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至关重要的作用。然而，人们对真菌种群动态和物种形成所涉及的因素知之甚少。杂交孢子致死是真菌合子后生殖隔离和物种形成的标志。然而，关于真菌合子后生殖隔离的遗传学信息很少。人致病酵母新生隐球菌具两极交配系统，有两种交配类型：MATA和MATAlpha，并含有两个不同的变种Var。新陈代谢和变异体。格鲁比。这两个品种之间的杂交产生的担孢子萌发率为20%，而品种内杂交的担孢子发芽率为80%。在这里，我们建议使用微生物学、遗传学和基因组学方法来研究导致新生隐孢子菌低杂交孢子活力的因素。该提案有四个具体目标： 目的1：鉴定JEC20株和CDC15株的染色体结构差异。这两个菌株在我们的杂交组合中被用作亲本，用于研究杂交连锁定位、超亲分离、线粒体遗传以及减数分裂和有丝分裂重组。我们最近已经获得了JEC20和CDC15的基因组序列，以帮助识别它们之间的染色体重排，这些重排将通过位点特异性PCR进一步单独确认。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。具体地说，我们将测试培养基、温度和孵化时间对担孢子萌发率的影响，并比较品种内杂交和品种间杂交的担孢子萌发率。我们将进一步对来自JEC20×CDC15杂交的约5000个担孢子进行显微解剖和分析，并在确认的重排染色体区域对所有萌发的孢子(估计约为900个)进行基因分型。 目的3：通过高密度连锁定位鉴定与杂种孢子致死相关的特异不亲和性基因区域。我们将利用更多的后代和更多的标记，对最近发现的候选不亲和基因座和AIM 2中包含候选不亲和基因座的基因区域进行高密度连锁定位。 目的4：通过基因敲除和替换来检测不亲和基因的作用。我们将利用分子生物学工具系统地敲除和替换候选不亲和基因，并确定它们对细胞活力和孢子萌发的影响。 我们的研究将为控制这种重要真菌合子后生殖隔离的机制提供重要的新信息。这项研究将极大地加深我们对真菌在人类寄主和农林生态系统中的分布和动态的了解。

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 <20%, in contrast to the >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.