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%。
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 Jiangshu
population, large in Shanghai population, and small in Vietnam population.
The genetic distance and the similarity between the populations of
Macrobrachium 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%.
引文
1.常青,周开亚,王义权,掌子凯,曹霄。太湖猪遗传多样性和系统发生关系的RAPD分析。遗传学报,1999,26(5):480~488。
2.陈学年,郭玉娟。彭泽鲫与兴国红鲤杂交优势的研究与利用。淡水渔业,2000,30(9):14~16。
3.陈自明,陈毅峰。用RAPD技术对特化等级裂腹鱼类亲缘关系的探讨。动物学研究,2000,21(4):262~268。
4.陈宗永。罗氏沼虾繁殖前的技术处理。淡水渔业,2000,30(6):14~15。
5.陈作红,张志光,张天晓,李敏。RAPD应用于蕈菌研究中的条件优化探讨。生命科学研究,1998,2(3):199~204。
6.邓怀,张四明,汪登强,邹素云。十种常见淡水鱼类的RAPD鉴定。淡水渔业,1998,2 (1):8~10。
7.常国斌,李慧芳,颜桂车,刘华雷。分子遗传标记的应用现状和展望。动物科学与动物医学,1999,16(2):17~18。
8.甘西,邓凤姣,陈晓汉,黎一键,张锡无,赵忠添,郑俊英。罗氏沼虾遗传多样性的RAPD研究。武汉大学学报(自然科学版),2000,46(6):215~218。
9.韩雅莉。生物遗传多样性和系统学研究中的RAPD分析。内蒙古农牧学院学报,1999,20(2):1~4。
10.何舜平,王伟,陈宜瑜。低等鲤科鱼类RAPD分析及系统发育研究。水生生物学报,2000,24(2):101~105。
11.洪水根。分子生物学技术在诊断养殖贝类传染病中的应用。福建水产,1996,3:55~61。
12.惠东威,陈受宜。RAPD技术及其应用。生物工程进展,1992,12(6):1~5。
13.李家乐,周志金。中国大陆奥利亚罗非鱼的引进和研究。浙江海洋学院学报,2000,19(3):261~265。
14.李健,王清印。中国对虾高健康养殖品种选育的初步研究。中山大学学报(自然科学版),2000,39(增刊):86~90。
15.李思发,邹曙明。中国大陆沿海六水系绒螯蟹(中华绒螯蟹和日本绒螯蟹)群体亲缘关系:RAPD指纹标记。水产学报,1999,23(4):325~330。
16.李向峰,隋正红,张学成。RAPD技术在龙须菜遗传多样性研究中的应用。青岛海洋大学学报,1998,28(2):293~298。
17.黎裕,贾继增,王天宇。分子标记的种类及其发展。生物技术通报,1999,4:1-4。
18.林万明。PCR技术操作和应用指南。人民军医出版社(北京),1993。
19.林小涛。不同光周期条件下罗氏沼虾幼体摄食量及发育的研究。海洋与湖沼,1997,28(1):13~20。
20.林小涛,黄长江,梁旭方,曹双俊。摄食、体重和光照条件下罗氏沼虾幼体氧代谢的影响。生态学杂志,1998,17(4):14~17。
21.刘必谦,戴继勋,喻子牛。RAPD标记在大连湾牡蛎种群研究中的应用。青岛海洋大学学报,1998,28(1):82~88。
22.刘德武,杨关福,李加琪。用RAPD方法分析六个品种猪的亲缘关系。华南农业大学学报,1997,18(增刊):85~89。
23.刘旭东,相建海。新的遗传标记技术——RAPD及其在遗传分析中的应用。海洋科学,1996,4:45~47。
24.卢建平,姜乃澄。罗氏沼虾胚胎发育过程中同工酶的研究。东海海洋,2000,18(3):34~39。
25.罗静,张亚平,朱春玲,肖武汉。鲫鱼遗传多样性的初步研究。遗传学报,1999,26(1):28~36。
26.罗静,杨君兴,张亚平。鱼类多样性的遗传基础。动物学研究,2000,21(2):158~164。
27.欧阳高亮,肖俐,李祺福,洪水根。分子生物学技术在水产养殖中的应用。海洋科学,2000,24(3):31~34。
28.潘莹,赵桂仿。分子水平的遗传多样性及其测量方法。西北植物学报,1998,18(4):645~653。
