利用pQTL与eQTL解析小麦赤霉病抗性候选基因

Fusarium head blight (FHB) is a devastating disease of wheat causing severe yield loss and significantly lowering grain quality. Breeding wheat cultivars possessing genes resistant to FHB is the preferable approach to minimize FHB damage. Numerous studies have been published on molecular mapping and phenotypic quantitative trait loci (pQTL) Fhb1 on chromosome 3B was identified as the major QTL for FHB resistance. To clone gene Fhb1, high resolution maps were constructed that flanking STS markers bracketing Fhb1 were within a 1.2-cM interval. A BAC contig spanning the Fhb1 region was constructed and Fhb1 was narrowed down to a 261 kb region with seven putative genes. Clones containing all candidate genes were transformed into FHB-susceptible cv. Bobwhite but none of transgenic plants were resistant to FHB. A BAC-based integrated physical map of chromosome 3B with 995 megabases also has been constructed. But till now, no exciting result on cloning FHB resistance gene with known function has been reported. .Resistance to FHB was a complex quantitative trait controled by multical genes. Classic pQTL mapping studies the relationship between a trait and markers in the genome to identify map location of pQTL associated with the trait. The ability to identify the molecular basis of quantitative traits is being enhanced by genomic methodologies such as transcriptomics, metabolomics, and proteomics. In some cases gene expression levels could be used as a surrogate/biomarker for classical phenotypes, and the gene expression QTL (eQTL) could be an excellent candidate for pQTL. Moreover, the locations of eQTL that regulate gene activity can be correlated with those of pQTL and so provide additional clues as to the genetic basis of quantitative genetic variation..Wheat variety Ning7840, a derivative of Sumai 3, has been used as the promising source in wheat breeding for FHB resistance. We have obtained the positive results on construction of recombinant inbred line (RIL) population and genetic map, identification of pQTL for FHB resistance and the significantly differentially expressed sequence tag (EST) between Ning 7840 and susceptible cultivars after Fusarium infection by microarray analysis, coupled with suppression subtractive hybridization technique. In the present study, we plan to localize those ESTs of Ning 7840 and other resistant lines by molecular markers and nulli-tetrasomes. The expression level of each gene then will be determined and eQTL mapping will be performed in the same RIL population under a controlled experiment conducted for measuring FHB resistance. Cis-eQTL and trans-eQTL will be revealed and the association of eQTL loci with pQTL loci will result in the identification of candidate genes or gene regulatory regions for FHB resistance within the genome. The results obtained in this study will provide more information on understanding the gene function and regulation system of FHB resistance in wheat.

赤霉病严重影响我国小麦产量和品质，利用抗性基因进行品种改良是减轻赤霉病危害的根本途径。利用连锁作图和表型鉴定已经定位了22个稳定的小麦赤霉病抗性pQTL，通过赤霉病接种后基因表达谱分析也获得了多个赤霉病抗性相关候选基因片段，但至今尚未通过图位克隆技术克隆出赤霉病抗性基因，也未能明确基因表达谱得到的候选基因片段与抗性pQTL的关系。本项目拟以宁7840及其作为抗性亲本的遗传群体为材料，在已经构建遗传图谱、定位抗性pQTL、利用基因芯片已获得赤霉病菌接种后基因表达谱的基础上，对病菌诱导条件下差异表达的145个基因EST片段进行染色体定位，通过real-time PCR技术进行基因表达量的eQTL作图。根据基因位点、eQTL区间定位以及与赤霉病抗性pQTL的比较，解析赤霉病抗性候选基因以及影响抗性基因表达的调控基因，以期进一步探明小麦赤霉病的抗性相关基因。

赤霉病严重影响我国小麦产量和品质，利用抗性基因进行品种改良是减轻赤霉病危害的根本途径。本项目以宁7840及其作为抗性亲本的遗传群体为材料，将赤霉病菌诱导条件下差异表达的110个基因定位于相应的染色体；依据geNorm，Delta CT, Best Keeper和Norm finder 等4种计算分析方法，确定了B-tubulin为本研究的最合适内参基因；通过种型与化学型分析确定了本研究的强致病力菌株F0609；对原有遗传连锁图进行了加密，分子标记数由原有的608个增加到1007个；通过real-time PCR技术，完成了2年度110个基因在117个重组自交系中的3次重复的基因表达量测定；利用QTL分析技术对84个抗性相关基因表达量进行eQTL定位，共发现25个抗性相关基因的eQTL位点，其中有6个基因为顺式位点，均位于染色体3B上，表明3BS染色体区域存在抗性候选基因。目前已克隆这6个抗性候选基因，并分析了所有基因的组织表达特异性和时空表达特异性。其中基因273和基因395已通过VIGS技术验证其抗赤霉病功能。本项目针对小麦赤霉病这一世界性难题，利用eQTL技术发现了3BS染色体区域存在6个抗性候选基因，相应基因的克隆与功能分析将有助于进一步探明小麦赤霉病的抗性机制。