【作者】 沈志军；

【导师】 章镇；

【作者基本信息】 南京农业大学 ， 果树学， 2003， 硕士

【摘要】 微卫星(simple sequence repeat或microsatellite，简称SSR)，是一种新的DNA标记方法，因其具有保守性、高多态性、共显性等特点深受广大研究者的青睐。基于微卫星研究在果梅上仍然是空白，以及核果类果树的亲缘关系仍然不很清晰，本文进行了以下几个方面的研究： 1．本研究采用链亲和素(streptavidin)与生物素(biotin)之间具有很强的亲和能力的原理，用链亲和素磁珠捕捉法构建了果梅品种‘甲州小梅’富含AG／TC重复的小片段基因组文库。通过直接对目的片段的测序获得了果梅微卫星序列共17条，并在Genebank上登录16条。根据这些微卫星序列用Primer5.0设计了9对微卫星引物，用于扩增微卫星区域。微卫星引物的种间保守性研究及多态性检测在包括‘甲州小梅’在内的核果类果树的23个种质材料上进行，发现有8对微卫星引物能够扩增出多态性高的PCR产物。微卫星跨属的保守性研究是通过对苹果品种“新红星”标记分析进行的，发现所设计的9对微卫星引物中有8对能在苹果上扩增出明显的条带，证明了这些微卫星引物的有效性。 2．以SSR标记和ISSR标记两种基于微卫星标记的方法对核果类果树的亲缘关系进行了研究，所选材料包含核果类果树的11个种，4个变种共23个种质，SSR标记除了采用果梅中新开发的8对微卫星引物，还采用已经在桃上报道的SSR引物19对、来自酸樱桃的2对、来自甜樱桃的1对，共30对微卫星引物。亲缘关系研究还采用ISSR的标记方法与SSR标记相结合，共用了22条ISSR引物。亲缘关系研究时首先建立了SSR和ISSR反应的最适反应体系，并通过单、双引物比较实验验证了该反应体系下对SSR双引物扩增的准确性。结果发现：30对SSR引物中有27对微卫星引物，22条ISSR引物中有8条能扩增出有多态性的产物。 3．通过SSR与ISSR标记的综合聚类发现核果类果树中梅和杏的亲缘关系最近(变异系数为0.522)，在聚类时首先聚为一类；梅、杏与李间的亲缘关系次之，在变异系数大于等于0.542时这三个种聚为独立的一类；而桃、樱桃与李、杏、梅之间在系统发育中处于同等的地位；变异系数大于0.555时核果类果树聚为一类。从核果类果树各种类内的聚类的结果可以发现桃的遗传变异系数最小(0.219)，次之为李(0.366)，梅(0.381)、杏(0.396)，种内变异最大的是樱桃(0.493)，推断桃种间遗传背景相对狭窄，而樱桃的遗传背景与其他核果类果树相比最复杂。更多还原

【Abstract】 Microsatellite or Simple Sequence Repeat is a newly appeared molecular marker. Because of its stabilization, high polymorphism, and co-dominate, it was used widely by investigators. Because no research was done about mume microsatellite, and because genetic relationship of peach, plum, apricot, mume and cherry was not clear, following investigations were done in this paper.1. Because of the strong affinity of biotin for streptavidin, we use Streptavidin MagneSphere Paramagenetic Particles combined with biotin-labbled simple sequence anneal with simple DNA digested by HindIII and BaniH I H to capture microsatellite in chromosome DNA from ’jiazhouxiaomei". In this way, microsatellite-enriched library was established and Microsatellite contained DNA was sequenced. we obtained 17 microsatellite sequences with GA/CT repeat. Nine primers were designed by soft Primer 5.0. Polymorphism of these primers were shown on 24 resources, include peach, plum, apricot, mume, sweet cherry and apple. Eight primers in nine showed polymorphism alleyes.2. Thirty pairs of SSR primers from peach, cherry, and mume also, and 22 ISSR primers were used to inverstaged relationship of ’stone-fruit’ trees. 27 of 30 SSR primers and 8 of 22 ISSR Primers showed polymorphism on these germplasms.3. Use soft SPSS11.5, a clustered dendrogram of ’stonefruits’ was obtained. The clustered dendrogram showed that mume and apricot was the nearest among the five, they was clustered into a group first when variation coefficients was 0.522, Plum, apricot and mume were clustered into one group when variation coefficients was 0.542. Cherry, peach and plum, apricot, mume was clustered into three independent group when variation coefficient was between 0.542 and 0.555. The five trees was clustered into one group when variation coefficient reached 0.555. We find that the genetic variation of peach was narrow, while cherry was wide.更多还原