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BACKGROUND: The investigation of plant genome structure and evolution requires comprehensive characterization of repetitive sequences that make up the majority of higher plant nuclear DNA. Since genome-wide characterization of repetitive elements is complicated by their high abundance and diversity, novel approaches based on massively-parallel sequencing are being adapted to facilitate the analysis. It has recently been demonstrated that the low-pass genome sequencing provided by a single 454 sequencing reaction is sufficient to capture information about all major repeat families, thus providing the opportunity for efficient repeat investigation in a wide range of species. However, the development of appropriate data mining tools is required in order to fully utilize this sequencing data for repeat characterization. RESULTS: We adapted a graph-based approach for similarity-based partitioning of whole genome 454 sequence reads in order to build clusters made of the reads derived from individual repeat families. The information about cluster sizes was utilized for assessing the proportion and composition of repeats in the genomes of two model species, Pisum sativum and Glycine max, differing in genome size and 454 sequencing coverage. Moreover, statistical analysis and visual inspection of the topology of the cluster graphs using a newly developed program tool, SeqGrapheR, were shown to be helpful in distinguishing basic types of repeats and investigating sequence variability within repeat families. CONCLUSIONS: Repetitive regions of plant genomes can be efficiently characterized by the presented graph-based analysis and the graph representation of repeats can be further used to assess the variability and evolutionary divergence of repeat families, discover and characterize novel elements, and aid in subsequent assembly of their consensus sequences.
BACKGROUND: The number and complexity of repetitive elements varies between species, being in general most represented in those with larger genomes. Combining the flow-sorted chromosome arms approach to genome analysis with second generation DNA sequencing technologies provides a unique opportunity to study the repetitive portion of each chromosome, enabling comparisons among them. Additionally, different sequencing approaches may produce different depth of insight to repeatome content and structure. In this work we analyze and characterize the repetitive sequences of Triticum aestivum cv. Chinese Spring homeologous group 4 chromosome arms, obtained through Roche 454 and Illumina sequencing technologies, hereinafter marked by subscripts 454 and I, respectively. Repetitive sequences were identified with the RepeatMasker software using the interspersed repeat database mips-REdat_v9.0p. The input sequences consisted of our 4DS454 and 4DL454 scaffolds and 4ASI, 4ALI, 4BSI, 4BLI, 4DSI and 4DLI contigs, downloaded from the International Wheat Genome Sequencing Consortium (IWGSC). RESULTS: Repetitive sequences content varied from 55% to 63% for all chromosome arm assemblies except for 4DLI, in which the repeat content was 38%. Transposable elements, small RNA, satellites, simple repeats and low complexity sequences were analyzed. SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher. Dinucleotides and trinucleotides were the most abundant SSR repeat units. (GA)n/(TC)n was the most abundant SSR except for 4DLI where the most frequently identified SSR was (CCG/CGG)n. Retrotransposons followed by DNA transposons were the most highly represented sequence repeats, mainly composed of CACTA/En-Spm and Gypsy superfamilies, respectively. This whole chromosome sequence analysis allowed identification of three new LTR retrotransposon families belonging to the Copia superfamily, one belonging to the Gypsy superfamily and two TRIM retrotransposon families. Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes. CONCLUSION: The presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4.
Structurally and functionally diverged sex chromosomes have evolved in many animals as well as in some plants. Sex chromosomes represent a specific genomic region(s) with locally suppressed recombination. As a consequence, repetitive sequences involving transposable elements, tandem repeats (satellites and microsatellites), and organellar DNA accumulate on the Y (W) chromosomes. In this paper, we review the main types of repetitive elements, their gathering on the Y chromosome, and discuss new findings showing that not only accumulation of various repeats in non-recombining regions but also opposite processes form Y chromosome. The aim of this review is also to discuss the mechanisms of repetitive DNA spread involving (retro) transposition, DNA polymerase slippage or unequal crossing-over, as well as modes of repeat removal by ectopic recombination. The intensity of these processes differs in non-recombining region(s) of sex chromosomes when compared to the recombining parts of genome. We also speculate about the relationship between heterochromatinization and the formation of heteromorphic sex chromosomes.
