWEB)

Skorokhod, Alexander [Division of Preventive Oncology, National Center for Tumor Diseases (NCT) Heidelberg, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg (Germany); Institute of Molecular Biology and Genetics, Ukrainian Academy of Sciences, Zabolotnogo str. 150, 03143, Kiev (Ukraine); Bachmann, Jeannine [Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675, Munich (Germany); Giese, Nathalia A [Department of General Surgery, University of Heidelberg, ImNeuenheimer Feld, 110 69120, Heidelberg (Germany); Martignoni, Marc E [Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675, Munich (Germany); Krakowski-Roosen, Holger [Division of Preventive Oncology, National Center for Tumor Diseases (NCT) Heidelberg, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg (Germany)

2012-06-21

Cancer cachexia is a progressive wasting syndrome and the most prevalent characteristic of cancer in patients with advanced pancreatic adenocarcinoma. We hypothesize that genes expressed in wasted skeletal muscle of pancreatic cancer patients may determine the initiation and severity of cachexia syndrome. We studied gene expression in skeletal muscle biopsies from pancreatic cancer patients with and without cachexia utilizing Real-Imaging cDNA-AFLP-based transcript profiling for genome-wide expression analysis. Our approach yielded 183 cachexia-associated genes. Ontology analysis revealed characteristic changes for a number of genes involved in muscle contraction, actin cytoskeleton rearrangement, protein degradation, tissue hypoxia, immediate early response and acute-phase response. We demonstrate that Real-Imaging cDNA-AFLP analysis is a robust method for high-throughput gene expression studies of cancer cachexia syndrome in patients with pancreatic cancer. According to quantitative RT-PCR validation, the expression levels of genes encoding the acute-phase proteins α-antitrypsin and fibrinogen α and the immediate early response genes Egr-1 and IER-5 were significantly elevated in the skeletal muscle of wasted patients. By immunohistochemical and Western immunoblotting analysis it was shown, that Egr-1 expression is significantly increased in patients with cachexia and cancer. This provides new evidence that chronic activation of systemic inflammatory response might be a common and unifying factor of muscle cachexia.

Real-imaging cDNA-AFLP transcript profiling of pancreatic cancer patients: Egr-1 as a potential key regulator of muscle cachexia

International Nuclear Information System (INIS)

Skorokhod, Alexander; Bachmann, Jeannine; Giese, Nathalia A; Martignoni, Marc E; Krakowski-Roosen, Holger

2012-01-01

Cancer cachexia is a progressive wasting syndrome and the most prevalent characteristic of cancer in patients with advanced pancreatic adenocarcinoma. We hypothesize that genes expressed in wasted skeletal muscle of pancreatic cancer patients may determine the initiation and severity of cachexia syndrome. We studied gene expression in skeletal muscle biopsies from pancreatic cancer patients with and without cachexia utilizing Real-Imaging cDNA-AFLP-based transcript profiling for genome-wide expression analysis. Our approach yielded 183 cachexia-associated genes. Ontology analysis revealed characteristic changes for a number of genes involved in muscle contraction, actin cytoskeleton rearrangement, protein degradation, tissue hypoxia, immediate early response and acute-phase response. We demonstrate that Real-Imaging cDNA-AFLP analysis is a robust method for high-throughput gene expression studies of cancer cachexia syndrome in patients with pancreatic cancer. According to quantitative RT-PCR validation, the expression levels of genes encoding the acute-phase proteins α-antitrypsin and fibrinogen α and the immediate early response genes Egr-1 and IER-5 were significantly elevated in the skeletal muscle of wasted patients. By immunohistochemical and Western immunoblotting analysis it was shown, that Egr-1 expression is significantly increased in patients with cachexia and cancer. This provides new evidence that chronic activation of systemic inflammatory response might be a common and unifying factor of muscle cachexia

Differential expression analysis of genic male sterility by cDNA-AFLP in maize

International Nuclear Information System (INIS)

Zhang Linbi; Rong Tingzhao; Pan Guangtang; Cao Moju

2009-01-01

The differential expression of male sterility induced by space flight with male fertility was studied using cDNA-AFLP technology. Total RNA was isolated from anther of male sterility and male fertility. Nine differential expression cDNA fragments were obtained with 16 primer combinations. The differential cDNA fragments were eluted, cloned and sequenced. Then half-quantitative RT-PCR was used to stuy the differential expressions of 4 development stages between sterility and fertility. Sequencing analysis shown 2 fragments from male sterility might be novel genes. Four fragments from male fertility were homology as chalcone and stilbene synthases, putative acyl CoA dehydrogenase, putative protein kinases and putative glycine decarboxylase. All these proteins might participate in the energy metabolisms, substance metabolisms or signal pollen development, Z8 took on increasing expression during the middle period of pollen development. These results just met the demand of more energy and more substance during the pollen development. (authors)

Transcriptome analysis by cDNA-AFLP of Suillus luteus Cd-tolerant and Cd-sensitive isolates.

Science.gov (United States)

Ruytinx, Joske; Craciun, Adrian R; Verstraelen, Karen; Vangronsveld, Jaco; Colpaert, Jan V; Verbruggen, Nathalie

2011-04-01

The ectomycorrhizal basidiomycete Suillus luteus (L.:Fr.), a typical pioneer species which associates with young pine trees colonizing disturbed sites, is a common root symbiont found at heavy metal contaminated sites. Three Cd-sensitive and three Cd-tolerant isolates of S. luteus, isolated respectively from non-polluted and a heavy metal-polluted site in Limburg (Belgium), were used for a transcriptomic analysis. We identified differentially expressed genes by cDNA-AFLP analysis. The possible roles of some of the encoded proteins in heavy metal (Cd) accumulation and tolerance are discussed. Despite the high conservation of coding sequences in S. luteus, a large intraspecific variation in the transcript profiles was observed. This variation was as large in Cd-tolerant as in sensitive isolates and may help this pioneer species to adapt to novel environments.

Risk preferences in strategic wildfire decision making: a choice experiment with U.S. wildfire managers.

Science.gov (United States)

Wibbenmeyer, Matthew J; Hand, Michael S; Calkin, David E; Venn, Tyron J; Thompson, Matthew P

2013-06-01

Federal policy has embraced risa management as an appropriate paradigm for wildfire management. Economic theory suggests that over repeated wildfire events, potential economic costs and risas of ecological damage are optimally balanced when management decisions are free from biases, risa aversion, and risa seeking. Of primary concern in this article is how managers respond to wildfire risa, including the potential effect of wildfires (on ecological values, structures, and safety) and the likelihood of different fire outcomes. We use responses to a choice experiment questionnaire of U.S. federal wildfire managers to measure attitudes toward several components of wildfire risa and to test whether observed risa attitudes are consistent with the efficient allocation of wildfire suppression resources. Our results indicate that fire managers' decisions are consistent with nonexpected utility theories of decisions under risa. Managers may overallocate firefighting resources when the likelihood or potential magnitude of damage from fires is low, and sensitivity to changes in the probability of fire outcomes depends on whether probabilities are close to one or zero and the magnitude of the potential harm. © 2012 Society for Risk Analysis.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB

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Lenice Roteia Cardoso Jung

2015-06-01

Full Text Available Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR. The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB).

Science.gov (United States)

Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

2015-06-01

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Variability among inbred lines and RFLP mapping of sunflower isozymes

Directory of Open Access Journals (Sweden)

Carrera Alicia D.

2002-01-01

Full Text Available Eight isozyme systems were used in this study: acid phosphatase (ACP, alcohol dehydrogenase (ADH, esterase (EST, glutamate dehydrogenase (GDH, malate dehydrogenase (MDH, phosphoglucoisomerase (PGI, 6-phosphogluconate dehydrogenase (PGD, and phosphoglucomutase (PGM. The polymorphism of these enzyme systems was studied in 25 elite inbred lines. A total of 19 loci were identified, but only eight of them were polymorphic in the germplasm tested. The polymorphic index for the eight informative markers ranged from 0.08 to 0.57, with a mean value of 0.36. Five isozyme loci were mapped in F2:3 populations with existing RFLP data. Est-1, Gdh-2 and Pgi-2 were mapped to linkage groups 3, 14 and 9, respectively. As in previous reports, an ACP locus and a PGD locus were found to be linked, both located in linkage group 2 of the public sunflower map.

The Variants Within the COL5A1 Gene are Associated with Reduced Risk of Anterior Cruciate Ligament Injury in Skiers

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Stępień-Słodkowska Marta

2015-03-01

Full Text Available The purpose of this study was to examine the association of the BstUI RFLP C/T (rs 12722 and DpnII RFLP C/T (rs 13946 COL5A1 polymorphisms, individually and as haplotypes, with anterior cruciate ligament ruptures in recreational skiers. Subjects were 138 male recreational skiers with surgically diagnosed primary anterior cruciate ligament ruptures. The control group consisted of 183 apparently healthy male recreational skiers, who were without any self-reported history of ligament or tendon injury. DNA was extracted from buccal cells donated by the subjects and genotyping was carried out using real-time PCR. The genotype distributions for both polymorphisms met Hardy- Weinberg expectations in both groups. There were no significant differences in genotype distribution of allele frequencies of COL5A1 BstUI RFLP C/T and COL5A1 DpnII RFLP C/T polymorphisms between the ACL rupture and control groups. The T-T (BstUI RFLP T, DpnII RFLP T haplotype was the most common (55.6%. The haplotype T-C was not present in any of the subjects. There was an underrepresentation tendency of the C-T haplotype in the study group compared to controls under recessive mode of inheritance. Higher frequency of the COL5A1 BstUI RFLP C/T and COL5A1DpnII RFLP C/T polymorphisms haplotype is associated with reduced risk of anterior cruciate ligament injury in a group of apparently healthy male recreational skiers.

Genetic relatedness between cassava (Manihot esculenta Crantz and M. flabellifolia and M. Peruviana based on both RAPD and AFLP markers

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Colombo Carlos

2000-01-01

Full Text Available The taxonomy of the genus Manihot is still uncertain and the genetic origin of cassava (M. esculenta Crantz continues to be controversial. We studied the degree of genetic relatedness between cassava and two naturally occurring species (M. flabellifolia and M. peruviana which are probably involved in the evolution of cassava, using RAPD and AFLP molecular markers. Thirty-three clonal accessions of cassava of known genetic diversity and 15 accessions of the wild species M. flabellifolia and M. peruviana were analyzed using 92 polymorphic RAPD bands and 73 polymorphic AFLP bands. The genetic markers were unable to differentiate the two wild species, which confirms their botanical similarity. Half of the total number of amplified bands were monomorphic in all of the genotypes evaluated. The mean genetic similarity (Jaccard between cassava and the species M. flabellifolia/M. peruviana was 0.59. A grouping analysis (neighbor-joining method with RAPD markers of cultivated cassava, M. flabellifolia/M. peruviana and the other wild species located the genotypes of cassava and M. flabellifolia/M. peruviana at one extremity and the three Mexican species (M. aesculifolia, M. michaelis and M. chlorostica at the other. An intermediate position between these groups was occupied by two wild species (M. glaziovii and M. reptans native to central and northeastern Brazil. These results are consistent with the hypothesis that the species M. flabellifolia and M. peruviana gave rise to the cultivated species.

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples.

Science.gov (United States)

Schmidt, H; Taniwaki, M H; Vogel, R F; Niessen, L

2004-01-01

The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.

PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus.

Science.gov (United States)

Nasri, Tuba; Hedayati, Mohammad Taghi; Abastabar, Mahdi; Pasqualotto, Alessandro C; Armaki, Mojtaba Taghizadeh; Hoseinnejad, Akbar; Nabili, Mojtaba

2015-10-01

Aspergillus species are important agents of life-threatening infections in immunosuppressed patients. Proper speciation in the Aspergilli has been justified based on varied fungal virulence, clinical presentations, and antifungal resistance. Accurate identification of Aspergillus species usually relies on fungal DNA sequencing but this requires expensive equipment that is not available in most clinical laboratories. We developed and validated a discriminative low-cost PCR-based test to discriminate Aspergillus isolates at the species level. The Beta tubulin gene of various reference strains of Aspergillus species was amplified using the universal fungal primers Bt2a and Bt2b. The PCR products were subjected to digestion with a single restriction enzyme AlwI. All Aspergillus isolates were subjected to DNA sequencing for final species characterization. The PCR-RFLP test generated unique patterns for six clinically important Aspergillus species, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus and Aspergillus nidulans. The one-enzyme PCR-RFLP on Beta tubulin gene designed in this study is a low-cost tool for the reliable and rapid differentiation of the clinically important Aspergillus species. Copyright © 2015 Elsevier B.V. All rights reserved.

Revisiting AFLP fingerprinting for an unbiased assessment of genetic structure and differentiation of taurine and zebu cattle

Science.gov (United States)

2014-01-01

Background Descendants from the extinct aurochs (Bos primigenius), taurine (Bos taurus) and zebu cattle (Bos indicus) were domesticated 10,000 years ago in Southwestern and Southern Asia, respectively, and colonized the world undergoing complex events of admixture and selection. Molecular data, in particular genome-wide single nucleotide polymorphism (SNP) markers, can complement historic and archaeological records to elucidate these past events. However, SNP ascertainment in cattle has been optimized for taurine breeds, imposing limitations to the study of diversity in zebu cattle. As amplified fragment length polymorphism (AFLP) markers are discovered and genotyped as the samples are assayed, this type of marker is free of ascertainment bias. In order to obtain unbiased assessments of genetic differentiation and structure in taurine and zebu cattle, we analyzed a dataset of 135 AFLP markers in 1,593 samples from 13 zebu and 58 taurine breeds, representing nine continental areas. Results We found a geographical pattern of expected heterozygosity in European taurine breeds decreasing with the distance from the domestication centre, arguing against a large-scale introgression from European or African aurochs. Zebu cattle were found to be at least as diverse as taurine cattle. Western African zebu cattle were found to have diverged more from Indian zebu than South American zebu. Model-based clustering and ancestry informative markers analyses suggested that this is due to taurine introgression. Although a large part of South American zebu cattle also descend from taurine cows, we did not detect significant levels of taurine ancestry in these breeds, probably because of systematic backcrossing with zebu bulls. Furthermore, limited zebu introgression was found in Podolian taurine breeds in Italy. Conclusions The assessment of cattle diversity reported here contributes an unbiased global view to genetic differentiation and structure of taurine and zebu cattle

Further studies of the influence of apolipoprotein B alleles on glucose and lipid metabolism

DEFF Research Database (Denmark)

Bentzen, J.; Poulsen, P.; Vaag, A.

2003-01-01

The effect of five genetic polymorphisms in the apolipoprotein B gene on parameters of lipid and glucose metabolism was assessed in 564 Danish mono- and dizygotic twins. Genotypes in apolipoprotein B T71I (ApaLI RFLP), A591V (AluI RFLP), L2712P (MvaI RFLP), R3611Q (MspI RFLP), and E4154K (Eco...... on the insulin-to-glucose ratio (p = 0.04), and E4154K (EcoRI RFLP) influenced HOMAbeta (p = 0.04). Significant interactions were observed between genotype in T71I (ApaLI RFLP), A591V (AluI RFLP), R3611Q (MspI RFLP), and E4154K (EcoRI RFLP) and glucose tolerance on lipid-related parameters (0.03

Styl RFLP recognized by a human IRBP cDNA localized to chromosome 10

Energy Technology Data Exchange (ETDEWEB)

Chin, K S; Mathew, C G.P.; Fong, S L; Bridges, C D; Ponder, B A.J.

1988-02-25

A 2184 bp cDNA (H.4 IRBP) encoding human interstitial retinol-biding protein isolated from a human retina cDNA library in lambdagt10 by screening with a bovine IRBP cDNA probe. Styl identifies a 2-allele polymorphism with bands at 2.3 kb (Cl) and 1.95 kb (C2) and invariant bands at 1.1, 1.0 and 0.8kb. Codominant segregation was observed in two informative families. The RFLP was mapped to chromosome 10 using somatic cell hybrids. In situ hybridization suggests regional assignments near p11.2 -q11.2 with a secondary site of hybridization at q24-25.

A BanI RFLP at a deletion hotspot in the human dystrophin gene

Energy Technology Data Exchange (ETDEWEB)

Read, A.P.; Mountford, R. (St. Mary' s Hospital, Manchester (England))

1990-01-25

Cf56a is a 0.9 kb EcoRI fragment of dystrophin cDNA in pUC13. Cf56a is identical to Kunkel's cDNA probe 8. Constant bands of 14.4, 11.0, 8.1, 6.2 and 1.3 kb correspond to exons I, N, L, N and K respectively. The polymorphic band is exon J (exon 48, 1.2+3.9 kb HindIII bands). This exon is deleted in 25% of all Duchenne/Becker dystrophy boys. Therefore this RFLP is useful for determining carrier status of at-risk females by showing heterozygosity or apparent non-maternity.

Identification of QTLs Associated with Callogenesis and Embryogenesis in Oil Palm Using Genetic Linkage Maps Improved with SSR Markers

Science.gov (United States)

Ting, Ngoot-Chin; Jansen, Johannes; Nagappan, Jayanthi; Ishak, Zamzuri; Chin, Cheuk-Weng; Tan, Soon-Guan; Cheah, Suan-Choo; Singh, Rajinder

2013-01-01

Clonal reproduction of oil palm by means of tissue culture is a very inefficient process. Tissue culturability is known to be genotype dependent with some genotypes being more amenable to tissue culture than others. In this study, genetic linkage maps enriched with simple sequence repeat (SSR) markers were developed for dura (ENL48) and pisifera (ML161), the two fruit forms of oil palm, Elaeis guineensis. The SSR markers were mapped onto earlier reported parental maps based on amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers. The new linkage map of ENL48 contains 148 markers (33 AFLPs, 38 RFLPs and 77 SSRs) in 23 linkage groups (LGs), covering a total map length of 798.0 cM. The ML161 map contains 240 markers (50 AFLPs, 71 RFLPs and 119 SSRs) in 24 LGs covering a total of 1,328.1 cM. Using the improved maps, two quantitative trait loci (QTLs) associated with tissue culturability were identified each for callusing rate and embryogenesis rate. A QTL for callogenesis was identified in LGD4b of ENL48 and explained 17.5% of the phenotypic variation. For embryogenesis rate, a QTL was detected on LGP16b in ML161 and explained 20.1% of the variation. This study is the first attempt to identify QTL associated with tissue culture amenity in oil palm which is an important step towards understanding the molecular processes underlying clonal regeneration of oil palm. PMID:23382832

Use of AFLP and RAPD molecular genetic markers and cytogenetic analysis to explore relationships among taxa of the Patagonian Bromus setifolius complex

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Ana M. García

2009-01-01

Full Text Available Bromus setifolius var. pictus (Hook Skottsb., B. setifolius var. setifolius Presl. and B. setifolius var. brevifolius Ness are three native Patagonian taxa in the section Pnigma Dumort of the genus Bromus L. AFLP and RAPD analysis, in conjunction with genetic distance measurements and statistical techniques, revealed variation within this group and indicated that B. setifolius var. brevifolius was closely related to B. setifolius var. pictus, with both taxa being more distantly related to B. setifolius var. setifolius. Cytogenetic analysis confirmed the chromosomal number of B. setifolius var. pictus (2n = 70 and B. setifolius var. setifolius (2n = 28 and showed for the first time that B. setifolius var. brevifolius had 2n = 70. The combination of molecular genetic and cytogenetic evidence supported a species status for two of the three taxa and suggested hypotheses for the evolutionary origin of these complex taxa. Species status was also indicated for B. setifolius var. setifolius. Based on these findings, we suggest that B. setifolius var. pictus be referred to as B. pictus Hook var. pictus, and B. setifolius var brevifolius as B. pictus Hook var brevifolius. The correlation between AFLP diversity and variation in ecological parameters suggested that this marker system could be used to assess breeding progress and to monitor the domestication of Patagonian Bromus species for agronomic use.

AFLP and AMP Fingerprints as Markers to Evaluate Genetic Differences between Medicago truncatula Line Jemalong and 2HA, a New Line Produced by in vitro Culture Selection

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R.R. Irwanto

2008-09-01

Full Text Available A new line, Medicago truncatula cv. Jemalong 2HA (herein known as 2HA has been developed via repetitive regeneration and selection of M. truncatula cv. Jemalong. During somatic embryogenesis, 2HA produces 500 times more embryos than its progenitor, Jemalong. It is interesting to study if those two lines are isogenic or has genetic differences. The main objectives of the study was to evaluate the genotypic differences between Jemalong and 2HA also to evaluate the methylation event in 2HA utilized two DNA fingerprinting techniques, i.e AFLP fingerprints (Amplified Length of Polymorphism and AMP (Amplified Methylation Polymorphism. The results showed that AFLP analysis using eight primers combinations could not detect any differences between Jemalong and 2HA. However, using AMP methylation sensitive primers it could detect 15 polymorphisms out of 840 markers. These results lead to a conclusion that Jemalong and 2HA are isogenic lines. 2HA may have higher regeneration capacities due to methylation process which occurs during the production of 2HA through repetitive regeneration cycles.

Microbial Community Analysis of Field-Grown Soybeans with Different Nodulation Phenotypes▿

OpenAIRE

Ikeda, Seishi; Rallos, Lynn Esther E.; Okubo, Takashi; Eda, Shima; Inaba, Shoko; Mitsui, Hisayuki; Minamisawa, Kiwamu

2008-01-01

Microorganisms associated with the stems and roots of nonnodulated (Nod−), wild-type nodulated (Nod+), and hypernodulated (Nod++) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod+ soybe...

Identificação de híbridos de citros resistentes à mancha-marrom-de-alternária por meio de fAFLP e testes de patogenicidade Alternaria brown spot resistant citrus hybrid identification by means of fAFLP and pathogenicity tests

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Edvan Alves Chagas

2007-07-01

Full Text Available O objetivo deste trabalho foi identificar híbridos, oriundos de hibridações controladas entre 'Folha Murcha' x 'Ponkan' e testá-los quanto à resistência a Alternaria alternata f. sp. citri. As plântulas foram obtidas via cultura in vitro de embriões. Utilizou-se o marcador molecular fAFLP para identificação dos híbridos e, em seguida, realizou-se o teste de patogenicidade nos híbridos com isolados de Alternaria alternata f. sp. citri, em condições de laboratório. Os pares de primers EcoRI AAG - MseI CAG e EcoRI ACC - MseI CAA foram os mais eficientes na identificação dos híbridos, os quais identificaram 48,5% de híbridos. Os híbridos F64, F108, F111, F113, F131 e F139 são potencialmente resistentes a Alternaria alternata f. sp. citri.The objective of this work was to identify hybrids obtained from controlled crossings between 'Folha Murcha' x 'Ponkan', and to check their resistance to Alternaria alternata f. sp. citri. The seedlings were obtained by in vitro embryo culture. The fAFLP marker technique was used to identify the hybrids, then laboratory pathogenicity test of the hybrids was accomplished with Alternaria alternata f. sp. citri isolates. The pairs of primers EcoRI AAG - Msel CAG and EcoRI ACC - Msel CAA were the most efficient for hybrids identification, and distinguished 48.5% of hybrids. Hybrids F64, F108, F111, F113, F131 and F139 showed potential resistance to the Alternaria alternata f. sp. citri.

IGS-RFLP analysis and development of molecular markers for identification of Fusarium poae, Fusarium langsethiae, Fusarium sporotrichioides and Fusarium kyushuense

NARCIS (Netherlands)

Konstantinova, P.S.; Yli-Mattila, T.

2004-01-01

The intergenic spacer (IGS) regions of the rDNA of several Fusarium spp. strains obtained from the collaborative researchers (Int. J. Food Microbiol. (2003)) were amplified by polymerase chain reaction (PCR), and an IGS¿RFLP analysis was performed. Restriction digestion with AluI, MspI and PstI

Intraarticular volume and clearance in human synovial effusions

International Nuclear Information System (INIS)

Wallis, W.J.; Simkin, P.A.; Nelp, W.B.; Foster, D.M.

1985-01-01

Intraarticular volumes were measured by radiolabeled albumin (RISA) distribution in chronic knee effusions from 11 rheumatoid arthritis patients and 9 osteoarthritis patients. Volumes of synovial fluid obtained at joint aspiration were substantially less than those found by RISA dilution. Up to 24 hours was needed for full distribution of RISA throughout the intraarticular compartment. Measured 123I and RISA radioactivity over the knee described monoexponential rate constants, lambda (minute-1). The clearance of 123I and RISA from synovial effusions was derived by the formulation volume (ml) X lambda (minute-1) = clearance (ml/minute). RISA clearance in rheumatoid effusions was significantly greater than that found in osteoarthritis effusions. Intraarticular volume and isotope clearance were easily quantified and provide measures for further evaluating the microvascular physiology of synovial effusions

Development of a PCR-RFLP assay for the detection and differentiation of canine parvovirus and mink enteritis virus.

Science.gov (United States)

Zhang, Chuanmei; Yu, Yongle; Yang, Haiyan; Li, Guimei; Yu, Zekun; Zhang, Hongliang; Shan, Hu

2014-12-15

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay has been developed to detect and differentiate between canine parvovirus (CPV) and mink enteritis virus (MEV). Eight CPV and three MEV epidemic strains isolated from 28 pathological samples from dogs and minks suspected of being infected with parvovirus were amplified by PCR using a pair of specific primers designed based on the CPV-N strain (M19296). PCR amplified a fragment of 1016bp from the genomic DNA of both MEV and CPV. The MEV-derived fragment could be digested with the restriction enzyme BSP1407I into three fragments of 102bp, 312bp and 602bp, while the fragment amplified from the CPV genomic DNA was digested into only two fragments of 414bp and 602bp. The lowest DNA concentration of CPV and MEV that could be detected using this assay was 0.004μg/ml and 0.03μg/ml, respectively. The PCR-RFLP assay developed in the present study can, therefore, be used to detect and differentiate MEV from CPV with high specificity and sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of Eastern United States Reticulitermes Termite Species via PCR-RFLP, Assessed Using Training and Test Data

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Ryan C. Garrick

2015-06-01

Full Text Available Reticulitermes termites play key roles in dead wood decomposition and nutrient cycling in forests. They also damage man-made structures, resulting in considerable economic loss. In the eastern United States, five species (R. flavipes, R. virginicus, R. nelsonae, R. hageni and R. malletei have overlapping ranges and are difficult to distinguish morphologically. Here we present a molecular tool for species identification. It is based on polymerase chain reaction (PCR amplification of a section of the mitochondrial cytochrome oxidase subunit II gene, followed by a three-enzyme restriction fragment length polymorphism (RFLP assay, with banding patterns resolved via agarose gel electrophoresis. The assay was designed using a large set of training data obtained from a public DNA sequence database, then evaluated using an independent test panel of Reticulitermes from the Southern Appalachian Mountains, for which species assignments were determined via phylogenetic comparison to reference sequences. After refining the interpretive framework, the PCR-RFLP assay was shown to provide accurate identification of four co-occurring species (the fifth species, R. hageni, was absent from the test panel, so accuracy cannot yet be extended to training data. The assay is cost- and time-efficient, and will help improve knowledge of Reticulitermes species distributions.

COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

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Tülin GÜVEN GÖKMEN

Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

Preliminary genetic linkage maps of Chinese herb Dendrobium ...

Indian Academy of Sciences (India)

2013-08-05

Aug 5, 2013 ... wild Dendrobium germplasm resources only survive in cer- tain habitats and are rapidly ..... cardunculus var. scolymus L.) based on AFLP, S-SAP, M-AFLP ... Weber J. L. 1990 Informativeness of human (dC-dA) n-(dG-dT) n.

