Progress in Oat FISHIS and Chromosome Specific Sequencing

FISHIS has been developed and validated using tetraploid and hexaploid wheat (Giorgi et al. 2013), showing that appropriate use of labelled oligonucleotides allows single chromosomes and whole sub-genomes from cereals to be separated in sufficient quantity and purity for sequencing (Lucretti et al. 2014; Abstr 16488). We have now begun to adapt the method to allow purification of hexaploid oat chromosomes, and have shown that FISHIS-based flow-sorting allows separation of chromosomes containing specific trinucleotide repeats which would otherwise be indistinguishable by size alone. Using the spring oat cultivar Firth we have established conditions that allow a clear separation of four specific chromosomes, two (18D and 1C) on the basis of size alone and two (9D and 2C) by combination of size and FISHIS with a simple repeat oligonucleotide. DNA from flow-sorted chromosomes can be easily purified in 10-20ng quantities, sufficient to allow Nextera library preparation and comprehensive Illumina sequencing. Genic regions of the sorted chromosomes have been assembled to N50s in excess of 2kb using diploid genome contigs as guides. FISHIS is expected to foster a rapid listing of gene content in these chromosomes.