基于RAD-seq技术的长体圆鲹二、三核苷酸重复微卫星标记开发与评价
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摘要
通过对长体圆鲹(Decapterus macrosoma)基因组进行RAD-Seq高通量测序,共获得58 180条微卫星序列,选取112条二、三核苷酸重复的微卫星序列设计引物,经筛选后,共获得27个具有多态性的微卫星标记。利用一个长体圆鲹群体对通过筛选的微卫星标记的种群遗传学特征进行评价。结果显示,27对引物扩增的序列中18个位点为二核苷酸重复,重复次数为9～14次,9个位点为三核苷酸重复,重复次数为6～10次,等位基因数(Na)为5～17 (平均10.6),表观杂合度(Ho)为0.342 9～0.857 1 (平均0.631 7),期望杂合度(He)为0.538 3～0.911 8(平均0.796 8),多肽信息含量(PIC)为0.497～0.886 (平均0.780 9),除1个位点外,其他位点PIC值均大于0.500,表明开发的微卫星位点具有较高的多态性。"哈迪-温伯格"平衡(HWE)检测结果显示,19个标记等位基因频率符合HWE。连锁不平衡检测表明各位点间无连锁不平衡现象。该研究开发的27个微卫星标记可为长体圆鲹种群遗传学研究提供基础。
A total of 58 180 microsatellites were developed by RAD-Seq high-throughput sequencing technology in Decapterus macrosoma. One hundred and twelve microsatellite loci were randomly selected from di-/tri-nucleotide repeat microsatellite loci. Finally, twenty-seven highly polymorphic markers were developed. The population genetic analysis finds 18 dinucleotide-repeated microsatellite loci(9-14 repeated) and 9 trinucleotide-repeated microsatellite loci(6-10 repeated). The number of alleles(Na) ranged from 5 to 17(mean 10.6). The observed and expected heterozygosities ranged from 0.342 9 to 0.857 1(mean 0.631 7) and 0.538 3 to0.911 8(mean 0.796 8), respectively. The polymorphism information content(PIC) ranged from 0.497 to 0.886(mean 0.780 9), and the PIC at all loci except for one was geater than 0.500,indicating that the microsatellites were highly polymorphic. The HardyWeinberg equilibrium(HWE) test shows that 19 microsatellite markers conformed to HWE. There is no linkage imbalance among the 27 loci. The 27 microsatellites developed by this study will be useful for further population genetic analysis.
A total of 58 180 microsatellites were developed by RAD-Seq high-throughput sequencing technology in Decapterus macrosoma. One hundred and twelve microsatellite loci were randomly selected from di-/tri-nucleotide repeat microsatellite loci. Finally, twenty-seven highly polymorphic markers were developed. The population genetic analysis finds 18 dinucleotide-repeated microsatellite loci(9-14 repeated) and 9 trinucleotide-repeated microsatellite loci(6-10 repeated). The number of alleles(Na) ranged from 5 to 17(mean 10.6). The observed and expected heterozygosities ranged from 0.342 9 to 0.857 1(mean 0.631 7) and 0.538 3 to0.911 8(mean 0.796 8), respectively. The polymorphism information content(PIC) ranged from 0.497 to 0.886(mean 0.780 9), and the PIC at all loci except for one was geater than 0.500,indicating that the microsatellites were highly polymorphic. The HardyWeinberg equilibrium(HWE) test shows that 19 microsatellite markers conformed to HWE. There is no linkage imbalance among the 27 loci. The 27 microsatellites developed by this study will be useful for further population genetic analysis.
引文
[1]中国科学院动物研究所,中国科学院海洋研究所,上海水产学院.南海鱼类志[M].北京:科学出版社,1962:387-389.
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[3]张立,李渊,林龙山,等.南海中南部主要经济种类渔业资源声学评估[J].海洋渔业,2016,38(6):577-587.
[4]张俊,张鹏,陈作志,等.南海外海鲹科鱼类资源量及其分布[J].南方水产科学,2016,12(4):38-48.
[5]张鹏,张俊,李渊,等.秋季南海中南部海域的一次灯光罩网探捕调查[J].南方水产科学,2016,12(2):67-74.
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[7]BALASUBRAMANIAN N K,NATARAJAN P.Resource characteristics of the scads,Decapterus russelli and D.macrosoma of the Vizhinjam area,southwest coast of India[J].Indian J Fish,1999,46(2):111-122.
