水稻柱头长度qSTL3.2的精细定位和候选基因分析及制种应用评价

Rice stigma length was high significantly correlated with percentage of exserted stigma(PES), while PES played an important role in hybrid rice seed production. Using a set of chromosome segment substitute line population (NKNCSSSL) derived from Nipponbare (receptor)/Kasalath (donor) and its parents, a QTL (named qSTL3.2) was located to C136-C746 on chromosome 3. CSSL15, the single segment substitution line of NKNCSSL containing donor fragments in the region of C136-C746 (flanking region of qSTL3.2) only, was observed significantly longer stigma than that of Nipponbare across four years. Based on the preparatory work, the fine mapping of qSTL3.2 locus will be conducted by using a secondary segregating population derived from a cross of CSSL15/Nipponbare. The annotated genes (candidate genes of qSTL3.2) in the finely mapped region can be acquired by searching against the related rice genome annotation database. According to the available genome sequence of Nipponbare and Kasalath, sequence differences of the candidate genes between the parents can be found by pairwise sequence alignment. During the development of stigma, expression differences of candidate genes between CSSL15 and Nipponbare should be conducted by real time quantitative PCR. With the analysis of T-DNA or Tos insertion mutants and transgenic complementation plants, candidate gene could be confirmed finally. It is anticipated that the molecular marker tightly linked to qSTL3.2 can be used to improve the stigma length of the maternal parent, thereby increasing outcrossing rate of maternal parent in rice hybrid seed production field. The application value of qSTL3.2 in hybrid seed production will be evaluated according to the frequencies of plants with purple leaf in the F1 populations obtained by crossing the transgenic and non-transgenic materials with pollen parent with dominant purple leaf trait.

水稻柱头长度是影响不育系繁殖与制种产量的重要性状。申请者前期以Nipponbare/Kasalath//Nipponbare CSSL及其亲本为材料，在第3染色体上检测到控制柱头长度的位点qSTL3.2。比较各代换系和亲本发现，CSSL15株系仅含有qSTL3.2目标片段的单片段置换系。本研究拟在前期基础上，利用CSSL15与Nipponbare杂交构建次级分离群体精细定位qSTL3.2；再在精细定位区间中查找候选基因，比较各候选基因双亲间的序列差异，通过qRT-PCR调查双亲间各候选基因的表达差异以及T-DNA/Tos插入突变体和转基因互补试验确定候选基因。利用具有显性的紫色叶片性状的品种为父本，与转基因和非转基因材料相间种植，根据F1代中紫叶出现频率，评价qSTL3.2的繁殖制种利用价值。

柱头长度是影响水稻不育系异交繁殖和杂交水稻制种产量的重要性状。本研究以前期检测到的1个稳定表达的控制柱头长度的QTL-qSTL3.2及携有其供体基因组的单片段置换系CSSL15为基础，通过构建CSSL15/Nipponbare F2次级分离群体对qSTL3.2精细定位。结果发现，在由240个F2单株构成的POP1群体中，柱头长度的分离比符合1:2:1，说明本组合中的柱头长度为单基因控制。利用CSSL15/Nipponbare F2群体的7701个单株，将qSTL3.2精细定位在第3染色体长臂中部LM10~RM7117的51.9kb区域。查找水稻基因组注释数据库发现，精细定位区间共有9个注释基因。9个注释基因在Nipponbare和Kasalath中的序列比对发现，Kasalath的LOC_Os03g50400编码序列在530bp处存在1个CC双碱基的插入，造成后续密码子移位，从而导致后续氨基酸的连续差异且不能及时终止翻译，而其余8个注释基因Kasalath编码序列的改变只是造成部分非连续氨基酸（1-9个）的差异。qSTL3.2精细定位的结果将为分子标记辅助选择改良水稻不育系柱头长度提供技术支撑，同时也为下一步柱头长度基因的图位克隆奠定基础。