Pacurar Daniel I, Pacurar Monica L, Pacurar Andrea M, Gutierrez Laurent, Bellini Catherine
Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden.
Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden; Faculty of Horticulture, University of Agricultural Sciences and Veterinary Medicine, Cluj Napoca, Romania; Present address: SweTree Technologies AB, Umeå, Sweden.
PLoS One. 2014 Jun 23;9(6):e100846. doi: 10.1371/journal.pone.0100846. eCollection 2014.
Map-based cloning (MBC) is the conventional approach for linking phenotypes to genotypes, and has been successfully used to identify causal mutations in diverse organisms. Next-generation sequencing (NGS) technologies offer unprecedented possibilities to sequence the entire genomes of organisms, thereby in principle enabling direct identification of causal mutations without mapping. However, although mapping-by-sequencing has proven to be a cost effective alternative to classical MBC in particular situations, methods based solely on NGS still have limitations and need to be refined. Aiming to identify the causal mutations in suppressors of Arabidopsis thaliana superroot2 phenotype, generated by ethyl methane sulfonate (EMS) treatment, we combined NGS and classical mapping, to rapidly identify the point mutations and restrict the number of testable candidates by defining the chromosomal intervals containing the causal mutations, respectively. The NGS-assisted mapping approach we describe here facilitates unbiased identification of virtually any causal EMS-generated mutation by overlapping the identification (deep sequencing) and validation (mapping) steps. To exemplify the useful marriage of the two approaches we discuss the strategy used to identify a new viable recessive allele of the Arabidopsis CULLIN1 gene in the non-reference Wassilewskija (Ws-4) accession.
基于图谱的克隆（MBC）是将表型与基因型联系起来的传统方法，已成功用于鉴定多种生物体中的因果突变。新一代测序（NGS）技术为对生物体的全基因组进行测序提供了前所未有的可能性，从而原则上能够直接鉴定因果突变而无需进行图谱构建。然而，尽管在特定情况下，测序图谱法已被证明是经典MBC的一种经济有效的替代方法，但仅基于NGS的方法仍有局限性，需要进一步完善。为了鉴定由甲基磺酸乙酯（EMS）处理产生的拟南芥超根2表型抑制子中的因果突变，我们将NGS与经典图谱构建相结合，分别通过定义包含因果突变的染色体区间来快速鉴定点突变并限制可测试候选基因的数量。我们在此描述的NGS辅助图谱构建方法通过重叠鉴定（深度测序）和验证（图谱构建）步骤，有助于无偏倚地鉴定几乎任何由EMS产生的因果突变。为了举例说明这两种方法的有效结合，我们讨论了在非参考生态型瓦西列夫斯基（Ws-4）中鉴定拟南芥CULLIN1基因一个新的可行隐性等位基因所采用的策略。
