线粒体COI基因条形码在鲿科鱼类物种鉴定中的应用
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摘要
为了探讨线粒体COI基因作为DNA条形码在中国鲿科(Bagridae)鱼类物种鉴定中的有效性,以及系统发育中的适用性,本研究对4属11种鲿科鱼类进行PCR扩增,获得48条线粒体COI基因序列,同时从Gen Bank筛选获得8种鲿科鱼类的12条COI基因序列进行分析。19种鲿科鱼类的COI基因序列特征显示:长度为674 bp的COI序列片段平均碱基组成为24.82%A,30.44%T,27.10%C和17.64%G,碱基组成呈现明显的AT偏倚性(55.26%),具有硬骨鱼类的线粒体COI基因的碱基组成的典型特征。核苷酸位点中有变异位点226个,简约信息位点195个,单一信息位点31个,转换颠换比为3.35。19种鲿科鱼类的种内、种间和属间平均遗传距离分别为0.0041、0.1136和0.1268,种间遗传距离平均为种内遗传距离的27.7倍。在本研究中,鲿科鱼类所有的物种均形成单系,在物种鉴别上与形态学分类结果基本一致,然而鲿科的4个属中只有鱯属形成单系,黄颡鱼属、属和拟鲿属均未形成单系,其进化地位需要进一步研究。线粒体COI基因作为条形码可有效对鲿科鱼类进行物种鉴定,也为鲿科的系统发育提供了参考。
The Bagridae catfishes, belonging to the order Siluriformes and class Osteichthyes, are widely distributed in freshwater areas in Asia, such as China, Japan, Korean peninsula, and Vietnam. In China, Bagridae catfishes include more than 30 species across 4 genera. In recent years, due to environmental deterioration and human disturbance, populations of the native Bagridae had sharply decreased and some of them were difficult to collect. However, the classification of some Bagridae catfishes is ambiguous due to the difference of individuals, genders, or habitat. A species identification technique for various fishes based on DNA barcoding has been rapidly developed and widely employed. For many fishes, DNA barcoding requires additional validation before use. Herein, we have developed and evaluated DNA barcoding based on COI gene sequences for identifying Bagridae species. Altogether, 11 Bagridae catfish species were collected and sequenced with the standard DNA barcoding protocols, then combined with 8 available species acquired from the Gen Bank. A total of 60 sequences from 19 species pertaining to 4 genera were analyzed. Dna SP 5 and MEGA7.0 were employed to calculate the base composition, sequence characterization, and genetic distance, and construct the neighbor-joining phylogenetic tree. The mrbayes software was used to construct the Bayesian tree. The result showed that the mean base contents were: 24.82% A, 30.44% T, 27.10% C, and 17.64% T. AT contents(55.26) were higher and the base composition was biased as others teleost fishes were also commonly observed. The nucleotide sequences included 226 variant sites, 195 parsimony informative sites, and 31 singleton sites. The average transition/transversion ratio was 3.35. Genetic distances within species, genus, and families were 0.0041, 0.1136, and 0.1268, respectively. The ratio of genetic distances between interspecies and intraspecies was 27.7, which indicated a notable genetic difference of the COI gene sequence. The efficiency of DNA barcoding based on the COI gene was reaffirmed from its successful identification of all 19 Bagridae catfishes in this study. This classification result is consistent with their morphologies. However, only Hemibagrus catfishes can be clustered into a single phylogenetic tree branch. Other Bagridae species were intertwined, and their phylogenetic status needs further research combined with data from more samples. The results revealed that the COI gene was suitable as a DNA barcoding parameter for identifying species; it provided a useful reference for the phylogenetic relationships of Bagridae.
