基于DNA条形码技术的湖南省柑橘主产区实蝇幼虫分子鉴定
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摘要
为明确湖南省柑橘主产区的实蝇入侵为害现状,从该省7个市(自治州)22个地点收集柑橘蛆果中的幼虫,利用DNA条形码技术对其进行分子鉴定,并以DNA条形码序列作为分子标记探究橘大实蝇Bactrocera minax的20个中国地理种群(湖南省6个组群共16个种群、其它3省市4个种群)以及1个印度地理种群间的亲缘地理关系,分析橘大实蝇在我国的遗传进化关系。结果表明,仅采集自邵阳市城步苗族自治县柑橘蛆果中的5头幼虫被鉴定为蜜柑大实蝇B. tsuneonis,其余21个地点采集的595头幼虫均被鉴定为橘大实蝇。21个橘大实蝇地理种群的平均单倍型多样性为0.75,核苷酸多样性为0.0032,核苷酸差异数为2.13,中国所有地理种群均具有较高的遗传多样性;单倍型网络进化图显示湖南、重庆、贵州种群共享的单倍型H3为原始单倍型,表明其为比较原始的种群;AMOVA分析结果显示种群内个体间遗传变异占总体变异的59.04%,是遗传变异的主要来源;遗传分化结果表明湖南省6个组群间均出现了中度至高度的遗传分化,FST在0.0521～0.7795之间。表明DNA条形码技术可用于蜜柑大实蝇和橘大实蝇幼虫的分子鉴定及其种群遗传进化分析。
To investigate the invasion and damage of fruit flies in major citrus main production areas in Hunan Province, the larvae from 22 sites of seven cities or autonomic prefectures in Hunan were collected and identified with DNA barcoding. The DNA barcoding sequences were used as molecular markers to explore the phylogeography of Bactrocera minax, including 20 geographical populations from China(16 geographical populations in six groups of Hunan Province and four geographical populations of other three provinces or cities) and one geographical population from India, and analyze the population genetic structure. The results showed that five individuals collected from Chengbu Miao Autonomous County of Shaoyang City were identified as B. tsuneonis, and 595 individuals collected from the other21 sites were identified as B. minax. The average haplotype diversity, nucleotide diversity and numbers of nucleotide differences of the 21 geographical populations were 0.75, 0.0032 and 2.13, respectively.All the populations from China had high genetic diversity. Haplotype network revealed that H3 shared by Hunan, Chongqing, Guizhou populations was the original haplotype, which implied that the above three provinces or city might share the same origin. AMOVA analysis showed that the variation within populations was the main source of variation, contributing 59.04% of total genetic variation. Genetic differentiation analysis indicted that the six groups in Hunan had moderate to high genetic differentiation, with the FSTvalues between 0.0521 and 0.7795. The results indicated that DNA barcodes could be an effective method for the molecular identification and population genetic structure analysis of B.minax and B. tsuneonis.
To investigate the invasion and damage of fruit flies in major citrus main production areas in Hunan Province, the larvae from 22 sites of seven cities or autonomic prefectures in Hunan were collected and identified with DNA barcoding. The DNA barcoding sequences were used as molecular markers to explore the phylogeography of Bactrocera minax, including 20 geographical populations from China(16 geographical populations in six groups of Hunan Province and four geographical populations of other three provinces or cities) and one geographical population from India, and analyze the population genetic structure. The results showed that five individuals collected from Chengbu Miao Autonomous County of Shaoyang City were identified as B. tsuneonis, and 595 individuals collected from the other21 sites were identified as B. minax. The average haplotype diversity, nucleotide diversity and numbers of nucleotide differences of the 21 geographical populations were 0.75, 0.0032 and 2.13, respectively.All the populations from China had high genetic diversity. Haplotype network revealed that H3 shared by Hunan, Chongqing, Guizhou populations was the original haplotype, which implied that the above three provinces or city might share the same origin. AMOVA analysis showed that the variation within populations was the main source of variation, contributing 59.04% of total genetic variation. Genetic differentiation analysis indicted that the six groups in Hunan had moderate to high genetic differentiation, with the FSTvalues between 0.0521 and 0.7795. The results indicated that DNA barcodes could be an effective method for the molecular identification and population genetic structure analysis of B.minax and B. tsuneonis.
