应用单核苷酸多态性(SNP)标记鉴定短颌鲚、湖鲚和刀鲚
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摘要
为了快速区分长江流域各水体中的鲚属鱼类,利用单核苷酸多态性(Single nucleotide polymorphism,SNP)标记对短颌鲚(Coilia brachygnathus)、湖鲚(C. nasus taihuensis)和刀鲚(C. nasus)进行物种鉴定。从120个在短颌鲚和刀鲚之间分化指数(Fst)为1的SNP位点中随机挑选出20个用于引物设计,从21个在湖鲚和刀鲚之间分化指数较高的SNP位点中随机挑选出10个设计引物。随机挑20尾短颌鲚、20尾湖鲚和12尾刀鲚用设计的引物扩增、测序。结果表明:洞庭湖和鄱阳湖的短颌鲚与刀鲚可以使用以上19个SNP分子标记中的任何一个完全分开,剩下1个SNP标记在所扩增的样本中没有发现多样性变异而被弃用。用于区分湖鲚与刀鲚的10个SNP分子标记中有8个在所测样本中可以扩增并用于物种鉴定。其中单独使用Ct-Cn_wtap位点时,湖鲚和刀鲚的基因频率相差最大,当使用Ct-Cn_wtap和Ct-Cn_eif2b4位点时,对湖鲚和刀鲚的鉴别准确率可以达到100%。本研究提供了稳定、高效和低成本识别短颌鲚和刀鲚、湖鲚和刀鲚的方法,为今后鲚属鱼类的物种鉴定和资源保护提供了有效的工具。
To identify the species of the tapertail anchovies rapidly,Coilia brachygnathus,C. nasus and C.nasus taihuensis distributed in freshwater lakes of the Yangtze River Basin,single nucleotide polymorphism( SNP) molecular markers were developed to distinguish them. Twenty SNP markers were randomly selected with fixation index( Fst) of 1 from the 120 loci found between C. brachygnathus and C. nasus; and 10 SNP markers were ramdomly selected with high Fstvalue from the 21 loci found between C. nasus and C. nasus taihuensis for primers designing. Twenty C. brachygnathus,20 C. nasus taihuensis and 12 C. nasus were randomly amplified and sequenced using the primers designed. The results showed that each of the 19 SNP markers can be used to completely distinguish C. brachygnathus and C. nasus,and no polymorphic sites were found in the other locus in the samples tested. Eight SNP makers were successfully amplified and used to distinguish C. nasus and C. nasus taihuensis. Marker Ct-Cn_wta showed the highest difference in genotype frequency between C. nasus and C. nasus taihuensis. When marker Ct-Cn_wtap and Ct-Cn_eif2b4 SNP were used,the successful identification rate on C. nasus and C. nasus taihuensis reached 100%. In conclusion,a robust and efficient method is provided to identify the tapertail anchovies,C. brachygnathus,C. nasus and C. nasus taihuensis using SNP molecular marker,which would provide an important tool for identification and conservation of the tapertail anchovies.
To identify the species of the tapertail anchovies rapidly,Coilia brachygnathus,C. nasus and C.nasus taihuensis distributed in freshwater lakes of the Yangtze River Basin,single nucleotide polymorphism( SNP) molecular markers were developed to distinguish them. Twenty SNP markers were randomly selected with fixation index( Fst) of 1 from the 120 loci found between C. brachygnathus and C. nasus; and 10 SNP markers were ramdomly selected with high Fstvalue from the 21 loci found between C. nasus and C. nasus taihuensis for primers designing. Twenty C. brachygnathus,20 C. nasus taihuensis and 12 C. nasus were randomly amplified and sequenced using the primers designed. The results showed that each of the 19 SNP markers can be used to completely distinguish C. brachygnathus and C. nasus,and no polymorphic sites were found in the other locus in the samples tested. Eight SNP makers were successfully amplified and used to distinguish C. nasus and C. nasus taihuensis. Marker Ct-Cn_wta showed the highest difference in genotype frequency between C. nasus and C. nasus taihuensis. When marker Ct-Cn_wtap and Ct-Cn_eif2b4 SNP were used,the successful identification rate on C. nasus and C. nasus taihuensis reached 100%. In conclusion,a robust and efficient method is provided to identify the tapertail anchovies,C. brachygnathus,C. nasus and C. nasus taihuensis using SNP molecular marker,which would provide an important tool for identification and conservation of the tapertail anchovies.
