小麦K-CMS育性恢复基因Rfk1精细定位与其候选基因克隆验证

Identifying and cloning fertility restorer gene are very important for revealing the mechanism of fertility restoration. Rfk1, a restore gene against Aegilops kotschyi cytoplasm and located on 1BS chromosome, was detected in a wheat cultivar LK783. To simplify the genetic background, one precious couple of near-isogenic lines of rfk1 Vs Rfk1 referred to as KTP116A and KTP116R, was developed. To further delineate its gene locus and restoration mechanism, we plan to construct a fine mapping of the restorer gene using an excellent BC1 and F2 population from a cross between KTP116A and KTP116R in this project. Firstly, various markers (markers mapped to 1BS BAC clones, SSR markers, wheat 1BS chromosome gene-related putative markers developed based on homology of 1BS sequences with Brachypodium Bd2, Barley 1H unigenes, and putative insertion-site based polymorphism markers developed based on 1BS chromosome 454 sequencing reads and 1BS chromosome BAC-end sequences) primer developed by 1BS physical mapping of Triticum aestivum and located in the physical bin mapping (covering 1BS9-0.84-sat-1.00), and KASP SNP primers developed from BSR-Seq will be employed to screen the population and then obtain the fine mapping of Rfk1. Secondly, the cosegregative markers were selected, the corresponding EST will be alignmented and verified with wheat genomes draft information and fertility differentially expressed sequences from RNA-Seq to clone the candidate restorer gene, then the full length gene will be isolated with In Silico and Race schematic. Thirdly, the expression profile, subcellular localization of candidate restorer gene in the presence of restoration gene will be delineated and its function in KTP116R and KTP116A will be tested by gene silencing and super-expressing, respctivly. In a word, this study will be helpful to enrich the gene bank of restorer gene, the detail location of Rfk1, to isolate the candidate restoring gene, provide reference sequences for molecular mechanism of fertility restoration. Additionally, these new closely linkage or cosegregative markers can be used for marker-assistant selection to develop wheat cultivars pyramiding of K-CMS restoration gene, and accelerate and improve utilization of the gene.

发掘和研究小麦K型细胞质雄性不育（K-CMS）育性恢复关键基因及其网络通路对揭示小麦育性恢复机制，促进杂交小麦研究具有重要意义。小麦LK783 1BS染色体上携带有K-CMS育性恢复基因Rfk1，对K-CMS小麦具有较强的恢复能力（恢复度达90%以上）。本研究欲以携带/缺失Rfk1基因的近等基因系KTP116R和KTP116A为材料，借助基于小麦1BS染色体物理图谱所开发的各类标记引物，结合新型SNP分子标记技术（BSR-Seq）对育性恢复基因Rfk1进行精细作图定位，获得共分离标记；其次，利用比较基因组学和转录组数据分析技术获得育性恢复候选基因；最后利用基因操作技术克隆育性恢复候选基因并进行功能验证。以期为该基因在育种和生产上的利用提供标记，为研究K型雄性不育小麦育性恢复基因的作用机制和促进该恢复基因在杂交小麦育种的应用奠定基础。

K型细胞质温敏雄性不育（TCMS-K）小麦遗传稳定，适应性广泛，可“一系两用”。在国内多个麦区正季播种完全雄性不育、反季播种稳定可育，在小麦杂种优势利用和杂交小麦商业化育种中具有广泛的应用潜力和价值。.本项目利用组合KTP116A//L783/TP116B为作图群体，采用SSR、EST-STS和KASP标记，将位于小麦1BS染色体上的恢复基因位点Rfk1定位在Xnwafu_6与Xnwafu_1B32之间；综合RNA-Seq、qRT-PCR、BSMV-VIGS试验结果，鉴定编码果胶脂酶的基因可能为Rfk1的候选基因；并根据基因序列差异开发的用于辅助选择的PCR标记。.基因过表达和CRISPR/Cas9基因诱导突变试验进一步明确了Rfk1基因，暂命名为TaPME。该基因（1350bp）仅有一个外显子，LK783和KTP116A序列间有7bp的缺失差别，可导致移码突变。TaPME被定位在花粉壁上，且在可育花粉上调表达，正向调控TCMS-K小麦的育性；该基因的沉默或突变，均导致花粉发育异常，如花药不开裂、小孢子皱缩、绒毡层降解异常等，最终导致育性降低或不育。.另外，为加深对TCMS-K小麦的育性恢复机制的理解，从细胞学和生理生化层面探索了花粉败育的特征，如花药绒毡层PCD和果胶含量的异常等。转录组和蛋白质组数据同样揭示，育性相关基因差异表达影响着TCMS-K小麦的育性。项目的实施与完成，将为丰富小麦雄性不育相关分子生物学理论，小麦杂种优势利用和杂交小麦商业化育种提供重要的理论依据。