DNA条形码快速鉴定广州常见菊花蚜虫
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摘要
为了探讨DNA条形码技术在花卉害虫防治时用于蚜虫物种的快速、准确鉴定的可行性,本研究利用条形码通用引物扩增了危害广州市常见菊花品种的2种蚜虫,桃蚜Myzus persicae(Sulzer)和棉蚜Aphis gossypii Glover的DNA条形码。通过分析获取的2种43个样本的线粒体细胞色素C氧化酶亚基I(mitochondrial cytochrome c oxidase subunit I,COI)基因658 bp序列后发现,碱基平均含量为39.8%T,14.3%C,35.3%A,10.6%G,其中A+T含量为75.1%,C+G含量为24.9%,存在明显的A、T碱基偏好性,符合昆虫线粒体基因碱基组成的基本特征;有保守位点551个,可变位点62个,简约位点56个,单个突变位点6个。序列饱和性检验分析结果显示该序列转换与颠换未达到饱和,因此可以进行基于Kimura双参数模型的种内种间遗传距离分析。所有样品的种内平均差异为0.08%(0.00%~1.20%±0.18%),种间平均序列差异为9.69%(9.60%~10.30%±0.13%)。同时,邻接法(neighbor-joining,NJ)构建的系统发育树显示采自不同地区不同菊花品种上的桃蚜和棉蚜分别聚为一支。因此,本研究证明应用基于COI基因片段的DNA条形码进行花卉常见蚜虫类害虫的快速鉴定具有可行性。
In order to identify the aphids rapidly and accurately in flower pest control. DNA barcoding based on COI( mitochondrial cytochrome c oxidase subunit I) genes were used to analyze aphids on Chrysanthemum plants in Guangzhou. The 658 bp COI sequences of 43 samples belonging to 2 species, Myzus persicae( Sulzer) and Aphis gossypii Glover, in Guangzhou were amplified using universal barcoding primers. Among all sites,the average proportions of base were 39. 8% T,14. 3% C,35. 3% A,and 10. 6% G respectively. It showed an obvious preference to A + T in the sequence,that A + T base pair accounts( 75. 1%) was significantly higher than that of G + C base pair( 24. 9%). The percentage of A and T was conforming to the basic feature of base composition in insect mitochondria. The results of sequence analysis revealed that there are 551 conserved sites,62 mutation sites,56 parsimony- informative sites,and 6 singletons in this gene. The results of transition and transversion showed that the sequence did not reach the saturation and the evolution analysis can be achieved accurately. Model Kimura 2- parameter was used to analyze the genetic distance,and the results showed that the average intraspecies sequence divergences is 9. 69%( 9. 60% ~ 10. 30% ± 0. 13%),while the average interspecies divergences is 0. 08%( 0. 00% ~ 1. 20% ± 0. 18%),confirming that the genetic distance can be used to discriminate different aphid species. Neighbor- joining phylogenetic tree was built based on the COI gene sequences,and the results indicated that the same species was gathered in the same branch. It suggested that DNA barcoding based on mitochondrial COI gene sequences is applicable in the classification and identification of common Chrysanthemum aphid species in Guangzhou City.
In order to identify the aphids rapidly and accurately in flower pest control. DNA barcoding based on COI( mitochondrial cytochrome c oxidase subunit I) genes were used to analyze aphids on Chrysanthemum plants in Guangzhou. The 658 bp COI sequences of 43 samples belonging to 2 species, Myzus persicae( Sulzer) and Aphis gossypii Glover, in Guangzhou were amplified using universal barcoding primers. Among all sites,the average proportions of base were 39. 8% T,14. 3% C,35. 3% A,and 10. 6% G respectively. It showed an obvious preference to A + T in the sequence,that A + T base pair accounts( 75. 1%) was significantly higher than that of G + C base pair( 24. 9%). The percentage of A and T was conforming to the basic feature of base composition in insect mitochondria. The results of sequence analysis revealed that there are 551 conserved sites,62 mutation sites,56 parsimony- informative sites,and 6 singletons in this gene. The results of transition and transversion showed that the sequence did not reach the saturation and the evolution analysis can be achieved accurately. Model Kimura 2- parameter was used to analyze the genetic distance,and the results showed that the average intraspecies sequence divergences is 9. 69%( 9. 60% ~ 10. 30% ± 0. 13%),while the average interspecies divergences is 0. 08%( 0. 00% ~ 1. 20% ± 0. 18%),confirming that the genetic distance can be used to discriminate different aphid species. Neighbor- joining phylogenetic tree was built based on the COI gene sequences,and the results indicated that the same species was gathered in the same branch. It suggested that DNA barcoding based on mitochondrial COI gene sequences is applicable in the classification and identification of common Chrysanthemum aphid species in Guangzhou City.
