双壳纲贝类rDNA分子系统学及马氏珠母贝遗传多样性研究
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摘要
我国近海海域包括渤、黄、东、南四海，地跨温带、亚热带和热带海区，海洋双壳纲贝类极为丰富，绝大部分种类具有很高的经济价值。近十几年来，近海贝类养殖业发展迅速，已成为沿海地区海水养殖的重要支柱产业之一。然而有关海洋经济贝类各科属种间的分子系统发育关系研究报道甚少。本研究测定了该纲11个不同种(分属于5科、6属)的核糖体18S基因部分序列及转录间隔区1(ITS1)全序列，所得序列已全部登录到GenBank数据库。结果表明，该序列在种间存在很高的多态性，长度从558 bp到784 bp不等，碱基差异百分比在3.8％～60.4％之间，种间ITS1序列同源性很低，有片段的插入与缺失。不同种间的18S基因部分序列同源性相对较高，差异主要表现在碱基的转换。用邻接法(NJ)构建了14个种的18S部分序列(约240 bp)的系统发育树，首次从分子水平上确定这些种在该纲中的分类地位，其系统关系与传统形态学分类结果基本一致。对取自四个不同地域群体的马氏珠母贝(Pinctada martensi)个体的18S-ITS1序列进行了测定，该序列在种内个体间的差异百分比在0.6％～1.9％之间。分析指出：18S基因可以作为双壳纲动物高阶元阶系统发育的分子标记；ITS1序列种间变化很大，可以应用于该纲物种的分类及鉴别，在亲缘关系相近种及种内变异相对较小，但核苷酸变异位点信息量丰富，可用于属内种间、亚种和群体间的遗传多样性研究。
另外，本论文运用27个10碱基随机引物对不同地域的马氏珠母贝种群间交配获得的子一代六个群体进行了RAPD分析。结果表明，群体间交配子代的Shannon多样性指数分别高于群体内交配所得子代的指数。最后讨论了不同地域之间进行马氏珠母贝育种及遗传多样性保护工作的可行性。
Bivalves constitute a dominant and divers group of marine animals in our country. Most of them are of major commercial important species, and studies on their genetic diversity are necessary for the sustainable exploitation and conservation of these bioresources. The objective of the present study is to explore the feasibility of using the ribosomal DNA as a molecular marker for studying the interspecific phylogenetic relationship and intraspecific genetic variations among bivalves. The 18S-ITS1 sequences of 11 species at differing taxonomic levels were determined. The sequences were found to exhibit a high degree of length polymorphism among different species, ranging from 558 bp (Chlamys farreri) to 784 bp in (Ostrea rivulahs), mainly resulting from the variation of ITS1. The percent divergence of sequences ranged from 3.8% to 60.4%. The NJ (neighbor-joining) tree inferred from 18S fragment of 14 species agree with the previous based on morphologies and chemical analysis well. The sequence was found to var
y among 4 geographical sites of Pinctada martensi, ranging from 0.6% to 1.9%. The variations involved substitutions, transversion as well as insertions and deletions among them in P.martensi. All these results show that ITS1 is highly divergent among different species of bivalves and could be used in classification and distinguishing closely related species, and also could be used for molecular systematic studies at relative species, subspecies and population levels.
Pinctata martensi is one of the most important aquacultural seashells in the southeast of China. Wild individuals were collected from Sanya (Hainan province), Beihai (Guangxi province) and Daya Bay (Guangdong province) randomly, then mated and cultured in the same condition. In order to get a deep understanding of it and improve efficiency of breeding by marker-assisted selection, we using 27 primers tested different filial populations by RAPD
technique. The results showed that the diversity Shannon index of filial population mated from inter-populations was higher than filial populations mated from inner-populations. Then we analyzed the feasibility of breeding from two different areas.
另外，本论文运用27个10碱基随机引物对不同地域的马氏珠母贝种群间交配获得的子一代六个群体进行了RAPD分析。结果表明，群体间交配子代的Shannon多样性指数分别高于群体内交配所得子代的指数。最后讨论了不同地域之间进行马氏珠母贝育种及遗传多样性保护工作的可行性。
Bivalves constitute a dominant and divers group of marine animals in our country. Most of them are of major commercial important species, and studies on their genetic diversity are necessary for the sustainable exploitation and conservation of these bioresources. The objective of the present study is to explore the feasibility of using the ribosomal DNA as a molecular marker for studying the interspecific phylogenetic relationship and intraspecific genetic variations among bivalves. The 18S-ITS1 sequences of 11 species at differing taxonomic levels were determined. The sequences were found to exhibit a high degree of length polymorphism among different species, ranging from 558 bp (Chlamys farreri) to 784 bp in (Ostrea rivulahs), mainly resulting from the variation of ITS1. The percent divergence of sequences ranged from 3.8% to 60.4%. The NJ (neighbor-joining) tree inferred from 18S fragment of 14 species agree with the previous based on morphologies and chemical analysis well. The sequence was found to var
y among 4 geographical sites of Pinctada martensi, ranging from 0.6% to 1.9%. The variations involved substitutions, transversion as well as insertions and deletions among them in P.martensi. All these results show that ITS1 is highly divergent among different species of bivalves and could be used in classification and distinguishing closely related species, and also could be used for molecular systematic studies at relative species, subspecies and population levels.
