Data_Sheet_5_Discovery of miRNAs and Development of Heat-Responsive miRNA-SSR Markers for Characterization of Wheat Germplasm for Terminal Heat Tolerance Breeding.docx

A large proportion of the Asian population fulfills their energy requirements from wheat (Triticum aestivum L.). Wheat quality and yield are critically affected by the terminal heat stress across the globe. It affects approximately 40% of the wheat-cultivating regions of the world. Therefore, there is a critical need to develop improved terminal heat-tolerant wheat varieties. Marker-assisted breeding with genic simple sequence repeats (SSR) markers have been used for developing terminal heat-tolerant wheat varieties; however, only few studies involved the use of microRNA (miRNA)-based SSR markers (miRNA-SSRs) in wheat, which were found as key players in various abiotic stresses. In the present study, we identified 104 heat-stress-responsive miRNAs reported in various crops. Out of these, 70 miRNA-SSR markers have been validated on a set of 20 terminal heat-tolerant and heat-susceptible wheat genotypes. Among these, only 19 miRNA-SSR markers were found to be polymorphic, which were further used to study the genetic diversity and population structure. The polymorphic miRNA-SSRs amplified 61 SSR loci with an average of 2.9 alleles per locus. The polymorphic information content (PIC) value of polymorphic miRNA-SSRs ranged from 0.10 to 0.87 with a mean value of 0.48. The dendrogram constructed using unweighted neighbor-joining method and population structure analysis clustered these 20 wheat genotypes into 3 clusters. The target genes of these miRNAs are involved either directly or indirectly in providing tolerance to heat stress. Furthermore, two polymorphic markers miR159c and miR165b were declared as very promising diagnostic markers, since these markers showed specific alleles and discriminated terminal heat-tolerant genotypes from the susceptible genotypes. Thus, these identified miRNA-SSR markers will prove useful in the characterization of wheat germplasm through the study of genetic diversity and population structural analysis and in wheat molecular breeding programs aimed at terminal heat tolerance of wheat varieties.

全球范围内有相当比例的亚洲人口通过普通小麦（Triticum aestivum L.）满足自身的能量需求。全球范围内的终期热胁迫（terminal heat stress）对小麦的品质与产量造成了严重影响，该胁迫波及全球约40%的小麦种植区域。因此，培育耐终期热胁迫的优良小麦品种刻不容缓。此前，研究人员已通过基于基因简单序列重复（genic simple sequence repeats, SSR）标记的标记辅助育种技术开发耐终期热胁迫小麦品种，但仅有少数研究将基于微小RNA（microRNA, miRNA）的SSR标记（miRNA-SSRs）应用于小麦研究中——这类标记被证实是多种非生物胁迫响应的关键调控因子。本研究从多种作物已报道的热胁迫响应miRNA中，共鉴定出104个相关miRNA。其中70个miRNA-SSR标记在20份耐终期热胁迫与热敏感小麦基因型的群体中得到验证。经筛选，其中19个miRNA-SSR标记呈现多态性，被用于后续的遗传多样性与群体结构分析。这些多态性miRNA-SSR标记共扩增得到61个SSR位点，平均每个位点扩增出2.9个等位基因。多态性miRNA-SSR标记的多态信息含量（polymorphic information content, PIC）值范围为0.10至0.87，平均值为0.48。通过非加权邻接法（unweighted neighbor-joining method）构建的系统发育树以及群体结构分析，将20份小麦基因型划分为3个类群。上述miRNA的靶基因直接或间接参与了热胁迫耐受性的调控过程。此外，标记miR159c与miR165b因可扩增出特异性等位基因、能有效区分耐终期热胁迫与热敏感基因型，被认定为极具应用潜力的诊断标记。综上，本研究鉴定得到的miRNA-SSR标记可有效用于小麦种质资源的遗传多样性与群体结构分析，以及面向小麦耐终期热胁迫品种培育的分子育种项目。