A set of 14 DIP-SNP markers to detect unbalanced DNA mixtures.

Science.gov (United States)

Liu, Zhizhen; Liu, Jinding; Wang, Jiaqi; Chen, Deqing; Liu, Zidong; Shi, Jie; Li, Zeqin; Li, Wenyan; Zhang, Gengqian; Du, Bing

2018-03-04

Unbalanced DNA mixture is still a difficult problem for forensic practice. DIP-STRs are useful markers for detection of minor DNA but they are not widespread in the human genome and having long amplicons. In this study, we proposed a novel type of genetic marker, termed DIP-SNP. DIP-SNP refers to the combination of INDEL and SNP in less than 300bp length of human genome. The multiplex PCR and SNaPshot assay were established for 14 DIP-SNP markers in a Chinese Han population from Shanxi, China. This novel compound marker allows detection of the minor DNA contributor with sensitivity from 1:50 to 1:1000 in a DNA mixture of any gender with 1 ng-10 ng DNA template. Most of the DIP-SNP markers had a relatively high probability of informative alleles with an average I value of 0.33. In all, we proposed DIP-SNP as a novel kind of genetic marker for detection of minor contributor from unbalanced DNA mixture and established the detection method by associating the multiplex PCR and SNaPshot assay. DIP-SNP polymorphisms are promising markers for forensic or clinical mixture examination because they are shorter, widespread and higher sensitive. Copyright © 2018 Elsevier Inc. All rights reserved.

Random amplified polymorphic DNA (RAPD) markers reveal genetic ...

African Journals Online (AJOL)

The present study evaluated genetic variability of superior bael genotypes collected from different parts of Andaman Islands, India using fruit characters and random amplified polymorphic DNA (RAPD) markers. Genomic DNA extracted from leaf material using cetyl trimethyl ammonium bromide (CTAB) method was ...

Assessment of genome origins and genetic diversity in the genus Eleusine with DNA markers.

Science.gov (United States)

Salimath, S S; de Oliveira, A C; Godwin, I D; Bennetzen, J L

1995-08-01

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.

Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes

Science.gov (United States)

Kitajima, Akira; Nonaka, Keisuke; Yoshioka, Terutaka; Ohta, Satoshi; Goto, Shingo; Toyoda, Atsushi; Fujiyama, Asao; Mochizuki, Takako; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2016-01-01

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy–Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies. PMID:27902727

Prenatal exclusion of Norrie disease with flanking DNA markers.

Science.gov (United States)

Gal, A; Uhlhaas, S; Glaser, D; Grimm, T

1988-10-01

Three polymorphic DNA markers linked to the locus of Norrie disease were used for indirect genotype analysis in a ten-wk-old fetus at risk for the disease. When haplotypes of the family members and the estimated recombination frequency between Norrie gene and each of the DNA marker loci DXS7, DXS84, and DXS146 were taken into account, the risk that the fetus had inherited the mutation was about 1%.

Optimization of ISSR Markers for Molecular DNA Fingerprinting in Aquilaria sp

International Nuclear Information System (INIS)

Azhar Mohamad; Muhammad Hanif Azhari; Siti Norhayati Ismail; Parween, K.S.A.S.

2013-01-01

Aquilaria sp. belongs to the Thymelaeaceae family and well distributed to Asia region. The species is a multipurpose use from root to shoot and becoming an economic important crop, which generates wide interest in understanding the genetic diversity of the species. Understanding of the effectiveness in differentiating DNA-based markers is an important step towards plant germplasm characterization and evaluation. It is becoming a prerequisite for more effective application of molecular marker techniques in breeding and mapping programs. Polymerase Chain Reaction (PCR)-based approaches are in demanding as its simplicity and requirement for only small quantities of sample genomic DNA. Inter-simple sequence repeats (ISRR) requires no prior genomic information as anchor template in producing multi-loci markers of tandem repeats for polymorphic patterns by PCR amplification which becoming a key of advantageous of ISSR primers. ISSR markers have shown rapid, simple, reproducible and inexpensive means in molecular taxonomy, conservation breeding and genetic diversity analysis. The ISSR for marker applications are essential to facilitate management, conservation and genetic improvement programs towards improvement of standard resin quality for perfume and or pharmaceutical industries. In this paper, a total of 100 ISSR primers were optimized by using Aquilaria malaccensis. Primers optimization resulted, 38 ISSR primers affirmative for the polymorphism evaluation study, which encountered both from specific and degenerate ISSR primers. Marker derived from ISSR profiling is a powerful method for identification and molecular classification of Aquilaria sp from species to accessions and further will useful in identifying any mutant lines derived from nature and/or mutagenesis activities. (author)

COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC MICROBIAL GENETIC MARKERS IN COW FECAL SAMPLES

Science.gov (United States)

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host specific markers. Here, we describe the application of a genome fragment enrichment met...

An annotated genetic map of loblolly pine based on microsatellite and cDNA markers

Directory of Open Access Journals (Sweden)

Wimalanathan Kokulapalan

2011-01-01

Full Text Available Abstract Background Previous loblolly pine (Pinus taeda L. genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats, also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety of sources and published cDNA markers into a composite P. taeda genetic map constructed from two reference mapping pedigrees. A dense genetic map that incorporates SSR loci will benefit complete pine genome sequencing, pine population genetics studies, and pine breeding programs. Careful marker annotation using a variety of references further enhances the utility of the integrated SSR map. Results The updated P. taeda genetic map, with an estimated genome coverage of 1,515 cM(Kosambi across 12 linkage groups, incorporated 170 new SSR markers and 290 previously reported SSR, RFLP, and ESTP markers. The average marker interval was 3.1 cM. Of 233 mapped SSR loci, 84 were from cDNA-derived sequences (EST-SSRs and 149 were from non-transcribed genomic sequences (genomic-SSRs. Of all 311 mapped cDNA-derived markers, 77% were associated with NCBI Pta UniGene clusters, 67% with RefSeq proteins, and 62% with functional Gene Ontology (GO terms. Duplicate (i.e., redundant accessory and paralogous markers were tentatively identified by evaluating marker sequences by their UniGene cluster IDs, clone IDs, and relative map positions. The average gene diversity, He, among polymorphic SSR loci, including those that were not mapped, was 0.43 for 94 EST-SSRs and 0.72 for 83 genomic-SSRs. The genetic map can be viewed and queried at http://www.conifergdb.org/pinemap. Conclusions Many polymorphic and genetically mapped SSR markers are now available for use in P. taeda population genetics, studies of adaptive traits, and various germplasm management applications. Annotating mapped

Marker chromosome 21 identified by microdissection and FISH

Energy Technology Data Exchange (ETDEWEB)

Sun, Y.; Palmer, C.G. [Indiana Univ. School of Medicine, Indianapolis, IN (United States); Rubinstein, J. [Univ. Affiliated Cincinnati Center for Developmental Disorders, OH (United States)] [and others

1995-03-27

A child without Down`s syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with {alpha}-satellite DNA probes for acrocentric chromosomes and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21. 14 refs., 3 figs.

Identification of body fluid-specific DNA methylation markers for use in forensic science.

Science.gov (United States)

Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

2014-11-01

DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Molecular marker to identify radiolarian species -toward establishment of paleo-environmental proxy-

Science.gov (United States)

Ishitani, Y.

2017-12-01

Marine fossilized unicellular plankton are known to have many genetically divergent species (biological species) in the single morphological species and these biological species show the species-specific environments much more precisely than that of morphological species. Among these plankton, Radiolaria are one of the best candidates for time- and environmental-indicators in the modern and past oceans, because radiolarians are the only group which represent entire water column from shallow to deep waters. However, the ecology and evolution of radiolarian were traditionally studied in paleontology and paleoceanography by morphological species. Even Radiolaria has a huge potential for novel proxy of wide and deep environments, there is no criterion to identify the biological species. The motivation for this study is setting the quantitative delimitation to establish the biological species of radiolarians based on molecular data, for leading the future ecological and paleo-environmental study. Identification of the biological species by ribosomal DNA sequences are mainly based on two ways: one is the evolutionary distance of the small subunit (SSU) rDNA, the internal transcribed spacer region of ribosomal DNA (ITS1 and 2), and the large subunit (LSU) rDNA; and the other is the secondary structure of ITS2. In the present study, all four possible genetic markers (SSU, ITS1, ITS2, and LSU rDNA) were amplified from 232 individuals of five radiolarian morphological species and applied to examine the evolutionary distance and secondary structure of rDNA. Comprehensive survey clearly shows that evolutionary distance of ITS1 rDNA and the secondary structure of ITS2 is good to identify the species. Notably, evolutionary distance of ITS1 rDNA is possible to set the common delimitation to identify the biological species, as 0.225 substitution per site. The results show that the ITS1 and ITS 2 rDNA could be the criterion for radiolarian species identification.

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples.

Science.gov (United States)

Westen, Antoinette A; Matai, Anuska S; Laros, Jeroen F J; Meiland, Hugo C; Jasper, Mandy; de Leeuw, Wiljo J F; de Knijff, Peter; Sijen, Titia

2009-09-01

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.

Forensic botany II, DNA barcode for land plants: Which markers after the international agreement?

Science.gov (United States)

Ferri, G; Corradini, B; Ferrari, F; Santunione, A L; Palazzoli, F; Alu', M

2015-03-01

The ambitious idea of using a short piece of DNA for large-scale species identification (DNA barcoding) is already a powerful tool for scientists and the application of this standard technique seems promising in a range of fields including forensic genetics. While DNA barcoding enjoyed a remarkable success for animal identification through cytochrome c oxidase I (COI) analysis, the attempts to identify a single barcode for plants remained a vain hope for a longtime. From the beginning, the Consortium for the Barcode of Life (CBOL) showed a lack of agreement on a core plant barcode, reflecting the diversity of viewpoints. Different research groups advocated various markers with divergent set of criteria until the recent publication by the CBOL-Plant Working Group. After a four-year effort, in 2009 the International Team concluded to agree on standard markers promoting a multilocus solution (rbcL and matK), with 70-75% of discrimination to the species level. In 2009 our group firstly proposed the broad application of DNA barcoding principles as a tool for identification of trace botanical evidence through the analysis of two chloroplast loci (trnH-psbA and trnL-trnF) in plant species belonging to local flora. Difficulties and drawbacks that were encountered included a poor coverage of species in specific databases and the lack of authenticated reference sequences for the selected markers. Successful preliminary results were obtained providing an approach to progressively identify unknown plant specimens to a given taxonomic rank, usable by any non-specialist botanist or in case of a shortage of taxonomic expertise. Now we considered mandatory to update and to compare our previous findings with the new selected plastid markers (matK+rbcL), taking into account forensic requirements. Features of all the four loci (the two previously analyzed trnH-psbA+trnL-trnF and matK+rbcL) were compared singly and in multilocus solutions to assess the most suitable combination for

ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts.

Science.gov (United States)

Wei, Guifang; Pan, Li; Du, Huimin; Chen, Junyi; Zhao, Liping

2004-10-01

Bacterial populations common to healthy human guts may play important roles in human health. A new strategy for discovering genomic sequences as markers for these bacteria was developed using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting. Structural features within microbial communities are compared with ERIC-PCR followed by DNA hybridization to identify genomic fragments shared by samples from healthy human individuals. ERIC-PCR profiles of fecal samples from 12 diseased or healthy human and piglet subjects demonstrated stable, unique banding patterns for each individual tested. Sequence homology of DNA fragments in bands of identical size was examined between samples by hybridization under high stringency conditions with DIG-labeled ERIC-PCR products derived from the fecal sample of one healthy child. Comparative analysis of the hybridization profiles with the original agarose fingerprints identified three predominant bands as signatures for populations associated with healthy human guts with sizes of 500, 800 and 1000 bp. Clone library profiling of the three bands produced 17 genome fragments, three of which showed high similarity only with regions of the Bacteroides thetaiotaomicron genome, while the remainder were orphan sequences. Association of these sequences with healthy guts was validated by sequence-selective PCR experiments, which showed that a single fragment was present in all 32 healthy humans and 13 healthy piglets tested. Two fragments were present in the healthy human group and in 18 children with non-infectious diarrhea but not in eight children with infectious diarrhea. Genome fragments identified with this novel strategy may be used as genome-specific markers for dynamic monitoring and sequence-guided isolation of functionally important bacterial populations in complex communities such as human gut microflora.

Genetic diversity and DNA fingerprinting in jute (Corchorus spp. based on SSR markers

Directory of Open Access Journals (Sweden)

Liwu Zhang

2015-10-01

Full Text Available Genetic diversity analysis and DNA finger printing are very useful in breeding programs, seed conservation and management. Jute (Corchorus spp. is the second most important natural fiber crop after cotton. DNA fingerprinting studies in jute using SSR markers are limited. In this study, 58 jute accessions, including two control varieties (Huangma 179 and Kuanyechangguo from the official variety registry in China were evaluated with 28 pairs of SSR primers. A total of 184 polymorphic loci were identified. Each primer detected 3 to 15 polymorphic loci, with an average of 6.6. The 58 jute accessions were DNA-fingerprinted with 67 SSR markers from the 28 primer pairs. These markers differentiated the 58 jute accessions from one another, with CoSSR305-120 and CoSSR174-195 differentiating Huangma 179 and Kuanyechangguo, respectively. NTSYS-pc2.10 software was used to analyze the genetic diversity in the 58 jute accessions. Their genetic similarity coefficients ranged from 0.520 to 0.910 with an average of 0.749, indicating relatively great genetic diversity among them. The 58 jute accessions were divided into four groups with the coefficient 0.710 used as a value for classification, consistent with their species and pedigrees. All these results may be useful both for protection of intellectual property rights of jute accessions and for jute improvement.

Genetic diversity and DNA fingerprinting in jute(Corchorus spp.) based on SSR markers

Institute of Scientific and Technical Information of China (English)

Liwu; Zhang; Rongrong; Cai; Minhang; Yuan; Aifen; Tao; Jiantang; Xu; Lihui; Lin; Pingping; Fang; Jianmin; Qi

2015-01-01

Genetic diversity analysis and DNA finger printing are very useful in breeding programs,seed conservation and management. Jute(Corchorus spp.) is the second most important natural fiber crop after cotton. DNA fingerprinting studies in jute using SSR markers are limited. In this study, 58 jute accessions, including two control varieties(Huangma 179 and Kuanyechangguo) from the official variety registry in China were evaluated with 28 pairs of SSR primers. A total of 184 polymorphic loci were identified. Each primer detected 3 to 15 polymorphic loci, with an average of 6.6. The 58 jute accessions were DNA-fingerprinted with 67 SSR markers from the 28 primer pairs. These markers differentiated the 58 jute accessions from one another, with Co SSR305-120 and Co SSR174-195 differentiating Huangma 179 and Kuanyechangguo, respectively. NTSYS-pc2.10 software was used to analyze the genetic diversity in the 58 jute accessions. Their genetic similarity coefficients ranged from 0.520 to 0.910 with an average of 0.749, indicating relatively great genetic diversity among them. The 58 jute accessions were divided into four groups with the coefficient 0.710 used as a value for classification, consistent with their species and pedigrees. All these results may be useful both for protection of intellectual property rights of jute accessions and for jute improvement.

Validation and use of DNA markers for sex determination in papaya (Carica papaya)

International Nuclear Information System (INIS)

Ejaz, M.; Iqbal, M.; Ahmed, I.

2015-01-01

Profitable papaya production requires female and hermaphrodite plants in higher number than male plants. This is only possible if sex of plants is determined at an early growth stage. The present study was conducted to validate sex-linked DNA markers using plants from two Pakistani papaya varieties and subsequently utilize them for determination of sex in juvenile papaya plants. One hundred and five plants (including 49 male and 56 female) of two Pakistani Papaya varieties at flowering stage were screened with six DNA markers viz., W-11, T12, SDP, Napf-76Napf-76, PKBT4 and PKBT5. All male plants exhibited amplification of sex-linked alleles with markers T12 and W11, whereas, 96% and 95% of female plants showed the absence of sex-linked allele with these markers, respectively. Markers SDP, PKBT5 and Napf-76 showed the presence of sex-linked alleles in 98%, 96% and 93% of male plants, respectively, whereas the same markers showed the absence of sex-linked alleles in 100%, 96% and 94% of female plants. One marker, PKBT4 could not produce expected PCR amplification reported previously. The five DNA markers were further used to screen 171 papaya seedlings. These markers clearly differentiated male and female sex types in the studied papaya plants. Results of our study are likely to facilitate Pakistani papaya breeders and growers to incorporate DNA based screening at juvenile stage to determine sex at early stage and to ensure profitable papaya production. (author)

Divergent evolutionary histories of DNA markers in a Hawaiian population of the coral Montipora capitata

OpenAIRE

Hollie M. Putnam; Diane K. Adams; Ehud Zelzion; Nicole E. Wagner; Huan Qiu; Tali Mass; Paul G. Falkowski; Ruth D. Gates; Debashish Bhattacharya

2017-01-01

We investigated intra- and inter-colony sequence variation in a population of the dominant Hawaiian coral Montipora capitata by analyzing marker gene and genomic data. Ribosomal ITS1 regions showed evidence of a reticulate history among the colonies, suggesting incomplete rDNA repeat homogenization. Analysis of the mitochondrial genome identified a major (M. capitata) and a minor (M. flabellata) haplotype in single polyp-derived sperm bundle DNA with some colonies containing 2?3 different mtD...

Effectiveness of ITS and sub-regions as DNA barcode markers for the identification of Basidiomycota (Fungi).

Science.gov (United States)

Badotti, Fernanda; de Oliveira, Francislon Silva; Garcia, Cleverson Fernando; Vaz, Aline Bruna Martins; Fonseca, Paula Luize Camargos; Nahum, Laila Alves; Oliveira, Guilherme; Góes-Neto, Aristóteles

2017-02-23

Fungi are among the most abundant and diverse organisms on Earth. However, a substantial amount of the species diversity, relationships, habitats, and life strategies of these microorganisms remain to be discovered and characterized. One important factor hindering progress is the difficulty in correctly identifying fungi. Morphological and molecular characteristics have been applied in such tasks. Later, DNA barcoding has emerged as a new method for the rapid and reliable identification of species. The nrITS region is considered the universal barcode of Fungi, and the ITS1 and ITS2 sub-regions have been applied as metabarcoding markers. In this study, we performed a large-scale analysis of all the available Basidiomycota sequences from GenBank. We carried out a rigorous trimming of the initial dataset based in methodological principals of DNA Barcoding. Two different approaches (PCI and barcode gap) were used to determine the performance of the complete ITS region and sub-regions. For most of the Basidiomycota genera, the three genomic markers performed similarly, i.e., when one was considered a good marker for the identification of a genus, the others were also; the same results were observed when the performance was insufficient. However, based on barcode gap analyses, we identified genomic markers that had a superior identification performance than the others and genomic markers that were not indicated for the identification of some genera. Notably, neither the complete ITS nor the sub-regions were useful in identifying 11 of the 113 Basidiomycota genera. The complex phylogenetic relationships and the presence of cryptic species in some genera are possible explanations of this limitation and are discussed. Knowledge regarding the efficiency and limitations of the barcode markers that are currently used for the identification of organisms is crucial because it benefits research in many areas. Our study provides information that may guide researchers in choosing

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.

Elucidating polyploidization of bermudagrasses as assessed by organelle and nuclear DNA markers.

Science.gov (United States)

Gulsen, Osman; Ceylan, Ahmet

2011-12-01

Clarification of relationships among ploidy series of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to elucidate polyploidization among Cynodon accessions with different ploidy series collected from Turkey based on chloroplast and nuclear DNA. Forty Cynodon accessions including 7 diploids, 3 triploids, 10 tetraploids, 11 pentaploids, and 9 hexaploids were analyzed using chloroplast DNA restriction fragment-length polymorphism (cpDNA RFLP), chloroplast DNA simple sequence repeat (cpDNA SSR), and nuclear DNA markers based on neighbor-joining (NJ) and principle component analyses (PCA). All three-marker systems with two statistical algorithms clustered the diploids apart from the other ploidy levels. Assuming autopolyploidy, spontaneous polyploidization followed by rapid diversification among the higher ploidy levels than the diploids is likely in Cynodon's evolution. Few tetraploid and hexaploid accessions were clustered with or closely to the group of diploids, supporting the hypothesis above. Eleven haplotypes as estimated by cpDNA RFLP and SSR markers were detected. This study indicated that the diploids had different organelle genome from the rest of the ploidy series and provided valuable insight into relationships among ploidy series of Cynodon accessions based on cp and nuclear DNAs.

Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma

Directory of Open Access Journals (Sweden)

Hagen Jeffrey A

2007-10-01

Full Text Available Abstract Background Lung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients. Results We identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value Conclusion The identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.

Salivary DNA and markers of oxidative stress in patients with chronic periodontitis.

Science.gov (United States)

Baňasová, Lenka; Kamodyová, Natália; Janšáková, Katarína; Tóthová, Ľubomíra; Stanko, Peter; Turňa, Ján; Celec, Peter

2015-03-01

Previous observational studies have shown that periodontal status is associated with salivary markers of oxidative damage. A direct comparison of periodontitis patients and controls using a wide palette of salivary markers of oxidative stress is lacking. Characteristics of salivary DNA in periodontitis are unknown. The aim of this study was to compare the salivary markers of oxidative stress and characteristics of salivary DNA between patients with chronic periodontitis and periodontitis-free controls. Saliva was collected from 23 patients with chronic periodontitis and 19 periodontitis-free controls. All participants underwent a clinical periodontal examination. Markers of oxidative and carbonyl stress were measured in saliva. Human and bacterial DNA was quantified, and human DNA integrity was assessed. Salivary thiobarbituric acid-reacting substances were higher in patients than in controls; at least in men, the difference was significant (p periodontitis patients. The results confirmed the association of salivary thiobarbituric acid-reacting substances with periodontitis. Lipid peroxidation in periodontitis seems to be caused by increased production of reactive oxygen species in men and by decreased antioxidant status in women. Whether lower salivary DNA integrity is involved in the pathogenesis of periodontitis remains to be elucidated. Salivary thiobarbituric acid-reacting substances are associated with periodontitis at least on a population level. Sex-specific causes of lipid peroxidation might point towards different pathogenic mechanisms.

Application of DNA markers against illegal logging as a new tool for the Forest Guard Service

OpenAIRE

Nowakowska, Justyna A.

2011-01-01

DNA markers are currently the most precise tool for forest tree species identification and can be used for comparative analyses of plant material. Molecular diagnosis of evidence and reference material is based on comparing the structure of DNA markers duplicated in the PCR reaction and estimation of the DNA profiles obtained in studied wood samples. For this purpose, the microsatellite DNA markers are the most suitable tool because of their high polymorphism and accurate detection of structu...

Filipino DNA variation at 12 X-chromosome short tandem repeat markers.

Science.gov (United States)

Salvador, Jazelyn M; Apaga, Dame Loveliness T; Delfin, Frederick C; Calacal, Gayvelline C; Dennis, Sheila Estacio; De Ungria, Maria Corazon A

2018-06-08

Demands for solving complex kinship scenarios where only distant relatives are available for testing have risen in the past years. In these instances, other genetic markers such as X-chromosome short tandem repeat (X-STR) markers are employed to supplement autosomal and Y-chromosomal STR DNA typing. However, prior to use, the degree of STR polymorphism in the population requires evaluation through generation of an allele or haplotype frequency population database. This population database is also used for statistical evaluation of DNA typing results. Here, we report X-STR data from 143 unrelated Filipino male individuals who were genotyped via conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) using the 12 X-STR loci included in the Investigator ® Argus X-12 kit (Qiagen) and via massively parallel sequencing (MPS) of seven X-STR loci included in the ForenSeq ™ DNA Signature Prep kit of the MiSeq ® FGx ™ Forensic Genomics System (Illumina). Allele calls between PCR-CE and MPS systems were consistent (100% concordance) across seven overlapping X-STRs. Allele and haplotype frequencies and other parameters of forensic interest were calculated based on length (PCR-CE, 12 X-STRs) and sequence (MPS, seven X-STRs) variations observed in the population. Results of our study indicate that the 12 X-STRs in the PCR-CE system are highly informative for the Filipino population. MPS of seven X-STR loci identified 73 X-STR alleles compared with 55 X-STR alleles that were identified solely by length via PCR-CE. Of the 73 sequence-based alleles observed, six alleles have not been reported in the literature. The population data presented here may serve as a reference Philippine frequency database of X-STRs for forensic casework applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and Molecular Characterization of Novel Polymorphic Genomic DNA SSR Markers in Lentinula edodes.

Science.gov (United States)

Moon, Suyun; Lee, Hwa-Yong; Shim, Donghwan; Kim, Myungkil; Ka, Kang-Hyeon; Ryoo, Rhim; Ko, Han-Gyu; Koo, Chang-Duck; Chung, Jong-Wook; Ryu, Hojin

2017-06-01

Sixteen genomic DNA simple sequence repeat (SSR) markers of Lentinula edodes were developed from 205 SSR motifs present in 46.1-Mb long L. edodes genome sequences. The number of alleles ranged from 3-14 and the major allele frequency was distributed from 0.17-0.96. The values of observed and expected heterozygosity ranged from 0.00-0.76 and 0.07-0.90, respectively. The polymorphic information content value ranged from 0.07-0.89. A dendrogram, based on 16 SSR markers clustered by the paired hierarchical clustering' method, showed that 33 shiitake cultivars could be divided into three major groups and successfully identified. These SSR markers will contribute to the efficient breeding of this species by providing diversity in shiitake varieties. Furthermore, the genomic information covered by the markers can provide a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection in the shiitake mushroom.

Supplementary data: Development of nuclear DNA markers for ...

Indian Academy of Sciences (India)

Supplementary data: Development of nuclear DNA markers for evolutionary studies in Plasmodium falciparum. Celia Thomas, Sneh Shalini, N. Raghavendra, Meenakshi Choudhary, Anju Verma, Hema Joshi,. A. P. Dash and Aparup Das. J. Genet. 86, 65–68. Primer sequences for amplification of putatively neutral ...

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding.

Science.gov (United States)

Hernández, P; Dorado, G; Ramírez, M C; Laurie, D A; Snape, J W; Martín, A

2003-01-01

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.

Identification of three randomly amplified polymorphic DNA-polymerase chain reaction markers for distinguishing Asian and North American Gypsy Moths (Lepidoptera: Lymantriidae)

Science.gov (United States)

David E. Schreiber; Karen J. Garner; James M. Slavicek

1997-01-01

Gypsy moths originating in Asia have recently been introduced into North America, making it necessary to develop markers for distinguishing the Asian strain from the established North American population. We have identified 3 randomly amplified polymorphic DNA-polymerase chain reaction generated (RAPD-PCR) markers which are specific for either Asian or North American...

Epigenetic markers in circulating cell-free DNA as prognostic markers for survival of castration-resistant prostate cancer patients.

Science.gov (United States)

Hendriks, Rianne J; Dijkstra, Siebren; Smit, Frank P; Vandersmissen, Johan; Van de Voorde, Hendrik; Mulders, Peter F A; van Oort, Inge M; Van Criekinge, Wim; Schalken, Jack A

2018-04-01

Noninvasive biomarkers to guide personalized treatment for castration-resistant prostate cancer (CRPC) are needed. In this study, we analyzed hypermethylation patterns of two genes (GSTP1 and APC) in plasma cell-free DNA (cfDNA) of CRPC patients. The aim of this study was to analyze the cfDNA concentrations and levels of the epigenetic markers and to assess the value of these biomarkers for prognosis. In this prospective study, patients were included before starting new treatment after developing CRPC. The blood samples were collected prior to start of the treatment and at three time points thereafter. cfDNA was extracted from 1.5 mL of plasma and before performing a methylation-specific PCR, bisulfate modification was carried out. The median levels of cfDNA, GSTP1, and APC copies in the baseline samples of CRPC patients (n = 47) were higher than in controls (n = 30). In the survival analysis, the group with baseline marker levels below median had significant less PCa-related deaths (P-values Prostate Published by Wiley Periodicals, Inc.

DNA markers linked to the major salinity tolerance locus of traditional rice, Pokkali (abstract)

International Nuclear Information System (INIS)

Rehman, S.; Seraj, Z.I.; Das, D.K.; Salam, M.A.

2005-01-01

The major QTL for salinity tolerance traits, of the traditional rice salt tolerant benchmark Pokkali, referred to as 'Saltol' was located within a large 16cM loci of rice chromosome 1 by previous workers at IRRI. This was done by using a recombinant inbred population between Pokkali and sensitive IR29 (Total RILs=275). These workers had identified the flanking markers, RM23 and RM9, as the limits of 'Saltol'. By designing primers between these two markers, and using a subset of the same RILs, we were able to identify a 5cM region, which was completely linked to the tolerance of seedlings. Further work with a subset of another NIL population raised at IRRI between Pokkali and recurring IR29 at the BC/sub 3/F/sub 2/ stage has narrowed down the linked region to about 0.3cM, each at 4 different locations within the 5cM loc. This was done by scoring the tolerance of the seedlings and determining the percent of progeny that showed the tolerant allele at the specified maker locus. Thirty seedlings from each of 10 BC/sub 3/F/sub 2/ progeny were scored. Only the most tolerant and sensitive seedlings were used for DNA isolation and amplification. The work was derived from complex crosses involving Pokkali as the tolerance donor. Three common loci linked to salinity tolerance were found to be the same in the NILs and the breeding population. DNA markers homologous to these 3 loci will be confirmed for their ability to identify tolerant progeny in breeding populations. (author)

Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

Science.gov (United States)

Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

2017-04-01

Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

Science.gov (United States)

Torelli, A; Soragni, E; Bolchi, A; Petrucco, S; Ottonello, S; Branca, C

1996-12-01

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Selection Of Drought Resistant Mutants In Rice Using DNA Markers

International Nuclear Information System (INIS)

Nguyen Duc Thanh; Le Thi Bich Thuy; Dang Thi Minh Lua

2008-01-01

In recent years, the marker - assisted selection (MAS) strategy have been used for selection of traits that are difficult and costly performed measurement and score. Selection for a well-developed root system could improve the drought resistance of rice as the plant would avoid water stress by absorbing water from the soil. There were several reports on map construction and identification of the markers tightly linked to morphological and physiological traits related to drought resistance in rice, in particular, root traits in upland and lowland rice (Champoux et al., 1995; Ray et al., 1996; Price et al., 1997, 2000; Yadav et al., 1997). In this report, we present the results on selection of drought resistance mutants in rice using the DNA markers tightly linked to root traits favorable for drought resistance. The mutant rice lines were obtained from irradiated seeds and calluses by gamma ray. The selection was performed at M2 mutants using the DNA markers linked to maximum root length (MRL), root weight to shoot weight ratio (RW/SR), and weight of deep root to shoot weight ratio (DRW/SR). The obtained results showed that there were many lines possessed drought resistant markers. In addition, there is a number of lines have altered genome. Several lines having drought markers proved to be more resistant to drought in green-house test. These lines could be useful for further test and development of drought resistant varieties. (author)

Identifying Contributors of DNA Mixtures by Means of Quantitative Information of STR Typing

DEFF Research Database (Denmark)

Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

2012-01-01

identified using polymorphic genetic markers. However, modern typing techniques supply additional quantitative data, which contain very important information about the observed evidence. This is particularly true for cases of DNA mixtures, where more than one individual has contributed to the observed......Abstract Estimating the weight of evidence in forensic genetics is often done in terms of a likelihood ratio, LR. The LR evaluates the probability of the observed evidence under competing hypotheses. Most often, probabilities used in the LR only consider the evidence from the genomic variation...... biological stain. This article presents a method for including the quantitative information of short tandem repeat (STR) DNA mixtures in the LR. Also, an efficient algorithmic method for finding the best matching combination of DNA mixture profiles is derived and implemented in an on-line tool for two...

A case study characterizing animal fecal sources in surface water using a mitochondrial DNA marker.

Science.gov (United States)

Bucci, John P; Shattuck, Michelle D; Aytur, Semra A; Carey, Richard; McDowell, William H

2017-08-01

Water quality impairment by fecal waste in coastal watersheds is a public health issue. The present study provided evidence for the use of a mitochondrial (mtDNA) marker to detect animal fecal sources in surface water. The accurate identification of fecal pollution is based on the notion that fecal microorganisms preferentially inhabit a host animal's gut environment. In contrast, mtDNA host-specific markers are inherent to eukaryotic host cells, which offers the advantage by detecting DNA from the host rather than its fecal bacteria. The present study focused on sampling water presumably from non-point sources (NPS), which can increase bacterial and nitrogen concentrations to receiving water bodies. Stream sampling sites located within the Piscataqua River Watershed (PRW), New Hampshire, USA, were sampled from a range of sites that experienced nitrogen inputs such as sewer and septic systems and suburban runoff. Three mitochondrial (mtDNA) gene marker assays (human, bovine, and canine) were tested from surface water. Nineteen sites were sampled during an 18-month period. Analyses of the combined single and multiplex assay results showed that the proportion of occurrence was highest for bovine (15.6%; n = 77) compared to canine (5.6%; n = 70) and human (5.7%; n = 107) mtDNA gene markers. For the human mtDNA marker, there was a statistically significant relationship between presence vs. absence and land use (Fisher's test p = 0.0031). This result was evident particularly for rural suburban septic, which showed the highest proportion of presence (19.2%) compared to the urban sewered (3.3%), suburban sewered (0%), and agricultural (0%) as well as forested septic (0%) sites. Although further testing across varied land use is needed, our study provides evidence for using the mtDNA marker in large watersheds.

An environmental DNA marker for detecting nonnative brown trout (Salmo trutta)

Science.gov (United States)

K. J. Carim; T. M. Wilcox; M. Anderson; D. Lawrence; Michael Young; Kevin McKelvey; Michael Schwartz

2016-01-01

Brown trout (Salmo trutta) are widely introduced in western North America where their presence has led to declines of several native species. To assist conservation efforts aimed at early detection and eradication of this species, we developed a quantitative PCR marker to detect the presence of brown trout DNA in environmental samples. The marker strongly...

Development of swine-specific DNA markers for biosensor-based halal authentication.

Science.gov (United States)

Ali, M E; Hashim, U; Kashif, M; Mustafa, S; Che Man, Y B; Abd Hamid, S B

2012-06-29

The pig (Sus scrofa) mitochondrial genome was targeted to design short (15-30 nucleotides) DNA markers that would be suitable for biosensor-based hybridization detection of target DNA. Short DNA markers are reported to survive harsh conditions in which longer ones are degraded into smaller fragments. The whole swine mitochondrial-genome was in silico digested with AluI restriction enzyme. Among 66 AluI fragments, five were selected as potential markers because of their convenient lengths, high degree of interspecies polymorphism and intraspecies conservatism. These were confirmed by NCBI blast analysis and ClustalW alignment analysis with 11 different meat-providing animal and fish species. Finally, we integrated a tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in deliberately contaminated autoclaved pork-beef binary mixtures and no cross-species detection was recorded, demonstrating the feasibility of this type of probe for biosensor-based detection of pork adulteration of halal and kosher foods.

Antibody Arrays Identify Potential Diagnostic Markers of Hepatocellular Carcinoma

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Brian J. Peter

2008-01-01

Full Text Available Hepatocellular carcinoma (HCC is the third leading cause of cancer deaths worldwide. Effective treatment of HCC patients is hampered by the lack of sensitive and specific diagnostic markers of HCC. Alpha-fetoprotein (AFP, the currently used HCC marker, misses 30%–50% of HCC patients, who therefore remain undiagnosed and untreated. In order to identify novel diagnostic markers that can be used individually or in combination with AFP, we used an antibody array platform to detect the levels of candidate proteins in the plasma of HCC patients (n = 48 and patients with chronic hepatitis B or C viral infections (n = 19 (both of which are the major risk factors of HCC. We identified 7 proteins that significantly differentiate HCC patients from hepatitis patients (p < 0.05 (AFP, CTNNB, CSF1, SELL, IGFBP6, IL6R, and VCAM1.Importantly, we also identified 8 proteins that significantly differentiate HCC patients with ‘normal’ levels of AFP (<20 ng/ml from hepatitis patients (p < 0.05 (IL1RN, IFNG, CDKN1A, RETN, CXCL14, CTNNB, FGF2, and SELL. These markers are potentially important complementary markers to AFP. Using an independent immunoassay method in an independent group of 23 HCC patients and 22 hepatitis patients, we validated that plasma levels of CTNNB were significantly higher in the HCC group (p = 0.020. In conclusion, we used an antibody array platform to identify potential circulating diagnostic markers of HCC, some of which may be valuable when used in combination with AFP. The clinical utility of these newly identified HCC diagnostic markers needs to be systematically evaluated.

DNA damage markers in dermal fibroblasts in vitro reflect chronological donor age

DEFF Research Database (Denmark)

Waaijer, Mariëtte E C; Croco, Eleonora; Westendorp, Rudi G J

2016-01-01

The aging process is accompanied by an accumulation of cellular damage, which compromises the viability and function of cells and tissues. We aim to further explore the association between in vitro DNA damage markers and the chronological age of the donor, as well as long-lived family membership...... markers and long-lived family membership or cardiovascular disease. Results were comparable when fibroblasts were stressed in vitro with rotenone. In conclusion, we found that DNA damage foci of cultured fibroblasts are significantly associated with the chronological age, but not biological age...

The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae

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Marco Ballardini

2013-12-01

Full Text Available The genus Phoenix (Arecaceae comprises 14 species distributed from Cape Verde Islands to SE Asia. It includes the economically important species Phoenix dactylifera. The paucity of differential morphological and anatomical useful characters, and interspecific hybridization, make identification of Phoenix species difficult. In this context, the development of reliable DNA markers for species and hybrid identification would be of great utility. Previous studies identified a 12 bp polymorphic chloroplast minisatellite in the trnG(GCC-trnfM(CAU spacer, and showed its potential for species identification in Phoenix. In this work, in order to develop an efficient DNA barcode marker for Phoenix, a longer cpDNA region (700 bp comprising the mentioned minisatellite, and located between the psbZ and trnfM(CAU genes, was sequenced. One hundred and thirty-six individuals, representing all Phoenix species except P. andamanensis, were analysed. The minisatellite showed 2-7 repetitions of the 12 bp motif, with 1-3 out of seven haplotypes per species. Phoenix reclinata and P. canariensis had species-specific haplotypes. Additional polymorphisms were found in the flanking regions of the minisatellite, including substitutions, indels and homopolymers. All this information allowed us to identify unambiguously eight out of the 13 species, and overall 80% of the individuals sampled. Phoenix rupicola and P. theophrasti had the same haplotype, and so had P. atlantica, P. dactylifera, and P. sylvestris (the “date palm complex” sensu Pintaud et al. 2013. For these species, additional molecular markers will be required for their unambiguous identification. The psbZ-trnfM(CAU region therefore could be considered as a good basis for the establishment of a DNA barcoding system in Phoenix, and is potentially useful for the identification of the female parent in Phoenix hybrids.

DNA Fingerprinting of Olive Varieties by Microsatellite Markers

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Dunja Bandelj

2002-01-01

Full Text Available Microsatellites combine several features of an ultimate molecular marker and they are used increasingly in various plant genetic studies and applications. In this work we report on the utilisation of fourteen previously developed olive microsatellite markers for the identification and differentiation of a set of nineteen olive varieties. All analysed microsatellite markers revealed a high level of polymorphism that allowed unique genotyping of the examined varieties. Ninety-six alleles were detected at all 14 loci, which multiplied into a large number of observed genotypes, giving high discrimination value for varietal identification. A minimum number of three microsatellite markers was chosen for the rapid and unambiguous varietal identification of nineteen olive varieties and only two markers were sufficient for differentiation of five local varieties. DNA fingerprints of olive cultivars by means of microsatellites provided meaningful data, which can be extended by additional olive varieties or new microsatellites and used for accurate inter-laboratory comparison. The data obtained can be used for the varietal survey and construction of a database of all olive varieties grown in Slovenia providing also additional genetic information on the agronomic and quality characteristics of the olive varieties.

DNA-based genetic markers for Rapid Cycling Brassica rapa (Fast Plants type designed for the teaching laboratory.

Directory of Open Access Journals (Sweden)

Eryn E. Slankster

2012-06-01

Full Text Available We have developed DNA-based genetic markers for rapid-cycling Brassica rapa (RCBr, also known as Fast Plants. Although markers for Brassica rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP based markers and 14 variable number tandem repeat (VNTR-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a nongenic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology.

IMPLEMENTATION OF DNA MARKERS TO IMPROVE BREEDING OF FORAGE LEGUMES

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S. Grljušić

2008-09-01

Full Text Available The low rates of estimated genetic gains in forage legumes breeding have emphasized the need for new breeding methods that would increase efficiency in forage selection and provide reliable improvement. Information on application of molecular methodologies and tools for the enhancement of the current empirical phenotype-based selection moved us toward implementation of DNA markers to our breeding activities. Firstly, attention was given to identification of genetic variability within the forage species involved in program and comparison of conventional and molecular marker efficiency in variability evaluation. RAPDs were used (i to estimate availability of alfalfa (Medicago sativa L. and Medicago falcata L. genetic variation and (ii to identify changes of red clover (Trifolium pratense L. variability after natural selection. SSRs were applied to evaluate diversity within and among field pea (Pisum sativum L. var. arvense and sativum groups/varieties. A total of 90 (alfalfa or 92 (red clover polymorphic bands was found by RAPDs. Total number of SSR alleles recorded was 118. The average Roger's distance per species/genus estimated was 0.29 (red clover, 0.33 (alfalfa and 0.51 (field pea. 2D PCo analysis of each species/genus separated materials into respective groups. A high degree of genetic variation within populations/varieties of each investigated species was found by AMOVA. The correspondence between pairs of matrices based on the morphological and molecular data was significant (p=0.95 only for red clover. RAPD and SSR data have given valuable information on genetic structure of materials and provided a description that determines heterogeneity. Further studies will be focused on identifying quantitative trait loci and marker assisted selection.

The use of DNA markers for rapid improvement of crops in Africa ...

African Journals Online (AJOL)

Genetic engineering and biotechnology are providing new tools for genetic improvement of food crops. Molecular DNA markers are some of these tools which can be used in various fields of plant breeding and germplasm management. For example, molecular markers have been used to confirm the identity of hybrids in ...

Analysis of relationship between tumor markers and quantification of free DNA in serum of lung cancer patients

International Nuclear Information System (INIS)

Yang Shunfang; Zhang Peiling; Cao Jie; Zeng Jun; Dong Qianggang

2006-01-01

To evaluate the diagnostic value and relationship between five tumor markers (CA19- 9,CA125,CYFRA21-1 ,CEA,NSE) and free DNA in serum for lung cancer detection and try to find a new and more efficient tumor marker, the amounts of CA19-9, CA125, CYFRA21-1, CEA, NSE were determined by RIA and free DNA was determined by the use of quantitative real time PCR amplification of the human epidermal growth factor receptor (EGFR) in 52 lung cancer patients and 8 cases of benign pulmonary disease and 10 healthy controls. The resulls showed that average concentration of free DNA in serum of lung cancer patients, benign pulmo- nary disease and healthy controls was 107.6ng/mL, 76.86ng/mL and 18.8ng/mL, respective- ly. The diagnostic sensitivity, specificity and accuracy of free DNA for lung cancer were 71. 2%, 50% and 68.3%, same as the diagnostic value of combined detection of five tumor markers. The sensitivity, specificity and accuracy of the five tumor markers and free DNA combinend detection for lung cancer were 94.2%, 25% and 85%, respectively. The free DNA in the serum of lung cancer patients may be a new and better tumor marker. (authors)

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.

[Norrie syndrome: identification of carriers by segregation analysis with flanking DNA markers].

Science.gov (United States)

Körner, J; Uhlhaas, S; Neugebauer, M; Gal, A

1989-01-01

Norrie disease is an X-linked recessive disorder. Affected males present with congenital blindness. Additionally, hearing loss and psychotic behavior may occur at any time. Since carriers are clinically healthy, they can only be identified by genetic means. Daughters of carriers or sisters of affected males have an à priori 50% risk of being carriers themselves. Close linkage has been found between the Norrie disease locus (NDP) and the DNA locus DXS7 mapped to Xp11.3. For genetic counselling, this linkage relationship allows carriers of the disease to be identified in informative families. We describe a large pedigree with Norrie disease. Segregation analysis was carried out with DXS7 and a second flanking marker, DXS255, both linked to NDP. In this way, three females at risk were identified who had a high probability of being carriers for Norrie disease.

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

Science.gov (United States)

2010-01-01

Background Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression. Results The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system. Conclusions In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts

Circulating, cell-free DNA as a marker for exercise load in intermittent sports.

