澳洲棉渐渗的棉花黄萎病广谱抗性遗传解析及其育种利用研究

The key challenge for making breakthrough in Verticillium wilt resistance breeding is the lack of resistance source, particularly short of broad-spectrum resistance to multiple biotypes of Verticillium in cotton cultivars, which necessitates to mine and study broad-spectrum resistance source from wild species. In previous research supported by National Natural Science Foundation of China, new germplasms, such chromosome translocation lines and chromosome addition lines, with broad-spectrum resistance were developed by introgression of wild diploid species, Gossypium australe, through the combination of chromosome engineering and molecular markers. The broad-spectrum resistance to Verticillium wilt has not been reported before, and the genes conferring broad-spectrum resistance are unable to be fine-mapped by conventional genetic methods due to the lack of necessary genetic recombination led by the distant relationship between G.australe and G. hirsutum. In this project, studies on the broad-spectrum resistance to Verticillium wilt introgressed from G. australe in G. hirsutum will be performed to obtain the candidate genes conferring the resistance by the combinations of transcriptome, genome, and bioinformatics analysis firstly. And then, using virus-inducing gene silencing (VIGS) and gene transformation techniques, analyses on functions and networks of the candidate genes conferring the resistance are to be carried to elucidate functions and gene regulatory networks in metabolic pathways, lay the foundations for the usage of the broad-spectrum resistance to Verticillium wilt from G. australe in cotton breeding, to develop novel high resistant germplasms, and to provide novel genes for the development of cotton varieties with sustainable resistance to Verticillium wilt.

棉花抗黄萎病育种难以取得突破性进展的关键是栽培棉中缺乏抗源，尤其是同时对黄萎病多个不同生态型表现高抗的广谱抗性，因此发掘和研究广谱抗性基因显得十分必要。本项目在前期国家自然科学基金资助下，利用染色体工程技术和分子标记相结合，创制获得了野生澳洲棉渐渗的陆地棉TM-1为遗传背景的对黄萎病广谱抗性的易位系和异附加系种质，这种广谱抗性在棉花上尚未见报道，是一种全新的抗源。由于澳洲棉与陆地棉亲缘关系远，很少发生遗传物质的交换重组,无法采用经典遗传学方法进行精细定位其抗病基因。本研究拟对澳洲棉渐渗于陆地棉的黄萎病广谱抗性性状，采用转录组、基因组、生物信息等方法，获得广谱抗性候选基因，对候选基因采用病毒介导基因沉默技术、转基因技术等进行基因功能验证和基因调控分析，阐明澳洲棉黄萎病广谱抗性基因的调控网络，奠定其在棉花抗黄萎病育种的理论基础，获得棉花黄萎病广谱抗性新种质，为棉花黄萎病持久抗性育种提供新基因。

由大丽轮枝菌(Verticillium dahliae Kleb)引起的黄萎病是我国近30年来棉花生产的主要障碍之一。棉花抗病育种难以取得突破的关键是四倍体栽培种陆地棉(Gossypium hirsutum，AD1)缺乏抗源，因此迫切需要发掘抗黄萎病基因。本项目利用分子细胞遗传学、基因组学、转录组学、BiFC、Luc实验、转基因、基因编辑等技术，以高抗黄萎病的陆地棉-澳洲棉（Gossypium australe，G2）易位系为材料，开展了抗黄萎病基因挖掘与功能研究。重要结果如下：将澳洲棉抗病片段向泗棉3号、鄂荆1号等骨干品种转育，片段传递率由10%提高到30%以上，且表型也得到明显改善,为进一步利用奠定了基础。经转录组、转基因和VIGS等分析，获得了GauREV2、Gau14-3-3、GauACLB等3个感兴趣的抗病候选基因，研究发现GauREV2是抗性最好的候选基因。GauREV2基因沉默后抗病性显著下降，茉莉酸（JA）通路相关基因表达也显著下降，JA含量及外施MeJA影响棉花抗病性，BiFC发现GausREV2与GhTPL和GhTPRs存在互作，表明GausREV2介导的抗病性依赖于 JA 信号通路。REV2基因结构分析，发现GausREV2与海7124的GbREV2_At/Dt、TM-1的GhREV2_At是相同的，但GhREV2_Dt则发生了点突变导致提前终止，GhREV2_Dt编码蛋白C-端缺失，亚细胞定位发现GhREV2_Dt失去了核定位信号。基因共表达分析发现只有完整结构的REV2基因才对黄萎病具有抗性；其次，GhREV2_Dt不但本身失去了抗病性，还严重干扰了GhREV2_At介导的抗病性，但对GbREV2_At/Dt介导的抗性不起作用，且干扰的抗性可被GausREV2恢复；对130份陆地棉和70份海岛棉以及毛棉、黄褐棉、达尔文氏棉等3个野生种重测序分析，发现所有棉种REV2_At结构都一样，130陆地棉GhREV2_Dt全都失去了C-端，而60份海岛棉、3个野生种的GhREV2_Dt都是完整的，该结果说明了海岛棉抗病性普遍强于陆地棉的可能原因之一，也说明了结构分化可能在四倍体棉种形成后。为进一步研究该基因功能，已创建获得了转基因过表达棉花T0代和基因编辑的T0代植株。本研究为创制抗黄萎病棉花品种提供了新材料、新基因和新理论。