棉铃虫种群遗传变化的研究_英文_

棉铃虫种群遗传变化的研究_英文_ STUDY O N THE GENETIC VARIATIO N OF THE COTTO N BOLL WO RM H EL I COV E R PA A R M I G E RA ( HΒBNER) 3 POP UL ATIO NS IN CHINA CH EN Xiao2feng , TAN Sheng2jiang , L IU Ren2yi , WAN G Ying and L I Dian2mo S t ate Key L ab of I nteg rated M an agement of I nsect Pests & Rodent , I nst i t ute of Zoology , Chi nese A cadem y of S ciences , Beijing 100080 , China ( )Received Sep . 17 , 1999 ; accep ted J un. 19 , 2000 ( ) Abstract Pop ulatio n genetic variatio ns of Hel icover pa a r m i gera Hbünerover different major cot to n grow2 ing regio ns were analyzed by DNA polymorp hism amplified wit h four simple repetitive sequence p rimers. The re2 sult s showed t hat t he laboratory pop ulatio n had relatively lower genetic variatio n t han nat ural pop ulatio ns. The genetic variatio n bet ween nat ural pop ulatio ns was not significant and genetic variatio n existed in t he same locatio n f ro m different years , indicating f requent migratio n amo ng nat ural cot to n bollworm pop ulatio ns. Cluster analysis ( ) ( ) indicated t hat individuals f ro m Chaoyang of Liao ning Province C Y, Gaotang of Shando ng Province GTand () Dafeng of J iangsu Province D Fwere more mixed each ot her , which suggested t hat C Y pop ulatio n might have higher gene flow wit h GT and D F pop ulatio ns , especially wit h GT pop ulatio n. It support s t he t heory t hat t he cot to n bollworm in Nort heast China came f ro m Shando ng and Hebei Provinces. This result also demo nst rated t hat t he molecular makers in t his st udy are sensitive to detect pop ulatio n genetic st ruct ure changes. ( ) Key words Hel icover pa a r m i gera Hbüner, genetic variatio n , repeat s sequence p rimer INTROD UCTIO N 1 ) () ( The cot to n bollwo r m Hel icoverpa a r m i gera HbünerL epitop tera : Noct uidaefeeds o n many ho st plant s and occurs i n Asia , Europe , Af rica and Aust ralia . It is o ne of t he mo st serio us agricult ural pest s i n Chi na , havi ng caused huge damage to cot to n p ro ductio n i n re2 cent years. Alt ho ugh many researches have been co nducted o n t he ecolo gy and pop ulatio n biology of t his pest species , it s pop ulatio n genetic variatio n , dispersal and migratio n pat ter ns are not well understoo d. It caused great difficulties i n p redicti ng pop ulatio n dynamics and () maki ng p roper management decisio ns Li et al . 1995 , Guo 1997. Insect species might be divided i nto genetically diverse pop ulatio ns wit h li mited gene flow amo ng t hem , especially t ho se coveri ng large and hetero geneo us geo grap hical regio ns. Pop ulatio ns co uld also accumulate genetic differentiatio n due to local adap tatio n o r rando m drif t . Fo r pest i nsect s , t he genetic variatio ns amo ng pop ulatio ns co uld have significant i nflu2 ence o n regio nal o ut break pat ter n and maki ng p roper pest management st rategies. Pesticide resistance , fo r example , co uld be developed at different rate i n different pop ulatio ns , and ( biot ypes wit h different adap tive st rategy might occur i n different pop ulatio ns Rui et al . ) 1996. It was fo und t hat so me pop ulatio ns f ro m different areas differed si gnificantly i n dis2 persal pat ter n , pesticide resistance and allele f requency. However , pop ulatio n genetic varia2 ( tio n pat ter n and it s i mpact o n t he pest o ut break are not well understoo d Wu and Guo 1995 , ) 1996 , 1997 ; L ei et al . 