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Nils Haller

Full Text Available Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of cfDNA due to repeated maximal sprints and due to a professional football game.Nine participants were subjected to a standardised sprint training session with cross-over design of five maximal sprints of 40 meters with either "short" (1 minute or "long" pauses (5 minutes. Capillary cfDNA and lactate were measured after every sprint and venous cfDNA before and after each series of sprints. Moreover, capillary cfDNA and lactate values were taken in 23 professional football players before and after incremental exercise testing, during the course of a training week at rest (baseline and in all 17 enrolled players following a season game.Lactate and venous cfDNA increased more pronounced during "short" compared to "long" (1.4-fold, p = 0.032 and 1.7-fold, p = 0.016 and cfDNA correlated significantly with lactate (r = 0.69; p<0.001. Incremental exercise testing increased cfDNA 7.0-fold (p<0.001. The season game increased cfDNA 22.7-fold (p<0.0001, while lactate showed a 2.0-fold (p = 0.09 increase compared to baseline. Fold-changes in cfDNA correlated with distance covered during game (spearman's r = 0.87, p = 0.0012, while no correlation between lactate and the tracking data could be found.We show for the first time that cfDNA could be an objective marker for distance covered in elite intermittent sports. In contrast to the potential of more established blood-based markers like IL-6, CK, or CRP, cfDNA shows by far the strongest fold-change and a high correlation with a

Integrating microsatellite DNA markers and otolith geochemistry to assess population structure of European hake (Merluccius merluccius)

Science.gov (United States)

Tanner, Susanne E.; Pérez, Montse; Presa, Pablo; Thorrold, Simon R.; Cabral, Henrique N.

2014-04-01

Population structure and natal origins of European hake were investigated using microsatellite DNA markers and otolith geochemistry data. Five microsatellites were sequenced and otolith core geochemical composition was determined from age-1 hake collected in the northeast Atlantic Ocean and the Mediterranean Sea. Microsatellites provided evidence of a major genetic split in the vicinity of the Strait of Gibraltar, separating the Atlantic and the Mediterranean populations, with the exception of the Gulf of Cádiz. Based on classification models using otolith core geochemical values, individual natal origins were identified, although with an increased error rate. Coupling genotype and otolith data increased the classification accuracy of individuals to their potential natal origins while providing evidence of movement between the northern and southern stock units in the Atlantic Ocean. Information obtained by the two natural markers on population structure of European hake was complementary as the two markers act at different spatio-temporal scales. Otolith geochemistry provides information over an ecological time frame and on a fine spatial scale, while microsatellite DNA markers report on gene flow over evolutionary time scales and therefore act on a broader spatio-temporal resolution. Thus, this study confirmed the value of otolith geochemistry to complement the assessment of early life stage dispersal in populations with high gene flow and low genetic divergence.

A novel PCR-based marker for identifying Ns chromosomes in wheat-Psathyrostachys huashanica Keng derivative lines

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J. Wang

2013-10-01

Full Text Available Psathyrostachys huashanica Keng is an endangered species that is endemic to China, which provides an important gene pool for wheat improvement. We developed a quick and reliable PCR-based diagnostic assay to accurately and efficiently detect P. huashanica DNA sequences from introgression lines, which was based on a species-specific marker derived from genomic DNA. The 900-bp PCR-amplified band used as a P. huashanica-specific RAPD marker was tested with 21 different plant species and was converted into a sequence-characterized amplified region (SCAR marker by cloning and sequencing the selected fragments (pHs11. This SCAR marker, which was designated as RHS23, could clearly distinguish the presence of P. huashanica DNA repetitive sequences in wheat-P. huashanica derivative lines. The specificity of the marker was validated using 21 different plant species and a complete set of wheat-P. huashanica disomic addition lines (1Ns–7Ns, 2n=44=22II. This specific sequence targeted the Ns genome of P. huashanica and it was present in all the seven P. huashanica chromosomes. Therefore, this SCAR marker is specific for P. huashanica chromosomes and may be used in the identification of alien repetitive sequences in large gene pools. This diagnostic PCR assay for screening the target genetic material may play a key role in marker-assisted selective breeding programs.

Genetic diversity of sago palm in Indonesia based on chloroplast DNA (cpDNA markers

Directory of Open Access Journals (Sweden)

MEMEN SURAHMAN

2010-07-01

Full Text Available Abbas B, Renwarin Y, Bintoro MH, Sudarsono, Surahman M, Ehara H (2010 Genetic diversity of sago palm in Indonesia based on chloroplast DNA (cpDNA markers. Biodiversitas 11: 112-117. Sago palm (Metroxylon sagu Rottb. was believed capable to accumulate high carbohydrate content in its trunk. The capability of sago palm producing high carbohydrate should be an appropriate criterion for defining alternative crops in anticipating food crisis. The objective of this research was to study genetic diversity of sago palm in Indonesia based on cpDNA markers. Total genome extraction was done following the Qiagen DNA isolation protocols 2003. Single Nucleotide Fragments (SNF analyses were performed by using ABI Prism GeneScanR 3.7. SNF analyses detected polymorphism revealing eleven alleles and ten haplotypes from total 97 individual samples of sago palm. Specific haplotypes were found in the population from Papua, Sulawesi, and Kalimantan. Therefore, the three islands will be considered as origin of sago palm diversities in Indonesia. The highest haplotype numbers and the highest specific haplotypes were found in the population from Papua suggesting this islands as the centre and the origin of sago palm diversities in Indonesia. The research had however no sufficient data yet to conclude the Papua origin of sago palm. Genetic hierarchies and differentiations of sago palm samples were observed significantly different within populations (P=0.04574, among populations (P=0.04772, and among populations within the island (P=0.03366, but among islands no significant differentiations were observed (P= 0.63069.

Nuclear internal transcribed spacer-1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio.

Science.gov (United States)

Minamoto, Toshifumi; Uchii, Kimiko; Takahara, Teruhiko; Kitayoshi, Takumi; Tsuji, Satsuki; Yamanaka, Hiroki; Doi, Hideyuki

2017-03-01

The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio. © 2016 John Wiley & Sons Ltd.

DNA marker mining of ILSTS035 microsatellite locus on ...

Indian Academy of Sciences (India)

Unknown

We describe tests for detecting and locating quantitative trait loci (QTL) for traits in Hanwoo cattle. From results of a permutation test to detect QTL for marbling, we selected the microsatellite locus ILSTS035 on chromosome 6 for further analysis. K-means clustering analysis applied to five traits and nine DNA markers in ...

Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos genome and cross-amplification in other birds

Directory of Open Access Journals (Sweden)

Xu Ke

2005-07-01

Full Text Available Abstract In order to study duck microsatellites, we constructed a library enriched for (CAn, (CAGn, (GCCn and (TTTCn. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97 was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04 at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%. The polymorphism information content (PIC of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose.

Classification of plant associated bacteria using RIF, a computationally derived DNA marker.

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Kevin L Schneider

Full Text Available A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF. Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS. Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF

Circulating, cell-free DNA as a marker for exercise load in intermittent sports

OpenAIRE

Haller, Nils; Helmig, Susanne; Taenny, Pascal; Petry, Julian; Schmidt, Sebastian; Simon, Perikles

2018-01-01

Background Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA) have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of ...

SMM-system: A mining tool to identify specific markers in Salmonella enterica.

Science.gov (United States)

Yu, Shuijing; Liu, Weibing; Shi, Chunlei; Wang, Dapeng; Dan, Xianlong; Li, Xiao; Shi, Xianming

2011-03-01

This report presents SMM-system, a software package that implements various personalized pre- and post-BLASTN tasks for mining specific markers of microbial pathogens. The main functionalities of SMM-system are summarized as follows: (i) converting multi-FASTA file, (ii) cutting interesting genomic sequence, (iii) automatic high-throughput BLASTN searches, and (iv) screening target sequences. The utility of SMM-system was demonstrated by using it to identify 214 Salmonella enterica-specific protein-coding sequences (CDSs). Eighteen primer pairs were designed based on eighteen S. enterica-specific CDSs, respectively. Seven of these primer pairs were validated with PCR assay, which showed 100% inclusivity for the 101 S. enterica genomes and 100% exclusivity of 30 non-S. enterica genomes. Three specific primer pairs were chosen to develop a multiplex PCR assay, which generated specific amplicons with a size of 180bp (SC1286), 238bp (SC1598) and 405bp (SC4361), respectively. This study demonstrates that SMM-system is a high-throughput specific marker generation tool that can be used to identify genus-, species-, serogroup- and even serovar-specific DNA sequences of microbial pathogens, which has a potential to be applied in food industries, diagnostics and taxonomic studies. SMM-system is freely available and can be downloaded from http://foodsafety.sjtu.edu.cn/SMM-system.html. Copyright © 2011 Elsevier B.V. All rights reserved.

DNA markers for forensic identification of non-human biological traces

NARCIS (Netherlands)

Wesselink, M.

2018-01-01

In this thesis, DNA markers are described that enable forensically relevant classification of three groups of non-human biological traces: fungi (Chapter 1), domestic cats (Chapters 2, 3 an d 4) and birch trees (Chapters 5 and 6). Because the forensic questions associated with these traces require

Circulating, cell-free DNA as a marker for exercise load in intermittent sports.

Science.gov (United States)

Haller, Nils; Helmig, Susanne; Taenny, Pascal; Petry, Julian; Schmidt, Sebastian; Simon, Perikles

2018-01-01

Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA) have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of cfDNA due to repeated maximal sprints and due to a professional football game. Nine participants were subjected to a standardised sprint training session with cross-over design of five maximal sprints of 40 meters with either "short" (1 minute) or "long" pauses (5 minutes). Capillary cfDNA and lactate were measured after every sprint and venous cfDNA before and after each series of sprints. Moreover, capillary cfDNA and lactate values were taken in 23 professional football players before and after incremental exercise testing, during the course of a training week at rest (baseline) and in all 17 enrolled players following a season game. Lactate and venous cfDNA increased more pronounced during "short" compared to "long" (1.4-fold, p = 0.032 and 1.7-fold, p = 0.016) and cfDNA correlated significantly with lactate (r = 0.69; psports. In contrast to the potential of more established blood-based markers like IL-6, CK, or CRP, cfDNA shows by far the strongest fold-change and a high correlation with a particular load related aspect in professional football.

MPO cDNA clone identifies an RFLP with PstI

Energy Technology Data Exchange (ETDEWEB)

Miki, T; Weil, S C; Rosner, G L; Reid, M S; Kidd, K K

1988-02-25

A myeloperoxidase (MPO) cDNA clone (pHMP7: 270 base pair insert in the vector pGEM-1reverse arrow was isolated from a library created from human promyelocytic (HL-60) cell mRNA. PstI (CTGCA/G) (New England Biolabs) identifies a simple two-allele polymorphism with bands at either 2.2 kb (Al) or 2.0 kb (A2). There are three constant bands at 2.8 kb, 0.95 kb and 0.6 kb. Preliminary family data show evidence of linkage to several markers in proximal 17q, with MPO closest to the Growth Hormone cluster at 17q22-q24. Autosomal condominant segregation was observed in four large reference pedigrees with several informative matings.

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)

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers.

Science.gov (United States)

Siew, Ging Yang; Ng, Wei Lun; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Tan, Soon Guan; Yeap, Swee Keong

2018-01-01

Durian ( Durio zibethinus ) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, H E  = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10 -3 . Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called "clones", "varieties", or "cultivars". Such matters have a direct impact on the regulation and management of durian genetic resources in the region.

Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

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Vasco Elbrecht

2016-04-01

Full Text Available Cytochrome c oxidase I (COI is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate.

Simple sequence repeat (SSR) markers are effective for identifying ...

African Journals Online (AJOL)

DNA was extracted from newly formed leaves and amplified using 21 simple sequence repeat (SSR) markers (NH001c, NH002b, NH005b, NH007b, NH008b, NH009b, NH011b, NH013b, NH012a, NH014a, NH015a, NH017a, KA4b, KA5, KA14, KA16, KB16, KU10, BGA35, BGT23b and HGA8b). The data was analyzed by ...

New STS molecular markers for assessment of genetic diversity and DNA fingerprinting in hop (Humulus lupulus L.).

Science.gov (United States)

Patzak, Josef; Vrba, Lukás; Matousek, Jaroslav

2007-01-01

Molecular markers have been increasingly used in genetic studies of crop species for their applicability in breeding programs. In this work, we report on the development of new sequence-tagged site (STS) markers based on sequence information from several identified hop (Humulus lupulus L.) genes. We demonstrate the usefulness of these STS markers and compare them to SSRs for identifying hop genotypes and estimating genetic diversity in a collection of 68 hop cultivars from around the world. We found 3 individual gene variants (A, B, C) of the chs_H1 gene in this collection. The most frequent gene variant, B (AJ304877), was not detected in Mt. Hood, Glacier, and Horizon (US) cultivars. Gene variant A came from an American germplasm through wild hops. We found length polymorphism in intron 1 of the chs2 gene, and 4 different amplified markers were detected in PCRs. The chs3 gene was found in only one third of the cultivars. None of the variants of the studied CHS genes were found in Humulus japonicus. We detected 5 major gene variants of DNA-binding protein in the collection of H. lupulus cultivars and 2 others in H. japonicus. We also found 3 individual gene variants of an endochitinase gene. The distribution of gene variants did not correlate with any resistance. We proved that developed STS markers can be successfully used for the analysis of genetic diversity and can substitute and supplement SSR markers in hop.

Optimization Of ISSR Markers For DNA Fingerprinting In Stevia Rebaudiana Bertoni

International Nuclear Information System (INIS)

Lyena Watty Zuraine Ahmad; Lyena Watty Zuraine Ahmad; Azhar Mohamad; Mohamad Osman; Zarina Zainuddin; Fatin Izzati Mohd Khari

2014-01-01

ISSR or inter-simple sequence repeat is PCR based markers which required no prior DNA sequence knowledge of the studied organism. It has been proved to overcome limitations in other genetic marker techniques. In this study, 100 ISSR primers which comprised of 80 specific primers and 20 degenerate primers were used. All of the primers were tested on gradient temperatures from 45-55 degree Celsius. For positive amplification, 62 specific primers (77.5 %) and 18 degenerate primers (90.0 %) were recorded as working primers. The most efficient temperature for 25 primers was 55 degree Celsius. Marker derived from ISSR profiling is a powerful approach for identification and molecular classification of Stevia rebaudiana bertoni. (author)

PineElm_SSRdb: a microsatellite marker database identified from genomic, chloroplast, mitochondrial and EST sequences of pineapple (Ananas comosus (L.) Merrill).

Science.gov (United States)

Chaudhary, Sakshi; Mishra, Bharat Kumar; Vivek, Thiruvettai; Magadum, Santoshkumar; Yasin, Jeshima Khan

2016-01-01

Simple Sequence Repeats or microsatellites are resourceful molecular genetic markers. There are only few reports of SSR identification and development in pineapple. Complete genome sequence of pineapple available in the public domain can be used to develop numerous novel SSRs. Therefore, an attempt was made to identify SSRs from genomic, chloroplast, mitochondrial and EST sequences of pineapple which will help in deciphering genetic makeup of its germplasm resources. A total of 359511 SSRs were identified in pineapple (356385 from genome sequence, 45 from chloroplast sequence, 249 in mitochondrial sequence and 2832 from EST sequences). The list of EST-SSR markers and their details are available in the database. PineElm_SSRdb is an open source database available for non-commercial academic purpose at http://app.bioelm.com/ with a mapping tool which can develop circular maps of selected marker set. This database will be of immense use to breeders, researchers and graduates working on Ananas spp. and to others working on cross-species transferability of markers, investigating diversity, mapping and DNA fingerprinting.

Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina).

Science.gov (United States)

Dentinger, Bryn T M; Didukh, Maryna Y; Moncalvo, Jean-Marc

2011-01-01

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (~450 bp) representing ~100 morphospecies from ~650 collections of Agaricomycotina using several sets of new primers. Large introns (~1500 bp) at variable locations were detected in ~5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (~30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.

DNA-based molecular markers as tools for the discovery of γ-induced mutants in cereals and soybean

International Nuclear Information System (INIS)

Bondarenco, E.; Bondarenco, V.; Barbacar, N.; Coretchi, L.

2009-01-01

γ-induced mutagenesis is one of the present techniques effective in producing crops with enhanced quality and novel properties. The fast detection of mutants can be nowadays assured by the employment of DNA-based molecular markers. Different kinds of molecular markers are being widely used all over the world to monitor DNA sequence variation and identification of desired traits. In the given paper we present a short overview of the types of molecular markers and the first steps of the attempt of their use for mutants' characterization in the Republic of Moldova (authors)

Use of a D17Z1 oligonucleotide probe for human DNA quantitation prior to PCR analysis of polymorphic DNA markers

Energy Technology Data Exchange (ETDEWEB)

Walsh, S.; Alavaren, M.; Varlaro, J. [Roche Molecular Systems, Alameda, CA (United States)] [and others

1994-09-01

The alpha-satellite DNA locus D17Z1 contains primate-specific sequences which are repeated several hundred times per chromosome 17. A probe that was designed to hybridize to a subset of the D17Z1 sequence can be used for very sensitive and specific quantitation of human DNA. Sample human genomic DNA is immobilized on nylon membrane using a slot blot apparatus, and then hybridized with a biotinylated D17Z1 oligonucleotide probe. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for either calorimetric (TMB) or chemiluminescent (ECL) detection. Signals obtained for sample DNAs are then compared to the signals obtained for a series of human DNA standards. For either detection method, forty samples can be quantitated in less than two hours, with a sensitivity of 150 pg. As little as 20 pg of DNA can be quantitated when using chemiluminescent detection with longer film exposures. PCR analysis of several VNTR and STR markers has indicated that optimal typing results are generally obtained within a relatively narrow range of input DNA quantities. Too much input DNA can lead to PCR artifacts such as preferential amplification of smaller alleles, non-specific amplification products, and exaggeration of the DNA synthesis slippage products that are seen with STR markers. Careful quantitation of human genomic DNA prior to PCR can avoid or minimize these problems and ultimately give cleaner, more unambiguous PCR results.

Interspecific introgression in cetaceans: DNA markers reveal post-F1 status of a pilot whale.

Directory of Open Access Journals (Sweden)

Laura Miralles

Full Text Available Visual species identification of cetacean strandings is difficult, especially when dead specimens are degraded and/or species are morphologically similar. The two recognised pilot whale species (Globicephala melas and Globicephala macrorhynchus are sympatric in the North Atlantic Ocean. These species are very similar in external appearance and their morphometric characteristics partially overlap; thus visual identification is not always reliable. Genetic species identification ensures correct identification of specimens. Here we have employed one mitochondrial (D-Loop region and eight nuclear loci (microsatellites as genetic markers to identify six stranded pilot whales found in Galicia (Northwest Spain, one of them of ambiguous phenotype. DNA analyses yielded positive amplification of all loci and enabled species identification. Nuclear microsatellite DNA genotypes revealed mixed ancestry for one individual, identified as a post-F1 interspecific hybrid employing two different Bayesian methods. From the mitochondrial sequence the maternal species was Globicephala melas. This is the first hybrid documented between Globicephala melas and G. macrorhynchus, and the first post-F1 hybrid genetically identified between cetaceans, revealing interspecific genetic introgression in marine mammals. We propose to add nuclear loci to genetic databases for cetacean species identification in order to detect hybrid individuals.

DNA typing of ancient parasite eggs from environmental samples identifies human and animal worm infections in viking-age settlement

DEFF Research Database (Denmark)

Søe, Martin Jensen; Nejsum, Peter; Fredensborg, Brian Lund

2015-01-01

Ancient parasite eggs were recovered from environmental samples collected at a Viking-age settlement in Viborg, Denmark, dated 1018-1030 A.D. Morphological examination identified Ascaris sp., Trichuris sp., and Fasciola sp. eggs, but size and shape did not allow species identification. By carefully...... selecting genetic markers, PCR amplification and sequencing of ancient DNA (aDNA) isolates resulted in identification of: the human whipworm, Trichuris trichiura, using SSUrRNA sequence homology; Ascaris sp. with 100% homology to cox1 haplotype 07; and Fasciola hepatica using ITS1 sequence homology...

Development and small-scale validation of a novel pigeon-associated mitochondrial DNA source tracking marker for the detection of fecal contamination in harvested rainwater.

Science.gov (United States)

Waso, M; Khan, S; Khan, W

2018-02-15

The current study was aimed at designing and validating (on a small-scale) a novel pigeon mitochondrial DNA (mtDNA) microbial source tracking (MST) marker for the detection of pigeon fecal matter in harvested rainwater. The pigeon mtDNA MST marker was designed to target the mtDNA Cytochrome b gene by employing mismatch amplification mutation assay kinetics. The pigeon marker was validated by screening 69 non-pigeon and 9 pigeon fecal samples. The host-sensitivity of the assay was determined as 1.00 while the host-specificity of the assay was 0.96. Harvested rainwater samples (n=60) were screened for the prevalence of the marker with the mtDNA Cytochrome b marker detected in 78% of the samples. Bayes' theorem was applied to calculate the conditional probability of the marker detecting true pigeon contamination and the marker subsequently displayed a 99% probability of detecting true pigeon contamination in the harvested rainwater samples. In addition, the mtDNA Cytochrome b marker displayed high concurrence frequencies versus heterotrophic bacteria (78.3%), E. coli (73.3%), total coliforms (71.1%) and fecal coliforms (66.7%). This study thus validates that targeting mtDNA for the design of source tracking markers may be a valuable tool to detect avian fecal contamination in environmental waters. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of microsatellite markers for identifying Brazilian coffee arabica varieties

NARCIS (Netherlands)

Vieira, E.S.N.; Pinho, Von E.V.R.; Carvalho, M.G.G.; Esselink, G.; Vosman, B.

2010-01-01

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of

Autosomal and mtDNA Markers Affirm the Distinctiveness of Lions in West and Central Africa.

Science.gov (United States)

Bertola, Laura D; Tensen, Laura; van Hooft, Pim; White, Paula A; Driscoll, Carlos A; Henschel, Philipp; Caragiulo, Anthony; Dias-Freedman, Isabela; Sogbohossou, Etotépé A; Tumenta, Pricelia N; Jirmo, Tuqa H; de Snoo, Geert R; de Iongh, Hans H; Vrieling, Klaas

2015-01-01

The evolutionary history of a species is key for understanding the taxonomy and for the design of effective management strategies for species conservation. The knowledge about the phylogenetic position of the lion (Panthera leo) in West/Central Africa is largely based on mitochondrial markers. Previous studies using mtDNA only have shown this region to hold a distinct evolutionary lineage. In addition, anthropogenic factors have led to a strong decline in West/Central African lion numbers, thus, the conservation value of these populations is particularly high. Here, we investigate whether autosomal markers are concordant with previously described phylogeographic patterns, and confirm the unique position of the West/Central African lion. Analysis of 20 microsatellites and 1,454 bp of the mitochondrial DNA in 16 lion populations representing the entire geographic range of the species found congruence in both types of markers, identifying four clusters: 1) West/Central Africa, 2) East Africa, 3) Southern Africa and 4) India. This is not in line with the current taxonomy, as defined by the IUCN, which only recognizes an African and an Asiatic subspecies. There are no indications that genetic diversity in West/Central Africa lions is lower than in either East or Southern Africa, however, given this genetic distinction and the recent declines of lion numbers in this region, we strongly recommend prioritization of conservation projects in West/Central Africa. As the current taxonomic nomenclature does not reflect the evolutionary history of the lion, we suggest that a taxonomic revision of the lion is warranted.

Autosomal and mtDNA Markers Affirm the Distinctiveness of Lions in West and Central Africa.

Directory of Open Access Journals (Sweden)

Laura D Bertola

Full Text Available The evolutionary history of a species is key for understanding the taxonomy and for the design of effective management strategies for species conservation. The knowledge about the phylogenetic position of the lion (Panthera leo in West/Central Africa is largely based on mitochondrial markers. Previous studies using mtDNA only have shown this region to hold a distinct evolutionary lineage. In addition, anthropogenic factors have led to a strong decline in West/Central African lion numbers, thus, the conservation value of these populations is particularly high. Here, we investigate whether autosomal markers are concordant with previously described phylogeographic patterns, and confirm the unique position of the West/Central African lion. Analysis of 20 microsatellites and 1,454 bp of the mitochondrial DNA in 16 lion populations representing the entire geographic range of the species found congruence in both types of markers, identifying four clusters: 1 West/Central Africa, 2 East Africa, 3 Southern Africa and 4 India. This is not in line with the current taxonomy, as defined by the IUCN, which only recognizes an African and an Asiatic subspecies. There are no indications that genetic diversity in West/Central Africa lions is lower than in either East or Southern Africa, however, given this genetic distinction and the recent declines of lion numbers in this region, we strongly recommend prioritization of conservation projects in West/Central Africa. As the current taxonomic nomenclature does not reflect the evolutionary history of the lion, we suggest that a taxonomic revision of the lion is warranted.

Autosomal and mtDNA Markers Affirm the Distinctiveness of Lions in West and Central Africa

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Bertola, Laura D.; Tensen, Laura; van Hooft, Pim; White, Paula A.; Driscoll, Carlos A.; Henschel, Philipp; Caragiulo, Anthony; Dias-Freedman, Isabela; Sogbohossou, Etotépé A.; Tumenta, Pricelia N.; Jirmo, Tuqa H.; de Snoo, Geert R.

2015-01-01

The evolutionary history of a species is key for understanding the taxonomy and for the design of effective management strategies for species conservation. The knowledge about the phylogenetic position of the lion (Panthera leo) in West/Central Africa is largely based on mitochondrial markers. Previous studies using mtDNA only have shown this region to hold a distinct evolutionary lineage. In addition, anthropogenic factors have led to a strong decline in West/Central African lion numbers, thus, the conservation value of these populations is particularly high. Here, we investigate whether autosomal markers are concordant with previously described phylogeographic patterns, and confirm the unique position of the West/Central African lion. Analysis of 20 microsatellites and 1,454 bp of the mitochondrial DNA in 16 lion populations representing the entire geographic range of the species found congruence in both types of markers, identifying four clusters: 1) West/Central Africa, 2) East Africa, 3) Southern Africa and 4) India. This is not in line with the current taxonomy, as defined by the IUCN, which only recognizes an African and an Asiatic subspecies. There are no indications that genetic diversity in West/Central Africa lions is lower than in either East or Southern Africa, however, given this genetic distinction and the recent declines of lion numbers in this region, we strongly recommend prioritization of conservation projects in West/Central Africa. As the current taxonomic nomenclature does not reflect the evolutionary history of the lion, we suggest that a taxonomic revision of the lion is warranted. PMID:26466139

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

NARCIS (Netherlands)

Schoch, C.L.; Seifert, K.A.; Huhndorf, S.; Robert, V.; Spouge, J.L.; Levesque, C.A.; Chen, W.; Crous, P.W.; Boekhout, T.; Damm, U.; Hoog, de G.S.; Eberhardt, U.; Groenewald, J.Z.; Groenewald, M.; Hagen, F.; Houbraken, J.; Quaedvlieg, W.; Stielow, B.; Vu, T.D.; Walther, G.

2012-01-01

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it

A leukocyte activation test identifies food items which induce release of DNA by innate immune peripheral blood leucocytes.

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Garcia-Martinez, Irma; Weiss, Theresa R; Yousaf, Muhammad N; Ali, Ather; Mehal, Wajahat Z

2018-01-01

Leukocyte activation (LA) testing identifies food items that induce a patient specific cellular response in the immune system, and has recently been shown in a randomized double blinded prospective study to reduce symptoms in patients with irritable bowel syndrome (IBS). We hypothesized that test reactivity to particular food items, and the systemic immune response initiated by these food items, is due to the release of cellular DNA from blood immune cells. We tested this by quantifying total DNA concentration in the cellular supernatant of immune cells exposed to positive and negative foods from 20 healthy volunteers. To establish if the DNA release by positive samples is a specific phenomenon, we quantified myeloperoxidase (MPO) in cellular supernatants. We further assessed if a particular immune cell population (neutrophils, eosinophils, and basophils) was activated by the positive food items by flow cytometry analysis. To identify the signaling pathways that are required for DNA release we tested if specific inhibitors of key signaling pathways could block DNA release. Foods with a positive LA test result gave a higher supernatant DNA content when compared to foods with a negative result. This was specific as MPO levels were not increased by foods with a positive LA test. Protein kinase C (PKC) inhibitors resulted in inhibition of positive food stimulated DNA release. Positive foods resulted in CD63 levels greater than negative foods in eosinophils in 76.5% of tests. LA test identifies food items that result in release of DNA and activation of peripheral blood innate immune cells in a PKC dependent manner, suggesting that this LA test identifies food items that result in release of inflammatory markers and activation of innate immune cells. This may be the basis for the improvement in symptoms in IBS patients who followed an LA test guided diet.

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

Science.gov (United States)

Six DNA regions were evaluated in a multi-national, multi-laboratory consortium as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it...

Extraction of Total DNA and RNA from Marine Filter Samples and Generation of a cDNA as Universal Template for Marker Gene Studies.

Science.gov (United States)

Schneider, Dominik; Wemheuer, Franziska; Pfeiffer, Birgit; Wemheuer, Bernd

2017-01-01

Microbial communities play an important role in marine ecosystem processes. Although the number of studies targeting marker genes such as the 16S rRNA gene has been increased in the last few years, the vast majority of marine diversity is rather unexplored. Moreover, most studies focused on the entire bacterial community and thus disregarded active microbial community players. Here, we describe a detailed protocol for the simultaneous extraction of DNA and RNA from marine water samples and for the generation of cDNA from the isolated RNA which can be used as a universal template in various marker gene studies.

DNA fingerprinting of some pakistani date palm (phoenix dactylifera L.) cultive ARS using issr markers

International Nuclear Information System (INIS)

Mirbahar, A.; Khan, S.; Markhand, G.S.

2016-01-01

Date palm is one of the oldest cultivated and economically important fruit trees. First time Inter Simple Sequence Repeats (ISSR) markers were used with twenty five economically important date palm cultivars of Pakistan for DNA fingerprinting analysis. Samples were collected from four provinces of Pakistan i.e., Sindh, Punjab, Khyber Pakhtoonkhwa and Balochistan. The study was carried out using seven ISSR markers. The twenty five date palm cultivars showed variation at the DNA level. The ISSR primers showed high polymorphism (84%) in the studied date palm cultivars. Dice similarity index was in range from 0.608 to 0.980 and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) divided twenty five date palm cultivars into two main clusters and sub-clusters. However ISSR markers efficiently discriminated for assessing genetic diversity among commercial Pakistani date palm cultivars. (author)

Soil Fungal Community Associated with Peat in Sarawak Identified Using 18S rDNA Marker

International Nuclear Information System (INIS)

Siti Ramlah Ahmad Ali; Sakinah Safari; Mohd Shawal Thakib; Shamsilawani Ahamed Bakeri; Nur Aziemah Ab Ghani

2016-01-01

Fungi are principal decomposing microorganisms in acidic environment of peat lands. A useful tool for molecular screening of soil fungal communities using the 18S ribosomal DNA primer has been proven capable of identifying a broad range of fungi species within Ascomycota, Basidiomycota, Zygomycota and Chytridiomycota. Currently, very little information is available on fungal communities in deep peat of Sarawak, Malaysia. In this study, we have isolated the fungi from soil samples taken in deep peat forests and oil palm cultivated areas. The fungal identity was undertaken using 18S ribosomal DNA primer which is EF4-F/ fung5-R. The microscopic structures were conducted to confirm the identity of the isolates. Based on this study, the fungal division most commonly found in deep peat is the Ascomycota. Aspergillus fumigatus was the most common species and more dominant in oil palm cultivated areas and logged-over forest than in primary forest. In the primary forest, the dominant species was the A. flavus, while Hypocrea atroviridis was commonly associated with oil palm cultivated areas and logged-over forest. Other species of fungi isolated in peat primary forests were Penicillium chrysogenum, Trichoderma sp., Phanerochaete sp., Mortierella chlamydospora, A. niger, A. alliaceus, etc. The in-depth difference in the fungal communities for the different sites will be further investigated using the next generation sequencing technology. (author)

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

Mitochondrial genome sequencing and development of genetic markers for the detection of DNA of invasive bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix in environmental water samples from the United States.

Directory of Open Access Journals (Sweden)

Heather L Farrington

Full Text Available Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix pose a substantial threat to North American aquatic ecosystems. Recently, environmental DNA (eDNA, genetic material shed by organisms into their environment that can be detected by non-invasive sampling strategies and genetic assays, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR and quantitative (qPCR markers for detection of these species in North American surface waters. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with those from other common and closely related fish species from the Illinois River watershed to identify and design new species-specific markers for the detection of bighead and silver carp DNA in environmental water samples. We then tested these genetic markers in the laboratory for species-specificity and sensitivity. Newly developed markers performed well in field trials, did not have any false positive detections, and many markers had much higher detection rates and sensitivity compared to the markers currently used in eDNA surveillance programs. We also explored the use of multiple genetic markers to determine whether it would improve detection rates, results of which showed that using multiple highly sensitive markers should maximize detection rates in environmental samples. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp and will improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.

Serum ALT levels as a surrogate marker for serum HBV DNA levels in HBeAg-negative pregnant women.

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Sangfelt, Per; Von Sydow, Madeleine; Uhnoo, Ingrid; Weiland, Ola; Lindh, Gudrun; Fischler, Björn; Lindgren, Susanne; Reichard, Olle

2004-01-01

In Stockholm, Sweden, the majority of pregnant women positive for hepatitis B surface antigen (HBsAg) are hepatitis Be antigen (HBeAg) negative. Newborns to HBeAg positive mothers receive vaccination and hepatitis B immunoglobulin (HBIg). Newborns to HBeAg negative mothers receive vaccine and HBIg only if the mothers have elevated ALT levels. The aim of this study was to retrospectively evaluate ALT levels as a surrogate marker for HBV DNA levels in HBeAg negative carrier mothers. Altogether 8947 pregnant women were screened for HBV markers from 1999 to 2001 at the Virology Department, Karolinska Hospital. Among mothers screened 192 tested positive for HBsAg (2.2%). 13 of these samples could not be retrieved. Of the remaining 179 sera, 8 (4%) tested positive for HBeAg and 171 (95.5%) were HBeAg negative. Among the HBeAg negative mothers, 9 had HBV DNA levels > 10(5) copies/ml, and of these 7 had normal ALT levels indicating low sensitivity of an elevated ALT level as a surrogate marker for high HBV DNA level. Furthermore, no correlation was found between ALT and HBV DNA levels. Hence, it is concluded that the use of ALT as a surrogate marker for high viral replication in HBeAg negative mothers could be questioned.

Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.).

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Zhang, Xiaofei; Liu, Dongcheng; Yang, Wenlong; Liu, Kunfan; Sun, Jiazhu; Guo, Xiaoli; Li, Yiwen; Wang, Daowen; Ling, Hongqing; Zhang, Aimin

2011-05-01

Low-molecular-weight glutenin subunits (LMW-GSs) play an important role in determining the bread-making quality of bread wheat. However, LMW-GSs display high polymorphic protein complexes encoded by multiple genes, and elucidating the complex LMW-GS gene family in bread wheat remains challenging. In the present study, using conventional polymerase chain reaction (PCR) with conserved primers and high-resolution capillary electrophoresis, we developed a new molecular marker system for identifying LMW-GS gene family members. Based on sequence alignment of 13 LMW-GS genes previously identified in the Chinese bread wheat variety Xiaoyan 54 and other genes available in GenBank, PCR primers were developed and assigned to conserved sequences spanning the length polymorphism regions of LMW-GS genes. After PCR amplification, 17 DNA fragments in Xiaoyan 54 were detected using capillary electrophoresis. In total, 13 fragments were identical to previously identified LMW-GS genes, and the other 4 were derived from unique LMW-GS genes by sequencing. This marker system was also used to identify LMW-GS genes in Chinese Spring and its group 1 nulli-tetrasomic lines. Among the 17 detected DNA fragments, 4 were located on chromosome 1A, 5 on 1B, and 8 on 1D. The results suggest that this marker system is useful for large-scale identification of LMW-GS genes in bread wheat varieties, and for the selection of desirable LMW-GS genes to improve the bread-making quality in wheat molecular breeding programmes.

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME, SINGLE-COPY NUCLEAR DNA, AND MITOCHONDRIAL DNA MARKERS.

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Buonaccorsi, Vincent P; Reece, Kimberly S; Morgan, Lee W; Graves, John E

1999-04-01

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θ O ) ranged from 0.00 to 0.15, with a mean of 0.08. The θ O values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θ O = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04-0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates. © 1999 The Society for the Study of Evolution.

Methylated Glutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic marker of prognosis and response to chemotherapy in castrate-resistant prostate cancer.

Science.gov (United States)

Mahon, K L; Qu, W; Devaney, J; Paul, C; Castillo, L; Wykes, R J; Chatfield, M D; Boyer, M J; Stockler, M R; Marx, G; Gurney, H; Mallesara, G; Molloy, P L; Horvath, L G; Clark, S J

2014-10-28

Glutathione S-transferase 1 (GSTP1) inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). This study assessed whether the level of circulating methylated GSTP1 (mGSTP1) in plasma DNA is associated with chemotherapy response and overall survival (OS). Plasma samples were collected prospectively from a Phase I exploratory cohort of 75 men with castrate-resistant PC (CRPC) and a Phase II independent validation cohort (n=51). mGSTP1 levels in free DNA were measured using a sensitive methylation-specific PCR assay. The Phase I cohort identified that detectable baseline mGSTP1 DNA was associated with poorer OS (HR, 4.2 95% CI 2.1-8.2; P<0.0001). A decrease in mGSTP1 DNA levels after cycle 1 was associated with a PSA response (P=0.008). In the Phase II cohort, baseline mGSTP1 DNA was a stronger predictor of OS than PSA change after 3 months (P=0.02). Undetectable plasma mGSTP1 after one cycle of chemotherapy was associated with PSA response (P=0.007). We identified plasma mGSTP1 DNA as a potential prognostic marker in men with CRPC as well as a potential surrogate therapeutic efficacy marker for chemotherapy and corroborated these findings in an independent Phase II cohort. Prospective Phase III assessment of mGSTP1 levels in plasma DNA is now warranted.

Phenotypic consequences of a mosaic marker chromosome identified by fluorescence in situ hybridization (FISH) as being derived from chromosome 16

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Ray, J.H.; Zhou, X.; Pletcher, B.A. [Cornell Univ. Medical College, Manhasset, NY (United States)] [and others

1994-09-01

De novo marker chromosomes are detected in 1 in 2500 amniotic fluid samples and are associated with a 10-15% risk for phenotypic abnormality. FISH can be utilized as a research tool to identify the origins of marker chromosomes. The phenotypic consequences of a marker chromosome derived from the short arm of chromosome 16 are described. A 26-year-old woman underwent amniocentesis at 28 weeks gestation because of a prenatally diagnosed tetralogy of Fallot. Follow-up ultrasounds also showed ventriculomegaly and cleft lip and palate. 32 of 45 cells had the karyotype 47,XY,+mar; the remaining cells were 46,XY. The de novo marker chromosome was C-band positive and non-satellited and failed to stain with distamycin A/DAPI. At birth the ultrasound findings were confirmed and dysmorphic features and cryptorchidism were noted. Although a newborn blood sample contained only normal cells, mosaicism was confirmed in 2 skin biopsies. FISH using whole-chromosome painting and alpha-satellite DNA probes showed that the marker chromosome had originated from chromosome 16. As proximal 16q is distamycin A/DAPI positive, the marker is apparently derived from proximal 16p. At 15 months of age, this child is hypotonic, globally delayed and is gavage-fed. His physical examination is significant for microbrachycephaly, a round face, sparse scalp hair, ocular hypertelorism, exotropia, a flat, wide nasal bridge and tip, mild micrognathia, and tapered fingers with lymphedema of hands and feet. Inguinal hernias have been repaired. His features are consistent with those described for patients trisomic for most or all of the short arm of chromosome 16. Marker chromosomes derived from the short arm of chromosome 16 appear to have phenotypic consequences. As the origin of more marker chromosomes are identified using FISH, their karyotype/phenotype correlations will become more apparent, which will permit more accurate genetic counseling.

Is ITS-2 rDNA suitable marker for genetic characterization of Sarcoptes mites from different wild animals in different geographic areas?

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Alasaad, S; Soglia, D; Spalenza, V; Maione, S; Soriguer, R C; Pérez, J M; Rasero, R; Degiorgis, M P Ryser; Nimmervoll, H; Zhu, X Q; Rossi, L

2009-02-05

The present study examined the relationship among individual Sarcoptes scabiei mites from 13 wild mammalian populations belonging to nine species in four European countries using the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) as genetic marker. The ITS-2 plus primer flanking 5.8S and 28S rDNA (ITS-2+) was amplified from individual mites by polymerase chain reaction (PCR) and the amplicons were sequenced directly. A total of 148 ITS-2+ sequences of 404bp in length were obtained and 67 variable sites were identified (16.59%). UPGMA analyses did not show any geographical or host-specific clustering, and a similar outcome was obtained using population pairwise Fst statistics. These results demonstrated that ITS-2 rDNA does not appear to be suitable for examining genetic diversity among mite populations.

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers.

Science.gov (United States)

Akdemir, Hülya; Suzerer, Veysel; Tilkat, Engin; Onay, Ahmet; Çiftçi, Yelda Ozden

2016-12-01

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in "reasonable polymorphism" among donor plant and its 18 clones. Mantel's test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.

Development of Insertion and Deletion Markers for Bottle Gourd Based on Restriction Site-associated DNA Sequencing Data

Directory of Open Access Journals (Sweden)

Xinyi WU

2017-01-01

Full Text Available Bottle gourd is an important cucurbit crop worldwide. To provide more available molecular markers for this crop, a bioinformatic approach was employed to develop insertion–deletions (InDels markers in bottle gourd based on restriction site-associated DNA sequencing (RAD-Seq data. A total of 892 Indels were predicted, with the length varying from 1 bp to 167 bp. Single-nucleotide InDels were the predominant types of InDels. To validate these InDels, PCR primers were designed from 162 loci where InDels longer than 2 bp were predicated. A total of 112 InDels were found to be polymorphic among 9 bottle gourd accessions under investigation. The rate of prediction accuracy was thus at a high level of 72.7%. DNA fingerprinting for 4 cultivars were performed using 8 selected Indels markers, demonstrating the usefulness of these markers.

Molecular characterization of Anthurium genotypes by using DNA fingerprinting and SPAR markers.

Science.gov (United States)

Souza Neto, J D; Soares, T C B; Motta, L B; Cabral, P D S; Silva, J A

2014-07-02

We characterized single primer amplification reaction (SPAR) molecular markers from 20 genotypes of Anthurium andraeanum Lind., including 3 from commercial varieties and 17 from 2 communities in the State of Espírito Santo, Brazil. Twenty-four SPAR, consisting of 7 random amplified polymorphic DNA and 17 inter-simple sequence repeat markers were used to estimate the genetic diversity of 20 Anthurium accessions. The set of SPAR markers generated 288 bands and showed an average polymorphism percentage of 93.39%, ranging from 71.43 to 100%. The polymorphism information content (PIC) of the random amplified polymorphic DNA primers averaged 0.364 and ranged from 0.258 to 0.490. Primer OPF 06 showed the lowest PIC, while OPAM 14 was the highest. The average PIC of the inter-simple sequence repeat primers was 0.299, with values ranging from 0.196 to 0.401. Primer UBC 845 had the lowest PIC (0.196), while primer UCB 810 had the highest (0.401). By using the complement of Jaccard's similarity index and unweighted pair group method with arithmetic mean clustering, 5 clusters were formed with a cophenetic correlation coefficient of 0.8093, indicating an acceptable clustering consistency. However, no genotype clustering patterns agreed with the morphological data. The Anthurium genotypes investigated in this study are a germplasm source for conservational research and may be used in improvement programs for this species.

Use of DNA barcodes to identify flowering plants

OpenAIRE

Kress, W. John; Wurdack, Kenneth J.; Zimmer, Elizabeth A.; Weigt, Lee A.; Janzen, Daniel H.

2005-01-01

Methods for identifying species by using short orthologous DNA sequences, known as “DNA barcodes,” have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We ther...

DNA barcoding as a means for identifying medicinal plants of Pakistan

International Nuclear Information System (INIS)

Schori, M.; Showalter, A.M.

2011-01-01

DNA barcoding involves the generation of DNA sequencing data from particular genetic regions in an organism and the use of these sequence data to identify or 'barcode' that organism and distinguish it from other species. Here, DNA barcoding is being used to identify several medicinal plants found in Pakistan and distinguished them from other similar species. Several challenges to the successful implementation of plant DNA barcoding are presented and discussed. Despite these challenges, DNA barcoding has the potential to uniquely identify medicinal plants and provide quality control and standardization of the plant material supplied to the pharmaceutical industry. (author)

Application of plant DNA markers in forensic botany: genetic comparison of Quercus evidence leaves to crime scene trees using microsatellites.