Determination of Molecular Genotyping of Ureaplasma SPP in Women with Genital Infections by 16S–23S rDNA PCR-RFLP Method

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R. Mirnejad

2011-04-01

Full Text Available Introduction & Objective: So far, despite the wide range of methods such as analytic methods used for differentiation of Mycoplasma, the diagnosis of Mycoplasma species is still difficult. Generally the low-level discriminatory power of serological methods because of the rapid changes in size and phase of the dominant antigens in the immune cell surface of Mycoplasmas greatly limits their applicability to the typing of Mycoplasmas. On the contrary,molecular methods do not suffer from these drawbacks and can be used for typing of Mycoplasmas. The aim of this investigation was molecular identification and genotyping of ureaplasma SPP in women with genital infections by 16S–23S rDNA PCR-RFLP.Materials & Methods: Genital swabs were taken from 210 patients who referred to gynecology clinic of Rasool hospital in Tehran, Iran during December 2007 until June 2008. The swabs suspended in PBS, were immediately transferred to laboratory .Following DNA extraction, PCR assay was performed using a genus specific primer pair. These primer sets amplified a 559 bp fragment for Ureaplasma Spp. Samples containing bands of the expected size for Ureaplasma strains were subjected to digestion with different restriction endonuclease enzymes (AluI, Taq I, CacI8, BbsI, EcoRI. Results: Of the 210 samples, Ureaplasma Spp was isolated from 93 patients (44.3% by PCR and 69 samples by culture. In the present study only Biovar 1 (Ureaplasma parvum was isolated from clinical specimens and the results were confirmed using a cutting enzyme TaqI (enzyme specific species of ureaplasma SPP. The results of this analysis using PCR-RFLP and sequencing showed that all had the same genotype and shared identical sequence with the genome sequence of serovar 3 Ureaplasma parvum.Conclusion: Ureaplasma parvum is generally isolated from the genital samples. In this study all isolates were identical and no difference was found among the enzyme patterns of the bacteria after PCR-RFLP .So

Status of biotechnology with emphasis on molecular techniques for mutation breeding in the Philippines

Energy Technology Data Exchange (ETDEWEB)

Lapade, A.G.; Nazarea, T.Y.; Veluz, A.M.S.; Marbella, L.J.; Nato, A.Q.; Coloma, C.B. Jr.; Asencion, A.B. [Philippine Nuclear Research Institute, Commonwealth Avenue, Quezon (Philippines)

2002-02-01

This paper summarizes the status of biotechnology with emphasis on molecular techniques for plant breeding in the Philippines. Several molecular and in-vitro culture techniques are integrated in plant breeding for crop improvement at PNRI, UPLB, IRRI and PhilRice. At IRRI, PCR techniques, RFLP and RAPD, PCR techniques, RFLP and RAPD were developed to establish high density molecular maps, determine breadth and diversity of germplasm and characterize alien introgression. The molecular maps have identified DNA sequence of resistance genes of HYVs and NPTs to abiotic and biotic stresses, the major achievement is the development of high density molecular maps in rice with at least 2000 markers. The biotechnology program at PhilRice for varietal improvement includes: (1) utilization of molecular marker technology such gene mapping of desired traits in rice, analysis of genetic relationships of germplasm materials and breeding lines through DNA fingerprinting and genetic diversity studies and development and application of marker aided selection for disease resistance (RTD and BLB); (2) application of in-vitro techniques in the development of lines with tolerance to adverse conditions; (3) molecular cloning of important genes for RTD resistance; (4) genetic transformation for male sterility and resistance to sheath blight and stem borers; and (5) transfer of disease resistance from wild species to cultivated varieties. In IPB, molecular markers:microsatellites or SSR, AFLP and RGA are being used for mapping and diversity studies in coconut, mango, banana, mungbean, corn and tomato. Mutation breeding at PNRI using gamma radiation has resulted in the development of crop varieties with desirable traits. The use of AFLP coupled to PCR is being used to study polymorphism in plant variants of radiation-induced mutants of rice, pineapple and ornamentals. (author)

Molecular characterization of Cryptococcus neoformans isolated from the environment in Beijing, China.

Science.gov (United States)

Dou, Hongtao; Wang, Huizhu; Xie, Shaowei; Chen, Xinxin; Xu, Zhipeng; Xu, Yingchun

2017-10-01

The molecular type of environmental Cryptococcus neoformans in Beijing was not clear. Our study aims to reveal the molecular characterization of C. neoformans complex from environment in Beijing, China. A total of 435 samples of pigeon droppings from 11 different homes in Beijing were collected from August to November in 2015. Pigeon droppings were inoculated onto caffeic acid cornmeal agar (CACA) to screen C. neoformans complex. Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed for species identification. Serotype and mating type was determined by specific primers. Restriction fragment length polymorphisms of URA5 (URA5-RFLP) were applied to genotype. Multi-locus sequence typing (MLST) was done for further identification and sequence type (ST) determination. Altogether, 81 isolates of C. neoformans AFLP1/VNI were recognized from 435 pigeon droppings in this study. The positive rate for C. neoformans AFLP1/VNI from pigeon droppings in different homes varied from 5.0% to 52.6%, the average was 20.2%. All of these cryptococcal strains were serotype A, MATα. They were genotyped as VNI by URA5-RFLP and were confirmed by MLST. No other molecular types of C. neoformans and Cryptococcus gattii isolates were isolated. Their STs were identified as ST 31 (n = 54, 66.7%), followed by ST 53 (n = 10), ST 191 (n = 8), ST 5 (n = 5), ST 57 (n = 3), and ST 38 (n = 1). We concluded that not only clinical but also environmental isolates of C. neoformans need to be investigated more deeply and more extensively. The virulence difference between ST 5 and ST 31 need to be explored in the future. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene

OpenAIRE

Kumar, Deepak; Singh, S. P.; Karabasanavar, Nagappa S.; Singh, Rashmi; Umapathi, V.

2012-01-01

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168–776 b...

Metabolic profiles and cDNA-AFLP analysis of Salvia miltiorrhiza and Salvia castanea Diel f. tomentosa Stib.

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Dongfeng Yang

Full Text Available Plants of the genus Salvia produce various types of phenolic compounds and tanshinones which are effective for treatment of coronary heart disease. Salvia miltiorrhiza and S. castanea Diels f. tomentosa Stib are two important members of the genus. In this study, metabolic profiles and cDNA-AFLP analysis of four samples were employed to identify novel genes potentially involved in phenolic compounds and tanshinones biosynthesis, including the red roots from the two species and two tanshinone-free roots from S. miltiorrhiza. The results showed that the red roots of S. castanea Diels f. tomentosa Stib produced high contents of rosmarinic acid (21.77 mg/g and tanshinone IIA (12.60 mg/g, but low content of salvianolic acid B (1.45 mg/g. The red roots of S. miltiorrhiza produced high content of salvianolic acid B (18.69 mg/g, while tanshinones accumulation in this sample was much less than that in S. castanea Diels f. tomentosa Stib. Tanshinones were not detected in the two tanshinone-free samples, which produced high contents of phenolic compounds. A cDNA-AFLP analysis with 128 primer pairs revealed that 2300 transcript derived fragments (TDFs were differentially expressed among the four samples. About 323 TDFs were sequenced, of which 78 TDFs were annotated with known functions through BLASTX searching the Genbank database and 14 annotated TDFs were assigned into secondary metabolic pathways through searching the KEGGPATHWAY database. The quantitative real-time PCR analysis indicated that the expression of 9 TDFs was positively correlated with accumulation of phenolic compounds and tanshinones. These TDFs additionally showed coordinated transcriptional response with 6 previously-identified genes involved in biosynthesis of tanshinones and phenolic compounds in S. miltiorrhiza hairy roots treated with yeast extract. The sequence data in the present work not only provided us candidate genes involved in phenolic compounds and tanshinones biosynthesis

Prognostic value of IDH1 mutations identified with PCR-RFLP assay in acute myeloid leukemia patients

International Nuclear Information System (INIS)

Elsayed, Gh.M.; Zaher, A.; Elnoshokaty, E.H.; Nassar, H.R.; Moneer, M.M.

2014-01-01

Background: Somatic mutations in isocitrate dehydrogenase 1 (1DH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique. Aim of the work: The aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML. Methods: Residual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was adapted to IDHl gene, codon 132 mutations screening. Results: The frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p = 0.03). Patients with 1DH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p = 0.08). Conclusion: IDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDF11 mutations in AML.

Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP.

Science.gov (United States)

Montalvo Alvarez, Ana Margarita; Nodarse, Jorge Fraga; Goodridge, Ivón Montano; Fidalgo, Lianet Monzote; Marin, Marcel; Van Der Auwera, Gert; Dujardin, Jean-Claude; Bernal, Iván Darío Velez; Muskus, Carlos

2010-05-01

Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist. Copyright 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

RFLP Analysis and Allelic Discrimination with Real-Time PCR Using the Human Lactase Persistence Trait: A Pair of Molecular Genetic Investigations

Science.gov (United States)

Weinlander, Kenneth M.; Hall, David J.; De Stasio, Elizabeth A.

2010-01-01

We describe here two open-ended laboratory investigations for an undergraduate laboratory course that uses students' DNA as templates for quantitative real-time PCR and for traditional PCR followed by RFLP analysis. Students are captivated by the immediacy of the application and the relevance of the genotypes and traits, lactase persistence or…

Identification of QTLs associated with callogenesis and embryogenesis in oil palm using genetic linkage maps improved with SSR markers.

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Ngoot-Chin Ting

Full Text Available Clonal reproduction of oil palm by means of tissue culture is a very inefficient process. Tissue culturability is known to be genotype dependent with some genotypes being more amenable to tissue culture than others. In this study, genetic linkage maps enriched with simple sequence repeat (SSR markers were developed for dura (ENL48 and pisifera (ML161, the two fruit forms of oil palm, Elaeis guineensis. The SSR markers were mapped onto earlier reported parental maps based on amplified fragment length polymorphism (AFLP and restriction fragment length polymorphism (RFLP markers. The new linkage map of ENL48 contains 148 markers (33 AFLPs, 38 RFLPs and 77 SSRs in 23 linkage groups (LGs, covering a total map length of 798.0 cM. The ML161 map contains 240 markers (50 AFLPs, 71 RFLPs and 119 SSRs in 24 LGs covering a total of 1,328.1 cM. Using the improved maps, two quantitative trait loci (QTLs associated with tissue culturability were identified each for callusing rate and embryogenesis rate. A QTL for callogenesis was identified in LGD4b of ENL48 and explained 17.5% of the phenotypic variation. For embryogenesis rate, a QTL was detected on LGP16b in ML161 and explained 20.1% of the variation. This study is the first attempt to identify QTL associated with tissue culture amenity in oil palm which is an important step towards understanding the molecular processes underlying clonal regeneration of oil palm.

Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism.

Science.gov (United States)

Jernberg, Cecilia; Sullivan, Asa; Edlund, Charlotta; Jansson, Janet K

2005-01-01

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

Covariance of bacterioplankton composition and environmental variables in a temperate delta system

Science.gov (United States)

Stepanauskas, R.; Moran, M.A.; Bergamaschi, B.A.; Hollibaugh, J.T.

2003-01-01

We examined seasonal and spatial variation in bacterioplankton composition in the Sacramento-San Joaquin River Delta (CA) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Cloned 16S rRNA genes from this system were used for putative identification of taxa dominating the T-RFLP profiles. Both cloning and T-RFLP analysis indicated that Actinobacteria, Verrucomicrobia, Cytophaga-Flavobacterium and Proteobacteria were the most abundant bacterioplankton groups in the Delta. Despite the broad variety of sampled habitats (deep water channels, lakes, marshes, agricultural drains, freshwater and brackish areas), and the spatial and temporal differences in hydrology, temperature and water chemistry among the sampling campaigns, T-RFLP electropherograms from all samples were similar, indicating that the same bacterioplankton phylotypes dominated in the various habitats of the Delta throughout the year. However, principal component analysis (PCA) and partial least-squares regression (PLS) of T-RFLP profiles revealed consistent grouping of samples on a seasonal, but not a spatial, basis. ??-Proteobacteria related to Ralstonia, Actinobacteria related to Microthrix, and ??-Proteobacteria identical to the environmental Clone LD12 had the highest relative abundance in summer/fall T-RFLP profiles and were associated with low river flow, high pH, and a number of optical and chemical characteristics of dissolved organic carbon (DOC) indicative of an increased proportion of phytoplankton-produced organic material as opposed to allochthonous, terrestrially derived organic material. On the other hand, Geobacter-related ??-Proteobacteria showed a relative increase in abundance in T-RFLP analysis during winter/spring, and probably were washed out from watershed soils or sediment. Various phylotypes associated with the same phylogenetic division, based on tentative identification of T-RFLP fragments, exhibited diverse seasonal patterns, suggesting that ecological

Genetic variation within and among Danish brown trout ( Salmo trutta L) hatchery strains, assessed by PCR-RFLP analysis of mitochondrial DNA segments

DEFF Research Database (Denmark)

Hansen, Michael Møller; Mensberg, Karen-Lise Dons; Rasmussen, Gorm

1997-01-01

Eleven Danish brown trout hatchery strains were studied by PCR- RFLP analysis of the ND-I and ND-5/6 segments of the mitochondrial genome. For comparison, data from wild trout representing three Danish river systems also were included. Reduced variability in terms of nucleon diversity and number...

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

Science.gov (United States)

Cervera, M T; Ruiz-García, L; Martínez-Zapater, J M

2002-12-01

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

Searching for RFLP markers to identify genes for aluminum tolerance in maize

International Nuclear Information System (INIS)

Paiva, E.; Lopes, M.A.; Parentoni, S.N.; Martins, P.R.; Torres, G.A.

1998-01-01

The objective of this study was to identify restriction fragment length polymorphism (RFLP) markers linked to Quantitative Trait Loci (QTL) that control aluminum (Al) tolerance in maize. The strategy used was bulked segregant analysis (BSA) and the genetic materials utilized were the F 2 , F 3 and F 4 populations derived from a cross between the Al-susceptible inbred line L53 and Al-tolerant inbred line L1327. The populations were evaluated in a nutrient solution containing a toxic concentration of Al (6 ppm) and relative seminal root length (RSRL) was used as a phenotypic measure of tolerance. Seedlings of the F 2 population with the highest and lowest RSRL values were transplanted to the field and subsequently selfed to obtain F 3 and F 4 families. The efficiency of the phenotypic index for selection was found to be greater when mean values were used instead of individual RSRL values. F 3 and F 4 families were then evaluated in nutrient solution to identify those that were not segregating. One hundred and thirteen probes, with an average interval of 30 cM, covering the 10 maize chromosomes were tested for their ability to discriminate the parental lines. Fifty four of these probes were polymorphic with 46 showing codominance. These probes were hybridized with DNA from two F 3 contrasting, bulks and three probes on chromosome 8 were found to be able distinguish the F 3 contrasting bulks on the basis of band position and intensity. DNA of families from the F 3 bulks hybridized with these probes showed the presence of heterozygous individuals. These three selected probes were also hybridized with DNA from F 2 individuals. Two of them showed a significant regression coefficient with the character. However, each of these probes explained only about 10% of the phenotypic variance observed in 70 F 2 individuals. One of the probes UMC 103 was hybridized with DNA from 168 F 4 families and the regression analysis of RFLP data showed a significant regression coefficient

Genomic diversity among Danish field strains of Mycoplasma hyosynoviae assessed by amplified fragment length polymorphism analysis

DEFF Research Database (Denmark)

Kokotovic, Branko; Friis, Niels F.; Nielsen, Elisabeth O.

2002-01-01

Genomic diversity among strains of Mycoplasma hyosynoviae isolated in Denmark was assessed by using amplified fragment length polymorphism (AFLP) analysis. Ninety-six strains, obtained from different specimens and geographical locations during 30 years and the type strain of M. hyosynoviae S16(T......) were concurrently examined for variance in BglII-MfeI and EcoRI-Csp6I-A AFLP markers. A total of 56 different genomic fingerprints having an overall similarity between 77 and 96% were detected. No correlation between AFLP variability and period of isolation or anatomical site of isolation could...

Diversidad genética intra e inter-específica de ñame (Dioscorea spp. de la región Caribe de Colombia mediante marcadores AFLP

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Hernando Javier Rivera-Jiménez

2011-12-01

Full Text Available Conocer la variabilidad genética del ñame, Dioscorea spp., permite apoyar estrategias de mejoramiento y conservación de este recurso fitogenético. El objetivo de este estudio fue la caracterización molecular de 20 accesiones de Dioscorea spp. mediante la técnica molecular de AFLP para determinar cómo se distribuye la variabilidad genética de manera intra e inter-específica. Los datos fueron analizados mediante los métodos de agrupación de correspondencia múltiple y análisis de similaridad de Dice, estableciendo los niveles de confiabilidad de los grupos genéticos mediante remuestreos. En términos de diversidad interespecífica, los valores promedios de similitud variaron entre 41.81% entre D. alata L. y D. rotundata Poir., y 33.51% entre D. trifida L.f. y D. esculenta (Lour. Burkill, lo que sugiere alta diversidad genética entre las accesiones estudiadas, que formaron cuatro grupos genéticos: D. alata, D. rotundata, D. esculenta y D. trifida, confirmando correspondencia entre la caracterización morfológica, clasificación botánica y la caracterización molecular. En términos de diversidad intraespecífica para la especie D. alata, el análisis también reveló una composición heterogénea en la región Caribe colombiana. Estos estudios ayudarán a definir una estrategia adecuada para fines de conservación y apoyar los esfuerzos futuros en los programas de mejoramiento genético.

Relation of polymorphism C1236T and C3435T in ABCB1 gene with bone marrow suppression in chemotherapy-treated breast cancer patients

Science.gov (United States)

Syarifah, S.; Hamdi, T.; Widyawati, T.; Sari, M. I.; Anggraini, D. R.

2018-03-01

ABCB1 is agene that encoded P-glycoprotein (P-gp), a transmembrane active efflux pump for a variety of carcinogens and cytostatics.ABCB1 polymorphisms C1236T and C3435T contribute to the variability oftherapeutic outcome and side effects.The present study was conducted to investigatethe relation of C1236T and C3435T polymorphisms in ABCB1 gene with bone marrow suppression in breast cancer patients treated withchemotherapy72 Indonesian womens isolated DNA sampleswere amplified using the PCR method. The analysis process of ABCB1 C1236T and C3435T polymorphism was by using thePCR-RFLP method. The frequencies of ABCB1 C1236T genotype for homozygous CC,heterozygous CT and variant TT was 11(15.28%), 42(58.33%), 19(26.39%), respectively. No associationwas between ABCB1 C1236T and C3435T polymorphisms in both individually and haplotypes with bone marrow suppression event (p > 0.05). There was no specific deviation of allele and genotype frequency from Hardy-Weinberg Equilibrium. There was a linkage between heterozygous CT-heterozygous CT in position 1236 and 3435 within 25 people (35%).

Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing--a continuous attempt at species and serovar differentiation.

Science.gov (United States)

Moreno, Luisa Z; Miraglia, Fabiana; Lilenbaum, Walter; Neto, José S F; Freitas, Julio C; Morais, Zenaide M; Hartskeerl, Rudy A; da Costa, Barbara L P; Vasconcellos, Silvio A; Moreno, Andrea M

2016-03-09

Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing.

Molecular detection of interleukin-1A +4845 G→T gene in aggresive periodontitis patients

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Chiquita Prahasanti

2012-12-01

Full Text Available Background: Abundant researches had been conducted based on the clinical and histopathological pathogenesis of aggresive periodontitis. Nevertheless, there were still few researches which based on molecular biology, and especially related to gene polymorphism. This study was done based on IL-1A +4845G→T gene polymorphism in aggressive periodontitis patients. Purpose: The purpose of this tudy was to characterized the generic variation of IL-1A +4845G→T as a risk factor aggressive periodontitis and chronic periodontitis. Methods: DNA from patients with aggressive periodontitis and chronic periodontitis was taken determination of IL-1A +4845 G→T polimorphism was conducted with PCR-RFLP technique. Results: Homozygous allele TT polymorphism was not found in all samples, only allele GG (wild type and allele GT (heterozygous mutant were not affect aggressive periodontitis and chronic periodontitis. Conclusion: The study showed there was no significant association between IL-1A +4845G→T gene polymorphism and aggressive periodontitis and chronic periodontitis. Latar belakang: Penelitian tentang patogenesa periodontitis agresif berdasar klinis dan histopatologi telah banyak dilakukan, akan tetapi penelitian berdasar biologimolekuler terutama polimorfisme gen masih sangat jarang dilakukan. Penelitian ini dilakukan berdasarkan pada polimorfisme gen IL-1A +4845G→T pada penderita periodontitis agresif. Tujuan: Tujuan dari penelitian ini adalah untuk mengetahui variasi genetik dari IL-1A +4845G→T yang merupakan faktor risiko periodontitis agresif dan periodontitis kronis. Metode: DNA dari penderita periodontitis agresif dan periodontitis kronis diisolasi, selanjutnya dilakukan determinasi dari polimorfisme gen IL-1A +4845G→T dengan menggunakan teknik PCR-RFLP. Hasil: Pada seluruh sampel penelitian ini tidak dijumpai polimorfisme allel TT (homosigot mutan, yang didapat adalah jenis allel GG (wild type dan allel GT (heterosigot mutan yang tidak

Diversity analysis of Bemisia tabaci biotypes: RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region

OpenAIRE

Rabello, Aline R.; Queiroz, Paulo R.; Simões, Kenya C.C.; Hiragi, Cássia O.; Lima, Luzia H.C.; Oliveira, Maria Regina V.; Mehta, Angela

2008-01-01

The Bemisia tabaci complex is formed by approximately 41 biotypes, two of which (B and BR) occur in Brazil. In this work we aimed at obtaining genetic markers to assess the genetic diversity of the different biotypes. In order to do that we analyzed Bemisia tabaci biotypes B, BR, Q and Cassava using molecular techniques including RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region. The analyses revealed a high similarity between the individuals of the B and Q biotypes, which could be distin...

Diversidad genética intra e inter-específica de ñame (Dioscorea spp. de la región Caribe de Colombia mediante marcadores AFLP

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Alvarez Soto Andres

2011-12-01

Full Text Available Conocer la variabilidad genética del ñame, Dioscorea spp., permite apoyar estrategias de mejoramiento y conservación de este recurso fitogenético. El objetivo de este estudio fue la caracterización molecular de 20 accesiones de Dioscorea spp. mediante la técnica molecular de AFLP para determinar cómo se distribuye la variabilidad genética de manera intra e inter-específica. Los datos fueron analizados mediante los métodos de agrupación de correspondencia múltiple y análisis de similaridad de Dice, estableciendo los niveles de confiabilidad de los grupos genéticos mediante remuestreos. En términos de diversidad interespecífica, los valores promedios de similitud variaron entre 41.81% entre D. alata L. y D. rotundata Poir., y 33.51% entre D. trifida L.f. y D. esculenta (Lour. Burkill, lo que sugiere alta diversidad genética entre las accesiones estudiadas, que formaron cuatro grupos genéticos: D. alata, D. rotundata, D. esculenta y D. trifida, confirmando correspondencia entre la caracterización morfológica, clasificación botánica y la caracterización molecular. En términos de diversidad intraespecífica para la especie D. alata, el análisis también reveló una composición heterogénea en la región Caribe colombiana. Estos estudios ayudarán a definir una estrategia adecuada para fines de conservación y apoyar los esfuerzos futuros en los programas de mejoramiento genético.

Preliminary assessment of AFLP fingerprinting of Rubus glaucus Benth. elite genotypes

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Duarte Delgado Diana

2011-04-01

Full Text Available

The Andean blackberry (Rubus glaucus Benth. is a promissory fruit crop for Colombia with potential to become an international commodity due to its high nutritional and nutraceutical value. Farmer genotypes from the national R. glaucus collection were selected from eight outstanding accessions according to their nutritional and agronomic value, for distribution among local producers. The goal of this work is to evaluate the genomic fingerprint by AFLP analysis of these elite genotypes using three primer combinations. From 179 total amplified loci produced by the three combinations, 20% resulted polymorphic. The EAGG/MCTT combination was the most informative with a 32% polymorphism and greater discrimination power. The genotypes tested showed a high average similarity (96% and the accessions San Antonio and ILS-1863 formed independent groups with good statistical support in the clustering analysis. The remaining accessions did not form discrete groups with good support (<50%, probably due to genetic homogeneity among them and/or low resolving power of markers. This study is one of the first attempts to generate a genomic fingerprint of these farmer elite genotypes for protection, seed certification and future support to breeding programs.

The effect of antimicrobial photodynamic therapy on the expression of novel methicillin resistance markers determined using cDNA-AFLP approach in Staphylococcus aureus.

Science.gov (United States)

Hoorijani, Mohammad Neshvan; Rostami, Hosein; Pourhajibagher, Maryam; Chiniforush, Nasim; Heidari, Mansour; Pourakbari, Babak; Kazemian, Hossein; Davari, Kambiz; Amini, Vahid; Raoofian, Reza; Bahador, Abbas

2017-09-01

Widespread methicillin resistant Staphylococcus aureus (MRSA) and absence of effective antimicrobial agents has led to limited therapeutic options for treating MRSA infection. We aimed to evaluate the effect of antimicrobial photodynamic therapy (aPDT) on the expression of novel identified methicillin resistance markers (NIMRMs) in S. aureus using complementary DNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) approaches to address the therapeutic alternatives for MRSA infections. We used cDNA-AFLP to compare MRSA and methicillin susceptible S. aureus (MSSA) for identification of target genes implicated in methicillin resistance. To determine the sub-lethal aPDT (sPDT), MRSA and MSSA clinical isolates photosensitized with toluidine blue O (TBO), and then were irradiated with diode laser. After sPDT, the colony forming units/mL was quantified. Antimicrobial susceptibility against methicillin was assessed for cell-surviving aPDT. Effects of sPDT on the expression of NIMRMs were evaluated by real-time quantitative reverse transcription PCR. According to our results, serine hydrolase family protein (Shfp) encoding gene and a gene encoding a conserved hypothetical protein (Chp) were implicated in methicillin resistance in MRSA. sPDT reduced the minimum inhibitory concentrations of methicillin by 3-fold in MRSA. sPDT could lead to about 10- and 6.2- fold suppression of expression of the Chp and Shfp encoding genes, respectively. sPDT would lead to reduction in resistance to methicillin of MRSA in surviving cells by suppressing the expression of the Shfp and Chp encoding genes associated with methicillin resistance. This may have potential implications of aPDT for the treatment of MRSA infections. Copyright © 2017 Elsevier B.V. All rights reserved.

Increased pulmonary vascular permeability as a cause of re-expansion edema in rabbits

International Nuclear Information System (INIS)

Pavlin, D.J.; Nessly, M.L.; Cheney, F.W.

1981-01-01

In order to study the mechanism(s) underlying re-expansion edema, we measured the concentration of labeled albumin (RISA) in the extravascular, extracellular water (EVECW) of the lung as a measure of pulmonary vascular permeability. Re-expansion edema was first induced by rapid re-expansion of rabbit lungs that had been collapsed for 1 wk by pneumothorax. The RISA in EVECW was expressed as a fraction of its plasma concentration: (RISA)L/(RISA)PL. The volume of EVECW (ml/gm dry lung) was measured using a 24 Na indicator. Results in re-expansion edema were compared with normal control lungs and with oleic acid edema as a model of permeability edema. In re-expanded lungs, EVECW (3.41 +/- SD 1.24 ml/g) and (RISA)L/(RISA)PL 0.84 +/- SD 0.15) were significantly increased when compared with normal control lungs (2.25 +/- 0.41 ml/g and 0.51 +/- 0.20, respectively). Results in oleic acid edema (5.66 +/- 2.23 ml/g and 0.84 +/- 0.23) were similar to re-expansion edema. This suggested that re-expansion edema is due to increased pulmonary vascular permeability caused by mechanical stresses applied to the lung during re-expansion

A combined approach of DNA probe and RFLP for family and species identification of larval stages of commercially important aquatic species: A study on the surfclam Spisula solidissima

Digital Repository Service at National Institute of Oceanography (India)

Achuthankutty, C.T.

fragment length polymorphism (RFLP) analysis. An oilgonucleotide sequence designed from the 18S ribosomal RNA gene (nucleotide position 259-276) provided a sensitive probe for the Family Mactridae, to which S. solidissima belongs. DNA of unknown larvae...

Rapid detection of SNP (c.309T>G in the MDM2 gene by the Duplex SmartAmp method.

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Yasuaki Enokida

Full Text Available BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

Women even the odds. Healthcare isn't an "old boys' network" anymore, as women catch up to men in leadership positions.

Science.gov (United States)

Szot, Alison

2005-04-18

Women are increasingly taking influential jobs in healthcare, whether it's Carolyn Clancy at the AHRQ helping lead the way on improving quality through IT or Risa Lavizzo-Mourey helping dole out millions of dollars in grants at the Robert Wood Johnson Foundation. Both won spots on Modern Healthcare's inaugural list of the Top 25 Women in Healthcare.

Delimitation of Umbelopsis (Mucorales, Umbelopsidaceae fam. nov.) based on ITS sequence and RFLP data.

Science.gov (United States)

Meyer, Wieland; Gams, Walter

2003-03-01

In a continuation of studies started by de Ruiter et al. (1993), all known species of the Mortierella isabellina-group (Micromucor/Umbelopsis clade of O'Donnell et al. 2001) and a few other Mucorales and species of Mortierella were investigated by RFLP (including ITS1, 5.8S, ITS2 and the 5' end of the large subunit rDNA gene) and ITS1 sequence analyses. This monophyletic group is unrelated to Mortierella and is only distantly related to the core group of the Mucoraceae. M. longicollis falls outside the Umbelopsis clade. Molecular data resolved two subclades within the M. isabellina-group; however, they are not correlated with any differences in sporangial wall and shape, spore pigmentation and shape, or sporangiophore branching. Therefore we subsume all taxa in one genus, Umbelopsis. The new family Umbelopsidaceae and the new combinations U. isabellina, U. ramanniana, and U. autotrophica are proposed.

Improved primer sequences for the mitochondrial ND1, ND3/4 and ND5/6 segments in salmonid fishes : application to RFLP analysis of Atlantic salmon

DEFF Research Database (Denmark)

Eg Nielsen, Einar; Hansen, Michael Møller; Mensberg, Karen-Lise Dons

1998-01-01

New specific primers for the mtDNA segments ND1, ND3/4 and ND5/6 designed from the rainbow trout sequence, improved PCR amplification for salmonid fishes. RFLP analysis revealed restriction site variation for all three segments in Atlantic salmon. Eleven haplotypes were detected in a screening...

Seropositivity of Toxoplasma gondii in domestic donkeys (Equus asinus) and isolation of T. gondii from farm cats.

