控制油菜菌核病抗性和早开花的主效QTL精细定位与基因克隆

Sclerotinia disease is a major disease in oilseed rape and one of main limiting factors affecting rapeseed production, and early flowering is a main objective of rapeseed breeding in China. Our previous studies have found overlapping or possibly linked major QTLs on the chromosome A2 for the disease susceptibility and early flowering in a RIL population through integrating phenotyping data of 9 environments/experiments and genotyping data from a 60K Illumina?infinium SNP array. This major QTL accounts for 27-43% of phenotypic variation and falls in a genomic region of about 380Kb. This study will further fine map this region for these two traits by constructing large secondary populations and employing new markers in the target region. Markers such as InDels and SNPs will mainly be developed through genome re-sequencing strategy from the parents and "extreme" lines with susceptibility + early flowering and resistance + late flowering (carrying the already developed target markers, respectively). The said new markers closer linking to the two target genes/traits will be used to narrow the window of candidate genes which will be subjected to functional validation by bioinformatic approach and a series of experimental methods. The said markers closer linking to the target genes or the markers to be developed from the gene(s) itself will serve as a useful and powerful molecular tool to break down link of the two traits for breeding of varieties with early flowering and resistance. If the two traits are controlled by one single gene, elucidation of this gene function will be of significance.

油菜菌核病是油菜的主要病害和油菜生产的主要限制因子之一，油菜早花早熟是目前我国油菜的主要育种目标。我们前期利用重组自交系（RILs）的9个环境或实验的鉴定数据和高通量的60K全基因组SNP芯片分型数据，获得了抗病性和开花时间的QTLs，其中在A2染色体的一个区域感病和早花主效QTLs对表型贡献为27-43%，且重叠或连锁，二者同在一个约380Kb的基因组区域。在此基础上，本研究主要开展次级大群体构建，通过基因组重测序，在目标QTL区域开发Indel等标记，精细定位和克隆感病和早花基因，并通过生物信息学和实验方法验证紧密连锁的标记和所克隆的基因。本项目将为育种提供打破这两个性状连锁、而又分别与两个基因紧密连锁的分子标记，为培育抗病、早花油菜品种提供必要的、有效的分子标记，具有重大的应用价值；如果感病和早花为同一基因，则对该基因功能的发现和阐述还具有重要的理论意义。

由核盘菌引起的菌核病是油菜最重要的真菌病害，也是影响油菜产量的主要限制因子。研究发现油菜的花期和成熟期与菌核病抗性高度相关，早花品种表现出较高的菌核病抗性水平，因此培育早花早熟的品种是目前油菜的主要育种目标之一，同时研究花期与菌核病的关系以及分子机制也有助于我们进一步拓展调控花期来防控油菜菌核病。本项目利用重组自交系的9个环境或实验的花期以及抗菌核病表型数据和高通量的60K全基因组SNP芯片分型数据，最终定位到30个主效抗病性QTL和22个开花主效QTL，两个性状的部分QTL存在共定位。在A02染色体上定位到一个感病和早花重叠的主效QTL, 其对表型贡献率为27%-43%。我们根据基因组的共线性特点开发了一种能够快速筛选候选基因的方法，根据此方法，我们在A2-C2 QTL区域中185个候选基因中成功排除了152个基因。此外，我们还在A04上的主效QTL区域筛选到候选基因BnLTP1基因。该基因属于植物病程相关蛋白PR14家族。我们进一步通过酵母双杂和双分子荧光互补实验表明AtLTP1与RDR蛋白互作，而RDR基因的实验结果表明它是一个调控开花的正调控因子，而RDR同时与抗病防御基因PDF2.2存在互作，因此我们推测深入解析LTP、RDR以及PDF2.2的生物学功能能够解析花期与菌核病之间的潜在关系。