Simplifying Complex Genomes: Flow Sorting Single Chromosomes and Whole Genomes after Fluorescent in situ Hybridization in Suspension - FISHIS

Genome complexity of major crop plants, which results from polyploidy and high abundance of non-genic repetitive sequences, has slow down the progress in the exploitation of genomic technologies. By enabling genome dissection into single chromosomes or chromosome arms via flow-sorting, the ‘chromosome approach’ can contribute to reduce genome complexity. It proved to be very effective in flow-sorting the large bread wheat genome in small and significant parts, being all the chromosome-size cytogenetic mutants available in the genetic background of a specific variety (e.g. T. aestivum Chinese Spring), not one of major agricultural interest. Such an approach is unpractical for the most of plants and animals, precluding an easier development of genome sequencing and assembling, even in the case of complex genomes. A new method has been developed named FISHIS ‘‘Fluorescence In Situ Hybridization In Suspension’’, allowing the coupling of the high discriminatory capabilities of FISH labeling and the high-throughput of flow-sorting to isolate pure chromosome and nuclei fractions, regardless of the availability of cytogenetic stocks. FISHIS is a easy method that relies on readily available fluorescent labeled DNA simple sequence repeats (e.g. microsatellites SSR) and employs alkaline DNA denaturation. This new technique allowed the isolation of i) the entire chromosome set of a single genome from the homeologus ones in polyploid pasta (genome A and B) and bread wheat (genome A, B and D, with the exception of a contamination from chromosome 1A in the latter); ii) several single-type chromosomes in a number of Triticeae (e.g. the entire chromosome complement of the wild grain mosquitograss); iii) the isolation of each of the three genomes present in the hexaploid Triticale cv Oceano