小麦粒重主效QTL qTkw-2D的图位克隆与单元型分析

qTkw-2D, a major stable QTL for common wheat (Triticum aestivum L.) thousand-kernel weight (TKW), has been confirmed in ten different environments using a recombinant inbred line (RIL) population derived from the cross between Kn9204 and J411. In this project, we proposed to do fine mapping, map-based cloning and haplotype analysis of qTkw-2D using forward genetics approach. Using the RIL and advanced backcrossing segregating populations, a high-density fine genetic linkage map of qTkw-2D will be constructed via Affymetrix wheat 660K SNP array and sequence analysis of the Aegilops tauschii chromosome 2D BAC physical map and reference sequences. The molecular markers cosegregated or tightly linked to qTkw-2D will be used to screen the Kenong9204 BAC library to construct a physical map of qTkw-2D in Kenong9204. The candidate gene of qTkw-2D will be speculated after sequence annotation. The effects of qTkw-2D on yield-related traits will be characterized by phenotyping the NILs in multi-environments. The haplotypes and allelic variation of qTkw-2D will be studied using Chinese wheat micro-core collections (MCC), cultivars, as well as the Kenong 9204-derived advanced lines. The elite alleles and excellent haplotypes of qTkw-2D will be clarified via candidate gene association mapping analysis. The functional molecular markers of the excellent haplotypes of qTkw-2D will be developed and used in wheat molecular breeding programs designed to improve wheat kernel weight.

本课题以科农9204×京411重组自交系群体检测到的控制粒重的多环境稳定主效QTL qTkw-2D为对象，利用660K SNP芯片构建了高密度遗传连锁图谱。应用粗山羊草2D染色体物理图谱和BAC序列信息，开发与qTkw-2D紧密连锁和共分离的分子标记，构建靶区段饱和遗传连锁图谱；结合NIL家系表型数据和靶区段次级作图大群体，实现靶基因精细作图。利用与靶基因紧密连锁和共分离的分子标记筛选科农9204基因组BAC文库，对阳性克隆进行测序、组装和注释，确定qTkw-2D候选基因。对靶基因NILs家系产量相关性状进行多环境表型鉴定，解析其对产量相关性状的影响。利用小麦微核心种质（MCC）、育成品种及科农9204衍生材料，研究qTkw-2D在MCC和现代品种的遗传多样性；通过候选基因关联分析明确靶基因优异单元型，开发其功能性标记用于分子育种。

小麦是世界主要粮食作物，高产是小麦生产和育种研究的首要目标。在亩穗数、穗粒数和千粒重产量三要素中，粒重提高一直是我国小麦增产的驱动因素。深入解析产量性状形成的遗传基础，挖掘控制籽粒性状关键基因对小麦遗传改良十分重要。qTkw-2D是一个控制小麦粒重的主效稳定QTL。本项目利用KJ-RIL群体的剩余杂合体构建qTkw-2D靶区间近等基因系和次级作图群体，开展qTkw-2D精细定位。基于中国春2D染色体物理序列，开发了46个多态性SSR标记，对基于Affymetrix Wheat 660K SNP芯片构建的高密度遗传图谱进行标记加密。根据科农9204基因组序列和京411重测序结果，进一步开发新的标记，构建qTkw-2D靶区间高密度饱和遗传图谱，将qTkw-2D精细定位到2.05Mb的区间。通过近等基因系不同籽粒发育时期的转录组测序，筛选到靶区间差异表达基因；结合科农9204基因组组装序列和基因注释，获得了候选基因。利用石新828×科农2007重组自交系群体开展连锁分析，对qTkw-2D再确认。通过132个品种构成的自然群体的关联分析，挖掘到一批小麦产量性状显著关联位点。针对qTkw-2D靶区间开发了4个紧密连锁的KASP标记，在不同群体中得以验证。所得结果为克隆籽粒性状关键基因奠定了基础，为产量性状分子设计育种提供了基因资源。