油菜无花瓣性状主效QTL qAP8的精细定位与候选基因克隆

Apetalous was considered as one of the important traits in high yield and disease resistance breeding in rapeseed (Brassica napus L), owing to it's significantly decreasing the consumption of energy in plant, increasing the efficiency of solar energy utilization and reducing the incidence of sclerotinia. Former research indicated that the inheritance of apetalous in rapeseed line APL01 was controlled by two recessive major genes, and qAP8 was one of the two main effect QTLs affecting this trait in APL01. In this research, the line with qAP8 locus and similar background as apetalous parent APL01 was screened from the (APL01/M083)F6 recombinant inbred lines, which delivered from the preliminary mapping population. This line was crossed with APL01 to get a large F2 segregation population, which could be used in qAP8 fine mapping. The genome sequence of Brassica napus, Brassica rapa and Arabidopsis thaliana, and BAC end sequence of Brassica napus were used to design primer, develop new markers and map qAP8 within 1cM region. Sequence near qAP8 was used to screen the BAC pools of Brassica napus, and physical map of qAP8 was constructed by these BACs. The candidate BAC was sequenced, and candidate gene of qAP8 was confirmed based on it's function and transgenic complementation. The results in this research will be helpful in elucidating the mechanism of petal development and assisting apetalous breeding in rapeseed.

油菜无花瓣性状可减少个体能量消耗、提高光能利用、降低病害损失，是油菜高产抗病育种的潜在优异性状。研究表明，无花瓣油菜APL01的无花瓣性状受2对隐性主基因控制，qAP8是控制该性状的主效QTL。本研究以（APL01/M083）F6重组近交系为材料，利用油菜60K SNP基因分型芯片，结合表型鉴定，筛选重组近交系中与APL01遗传背景相近且带有qAP8的株系与亲本杂交，构建qAP8位点的F2分离大群体；利用甘蓝型油菜、白菜型油菜和拟南芥相关基因组序列，设计引物，开发新标记，加密qAP8区域，将qAP8精细定位于1cM遗传距离以内；用获得的qAP8两侧标记筛选油菜BAC文库，构建覆盖qAP8位点的物理图谱；对候选BAC进行序列分析，根据基因功能确定候选基因，并进行转基因验证与基因表达分析。本项目研究结果可揭示油菜花瓣发育的调控机制，也可用于无花瓣性状的标记辅助选择，加速高产抗病油菜育种进程。

甘蓝型油菜无花瓣性状具有低能耗、高光效及避病等特点，得到广泛重视。本研究以无花瓣材料APL01和有花瓣材料Holly衍生的重组自交系（AH群体）为材料，利用油菜60 K芯片，构建高密度图谱，开展QTL定位，并进行基因的表达分析，筛选差异基因，解析无花瓣性状的发生机理。主要结果：.（1）构建了油菜高密度SNP遗传图谱，定位了无花瓣性状主效QTL。该图谱包含2755个SNP-bin (包含11458个SNP)和57个SSR标记，总长2027.53 cM，平均间距0.72 cM。图谱与甘蓝型油菜参考基因组物理图谱共线性良好。考察AH群体在5个环境下的花瓣度，定位了9个无花瓣性状的QTL，其中qPD.C8-2、qPD.A9-2和qPD.C8-3在多个环境中能检测到，是稳定的主效QTL。.（2）明确了油菜无花瓣性状形成的关键时期，并开展了无花瓣性状形成的转录组学分析。花发育第5阶段末期是决定无花瓣油菜APL01无花瓣表型的关键时期。无花瓣性状形成的转录组学分析发现，36个花瓣调控基因在无花瓣品系APL01中差异表达，这些基因分别在转录调控、表观修饰、蛋白质泛素化及蛋白质法尼基化过程起作用，通过调控基本细胞学过程相关基因表达参与APL01的无花瓣性状发育。QTL定位与RNA-seq分析结果相结合，进一步结合同源基因功能分析，预测了8个无瓣性状的候选基因CG1-CG8。.（3）无花瓣性状形成的基因调控网络初步分析。以13个花瓣调控基因和无花瓣性状QTL定位区间的1个候选基因CG1为目标基因，进行基因表达量与花瓣度间的关联分析以及基因表达量间的关联分析，初步构建了无花瓣性状发育的调控网络。上述14个花瓣调控基因主要通过CHR11-PLP途径参与APL01的无花瓣性状调控，PLP在该途径中作为终端信号整合因子，直接负调控花瓣发育。