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[9]翟云,吴仁协,牛素芳,等.基于SLAF-seq技术开发蓝圆鲹微卫星标记及跨物种扩增检测[J].应用海洋学学报,2018,37(3):426-434.
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[11]EUSTICE M,YU Q Y,LAI C W.Development and application of microsatellite markers for genomic analysis of papaya[J].Tree Genet Genome,2008,4(2):333-341.
[12]VARELA A I,RITCHIE P A,SMITH P J.Global genetic population structure in the commercially exploited deep-sea teleost orange roughy(Hoplostethus atlanticus)based on microsatellite DNA analyses[J].Fish Res,2013,140:83-90.
[13]海萨,李家乐,郭焱,等.河鲈微卫星引物筛选[J].水生态学杂志,2008,1(6):90-94.
[14]赵哲霞,蒋珊,王滨花,等.黄颡鱼属SSR分子鉴定及其遗传多样性[J].南昌大学学报(理科版),2014,38(5):498-501.
[15]翟云,吴仁协,牛素芳,等.采用高通量技术开发花鲈二碱基重复微卫星标记[J].基因组学与应用生物学,2018(9):1-11.
[16]CASTOE T A,STREICHER J W,MEIK J M,et al.Thousands of microsatellite loci from the venomous coralsnake Micrurus fulvius and variability of select loci across populations and related species[J].Mol Ecol Resour,2012,12(6):1105-1113.
[17]鲁翠云,毛瑞鑫,李鸥,等.鲤鱼三、四核苷酸重复微卫星座位的筛选及特征分析[J].农业生物技术学报,2009,17(6):979-987.
[18]谭照君,张天奇,鲁翠云,等.鲢三、四核苷酸重复微卫星标记的筛选及其特征分析[J].上海海洋大学学报,2011,20(3):328-335.
[19]李文升,刘翠,鲁翠云,等.草鱼三、四核苷酸重复微卫星标记的分离与特征分析[J].中国水产科学,2011,18(4):742-750.
[20]THIEL T,MICHALEK W,VARSHNEY R K,et al.Exploiting EST databases for the development and characterization of genederived SSR-markers in barley(Hordeum vulgare L.)[J].Theor Appl Genet,2003,106(3):411-422.
[21]SCHUELKE M.An economic method for the fluorescent labeling of PCR fragments[J].Nat Biotechnol,2000,18(2):233-234.
[22]ROUSSET F.genepop'007:a complete re-implementation of the genepop software for Windows and Linux[J].Mol Ecol Resour,2008,8(1):103-106.
[23]KALINOWSKI S T,TAPER M L,MARSHALL T C.Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment[J].Mol Ecol,2007,16(5):1099-1106.
[24]杨兵,林琳,李纯厚,等.基于高通量测序的二长棘鲷微卫星标记开发与评价[J].南方水产科学,2015,11(4):116-120.
[25]秦海峰,龙宁,吴建国,等.甜叶菊微卫星富集文库的构建与多态性标记的筛选[J].作物学报,2014,40(3):447-456.
[26]LIN L,LI C H,CHEN Z Z,et al.Development and characterization of twenty-three microsatellite markers for the purpleback flying squid(Symplectoteuthis oualaniensis)[J].Conserv Genet Resour,2015,7(1):161-163.
[27]KONG X L,CHEN Z Z,LIN L,et al.Polymorphic micro-satellite loci isolated from the yellowbelly threadfin bream,Nemipterus bathybius[J].Genet Mol Res,2014,13(3):5254-5257.
[28]房祖业,陈晓东,吴咏诗,等.大刺鳅(Mastacembelus armatus)二、三、四碱基重复微卫星标记的筛选和特征分析[J].海洋与湖沼,2018,49(1):174-182.
[29]高峰涛,邵长伟,崔忠凯,等.基于高通量测序的青石斑鱼基因组微卫星开发及评价[J].中国海洋大学学报(自然科学版),2017,47(4):52-57.
[30]SONG W T,PANG R Y,NIU Y Z,et al.Construction of highdensity genetic linkage maps and mapping of growth-related quantitative trail loci in the Japanese flounder(Paralichthys olivaceus)[J].PLoS One,2012,7(11):50404.
[31]SONG W,LI Y,ZHAO Y,et al.Construction of high-density microsatellite genetic linkage maps and mapping of sexual and growthrelated traits in half-smooth tongue sole(Cynoglossus semilaevis)[J].PLoS One,2012,7(12):e52097.
