How eukaryotic pathogens explore the fitness landscape by mitotic recombination

描述（由申请人提供）： 有丝分裂重组发生在所有二倍体生物中，但其进化意义在很大程度上被忽视了。有丝分裂重组导致杂合性丢失（洛），这使得它可能是适应性的违反直觉。在大型多细胞生物体中，洛缺失被认为主要是适应不良，并经常与肿瘤发生有关。然而，在缺乏高遗传负荷的自由生活的单倍体阶段的生物体中，例如许多单细胞真菌和原生动物病原体，洛缺失可能是加速有益隐性等位基因固定的重要机制。对二倍体病原体自然种群的研究加强了这一假设，这些研究一致显示了多态性洛基因组区域的证据。这些观察结果表明，洛是普遍存在的，但有一个关键的需要，以解决是否，在什么条件下，以及通过什么机制洛是二倍体病原体如何探索其健身景观的一个重要组成部分。我们的中心假设是洛缺失是正向选择进化的一个重要组成部分，但洛作为一种适应力的相对重要性将与初始基因型或群体的杂合性呈正相关。我们特别感兴趣的是将其应用于病原体的适应度景观，因为它们是众所周知的克隆，至少有1,500种描述的病原性原生动物物种是二倍体，并且病原体似乎具有体内洛率增加。在这里，我们提出了一种新的方法来测试是否杂合性速度的适应率有丝分裂重组酵母蜡蠕虫（Saccharomyces galleria mellonella）的发病机制模型。目的1将克隆进化重复种群内生长的蜡虫幼虫开始从单一的亲本基因型不同的杂合性超过32倍的范围。使用基于绿色荧光蛋白标记的酵母的荧光细胞分选，我们将能够在体内生长48小时后从感染的幼虫中提取纯酵母群体，并且将重复该过程100次连续转移。细胞分选还允许在每次转移时估计病原体适应性，使我们能够测试适应率是否与初始杂合性相关。目标2将使用下一代测序对进化株系进行基因分型，以鉴定重复群体中的平行洛缺失事件，这表明正选择的作用，并鉴定毒力基因。使用这种实验进化方法将使我们能够避免由于小种群规模而导致的遗传漂变和突变积累相关问题。将酵母-Galleria感染模型发展为实验进化系统将提供一种手段，以比标准反向遗传方法更强大的方式来映射病原体基因型、毒力和适应性之间的关系。

DESCRIPTION (provided by applicant): Mitotic recombination occurs in all diploid organisms, but its evolutionary significance has largely been ignored. Mitotic recombination causes loss of heterozygosity (LOH), making it counterintuitive that it could be adaptive. In large multicellular organisms, LOH is considered primarily maladaptive and is frequently associated with tumorigenesis. Yet in organisms with a free-living haploid stage that lack a high genetic load, such as many unicellular fungal and protozoan pathogens, LOH it is likely to be an important mechanism speeding the fixation of beneficial recessive alleles. This assumption is reinforced by studies of natural populations of diploid pathogens that consistently show evidence for polymorphic LOH genomic regions. These observations indicate that LOH is pervasive, however there is a critical need to address whether, under what conditions, and by what mechanisms LOH is an essential component of how diploid pathogens explore their fitness landscapes. Our central hypothesis is that LOH is an important component of evolution by positive selection, but the relative importance of LOH as an adaptive force will be positively correlated with the heterozygosity of the initial genotype or population. We are particularly interested in applying this to the fitness landscape of pathogens because they are notoriously clonal, at least 1,500 described pathogenic protozoon species are diploid, and pathogens appear to have increased rates of LOH in vivo. Here we propose a novel method to test whether heterozygosity speeds the rate of adaptation by mitotic recombination using a yeast-wax worm (Saccharomyces cerevisiae-Galleria mellonella) pathogenesis model. Aim 1 will clonally evolve replicate populations growing inside waxworm larvae initiated from single parental genotypes differing over a 32-fold range of heterozygosity. Using fluorescent cell sorting based on a green fluorescent protein tagged yeast, we will be able to extract pure yeast populations from the infected larvae after 48 hrs of in vivo growth, and this process will be repeated for 100 serial transfers. Cell sorting also allows pathogen fitness to be estimated at each transfer, allowing us to test whether the rate of adaptation is correlated with initial heterozygosity. Aim 2 will use next generation sequencing to genotype the evolved lines to identify parallel LOH events among replicate populations that indicate the action of positive selection and identify virulence genes. Using this experimental evolution approach will allow us to avoid the problems associated genetic drift and mutation accumulation due to small population sizes. Development of the yeast-Galleria infection model into an experimental evolution system will provide a means to map the relationship between pathogen genotype, virulence, and fitness in more powerful way than standard reverse genetic approaches.