High throughput measuring of meiotic recombination rates in barley pollen nuclei using Crystal Digital PCRTM.

Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measuring meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far established methods rely on whole-genome amplification (WGA) of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, we measured meiotic recombination rates in barley pollen nuclei without WGA in high-throughput through a Crystal Digital PCRTM-based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping in total more than 42,000 individual pollen nuclei (up to 4,900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCRTM enables reliable, rapid and high-throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from different tissues from a range of species with different nuclear and genome sizes suggests, that the proposed method is broadly applicable to genotype single nuclei. This article is protected by copyright. All rights reserved.