[32]曾晓芸,杨宗英,田辉伍,等.基于Mi-Seq高通量测序分析裸体异鳔鳅鮀微卫星组成[J].淡水渔业,2015,45(1):3-7.
[33]熊良伟,王帅兵,封琦,等.基于高通量测序的中华鳑鲏基因组微卫星特征分析及标记开发[J].江苏农业科学,2018,46(18):164-168.
[34]XIONG L W,WANG Q,QIU G F.Large-scale isolation of microsatellites from Chinese mitten crab Eriocheir sinensis via a solexa genomic survey[J].Int J Mol Sci,2012,13(12):16333-16345.
[35]高焕,孔杰.串联重复序列的物种差异及其生物功能[J].动物学研究,2005,26(5):555-564.
[36]BOTSTEIN D,WHITE R L,SKOLNICK M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J].Am J Hum Genet,1980,32(3):314-331.
[37]ELLEGREN H.Microsatellite evolution:a battle between replication slippage and point mutation[J].Trends Genet,2002,18(2):70.
[38]WEBER J L.Informativeness of human(dC-dA)n·(dG-dT)n polymorphisms[J].Genomics,1990,7(4):524-530.
[39]龚小玲,李思发,蔡完其,等.澳洲鳗鲡微卫星分子标记的筛选与检测[J].中国水产科学,2009,16(1):133-138.
[2]杨吝,张旭丰,谭永光,等.南海北部灯光围网渔获组成分析[J].南方水产,2009,5(6):65-70.
[3]张立,李渊,林龙山,等.南海中南部主要经济种类渔业资源声学评估[J].海洋渔业,2016,38(6):577-587.
[4]张俊,张鹏,陈作志,等.南海外海鲹科鱼类资源量及其分布[J].南方水产科学,2016,12(4):38-48.
[5]张鹏,张俊,李渊,等.秋季南海中南部海域的一次灯光罩网探捕调查[J].南方水产科学,2016,12(2):67-74.
[6]SHIRAISHI T,TANAKA H,OHSHIMO S,et al.Age,growth and reproduction of two species of scad,Decapterus macrosoma and D.macarellus in the waters off southern Kyushu[J].Jpn Agr Res Q,2010,44(2):197-206.
[7]BALASUBRAMANIAN N K,NATARAJAN P.Resource characteristics of the scads,Decapterus russelli and D.macrosoma of the Vizhinjam area,southwest coast of India[J].Indian J Fish,1999,46(2):111-122.
[8]POTIE M,DRAPEAU L.Modelling and forecasting the catch of the scads(Decapterus macrosoma,Decapterus russellii)in the Javanese purse seine fishery using ARIMA time series models[J].Asian Fish Sci,2000,13:75-85.
[9]翟云,吴仁协,牛素芳,等.基于SLAF-seq技术开发蓝圆鲹微卫星标记及跨物种扩增检测[J].应用海洋学学报,2018,37(3):426-434.
[10]DEWOODY J A,AVISE J C.Microsatellite variation in marine,freshwater and anadromous fishes compared with other animals[J].J Fish Biol,2000,56(3):461-473.
[11]EUSTICE M,YU Q Y,LAI C W.Development and application of microsatellite markers for genomic analysis of papaya[J].Tree Genet Genome,2008,4(2):333-341.
[12]VARELA A I,RITCHIE P A,SMITH P J.Global genetic population structure in the commercially exploited deep-sea teleost orange roughy(Hoplostethus atlanticus)based on microsatellite DNA analyses[J].Fish Res,2013,140:83-90.
[13]海萨,李家乐,郭焱,等.河鲈微卫星引物筛选[J].水生态学杂志,2008,1(6):90-94.
[14]赵哲霞,蒋珊,王滨花,等.黄颡鱼属SSR分子鉴定及其遗传多样性[J].南昌大学学报(理科版),2014,38(5):498-501.
[15]翟云,吴仁协,牛素芳,等.采用高通量技术开发花鲈二碱基重复微卫星标记[J].基因组学与应用生物学,2018(9):1-11.
[16]CASTOE T A,STREICHER J W,MEIK J M,et al.Thousands of microsatellite loci from the venomous coralsnake Micrurus fulvius and variability of select loci across populations and related species[J].Mol Ecol Resour,2012,12(6):1105-1113.
