小麦矮秆新基因的精细定位与效应分析

Plant height is closely related to the yield in crops. Development of dwarf and semi-dwarf wheat significantly improves yield in wheat. Currently, the available dwarfing genes of wheat in our country are very limited and the genetic resources are very poor. The potential for increase of yield is thus limited. Most of the discovered dwarfing genes have some negative effects on yield. Therefore, the identification of wheat dwarfing genes is needed to study. Previous study has identified an elite dwarf mutant (named Rht6621) with high-yield potential from the wheat mutant library. The novel dwarfing gene was mapped within the interval of 31 Mb on chromosome 5A by the SNP microarray screening and development of genetic linkage map. On this basis, this project is intended to fine mapping of the dwarfing gene in the mutant by using the flanking marker approaches based on the secondary segregation population, development of high-density molecular markers within the mapped region; and also using the MutMap method on F2 population crossing from mutant and wild type, combining with the mapped region, to fine mapping the mutated gene; and then the candidate genes for dwarf phenotype in the mutant within the fine-mapped region will be identified; Additionally, the other important content of this project is to evaluate the effects of Rht6621 on plant height-reduced and yield-related components by the near-isogenic lines of Rht-B1b, Rht-D1b and Rht6621. This research is aimed at providing important information for genetic regulation mechanisms study in wheat plant height, and also supplying the novel gene for development of high-yield dwarf wheat varieties.

株高与作物产量密切相关，矮秆和半矮秆小麦品种的培育推动了小麦产量革命性提升。目前，我国小麦育种可利用的矮秆基因源单一，严重限制了高产小麦新品种培育，因此亟待发掘和鉴定新的小麦矮秆优异基因资源。申请人所在课题组前期通过构建小麦诱变突变体库，从中筛选了一个产量性状优良的矮秆突变体（暂命名为Rht6621）。通过基因芯片扫描和遗传图谱构建，将小麦矮秆新基因定位在5A染色体31Mb区间内。本项目拟在此基础上，通过构建的次级分离群体，在初定位区段开发高密度分子标记，根据侧翼标记逼近法进行精细定位；同时利用突变体和野生型杂交获得的F2群体，采用MutMap方法，精细定位突变基因；并预测候选基因；构建Rht-B1b、Rht-D1b及Rht6621矮秆基因近等基因系，通过调查产量性状，评价其在小麦育种中应用潜力。研究结果不仅为揭示小麦株高遗传调控机制提供参考，而且将为培育高产矮秆新品种提供新基因。

株高决定了小麦的抗倒伏性，从而影响小麦的产量和品质。利用小麦矮秆突变体挖掘矮秆基因具有重要的研究价值。本项目前期利用筛选到的产量性状优良的矮秆突变体，分别构建了遗传分离群体，后续通过F3代多点表型鉴定，结合基因型分析，确定了矮秆基因定位于2D染色体（分析后确定为Rht8矮秆基因位点），因此本项目调整为针对该位点进行基因精细定位、候选基因预测与育种利用价值评估分析。通过构建的2个扩大的遗传分离群体，将矮秆基因分别定位在2D染色体约700kb和170kb物理区间内。该区段在当时的中国春参考基因组存在较大的未知序列区，进一步通过三代测序组装野生型京411，发现编码含有RNase H结构域的基因位于该定位区段。基因克隆和测序分析发现该基因在两个突变体中分别产生移码和提前终止突变，最终导致蛋白编码不完整。不同组织基因表达分析表明该基因主要在茎秆的节中表达，并且在野生型中的表达量较高。亚细胞定位分析发现该基因表达的蛋白定位于细胞核中。基因编辑分析显示敲除该基因的突变株系株高显著低于野生型，表明该基因为导致株高降低的目标基因。赤霉素喷施、含量测定及表达分析表明，该基因通过调节赤霉素合成相关基因，降低活性赤霉素GA3的含量，同时增加GA4的含量，从而降低株高。近等基因系分析结果表明该基因在降低株高的同时对千粒重以及穗粒数没有影响。经对Rht8来源的赤小麦品种中该基因序列分析发现目标基因为Rht8矮秆基因。该矮秆基因的克隆对于小麦矮化育种以及培育适宜株高的小麦新品种具有重要的意义。同时，利用一个茎秆抗折断力提高的矮秆突变体构建遗传分离群体，将矮秆基因定位在2D染色体长臂0.85 cM区间，分析表明该定位区间为一个新的矮秆基因位点，研究结果将丰富小麦矮化育种中的遗传资源。在本项目的资助下，发表第一标注SCI论文1篇，第二及第三标注SCI论文4篇，第一标注中文核心期刊论文1篇，申请专利1项。