Science.gov (United States)

Craft, Kathleen J; Owens, Jeffrey D; Ashley, Mary V

2007-01-05

As highly polymorphic DNA markers become increasingly available for a wide range of plant and animal species, there will be increasing opportunities for applications to forensic investigations. To date, however, relatively few studies have reported using DNA profiles of non-human species to place suspects at or near crime scenes. Here we describe an investigation of a double homicide of a female and her near-term fetus. Leaf material taken from a suspect's vehicle was identified to be that of sand live oak, Quercus geminata, the same tree species that occurred near a shallow grave where the victims were found. Quercus-specific DNA microsatellites were used to genotype both dried and fresh material from trees located near the burial site and from the material taken from the suspect's car. Samples from the local population of Q. geminata were also collected and genotyped in order to demonstrate that genetic variation at four microsatellite loci was sufficient to assign leaves to an individual tree with high statistical certainty. The cumulative average probability of identity for these four loci was 2.06x10(-6). DNA was successfully obtained from the dried leaf material although PCR amplification was more difficult than amplification of DNA from fresh leaves. The DNA profiles of the dried leaves from the suspect's car did not match those of the trees near the crime scene. Although this investigation did not provide evidence that could be used against the suspect, it does demonstrate the potential for plant microsatellite markers providing physical evidence that links plant materials to live plants at or near crime scenes.

Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region.

Science.gov (United States)

Ma, Xin-Ye; Xie, Cai-Xiang; Liu, Chang; Song, Jing-Yuan; Yao, Hui; Luo, Kun; Zhu, Ying-Jie; Gao, Ting; Pang, Xiao-Hui; Qian, Jun; Chen, Shi-Lin

2010-01-01

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.

Recent innovation in microbial source tracking using bacterial real-time PCR markers in shellfish

International Nuclear Information System (INIS)

Mauffret, A.; Mieszkin, S.; Morizur, M.; Alfiansah, Y.; Lozach, S.; Gourmelon, M.

2013-01-01

Highlights: ► DNA extraction from intravalvular liquid is promising for microbial source tracking in oysters. ► Host-associated bacterial markers in shellfish digestive tissues were difficult to assess with real-time PCR. ► DNA extracts from shellfish flesh appeared to have low inhibitor levels but low marker levels. ► Protocol transfer from one shellfish species to another does not appear possible. -- Abstract: We assessed the capacity of real-time PCR markers to identify the origin of contamination in shellfish. Oyster, cockles or clams were either contaminated with fecal materials and host-associated markers designed from Bacteroidales or Catellicoccus marimammalium 16S RNA genes were extracted from their intravalvular liquid, digestive tissues or shellfish flesh. Extraction of bacterial DNA from the oyster intravalvular liquid with FastDNA spin kit for soil enabled the selected markers to be quantified in 100% of artificially contaminated samples, and the source of contamination to be identified in 13 out of 38 naturally contaminated batches from European Class B and Class C areas. However, this protocol did not enable the origin of the contamination to be identified in cockle or clam samples. Although results are promising for extracts from intravalvular liquid in oyster, it is unlikely that a single protocol could be the best across all bacterial markers and types of shellfish

Development of genomic SSR and potential EST-SSR markers in ...

African Journals Online (AJOL)

In addition, forty four EST-SSRs which can be amplified with expected sizes were identified from a B. chinense root cDNA library. The genomic SSR markers and potential EST-SSR markers developed in the present study should be useful for genetic diversity and molecular marker assistant selection breeding research in ...

Assessment of four molecular markers as potential DNA barcodes for red algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta).

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Tan, Ji; Lim, Phaik-Eem; Phang, Siew-Moi; Hong, Dang Diem; Sunarpi, H; Hurtado, Anicia Q

2012-01-01

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.

Genetic Confirmation of Mungbean (Vigna radiata) and Mashbean (Vigna mungo) Interspecific Recombinants using Molecular Markers.

Science.gov (United States)

Abbas, Ghulam; Hameed, Amjad; Rizwan, Muhammad; Ahsan, Muhammad; Asghar, Muhammad J; Iqbal, Nayyer

2015-01-01

Molecular confirmation of interspecific recombinants is essential to overcome the issues like self-pollination, environmental influence, and inadequacy of morphological characteristics during interspecific hybridization. The present study was conducted for genetic confirmation of mungbean (female) and mashbean (male) interspecific crosses using molecular markers. Initially, polymorphic random amplified polymorphic DNA (RAPD), universal rice primers (URP), and simple sequence repeats (SSR) markers differentiating parent genotypes were identified. Recombination in hybrids was confirmed using these polymorphic DNA markers. The NM 2006 × Mash 88 was most successful interspecific cross. Most of true recombinants confirmed by molecular markers were from this cross combination. SSR markers were efficient in detecting genetic variability and recombination with reference to specific chromosomes and particular loci. SSR (RIS) and RAPD identified variability dispersed throughout the genome. In conclusion, DNA based marker assisted selection (MAS) efficiently confirmed the interspecific recombinants. The results provided evidence that MAS can enhance the authenticity of selection in mungbean improvement program.

[Genomic DNA fingerprints of Legionella pneumophila serogroup 2 strains as an epidemiologic marker].

Science.gov (United States)

Bender-Beck, L; Mühlenberg, W; Lück, P C; Ott, M; Horbach, I; Fehrenbach, F J; Wewalka, G; Hacker, J

1995-08-01

Using pulsed-field gel electrophoresis, DNA fingerprints of eleven Legionella pneumophila isolates of serogroup 2 were generated. It was shown that two strains from a patient suffering from pneumonia as well as three environmental strains isolated from the shower in the hotel where the patient stayed 5 days before his illness were identical. Six strains of the same serogroup isolated from other sources were clearly separated. Thus, DNA fingerprints by pulsed-field gel electrophoresis are excellent epidemiological markers for the rarely occurring serogroup 2 of Legionella pneumophila.

Methylcap-seq reveals novel DNA methylation markers for the diagnosis and recurrence prediction of bladder cancer in a Chinese population.

Directory of Open Access Journals (Sweden)

Yangxing Zhao

Full Text Available PURPOSE: There is a need to supplement or supplant the conventional diagnostic tools, namely, cystoscopy and B-type ultrasound, for bladder cancer (BC. We aimed to identify novel DNA methylation markers for BC through genome-wide profiling of BC cell lines and subsequent methylation-specific PCR (MSP screening of clinical urine samples. EXPERIMENTAL DESIGN: The methyl-DNA binding domain (MBD capture technique, methylCap/seq, was performed to screen for specific hypermethylated CpG islands in two BC cell lines (5637 and T24. The top one hundred hypermethylated targets were sequentially screened by MSP in urine samples to gradually narrow the target number and optimize the composition of the diagnostic panel. The diagnostic performance of the obtained panel was evaluated in different clinical scenarios. RESULTS: A total of 1,627 hypermethylated promoter targets in the BC cell lines was identified by Illumina sequencing. The top 104 hypermethylated targets were reduced to eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A after the urine DNA screening in a small sample size of 8 normal control and 18 BC subjects. Validation in an independent sample of 212 BC patients enabled the optimization of five methylation targets, including VAX1, KCNV1, TAL1, PPOX1, and CFTR, which was obtained in our previous study, for BC diagnosis with a sensitivity and specificity of 88.68% and 87.25%, respectively. In addition, the methylation of VAX1 and LMX1A was found to be associated with BC recurrence. CONCLUSIONS: We identified a promising diagnostic marker panel for early non-invasive detection and subsequent BC surveillance.

Mitochondrial DNA Marker EST00083 Is Not Associated with High vs. Average IQ in a German Sample.

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Moises, Hans W.; Yang, Liu; Kohnke, Michael; Vetter, Peter; Neppert, Jurgen; Petrill, Stephen A.; Plomin, Robert

1998-01-01

Tested the association of a mitochondrial DNA marker (EST00083) with high IQ in a sample of 47 German adults with high IQ scores and 77 adults with IQs estimated at lower than 110. Results do not support the hypothesis that high IQ is associated with this marker. (SLD)

Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.

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Breitbach, Sarah; Tug, Suzan; Simon, Perikles

2012-07-01

The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer. In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15-20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis

The value of using DNA markers for beef bull selection in the seedstock sector.

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Van Eenennaam, A L; van der Werf, J H J; Goddard, M E

2011-02-01

The objective of this study was to estimate the value derived from using DNA information to increase the accuracy of beef sire selection in a closed seedstock herd. Breeding objectives for commercial production systems targeting 2 diverse markets were examined using multiple-trait selection indexes developed for the Australian cattle industry. Indexes included those for both maternal (self-replacing) and terminal herds targeting either a domestic market, where steers are finished on pasture, or the export market, where steers are finished on concentrate rations in feedlots and marbling has a large value. Selection index theory was used to predict the response to conventional selection based on phenotypic performance records, and this was compared with including information from 2 hypothetical marker panels. In 1 case the marker panel explained a percentage of additive genetic variance equal to the heritability for all traits in the breeding objective and selection criteria, and in the other case to one-half of this amount. Discounted gene flow methodology was used to calculate the value derived from the use of superior bulls selected using DNA test information and performance recording over that derived from conventional selection using performance recording alone. Results were ultimately calculated as discounted returns per DNA test purchased by the seedstock operator. The DNA testing using these hypothetical marker panels increased the selection response between 29 to 158%. The value of this improvement above that obtained using traditional performance recording ranged from $89 to 565 per commercial bull, and $5,332 to 27,910 per stud bull. Assuming that the entire bull calf crop was tested to achieve these gains, the value of the genetic gain derived from DNA testing ranged from $204 to 1,119 per test. All values assumed that the benefits derived from using superior bulls were efficiently transferred along the production chain to the seedstock producer incurring

Further evidence for population specific differences in the effect of DNA markers and gender on eye colour prediction in forensics

OpenAIRE

Po?piech, Ewelina; Kar?owska-Pik, Joanna; Ziemkiewicz, Bartosz; Kukla, Magdalena; Skowron, Ma?gorzata; Wojas-Pelc, Anna; Branicki, Wojciech

2016-01-01

The genetics of eye colour has been extensively studied over the past few years, and the identified polymorphisms have been applied with marked success in the field of Forensic DNA Phenotyping. A picture that arises from evaluation of the currently available eye colour prediction markers shows that only the analysis of HERC2-OCA2 complex has similar effectiveness in different populations, while the predictive potential of other loci may vary significantly. Moreover, the role of gender in the ...

DNA Fingerprinting Eastern Redbud Cultivars (Cercis canadensis) Using SSR Markers

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In this study we present data for a subset of SSR loci, 76 out of the 130 high-quality loci, which were selected out of hundreds of SSR loci identified from a SSR-enriched library. SSR markers are abundant in eukaryotic genomes and are highly reproducible. Previously, we have used SSR markers to e...

Diagnostic value of stool DNA testing for multiple markers of colorectal cancer and advanced adenoma: a meta-analysis.

Science.gov (United States)

Yang, Hua; Xia, Bing-Qing; Jiang, Bo; Wang, Guozhen; Yang, Yi-Peng; Chen, Hao; Li, Bing-Sheng; Xu, An-Gao; Huang, Yun-Bo; Wang, Xin-Ying

2013-08-01

The diagnostic value of stool DNA (sDNA) testing for colorectal neoplasms remains controversial. To compensate for the lack of large-scale unbiased population studies, a meta-analysis was performed to evaluate the diagnostic value of sDNA testing for multiple markers of colorectal cancer (CRC) and advanced adenoma. The PubMed, Science Direct, Biosis Review, Cochrane Library and Embase databases were systematically searched in January 2012 without time restriction. Meta-analysis was performed using a random-effects model using sensitivity, specificity, diagnostic OR (DOR), summary ROC curves, area under the curve (AUC), and 95% CIs as effect measures. Heterogeneity was measured using the χ(2) test and Q statistic; subgroup analysis was also conducted. A total of 20 studies comprising 5876 individuals were eligible. There was no heterogeneity for CRC, but adenoma and advanced adenoma harboured considerable heterogeneity influenced by risk classification and various detection markers. Stratification analysis according to risk classification showed that multiple markers had a high DOR for the high-risk subgroups of both CRC (sensitivity 0.759 [95% CI 0.711 to 0.804]; specificity 0.883 [95% CI 0.846 to 0.913]; AUC 0.906) and advanced adenoma (sensitivity 0.683 [95% CI 0.584 to 0.771]; specificity 0.918 [95% CI 0.866 to 0.954]; AUC 0.946) but not for the average-risk subgroups of either. In the methylation subgroup, sDNA testing had significantly higher DOR for CRC (sensitivity 0.753 [95% CI 0.685 to 0.812]; specificity 0.913 [95% CI 0.860 to 0.950]; AUC 0.918) and advanced adenoma (sensitivity 0.623 [95% CI 0.527 to 0.712]; specificity 0.926 [95% CI 0.882 to 0.958]; AUC 0.910) compared with the mutation subgroup. There was no significant heterogeneity among studies for subgroup analysis. sDNA testing for multiple markers had strong diagnostic significance for CRC and advanced adenoma in high-risk subjects. Methylation makers had more diagnostic value than mutation

Elevated levels of urinary markers of oxidatively generated DNA and RNA damage in bipolar disorder

DEFF Research Database (Denmark)

Munkholm, Klaus; Poulsen, Henrik Enghusen; Kessing, Lars Vedel

2015-01-01

OBJECTIVES: The pathophysiological mechanisms underlying bipolar disorder and its multi-system nature are unclear. Oxidatively generated damage to nucleosides has been demonstrated in metabolic disorders; however, the extent to which this occurs in bipolar disorder in vivo is unknown. We...... investigated oxidatively generated damage to DNA and RNA in patients with bipolar disorder and its relationship with the affective phase compared with healthy control subjects. METHODS: Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), markers...... of oxidatively generated DNA and RNA damage, respectively, was measured in 37 rapid cycling patients with bipolar disorder and in 40 age- and gender-matched healthy control subjects. Employing a longitudinal design, repeated measurements of both markers were evaluated in various affective phases in patients...

Whole genome DNA copy number changes identified by high density oligonucleotide arrays

Directory of Open Access Journals (Sweden)

Huang Jing

2004-05-01

Full Text Available Abstract Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs. Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA, to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM and mismatch (MM probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number

Applicability of SCAR markers to food genomics: olive oil traceability.

Science.gov (United States)

Pafundo, Simona; Agrimonti, Caterina; Maestri, Elena; Marmiroli, Nelson

2007-07-25

DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.

Reconstruction of molecular phylogeny of closely related Amorphophallus species of India using plastid DNA marker and fingerprinting approaches.

Science.gov (United States)

Gholave, Avinash R; Pawar, Kiran D; Yadav, Shrirang R; Bapat, Vishwas A; Jadhav, Jyoti P

2017-01-01

Plastid DNA markers sequencing and DNA fingerprinting approaches were used and compared for resolving molecular phylogeny of closely related, previously unexplored Amorphophallus species of India. The utility of individual plastid markers namely rbcL , matK , trnH - psbA , trnLC - trnLD , their combined dataset and two fingerprinting techniques viz. RAPD and ISSR were tested for their efficacy to resolves Amorphophallus species into three sections specific clades namely Rhaphiophallus , Conophallus and Amorphophallus . In the present study, sequences of these four plastid DNA regions as well as RAPD and ISSR profiles of 16 Amorphophallus species together with six varieties of two species were generated and analyzed. Maximum likelihood and Bayesian Inference based construction of phylogenetic trees indicated that among the four plastid DNA regions tested individually and their combined dataset, rbcL was found best suited for resolving closely related Amorphophallus species into section specific clades. When analyzed individually, rbcL exhibited better discrimination ability than matK , trnH - psbA , trnLC - trnLD and combination of all four tested plastid markers. Among two fingerprinting techniques used, the resolution of Amorphophallus species using RAPD was better than ISSR and combination of RAPD +ISSR and in congruence with resolution based on rbcL .

Marker-assisted selection in forestry species

International Nuclear Information System (INIS)

Butcher, P.; Southerton, S.

2007-01-01

The primary goal of tree breeding is to increase the quantity and quality of wood products from plantations. Major gains have been achieved using recurrent selection in genetically diverse breeding populations to capture additive variation. However, the long generation times of trees, together with poor juvenile-mature trait correlations, have promoted interest in marker-assisted selection (MAS) to accelerate breeding through early selection. MAS relies on identifying DNA markers, which explain a high proportion of variation in phenotypic traits. Genetic linkage maps have been developed for most commercial tree species and these can be used to locate chromosomal regions where DNA markers co-segregate with quantitative traits (quantitative trait loci, QTL). MAS based on QTL is most likely to be used for within-family selection in a limited number of elite families that can be clonally propagated. Limitations of the approach include the low resolution of marker-trait associations, the small proportion of phenotypic variation explained by QTL and the low success rate in validating QTL in different genetic backgrounds and environments. This has led to a change in research focus towards association mapping to identify variation in the DNA sequence of genes directly controlling phenotypic variation (gene-assisted selection, GAS). The main advantages of GAS are the high resolution of marker-trait associations and the ability to transfer markers across families and even species. Association studies are being used to examine the adaptive significance of variation in genes controlling wood formation and quality, pathogen resistance, cold tolerance and drought tolerance. Single nucleotide polymorphisms (SNPs) in these gene sequences that are significantly associated with trait variation can then be used for early selection. Markers for SNPs can be transferred among individuals regardless of pedigree or family relationship, increasing opportunities for their application in

Microsatellite DNA markers for delineating population structure and kinship among the endangered Kirtland’s warbler (Dendroica kirtlandii)

Science.gov (United States)

TIM L. KING; MICHAEL S. EACKLES; ANNE P. HENDERSON; CAROL I. BOCETTI; DAVE CURRIE; JR WUNDERLE

2005-01-01

We document the isolation and characterization of 23 microsatellite DNA markers for the endangered Kirtland’s warbler (Dendroica kirtlandii), a Nearctic/Neotropical migrant passerine. This suite of markers revealed moderate to high levels of allelic diversity (averaging 7.7 alleles per locus) and heterozygosity (averaging 72%). Genotypic frequencies at 22 of 23 (95%)...

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

Science.gov (United States)

Arulandhu, Alfred J; Staats, Martijn; Hagelaar, Rico; Voorhuijzen, Marleen M; Prins, Theo W; Scholtens, Ingrid; Costessi, Adalberto; Duijsings, Danny; Rechenmann, François; Gaspar, Frédéric B; Barreto Crespo, Maria Teresa; Holst-Jensen, Arne; Birck, Matthew; Burns, Malcolm; Haynes, Edward; Hochegger, Rupert; Klingl, Alexander; Lundberg, Lisa; Natale, Chiara; Niekamp, Hauke; Perri, Elena; Barbante, Alessandra; Rosec, Jean-Philippe; Seyfarth, Ralf; Sovová, Tereza; Van Moorleghem, Christoff; van Ruth, Saskia; Peelen, Tamara; Kok, Esther

2017-10-01

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance. © The Authors 2017. Published by Oxford University Press.

DNA typing of ancient parasite eggs from environmental samples identifies human and animal worm infections in Viking-age settlement.

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Søe, Martin Jensen; Nejsum, Peter; Fredensborg, Brian Lund; Kapel, Christian Moliin Outzen

2015-02-01

Ancient parasite eggs were recovered from environmental samples collected at a Viking-age settlement in Viborg, Denmark, dated 1018-1030 A.D. Morphological examination identified Ascaris sp., Trichuris sp., and Fasciola sp. eggs, but size and shape did not allow species identification. By carefully selecting genetic markers, PCR amplification and sequencing of ancient DNA (aDNA) isolates resulted in identification of: the human whipworm, Trichuris trichiura , using SSUrRNA sequence homology; Ascaris sp. with 100% homology to cox1 haplotype 07; and Fasciola hepatica using ITS1 sequence homology. The identification of T. trichiura eggs indicates that human fecal material is present and, hence, that the Ascaris sp. haplotype 07 was most likely a human variant in Viking-age Denmark. The location of the F. hepatica finding suggests that sheep or cattle are the most likely hosts. Further, we sequenced the Ascaris sp. 18S rRNA gene in recent isolates from humans and pigs of global distribution and show that this is not a suited marker for species-specific identification. Finally, we discuss ancient parasitism in Denmark and the implementation of aDNA analysis methods in paleoparasitological studies. We argue that when employing species-specific identification, soil samples offer excellent opportunities for studies of human parasite infections and of human and animal interactions of the past.

Association between Urinary Excretion of Cortisol and Markers of Oxidatively Damaged DNA and RNA in Humans

DEFF Research Database (Denmark)

Joergensen, Anders; Broedbaek, Kasper; Weimann, Allan

2011-01-01

Chronic psychological stress is associated with accelerated aging, but the underlying biological mechanisms are not known. Prolonged elevations of the stress hormone cortisol is suspected to play a critical role. Through its actions, cortisol may potentially induce oxidatively generated damage...... to cellular constituents such as DNA and RNA, a phenomenon which has been implicated in aging processes. We investigated the relationship between 24 h excretion of urinary cortisol and markers of oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine......, in a sample of 220 elderly men and women (age 65 - 83 years). We found a robust association between the excretion of cortisol and the oxidation markers (R(2)¿=¿0.15, P...

Rust resistance evaluation of advanced wheat (triticum aestivum l.) genotypes using pcr-based dna markers

International Nuclear Information System (INIS)

Rahman, S.U.; Younis, M.; Iqbal, M.Z.; Nawaz, M.

2014-01-01

The most effective and environmental friendly approach for the control of wheat rust disease is the use of resistant genotypes. The present study was conducted to explore rust resistance potential of 85 elite wheat genotypes (36 varieties and 49 advanced lines) using various types of DNA markers like STS, SCAR and SSR. DNA markers linked with different genes conferring resistance to rusts (Leaf rust=Lr, Yellow rust=Yr and Stem rust=Sr) were employed in this study. A total of 18 genes, consisting of eleven Lr (lr1, lr10, lr19, lr21, lr28, lr34, lr39, lr46, lr47, lr51 and lr52), four Yr (yr5, yr18, yr26 and yr29) and three Sr genes (sr2, sr29, and sr36) were studied through linked DNA markers. Maximum number of Lr genes was found in 17 advanced lines and 9 varieties, Yr genes in 26 advanced lines and 20 wheat varieties, and Sr genes in 43 advanced lines and 27 varieties. Minimum number of Lr genes was found in advanced line D-97 and variety Kohinoor-83, Yr genes in wheat variety Bwp-97 and Sr genes in 6 advanced lines and 8 varieties. Molecular data revealed that genotypes having same origin, from a specified area showed resistance for similar type of genes. In this study, an average similarity of 84% was recorded among wheat genotypes. Out of 18 loci, 15 were found to be polymorphic. (author)

Viral-Cellular DNA Junctions as Molecular Markers for Assessing Intra-Tumor Heterogeneity in Cervical Cancer and for the Detection of Circulating Tumor DNA

Directory of Open Access Journals (Sweden)

Katrin Carow

2017-09-01

Full Text Available The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.

DNA Profiles of MTG (Moderat Tahan Gano) Oil Palm Variety Based on SSR Marker

Science.gov (United States)

Putri, L. A. P.; Setiado, H.; Hardianti, R.

2017-03-01

The oil palm, an economically important tree in Indonesia, has been one of the world’s major sources of edible oil and a significant precursor of biodiesel fuel. The objectives of this study were to know DNA profile of commercial MTG (Moderat Tahan Gano) oil palm variety collections. A total of 10 trees MTG oil palm variety were used for analysis. In this experiment, the DNA profile diversity was assessed using mEgCIR0174 and SSR-1 loci of oil palm’s specific SSR markers. The results of the experiment indicated out of 3 alleles of pcr product of mEgCIR0174 (198, 203 and 208 bp) and SSR-1 (201, 217 and 232 bp). These preliminary results demonstrated SSR marker can be used to evaluate genetic relatedness among trees of MTG (Moderat Tahan Gano) oil palm variety derived from different crossing or difference to desease resistance trait or misslabeled.

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada.

Science.gov (United States)

Braukmann, Thomas W A; Kuzmina, Maria L; Sills, Jesse; Zakharov, Evgeny V; Hebert, Paul D N

2017-01-01

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is

Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker

Czech Academy of Sciences Publication Activity Database

Havran, Luděk; Vacek, Jan; Cahová, Kateřina; Fojta, Miroslav

2008-01-01

Roč. 391, č. 5 (2008), s. 1751-1758 ISSN 1618-2642 R&D Projects: GA AV ČR(CZ) IAA4004402; GA AV ČR(CZ) IAA400040611; GA ČR(CZ) GA203/07/1195; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA damage * electroactive marker * carbon electrodes Subject RIV: BO - Biophysics Impact factor: 3.328, year: 2008

Concordance of Hypermethylated DNA and the Tumor Markers CA 15-3, CEA, and TPA in Serum during Monitoring of Patients with Advanced Breast Cancer

Directory of Open Access Journals (Sweden)

Søren Kristiansen

2015-01-01

Full Text Available The serological protein tumor markers CA 15-3, CEA, and TPA are frequently used to monitor tumor burden among metastatic breast cancer patients. Breast cancer is associated with global DNA hypomethylation and hypermethylation of some promoter regions. No monitoring study has yet investigated the interrelationship between protein tumor markers, the global DNA hypomethylation, and hypermethylated genes in serum from patients with advanced disease. Twenty-nine patients with histologically proven advanced breast cancer received first-line chemotherapy with epirubicin. Samples were collected prior to each treatment and prospectively analyzed for CA 15-3, CEA, and TPA. The same samples were retrospectively analyzed for the concentration of hypermethylated RASSF1A and for global DNA hypomethylation using LINE-1. Among patients with elevated concentrations of the protein markers, concordance could be observed between serial changes of the hypermethylated RASSF1A gene and the protein markers. Among patients with lower concentrations, RASSF1A could only be detected periodically. There was discordance between changes of the hypomethylated LINE-1 as compared to the protein markers. Circulating hypermethylated RASSF1A and protein markers may have similar kinetics during monitoring of tumor burden. Further investigations are needed to determine whether any of the hypermethylated DNA genes may provide predictive information during monitoring.

Concordance of Hypermethylated DNA and the Tumor Markers CA 15-3, CEA, and TPA in Serum during Monitoring of Patients with Advanced Breast Cancer.

Science.gov (United States)

Kristiansen, Søren; Jørgensen, Lars Mønster; Hansen, Morten Høgh; Nielsen, Dorte; Sölétormos, György

2015-01-01

The serological protein tumor markers CA 15-3, CEA, and TPA are frequently used to monitor tumor burden among metastatic breast cancer patients. Breast cancer is associated with global DNA hypomethylation and hypermethylation of some promoter regions. No monitoring study has yet investigated the interrelationship between protein tumor markers, the global DNA hypomethylation, and hypermethylated genes in serum from patients with advanced disease. Twenty-nine patients with histologically proven advanced breast cancer received first-line chemotherapy with epirubicin. Samples were collected prior to each treatment and prospectively analyzed for CA 15-3, CEA, and TPA. The same samples were retrospectively analyzed for the concentration of hypermethylated RASSF1A and for global DNA hypomethylation using LINE-1. Among patients with elevated concentrations of the protein markers, concordance could be observed between serial changes of the hypermethylated RASSF1A gene and the protein markers. Among patients with lower concentrations, RASSF1A could only be detected periodically. There was discordance between changes of the hypomethylated LINE-1 as compared to the protein markers. Circulating hypermethylated RASSF1A and protein markers may have similar kinetics during monitoring of tumor burden. Further investigations are needed to determine whether any of the hypermethylated DNA genes may provide predictive information during monitoring.

The dual challenges of generality and specificity when developing environmental DNA markers for species and subspecies of Oncorhynchus

Science.gov (United States)

Taylor M. Wilcox; Kellie J. Carim; Kevin S. McKelvey; Michael Young; Michael K. Schwartz

2015-01-01

Environmental DNA (eDNA) sampling is a powerful tool for detecting invasive and native aquatic species. Often, species of conservation interest co-occur with other, closely related taxa. Here, we developed qPCR (quantitative PCR) markers which distinguish westslope cutthroat trout (Oncorhynchus clarkii lewsi), Yellowstone cutthroat trout (O. clarkii bouvieri...

Short tandem repeat (STR) DNA markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations.

Science.gov (United States)

Gilmore, Simon; Peakall, Rod; Robertson, James

2003-01-09

Short tandem repeat (STR) markers are the DNA marker of choice in forensic analysis of human DNA. Here we extend the application of STR markers to Cannabis sativa and demonstrate their potential for forensic investigations. Ninety-three individual cannabis plants, representing drug and fibre accessions of widespread origin were profiled with five STR makers. A total of 79 alleles were detected across the five loci. All but four individuals from a single drug-type accession had a unique multilocus genotype. An analysis of molecular variance (AMOVA) revealed significant genetic variation among accessions, with an average of 25% genetic differentiation. By contrast, only 6% genetic difference was detected between drug and fibre crop accessions and it was not possible to unequivocally assign plants as either drug or fibre type. However, our results suggest that drug strains may typically possess lower genetic diversity than fibre strains, which may ultimately provide a means of genetic delineation. Our findings demonstrate the promise of cannabis STR markers to provide information on: (1) agronomic type, (2) the geographical origin of drug seizures, and (3) evidence of conspiracy in production of clonally propagated drug crops.

COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC GENETIC MARKERS IN HUMAN FECAL MICROBIAL COMMUNITIES

Science.gov (United States)

Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, the use of these approaches to discern key genomic differences between natural microbial communities remains prohibitively expensive for mo...

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

Science.gov (United States)

Yao, Jia-Long; Tomes, Sumathi; Gleave, Andrew P

2013-05-01

Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

Gender Identification in Date Palm Using Molecular Markers.

Science.gov (United States)

Awan, Faisal Saeed; Maryam; Jaskani, Muhammad J; Sadia, Bushra

2017-01-01

Breeding of date palm is complicated because of its long life cycle and heterozygous nature. Sexual propagation of date palm does not produce true-to-type plants. Sex of date palms cannot be identified until the first flowering stage. Molecular markers such as random amplified polymorphic DNA (RAPD), sequence-characterized amplified regions (SCAR), and simple sequence repeats (SSR) have successfully been used to identify the sex-linked loci in the plant genome and to isolate the corresponding genes. This chapter highlights the use of three molecular markers including RAPD, SCAR, and SSR to identify the gender of date palm seedlings.

ITS and trnH-psbA as Efficient DNA Barcodes to Identify Threatened Commercial Woody Angiosperms from Southern Brazilian Atlantic Rainforests.

Science.gov (United States)

Bolson, Mônica; Smidt, Eric de Camargo; Brotto, Marcelo Leandro; Silva-Pereira, Viviane

2015-01-01

The Araucaria Forests in southern Brazil are part of the Atlantic Rainforest, a key hotspot for global biodiversity. This habitat has experienced extensive losses of vegetation cover due to commercial logging and the intense use of wood resources for construction and furniture manufacturing. The absence of precise taxonomic tools for identifying Araucaria Forest tree species motivated us to test the ability of DNA barcoding to distinguish species exploited for wood resources and its suitability for use as an alternative testing technique for the inspection of illegal timber shipments. We tested three cpDNA regions (matK, trnH-psbA, and rbcL) and nrITS according to criteria determined by The Consortium for the Barcode of Life (CBOL). The efficiency of each marker and selected marker combinations were evaluated for 30 commercially valuable woody species in multiple populations, with a special focus on Lauraceae species. Inter- and intraspecific distances, species discrimination rates, and ability to recover species-specific clusters were evaluated. Among the regions and different combinations, ITS was the most efficient for identifying species based on the 'best close match' test; similarly, the trnH-psbA + ITS combination also demonstrated satisfactory results. When combining trnH-psbA + ITS, Maximum Likelihood analysis demonstrated a more resolved topology for internal branches, with 91% of species-specific clusters. DNA barcoding was found to be a practical and rapid method for identifying major threatened woody angiosperms from Araucaria Forests such as Lauraceae species, presenting a high confidence for recognizing members of Ocotea. These molecular tools can assist in screening those botanical families that are most targeted by the timber industry in southern Brazil and detecting certain species protected by Brazilian legislation and could be a useful tool for monitoring wood exploitation.

ITS and trnH-psbA as Efficient DNA Barcodes to Identify Threatened Commercial Woody Angiosperms from Southern Brazilian Atlantic Rainforests.

Directory of Open Access Journals (Sweden)

Mônica Bolson

Full Text Available The Araucaria Forests in southern Brazil are part of the Atlantic Rainforest, a key hotspot for global biodiversity. This habitat has experienced extensive losses of vegetation cover due to commercial logging and the intense use of wood resources for construction and furniture manufacturing. The absence of precise taxonomic tools for identifying Araucaria Forest tree species motivated us to test the ability of DNA barcoding to distinguish species exploited for wood resources and its suitability for use as an alternative testing technique for the inspection of illegal timber shipments. We tested three cpDNA regions (matK, trnH-psbA, and rbcL and nrITS according to criteria determined by The Consortium for the Barcode of Life (CBOL. The efficiency of each marker and selected marker combinations were evaluated for 30 commercially valuable woody species in multiple populations, with a special focus on Lauraceae species. Inter- and intraspecific distances, species discrimination rates, and ability to recover species-specific clusters were evaluated. Among the regions and different combinations, ITS was the most efficient for identifying species based on the 'best close match' test; similarly, the trnH-psbA + ITS combination also demonstrated satisfactory results. When combining trnH-psbA + ITS, Maximum Likelihood analysis demonstrated a more resolved topology for internal branches, with 91% of species-specific clusters. DNA barcoding was found to be a practical and rapid method for identifying major threatened woody angiosperms from Araucaria Forests such as Lauraceae species, presenting a high confidence for recognizing members of Ocotea. These molecular tools can assist in screening those botanical families that are most targeted by the timber industry in southern Brazil and detecting certain species protected by Brazilian legislation and could be a useful tool for monitoring wood exploitation.

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

Science.gov (United States)

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-01-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

Environmental DNA (eDNA metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

Directory of Open Access Journals (Sweden)

Katy E Klymus

Full Text Available Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05 and high coefficients of determination (R2 for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

Characterization of genetic diversity in chickpea using SSR markers, Start Codon Targeted Polymorphism (SCoT) and Conserved DNA-Derived Polymorphism (CDDP).

Science.gov (United States)

Hajibarat, Zahra; Saidi, Abbas; Hajibarat, Zohreh; Talebi, Reza

2015-07-01

To evaluate the genetic diversity among 48 genotypes of chickpea comprising cultivars, landraces and internationally developed improved lines genetic distances were evaluated using three different molecular marker techniques: Simple Sequence Repeat (SSR); Start Codon Targeted (SCoT) and Conserved DNA-derived Polymorphism (CDDP). Average polymorphism information content (PIC) for SSR, SCoT and CDDP markers was 0.47, 0.45 and 0.45, respectively, and this revealed that three different marker types were equal for the assessment of diversity amongst genotypes. Cluster analysis for SSR and SCoT divided the genotypes in to three distinct clusters and using CDDP markers data, genotypes grouped in to five clusters. There were positive significant correlation (r = 0.43, P SSR markers. These results suggest that efficiency of SSR, SCOT and CDDP markers was relatively the same in fingerprinting of chickpea genotypes. To our knowledge, this is the first detailed report of using targeted DNA region molecular marker (CDDP) for genetic diversity analysis in chickpea in comparison with SCoT and SSR markers. Overall, our results are able to prove the suitability of SCoT and CDDP markers for genetic diversity analysis in chickpea for their high rates of polymorphism and their potential for genome diversity and germplasm conservation.

A suite of microsatellite markers optimized for amplification of DNA from Addax (Addax nasomaculatus) blood preserved on FTA cards.

Science.gov (United States)

Heim, Brett C; Ivy, Jamie A; Latch, Emily K

2012-01-01

The addax (Addax nasomaculatus) is a critically endangered antelope that is currently maintained in zoos through regional, conservation breeding programs. As for many captive species, incomplete pedigree data currently impedes the ability of addax breeding programs to confidently manage the genetics of captive populations and to select appropriate animals for reintroduction. Molecular markers are often used to improve pedigree resolution, thereby improving the long-term effectiveness of genetic management. When developing a suite of molecular markers, it is important to consider the source of DNA, as the utility of markers may vary across DNA sources. In this study, we optimized a suite of microsatellite markers for use in genotyping captive addax blood samples collected on FTA cards. We amplified 66 microsatellite loci previously described in other Artiodactyls. Sixteen markers amplified a single product in addax, but only 5 of these were found to be polymorphic in a sample of 37 addax sampled from a captive herd at Fossil Rim Wildlife Center in the US. The suite of microsatellite markers developed in this study provides a new tool for the genetic management of captive addax, and demonstrates that FTA cards can be a useful means of sample storage, provided appropriate loci are used in downstream analyses. © 2011 Wiley Periodicals, Inc.

A novel two T-DNA binary vector allows efficient generation of marker-free transgenic plants in three elite cultivars of rice (Oryza sativa L.).

Science.gov (United States)

Breitler, Jean-Christophe; Meynard, Donaldo; Van Boxtel, Jos; Royer, Monique; Bonnot, François; Cambillau, Laurence; Guiderdoni, Emmanuel

2004-06-01

A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82-90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.

X linked neonatal centronuclear/myotubular myopathy: evidence for linkage to Xq28 DNA marker loci.

OpenAIRE

Thomas, N S; Williams, H; Cole, G; Roberts, K; Clarke, A; Liechti-Gallati, S; Braga, S; Gerber, A; Meier, C; Moser, H

1990-01-01

We have studied the inheritance of several polymorphic Xq27/28 DNA marker loci in two three generation families with the X linked neonatal lethal form of centronuclear/myotubular myopathy (XL MTM). We found complete linkage of XLMTM to all four informative Xq28 markers analysed, with GCP/RCP (Z = 3.876, theta = 0.00), with DXS15 (Z = 3.737, theta = 0.00), with DXS52 (Z = 2.709, theta = 0.00), and with F8C (Z = 1.020, theta = 0.00). In the absence of any observable recombination, we are unable...

Molecular Markers of Metastasis in Ductal Mammary Carcinoma

National Research Council Canada - National Science Library

Achary, Patnala

2002-01-01

...% of those patients, however, the disease spreads, and they are at risk of death. Our goal is to develop DNA markers that could be reliably used to identify the ductal mammary carcinomas that are prone to develop metastasis...

Evaluation of Faecalibacterium 16S rDNA genetic markers for accurate identification of swine faecal waste by quantitative PCR.

Science.gov (United States)

Duan, Chuanren; Cui, Yamin; Zhao, Yi; Zhai, Jun; Zhang, Baoyun; Zhang, Kun; Sun, Da; Chen, Hang

2016-10-01

A genetic marker within the 16S rRNA gene of Faecalibacterium was identified for use in a quantitative PCR (qPCR) assay to detect swine faecal contamination in water. A total of 146,038 bacterial sequences were obtained using 454 pyrosequencing. By comparative bioinformatics analysis of Faecalibacterium sequences with those of numerous swine and other animal species, swine-specific Faecalibacterium 16S rRNA gene sequences were identified and Polymerase Chain Okabe (PCR) primer sets designed and tested against faecal DNA samples from swine and non-swine sources. Two PCR primer sets, PFB-1 and PFB-2, showed the highest specificity to swine faecal waste and had no cross-reaction with other animal samples. PFB-1 and PFB-2 amplified 16S rRNA gene sequences from 50 samples of swine with positive ratios of 86 and 90%, respectively. We compared swine-specific Faecalibacterium qPCR assays for the purpose of quantifying the newly identified markers. The quantification limits (LOQs) of PFB-1 and PFB-2 markers in environmental water were 6.5 and 2.9 copies per 100 ml, respectively. Of the swine-associated assays tested, PFB-2 was more sensitive in detecting the swine faecal waste and quantifying the microbial load. Furthermore, the microbial abundance and diversity of the microbiomes of swine and other animal faeces were estimated using operational taxonomic units (OTUs). The species specificity was demonstrated for the microbial populations present in various animal faeces. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative polymorphism of sterlet fertilizers (Acipenser Ruthenus for microsatellite DNA markers

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Ольга Олексіївна Малишева

2015-11-01

Full Text Available Based on microsatellite DNA markers in three (LG-19, LS-68 and LS-39 it is examined intraspecific genetic structure of sterlet fertilizers. As a result of this work it was found that this group of fish is in a balanced state in terms of the genetic polymorphism. On the basis of certain individual differences in allelic variants have been selected and combined the parental pairs for alternative genotypes. The results of the research allow optimize further work on the reproduction of sturgeon under artificial cultivation

Identify super quality markers from prototype-based pharmacokinetic markers of Tangzhiqing tablet (TZQ) based on in vitro dissolution/ permeation and in vivo absorption correlations.

Science.gov (United States)

Li, Ziqiang; Liu, Jia; Li, Yazhuo; Du, Xi; Li, Yanfen; Wang, Ruihua; Lv, Chunxiao; He, Xin; Wang, Baohe; Huang, Yuhong; Zhang, Deqin

2018-06-01

A quality marker (Q-marker) is defined as an inherent chemical compound that is used for the quality control of a drug. Its biological activities are closely related to safety and therapeutic effects. Generally, a multiple-component herbal medicine may have many Q-markers. We therefore proposed a concept of "super Q-marker" satisfying both the criterion of Q-markers and PK-markers to be used in more effective quality control of herbal medicine. The first aim was to find suitable prototype-based PK-markers from Tangzhiqing tablets (TZQ), a Chinese patent medicine. Then super Q-markers were expected to be identified from the prototype-based PK-markers based on an in vitro-in vivo correlation study. Potentially eligible prototype-based PK-markers were identified in a single- and multiple-dose pharmacokinetic study on TZQ in 30 healthy volunteers. The in vitro dissolution and permeation profiles of the prototype-based PK-markers of TZQ were evaluated by the physiologically-based drug dissolution/absorption simulating system (DDASS). An in vitro-in vivo correlation analysis was conducted between the dissolution/permeation behaviors in DDASS and the actual absorption profiles in human to test the transferability and traceability of the promising super Q-markers for TZQ. In human, plasma paeoniflorin and nuciferine as prototype-based PK-markers exhibited the appropriate pharmacokinetic properties, including dose-dependent systemic exposure (AUC, C max ) and a proper elimination half-life (1∼3h). In DDASS, it was predicted that paeoniflorin and nuciferine are highly permeable but the absorption rates are primarily limited by the dissolution rates. Moreover, the established in vitro-in vivo correlations of paeoniflorin and nuciferine were in support of the super Q-markers features. Paeoniflorin and nuciferine are identified as the super Q-markers from the prototype-based PK-markers of TZQ based on findings from a combination of in vitro, in vivo, and in vitro-in vivo

Polymorphic DNA microsatellite markers for forensic individual identification and parentage analyses of seven threatened species of parrots (family Psittacidae).

Science.gov (United States)

Jan, Catherine; Fumagalli, Luca

2016-01-01

The parrot family represents one of the bird group with the largest number of endangered species, as a result of habitat destruction and illegal trade. This illicit traffic involves the smuggling of eggs and animals, and the laundering through captive breeding facilities of wild-caught animals. Despite the huge potential of wildlife DNA forensics to determine with conclusive evidence illegal trade, current usage of DNA profiling approaches in parrots has been limited by the lack of suitable molecular markers specifically developed for the focal species and by low cross-species polymorphism. In this study, we isolated DNA microsatellite markers in seven parrot species threatened with extinction (Amazona brasiliensis, A. oratrix, A. pretrei, A. rhodocorytha, Anodorhynchus leari, Ara rubrogenys and Primolius couloni). From an enriched genomic library followed by 454 pyrosequencing, we characterized a total of 106 polymorphic microsatellite markers (mostly tetranucleotides) in the seven species and tested them across an average number of 19 individuals per species. The mean number of alleles per species and across loci varied from 6.4 to 8.3, with the mean observed heterozygosities ranging from 0.65 to 0.84. Identity and parentage exclusion probabilities were highly discriminatory. The high variability displayed by these microsatellite loci demonstrates their potential utility to perform individual genotyping and parentage analyses, in order to develop a DNA testing framework to determine illegal traffic in these threatened species.

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

OpenAIRE

Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schr?der, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinh?usel, Andreas; Egger, Gerda

2015-01-01

Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-...

