Measuring Meiotic Crossovers via Multi-Locus Genotyping of Single Pollen Grains in Barley.

Steven Dreissig,Andreas Houben,Ed Byrne,Jörg Fuchs,Nicola Kettles,Petr Cápal,Michael Lichten

doi:10.1371/journal.pone.0137677

Abstract

The detection of meiotic crossovers in crop plants currently relies on scoring DNA markers in a segregating population or cytological visualization. We investigated the feasibility of using flow-sorted haploid nuclei, Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multi-locus KASP-genotyping to measure meiotic crossovers in individual barley pollen grains. To demonstrate the proof of concept, we used 24 gene-based physically mapped single nucleotide polymorphisms to genotype the WGA products of 50 single pollen nuclei. The number of crossovers per chromosome, recombination frequencies along chromosome 3H and segregation distortion were analysed and compared to a doubled haploid (DH) population of the same genotype. The number of crossovers and chromosome wide recombination frequencies show that this approach is able to produce results that resemble those obtained from other methods in a biologically meaningful way. Only the segregation distortion was found to be lower in the pollen population than in DH plants.

Highlights

Meiotic recombination is the primary mechanism of generating novel allelic combinations and introducing genetic diversity
The ability to induce an increase in meiotic recombination is so far limited to the model species Arabidopsis thaliana via a mutation of the FANCM helicase [3]
We describe a strategy to perform a parallel analysis of individual haploid nuclei derived from pollen grains by utilizing fluorescence activated cell sorting (FACS) coupled with Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multilocus KASP genotyping

Summary

Introduction

Meiotic recombination is the primary mechanism of generating novel allelic combinations and introducing genetic diversity. They can be identified using molecular markers in a segregating population [5], or alternatively the frequency and distribution of crossovers can be visualized by cytological means like analysis of pairing configurations [6] or immunolabeling of proteins involved in meiotic recombination such as the barley Chen et al [10] developed a method using pollen grains of several plant species for molecular analysis utilizing randomly amplified polymorphic DNA and simple sequence repeat markers.

Results

Conclusion