青鱼微卫星标记的开发与特性分析
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摘要
青鱼(Mylopharyngodon piceus)是中国最为重要的淡水养殖鱼类。开发青鱼的微卫星标记能为青鱼的遗传多样性分析提供更多工具。本研究使用磁珠富集法,利用生物素探针(CA)10和(GACA)6,富集得到青鱼基因组微卫星片段,进一步通过设计微卫星引物检验其在青鱼原种群体中的有效性和多态性水平。结果显示,所构建文库中849个克隆含有微卫星序列,通过利用PCR技术在吴江原种青鱼36个个体中进行多态性筛选,获得了25个多态性微卫星位点。其平均等位基因数(Na)和有效等位基因数(Ne)分别为7.08和3.526,平均观测杂合度(Ho)和期望杂合度(He)分别为0.602和0.619,平均多态信息含量(PIC)为0.568。其中,Mp23、Mp27和Mp35这3个位点极显著偏离哈迪-温伯格平衡(P <0.01)。本研究开发的微卫星标记能为青鱼种质资源的评价和保护等研究提供工具。
Black Carp(Mylopharyngodon piceus) is an important aquaculture species in China. Developing microsatellite DNA markers from genome of M. piceus will provide a tool for its genetic diversity analysis. In this study, two biotin probes(CA)10 and(GACA)6 were used to construct two microsatellite enrichment libraries of M. piceus by magnetic beads enrichment. A series of microsatellite primers was designed and their polymorphisms were tested in a population of M. piceus. Softwares GeneMapperv4.0, Cervus3.0 and PopGen32 were used to analyze their genetic diversity information. Microsatellite markers with high polymorphism and rich genetic diversity were screene. Through analysis, 849 sequences containing microsatellite loci were obtained from 993 positive clones. The repeat number of the repeating unit was then counted(Fig. 1). The statistics showed that most numbers of the repeats were between 5 and 21. The repeating units were classified according to the number of nucleotides in each repeating unit. The results showed that the number of dinucleotide microsatellite loci was the most, as high as 1 076. It accounted for 82.45% of the total. There were 217 trinucleotide type microsatellite loci, accounting for 16.63% of the total. In trinucleotide type, only 0.77% of all 39 microsatellite markers were designed and assessed in 36 individuals of black carp from Wujiang population. Results showed that 25 microsatellite loci showed polymorphism(Table 1). The mean number of alleles(Na) and effective number of alleles(Ne) were 7.08 and 3.526, respectively. The mean value of observed heterozygosity(Ho) and expected heterozygosity(He) were 0.602 and 0.619, respectively. The mean value of polymorphic information content was 0.568. Mp23, Mp27 and Mp35 were extremely significant deviating from Hardy-Weinberg equilibrium(P < 0.01)(Table 2). The microsatellite primers developed in this study will provide a tool and theoretical basis for evaluation and protection of germplasm resources, and genetic diversity analysis in M piceus.
Black Carp(Mylopharyngodon piceus) is an important aquaculture species in China. Developing microsatellite DNA markers from genome of M. piceus will provide a tool for its genetic diversity analysis. In this study, two biotin probes(CA)10 and(GACA)6 were used to construct two microsatellite enrichment libraries of M. piceus by magnetic beads enrichment. A series of microsatellite primers was designed and their polymorphisms were tested in a population of M. piceus. Softwares GeneMapperv4.0, Cervus3.0 and PopGen32 were used to analyze their genetic diversity information. Microsatellite markers with high polymorphism and rich genetic diversity were screene. Through analysis, 849 sequences containing microsatellite loci were obtained from 993 positive clones. The repeat number of the repeating unit was then counted(Fig. 1). The statistics showed that most numbers of the repeats were between 5 and 21. The repeating units were classified according to the number of nucleotides in each repeating unit. The results showed that the number of dinucleotide microsatellite loci was the most, as high as 1 076. It accounted for 82.45% of the total. There were 217 trinucleotide type microsatellite loci, accounting for 16.63% of the total. In trinucleotide type, only 0.77% of all 39 microsatellite markers were designed and assessed in 36 individuals of black carp from Wujiang population. Results showed that 25 microsatellite loci showed polymorphism(Table 1). The mean number of alleles(Na) and effective number of alleles(Ne) were 7.08 and 3.526, respectively. The mean value of observed heterozygosity(Ho) and expected heterozygosity(He) were 0.602 and 0.619, respectively. The mean value of polymorphic information content was 0.568. Mp23, Mp27 and Mp35 were extremely significant deviating from Hardy-Weinberg equilibrium(P < 0.01)(Table 2). The microsatellite primers developed in this study will provide a tool and theoretical basis for evaluation and protection of germplasm resources, and genetic diversity analysis in M piceus.
引文
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Cheng L,Liu L,Yu X,et al.2008.Sixteen polymorphic microsatellites in bighead carp(Aristichthys nobilis)and cross-amplification in silver carp(Hypophthalmichthys molitrix).Molecular Ecology Resources,8(3):656-658.