29.乔德亮。RAPD技术在水产动物种质资源研究中的应用。水产科技情报,2001,28(3):112~114。
30.邱高峰。虾蟹类遗传育种学研究。水产学报,1998,22(3):265~274。
31.邱高峰。分析基因组DNA多态性的RAPD技术在水产生物技术研究中的应用前景。上海水产大学学报,1996,5(2):102~105。
32.邱高峰,常林瑞。我国近海中国对虾种群遗传差异的RAPD分析。上海水产大学学报,2001,10(1):1~5。
33.邱涛,陆仁后,项超美,张菁。RAPD方法对中华绒螯蟹长江、辽河、瓯江三群体的遗传多样性分析。淡水渔业,1997,27(5):3~6。
34.权洁霞,戴继勋,尚迅。海洋生物遗传多样性研究现状。青岛海洋大学学报,1999,29(2):283~288。
35.石拓,孔杰,刘萍,韩玲玲,庄志猛,邓景耀。中国对虾遗传多样性的RAPD分析——朝鲜半岛西海岸群体的DNA多态性。海洋与湖沼,1999,30(6):609-614。
36.史建华,肖雨,徐琴英。罗氏沼虾引种复壮技术的研究。水产科技情报,2001,28(2):64~67。
37.宋林生,相建海,李晨曦,周岭华,刘保忠,刘瑞玉。日本对虾野生种群和养殖种群遗传结构的RAPD标记研究。海洋与湖沼,1999,30(3):261~265。
38.宋林生,相建海,李晨曦,周岭华,刘瑞玉。用RAPD标记研究对虾属六个种间的亲缘关系。动物学报,1998,44(3):353~359。
39.谭远德。杂种优势的一种可能的分子机理——杂合酶的协同效应。南京师大学报(白然科学版),1998,21(3):80~87。
40.田义轲,王彩虹,孟凡佳。DNA分子标记技术及其原理。莱阳农学院学报,2001,7(3):176~179。
41.王慧,杜立新,曾勇庆。RAPD技术及其在动物育种中的应用。山东农业大学学报,1997,28(1):79~82。
42.王家玉译。分子群体遗传学和进化论。农业出版社(北京),1975:121~203。
43.王可玲。增养殖生物种质资源的保护。海洋科学,1998,4:30~34。
44.王晓梅,宋文芹,李秀兰,陈瑞阳。用RAPD技术检测野生鲫鱼和四个金鱼代表品种的基因组DNA多态性。遗传,1998,20(5):7~11。
45.汪小全等。银杉遗传多样性的RAPD分析。中国科学(C辑),1996,26(5):436~441。
46.王艺磊,张子平,王志勇,闵志勇。水产生物技术概述。集美大学学报(自然科学版),1999,4(1):28~32。
47.王玉堂。现代生物技术及其在渔业中的应用。科学养鱼,2000,8:5-6。
48.吴丰春,魏泓。小鼠RAPD-PCR反应体系及扩增程序的筛选。第三军医大学学报,1999,1:19~21。
49.夏德全,曹萤,吴婷婷,王涛。用RAPD分析对罗非鱼遗传变异的研究及其对杂种优势的应用。水产学报,1999,23(1):27~31。
50.夏铭。生物多样性研究进展。东北农业大学学报,1999,30(1):94~100。
51.夏铭,栾非时,李景鹏。RAPD影响因素的研究及实验条件的优化。植物研究,1999,19(2):195~201。
52.项超美,陆仁后,谢浩,刘向宇,张菁。四种十足目甲壳动物遗传差异的RAPD分析。水生生物学报,1998,22(3):251~256。
53.谢浩,陆仁后,项超美,张菁,邱涛。利用RAPD技术对绒螯蟹亲缘关系的研究。水生生物学报,1999,23(2):120-125。
54.颜超群。罗氏沼虾病害防治技术。淡水渔业,2000,30(1):40。
55.杨宁,严华祥,吴常信。DNA分子标记及其在植物遗传变异的抽样统计研究。中国遗传育种研究——第八次全国遗传育种学术讨论会论文集。中国农业出版社(北京),1995:291~294。
56.阎文昭,蔡平钟,宣朴。RAPD技术在生物学研究中的应用。21世纪青年学者论坛,21(3):82~85。
57.张四明,邓怀,晏勇,汪登强,吴清江。中华鲟随机扩增多态性DNA及遗传多样性研究。海洋与湖沼,2000,31(1):1~6。
58.张跃,郑光明,朱新平。PCR技术新应用与水产分子生物学研究。农业生物技术学报,1999,7(2):193~200。
59.张跃,朱新平,罗建仁,夏仁玲。DNA指纹技术及其在水产上的应用。中国水产科学,1998,5(1):79~83。
60.章怀云,刘荣宗,张学文,陈韬,肖调义,李晋衡。草鱼和鲤鱼RAPD引物筛选及鱼特异性分子标记。湖南农业大学学报,1998,24(2):137~142。