Science.gov (United States)

Dubey, J P; Ness, S L; Kwok, O C H; Choudhary, S; Mittel, L D; Divers, T J

2014-01-17

Donkeys (Equus asinus) are used as both companion and working animals throughout the world and in some countries, their meat and milk are used for human consumption. Here we report the first serological survey of Toxoplasma gondii in donkeys in the United States. Serum samples from 373 donkeys from eight farms in five states were tested for T. gondii antibodies by the modified agglutination test (MAT). Twenty-four of 373 (6.4%) of donkeys were seropositive, with MAT titers ranging from 25 to ≥ 200. All seropositive donkeys were Miniature breed. Seropositivity prevalence was 7.0% in female donkeys (20/282) and 4.1% in male donkeys (4/91). No donkeys less than 24 months of age (129) were seropositive, suggesting postnatal transmission of infection. Domestic cats were present on six of the eight farms. Three cats from one farm had MAT titers of 200. Viable T. gondii was isolated from the hearts of two cats, but not from brain tissues. Genotyping of isolate DNA extracted from culture-derived tachyzoites using 10 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico loci) revealed that both isolates were clonal Type II (ToxoDB PCR-RFLP genotype #1). This is the first serological survey for T. gondii in donkeys in the United States, and suggests that donkey milk and meat should be considered as a potential source for human infection. The role of barn cats in the transmission of T. gondii to donkeys on farms warrents further investigation. Copyright © 2013 Elsevier B.V. All rights reserved.

Development, distribution and application of DNA markers for cereal research

International Nuclear Information System (INIS)

Qi, X.; Stephenson, P.; Devos, K.M.; Gale, M.D.

2001-01-01

DNA probes and primers are important resources for molecular genetic research and molecular breeding. Presently, more than 2500 wheat probes, 400 barley probes, 800 foxtail, pearl millet and finger millet probes, and approximately 150 wheat microsatellite (SSR) primer pairs have been developed and maintained in our DNA Resource Centre at the John Innes Centre (JIC). To accelerate probe and primer distribution, an 'anchor set' and a 'supplementary anchor set', containing 73 and 31 wheat RFLP probes, respectively, and a standard set of 42 primer pairs for wheat SSR markers were selected. Similarly, a set of 52 pearl millet probes has been selected for distribution. More than 8000 wheat RFLP probes, 2000 wheat SSR primer pairs, 700 millet probes and 200 barley probes have been distributed to more than 250 research groups in 40 countries. Our wheat and millet probes and other grass cDNA probes have been used for comparative genetic studies. The revealed conservation of gene content and gene order has been used to construct maps of many grass species and to predict the locations of key genes from one crop species to another. Developed SSR and AFLP markers in wheat, barley and millet are particularly suited for genetic diversity analyses and map construction. (author)

Detection and discrimination of two Brucella species by multiplex real-time PCR and high-resolution melt analysis curve from human blood and comparison of results using RFLP

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Vahhab Piranfar

2015-09-01

Conclusion: The present study showed that this technique, which scans gene segments and creates an analysis pattern for detection of clinical samples, is useful and more dominant compared with PCR-RFLP. Thus, this method can be used for brucellosis detection, and clinical and epidemiological research.

Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP.

Science.gov (United States)

Cirovic, Natasa; Kecmanovic, Miljana; Keckarevic, Dusan; Keckarevic Markovic, Milica

2017-10-01

Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana (C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene, responsible for THC production, and only one active copy of the gene, distinctive for marijuana, is capable of producing THC in concentration more then 0,3% in dried plants, usually punishable by the law. Twenty different samples of marijuana that contain THC in concentration more then 0,3% and three varieties of industrial hemp were analyzed for presence of an active copy of THCA synthase gene using in-house developed restriction fragment length polymorphism (RFLP) method All twenty samples of marijuana were positive for the active copy of THCA synthase gene, 16 of them heterozygous. All three varieties of industrial hemp were homozygous for inactive copy. An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of producing THC in concentrations higher than 0.3%. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines

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Asing; Ali, Md. Eaqub; Abd Hamid, Sharifah Bee; Hossain, M. A. Motalib; Mustafa, Shuhaimi; Kader, Md. Abdul; Zaidul, I. S. M.

2016-01-01

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition. PMID:27716792

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

Science.gov (United States)

Asing; Ali, Md Eaqub; Abd Hamid, Sharifah Bee; Hossain, M A Motalib; Mustafa, Shuhaimi; Kader, Md Abdul; Zaidul, I S M

2016-01-01

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

Vitamin D Receptor Gene Polymorphisms Influence T1D Susceptibility among Pakistanis

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Maryam Mukhtar

2017-01-01

Full Text Available Background. The vitamin D receptor (VDR gene regulates insulin secretion from the pancreas and acts as a mediator of the immune response through vitamin D. Polymorphism in VDR causes alterations in the functioning of vitamin D, leading to type 1 diabetes (T1D predisposition. The aim of the present study was to determine VDR gene polymorphism in association with T1D in Pakistanis. Methods. The association was evaluated by selecting rs2228570 (FokΙ, rs7975232 (ApaΙ, and rs731236 (TaqΙ polymorphic sites in 102 patients and 100 controls. Genotypes were identified by DNA sequencing and PCR-RFLP. Results. The allelic and genotypic frequencies of FokΙ and ApaI were significantly associated with T1D (p0.05. CCGC, CCGG, CCTC, and CCTG haplotypes were significantly associated with disease development (p<0.05. However, CTGG haplotype was protective towards T1D (p<0.01. Conclusion. VDR polymorphisms were identified as susceptible regions for T1D development in the Pakistani population.

DETECTING PRESENCE OF C/T POLYMORPHISM AT POSITION 34 SECOND INTRON OF THE MYOSTATIN GENE IN RABBITS

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Agnieszka MARKOWSKA

2011-01-01

Full Text Available Myostatin gene is a negative regulator of skeletal muscles growth. It is responsible for normal development of skeletal muscles. The objective of the research was to detect variation of C/T at position 34 of the second intron of the MNST gene in rabbits. The research included 114 rabbits: 54 of them Polish Rabbits, and 60 of them White Flemish Giants, examined by means of the PCR-RFLP method using AluI restriction enzyme. We found allele C with a frequency of 0.6184 of the examined rabbit population, and allele T with a frequency of 0.3816 of the examined rabbits.

Clinical characteristics and persistence of bovine mastitis caused by different species of coagulase-negative staphylococci identified with API or AFLP

DEFF Research Database (Denmark)

Taponen, S.; Simojoki, H.; Haveri, M.

2006-01-01

The coagulase-negative staphylococcal species causing mastitis in lactating cattle were identified and possible differences in the clinical characteristics or persistence of mastitis caused by different CNS were evaluated. The effect of antimicrobial treatment was also assessed. In addition, AFLP...... of these species. Approximately half of the mastitis cases were clinical, and in the majority clinical signs were mild. The severity and persistence of intramammary infection were unaffected by CNS species. Fifty-nine percent of the quarter cases were treated with antimicrobials, and the rest were left without...... treatment. Mastitis due to P-lactamase-negative CNS was treated with penicillin G and that due to beta-lactamase-positive CNS with cloxacillin. Nineteen percent of the isolates were P-lactamase-positive. The bacterial cure rate for quarters treated with antimicrobials was high, 85.9%, as opposed to only 45...

Distribution and differential diagnosis of Entamoeba Histolytica from Entamoeba Dispar by the PCR-RFLP method in central Iran

International Nuclear Information System (INIS)

Hooshyar, Hossein; Rezian, Mostafa; Kazemi, Bahram

2003-01-01

Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable human protozoan parasites that are genetically distinct species. The potential invasive pathogenic Entamoeba histolytica and non-invasive parasites Entamoeba dispar can be differentiated by molecular and other methods. We used Polymer Chain Reaction (PCR) to determine the ratio of two species in a population in central Iran.Human stool samples(n=12 148) were randomly collected in Tehran and Karaj and examined for E.histolytica/E.dispar cysts with direct and formalin-ether methods.Eighty -seven (0.7%) cases were positive, of which 49 (62.8%) isolates were successfully cultured in Robison's medium. A pair of oligonucleotide primers designed from sequence data for genomic DNA coding the 30-KD surface antigen of E.histolytica/E.dipar was used to amplify a 374 base-pair (bp) fragment. The restriction fragment length polymorphism (RFLP) pattern obtained from a standard E.histolytica isolate had had two fragments (219 bp and 155bp), but the standard isolate of E. disparshowed three fragments (155, 152 and 67bp ). Differential diagonosis of 49 isolates of E. histolytica/ E. dispar from Tehran and Karaj using PCR-RFLP revealed that 46(93.9%) were E. were E. dispar while only 2(4.1%) were E. histolytica .One person (2%) had a mixed infection and showed both patterns The differential diagonosis of the potentially pathogenic parasite E.histolytica from the non-pathogenic E. dispar is of clinical and epidemiological importance. This study demonstrated E. dispar is much more prevalent than E.histolytica among the c yst passersin Tehran and Kraj in Central Iran. (author)

cDNA-AFLP analysis of differential gene expression related to cell chemotactic and encystment of Azospirillum brasilense.

Science.gov (United States)

Li, Huamin; Cui, Yanhua; Wu, Lixian; Tu, Ran; Chen, Sanfeng

2011-12-20

Our previous study indicated org35 was involved in chemotaxis and interacted with nitrogen fixation transcriptional activator NifA via PAS domain. In order to reveal the role of org35 in nitrogen regulation, the downstream target genes of org35 were identified. We here report differentially expressed genes in org35 mutants comparing with wild type Sp7 by means of cDNA-AFLP. Four up-regulated transcript-derived fragments (TDFs) homologues of chemotaxis transduction proteins were found, including CheW, methyl-accepting chemotaxis protein and response regulator CheY-like receiver. Three distinct TDFs (AB46, AB58 and AB63) were similar to PHB de-polymerase C-terminus, cell shape-determining protein and flagellin domain protein. And 11 TDFs showed similarities with signal transduction proteins, including homologous protein of the nitrogen regulation protein NtrY and nitrate/nitrite response regulator protein NarL. These data suggested that the Azospirillum brasilense org35 was a multi-effecter and involved in chemotaxis, cyst development and regulation of nitrogen fixation. Copyright © 2010 Elsevier GmbH. All rights reserved.

Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece

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Kizis, Dimosthenis; Natskoulis, Pantelis; Nychas, George-John E.; Panagou, Efstathios Z.

2014-01-01

A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS). Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP), four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immuno Sorbent Assay (ELISA), resulting in significant interspecies differences in OTA production. PMID:24710283

Molecular identification of Indian crocodile species: PCR-RFLP method for forensic authentication*.

Science.gov (United States)

Meganathan, P R; Dubey, Bhawna; Haque, Ikramul

2009-09-01

South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile-based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA-based identification of species using PCR-RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species-specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.

Differentiation of strains from the Bacillus cereus group by RFLP-PFGE genomic fingerprinting.

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Otlewska, Anna; Oltuszak-Walczak, Elzbieta; Walczak, Piotr

2013-11-01

Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus belong to the B. cereus group. The last three species are characterized by different phenotype features and pathogenicity spectrum, but it has been shown that these species are genetically closely related. The macrorestriction analysis of the genomic DNA with the NotI enzyme was used to generate polymorphism of restriction profiles for 39 food-borne isolates (B. cereus, B. mycoides) and seven reference strains (B. mycoides, B. thuringiensis, B. weihenstephanensis, and B. cereus). The PFGE method was applied to differentiate the examined strains of the B. cereus group. On the basis of the unweighted pair group method with the arithmetic mean method and Dice coefficient, the strains were divided into five clusters (types A-E), and the most numerous group was group A (25 strains). A total of 21 distinct pulsotypes were observed. The RFLP-PFGE analysis was successfully used for the differentiation and characterization of B. cereus and B. mycoides strains isolated from different food products. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular Fingerprints of the Human Fecal Microbiota From 9 to 18 Months Old and the Effect of Fish Oil Supplementation

DEFF Research Database (Denmark)

Andersen, Anders Daniel; Mølbak, Lars; Michaelsen, Kim Fleischer

2011-01-01

supplementation with 5mL of fish oil (FO) or sunflower oil (SO) from 9 to 18 months of age, stool samples were collected from 132 healthy Danish infants. Molecular fingerprints of the bacterial DNA were obtained by terminal restriction fragment length polymorphism (T-RFLP). Results: The T-RFLP profiles indicated...

Genetic maps of Saccharum officinarum L. and Saccharum robustum Brandes & Jew. ex grassl

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Claudia T. Guimarães

1999-03-01

Full Text Available Genetic analysis was performed in a population composed of 100 F1 individuals derived from a cross between a cultivated sugarcane (S. officinarum `LA Purple' and its proposed progenitor species (S. robustum `Mol 5829'. Various types (arbitrarily primed-PCR, RFLPs, and AFLPs of single-dose DNA markers (SDMs were used to construct genetic linkage maps for both species. The LA Purple map was composed of 341 SDMs, spanning 74 linkage groups and 1,881 cM, while the Mol 5829 map contained 301 SDMs, spanning 65 linkage groups and 1,189 cM. Transmission genetics in these two species showed incomplete polysomy based on the detection of 15% of SDMs linked in repulsion in LA Purple and 13% of these in Mol 5829. Because of this incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. Due to inclusion of RFLP anchor probes, conserved in related species, the resulting maps will serve as useful tools for breeding, ecology, evolution, and molecular biology studies within the Andropogoneae.Uma progênie de 100 indivíduos F1 obtidos de um cruzamento entre cana-de-açúcar (S. officinarum `LA Purple' e seu suposto progenitor (S. robustum `Mol 5829' foi analisada utilizando marcadores moleculares em dose única. Marcadores do tipo AP-PCR, RFLP e AFLP, gerando um total de 642 polimorfismos, foram mapeados em ambas espécies. O mapa genético de LA Purple foi composto de 341 marcadores, distribuídos em 74 grupos de ligação e 1.881 cM, enquanto que o mapa de ligação de Mol 5829 continha 301 marcadores ao longo de 65 grupos de ligação e 1.189 cM. A transmissão genética nessas duas espécies apresentou polissomia incompleta devido a detecção de 15% dos marcadores em dose simples ligados em fase de repulsão e 13% desses em Mol 5829. Devido a essa polissomia incompleta, os marcadores em dose múltipla não puderam ser mapeados por falta de um modelo genético para descrever tal segregação. O

Similaridade genética entre cultivares de cebola de diferentes tipos e origens, baseada em marcadores AFLP Genetic similarity among onion cultivars of different types and origins, based on AFLP markers

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CAF Santos

2011-03-01

Full Text Available Foi estimada a similaridade genética entre cultivares de cebola de diferentes tipos e regiões geográficas, de forma a orientar programas de recursos genéticos e melhoramento da espécie no Nordeste brasileiro. Foram analisadas 41 cultivares, adotando-se para a visualização da similaridade genética o fenograma UPGMA gerado da matriz de distâncias genéticas estimadas pelo coeficiente de Jaccard e baseadas em 146 bandas polimórficas de Pst1 e Mse1 de AFLP. A correlação cofenética foi de 0,91, indicando boa confiabilidade da representação gráfica para a interpretação dos resultados. Foram observados dois grupos principais no fenograma, no ponto de corte de 0,55 de similaridade: 1 grupo formado por cultivares predominantemente brasileiras, com algumas inclusões de cultivares estrangeiras; e 2 grupo formado por três cultivares estrangeiras (Mercedes, Perfect e TEG 502 PRR. Rijnsburger Jumbo e IPA 8 apresentaram a maior similaridade (85%, enquanto Madrugada foi a mais divergente em relação às demais cultivares. As cultivares da série IPA se dividiram em subgrupos no grupo das cultivares brasileiras (IPA 8, IPA 10 e IPA 11; IPA 12, IPA 7, IPA 2 e IPA 6; IPA 3, IPA 4 e IPA 9, indicando haver variabilidade genética a ser explorada entre aquelas situadas em subgrupos distintos. Bola Precoce e BRS Cascata apresentaram a maior similaridade entre as cultivares de origem brasileira. Foi observada similaridade superior a 39%, refletindo a alta variabilidade genética da coleção de cebola estudada. A introdução de novos acessos deve considerar procedências outras que não norte americanas, para aumentar a variabilidade de germoplasma de cebola disponível no Nordeste do Brasil.The genetic similarity among onion cultivars of different origins was evaluated, in order to carry out genetic resources and breeding programs for this species on the Brazilian Northeast. Forty-one onion cultivars were analyzed for 146 polymorphic Pst1/Mse1

Karakterisasi Molekuler Ikan Gurami Soang (Osphronemus goramy Lac. berbeda uuran menggunakan Pcr-Rflp Gen Sitokrom C Oksidase 1

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Siti Nur Azizah

2017-01-01

Full Text Available Gourami (Osprhronemus goramy Lac. is a  freshwater fish that is  widely cultivated in Indonesia, particularly by fish farmer in Java. Soang strain has a faster growth rate than other strains. However, the fry that derived from a single cohort (generated from a single spawning shows various sizes even in the same age because they have different growth rate. Differences in growth rate may occur due to differences in metabolic capabilities especially cellular respiration. Different rate of respiration can be assumed to be due to differences in the genetic component, especially on their cytochrome c oxidase 1 gene (CO1. Therefore this study aimed to determine whether there are differences in PCR-RFLP marker CO1 gene among different sizes of gourami soang strain from the same cohort so that can be used to analyze genetic diversity. This study used survey method by applying purposive random sampling. Four restriction enzymes were used  during the  research. Molecular characters were defined descriptively based on the appearance of restriction fragment on agarose gel. The result showed that soang strain which used in this study had monomorphics allele on their CO1-HindIII. PstI, BamHI, and EcoRI, could not cut the PCR products and results no RFLP markers. So that genetic variation in the used CO1 gene fragment could not be detected using HindIII, Pst1, BamH1 and EcoR1 enzymes. The three different sizes of soang strain had similar alleles or homozygous, meaning that heterozigocity value was 0 (zero. Therefore, the CO1 gene in this study could not be used as molecular character to differentiate different sizes of gourami soang strain from the same cohort.

Principal Component Analysis of Microbial Community Data from an Accelerated Decay Cellar Test

Science.gov (United States)

Grant T. Kirker; Patricia K. Lebow

2014-01-01

Analysis of microbial communities is a valuable tool for characterization and identification of microbes in a myriad of environments. We are currently using the molecular method terminal restriction fragment length polymorphism (T-RFLP) analysis to characterize changes in bacterial and fungal communities on treated and untreated wood in soil. T-RFLP uses fluorescently...

Comparison of Enzymatic Method Rapid Yeast Plus System with RFLP-PCR for Identification of Isolated Yeast from Vulvovaginal Candidiasis.

Science.gov (United States)

Hossein, Moallaei; Mirhendi, Seied Hossein; Brandão, João; Mirdashti, Reza; Rosado, Laura

2011-09-01

To compare two identification methods, i.e., restriction fragment length polymorphism (RFLP)-PCR analysis and enzymatic method Rapid TM Yeast Plus System to identify different species causing vulvovaginal candidiasis (VVC). Vaginal discharges of women who had attended the gynecology outpatient clinic of Mobini Hospital in Sabzevar, Iran were collected using cotton swabs and were cultured on Sabouraud dextrose agar. Isolated yeasts were identified by germ-tube testing and Rapid TM Yeast Plus System (Remel USA). For molecular identification, the isolated DNA was amplified with ITS1 and ITS4 universal primers and PCR products digested with the enzyme HpaІІ followed by agarose gel electrophoresis. Epidemiological and clinical features of women with respect to identified species were also evaluated. Out of 231 subjects enrolled, 62 VVC cases were detected. The isolated species were identified as follows: Candida albicans, 24 (38.7%), C. glabrata, 15 (24.2%), C. kefyr, 13 (21.0%) C. krusei, 9 (14.5%), and Saccharomyces cerevisiae, 1 (1.6%) by RFLP-PCR method; whereas findings by Rapid TM Yeast Plus System were C. albicans, 24 (38.7%), C. glabrata, 5 (8%), C. kefyr, 11 (17.7%) C. krusei, 2 (3.2%), S. cerevisiae, 9 (14.5%), and C. tropicalis, 6 (9.6%) as well as other nonpathogenic yeasts, 4 (6.9%). Statistical comparison showed that there is no significant difference in identification of C. albicans by the two methods; although, in this study, it was not true about other species of yeasts. A correlation between clinical and laboratory findings is important as it enables us to administer an appropriate treatment on time.

Effect of lifestyle factors on plasma total homocysteine concentrations in relation to MTHFR(C677T) genotype. Inter99 (7)

DEFF Research Database (Denmark)

Husemoen, L L N; Thomsen, T F; Fenger, M

2004-01-01

a Fluorescent Polarization Immuno Assay. MTHFR-genotype was determined by PCR and RFLP analysis. Information about lifestyle factors was obtained from a self-administered questionnaire. RESULTS: Daily smoking, less healthy dietary habits, and coffee drinking were associated with elevated tHcy concentrations......OBJECTIVE: To examine the associations between various lifestyle factors--smoking habits, physical activity, dietary habits, coffee, tea, and alcohol consumption--and homocysteine (tHcy) in relation to MTHFR(C677T) genotype. DESIGN: Cross-sectional population-based study. SETTING: Residents....... The effect of smoking was more pronounced in persons with the TT genotype and in women. The effect of beer consumption was more pronounced at younger than at older ages. CONCLUSIONS: Smoking status, dietary habits, coffee intake, wine, and beer consumption were major lifestyle determinants of tHcy. Changes...

Differentiation between Aspergillus flavus and Aspergillus parasiticus from Pure Culture and Aflatoxin-Contaminated Grapes Using PCR-RFLP Analysis of aflR-aflJ Intergenic Spacer

International Nuclear Information System (INIS)

El Khoury, A.; Atoui, A.; Lebrihi, A.; Rizk, T.; Lteif, R.; Kallassy, M.

2011-01-01

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification. (author)

Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species

OpenAIRE

ŞAKALAR, Çağrı; KUK, Salih; ERENSOY, Ahmet; DAĞLI, Adile Ferda; ÖZERCAN, İbrahim Hanifi

2015-01-01

To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. Materials and methods: DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial...

Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities.

Science.gov (United States)

Blackwood, Christopher B; Hudleston, Deborah; Zak, Donald R; Buyer, Jeffrey S

2007-08-01

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E(var)) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E(var)). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.

Bovine Papillomavirus in Brazil: Detection of Coinfection of Unusual Types by a PCR-RFLP Method

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R. F. Carvalho

2013-01-01

Full Text Available Bovine papillomavirus (BPV is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil.

Genotyping of the Holstein-Friesian crossbred cattle for CD18 gene using PCR-RFLP

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A. S. Khade

2014-05-01

Full Text Available Aim: The present study was undertaken in Holstein-Friesian (HF crossbred cattle with the objective to find out genotype of HF crossbred cattle for Bovine Leucocyte Adhesion Deficiency (BLAD by using PCR-RFLP. Materials and Methods: 50 blood samples were collected from HF crossbred cattle and subjected to PCR. The amplified PCR products were digested using Taq I restriction enzyme at 65 oC overnight. After restriction digestion, the final PCR products were electrophoresed on 2.5 % agarose gel. Results: All the 50 animals under present investigation were found to be normal as the amplified PCR product upon digestion with Taq I restriction enzyme, revealed two bands of 313 bp and 54 bp for normal animals. Conclusions: In the present investigation D128G carrier frequency was found to be 0 %. However, recent reports suggest that the mutant gene has already been observed in the HF crossbred cattle population of India, which makes it necessary to screen the animals to avoid the risk of spreading BLAD in the breeding cattle population.

AFLP analysis of genetic diversity in leafy kale (Brassica oleracea L. convar. acephala (DC.) Alef.) landraces, cultivars and wild populations in Europe

DEFF Research Database (Denmark)

Christensen, Stina; von Bothmer, Roland; Poulsen, G.

2011-01-01

AFLP markers were used to characterize diversity and asses the genetic structure among 17 accessions of kale landraces, cultivars and wild populations from Europe. The range of average gene diversity in accessions was 0.11–0.27. Several landraces showed higher levels of diversity than the wild...... populations and one cultivar had the lowest diversity measures. The landraces that were most genetically diverse were from areas where kales are known to be extensively grown, suggesting in situ conservation in these areas as a supplement to storage of seeds in gene banks. An analysis of molecular variance...... the relationship among them is due to seed dispersal through human interactions. Our results indicate that a kale population found in a natural habitat in Denmark was probably not truly wild but most likely an escape from a cultivated Danish kale that had subsequently become naturalized....

Molecular analysis of point mutations in a barley genome exposed to MNU and gamma rays

Energy Technology Data Exchange (ETDEWEB)

Kurowska, Marzena, E-mail: mkurowsk@us.edu.pl [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland); Labocha-Pawlowska, Anna; Gnizda, Dominika; Maluszynski, Miroslaw; Szarejko, Iwona [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland)

2012-10-15

We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M{sub 2} populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117 bp in the MNU population, the average mutation density was estimated as 1/504 kb. Only one nucleotide change was found after an analysis of 3,207,444 bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G > A and C > T substitutions. The similar share of G > A and C > T transitions indicates a lack of bias in the repair of O{sup 6}-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O{sup 6}-meG at the -1 position. Purines formed 81% of nucleotides observed at the -1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNU-treated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome.

Universal LD50 predictions using deep learning

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NICEATM Predictive Models for Acute Oral Systemic Toxicity LD50 entry Risa R. Sayre (sayre.risa@epa.gov) & Christopher M. Grulke Our approach uses an ensemble of multilayer perceptron regressions to predict rat acute oral LD50 values from chemical features. Features were genera...

SC*994C>T causes the Sc(null) phenotype in Pacific Islanders and successful transfusion of Sc3+ blood to a patient with anti-Sc3.

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Reid, Marion E; Hue-Roye, Kim; Velliquette, Randall W; Larimore, Kathleen; Moscarelli, Sue; Ohswaldt, Nicolas; Lomas-Francis, Christine

2013-01-01

Antigens in the SC blood group system are expressed by the human erythrocyte membrane-associated protein (ERMAP).Two molecular bases have been reported for the Sc,un phenotype:SC*307del2 and SC*994C>T. We report our investigation of the molecular background of five Sc,n1 individuals from the Pacific Islands and describe the successful transfusion of Sc3+ blood to a patient with anti-Sc3 in her plasma. SC (ERMAP) exons 2,3, and 12 and their flanking intronic regions were analyzed. TheSC*994C>T change introduces a restriction enzyme cleavage site for Tsp45I, and polymerase chain reaction (PCR) products from exon 12 were subjected to this PCR-restriction fragment length polymorphism (RFLP) assay. The five samples had the variant SC*994T/T. One sample, from a first cousin of one Marshallese proband, was heterozygous for SC*1514C/T (in the 3' untranslated region); the other four samples were SC*1514C/C(consensus sequence). Samples from white donors (n = 100) and African American donors (n = 99) were tested using the Tsp45IPCR-RFLP assay; all gave a banding pattern that was consistent with the SC*994C/C consensus sequence. In all five samples,our analyses showed homozygosity for the nonsense nucleotide change SC*994C>Tin an allele carrying the nucleotide associated with SLd. Further investigation determined that one of the probands reported previously with the SC*994C>T change was from the Marshall Islands (which form part of the Micronesian Pacific Islands) and the other was from an unspecified location within the large collection of Pacific Islands. Taken together, the five known probands with the SC*994C>T silencing nucleotide change were from the Pacific Islands.

AFLP-Based Analysis of Genetic Diversity, Population Structure, and Relationships with Agronomic Traits in Rice Germplasm from North Region of Iran and World Core Germplasm Set.

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Sorkheh, Karim; Masaeli, Mohammad; Chaleshtori, Maryam Hosseini; Adugna, Asfaw; Ercisli, Sezai

2016-04-01

Analysis of the genetic diversity and population structure of crops is very important for use in breeding programs and for genetic resources conservation. We analyzed the genetic diversity and population structure of 47 rice genotypes from diverse origins using amplified fragment length polymorphism (AFLP) markers and morphological characters. The 47 genotypes, which were composed of four populations: Iranian native varieties, Iranian improved varieties, International Rice Research Institute (IRRI) rice varieties, and world rice collections, were analyzed using ten primer combinations. A total of 221 scorable bands were produced with an average of 22.1 alleles per pair of primers, of which 120 (54.30%) were polymorphic. The polymorphism information content (PIC) values varied from 0.32 to 0.41 with an average of 0.35. The high percentage of polymorphic bands (%PB) was found to be 64.71 and the resolving power (R p) collections were 63.36. UPGMA clustering based on numerical data from AFLP patterns clustered all 47 genotypes into three large groups. The genetic similarity between individuals ranged from 0.54 to 0.94 with an average of 0.74. Population genetic tree showed that Iranian native cultivars formed far distant cluster from the other populations, which may indicate that these varieties had minimal genetic change over time. Analysis of molecular variance (AMOVA) revealed that the largest proportion of the variation (84%) to be within populations showing the inbreeding nature of rice. Therefore, Iranian native varieties (landraces) may have unique genes, which can be used for future breeding programs and there is a need to conserve this unique diversity. Furthermore, crossing of Iranian genotypes with the genetically distant genotypes in the other three populations may result in useful combinations, which can be used as varieties and/or lines for future rice breeding programs.

