葡萄白腐病抗性QTL定位及候选基因研究

发布时间：2018-03-29 07:00

本文选题：葡萄　切入点：白腐病　出处：《沈阳农业大学》2017年硕士论文

【摘要】：白腐病是葡萄主要病害之一,是由白腐病原菌(Coniothyrium diplodiella(Speq.)Sacc)引起的。多数栽培葡萄品种易感染白腐病,葡萄属野生种是抗白腐病的重要种质资源,其中起源于我国的山葡萄(Va.armuRENSIS Rupr.)'双优'具有抗白腐病特性。本研究以'红地球'×'双优'的杂交后代群体作为试验材料,在本试验室已构建的葡萄分子遗传连锁图谱上,开展葡萄白腐病的抗性鉴定,并进行白腐病抗性的QTL定位,最终对抗白腐病候选基因进行荧光定量PCR分析。主要研究结果如下:利用rDNA-ITS序列分析技术分析培养的供试菌株,将获得的ITS序列与GenBank中知序列进行Blast比对,并利用MEGA4.1软件进行ITS序列聚类分析。本试验培养所得菌株为葡萄白腐菌,可以开展后续白腐病原菌侵染离体叶片试验。连续两年采用室内离体叶片鉴定的方法,对'红地球'×'双优'杂交产生的149株F1代群体开展白腐病抗性评价。试验群体单株发病情况存在明显差异,后代群体频率分布呈现出单峰分布,遗传特征表现为连续分布,符合数量性状遗传的规律,抗性评价表型值可用于后续的白腐病QTL定位。结合试验室前期构建的'红地球'×'双优'分子遗传图谱,应用MapQTL 5.0对葡萄白腐病抗性进行QTL定位,共检测到17个与白腐病抗性相关的位点。在双亲整合图谱上共检测到6个QTLs,定位在LG1、LG7、LG11和LG15连锁群上;在母本'红地球'遗传连锁图谱上共检测到5个QTLs,定位在LG1、LG7、LG11和LG15连锁群上;在父本'双优'遗传连锁图谱上共检测到6个QTLs,定位在LG1、LG7、LG11和LG16连锁群上。在白腐病抗性QTL初定位结果的基础上,对定位区段内的基因进行生物信息学分析,筛选出抗病候选基因7IFY6B,应用荧光定量PCR技术分析基因在抗病和感病葡萄叶片中的表达。无论在葡萄抗病或感病葡萄叶片中,TIFY6B基因都受到了白腐菌的诱导,说明TIFY6B基因可能为潜在的葡萄抗白腐病基因,为分子标记辅助选择育种奠定理论基础。
[Abstract]:White rot is one of the main diseases of grape, which is caused by Coniothyrium diplodiella.Speq.Sacc. most cultivated grape varieties are susceptible to white rot, and wild grape species are important germplasm resources against white rot. Va.armuRENSIS Rupr.'Shuangyou', which originated from China, has the characteristics of resistance to white rot. In this study, the hybrid progenies of 'Red Earth' 脳 'Shuangyou' were used as experimental materials on the molecular genetic linkage map of grape, which has been constructed in our laboratory. The identification of resistance to white rot of grape was carried out, and the QTL mapping of resistance to white rot was carried out. Finally, fluorescent quantitative PCR analysis of candidate genes against white rot was carried out. The main results were as follows: the tested strains were analyzed by rDNA-ITS sequence analysis technique. The obtained ITS sequence was compared with the known GenBank sequence by Blast, and the ITS sequence cluster analysis was carried out by using MEGA4.1 software. The strain cultured in this experiment was white rot fungus of grape. It is possible to carry out subsequent experiments on the infection of white rot pathogens against leaves in vitro. For two consecutive years, the method of identification of isolated leaves in laboratory was used. White rot resistance was evaluated in 149 F1 populations from 'Red Earth' 脳 'double you' hybrids. There were significant differences in the incidence of single plant in the tested populations. The frequency distribution of progenies showed a single peak distribution, and the genetic characteristics showed a continuous distribution. In accordance with the law of inheritance of quantitative traits, the evaluation of resistance to white rot can be used for the subsequent QTL mapping of white rot. Combined with the molecular genetic map of 'Red Earth' 脳 'double you' constructed in the early stage of laboratory, MapQTL 5.0 was used to map the resistance to white rot of grape by QTL. A total of 17 loci associated with white rot resistance were detected. Six QTLs were detected on the amphiphilic integration map, which were located in the LG1LLG7LG11 and LG15 linkage groups, and 5 QTLswere detected on the genetic linkage map of the female parent 'Red Earth', which were located in the LG1LL7LG11 and LG15 linkage groups. A total of 6 QTLs were detected on the genetic linkage map of male parent 'Shuangyou', located in LG1 / LG7 / LG11 and LG16 linkage groups. Based on the results of QTL initial mapping of resistance to white rot, bioinformatics analysis was carried out on the genes in the locus. The candidate gene 7IFY6B was screened, and the expression of 7IFY6B gene in disease-resistant and disease-susceptible grape leaves was analyzed by fluorescence quantitative PCR. The TIFY6B gene was induced by white rot fungus in both disease-resistant and disease-susceptible grape leaves. The results suggested that TIFY6B gene might be a potential gene for resistance to white rot in grape and lay a theoretical foundation for molecular marker-assisted selection breeding.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.631

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