引文
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[3]YANG Q L,GAO T X,MIAO Z Q.Differentiation between populations of Japanese grenadier anchovy(Coilia nasus)in Northwestern Pacific based on ISSR markers:implications for biogeography[J].Biochemical Systematics and Ecology,2011,39(4/6):286-296.
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[8]顾海龙,冯亚明,游华斌,等.长江刀鲚资源调查与人工养殖研究进展[J].江苏农业科学,2016,44(3):265-267.GU H L,FENG Y M,YOU H B,et al.Research progress of resource investigation and artificial breeding of Coilia nasus of the Yangtze River[J].Jiangsu Agricultural Sciences,2016,44(3):265-267.
[9]王庆萍,甘江英.人类活动对鄱阳湖刀鲚的影响及其保护性开发对策[J].渔业致富指南,2017(7):18-21.WANG Q P,GAN J Y.Effects of human activities on Coilia nasus in Poyang Lake and protective development strategies[J].Fishery Guide to be Rich,2017(7):18-21.
[10]ERGUDEN D,TURAN C.Examination of genetic and morphologic structure of seabass(Dicentrarchus labrax L.,1758)populations in Turkish coastal waters[J].Turkish Journal of Veterinary&Animal Sciences,2005,29(3):727-733.
[11]程起群,李思发.刀鲚和湖鲚种群的形态判别[J].海洋科学,2004,28(11):39-44.CHENG Q Q,LI S F.Morphological discrimination between two populations of Coilia ectenes[J].Marine Sciences,2004,28(11):39-44.
[12]向文殿,谢佳燕,林佳.不同湖泊湖鲚种群形态差异的研究[J].湖北农业科学,2011,50(21):4445-4447.XIANG W D,XIE J Y,LIN J.Morphological variations of Coilia ectenes in different lakes[J].Hubei Agricultural Sciences,2011,50(21):4445-4447.
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[14]TANG W Q,HU X L,YANG J Q.Species validities of Coilia brachygnathus and C.nasus taihuensis based on sequence variations of complete mt DNA control region[J].Biodiversity Science,2007,15(3):224-231.
[15]王倩,程方圆,李晨虹.鲚属单核苷酸多态性位点(SNPs)标记开发及在物种界定中的应用初探[J].上海海洋大学学报,2017,26(1):8-16.WANG Q,CHENG F Y,LI C H.Developing SNP markers for Coilia and its application in species delimitation[J].Journal of Shanghai Ocean University,2017,26(1):8-16.
[16]YANG Q L,HAN Z Q,SUN D R,et al.Genetics and phylogeny of genus Coilia in China based on AFLP markers[J].Chinese Journal of Oceanology and Limnology,2010,28(4):795-801.
[17]杨金权,胡雪莲,唐文乔.长江及其南部邻近水域刀鲚的种群遗传结构及种群历史[J].上海水产大学学报,2008,17(5):513-519.YANG J Q,HU X L,TANG W Q.Genetic structure and population history of Coilia nasus in Yangtze River and its south adjacent water[J].Journal of Shanghai Fisheries University,2008,17(5):513-519.
[18]张燕萍,贺刚,王生,等.基于线粒体DNA D-loop序列分析刀鲚与短颌鲚的遗传多样性[J].江苏农业科学,2017,45(20):59-64.ZHANG Y P,HE G,WANG S,et al.Genetic diversity analysis of Coilia nasus and Coilia brachynathus based on mitochondrial DNA D-loop sequence[J].Jiangsu Agricultural Sciences,2017,45(20):59-64.
[19]姜涛,刘洪波,李孟孟,等.溯河洄游长江刀鲚(Coilia nasus)摄食虾类的调查[J].湖泊科学,2018,30(2):458-463.JIANG T,LIU H B,LI M M,et al.Investigation on shrimp feeding of Coilia nasus during its anadromous migration along the Yangtze River[J].Journal of Lake Sciences,2018,30(2):458-463.
[20]李孟孟,姜涛,KHUMBANYIWA D D,等.基于耳石微化学的长江安徽和县江段刀鲚生境履历重建[J].水生生物学报,2017,41(5):1054-1061.LI M M,JIANG T,KHUMBANYIWA D D,et al.Reconstructing habitat history of Coilia nasus from the Hexian section of the Yangtze river in Anhui province by otolith microchemistry[J].Acta Hydrobiologica Sinica,2017,41(5):1054-1061.