Effect of lifestyle factors on plasma total homocysteine concentrations in relation to MTHFR(C677T) genotype. Inter99 (7)

DEFF Research Database (Denmark)

Husemoen, L L N; Thomsen, T F; Fenger, M

2004-01-01

a Fluorescent Polarization Immuno Assay. MTHFR-genotype was determined by PCR and RFLP analysis. Information about lifestyle factors was obtained from a self-administered questionnaire. RESULTS: Daily smoking, less healthy dietary habits, and coffee drinking were associated with elevated tHcy concentrations...... independent of other determinants. Wine consumption was related to tHcy in a J-shaped manner, whereas beer consumption was negatively associated with tHcy after multiple adjustments. Interaction was observed between smoking status and MTHFR-genotype, smoking status and sex, and beer consumption and age....... The effect of smoking was more pronounced in persons with the TT genotype and in women. The effect of beer consumption was more pronounced at younger than at older ages. CONCLUSIONS: Smoking status, dietary habits, coffee intake, wine, and beer consumption were major lifestyle determinants of tHcy. Changes...

Application of PCR – RFLP markers for identification of genetically delimited groups of the Calypogeia fissa complex (Jungermanniopsida, Calypogeiaceae

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Buczkowska Katarzyna

2015-06-01

Full Text Available Currently, two subspecies are formally recognized within Calypogeia fissa: C. fissa subsp. fissa occurring in Europe and C. fissa subsp. neogea known from North America. Genetic studies have revealed a complex structure of this species. Within the European part of distribution, three genetically distinct groups PS, PB and G are distinguished. The combination of the SCAR marker Cal04 and PCR-RFLP markers with three restriction enzymes (SmaI, TaqI and TspGWI allowed the recognition of all groups within the C. fissa complex. The TaqI enzyme recognizing the restriction sites in the PCR product of SCAR marker Ca104 turned out to be the best marker

A Genetic Linkage Map of Mycosphaerella Fijiensis, using SSR and DArT Markers

Science.gov (United States)

Mycosphaerella fijiensis is the causal agent of black leaf streak or Black Sigatoka disease in bananas. This pathogen threatens global banana production as the main export Cavendish cultivars are highly susceptible. Previously a genetic linkage map was generated predominantly using anonymous AFLP ma...

Genetic diversity and structure of Brazilian ginger germplasm (Zingiber officinale) revealed by AFLP markers.

Science.gov (United States)

Blanco, Eleonora Zambrano; Bajay, Miklos Maximiliano; Siqueira, Marcos Vinícius Bohrer Monteiro; Zucchi, Maria Imaculada; Pinheiro, José Baldin

2016-12-01

Ginger is a vegetable with medicinal and culinary properties widely cultivated in the Southern and Southeastern Brazil. The knowledge of ginger species' genetic variability is essential to direct correctly future studies of conservation and genetic improvement, but in Brazil, little is known about this species' genetic variability. In this study, we analyzed the genetic diversity and structure of 55 Brazilian accessions and 6 Colombian accessions of ginger, using AFLP (Amplified Fragment Length Polymorphism) molecular markers. The molecular characterization was based on 13 primers combinations, which generated an average of 113.5 polymorphic loci. The genetic diversity estimates of Nei (Hj), Shannon-Weiner index (I) and an effective number of alleles (n e ) were greater in the Colombian accessions in relation to the Brazilian accessions. The analysis of molecular variance showed that most of the genetic variation occurred between the two countries while in the Brazilian populations there is no genetic structure and probably each region harbors 100 % of genetic variation found in the samples. The bayesian model-based clustering and the dendrogram using the dissimilarity's coefficient of Jaccard were congruent with each other and showed that the Brazilian accessions are highly similar between themselves, regardless of the geographic region of origin. We suggested that the exploration of the interspecific variability and the introduction of new varieties of Z.officinale are viable alternatives for generating diversity in breeding programs in Brazil. The introduction of new genetic materials will certainly contribute to a higher genetic basis of such crop.

Molecular Identification of Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) Markers.

Science.gov (United States)

Al-Khalifah, Nasser S; Shanavaskhan, A E

2017-01-01

Ambiguity in the total number of date palm cultivars across the world is pointing toward the necessity for an enumerative study using standard morphological and molecular markers. Among molecular markers, DNA markers are more suitable and ubiquitous to most applications. They are highly polymorphic in nature, frequently occurring in genomes, easy to access, and highly reproducible. Various molecular markers such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) markers have been successfully used as efficient tools for analysis of genetic variation in date palm. This chapter explains a stepwise protocol for extracting total genomic DNA from date palm leaves. A user-friendly protocol for RAPD analysis and a table showing the primers used in different molecular techniques that produce polymorphisms in date palm are also provided.

Prevalence of renal lesions in slaughtered cattle in Shiraz, Iran, and detection of Leptospira in them by nested PCR-RFLP.

Science.gov (United States)

Taghadosi, Vahideh; Hosseinzadeh, Saeid; Shekarforoush, Seyed Shahram; Samiei, Azadeh

2016-12-01

Renal diseases in cattle are frequently not recognized due to the subclinical conditions. Some species of Leptospira are the main cause of infectious agents that damage the kidneys and lead to abortion and economic losses in cattle and are also of major concern in the public health. This study was aimed to assess the prevalence of renal lesions of slaughtered cattle in the Shiraz abattoir and to determine the correlation between rejected kidneys and infection with Leptospira using nested PCR-restriction fragment length polymorphism (RFLP) techniques. Out of 1000 inspected animals, 205 (20.5 %) revealed the renal lesions. Chronic nephritis (7.5 %), white-spotted kidney (7.3 %), and petechial hemorrhage (3.5 %) were the most prevalent forms of the lesions. A direct correlation between increasing the age and significant increase in the rate of lesions was also observed (P = 0.03). Using nested PCR-RFLP assay, 40.8 % of the tested kidneys were turned to be infected to the pathogenic species of Leptospira. The risk of infection of the kidneys with white spot to pathogenic species of Leptospira (53.8 %) was more than that of the kidneys with other lesions (25.0 %) (P = 0.014). The odd ratio indicates that the kidneys with white spot lesions are likely to be infected with pathogenic species of Leptospira, five times greater than other lesions. This study showed that renal lesions especially white-spotted kidney, which were considerably associated with Leptospira in slaughtered cattle in Shiraz, were very high. This is important in terms of public health and in particular, increases the risk of transmission of disease to human specially in the high-risk careers including farmers, veterinarians, and abattoir workers.

Genetic diversity of populations and clones of Rhopilema esculentum in China based on AFLP analysis

Science.gov (United States)

Qiao, Hongjin; Liu, Xiangquan; Zhang, Xijia; Jiang, Haibin; Wang, Jiying; Zhang, Limin

2013-03-01

Amplified fragment length polymorphisms (AFLP) markers were developed to assess the genetic variation of populations and clones of Rhopilema esculentum Kishinouye (Scyphozoa, Rhizostomatidae). One hundred and seventy-nine loci from 56 individuals of two hatchery populations and two wild populations were genotyped with five primer combinations. The polymorphic ratio, Shannon's diversity index and average heterozygosity were 70.3%, 0.346 and 0.228 for the white hatchery population, 74.3%, 0.313, and 0.201 for the red hatchery population, 79.3%, 0.349, and 0.224 for the Jiangsu wild population, and 74.9%, 0.328 and 0.210 for the Penglai wild population, respectively. Thus, all populations had a relatively high level of genetic diversity. A specific band was identified that could separate the white from the red hatchery population. There was 84.85% genetic differentiation within populations. Individual cluster analysis using unweighted pair-group method with arithmetic mean (UPGMA) suggested that hatchery populations and wild populations could be divided. For the hatchery populations, the white and red populations clustered separately; however, for the wild populations, Penglai and Jiangsu populations clustered together. The genetic diversity at the clone level was also determined. Our data suggest that there are relatively high genetic diversities within populations but low genetic differentiation between populations, which may be related to the long-term use of germplasm resources from Jiangsu Province for artificial seeding and releasing. These findings will benefit the artificial seeding and conservation of the germplasm resources.

Comparative molecular analysis of Herbaspirillum strains by RAPD, RFLP, and 16S rDNA sequencing

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Soares-Ramos Juliana R.L.

2003-01-01

Full Text Available Herbaspirillum spp. are endophytic diazotrophic bacteria associated with important agricultural crops. In this work, we analyzed six strains of H. seropedicae (Z78, M2, ZA69, ZA95, Z152, and Z67 and one strain of H. rubrisubalbicans (M4 by restriction fragment length polymorphism (RFLP using HindIII or DraI restriction endonucleases, random amplified polymorphic DNA (RAPD, and partial sequencing of 16S rDNA. The results of these analyses ascribed the strains studied to three distinct groups: group I, consisting of M2 and M4; group II, of ZA69; and group III, of ZA95, Z78, Z67, and Z152. RAPD fingerprinting showed a higher variability than the other methods, and each strain had a unique electrophoretic pattern with five of the six primers used. Interestingly, H. seropedicae M2 was found by all analyses to be genetically very close to H. rubrisubalbicans M4. Our results show that RAPD can distinguish between all Herbaspirillum strains tested.

ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and Strains from Cases of Human Cutaneous Leishmaniasis in States of the Mexican Southeast

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Amalia Monroy-Ostria

2014-01-01

Full Text Available American cutaneous leishmaniasis includes a spectrum of clinical forms localized cutaneous, diffuse cutaneous, and mucocutaneous leishmaniasis which can be caused by different strains of Leishmania belonging to the L. mexicana or L. braziliensis complexes which may coexist in the same endemic area. We evaluated the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 163 clinical samples and 21 Mexican isolates of Leishmania. In relation to the Mexican isolates of Leishmania 52% displayed a pattern similar to the L. (L. mexicana, 5% showed a mixed pattern compatible with L. (L. mexicana and L. (V. braziliensis, eight with L. (L. amazonensis and L. (L. mexicana, and one to L. (V. braziliensis. Most of the clinical samples, 109/116 (94%, gave a pattern similar to that of the L. mexicana, two clinical samples gave similar patterns to that of Leishmania braziliensis, and 5 samples gave patterns that suggest a coinfection of L. (L. mexicana and L. (V. braziliensis or L. (L. mexicana and L. (L. amazonensis. The ITS1 PCR-RFLP assay is a multipurpose tool for diagnosis of Leishmania from clinical samples and enables determination of the infecting species of New World Leishmania in the field in relatively short time and low cost.

Use of AFLPS to distinguish landraces of pejibaye (Bactris gasipaes in brazilian Amazonia Uso de AFLPS para discriminar raças primitivas de pupunha (Bactris gasipaes na Amazônia brasileira

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Charles R. Clement

2002-12-01

Full Text Available Although the first inhabitants of western Amazonia domesticated pejibaye (Bactris gasipaes Kunth, Palmae or peach palm for its fruits, today it is widely planted for its heart-of-palm. Like other domesticates, pejibaye presents a complex hierarchy of landraces developed before the conquest of the Americas. The existence of three landraces (Pará, Solimões, Putumayo was proposed along the Amazonas and Solimões Rivers, Brazil, based on morphological characteristics. There are some questions remaining about the intermediate landrace being an artifact of the morphometric analysis. AFLPs were used to evaluate the relationships among samples of these putative landraces. DNA was extracted from 99 plants representing 13 populations maintained in the Pejibaye Germplasm Bank, Manaus, AM; six primer combinations generated 245 markers via PCR, which were scored in an ABI Prism 310 sequencer and analyzed with GeneScan Software; Jaccard similarities were estimated and a dendrogram was generated with UPGMA. Two groups of plants were observed in the dendrogram instead of three, and were similar at 0.795. Each group contained two subgroups, similar at 0.815. One group (n=41 contained 73% Pará landrace plants, with one subgroup (n=22 containing 91% Pará, and the other (n=19 containing 53% Pará. The other group (n=58 contained 53% Solimões and 40% Putumayo landrace plants, with one subgroup (n=21 containing 52% Solimões and 43% Putumayo, and the other (n=35 containing 57% Solimões and 37% Putumayo. The first group confirmed the Pará landrace. The second group suggested that the Solimões landrace does not exist, but that the Putumayo landrace extends along the Solimões River to Central Amazonia.Os primeiros povos da Amazônia ocidental domesticaram a pupunha (Bactris gasipaes Kunth, Palmae por seu fruto, embora hoje seja muito plantada por seu palmito. Como outros cultivos domesticados, a pupunha apresenta uma hierarquia complexa de raças primitivas

Interleukin-1β-31C/T and -511T/C polymorphisms were associated with preeclampsia in Chinese Han population.

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Xuefeng Wang

Full Text Available OBJECTIVE: The purpose of our study is to investigate the relationship between IL-1β -31C/T (rs1143627 and -511T/C (rs16944 polymorphisms and the preeclampsia (PE, and analyze the Linkage disequilibrium (LD and haplotype frequency of the two polymorphism loci. METHODS: Polymorphisms at -31C/T and -511T/C of IL-1β were genotyped with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP in 232 PE and 447 control subjects. Genotype and allele frequencies between case-control groups were compared by chi-square(X2 tests. Two-point LD and haplotype frequency analyses were done with the software Haploview4.2. RESULTS: Significant statistical differences were found between PE and control groups regarding genotype and allele frequencies of the two polymorphisms of IL-1β (For IL-1β -31C/T: X2 = 11.478, P = 0.003; For IL-1β-511T/C: X2 = 9.687, P = 0.008. LD analysis revealed that the IL-1β -31C/T SNP was in high LD with the IL-1β-511C/T SNP(D' = 0.92, r2 = 0.79. Both CT and TC haplotypes showed significant differences between case and control groups. Only the plasma level of Prothrombin Time had a significantly statistical difference among TT, CT and CC groups of the preeclamptic two polymorphisms of IL-1β-31C/T and -511T/C (for IL-1β-31C/T, F = 1.644, P = 0.01; F = 1.587, P = 0.016. CONCLUSION: Our results revealed IL-1β was associated with the PE in Chinese Han population. The CT haplotype may increase the risk of PE, while haplotype TC could be considered as a protective haplotype of PE.

Gene expression analysis by cDNA-AFLP highlights a set of new signaling networks and translational control during seed dormancy breaking in Nicotiana plumbaginifolia.

Science.gov (United States)

Bove, Jérôme; Lucas, Philippe; Godin, Béatrice; Ogé, Laurent; Jullien, Marc; Grappin, Philippe

2005-03-01

Seed dormancy in Nicotiana plumbaginifolia is characterized by an abscisic acid accumulation linked to a pronounced germination delay. Dormancy can be released by 1 year after-ripening treatment. Using a cDNA-amplified fragment length polymorphism (cDNA-AFLP) approach we compared the gene expression patterns of dormant and after-ripened seeds, air-dry or during one day imbibition and analyzed 15,000 cDNA fragments. Among them 1020 were found to be differentially regulated by dormancy. Of 412 sequenced cDNA fragments, 83 were assigned to a known function by search similarities to public databases. The functional categories of the identified dormancy maintenance and breaking responsive genes, give evidence that after-ripening turns in the air-dry seed to a new developmental program that modulates, at the RNA level, components of translational control, signaling networks, transcriptional control and regulated proteolysis.

A specific PCR-assay for resistance to Biotypes 1 and 2 of the rosy leaf curling aphid in apple based on an RFLP marker closely linked to the Sd1 gene

NARCIS (Netherlands)

Roche, P.; Arkel, van G.; Heusden, van A.W.

1997-01-01

This report describes the conversion of a restriction fragment length polymorphism (RFLP) marker (the 2B12a locus), linked to the Sd1 aphid resistance gene, to a polymerase chain reaction (PCR) based marker. A section of the 2B12 probe was sequenced and two primers were designed to amplify this

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico.

Science.gov (United States)

Erwin, Patrick M; Olson, Julie B; Thacker, Robert W

2011-01-01

Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP), might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods. We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6%) of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil). The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the ambient

LD2SNPing: linkage disequilibrium plotter and RFLP enzyme mining for tag SNPs

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Cheng Yu-Huei

2009-06-01

Full Text Available Abstract Background Linkage disequilibrium (LD mapping is commonly used to evaluate markers for genome-wide association studies. Most types of LD software focus strictly on LD analysis and visualization, but lack supporting services for genotyping. Results We developed a freeware called LD2SNPing, which provides a complete package of mining tools for genotyping and LD analysis environments. The software provides SNP ID- and gene-centric online retrievals for SNP information and tag SNP selection from dbSNP/NCBI and HapMap, respectively. Restriction fragment length polymorphism (RFLP enzyme information for SNP genotype is available to all SNP IDs and tag SNPs. Single and multiple SNP inputs are possible in order to perform LD analysis by online retrieval from HapMap and NCBI. An LD statistics section provides D, D', r2, δQ, ρ, and the P values of the Hardy-Weinberg Equilibrium for each SNP marker, and Chi-square and likelihood-ratio tests for the pair-wise association of two SNPs in LD calculation. Finally, 2D and 3D plots, as well as plain-text output of the results, can be selected. Conclusion LD2SNPing thus provides a novel visualization environment for multiple SNP input, which facilitates SNP association studies. The software, user manual, and tutorial are freely available at http://bio.kuas.edu.tw/LD2NPing.

COMPARATIVE STUDIES OF THREE METHODS FOR MEASURING PEPSIN ACTIVITY

Science.gov (United States)

Loken, Merle K.; Terrill, Kathleen D.; Marvin, James F.; Mosser, Donn G.

1958-01-01

Comparison has been made of a simple method originated by Absolon and modified in our laboratories for assay of proteolytic activity using RISA (radioactive iodinated serum albumin—Abbott Laboratories), with the commonly used photometric methods of Anson and Kunitz. In this method, pepsin was incubated with an albumin substrate containing RISA, followed by precipitation of the undigested substrate with trichloroacetic acid and measurement of radioactive digestion products in the supernatant fluid. The I131—albumin bond was shown in the present studies to be altered only by the proteolytic activity, and not by the incubation procedures at various values of pH. Any free iodine present originally in the RISA was removed by a single passage through a resin column (amberlite IRA-400-C1). Pepsin was shown to be most stable in solution at a pH of 5.5. Activity of pepsin was shown to be maximal when it was incubated with albumin at a pH of 2.5. Pepsin activity was shown to be altered in the presence of various electrolytes. Pepsin activity measured by the RISA and Anson methods as a function of concentration or of time of incubation indicated that these two methods are in good agreement and are equally sensitive. Consistently smaller standard errors were obtained by the RISA method of pepsin assay than were obtained with either of the other methods. PMID:13587910

MTHFR Gene C677T Polymorphism in Autism Spectrum Disorders

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Elif Funda Sener

2014-01-01

Full Text Available Aim. Autism is a subgroup of autism spectrum disorders, classified as a heterogeneous neurodevelopmental disorder and symptoms occur in the first three years of life. The etiology of autism is largely unknown, but it has been accepted that genetic and environmental factors may both be responsible for the disease. Recent studies have revealed that the genes involved in the folate/homocysteine pathway may be risk factors for autistic children. In particular, C677T polymorphism in the MTHFR gene as a possible risk factor for autism is still controversial. We aimed to investigate the possible effect of C677T polymorphism in a Turkish cohort. Methods. Autism patients were diagnosed by child psychiatrists according to DSM-IV and DSM-V criteria. A total of 98 children diagnosed as autistic and 70 age and sex-matched children who are nonautistic were tested for C677T polymorphism. This polymorphism was studied by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP methods. Results. MTHFR 677T-allele frequency was found to be higher in autistic children compared with nonautistic children (29% versus 24%, but it was not found statistically significant. Conclusions. We conclude that other MTHFR polymorphisms such as A1298C or other folate/homocysteine pathway genes may be studied to show their possible role in autism.

AFLP analysis of Genetic Diversity Among Different Jatropha curcas L. Genotypes from Africa and Ecuador

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Konan, NO.

2018-01-01

Full Text Available Six populations amounting to a total number of seventy genotypes of Jatropha curcas L. originating from Africa (Senegal, Mali, Burkina Faso and Madagascar and Ecuador were investigated for genetic diversity using two AFLP primer combinations. The results revealed a high genetic diversity in the populations studied. The population with greatest genetic diversity was Madagascar (He = 0.2638 and I = 0.4066 and the least diverse was Senegal-Tamba (He = 0.1962 and I = 0.3079. AMOVA (analysis of molecular variance detected the highest proportion of variation within populations (81% of the total molecular variation. This may be attributed to the high level of allogamy observed in this species. The Nei's standard unbiased genetic distance (D between the populations ranged from 0.010 (Senegal-Tamba and Burkina Faso to 0.131 (Mali and Ecuador; the average was 0.063. Analysis of the genetic relationships among the 6 populations using both neighbor-joining cluster analysis and principal component analysis (PCoA showed five clusters with globally, groupings of i most of Burkina Faso and Senegal-Tamba genotypes, ii most of Mali and Senegal-Diobass genotypes , iii most of Madagascar and Ecuador genotypes, and iv some mixings of genotypes with different origins. Considering the distance existing between the different origins there are prospects to develop F1 hybrids. The greatest heterosis might be expected from crossing involving genotypes of cluster I and cluster V which group the more distant genotypes. Such crossing schemes might produce greater success in the production of genetic variability and might maximize the exploitation of heterosis and segregation.

MTHFR C667T polymorphism association with male infertility risk in pakistan

International Nuclear Information System (INIS)

Naz, M.; Siddiqui, R.T.

2012-01-01

The methylenetetrahydrofolate reductase (MTHFR) gene is one of the main regulatory enzymes involved in folate metabolism, DNA synthesis and remethylation reactions. The objective of this study was to analyze the distribution of the MTHFR C677T variants using PCR-Restriction Fragment Length Polymorphism (RFLP) in a cross sectional study consisting of 160 infertile men including azoospermia, oligospermia, severe-oligospermia and normospermia infertile subjects compared to 90 ancestry-matched fertile normozoospermic controls. The genotype CT of C677T was highly significant frequency in controls and all infertility groups (28.75%; OR 1.983; 95% CI 1.117-3.522; P 0.012; chi-square 6.262) while TT homozygous variant is present statistically significant frequency in controls and azoospermic subjects and severe-oligozoospermic subjects with (P 0.065; chi-square 3.406 in azoosprimic) and ( P 0.071; chi-square 3.267 in severe-oligospermic). The prevalence of C677T genotypes TT between azoospermic and severeoligozoospermic patients and controls was almost similar 6.67% and 7.4% respectively but high as compared to controls (1.11%). In conclusion, this analysis supports that the MTHFR C677T polymorphism acts as a genetic mutation risk factor and is capable of causing male infertility susceptibility in Pakistani population. (author)

Incorporation of conventional genetic markers and RAPD markers into an RFLP based map in maize

International Nuclear Information System (INIS)

Coe, E.H. Jr.; McMullen, M.D.; Polacco, M.; Davis, G.L.; Chao, S.

1998-01-01

Integration of classical genetic markers, in particular mutants, onto the maize Restriction Fragment Length Polymorphism (RFLP) map will provide the tools necessary to further our understanding of plant development and of complex traits. Initially integration was accomplished by visual alignment of common markers and sometimes involved the use of information from several different molecular maps to determine the relative placement of a single mutant. The maize core marker set was designed to provide a common set of markers which could be used for integration of map data. We have completed the mapping, of 56 mutants on chromosome one relative to the core marker set. Phenotypes included whole plant, seedling, and kernel effects and represented a variety of biological processes. Since these mutants were previously located to chromosome arm, mapping required the use of only seven markers per mutant to define the correct bin location. Two mistakes in marker order relative to the classical map were identified, as well as, six groups of mutants which require allelism testing. Placement of mutants and cDNAs into bins using, the core markers provides a necessary resource for identification of gene function in maize. (author)

Discrimination of the Thai rejuvenating herbs Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna collettii (Black Kwao Khruea) using PCR-RFLP.

Science.gov (United States)

Wiriyakarun, Suchaya; Yodpetch, Woraluk; Komatsu, Katsuko; Zhu, Shu; Ruangrungsi, Nijsiri; Sukrong, Suchada

2013-07-01

The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name "Kwao Khruea" is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating "Kwao Khruea" species.

Assessment of genetic diversity among accessions of two traditional ...

African Journals Online (AJOL)

Assessment of genetic diversity among accessions of two traditional leafy vegetables (Acmella uliginosa (L.) and Justicia tenella (Nees) T.) consumed in Benin using amplified fragment length polymorphism (AFLP) markers. K Adéoti, A Rival, A Dansi, BS Ahohuendo, S Santoni, T Beule, A Nato, Y Henry, A Ahanchédé, ...

Alpha-synuclein A53T mutation is not frequent on a sample of Brazilian Parkinson’s disease patients

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Gabriela S. Longo

2015-06-01

Full Text Available Introduction The pathogenesis of Parkinson’s disease (PD involves both genetic susceptibility and environmental factors, with focus on the mutation in the alpha-synuclein gene (SNCA.Objective To analyse the polymorphism SNCA-A53T in patients with familial PD (FPD and sporadic PD (SPD.Method A total of 294 individuals were studied, regardless of sex and with mixed ethnicity. The study group with 154 patients with PD, and the control group included 140 individuals without PD. The genotyping of SNCA-A53T was performed by PCR/RFLP. Significance level was p < 0.05.Results Among all patients, 37 (24% had FPD and 117 (75.9% had SPD. The absence of SNCA-A53T mutation was observed in all individuals.Conclusion SPD is notably observed in patients. However, the SNCA-A53T mutation was absent in all individuals, which does not differ controls from patients. This fact should be confirmed in a Brazilian study case with a more numerous and older population.

Towards an optimal sampling strategy for assessing genetic variation within and among white clover (Trifolium repens L. cultivars using AFLP

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Khosro Mehdi Khanlou

2011-01-01

Full Text Available Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He and Shannon diversity index (I were greatly underestimated, whereas with 20, more than 95% of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation.

Genotypic frequency of Caveolin-1 (CAV1 T29107A polymorphism in the Iranian patients with breast cancer

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Ahmad Hamta

2016-09-01

Full Text Available Introduction: Caveolin-1 (Cav-1 is a scaffolding protein found in special structures of plasma membrane, known as Caveolae. Cav-1 can regulate many intracellular processes, including signal transmission and cholesterol metabolism. This protein plays an important role in the growth and differentiation of breast tissue and acts as a tumor suppressor gene as well. The aim of this study was to determine the genotypic frequency of Cav-1 T29107A (rs7804372 polymorphism and its association with susceptibility to breast cancer among the female population in Kermanshah, Iran. Methods: A total of 120 patients with breast cancer and an equal number of non-cancer individuals (control group, matched for age and gender with the patients, were selected in this study. The paraffin tissues of the patients from 2006 to 2013 were collected from Imam Reza hospital, Kermanshah, and 2.5 cc blood sample was taken from non-cancer individuals. The genomic DNA was extracted from paraffin tissues and blood by salting out method. The genotype of samples was determined by RFLP-PCR method, and Sau3A1 enzyme was used for RFLP analysis. Results: The distribution of Cav-1 T29107A genotype was found to be significantly different between breast cancer patients and control group (p=0.004. Among the patients, 84 (70% samples had genotype TT, 29 (24.78% genotype AT and 7 (5.83% genotype AA. As for the control group, however, 59 (49.17% samples had genotype TT, 49 (40.83% genotype AT and 12 (10% genotype AA. Conclusion: The results of this study showed that genotype TT is associated with an increased risk of susceptibility to breast cancer.

Comparison of different volume markers in peritoneal dialysis

International Nuclear Information System (INIS)

De Paepe, M.; Belpaire, F.; Schelstraete, K.; Lameire, N.

1988-01-01

Four peritoneal volume markers (carbon 14 -labeled dextran, dextran blue, radioactive albumin, and hemoglobin) were compared. In six rabbits 14 C-dextran was compared with dextran blue during a 4-hour dwell with a 4.25% dextrose solution. The recovery of 14 C-dextran at the end of the dwell was 71% +/- 3% vs. 92% +/- 1% for dextran blue (P less than 0.001). In six other rabbits, radioactive albumin (RISA) was compared with dextran blue. The recovery of RISA was 78% +/- 4%, compared with 85% +/- 2% for dextran blue (P less than 0.05). The calculated peritoneal volumes, uncorrected for disappearance of the markers, were consistently higher than when correction was made. After correction, the calculated end volumes were similar to actually measured end volumes. In six patients with chronic ambulatory peritoneal dialysis, the intraperitoneal volume during a single dwell of 6 hours was estimated in paired observations with lactated Ringer's solution and 1.5% dextrose dialysate, using simultaneously autologous hemoglobin and RISA. In eight additional patients, a single dwell with 4.25% dextrose dialysate was studied. The recoveries of both markers were related to the osmotic strength of the dialysate. Recoveries were 66.7% +/- 2.3% and 69.6% +/- 0.9% in lactated Ringer's solution, and increased to 81% +/- 3% and 82% +/- 2% in 4.25% dextrose for hemoglobin and RISA, respectively. With each dialysate, after correction for disappearance of the marker, no differences in volume profiles or between calculated or measured end volumes could be found with either hemoglobin or RISA

Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

Science.gov (United States)

Wood, S; Starr, T V; Shukin, R J

1986-01-01

We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome. Images Fig. 1 PMID:2879441

Comparison of bacteroides-prevotella 16S rRNA genetic markers for fecal samples from different animal species.

Science.gov (United States)

Fogarty, Lisa R; Voytek, Mary A

2005-10-01

To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.

