DNA condensation with polyamines. II. Electron microscopic studies

Approximately 75% of the wheat and rye genomes consist of repeated sequence DNA. Three-quarters of the non-repeated or few copy sequences in wheat are less than 1000 base-pairs long, whilst in rye approximately half of the non-repeated or few copy sequences are in this size class. Most of the remaining non-repeated or few copy sequences appear to be a few thousand base-pairs long.In this paper a somewhat novel approach has been used to quantitatively analyse the linear organisation of the large proportion of repeated sequence DNA as well as the non-repeated DNA in the wheat and rye genomes. Repeated sequences in the genomes of oats, barley, wheat and rye have been used as probes to distinguish and isolate four different groups of repeated sequences and their neighbouring sequences from the wheat and rye genomes. Radioactively labelled wheat or rye DNA fragments ranging from 200 to over 9000 nucleotides long were incubated separately with large excesses of denatured unlabelled oats, barley, wheat and rye DNAs to C_ot values which enable all the repeated sequences of the unlabelled DNA to renature. The following parameters were then determined from the proportions of total labelled DNA in fragments which had at least partially renatured. (1) The proportions of the repeated sequences in the labelled DNAs that were able to hybridise to each unlabelled DNA; (2) the mean distance apart of the hybridising sequences on the longer labelled fragments; and (3) the proportion of the genome in which the hybridising sequences were concentrated. Analysis of these results, together with those of separate experiments designed to quantitatively estimate the nature of sequences unable to reanneal with the repeated sequences of each of the probe DNAs, have enabled schematic maps to be drawn which show how the repeated and non-repeated sequences are arranged in the wheat and rye genomes.Both genomes are constructed from millions of relatively short sequences, most of them considerably shorter than 3000 base-pairs. This structure was recognised because adjacent sequences can be distinguished by their frequency of repetition (i.e. repeated or non-repeated) or by their evolutionary origin. Approximately 40 to 45% of the wheat genome and 30 to 35% of the rye genome consists of short non-repeated sequences interspersed between short repeated sequences. Approximately 50% of the wheat genome and 60% of the rye genome consists of tandemly arranged repeated sequences of different evolutionary origins. It is postulated that much of this complex repeated sequence DNA could have arisen from amplification of compound sequences, each containing repeated and non-repeated sequence DNA.Short repeated sequences with a number average length of around 200 base-pairs and which occupy about 20% of the wheat and rye genomes are related to repeated sequences also found in oats and barley. They are concentrated in 60 to 70% of the wheat and rye genomes, being interspersed with different short repeated sequences and a significant proportion of the short non-repeated sequences.Rye chromosomes contain more DNA than wheat chromosomes. This is principally, but not entirely, due to additional repeated sequence DNA. Many quantitative changes appear to have occurred in both genomes, possibly affecting most families of repeated sequences, since wheat and rye diverged from a common ancestor. Both species contain species-specific repeated sequences (24% of rye genome; 16% of wheat genome) but a large proportion of these are closely interspersed with repeated sequences found in both genomes.     

Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron

Standard plant DNA barcodes based on 2–3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near-complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145 species of this species-rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya-Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH-psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3–4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants.     

DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

DNA条形码分析方法研究进展

DNA条形码是一段短的、标准化的DNA序列,DNA条形码技术通过对DNA条形码序列分析实现物种的有效鉴定.随着生物DNA条形码序列的大量测定,DNA条形码分析方法得到迅速发展,推动了其在生物分子鉴定中的应用.2003年以来,DNA条形码技术已广泛应用于动物、植物和真菌等物种的鉴定,并有力地推动了生物分类学、生物多样性和生态学等学科的发展.本文在综述DNA条形码技术的基础上,总结了5类主要的DNA条形码分析方法,即基于遗传距离的分析、基于遗传相似度的分析、基于系统发育树的分析、基于序列特征的分析和基于统计分类法的分析,并进一步展望了DNA条形码技术的发展与应用.     

Plant DNA barcoding: from gene to genome

DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single‐locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole‐chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource‐effective and does not yet offer the speed of analysis provided by single‐locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super‐barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single‐locus barcodes and super‐barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.     

Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies

DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648‐bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein‐encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most – possibly all – synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well‐curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.     

