A simple and high-throughput single pollen genotyping pipeline to study meiosis in apple (Malus x domestica).

Abstract

<h4>Background</h4>Pollen are the primary carrier of male genetic information during sexual reproduction. In Angiosperms, pollen are produced in the anthers following the process of meiosis, during which ploidy halving and genetic recombination occurs, mixing the parental genomes and creating allelic variation. Therefore, the genetic configuration of pollen is of great interest for the study of meiotic recombination and cell division as well as genetic variation in general, having applications in plant genetics, breeding, and evolution. Single pollen genotyping recently emerged as a powerful tool for the genetic characterization of the male germline, allowing a more detailed analysis of genetic diversity and reproductive behavior down to the level of the individual gamete. However, current methods for single pollen genotyping rely on manual sorting and use of expensive whole genome amplification technology.<h4>Results</h4>We developed a simple and straightforward pipeline for single pollen genotyping in apple, an economically important fruit crop. In brief, this method includes filter bursting to efficiently release pollen nuclei followed by isolation of single nuclei via fluorescence-activated cell sorting. Subsequently, single nuclei are directly PCR genotyped, resulting in up to 85% amplification success without the use of whole genome amplification. Using this optimized pipeline, we developed a multiplex genotyping toolbox for apple which can be used to study meiotic recombination, meiotic restitution, and cross-over interference.<h4>Conclusions</h4>Overall, this new single pollen genotyping procedure for apple enables high-throughput genotyping of large pollen populations in a single cell manner, facilitating reproductive research in apple as a non-model crop species with potential for translation to other fruit species.