title: 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 F1heterozygous 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.
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK.
authors.forename: Kyuha
authors.initials: K
authors.lastname: Choi
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK.
authors.forename: Nataliya E
authors.initials: NE
authors.lastname: Yelina
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK.
authors.forename: Heïdi
authors.initials: H
authors.lastname: Serra
authors.affiliation: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK. irh25@cam.ac.uk.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Henderson
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D003434:Crossing Over, Genetic / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics*; D008540:Meiosis / Q000235:genetics; D011058:Pollen / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant
keywords: Allele-specific PCR; Crossover; Meiosis; Pollen DNA; Recombination; Titration