Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the progenitor of wheat. We performed chromosome-based survey sequencing of the 14 chromosomes, examining repetitive sequences, protein-coding genes, miRNA/target pairs and tRNA genes, as well as syntenic relationships with related grasses. We found considerable differences in the content and distribution of repetitive sequences between the A and B subgenomes. The gene contents of individual chromosomes varied widely, not necessarily correlating with chromosome size. We catalogued candidate agronomically important loci, along with new alleles and flanking sequences that can be used to design exome sequencing. Syntenic relationships and virtual gene orders revealed several small-scale evolutionary rearrangements, in addition to providing evidence for the 4AL-5AL-7BS translocation in wild emmer wheat. Chromosome-based sequence assemblies contained five novel miRNA families, among 59 families putatively encoded in the entire genome which provide insight into the domestication of wheat and an overview of the genome content and organization.

• Klíčová slova
• chromosome sorting, comparative genomics, hexaploid wheat, next-generation sequencing, wild emmer wheat,
• MeSH
• chromozomy rostlin genetika   MeSH
• genetické lokusy genetika   MeSH
• genom rostlinný genetika   MeSH
• konzervovaná sekvence genetika   MeSH
• lipnicovité genetika   MeSH
• mikro RNA genetika   MeSH
• nekódující RNA genetika   MeSH
• polyploidie MeSH
• průtoková cytometrie MeSH
• pšenice genetika   MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• rostlinné geny genetika   MeSH
• tetraploidie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH
• nekódující RNA MeSH

BACKGROUND: Haynaldia villosa (H. villosa) has been recognized as a species potentially useful for wheat improvement. The availability of its genomic sequences will boost its research and application. RESULTS: In this work, the short arm of H. villosa chromosome 4V (4VS) was sorted by flow cytometry and sequenced using Illumina platform. About 170.6 Mb assembled sequences were obtained. Further analysis showed that repetitive elements accounted for about 64.6% of 4VS, while the coding fraction, which is corresponding to 1977 annotated genes, represented 1.5% of the arm. The syntenic regions of the 4VS were searched and identified on wheat group 4 chromosomes 4AL, 4BS, 4DS, Brachypodium chromosomes 1 and 4, rice chromosomes 3 and 11, and sorghum chromosomes 1, 5 and 8. Based on genome-zipper analysis, a virtual gene order comprising 735 gene loci on 4VS genome was built by referring to the Brachypodium genome, which was relatively consistent with the scaffold order determined for Ae. tauschii chromosome 4D. The homologous alleles of several cloned genes on wheat group 4 chromosomes including Rht-1 gene were identified. CONCLUSIONS: The sequences provided valuable information for mapping and positional-cloning genes located on 4VS, such as the wheat yellow mosaic virus resistance gene Wss1. The work on 4VS provided detailed insights into the genome of H. villosa, and may also serve as a model for sequencing the remaining parts of H. villosa genome.

• Klíčová slova
• Chromosome arm 4VS, Flow sorting, Genome zipper, Haynaldia villosa, Scaffold,
• MeSH
• chromozomy rostlin genetika   MeSH
• druhová specificita MeSH
• genomika MeSH
• lipnicovité genetika   MeSH
• mapování chromozomů MeSH
• pořadí genů genetika   MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• sekvenční analýza DNA * MeSH
• Publikační typ
• časopisecké články MeSH

Wild emmer wheat, Triticum turgidum ssp. dicoccoides is the wild relative of Triticum turgidum, the progenitor of durum and bread wheat, and maintains a rich allelic diversity among its wild populations. The lack of adequate genetic and genomic resources, however, restricts its exploitation in wheat improvement. Here, we report next-generation sequencing of the flow-sorted chromosome 5B of T. dicoccoides to shed light into its genome structure, function and organization by exploring the repetitive elements, protein-encoding genes and putative microRNA and tRNA coding sequences. Comparative analyses with its counterparts in modern and wild wheats suggest clues into the B-genome evolution. Syntenic relationships of chromosome 5B with the model grasses can facilitate further efforts for fine-mapping of traits of interest. Mapping of 5B sequences onto the root transcriptomes of two additional T. dicoccoides genotypes, with contrasting drought tolerances, revealed several thousands of single nucleotide polymorphisms, of which 584 shared polymorphisms on 228 transcripts were specific to the drought-tolerant genotype. To our knowledge, this study presents the largest genomics resource currently available for T. dicoccoides, which, we believe, will encourage the exploitation of its genetic and genomic potential for wheat improvement to meet the increasing demand to feed the world.

