Assaying functional divergence in the hybrid incompatibility gene Hybrid male rescue (Hmr) between D. melanogaster and D. simulans. Tawny N. Cuykendall, Daniel A. Barbash. Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY.

   Hybrid lethality and sterility are examples of post-zygotic reproductive isolating barriers between species. Crosses between Drosophila melanogaster females and D. simulans males, which diverged ~3 MYA, produce viable but sterile daughters, and sons which die as 3^rd instar larvae. Lethality is caused by an epistatic interaction in the hybrid background between D. melanogaster Hybrid male rescue (Hmr) and D. simulans Lethal hybrid rescue (Lhr). The hybrid lethal activity of Hmr is unique to the D. melanogaster ortholog. We are interested in explaining this functional divergence of Hmr between D. melanogaster and D. simulans. Our experiments have two goals: 1) to determine the intraspecific function of HMR and 2) to identify functions of Hmr that have diverged between orthologs. Using a 3X-HA epitope-tagged Hmr transgene injected in D. melanogaster, we find that HMR, like LHR, localizes to heterochromatin. We have used fluorescent in situ hybridization (FISH) to more finely map HMR to specific satellite sequences in the D. melanogaster genome, including dodeca, 2L3L, and AG-rich repeats. We have also generated a transgenic line expressing D. simulans Hmr in a D. melanogaster background and are currently testing whether D. simulans HMR localizes to heterochromatin in a foreign species background. Additionally, we are performing RNA-seq on transgenic lines expressing either D. melanogaster Hmr or D. simulans Hmr in an Hmr mutant background to investigate how HMR affects gene expression and whether it has diverged between these two species with respect to its function in gene regulation.