Genetic Polymorphisms in the EGFR (R521K and Estrogen Receptor (T594T Genes, EGFR and ErbB-2 Protein Expression, and Breast Cancer Risk in Tunisia

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Imen Kallel

2009-01-01

Full Text Available We evaluated the association of epidermal growth factor receptor (EGFR 142285G>A (R521K and estrogen receptor alpha (ESR1 2014G>A (T594T single nucleotide polymorphisms with breast cancer risk and prognosis in Tunisian patients. EGFR 142285G>A and ESR1 2014G>A were genotyped in a sample of 148 Tunisian breast cancer patients and 303 controls using PCR-RFLP method. Immunohistochemitsry was used to evaluate the expression levels of EGFR, HER2, ESR1, progesterone receptor and BCL2 in tumors. We found no evidence for an association between EGFR R521K polymorphism and breast cancer risk. However, we found that the homozygous GG (Arg genotype was more prevalent in patients with lymph node metastasis (=.03 and high grade tumors (=.011. The ESR1 2014G allele showed significant association with breast cancer risk (=.025. The GG genotype was associated with HER2 overexpression and this association withstood univariate and multivariate analyses (=.009; =.021, resp.. These data suggest that the R521K might be a prognostic factor, because it correlates with both tumor grade and nodule status. The higher expression of HER2 in ESR1 T594T GG patients suggests the possibility that ESR1 gene polymorphisms accompanied by HER2 expression might influence the pathogenesis of breast cancers.

AFLP analysis reveals a lack of phylogenetic structure within Solanum section Petota

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Vleeshouwers Vivianne GAA

2008-05-01

Full Text Available Abstract Background The secondary genepool of our modern cultivated potato (Solanum tuberosum L. consists of a large number of tuber-bearing wild Solanum species under Solanum section Petota. One of the major taxonomic problems in section Petota is that the series classification (as put forward by Hawkes is problematic and the boundaries of some series are unclear. In addition, the classification has received only partial cladistic support in all molecular studies carried out to date. The aim of the present study is to describe the structure present in section Petota. When possible, at least 5 accessions from each available species and 5 individual plants per accession (totally approx. 5000 plants were genotyped using over 200 AFLP markers. This resulted in the largest dataset ever constructed for Solanum section Petota. The data obtained are used to evaluate the 21 series hypothesis put forward by Hawkes and the 4 clade hypothesis of Spooner and co-workers. Results We constructed a NJ tree for 4929 genotypes. For the other analyses, due to practical reasons, a condensed dataset was created consisting of one representative genotype from each available accession. We show a NJ jackknife and a MP jackknife tree. A large part of both trees consists of a polytomy. Some structure is still visible in both trees, supported by jackknife values above 69. We use these branches with >69 jackknife support in the NJ jackknife tree as a basis for informal species groups. The informal species groups recognized are: Mexican diploids, Acaulia, Iopetala, Longipedicellata, polyploid Conicibaccata, diploid Conicibaccata, Circaeifolia, diploid Piurana and tetraploid Piurana. Conclusion Most of the series that Hawkes and his predecessors designated can not be accepted as natural groups, based on our study. Neither do we find proof for the 4 clades proposed by Spooner and co-workers. A few species groups have high support and their inner structure displays also

Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes psient1 and psient2 and the selu or seluv gene using PCR-RFLP.

Science.gov (United States)

Collery, Mark M; Smyth, Cyril J

2007-02-01

The egc locus of Staphylococus aureus harbours two enterotoxin genes (seg and sei) and three enterotoxin-like genes (selm, seln and selo). Between the sei and seln genes are located two pseudogenes, psient1 and psient2, or the selu or seluv gene. While these two alternative sei-seln intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between egc loci bearing the pseudogenes and the selu or seluv gene. In silico restriction enzyme digestion of genomic regions encompassing the egc locus from the 3' end of the sei gene through the 5' first quarter of the seln gene allowed pseudogene- and selu- or seluv-bearing egc loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease HindIII cleaved PCR amplimers bearing pseudogenes but not those with a selu or seluv gene, while selu- or seluv-bearing amplimers were susceptible to cleavage by endonuclease HphI, but not by endonuclease HindIII. The restriction enzyme BccI cleaved selu- or seluv-harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these egc loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an egc locus type, and can also be used for the primary identification of the intergenic sei-seln egc locus type.

Influence of nitrogen fertilization on diazotrophic communities in the rhizosphere of the Jerusalem artichoke (Helianthus tuberosus L.).

Science.gov (United States)

Meng, Xianfa; Wang, Lin; Long, Xiaohua; Liu, Zhaopu; Zhang, Zhenhua; Zed, Rengel

2012-06-01

Diazotrophs in the soil may be influenced by plant factors as well as nitrogen (N) fertilization. In this study, we investigated potential diazotrophic communities in the rhizosphere of the Jerusalem artichoke (Helianthus tuberosus L.) supplied with differing amounts of N. The community structure of N(2)-fixing bacteria was profiled using the length heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) based on a variation in the nifH gene. Higher numbers of diazotrophs were detected by T-RFLP compared to LH-PCR. The lowest number of N(2)-fixing bacteria was observed in the rhizosphere soil with high N fertilization. T-RFLP was a better method than LH-PCR for profiling microbial diversity of diazotrophs using multidimensional scaling (MDS) and analysis of similarity (ANOSIM) of fingerprints as well as diversity measures. The supply of N fertilizer appeared to negatively influence the abundance of diazotrophs in the rhizophere of the Jerusalem artichoke. Copyright © 2012 Institut Pasteur. All rights reserved.

Restriction fragment length polymorphism (RFLP) of two HLA-B-associated transcripts (BATs) genes in healthy Danes

DEFF Research Database (Denmark)

Fugger, L; Morling, N; Ryder, L P

1990-01-01

The restriction fragment length polymorphism (RFLP) of the two human HLA-B-associated transcripts (BATs) genes, BAT1 and BAT2, was investigated using 5 different restriction enzymes and two human BAT1 and BAT2 cDNA probes. Two of the enzymes, NcoI and RsaI, revealed polymorphic patterns which were...... investigated in healthy Danes. The cDNA/restriction enzyme combination BAT1/NcoI identifies polymorphic bands at 12 kb, 8 kb, 2.5 kb, and 1.1 kb, while the BAT2/RsaI combination identifies polymorphic bands at 3.3 kb, 2.7 kb, 2.3 kb, and 0.9 kb. The frequencies of these markers were determined in 90 unrelated...... Danes. Co-dominant segregation and allelic behavior was seen for the BAT1/NcoI 12 kb and 8 kb bands and the BAT2/RsaI 2.7 kb and 2.3 kb bands, respectively. It is possible that the BAT2/RsaI 3.3 kb band represents a rare allele of the BAT2/RsaI system. The BAT2/RsaI 2.3 kb marker was strongly negatively...

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico.

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Patrick M Erwin

Full Text Available Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP, might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods.We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6% of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil.The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the

Trichoderma stromaticum and its overseas relatives

Science.gov (United States)

Trichoderma stromaticum, T. rossicum and newly discovered species form a new lineage in Trichoderma. Phylogenetic and phenotypic diversity in Trichoderma stromaticum are examined in light of reported differences in ecological parameters and AFLP patterns. Multilocus phylogenetic analysis using 4 gen...

Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae

DEFF Research Database (Denmark)

Leser, Thomas D.; Lindecrona, Rikke Hvid; Jensen, Tim Kåre

2000-01-01

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with die......Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented...

Microbial community analysis of field-grown soybeans with different nodulation phenotypes.

Science.gov (United States)

Ikeda, Seishi; Rallos, Lynn Esther E; Okubo, Takashi; Eda, Shima; Inaba, Shoko; Mitsui, Hisayuki; Minamisawa, Kiwamu

2008-09-01

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.

Microbial Community Analysis of Field-Grown Soybeans with Different Nodulation Phenotypes▿

Science.gov (United States)

Ikeda, Seishi; Rallos, Lynn Esther E.; Okubo, Takashi; Eda, Shima; Inaba, Shoko; Mitsui, Hisayuki; Minamisawa, Kiwamu

2008-01-01

Microorganisms associated with the stems and roots of nonnodulated (Nod−), wild-type nodulated (Nod+), and hypernodulated (Nod++) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod+ soybean roots. Fusarium solani was stably associated with nodulated (Nod+ and Nod++) roots and less abundant in Nod− soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod−, Nod+, or Nod++). The analysis of root samples indicated that the microbial community in Nod− soybeans was more similar to that in Nod++ soybeans than to that in Nod+ soybeans. PMID:18658280

Fantasías de nubes al viento: primera ronda

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León de Greiff

1969-10-01

Full Text Available Oiga entonces, óye, oíd palabras sin sentido conocido: Las otras son tan huecas si sonoras (dice mi risa como tambor de feria (añejo símil de Perogrullo "profundas", "salomónicas" (torna a decir Mi Risa o Mi Sonrisa. -Tan profundas que no se toca fondo- (símil vetusto asaz, no nada gris, mas sí del buen Monsieur de la Palice...

Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: Isolation, RFLP marker development, and physical mapping

Directory of Open Access Journals (Sweden)

Souza Manoel T

2008-01-01

Full Text Available Abstract Background Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes. The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS and C-terminal leucine-rich repeat (LRR domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence. Results A computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs, together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities. Conclusion This is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were

Analysis of genetic diversity in pigeon pea germplasm using ...

Indian Academy of Sciences (India)

MANEESHA

2017-08-16

Aug 16, 2017 ... fied polymorphic DNA (RAPD), simple sequence repeats. (SSR), amplified fragment length polymorphism (AFLP), single-nucleotide polymorphisms (SNPs), diversity array technology (DArT), genic-simple sequence repeats (genic-. SSR) etc. (see review by Varshney et al. 2013). Since retrotransposons are ...

RFLP-facilitated investigation of the quantitative resistance of rice to brown planthopper ( Nilaparvata lugens).

Science.gov (United States)

Xu, X. F.; Mei, H. W.; Luo, L. J.; Cheng, X. N.; Li, Z. K.

2002-02-01

Quantitative trait loci (QTLs), conferring quantitative resistance to rice brown planthopper (BPH), were investigated using 160 F(11) recombinant inbred lines (RILs) from the Lemont/Teqing cross, a complete RFLP map, and replicated phenotyping of seedbox inoculation. The paternal indica parent, Teqing, was more-resistant to BPH than the maternal japonica parent, Lemont. The RILs showed transgressive segregation for resistance to BPH. Seven main-effect QTLs and many epistatic QTL pairs were identified and mapped on the 12 rice chromosomes. Collectively, the main-effect and epistatic QTLs accounted for over 70% of the total variation in damage scores. Teqing has the resistance allele at four main-effect QTLs, and the Lemont allele resulted in resistance at the other three. Of the main-effect QTLs identified, QBphr5b was mapped to the vicinity of gl1, a major gene controlling leaf and stem pubescence. The Teqing allele controlling leaf and stem pubescence was associated with resistance, while the Lemont allele for glabrous stem and leaves was associated with susceptibility, indicating that this gene may have contributed to resistance through antixenosis. Similar to the reported BPH resistance genes, the other six detected main-effect QTLs were all mapped to regions where major disease resistance genes locate, suggesting they might have contributed either to antibiosis or tolerance. Our results indicated that marker-aided pyramiding of major resistance genes and QTLs should provide effective and stable control over this devastating pest.

Leaf cDNA-AFLP analysis reveals novel mechanisms for boron-induced alleviation of aluminum-toxicity in Citrus grandis seedlings.

Science.gov (United States)

Wang, Liu-Qing; Yang, Lin-Tong; Guo, Peng; Zhou, Xin-Xing; Ye, Xin; Chen, En-Jun; Chen, Li-Song

2015-10-01

Little information is available on the molecular mechanisms of boron (B)-induced alleviation of aluminum (Al)-toxicity. 'Sour pummelo' (Citrus grandis) seedlings were irrigated for 18 weeks with nutrient solution containing different concentrations of B (2.5 or 20μM H3BO3) and Al (0 or 1.2mM AlCl3·6H2O). B alleviated Al-induced inhibition in plant growth accompanied by lower leaf Al. We used cDNA-AFLP to isolate 127 differentially expressed genes from leaves subjected to B and Al interactions. These genes were related to signal transduction, transport, cell wall modification, carbohydrate and energy metabolism, nucleic acid metabolism, amino acid and protein metabolism, lipid metabolism and stress responses. The ameliorative mechanisms of B on Al-toxicity might be related to: (a) triggering multiple signal transduction pathways; (b) improving the expression levels of genes related to transport; (c) activating genes involved in energy production; and (d) increasing amino acid accumulation and protein degradation. Also, genes involved in nucleic acid metabolism, cell wall modification and stress responses might play a role in B-induced alleviation of Al-toxicity. To conclude, our findings reveal some novel mechanisms on B-induced alleviation of Al-toxicity at the transcriptional level in C. grandis leaves. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular epidemiology of contagious bovine pleuropneumonia in Tanzania based on amplified fragment length polymorphism and pulsed-field gel electrophoresis analysis

DEFF Research Database (Denmark)

Kusiluka, L.J.M.; Ojeniyi, B.; Friis, N.F.

2001-01-01

anti vaccine strains. The strong genomic homogeneity among, M. mycoides SC strains associated with outbreaks of contagious bovine pleuropneumonia in different regions of Tanzania suggests that the outbreaks of the disease in the 1990-99 period might have been caused Ly a single epidemic clone. Moreover......The genetic diversity of 60 field strains of Mycoplasma mycoides ssp. mycoides, small colony type (M,. mycoides), comprising 56 isolates from cattle in Tanzania, one from Kenya, two from Botswana and one from Portugal, as well as the type (PG1(T)) and vaccine (T-1-SR49) strains, was ivestigated...... strains. The AFLP profiles of the type and vaccine strains were indistingiuishable from each other. Indistinguishable AFLP profiles were found for 55 Tanzanian held strains, one of them isolated in 1990 and the other 54 isolated in 1998/1999), although one strain isolated in 1999 showed a different...

Long-term boron-deficiency-responsive genes revealed by cDNA-AFLP differ between Citrus sinensis roots and leaves

Science.gov (United States)

Lu, Yi-Bin; Qi, Yi-Ping; Yang, Lin-Tong; Lee, Jinwook; Guo, Peng; Ye, Xin; Jia, Meng-Yang; Li, Mei-Li; Chen, Li-Song

2015-01-01

Seedlings of Citrus sinensis (L.) Osbeck were supplied with boron (B)-deficient (without H3BO3) or -sufficient (10 μM H3BO3) nutrient solution for 15 weeks. We identified 54 (38) and 38 (45) up (down)-regulated cDNA-AFLP bands (transcript-derived fragments, TDFs) from B-deficient leaves and roots, respectively. These TDFs were mainly involved in protein and amino acid metabolism, carbohydrate and energy metabolism, nucleic acid metabolism, cell transport, signal transduction, and stress response and defense. The majority of the differentially expressed TDFs were isolated only from B-deficient roots or leaves, only seven TDFs with the same GenBank ID were isolated from the both. In addition, ATP biosynthesis-related TDFs were induced in B-deficient roots, but unaffected in B-deficient leaves. Most of the differentially expressed TDFs associated with signal transduction and stress defense were down-regulated in roots, but up-regulated in leaves. TDFs related to protein ubiquitination and proteolysis were induced in B-deficient leaves except for one TDF, while only two down-regulated TDFs associated with ubiquitination were detected in B-deficient roots. Thus, many differences existed in long-term B-deficiency-responsive genes between roots and leaves. In conclusion, our findings provided a global picture of the differential responses occurring in B-deficient roots and leaves and revealed new insight into the different adaptive mechanisms of C. sinensis roots and leaves to B-deficiency at the transcriptional level. PMID:26284101

Identification of Ancylostoma ceylanicum in children from a tribal community in Tamil Nadu, India using a semi-nested PCR-RFLP tool.

Science.gov (United States)

George, Santosh; Kaliappan, Saravanakumar Puthupalayam; Kattula, Deepthi; Roy, Sheela; Geldhof, Peter; Kang, Gagandeep; Vercruysse, Jozef; Levecke, Bruno

2015-04-01

It is generally assumed that hookworm infections in humans are caused by Necator americanus and Ancylostoma duodenale. However, previous studies have also reported the presence of the animal hookworm A. ceylanicum in human stools. We determined hookworm infections in children in a tribal community in Tamil Nadu, India, using a semi-nested PCR-RFLP approach. The results indicate that human species account for a majority of the hookworm infections (N. americanus 39/41 [95%]; A. duodenale 6/41 [15%]), whereas the animal hookworm A. ceylanicum only accounts for a minority of the infections (5%; 2/41). The results emphasize the need to consider zoonotic ancylostomiasis while developing strategies to control hookworm infections. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Sensitive radioimmunosorbent assay for the detection of plant viruses. [Cauliflower mosaic virus, lettuce mosaic virus

Energy Technology Data Exchange (ETDEWEB)

Ghabrial, S A; Shepherd, R J [Kentucky Univ., Lexington (USA); California Univ., Davis (USA))

1980-06-01

A simple and highly sensitive radioimmunosorbent assay (RISA) for the detection of plant viruses is described. The RISA procedure is a microplate method based on the principle of 'double-antibody sandwich' and follows essentially the protocol of the enzyme-linked immunosorbent assay (ELISA) (Clark and Adams, 1977), with the exception that /sup 125/I-labelled ..gamma..-globulin is substituted for the ..gamma..-globulin enzyme conjugate; the bound /sup 125/I-..gamma..-globulin is dissociated by acidification from the double-antibody sandwich. The radioactivity is proportional to virus concentration, and cauliflower mosaic virus (CaMV) and lettuce mosaic virus (LMV) could be detected at concentrations as low as 5 and 2 ng/ml, respectively. Direct evidence of the adverse effects of conjugation with enzyme on the binding abilities of antibodies is presented. The RISA procedure should prove valuable with viruses for which the ELISA values are too low to be dependable.

A sensitive radioimmunosorbent assay for the detection of plant viruses

International Nuclear Information System (INIS)

Ghabrial, S.A.; Shepherd, R.J.

1980-01-01

A simple and highly sensitive radioimmunosorbent assay (RISA) for the detection of plant viruses is described. The RISA procedure is a microplate method based on the principle of 'double-antibody sandwich' and follows essentially the protocol of the enzyme-linked immunosorbent assay (ELISA) (Clark and Adams, 1977), with the exception that 125 I-labelled γ-globulin is substituted for the γ-globulin enzyme conjugate; the bound 125 I-γ-globulin is dissociated by acidification from the double-antibody sandwich. The radioactivity is proportional to virus concentration, and cauliflower mosaic virus (CaMV) and lettuce mosaic virus (LMV) could be detected at concentrations as low as 5 and 2 ng/ml, respectively. Direct evidence of the adverse effects of conjugation with enzyme on the binding abilities of antibodies is presented. The RISA procedure should prove valuable with viruses for which the ELISA values are too low to be dependable. (author)

Assessment of genetic diversity among accessions of two traditional ...

African Journals Online (AJOL)

user

2011-04-11

Apr 11, 2011 ... Adéoti K.1, Rival A.2, Dansi A.3*, Ahohuendo B.C4, Santoni S.5, Beule T.2, Nato ... Acmella accessions although three distinct clusters could be differentiated. ..... length polymorphism (AFLP): A review of the procedure and its.

Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay.

Science.gov (United States)

Acardi, Soraya Alejandra; Liotta, Domingo Javier; Santini, María Soledad; Romagosa, Carlo Mariano; Salomón, Oscar Daniel

2010-09-01

In this study, a genotypification of Leishmania was performed using polimerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing techniques to identify species of Leishmania parasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpis were grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantum by PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpis among the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantum in the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

Interlukin-10 gene polymorphisms (-819T/C and -1082A/G and Type 2 diabetes mellitus in North Indian population

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Sushma Verma

2016-12-01

Full Text Available Diabetes is a metabolic disorder characterized by chronic hyperglycemia and impaired cytokine levels leading to inflammation. Interlukin-10 (IL-10 is an anti-inflammatory cytokine which acts as macrophage deactivator affecting the synthesis of TNF-a, IL-1, IL-6, IL-8 and GM-CSF. Single-nucleotide polymorphisms (SNPs viz. -592A/C, -819T/C and -1082A/G in IL-10 promoter are associated with IL-10 production. Low IL-10 levels in T2DM cases may be regulated by such gene variants. The present study was undertaken to evaluate the association of two genetic polymorphisms viz. IL-10 -819T/C and -1082A/G with T2DM in a North Indian population. Blood samples from 402 subjects (201 each of controls and T2DM cases were collected after ethical approval and individual written consent. All subjects were genotyped by polymerase chain reaction-restriction length polymorphism (PCR-RFLP using specific primers and restriction enzymes. Genotypic, allelic, carriage rate frequencies were calculated and haplotypic analysis performed by SPSS (version 21.0 and SHEsis (online version. All biochemical parameters except WHR and TG showed significant association with T2DM (P [Dis Mol Med 2016; 4(4.000: 68-76

PCR and microsatellite analysis of diminazene aceturate resistance of bovine trypanosomes correlated to knowledge, attitude and practice of livestock keepers in South-Western Ethiopia.

Science.gov (United States)

Moti, Y; De Deken, R; Thys, E; Van Den Abbeele, J; Duchateau, L; Delespaux, V

2015-06-01

African Animal Trypanosomosis is threatening the agricultural production and cattle breeding more severely than any other livestock disease in the continent, even more since the advent of drug resistance. A longitudinal study was conducted from November 2012 to May 2013 in the Ghibe valley to evaluate diminazene aceturate (DA) resistance and assess livestock owner's perception of trypanocidal drug use. Four Peasant Associations (PAs) were purposively selected and the cattle randomly sampled in each PAs. At the beginning of the study (t0), 106 bovines positive for trypanosomes by the haematocrit centrifugation technique (HCT) and 119 negative control animals were recruited for six months follow-up using HCT, 18S-PCR-RFLP, DpnII-PCR-RFLP and microsatellite analysis. Prevalence of trypanosomosis was 18.1% based on the HCT technique and the mean PCV value was 23.6±5.1% for the 587 sampled cattle. Out of the 106 HCT positive, 64 (60.4%) were positive for the presence of trypanosomes using the 18S-PCR-RFLP. Species detection showed 38 (59.4%) Trypanosoma congolense savannah, 18 (28.1%) Trypanosoma vivax, 5 (7.8%) Trypanosoma theileri and 3 (4.7%) T. congolense Kilifi. Among the T. congolense savannah samples, 31 (81.6%) showed a DA resistant RFLP profile, 2 (5.3%) a mixed profile and 5 did not amplify using the DpnII-PCR-RFLP. A positive HCT had a significant effect on PCV (pmessages should be delivered to promote a rational drug use, improved livestock management and the application of strategic vector control methods. Copyright © 2015 Elsevier B.V. All rights reserved.

[Recent advances of amplified fragment length polymorphism and its applications in forensic botany].

Science.gov (United States)

Li, Cheng-Tao; Li, Li

2008-10-01

Amplified fragment length polymorphism (AFLP) is a new molecular marker to detect genomic polymorphism. This new technology has advantages of high resolution, good stability, and reproducibility. Great achievements have been derived in recent years in AFLP related technologies with several AFLP expanded methodologies available. AFLP technology has been widely used in the fields of plant, animal, and microbes. It has become one of the hotspots in Forensic Botany. This review focuses on the recent advances of AFLP and its applications in forensic biology.

SPO11-C631T Gene Polymorphism: Association With Male Infertility and an in Silico-Analysis

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Mohammad Karimian

2016-04-01

Full Text Available Objective: To investigate the association of C631T single nucleotide polymorphisms in SPO11 gene with male infertilityfollowed by an in silico approach. SPO11 is a gene involved in meiosis and spermatogenesis process, which in humans, this gene is located on chromosome 20 (20q13.2-13.3 with 13 exons.Materials and methods: In a case-control study, 200 blood samples were collected from the IVF center (Kashan, Iran including; 100 infertile and 100 healthy control men. SPO11-C631T were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method.The effects of C631T transition on the structure of mRNA and protein of SPO11 was evaluated by bioinformatics tools.Results: Our data revealed that all subjects were wild-type homozygous inC631T positionsand just a sample from fertile group was heterozygousin C631T (OR: 0.3300, 95% CI: 0.0133 to 8.1992, p = 0.4988.Our in silico-analysis revealed that C631T transition could make fundamental changes in the structure of the mRNA (Score: 0.1983 and protein (PROVEAN Score: -3.371; Reliability Index: 4; Expected Accuracy: 82% of SPO11. Also, C631T substitution could change the aggregation prone regions of the SPO11 protein (dTANGO = 209.99.Conclusion: So even though the SPO11-C631T don’t increase the risk of male infertility, it could be deleterious for themRNA and protein.

AFLP marker analysis revealing genetic structure of the tree Parapiptadenia rigida (Benth. Brenan (Leguminosae-Mimosoideae in the southern Brazilian Tropical Rainforest

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Laís Bérgamo de Souza

2013-01-01

Full Text Available Parapiptadenia rigida is a tropical early secondary succession tree characteristic of the Tropical Atlantic Rainforest. This species is of great ecological importance in the recovery of degraded areas. In this study we investigated the variability and population genetic structure of eight populations of P. rigida. Five AFLP primer combinations were used in a sample of 159 individuals representing these eight populations, rendering a total of 126 polymorphic fragments. The averages of percentage of polymorphic loci, gene diversity, and Shannon index were 60.45%, 0.217, and 0.322, respectively. A significant correlation between the population genetic variability and the population sizes was observed. The genetic variability within populations (72.20% was higher than between these (22.80%. No perfect correlation was observed between geographic and genetic distances, which might be explained by differences in deforestation intensities that occurred in these areas. A dendrogram constructed by the UPGMA method revealed the formation of two clusters, these also confirmed by Bayesian analysis for the number of K cluster. These results show that it is necessary to develop urgent management strategies for the conservation of certain populations of P. rigida, while other populations still preserve reasonably high levels of genetic variability.

Índice baseado em RFLPs para seleção de linhagens visando sintéticos de milho RFLP marker index for selection of inbred lines aimed at synthetic maize populations

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Glauce Cristina Ricardo Rumin

2001-06-01

Full Text Available Marcadores moleculares têm sido sugeridos como ferramentas úteis em programas de melhoramento de plantas. Assim, este trabalho teve como objetivo propor um índice de seleção de linhagens visando a geração de populações sintéticas de milho, baseado em marcadores moleculares e dados de campo. Para tanto foram utilizados valores fenotípicos e genotípicos, oriundos de experimentos convencionais de campo e através da genotipagem das plantas-mãe das linhagens por marcadores RFLP, respectivamente. O índice foi examinado quanto à sua capacidade de originar sintéticos com alta freqüência de alelos favoráveis, em comparação com os obtidos baseando-se na seleção convencional. Utilizaram-se inicialmente 157 locos marcadores com distância média de 15,30 cM entre eles. Sessenta e oito linhagens S2, provenientes da geração F2 do cruzamento entre duas linhagens homozigóticas foram cruzadas em topcross e avaliadas em quatro locais de Iowa, EUA, em 1996. Dos 157 locos foram selecionados 18 que explicaram 74,70% da variância genética entre progênies, na média dos locais. O índice proposto leva em conta o comportamento próprio (per se das linhagens e a complementaridade genotípica entre elas, o que não é conseguido utilizando-se apenas dados dos topcrosses. O uso do índice proposto permite obter homólogo de tabela dialélica tendo-se apenas dados de médias de topcrosses. O índice levou à seleção de sintéticos com propriedades superiores às esperadas pela seleção baseada apenas nas médias das progênies topcross. Essa conclusão, no entanto, só é válida para os QTL's abrangidos pelas marcas. A metodologia proposta permite monitoramento muito adequado e útil das propriedades genéticas dos sintéticos a serem obtidos.Molecular markers have been suggested as useful tools in breeding programmes. The purpose of this work was to propose and apply an index for inbred line selection to be used for generating synthetic

Incidence Assessment of MTHFR C677T and A1298C Polymorphisms in Iranian Non-syndromic Cleft Lip and/or Palate Patients

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Asghar Ebadifar

2015-06-01

Full Text Available Background and aims. The aim of the present study is to determine the incidence of MTHFR C677 T and A1298C muta-tions in Iranian patients with cleft lip and/or cleft palate. Materials and methods. We screened 61 Iranian patients with cleft lip and/or cleft palate for mutations in the two alleles of MTHFR gene associated with cleft lip and/or palate: A1298C and C677T, using Polymerase Chain Reaction following by RFLP. Results. The 677T and 1298C homozygote genotypes showed a frequency of 36.1% and 11.4%, respectively. Combined genotype frequencies in newborns having oral clefts showed that the highest genotype was 677TT/1298AA (22.9% and 677TT/1298CC genotypes were not observed. Conclusion. The results showed that 65.6% of all patients had at least one T mutant allele in C677T and 58.9% C mutant allele for A1298C. According to the frequencies of homozygosity of mutant alleles, it could be said that MTHFRgenotype of 677TT shows a greater role in having oral clefts.