• MeSH
• chromozomy rostlin genetika   MeSH
• mikro RNA genetika   MeSH
• molekulární evoluce * MeSH
• pšenice genetika   MeSH
• RNA rostlin genetika   MeSH
• RNA transferová genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• mikro RNA MeSH
• RNA rostlin MeSH
• RNA transferová MeSH

BACKGROUND: The substantially large bread wheat genome, organized into highly similar three sub-genomes, renders genomic research challenging. The construction of BAC-based physical maps of individual chromosomes reduces the complexity of this allohexaploid genome, enables elucidation of gene space and evolutionary relationships, provides tools for map-based cloning, and serves as a framework for reference sequencing efforts. In this study, we constructed the first comprehensive physical map of wheat chromosome arm 5DS, thereby exploring its gene space organization and evolution. RESULTS: The physical map of 5DS was comprised of 164 contigs, of which 45 were organized into 21 supercontigs, covering 176 Mb with an N50 value of 2,173 kb. Fifty-eight of the contigs were larger than 1 Mb, with the largest contig spanning 6,649 kb. A total of 1,864 molecular markers were assigned to the map at a density of 10.5 markers/Mb, anchoring 100 of the 120 contigs (>5 clones) that constitute ~95 % of the cumulative length of the map. Ordering of 80 contigs along the deletion bins of chromosome arm 5DS revealed small-scale breaks in syntenic blocks. Analysis of the gene space of 5DS suggested an increasing gradient of genes organized in islands towards the telomere, with the highest gene density of 5.17 genes/Mb in the 0.67-0.78 deletion bin, 1.4 to 1.6 times that of all other bins. CONCLUSIONS: Here, we provide a chromosome-specific view into the organization and evolution of the D genome of bread wheat, in comparison to one of its ancestors, revealing recent genome rearrangements. The high-quality physical map constructed in this study paves the way for the assembly of a reference sequence, from which breeding efforts will greatly benefit.

• MeSH
• chromozomy rostlin MeSH
• DNA rostlinná analýza   MeSH
• duplikace genu * MeSH
• fyzikální mapování chromozomů metody   MeSH
• genová přestavba * MeSH
• kontigové mapování metody   MeSH
• molekulární evoluce MeSH
• pšenice genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

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.

• MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná analýza   MeSH
• fyzikální mapování chromozomů MeSH
• pšenice genetika   MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• sekvenční analýza DNA metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

Survey sequencing of the bread wheat (Triticum aestivum L.) genome (AABBDD) has been approached through different strategies delivering important information. However, the current wheat sequence knowledge is not complete. The aim of our study is to provide different and complementary set of data for chromosome 4D. A survey sequence was obtained by pyrosequencing of flow-sorted 4DS (7.2×) and 4DL (4.1×) arms. Single ends (SE) and long mate pairs (LMP) reads were assembled into contigs (223Mb) and scaffolds (65Mb) that were aligned to Aegilops tauschii draft genome (DD), anchoring 34Mb to chromosome 4. Scaffolds annotation rendered 822 gene models. A virtual gene order comprising 1973 wheat orthologous gene loci and 381 wheat gene models was built. This order was largely consistent with the scaffold order determined based on a published high density map from the Ae. tauschii chromosome 4, using bin-mapped 4D ESTs as a common reference. The virtual order showed a higher collinearity with homeologous 4B compared to 4A. Additionally, a virtual map was constructed and ∼5700 genes (∼2200 on 4DS and ∼3500 on 4DL) predicted. The sequence and virtual order obtained here using the 454 platform were compared with the Illumina one used by the IWGSC, giving complementary information.

• Klíčová slova
• Chromosome 4D survey sequence, Gene annotation, Gene content, Synteny, Triticum aestivum, Virtual gene order,
• MeSH
• chromozomy rostlin * MeSH
• exprimované sekvenční adresy chemie   MeSH
• mapování chromozomů MeSH
• molekulární sekvence - údaje MeSH
• pořadí genů * MeSH
• pšenice genetika   MeSH
• sekvenční analýza DNA MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.

• MeSH
• chromozomy rostlin genetika   MeSH
• Festuca genetika   MeSH
• genom rostlinný genetika   MeSH
• genomika metody   MeSH
• hybridizace in situ fluorescenční MeSH
• ječmen (rod) genetika   MeSH
• karyotypizace metody   MeSH
• mapování chromozomů MeSH
• molekulární sekvence - údaje MeSH
• pořadí genů MeSH
• reprodukovatelnost výsledků MeSH
• rýže (rod) MeSH
• sekvenční analýza DNA metody   MeSH
• Sorghum genetika   MeSH
• Southernův blotting MeSH
• syntenie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.

• MeSH
• chromozomy chemie   genetika   MeSH
• fyzikální mapování chromozomů metody   MeSH
• genom lidský MeSH
• genomika metody   MeSH
• genová knihovna MeSH
• karyotyp MeSH
• lidé MeSH
• malování chromozomů metody   MeSH
• mitóza MeSH
• průtoková cytometrie metody   MeSH
• rostliny chemie   genetika   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   MeSH
• struktury chromozomu chemie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH