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.
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
Species belonging to the Festuca-Lolium complex are important forage and turf species and as such, have been studied intensively. However, their out-crossing nature and limited availability of molecular markers make genetic studies difficult. Here, we report on saturation of F. pratensis and L. multiflorum genetic maps using Diversity Array Technology (DArT) markers and the DArTFest array.The 530 and 149 DArT markers were placed on genetic maps of L. multiflorum and F. pratensis, respectively, with overlap of 20 markers, which mapped in both species. The markers were sequenced and comparative sequence analysis was performed between L. multiflorum, rice and Brachypodium. The utility of the DArTFest array was then tested on a Festulolium population FuRs0357 in an integrated analysis using the DArT marker map positions to study associations between markers and freezing tolerance. Ninety six markers were significantly associated with freezing tolerance and five of these markers were genetically mapped to chromosomes 2, 4 and 7. Three genomic loci associated with freezing tolerance in the FuRs0357 population co-localized with chromosome segments and QTLs previously identified to be associated with freezing tolerance. The present work clearly confirms the potential of the DArTFest array in genetic studies of the Festuca-Lolium complex. The annotated DArTFest array resources could accelerate further studies and improvement of desired traits in Festuca-Lolium species.
- MeSH
- Brachypodium genetika MeSH
- chromozomy rostlin MeSH
- Festuca genetika MeSH
- fyziologická adaptace genetika MeSH
- genetická vazba MeSH
- genetické markery MeSH
- jílek genetika MeSH
- lidé MeSH
- mapování chromozomů metody MeSH
- molekulární sekvence - údaje MeSH
- polymorfismus genetický MeSH
- rýže (rod) genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- syntenie MeSH
- zmrazování MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