[21]HATCHER P E,WILKINSON M J,ALBANI M C,et al.Conserving marginal populations of the food plant(Impatiens noli-tangere)of an endangered moth(Eustroma reticulatum)in a changing climate[J].Biological Conservation,2004,116(3):305-317.
[22]马春艳,刘敏,马凌波,等.长江口刀鲚遗传多样性的随机扩增多态DNA(RAPD)分析[J].海洋水产研究,2004,25(5):19-24.MA C Y,LIU M,MA L B,et al.Genetic diversity in Coilia ectenes by RAPD analysis[J].Marine Fisheries Research,2004,25(5):19-24.
[23]王美垚,管建洪.刀鲚遗传多样性研究进展[J].安徽农业科学,2015,43(10):149-151.WANG M Y,GUAN H J.Research progress on genetic diversity of Coilia nasus[J].Journal of Anhui Agricultural Sciences,2015,43(10):149-151.
[24]LI C H,HOFREITER M,STRAUBE N,et al.Capturing protein-coding genes across highly divergent species[J].BioTechniques,2013,54(6):321-326.
[25]程起群,温俊娥,王云龙,等.刀鲚与湖鲚线粒体细胞色素b基因片段多态性及遗传关系[J].湖泊科学,2006,18(4):425-430.CHENG Q Q,WEN J E,WANG Y L,et al.Genetic diversity and genetic differentiation between Coilia ectenes and Coilia ectenes taihuensis inferred from cytochrome b gene segment sequence of mitochondrial DNA[J].Journal of Lake Sciences,2006,18(4):425-430.
[26]ZHOU X D,YANG J Q,TANG W Q.Species validities analyses of Chinese Coilia fishes based on mt DNA COIbarcoding[J].Acta Zootaxonomica Sinica,2010,35(4):819-826.
[27]JORDAN D S,SEALE A.List of fishes collected in 1882-83by Pierre Louis Jouy at Shanghai and Hong Kong,China[J].Proceedings of the United States National Museum,1905,29(1433):517-29.
[28]葛家春,曹廷,陈婵娟,等.利用扩增片断长度多态性技术分析长江刀鲚的遗传多样性[J].南京大学学报(自然科学版),2008,44(3):332-338.GE J G,CAO Y,CHEN C J,et al.Analysis of genetic diversity based on amplified fragment length polymorphism fingerprint of Coilia nasus from Yangtze River[J].Journal of Nanjing University(Natural Sciences),2008,44(3):332-338.
[29]李红东.不同年间长江刀鲚形态差异及其遗传多样性研究[D].扬州:扬州大学,2011:1-43.LI H D.Morphological variations and genetic diversity of Coilia ectenes in different years[D].Yangzhou:Yangzhou University,2011:1-43.
[30]CHENG Q Q,ZHANG Q Y,MA C Y,et al.Genetic structure and differentiation of four lake populations of Coilia ectenes(Clupeiformes:Engraulidae)based on mt DNAcontrol region sequences[J].Biochemical Systematics and Ecology,2011,39(4/5):544-552.
[31]姜涛,周昕期,刘洪波,等.鄱阳湖刀鲚耳石的两种微化学特征[J].水产学报,2013,37(2):239-244.JIANG T,ZHOU X Q,LIU H B,et al.Two microchemistry patterns in otoliths of Coilia nasus from Poyang Lake,China[J].Journal of Fisheries of China,2013,37(2):239-244.
[32]田思泉,田芝清,高春霞,等.长江口刀鲚汛期特征及其资源状况的年际变化分析[J].上海海洋大学学报,2014,23(2):245-250.TIAN S Q,TIAN Z Q,GAO C X,et al.Analyzing of annual changes for the stock status of Coilia nasus in fishing season in Yangtze River estuary[J].Journal of Shanghai Ocean University,2014,23(2):245-250.
[2]LIU D,LI Y,LI Y Y,TANG W Q,et al.Population structure of Coilia nasus in the Yangtze River revealed by insertion of short interspersed elements[J].Biochemical Systematics and Ecology,2014,54:103-112.
