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Sample records for chloroplast dna sequences

  1. Cloning and molecular genetics analyses of Deschampsia antarctica Desv. chloroplast and mitochondrial DNA sequence

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    O.P. Savchuk

    2012-03-01

    Full Text Available Chloroplast and mitochondrial DNA sequences of Deschampsia antarctica were studied. We had made comparison analysis with completely sequenced genomes of other temperateness plants to find homology.

  2. An improved chloroplast DNA extraction procedure for whole plastid genome sequencing.

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    Chao Shi

    Full Text Available BACKGROUND: Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs. METHODOLOGY/PRINCIPAL FINDINGS: We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40-50% cpDNA purity is achieved with our method. CONCLUSION: Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

  3. Complete genome sequence of chloroplast DNA (cpDNA) of Chlorella sorokiniana.

    Science.gov (United States)

    Orsini, Massimiliano; Cusano, Roberto; Costelli, Cristina; Malavasi, Veronica; Concas, Alessandro; Angius, Andrea; Cao, Giacomo

    2016-01-01

    The complete chloroplast genome sequence of Chlorella sorokiniana strain (SAG 111-8 k) is presented in this study. The genome consists of circular chromosomes of 109,811 bp, which encode a total of 109 genes, including 74 proteins, 3 rRNAs and 31 tRNAs. Moreover, introns are not detected and all genes are present in single copy. The overall AT contents of the C. sorokiniana cpDNA is 65.9%, the coding sequence is 59.1% and a large inverted repeat (IR) is not observed.

  4. An optimized chloroplast DNA extraction protocol for grasses (Poaceae proves suitable for whole plastid genome sequencing and SNP detection.

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    Kerstin Diekmann

    Full Text Available BACKGROUND: Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge. CONCLUSIONS/SIGNIFICANCE: The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus x giganteus, Panicoideae. The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.

  5. Conflict amongst chloroplast DNA sequences obscures the phylogeny of a group of Asplenium ferns.

    Science.gov (United States)

    Shepherd, Lara D; Holland, Barbara R; Perrie, Leon R

    2008-07-01

    A previous study of the relationships amongst three subgroups of the Austral Asplenium ferns found conflicting signal between the two chloroplast loci investigated. Because organelle genomes like those of chloroplasts and mitochondria are thought to be non-recombining, with a single evolutionary history, we sequenced four additional chloroplast loci with the expectation that this would resolve these relationships. Instead, the conflict was only magnified. Although tree-building analyses favoured one of the three possible trees, one of the alternative trees actually had one more supporting site (six versus five) and received greater support in spectral and neighbor-net analyses. Simulations suggested that chance alone was unlikely to produce strong support for two of the possible trees and none for the third. Likelihood permutation tests indicated that the concatenated chloroplast sequence data appeared to have experienced recombination. However, recombination between the chloroplast genomes of different species would be highly atypical, and corollary supporting observations, like chloroplast heteroplasmy, are lacking. Wider taxon sampling clarified the composition of the Austral group, but the conflicting signal meant analyses (e.g., morphological evolution, biogeographic) conditional on a well-supported phylogeny could not be performed.

  6. The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

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    Lemieux Claude

    2006-02-01

    Full Text Available Abstract Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae, in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR featuring an inverted rRNA operon and a small single-copy (SSC region containing 14 genes normally found in the large single-copy (LSC region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage. Results The 151,933 bp IR-containing genome of Oltmannsiellopsis differs considerably from Pseudendoclonium and other chlorophyte cpDNAs in intron content and gene order, but shares close similarities with its ulvophyte homologue at the levels of quadripartite architecture, gene content and gene density. Oltmannsiellopsis cpDNA encodes 105 genes, contains five group I introns, and features many short dispersed repeats. As in Pseudendoclonium cpDNA, the rRNA genes in the IR are transcribed toward the single copy region featuring the genes typically found in the ancestral LSC region, and the opposite single copy region harbours genes characteristic of both the ancestral SSC and LSC regions. The 52 genes that were transferred from the ancestral LSC to SSC region include 12 of those observed in Pseudendoclonium cpDNA. Surprisingly, the overall gene organization of

  7. A hybrid swarm population of Pinus densiflora x P. sylvestris hybrids inferred from sequence analysis of chloroplast DNA and morphological characters

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    To confirm a hybrid swarm population of Pinus densiflora × P. sylvestris in Jilin, China and to study whether shoot apex morphology of 4-year old seedlings can be correlated with the sequence of a chloroplast DNA simple sequence repeat marker (cpDNA SSR), needles and seeds from P. densiflora, P. syl...

  8. Direct chloroplast sequencing: comparison of sequencing platforms and analysis tools for whole chloroplast barcoding.

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    Marta Brozynska

    Full Text Available Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina and Ion Torrent (Life Technology sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare. Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.

  9. Phylogeny of Ptychostomum (Bryaceae, Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

    Institute of Scientific and Technical Information of China (English)

    Chen-Ying WANG; Jian-Cheng ZHAO

    2009-01-01

    The phylogeny of Ptychostomum was first undertaken based on analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal (nr) DNA and by combining data from nrDNA ITS and chloroplast DNA rps4 sequences. Maximum parsimony, maximum likelihood, and Bayesian analyses all support the conclu-sion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii (Schwagr.) J. R. Spence (= Bryum funkii Schwagr.) is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwagr., and Bryum pallens Sw. to Ptychostomum.

  10. A hybrid swarm population of Pinus densiflora × P. sylvestris inferred from sequence analysis of chloroplast DNA and morphological characters

    Institute of Scientific and Technical Information of China (English)

    Young Hee Joung; Jerry L.Hill; Jung Oh Hyun; Ding Mu; Juchun Luo; Do Hyung Lee; Takayuki Kawahara; Jeung Keun Suh; Mark S.Roh

    2013-01-01

    To confirm a hybrid swarm population ofPinus densiflora × P.sylvestris in Jilin,China,we used needles and seeds from P.densiflora,P.sylvestris,and P.densiflora × P.sylvestris collected from natural stands or experimental stations to study whether shoot apex morphology of 4-year old seedlings can be correlated with the sequence of a chloroplast DNA simple sequence repeat marker (cpDNA SSRs).Total genomic DNA was extracted and subjected to sequence analysis of the pine cpDNA SSR marker Pt15169.Results show that morphological characters from 4-year old seedlings did not correlate with sequence variants of this marker.Marker haplotypes from all P.sylvestris trees had a CTAT element that was absent from all sampled P.densiflora trees.However,both haplotype classes involving this insertion/deletion element were found in a P.densiflora × P.sylvestris population and its seedling progeny.It was concluded that the P.densiflora × P.sylvestris accessions sampled from Jilin,China resulted from bi-directional crosses,as evidenced by both species' cpDNA haplotypes within the hybrid swarm population.

  11. Phylogeography of Cyananthus delavayi (Campanulaceae) in Hengduan Mountains inferred from variation in nuclear and chloroplast DNA sequences

    Institute of Scientific and Technical Information of China (English)

    Guo-Dong LI; Liang-Liang YUE; Hang SUN; Zi-Gang QIAN

    2012-01-01

    Phylogeographic studies on alpine plants endemic to the Hengduan Mountains of the southeastern Qinghai-Tibet Plateau are still limited in number.In this study,we used sequence variation of one nuclear gene (ncpGS,which encodes the chloroplastic glutamine synthetase) and in two chloroplast DNA segments to investigate the phylogeographic structure and population demographic history of Cyananthus delavayi,a narrow-range species endemic to this region.We identified eight chlorotypes and 16 nuclear genotypes in a survey of 10 populations sampled throughout the range of the species.The results of both phylogenetic and network analyses suggested that the genealogical relationships of both chlorotypes and nuclear genotypes showed a clear geographical correlation.High total genetic diversity,low levels of within-population diversity,and strong population differentiation (chloroplast DNA:hT =0.827,hS =0.087,NST =0.899,GST =0.895; nuclear DNA:hT =0.910,hS =0.348,NST =0719,GST =0.618) were identified.Based on the mismatch distribution analyses,no evidence of recent demographic population expansion was found for this species.Nested clade analyses of both chlorotypes and nuclear genotypes indicated that restricted gene flow resulting from isolation by distance and allopatrc fragmentation were likely to have been the major processes that shaped their present-day spatial distribution.Our dating of the genetic divergences between three major geographic lineages suggested that the largest glaciation of the early Quaternary developed in the Qinghai-Tibet Plateau and mountainous isolation may have together led to deep intraspecific vicariance within this species.

  12. Phylogeography of Spiraea alpina (Rosaceae) in the Qinghai-Tibetan Plateau inferred from chloroplast DNA sequence variations

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    Fa-Qi ZHANG; Qing-Bo GAO; De-Jun ZHANG; Yi-Zhong DUAN; Yin-Hu LI; Peng-Cheng FU; Rui XING; Khan GULZAR; Shi-Long CHEN

    2012-01-01

    The aim of the present study was to investigate the phylogeographic patterns of Spiraea alpina (Rosaceae) and clarify its response to past climatic changes in the climate-sensitive Qinghai-Tibetan Plateau (QTP).We sequenced a chloroplast DNA fragment (trnL-trnF) from 528 individuals representing 43 populations.We identified 10 haplotypes,which were tentatively divided into three groups.These haplotypes or groups were distributed in the different regions of the QTP.Only half the populations were fixed by a single haplotype,whereas the others contained two or more.In the central and eastern regions,adjacent populations at the local scale shared the same haplotype.Our phylogeographic analyses suggest that this alpine shrub survived in multiple refugia during the Last Glacial Maximum and that earlier glaciations may have trigged deep intraspecific divergences.Post-glacial expansions occurred only within populations or across multiple populations within a local range.The findings of the present study together with previous phylogeographic reports suggest that evolutionary histories of plants in the QTP are complex and variable depending on the species investigated.

  13. Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae: Unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer

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    Lemieux Claude

    2008-06-01

    Full Text Available Abstract Background To gain insight into the branching order of the five main lineages currently recognized in the green algal class Chlorophyceae and to expand our understanding of chloroplast genome evolution, we have undertaken the sequencing of chloroplast DNA (cpDNA from representative taxa. The complete cpDNA sequences previously reported for Chlamydomonas (Chlamydomonadales, Scenedesmus (Sphaeropleales, and Stigeoclonium (Chaetophorales revealed tremendous variability in their architecture, the retention of only few ancestral gene clusters, and derived clusters shared by Chlamydomonas and Scenedesmus. Unexpectedly, our recent phylogenies inferred from these cpDNAs and the partial sequences of three other chlorophycean cpDNAs disclosed two major clades, one uniting the Chlamydomonadales and Sphaeropleales (CS clade and the other uniting the Oedogoniales, Chaetophorales and Chaetopeltidales (OCC clade. Although molecular signatures provided strong support for this dichotomy and for the branching of the Oedogoniales as the earliest-diverging lineage of the OCC clade, more data are required to validate these phylogenies. We describe here the complete cpDNA sequence of Oedogonium cardiacum (Oedogoniales. Results Like its three chlorophycean homologues, the 196,547-bp Oedogonium chloroplast genome displays a distinctive architecture. This genome is one of the most compact among photosynthetic chlorophytes. It has an atypical quadripartite structure, is intron-rich (17 group I and 4 group II introns, and displays 99 different conserved genes and four long open reading frames (ORFs, three of which are clustered in the spacious inverted repeat of 35,493 bp. Intriguingly, two of these ORFs (int and dpoB revealed high similarities to genes not usually found in cpDNA. At the gene content and gene order levels, the Oedogonium genome most closely resembles its Stigeoclonium counterpart. Characters shared by these chlorophyceans but missing in members

  14. Phylogeography of Thlaspi arvense (Brassicaceae in China Inferred from Chloroplast and Nuclear DNA Sequences and Ecological Niche Modeling

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    Miao An

    2015-06-01

    Full Text Available Thlaspi arvense is a well-known annual farmland weed with worldwide distribution, which can be found from sea level to above 4000 m high on the Qinghai-Tibetan Plateau (QTP. In this paper, a phylogeographic history of T. arvense including 19 populations from China was inferred by using three chloroplast (cp DNA segments (trnL-trnF, rpl32-trnL and rps16 and one nuclear (n DNA segment (Fe-regulated transporter-like protein, ZIP. A total of 11 chloroplast haplotypes and six nuclear alleles were identified, and haplotypes unique to the QTP were recognized (C4, C5, C7 and N4. On the basis of molecular dating, haplotypes C4, C5 and C7 have separated from others around 1.58 Ma for cpDNA, which corresponds to the QTP uplift. In addition, this article suggests that the T. arvense populations in China are a mixture of diverged subpopulations as inferred by hT/vT test (hT ≤ vT, cpDNA and positive Tajima’s D values (1.87, 0.05 < p < 0.10 for cpDNA and 3.37, p < 0.01 for nDNA. Multimodality mismatch distribution curves and a relatively large shared area of suitable environmental conditions between the Last Glacial Maximum (LGM as well as the present time recognized by MaxEnt software reject the sudden expansion population model.

  15. Intraspecific sequence variation of chloroplast DNA among the component species of deciduous broad-leaved forests in Japan.

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    Iwasaki, Takaya; Aoki, Kyoko; Seo, Akihiro; Murakami, Noriaki

    2006-09-01

    To select appropriate plant materials for a phylogeography of deciduous broad-leaved forests in Japan, we surveyed intraspecific chloroplast DNA variation in 34 species found in these forests. A relatively large number of intraspecific cpDNA variations were detected in ten species: Carpinus japonica (nucleotide diversity pi=0.00083), C. laxiflora (pi=0.00221), Magnolia obovata (pi=0.00134), Lindera triloba (pi=0.00255), L. obtusiloba (pi=0.00289), Pourthiaea villosa var. leavis (pi=0.00263), Acer japonicum (pi=0.00170), A. micranthum (pi=0.00237), Euonymus oxyphyllus (pi=0.00322) and Styrax obassia (pi=0.00100).

  16. Local repeat sequence organization of an intergenic spacer in the chloroplast genome of Chlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: a complex mode of “copy-choice replication”?

    Indian Academy of Sciences (India)

    Mahendra D Wagle; Subhojit Sen; Basuthkar J Rao

    2001-12-01

    Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA of Chlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt+) in a cross between a pair of polymorphic interfertile strains of Chlamydomonas (C. reinhardtii and C. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, ``P2” (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3′ end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, ``P2” seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely ``sequence-scrambled” product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a ``unique” new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a ``complex path” of copy-choice replication.

  17. Phylogeny and divergence of Chinese Angiopteridaceae based on chloroplast DNA sequence data (rbcL and trnL-F)

    Institute of Scientific and Technical Information of China (English)

    LI ChunXiang; LU ShuGang

    2007-01-01

    Marattioid ferns are an ancient lineage of primitive vascular plants that first appeared in the middle Carboniferous. Extant members are almost exclusively restricted to tropical regions, and the species-rich family Angiopteridaceae are limited in their distribution to the eastern hemisphere; relationships within the group are currently vague. Here the phylogenetic relationship between Angiopteris Hoffm. and Archangiopteris Christ et Gies. was evaluated based on the sequence analysis of chloroplast rbcL gene and trnL-F intergenic spacer with MEGA2 and MrBayes v3.0b4. On the basis of the phylogenetic pattern and fossil record, we further estimated the divergence time for the two genera. The phylogenetic trees revealed that all species of Angiopteris and Archangiopteris in this study formed a monophyletic group with strong statistical support, but the relationship between the two genera remained unresolved based on individual sequence analysis. On the other hand, the sequence analyses of combined data set revealed that Archangiopteris species diverged first, indicating that Archangiopteris may not be a direct derivative as traditionally assumed. The clade of Angiopteris and Archangiopteris appears to have diversified in the late Oligocene (≈26 Ma) based on the molecular estimate. Thus, the evolutionary history of extant Angiopteris and Archangiopteris has been characterized by ancient origin and recent diversification, and these groups are not relic and endangered lineages as traditionally considered.

  18. Mitochondrial and chloroplast DNA based phylogeny of Pelargonium (Geraniaceae)

    NARCIS (Netherlands)

    Bakker, F.T.; Culham, A.; Pankhurst, C.E.; Gibby, M.

    2000-01-01

    Overall phylogenetic relationships within the genus Pelargonium (Geraniaceae) were inferred based on DNA sequences from mitochondrial(mt)-encoded nad1 b/c exons and from chloroplast(cp)-encoded trnL (UAA) 5' exon-trnF (GAA) exon regions using two species of Geranium and Sarcocaulon vanderetiae as ou

  19. Molecular phylogeography and evolutionary history of Picea likiangensis in the Qinghai-Tibetan Plateau inferred from mitochondrial and chloroplast DNA sequence variation

    Institute of Scientific and Technical Information of China (English)

    Jia-Bin ZOU; Xiao-Li PENG; Long LI; Jian-Quan LIU; Georg MIEHE; Lars OPGENOORTH

    2012-01-01

    The aim of the present study was to examine the phylogeographic and evolutionary history of Picea likiangensis,a dominant species of the conifer forests in the eastern declivity of the Qinghai-Tibetan Plateau.We collected 422 individuals from 42 natural populations of three major varieties classified under this species.In conifers,mitochondrial (mt) DNA and chloroplast (cp) DNA dispersed by seeds or pollen experience very different levels of gene flow.To this end,we examined the sequence variation of two mtDNA fragments (nad5 intron 1 and nadl intron b/c) and three cpDNA fragments (trnL-trnF,trnS-trnG and nadhK/C).We found that cpDNA probably introgressed from P.purpurea into remote populations of P.likiangensis through long-distance dispersal.Multiple refugia seem to have been maintained for P.likiangensis during the Last Glacial Maximum because the cpDNA and mtDNA haplotypes recovered were fixed in the different regions.Postglacial expansions were only detected at the distributional edges of this species where a single cpDNA or mtDNA haplotype was fixed in adjacent populations.However,genetic imprints of postglacial expansions from these two sets of markers were different in the western and southeastern regions,which may result from the long-distance dispersal of the cpDNA,as well as its fast lineage sorting during intraspecific divergences.Analysis of molecular variance further suggested that genetic differentiation between the three varieties is higher at cpDNA markers than at mtDNA markers,which supports the previous viewpoint that cpDNA markers with a high rate of gene flow may be more effective in delimitating closely related taxa.Together,the results of the present study highlight the evolutionary complexity of a widely distributed species owing to interactions among local and edge expansion,long-distance dispersal,and intraspecific divergences at two sets of DNA genomes with different rates of gene flow.

  20. Chloroplast DNA analysis of Tunisian cork oak populations (Quercus suber L.): sequence variations and molecular evolution of the trnL (UAA)-trnF (GAA) region.

    Science.gov (United States)

    Abdessamad, A; Baraket, G; Sakka, H; Ammari, Y; Ksontini, M; Hannachi, A Salhi

    2016-10-24

    Sequences of the trnL-trnF spacer and combined trnL-trnF region in chloroplast DNA of cork oak (Quercus suber L.) were analyzed to detect polymorphisms and to elucidate molecular evolution and demographic history. The aligned sequences varied in length and nucleotide composition. The overall ratio of transition/transversion (ti/tv) of 0.724 for the intergenic spacer and 0.258 for the pooled sequences were estimated, and indicated that transversions are more frequent than transitions. The molecular evolution and demographic history of Q. suber were investigated. Neutrality tests (Tajima's D and Fu and Li) ruled out the null hypothesis of a strictly neutral model, and Fu's Fs and Ramos-Onsins and Rozas' R2 confirmed the recent expansion of cork oak trees, validating its persistency in North Africa since the last glaciation during the Quaternary. The observed uni-modal mismatch distribution and the Harpending's raggedness index confirmed the demographic history model for cork oak. A phylogenetic dendrogram showed that the distribution of Q. suber trees occurs independently of geographical origin, the relief of the population site, and the bioclimatic stages. The molecular history and cytoplasmic diversity suggest that in situ and ex situ conservation strategies can be recommended for preserving landscape value and facing predictable future climatic changes.

  1. SEQUENCE POLYMORPHISMS OF FOUR CHLOROPLAST GENES IN FOUR ACACIA SPECIES

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    Anthonius Y.P.B.C. Widyatmoko

    2011-06-01

    Full Text Available Sequence polymorphisms among and within four Acacia species,  A. aulacocarpa, A. auriculiformis, A. crassicarpa, and A. mangium, were investigated using four chloroplast DNA genes (atpA, petA, rbcL, and rpoA. The phylogenetic relationship among these species is discussed in light of the results of the sequence information. No intraspecific sequence variation was found in the four genes of the four species, and a conservative rate of mutation of the chloroplast DNA genes was also confirmed in the Acacia species. In the atpA and petA of the four genes, all four species possessed identical sequences, and no sequence variation was found among the four Acacia species. In the rbcL and rpoA genes, however, sequence polymorphisms were revealed among these species. Acacia aulacocarpa and A. crassicarpa shared an identical sequence, and A. auriculiformis and A. mangium also showed no sequence variation.  The fact that A. mangium and A. auriculiformis shared identical sequences as did A. aulacocarpa and A. crassicarpa indicated that the two respective species were extremely closely related. Although a putative natural hybrid of A. aulacocarpa and A. auriculiformis has been reported, our results suggested that natural hybridization should be further verified using molecular markers.

  2. The historical demography and genetic variation of the endangered Cycas multipinnata (Cycadaceae in the red river region, examined by chloroplast DNA sequences and microsatellite markers.

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    Yi-Qing Gong

    Full Text Available Cycas multipinnata C.J. Chen & S.Y. Yang is a cycad endemic to the Red River drainage region that occurs under evergreen forest on steep limestone slopes in Southwest China and northern Vietnam. It is listed as endangered due to habitat loss and over-collecting for the ornamental plant trade, and only several populations remain. In this study, we assess the genetic variation, population structure, and phylogeography of C. multipinnata populations to help develop strategies for the conservation of the species. 60 individuals from six populations were used for chloroplast DNA (cpDNA sequencing and 100 individuals from five populations were genotyped using 17 nuclear microsatellites. High genetic differentiation among populations was detected, suggesting that pollen or seed dispersal was restricted within populations. Two main genetic clusters were observed in both the cpDNA and microsatellite loci, corresponding to Yunnan China and northern Vietnam. These clusters indicated low levels of gene flow between the regions since their divergence in the late Pleistocene, which was inferred from both Bayesian and coalescent analysis. In addition, the result of a Bayesian skyline plot based on cpDNA portrayed a long history of constant population size followed by a decline in the last 50,000 years of C. multipinnata that was perhaps affected by the Quaternary glaciations, a finding that was also supported by the Garza-Williamson index calculated from the microsatellite data. The genetic consequences produced by climatic oscillations and anthropogenic disturbances are considered key pressures on C. multipinnata. To establish a conservation management plan, each population of C. multipinnata should be recognized as a Management Unit (MU. In situ and ex situ actions, such as controlling overexploitation and creating a germplasm bank with high genetic diversity, should be urgently implemented to preserve this species.

  3. The Historical Demography and Genetic Variation of the Endangered Cycas multipinnata (Cycadaceae) in the Red River Region, Examined by Chloroplast DNA Sequences and Microsatellite Markers

    Science.gov (United States)

    Gong, Yi-Qing; Zhan, Qing-Qing; Nguyen, Khang Sinh; Nguyen, Hiep Tien; Wang, Yue-Hua; Gong, Xun

    2015-01-01

    Cycas multipinnata C.J. Chen & S.Y. Yang is a cycad endemic to the Red River drainage region that occurs under evergreen forest on steep limestone slopes in Southwest China and northern Vietnam. It is listed as endangered due to habitat loss and over-collecting for the ornamental plant trade, and only several populations remain. In this study, we assess the genetic variation, population structure, and phylogeography of C. multipinnata populations to help develop strategies for the conservation of the species. 60 individuals from six populations were used for chloroplast DNA (cpDNA) sequencing and 100 individuals from five populations were genotyped using 17 nuclear microsatellites. High genetic differentiation among populations was detected, suggesting that pollen or seed dispersal was restricted within populations. Two main genetic clusters were observed in both the cpDNA and microsatellite loci, corresponding to Yunnan China and northern Vietnam. These clusters indicated low levels of gene flow between the regions since their divergence in the late Pleistocene, which was inferred from both Bayesian and coalescent analysis. In addition, the result of a Bayesian skyline plot based on cpDNA portrayed a long history of constant population size followed by a decline in the last 50,000 years of C. multipinnata that was perhaps affected by the Quaternary glaciations, a finding that was also supported by the Garza-Williamson index calculated from the microsatellite data. The genetic consequences produced by climatic oscillations and anthropogenic disturbances are considered key pressures on C. multipinnata. To establish a conservation management plan, each population of C. multipinnata should be recognized as a Management Unit (MU). In situ and ex situ actions, such as controlling overexploitation and creating a germplasm bank with high genetic diversity, should be urgently implemented to preserve this species. PMID:25689828

  4. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  5. Phylogeography of an alpine plant (Bupleurum smithii, Apiaceae) endemic to the Qinghai-Tibetan Plateau and adjacent regions inferred from chloroplast DNA sequence variation

    Institute of Scientific and Technical Information of China (English)

    Cai ZHAO; Xiang-Guang MA; Qian-Long LIANG; Chang-Bao WANG; Xing-Jin HE

    2013-01-01

    To obtain a better understanding of how Quaternary climatic oscillations influenced range distributions and intraspecific split of alpine plants on the Qinghai-Tibetan Plateau (QTP) and in adjacent regions,we investigated the extant phylogeographical structure of Bupleurum smithii in this area based on 22 populations and 103 individuals spanning the entire distribution region of this species using chloroplast DNA sequences.Two major haplotype lineages were identified,and at least two corresponding glacial refugia maintaining in the northeastern and eastern edge of the QTP during the Last Glacial Maximum were revealed.Secondary contact between populations and efficient gene flow were also found between two major haplotype lineages.In addition,based on the geographic distribution of haplotypes,we found that populations on the platform derived from individuals that recolonized this area from refugia situated at the northeastem and eastern edges of the QTP,and that B.smithii recolonized from southern to northern China during inter-and post-glacial periods.

  6. Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Angelova, Angelina [University of Arizona; Park, Sang-Hycuk [University of Arizona; Kyndt, John [Bellevue University; Fitzsimmons, Kevin [University of Arizona; Brown, Judith K [University of Arizona

    2013-09-01

    With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

  7. Phylogeny and character evolution of the fern genus Tectaria (Tectariaceae) in the Old World inferred from chloroplast DNA sequences.

    Science.gov (United States)

    Ding, Hui-Hui; Chao, Yi-Shan; Callado, John Rey; Dong, Shi-Yong

    2014-11-01

    In this study we provide a phylogeny for the pantropical fern genus Tectaria, with emphasis on the Old World species, based on sequences of five plastid regions (atpB, ndhF plus ndhF-trnL, rbcL, rps16-matK plus matK, and trnL-F). Maximum parsimony, maximum likelihood, and Bayesian inference are used to analyze 115 individuals, representing ca. 56 species of Tectaria s.l. and 36 species of ten related genera. The results strongly support the monophyly of Tectaria in a broad sense, in which Ctenitopsis, Hemigramma, Heterogonium, Psomiocarpa, Quercifilix, Stenosemia, and Tectaridium should be submerged. Such broadly circumscribed Tectaria is supported by the arising pattern of veinlets and the base chromosome number (x=40). Four primary clades are well resolved within Tectaria, one from the Neotropic (T. trifoliata clade) and three from the Old World (T. subtriphylla clade, Ctenitopsis clade, and T. crenata clade). Tectaria crenata clade is the largest one including six subclades. Of the genera previously recognized as tectarioid ferns, Ctenitis, Lastreopsis, and Pleocnemia, are confirmed to be members in Dryopteridaceae; while Pteridrys and Triplophyllum are supported in Tectariaceae. To infer morphological evolution, 13 commonly used characters are optimized on the resulting phylogenetic trees and in result, are all homoplastic in Tectaria.

  8. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

    Science.gov (United States)

    Gaynor, J. J.

    1984-01-01

    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  9. The complete chloroplast genome sequence of Dianthus superbus var. longicalycinus.

    Science.gov (United States)

    Gurusamy, Raman; Lee, Do-Hyung; Park, SeonJoo

    2016-05-01

    The complete chloroplast genome (cpDNA) sequence of Dianthus superbus var. longicalycinus is an economically important traditional Chinese medicine was reported and characterized. The cpDNA of Dianthus superbus var. longicalycinus is 149,539 bp, with 36.3% GC content. A pair of inverted repeats (IRs) of 24,803 bp is separated by a large single-copy region (LSC, 82,805 bp) and a small single-copy region (SSC, 17,128 bp). It encodes 85 protein-coding genes, 36 tRNA genes and 8 rRNA genes. Of 129 individual genes, 13 genes encoded one intron and three genes have two introns.

  10. Complete sequencing of five araliaceae chloroplast genomes and the phylogenetic implications.

    Directory of Open Access Journals (Sweden)

    Rong Li

    Full Text Available BACKGROUND: The ginseng family (Araliaceae includes a number of economically important plant species. Previously phylogenetic studies circumscribed three major clades within the core ginseng plant family, yet the internal relationships of each major group have been poorly resolved perhaps due to rapid radiation of these lineages. Recent studies have shown that phyogenomics based on chloroplast genomes provides a viable way to resolve complex relationships. METHODOLOGY/PRINCIPAL FINDINGS: We report the complete nucleotide sequences of five Araliaceae chloroplast genomes using next-generation sequencing technology. The five chloroplast genomes are 156,333-156,459 bp in length including a pair of inverted repeats (25,551-26,108 bp separated by the large single-copy (86,028-86,566 bp and small single-copy (18,021-19,117 bp regions. Each chloroplast genome contains the same 114 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 80 protein coding genes. Gene size, content, and order, AT content, and IR/SC boundary structure are similar among all Araliaceae chloroplast genomes. A total of 140 repeats were identified in the five chloroplast genomes with palindromic repeat as the most common type. Phylogenomic analyses using parsimony, likelihood, and Bayesian inference based on the complete chloroplast genomes strongly supported the monophyly of the Asian Palmate group and the Aralia-Panax group. Furthermore, the relationships among the sampled taxa within the Asian Palmate group were well resolved. Twenty-six DNA markers with the percentage of variable sites higher than 5% were identified, which may be useful for phylogenetic studies of Araliaceae. CONCLUSION: The chloroplast genomes of Araliaceae are highly conserved in all aspects of genome features. The large-scale phylogenomic data based on the complete chloroplast DNA sequences is shown to be effective for the phylogenetic reconstruction of Araliaceae.

  11. Genome Sequences of Populus tremula Chloroplast and Mitochondrion: Implications for Holistic Poplar Breeding.

    Directory of Open Access Journals (Sweden)

    Birgit Kersten

    Full Text Available Complete Populus genome sequences are available for the nucleus (P. trichocarpa; section Tacamahaca and for chloroplasts (seven species, but not for mitochondria. Here, we provide the complete genome sequences of the chloroplast and the mitochondrion for the clones P. tremula W52 and P. tremula x P. alba 717-1B4 (section Populus. The organization of the chloroplast genomes of both Populus clones is described. A phylogenetic tree constructed from all available complete chloroplast DNA sequences of Populus was not congruent with the assignment of the related species to different Populus sections. In total, 3,024 variable nucleotide positions were identified among all compared Populus chloroplast DNA sequences. The 5-prime part of the LSC from trnH to atpA showed the highest frequency of variations. The variable positions included 163 positions with SNPs allowing for differentiating the two clones with P. tremula chloroplast genomes (W52, 717-1B4 from the other seven Populus individuals. These potential P. tremula-specific SNPs were displayed as a whole-plastome barcode on the P. tremula W52 chloroplast DNA sequence. Three of these SNPs and one InDel in the trnH-psbA linker were successfully validated by Sanger sequencing in an extended set of Populus individuals. The complete mitochondrial genome sequence of P. tremula is the first in the family of Salicaceae. The mitochondrial genomes of the two clones are 783,442 bp (W52 and 783,513 bp (717-1B4 in size, structurally very similar and organized as single circles. DNA sequence regions with high similarity to the W52 chloroplast sequence account for about 2% of the W52 mitochondrial genome. The mean SNP frequency was found to be nearly six fold higher in the chloroplast than in the mitochondrial genome when comparing 717-1B4 with W52. The availability of the genomic information of all three DNA-containing cell organelles will allow a holistic approach in poplar molecular breeding in the future.

  12. Chloroplast DNA evolution and phylogenetic relationships in Lycopersicon.

    Science.gov (United States)

    Palmer, J D; Zamir, D

    1982-08-01

    Chloroplast DNA was purified from 12 accessions that represent most of the species diversity in the genus Lycopersicon (family Solanaceae) and from 3 closely related species in the genus Solanum. Fragment patterns produced by digestion of these DNAs with 25 different restriction endonucleases were analyzed by agarose gel electrophoresis. In all 15 DNAs, a total of only 39 restriction site mutations were detected among 484 restriction sites surveyed, representing 2,800 base pairs of sequence information. This low rate of base sequence change is paralleled by an extremely low rate of convergent change in restriction sites; only 1 of the 39 mutations appears to have occurred independently in two different lineages. Parsimony analysis of shared mutations has allowed the construction of a maternal phylogeny for the 15 accessions. This phylogeny is generally consistent with relationships based on morphology and crossability but provides more detailed resolution at several places. All accessions within Lycopersicon form a coherent group, with two of the three species of Solanum as outside reference points. Chloroplast DNA analysis places S. pennellii firmly within Lycopersicon, confirming recent studies that have removed it from Solanum. Red-orange fruit color is shown to be a monophyletic trait in three species of Lycopersicon, including the cultivated tomato, L. esculentum. Analysis of six accessions within L. peruvianum reveals a limited amount of intraspecific polymorphism which, however, encompasses all the variation observed in L. chilense and L. chmielewskii. It is suggested that these latter two accessions be relegated to positions within the L. peruvianum complex.

  13. Identifying the North American plum species phylogenetic signal using nuclear, mitochondrial, and chloroplast DNA markers

    Science.gov (United States)

    Premise of the study: Prunus L. phylogeny has extensively studied using cpDNA sequences. CpDNA has a slow rate of evolution which is beneficial to determine species relationships at a deeper level. However, a limitation of the chloroplast based phylogenies is its transfer by interspecific hybridizat...

  14. The complete chloroplast genome sequence of Abies nephrolepis (Pinaceae: Abietoideae

    Directory of Open Access Journals (Sweden)

    Dong-Keun Yi

    2016-06-01

    Full Text Available The plant chloroplast (cp genome has maintained a relatively conserved structure and gene content throughout evolution. Cp genome sequences have been used widely for resolving evolutionary and phylogenetic issues at various taxonomic levels of plants. Here, we report the complete cp genome of Abies nephrolepis. The A. nephrolepis cp genome is 121,336 base pairs (bp in length including a pair of short inverted repeat regions (IRa and IRb of 139 bp each separated by a small single copy (SSC region of 54,323 bp (SSC and a large single copy region of 66,735 bp (LSC. It contains 114 genes, 68 of which are protein coding genes, 35 tRNA and four rRNA genes, six open reading frames, and one pseudogene. Seventeen repeat units and 64 simple sequence repeats (SSR have been detected in A. nephrolepis cp genome. Large IR sequences locate in 42-kb inversion points (1186 bp. The A. nephrolepis cp genome is identical to Abies koreana’s which is closely related to taxa. Pairwise comparison between two cp genomes revealed 140 polymorphic sites in each. Complete cp genome sequence of A. nephrolepis has a significant potential to provide information on the evolutionary pattern of Abietoideae and valuable data for development of DNA markers for easy identification and classification.

  15. Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes

    Directory of Open Access Journals (Sweden)

    Wolf Paul G

    2010-02-01

    Full Text Available Abstract Background Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant. Results The Tortula chloroplast genome is ~123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the ~71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered. Conclusions Although the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations.

  16. Chloroplast DNA phylogeography and cytotype geography in autopolyploid Plantago media

    NARCIS (Netherlands)

    Van Dijk, P.J.; Bakx-Schotman, Tanja

    1997-01-01

    In order to gain insight into the causes of parapatric diploid and tetraploid distributions in Plantago media chloroplast DNA (cpDNA) restriction site polymorphism was studied in 36 European populations. Parapatric distributions are often explained by adaptive differences between cytotypes to an und

  17. Complete Chloroplast Genome Sequence of Dendrobium nobile from Northeastern India

    Science.gov (United States)

    Parameswaran, Sriram; Sundar, Durai

    2016-01-01

    The orchid species Dendrobium nobile belonging to the family Orchidaceae and genus Dendrobium (a vast genus that encompasses nearly 1,200 species) has an herbal medicinal history of about 2000 years in east and south Asian countries. Here, we report the complete chloroplast genome sequence of D. nobile from northeastern India for the first time.

  18. Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba).

    Science.gov (United States)

    Kestermann, M; Neukirchen, S; Kloppstech, K; Link, G

    1998-06-01

    Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors. We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors. The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide. An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed. The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified. It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter. Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard. RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.

  19. Chloroplast DNA Diversity of Oak Species in Eastern Romania

    Directory of Open Access Journals (Sweden)

    Ioan Calin MOLDOVAN

    2010-12-01

    Full Text Available The chloroplast DNA of 34 sessile oak (Quercus petraea and 27 pedunculate oak (Q. robur populations covering the entire natural distribution of the two oak species in Eastern Romania was investigated using four large regions of the chloroplast genome by PCR and RFLP technique. A total of seven chloroplast DNA haplotypes sensu lato have been observed by analysing 305 mature trees. However, due to the high resolution of the electrophoresis method a total of 22 chloroplast variants could have been detected, with new mutations and fragment combinations in two of the amplified regions: psbC/trnD and trnT/trnF. All of the haplotypes belong to the phylogenetic lineages A and E, which originate from the Balkan Peninsula. Most of genetic diversity is distributed among populations (GST=0.779. The chloroplast DNA haplotypes are shared by the two oak species. Different dispersal abilities may explain the higher value of genetic differentiation among populations in sessile oak than in pedunculate oak.

  20. A set of 100 chloroplast DNA primer pairs to study population genetics and phylogeny in monocotylenons

    DEFF Research Database (Denmark)

    Scarcelli, Nora; Bernaud, Adeline; Eiserhardt, Wolf L.;

    2011-01-01

    Chloroplast DNA sequences are of great interest for population genetics and phylogenetic studies. However, only a small set of markers are commonly used. Most of them have been designed for amplification in a large range of Angiosperms and are located in the Large Single Copy (LSC). Here we...... developed a new set of 100 primer pairs optimized for amplification in Monocotyledons. Primer pairs amplify coding (exon) and non-coding regions (intron and intergenic spacer). They span the different chloroplast regions: 72 are located in the LSC, 13 in the Small Single Copy (SSC) and 15 in the Inverted...

  1. A fragment of chloroplast DNA was transferred horizontally, probably from non-eudicots, to mitochondrial genome of Phaseolus.

    Science.gov (United States)

    Woloszynska, Magdalena; Bocer, Tomasz; Mackiewicz, Pawel; Janska, Hanna

    2004-11-01

    The mitochondrial genomes of some Phaseolus species contain a fragment of chloroplast trnA gene intron, named pvs-trnA for its location within the Phaseolus vulgaris sterility sequence (pvs). The purpose of this study was to determine the type of transfer (intracellular or horizontal) that gave rise to pvs-trnA. Using a PCR approach we could not find the respective portion of the trnA gene as a part of pvs outside the Phaseolus genus. However, a BLAST search revealed longer fragments of trnA present in the mitochondrial genomes of some Citrus species, Helianthus annuus and Zea mays. Basing on the identity or near-identity between these mitochondrial sequences and their chloroplast counterparts we concluded that they had relocated from chloroplasts to mitochondria via recent, independent, intracellular DNA transfers. In contrast, pvs-trnA displayed a relatively higher sequence divergence when compared with its chloroplast counterpart from Phaseolus vulgaris. Alignment of pvs-trnA with corresponding trnA fragments from 35 plant species as well as phylogenetic analysis revealed that pvs-trnA grouped with non-eudicot sequences and was well separated from all Fabales sequences. In conclusion, we propose that pvs-trnA arose via horizontal transfer of a trnA intron fragment from chloroplast of a non-eudicot plant to Phaseolus mitochondria. This is the first example of horizontal transfer of a chloroplast sequence to the mitochondrial genome in higher plants.

  2. A Comparison of the First Two Sequenced Chloroplast Genomes in Asteraceae: Lettuce and Sunflower

    Energy Technology Data Exchange (ETDEWEB)

    Timme, Ruth E.; Kuehl, Jennifer V.; Boore, Jeffrey L.; Jansen, Robert K.

    2006-01-20

    Asteraceae is the second largest family of plants, with over 20,000 species. For the past few decades, numerous phylogenetic studies have contributed to our understanding of the evolutionary relationships within this family, including comparisons of the fast evolving chloroplast gene, ndhF, rbcL, as well as non-coding DNA from the trnL intron plus the trnLtrnF intergenic spacer, matK, and, with lesser resolution, psbA-trnH. This culminated in a study by Panero and Funk in 2002 that used over 13,000 bp per taxon for the largest taxonomic revision of Asteraceae in over a hundred years. Still, some uncertainties remain, and it would be very useful to have more information on the relative rates of sequence evolution among various genes and on genome structure as a potential set of phylogenetic characters to help guide future phylogenetic structures. By way of contributing to this, we report the first two complete chloroplast genome sequences from members of the Asteraceae, those of Helianthus annuus and Lactuca sativa. These plants belong to two distantly related subfamilies, Asteroideae and Cichorioideae, respectively. In addition to these, there is only one other published chloroplast genome sequence for any plant within the larger group called Eusterids II, that of Panax ginseng (Araliaceae, 156,318 bps, AY582139). Early chloroplast genome mapping studies demonstrated that H. annuus and L. sativa share a 22 kb inversion relative to members of the subfamily Barnadesioideae. By comparison to outgroups, this inversion was shown to be derived, indicating that the Asteroideae and Cichorioideae are more closely related than either is to the Barnadesioideae. Later sequencing study found that taxa that share this 22 kb inversion also contain within this region a second, smaller, 3.3 kb inversion. These sequences also enable an analysis of patterns of shared repeats in the genomes at fine level and of RNA editing by comparison to available EST sequences. In addition, since

  3. Characterization of the chloroplast genome sequence of oil palm (Elaeis guineensis Jacq.).

    Science.gov (United States)

    Uthaipaisanwong, P; Chanprasert, J; Shearman, J R; Sangsrakru, D; Yoocha, T; Jomchai, N; Jantasuriyarat, C; Tragoonrung, S; Tangphatsornruang, S

    2012-06-01

    Oil palm (Elaeis guineensis Jacq.) is an economically important crop, which is grown for oil production. To better understand the molecular basis of oil palm chloroplasts, we characterized the complete chloroplast (cp) genome sequence obtained from 454 pyrosequencing. The oil palm cp genome is 156,973 bp in length consisting of a large single-copy region of 85,192 bp flanked on each side by inverted repeats of 27,071 bp with a small single-copy region of 17,639 bp joining the repeats. The genome contains 112 unique genes: 79 protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. By aligning the cp genome sequence with oil palm cDNA sequences, we observed 18 non-silent and 10 silent RNA editing events among 19 cp protein-coding genes. Creation of an initiation codon by RNA editing in rpl2 has been reported in several monocots and was also found in the oil palm cp genome. Fifty common chloroplast protein-coding genes from 33 plant taxa were used to construct ML and MP phylogenetic trees. Their topologies are similar and strongly support for the position of E. guineensis as the sister of closely related species Phoenix dactylifera in Arecaceae (palm families) of monocot subtrees.

  4. Assembly of the Complete Sitka Spruce Chloroplast Genome Using 10X Genomics’ GemCode Sequencing Data

    Science.gov (United States)

    Coombe, Lauren; Jackman, Shaun D.; Yang, Chen; Vandervalk, Benjamin P.; Moore, Richard A.; Pleasance, Stephen; Coope, Robin J.; Bohlmann, Joerg; Holt, Robert A.; Jones, Steven J. M.; Birol, Inanc

    2016-01-01

    The linked read sequencing library preparation platform by 10X Genomics produces barcoded sequencing libraries, which are subsequently sequenced using the Illumina short read sequencing technology. In this new approach, long fragments of DNA are partitioned into separate micro-reactions, where the same index sequence is incorporated into each of the sequencing fragment inserts derived from a given long fragment. In this study, we exploited this property by using reads from index sequences associated with a large number of reads, to assemble the chloroplast genome of the Sitka spruce tree (Picea sitchensis). Here we report on the first Sitka spruce chloroplast genome assembled exclusively from P. sitchensis genomic libraries prepared using the 10X Genomics protocol. We show that the resulting 124,049 base pair long genome shares high sequence similarity with the related white spruce and Norway spruce chloroplast genomes, but diverges substantially from a previously published P. sitchensis- P. thunbergii chimeric genome. The use of reads from high-frequency indices enabled separation of the nuclear genome reads from that of the chloroplast, which resulted in the simplification of the de Bruijn graphs used at the various stages of assembly. PMID:27632164

  5. Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding.

    Directory of Open Access Journals (Sweden)

    Wenpan Dong

    Full Text Available BACKGROUND: At present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species. METHODOLOGY/PRINCIPAL FINDINGS: We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnS(UGA-trnG(UCC, petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnS(UGA-trnG(UCC, trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species. SIGNIFICANCE/CONCLUSIONS: Chloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve

  6. DNA sequences encoding erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, F.K.

    1987-10-27

    A purified and isolated DNA sequence is described consisting essentially of a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of that of erythropoietin to allow possession of the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells, and to increase hemoglobin synthesis or iron uptake.

  7. Identification of Orchidaceae species from Northern West of Syria based on chloroplast DNA.

    Science.gov (United States)

    Haider, N; Nabulsi, I; Kamary, Y

    2010-08-01

    The plant family Orchidaceae has a great economic value (ornamental and medical uses, beside the aromatic features). Traditionally, identification of orchid species has relied heavily on morphological features. These features, however, are either not variable enough between species or too plastic to be used for identification at the species level. DNA-based markers could be the alternative strategy towards an accurate and robust identification of those species. Since the chloroplast DNA has a lower level of evolution compared to the nuclear genome, an attempt was made in this study to investigate polymorphism in the chloroplast DNA among orchid species distributed in North-West region of Syria using Cleaved Amplified Polymorphic Sequence (CAPS) technique for developing markers for the diagnosis of targeted species. CAPS analysis was carried out on 34 orchid samples that represent all species observed in the region. Universal primers were used to amplify targeted chloroplast regions. Generated PCR products were digested with various restriction enzymes. CAPS results revealed high polymorphism among species examined. This polymorphism was suffiecient for the diagnosis of all of those species apart from five species (Ophrys fuciflora (one sample), Oph. bornmuelleri, Ophrys sp., Oph. scolopax and Oph. argolica). Availability of such species-specific markers would ensure more authentic identification of orchid species compared to morphological characters and can be regarded as a valuable tool to guide in conservation programs of orchid species in Syria. CAPS data generated were converted to an identification key for orchid species studied.

  8. CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences

    Directory of Open Access Journals (Sweden)

    Liu Chang

    2012-12-01

    Full Text Available Abstract Background The complete sequences of chloroplast genomes provide wealthy information regarding the evolutionary history of species. With the advance of next-generation sequencing technology, the number of completely sequenced chloroplast genomes is expected to increase exponentially, powerful computational tools annotating the genome sequences are in urgent need. Results We have developed a web server CPGAVAS. The server accepts a complete chloroplast genome sequence as input. First, it predicts protein-coding and rRNA genes based on the identification and mapping of the most similar, full-length protein, cDNA and rRNA sequences by integrating results from Blastx, Blastn, protein2genome and est2genome programs. Second, tRNA genes and inverted repeats (IR are identified using tRNAscan, ARAGORN and vmatch respectively. Third, it calculates the summary statistics for the annotated genome. Fourth, it generates a circular map ready for publication. Fifth, it can create a Sequin file for GenBank submission. Last, it allows the extractions of protein and mRNA sequences for given list of genes and species. The annotation results in GFF3 format can be edited using any compatible annotation editing tools. The edited annotations can then be uploaded to CPGAVAS for update and re-analyses repeatedly. Using known chloroplast genome sequences as test set, we show that CPGAVAS performs comparably to another application DOGMA, while having several superior functionalities. Conclusions CPGAVAS allows the semi-automatic and complete annotation of a chloroplast genome sequence, and the visualization, editing and analysis of the annotation results. It will become an indispensible tool for researchers studying chloroplast genomes. The software is freely accessible from http://www.herbalgenomics.org/cpgavas.

  9. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  10. In silico analysis of Simple Sequence Repeats from chloroplast genomes of Solanaceae species

    Directory of Open Access Journals (Sweden)

    Evandro Vagner Tambarussi

    2009-01-01

    Full Text Available The availability of chloroplast genome (cpDNA sequences of Atropa belladonna, Nicotiana sylvestris, N.tabacum, N. tomentosiformis, Solanum bulbocastanum, S. lycopersicum and S. tuberosum, which are Solanaceae species,allowed us to analyze the organization of cpSSRs in their genic and intergenic regions. In general, the number of cpSSRs incpDNA ranged from 161 in S. tuberosum to 226 in N. tabacum, and the number of intergenic cpSSRs was higher than geniccpSSRs. The mononucleotide repeats were the most frequent in studied species, but we also identified di-, tri-, tetra-, pentaandhexanucleotide repeats. Multiple alignments of all cpSSRs sequences from Solanaceae species made the identification ofnucleotide variability possible and the phylogeny was estimated by maximum parsimony. Our study showed that the plastomedatabase can be exploited for phylogenetic analysis and biotechnological approaches.

  11. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    Directory of Open Access Journals (Sweden)

    Oldenburg Delene J

    2007-03-01

    Full Text Available Abstract Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI, SYBR Green I (SG, SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content.

  12. Complete chloroplast genome sequence of a major invasive species, crofton weed (Ageratina adenophora.

    Directory of Open Access Journals (Sweden)

    Xiaojun Nie

    Full Text Available BACKGROUND: Crofton weed (Ageratina adenophora is one of the most hazardous invasive plant species, which causes serious economic losses and environmental damages worldwide. However, the sequence resource and genome information of A. adenophora are rather limited, making phylogenetic identification and evolutionary studies very difficult. Here, we report the complete sequence of the A. adenophora chloroplast (cp genome based on Illumina sequencing. METHODOLOGY/PRINCIPAL FINDINGS: The A. adenophora cp genome is 150, 689 bp in length including a small single-copy (SSC region of 18, 358 bp and a large single-copy (LSC region of 84, 815 bp separated by a pair of inverted repeats (IRs of 23, 755 bp. The genome contains 130 unique genes and 18 duplicated in the IR regions, with the gene content and organization similar to other Asteraceae cp genomes. Comparative analysis identified five DNA regions (ndhD-ccsA, psbI-trnS, ndhF-ycf1, ndhI-ndhG and atpA-trnR containing parsimony-informative characters higher than 2%, which may be potential informative markers for barcoding and phylogenetic analysis. Repeat structure, codon usage and contraction of the IR were also investigated to reveal the pattern of evolution. Phylogenetic analysis demonstrated a sister relationship between A. adenophora and Guizotia abyssinica and supported a monophyly of the Asterales. CONCLUSION: We have assembled and analyzed the chloroplast genome of A. adenophora in this study, which was the first sequenced plastome in the Eupatorieae tribe. The complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies within this invasive species and also within the Asteraceae family.

  13. Automated DNA Sequencing System

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  14. Expression of Amyloplast and Chloroplast DNA in Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ngernprasirtsiri, J; Macherel, D; Kobayashi, H; Akazawa, T

    1988-01-01

    Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

  15. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    Directory of Open Access Journals (Sweden)

    Zdepski Anna

    2011-05-01

    Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

  16. Chloroplast DNA Copy Number May Link to Sex Determination in Leucadendron (Proteaceae

    Directory of Open Access Journals (Sweden)

    MADE PHARMAWATI

    2009-03-01

    Full Text Available Leucadendron (Proteaceae is a South African genus, the flowers of which have become a popular item in the Australian cut-flower industry. All species are dioecious. In general the female flowers are the more desirable as cut flowers. The availability of a molecular marker linked to sex determination is therefore needed both to maximize the efficiency of breeding programs and to supply markets with flowers from the preferred sex. The polymerase chain reaction-based method of suppression subtractive hybridization (SSH combined with mirror orientation selection (MOS were applied in an attempt to identify genome differences between male and female plants of Leucadendron discolor. Screening of 416 clones from a male-subtracted genomic DNA library and 282 clones from a female-subtracted library identified 13 candidates for male-specific genomic fragments. Sequence analyses of the 13 candidate DNA fragments showed that they were fragments of the chloroplast DNA, raising the possibility that chloroplast DNA copy number is linked to sex determination in Leucadendron.

  17. The complete chloroplast genome sequence of Dendropanax morbifera (Léveillé).

    Science.gov (United States)

    Kim, Kyunghee; Lee, Sang-Choon; Yang, Tae-Jin

    2016-07-01

    The complete chloroplast genome sequence of Dendropanax morbifera, an economically and medicinally important endemic tree species in Korea, was obtained by de novo assembly with whole-genome sequence data and manual correction. A circular 156 366-bp chloroplast genome showed typical chloroplast genome structure comprising a large single copy region of 86 475 bp, a small single copy region of 18 125 bp, and a pair of inverted repeats of 25 883 bp. The chloroplast genome harbored 87 protein-coding genes. Phylogenetic analysis with the chloroplast genome revealed that D. morbifera is most closely related to Dendropanax dentiger, an evergreen tree species in China and Southeastern Asia.

  18. The Complete Chloroplast Genome of Capsicum annuum var. glabriusculum Using Illumina Sequencing

    Directory of Open Access Journals (Sweden)

    Sebastin Raveendar

    2015-07-01

    Full Text Available Chloroplast (cp genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs of 50,284 bp were separated by a small single copy (SSC; 18,948 bp and a large single copy (LSC; 87,446 bp. The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC; SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

  19. The complete chloroplast genome of Capsicum annuum var. glabriusculum using Illumina sequencing.

    Science.gov (United States)

    Raveendar, Sebastin; Na, Young-Wang; Lee, Jung-Ro; Shim, Donghwan; Ma, Kyung-Ho; Lee, Sok-Young; Chung, Jong-Wook

    2015-07-20

    Chloroplast (cp) genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum) using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs) of 50,284 bp were separated by a small single copy (SSC; 18,948 bp) and a large single copy (LSC; 87,446 bp). The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC; SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR) and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

  20. Topological incongruence between nuclear and chloroplast DNA trees suggesting hybridization in the urophyllum group of the genus Fagopyrum (Polygonaceae).