Pinctata martensi is one of the most important aquacultural seashells in the southeast of China. Wild individuals were collected from Sanya (Hainan province), Beihai (Guangxi province) and Daya Bay (Guangdong province) randomly, then mated and cultured in the same condition. In order to get a deep understanding of it and improve efficiency of breeding by marker-assisted selection, we using 27 primers tested different filial populations by RAPD
technique. The results showed that the diversity Shannon index of filial population mated from inter-populations was higher than filial populations mated from inner-populations. Then we analyzed the feasibility of breeding from two different areas.
引文
蔡英亚，张英，魏若飞．见：贝类学概论(第一版) ，上海：上海科学技术出版社，1979，16-21．
陈来钊，王子臣，1994．温度对海湾扇贝与虾夷扇贝极其杂交受精、胚胎和早期幼体发育的影响．大连水产学院学报，1994，9(4) 1-9．
巩宁，阙华勇，张国范．贝类非整倍体研究进展．生命科学研究，2001．5：68-72．
何毛贤，林岳光等．合浦珠母贝四倍体诱导过程中非整倍体的产生．热带海洋，2000，19：59-64．
何毛贤，林岳光，姜卫国．三倍体合浦珠母贝不育性研究．热带海洋，1996，15，17-21．
金启增，姜卫国，谢玉坎等．水温变化对合浦珠母贝心脏搏动的影响．中国贝类学会编辑，贝类学论文集，第二辑，北京：科学出版社．1984．24-29．
金冬燕，黎孟枫等译，1992，分子克隆实验指南，科学出版社．
孔晓瑜，刘亚军，喻子牛等．栉孔扇贝和海湾扇贝线粒体16S rRNA基因片段的比较研究．贝类学论文集，第Ⅸ辑，中国贝类学会编，海洋出版社2001．
李刚，姜卫国等．合浦珠母贝、长耳珠母贝合大珠母贝种间人工杂交的研究Ⅱ．受精过程和杂交后代的染色体观察．热带海洋，1983，2(4) ：316-320．
李刚，金启增等．合浦珠母贝和长耳珠母贝的生化遗传变异．遗传学报，1985，12：204-212．
李广丽，叶富良．马氏珠母贝不同组织同工酶的比较．水产学报，2000，24 (5) ：417-521．
刘景旋．台山川山群岛沿岸软体动物名录口．辦鰓纲(Lamellibranchia)．生态科学，1994，2：50-55．
缪炜，余育和等．上海四膜虫和两株嗜热四膜虫的rRNA基因序列分子系统关系．动物学研究，2001，22：265-269．
缪炜，余育和等．6种累枝虫(Epistylis) rRNA基因18S-ITS1序列及其分子系
统发育关系．中国科学(C辑) ，2002，2：138-145．
施立明．遗传多样性及其保存．生物科学信息，1990，2：118-164．
唐伯平，周开亚，宋大祥．核rDNA ITS序列在无脊椎动物分子系统学研究中的应用．动物学杂志，2002，37(4) ：67-73．
苏天凤，蔡云川等．合浦珠母贝三个养殖群体的RAPD分析．中国水产科学，2002，9(2) ： 106-109．
王爱民，邓凤姣，张锡元等．马氏珠母贝遗传多样性的RAPD分析．武汉大学学报，2000，46(4) ：467-470．
王梅林，郑家声等．我国海洋鱼类和贝类染色体组型研究进展．青岛海洋大学学报，2000，30：278-284．
王华接，严文侠等．珍珠贝在南海北部海洋结构物上的分布．热带海洋，2000，19：64-69．
王清印，杨爱国．栉孔扇贝三倍体研究进展和展望．中国水产科学，2000，7： 93-96．
魏贻尧 姜卫国．三种珠母贝种间杂交的初步研究．中国贝类学会编辑，贝类学论文集，第二辑，北京：科学出版社．1984．215-216．
杨俊毅，高爱跟等．台州列岛海域贝类种类组成与分布特征．东海海洋，2000，18：37-42．
俞海箐，张亚平，林飞栈等．探讨rDNA ITS区作为果蝇drosphila nasuta分子系统发育研究的价值．遗传学报，2000，27：18-28．
章超桦，吴红棉等．马氏珠母贝肉的营养成分及游离氨基酸组成．水产学报，2000，24(2) ，180-184．
郑成兴，黄宗国等．1997大亚湾核电站附近水域马氏珠母贝种群生态．动物学报，1997，43(3) 271-278．
张亚平．从DNA序列到物种树．动物学研究，1996，17：247-252．
Allendof. F. W et al., Genetic variation in Scandinavian brown trout(Salmo trutta L) : Evidence of distinct sympatric population. 1976, Hereditas, 83: 73-82.