1997. Integrated management st rategies of t he cot to n bollwo r m ( ) ( 3 This st udy was suppo rted by t he Natio nal Nat ural Science Fo undatio n of China No . 39670129, IFS grant No . C/ ) 660221, and State Pan2Deng p rogram. sho uld be based o n regio nal pop ulatio n characteristics and habitat st ruct ures ; t hus mo re i n2 fo r matio n abo ut t he pop ulatio n genetic variatio n pat ter ns wo uld be very usef ul . Knowledge abo ut pop ulatio n genetic variatio n pat ter n of t he cot to n bollwo r m is t hus very i mpo rtant fo r understandi ng po ssible local adap tatio n and migratio n pat ter ns of t he pest , and might p rovide usef ul data fo r pest co nt rol . Molecular genetic mar ker technolo gy has beco me a very i mpo rtant tool i n st udyi ng ani mal pop ulatio n genetic st ruct ure , gene flow ( ) and migratio n pat ter ns amo ng pop ulatio ns i n recent years Avise 1994 , Ro derick 1996 . This paper repo rt s t he result s of DNA polymo rp hism st udies o n t he cot to n bollwo r m , to an2 alyze t he pop ulatio n genetic variatio ns , dispersal and migratio n pat ter ns of H . a r m i gera over different majo r cot to n growi ng regio ns i n Chi na . 2 MATERIALS AND METHODS 211 Sa mpl ing of the cotton boll worm ( )Geograp hic locatio ns of sampli ng sites shown i n Fig. 1 o n t he were selected based cot to n bollwo r m o ut break histo r y and expected migrati ng ro ute . The sampli ng sites i nclude : ) ( ) ( Chaoyang of Liao ni ng Provi nce C Y, Gaotang of Shando ng Provi nce GT, Handan of ( ) ( ) Hebei Provi nce HD, Xi nxiang of Henan Provi nce XX, and Dafeng of J iangsu Provi nce () D F. The number followi ng t he abbreviatio n of t he sites i ndicates t he year w hen t he sam2 ples were collected. Fo r example , sample collected f ro m Chao yang i n 1995 was writ ten as Fig. 1 Sampling sites of Hel icover pa a r m i gera. C Y ,Chaoyang of Liao ning Province ; GT , Gaotang of Shando ng Province ; HD , Handan of Hebei Province ; XX , Xinxiang of Henan Province ; D F , Dafeng of J iangsu Province . C Y951 L arvae i n t he si xt h i nstar were collected f ro m t he field ; each i ndividual was f ro m different cot to n plant i n t he same field. The larvae were fed o n t he artificial diet o r p re2 ( ) served i n et hanol 95 %. A labo rato ry pop ulatio n was used as a co nt rol , w hich showed si g2 ( ) nificant genetic differentatio n f ro m nat ural pop ulatio ns Chen et al . 1997. 212 Preparation of genome D NA ( ) Geno me DNA was ext racted acco rdi ng to Chen et al . 1996. Individuals of t he wo r m ( were ho mo genized i n 4 mL S T E 012 mol/ L sucro se , 50 mmol/ L ED TA , 100 mmol/ L μ) Tris , 015 % SDS. Then 80g p rotei nase K was added and t he mi xt ure was kep t at 60 ? fo r o ne ho ur . Af ter t wo volumes of et hanol was added , t he mi xt ure was kep t i n - 20 ?fo r30 mi nutes. The sedi ment material af ter cent rif ugatio n at 12 000 g , was dried i n roo m tem2 μ perat ure. One milliliter T E wit h 20 g RNase A was added and t he mi xt ure was kep t at 37 ?fo r o ne ho ur . Then p henol/ chlo rofo r m/ isoamyl alco hol ext ractio n was followed to get ( ) p urified DNA Sambroo k et al . 1989. The DNA was t hen redissolved i n T E and kep t at 4 ?af ter exami natio n of t he co ncent ratio n and p urit y. 213 PCR ( ) ( ) ( ) Fo ur si mple repetitive sequence DNA p ri mers , GT G, GT , ACC, and a 5 8 5 ( )16 bp lo ng co re sequence 5’A GA GGT GGGCA GGT G 3’ of human 33115 micro satellite DNA were used to amplif y t he geno me DNA of H . a r m i gera . The reactio n volume was 25 μμμμ L , which co ntai ned 215 L 10 ×PCR buffer , 10 mol/ L p ri mer , 215 mol/ L dN TPs ,1 U T aq DNA Polymerase , 50 ng template DNA . The reactio n was carried o ut o n a Per ki n El mer 480 DNA Ther mal Cycler , wit h p rograms set as 94 ? 