Fluorescent In Situ Hybridization (FISH) on pachytene chromosomes as a tool for genome characterization

A growing number of international genome consortia have initiated large-scale sequencing projects for most of the major crop species. This huge amount of information not only boosted genetic and physical mapping research, but it also enabled novel applications on the level of chromosome biology including molecular cytogenetics that supports plant genetics, genomics, and breeding programs. The simultaneous detection of a large number of BAC-based probes by multicolor fluorescent in situ hybridization (FISH) can provide a rapid overview of super-contig and gap distribution on euchromatin chromosome areas and will display directly and precisely the positions of chromosome rearrangements. Furthermore, hybridizations of BACs on the chromosomes of related species can confirm genomic colinearity, or the occurrence of inversions and translocations events. This cross-species FISH together with meiotic pairing studies is a powerful source of information that elucidates the nature of genome rearrangements, and the consequences of such rearrangements for introgressive hybridizations. In this chapter we describe a general-purpose protocol for FISH on pachytene chromosomes.