Ankeim N. Concerted evolution of multigene families, in: Nei M Koehm R K eds. Evolution of genes and protein. Sinauer, Sunderland MA, 1983, 38-61.
Arnaud. S, Monteforte. M, Galtier. N et al., Population structure and genetic variability of pearl oyster Pinctada mazatlanica along Pacific coasts from Mexico to Panama. Conservation Genetics, 2000, 1: 299-307.
Bargues MD, Mas-Coma S. Phylogenetic analysis of Lyrnnaeid snails based on 18S rDNA sequences. Molecular Biology and Evolution. 1997, 14: 569-577.
Benjamin K D, Robert D A. Intraspecific variation in Cryptocaryon irritans. J Euk Microbiol. 1997, 44: 25-32.
Blankenship H L, et al. A responsible approach to marine stock enhancement. American fisheries Society Symposium, 1995, 15:001.
Bowles J, Blair D, McManus D P. A molecular phylogeny of the human schistosomes. Mol Phylogenet Evol, 1995a, 4:103-109.
Bowles J, Blair D, MeManus D P. A molecular phylogeny of the genus Echinococcus. Parasitoltogy, 1995b, 110:317-328.
Campbell B C, Steffen-Campbell J D, Werren H J. Phylogeny of the Nasonia species compex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences, Insect Mol Biol, 1993, 2 (4) : 225-237.
Despres L, Kruger F J, Imbert-Establet D et al. ITS2 ribosomal RNA indicates Schistosoma hippopotami is a distinct species, Int J Parasitol, 1955, 25: 1509-1514.
Dickerson. R. E, The structure of eytochrome C and the rates of molecular evolution. J. Mol. Evol, 1971, 1:26-45.
Dover G A, Molecular drive: a cohesive mode of species evolution. Nature, 1982, 299: 111-117.
Dmitrv Mukha, Brian M. Wiegmarm, Coby Schal. Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta) . Insect Biochemistry and Molecular Biology, 2002, 32:951-960.
Felsenstein. J, PHYLIP: Phylogenetic inference package, ver. 3. 5c. 1993, Washington: University of Washington.
Felsenstein. J, Confidence limits on phylogenies: an approach using bootstrap. Evolution, 1985, 39: 783-791.
Fenton B, Malloch G, Germa F. A study of variation in rDNA ITS region showa that two haplotypes coexist within a single aphed genome. Genome, 1998, 41: 338-344.
Gomez-Zurita J, Juan C, Petitpierre E. Sequence, secondary structure and phylogenetic analyses of the internal transcribed spacer 2 (ITS2) in the Timarcha leaf beetles (Coleoptera: Chrysomelidae) . Insect Mol Biol, 2000, 9: 591-604.
Gotoh T, Gutienze J, Navajas M, Molecular comparison of the sibling species Tetranychus puerasicola Ehora et Gotoh and T. urticae Koch (Acari: Tetranychidae) . Entomological Science, 1998, 1 : 55-57.
Haertl. B, Bonndlow, G. Epidemiologieal fingerprinting of Enterobactel cloacae by small-fragment restriction endonudease anaiysis and pulsed field gel electropdoresis of genomic restriction fragments. J. Clin. Microbiol. 1993, 31 : 128-133.
Harris D J, Crandall K A. intragenomic variation within ITS1 and ITS2 of freshwater crayfishes (Deeapioda: Cambaridae) : implications for phylogenetic and microsatellite studies. Mol Biol Evol, 2000, 17(2) : 284-291.
Hillis M D M, Davis S K, Ribosomal DNA: Intraspecific polyporphism, concered evolution and phylogeny reconstruction. Syst Zool, 1988, 37: 63-66.
Homan W L, Verlaan M et al. Comparison of the internal transcribed spacer, ITS1, from Toxoplasma gondii isolates and Neospora caninum. Parasital Res, 1997, 83: 285-289.
Hunter. R & C. Marker. Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science, 1957, 125:1294.
Jeffreys. A. J, Individual specific "fingerprints" of human DNA. Nature. 1985, 316: 76-79.
Jeffreys. A. J et al., DNA fingerprints and segregation analysis of multiple
markers in human pedigrees. Am J. Hum. Genet, 1986, 39:11-24.