引文
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Excoffier L,Lischer HE.2010.Arlequin suite ver 3.5:a new series of programs to perform population genetics analyses under Linux and Windows.Molecular Ecology Resources,10(3):564-567
Folmer O,Black M,Hoeh W,Lutz R,Vrijenhoek R.1994.DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.Molecular Marine Biology and Biotechnology,3(5):294-299
Gao LZ,Liu YH,Wan XW,Tu ZX,Pu P,Chen Y.2016.Genetic diversity analysis of Bactrocera minax(Diptera:Tephritidae)in China.Plant Protection,42(1):55-55,80(in Chinese)[高立志,刘映红,万宣伍,涂祖霞,蒲颇,陈媛.2016.中国柑橘大实蝇遗传多样性分析.植物保护,42(1):55-55,80]
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Hebert PDN,Ratnasingham S,deWaard JR.2003b.Barcoding animal life:cytochrome c oxidase subunit 1 divergences among closely related species.Proceedings of the Royal Society of London Series B:Biological Sciences,270(S1):96-99
Hou BH,Ouyang GC,Lu HL,Ma J,Lu YY,Xia YL.2018.First detection of Bactrocera tsuneonis(Diptera:Tephritidae)in Guangdong Province of China.Florida Entomologist,101(3):533-535
Jiang F.2015.Technique system for molecular identification of quarantine fruit flies in China.Ph.D Thesis.Beijing:China Agricultural University(in Chinese)[姜帆.2015.我国检疫性实蝇分子鉴定技术体系的研究.博士学位论文.北京:中国农业大学]
Krosch MN,Schutze MK,Armstrong KF,Boontop Y,Boykin LM,Chapman TA,Englezou A,Cameron SL,Clarke AR.2013.Piecing together an integrative taxonomic puzzle:microsatellite,wing shape and aedeagus length analyses of Bactrocera dorsalis s.l.(Diptera:Tephritidae)find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay Peninsula.Systematic Entomology,38(1):2-13
Krosch MN,Strutt F,Blacket MJ,Batovska J,Starkie M,Clarke AR,Cameron SL,Schutze MK.2018.Development of internal COIprimers to improve and extend barcoding of fruit flies(Diptera:Tephritidae:Dacini).Insect Science,doi:10.1111/1744-7917.12612
Kwong S,Srivathsan A,Meier R.2012.An update on DNA barcoding:low species coverage and numerous unidentified sequences.Cladistics,28(6):639-644
Leigh JW,Bryant D.2015.POPART:full-feature software for haplotype network construction.Methods in Ecology and Evolution,6(9):1110-1116
Li YL.2009.Population genetic relationship of Bactrocera dorsalis(Diptera:Tephritidae)inferred from mtDNA COI and ND6genes.Ph.D Thesis.Beijing:China Agricultural University(in Chinese)[李云龙.2009.基于线粒体COI和ND6基因的橘小实蝇种群遗传关系研究.博士学位论文.北京:中国农业大学]
Li ZH.2015.Prevention and control of biological invasions:potential geographical distribution of economic important fruit flies.Beijing:China Agricultural University Press(in Chinese)[李志红.2015.生物入侵防控:重要经济实蝇潜在地理分布研究.北京:中国农业大学出版社]
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Liang GQ,Liang F,Wu JJ,Shao QH,Wu SH,Chen QS.2003.Controlling principle and measures of Tephritidae.Guangdong Agricultural Sciences,(1):36-38(in Chinese)[梁广勤,梁帆,吴佳教,邵庆华,吴士豪,陈其生.2003.实蝇的防治原理及防治措施.广东农业科学,(1):36-38]
Liang GQ,Liang F,Zhao JP,Hu XN,Wu JJ.2008.Overview of the study on fruit flies quarantine in China.Journal of Environmental Entomology,30(4):361-369(in Chinese)[梁广勤,梁帆,赵菊鹏,胡学难,吴佳教.2008.中国实蝇检疫研究概况.环境昆虫学报,30(4):361-369]
Librado P,Rozas J.2009.DnaSP v5:a software for comprehensive analysis of DNA polymorphism data.Bioinformatics,25(11):1451-1452
Lin WL,Xiao FL,Yang SZ.2013.A preliminary study on Chinese citrus fly in Hunan Province.Hunan Agricultural Sciences,(15):84-86(in Chinese)[林文力,肖伏莲,杨水芝.2013.湖南省柑橘大实蝇研究初报.湖南农业科学,(15):84-86]
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