higher crossover rate (hcr) mutant
High-throughput genetic screen for crossover frequency mutants. (A) Segregations of cis-fluorescent GFP and RFP (5 Mb, physical distance) on chromosome 3 during meiosis to produce parental gametes or recombinants. (B) A scheme of high-throughput forward genetic screening to isolate new recombination mutants by using seed-based recombination assay system. (C) Sensitive high-throughput crossover measurement of fluorescent seeds from individual M2 plants for mutant screening by running a Cell Profiler software. (D) Isolation of altered recombination rate mutants from M2 plants by the sensitive high-throughput genetic screen.
New high-throughput genetic screening: Arabidopsis thaliana has proven to be a successful model plant for forward genetics. The Arabidopsis genes involved in meiotic recombination have been identified through genetic screen of mutants with reduced fertility or characterized by reverse genetics using meiotic genes identified in yeast and mammals. However, forward genetic screening for altered recombination rate mutants has not yet been performed, due to a limitation robustly of measuring recombination from a single plant in a high-throughput manner.
The seed-based fluorescent reporter (420 GFP-RFP (GR)/++) system has been used to systemically detect significant changes of recombination rate in mutants such as arp6 (16.5 cM), met1 (25 cM) and fancm (35 cM), compared to wild type plants (20.5 cM) (Choi et al., 2013). Because this seed-based system is so sufficiently sensitive to detect a broad range of recombination rate changes, it will allow to discover many mutants with altered recombination rates (Figure). We anticipate this screen will reveal new meiotic factors which are associated with chromatin accessibility of the DSB machinery, chromosome organization, homolog pairing, DSBs repair processes, resolution of intermediate molecules and crossover interference for regulation of meiotic recombination in plants and potentially other species.
Isolation of higher crossover rate (hcr) mutants: The fluorescent seed-based system requires the hemizygote state of two cis-reporters for scoring, thus 10,000 M0 hemizygote seeds (420 GR/++) were prepared for ethyl-methanesulfonate (EMS) mutagenesis (Figure). The M2 seeds had been harvested from 7,000 EMS-treated M1 plants, pooled into 600 groups of 12 plants. In a preliminary screening, we have isolated and characterized three higher crossover rate mutants (hcr1 (27 cM), hcr2 (40 cM), hcr3 (30 cM)). We have mapped the hcr1 by sequencing-by-mapping in Dr Henderson laboratory. HCR1 encodes a novel protein limiting crossovers in Arabidopsis, further demonstrating that the genetic screening system is successful. The hcr2 and hcr3 mutants will be intensively characterized in Kyuha’s lab within the first 1-3 years, from mapping of the causal mutation site to molecular and genomic analyses. Studies on hcr2 and hcr3 will elucidate novel factors which limit crossovers, and the information can be transferred to a plant molecular breeding program for recombining or mapping of useful variations in crops.