Katsuhiko T. Wada. Aquaculture genetics of bivalve molluscs: a review. National Journal of Ocean University of Qingdao. 2000, 30:107-114.
K H Chu, C P Li, H Y. Ho. The first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for phylogenetic and population analysisin crustacea. Mar biotechnol, 2001, 3: 355-361.
Kojima. S, Segawa. R, Hashimoto, Molecular phylogeny of vestimentiferans collected around Japan, revealed by the nucleotide sequences of mitochondrial DNA. Marine Biology. 1997, 127: 507-513.
Lindsay D S, Upton S J, Owens D S et al. Cryptosporidium andersons n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus. J Eukaryot Microbiol, 2000, 47 (1) : 91-95.
Lounibos L P, Wilkerson R C, Conn J E et al. Morphological, molecular and chromosomal discrimination of cryptic Anopheles (Nyssorhynehus) (Diptera: Culicidae) from south America. J Med Entomal, 1998, 35:830-838.
Lynch M, The similarity index and DNA fingerpringting. Mol Biol Evol, 1990, 7: 478.
Mazureak. G. H et al., Large DNA restriction fragment polymorphism in the Mycobacterium avium-M, intracellulare complex: a potential epidemi olgical tool. J. Clin. Microbiol. 1993, 31:390-394.
Miesfeld et al., A member of a new repeated sequence family which is conserved throughout the human δ and β globin genes. NucL Acids. Res, 1981, 9:5931-5948.
M. J. López-Pion, A. Insua. Identification of four scallop species using PCR and restrication analysis of the ribosomal DNA internal transcribed spacer region. Marine Bioteehnology, 2002, 4: 495-502.
Moller. D, Genetic diversity in spawning cod along the Norwegian cast. Hereditas: 1968, 60:1～82.
Morgan J A T, Blair D, Nuclear rDNA ITS sequence variation in the trematode
genus Echinostoma: an aid to establishing relationships within the 37-collar-spine group. Parasitology, 1995, 111: 609-615.
Muster W, Boon K, ven der Sande C A F M et al., Functional analysis of transcribed spacers of yeast ribosomal DNA. EMBO, 1990, 9: 3989-3996.
Navajas M, Lagnel J, Gutierrez J et al., Species-wide homogeneity of nuclear ribosomal ITS2 sequences in the spider mite Tetranychus urticae contrasts with extensive mitochondrial COI polymorphism. Heredity, 1998, 80 (Pt6) : 742-752.
Odorica D M, Miller D J. Variation in the ribosomal internal transcribed spacers and 5. 8S rDNA among five species of Acropora (Cnidaria; Scleractinia) : patterns of variation consistent with reticulate evolution. Mol Biol Evol, 1997, 14(5) : 465-473.
Okamoto M, Agatsuma T, Kurosawa T et al., Phylogenetic relationships of three hymenolepidid species inferred from nuclear ribosomal and mitochondrial DNA sequences. Parasitology, 1997, 115: 661-666.
Park. L. K & P. Moran, Developments in molecular genetic techniques in fisheries. Rev. Fish, Biol, Fish. 1994. 4:272-289.
Paskewitz S M, Wesson D M, Collins F H. The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae complex. Insect Mol Biol, 1993, 2: 247-357.
Peyretaillade E, Biderre C, Peyret P et al., Microsporidian Encephalitozoon cuniculi, a unicellular with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core. Ucleic Acids Res, 1998, 26: 3513-3520.
Pleytc K A, Duncan S D et al. Evolutionary relationships of the samonid fish genus Salvelinus inferred from DNA sequences of the first internal transcribed spacer (ITS1) ofrDNA. Mol Phylogen Evol. 1992, 1: 223-230.
Power. A. G, Entomoh Exp. Appl. 1988, 49:213-219. Rasmussen S W. 2001, DNAtools, a software package for sequence analysis.
The carlsbeg laboratory, Copenhagen, Http://www. dnatool. dk.
Schlouerer C, Hauser M et al. Comparative evolutionary analysis of rDNA ITS regions in Drosophila. Mol Biol Evol. 1994, 11: 513-522.
Smith, D. R. and T. C. Glenn. Allozyme polymorphisms in Spanish Honey bees (Apis mellifera iberica) . The Journal of Heredity, 1996, 86: 12-14.
Schilthuizen M, Gettenberger E, Gultyaev A P. phyogenetic relationships inferred from the sequence and second structure of ITS1 rRNA in Albinaria and putative lsabellaria species (Gastropoda, Pulmonata, Clausiliidae) . Mol Phylogenet Evol, 1995, 4 (4) : 157-462.
Schulenburg J H G, Englisch U, Wagele J W. Evolution of ITS1 rDNA in the Digenea (Platyhelminthes: Trematoda) : 3' end sequence conservation and its phylogenetic utility. J. Mol. Evol, 1999, 48, 2-12.
Schwarz. D. C, Cantor. C. R, Separation of yeast chromosome—sized DNAs by pulsed-field gel electrophoresis, Cell, 1984, 37: 67-75.
Sollner-Webb B, Mougey E B. News from the nueleolus: rRNA gene expression. Trands. Bochem, Sci., 1991, 16: 58-62.
Stimmer. K, von Haeseler. A, Quartet puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 1996, 13: 964-969.
Suneson, C. A et al., Agron. Jour, 1960, 52: 319-321.
Tang J, Toe L, Back C et al., Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simulliidae) complex. Mol Biol Evol, 1996, 13: 244-252.
Teutsch. M. R et al., Anim Biotechnol, 1990, 1: 185-199.
Van der Sande C A F M, Kwa M, van Nues R Wet al., Functional analysis of internal transcribed spacer2 of Saccharomyces cerevisine ribosomal DNA. J Mo Bio, 1996, 223: 899-910.
Van Herwerden L, Blair D, Agatsuma T. Intra-and specific variation in nuclear ribosomal internal transcribed spacer 1 of the Schistosoma japonicum species
complex. Parasitology, 1998, 116:311-317.
Van Herwerden L, Blair D, Agatsuma T. Intra-and inter-individual variation in ITS1 of Paragonimus westermani (Tremotoda: Digenea) and related species: implication for phylogenetic studies. Mol Phylogenet Evol, 1999, 12: 67-73.
Verneau O, Renaud F, Catzeflis F. Evolutionary relationships of sibling tapeworm species (Cestoda) parasitizing teleost fishes. Mol Biol Evol, 1997, 14: 630-636.
Wachira F N et al, Detection of genetic in tea (Camellia sinensis) using RAPD markers. Genome, 1995, 38:201.
Welsh. J, McClelland. M, Fingerprinting genomes using PCR with arbitary primers. Nucl, Acids. Res, 1990, 18: 7213-7218.
Vidigal T H, Kissingler J C, Caldeira R L et al. Phylogenetic relationships among Brazilian Biomphalaria species (Mollusca: Planorbridae) based upon analysis of ribosomal ITS2 sequences. Parasitology, 2000, 121:611-620.
Williams. J et al., DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nuch Acids. Res, 1990, 18:6531-6535.
Winans. G., Geographic varication in the milkfish Chanos chanoa, Ⅰ, Bio-ehomieal evidence. Evolution, 1980, 34: 558-574.
Yasuo Kiluchi, Nubuo Tamiya. Chemical taxonomy of the hinge-ligament proteins of bivalves according to their amino acid compositions. Biochem. J, 1987, 242:505-510.
Zahler M, Schein E, Rinder H et al. Characteristic genotypes discriminate between Babesia isolates of differing vector specificity and pathogenicity to dogs. Parasitol Res, 1998, 84: 544-548.
陈来钊，王子臣，1994．温度对海湾扇贝与虾夷扇贝极其杂交受精、胚胎和早期幼体发育的影响．大连水产学院学报，1994，9(4) 1-9．
巩宁，阙华勇，张国范．贝类非整倍体研究进展．生命科学研究，2001．5：68-72．
何毛贤，林岳光等．合浦珠母贝四倍体诱导过程中非整倍体的产生．热带海洋，2000，19：59-64．
何毛贤，林岳光，姜卫国．三倍体合浦珠母贝不育性研究．热带海洋，1996，15，17-21．
金启增，姜卫国，谢玉坎等．水温变化对合浦珠母贝心脏搏动的影响．中国贝类学会编辑，贝类学论文集，第二辑，北京：科学出版社．1984．24-29．
金冬燕，黎孟枫等译，1992，分子克隆实验指南，科学出版社．
孔晓瑜，刘亚军，喻子牛等．栉孔扇贝和海湾扇贝线粒体16S rRNA基因片段的比较研究．贝类学论文集，第Ⅸ辑，中国贝类学会编，海洋出版社2001．
李刚，姜卫国等．合浦珠母贝、长耳珠母贝合大珠母贝种间人工杂交的研究Ⅱ．受精过程和杂交后代的染色体观察．热带海洋，1983，2(4) ：316-320．
李刚，金启增等．合浦珠母贝和长耳珠母贝的生化遗传变异．遗传学报，1985，12：204-212．
李广丽，叶富良．马氏珠母贝不同组织同工酶的比较．水产学报，2000，24 (5) ：417-521．
刘景旋．台山川山群岛沿岸软体动物名录口．辦鰓纲(Lamellibranchia)．生态科学，1994，2：50-55．
缪炜，余育和等．上海四膜虫和两株嗜热四膜虫的rRNA基因序列分子系统关系．动物学研究，2001，22：265-269．
缪炜，余育和等．6种累枝虫(Epistylis) rRNA基因18S-ITS1序列及其分子系
统发育关系．中国科学(C辑) ，2002，2：138-145．
施立明．遗传多样性及其保存．生物科学信息，1990，2：118-164．
唐伯平，周开亚，宋大祥．核rDNA ITS序列在无脊椎动物分子系统学研究中的应用．动物学杂志，2002，37(4) ：67-73．
苏天凤，蔡云川等．合浦珠母贝三个养殖群体的RAPD分析．中国水产科学，2002，9(2) ： 106-109．
王爱民，邓凤姣，张锡元等．马氏珠母贝遗传多样性的RAPD分析．武汉大学学报，2000，46(4) ：467-470．
王梅林，郑家声等．我国海洋鱼类和贝类染色体组型研究进展．青岛海洋大学学报，2000，30：278-284．
王华接，严文侠等．珍珠贝在南海北部海洋结构物上的分布．热带海洋，2000，19：64-69．
王清印，杨爱国．栉孔扇贝三倍体研究进展和展望．中国水产科学，2000，7： 93-96．
魏贻尧 姜卫国．三种珠母贝种间杂交的初步研究．中国贝类学会编辑，贝类学论文集，第二辑，北京：科学出版社．1984．215-216．
杨俊毅，高爱跟等．台州列岛海域贝类种类组成与分布特征．东海海洋，2000，18：37-42．
俞海箐，张亚平，林飞栈等．探讨rDNA ITS区作为果蝇drosphila nasuta分子系统发育研究的价值．遗传学报，2000，27：18-28．
章超桦，吴红棉等．马氏珠母贝肉的营养成分及游离氨基酸组成．水产学报，2000，24(2) ，180-184．
郑成兴，黄宗国等．1997大亚湾核电站附近水域马氏珠母贝种群生态．动物学报，1997，43(3) 271-278．
张亚平．从DNA序列到物种树．动物学研究，1996，17：247-252．
Allendof. F. W et al., Genetic variation in Scandinavian brown trout(Salmo trutta L) : Evidence of distinct sympatric population. 1976, Hereditas, 83: 73-82.
Ankeim N. Concerted evolution of multigene families, in: Nei M Koehm R K eds. Evolution of genes and protein. Sinauer, Sunderland MA, 1983, 38-61.
Arnaud. S, Monteforte. M, Galtier. N et al., Population structure and genetic variability of pearl oyster Pinctada mazatlanica along Pacific coasts from Mexico to Panama. Conservation Genetics, 2000, 1: 299-307.
Bargues MD, Mas-Coma S. Phylogenetic analysis of Lyrnnaeid snails based on 18S rDNA sequences. Molecular Biology and Evolution. 1997, 14: 569-577.
Benjamin K D, Robert D A. Intraspecific variation in Cryptocaryon irritans. J Euk Microbiol. 1997, 44: 25-32.
Blankenship H L, et al. A responsible approach to marine stock enhancement. American fisheries Society Symposium, 1995, 15:001.
Bowles J, Blair D, McManus D P. A molecular phylogeny of the human schistosomes. Mol Phylogenet Evol, 1995a, 4:103-109.
Bowles J, Blair D, MeManus D P. A molecular phylogeny of the genus Echinococcus. Parasitoltogy, 1995b, 110:317-328.
Campbell B C, Steffen-Campbell J D, Werren H J. Phylogeny of the Nasonia species compex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences, Insect Mol Biol, 1993, 2 (4) : 225-237.
Despres L, Kruger F J, Imbert-Establet D et al. ITS2 ribosomal RNA indicates Schistosoma hippopotami is a distinct species, Int J Parasitol, 1955, 25: 1509-1514.
Dickerson. R. E, The structure of eytochrome C and the rates of molecular evolution. J. Mol. Evol, 1971, 1:26-45.
Dover G A, Molecular drive: a cohesive mode of species evolution. Nature, 1982, 299: 111-117.
Dmitrv Mukha, Brian M. Wiegmarm, Coby Schal. Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta) . Insect Biochemistry and Molecular Biology, 2002, 32:951-960.
Felsenstein. J, PHYLIP: Phylogenetic inference package, ver. 3. 5c. 1993, Washington: University of Washington.
Felsenstein. J, Confidence limits on phylogenies: an approach using bootstrap. Evolution, 1985, 39: 783-791.
Fenton B, Malloch G, Germa F. A study of variation in rDNA ITS region showa that two haplotypes coexist within a single aphed genome. Genome, 1998, 41: 338-344.
