导　　师： 宋尚伟

学科专业： 090201

授予学位： 硕士

作　　者： ;

机构地区： 河南农业大学

摘　　要： 苹果属/(Malus Mill./)植物分布广泛,栽培历史悠久,种质资源极为丰富。苹果属植物基因型高度杂合,种质之间的遗传关系和演化过程等许多理论问题利用传统的分析方法难以解决。开发适用于苹果的EST-SSR标记对于研究苹果属种质资源的起源演化、亲缘关系以及遗传多样性等具有重要意义。本研究利用NCBI数据库中苹果EST的SSR位点,开发了35对EST-SSR引物,建立了苹果EST-SSR体系,并利用筛选出的16对引物对118份苹果资源的亲缘关系进行了研究。研究结果如下: 1.利用SSRIT软件,在NCBI数据库中的86228条单一苹果属EST序列中查找出13259条含SSR的EST,其中有8217条符合可利用性筛选条件。在所有可用的EST-SSR序列中,以二核苷酸为重复单元的序列最多,为4421条,在二核苷酸重复类型中又以CT//TC形式的SSR最为丰富,其次为AG//GA。 2.从筛选出的8217条可利用的EST-SSR序列中随机选取部分序列,依据引物设计原则,利用Primer Premier5.0软件设计出35对EST-SSR引物。 3.通过对苹果EST-SSR反应体系进行优化,确立了最佳体系。即在25μL体系中,MgCl2浓度为2.0mmol·L-1,dNTPs浓度为0.3mmol·L-1,TaqDNA聚合酶用量为1.0U,DNA模板用量为30ng,引物浓度为0.4μmol·L-1。扩增程序为94℃预变性5min;94℃变性45s,52℃退火45s,72℃延伸1min,进行30个循环;72℃延伸10min;4℃保存。该体系稳定可靠,适用于苹果SSR分析。 4.利用8/%的聚丙烯酰胺凝胶电泳对35对引物进行筛选,以条带的多态性和稳定性为标准筛选出16对多态性较好的引物。利用这16对引物对118份苹果品种基因组DNA扩增,共扩增出等位基因位点138个,位点数的变化从3到13不等,平均每对引物检测到8.62个位点,总的多态性比率为94.2/%,各引物的多态性比例分布于81.8/%～100.0/%。 5.根据EST-SSR谱带数据统计结果,应用NTSYS-pc2.0e软件进行遗传相似系数计算,118份苹果品� Malus Mill., which contains a large number of species, widely distributed in world with long cultivated history. Because of the highly heterozygous genotype, many theoretical problems, such as genetic relationships, process of evolution among the germplasms, could not be solved by traditional methods. As a new molecular marker technique, EST-SSR are very suitable for the research on origin and evolution, phylogenetic relationship and genetic diversity of Malus species. In this study, 35 pairs of EST-SSR primers were develped by using SSR locus located in Malus EST from NCBI database, and Malus EST-SSR system was constructed. Sixteen pairs of primers were selected to investigate genetic relationships of 118 apple resources. The main results as follows: 1. In this study, 8217 available EST-SSR were selected from 86228 single Malus EST in NCBI database by using SSRIT software. In the 8217 EST-SSR, there were 4421 dinucleotide repeating units, in which the number of CT//TC repeating pattern EST-SSR was maximum, secondly for AG//GA. 2. Partial EST-SSR were randomly selected from the 8217 available sequences, and 35 pairs of primers were exploited by using Primer Premier 5.0 software according to primer designed principle. 3. The factors which affected on the EST-SSR results of apple were studied, and the optimizing SSR reaction system was established. In 25μL amplification reaction system, solution was composed of 2.0mmo1·L-1 MgCl2, 0.30 mmo1·L-1dNTPs, 1U Taq DNA polymerase, 30ng emplate DNA, 0 .4μmol·L-1primer. The amplification program was a pre-denaturalization step of 5 minutes at 94℃followed by the step which consists of denaturalization 45 seconds at 94℃, annealing 45 seconds at 52℃, extension 1 minute at 72℃for 30 cycles, and a final step of extension at 72℃for 10 minutes. Then PCR Products stored at 4℃for electrophoresis.The system had a high stability, and could be applied to SSR analysis. 4. Sixteen pairs of primers were selected by Polyacrylamide gel electrophoresis

关 键 词： 苹果 引物 亲缘关系 聚类分析 相似性系数

领　　域： [农业科学] [农业科学]