Development of a molecular cytogenetic recombination assay for barley

Distal localisation of chiasmata is common to many cereals and grasses, which consigns many genes of the complement to recombination backwaters. Releasing this potential untapped genetic variation for use in advanced breeding programmes is an ambitious and technically demanding challenge, necessitating controlled shifts in the distribution of crossover events. As part of a collaborative programme to manipulate recombination in barley, we are developing a robust and reliable molecular cytogenetic assay for recombination in this species, which will be used to gauge the success of our forward and reverse genetic interventions. Single-locus bacterial artificial chromosome clones and rDNA probes identify the 7 somatic chromosomes of the complement. Meiocytes at pachytene of meiosis were embedded in polyacrylamide and hybridised in situ with centromere and telomere probes, followed by immunolocalisation of the synaptonemal complex-associated protein Asy1 which highlights the bivalents' axes. Optical sectioning, deconvolution and image analysis of the z-stacks of the nuclei allowed the disentanglement of each bivalent and the construction of an accurate meiotic ideogram. The landing of single-locus bacterial artificial chromosomes and the detection of late recombination proteins will complete the assay and provide a means of discerning subtle changes in recombination in this species.