qBW12，一个影响棉花海陆种间杂交单铃重QTL的克隆与功能验证

Cotton (Gossypium L.) is an importantly economic and strategic crop, which provides the most natural textile fiber throughout the world. Upland cotton (G. hirsutum) is widely cultivated in the world, which is planted in more than 70 countries, and contributes to over 95% of the total cotton yield worldwide. However, the improvement of cotton fiber quality is slow, which can not meet the demands of the modern textile industry. What’s more, limited breeding parents and long-term direct selections result in the narrow genetic diversity of upland cotton. Another tetraploid cultivated species, G. barbadense or Sea-island cotton, is characterized as fine fiber quality but low yield and planted in limited areas. In order to improve the fiber quality of upland cotton, cotton breeders have tried to introduce elite fiber genes into upland cotton. But, the negative correlations between yield and fiber quality make it difficult to improve these traits simultaneously. In order to achieve this goal, we developed introgression lines population by backcrossing and genome-wide marker assisted selection with ‘Emian22’, a high yield inbred in Hubei province, as receptor and G. barbadense acc. 3-79 as donor. We aimed to introgress favorite traits of sea-island cotton into upland cotton and keep its high yield traits. Among the introgression lines, BS41, a line showed better fiber quality but low boll weight with other yield traits no obvious difference to ‘Emian22’. We speculated that this line carry gene loci which result in hybrid breakdown between sea-island cotton and upland cotton. Recently, we have mapped a major QTL on Chromosome 12 with 3.3 cM interval and 0.9 Mb region on D5 genome. In this project, we plan to (1) fine map the QTL by large segregation population, developing markers from the candidate region on the reference genome and construction near-isogenic lines; (2) obtain the candidate gene via screening 3-79 genome scaffolds and BAC clones and confirm its function by transgene; and (3) detect allele variation of the candidate gene in different upland cottons and sea-island cottons to find out the relationship between the alleles and the two species differentiation.

棉花是世界第一大天然纤维作物，陆地棉产量高而被广泛栽培；但其纤维改良缓慢无法满足纺织工业的发展要求，且遗传多样性狭窄。海岛棉纤维品质优良但产量较低。产量和纤维性状的负相关使陆地棉的改良困难。我们以高产常规种‘鄂棉22’为受体，海岛棉标准系3-79为供体，通过回交和MAS创造导入系群体，在导入海岛棉优良性状的同时，最大限度地保持陆地棉的高产性状。其中一个导入系纤维品质较好，但单铃重较低，其它产量性状变化不大。该株系可能携带导致两个棉种杂交后代产量和纤维性状同步改良困难的基因位点。我们以该材料与‘鄂棉22’杂交的F2群体实现了该位点的初步定位。本项目拟通过大分离群体、参照基因组候选区段开发标记、构建近等基因系以及海岛棉scaffolds、BAC文库筛选等方法对该QTL进行精细定位和克隆并通过转基因验证其功能。此外，在不同海陆品种（系）中检测候选基因的等位基因变异，探讨其与海陆种间分化的关系。

棉花是世界上最重要的农作物之一，单铃重是棉花产量构成的重要因素。本实验室前期通过海陆种间杂交获得一个棉铃变小、单铃重和皮棉重变低、衣分变低、株高变矮的突变体BS41。我们以Emian22和BS41为亲本构建F2、F3和F4的定位群体，检测到一个位于A12染色体上的单铃重QTL（qBWT-c12）。该QTL在陆地棉参考基因组中区间大小为180 kb，包含有11个候选基因，表达量分析表明一个油菜素内酯（BR）响应基因GhBRH1_A12在两亲本0 DPA胚珠中表达量存在显著差异，暗示该基因可能参与了早期棉铃发育的调控。激素相关实验表明BS41是一个油菜素内酯不敏感突变体，且突变体中BR的生物合成和信号通路基因在不同组织中均上调表达。. 控制单铃重的关键候选基因GhBRH1_A12与AtBRH1高度同源，具有保守的RING结构域，编码一类假定的E3泛素连接酶。亚细胞定位结果显示它在细胞核和细胞质内均有表达。陆地棉和海岛棉中分别具有16和18个BRH1家族基因成员，TM-1的表达谱数据表明，与BRH1家族其它成员相比，GhBRH1_A12在各组织中表达量相对较高。. 为了探究GhBRH1_A12在调节BR稳态及调节棉花生长和棉铃发育中的作用，我们分别克隆了Emian22和BS41中基因CDS及其上下游序列，在BS41中该基因CDS区存在3个SNP，其中第244位的SNP导致氨基酸的非同义突变（亮氨酸变为缬氨酸），同时基因上下游分别存在2个和1个InDel。我们构建了4个CaMV35S超表达载体（BOX，EOX，gBOX和gEOX）和1个敲除载体并转化棉花。除BOX载体以外，其余载体目前拿到了部分T0代材料。在表达量相对较高的BOX3的T2代超表达株系中，产量相关性状都显著降低，暗示了GhBRH1_A12对棉花产量性状潜在的调控作用。通过酵母双杂系统，以GhBRH1_A12作为诱饵蛋白筛选0 DPA胚珠及花药酵母双杂文库，点对点验证实验表明它能与COP9信号复合体亚基GhCSN5A互作。. 本研究主要通过图位克隆了一个铃重相关的候选基因GhBRH1_A12，并致力于探索油菜素内酯信号响应基因GhBRH1_A12的生物学功能，阐明GhBRH1_A12参与早期棉铃发育和纤维发育的调控网络及作用机制，为后续棉花的遗传改良奠定基础。