小麦YrSM139-1B免疫条锈病基因精细作图和候选基因克隆验证

A broad immunity resistance to stripe rust was detected in a new variety-Shaanmai 139, which was an introgressed line derived from triticum dicoccoide. To simplify the genetic background, three precious couples of near-isogenic lines of YrSM139-1B Vs yryr referred to as N152-1013R/S, N152-185R/S and N152-1812R/S separately, were developed during last seven years. To further delineate its gene locus and immune resistance mechanism, we plan to construct a fine mapping of the immunity resistance gene using an excellent F2 large population from a cross between R and S in this project. Firstly, referencing to Graingene 2.0, various markers primer located in physical mapping of triticun aestivum and poaceae model plant species Brachypodium distachyon, the EST-STS and RGA marker primers flanked SSR marker Xgwm273 (covering 1BS10-0.5 and 1BS10-0.5-0.84) and AS-PCR primers developed from wheat 90K iSelect chips will be employed to screen the population and then detail the fine mapping location of YrSM139-1B. Secondly, the cosegregativ markers were selected, the corresponding EST will be alignmented with dbEST of Brachypodium distachyon to clone the candidate resistance gene. And then the full length gene will be isolated with In Silico and Race schematic. Thirdly, the expression profile, subcellular localization of candidate resistance gene in the presence of Puccinia striiformis infection will be delineated and its function in response to P. striiformis will be tested using the suppression of candidate gene by virus-induced gene silencing. In a word, this study will be helpful to enrich the gene bank of resistance gene, detail the location of YrSM139-1B, to isolate the candidate resistance gene, provide reference sequences for molecular mechanism of stripe rust resistance, and give more useful information to explore the mechanism of gene expression to defend against biotic stress. Additionally, these new closely linkage or cosegregation markers can be used for marker-assistant selection to develop wheat cultivars pyramiding of stripe rust resistance gene.

本研究以创制的携带/缺失野生二粒小麦广谱免疫条锈病基因YRSM139-1B的近等基因系N152YR1B和N152yr为材料，构建高密度作图群体；借助生物信息学获得1B染色体273位点附近的各类标记引物、物理图谱和禾本科模式植物二穗短柄草数据库EST-STS引物；利用90K小麦SNP芯片分析获得近等基因系间差异SNP位点，并进一步开发AS-PCR引物；综合经典遗传学、新型SNP分子标记技术（高通量90K芯片）、生物信息学对免疫条锈病基因YRSM139-1B进行精细作图定位，获得共分离标记3-5个。 同时，利用比较基因组学方法结合基因操作技术克隆抗病候选基因并进行细胞定位分析和功能验证。以期为该基因在育种和生产上的利用提供标记，为研究抗病基因作用机理和促进该免疫抗性基因在抗病育种的应用奠定基础。

本研究在综合利用简化基因组测序、90K小麦SNP芯片分型和生物学共线性基因序列信息方法，设计开发新型小麦STS、AS-PCR标记，并配合经典SSR标记，在抗病近等基因系培育鉴选的基础上，对小麦抗条锈病基因YrSM139-1B开展了精细遗传图谱绘制。进而在条锈病菌诱导抗病种质陕麦139亲本N9134大规模转录组测序的基础上，克隆了抗病相关基因，同时对其功能进行了验证。取得以下主要研究结果：①证实该基因为一个新基因。②开发SNP和STS新型标记18个，并将YrSM139-1B基因定位于SNP43和gBRG标记之间。③通过转录组和蛋白质组学关联分析预测了条锈病抗病候选基因，获得该基因两侧最近EST标记之间的候选基因及新功能标记16个，标记平均间距0.5cM，最小间距仅为0.2cM。④分离克隆了TaNRC1和Tap23基因，证明TaNRC1基因在条锈病胁迫条件下通过调节剪切体活性激活具有自发激活细胞凋亡功能基因转录剪切变体的转录，实现其非专化性抗病功能。综上所述，项目圆满的完成了任务目标， 为后续该基因克隆奠定了基础。