Gomez-Zurita J, Juan C, Petitpierre E. Sequence, secondary structure and phylogenetic analyses of the internal transcribed spacer 2 (ITS2) in the Timarcha leaf beetles (Coleoptera: Chrysomelidae) . Insect Mol Biol, 2000, 9: 591-604.
Gotoh T, Gutienze J, Navajas M, Molecular comparison of the sibling species Tetranychus puerasicola Ehora et Gotoh and T. urticae Koch (Acari: Tetranychidae) . Entomological Science, 1998, 1 : 55-57.
Haertl. B, Bonndlow, G. Epidemiologieal fingerprinting of Enterobactel cloacae by small-fragment restriction endonudease anaiysis and pulsed field gel electropdoresis of genomic restriction fragments. J. Clin. Microbiol. 1993, 31 : 128-133.
Harris D J, Crandall K A. intragenomic variation within ITS1 and ITS2 of freshwater crayfishes (Deeapioda: Cambaridae) : implications for phylogenetic and microsatellite studies. Mol Biol Evol, 2000, 17(2) : 284-291.
Hillis M D M, Davis S K, Ribosomal DNA: Intraspecific polyporphism, concered evolution and phylogeny reconstruction. Syst Zool, 1988, 37: 63-66.
Homan W L, Verlaan M et al. Comparison of the internal transcribed spacer, ITS1, from Toxoplasma gondii isolates and Neospora caninum. Parasital Res, 1997, 83: 285-289.
Hunter. R & C. Marker. Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science, 1957, 125:1294.
Jeffreys. A. J, Individual specific "fingerprints" of human DNA. Nature. 1985, 316: 76-79.
Jeffreys. A. J et al., DNA fingerprints and segregation analysis of multiple
markers in human pedigrees. Am J. Hum. Genet, 1986, 39:11-24.
Katsuhiko T. Wada. Aquaculture genetics of bivalve molluscs: a review. National Journal of Ocean University of Qingdao. 2000, 30:107-114.
K H Chu, C P Li, H Y. Ho. The first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for phylogenetic and population analysisin crustacea. Mar biotechnol, 2001, 3: 355-361.
Kojima. S, Segawa. R, Hashimoto, Molecular phylogeny of vestimentiferans collected around Japan, revealed by the nucleotide sequences of mitochondrial DNA. Marine Biology. 1997, 127: 507-513.
Lindsay D S, Upton S J, Owens D S et al. Cryptosporidium andersons n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus. J Eukaryot Microbiol, 2000, 47 (1) : 91-95.
Lounibos L P, Wilkerson R C, Conn J E et al. Morphological, molecular and chromosomal discrimination of cryptic Anopheles (Nyssorhynehus) (Diptera: Culicidae) from south America. J Med Entomal, 1998, 35:830-838.
Lynch M, The similarity index and DNA fingerpringting. Mol Biol Evol, 1990, 7: 478.
Mazureak. G. H et al., Large DNA restriction fragment polymorphism in the Mycobacterium avium-M, intracellulare complex: a potential epidemi olgical tool. J. Clin. Microbiol. 1993, 31:390-394.
Miesfeld et al., A member of a new repeated sequence family which is conserved throughout the human δ and β globin genes. NucL Acids. Res, 1981, 9:5931-5948.
M. J. López-Pion, A. Insua. Identification of four scallop species using PCR and restrication analysis of the ribosomal DNA internal transcribed spacer region. Marine Bioteehnology, 2002, 4: 495-502.
Moller. D, Genetic diversity in spawning cod along the Norwegian cast. Hereditas: 1968, 60:1～82.
Morgan J A T, Blair D, Nuclear rDNA ITS sequence variation in the trematode
genus Echinostoma: an aid to establishing relationships within the 37-collar-spine group. Parasitology, 1995, 111: 609-615.
Muster W, Boon K, ven der Sande C A F M et al., Functional analysis of transcribed spacers of yeast ribosomal DNA. EMBO, 1990, 9: 3989-3996.
Navajas M, Lagnel J, Gutierrez J et al., Species-wide homogeneity of nuclear ribosomal ITS2 sequences in the spider mite Tetranychus urticae contrasts with extensive mitochondrial COI polymorphism. Heredity, 1998, 80 (Pt6) : 742-752.
Odorica D M, Miller D J. Variation in the ribosomal internal transcribed spacers and 5. 8S rDNA among five species of Acropora (Cnidaria; Scleractinia) : patterns of variation consistent with reticulate evolution. Mol Biol Evol, 1997, 14(5) : 465-473.
Okamoto M, Agatsuma T, Kurosawa T et al., Phylogenetic relationships of three hymenolepidid species inferred from nuclear ribosomal and mitochondrial DNA sequences. Parasitology, 1997, 115: 661-666.
