Weiss Carly V, Chuong Julie N, Brem Rachel B
Department of Plant and Microbial Biology, University of California Berkeley; Department of Biology, Stanford University.
Buck Institute for Research on Aging.
J Vis Exp. 2019 Aug 12(150). doi: 10.3791/59972.
A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.
现代遗传学的一个核心目标是了解野生生物在表型上存在差异的方式和原因。迄今为止，该领域的进展主要依赖于连锁和关联图谱绘制方法，这些方法追踪物种个体杂交产生的重组后代中DNA序列变异与表型之间的关系。这些方法虽然强大，但并不适合用于研究生殖隔离物种之间的性状差异。在此，我们描述了一种新的全基因组剖析自然性状变异的方法，该方法可轻松应用于不兼容的物种。我们的策略RH-seq是相互半合子测试在全基因组层面的应用。我们利用它来鉴定酿酒酵母相对于其姊妹物种奇异酵母在高温下显著生长的相关基因。RH-seq利用转座子诱变创建一组相互半合子，然后通过高通量测序在高温竞争中对其进行追踪。我们在此阐述的RH-seq工作流程为剖析芽殖酵母进化枝中古老、复杂的性状提供了一种严谨、无偏的方法，但需要进行资源密集型的深度测序以确保遗传图谱的基因组覆盖范围。随着测序成本的下降，这种方法在未来真核生物研究中具有巨大的应用前景。