    Science.gov (United States)

    Nishimoto, Yuriko; Ohnishi, Ohmi; Hasegawa, Masami

    2003-04-01

    We performed phylogenetic analyses of Fagopyrum species in the urophyllum group based on nucleotide sequences of two nuclear genes, FLORICAULA/LEAFY (FLO/LFY) and AGAMOUS (AG), and three segments of chloroplast DNA (cpDNA), rbcL-accD, trnK intron, and trnC-rpoB spacer. The FLO/LFY and AG sequences turned out to be phylogenetically more informative at the intrageneric level than the cpDNA sequences. Congruence among these gene trees, inferred by a maximum-likelihood (ML) method, demonstrated that topologies were partially incongruent between the nuclear and chloroplast DNA phylogenies. The nuclear DNA sequence data supported a monophyletic relation of F. statice, F. gilesii, and F. jinshaense, whereas the former two species formed another monophyletic relation with the F. capillatum-F. gracilipes-F. gracilipedoides-F. rubifolium clade excluding F. jinshaense in the synthetic cpDNA phylogeny. In addition, two divergent sequences of FLO/LFY were found in F. rubifolium (tetraploid). One of these was sister to F. gracilipedoides and another was sister to F. statice, and a monophyletic relation of these two genes was rejected by a bootstrap analysis. These results suggest that hybridization may have occurred during diversification of Fagopyrum species in the urophyllum group, and that F. rubifolium is possibly allotetraploid species.

  1. Diversification and genetic differentiation of cultivated melon inferred from sequence polymorphism in the chloroplast genome

    OpenAIRE

    Tanaka, Katsunori; Akashi, Yukari; FUKUNAGA, Kenji; Yamamoto, Tatsuya; Aierken, Yasheng; Nishida, Hidetaka; Long, Chun Lin; Yoshino, Hiromichi; Sato, Yo-Ichiro; KATO, Kenji

    2013-01-01

    Molecular analysis encouraged discovery of genetic diversity and relationships of cultivated melon (Cucumis melo L.). We sequenced nine inter- and intra-genic regions of the chloroplast genome, about 5500 bp, using 60 melon accessions and six reference accessions of wild species of Cucumis to show intra-specific variation of the chloroplast genome. Sequence polymorphisms were detected among melon accessions and other Cucumis species, indicating intra-specific diversification of the chloroplas...

  2. Phylogenetic relationships in Nuphar (Nymphaeaceae): evidence from morphology, chloroplast DNA, and nuclear ribosomal DNA.

    Science.gov (United States)

    Padgett, D J; Les, D H; Crow, G E

    1999-09-01

    The genus Nuphar consists of yellow-flowered waterlilies and is widely distributed in north-temperate bodies of water. Despite regular taxonomic evaluation of these plants, no explicit phylogenetic hypotheses have been proposed for the genus. We investigated phylogenetic relationships in Nuphar using morphology and sequences of the chloroplast gene matK and of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Two major lineages within Nuphar are consistently resolved with the morphological and molecular data sets. One lineage comprises New World taxa and the other represents a primarily Old World lineage. Relationships within the major lineages were poorly resolved by morphology and ITS, yet certain relationships were elucidated by all analyses. Most notable is the strong support for a monophyletic lineage of dwarf taxa and the alliance of the North American N. microphylla with the Eurasian taxa. Minor discordance between the independent cladograms is accounted for by hybridization. The common taxonomic practice of uniting all North American and Eurasian taxa under one species is not supported phylogenetically.

  3. Molecular authentication of the medicinal herb Ruta graveolens (Rutaceae) and an adulterant using nuclear and chloroplast DNA markers.

    Science.gov (United States)

    Al-Qurainy, F; Khan, S; Tarroum, M; Al-Hemaid, F M; Ali, M A

    2011-11-10

    Dried parts of different plant species often look alike, especially in powdered form, making them very difficult to identify. Ruta graveolens, sold as a dried medicinal herb, can be adulterated with Euphorbia dracunculoides. The genomic DNA was isolated from the leaf powder (100 mg each) using the modified CTAB method. Internal transcribed spacer sequences of nuclear ribosomal DNA (nrDNA-ITS), and chloroplast spacer sequences (rpoB and rpoC1) are regarded as potential genes for plant DNA barcoding. We amplified and sequenced these spacer sequences and confirmed the sequences with a BLAST search. Sequence alignment was performed using ClustalX to look for differences in the sequences. A DNA marker was developed based on rpoB and rpoC1 of the nrDNA-ITS for the identification of the adulterant E. dracunculoides in samples of R. graveolens that are sold in local herbal markets. Sequence-characterized amplified region markers of 289 and 264 bp for R. graveolens and 424 bp for E. dracunculoides were developed from dissimilar sequences of this nrDNA-ITS to speed up the authentication process. This marker successfully distinguished these species in extracted samples with as little as 5 ng DNA/μL extract.

  4. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  5. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  6. Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes

    OpenAIRE

    Wolf Paul G; Everett Karin DE; Mandoli Dina F; Boore Jeffrey L; Kuehl Jennifer V; Mishler Brent D; Murdock Andrew G; Oliver Melvin J; Duffy Aaron M; Karol Kenneth G

    2010-01-01

    Abstract Background Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-t...

  7. The complete chloroplast genome sequence of sugar beet (Beta vulgaris ssp. vulgaris).

    Science.gov (United States)

    Li, Han; Cao, Hua; Cai, Yan-Fei; Wang, Ji-Hua; Qu, Su-Ping; Huang, Xing-Qi

    2014-06-01

    The complete nucleotide sequence of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome (cpDNA) was determined in this study. The cpDNA was 149,637 bp in length, containing a pair of 24,439 bp inverted repeat regions (IR), which were separated by small and large single copy regions (SSC and LSC) of 17,701 and 83,057 bp, respectively. 53.4% of the sugar beet cpDNA consisted of gene coding regions (protein coding and RNA genes). The gene content and relative positions of 113 individual genes (79 protein encoding genes, 30 tRNA genes, 4 rRNA genes) were almost identical to those of tobacco cpDNA. The overall AT contents of the sugar beet cpDNA were 63.6% and in the LSC, SSC and IR regions were 65.9%, 70.8% and 57.8%, respectively. Fifteen genes contained one intron, while three genes had two introns.

  8. A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited

    Energy Technology Data Exchange (ETDEWEB)

    Sutton, C.A.; Hanson, M.R. [Cornell Univ., Ithaca, NY (United States); Zoubenko, O.V.; Maliga, P. [State Univ. of New Jersey, Piscataway, NJ (United States)

    1995-03-01

    RNA editing occurs in two higher-plant organelles, chloroplasts, and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components. 33 refs., 5 figs.

  9. DNA sequencing by CE.

    Science.gov (United States)

    Karger, Barry L; Guttman, András

    2009-06-01

    Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA-sequencing methods have evolved from the labor-intensive slab gel electrophoresis, through automated multiCE systems using fluorophore labeling with multispectral imaging, to the "next-generation" technologies of cyclic-array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes were only possible with the advent of modern sequencing technologies that were a result of step-by-step advances with a contribution of academics, medical personnel and instrument companies. While next-generation sequencing is moving ahead at breakneck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of CE in DNA sequencing based in part of several of our articles in this journal.

  10. Phylogeny of Pelargonium (Geraniaceae) based on DNA sequences from three genomes

    NARCIS (Netherlands)

    Bakker, F.T.; Culham, A.; Hettiarachi, P.; Touloumendidou, T.; Gibby, M.

    2004-01-01

    Phylogenetic hypotheses for the largely South African genus Pelargonium L'Hér. (Geraniaceae) were derived based on DNA sequence data from nuclear, chloroplast and mitochondrial encoded regions. The datasets were unequally represented and comprised cpDNA trnL-F sequences for 152 taxa, nrDNA ITS seque

  11. A set of 100 chloroplast DNA primer pairs to study population genetics and phylogeny in monocotyledons.

    Directory of Open Access Journals (Sweden)

    Nora Scarcelli

    Full Text Available Chloroplast DNA sequences are of great interest for population genetics and phylogenetic studies. However, only a small set of markers are commonly used. Most of them have been designed for amplification in a large range of Angiosperms and are located in the Large Single Copy (LSC. Here we developed a new set of 100 primer pairs optimized for amplification in Monocotyledons. Primer pairs amplify coding (exon and non-coding regions (intron and intergenic spacer. They span the different chloroplast regions: 72 are located in the LSC, 13 in the Small Single Copy (SSC and 15 in the Inverted Repeat region (IR. Amplification and sequencing were tested in 13 species of Monocotyledons: Dioscorea abyssinica, D. praehensilis, D. rotundata, D. dumetorum, D. bulbifera, Trichopus sempervirens (Dioscoreaceae, Phoenix canariensis, P. dactylifera, Astrocaryum scopatum, A. murumuru, Ceroxylon echinulatum (Arecaceae, Digitaria excilis and Pennisetum glaucum (Poaceae. The diversity found in Dioscorea, Digitaria and Pennisetum mainly corresponded to Single Nucleotide Polymorphism (SNP while the diversity found in Arecaceae also comprises Variable Number Tandem Repeat (VNTR. We observed that the most variable loci (rps15-ycf1, rpl32-ccsA, ndhF-rpl32, ndhG-ndhI and ccsA are located in the SSC. Through the analysis of the genetic structure of a wild-cultivated species complex in Dioscorea, we demonstrated that this new set of primers is of great interest for population genetics and we anticipate that it will also be useful for phylogeny and bar-coding studies.

  12. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes

    Directory of Open Access Journals (Sweden)

    Cronn Richard

    2009-12-01

    Full Text Available Abstract Background Molecular evolutionary studies share the common goal of elucidating historical relationships, and the common challenge of adequately sampling taxa and characters. Particularly at low taxonomic levels, recent divergence, rapid radiations, and conservative genome evolution yield limited sequence variation, and dense taxon sampling is often desirable. Recent advances in massively parallel sequencing make it possible to rapidly obtain large amounts of sequence data, and multiplexing makes extensive sampling of megabase sequences feasible. Is it possible to efficiently apply massively parallel sequencing to increase phylogenetic resolution at low taxonomic levels? Results We reconstruct the infrageneric phylogeny of Pinus from 37 nearly-complete chloroplast genomes (average 109 kilobases each of an approximately 120 kilobase genome generated using multiplexed massively parallel sequencing. 30/33 ingroup nodes resolved with ≥ 95% bootstrap support; this is a substantial improvement relative to prior studies, and shows massively parallel sequencing-based strategies can produce sufficient high quality sequence to reach support levels originally proposed for the phylogenetic bootstrap. Resampling simulations show that at least the entire plastome is necessary to fully resolve Pinus, particularly in rapidly radiating clades. Meta-analysis of 99 published infrageneric phylogenies shows that whole plastome analysis should provide similar gains across a range of plant genera. A disproportionate amount of phylogenetic information resides in two loci (ycf1, ycf2, highlighting their unusual evolutionary properties. Conclusion Plastome sequencing is now an efficient option for increasing phylogenetic resolution at lower taxonomic levels in plant phylogenetic and population genetic analyses. With continuing improvements in sequencing capacity, the strategies herein should revolutionize efforts requiring dense taxon and character sampling

  13. The complete chloroplast genome sequence of Helwingia himalaica (Helwingiaceae, Aquifoliales and a chloroplast phylogenomic analysis of the Campanulidae

    Directory of Open Access Journals (Sweden)

    Xin Yao

    2016-11-01

    Full Text Available Complete chloroplast genome sequences have been very useful for understanding phylogenetic relationships in angiosperms at the family level and above, but there are currently large gaps in coverage. We report the chloroplast genome for Helwingia himalaica, the first in the distinctive family Helwingiaceae and only the second genus to be sequenced in the order Aquifoliales. We then combine this with 36 published sequences in the large (c. 35,000 species subclass Campanulidae in order to investigate relationships at the order and family levels. The Helwingia genome consists of 158,362 bp containing a pair of inverted repeat (IR regions of 25,996 bp separated by a large single-copy (LSC region and a small single-copy (SSC region which are 87,810 and 18,560 bp, respectively. There are 142 known genes, including 94 protein-coding genes, eight ribosomal RNA genes, and 40 tRNA genes. The topology of the phylogenetic relationships between Apiales, Asterales, and Dipsacales differed between analyses based on complete genome sequences and on 36 shared protein-coding genes, showing that further studies of campanulid phylogeny are needed.

  14. Chloroplast microsatellite markers for Artocarpus (Moraceae) developed from transcriptome sequences

    Science.gov (United States)

    Premise of the study: Chloroplast microsatellite loci were characterized from transcriptomes of Artocarpus (A.) altilis (breadfruit) and A. camansi (breadnut). They were tested in A. odoratissimus (terap) and A. altilis and evaluated in silico for two congeners. Methods and Results: 15 simple seque...

  15. The complete chloroplast genome sequence of Brachypodium distachyon: sequence comparison and phylogenetic analysis of eight grass plastomes

    Directory of Open Access Journals (Sweden)

    Anderson Olin D

    2008-07-01

    Full Text Available Abstract Background Wheat, barley, and rye, of tribe Triticeae in the Poaceae, are among the most important crops worldwide but they present many challenges to genomics-aided crop improvement. Brachypodium distachyon, a close relative of those cereals has recently emerged as a model for grass functional genomics. Sequencing of the nuclear and organelle genomes of Brachypodium is one of the first steps towards making this species available as a tool for researchers interested in cereals biology. Findings The chloroplast genome of Brachypodium distachyon was sequenced by a combinational approach using BAC end and shotgun sequences derived from a selected BAC containing the entire chloroplast genome. Comparative analysis indicated that the chloroplast genome is conserved in gene number and organization with respect to those of other cereals. However, several Brachypodium genes evolve at a faster rate than those in other grasses. Sequence analysis reveals that rice and wheat have a ~2.1 kb deletion in their plastid genomes and this deletion must have occurred independently in both species. Conclusion We demonstrate that BAC libraries can be used to sequence plastid, and likely other organellar, genomes. As expected, the Brachypodium chloroplast genome is very similar to those of other sequenced grasses. The phylogenetic analyses and the pattern of insertions and deletions in the chloroplast genome confirmed that Brachypodium is a close relative of the tribe Triticeae. Nevertheless, we show that some large indels can arise multiple times and may confound phylogenetic reconstruction.

  16. Phylogeny of Catalpa (Bignoniaceae) inferred from sequences of chloroplast ndhF and nuclear ribosomal DNA%根据叶绿体ndhF和核核糖体DNA序列推断梓属(紫葳科)的系统发育

    Institute of Scientific and Technical Information of China (English)

    李建华

    2008-01-01

    Phylogenetics of Chilopsis and Catalpa (Bignoniaceae) was elucidated based on sequences of chloroplast ndhF and the nrDNA ITS region. In Bignoniaceae, Chilopsis and Catalpa are most closely related as sister genera. Our data supported section Macrocatalpa of the West Indies and section Catalpa of eastern Asian and North American continents. Within section Catalpa, Catalpa ovata of eastern Asia form a clade with North American species, C. speciosa and C. bignonioides, while the other eastern Asian species comprise a clade where C. duclouxii is sister to the clade of C. bungei and C. fargesii. The Caribbean species of Catalpa diverged early from the continental species. More studies are needed to test whether the phylogenetic pattern is common in eastern Asian-North American disjunct genera with species in the West Indies.

  17. Chloroplast genome sequence confirms distinctness of Australian and Asian wild rice.

    Science.gov (United States)

    Waters, Daniel L E; Nock, Catherine J; Ishikawa, Ryuji; Rice, Nicole; Henry, Robert J

    2012-01-01

    Cultivated rice (Oryza sativa) is an AA genome Oryza species that was most likely domesticated from wild populations of O. rufipogon in Asia. O. rufipogon and O. meridionalis are the only AA genome species found within Australia and occur as widespread populations across northern Australia. The chloroplast genome sequence of O. rufipogon from Asia and Australia and O. meridionalis and O. australiensis (an Australian member of the genus very distant from O. sativa) was obtained by massively parallel sequencing and compared with the chloroplast genome sequence of domesticated O. sativa. Oryza australiensis differed in more than 850 sites single nucleotide polymorphism or indel from each of the other samples. The other wild rice species had only around 100 differences relative to cultivated rice. The chloroplast genomes of Australian O. rufipogon and O. meridionalis were closely related with only 32 differences. The Asian O. rufipogon chloroplast genome (with only 68 differences) was closer to O. sativa than the Australian taxa (both with more than 100 differences). The chloroplast sequences emphasize the genetic distinctness of the Australian populations and their potential as a source of novel rice germplasm. The Australian O. rufipogon may be a perennial form of O. meridionalis.

  18. Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

    Science.gov (United States)

    Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

    2016-03-01

    Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

  19. Comparative genomics of four Liliales families inferred from the complete chloroplast genome sequence of Veratrum patulum O. Loes. (Melanthiaceae).

    Science.gov (United States)

    Do, Hoang Dang Khoa; Kim, Jung Sung; Kim, Joo-Hwan

    2013-11-10

    The sequence of the chloroplast genome, which is inherited maternally, contains useful information for many scientific fields such as plant systematics, biogeography and biotechnology because its characteristics are highly conserved among species. There is an increase in chloroplast genomes of angiosperms that have been sequenced in recent years. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Veratrum patulum Loes. (Melanthiaceae, Liliales) was analyzed completely. The circular double-stranded DNA of 153,699 bp consists of two inverted repeat (IR) regions of 26,360 bp each, a large single copy of 83,372 bp, and a small single copy of 17,607 bp. This plastome contains 81 protein-coding genes, 30 distinct tRNA and four genes of rRNA. In addition, there are six hypothetical coding regions (ycf1, ycf2, ycf3, ycf4, ycf15 and ycf68) and two open reading frames (ORF42 and ORF56), which are also found in the chloroplast genomes of the other species. The gene orders and gene contents of the V. patulum plastid genome are similar to that of Smilax china, Lilium longiflorum and Alstroemeria aurea, members of the Smilacaceae, Liliaceae and Alstroemeriaceae (Liliales), respectively. However, the loss rps16 exon 2 in V. patulum results in the difference in the large single copy regions in comparison with other species. The base substitution rate is quite similar among genes of these species. Additionally, the base substitution rate of inverted repeat region was smaller than that of single copy regions in all observed species of Liliales. The IR regions were expanded to trnH_GUG in V. patulum, a part of rps19 in L. longiflorum and A. aurea, and whole sequence of rps19 in S. china. Furthermore, the IGS lengths of rbcL-accD-psaI region were variable among Liliales species, suggesting that this region might be a hotspot of indel events and the informative site for phylogenetic studies in Liliales. In general, the whole chloroplast genome of V. patulum, a

  20. Two major groups of chloroplast DNA haplotypes in diploid and tetraploid Aconitum subgen. Aconitum (Ranunculaceae in the Carpathians

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    J. Mitka

    2016-04-01

    Full Text Available Aconitum in Europe is represented by ca. 10% of the total number of species and the Carpathian Mts. are the center of the genus variability in the subcontinent. We studied the chloroplast DNA intergenic spacer trnL(UAG-rpl32- ndhF (cpDNA variability of the Aconitum subgen. Aconitum in the Carpathians: diploids (2n=16, sect. Cammarum, tetraploids (2n=32, sect. Aconitum and triploids (2n=24, nothosect. Acomarum. Altogether 25 Aconitum accessions representing the whole taxonomic variability of the subgenus were sequenced and subjected to phylogenetic analyses. Both parsimony, Bayesian and character network analyses showed the two distinct types of the cpDNA chloroplast, one typical of the diploid and the second of the tetraploid groups. Some specimens had identical cpDNA sequences (haplotypes and scattered across the whole mountain arch. In the sect. Aconitum 9 specimens shared one haplotype, while in the sect. Camarum one haplotype represents 4 accessions and the second – 5 accessions. The diploids and tetraploids were diverged by 6 mutations, while the intrasectional variability amounted maximally to 3 polymorphisms. Taking into consideration different types of cpDNA haplotypes and ecological profiles of the sections (tetraploids – high‑mountain species, diploids – species from forest montane belt we speculate on the different and independent history of the sections in the Carpathians.

  1. 中国野生葡萄叶绿体分离及叶绿体DNA 提取的研究%An Optimized Chloroplast Isolation and Chloroplast DNA Extraction Protocol for Chinese Wild Grapes

    Institute of Scientific and Technical Information of China (English)

    谢海坤; 焦健; 樊秀彩; 张颖; 姜建福; 孙海生; 刘崇怀

    2016-01-01

    Mature leaves collected from Vitis davidii ,V .amurensis ,V .heyneana and V .chunganensis were used for chloroplast isolation and cpDNA extraction in this study .The two methods were the column plant chloroplast DNAout and modified high-salt low-pH method ,and the results were compared with each other .(1) Both methods had separated the chloroplast of Chinese wild grapes ,but the modified high-salt low-pH method obtained higher concentration and less impurity of chloroplast than that of column plant chloroplast DNAout .So the modified high-salt low-pH method was more suitable for chloroplast isolation . (2) The value of OD260/OD280 of cpDNA extracted by the column plant chloroplast DNAout was between 1 .28 and 1 .36 ,and the concentration was between 4 .2 ng・μL -1 and 7 .8 ng・μL -1 ,which did not meet the demand of subsequent chloroplast genome sequencing .In contrast ,the value of OD260/OD280 of cpDNA extracted by the modified high-salt low-pH method was between 1 .84 and 1 .90 and the concentration was between 2 514 .4 ng ・ μL -1 and 4 133 .7 ng・ μL -1 ,so the cpDNA extracted in this way was extremely high-quality and pure .As a result ,the cpDNA extracted by the modified high-salt low-pH method meet the demand of subsequent chloroplast genome sequencing .As a conclusion ,the modified high-salt low-pH method isolated intact chloroplast and extract high-quality cpDNA of Chinese wild grapes simply and quickly .And the cpDNA meet the demand of subsequent chloroplast genome sequencing .It was also a critical step to make further research of chloroplast genomes of V itis L .%以中国野生刺葡萄、山葡萄、桑叶葡萄和东南葡萄的成熟叶片为材料,比较柱式植物叶绿体DNAout试剂盒和改良的高盐-低pH法分离叶绿体及提取cpDNA效果。结果显示:(1)2种方法均分离得到了中国野生葡萄的叶绿体,但与柱式植物叶绿体DNAout试剂盒相比,改良的高盐-低pH法得到的叶绿体浓度高

  2. Utilization of complete chloroplast genomes for phylogenetic studies

    NARCIS (Netherlands)

    Ramlee, Shairul Izan Binti

    2016-01-01

    Chloroplast DNA sequence polymorphisms are a primary source of data in many plant phylogenetic studies. The chloroplast genome is relatively conserved in its evolution making it an ideal molecule to retain phylogenetic signals. The chloroplast genome is also largely, but not completely, free from ot

  3. The Complete Chloroplast Genome Sequences of the Medicinal Plant Pogostemon cablin

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    Yang He

    2016-06-01

    Full Text Available Pogostemon cablin, the natural source of patchouli alcohol, is an important herb in the Lamiaceae family. Here, we present the entire chloroplast genome of P. cablin. This genome, with 38.24% GC content, is 152,460 bp in length. The genome presents a typical quadripartite structure with two inverted repeats (each 25,417 bp in length, separated by one small and one large single-copy region (17,652 and 83,974 bp in length, respectively. The chloroplast genome encodes 127 genes, of which 107 genes are single-copy, including 79 protein-coding genes, four rRNA genes, and 24 tRNA genes. The genome structure, GC content, and codon usage of this chloroplast genome are similar to those of other species in the family, except that it encodes less protein-coding genes and tRNA genes. Phylogenetic analysis reveals that P. cablin diverged from the Scutellarioideae clade about 29.45 million years ago (Mya. Furthermore, most of the simple sequence repeats (SSRs are short polyadenine or polythymine repeats that contribute to high AT content in the chloroplast genome. Complete sequences and annotation of P. cablin chloroplast genome will facilitate phylogenic, population and genetic engineering research investigations involving this particular species.

  4. IDENTIFICATION OF GYMNEMA SPECIES BY RANDOM AMPLIFIED POLYMORPHIC DNA TECHNIQUE AND CHLOROPLAST trnK GENE

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    Subashini Sekar

    2014-12-01

    Full Text Available Gymnema is one of the important anti-diabetic medicinal plants used from ancient times and is commonly known as ‘sugar killer’. Most of its species have been used in many applications in Indian traditional medicine. Nevertheless, their efficiency is critically dependent on the use of the correct material. The sharing of similar vernacular name and morphological features make confusion in the usage of Gymnema species. In the present study, Gymnema sp. were identified through random amplified polymorphic DNA (RAPD technique and species specific markers were generated for easy identification of G. elegans, G. montanum and G. sylvestre. Using the RAPD techniques of 3 species specific markers for G. sylvestre, 7 markers for G. elegans and 4 markers for G. montanum had been generated. Highest genetic identity was found between G. sylvestre and G. montanum and highest genetic distance was found between G. sylvestre and G. elegans. Further, DNA barcode was developed by sequencing chloroplast partial trnK DNA of these three species. No significant variation was found in partial trnK gene sequences between Gymnema species. But these sequences can efficiently differentiate the Gymnema and Mandevilla species. In-silico sequence–restriction fragment length polymorphism (RFLP analysis revealed three fragments measuring G. sylvestre - 204, G. elegans - 174, and G. montanum - 168 bp Gymnema species. The present study concluded that RAPD markers were highly efficient for species detection than the partial trnK gene sequences. This could be used to confirm the Gymnema sp. identities and to ensure their safe application in pharmaceuticals.

  5. Information Theory of DNA Sequencing

    CERN Document Server

    Motahari, Abolfazl; Tse, David

    2012-01-01

    DNA sequencing is the basic workhorse of modern day biology and medicine. Shotgun sequencing is the dominant technique used: many randomly located short fragments called reads are extracted from the DNA sequence, and these reads are assembled to reconstruct the original sequence. By drawing an analogy between the DNA sequencing problem and the classic communication problem, we define an information theoretic notion of sequencing capacity. This is the maximum number of DNA base pairs that can be resolved reliably per read, and provides a fundamental limit to the performance that can be achieved by any assembly algorithm. We compute the sequencing capacity explicitly for a simple statistical model of the DNA sequence and the read process. Using this framework, we also study the impact of noise in the read process on the sequencing capacity.

  6. Sequence characteristics and divergent evolution of the chloroplast psbA-trnH noncoding region in gymnosperms.

    Science.gov (United States)

    Hao, D C; Chen, S L; Xiao, P G

    2010-01-01

    The psbA-trnH intergenic region is among the most variable regions in the gymnosperm chloroplast genome. It is proposed as suitable for DNA barcoding studies and is useful in phylogenetics at the species level. This region consists of two parts differing in their evolutionary characteristics: 1) the psbA 3’UTR (untranslated region) and 2) the psbA-trnH intergenic spacer. We compared the sequence and RNA secondary structure of the psbA 3’ UTR across gymnosperms and found consensus motifs corresponding to the stem portions of the RNA stem-loop structures and a consensus TGGATTGTTATGT box. The psbA-trnH spacer is highly variable in length and composition. Tandem repeats that form stem-loop structures were detected in both the psbA 3’ UTR and the psbA-trnH spacer. The presence of promoters and stem-loop structures in the psbA-trnH spacer and high sequence variation in this region suggest that psbA and trnH in some gymnosperms are independently transcribed. A comparison of chloroplast UTRs across gymnosperms offer clues to the identity of putative regulatory elements and information on selective constraints imposed on the chloroplast non-coding regions. The present study should inspire researchers to explore the full potential of the psbA-trnH non-coding sequence and to further stimulate its application in a broader spectrum of studies, not limited to phylogenetics and DNA barcoding.

  7. Molecular polymorphism in Pistacia vera L. using non-coding regions of chloroplast DNA

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    Majid Talebi

    2016-06-01

    Full Text Available The present study describes plastid DNA polymorphism and reports a comparative analysis of two non-coding cpDNA regions (trnC–trnD and atpB–rbcL in pistachio. Seventeen different genotypes of domestic and wild pistachio from Iran, Syria, Turkey and America were sampled. Total genomic DNA was extracted and amplified with trnC–trnD and atpB–rbcL specific primers and then were sequenced. Phylogenetic relationships and depiction of phylogenetic trees were conducted. Cultivated genotypes of Pistacia vera were classified in a group regardless of their geographic location. P. vera was isolated from Sarakhs but they placed in the two close groups. Among cultivated genotypes, Jalab was separated from other cultivated genotypes. Pistacia Khinjuk was classified with Pistacia atlantica subsp. mutica. The findings confirm the common splitting hypothesis for commercial pistachio genotypes of the P. vera wild-type and also indicated the direct impact of Iranian genotypes in the evolutionary process of cultivated pistachios in other parts of the world. In conclusion it can be inferred that cultivated varieties of pistachio and P. vera var. sarakhs have the same origin, moreover genomic chloroplast could appropriately identify the interspecies relationships of pistachios.

  8. Polymerase chain reaction-single strand conformation polymorphism analyses of nuclear and chloroplast DNA provide evidence for recombination, multiple introductions and nascent speciation in the Caulerpa taxifolia complex.

    Science.gov (United States)

    Meusnier, I; Valero, M; Destombe, C; Godé, C; Desmarais, E; Bonhomme, F; Stam, W T; Olsen, J L

    2002-11-01

    Independent lines of evidence support an Australian origin for the Mediterranean populations of the tropical alga Caulerpa taxifolia. To complement previous biogeographical studies based on nuclear rDNA internal transcribed spacer (ITS), a new chloroplast marker was developed--the cp 16S rDNA intron-2. Sequence variability for both nuclear and chloroplast markers were assessed in 110 individuals using single strand conformation polymorphism. Comparison of intrapopulation genetic diversity between invasive Mediterranean and 'native' Australian populations revealed the occurrence of two divergent and widespread clades. The first clade grouped nontropical invasive populations with inshore-mainland populations from Australia, while the second clustered all offshore-island populations studied so far. Despite our finding of nine distinct nuclear and five distinct chloroplast profiles, a single nucleocytoplasmic combination was characteristic of the invasive populations and sexual reproduction was found to be very rare. C. taxifolia is clearly a complex of genetically and ecologically differentiated sibling species or subspecies.

  9. Phylogenetic relationships within Pelargonium section Peristera (Geraniaceae) inferred from nrDNA and cpDNA sequence comparisons.

    NARCIS (Netherlands)

    Bakker, F.T.; Helbrugge, D.; Culham, A.; Gibby, M.

    1998-01-01

    Phylogenetic analysis of nrDNA ITS and tmL (UAA) 5' exon-tmF (GAA) chloroplast DNA sequences from 17 species of Pelargonium sect. Peristera, together with nine putative outgroups, suggests paraphyly for the section and a close relationship between the highly disjurmt South African and Australian spe

  10. Complete chloroplast genome sequence of Omani lime (Citrus aurantiifolia and comparative analysis within the rosids.

    Directory of Open Access Journals (Sweden)

    Huei-Jiun Su

    Full Text Available The genus Citrus contains many economically important fruits that are grown worldwide for their high nutritional and medicinal value. Due to frequent hybridizations among species and cultivars, the exact number of natural species and the taxonomic relationships within this genus are unclear. To compare the differences between the Citrus chloroplast genomes and to develop useful genetic markers, we used a reference-assisted approach to assemble the complete chloroplast genome of Omani lime (C. aurantiifolia. The complete C. aurantiifolia chloroplast genome is 159,893 bp in length; the organization and gene content are similar to most of the rosids lineages characterized to date. Through comparison with the sweet orange (C. sinensis chloroplast genome, we identified three intergenic regions and 94 simple sequence repeats (SSRs that are potentially informative markers with resolution for interspecific relationships. These markers can be utilized to better understand the origin of cultivated Citrus. A comparison among 72 species belonging to 10 families of representative rosids lineages also provides new insights into their chloroplast genome evolution.

  11. Complete chloroplast genome sequence of Omani lime (Citrus aurantiifolia) and comparative analysis within the rosids.

    Science.gov (United States)

    Su, Huei-Jiun; Hogenhout, Saskia A; Al-Sadi, Abdullah M; Kuo, Chih-Horng

    2014-01-01

    The genus Citrus contains many economically important fruits that are grown worldwide for their high nutritional and medicinal value. Due to frequent hybridizations among species and cultivars, the exact number of natural species and the taxonomic relationships within this genus are unclear. To compare the differences between the Citrus chloroplast genomes and to develop useful genetic markers, we used a reference-assisted approach to assemble the complete chloroplast genome of Omani lime (C. aurantiifolia). The complete C. aurantiifolia chloroplast genome is 159,893 bp in length; the organization and gene content are similar to most of the rosids lineages characterized to date. Through comparison with the sweet orange (C. sinensis) chloroplast genome, we identified three intergenic regions and 94 simple sequence repeats (SSRs) that are potentially informative markers with resolution for interspecific relationships. These markers can be utilized to better understand the origin of cultivated Citrus. A comparison among 72 species belonging to 10 families of representative rosids lineages also provides new insights into their chloroplast genome evolution.

  12. The complete chloroplast genome sequences for four Amaranthus species (Amaranthaceae)1

    Science.gov (United States)

    Chaney, Lindsay; Mangelson, Ryan; Ramaraj, Thiruvarangan; Jellen, Eric N.; Maughan, Peter J.

    2016-01-01

    Premise of the study: The amaranth genus contains many important grain and weedy species. We further our understanding of the genus through the development of a complete reference chloroplast genome. Methods and Results: A high-quality Amaranthus hypochondriacus (Amaranthaceae) chloroplast genome assembly was developed using long-read technology. This reference genome was used to reconstruct the chloroplast genomes for two closely related grain species (A. cruentus and A. caudatus) and their putative progenitor (A. hybridus). The reference genome was 150,518 bp and possesses a circular structure of two inverted repeats (24,352 bp) separated by small (17,941 bp) and large (83,873 bp) single-copy regions; it encodes 111 genes, 72 for proteins. Relative to the reference chloroplast genome, an average of 210 single-nucleotide polymorphisms (SNPs) and 122 insertion/deletion polymorphisms (indels) were identified across the analyzed genomes. Conclusions: This reference chloroplast genome, along with the reported simple sequence repeats, SNPs, and indels, is an invaluable genetic resource for studying the phylogeny and genetic diversity within the amaranth genus. PMID:27672525

  13. Biosensors for DNA sequence detection

    Science.gov (United States)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  14. Unique haplotypes of cacao trees as revealed by trnH-psbA chloroplast DNA.

    Science.gov (United States)

    Gutiérrez-López, Nidia; Ovando-Medina, Isidro; Salvador-Figueroa, Miguel; Molina-Freaner, Francisco; Avendaño-Arrazate, Carlos H; Vázquez-Ovando, Alfredo

    2016-01-01

    Cacao trees have been cultivated in Mesoamerica for at least 4,000 years. In this study, we analyzed sequence variation in the chloroplast DNA trnH-psbA intergenic spacer from 28 cacao trees from different farms in the Soconusco region in southern Mexico. Genetic relationships were established by two analysis approaches based on geographic origin (five populations) and genetic origin (based on a previous study). We identified six polymorphic sites, including five insertion/deletion (indels) types and one transversion. The overall nucleotide diversity was low for both approaches (geographic = 0.0032 and genetic = 0.0038). Conversely, we obtained moderate to high haplotype diversity (0.66 and 0.80) with 10 and 12 haplotypes, respectively. The common haplotype (H1) for both networks included cacao trees from all geographic locations (geographic approach) and four genetic groups (genetic approach). This common haplotype (ancient) derived a set of intermediate haplotypes and singletons interconnected by one or two mutational steps, which suggested directional selection and event purification from the expansion of narrow populations. Cacao trees from Soconusco region were grouped into one cluster without any evidence of subclustering based on AMOVA (F ST = 0) and SAMOVA (F ST = 0.04393) results. One population (Mazatán) showed a high haplotype frequency; thus, this population could be considered an important reservoir of genetic material. The indels located in the trnH-psbA intergenic spacer of cacao trees could be useful as markers for the development of DNA barcoding.

  15. Unique haplotypes of cacao trees as revealed by trnH-psbA chloroplast DNA

    Directory of Open Access Journals (Sweden)

    Nidia Gutiérrez-López

    2016-04-01

    Full Text Available Cacao trees have been cultivated in Mesoamerica for at least 4,000 years. In this study, we analyzed sequence variation in the chloroplast DNA trnH-psbA intergenic spacer from 28 cacao trees from different farms in the Soconusco region in southern Mexico. Genetic relationships were established by two analysis approaches based on geographic origin (five populations and genetic origin (based on a previous study. We identified six polymorphic sites, including five insertion/deletion (indels types and one transversion. The overall nucleotide diversity was low for both approaches (geographic = 0.0032 and genetic = 0.0038. Conversely, we obtained moderate to high haplotype diversity (0.66 and 0.80 with 10 and 12 haplotypes, respectively. The common haplotype (H1 for both networks included cacao trees from all geographic locations (geographic approach and four genetic groups (genetic approach. This common haplotype (ancient derived a set of intermediate haplotypes and singletons interconnected by one or two mutational steps, which suggested directional selection and event purification from the expansion of narrow populations. Cacao trees from Soconusco region were grouped into one cluster without any evidence of subclustering based on AMOVA (FST = 0 and SAMOVA (FST = 0.04393 results. One population (Mazatán showed a high haplotype frequency; thus, this population could be considered an important reservoir of genetic material. The indels located in the trnH-psbA intergenic spacer of cacao trees could be useful as markers for the development of DNA barcoding.

  16. Comparative analysis of the complete chloroplast genome sequences in psammophytic Haloxylon species (Amaranthaceae

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    Wenpan Dong

    2016-11-01

    Full Text Available The Haloxylon genus belongs to the Amaranthaceae (formerly Chenopodiaceae family. The small trees or shrubs in this genus are referred to as the King of psammophytic plants, and perform important functions in environmental protection, including wind control and sand fixation in deserts. To better understand these beneficial plants, we sequenced the chloroplast (cp genomes of Haloxylon ammodendron (HA and Haloxylon persicum (HP and conducted comparative genomic analyses on these and two other representative Amaranthaceae species. Similar to other higher plants, we found that the Haloxylon cp genome is a quadripartite, double-stranded, circular DNA molecule of 151,570 bp in HA and 151,586 bp in HP. It contains a pair of inverted repeats (24,171 bp in HA and 24,177 bp in HP that separate the genome into a large single copy region of 84,214 bp in HA and 84,217 bp in HP, and a small single copy region of 19,014 bp in HA and 19,015 bp in HP. Each Haloxylon cp genome contains 112 genes, including 78 coding, 30 tRNA, and four ribosomal RNA genes. We detected 59 different simple sequence repeat loci, including 44 mono-nucleotide, three di-nucleotide, one tri-nucleotide, and 11 tetra-nucleotide repeats. Comparative analysis revealed only 67 mutations between the two species, including 44 substitutions, 23 insertions/deletions, and two micro-inversions. The two inversions, with lengths of 14 and 3 bp, occur in the petA-psbJ intergenic region and rpl16 intron, respectively, and are predicted to form hairpin structures with repeat sequences of 27 and 19 bp, respectively, at the two ends. The ratio of transitions to transversions was 0.76. These results are valuable for future studies on Haloxylon genetic diversity and will enhance our understanding of the phylogenetic evolution of Amaranthaceae.

  17. The chloroplast genome sequence of the green alga Leptosira terrestris: multiple losses of the inverted repeat and extensive genome rearrangements within the Trebouxiophyceae

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    Turmel Monique

    2007-07-01

    Full Text Available Abstract Background In the Chlorophyta – the green algal phylum comprising the classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae – the chloroplast genome displays a highly variable architecture. While chlorophycean chloroplast DNAs (cpDNAs deviate considerably from the ancestral pattern described for the prasinophyte Nephroselmis olivacea, the degree of remodelling sustained by the two ulvophyte cpDNAs completely sequenced to date is intermediate relative to those observed for chlorophycean and trebouxiophyte cpDNAs. Chlorella vulgaris (Chlorellales is currently the only photosynthetic trebouxiophyte whose complete cpDNA sequence has been reported. To gain insights into the evolutionary trends of the chloroplast genome in the Trebouxiophyceae, we sequenced cpDNA from the filamentous alga Leptosira terrestris (Ctenocladales. Results The 195,081-bp Leptosira chloroplast genome resembles the 150,613-bp Chlorella genome in lacking a large inverted repeat (IR but differs greatly in gene order. Six of the conserved genes present in Chlorella cpDNA are missing from the Leptosira gene repertoire. The 106 conserved genes, four introns and 11 free standing open reading frames (ORFs account for 48.3% of the genome sequence. This is the lowest gene density yet observed among chlorophyte cpDNAs. Contrary to the situation in Chlorella but similar to that in the chlorophycean Scenedesmus obliquus, the gene distribution is highly biased over the two DNA strands in Leptosira. Nine genes, compared to only three in Chlorella, have significantly expanded coding regions relative to their homologues in ancestral-type green algal cpDNAs. As observed in chlorophycean genomes, the rpoB gene is fragmented into two ORFs. Short repeats account for 5.1% of the Leptosira genome sequence and are present mainly in intergenic regions. Conclusion Our results highlight the great plasticity of the chloroplast genome in the Trebouxiophyceae and indicate

  18. Analysis of complete nucleotide sequences of 12 Gossypium chloroplast genomes: origin and evolution of allotetraploids.

    Directory of Open Access Journals (Sweden)

    Qin Xu

    Full Text Available BACKGROUND: Cotton (Gossypium spp. is a model system for the analysis of polyploidization. Although ascertaining the donor species of allotetraploid cotton has been intensively studied, sequence comparison of Gossypium chloroplast genomes is still of interest to understand the mechanisms underlining the evolution of Gossypium allotetraploids, while it is generally accepted that the parents were A- and D-genome containing species. Here we performed a comparative analysis of 13 Gossypium chloroplast genomes, twelve of which are presented here for the first time. METHODOLOGY/PRINCIPAL FINDINGS: The size of 12 chloroplast genomes under study varied from 159,959 bp to 160,433 bp. The chromosomes were highly similar having >98% sequence identity. They encoded the same set of 112 unique genes which occurred in a uniform order with only slightly different boundary junctions. Divergence due to indels as well as substitutions was examined separately for genome, coding and noncoding sequences. The genome divergence was estimated as 0.374% to 0.583% between allotetraploid species and A-genome, and 0.159% to 0.454% within allotetraploids. Forty protein-coding genes were completely identical at the protein level, and 20 intergenic sequences were completely conserved. The 9 allotetraploids shared 5 insertions and 9 deletions in whole genome, and 7-bp substitutions in protein-coding genes. The phylogenetic tree confirmed a close relationship between allotetraploids and the ancestor of A-genome, and the allotetraploids were divided into four separate groups. Progenitor allotetraploid cotton originated 0.43-0.68 million years ago (MYA. CONCLUSION: Despite high degree of conservation between the Gossypium chloroplast genomes, sequence variations among species could still be detected. Gossypium chloroplast genomes preferred for 5-bp indels and 1-3-bp indels are mainly attributed to the SSR polymorphisms. This study supports that the common ancestor of diploid A

  19. Graphene nanodevices for DNA sequencing

    Science.gov (United States)

    Heerema, Stephanie J.; Dekker, Cees

    2016-02-01

    Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.

  20. Complex interplay among DNA modification, noncoding RNA expression and protein-coding RNA expression in Salvia miltiorrhiza chloroplast genome.

    Directory of Open Access Journals (Sweden)

    Haimei Chen

    Full Text Available Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA genes. Comparison of the abundance of protein-coding transcripts (cRNA with and without overlapping antisense ncRNAs (asRNA suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05. Using the SMRT Portal software (v1.3.2, 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box-like motif (CPGDMM1, "TATANNNATNA", and an unknown motif (CPGDMM2 "WNYANTGAW". Specifically, 35 of the 97 CPGDMM1 motifs (36.1% and 91 of the 369 CPGDMM2 motifs (24.7% were found to be significantly modified (p<0.01. Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01. Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome.

  1. The first complete chloroplast genome sequence of a lycophyte,Huperzia lucidula (Lycopodiaceae)

    Energy Technology Data Exchange (ETDEWEB)

    Wolf, Paul G.; Karol, Kenneth G.; Mandoli, Dina F.; Kuehl,Jennifer V.; Arumuganathan, K.; Ellis, Mark W.; Mishler, Brent D.; Kelch,Dean G.; Olmstead, Richard G.; Boore, Jeffrey L.

    2005-02-01

    We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8x depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,671 bp. Gene order is more similar to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperziachloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophytechloroplast genome data also enable a better reconstruction of the basaltracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.

  2. Complete sequence and comparative analysis of the chloroplast genome of coconut palm (Cocos nucifera.

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    Ya-Yi Huang

    Full Text Available Coconut, a member of the palm family (Arecaceae, is one of the most economically important trees used by mankind. Despite its diverse morphology, coconut is recognized taxonomically as only a single species (Cocos nucifera L.. There are two major coconut varieties, tall and dwarf, the latter of which displays traits resulting from selection by humans. We report here the complete chloroplast (cp genome of a dwarf coconut plant, and describe the gene content and organization, inverted repeat fluctuations, repeated sequence structure, and occurrence of RNA editing. Phylogenetic relationships of monocots were inferred based on 47 chloroplast protein-coding genes. Potential nodes for events of gene duplication and pseudogenization related to inverted repeat fluctuation were mapped onto the tree using parsimony criteria. We compare our findings with those from other palm species for which complete cp genome sequences are available.

  3. Complete sequence and comparative analysis of the chloroplast genome of coconut palm (Cocos nucifera).

    Science.gov (United States)

    Huang, Ya-Yi; Matzke, Antonius J M; Matzke, Marjori

    2013-01-01

    Coconut, a member of the palm family (Arecaceae), is one of the most economically important trees used by mankind. Despite its diverse morphology, coconut is recognized taxonomically as only a single species (Cocos nucifera L.). There are two major coconut varieties, tall and dwarf, the latter of which displays traits resulting from selection by humans. We report here the complete chloroplast (cp) genome of a dwarf coconut plant, and describe the gene content and organization, inverted repeat fluctuations, repeated sequence structure, and occurrence of RNA editing. Phylogenetic relationships of monocots were inferred based on 47 chloroplast protein-coding genes. Potential nodes for events of gene duplication and pseudogenization related to inverted repeat fluctuation were mapped onto the tree using parsimony criteria. We compare our findings with those from other palm species for which complete cp genome sequences are available.

  4. Chloroplast genome sequence confirms distinctness of Australian and Asian wild rice

    OpenAIRE

    Waters, Daniel L. E.; Nock, Catherine J; Ishikawa, Ryuji; Rice, Nicole; Henry, Robert J.

    2012-01-01

    Cultivated rice (Oryza sativa) is an AA genome Oryza species that was most likely domesticated from wild populations of O. rufipogon in Asia. O. rufipogon and O. meridionalis are the only AA genome species found within Australia and occur as widespread populations across northern Australia. The chloroplast genome sequence of O. rufipogon from Asia and Australia and O. meridionalis and O. australiensis (an Australian member of the genus very distant from O. sativa) was obtained by massively pa...

  5. The first complete chloroplast genome sequences of Ulmus species by de novo sequencing: Genome comparative and taxonomic position analysis

    Science.gov (United States)

    Zhang, Shuang; Yu, Xiao-Yue; Ren, Ya-Chao; Yang, Min-Sheng; Wang, Jin-Mao

    2017-01-01

    Elm (Ulmus) has a long history of use as a high-quality heavy hardwood famous for its resistance to drought, cold, and salt. It grows in temperate, warm temperate, and subtropical regions. This is the first report of Ulmaceae chloroplast genomes by de novo sequencing. The Ulmus chloroplast genomes exhibited a typical quadripartite structure with two single-copy regions (long single copy [LSC] and short single copy [SSC] sections) separated by a pair of inverted repeats (IRs). The lengths of the chloroplast genomes from five Ulmus ranged from 158,953 to 159,453 bp, with the largest observed in Ulmus davidiana and the smallest in Ulmus laciniata. The genomes contained 137–145 protein-coding genes, of which Ulmus davidiana var. japonica and U. davidiana had the most and U. pumila had the fewest. The five Ulmus species exhibited different evolutionary routes, as some genes had been lost. In total, 18 genes contained introns, 13 of which (trnL-TAA+, trnL-TAA−, rpoC1-, rpl2-, ndhA-, ycf1, rps12-, rps12+, trnA-TGC+, trnA-TGC-, trnV-TAC-, trnI-GAT+, and trnI-GAT) were shared among all five species. The intron of ycf1 was the longest (5,675bp) while that of trnF-AAA was the smallest (53bp). All Ulmus species except U. davidiana exhibited the same degree of amplification in the IR region. To determine the phylogenetic positions of the Ulmus species, we performed phylogenetic analyses using common protein-coding genes in chloroplast sequences of 42 other species published in NCBI. The cluster results showed the closest plants to Ulmaceae were Moraceae and Cannabaceae, followed by Rosaceae. Ulmaceae and Moraceae both belonged to Urticales, and the chloroplast genome clustering results were consistent with their traditional taxonomy. The results strongly supported the position of Ulmaceae as a member of the order Urticales. In addition, we found a potential error in the traditional taxonomies of U. davidiana and U. davidiana var. japonica, which should be confirmed with a

  6. The DnaJ OsDjA7/8 is essential for chloroplast development in rice (Oryza sativa).

    Science.gov (United States)

    Zhu, Xiaobo; Liang, Sihui; Yin, Junjie; Yuan, Can; Wang, Jing; Li, Weitao; He, Min; Wang, Jichun; Chen, Weilan; Ma, Bingtian; Wang, Yuping; Qin, Peng; Li, Shigui; Chen, Xuewei

    2015-12-10

    DnaJ proteins belong to chaperones of Hsp40 family that ubiquitously participate in various cellular processes. Previous studies have shown chloroplast-targeted DnaJs are involved in the development of chloroplast in some plant species. However, little is known about the function of DnaJs in rice, one of the main staple crops. In this study, we characterized a type I DnaJ protein OsDjA7/8. We found that the gene OsDjA7/8 was expressed in all collected tissues, with a priority in the vigorous growth leaf. Subcellular localization revealed that the protein OsDjA7/8 was mainly distributed in chloroplast. Reduced expression of OsDjA7/8 in rice led to albino lethal at the seedling stage. Transmission electron microscopy observation showed that the chloroplast structures were abnormally developed in the plants silenced for OsDjA7/8. In addition, the transcriptional expression of the genes tightly associated with the development of chloroplast was deeply reduced in the plants silenced for OsDjA7/8. Collectively, our study reveals that OsDjA7/8 encodes a chloroplast-localized protein and is essential for chloroplast development and differentiation in rice.

  7. The complete chloroplast genome sequence of American bird pepper (Capsicum annuum var. glabriusculum).

    Science.gov (United States)

    Zeng, Fan-chun; Gao, Cheng-wen; Gao, Li-zhi

    2016-01-01

    The complete chloroplast genome sequence of American bird pepper (Capsicum annuum var. glabriusculum) is reported and characterized in this study. The genome size is 156,612 bp, containing a pair of inverted repeats (IRs) of 25,776 bp separated by a large single-copy region of 87,213 bp and a small single-copy region of 17,851 bp. The chloroplast genome harbors 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes, and 37 tRNA genes. A total of 18 of these genes are duplicated in the inverted repeat regions, 16 genes contain 1 intron, and 2 genes and one ycf have 2 introns.

  8. Chloroplast DNA evidence for the origin of the genus Heterogaura from a species of Clarkia (Onagraceae)

    OpenAIRE

    1986-01-01

    Restriction-site variation in chloroplast DNA was examined in the morphologically distinct and monotypic genus Heterogaura and the related speciose genus Clarkia (Onagraceae), both native to California. Of the 605 restriction sites surveyed, a total of 119 mutations were identified. Of these, 55 were shared by at least two species and were used to construct a most parsimonious phylogenetic tree. This analysis, as well as one based on a distance metric, provided evidence that Heterogaura and C...

  9. Duplication in DNA Sequences

    Science.gov (United States)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  10. Chloroplast DNA inversions and the origin of the grass family (Poaceae).

    OpenAIRE

    Doyle, J.J.; Davis, J I; Soreng, R J; Garvin, D; Anderson, M J

    1992-01-01

    The phylogenetic affinities of the grass family (Poaceae) have long been debated. The chloroplast genomes of at least some grasses have been known to possess three inversions relative to the typical gene arrangement found in most flowering plants. We have surveyed for the presence of these inversions in grasses and other monocots by polymerase chain reaction amplification with primers constructed from sequences flanking the inversion end points. Amplification phenotypes diagnostic for the lar...

  11. The First Chloroplast Genome Sequence of Boswellia sacra, a Resin-Producing Plant in Oman

    Science.gov (United States)

    Khan, Abdul Latif; Al-Harrasi, Ahmed; Asaf, Sajjad; Park, Chang Eon; Park, Gun-Seok; Khan, Abdur Rahim; Lee, In-Jung; Al-Rawahi, Ahmed; Shin, Jae-Ho

    2017-01-01

    Boswellia sacra (Burseraceae), a keystone endemic species, is famous for the production of fragrant oleo-gum resin. However, the genetic make-up especially the genomic information about chloroplast is still unknown. Here, we described for the first time the chloroplast (cp) genome of B. sacra. The complete cp sequence revealed a circular genome of 160,543 bp size with 37.61% GC content. The cp genome is a typical quadripartite chloroplast structure with inverted repeats (IRs 26,763 bp) separated by small single copy (SSC; 18,962 bp) and large single copy (LSC; 88,055 bp) regions. De novo assembly and annotation showed the presence of 114 unique genes with 83 protein-coding regions. The phylogenetic analysis revealed that the B. sacra cp genome is closely related to the cp genome of Azadirachta indica and Citrus sinensis, while most of the syntenic differences were found in the non-coding regions. The pairwise distance among 76 shared genes of B. sacra and A. indica was highest for atpA, rpl2, rps12 and ycf1. The cp genome of B. sacra reveals a novel genome, which could be used for further studied to understand its diversity, taxonomy and phylogeny. PMID:28085925

  12. DNA Sequencing Sensors: An Overview

    Directory of Open Access Journals (Sweden)

    Jose Antonio Garrido-Cardenas

    2017-03-01

    Full Text Available The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years.

  13. Molecular evidence for the hybrid origin of Paulownia Taiwaniana based on RAPD markers and RFLP of chloroplast DNA.

    Science.gov (United States)

    Wang, W Y; Pai, R C; Lai, C C; Lin, T P

    1994-10-01

    Genomic DNA of Paulownia fortunei, P. kawakamii and P. taiwaniana were amplified with 10-base primers of arbitrary sequences using the polymerase chain reaction (PCR). A total of 351 DNA fragments were amplified from 23 primers and of these 265 fragments (75.5%) were polymorphic. Almost all of the PCR-amplified products of P. taiwaniana were shared by either P. fortunei or P. kawakamii, or both, and the number of polymorphic fragments shared by P. taiwaniana and P. fortunei was about equivalent to those shared by P. taiwaniana and P. kawakamii. Restriction fragments of chloroplast DNA (cpDNA) purified from Paulownia species and from reciprocal crosses between P. fortunei and P. kawakamii were analyzed. Restriction enzyme SalI-digested cpDNA showed an identical pattern in both P. kawakamii and P. taiwaniana. These results further support the hypothesis that P. taiwaniana is the natural hybrid between P. fortunei and P. kawakamii and that the maternal parent of P. taiwaniana is P. kawakamii.