5 mi n , 45 cycles of reactio n co nsisted of 94 ? 1 mi n , 56 ? 1 mi n , 72 ? 2 mi n , and t hen 72 ? 5 mi n . The p ro duct of PCR was run o n 112 % agaro se gel stai ned wit h et hidium bro mide , t hen visualized and p ho2 tograp hed under U V light . Molecular weight was sco red usi ng a St rategene Image Analysis system . 214 Data analysis Three i ndexes of genetic variatio n were calculated. The average band shari ng rate wit hi n a pop ulatio n was calculated as t he average of band shari ng rates bet ween each t wo i n2 dividuals wit hi n t he pop ulatio n . Gene diversit y wit hi n pop ulatio ns was calculated acco rdi ng ( ) to L ynch and Milligan 1994. The polymo rp hic loci were defi ned as t ho se wit h t he mo st f requent allele bei ng less t han 95 % , and t he p ropo rtio n of polymo rp hic loci was calculated fo r each pop ulatio n . U P GMA met ho d was used to perfo r m clusteri ng analysis of i ndividuals o r pop ulatio ns based o n t ho se genetic variatio n i ndexes. Band shari ng data and gene diversi2 ( t y i ndex were t ransferred as t he arcsi ne of it s square root , befo re co nducti ng ANOVA SAS ) Instit ute 1996. 3 RES UL TS 311 PCR result ( ) ( ) ( ) In t his st udy , t he si mple repetitive sequence GT G, GT, ACC, and a 16 bp 5 8 5 () lo ng co re sequence 5’A GA GGT GGGCA GGT G 3 ’of human 33115 micro satellite DNA were used as p ri mers i n t he PCR to generate i ndividual PCR fi ngerp ri nt s. This technique , ter med PCR fi ngerp ri nti ng , has been applied successf ully to amplif y hypervariable repetitive ( ) DNA sequence i n a wide range of f ungal geno mes Meyer and Mitchell 1995 . Clear and stable amplificatio n result s were got wit h t his technique . We detected 64 sites usi ng t he fo ur p ri mers , and 9614 % of t hem are polymo rp hic sites. Figure 2 showes t he result s amplified ( ) wit h p ri mer ACC. 5 λ() ( Fig. 2 Amplificatio n result s wit h ACCp rimer . Mar k is t he standard molecular weight mar kerDNA/ 5 Hi nd ?+ EcoR ?. Number 1 to 5 were individuals f ro m GT95 pop ulatio n , and 6 —10 were f ro m D F95 , ) 11 —15 were f ro m C Y95 pop ulatio n 312 Population genetic variation Genetic diversit y wit hi n pop ulatio n , gene diversit y , band shari ng i ndex and t he p ro2 po rtio n of polymo rp hic loci are p rovided i n Table 11 Table 2 is t he average bands shari ng i n2 dexes amo ng pop ulatio ns. Ta ble 1 Genetic variatio n wit hi n pop ulatio ns of t he cot to n bollwo r m . Sample Average band Gene diversity Proportio n of Pop ulatio n ( )( )size sharing rate SD SD polymorp hic loci ()()C Y95 153 0102 125 0102 174 8 000 ()()0161 0101 0125 0102 0175 GT95 8 ()()D F95 8 0161 0101 0124 0103 0172 ()()0164 0102 0120 0103 0157 HD95 8 ()()0167 0102 0116 0103 L AB 8 0143 ()()0148 0102 0126 0102 0177 C Y96 8 ()()0157 0101 0124 0102 0172 8 GT96 ()() 16 0154 0101 0126 0102 0175 XX96 ( ANOVA , F = Band shari ng i ndexes were significantly different amo ng pop ulatio ns ) 1117 , P < 01001. The average bands shari ng rate of t he L AB pop ulatio n has t he lar gest value . Bot h C Y95 and C Y96 pop ulatio ns have t he lowest average bands shari ng rate amo ng t he field samples i n 1995 and 1996 , respectively , i ndicati ng relatively high genetic varia2 tio n . However , when