TERAD: Extraction of transposable element composition from RADseq data

Transposable elements (TEs) – selfish DNA sequences that can move within the genome – comprise a large proportion of the genomes of many organisms. Although low‐coverage whole‐genome sequencing can be used to survey TE composition, it is noneconomical for species with large quantities of DNA. Here, we utilize restriction‐site associated DNA sequencing (RADSeq) as an alternative method to survey TE composition. First, we demonstrate in silico that double digest restriction‐site associated DNA sequencing (ddRADseq) markers contain the same TE compositions as whole genome assemblies across arthropods. Next, we show empirically using eight Synalpheus snapping shrimp species with large genomes that TE compositions from ddRADseq and low‐coverage whole‐genome sequencing are comparable within and across species. Finally, we develop a new bioinformatic pipeline, TERAD, to extract TE compositions from RADseq data. Our study expands the utility of RADseq to study the repeatome, making comparative studies of genome structure for species with large genomes more tractable and affordable.     

Short repetitive sequences in green algal mitochondrial genomes: potential roles in mitochondrial genome evolution

Current data on green algal mitochondrial genomes suggest an unexpected dichotomy within the group with respect to genome structure, organization, and sequence affiliations. The present study suggests that there is a correlation between this dichotomy on one hand and the differences in the abundance, base composition, and distribution of short repetitive sequences we observed among green algal mitochondrial genomes on the other. It is conceivable that the accumulation of GC- rich short repeated sequences in the Chlamydomonas-like but not Prototheca-like mitochondrial genomes might have triggered evolutionary events responsible for the distinct series of evolutionary changes undergone by the two green algal mitochondrial lineages. The similarity in base composition, nucleotide sequence, abundance, and mode of organization we observed between the short repetitive sequences present in Chlamydomonas-like mitochondrial genomes on one hand and fungal and vertebrate homologs on the other might extend to some of the roles that the short repetitive sequences have been shown to have in the latter. Potential involvements we propose for the short repetitive sequences in the evolution of Chlamydomonas-like mitochondrial genomes include fragmentation and scrambling of the ribosomal-RNA-coding regions, extensive gene rearrangements, coding-region deletions, surrogate origins of replication, and chromosomal linearization.      

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.     

ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae)

The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. Here we show that second internal transcribed spacer of nuclear ribosomal DNA (rDNA-ITS2) barcodes effectively discriminate among 16 species of spider mites (Acari: Tetranychidae) from Israel. The barcode sequences of each species were unambiguously distinguishable from all other species and formed distinct, nonoverlapping monophyletic groups in the maximum-parsimony tree. Sequence divergences were generally much greater between species than within them. Using a 0.02 (2%) threshold for species diagnosis in our data set, 14 out of 16 species recognized by morphological criteria would be accurately identified. The only exceptions involved the low divergence, 0.011–0.015 (1.1–1.5%), between Tetranychus urticae and Tetranychus turkestani, where speciation may have occurred only recently. Still, these species had fixed alternative rDNA-ITS2 variants, with five diagnostic nucleotide substitutions. As a result, we tentatively conclude that rDNA-ITS2 sequence barcodes may serve as an effective tool for the identification of spider mite species and can be applicable as a diagnostic tool for quarantine and other pest management activities and decision-making. We predict that our work, together with similar efforts, will provide in the future the platform for a uniform, accurate, practical and easy-to-use method of spider mite species identification.     

A Novel Construction of Genome Space with Biological Geometry

A genome space is a moduli space of genomes. In this space, each point corresponds to a genome. The natural distance between two points in the genome space reflects the biological distance between these two genomes. Currently, there is no method to represent genomes by a point in a space without losing biological information. Here, we propose a new graphical representation for DNA sequences. The breakthrough of the subject is that we can construct the moment vectors from DNA sequences using this new graphical method and prove that the correspondence between moment vectors and DNA sequences is one-to-one. Using these moment vectors, we have constructed a novel genome space as a subspace in R^N. It allows us to show that the SARS-CoV is most closely related to a coronavirus from the palm civet not from a bird as initially suspected, and the newly discovered human coronavirus HCoV-HKU1 is more closely related to SARS than to any other known member of group 2 coronavirus. Furthermore, we reconstructed the phylogenetic tree for 34 lentiviruses (including human immunodeficiency virus) based on their whole genome sequences. Our genome space will provide a new powerful tool for analyzing the classification of genomes and their phylogenetic relationships.     

An investigation of genomic base distribution

While veritable oceans of ink have been spilled over the base distributions within genes, the literature is virtually silent on large scale intra genomic base distribution. To address this issue, we have examined approximately 3400 chromosomal sequences from approximately 2000 entire genomes-including DNA and RNA, single- and double-stranded, coding and non-coding genomes. For each sequence the mean, variance, skewness, and kurtosis for each base were computed along with the genome base composition. The main findings are: (1) there is no simple relationship between these statistics and the base composition of the genome, (2) in non-viral genomes, base distribution is non-uniform, (3) base distribution in non-eukaryotic genomes obeys a number of simple rules, (4) these rules are not dependent on the presence of coding sequences, (5) bacterial genomes in particular are unusually compliant with these rules, and (6) eukaryotes have a unique pattern of base distribution.     

Deltarho-web, an online tool to assess composition similarity of individual nucleic acid sequences

SUMMARY: Although whole-genome sequences have been analysed for the presence of anomalous DNA, no dedicated application is currently available to analyse the composition of individual sequence entries, for instance those derived by experimental techniques, such as subtractive hybridization. Since genomic dinucleotide frequency values are conserved between related species, a representative genome sequence can often be found to score for anomalous sequence composition for many of these putative horizontally transferred sequences. We developed the application deltarho-web, which enables the determination of the differences between the dinucleotide composition of an input sequence and that of a selected genome in a size-dependent manner. A feature allowing batch comparisons is included as well. In addition, deltarho-web allows the analysis of the dinucleotide composition of complete genomes. This provides complementary information for the identification of large anomalous gene clusters.     

Organizational heterogeneity of vertebrate genomes

Genomes of higher eukaryotes are mosaics of segments with various structural, functional, and evolutionary properties. The availability of whole-genome sequences allows the investigation of their structure as "texts" using different statistical and computational methods. One such method, referred to as Compositional Spectra (CS) analysis, is based on scoring the occurrences of fixed-length oligonucleotides (k-mers) in the target DNA sequence. CS analysis allows generating species- or region-specific characteristics of the genome, regardless of their length and the presence of coding DNA. In this study, we consider the heterogeneity of vertebrate genomes as a joint effect of regional variation in sequence organization superimposed on the differences in nucleotide composition. We estimated compositional and organizational heterogeneity of genome and chromosome sequences separately and found that both heterogeneity types vary widely among genomes as well as among chromosomes in all investigated taxonomic groups. The high correspondence of heterogeneity scores obtained on three genome fractions, coding, repetitive, and the remaining part of the noncoding DNA (the genome dark matter--GDM) allows the assumption that CS-heterogeneity may have functional relevance to genome regulation. Of special interest for such interpretation is the fact that natural GDM sequences display the highest deviation from the corresponding reshuffled sequences.     

A whole-genome snapshot of 454 sequences exposes the composition of the barley genome and provides evidence for parallel evolution of genome size in wheat and barley

The genomes of barley and wheat, two of the world's most important crops, are very large and complex due to their high content of repetitive DNA. In order to obtain a whole-genome sequence sample, we performed two runs of 454 (GS20) sequencing on genomic DNA of barley cv. Morex, which yielded approximately 1% of a haploid genome equivalent. Almost 60% of the sequences comprised known transposable element (TE) families, and another 9% represented novel repetitive sequences. We also discovered high amounts of low-complexity DNA and non-genic low-copy DNA. We identified almost 2300 protein coding gene sequences and more than 660 putative conserved non-coding sequences. Comparison of the 454 reads with previously published genomic sequences suggested that TE families are distributed unequally along chromosomes. This was confirmed by in situ hybridizations of selected TEs. A comparison of these data for the barley genome with a large sample of publicly available wheat sequences showed that several TE families that are highly abundant in wheat are absent from the barley genome. This finding implies that the TE composition of their genomes differs dramatically, despite their very similar genome size and their close phylogenetic relationship.     

Molecular cytogenetics of Leymus: Mapping the Ns genome-specific repetitive sequences

The objective of this paper is to summarize the work in my group on FISH (fluorescent in situ hybridization) mapping of Ns-specific repetitive DNA sequences fromLeymus and discuss the results in the context of classification based on the genome system currently used among Triticeae researchers. The key question here is whether the genome composition of a tetraploid Leymus species should be NsXm or NsNs (Ns₁Ns₂). Different types of Leymus-specific dispersed retroelement-like repeats have been isolated and characterized. Because the sequences occur in significantly high copy number in Leymus, based on strong hybridization signal in Southern blots, they are considered essentially specific to Leymus. They are also abundant in Psathyrostachys, the progenitor of Ns genome in Leymus. These dispersed repeats are found to distribute over the whole of all Leymus chromosomes, without any differentiation between chromosomes that have been suggested to be of different genomic origins, meaning that all genomes in Leymus are the same. GISH (genomic in situ hybridization) experiments on Leymus chromosomes using Psathyrostachys genomic DNA as probes further support the NsNs (Ns₁Ns₂) genome constitution for Leymus. The Xm genome of an unknown origin might have been there in the beginning of the allopolyploidization process, but the Ns genome-specific elements must have spread predominantly and rapidly across genomes, thus homogenizing the nuclear genomes of Leymus. I present here for the first time evidence that Ns-specific dispersed repeats can spread in a very short time, from Leymus over to wheat in Triticum × Leymus hybrids growing in artificial conditions.     