Association between methylene tetrahydrofolate reductase gene C677T polymorphism with preeclampsia in south-east of Iran

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Mohsen Saravani

2011-12-01

Full Text Available Background: Preeclampsia (PE is a serious problem of pregnancy and its etiology is still unknown. There are some evidence that vascular complications play an important role in development of PE. Methylenetetrahydrofolate reductase (MTHFR is a key enzyme in folate metabolism and the C677T polymorphism of the MTHFR gene is associated with decreased MTHFR activity. The aim of this study was to evaluate the relationship between MTHFR gene C677T polymorphism with PE development in south-east of Iran.Materials and method: This study was performed in 106 preeclamptic pregnant women and 107 control individuals. The C677T polymorphism of the MTHFR gene was determined by PCR-RFLP method. Results: The CC, CT and TT genotypes frequency of C677T polymorphism of MTHFR gene were 77, 22 and 1 percent in preeclamptic women and 73, 19.5 and 7.5 percent in control group. They were not significantly different (p=0.06. However, the frequency of TT genotype was significantly higher in control group (OR=8.5; 95% CI 1.1-71; p=.018. There was not any significant difference in T allele distribution between preeclamptic women (12% and control group (17%. Conclusion: Our results showed that there was not any correlation between the C677T polymorphism and PE but the TT genotype of C677T polymorphism seems to be a protective factor for preeclampsia

Prevalence of the N-Acetyltransferase (NAT2 gene polymorphism 282C>T in Peruvian population and health implications

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Salazar-Granara Alberto

2016-03-01

Full Text Available Objective: To determine the frequency of the C282T polymorphism of the NAT2 gene (N acetyltransferase in Peruvian populations. Field work, focused on exploring genetic risk factor in Peruvian populations, which has influence in the response to drugs and malignancies aetiology. Material and Methods: Cross-sectional study. 166 voluntaries from Lima, Lambayeque, Apurimac, Puno, San Martin, Amazonas and Loreto were enrolled. The sampling was done by convenience and it was use the RFLP-PCR conventional technique was used. Results: The allele frequency were 54% (n=126 for C282 and 46% (n=106 for T282. For the T allele, by its orign , stand out 2 those which origins were Lima 42% (n=25, Amazonas 47% (n=16, San Martin 74% (n=28 and Apurimac 50% (n=13 (X , p>0.05. A global genotype frequency were 26.7% (n=31 for C282/C282, 56.0% (n=65 for C282/T282 and 17.2% (n=20 for T282/T282 (Hardy Weinberg Test p>0.05. By origin, Puno presented allelic imbalance (Hardy Weinberg test p0.05. Conclusion: The overall frequency of NAT2 allele T282 was 46%; San Martin had the highest prevalence (74%. The T282 allele is linked to neoplastic diseases and adverse reactions to anti-TB drugs, these results will be used for the application of pharmacogenetics in Peru

Separation of the genera in the subtribe Cassiinae (Leguminosae: Caesalpinioidae using molecular markers Separação dos gêneros na subtribo Cassiinae (Leguminosae: Caesalpinioidae utilizando marcadores moleculares

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Laxmikanta Acharya

2011-03-01

Full Text Available Random amplified polymorphic DNA (RAPD, Inter simple sequence repeat (ISSR and Amplified fragment length polymorphism (AFLP markers were used to verify the segregation of the genus Cassia L. senso lato into three distinct genera namely Chamaecrista Moench., Senna P. Mill. and Cassia L. sensostricto Eighteen representatives of the three taxa were characterized using the molecular markers. 25 RAPD, six ISSR primers and six AFLP primer combinations resulted in the amplification of 612, 115 and 622 bands (loci respectively. Most of the loci are found to be polymorphic, showing high degrees of genetic diversity among the different taxa studied. The dendrogram constructed on the basis of the RAPD, ISSR and AFLP data using SHAN clustering, divided Cassia L. senso lato. into three different clusters as Chamaecrista Moench. Senna P. Mill. and Cassia L. senso stricto High bootstrap value revealed that all the clusters were stable and robust. It was observed from the present investigation that these genera have their identity at molecular level, which supports the elevation of the genus Cassia L. senso lato to the level of subtribe Cassiinae and segregation into three distinct genera instead of intrageneric categories.Técnicas de Random amplified polymorphic DNA (RAPD, Inter simple sequence repeat (ISSR e Amplified Fragment Length Polymorphism markers (AFLP foram utilizadas para verificar a segregação do gênero Cassia L. senso lato em três diferentes gêneros, Chamaecrista Moench., Senna P. Mill. e Cassia L. senso stricto Dezoito representantes dos três táxons foram caracterizados com o uso de marcadores moleculares: 25 RAPD, seis iniciadores ("primers" ISSR e seis AFLP combinações de iniciadores, resultando na amplificação de 612, 115 e 622 bandas (loci, respectivamente. A maioria dos loci apresentou-se como polimórfico, mostrando um alto grau de diversidade genética entre os táxons estudados. O dendrograma construído com base nos dados de

A comprehensive experiment for molecular biology: Determination of single nucleotide polymorphism in human REV3 gene using PCR-RFLP.

Science.gov (United States)

Zhang, Xu; Shao, Meng; Gao, Lu; Zhao, Yuanyuan; Sun, Zixuan; Zhou, Liping; Yan, Yongmin; Shao, Qixiang; Xu, Wenrong; Qian, Hui

2017-07-08

Laboratory exercise is helpful for medical students to understand the basic principles of molecular biology and to learn about the practical applications of molecular biology. We have designed a lab course on molecular biology about the determination of single nucleotide polymorphism (SNP) in human REV3 gene, the product of which is a subunit of DNA polymerase ζ and SNPs in this gene are associated with altered susceptibility to cancer. This newly designed experiment is composed of three parts, including genomic DNA extraction, gene amplification by PCR, and genotyping by RFLP. By combining these activities, the students are not only able to learn a series of biotechniques in molecular biology, but also acquire the ability to link the learned knowledge with practical applications. This comprehensive experiment will help the medical students improve the conceptual understanding of SNP and the technical understanding of SNP detection. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):299-304, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Conditioners and significance of t-RFLP profile of the assemblage of ...

African Journals Online (AJOL)

Following an increased interest in management practices designed to reduce posed phytotoxicity during phytoremediation experiments, crude oil polluted soil and ... Genetic fingerprinting showed that hydrocarbons stress led to depletion of the genetic resources of soil microflora and to a radical change in its qualitative ...

Role of Hyperhomocysteinemia and Methylene Tetrahydrofolate Reductase C677T Polymorphism in Idiopathic Portal Vein Thrombosis.

Science.gov (United States)

Ghaznavi, Habib; Soheili, Zahra; Samiei, Shahram; Soltanpour, Mohammad Soleiman

2016-03-01

Portal vein thrombosis (PVT) is a rare and life-threatening vascular disorder characterized by obstruction or narrowing of the portal vein. Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been studied in PVT patients with conflicting results. In the present study the association of hyperhomocysteinemia and MTHFR C677T polymorphism with PVT risk was investigated in Iranians. Our study population consisted of 10 idiopathic PVT patients and 80 healthy control subjects matched for age and sex. MTHFR C677T polymorphism was genotyped by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) technique and plasma total homocysteine (tHcy) levels were determined by enzyme immunoassay method. Mean plasma tHcy levels were significantly higher in PVT patients (20.2±6.8) than control subjects (10.9±4.7) (P=0.001). Moreover, plasma tHcy levels were significantly higher in 677T allele carriers relative to 677C allele carriers in both PVT patients (P=0.01) and control subjects (P=0.03). Neither homozygote nor heterozygote genotypes of MTHFR C677T polymorphism correlated significantly with PVT risk (P>0.05). Moreover, MTHFR C677T polymorphism didn't increase the risk of PVT under dominant (CT+TT vs. CC) or recessive (TT vs. CC+CT) genetic models analyzed (P>0.05). The difference in frequency of minor 677T allele between PVT patients and control subjects was not statistically significant (P>0.05). Based on the current study, we suggest that hyperhomocysteinemia constitutes a significant and common risk factor for PVT. Also, MTHFR C677T polymorphism is not a risk factor for PVT but is a contributing factor for elevated plasma tHcy levels.

Aspects pertinent to the usefulness of a solid phase radio-immuno-sorbent assay for the detection of spermatozoa antibodies in sera of infertility patients

International Nuclear Information System (INIS)

Hinrichs-Reiche, I.

1987-01-01

The solid phase Radio-Immuno-Sorbent Assay (RISA) is a highly sensitive and valid test to detect 125-iodinetagged antibodies to spermatozoa that allows qualitative and quantitative evaluations of sperm-incapacitating immunglobulin Ig G in sera from patients believed to be infertile for immunological reasons. The study failed to reveal any correlations between the results of RISA and those of micro-sperm-agglutination or micro-sperm-immobilisation tests. There was a major body of evidence pointing to possible links between female isoimmunity and male autoimmunity. (TRV) [de

Identification of aluminum-regulated genes by cDNA-AFLP analysis of roots in two contrasting genotypes of highbush blueberry (Vaccinium corymbosum L.).

Science.gov (United States)

Inostroza-Blancheteau, Claudio; Aquea, Felipe; Reyes-Díaz, Marjorie; Alberdi, Miren; Arce-Johnson, Patricio

2011-09-01

To investigate the molecular mechanisms of Al(3+)-stress in blueberry, a cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was employed to identify Al-regulated genes in roots of contrasting genotypes of highbush blueberry (Brigitta, Al(3+)-resistant and Bluegold, Al(3+)-sensitive). Plants grown in hydroponic culture were treated with 0 and 100 μM Al(3+) and collected at different times over 48 h. Seventy transcript-derived fragments (TDFs) were identified as being Al(3+) responsive, 31 of which showed significant homology to genes with known or putative functions. Twelve TDFs were homologous to uncharacterized genes and 27 did not have significant matches. The expression pattern of several of the genes with known functions in other species was confirmed by quantitative relative real-time RT-PCR. Twelve genes of known or putative function were related to cellular metabolism, nine associated to stress responses and other transcription and transport facilitation processes. Genes involved in signal transduction, photosynthetic and energy processes were also identified, suggesting that a multitude of processes are implicated in the Al(3+)-stress response as reported previously for other species. The Al(3+)-stress response genes identified in this study could be involved in Al(3+)-resistance in woody plants.

Distinct colonization patterns and cDNA-AFLP transcriptome profiles in compatible and incompatible interactions between melon and different races of Fusarium oxysporum f. sp. melonis

Science.gov (United States)

2011-01-01

Background Fusarium oxysporum f. sp. melonis Snyd. & Hans. (FOM) causes Fusarium wilt, the most important infectious disease of melon (Cucumis melo L.). The four known races of this pathogen can be distinguished only by infection on appropriate cultivars. No molecular tools are available that can discriminate among the races, and the molecular basis of compatibility and disease progression are poorly understood. Resistance to races 1 and 2 is controlled by a single dominant gene, whereas only partial polygenic resistance to race 1,2 has been described. We carried out a large-scale cDNA-AFLP analysis to identify host genes potentially related to resistance and susceptibility as well as fungal genes associated with the infection process. At the same time, a systematic reisolation procedure on infected stems allowed us to monitor fungal colonization in compatible and incompatible host-pathogen combinations. Results Melon plants (cv. Charentais Fom-2), which are susceptible to race 1,2 and resistant to race 1, were artificially infected with a race 1 strain of FOM or one of two race 1,2 w strains. Host colonization of stems was assessed at 1, 2, 4, 8, 14, 16, 18 and 21 days post inoculation (dpi), and the fungus was reisolated from infected plants. Markedly different colonization patterns were observed in compatible and incompatible host-pathogen combinations. Five time points from the symptomless early stage (2 dpi) to obvious wilting symptoms (21 dpi) were considered for cDNA-AFLP analysis. After successful sequencing of 627 transcript-derived fragments (TDFs) differentially expressed in infected plants, homology searching retrieved 305 melon transcripts, 195 FOM transcripts expressed in planta and 127 orphan TDFs. RNA samples from FOM colonies of the three strains grown in vitro were also included in the analysis to facilitate the detection of in planta-specific transcripts and to identify TDFs differentially expressed among races/strains. Conclusion Our data

Genetic characterization of two traditional leafy vegetables ...

African Journals Online (AJOL)

Genetic characterization of two traditional leafy vegetables (Sesamum radiatum Thonn. ex Hornem and Ceratotheca sesamoides Endl.) of Benin, using flow cytometry and amplified fragment length polymorphism (AFLP) markers. K Adéoti, A Rival, A Dansi, S Santoni, S Brown, T Beule, A Nato, Y Henry, R Vodouhe, L Loko, ...

Association of the TCF7L2 rs12255372 (G/T variant with type 2 diabetes mellitus in an Iranian population

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Faranak Mahmoudi Alami

2012-01-01

Full Text Available In various populations worldwide, common variants of the TCF7L2 (Transcription factor 7-like 2 gene are associated with the risk of type 2 diabetes mellitus (T2DM. The aim was to investigate the association between rs12255372 (G/T polymorphism in the TCF7L2 gene and T2DM in an Iranian population. 236 unrelated patients with T2DM, and 255 normoglycemic controls without diabetes were studied. The PCR-RFLP method was used for genotyping rs12255372 (G/T polymorphism, and the SPSS version 18.0 for Windows for statistical analysis. The minor T allele of TCF7L2 rs12255372 was found to significantly increase the risk of T2DM, with an allelic odds ratio (OR of 1.458 (95% CI 1.108-1.918, p = 0.007. A significant difference in TT genotype was observed between T2DM patients and normoglycemic controls (OR 2.038, 95% CI 1.147-3.623; p = 0.014. On assuming dominant and recessive models, ORs of 1.52 [95% CI (1.05-2.21 p = 0.026] and 1.74 [95% CI (1.01-3.00 p = 0.043] were obtained, respectively, thereby implying that the co-dominant model would best fit the susceptible gene effect. This study further confirms the TCF7L2 gene as enhancing susceptibility to the development of T2DM.

Mapping of a rice thermosensitive genic male sterility gene from a TGMS mutant line

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Vu Duc Quang; Nguyen Van Dong; Pham Ngoc Luong; Tran Duy Quy [Argicultural Genetics Institute, Hanoi (Viet Nam); Nguyen, Henry T. [Texas Tech Univ., Department of Plant and Soil Science, Lubbock TX (United States)

2001-03-01

At the Agricultural Genetics Institute (AGI), Hanoi, Vietnam, a number of thermo-sensitive genic male sterility (TGMS) homozygous rice lines have been developed by means of experimental mutagenesis followed by anther culture techniques. One of them (TGMS-1 indica mutant line) was used in this research. The critical temperature (at the period from pollen mother cell formation to the beginning of meiotic division) for TGMS-1 sterility was 24-25degC, below which the plants were fertile and above which the plants became sterile. Segregation analysis showed that the TGMS trait of the TGMS-1 mutant line was controlled by a single recessive gene. An F{sub 2} mapping population from a cross between TGMS-1 mutant line and CH1 (a fertile indica line) was developed for tagging and mapping the TGMS gene. From survey of 200 AFLP primer combinations in a bulked segregant analysis, 4 AFLP markers (E2/M5-200, E3/M16-400, E5/M12-600 and E5/M12-200) linked to TGMS-1 gene were identified and cloned. All except E2/M5-200 were found to be low-copy number sequences. The marker E5/M12-600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64xAzucena and CT9993xIR62666. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12-600, was sequenced so that a PCR marker can be developed for the use in marker-assisted breeding. The application of TGMS genes to the commercial two-line hybrid rice breeding system was discussed. (author)

Mapping of a rice thermosensitive genic male sterility gene from a TGMS mutant line

International Nuclear Information System (INIS)

Vu Duc Quang; Nguyen Van Dong; Pham Ngoc Luong; Tran Duy Quy; Nguyen, Henry T.

2001-01-01

At the Agricultural Genetics Institute (AGI), Hanoi, Vietnam, a number of thermo-sensitive genic male sterility (TGMS) homozygous rice lines have been developed by means of experimental mutagenesis followed by anther culture techniques. One of them (TGMS-1 indica mutant line) was used in this research. The critical temperature (at the period from pollen mother cell formation to the beginning of meiotic division) for TGMS-1 sterility was 24-25degC, below which the plants were fertile and above which the plants became sterile. Segregation analysis showed that the TGMS trait of the TGMS-1 mutant line was controlled by a single recessive gene. An F 2 mapping population from a cross between TGMS-1 mutant line and CH1 (a fertile indica line) was developed for tagging and mapping the TGMS gene. From survey of 200 AFLP primer combinations in a bulked segregant analysis, 4 AFLP markers (E2/M5-200, E3/M16-400, E5/M12-600 and E5/M12-200) linked to TGMS-1 gene were identified and cloned. All except E2/M5-200 were found to be low-copy number sequences. The marker E5/M12-600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64xAzucena and CT9993xIR62666. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12-600, was sequenced so that a PCR marker can be developed for the use in marker-assisted breeding. The application of TGMS genes to the commercial two-line hybrid rice breeding system was discussed. (author)

Association of Methylenetetrahydrofolate Reductase C677T Polymorphism with Hyperhomocysteinemia and Deep Vein Thrombosis in the Iranian Population.

Science.gov (United States)

Ghaznavi, Habib; Soheili, Zahra; Samiei, Shahram; Soltanpour, Mohammad Soleiman

2015-12-01

Deep venous thrombosis (DVT) is a common but elusive condition characterized by a high morbidity and mortality rate. The aim of the present study was to investigate the correlation between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with plasma total homocysteine (tHcy) levels and DVT risk in an Iranian population. Our study population consisted of 67 patients with a diagnosis of DVT and 67 healthy subjects as controls. Genotyping of MTHFR C677T polymorphism was performed by the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP) and measurement of tHcy levels was done by enzyme immunoassay method. Plasma tHcy levels were significantly higher in DVT patients than controls (18.09±7.6 vs. 10.5±4.3, P=0.001). Also, plasma tHcy levels were significantly higher in MTHFR 677TT genotypes compared to 677CC genotypes in both DVT patients (P=0.016) and controls (P=0.03). Neither heterozygote nor homozygote genotypes of MTHFR C677T polymorphism was significantly correlated with DVT (P>0.05). The distribution of MTHFR C677T genotypes was similar between men and women in both DVT patients and controls (P>0.05). Moreover, the frequency of mutant 677T allele did not differ significantly between the two groups (28.3% vs. 21.6%, P=0.15). Based on this study, we propose that hyperhomocysteinemia but not homozygosity for MTHFR C677T polymorphism is a significant risk factor for DVT in the Iranian population. Also, MTHFR 677TT genotype is a determinant of elevated plasma tHcy levels.

Uterine bacterial flora in postpartum Danish Holstein dairy cows determined using DNA-based fingerprinting: Correlation to uterine condition and calving management

DEFF Research Database (Denmark)

Elkjær, K.; Ancker, M.-L.; Gustafsson, H.

2013-01-01

The overall aim of this study was to describe uterine bacterial flora during the postpartum period in Danish Holstein cows using the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method. This method produces a pattern of nucleic acid fragments from the microorganisms present, reflect......The overall aim of this study was to describe uterine bacterial flora during the postpartum period in Danish Holstein cows using the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method. This method produces a pattern of nucleic acid fragments from the microorganisms present......, reflecting the “fingerprint” of the actual microbial flora. As well as characterizing changes in flora with time from calving and between herds, data were examined for strong relations between uterine bacterial flora, calving management and uterine condition. In total 125 Holstein cows from five herds were...... included, and for each cow calving management was recorded. Cows were clinically examined on average 8 (range 0–19) and 28 (range 22–38) days after calving, and a uterine sample was taken for bacterial identification using T-RFLP. Milk samples were taken weekly for progesterone analysis. Bacteria were...

D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.

Science.gov (United States)

Dagar, Sumit S; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K

2011-09-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp.▿

Science.gov (United States)

Dagar, Sumit S.; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K.

2011-01-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation. PMID:21784906

Association of aldose reductase gene polymorphism (C-106T) in susceptibility of diabetic peripheral neuropathy among north Indian population.

Science.gov (United States)

Gupta, Balram; Singh, S K

2017-07-01

Polymorphism in aldose reductase (ALR) gene at nucleotide C(-106)T (rs759853) in the promoter region is associated with susceptibility to development of diabetic peripheral neuropathy. The aim of this study was to detect the association of the C (-106)T polymorphism of ALR gene and its frequency among patients with type 2 diabetes mellitus with and without peripheral neuropathy. The study subjects were divided into three groups. Group I included 356 patients with diabetes having peripheral neuropathy. Group II included 294 patients with diabetes without peripheral neuropathy and group III included 181 healthy subjects. Genotyping of ALR C(-106)T SNPs was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods. The genetic risk among the groups was compared and tested by calculating odds ratio with 95% class interval. ALR 106TT genotype was significantly higher in group I compared to group II with an odds ratio of 2.12 (95% CI: 1.22-3.67; pneuropathy with relative risk of 1.97 (95% CI: 1.16-3.35; pperipheral neuropathy in patients with type 2 diabetes mellitus. Copyright © 2017 Elsevier Inc. All rights reserved.

An AFLP marker linked to the Pm-1 gene that confers resistance to Podosphaera xanthii race 1 in Cucumis melo

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Ana Paula Matoso Teixeira

2008-01-01

Full Text Available Brazil produced 330,000 metric tons of melons in 2005, principally in the Northeast region where one of the most important melon pathogens is the powdery mildew fungus Podosphaera xanthii. The disease is controlled mainly by incorporating single dominant resistance genes into commercial hybrids. We report on linkage analysis of the Pm-1 resistance gene, introgressed from the AF125Pm-1 Cantalupensis Charentais-type breeding line into the yellow-fleshed melon (Group Inodorus breeding line AF426-S by backcrossing to produce the resistant line AF426-R, and the amplified fragment length polymorphism (AFLP marker M75/H35_155 reported to be polymorphic between AF426-S and AF426-R. Segregation analysis of M75/H35_155 using a backcross population of 143 plants derived from [AF426-R x AF426-S] x AF426-S and screened for resistance to P. xanthii race 1 produced a recombination frequency of 4.9%, indicating close linkage between M75/H35_155 and Pm-1. Using the same segregating population, the M75/H35_155 marker had previously been reported to be distantly linked to Prv¹, a gene conferring resistance to papaya ringspot virus-type W. Since M75/H35_155 is linked to Prv¹ at a distance of 40.9 cM it is possible that Pm-1 and Prv¹ are also linked.

DNMT3B -579 G>T Promoter Polymorphism and the Risk of Gastric Cancer in the West of Iran.

Science.gov (United States)

Ahmadi, Kulsom; Soleimani, Azam; Irani, Shiva; Kiani, Aliasghar; Ghanadi, Kourosh; Noormohamadi, Zahra; Sakinejad, Foroozan

2018-06-01

Many studies have suggested that modulation of DNMT3B function caused by single nucleotide polymorphisms of the DNMT3B promoter region may underlie the susceptibility to various cancers such as tumors of the digestive system. The aim of this study was to investigate the effect of -579 G>T polymorphism in the promoter of the DNMT3B gene on risk of gastric cancer in a population from West Iran. We conducted a case-control study in 100 gastric cancer patients and 112 cancer-free controls to assess the correlation between DNMT3B -579 G>T (rs1569686) polymorphism and the risk of gastric cancer. Detection of genotypes of DNMT3B G39179T polymorphism was analyzed by PCR-RFLP. There was no significant difference in the distribution of DNMT3B -579 G>T genotypes between the cases and controls. However, in the stratified analysis by clinicopathological characteristic types, we found that statistically, the risk susceptibility to gastric cancer was significantly associated with tumor grade II and GT/TT genotype of patients, compared to patients having GG genotype, (OR = 5.4737, 95% CI = 1.4746. 20.3184, P = 0.01). Our study suggested that the -579 T allele may increase the relative risk for the progression of clinicopathological characteristic of tumor grade of gastric cancer patients.

Norrie disease: linkage analysis using a 4.2-kb RFLP detected by a human ornithine aminotransferase cDNA probe.

Science.gov (United States)

Ngo, J T; Bateman, J B; Cortessis, V; Sparkes, R S; Mohandas, T; Inana, G; Spence, M A

1989-05-01

Previous study has shown that the usual DNA marker for Norrie disease, the L1.28 probe which identifies the DXS7 locus, can recombine with the disease locus. In this study, we used a human ornithine aminotransferase (OAT) cDNA which detects OAT-related DNA sequences mapped to the same region on the X chromosome as that of the L1.28 probe to investigate the family with Norrie disease who exhibited the recombinational event. When genomic DNA from this family was digested with the PvuII restriction endonuclease, we found a restriction fragment length polymorphism (RFLP) of 4.2 kb in size. This fragment was absent in the affected males and cosegregated with the disease locus; we calculated a lod score of 0.602, at theta = 0.00. No deletion could be detected by chromosomal analysis or on Southern blots with other enzymes. These results suggest that one of the OAT-related sequences on the X chromosome may be in close proximity to the Norrie disease locus and represent the first report which indicates that the OAT cDNA may be useful for the identification of carrier status and/or prenatal diagnosis.

Evaluation of gestational diabetes mellitus risk in South Indian women based on MTHFR (C677T and FVL (G1691A mutations

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Imran Ali Khan

2015-05-01

Full Text Available We aimed to scrutinize the extent to which single amino acid substitutions in the MTHFR and FVL genes affect the risk of GDM in pregnant women of South Indian descendant. This case-control study was implemented once the ethical approval has been obtained. Overall 237 women were recruited in this study: 137 had been diagnosed with GDM and the remaining 100 women were used as normal controls or non-GDM. The diagnosis of GDM was confirmed with biochemical analysis i.e., GCT and OGTT tests. Five milliliters of peripheral blood was collected and used for biochemical and molecular analyses. DNA was isolated and genotyping for MTHFR (C677T and FVL (G1691A mutations was performed using PCR-RFLP. FVL (G1691A locus was not polymorphic in the investigated sample. There was no significant difference in the allele and genotype frequencies of C677T polymorphism between GDM and non-GDM women (p=0.8892

Differentiation of sheep pox and goat poxviruses by sequence analysis and PCR-RFLP of P32 gene.

Science.gov (United States)

Hosamani, Madhusudan; Mondal, Bimalendu; Tembhurne, Prabhakar A; Bandyopadhyay, Santanu Kumar; Singh, Raj Kumar; Rasool, Thaha Jamal

2004-08-01

Sheep pox and Goat pox are highly contagious viral diseases of small ruminants. These diseases were earlier thought to be caused by a single species of virus, as they are serologically indistinguishable. P32, one of the major immunogenic genes of Capripoxvirus, was isolated and Sequenced from two Indian isolates of goat poxvirus (GPV) and a vaccine strain of sheep poxvirus (SPV). The sequences were compared with other P32 sequences of capripoxviruses available in the database. Sequence analysis revealed that sheep pox and goat poxviruses share 97.5 and 94.7% homology at nucleotide and amino acid level, respectively. A major difference between them is the presence of an additional aspartic acid at 55th position of P32 of sheep poxvirus that is absent in both goat poxvirus and lumpy skin disease virus. Further, six unique neutral nucleotide substitutions were observed at positions 77, 275, 403, 552, 867 and 964 in the sequence of goat poxvirus, which can be taken as GPV signature residues. Similar unique nucleotide signatures could be identified in SPV and LSDV sequences also. Phylogenetic analysis showed that members of the Capripoxvirus could be delineated into three distinct clusters of GPV, SPV and LSDV based on the P32 genomic sequence. Using this information, a PCR-RFLP method has been developed for unequivocal genomic differentiation of SPV and GPV.

Analysis of Single Nucleotide Polymorphism (SNP rs22114085 Associated with Canine Atopic Dermatitis by PCR-RFLP Method

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Martina Miluchová

2012-05-01

Full Text Available Canine atopic dermatitis (cAD is a common inflammatory skin disease that is considered to be a naturally occurring, spontaneous model of human atopic dermatitis (eczema. The aim of the paper was to identify of the SNP rs22114085 in different dog breeds. The material involved 52 dogs from 5 different breeds. Canine genomic DNA was isolated from saliva by modified method with using DNAzol® and linear polyacrylamide (LPA carrier and from blood by using commercial kit NucleospinBlood and used in order to estimate rs22114085 SNP genotypes by PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. The C allele was distributed in Czech Pointer, Chihuahua, German Wirehaired Pointer with an allele frequency ranging from 0.4545 to 1.00. In the population of Czech Pointer we detected all genotypes CC, CT and TT with frequency in male 0.25, 0.5833 and 0.1667, and in female 0.2728, 0.3636 and 0.3636, subsequently. In German Wirehaired Pointer was detected homozygote genotype CC in male and heterozygote genotype CT in female with frequency 1 and 1. In Chihuahua was observed homozygote genotype CC and heterozygote genotype CT with frequency 0.3333 and 0.6667, subsequently. In Golden retriever and Pincher we detected genotype TT with frequency 1.

Association Analysis between g.18873C>T and g.27522G>A Genetic Polymorphisms of OPG and Bone Mineral Density in Chinese Postmenopausal Women

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Fei Wang

2014-01-01

Full Text Available Several studies report that the OPG is an important candidate gene in the pathogenesis of osteoporosis. This study aimed to detect the potential association of OPG gene polymorphisms with osteoporosis in postmenopausal women. We recruited 928 subjects containing 463 with primary postmenopausal osteoporosis and 465 healthy volunteers as controls. The BMD of neck hip, lumbar spine (L2–4, and total hip were assessed by dual-energy X-ray absorptiometry (DEXA. Through the created restriction site-polymerase chain reaction (CRS-PCR, PCR-restriction fragment length polymorphism (PCR-RFLP, and DNA sequencing methods, the g.18873C>T and g.27522G>A have been investigated. As for g.18873C>T, our data indicated that subjects with CC genotype have significantly higher BMD value than those of CT and TT genotypes (all P values A, the BMD values of subjects with GG genotype were significantly higher than those of GA and AA genotypes (all P values T and g.27522G>A genetic polymorphisms are associated with the decreased risk for osteoporosis in Chinese postmenopausal women.