DNA Methylation as a Biomarker for Body Fluid Identification

Directory of Open Access Journals (Sweden)

Rania Gomaa

2017-12-01

Full Text Available Currently, available identification techniques for forensic samples are either enzyme or protein based, which can be subjected to degradation, thus limiting its storage potentials. Epigenetic changes arising due to DNA methylation and histone acetylation can be used for body fluid identification. Markers DACT1, USP49, ZC3H12D, FGF7, cg23521140, cg17610929, chromosome 4 (25287119–25287254, chromosome 11 (72085678–72085798, 57171095–57171236, 1493401–1493538, and chromosome 19 (47395505–47395651 are currently being used for semen identification. Markers cg26107890, cg20691722, cg01774894 and cg14991487 are used to differentiate saliva and vaginal secretions from other body fluids. However, such markers show overlapping methylation pattern. This review article aimed to highlight the feasibility of using DNA methylation of certain genetic markers in body fluid identification and its implications for forensic investigations. The reviewed articles have employed molecular genetics techniques such as Bisulfite sequencing PCR (BSP, methylation specific PCR (MSP, Pyrosequencing, Combined Bisulfite Restriction Analysis (COBRA, Methylation-sensitive Single Nucleotide Primer Extension (SNuPE, and Multiplex SNaPshot Microarray. Bioinformatics software such as MATLAB and BiQ Analyzer has been used. Biological fluids have different methylation patterns and thus, this difference can be used to identify the nature of the biological fluid found at the crime scene. Using DNA methylation to identify the body fluids gives accurate results without consumption of the trace evidence and requires a minute amount of DNA for analysis. Recent studies have incorporated next-generation sequencing aiming to find out more reliable markers that can differentiate between different body fluids. Nonetheless, new DNA methylation markers are yet to be discovered to accurately differentiate between saliva and vaginal secretions with high confidence. Epigenetic changes are

Investigation of the somaclonal and mutagen induced variability in barley by the application of protein and DNA markers

International Nuclear Information System (INIS)

Atanassov, A.; Todorovska, E.; Trifonova, A.; Petrova, M.; Marinova, E.; Gramatikova, M.; Valcheva, D.; Zaprianov, S.; Mersinkov, N.

1998-01-01

Barley, Hordeum vulgare L., is one of the most important crop species for Bulgaria. The characterisation of the genetic pool is of great necessity for the Bulgarian barley breeding programme which is directed toward improving quantitative and qualitative traits. Molecular markers [protein, restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNA (RAPD)] have been applied to characterise the Bulgarian barley cultivars and their regenerants. The changes in DNA loci coding for 26S, 5.8S and 18S rRNA repeats, C hordein locus and mitochondrial DNA organisation have been investigated. The potential for ribosomal DNA length polymorphism in Bulgarian barley cultivars appear to be limited to three different repeat lengths (10.2, 9.5 and 9.0kb) and three plant rDNA phenotypes. Polymorphism was not observed in ribosomal DNA repeat units in somaclonal variants. Variation concerning C hordein electrophoretic pattern was observed in one line from cultivar Jubiley. Analysis of the HorI locus reveals RFLPs in sequences coding for C hordeins in this line. Mitochondrial molecular markers are convenient for detection of DNA polymorphisms in the variant germplasm as well as for the somaclonal variants derived from it. Two lines from Ruen revealed polymorphic bands after hybridisation with mitochondrial DNA probe. RAPD assays have been carried out by using 20 different 10-mer primers. Heritable polymorphism in several tissue culture derived (TCD) lines was observed. RAPD assay is a sensitive and representative approach to distinguish the variability created by tissue culture and mutagenesis

Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells

DEFF Research Database (Denmark)

Vindigni, Giulia; Raniolo, Sofia; Ottaviani, Alessio

2016-01-01

, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin–streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological...

Identification of Amazonian trees with DNA barcodes.

Science.gov (United States)

Gonzalez, Mailyn Adriana; Baraloto, Christopher; Engel, Julien; Mori, Scott A; Pétronelli, Pascal; Riéra, Bernard; Roger, Aurélien; Thébaud, Christophe; Chave, Jérôme

2009-10-16

Large-scale plant diversity inventories are critical to develop informed conservation strategies. However, the workload required for classic taxonomic surveys remains high and is particularly problematic for megadiverse tropical forests. Based on a comprehensive census of all trees in two hectares of a tropical forest in French Guiana, we examined whether plant DNA barcoding could contribute to increasing the quality and the pace of tropical plant biodiversity surveys. Of the eight plant DNA markers we tested (rbcLa, rpoC1, rpoB, matK, ycf5, trnL, psbA-trnH, ITS), matK and ITS had a low rate of sequencing success. More critically, none of the plastid markers achieved a rate of correct plant identification greater than 70%, either alone or combined. The performance of all barcoding markers was noticeably low in few species-rich clades, such as the Laureae, and the Sapotaceae. A field test of the approach enabled us to detect 130 molecular operational taxonomic units in a sample of 252 juvenile trees. Including molecular markers increased the identification rate of juveniles from 72% (morphology alone) to 96% (morphology and molecular) of the individuals assigned to a known tree taxon. We conclude that while DNA barcoding is an invaluable tool for detecting errors in identifications and for identifying plants at juvenile stages, its limited ability to identify collections will constrain the practical implementation of DNA-based tropical plant biodiversity programs.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples Marcadores de DNA nuclear e mitocondrial para rastreabilidade da carne bovina comercializada

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Aline S.M. Cesar

2010-09-01

Full Text Available Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male. For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.Várias características são importantes no sistema de rastreabilidade, como o sexo, a idade, a raça e/ou a composição racial dos animais, al

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.

Science.gov (United States)

Schoch, Conrad L; Seifert, Keith A; Huhndorf, Sabine; Robert, Vincent; Spouge, John L; Levesque, C André; Chen, Wen

2012-04-17

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes.

Science.gov (United States)

Benz, Karin; Breit, Stephen; Lukoschek, Martin; Mau, Hans; Richter, Wiltrud

2002-04-26

This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant down-regulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.

Inferring Genetic Variation and Demographic History of Michelia yunnanensis Franch. (Magnoliaceae from Chloroplast DNA Sequences and Microsatellite Markers

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Shikang Shen

2017-04-01

Full Text Available Michelia yunnanensis Franch., is a traditional ornamental, aromatic, and medicinal shrub that endemic to Yunnan Province in southwest China. Although the species has a large distribution pattern and is abundant in Yunnan Province, the populations are dramatically declining because of overexploitation and habitat destruction. Studies on the genetic variation and demography of endemic species are necessary to develop effective conservation and management strategies. To generate such knowledge, we used 3 pairs of universal cpDNA markers and 10 pairs of microsatellite markers to assess the genetic diversity, genetic structure, and demographic history of 7 M. yunnanensis populations. We calculated a total of 88 alleles for 10 polymorphic loci and 10 haplotypes for a combined 2,089 bp of cpDNA. M. yunnanensis populations showed high genetic diversity (Ho = 0.551 for nuclear markers and Hd = 0.471 for cpDNA markers and low genetic differentiation (FST = 0.058. Geographical structure was not found among M. yunnanensis populations. Genetic distance and geographic distance were not correlated (P > 0.05, which indicated that geographic isolation is not the primary cause of the low genetic differentiation of M. yunnanensis. Additionally, M. yunnanensis populations contracted ~20,000–30,000 years ago, and no recent expansion occurred in current populations. Results indicated that the high genetic diversity of the species and within its populations holds promise for effective genetic resource management and sustainable utilization. Thus, we suggest that the conservation and management of M. yunnanensis should address exotic overexploitation and habitat destruction.

Use of DNA Markers for Investigating Sources of Bacteria in Contaminated Ground Water: Wooster Township, Wayne County, Ohio

Science.gov (United States)

Dumouchelle, Denise H.

2006-01-01

In 2004, a public-health nuisance was declared by the Wayne County Board of Health in the Scenic Heights Drive-Batdorf Road area of Wooster Township, Wayne County, Ohio, because of concerns about the safety of water from local wells. Repeated sampling had detected the presence of fecal-indicator bacteria and elevated nitrate concentrations. In June 2006, the U.S. Geological Survey (USGS), in cooperation with the Ohio Environmental Protection Agency (Ohio EPA), collected and analyzed samples from some of the affected wells to help investigate the possibility of human-origin bacterial contamination. Water samples from 12 wells and 5 home sewage-treatment systems (HSTS) were collected. Bromide concentrations were determined in samples from the 12 wells. Samples from 5 of the 12 wells were analyzed for wastewater compounds. Total coliform, enterococci and Escherichia coli (E. coli) bacteria concentrations were determined for samples from 8 of the 12 wells. In addition, two microbial source-tracking tools that employ DNA markers were used on samples from several wells and a composite sample of water from five septic tanks. The DNA markers from the Enterococcus faecium species and the order Bacteroidales are associated with specific sources, either human or ruminant sources. Bromide concentrations ranged from 0.04 to 0.18 milligrams per liter (mg/L). No wastewater compounds were detected at concentrations above the reporting limits. Samples from the 12 wells also were collected by Ohio EPA and analyzed for chloride and nitrate. Chloride concentrations ranged from 12.6 to 61.6 mg/L and nitrate concentrations ranged from 2.34 to 11.9 mg/L (as N). Total coliforms and enterococci were detected in samples from 8 wells, at concentrations from 2 to 200 colony-forming units per 100 milliliters (CFU/100 mL) and 0.5 to 17 CFU/100 mL, respectively. E. coli were detected in samples from three of the eight wells, at concentrations of 1 or 2 CFU/100 mL. Tests for the human

Identification and reproducibility of diagnostic DNA markers for tuber starch and yield optimization in a novel association mapping population of potato (Solanum tuberosum L.).

Science.gov (United States)

Schönhals, E M; Ortega, F; Barandalla, L; Aragones, A; Ruiz de Galarreta, J I; Liao, J-C; Sanetomo, R; Walkemeier, B; Tacke, E; Ritter, E; Gebhardt, C

2016-04-01

SNPs in candidate genes Pain - 1, InvCD141 (invertases), SSIV (starch synthase), StCDF1 (transcription factor), LapN (leucine aminopeptidase), and cytoplasm type are associated with potato tuber yield, starch content and/or starch yield. Tuber yield (TY), starch content (TSC), and starch yield (TSY) are complex characters of high importance for the potato crop in general and for industrial starch production in particular. DNA markers associated with superior alleles of genes that control the natural variation of TY, TSC, and TSY could increase precision and speed of breeding new cultivars optimized for potato starch production. Diagnostic DNA markers are identified by association mapping in populations of tetraploid potato varieties and advanced breeding clones. A novel association mapping population of 282 genotypes including varieties, breeding clones and Andean landraces was assembled and field evaluated in Northern Spain for TY, TSC, TSY, tuber number (TN) and tuber weight (TW). The landraces had lower mean values of TY, TW, TN, and TSY. The population was genotyped for 183 microsatellite alleles, 221 single nucleotide polymorphisms (SNPs) in fourteen candidate genes and eight known diagnostic markers for TSC and TSY. Association test statistics including kinship and population structure reproduced five known marker-trait associations of candidate genes and discovered new ones, particularly for tuber yield and starch yield. The inclusion of landraces increased the number of detected marker-trait associations. Integration of the present association mapping results with previous QTL linkage mapping studies for TY, TSC, TSY, TW, TN, and tuberization revealed some hot spots of QTL for these traits in the potato genome. The genomic positions of markers linked or associated with QTL for complex tuber traits suggest high multiplicity and genome wide distribution of the underlying genes.

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

Science.gov (United States)

Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

Establishment of dna fingerprinting in clonal tea improved cultivars from yunnan of china using issr markers

International Nuclear Information System (INIS)

Liu, B.Y.; Zhao, C.M.; Sun, X.M.; Jiang, H.B.

2015-01-01

In this study, DNA fingerprints were constructed by using ISSR markers for 20 clonal improved varieties developed by two breeding institutes in Yunnan province. Seven core ISSR primers were selected from 15 primers. A total of 110 bands were generated by PAGE with seven core primers, 93 of which were polymorphic bands, the percentage of polymorphic band (PPB) was 84.54%, and the mean value of polymorphism information content (PIC) reached 0.417; the genetic similarity coefficient of the cultivars was 0.574-0.854. The two primers, UBC835 and ISSR2, had high PIC values, and could be used to distinguish all cultivars, presenting the most efficient single primers. Among the all of primer combinations from the seven core primers, the three combinations, UBC835/UBC811, UBC835/ISSR2, and UBC835/ISSR3 showed lower similar coefficients, and more efficient in identifying the 20 improved varieties than the other primer combinations. Then these three primer combinations were further scored in 15 traditional cultivars. The results showed that UBC835/ISSR2 was the optimal primer combination, which could be used to distinguish each material among the 20 clonal improved varieties and 15 traditional cultivals. Finally, the DNA fingerprints of the 20 clonal improved varieties were constructed based on country and region code, breeding institute, core primer name and ISSR marker data. The established fingerprints could provide reliable scientific base for the protection of intellectual property right for these clonal improved varieties, and the important molecular information contained in these fingerprints would be useful for the authenticity identification and genetic relationship analysis of tea varieties. (author)

DNA barcodes for ecology, evolution, and conservation.

Science.gov (United States)

Kress, W John; García-Robledo, Carlos; Uriarte, Maria; Erickson, David L

2015-01-01

The use of DNA barcodes, which are short gene sequences taken from a standardized portion of the genome and used to identify species, is entering a new phase of application as more and more investigations employ these genetic markers to address questions relating to the ecology and evolution of natural systems. The suite of DNA barcode markers now applied to specific taxonomic groups of organisms are proving invaluable for understanding species boundaries, community ecology, functional trait evolution, trophic interactions, and the conservation of biodiversity. The application of next-generation sequencing (NGS) technology will greatly expand the versatility of DNA barcodes across the Tree of Life, habitats, and geographies as new methodologies are explored and developed. Published by Elsevier Ltd.

Introgression evidence and phylogenetic relationships among three (ParaMisgurnus species as revealed by mitochondrial and nuclear DNA markers

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Jakovlić I.

2013-01-01

Full Text Available The taxonomy of (ParaMisgurnus genera is still debated. We therefore used mitochondrial and nuclear DNA markers to analyze the phylogenetic relationships among Misgurnus anguillicaudatus, Paramisgurnus dabryanus and Misgurnus fossilis. Differing phylogenetic signals from mitochondrial and nuclear marker data suggest an introgression event in the history of M. anguillicaudatus and M. mohoity. No substantial genetic evidence was found that Paramisgurnus dabryanus should be classified as a separate genus.

Polymorphic DNA microsatellite markers for forensic individual identification and parentage analyses of seven threatened species of parrots (family Psittacidae

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Catherine Jan

2016-09-01

Full Text Available The parrot family represents one of the bird group with the largest number of endangered species, as a result of habitat destruction and illegal trade. This illicit traffic involves the smuggling of eggs and animals, and the laundering through captive breeding facilities of wild-caught animals. Despite the huge potential of wildlife DNA forensics to determine with conclusive evidence illegal trade, current usage of DNA profiling approaches in parrots has been limited by the lack of suitable molecular markers specifically developed for the focal species and by low cross-species polymorphism. In this study, we isolated DNA microsatellite markers in seven parrot species threatened with extinction (Amazona brasiliensis, A. oratrix, A. pretrei, A. rhodocorytha, Anodorhynchus leari, Ara rubrogenys and Primolius couloni. From an enriched genomic library followed by 454 pyrosequencing, we characterized a total of 106 polymorphic microsatellite markers (mostly tetranucleotides in the seven species and tested them across an average number of 19 individuals per species. The mean number of alleles per species and across loci varied from 6.4 to 8.3, with the mean observed heterozygosities ranging from 0.65 to 0.84. Identity and parentage exclusion probabilities were highly discriminatory. The high variability displayed by these microsatellite loci demonstrates their potential utility to perform individual genotyping and parentage analyses, in order to develop a DNA testing framework to determine illegal traffic in these threatened species.

Next generation DNA sequencing technology delivers valuable genetic markers for the genomic orphan legume species, Bituminaria bituminosa

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Pazos-Navarro María

2011-12-01

Full Text Available Abstract Background Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species. Results 432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total. Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2% successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme. Conclusions B. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This

Promoter DNA methylation pattern identifies prognostic subgroups in childhood T-cell acute lymphoblastic leukemia.

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Magnus Borssén

Full Text Available BACKGROUND: Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in T-ALL is hitherto undecided. DESIGN AND METHODS: Genome wide promoter DNA methylation analysis was performed in pediatric T-ALL samples (n = 43 using arrays covering >27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary T-ALL group not tested for methylation (n = 32. RESULTS: Based on CpG island methylator phenotype (CIMP, T-ALL samples were subgrouped as CIMP+ (high methylation and CIMP- (low methylation. CIMP- T-ALL patients had significantly worse overall and event free survival (p = 0.02 and p = 0.001, respectively compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. CONCLUSIONS: We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric T-ALL.

An annotated genetic map of loblolly pine based on microsatellite and cDNA markers

Science.gov (United States)

Craig S. Echt; Surya Saha; Konstantin V. Krutovsky; Kokulapalan Wimalanathan; John E. Erpelding; Chun Liang; C Dana Nelson

2011-01-01

Previous loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety...

Natural hybridization in tropical spikerushes of Eleocharis subgenus Limnochloa (Cyperaceae): Evidence from morphology and DNA markers

Czech Academy of Sciences Publication Activity Database

Košnar, J.; Košnar, Ji.; Macek, Petr; Herbstová, Miroslava; Rejmánková, E.; Stech, M.

2010-01-01

Roč. 97, č. 7 (2010), s. 1229-1240 ISSN 0002-9122 Institutional research plan: CEZ:AV0Z60050516; CEZ:AV0Z50510513 Keywords : Belize * Cyperaceae * DNA markers * hybridization Subject RIV: EB - Genetics ; Molecular Biology; EB - Genetics ; Molecular Biology (BU-J) Impact factor: 3.052, year: 2010

A suite of molecular markers for identifying species, detecting introgression and describing population structure in spadefoot toads (Spea spp.).

Science.gov (United States)

Pfennig, Karin S; Allenby, Ashley; Martin, Ryan A; Monroy, Anaïs; Jones, Corbin D

2012-09-01

Two congeneric species of spadefoot toad, Spea multiplicata and Spea bombifrons, have been the focus of hybridization studies since the 1970s. Because complex hybrids are not readily distinguished phenotypically, genetic markers are needed to identify introgressed individuals. We therefore developed a set of molecular markers (amplified fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and single nucleotide polymorphism) for identifying pure-species, F1 hybrids and more complex introgressed types. To do so, we tested a series of markers across both species and known hybrids using populations in both allopatry and sympatry. We retained those markers that differentiated the two pure-species and also consistently identified known species hybrids. These markers are well suited for identifying hybrids between these species. Moreover, those markers that show variation within each species can be used in conjunction with existing molecular markers in studies of population structure and gene flow. © 2012 Blackwell Publishing Ltd.

Validation of eDNA markers for New Zealand mudsnail surveillance and initial eDNA monitoring at Mississippi River Basin sites

Science.gov (United States)

Merkes, Christopher; Turnquist, Keith N.; Rees, Christopher B.; Amberg, Jon J.

2015-01-01

The performance of newly developed New Zealand mudsnail (Potamopyrgus antipodarum; NZMS) genetic markers for environmental (eDNA) analysis of water were compared across two laboratories. The genetic markers were tested in four quantitative polymerase chain reaction assays targeting two regions of the NZMS mitochondrial genome, specifically the cytochrome c oxidase subunit 1 (coi) and cytochrome b (cytb) genes. In a blind study, analysts tested each sample eight times with each assay. There were 10 expected-negative samples from the Black River in La Crosse, Wisconsin, 10 expected-positive samples from the Black Earth Creek in Black Earth, Wisconsin, and 10 known-positive samples from the Black River spiked with NZMS DNA. Previously extracted samples, kept at the Upper Midwest Environmental Sciences Center, were pooled by sample location and then equal quantities were distributed between the Upper Midwest Environmental Sciences Center and the Molecular Conservation Genetics Laboratory at the University of Wisconsin-Stevens Point for analysis. The assays tested were (1) the assay targeting cytb with a minor groove binder probe described by Goldberg and others (2013), (2) the cytb assay with a modified double-quenched probe, (3) an assay targeting coi with a double-quenched probe, and (4) a duplex reaction combining the modified cytb assay and the coi assay. Samples were considered positive for the presence of NZMS DNA when quantitative polymerase chain reaction amplification and probe signal was higher than the normalized threshold value above baseline fluorescence. For the duplex assay, samples were considered positive only when both probe signals were higher than the normalized threshold value above baseline fluorescence. Positive results were then confirmed by sequencing the products.

Prenatal Screening Using Maternal Markers

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Howard Cuckle

2014-05-01

Full Text Available Maternal markers are widely used to screen for fetal neural tube defects (NTDs, chromosomal abnormalities and cardiac defects. Some are beginning to broaden prenatal screening to include pregnancy complications such as pre-eclampsia. The methods initially developed for NTDs using a single marker have since been built upon to develop high performance multi-maker tests for chromosomal abnormalities. Although cell-free DNA testing is still too expensive to be considered for routine application in public health settings, it can be cost-effective when used in combination with existing multi-maker marker tests. The established screening methods can be readily applied in the first trimester to identify pregnancies at high risk of pre-eclampsia and offer prevention though aspirin treatment. Prenatal screening for fragile X syndrome might be adopted more widely if the test was to be framed as a form of maternal marker screening.

Molecular species identification of Central European ground beetles (Coleoptera: Carabidae using nuclear rDNA expansion segments and DNA barcodes

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Raupach Michael J

2010-09-01

Full Text Available Abstract Background The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous. Results We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97% of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95% of the studied Carabidae. Conclusion Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.

Molecular species identification of Central European ground beetles (Coleoptera: Carabidae) using nuclear rDNA expansion segments and DNA barcodes.

Science.gov (United States)

Raupach, Michael J; Astrin, Jonas J; Hannig, Karsten; Peters, Marcell K; Stoeckle, Mark Y; Wägele, Johann-Wolfgang

2010-09-13

The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI) gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous. We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97%) of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95%) of the studied Carabidae. Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.

DNA fingerprinting in forensics: past, present, future.

Science.gov (United States)

Roewer, Lutz

2013-11-18

DNA fingerprinting, one of the great discoveries of the late 20th century, has revolutionized forensic investigations. This review briefly recapitulates 30 years of progress in forensic DNA analysis which helps to convict criminals, exonerate the wrongly accused, and identify victims of crime, disasters, and war. Current standard methods based on short tandem repeats (STRs) as well as lineage markers (Y chromosome, mitochondrial DNA) are covered and applications are illustrated by casework examples. Benefits and risks of expanding forensic DNA databases are discussed and we ask what the future holds for forensic DNA fingerprinting.

Assessing Date Palm Genetic Diversity Using Different Molecular Markers.

Science.gov (United States)

Atia, Mohamed A M; Sakr, Mahmoud M; Adawy, Sami S

2017-01-01

Molecular marker technologies which rely on DNA analysis provide powerful tools to assess biodiversity at different levels, i.e., among and within species. A range of different molecular marker techniques have been developed and extensively applied for detecting variability in date palm at the DNA level. Recently, the employment of gene-targeting molecular marker approaches to study biodiversity and genetic variations in many plant species has increased the attention of researchers interested in date palm to carry out phylogenetic studies using these novel marker systems. Molecular markers are good indicators of genetic distances among accessions, because DNA-based markers are neutral in the face of selection. Here we describe the employment of multidisciplinary molecular marker approaches: amplified fragment length polymorphism (AFLP), start codon targeted (SCoT) polymorphism, conserved DNA-derived polymorphism (CDDP), intron-targeted amplified polymorphism (ITAP), simple sequence repeats (SSR), and random amplified polymorphic DNA (RAPD) to assess genetic diversity in date palm.

DNA methylation signatures for prediction of biochemical recurrence after radical prostatectomy of clinically localized prostate cancer

DEFF Research Database (Denmark)

Haldrup, Christa; Mundbjerg, Kamilla; Vestergaard, Else Marie

2013-01-01

Purpose Diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, causing overtreatment of indolent PC and risk of delayed treatment of aggressive PC. Here, we identify six novel candidate DNA methylation markers for PC with promising diagnostic and prognostic potential. Methods...... Microarray-based screening and bisulfite sequencing of 20 nonmalignant and 29 PC tissue specimens were used to identify new candidate DNA hypermethylation markers for PC. Diagnostic and prognostic potential was evaluated in 35 nonmalignant prostate tissue samples, 293 radical prostatectomy (RP) samples...... into low- and high-methylation subgroups, was trained in cohort 1 (HR, 1.91; 95% CI, 1.26 to 2.90) and validated in cohort 2 (HR, 2.33; 95% CI, 1.31 to 4.13). Conclusion We identified six novel candidate DNA methylation markers for PC. C1orf114 hypermethylation and a three-gene methylation signature were...

Changes in markers of oxidative stress and DNA damage in human visceral adipose tissue from subjects with obesity and type 2 diabetes.

Science.gov (United States)

Jones, D A; Prior, S L; Barry, J D; Caplin, S; Baxter, J N; Stephens, J W

2014-12-01

In the past 30 years, prevalence of obesity has almost trebled resulting in an increased incidence of type 2 diabetes mellitus and other co-morbidities. Visceral adipose tissue is believed to play a vital role, but underlying mechanisms remain unclear. Our aim was to investigate changes in markers of oxidative damage in human visceral adipose tissue to determine levels of oxidative burden that may be attributed to obesity and/or diabetes. Visceral adipose tissue samples from 61 subjects undergoing abdominal surgery grouped as lean, obese and obese with type 2 diabetes mellitus, were examined using 3 different markers of oxidative stress. Malondialdehyde (MDA) concentration was measured as a marker of lipid peroxidation, telomere length and Comet assay as markers of oxidative DNA damage. No significant difference in MDA concentration, telomere length and DNA damage was observed between groups, although longer telomere lengths were seen in the obese with diabetes group compared to the obese group (Pstress and DNA damage was observed in samples from subjects with type 2 diabetes mellitus. Further work is required to investigate this further, however this phenomenon may be due to an up regulation of antioxidant defences in adipose tissue. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Quantification of Circulating Free DNA as a Diagnostic Marker in Gall Bladder Cancer.

Science.gov (United States)

Kumari, Swati; Tewari, Shikha; Husain, Nuzhat; Agarwal, Akash; Pandey, Anshuman; Singhal, Ashish; Lohani, Mohtashim

2017-01-01

Gall bladder Carcinoma (GBC) is the fifth most common cancer of the digestive tract and frequently diagnosed in late stage of disease. Estimation of circulating free DNA (cfDNA) in serum has been applied as a "liquid biopsy" in several deep seated malignancies. Its value in diagnosis of gall bladder carcinoma has not been studied. The present study was designed to assess the role of cfDNA in the diagnosis of GBC and correlate levels with the TNM stage. Serum was collected from 34 patients with GBC and 39 age and sex matched controls including 22 cholecystitis and 17 healthy individuals. Serum cfDNA levels were measured through quantitative polymerase chain reaction (qPCR) by amplification of β-globin gene. Performance of the assay was calculated through the receiver operating characteristic (ROC) curve. The cfDNA level was significantly lower in healthy controls and cholecystitis (89.32 ± 59.76 ng/ml, 174.21 ± 99.93 ng/ml) compared to GBC (1245.91 ± 892.46 ng/ml, p = <0.001). The cfDNA level was significantly associated with TNM stage, lymph node involvement and jaundice (0.002, 0.027, and 0.041, respectively). Area under curve of ROC analysis for cancer group versus healthy and cholecystitis group was 1.00 and 0.983 with sensitivity of 100 %, 88.24 % and specificity of 100 % respectively. Quantitative analysis of cfDNA may distinguish cholecystitis and gall bladder carcinoma and may serve as new diagnostic, noninvasive marker adjunct to imaging for the diagnosis of GBC.

Inheritance of blast resistance and identification of SSR marker ...

Indian Academy of Sciences (India)

2013-08-02

Aug 2, 2013 ... arm of chromosome 6 and in close proximity of blast resis- ... resistance to a M. oryzae race in a gene-for-gene manner. DNA markers have been ... order to identify the new sources of resistance against blast, there is need for ...

Identification of apple cultivars on the basis of simple sequence repeat markers.

Science.gov (United States)

Liu, G S; Zhang, Y G; Tao, R; Fang, J G; Dai, H Y

2014-09-12

DNA markers are useful tools that play an important role in plant cultivar identification. They are usually based on polymerase chain reaction (PCR) and include simple sequence repeats (SSRs), inter-simple sequence repeats, and random amplified polymorphic DNA. However, DNA markers were not used effectively in the complete identification of plant cultivars because of the lack of known DNA fingerprints. Recently, a novel approach called the cultivar identification diagram (CID) strategy was developed to facilitate the use of DNA markers for separate plant individuals. The CID was designed whereby a polymorphic maker was generated from each PCR that directly allowed for cultivar sample separation at each step. Therefore, it could be used to identify cultivars and varieties easily with fewer primers. In this study, 60 apple cultivars, including a few main cultivars in fields and varieties from descendants (Fuji x Telamon) were examined. Of the 20 pairs of SSR primers screened, 8 pairs gave reproducible, polymorphic DNA amplification patterns. The banding patterns obtained from these 8 primers were used to construct a CID map. Each cultivar or variety in this study was distinguished from the others completely, indicating that this method can be used for efficient cultivar identification. The result contributed to studies on germplasm resources and the seedling industry in fruit trees.

Preliminary Comparative Analysis of Phenological Varieties of Quercus robur by ISSR-Markers

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Vasiliy Chokheli

2016-01-01

Full Text Available Quercus robur L. is a valuable wood species having long ontogeny and promising to create long-living artificial plantings of recreational and ameliorative purposes in the steppes zone of Russia and other countries. In this work we have performed the genotyping of varieties of Quercus robur L. obtained from collection of Botanical Garden of Southern Federal University using intersimple sequence repeat (ISSR molecular markers. The most polymorphic ISSR-marker (GA 8YC was found in the collection. The polymorphic DNA markers identified in the present study can be used for the future breeding works to obtain valuable genotypes of Quercus genus. In addition we have performed DNA fingerprinting of the prospective sample of the variety Q. robur var. tardiflora Czern.

DNA markers provide insight about common lime in historicalplantings

DEFF Research Database (Denmark)

Hansen, Ole Kim; Thomsen, Pernille; Rasmussen, Christine Waage

2014-01-01

As part of the restoration process of an avenue of common lime (Tilia × europaea) from 1760 in the Royal Danish Gardens, all remaining trees were genotyped with DNA markers before they were felled. As such, information about the nature of the plant material (clonal versus non-clonal) and mode...... of propagation was obtained, revealing that a single clone constituted 92% of the remaining trees (106 out of 115). Five trees were of another clone, while the remaining four trees had unique genotypes. Mode of clonal propagation was most likely layering since the genotype of the crown and the roots...... of a subsample of the trees had the same genotype. Trees from four other locations with historical avenues/plantings from the 17th century were also genotyped. The two clones registered in the first location were also found at the other four locations. Of 76 trees from the other historical avenues...

Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B.

Science.gov (United States)

Chen, En-Qiang; Feng, Shu; Wang, Meng-Lan; Liang, Ling-Bo; Zhou, Ling-Yun; Du, Ling-Yao; Yan, Li-Bo; Tao, Chuan-Min; Tang, Hong

2017-03-14

Recently, hepatitis B core-related antigen (HBcrAg) has been suggested as an additional marker of hepatitis B virus (HBV) infection. This study aimed to investigate whether serum quantitative HBcrAg (qHBcrAg) was a satisfactory surrogate marker of intrahepatic covalently closed circular DNA (cccDNA). A total of 139 patients with liver biopsy were enrolled, consisting of 59 patients in immune tolerance (IT) phase, 52 patients in immune clearance (IC) phase, 18 patients in low-replication (LR) phase, and 10 patients in reactivation phase. All patients in IC phase have received entecavir (ETV) therapy, and 32 of them undergone a second liver biopsy at 24 months. Among those patients, qHBcrAg was strongly correlated with intrahepatic cccDNA, which is superior to that of qHBsAg and HBV DNA. And similar findings were also observed in patients in IT, IC, LR and reactivation phases. Among the 32 ETV-treated patients with a second liver biopsy in IC phase, the decline of intrahepatic cccDNA was accompanied by changes in both qHBcrAg and qHBsAg. However, as compared to qHBsAg, the change of qHBcrAg was more strongly associated with intrahepatic cccDNA-decline. In summary, serum qHBcrAg should be a satisfactory surrogate of intrahepatic HBV cccDNA in CHB patients.

Dine marker har DNA

DEFF Research Database (Denmark)

Eckholdt, Annette; Winding, Anne; Krogh, Paul Henning

2017-01-01

Ordet "biodiversitet" og at det er noget, vi skal have mere af, nævnes hyppigt. Men hvad er biodiversitet, og hvordan måles det? Agrologisk har bedt et par eksperter fra Aarhus Universitet forklare, hvordan et DNA-aftryk af jord og vand kan erstatte optællinger i felten og sige noget om biodivers......Ordet "biodiversitet" og at det er noget, vi skal have mere af, nævnes hyppigt. Men hvad er biodiversitet, og hvordan måles det? Agrologisk har bedt et par eksperter fra Aarhus Universitet forklare, hvordan et DNA-aftryk af jord og vand kan erstatte optællinger i felten og sige noget om...

DNA fingerprinting and diversity analysis in Aus genotypes using microsatellite markers

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MD. MONIRUL ISLAM

2015-08-01

Full Text Available DNA fingerprinting and genetic diversity of 94 Aus (6 BRRI released Aus variety and 88 local Aus landraces genotypes were carried out to protect the Aus landraces from biopiracy. A total of 91 microsatellite markers were tested for screening the genotypes. Among 91 amplified products, 56% have polymorphic bands giving 195 alleles. The number of alleles per locus ranged from four (RM25 and RM147 to twenty seven (RM519, where average allele number was 9.76. The Polymorphism Information Contents (PIC lied between 0.455 (RM5 to 0.934 (RM519. Most robust marker was found RM519 since it provided the highest PIC value (0.934. Pair-wise genetic dissimilarity co-efficient showed the lowest genetic dissimilarity was found BRRI dhan42 and BRRI dhan43 and the highest genetic dissimilarity was found local landraces each other. Here it is shown that most Aus landraces is recognized to have broad genetic base. Thus it is recommended to use these landraces for future breeding program or include new and untouched local landraces to incorporate new genes and broaden genetic base.

Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities

Science.gov (United States)

2017-10-01

15.3%) NA 6 (6%) 6 (5.4%) Prostate - specific Antigen (PSA) ng/mL 76.7 (42.9) 78.2 (40.7) pTNM Stage T2 68 (67.3%) 48 (43.2%) T3 29 (28.7%) 58...Profiles Primary Aim #1: Determine if methylation profiles differ by race/ancestry Primary Aim #2: Identify ethnicity- specific markers of prostate ...by ethnicity and to identify ethnicity- specific methylation features of prostate cancer that could contribute the racial disparities that exist in

Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality.

Science.gov (United States)

Li, Li; Tacke, Eckhard; Hofferbert, Hans-Reinhardt; Lübeck, Jens; Strahwald, Josef; Draffehn, Astrid M; Walkemeier, Birgit; Gebhardt, Christiane

2013-04-01

Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were

Intelligent DNA-based molecular diagnostics using linked genetic markers

Energy Technology Data Exchange (ETDEWEB)

Pathak, D.K.; Perlin, M.W.; Hoffman, E.P.

1994-12-31

This paper describes a knowledge-based system for molecular diagnostics, and its application to fully automated diagnosis of X-linked genetic disorders. Molecular diagnostic information is used in clinical practice for determining genetic risks, such as carrier determination and prenatal diagnosis. Initially, blood samples are obtained from related individuals, and PCR amplification is performed. Linkage-based molecular diagnosis then entails three data analysis steps. First, for every individual, the alleles (i.e., DNA composition) are determined at specified chromosomal locations. Second, the flow of genetic material among the individuals is established. Third, the probability that a given individual is either a carrier of the disease or affected by the disease is determined. The current practice is to perform each of these three steps manually, which is costly, time consuming, labor-intensive, and error-prone. As such, the knowledge-intensive data analysis and interpretation supersede the actual experimentation effort as the major bottleneck in molecular diagnostics. By examining the human problem solving for the task, we have designed and implemented a prototype knowledge-based system capable of fully automating linkage-based molecular diagnostics in X-linked genetic disorders, including Duchenne Muscular Dystrophy (DMD). Our system uses knowledge-based interpretation of gel electrophoresis images to determine individual DNA marker labels, a constraint satisfaction search for consistent genetic flow among individuals, and a blackboard-style problem solver for risk assessment. We describe the system`s successful diagnosis of DMD carrier and affected individuals from raw clinical data.

Development and validation of new SSR markers from expressed regions in the garlic genome

Directory of Open Access Journals (Sweden)

Meryem Ipek

2015-02-01

Full Text Available Only a limited number of simple sequence repeat (SSR markers is available for the genome of garlic (Allium sativum L. despite the fact that SSR markers have become one of the most preferred DNA marker systems. To develop new SSR markers for the garlic genome, garlic expressed sequence tags (ESTs at the publicly available GarlicEST database were screened for SSR motifs and a total of 132 SSR motifs were identified. Primer pairs were designed for 50 SSR motifs and 24 of these primer pairs were selected as SSR markers based on their consistent amplification patterns and polymorphisms. In addition, two SSR markers were developed from the sequences of garlic cDNA-AFLP fragments. The use of 26 EST-SSR markers for the assessment of genetic relationship was tested using 31 garlic genotypes. Twenty six EST-SSR markers amplified 130 polymorphic DNA fragments and the number of polymorphic alleles per SSR marker ranged from 2 to 13 with an average of 5 alleles. Observed heterozygosity and polymorphism information content (PIC of the SSR markers were between 0.23 and 0.88, and 0.20 and 0.87, respectively. Twenty one out of the 31 garlic genotypes were analyzed in a previous study using AFLP markers and the garlic genotypes clustered together with AFLP markers were also grouped together with EST-SSR markers demonstrating high concordance between AFLP and EST-SSR marker systems and possible immediate application of EST-SSR markers for fingerprinting of garlic clones. EST-SSR markers could be used in genetic studies such as genetic mapping, association mapping, genetic diversity and comparison of the genomes of Allium species.

Evaluation of powdery mildew-resistance of grape germplasm and rapid amplified polymorphic DNA markers associated with the resistant trait in Chinese wild Vitis.

Science.gov (United States)

Zhang, J; Zhang, Y; Yu, H; Wang, Y

2014-05-09

The resistance of wild Vitis germplasm, including Chinese and American wild Vitis and Vitis vinifera cultivars, to powdery mildew (Uncinula necator Burr.) was evaluated for two consecutive years under natural conditions. Most of the Chinese and North American species displayed a resistant phenotype, whereas all of the European species were highly susceptible. The Alachua and Conquistador accessions of Vitis rotundifolia species, which originated in North America, were immune to the disease, while Baihe-35-1, one of the accessions of Vitis pseudoreticulata, showed the strongest resistance among all Chinese accessions evaluated. Three rapid amplified polymorphic DNA (RAPD) markers, OPW02-1756, OPO11-964, and OPY13-661, were obtained after screening 520 random primers among various germplasm, and these markers were found to be associated with powdery mildew resistance in Baihe-35-1 and in some Chinese species, but not in any European species. Analysis of F₁ and F₂ progenies of a cross between resistant Baihe-35-1 and susceptible Carignane (V. vinifera) revealed that the three RAPD markers were linked to the powdery resistant trait in Baihe-35-1 plants. Potential applications of the identified RAPD markers for gene mapping, marker-assisted selection, and breeding were investigated in 168 F₂ progenies of the same cross. Characterization of the resistant phenotype of the selected F₂ seedlings for breeding a new disease-resistant grape cultivar is in progress.

Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p,p'-DDE in European and Inuit populations

DEFF Research Database (Denmark)

Stronati, A; Manicardi, G C; Cecati, M

2006-01-01

Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652...... adult males (200 Inuits from Greenland, 166 Swedish, 134 Polish and 152 Ukrainian). Serum levels of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (CB-153), as a proxy of the total POP burden, and of 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE), as a proxy of the total DDT exposure were determined...... neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas...

X linked neonatal centronuclear/myotubular myopathy: evidence for linkage to Xq28 DNA marker loci.

Science.gov (United States)

Thomas, N S; Williams, H; Cole, G; Roberts, K; Clarke, A; Liechti-Gallati, S; Braga, S; Gerber, A; Meier, C; Moser, H

1990-05-01

We have studied the inheritance of several polymorphic Xq27/28 DNA marker loci in two three generation families with the X linked neonatal lethal form of centronuclear/myotubular myopathy (XL MTM). We found complete linkage of XLMTM to all four informative Xq28 markers analysed, with GCP/RCP (Z = 3.876, theta = 0.00), with DXS15 (Z = 3.737, theta = 0.00), with DXS52 (Z = 2.709, theta = 0.00), and with F8C (Z = 1.020, theta = 0.00). In the absence of any observable recombination, we are unable to sublocalise the XLMTM locus further within the Xq28 region. This evidence for an Xq28 localisation may allow us to carry out useful genetic counselling within such families.

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

Directory of Open Access Journals (Sweden)

Yang Huaan

2012-07-01

Full Text Available Abstract Background In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Results Twenty informative plants from a cross of RxS (disease resistant x susceptible in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM, and are now replacing the markers previously developed by a traditional DNA

Toward a DNA taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae using a mixed Yule-coalescent analysis of mitochondrial and nuclear DNA.

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Laurent Vuataz

Full Text Available Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1 marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe.

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

Science.gov (United States)

M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

2009-01-01

The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

Genetic characterization of Moroccan and the exotic bread wheat cultivars using functional and random DNA markers linked to the agronomic traits for genomics-assisted improvement.

Science.gov (United States)

Henkrar, Fatima; El-Haddoury, Jamal; Ouabbou, Hassan; Bendaou, Najib; Udupa, Sripada M

2016-06-01

Genetic characterization, diversity analysis and estimate of the genetic relationship among varieties using functional and random DNA markers linked to agronomic traits can provide relevant guidelines in selecting parents and designing new breeding strategies for marker-assisted wheat cultivar improvement. Here, we characterize 20 Moroccan and 19 exotic bread wheat (Triticum aestivum L.) cultivars using 47 functional and 7 linked random DNA markers associated with 21 loci of the most important traits for wheat breeding. The functional marker analysis revealed that 35, 45, and 10 % of the Moroccan cultivars, respectively have the rust resistance genes (Lr34/Yr18/Pm38), dwarfing genes (Rht1b or Rht2b alleles) and the leaf rust resistance gene (Lr68). The marker alleles for genes Lr37/Yr17/Sr38, Sr24 and Yr36 were present only in the exotic cultivars and absent in Moroccan cultivars. 25 % of cultivars had 1BL.1RS translocation. 70 % of the wheat cultivars had Ppo-D1a and Ppo-A1b associated with low polyphenol oxidase activity. 10 % of cultivars showed presence of a random DNA marker allele (175 bp) linked to Hessian fly resistance gene H22. The majority of the Moroccan cultivars were carrying alleles that impart good bread making quality. Neighbor joining (NJ) and principal coordinate analysis based on the marker data revealed a clear differentiation between elite Moroccan and exotic wheat cultivars. The results of this study are useful for selecting suitable parents for making targeted crosses in marker-assisted wheat breeding and enhancing genetic diversity in the wheat cultivars.

DNA landmarks for genetic relatedness and diversity assessment in Pakistani wheat genotypes using RAPD markers

International Nuclear Information System (INIS)

Siddiqui, M.F.; Iqbal, S.; Naz, N.; Khan, S.; Erum, S.

2010-01-01

DNA profiles from 10 Pakistani wheat genotypes were evaluated for diversity assessment based on RAPD markers. A total of 79 DNA fragments were generated by 10 RAPD primers, with an average of 7.9 bands primer-1. Of these, 64 fragments (81%) were polymorphic among 10 genotypes. Genetic diversity was evaluated via UPGMA cluster analysis by constructing dendrogram, which were used for the calculation of similarity coefficients between these genotypes. The greatest similarity (95%) was observed between PR-94 and PR-95, whereas PR-96 with PR-90 showed the lowest similarity (60%). Adoption of this technology would be useful to the plant protection regulatory systems, especially for plant variety identification and registration of new plant varieties, breeding programs and protection purposes. (author)

DNA landmarks for genetic relatedness and diversity assessment in Pakistani wheat genotypes using RAPD markers

Energy Technology Data Exchange (ETDEWEB)

Siddiqui, M F; Iqbal, S; Naz, N; Khan, S [Federal Seed Certification and Registration Dept., Islamabad (Pakistan); Erum, S [National Agricultural Research Centre, Islamabad (Pakistan). Plant Genetic Resources Inst.