  14. Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Binladen, Jonas; Miller, Webb;

    2007-01-01

    , and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390,965 bp of modern chloroplast and 131,474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved...

  15. Complete chloroplast genome sequence of an orchid model plant candidate: Erycina pusilla apply in tropical Oncidium breeding.

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    I-Chun Pan

    Full Text Available Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC clones and next-generation sequencing (NGS, the chloroplast (cp genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding.

  16. The complete chloroplast genome sequence of date palm (Phoenix dactylifera L..

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    Meng Yang

    Full Text Available BACKGROUND: Date palm (Phoenix dactylifera L., a member of Arecaceae family, is one of the three major economically important woody palms--the two other palms being oil palm and coconut tree--and its fruit is a staple food among Middle East and North African nations, as well as many other tropical and subtropical regions. Here we report a complete sequence of the data palm chloroplast (cp genome based on pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: After extracting 369,022 cp sequencing reads from our whole-genome-shotgun data, we put together an assembly and validated it with intensive PCR-based verification, coupled with PCR product sequencing. The date palm cp genome is 158,462 bp in length and has a typical quadripartite structure of the large (LSC, 86,198 bp and small single-copy (SSC, 17,712 bp regions separated by a pair of inverted repeats (IRs, 27,276 bp. Similar to what has been found among most angiosperms, the date palm cp genome harbors 112 unique genes and 19 duplicated fragments in the IR regions. The junctions between LSC/IRs and SSC/IRs show different features of sequence expansion in evolution. We identified 78 SNPs as major intravarietal polymorphisms within the population of a specific cp genome, most of which were located in genes with vital functions. Based on RNA-sequencing data, we also found 18 polycistronic transcription units and three highly expression-biased genes--atpF, trnA-UGC, and rrn23. CONCLUSIONS: Unlike most monocots, date palm has a typical cp genome similar to that of tobacco--with little rearrangement and gene loss or gain. High-throughput sequencing technology facilitates the identification of intravarietal variations in cp genomes among different cultivars. Moreover, transcriptomic analysis of cp genes provides clues for uncovering regulatory mechanisms of transcription and translation in chloroplasts.

  17. Fractals in DNA sequence analysis

    Institute of Scientific and Technical Information of China (English)

    Yu Zu-Guo(喻祖国); Vo Anh; Gong Zhi-Min(龚志民); Long Shun-Chao(龙顺潮)

    2002-01-01

    Fractal methods have been successfully used to study many problems in physics, mathematics, engineering, finance,and even in biology. There has been an increasing interest in unravelling the mysteries of DNA; for example, how can we distinguish coding and noncoding sequences, and the problems of classification and evolution relationship of organisms are key problems in bioinformatics. Although much research has been carried out by taking into consideration the long-range correlations in DNA sequences, and the global fractal dimension has been used in these works by other people, the models and methods are somewhat rough and the results are not satisfactory. In recent years, our group has introduced a time series model (statistical point of view) and a visual representation (geometrical point of view)to DNA sequence analysis. We have also used fractal dimension, correlation dimension, the Hurst exponent and the dimension spectrum (multifractal analysis) to discuss problems in this field. In this paper, we introduce these fractal models and methods and the results of DNA sequence analysis.

  18. DNA Sequencing Using capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Barry Karger

    2011-05-09

    The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linked polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other

  19. The complete chloroplast genome sequence of Podocarpus lambertii: genome structure, evolutionary aspects, gene content and SSR detection.

    Directory of Open Access Journals (Sweden)

    Leila do Nascimento Vieira

    Full Text Available BACKGROUND: Podocarpus lambertii (Podocarpaceae is a native conifer from the Brazilian Atlantic Forest Biome, which is considered one of the 25 biodiversity hotspots in the world. The advancement of next-generation sequencing technologies has enabled the rapid acquisition of whole chloroplast (cp genome sequences at low cost. Several studies have proven the potential of cp genomes as tools to understand enigmatic and basal phylogenetic relationships at different taxonomic levels, as well as further probe the structural and functional evolution of plants. In this work, we present the complete cp genome sequence of P. lambertii. METHODOLOGY/PRINCIPAL FINDINGS: The P. lambertii cp genome is 133,734 bp in length, and similar to other sequenced cupressophytes, it lacks one of the large inverted repeat regions (IR. It contains 118 unique genes and one duplicated tRNA (trnN-GUU, which occurs as an inverted repeat sequence. The rps16 gene was not found, which was previously reported for the plastid genome of another Podocarpaceae (Nageia nagi and Araucariaceae (Agathis dammara. Structurally, P. lambertii shows 4 inversions of a large DNA fragment ∼20,000 bp compared to the Podocarpus totara cp genome. These unexpected characteristics may be attributed to geographical distance and different adaptive needs. The P. lambertii cp genome presents a total of 28 tandem repeats and 156 SSRs, with homo- and dipolymers being the most common and tri-, tetra-, penta-, and hexapolymers occurring with less frequency. CONCLUSION: The complete cp genome sequence of P. lambertii revealed significant structural changes, even in species from the same genus. These results reinforce the apparently loss of rps16 gene in Podocarpaceae cp genome. In addition, several SSRs in the P. lambertii cp genome are likely intraspecific polymorphism sites, which may allow highly sensitive phylogeographic and population structure studies, as well as phylogenetic studies of species of

  20. The Complete Chloroplast Genome Sequence of Podocarpus lambertii: Genome Structure, Evolutionary Aspects, Gene Content and SSR Detection

    Science.gov (United States)

    Vieira, Leila do Nascimento; Faoro, Helisson; Rogalski, Marcelo; Fraga, Hugo Pacheco de Freitas; Cardoso, Rodrigo Luis Alves; de Souza, Emanuel Maltempi; de Oliveira Pedrosa, Fábio; Nodari, Rubens Onofre; Guerra, Miguel Pedro

    2014-01-01

    Background Podocarpus lambertii (Podocarpaceae) is a native conifer from the Brazilian Atlantic Forest Biome, which is considered one of the 25 biodiversity hotspots in the world. The advancement of next-generation sequencing technologies has enabled the rapid acquisition of whole chloroplast (cp) genome sequences at low cost. Several studies have proven the potential of cp genomes as tools to understand enigmatic and basal phylogenetic relationships at different taxonomic levels, as well as further probe the structural and functional evolution of plants. In this work, we present the complete cp genome sequence of P. lambertii. Methodology/Principal Findings The P. lambertii cp genome is 133,734 bp in length, and similar to other sequenced cupressophytes, it lacks one of the large inverted repeat regions (IR). It contains 118 unique genes and one duplicated tRNA (trnN-GUU), which occurs as an inverted repeat sequence. The rps16 gene was not found, which was previously reported for the plastid genome of another Podocarpaceae (Nageia nagi) and Araucariaceae (Agathis dammara). Structurally, P. lambertii shows 4 inversions of a large DNA fragment ∼20,000 bp compared to the Podocarpus totara cp genome. These unexpected characteristics may be attributed to geographical distance and different adaptive needs. The P. lambertii cp genome presents a total of 28 tandem repeats and 156 SSRs, with homo- and dipolymers being the most common and tri-, tetra-, penta-, and hexapolymers occurring with less frequency. Conclusion The complete cp genome sequence of P. lambertii revealed significant structural changes, even in species from the same genus. These results reinforce the apparently loss of rps16 gene in Podocarpaceae cp genome. In addition, several SSRs in the P. lambertii cp genome are likely intraspecific polymorphism sites, which may allow highly sensitive phylogeographic and population structure studies, as well as phylogenetic studies of species of this genus. PMID

  1. An internal part of the chloroplast atpA gene sequence is present in the mitochondrial genome of Triticum aestivum: molecular organisation and evolutionary aspects.

    Science.gov (United States)

    Jubier, M F; Lucas, H; Delcher, E; Hartmann, C; Quétier, F; Lejeune, B

    1990-06-01

    An internal part of the chloroplast atpA gene has been identified in the mitochondrial DNA of Triticum aestivum. It is located near the 18S-5S ribosomal genes and partially contained within a repeated sequence. Comparison of the transferred sequence with the original ct sequence reveals several nucleotide changes and shows that neither 5' nor 3' ends are present in the mt genome. No transcript of this region could be detected by Northern analysis. This sequence is present in mitochondrial genomes of other tetraploid and diploid species of Triticum, also in the vicinity of the 18S-5S ribosomal genes, suggesting a unique transfer event. The date of this event is discussed.

  2. Phylogeny of the basal angiosperm genus Pseuduvaria (Annonaceae) inferred from five chloroplast DNA regions, with interpretation of morphological character evolution.

    Science.gov (United States)

    Su, Yvonne C F; Smith, Gavin J D; Saunders, Richard M K

    2008-07-01

    Phylogenetic relationships within the magnoliid basal angiosperm genus Pseuduvaria (Annonaceae) are investigated using chloroplast DNA sequences from five regions: psbA-trnH spacer, trnL-F, matK, rbcL, and atpB-rbcL spacer. Over 4000 nucleotides from 51 species (of the total 53) were sequenced. The five cpDNA datasets were analyzed separately and in combination using maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. The phylogenetic trees constructed using all three phylogenetic methods, based on the combined data, strongly support the monophyly of Pseuduvaria following the inclusion of Craibella phuyensis. The trees generated using MP were less well resolved, but relationships are similar to those obtained using the other methods. ML and Bayesian analyses recovered trees with short branch lengths, showing five main clades. This study highlights the evolutionary changes in seven selected morphological characters (floral sex, stamen and carpel numbers, inner petal color, presence of inner petal glands, flowering peduncle length, and monocarp size). Although floral unisexuality is ancestral within the genus, several evolutionary lineages reveal reversal to bisexuality. Other phylogenetic transitions include the evolution of sapromyophily, and fruit-bat frugivory and seed dispersal, thus allowing a wide range of adaptations for species survival.

  3. The complete chloroplast genome sequence of an endemic monotypic genus Hagenia (Rosaceae: structural comparative analysis, gene content and microsatellite detection

    Directory of Open Access Journals (Sweden)

    Andrew W. Gichira

    2017-01-01

    Full Text Available Hagenia is an endangered monotypic genus endemic to the topical mountains of Africa. The only species, Hagenia abyssinica (Bruce J.F. Gmel, is an important medicinal plant producing bioactive compounds that have been traditionally used by African communities as a remedy for gastrointestinal ailments in both humans and animals. Complete chloroplast genomes have been applied in resolving phylogenetic relationships within plant families. We employed high-throughput sequencing technologies to determine the complete chloroplast genome sequence of H. abyssinica. The genome is a circular molecule of 154,961 base pairs (bp, with a pair of Inverted Repeats (IR 25,971 bp each, separated by two single copies; a large (LSC, 84,320 bp and a small single copy (SSC, 18,696. H. abyssinica’s chloroplast genome has a 37.1% GC content and encodes 112 unique genes, 78 of which code for proteins, 30 are tRNA genes and four are rRNA genes. A comparative analysis with twenty other species, sequenced to-date from the family Rosaceae, revealed similarities in structural organization, gene content and arrangement. The observed size differences are attributed to the contraction/expansion of the inverted repeats. The translational initiation factor gene (infA which had been previously reported in other chloroplast genomes was conspicuously missing in H. abyssinica. A total of 172 microsatellites and 49 large repeat sequences were detected in the chloroplast genome. A Maximum Likelihood analyses of 71 protein-coding genes placed Hagenia in Rosoideae. The availability of a complete chloroplast genome, the first in the Sanguisorbeae tribe, is beneficial for further molecular studies on taxonomic and phylogenomic resolution within the Rosaceae family.

  4. The complete chloroplast genome sequence of an endemic monotypic genus Hagenia (Rosaceae): structural comparative analysis, gene content and microsatellite detection.

    Science.gov (United States)

    Gichira, Andrew W; Li, Zhizhong; Saina, Josphat K; Long, Zhicheng; Hu, Guangwan; Gituru, Robert W; Wang, Qingfeng; Chen, Jinming

    2017-01-01

    Hagenia is an endangered monotypic genus endemic to the topical mountains of Africa. The only species, Hagenia abyssinica (Bruce) J.F. Gmel, is an important medicinal plant producing bioactive compounds that have been traditionally used by African communities as a remedy for gastrointestinal ailments in both humans and animals. Complete chloroplast genomes have been applied in resolving phylogenetic relationships within plant families. We employed high-throughput sequencing technologies to determine the complete chloroplast genome sequence of H. abyssinica. The genome is a circular molecule of 154,961 base pairs (bp), with a pair of Inverted Repeats (IR) 25,971 bp each, separated by two single copies; a large (LSC, 84,320 bp) and a small single copy (SSC, 18,696). H. abyssinica's chloroplast genome has a 37.1% GC content and encodes 112 unique genes, 78 of which code for proteins, 30 are tRNA genes and four are rRNA genes. A comparative analysis with twenty other species, sequenced to-date from the family Rosaceae, revealed similarities in structural organization, gene content and arrangement. The observed size differences are attributed to the contraction/expansion of the inverted repeats. The translational initiation factor gene (infA) which had been previously reported in other chloroplast genomes was conspicuously missing in H. abyssinica. A total of 172 microsatellites and 49 large repeat sequences were detected in the chloroplast genome. A Maximum Likelihood analyses of 71 protein-coding genes placed Hagenia in Rosoideae. The availability of a complete chloroplast genome, the first in the Sanguisorbeae tribe, is beneficial for further molecular studies on taxonomic and phylogenomic resolution within the Rosaceae family.

  5. The complete chloroplast genome sequence of an endemic monotypic genus Hagenia (Rosaceae): structural comparative analysis, gene content and microsatellite detection

    Science.gov (United States)

    Saina, Josphat K.; Long, Zhicheng; Hu, Guangwan; Gituru, Robert W.

    2017-01-01

    Hagenia is an endangered monotypic genus endemic to the topical mountains of Africa. The only species, Hagenia abyssinica (Bruce) J.F. Gmel, is an important medicinal plant producing bioactive compounds that have been traditionally used by African communities as a remedy for gastrointestinal ailments in both humans and animals. Complete chloroplast genomes have been applied in resolving phylogenetic relationships within plant families. We employed high-throughput sequencing technologies to determine the complete chloroplast genome sequence of H. abyssinica. The genome is a circular molecule of 154,961 base pairs (bp), with a pair of Inverted Repeats (IR) 25,971 bp each, separated by two single copies; a large (LSC, 84,320 bp) and a small single copy (SSC, 18,696). H. abyssinica’s chloroplast genome has a 37.1% GC content and encodes 112 unique genes, 78 of which code for proteins, 30 are tRNA genes and four are rRNA genes. A comparative analysis with twenty other species, sequenced to-date from the family Rosaceae, revealed similarities in structural organization, gene content and arrangement. The observed size differences are attributed to the contraction/expansion of the inverted repeats. The translational initiation factor gene (infA) which had been previously reported in other chloroplast genomes was conspicuously missing in H. abyssinica. A total of 172 microsatellites and 49 large repeat sequences were detected in the chloroplast genome. A Maximum Likelihood analyses of 71 protein-coding genes placed Hagenia in Rosoideae. The availability of a complete chloroplast genome, the first in the Sanguisorbeae tribe, is beneficial for further molecular studies on taxonomic and phylogenomic resolution within the Rosaceae family.

  6. Phylogenetic placement of Cynomorium in Rosales inferred from sequences of the inverted repeat region of the chloroplast genome

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hong ZHANG; Chun-Qi LI; Jian-hua LI

    2009-01-01

    Cynomorium is a herbaceous holoparasite that has been placed in Santalales, Saxifragales, Myrtales, or Sapindales. The inverted repeat (IR) region of the chloroplast genome region is slow evolving and, unlike mitochondrial genes, the chloroplast genome experiences few horizontal gene transfers between the host and parasite. Thus, in the present study, we used sequences of the IR region to test the phylogenetic placements of Cynomorium. Phylogenetic analyses of the chloroplast IR sequences generated largely congruent ordinal relationships with those from previous studies of angiosperm phylogeny based on single or multiple genes. Santalales was closely related to Caryophyllales and asterids. Saxifragales formed a clade where Peridiscus was sister to the remainder of the order, whereas Paeonia was sister to the woody clade of Saxifragales. Cynomorium is not closely related to Santalales, Saxifragales, Myrtales, or Sapindales; instead, it is included in Rosales and sister to Rosaceae. The various placements of the holoparasite on the basis of different regions of the mitochondrial genome may indicate the heterogeneous nature of the genome in the parasite. However, it is unlikely that the placement of Cynomorium in Rosales is the result of chloroplast gene transfer because Cynomorium does not parasitize on rosaceous plants and there is no chloroplast gene transfer between Cynomorium and Nitraria, a confirmed host of Cynomorium and a member of Sapindales.

  7. DNA Sequencing Using capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Barry Karger

    2011-05-09

    The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linked polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other

  8. The complete chloroplast genome sequences of five Epimedium species: lights into phylogenetic and taxonomic analyses

    Directory of Open Access Journals (Sweden)

    Yanjun eZhang

    2016-03-01

    Full Text Available Epimedium L. is a phylogenetically and economically important genus in the family Berberidaceae. We here sequenced the complete chloroplast (cp genomes of four Epimedium species using Illumina sequencing technology via a combination of de novo and reference-guided assembly, which was also the first comprehensive cp genome analysis on Epimedium combining the cp genome sequence of E. koreanum previously reported. The five Epimedium cp genomes exhibited typical quadripartite and circular structure that was rather conserved in genomic structure and the synteny of gene order. However, these cp genomes presented obvious variations at the boundaries of the four regions because of the expansion and contraction of the inverted repeat (IR region and the single-copy (SC boundary regions. The trnQ-UUG duplication occurred in the five Epimedium cp genomes, which was not found in the other basal eudicotyledons. The rapidly evolving cp genome regions were detected among the five cp genomes, as well as the difference of simple sequence repeats (SSR and repeat sequence were identified. Phylogenetic relationships among the five Epimedium species based on their cp genomes showed accordance with the updated system of the genus on the whole, but reminded that the evolutionary relationships and the divisions of the genus need further investigation applying more evidences. The availability of these cp genomes provided valuable genetic information for accurately identifying species, taxonomy and phylogenetic resolution and evolution of Epimedium, and assist in exploration and utilization of Epimedium plants.

  9. Noncoding sequences from the slowly evolving chloroplast inverted repeat in addition to rbcL data do not support gnetalean affinities of angiosperms.

    Science.gov (United States)

    Goremykin, V; Bobrova, V; Pahnke, J; Troitsky, A; Antonov, A; Martin, W

    1996-02-01

    We developed PCR primers against highly conserved regions of the rRNA operon located within the inverted repeat of the chloroplast genome and used these to amplify the region spanning from the 3' terminus of the 23S rRNA gene to the 5' terminus of the 5S rRNA gene. The sequence of this roughly 500-bp region, which includes the 4.5S rRNA gene and two chloroplast intergenic transcribed spacer regions (cpITS2 and cpITS3), was determined from 20 angiosperms, 7 gymnosperms, and 16 ferns (21,700 bp). Sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) from the same or confamilial genera were analyzed in both separate and combined data sets. Due to the low substitution rate in the inverted repeat region, noncoding sequences in the cpITS region are not saturated with substitutions, in contrast to synonymous sites in rbcL, which are shown to evolve roughly six times faster than noncoding cpITS sequences. Several length polymorphisms with very clear phylogenetic distributions were detected in the data set. Results of phylogenetic analyses provide very strong bootstrap support for monophyly of both spermatophytes and angiosperms. No support for a sister group relationship between Gnetales and angiosperms in either cpITS or rbcL data was found. Rather, weak bootstrap support for monophyly of gymnosperms studied and for a basal position for the aquatic angiosperm Nymphaea among angiosperms studied was observed. Noncoding sequences from the inverted repeat region of chloroplast DNA appear suitable for study of land plant evolution.

  10. The nucleotide sequence of 4.5S ribosomal RNA from tobacco chloroplasts.

    OpenAIRE

    Takaiwa, F; Sugiura, M

    1980-01-01

    The nucleotide sequence of tobacco chloroplast 4.5S ribosomal RNA has been determined to be: OHG-A-A-G-G-U-C-A-C-G-G-C-G-A-G-A-C-G-A-G-C-C-G-U-U-U-A-U-C-A-U-U-A-C-G-A-U-A-G-G-U-G-U-C-A-A-G-U-G-G-A-A-G-U-G-C-A-G-U-G-A-U-G-U-A-U-G-C-(G-A)-C-U-G-A-G-G-C-A-U-C-C-U-A-A-C-A-G-A-C-C-G-G-U-A-G-A-C-U-U-G-A-A-COH. The 4.5S RNA is 103 nucleotides long and its 5'-terminus is not phosphorylated.

  11. The complete chloroplast genome sequence of the medicinal plant Salvia miltiorrhiza.

    Science.gov (United States)

    Qian, Jun; Song, Jingyuan; Gao, Huanhuan; Zhu, Yingjie; Xu, Jiang; Pang, Xiaohui; Yao, Hui; Sun, Chao; Li, Xian'en; Li, Chuyuan; Liu, Juyan; Xu, Haibin; Chen, Shilin

    2013-01-01

    Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.

  12. The complete chloroplast genome sequence of the medicinal plant Salvia miltiorrhiza.

    Directory of Open Access Journals (Sweden)

    Jun Qian

    Full Text Available Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp and small (SSC, 17,555 bp single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp. It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.

  13. Information Analysis of DNA Sequences

    CERN Document Server

    Mohammed, Riyazuddin

    2010-01-01

    The problem of differentiating the informational content of coding (exons) and non-coding (introns) regions of a DNA sequence is one of the central problems of genomics. The introns are estimated to be nearly 95% of the DNA and since they do not seem to participate in the process of transcription of amino-acids, they have been termed "junk DNA." Although it is believed that the non-coding regions in genomes have no role in cell growth and evolution, demonstration that these regions carry useful information would tend to falsify this belief. In this paper, we consider entropy as a measure of information by modifying the entropy expression to take into account the varying length of these sequences. Exons are usually much shorter in length than introns; therefore the comparison of the entropy values needs to be normalized. A length correction strategy was employed using randomly generated nucleonic base strings built out of the alphabet of the same size as the exons under question. Our analysis shows that intron...

  14. Chloroplast DNA variation and phylogeography of Ligularia tongolensis (Asteraceae), a species endemic to the Hengduan Mountains region of China

    Institute of Scientific and Technical Information of China (English)

    Jin-Feng WANG; Yue-Zhi PAN; Xun GONG; Yu-Chung CHIANG; Chiaki KURODA

    2011-01-01

    In this research, we aimed to study the genetic variation and phylogeographic pattern of Ligularia tongolensis, a perennial herb endemic to the Hengduan Mountains region of China. We sequenced two chloroplast DNA (cpDNA) intergenic spacers (trnQ-5'rps 16, trnL-rpl32) in 140 individuals from 14 populations of three groups (Jinshajiang vs. Yalongjiang vs. Wumeng) within this species range. High levels of haplotype diversity (Hd= 0.814)and total genetic diversity (Ht = 0.862) were detected at the species level, based on a total of 12 haplotypes identified.Low levels of intrapopulation diversity (Hs = 0.349), high levels of genetic divergence (Gst = 0.595, Nst = 0.614,Fst = 0.597), and the absence of isolation by distance tests were also found in L. tongolensis. Furthermore, H2 and H5, the dominant haplotypes that located at internal nodes and deviated from extinct ancestral haplotype in the network, were found to be shared between Jinshajiang and Yalongjiang groups. These results indicate that past fragmentation may be the important factor responsible for the present phylogeographical pattern of L. tongolensis.Meanwhile, the locations occupied by each group might have served as independent refugia for L. tongolensis during the Quaternary glaciation. Unimodal mismatch distribution and star-like genealogies indicated this species underwent past demographic expansion events, with expansion ages of 274 ka BP.

  15. High-throughput sequencing of six bamboo chloroplast genomes: phylogenetic implications for temperate woody bamboos (Poaceae: Bambusoideae.

    Directory of Open Access Journals (Sweden)

    Yun-Jie Zhang

    Full Text Available BACKGROUND: Bambusoideae is the only subfamily that contains woody members in the grass family, Poaceae. In phylogenetic analyses, Bambusoideae, Pooideae and Ehrhartoideae formed the BEP clade, yet the internal relationships of this clade are controversial. The distinctive life history (infrequent flowering and predominance of asexual reproduction of woody bamboos makes them an interesting but taxonomically difficult group. Phylogenetic analyses based on large DNA fragments could only provide a moderate resolution of woody bamboo relationships, although a robust phylogenetic tree is needed to elucidate their evolutionary history. Phylogenomics is an alternative choice for resolving difficult phylogenies. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the complete nucleotide sequences of six woody bamboo chloroplast (cp genomes using Illumina sequencing. These genomes are similar to those of other grasses and rather conservative in evolution. We constructed a phylogeny of Poaceae from 24 complete cp genomes including 21 grass species. Within the BEP clade, we found strong support for a sister relationship between Bambusoideae and Pooideae. In a substantial improvement over prior studies, all six nodes within Bambusoideae were supported with ≥0.95 posterior probability from Bayesian inference and 5/6 nodes resolved with 100% bootstrap support in maximum parsimony and maximum likelihood analyses. We found that repeats in the cp genome could provide phylogenetic information, while caution is needed when using indels in phylogenetic analyses based on few selected genes. We also identified relatively rapidly evolving cp genome regions that have the potential to be used for further phylogenetic study in Bambusoideae. CONCLUSIONS/SIGNIFICANCE: The cp genome of Bambusoideae evolved slowly, and phylogenomics based on whole cp genome could be used to resolve major relationships within the subfamily. The difficulty in resolving the diversification among

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

    Directory of Open Access Journals (Sweden)

    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.

  17. The complete chloroplast genome sequence of Ampelopsis: gene organization, comparative analysis and phylogenetic relationships to other angiosperms

    Directory of Open Access Journals (Sweden)

    Gurusamy eRaman

    2016-03-01

    Full Text Available Ampelopsis brevipedunculata is an economically important plant that belongs to the Vitaceae family of angiosperms. The phylogenetic placement of Vitaceae is still unresolved. Recent phylogenetic studies suggested that it should be placed in various alternative families including Caryophyllaceae, asteraceae, Saxifragaceae, Dilleniaceae, or with the rest of the rosid families. However, these analyses provided weak supportive results because they were based on only one of several genes. Accordingly, complete chloroplast genome sequences are required to resolve the phylogenetic relationships among angiosperms. Recent phylogenetic analyses based on the complete chloroplast genome sequence suggested strong support for the position of Vitaceae as the earliest diverging lineage of rosids and placed it as a sister to the remaining rosids. These studies also revealed relationships among several major lineages of angiosperms; however, they highlighted the significance of taxon sampling for obtaining accurate phylogenies. In the present study, we sequenced the complete chloroplast genome of A. brevipedunculata and used these data to assess the relationships among 32 angiosperms, including 18 taxa of rosids. The Ampelopsis chloroplast genome is 161,090 bp in length, and includes a pair of inverted repeats of 26,394 bp that are separated by small and large single copy regions of 19,036 bp and 89,266 bp, respectively. The gene content and order of Ampelopsis is identical to many other unrearranged angiosperm chloroplast genomes, including Vitis and tobacco. A phylogenetic tree constructed based on 70 protein-coding genes of 33 angiosperms showed that both Saxifragales and Vitaceae diverged from the rosid clade and formed two clades with 100% bootstrap value. The position of the Vitaceae is sister to Saxifragales, and both are the basal and earliest diverging lineages. Moreover, Saxifragales forms a sister clade to Vitaceae of rosids. Overall, the results of

  18. Systematic positions of Lamiophiomis and Paraphlomis (Lamiaceae) based on nuclear and chloroplast sequences

    Institute of Scientific and Technical Information of China (English)

    Yue-Zhi PAN; Li-Qin FANG; Gang HAO; Jie CAI; Xun GONG

    2009-01-01

    Genera Lamiophlomis and Paraphlomis were originally separated from genus Phlomis s.l. on the basis of particular morphological characteristics. However, their relationship was highly contentious, as evidenced by the literature. In the present paper, the systematic positions of Lamiophlomis, Paraphlomis, and their related genera were assessed based on nuclear internal transcribed spacer (ITS) and chloroplast rpl16 and trnL-F sequence data using maximum parsimony (MP) and Bayesian methods. In total, 24 species representing six genera of the ingroup and outgroup were sampled. Analyses of both separate and combined sequence data were conducted to resolve the systematic relationships of these genera. The results reveal that Lamiophlomis is nested within Phlomis sect. Phlomoides and its genetic status is not supported. With the inclusion of Lamiophlomis rotata in sect. Phlomoides, sections Phlomis and Phlomoides of Phlomis were resolved as monophyletic. Paraphlomis was supported as an inde-pendent genus. However, the resolution of its monophyly conflicted between MP and Bayesian analyses, suggesting the need for expended sampling and further evidence.

  19. The Chloroplast Genome of Passiflora edulis (Passifloraceae) Assembled from Long Sequence Reads: Structural Organization and Phylogenomic Studies in Malpighiales

    Science.gov (United States)

    Cauz-Santos, Luiz A.; Munhoz, Carla F.; Rodde, Nathalie; Cauet, Stephane; Santos, Anselmo A.; Penha, Helen A.; Dornelas, Marcelo C.; Varani, Alessandro M.; Oliveira, Giancarlo C. X.; Bergès, Hélène; Vieira, Maria Lucia C.

    2017-01-01

    The family Passifloraceae consists of some 700 species classified in around 16 genera. Almost all its members belong to the genus Passiflora. In Brazil, the yellow passion fruit (Passiflora edulis) is of considerable economic importance, both for juice production and consumption as fresh fruit. The availability of chloroplast genomes (cp genomes) and their sequence comparisons has led to a better understanding of the evolutionary relationships within plant taxa. In this study, we obtained the complete nucleotide sequence of the P. edulis chloroplast genome, the first entirely sequenced in the Passifloraceae family. We determined its structure and organization, and also performed phylogenomic studies on the order Malpighiales and the Fabids clade. The P. edulis chloroplast genome is characterized by the presence of two copies of an inverted repeat sequence (IRA and IRB) of 26,154 bp, each separating a small single copy region of 13,378 bp and a large single copy (LSC) region of 85,720 bp. The annotation resulted in the identification of 105 unique genes, including 30 tRNAs, 4 rRNAs, and 71 protein coding genes. Also, 36 repetitive elements and 85 SSRs (microsatellites) were identified. The structure of the complete cp genome of P. edulis differs from that of other species because of rearrangement events detected by means of a comparison based on 22 members of the Malpighiales. The rearrangements were three inversions of 46,151, 3,765 and 1,631 bp, located in the LSC region. Phylogenomic analysis resulted in strongly supported trees, but this could also be a consequence of the limited taxonomic sampling used. Our results have provided a better understanding of the evolutionary relationships in the Malpighiales and the Fabids, confirming the potential of complete chloroplast genome sequences in inferring evolutionary relationships and the utility of long sequence reads for generating very accurate biological information. PMID:28344587

  20. The complete chloroplast genome sequence of Citrus sinensis (L. Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms

    Directory of Open Access Journals (Sweden)

    Jansen Robert K

    2006-09-01

    Full Text Available Abstract Background The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over nuclear transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms. Results The complete chloroplast genome sequence of Citrus sinensis is 160,129 bp in length, and contains 133 genes (89 protein-coding, 4 rRNAs and 30 distinct tRNAs. Genome organization is very similar to the inferred ancestral angiosperm chloroplast genome. However, in Citrus the infA gene is absent. The inverted repeat region has expanded to duplicate rps19 and the first 84 amino acids of rpl22. The rpl22 gene in the IRb region has a nonsense mutation resulting in 9 stop codons. This was confirmed by PCR amplification and sequencing using primers that flank the IR/LSC boundaries. Repeat analysis identified 29 direct and inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Comparison of protein-coding sequences with expressed sequence tags revealed six putative RNA edits, five of which resulted in non-synonymous modifications in petL, psbH, ycf2 and ndhA. Phylogenetic analyses using maximum parsimony (MP and maximum likelihood (ML methods of a dataset composed of 61 protein-coding genes for 30 taxa provide strong support for the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids and asterids. The MP and ML trees are incongruent in three areas: the position of Amborella and

  1. The complete chloroplast genome sequence of Pelargonium x hortorum: organization and evolution of the largest and most highly rearranged chloroplast genome of land plants.

    Science.gov (United States)

    Chumley, Timothy W; Palmer, Jeffrey D; Mower, Jeffrey P; Fourcade, H Matthew; Calie, Patrick J; Boore, Jeffrey L; Jansen, Robert K

    2006-11-01

    The chloroplast genome of Pelargonium x hortorum has been completely sequenced. It maps as a circular molecule of 217,942 bp and is both the largest and most rearranged land plant chloroplast genome yet sequenced. It features 2 copies of a greatly expanded inverted repeat (IR) of 75,741 bp each and, consequently, diminished single-copy regions of 59,710 and 6,750 bp. Despite the increase in size and complexity of the genome, the gene content is similar to that of other angiosperms, with the exceptions of a large number of pseudogenes, the recognition of 2 open reading frames (ORF56 and ORF42) in the trnA intron with similarities to previously identified mitochondrial products (ACRS and pvs-trnA), the losses of accD and trnT-ggu and, in particular, the presence of a highly divergent set of rpoA-like ORFs rather than a single, easily recognized gene for rpoA. The 3-fold expansion of the IR (relative to most angiosperms) accounts for most of the size increase of the genome, but an additional 10% of the size increase is related to the large number of repeats found. The Pelargonium genome contains 35 times as many 31 bp or larger repeats than the unrearranged genome of Spinacia. Most of these repeats occur near the rearrangement hotspots, and 2 different associations of repeats are localized in these regions. These associations are characterized by full or partial duplications of several genes, most of which appear to be nonfunctional copies or pseudogenes. These duplications may also be linked to the disruption of at least 1 but possibly 2 or 3 operons. We propose simple models that account for the major rearrangements with a minimum of 8 IR boundary changes and 12 inversions in addition to several insertions of duplicated sequence.

  2. Chloroplast DNA diversity reveals the contribution of two wild species to the origin and evolution of diploid safflower (Carthamus tinctorius L.).

    Science.gov (United States)

    Sehgal, Deepmala; Rajpal, Vijay Rani; Raina, Soom Nath

    2008-08-01

    The identity of the wild progenitor of one of the most important oil crop species, Carthamus tinctorius (2n = 2x = 24), commonly known as safflower, has been the subject of numerous studies at morphological, biochemical, cytogenetic, and biosystematic levels, but no definitive conclusions have been made. The nuclear, mitochondrial, and chloroplast genomes of the two botanical varieties of C. tinctorius, C. tinctorius var. tinctorius and C. tinctorius var. inermis, and two wild species, C. palaestinus and C. oxyacantha, were assayed at the nucleotide sequence level and by DNA markers. The nuclear and mitochondrial DNA assays were not helpful in conclusively identifying the diploid ancestor of C. tinctorius. The chloroplast DNA diversity, on the other hand, unambiguously provided new and novel evidence that C. palaestinus and C. oxyacantha contributed their plastomes to the evolution of C. tinctorius var. inermis and C. tinctorius var. tinctorius, respectively. This study, therefore, affirms a startling revelation of a rare event of two wild species contributing to the origin and evolution of safflower, a major world oilseed crop about whose genetics very little is known.

  3. Comparative chloroplast genomics: analyses including new sequences from the angiosperms Nuphar advena and Ranunculus macranthus

    Directory of Open Access Journals (Sweden)

    Boore Jeffrey L

    2007-06-01

    Full Text Available Abstract Background The number of completely sequenced plastid genomes available is growing rapidly. This array of sequences presents new opportunities to perform comparative analyses. In comparative studies, it is often useful to compare across wide phylogenetic spans and, within angiosperms, to include representatives from basally diverging lineages such as the genomes reported here: Nuphar advena (from a basal-most lineage and Ranunculus macranthus (a basal eudicot. We report these two new plastid genome sequences and make comparisons (within angiosperms, seed plants, or all photosynthetic lineages to evaluate features such as the status of ycf15 and ycf68 as protein coding genes, the distribution of simple sequence repeats (SSRs and longer dispersed repeats (SDR, and patterns of nucleotide composition. Results The Nuphar [GenBank:NC_008788] and Ranunculus [GenBank:NC_008796] plastid genomes share characteristics of gene content and organization with many other chloroplast genomes. Like other plastid genomes, these genomes are A+T-rich, except for rRNA and tRNA genes. Detailed comparisons of Nuphar with Nymphaea, another Nymphaeaceae, show that more than two-thirds of these genomes exhibit at least 95% sequence identity and that most SSRs are shared. In broader comparisons, SSRs vary among genomes in terms of abundance and length and most contain repeat motifs based on A and T nucleotides. Conclusion SSR and SDR abundance varies by genome and, for SSRs, is proportional to genome size. Long SDRs are rare in the genomes assessed. SSRs occur less frequently than predicted and, although the majority of the repeat motifs do include A and T nucleotides, the A+T bias in SSRs is less than that predicted from the underlying genomic nucleotide composition. In codon usage third positions show an A+T bias, however variation in codon usage does not correlate with differences in A+T-richness. Thus, although plastome nucleotide composition shows "A

  4. Phylogenetic relationships and generic delimitation in Inuleae subtribe Inulinae (Asteraceae) based on ITS and cpDNA sequence data

    DEFF Research Database (Denmark)

    Englund, Marcus; Pornpongrungrueng, Pimwadee; Gustafsson, Mats

    2009-01-01

    Phylogenetic relationships in Inuleae subtribe Inulinae (Asteraceae) were investigated. DNA sequence data from three chloroplast regions (ndhF, trnL-F and psbA-trnH) and the nuclear ribosomal internal transcribed spacer (ITS) region were analysed separately and in combination using parsimony...

  5. Relationships in Ananas and other related genera using chloroplast DNA restriction site variation.

    Science.gov (United States)

    Duval, M F; Buso, G S C; Ferreira, F R; Noyer, J L; Coppens d'Eeckenbrugge, G; Hamon, P; Ferreira, M E

    2003-12-01

    Chloroplast DNA (cpDNA) diversity was examined using PCR-RFLP to study phylogenetic relationships in Ananas and related genera. One hundred fifteen accessions representing the seven Ananas species and seven other Bromelioideae including the neighboring monospecific genus Pseudananas, two Pitcairnioideae, and one Tillandsioideae were included in the study. Eight primers designed from cpDNA were used for generating fragments. Restriction by 18 endonucleases generated 255 variable fragments. Dissimilarities were calculated from the resulting matrix using the Sokal and Michener index and the neighbor-joining method was used to reconstruct the diversity tree. Phylogenetic reconstruction was attempted using Wagner parsimony. Phenetic and cladistic analyses gave consistent results. They confirm the basal position of Bromelia in the Bromelioideae. Ananas and Pseudananas form a monophyletic group, with three strongly supported sub-groups, two of which are geographically consistent. The majority of Ananas parguazensis accessions constitute a northern group restricted to the Rio Negro and Orinoco basins in Brazil. The tetraploid Pseudananas sagenarius joins the diploid Ananas fritzmuelleri to constitute a southern group. The third and largest group, which includes all remaining species plus some accessions of A. parguazensis and intermediate phenotypes, is the most widespread and its distribution overlaps those of the northern and southern groups. Ananas ananassoides is dominant in this sub-group and highly variable. Its close relationship to all cultivated species supports the hypothesis that this species is the wild ancestor of the domesticated pineapple. The data indicate that gene flow is common within this group and scarcer with both the first and second groups. Comparison of cpDNA data with published genomic DNA data point to the hybrid origin of Ananas bracteatus and support the autopolyploidy of Pseudananas. The Ananas-Pseudananas group structure and distribution are

  6. Chloroplast and microsatellite DNA diversities reveal the introduction history of Brazilian peppertree (Schinus terebinthifolius) in Florida.

    Science.gov (United States)

    Williams, Dean A; Overholt, William A; Cuda, James P; Hughes, Colin R

    2005-10-01

    Brazilian peppertree (Schinus terebinthifolius) is a woody perennial that has invaded much of Florida. This native of northeastern Argentina, Paraguay, and Brazil was brought as an ornamental to both the west and east coasts of Florida at the end of the 19th century. It was recorded as an invader of natural areas in the 1950s, and has since extended its range to cover over 280 000 ha. Our goals were to understand the history of this invasion, as one step toward understanding why this exotic was so successful, and ultimately to improve development of biological control agents. We sampled plants from the native and exotic ranges, particularly Florida, and genotyped these individuals at nuclear and chloroplast loci. Nuclear microsatellite and cpDNA loci reveal strong genetic population structure consistent with limited dispersal in the introduced and native ranges. Bayesian clustering of microsatellite data separates the east and west coast plants in Florida into distinct populations. The two chloroplast haplotypes found in Florida are also concordant with this separation: one predominates on the east coast, the other on the west coast. Analysis of samples collected in South America shows that haplotypes as distinct as the two in Florida are unlikely to have come from a single source population. We conclude that the genetic evidence supports two introductions of Brazilian peppertree into Florida and extensive hybridization between them. The west coast genotype likely came from coastal Brazil at about 27 degrees south, whereas the east coast genotype probably originated from another, as yet unidentified site. As a result of hybridization, the Florida population does not exhibit low genetic variation compared to populations in the native range, possibly increasing its ability to adapt to novel environments. Hybridization also has important consequences for the selection of biocontrol agents since it will not be possible to identify closely co-adapted natural enemies in

  7. The complete chloroplast genome sequence of Pelargonium xhortorum: Or ganization and evolution of the largest and most highlyrearranged chloroplast genome of land plants

    Energy Technology Data Exchange (ETDEWEB)

    Chumley, Timothy W.; Palmer, Jeffrey D.; Mower, Jeffrey P.; Fourcade, H. Matthew; Calie, Patrick J.; Boore, Jeffrey L.; Jansen,Robert K.

    2006-01-20

    The chloroplast genome of Pelargonium e hortorum has beencompletely sequenced. It maps as a circular molecule of 217,942 bp, andis both the largest and most rearranged land plant chloroplast genome yetsequenced. It features two copies of a greatly expanded inverted repeat(IR) of 75,741 bp each, and consequently diminished single copy regionsof 59,710 bp and 6,750 bp. It also contains two different associations ofrepeated elements that contribute about 10 percent to the overall sizeand account for the majority of repeats found in the genome. Theyrepresent hotspots for rearrangements and gene duplications and include alarge number of pseudogenes. We propose simple models that account forthe major rearrangements with a minimum of eight IR boundary changes and12 inversions in addition to a several insertions of duplicated sequence.The major processes at work (duplication, IR expansion, and inversion)have disrupted at least one and possibly two or three transcriptionaloperons, and the genes involved in these disruptions form the core of thetwo major repeat associations. Despite the vast increase in size andcomplexity of the genome, the gene content is similar to that of otherangiosperms, with the exceptions of a large number of pseudogenes as partof the repeat associations, the recognition of two open reading frames(ORF56 and ORF42) in the trnA intron with similarities to previouslyidentified mitochondrial products (ACRS and pvs-trnA), the loss of accDand trnT-GGU, and in particular, the lack of a recognizably functionalrpoA. One or all of three similar open reading frames may possibly encodethe latter, however.

  8. Complete Chloroplast Genome Sequence of Aquilaria sinensis (Lour. Gilg and the Evolution Analysis within the Malvalesorder

    Directory of Open Access Journals (Sweden)

    Ying eWang

    2016-03-01

    Full Text Available Aquilaria sinensis (Lour. Gilg is an important medicinal woody plant producing agarwood, which is widely used in traditional Chinese medicine. High-throughput sequencing of chloroplast (cp genomes enhanced the understanding about evolutionary relationships within plant families. In this study, we determined the complete cp genome sequences for A. sinensis. The size of the A.sinensis cp genome was 159,565 bp. This genome included a large single-copy region of 87,482 bp, a small single-copy region of 19,857 bp, and a pair of inverted repeats (IRa and IRb of 26,113 bp each. The GC content of the genome was 37.11%. The A.sinensis cp genome encoded 113 functional genes, including 82 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. Seven genes were duplicated in the protein-coding genes, whereas 11 genes were duplicated in the RNA genes. A total of 45 polymorphic simple-sequence repeat loci and 60 pairs of large repeats were identified. Most simple-sequence repeats were located in the noncoding sections of the large single-copy/small single-copy region and exhibited high A/T content. Moreover, 33 pairs of large repeat sequences were located in the protein-coding genes, whereas 27 pairs were located in the intergenic regions. Aquilaria sinensis cp genome bias ended with A/T on the basis of codon usage. The distribution of codon usage in A.sinensis cp genome was most similar to that in the Gonystylus bancanus cp genome. Comparative results of 82 protein-coding genes from 29 species of cp genomes demonstrated that A.sinensis was a sister species to G. bancanus within the Malvales order. Aquilaria sinensis cp genome presented the highest sequence similarity of >90% with the G. bancanus cp genome by using CGView Comparison Tool. This finding strongly supports the placement of A.sinensis as a sister to G. bancanus within the Malvales order. The complete A.sinensis cp genome information will be highly beneficial for further studies on this traditional

  9. [DNA sequencing technology and automatization of it].

    Science.gov (United States)

    Kraev, A S

    1991-01-01

    Precise manipulations with genetic material, typical for modern experiments in molecular biology and in new biotechnology, require a capability to determine DNA base sequence. This capability enables today to exploit specific genetic knowledge for the dissection of complex cell processes and for modulation of cell metabolism in transgenic organisms. The review focuses on such DNA sequencing technologies that are widespread in general laboratory practice. They can safely be called, with the availability of commercial reagents, industrial techniques. Modern DNA sequencing requires recurrent breakdown of large genomic DNA into smaller pieces, that are then amplified, sequenced and the initial long stretch reconstructed via overlap of small pieces. The DNA sequencing process has several steps: a DNA fragment is obtained in sufficient quantity and purity, it is converted to a form suitable for a particular sequencing method, a sequencing reaction is performed and its products fractionated; and finally the resultant data are interpreted (i.e. an autoradiograph is read into a computer memory) and a long sequence in reconstructed via overlap of short stretches. These steps are considered in separate parts; an accent is made on sequencing strategies with respect to their biological task. In the last part, possibilities for automation of sequencing experiment are considered, followed by a discussion of domestic problems in DNA sequencing.

  10. OrgConv: detection of gene conversion using consensus sequences and its application in plant mitochondrial and chloroplast homologs

    Directory of Open Access Journals (Sweden)

    Hao Weilong

    2010-03-01

    Full Text Available Abstract Background The ancestry of mitochondria and chloroplasts traces back to separate endosymbioses of once free-living bacteria. The highly reduced genomes of these two organelles therefore contain very distant homologs that only recently have been shown to recombine inside the mitochondrial genome. Detection of gene conversion between mitochondrial and chloroplast homologs was previously impossible due to the lack of suitable computer programs. Recently, I developed a novel method and have, for the first time, discovered recurrent gene conversion between chloroplast mitochondrial genes. The method will further our understanding of plant organellar genome evolution and help identify and remove gene regions with incongruent phylogenetic signals for several genes widely used in plant systematics. Here, I implement such a method that is available in a user friendly web interface. Results OrgConv (Organellar Conversion is a computer package developed for detection of gene conversion between mitochondrial and chloroplast homologous genes. OrgConv is available in two forms; source code can be installed and run on a Linux platform and a web interface is available on multiple operating systems. The input files of the feature program are two multiple sequence alignments from different organellar compartments in FASTA format. The program compares every examined sequence against the consensus sequence of each sequence alignment rather than exhaustively examining every possible combination. Making use of consensus sequences significantly reduces the number of comparisons and therefore reduces overall computational time, which allows for analysis of very large datasets. Most importantly, with the significantly reduced number of comparisons, the statistical power remains high in the face of correction for multiple tests. Conclusions Both the source code and the web interface of OrgConv are available for free from the OrgConv website http

  11. Fibonacci Sequence and Supramolecular Structure of DNA.

    Science.gov (United States)

    Shabalkin, I P; Grigor'eva, E Yu; Gudkova, M V; Shabalkin, P I

    2016-05-01

    We proposed a new model of supramolecular DNA structure. Similar to the previously developed by us model of primary DNA structure [11-15], 3D structure of DNA molecule is assembled in accordance to a mathematic rule known as Fibonacci sequence. Unlike primary DNA structure, supramolecular 3D structure is assembled from complex moieties including a regular tetrahedron and a regular octahedron consisting of monomers, elements of the primary DNA structure. The moieties of the supramolecular DNA structure forming fragments of regular spatial lattice are bound via linker (joint) sequences of the DNA chain. The lattice perceives and transmits information signals over a considerable distance without acoustic aberrations. Linker sequences expand conformational space between lattice segments allowing their sliding relative to each other under the action of external forces. In this case, sliding is provided by stretching of the stacked linker sequences.

  12. Complete chloroplast genome sequences of Drimys, Liriodendron, andPiper: Implications for the phylogeny of magnoliids and the evolution ofGC content

    Energy Technology Data Exchange (ETDEWEB)

    Zhengqiu, C.; Penaflor, C.; Kuehl, J.V.; Leebens-Mack, J.; Carlson, J.; dePamphilis, C.W.; Boore, J.L.; Jansen, R.K.

    2006-06-01

    the inverted repeat due to the presence of rRNA genes and lowest in the small single copy region where most NADH genes are located. Phylogenetic analyses using maximum parsimony and maximum likelihood methods were performed on DNA sequences of 61 protein-coding genes. Trees from both analyses provided strong support for the monophyly of magnoliids and two strongly supported groups were identified, the Canellales/Piperales and the Laurales/Magnoliales. The phylogenies also provided moderate to strong support for the basal position of Amborella, and a sister relationship of magnoliids to a clade that includes monocots and eudicots. The complete sequences of three magnoliid chloroplast genomes provide new data from the largest basal angiosperm clade. Evolutionary comparisons of these new genome sequences, combined with other published angiosperm genome, confirm that GC content is unevenly distributed across the genome by location, codon position, and functional group. Furthermore, phylogenetic analyses provide the strongest support so far for the hypothesis that the magnoliids are sister to a large clade that includes both monocots and eudicots.

  13. Complete Chloroplast Genome Sequence of Omani Lime (Citrus aurantiifolia) and Comparative Analysis within the Rosids

    OpenAIRE

    Huei-Jiun Su; Hogenhout, Saskia A.; Al-Sadi, Abdullah M.; Chih-Horng Kuo

    2014-01-01

    The genus Citrus contains many economically important fruits that are grown worldwide for their high nutritional and medicinal value. Due to frequent hybridizations among species and cultivars, the exact number of natural species and the taxonomic relationships within this genus are unclear. To compare the differences between the Citrus chloroplast genomes and to develop useful genetic markers, we used a reference-assisted approach to assemble the complete chloroplast genome of Omani lime (C....

  14. Cloning and Analysis of a cDNA Encoding psbL and psbJ Gene in Rice Chloroplast Genome%水稻叶绿体基因组中一个编码psbL 和psbJ基因cDNA的克隆与分析

    Institute of Scientific and Technical Information of China (English)

    顾克余; 罗林广; 苏昌潮; 翟虎渠

    2001-01-01

    A 505 bp cDNA was cloned from the leaves of rice (Oryza sativaL.) Shanyou 63 combination. DNA sequence analysis showed that it is a part of rice chloroplast genome. Its homology comparison with those known in GenBank found that it encodes 38 amino acid peptide deduced from psbL gene and 40 amino acid peptide deduced from psbJ gene in rice chloroplast PSⅡ. Northern hybridization showed that the cDNA was differentially displayed in hybrid F1 and its parental lines.

  15. Mitochondrial DNA sequence evolution in shorebird populations.

    NARCIS (Netherlands)

    Wenink, P.W.

    1994-01-01

    This thesis describes the global molecular population structure of two shorebird species, in particular of the dunlin, Calidris alpina, by means of comparative sequence analysis of the most variable part of the mitochondrial DNA (mtDNA) genome. There are several reasons why mtDNA is the molecule of

  16. The complete chloroplast genome sequence of Cephalotaxus oliveri (Cephalotaxaceae): evolutionary comparison of cephalotaxus chloroplast DNAs and insights into the loss of inverted repeat copies in gymnosperms.

    Science.gov (United States)

    Yi, Xuan; Gao, Lei; Wang, Bo; Su, Ying-Juan; Wang, Ting

    2013-01-01

    We have determined the complete chloroplast (cp) genome sequence of Cephalotaxus oliveri. The genome is 134,337 bp in length, encodes 113 genes, and lacks inverted repeat (IR) regions. Genome-wide mutational dynamics have been investigated through comparative analysis of the cp genomes of C. oliveri and C. wilsoniana. Gene order transformation analyses indicate that when distinct isomers are considered as alternative structures for the ancestral cp genome of cupressophyte and Pinaceae lineages, it is not possible to distinguish between hypotheses favoring retention of the same IR region in cupressophyte and Pinaceae cp genomes from a hypothesis proposing independent loss of IRA and IRB. Furthermore, in cupressophyte cp genomes, the highly reduced IRs are replaced by short repeats that have the potential to mediate homologous recombination, analogous to the situation in Pinaceae. The importance of repeats in the mutational dynamics of cupressophyte cp genomes is also illustrated by the accD reading frame, which has undergone extreme length expansion in cupressophytes. This has been caused by a large insertion comprising multiple repeat sequences. Overall, we find that the distribution of repeats, indels, and substitutions is significantly correlated in Cephalotaxus cp genomes, consistent with a hypothesis that repeats play a role in inducing substitutions and indels in conifer cp genomes.

  17. LeCDJ1, a chloroplast DnaJ protein, facilitates heat tolerance in transgenic tomatoes

    Institute of Scientific and Technical Information of China (English)

    Fanying Kong; Yongsheng Deng; Guodong Wang; Jieru Wang; Xiaoqing Liang; Qingwei Meng

    2014-01-01

    The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1) were investigat-ed using wild-type (WT) and sense transgenic tomatoes. The LeCDJ1 expression was upregulated by 38 °C, 42 °C, 45 °C, NaCl, PEG, methyl viologen (MV) and hydrogen peroxide (H2O2), but not by 30 °C and 35 °C. Meanwhile, LeCDJ1 was involved in the response of plants to abscisic acid (ABA). Under heat stress, the sense plants showed better growth, higher chlorophyll content, lower malondialdehyde (MDA) accumulation and relative electrical conductivity (REC), and also less PSII photoinhibition than WT. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher maximum photo-chemistry efficiency of PSII (Fv/Fm) and D1 protein levels than the SM-untreated WT, suggesting that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, the relatively lower super-oxide radical (O2•?) and H2O2 levels in the sense plants were considered to be due to the higher ascorbate peroxidase (APX) and superoxide dismutase (SOD) activity, which seemed unlikely dependent on their transcription level. These results indicated that LeCDJ1 overexpression facilitated heat tolerance in transgenic tomatoes.