Monitoring of microbial communities in anaerobic digestion sludge for biogas optimisation.

Science.gov (United States)

Lim, Jun Wei; Ge, Tianshu; Tong, Yen Wah

2018-01-01

This study characterised and compared the microbial communities of anaerobic digestion (AD) sludge using three different methods - (1) Clone library; (2) Pyrosequencing; and (3) Terminal restriction fragment length polymorphism (T-RFLP). Although high-throughput sequencing techniques are becoming increasingly popular and affordable, the reliance of such techniques for frequent monitoring of microbial communities may be a financial burden for some. Furthermore, the depth of microbial analysis revealed by high-throughput sequencing may not be required for monitoring purposes. This study aims to develop a rapid, reliable and economical approach for the monitoring of microbial communities in AD sludge. A combined approach where genetic information of sequences from clone library was used to assign phylogeny to T-RFs determined experimentally was developed in this study. In order to assess the effectiveness of the combined approach, microbial communities determined by the combined approach was compared to that characterised by pyrosequencing. Results showed that both pyrosequencing and clone library methods determined the dominant bacteria phyla to be Proteobacteria, Firmicutes, Bacteroidetes, and Thermotogae. Both methods also found that sludge A and B were predominantly dominated by acetogenic methanogens followed by hydrogenotrophic methanogens. The number of OTUs detected by T-RFLP was significantly lesser than that detected by the clone library. In this study, T-RFLP analysis identified majority of the dominant species of the archaeal consortia. However, many of the more highly diverse bacteria consortia were missed. Nevertheless, the combined approach developed in this study where clone sequences from the clone library were used to assign phylogeny to T-RFs determined experimentally managed to accurately predict the same dominant microbial groups for both sludge A and sludge B, as compared to the pyrosequencing results. Results showed that the combined approach of

AFLPMax: a user-friendly application for computing the optimal number of amplified fragment length polymorphism markers needed in phylogenetic reconstruction.

Science.gov (United States)

García-Pereira, M J; Quesada, H; Caballero, A; Carvajal-Rodríguez, A

2012-05-01

Amplified fragment length polymorphisms (AFLPs) are widely used for phylogenetic inference especially in non-model species. Frequently, trees obtained with other nuclear or mitochondrial markers or with morphological information need additional resolution, increased branch support, or independent data sources (i.e. unlinked loci). In such cases, the use of AFLPs is a quick and cheap option. Computer simulation has shown that dominant AFLP markers lead to less accurate tree topologies than bi-allelic codominant markers such as SNPs, but this difference becomes negligible for shallow trees when using AFLP data sets that include a sufficiently large number of characters. Thus, determining how many AFLP characters are required to recover a given phylogeny is a key issue regarding the appropriateness of AFLPs for phylogenetic reconstruction. Here, we present a user-friendly, java-based graphical interface, AFLPMax, which executes an automatic pipeline of different programs providing the user with the optimal number of AFLP characters needed to recover a given phylogeny with high accuracy and support. Executables for Windows, linux and MacOS X operating systems, source code and user manual are available from: http://webs.uvigo.es/acraaj/AFLPMax.htm. © 2012 Blackwell Publishing Ltd.

Violencia y memoria en Milan Kundera

OpenAIRE

Tafalla, Marta

2007-01-01

Partiendo de la filosofía de la memoria de T. W. Adorno, este artículo defiende la tesis de que la literatura tiene una mayor capacidad que la historia o la filosofía para explicar en qué consisten los regímenes totalitarios y para salvaguardar la memoria de sus víctimas. Con este propósito, toma como ejemplo la literatura del escritor checo Milan Kundera y analiza algunas de sus obras, especialmente El libro de la risa y el olvido, La insoportable levedad del ser y su ensayo El arte de la no...

Laugh and Interview with Antonia Baehr

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Alicia G. Hierro

2013-12-01

Full Text Available Con motivo del programa Secciò Irregular celebrado en el Mercat de les Flors en Barcelona el pasado 16 de enero, la artista alemana Antonia Baehr2 mostró el resultado de una extensa investigación sobre la risa que ha ocupado los últimos siete años de su carrera. Rire, Laugh, Lachen3 (título original en tres idiomas se presentó dentro del circuito performativo en Madrid en el año 2010 dentro de Escena Contemporánea. En esta ocasión, en 2013 repite la misma performance aplaudida por la escena internacional.

Vadose zone microbial community structure and activity in metal/radionuclide contaminated sediments. Final technical report

Energy Technology Data Exchange (ETDEWEB)

Balkwill, David L.

2002-08-17

This final technical report describes the research carried out during the final two months of the no-cost extension ending 11/14/01. The primary goals of the project were (1) to determine the potential for transformation of Cr(VI) (oxidized, mobile) to Cr(III) (reduced, immobile) under unsaturated conditions as a function of different levels and combinations of (a) chromium, (b) nitrate (co-disposed with Cr), and (c) molasses (inexpensive bioremediation substrate), and (2) to determine population structure and activity in experimental treatments by characterization of the microbial community by signature biomarker analysis and by RT-PCR and terminal restriction fragment length polymorphism (T-RFLP) and 16S ribosomal RNA genes. It was determined early in the one-year no-cost extension period that the T-RFLP approach was problematic in regard to providing information on the identities of microorganisms in the samples examined. As a result, it could not provide the detailed information on microbial community structure that was needed to assess the effects of treatments with chromium, nitrate, and/or molasses. Therefore, we decided to obtain the desired information by amplifying (using TR-PCR, with the same primers used for T-RFLP) and cloning 16S rRNA gene sequences from the same RNA extracts that were used for T-RFLP analysis. We also decided to use a restriction enzyme digest procedure (fingerprinting procedure) to place the clones into types. The primary focus of the research carried out during this report period was twofold: (a) to complete the sequencing of the clones, and (b) to analyze the clone sequences phylogenetically in order to determine the relatedness of the bacteria detected in the samples to each other and to previously described genera and species.

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

Science.gov (United States)

Qin, L; Overmars, H; Helder, J; Popeijus, H; van der Voort, J R; Groenink, W; van Koert, P; Schots, A; Bakker, J; Smant, G

2000-08-01

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

cDNA-AFLP analysis reveals the adaptive responses of citrus to long-term boron-toxicity.

Science.gov (United States)

Guo, Peng; Qi, Yi-Ping; Yang, Lin-Tong; Ye, Xin; Jiang, Huan-Xin; Huang, Jing-Hao; Chen, Li-Song

2014-10-28

Boron (B)-toxicity is an important disorder in agricultural regions across the world. Seedlings of 'Sour pummelo' (Citrus grandis) and 'Xuegan' (Citrus sinensis) were fertigated every other day until drip with 10 μM (control) or 400 μM (B-toxic) H3BO3 in a complete nutrient solution for 15 weeks. The aims of this study were to elucidate the adaptive mechanisms of citrus plants to B-toxicity and to identify B-tolerant genes. B-toxicity-induced changes in seedlings growth, leaf CO2 assimilation, pigments, total soluble protein, malondialdehyde (MDA) and phosphorus were less pronounced in C. sinensis than in C. grandis. B concentration was higher in B-toxic C. sinensis leaves than in B-toxic C. grandis ones. Here we successfully used cDNA-AFLP to isolate 67 up-regulated and 65 down-regulated transcript-derived fragments (TDFs) from B-toxic C. grandis leaves, whilst only 31 up-regulated and 37 down-regulated TDFs from B-toxic C. sinensis ones, demonstrating that gene expression is less affected in B-toxic C. sinensis leaves than in B-toxic C. grandis ones. These differentially expressed TDFs were related to signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein and amino acid metabolism, lipid metabolism, cell wall and cytoskeleton modification, stress responses and cell transport. The higher B-tolerance of C. sinensis might be related to the findings that B-toxic C. sinensis leaves had higher expression levels of genes involved in photosynthesis, which might contribute to the higher photosyntheis and light utilization and less excess light energy, and in reactive oxygen species (ROS) scavenging compared to B-toxic C. grandis leaves, thus preventing them from photo-oxidative damage. In addition, B-toxicity-induced alteration in the expression levels of genes encoding inorganic pyrophosphatase 1, AT4G01850 and methionine synthase differed between the two species, which might play a role in the B-tolerance of C. sinensis. C. sinensis

A comparative genetic diversity analysis in mungbean ( Vigna ...

African Journals Online (AJOL)

Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite mungbean genotypes. A total of nine AFLP primer combination and 22 ISSR primers were used. Amplification of genomic DNA of the 30 genotypes, using AFLP analysis, ...

cDNA-AFLP analysis reveals differential gene expression in response to salt stress in foxtail millet (Setaria italica L.).

Science.gov (United States)

Jayaraman, Ananthi; Puranik, Swati; Rai, Neeraj Kumar; Vidapu, Sudhakar; Sahu, Pranav Pankaj; Lata, Charu; Prasad, Manoj

2008-11-01

Plant growth and productivity are affected by various abiotic stresses such as heat, drought, cold, salinity, etc. The mechanism of salt tolerance is one of the most important subjects in plant science as salt stress decreases worldwide agricultural production. In our present study we used cDNA-AFLP technique to compare gene expression profiles of a salt tolerant and a salt-sensitive cultivar of foxtail millet (Seteria italica) in response to salt stress to identify early responsive differentially expressed transcripts accumulated upon salt stress and validate the obtained result through quantitative real-time PCR (qRT-PCR). The expression profile was compared between a salt tolerant (Prasad) and susceptible variety (Lepakshi) of foxtail millet in both control condition (L0 and P0) and after 1 h (L1 and P1) of salt stress. We identified 90 transcript-derived fragments (TDFs) that are differentially expressed, out of which 86 TDFs were classified on the basis of their either complete presence or absence (qualitative variants) and 4 on differential expression pattern levels (quantitative variants) in the two varieties. Finally, we identified 27 non-redundant differentially expressed cDNAs that are unique to salt tolerant variety which represent different groups of genes involved in metabolism, cellular transport, cell signaling, transcriptional regulation, mRNA splicing, seed development and storage, etc. The expression patterns of seven out of nine such genes showed a significant increase of differential expression in tolerant variety after 1 h of salt stress in comparison to salt-sensitive variety as analyzed by qRT-PCR. The direct and indirect relationship of identified TDFs with salinity tolerance mechanism is discussed.

The T -786C, G894T, and Intron 4 VNTR (4a/b) Polymorphisms of the Endothelial Nitric Oxide Synthase Gene in Prostate Cancer Cases.

Science.gov (United States)

Diler, S B; Öden, A

2016-02-01

In previously conducted some studies it has been revealed that nitric oxide (NO) and nitric oxide synthase (NOS) system play a significant role in carcinogenesis. Nitric oxide (NO) is regulated by endothelial nitric oxide synthase (eNOS) enzyme which is one of the isoenzymes of NO synthase (NOS). In this study we have tried to come to a conclusion about whether eNOS gene T -786C, G894T and Intron 4 VNTR (4a/b) polymorphisms might be considered as a risk factor causing prostate cancer (PCa) or not. A total of 200 subjects were included in this research. 84 patients with PCa (mean age 70.0 ± 6.4) and 116 healthy controls (mean age 69.9 ± 7.5) were recruited in this case-control study. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Maryland, USA), according to the manufacturer's guidelines. The T-786C, G894T and Intron 4 VNTR (4a/b) polymorphisms were amplified using polymerase chain reation (PCR), detected by restriction fragment length polymorphism (RFLP). For T -786C polymorphism CC genotype [odds ratio (OR): 0.34, 95% confidence interval (CI): 0.15-0.78, P = 0.009)] and allele frequency (OR: 0.631, CI: 0.421-0.946, P = 0.026) is significant for control. In patients with PCa eNOS G894T polymorphism, both GT (OR: 0.069, CI: 0.027-0.174; P = 0.0001) and TT (OR: 0.040, CI: 0.013-0.123; P = = 0.0001) genotype distribution, and also T allele frequency (OR: 0.237, CI: 0.155-0.362, P = 0.0001) were considered significant statistically. While genotype distribution for the other polymorphism eNOS, intron 4 VNTR (4a/b), is insignificant statistically, "a" allele frequency was found out to be significant (OR: 2.223, CI: 1.311-3.769, P = 0.003). In this study we indicated that genotype and allele frequencies of eNOS T -786C and G894T polymorphisms are statistically significant in patients with PCa. eNOS T -786C and G894T polymorphisms may be associated with PCa susceptibility in the Turkish population. In contrast, intron 4 VNTR (4a

Molecular Analysis of Bacterial Communities in Biofilms of a Drinking Water Clearwell

Science.gov (United States)

Zhang, Minglu; Liu, Wenjun; Nie, Xuebiao; Li, Cuiping; Gu, Junnong; Zhang, Can

2012-01-01

Microbial community structures in biofilms of a clearwell in a drinking water supply system in Beijing, China were examined by clone library, terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing of the amplified 16S rRNA gene. Six biofilm samples (designated R1–R6) collected from six locations (upper and lower sites of the inlet, middle and outlet) of the clearwell revealed similar bacterial patterns by T-RFLP analysis. With respect to the dominant groups, the phylotypes detected by clone library and T-RFLP generally matched each other. A total of 9,543 reads were obtained from samples located at the lower inlet and the lower outlet sites by pyrosequencing. The bacterial diversity of the two samples was compared at phylum and genus levels. Alphaproteobacteria dominated the communities in both samples and the genus of Sphingomonas constituted 75.1%–99.6% of this phylum. A high level of Sphingomonas sp. was first observed in the drinking water biofilms with 0.6–1.0 mg L−1 of chlorine residual. Disinfectant-resistant microorganisms deserve special attention in drinking water management. This study provides novel insights into the microbial populations in drinking water systems and highlights the important role of Sphingomonas species in biofilm formation. PMID:23059725

Association SOD2 Polymorphism(-9C/T and Senile Cataract

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A.R. Nakhaee

2017-01-01

Full Text Available Introduction: One of the most common causes of blindness around the world is cataract, which is a multifactorial eye disease and a major cause the loss lens transparency in the aging population. Oxidative stress is a major factor that often leads to cataract formation. Oxidative stress is defined as a disturbance in the balance of reactive oxygen species (ROS production  and antioxidant defenses, including enzymatic and non-enzymatic systems. One of the defense systems against free radicals is superoxide dismutase II (Mn SOD enzyme. SOD enzyme catalyses the dismutation of superoxide anion to O2 and H2O2. Several polymorphism  have been found associated with SOD2 gene. Present study has been done to evaluaet effects of genetic polymorphism, including SOD2 C/T polymorphism in the -9 position in senile cataract patiens and normal individuals. Material and methods: in this case- control study, there are 120 patients with senile cataract and 104 healthy people. We collected 2ml of whole blood in tubes containing EDTA, and then DNA extraction was performed. Polymorphisms were detected by PCR–RFLP technique. Findings: The distribution of CC, CT, TT genotypes of SOD2 gene were 28.3%, 43.3% and 28.3% in the patient group and 24%, 48.1% and 27.9% in the healthy group, respectively. Conclusion: No significant difference in the distribution SOD2 C/T polymorphism was observed between cases and controls. 

Role of aldose reductase C-106T polymorphism among diabetic Egyptian patients with different microvascular complications

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Nermine Hossam Zakaria

2014-04-01

Full Text Available The aldose reductase pathway proves that elevated blood glucose promotes cellular dysfunction. The polyol pathway converts excess intracellular glucose into alcohols via activity of the aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol which triggers variety of intracellular changes in the tissues. Among diabetes, activity is drastically increased in association with three main consequences inside the cells. The aim of this study was to detect the association of the C-106 T polymorphism of the aldose reductase gene and its frequency among a sample of 150 Egyptian adults with type 2 diabetic patients having diabetic microvascular. The detection of the aldose reductase C-106 T polymorphism gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The genotype distribution of the C-106 T polymorphism showed that CC genotype was statistically significantly higher among patients with retinopathy compared to nephropathy. Patients with nephropathy had significant association with the TT genotype when compared with diabetic retinopathy patients. Follow up study after the genotype detection among recently diagnosed diabetic patients in order to give a prophylactic aldose reductase inhibitors; studying the microvascular complications and its relation to the genotype polymorphisms. The study may include multiple gene polymorphisms to make the relation between the gene and the occurrence of these complications more evident.

PCR-RFLP analyses for studying the diversity of GH and Pit-1 genes in Slovak Simmental cattle

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Anna Trakovická

2013-10-01

Full Text Available The aim of this study was evaluation of growth hormone (GH and specific pituitary transcription factor (Pit-1 genes diversity in population of 353 Slovak Simmental cows. The analyses were based on single nucleotide polymorphisms GH/AluI and Pit-1/HinfI detections. A polymorphic site of GH gene (AluI has been linked to differences in circulating metabolites, metabolic hormones and milk yield. Bovine Pit-1 is responsible for pituitary development and hormone secreting gene expression, including GH gene. The Pit-1/HinfI locus was associated with growth, milk production and reproduction performance in cattle. Samples of genomic DNA were analyzed by PCR-RFLP method. Digestion of GH gene PCR products with restriction enzyme AluI revealed allele L and V with frequency 0.695 and 0.305, respectively. The digested Pit-1 gene PCR products with enzyme HinfI revealed alleles A (0.249 and B (0.751. Dominant genotypes were for GH gene heterozygous LV (0.47 and for Pit-1 gene homozygous BB (0.56 animals. The observed heterozygosity, effective allele numbers and polymorphism information content of GH/AluI and Pit-1/HinfI bovine loci population were 0.42/0.37, 1.73/1.59 and 0.33/0.30, respectively. The median polymorphic information content of loci was also transferred to the higher observed homozygosity in population (0.58/0.63. Keywords: cattle, growth hormone, leptin, PCR, Pit-1, polymorphism.

Association between RAS Gene Polymorphisms (ACE I/D, AGT M235T and Henoch-Schönlein Purpura in a Turkish Population

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Sinem Nalbantoglu

2013-01-01

Full Text Available Henoch-Schönlein purpura (HSP is a small-vessel vasculitis of autoimmune hypersensitivity, and renin-angiotensin system (RAS regulates vascular homeostasis and inflammation with activation of cytokine release. Thus, we aimed to investigate the association between HSP and ACE I/D and AGT M235T polymorphisms. Genotyping was determined by allele specific PCR and PCR-RFLP. We obtained a significant difference in genotype distribution (p = 0.003 and allele frequencies (p 0.05 and allele frequencies (p > 0.05 of the AGT M235T polymorphism. Risk assessment showed significant risk for HSP in the subjects both with the ID + DD genotype (p = 0.019, OR: 2.288, 95% CI: 1.136–4.609 and D allele (OR: D vs. I: 2.0528, 95% CI: 1.3632–3.0912, p = 0.001 while no significant risk was obtained for HSP in the subjects both with the MT + TT genotype (p = 0.312, OR: 1.3905, 95% CI: 0.7326–2.6391 and T allele (OR: T vs. M: 1.065, 95% CI: 0.729–1.557, p = 0.743. Furthermore, when patients were stratified by the presence of certain systemic complications of HSP, no significant association was detected with ACE I/D, and AGT M235T polymorphisms. Our findings suggest that ACE I/D polymorphism is significantly associated with HSP susceptibility.

Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure

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Maria Lodovica Gullino

2012-01-01

Full Text Available A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP and tested for ochratoxin A (OTA production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%, while A. carbonarius was the major species during 2007 (46.6%. All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES, as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7% isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46 in both years, but also some strains of A. tubingensis (11 and A. japonicus (6 produced lower amounts of OTA.

Techniques for characterization and eradication of potato cyst nematode: a review.

Science.gov (United States)

Bairwa, Aarti; Venkatasalam, E P; Sudha, R; Umamaheswari, R; Singh, B P

2017-09-01

Correct identification of species and pathotypes is must for eradication of potato cyst nematodes (PCN). The identification of PCN species after completing the life cycle is very difficult because it is based on morphological and morphometrical characteristics. Genetically different populations of PCN are morphologically same and differentiated based on the host differential study. Later on these traditional techniques have been replaced by biochemical techniques viz, one and two dimensional gel electrophoresis, capillary gel electrophoresis, isozymes, dot blot hybridization and isoelectric focusing etc. to distinguish both the species. One and two dimensional gel electrophoresis has used to examine inter- and intra-specific differences in proteins of Globodera rostochiensis and G. pallida . Now application of PCR and DNA based characterization techniques like RAPD, AFLP and RFLP are the important tools for differentiating inter- and intra specific variation in PCN and has given opportunities to accurate identification of PCN. For managing the PCN, till now we are following integrated pest management (IPM) strategies, however these strategies are not effective to eradicate the PCN. Therefore to eradicate the PCN we need noval management practices like RNAi (RNA interference) or Gene silencing.

Application of molecular markers in wheat breeding: Reality or delusion?

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Kobiljski Borislav

2004-01-01

Full Text Available Conventional plant breeding use morphological and phenotypic markers for the identification of important agronomic traits. Plant breeders and scientists continuously seek to develop new techniques, which can be used for faster and more accurate introgression of desirable traits into plants. Over the last several years there has been significant increase in the application of molecular markers in the breeding programmes of different species. So far, detected level of polymorphism and informatitivnes of different molecular marker methods applied in MAS (Marker Assisted Selection studies (RFLP, AFLP, etc. were insufficient either to validate their further use or there were very expensive and of huge healthy risk. Fortunately for wheat (and other crops breeders, the new class of molecular markers - microsatellites have prove recently to be most powerful for MAS. But, due to lack of the knowledge, experience, valid informations and even tradition and habits, many breeders have either negative or repulsive attitude towards implementation of MAS in breeding programes. In this paper the relevant facts regarding implementation of MAS in breeding are discussed in general, and for wheat breeding in particular, in order to summarize merits and limitations in application of microsatellites in MAS selection. .

MTHFR gene C677T and A1298C variants are associated with FMF risk in a Turkish cohort.

Science.gov (United States)

Nursal, Ayse Feyda; Kaya, Süheyla; Sezer, Ozlem; Karakus, Nevin; Yigit, Serbulent

2018-02-01

Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in homocysteine (Hcy) metabolism. We aimed to evaluate a possible relationship between MTHFR gene C677T (rs 1801133), A1298C (rs 1801131) variants and susceptibility to FMF in a Turkish cohort. This case-control study included 198 Turkish FMF patients and 100 healthy subjects as controls. MTHFR C677T and A1298C were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. The genotype distribution and allele frequency of the MTHFR C677T were statistically different between the patients and the control group (P=.006, P=.001, respectively). The frequency of the TT genotype and T allele of MTHFR C677T was significantly higher in the patients than in the controls. The genotype distribution of MTHFR A1298C variant did not show any statistically significant difference between the patients and the controls (P›.05). The patients had statistically different frequencies in allele C of MTHFR A1298C variant compared with the control (P=.032). We also examined the risk associated with inheriting the combined genotypes for the two MTHFR variants. According to these results, individuals who were CC homozygous at C677T locus and AA homozygous at A1298C locus have a lower risk of developing FMF (P=.002). Individuals who were TT homozygous at C677T locus and AC heterozygous at A1298C locus have higher risk of developing FMF (P=.033). Our findings clearly showed there was an association the MTHFR C677T/A1298C variants and susceptibility to FMF in the Turkish sample. © 2017 Wiley Periodicals, Inc.

Molecular markers. Amplified fragment length polymorphism

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Pržulj Novo

2005-01-01

Full Text Available Amplified Fragment Length Polymorphism molecular markers (AFLPs has been developed combining procedures of RFLPs and RAPDs molekular markers, i.e. the first step is restriction digestion of the genomic DNA that is followed by selective amplification of the restricted fragments. The advantage of the AFLP technique is that it allows rapid generation of a large number of reproducible markers. The reproducibility of AFLPs markers is assured by the use of restriction site-specific adapters and adapter-specific primers for PCR reaction. Only fragments containing the restriction site sequence plus the additional nucleotides will be amplified and the more selected nucleotides added on the primer sequence the fewer the number of fragments amplified by PCR. The amplified products are normally separated on a sequencing gel and visualized after exposure to X-ray film or by using fluorescent labeled primers. AFLP shave proven to be extremely proficient in revealing diversity at below the species level. A disadvantage of AFLP technique is that AFLPs are essentially a dominant marker system and not able to identify heterozygotes.

Geographical distribution of a specific mitochondrial haplotype of Zymoseptoria tritici

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Sameh BOUKEF

2014-01-01

Full Text Available Severity of disease caused by the fungus Zymoseptoria tritici throughout world cereal growing regions has elicited much debate on the potential evolutionary mechanism conferring high adaptability of the pathogen to diverse climate conditions and different wheat hosts (Triticum durum and T. aestivum. Specific mitochondrial DNA sequence was used to investigate geographic distribution of the type 4 haplotype (mtRFLP4 within 1363 isolates of Z. tritici originating from 21 countries. The mtRFLP4 haplotype was detected from both durum and bread wheat hosts with greater frequency on durum wheat. The distribution of mtRFLP4 was limited to populations sampled from the Mediterranean and the Red Sea region. Greater frequencies of mtRFLP4 were found in Tunisia (87% and Algeria (60%. The haplotype was absent within European, Australian, North and South American populations except Argentina. While alternative hypotheses such as climatic adaptation could not be ruled out, it is postulated that mtRFLP4 originated in North Africa (e.g. Tunisia or Algeria as an adaptation to durum wheat as the prevailing cereal crop. The specialized haplotype has subsequently spread as indicated by lower frequency of occurrence in the surrounding Mediterranean countries and on bread wheat hosts.

Phylogeny of the pollinating yucca moths, with revision of Mexican species (Tegeticula and Parategeticula; Lepidoptera, Prodoxidae)

Energy Technology Data Exchange (ETDEWEB)

Pellmyr, Olof; Balcazar-Lara, Manuel; Segraves, Kari A.; Althoff, David M.; Littlefield, Rik J.

2008-02-01

ABSTRACT The yucca moths (Tegeticula and Parategeticula; Lepidoptera, Prodoxidae) are well-known for their obligate relationship as exclusive pollinators of yuccas. Revisionary work in recent years has revealed far higher species diversity than historically recognized, increasing the number of described species from four to 21. Based on field surveys in Mexico and examination of collections, we describe five additional species: T. californica Pellmyr sp. nov., T. tehuacana Pellmyr & Balcázar-Lara sp. nov., T. tambasi Pellmyr & Balcázar-Lara sp. nov., T. baja Pellmyr & Balcázar-Lara sp. nov., and P. californica Pellmyr & Balcázar-Lara sp. nov. Tegeticula treculeanella Pellmyr is identified as a junior synonym of T. mexicana Bastida. A diagnostic key to the adults of all species of the T. yuccasella complex is provided. A phylogeny based on a 2104-bp segment of mitochondrial DNA (mtDNA) in the cytochrome oxidase I and II region supported monophyly of the two pollinator genera, and strongly supported monophyly of the 17 recognized species of the T. yuccasella complex. Most relationships are well-supported, but some relationships within a recent and rapidly diversified group of 11 taxa are less robust, and in one case conflicts with a whole-genome data set (AFLP). The current mtDNA-based analyses, together with previously published AFLP data, provide a robust phylogenetic foundation for future studies of life history evolution and host interactions in one of the classical models of coevolution and obligate mutualism. ADDITIONAL KEY WORDS: mutualism, pollination, molecular phylogenetics, mitochondrial DNA

Identificação de marcas moleculares associadas à ausência de sementes em videira Identification of molecular markers associated to the absence of seeds in grapevine

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Ana Veruska Cruz da Silva

2006-06-01

Full Text Available A ausência de sementes tem sido uma característica bastante exigida pelos consumidores de uvas de mesa. O objetivo deste trabalho foi identificar marcas moleculares associadas à ausência de sementes, utilizando as técnicas RAPD e fAFLP. Foram utilizadas folhas jovens de 19 cultivares. Na análise RAPD 30, iniciadores possibilitaram amplificação de todas as amostras, produzindo 392 bandas polimórficas. Foi possível encontrar uma marca específica para a ausência de sementes, utilizando o iniciador UBC 443, que poderá futuramente ser utilizado para o desenvolvimento de marcadores SCAR, possibilitando a criação de um teste de identificação rápida e precoce de apirenia em videira. A análise fAFLP proporcionou a visualização de um dendrograma com grupos específicos de cultivares com sementes, sem sementes e porta enxertos.Seedless has been an important characteristic of table grapes required by consumers. The objective was to identify molecular markers associated to seedless, by RAPD and fAFLP techniques with young leaves samples of 19 cultivars. Thirty primers were used for RAPD analysis, producing a total of 337 polymorphic bands. It was also to find a specific mark for seedless, using UBC 443 primer. This mark would be transformed in a scar marker, making possible the early identification of seedless grape possible. The fAFLP analysis provided a visualization of a dendrogram with specific groups, separated in three different cultivars: with seeds, without seeds and the rootstocks.

Molecular characterization and antifungal susceptibility of Cryptococcus neoformans strains collected from a single institution in Lima, Peru.