2010-04-15

DNA profiles from 10 Pakistani wheat genotypes were evaluated for diversity assessment based on RAPD markers. A total of 79 DNA fragments were generated by 10 RAPD primers, with an average of 7.9 bands primer-1. Of these, 64 fragments (81%) were polymorphic among 10 genotypes. Genetic diversity was evaluated via UPGMA cluster analysis by constructing dendrogram, which were used for the calculation of similarity coefficients between these genotypes. The greatest similarity (95%) was observed between PR-94 and PR-95, whereas PR-96 with PR-90 showed the lowest similarity (60%). Adoption of this technology would be useful to the plant protection regulatory systems, especially for plant variety identification and registration of new plant varieties, breeding programs and protection purposes. (author)

Molecular markers. Amplified fragment length polymorphism

Directory of Open Access Journals (Sweden)

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.

Viability of human dental pulp in determination of sex of an individual by identifying srygene through DNA analysis: A single blind pilot study

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Prachi Ravikant Naik

2012-01-01

Full Text Available Recognition of importance of human teeth in personal identification has been recognized from time immemorial. In any natural calamity or man-made catastrophe identification of an individual is of paramount importance. Here tooth plays an important role as it is the last one to get affected in a disaster due to its durable nature and good survival rate. This information comes under the aegis of forensic odontology and is of paramount importance from legal and social viewpoints. This analysis uses highly informative genetic markers and can be carried out easily in a typical forensic lab oratory. The SRY gene marker (sex determining region Y is a sex-determining gene on the Y chromosome in the therians (placental mammals and marsupials and this gene marker is considered as a signature gene to differentiate the male from female sex chromosome. The detection of SRY gene in the DNA from a forensic sample can be confirmatory to type the gender as male. This study was taken up to identify the viability of human tooth pulp by identification of SRY gene in gender determination.

Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury

Science.gov (United States)

Dutta, Suhrid R.; Kar, Prasanta K.; Srivastava, Ashok K.; Sinha, Manoj K.; Shankar, Jai; Ghosh, Ananta K.

2012-01-01

The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F2 progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16905 bp showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F2 progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16826 bp). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk. PMID:23271934

Mitochondrial DNA variation in brood stocks of the lake trout

International Nuclear Information System (INIS)

Grewe, P.M.; Hebert, P.D.N.

1986-01-01

Efforts are in progress to restore lake trout populations in the Great Lakes from hatchery stocks. In most cases, plantings include a variety of brood stocks that originated from different portions of the Great Lakes. Members of the various stocks can be differentially fin clipped to permit comparison of their survival success, but this does not allow assessment of their reproductive capability in the wild. Assessment of reproductive success requires the existence of genetic markers between brook stocks which will ideally persist over many generations. Efforts to identify allozyme differences between brood stocks have met with little success. The present investigation has employed an alternative technique to identify genetic markers--the restriction analysis of mitochondrial DNA. Mitochondiral DNA analysis of 7 lake trout brood stocks has revealed the existence of 10 mitochondrial clones falling into 3 major groups. The results indicate that mt-DNA markers have great potential for brood stock management. Genetic variability in the nuclear genome of each stock can be maintained by utilizing a large number of male parents, while restricting female parents to members of a single mitochondrial clone. Genetically marked fry could then be produced with only minor shifts in hatchery management

Use of Simple Sequence Repeat (SSR) markers for DNA fingerprinting and diversity analysis of sugarcane (Saccharum spp.) cultivars resistant and susceptible to red rot

Science.gov (United States)

In recent years SSR markers have been used widely for the genetic analysis. The objective of present research was to use SSR markers to develop DNA-based genetic identification and analyze genetic relationship of sugarcane cultivars grown in Pakistan either resistant or susceptible to red rot. Twent...

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.

Modeling of genetic gain for single traits from marker-assisted seedling selection in clonally propagated crops

Science.gov (United States)

Ru, Sushan; Hardner, Craig; Carter, Patrick A; Evans, Kate; Main, Dorrie; Peace, Cameron

2016-01-01

Seedling selection identifies superior seedlings as candidate cultivars based on predicted genetic potential for traits of interest. Traditionally, genetic potential is determined by phenotypic evaluation. With the availability of DNA tests for some agronomically important traits, breeders have the opportunity to include DNA information in their seedling selection operations—known as marker-assisted seedling selection. A major challenge in deploying marker-assisted seedling selection in clonally propagated crops is a lack of knowledge in genetic gain achievable from alternative strategies. Existing models based on additive effects considering seed-propagated crops are not directly relevant for seedling selection of clonally propagated crops, as clonal propagation captures all genetic effects, not just additive. This study modeled genetic gain from traditional and various marker-based seedling selection strategies on a single trait basis through analytical derivation and stochastic simulation, based on a generalized seedling selection scheme of clonally propagated crops. Various trait-test scenarios with a range of broad-sense heritability and proportion of genotypic variance explained by DNA markers were simulated for two populations with different segregation patterns. Both derived and simulated results indicated that marker-based strategies tended to achieve higher genetic gain than phenotypic seedling selection for a trait where the proportion of genotypic variance explained by marker information was greater than the broad-sense heritability. Results from this study provides guidance in optimizing genetic gain from seedling selection for single traits where DNA tests providing marker information are available. PMID:27148453

Taxonomic confirmation of mud crab species (genus Scylla) in Bangladesh by nuclear and mitochondrial DNA markers.

Science.gov (United States)

Sarower, Mohammed Golam; Shahriar, Sheik Istiak Md; Nakamura, Hiromasa; Rouf, Muhammad Abdur; Okada, Shigeru

2017-11-01

Taxonomy of mud crabs genus Scylla has been misidentified for several years due to their high morphological plasticity. Several reports concerning mud crab have been published with misleading identification in Bangladesh. In this study, partial fragments of nuclear and mitochondrial DNA of Scylla species obtained from four locations along the Bangladesh coast were used to resolve taxonomical ambiguity of mud crab species. A single PCR product from the nuclear first internal transcribed spacer (ITS-1) marker and phylogenetic trees constructed based on 16S rDNA sequences indicated that all Scylla species obtained in this study were S. olivacea. Both molecular data and morphological characters revealed that S. olivacea is the only major species in Bangladesh coastal waters. Further, the 16S rDNA haplotypes significantly differed with known S. serrata by 33%. From this study it is clear that 'S. serrata' commonly reported from Bangladesh should be S. olivacea.

Identification of RAPD marker associated with brown rust resistance in sugarcane

Science.gov (United States)

Susceptibility to brown rust caused by Puccinia melanocephala is a major reason for the withdrawal of sugarcane cultivars from production. An efficient way to control the disease is to breed cultivars with durable resistance. Our aim was to identify random amplified polymorphic DNA (RAPD) markers ...

Identification and Validation of a New Male Sex-Specific ISSR Marker in Pointed Gourd (Trichosanthes dioica Roxb.

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Sinchan Adhikari

2014-01-01

Full Text Available The aim of the present study was to develop a genetic sex marker for the pointed gourd (Trichosanthes dioica Roxb. to allow gender determination at any stage in the life cycle. Screening of genomic DNA with intersimple sequence repeat (ISSR primers was used to discover sex-specific touch-down polymerase chain reaction (Td-PCR amplification products. Using pooled DNA from male and female genotypes and 42 ISSR primers, a putative male specific marker (~550 bp was identified. DNA marker specific to male is an indication of existence of nonepigenetic factors involved in gender development in pointed gourd. The ISSR technique has proved to be a reliable technique in gender determination of pointed gourd genotypes at the seedling phenophase. The sex marker developed here could also be used as a starting material towards sequence characterization of sex linked genes for better understanding the developmental as well as evolutionary pathways in sexual dimorphism.

A non-destructive genotyping system from a single seed for marker-assisted selection in watermelon.

Science.gov (United States)

Meru, G; McDowell, D; Waters, V; Seibel, A; Davis, J; McGregor, C

2013-03-11

Genomic tools for watermelon breeding are becoming increasingly available. A high throughput genotyping system would facilitate the use of DNA markers in marker-assisted selection. DNA extraction from leaf material requires prior seed germination and is often time-consuming and cost prohibitive. In an effort to develop a more efficient system, watermelon seeds of several genotypes and various seed sizes were sampled by removing ⅓ or ½ sections from the distal ends for DNA extraction, while germinating the remaining proximal parts of the seed. Removing ⅓ of the seed from the distal end had no effect on seed germination percentage or seedling vigor. Different DNA extraction protocols were tested to identify a method that could yield DNA of sufficient quality for amplification by polymerase chain reaction. A sodium dodecyl sulfate extraction protocol with 1% polyvinylpyrrolidone yielded DNA that could be amplified with microsatellite primers and was free of pericarp contamination. In this study, an efficient, non-destructive genotyping protocol for watermelon seed was developed.

Molecular markers of carcinogenesis for risk stratification of individuals with colorectal polyps: a case-control study.

Science.gov (United States)

Gupta, Samir; Sun, Han; Yi, Sang; Storm, Joy; Xiao, Guanghua; Balasubramanian, Bijal A; Zhang, Song; Ashfaq, Raheela; Rockey, Don C

2014-10-01

Risk stratification using number, size, and histology of colorectal adenomas is currently suboptimal for identifying patients at increased risk for future colorectal cancer. We hypothesized that molecular markers of carcinogenesis in adenomas, measured via immunohistochemistry, may help identify high-risk patients. To test this hypothesis, we conducted a retrospective, 1:1 matched case-control study (n = 216; 46% female) in which cases were patients with colorectal cancer and synchronous adenoma and controls were patients with adenoma but no colorectal cancer at baseline or within 5 years of follow-up. In phase I of analyses, we compared expression of molecular markers of carcinogenesis in case and control adenomas, blind to case status. In phase II of analyses, patients were randomly divided into independent training and validation groups to develop a model for predicting case status. We found that seven markers [p53, p21, Cox-2, β-catenin (BCAT), DNA-dependent protein kinase (DNApkcs), survivin, and O6-methylguanine-DNA methyltransferase (MGMT)] were significantly associated with case status on unadjusted analyses, as well as analyses adjusted for age and advanced adenoma status (P marker component). When applied to the validation set, a predictive model using these seven markers showed substantial accuracy for identifying cases [area under the receiver operation characteristic curve (AUC), 0.83; 95% confidence interval (CI), 0.74-0.92]. A parsimonious model using three markers performed similarly to the seven-marker model (AUC, 0.84). In summary, we found that molecular markers of carcinogenesis distinguished adenomas from patients with and without colorectal cancer. Furthermore, we speculate that prospective studies using molecular markers to identify individuals with polyps at risk for future neoplasia are warranted. ©2014 American Association for Cancer Research.

Impact of deep coalescence on the reliability of species tree inference from different types of DNA markers in mammals.

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Alejandro Sánchez-Gracia

Full Text Available An important challenge for phylogenetic studies of closely related species is the existence of deep coalescence and gene tree heterogeneity. However, their effects can vary between species and they are often neglected in phylogenetic analyses. In addition, a practical problem in the reconstruction of shallow phylogenies is to determine the most efficient set of DNA markers for a reliable estimation. To address these questions, we conducted a multilocus simulation study using empirical values of nucleotide diversity and substitution rates obtained from a wide range of mammals and evaluated the performance of both gene tree and species tree approaches to recover the known speciation times and topological relationships. We first show that deep coalescence can be a serious problem, more than usually assumed, for the estimation of speciation times in mammals using traditional gene trees. Furthermore, we tested the performance of different sets of DNA markers in the determination of species trees using a coalescent approach. Although the best estimates of speciation times were obtained, as expected, with the use of an increasing number of nuclear loci, our results show that similar estimations can be obtained with a much lower number of genes and the incorporation of a mitochondrial marker, with its high information content. Thus, the use of the combined information of both nuclear and mitochondrial markers in a species tree framework is the most efficient option to estimate recent speciation times and, consequently, the underlying species tree.

Molecular markers for tolerance of European ash (Fraxinus excelsior) to dieback disease identified using Associative Transcriptomics

DEFF Research Database (Denmark)

Harper, Andrea L.; McKinney, Lea Vig; Nielsen, Lene Rostgaard

2016-01-01

panel scored for disease symptoms and identified markers strongly associated with canopy damage in infected trees. Using these markers we predicted phenotypes in a test panel of unrelated trees, successfully identifying individuals with a low level of susceptibility to the disease. Co......Tree disease epidemics are a global problem, impacting food security, biodiversity and national economies. The potential for conservation and breeding in trees is hampered by complex genomes and long lifecycles, with most species lacking genomic resources. The European Ash tree Fraxinus excelsior...

Using the Developmental Gene Bicoid to Identify Species of Forensically Important Blowflies (Diptera: Calliphoridae

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Seong Hwan Park

2013-01-01

Full Text Available Identifying species of insects used to estimate postmortem interval (PMI is a major subject in forensic entomology. Because forensic insect specimens are morphologically uniform and are obtained at various developmental stages, DNA markers are greatly needed. To develop new autosomal DNA markers to identify species, partial genomic sequences of the bicoid (bcd genes, containing the homeobox and its flanking sequences, from 12 blowfly species (Aldrichina grahami, Calliphora vicina, Calliphora lata, Triceratopyga calliphoroides, Chrysomya megacephala, Chrysomya pinguis, Phormia regina, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Hemipyrellia ligurriens and Lucilia sericata; Calliphoridae: Diptera were determined and analyzed. This study first sequenced the ten blowfly species other than C. vicina and L. sericata. Based on the bcd sequences of these 12 blowfly species, a phylogenetic tree was constructed that discriminates the subfamilies of Calliphoridae (Luciliinae, Chrysomyinae, and Calliphorinae and most blowfly species. Even partial genomic sequences of about 500 bp can distinguish most blowfly species. The short intron 2 and coding sequences downstream of the bcd homeobox in exon 3 could be utilized to develop DNA markers for forensic applications. These gene sequences are important in the evolution of insect developmental biology and are potentially useful for identifying insect species in forensic science.

Using the Developmental Gene Bicoid to Identify Species of Forensically Important Blowflies (Diptera: Calliphoridae)

Science.gov (United States)

Park, Seong Hwan; Park, Chung Hyun; Zhang, Yong; Piao, Huguo; Chung, Ukhee; Kim, Seong Yoon; Ko, Kwang Soo; Yi, Cheong-Ho; Jo, Tae-Ho; Hwang, Juck-Joon

2013-01-01

Identifying species of insects used to estimate postmortem interval (PMI) is a major subject in forensic entomology. Because forensic insect specimens are morphologically uniform and are obtained at various developmental stages, DNA markers are greatly needed. To develop new autosomal DNA markers to identify species, partial genomic sequences of the bicoid (bcd) genes, containing the homeobox and its flanking sequences, from 12 blowfly species (Aldrichina grahami, Calliphora vicina, Calliphora lata, Triceratopyga calliphoroides, Chrysomya megacephala, Chrysomya pinguis, Phormia regina, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Hemipyrellia ligurriens and Lucilia sericata; Calliphoridae: Diptera) were determined and analyzed. This study first sequenced the ten blowfly species other than C. vicina and L. sericata. Based on the bcd sequences of these 12 blowfly species, a phylogenetic tree was constructed that discriminates the subfamilies of Calliphoridae (Luciliinae, Chrysomyinae, and Calliphorinae) and most blowfly species. Even partial genomic sequences of about 500 bp can distinguish most blowfly species. The short intron 2 and coding sequences downstream of the bcd homeobox in exon 3 could be utilized to develop DNA markers for forensic applications. These gene sequences are important in the evolution of insect developmental biology and are potentially useful for identifying insect species in forensic science. PMID:23586044

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

Science.gov (United States)

Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schröder, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinhäusel, Andreas; Egger, Gerda

2015-01-01

Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. Results: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. Conclusions: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer. PMID:26335606

Development of Multiplexed Marker Sets to Identify the Most Relevant Poplar Species for Breeding

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Hilke Schroeder

2017-12-01

Full Text Available Within the genus Populus, about 30 species are classified into six sections, of which some are cross-compatible. Besides naturally occurring hybrids, huge breeding programs have led to a high number of artificially produced hybrids, for which the determination of genetically involved species by morphological characteristics is often difficult. This necessitates the use of molecular markers for the identification of both maternal as well as paternal species, and in the case of complex hybrids, the genealogy. For this reason, we developed new chloroplast and nuclear markers for the differentiation of up to 19 poplar species, with one to 32 individuals per species regularly used in breeding programs based on already known barcoding, other chloroplast regions, and nuclear genes of interest. We developed methods to identify species by either species-specific nucleotide variations or, when no initial information for the species was given, by using a set of markers either in a procedure of exclusion or in a multiplexed marker set. The developed markers can all be used with low-cost equipment, and some can additionally be applied using a genetic analyzer. We combined these markers in multiplexes for a very fast and easy-to-use application for the identification of poplar species and their hybrids.

Using RAD-seq to recognize sex-specific markers and sex chromosome systems.

Science.gov (United States)

Gamble, Tony

2016-05-01

Next-generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. ). Among the most impressive of these sequencing innovations is restriction site-associated DNA sequencing or RAD-seq (Baird et al. ; Andrews et al. ). RAD-seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD-seq data has been to identify sex-specific genetic markers, markers found in one sex but not the other (Baxter et al. ; Gamble & Zarkower ). Sex-specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon ; Mossman & Waser ), the management and breeding of endangered species (Taberlet et al. ; Griffiths & Tiwari ; Robertson et al. ) and sexing embryonic material (Hacker et al. ; Smith et al. ). Furthermore, sex-specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank ; Gamble & Zarkower ). Thus, species with male-specific markers have male heterogamety (XY) while species with female-specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi () illustrate the ease by which RAD-seq data can generate sex-specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD-seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig. ), Fowler & Buonaccorsi () uncover shared sex-specific markers and a conserved sex chromosome system. © 2016 John Wiley & Sons Ltd.

A transcriptome derived female-specific marker from the invasive Western mosquitofish (Gambusia affinis.

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Dunja K Lamatsch

Full Text Available Sex-specific markers are a prerequisite for understanding reproductive biology, genetic factors involved in sex differences, mechanisms of sex determination, and ultimately the evolution of sex chromosomes. The Western mosquitofish, Gambusia affinis, may be considered a model species for sex-chromosome evolution, as it displays female heterogamety (ZW/ZZ, and is also ecologically interesting as a worldwide invasive species. Here, de novo RNA-sequencing on the gonads of sexually mature G. affinis was used to identify contigs that were highly transcribed in females but not in males (i.e., transcripts with ovary-specific expression. Subsequently, 129 primer pairs spanning 79 contigs were tested by PCR to identify sex-specific transcripts. Of those primer pairs, one female-specific DNA marker was identified, Sanger sequenced and subsequently validated in 115 fish. Sequence analyses revealed a high similarity between the identified sex-specific marker and the 3´ UTR of the aminomethyl transferase (amt gene of the closely related platyfish (Xiphophorus maculatus. This is the first time that RNA-seq has been used to successfully characterize a sex-specific marker in a fish species in the absence of a genome map. Additionally, the identified sex-specific marker represents one of only a handful of such markers in fishes.

Using DNA Metabarcoding to Identify the Floral Composition of Honey: A New Tool for Investigating Honey Bee Foraging Preferences.

Science.gov (United States)

Hawkins, Jennifer; de Vere, Natasha; Griffith, Adelaide; Ford, Col R; Allainguillaume, Joel; Hegarty, Matthew J; Baillie, Les; Adams-Groom, Beverley

2015-01-01

Identifying the floral composition of honey provides a method for investigating the plants that honey bees visit. We compared melissopalynology, where pollen grains retrieved from honey are identified morphologically, with a DNA metabarcoding approach using the rbcL DNA barcode marker and 454-pyrosequencing. We compared nine honeys supplied by beekeepers in the UK. DNA metabarcoding and melissopalynology were able to detect the most abundant floral components of honey. There was 92% correspondence for the plant taxa that had an abundance of over 20%. However, the level of similarity when all taxa were compared was lower, ranging from 22-45%, and there was little correspondence between the relative abundance of taxa found using the two techniques. DNA metabarcoding provided much greater repeatability, with a 64% taxa match compared to 28% with melissopalynology. DNA metabarcoding has the advantage over melissopalynology in that it does not require a high level of taxonomic expertise, a greater sample size can be screened and it provides greater resolution for some plant families. However, it does not provide a quantitative approach and pollen present in low levels are less likely to be detected. We investigated the plants that were frequently used by honey bees by examining the results obtained from both techniques. Plants with a broad taxonomic range were detected, covering 46 families and 25 orders, but a relatively small number of plants were consistently seen across multiple honey samples. Frequently found herbaceous species were Rubus fruticosus, Filipendula ulmaria, Taraxacum officinale, Trifolium spp., Brassica spp. and the non-native, invasive, Impatiens glandulifera. Tree pollen was frequently seen belonging to Castanea sativa, Crataegus monogyna and species of Malus, Salix and Quercus. We conclude that although honey bees are considered to be supergeneralists in their foraging choices, there are certain key species or plant groups that are particularly

Using DNA Metabarcoding to Identify the Floral Composition of Honey: A New Tool for Investigating Honey Bee Foraging Preferences.

Directory of Open Access Journals (Sweden)

Jennifer Hawkins

Full Text Available Identifying the floral composition of honey provides a method for investigating the plants that honey bees visit. We compared melissopalynology, where pollen grains retrieved from honey are identified morphologically, with a DNA metabarcoding approach using the rbcL DNA barcode marker and 454-pyrosequencing. We compared nine honeys supplied by beekeepers in the UK. DNA metabarcoding and melissopalynology were able to detect the most abundant floral components of honey. There was 92% correspondence for the plant taxa that had an abundance of over 20%. However, the level of similarity when all taxa were compared was lower, ranging from 22-45%, and there was little correspondence between the relative abundance of taxa found using the two techniques. DNA metabarcoding provided much greater repeatability, with a 64% taxa match compared to 28% with melissopalynology. DNA metabarcoding has the advantage over melissopalynology in that it does not require a high level of taxonomic expertise, a greater sample size can be screened and it provides greater resolution for some plant families. However, it does not provide a quantitative approach and pollen present in low levels are less likely to be detected. We investigated the plants that were frequently used by honey bees by examining the results obtained from both techniques. Plants with a broad taxonomic range were detected, covering 46 families and 25 orders, but a relatively small number of plants were consistently seen across multiple honey samples. Frequently found herbaceous species were Rubus fruticosus, Filipendula ulmaria, Taraxacum officinale, Trifolium spp., Brassica spp. and the non-native, invasive, Impatiens glandulifera. Tree pollen was frequently seen belonging to Castanea sativa, Crataegus monogyna and species of Malus, Salix and Quercus. We conclude that although honey bees are considered to be supergeneralists in their foraging choices, there are certain key species or plant groups that

Identification of RAPD markers linked to salinity tolerance in wheat ...

African Journals Online (AJOL)

Genetic diversity can be measured by a number of ways, including pedigree, phenotype and allelic diversity at loci controlling phenotypes of interest. A DNA marker for root length in wheat (Triticum aestivum L.) was identified. The individual plants from F2 population segregation for salinity tolerance and the parents (S-24 ...

MethylMix 2.0: an R package for identifying DNA methylation genes.

Science.gov (United States)

Cedoz, Pierre-Louis; Prunello, Marcos; Brennan, Kevin; Gevaert, Olivier

2018-04-14

DNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes. Here we present a new version of MethylMix that automates the construction of DNA-methylation and gene expression datasets from The Cancer Genome Atlas (TCGA). More precisely, MethylMix 2.0 incorporates two major updates: the automated downloading of DNA methylation and gene expression datasets from TCGA and the automated preprocessing of such datasets: value imputation, batch correction and CpG sites clustering within each gene. The resulting datasets can subsequently be analyzed with MethylMix to identify transcriptionally predictive methylation states. We show that the Differential Methylation Values created by MethylMix can be used for cancer subtyping. olivier.gevaert@stanford.edu. https://bioconductor.org/packages/release/bioc/manuals/MethylMix/man/MethylMix.pdf. MethylMix 2.0 was implemented as an R package and is available in bioconductor.

Identification and evaluation of two diagnostic markers linked to Fusarium wilt resistance (race 4) in banana (Musa spp.).

Science.gov (United States)

Wang, Wei; Hu, Yulin; Sun, Dequan; Staehelin, Christian; Xin, Dawei; Xie, Jianghui

2012-01-01

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (FOC4) results in vascular tissue damage and ultimately death of banana (Musa spp.) plants. Somaclonal variants of in vitro micropropagated banana can hamper success in propagation of genotypes resistant to FOC4. Early identification of FOC4 resistance in micropropagated banana plantlets is difficult, however. In this study, we identified sequence-characterized amplified region (SCAR) markers of banana associated with resistance to FOC4. Using pooled DNA from resistant or susceptible genotypes and 500 arbitrary 10-mer oligonucleotide primers, 24 random amplified polymorphic DNA (RAPD) products were identified. Two of these RAPD markers were successfully converted to SCAR markers, called ScaU1001 (GenBank accession number HQ613949) and ScaS0901 (GenBank accession number HQ613950). ScaS0901 and ScaU1001 could be amplified in FOC4-resistant banana genotypes ("Williams 8818-1" and Goldfinger), but not in five tested banana cultivars susceptible to FOC4. The two SCAR markers were then used to identify a somaclonal variant of the genotype "Williams 8818-1", which lost resistance to FOC4. Hence, the identified SCAR markers can be applied for a rapid quality control of FOC4-resistant banana plantlets immediately after the in vitro micropropagation stage. Furthermore, ScaU1001 and ScaS0901 will facilitate marker-assisted selection of new banana cultivars resistant to FOC4.

DNA barcodes identify Central Asian Colias butterflies (Lepidoptera, Pieridae).

Science.gov (United States)

Laiho, Juha; Ståhls, Gunilla

2013-12-30

A majority of the known Colias species (Lepidoptera: Pieridae, Coliadinae) occur in the mountainous regions of Central-Asia, vast areas that are hard to access, rendering the knowledge of many species limited due to the lack of extensive sampling. Two gene regions, the mitochondrial COI 'barcode' region and the nuclear ribosomal protein RpS2 gene region were used for exploring the utility of these DNA markers for species identification. A comprehensive sampling of COI barcodes for Central Asian Colias butterflies showed that the barcodes facilitated identification of most of the included species. Phylogenetic reconstruction based on parsimony and Neighbour-Joining recovered most species as monophyletic entities. For the RpS2 gene region species-specific sequences were registered for some of the included Colias spp. Nevertheless, this gene region was not deemed useful as additional molecular 'barcode'. A parsimony analysis of the combined COI and RpS2 data did not support the current subgeneric classification based on morphological characteristics.

Application of DNA based marker mutations for improvement of cereals and other sexually reproduced crop plants. Proceedings of a final research co-ordination meeting

Energy Technology Data Exchange (ETDEWEB)

NONE

1998-03-01

The Co-ordinated Research Programme (CRP) on the Application of DNA Based Marker Mutations for Improvement of Cereals and Other Sexually Reproduced Crop Plants represents the first of three CRPs dealing with the application of molecular markers to mutations and plant breeding and was implemented between 1992 and 1996. A second companion CRP entitled Use of Novel DNA Fingerprinting Techniques for the Detection and Characterization of Genetic Variation in Vegetatively Propagated Crops devoted to the application of molecular markers in vegetatively propagated crops species was implemented between 1993 and 1997. One positive consequence of these two CRPs has been the implementation of a third CRP entitled Radioactively Labeled DNA Probes for Crop Improvement, which began in 1995 and aims to provide enabling technologies, in the form of probes and primers, to laboratories in developing countries. The rapid development of molecular marker technologies has also resulted in a dramatic increase in request from developing Member States for technical co-operation projects utilizing molecular markers to improve local varieties for biotic and abiotic stresses and other traits of relevance. With the intensified use of induced mutations in genetic studies, it will be important to continue the important work of understanding induced mutations at the molecular level. Evidence of the progress made in implementing molecular marker technologies in laboratories around the world is presented in this publication, which contains the results presented by the participants at the fourth and final Research Co-ordination Meeting of the CRP held in Vienna, 4-8 November 1996. The FAO and IAEA wish to express their sincere appreciation to the participants of the meeting for their work during the project period resulting in the summary and scientific reports presented in this publication. Refs, figs, tabs.

Application of DNA based marker mutations for improvement of cereals and other sexually reproduced crop plants. Proceedings of a final research co-ordination meeting

International Nuclear Information System (INIS)

1998-03-01

The Co-ordinated Research Programme (CRP) on the Application of DNA Based Marker Mutations for Improvement of Cereals and Other Sexually Reproduced Crop Plants represents the first of three CRPs dealing with the application of molecular markers to mutations and plant breeding and was implemented between 1992 and 1996. A second companion CRP entitled Use of Novel DNA Fingerprinting Techniques for the Detection and Characterization of Genetic Variation in Vegetatively Propagated Crops devoted to the application of molecular markers in vegetatively propagated crops species was implemented between 1993 and 1997. One positive consequence of these two CRPs has been the implementation of a third CRP entitled Radioactively Labeled DNA Probes for Crop Improvement, which began in 1995 and aims to provide enabling technologies, in the form of probes and primers, to laboratories in developing countries. The rapid development of molecular marker technologies has also resulted in a dramatic increase in request from developing Member States for technical co-operation projects utilizing molecular markers to improve local varieties for biotic and abiotic stresses and other traits of relevance. With the intensified use of induced mutations in genetic studies, it will be important to continue the important work of understanding induced mutations at the molecular level. Evidence of the progress made in implementing molecular marker technologies in laboratories around the world is presented in this publication, which contains the results presented by the participants at the fourth and final Research Co-ordination Meeting of the CRP held in Vienna, 4-8 November 1996. The FAO and IAEA wish to express their sincere appreciation to the participants of the meeting for their work during the project period resulting in the summary and scientific reports presented in this publication

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.

Application of microsatellite markers as potential tools for traceability of Girgentana goat breed dairy products.

Science.gov (United States)

Sardina, Maria Teresa; Tortorici, Lina; Mastrangelo, Salvatore; Di Gerlando, Rosalia; Tolone, Marco; Portolano, Baldassare

2015-08-01

In livestock, breed assignment may play a key role in the certification of products linked to specific breeds. Traceability of farm animals and authentication of their products can contribute to improve breed profitability and sustainability of animal productions with significant impact on the rural economy of particular geographic areas and on breed and biodiversity conservation. With the goal of developing a breed genetic traceability system for Girgentana dairy products, the aim of this study was to identify specific microsatellite markers able to discriminate among the most important Sicilian dairy goat breeds, in order to detect possible adulteration in Girgentana dairy products. A total of 20 microsatellite markers were analyzed on 338 individual samples from Girgentana, Maltese, and Derivata di Siria goat breeds. Specific microsatellite markers useful for traceability of dairy products were identified. Eight microsatellite markers showed alleles present at the same time in Maltese and Derivata di Siria and absent in Girgentana and, therefore, they were tested on DNA pools of the three breeds. Considering the electropherograms' results, only FCB20, SRCRSP5, and TGLA122 markers were tested on DNA samples extracted from cheeses of Girgentana goat breed. These three microsatellite markers could be applied in a breed genetic traceability system of Girgentana dairy products in order to detect adulteration due to Maltese and Derivata di Siria goat breeds. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evolution and perspectives of cultivar identification and traceability from tree to oil and table olives by means of DNA markers.

Science.gov (United States)

Pasqualone, Antonella; Montemurro, Cinzia; di Rienzo, Valentina; Summo, Carmine; Paradiso, Vito Michele; Caponio, Francesco

2016-08-01

In recent years, an increasing number of typicality marks has been awarded to high-quality olive oils produced from local cultivars. In this case, quality control requires effective varietal checks of the starting materials. Moreover, accurate cultivar identification is essential in vegetative-propagated plants distributed by nurseries and is a pre-requisite to register new cultivars. Food genomics provides many tools for cultivar identification and traceability from tree to oil and table olives. The results of the application of different classes of DNA markers to olive with the purpose of checking cultivar identity and variability of plant material are extensively discussed in this review, with special regard to repeatability issues and polymorphism degree. The characterization of olive germplasm from all countries of the Mediterranean basin and from less studied geographical areas is described and innovative high-throughput molecular tools to manage reference collections are reviewed. Then the transferability of DNA markers to processed products - virgin olive oils and table olives - is overviewed to point out strengths and weaknesses, with special regard to (i) the influence of processing steps and storage time on the quantity and quality of residual DNA, (ii) recent advances to overcome the bottleneck of DNA extraction from processed products, (iii) factors affecting whole comparability of DNA profiles between fresh plant materials and end-products, (iv) drawbacks in the analysis of multi-cultivar versus single-cultivar end-products and (v) the potential of quantitative polymerase chain reaction (PCR)-based techniques. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Staining Against Phospho-H2AX (gamma-H2AX) as a Marker for DNA Damage and Genomic Instability in Cancer Tissues and Cells

NARCIS (Netherlands)

Nagelkerke, A.P.; Span, P.N.

2016-01-01

Phospho-H2AX or gamma-H2AX- is a marker of DNA double-stranded breaks and can therefore be used to monitor DNA repair after, for example, irradiation. In addition, positive staining for phospho-H2AX may indicate genomic instability and telomere dysfunction in tumour cells and tissues. Here, we

Using optical markers of nondysplastic rectal epithelial cells to identify patients with ulcerative colitis-associated neoplasia.

Science.gov (United States)

Bista, Rajan K; Brentnall, Teresa A; Bronner, Mary P; Langmead, Christopher J; Brand, Randall E; Liu, Yang

2011-12-01

Current surveillance guidelines for patients with long-standing ulcerative colitis (UC) recommend repeated colonoscopy with random biopsies, which is time-consuming, discomforting, and expensive. A less invasive strategy is to identify neoplasia by analyzing biomarkers from the more accessible rectum to predict the need for a full colonoscopy. The goal of this pilot study was to evaluate whether optical markers of rectal mucosa derived from a novel optical technique, partial-wave spectroscopic microscopy (PWS), could identify UC patients with high-grade dysplasia (HGD) or cancer (CA) present anywhere in their colon. Banked frozen nondysplastic mucosal rectal biopsies were used from 28 UC patients (15 without dysplasia and 13 with concurrent HGD or CA). The specimen slides were made using a touch prep method and underwent PWS analysis. We divided the patients into two groups: 13 as a training set and an independent 15 as a validation set. We identified six optical markers, ranked by measuring the information gain with respect to the outcome of cancer. The most effective markers were selected by maximizing the cross-validated training accuracy of a Naive Bayes classifier. The optimal classifier was applied to the validation data yielding 100% sensitivity and 75% specificity. Our results indicate that the PWS-derived optical markers can accurately predict UC patients with HGD/CA through assessment of rectal epithelial cells. By aiming for high sensitivity, our approach could potentially simplify the surveillance of UC patients and improve overall resource utilization by identifying patients with HGD/CA who should proceed with colonoscopy. Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.

DNA fingerprinting based on simple sequence repeat (SSR ...

African Journals Online (AJOL)

New varieties of sugarcane are protected using morphological descriptors, which have limitations in identifying morphologically similar cultivars. Development of a reliable DNA fingerprint system for identification of new varieties would contribute greatly to the breeding of these species. Microsatellite markers are tools with ...

Molecular identification and phylogenetic analysis of important medicinal plant species in genus Paeonia based on rDNA-ITS, matK, and rbcL DNA barcode sequences.

Science.gov (United States)

Kim, W J; Ji, Y; Choi, G; Kang, Y M; Yang, S; Moon, B C

2016-08-05

This study was performed to identify and analyze the phylogenetic relationship among four herbaceous species of the genus Paeonia, P. lactiflora, P. japonica, P. veitchii, and P. suffruticosa, using DNA barcodes. These four species, which are commonly used in traditional medicine as Paeoniae Radix and Moutan Radicis Cortex, are pharmaceutically defined in different ways in the national pharmacopoeias in Korea, Japan, and China. To authenticate the different species used in these medicines, we evaluated rDNA-internal transcribed spacers (ITS), matK and rbcL regions, which provide information capable of effectively distinguishing each species from one another. Seventeen samples were collected from different geographic regions in Korea and China, and DNA barcode regions were amplified using universal primers. Comparative analyses of these DNA barcode sequences revealed species-specific nucleotide sequences capable of discriminating the four Paeonia species. Among the entire sequences of three barcodes, marker nucleotides were identified at three positions in P. lactiflora, eleven in P. japonica, five in P. veitchii, and 25 in P. suffruticosa. Phylogenetic analyses also revealed four distinct clusters showing homogeneous clades with high resolution at the species level. The results demonstrate that the analysis of these three DNA barcode sequences is a reliable method for identifying the four Paeonia species and can be used to authenticate Paeoniae Radix and Moutan Radicis Cortex at the species level. Furthermore, based on the assessment of amplicon sizes, inter/intra-specific distances, marker nucleotides, and phylogenetic analysis, rDNA-ITS was the most suitable DNA barcode for identification of these species.

A Mononucleotide Markers Panel to Identify hMLH1/hMSH2 Germline Mutations

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M. Pedroni

2007-01-01

Full Text Available Hereditary NonPolyposis Colorectal Cancer (Lynch syndrome is an autosomal dominant disease caused by germline mutations in a class of genes deputed to maintain genomic integrity during cell replication, mutations result in a generalized genomic instability, particularly evident at microsatellite loci (Microsatellite Instability, MSI. MSI is present in 85–90% of colorectal cancers that occur in Lynch Syndrome. To standardize the molecular diagnosis of MSI, a panel of 5 microsatellite markers was proposed (known as the “Bethesda panel”. Aim of our study is to evaluate if MSI testing with two mononucleotide markers, such as BAT25 and BAT26, was sufficient to identify patients with hMLH1/hMSH2 germline mutations. We tested 105 tumours for MSI using both the Bethesda markers and the two mononucleotide markers BAT25 and BAT26. Moreover, immunohistochemical evaluation of MLH1 and MSH2 proteins was executed on the tumours with at least one unstable microsatellite, whereas germline hMLH1/hMSH2 mutations were searched for all cases showing two or more unstable microsatellites.

DNA replication stress restricts ribosomal DNA copy number.

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Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

DNA replication stress restricts ribosomal DNA copy number

Science.gov (United States)

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

DNA replication stress restricts ribosomal DNA copy number.

Directory of Open Access Journals (Sweden)

Devika Salim

2017-09-01

Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Identification of mitochondrial DNA sequence variation and development of single nucleotide polymorphic markers for CMS-D8 in cotton.

Science.gov (United States)

Suzuki, Hideaki; Yu, Jiwen; Wang, Fei; Zhang, Jinfa

2013-06-01

Cytoplasmic male sterility (CMS), which is a maternally inherited trait and controlled by novel chimeric genes in the mitochondrial genome, plays a pivotal role in the production of hybrid seed. In cotton, no PCR-based marker has been developed to discriminate CMS-D8 (from Gossypium trilobum) from its normal Upland cotton (AD1, Gossypium hirsutum) cytoplasm. The objective of the current study was to develop PCR-based single nucleotide polymorphic (SNP) markers from mitochondrial genes for the CMS-D8 cytoplasm. DNA sequence variation in mitochondrial genes involved in the oxidative phosphorylation chain including ATP synthase subunit 1, 4, 6, 8 and 9, and cytochrome c oxidase 1, 2 and 3 subunits were identified by comparing CMS-D8, its isogenic maintainer and restorer lines on the same nuclear genetic background. An allelic specific PCR (AS-PCR) was utilized for SNP typing by incorporating artificial mismatched nucleotides into the third or fourth base from the 3' terminus in both the specific and nonspecific primers. The result indicated that the method modifying allele-specific primers was successful in obtaining eight SNP markers out of eight SNPs using eight primer pairs to discriminate two alleles between AD1 and CMS-D8 cytoplasms. Two of the SNPs for atp1 and cox1 could also be used in combination to discriminate between CMS-D8 and CMS-D2 cytoplasms. Additionally, a PCR-based marker from a nine nucleotide insertion-deletion (InDel) sequence (AATTGTTTT) at the 59-67 bp positions from the start codon of atp6, which is present in the CMS and restorer lines with the D8 cytoplasm but absent in the maintainer line with the AD1 cytoplasm, was also developed. A SNP marker for two nucleotide substitutions (AA in AD1 cytoplasm to CT in CMS-D8 cytoplasm) in the intron (1,506 bp) of cox2 gene was also developed. These PCR-based SNP markers should be useful in discriminating CMS-D8 and AD1 cytoplasms, or those with CMS-D2 cytoplasm as a rapid, simple, inexpensive, and

Epigenetic subgroups of esophageal and gastric adenocarcinoma with differential GATA5 DNA methylation associated with clinical and lifestyle factors.

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Xinhui Wang

Full Text Available BACKGROUND: Adenocarcinomas located near the gastroesophageal junction have unclear etiology and are difficult to classify. We used DNA methylation analysis to identify subtype-specific markers and new subgroups of gastroesophageal adenocarcinomas, and studied their association with epidemiological risk factors and clinical outcomes. METHODOLOGY/PRINCIPAL FINDINGS: We used logistic regression models and unsupervised hierarchical cluster analysis of 74 DNA methylation markers on 45 tumor samples (44 patients of esophageal and gastric adenocarcinomas obtained from a population-based case-control study to uncover epigenetic markers and cluster groups of gastroesophageal adenocarcinomas. No distinct epigenetic differences were evident between subtypes of gastric and esophageal cancers. However, we identified two gastroesophageal adenocarcinoma subclusters based on DNA methylation profiles. Group membership was best predicted by GATA5 DNA methylation status. We analyzed the associations between these two epigenetic groups and exposure using logistic regression, and the associations with survival time using Cox regression in a larger set of 317 tumor samples (278 patients. There were more males with esophageal and gastric cardia cancers in Cluster Group 1 characterized by higher GATA5 DNA methylation values (all p<0.05. This group also showed associations of borderline statistical significance with having ever smoked (p-value = 0.07, high body mass index (p-value = 0.06, and symptoms of gastroesophageal reflux (p-value = 0.07. Subjects in cluster Group 1 showed better survival than those in Group 2 after adjusting for tumor differentiation grade, but this was not found to be independent of tumor stage. CONCLUSIONS/SIGNIFICANCE: DNA methylation profiling can be used in population-based studies to identify epigenetic subclasses of gastroesophageal adenocarcinomas and class-specific DNA methylation markers that can be linked to

Reduced Representation Libraries from DNA Pools Analysed with Next Generation Semiconductor Based-Sequencing to Identify SNPs in Extreme and Divergent Pigs for Back Fat Thickness

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Samuele Bovo

2015-01-01

Full Text Available The aim of this study was to identify single nucleotide polymorphisms (SNPs that could be associated with back fat thickness (BFT in pigs. To achieve this goal, we evaluated the potential and limits of an experimental design that combined several methodologies. DNA samples from two groups of Italian Large White pigs with divergent estimating breeding value (EBV for BFT were separately pooled and sequenced, after preparation of reduced representation libraries (RRLs, on the Ion Torrent technology. Taking advantage from SNAPE for SNPs calling in sequenced DNA pools, 39,165 SNPs were identified; 1/4 of them were novel variants not reported in dbSNP. Combining sequencing data with Illumina PorcineSNP60 BeadChip genotyping results on the same animals, 661 genomic positions overlapped with a good approximation of minor allele frequency estimation. A total of 54 SNPs showing enriched alleles in one or in the other RRLs might be potential markers associated with BFT. Some of these SNPs were close to genes involved in obesity related phenotypes.

Lung cancer and risk factors: how to identify phenotypic markers?

International Nuclear Information System (INIS)

Clement-Duchene, Christelle

2009-01-01

Lung cancer is the leading cause of death in the world. Most lung cancer are diagnosed at an advanced stage (IIIB and IV), with a poor prognosis. The main risk factors are well known like active smoking, and occupational exposure (asbestos), but 10 a 20% occur in never smokers. In this population, various studies have been conducted in order to identify possible risk factors, and although many have been identified, none seem to explain more than a small percentage of the cases. According to the histological types, adenocarcinoma is now the more frequent type, and its association with the main risk factors (tobacco exposure, asbestos exposure) is still studied. The tumoral location is associated with the exposure to the risk factors. Finally, the survival seems to be different between gender, and between smokers, and never smokers. All these characteristics are perhaps associated with different pathways of carcinogenesis. In this context, we have analyzed a cohort of 1493 patients with lung cancer in order to identify phenotypic markers, and to understand the mechanisms of the lung carcinogenesis. (author) [fr

Molecular markers in bladder cancer: Novel research frontiers.