61.章怀云,刘荣宗,张学文,陈韬,肖调义,李晋衡。草鱼和鲤鱼群体遗传变异的RAPD指纹分析。水生生物学报,1998,22(2):168~173。
62.赵金良,李思发。中国大陆沿海六水系绒螯蟹(中华绒螯蟹和日本绒螯蟹)群体亲缘关系:生化遗传差异分析。水产学报,1999,23(4):331~336。
63.郑国兴。不同家系的万氏对虾对对虾杆状病毒抗病力的差异。水产学报,1996,18(2):148~152。
64.周开亚,高志千。RAPD标记鉴别中华绒螯蟹种群的初步研究。应用与环境生物学报,1999,5(2):176~180。
65.周莉,樊连春,桂建芳。银鲫复合种外源遗传物质整入的RAPD分析。水生生物学报,1998,22(4):301~305。
66.邹喻苹。RAPD分子标记简介。生物多样性,1995,3(2):104~108。
67.邹曙明,楼允东,孙效文,沈俊宝。用RAPD方法研究草鱼、柏氏鲤和3个地理种群鲤的亲缘关系。中国水产科学,2000,7(3):6~11。
68. Adams, R.P., Demeke, T. systematic relationships in Juniperus based on random amplified polymorphic DNAs (RAPDs). Taxon, 1993, 42(3): 553~571.
69.Ausubel F.M.等著,颜子颖,王海林译。精编分子生物学实验指南。科学出版社,1998。
70. Bardakci F. and Skibinski D.O.F. Application of the RAPD technique in tilapia fish: species and subspecies identification. Heredity, 1994, 73: 117~123.
71. Benzie. J A H. Penaeid genetics and biotechnology. Aquaculture, 1998, 164:23-47.
72. Bernard Chevassus and Michel Dorson. Genetics of Resistance to Disease in Fishes. Aquaculture, 1990, 85: 83~107.
73. Brad J. Argue, Steve M. Arce, Jeffrey M. Lotz, Shaun M. Moss. Selective breeding of Pacific white shrimp (Litopenaeus Vannamei) for growth and resistance to Taura Syndrome Virus. Aquaculture, 2002, 204: 447-460.
74. Caetano-Anolles G.. MAAP: a versatile and universal tool for genome
analysis. Plant Mole Bio, 1994, 25: 1011-477.
75. Dinesh K R. Lim T M, Chan W K, et al. Genetic variation inferred from RAPD fingerprinting in three species of Tilapia. Aquaculture Int., 1996, 4(1) : 19-30.
76. Garcia D. K., Benzie J. A. H. RAPD markers of potential use in penaeid prawn (Penaeus monodon) breeding programs. Aquaculture, 1995, 130: 137-144.
77. Goyard, E., Patrois, J., Reignon, J. M., Vanaa, V., Dufour, R., Bedier, E.. IFREMER's shrimp genetics program. Global Aquacult. Advocate, 1999 26: 26-28.
78. Hedgecock D, Tracey M, Nelson K. Genetics. In: Bliss D E.. The biology of Crustacea. Embryology, morphology, and genetics. Acad Press. (New york) 1982,2:283-403.