Science.gov (United States)

Bejar, Vilma; Tello, Mercedes; García, Ruth; Guevara, José M; Gonzales, Sofia; Vergaray, German; Valencia, Esther; Abanto, Enma; Ortega-Loayza, Alex G; Hagen, Ferry; Gutierrez, Ericson L

2015-01-01

Cryptococcosis is a fungal infection with a worldwide distribution, mainly caused by Cryptococcus neoformans and Cryptococcus gattii. To molecularly characterize the mating-types, serotypes, genotypes and antifungal susceptibility profiles of a set of retrospectively isolated C. neoformans strains from Lima, Peru. A set of 32 Cryptococcus spp. strains from the Institute of Tropical Medicine of the National University of San Marcos, Lima, Peru, were included in this retrospective study. Twenty-four strains were isolated from patients, while the remaining 8 were isolated from the environment. Using conventional PCR, 27 (84.4%) of the isolates were identified as C. neoformans var. grubii mating-type alpha and serotype A. Using the AFLP fingerprinting, it was shown that 16 (50%) of the C. neoformans strains were genotype AFLP1, 13 (40.6%) were genotype AFLP1B, 2 (6.3%) were genotype AFLP2, and 1 (3.1%) was found to be a hybrid between both C. neoformans varieties (genotype AFLP3). The antifungal susceptibility profiles for amphotericin B, fluconazole and voriconazole showed that all the 32 C. neoformans are sensitive to these antifungal compounds. In this study we observed that C. neoformans var. grubii (AFLP1 and AFLP1B) and C. neoformans var. neoformans (AFLP2) were the only cryptococcal varieties involved. All strains were found to be sensitive to the antifungals tested, results that are consistent with those found in the international literature. Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Mechanisms on boron-induced alleviation of aluminum-toxicity in Citrus grandis seedlings at a transcriptional level revealed by cDNA-AFLP analysis.

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Xin-Xing Zhou

Full Text Available The physiological and biochemical mechanisms on boron (B-induced alleviation of aluminum (B-toxicity in plants have been examined in some details, but our understanding of the molecular mechanisms underlying these processes is very limited. In this study, we first used the cDNA-AFLP to investigate the gene expression patterns in Citrus grandis roots responsive to B and Al interactions, and isolated 100 differentially expressed genes. Results showed that genes related to detoxification of reactive oxygen species (ROS and aldehydes (i.e., glutathione S-transferase zeta class-like isoform X1, thioredoxin M-type 4, and 2-alkenal reductase (NADP+-dependent-like, metabolism (i.e., carboxylesterases and lecithin-cholesterol acyltransferase-like 4-like, nicotianamine aminotransferase A-like isoform X3, thiosulfate sulfurtransferase 18-like isoform X1, and FNR, root isozyme 2, cell transport (i.e., non-specific lipid-transfer protein-like protein At2g13820-like and major facilitator superfamily protein, Ca signal and hormone (i.e., calcium-binding protein CML19-like and IAA-amino acid hydrolase ILR1-like 4-like, gene regulation (i.e., Gag-pol polyprotein and cell wall modification (i.e., glycosyl hydrolase family 10 protein might play a role in B-induced alleviation of Al-toxicity. Our results are useful not only for our understanding of molecular processes associated with B-induced alleviation of Al-toxicity, but also for obtaining key molecular genes to enhance Al-tolerance of plants in the future.

Mechanisms on boron-induced alleviation of aluminum-toxicity in Citrus grandis seedlings at a transcriptional level revealed by cDNA-AFLP analysis.

Science.gov (United States)

Zhou, Xin-Xing; Yang, Lin-Tong; Qi, Yi-Ping; Guo, Peng; Chen, Li-Song

2015-01-01

The physiological and biochemical mechanisms on boron (B)-induced alleviation of aluminum (B)-toxicity in plants have been examined in some details, but our understanding of the molecular mechanisms underlying these processes is very limited. In this study, we first used the cDNA-AFLP to investigate the gene expression patterns in Citrus grandis roots responsive to B and Al interactions, and isolated 100 differentially expressed genes. Results showed that genes related to detoxification of reactive oxygen species (ROS) and aldehydes (i.e., glutathione S-transferase zeta class-like isoform X1, thioredoxin M-type 4, and 2-alkenal reductase (NADP+-dependent)-like), metabolism (i.e., carboxylesterases and lecithin-cholesterol acyltransferase-like 4-like, nicotianamine aminotransferase A-like isoform X3, thiosulfate sulfurtransferase 18-like isoform X1, and FNR, root isozyme 2), cell transport (i.e., non-specific lipid-transfer protein-like protein At2g13820-like and major facilitator superfamily protein), Ca signal and hormone (i.e., calcium-binding protein CML19-like and IAA-amino acid hydrolase ILR1-like 4-like), gene regulation (i.e., Gag-pol polyprotein) and cell wall modification (i.e., glycosyl hydrolase family 10 protein) might play a role in B-induced alleviation of Al-toxicity. Our results are useful not only for our understanding of molecular processes associated with B-induced alleviation of Al-toxicity, but also for obtaining key molecular genes to enhance Al-tolerance of plants in the future.

Association of kynurenine aminotransferase II gene C401T polymorphism with immune response in patients with meningitis

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Leib Stephen L

2011-04-01

Full Text Available Abstract Background The kynurenine (KYN pathway has been shown to be altered in several diseases which compromise the central nervous system (CNS including infectious diseases such as bacterial meningitis (BM. The aim of this study was to assess single nucleotide polymorphisms (SNPs in four genes of KYN pathway in patients with meningitis and their correlation with markers of immune response in BM. Methods One hundred and one individuals were enrolled in this study to investigate SNPs in the following genes: indoleamine-2,3-dioxygenase (IDO1 gene, kynureninase (KYNU gene, kynurenine aminotransferase I (CCBL1 gene, and kynurenine aminotransferase II (AADAT gene. SNP analyses were performed by primer-introduced restriction analysis-PCR (PIRA-PCR followed by RFLP. Cytokines were measured using multiplex bead assay while immunoglobulins (IG by immunodiffusion plates and NF-kappaB and c-Jun by dot blot assay. Results The variant allele of SNP AADAT+401C/T showed prevalent frequency in patients with BM. A significant decrease (p AADAT+401C/T. Furthermore, a significant (p p KYNU+715G/A was found with low frequency in the groups, and the SNPs in IDO1+434T/G, KYNU+693G/A, CCBL1+164T/C, and AADAT+650C/T had no frequency in this population. Conclusions This study is the first report of an association of SNP AADAT+401C/T with the host immune response to BM, suggesting that this SNP may affect the host ability in recruitment of leukocytes to the infection site. This finding may contribute to identifying potential targets for pharmacological intervention as adjuvant therapy for BM.

Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Science.gov (United States)

Braker, Gesche; Ayala-del-Río, Héctor L.; Devol, Allan H.; Fesefeldt, Andreas; Tiedje, James M.

2001-01-01

Steep vertical gradients of oxidants (O2 and NO3−) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities. PMID:11282647

ANÁLISIS GENÓMICO DE PARVOVIRUS CANINO POR PCR - RFLP A PARTIR DE AISLAMIENTOS DE CASOS CLÍNICOS SINTOMÁTICOS TOMADOS EN BOGOTÁ - COLOMBIA -ESTUDIO PRELIMINAR-

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Ángela Castillo

2001-12-01

Full Text Available Con el objetivo de detemúnar el genotipo del parvovirus canino (CPV circulante en Bogotá, se analizaron 56 muestras tomadas a conveniencia, de materia fecal y sangre de perros menores a un año con gastroenteritis hemorrágica presuntiva de parvovirosis, por reacción en cadena de la polirnerasa - huella genórnica (PCR - RFLP. Se obtuvieron fragmentos amplificados de DNA de 2.2 Kb por PCR de la cepa vacunal de CPV-2 y de cuatro muestras de heces, no se obtuvieron amplificados de las muestras de sangre. Los productos amplificados fueron digeridos por la enzima de restricción Rsa I pemútiendo la identificación de los genotipos CPV-2a y CPV-2b así como su diferenciación con el parvovirus tipo - 2 (CPV-2.

Identification of Pit-1 Gen Using PCR-RFLP of Padjadjaran Sheep and Evaluate of Growth Rate

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Prajoga, S. B. K.; Andriani, L.; Subhandiana, H.

2018-02-01

The objectives of this research were to evaluate variation of Pit-1 gen of Padjadjaran Sheep and evaluate of their growth rate. The data comprised of 15 lambs female and 15 lambs male records of 0 - 12 mouths old lambs. Variation of Padjadjaran Sheep PIT-1 gen was analyzed using PCR-RFLP and used primer by 5’-GAGGGATAATTACAAATGGTCC-3’ and 5’-TGTTAACAGCTGTGGGACACAC-3’, length fragment analyse using HinfI restriction enzyme. Presence or absence of deference bands in a PCR fragment of the result can be distinguished by differences in the electrophoretic migration of the fragment. The result showed that the variation Pit1 gene showed that there was in the position 345 bp, 137 bp and 115 bp. In this experiment the difference migration did occur in all samples (monomorphic), that was amplificated with reverse and forward primer. The average male birth weight (BW), weaning weight (WN) and Yearling Weight (YW) was 2.29 ±0.42kg 8.96 ±1,89 kg and 30.12 ±5,65 kg. The average female birth weight (BW), weaning weight (WN) and Yearling Weight (YW) was 2.33 ±0.49 kg; 8.23 ±1.99 kg and 26.11 ±5.50 kg. Correlation value between age and body weight was 0,99 for female and 0,97 for male. The highest body weight gain per month was 2.85 kg for female at the age of six months and 3.4 kg for male at the age of one year. The best equition of growth rate was Ŷ = 1,862 + 0,127X1 0,076X2

Keragaman Genetik Gen Hormon Pertumbuhan (GH|MboII pada Itik Sikumbang Janti Menggunakan Penciri PCR-RFLP

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T.D. Nova

2016-02-01

Full Text Available Penelitian ini bertujuan untuk mengetahui keragaman gen hormon pertumbuhan (GH dengan enzim MboII pada itik Sikumbang Janti dengan menggunakan penciri PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism. Penelitian ini menggunakan sebanyak 50 sampel darah itik Sikumbang Janti. Sampel darah itik Sikumbang Janti diambil melalui vena achilaris sebanyak ± 1 ml. DNA sampel darah diisolasi menggunakan protocol Genomik DNA Purification Kit (Promega. DNA total diamplifikasi menggunakan sepasang primer F : 5’-CTG GAG CAG GCA GGA AAA TT-3’ dan R: 5’-TCC AGG GAC AGT GAC TCA AC-3’ yang menghasilkan fragmen exon 1 gen GH dengan panjang 801 bp. Produk amplifikasi direstriksi dengan menggunakan MboII yang mengenali situs pemotongan GAAGA (N/8↓ . Dari 46 sampel hasil restriksi diperoleh dua posisi. Pada posisi 618 bp dengan genotip yaitu genotip heterozigot (+/- yang terdiri dari 3 pita (266 bp, 535 bp dan 801 bp, genotip homozigot (+/+ yang terdiri dari 3 pita (109 bp, 266 bp, 426 bp dan genotip homozigot (-/- yang terdiri dari 1 pita ( 801 dan terdapat dua tipe alel, yaitu alel (+ dan all (-, all (+ sebesar 0,79 dan alel (- sebesar 0,21. Sedangkan pada posisi 727 bp memiliki genotip yaitu genotip heterozigot (+/- yang terdiri dari 3 pita (109 bp, 266 bp, 426 bp, dan genotip homozigot (-/- yang terdiri dari 3 pita dan terdapat dua tipe alel, yaitu frekuensi alel (+ sebesar 0,61 dan frekuensi alel (- sebesar 0,39. Dari hasil penelitian ini dapat disimpulkan bahwa gen GH-MboII memiliki keragamanan yang tinggi serta menunjukkan adanya keseimbangan atau tidak menyimpang dari keseimbangan Hardy Weinberg pada posisi keragaman 618 bp dan pada posisi 727 dalam ketidakseimbangan Hardy Weinberg.

Quantitative trait locus (QTL) analysis of percentage grains ...

African Journals Online (AJOL)

user

2011-03-28

Mar 28, 2011 ... ATA/M-CGT; (B) AFLP results using primer E-AAA/M-CTC; (C) AFLP results using primer E-AAA/M-CTA. 1,. Minghui63; 2, Zhengshan97A; 3, low PGC bulk; 4, high PGC bulk. The arrow show linkage segments of percentage chalky grain in rice. Table 1. Chromosomal location of AFLP segments linked to ...

Is GDF5 gene promoter polymorphism +104T/C associated with osteoarthritis in the Eastern of Turkey population?

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Tülüce, Y; Yildirim, I H; Özkol, H; Edi Z, L; Delen, V

2017-08-30

Osteoarthritis (OA) is the most common form of arthritis. Genetic factors have been shown to play important roles in the etiology of OA. The gene growth differentiation factor 5 (GDF5) has been implicated in skeletal development and joint morphogenesis in human and mice. A functional single nucleotide polymorphism (SNP) +104T/C in the 5'-UTR of GDF5 (rs143383) was reported to be associated with osteoarthritis susceptibility in Han Chinese and Japanese populations. Our objective was to assess whether this SNP was also associated with OA in the Eastern Turkey population.A total of 172 cases including 95 patients with idiopathic OA and 77 control cases were recruited into the study. DNA samples were extracted from peripheral blood lymphocytes of all cases by using salting out method. The +104T/C polymorphism was genotyped by PCR-RFLP method. In terms of genotype comparison there wasn't any correlation between patient and control groups. Frequency of C allele was found to be higher in-patient group than control group and statistical analysis showed a poor correlation in allele frequencies of the +104T/C SNP of GDF5 gene between cases and controls (p<0.05). Significant correlation between GDF5 and OA has been reported in Asian population, especially T alleles were found in higher frequencies and related to OA.  Our study did not confirm this association and also in term of T allele. Interestingly, we found higher frequency of C allele in patient group than control group and our results are compatible with the study carried out in Greek population.

Trypanosoma cruzi infection in neotropical wild carnivores (Mammalia: Carnivora: at the top of the T. cruzi transmission chain.

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Fabiana Lopes Rocha

Full Text Available Little is known on the role played by Neotropical wild carnivores in the Trypanosoma cruzi transmission cycles. We investigated T. cruzi infection in wild carnivores from three sites in Brazil through parasitological and serological tests. The seven carnivore species examined were infected by T. cruzi, but high parasitemias detectable by hemoculture were found only in two Procyonidae species. Genotyping by Mini-exon gene, PCR-RFLP (1f8/Akw21I and kDNA genomic targets revealed that the raccoon (Procyon cancrivorus harbored TcI and the coatis (Nasua nasua harbored TcI, TcII, TcIII-IV and Trypanosoma rangeli, in single and mixed infections, besides four T. cruzi isolates that displayed odd band patterns in the Mini-exon assay. These findings corroborate the coati can be a bioaccumulator of T. cruzi Discrete Typing Units (DTU and may act as a transmission hub, a connection point joining sylvatic transmission cycles within terrestrial and arboreal mammals and vectors. Also, the odd band patterns observed in coatis' isolates reinforce that T. cruzi diversity might be much higher than currently acknowledged. Additionally, we assembled our data with T. cruzi infection on Neotropical carnivores' literature records to provide a comprehensive analysis of the infection patterns among distinct carnivore species, especially considering their ecological traits and phylogeny. Altogether, fifteen Neotropical carnivore species were found naturally infected by T. cruzi. Species diet was associated with T. cruzi infection rates, supporting the hypothesis that predator-prey links are important mechanisms for T. cruzi maintenance and dispersion in the wild. Distinct T. cruzi infection patterns across carnivore species and study sites were notable. Musteloidea species consistently exhibit high parasitemias in different studies which indicate their high infectivity potential. Mesocarnivores that feed on both invertebrates and mammals, including the coati, a host that

Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

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Dinka eMandakovic

2016-05-01

Full Text Available The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS, a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination and fish samples (coinfection, aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants. Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

Energy Technology Data Exchange (ETDEWEB)

Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

2009-09-01

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Genetic analysis of autoimmune gld mice. I. Identification of a restriction fragment length polymorphism closely linked to the gld mutation within a conserved linkage group

Science.gov (United States)

1988-01-01

A linkage map of distal mouse chromosome 1 was generated using restriction fragment length polymorphism (RFLP) analysis of DNA prepared from 95 [C3H-gld/gld X Mus spretus)F1 X C3H-gld/gld] backcross mice. The gene order was: (centromere) C4bp, Ren-1,2, Ly-5, [At-3/gld], Apoa-2/Ly-17, Spna-1 (telomere). All mice expressing the phenotype of gld homozygotes were homozygous for the At-3 RFLP characteristic of C3H mice and none of the mice heterozygous for At-3 RFLPs had characteristics of gld homozygotes, demonstrating close linkage between these genes. The identification of an RFLP closely linked to the gld gene provides a starting point for the identification of a genetic defect that results in abnormal T cells and autoimmune disease. PMID:2894402

Cytogenetic characterization and AFLP-based genetic linkage mapping for the butterfly Bicyclus anynana, covering all 28 karyotyped chromosomes.

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Arjen E Van't Hof

Full Text Available BACKGROUND: The chromosome characteristics of the butterfly Bicyclus anynana, have received little attention, despite the scientific importance of this species. This study presents the characterization of chromosomes in this species by means of cytogenetic analysis and linkage mapping. METHODOLOGY/PRINCIPAL FINDINGS: Physical genomic features in the butterfly B. anynana were examined by karyotype analysis and construction of a linkage map. Lepidoptera possess a female heterogametic W-Z sex chromosome system. The WZ-bivalent in pachytene oocytes of B. anynana consists of an abnormally small, heterochromatic W-chromosome with the Z-chromosome wrapped around it. Accordingly, the W-body in interphase nuclei is much smaller than usual in Lepidoptera. This suggests an intermediate stage in the process of secondary loss of the W-chromosome to a ZZ/Z sex determination system. Two nucleoli are present in the pachytene stage associated with an autosome and the WZ-bivalent respectively. Chromosome counts confirmed a haploid number of n = 28. Linkage mapping had to take account of absence of crossing-over in females, and of our use of a full-sib crossing design. We developed a new method to determine and exclude the non-recombinant uninformative female inherited component in offspring. The linkage map was constructed using a novel approach that uses exclusively JOINMAP-software for Lepidoptera linkage mapping. This approach simplifies the mapping procedure, avoids over-estimation of mapping distance and increases the reliability of relative marker positions. A total of 347 AFLP markers, 9 microsatellites and one single-copy nuclear gene covered all 28 chromosomes, with a mapping distance of 1354 cM. Conserved synteny of Tpi on the Z-chromosome in Lepidoptera was confirmed for B. anynana. The results are discussed in relation to other mapping studies in Lepidoptera. CONCLUSIONS/SIGNIFICANCE: This study adds to the knowledge of chromosome structure and

Discrimination among individuals using terminal restriction fragment length polymorphism profiling of bacteria derived from forensic evidence.

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Nishi, Eiji; Tashiro, Yukihiro; Sakai, Kenji

2015-05-01

DNA typing from forensic evidence is commonly used to identify individuals. However, when the quantity of the forensic evidence is insufficient, successful identification using DNA typing is impossible. Such evidence may also contain DNA from bacteria that occur naturally on the skin. In this study, we aimed to establish a profiling method using terminal restriction fragment length polymorphisms (T-RFLPs) of the amplified bacterial 16S ribosomal RNA (rRNA) gene. First, the extraction and digestion processes were investigated, and the T-RFLP profiling method using the 16S rRNA gene amplicon was optimized. We then used this method to compare the profiles of bacterial flora from the hands of 12 different individuals. We found that the T-RFLP profiles from one person on different days displayed higher similarity than those between individuals. In a principal component analysis (PCA), T-RFLPs from each individual were closely clustered in 11 out of 12 cases. The clusters could be distinguished from each other, even when the samples were collected from different conditions. No major change of the profile was observed after six months except in two cases. When handprints on glass plates were compared, 11 of 12 individuals were assigned to a few clusters including the cluster corresponding to the correct individual. In conclusion, a method for reproducible T-RFLP profiling of bacteria from trace amounts of handprints was established. The profiles were obtained for particular individuals clustered in PCA and were experimentally separable from other individuals in most cases. This technique could provide useful information for narrowing down a suspect in a criminal investigation.

Association of the IFN-γ (+874A/T) Genetic Polymorphism with Paranoid Schizophrenia in Tunisian Population.

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Jemli, Achraf; Eshili, Awatef; Trifa, Fatma; Mechri, Anouar; Zaafrane, Ferid; Gaha, Lotfi; Juckel, George; Tensaout, Besma Bel Hadj Jrad

2017-02-01

Since growing evidence suggests a significant role of chronic low-grade inflammation in the physiopathology of schizophrenia, we have hypothesized that functional genetic variant of the IFN gamma (IFN-γ; +874A/T; rs2430561) gene may be involved in the predisposition to schizophrenia. This research is based on a case-control study which aims to identify whether polymorphism of the IFN-γ gene is a risk factor for the development of schizophrenia. The RFLP-PCR genotyping of the IFN-γ gene was conducted on a Tunisian population composed of 218 patients and 162 controls. The IFN-γ (+874A/T) polymorphism analysis showed higher frequencies of minor homozygous genotype (TT) and allele (T) in all patients compared with controls (11.5 vs. 4.9%; p = 0.03, OR = 2.64 and 30.7 vs. 24.1%, p = 0.04, OR = 1.4, respectively). This correlation was confirmed for male but not for female patients. Also, the T allele was significantly more common among patients with paranoid schizophrenia when compared with controls (25.8 vs. 4.9%, p = 0.0001; OR = 6.7). Using the binary regression analysis to eliminate confounding factors as age and sex, only this last association remained significant (p = 0.03; OR = 1.76, CI = 1.05-2.93). In conclusion, our results showed a significant association between +874A/T polymorphism of IFN-γ and paranoid schizophrenia, suggesting that this single nucleotide polymorphism (SNP) or another at proximity could predispose to paranoid schizophrenia. Since the minor allele of this polymorphism was correlated with an increased expression of their product, our study validates the hypothesis of excessive pro-inflammatory cytokine in the physiopathology of paranoid schizophrenia.

Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP Analysis

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Yuny Erwanto

2014-10-01

Full Text Available This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp. Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis.

Science.gov (United States)

Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

2014-10-01

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

Methanogenic community changes, and emissions of methane and other gases, during storage of acidified and untreated pig slurry

DEFF Research Database (Denmark)

Petersen, Søren O; Højberg, Ole; Poulsen, Morten

2014-01-01

H was nearly constant at values of 5.5 and 6.5. Ammonia losses were reduced by 84 and 49%, respectively, while CH4 emission with both acidification techniques was reduced by >90%. T-RFLP fingerprints showed little effect of acidification or storage time. A major T-RF of 105 bp could represent methanogens...

Bcıı--RFLP profiles for serum amiloid A1 and mutated MEFV gene prevalence in chronic renal failure patients requiring long-term hemodialysis.

Science.gov (United States)

Ozdemir, Ozturk; Kayatas, Mansur; Cetinkaya, Selma; Yildirim, Malik Ejder; Silan, Fatma; Kurtulgan, Hande Kucuk; Koksal, Binnur; Urfali, Mine; Candan, Ferhan

2015-03-01

There is an increased mortality risk in long-term hemodialysis patients of renal failure due to the chronic inflammation. The relationship between the chronic renal failure (CRF) and the role of familial genetic markers remains incompletely understood. In the current study, it was aimed to find out the prevalence of common MEFV gene mutations and BcII polymorphism in serum amyloid A1 (SAA1) gene in chronic renal patients (CRF) who require long-term hemodialysis. Current cohort includes 242 CRF patients and 245 healthy individuals from the same population. Total genomic DNA was isolated from peripheral blood-EDTA samples and genotyping of target MEFV gene was carried out by reverse hybridization Strip Assay and real-time techniques. The SAA1 gene was genotyped by the BclI-RFLP method. Increased mutated MEFV genotypes were found in current CRF patients when compared with the control group from the same ethnicity and the difference was statistically significant (Table 2) (OR: 4.9401, 95% CI: 3.0694-7.9509), pchronic inflammation.

Silver relase from decomposed hyperaccumulating Amanita solitaria fruit-body biomass strongly affects soil microbial community

Czech Academy of Sciences Publication Activity Database

Gryndler, M.; Hršelová, H.; Soukupová, L.; Borovička, Jan

2012-01-01

Roč. 25, č. 5 (2012), s. 987-993 ISSN 0966-0844 Institutional support: RVO:67985831 Keywords : soil fungi * bacteria * microbiota * heavy metals * toxicity * T-RFLP Subject RIV: DD - Geochemistry Impact factor: 3.284, year: 2012

An extremely sensitive nested PCR-RFLP mitochondrial marker for detection and identification of salmonids in eDNA from water samples

Directory of Open Access Journals (Sweden)

Laura Clusa

2017-02-01

Full Text Available Background Salmonids are native from the North Hemisphere but have been introduced for aquaculture and sport fishing in the South Hemisphere and inhabit most rivers and lakes in temperate and cold regions worldwide. Five species are included in the Global Invasive Species Database: rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar, brown trout Salmo trutta, brook trout Salvelinus fontinalis, and lake trout Salvelinus namaycush. In contrast, other salmonids are endangered in their native settings. Methods Here we have developed a method to identify salmonid species directly from water samples, focusing on the Iberian Peninsula as a case study. We have designed nested Salmonidae-specific primers within the 16S rDNA region. From these primers and a PCR-RFLP procedure the target species can be unequivocally identified from DNA extracted from water samples. Results The method was validated in aquarium experiments and in the field with water from watersheds with known salmonid populations. Finally, the method was applied to obtain a global view of the Salmonidae community in Nalón River (north coast of Spain. Discussion This new powerful, very sensitive (identifying the species down to 10 pg DNA/ml water and economical tool can be applied for monitoring the presence of salmonids in a variety of situations, from checking upstream colonization after removal of river barriers to monitoring potential escapes from fish farms.

Genetic divergence and phylogenetic relationships in grey mullets (Teleostei: Mugilidae) based on PCR-RFLP analysis of mtDNA segments.

Science.gov (United States)

Papasotiropoulos, V; Klossa-Kilia, E; Kilias, G; Alahiotis, S

2002-04-01

The genetic differentiation and phylogenetic relationships among five species of the Mugilidae family (Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens) were investigated at the mtDNA level, on samples taken from Messolongi lagoon-Greece. RFLP analysis of three PCR-amplified mtDNA gene segments (12s rRNA, 16s rRNA, and CO I) was used. Ten, eight, and nine restriction enzymes were found to have at least one recognition site at 12s rRNA, 16s rRNA, and CO I genes, respectively. Several fragment patterns were revealed to be species-specific, and thus they could be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. Seven different haplotypes were detected. The greatest amount of genetic differentiation was observed at the interspecific level, while little variation was revealed at the intraspecific level. The highest values of nucleotide sequence divergence were observed between M. cephalus and all the other species, while the lowest was found between C. labrosus and L. saliens. Dendrograms obtained by the three different methods (UPGMA, Neighbor-Joining, and Dollo parsimony), were found to exhibit in all cases the same topology. According to this, the most distinct species is M. cephalus, while the other species are clustered in two separate groups, thefirst one containing L. aurata and L. ramada, the other L. saliens and C. labrosus. This last clustering makes the monophyletic origin of the genus Liza questionable.

The neurovascular relation in oxygen-induced retinopathy.

Science.gov (United States)

Akula, James D; Mocko, Julie A; Benador, Ilan Y; Hansen, Ronald M; Favazza, Tara L; Vyhovsky, Tanya C; Fulton, Anne B

2008-01-01

Longitudinal studies in rat models of retinopathy of prematurity (ROP) have demonstrated that abnormalities of retinal vasculature and function change hand-in-hand. In the developing retina, vascular and neural structures are under cooperative molecular control. In this study of rats with oxygen-induced retinopathy (OIR) models of ROP, mRNA expression of vascular endothelial growth factor (VEGF), semaphorin (Sema), and their neuropilin receptor (NRP) were examined during the course of retinopathy to evaluate their roles in the observed neurovascular congruency. Oxygen exposures designed to induce retinopathy were delivered to Sprague-Dawley rat pups (n=36) from postnatal day (P) 0 to P14 or from P7 to P14. Room-air-reared controls (n=18) were also studied. Sensitivities of the rod photoreceptors (S(rod)) and the postreceptor cells (Sm) were derived from electroretinographic (ERG) records. Arteriolar tortuosity, T(A), was derived from digital fundus images using Retinal Image multi-Scale Analysis (RISA) image analysis software. mRNA expression of VEGF(164), semaphorin IIIA (Sema3A), and neuropilin-1 (NRP-1) was evaluated by RT-PCR of retinal extracts. Tests were performed at P15-P16, P18-P19, and P25-P26. Relations among ERG, RISA, and PCR parameters were evaluated using linear regression on log transformed data. Sm was low and T(A) was high at young ages, then both resolved by P25-P26. VEGF(164) and Sema3A mRNA expression were also elevated early and decreased with age. Low Sm was significantly associated with high VEGF(164) and Sema3A expression. Low S(rod) was also significantly associated with high VEGF(164). S(rod) and Sm were both correlated with T(A). NRP-1 expression was little affected by OIR. The postreceptor retina appears to mediate the vascular abnormalities that characterize OIR. Because of the relationships revealed by these data, early treatment that targets the neural retina may mitigate the effects of ROP.