Science.gov (United States)

Sanguedolce, Francesca; Cormio, Antonella; Bufo, Pantaleo; Carrieri, Giuseppe; Cormio, Luigi

2015-01-01

Bladder cancer (BC) is a heterogeneous disease encompassing distinct biologic features that lead to extremely different clinical behaviors. In the last 20 years, great efforts have been made to predict disease outcome and response to treatment by developing risk assessment calculators based on multiple standard clinical-pathological factors, as well as by testing several molecular markers. Unfortunately, risk assessment calculators alone fail to accurately assess a single patient's prognosis and response to different treatment options. Several molecular markers easily assessable by routine immunohistochemical techniques hold promise for becoming widely available and cost-effective tools for a more reliable risk assessment, but none have yet entered routine clinical practice. Current research is therefore moving towards (i) identifying novel molecular markers; (ii) testing old and new markers in homogeneous patients' populations receiving homogeneous treatments; (iii) generating a multimarker panel that could be easily, and thus routinely, used in clinical practice; (iv) developing novel risk assessment tools, possibly combining standard clinical-pathological factors with molecular markers. This review analyses the emerging body of literature concerning novel biomarkers, ranging from genetic changes to altered expression of a huge variety of molecules, potentially involved in BC outcome and response to treatment. Findings suggest that some of these indicators, such as serum circulating tumor cells and tissue mitochondrial DNA, seem to be easily assessable and provide reliable information. Other markers, such as the phosphoinositide-3-kinase (PI3K)/AKT (serine-threonine kinase)/mTOR (mammalian target of rapamycin) pathway and epigenetic changes in DNA methylation seem to not only have prognostic/predictive value but also, most importantly, represent valuable therapeutic targets. Finally, there is increasing evidence that the development of novel risk assessment tools

A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks

Science.gov (United States)

Schär, Primo; Herrmann, Gernot; Daly, Graham; Lindahl, Tomas

1997-01-01

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis. PMID:9271115

8-oxo-7,8-dihydroguanine level - the DNA oxidative stress marker - recognized by fluorescence image analysis in sporadic uterine adenocarcinomas in women.

Science.gov (United States)

Postawski, Krzysztof; Przadka-Rabaniuk, Dorota; Piersiak, Tomasz

2013-01-01

In the case of carcinogenesis in human endometrium no information exists on tissue concentration of 8-oxo-7,8-dihydroguanine, the DNA oxidative stress marker This was the main reason to undertake the investigation of this DNA modification in human uterine estrogen-dependent tissue cancers. In order to estimate the level of oxidative damage, 8-oxo-7,8-dihydroguanine was determined directly in cells of tissue microscope slides using OxyDNA Assay Kit, Fluorometric. Cells were investigated under confocal microscope. Images of individual cells were captured by computer-interfaced digital photography and analyzed for fluorescence intensities (continuous inverted 8-bit gray-scale = 0 [black]-255 [white]). Fluorescence scores were calculated for each of 13 normal endometrial samples and 31 uterine adenocarcinoma specimens. Finally the level of the oxidative stress marker was also analyzed according to histological and clinical features of the neoplasms. The obtained data revealed that: 8-oxo-7,8-dihydroguanine levels were higher in uterine adenocarcinomas than in normal endometrial samples (48,32 vs. 38,64; p<0,001); in contrast to normal endometrium there was no correlation between age and DNA oxidative modification content in uterine cancer; highest mean fluorescence intensity was recognized in G2 endometrial adenocarcinomas; level of 8-oxo-7,8-dihydroguanine does not depend on Body Mass Index (BMI) and cancer uterine wall infiltration or tumor FIGO stage. Our study indicates that accumulation of the oxidized DNA base may contribute to the development of endometrial neoplasia, however oxidative DNA damage does not seem to increase with tumor progression.

Identification of fungal DNA barcode targets and PCR primers based on Pfam protein families and taxonomic hierarchy

NARCIS (Netherlands)

Lewis, C.T.; Bilkhu, S.; Robert, V.; Eberhardt, U.; Szoke, S.; Seifert, K.A.; Lévesque, C.A.

2011-01-01

Abstract: DNA barcoding is the application of DNA sequences of standardized genetic markers for the identification of eukaryotic organisms. We attempted to identify alternative candidate barcode gene targets for the fungal biota from available fungal genomes using a taxonomy-aware processing

Generation and application of SSR markers in avocado

International Nuclear Information System (INIS)

Sharon, D.; Lavi, U.; Cregan, P.B.; Hillel, J.

1998-01-01

Simple Sequence Repeat (SSR) DNA markers were generated and applied to avocado. An SSR marker is based on a pair of primers which are synthesized on the basis of DNA sequences flanking a micro satellite. These markers are PCR based, quite polymorphic and abundant in several species. These are the markers, of choice in the human genome. The number of SSR markers in the avocado genome was calculated to be about 45,000, with the A/T micro satellite being the most frequent (1 in 40 kb). SSR markers are quite expensive to generate due to the required multi-step procedure; Screening a genomic library, about 66% of the positive clones turned out after sequencing to be SSR containing clones. In only about 55% of these, was it possible to synthesize primers and, of this group, only about 50% of the markers were useful for typing a specific family. Typing of five avocado cultivars using 59 SSR markers results in one to eight alleles per locus, mean heterozygosity ranging between 0.51 and 0.66 and gene diversity ranging between 0.42 and 0.66. The SSR markers were used to estimate the genetic relationships between various Persea species. The number of alleles in these species ranged between five and twelve with heterozygosity levels between 0.11-0.78 and gene diversity between 0.69-0.89. A preliminary genetic map, based on these SSR markers together with some DNA fingerprints (DFP) and randomly amplified polymorphic DNA (RAPD) markers, was drawn. The map consists of 12 linkage group having two to five markers each. Linkage analysis with several quantitative trait loci (QTLs) was performed by genetic typing and phenotypic assessment of the progeny of a controlled cross. The results of the interval mapping suggest that the gene(s) coding for the existence of fibers in the flesh, are probably linked to linkage group 3. (author)

Generation and application of SSR markers in avocado

Energy Technology Data Exchange (ETDEWEB)

Sharon, D; Lavi, U [Institute of Horticulture, ARO Volcani Center, Bet-Dagan (Israel); Cregan, P B [United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland (United States); Hillel, J [Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot (Israel)

1998-10-01

Simple Sequence Repeat (SSR) DNA markers were generated and applied to avocado. An SSR marker is based on a pair of primers which are synthesized on the basis of DNA sequences flanking a micro satellite. These markers are PCR based, quite polymorphic and abundant in several species. These are the markers, of choice in the human genome. The number of SSR markers in the avocado genome was calculated to be about 45,000, with the A/T micro satellite being the most frequent (1 in 40 kb). SSR markers are quite expensive to generate due to the required multi-step procedure; Screening a genomic library, about 66% of the positive clones turned out after sequencing to be SSR containing clones. In only about 55% of these, was it possible to synthesize primers and, of this group, only about 50% of the markers were useful for typing a specific family. Typing of five avocado cultivars using 59 SSR markers results in one to eight alleles per locus, mean heterozygosity ranging between 0.51 and 0.66 and gene diversity ranging between 0.42 and 0.66. The SSR markers were used to estimate the genetic relationships between various Persea species. The number of alleles in these species ranged between five and twelve with heterozygosity levels between 0.11-0.78 and gene diversity between 0.69-0.89. A preliminary genetic map, based on these SSR markers together with some DNA fingerprints (DFP) and randomly amplified polymorphic DNA (RAPD) markers, was drawn. The map consists of 12 linkage group having two to five markers each. Linkage analysis with several quantitative trait loci (QTLs) was performed by genetic typing and phenotypic assessment of the progeny of a controlled cross. The results of the interval mapping suggest that the gene(s) coding for the existence of fibers in the flesh, are probably linked to linkage group 3. (author) 20 refs, 3 figs, 8 tabs

Comparative analysis of chromosomal localization of ribosomal and telomeric DNA markers in three species of Pyrgomorphidae grasshoppers

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Olesya G. Buleu

2017-09-01

Full Text Available The karyotypes of three species of Pyrgomorphidae grasshoppers were studied: Zonocerus elegans (Thunberg, 1815, Pyrgomorpha guentheri (Burr, 1899 and Atractomorpha lata (Mochulsky, 1866. Data on karyotypes of P. guentheri and Z. elegans are reported here for the first time. All species have karyotypes consisting of 19 acrocentric chromosomes in males and 20 acrocentric chromosomes in females (2n♂=19, NF=19; 2n♀=20, NF=20 and X0/XX sex determination system. A comparative analysis of the localization of C-heterochromatin, clusters of ribosomal DNA, and telomere repeats revealed inter-species diversity in these cytogenetic markers. These differences indicate that the karyotype divergence in the species studied is not associated with structural chromosome rearrangements, but with the evolution of repeated DNA sequences.

MtDNA COI-COII marker and drone congregation area: an efficient method to establish and monitor honeybee (Apis mellifera L.) conservation centres.

Science.gov (United States)

Bertrand, Bénédicte; Alburaki, Mohamed; Legout, Hélène; Moulin, Sibyle; Mougel, Florence; Garnery, Lionel

2015-05-01

Honeybee subspecies have been affected by human activities in Europe over the past few decades. One such example is the importation of nonlocal subspecies of bees which has had an adverse impact on the geographical repartition and subsequently on the genetic diversity of the black honeybee Apis mellifera mellifera. To restore the original diversity of this local honeybee subspecies, different conservation centres were set up in Europe. In this study, we established a black honeybee conservation centre Conservatoire de l'Abeille Noire d'Ile de France (CANIF) in the region of Ile-de-France, France. CANIF's honeybee colonies were intensively studied over a 3-year period. This study included a drone congregation area (DCA) located in the conservation centre. MtDNA COI-COII marker was used to evaluate the genetic diversity of CANIF's honeybee populations and the drones found and collected from the DCA. The same marker (mtDNA) was used to estimate the interactions and the haplotype frequency between CANIF's honeybee populations and 10 surrounding honeybee apiaries located outside of the CANIF. Our results indicate that the colonies of the conservation centre and the drones of the DCA show similar stable profiles compared to the surrounding populations with lower level of introgression. The mtDNA marker used on both DCA and colonies of the conservation centre seems to be an efficient approach to monitor and maintain the genetic diversity of the protected honeybee populations. © 2014 John Wiley & Sons Ltd.

Y-chromosomal DNA markers for discrimination of chemical substance and effluent effects on sexual differentiation in salmon.

OpenAIRE

Afonso, Luis O B; Smith, Jack L; Ikonomou, Michael G; Devlin, Robert H

2002-01-01

Chinook salmon alevins were exposed during their labile period for sex differentiation to different concentrations of bleached kraft mill effluent (BKME), primary sewage effluent, secondary sewage effluent (SE), 17ss-estradiol, testosterone, and nonylphenol. After exposure for 29 days post hatching (DPH), fish were allowed to grow until 103 and 179 DPH, at which time their genetic sex was determined using Y-chromosomal DNA markers and their gonadal sex was determined by histology. Independent...

DNA barcode goes two-dimensions: DNA QR code web server.

Science.gov (United States)

Liu, Chang; Shi, Linchun; Xu, Xiaolan; Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

DNA barcode goes two-dimensions: DNA QR code web server.

Directory of Open Access Journals (Sweden)

Chang Liu

Full Text Available The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

DNA in the conservation and management of African antelope

DEFF Research Database (Denmark)

Lorenzen, Eline

2016-01-01

tool in informed species conservation and sustainable wildlife management. The movement of antelope through translocations, reintroductions, and population augmentations is common practice in wildlife management. DNA-led species identification using genetic barcoding is an effective use of genetic data...... within forensics. DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species....... databases, and represents a valuable reference database of antelope DNA diversity. For the evolution of antelope, sub-Saharan Africa is a region of particular intrigue. The geographic regions of sub-Saharan Africa represent unique evolutionary scenarios. Molecular data have become an increasingly important...

Modeling the Sensitivity of Field Surveys for Detection of Environmental DNA (eDNA.

Directory of Open Access Journals (Sweden)

Martin T Schultz

Full Text Available The environmental DNA (eDNA method is the practice of collecting environmental samples and analyzing them for the presence of a genetic marker specific to a target species. Little is known about the sensitivity of the eDNA method. Sensitivity is the probability that the target marker will be detected if it is present in the water body. Methods and tools are needed to assess the sensitivity of sampling protocols, design eDNA surveys, and interpret survey results. In this study, the sensitivity of the eDNA method is modeled as a function of ambient target marker concentration. The model accounts for five steps of sample collection and analysis, including: 1 collection of a filtered water sample from the source; 2 extraction of DNA from the filter and isolation in a purified elution; 3 removal of aliquots from the elution for use in the polymerase chain reaction (PCR assay; 4 PCR; and 5 genetic sequencing. The model is applicable to any target species. For demonstration purposes, the model is parameterized for bighead carp (Hypophthalmichthys nobilis and silver carp (H. molitrix assuming sampling protocols used in the Chicago Area Waterway System (CAWS. Simulation results show that eDNA surveys have a high false negative rate at low concentrations of the genetic marker. This is attributed to processing of water samples and division of the extraction elution in preparation for the PCR assay. Increases in field survey sensitivity can be achieved by increasing sample volume, sample number, and PCR replicates. Increasing sample volume yields the greatest increase in sensitivity. It is recommended that investigators estimate and communicate the sensitivity of eDNA surveys to help facilitate interpretation of eDNA survey results. In the absence of such information, it is difficult to evaluate the results of surveys in which no water samples test positive for the target marker. It is also recommended that invasive species managers articulate concentration

Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.

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Zhao Yongli

2012-12-01

Full Text Available Abstract Background Date palm (Phoenix dactylifera L. is an important tree in the Middle East and North Africa due to the nutritional value of its fruit. Molecular Breeding would accelerate genetic improvement of fruit tree through marker assisted selection. However, the lack of molecular markers in date palm restricts the application of molecular breeding. Results In this study, we analyzed 28,889 EST sequences from the date palm genome database to identify simple-sequence repeats (SSRs and to develop gene-based markers, i.e. expressed sequence tag-SSRs (EST-SSRs. We identified 4,609 ESTs as containing SSRs, among which, trinucleotide motifs (69.7% were the most common, followed by tetranucleotide (10.4% and dinucleotide motifs (9.6%. The motif AG (85.7% was most abundant in dinucleotides, while motifs AGG (26.8%, AAG (19.3%, and AGC (16.1% were most common among trinucleotides. A total of 4,967 primer pairs were designed for EST-SSR markers from the computational data. In a follow up laboratory study, we tested a sample of 20 random selected primer pairs for amplification and polymorphism detection using genomic DNA from date palm cultivars. Nearly one-third of these primer pairs detected DNA polymorphism to differentiate the twelve date palm cultivars used. Functional categorization of EST sequences containing SSRs revealed that 3,108 (67.4% of such ESTs had homology with known proteins. Conclusion Date palm EST sequences exhibits a good resource for developing gene-based markers. These genic markers identified in our study may provide a valuable genetic and genomic tool for further genetic research and varietal development in date palm, such as diversity study, QTL mapping, and molecular breeding.

Transcription analysis of apple fruit development using cDNA microarrays

NARCIS (Netherlands)

Soglio, V.; Costa, F.; Molthoff, J.W.; Weemen-Hendriks, M.; Schouten, H.J.; Gianfranceschi, L.

2009-01-01

The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and

DNA fingerprinting sets for four southern pines

Science.gov (United States)

Craig Echt; Sedley Josserand

2018-01-01

DNA markers can provide valuable genetic information for forest tree research, breeding, conservation, and restoration programs. When properly evaluated, selected sets of DNA markers can be used to efficiently get information about genetic diversity in regions, forests, or stands, or in seed lots and orchards. Selected markers also can be used to determine parentage or...

DNA barcoding of invasive plants in China: A resource for identifying invasive plants.

Science.gov (United States)

Xu, Song-Zhi; Li, Zhen-Yu; Jin, Xiao-Hua

2018-01-01

Invasive plants have aroused attention globally for causing ecological damage and having a negative impact on the economy and human health. However, it can be extremely challenging to rapidly and accurately identify invasive plants based on morphology because they are an assemblage of many different families and many plant materials lack sufficient diagnostic characteristics during border inspections. It is therefore urgent to evaluate candidate loci and build a reliable genetic library to prevent invasive plants from entering China. In this study, five common single markers (ITS, ITS2, matK, rbcL and trnH-psbA) were evaluated using 634 species (including 469 invasive plant species in China, 10 new records to China, 16 potentially invasive plant species around the world but not introduced into China yet and 139 plant species native to China) based on three different methods. Our results indicated that ITS2 displayed largest intra- and interspecific divergence (1.72% and 91.46%). Based on NJ tree method, ITS2, ITS, matK, rbcL and trnH-psbA provided 76.84%, 76.5%, 63.21%, 52.86% and 50.68% discrimination rates, respectively. The combination of ITS + matK performed best and provided 91.03% discriminatory power, followed by ITS2 + matK (85.78%). For identifying unknown individuals, ITS + matK had 100% correct identification rate based on our database, followed by ITS/ITS2 (both 93.33%) and ITS2 + matK (91.67%). Thus, we propose ITS/ITS2 + matK as the most suitable barcode for invasive plants in China. This study also demonstrated that DNA barcoding is an efficient tool for identifying invasive species. © 2017 John Wiley & Sons Ltd.

DNA fingerprinting of Chinese melon provides evidentiary support of seed quality appraisal.

Science.gov (United States)

Gao, Peng; Ma, Hongyan; Luan, Feishi; Song, Haibin

2012-01-01

Melon, Cucumis melo L. is an important vegetable crop worldwide. At present, there are phenomena of homonyms and synonyms present in the melon seed markets of China, which could cause variety authenticity issues influencing the process of melon breeding, production, marketing and other aspects. Molecular markers, especially microsatellites or simple sequence repeats (SSRs) are playing increasingly important roles for cultivar identification. The aim of this study was to construct a DNA fingerprinting database of major melon cultivars, which could provide a possibility for the establishment of a technical standard system for purity and authenticity identification of melon seeds. In this study, to develop the core set SSR markers, 470 polymorphic SSRs were selected as the candidate markers from 1219 SSRs using 20 representative melon varieties (lines). Eighteen SSR markers, evenly distributed across the genome and with the highest contents of polymorphism information (PIC) were identified as the core marker set for melon DNA fingerprinting analysis. Fingerprint codes for 471 melon varieties (lines) were established. There were 51 materials which were classified into17 groups based on sharing the same fingerprint code, while field traits survey results showed that these plants in the same group were synonyms because of the same or similar field characters. Furthermore, DNA fingerprinting quick response (QR) codes of 471 melon varieties (lines) were constructed. Due to its fast readability and large storage capacity, QR coding melon DNA fingerprinting is in favor of read convenience and commercial applications.

DNA Fingerprinting of Chinese Melon Provides Evidentiary Support of Seed Quality Appraisal

Science.gov (United States)

Gao, Peng; Ma, Hongyan; Luan, Feishi; Song, Haibin

2012-01-01

Melon, Cucumis melo L. is an important vegetable crop worldwide. At present, there are phenomena of homonyms and synonyms present in the melon seed markets of China, which could cause variety authenticity issues influencing the process of melon breeding, production, marketing and other aspects. Molecular markers, especially microsatellites or simple sequence repeats (SSRs) are playing increasingly important roles for cultivar identification. The aim of this study was to construct a DNA fingerprinting database of major melon cultivars, which could provide a possibility for the establishment of a technical standard system for purity and authenticity identification of melon seeds. In this study, to develop the core set SSR markers, 470 polymorphic SSRs were selected as the candidate markers from 1219 SSRs using 20 representative melon varieties (lines). Eighteen SSR markers, evenly distributed across the genome and with the highest contents of polymorphism information (PIC) were identified as the core marker set for melon DNA fingerprinting analysis. Fingerprint codes for 471 melon varieties (lines) were established. There were 51 materials which were classified into17 groups based on sharing the same fingerprint code, while field traits survey results showed that these plants in the same group were synonyms because of the same or similar field characters. Furthermore, DNA fingerprinting quick response (QR) codes of 471 melon varieties (lines) were constructed. Due to its fast readability and large storage capacity, QR coding melon DNA fingerprinting is in favor of read convenience and commercial applications. PMID:23285039

DNA fingerprinting of Chinese melon provides evidentiary support of seed quality appraisal.

Directory of Open Access Journals (Sweden)

Peng Gao

Full Text Available Melon, Cucumis melo L. is an important vegetable crop worldwide. At present, there are phenomena of homonyms and synonyms present in the melon seed markets of China, which could cause variety authenticity issues influencing the process of melon breeding, production, marketing and other aspects. Molecular markers, especially microsatellites or simple sequence repeats (SSRs are playing increasingly important roles for cultivar identification. The aim of this study was to construct a DNA fingerprinting database of major melon cultivars, which could provide a possibility for the establishment of a technical standard system for purity and authenticity identification of melon seeds. In this study, to develop the core set SSR markers, 470 polymorphic SSRs were selected as the candidate markers from 1219 SSRs using 20 representative melon varieties (lines. Eighteen SSR markers, evenly distributed across the genome and with the highest contents of polymorphism information (PIC were identified as the core marker set for melon DNA fingerprinting analysis. Fingerprint codes for 471 melon varieties (lines were established. There were 51 materials which were classified into17 groups based on sharing the same fingerprint code, while field traits survey results showed that these plants in the same group were synonyms because of the same or similar field characters. Furthermore, DNA fingerprinting quick response (QR codes of 471 melon varieties (lines were constructed. Due to its fast readability and large storage capacity, QR coding melon DNA fingerprinting is in favor of read convenience and commercial applications.

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.

Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas

International Nuclear Information System (INIS)

Sriram, Krishna B; Courtney, Deborah; Yang, Ian A; Bowman, Rayleen V; Fong, Kwun M; Relan, Vandana; Clarke, Belinda E; Duhig, Edwina E; Windsor, Morgan N; Matar, Kevin S; Naidoo, Rishendran; Passmore, Linda; McCaul, Elizabeth

2012-01-01

The diagnosis of malignant pleural effusions (MPE) is often clinically challenging, especially if the cytology is negative for malignancy. DNA integrity index has been reported to be a marker of malignancy. The aim of this study was to evaluate the utility of pleural fluid DNA integrity index in the diagnosis of MPE. We studied 75 pleural fluid and matched serum samples from consecutive subjects. Pleural fluid and serum ALU DNA repeats [115bp, 247bp and 247bp/115bp ratio (DNA integrity index)] were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were quantified using ELISA. Based on clinico-pathological evaluation, 52 subjects had MPE (including 16 mesotheliomas) and 23 had benign effusions. Pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs. 0.8; p<0.001). Cytology had a sensitivity of 55% in diagnosing MPE. If cytology and pleural fluid DNA integrity index were considered together, they exhibited 81% sensitivity and 87% specificity in distinguishing benign and malignant effusions. In cytology-negative pleural effusions (35 MPE and 28 benign effusions), elevated pleural fluid DNA integrity index had an 81% positive predictive value in detecting MPEs. In the detection of mesothelioma, at a specificity of 90%, pleural fluid DNA integrity index had similar sensitivity to pleural fluid and serum mesothelin (75% each respectively). Pleural fluid DNA integrity index is a promising diagnostic biomarker for identification of MPEs, including mesothelioma. This biomarker may be particularly useful in cases of MPE where pleural aspirate cytology is negative, and could guide the decision to undertake more invasive definitive testing. A prospective validation study is being undertaken to validate our findings and test the clinical utility of this biomarker for altering clinical practice

Development and mapping of SSR markers linked to resistance-gene homologue clusters in common bean

Institute of Scientific and Technical Information of China (English)

Luz; Nayibe; Garzon; Matthew; Wohlgemuth; Blair

2014-01-01

Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.

Development and assessment of microarray-based DNA fingerprinting in Eucalyptus grandis.

Science.gov (United States)

Lezar, Sabine; Myburg, A A; Berger, D K; Wingfield, M J; Wingfield, B D

2004-11-01

Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R2>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.

Evaluating the Feasibility of Five Candidate DNA Barcoding Loci for Philippine Lasianthus Jack (Lasiantheae: Rubiaceae).

Science.gov (United States)

Arshed, Muhammad Jefte C; Valdez, Marcos B; Alejandro, Grecebio Jonathan D

2017-01-01

The pantropical genus Lasianthus Jack is identified for high phenotypic plasticity making traditional taxonomic identification difficult. Having some members with important medicinal properties, a precise complimentary identification through DNA barcoding is needed for species delineation. In this study, 12 samples representing six Philippine Lasianthus species were used to determine the most efficient barcoding loci among the cpDNA markers ( mat K, rbc L, rps 16, and trn T-F) and nrDNA (ITS) based on the criteria of universality, discriminatory power, and resolution of species. The results revealed that ITS has the recommended primer universality, greatest interspecific divergences, and average resolution of species. Among the cpDNA markers, mat K and rbc L are recommended but with minimal resolution of species. While trn T-F showed moderate interspecific variations and resolution of Lasianthus species, rps 16 has the lowest interspecific divergence and resolution of species. Consequently, ITS is the potential ideal DNA barcode for Lasianthus species. ITS, mat K, and rps 16 markers have the excellent amplification and sequence qualityITS marker has the highest interspecific divergence with the maximum values, followed by mat K, rbc L, trn T-F, and rps 16, respectivelyAll markers except rps 16 yielded average resolution to Lasianthus speciesITS marker is the most ideal locus in terms of excellent universality, high interspecific discriminatory ability, and average species resolution. Abbreviations used: ITS: Internal Transcribe Spacer, mat K: maturase K, rbc L: ribulose-1,5-biphospahte-carboxylase, rps 16: ribosomal protein 16 small subunit gene.

Status and prospects of DNA barcoding in medically important parasites and vectors.

Science.gov (United States)

Ondrejicka, Danielle A; Locke, Sean A; Morey, Kevin; Borisenko, Alex V; Hanner, Robert H

2014-12-01

For over 10 years, DNA barcoding has been used to identify specimens and discern species. Its potential benefits in parasitology were recognized early, but its utility and uptake remain unclear. Here we review studies using DNA barcoding in parasites and vectors affecting humans and find that the technique is accurate (accords with author identifications based on morphology or other markers) in 94-95% of cases, although aspects of DNA barcoding (vouchering, marker implicated) have often been misunderstood. In a newly compiled checklist of parasites, vectors, and hazards, barcodes are available for 43% of all 1403 species and for more than half of 429 species of greater medical importance. This is encouraging coverage that would improve with an active campaign targeting parasites and vectors. Copyright © 2014 Elsevier Ltd. All rights reserved.

A 2-megabase physical contig incorporating 43 DNA markers on the human X chromosome at p11.23-p11.22 from ZNF21 to DXS255

Energy Technology Data Exchange (ETDEWEB)

Boycott, K.M.; Bech-Hansen, N.T. [Univ. of Calgary, Alberta (Canada); Halley, G.R.; Schlessinger, D. [Washington Univ. School of Medicine, St. Louis, MO (United States)

1996-05-01

A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23-p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internal Alu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known gene and expressed sequence tags in this region is predicted to be Xpter-ZNF21-DXS7465E (MG66)-DXS7927E (MG81)-WASP, DXS1011E, DXS7467E (MG21)-DXS-7466E (MG44)-GATA1-DXS7469E (Xp664)-TFE3-SYP (DXS1007E)-Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates. 47 refs., 1 fig., 4 tabs.

Aberrantly methylated DNA as a biomarker in breast cancer.

Science.gov (United States)

Kristiansen, Søren; Jørgensen, Lars M; Guldberg, Per; Sölétormos, György

2013-01-01

Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.

Identification of hybrids of painted and milky storks using FTA card-collected blood, molecular markers, and morphologies.

Science.gov (United States)

Yee, Elsie Yoke Sim; Zainuddin, Zainal Zahari; Ismail, Ahmad; Yap, Chee Kong; Tan, Soon Guan

2013-10-01

Suspicious hybrids of painted storks and milky storks were found in a Malaysian zoo. Blood of these birds was sampled on FTA cards for DNA fingerprinting. Of 44 optimized primers, 6 produced diagnostic markers that could identify hybrids. The markers were based on simple, direct PCR-generated multilocus banding patterns that provided two sets of genetic data, one for each of the two stork species and another for the hybrids. It also revealed that large DNA fragments (3,000 bp) could be amplified from blood collected on FTA cards. When the results of each individual bird's DNA fingerprint were compared with plumage characters, the hybrids were found to express a range of intermediate phenotypic traits of the pure breeds with no dominant plumage characteristic from either parental species.

Covariance Association Test (CVAT) Identifies Genetic Markers Associated with Schizophrenia in Functionally Associated Biological Processes.

Science.gov (United States)

Rohde, Palle Duun; Demontis, Ditte; Cuyabano, Beatriz Castro Dias; Børglum, Anders D; Sørensen, Peter

2016-08-01

Schizophrenia is a psychiatric disorder with large personal and social costs, and understanding the genetic etiology is important. Such knowledge can be obtained by testing the association between a disease phenotype and individual genetic markers; however, such single-marker methods have limited power to detect genetic markers with small effects. Instead, aggregating genetic markers based on biological information might increase the power to identify sets of genetic markers of etiological significance. Several set test methods have been proposed: Here we propose a new set test derived from genomic best linear unbiased prediction (GBLUP), the covariance association test (CVAT). We compared the performance of CVAT to other commonly used set tests. The comparison was conducted using a simulated study population having the same genetic parameters as for schizophrenia. We found that CVAT was among the top performers. When extending CVAT to utilize a mixture of SNP effects, we found an increase in power to detect the causal sets. Applying the methods to a Danish schizophrenia case-control data set, we found genomic evidence for association of schizophrenia with vitamin A metabolism and immunological responses, which previously have been implicated with schizophrenia based on experimental and observational studies. Copyright © 2016 by the Genetics Society of America.

Development of New Microsatellite DNA Markers from Apostichopus japonicus and Their Cross-Species Application in Parastichopus parvimensis and Pathallus mollis

Directory of Open Access Journals (Sweden)

Guiping Chen

2011-09-01

Full Text Available Twenty microsatellite DNA markers were developed for sea cucumber and used to investigate polymorphisms of 60 wild Apostichopus japonicus individuals collected from China. It revealed that all the markers were polymorphic. A total of 164 alleles were detected at 20 loci. The number of alleles per locus varied from 3 to 17 with an average of 8.2, and the expected heterozygosities of each locus ranged from 0.03 to 0.89 with an average of 0.64. Cross-species amplification was also conducted in Parastichopus parvimensis collected from the United States and Pathallus mollis collected from Peru. The result showed that 17 loci amplified Parastichopus parvimensis DNAs while only 4 loci could amplify Pathallus mollis DNAs. All of the polymorphic markers would be useful for future genetic breeding and the assessment of genetic variation within sea cucumbers.

An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers.

Directory of Open Access Journals (Sweden)

Ajay Goel

2010-02-01

Full Text Available Microsatellite instability (MSI is used to screen colorectal cancers (CRC for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR to detect MSI.We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using > or = 2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1-98.1% and a positive predictive value of 100% (95% CI = 96.6%-100%. Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27 was comparable in sensitivity (97.4% and positive predictive value (96.5% to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.An optimized pentaplex (or triplex PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.

Mitochondrial DNA markers of loggerhead marine turtles (Caretta caretta (Testudines: Cheloniidae nesting at Kyparissia Bay, Greece, confirm the western Greece unit and regional structuring

Directory of Open Access Journals (Sweden)

Carlos Carreras

2014-03-01

Full Text Available Genetic markers have been widely used in marine turtles to assess population structuring and origin of individuals in common feeding grounds, which are key elements for understanding their ecology and for developing conservation strategies. However, these analyses are very sensitive to missing information, especially from abundant nesting sites. Kyparissia Bay (western Greece hosts the second largest Mediterranean nesting aggregation of the loggerhead turtle (Caretta caretta, but the genetic profile of this nesting site has not, as yet, been described using the extended version of the historically used mitochondrial DNA (mtDNA marker. This marker was genotyped for 36 individuals nesting at Kyparissia Bay and haplotype frequencies obtained were compared with published data from other Mediterranean nesting sites. The results confirmed the connection between Kyparissia and other western Greek nesting sites and the isolation of this western Greek group from other Mediterranean nesting areas. As a consequence of this isolation, this abundant group of nesting aggregations (almost 30% of the Mediterranean stock is not likely to significantly contribute to the recovery of other declining Mediterranean units.

DNA tagging of blast resistant gene(s in three Brazilian rice cultivars

Directory of Open Access Journals (Sweden)

S.S. Sandhu

2003-12-01

Full Text Available Rice blast is the most important fungal disease of rice and is caused by Pyricularia oryzae Sacc. (Telomorph Magnoporthe grisea Barr.. Seven randomly amplified polymorphic DNA (RAPD markers OPA5, OPG17, OPG18, OPG19, OPF9, OPF17 and OPF19 showed very clear polymorphism in resistant cultivar lines which differed from susceptible lines. By comparing different susceptible lines, nine DNA amplifications of seven primers (OPA5(1000, OPA5(1200, OPG17(700, OPG18(850, OPG19(500, OPG19(600, OPF9(600, OPF17(1200 and OPF19(600 were identified as dominant markers for the blast resistant gene in resistant cultivar lines. These loci facilitate the indirect scoring of blast resistant and blast susceptible genotypes. The codomine RAPDs markers will facilitate marker-assisted selection of the blast resistant gene in two blast resistant genotypes of rice (Labelle and Line 11 and will be useful in rice breeding programs.

Early markers of autism spectrum disorders in infants and toddlers prospectively identified in the Social Attention and Communication Study.

Science.gov (United States)

Barbaro, Josephine; Dissanayake, Cheryl

2013-01-01

The Social Attention and Communication Study involved the successful implementation of developmental surveillance of the early markers of autism spectrum disorders in a community-based setting. The objective in the current study was to determine the most discriminating and predictive markers of autism spectrum disorders used in the Social Attention and Communication Study at 12, 18 and 24 months of age, so that these could be used to identify children with autism spectrum disorders with greater accuracy. The percentage of 'yes/no' responses for each behavioural marker was compared between children with autistic disorder (n = 39), autism spectrum disorder (n = 50) and developmental and/or language delay (n = 20) from 12 to 24 months, with a logistic regression also conducted at 24 months. Across all ages, the recurring key markers of both autistic disorder and autism spectrum disorder were deficits in eye contact and pointing, and from 18 months, deficits in showing became an important marker. In combination, these behaviours, along with pretend play, were found to be the best group of predictors for a best estimate diagnostic classification of autistic disorder/autism spectrum disorder at 24 months. It is argued that the identified markers should be monitored repeatedly during the second year of life by community health-care professionals.

Prospects of molecular markers in Fusarium species diversity

DEFF Research Database (Denmark)

Nayaka, S. Chandra; Wulff, Ednar Gadelha; Udayashankar, A.C.

2011-01-01

focuses of various molecular-based techniques employed to study the diversity of Fusarium species causing diseases in major food crops. An introduction of fusarial diseases and their mycotoxins and molecular-marker-based methods for detection introduce the concept of marker application. Various well...... for generation of probes and their use in phylogeny of Fusarium spp. are also presented. The concluding part emphasizes the value of molecular markers for assessing genetic variability and reveals that molecular tools are indispensable for providing information not only of one Fusarium species but on whole......-known molecular techniques such as random amplified polymorphic DNA, amplification fragment length polymorphism, etc. to more modern ones such as DNA microarrays, DNA barcoding, and pyrosequencing and their application form the core of the review. Target regions in the genome which can be potential candidates...

Identification of Fabaceae plants using the DNA barcode matK.

Science.gov (United States)

Gao, Ting; Sun, Zhiying; Yao, Hui; Song, Jingyuan; Zhu, Yingjie; Ma, Xinye; Chen, Shilin

2011-01-01

In this study, we tested the applicability of the core DNA barcode MATK for identifying species within the Fabaceae family. Based on an evaluation of genetic variation, DNA barcoding gaps, and species discrimination power, MATK is a useful barcode for Fabaceae species. Of 1355 plant samples collected from 1079 species belonging to 409 diverse genera, MATK precisely identified approximately 80 % and 96 % of them at the species and genus levels, respectively. Therefore, our research indicates that the MATK region is a valuable marker for plant species within Fabaceae. © Georg Thieme Verlag KG Stuttgart · New York.

Characterization of Fasciola spp. in Myanmar on the basis of spermatogenesis status and nuclear and mitochondrial DNA markers.

Science.gov (United States)

Ichikawa, Madoka; Bawn, Saw; Maw, Ni Ni; Htun, Lat Lat; Thein, Myint; Gyi, Aung; Sunn, Kyaw; Katakura, Ken; Itagaki, Tadashi

2011-12-01

Fasciola spp. in Myanmar were characterized on the basis of spermatogenesis status and DNA markers of nuclear internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1). We collected 88 adult flukes from Yangon, Lashio, and Myitkyina. Spermatogenesis status was analyzed by the presence of sperm in the seminal vesicles, and 8 aspermic and 80 spermic flukes were detected. The flukes were identified on the basis of spermatogenesis status and ITS1 types which were analyzed by a PCR-RFLP method, and 80 spermic flukes were identified as F. gigantica. A very low detection rate of aspermic Fasciola sp. indicated that they are not established in Myanmar. In phylogenetic analyses, the 7 aspermic Fasciola sp. from Myitkyina displayed a haplotype in nad1 sequence, which was identical to that of aspermic Fasciola sp. from other Asian countries including China. Therefore, they were probably introduced from China through an infected domestic ruminant. On the other hand, 17 nad1 haplotypes detected in F. gigantica belonged to 2 clades unique to Myanmar, each with a distinct founder haplotype in a network analysis. This indicated a unique history of F. gigantica introduction into Myanmar involving ancient artificial movements of domestic ruminants. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Development of Genetic Markers in Eucalyptus Species by Target Enrichment and Exome Sequencing

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Dasgupta, Modhumita Ghosh; Dharanishanthi, Veeramuthu; Agarwal, Ishangi; Krutovsky, Konstantin V.

2015-01-01

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family– based QTL and association analysis in Eucalyptus. PMID:25602379

Development of genetic markers in Eucalyptus species by target enrichment and exome sequencing.

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Modhumita Ghosh Dasgupta

Full Text Available The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs and insertions/ deletions (InDels were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family- based QTL and association analysis in Eucalyptus.

Meta-analysis of the predictive value of DNA aneuploidy in malignant transformation of oral potentially malignant disorders.

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Alaizari, Nader A; Sperandio, Marcelo; Odell, Edward W; Peruzzo, Daiane; Al-Maweri, Sadeq A

2018-02-01

DNA aneuploidy is an imbalance of chromosomal DNA content that has been highlighted as a predictor of biological behavior and risk of malignant transformation. To date, DNA aneuploidy in oral potentially malignant diseases (OPMD) has been shown to correlate strongly with severe dysplasia and high-risk lesions that appeared non-dysplastic can be identified by ploidy analysis. Nevertheless, the prognostic value of DNA aneuploidy in predicting malignant transformation of OPMD remains to be validated. The aim of this meta-analysis was to assess the role of DNA aneuploidy in predicting malignant transformation in OPMD. The questions addressed were (i) Is DNA aneuploidy a useful marker to predict malignant transformation in OPMD? (ii) Is DNA diploidy a useful negative marker of malignant transformation in OPMD? These questions were addressed using the PECO method. Five studies assessing aneuploidy as a risk marker of malignant change were pooled into the meta-analysis. Aneuploidy was found to be associated with a 3.12-fold increased risk to progress into cancer (RR=3.12, 95% CI 1.86-5.24). Based on the five studies meta-analyzed, "no malignant progression" was more likely to occur in DNA diploid OPMD by 82% when compared to aneuploidy (RR=0.18, 95% CI 0.08-0.41). In conclusion, aneuploidy is a useful marker of malignant transformation in OPMD, although a diploid result should be interpreted with caution. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Identifying the Genotypes of Hepatitis B Virus (HBV) with DNA Origami Label.

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Liu, Ke; Pan, Dun; Wen, Yanqin; Zhang, Honglu; Chao, Jie; Wang, Lihua; Song, Shiping; Fan, Chunhai; Shi, Yongyong

2018-02-01

The hepatitis B virus (HBV) genotyping may profoundly affect the accurate diagnosis and antiviral treatment of viral hepatitis. Existing genotyping methods such as serological, immunological, or molecular testing are still suffered from substandard specificity and low sensitivity in laboratory or clinical application. In a previous study, a set of high-efficiency hybridizable DNA origami-based shape ID probes to target the templates through which genetic variation could be determined in an ultrahigh resolution of atomic force microscopy (AFM) nanomechanical imaging are established. Here, as a further confirmatory research to explore the sensitivity and applicability of this assay, differentially predesigned DNA origami shape ID probes are also developed for precisely HBV genotyping. Through the specific identification of visualized DNA origami nanostructure with clinical HBV DNA samples, the genetic variation information of genotypes can be directly identified under AFM. As a proof-of-concept, five genotype B and six genotype C are detected in 11 HBV-infected patients' blood DNA samples of Han Chinese population in the single-blinded test. The AFM image-based DNA origami shape ID genotyping approach shows high specificity and sensitivity, which could be promising for virus infection diagnosis and precision medicine in the future. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar ?Koshihikari?

OpenAIRE

Yonemaru, Jun-ichi; Choi, Sun Hee; Sakai, Hiroaki; Ando, Tsuyu; Shomura, Ayahiko; Yano, Masahiro; Wu, Jianzhong; Fukuoka, Shuichi

2015-01-01

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultiva...

Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

DEFF Research Database (Denmark)

Reisner, A.; Molin, Søren; Zechner, E.L.

2002-01-01

An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

Using Optical Markers of Non-dysplastic Rectal Epithelial Cells to Identify Patients With Ulcerative Colitis (UC) - Associated Neoplasia

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Bista, Rajan K.; Brentnall, Teresa A.; Bronner, Mary P.; Langmead, Christopher J.; Brand, Randall E.; Liu, Yang

2011-01-01

BACKGROUND Current surveillance guidelines for patients with long-standing ulcerative colitis (UC) recommend repeated colonoscopy with random biopsies, which is time-consuming, discomforting and expensive. A less invasive strategy is to identify neoplasia by analyzing biomarkers from the more accessible rectum to predict the need for a full colonoscopy. The goal of this pilot study is to evaluate whether optical markers of rectal mucosa derived from a novel optical technique – partial-wave spectroscopic microscopy (PWS) could identify UC patients with high-grade dysplasia (HGD) or cancer (CA) present anywhere in their colon. METHODS Banked frozen non-dysplastic mucosal rectal biopsies were used from 28 UC patients (15 without dysplasia and 13 with concurrent HGD or CA). The specimen slides were made using a touch prep method and underwent PWS analysis. We divided the patients into two groups: 13 as a training set and an independent 15 as a validation set. RESULTS We identified six optical markers, ranked by measuring the information gain with respect to the outcome of cancer. The most effective markers were selected by maximizing the cross validated training accuracy of a Naive Bayes classifier. The optimal classifier was applied to the validation data yielding 100% sensitivity and 75% specificity. CONCLUSIONS Our results indicate that the PWS-derived optical markers can accurately predict UC patients with HGD/CA through assessment of rectal epithelial cells. By aiming for a high sensitivity, our approach could potentially simplify the surveillance of UC patients and improve overall resource utilization by identifying patients with HGD/CA who should proceed with colonoscopy. PMID:21351200

Phylogeography of the common vampire bat (Desmodus rotundus: Marked population structure, Neotropical Pleistocene vicariance and incongruence between nuclear and mtDNA markers

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Morgante João S

2009-12-01

Full Text Available Abstract Background The common vampire bat Desmodus rotundus is an excellent model organism for studying ecological vicariance in the Neotropics due to its broad geographic range and its preference for forested areas as roosting sites. With the objective of testing for Pleistocene ecological vicariance, we sequenced a mitocondrial DNA (mtDNA marker and two nuclear markers (RAG2 and DRB to try to understand how Pleistocene glaciations affected the distribution of intraspecific lineages in this bat. Results Five reciprocally monophyletic clades were evident in the mitochondrial gene tree, and in most cases with high bootstrap support: Central America (CA, Amazon and Cerrado (AMC, Pantanal (PAN, Northern Atlantic Forest (NAF and Southern Atlantic Forest (SAF. The Atlantic forest clades formed a monophyletic clade with high bootstrap support, creating an east/west division for this species in South America. On the one hand, all coalescent and non-coalescent estimates point to a Pleistocene time of divergence between the clades. On the other hand, the nuclear markers showed extensive sharing of haplotypes between distant localities, a result compatible with male-biased gene flow. In order to test if the disparity between the mitochondrial and nuclear markers was due to the difference in mutation rate and effective size, we performed a coalescent simulation to examine the feasibility that, given the time of separation between the observed lineages, even with a gene flow rate close to zero, there would not be reciprocal monophyly for a neutral nuclear marker. We used the observed values of theta and an estimated mutation rate for the nuclear marker gene to perform 1000 iterations of the simulation. The results of this simulation were inconclusive: the number of iterations with and without reciprocal monophyly of one or more clades are similar. Conclusions We therefore conclude that the pattern exhibited by the common vampire bat, with marked

Current Status of Marker Genes of Bacteroides and Related Taxa for Identifying Sewage Pollution in Environmental Waters

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Warish Ahmed

2016-05-01

Full Text Available Microbial source tracking (MST endeavors to determine sources of fecal pollution in environmental waters by capitalizing on the association of certain microorganisms with the gastrointestinal tract and feces of specific animal groups. Several decades of research have shown that bacteria belonging to the gut-associated order Bacteroidales, and particularly the genus Bacteroides, tend to co-evolve with the host, and are, therefore, particularly suitable candidates for MST applications. This review summarizes the current research on MST methods that employ genes belonging to Bacteroidales/Bacteroides as tracers or “markers” of sewage pollution, including known advantages and deficiencies of the many polymerase chain reaction (PCR-based methods that have been published since 2000. Host specificity is a paramount criterion for confidence that detection of a marker is a true indicator of the target host. Host sensitivity, or the prevalence of the marker in feces/waste from the target host, is necessary for confidence that absence of the marker is indicative of the absence of the pollution source. Each of these parameters can vary widely depending on the type of waste assessed and the geographic location. Differential decay characteristics of bacterial targets and their associated DNA contribute to challenges in interpreting MST results in the context of human health risks. The HF183 marker, derived from the 16S rRNA gene of Bacteroides dorei and closely related taxa, has been used for almost two decades in MST studies, and is well characterized regarding host sensitivity and specificity, and in prevalence and concentration in sewage in many countries. Other markers such as HumM2 and HumM3 show promise, but require further performance testing to demonstrate their widespread utility. An important limitation of the one-marker-one-assay approach commonly used for MST is that given the complexities of microbial persistence in environmental waters, and

Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis

International Nuclear Information System (INIS)

Weinrauch, Y.; Dubnau, D.

