Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA

Abstract

During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp-kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions.

Interhomolog polymorphism shapes meiotic crossover within the Arabidopsis RAC1 and RPP13 disease resistance genes

“…In order to experimentally measure crossovers within RAC1 and RPP13 we used pollen-typing [68,69]. This method uses allele-specific PCR amplification from F 1 hybrid genomic DNA extracted from gametes, in order to quantify and sequence crossover molecules (Fig 2A and S1 Fig) [68,69]. This method is directly analogous to mammalian sperm-typing methods [70–73].…”

“…Sanger sequencing of PCR products amplified from single crossover molecules is performed to identify internal recombination points, to the resolution of individual polymorphisms (Fig 2A). Together this information describes the recombination rate (cM/Mb) topology throughout the PCR amplified region [68,69]. It is also possible to mass amplify crossover molecules, which may be pooled and sequenced using paired-end reads to identify crossover locations (Fig 2A) [68].…”

Interhomolog polymorphism shapes meiotic crossover withinRAC1andRPP13disease resistance genes

“…2A). Together this information 221 describes the recombination rate (cM/Mb) topology throughout the PCR amplified region 222 Choi et al, 2017). It is also possible to mass amplify crossover 223 molecules, which may be pooled and then used with high throughput paired-end sequencing 224…”

“…performed pollen-seq (Choi et al, 2017(Choi et al, , 2016. In this approach, allele-specific PCR 343…”

“…Pollen-typing was performed as described (Choi et al, 2017 (crossovers/(parentals+crossovers))×100. Amplified single crossover molecules were treated 549 with exonuclease I (NEB, M0293) and shrimp alkaline phosphatase (Amersham, E70092), 550…”