Identification of EMS-Induced Causal Mutations in a Non-Reference Arabidopsis thaliana Accession by Whole Genome Sequencing
Identification of EMS-Induced Causal Mutations in a Non-Reference Arabidopsis thaliana Accession by Whole Genome Sequencing
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DOI：
10.1093/pcp/pcr029
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发表时间：
2011-04-01
影响因子：
4.9
通讯作者：
Tasaka, Masao
中科院分区：
文献类型：
--
作者：
Uchida, Naoyuki;Sakamoto, Tomoaki;Tasaka, Masao
图位克隆法是鉴定由EMS（甲基磺酸乙酯）诱变拟南芥突变的最常用的方法。该方法的第一步是将突变体与另一个登录号的植物杂交，因为它需要登录号之间的多态性用于连锁分析。因此，为了常规地进行该方法，非常优选使用已经知道足够多态性的登录组合。此外，需要使用许多标记对大量F（2）重组体进行费力的检查以检测每个多态性。在连锁分析缩小了含有致病突变的染色体区域后，必须在该区域内逐个对候选基因进行测序，直到最终确定突变。总的来说，这种方法通常是耗时和劳动密集型的，当多个基因座参与表型时，它变得更难。最近的一些报道表明，当在基因组结构众所周知的拟南芥参考哥伦比亚背景中产生突变体时，与常规方法相比，通过深度测序技术可以以更少的劳动来鉴定EMS诱导的因果突变。在这里，我们报告说，我们成功地快速识别EMS诱导的因果突变的非参考登录背景下，其全基因组序列是不公开的，使用一轮全基因组测序。此外，在我们的案例中，我们可以同时监测因果基因座和转基因报告基因座，这意味着这种方法理论上可以适用于分析甚至复杂的性状。我们描述了这种方法的管道，并讨论了它的特点。
The most frequently used method to identify mutations induced by a commonly used mutagen, EMS (ethyl methane sulfonate), in Arabidopsis thaliana has been map-based cloning. The first step of this method is crossing a mutant with a plant of another accession as it requires polymorphisms between accessions for linkage analysis. Therefore, to perform the method routinely, it is greatly preferred to use accession combinations between which enough polymorphisms are already known. Further, it requires laborious examination of a large number of F(2) recombinants using many markers to detect each polymorphism. After linkage analysis narrows down the chromosomal region containing the causal mutation, sequencing candidate genes one by one within the region is necessary until the mutation is finally identified. Overall, this method is generally time-consuming and labor intensive, and it becomes harder when multiple loci are involved in phenotypes. A few recent reports showed that causal mutations induced by EMS could be identified by deep-sequencing technologies with less labor compared with the conventional method when mutants were generated in the Arabidopsis reference Columbia background whose genome organization is well known. Here we report that we succeeded in rapid identification of EMS-induced causal mutations in a non-reference accession background, whose whole genome sequence is not publicly available, using one round of whole genome sequencing. Moreover, in our case, we could monitor the causal locus and the transgenic reporter locus simultaneously, implying that this methodology could theoretically be applicable to analyzing even complex traits. We describe the pipeline of this methodology and discuss its characteristics.