基于染色体片段置换系的野生稻粒长QTL——qGL12的精细定位

【目的】利用野生稻染色体片段置换系以及次级群体,精细定位与水稻粒长相关的QTL,发掘野生稻中影响粒长的新基因,为水稻育种提供遗传材料和基因资源。【方法】利用中国农业科学院作物科学研究所野生稻实验室已构建的野生稻染色体片段置换系群体,定位到一个与粒长相关的QTL——qGL12。在此基础上,选择粒长与受体亲本9311差异显著,且携带qGL12片段的置换系CSSL141与9311回交构建次级分离群体,设计区间内分子标记引物,筛选交换单株,结合粒型表型分析与基因型鉴定,对qGL12进行精细定位。通过扫描电子显微镜检测颖壳细胞,观察细胞的长宽变化,对定位区间内的基因进行基因注释以及测序分析,预测候选基因。【结果】根据整套置换系多个环境下的表型鉴定,qGL12初定位于第12染色体标记RM28621附近;选取携带qGL12的置换系CSSL141作为精细定位的亲本,CSSL141携带4个野生稻导入片段,在多年多点的田间试验中,CSSL141粒长、粒宽、粒重均明显高于9311;利用构建的CSSL141/9311 F2群体将qGL12定位于第12染色体标记RM5479与RM28621之间,影响粒长、粒宽以及粒重,对粒长的贡献率最高,为44.61%。在定位区间内设计了7个多态性分子标记引物,选择目标区间基因型杂合的植株种植F3,通过筛选交换单株,结合交换单株的基因型与表型,将qGL12定位于RM5479与RM28586之间50 kb区间内,为进一步缩短定位区间,在此区间内设计了4个多态性分子标记引物,选择交换单株种植下一代,筛选后得到20株交换单株,结合交换单株基因型与籽粒表型,最终将qGL12定位到第12染色体15.69 kb区间,该区间内有3个候选基因,其中Os12g39650编码一种微管蛋白,Os12g39660编码一种质膜钙转运ATP酶,Os12g39670尚未有明确功能,通过测序分析发现,Os12g39650、Os12g39660在编码区内存在变异;对亲本以及后代交换单株的颖壳细胞进行电镜扫描,发现9311颖壳细胞的长度与宽度均比CSSL141小,CSSL141/9311 F4代群体中,目标区间为野生稻基因型的交换单株颖壳细胞的长度与宽度均比目标区间为9311基因型的交换单株大,表明qGL12通过调控颖壳细胞的大小影响水稻粒长。【结论】利用野生稻染色体片段置换系,将野生稻粒长QTL——qGL12定位于第12染色体15.69 kb区间内,通过调控水稻颖壳细胞的大小影响粒长。该区间内有3个基因。来自野生稻的Os12g39650与Os12g39660与栽培稻等位基因相比,存在自然变异,确定为qGL12的候选基因。

Fine Mapping of Grain Length Associated QTL,qGL12 in Wild Rice (Oryza sativa L.) Using a Chromosome Segment Substitution Line

【Objective】Fine mapping of a grain length QTL from wild rice using a chromosome segment substitution line and secondary population, exploring new genes affecting grain length and providing genetic materials and gene resources for rice breeding.【Method】Our laboratory had preliminary mapped a grain length related QTL, qGL12 using the chromosome segment substitution line (CSSL) population. On this basis, we choose one CSSL, CSSL141 which harbors qGL12 substitution segment and has significant difference of grain length compared with receptor parent 9311. CSSL141 was backcrossed with 9311. The secondary separation population was constructed for fine mapping of qGL12. Detection of length of glumes cells was performed by scanning electron microscope.【Result】CSSL141 has 4 introgressive segments from wild rice, its grain length, grain width and grain weight was significantly higher than 9311 under multi-conditions. Using CSSL141/9311 F₂ population, qGL12 was localized to the interval between RM5479 and RM28621 on chromosome 12. qGL12 affected grain length, grain width and grain weight, and the phenotypic variation explained of grain length was 44.61%. Seven polymorphic molecular marker primers in the location interval were designed, F₃ plants which had heterozygous genotype in the target interval were investigated. qGL12 was mapped to a 50 kb region between RM5479 and RM28586. Four polymorphic molecular marker primers were designed in this interval, individuals which harbor heterozygous genotype in this interval were selected for next generation F₄. Eventually qGL12 was narrowed to a 15.69 kb region between DYB9.1 and RM28586 on chromosome 12. There are 3 genes in the interval. Two candidate genes, Os12g39650 and Os12g39660, which encode a tubulin protein and a calcium-transporting ATPase respectively, with variations in their coding regions. The results of electron microscopes scanning of glumes cell showed that the length and width of 9311’s glumes cell was smaller than CSSL141, indicated that qGL12 regulate grain size of rice by controlling the size of glumes cells.【Conclusion】The wild rice grain length associated QTL qGL12 was fine mapped to a 15.69 kb region on chromosome12. qGL12 control grain length via regulate the size of glumes cells. Two candidate genes, Os12g39650 and Os12g39660, were found and would be used for further research.