Flow sorting and sequencing meadow fescue chromosome 4F.

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.

Kopecký D ，Martis M ，Číhalíková J ，Hřibová E ，Vrána J ，Bartoš J ，Kopecká J ，Cattonaro F ，Stočes Š ，Novák P ，Neumann P ，Macas J ，Šimková H ，Studer B ，Asp T ，Baird JH ，Navrátil P ，Karafiátová M ，Kubaláková M ，Šafář J ，Mayer K ，Doležel J ... -  《-》

Exploring the tertiary gene pool of bread wheat: sequence assembly and analysis of chromosome 5M(g) of Aegilops geniculata.

Next-generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole-genome shotgun sequencing is cost-prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow-sorted chromosome 5M(g) from a wheat/Aegilops geniculata disomic substitution line [DS5M(g) (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired-end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M(g) , in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single-gene FISH indicated no major chromosomal rearrangements between chromosomes 5M(g) and 5D. Comparing chromosome 5M(g) with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M(g) -specific SNPs and cytogenetic probe-based resources were developed and validated. Deletion bin-mapped and ordered 5M(g) SNP markers will be useful to track 5M-specific introgressions and translocations. This study provides a detailed sequence-based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.

Tiwari VK ，Wang S ，Danilova T ，Koo DH ，Vrána J ，Kubaláková M ，Hribova E ，Rawat N ，Kalia B ，Singh N ，Friebe B ，Doležel J ，Akhunov E ，Poland J ，Sabir JS ，Gill BS ... -  《-》

Gene content and virtual gene order of barley chromosome 1H.

Chromosome 1H (approximately 622 Mb) of barley (Hordeum vulgare) was isolated by flow sorting and shotgun sequenced by GSFLX pyrosequencing to 1.3-fold coverage. Fluorescence in situ hybridization and stringent sequence comparison against genetically mapped barley genes revealed 95% purity of the sorted chromosome 1H fraction. Sequence comparison against the reference genomes of rice (Oryza sativa) and sorghum (Sorghum bicolor) and against wheat (Triticum aestivum) and barley expressed sequence tag datasets led to the estimation of 4,600 to 5,800 genes on chromosome 1H, and 38,000 to 48,000 genes in the whole barley genome. Conserved gene content between chromosome 1H and known syntenic regions of rice chromosomes 5 and 10, and of sorghum chromosomes 1 and 9 was detected on a per gene resolution. Informed by the syntenic relationships between the two reference genomes, genic barley sequence reads were integrated and ordered to deduce a virtual gene map of barley chromosome 1H. We demonstrate that synteny-based analysis of low-pass shotgun sequenced flow-sorted Triticeae chromosomes can deliver linearly ordered high-resolution gene inventories of individual chromosomes, which complement extensive Triticeae expressed sequence tag datasets. Thus, integration of genomic, transcriptomic, and synteny-derived information represents a major step toward developing reference sequences of chromosomes and complete genomes of the most important plant tribe for mankind.

Mayer KF ，Taudien S ，Martis M ，Simková H ，Suchánková P ，Gundlach H ，Wicker T ，Petzold A ，Felder M ，Steuernagel B ，Scholz U ，Graner A ，Platzer M ，Dolezel J ，Stein N ... -  《-》

The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics.

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.

Pfeifer M ，Martis M ，Asp T ，Mayer KF ，Lübberstedt T ，Byrne S ，Frei U ，Studer B ... -  《-》

Repetitive DNA: A Versatile Tool for Karyotyping in Festuca pratensis Huds.

FISH is a useful method to identify individual chromosomes in a karyotype and to discover their structural changes accompanying genome evolution and speciation. DNA probes for FISH should be chromosome specific and/or exhibit specific patterns of distribution along each chromosome. Such probes are not available in many plants including meadow fescue (Festuca pratensis Huds.), an important forage grass species. In the present study, various DNA repeats identified in Illumina shotgun sequences specific to chromosome 4F of F. pratensis were used as probes for FISH to develop the molecular karyotype of meadow fescue and to reveal a long-range molecular organization of its chromosomes. Five tandem repeats produced specific patterns on individual chromosomes. Their use in combination with probes for rRNA genes enabled the establishment of the molecular karyotype of meadow fescue. Most of the mobile genetic elements were dispersed along all the chromosomes except for the DNA transposon CACTA, which was localized preferentially to telomeric and subtelomeric regions, and a putative LTR element, which was localized to (peri)centromeric regions. Cytogenetic mapping of the 5 tandem repeats in other accessions of meadow fescue showed a highly similar distribution and confirmed the versatility and robustness of these probes.

Křivánková A ，Kopecký D ，Stočes Š ，Doležel J ，Hřibová E ... -  《-》