- MeSH
- chromozomy rostlin * MeSH
- DNA rostlinná * MeSH
- molekulární evoluce * MeSH
- pohlavní chromozomy genetika MeSH
- regulace genové exprese u rostlin MeSH
- repetitivní sekvence nukleových kyselin * MeSH
- rostliny genetika MeSH
- transpozibilní elementy DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Silene latifolia is a dioecious [corrected] plant with well distinguished X and Y chromosomes that is used as a model to study sex determination and sex chromosome evolution in plants. However, efficient utilization of this species has been hampered by the lack of large-scale sequencing resources and detailed analysis of its genome composition, especially with respect to repetitive DNA, which makes up the majority of the genome. METHODOLOGY/PRINCIPAL FINDINGS: We performed low-pass 454 sequencing followed by similarity-based clustering of 454 reads in order to identify and characterize sequences of all major groups of S. latifolia repeats. Illumina sequencing data from male and female genomes were also generated and employed to quantify the genomic proportions of individual repeat families. The majority of identified repeats belonged to LTR-retrotransposons, constituting about 50% of genomic DNA, with Ty3/gypsy elements being more frequent than Ty1/copia. While there were differences between the male and female genome in the abundance of several repeat families, their overall repeat composition was highly similar. Specific localization patterns on sex chromosomes were found for several satellite repeats using in situ hybridization with probes based on k-mer frequency analysis of Illumina sequencing data. CONCLUSIONS/SIGNIFICANCE: This study provides comprehensive information about the sequence composition and abundance of repeats representing over 60% of the S. latifolia genome. The results revealed generally low divergence in repeat composition between the sex chromosomes, which is consistent with their relatively recent origin. In addition, the study generated various data resources that are available for future exploration of the S. latifolia genome.
RepeatExplorer2 is a novel version of a computational pipeline that uses graph-based clustering of next-generation sequencing reads for characterization of repetitive DNA in eukaryotes. The clustering algorithm facilitates repeat identification in any genome by using relatively small quantities of short sequence reads, and additional tools within the pipeline perform automatic annotation and quantification of the identified repeats. The pipeline is integrated into the Galaxy platform, which provides a user-friendly web interface for script execution and documentation of the results. Compared to the original version of the pipeline, RepeatExplorer2 provides automated annotation of transposable elements, identification of tandem repeats and enhanced visualization of analysis results. Here, we present an overview of the RepeatExplorer2 workflow and provide procedures for its application to (i) de novo repeat identification in a single species, (ii) comparative repeat analysis in a set of species, (iii) development of satellite DNA probes for cytogenetic experiments and (iv) identification of centromeric repeats based on ChIP-seq data. Each procedure takes approximately 2 d to complete. RepeatExplorer2 is available at https://repeatexplorer-elixir.cerit-sc.cz .
- MeSH
- DNA sondy chemie genetika MeSH
- DNA chemie genetika MeSH
- genomika metody MeSH
- lidé MeSH
- repetitivní sekvence nukleových kyselin MeSH
- sekvenční analýza DNA metody MeSH
- shluková analýza MeSH
- software MeSH
- transpozibilní elementy DNA MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Festuca genus is thought to be the most numerous genus of the Poaceae family. One of the most agronomically important forage grasses, Festuca pratensis Huds. is treated as a model plant to study the molecular mechanisms associated with tolerance to winter stresses, including frost. However, the precise mapping of the genes governing stress tolerance in this species is difficult as its karyotype remains unrecognized. Only two F. pratensis chromosomes with 35S and 5S rDNA sequences can be easily identified, but its remaining chromosomes have not been distinguished to date. Here, two libraries derived from F. pratensis nuclear DNA with various contents of repetitive DNA sequences were used as sources of molecular probes for fluorescent in situ hybridisation (FISH), a BAC library and a library representing sequences most frequently present in the F. pratensis genome. Using FISH, six groups of DNA sequences were revealed in chromosomes on the basis of their signal position, including dispersed-like sequences, chromosome painting-like sequences, centromeric-like sequences, knob-like sequences, a group without hybridization signals, and single locus-like sequences. The last group was exploited to develop cytogenetic maps of diploid and tetraploid F. pratensis, which are presented here for the first time and provide a remarkable progress in karyotype characterization.
- MeSH
- chromozomy rostlin genetika MeSH
- diploidie MeSH
- Festuca genetika růst a vývoj MeSH
- fyziologický stres genetika MeSH
- genová knihovna MeSH
- hybridizace genetická MeSH
- hybridizace in situ fluorescenční MeSH
- karyotypizace MeSH
- nízká teplota MeSH
- repetitivní sekvence nukleových kyselin genetika MeSH
- RNA ribozomální 5S genetika MeSH
- tetraploidie MeSH
- Publikační typ
- časopisecké články MeSH
We applied comparative genomic hybridization (CGH) and genomic in situ hybridization (GISH) to examine genomes of artificially produced sturgeon hybrids between sterlet, Acipenser ruthenus female (∼120 chromosomes) or Russian sturgeon, A. gueldenstaedtii female (∼240 chromosomes) and a spontaneous triploid Siberian sturgeon A. baerii male (∼360 chromosomes), respectively. The ploidy levels of progenies were analyzed by karyotyping and flow cytometry. We found that the species-specific regions were surprisingly identifiable only on some micro- and small(er) macrochromosomes in hybrid metaphases. We hypothesize that these distinguishable regions are represented by species-specific repetitive sequences driven by more dynamic molecular evolutionary mechanisms. On larger chromosomes, GISH faintly visualized only blocks of pericentromeric and telomeric repetitive sequences, remaining regions were equally shared by both parental species. We concluded that the interspecies hybridization producing viable and even fertile progeny is enabled by the fact that genomes of the species involved are likely divergent at the level of the repetitive sequences only and probably highly conserved in the coding sequences. These small differences of coding sequences are in concordance with previous estimations of relatedness of examined species producing artificial as well as natural hybrids. CGH and GISH represent a challenge in sturgeon cytogenetics as a valuable though technically not simple tool to discriminate chromosomes of parental species in hybrids. The potentials and drawbacks of CGH and GISH application in sturgeons are discussed and further experimental possibilities are proposed.
- MeSH
- chromozomy MeSH
- genom MeSH
- karyotypizace MeSH
- molekulární evoluce * MeSH
- polyploidie * MeSH
- repetitivní sekvence nukleových kyselin * MeSH
- ryby genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIMS: The aim of this study was to develop a simple protocol for a PCR-based fingerprinting of Stenotrophomonas maltophilia (SmrepPCR) that utilizes primers complementary to repetitive extragenic palindromic elements (REPs) of this micro-organism. METHODS AND RESULTS: The relatedness of 34 isolates of environmental and clinical origin was investigated by two SmrepPCRs with two different primers, gyrB sequencing and XbaI macrorestriction followed by pulsed-field gel electrophoresis. While SmrepPCR (with primer DIR) results matched data obtained from the analysis of gyrB nucleotide sequences and identified several clonal complexes, XbaI macrorestriction showed high level of heterogeneity between isolates. The macrorestriction-based clustering of isolates did not correspond to both gyrB and DIR-SmrepPCR grouping. CONCLUSIONS: Our results show that SmrepPCR-inferred relationship of isolates is in a good agreement with sequence-based methods. The combined information from all methods used suggests that rapid evolution of S. maltophilia genomes might be predominantly due to high rate of rearrangements caused by mobile genetic elements. SIGNIFICANCE AND IMPACT OF THE STUDY: The presented method is an inexpensive and easy to perform alternative to genotype S. maltophilia isolates and to study their population genetics. SmrepPCR demonstrates the usefulness of species-specific repetitive elements in genomic analyses.
- MeSH
- DNA bakterií analýza genetika MeSH
- DNA fingerprinting MeSH
- druhová specificita MeSH
- fylogeneze MeSH
- lidé MeSH
- obrácené repetice MeSH
- polymerázová řetězová reakce metody MeSH
- pulzní gelová elektroforéza MeSH
- Stenotrophomonas maltophilia genetika izolace a purifikace MeSH
- variabilita počtu kopií segmentů DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