Curriefraser E,Shah P,True S.2010.Data analysis using GeneMapper?v4.1:Comparing the Newest Generation of GeneMapper Software to Legacy Genescan?and Genotyper?Software.Journal of Biomolecular Techniques,21(Suppl 3):S31.
Duan X B,Liu S P,Huang M G,et al.2010.Changes in abundance of larvae of the four domestic Chinese carps in the middle reach of the Yangtze River,China,before and after closing of the Three Gorges Dam.Environmental Biology of Fishes,86(1):13-22.
Kalinowski S T,Taper M L,Marshall T C.2007.Revising how the computer program cervus accommodates genotyping error increases success in paternity assignment.Molecular Ecology,16(5):1099-1106.
Lalitha,S.2000.Primer Premier 5.Biotech Software&Internet Report,1(6):270-272.
Li D,Shen Y B,Fu J J,et al.2013.Isolation and characterization of25 novel polymorphic microsatellite markers from grass carp(Ctenopharyngodon idella).Conservation Genetics Resources,5(3):745-748.
Li L,Lin H,Tang W,et al.2017.Population genetic structure in wild and aquaculture populations of Hemibarbus maculates inferred from microsatellites markers.Aquaculture and Fisheries,2:78-83.
Li Q,Wan J M.2005,SSRHunter:development of a local searching software for SSR sites.Hereditas,27(5):808-810.
Sahu B P,Sahoo L,Joshi C G,et al.2014.Isolation and characterization of polymorphic microsatellite loci in Indian major carp,Catla catla using next-generation sequencing platform.Biochemical Systematics&Ecology,57(3):357-362.
Yeh F C.1997.Population genetic analysis of codominant and dominant markers and quantitative traits.Belgian Journal of Botany,129:157.
Yue G H,Zhu Z Y,Wang C M,et al.2009.A simple and efficient method for isolating polymorphic microsatellites from c DNA.Bmc Genomics,10(1):125.
Zhu X,Zhang F,Cao W.2013.Isolation and characterization of 28polymorphic microsatellite loci in black carp(Mylopharyngodon piceus).Conservation Genetics Resources,5(2):469-472.
蔡磊,陈小曲,郑伟强,等.2015.诸氏鲻虾虎鱼多态性微卫星标记的开发及评价.中国实验动物学报,23(1):57-62.
孔杰,蒋晓红,周洲,等.2016.磁珠富集法开发长臀鮠微卫星分子标记.贵州农业科学,44(7):14-17.
李达.2014.草鱼微卫星标记开发及9个养殖群体遗传多样性分析.上海:上海海洋大学硕士学位论文.
刘红艳,熊飞,宋丽香.2017.长江上游特有鱼类红唇薄鳅微卫星DNA分离及序列特征分析.江西农业大学学报,39(1):145-152.
刘佳瑶2012.鲮微卫星分子标记的筛选及遗传多样性分析.上海:上海海洋大学硕士学位论文.
刘连为,陈新军,许强华,等.2014.北太平洋柔鱼微卫星标记的筛选及遗传多样性.生态学报,34(23):6847-6854.
刘瑞成,张富铁,但胜国,等.2013.宽口光唇鱼微卫星位点的筛选与特征分析.四川动物,32(2):161-166.
马海涛,鲁翠云,于冬梅,等.2007.草鱼基因组中微卫星分子标记的制备及筛选.上海海洋大学学报,16(4):389-393.
裴宋雨,任志勇,焦振彬,等.2015.香椿(Toona sinensis)微卫星富集文库构建及特性分析.种子,34(2):13-16.
帅方敏,李新辉,黄艳飞,等.2017.珠江水系四大家鱼资源现状及空间分布特征研究.水生生物学报,41(6):1336-1344.
孙建华,马爱军,崔文晓,等.2017.利用微卫星标记技术对红鳍东方鲀(Takifugu rubripes)家系系谱认证的研究.海洋科学进展,35(3):392-403.
孙效文,鲁翠云,梁利群.2005.磁珠富集法分离草鱼微卫星分子标记.水产学报,29(4):482-486.
谭照君,张天奇,鲁翠云,等.2011.鲢三、四核苷酸重复微卫星标记的筛选及其特征分析.上海海洋大学学报,20(3):328-335.
杨万云,郑军军,贾博寅,等.2017.微卫星分子标记及其在动物遗传育种中的研究进展.基因组学与应用生物学,2017(11):4644-4649.
郑基阳,陈乃富,王晖,等.2011.霍山石斛微卫星标记的筛选及遗传多样性分析.中国中药杂志,36(21):2926-2931.
朱传坤,潘正军,王辉,等.2017.乌苏里拟鲿微卫星文库的构建及分析.水生生物学报,41(2):473-478.
