棉花纤维比强度主效QTL（qFS15.1）的精细定位与候选基因鉴定

Cotton is a leading fiber crop. Fine-mapping QTL controlling fiber quality trait is important for cotton molecular breeding. Our previous study showed that the fiber strength major QTL (qFS15.1）on chromosome 15 were stable in different environments in upland cotton (Yumian 1×Acala Maxxa) F2 and recombianant inbred line populations, and Yumian 1 had favorable QTL allele. For fine-mapping and candidate gene identification of fiber strength major QTL (qFS15.1) on chromosome 15, an isogene line population with about 5000 individual plants is produced from the residual hybrid line RIL011 at the QTL region by two-generation selfing. The SSR markers mapped on chromosome 15 in recombinant inbred line population (Yumian 1×Acala Maxxa), and SSR designed from G. raimondii and G. arboretum genome sequences which corresponds the QTL region are used to construct a saturated genetic map of QTL region. The F2 individuals, F3 and F4 recombinant lines from isogene line population are used to fine-map major QTL for fiber strength through substitution mapping. SSR markers at the QTL region are used to screen the BAC bank developped from Yumian 1 genome, the positive clones are sequenced and annoted. RNA-seq was used to analyze the differential expression genes at the QTL region with RNA samples from different development stage fiber of Acala Maxxa, Yumian 1 and recombinant lines. Based on the annotated genes of positive BAC clones and the tissue expression analysis of differential genes within the QTL region, the major QTL candidate gene is confirmed and cloned.

棉花是首要的纤维作物，精细定位纤维品质QTL对棉花分子育种具有重要意义。项目组前期利用（渝棉1号×Acala Maxxa）F2和RIL群体，检测到环境稳定的纤维比强度主效QTL（qFS15.1），有利等位基因来源于渝棉1号。为精细定位纤维比强度QTL（qFS15.1）和鉴定其候选基因，本研究选择QTL区域剩余杂合株RIL011自交，自交后代选择杂合株再次自交建立包括5000个单株的近等基因系F2群体。利用第15染色体图谱的SSR、QTL区域对应的雷蒙德氏棉和亚洲棉基因组SSR标记构建遗传图谱。以F2单株、F3和F4重组系采用替换作图精细定位纤维比强度QTL。利用QTL区域标记筛选渝棉1号BAC库，对阳性克隆测序和基因注释。采用RNA-seq分析Acala Maxxa、渝棉1号与重组株不同发育时期纤维的差异表达基因。根据BAC克隆注释基因和差异表达基因的组织表达分析结果，确定并克隆候选基因。

选择第15来源渝棉1号的重组系RIL138与Acala Maxxa杂交建立(RIL138×Acala Maxxa) F2精细定位群体，利用SSR和InDel标记检测(RIL138×Acala Maxxa) F2精细定位群体的基因型，构建的第15染色体高密度遗传图谱包括93个标记，重组长度为160.03 cM，标记间平均遗传距离为1.72 cM。利用(RIL138×Acala Maxxa) F2精细定位群体高密度遗传图谱及纤维品质表型数据，在第15染色体精细定位到2个纤维比强度QTL, QTL有利等位基因都来源于Acala Maxxa。其中，qFS15.1精细定位在6.40 cM区间，加性效应0.84 cN/tex，对应陆地棉TM-1参考基因组(1767206 bp)，该区域包括了69个注释基因；qFS15.2精细定位在7.47cM区间，加性效应为0.96 cN/tex，对应陆地棉TM-1参考基因组(648288 bp)包括了43个注释基因。亲本数字表达谱测序和重测序结果显示，编码细胞壁松弛蛋白(expansin-like A2)的 Gh_D01G1679可能为qFS15.1的候选基，编码TPX2结构域蛋白（微管结合蛋白）的Gh_D01G1953可能为qFS15.2的候选基因。研究结果将为陆地棉分子标记辅助选择育种和棉花纤维发育机制研究奠定基础。