High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana

Abstract

During meiosis, reciprocal exchange between homologous chromosomes occurs as a result of crossovers (COs). CO frequency varies within genomes and is subject to genetic, epigenetic and environmental control. As robust measurement of COs is limited by their low numbers, typically 1-2 per chromosome, we adapted flow cytometry for use with Arabidopsis transgenic fluorescent protein-tagged lines (FTLs) that express eCFP, dsRed or eYFP fluorescent proteins in pollen. Segregation of genetically linked transgenes encoding fluorescent proteins of distinct colors can be used to detect COs. The fluorescence of up to 80,000 pollen grains per individual plant can be measured in 10-15 min using this protocol. A key element of CO control is interference, which inhibits closely spaced COs. We describe a three-color assay for the measurement of CO frequency in adjacent intervals and calculation of CO interference. We show that this protocol can be used to detect changes in CO frequency and interference in the fancm zip4 double mutant. By enabling high-throughput measurement of CO frequency and interference, these methods will facilitate genetic dissection of meiotic recombination control.

DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis

“…In contrast, elevated I1b crossovers observed in fancm are not suppressed in fancm zip4 double mutants due to this increase being dependent on noninterfering crossovers ( Fig. 4G; Supplemental Table 17; Crismani et al 2012;Knoll et al 2012;Yelina et al 2013). The fancm met1/+ double mutant showed a further significant increase over fancm alone (GLM, P = 2.19 × 10 −6 ) ( Fig.…”

“…Therefore, we next tested whether interference strength was significantly altered in met1 mutants compared with wild type. Arrangement of three linked FTL transgenes expressing different colors of fluorescent protein allows simultaneous measurement of crossovers in adjacent intervals and calculation of interference (Berchowitz and Copenhaver 2008;Yelina et al 2013). Therefore, we crossed met1 to two independent three-color intervals on chromosomes 1 (I1bc) and 5 (I5ab), both of which are located interstitially.…”

Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination

“…1; Emmanuel et al 2006;Berchowitz and Copenhaver 2008;Yelina et al 2013;Ziolkowski et al 2015). We previously analyzed crossovers in an F 2 population derived from crosses between the Col-420 subtelomeric FTL and Catania-1 (Ct) parents, which did not identify significant trans-acting recombination modifier loci (Ziolkowski et al 2015).…”

“…Berchowitz and Copenhaver 2008;Yelina et al 2013;Ziolkowski et al 2015). In F 2 populations derived from FTL hemizygotes, only a subset of progeny will contain the fluorescent protein-encoding transgenes also in a hemizygous state, which is necessary for crossover measurement.…”

DNA Crossover Motifs Associated with Epigenetic Modifications Delineate Open Chromatin Regions in Arabidopsis

“…One approach is high-throughput screening of many recombination events in a defined region. In this approach, two notable methods are pollen typing (Drouaud et al, 2013) and the use of fluorescent markers in seed (Melamed-Bessudo et al, 2005) and in pollen tetrads (Preuss et al, 1994;Francis et al, 2006Francis et al, , 2007Berchowitz and Copenhaver, 2008;Sun et al, 2012;Yelina et al, 2013). With the recent advent of high-throughput sequencing technologies, it became possible to map historical recombination events using linkage disequilibrium, as has been done for human, mice, Arabidopsis thaliana, and maize (Zea mays) (Myers et al, 2008;Gore et al, 2009;Brunschwig et al, 2012;Choi et al, 2013).…”