  18. Glacial Refugia of Ginkgo biloba and Human Impact on Its Genetic Diversity: Evidence from Chloroplast DNA

    Institute of Scientific and Technical Information of China (English)

    Wei Gong; Zhen Zeng; Ye-Ye Chen; Chuan Chen; Ying-Xiong Qiu; Cheng-Xin Fu

    2008-01-01

    Variations in the trnK region of chloroplast DNA were investigated in the present study using polymerase chain reaction-restriction fragment length polymorphism to detect the genetic structure and to infer the possible glacial refugia of Ginkgo biloba L. in China. In total, 220 individuals from 12 populations in China and three populations outside China were analyzed, representing the largest number of populations studied by molecular markers to date. Nineteen haplotypes were produced and haplotype A was found in all populations. Populations in south-western China, including WC, JF, PX, and SP, contained 14 of the 19 haplotypes and their genetic diversity ranged from 0.771 4 to 0.867 6. The TM population from China also showed a high genetic diversity (H=0.848 5). Most of the genetic variation existed within populations and the differentiation among populations was low (GST>=0.2). According to haplotype distribution and the historical record, we suggest that populations of G. biloba have been subjected to extensive human impact, which has compounded our attempt to infer glacial refugia for Ginkgo. Nevertheless, the present results suggest that the center of genetic diversity of Ginkgo is mainly in south-western China and in situ conservation is needed to protect and preserve the genetic resources.

  19. The complete chloroplast and mitochondrial genome sequences of Boea hygrometrica: insights into the evolution of plant organellar genomes.

    Directory of Open Access Journals (Sweden)

    Tongwu Zhang

    Full Text Available The complete nucleotide sequences of the chloroplast (cp and mitochondrial (mt genomes of resurrection plant Boea hygrometrica (Bh, Gesneriaceae have been determined with the lengths of 153,493 bp and 510,519 bp, respectively. The smaller chloroplast genome contains more genes (147 with a 72% coding sequence, and the larger mitochondrial genome have less genes (65 with a coding faction of 12%. Similar to other seed plants, the Bh cp genome has a typical quadripartite organization with a conserved gene in each region. The Bh mt genome has three recombinant sequence repeats of 222 bp, 843 bp, and 1474 bp in length, which divide the genome into a single master circle (MC and four isomeric molecules. Compared to other angiosperms, one remarkable feature of the Bh mt genome is the frequent transfer of genetic material from the cp genome during recent Bh evolution. We also analyzed organellar genome evolution in general regarding genome features as well as compositional dynamics of sequence and gene structure/organization, providing clues for the understanding of the evolution of organellar genomes in plants. The cp-derived sequences including tRNAs found in angiosperm mt genomes support the conclusion that frequent gene transfer events may have begun early in the land plant lineage.

  20. The complete chloroplast and mitochondrial genome sequences of Boea hygrometrica: insights into the evolution of plant organellar genomes.

    Science.gov (United States)

    Zhang, Tongwu; Fang, Yongjun; Wang, Xumin; Deng, Xin; Zhang, Xiaowei; Hu, Songnian; Yu, Jun

    2012-01-01

    The complete nucleotide sequences of the chloroplast (cp) and mitochondrial (mt) genomes of resurrection plant Boea hygrometrica (Bh, Gesneriaceae) have been determined with the lengths of 153,493 bp and 510,519 bp, respectively. The smaller chloroplast genome contains more genes (147) with a 72% coding sequence, and the larger mitochondrial genome have less genes (65) with a coding faction of 12%. Similar to other seed plants, the Bh cp genome has a typical quadripartite organization with a conserved gene in each region. The Bh mt genome has three recombinant sequence repeats of 222 bp, 843 bp, and 1474 bp in length, which divide the genome into a single master circle (MC) and four isomeric molecules. Compared to other angiosperms, one remarkable feature of the Bh mt genome is the frequent transfer of genetic material from the cp genome during recent Bh evolution. We also analyzed organellar genome evolution in general regarding genome features as well as compositional dynamics of sequence and gene structure/organization, providing clues for the understanding of the evolution of organellar genomes in plants. The cp-derived sequences including tRNAs found in angiosperm mt genomes support the conclusion that frequent gene transfer events may have begun early in the land plant lineage.

  1. A Long PCR–Based Approach for DNA Enrichment Prior to Next-Generation Sequencing for Systematic Studies

    Directory of Open Access Journals (Sweden)

    Simon Uribe-Convers

    2014-01-01

    Full Text Available Premise of the study: We present an alternative approach for molecular systematic studies that combines long PCR and next-generation sequencing. Our approach can be used to generate templates from any DNA source for next-generation sequencing. Here we test our approach by amplifying complete chloroplast genomes, and we present a set of 58 potentially universal primers for angiosperms to do so. Additionally, this approach is likely to be particularly useful for nuclear and mitochondrial regions. Methods and Results: Chloroplast genomes of 30 species across angiosperms were amplified to test our approach. Amplification success varied depending on whether PCR conditions were optimized for a given taxon. To further test our approach, some amplicons were sequenced on an Illumina HiSeq 2000. Conclusions: Although here we tested this approach by sequencing plastomes, long PCR amplicons could be generated using DNA from any genome, expanding the possibilities of this approach for molecular systematic studies.

  2. Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown algae (Phaeophyceae) in China

    Institute of Scientific and Technical Information of China (English)

    JIA Shangang; LIU Tao; WU Shuangxiu; WANG Xumin; LI Tianyong; QIAN Hao; SUN Jing; WANG Liang; YU Jun; REN Lufeng; YIN Jinlong

    2014-01-01

    The chloroplast and mitochondrion of brown algae (Class Phaeophyceae of Phylum Ochrophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others (Phylum Cryptophyta and Haptophyta) in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassi-folia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.

  3. Long range correlations in DNA sequences

    CERN Document Server

    Mohanty, A K

    2002-01-01

    The so called long range correlation properties of DNA sequences are studied using the variance analyses of the density distribution of a single or a group of nucleotides in a model independent way. This new method which was suggested earlier has been applied to extract slope parameters that characterize the correlation properties for several intron containing and intron less DNA sequences. An important aspect of all the DNA sequences is the properties of complimentarity by virtue of which any two complimentary distributions (like GA is complimentary to TC or G is complimentary to ATC) have identical fluctuations at all scales although their distribution functions need not be identical. Due to this complimentarity, the famous DNA walk representation whose statistical interpretation is still unresolved is shown to be a special case of the present formalism with a density distribution corresponding to a purine or a pyrimidine group. Another interesting aspect of most of the DNA sequences is that the factorial m...

  4. Dynamics and Control of DNA Sequence Amplification

    CERN Document Server

    Marimuthu, Karthikeyan

    2014-01-01

    DNA amplification is the process of replication of a specified DNA sequence \\emph{in vitro} through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction (PCR) as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal tempe...

  5. Small chloroplast-targeted DnaJ proteins are involved in optimization of photosynthetic reactions in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Piippo Mirva

    2010-03-01

    Full Text Available Abstract Background DnaJ proteins participate in many metabolic pathways through dynamic interactions with various components of these processes. The role of three small chloroplast-targeted DnaJ proteins, AtJ8 (At1 g80920, AtJ11 (At4 g36040 and AtJ20 (At4 g13830, was investigated here using knock-out mutants of Arabidopsis thaliana. Photochemical efficiency, capacity of CO2 assimilation, stabilization of Photosystem (PS II dimers and supercomplexes under high light illumination, energy distribution between PSI and PSII and phosphorylation of PSII-LHCII proteins, global gene expression profiles and oxidative stress responses of these DnaJ mutants were analyzed. Results Knockout of one of these proteins caused a series of events including a decrease in photosynthetic efficiency, destabilization of PSII complexes and loss of control for balancing the redox reactions in chloroplasts. Data obtained with DNA microarray analysis demonstrated that the lack of one of these DnaJ proteins triggers a global stress response and therefore confers the plants greater tolerance to oxidative stress induced by high light or methyl viologen treatments. Expression of a set of genes encoding enzymes that detoxify reactive oxygen species (ROS as well as a number of stress-related transcription factors behaved in the mutants at growth light similarly to that when wild-type (WT plants were transferred to high light. Also a set of genes related to redox regulation were upregulated in the mutants. On the other hand, although the three DnaJ proteins reside in chloroplasts, the expression of most genes encoding thylakoid membrane proteins was not changed in the mutants. Conclusion It is proposed that the tolerance of the DnaJ protein knockout plants to oxidative stress occurs at the expense of the flexibility of photosynthetic reactions. Despite the fact that the effects of the individual protein knockout on the response of plants to high light treatment are quite similar

  6. DNA display I. Sequence-encoded routing of DNA populations.

    Directory of Open Access Journals (Sweden)

    David R Halpin

    2004-07-01

    Full Text Available Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools based on sequence. Partitioning steps can be iterated indefinitely, with worst-case yields of 85% per step. These techniques facilitate DNA-programmed chemical synthesis, and thus enable a materials biology that could revolutionize drug discovery.

  7. Visible periodicity of strong nucleosome DNA sequences.

    Science.gov (United States)

    Salih, Bilal; Tripathi, Vijay; Trifonov, Edward N

    2015-01-01

    Fifteen years ago, Lowary and Widom assembled nucleosomes on synthetic random sequence DNA molecules, selected the strongest nucleosomes and discovered that the TA dinucleotides in these strong nucleosome sequences often appear at 10-11 bases from one another or at distances which are multiples of this period. We repeated this experiment computationally, on large ensembles of natural genomic sequences, by selecting the strongest nucleosomes--i.e. those with such distances between like-named dinucleotides, multiples of 10.4 bases, the structural and sequence period of nucleosome DNA. The analysis confirmed the periodicity of TA dinucleotides in the strong nucleosomes, and revealed as well other periodic sequence elements, notably classical AA and TT dinucleotides. The matrices of DNA bendability and their simple linear forms--nucleosome positioning motifs--are calculated from the strong nucleosome DNA sequences. The motifs are in full accord with nucleosome positioning sequences derived earlier, thus confirming that the new technique, indeed, detects strong nucleosomes. Species- and isochore-specific variations of the matrices and of the positioning motifs are demonstrated. The strong nucleosome DNA sequences manifest the highest hitherto nucleosome positioning sequence signals, showing the dinucleotide periodicities in directly observable rather than in hidden form.

  8. Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

    Directory of Open Access Journals (Sweden)

    Ching-Ping Lin

    Full Text Available We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911. In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum, two monocots (Oryza sativa and Zea mays, two gymnosperms (Pinus taeda and Ginkgo biloba and one moss (Physcomitrella patens. Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

  9. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. (Oak Ridge National Lab., TN (United States)); Arlinghaus, H.F. (Atom Sciences, Inc., Oak Ridge, TN (United States))

    1993-01-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  10. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. [Oak Ridge National Lab., TN (United States); Arlinghaus, H.F. [Atom Sciences, Inc., Oak Ridge, TN (United States)

    1993-06-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  11. EGNAS: an exhaustive DNA sequence design algorithm

    Directory of Open Access Journals (Sweden)

    Kick Alfred

    2012-06-01

    Full Text Available Abstract Background The molecular recognition based on the complementary base pairing of deoxyribonucleic acid (DNA is the fundamental principle in the fields of genetics, DNA nanotechnology and DNA computing. We present an exhaustive DNA sequence design algorithm that allows to generate sets containing a maximum number of sequences with defined properties. EGNAS (Exhaustive Generation of Nucleic Acid Sequences offers the possibility of controlling both interstrand and intrastrand properties. The guanine-cytosine content can be adjusted. Sequences can be forced to start and end with guanine or cytosine. This option reduces the risk of “fraying” of DNA strands. It is possible to limit cross hybridizations of a defined length, and to adjust the uniqueness of sequences. Self-complementarity and hairpin structures of certain length can be avoided. Sequences and subsequences can optionally be forbidden. Furthermore, sequences can be designed to have minimum interactions with predefined strands and neighboring sequences. Results The algorithm is realized in a C++ program. TAG sequences can be generated and combined with primers for single-base extension reactions, which were described for multiplexed genotyping of single nucleotide polymorphisms. Thereby, possible foldback through intrastrand interaction of TAG-primer pairs can be limited. The design of sequences for specific attachment of molecular constructs to DNA origami is presented. Conclusions We developed a new software tool called EGNAS for the design of unique nucleic acid sequences. The presented exhaustive algorithm allows to generate greater sets of sequences than with previous software and equal constraints. EGNAS is freely available for noncommercial use at http://www.chm.tu-dresden.de/pc6/EGNAS.

  12. Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines

    Directory of Open Access Journals (Sweden)

    Cornish Katrina

    2009-11-01

    Full Text Available Abstract Background Parthenium argentatum (guayule is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of P. argentatum was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, Lactuca sativa, Guitozia abyssinica and Helianthus annuus revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of Parthenium species and lines. Results The complete plastid genome of P. argentatum is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in L. sativa, G. abyssinica and H. annuus, we demonstrate that the P. argentatum chloroplast genome sequence is most closely related to that of H. annuus. Similar to chloroplast genomes in G. abyssinica, L. sativa and H. annuus, the plastid genome of P. argentatum has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the matK and psbA-trnH spacer chloroplast DNA barcodes, three of the four Parthenium species tested, P. tomentosum, P. hysterophorus and P. schottii, can be differentiated from P. argentatum. In addition, we identified lines within P. argentatum. Conclusion The genome sequence of the P. argentatum chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate transformation efficiency by using the precise sequence of endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially significant lines of P. argentatum that are important for producing latex.

  13. A phylogeny of cycads (Cycadales) inferred from chloroplast matK gene, trnK intron, and nuclear rDNA ITS region.

    Science.gov (United States)

    Chaw, Shu-Miaw; Walters, Terrence W; Chang, Chien-Chang; Hu, Shu-Hsuan; Chen, Shin-Hsiao

    2005-10-01

    Phylogenetic relationships among the three families and 12 living genera of cycads were reconstructed by distance and parsimony criteria using three markers: the chloroplast matK gene, the chloroplast trnK intron and the nuclear ITS/5.8S rDNA sequence. All datasets indicate that Cycadaceae (including only the genus Cycas) is remotely related to other cycads, in which Dioon was resolved as the basal-most clade, followed by Bowenia and a clade containing the remaining nine genera. Encephalartos and Lepidozamia are closer to each other than to Macrozamia. The African genus Stangeria is embedded within the New World subfamily Zamiodeae. Therefore, Bowenia is an unlikely sister to Stangeria, contrary to the view that they form the Stangeriaceae. The generic status of Dyerocycas and Chigua is unsupportable as they are paraphyletic with Cycas and the Zamia, respectively. Nonsense mutations in the matK gene and indels in the other two datasets lend evidence to reinforce the above conclusions. According to the phylogenies, the past geography of the genera of cycads and the evolution of character states are hypothesized and discussed. Within the suborder Zamiieae, Stangeria, and the tribe Zamieae evolved significantly faster than other genera. The matK gene and ITS/5.8S region contain more useful information than the trnK intron in addressing phylogeny. Redelimitations of Zamiaceae, Stangeriaceae, subfamily Encephalartoideae and subtribe Macrozamiineae are necessary.

  14. Unexpected Diversity of Chloroplast Noncoding RNAs as Revealed by Deep Sequencing of the Arabidopsis Transcriptome.

    Science.gov (United States)

    Hotto, Amber M; Schmitz, Robert J; Fei, Zhangjun; Ecker, Joseph R; Stern, David B

    2011-12-01

    Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18-25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5' end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.

  15. Extracting biological knowledge from DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    De La Vega, F.M. [CINVESTAV-IPN (Mexico); Thieffry, D. [Universite Libre de Bruxelles, Rhode-Saint-Genese (Belgium)]|[Universidad Nacional Autonoma de Mexico, Morelos (Mexico); Collado-Vides, J. [Universidad Nacional Autonoma de Mexico, Morelos (Mexico)

    1996-12-31

    This session describes the elucidation of information from dna sequences and what challenges computational biologists face in their task of summarizing and deciphering the human genome. Techniques discussed include methods from statistics, information theory, artificial intelligence and linguistics. 1 ref.

  16. Nanopore DNA sequencing using kinetic proofreading

    Science.gov (United States)

    Ling, Xinsheng

    We propose a method of DNA sequencing by combining the physical method of nanopore electrical measurements and Southern's sequencing-by-hybridization. The new key ingredient, essential to both lowering the costs and increasing the precision, is an asymmetric nanopore sandwich device capable of measuring the DNA hybridization probe twice separated by a designed waiting time. Those incorrect probes appearing only once in nanopore ionic current traces are discriminated from the correct ones that appear twice. This method of discrimination is similar to the principle of kinetic proofreading proposed by Hopfield and Ninio in gene transcription and translation processes. An error analysis is of this nanopore kinetic proofreading (nKP) technique for DNA sequencing is carried out in comparison with the most precise 3' dideoxy termination method developed by Sanger. Nanopore DNA sequencing using kinetic proofreading.

  17. An approach to sequence DNA without tagging

    Science.gov (United States)

    Niu, Sanjun; Saraf, Ravi F.

    2002-10-01

    Microarray technology is playing an increasingly important role in biology and medicine and its application to genomics for gene expression analysis has already reached the market with a variety of commercially available instruments. In these combinatorial analysis methods, known probe single-strand DNA (ssDNA) 'primers' are attached in clusters of typically 100 µm × 100 µm pixels. Each pixel of the array has a slightly different sequence. On exposure to 'unknown' target ssDNA, the pixels with the right complementary probe ssDNA sequence convert to double-stranded DNA (dsDNA) by a hybridization reaction. To transduct the conversion of the pixel to dsDNA, the target ssDNA is labelled with a photoluminescent tag during the polymerase chain reaction (PCR) amplification process. Due to the statistical distribution of the tags in the target ssDNA, it becomes significantly difficult to implement these methods as a diagnostic tool in a pathology laboratory. A method to sequence DNA without tagging the molecule is developed. The fabrication process is compatible with current microelectronics and (emerging) soft-material fabrication technologies, allowing the method to be integrable with micro-electromechanical systems (MEMS) and lab-on-a-chip devices. An estimated sensitivity of 10-12 g on a 1 cm2 device area is obtained.

  18. gargammel: a sequence simulator for ancient DNA.

    Science.gov (United States)

    Renaud, Gabriel; Hanghøj, Kristian; Willerslev, Eske; Orlando, Ludovic

    2016-10-29

    Ancient DNA has emerged as a remarkable tool to infer the history of extinct species and past populations. However, many of its characteristics, such as extensive fragmentation, damage and contamination, can influence downstream analyses. To help investigators measure how these could impact their analyses in silico, we have developed gargammel, a package that simulates ancient DNA fragments given a set of known reference genomes. Our package simulates the entire molecular process from post-mortem DNA fragmentation and DNA damage to experimental sequencing errors, and reproduces most common bias observed in ancient DNA datasets.

  19. Inconsistencies in Neanderthal genomic DNA sequences.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Wall

    2007-10-01

    Full Text Available Two recently published papers describe nuclear DNA sequences that were obtained from the same Neanderthal fossil. Our reanalyses of the data from these studies show that they are not consistent with each other and point to serious problems with the data quality in one of the studies, possibly due to modern human DNA contaminants and/or a high rate of sequencing errors.

  20. Sequencing intractable DNA to close microbial genomes.

    Directory of Open Access Journals (Sweden)

    Richard A Hurt

    Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  1. Sequencing intractable DNA to close microbial genomes.

    Science.gov (United States)

    Hurt, Richard A; Brown, Steven D; Podar, Mircea; Palumbo, Anthony V; Elias, Dwayne A

    2012-01-01

    Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  2. Nanopore-CMOS Interfaces for DNA Sequencing.

    Science.gov (United States)

    Magierowski, Sebastian; Huang, Yiyun; Wang, Chengjie; Ghafar-Zadeh, Ebrahim

    2016-08-06

    DNA sequencers based on nanopore sensors present an opportunity for a significant break from the template-based incumbents of the last forty years. Key advantages ushered by nanopore technology include a simplified chemistry and the ability to interface to CMOS technology. The latter opportunity offers substantial promise for improvement in sequencing speed, size and cost. This paper reviews existing and emerging means of interfacing nanopores to CMOS technology with an emphasis on massively-arrayed structures. It presents this in the context of incumbent DNA sequencing techniques, reviews and quantifies nanopore characteristics and models and presents CMOS circuit methods for the amplification of low-current nanopore signals in such interfaces.

  3. Osmylated DNA, a novel concept for sequencing DNA using nanopores

    Science.gov (United States)

    Kanavarioti, Anastassia

    2015-03-01

    Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. ‘Base calling’ becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.

  4. Polymerase chain reaction-single strand conformation polymorphism analyses of nuclear and chloroplast DNA provide evidence for recombination, multiple introductions and nascent speciation in the Caulerpa taxifolia complex

    NARCIS (Netherlands)

    Meusnier, I; Valero, M; Destombe, C; Gode, E.; Desmarais, E.; Bonhomme, F.; Stam, W.T.; Olsen, J.L.

    2002-01-01

    Independent lines of evidence support an Australian origin for the Mediterranean populations of the tropical alga Caulerpa taxifolia. To complement previous biogeographical studies based on nuclear rDNA internal transcribed spacer (ITS), a new chloroplast marker was developed - the cp 16S rDNA intro

  5. Female-specific DNA sequences in geese.

    Science.gov (United States)

    Huang, M C; Lin, W C; Horng, Y M; Rouvier, R; Huang, C W

    2003-07-01

    1. The OPAE random primers (Operon Technologies, Inc., CA) were used for random amplified polymorphic DNA (RAPD) fingerprinting in Chinese, White Roman and Landaise geese. One of these primers, OPAE-06, produced a 938-bp sex-specific fragment in all females and in no males of Chinese geese only. 2. A novel female-specific DNA sequence in Chinese goose was cloned and sequenced. Two primers, CGSex-F and CGSex-R, were designed in order to amplify a 912-bp sex-specific polymerase chain reaction (PCR) fragment on genomic DNA from female geese. 3. It was shown that a simple and effective PCR-based sexing technique could be used in the three goose breeds studied. 4. Nucleotide sequencing of the sex-specific fragments in White Roman and Landaise geese was performed and sequence differences were observed among these three breeds.

  6. Dynamics and control of DNA sequence amplification

    Energy Technology Data Exchange (ETDEWEB)

    Marimuthu, Karthikeyan [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054 (United States)

    2014-10-28

    DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.

  7. Compressing DNA sequence databases with coil

    Directory of Open Access Journals (Sweden)

    Hendy Michael D

    2008-05-01

    Full Text Available Abstract Background Publicly available DNA sequence databases such as GenBank are large, and are growing at an exponential rate. The sheer volume of data being dealt with presents serious storage and data communications problems. Currently, sequence data is usually kept in large "flat files," which are then compressed using standard Lempel-Ziv (gzip compression – an approach which rarely achieves good compression ratios. While much research has been done on compressing individual DNA sequences, surprisingly little has focused on the compression of entire databases of such sequences. In this study we introduce the sequence database compression software coil. Results We have designed and implemented a portable software package, coil, for compressing and decompressing DNA sequence databases based on the idea of edit-tree coding. coil is geared towards achieving high compression ratios at the expense of execution time and memory usage during compression – the compression time represents a "one-off investment" whose cost is quickly amortised if the resulting compressed file is transmitted many times. Decompression requires little memory and is extremely fast. We demonstrate a 5% improvement in compression ratio over state-of-the-art general-purpose compression tools for a large GenBank database file containing Expressed Sequence Tag (EST data. Finally, coil can efficiently encode incremental additions to a sequence database. Conclusion coil presents a compelling alternative to conventional compression of flat files for the storage and distribution of DNA sequence databases having a narrow distribution of sequence lengths, such as EST data. Increasing compression levels for databases having a wide distribution of sequence lengths is a direction for future work.

  8. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data Description of data contents Phred's quality score. PHD format, one file to a single cDNA data, and co...ription Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive ...

  9. DNA Sequencing in Cultural Heritage.

    Science.gov (United States)

    Vai, Stefania; Lari, Martina; Caramelli, David

    2016-02-01

    During the last three decades, DNA analysis on degraded samples revealed itself as an important research tool in anthropology, archaeozoology, molecular evolution, and population genetics. Application on topics such as determination of species origin of prehistoric and historic objects, individual identification of famous personalities, characterization of particular samples important for historical, archeological, or evolutionary reconstructions, confers to the paleogenetics an important role also for the enhancement of cultural heritage. A really fast improvement in methodologies in recent years led to a revolution that permitted recovering even complete genomes from highly degraded samples with the possibility to go back in time 400,000 years for samples from temperate regions and 700,000 years for permafrozen remains and to analyze even more recent material that has been subjected to hard biochemical treatments. Here we propose a review on the different methodological approaches used so far for the molecular analysis of degraded samples and their application on some case studies.

  10. The DnaJ-like zinc finger domain protein PSA2 affects light acclimation and chloroplast development in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yan-Wen eWang

    2016-03-01

    Full Text Available The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development.

  11. Chloroplast Genome Sequence of pigeonpea (Cajanus cajan (L. Millspaugh and Cajanus scarabaeoides: Genome organization and Comparison with other legumes

    Directory of Open Access Journals (Sweden)

    Tanvi Kaila

    2016-12-01

    Full Text Available Pigeonpea (Cajanus cajan (L. Millspaugh, a diploid (2n = 22 legume crop with a genome size of 852 Mbp, serves as an important source of human dietary protein especially in South East Asian and African regions. In this study, the draft chloroplast genomes of Cajanus cajan and Cajanus scarabaeoides were sequenced. Cajanus scarabaeoides is an important species of the Cajanus gene pool and has also been used for developing promising CMS system by different groups. A male sterile genotype harbouring the Cajanus scarabaeoides cytoplasm was used for sequencing the plastid genome. The cp genome of Cajanus cajan is 152,242bp long, having a quadripartite structure with LSC of 83,455 bp and SSC of 17,871 bp separated by IRs of 25,398 bp. Similarly, the cp genome of Cajanus scarabaeoides is 152,201bp long, having a quadripartite structure in which IRs of 25,402 bp length separates 83,423 bp of LSC and 17,854 bp of SSC. The pigeonpea cp genome contains 116 unique genes, including 30 tRNA, 4 rRNA, 78 predicted protein coding genes and 5 pseudogenes. A 50kb inversion was observed in the LSC region of pigeonpea cp genome, consistent with other legumes. Comparison of cp genome with other legumes revealed the contraction of IR boundaries due to the absence of rps19 gene in the IR region. Chloroplast SSRs were mined and a total of 280 and 292 cpSSRs were identified in Cajanus scarabaeoides and Cajanus cajan respectively. RNA editing was observed at 37 sites in both Cajanus scarabaeoides and Cajanus cajan, with maximum occurrence in the ndh genes. The pigeonpea cp genome sequence would be beneficial in providing informative molecular markers which can be utilized for genetic diversity analysis and aid in understanding the plant systematics studies among major grain legumes.

  12. DNA sequencing by synthesis with degenerate primers

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The degenerate primer-based sequencing Was developed by a synthesis method(DP-SBS)for high-throughput DNA sequencing,in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays.In this method,adifferent set of degenerate primers containing a give nnumber(n)of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface.The nucleotides(n+1)on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides.The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately.The main advanmge of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension.From the present study,it is found that the DP-SBS method is reliable,simple,and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

  13. Glycome mapping on DNA sequencing equipment.

    Science.gov (United States)

    Laroy, Wouter; Contreras, Roland; Callewaert, Nico

    2006-01-01

    Here we provide a detailed protocol for the analysis of protein-linked glycans on DNA sequencing equipment. This protocol satisfies the glyco-analytical needs of many projects and can form the basis of 'glycomics' studies, in which robustness, high throughput, high sensitivity and reliable quantification are of paramount importance. The protocol routinely resolves isobaric glycan stereoisomers, which is much more difficult by mass spectrometry (MS). Earlier methods made use of polyacrylamide gel-based sequencers, but we have now adapted the technique to multicapillary DNA sequencers, which represent the state of the art today. In addition, we have integrated an option for HPLC-based fractionation of highly anionic 8-amino-1,3,6-pyrenetrisulfonic acid (APTS)-labeled glycans before rapid capillary electrophoretic profiling. This option facilitates either two-dimensional profiling of complex glycan mixtures and exoglycosidase sequencing, or MS analysis of particular compounds of interest rather than of the total pool of glycans in a sample.

  14. The complete DNA sequence of vaccinia virus.

    Science.gov (United States)

    Goebel, S J; Johnson, G P; Perkus, M E; Davis, S W; Winslow, J P; Paoletti, E

    1990-11-01

    The complete DNA sequence of the genome of vaccinia virus has been determined. The genome consisted of 191,636 bp with a base composition of 66.6% A + T. We have identified 198 "major" protein-coding regions and 65 overlapping "minor" regions, for a total of 263 potential genes. Genes encoded by the virus were located by examination of DNA sequence characteristics and compared with existing vaccinia virus mapping analyses, sequence data, and transcription data. These genes were found to be compactly organized along the genome with relatively few regions of noncoding sequences. Whereas several similarities to proteins of known function were discerned, the function of the majority of proteins encoded by these open reading frames is as yet undetermined.

  15. Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum and Comparative Analysis with Common Buckwheat (F. esculentum.

    Directory of Open Access Journals (Sweden)

    Kwang-Soo Cho

    Full Text Available We report the chloroplast (cp genome sequence of tartary buckwheat (Fagopyrum tataricum obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats and F. esculentum (one repeat, and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes--rpoC2, ycf3, accD, and clpP--have high synonymous (Ks value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum.

  16. Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum).

    Science.gov (United States)

    Cho, Kwang-Soo; Yun, Bong-Kyoung; Yoon, Young-Ho; Hong, Su-Young; Mekapogu, Manjulatha; Kim, Kyung-Hee; Yang, Tae-Jin

    2015-01-01

    We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes--rpoC2, ycf3, accD, and clpP--have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum.

  17. Chapter 2: Genetic Variability in Nuclear Ribosomal and Chloroplast DNA in Utah (Juniperus Osteosperma) and Western (J. Occidentalis) Juniper (Cupressaceae): Evidence for Interspecific Gene Flow1

    Energy Technology Data Exchange (ETDEWEB)

    Terry, Randall G.; Tausch, Robin J.; Nowak, Robert S.

    1998-02-14

    Early studies of evolutionary change in chloroplast DNA indicated limited variability within species. This finding has been attributed to relatively low rates of sequence evolution and has been maintained as justification for the lack of intraspecific sampling in studies examining, relationships at the species level and above. However, documentation of intraspecific variation in cpDNA has become increasingly common and has been attributed in many cases to ''chloroplast capture'' following genetic exchange across species boundaries. Rleseberg and Wendel (1993) list 37 cases of proposed hybridization in plants that include intraspecific variation in cpDNA, 24 (65%) of which they considered to be probable instances of introgression. Rieseberg (1995) suspected that a review of the literature at that time would reveal over 100 cases of intraspecific variation in CPDNA that could be attributed to hybridization and introgression. That intraspecific variation in cpDNA is potentially indicative of hybridization is founded on the expectation that slowly evolving loci or genomes will produce greater molecular variation between than within species. In cases where a species is polymorphic for CPDNA and at least one of the molecular variants is diagnostic for a second species, interspecific hybridization is a plausible explanation. Incongruence between relationships suggested by cpDNA variation and those supported by other types of data (e.g., morphology or molecular data from an additional locus) provides additional support for introgression. One aspect of hybridization in both animals and plants that has become increasingly evident is incongruence in the phylogenetic and geographic distribution of cytoplasmic and nuclear markers. In most cases cytoplasmic introgression appears to be more pervasive than nuclear exchange. This discordance appears attributable to several factors including differences in the mutation rate, number of effective alleles, and modes

  18. Phylogeny of the paleotropical fern genus Lepisorus (Polypodiaceae, Polypodiopsida) inferred from four chloroplast DNA regions.

    Science.gov (United States)

    Wang, Li; Qi, Xin-ping; Xiang, Qiao-ping; Heinrichs, Jochen; Schneider, Harald; Zhang, Xian-chun

    2010-01-01

    Phylogenetic relationships within the paleotropical genus Lepisorus (Polypodiaceae) were investigated using plastid DNA sequences from four regions: rbcL, rps4 and rps4-trnS IGS, trnL intron plus trnL-F IGS, rbcL-atpB IGS. Over 4000 nucleotides were sequenced for 77 specimens belonging to 54 species. Each cpDNA region was analyzed separately and combined into a single dataset. All phylogenetic analyses, maximum parsimony, maximum likelihood and Bayesian Inference of phylogeny, revealed the paraphyly of Lepisorus with the monotypic Drymotaenium miyoshianum and of the paleotropical genus Belvisia nested within the Lepisorus clade. Nine well-supported major clades were found. The phylogenetic results provided new evidence for the sectional classification of Lepisorus. The evolution of three morphological characters, clathrateness of rhizome scales, margin of rhizome scales and defoliated leaves, and the evolution of the karyotype, were reconstructed to identify lineage specific phenotypic character states or combination of characters. Unique character combinations, rather than synapomorphies, were found to be of systematic value in sectional delimitation. The variation of chromosome numbers is largely due to a single aneuploidy event instead of a stepwise reduction during the evolutionary history of this genus.

  19. DNA Sequence Alignment during Homologous Recombination.

    Science.gov (United States)

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination.

  20. Automated Template Quantification for DNA Sequencing Facilities

    Science.gov (United States)

    Ivanetich, Kathryn M.; Yan, Wilson; Wunderlich, Kathleen M.; Weston, Jennifer; Walkup, Ward G.; Simeon, Christian

    2005-01-01

    The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n = 198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/μL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by ≥ 20% from the requested concentration (500 ng/μL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/μL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown. PMID:16461949

  1. The first determination of DNA sequence of a specific gene.

    Science.gov (United States)

    Inouye, Masayori

    2016-05-10

    How and when the first DNA sequence of a gene was determined? In 1977, F. Sanger came up with an innovative technology to sequence DNA by using chain terminators, and determined the entire DNA sequence of the 5375-base genome of bacteriophage φX 174 (Sanger et al., 1977). While this Sanger's achievement has been recognized as the first DNA sequencing of genes, we had determined DNA sequence of a gene, albeit a partial sequence, 11 years before the Sanger's DNA sequence (Okada et al., 1966).

  2. DNA sequencing by nanopores: advances and challenges

    Science.gov (United States)

    Agah, Shaghayegh; Zheng, Ming; Pasquali, Matteo; Kolomeisky, Anatoly B.

    2016-10-01

    Developing inexpensive and simple DNA sequencing methods capable of detecting entire genomes in short periods of time could revolutionize the world of medicine and technology. It will also lead to major advances in our understanding of fundamental biological processes. It has been shown that nanopores have the ability of single-molecule sensing of various biological molecules rapidly and at a low cost. This has stimulated significant experimental efforts in developing DNA sequencing techniques by utilizing biological and artificial nanopores. In this review, we discuss recent progress in the nanopore sequencing field with a focus on the nature of nanopores and on sensing mechanisms during the translocation. Current challenges and alternative methods are also discussed.

  3. Chloroplast DNA phylogeography reveals repeated range expansion in a widespread aquatic herb Hippuris vulgaris in the Qinghai-Tibetan Plateau and adjacent areas.

    Directory of Open Access Journals (Sweden)

    Jin-Ming Chen

    Full Text Available BACKGROUND: The Qinghai-Tibetan Plateau (QTP is one of the most extensive habitats for alpine plants in the world. Climatic oscillations during the Quaternary ice age had a dramatic effect on species ranges on the QTP and the adjacent areas. However, how the distribution ranges of aquatic plant species shifted on the QTP in response to Quaternary climatic changes remains almost unknown. METHODOLOGY AND PRINCIPAL FINDINGS: We studied the phylogeography and demographic history of the widespread aquatic herb Hippuris vulgaris from the QTP and adjacent areas. Our sampling included 385 individuals from 47 natural populations of H. vulgaris. Using sequences from four chloroplast DNA (cpDNA non-coding regions, we distinguished eight different cpDNA haplotypes. From the cpDNA variation in H. vulgaris, we found a very high level of population differentiation (G ST = 0.819 but the phylogeographical structure remained obscure (N ST = 0.853>G ST = 0.819, P>0.05. Phylogenetic analyses revealed two main cpDNA haplotype lineages. The split between these two haplotype groups can be dated back to the mid-to-late Pleistocene (ca. 0.480 Myr. Mismatch distribution analyses showed that each of these had experienced a recent range expansion. These two expansions (ca. 0.12 and 0.17 Myr might have begun from the different refugees before the Last Glacial Maximum (LGM. CONCLUSIONS/SIGNIFICANCE: This study initiates a research on the phylogeography of aquatic herbs in the QTP and for the first time sheds light on the response of an alpine aquatic seed plant species in the QTP to Quaternary climate oscillations.

  4. Mitochondrial DNA sequence variation in Greeks.

    Science.gov (United States)

    Kouvatsi, A; Karaiskou, N; Apostolidis, A; Kirmizidis, G

    2001-12-01

    Mitochondrial DNA (mtDNA) control region sequences were determined in 54 unrelated Greeks, coming from different regions in Greece, for both segments HVR-I and HVR-II. Fifty-two different mtDNA haplotypes were revealed, one of which was shared by three individuals. A very low heterogeneity was found among Greek regions. No one cluster of lineages was specific to individuals coming from a certain region. The average pairwise difference distribution showed a value of 7.599. The data were compared with that for other European or neighbor populations (British, French, Germans, Tuscans, Bulgarians, and Turks). The genetic trees that were constructed revealed homogeneity between Europeans. Median networks revealed that most of the Greek mtDNA haplotypes are clustered to the five known haplogroups and that a number of haplotypes are shared among Greeks and other European and Near Eastern populations.

  5. Local Renyi entropic profiles of DNA sequences

    Directory of Open Access Journals (Sweden)

    Vinga Susana

    2007-10-01

    Full Text Available Abstract Background In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM. Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs. Results The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at http://kdbio.inesc-id.pt/~svinga/ep/. Conclusion The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures.

  6. Sequence-specific recognition of DNA nanostructures.

    Science.gov (United States)

    Rusling, David A; Fox, Keith R

    2014-05-15

    DNA is the most exploited biopolymer for the programmed self-assembly of objects and devices that exhibit nanoscale-sized features. One of the most useful properties of DNA nanostructures is their ability to be functionalized with additional non-nucleic acid components. The introduction of such a component is often achieved by attaching it to an oligonucleotide that is part of the nanostructure, or hybridizing it to single-stranded overhangs that extend beyond or above the nanostructure surface. However, restrictions in nanostructure design and/or the self-assembly process can limit the suitability of these procedures. An alternative strategy is to couple the component to a DNA recognition agent that is capable of binding to duplex sequences within the nanostructure. This offers the advantage that it requires little, if any, alteration to the nanostructure and can be achieved after structure assembly. In addition, since the molecular recognition of DNA can be controlled by varying pH and ionic conditions, such systems offer tunable properties that are distinct from simple Watson-Crick hybridization. Here, we describe methodology that has been used to exploit and characterize the sequence-specific recognition of DNA nanostructures, with the aim of generating functional assemblies for bionanotechnology and synthetic biology applications.

  7. DNA Barcoding: Amplification and sequence analysis of rbcl and matK genome regions in three divergent plant species

    Directory of Open Access Journals (Sweden)

    Javed Iqbal Wattoo

    2016-11-01

    Full Text Available Background: DNA barcoding is a novel method of species identification based on nucleotide diversity of conserved sequences. The establishment and refining of plant DNA barcoding systems is more challenging due to high genetic diversity among different species. Therefore, targeting the conserved nuclear transcribed regions would be more reliable for plant scientists to reveal genetic diversity, species discrimination and phylogeny. Methods: In this study, we amplified and sequenced the chloroplast DNA regions (matk+rbcl of Solanum nigrum, Euphorbia helioscopia and Dalbergia sissoo to study the functional annotation, homology modeling and sequence analysis to allow a more efficient utilization of these sequences among different plant species. These three species represent three families; Solanaceae, Euphorbiaceae and Fabaceae respectively. Biological sequence homology and divergence of amplified sequences was studied using Basic Local Alignment Tool (BLAST. Results: Both primers (matk+rbcl showed good amplification in three species. The sequenced regions reveled conserved genome information for future identification of different medicinal plants belonging to these species. The amplified conserved barcodes revealed different levels of biological homology after sequence analysis. The results clearly showed that the use of these conserved DNA sequences as barcode primers would be an accurate way for species identification and discrimination. Conclusion: The amplification and sequencing of conserved genome regions identified a novel sequence of matK in native species of Solanum nigrum. The findings of the study would be applicable in medicinal industry to establish DNA based identification of different medicinal plant species to monitor adulteration.

  8. New stopping criteria for segmenting DNA sequences

    CERN Document Server

    Li, W

    2001-01-01

    We propose a solution on the stopping criterion in segmenting inhomogeneous DNA sequences with complex statistical patterns. This new stopping criterion is based on Bayesian Information Criterion (BIC) in the model selection framework. When this stopping criterion is applied to a left telomere sequence of yeast Saccharomyces cerevisiae and the complete genome sequence of bacterium Escherichia coli, borders of biologically meaningful units were identified (e.g. subtelomeric units, replication origin, and replication terminus), and a more reasonable number of domains was obtained. We also introduce a measure called segmentation strength which can be used to control the delineation of large domains. The relationship between the average domain size and the threshold of segmentation strength is determined for several genome sequences.

  9. Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

    Institute of Scientific and Technical Information of China (English)

    L(U) Fang; WANG Guangce

    2011-01-01

    Using shotgun sequencing data,the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao,China).There are two group Ⅰ introns in the 16S rRNA gene,which is structurally similar to that of Caulerpa sertularioides (Bryopsidales,Chlorophyta).The chloroplast-encoded tufA gene sequence is 1230 bp long,very AT-rich (61.5%),and is similar to previously published 16S rRNA sequences of bryopsidinean algae.Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae,Halimedineae,and Ostreobidineae are three distinct lineages.These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae.Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however,this clade lacked robust nodal support.Moreover,the phylogenetic tree inferred from rbcL GenBank sequences,combined with the geographical distributions of Bryopsis species,identified a strongly supportive clade for three differently distributed Asian Bryopsis species.The preliminary results suggesting that these organisms are of distinct regional endemism.

  10. The Origin of Garden Chrysanthemums and Molecular Phylogeny of Dendranthema in China based on Nucleotide Sequences of nrDNA ITS, trnT-trnL and trnL-trnF Intergenic Spacer Regions in cpDNA%基于核糖体DNA的ITS序列和叶绿体trnT-trnL及trnL-trnF基因间区的菊花起源与中国菊属植物分子系统学研究

    Institute of Scientific and Technical Information of China (English)

    赵惠恩; 汪小全; 陈俊愉; 洪德元

    2003-01-01

    Several sequences were applied to resolve phylogenetic relationships within Dendranthema and clarify the origin of garden chrysanthemums including sequences of nrDNA ITS, trnT-trnL and trnL-trnF intergenic spacer regions in cpDNA. The relationships among the species are so close that the three sequences could only provide limited information. From the evidence presented, we suggest that: ① D.rhmobifolium be the chloroplast donor of D.vestitum (HN) with the resembling morphology and the same trnT/L IGS sequence. ② D.vestitum, a putative ancestor, may be not the chloroplast donor of garden chrysanthemums. D.lavand-ulifolium might be the chloroplast donor of the type population of D.indicum (HN) or the direct chloroplast donor of the ancient garden chrysanthemum cultivar. ③ D.zawaskii might be not the ancestor of garden chrysanthemums.

  11. Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants

    Institute of Scientific and Technical Information of China (English)

    LI Yi-nü; SUN Bing-yao; SU Ning; MENG Xiang-xun; ZHANG Zhi-fang; SHEN Gui-fang

    2009-01-01

    In contrast to the situation of random integration of foreign genes in nuclear transformation,the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination.To establish an expression system for alien genes in rice chloroplast,the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study.Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique,and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator of psbA gene 3'sequence.Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct.Subsequently,the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained.Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome.Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome.Thus,the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome,but PPT-resistant trait for rice plants as well.It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.

  12. Next-generation sequencing offers new insights into DNA degradation

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Orlando, Ludovic Antoine Alexandre; Willerslev, Eske

    2012-01-01

    The processes underlying DNA degradation are central to various disciplines, including cancer research, forensics and archaeology. The sequencing of ancient DNA molecules on next-generation sequencing platforms provides direct measurements of cytosine deamination, depurination and fragmentation r...

  13. Random Coding Bounds for DNA Codes Based on Fibonacci Ensembles of DNA Sequences

    Science.gov (United States)

    2008-07-01

    COVERED (From - To) 6 Jul 08 – 11 Jul 08 4. TITLE AND SUBTITLE RANDOM CODING BOUNDS FOR DNA CODES BASED ON FIBONACCI ENSEMBLES OF DNA SEQUENCES ... sequences which are generalizations of the Fibonacci sequences . 15. SUBJECT TERMS DNA Codes, Fibonacci Ensembles, DNA Computing, Code Optimization 16...coding bound on the rate of DNA codes is proved. To obtain the bound, we use some ensembles of DNA sequences which are generalizations of the Fibonacci

  14. Phylogenomic relationship of feijoa (Acca sellowiana (O.Berg) Burret) with other Myrtaceae based on complete chloroplast genome sequences.

    Science.gov (United States)

    Machado, Lilian de Oliveira; Vieira, Leila do Nascimento; Stefenon, Valdir Marcos; Oliveira Pedrosa, Fábio de; Souza, Emanuel Maltempi de; Guerra, Miguel Pedro; Nodari, Rubens Onofre

    2017-04-01

    Given their distribution, importance, and richness, Myrtaceae species comprise a model system for studying the evolution of tropical plant diversity. In addition, chloroplast (cp) genome sequencing is an efficient tool for phylogenetic relationship studies. Feijoa [Acca sellowiana (O. Berg) Burret; CN: pineapple-guava] is a Myrtaceae species that occurs naturally in southern Brazil and northern Uruguay. Feijoa is known for its exquisite perfume and flavorful fruits, pharmacological properties, ornamental value and increasing economic relevance. In the present work, we reported the complete cp genome of feijoa. The feijoa cp genome is a circular molecule of 159,370 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC 88,028 bp) and a Small Single Copy region (SSC 18,598 bp) separated by Inverted Repeat regions (IRs 26,372 bp). The genome structure, gene order, GC content and codon usage are similar to those of typical angiosperm cp genomes. When compared to other cp genome sequences of Myrtaceae, feijoa showed closest relationship with pitanga (Eugenia uniflora L.). Furthermore, a comparison of pitanga synonymous (Ks) and nonsynonymous (Ka) substitution rates revealed extremely low values. Maximum Likelihood and Bayesian Inference analyses produced phylogenomic trees identical in topology. These trees supported monophyly of three Myrtoideae clades.

  15. An oligonucleotide hybridization approach to DNA sequencing.

    Science.gov (United States)

    Khrapko, K R; Lysov YuP; Khorlyn, A A; Shick, V V; Florentiev, V L; Mirzabekov, A D

    1989-10-09

    We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

  16. Phylogenetic Relationships of the Fern Cyrtomium falcatum (Dryopteridaceae) from Dokdo Island Based on Chloroplast Genome Sequencing.

    Science.gov (United States)

    Raman, Gurusamy; Choi, Kyoung Su; Park, SeonJoo

    2016-12-02

    Cyrtomium falcatum is a popular ornamental fern cultivated worldwide. Native to the Korean Peninsula, Japan, and Dokdo Island in the Sea of Japan, it is the only fern present on Dokdo Island. We isolated and characterized the chloroplast (cp) genome of C. falcatum, and compared it with those of closely related species. The genes trnV-GAC and trnV-GAU were found to be present within the cp genome of C. falcatum, whereas trnP-GGG and rpl21 were lacking. Moreover, cp genomes of Cyrtomium devexiscapulae and Adiantum capillus-veneris lack trnP-GGG and rpl21, suggesting these are not conserved among angiosperm cp genomes. The deletion of trnR-UCG, trnR-CCG, and trnSeC in the cp genomes of C. falcatum and other eupolypod ferns indicates these genes are restricted to tree ferns, non-core leptosporangiates, and basal ferns. The C. falcatum cp genome also encoded ndhF and rps7, with GUG start codons that were only conserved in polypod ferns, and it shares two significant inversions with other ferns, including a minor inversion of the trnD-GUC region and an approximate 3 kb inversion of the trnG-trnT region. Phylogenetic analyses showed that Equisetum was found to be a sister clade to Psilotales-Ophioglossales with a 100% bootstrap (BS) value. The sister relationship between Pteridaceae and eupolypods was also strongly supported by a 100% BS, but Bayesian molecular clock analyses suggested that C. falcatum diversified in the mid-Paleogene period (45.15 ± 4.93 million years ago) and might have moved from Eurasia to Dokdo Island.

  17. Modified Genetic Algorithm for DNA Sequence Assembly by Shotgun and Hybridization Sequencing Techniques

    Directory of Open Access Journals (Sweden)

    Prof.Narayan Kumar Sahu

    2012-09-01

    Full Text Available Since the advent of rapid DNA sequencing methods in 1976, scientists have had the problem of inferring DNA sequences from sequenced fragments. Shotgun sequencing is a well-established biological and computational method used in practice. Many conventional algorithms for shotgun sequencing are based on the notion of pair wise fragment overlap. While shotgun sequencing infers a DNA sequence given the sequences of overlapping fragments, a recent and complementary method, called sequencing by hybridization (SBH, infers a DNA sequence given the set of oligomers that represents all sub words of some fixed length, k. In this paper, we propose a new computer algorithm for DNA sequence assembly that combines in a novel way the techniques of both shotgun and SBH methods. Based on our preliminary investigations, the algorithm promises- to be very fast and practical for DNA sequence assembly [1].

  18. Nucleosome DNA sequence structure of isochores

    Directory of Open Access Journals (Sweden)

    Trifonov Edward N

    2011-04-01

    Full Text Available Abstract Background Significant differences in G+C content between different isochore types suggest that the nucleosome positioning patterns in DNA of the isochores should be different as well. Results Extraction of the patterns from the isochore DNA sequences by Shannon N-gram extension reveals that while the general motif YRRRRRYYYYYR is characteristic for all isochore types, the dominant positioning patterns of the isochores vary between TAAAAATTTTTA and CGGGGGCCCCCG due to the large differences in G+C composition. This is observed in human, mouse and chicken isochores, demonstrating that the variations of the positioning patterns are largely G+C dependent rather than species-specific. The species-specificity of nucleosome positioning patterns is revealed by dinucleotide periodicity analyses in isochore sequences. While human sequences are showing CG periodicity, chicken isochores display AG (CT periodicity. Mouse isochores show very weak CG periodicity only. Conclusions Nucleosome positioning pattern as revealed by Shannon N-gram extension is strongly dependent on G+C content and different in different isochores. Species-specificity of the pattern is subtle. It is reflected in the choice of preferentially periodical dinucleotides.

  19. Molecular phylogenetics of the species-rich angiosperm genus Goniothalamus (Annonaceae) inferred from nine chloroplast DNA regions: Synapomorphies and putative correlated evolutionary changes in fruit and seed morphology.

    Science.gov (United States)

    Tang, Chin Cheung; Thomas, Daniel C; Saunders, Richard M K

    2015-11-01

    A phylogenetic study of the genus Goniothalamus (Annonaceae) is presented using maximum parsimony, maximum likelihood and Bayesian approaches, with 65 species sampled (48.5% of the genus) based on sequences of nine chloroplast DNA regions (11,214 aligned positions). The resultant phylogeny clearly indicates that Goniothalamus is monophyletic. Preliminary research initially focused on identifying synapomorphies and estimating the phylogenetic signal of selected morphological characters based on parsimony and likelihood ancestral character state reconstructions. This prescreening of characters enabled 40 to be selected for further study, and of these 15 are shown here to demonstrate significant phylogenetic signal and to provide clear synapomorphies for several infrageneric clades. Although floral structure in Goniothalamus is comparatively uniform, suggesting a common basic pattern of pollination ecology, fruit and seed morphology in the genus is very diverse and is presumably associated with different patterns of frugivory. The present study assesses correlations amongst fruit and seed characters which are putatively of functional importance with regard to frugivory and dispersal. One-way phylogenetic ANOVA indicates significant phylogenetically independent correlation between the following fruit and seed characters: fruits borne on older branches and/or on the main trunk have larger monocarps than fruits borne on young branches; and monocarps that contain seeds with a hairy testa are larger than those with glabrous seeds. We discuss fruit morphologies and potential explanations for the inferred correlations, and suggest that they may be the result of adaptation to different frugivores (birds, larger non-volant animal and primate seed dispersers, respectively).

  20. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Winston Chen, C.H.; Taranenko, N.I.; Zhu, Y.F.; Chung, C.N.; Allman, S.L.

    1997-03-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, the authors recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Snager`s enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. The preliminary results indicate laser mass spectrometry can possibly be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, the authors applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  1. Chloroplast movement.

    Science.gov (United States)

    Wada, Masamitsu

    2013-09-01

    Chloroplast movement is important for plant survival under high light and for efficient photosynthesis under low light. This review introduces recent knowledge on chloroplast movement and shows how to analyze the responses and the moving mechanisms, potentially inspiring research in this field. Avoidance from the strong light is mediated by blue light receptor phototropin 2 (phot2) plausibly localized on the chloroplast envelop and accumulation at the week light-irradiated area is mediated by phot1 and phot2 localized on the plasma membrane. Chloroplasts move by chloroplast actin (cp-actin) filaments that must be polymerized by Chloroplast Unusual Positioning1 (CHUP1) at the front side of moving chloroplast. To understand the signal transduction pathways and the mechanism of chloroplast movement, that is, from light capture to motive force-generating mechanism, various methods should be employed based on the various aspects. Observation of chloroplast distribution pattern under different light condition by fixed cell sectioning is somewhat an old-fashioned technique but the most basic and important way. However, most importantly, precise chloroplast behavior during and just after the induction of chloroplast movement by partial cell irradiation using an irradiator with either low light or strong light microbeam should be recorded by time lapse photographs under infrared light and analyzed. Recently various factors involved in chloroplast movement, such as cp-actin filaments and CHUP1, could be traced in Arabidopsis transgenic lines with fluorescent protein tags under a confocal laser scanning microscope (CLSM) and/or a total internal reflection fluorescence microscope (TIRFM). These methods are listed and their advantages and disadvantages are evaluated.