Metazoa-level USCOs as markers in species delimitation and classification

Metazoa-level universal single-copy orthologs (mzl-USCOs) are universally applicable markers for DNA taxonomy in animals that can replace or supplement single-gene barcodes. Previously, mzl-USCOs from target enrichment data were shown to reliably distinguish species. Here, we tested whether USCOs are an evenly distributed, representative sample of a given metazoan genome and therefore able to cope with past hybridization events and incomplete lineage sorting. This is relevant for coalescent-based species delimitation approaches, which critically depend on the assumption that the investigated loci do not exhibit autocorrelation due to physical linkage. Based on 239 chromosome-level assembled genomes, we confirmed that mzl-USCOs are genetically unlinked for practical purposes and a representative sample of a genome in terms of reciprocal distances between USCOs on a chromosome and of distribution across chromosomes. We tested the suitability of mzl-USCOs extracted from genomes for species delimitation and phylogeny in four case studies: Anopheles mosquitos, Drosophila fruit flies, Heliconius butterflies and Darwin's finches. In almost all instances, USCOs allowed delineating species and yielded phylogenies that corresponded to those generated from whole genome data. Our phylogenetic analyses demonstrate that USCOs may complement single-gene DNA barcodes and provide more accurate taxonomic inferences. Combining USCOs from sources that used different versions of ortholog reference libraries to infer marker orthology may be challenging and, at times, impact taxonomic conclusions. However, we expect this problem to become less severe as the rapidly growing number of reference genomes provides a better representation of the number and diversity of organismal lineages.     

Differential distribution of simple sequence repeats in eukaryotic genome sequences.

Complete chromosome/genome sequences available from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cerevisiae were analyzed for the occurrence of mono-, di-, tri-, and tetranucleotide repeats. In all of the genomes studied, dinucleotide repeat stretches tended to be longer than other repeats. Additionally, tetranucleotide repeats in humans and trinucleotide repeats in Drosophila also seemed to be longer. Although the trends for different repeats are similar between different chromosomes within a genome, the density of repeats may vary between different chromosomes of the same species. The abundance or rarity of various di- and trinucleotide repeats in different genomes cannot be explained by nucleotide composition of a sequence or potential of repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication/repair/recombination machinery might play an important role in the genesis of repeats. Moreover, analysis of complete genome coding DNA sequences of Drosophila, C. elegans, and yeast indicated that expansions of codon repeats corresponding to small hydrophilic amino acids are tolerated more, while strong selection pressures probably eliminate codon repeats encoding hydrophobic and basic amino acids. The locations and sequences of all of the repeat loci detected in genome sequences and coding DNA sequences are available at http://www.ncl-india.org/ssr and could be useful for further studies.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

Repetitive sequences in complete and defective genomes of Herpesvirus saimiri.

Two types of Herpesvirus saimiri genomes can be isolated from purified virions: (i) the M genome is a double-stranded, liniear DNA molecule with a mean contour length corresponding to 89 times 10-6 daltons. The M genome contains about 70% of unique sequences (light DNA, 36% guanine plus cytosine) and 30% reiterated sequences (heavy DNA, 71% guanine plus cytosine). (ii) the H genome is composed of heavy DNA only and is more heterogeneous in size. The sequences in the H genome are up to 40-fold reiterated, indicating defectiveness of this type of genome. The repetitions in the H genome and the M genome cross-hybridize almost completely and have identical kinetic complexity (2.8 times 10-6 daltons). DNA infectivity studies by using the calcium phosphate and the DEAE-dextran method gave further evidence that H genomes are defective: no infectious virus was recovered from permissive cells treated with heavy DNA, whereas M genome-infected cells developed cytopathic changes after 11 to 56 days. Defective H genomes were present in the progeny virus two passages after transfection.