Speciation genetics and genomics in a basidiomycete yeast

真菌在人类健康、农业、林业和环境中发挥着关键作用。然而，对真菌种群动态和物种形成的相关因素知之甚少。杂交孢子致死率是真菌合子后生殖分离和物种形成的标志。然而，关于真菌合子后生殖分离的遗传学信息很少。人致病性新隐球菌具有MATa和matα两种交配类型的双极交配系统，包含新隐球菌变种和格鲁比变种两个不同的品种。两个品种之间的杂交产生担子孢子，在品种内杂交中萌发率为80%。在此，我们建议采用微生物学、遗传学和基因组学的方法来研究导致新生芽孢杆菌杂交孢子活力低的因素。目的1：鉴定菌株JEC20和CDC15的染色体结构差异。我们将这两个菌株作为杂交亲本，研究杂交连锁定位、越界分离、线粒体遗传以及减数分裂和有丝分裂重组。我们最近获得了JEC20和CDC15的基因组序列，以帮助鉴定它们之间的染色体重排，并将使用位点特异性PCR进一步单独确认。我们将分析这些重排在自然菌株中的分布。***目的2：评价环境因素和个体染色体重排对杂交孢子活力的影响。具体来说，我们将测试培养基、温度和培养时间对担子孢子发芽率的影响，并比较品种内杂交和品种间杂交的结果。我们将进一步显微解剖和分析来自JEC20与CDC15杂交的约5000个担子孢子，并在确认的重排染色体区域对所有发芽孢子（估计约900个）进行基因型分析。***目的3：通过高密度连锁作图，确定与杂交孢子致死性相关的特异性不亲和性基因区域。我们将利用更多的后代和更多的标记，对最近发现的和AIM 2中含有候选不亲和性位点的基因区域进行高密度连锁定位。***目的4：通过基因敲除和置换检测不亲和基因的作用。我们将使用分子生物学工具系统地敲除和替换候选不相容基因，并确定它们对细胞活力和孢子萌发的影响。******我们的研究将为这种重要真菌的合子后生殖分离控制机制提供重要的新信息。该研究将大大提高我们对人类宿主和农林生态系统中真菌种类和基因型的分布和动态的认识

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.**