[3]YANG Q L,GAO T X,MIAO Z Q.Differentiation between populations of Japanese grenadier anchovy(Coilia nasus)in Northwestern Pacific based on ISSR markers:implications for biogeography[J].Biochemical Systematics and Ecology,2011,39(4/6):286-296.
[4]袁传宓,秦安黔,刘仁华,等.关于长江中下游及东南沿海各省的鲚属鱼类种下分类的探讨[J].南京大学学报(自然科学版),1980(3):67-82.YUAN C M,QING A Q,LIU R H,et al.On the classification of the anchovies,Coilia from the lower Yangtze River and the southeast coast of China[J].Journal of Nanjing University(Natural Sciences),1980(3):67-82.
[5]KREYENBERG W,PAPPENHEIM P.Ein Beitrag zur Kenntnis der Fische der Jangtze und seiner Zuflüsse[J].Sber Ges Naturf Freunde Berl,1908:95-109.
[6]吴斌,方春林,傅培峰,等.鄱阳湖通江水道短颌鲚生长特性初探[J].水生态学杂志,2015,36(3):51-55.WU B,FANG C L,FU P F,et al.Growth characteristics of Coilia brachygnathus in the Poyang Lake-Yangtze River waterway[J].Journal of Hydroecology,2015,36(3):51-55.
[7]董文霞,唐文乔,王磊.长江刀鲚繁殖群体的生长特性[J].上海海洋大学学报,2014,23(5):669-674.DONG W X,TANG W Q,WANG L.Growth characteristics of reproductive population of Coilia nasus in the Yangtze River[J].Journal of Shanghai Ocean University,2014,23(5):669-674.
[8]顾海龙,冯亚明,游华斌,等.长江刀鲚资源调查与人工养殖研究进展[J].江苏农业科学,2016,44(3):265-267.GU H L,FENG Y M,YOU H B,et al.Research progress of resource investigation and artificial breeding of Coilia nasus of the Yangtze River[J].Jiangsu Agricultural Sciences,2016,44(3):265-267.
[9]王庆萍,甘江英.人类活动对鄱阳湖刀鲚的影响及其保护性开发对策[J].渔业致富指南,2017(7):18-21.WANG Q P,GAN J Y.Effects of human activities on Coilia nasus in Poyang Lake and protective development strategies[J].Fishery Guide to be Rich,2017(7):18-21.
[10]ERGUDEN D,TURAN C.Examination of genetic and morphologic structure of seabass(Dicentrarchus labrax L.,1758)populations in Turkish coastal waters[J].Turkish Journal of Veterinary&Animal Sciences,2005,29(3):727-733.
[11]程起群,李思发.刀鲚和湖鲚种群的形态判别[J].海洋科学,2004,28(11):39-44.CHENG Q Q,LI S F.Morphological discrimination between two populations of Coilia ectenes[J].Marine Sciences,2004,28(11):39-44.
[12]向文殿,谢佳燕,林佳.不同湖泊湖鲚种群形态差异的研究[J].湖北农业科学,2011,50(21):4445-4447.XIANG W D,XIE J Y,LIN J.Morphological variations of Coilia ectenes in different lakes[J].Hubei Agricultural Sciences,2011,50(21):4445-4447.
[13]倪勇,伍汉霖.江苏鱼类志[M].北京:中国农业出版社,2006.NI Y,WU H L.Fishes of Jiangsu Province[M].Beijing:China Agriculture Press,2006.
[14]TANG W Q,HU X L,YANG J Q.Species validities of Coilia brachygnathus and C.nasus taihuensis based on sequence variations of complete mt DNA control region[J].Biodiversity Science,2007,15(3):224-231.
[15]王倩,程方圆,李晨虹.鲚属单核苷酸多态性位点(SNPs)标记开发及在物种界定中的应用初探[J].上海海洋大学学报,2017,26(1):8-16.WANG Q,CHENG F Y,LI C H.Developing SNP markers for Coilia and its application in species delimitation[J].Journal of Shanghai Ocean University,2017,26(1):8-16.
[16]YANG Q L,HAN Z Q,SUN D R,et al.Genetics and phylogeny of genus Coilia in China based on AFLP markers[J].Chinese Journal of Oceanology and Limnology,2010,28(4):795-801.