1987-01-01

Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2 H and 15 N, in the presence of 32 Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases

The Comparison of Biochemical and Sequencing 16S rDNA Gene Methods to Identify Nontuberculous Mycobacteria

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Shafipour1, M.

2014-11-01

Full Text Available The identification of Mycobacteria in the species level has great medical importance. Biochemical tests are laborious and time-consuming, so new techniques could be used to identify the species. This research aimed to the comparison of biochemical and sequencing 16S rDNA gene methods to identify nontuberculous Mycobacteria in patients suspected to tuberculosis in Golestan province which is the most prevalent region of tuberculosis in Iran. Among 3336 patients suspected to tuberculosis referred to hospitals and health care centres in Golestan province during 2010-2011, 319 (9.56% culture positive cases were collected. Identification of species by using biochemical tests was done. On the samples recognized as nontuberculous Mycobacteria, after DNA extraction by boiling, 16S rDNA PCR was done and their sequencing were identified by NCBI BLAST. Of the 319 positive samples in Golestan Province, 300 cases were M.tuberculosis and 19 cases (5.01% were identified as nontuberculous Mycobacteria by biochemical tests. 15 out of 19 nontuberculous Mycobacteria were identified by PCR and sequencing method as similar by biochemical methods (similarity rate: 78.9%. But after PCR, 1 case known as M.simiae by biochemical test was identified as M. lentiflavum and 3 other cases were identified as Nocardia. Biochemical methods corresponded to the 16S rDNA PCR and sequencing in 78.9% of cases. However, in identification of M. lentiflavum and Nocaria sp. the molecular method is better than biochemical methods.

Consequences of low birthweight on urinary excretion of DNA markers of oxidative stress in young men

DEFF Research Database (Denmark)

Hillestrøm, P R; Weimann, A; Jensen, C B

2006-01-01

OBJECTIVE: Low birthweight (LBW) has been associated with an increased risk of development of type 2 diabetes in adult life. Both type 1 and type 2 diabetes mellitus are characterized by increased oxidative stress. The purpose of this study was to investigate whether young healthy adults born...... with LBW showed differences in oxidative stress under normal conditions and during the added challenge of a physiological Intralipid infusion. MATERIAL AND METHODS: Urinary excretion of DNA markers of oxidative stress were analyzed by LC-MS/MS in 19 men (aged 19 years) with LBW and in 19 age matched...... with LBW and NBW (66.9 versus 73.9 nmol/15 h, 17.8 versus 18.5 nmol/15 h, 11.9 versus 14.4 nmol/15 h and 44.0 versus 43.2 pmol/15 h, respectively). Furthermore, Intralipid infusion did not affect excretion of DNA adducts in LBW or NBW subjects. Statistically significant correlations were found between body...

Microsatellite marker development by partial sequencing of the sour passion fruit genome (Passiflora edulis Sims).

Science.gov (United States)

Araya, Susan; Martins, Alexandre M; Junqueira, Nilton T V; Costa, Ana Maria; Faleiro, Fábio G; Ferreira, Márcio E

2017-07-21

The Passiflora genus comprises hundreds of wild and cultivated species of passion fruit used for food, industrial, ornamental and medicinal purposes. Efforts to develop genomic tools for genetic analysis of P. edulis, the most important commercial Passiflora species, are still incipient. In spite of many recognized applications of microsatellite markers in genetics and breeding, their availability for passion fruit research remains restricted. Microsatellite markers in P. edulis are usually limited in number, show reduced polymorphism, and are mostly based on compound or imperfect repeats. Furthermore, they are confined to only a few Passiflora species. We describe the use of NGS technology to partially assemble the P. edulis genome in order to develop hundreds of new microsatellite markers. A total of 14.11 Gbp of Illumina paired-end sequence reads were analyzed to detect simple sequence repeat sites in the sour passion fruit genome. A sample of 1300 contigs containing perfect repeat microsatellite sequences was selected for PCR primer development. Panels of di- and tri-nucleotide repeat markers were then tested in P. edulis germplasm accessions for validation. DNA polymorphism was detected in 74% of the markers (PIC = 0.16 to 0.77; number of alleles/locus = 2 to 7). A core panel of highly polymorphic markers (PIC = 0.46 to 0.77) was used to cross-amplify PCR products in 79 species of Passiflora (including P. edulis), belonging to four subgenera (Astrophea, Decaloba, Distephana and Passiflora). Approximately 71% of the marker/species combinations resulted in positive amplicons in all species tested. DNA polymorphism was detected in germplasm accessions of six closely related Passiflora species (P. edulis, P. alata, P. maliformis, P. nitida, P. quadrangularis and P. setacea) and the data used for accession discrimination and species assignment. A database of P. edulis DNA sequences obtained by NGS technology was examined to identify microsatellite repeats in

Alpha-Fetoprotein, Identified as a Novel Marker for the Antioxidant Effect of Placental Extract, Exhibits Synergistic Antioxidant Activity in the Presence of Estradiol

Science.gov (United States)

Choi, Hye Yeon; Kim, Seung Woo; Kim, BongWoo; Lee, Hae Na; Kim, Su-Jeong; Song, Minjung; Kim, Sol; Kim, Jungho; Kim, Young Bong; Kim, Jin-Hoi; Cho, Ssang-Goo

2014-01-01

Placenta, as a reservoir of nutrients, has been widely used in medical and cosmetic materials. Here, we focused on the antioxidant properties of placental extract and attempted to isolate and identify the main antioxidant factors. Porcine placental extracts were prepared through homogenization or acid hydrolysis, and their antioxidant activity was investigated in the human keratinocyte HaCaT cell line. Treatment with homogenized placental extract (H-PE) increased the cell viability of H2O2-treated HaCaT cells more than two-fold. H-PE treatment suppressed H2O2-induced apoptotic and necrotic cell death and decreased intracellular ROS levels in H2O2-treated HaCaT cells. The antioxidant factors in H-PE were found to be thermo-unstable and were thus expected to include proteins. The candidate antioxidant proteins were fractionated with cation-exchange, anion-exchange, and size-exclusion chromatography, and the antioxidant properties of the chromatographic fractions were investigated. We obtained specific antioxidant fractions that suppressed ROS generation and ROS-induced DNA strand breaks. From silver staining and MALDI-TOF analyses, alpha-fetoprotein (AFP) precursor was identified as a main marker for the antioxidant effect of H-PE. Purified AFP or ectopically expressed AFP exhibited synergistic antioxidant activity in the presence of estradiol. Taken together, our data suggest that AFP, a serum glycoprotein produced at high levels during fetal development, is a novel marker protein for the antioxidant effect of the placenta that exhibits synergistic antioxidant activity in the presence of estradiol. PMID:24922551

Alpha-fetoprotein, identified as a novel marker for the antioxidant effect of placental extract, exhibits synergistic antioxidant activity in the presence of estradiol.

Directory of Open Access Journals (Sweden)

Hye Yeon Choi

Full Text Available Placenta, as a reservoir of nutrients, has been widely used in medical and cosmetic materials. Here, we focused on the antioxidant properties of placental extract and attempted to isolate and identify the main antioxidant factors. Porcine placental extracts were prepared through homogenization or acid hydrolysis, and their antioxidant activity was investigated in the human keratinocyte HaCaT cell line. Treatment with homogenized placental extract (H-PE increased the cell viability of H2O2-treated HaCaT cells more than two-fold. H-PE treatment suppressed H2O2-induced apoptotic and necrotic cell death and decreased intracellular ROS levels in H2O2-treated HaCaT cells. The antioxidant factors in H-PE were found to be thermo-unstable and were thus expected to include proteins. The candidate antioxidant proteins were fractionated with cation-exchange, anion-exchange, and size-exclusion chromatography, and the antioxidant properties of the chromatographic fractions were investigated. We obtained specific antioxidant fractions that suppressed ROS generation and ROS-induced DNA strand breaks. From silver staining and MALDI-TOF analyses, alpha-fetoprotein (AFP precursor was identified as a main marker for the antioxidant effect of H-PE. Purified AFP or ectopically expressed AFP exhibited synergistic antioxidant activity in the presence of estradiol. Taken together, our data suggest that AFP, a serum glycoprotein produced at high levels during fetal development, is a novel marker protein for the antioxidant effect of the placenta that exhibits synergistic antioxidant activity in the presence of estradiol.

SCAR marker specific to detect Magnaporthe grisea infecting finger millets (Eleusine coracana).

Science.gov (United States)

Gnanasing Jesumaharaja, L; Manikandan, R; Raguchander, T

2016-09-01

To determine the molecular variability and develop specific Sequence Characterized Amplified Region (SCAR) marker for the detection of Magnaporthe grisea causing blast disease in finger millet. Random amplified polymorphic DNA (RAPD) was performed with 14 isolates of M. grisea using 20 random primers. SCAR marker was developed for accurate and specific detection of M. grisea infecting only finger millets. The genetic similarity coefficient within each group and variation between the groups was observed. Among the primers, OPF-08 generated a RAPD polymorphic profile that showed common fragment of 478 bp in all the isolates. This fragment was cloned and sequenced. SCAR primers, Mg-SCAR-FP and Mg-SCAR-RP, were designed using sequence of the cloned product. The specificity of the SCAR primers was evaluated using purified DNA from M. grisea isolates from finger millets and other pathogens viz., Pyricularia oryzae, Colletotrichum gloeosporioides, Colletotrichum falcatum and Colletotrichum capcisi infecting different crops. The SCAR primers amplified only specific 460 bp fragment from DNA of M. grisea isolates and this fragment was not amplified in other pathogens tested. SCAR primers distinguish blast disease of finger millet from rice as there is no amplification in the rice blast pathogen. PCR-based SCAR marker is a convenient tool for specific and rapid detection of M. grisea in finger millets. Genetic diversity in fungal population helps in developing a suitable SCAR marker to identify the blast pathogen at the early stage of infection. © 2016 The Society for Applied Microbiology.

Screening of specific nucleic acid aptamers binding tumor markers in the serum of the lung cancer patients and identification of their activities.

Science.gov (United States)

Li, Kun; Xiu, Chen-Lin; Gao, Li-Ming; Liang, Hua-Gang; Xu, Shu-Feng; Shi, Ming; Li, Jian; Liu, Zhi-Wei

2017-07-01

Lung cancer is by far the leading cause of cancer death in the world. Despite the improvements in diagnostic methods, the status of early detection was not achieved. So, a new diagnostic method is needed. The aim of this study is to obtain the highly specific nucleic acid aptamers with strong affinity to tumor markers in the serum of the lung cancer patients for targeting the serum. Aptamers specifically binding to tumor markers in the serum of the lung cancer patients were screened from the random single-stranded DNA library with agarose beads as supports and the serum as a target by target-substituting subtractive SELEX technique and real-time quantitative polymerase chain reaction technique. Subsequently, the secondary single-stranded DNA library obtained by 10 rounds of screening was amplified to double-stranded DNA, followed by high-throughput genome sequence analysis to screen aptamers with specific affinity to tumor markers in the serum of the lung cancer patients. Finally, six aptamers obtained by 10 rounds of screening were identified with high specific affinity to tumor markers in the serum of the lung cancer patients. Compared with other five aptamers, the aptamer 43 was identified both with the highest specificity to bind target molecule and without any obvious affinity to non-specific proteins. The screened aptamers have relatively high specificity to combine tumor markers in the serum of the lung cancer patients, which provides breakthrough points for early diagnosis and treatment of lung cancer.

Short Communication: Genetic linkage map of Cucurbita maxima with molecular and morphological markers.

Science.gov (United States)

Ge, Y; Li, X; Yang, X X; Cui, C S; Qu, S P

2015-05-22

Cucurbita maxima is one of the most widely cultivated vegetables in China and exhibits distinct morphological characteristics. In this study, genetic linkage analysis with 57 simple-sequence repeats, 21 amplified fragment length polymorphisms, 3 random-amplified polymorphic DNA, and one morphological marker revealed 20 genetic linkage groups of C. maxima covering a genetic distance of 991.5 cM with an average of 12.1 cM between adjacent markers. Genetic linkage analysis identified the simple-sequence repeat marker 'PU078072' 5.9 cM away from the locus 'Rc', which controls rind color. The genetic map in the present study will be useful for better mapping, tagging, and cloning of quantitative trait loci/gene(s) affecting economically important traits and for breeding new varieties of C. maxima through marker-assisted selection.

Serum amyloid A as a prognostic marker in melanoma identified by proteomic profiling.

Science.gov (United States)

Findeisen, Peter; Zapatka, Marc; Peccerella, Teresa; Matzk, Heike; Neumaier, Michael; Schadendorf, Dirk; Ugurel, Selma

2009-05-01

Currently known prognostic serum biomarkers of melanoma are powerful in metastatic disease, but weak in early-stage patients. This study was aimed to identify new prognostic biomarkers of melanoma by serum mass spectrometry (MS) proteomic profiling, and to validate candidates compared with established markers. Two independent sets of serum samples from 596 melanoma patients were investigated. The first set (stage I = 102; stage IV = 95) was analyzed by matrix assisted laser desorption and ionization time of flight (MALDI TOF) MS for biomarkers differentiating between stage I and IV. In the second set (stage I = 98; stage II = 91; stage III = 87; stage IV = 103), the serum concentrations of the candidate marker serum amyloid A (SAA) and the known biomarkers S100B, lactate dehydrogenase, and C reactive protein (CRP) were measured using immunoassays. MALDI TOF MS revealed a peak at m/z 11.680 differentiating between stage I and IV, which could be identified as SAA. High peak intensities at m/z 11.680 correlated with poor survival. In univariate analysis, SAA was a strong prognostic marker in stage I to III (P = .043) and stage IV (P = .000083) patients. Combination of SAA and CRP increased the prognostic impact to P = .011 in early-stage (I to III) patients. Multivariate analysis revealed sex, stage, tumor load, S100B, SAA, and CRP as independent prognostic factors, with an interaction between SAA and CRP. In stage I to III patients, SAA combined with CRP was superior to S100B in predicting patients' progression-free and overall survival. SAA combined with CRP might be used as prognostic serological biomarkers in early-stage melanoma patients, helping to discriminate low-risk patients from high-risk patients needing adjuvant treatment.

New approach for isolation of VNTR markers.

OpenAIRE

Nakamura, Y; Carlson, M; Krapcho, K; Kanamori, M; White, R

1988-01-01

Elsewhere we have reported an efficient method for isolating VNTR (Variable Number of Tandem Repeats) markers. Several of the VNTR markers isolated in those experiments were sequenced, and a DNA sequence of 9 bp (GNNGTGGG) emerged as an apparent consensus sequence for VNTR markers. To confirm this result and to develop more VNTR markers, we synthesized nine different 18-base-long oligonucleotides whose sequences each included GNNGTGGG. When 102 cosmid clones selected by these oligonucleotides...

The DNA-instability test as a specific marker of malignancy and its application to detect cancer clones in borderline malignancy

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M Fukuda

2009-06-01

Full Text Available Recent progress in cytogenetic and biochemical mutator assay technologies has enabled us to detect single gene alterations and gross chromosomal rearrangements, and it became clear that all cancer cells are genetically unstable. In order to detect the genome-wide instability of cancer cells, a new simple method, the DNA-instability test, was developed. The methods to detect genomic instability so far reported have only demonstrated the presence of qualitative and quantitative alterations in certain specific genomic loci. In contrast to these commonly used methods to reveal the genomic instability at certain specific DNA regions, the newly introduced DNA-instability test revealed the presence of physical DNA-instability in the entire DNA molecule of a cancer cell nucleus as revealed by increased liability to denature upon HCl hydrolysis or formamide exposure. When this test was applied to borderline malignancies, cancer clones were detected in all cases at an early-stage of cancer progression. We proposed a new concept of “procancer” clones to define those cancer clones with “functional atypia” showing positivities for various cancer markers, as well as DNA-instability testing, but showing no remarkable ordinary “morphological atypia” which is commonly used as the basis of histopathological diagnosis of malignancy.

Epigenetic Markers of Renal Function in African Americans

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Samantha M. Bomotti

2013-01-01

Full Text Available Chronic kidney disease (CKD is an increasing concern in the United States due to its rapidly rising prevalence, particularly among African Americans. Epigenetic DNA methylation markers are becoming important biomarkers of chronic diseases such as CKD. To better understand how these methylation markers play a role in kidney function, we measured 26,428 DNA methylation sites in 972 African Americans from the Genetic Epidemiology Network of Arteriopathy (GENOA study. We then evaluated (1 whether epigenetic markers are associated with estimated glomerular filtration rate (eGFR, (2 whether the significantly associated markers are also associated with traditional risk factors and/or novel biomarkers for eGFR, and (3 how much additional variation in eGFR is explained by epigenetic markers beyond established risk factors and biomarkers. The majority of methylation markers most significantly associated with eGFR (24 out of the top 30 appeared to function, at least in part, through pathways related to aging, inflammation, or cholesterol. However, six epigenetic markers were still able to significantly predict eGFR after adjustment for other risk factors. This work shows that epigenetic markers may offer valuable new insight into the complex pathophysiology of CKD in African Americans.

Genetic diversity and differentiation in Prunus species (Rosaceae) using chloroplast and mitochondrial DNA CAPS markers.

Science.gov (United States)

Ben Mustapha, S; Ben Tamarzizt, H; Baraket, G; Abdallah, D; Salhi Hannachi, A

2015-04-27

Chloroplast (cpDNA) and mitochondrial DNA (mtDNA) were analyzed to establish genetic relationships among Tunisian plum cultivars using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Two mtDNA regions (nad 1 b/c and nad 4 1/2) and a cpDNA region (trnL-trnF) were amplified and digested using restriction enzymes. Seventy and six polymorphic sites were revealed in cpDNA and mtDNA, respectively. As a consequence, cpDNA appears to be more polymorphic than mtDNA. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed that accessions were distributed independently of their geographical origin, and introduced and local cultivars appear to be closely related. Both UPGMA and principal component analysis grouped Tunisian plum accessions into similar clusters. The analysis of the pooled sequences allowed the detection of 17 chlorotypes and 12 mitotypes. The unique haplotypes detected for cultivars are valuable for management and preservation of the plum local resources. From this study, PCR-RFLP analysis appears to be a useful approach to detect and identify cytoplasmic variation in plum trees. Our results also provide useful information for the management of genetic resources and to establish a program to improve the genetic resources available for plums.

Probabilistic analysis showing that a combination of bacteroides and methanobrevibacter source tracking markers is effective for identifying waters contaminated by human fecal pollution

Science.gov (United States)

Johnston, Christopher; Byappanahalli, Muruleedhara N.; Gibson, Jacqueline MacDonald; Ufnar, Jennifer A.; Whitman, Richard L.; Stewart, Jill R.

2013-01-01

Microbial source tracking assays to identify sources of waterborne contamination typically target genetic markers of host-specific microorganisms. However, no bacterial marker has been shown to be 100% host-specific, and cross-reactivity has been noted in studies evaluating known source samples. Using 485 challenge samples from 20 different human and animal fecal sources, this study evaluated microbial source tracking markers including the Bacteroides HF183 16S rRNA, M. smithii nifH, and Enterococcus esp gene targets that have been proposed as potential indicators of human fecal contamination. Bayes' Theorem was used to calculate the conditional probability that these markers or a combination of markers can correctly identify human sources of fecal pollution. All three human-associated markers were detected in 100% of the sewage samples analyzed. Bacteroides HF183 was the most effective marker for determining whether contamination was specifically from a human source, and greater than 98% certainty that contamination was from a human source was shown when both Bacteroides HF183 and M. smithii nifH markers were present. A high degree of certainty was attained even in cases where the prior probability of human fecal contamination was as low as 8.5%. The combination of Bacteroides HF183 and M. smithii nifH source tracking markers can help identify surface waters impacted by human fecal contamination, information useful for prioritizing restoration activities or assessing health risks from exposure to contaminated waters.

A DNA methylation-based definition of biologically distinct breast cancer subtypes.

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Stefansson, Olafur A; Moran, Sebastian; Gomez, Antonio; Sayols, Sergi; Arribas-Jorba, Carlos; Sandoval, Juan; Hilmarsdottir, Holmfridur; Olafsdottir, Elinborg; Tryggvadottir, Laufey; Jonasson, Jon G; Eyfjord, Jorunn; Esteller, Manel

2015-03-01

In cancer, epigenetic states are deregulated and thought to be of significance in cancer development and progression. We explored DNA methylation-based signatures in association with breast cancer subtypes to assess their impact on clinical presentation and patient prognosis. DNA methylation was analyzed using Infinium 450K arrays in 40 tumors and 17 normal breast samples, together with DNA copy number changes and subtype-specific markers by tissue microarrays. The identified methylation signatures were validated against a cohort of 212 tumors annotated for breast cancer subtypes by the PAM50 method (The Cancer Genome Atlas). Selected markers were pyrosequenced in an independent validation cohort of 310 tumors and analyzed with respect to survival, clinical stage and grade. The results demonstrate that DNA methylation patterns linked to the luminal-B subtype are characterized by CpG island promoter methylation events. In contrast, a large fraction of basal-like tumors are characterized by hypomethylation events occurring within the gene body. Based on these hallmark signatures, we defined two DNA methylation-based subtypes, Epi-LumB and Epi-Basal, and show that they are associated with unfavorable clinical parameters and reduced survival. Our data show that distinct mechanisms leading to changes in CpG methylation states are operative in different breast cancer subtypes. Importantly, we show that a few selected proxy markers can be used to detect the distinct DNA methylation-based subtypes thereby providing valuable information on disease prognosis. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Application of next-generation sequencing technology to study genetic diversity and identify unique SNP markers in bread wheat from Kazakhstan.

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Shavrukov, Yuri; Suchecki, Radoslaw; Eliby, Serik; Abugalieva, Aigul; Kenebayev, Serik; Langridge, Peter

2014-09-28

New SNP marker platforms offer the opportunity to investigate the relationships between wheat cultivars from different regions and assess the mechanism and processes that have led to adaptation to particular production environments. Wheat breeding has a long history in Kazakhstan and the aim of this study was to explore the relationship between key varieties from Kazakhstan and germplasm from breeding programs for other regions. The study revealed 5,898 polymorphic markers amongst ten cultivars, of which 2,730 were mapped in the consensus genetic map. Mapped SNP markers were distributed almost equally across the A and B genomes, with between 279 and 484 markers assigned to each chromosome. Marker coverage was approximately 10-fold lower in the D genome. There were 863 SNP markers identified as unique to specific cultivars, and clusters of these markers (regions containing more than three closely mapped unique SNPs) showed specific patterns on the consensus genetic map for each cultivar. Significant intra-varietal genetic polymorphism was identified in three cultivars (Tzelinnaya 3C, Kazakhstanskaya rannespelaya and Kazakhstanskaya 15). Phylogenetic analysis based on inter-varietal polymorphism showed that the very old cultivar Erythrospermum 841 was the most genetically distinct from the other nine cultivars from Kazakhstan, falling in a clade together with the American cultivar Sonora and genotypes from Central and South Asia. The modern cultivar Kazakhstanskaya 19 also fell into a separate clade, together with the American cultivar Thatcher. The remaining eight cultivars shared a single sub-clade but were categorised into four clusters. The accumulated data for SNP marker polymorphisms amongst bread wheat genotypes from Kazakhstan may be used for studying genetic diversity in bread wheat, with potential application for marker-assisted selection and the preparation of a set of genotype-specific markers.

Identification of DNA repair genes in the human genome

International Nuclear Information System (INIS)

Hoeijmakers, J.H.J.; van Duin, M.; Westerveld, A.; Yasui, A.; Bootsma, D.

1986-01-01

To identify human DNA repair genes we have transfected human genomic DNA ligated to a dominant marker to excision repair deficient xeroderma pigmentosum (XP) and CHO cells. This resulted in the cloning of a human gene, ERCC-1, that complements the defect of a UV- and mitomycin-C sensitive CHO mutant 43-3B. The ERCC-1 gene has a size of 15 kb, consists of 10 exons and is located in the region 19q13.2-q13.3. Its primary transcript is processed into two mRNAs by alternative splicing of an internal coding exon. One of these transcripts encodes a polypeptide of 297 aminoacids. A putative DNA binding protein domain and nuclear location signal could be identified. Significant AA-homology is found between ERCC-1 and the yeast excision repair gene RAD10. 58 references, 6 figures, 1 table

Genetic diversity analysis of chewing sugarcane (Saccharum officinarum L. varieties by using RAPD markers

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S. M. Sarid Ullah

2013-01-01

Full Text Available In the present study an efficient and easy method was followed for the isolation of DNA from meristem cylinder in five chewing sugarcane varieties, namely Amrita, Bomaby, Babulal (Co.527, Q83 and Misrimala. The quality and quantity of DNA were assured by visual estimation using agarose gel electrophoresis and UV spectrophotometry. The highest amount of DNA was retrieved from the Amrita (3250 ng/ml and the lowest amount was attained from the variety Q83 (1450 ng/ml. The amount of recovered DNA was enough for PCR amplification and marker studies such as random amplified polymorphic DNA (RAPD. Using RAPD markers, bands obtained from fingerprinting (190 bp to 1200 bp showed 73.5% polymorphism. The dendrogram, based on linkage distance using unweighted pair group method of arithmetic means (UPGMA, indicated segregation of the five chewing varieties of sugarcane into two main clusters. Amrita, Bombay and Misrimala were grouped in cluster 1 (C1 followed by sub-clusters. Babulal and Q83 were grouped in cluster 2 (C2. The results of the present investigation also revealed that the twenty RAPD primers were able to identify and classify the chewing sugarcane varieties based on their genetic relationship.

Molecular approaches for forensic cell type identification: On mRNA, miRNA, DNA methylation and microbial markers.

Science.gov (United States)

Sijen, Titia

2015-09-01

Human biological traces have the potential to present strong evidence for placing a suspect at a crime scene. In cases, the activity that led to deposition of an individual's cellular material is increasingly disputed, for which the identification of cell types could be crucial. This review aims to give an overview of the possibilities of the employment of mRNA, miRNA, DNA methylation and microbial markers for tissue identification in a forensic context. The biological background that renders these markers tissue-specificity is considered, as this can affect data interpretation. Furthermore, the forensic relevance of inferring certain cell types is discussed, as are the various methodologies that can be applied. Forensic stains can carry minute amounts of cell material that may be degraded or polluted and most likely cell material of multiple sources will be present. The interpretational challenges that are imposed by this compromised state will be discussed as well. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Species-specific identification from incomplete sampling: applying DNA barcodes to monitoring invasive solanum plants.

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Zhang, Wei; Fan, Xiaohong; Zhu, Shuifang; Zhao, Hong; Fu, Lianzhong

2013-01-01

Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling-through this, DNA barcoding will greatly benefit the current fields of its application.

Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology.

Science.gov (United States)

de Souza, Aracele M; de Araújo, Flávia C F; Fontes, Cor J F; Carvalho, Luzia H; de Brito, Cristiana F A; de Sousa, Taís N

2015-08-25

Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones. The detection of multiple-clone infections depends on several factors, such as the accuracy of the genotyping method, and the type and number of the molecular markers analysed. Characterizing the multiplicity of infection has broad implications that range from population genetic studies of the parasite to malaria treatment and control. This study compared and evaluated the efficiency of neutral and non-neutral markers that are widely used in studies of molecular epidemiology to detect the multiplicity of P. vivax infection. The performance of six markers was evaluated using 11 mixtures of DNA with well-defined proportions of two different parasite genotypes for each marker. These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA. In addition, 51 samples of natural infections from the Brazil were genotyped for all markers. The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers. The criteria for differentiating minor peaks from artifacts were also evaluated. The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations. Nevertheless, msp1B2 was not able to detect the majority of multiple-clone infections in field samples; it identified only 6 % of these infections. The merozoite surface protein-3 alpha and microsatellites (PvMS6 and PvMS7) did not accurately estimate the relative clonal proportions in artificial mixtures, but the microsatellites performed well in detecting natural multiple-clone infections. Notably, the use of a less stringent criterion to score rare alleles significantly increased the sensitivity of the detection of multi-clonal infections. Depending on the type of marker used, a considerable

Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes.

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Vardaki, Ioulia; Ceder, Sophia; Rutishauser, Dorothea; Baltatzis, George; Foukakis, Theodoros; Panaretakis, Theocharis

2016-11-15

Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis.

Genotyping by sequencing (GBS, an ultimate marker-assisted selection (MAS tool to accelerate plant breeding

Directory of Open Access Journals (Sweden)

Jiangfeng eHe

2014-09-01

Full Text Available Marker-assisted selection (MAS refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP, have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping by sequencing (GBS has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS, genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection (GS under a large scale of plant breeding programs.

Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding.

Science.gov (United States)

He, Jiangfeng; Zhao, Xiaoqing; Laroche, André; Lu, Zhen-Xiang; Liu, HongKui; Li, Ziqin

2014-01-01

Marker-assisted selection (MAS) refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP), have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS) technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping-by-sequencing (GBS) has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs.

Identifying the ichthyoplankton of a coral reef using DNA barcodes.

Science.gov (United States)

Hubert, Nicolas; Espiau, Benoit; Meyer, Christopher; Planes, Serge

2015-01-01

Marine fishes exhibit spectacular phenotypic changes during their ontogeny, and the identification of their early stages is challenging due to the paucity of diagnostic morphological characters at the species level. Meanwhile, the importance of early life stages in dispersal and connectivity has recently experienced an increasing interest in conservation programmes for coral reef fishes. This study aims at assessing the effectiveness of DNA barcoding for the automated identification of coral reef fish larvae through large-scale ecosystemic sampling. Fish larvae were mainly collected using bongo nets and light traps around Moorea between September 2008 and August 2010 in 10 sites distributed in open waters. Fish larvae ranged from 2 to 100 mm of total length, with the most abundant individuals being <5 mm. Among the 505 individuals DNA barcoded, 373 larvae (i.e. 75%) were identified to the species level. A total of 106 species were detected, among which 11 corresponded to pelagic and bathypelagic species, while 95 corresponded to species observed at the adult stage on neighbouring reefs. This study highlights the benefits and pitfalls of using standardized molecular systems for species identification and illustrates the new possibilities enabled by DNA barcoding for future work on coral reef fish larval ecology. © 2014 John Wiley & Sons Ltd.

Genetic variations and relationships of cultivated and weedy types of perilla species in Korea and Japan using multi DNA markers

International Nuclear Information System (INIS)

Sun, Y.; Zheng, S.; Lee, J.; Hong, S.

2017-01-01

The genus Perilla, known as an oil crop or a Chinese medicine, vegetable crop, is widely cultivated in East Asia. It occurs in two distinct varieties, var. frutescens and var. crispa, and their genetic relationship is still obscure. To understand the genetic diversity and relationships of the cultivated and weedy types of Perilla crops in Korea, Japan and China, we evaluated the genetic variation of 20 accessions by 3 rDNA markers. Among these three markers, the nuclear ribosomal DNA (nrDNA) internal transcribed spacers (ITS) region of Perilla crops showed less sequence variations than the 5S and 18S genes. There were abundant variable nucleotide sites appearing in the 5S and 18S genes. Especially in the 18S gene, the variable nucleotide sites showed specificity between some Perilla type and other varieties. JPN1 showed 6 special variable nucleotide sites differing from other varieties, resulting in the farthest phylogenetic relationship in the 18S tree. CHI15 shared 8 special variable sites, also showing far phylogenetic relationship with other varieties. According to the sequence analysis result, the cultivated types of Korean var. frutescens showed relatively more genetic diversity than those of Japanese var. frutescens, while Korean var. crispa showed lower genetic diversity than those of Japanese var. crispa. However, the intra- or inter-variety genetic distance did not have significant difference. This work provided more sequence resources of Perilla crops and evidences to evaluate the genetic variation and relationships. Our result would help molecular type identification, functional plant breeding and trait improvement of Perilla crops. (author)

Concordance of Hypermethylated DNA and the Tumor Markers CA 15-3, CEA, and TPA in Serum during Monitoring of Patients with Advanced Breast Cancer

DEFF Research Database (Denmark)

Kristiansen, Søren; Jørgensen, Lars Mønster; Høgh Hansen, Morten

2015-01-01

The serological protein tumor markers CA 15-3, CEA, and TPA are frequently used to monitor tumor burden among metastatic breast cancer patients. Breast cancer is associated with global DNA hypomethylation and hypermethylation of some promoter regions. No monitoring study has yet investigated the ...

A Dynamic Combinatorial Approach for Identifying Side Groups that Stabilize DNA-Templated Supramolecular Self-Assemblies

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Delphine Paolantoni

2015-02-01

Full Text Available DNA-templated self-assembly is an emerging strategy for generating functional supramolecular systems, which requires the identification of potent multi-point binding ligands. In this line, we recently showed that bis-functionalized guanidinium compounds can interact with ssDNA and generate a supramolecular complex through the recognition of the phosphodiester backbone of DNA. In order to probe the importance of secondary interactions and to identify side groups that stabilize these DNA-templated self-assemblies, we report herein the implementation of a dynamic combinatorial approach. We used an in situ fragment assembly process based on reductive amination and tested various side groups, including amino acids. The results reveal that aromatic and cationic side groups participate in secondary supramolecular interactions that stabilize the complexes formed with ssDNA.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

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Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

2017-01-01

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

DNA marker-assisted evaluation of potato genotypes for potential resistance to potato cyst nematode pathotypes not yet invading into Japan.

Science.gov (United States)

Asano, Kenji; Kobayashi, Akira; Tsuda, Shogo; Nishinaka, Mio; Tamiya, Seiji

2012-06-01

One of major objectives of crop breeding is conferring resistance to diseases and pests. However, large-scale phenotypic evaluation for many diseases and pests is difficult because strict controls are required to prevent their spread. Detection of disease resistance genes by using DNA markers may be an alternative approach to select potentially resistant accessions. Potato (Solanum tuberosum L.) breeders in Japan extensively use resistance gene H1, which confers nearly absolute resistance to potato cyst nematode (Globodera rostochiensis) pathotype Ro1, the only pathotype found in Japan. However, considering the possibility of accidental introduction of the other pathotypes, breeding of resistant varieties is an important strategy to prevent infestation by non-invading pathotypes in Japan. In this study, to evaluate the prevalence of resistance genes in Japanese genetic resources, we developed a multiplex PCR method that simultaneously detects 3 resistance genes, H1, Gpa2 and Gro1-4. We revealed that many Japanese varieties possess not only H1 but Gpa2, which are potentially resistant to other pathotypes of potato cyst nematode. On the other hand, no genotype was found to have the Gro1-4, indicating importance of introduction of varieties having Gro1-4. Our results demonstrate the applicability of DNA-marker assisted evaluation of resistant potato genotypes without phenotypic evaluation.

Next-generation sequencing to identify candidate genes and develop diagnostic markers for a novel Phytophthora resistance gene, RpsHC18, in soybean.

Science.gov (United States)

Zhong, Chao; Sun, Suli; Li, Yinping; Duan, Canxing; Zhu, Zhendong

2018-03-01

A novel Phytophthora sojae resistance gene RpsHC18 was identified and finely mapped on soybean chromosome 3. Two NBS-LRR candidate genes were identified and two diagnostic markers of RpsHC18 were developed. Phytophthora root rot caused by Phytophthora sojae is a destructive disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Phytophthora-resistant Rps genes. The soybean cultivar Huachun 18 has a broad and distinct resistance spectrum to 12 P. sojae isolates. Quantitative trait loci sequencing (QTL-seq), based on the whole-genome resequencing (WGRS) of two extreme resistant and susceptible phenotype bulks from an F 2:3 population, was performed, and one 767-kb genomic region with ΔSNP-index ≥ 0.9 on chromosome 3 was identified as the RpsHC18 candidate region in Huachun 18. The candidate region was reduced to a 146-kb region by fine mapping. Nonsynonymous SNP and haplotype analyses were carried out in the 146-kb region among ten soybean genotypes using WGRS. Four specific nonsynonymous SNPs were identified in two nucleotide-binding sites-leucine-rich repeat (NBS-LRR) genes, RpsHC18-NBL1 and RpsHC18-NBL2, which were considered to be the candidate genes. Finally, one specific SNP marker in each candidate gene was successfully developed using a tetra-primer ARMS-PCR assay, and the two markers were verified to be specific for RpsHC18 and to effectively distinguish other known Rps genes. In this study, we applied an integrated genomic-based strategy combining WGRS with traditional genetic mapping to identify RpsHC18 candidate genes and develop diagnostic markers. These results suggest that next-generation sequencing is a precise, rapid and cost-effective way to identify candidate genes and develop diagnostic markers, and it can accelerate Rps gene cloning and marker-assisted selection for breeding of P. sojae-resistant soybean cultivars.

Mitochondrial DNA paradox: sex-specific genetic structure in a marine mussel – despite maternal inheritance and passive dispersal

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Teske Peter R

2012-06-01

Full Text Available Abstract Background When genetic structure is identified using mitochondrial DNA (mtDNA, but no structure is identified using biparentally-inherited nuclear DNA, the discordance is often attributed to differences in dispersal potential between the sexes. Results We sampled the intertidal rocky shore mussel Perna perna in a South African bay and along the nearby open coast, and sequenced maternally-inherited mtDNA (there is no evidence for paternally-inherited mtDNA in this species and a biparentally-inherited marker. By treating males and females as different populations, we identified significant genetic structure on the basis of mtDNA data in the females only. Conclusions This is the first study to report sex-specific differences in genetic structure based on matrilineally-inherited mtDNA in a passively dispersing species that lacks social structure or sexual dimorphism. The observed pattern most likely stems from females being more vulnerable to selection in habitats from which they did not originate, which also manifests itself in a male-biased sex ratio. Our results have three important implications for the interpretation of population genetic data. First, even when mtDNA is inherited exclusively in the female line, it also contains information about males. For that reason, using it to identify sex-specific differences in genetic structure by contrasting it with biparentally-inherited markers is problematic. Second, the fact that sex-specific differences were found in a passively dispersing species in which sex-biased dispersal is unlikely highlights the fact that significant genetic structure is not necessarily a function of low dispersal potential or physical barriers. Third, even though mtDNA is typically used to study historical demographic processes, it also contains information about contemporary processes. Higher survival rates of males in non-native habitats can erase the genetic structure present in their mothers within a single

Novel tetra-nucleotide microsatellite DNA markers for assessing the evolutionary genetics and demographics of Northern Snakehead (Channa argus) invading North America

Science.gov (United States)

King, Timothy L.; Johnson, Robin L.

2011-01-01

We document the isolation and characterization of 19 tetra-nucleotide microsatellite DNA markers in northern snakehead (Channa argus) fish that recently colonized Meadow Lake, New York City, New York. These markers displayed moderate levels of allelic diversity (averaging 6.8 alleles/locus) and heterozygosity (averaging 74.2%). Demographic analyses suggested that the Meadow Lake collection has not achieved mutation-drift equilibrium. These results were consistent with instances of deviations from Hardy–Weinberg equilibrium and the presence of some linkage disequilibrium. A comparison of individual pair-wise distances suggested the presence of multiple differentiated groups of related individuals. Results of all analyses are consistent with a pattern of multiple, recent introductions. The microsatellite markers developed for C. argus yielded sufficient genetic diversity to potentially: (1) delineate kinship; (2) elucidate fine-scale population structure; (3) define management (eradication) units; (4) estimate dispersal rates; (5) estimate population sizes; and (6) provide unique demographic perspectives of control or eradication effectiveness.

Targeting the Sources of Fecal Contamination using Dog-, Human-, and Ruminant- Specific Markers in the Lake Herrick Watershed, Georgia.

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Saintil, T.; Radcliffe, D. E.; Rasmussen, T. C.; Habteselassie, M.; Sowah, R.; Kannan, A.

2016-12-01

The Lake Herrick Watershed is about 1.5 km2 and covers portions of the University of Georgia's East campus, the Oconee Forest, residential and commercial landuse. Lake Herrick, a recreational site on the University of Georgia campus, was closed in 2002 due to fecal contamination. Subsequent monitoring confirmed persistent contamination, which led to a permanent closure to swimming, boating, and fishing. While fecal coliform abundance is a standard metric for determining human health risks, Geldreich (1970) showed that fecal abundance does not necessarily correlate with the presence of pathogens. Nor does it identify pollution sources, which are needed to mitigate health risks. Two inflow tributaries and the outlet stream were monitored for discharge, fecal coliform, forms of nitrogen and phosphorus and other water-quality data to quantify lake influent and effluent bacteria loads. Fecal sources were identified using the human HF183 genetic marker (Seurinck et al., 2005), the ruminant BacR marker (Reischer et al., 2006), and the dog mitochondrial DNA (mtDNA) marker (Tambalo et al., 2012). Preliminary results confirm high concentrations of E. coli and Enterococci, above the State's limit of 124 MPN/100 mL, in both baseflows and stormflows. The findings also suggest that the E. coli and Enterococci loads from the inlet tributaries are on average higher compared to the bacteria loads coming out of the outlet stream. The human markers were detectable at all three sites but most of the samples were not quantifiable. The ruminant markers were quantifiable at both inlets but no ruminant markers were found at the outlet. The dog markers were detectable but not quantifiable at both inlets and no dog markers were detected at the outlet. Statistical analyses will be used to establish relationships between the nutrients data, the fecal concentrations, and the gene-specific markers.

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis.

Science.gov (United States)

Konakandla, Bhanu; Park, Yoonseong; Margolies, David

2006-01-01

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.

Screening of Genes Specifically Expressed in Males of Fenneropenaeus chinensis and Their Potential as Sex Markers

Directory of Open Access Journals (Sweden)

Shihao Li

2013-01-01

Full Text Available The androgenic gland (AG, playing an important role in sex differentiation of male crustacean, is a target candidate to understand the mechanism of male development and to mine male-specific sex markers. An SSH library (designated as male reproduction-related tissues—SSH library, MRT-SSH library for short was constructed using cDNA from tissues located at the basal part of the 5th pereiopods, including AG and part of spermatophore sac, as tester, and the cDNA from the basal part of the 4th pereiopods of these male shrimp as driver. 402 ESTs from the SSH library were sequenced and assembled into 48 contigs and 104 singlets. Twelve contigs and 14 singlets were identified as known genes. The proteins encoded by the identified genes were categorized, according to their proposed functions, into neuropeptide hormone and hormone transporter, RNA posttranscriptional regulation, translation, cell growth and death, metabolism, genetic information processing, signal transduction/transport, or immunity-related proteins. Eleven highly expressed contigs in the SSH library were selected for validation of the MRT-SSH library and screening sex markers of shrimp. One contig, specifically expressed in male shrimp, had a potential to be developed as a transcriptomic sex marker in shrimp.

