Flow sorting and sequencing meadow fescue chromosome 4F.

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 it has been used in intergeneric hybridization with ryegrasses to produce Festulolium cultivars. In this work we describe a new approach to analyze the large genome of meadow fescue, which involves 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 ~50x coverage. This provided the first insight into the composition of the fescue genome, enabled the construction of virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice, Brachypodium, Sorghum and barley. Using GenomeZipper, we were able to confirm collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H (5HL). Several new tandem repeats were identified and physically mapped using FISH. 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 a 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 on chromosome structure and evolution at a resolution never achieved before