  2. DNA barcoding of arid wild plants using rbcL gene sequences.

    Science.gov (United States)

    Bafeel, S O; Arif, I A; Bakir, M A; Al Homaidan, A A; Al Farhan, A H; Khan, H A

    2012-07-19

    DNA barcoding is currently gaining popularity due to its simplicity and high accuracy as compared to the complexity and subjective biases associated with morphology-based identification of taxa. The standard chloroplast DNA barcode for land plants recommended by the Consortium for the Barcode of Life (CBOL) plant working group needs to be evaluated for a wide range of plant species. We therefore tested the potential of the rbcL marker for the identification of wild plants belonging to diverse families of arid regions. Maximum likelihood tree analysis was performed to evaluate the discriminatory power of the rbcL gene. Our findings showed that using rbcL gene sequences enabled identification of the majority of the samples (92%) to genus level and only 17% to species level.

  3. The complete chloroplast genome sequence of Gentiana lawrencei var. farreri (Gentianaceae and comparative analysis with its congeneric species

    Directory of Open Access Journals (Sweden)

    Peng-Cheng Fu

    2016-09-01

    Full Text Available Background The chloroplast (cp genome is useful in plant systematics, genetic diversity analysis, molecular identification and divergence dating. The genus Gentiana contains 362 species, but there are only two valuable complete cp genomes. The purpose of this study is to report the characterization of complete cp genome of G. lawrencei var. farreri, which is endemic to the Qinghai-Tibetan Plateau (QTP. Methods Using high throughput sequencing technology, we got the complete nucleotide sequence of the G. lawrencei var. farreri cp genome. The comparison analysis including genome difference and gene divergence was performed with its congeneric species G. straminea. The simple sequence repeats (SSRs and phylogenetics were studied as well. Results The cp genome of G. lawrencei var. farreri is a circular molecule of 138,750 bp, containing a pair of 24,653 bp inverted repeats which are separated by small and large single-copy regions of 11,365 and 78,082 bp, respectively. The cp genome contains 130 known genes, including 85 protein coding genes (PCGs, eight ribosomal RNA genes and 37 tRNA genes. Comparative analyses indicated that G. lawrencei var. farreri is 10,241 bp shorter than its congeneric species G. straminea. Four large gaps were detected that are responsible for 85% of the total sequence loss. Further detailed analyses revealed that 10 PCGs were included in the four gaps that encode nine NADH dehydrogenase subunits. The cp gene content, order and orientation are similar to those of its congeneric species, but with some variation among the PCGs. Three genes, ndhB, ndhF and clpP, have high nonsynonymous to synonymous values. There are 34 SSRs in the G. lawrencei var. farreri cp genome, of which 25 are mononucleotide repeats: no dinucleotide repeats were detected. Comparison with the G. straminea cp genome indicated that five SSRs have length polymorphisms and 23 SSRs are species-specific. The phylogenetic analysis of 48 PCGs from 12 Gentianales

  4. Sequence dependent hole evolution in DNA.

    Science.gov (United States)

    Lakhno, V D

    2004-06-01

    The paper examines thedynamical behavior of a radical cation(G(+*)) generated in adouble stranded DNA for differentoligonucleotide sequences. The resonancehole tunneling through an oligonucleotidesequence is studied by the method ofnumerical integration of self-consistentquantum-mechanical equations. The holemotion is considered quantum mechanicallyand nucleotide base oscillations aretreated classically. The results obtaineddemonstrate a strong dependence of chargetransfer on the type of nucleotidesequence. The rates of the hole transferare calculated for different nucleotidesequences and compared with experimentaldata on the transfer from (G(+*))to a GGG unit.

  5. Evolutionary processes in a continental island system: molecular phylogeography of the Aegean Nigella arvensis alliance (Ranunculaceae) inferred from chloroplast DNA.

    Science.gov (United States)

    Bittkau, C; Comes, H P

    2005-11-01

    Continental shelf island systems, created by rising sea levels, provide a premier setting for studying the effects of past fragmentation, dispersal, and genetic drift on taxon diversification. We used phylogeographical (nested clade) and population genetic analyses to elucidate the relative roles of these processes in the evolutionary history of the Aegean Nigella arvensis alliance (= 'coenospecies'). We surveyed chloroplast DNA (cpDNA) variation in 455 individuals from 47 populations (nine taxa) of the alliance throughout its core range in the Aegean Archipelago and surrounding mainland areas of Greece and Turkey. The study revealed the presence of three major lineages, with largely nonoverlapping distributions in the Western, Central, and Eastern Aegean. There is evidence supporting the idea that these major lineages evolved in situ from a widespread (pan-Aegean) ancestral stock as a result of multiple fragmentation events, possibly due to the influence of post-Messinian sea flooding, Pleistocene eustatic changes and corresponding climate fluctuations. Over-sea dispersal and founder events appear to have played a rather insignificant role in the group's history. Rather, all analytical approaches identified the alliance as an organism group with poor seed dispersal capabilities and a susceptibility to genetic drift. In particular, we inferred that the observed level of cpDNA differentiation between Kikladian island populations of Nigella degenii largely reflects population history, (viz. Holocene island fragmentation) and genetic drift in the near absence of seed flow since their time of common ancestry. Overall, our cpDNA data for the N. arvensis alliance in general, and N. degenii in particular, indicate that historical events were important in determining the phylogeographical patterns seen, and that genetic drift has historically been relatively more influential on population structure than has cytoplasmic gene flow.

  6. Transverse Electronic Signature of DNA for Electronic Sequencing

    Science.gov (United States)

    Xu, Mingsheng; Endres, Robert G.; Arakawa, Yasuhiko

    In recent years, the proliferation of large-scale DNA sequencing projects for applications in clinical medicine and health care has driven the search for new methods that could reduce the time and cost. The commonly used Sanger sequencing method relies on the chemistry to read the bases in DNA and is far too slow and expensive for reading personal genetic codes. There were earlier attempts to sequence DNA by directly visualizing the nucleotide composition of the DNA molecules by scanning tunneling microscopy (STM). However, sequencing DNA based on directly imaging DNA's atomic structure has not yet been successful. In Chap. 9, Xu, Endres, and Arakawa report a potential physical alternative by detecting unique transverse electronic signatures of DNA bases using ultrahigh vacuum STM. Supported by the principles, calculations and statistical analyses, these authors argue that it would be possible to directly sequence DNA by the STM-based technology without any modification of the DNA.

  7. Understanding Long-Range Correlations in DNA sequences

    CERN Document Server

    Li, W; Kaneko, K; Wentian Li; Thomas G Marr; Kunihiko Kaneko

    1994-01-01

    Abstract: In this paper, we review the literature on statistical long-range correlation in DNA sequences. We examine the current evidence for these correlations, and conclude that a mixture of many length scales (including some relatively long ones) in DNA sequences is responsible for the observed 1/f-like spectral component. We note the complexity of the correlation structure in DNA sequences. The observed complexity often makes it hard, or impossible, to decompose the sequence into a few statistically stationary regions. We suggest that, based on the complexity of DNA sequences, a fruitful approach to understand long-range correlation is to model duplication, and other rearrangement processes, in DNA sequences. One model, called ``expansion-modification system", contains only point duplication and point mutation. Though simplistic, this model is able to generate sequences with 1/f spectra. We emphasize the importance of DNA duplication in its contribution to the observed long-range correlation in DNA sequen...

  8. Intra-individual polymorphism in chloroplasts from NGS data: where does it come from and how to handle it?

    Science.gov (United States)

    Scarcelli, N; Mariac, C; Couvreur, T L P; Faye, A; Richard, D; Sabot, F; Berthouly-Salazar, C; Vigouroux, Y

    2016-03-01

    Next-generation sequencing allows access to a large quantity of genomic data. In plants, several studies used whole chloroplast genome sequences for inferring phylogeography or phylogeny. Even though the chloroplast is a haploid organelle, NGS plastome data identified a nonnegligible number of intra-individual polymorphic SNPs. Such observations could have several causes such as sequencing errors, the presence of heteroplasmy or transfer of chloroplast sequences in the nuclear and mitochondrial genomes. The occurrence of allelic diversity has practical important impacts on the identification of diversity, the analysis of the chloroplast data and beyond that, significant evolutionary questions. In this study, we show that the observed intra-individual polymorphism of chloroplast sequence data is probably the result of plastid DNA transferred into the mitochondrial and/or the nuclear genomes. We further assess nine different bioinformatics pipelines' error rates for SNP and genotypes calling using SNPs identified in Sanger sequencing. Specific pipelines are adequate to deal with this issue, optimizing both specificity and sensitivity. Our results will allow a proper use of whole chloroplast NGS sequence and will allow a better handling of NGS chloroplast sequence diversity.

  9. A new DNA sequence assembly program.

    Science.gov (United States)

    Bonfield, J K; Smith, K f; Staden, R

    1995-01-01

    We describe the Genome Assembly Program (GAP), a new program for DNA sequence assembly. The program is suitable for large and small projects, a variety of strategies and can handle data from a range of sequencing instruments. It retains the useful components of our previous work, but includes many novel ideas and methods. Many of these methods have been made possible by the program's completely new, and highly interactive, graphical user interface. The program provides many visual clues to the current state of a sequencing project and allows users to interact in intuitive and graphical ways with their data. The program has tools to display and manipulate the various types of data that help to solve and check difficult assemblies, particularly those in repetitive genomes. We have introduced the following new displays: the Contig Selector, the Contig Comparator, the Template Display, the Restriction Enzyme Map and the Stop Codon Map. We have also made it possible to have any number of Contig Editors and Contig Joining Editors running simultaneously even on the same contig. The program also includes a new 'Directed Assembly' algorithm and routines for automatically detecting unfinished segments of sequence, to which it suggests experimental solutions. Images PMID:8559656

  10. Phylogenetic analysis of Rutaceous plants based on single nucleotide polymorphism in chloroplast and nuclear gene sequences

    Science.gov (United States)

    The family Rutaceae encompasses several genera including the economically important genus Citrus. In this study, we selected 22 citrus relatives belonging to the various sub groups of Rutaceae and compared the sequences of three gene fragments. The accessions selected belong to the subfamily Rutoide...

  11. SIMPLE PROCEDURES FOR EXTRACTION OF TOTAL AND CHLOROPLAST DNA FROM Passiflora SPECIES

    OpenAIRE

    Góngora F., Gustavo; Departamento de Biología Facultad de Ciencias Pontificia Universidad Javeriana; Hodson de Jaramillo, Elizabeth; Unidad de Biología Vegetal, Departamento de Biología, Pontificia Universidad Javeriana, Bogotá

    2013-01-01

    Se describe una técnica sencilla para la extracción de DNA total y ctDNA de especies del género Passiflora. El DNA total fue purificado a partir de bajas cantidades (40 mg) de material vegetal de diferentes fuentes (brotes, zarcillos, estípulas, nudos y hojas) en presencia de PVPP con el fin de evitar la acción deletérea de los polifenoles. El aislamiento del ctDNA se realizó a través de la lisis de cloroplastos purificados en gradientes discontinuos de sacarosa. Se obtuvo alrededor de 50 mg ...

  12. Improved taboo search algorithm for designing DNA sequences

    Institute of Scientific and Technical Information of China (English)

    Kai Zhang; Jin Xu; Xiutang Geng; Jianhua Xiao; Linqiang Pan

    2008-01-01

    The design of DNA sequences is one of the most practical and important research topics in DNA computing.We adopt taboo search algorithm and improve the method for the systematic design of equal-length DNA sequences,which can satisfy certain combinatorial and thermodynamic constraints.Using taboo search algorithm,our method can avoid trapping into local optimization and can find a set of good DNA sequences satisfying required constraints.

  13. Diversification, biogeographic pattern, and demographic history of Taiwanese Scutellaria species inferred from nuclear and chloroplast DNA.

    Directory of Open Access Journals (Sweden)

    Yu-Chung Chiang

    Full Text Available The ragged topography created by orogenesis generates diversified habitats for plants in Taiwan. In addition to colonization from nearby mainland China, high species diversity and endemism of plants is also present in Taiwan. Five of the seven Scutellaria species (Lamiaceae in Taiwan, for example, are endemic to the island. Hypotheses of multiple sources or in situ radiation have arisen to explain the high endemism of Taiwanese species. In this study, phylogenetic analyses using both nuclear and chloroplast markers revealed the multiple sources of Taiwanese Scutellaria species and confirmed the rapid and recent speciation of endemic species, especially those of the "indica group" composed of S. indica, S. austrotaiwanensis, S. tashiroi, and S. playfairii. The common ancestors of the indica group colonized first in northern Taiwan and dispersed regionally southward and eastward. Climate changes during glacial/interglacial cycles led to gradual colonization and variance events in the ancestors of these species, resulting in the present distribution and genetic differentiation of extant populations. Population decline was also detected in S. indica, which might reflect a bottleneck effect from the glacials. In contrast, the recently speciated endemic members of the indica group have not had enough time to accumulate much genetic variation and are thus genetically insensitive to demographic fluctuations, but the extant lineages were spatially expanded in the coalescent process. This study integrated phylogenetic and population genetic analyses to illustrate the evolutionary history of Taiwanese Scutellaria of high endemism and may be indicative of the diversification mechanism of plants on continental islands.

  14. Diversification, biogeographic pattern, and demographic history of Taiwanese Scutellaria species inferred from nuclear and chloroplast DNA.

    Science.gov (United States)

    Chiang, Yu-Chung; Huang, Bing-Hong; Liao, Pei-Chun

    2012-01-01

    The ragged topography created by orogenesis generates diversified habitats for plants in Taiwan. In addition to colonization from nearby mainland China, high species diversity and endemism of plants is also present in Taiwan. Five of the seven Scutellaria species (Lamiaceae) in Taiwan, for example, are endemic to the island. Hypotheses of multiple sources or in situ radiation have arisen to explain the high endemism of Taiwanese species. In this study, phylogenetic analyses using both nuclear and chloroplast markers revealed the multiple sources of Taiwanese Scutellaria species and confirmed the rapid and recent speciation of endemic species, especially those of the "indica group" composed of S. indica, S. austrotaiwanensis, S. tashiroi, and S. playfairii. The common ancestors of the indica group colonized first in northern Taiwan and dispersed regionally southward and eastward. Climate changes during glacial/interglacial cycles led to gradual colonization and variance events in the ancestors of these species, resulting in the present distribution and genetic differentiation of extant populations. Population decline was also detected in S. indica, which might reflect a bottleneck effect from the glacials. In contrast, the recently speciated endemic members of the indica group have not had enough time to accumulate much genetic variation and are thus genetically insensitive to demographic fluctuations, but the extant lineages were spatially expanded in the coalescent process. This study integrated phylogenetic and population genetic analyses to illustrate the evolutionary history of Taiwanese Scutellaria of high endemism and may be indicative of the diversification mechanism of plants on continental islands.

  15. Sequencing of chloroplast genomes from wheat, barley, rye and their relatives provides a detailed insight into the evolution of the Triticeae tribe.

    Directory of Open Access Journals (Sweden)

    Christopher P Middleton

    Full Text Available Using Roche/454 technology, we sequenced the chloroplast genomes of 12 Triticeae species, including bread wheat, barley and rye, as well as the diploid progenitors and relatives of bread wheat Triticum urartu, Aegilops speltoides and Ae. tauschii. Two wild tetraploid taxa, Ae. cylindrica and Ae. geniculata, were also included. Additionally, we incorporated wild Einkorn wheat Triticum boeoticum and its domesticated form T. monococcum and two Hordeum spontaneum (wild barley genotypes. Chloroplast genomes were used for overall sequence comparison, phylogenetic analysis and dating of divergence times. We estimate that barley diverged from rye and wheat approximately 8-9 million years ago (MYA. The genome donors of hexaploid wheat diverged between 2.1-2.9 MYA, while rye diverged from Triticum aestivum approximately 3-4 MYA, more recently than previously estimated. Interestingly, the A genome taxa T. boeoticum and T. urartu were estimated to have diverged approximately 570,000 years ago. As these two have a reproductive barrier, the divergence time estimate also provides an upper limit for the time required for the formation of a species boundary between the two. Furthermore, we conclusively show that the chloroplast genome of hexaploid wheat was contributed by the B genome donor and that this unknown species diverged from Ae. speltoides about 980,000 years ago. Additionally, sequence alignments identified a translocation of a chloroplast segment to the nuclear genome which is specific to the rye/wheat lineage. We propose the presented phylogeny and divergence time estimates as a reference framework for future studies on Triticeae.

  16. Generic boundaries and evolution of characters in the Arctium group: a nuclear and chloroplast DNA analysis

    Directory of Open Access Journals (Sweden)

    Susanna, A.

    2003-12-01

    Full Text Available Generic delineation within the Arctium group (Compositae, Carduceae-Carduinae, formed by the genera Arctium, Cousinia, Hypacanthium and Schamalhausenia, has proven a complicated task. In particular, the precise limits between Arctium and Cousinia are very difficult to establish. Therefore, we have carried out a molecular survey of DNA sequences of two regions, the chloroplast gene matK and the nuclear-ribosomal spacers ITS 1 and 2, of a representation of all the genera of the group (in the case of Cousinia, centered in the species more obviously related to Arctiium. Our results show a precise correlation between molecular phylogeny and two very important characters, pollen type and chromosome numbers: all the investigated species with the Arctiastrum pollen type and x= 18, characteristics of Arctium sensu stricto, form a monophyletic clade, sister of another monophyletic clade formed by all the investigated species of Cousinia sensu slricto. However, the resulting "Arctioid" clade cannot be defined on macroscopic morphologic characters, because the main trait for segregating Arctium and Cousinia, the spiny pinnatifid-pinnatisect leaves of Cousinia, is adaptative and of scarce systematic relevance. In fact, our results suggest that spines have appeared at least in two different lineages: the genera Hypacanthium and Schamalhausenia, spiny and thus morphologically closer to Cousinia, are unambiguously related to the unarmed genus Arctium. An hypothesis on the evolution of morphology, pollen and chromosome numbers in the group is formulated. The systematic implications of this incongruence between molecular, pollen and karyology, on the one hand, and morphology, on the other hand, are evaluated. Some possible solutions are proposed, but none of them is totally satisfactory: more studies are necessary with the inclusion

  17. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors

    OpenAIRE

    2009-01-01

    This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein–DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are esse...

  18. Complete Chloroplast Genome of Tanaecium tetragonolobum: The First Bignoniaceae Plastome.

    Directory of Open Access Journals (Sweden)

    Alison Gonçalves Nazareno

    Full Text Available Bignoniaceae is a Pantropical plant family that is especially abundant in the Neotropics. Members of the Bignoniaceae are diverse in many ecosystems and represent key components of the Tropical flora. Despite the ecological importance of the Bignoniaceae and all the efforts to reconstruct the phylogeny of this group, whole chloroplast genome information has not yet been reported for any members of the family. Here, we report the complete chloroplast genome sequence of Tanaecium tetragonolobum (Jacq. L.G. Lohmann, which was reconstructed using de novo and referenced-based assembly of single-end reads generated by shotgun sequencing of total genomic DNA in an Illumina platform. The gene order and organization of the chloroplast genome of T. tetragonolobum exhibits the general structure of flowering plants, and is similar to other Lamiales chloroplast genomes. The chloroplast genome of T. tetragonolobum is a circular molecule of 153,776 base pairs (bp with a quadripartite structure containing two single copy regions, a large single copy region (LSC, 84,612 bp and a small single copy region (SSC, 17,586 bp separated by inverted repeat regions (IRs, 25,789 bp. In addition, the chloroplast genome of T. tetragonolobum has 38.3% GC content and includes 121 genes, of which 86 are protein-coding, 31 are transfer RNA, and four are ribosomal RNA. The chloroplast genome of T. tetragonolobum presents a total of 47 tandem repeats and 347 simple sequence repeats (SSRs with mononucleotides being the most common and di-, tri-, tetra-, and hexanucleotides occurring with less frequency. The results obtained here were compared to other chloroplast genomes of Lamiales available to date, providing new insight into the evolution of chloroplast genomes within Lamiales. Overall, the evolutionary rates of genes in Lamiales are lineage-, locus-, and region-specific, indicating that the evolutionary pattern of nucleotide substitution in chloroplast genomes of flowering

  19. DNA Sequence Optimization Based on Continuous Particle Swarm Optimization for Reliable DNA Computing and DNA Nanotechnology

    Directory of Open Access Journals (Sweden)

    N. K. Khalid

    2008-01-01

    Full Text Available Problem statement: In DNA based computation and DNA nanotechnology, the design of good DNA sequences has turned out to be an essential problem and one of the most practical and important research topics. Basically, the DNA sequence design problem is a multi-objective problem and it can be evaluated using four objective functions, namely, Hmeasure, similarity, continuity and hairpin. Approach: There are several ways to solve multi-objective problem, however, in order to evaluate the correctness of PSO algorithm in DNA sequence design, this problem is converted into single objective problem. Particle Swarm Optimization (PSO is proposed to minimize the objective in the problem, subjected to two constraints: melting temperature and GCcontent. A model is developed to present the DNA sequence design based on PSO computation. Results: Based on experiments and researches done, 20 particles are used in the implementation of the optimization process, where the average values and the standard deviation for 100 runs are shown along with comparison to other existing methods. Conclusion: The results achieve verified that PSO can suitably solves the DNA sequence design problem using the proposed method and model, comparatively better than other approaches.

  20. A DNA Structure-Based Bionic Wavelet Transform and Its Application to DNA Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2003-01-01

    Full Text Available DNA sequence analysis is of great significance for increasing our understanding of genomic functions. An important task facing us is the exploration of hidden structural information stored in the DNA sequence. This paper introduces a DNA structure-based adaptive wavelet transform (WT – the bionic wavelet transform (BWT – for DNA sequence analysis. The symbolic DNA sequence can be separated into four channels of indicator sequences. An adaptive symbol-to-number mapping, determined from the structural feature of the DNA sequence, was introduced into WT. It can adjust the weight value of each channel to maximise the useful energy distribution of the whole BWT output. The performance of the proposed BWT was examined by analysing synthetic and real DNA sequences. Results show that BWT performs better than traditional WT in presenting greater energy distribution. This new BWT method should be useful for the detection of the latent structural features in future DNA sequence analysis.

  1. Comparison of intraspecific, interspecific and intergeneric chloroplast diversity in Cycads

    Science.gov (United States)

    Jiang, Guo-Feng; Hinsinger, Damien Daniel; Strijk, Joeri Sergej

    2016-01-01

    Cycads are among the most threatened plant species. Increasing the availability of genomic information by adding whole chloroplast data is a fundamental step in supporting phylogenetic studies and conservation efforts. Here, we assemble a dataset encompassing three taxonomic levels in cycads, including ten genera, three species in the genus Cycas and two individuals of C. debaoensis. Repeated sequences, SSRs and variations of the chloroplast were analyzed at the intraspecific, interspecific and intergeneric scale, and using our sequence data, we reconstruct a phylogenomic tree for cycads. The chloroplast was 162,094 bp in length, with 133 genes annotated, including 87 protein-coding, 37 tRNA and 8 rRNA genes. We found 7 repeated sequences and 39 SSRs. Seven loci showed promising levels of variations for application in DNA-barcoding. The chloroplast phylogeny confirmed the division of Cycadales in two suborders, each of them being monophyletic, revealing a contradiction with the current family circumscription and its evolution. Finally, 10 intraspecific SNPs were found. Our results showed that despite the extremely restricted distribution range of C. debaoensis, using complete chloroplast data is useful not only in intraspecific studies, but also to improve our understanding of cycad evolution and in defining conservation strategies for this emblematic group. PMID:27558458

  2. Chloroplast DNA phylogeography suggests a West African centre of origin for the baobab, Adansonia digitata L. (Bombacoideae, Malvaceae).

    Science.gov (United States)

    Leong Pock Tsy, Jean-Michel; Lumaret, Roselyne; Mayne, Diana; Vall, Abdallahi Ould Mohamed; Abutaba, Yahia I M; Sagna, Maurice; Rakotondralambo Raoseta, Soaharin'ny Ony; Danthu, Pascal

    2009-04-01

    The African baobab (Adansonia digitata L.) is an emblematic, culturally important, and physically huge tropical tree species whose natural geographical distribution comprises most of tropical Africa, but also small patches of southern Arabia, and several Atlantic and Indian Ocean islands surrounding the African continent, notably including Madagascar. We analysed the polymerase chain reaction-restriction fragment length polymorphism of five chloroplast DNA fragments obtained from 344 individuals of A. digitata collected from 74 populations covering the entire extant distribution range of the species. Our goal was to reconstruct the phylogeographical history of the species and, if possible, to identify its centre of origin, which has been a subject of controversy for many decades. We identified five haplotypes whose distribution is clearly geographically structured. Using several species of Adansonia and of closely related genera as outgroups, the haplotypes showed a clear phylogeographical pattern of three groups. Two are phylogenetically related to the outgroup taxa, and are distributed in West Africa. The third group is substantially more differentiated genetically from outgroup species, and it corresponds to southern and eastern Africa, Arabia and the Indian Ocean islands, including Madagascar. According to our results, the tetraploid A. digitata, or its diploid progenitor, probably originated in West Africa and migrated subsequently throughout the tropical parts of that continent, and beyond, by natural and human-mediated terrestrial and overseas dispersal.

  3. Complete chloroplast genome sequence of poisonous and medicinal plant Datura stramonium: organizations and implications for genetic engineering.

    Science.gov (United States)

    Yang, Yang; Dang, Yuanye; Yuanye, Dang; Li, Qing; Qing, Li; Lu, Jinjian; Jinjian, Lu; Li, Xiwen; Xiwen, Li; Wang, Yitao; Yitao, Wang

    2014-01-01

    Datura stramonium is a widely used poisonous plant with great medicinal and economic value. Its chloroplast (cp) genome is 155,871 bp in length with a typical quadripartite structure of the large (LSC, 86,302 bp) and small (SSC, 18,367 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,601 bp). The genome contains 113 unique genes, including 80 protein-coding genes, 29 tRNAs and four rRNAs. A total of 11 forward, 9 palindromic and 13 tandem repeats were detected in the D. stramonium cp genome. Most simple sequence repeats (SSR) are AT-rich and are less abundant in coding regions than in non-coding regions. Both SSRs and GC content were unevenly distributed in the entire cp genome. All preferred synonymous codons were found to use A/T ending codons. The difference in GC contents of entire genomes and of the three-codon positions suggests that the D. stramonium cp genome might possess different genomic organization, in part due to different mutational pressures. The five most divergent coding regions and four non-coding regions (trnH-psbA, rps4-trnS, ndhD-ccsA, and ndhI-ndhG) were identified using whole plastome alignment, which can be used to develop molecular markers for phylogenetics and barcoding studies within the Solanaceae. Phylogenetic analysis based on 68 protein-coding genes supported Datura as a sister to Solanum. This study provides valuable information for phylogenetic and cp genetic engineering studies of this poisonous and medicinal plant.

  4. Solid-Phase Purification of Synthetic DNA Sequences.

    Science.gov (United States)

    Grajkowski, Andrzej; Cieslak, Jacek; Beaucage, Serge L

    2016-08-05

    Although high-throughput methods for solid-phase synthesis of DNA sequences are currently available for synthetic biology applications and technologies for large-scale production of nucleic acid-based drugs have been exploited for various therapeutic indications, little has been done to develop high-throughput procedures for the purification of synthetic nucleic acid sequences. An efficient process for purification of phosphorothioate and native DNA sequences is described herein. This process consists of functionalizing commercial aminopropylated silica gel with aminooxyalkyl functions to enable capture of DNA sequences carrying a 5'-siloxyl ether linker with a "keto" function through an oximation reaction. Deoxyribonucleoside phosphoramidites functionalized with the 5'-siloxyl ether linker were prepared in yields of 75-83% and incorporated last into the solid-phase assembly of DNA sequences. Capture of nucleobase- and phosphate-deprotected DNA sequences released from the synthesis support is demonstrated to proceed near quantitatively. After shorter than full-length DNA sequences were washed from the capture support, the purified DNA sequences were released from this support upon treatment with tetra-n-butylammonium fluoride in dry DMSO. The purity of released DNA sequences exceeds 98%. The scalability and high-throughput features of the purification process are demonstrated without sacrificing purity of the DNA sequences.

  5. Affinity purification of sequence-specific DNA binding proteins.

    OpenAIRE

    1986-01-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed t...

  6. The toxic dinoflagellate Dinophysis acuminata harbors permanent chloroplasts of cryptomomad prigin, not kleptochloroplasts

    DEFF Research Database (Denmark)

    Garcia, Lydia; Moestrup, Øjvind; Hansen, Per Juel;

    2010-01-01

    Most species belonging to the toxigenic genus Dinophysis have chloroplasts of cryptophyte origin. Whether these chloroplasts are temporarily sequestered from the prey, or permanently established under the control of the dinoflagellate is currently disputed. To investigate this, a culture...... of Dinophysis acuminata was established by feeding it the phototrophic ciliate Mesodinium rubrum (= Myrionecta rubra), which again was fed the cryptophyte Teleaulax amphioxeia. Molecular analysis comprising the nucleomorph LSU and two chloroplast markers (tufA gene and a fragment from the end of 16S r......DNA to the beginning of 23S rDNA) resulted in identical sequences for the three organisms. Yet, transmission electron microscopy of the three organisms revealed that several chloroplast features separated D. acuminata from both T. amphioxeia and M. rubrum. The thylakoid arrangement, the number of membranes around...

  7. Phototropins mediate blue and red light-induced chloroplast movements in Physcomitrella patens.

    Science.gov (United States)

    Kasahara, Masahiro; Kagawa, Takatoshi; Sato, Yoshikatsu; Kiyosue, Tomohiro; Wada, Masamitsu

    2004-07-01

    Phototropin is the blue-light receptor that mediates phototropism, chloroplast movement, and stomatal opening in Arabidopsis. Blue and red light induce chloroplast movement in the moss Physcomitrella patens. To study the photoreceptors for chloroplast movement in P. patens, four phototropin genes (PHOTA1, PHOTA2, PHOTB1, and PHOTB2) were isolated by screening cDNA libraries. These genes were classified into two groups (PHOTA and PHOTB) on the basis of their deduced amino acid sequences. Then phototropin disruptants were generated by homologous recombination and used for analysis of chloroplast movement. Data revealed that blue light-induced chloroplast movement was mediated by phototropins in P. patens. Both photA and photB groups were able to mediate chloroplast avoidance, as has been reported for Arabidopsis phot2, although the photA group contributed more to the response. Red light-induced chloroplast movement was also significantly reduced in photA2photB1photB2 triple disruptants. Because the primary photoreceptor for red light-induced chloroplast movement in P. patens is phytochrome, phototropins may be downstream components of phytochromes in the signaling pathway. To our knowledge, this work is the first to show a function for the phototropin blue-light receptor in a response to wavelengths that it does not absorb.

  8. SWORDS: A statistical tool for analysing large DNA sequences

    Indian Academy of Sciences (India)

    Probal Chaudhuri; Sandip Das

    2002-02-01

    In this article, we present some simple yet effective statistical techniques for analysing and comparing large DNA sequences. These techniques are based on frequency distributions of DNA words in a large sequence, and have been packaged into a software called SWORDS. Using sequences available in public domain databases housed in the Internet, we demonstrate how SWORDS can be conveniently used by molecular biologists and geneticists to unmask biologically important features hidden in large sequences and assess their statistical significance.

  9. DNA Sequence Determination by Hybridization: A Strategy for Efficient Large-Scale Sequencing

    Science.gov (United States)

    Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoddy, J.; Funkhouser, W. K.; Koop, B.; Hood, L.; Crkvenjakov, R.

    1993-06-01

    The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.

  10. A novel constraint for thermodynamically designing DNA sequences.

    Directory of Open Access Journals (Sweden)

    Qiang Zhang

    Full Text Available Biotechnological and biomolecular advances have introduced novel uses for DNA such as DNA computing, storage, and encryption. For these applications, DNA sequence design requires maximal desired (and minimal undesired hybridizations, which are the product of a single new DNA strand from 2 single DNA strands. Here, we propose a novel constraint to design DNA sequences based on thermodynamic properties. Existing constraints for DNA design are based on the Hamming distance, a constraint that does not address the thermodynamic properties of the DNA sequence. Using a unique, improved genetic algorithm, we designed DNA sequence sets which satisfy different distance constraints and employ a free energy gap based on a minimum free energy (MFE to gauge DNA sequences based on set thermodynamic properties. When compared to the best constraints of the Hamming distance, our method yielded better thermodynamic qualities. We then used our improved genetic algorithm to obtain lower-bound DNA sequence sets. Here, we discuss the effects of novel constraint parameters on the free energy gap.

  11. Analysis of gene sequences indicates that quantity not quality of chloroplast small HSPs improves thermotolerance in C4 and CAM plants.

    Science.gov (United States)

    Shakeel, Samina N; Ul Haq, Noor; Heckathorn, Scott; Luthe, D S

    2012-10-01

    Chloroplast-localized small heat-shock proteins (Cp-sHSP) protect Photosystem II and thylakoid membranes during heat and other stresses, and Cp-sHSP production levels are related to plant thermotolerance. However, to date, a paucity of Cp-sHSP sequences from C4 or CAM species, or from other extremely heat-tolerant species, has precluded an examination to determine if Cp-sHSP genes or proteins might differ among plants with photosynthetic pathways or between heat-sensitive and heat-tolerant species. To investigate this, we isolated and characterized novel Cp-sHSP genes in four plant species: two moderately heat-tolerant C4 species, Spartina alterniflora (monocot) and Amaranthus retroflexus (eudicot), and two very heat-tolerant CAM species, Agave americana (monocot) and Ferocactus wislizenii (eudicot) (respective genes: SasHSP27.12, ArsHSP26.43, AasHSP26.85 and FwsHSP27.52) by PCR-based genome walking and cDNA RACE. Analysis of these Cp-sHSPs has confirmed the presence of conserved domains common to previously examined species. As expected, the transit peptide was found to be the most variable part of these proteins. Promoter analysis of these genes revealed differences in CAM versus C3 and C4 species that were independent of a general difference between monocots and eudicots observed for the entire protein. Heat-induced gene and protein expression indicated that Cp-sHSP protein levels were correlated with thermotolerance of photosynthetic electron transport, and that in most cases protein and transcript levels were correlated. Thus, available evidence indicates little variation in the amino acid sequence of Cp-sHSP mature proteins between heat-sensitive and -tolerant species, but that variation in Cp-sHSP protein production is related to heat tolerance or photosynthetic pathway (CAM vs. C3 and C4) and is driven by promoter differences. Key message We isolated and characterized four novel Cp-sHSP genes with promoters from wild plants, analysis has shown qualitative

  12. Mechanisms of Protein Synthesis in Chloroplasts: How to Design Translatable mRNAs in Chloroplasts

    Institute of Scientific and Technical Information of China (English)

    M. Sugiura

    2007-01-01

    @@ Chloroplast transformation provides a powerful tool to produce useful proteins in plants. After completion of the chloroplast genome sequencing from tobacco plants (Shinozaki et al., 1986, Yukawa et al., 2005), Pal Maliga group developed the high-frequency chloroplast transformation system in tobacco (Svab and Maliga, 1993).

  13. Rapid plant identification using species- and group-specific primers targeting chloroplast DNA.

    Directory of Open Access Journals (Sweden)

    Corinna Wallinger

    Full Text Available Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae, the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory.

  14. Preparing DNA libraries for multiplexed paired-end deep sequencing for Illumina GA sequencers.

    Science.gov (United States)

    Son, Mike S; Taylor, Ronald K

    2011-02-01

    Whole-genome sequencing, also known as deep sequencing, is becoming a more affordable and efficient way to identify SNP mutations, deletions, and insertions in DNA sequences across several different strains. Two major obstacles preventing the widespread use of deep sequencers are the costs involved in services used to prepare DNA libraries for sequencing and the overall accuracy of the sequencing data. This unit describes the preparation of DNA libraries for multiplexed paired-end sequencing using the Illumina GA series sequencer. Self-preparation of DNA libraries can help reduce overall expenses, especially if optimization is required for the different samples, and use of the Illumina GA Sequencer can improve the quality of the data.

  15. New method to study DNA sequences: the languages of evolution.

    Science.gov (United States)

    Spinelli, Gino; Mayer-Foulkes, David

    2008-04-01

    Recently, several authors have reported statistical evidence for deterministic dynamics in the flux of genetic information, suggesting that evolution involves the emergence and maintenance of a fractal landscape in DNA chains. Here we examine the idea that motif repetition lies at the origin of these statistical properties of DNA. To analyse repetition patterns we apply a modification of the BDS statistic, devised to analyze complex economic dynamics and adapted here to DNA sequence analysis. This provides a new method to detect structured signals in genetic information. We compare naturally occurring DNA sequences along the evolutionary tree with randomly generated sequences and also with simulated sequences with repetition motifs. For easier understanding, we also define a new statistic for a DNA sequence that constitutes a specific fingerprint. The new methods are applied to exon and intron DNA sequences, finding specific statistical differences. Moreover, by analysing DNA sequences of different species from Bacteria to Man, we explore the evolution of these linguistic DNA features along the evolutionary tree. The results are consistent with the idea that all the flux of DNA information need not be random, but may be structured along the evolutionary tree. The implications for evolutionary theory are discussed.

  16. Statistical assignment of DNA sequences using Bayesian phylogenetics

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Huelsenbeck, John P.;

    2008-01-01

    -analysis of previously published ancient DNA data and show that, with high statistical confidence, most of the published sequences are in fact of Neanderthal origin. However, there are several cases of chimeric sequences that are comprised of a combination of both Neanderthal and modern human DNA....

  17. Levenshtein error-correcting barcodes for multiplexed DNA sequencing

    NARCIS (Netherlands)

    Buschmann, Tilo; Bystrykh, Leonid V.

    2013-01-01

    Background: High-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multi

  18. Primers for the Amplification of the Circular Chloroplast DNA from the A-genome Group of Cultivated Cotton

    Institute of Scientific and Technical Information of China (English)

    IBRAHIM Rashid Ismael Hag; AZUMA Jun-Ichi; SAKAMOTO Masahiro

    2008-01-01

    @@ The availability of the plastid genome sequences is one of the bases for comparative,functional,and structural genomic studies of plastid-containing living organisms,in addition to the application of plastid genetic engineering technology.The past efforts to sequence plastid genomes involve complicated preparation protocols.One procedure starts with the isolation of plastids,which was tiresome and time wasting that followed by a second step to extract plastid DNA from the isolated plastids,then finally the build up of plasmid or bacterial artificial chromosome (BAC) library.

  19. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  20. Cloning and sequencing of mouse GABA transporter complementary DNA

    Institute of Scientific and Technical Information of China (English)

    TAMANTHONYC.W.; LIHEGUO; 等

    1994-01-01

    A cDNA encoding the mouse GABA transporter has been isolated and sequenced.The results show that the mouse GABA transporter cDNA differs from that of the rat by 60 base pairs at the open reading frame region but the deduced amino acid sequences of the two cDNAs are identical and both composed of 599 amino acids.However,the amino acid sequence is different from the sequence deduced from a recently published mouse GABA transporter cDNA.

  1. Affordable Hands-On DNA Sequencing and Genotyping: An Exercise for Teaching DNA Analysis to Undergraduates

    Science.gov (United States)

    Shah, Kushani; Thomas, Shelby; Stein, Arnold

    2013-01-01

    In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C…

  2. 基于柑橘及其近缘属植物DNA条形码的叶绿体编码序列筛选%Screening Potential DNA Barcode Regions of Chloroplast Coding Genome for Citrus and Its Related Genera

    Institute of Scientific and Technical Information of China (English)

    于杰; 闫化学; 鲁振华; 周志钦

    2011-01-01

    [Objective] Four coding regions of chloroplast genome of Citrus and its close relatives were analyzed in an attempt to find suitable DNA barcoding markers for species identification and lay a foundation for further study of non-coding region.[ Method ] Four chloroplast DNA regions (matK, rpoB, rpoC1 and rbcL ) of 59 Citrus accessions were sequenced, the intergeneric,interspecific, intraspecific genetic distances were calculated, and the phylogenetic tree of all the accessions tested was built based on the distance data obtained. [Result] The intergeneric and interspecific sequence variations of matK were the highest among four coding regions tested, and had significant difference from other regions studied. On the contrary, no obvious variations were found in the rpoB and rpoC1 regions. The sequence variation of rbcL was medium among the fragments sequenced. [Conclusion] The matK sequence could be used as potential candidate fragment for future DNA barcoding study of Citrus and its closely related genera.%[目的]通过对柑橘及其近缘属植物叶绿体4种编码序列的测定分析,获得能进行DNA条形编码的特征序列,为进一步研究叶绿体非编码区序列奠定基础.[方法]对柑橘及其近缘属植物59份样品进行matK、rpoB、rpoC1、rbcL测序,序列比对与人工校正,计算属间,种同、种内的遗传距离,比较序列间的差异,建立系统发育树.[结果]4种序列中,matK序列在属间、种间差异最大,与其它序列相比具有显著性差异,rbcL序列次之,而rpoB、rpoC1序列两者间没有显著性差异.[结论]matK序列是柑橘及其近缘属植物DNA条形码的未来研究中一个重要的候选片段.

  3. DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

    Directory of Open Access Journals (Sweden)

    Cheng-Yao eChen

    2014-06-01

    Full Text Available Next-generation sequencing (NGS technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

  4. Effects of Sequence on Transmission Properties of DNA Molecules

    Institute of Scientific and Technical Information of China (English)

    DONG Rui-Xin; YAN Xun-Ling; YANG Bing

    2008-01-01

    A double helix model of charge transport in DNA molecule is given and the transmission spectra of four DNA sequences are obtained. The calculated results show that the transmission characteristics of DNA are not only related to the longitudinal transport but also to the transverse transport of molecule. The periodic sequence with the same composition has stronger conduction ability. With the increasing of bases composition, the conductive ability reduces, but the weight of θ direction rises in charge transfer.

  5. Phylogeography of Japanese horse chestnut (Aesculus turbinata) in the Japanese Archipelago based on chloroplast DNA haplotypes.

    Science.gov (United States)

    Sugahara, Kanako; Kaneko, Yuko; Ito, Satoshi; Yamanaka, Keisuke; Sakio, Hitoshi; Hoshizaki, Kazuhiko; Suzuki, Wajiro; Yamanaka, Norikazu; Setoguchi, Hiroaki

    2011-01-01

    Japanese horse chestnut (Aesculus turbinata: Hippocastanaceae) is one of the typical woody plants that grow in temperate riparian forests in the Japanese Archipelago. To analyze the phylogeography of this plant in the Japanese Archipelago, we determined cpDNA haplotypes for 337 samples from 55 populations covering the entire distribution range. Based on 1,313 bp of two spacers, we determined ten haplotypes that are distinguished from adjacent haplotypes by one or two steps. Most of the populations had a single haplotype, suggesting low diversity. Spatial analysis of molecular variance suggested three obvious phylogeographic structures in western Japan, where Japanese horse chestnut is scattered and isolated in mountainous areas. Conversely, no clear phylogeographic structure was observed from the northern to the southern limit of this species, including eastern Japan, where this plant is more common. Rare and private haplotypes were also found in southwestern Japan, where Japanese horse chestnuts are distributed sparsely. These findings imply that western Japan might have maintained a relatively large habitat for A. turbinata during the Quaternary climatic oscillations, while northerly regions could not.

  6. Code domains in tandem repetitive DNA sequence structures.

    Science.gov (United States)

    Vogt, P

    1992-10-01

    Traditionally, many people doing research in molecular biology attribute coding properties to a given DNA sequence if this sequence contains an open reading frame for translation into a sequence of amino acids. This protein coding capability of DNA was detected about 30 years ago. The underlying genetic code is highly conserved and present in every biological species studied so far. Today, it is obvious that DNA has a much larger coding potential for other important tasks. Apart from coding for specific RNA molecules such as rRNA, snRNA and tRNA molecules, specific structural and sequence patterns of the DNA chain itself express distinct codes for the regulation and expression of its genetic activity. A chromatin code has been defined for phasing of the histone-octamer protein complex in the nucleosome. A translation frame code has been shown to exist that determines correct triplet counting at the ribosome during protein synthesis. A loop code seems to organize the single stranded interaction of the nascent RNA chain with proteins during the splicing process, and a splicing code phases successive 5' and 3' splicing sites. Most of these DNA codes are not exclusively based on the primary DNA sequence itself, but also seem to include specific features of the corresponding higher order structures. Based on the view that these various DNA codes are genetically instructive for specific molecular interactions or processes, important in the nucleus during interphase and during cell division, the coding capability of tandem repetitive DNA sequences has recently been reconsidered.

  7. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors.

    Science.gov (United States)

    Chu, Wen-Yi; Huang, Yu-Feng; Huang, Chun-Chin; Cheng, Yi-Sheng; Huang, Chien-Kang; Oyang, Yen-Jen

    2009-07-01

    This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein-DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are essential for correct gene regulation. In this respect, ProteDNA is distinctive since it has been designed to identify sequence-specific binding residues. In order to accommodate users with different application needs, ProteDNA has been designed to operate under two modes, namely, the high-precision mode and the balanced mode. According to the experiments reported in this article, under the high-precision mode, ProteDNA has been able to deliver precision of 82.3%, specificity of 99.3%, sensitivity of 49.8% and accuracy of 96.5%. Meanwhile, under the balanced mode, ProteDNA has been able to deliver precision of 60.8%, specificity of 97.6%, sensitivity of 60.7% and accuracy of 95.4%. ProteDNA is available at the following websites: http://protedna.csbb.ntu.edu.tw/, http://protedna.csie.ntu.edu.tw/, http://bio222.esoe.ntu.edu.tw/ProteDNA/.

  8. DNA Shape Dominates Sequence Affinity in Nucleosome Formation

    Science.gov (United States)

    Freeman, Gordon S.; Lequieu, Joshua P.; Hinckley, Daniel M.; Whitmer, Jonathan K.; de Pablo, Juan J.

    2014-10-01

    Nucleosomes provide the basic unit of compaction in eukaryotic genomes, and the mechanisms that dictate their position at specific locations along a DNA sequence are of central importance to genetics. In this Letter, we employ molecular models of DNA and proteins to elucidate various aspects of nucleosome positioning. In particular, we show how DNA's histone affinity is encoded in its sequence-dependent shape, including subtle deviations from the ideal straight B-DNA form and local variations of minor groove width. By relying on high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome positioning.

  9. Next Generation Sequencing of Ancient DNA: Requirements, Strategies and Perspectives

    Directory of Open Access Journals (Sweden)

    Michael Knapp

    2010-07-01

    Full Text Available The invention of next-generation-sequencing has revolutionized almost all fields of genetics, but few have profited from it as much as the field of ancient DNA research. From its beginnings as an interesting but rather marginal discipline, ancient DNA research is now on its way into the centre of evolutionary biology. In less than a year from its invention next-generation-sequencing had increased the amount of DNA sequence data available from extinct organisms by several orders of magnitude. Ancient DNA  research is now not only adding a temporal aspect to evolutionary studies and allowing for the observation of evolution in real time, it also provides important data to help understand the origins of our own species. Here we review progress that has been made in next-generation-sequencing of ancient DNA over the past five years and evaluate sequencing strategies and future directions.

  10. No genome barriers to promiscuous DNA

    Science.gov (United States)

    Lewin, R.

    1984-06-01

    Farrelly and Butow (1983) used the term 'promiscuous DNA' in their report of the apparent natural transfer of yeast mitochondrial DNA sequences into the nuclear genome. Ellis (1982) applied the same term in an editorial comment. It is pointed out since that time the subject of DNA's promiscuity has exploded with a series of reports. According to a report by Stern (1984), movement of DNA sequences between chloroplasts and mitochondria is not just a rare event but is a rampant process. It was recently concluded that 'the widespread presence of ctDNA sequences in plant mtDNA is best regarded as a dramatic demonstration of the dynamo nature of interactions between the chloroplast and the mitochondrion, similar to the ongoing process of interorganellar DNA transfer already documented between mitochondrion and nucleus and between chloroplast and nucleus'.

  11. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group......Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within...

  12. DNA splice site sequences clustering method for conservativeness analysis

    Institute of Scientific and Technical Information of China (English)

    Quanwei Zhang; Qinke Peng; Tao Xu

    2009-01-01

    DNA sequences that are near to splice sites have remarkable conservativeness,and many researchers have contributed to the prediction of splice site.In order to mine the underlying biological knowledge,we analyze the conservativeness of DNA splice site adjacent sequences by clustering.Firstly,we propose a kind of DNA splice site sequences clustering method which is based on DBSCAN,and use four kinds of dissimilarity calculating methods.Then,we analyze the conservative feature of the clustering results and the experimental data set.

  13. PNA Directed Sequence Addressed Self-Assembly of DNA Nanostructures

    Science.gov (United States)

    Nielsen, Peter E.

    2008-10-01

    Peptide nucleic acids (PNA) can be designed to target duplex DNA with very high sequence specificity and efficiency via various binding modes. We have designed three domain PNA clamps, that bind stably to predefined decameric homopurine targets in large dsDNA molecules and via a third PNA domain sequence specifically recognize another PNA oligomer. We describe how such three domain PNAs have utility for assembling dsDNA grid and clover leaf structures, and in combination with SNAP-tag technology of protein dsDNA structures.

  14. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

  15. Extracting DNA from submerged pine wood.

    Science.gov (United States)

    Reynolds, M Megan; Williams, Claire G

    2004-10-01

    A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.

  16. Current-voltage characteristics of double-strand DNA sequences

    Science.gov (United States)

    Bezerril, L. M.; Moreira, D. A.; Albuquerque, E. L.; Fulco, U. L.; de Oliveira, E. L.; de Sousa, J. S.

    2009-09-01

    We use a tight-binding formulation to investigate the transmissivity and the current-voltage (I-V) characteristics of sequences of double-strand DNA molecules. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of artificial sequences (the long-range correlated Fibonacci and Rudin-Shapiro one) and a random sequence, which is a kind of prototype of a short-range correlated system. The random sequence is presented here with the same first neighbors pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the transmissivity spectra, although the I-V curves seem to be mostly influenced by the short-range correlations.

  17. Current-voltage characteristics of double-strand DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Bezerril, L.M.; Moreira, D.A. [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Albuquerque, E.L., E-mail: eudenilson@dfte.ufrn.b [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Fulco, U.L. [Departamento de Biofisica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Oliveira, E.L. de; Sousa, J.S. de [Departamento de Fisica, Universidade Federal do Ceara, 60455-760, Fortaleza-CE (Brazil)

    2009-09-07

    We use a tight-binding formulation to investigate the transmissivity and the current-voltage (I-V) characteristics of sequences of double-strand DNA molecules. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of artificial sequences (the long-range correlated Fibonacci and Rudin-Shapiro one) and a random sequence, which is a kind of prototype of a short-range correlated system. The random sequence is presented here with the same first neighbors pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the transmissivity spectra, although the I-V curves seem to be mostly influenced by the short-range correlations.

  18. Characteristics of alternating current hopping conductivity in DNA sequences

    Institute of Scientific and Technical Information of China (English)

    Ma Song-Shan; Xu Hui; Wang Huan-You; Guo Rui

    2009-01-01

    This paper presents a model to describe alternating current (AC) conductivity of DNA sequences,in which DNA is considered as a one-dimensional (1D) disordered system,and electrons transport via hopping between localized states.It finds that AC conductivity in DNA sequences increases as the frequency of the external electric field rises,and it takes the form of σac(ω)~ω2 ln2(1/ω).Also AC conductivity of DNA sequences increases with the increase of temperature,this phenomenon presents characteristics of weak temperature-dependence.Meanwhile,the AC conductivity in an off diagonally correlated case is much larger than that in the uncorrelated case of the Anderson limit in low temperatures,which indicates that the off-diagonal correlations in DNA sequences have a great effect on the AC conductivity,while at high temperature the off-diagonal correlations no longer play a vital role in electric transport. In addition,the proportion of nucleotide pairs p also plays an important role in AC electron transport of DNA sequences.For p<0.5,the conductivity of DNA sequence decreases with the increase of p,while for p > 0.5,the conductivity increases with the increase of p.

  19. Next-Gen phylogeography of rainforest trees: exploring landscape-level cpDNA variation from whole-genome sequencing.

    Science.gov (United States)

    van der Merwe, M; McPherson, H; Siow, J; Rossetto, M

    2014-01-01

    Standardized phylogeographic studies across codistributed taxa can identify important refugia and biogeographic barriers, and potentially uncover how changes in adaptive constraints through space and time impact on the distribution of genetic diversity. The combination of next-generation sequencing and methodologies that enable uncomplicated analysis of the full chloroplast genome may provide an invaluable resource for such studies. Here, we assess the potential of a shotgun-based method across twelve nonmodel rainforest trees sampled from two evolutionary distinct regions. Whole genomic shotgun sequencing libraries consisting of pooled individuals were used to assemble species-specific chloroplast references (in silicio). For each species, the pooled libraries allowed for the detection of variation within and between data sets (each representing a geographic region). The potential use of nuclear rDNA as an additional marker from the NGS libraries was investigated by mapping reads against available references. We successfully obtained phylogeographically informative sequence data from a range of previously unstudied rainforest trees. Greater levels of diversity were found in northern refugial rainforests than in southern expansion areas. The genetic signatures of varying evolutionary histories were detected, and interesting associative patterns between functional characteristics and genetic diversity were identified. This approach can suit a wide range of landscape-level studies. As the key laboratory-based steps do not require prior species-specific knowledge and can be easily outsourced, the techniques described here are even suitable for researchers without access to wet-laboratory facilities, making evolutionary ecology questions increasingly accessible to the research community.

  20. Nuclear and Chloroplast DNA Variation Provides Insights into Population Structure and Multiple Origin of Native Aromatic Rices of Odisha, India

    Science.gov (United States)

    Roy, Pritesh Sundar; Rao, Gundimeda Jwala Narasimha; Patnaik, Ashok; Patnaik, Sasank Sekhar Chyau; Jambhulkar, Nitiprasad Namdeorao; Sharma, Srigopal; Mohapatra, Trilochan

    2016-01-01

    A large number of short grain aromatic rice suited to the agro-climatic conditions and local preferences are grown in niche areas of different parts of India and their diversity is evolved over centuries as a result of selection by traditional farmers. Systematic characterization of these specialty rices has not been attempted. An effort was made to characterize 126 aromatic short grain rice landraces, collected from 19 different districts in the State of Odisha, from eastern India. High level of variation for grain quality and agronomic traits among these aromatic rices was observed and genotypes having desirable phenotypic traits like erect flag leaf, thick culm, compact and dense panicles, short plant stature, early duration, superior yield and grain quality traits were identified. A total of 24 SSR markers corresponding to the hyper variable regions of rice chromosomes were used to understand the genetic diversity and to establish the genetic relationship among the aromatic short grain rice landraces at nuclear genome level. SSR analysis of 126 genotypes from Odisha and 10 genotypes from other states revealed 110 alleles with an average of 4.583 and the Nei’s genetic diversity value (He) was in the range of 0.034–0.880 revealing two sub-populations SP 1 (membership percentage-27.1%) and SP 2 (72.9%). At the organelle genomic level for the C/A repeats in PS1D sequence of chloroplasts, eight different plastid sub types and 33 haplotypes were detected. The japonica (Nipponbare) subtype (6C7A) was detected in 100 genotypes followed by O. rufipogon (KF428978) subtype (6C6A) in 13 genotypes while indica (93–11) sub type (8C8A) was seen in 14 genotypes. The tree constructed based on haplotypes suggests that short grain aromatic landraces might have independent origin of these plastid subtypes. Notably a wide range of diversity was observed among these landraces cultivated in different parts confined to the State of Odisha. PMID:27598392

  1. Spectroscopic investigation on the telomeric DNA base sequence repeat

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Telomeres are protein-DNA complexes at the terminals of linear chromosomes, which protect chromosomal integrity and maintain cellular replicative capacity.From single-cell organisms to advanced animals and plants,structures and functions of telomeres are both very conservative. In cells of human and vertebral animals, telomeric DNA base sequences all are (TTAGGG)n. In the present work, we have obtained absorption and fluorescence spectra measured from seven synthesized oligonucleotides to simulate the telomeric DNA system and calculated their relative fluorescence quantum yields on which not only telomeric DNA characteristics are predicted but also possibly the shortened telomeric sequences during cell division are imrelative fluorescence quantum yield and remarkable excitation energy innerconversion, which tallies with the telomeric sequence of (TTAGGG)n. This result shows that telomeric DNA has a strong non-radiative or innerconvertible capability.``

  2. ATRF Houses the Latest DNA Sequencing Technologies | Poster

    Science.gov (United States)

    By Ashley DeVine, Staff Writer By the end of October, the Advanced Technology Research Facility (ATRF) will be one of the few facilities in the world to house all of the latest DNA sequencing technologies.

  3. Which Are More Random: Coding or Noncoding DNA Sequences?

    Institute of Scientific and Technical Information of China (English)

    WU Fang; ZHENG Wei-Mou

    2002-01-01

    Evidence seems to show that coding DNA is more random than noncoding DNA, but other conflictingevidence also exists. Based on the third-base degeneracy of codons, we regard the third position of codons as a 'noisy'position. By deleting one fixed position of non-overlapping triplets in a given sequence, three masked sequences may bededuced from the sequence. We have investigated the block-to-site mutual information functions of coding and noncodingsequences in yeast without and with the masking. Characteristics that distinguish coding from noncoding DNA havebeen found. It is observed that the strong correlations in the coding regions may be blocked by the third base of codons,and the proper masking can extract the correlations. Distribution of dimeric tandem repeats of unmasked sequences isalso compared with that of masked sequences.

  4. Repetitive DNA Sequences in Wheat and Its Relatives

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xue-yong; LI Da-yong

    2001-01-01

    Repetitive DNA sequences form a large portion of eukaryote genomes. Using wheat ( Triticum )as a model, the classification, features and functions of repetitive DNA sequences in the Tritieeae grass tribe is reviewed as well as the role of these sequences in genome differentiation, control and regulation of homologous chromosome synapsis and pairing. Transposable elements, as an important portion of dispersed repetitives,may play an essential role in gene mutation of the host. Dynamic models for change of copy number and sequences of the repetitive family are also presented after the models of Charlesworth et al. Application of repetitive DNA sequences in the study of evolution, chromosome fingerprinting and marker assisted gene transfer and breeding are described by taking wheat as an example.

  5. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-06-01

    Activities of DNA are to a great extent controlled epigenetically through the internal struc- ture of chromatin. This structure is dynamic and is influenced by different modifications of histone proteins. Various combinations of epigenetic modification of histones pinpoint to different functional regions of the DNA determining the so-called chromatin states. How- ever, the characterization of chromatin states by the DNA sequence properties remains largely unknown. In this study we aim to explore whether DNA sequence patterns in the human genome can characterize different chromatin states. Using DNA sequence motifs we built binary classifiers for each chromatic state to eval- uate whether a given genomic sequence is a good candidate for belonging to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results suggest that in general these models lack sufficient predictive power, although for four chromatin states (insulators, het- erochromatin, and two types of copy number variation) we found that presence of certain motifs in DNA sequences does imply an increased probability that such a sequence is one of these chromatin states.

  6. PNA Directed Sequence Addressed Self-Assembly of DNA Nanostructures

    DEFF Research Database (Denmark)

    Nielsen, Peter E.

    2008-01-01

    sequence specifically recognize another PNA oligomer. We describe how such three domain PNAs have utility for assembling dsDNA grid and clover leaf structures, and in combination with SNAP-tag technol. of protein dsDNA structures. (c) 2008 American Institute of Physics. [on SciFinder (R)] Udgivelsesdato...

  7. CHUP1 mediates actin-based light-induced chloroplast avoidance movement in the moss Physcomitrella patens.

    Science.gov (United States)

    Usami, Hiroka; Maeda, Takuma; Fujii, Yusuke; Oikawa, Kazusato; Takahashi, Fumio; Kagawa, Takatoshi; Wada, Masamitsu; Kasahara, Masahiro

    2012-12-01

    Chloroplasts change their intracellular distribution in response to light intensity. CHUP1 (CHLOROPLAST UNUSUAL POSITIONING1) is indispensable for this response in Arabidopsis thaliana. However, involvement of CHUP1 in light-induced chloroplast movement is unknown in other plants. In this study, CHUP1 orthologues were isolated from a moss, Physcomitrella patens, and a fern, Adiantum capillus-veneris, by cDNA library screening and PCR cloning based on the P. patens genome sequence. Functional motifs found in CHUP1 of A. thaliana were conserved among the CHUP1 orthologues. In addition to the putative functional regions, the C-terminal regions (approximately 250 amino acids), which are unique in CHUP1s, were highly conserved. Green fluorescent protein (GFP) fusions of P. patens CHUP1s (PpCHUP1A, PpCHUP1B and PpCHUP1C) were transiently expressed in protoplast cells. All GFP fusions were localized on the chloroplasts. Light-induced chloroplast avoidance movement of chup1 disruptants of P. patens was examined in the presence of cytoskeletal inhibitors because of the utilization of both microtubules and actin filaments for the movement in P. patens. When actin filaments were disrupted by cytochalasin B, the wild type (WT) and all chup1 disruptants showed chloroplast avoidance movement. However, when microtubules were disrupted by Oryzalin, chloroplasts in ∆chup1A and ∆chup1A/B rarely moved and stayed in the strong light-irradiated area. On the other hand, WT, ∆chup1B and ∆chup1C showed chloroplast avoidance movement. These results suggest that PpCHUP1A predominantly mediates the actin-based light-induced chloroplast avoidance movement. This study reveals that CHUP1 functions on the chloroplasts and is involved in the actin-based light-induced chloroplast avoidance movement in P. patens.

  8. Biometric Authentication Using ElGamal Cryptosystem And DNA Sequence

    Directory of Open Access Journals (Sweden)

    V.SAMUEL SUSAN

    2010-06-01

    Full Text Available Biometrics are automated methods of identifying a person or verifying the identity of a person based on a Physiological or behavioral characteristic. Physiological haracteristics include hand or finger images, facial characteristics and iris recognition. Behavioral characteristics include dynamic signature verification, speaker verification and keystroke dynamics. DNA is unique feature among individuals. DNA provides high security level, long term stability, user acceptance and is intrusive. Combining ElGamal cryptosystem and DNA sequence, a novel biometric authentication scheme is proposed.

  9. Protein sequence for clustering DNA based on Artificial Neural Networks

    Directory of Open Access Journals (Sweden)

    Gamal. F. Elhadi

    2012-01-01

    Full Text Available DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. Clustering is a process that groups a set of objects into clusters so that the similarity among objects in the same cluster is high, while that among the objects in different clusters is low. In this paper, we proposed an approach for clustering DNA sequences using Self-Organizing Map (SOM algorithm and Protein Sequence. The main objective is to analyze biological data and to bunch DNA to many clusters more easily and efficiently. We use the proposed approach to analyze both large and small amount of input DNA sequences. The results show that the similarity of the sequences does not depend on the amount of input sequences. Our approach depends on evaluating the degree of the DNA sequences similarity using the hierarchal representation Dendrogram. Representing large amount of data using hierarchal tree gives the ability to compare large sequences efficiently

  10. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

    Science.gov (United States)

    Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

    2012-01-01

    DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

  11. Sequencing and Analysis of Neanderthal Genomic DNA

    OpenAIRE

    Noonan, James P.; Coop, Graham; Kudaravalli, Sridhar; Smith, Doug; Krause, Johannes; Alessi, Joe; Chen, Feng; Platt, Darren; Paabo, Svante; Pritchard, Jonathan K; Rubin, Edward M.

    2006-01-01

    Our knowledge of Neanderthals is based on a limited number of remains and artifacts from which we must make inferences about their biology, behavior, and relationship to ourselves. Here, we describe the characterization of these extinct hominids from a new perspective, based on the development of a Neanderthal metagenomic library and its high-throughput sequencing and analysis. Several lines of evidence indicate that the 65,250 base pairs of hominid sequence so far identified in the library a...

  12. Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

    DEFF Research Database (Denmark)

    Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie;

    2014-01-01

    sections for electron induced single strand breaks in specific 13 mer oligonucleotides we used atomic force microscopy analysis of DNA origami based DNA nanoarrays. We investigated the DNA sequences 5'-TT(XYX)3TT with X = A, G, C and Y = T, BrU 5-bromouracil and found absolute strand break cross sections...

  13. Zinc finger recombinases with adaptable DNA sequence specificity.

    Directory of Open Access Journals (Sweden)

    Chris Proudfoot

    Full Text Available Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural 'pseudosites' are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine β-casein gene, mediated by zinc finger recombinases (ZFRs, chimaeric enzymes with linked zinc finger (DNA recognition and recombinase (catalytic domains. In the "Z-sites" tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences.

  14. cDNA cloning and sequencing of ostrich Growth hormone

    Directory of Open Access Journals (Sweden)

    Doosti Abbas

    2012-01-01

    Full Text Available In recent years, industrial breeding of ostrich (Struthio camelus has been widely developed in Iran. Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in different animals. The aim of this study was to clone and sequence the ostrich growth hormone gene in E. coli, done for the first time in Iran. The cDNA that encodes ostrich growth hormone was isolated from total mRNA of the pituitary gland and amplified by RT-PCR using GH specific PCR primers. Then GH cDNA was cloned by T/A cloning technique and the construct was transformed into E. coli. Finally, GH cDNA sequence was submitted to the GenBank (Accession number: JN559394. The results of present study showed that GH cDNA was successfully cloned in E. coli. Sequencing confirmed that GH cDNA was cloned and that the length of ostrich GH cDNA was 672 bp; BLAST search showed that the sequence of growth hormone cDNA of the ostrich from Iran has 100% homology with other records existing in GenBank.

  15. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    Science.gov (United States)

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

  16. Bayesian classification for promoter prediction in human DNA sequences

    Science.gov (United States)

    Bercher, J.-F.; Jardin, P.; Duriez, B.

    2006-11-01

    Many Computational methods are yet available for data retrieval and analysis of genomic sequences, but some functional sites are difficult to characterize. In this work, we examine the problem of promoter localization in human DNA sequences. Promoters are regulatory regions that governs the expression of genes, and their prediction is reputed difficult, so that this issue is still open. We present the Chaos Game representation (CGR) of DNA sequences which has many interesting properties, and the notion of `genomic signature' that proved relevant in phylogeny applications. Based on this notion, we develop a (naïve) bayesian classifier, evaluate its performances, and show that its adaptive implementation enable to reveal or assess core-promoter positions along a DNA sequence.

  17. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  18. Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis

    Directory of Open Access Journals (Sweden)

    Zhou Xiangjun

    2011-11-01

    Full Text Available Abstract Background Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Results Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5 was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. Conclusions The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant.

  19. Recognizing a Single Base in an Individual DNA Strand: A Step Toward Nanopore DNA Sequencing**

    Science.gov (United States)

    Ashkenasy, N.; Sánchez-Quesada, J.; Ghadiri, M. R.; Bayley, H.

    2007-01-01

    Functional supramolecular chemistry at the single-molecule level. Single strands of DNA can be captured inside α-hemolysin transmembrane pore protein to form single-species α-HL·DNA pseudorotaxanes. This process can be used to identify a single adenine nucleotide at a specific location on a strand of DNA by the characteristic reductions in the α-HL ion conductance. This study suggests that α-HL-mediated single-molecule DNA sequencing might be fundamentally feasible. PMID:15666419

  20. Comparative analysis of microsatellites in chloroplast genomes of lower and higher plants.

    Science.gov (United States)

    George, Biju; Bhatt, Bhavin S; Awasthi, Mayur; George, Binu; Singh, Achuit K

    2015-11-01

    Microsatellites, or simple sequence repeats (SSRs), contain repetitive DNA sequence where tandem repeats of one to six base pairs are present number of times. Chloroplast genome sequences have been  shown to possess extensive variations in the length, number and distribution of SSRs. However, a comparative analysis of chloroplast microsatellites is not available. Considering their potential importance in generating genomic diversity, we have systematically analysed the abundance and distribution of simple and compound microsatellites in 164 sequenced chloroplast genomes from wide range of plants. The key findings of these studies are (1) a large number of mononucleotide repeats as compared to SSR(2-6)(di-, tri-, tetra-, penta-, hexanucleotide repeats) are present in all chloroplast genomes investigated, (2) lower plants such as algae show wide variation in relative abundance, density and distribution of microsatellite repeats as compared to flowering plants, (3) longer SSRs are excluded from coding regions of most chloroplast genomes, (4) GC content has a weak influence on number, relative abundance and relative density of mononucleotide as well as SSR(2-6). However, GC content strongly showed negative correlation with relative density (R (2) = 0.5, P plants possesses relatively more genomic diversity compared to higher plants.

  1. Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence.

    Science.gov (United States)

    Ngarmamonpirat, Charinthon; Waikagul, Jitra; Petmitr, Songsak; Dekumyoy, Paron; Rojekittikhun, Wichit; Anantapruti, Malinee T

    2005-03-01

    Morphological variations were observed in the advance third stage larvae of Gnathostoma spinigerum collected from swamp eel (Fluta alba), the second intermediate host. Larvae with typical and three atypical types were chosen for partial cytochrome c oxidase subunit I (COI) gene sequence analysis. A 450 bp polymerase chain reaction product of the COI gene was amplified from mitochondrial DNA. The variations were analyzed by single-strand conformation polymorphism and DNA sequencing. The nucleotide variations of the COI gene in the four types of larvae indicated the presence of an intra-specific variation of mitochondrial DNA in the G. spinigerum population.

  2. LONG-RANGE CORRELATIONS IN DNA SEQUENCES USING TWO-DIMENSIONAL DNA WALKS

    Institute of Scientific and Technical Information of China (English)

    Jin Chen; Lin-xi Zhang; De-lu Zhao

    2005-01-01

    The characterization of long-range correlations and fractal properties of DNA sequences has proved to be a difficult though rewarding task mainly due to the mosaic character of DNA consisting of many patches of various lengths with different nucleotide constitutions. In this paper we investigate statistical correlations among different positions in DNA sequences using the two-dimensional DNA walk. The root-mean-square fluctuation F(l) is described by a power law. The autocorrelation function C(l), which is used to measure the linear dependence and periodicity, exists a power law of C(l) -τμ. We also calculate the mean-square distance <R2(l)> along the DNA chain, and it may be expressed as <R2(l)> - l r with 2 >γ> 1. Our investigations can provide some insights into long-range correlations in DNA sequences.

  3. [Nuclear-cytoplasmic compatibility and the state of mitochondrial and chloroplast DNA regions in alloplasmic recombinant and introgressive lines (H. vulgare)-T. aestivum].

    Science.gov (United States)

    Pershina, L A; Trubacheva, N V; Sinyavskaya, M G; Devyatkina, E P; Kravtsova, L A

    2014-10-01

    Alloplasmic lines combining alien nuclear and cytoplasmic genomes are convenient models for studying the mechanisms of nuclear-cytoplasmic compatibility/incompatibility. In the.present study, we have investigated the correlation between the characters and state of mitochondrial (mt) and chloroplast (cp) DNA regions in alloplasmic recombinant common wheat lines with barley cytoplasm characterized by partial or total fertility. Fertility restoration in the studied lines (Hordeum vulgare)-Triticum aestivum is determined by different ratios of the genetic material of common wheat variety Pyrotrix 28, which is a fertility restorer in the cytoplasm of barley, and varietySaratovskaya 29, which is a fixer of sterility. In partially fertile lines with nuclear genomes dominated by the genetic material of Saratovskaya 29, plant growth and development are suppressed. In these lines we have identified the barley homoplasmy of cpDNA regions infA and rpoB and the heteroplasmy of the 18S/5S mt repeat and the cpDNA ycf5 region. Nuclear-cytoplasmic compatibility in lines with reduced fertility (the genetic material of Pyrotrix 28 predominates in their nuclear genomes) is associated with restoration of normal plant growth and development and the changes in thestate of the studied cpDNA and mtDNA regions towards the wheat type. Thus, in fertile lines, the cpDNA regions (infA, rpoB) and the 18S/5S mt repeat were identified in the homoplasmic wheat state; though the cpDNAycf5 region was in the heteroplasmic state, it was dominated by the wheat type of the copies. The nuclearicytoplasmic compatibility is not broken as a result of introgression of the alien genetic material into the nuclear genome of one of the fertile lines; the plants of introgressive lines are fertile and normally developed, and the states of the cpDNA and mtDNA regions correspond to their states in fertile recombinant lines.

  4. Probabilistic models for semisupervised discriminative motif discovery in DNA sequences.

    Science.gov (United States)

    Kim, Jong Kyoung; Choi, Seungjin

    2011-01-01

    Methods for discriminative motif discovery in DNA sequences identify transcription factor binding sites (TFBSs), searching only for patterns that differentiate two sets (positive and negative sets) of sequences. On one hand, discriminative methods increase the sensitivity and specificity of motif discovery, compared to generative models. On the other hand, generative models can easily exploit unlabeled sequences to better detect functional motifs when labeled training samples are limited. In this paper, we develop a hybrid generative/discriminative model which enables us to make use of unlabeled sequences in the framework of discriminative motif discovery, leading to semisupervised discriminative motif discovery. Numerical experiments on yeast ChIP-chip data for discovering DNA motifs demonstrate that the best performance is obtained between the purely-generative and the purely-discriminative and the semisupervised learning improves the performance when labeled sequences are limited.

  5. Chaos game representation (CGR)-walk model for DNA sequences

    Institute of Scientific and Technical Information of China (English)

    Gao Jie; Xu Zhen-Yuan

    2009-01-01

    Chaos game representation (CGR) is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to determine the coordinates of their positions in a continuous space. This distribution of positions has two features: one is unique, and the other is source sequence that can be recovered from the coordinates so that the distance between positions may serve as a measure of similarity between the corresponding sequences. A CGR-walk model is proposed based on CGR coordinates for the DNA sequences. The CGR coordinates are converted into a time series, and a long-memory ARFIMA (p, d, q) model, where ARFIMA stands for autoregressive fractionally integrated moving average, is introduced into the DNA sequence analysis. This model is applied to simulating real CGR-walk sequence data of ten genomic sequences. Remarkably long-range correlations are uncovered in the data, and the results from these models are reasonably fitted with those from the ARFIMA (p, d, q) model.

  6. Applications of recursive segmentation to the analysis of DNA sequences.

    Science.gov (United States)

    Li, Wentian; Bernaola-Galván, Pedro; Haghighi, Fatameh; Grosse, Ivo

    2002-07-01

    Recursive segmentation is a procedure that partitions a DNA sequence into domains with a homogeneous composition of the four nucleotides A, C, G and T. This procedure can also be applied to any sequence converted from a DNA sequence, such as to a binary strong(G + C)/weak(A + T) sequence, to a binary sequence indicating the presence or absence of the dinucleotide CpG, or to a sequence indicating both the base and the codon position information. We apply various conversion schemes in order to address the following five DNA sequence analysis problems: isochore mapping, CpG island detection, locating the origin and terminus of replication in bacterial genomes, finding complex repeats in telomere sequences, and delineating coding and noncoding regions. We find that the recursive segmentation procedure can successfully detect isochore borders, CpG islands, and the origin and terminus of replication, but it needs improvement for detecting complex repeats as well as borders between coding and noncoding regions.

  7. How effective is graphene nanopore geometry on DNA sequencing?

    CERN Document Server

    Satarifard, Vahid; Ejtehadi, Mohammad Reza

    2015-01-01

    In this paper we investigate the effects of graphene nanopore geometry on homopolymer ssDNA pulling process through nanopore using steered molecular dynamic (SMD) simulations. Different graphene nanopores are examined including axially symmetric and asymmetric monolayer graphene nanopores as well as five layer graphene polyhedral crystals (GPC). The pulling force profile, moving fashion of ssDNA, work done in irreversible DNA pulling and orientations of DNA bases near the nanopore are assessed. Simulation results demonstrate the strong effect of the pore shape as well as geometrical symmetry on free energy barrier, orientations and dynamic of DNA translocation through graphene nanopore. Our study proposes that the symmetric circular geometry of monolayer graphene nanopore with high pulling velocity can be used for DNA sequencing.

  8. Qualitatively predicting acetylation and methylation areas in DNA sequences.

    Science.gov (United States)

    Pham, Tho Hoan; Tran, Dang Hung; Ho, Tu Bao; Satou, Kenji; Valiente, Gabriel

    2005-01-01

    Eukaryotic genomes are packaged by the wrapping of DNA around histone octamers to form nucleosomes. Nucleosome occupancy, acetylation, and methylation, which have a major impact on all nuclear processes involving DNA, have been recently mapped across the yeast genome using chromatin immunoprecipitation and DNA microarrays. However, this experimental protocol is laborious and expensive. Moreover, experimental methods often produce noisy results. In this paper, we introduce a computational approach to the qualitative prediction of nucleosome occupancy, acetylation, and methylation areas in DNA sequences. Our method uses support vector machines to discriminate between DNA areas with high and low relative occupancy, acetylation, or methylation, and rank k-gram features based on their support for these DNA modifications. Experimental results on the yeast genome reveal genetic area preferences of nucleosome occupancy, acetylation, and methylation that are consistent with previous studies. Supplementary files are available from http://www.jaist.ac.jp/~tran/nucleosome/.

  9. Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

    Science.gov (United States)

    Jaffé, Benjamin; Kovács, Katalin; Andras, Calin; Bódi, Zsuzsanna; Liu, Zheng

    2008-01-01

    We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background. PMID:18536921

  10. PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities

    Directory of Open Access Journals (Sweden)

    Baldwin Stephen A

    2011-03-01

    Full Text Available Abstract Background Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. Results The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. Conclusions PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/.

  11. PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities

    Science.gov (United States)

    2011-01-01

    Background Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS) that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. Results The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. Conclusions PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/. PMID:21385349

  12. Label-free DNA sequencing using Millikan detection.

    Science.gov (United States)

    Dettloff, Roger; Leiske, Danielle; Chow, Andrea; Farinas, Javier

    2015-10-15

    A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.

  13. Nonlinear Aspects of Coding and Noncoding DNA Sequences

    Science.gov (United States)

    Stanley, H. Eugene

    2001-03-01

    One of the most remarkable features of human DNA is that 97 percent is not coding for proteins. Studying this noncoding DNA is important both for practical reasons (to distinguish it from the coding DNA as the human genome is sequenced), and for scientific reasons (why is the noncoding DNA present at all, if it appears to have little if any purpose?). In this talk we discuss new methods of analyzing coding and noncoding DNA in parallel, with a view to uncovering different statistical properties of the two kinds of DNA. We also speculate on possible roles of noncoding DNA. The work reported here was carried out primarily by P. Bernaola-Galvan, S. V. Buldyrev, P. Carpena, N. Dokholyan, A. L. Goldberger, I. Grosse, S. Havlin, H. Herzel, J. L. Oliver, C.-K. Peng, M. Simons, H. E. Stanley, R. H. R. Stanley, and G. M. Viswanathan. [1] For a brief overview in language that physicists can understand, see H. E. Stanley, S. V. Buldyrev, A. L. Goldberger, S. Havlin, C.-K. Peng, and M. Simons, "Scaling Features of Noncoding DNA" [Proc. XII Max Born Symposium, Wroclaw], Physica A 273, 1-18 (1999). [2] I. Grosse, H. Herzel, S. V. Buldyrev, and H. E. Stanley, "Species Independence of Mutual Information in Coding and Noncoding DNA," Phys. Rev. E 61, 5624-5629 (2000). [3] P. Bernaola-Galvan, I. Grosse, P. Carpena, J. L. Oliver, and H. E. Stanley, "Identification of DNA Coding Regions Using an Entropic Segmentation Method," Phys. Rev. Lett. 84, 1342-1345 (2000). [4] N. Dokholyan, S. V. Buldyrev, S. Havlin, and H. E. Stanley, "Distributions of Dimeric Tandem Repeats in Non-coding and Coding DNA Sequences," J. Theor. Biol. 202, 273-282 (2000). [5] R. H. R. Stanley, N. V. Dokholyan, S. V. Buldyrev, S. Havlin, and H. E. Stanley, "Clumping of Identical Oligonucleotides in Coding and Noncoding DNA Sequences," J. Biomol. Structure and Design 17, 79-87 (1999). [6] N. Dokholyan, S. V. Buldyrev, S. Havlin, and H. E. Stanley, "Distribution of Base Pair Repeats in Coding and Noncoding DNA

  14. Inverted repeat of Olisthodiscus luteus chloroplast DNA contains genes for both subunits of ribulose-1,5-bisphosphate carboxylase and the 32,000-dalton QB protein: Phylogenetic implications

    Science.gov (United States)

    Reith, Michael; Cattolico, Rose Ann

    1986-01-01

    The chloroplast DNA of the chromophytic alga Olisthodiscus luteus has been physically mapped with four restriction enzymes. An inverted repeat of 22 kilobase pairs is present in this 150-kilobase-pair plastid genome. The inverted repeat contains the genes for the large and small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and also codes for the 32,000-dalton QB protein. These observations demonstrate that significant differences exist in chloroplast genome structure and organization among major plant taxa. Images PMID:16578794

  15. Authentication of Curcuma species (Zingiberaceae) based on nuclear 18S rDNA and plastid trnK sequences.

    Science.gov (United States)

    Cao, Hui; Sasaki, Yohei; Fushimi, Hirotoshi; Komatsu, Katsuko

    2010-07-01

    Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. Comparative sequencing of the 18S rRNA gene in nuclear ribosomal DNA (rDNA) and trnK gene in chloroplast DNA (cpDNA) was carried out in order to examine interspecies phylogeny and to identify ultimately Curcuma species. A total of a hundred of accessions of eighteen species were analyzed. This resulted in an aligned matrix of 1810 bp for 18S rDNA and 2 800 bp for trnK. 18S rDNA sequence divergence within the ingroup ranged from 0-0.05%, trnK ranged from 0-0.19%. One base transversion-substituted site (from cytosine to thymine) was observed from the upstream of 18S rDNA at nucleotide position 234 in C. kwangsiensis and Japanese population of C. zedoaria which have separated genetic distance to other Curcuma taxa. Two noncoding regions embedded in trnK intron showed higher variability, including nucleotide substitutions, repeat insertion and deletions. Based on consensus of relationship, eighteen major lineages within Curcuma are recognized at the species level. The results suggest that Curcuma is monophyletic with 100% bootstrap support and sister to the genera Hedychium and Zingiber. The trnK sequences showed considerable variations between Curcuma species and thus were revealed as a promising candidate for barcoding of Curcuma species, which provide valuable characters for inferring relationship within species but are insufficient to resolve relationships among closely related taxa.

  16. Adaptive Evolutionary Analysis of Chloroplast Genes in Euphyllophytes Based on Complete Chloroplast Genome Sequences%基于叶绿体基因组全序列分析真叶植物叶绿体基因的适应性进化

    Institute of Scientific and Technical Information of China (English)

    王博; 高磊; 苏应娟; 王艇

    2012-01-01

    Euphyllophytes comprise fems, gymnosperms, and angiosperms. Relatively abundant chloro-plast genome sequence data has been available for them. In this research, chloroplast gene sequences of 29 euphyllophyte species were extracted from their completely sequenced chloroplast genomes; then an a-daptive evolutionary analysis was performed on the chloroplast genes by running PAML under models allowing w (nonsynonymous/synonymous rate ratio) to vary among sites. The results showed that: ①The percentage of chloroplast genes under positive selection in ferns, gymnosperms, and angiosperms were 6. 5%, 7.5% and 19. 2% , respectively. The number of positively selected genes in angiosperms appeared significantly larger than that of ferns and gymnosperms. ②Most positively selected genes were genetic system or photosynthesis-related genes. Their coding proteins often functioned in chloroplast protein synthesis, gene transcription, energy transformation and regulation, and photosynthesis. We infer that the chloroplast functional genes may have played key roles during the adaptation of euphyllophytes to terrestrial ecosystems.%真叶植物包括蕨类、裸子植物和被子植物.迄今已积累有较为丰富的真叶植物叶绿体基因组全序列数据.选取了29种真叶植物的叶绿体基因组全序列,采用PAML软件基于位点间可变ω模型,分别分析了蕨类、裸子植物和被子植物叶绿体基因的适应性进化.结果显示:①蕨类、裸子植物和被子植物各有6.5%、7.5%和19.2%的叶绿体基因受正选择作用;被子植物经历正选择的叶绿体基因明显比蕨类和裸子植物为多;②被正选择作用的叶绿体基因主要是遗传系统和光合系统基因,它们的编码产物涉及叶绿体蛋白质合成、基因转录、能量转化与调节及光合作用等过程.推测叶绿体功能基因可能在真叶植物对陆生生态环境的适应过程中起着重要作用.

  17. Dialects of the DNA uptake sequence in Neisseriaceae.

    Directory of Open Access Journals (Sweden)

    Stephan A Frye

    2013-04-01

    Full Text Available In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS, which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS-dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5'-CTG-3' is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS-dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic

  18. Mitochondrial DNA sequence of Onychostoma rara.

    Science.gov (United States)

    Zeng, Chun-Fang; Li, Xiao-Ling; Li, Chuan-Wu; Huang, Xiang-Rong; Wan, Yi-Wen

    2015-01-01

    The complete mitochondrial genome sequence of Onychostoma rara was determined to be 16,590 bp in length and contains 13 protein-coding genes (PCGs), 22 tRNA genes, large (rrnL) and small (rrnS) rRNA and the non-coding control region. Its total A + T content is 55.65%. We also analyzed the structure of control region, 6 CSBs (CSB-1, CSB-2, CSB-3, CSB-D, CSB-E and CSB-F) and 2 bp tandem repeat were detected.

  19. Noninvasive prenatal paternity testing (NIPAT) through maternal plasma DNA sequencing

    DEFF Research Database (Denmark)

    Jiang, Haojun; Xie, Yifan; Li, Xuchao

    2016-01-01

    Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we...... developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels...... paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future....

  20. Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.

    Science.gov (United States)

    Brumley, R L; Smith, L M

    1991-01-01

    A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads. Images PMID:1870968

  1. Facilitated diffusion on mobile DNA: configurational traps and sequence heterogeneity

    CERN Document Server

    Brackley, C A; Marenduzzo, D; 10.1103/PhysRevLett.109.168103

    2012-01-01

    We present Brownian dynamics simulations of the facilitated diffusion of a protein, modelled as a sphere with a binding site on its surface, along DNA, modelled as a semi-flexible polymer. We consider both the effect of DNA organisation in 3D, and of sequence heterogeneity. We find that in a network of DNA loops, as are thought to be present in bacterial DNA, the search process is very sensitive to the spatial location of the target within such loops. Therefore, specific genes might be repressed or promoted by changing the local topology of the genome. On the other hand, sequence heterogeneity creates traps which normally slow down facilitated diffusion. When suitably positioned, though, these traps can, surprisingly, render the search process much more efficient.

  2. Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...od - Number of data entries 7 entries - Joomla SEF URLs by Artio About This Database Database Description Download License Update His...tory of This Database Site Policy | Contact Us Vector sequences - Budding yeast cDNA sequencing project | LSDB Archive ...

  3. Label-Free DNA Sequencing Using Millikan Detection

    OpenAIRE

    Dettloff, Roger; Leiske, Danielle; Chow, Andrea; Farinas, Javier

    2015-01-01

    A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucl...

  4. Anaplasma phagocytophilum in Danish sheep: confirmation by DNA sequencing

    Directory of Open Access Journals (Sweden)

    Thamsborg Stig M

    2009-12-01

    Full Text Available Abstract Background The presence of Anaplasma phagocytophilum, an Ixodes ricinus transmitted bacterium, was investigated in two flocks of Danish grazing lambs. Direct PCR detection was performed on DNA extracted from blood and serum with subsequent confirmation by DNA sequencing. Methods 31 samples obtained from clinically normal lambs in 2000 from Fussingø, Jutland and 12 samples from ten lambs and two ewes from a clinical outbreak at Feddet, Zealand in 2006 were included in the study. Some of the animals from Feddet had shown clinical signs of polyarthritis and general unthriftiness prior to sampling. DNA extraction was optimized from blood and serum and detection achieved by a 16S rRNA targeted PCR with verification of the product by DNA sequencing. Results Five DNA extracts were found positive by PCR, including two samples from 2000 and three from 2006. For both series of samples the product was verified as A. phagocytophilum by DNA sequencing. Conclusions A. phagocytophilum was detected by molecular methods for the first time in Danish grazing lambs during the two seasons investigated (2000 and 2006.

  5. Preparation of next-generation sequencing libraries from damaged DNA.

    Science.gov (United States)

    Briggs, Adrian W; Heyn, Patricia

    2012-01-01

    Next-generation sequencing (NGS) has revolutionized ancient DNA research, especially when combined with high-throughput target enrichment methods. However, attaining high sequencing depth and accuracy from samples often remains problematic due to the damaged state of ancient DNA, in particular the extremely low copy number of ancient DNA and the abundance of uracil residues derived from cytosine deamination that lead to miscoding errors. It is therefore critical to use a highly efficient procedure for conversion of a raw DNA extract into an adaptor-ligated sequencing library, and equally important to reduce errors from uracil residues. We present a protocol for NGS library preparation that allows highly efficient conversion of DNA fragments into an adaptor-ligated form. The protocol incorporates an option to remove the vast majority of uracil miscoding lesions as part of the library preparation process. The procedure requires only two spin column purification steps and no gel purification or bead handling. Starting from an aliquot of DNA extract, a finished, highly amplified library can be generated in 5 h, or under 3 h if uracil removal is not required.

  6. Sequence-selective DNA recognition with peptide-bisbenzamidine conjugates.

    Science.gov (United States)

    Sánchez, Mateo I; Vázquez, Olalla; Vázquez, M Eugenio; Mascareñas, José L

    2013-07-22

    Transcription factors (TFs) are specialized proteins that play a key role in the regulation of genetic expression. Their mechanism of action involves the interaction with specific DNA sequences, which usually takes place through specialized domains of the protein. However, achieving an efficient binding usually requires the presence of the full protein. This is the case for bZIP and zinc finger TF families, which cannot interact with their target sites when the DNA binding fragments are presented as isolated monomers. Herein it is demonstrated that the DNA binding of these monomeric peptides can be restored when conjugated to aza-bisbenzamidines, which are readily accessible molecules that interact with A/T-rich sites by insertion into their minor groove. Importantly, the fluorogenic properties of the aza-benzamidine unit provide details of the DNA interaction that are eluded in electrophoresis mobility shift assays (EMSA). The hybrids based on the GCN4 bZIP protein preferentially bind to composite sequences containing tandem bisbenzamidine-GCN4 binding sites (TCAT⋅AAATT). Fluorescence reverse titrations show an interesting multiphasic profile consistent with the formation of competitive nonspecific complexes at low DNA/peptide ratios. On the other hand, the conjugate with the DNA binding domain of the zinc finger protein GAGA binds with high affinity (KD≈12 nM) and specificity to a composite AATTT⋅GAGA sequence containing both the bisbenzamidine and the TF consensus binding sites.

  7. Perspectives of DNA microarray and next-generation DNA sequencing technologies

    Institute of Scientific and Technical Information of China (English)

    TENG XiaoKun; XIAO HuaSheng

    2009-01-01

    DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequenc-ing method was first introduced by the 454 Company in 2003, immediately followed by the establish-ment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.

  8. Real-time DNA sequencing from single polymerase molecules.

    Science.gov (United States)

    Eid, John; Fehr, Adrian; Gray, Jeremy; Luong, Khai; Lyle, John; Otto, Geoff; Peluso, Paul; Rank, David; Baybayan, Primo; Bettman, Brad; Bibillo, Arkadiusz; Bjornson, Keith; Chaudhuri, Bidhan; Christians, Frederick; Cicero, Ronald; Clark, Sonya; Dalal, Ravindra; Dewinter, Alex; Dixon, John; Foquet, Mathieu; Gaertner, Alfred; Hardenbol, Paul; Heiner, Cheryl; Hester, Kevin; Holden, David; Kearns, Gregory; Kong, Xiangxu; Kuse, Ronald; Lacroix, Yves; Lin, Steven; Lundquist, Paul; Ma, Congcong; Marks, Patrick; Maxham, Mark; Murphy, Devon; Park, Insil; Pham, Thang; Phillips, Michael; Roy, Joy; Sebra, Robert; Shen, Gene; Sorenson, Jon; Tomaney, Austin; Travers, Kevin; Trulson, Mark; Vieceli, John; Wegener, Jeffrey; Wu, Dawn; Yang, Alicia; Zaccarin, Denis; Zhao, Peter; Zhong, Frank; Korlach, Jonas; Turner, Stephen

    2009-01-02

    We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

  9. Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

    Science.gov (United States)

    Chen, K.

    2017-01-01

    With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

  10. DNA qualification workflow for next generation sequencing of histopathological samples.

    Directory of Open Access Journals (Sweden)

    Michele Simbolo

    Full Text Available Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF tissues, 6 formalin-fixed paraffin-embedded (FFPE tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard

  11. DNA qualification workflow for next generation sequencing of histopathological samples.

    Science.gov (United States)

    Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T; Scarpa, Aldo

    2013-01-01

    Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

  12. Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses.

    Science.gov (United States)

    Li, Dawei; Qi, Xiaoqiong; Li, Xinwei; Li, Li; Zhong, Caihong; Huang, Hongwen

    2013-04-01

    The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.

  13. Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes

    Institute of Scientific and Technical Information of China (English)

    LIU Nian; ZHU Yong; WEI ZongXian; CHEN Jie; WANG QingBiao; JIAN ShuGuang; ZHOU DangWei; SHI Jing; YANG Yong; ZHONG Yang

    2009-01-01

    Araucariaceae is one of the most primitive families of the living conifers,and its phylogenetic relationships and divergence times are critically important issues.The DNA sequences of 8 genes,i.e.,nuclear ribosomal 18S and 26S rRNA,chloroplast 16S rRNA,rbcL,mafK and rps4,and mitochondrial coxl and atp1,obtained from this study and GenBank were used for constructing the molecular phylogenetic trees of Araucariaceae,indicating that the phylogenetic relationships among the three genera of this family should be ((Wollemia,Agathis),Araucaria).On the basis of the fossil calibrations of Wollemia and the two tribes Araucaria and Eutacta of the genus Araucaria,the divergence time of Araucariaceae was estimated to be (308±53) million years ago,that is,the origin of the family was in the Late Carboniferous rather than Triassic as a traditional view.With the same gene combination,the divergence times of the genera Araucaria and Agathis were (246 ±47) and (61±5) Ma,respectively.Statistical analyses on the phylogenetic trees generated by using different genes and comparisons of thedivergence times estimated by using those genes suggested that the chloroplast mafK and rps4 genes are most suitable for investigating the phylogenetic relationships and divergence times of the family Araucariaceae.

  14. High-throughput DNA sequencing: a genomic data manufacturing process.

    Science.gov (United States)

    Huang, G M

    1999-01-01

    The progress trends in automated DNA sequencing operation are reviewed. Technological development in sequencing instruments, enzymatic chemistry and robotic stations has resulted in ever-increasing capacity of sequence data production. This progress leads to a higher demand on laboratory information management and data quality assessment. High-throughput laboratories face the challenge of organizational management, as well as technology management. Engineering principles of process control should be adopted in this biological data manufacturing procedure. While various systems attempt to provide solutions to automate different parts of, or even the entire process, new technical advances will continue to change the paradigm and provide new challenges.

  15. Phylogenetic Relationships of the Fern Cyrtomium falcatum (Dryopteridaceae) from Dokdo Island, Sea of East Japan, Based on Chloroplast Genome Sequencing.

    Science.gov (United States)

    Raman, Gurusamy; Choi, Kyoung Su; Park, SeonJoo

    2016-12-02

    Cyrtomium falcatum is a popular ornamental fern cultivated worldwide. Native to the Korean Peninsula, Japan, and Dokdo Island in the Sea of Japan, it is the only fern present on Dokdo Island. We isolated and characterized the chloroplast (cp) genome of C. falcatum, and compared it with those of closely related species. The genes trnV-GAC and trnV-GAU were found to be present within the cp genome of C. falcatum, whereas trnP-GGG and rpl21 were lacking. Moreover, cp genomes of Cyrtomium devexiscapulae and Adiantum capillus-veneris lack trnP-GGG and rpl21, suggesting these are not conserved among angiosperm cp genomes. The deletion of trnR-UCG, trnR-CCG, and trnSeC in the cp genomes of C. falcatum and other eupolypod ferns indicates these genes are restricted to tree ferns, non-core leptosporangiates, and basal ferns. The C. falcatum cp genome also encoded ndhF and rps7, with GUG start codons that were only conserved in polypod ferns, and it shares two significant inversions with other ferns, including a minor inversion of the trnD-GUC region and an approximate 3 kb inversion of the trnG-trnT region. Phylogenetic analyses showed that Equisetum was found to be a sister clade to Psilotales-Ophioglossales with a 100% bootstrap (BS) value. The sister relationship between Pteridaceae and eupolypods was also strongly supported by a 100% BS, but Bayesian molecular clock analyses suggested that C. falcatum diversified in the mid-Paleogene period (45.15 ± 4.93 million years ago) and might have moved from Eurasia to Dokdo Island.

  16. RNA-DNA sequence differences spell genetic code ambiguities

    DEFF Research Database (Denmark)

    Bentin, Thomas; Nielsen, Michael L

    2013-01-01

    A recent paper in Science by Li et al. 2011(1) reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized. ...

  17. Decoding long nanopore sequencing reads of natural DNA.

    Science.gov (United States)

    Laszlo, Andrew H; Derrington, Ian M; Ross, Brian C; Brinkerhoff, Henry; Adey, Andrew; Nova, Ian C; Craig, Jonathan M; Langford, Kyle W; Samson, Jenny Mae; Daza, Riza; Doering, Kenji; Shendure, Jay; Gundlach, Jens H

    2014-08-01

    Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

  18. Functionalized nanopore-embedded electrodes for rapid DNA sequencing

    CERN Document Server

    He, Haiying; Pandey, Ravindra; Rocha, Alexandre Reily; Sanvito, Stefano; Grigoriev, Anton; Ahuja, Rajeev; Karna, Shashi P

    2007-01-01

    The determination of a patient's DNA sequence can, in principle, reveal an increased risk to fall ill with particular diseases [1,2] and help to design "personalized medicine" [3]. Moreover, statistical studies and comparison of genomes [4] of a large number of individuals are crucial for the analysis of mutations [5] and hereditary diseases, paving the way to preventive medicine [6]. DNA sequencing is, however, currently still a vastly time-consuming and very expensive task [4], consisting of pre-processing steps, the actual sequencing using the Sanger method, and post-processing in the form of data analysis [7]. Here we propose a new approach that relies on functionalized nanopore-embedded electrodes to achieve an unambiguous distinction of the four nucleic acid bases in the DNA sequencing process. This represents a significant improvement over previously studied designs [8,9] which cannot reliably distinguish all four bases of DNA. The transport properties of the setup investigated by us, employing state-o...

  19. DNA sequence handling programs in BASIC for home computers.

    OpenAIRE

    Biro, P A

    1984-01-01

    This paper describes a DNA sequence handling program written entirely in BASIC and designed to be run on an Atari home computer. Many of the features common to more sophisticated programs have been included. The advantage of this program are its convenience, its transportability and its potential for user modification. The disadvantages are lack of sophistication and speed.

  20. POSA : Perl objects for DNA sequencing data analysis

    NARCIS (Netherlands)

    Aerts, JA; Jungerius, BJ; Groenen, MA

    2004-01-01

    Background: Capillary DNA sequencing machines allow the generation of vast amounts of data with little hands-on time. With this expansion of data generation, there is a growing need for automated data processing. Most available software solutions, however, still require user intervention or provide

  1. A simple method encoding linear single strain DNA sequence with natural numbers

    Institute of Scientific and Technical Information of China (English)

    LI Jiye; XU Yuan; ZHANG Wang

    2008-01-01

    A simple method presenting linear single strain DNA (LssDNA) sequence with natural numbers is introduced in this paper. The method presents LssDNA correspondingly with the numerals 1, 2, 3 and 4. After calculation, the sequence can be coded in natural numbers which can also be decoded into the DNA sequence. Thus, an LssDNA sequence can be expressed in a natural number and a dot at coordinate axes. In the future, a new LssDNA sequences database termed "DotBank" would be realized in which each LssDNA sequence is determined as a dot.

  2. Polyphyly of the fern family Tectariaceae sensu Ching: Insights from cpDNA sequence data

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Tectariaceae are a pantropical fern family of about 20 genera, among which 8 are distributed in China. The morphological distinctiveness of the family is widely recognized, yet relatively little systematic research has been conducted on members of Tectariaceae. Phylogenetic analyses of chloroplast DNA sequence data (rbcL and atpB) from 15 species representing all 8 genera in China were carried out under parsimony criteria and Bayesian inference. The phylogenetic reconstructions indicated that the fern family Tectariaceae as traditionally circumscribed are polyphyletic. Ctenitis, Dryopsis, Lastreopsis clustered with and should be included within the newly-defined Dryopteridaceae, and Pleocnemia is also tentatively assigned to it. A narrowly monophyletic Tectariaceae is identified, which includes Ctenitopsis, Hemigramma, Pteridrys, Quercifilix, and Tectaria. In the single rbcL analysis, Arthropteris clustered with the above-mentioned monophyletic Tectariaceae. Although further investigations are still needed to identify infrafamilial relationships within the monophyletic Tectariaceae and to redefine several problematic genera, we propose a working concept here that better reflects the inferred evolutionary history of this group.

  3. Polyphyly of the fern family Tectariaceae sensu Ching: insights from cpDNA sequence data.

    Science.gov (United States)

    Liu, HongMei; Zhang, XianChun; Chen, ZhiDuan; Dong, ShiYong; Qiu, YinLong

    2007-12-01

    Tectariaceae are a pantropical fern family of about 20 genera, among which 8 are distributed in China. The morphological distinctiveness of the family is widely recognized, yet relatively little systematic research has been conducted on members of Tectariaceae. Phylogenetic analyses of chloroplast DNA sequence data (rbcL and atpB) from 15 species representing all 8 genera in China were carried out under parsimony criteria and Bayesian inference. The phylogenetic reconstructions indicated that the fern family Tectariaceae as traditionally circumscribed are polyphyletic. Ctenitis, Dryopsis, Lastreopsis clustered with and should be included within the newly-defined Dryopteridaceae, and Pleocnemia is also tentatively assigned to it. A narrowly monophyletic Tectariaceae is identified, which includes Ctenitopsis, Hemigramma, Pteridrys, Quercifilix, and Tectaria. In the single rbcL analysis, Arthropteris clustered with the above-mentioned monophyletic Tectariaceae. Although further investigations are still needed to identify infrafamilial relationships within the monophyletic Tectariaceae and to redefine several problematic genera, we propose a working concept here that better reflects the inferred evolutionary history of this group.

  4. Solid-State Nanopore-Based DNA Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Zewen Liu

    2016-01-01

    Full Text Available The solid-state nanopore-based DNA sequencing technology is becoming more and more attractive for its brand new future in gene detection field. The challenges that need to be addressed are diverse: the effective methods to detect base-specific signatures, the control of the nanopore’s size and surface properties, and the modulation of translocation velocity and behavior of the DNA molecules. Among these challenges, the realization of the high-quality nanopores with the help of modern micro/nanofabrication technologies is a crucial one. In this paper, typical technologies applied in the field of solid-state nanopore-based DNA sequencing have been reviewed.

  5. Sequence heterogeneity accelerates protein search for targets on DNA

    Energy Technology Data Exchange (ETDEWEB)

    Shvets, Alexey A.; Kolomeisky, Anatoly B., E-mail: tolya@rice.edu [Department of Chemistry and Center for Theoretical Biological Physics, Rice University, Houston, Texas 77005 (United States)

    2015-12-28

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome.

  6. Electronic density of states in sequence dependent DNA molecules

    Science.gov (United States)

    de Oliveira, B. P. W.; Albuquerque, E. L.; Vasconcelos, M. S.

    2006-09-01

    We report in this work a numerical study of the electronic density of states (DOS) in π-stacked arrays of DNA single-strand segments made up from the nucleotides guanine G, adenine A, cytosine C and thymine T, forming a Rudin-Shapiro (RS) as well as a Fibonacci (FB) polyGC quasiperiodic sequences. Both structures are constructed starting from a G nucleotide as seed and following their respective inflation rules. Our theoretical method uses Dyson's equation together with a transfer-matrix treatment, within an electronic tight-binding Hamiltonian model, suitable to describe the DNA segments modelled by the quasiperiodic chains. We compared the DOS spectra found for the quasiperiodic structure to those using a sequence of natural DNA, as part of the human chromosome Ch22, with a remarkable concordance, as far as the RS structure is concerned. The electronic spectrum shows several peaks, corresponding to localized states, as well as a striking self-similar aspect.

  7. A blind testing design for authenticating ancient DNA sequences.

    Science.gov (United States)

    Yang, H; Golenberg, E M; Shoshani, J

    1997-04-01

    Reproducibility is a serious concern among researchers of ancient DNA. We designed a blind testing procedure to evaluate laboratory accuracy and authenticity of ancient DNA obtained from closely related extant and extinct species. Soft tissue and bones of fossil and contemporary museum proboscideans were collected and identified based on morphology by one researcher, and other researchers carried out DNA testing on the samples, which were assigned anonymous numbers. DNA extracted using three principal isolation methods served as template in PCR amplifications of a segment of the cytochrome b gene (mitochondrial genome), and the PCR product was directly sequenced and analyzed. The results show that such a blind testing design performed in one laboratory, when coupled with phylogenetic analysis, can nonarbitrarily test the consistency and reliability of ancient DNA results. Such reproducible results obtained from the blind testing can increase confidence in the authenticity of ancient sequences obtained from postmortem specimens and avoid bias in phylogenetic analysis. A blind testing design may be applicable as an alternative to confirm ancient DNA results in one laboratory when independent testing by two laboratories is not available.

  8. DNA watermarks in non-coding regulatory sequences

    Directory of Open Access Journals (Sweden)

    Pyka Martin

    2009-07-01

    Full Text Available Abstract Background DNA watermarks can be applied to identify the unauthorized use of genetically modified organisms. It has been shown that coding regions can be used to encrypt information into living organisms by using the DNA-Crypt algorithm. Yet, if the sequence of interest presents a non-coding DNA sequence, either the function of a resulting functional RNA molecule or a regulatory sequence, such as a promoter, could be affected. For our studies we used the small cytoplasmic RNA 1 in yeast and the lac promoter region of Escherichia coli. Findings The lac promoter was deactivated by the integrated watermark. In addition, the RNA molecules displayed altered configurations after introducing a watermark, but surprisingly were functionally intact, which has been verified by analyzing the growth characteristics of both wild type and watermarked scR1 transformed yeast cells. In a third approach we introduced a second overlapping watermark into the lac promoter, which did not affect the promoter activity. Conclusion Even though the watermarked RNA and one of the watermarked promoters did not show any significant differences compared to the wild type RNA and wild type promoter region, respectively, it cannot be generalized that other RNA molecules or regulatory sequences behave accordingly. Therefore, we do not recommend integrating watermark sequences into regulatory regions.

  9. DNA sequence analysis of newly formed telomeres in yeast.

    Science.gov (United States)

    Wang, S S; Pluta, A F; Zakian, V A

    1989-01-01

    A plasmid can be maintained in linear form in baker's yeast if it bears telomeric sequences at each end. Linear plasmids bearing cloned telomeric C4A4 repeats at one end (test end) and a natural DNA terminus with approximately 300 bps of C4A2 repeats at the other or control end were introduced by transformation into yeast. Test-end termini of 28 to 112 bps supported telomere formation. During telomere formation, C4A2 repeats were often transferred to test-end termini. To determine in greater detail the fate of test-end sequences on these plasmids after propagation in yeast, test-end telomeres were subcloned into E. coli and sequenced. DNA sequencing established a number of points about the molecular events involved in telomere formation in yeast. The results suggest that there are at least two mechanisms for telomere formation in yeast. One is mediated by a recombination event that requires neither a long stretch of homology nor the RAD52 gene product. The other mechanism is by addition of C1-3A repeats to the termini of linear DNA molecules. The telomeric sequence required to support C1-3A addition need not be at the very end of a molecule for telomere formation.

  10. A Nano-Biosensor for DNA Sequence Detection Using Absorption Spectra of SWNT-DNA Composite

    Directory of Open Access Journals (Sweden)

    J. Bansal

    2011-01-01

    Full Text Available A biosensor based on Single Walled Carbon Nanotube (SWNT-Poly (GTn ssDNA hybrid has been developed for medical diagnostics. The absorption spectrum of this assay is determined with the help of a Shimadzu UV-VIS-NIR spectrophotometer. Two distinct bands each containing three peaks corresponding to first and second van Hove singularities in the density of states of the nanotubes were observed in the absorption spectrum. When a single-stranded DNA (ssDNA having a sequence complementary to probic DNA is added to the ssDNA-SWNT conjugates, hybridization takes place, which causes the red shift of absorption spectrum of nanotubes. On the other hand, when the DNA is noncomplementary, no shift in the absorption spectrum occurs since hybridization between the DNA and probe does not take place. The red shifting of the spectrum is considered to be due to change in the dielectric environment around nanotubes.

  11. Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers

    Directory of Open Access Journals (Sweden)

    Martin Andrew P

    2009-12-01

    Full Text Available Abstract Background The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms. Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers. Results The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism. Conclusion The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of

  12. Spectral sum rules and search for periodicities in DNA sequences

    Science.gov (United States)

    Chechetkin, V. R.

    2011-04-01

    Periodic patterns play the important regulatory and structural roles in genomic DNA sequences. Commonly, the underlying periodicities should be understood in a broad statistical sense, since the corresponding periodic patterns have been strongly distorted by the random point mutations and insertions/deletions during molecular evolution. The latent periodicities in DNA sequences can be efficiently displayed by Fourier transform. The criteria of significance for observed periodicities are obtained via the comparison versus the counterpart characteristics of the reference random sequences. We show that the restrictions imposed on the significance criteria by the rigorous spectral sum rules can be rationally described with De Finetti distribution. This distribution provides the convenient intermediate asymptotic form between Rayleigh distribution and exact combinatoric theory.

  13. Cloning, characterization, and properties of seven triplet repeat DNA sequences.

    Science.gov (United States)

    Ohshima, K; Kang, S; Larson, J E; Wells, R D

    1996-07-12

    Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG.CAG, CGG.CCG, or AAG.CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA.TAC, GAT.ATC, GTT.AAC, CAC.GTG, AGG.CCT, TCG.CGA, and AAG.CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D.(1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA.TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of approximately 16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG.CTT and AGG.CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG.CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG.CAG and CGG.CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D.(1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D.(1996) J. Biol. Chem. 271, 2875-2878) on CTG.CAG, CGG.CCG, and TTA.TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.

  14. VoSeq: a voucher and DNA sequence web application.

    Directory of Open Access Journals (Sweden)

    Carlos Peña

    Full Text Available There is an ever growing number of molecular phylogenetic studies published, due to, in part, the advent of new techniques that allow cheap and quick DNA sequencing. Hence, the demand for relational databases with which to manage and annotate the amassing DNA sequences, genes, voucher specimens and associated biological data is increasing. In addition, a user-friendly interface is necessary for easy integration and management of the data stored in the database back-end. Available databases allow management of a wide variety of biological data. However, most database systems are not specifically constructed with the aim of being an organizational tool for researchers working in phylogenetic inference. We here report a new software facilitating easy management of voucher and sequence data, consisting of a relational database as back-end for a graphic user interface accessed via a web browser. The application, VoSeq, includes tools for creating molecular datasets of DNA or amino acid sequences ready to be used in commonly used phylogenetic software such as RAxML, TNT, MrBayes and PAUP, as well as for creating tables ready for publishing. It also has inbuilt BLAST capabilities against all DNA sequences stored in VoSeq as well as sequences in NCBI GenBank. By using mash-ups and calls to web services, VoSeq allows easy integration with public services such as Yahoo! Maps, Flickr, Encyclopedia of Life (EOL and GBIF (by generating data-dumps that can be processed with GBIF's Integrated Publishing Toolkit.

  15. Perspectives of DNA microarray and next-generation DNA sequencing technologies

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research,in revealing both the structural and functional characteristics of genomes.In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics,systems biology and pharmacogenomics.The next-generation DNA sequencing method was first introduced by the 454 Company in 2003,immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies.Though it has not been long since the first emergence of this technology,with the fast and impressive improvement,the application of this technology has extended to almost all fields of genomics research,as a rival challenging the existing DNA microarray technology.This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.

  16. Recent developments in sequence selective minor groove DNA effectors.

    Science.gov (United States)

    Reddy, B S; Sharma, S K; Lown, J W

    2001-04-01

    DNA is a well characterized intracellular target but its large size and sequential nature make it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this respect the main consideration is given to recent developments in DNA sequence selective binding agents bearing conjugated effectors because of their potential application in diagnosis and treatment of cancers as well as in molecular biology. Recent progress in the development of cross linked lexitropsin oligopeptides and hairpins, which bind selectively to the minor groove of duplex DNA, is discussed. Bis-distamycins and related lexitropsins show inhibitory activity against HIV-1 and HIV-2 integrases at low nanomolar concentrations. Benzoyl nitrogen mustard analogs of lexitropsins are active against a variety of tumor models. Certain of the bis-benzimidazoles show altered DNA sequence preference and bind to DNA at 5'CG and TG sequences rather than at the preferred AT sites of the parent drug. A comparison of bifunctional bizelesin with monoalkylating adozelesin shows that it appears to have an increased sequence selectivity such that monoalkylating compounds react at more than one site but bizelesin reacts only at sites where there are two suitably positioned alkylation sites. Adozelesin, bizelesin and carzelesin are far more potent as cytotoxic agents than cisplatin or doxorubicin. A new class of 1,2,9,9a-tetrahydrocyclo-propa[c]benz[e]indole-4-one (CBI) analogs i.e., CBI-lexitropsin conjugates arising from the latter leads are also discussed.A number of cyclopropylpyrroloindole (CPI) and CBI-lexitropsin conjugates related to CC-1065 alkylate at the N3 position of adenine in the minor groove of DNA in a sequence specific manner, and also show cytotoxicities in the femtomolar range. The cross linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin, and melphalan. A new

  17. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    Energy Technology Data Exchange (ETDEWEB)

    1992-01-01

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3' to 5' exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  18. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    Energy Technology Data Exchange (ETDEWEB)

    1992-12-31

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3` to 5` exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  19. Next generation sequencing of DNA-launched Chikungunya vaccine virus

    Energy Technology Data Exchange (ETDEWEB)

    Hidajat, Rachmat; Nickols, Brian [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Forrester, Naomi [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Tretyakova, Irina [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Weaver, Scott [Institute for Human Infections and Immunity, Sealy Center for Vaccine Development and Department of Pathology, University of Texas Medical Branch, GNL, 301 University Blvd., Galveston, TX 77555 (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

    2016-03-15

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.

  20. Delineating relative homogeneous G+C domains in DNA sequences.

    Science.gov (United States)

    Li, W

    2001-10-03

    The concept of homogeneity of G+C content is always relative and subjective. This point is emphasized and quantified in this paper using a simple example of one sequence segmented into two subsequences. Whether the sequence is homogeneous or not can be answered by whether the two-subsequence model describes the DNA sequence better than the one-sequence model. There are at least three equivalent ways of looking at the 1-to-2 segmentation: Jensen-Shannon divergence measure, log likelihood ratio test, and model selection using Bayesian information criterion. Once a criterion is chosen, a DNA sequence can be recursively segmented into multiple domains. We use one subjective criterion called segmentation strength based on the Bayesian information criterion. Whether or not a sequence is homogeneous and how many domains it has depend on this criterion. We compare six different genome sequences (yeast S. cerevisiae chromosome III and IV, bacterium M. pneumoniae, human major histocompatibility complex sequence, longest contigs in human chromosome 21 and 22) by recursive segmentations at different strength criteria. Results by recursive segmentation confirm that yeast chromosome IV is more homogeneous than yeast chromosome III, human chromosome 21 is more homogeneous than human chromosome 22, and bacterial genomes may not be homogeneous due to short segments with distinct base compositions. The recursive segmentation also provides a quantitative criterion for identifying isochores in human sequences. Some features of our recursive segmentation, such as the possibility of delineating domain borders accurately, are superior to those of the moving-window approach commonly used in such analyses.

  1. Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp from the Coastal Waters off Western Korea: Morphology, Pigments, and Ribosomal DNA Gene Sequence

    DEFF Research Database (Denmark)

    Kang, Nam Seon; Jeong, Hae Jin; Moestrup, Øjvind

    2010-01-01

    The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic...... pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate-like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal...... fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium...

  2. Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq).

    Science.gov (United States)

    Graham, Carly F; Glenn, Travis C; McArthur, Andrew G; Boreham, Douglas R; Kieran, Troy; Lance, Stacey; Manzon, Richard G; Martino, Jessica A; Pierson, Todd; Rogers, Sean M; Wilson, Joanna Y; Somers, Christopher M

    2015-11-01

    Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

  3. Automated parallel DNA sequencing on multiple channel microchips.

    Science.gov (United States)

    Liu, S; Ren, H; Gao, Q; Roach, D J; Loder, R T; Armstrong, T M; Mao, Q; Blaga, I; Barker, D L; Jovanovich, S B

    2000-05-09

    We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

  4. Assessing the fidelity of ancient DNA sequences amplified from nuclear genes

    DEFF Research Database (Denmark)

    Binladen, Jonas; Wiuf, Carsten Henrik; Gilbert, M. Thomas P.

    2006-01-01

    To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in ph...

  5. The influence of DNA sequence on epigenome-induced pathologies

    Directory of Open Access Journals (Sweden)

    Meagher Richard B

    2012-07-01

    Full Text Available Abstract Clear cause-and-effect relationships are commonly established between genotype and the inherited risk of acquiring human and plant diseases and aberrant phenotypes. By contrast, few such cause-and-effect relationships are established linking a chromatin structure (that is, the epitype with the transgenerational risk of acquiring a disease or abnormal phenotype. It is not entirely clear how epitypes are inherited from parent to offspring as populations evolve, even though epigenetics is proposed to be fundamental to evolution and the likelihood of acquiring many diseases. This article explores the hypothesis that, for transgenerationally inherited chromatin structures, “genotype predisposes epitype”, and that epitype functions as a modifier of gene expression within the classical central dogma of molecular biology. Evidence for the causal contribution of genotype to inherited epitypes and epigenetic risk comes primarily from two different kinds of studies discussed herein. The first and direct method of research proceeds by the examination of the transgenerational inheritance of epitype and the penetrance of phenotype among genetically related individuals. The second approach identifies epitypes that are duplicated (as DNA sequences are duplicated and evolutionarily conserved among repeated patterns in the DNA sequence. The body of this article summarizes particularly robust examples of these studies from humans, mice, Arabidopsis, and other organisms. The bulk of the data from both areas of research support the hypothesis that genotypes predispose the likelihood of displaying various epitypes, but for only a few classes of epitype. This analysis suggests that renewed efforts are needed in identifying polymorphic DNA sequences that determine variable nucleosome positioning and DNA methylation as the primary cause of inherited epigenome-induced pathologies. By contrast, there is very little evidence that DNA sequence directly

  6. Spectral sum rules and search for periodicities in DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Chechetkin, V.R., E-mail: chechet@biochip.r [Theoretical Department of Division for Perspective Investigations, Troitsk Institute of Innovation and Thermonuclear Investigations (TRINITI), Troitsk, 142190 Moscow Region (Russian Federation)

    2011-04-18

    Periodic patterns play the important regulatory and structural roles in genomic DNA sequences. Commonly, the underlying periodicities should be understood in a broad statistical sense, since the corresponding periodic patterns have been strongly distorted by the random point mutations and insertions/deletions during molecular evolution. The latent periodicities in DNA sequences can be efficiently displayed by Fourier transform. The criteria of significance for observed periodicities are obtained via the comparison versus the counterpart characteristics of the reference random sequences. We show that the restrictions imposed on the significance criteria by the rigorous spectral sum rules can be rationally described with De Finetti distribution. This distribution provides the convenient intermediate asymptotic form between Rayleigh distribution and exact combinatoric theory. - Highlights: We study the significance criteria for latent periodicities in DNA sequences. The constraints imposed by sum rules can be described with De Finetti distribution. It is intermediate between Rayleigh distribution and exact combinatoric theory. Theory is applicable to the study of correlations between different periodicities. The approach can be generalized to the arbitrary discrete Fourier transform.

  7. Early Lyme disease with spirochetemia - diagnosed by DNA sequencing

    Directory of Open Access Journals (Sweden)

    Jones William

    2010-11-01

    Full Text Available Abstract Background A sensitive and analytically specific nucleic acid amplification test (NAAT is valuable in confirming the diagnosis of early Lyme disease at the stage of spirochetemia. Findings Venous blood drawn from patients with clinical presentations of Lyme disease was tested for the standard 2-tier screen and Western Blot serology assay for Lyme disease, and also by a nested polymerase chain reaction (PCR for B. burgdorferi sensu lato 16S ribosomal DNA. The PCR amplicon was sequenced for B. burgdorferi genomic DNA validation. A total of 130 patients visiting emergency room (ER or Walk-in clinic (WALKIN, and 333 patients referred through the private physicians' offices were studied. While 5.4% of the ER/WALKIN patients showed DNA evidence of spirochetemia, none (0% of the patients referred from private physicians' offices were DNA-positive. In contrast, while 8.4% of the patients referred from private physicians' offices were positive for the 2-tier Lyme serology assay, only 1.5% of the ER/WALKIN patients were positive for this antibody test. The 2-tier serology assay missed 85.7% of the cases of early Lyme disease with spirochetemia. The latter diagnosis was confirmed by DNA sequencing. Conclusion Nested PCR followed by automated DNA sequencing is a valuable supplement to the standard 2-tier antibody assay in the diagnosis of early Lyme disease with spirochetemia. The best time to test for Lyme spirochetemia is when the patients living in the Lyme disease endemic areas develop unexplained symptoms or clinical manifestations that are consistent with Lyme disease early in the course of their illness.

  8. The closest relatives of cacti: insights from phylogenetic analyses of chloroplast and mitochondrial sequences with special emphasis on relationships in the tribe Anacampseroteae.

    Science.gov (United States)

    Nyffeler, Reto

    2007-01-01

    Recent molecular and morphological systematic investigations revealed that the cacti are most closely related to Anacampseroteae, Portulaca and Talinum of the family Portulacaceae (ACPT clade of suborder Portulacineae). A combined analysis of ndhF, matK, and nad1 sequence data from the chloroplast and the mitochondrial genomes indicates that the tribe Anacampseroteae is the sister group of the family Cactaceae. This clade, together with Portulaca, is well characterized by the presence of axillary hairs or scales. Relationships within Anacampseroteae are characterized by a grade of five species of Grahamia s.l. from North and South America, and Grahamia australiana is found to be sister to the genera Anacampseros and Avonia. A comparison of vegetative characteristics indicates an evolutionary transition from woody subshrubs to dwarf perennial and highly succulent herbs during the diversification of Anacampseroteae. Available evidence from the present investigation as well as from previously published studies suggests that a revised classification of Portulacineae on the basis of inferred phylogenetic relationships might consist of a superfamily that includes Cactaceae and the three genera Anacampseros s.l. (including Avonia and Grahamia s.l.), Portulaca, and Talinum (including Talinella), either referred to three monogeneric families or to a paraphyletic family Portulacaceae*.

  9. A CLIQUE algorithm using DNA computing techniques based on closed-circle DNA sequences.

    Science.gov (United States)

    Zhang, Hongyan; Liu, Xiyu

    2011-07-01

    DNA computing has been applied in broad fields such as graph theory, finite state problems, and combinatorial problem. DNA computing approaches are more suitable used to solve many combinatorial problems because of the vast parallelism and high-density storage. The CLIQUE algorithm is one of the gird-based clustering techniques for spatial data. It is the combinatorial problem of the density cells. Therefore we utilize DNA computing using the closed-circle DNA sequences to execute the CLIQUE algorithm for the two-dimensional data. In our study, the process of clustering becomes a parallel bio-chemical reaction and the DNA sequences representing the marked cells can be combined to form a closed-circle DNA sequences. This strategy is a new application of DNA computing. Although the strategy is only for the two-dimensional data, it provides a new idea to consider the grids to be vertexes in a graph and transform the search problem into a combinatorial problem.

  10. Short sequence effect of ancient DNA on mammoth phylogenetic analyses

    Institute of Scientific and Technical Information of China (English)

    Guilian SHENG; Lianjuan WU; Xindong HOU; Junxia YUAN; Shenghong CHENG; Bojian ZHONG; Xulong LAI

    2009-01-01

    The evolution of Elephantidae has been intensively studied in the past few years, especially after 2006. The molecular approaches have made great contribution to the assumption that the extinct woolly mammoth has a close relationship with the Asian elephant instead of the African elephant. In this study, partial ancient DNA sequences of cytochrome b (cyt b) gene in mitochondrial genome were successfully retrieved from Late Pleistocene Mammuthus primigenius bones collected from Heilongjiang Province in Northeast China. Both the partial and complete homologous cyt b gene sequences and the whole mitochondrial genome sequences extracted from GenBank were aligned and used as datasets for phylogenetic analyses. All of the phylogenetic trees, based on either the partial or the complete cyt b gene, reject the relationship constructed by the whole mitochondrial genome, showing the occurrence of an effect of sequence length of cyt b gene on mammoth phylogenetic analyses.

  11. Phylogenetic relationships and speciation in the genus Passerina L.(Thymelaeaceae)inferred from chloroplast and nuclear sequence data.

    OpenAIRE

    2008-01-01

    The Eastern Cape is regarded as the centre of diversity for Passerina, except for two species occurring in the outliers of eastern Africa. Ten species are endemic to the Cape Floristic Region and four are regarded as near endemics. A complete species-level phylogeny for Passerina utilising sequences from three plastid and one nuclear gene is presented. The loci sequences were rbcL, trnL-F, rps16 and ITS. Parsimony and Bayesian analysis yield identical relationships and two informal groupings ...

  12. DNA sequence representation by trianders and determinative degree of nucleotides

    Institute of Scientific and Technical Information of China (English)

    DUPLIJ Diana; DUPLIJ Steven

    2005-01-01

    A new version of DNA walks, where nucleotides are regarded unequal in their contribution to a walk is introduced,which allows us to study thoroughly the "fine structure" of nucleotide sequences. The approach is based on the assumption that nucleotides have an inner abstract characteristic, the determinative degree, which reflects genetic code phenomenological properties and is adjusted to nucleotides physical properties. We consider each codon position independently, which gives three separate walks characterized by different angles and lengths, and that such an object is called triander which reflects the "strength"of branch. A general method for identifying DNA sequence "by triander" which can be treated as a unique "genogram" (or "gene passport") is proposed. The two- and three-dimensional trianders are considered. The difference of sequences fine structure in genes and the intergenic space is shown. A clear triplet signal in coding sequences was found which is absent in the intergenic space and is independent from the sequence length. This paper presents the topological classification oftrianders which can allow us to provide a detailed working out signatures of functionally different genomic regions.

  13. Sequences sufficient for programming imprinted germline DNA methylation defined.

    Directory of Open Access Journals (Sweden)

    Yoon Jung Park

    Full Text Available Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC; the second carries only the DMD and repeats (DR from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.

  14. Evaluation of intra- and interspecific divergence of satellite DNA sequences by nucleotide frequency calculation and pairwise sequence comparison

    Directory of Open Access Journals (Sweden)

    Kato Mikio

    2003-01-01

    Full Text Available Satellite DNA sequences are known to be highly variable and to have been subjected to concerted evolution that homogenizes member sequences within species. We have analyzed the mode of evolution of satellite DNA sequences in four fishes from the genus Diplodus by calculating the nucleotide frequency of the sequence array and the phylogenetic distances between member sequences. Calculation of nucleotide frequency and pairwise sequence comparison enabled us to characterize the divergence among member sequences in this satellite DNA family. The results suggest that the evolutionary rate of satellite DNA in D. bellottii is about two-fold greater than the average of the other three fishes, and that the sequence homogenization event occurred in D. puntazzo more recently than in the others. The procedures described here are effective to characterize mode of evolution of satellite DNA.

  15. Effect of dephasing on DNA sequencing via transverse electronic transport

    Energy Technology Data Exchange (ETDEWEB)

    Zwolak, Michael [Los Alamos National Laboratory; Krems, Matt [NON LANL; Pershin, Yuriy V [NON LANL; Di Ventra, Massimiliano [NON LANL

    2009-01-01

    We study theoretically the effects of dephasing on DNA sequencing in a nanopore via transverse electronic transport. To do this, we couple classical molecular dynamics simulations with transport calculations using scattering theory. Previous studies, which did not include dephasing, have shown that by measuring the transverse current of a particular base multiple times, one can get distributions of currents for each base that are distinguishable. We introduce a dephasing parameter into transport calculations to simulate the effects of the ions and other fluctuations. These effects lower the overall magnitude of the current, but have little effect on the current distributions themselves. The results of this work further implicate that distinguishing DNA bases via transverse electronic transport has potential as a sequencing tool.

  16. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA

    DEFF Research Database (Denmark)

    Alquezar-Planas, David E; Fordyce, Sarah Louise

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure tha...

  17. A large population of small chloroplasts in tobacco leaf cells allows more effective chloroplast movement than a few enlarged chloroplasts.

    Science.gov (United States)

    Jeong, Won Joong; Park, Youn-Il; Suh, KyeHong; Raven, John A; Yoo, Ook Joon; Liu, Jang Ryol

    2002-05-01

    We generated transgenic tobacco (Nicotiana tabacum cv Xanthi) plants that contained only one to three enlarged chloroplasts per leaf mesophyll cell by introducing NtFtsZ1-2, a cDNA for plastid division. These plants were used to investigate the advantages of having a large population of small chloroplasts rather than a few enlarged chloroplasts in a leaf mesophyll cell. Despite the similarities in photosynthetic components and ultrastructure of photosynthetic machinery between wild-type and transgenic plants, the overall growth of transgenic plants under low- and high-light conditions was retarded. In wild-type plants, the chloroplasts moved toward the face position under low light and toward the profile position under high-light conditions. However, chloroplast rearrangement in transgenic plants in response to light conditions was not evident. In addition, transgenic plant leaves showed greatly diminished changes in leaf transmittance values under both light conditions, indicating that chloroplast rearrangement was severely retarded. Therefore, under low-light conditions the incomplete face position of the enlarged chloroplasts results in decreased absorbance of light energy. This, in turn, reduces plant growth. Under high-light conditions, the amount of absorbed light exceeds the photosynthetic utilization capacity due to the incomplete profile position of the enlarged chloroplasts, resulting in photodamage to the photosynthetic machinery, and decreased growth. The presence of a large number of small and/or rapidly moving chloroplasts in the cells of higher land plants permits more effective chloroplast phototaxis and, hence, allows more efficient utilization of low-incident photon flux densities. The photosynthetic apparatus is, consequently, protected from damage under high-incident photon flux densities.

  18. Measuring cation dependent DNA polymerase fidelity landscapes by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Bradley Michael Zamft

    Full Text Available High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP on a known template, allowing DNA sequences to encode information about the local cation concentration. In this work we quantify the cation sensitivity of DNAP misincorporation rates, making possible the indirect readout of cation concentration by DNA sequencing. Using multiplexed deep sequencing, we quantify the misincorporation properties of two DNA polymerases--Dpo4 and Klenow exo(---obtaining the probability and base selectivity of misincorporation at all positions within the template. We find that Dpo4 acts as a DNA recording device for Mn(2+ with a misincorporation rate gain of ∼2%/mM. This modulation of misincorporation rate is selective to the template base: the probability of misincorporation on template T by Dpo4 increases >50-fold over the range tested, while the other template bases are affected less strongly. Furthermore, cation concentrations act as scaling factors for misincorporation: on a given template base, Mn(2+ and Mg(2+ change the overall misincorporation rate but do not alter the relative frequencies of incoming misincorporated nucleotides. Characterization of the ion dependence of DNAP misincorporation serves as the first step towards repurposing it as a molecular recording device.

  19. An automated annotation tool for genomic DNA sequences using GeneScan and BLAST

    Indian Academy of Sciences (India)

    Andrew M. Lynn; Chakresh Kumar Jain; K. Kosalai; Pranjan Barman; Nupur Thakur; Harish Batra; Alok Bhattacharya

    2001-04-01

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

  20. Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome.

    Science.gov (United States)

    Azim, M Kamran; Khan, Ishtaiq A; Zhang, Yong

    2014-05-01

    We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.

  1. Computational optimisation of targeted DNA sequencing for cancer detection

    DEFF Research Database (Denmark)

    Martinez, Pierre; McGranahan, Nicholas; Birkbak, Nicolai Juul

    2013-01-01

    detection. Dividing 4,467 samples into one discovery and two independent validation cohorts, we show that up to 76% of 10 cancer types harbour at least one mutation in a panel of only 25 genes, with high sensitivity across most tumour types. Our analyses demonstrate that targeting "hotspot" regions would......Despite recent progress thanks to next-generation sequencing technologies, personalised cancer medicine is still hampered by intra-tumour heterogeneity and drug resistance. As most patients with advanced metastatic disease face poor survival, there is need to improve early diagnosis. Analysing...... circulating tumour DNA (ctDNA) might represent a non-invasive method to detect mutations in patients, facilitating early detection. In this article, we define reduced gene panels from publicly available datasets as a first step to assess and optimise the potential of targeted ctDNA scans for early tumour...

  2. Similarity Estimation Between DNA Sequences Based on Local Pattern Histograms of Binary Images

    Institute of Scientific and Technical Information of China (English)

    Yusei Kobori; Satoshi Mizuta

    2016-01-01

    Graphical representation of DNA sequences is one of the most popular techniques for alignment-free sequence comparison. Here, we propose a new method for the feature extraction of DNA sequences represented by binary images, by estimating the similarity between DNA sequences using the frequency histograms of local bitmap patterns of images. Our method shows linear time complexity for the length of DNA sequences, which is practical even when long sequences, such as whole genome sequences, are compared. We tested five distance measures for the estimation of sequence similarities, and found that the histogram intersection and Manhattan distance are the most appropriate ones for phylogenetic analyses.

  3. Nuclear and chloroplast DNA reassessment of the origin of Indian potato varieties and its implications for the origin of the early European potato.

    Science.gov (United States)

    Spooner, D M; Nuñez, J; Rodríguez, F; Naik, P S; Ghislain, M

    2005-04-01

    The modern cultivated potato was first recorded in Europe in 1562, but its area(s) of exportation has long been in dispute. Two competing hypotheses have proposed an "Andean" area (somewhere from upland Venezuela to northern Argentina) or a lowland south central "Chilean" area. Potato landraces from these two areas can be distinguished, although sometimes with difficulty, by (1) cytoplasmic sterility factors, (2) morphological traits, (3) daylength adaptation, (4) microsatellite markers, and (5) co-evolved chloroplast (cp) and mitochondria (mt) DNA. The Chilean introduction hypothesis originally was proposed because of similarities among Chilean landraces and modern "European" cultivars with respect to traits 2 and 3. Alternatively, the Andean introduction hypothesis suggests that (1) traits 2 and 3 of European potato evolved rapidly, in parallel, from Andean landraces to a Chilean type through selection following import to Europe, and (2) the worldwide late blight epidemics beginning in 1845 in the United Kingdom displaced most existing European cultivars and the potato was subsequently improved by importations of Chilean landraces. We reassess these two competing hypotheses with nuclear microsatellite and cpDNA analyses of (1) 32 Indian cultivars, some of which are thought to preserve putatively remnant populations of Andean landraces, (2) 12 Andean landraces, and (3) five Chilean landraces. Our microsatellite results cluster all Indian cultivars, including putatively remnant Andean landrace populations, with the Chilean landraces, and none with the "old Andigenum" landraces. Some of these Indian landraces, however, lack the cpDNA typical of Chilean landraces and advanced cultivars, indicating they likely are hybrids of Andean landraces with Chilean clones or more advanced cultivars. These results lead us to reexamine the hypothesis that early introductions of potato to Europe were solely from the Andes.

  4. Rapid sequencing of DNA based on single-molecule detection

    Science.gov (United States)

    Soper, Steven A.; Davis, Lloyd M.; Fairfield, Frederick R.; Hammond, Mark L.; Harger, Carol A.; Jett, James H.; Keller, Richard A.; Marrone, Babetta L.; Martin, John C.; Nutter, Harvey L.; Shera, E. Brooks; Simpson, Daniel J.

    1991-07-01

    Sequencing the human genome is a major undertaking considering the large number of nucleotides present in the genome and the slow methods currently available to perform the task. The authors have recently reported on a scheme to sequence DNA rapidly using a non-gel based technique. The concept is based upon the incorporation of fluorescently labeled nucleotides into a strand of DNA, isolation and manipulation of a labeled DNA fragment and the detection of single nucleotides using ultra-sensitive laser-induced fluorescence detection following their cleavage from the fragment. Detection of individual fluorophores in the liquid phase was accomplished with time-gated detection following pulsed-laser excitation. The photon bursts from individual rhodamine 6G (R6G) molecules travelling through a laser beam have been observed, as have bursts from single fluorescently modified nucleotides. Using two different biotinylated nucleotides as a model system for fluorescently labeled nucleotides, the authors have observed synthesis of the complementary copy of M13 bacteriophage. Work with fluorescently labeled nucleotides is underway. Individual molecules of DNA attached to a microbead have been observed and manipulated with an epifluorescence microscope.

  5. Human mitochondrial DNA complete amplification and sequencing: a new validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification.

    Science.gov (United States)

    Ramos, Amanda; Santos, Cristina; Alvarez, Luis; Nogués, Ramon; Aluja, Maria Pilar

    2009-05-01

    To date, there are no published primers to amplify the entire mitochondrial DNA (mtDNA) that completely prevent the amplification of nuclear DNA (nDNA) sequences of mitochondrial origin. The main goal of this work was to design, validate and describe a set of primers, to specifically amplify and sequence the complete human mtDNA, allowing the correct interpretation of mtDNA heteroplasmy in healthy and pathological samples. Validation was performed using two different approaches: (i) Basic Local Alignment Search Tool and (ii) amplification using isolated nDNA obtained from sperm cells by differential lyses. During the validation process, two mtDNA regions, with high similarity with nDNA, represent the major problematic areas for primer design. One of these could represent a non-published nuclear DNA sequence of mitochondrial origin. For two of the initially designed fragments, the amplification results reveal PCR artifacts that can be attributed to the poor quality of the DNA. After the validation, nine overlapping primer pairs to perform mtDNA amplification and 22 additional internal primers for mtDNA sequencing were obtained. These primers could be a useful tool in future projects that deal with mtDNA complete sequencing and heteroplasmy detection, since they represent a set of primers that have been tested for the non-amplification of nDNA.

  6. A DNA sequence alignment algorithm using quality information and a fuzzy inference method

    Institute of Scientific and Technical Information of China (English)

    Kwangbaek Kim; Minhwan Kim; Youngwoon Woo

    2008-01-01

    DNA sequence alignment algorithms in computational molecular biology have been improved by diverse methods.In this paper.We propose a DNA sequence alignment that Uses quality information and a fuzzy inference method developed based on the characteristics of DNA fragments and a fuzzy logic system in order to improve conventional DNA sequence alignment methods that uses DNA sequence quality information.In conventional algorithms.DNA sequence alignment scores are calculated by the global sequence alignment algorithm proposed by Needleman-Wunsch,which is established by using quality information of each DNA fragment.However,there may be errors in the process of calculating DNA sequence alignment scores when the quality of DNA fragment tips is low.because only the overall DNA sequence quality information are used.In our proposed method.an exact DNA sequence alignment can be achieved in spite of the low quality of DNA fragment tips by improvement of conventional algorithms using quality information.Mapping score parameters used to calculate DNA sequence alignment scores are dynamically adjusted by the fuzzy logic system utilizing lengths of DNA fragments and frequencies of low quality DNA bases in the fragments.From the experiments by applying real genome data of National Center for Bioteclmology Information,we could see that the proposed method is more efficient than conventional algorithms.

  7. Artificial intelligence approach in analysis of DNA sequences.

    Science.gov (United States)

    Brézillon, P J; Zaraté, P; Saci, F

    1993-01-01

    We present an approach for designing a knowledge-based system, called Sequence Acquisition In Context (SAIC), that will be able to cooperate with a biologist in the analysis of DNA sequences. The main task of the system is the acquisition of the expert knowledge that the biologist uses for solving ambiguities from gel autoradiograms, with the aim of re-using it later for solving similar ambiguities. The various types of expert knowledge constitute what we call the contextual knowledge of the sequence analysis. Contextual knowledge deals with the unavoidable problems that are common in the study of the living material (eg noise on data, difficulties of observations). Indeed, the analysis of DNA sequences from autoradiograms belongs to an emerging and promising area of investigation, namely reasoning with images. The SAIC project is developed in a theoretical framework that is shared with other applications. Not all tasks have the same importance in each application. We use this observation for designing an intelligent assistant system with three applications. In the SAIC project, we focus on knowledge acquisition, human-computer interaction and explanation. The project will benefit research in the two other applications. We also discuss our SAIC project in the context of large international projects that aim to re-use and share knowledge in a repository.

  8. Model identification for DNA sequence-structure relationships.

    Science.gov (United States)

    Hawley, Stephen Dwyer; Chiu, Anita; Chizeck, Howard Jay

    2006-11-01

    We investigate the use of algebraic state-space models for the sequence dependent properties of DNA. By considering the DNA sequence as an input signal, rather than using an all atom physical model, computational efficiency is achieved. A challenge in deriving this type of model is obtaining its structure and estimating its parameters. Here we present two candidate model structures for the sequence dependent structural property Slide and a method of encoding the models so that a recursive least squares algorithm can be applied for parameter estimation. These models are based on the assumption that the value of Slide at a base-step is determined by the surrounding tetranucleotide sequence. The first model takes the four bases individually as inputs and has a median root mean square deviation of 0.90 A. The second model takes the four bases pairwise and has a median root mean square deviation of 0.88 A. These values indicate that the accuracy of these models is within the useful range for structure prediction. Performance is comparable to published predictions of a more physically derived model, at significantly less computational cost.

  9. DNA sequence chromatogram browsing using JAVA and CORBA.

    Science.gov (United States)

    Parsons, J D; Buehler, E; Hillier, L

    1999-03-01

    DNA sequence chromatograms (traces) are the primary data source for all large-scale genomic and expressed sequence tags (ESTs) sequencing projects. Access to the sequencing trace assists many later analyses, for example contig assembly and polymorphism detection, but obtaining and using traces is problematic. Traces are not collected and published centrally, they are much larger than the base calls derived from them, and viewing them requires the interactivity of a local graphical client with local data. To provide efficient global access to DNA traces, we developed a client/server system based on flexible Java components integrated into other applications including an applet for use in a WWW browser and a stand-alone trace viewer. Client/server interaction is facilitated by CORBA middleware which provides a well-defined interface, a naming service, and location independence. [The software is packaged as a Jar file available from the following URL: http://www.ebi.ac.uk/jparsons. Links to working examples of the trace viewers can be found at http://corba.ebi.ac.uk/EST. All the Washington University mouse EST traces are available for browsing at the same URL.

  10. DNA Sequencing via Quantum Mechanics and Machine Learning

    CERN Document Server

    Yuen, Henry; Zhang, Kevin J; Nomura, Ken-ichi; Kalia, Rajiv K; Nakano, Aiichiro; Vashishta, Priya

    2010-01-01

    Rapid sequencing of individual human genome is prerequisite to genomic medicine, where diseases will be prevented by preemptive cures. Quantum-mechanical tunneling through single-stranded DNA in a solid-state nanopore has been proposed for rapid DNA sequencing, but unfortunately the tunneling current alone cannot distinguish the four nucleotides due to large fluctuations in molecular conformation and solvent. Here, we propose a machine-learning approach applied to the tunneling current-voltage (I-V) characteristic for efficient discrimination between the four nucleotides. We first combine principal component analysis (PCA) and fuzzy c-means (FCM) clustering to learn the "fingerprints" of the electronic density-of-states (DOS) of the four nucleotides, which can be derived from the I-V data. We then apply the hidden Markov model and the Viterbi algorithm to sequence a time series of DOS data (i.e., to solve the sequencing problem). Numerical experiments show that the PCA-FCM approach can classify unlabeled DOS ...

  11. Mixed Sequence Reader: A Program for Analyzing DNA Sequences with Heterozygous Base Calling

    Science.gov (United States)

    Chang, Chun-Tien; Tsai, Chi-Neu; Tang, Chuan Yi; Chen, Chun-Houh; Lian, Jang-Hau; Hu, Chi-Yu; Tsai, Chia-Lung; Chao, Angel; Lai, Chyong-Huey; Wang, Tzu-Hao; Lee, Yun-Shien

    2012-01-01

    The direct sequencing of PCR products generates heterozygous base-calling fluorescence chromatograms that are useful for identifying single-nucleotide polymorphisms (SNPs), insertion-deletions (indels), short tandem repeats (STRs), and paralogous genes. Indels and STRs can be easily detected using the currently available Indelligent or ShiftDetector programs, which do not search reference sequences. However, the detection of other genomic variants remains a challenge due to the lack of appropriate tools for heterozygous base-calling fluorescence chromatogram data analysis. In this study, we developed a free web-based program, Mixed Sequence Reader (MSR), which can directly analyze heterozygous base-calling fluorescence chromatogram data in .abi file format using comparisons with reference sequences. The heterozygous sequences are identified as two distinct sequences and aligned with reference sequences. Our results showed that MSR may be used to (i) physically locate indel and STR sequences and determine STR copy number by searching NCBI reference sequences; (ii) predict combinations of microsatellite patterns using the Federal Bureau of Investigation Combined DNA Index System (CODIS); (iii) determine human papilloma virus (HPV) genotypes by searching current viral databases in cases of double infections; (iv) estimate the copy number of paralogous genes, such as β-defensin 4 (DEFB4) and its paralog HSPDP3. PMID:22778697

  12. Mixed Sequence Reader: A Program for Analyzing DNA Sequences with Heterozygous Base Calling

    Directory of Open Access Journals (Sweden)

    Chun-Tien Chang

    2012-01-01

    Full Text Available The direct sequencing of PCR products generates heterozygous base-calling fluorescence chromatograms that are useful for identifying single-nucleotide polymorphisms (SNPs, insertion-deletions (indels, short tandem repeats (STRs, and paralogous genes. Indels and STRs can be easily detected using the currently available Indelligent or ShiftDetector programs, which do not search reference sequences. However, the detection of other genomic variants remains a challenge due to the lack of appropriate tools for heterozygous base-calling fluorescence chromatogram data analysis. In this study, we developed a free web-based program, Mixed Sequence Reader (MSR, which can directly analyze heterozygous base-calling fluorescence chromatogram data in .abi file format using comparisons with reference sequences. The heterozygous sequences are identified as two distinct sequences and aligned with reference sequences. Our results showed that MSR may be used to (i physically locate indel and STR sequences and determine STR copy number by searching NCBI reference sequences; (ii predict combinations of microsatellite patterns using the Federal Bureau of Investigation Combined DNA Index System (CODIS; (iii determine human papilloma virus (HPV genotypes by searching current viral databases in cases of double infections; (iv estimate the copy number of paralogous genes, such as β-defensin 4 (DEFB4 and its paralog HSPDP3.

  13. Mixed sequence reader: a program for analyzing DNA sequences with heterozygous base calling.

    Science.gov (United States)

    Chang, Chun-Tien; Tsai, Chi-Neu; Tang, Chuan Yi; Chen, Chun-Houh; Lian, Jang-Hau; Hu, Chi-Yu; Tsai, Chia-Lung; Chao, Angel; Lai, Chyong-Huey; Wang, Tzu-Hao; Lee, Yun-Shien

    2012-01-01

    The direct sequencing of PCR products generates heterozygous base-calling fluorescence chromatograms that are useful for identifying single-nucleotide polymorphisms (SNPs), insertion-deletions (indels), short tandem repeats (STRs), and paralogous genes. Indels and STRs can be easily detected using the currently available Indelligent or ShiftDetector programs, which do not search reference sequences. However, the detection of other genomic variants remains a challenge due to the lack of appropriate tools for heterozygous base-calling fluorescence chromatogram data analysis. In this study, we developed a free web-based program, Mixed Sequence Reader (MSR), which can directly analyze heterozygous base-calling fluorescence chromatogram data in .abi file format using comparisons with reference sequences. The heterozygous sequences are identified as two distinct sequences and aligned with reference sequences. Our results showed that MSR may be used to (i) physically locate indel and STR sequences and determine STR copy number by searching NCBI reference sequences; (ii) predict combinations of microsatellite patterns using the Federal Bureau of Investigation Combined DNA Index System (CODIS); (iii) determine human papilloma virus (HPV) genotypes by searching current viral databases in cases of double infections; (iv) estimate the copy number of paralogous genes, such as β-defensin 4 (DEFB4) and its paralog HSPDP3.

  14. From DNA sequence to transcriptional behaviour: a quantitative approach.

    Science.gov (United States)

    Segal, Eran; Widom, Jonathan

    2009-07-01

    Complex transcriptional behaviours are encoded in the DNA sequences of gene regulatory regions. Advances in our understanding of these behaviours have been recently gained through quantitative models that describe how molecules such as transcription factors and nucleosomes interact with genomic sequences. An emerging view is that every regulatory sequence is associated with a unique binding affinity landscape for each molecule and, consequently, with a unique set of molecule-binding configurations and transcriptional outputs. We present a quantitative framework based on existing methods that unifies these ideas. This framework explains many experimental observations regarding the binding patterns of factors and nucleosomes and the dynamics of transcriptional activation. It can also be used to model more complex phenomena such as transcriptional noise and the evolution of transcriptional regulation.

  15. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    Science.gov (United States)

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

  16. Chimeric TALE recombinases with programmable DNA sequence specificity.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Fuller, Roberta P; Barbas, Carlos F

    2012-11-01

    Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

  17. Bacterial DNA Sequence Compression Models Using Artificial Neural Networks

    Directory of Open Access Journals (Sweden)

    Armando J. Pinho

    2013-08-01

    Full Text Available It is widely accepted that the advances in DNA sequencing techniques have contributed to an unprecedented growth of genomic data. This fact has increased the interest in DNA compression, not only from the information theory and biology points of view, but also from a practical perspective, since such sequences require storage resources. Several compression methods exist, and particularly, those using finite-context models (FCMs have received increasing attention, as they have been proven to effectively compress DNA sequences with low bits-per-base, as well as low encoding/decoding time-per-base. However, the amount of run-time memory required to store high-order finite-context models may become impractical, since a context-order as low as 16 requires a maximum of 17.2 x 109 memory entries. This paper presents a method to reduce such a memory requirement by using a novel application of artificial neural networks (ANN to build such probabilistic models in a compact way and shows how to use them to estimate the probabilities. Such a system was implemented, and its performance compared against state-of-the art compressors, such as XM-DNA (expert model and FCM-Mx (mixture of finite-context models , as well as with general-purpose compressors. Using a combination of order-10 FCM and ANN, similar encoding results to those of FCM, up to order-16, are obtained using only 17 megabytes of memory, whereas the latter, even employing hash-tables, uses several hundreds of megabytes.

  18. A comparison of rice chloroplast genomes

    DEFF Research Database (Denmark)

    Tang, Jiabin; Xia, Hong'ai; Cao, Mengliang

    2004-01-01

    ), which are both parental varieties of the super-hybrid rice, LYP9. Based on the patterns of high sequence coverage, we partitioned chloroplast sequence variations into two classes, intravarietal and intersubspecific polymorphisms. Intravarietal polymorphisms refer to variations within 93-11 or PA64S...... to intersubspecific polymorphisms. In our study, we found that the intersubspecific variations of 93-11 (indica) and PA64S (japonica) chloroplast genomes consisted of 72 single nucleotide polymorphisms and 27 insertions or deletions. The intersubspecific polymorphism rates between 93-11 and PA64S were 0.......05% for single nucleotide polymorphisms and 0.02% for insertions or deletions, nearly 8 and 10 times lower than their respective nuclear genomes. Based on the total number of nucleotide substitutions between the two chloroplast genomes, we dated the divergence of indica and japonica chloroplast genomes...

  19. Stability of capillary gels for automated sequencing of DNA.

    Science.gov (United States)

    Swerdlow, H; Dew-Jager, K E; Brady, K; Grey, R; Dovichi, N J; Gesteland, R

    1992-08-01

    Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Choosing the best heuristic for seeded alignment of DNA sequences

    Directory of Open Access Journals (Sweden)

    Buhler Jeremy

    2006-03-01

    Full Text Available Abstract Background Seeded alignment is an important component of algorithms for fast, large-scale DNA similarity search. A good seed matching heuristic can reduce the execution time of genomic-scale sequence comparison without degrading sensitivity. Recently, many types of seed have been proposed to improve on the performance of traditional contiguous seeds as used in, e.g., NCBI BLASTN. Choosing among these seed types, particularly those that use information besides the presence or absence of matching residue pairs, requires practical guidance based on a rigorous comparison, including assessment of sensitivity, specificity, and computational efficiency. This work performs such a comparison, focusing on alignments in DNA outside widely studied coding regions. Results We compare seeds of several types, including those allowing transition mutations rather than matches at fixed positions, those allowing transitions at arbitrary positions ("BLASTZ" seeds, and those using a more general scoring matrix. For each seed type, we use an extended version of our Mandala seed design software to choose seeds with optimized sensitivity for various levels of specificity. Our results show that, on a test set biased toward alignments of noncoding DNA, transition information significantly improves seed performance, while finer distinctions between different types of mismatches do not. BLASTZ seeds perform especially well. These results depend on properties of our test set that are not shared by EST-based test sets with a strong bias toward coding DNA. Conclusion Practical seed design requires careful attention to the properties of the alignments being sought. For noncoding DNA sequences, seeds that use transition information, especially BLASTZ-style seeds, are particularly useful. The Mandala seed design software can be found at http://www.cse.wustl.edu/~yanni/mandala/.

  1. Complete genome sequence of mitochondrial DNA (mtDNA) of Chlorella sorokiniana.

    Science.gov (United States)

    Orsini, Massimiliano; Costelli, Cristina; Malavasi, Veronica; Cusano, Roberto; Concas, Alessandro; Angius, Andrea; Cao, Giacomo

    2016-01-01

    The complete sequence of mitochondrial genome of the Chlorella sorokiniana strain (SAG 111-8 k) is presented in this work. Within the Chlorella genus, it represents the second species with a complete sequenced and annotated mitochondrial genome (GenBank accession no. KM241869). The genome consists of circular chromosomes of 52,528 bp and encodes a total of 31 protein coding genes, 3 rRNAs and 26 tRNAs. The overall AT contents of the C. sorokiniana mtDNA is 70.89%, while the coding sequence is of 97.4%.

  2. Sequencing of mitochondrial HV1 and HV2 DNA with length heteroplasmy

    DEFF Research Database (Denmark)

    Rasmussen, E. Michael; Eriksen, Birthe; Larsen, Hans Jakob;

    2003-01-01

    This study presents a fast method for sequencing the poly C/G regions in HV1 and HV2 in the mitochondrial DNA (mtDNA)......This study presents a fast method for sequencing the poly C/G regions in HV1 and HV2 in the mitochondrial DNA (mtDNA)...

  3. Developmentally programmed excision of internal DNA sequences in Paramecium aurelia.

    Science.gov (United States)

    Gratias, A; Bétermier, M

    2001-01-01

    The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.

  4. Genetic variability of Taenia saginata inferred from mitochondrial DNA sequences.

    Science.gov (United States)

    Rostami, Sima; Salavati, Reza; Beech, Robin N; Babaei, Zahra; Sharbatkhori, Mitra; Harandi, Majid Fasihi

    2015-04-01

    Taenia saginata is an important tapeworm, infecting humans in many parts of the world. The present study was undertaken to identify inter- and intraspecific variation of T. saginata isolated from cattle in different parts of Iran using two mitochondrial CO1 and 12S rRNA genes. Up to 105 bovine specimens of T. saginata were collected from 20 slaughterhouses in three provinces of Iran. DNA were extracted from the metacestode Cysticercus bovis. After PCR amplification, sequencing of CO1 and 12S rRNA genes were carried out and two phylogenetic analyses of the sequence data were generated by Bayesian inference on CO1 and 12S rRNA sequences. Sequence analyses of CO1 and 12S rRNA genes showed 11 and 29 representative profiles respectively. The level of pairwise nucleotide variation between individual haplotypes of CO1 gene was 0.3-2.4% while the overall nucleotide variation among all 11 haplotypes was 4.6%. For 12S rRNA sequence data, level of pairwise nucleotide variation was 0.2-2.5% and the overall nucleotide variation was determined as 5.8% among 29 haplotypes of 12S rRNA gene. Considerable genetic diversity was found in both mitochondrial genes particularly in 12S rRNA gene.

  5. DNA sequencing technology, walking with modular primers. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Ulanovsky, L.

    1996-12-31

    The success of the Human Genome Project depends on the development of adequate technology for rapid and inexpensive DNA sequencing, which will also benefit biomedical research in general. The authors are working on DNA technologies that eliminate primer synthesis, the main bottleneck in sequencing by primer walking. They have developed modular primers that are assembled from three 5-mer, 6-mer or 7-mer modules selected from a presynthesized library of as few as 1,000 oligonucleotides ({double_bond}4, {double_bond}5, {double_bond}7). The three modules anneal contiguously at the selected template site and prime there uniquely, even though each is not unique for the most part when used alone. This technique is expected to speed up primer walking 30 to 50 fold, and reduce the sequencing cost by a factor of 5 to 15. Time and expensive will be saved on primer synthesis itself and even more so due to closed-loop automation of primer walking, made possible by the instant availability of primers. Apart from saving time and cost, closed-loop automation would also minimize the errors and complications associated with human intervention between the walks. The author has also developed two additional approaches to primer-library based sequencing. One involves a branched structure of modular primers which has a distinctly different mechanism of achieving priming specificity. The other introduces the concept of ``Differential Extension with Nucleotide Subsets`` as an approach increasing priming specificity, priming strength and allowing cycle sequencing. These approaches are expected to be more robust than the original version of the modular primer technique.

  6. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

    Directory of Open Access Journals (Sweden)

    Petitjean Céline

    2012-11-01

    Full Text Available Abstract Background In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants. Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Results Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. Conclusions We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  7. Genetic population structure of the alpine species Rhododendron pseudochrysanthum sensu lato (Ericaceae inferred from chloroplast and nuclear DNA

    Directory of Open Access Journals (Sweden)

    Wang Wei-Kuang

    2011-04-01

    Full Text Available Abstract Background A complex of incipient species with different degrees of morphological or ecological differentiation provides an ideal model for studying species divergence. We examined the phylogeography and the evolutionary history of the Rhododendron pseudochrysanthum s. l. Results Systematic inconsistency was detected between gene genealogies of the cpDNA and nrDNA. Rooted at R. hyperythrum and R. formosana, both trees lacked reciprocal monophyly for all members of the complex. For R. pseudochrysanthum s.l., the spatial distribution of the cpDNA had a noteworthy pattern showing high genetic differentiation (FST = 0.56-0.72 between populations in the Yushan Mountain Range and populations of the other mountain ranges. Conclusion Both incomplete lineage sorting and interspecific hybridization/introgression may have contributed to the lack of monophyly among R. hyperythrum, R. formosana and R. pseudochrysanthum s.l. Independent colonizations, plus low capabilities of seed dispersal in current environments, may have resulted in the genetic differentiation between populations of different mountain ranges. At the population level, the populations of Central, and Sheishan Mountains may have undergone postglacial demographic expansion, while populations of the Yushan Mountain Range are likely to have remained stable ever since the colonization. In contrast, the single population of the Alishan Mountain Range with a fixed cpDNA haplotype may have experienced bottleneck/founder's events.

  8. Discovering motifs in ranked lists of DNA sequences.

    Directory of Open Access Journals (Sweden)

    Eran Eden

    2007-03-01

    Full Text Available Computational methods for discovery of sequence elements that are enriched in a target set compared with a background set are fundamental in molecular biology research. One example is the discovery of transcription factor binding motifs that are inferred from ChIP-chip (chromatin immuno-precipitation on a microarray measurements. Several major challenges in sequence motif discovery still require consideration: (i the need for a principled approach to partitioning the data into target and background sets; (ii the lack of rigorous models and of an exact p-value for measuring motif enrichment; (iii the need for an appropriate framework for accounting for motif multiplicity; (iv the tendency, in many of the existing methods, to report presumably significant motifs even when applied to randomly generated data. In this paper we present a statistical framework for discovering enriched sequence elements in ranked lists that resolves these four issues. We demonstrate the implementation of this framework in a software application, termed DRIM (discovery of rank imbalanced motifs, which identifies sequence motifs in lists of ranked DNA sequences. We applied DRIM to ChIP-chip and CpG methylation data and obtained the following results. (i Identification of 50 novel putative transcription factor (TF binding sites in yeast ChIP-chip data. The biological function of some of them was further investigated to gain new insights on transcription regulation networks in yeast. For example, our discoveries enable the elucidation of the network of the TF ARO80. Another finding concerns a systematic TF binding enhancement to sequences containing CA repeats. (ii Discovery of novel motifs in human cancer CpG methylation data. Remarkably, most of these motifs are similar to DNA sequence elements bound by the Polycomb complex that promotes histone methylation. Our findings thus support a model in which histone methylation and CpG methylation are mechanistically linked

  9. A MapReduce Framework for DNA Sequencing Data Processing

    Directory of Open Access Journals (Sweden)

    Samy Ghoneimy

    2016-12-01

    Full Text Available Genomics and Next Generation Sequencers (NGS like Illumina Hiseq produce data in the order of ‎‎200 billion base pairs in a single one-week run for a 60x human genome coverage, which ‎requires modern high-throughput experimental technologies that can ‎only be tackled with high performance computing (HPC and specialized software algorithms called ‎‎“short read aligners”. This paper focuses on the implementation of the DNA sequencing as a set of MapReduce programs that will accept a DNA data set as a FASTQ file and finally generate a VCF (variant call format file, which has variants for a given DNA data set. In this paper MapReduce/Hadoop along with Burrows-Wheeler Aligner (BWA, Sequence Alignment/Map (SAM ‎tools, are fully utilized to provide various utilities for manipulating alignments, including sorting, merging, indexing, ‎and generating alignments. The Map-Sort-Reduce process is designed to be suited for a Hadoop framework in ‎which each cluster is a traditional N-node Hadoop cluster to utilize all of the Hadoop features like HDFS, program ‎management and fault tolerance. The Map step performs multiple instances of the short read alignment algorithm ‎‎(BoWTie that run in parallel in Hadoop. The ordered list of the sequence reads are used as input tuples and the ‎output tuples are the alignments of the short reads. In the Reduce step many parallel instances of the Short ‎Oligonucleotide Analysis Package for SNP (SOAPsnp algorithm run in the cluster. Input tuples are sorted ‎alignments for a partition and the output tuples are SNP calls. Results are stored via HDFS, and then archived in ‎SOAPsnp format. ‎ The proposed framework enables extremely fast discovering somatic mutations, inferring population genetical ‎parameters, and performing association tests directly based on sequencing data without explicit genotyping or ‎linkage-based imputation. It also demonstrate that this method achieves comparable

  10. PCR master mixes harbour murine DNA sequences. Caveat emptor!

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    Philip W Tuke

    Full Text Available BACKGROUND: XMRV is the most recently described retrovirus to be found in Man, firstly in patients with prostate cancer (PC and secondly in 67% of patients with chronic fatigue syndrome (CFS and 3.7% of controls. Both disease associations remain contentious. Indeed, a recent publication has concluded that "XMRV is unlikely to be a human pathogen". Subsequently related but different polytropic MLV (pMLV sequences were also reported from the blood of 86.5% of patients with CFS. and 6.8% of controls. Consequently we decided to investigate blood donors for evidence of XMRV/pMLV. METHODOLOGY/PRINCIPAL FINDINGS: Testing of cDNA prepared from the whole blood of 80 random blood donors, generated gag PCR signals from two samples (7C and 9C. These had previously tested negative for XMRV by two other PCR based techniques. To test whether the PCR mix was the source of these sequences 88 replicates of water were amplified using Invitrogen Platinum Taq (IPT and Applied Biosystems Taq Gold LD (ABTG. Four gag sequences (2D, 3F, 7H, 12C were generated with the IPT, a further sequence (12D by ABTG re-amplification of an IPT first round product. Sequence comparisons revealed remarkable similarities between these sequences, endogeous MLVs and the pMLV sequences reported in patients with CFS. CONCLUSIONS/SIGNIFICANCE: Methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

  11. Peptide Synthesis on a Next-Generation DNA Sequencing Platform.

    Science.gov (United States)

    Svensen, Nina; Peersen, Olve B; Jaffrey, Samie R

    2016-09-01

    Methods for displaying large numbers of peptides on solid surfaces are essential for high-throughput characterization of peptide function and binding properties. Here we describe a method for converting the >10(7) flow cell-bound clusters of identical DNA strands generated by the Illumina DNA sequencing technology into clusters of complementary RNA, and subsequently peptide clusters. We modified the flow-cell-bound primers with ribonucleotides thus enabling them to be used by poliovirus polymerase 3D(pol) . The primers hybridize to the clustered DNA thus leading to RNA clusters. The RNAs fold into functional protein- or small molecule-binding aptamers. We used the mRNA-display approach to synthesize flow-cell-tethered peptides from these RNA clusters. The peptides showed selective binding to cognate antibodies. The methods described here provide an approach for using DNA clusters to template peptide synthesis on an Illumina flow cell, thus providing new opportunities for massively parallel peptide-based assays.