[cDNA library construction from panicle meristem of finger millet].

Science.gov (United States)

Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

2014-01-01

The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

Marker evaluation for differentiation of blood and menstrual fluid by methylation-sensitive SNaPshot analysis.

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Holtkötter, Hannah; Schwender, Kristina; Wiegand, Peter; Pfeiffer, Heidi; Vennemann, Marielle

2018-03-01

The differentiation of blood and menstrual fluid is especially important in cases of alleged sexual assault. While the identification of blood is relatively straightforward, the identification of menstrual fluid in trace evidence has been shown to be more challenging. This may be due to the complex nature of menstrual fluid that leads to intra- and inter-individual differences in composition. Nevertheless, recent advances in DNA methylation profiling have revealed promising markers for the differentiation of the two body fluids and furthermore, markers to distinguish menstrual fluid from vaginal fluid. A literature study was performed and in total, 11 markers were evaluated in this study of which seven could be validated for menstrual fluid and blood identification purposes. Marker "BLU2" (chr16:29757334) was identified as most suitable for differentiation of blood and menstrual fluid.

Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library

DEFF Research Database (Denmark)

Petersen, L. K.; Blakskjær, P.; Chaikuad, A.

2016-01-01

A highly specific and potent (7 nM cellular IC50) inhibitor of p38α kinase was identified directly from a 12.6 million membered DNA-encoded small molecule library. This was achieved using the high fidelity yoctoReactor technology (yR) for preparing the DNA-encoded library, and a homogeneous...... interactions. Moreover, the crystal structure showed, that although buried in the p38α active site, the original DNA attachment point of the compound was accessible through a channel created by the distorted P-loop conformation. This study demonstrates the usability of DNA-encoded library technologies...

Forensic DNA methylation profiling from evidence material for investigative leads

Science.gov (United States)

Lee, Hwan Young; Lee, Soong Deok; Shin, Kyoung-Jin

2016-01-01

DNA methylation is emerging as an attractive marker providing investigative leads to solve crimes in forensic genetics. The identification of body fluids that utilizes tissue-specific DNA methylation can contribute to solving crimes by predicting activity related to the evidence material. The age estimation based on DNA methylation is expected to reduce the number of potential suspects, when the DNA profile from the evidence does not match with any known person, including those stored in the forensic database. Moreover, the variation in DNA implicates environmental exposure, such as cigarette smoking and alcohol consumption, thereby suggesting the possibility to be used as a marker for predicting the lifestyle of potential suspect. In this review, we describe recent advances in our understanding of DNA methylation variations and the utility of DNA methylation as a forensic marker for advanced investigative leads from evidence materials. [BMB Reports 2016; 49(7): 359-369] PMID:27099236

Development and characterization of genomic SSR markers in Cynodon transvaalensis Burtt-Davy.

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Tan, Chengcheng; Wu, Yanqi; Taliaferro, Charles M; Bell, Greg E; Martin, Dennis L; Smith, Mike W

2014-08-01

Simple sequence repeat (SSR) markers are a major molecular tool for genetic and genomic research that have been extensively developed and used in major crops. However, few are available in African bermudagrass (Cynodon transvaalensis Burtt-Davy), an economically important warm-season turfgrass species. African bermudagrass is mainly used for hybridizations with common bermudagrass [C. dactylon var. dactylon (L.) Pers.] in the development of superior interspecific hybrid turfgrass cultivars. Accordingly, the major objective of this study was to develop and characterize a large set of SSR markers. Genomic DNA of C. transvaalensis '4200TN 24-2' from an Oklahoma State University (OSU) turf nursery was extracted for construction of four SSR genomic libraries enriched with [CA](n), [GA](n), [AAG](n), and [AAT](n) as core repeat motifs. A total of 3,064 clones were sequenced at the OSU core facility. The sequences were categorized into singletons and contiguous sequences to exclude redundancy. From the two sequence categories, 1,795 SSR loci were identified. After excluding duplicate SSRs by comparison with previously developed SSR markers using a nucleotide basic local alignment tool, 1,426 unique primer pairs (PPs) were designed. Out of the 1,426 designed PPs, 981 (68.8 %) amplified alleles of the expected size in the donor DNA. Polymorphisms of the SSR PPs tested in eight C. transvaalensis plants were 93 % polymorphic with 544 markers effective in all genotypes. Inheritance of the SSRs was examined in six F(1) progeny of African parents 'T577' × 'Uganda', indicating 917 markers amplified heritable alleles. The SSR markers developed in the study are the first large set of co-dominant markers in African bermudagrass and should be highly valuable for molecular and traditional breeding research.

Segregation analysis of microsatellite (SSR) markers in sugarcane polyploids.

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Lu, X; Zhou, H; Pan, Y-B; Chen, C Y; Zhu, J R; Chen, P H; Li, Y-R; Cai, Q; Chen, R K

2015-12-28

No information is available on segregation analysis of DNA markers involving both pollen and self-progeny. Therefore, we used capillary electrophoresis- and fluorescence-based DNA fingerprinting together with single pollen collection and polymerase chain reaction (PCR) to investigate simple sequence repeat (SSR) marker segregation among 964 single pollens and 288 self-progenies (S1) of sugarcane cultivar LCP 85-384. Twenty SSR DNA fragments (alleles) were amplified by five polymorphic SSR markers. Only one non-parental SSR allele was observed in 2392 PCRs. SSR allele inheritance was in accordance with Mendelian laws of segregation and independent assortment. Highly significant correlation coefficients were found between frequencies of observed and expected genotypes in pollen and S1 populations. Within the S1 population, the most frequent genotype of each SSR marker was the parental genotype of the same marker. The number of genotypes was higher in pollen than S1 population. PIC values of the five SSR markers were greater in pollen than S1 populations. Eleven of 20 SSR alleles (55%) were segregated in accordance with Mendelian segregation ratios expected from pollen and S1 populations of a 2n = 10x polyploid. Six of 20 SSR alleles were segregated in a 3:1 (presence:absence) ratio and were simplex markers. Four and one alleles were segregated in 77:4 and 143:1 ratios and considered duplex and triplex markers, respectively. Segregation ratios of remaining alleles were unexplainable. The results provide information about selection of crossing parents, estimation of seedling population optimal size, and promotion of efficient selection, which may be valuable for sugarcane breeders.

Phase angle as bioelectrical marker to identify elderly patients at risk of sarcopenia.

Science.gov (United States)

Basile, Claudia; Della-Morte, David; Cacciatore, Francesco; Gargiulo, Gaetano; Galizia, Gianluigi; Roselli, Mario; Curcio, Francesco; Bonaduce, Domenico; Abete, Pasquale

2014-10-01

Several markers have been associated with sarcopenia in the elderly, including bioelectrical indices. Phase angle (PhA) is an impedance parameter and it has been suggested as an indicator of cellular death. Thus, the relationship between PhA and muscle mass and strength was investigated in 207 consecutively elderly participants (mean age 76.2±6.7years) admitted for multidimensional geriatric evaluation. Muscle strength by grip strength using a hand-held dynamometer and muscle mass was measured by bioimpedentiometer. PhA was calculated directly with its arctangent (resistance/reactance×180°/π). Linear relationship among muscular mass and strength and with clinical and biochemical parameters, including PhA at uni- and multivariate analysis were performed. Linear regression analysis demonstrated that lower level of PhA is associated with reduction in grip strength (y=3.16+0.08x; r=0.49; pelderly subjects and it may be considered a good bioelectrical marker to identify elderly patients at risk of sarcopenia. Copyright © 2014 Elsevier Inc. All rights reserved.

Public involvement in pharmacogenomics research: a national survey on public attitudes towards pharmacogenomics research and the willingness to donate DNA samples to a DNA bank in Japan.

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Kobayashi, Eriko; Satoh, Nobunori

2009-11-01

To assess the attitudes of the Japanese general public towards pharmacogenomics research and a DNA bank for identifying genomic markers associated with ADRs and their willingness to donate DNA samples, we conducted a national survey for 1,103 Japanese adults from the general public, not a patient population. The response rate was 36.8%. The majority of the respondents showed a positive attitude towards pharmacogenomics research (81.0%) and a DNA bank (70.4%). Considering fictitious clinical situations such as taking medications and experiencing ADRs, the willingness to donate DNA samples when experiencing ADRs (61.7%) was higher than when taking medications (45.3%). Older generations were significantly associated with a decreased willingness to donate (OR = 0.45, CI 0.28-0.72 in 50s. OR = 0.49, CI: 0.31-0.77 in 60s). Positive attitudes towards pharmacogenomics research, a DNA bank, blood/bone marrow/organ donation were significantly associated with an increased willingness. However, the respondents had the following concerns regarding a DNA bank: the confidentiality of their personal information, the manner by which research results were utilized and simply the use of their own DNA for research. In order to attain public understanding to overcome these concerns, a process of public awareness should be put into place to emphasize the beneficial aspects of identifying genomic markers associated with ADRs and to address these concerns raised in our study. Further study is needed to assess the willingness of actual patients taking medications in real situations, since the respondents in our study were from the general public, not a patient population, and their willingness was assessed on the condition of assuming that they were patients taking medications.

Data analytics identify glycated haemoglobin co-markers for type 2 diabetes mellitus diagnosis.

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Jelinek, Herbert F; Stranieri, Andrew; Yatsko, Andrew; Venkatraman, Sitalakshmi

2016-08-01

Glycated haemoglobin (HbA1c) is being more commonly used as an alternative test for the identification of type 2 diabetes mellitus (T2DM) or to add to fasting blood glucose level and oral glucose tolerance test results, because it is easily obtained using point-of-care technology and represents long-term blood sugar levels. HbA1c cut-off values of 6.5% or above have been recommended for clinical use based on the presence of diabetic comorbidities from population studies. However, outcomes of large trials with a HbA1c of 6.5% as a cut-off have been inconsistent for a diagnosis of T2DM. This suggests that a HbA1c cut-off of 6.5% as a single marker may not be sensitive enough or be too simple and miss individuals at risk or with already overt, undiagnosed diabetes. In this study, data mining algorithms have been applied on a large clinical dataset to identify an optimal cut-off value for HbA1c and to identify whether additional biomarkers can be used together with HbA1c to enhance diagnostic accuracy of T2DM. T2DM classification accuracy increased if 8-hydroxy-2-deoxyguanosine (8-OhdG), an oxidative stress marker, was included in the algorithm from 78.71% for HbA1c at 6.5% to 86.64%. A similar result was obtained when interleukin-6 (IL-6) was included (accuracy=85.63%) but with a lower optimal HbA1c range between 5.73 and 6.22%. The application of data analytics to medical records from the Diabetes Screening programme demonstrates that data analytics, combined with large clinical datasets can be used to identify clinically appropriate cut-off values and identify novel biomarkers that when included improve the accuracy of T2DM diagnosis even when HbA1c levels are below or equal to the current cut-off of 6.5%. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isolation and identification of female DNA on postcoital penile swabs.

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Cina, S J; Collins, K A; Pettenati, M J; Fitts, M

2000-06-01

After sexual assault, cells originating from the assailant may be recovered from the victim. Through polymerase chain reaction (PCR)-based technology, positive scientific identification of the assailant may be made from these cells. Described is a prospective study describing a method for positively identifying cells from a female sex partner obtained from postcoital swabs of the penis of the male sex partner. Swabs were taken from the penis of a man at 1- to 24-hour intervals after coitus. DNA was isolated from each swab through standard organic extraction methods. The presence of female DNA was detected using the gender-specific amelogenin marker. Extracted DNA was amplified for eight different genetic loci using the Promega PowerPlex kit (Promega) and Amplitaq Gold (Perkin Elmer). Amplified samples were electrophoresed on precast sequencing gels (Hitachi) and were analyzed fluorescently using Hitachi's FMBIO 2 fluorescent scanner and software. Each sample obtained from a penile swab or condom was compared to male and female buccal controls. Female DNA was isolated from all postcoital penile swabs as determined by exclusive amplification of the X-chromosome specific 212 base pair amelogenin marker. In all cases, scientific identification of the female DNA from the swabs was determined by coamplification of eight STR loci (PowerPlex) and was compared to female and male control profiles. Cells shed from a female victim during sexual intercourse can be retrieved from the penis of a male offender after sexual intercourse during a 1- to 24-hour postcoital interval. DNA can be extracted from these cells and can be used to scientifically identify the female sexual participant through PCR-based technology. It is suggested that penile swabs be taken from alleged perpetrators of sexual assaults to associate them with a female victim.

Selection of microsatellite markers for bladder cancer diagnosis without the need for corresponding blood.

Directory of Open Access Journals (Sweden)

Angela A G van Tilborg

Full Text Available Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.

Characterization and cross-amplification of microsatellite markers in four species of anemonefish (Pomacentridae, Amphiprion spp.)

KAUST Repository

Bonin, Mary C.

2015-04-09

Anemonefish are iconic symbols of coral reefs and have become model systems for research on larval dispersal and population connectivity in coral reef fishes. Here we present 24 novel microsatellite markers across four species of anemonefish and also test 35 previously published markers for cross-amplification on two anemonefish species in order to facilitate further research on their population genetics and phylogenetics. Novel loci were isolated from sequences derived from microsatellite-enriched or 454 GS-FLX shotgun sequence libraries developed using congeneric DNA. Primer testing successfully identified 15 new microsatellite loci for A. percula, 4 for A. melanopus, 3 for A. akindynos, and 2 for A. omanensis. These novel microsatellite loci were polymorphic with a mean of 10 ± 1.6 SE (standard error) alleles per locus and an average observed heterozygosity of 0.647 ± 0.032 SE. Reliable cross-amplification of 12 and 26 of the 35 previously published Amphiprion markers was achieved for A. melanopus and A. akindynos, respectively, suggesting that the use of markers developed from the DNA of congeners can provide a quick and cost-effective alternative to the isolation of new loci. Together, the markers presented here provide an important resource for ecological, evolutionary, and conservation genetic research on anemonefishes that will inform broader conservation and management actions for coral reef fishes. © 2015 Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg

Characterization and cross-amplification of microsatellite markers in four species of anemonefish (Pomacentridae, Amphiprion spp.)

KAUST Repository

Bonin, Mary C.; Saenz Agudelo, Pablo; Harrison, Hugo B.; Nanninga, Gerrit B.; Van Der Meer, Martin H.; Mansour, Hicham; Perumal, Sadhasivam; Jones, Geoffrey P.; Berumen, Michael L.

2015-01-01

Anemonefish are iconic symbols of coral reefs and have become model systems for research on larval dispersal and population connectivity in coral reef fishes. Here we present 24 novel microsatellite markers across four species of anemonefish and also test 35 previously published markers for cross-amplification on two anemonefish species in order to facilitate further research on their population genetics and phylogenetics. Novel loci were isolated from sequences derived from microsatellite-enriched or 454 GS-FLX shotgun sequence libraries developed using congeneric DNA. Primer testing successfully identified 15 new microsatellite loci for A. percula, 4 for A. melanopus, 3 for A. akindynos, and 2 for A. omanensis. These novel microsatellite loci were polymorphic with a mean of 10 ± 1.6 SE (standard error) alleles per locus and an average observed heterozygosity of 0.647 ± 0.032 SE. Reliable cross-amplification of 12 and 26 of the 35 previously published Amphiprion markers was achieved for A. melanopus and A. akindynos, respectively, suggesting that the use of markers developed from the DNA of congeners can provide a quick and cost-effective alternative to the isolation of new loci. Together, the markers presented here provide an important resource for ecological, evolutionary, and conservation genetic research on anemonefishes that will inform broader conservation and management actions for coral reef fishes. © 2015 Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg

HIV-1 DNA predicts disease progression and post-treatment virological control

Science.gov (United States)

Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

2014-01-01

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

Co-expression modules construction by WGCNA and identify potential prognostic markers of uveal melanoma.

Science.gov (United States)

Wan, Qi; Tang, Jing; Han, Yu; Wang, Dan

2018-01-01

Uveal melanoma is an aggressive cancer which has a high percentage recurrence and with a worse prognosis. Identify the potential prognostic markers of uveal melanoma may provide information for early detection of recurrence and treatment. RNA sequence data of uveal melanoma and patient clinic traits were obtained from The Cancer Genome Atlas (TCGA) database. Co-expression modules were built by weighted gene co -expression network analysis (WGCNA) and applied to investigate the relationship underlying modules and clinic traits. Besides, functional enrichment analysis was performed on these co-expression genes from interested modules. First, using WGCNA, identified 21 co-expression modules were constructed by the 10975 genes from the 80 human uveal melanoma samples. The number of genes in these modules ranged from 42 to 5091. Found four co -expression modules significantly correlated with three clinic traits (status, recurrence and recurrence Time). Module red, and purple positively correlated with patient's life status and recurrence Time. Module green positively correlates with recurrence. The result of functional enrichment analysis showed that the module magenta was mainly enriched genetic material assemble processes, the purple module was mainly enriched in tissue homeostasis and melanosome membrane and the module red was mainly enriched metastasis of cell, suggesting its critical role in the recurrence and development of the disease. Additionally, identified the hug gene (top connectivity with other genes) in each module. The hub gene SLC17A7, NTRK2, ABTB1 and ADPRHL1 might play a vital role in recurrence of uveal melanoma. Our findings provided the framework of co-expression gene modules of uveal melanoma and identified some prognostic markers might be detection of recurrence and treatment for uveal melanoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification and characterization of a RAPD-PCR marker for distinguishing Asian and North American gypsy moths

Science.gov (United States)

K.J. Garner; J.M. Slavicek

1996-01-01

The recent introduction of the Asian gypsy moth (Lymantria dispar L.) into North America has necessitated the development of genetic markers to distinguish Asian moths from the established North American population, which originated in Europe. We used RAPD-PCR to identify a DNA length polymorphism that is diagnostic for the two moth strains. The...

Cortical GABA markers identify a molecular subtype of psychotic and bipolar disorders.

Science.gov (United States)

Volk, D W; Sampson, A R; Zhang, Y; Edelson, J R; Lewis, D A

2016-09-01

Deficits in gamma aminobutyric acid (GABA) neuron-related markers, including the GABA-synthesizing enzyme GAD67, the calcium-binding protein parvalbumin, the neuropeptide somatostatin, and the transcription factor Lhx6, are most pronounced in a subset of schizophrenia subjects identified as having a 'low GABA marker' (LGM) molecular phenotype. Furthermore, schizophrenia shares degrees of genetic liability, clinical features and cortical circuitry abnormalities with schizoaffective disorder and bipolar disorder. Therefore, we determined the extent to which a similar LGM molecular phenotype may also exist in subjects with these disorders. Transcript levels for GAD67, parvalbumin, somatostatin, and Lhx6 were quantified using quantitative PCR in prefrontal cortex area 9 of 184 subjects with a diagnosis of schizophrenia (n = 39), schizoaffective disorder (n = 23) or bipolar disorder (n = 35), or with a confirmed absence of any psychiatric diagnoses (n = 87). A blinded clustering approach was employed to determine the presence of a LGM molecular phenotype across all subjects. Approximately 49% of the subjects with schizophrenia, 48% of the subjects with schizoaffective disorder, and 29% of the subjects with bipolar disorder, but only 5% of unaffected subjects, clustered in the cortical LGM molecular phenotype. These findings support the characterization of psychotic and bipolar disorders by cortical molecular phenotype which may help elucidate more pathophysiologically informed and personalized medications.

Prevalence of Helicobacter pylori vacuolating cytotoxin and its allelic mosaicism as a predictive marker for Iranian dyspeptic patients

DEFF Research Database (Denmark)

Mohammadi, M; Oghalaie, A; Mohajerani, N

2003-01-01

can serve as screening markers for such a population, H. pylori strains were isolated from one hundred and thirty two dyspeptic patients. H. pylori genomic DNA was extracted and underwent PCR-amplification for the cytotoxin alleles. Genotyping of the signal sequence region of the vacA gene identified...

Use of toxicogenomics for identifying genetic markers of pulmonary oedema

International Nuclear Information System (INIS)

Balharry, Dominique; Oreffo, Victor; Richards, Roy

2005-01-01

This study was undertaken primarily to identify genetic markers of oedema and inflammation. Mild pulmonary injury was induced following the instillation of the oedema-producing agent, bleomycin (0.5 units). Oedema was then confirmed by conventional toxicology (lavage protein levels, free cell counts and lung/body weight ratios) and histology 3 days post-bleomycin instillation.The expression profile of 1176 mRNA species was determined for bleomycin-exposed lung (Clontech Atlas macroarray, n = 9). To obtain pertinent results from these data, it was necessary to develop a simple, effective method for bioinformatic analysis of altered gene expression. Data were log 10 transformed followed by global normalisation. Differential gene expression was accepted if: (a) genes were statistically significant (P ≤ 0.05) from a two-tailed t test; (b) genes were consistently outside a two standard deviation (SD) range from control levels. A combination of these techniques identified 31 mRNA transcripts (approximately 3%) which were significantly altered in bleomycin treated tissue. Of these genes, 26 were down-regulated whilst only five were up-regulated. Two distinct clusters were identified, with 17 genes classified as encoding hormone receptors, and nine as encoding ion channels. Both these clusters were consistently down-regulated.The magnitude of the changes in gene expression were quantified and confirmed by Q-PCR (n = 6), validating the macroarray data and the bioinformatic analysis employed.In conclusion, this study has developed a suitable macroarray analysis procedure and provides the basis for a better understanding of the gene expression changes occurring during the early phase of drug-induced pulmonary oedema

Algorithm for post-clustering curation of DNA amplicon data yields reliable biodiversity estimates

DEFF Research Database (Denmark)

Froslev, Tobias Guldberg; Kjoller, Rasmus; Bruun, Hans Henrik

2017-01-01

by high-throughput sequencing of amplified marker genes. LULU identifies errors by combining sequence similarity and co-occurrence patterns. To validate the LULU method, we use a unique data set of high quality survey data of vascular plants paired with plant ITS2 metabarcoding data of DNA extracted from...

RAPD-SCAR marker and genetic relationship analysis of three Demodex species (Acari: Demodicidae).

Science.gov (United States)

Zhao, Ya-E; Wu, Li-Ping

2012-06-01

For a long time, classification of Demodex mites has been mainly based on their hosts and phenotype characteristics. The study was the first to conduct molecular identification and genetic relationship analysis for six isolates of three Demodex species by random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) marker. Totally, 239 DNA fragments were amplified from six Demodex isolates with 10 random primers in RAPD, of which 165 were polymorphic. Using a single primer, at least five fragments and at most 40 in the six isolates were amplified, whereas within a single isolate, a range of 35-49 fragments were amplified. DNA fingerprints of primers CZ 1-9 revealed intra- and interspecies difference in six Demodex isolates, whereas primer CZ 10 only revealed interspecies difference. The genetic distance and dendrogram showed the intraspecific genetic distances were closer than the interspecific genetic distances. The interspecific genetic distances of Demodex folliculorum and Demodex canis (0.7931-0.8140) were shorter than that of Demodex brevis and D. canis (0.8182-0.8987). The RAPD-SCAR marker displayed primer CZ 10 could be applied to identify the three Demodex species. The 479-bp fragment was specific for D. brevis, and the 261-bp fragment was specific for D. canis. The conclusion was that the RAPD-SCAR multi-marker was effective in molecular identification of three Demodex species. The genetic relationship between D. folliculorum and D. canis was nearer than that between D. folliculorum and D. brevis.

A Fluorescent Tile DNA Diagnocode System for In Situ Rapid and Selective Diagnosis of Cytosolic RNA Cancer Markers

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Park, Kyung Soo; Shin, Seung Won; Jang, Min Su; Shin, Woojung; Yang, Kisuk; Min, Junhong; Cho, Seung-Woo; Oh, Byung-Keun; Bae, Jong Wook; Jung, Sunghwan; Choi, Jeong-Woo; Um, Soong Ho

2015-01-01

Accurate cancer diagnosis often requires extraction and purification of genetic materials from cells, and sophisticated instrumentations that follow. Otherwise in order to directly treat the diagnostic materials to cells, multiple steps to optimize dose concentration and treatment time are necessary due to diversity in cellular behaviors. These processes may offer high precision but hinder fast analysis of cancer, especially in clinical situations that need rapid detection and characterization of cancer. Here we present a novel fluorescent tile DNA nanostructure delivered to cancer cytosol by employing nanoparticle technology. Its structural anisotropicity offers easy manipulation for multifunctionalities, enabling the novel DNA nanostructure to detect intracellular cancer RNA markers with high specificity within 30 minutes post treatment, while the nanoparticle property bypasses the requirement of treatment optimization, effectively reducing the complexity of applying the system for cancer diagnosis. Altogether, the system offers a precise and rapid detection of cancer, suggesting the future use in the clinical fields. PMID:26678430

The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.

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Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Kaleci, Shaniko; Mandrioli, Mauro; Pellacani, Giovanni; Ozben, Tomris; Depenni, Roberta; Bianchi, Giampaolo; Pirola, Giacomo Maria; Tomasi, Aldo

2018-04-01

Circulating cell-free tumor DNA (cfDNA) is of crucial interest in oncology. cfDNA constitutes a potential prognostic and therapeutic marker for different solid tumors and can be used in the diagnostic and therapeutic management of cancer patients for which nowadays there are no valid laboratory markers. In the present study, the quality and quantity of the cfDNA were assessed by different quantification procedures, in order to identify the potential applications of these techniques in the preliminary cfDNA quantification. Qubit with single (ss) and double strand (ds) DNA assay kits, NanoDrop and quantitative Real Time PCR (qPCR), were adopted to assess the cfDNA in the blood samples of 18 melanoma patients, 67 prostate cancer patients and 15 healthy controls. The quantification by NanoDrop (average value 8.48ng/μl, 95% confidence limit (CL)=7.23-9.73), Qubit ssDNA (average value 23.08ng/μl, CL=19.88-26.28), dsDNA (average value 4.32ng/μl, CL=3.52-5.12) assay kits and qPCR (average value 0.39ng/μl, CL=0.31-0.47) revealed differences among the four procedures. Qubit 2.0 ss-DNA kit gave higher cfDNA concentration values for all the samples analyzed. In detail, Qubit ssDNA assay revealed higher sensitivity in the quantification of small amounts of pure ss-DNA and ds-DNA, while NanoDrop allowed the assessment of the purity of cfDNA samples. The NanoDrop and Qubit 2.0 measurements were analyzed in order to define their correlation with qPCR cfDNA assessment, showing good correlation values with the qPCR that should be considered the "gold standard". In our proposal, the sequential combination of NanoDrop and Qubit ssDNA methods should be adopted for a cost-effective preliminary assessment of total circulating cfDNA in melanoma and prostate cancer patients, and only discordant values should undergo qPCR assessment. Copyright © 2018 Elsevier B.V. All rights reserved.

Identifying Quantitative Trait Loci (QTLs) and Developing Diagnostic Markers Linked to Orange Rust Resistance in Sugarcane (Saccharum spp.).

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Yang, Xiping; Islam, Md S; Sood, Sushma; Maya, Stephanie; Hanson, Erik A; Comstock, Jack; Wang, Jianping

2018-01-01

Sugarcane ( Saccharum spp.) is an important economic crop, contributing up to 80% of table sugar used in the world and has become a promising feedstock for biofuel production. Sugarcane production has been threatened by many diseases, and fungicide applications for disease control have been opted out for sustainable agriculture. Orange rust is one of the major diseases impacting sugarcane production worldwide. Identifying quantitative trait loci (QTLs) and developing diagnostic markers are valuable for breeding programs to expedite release of superior sugarcane cultivars for disease control. In this study, an F 1 segregating population derived from a cross between two hybrid sugarcane clones, CP95-1039 and CP88-1762, was evaluated for orange rust resistance in replicated trails. Three QTLs controlling orange rust resistance in sugarcane (qORR109, qORR4 and qORR102) were identified for the first time ever, which can explain 58, 12 and 8% of the phenotypic variation, separately. We also characterized 1,574 sugarcane putative resistance ( R ) genes. These sugarcane putative R genes and simple sequence repeats in the QTL intervals were further used to develop diagnostic markers for marker-assisted selection of orange rust resistance. A PCR-based Resistance gene-derived maker, G1 was developed, which showed significant association with orange rust resistance. The putative QTLs and marker developed in this study can be effectively utilized in sugarcane breeding programs to facilitate the selection process, thus contributing to the sustainable agriculture for orange rust disease control.

germplasm using ISSR markers and their relationships

African Journals Online (AJOL)

STORAGESEVER

2008-12-17

Dec 17, 2008 ... (MAS) is the current trend in 'Modern Agriculture'. These. DNA markers allow the construction of ... are inherited in Mendelian fashion and are scored as dominant markers (Ratnaparkhe et al., 1998) ... ISSR amplified PCR products were resolved on 2% agarose gel in. 1X TBE buffer (89 mM Tris-Hcl, pH 8.3, ...

Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data

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Tsang Percy CK

2006-08-01

Full Text Available Abstract Background Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. Methods For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using χ2 or Fisher's exact test. Results APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2, endometrial cancer (CASP8, CDH13, hMLH1 and p73, and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1. The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer to the highest 56

Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data

International Nuclear Information System (INIS)

Yang, Hui-Juan; Liu, Vincent WS; Wang, Yue; Tsang, Percy CK; Ngan, Hextan YS

2006-01-01

Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers) was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using χ 2 or Fisher's exact test. APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2), endometrial cancer (CASP8, CDH13, hMLH1 and p73), and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1). The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers) were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer) to the highest 56.7% (DAPK in cervical cancer). Aberrant methylation

Comparison of genetic detection efficiency of different markers ...

African Journals Online (AJOL)

STORAGESEVER

2009-06-03

Jun 3, 2009 ... Chinese native sheep populations, Hu sheep, Tong sheep, Small-tailed Han sheep and Tan sheep were used to study the efficiency of genetic markers. The genetic markers used in this study include morphological and ecological indices, blood protein enzyme, microsatellite DNA and the combination of.

Genome-scale analysis of aberrant DNA methylation in colorectal cancer

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Hinoue, Toshinori; Weisenberger, Daniel J.; Lange, Christopher P.E.; Shen, Hui; Byun, Hyang-Min; Van Den Berg, David; Malik, Simeen; Pan, Fei; Noushmehr, Houtan; van Dijk, Cornelis M.; Tollenaar, Rob A.E.M.; Laird, Peter W.

2012-01-01

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation–based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAFV600E mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent ∼7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing. PMID:21659424

A study of autoimmune markers in hepatitis C infection.

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Agarwal, N; Handa, R; Acharya, S K; Wali, J P; Dinda, A K; Aggarwal, P

2001-05-01

Hepatitis C virus (HCV) infection is associated with several autoimmune markers. Despite HCV being common in India, no information on this aspect is available. This study was undertaken to ascertain the frequency and clinical significance of autoimmune markers like rheumatoid factor (RF), antinuclear antibodies (ANA), antibodies to double stranded deoxyribonucleic acid (dsDNA), anti neutrophil cytoplasmic antibody (ANCA), anti smooth muscle antibodies (ASMA), anti liver kidney microsomal 1 antibodies (anti LKM1), anti gastric parietal cell antibodies (anti GPCA), anti mitochondrial antibodies (AMA), anti cardiolipin antibodies (ACL) and cryoglobulins in HCV infection and to determine the effect of treatment on these markers. Twenty five patients with chronic hepatitis C and 25 healthy controls were studied. Cryoglobulins were detected by cryoprecipitation, RF by latex agglutination, anti dsDNA and ACL by ELISA while indirect immunofluorescence was used to detect all other autoantibodies. Eighteen patients (72%) demonstrated autoimmune markers. RF, cryoglobulins and anti LKM1 antibodies were the most frequently detected markers (in 32% patients each). ASMA, perinuclear ANCA (pANCA), ANA and anti GPCA were seen in 24, 20, 12 and 4 per cent patients respectively. None of the patients exhibited ACL, AMA or antibodies to dsDNA. No antibodies were detected in healthy controls. Sixty per cent of the patients had rheumatological symptoms. Of the seven patients followed up after treatment with alpha interferon, only two exhibited persistence of RF, while symptoms and other markers disappeared. Rheumatological symptoms and autoimmune markers are common in HCV infection and are usually overlooked. Patients with unexplained joint pains and/or palpable purpura should be screened for HCV. Further studies are needed to delineate fully the link between infection and autoimmunity.

Potential of Start Codon Targeted (SCoT) markers for DNA fingerprinting of newly synthesized tritordeums and their respective parents.

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Cabo, Sandra; Ferreira, Luciana; Carvalho, Ana; Martins-Lopes, Paula; Martín, António; Lima-Brito, José Eduardo

2014-08-01

Hexaploid tritordeum (H(ch)H(ch)AABB; 2n = 42) results from the cross between Hordeum chilense (H(ch)H(ch); 2n = 14) and cultivated durum wheat (Triticum turgidum ssp. durum (AABB; 2n = 28). Morphologically, tritordeum resembles the wheat parent, showing promise for agriculture and wheat breeding. Start Codon Targeted (SCoT) polymorphism is a recently developed technique that generates gene-targeted markers. Thus, we considered it interesting to evaluate its potential for the DNA fingerprinting of newly synthesized hexaploid tritordeums and their respective parents. In this study, 60 SCoT primers were tested, and 18 and 19 of them revealed SCoT polymorphisms in the newly synthesized tritordeum lines HT27 and HT22, respectively, and their parents. An analysis of the presence/absence of bands among tritordeums and their parents revealed three types of polymorphic markers: (i) shared by tritordeums and one of their parents, (ii) exclusively amplified in tritordeums, and (iii) exclusively amplified in the parents. No polymorphism was detected among individuals of each parental species. Three SCoT markers were exclusively amplified in tritordeums of lines HT22 and HT27, being considered as polyploidization-induced rearrangements. About 70% of the SCoT markers of H. chilense origin were not transmitted to the allopolyploids of both lines, and most of the SCoTs scored in the newly synthesized allopolyploids originated from wheat, reinforcing the potential use of tritordeum as an alternative crop.

Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

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Chiari Ylenia

2005-03-01

Full Text Available Abstract Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%, with low degrees of pairwise haplotype divergence within populations (0–1%. Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.

How effective are DNA barcodes in the identification of African rainforest trees?

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Parmentier, Ingrid; Duminil, Jérôme; Kuzmina, Maria; Philippe, Morgane; Thomas, Duncan W; Kenfack, David; Chuyong, George B; Cruaud, Corinne; Hardy, Olivier J

2013-01-01

DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species. We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95-100% success), but less for species identification (71-88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84-90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA. Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications.

Conversion of the random amplified polymorphic DNA (RAPD ...

African Journals Online (AJOL)

Conversion of the random amplified polymorphic DNA (RAPD) marker UBC#116 linked to Fusarium crown and root rot resistance gene (Frl) into a co-dominant sequence characterized amplified region (SCAR) marker for marker-assisted selection of tomato.

Identifying the true oysters (Bivalvia: Ostreidae) with mitochondrial phylogeny and distance-based DNA barcoding.

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Liu, Jun; Li, Qi; Kong, Lingfeng; Yu, Hong; Zheng, Xiaodong

2011-09-01

Oysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. As a useful tool for molecular species identification, DNA barcoding might offer significant potential for oyster identification and taxonomy. This study used two mitochondrial fragments, cytochrome c oxidase I (COI) and the large ribosomal subunit (16S rDNA), to assess whether oyster species could be identified by phylogeny and distance-based DNA barcoding techniques. Relationships among species were estimated by the phylogenetic analyses of both genes, and then pairwise inter- and intraspecific genetic divergences were assessed. Species forming well-differentiated clades in the molecular phylogenies were identical for both genes even when the closely related species were included. Intraspecific variability of 16S rDNA overlapped with interspecific divergence. However, average intra- and interspecific genetic divergences for COI were 0-1.4% (maximum 2.2%) and 2.6-32.2% (minimum 2.2%), respectively, indicating the existence of a barcoding gap. These results confirm the efficacy of species identification in oysters via DNA barcodes and phylogenetic analysis. © 2011 Blackwell Publishing Ltd.

Fine mapping of the rice Bph1 gene, which confers resistance to the brown planthopper (Nilaparvata lugens stal), and development of STS markers for marker-assisted selection.

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Cha, Young-Soon; Ji, Hyeonso; Yun, Doh-Won; Ahn, Byoung-Ohg; Lee, Myung Chul; Suh, Seok-Cheol; Lee, Chun Seok; Ahn, Eok Keun; Jeon, Yong-Hee; Jin, Il-Doo; Sohn, Jae-Keun; Koh, Hee-Jong; Eun, Moo-Young

2008-08-31

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.

Variabilidade genética de etnovariedades de mandioca, avaliada por marcadores de DNA Genetic diversity of cassava folk varieties assessed by DNA markers

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Gilda Santos Mühlen

2000-06-01

reference. Among these, 38 were bitter varieties and 17 sweet. Three different types of DNA markers were used: RAPD (randomly amplified polymorphic DNA, AFLP (amplified fragment length polymorphism and microsatellites. Analysis of the results consisted of a description of band patterns, a calculation of similarity indexes (Nei & Li and a principal coordinate analysis (PCoA for each marker type. Heterozygosity, diversity indexes (DI, Weir and genetic differentiation coefficients (G ST were calculated for the microsatellite loci.Genetic variability was more concentrated within regions, then among regions (G ST = 0.07. Mean heterozygosity was 56%. Mean similarity indexes were dependent on the marker used: S = 0.89 for RAPD, S = 0.85 for AFLP and S = 0.59 for microsatellites. PCoA analysis revealed groups, distinguishing bitter from sweet varieties.

Uniparental genetic markers in South Amerindians

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Rafael Bisso-Machado

2012-01-01

Full Text Available A comprehensive review of uniparental systems in South Amerindians was undertaken. Variability in the Y-chromosome haplogroups were assessed in 68 populations and 1,814 individuals whereas that of Y-STR markers was assessed in 29 populations and 590 subjects. Variability in the mitochondrial DNA (mtDNA haplogroup was examined in 108 populations and 6,697 persons, and sequencing studies used either the complete mtDNA genome or the highly variable segments 1 and 2. The diversity of the markers made it difficult to establish a general picture of Y-chromosome variability in the populations studied. However, haplogroup Q1a3a* was almost always the most prevalent whereas Q1a3* occurred equally in all regions, which suggested its prevalence among the early colonizers. The STR allele frequencies were used to derive a possible ancient Native American Q-clade chromosome haplotype and five of six STR loci showed significant geographic variation. Geographic and linguistic factors moderately influenced the mtDNA distributions (6% and 7%, respectively and mtDNA haplogroups A and D correlated positively and negatively, respectively, with latitude. The data analyzed here provide rich material for understanding the biological history of South Amerindians and can serve as a basis for comparative studies involving other types of data, such as cultural data.

Molecular Markers Useful for Intraspecies Subtyping and Strain Differentiation of Dermatophytes.

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Mochizuki, Takashi; Takeda, Kiminobu; Anzawa, Kazushi

2017-02-01

Dermatophytosis is a very common skin disorder and the most frequent infection encountered by practicing dermatologists. The identification, pathogenicity, biology, and epidemiology of dermatophytes, the causative agents of dermatophytosis, are of interest for both dermatologists and medical mycologists. Recent advances in molecular methods have provided new techniques for identifying dermatophytes, including intraspecies variations. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections, recurrence or reinfection after treatment, and analysis of strain virulence and drug resistance. This review describes molecular methods of intraspecies subtyping and strain differentiation, including analyses of mitochondrial DNA and non-transcribed spacer regions of ribosomal RNA genes, random amplification of polymorphic DNA, and microsatellite markers, along with their advantages and limitations.

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population

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Haryana Sofia M

2011-05-01

Full Text Available Abstract Background Undifferentiated nasopharyngeal carcinoma (NPC is strongly related to Epstein-Barr virus (EBV infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection. Methods Nasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP. Ten TSGs were studied. Results NPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%. High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1 with detection rate of 98%. Conclusion Multiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.

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

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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.

An efficient identification strategy of clonal tea cultivars using long-core motif SSR markers.

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Wang, Rang Jian; Gao, Xiang Feng; Kong, Xiang Rui; Yang, Jun

2016-01-01

Microsatellites, or simple sequence repeats (SSRs), especially those with long-core motifs (tri-, tetra-, penta-, and hexa-nucleotide) represent an excellent tool for DNA fingerprinting. SSRs with long-core motifs are preferred since neighbor alleles are more easily separated and identified from each other, which render the interpretation of electropherograms and the true alleles more reliable. In the present work, with the purpose of characterizing a set of core SSR markers with long-core motifs for well fingerprinting clonal cultivars of tea (Camellia sinensis), we analyzed 66 elite clonal tea cultivars in China with 33 initially-chosen long-core motif SSR markers covering all the 15 linkage groups of tea plant genome. A set of 6 SSR markers were conclusively selected as core SSR markers after further selection. The polymorphic information content (PIC) of the core SSR markers was >0.5, with ≤5 alleles in each marker containing 10 or fewer genotypes. Phylogenetic analysis revealed that the core SSR markers were not strongly correlated with the trait 'cultivar processing-property'. The combined probability of identity (PID) between two random cultivars for the whole set of 6 SSR markers was estimated to be 2.22 × 10(-5), which was quite low, confirmed the usefulness of the proposed SSR markers for fingerprinting analyses in Camellia sinensis. Moreover, for the sake of quickly discriminating the clonal tea cultivars, a cultivar identification diagram (CID) was subsequently established using these core markers, which fully reflected the identification process and provided the immediate information about which SSR markers were needed to identify a cultivar chosen among the tested ones. The results suggested that long-core motif SSR markers used in the investigation contributed to the accurate and efficient identification of the clonal tea cultivars and enabled the protection of intellectual property.

Identifying DNA Methylation Biomarkers for Non-Endoscopic Detection of Barrett’s Esophagus

Science.gov (United States)

Moinova, Helen R.; LaFramboise, Thomas; Lutterbaugh, James D.; Chandar, Apoorva Krishna; Dumot, John; Faulx, Ashley; Brock, Wendy; De la Cruz Cabrera, Omar; Guda, Kishore; Barnholtz-Sloan, Jill S.; Iyer, Prasad G.; Canto, Marcia I.; Wang, Jean S.; Shaheen, Nicholas J.; Thota, Prashanti N.; Willis, Joseph E.; Chak, Amitabh; Markowitz, Sanford D.

2018-01-01

We report a biomarker-based non-endoscopic method for detecting Barrett’s esophagus (BE), based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma (EAC). Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve (AUC)=0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals, who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 minutes. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE. PMID:29343623

Replication stress, DNA damage signalling, and cytomegalovirus infection in human medulloblastomas

DEFF Research Database (Denmark)

Bartek, Jiri; Fornara, Olesja; Merchut-Maya, Joanna Maria

2017-01-01

suppressor activation, across our medulloblastoma cohort. Most tumours showed high proliferation (Ki67 marker), variable oxidative DNA damage (8-oxoguanine lesions) and formation of 53BP1 nuclear 'bodies', the latter indicating (along with ATR-Chk1 signalling) endogenous replication stress. The bulk...... cell replication stress and DNA repair. Collectively, the scenario we report here likely fuels genomic instability and evolution of medulloblastoma resistance to standard-of-care genotoxic treatments....... eight established immunohistochemical markers to assess the status of the DDR machinery, we found pronounced endogenous DNA damage signalling (γH2AX marker) and robust constitutive activation of both the ATM-Chk2 and ATR-Chk1 DNA damage checkpoint kinase cascades, yet unexpectedly modest p53 tumour...

An overview of molecular marker methods for plants | Semagn ...

African Journals Online (AJOL)

The development and use of molecular markers for the detection and exploitation of DNA polymorphism is one of the most significant developments in the field of molecular genetics. The presence of various types of molecular markers, and differences in their principles, methodologies, and applications require careful ...

A quick DNA extraction protocol: Without liquid nitrogen in ambient ...

African Journals Online (AJOL)

Marker assisted selection is an effective technique for quality traits selection in breeding program which are impossible by visual observation. Marker assisted selection in early generation requires rapid DNA extraction protocol for large number of samples in a low cost approach. A rapid and inexpensive DNA extraction ...

Detection of Ribosomal DNA Sequence Polymorphisms in the Protist Plasmodiophora brassicae for the Identification of Geographical Isolates

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Rawnak Laila

2017-01-01

Full Text Available Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae. It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates, collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY. The nuclear ribosomal DNA (rDNA sequence of P. brassicae, comprising 6932 base pairs (bp, was cloned and sequenced and found to include the small subunits (SSUs and a large subunit (LSU, internal transcribed spacers (ITS1 and ITS2, and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs, oligonucleotide polymorphisms, and insertions/deletions (InDels. A combination of three markers was able to distinguish the geographical isolates into two groups.