红白锦鲤人工雌核发育纯系的微卫星标记分析
MICROSATELLITE MARKER ANALYSIS ON ARTIFICIALLY GYNOGENETIC PURE LINE OF RED-WHITE ORNAMENTAL CARP
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摘要: 利用连续两代人工雌核发育操作,构建了极具观赏价值的红白锦鲤人工雌核发育系。应用对微卫星引物进行遗传标记研究,检测分析了该雌核发育系内个体间的遗传同质性及其基因座位的纯合度。实验结果显示,11尾经人工挑选具有相同表形、极具观赏价值的F1代性成熟个体的微卫星扩增图谱呈现出高度的一致性。同时,随机挑选的人工雌核发育F2代个体在所检测的基因座位均呈现纯合,扩增图谱呈现了高度的一致,从分子水平上证实了本实验所获得的红白人工雌核发育F2代是一个纯系。Abstract: One pure line of red white ornamental carp( Cyprinus carpio L.) was produced by two successive artificial gynogenesis with the combination treatment of UV irradiation sperm and cold shock (3-5℃) to block second polar body extrusion.The used heterozygous sperm came from bluntnose black bream( Megalobrama amblycephala Yih).Six microsatellite markers of the poly (CA) type isolated from common carp were used to assess genetic homogeneity among individuals of the artificially gynogenetic Red Write ornamental carp pure line.Electrophoretic patterns showed that the six pairs of microsatellite primers all produced well identifiable DNA fragments,and their size ranged from 100bp to 300bp.In this study,11 individuals similar to their mother in the color phenotype were selected from the artificially gynogenetic F1 progeny of Red-Write ornamental carps.The DNA fragment patterns of 11 individuals amplified by six primers were highly identical.One of them was successfully induced to spawn,and the ovulated eggs were used to produce artificial gynogenetic F2 progeny.Six individuals were randomly sampled from the F2 gynogenetic ornamental carps to analyze their genetic homogeneity.As a control,8 individuals reproduced from bisexual mating were also analyzed by the six microsatellite markers.The results indicated that the 6F2 gynogenetic individuals showed highly identical DNA patterns to their mother,and Mendel's genetic law of the six microsatellite markers were demonstrated by the control group.The current study provides a new technique to produce pure line in ornamental carp.Moreover,the artificially gynogenetic pure ling of red white ornamental carp will be useful for further studying on genetic mechanism of body color types in ornamental carp,and the molecular markers will be able to provide some valuable tools for marker-assisted selection breeding or gene mapping in the ornamental carp.
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Keywords:
- Ornamental carp /
- Artificial gynogenesis /
- Microsatellite
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Journal of Experimental Zoology. 1997,277:72-79[8] Taniguchi N, Kijima A, Tamura T,et al. Color, growth and maturation in ploidy-manipulated fancy carp[J]. Aquaculture. 1986,57:321-328[9] Gomelsky B,Cherfas N B, Bendom N et al. Color inheritance in ornamental (koi) carp (Cyprinus carpio L) inferred from color variability in normal and gynogenetic progenies[J]. Isreali of Journal of Aquacuture.1996,48:219-230[10] Gomelsky B, Cherfas N, Hulata G. Studies on the inheritance of black patches in ornamental (koi) carp[J]. Isreali of Journal of Aquacuture. 1998,50:134-139[11] Zhang H, Zhang J Q, Li T P. Studies on the polymorphism of transferrin and serum esterase in common carp[j]. Acta Hydrobiologica Sinica, 1993, 17(30):278-281.