Crystal Digital PCR™-Based Single Nucleus Pollen Genotyping in Barley (Hordeum vulgare L.) to Measure Meiotic...

Genotyping-based meiotic crossover (CO) rate measurement within defined chromosomal regions typically relies on the segregation analysis of linked markers (polymorphism) in hybrid offspring populations. Pollen (male gametes) nucleus genotyping can be an alternative as pollen is abundant even from single individuals. Hence, CO rates can be measured without segregation distortion and growing offspring populations. Here, we present a protocol for meiotic recombination and CO interference rate measurements within two-linked chromosomal regions in hybrid barley pollen nuclei using multiplex Crystal digital PCR (dPCR)-based single pollen nucleus genotyping on the Naica™ Crystal Digital PCR platform. It consists of hybrid pollen isolation, flow sorting of single pollen nuclei, and their genotyping through dPCR.