利用野生大豆染色体片段代换系精细定位三粒荚数QTL及候选基因挖掘

The three-seeded pod number was closely related to yield on soybean. The wild soybean genome contained excellent three-seeded pod gene need to be further mined. So in this research, a chromsome segment substitution lines (CSSLs) covering 93.35 percent of the whole genome of wild soybean parent, was constructed by repeatedly backcross with ZYD00006 (Glycine soja, donor parent) and cultivar Suinong 14 (Glycine max, recurrent parent). The CSSLs included 220 individuals and two parents had been sequenced. After continuous phenotype identification in six years, the CSSL-57 was filtrated with extreme three-seeded pod number. A F2 segregation population contained 970 individuals were constructed by more once time backcrossing. The primary QTL mapping was done by method of BSA sequencing. Three tagged QTL qTh03-1, qTh12-1 and qTh12-2 were identified with fragment length 2.15, 3.63 and 3.69Mb, respectively. Residual eterozygous Lines (RHL) and Single Segment Substitution Lines (SSSL) were constructed by more backcrossing and selfing base on the CSSLs contained tagged segment. So the QTL fine mapping could progressively realized. The candidate genes could been annotated according to the fine tagged segment. Primary functional verification of candidate genes was done by the quantitative Real-Time PCR technology and further verification for authenticity and universality by the variety population with extreme three-seeded pod number. The research results would provid theoretical foundation for gene clone.

大豆三粒荚数与产量密切相关。野生大豆基因组蕴含丰富的三粒荚数优异基因，有待于挖掘。本研究前期构建了覆盖野生大豆ZYD00006基因组93.35%的染色体片段代换系220份（两亲本及后代均已重测序），经连续6年表型鉴定筛选出三粒荚数极端多代换系个体CSSL-57，通过回交创建了目标性状F2分离大群体（970株），采用BSA法获得了3个QTL初步位点：qTh03-1（2.15Mb）、qTh12-1（3.63Mb）及qTh12-2（3.69Mb）；以此为目标区域，通过继续回交及自交创建筛选仅含目标片段寡片段或单片段代换系及目标区间残留异质系（RHL），进而逐步缩小目标区间实现QTL精细定位（300kb之内）；结合亲本重测序信息对精细区间基因预测，采用qRT-PCR技术验证候选基因功能，并结合生产上三粒荚多与少的大豆品种资源进一步验证候选基因的真实性与普遍性，为大豆三粒荚数基因克隆提供理论依据。

大豆（Glycine max）起源于中国，是重要的油料、饲料及豆制品加工原料。大豆荚粒数性状是重要的产量构成因子，尤其是三四粒荚数对大豆产量具有积极促进作用。随着分子育种的迅速发展，对大豆三四粒荚进行QTL精细定位及候选基因挖掘具有重要意义。主要研究内容：一是大豆三四粒荚数QTL初步定位，以含有147个家系的重组自交系群体（RILs）为材料，结合四年表型数据及SLAF-seq 高密度遗传图谱，对大豆三四粒荚数进行了QTL初步定位；二是对QTL位点进行Meta分析，获得了三四粒荚数通用QTL区间；三是以一套全基因组导入系（CSSLs）为材料，通过筛选含有目标区间导入片段的个体对QTL通用区间进行表型验证，进一步验证通用区间的准确性；四是以含有目标区间导入片段的导入系为材料，采用与轮回亲本回交的方式构建次级分离群体，采用BSA重测序等方法进行QTL精细定位，其中三粒荚数定位了4个QTL精细区间，分别为GM03染色体上NThSPBSA03-1位点（区间长度为0.94Mbp），GM13染色体上的NThSPBSA13-1（区间长度为1.48Mbp）和NThSPBSA13-2（区间长度为1.50Mbp），GM17染色体上的NThSPBSA17-1（区间长度为1.34Mbp），四粒荚数在GM07染色体定位到一个含有6个SSR标记的位点，长度为321kb；五是针对候选区间进行基因注释及预测，筛选与荚粒发育相关的基因，经过在大豆及拟南芥基因组的同源基因分析，筛选与大豆荚粒发育密切相关的候选基因；六是对候选基因进行初步功能验证，包括大豆资源群体单倍型分析和极端材料荚粒不同发育时期的qRT-PCR表达量分析，以92份资源群体为材料，分析候选基因在资源群体中的单倍型，不同单倍型资源表型差异达到极显著水平说明候选基因对大豆三粒荚数具有一定影响，同时在次级分离群体中筛选表型极端个体，分析荚粒不同发育时期候选基因表达量差异，进一步验证候选基因，经过初步验证，与大豆三粒荚数密切相关的候选基因为Glyma.03G029800、Glyma.17G057300、Glyma.17G062600和Glyma.17G062000，四粒荚数候选基因为Glyma.07G200900和Glyma.07G201200。本项目开展了大豆三四粒荚数QTL初步定位、精细定位、基因挖掘与功能验证等工作，为大豆产量构成因子基因