不同DNA条形码基因在帘蛤目贝类分类鉴定中的比较分析
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摘要
DNA条形码基因已经广泛应用在海洋贝类的分类鉴定、系统发育进化、种群遗传分析等领域的研究。为进一步研究评估不同DNA条形码基因在海洋贝类鉴定中的作用,本研究利用从Gen Bank数据库随机下载的帘蛤目COI、16S r RNA、18S r RNA和28S r RNA基因序列,通过传统距离法和单系聚类法结合分析,比较了上述DNA条形码基因在鉴定物种及系统发育进化中的鉴定效率,并以本实验室已获得的部分贝类DNA序列进行了验证。结果表明,根据"10倍法则"和"2%"阈值标准,本研究中COI能够鉴定57.1%物种,16S r RNA能够鉴定60.9%,18S r RNA鉴定16.7%,而28S r RNA无法有效鉴定;多数种COI和16S r RNA基因序列的种间遗传距离和种内遗传距离存在"条形码间隙",而18S r RNA和28S r RNA序列的种间和种内的遗传距离存在显著重叠,没有明显"条形码间隙";聚类分析结果表明,基于COI基因序列,87.9%的个体与同种聚为单系,以16S r RNA序列,65.6%的个体与同种聚为单系,未聚成单系的个体则形成姐妹系,未出现不同种聚为单系现象,能够呈现与形态分类基本一致的系统发生关系;但18S r RNA和28S r RNA呈现的聚类关系相对混乱。相对而言,在鉴定帘蛤目物种时,COI和16S r RNA都能够作为条形码基因,且COI有效性更高,18S r RNA和28S r RNA基因由于种内变异较大,不适于作为条码基因。研究结果为科学选用DNA条形码基因进行帘蛤目贝类的鉴定提供了参考资料。
DNA barcoding has been widely used in the fields of taxonomy, identification, phylogenetic evolution, and population genetic analysis of marine shellfish. To further evaluate the identification validity of different DNA barcoding genes in marine shellfish, sequences of COI, 16 S rRNA, 18 S rRNA, and 28 S rRNA from Veneroida were randomly downloaded from the Gen Bank database, analyzed by the distance-and tree-based methods, and taxonomic relation established by the tree-based method, and compared the results with some sequences from our laboratory. According to the "10 × rule" and "2%" standard criterion, 57.1% of species could be distinguished using the COI gene, whereas 16 S rRNA identified 60.9%, 18 S rRNA identified 16.7%, and 28 S rRNA did not identify any species. We also found that there were significant barcode gaps between the genetic distances of pairwise-and within-species in most genera based on COI and 16 S rRNA genes; however, there were significant overlaps, instead of barcode gaps, based on 18 S rRNA and 28 S rRNA. The cluster analysis showed that 87.9% of individuals clustered to a monophyletic group, with other within-species individuals clustered on the COI; and 65.6% 16 S rRNA individuals clustered in monophyletic groups. Furthermore individuals without monophyletic groups clustered into sister groups, which revealed that the evolutionary relationships showed by the NJ tree constructed via COI and 16 S rRNA mainly agreed with that from the morphological classification, especially that of the COI gene. In contrast, the NJ tree constructed via 18 S rRNA and 28 S rRNA genes showed disordered clustering relationships, including some individuals from different species cluster to monophyletic groups. In the present study, the results clearly suggested that both COI and 16 S rRNA can be used as DNA barcodes in identifying species in Veneroida, particular COI, but 18 S rRNA and 28 S rRNA are not suitable because of the large intraspecific variation. This study provides new data for DNA barcode selection in Veneroida.
