【作者】 王明；

【导师】 郑用琏；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 玉米丝黑穗病是由丝轴黑粉菌sporisorium reilianum引起的真菌性病害,严重影响玉米的产量性状,是制约玉米发展的重要病害之一。利用分子育种提高玉米品种抗病性,是减少病害造成产量损失的有效途径,而抗病基因的挖掘和抗病功能标记的开发是抗病分子育种的理论前提。近年来,随着高通量基因型分型技术的发展和关联分析理论的不断完善,植物中全基因组关联分析的研究越来越多,但目前对玉米丝黑穗病的遗传研究仍为鲜见,本研究旨在通过高密度的基因分型技术结合全基因组的关联分析,定位玉米抗丝黑穗病相关的基因,并探讨可能存在的抗病机制,主要研究结果如下：1.以0.1kb,0.2kb,0.5kb, lkb,2kb,5kb,10kb,30kb,50kb,100kb,300kb,500kb,1Mb,3Mb,5Mb,40Mb,50Mb和100Mb为步长,研究144份玉米自交系的连锁不平衡(linkage disequilibrium,LD)的衰减距离。结果表明：不同染色体及全基因组染色体上,随着步长距离的增加,LD急剧下降。并在不同染色体上衰减速率不同,第一染色体衰减较快,第十染色体衰减较慢。当LD衰减cutoff值设定为0.1时,144份玉米自交系的LD平均衰减距离约为200kb；2.选取PCA+K, Q+K和K三种混合线性模型,只有Q的模型一般线性模型,只有PCA的模型的一般线性模型,没有Q也没有PCA的模型一般线性模型,总共六种模型,进行关联分析。通过模型的比较分析,选取Q+K模型为最适合的抗丝黑穗病的关联模型；3.对两年两地144份自交系人工接种条件下的抗性表型和MaizeSNP50芯片分析的SNP基因型进行全基因组关联分析,检测到19个SNPs与玉米抗丝黑穗病关联,这些SNPs位点分布于玉米十条染色体上,其中第一、第三、第四和第十染色体只检测到1个抗病位点,19个位点中有8个抗性位点与前人利用连锁作图得到的定位信息一致。大多数SNPs位于基因附近。这些基因可分为三类,一类为抗病基因,如第八染色体上与NBS-LRR类型抗病基因同源的基因；一类为抗病相关基因,如第四染色体上与编码tubby-like蛋白类似基因；一类为可能在抗病反应中发挥作用的基因,如2.04bin的Antifreeze基因。以上结果从一定程度上反应了玉米抗丝黑穗病机制的复杂性；4.对其中2.09bin位点进行了候选区段的丝黑穗病抗性基因的关联分析。在144份玉米自交系材料的目标区域内获得1.6Mb长度的序列结果,其中包括231个SNPs和82个Indels标记。候选区段关联分析表明基因GRMZM2G166566的9个位点SNPs对抗丝黑穗病起关键作用；5.对玉米全基因组关联分析中发现的NBS类型抗病基因,利用已经测序公共数据库,以玉米自交系B73基因组为参考序列,对NBS结构的候选抗病基因进行了基因总数、类型、系统进化关系等分析。更多还原

【Abstract】 Maize head smut, caused by Sporisorium reilianum f. sp. Zea, is a kind of fungal diseases, which has seriously limited the maize production and development. Molecular breeding for improving disease resistance against various pathogens is an effective approach to increase the quality and quantity of maize, and mine disease resistance genes and functional marker associated with resistance are the theoretical premises of disease resistance in molecular breeding.In recent years, with the development of high-throughput genotyping technology and the theory of association analysis, genome-wide association studies (GWAS) are performed in more and more plants. At present, little information is available about the genetic study against S. reilianum in maize. The present study aims at localization of genes involved in head smut resistance and exploration the possible mechanism of resistance against S. reilianum in maize, via combination high density analysis techniques in whole genome with genome-wide association analysis. The main results are as follows:1. When assessing linkage disequilibrium (LD), a serial spacing between two loci on the same chromosome of0.1kb,0.2kb,0.5kb,1kb,2kb,5kb,10kb,30kb,50kb,100kb,300kb,500kb,1Mb,3Mb,5Mb,40Mb,50Mb, and100Mb respectively, were considered. A sharp decline in the r2value was observed as the physical distance increased among each chromosome and all the chromosomes. Linkage disequilibrium decay for each chromosome is also different. The level of LD decay was faster in chr.10than in chr.l. The average r2for all chromosomes was estimated at-200kb, when the value of the cut off for r2was set to0.1.2. To correct for false positives, six models were selected, which referred to the population structure (Q, PCA) and kinship (K). The general linear model (GLM) included the Q model controlling for Q, the PCA model controlling for PCA, and a model that did not control for Q and PCA. The mixed linear model (MLM) comprised the K model controlling for K, the Q+K model controlling for both Q and K, and the PCA+K model controlling for both PCA and K. The results were better from the Q+k model than those from other models.3. In the present study,19association loci were found to be distributed over10chromosomes. One loci in chrl, chr3, chr4and chr10. Eight loci overlapped with previous studies. Most of SNP locations were adjacent to genes. These genes were classified into three groups including R genes such as NBS-LRR homologous gene in chr8, disease response genes such as encoding tubby-like protein gene in chr4, and other genes possibly functioning in plant disease resistance such as Antifreeze gene in2.04bin. These could reflect a complicated molecular mechanism of maize resistance to head smut.4. Candidate gene association in a region expanding SNP4to400kb from both ends was performed to ascertain whether a true association signal exists in2.09bin.1.6Mb of sequence from a panel of144maize inbred lines was obtained, including231SNPs and82Indels. Nine loci showed significant associations with resistance to head smut. These nine loci all belonged to the gene GRMZM2G166566.5. In the analysis of genome-wide association results, NBS type disease resistance genes were found. Therefore, in maize inbred line B73genome, gene number, type and phylogenetic relationship for NBS candidate disease resistance gene were analyzed, and these findings provided useful information on the understanding of the maize genome structure, genome expansion and new genes generation.更多还原