论文标题:银杏主要栽培品种的RAPD分子鉴别
Study on main cultivated varieties identification with RAPD marker technology
论文作者 冯晓黎
论文导师 谭晓凤；胡芳名,论文学位 硕士,论文专业 森林培育学
论文单位 中南林学院,点击次数 722,论文页数 50页File Size3020k
2002-05-01论文网 http://www.lw23.com/lunwen_543932/ 银杏;品种鉴别;CTAB法;SDS法;RAPD
Ginkgo biloba;cultivated breeds identification;CTAB method;SDS method; RAPD
银杏属于银杏科(Ginkgoaceae)银杏属(Ginkgo)，学名为Ginkgo biloba Linn．。其水平分布大体是在北纬22～24，东经97～124，年平均气温10～20℃的地区是银杏经济栽培区。我国除青海和黑龙江省外，其他大部分省区或多或少都有银杏分布。山东郯城、江苏泰兴、浙江诸暨、河南光山、广西兴安等都是本省的“银杏之乡”。银杏具有用材、果用、食用、药用、观赏于一体的价值，目前我国在产量上居世界首位。 RAPD是1990年Williams和Welsh创立的，这一分子标记建立在PCR基础之上，使用一系列具有10个碱基的单链随机引物，对基因组的DNA进行PCR扩增以检验多态性。银杏的RAPD分子鉴别的研究报道很少，大都以形态学特征和同工酶的方法对其品种进行分类和鉴别。鉴别银杏栽培品种不仅能够清理银杏栽培品种命名混乱的局面，尽早制定统一合理的分类标准，由此减少或避免银杏生产上的巨大损失，而且对开展银杏的进一步科研也有非常重要的意义。 本论文是导师谭晓风教授主持的国家林业局重点课题“银杏、香榧无性系及黄山松、黑松种子的分子鉴别”的部分内容。银杏栽培品种采自浙江临安银杏栽培基地和湖南东安县，共17个品种，分别为金兵卫、黄金丸、滕九郎、岭南、郯城5号、郯城9号、郯城长籽、郯城306号、广西灵川佛手、泰兴佛手、大梅核、大佛手(无雄花也结果)、大佛手(适合少雄树种植)、洞庭皇大佛手、郯城圆籽、东安佛手、东安梅核。以这些银杏品种为原料，利用RAPD技术进行分析鉴别，结果如下： 首先研究了改进CTAB法和改进SDS法抽提银杏胚乳DNA的优劣。结果表明，改良后的SDS法全过程所花费的时间只有5小时，比CTAB法缩短了半天。而CTAB法抽提银杏胚乳DNA纯度较高，含杂质少。通过RAPD实验扩增的指纹图谱可以看出，经SDS法抽提的样品其RAPD指纹图谱清晰可见，与CTAB法抽提样品所得到的图谱效果相差无几，因此能够得出SDS法抽提银杏胚乳来做RAPD实验是可行的。同时也可以得知样品纯度即使低于1.4，虽有大量的蛋白质和多糖物质，但是只要浸提的有机溶剂清除干净，将不会影响RAPD的实验结果。其中改进SDS法抽提银杏胚乳DNA为首次报道。 利用RAPD分子标记技术，对17种银杏栽培品种进行鉴别和分类。以单倍体胚乳DNA为材料，筛选出了OPAD-13、OPAF-16、OPAM-06、OPAN-07、OPAN-08、OPAY-12六个高效引物，确定了49个标记基因型，通过聚类分析软件分析，将这些银杏栽培品种分为两大类，第一类包括11种，分别为金兵卫、郯城5号、郯城9号、郯城长籽、临安大佛手(无雄花也结果)、岭南、圆籽、滕九郎、大梅核(千年古树)、东安梅核、东安佛手；第二大类包括6种栽培品种，分别为黄金丸、泰兴佛手、广西佛手、洞庭皇大佛手、临安大佛手(适合少雄树种植)、郯城306号。结果与传统形态分类有所出入，说明了从分子水平研究银杏栽培品种之间的亲缘关系更为准确可靠，为今后品种鉴别时采用分子标记和形态学相结合的方法提供了科学的理论依据。
Study on main cultivated varieties identification with RAPD marker technologyGinkgo biloba, a kind of remained tree, is belonged to Ginkgoaceae and Ginkgo. It is wide in its distribution. There are 30 provinces in China existed Ginkgo biloba, except for Hei longjiang and Qinghai. It is pretty well in its nutrition and medicine uses, it is also looked upon as beautiful art in landscape garden. So it is high value in research and exploitation. Now Ginkgo biloba plant area is increasing year after year. For resolving the question of how to precise determine the cloning varieties of Ginkgo biloba, the research on identification of main cultivated varieties of Ginkgo biloba with RAPD is made.RAPD is a kind of molecular marker based on PCR, set up by Williams and Welsh in 1990. It uses lObp random primer amplified the different DNA fragment in genome to show the polymorphism. It is often used configuration method to class the cultivated breeds of Ginkgo biloba and seldom reported with RAPD, which is built the theory on the DNA level. Do this research is not only provide with the scientific and reasonable result to make the agreeable norm in classification study, but also free of the economic loss of productive development.The paper is a part of China State Forestry Administration key subject chaired by professor Tan xiaofeng. The breeds of Ginkgo biloba come from the plant areas in Linan county, Zhejiang province and Dongan county, Hunan province. Altogether there are 17 breeds. They are Jinbingwei, Huangjinwan, Tengjiulang Lingnan, Tancheng number 5, Tancheng number 9, Tancheng number 306. Tancheng Changzi. Tancheng Yuanzi. Foushou in Lingchuan in Guangxi, Taixing in Shandong, Linan in Zhejiang, Dongan in Hunan, Da Meihe, Dongan Meihe, Dongtinghuang Da Foushou, separately. There are six efficient primer selected from 49 primers which is built the molecular genetic linkage map of Ginkgo biloba and use them to identify 17 species each other, then draw the assembling class map of cultivated varieties, the research result is as following:The first part is study on the effect of extraction endosperm DNA with improved CTAB and SDS method. It is showed that improved SDS method spent less time than improved CTAB method. It takes only 5 hours to finish the whole procedure, half day ahead of another. And the DNA concentration is higher than improved CTAB method. But the quality of purity of sample is not better with comparison. RAPD experiment gives a piece of proof that low purity DNA sample gets the same clear fingerprint as the high. Hence, we can draw a conclusion that improved SDS method is feasibly applied in endosperm DNA of Ginkgo biloba. At the same time, the content of protein and sugar is not influenced the result of RAPD. Improved SDS method is first reported here.It has found 49 marker sites with 6 efficient primers by RAPD. Based on it marker genotype is formed and the molecular identification system is established accordingly. 17 main cultivated varieties classified into two groups based on Nei"s (1978) Genetic distance. The result is a little different with conventional classification. Probably, it is more scientific to get the truth when configuration character is combined with DNA data. It is needed to do further work to confirm it.
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