【作者】 钟金香；

【导师】 林小涛； 章群；

【作者基本信息】 暨南大学 ， 水生生物学， 2002， 硕士

【摘要】 采用RAPD技术，对省级斗门罗氏沼虾良种场提供的四个种群的罗氏沼虾：广东本地人工多代繁殖种群(G)、自江苏吴江引进的种群(J)、自上海水产研究所引进的种群(S)以及自越南引进的野生种群(Y)的遗传多样性进行了研究。 各种群罗氏沼虾个体基因组DNA的提取和纯化：采用SDS-蛋白酶K法进行提取，利用酚-氯仿-异戊醇进行纯化。对所得到的DNA进行检测，20℃保存。 利用OPM组和OPB组随机引物对所提取的罗氏沼虾各种群的混合DNA和个体DNA进行扩增筛选，最后得到13个可用于正式扩增的引物。用下列反应条件：反应体系总体积为25μl，其中含10×buffer2.5μl(含2.0mmol／L镁离子浓度)，200μmol／L dNTP(脱氧核苷三磷酸)，1.5个单位Taq酶和20ng左右模板DNA。用下列PCR反应程序：反应过程包括47个循环：94℃变性40s，36℃退火50s，72℃延伸90s。首次循环前于94℃预变性5min，最后一个循环后于72℃延伸5min。 结果表明，用SDS-蛋白酶K法可以从罗氏沼虾的肌肉组织中提纯到较高质量的DNA，可以用于RAPD分析，所选用的RAPD反应体系和PCR反应程序可扩增出很好的RAPD带，重复性好。结果还表明在研究罗氏沼虾的遗传多样性方面RAPD是一种快捷而较有效的方法。 对于四个种群的种群内个体间遗传距离和Jaccard遗传相似度进行比较，结果发现，越南种的种群内个体间遗传距离最大，江苏种群次之，上海种群内个体间遗传距离较小，广东本地种群内个体间的遗传距离最小。而遗传相似度的排序则相反。 对四个种群间的遗传距离和遗传相似度进行比较，结果发现，各种群间的平均遗传距离为0.13，其中广东本地种群与其它几个种群间的遗传距离最远。而种群间的平均遗传相似度为79.3％。更多还原

【Abstract】 In order to have a sight into genetic diversity of Macrobrachium rosenbergii, Randomly Amplified Polymorphic DNA (RAPD) analysis was applied to the four Populations carried from Macrobrachium rosenbergii well-bred farm, which were the filial generation of the local population by artificial reproduction(G), the population introduced from WuJiang of Jiangshu(J), the one introduced from Shanghai fishery Institute and one natural population introduced from Vietnam(Y).The purification of each individual’s gene DNA from the four populations: extract the DNA with SDS-proteinase K, then purify them with hydroxybenzene-chloroform-Isoamyl alcohol. The purified DNA was tested by electrophbresis and then holded in 20℃ below zero.Thirteen 10-mer primers were selected from 40 primers. The following condition for amplification was used in this study: 25|il volume contained 10xbuffer2.5ul (contained 2.0mmol/L Mg2+) 200umol/L dNTP, 1.5U Taq polymerase. The following PCR procedure was selected: 47 circles in the whole amplification: Denaturalize in 94 ℃ for 40s, melt in 36℃ for 50s, extend in 72℃ for 90s. Predenaturalize in 94℃ for 5min before the first circle, and extend in 72℃ for 5min after the last circle.From the result we can conclude that high quality of genome DNA can be extracted and purified effectively with SDS-proteinase K from the muscle of each Macrobrachium rosenbergii individuals. They were enough to be used in RAPD analysis. Based on the selected RAPD amplification condition and PCR procedure, high quality RAPD fragments could be amplified and could be repeated. What’s more, the result suggested that RAPD analysis was a convenient, effective method.The genetic data were analysed using similarity and genetic distance between individuals of Macrobrachium rosenbergii. The result suggested that genetic similarity was the largest in the Guangdong native population, larger in Jiangshupopulation, large in Shanghai population, and small in Vietnam population.The genetic distance and the similarity between the populations ofMacrobrachium rosenbergii were also been analysed. The results suggested that the average genetic diversity of the four populations was 0.13, and the similarity was 79.3%.更多还原