Harrington Sophie A, Cobo Nicolas, Karafiátová Miroslava, Doležel Jaroslav, Borrill Philippa, Uauy Cristobal
John Innes Centre, Norwich, United Kingdom.
Department of Plant Sciences, University of California, Davis, Davis, CA, United States.
Front Plant Sci. 2019 Jul 24;10:963. doi: 10.3389/fpls.2019.00963. eCollection 2019.
Durum wheat () derives from a hybridization event approximately 400,000 years ago which led to the creation of an allotetraploid genome. The evolutionary recent origin of durum wheat means that its genome has not yet been fully diploidised. As a result, many of the genes present in the durum genome act in a redundant fashion, where loss-of-function mutations must be present in both gene copies to observe a phenotypic effect. Here, we use a novel set of induced variation within the cv. Kronos TILLING population to identify a locus controlling a dominant, environmentally dependent chlorosis phenotype. We carried out a forward screen of the sequenced cv. Kronos TILLING lines for senescence phenotypes and identified a line with a dominant early senescence and chlorosis phenotype. Mutant plants contained less chlorophyll throughout their development and displayed premature flag leaf senescence. A segregating population was classified into discrete phenotypic groups and subjected to bulked-segregant analysis using exome capture followed by next-generation sequencing. This allowed the identification of a single region on chromosome 3A, (), which was associated with the mutant phenotype. While this phenotype was consistent across 4 years of field trials in the United Kingdom, the mutant phenotype was not observed when grown in Davis, CA (United States). To obtain further SNPs for fine-mapping, we isolated chromosome 3A using flow sorting and sequenced the entire chromosome. By mapping these reads against both the cv. Chinese Spring reference sequence and the cv. Kronos assembly, we could identify high-quality, novel EMS-induced SNPs in non-coding regions within that were previously missed in the exome capture data. This allowed us to fine-map to 4.3 Mb, containing 59 genes. Our study shows that populations containing induced variation can be sources of novel dominant variation in polyploid crop species, highlighting their importance in future genetic screens. We also demonstrate the value of using cultivar-specific genome assemblies alongside the gold-standard reference genomes particularly when working with non-coding regions of the genome. Further fine-mapping of the locus will be pursued to identify the causal SNP underpinning this dominant, environmentally dependent phenotype.
硬粒小麦（）起源于约40万年前的一次杂交事件，该事件导致了异源四倍体基因组的产生。硬粒小麦进化起源较近，这意味着其基因组尚未完全二倍体化。因此，硬粒小麦基因组中存在的许多基因以冗余方式起作用，即功能丧失突变必须同时存在于两个基因拷贝中才能观察到表型效应。在此，我们利用硬粒小麦品种Kronos定向诱导基因组局部突变群体中的一组新的诱导变异，来鉴定一个控制显性、环境依赖性黄化表型的基因座。我们对已测序的Kronos定向诱导基因组局部突变系进行了衰老表型的正向筛选，鉴定出一个具有显性早期衰老和黄化表型的品系。突变植株在整个发育过程中叶绿素含量较低，并表现出旗叶过早衰老。一个分离群体被分类为离散的表型组，并使用外显子捕获随后进行下一代测序进行混合分组分析。这使得能够鉴定出3A染色体上的一个单一区域（），该区域与突变表型相关。虽然这种表型在英国的4年田间试验中是一致的，但在美国加利福尼亚州戴维斯种植时未观察到突变表型。为了获得更多用于精细定位的单核苷酸多态性（SNP），我们通过流式细胞分选分离出3A染色体并对整个染色体进行测序。通过将这些读数与中国春品种参考序列和Kronos品种组装序列进行比对，我们可以在以前外显子捕获数据中遗漏的内非编码区域中鉴定出高质量的、新的甲基磺酸乙酯（EMS）诱导的SNP。这使我们能够将精细定位到4.3兆碱基，包含59个基因。我们的研究表明，含有诱导变异的群体可以成为多倍体作物新显性变异的来源，突出了它们在未来遗传筛选中的重要性。我们还证明了在使用黄金标准参考基因组的同时使用品种特异性基因组组装的价值，特别是在处理基因组非编码区域时。将对基因座进行进一步的精细定位，以鉴定支撑这种显性、环境依赖性表型的因果SNP。