The Bagridae catfishes, belonging to the order Siluriformes and class Osteichthyes, are widely distributed in freshwater areas in Asia, such as China, Japan, Korean peninsula, and Vietnam. In China, Bagridae catfishes include more than 30 species across 4 genera. In recent years, due to environmental deterioration and human disturbance, populations of the native Bagridae had sharply decreased and some of them were difficult to collect. However, the classification of some Bagridae catfishes is ambiguous due to the difference of individuals, genders, or habitat. A species identification technique for various fishes based on DNA barcoding has been rapidly developed and widely employed. For many fishes, DNA barcoding requires additional validation before use. Herein, we have developed and evaluated DNA barcoding based on COI gene sequences for identifying Bagridae species. Altogether, 11 Bagridae catfish species were collected and sequenced with the standard DNA barcoding protocols, then combined with 8 available species acquired from the Gen Bank. A total of 60 sequences from 19 species pertaining to 4 genera were analyzed. Dna SP 5 and MEGA7.0 were employed to calculate the base composition, sequence characterization, and genetic distance, and construct the neighbor-joining phylogenetic tree. The mrbayes software was used to construct the Bayesian tree. The result showed that the mean base contents were: 24.82% A, 30.44% T, 27.10% C, and 17.64% T. AT contents(55.26) were higher and the base composition was biased as others teleost fishes were also commonly observed. The nucleotide sequences included 226 variant sites, 195 parsimony informative sites, and 31 singleton sites. The average transition/transversion ratio was 3.35. Genetic distances within species, genus, and families were 0.0041, 0.1136, and 0.1268, respectively. The ratio of genetic distances between interspecies and intraspecies was 27.7, which indicated a notable genetic difference of the COI gene sequence. The efficiency of DNA barcoding based on the COI gene was reaffirmed from its successful identification of all 19 Bagridae catfishes in this study. This classification result is consistent with their morphologies. However, only Hemibagrus catfishes can be clustered into a single phylogenetic tree branch. Other Bagridae species were intertwined, and their phylogenetic status needs further research combined with data from more samples. The results revealed that the COI gene was suitable as a DNA barcoding parameter for identifying species; it provided a useful reference for the phylogenetic relationships of Bagridae.
引文
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[26]Mao Y T,Gan X N,Wang X Z.DNA barcodes and molecular phylogeny of botiinae fishes based on the mitochondrial COI gene[J].Acta Hydrobilolgica Sinica,2014,38(4):737-744.[毛云涛,甘小妮,王绪祯.基于线粒体COI基因的沙鳅亚科鱼类DNA条形码及其分子系统发育研究[J].水生生物学报.2014,38(4):737-744.]
[27]Yu Y N,Song C,Hou J L,et al.System classification for Gobiidae derived from the Yangtze River based on mitochondrial COI gene partial sequence[J].Freshwater Fisheries,2014,44(5):3-8.[于亚男,宋超,侯俊利,等.基于线粒体COI基因部分序列的长江口虾虎鱼科鱼类系统分类[J].淡水渔业,2014,44(5):3-8.]
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[29]Meng W,Yang T Y,Hai S,et a1.Study of DNA barcoding based on the mitochondrial COI gene sequences in Salmotrutta fraio[J].Chinese Journal of Fisheries,2010,23(1):6-10.[孟玮,杨天燕,海萨,等.基于线粒体COI基因序列的亚东鲑DNA条形码研究[J].水产学杂志,2010,23(1):6-10.]
[30]Chen Z X,Guo S L,Gong Y Q.Progress on application of fish DNA barcoding:a review[J].Fisheries Science,2017,36(6):834-842.[陈忠信,郭书林,龚艳青.鱼类DNA条形码技术的应用进展[J].水产科学,2017,36(6):834-842.]
[31]Zhang Y G,Wang D S.Studies on the taxonomical position of Pseudobagrus brevicaudatus(Bagdidae,Siluriformes)[J].Acta Hydrobiologica Sinica,1996,20(4):379-382.[张耀光,王德寿.短尾拟鲿分类地位的探讨[J].水生生物学报,1996,20(4):379-382.]
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[2]Editorial Commitee of Fauna Sinica of China.Fauna Sinica,Class Teleostei,Siluriformes[M].Beijing:Science Press,1999.[中国科学院中国动物志编辑委员会.中国动物志·硬骨鱼纲·鲇形目[M].北京:科学出版社,1999.]