[张辉,张俊权,李太平.鲤转铁蛋白和血清脂酶多态性研究[J].水生生物学报,1993,17(30):278-281][12] Gross R, Klaus K, Kersten P. PCR-RFLP analysis of the mitochondrial ND-3/4 and ND-5/6 gene polymorphisms in the European and East Asian subspecies of common carp(Cyprinus carpio L.)[J]. Aquaculture.2002,204:507-516[13] Gui J F, Sun J M, Liang S C, et al, Studies on genome manipulation in fish:Ⅰ. Induction of tramsparent colored crucian carp(Carass ius auratus transparent colord variety) by hydtostatic pressure[J]. Acta Hydrobiologica Sinica, 1990, 14(4):336-344.[桂建芳,孙建民,梁绍昌,等.鱼类染色体操作研究,Ⅰ.静水压处理和静水压与冷休克结合处理诱导水晶彩鲫三倍体. 水生生物学报. 1990,14(4):336-344][14] Gui J F, Sun J M, Liang S C, et al, Studies on genome manipulation in fish:Ⅱ. Tetrapioidy induced by hydrostatic pressure treatment and a combination of hydrostatic pressure and cold treatments in transparent colored crucian carp[J]. Acta Hydrobiologica Sinica, 1991, 15(4):333-341. [桂建芳,孙建民,梁绍昌,等. 鱼类染色体操作研究,Ⅱ.静水压处理和静水压与冷休克结合处理诱导水晶彩鲫四倍体. 水生生物学报.1991,15(4):333-341][15] Jia H B, Zhou L, Gui J F. The establish of two artificial gynogenetic red-white ornamental carp and detection of RAPD markers[J]. Acta Hydrobiologica Sinica, 2002,26(1):1-7[贾海波,周莉,桂建芳. 两个不同锦鲤的红白雌核发育系的建立及其RAPD分子鉴别标记.水生生物学报.2002,26(1):1-7][16] Zhou L, Fan L C, Gui J F.RAPD analysis of incorporation of heterologous genetic materials inmultiple species of silver crucion carp[J]. Acta Hydrobiologica Sinica,1998,22(4):301-306.[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):301-306][17] Zhou L,Wang Y,Gui J F. Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch,based on detection of RAPD molecular markers[J]. Cytogenet.Cell Genet.2000a,88:133-139[18] Crooijmans R P M A,Bierbooms V A F,Komen J, et al. Microsatellite markers in common carp (Cyprinus carpio L.)[J]. Animal Genetics.1997,28:129-134[19] Young W P, Wheeler P A,Thorgaard G H.DNA fingerprinting confirms isogenicity of androgenetically derived rainbow trout lines[J].J Hered.1996,87(1):77-80[20] Yang S T, Gui J F. lsozyme analysis and preliminary confirmation of the genetic markers in two artificial gynogenetic populations of silver carp,H.molitrix[J]. Acta Hydrobiologica Sinica,23(3):264-268.[杨书婷,桂建芳. 两上雌核发育白鲢群体同工酶分析及遗传标记的确定.水生生物学报.1999,23(3):264-268][21] Nagy A,Rakji K,Bakos J, et al. Genetic analysis in carp(Cyprinus carpio)[J]. Heredity.1979,43:35-40 Gui J F. Ornamental carp[J].Reservoir Fisheries. 1986, (5):40-42.[桂建芳. 锦鲤欣赏. 水利渔业. 1986,(5):40-44][2] Kuroki T. Manual to Nishikigoi[M]. Shimonoseki: Shuji Fujita, 1990[3] Liu J X, Zhou L, Zhao Z S, et al. Studies on microsatellite markers of four artificially gynogenetic families in ornamental carp[J]. Zoological Research, 2002, 22(2):97-105. [刘静霞,周莉,赵振山等.4个人工雌核发育锦鲤的微卫星标记. 动物学研究,2002,22(2):97-105][4] Kataoka M. Nishikigoi Dangi(Short Stori:s)[M]. Kumagayashi: Takayoshi Kataoka, 1989[5] Streisinger G, Walker C, Dower N, et al,. Production of homozygous diploid zebra fish[J]. Nature. 1981, 291:293-296[6] Wu Q J, Ye Y Z,Cheng D R. Investigation on the carp gynogenesis with reference to establishing a pure line[J]. Acta Genetica Sinica 1981, 8(1):50-55. [吴清江,叶玉珍,陈德荣. 鲤鱼人工雌核发育及其作为建立近交系新途径的研究. 