Meiosis, the reductive division of the genome in preparation for fertilization, is a critical phase in the life-cycle of sexually reproducing organisms. During meiosis homologous chromosomes interact resulting in the heritable rearrangement of DNA, through reciprocal exchange between homologous chromosomes (crossing over, CO) or gene conversion (GC). In most eukaryotes these events ensure proper chromosome segregation, facilitate DNA repair and provide a basis for genetic diversity. Detailed models describing recombination mechanisms have been proposed and measurements of the frequency, and distribution of GC events can be used to test the accuracy of these models. Meiotic recombination also causes the breakdown linkage disequilibrium (LD), the non-random association of sets of alleles at linked loci, over evolutionary time which population geneticists use to better understand how species, their phenotypes, and their genomes evolve. Recent models suggest that GC may be as important as CO in breaking down LD in some genomic regions. The immediate goals of this project are to measure GC frequency at multiple loci in two distinct organisms, the plant Arabidopsis thaliana and the metazoan Drosophila melanogaster. The investigators will also make population genetic estimates of recombination at the same loci and compare these estimates with direct measurements of GC and CO in order to refine population genetic models of recombination and its effect on genome wide patterns of LD. The long-term goal of the project is to understand how meiotic recombination operates in multi-cellular organisms and how it influences genome variation and evolution. In addition to deepening our understanding of a classic genetic problem - gene conversion - this proposal also provides broader impact by building truly interdisciplinary collegial partnerships, creating a platform for participation by junior scientists from underrepresented groups and emphasizing graduate level education in the form of first-hand research and collaboration.
Project Summary: The immediate objectives of this grant were to measure meiotic gene conversion (GC) frequencies at multiple sites in the genome of two organisms, A. thaliana and D. melanogaster. Meiotic GC is the non-reciprocal exchange of sequence information between chromosomes during the production of gametes. GC is important because it influences allelic diversity, it is a conserved feature of recombination in all species that reproduce sexually and, understanding its molecular mechanisms may have practical implications for genetic engineering. In addition to deepening our understanding of a classic genetic problem – gene conversion – this proposal also provided broader impact by: building truly interdisciplinary collegial partnerships, creating a platform for participation by junior scientists from underrepresented groups and emphasizing graduate level education in the form of first-hand research and collaboration. There were three specific aims: Results From Aim 1: We created a novel visual assay that enabled us to measure GC directly in the gametes of Arabidopsis. Using this assay we scored 1,204,573 meioses and observed 202 GC events. Based on these observations we conclude the genome-wide average GC frequency is 1/2982 meioses. Of particular interest was a test locus on chromosomes 4 that experienced elevated GC (1/936 meioses). Because GC events are experienced frequently at this locus it will be useful for examining GC frequencies in mutant lines in future experiments. Previously, we had observed that crossing over is elevated when plants are heat stressed and on the secondary branches of plants (i.e. a developmental difference). We measured GC in both these contexts and found that GC was also elevated in heat-stressed plants but was diminished on secondary branches. This suggests that under heat stress more double strand breaks are created (since both crossing over and GC increased) whereas the developmental increase in cross overs came at the expense of GC. Results From Aim 2: We built an assay to measure crossover and non-crossover (observed as GC) meiotic recombination in Drosophila using a pair of transgenes, each carrying a mutated copy of the rosy (ry) gene, and each inserted into identical locations on the X chromosome. We first measured recombination at a site in the middle of the arm, where crossovers are typically high. From 867,000 larvae, we recovered 6 crossovers and 10 non-crossover GCs. We repeated this experiment at a site toward the end of the X chromosome, where meiotic crossovers are typically very low. The number of non-crossover gene conversions was similar to the other site (7 from 867,000 meioses). Surprisingly, the number of crossovers was also similar (9). This suggests that the recombination frequency is intrinsic to the DNA segment. We are currently testing two additional sites on the X chromosome. We have also inserted this assay onto the small chromosome 4. There are normally no meiotic crossovers on this chromosome, but population sequence analyses suggest that gene conversion might occur. Our experiments will be the first to directly measure gene conversion. It will be of great interest to determine whether our transgene carries the ability to make crossovers on this chromosome. Results From Aim 3: This aim focused on using genome wide sequence data to estimate the population genetic recombination rate (ρ) for a wild population of Drosophila. Our goal was to compare these estimates with the empirical estimates of crossing-over and GC estimated by Dr. Sekelsky. While we succeeded in estimating ρ for whole genome re-sequencing data from 47 Drosophila genomes, we are awaiting completion of the empirical estimates. Preliminary data suggest that ρ varies by up to three orders of magnitude across the genome. The rank correlation between regional rates of crossing over estimate from the D. melanogaster genetic map and the population genetic estimate of recombination, shows a relationship (rs = 0.29), but there is considerable variation around that trend. As a group, regions of extremely low crossing over appear to be driving the relationship. These same regions have some of the most intriguing disconnects. In centromeric regions and on the 4th chromosome, which are normally areas of very low crossing-over, ρ ranged from 0.0001-0.01, which was higher than expected (especially for the 4th chromosome which normally does not undergo crossovers in Drosophila). This suggests that gene-conversion maybe driving the disconnect between empirical estimates of crossing-over and ρ. Results from Broader Impacts and Outreach Efforts: We created a new course entitled "Meiosis, Recombination, and Sex" that emphasizes critical evaluation of literature on meiosis. We offered the course five times and had a total enrollment of 47 students. Each of the participating PIs recruited several undergraduates to work on the project. These students participated as research-for-credit, NSF- REU summer fellows, or SMART (Science and Math Achievement and Resourcefulness Track Program) awardees.