引文
Chang H,Hao DJ,Xiao RT,Liu Y,Qian L,An YL,Yang XJ,2012.DNA barcoding based on the mitochondrial COI gene sequences for Ips species(Coleoptera:Scolytidae).Acta Entomologica Sinica,55 (9):1075-1081.[常虹,郝德君,肖荣堂,刘勇,钱路,安榆林,杨晓军,2012.基于线粒体COI基因的齿小蠹属昆虫DNA条形码研究.昆虫学报,55(9):1075-1081]
Chen R,Jiang LY,Qiao GX,2013.DNA barcoding in rapid identification of aphids on Pinus armandii.Chinese Journal of Applied Entomology,50(1):50-60.[陈睿,姜立云,乔格侠,2013.华山松不同生态位上蚜虫的快速鉴定———基于DNA条形码.应用昆虫学报,50(1):50-60]
Ellis PR,Pink DAC,Phelps K,Jukes PL,Breeds SE,Pinnegar AE,1998.Evaluation of a core collection of Brassica oleracea accessions for resistance to Brevicoryne brassicae,the cabbage aphid.Euphytica,103(2):149-160.
Floyd RM,Wilson JJ,Hebert PDN,2009.DNA barcodes and insect biodiversity.In:Foottit RG,Adler PH,eds.Insect Biodiversity:Science and Society.Oxford:Wiley-Blackwell.417-432.
Foottit RG,Maw HEL,von Dohlen CD,Hebert PDN,2008.Species identification of aphids(Insecta:Hemiptera:Aphididae)through DNA barcodes.Mol.Ecol.Res.,8:1189-1201.
Hebert PDN,Cywinska A,Ball SL,de Waard JR,2003.Biological identifications through DNA barcodes.Proc.R.Soc.Lond.B,270:313-321.
Lee W,Kim H,Lim J,Choi HR,Kim Y,Kim YS,Ji JY,Foottit RG,Lee S,2011.Barcoding aphids(Hemiptera:Aphididae)of the Korean Peninsula:updating the global data set.Mol.Ecol.Resour,11:32-37.
Liu H,Beckenbach AT,1992.Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects.Molecular Phylogenetics and Evolution,1:41-52.
Liu SS,Zhang GF,Wu Q,Zhang AB,Wang JJ,Wan FH,2012.Establishment and application of DNA barcoding technology for identifi-cation of the immature and adult debris of Bactrocera dorsalis(Hendel)(Diptera:Tephritidae).Acta Entomologica Sinica,55(3):336-343.[刘慎思,张桂芬,武强,张爱兵,王进军,万方浩,2012.桔小实蝇幼体及成虫残体DNA条形码识别技术的建立与应用.昆虫学报,55(3):336-343]
Li HJ,1981.Chysanthemums in China.Nanjing:Jiangsu Science and Technology Press.59-63.
Pan CY,Hu J,Zhang X,Huang Y,2006.The DNA barcoding application of mtDNA COI genes in seven species of Catantopidae(Orthoptera).Entomotaxonomia,28(2):103-110.[潘程莹,胡婧,张霞,黄原.斑腿蝗科Catantopidae七种蝗虫线粒体COI基因的DNA条形码研究.昆虫分类学报,28(2):103-110]
Qiao WN,Wan FH,Zhang AB,Min L,Zhang GF,2012.Application of DNA barcoding technology for species identification of common thrips(Insecta:Thysanoptera)in China.Acta Entomologica Sinica,55(3):344-356.[乔玮娜,万方浩,张爱兵,闵亮,张桂芬,2012.DNA条形码技术在田间常见蓟马种类识别中的应用.昆虫学报,55(3):344-356]
Remaudière G,Remaudière M,1997.Catalogue of the World's Aphididae.Paris:Homoptera Aphidoidea.Institut National de la Recherche Agronomique.1-473.
Sauge MH,Lacroze JP,Poёssel JL,Pascal T,Kervella J,2002.Induced resistance by Myzus persicae in the peach cultivar‘Rubira'.Entomologia Experimentalis et Applicata,102(1):29-37.
Simon C,Frati F,Beckenbach A,Crespi B,Liu H,Flook P,1994.Evolution,weighting,and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers.Ann.Ent.Soc.Am.,87:651-701.
Tamura K,Nei M,1993.Estimation of the number nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.Mol.Biol.Evol.,10:512-526.
