Determination of the distribution of fitness effects of mutations in C. elegans

描述(由申请人提供)： 新突变的适应度效应分布在进化生物学中具有重要意义，在复杂疾病和癌症等生物医学中具有实际应用价值。有几种方法可以估计DFE，每种方法都有局限性。来自标准遗传变异的间接估计提供了关于非常弱到中等选择下突变的DFE的信息，但提供了关于强选择突变的很少的信息。相反，从实验数据对DFE的估计提供了有关相对强选择的突变的信息，但只能提供有关弱选择的突变的总体信息。对完整的DFE的全面描述必须综合这两种信息。重要的是，在人类中，除了MOS高度有害的突变外，所有其他突变对健康的影响都无法直接估计。从模式生物中直接估计DFE可以为模拟人类种群遗传过程的努力提供信息，并促进我们对进化的总体理解。线虫线虫提供了理想的模型系统来实验表征DFE：它的世代时间短，无与伦比的遗传资源，以及易于冷冻保存的股票，使其在多细胞生物中独一无二。在目标1和目标2中，将使用在最小自然选择下积累了250代突变的线虫种群来估计DFE，将经典数量遗传学方法与高通量基因分型和表型分析相结合。目标1的目标是(1)通过对一组突变累积(MA)线进行测序来表征全基因组的突变率，以及(2)通过大颗粒流式细胞术(又名“蠕虫分类器”)获得对MA适应性下降的准确估计，以测量非常大量的个体的终生繁殖。马系将与未突变的祖先交配，以产生F1杂合子，其适合性特征类似。在目标2中，一个大的重组近交系(RIAILs)将由两个完全测序的MA系杂交而成。每个RIAIL将在每个突变座位上进行基因分型，单个突变可以被处理为数量性状基因座(QTL)，由此可以统计出纯合子DFE。为了估计杂合DFE，每个RIAIL将与MA系的祖先交配，并在F1后代中确定适合度。此外，如果存在超显性突变(即杂合子优势)，这将是显而易见的；真正超显性的明确证据几乎不存在。在目标1和目标2中，适合性将在几个相关的环境条件下进行评估。在目标3中，将根据162个线虫野生分离物的位置频谱统计确定DFE。这项拟议的工作将提供任何多细胞生物体中最全面的DFE特征，并将为任何应用种群遗传学的研究提供信息。

DESCRIPTION (provided by applicant): The distribution of fitness effects (DFE) of new mutations is of fundamental importance in evolutionary biology and has practical applications in biomedicine, including complex disease and cancer. Several methods exist by which the DFE can be estimated, each of which has limitations. Indirect estimates derived from standing genetic variation provide information about the DFE of mutations under very weak to moderate selection but provide little information about strongly selected mutations. Conversely, estimates of the DFE from experimental data provide information about relatively strongly selected mutations but can only provide information about weakly selected mutations in aggregate. A comprehensive characterization of the full DFE must integrate both kinds of information. Importantly, in humans the fitness effects of all but the mos highly deleterious mutations cannot be estimated directly. Direct estimates of the DFE from model organisms can inform efforts to model human population genetic processes, as well as advance our understanding of evolution in general. The nematode C. elegans provides the ideal model system to in which to experimentally characterize the DFE: its short generation time, unparalleled genetic resources and ease with which stocks can be cryopreserved make it unique among multicellular organisms. In Aims 1 and 2, stocks of C. elegans that have accumulated mutations for 250 generations under minimal natural selection will be employed to estimate the DFE, combining the methods of classical quantitative genetics with high-throughput genotyping and phenotyping. The goals of Aim 1 are (1) characterize the genome-wide mutation rate by sequencing a set of mutation accumulation (MA) lines, and (2) obtain an accurate estimate of the decline in fitness with MA by large-particle flow cytometry (aka a "worm sorter") to measure lifetime reproduction of a very large number of individuals. MA lines will be mated to the unmutated ancestor to generate F1 heterozygotes, which will be similarly characterized for fitness. In Aim 2, a large panel of Recombinant Inbred Advanced Intercross Lines (RIAILs) will be constructed from a cross of two fully-sequenced MA lines. Each RIAIL will be genotyped at each mutant locus and individual mutations can be treated as quantitative trait loci (QTL), from which the homozygous DFE can be inferred statistically. To estimate the heterozygous DFE, each RIAIL will be mated to the ancestor of the MA lines and fitness determined in the F1 offspring. Further, it will be apparent if overdominant mutations (i.e., heterozygote advantage) are present; unambiguous evidence for true overdominance is almost non-existent. In both Aims 1 and 2, fitness will be assessed under several relevant environmental conditions. In Aim 3, the DFE will be determined statistically from the site-frequency spectrum in a set of 162 wild isolates of C. elegans. The proposed work will provide the most comprehensive characterization of the DFE in any multicellular organism, and will inform any study in applied population genetics.