[3]Yang J,Chen X Y,Yang J X.A new record of bagrid fish in Yunnan Pseudobagrus Kyphus Mai,1978[J].Zoological Research.2008,29(3):328-330.[杨剑,陈小勇,杨君兴.云南鲿科鱼类一新记录-越南拟鲿[J].动物学研究,2008,29(3):328-330.]
[4]Cheng J L,Ishihara H,Zhang E.Pseudobagrus brachyrhabdion,a new catfish(Teleostei:Bagridae)from the middle Yangtze River drainage,South China[J].Ichthyological Research,2008,55:112-123.
[5]Yu C F,Li D C,Li Z Q,et al.Studies on relationship of four bagridae catfishes by discontinous polyacrylamide gel electrophoresis[J].Journal of Sichuan Vocational and Technical College,2003,13(1):86-88.[俞纯方,刘大翠,刘中全,等.鲿科四种鱼LDH同工酶谱的研究[J].四川职业技术学院学报,2003,13(1):86-88.]
[6]Cheng J L,Zhang E.A taxonomic research situation of the bagrid catfish genus pseudobagrus[J].Journal of Jinggangshan University(Natural Science),2012,33(2):94-98.[程建丽,张鹗.拟鲿属鱼类分类学研究概况[J].井冈山大学学报(自然科学版),2012,33(2):94-98.]
[7]Ward R D,Hanner R,Hebert P D N.The campaign to DNA barcode all fishes,FISH-BOL[J].Journal of Fish Biology,2009,74:329-356.
[8]Ratnasingham S,Hebert P D N.BOLD:The barcode of life data system(www.barcodinglife.org)[J].Molecular Ecology Notes,2007,7:355-364.
[9]Pollack S J,Kawalek M D,Williams-Hill D M,et al.Evaluation of DNA barcoding methodologies for the identification of fish species in cooked products[J].Food Control,2018,84:297-304.
[10]Barco A,Raupach M J,Laakmann S,et al.Identification of north sea molluscs with DNA barcoding[J].Molecular Ecology Resources,2016,16:288-297.
[11]Cheng J,Sha Z L,Liu R Y.DNA barcoding of genus Metapenaeopsis(Decapoda:Penaeidae)and molecular phylogeny inferred from mitochondrial and nuclear DNA sequences[J].Biochemical Systematics and Ecology,2015,61:376-384.
[12]Hellberg R S,Kawalek M D,Van K T,et.al.Comparison of DNA extraction and PCR setup methods for use in highthroughput DNA barcoding of fish species[J].Food Analytical Methods,2014,7:1950-1959.
[13]Mueller S,Handy S M,Deeds J R,et al.Development of a COX1 based PCR-RFLP method for fish species identification[J].Food Control,2015,55:39-42.
[14]Shen Y J,Guan L H,Wang D Q,et al.DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River[J].Ecology Evolution,2016,6(9):2702-2713.
[15]Li X R,Liu S F,Li D,et al.Species identification and phylogenetic relationships in order Clupeiformes based on DNA barcoding[J].Journal of Fishery Sciences of China,2015,22(6):1133-1141.[李献儒,柳淑芳,李达,等.DNA条形码在鲱形目鱼类物种鉴定和系统进化分析中的应用[J].中国水产科学,2015,22(6):1133-1141.]
[16]Gao C X.DNA barcoding of Tetraodontiformes in coastal waters of China[D].Guangzhou:Jinan University,2015.[郜星晨.基于线粒体COI序列的中国沿海鲀形目鱼类DNA条形码研究[D].广州:暨南大学,2015.]
[17]Liu L,Sun D R,Li C H,et al.Application of DNA barcoding in classification of mugilidae fishes[J].Periodical of Ocean University of China,2016,46(11):178-186.[刘璐,孙典荣,李纯厚,等.DNA条形码技术在鲻科鱼类鉴定中的应用[J].中国海洋大学学报,2016,46(11):178-186.]
