德国白黑麦2R染色体上抗小麦叶锈病和白粉病新基因的定位及小片段易位系的创制

Rye (Secale cereal L.), a close relative of common wheat (Triticum aestivum L.), has already been proved to be an important and valuable gene sources for wheat improvement because of its multiple disease resistance. In the previous study, new wheat (Xiaoyan 6)-rye(German White) 2R (2D) substitution line WR91 and 2RL addition line WR56 were produced through distant hybridization and chromosome engineering protocols, then were identified using GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization) and rye chromosome arm-specific marker analysis. WR91 was highly resistant to leaf rust (Puccinia triticina) and powdery mildew (Blumeria graminis f. sp. tritici), while WR56 showed susceptible to leaf rust and resistant to powdery mildew. By using resistance spectrum analysis on WR91 and WR56, disease resistance evaluation, GISH and specific marker analysis on F2 population of WR91×Mingxian 169, the result demonstrated that leaf rust and powdery mildew resistance gene different from the known resistance genes, respectively, derived from rye chromosome 2RS and 2RL. This program plans to (i) develop 2R chromosome deletion lines and wheat-rye 2R translocation lines by inducing chromosome variations using 60Coγ radiation, gametocidal chromosome originating from Aegilops; (ii) develop a large number of markers specific for rye chromosome 2RS and 2RL by using SLAF-seq (specific-locus amplified fragment sequencing), and construct physical map of chromosome 2R of German White rye; (iii) locate leaf rust and powdery mildew resistance genes on the chromosome region of 2RS and 2RL, identify the closely associated molecular markers with resistance genes, and obtain small segment translocation lines with resistance genes. These results will provide new disease resistant germplasm and gene sources for wheat breeding.

小麦近缘属物种黑麦(Secale cereale L. 2n = 2x = 14, RR)高抗小麦多种病害，是改良小麦的重要基因源。前期申请者通过远缘杂交、分子细胞学检测鉴定出了高抗小麦叶锈病和白粉病的小偃6号-德国白黑麦2R(2D)异代换系WR91，以及感叶锈病和高抗白粉病的2RL双端体附加系WR56。抗谱分析、F2群体的抗病性鉴定及原位杂交GISH与分子标记检测的结果表明，黑麦2RS和2RL染色体分别携带有不同于现有已知基因的抗叶锈病和抗白粉病新基因。本项目拟计划：利用60Coγ辐射和3C杀配子染色体等手段，创制黑麦2RS和2RL染色体缺失系和易位系；并利用高通量的SLAF测序技术开发大量黑麦染色体的专化标记，构建2R的分子标记图谱；定位抗叶锈病和白粉病新基因的染色体区段，鉴定出能追踪抗病基因的专化标记，并获得高抗叶锈病和白粉病的2R小片段易位系，为小麦育种提供新的抗病种质基因资源。

白粉病、叶锈病是影响小麦稳产、高产的重要病害。小麦近缘属黑麦(Secale cereale L. RR)高抗小麦多种病害，是遗传改良小麦的重要基因源。随着病原菌和寄主抗性的协同进化，生产上大规模使用的源于黑麦的部分抗性基因陆续丧失抗性。因此，亟需从黑麦中发掘新的有效抗性基因，创制新的抗病种质以应用于小麦抗病育种。利用基因组原位杂交GISH、多色荧光原位杂交FISH鉴定及黑麦染色体特异分子标记分析，证明小麦骨干亲本小偃6号与德国白黑麦远缘杂交的后代材料WR91、WR56分别是小麦-黑麦2R(2D)异代换系和2RL双端体附加系。WR56成株期高抗白粉病，WR91成株期高抗白粉病与叶锈病，苗期高抗13个叶锈病生理小种，成株期抗白粉病新基因位于德国白黑麦2RL染色体上。利用60Coγ辐射、杀配子染色体等手段以及以小麦主栽品种回交诱导产生新的染色体结构变异，利用2R染色体的易位系与缺失系，通过GISH鉴定、黑麦染色体特异标记检测和抗病性鉴定，将成株期抗白粉病新基因定位到2RL染色体上763-896Mb的物理区间，并创制了高抗白粉病具有2RL不同黑麦染色体片段的易位系新种质。通过构建抗感池，利用小麦660K SNP芯片及RNA-Seq转录组测序技术，将WR91携带的抗叶锈病基因定位于6B染色体上660-716Mb的物理区间，与6BL末端的已知抗叶锈病基因Lr9位置相近。基于5个黑麦品种的SLAF-seq简化基因组测序结果，开发了57对2RL和2RS黑麦染色体特异PCR标记及3对特异KASP标记。本项目创制的抗病新种质基因资源及检测标记可用于小麦抗性的遗传改良，为抗病基因的精细物理定位和克隆奠定了基础。发表标注本项目资助的SCI论文8篇，授权发明专利2项，培养研究生3名。