Park. L. K & P. Moran, Developments in molecular genetic techniques in fisheries. Rev. Fish, Biol, Fish. 1994. 4:272-289.
Paskewitz S M, Wesson D M, Collins F H. The internal transcribed spacers of ribosomal DNA in five members of the Anopheles gambiae complex. Insect Mol Biol, 1993, 2: 247-357.
Peyretaillade E, Biderre C, Peyret P et al., Microsporidian Encephalitozoon cuniculi, a unicellular with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core. Ucleic Acids Res, 1998, 26: 3513-3520.
Pleytc K A, Duncan S D et al. Evolutionary relationships of the samonid fish genus Salvelinus inferred from DNA sequences of the first internal transcribed spacer (ITS1) ofrDNA. Mol Phylogen Evol. 1992, 1: 223-230.
Power. A. G, Entomoh Exp. Appl. 1988, 49:213-219. Rasmussen S W. 2001, DNAtools, a software package for sequence analysis.
The carlsbeg laboratory, Copenhagen, Http://www. dnatool. dk.
Schlouerer C, Hauser M et al. Comparative evolutionary analysis of rDNA ITS regions in Drosophila. Mol Biol Evol. 1994, 11: 513-522.
Smith, D. R. and T. C. Glenn. Allozyme polymorphisms in Spanish Honey bees (Apis mellifera iberica) . The Journal of Heredity, 1996, 86: 12-14.
Schilthuizen M, Gettenberger E, Gultyaev A P. phyogenetic relationships inferred from the sequence and second structure of ITS1 rRNA in Albinaria and putative lsabellaria species (Gastropoda, Pulmonata, Clausiliidae) . Mol Phylogenet Evol, 1995, 4 (4) : 157-462.
Schulenburg J H G, Englisch U, Wagele J W. Evolution of ITS1 rDNA in the Digenea (Platyhelminthes: Trematoda) : 3' end sequence conservation and its phylogenetic utility. J. Mol. Evol, 1999, 48, 2-12.
Schwarz. D. C, Cantor. C. R, Separation of yeast chromosome—sized DNAs by pulsed-field gel electrophoresis, Cell, 1984, 37: 67-75.
Sollner-Webb B, Mougey E B. News from the nueleolus: rRNA gene expression. Trands. Bochem, Sci., 1991, 16: 58-62.
Stimmer. K, von Haeseler. A, Quartet puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 1996, 13: 964-969.
Suneson, C. A et al., Agron. Jour, 1960, 52: 319-321.
Tang J, Toe L, Back C et al., Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simulliidae) complex. Mol Biol Evol, 1996, 13: 244-252.
Teutsch. M. R et al., Anim Biotechnol, 1990, 1: 185-199.
Van der Sande C A F M, Kwa M, van Nues R Wet al., Functional analysis of internal transcribed spacer2 of Saccharomyces cerevisine ribosomal DNA. J Mo Bio, 1996, 223: 899-910.
Van Herwerden L, Blair D, Agatsuma T. Intra-and specific variation in nuclear ribosomal internal transcribed spacer 1 of the Schistosoma japonicum species
complex. Parasitology, 1998, 116:311-317.
Van Herwerden L, Blair D, Agatsuma T. Intra-and inter-individual variation in ITS1 of Paragonimus westermani (Tremotoda: Digenea) and related species: implication for phylogenetic studies. Mol Phylogenet Evol, 1999, 12: 67-73.
Verneau O, Renaud F, Catzeflis F. Evolutionary relationships of sibling tapeworm species (Cestoda) parasitizing teleost fishes. Mol Biol Evol, 1997, 14: 630-636.
Wachira F N et al, Detection of genetic in tea (Camellia sinensis) using RAPD markers. Genome, 1995, 38:201.
Welsh. J, McClelland. M, Fingerprinting genomes using PCR with arbitary primers. Nucl, Acids. Res, 1990, 18: 7213-7218.
Vidigal T H, Kissingler J C, Caldeira R L et al. Phylogenetic relationships among Brazilian Biomphalaria species (Mollusca: Planorbridae) based upon analysis of ribosomal ITS2 sequences. Parasitology, 2000, 121:611-620.
Williams. J et al., DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nuch Acids. Res, 1990, 18:6531-6535.
Winans. G., Geographic varication in the milkfish Chanos chanoa, Ⅰ, Bio-ehomieal evidence. Evolution, 1980, 34: 558-574.
Yasuo Kiluchi, Nubuo Tamiya. Chemical taxonomy of the hinge-ligament proteins of bivalves according to their amino acid compositions. Biochem. J, 1987, 242:505-510.
Zahler M, Schein E, Rinder H et al. Characteristic genotypes discriminate between Babesia isolates of differing vector specificity and pathogenicity to dogs. Parasitol Res, 1998, 84: 544-548.