[18]Song C,Yu Y N,Zhang T,et al.Phylogenetic analysis of Cynoglossidae in the Yangtze Estuary based on partial sequence of mitochondrial COI[J].Chinese Journal of Zoology,2014,49(5):716-726.[宋超,于亚男,张涛,等.基于线粒体CO I基因部分序列的长江口舌鳎科鱼类系统分类研究[J].动物学杂志,2014,49(5):716-726.]
[19]Peng Z G,He S P,Zhang Y G.Cytochrome b gene variation and phylogeny of Asian Bagrid catfishes[J].Progress in Natural Science,2002,12(6):596-600.[彭作刚,何舜平,张耀光.细胞色素b基因序列变异与东亚鲿科鱼类系统发育[J].自然科学进展,2002,12(6):596-600.]
[20]Zhang Y,Zhang E,He S P.Studies on the structure of the control region of the bagridae in China and its phylogentic significance[J].Acta Hydrobiologica Sinica,2003,27(5):463-467.[张燕,张鹗,何舜平.中国鲿科鱼类线粒体DNA控制区结构及其系统发育分析[J].水生生物学报,2003,27(5):463-467.]
[21]Cheng Q T,Zheng B S.Systematic Synopsis of Chinese Fishes[M].Beijing:Science Press,1987.[成庆泰,郑葆珊.中国鱼类系统检索[M].北京:科学出版社,1987.]
[22]Meng Q W,Su J W,Miao X Z.Systematics of Fishes[M].Beijing:China Agriculture Press,1995:153-172.[孟庆闻,苏锦祥,缪学祖.鱼类分类学[M].北京:中国农业出版社,1995:153-172.]
[23]Liang H W.The complete mitochondrial genomes of six catfishes(Siluriformes)and the phylogenetic relationships analysis[D].Yangling:Northwest A&F University,2012.[梁宏伟.六种鲇形目鱼类线粒体基因组克隆及其系统发育研究[D].杨凌:西北农林科技大学,2012.]
[24]Duan X K.Study on complete mitochondrial genome of parabramis pekinesis strenosoma and codon usage patterns of Megalabrama amblycephala[D].Wuhan:Huazhong Agriculture University,2015.[段晓克.壮体长春鳊线粒体基因组全长测定及团头鲂密码子偏好模型分析[D].武汉:华中农业大学,2012.]
[25]Peng J L,Wang X Z,Wang D,et al.Application of DNA barcoding based on the mitochondrial COI gene sequences in classification of Culter(Pisces:Cyprinidae)[J].Acta Hydrobilolgica Sinica,2009,33(2):271-276.[彭居俐,王绪祯,王丁,等.基于线粒体COI基因序列的DNA条形码在鲤科鲌属鱼类物种鉴定中的应用[J].水生生物学报,2009,33(2):271-276.]
[26]Mao Y T,Gan X N,Wang X Z.DNA barcodes and molecular phylogeny of botiinae fishes based on the mitochondrial COI gene[J].Acta Hydrobilolgica Sinica,2014,38(4):737-744.[毛云涛,甘小妮,王绪祯.基于线粒体COI基因的沙鳅亚科鱼类DNA条形码及其分子系统发育研究[J].水生生物学报.2014,38(4):737-744.]
[27]Yu Y N,Song C,Hou J L,et al.System classification for Gobiidae derived from the Yangtze River based on mitochondrial COI gene partial sequence[J].Freshwater Fisheries,2014,44(5):3-8.[于亚男,宋超,侯俊利,等.基于线粒体COI基因部分序列的长江口虾虎鱼科鱼类系统分类[J].淡水渔业,2014,44(5):3-8.]
[28]Huang Y.Molecular Phylogenetics:Principle,Method and Application[M].Beijing:China Agriculture Press,1998.[黄原.分子系统学:原理、方法及应用[M].北京:中国农业出版社,1998.]
[29]Meng W,Yang T Y,Hai S,et a1.Study of DNA barcoding based on the mitochondrial COI gene sequences in Salmotrutta fraio[J].Chinese Journal of Fisheries,2010,23(1):6-10.[孟玮,杨天燕,海萨,等.基于线粒体COI基因序列的亚东鲑DNA条形码研究[J].水产学杂志,2010,23(1):6-10.]
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