DNA barcoding has been widely used in the fields of taxonomy, identification, phylogenetic evolution, and population genetic analysis of marine shellfish. To further evaluate the identification validity of different DNA barcoding genes in marine shellfish, sequences of COI, 16 S rRNA, 18 S rRNA, and 28 S rRNA from Veneroida were randomly downloaded from the Gen Bank database, analyzed by the distance-and tree-based methods, and taxonomic relation established by the tree-based method, and compared the results with some sequences from our laboratory. According to the "10 × rule" and "2%" standard criterion, 57.1% of species could be distinguished using the COI gene, whereas 16 S rRNA identified 60.9%, 18 S rRNA identified 16.7%, and 28 S rRNA did not identify any species. We also found that there were significant barcode gaps between the genetic distances of pairwise-and within-species in most genera based on COI and 16 S rRNA genes; however, there were significant overlaps, instead of barcode gaps, based on 18 S rRNA and 28 S rRNA. The cluster analysis showed that 87.9% of individuals clustered to a monophyletic group, with other within-species individuals clustered on the COI; and 65.6% 16 S rRNA individuals clustered in monophyletic groups. Furthermore individuals without monophyletic groups clustered into sister groups, which revealed that the evolutionary relationships showed by the NJ tree constructed via COI and 16 S rRNA mainly agreed with that from the morphological classification, especially that of the COI gene. In contrast, the NJ tree constructed via 18 S rRNA and 28 S rRNA genes showed disordered clustering relationships, including some individuals from different species cluster to monophyletic groups. In the present study, the results clearly suggested that both COI and 16 S rRNA can be used as DNA barcodes in identifying species in Veneroida, particular COI, but 18 S rRNA and 28 S rRNA are not suitable because of the large intraspecific variation. This study provides new data for DNA barcode selection in Veneroida.
引文
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[19]Chen A H,Li Z X,Feng G N.Phylogenetic relationships of the genus meretrix(Mollusca:Veneridae)based on mitochondrial COI gene sequences[J].Zoological Research,2009,30(3):233-239.
[20]Yang G,Yu J H,Xu P,et al.Phylogenetic relationship among 8 common species of catfish based on ribosome 18S and ITS sequences[J].Chinese Journal of Zoology,2010,45(4):110-117.[杨光,俞菊华,徐跑,等.利用18S和ITS序列揭示8种鲇形目鱼类的系统发育[J].动物学杂志,2010,45(4):110-117.]
[21]Luo J,Liu G Y,Tian Z C,et al.Taxonomic status of equine piroplasmid assays based on 18S r RNA gene sequencing in China[J].Zoological Systematics,2011,36(1):99-103.[罗金,刘光远,田占成,等.基于18S r RNA基因序列的我国马梨形虫分类学地位分析[J].动物分类学报,2011,6(1):99-103.]
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[23]Avise J C.Phylogeography:The History and Formation of Species[M].Cambridge:Harvard University Press,2000.
[2]Li Q,Zou S M,Zheng X D,et al.DNA barcoding and its application in marine organisms[J].Periodical of Ocean University of China,2010,40(8):43-47.[李琪,邹山梅,郑小东,等.DNA条形码及其在海洋生物中的应用[J].中国海洋大学学报(自然科学版),2010,40(8):43-47.]
[3]Liu J.DNA taxonomy in marine bivalves:DNA barcoding in Mytilidae and Ostreidae,and cryptic diversity in the pen shell Atrina pectinata[D].Qingdao:Ocean University of China,2012.[刘君.双壳贝类DNA分类:贻贝科和牡蛎科DNA条形码及栉江珧隐存种研究[D].青岛:中国海洋大学,2012.]
[4]Chen J X,Chang Y Q.Practical Manual of Double Shell Shellfish Seedling[M].Food and Agriculture Organization of the United Nations,2006.[陈家鑫,常亚青.双壳贝类育苗实用手册(译)[M],联合国粮食及农业组织,2006.]
[5]Ebach M C,Holdrege C.DNA barcoding is no substitute for taxonomy[J].Nature,2005,434(7034):697.
[6]Marshall E.Will DNA bar codes breathe life into classification?[J].Science,2005,307(5712):1037.
[7]Kim D W,Yoo W G,Park H C,et al.DNA barcoding of fish,insects,and shellfish in Korea[J].Genomics&Informatics,2012,10(3):206-211.
[8]Zhang J,Hanner R.Molecular approach to the identification of fish in the South China Sea[J].PLo S ONE,2012,7(2):e30621.
[9]Kundu S,Laskar B A,Venkataraman K,et al.DNA barcoding of Nilssonia congeners corroborates existence of wild N.nigricans in northeast India[J].Mitochondrial DNA,2016,27(4):2753-2756.
