【作者】 李开拓；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2008， 硕士

【摘要】 黄皮(Clausena lansium Lour.)Skeels属芸香科黄皮属,原产于我国南部,目前世界上发现黄皮属约有30多个种,在我国约有11个种,主要分布于福建、广东、广西、海南等省,是我国南方重要特产水果之一,已有1 500多年的栽培历史。广东和广西已经开展了相关的基础性研究,主要集中在栽培、管理、生理、形态及细胞学等方面,但从分子生物学方面进行研究的相对较少,对现有的资源遗传多样性及种质鉴定工作还缺乏系统的研究。本研究在开展黄皮栽培品种资源的调查基础上,运用ISSR分子标记,研究了40份黄皮材料的遗传多样性,为黄皮种质资源的有效保护和合理利用提供科学依据,为黄皮新品种的培育提供遗传背景。主要研究结果如下:1.针对黄皮富含酚类化合物和多糖等物质导致DNA提取和纯化困难的问题,采用经过改良的SDS法提取的黄皮DNA,不论从数量还是纯度上均可满足ISSR分析要求,摸索提出了合适的黄皮DNA提取方法。2.研究建立了黄皮ISSR-PCR反应的技术体系。在20μL的反应体系中,TaqDNA聚合酶的用量为1.5 U,引物浓度为0.4μmol/L,dNTP的浓度为300μmol/L,模板DNA浓度为30 ng/反应,10×Buffer 2μL。按照此体系,在94℃预变性5 min,94℃变性30 s,在最适退火温度下退火60 s,72℃延伸90 s,进行40个循环,循环结束后72℃延伸10 min。该体系扩增出的条带清晰,效果良好,能够满足ISSR分析。3.从96个引物中筛选出15个ISSR引物,对40份黄皮材料进行了ISSR分析。共扩增出165条带,其中多态性为100条,多态性百分率为60.6%。同时研究中发现含(AG)n、(GA)n序列的ISSR引物在黄皮上扩增的多态性水平较高,说明黄皮基因组中(TC)n/(CT)n含量丰富。4.依据ISSR分析结果,对供试的40个品种进行聚类分析,结果表明:它们之间的遗传相似系数在0.714-1.000之间,揭示了不同省(区)地方性黄皮品种之间遗传多样性、遗传相似性与亲缘关系,进一步明晰了品种间的遗传距离。为以后更有目的、有选择的引种、育种,缩短育种年限,提高育种效率奠定了一定的理论基础。更多还原

【Abstract】 Wampee(Clausena lansium Lour. Skeels) belongs to Rutacease, consists of about 30 species all over the world. It is one of the most well-known special fruit tree in south china, where is the native land of Wampee, there are 11 species, mainly distributing in Fujian, Guangdong, Guangxi, and Hainan. Wampee has been cultivated in china for 1500 years old. Guangdong and Guangxi have carried some basic studies accordingly, including cultivation, management, morphologically, physiologically and so on, seldom from the angle of molecular biology. The research on both genetic diversity investigation and idioplasm evaluation systematically are still absent.In this study, inter-simple sequence repeat(ISSR) method was applied to detect genetic variation of cultivated Wampee from 40 samples. The results could provide scientific basis for making right protection strategy and help to foster new cultivars. The main results were as follow:1. Faced the fact that it is difficulty to extract DNA from Wampee which contains phenolic compound and quite a lot of sugar, the researcher used improved SDS method to extract DNA of Wampee, which meets ISSR requirements in quantity and purity. So far it is quite a proper way to extract DNA from Wampee.2. The technological system of ISSR-PCR reaction of Wampee has been built up. In the 20μL reaction system, there is 1.5 U of TaqDNAenzyme, 0.4μmol/L of primer, 300μmol/L of dNTP, 30 ng/reaction of template DNA, 2.0μL 10×Buffer solution. According to the system, the experimental steps are as follows. 5 min predenaturation at 94℃, 30 seconds denaturation at 94℃, 60 seconds annealing, 90 seconds extension at 72℃. The cycle repeats forty times and ends in 10 min extention at 72℃.3. A total of 15 ISSR primers were employed on 40 individuals of cultivated Wampee from 96 primers, and 165 scorable amplified polymorphic were detected, of which 100(60.6%) were polymorphic loci. And this research found that primers containing ( AG) n, (GA) n sequence resulted in higher levels of amplified polymorphism of Wampee.4. According to the clustering analysis of ISSR results, it proved that the genetic similarity index was 0.714～1.000. From the result, genetic diversity and similarity and genetic relationships were revealed.更多还原