Tamura K,Dudley J,Nei M,Kumar S,2007.MEGA4:Molecular Evolutionary Genetics Analysis(MEGA)software version 4.0.Mol.Biol.Evol.,24:1596-1599.
Von Dohlen CD,Moran NA,2000.Molecular data support a rapid radiation of aphids in the Cretaceous and multiple origins of host alternation.Biol.J.Linn.Soc.,71:689-717.
Wang J,Hu HL,Zhang CL,Hu YB,Liu MF,2002.Integrated control of main diseases and insects of Dendranthema morifolium.Chinese Journal of Applied Ecology,13(4):444-448.
Wang JF,Jiang LY,Qiao GX,2011.Use of a mitochondrial COI sequence to identify species of the subtribe Aphidina(Hemiptera,Aphididae).ZooKeys,122:1-17.
Wen J,Chen R,Jiang LY,Qiao GX,2013.Rapid identification of aphids on flowering plants of Rosaceae in Beijing based on DNA barcoding.Chinese Journal of Applied Entomology,50(1):29-40 .[温娟,陈睿,姜立云,乔格侠,2013.基于DNA条形码对北京地区蔷薇科花卉上蚜虫的快速鉴定.应用昆虫学报,50(1):29-40]
Xia X,Xie Z,2001.DAMBE:Data analysis in molecular biology and evolution.J.Hered.,92:371-373.
Yang CH,Han HL,Chi MY,Jin Q,Wu CS,Zhu CD,Zhang AB,2012.Species identification of Noctuidae moths(Insecta:Lepidoptera)from Baihuashan,Beijing,China with DNA barcoding.Acta Entomologica Sinica,55(9):1082-1092.[杨聪慧,韩辉林,迟美妍,金倩,武春生,朱朝东,张爱兵,2012.DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用.昆虫学报,55(9):1082-1092]
Zhang GX,Zhong TS,1983.Economic Insect Fauna of China Fasc.25 .Homoptera:Aphidinea.Beijing:Science Press.1-387.[张广学,钟铁森,1983.中国经济昆虫志,第25册,同翅目,蚜虫类(一).北京:科学出版社.1-387]
Zhang HC,Qiao GX,2007a.Molecular phylogeny of Fordini(Hemiptera:Aphididae:Pemphiginae)inferred from nuclear gene EF-1αand mitochondrial gene COI.Bull.Entomol.Res.,97:379-386.
Zhang HC,Qiao GX,2007b.Systematic status of genus Formosaphis Takahashi and evolution of galls based on the molecular phylogeny of Pemphigini(Hemiptera:Aphididae:Eriosomatinae).Syst.Entomol.,32:690-699.
Chen R,Jiang LY,Qiao GX,2013.DNA barcoding in rapid identification of aphids on Pinus armandii.Chinese Journal of Applied Entomology,50(1):50-60.[陈睿,姜立云,乔格侠,2013.华山松不同生态位上蚜虫的快速鉴定———基于DNA条形码.应用昆虫学报,50(1):50-60]
Ellis PR,Pink DAC,Phelps K,Jukes PL,Breeds SE,Pinnegar AE,1998.Evaluation of a core collection of Brassica oleracea accessions for resistance to Brevicoryne brassicae,the cabbage aphid.Euphytica,103(2):149-160.
Floyd RM,Wilson JJ,Hebert PDN,2009.DNA barcodes and insect biodiversity.In:Foottit RG,Adler PH,eds.Insect Biodiversity:Science and Society.Oxford:Wiley-Blackwell.417-432.
Foottit RG,Maw HEL,von Dohlen CD,Hebert PDN,2008.Species identification of aphids(Insecta:Hemiptera:Aphididae)through DNA barcodes.Mol.Ecol.Res.,8:1189-1201.
Hebert PDN,Cywinska A,Ball SL,de Waard JR,2003.Biological identifications through DNA barcodes.Proc.R.Soc.Lond.B,270:313-321.
Lee W,Kim H,Lim J,Choi HR,Kim Y,Kim YS,Ji JY,Foottit RG,Lee S,2011.Barcoding aphids(Hemiptera:Aphididae)of the Korean Peninsula:updating the global data set.Mol.Ecol.Resour,11:32-37.
Liu H,Beckenbach AT,1992.Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects.Molecular Phylogenetics and Evolution,1:41-52.
