High‐throughput measuring of meiotic recombination rates in barley pollen nuclei using Crystal Digital PCR^TM

Abstract

Summary Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole‐genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high‐throughput measurements of meiotic recombination rates in barley pollen nuclei without whole‐genome amplification were performed 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 a total of >42 000 individual pollen nuclei (up to 4900 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 a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei.

Technology-driven approaches for meiosis research in tomato and wild relatives

“…Moreover, pollen profiling may be applied to reliably profile meiotic recombination in a wide variety of crop species, including in crops with relatively long generation periods, without the laborious and time-consuming production and screening of offspring populations, greatly benefitting introgression hybridization and precision breeding. A complementary approach to sequencing of pollen DNA is the targeted measurement of meiotic recombination rates in pollen nuclei using Crystal Digital PCR, as recently established in barley (Ahn et al 2021 ). Ultimately, genome-wide and targeted high throughput recombination profiling in pollen will help discover features that determine recombination rates.…”