[17]杨金权,胡雪莲,唐文乔.长江及其南部邻近水域刀鲚的种群遗传结构及种群历史[J].上海水产大学学报,2008,17(5):513-519.YANG J Q,HU X L,TANG W Q.Genetic structure and population history of Coilia nasus in Yangtze River and its south adjacent water[J].Journal of Shanghai Fisheries University,2008,17(5):513-519.
[18]张燕萍,贺刚,王生,等.基于线粒体DNA D-loop序列分析刀鲚与短颌鲚的遗传多样性[J].江苏农业科学,2017,45(20):59-64.ZHANG Y P,HE G,WANG S,et al.Genetic diversity analysis of Coilia nasus and Coilia brachynathus based on mitochondrial DNA D-loop sequence[J].Jiangsu Agricultural Sciences,2017,45(20):59-64.
[19]姜涛,刘洪波,李孟孟,等.溯河洄游长江刀鲚(Coilia nasus)摄食虾类的调查[J].湖泊科学,2018,30(2):458-463.JIANG T,LIU H B,LI M M,et al.Investigation on shrimp feeding of Coilia nasus during its anadromous migration along the Yangtze River[J].Journal of Lake Sciences,2018,30(2):458-463.
[20]李孟孟,姜涛,KHUMBANYIWA D D,等.基于耳石微化学的长江安徽和县江段刀鲚生境履历重建[J].水生生物学报,2017,41(5):1054-1061.LI M M,JIANG T,KHUMBANYIWA D D,et al.Reconstructing habitat history of Coilia nasus from the Hexian section of the Yangtze river in Anhui province by otolith microchemistry[J].Acta Hydrobiologica Sinica,2017,41(5):1054-1061.
[21]HATCHER P E,WILKINSON M J,ALBANI M C,et al.Conserving marginal populations of the food plant(Impatiens noli-tangere)of an endangered moth(Eustroma reticulatum)in a changing climate[J].Biological Conservation,2004,116(3):305-317.
[22]马春艳,刘敏,马凌波,等.长江口刀鲚遗传多样性的随机扩增多态DNA(RAPD)分析[J].海洋水产研究,2004,25(5):19-24.MA C Y,LIU M,MA L B,et al.Genetic diversity in Coilia ectenes by RAPD analysis[J].Marine Fisheries Research,2004,25(5):19-24.
[23]王美垚,管建洪.刀鲚遗传多样性研究进展[J].安徽农业科学,2015,43(10):149-151.WANG M Y,GUAN H J.Research progress on genetic diversity of Coilia nasus[J].Journal of Anhui Agricultural Sciences,2015,43(10):149-151.
[24]LI C H,HOFREITER M,STRAUBE N,et al.Capturing protein-coding genes across highly divergent species[J].BioTechniques,2013,54(6):321-326.
[25]程起群,温俊娥,王云龙,等.刀鲚与湖鲚线粒体细胞色素b基因片段多态性及遗传关系[J].湖泊科学,2006,18(4):425-430.CHENG Q Q,WEN J E,WANG Y L,et al.Genetic diversity and genetic differentiation between Coilia ectenes and Coilia ectenes taihuensis inferred from cytochrome b gene segment sequence of mitochondrial DNA[J].Journal of Lake Sciences,2006,18(4):425-430.
[26]ZHOU X D,YANG J Q,TANG W Q.Species validities analyses of Chinese Coilia fishes based on mt DNA COIbarcoding[J].Acta Zootaxonomica Sinica,2010,35(4):819-826.
[27]JORDAN D S,SEALE A.List of fishes collected in 1882-83by Pierre Louis Jouy at Shanghai and Hong Kong,China[J].Proceedings of the United States National Museum,1905,29(1433):517-29.
[28]葛家春,曹廷,陈婵娟,等.利用扩增片断长度多态性技术分析长江刀鲚的遗传多样性[J].南京大学学报(自然科学版),2008,44(3):332-338.GE J G,CAO Y,CHEN C J,et al.Analysis of genetic diversity based on amplified fragment length polymorphism fingerprint of Coilia nasus from Yangtze River[J].Journal of Nanjing University(Natural Sciences),2008,44(3):332-338.
[29]李红东.不同年间长江刀鲚形态差异及其遗传多样性研究[D].扬州:扬州大学,2011:1-43.LI H D.Morphological variations and genetic diversity of Coilia ectenes in different years[D].Yangzhou:Yangzhou University,2011:1-43.
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