Liu SS,Zhang GF,Wu Q,Zhang AB,Wang JJ,Wan FH,2012.Establishment and application of DNA barcoding technology for identifi-cation of the immature and adult debris of Bactrocera dorsalis(Hendel)(Diptera:Tephritidae).Acta Entomologica Sinica,55(3):336-343.[刘慎思,张桂芬,武强,张爱兵,王进军,万方浩,2012.桔小实蝇幼体及成虫残体DNA条形码识别技术的建立与应用.昆虫学报,55(3):336-343]
Li HJ,1981.Chysanthemums in China.Nanjing:Jiangsu Science and Technology Press.59-63.
Pan CY,Hu J,Zhang X,Huang Y,2006.The DNA barcoding application of mtDNA COI genes in seven species of Catantopidae(Orthoptera).Entomotaxonomia,28(2):103-110.[潘程莹,胡婧,张霞,黄原.斑腿蝗科Catantopidae七种蝗虫线粒体COI基因的DNA条形码研究.昆虫分类学报,28(2):103-110]
Qiao WN,Wan FH,Zhang AB,Min L,Zhang GF,2012.Application of DNA barcoding technology for species identification of common thrips(Insecta:Thysanoptera)in China.Acta Entomologica Sinica,55(3):344-356.[乔玮娜,万方浩,张爱兵,闵亮,张桂芬,2012.DNA条形码技术在田间常见蓟马种类识别中的应用.昆虫学报,55(3):344-356]
Remaudière G,Remaudière M,1997.Catalogue of the World's Aphididae.Paris:Homoptera Aphidoidea.Institut National de la Recherche Agronomique.1-473.
Sauge MH,Lacroze JP,Poёssel JL,Pascal T,Kervella J,2002.Induced resistance by Myzus persicae in the peach cultivar‘Rubira'.Entomologia Experimentalis et Applicata,102(1):29-37.
Simon C,Frati F,Beckenbach A,Crespi B,Liu H,Flook P,1994.Evolution,weighting,and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers.Ann.Ent.Soc.Am.,87:651-701.
Tamura K,Nei M,1993.Estimation of the number nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.Mol.Biol.Evol.,10:512-526.
Tamura K,Dudley J,Nei M,Kumar S,2007.MEGA4:Molecular Evolutionary Genetics Analysis(MEGA)software version 4.0.Mol.Biol.Evol.,24:1596-1599.
Von Dohlen CD,Moran NA,2000.Molecular data support a rapid radiation of aphids in the Cretaceous and multiple origins of host alternation.Biol.J.Linn.Soc.,71:689-717.
Wang J,Hu HL,Zhang CL,Hu YB,Liu MF,2002.Integrated control of main diseases and insects of Dendranthema morifolium.Chinese Journal of Applied Ecology,13(4):444-448.
Wang JF,Jiang LY,Qiao GX,2011.Use of a mitochondrial COI sequence to identify species of the subtribe Aphidina(Hemiptera,Aphididae).ZooKeys,122:1-17.
Wen J,Chen R,Jiang LY,Qiao GX,2013.Rapid identification of aphids on flowering plants of Rosaceae in Beijing based on DNA barcoding.Chinese Journal of Applied Entomology,50(1):29-40 .[温娟,陈睿,姜立云,乔格侠,2013.基于DNA条形码对北京地区蔷薇科花卉上蚜虫的快速鉴定.应用昆虫学报,50(1):29-40]
Xia X,Xie Z,2001.DAMBE:Data analysis in molecular biology and evolution.J.Hered.,92:371-373.
Yang CH,Han HL,Chi MY,Jin Q,Wu CS,Zhu CD,Zhang AB,2012.Species identification of Noctuidae moths(Insecta:Lepidoptera)from Baihuashan,Beijing,China with DNA barcoding.Acta Entomologica Sinica,55(9):1082-1092.[杨聪慧,韩辉林,迟美妍,金倩,武春生,朱朝东,张爱兵,2012.DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用.昆虫学报,55(9):1082-1092]
Zhang GX,Zhong TS,1983.Economic Insect Fauna of China Fasc.25 .Homoptera:Aphidinea.Beijing:Science Press.1-387.[张广学,钟铁森,1983.中国经济昆虫志,第25册,同翅目,蚜虫类(一).北京:科学出版社.1-387]
Zhang HC,Qiao GX,2007a.Molecular phylogeny of Fordini(Hemiptera:Aphididae:Pemphiginae)inferred from nuclear gene EF-1αand mitochondrial gene COI.Bull.Entomol.Res.,97:379-386.
Zhang HC,Qiao GX,2007b.Systematic status of genus Formosaphis Takahashi and evolution of galls based on the molecular phylogeny of Pemphigini(Hemiptera:Aphididae:Eriosomatinae).Syst.Entomol.,32:690-699.