【作者】 王彩红；

【导师】 魏兴华；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2014， 博士

【摘要】 水稻是重要的粮食作物。稻瘟病是全世界水稻主栽区发生最严重的病害之一，平均每年因稻瘟病损失的产量可供6千万人食用。防治稻瘟病最经济、安全的方法是培育含稻瘟病持久抗性基因的新品种。目前对稻瘟病抗性基因的克隆主要是通过构建双亲本遗传群体，运用图位克隆的方法实现。本研究首次应用805,158个SNP标记，对517份中国水稻地方品种资源的稻瘟病抗性进行全基因组关联分析。主要结果如下：1.运用混合线性模型，在P<10-7水平条件下，全基因组关联分析（Genome-Wide AssociatedStudy,GWAS）共检测到126个关联位点。在籼稻群体内共检测到51个与稻瘟病抗性相关联的SNP位点；在粳稻群体内共检测到5个与稻瘟病抗性相关联的SNP位点；在总样本中共检测到72个与稻瘟病抗性相关联的SNP位点。2.在所检测到的SNP位点中，有18个已报道位点，其中包含7个已克隆基因（Pia、Pik、Pi-1、Pik-h/Pi-54、Pik-m、Pik-p、Pi5/Pi3/Pi-i）和8个QTL位点（Pif、Pig、PiGD3、Pik-s、Pik-g（t）、Pitq5、Pitg6、Pi6）。除已知位点外，还发现18个新的SNP位点，共涉及25个尚未报道的R基因；籼稻群体中检测到17个，粳稻群体中仅检测到1个，总样本自然群体中检测到11个。其中有4个候选R基因同时在籼稻群体和总样本群体中均被检测到，1个在粳稻群体和总样本群体中被检测到。3.利用基因功能注释（gene ontology，GO）对其它候选基因进行功能分类，划分为53个功能亚范畴。在蛋白质类型范畴中，核苷酸结合（nucleic acid binding）和水解酶（hydrolase）所占比例最大；在生物途径范畴中，代谢途径（metabolic process）所占比例最大；在代谢途径范畴中，DNA复制（DNA replication）所占比例最大；在分子功能范畴中，催化过程（catalytic activity）和连接（binding）所占比例最大。4.本研究共选取4个显著关联位点：Chr11₆526998、Chr12₁4696604、Chr12₁3690289和Chr12₁3032951。运用实时定量PCR（Real-time quantitative PCR，RT-PCR）技术及基因表达谱信息，对候选基因进行检测和验证。结果显示：（1）Chr11₆526998位于稻瘟病抗性基因Pia的组成基因Os11g0225100的编码区；（2）Chr12₁4696604位于PiGD3（QTL）附近，研究发现基因Os12g0438300（含有NB-ARC和LRR结构域）很可能是PiGD3的最优候选基因；（3）对于Chr12₁3690289关联位点，基因Os12g0424700和基因Os12g0427000可能相互协作，共同完成稻瘟病抗性调控；（4）对于Chr12₁3032951关联位点，基因Os12g0416300、Os12g0417100和Os12g0417600被视为最优候选基因。5.候选基因单倍型分析表明大部分SNPs变异均属于非同义突变。6.本研究证实GWAS能够作为一种高效率挖掘抗性候选基因的方法。更多还原

【Abstract】 Rice is a vital and stable grain food, but rice blast disease is one of the most serious and recurrentproblems in rice-growing regions. Every year the rice capable of feeding60million people is destroyedby rice blast disease. Hence, cultivating rice varieties with highly efficient, durable resistance to blast isstill the most economically feasible and environmentally sound management approach in mostblast-prone rice ecosystems. Most previous studies cloned resistance genes were based on linkagemapping using bi-parental populations. Here, we extensively examined blast resistance in agenome-wide association study （GWAS） based on genotyping805,158SNPs variants across517diverse rice accessions. The main results were as follows:1. GWAS identified126associated loci at P<10-7in the compressed MLM. A total of51associatedSNPs were identified in indica panel. Only five associated loci were detected in japonica panel. AGWAS in the full panel was higher power with a total of72SNPs.2. Searching the flanking regions of the associated SNPs, we revealed18regions co-localized, withseven known cloned genes （Pia, Pik, Pi-1, Pik-h/Pi-54, Pik-m, Pik-p, Pi5/Pi3/Pi-i）, and eight QTLs （Pif,Pig, PiGD3, Pik-s, Pik-g（t）, Pitq5, Pitg6, Pi6）. In addition to the loci associated with the known, wealso found another18new loci referring to25previously unreported candidate genes, which describedas disease resistance protein and showed potential function for rice blast as R genes, separately17inindica panel, one in japonica panel and11in the full panel. Of25candidate genes，four simultaneouslyfound in indica and the full panel, and one simultaneously in japonica and the full panel.3. According to gene ontology （GO） analysis, the others encoding proteins involved53classifications.In the protein class, nucleic acid binding and hybrolase were the main. In the metabolic pathways, DNAreplication was major. In the biological process, metabolic process was the most. In the molecularfunction, catalytic activity and binding were the largest proportion.4. We chose four associated SNPs to identify the candidate genes, referring to Chr11₆526998,Chr12₁4696604, Chr12₁3690289and Chr12₁3032951. Through RT-PCR and expression profilesfrom microarray data, we observed:（1） Chr11₆526998was within the coding domain of the geneOs11g0225100, one of the rice Pia-blast resistance gene.（2） A signal, Chr12₁4696604, was detectedaround the QTL PiGD3. And, our study identified the gene Os12g0438300, a disease resistance proteincontaining NB-ARC and leucine-rich repeat domain, as the main candidate gene of PiGD3.（3） Forassociated SNP, Chr12₁3690289, the newly discovered R genes, Os12g0424700and Os12g0427000were presumed to be required for the full function of this locus.（4） In addition, for Chr12₁3032951,the region without R genes, Os12g0416300, Os12g0417100, and Os12g0417600were considered themost likely candidate genes for this region.5. Haplotype analysis around the candidate genes reflected that most of the SNPs were non-synonymousmutation. 6. Our results further confirmed that GWAS is a powerful complementary approach for dissecting thequantitative disease resistant genes to traditional QTL mapping.更多还原