遗传学报.1981,8(1):50-55][7] Bongers A B J, Ben-Ayed M Z, Zandieh-Doulabi B,et al. Origin of variation in isogenic gynogenetic and androgenetic strains of common carp Cyprinus carpio [J]. Journal of Experimental Zoology. 1997,277:72-79[8] Taniguchi N, Kijima A, Tamura T,et al. Color, growth and maturation in ploidy-manipulated fancy carp[J]. Aquaculture. 1986,57:321-328[9] Gomelsky B,Cherfas N B, Bendom N et al. Color inheritance in ornamental (koi) carp (Cyprinus carpio L) inferred from color variability in normal and gynogenetic progenies[J]. Isreali of Journal of Aquacuture.1996,48:219-230[10] Gomelsky B, Cherfas N, Hulata G. Studies on the inheritance of black patches in ornamental (koi) carp[J]. Isreali of Journal of Aquacuture. 1998,50:134-139[11] Zhang H, Zhang J Q, Li T P. Studies on the polymorphism of transferrin and serum esterase in common carp[j]. Acta Hydrobiologica Sinica, 1993, 17(30):278-281.[张辉,张俊权,李太平.鲤转铁蛋白和血清脂酶多态性研究[J].水生生物学报,1993,17(30):278-281][12] Gross R, Klaus K, Kersten P. PCR-RFLP analysis of the mitochondrial ND-3/4 and ND-5/6 gene polymorphisms in the European and East Asian subspecies of common carp(Cyprinus carpio L.)[J]. Aquaculture.2002,204:507-516[13] Gui J F, Sun J M, Liang S C, et al, Studies on genome manipulation in fish:Ⅰ. Induction of tramsparent colored crucian carp(Carass ius auratus transparent colord variety) by hydtostatic pressure[J]. Acta Hydrobiologica Sinica, 1990, 14(4):336-344.[桂建芳,孙建民,梁绍昌,等.鱼类染色体操作研究,Ⅰ.静水压处理和静水压与冷休克结合处理诱导水晶彩鲫三倍体. 水生生物学报. 1990,14(4):336-344][14] Gui J F, Sun J M, Liang S C, et al, Studies on genome manipulation in fish:Ⅱ. Tetrapioidy induced by hydrostatic pressure treatment and a combination of hydrostatic pressure and cold treatments in transparent colored crucian carp[J]. Acta Hydrobiologica Sinica, 1991, 15(4):333-341. [桂建芳,孙建民,梁绍昌,等. 鱼类染色体操作研究,Ⅱ.静水压处理和静水压与冷休克结合处理诱导水晶彩鲫四倍体. 水生生物学报.1991,15(4):333-341][15] Jia H B, Zhou L, Gui J F. The establish of two artificial gynogenetic red-white ornamental carp and detection of RAPD markers[J]. Acta Hydrobiologica Sinica, 2002,26(1):1-7[贾海波,周莉,桂建芳. 两个不同锦鲤的红白雌核发育系的建立及其RAPD分子鉴别标记.水生生物学报.2002,26(1):1-7][16] Zhou L, Fan L C, Gui J F.RAPD analysis of incorporation of heterologous genetic materials inmultiple species of silver crucion carp[J]. Acta Hydrobiologica Sinica,1998,22(4):301-306.[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):301-306][17] Zhou L,Wang Y,Gui J F. Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch,based on detection of RAPD molecular markers[J]. Cytogenet.Cell Genet.2000a,88:133-139[18] Crooijmans R P M A,Bierbooms V A F,Komen J, et al. Microsatellite markers in common carp (Cyprinus carpio L.)[J]. Animal Genetics.1997,28:129-134[19] Young W P, Wheeler P A,Thorgaard G H.DNA fingerprinting confirms isogenicity of androgenetically derived rainbow trout lines[J].J Hered.1996,87(1):77-80[20] Yang S T, Gui J F. lsozyme analysis and preliminary confirmation of the genetic markers in two artificial gynogenetic populations of silver carp,H.molitrix[J]. Acta Hydrobiologica Sinica,23(3):264-268.[杨书婷,桂建芳. 两上雌核发育白鲢群体同工酶分析及遗传标记的确定.水生生物学报.1999,23(3):264-268][21] Nagy A,Rakji K,Bakos J, et al. Genetic analysis in carp(Cyprinus carpio)[J]. Heredity.1979,43:35-40
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