[10]Zhao T,Wu Q,Pan B P.Molecular phylogeny of Veneridae(Mollusca,Bivalvia)based on 16S r RNA sequences[J].Oceanologia et Limnologia Sinica,2013,44(6):1500-1505.[赵婷,吴琪,潘宝平.我国重要帘蛤科(Veneridae)贝类的16S r RNA序列系统学分析[J].海洋与湖沼,2013,44(6):1500-1505.]
[11]Cui C J,Zhang L N,Wang N,et al.Progress of DNA barcoding in Algae[J].China Biotechnology,2012,32(9):113-117.[崔翠菊,张立楠,王娜,等.藻类DNA条形码研究进展[J].中国生物工程杂志,2012,32(9):113-117.]
[12]Feng Y W.DNA barcoding and molecular phylogeny of the Pteriina and Arcoida[D].Qingdao:Ocean University of China,2011.[冯艳微.珍珠贝亚目和蚶目DNA条形码与系统发生学研究[D].青岛:中国海洋大学,2012.]
[13]Moniz M B,Kaczmarska I.Barcoding diatoms:Is there a good marker?[J].Molecular Ecology Resources,2009,9(s1):65-74.
[14]Mao Y T,Gan X N,Wang X Z.DNA barcodes and molecular phylogeny of Botiinae fishes based on the mitochondrial CO I gene[J].Acta Hydrobiologica Sinia,2014,38(4):737-744.[毛云涛,甘小妮,王绪祯.基于线粒体CO I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.]
[15]Wu B,Yang A G,Dong Y H,et al.MSAP analysis on genome-wide DNA methylation in selected and wild Japanese scallop Patinopecten yessoensis[J].Oceanologia et Limnologia Sinica,2015,46(4):976-982.[吴彪,杨爱国,董迎辉,等.虾夷扇贝选育群体与野生群体基因组DNA甲基化的MSAP分析[J].海洋与湖沼,2015,46(4):976-982.]
[16]Hebert P D N,Stoeckle M Y,Zemlak T S,et al.Identification of Birds through DNA Barcodes[J].PLo S Biology,2004,2(10):e312.
[17]Hebert P D,Cywinska A,Ball S L,et al.Biological identifications through DNA barcodes[J].Proceedings of the Royal Society B:Biological Sciences,2003,270(1512):313-321.
[18]Feng J B,Sun Y N,Cheng X,et al.Sequence analysis of mitochondrial COI gene of Macrobrachiumni pponense from the five largest freshwater lakes in China[J].Journal of Fisheries of China,2008,32(4):517-525.[冯建彬,孙悦娜,程熙,等.我国五大淡水湖日本沼虾线粒体COI基因部分片段序列比较[J].水产学报,2008,32(4):517-525.]
[19]Chen A H,Li Z X,Feng G N.Phylogenetic relationships of the genus meretrix(Mollusca:Veneridae)based on mitochondrial COI gene sequences[J].Zoological Research,2009,30(3):233-239.
[20]Yang G,Yu J H,Xu P,et al.Phylogenetic relationship among 8 common species of catfish based on ribosome 18S and ITS sequences[J].Chinese Journal of Zoology,2010,45(4):110-117.[杨光,俞菊华,徐跑,等.利用18S和ITS序列揭示8种鲇形目鱼类的系统发育[J].动物学杂志,2010,45(4):110-117.]
[21]Luo J,Liu G Y,Tian Z C,et al.Taxonomic status of equine piroplasmid assays based on 18S r RNA gene sequencing in China[J].Zoological Systematics,2011,36(1):99-103.[罗金,刘光远,田占成,等.基于18S r RNA基因序列的我国马梨形虫分类学地位分析[J].动物分类学报,2011,6(1):99-103.]
[22]Yu Z Z.DNA barcoding and molecular phylogeny of Heterodonta[D].Qingdao:Ocean University of China,2014.[于贞贞.异齿亚纲贝类DNA条形码与系统发生学研究[D].青岛:中国海洋大学,2014.]
[23]Avise J C.Phylogeography:The History and Formation of Species[M].Cambridge:Harvard University Press,2000.