79. Hetzel, D. J. S., Crocos, P. J., Davis, G. P., Moore, S. S., Preston, N.C.,. Response to selection and heritability for growth in the Kuruma prawn, Penaeus japonicus. Aquaculture, 2000,181: 215-223.
80. Hirschfeld, B M; Dhar, A K; Rask, K; Alcivar-Warren, A. Genetic diversity in the eastern oyster (Crassostrea virginica) from Massachusetts using the RAPD technique. Journal of Shellfish Research, 1999,18(1) : 121-125.
81. Ilan Karplus, Gideon Hulata, Giora W. Wohlafarth and Amier Halevy. The effect of Size-Grading Juvenile Macrobrachium rosenbergii prior to stoking on their population structure and production in polyculture.Ⅱ. Dividing the population into three Fracitons. Aquacuture, 1987, 62: 85-95.
82. Inglis V et al. Analysis of DNAs relationships among Aeromonas Species by RAPD typing. Aquat. Int., 1996,4(1) : 43-53.
83. John G.K.Williams, Anne R.Kubelik, Kenneth J.Livak, J.Antoni Rafalski and Scott V. Tingey. DNA Polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 1990,18(22) : 6531-6535.
84. Kubota Y, et al. Detection of r-ray induced DNA damage in malformed dominant (Oryzias latips) using AP-pringting. Mutation Res. 1992, 283:
263-270.
85. Lester L J. Population genetics of penaeid shrimp from the Gulf of Mexico, J Heredity,1979,70:175~180.
86. Ling, S.W. and Merican. A.B.O. Note on the life and habits of the adults and larval stages of Macrobrachium(De Man). Indo-pacific Counc. Curr. Aff. Bull. 1961,9(2) : 55-91.
87. Michael B. New and Wagner C. Freshwater Prawn Culture. The Farming of Macrobrachium rosenbergii. Aquaculture, 2002, 203: 399-400.
88. Mulley J C, Latter B D. Genetics variation and evolutionary relationships within a group of thirteen species of penaeid prawns. Evolution, 1980, 34: 904-916.
89. Nei, M. and Li W.H. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. USA, 1979, 76(10) : 5269-5273.
90. Nelson K, Hedgecock D. Enzyme polymorphism and adaptive strategy in the decapod Crustacea. American Natutalist, 1980, 116:238-280.
91. Postiethwait J H, Johnson S. H. Midson C W, et al, A genetic linkage map for the zebrafish. Science. 1994, 264(29) : 699-703.
92. Sara L. Sunden and Scott K. Davis. Evaluation of genetic variation in a domestic population of Penaeus vannamei (Boone): a comparison with three natural populations. Aquculture, 1991,97: 131-134.
93. Sbordoni V, Matthaeis E, Cobdlis, et al. Bottle neck effect and the expression of genetic variability in hatchery tocks of Penaeus japonicus (Crustacea Decapoda) Aquaculture, 1986, 57: 239-251.
94. Stile, J. I., Lemme, C. Sondur, S. et al. Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars. Theor Appl. Genet, 1993,85:697-701.
95. Thorpe J P. The molecular clock hypothesis: Biochemicalevolution, genetic defferentiation and systematics. Ann Rev Evol Syst. 1982,13: 139-168.
96. Uno Yutaka and Kwon chin soo. Larval development of Macrobrachium
Rosenbergi(Da Man) reared in the laboratory. Journal of the Tokyo University of Fisheries. 1969, 55(2) : 179-190.
97. Welsh J. and McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research, 1990, 18(24) : 7213-7218.
98. Welsh J. and McClelland M. Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping. Nucleic Acids Research, 1991, 19(2) : 303-306.
99. Werner Steffens, et al. Pollibilities of sturge on culture in central Europes. Aquaculture, 1990, 89: 101-122.
100. Zhao Z T, Wu T J, Gan X. The experiment of interspecific hybridization in Macrobrach Iumrosenberigii. Science and Technology of Fisheries of Guangxi, 1999(3) : 21-23.
101. Zhuang Zhi-meng, Meng Xian-Hong et al. Genetic diversity in the wild population and hatchery stock of Penaeus japonicus shrimp by isoenzyme analysis. Zoological research. 2000, 21(4) : 323-326.