Quantification of Recombination Rate and Segregation Distortion by Genotyping and Sequencing of Single Pollen Nuclei

Meiosis is a specialized cell division during which homologous chromosomes can exchange genetic material through recombination. This mechanism generates novel allelic combinations, which can be exploited by plant breeders to achieve crop improvement. Pollen grains are the haploid products of meiosis required in fertilization. Here, we describe two approaches to measure meiotic recombination in single haploid pollen nuclei. Pollen nuclei are first separated by fluorescence-activated cell-sorting. Afterwards, the DNA of single pollen nuclei can be amplified by multiple-displacement-amplification using Phi29 DNA polymerase and meiotic recombination events can be measured using KASP markers. Alternatively, the PicoPLEX DNA-seq kit can be used to amplify the DNA of single pollen nuclei followed by library preparation for whole-genome sequencing and subsequent bioinformatic analysis.