  12. Mitochondrial DNA sequence analysis of patients with 'atypical psychosis'.

    Science.gov (United States)

    Kazuno, An-A; Munakata, Kae; Mori, Kanako; Tanaka, Masashi; Nanko, Shinichiro; Kunugi, Hiroshi; Umekage, Tadashi; Tochigi, Mamoru; Kohda, Kazuhisa; Sasaki, Tsukasa; Akiyama, Tsuyoshi; Washizuka, Shinsuke; Kato, Nobumasa; Kato, Tadafumi

    2005-08-01

    Although classical psychopathological studies have shown the presence of an independent diagnostic category, 'atypical psychosis', most psychotic patients are currently classified into two major diagnostic categories, schizophrenia and bipolar disorder, by the Diagnostic and Statistical Manual of Mental Disorders (4th edn; DSM-IV) criteria. 'Atypical psychosis' is characterized by acute confusion without systematic delusion, emotional instability, and psychomotor excitement or stupor. Such clinical features resemble those seen in organic mental syndrome, and differential diagnosis is often difficult. Because patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) sometimes show organic mental disorder, 'atypical psychosis' may be caused by mutations of mitochondrial DNA (mtDNA) in some patients. In the present study whole mtDNA was sequenced for seven patients with various psychotic disorders, who could be categorized as 'atypical psychosis'. None of them had known mtDNA mutations pathogenic for mitochondrial encephalopathy. Two of seven patients belonged to a subhaplogroup F1b1a with low frequency. These results did not support the hypothesis that clinical presentation of some patients with 'atypical psychosis' is a reflection of subclinical mitochondrial encephalopathy. However, the subhaplogroup F1b1a may be a good target for association study of 'atypical psychosis'.

  13. Entropy and long-range correlations in DNA sequences.

    Science.gov (United States)

    Melnik, S S; Usatenko, O V

    2014-12-01

    We analyze the structure of DNA molecules of different organisms by using the additive Markov chain approach. Transforming nucleotide sequences into binary strings, we perform statistical analysis of the corresponding "texts". We develop the theory of N-step additive binary stationary ergodic Markov chains and analyze their differential entropy. Supposing that the correlations are weak we express the conditional probability function of the chain by means of the pair correlation function and represent the entropy as a functional of the pair correlator. Since the model uses two point correlators instead of probability of block occurring, it makes possible to calculate the entropy of subsequences at much longer distances than with the use of the standard methods. We utilize the obtained analytical result for numerical evaluation of the entropy of coarse-grained DNA texts. We believe that the entropy study can be used for biological classification of living species.

  14. Phylogenetic position of Schnabelia, a genus endemic to China: Evidence from sequences of cpDNA matK gene and nrDNA ITS regions

    Institute of Scientific and Technical Information of China (English)

    SHI Suhua; DU Yaqing; D. E. Boufford; GONG Xun; HUANG Yelin; HE Hanghang; ZHONG Yang

    2003-01-01

    The chloroplast gene matK and the internal transcribed spacers (ITS) of the nuclear ribosomal DNA from Schnabelia, a genus endemic to China, and 6 genera of Verbenaceae and 13 genera of Lamiaceae were sequenced. The phylogenetic signal and validity outgroups were measured and evaluated by means of the relatively apparent synapomorphy analysis (RASA). Independent and combined phylogenetic analyses for the matK and ITS sequences were performed using the maximum parsimony (MP), neighbor- joining (NJ) and maximum likelihood (ML) methods, indicating that Schnabelia oligophylla and Caryopteris terniflora form a sister-group relationship. The Caryopteris complex is not shown to be a monophyly because Trichostema, C. paniculata and C. forrestii are paraphyletic to the clade containing the remaining members of the complex. A monophyly of Ajugoideae proposed by Cantino et al., including 8 genera in this study, is strongly supported and the closest relatives of Schnabelia are in the Ajugoideae (Lamiaceae), especially near Caryopteris terniflora. The polygenetic analyses also showed that the genera of Lamiaceae and Verbenaceae sampled in this tudy are phylogenetically mixed and the genus Avicennia is distant to other genera of Verbenaceae. RASA and combined analysis can be used as effective approaches to determining the relationships among phylogenetically complex groups.

  15. New scoring schema for finding motifs in DNA Sequences

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    Nowzari-Dalini Abbas

    2009-03-01

    Full Text Available Abstract Background Pattern discovery in DNA sequences is one of the most fundamental problems in molecular biology with important applications in finding regulatory signals and transcription factor binding sites. An important task in this problem is to search (or predict known binding sites in a new DNA sequence. For this reason, all subsequences of the given DNA sequence are scored based on an scoring function and the prediction is done by selecting the best score. By assuming no dependency between binding site base positions, most of the available tools for known binding site prediction are designed. Recently Tomovic and Oakeley investigated the statistical basis for either a claim of dependence or independence, to determine whether such a claim is generally true, and they presented a scoring function for binding site prediction based on the dependency between binding site base positions. Our primary objective is to investigate the scoring functions which can be used in known binding site prediction based on the assumption of dependency or independency in binding site base positions. Results We propose a new scoring function based on the dependency between all positions in biding site base positions. This scoring function uses joint information content and mutual information as a measure of dependency between positions in transcription factor binding site. Our method for modeling dependencies is simply an extension of position independency methods. We evaluate our new scoring function on the real data sets extracted from JASPAR and TRANSFAC data bases, and compare the obtained results with two other well known scoring functions. Conclusion The results demonstrate that the new approach improves known binding site discovery and show that the joint information content and mutual information provide a better and more general criterion to investigate the relationships between positions in the TFBS. Our scoring function is formulated by simple

  16. Next-generation DNA barcoding: using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens.

    Science.gov (United States)

    Shokralla, Shadi; Gibson, Joel F; Nikbakht, Hamid; Janzen, Daniel H; Hallwachs, Winnie; Hajibabaei, Mehrdad

    2014-09-01

    DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large-scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next-generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high-target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next-generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10-mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full-length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full-length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next-generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.

  17. Amplification of Chloroplast DNA Using the Polymerase Chain Reaction (PCR): A Practical Activity for Secondary School Students

    Science.gov (United States)

    Hamilton, Kenny; Barfoot, Jan; Crawford, Kathleen E.; Simpson, Craig G.; Beaumont, Paul C.; Bownes, Mary

    2006-01-01

    We describe a polymerase chain reaction (PCR) protocol suitable for use in secondary schools and colleges. This PCR protocol can be used to investigate genetic variation between plants. The protocol makes use of primers which are complementary to sequences of nucleotides that are highly conserved across different plant genera. The regions of…

  18. Indirect readout of DNA sequence by p22 repressor: roles of DNA and protein functional groups in modulating DNA conformation.

    Science.gov (United States)

    Harris, Lydia-Ann; Watkins, Derrick; Williams, Loren Dean; Koudelka, Gerald B

    2013-01-09

    The repressor of bacteriophage P22 (P22R) discriminates between its various DNA binding sites by sensing the identity of non-contacted base pairs at the center of its binding site. The "indirect readout" of these non-contacted bases is apparently based on DNA's sequence-dependent conformational preferences. The structures of P22R-DNA complexes indicate that the non-contacted base pairs at the center of the binding site are in the B' state. This finding suggests that indirect readout and therefore binding site discrimination depend on P22R's ability to either sense and/or impose the B' state on the non-contacted bases of its binding sites. We show here that the affinity of binding sites for P22R depends on the tendency of the central bases to assume the B'-DNA state. Furthermore, we identify functional groups in the minor groove of the non-contacted bases as the essential modulators of indirect readout by P22R. In P22R-DNA complexes, the negatively charged E44 and E48 residues are provocatively positioned near the negatively charged DNA phosphates of the non-contacted nucleotides. The close proximity of the negatively charged groups on protein and DNA suggests that electrostatics may play a key role in the indirect readout process. Changing either of two negatively charged residues to uncharged residues eliminates the ability of P22R to impose structural changes on DNA and to recognize non-contacted base sequence. These findings suggest that these negatively charged amino acids function to force the P22R-bound DNA into the B' state and therefore play a key role in indirect readout by P22R.

  19. Mitochondrial DNA sequence variation in the Anatolian Peninsula (Turkey)

    Indian Academy of Sciences (India)

    Hatice Mergen; Reyhan Öner; Cihan Öner

    2004-04-01

    Throughout human history, the region known today as the Anatolian peninsula (Turkey) has served as a junction connecting the Middle East, Europe and Central Asia, and, thus, has been subject to major population movements. The present study is undertaken to obtain information about the distribution of the existing mitochondrial D-loop sequence variations in the Turkish population of Anatolia. A few studies have previously reported mtDNA sequences in Turks. We attempted to extend these results by analysing a cohort that is not only larger, but also more representative of the Turkish population living in Anatolia. In order to obtain a descriptive picture for the phylogenetic distribution of the mitochondrial genome within Turkey, we analysed mitochondrial D-loop region sequence variations in 75 individuals from different parts of Anatolia by direct sequencing. Analysis of the two hypervariable segments within the noncoding region of the mitochondrial genome revealed the existence of 81 nucleotide mutations at 79 sites. The neighbour-joining tree of Kimura’s distance matrix has revealed the presence of six main clusters, of which H and U are the most common. The data obtained are also compared with several European and Turkic Central Asian populations.

  20. Complete chloroplast genome of Sedum sarmentosum and chloroplast genome evolution in Saxifragales.

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    Wenpan Dong

    Full Text Available Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC region, 16.670 bp of a small single-copy (SSC region, and a pair of 25,783 bp sequences of inverted repeats (IRs.The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.

  1. Autoantigenic proteins that bind recombinogenic sequences in Epstein-Barr virus and cellular DNA.

    OpenAIRE

    1994-01-01

    We have identified conserved autoantigenic cellular proteins that bind to G-rich sequence motifs in recombinogenic regions of Epstein-Barr virus (EBV) DNA. This binding activity, called TRBP, recognizes the EBV terminal repeats, a locus responsible for interconversion of linear and circular EBV DNA. We found that TRBP also binds to EBV DNA sequences involved in deletion of EBNA2, a gene product required for immortalization. We show that TRBP binds sequences present in repetitive cellular DNA,...

  2. Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements

    Directory of Open Access Journals (Sweden)

    Pareja Eduardo

    2006-03-01

    Full Text Available Abstract Background Mobile elements are involved in genomic rearrangements and virulence acquisition, and hence, are important elements in bacterial genome evolution. The insertion of some specific Insertion Sequences had been associated with repetitive extragenic palindromic (REP elements. Considering that there are a sufficient number of available genomes with described REPs, and exploiting the advantage of the traceability of transposition events in genomes, we decided to exhaustively analyze the relationship between REP sequences and mobile elements. Results This global multigenome study highlights the importance of repetitive extragenic palindromic elements as target sequences for transposases. The study is based on the analysis of the DNA regions surrounding the 981 instances of Insertion Sequence elements with respect to the positioning of REP sequences in the 19 available annotated microbial genomes corresponding to species of bacteria with reported REP sequences. This analysis has allowed the detection of the specific insertion into REP sequences for ISPsy8 in Pseudomonas syringae DC3000, ISPa11 in P. aeruginosa PA01, ISPpu9 and ISPpu10 in P. putida KT2440, and ISRm22 and ISRm19 in Sinorhizobium meliloti 1021 genome. Preference for insertion in extragenic spaces with REP sequences has also been detected for ISPsy7 in P. syringae DC3000, ISRm5 in S. meliloti and ISNm1106 in Neisseria meningitidis MC58 and Z2491 genomes. Probably, the association with REP elements that we have detected analyzing genomes is only the tip of the iceberg, and this association could be even more frequent in natural isolates. Conclusion Our findings characterize REP elements as hot spots for transposition and reinforce the relationship between REP sequences and genomic plasticity mediated by mobile elements. In addition, this study defines a subset of REP-recognizer transposases with high target selectivity that can be useful in the development of new tools for

  3. Noncontinuously binding loop-out primers for avoiding problematic DNA sequences in PCR and sanger sequencing.

    Science.gov (United States)

    Sumner, Kelli; Swensen, Jeffrey J; Procter, Melinda; Jama, Mohamed; Wooderchak-Donahue, Whitney; Lewis, Tracey; Fong, Michael; Hubley, Lindsey; Schwarz, Monica; Ha, Youna; Paul, Eleri; Brulotte, Benjamin; Lyon, Elaine; Bayrak-Toydemir, Pinar; Mao, Rong; Pont-Kingdon, Genevieve; Best, D Hunter

    2014-09-01

    We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.

  4. Two Dimensional Yau-Hausdorff Distance with Applications on Comparison of DNA and Protein Sequences.

    Directory of Open Access Journals (Sweden)

    Kun Tian

    Full Text Available Comparing DNA or protein sequences plays an important role in the functional analysis of genomes. Despite many methods available for sequences comparison, few methods retain the information content of sequences. We propose a new approach, the Yau-Hausdorff method, which considers all translations and rotations when seeking the best match of graphical curves of DNA or protein sequences. The complexity of this method is lower than that of any other two dimensional minimum Hausdorff algorithm. The Yau-Hausdorff method can be used for measuring the similarity of DNA sequences based on two important tools: the Yau-Hausdorff distance and graphical representation of DNA sequences. The graphical representations of DNA sequences conserve all sequence information and the Yau-Hausdorff distance is mathematically proved as a true metric. Therefore, the proposed distance can preciously measure the similarity of DNA sequences. The phylogenetic analyses of DNA sequences by the Yau-Hausdorff distance show the accuracy and stability of our approach in similarity comparison of DNA or protein sequences. This study demonstrates that Yau-Hausdorff distance is a natural metric for DNA and protein sequences with high level of stability. The approach can be also applied to similarity analysis of protein sequences by graphic representations, as well as general two dimensional shape matching.

  5. Two Dimensional Yau-Hausdorff Distance with Applications on Comparison of DNA and Protein Sequences.

    Science.gov (United States)

    Tian, Kun; Yang, Xiaoqian; Kong, Qin; Yin, Changchuan; He, Rong L; Yau, Stephen S-T

    2015-01-01

    Comparing DNA or protein sequences plays an important role in the functional analysis of genomes. Despite many methods available for sequences comparison, few methods retain the information content of sequences. We propose a new approach, the Yau-Hausdorff method, which considers all translations and rotations when seeking the best match of graphical curves of DNA or protein sequences. The complexity of this method is lower than that of any other two dimensional minimum Hausdorff algorithm. The Yau-Hausdorff method can be used for measuring the similarity of DNA sequences based on two important tools: the Yau-Hausdorff distance and graphical representation of DNA sequences. The graphical representations of DNA sequences conserve all sequence information and the Yau-Hausdorff distance is mathematically proved as a true metric. Therefore, the proposed distance can preciously measure the similarity of DNA sequences. The phylogenetic analyses of DNA sequences by the Yau-Hausdorff distance show the accuracy and stability of our approach in similarity comparison of DNA or protein sequences. This study demonstrates that Yau-Hausdorff distance is a natural metric for DNA and protein sequences with high level of stability. The approach can be also applied to similarity analysis of protein sequences by graphic representations, as well as general two dimensional shape matching.

  6. A trnI_CAU triplication event in the complete chloroplast genome of Paris verticillata M.Bieb. (Melanthiaceae, Liliales).

    Science.gov (United States)

    Do, Hoang Dang Khoa; Kim, Jung Sung; Kim, Joo-Hwan

    2014-06-19

    The chloroplast is an essential plant organelle responsible for photosynthesis. Gene duplication, relocation, and loss in the chloroplast genome (cpDNA) are useful for exploring the evolution and phylogeny of plant species. In this study, the complete chloroplast genome of Paris verticillata was sequenced using the 454 sequencing system and Sanger sequencing method to trace the evolutionary pattern in the tribe Parideae of the family Melanthiaceae (Liliales). The circular double-stranded cpDNA of P. verticillata (157,379 bp) consists of two inverted repeat regions each of 28,373 bp, a large single copy of 82,726 bp, and a small single copy of 17,907 bp. Gene content and order are generally similar to the previously reported cpDNA sequences within the order Liliales. However, we found that trnI_CAU was triplicated in P. verticillata. In addition, cemA is suspected to be a pseudogene due to the presence of internal stop codons created by poly(A) insertion and single small CA repeats. Such changes were not found in previously examined cpDNAs of the Melanthiaceae or other families of the Liliales, suggesting that such features are unique to the tribe Parideae of Melanthiaceae. The characteristics of P. verticillata cpDNA will provide useful information for uncovering the evolution within Paris and for further research of plastid genome evolution and phylogenetic studies in Liliales.

  7. Statistical methods for detecting periodic fragments in DNA sequence data

    Directory of Open Access Journals (Sweden)

    Ying Hua

    2011-04-01

    Full Text Available Abstract Background Period 10 dinucleotides are structurally and functionally validated factors that influence the ability of DNA to form nucleosomes, histone core octamers. Robust identification of periodic signals in DNA sequences is therefore required to understand nucleosome organisation in genomes. While various techniques for identifying periodic components in genomic sequences have been proposed or adopted, the requirements for such techniques have not been considered in detail and confirmatory testing for a priori specified periods has not been developed. Results We compared the estimation accuracy and suitability for confirmatory testing of autocorrelation, discrete Fourier transform (DFT, integer period discrete Fourier transform (IPDFT and a previously proposed Hybrid measure. A number of different statistical significance procedures were evaluated but a blockwise bootstrap proved superior. When applied to synthetic data whose period-10 signal had been eroded, or for which the signal was approximately period-10, the Hybrid technique exhibited superior properties during exploratory period estimation. In contrast, confirmatory testing using the blockwise bootstrap procedure identified IPDFT as having the greatest statistical power. These properties were validated on yeast sequences defined from a ChIP-chip study where the Hybrid metric confirmed the expected dominance of period-10 in nucleosome associated DNA but IPDFT identified more significant occurrences of period-10. Application to the whole genomes of yeast and mouse identified ~ 21% and ~ 19% respectively of these genomes as spanned by period-10 nucleosome positioning sequences (NPS. Conclusions For estimating the dominant period, we find the Hybrid period estimation method empirically to be the most effective for both eroded and approximate periodicity. The blockwise bootstrap was found to be effective as a significance measure, performing particularly well in the problem of

  8. Chloroplast DNA transgresses species boundaries and evolves at variable rates in the California closed-cone pines (Pinus radiata, P. muricata, and P. attenuata).

    Science.gov (United States)

    Hong, Y P; Krupkin, A B; Strauss, S H

    1993-12-01

    We studied phylogenetic relationships among populations and species in the California closed-cone pines (Pinus radiata D. Don, P. attenuata Lemm., and P. muricata D. Don) via chloroplast DNA restriction site analysis. Data on genetic polymorphism within and among 19 populations in the three species were collected using 9 to 20 restriction enzymes and 38 to 384 trees. Because only five clades and extremely low intraclade diversity were found, additional phylogenetic data were collected using a single representative per clade and two outgroup species, P. oocarpa Schiede and P. jeffreyi Loud. In total, 25 restriction enzymes were employed and approximately 2.7 kb surveyed (2.3% of genome). The five clades recognized were Monterey pine, knobcone pine, and the southern, intermediate, and northern races of bishop pine. On the basis of bootstrapping, both Wagner and Dollo parsimony analyses strongly separated the northern and intermediate races of bishop pine from the southern race; knobcone pine from Monterey and bishop pines; and the closed-cone pines from the two outgroups. Approximate divergence times were estimated for the lineages leading to knobcone pine and to the intermediate and northern populations of bishop pine. The position of Monterey pine relative to bishop pine within their monophyletic clade was unresolved. Surprisingly, Montery pine and the southern race of bishop pine were much more similar to one another than was the southern race of bishop pine to its conspecific intermediate and northern races. Both the Monterey and southern bishop pine lineages also evolved severalfold more slowly than did the knobcone pine and intermediate-northern bishop pine lineages.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Diversity of chloroplast genome among local clones of cocoa (Theobroma cacao, L.) from Central Sulawesi

    Science.gov (United States)

    Suwastika, I. Nengah; Pakawaru, Nurul Aisyah; Rifka, Rahmansyah, Muslimin, Ishizaki, Yoko; Cruz, André Freire; Basri, Zainuddin; Shiina, Takashi

    2017-02-01

    Chloroplast genomes typically range in size from 120 to 170 kilo base pairs (kb), which relatively conserved among plant species. Recent evaluation on several species, certain unique regions showed high variability which can be utilized in the phylogenetic analysis. Many fragments of coding regions, introns, and intergenic spacers, such as atpB-rbcL, ndhF, rbcL, rpl16, trnH-psbA, trnL-F, trnS-G, etc., have been used for phylogenetic reconstructions at various taxonomic levels. Based on that status, we would like to analysis the diversity of chloroplast genome within species of local cacao (Theobroma cacao L.) from Central Sulawesi. Our recent data showed, there were more than 20 clones from local farming in Central Sulawesi, and it can be detected based on phenotypic and nuclear-genome-based characterization (RAPD- Random Amplified Polymorphic DNA and SSR- Simple Sequences Repeat) markers. In developing DNA marker for this local cacao, here we also included analysis based on the variation of chloroplast genome. At least several regions such as rpl32-TurnL, it can be considered as chloroplast markers on our local clone of cocoa. Furthermore, we could develop phylogenetic analysis in between clones of cocoa.

  10. Chloroplast phylogeny of asplenioid ferns based on rbcL and trnL-F Spacer sequences (Polypodiidae, aspleniaceae) and its implications for biogeography

    NARCIS (Netherlands)

    Schneider, H.; Russell, S.J.; Cox, C.J.; Bakker, F.T.; Henderson, S.; Rumsey, F.; Barrett, J.; Gibby, M.; Vogel, J.C.

    2004-01-01

    Molecular phylogenies have been generated to investigate relationships among species and putative segregates in Asplenium, one of the largest genera in ferns. Of the ~700 described taxa, 71 are included in a phylogenetic analysis using the chloroplast rbcL gene and trnL-F spacer. Our results support

  11. Patterns of chloroplast DNA polymorphism in the endangered polyploid Centaurea borjae (Asteraceae): Implications for preserving genetic diversity

    Institute of Scientific and Technical Information of China (English)

    Lua LOPEZ; Rodolfo BARREIRO

    2013-01-01

    A previous study with amplified fragment length polymorphism (AFLP) fingerprints found no evidence of genetic impoverishment in the endangered Centaurea borjae and recommended that four management units (MUs) should be designated.Nevertheless,the high ploidy (6x) of this narrow endemic plant suggested that these conclusions should be validated by independent evidence derived from non-nuclear markers.Here,the variable trnT-F region of the plastid genome was sequenced to obtain this new evidence and to provide an historical background for the current genetic structure.Plastid sequences revealed little genetic variation; calling into question the previous conclusion that C.borjae does not undergo genetic impoverishment.By contrast,the conclusion that gene flow must be low was reinforced by the strong genetic differentiation detected among populations using plastid sequences (global FST =0.419).The spatial arrangement of haplotypes and diversity indicate that the populations currently located at the center of the species range are probable sites of long-persistence whereas the remaining sites may have derived from a latter colonization.From a conservation perspective,four populations contributed most to the allelic richness of the plastid genome of the species and should be given priority.Combined with previous AFLP results,these new data recommended that five,instead of four,MUs should be established.Altogether,our study highlights the benefits of combining markers with different modes of inheritance to design accurate conservation guidelines and to obtain clues on the evolutionary processes behind the present-day genetic structures.

  12. Anti-DNA antibodies: Sequencing, cloning, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.

    1992-01-01

    To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

  13. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    2001-01-01

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus whi

  14. Polymorphic DNA sequences and their application in paternity testing; Polimorficzne sekwencje DNA i ich zastosowanie w dochodzeniu spornego ojcostwa

    Energy Technology Data Exchange (ETDEWEB)

    Slomski, R. [Polska Akademia Nauk, Poznan (Poland). Zaklad Genetyki Czlowieka]|[Akademia Rolnicza, Poznan (Poland)]|[Laboratorium Genetyki Molekularnej, Poznan (Poland); Kwiatkowska, J.; Chlebowska, H. [Polska Akademia Nauk, Poznan (Poland). Zaklad Genetyki Czlowieka; Siemieniallo, B. [Akademia Rolnicza, Poznan (Poland); Slomska, M. [Laboratorium Genetyki Molekularnej, Poznan (Poland)

    1994-12-31

    Characteristics of polymorphic sequences of DNA, especially satellite, mini satellite and micro satellite sequences are presented. Own experience from the use of multi and single locus analysis of DNA in paternity testing has been compared with the results of research in other laboratories. Critical points of both types of analysis are discussed. (author). 53 refs, 4 figs, 2 tabs.

  15. The DNA sequence of the human X chromosome.

    Science.gov (United States)

    Ross, Mark T; Grafham, Darren V; Coffey, Alison J; Scherer, Steven; McLay, Kirsten; Muzny, Donna; Platzer, Matthias; Howell, Gareth R; Burrows, Christine; Bird, Christine P; Frankish, Adam; Lovell, Frances L; Howe, Kevin L; Ashurst, Jennifer L; Fulton, Robert S; Sudbrak, Ralf; Wen, Gaiping; Jones, Matthew C; Hurles, Matthew E; Andrews, T Daniel; Scott, Carol E; Searle, Stephen; Ramser, Juliane; Whittaker, Adam; Deadman, Rebecca; Carter, Nigel P; Hunt, Sarah E; Chen, Rui; Cree, Andrew; Gunaratne, Preethi; Havlak, Paul; Hodgson, Anne; Metzker, Michael L; Richards, Stephen; Scott, Graham; Steffen, David; Sodergren, Erica; Wheeler, David A; Worley, Kim C; Ainscough, Rachael; Ambrose, Kerrie D; Ansari-Lari, M Ali; Aradhya, Swaroop; Ashwell, Robert I S; Babbage, Anne K; Bagguley, Claire L; Ballabio, Andrea; Banerjee, Ruby; Barker, Gary E; Barlow, Karen F; Barrett, Ian P; Bates, Karen N; Beare, David M; Beasley, Helen; Beasley, Oliver; Beck, Alfred; Bethel, Graeme; Blechschmidt, Karin; Brady, Nicola; Bray-Allen, Sarah; Bridgeman, Anne M; Brown, Andrew J; Brown, Mary J; Bonnin, David; Bruford, Elspeth A; Buhay, Christian; Burch, Paula; Burford, Deborah; Burgess, Joanne; Burrill, Wayne; Burton, John; Bye, Jackie M; Carder, Carol; Carrel, Laura; Chako, Joseph; Chapman, Joanne C; Chavez, Dean; Chen, Ellson; Chen, Guan; Chen, Yuan; Chen, Zhijian; Chinault, Craig; Ciccodicola, Alfredo; Clark, Sue Y; Clarke, Graham; Clee, Chris M; Clegg, Sheila; Clerc-Blankenburg, Kerstin; Clifford, Karen; Cobley, Vicky; Cole, Charlotte G; Conquer, Jen S; Corby, Nicole; Connor, Richard E; David, Robert; Davies, Joy; Davis, Clay; Davis, John; Delgado, Oliver; Deshazo, Denise; Dhami, Pawandeep; Ding, Yan; Dinh, Huyen; Dodsworth, Steve; Draper, Heather; Dugan-Rocha, Shannon; Dunham, Andrew; Dunn, Matthew; Durbin, K James; Dutta, Ireena; Eades, Tamsin; Ellwood, Matthew; Emery-Cohen, Alexandra; Errington, Helen; Evans, Kathryn L; Faulkner, Louisa; Francis, Fiona; Frankland, John; Fraser, Audrey E; Galgoczy, Petra; Gilbert, James; Gill, Rachel; Glöckner, Gernot; Gregory, Simon G; Gribble, Susan; Griffiths, Coline; Grocock, Russell; Gu, Yanghong; Gwilliam, Rhian; Hamilton, Cerissa; Hart, Elizabeth A; Hawes, Alicia; Heath, Paul D; Heitmann, Katja; Hennig, Steffen; Hernandez, Judith; Hinzmann, Bernd; Ho, Sarah; Hoffs, Michael; Howden, Phillip J; Huckle, Elizabeth J; Hume, Jennifer; Hunt, Paul J; Hunt, Adrienne R; Isherwood, Judith; Jacob, Leni; Johnson, David; Jones, Sally; de Jong, Pieter J; Joseph, Shirin S; Keenan, Stephen; Kelly, Susan; Kershaw, Joanne K; Khan, Ziad; Kioschis, Petra; Klages, Sven; Knights, Andrew J; Kosiura, Anna; Kovar-Smith, Christie; Laird, Gavin K; Langford, Cordelia; Lawlor, Stephanie; Leversha, Margaret; Lewis, Lora; Liu, Wen; Lloyd, Christine; Lloyd, David M; Loulseged, Hermela; Loveland, Jane E; Lovell, Jamieson D; Lozado, Ryan; Lu, Jing; Lyne, Rachael; Ma, Jie; Maheshwari, Manjula; Matthews, Lucy H; McDowall, Jennifer; McLaren, Stuart; McMurray, Amanda; Meidl, Patrick; Meitinger, Thomas; Milne, Sarah; Miner, George; Mistry, Shailesh L; Morgan, Margaret; Morris, Sidney; Müller, Ines; Mullikin, James C; Nguyen, Ngoc; Nordsiek, Gabriele; Nyakatura, Gerald; O'Dell, Christopher N; Okwuonu, Geoffery; Palmer, Sophie; Pandian, Richard; Parker, David; Parrish, Julia; Pasternak, Shiran; Patel, Dina; Pearce, Alex V; Pearson, Danita M; Pelan, Sarah E; Perez, Lesette; Porter, Keith M; Ramsey, Yvonne; Reichwald, Kathrin; Rhodes, Susan; Ridler, Kerry A; Schlessinger, David; Schueler, Mary G; Sehra, Harminder K; Shaw-Smith, Charles; Shen, Hua; Sheridan, Elizabeth M; Shownkeen, Ratna; Skuce, Carl D; Smith, Michelle L; Sotheran, Elizabeth C; Steingruber, Helen E; Steward, Charles A; Storey, Roy; Swann, R Mark; Swarbreck, David; Tabor, Paul E; Taudien, Stefan; Taylor, Tineace; Teague, Brian; Thomas, Karen; Thorpe, Andrea; Timms, Kirsten; Tracey, Alan; Trevanion, Steve; Tromans, Anthony C; d'Urso, Michele; Verduzco, Daniel; Villasana, Donna; Waldron, Lenee; Wall, Melanie; Wang, Qiaoyan; Warren, James; Warry, Georgina L; Wei, Xuehong; West, Anthony; Whitehead, Siobhan L; Whiteley, Mathew N; Wilkinson, Jane E; Willey, David L; Williams, Gabrielle; Williams, Leanne; Williamson, Angela; Williamson, Helen; Wilming, Laurens; Woodmansey, Rebecca L; Wray, Paul W; Yen, Jennifer; Zhang, Jingkun; Zhou, Jianling; Zoghbi, Huda; Zorilla, Sara; Buck, David; Reinhardt, Richard; Poustka, Annemarie; Rosenthal, André; Lehrach, Hans; Meindl, Alfons; Minx, Patrick J; Hillier, Ladeana W; Willard, Huntington F; Wilson, Richard K; Waterston, Robert H; Rice, Catherine M; Vaudin, Mark; Coulson, Alan; Nelson, David L; Weinstock, George; Sulston, John E; Durbin, Richard; Hubbard, Tim; Gibbs, Richard A; Beck, Stephan; Rogers, Jane; Bentley, David R

    2005-03-17

    The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

  16. Construction of a Sequencing Library from Circulating Cell-Free DNA.

    Science.gov (United States)

    Fang, Nan; Löffert, Dirk; Akinci-Tolun, Rumeysa; Heitz, Katja; Wolf, Alexander

    2016-04-01

    Circulating DNA is cell-free DNA (cfDNA) in serum or plasma that can be used for non-invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High-throughput sequence analysis of the cfDNA with next-generation sequencing technologies has proven to be a highly sensitive and specific method in detecting and characterizing mutations in cancer and other diseases, as well as aneuploidy during pregnancy. This unit describes detailed procedures to extract circulating cfDNA from human serum and plasma and generate sequencing libraries from a wide concentration range of circulating DNA.

  17. Long-range correlations and charge transport properties of DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Liu Xiaoliang, E-mail: xlliucsu@yahoo.com.c [College of Physical Science and Technology and College of Metallurgical Science and Engineering, Central South University, Changsha 410083 (China); Ren, Yi [College of Physical Science and Technology and College of Metallurgical Science and Engineering, Central South University, Changsha 410083 (China); Xie, Qiong-tao [Key Laboratory of Low Dimensional Quantum Structures and Quantum Control of Ministry of Education (Hunan Normal University), Changsha 410081 (China); Deng, Chao-sheng; Xu, Hui [College of Physical Science and Technology and College of Metallurgical Science and Engineering, Central South University, Changsha 410083 (China)

    2010-04-26

    By using Hurst's analysis and transfer approach, the rescaled range functions and Hurst exponents of human chromosome 22 and enterobacteria phage lambda DNA sequences are investigated and the transmission coefficients, Landauer resistances and Lyapunov coefficients of finite segments based on above genomic DNA sequences are calculated. In a comparison with quasiperiodic and random artificial DNA sequences, we find that lambda-DNA exhibits anticorrelation behavior characterized by a Hurst exponent 0.5sequence displays a transition from correlation behavior to anticorrelation behavior. The resonant peaks of the transmission coefficient in genomic sequences can survive in longer sequence length than in random sequences but in shorter sequence length than in quasiperiodic sequences. It is shown that the genomic sequences have long-range correlation properties to some extent but the correlations are not strong enough to maintain the scale invariance properties.

  18. Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima

    DEFF Research Database (Denmark)

    Worning, Peder; Jensen, Lars Juhl; Nelson, K. E.;

    2000-01-01

    The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters......, which brings independent evidence for the lateral gene transfer in the genome of T.maritima, The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii. Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms...

  19. Thermochemical and kinetic evidence for nucleotide-sequence-dependent RecA-DNA interactions.

    Science.gov (United States)

    Wittung, P; Ellouze, C; Maraboeuf, F; Takahashi, M; Nordèn, B

    1997-05-01

    RecA catalyses homologous recombination in Escherichia coli by promoting pairing of homologous DNA molecules after formation of a helical nucleoprotein filament with single-stranded DNA. The primary reaction of RecA with DNA is generally assumed to be unspecific. We show here, by direct measurement of the interaction enthalpy by means of isothermal titration calorimetry, that the polymerisation of RecA on single-stranded DNA depends on the DNA sequence, with a high exothermic preference for thymine bases. This enthalpic sequence preference of thymines by RecA correlates with faster binding kinetics of RecA to thymine DNA. Furthermore, the enthalpy of interaction between the RecA x DNA filament and a second DNA strand is large only when the added DNA is complementary to the bound DNA in RecA. This result suggests a possibility for a rapid search mechanism by RecA x DNA filaments for homologous DNA molecules.

  20. Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

    Science.gov (United States)

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2005-03-01

    Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

  1. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human

    Science.gov (United States)

    Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

    2017-01-01

    DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation. PMID:28212312

  2. Structure and organization of Marchantia polymorpha chloroplast genome. I. Cloning and gene identification.

    Science.gov (United States)

    Ohyama, K; Fukuzawa, H; Kohchi, T; Sano, T; Sano, S; Shirai, H; Umesono, K; Shiki, Y; Takeuchi, M; Chang, Z

    1988-09-20

    We have determined the complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha, using a clone bank of chloroplast DNA fragments. The circular genome consists of 121,024 base-pairs and includes two large inverted repeats (IRA and IRB, each 10,058 base-pairs), a large single-copy region (LSC, 81,095 base-pairs), and a small single-copy region (SSC, 19,813 base-pairs). The nucleotide sequence was analysed with a computer to deduce the entire gene organization, assuming the universal genetic code and the presence of introns in the coding sequences. We detected 136 possible genes. 103 gene products of which are related to known stable RNA or protein molecules. Stable RNA genes for four species of ribosomal RNA and 32 species of tRNA were located, although one of the tRNA genes may be defective. Twenty genes encoding polypeptides involved in photosynthesis and electron transport were identified by comparison with known chloroplast genes. Twenty-five open reading frames (ORFs) show structural similarities to Escherichia coli RNA polymerase subunits, 19 ribosomal proteins and two related proteins. Seven ORFs are comparable with human mitochondrial NADH dehydrogenase genes. A computer-aided homology search predicted possible chloroplast homologues of bacterial proteins; two ORFs for bacterial 4Fe-4S-type ferredoxin, two for distinct subunits of a protein-dependent transport system, one ORF for a component of nitrogenase, and one for an antenna protein of a light-harvesting complex. The other 33 ORFs, consisting of 29 to 2136 codons, remain to be identified, but some of them seem to be conserved in evolution. Detailed information on gene identification is presented in the accompanying papers. We postulated that there were 22 introns in 20 genes (8 tRNA genes and 12 ORFs), which may be classified into the groups I and II found in fungal mitochondrial genes. The structural gene for ribosomal protein S12 is trans-split on the opposite DNA strand

  3. Speciation process of Salvia isensis (Lamiaceae), a species endemic to serpentine areas in the Ise-Tokai district, Japan, from the viewpoint of the contradictory phylogenetic trees generated from chloroplast and nuclear DNA.

    Science.gov (United States)

    Sudarmono; Okada, Hiroshi

    2007-07-01

    To understand the speciation process of Salvia isensis (Lamiaceae), a species endemic to a special environment (serpentine areas in the Ise-Tokai district, central Honshu, Japan), chloroplast DNA (cpDNA) and nuclear ribosomal DNA (nrDNA) were employed to analyze the phylogenetic relationships of S. isensis with related species in Japan. Allozymic polymorphisms were also used to analyze genetic relationships among Salvia species. A contradiction in the phylogenetic positions of species studied was detected when phylogenetic trees were constructed using cpDNA or nrDNA, i.e., S. isensis was a sister to the other species in phylogenetic trees generated from cpDNA, while S. japonica was a sister to the other species in the case of nrDNA. Genetic relationships between Salvia species estimated from allozymic polymorphisms did not contradict to the topology for nrDNA. Using the present results, the speciation process of S. isensis is discussed with regard to introgressive gene exchanges between related species.

  4. mapDamage: testing for damage patterns in ancient DNA sequences

    DEFF Research Database (Denmark)

    Ginolhac, Aurelien; Rasmussen, Morten; Gilbert, M Thomas P;

    2011-01-01

    Ancient DNA extracts consist of a mixture of contaminant DNA molecules, most often originating from environmental microbes, and endogenous fragments exhibiting substantial levels of DNA damage. The latter introduce specific nucleotide misincorporations and DNA fragmentation signatures in sequenci...... of the SAMtools suite and R environment and has been validated on both GNU/Linux and MacOSX operating systems....

  5. Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency.

    NARCIS (Netherlands)

    Hinttala, R.; Smeets, R.; Moilanen, J.S.; Ugalde, C.; Uusimaa, J.; Smeitink, J.A.M.; Majamaa, K.

    2006-01-01

    BACKGROUND: Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. OBJECTIVE: To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying g

  6. A discriminative approach for unsupervised clustering of DNA sequence motifs.

    Directory of Open Access Journals (Sweden)

    Philip Stegmaier

    Full Text Available Algorithmic comparison of DNA sequence motifs is a problem in bioinformatics that has received increased attention during the last years. Its main applications concern characterization of potentially novel motifs and clustering of a motif collection in order to remove redundancy. Despite growing interest in motif clustering, the question which motif clusters to aim at has so far not been systematically addressed. Here we analyzed motif similarities in a comprehensive set of vertebrate transcription factor classes. For this we developed enhanced similarity scores by inclusion of the information coverage (IC criterion, which evaluates the fraction of information an alignment covers in aligned motifs. A network-based method enabled us to identify motif clusters with high correspondence to DNA-binding domain phylogenies and prior experimental findings. Based on this analysis we derived a set of motif families representing distinct binding specificities. These motif families were used to train a classifier which was further integrated into a novel algorithm for unsupervised motif clustering. Application of the new algorithm demonstrated its superiority to previously published methods and its ability to reproduce entrained motif families. As a result, our work proposes a probabilistic approach to decide whether two motifs represent common or distinct binding specificities.

  7. Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing.

    Science.gov (United States)

    Marosy, Beth A; Craig, Brian D; Hetrick, Kurt N; Witmer, P Dane; Ling, Hua; Griffith, Sean M; Myers, Benjamin; Ostrander, Elaine A; Stanford, Janet L; Brody, Lawrence C; Doheny, Kimberly F

    2017-01-11

    This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc.

  8. True single-molecule DNA sequencing of a pleistocene horse bone

    DEFF Research Database (Denmark)

    Orlando, Ludovic Antoine Alexandre; Ginolhac, Aurélien; Raghavan, Maanasa

    2011-01-01

    -preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing...... to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by 3-fold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3' ends of ancient templates...

  9. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    Science.gov (United States)

    Verga, Daniela; Welter, Moritz; Marx, Andreas

    2016-02-01

    DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye.

  10. Plant DNA Detection from Grasshopper Guts: A Step-by-Step Protocol, from Tissue Preparation to Obtaining Plant DNA Sequences

    Directory of Open Access Journals (Sweden)

    Alina Avanesyan

    2014-02-01

    Full Text Available Premise of the study: A PCR-based method of identifying ingested plant DNA in gut contents of Melanoplus grasshoppers was developed. Although previous investigations have focused on a variety of insects, there are no protocols available for plant DNA detection developed for grasshoppers, agricultural pests that significantly influence plant community composition. Methods and Results: The developed protocol successfully used the noncoding region of the chloroplast trnL (UAA gene and was tested in several feeding experiments. Plant DNA was obtained at seven time points post-ingestion from whole guts and separate gut sections, and was detectable up to 12 h post-ingestion in nymphs and 22 h post-ingestion in adult grasshoppers. Conclusions: The proposed protocol is an effective, relatively quick, and low-cost method of detecting plant DNA from the grasshopper gut and its different sections. This has important applications, from exploring plant “movement” during food consumption, to detecting plant–insect interactions.

  11. Characterization of Expressed Sequence Tags From a Gallus gallus Pineal Gland cDNA Library

    OpenAIRE

    2005-01-01

    The pineal gland is the circadian oscillator in the chicken, regulating diverse functions ranging from egg laying to feeding. Here, we describe the isolation and characterization of expressed sequence tags (ESTs) isolated from a chicken pineal gland cDNA library. A total of 192 unique sequences were analysed and submitted to GenBank; 6% of the ESTs matched neither GenBank cDNA sequences nor the newly assembled chicken genomic DNA sequence, three ESTs aligned with sequences designated to be on...

  12. Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone (Haliotis discus hannat)

    Institute of Scientific and Technical Information of China (English)

    LI Qi; Kijima Akihiro

    2007-01-01

    The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (< 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.

  13. DUC-Curve, a highly compact 2D graphical representation of DNA sequences and its application in sequence alignment

    Science.gov (United States)

    Li, Yushuang; Liu, Qian; Zheng, Xiaoqi

    2016-08-01

    A highly compact and simple 2D graphical representation of DNA sequences, named DUC-Curve, is constructed through mapping four nucleotides to a unit circle with a cyclic order. DUC-Curve could directly detect nucleotide, di-nucleotide compositions and microsatellite structure from DNA sequences. Moreover, it also could be used for DNA sequence alignment. Taking geometric center vectors of DUC-Curves as sequence descriptor, we perform similarity analysis on the first exons of β-globin genes of 11 species, oncogene TP53 of 27 species and twenty-four Influenza A viruses, respectively. The obtained reasonable results illustrate that the proposed method is very effective in sequence comparison problems, and will at least play a complementary role in classification and clustering problems.

  14. Sequencing of megabase plus DNA by hybridization: Method development ENT. Final technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    Crkvenjakov, R.; Drmanac, R.

    1991-01-31

    Sequencing by hybridization (SBH) is the only sequencing method based on the experimental determination of the content of oligonucleotide sequences. The data acquisition relies on the natural process of base pairing. It is possible to determine the content of complementary oligosequences in the target DNA by the process of hybridization with oligonucleotide probes of known sequences.

  15. A 28,000 years old Cro-Magnon mtDNA sequence differs from all potentially contaminating modern sequences.

    Directory of Open Access Journals (Sweden)

    David Caramelli

    Full Text Available BACKGROUND: DNA sequences from ancient specimens may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal and early modern (Cro-Magnoid Europeans. METHODOLOGY/PRINCIPAL FINDINGS: We typed the mitochondrial DNA (mtDNA hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23 and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences. CONCLUSIONS/SIGNIFICANCE: The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans.

  16. One-way sequencing of multiple amplicons from tandem repetitive mitochondrial DNA control region.

    Science.gov (United States)

    Xu, Jiawu; Fonseca, Dina M

    2011-10-01

    Repetitive DNA sequences not only exist abundantly in eukaryotic nuclear genomes, but also occur as tandem repeats in many animal mitochondrial DNA (mtDNA) control regions. Due to concerted evolution, these repetitive sequences are highly similar or even identical within a genome. When long repetitive regions are the targets of amplification for the purpose of sequencing, multiple amplicons may result if one primer has to be located inside the repeats. Here, we show that, without separating these amplicons by gel purification or cloning, directly sequencing the mitochondrial repeats with the primer outside repetitive region is feasible and efficient. We exemplify it by sequencing the mtDNA control region of the mosquito Aedes albopictus, which harbors typical large tandem DNA repeats. This one-way sequencing strategy is optimal for population surveys.

  17. DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

    2010-06-01

    A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

  18. ASAP: Amplification, sequencing & annotation of plastomes

    Directory of Open Access Journals (Sweden)

    Folta Kevin M

    2005-12-01

    Full Text Available Abstract Background Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate ~1 – 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. Results 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and

  19. SeeDNA: A Visualization Tool for K-string Content of Long DNA Sequences and Their Randomized Counterparts

    Institute of Scientific and Technical Information of China (English)

    Junjie Shen; Shuyu Zhang; Hoong-Chien Lee; Bailin Hao

    2004-01-01

    An interactive tool to visualize the K-string composition of long DNA sequences including bacterial complete genomes is described. It is especially useful for exploring short palindromic structures in the sequences. The SeeDNA program runs on Red Hat Linux with GTK+ support. It displays two-dimensional (2D) or one-dimensional (1D) histograms of the K-string distribution of a given sequence and/or its randomized counterpart. It is also capable of showing the difference of K-string distributions between two sequences. The C source code using the GTK+package is freely available.

  20. Analysis and location of a rice BAC clone containing telomeric DNA sequences

    Institute of Scientific and Technical Information of China (English)

    翟文学; 陈浩; 颜辉煌; 严长杰; 王国梁; 朱立煌

    1999-01-01

    BAC2, a rice BAC clone containing (TTTAGGG)n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG) n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescence in situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.

  1. Auxin and chloroplast movements.

    Science.gov (United States)

    Eckstein, Aleksandra; Krzeszowiec, Weronika; Waligórski, Piotr; Gabryś, Halina

    2016-03-01

    Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins.

  2. Complex chloroplast RNA metabolism: just debugging the genetic programme?

    Directory of Open Access Journals (Sweden)

    Schmitz-Linneweber Christian

    2008-08-01

    Full Text Available Abstract Background The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity. Results We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants. Conclusion Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions.

  3. Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

    Science.gov (United States)

    Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

    2012-05-01

    The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.

  4. Chloroplast SSR polymorphisms in the Compositae and the mode of organellar inheritance in Helianthus annuus.

    Science.gov (United States)

    Wills, David M; Hester, Melissa L; Liu, Aizhong; Burke, John M

    2005-03-01

    Because organellar genomes are often uniparentally inherited, chloroplast (cp) and mitochondrial (mt) DNA polymorphisms have become the markers of choice for investigating evolutionary issues such as sex-biased dispersal and the directionality of introgression. To the extent that organellar inheritance is strictly maternal, it has also been suggested that the insertion of transgenes into either the chloroplast or mitochondrial genomes would reduce the likelihood of gene escape via pollen flow from crop fields into wild plant populations. In this paper we describe the adaptation of chloroplast simple sequence repeats (cpSSRs) for use in the Compositae. This work resulted in the identification of 12 loci that are variable across the family, seven of which were further shown to be highly polymorphic within sunflower (Helianthus annuus). We then used these markers, along with a novel mtDNA restriction fragment length polymorphism (RFLP), to investigate the mode of organellar inheritance in a series of experimental crosses designed to mimic the initial stages of crop-wild hybridization in sunflower. Although we cannot rule out the possibility of extremely rare paternal transmission, our results provide the best evidence to date of strict maternal organellar inheritance in sunflower, suggesting that organellar gene containment may be a viable strategy in sunflower. Moreover, the portability of these markers suggests that they will provide a ready source of cpDNA polymorphisms for use in evolutionary studies across the Compositae.

  5. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  6. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, Jane; Gordon, Laurie A.; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A.; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C.; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M.; Glavina, Tijana; Gomez, Maria; Gonzales, Eldelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Issac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Malfatti, Stephanie; Martinez, Diego; McCready, Paula; Medina, Catherine; Morgan, Jenna; Nelson, Kathryn; Nolan, Matt; Ovcharenko, Ivan; Pitluck, Sam; Pollard, Martin; Popkie, Anthony P.; Predki, Paul; Quan, Glenda; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanine; Salamov, Asaf; Salazar, Angelica; She, Xinwei; Smith, Doug; Slezak, Tom; Solovyev, Victor; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wagner, Mark; Wheeler, Jeremy; Wu, Kevin; Xie, Gary; Yang, Joan; Dubchak, Inna; Furey, Terrence S.; DeJong, Pieter; Dickson, Mark; Gordon, David; Eichler, Evan E.; Pennacchio, Len A.; Richardson, Paul; Stubbs, Lisa; Rokhsar, Daniel S.; Myers, Richard M.; Rubin, Edward M.; Lucas, Susan M.

    2003-09-15

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G1C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9 percent of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25 percent of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, a nd segments of coding and non-coding conservation with the distant fish species Takifugu.

  7. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, J; Gordon, L A; Olsen, A; Terry, A; Schmutz, J; Lamerdin, J; Hellsten, U; Goodstein, D; Couronne, O; Tran-Gyamfi, M

    2004-04-06

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high GC content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in Mendelian disorders, including familial hypercholesterolemia and insulin-resistant diabetes. Nearly one quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

  8. Silicon Nanopore Devices for DNA Translocation and Sequencing Studies

    Science.gov (United States)

    Ling, Sean

    2005-03-01

    In this talk, I will discuss the recent progress [1-3] in developing solid-state nanopore devices using silicon technology. We have demonstrated a novel technique for shaping nanopores in the range of 1-10 nm, using surface-tension-driven mass flow with single nanometer precision. This technique overcomes a major technical challenge in silicon technology. I will also discuss the current effort [3] in developing integrated nanopore silicon chips with electrically addressable nanopores. These devices are used for DNA translocation and sequencing studies. This work was done in collaboration with the group of Cees Dekker at TU-Delft with partial support from FOM and Guggenheim Foundation. The work at Brown was supported by NSF-NER and NSF-NIRT. [1] A.J. Storm, J.H. Chen, X.S. Ling, H. Zandbergen, and C. Dekker, ``Fabrication of Solid-State Nanopores with Single Nanometer Precision'', Nature Materials, 2, 537 (2003). [2] A.J. Storm, J.H. Chen, X.S. Ling, H. Zandbergen, and C. Dekker, ``Electron-Beam-Induced Deformations of SiO2 Nanostructures'', Journal of Applied Physics (submitted, 2004). [3] X.S. Ling, "Addressable nanopores and micropores" (patent pending).

  9. [Patentability of DNA sequences: the debate remains open].

    Science.gov (United States)

    Martín Uranga, Amelia

    2013-01-01

    The patentability of human genes was from the beginning of the discussion concerning the Directive on the legal protection of biotechnological inventions, an issue that provoked debates among politicians, scientists, lawyers and civil society itself. Although Directive 98/44 tried to settle the matter by stating that to support the patentability of human genes, it should know what role they fulfill, which protein they encode, all of this as an essential requirement to test its industrial application. However, following the judgment of 13 June 2013 (Supreme Court of the United States of America in the case of Association for Molecular Pathology et al. versus Myriad Genetics Inc.) the debate on this issue has been reopened. There are several issues to be considered, taking into account that the patents on DNA & Gene Sequences have played an important incentive to increase the interest in biotechnology applied to human health. On the other hand, this is a paradigm shift in the R & D of biopharmaceutical companies, and it has moved from an in house research model to a model of open innovation, a model of collaboration between large corporations with biotech SMEs and public and private research centers. This model of innovation, impacts on the issue of the industrial property, and therefore it will be necessary to clearly define what each party brings to the relationship and how they are expected to share the results. But all of this, with the ultimate goal that the patients have access to treatments and medications most innovative, safe and effective.

  10. The DNA sequence and biology of human chromosome 19.

    Science.gov (United States)

    Grimwood, Jane; Gordon, Laurie A; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Isaac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry,