一个新的油菜粒重主效位点qSW-A9-2的图位克隆及功能验证

Yield trait is a complicated quantitative ones, it is the precondition of high-yield molecular breeding to understand the genetic and molecular basics of seed yield in Brassica napus. Seed weight is one of the yield components, at the present time, the researches on seed weight still rest on the level of QTL analysis. A novel and major QTL qSW-A9-2 controlling seed weight has been mapped to an interval of 2.16cM and 3.5Mb in A9 linkage group using several populations derived from two DH lines with extremely different seed weight. In this project, three different approaches will be used to build several near isogenic lines (NIL) with target interval and obtain recombinant groups in target range respectively. Based on the corresponding section of Brassica rapa SSR markers, the target QTL site can be located in a small area firstly, then in a fine SNP map via high-throughput sequencing technology and will finally be targeted to the interval of several genes or even single gene. After isolating the candidate gene(s), the overexpression and inhibition of expression of candidate gene(s) will be studied to verify its function and ultimately determine the target gene. Separating the functional gene of this QTL site has potentially important value and great significance to carry out molecular breeding, molecular regulation mechanism of seed weight and gene evolution research.

油菜产量性状是一个复杂的数量性状，深入认识其遗传及分子调控机理是开展高产分子设计育种的前提。粒重是油菜产量主要构成因子之一，目前有关油菜粒重的研究仍停留在QTL定位分析水平。利用粒重差异极端的两个DH系亲本构建的多个群体，已将一个新的油菜粒重主效QTL位点qSW-A9-2重复定位在A9连锁群上，目标区间遗传距离2.16cM、物理距离3.5Mb。本项目通过三种不同的技术途径,构建目标区间的多个近等基因系,分别获得目标区间的重组群体，基于白菜对应区段的SSR标记,先将目标QTL定位在一个更小的区间,再利用高通量测序技术,构建此区间的SNP图谱,将目标QTL定位到少数几个甚至单个基因的区间,分离出候选基因后,在两个油菜品种中过量表达和抑制表达候选基因验证其功能,最终确定目的基因。该位点目的功能基因的分离对开展油菜分子育种研究、粒重调控的分子机制研究及基因进化研究等均将具有潜在的重要价值和意义。

本项目旨在精细定位和克隆甘蓝型油菜A9连锁群上qSW-A9-2位点一个控制油菜粒重的主效基因，并验证其功能。利用油菜大粒品系G-42与小粒品系7-9杂交后代DH系群体，选取46个极端大粒株系及47个极端小粒株系组成两个BSA混合池，基于高通量测序，通过SLAF-seq(specific locus amplified fragment sequencing)技术鉴定靶区间SNP标记，首先将目的基因定位在A09染色体上一个0.58Mb的物理区间(25,401,885-25,985,931)，基因注释显示该区间有四个粒重候选基因(GSBRNA2T00037136001, GSBRNA2T00037157001, GSBRNA2T00037129001及GSBRNA2T00069389001)。 取大粒品系G-42与小粒品系7-9未成熟种子进行转绿组分析，差异表达的基因中GSBRNA2T00037121001位于目标QTL区间(A09染色体0.58Mb的物理区间25,401,885-25,985,931)，鉴定为另一个调控粒重的候选基因。构建5个候选基因过量表达载体(pBI121)及抑制表达载体(pHANNIBAL)，转化油菜品种“中油821”，验证其功能，发现基因GSBRNA2T00037129001明显具有调节种子粒重功能。克隆和鉴定控制油菜千粒重的主效基因，是全面阐明油菜产量性状遗传基础及分子调控机制、开展油菜高产分子设计育种的前提。