PROJECT SUMMARY/ABSTRACT Over the four billion years that life has evolved on this planet, organisms have acquired amazing phenotypes. Some, like lions' manes and butterflies' wings, capture our attention by their sheer beauty. Others get us excited in a very different way—their relevance to biomedicine. Ecologists have catalogued remarkable disease and stress resistance traits in the plant and animal worlds, which have arisen to solve problems similar to those in human patients. We'd love to know the molecular basis of these natural resistance phenotypes, so that we can design drugs to mimic them in the biomedical context. However, most often, we know about a given trait because it is a defining feature of its respective species, acquired long ago to adapt to a unique niche. Now, millions of years later, the species usually has lost the ability to interbreed with relatives in other environments. And this reproductive isolation is a death knell for existing tools to map genotype to phenotype. The latter, which fill the pages of the modern genetics literature, rely on big panels of recombinant progeny from matings between distinct parents. These tools are no use in the study of species that can't mate to form progeny in the first place. We have developed a new strategy to break through this roadblock, and map the genetic basis of trait variation between long-diverged species. Our approach starts with a viable, but sterile, interspecific hybrid. In this hybrid, at a given gene, we introduce mutations to disrupt each of the two alleles in turn from the two species parents. These hemizygote mutants are identical with respect to background, except that at the target gene, each strain expresses a wild-type allele from only one of the parents. As such, if the hemizygotes differ with respect to a trait of interest, we infer that it must be because of functional allelic variation at the manipulated site. We have pioneered a genome-scale pipeline for this so-called reciprocal hemizygosity test, which we call RH-seq, using yeast as proof of concept. In the current proposal we describe experiments to port RH-seq to mammalian cells. We focus on a little-studied mouse species, M. castaneus, which can regrow axons of the central nervous system after injury. The genes we find in this pioneering study will serve as a springboard for drug design for stroke and brain trauma patients. And our metazoan RH-seq approach will pave the way for the genetic dissection of trait variation between species across Eukarya.