[17]鲁翠云,毛瑞鑫,李鸥,等.鲤鱼三、四核苷酸重复微卫星座位的筛选及特征分析[J].农业生物技术学报,2009,17(6):979-987.
[18]谭照君,张天奇,鲁翠云,等.鲢三、四核苷酸重复微卫星标记的筛选及其特征分析[J].上海海洋大学学报,2011,20(3):328-335.
[19]李文升,刘翠,鲁翠云,等.草鱼三、四核苷酸重复微卫星标记的分离与特征分析[J].中国水产科学,2011,18(4):742-750.
[20]THIEL T,MICHALEK W,VARSHNEY R K,et al.Exploiting EST databases for the development and characterization of genederived SSR-markers in barley(Hordeum vulgare L.)[J].Theor Appl Genet,2003,106(3):411-422.
[21]SCHUELKE M.An economic method for the fluorescent labeling of PCR fragments[J].Nat Biotechnol,2000,18(2):233-234.
[22]ROUSSET F.genepop'007:a complete re-implementation of the genepop software for Windows and Linux[J].Mol Ecol Resour,2008,8(1):103-106.
[23]KALINOWSKI S T,TAPER M L,MARSHALL T C.Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment[J].Mol Ecol,2007,16(5):1099-1106.
[24]杨兵,林琳,李纯厚,等.基于高通量测序的二长棘鲷微卫星标记开发与评价[J].南方水产科学,2015,11(4):116-120.
[25]秦海峰,龙宁,吴建国,等.甜叶菊微卫星富集文库的构建与多态性标记的筛选[J].作物学报,2014,40(3):447-456.
[26]LIN L,LI C H,CHEN Z Z,et al.Development and characterization of twenty-three microsatellite markers for the purpleback flying squid(Symplectoteuthis oualaniensis)[J].Conserv Genet Resour,2015,7(1):161-163.
[27]KONG X L,CHEN Z Z,LIN L,et al.Polymorphic micro-satellite loci isolated from the yellowbelly threadfin bream,Nemipterus bathybius[J].Genet Mol Res,2014,13(3):5254-5257.
[28]房祖业,陈晓东,吴咏诗,等.大刺鳅(Mastacembelus armatus)二、三、四碱基重复微卫星标记的筛选和特征分析[J].海洋与湖沼,2018,49(1):174-182.
[29]高峰涛,邵长伟,崔忠凯,等.基于高通量测序的青石斑鱼基因组微卫星开发及评价[J].中国海洋大学学报(自然科学版),2017,47(4):52-57.
[30]SONG W T,PANG R Y,NIU Y Z,et al.Construction of highdensity genetic linkage maps and mapping of growth-related quantitative trail loci in the Japanese flounder(Paralichthys olivaceus)[J].PLoS One,2012,7(11):50404.
[31]SONG W,LI Y,ZHAO Y,et al.Construction of high-density microsatellite genetic linkage maps and mapping of sexual and growthrelated traits in half-smooth tongue sole(Cynoglossus semilaevis)[J].PLoS One,2012,7(12):e52097.
[32]曾晓芸,杨宗英,田辉伍,等.基于Mi-Seq高通量测序分析裸体异鳔鳅鮀微卫星组成[J].淡水渔业,2015,45(1):3-7.
[33]熊良伟,王帅兵,封琦,等.基于高通量测序的中华鳑鲏基因组微卫星特征分析及标记开发[J].江苏农业科学,2018,46(18):164-168.
[34]XIONG L W,WANG Q,QIU G F.Large-scale isolation of microsatellites from Chinese mitten crab Eriocheir sinensis via a solexa genomic survey[J].Int J Mol Sci,2012,13(12):16333-16345.
[35]高焕,孔杰.串联重复序列的物种差异及其生物功能[J].动物学研究,2005,26(5):555-564.
[36]BOTSTEIN D,WHITE R L,SKOLNICK M,et al.Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J].Am J Hum Genet,1980,32(3):314-331.
[37]ELLEGREN H.Microsatellite evolution:a battle between replication slippage and point mutation[J].Trends Genet,2002,18(2):70.
[38]WEBER J L.Informativeness of human(dC-dA)n·(dG-dT)n polymorphisms[J].Genomics,1990,7(4):524-530.
[39]龚小玲,李思发,蔡完其,等.澳洲鳗鲡微卫星分子标记的筛选与检测[J].中国水产科学,2009,16(1):133-138.
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