PROJECT SUMMARY Loss of heterozygosity (LOH) by mitotic recombination is an inevitable outcome of asexual diploid reproduction, and it has been ubiquitously observed in experimental and natural populations of clonal unicellular pathogens. Yet, it is unclear how often these replication errors serve as a source of adaptive variation. In particular, because there is no estimate of the distribution of fitness effects (DFE) of spontaneous LOH events, it is unclear whether the population-level observations of frequent LOH indicate genotypic changes that were beneficial, deleterious, or neutral. The goal of this proposal is to directly address the potential and limitations of LOH to facilitate rapid adaptation of unicellular diploid pathogens by estimating the fitness and pleiotropy of LOH events. Because LOH occurs through a diversity of mechanisms, some of which are precise, such as gene conversion, while others are imprecise, such as nondisjunction, a central hypothesis is that long-distance LOH events are associated with antagonistic pleiotropy that limits adaptability to diverse environments and hosts. Saccharomyces cerevisiae is the ideal model for the study of the evolutionary impacts of LOH because of the wealth of knowledge on mitotic recombination and gene function in the species and the plethora of genetic tools available. The species is an opportunistic pathogen of increasing significance in the clinic that can be studied using simple invertebrate models of infection. To estimate the DFE of LOH events, a randomly-integrated transposon cassette will be used to trigger double strand DNA breaks throughout the genome that stimulate repair by mitotic recombination and LOH. After LOH events are characterized using genome resequencing, the fitness of the resulting transformants will be estimated using competition assays. These assays will be conducted in two animal models (waxworm and nematode) and multiple stressful and chemically varying pure culture environments, and by comparison to fully heterozygous genomes the fitness effect of specific LOH events estimated. These data will be the first robust DFE of LOH in any species, allowing a first approximation of the potential for LOH to drive evolution. The experiment will also explore the limits of adaptation by LOH by testing the relationship between LOH size in base pairs and fitness effect across environments to detect antagonistic pleiotropy. Positively correlated fitness effects across animal models and stressful nutrient conditions will inform the genetic bases of pathogenicity in S. cerevisiae and related fungi. Successfully implemented, this will be the most detailed look at the impact of LOH on rapid evolution of asexual diploids, a group of increasing importance in the clinic.

PROJECT NARRATIVE Loss of heterozygosity is an inevitable outcome of diploid mitosis that is associated with cancer in multicellular organisms and genotypic evolution in unicellular organisms, such as the myriad eukaryotic pathogens. This project will lead to a better understanding of the impact of loss of heterozygosity on the genome and its limitations and potential for driving evolution in pathogenic eukaryotes. 1