小麦抗叶枯病基因Sb2的图位克隆

Bread wheat is one of the most important food crops in the world. Wheat diseases are major constraint for large yield losses every year. Breeding resistant cultivars is the most economical and environmentally safe method to prevent the epidemic outbreaks of wheat disease. Discovering and cloning wheat disease resistance genes are the basis for studying resistance mechanism, host-pathogen interaction, allelic variation and evolution of wheat resistance gene, as well as molecular marker assisted selection in wheat breeding program. .Wheat spot blotch caused by Bipolaris sorokiniana is one of the most important floral diseases also causing root rot and black embryo in wheat grain which seriously reduced the yield and processing quality. By screening wheat germplasm, we found a spot blotch resistance common wheat line 621-7-1 and a highly susceptible wheat line 621-18-3. After performing genetic analysis using a F2 segregating population between B. sorokiniana susceptible line 621-18-3 and B. sorokiniana resistant line 621-7-1, a single dominant spot blotch resistance gene Sb2 was identified. Using bulk segregant analysis (BSA) and molecular marker analysis, the Sb2 gene was mapped on the short arm of chromosome 3B. Using available Aegilops tauschii SNP genetic linkage map and physical map and the recently released common wheat cv. Chinese Spring chromosome 3B reference sequence, the Sb2 gene was further mapped in an 0.232 cM genetic interval using 4320 F2 plants, corresponding to 747 kb physical distance with 30 putative genes. In this proposal, we are going to increase the fine mapping population to another 10000 F2 plants and develop more tightly or co-segregated markers using the Chinese spring 3B reference sequence to fine map the Sb2 gene in a small genetic and physical interval. The candidate gene of Sb2 will be identified and for leaf spot blotch and root rot resistance functional characterization using transgenic and EMS mutants approached. The Triticum dicoccoides 3BS homologous region will also be isolated by screening and sequencing the corresponding BACs for comparison analyses. Haplotypic variation analyses of the Sb2 alleles in wheat germplasm will also be performed and used for functional marker development.

小麦是我国的主要粮食作物之一，小麦叶枯病（Spot Blotch）近年来从原来的点片发生发展到较大地区范围，且危害逐年加重，在部分地区成为常发和重要病害。国内对小麦叶枯病抗性的遗传研究相对落后，尚未见基因克隆的研究。前期通过对小麦抗、感叶枯病种质资源的遗传分析，发现了一个显性抗叶枯病基因Sb2，并通过分离致病菌，明确了导致该叶枯病的平脐蠕孢菌（Bipolaris sorokiniana）。本研究拟在前期研究的基础上，通过构建大规模抗病性分离群体，利用已经完成的中国春3B染色体测序信息，进一步开发与目的基因紧密连锁或共分离的分子标记，精细定位和图位克隆抗叶枯病基因Sb2，通过转基因和EMS诱变的手段验证其抗叶枯病和根腐病功能，在小麦种质资源中分离Sb2等位基因，研究其单倍型变异和遗传进化特征，并开发功能标记，应用于分子标记辅助选择育种。

小麦是我国主要的粮食作物，其生产易受病虫害的影响，其中小麦蠕孢叶枯病（Spot blotch）是由平脐蠕孢菌（Bipolaris sorokiniana）引起的真菌性病害，在世界范围内分布广泛，能够严重降低小麦的产量和品质。近年来，随着全球变暖、半矮秆品种的推广和耕作制度的改变，小麦蠕孢叶枯病在我国的发病面积有逐渐扩大的趋势，危害程度日益加深，已经成为影响我国部分地区小麦生产的主要病害。挖掘抗蠕孢叶枯病基因，并建立与之紧密连锁的分子标记，将有助于多个抗病基因进行聚合，实现小麦品种抗病的广谱性与持久性。项目执行期间，本研究取得了以下成果：明确了叶枯病致病菌为平脐蠕孢菌（Bipolaris sorokiniana），并在离体无菌条件和大田人工接种下对抗病与感病亲本进行抗病性鉴定，确认了其致病性；遗传分析证实了抗病品系621-7-1的抗病性为显性单基因控制，被国际小麦基因命名协会正式命名为Sb3；利用分子标记进行精细定位，将Sb3定位在分子标记XWGGC9893和XWGGC12530之间0.094 cM的遗传区间，对应中国春参考基因组序列380 kb物理区间；利用化学诱变剂甲基磺酸乙酯(EMS)诱变创制出32个高感小麦蠕孢叶枯病的M2代纯合感病家系，对其中21个纯合的感病家系分别进行了RNA-Seq，在8个纯合突变体中候选区间的一个Gene7均发生了点突变，且导致了非同义氨基酸的变异，将其确定为候选基因；构建Sb3超表达和互补载体转化感叶枯病小麦品系621-7-1，超量表达转基因系验证了Sb3功能；根据抗病和感病基因序列特征开发了功能STS标记，对来自CIMMYT的小麦品系和中国小麦核心种质资源进行了单倍型分析，并与抗叶枯病鉴定结果进行了关联分析；利用遗传分离群体和BSR-Seq技术，发掘出小麦抗叶枯病新基因Sb4，开发分子标记将Sb4基因定位与4BL染色体臂上。