Project Summary Hybrid dysfunction offers an unparalleled window into the evolutionary drivers and genetic basis of divergence in key developmental processes. Hybrid dysfunction can also confer reproductive isolation and contribute to speciation. Intra- genomic conflicts may play a central role in this divergence, and underlie hybrid dysfunction, but empirical tests are few. A common source of conflict in viviparous organisms stems from conflict between dams and sires for resource allocation to developing offspring (e.g. parental conflict). Parental conflict may drive the evolution of genomic imprinting; an epigenetic phenomenon whereby alleles have parent-of-origin-specific expression. Under parental conflict, misregulation of imprinted genes causes abnormal placenta or endosperm development, and subsequently hybrid embryo death. This early onset inviability is common in both mammalian and plant hybrids, but the underlying genes are unknown. I will address this knowledge gap by pairing population and quantitative genomics, gene expression, and functional genetics to identify the genetic basis of hybrid seed inviability and test the role of parental conflict in generating this barrier. I recently discovered a species in one of the most widely studied models for genetics and evolution- the Mimulus guttatus species complex. Despite a recent split (~230KYA), M. decorus and M. guttatus are reproductively isolated via hybrid seed inviability caused by atypical endosperm development. Hybrid seed inviability has rapidly and repeatedly evolved in this group, with at least two independent incidences in ~230KYA. This exceptional diversity in reproductive isolation, paired with the tremendous genetic and genomic resources of M. guttatus makes it an ideal system to study the genetic basis of hybrid seed inviability. By quantifying expression differences among species, I will characterize the epigenomic landscape of imprinting and assess if divergence in imprinting is driven by natural selection. In tandem, I will map the genetic basis of multiple incidences of hybrid seed inviability to assess the extent of overlap in inviability loci and estimate how repeatable conflict-driven evolution is. I will then identify candidate genes and functionally test them by constructing transgenic lines that alter imprinted gene expression and quantify if abnormal imprinted expression causes inviability; a central prediction of parental conflict. Lastly, using replicated contact zones, I will assess the dynamics of introgression between these species to estimate the efficacy of hybrid seed inviability as a barrier to gene flow. By using patterns of ancestry disequilibrium, I can also identify other putative incompatibility alleles. As I have generated a set of immortal inbred lines from these contact zones, I can then resurrect specific genotypes, perform manipulative crosses, and identify putative incompatibility phenotypes, thus bridging long term patterns of selection wi...