Cheng L,Liu L,Yu X,et al.2008.Sixteen polymorphic microsatellites in bighead carp(Aristichthys nobilis)and cross-amplification in silver carp(Hypophthalmichthys molitrix).Molecular Ecology Resources,8(3):656-658.
Curriefraser E,Shah P,True S.2010.Data analysis using GeneMapper?v4.1:Comparing the Newest Generation of GeneMapper Software to Legacy Genescan?and Genotyper?Software.Journal of Biomolecular Techniques,21(Suppl 3):S31.
Duan X B,Liu S P,Huang M G,et al.2010.Changes in abundance of larvae of the four domestic Chinese carps in the middle reach of the Yangtze River,China,before and after closing of the Three Gorges Dam.Environmental Biology of Fishes,86(1):13-22.
Kalinowski S T,Taper M L,Marshall T C.2007.Revising how the computer program cervus accommodates genotyping error increases success in paternity assignment.Molecular Ecology,16(5):1099-1106.
Lalitha,S.2000.Primer Premier 5.Biotech Software&Internet Report,1(6):270-272.
Li D,Shen Y B,Fu J J,et al.2013.Isolation and characterization of25 novel polymorphic microsatellite markers from grass carp(Ctenopharyngodon idella).Conservation Genetics Resources,5(3):745-748.
Li L,Lin H,Tang W,et al.2017.Population genetic structure in wild and aquaculture populations of Hemibarbus maculates inferred from microsatellites markers.Aquaculture and Fisheries,2:78-83.
Li Q,Wan J M.2005,SSRHunter:development of a local searching software for SSR sites.Hereditas,27(5):808-810.
Sahu B P,Sahoo L,Joshi C G,et al.2014.Isolation and characterization of polymorphic microsatellite loci in Indian major carp,Catla catla using next-generation sequencing platform.Biochemical Systematics&Ecology,57(3):357-362.
Yeh F C.1997.Population genetic analysis of codominant and dominant markers and quantitative traits.Belgian Journal of Botany,129:157.
Yue G H,Zhu Z Y,Wang C M,et al.2009.A simple and efficient method for isolating polymorphic microsatellites from c DNA.Bmc Genomics,10(1):125.
Zhu X,Zhang F,Cao W.2013.Isolation and characterization of 28polymorphic microsatellite loci in black carp(Mylopharyngodon piceus).Conservation Genetics Resources,5(2):469-472.
蔡磊,陈小曲,郑伟强,等.2015.诸氏鲻虾虎鱼多态性微卫星标记的开发及评价.中国实验动物学报,23(1):57-62.
孔杰,蒋晓红,周洲,等.2016.磁珠富集法开发长臀鮠微卫星分子标记.贵州农业科学,44(7):14-17.
李达.2014.草鱼微卫星标记开发及9个养殖群体遗传多样性分析.上海:上海海洋大学硕士学位论文.
刘红艳,熊飞,宋丽香.2017.长江上游特有鱼类红唇薄鳅微卫星DNA分离及序列特征分析.江西农业大学学报,39(1):145-152.
刘佳瑶2012.鲮微卫星分子标记的筛选及遗传多样性分析.上海:上海海洋大学硕士学位论文.
刘连为,陈新军,许强华,等.2014.北太平洋柔鱼微卫星标记的筛选及遗传多样性.生态学报,34(23):6847-6854.
刘瑞成,张富铁,但胜国,等.2013.宽口光唇鱼微卫星位点的筛选与特征分析.四川动物,32(2):161-166.
马海涛,鲁翠云,于冬梅,等.2007.草鱼基因组中微卫星分子标记的制备及筛选.上海海洋大学学报,16(4):389-393.
裴宋雨,任志勇,焦振彬,等.2015.香椿(Toona sinensis)微卫星富集文库构建及特性分析.种子,34(2):13-16.
帅方敏,李新辉,黄艳飞,等.2017.珠江水系四大家鱼资源现状及空间分布特征研究.水生生物学报,41(6):1336-1344.
孙建华,马爱军,崔文晓,等.2017.利用微卫星标记技术对红鳍东方鲀(Takifugu rubripes)家系系谱认证的研究.海洋科学进展,35(3):392-403.
孙效文,鲁翠云,梁利群.2005.磁珠富集法分离草鱼微卫星分子标记.水产学报,29(4):482-486.
谭照君,张天奇,鲁翠云,等.2011.鲢三、四核苷酸重复微卫星标记的筛选及其特征分析.上海海洋大学学报,20(3):328-335.
杨万云,郑军军,贾博寅,等.2017.微卫星分子标记及其在动物遗传育种中的研究进展.基因组学与应用生物学,2017(11):4644-4649.
郑基阳,陈乃富,王晖,等.2011.霍山石斛微卫星标记的筛选及遗传多样性分析.中国中药杂志,36(21):2926-2931.
朱传坤,潘正军,王辉,等.2017.乌苏里拟鲿微卫星文库的构建及分析.水生生物学报,41(2):473-478.