西藏半野生小麦脆穗性状主效基因Br1的图位克隆及功能分析

Loss of brittle rachis is one of the most essential step in wheat domestication. The genes for brittle rachis have received many concerns all the time, but so far have not been fine mapped yet. Tibetan semi-wild wheat (T. aestivum ssp. tibetanum Shao) is an endemic hexaploid wheat group in China. Its brittle rachis is unique in wheat, since disarticulation occurs above the junction of the rachis with the rachilla. Br1 is the major gene responsible for brittle rachis in Tibetan semi-wild wheat, which was mapped to the short arm of chromosome 3D. However, it was difficult to perform fine mapping for Br1 due to multiple gene affected brittle rachis in Tibetan semi-wild wheat. In this project, we will construct the secondary population with single background for Br1; identify genes co-segregated with targeted character (brittle rachis) by the combination of fine mapping and candidate gene cloning; elevate the function of co-segregated gene by transgenic research; identify SNPs among Br1 in wheat and Aegilops tauschii accessions; measure the spatial and temporal expression pattern of Br1; clarify the cellular mechanism of brittle rachis. This study will certainly provide new knowledge for wheat domestication.

脆穗性的改良是小麦驯化中的关键步骤之一。小麦控制脆穗性的基因与驯化密切相关，一直以来都备受关注，但至今还没有被精细定位。西藏半野生小麦为我国所特有，与其他原始小麦材料不同，它是唯一的穗轴节片下节位断裂的断穗种质。Br1是其控制其脆穗性的主效基因，Br1被定位在3DS。由于西藏半野生小麦的脆穗性由多个位点控制，这给精细定位Br1基因的带来了困难。本研究基于前期研究筛选和构建的Br1基因单一背景的遗传群体，拟采用正反向遗传学方法，利用精细定位与候选基因克隆相结合，以筛选与脆穗性共分离的候选基因，利用转基因验证功能；明确Br1基因导致脆穗的SNP变异；检测Br1基因的时空表达特性；明确Br1基因导致脆穗的细胞学机理。本研究将为小麦驯化研究提供新的知识。

西藏半野生小麦是我国特有的六倍体小麦亚种，自然脆穗是其最典型的特征。挖掘西藏半野生小麦的特有基因和位点对科学认识小麦的驯化和演化，对丰富小麦育种的基因资源具有重要价值。本研究通过图位克隆、基因组重测序、捕获测序、转基因和酵母杂交等方法，系统研究了西藏半野生小麦关键脆穗性基因位点。1）图位克隆了Qbr.sau-5A位点，其脆穗效应来自于西藏半野生小麦的Q等位变异（Qt）；Qt具有正常的表达活性，但其第五外显子包含一个161 bp的转座子插入，这导致编码蛋白结构发生变化，仅有前端211个氨基酸与Q蛋白保持相同，缺失AP2 domain R2、Motif 3 以及AASSGF box等关键结构域；Qt蛋白无法与小麦穗部其他蛋白互作；基因敲除和过表达实验证明功能丧失是Qt导致小麦脆穗的关键分子机制；穗轴石蜡切片显示Qbr.sau-5A位点通过减弱穗轴的韧性增强脆穗性；捕获测序数表明Qt起源于六倍体小麦中驯化的Q基因的突变，这证明西藏半野生小麦是普通小麦去驯化的结果。2）精细定位了Br1位点，获得共分离的分子标记；重测序发现目标区域存在大片段缺失，目前已锁定基因，尚未完成功能分析；电镜观察发现Br1可导致断裂面光滑，并在断裂位点形成收缩凹槽；Br1的脆穗不是由于离层木质化而导致的，这与Qbr.sau-5A所控制的节间脱落机制不同。3）通过QTL分析，发掘了若干对小麦育种具有潜在价值的QTL位点。