Speciation genetics and genomics in a basidiomycete yeast

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Fungi play pivotal roles in human health, agriculture, forestry, and the environment. Yet, relatively little is known about the factors involved in fungal population dynamics and speciation. Hybrid spore lethality is the hallmark of post-zygotic reproductive isolation and speciation in fungi. However, there is little information about the genetics of post-zygotic reproductive isolation in fungi. The human pathogenic yeast Cryptococcus neoformans has a bipolar mating system with two mating types, MATa and MATalpha and contains two divergent varieties var. neoformans and var. grubii. Hybrids between the two varieties produce basidiospores that have germination rates 80% in intra-varietal crosses. Here we propose to use microbiological, genetic and genomic approaches to investigate the factors contributing to the low hybrid spore viability in C. neoformans. The proposal has four specific aims:***AIM 1: Identify the chromosomal structural differences between strains JEC20 and CDC15. These two strains have been used as parents in our hybrid cross to study hybrid linkage mapping, transgressive segregation, mitochondrial inheritance, and meiotic and mitotic recombination. We have recently obtained the genome sequences of JEC20 and CDC15 to help identify chromosomal rearrangements between them that will be further confirmed individually using locus specific PCR. The distributions of these rearrangements among natural strains will be analyzed.***AIM 2: Assess the effects of environmental factors and individual chromosome rearrangements on hybrid spore viability. Specifically, we will test the effects of media, temperature, and incubation time on basidiospore germination rate and compare those from intra-varietal crosses with hybrid inter-varietal crosses. We will further micro-dissect and analyze ~5000 basidiospores from the JEC20 x CDC15 cross and genotype all germinated spores (estimated at ~900) at the confirmed rearranged chromosomal regions.***AIM 3: Identify the specific incompatibility gene regions involved in hybrid spore lethality through high-density linkage mapping. We will conduct high-density linkage mapping for gene regions containing candidate incompatibility loci identified recently and in AIM 2 using more progeny and more markers.***AIM 4: Test the roles of incompatibility genes through gene knockouts and replacements. We will use molecular biological tools to systematically knockout and replace the candidate incompatibility genes and determine their effects on cell viability and spore germination.******Our research will generate significant new information about the mechanisms controlling post-zygotic reproductive isolation in this important fungus. This research will greatly enhance our understanding of the distributions and dynamics of fungal species and genotypes in human hosts and in agriculture and forestry ecosystems.**