t he L AB pop ulatio n is excluded , t he difference is not si gnificant . Gene diversit y wit hi n pop ulatio ns was also not fi nd to be si gnificantly different amo ng pop u2 ( ) latio ns ANOVA , F = 718 , P < 0107. The p ropo rtio n of polymo rp hic loci has t he si milar pat ter n as t he band shari ng data . These result s i ndicated t hat t he nat ural pop ulatio ns might migrate currently , and t here was f requently gene flow amo ng t hem. Ta ble 2 Average bands shari ng i ndexes amo ng pop ulatio ns. GT95 D F95 C Y95 HD95 L AB XX96 GT96 C Y96 ()GT95 0161 0101 () ()D F95 0160 01010161 0101 () () ()C Y95 0154 01010156 01010153 0102 () () () ()HD95 0154 01010152 01010151 01010164 0102 () () () () ()L AB 0146 01010149 01010144 01020157 01010167 0102 () () () () () ()XX96 0149 01020150 01020144 01020150 01050153 01010154 0101 () () () () () () ()GT96 0142 01010142 01010138 01020142 01020138 01010146 01010157 0101 () () () () () () () ()C Y96 0139 01010140 01020138 01030139 01030138 01010144 01010150 01010148 0102 313 Cl uster anal ysis on band sharing data of cotton boll worm population Cluster analysis of t he band shari ng data by U P GMA met ho d was co nducted to i nvesti2 ( ) gate t he genetic relatio nship amo ng i ndividuals f ro m different pop ulatio ns Fig. 3. Fig. 3 Cluster diagram of individuals f ro m different pop ulatio ns by U P GMA based o n band sharing rates be2 t ween individuals. Individuals f ro m different pop ulatio ns are labeled as : GT95 , a ; D F95 , b ; C Y95 , c ; HD95 , d ; L AB , e ; XX96 f ; GT96 , g ; C Y96 , k. ( ) () It co uld be fo und t hat t he i ndividuals f ro m L AB eand D F95 bpop ulatio ns were generally clustered to get her wit h i ndividuals f ro m t he same pop ulatio n . Individuals f ro m () (( ) () ) () ) (GT95 a, C Y95 c, XX96 f and HD95 d, i ndividuals f ro m GT96 gand C Y96 hwere mo re mi xed each ot her . It i ndicated t hat t he L AB , D F95 pop ulatio ns have higher ge2 ( ) ( ) netic si milarit y wit hi n pop ulatio n , w hile t he GT 95 and 96, C Y 95 and 96, HD and XX pop ulatio ns have higher i ndividual genetic si milarit y bet ween pop ulatio ns. It suggested t hat t he i ndividuals of t hese pop ulatio ns mi grated f requently and t here was st ro ng gene flow bet ween t hese pop ulatio ns. 4 CO NCL USIO NS A ND D ISCUSSIO N High genetic variatio n was fo und amo ng i ndividuals wit hi n all sampled nat ural pop ula2 ( ) tio ns. Inter2pop ulatio n genetic variatio n average bands shari ng , 0147 were larger t han i () nt ra2pop ulatio n genetic variatio n average bands shari ng , 0158, but t he genetic variatio n bet ween nat ural pop ulatio ns was not si gnificant as i ndicated by t he t hree i ndexes above . It was fo und t hat t he lab pop ulatio n has a relatively low genetic variatio n , as i ndicated ( ( ) ) by it s high band shari ng rate 0167, low gene diversit y 0116and lower p ropo rtio n of ( ) polymo rp hic loci 0143. And t he result s of cluster anal ysis showed t hat mo st i ndividuals of t he L AB pop ulatio n were firstly clustered to get her . These are w hat expected f ro m pop ula2 tio n genetic t heo ry , t hat small and i nbreedi ng pop ulatio n sho uld have low genetic variatio n . This result demo nst rates t hat t he molecular mar kers i n t his st udy are sensitive to detect pop2 ulatio n genetic st ruct ure changes. The result s of t he cluster analysis showed t hat i ndividuals f ro m C Y , GT and D F were () mo re mi xed each ot her . Half of C Y95 i ndividuals 5/ 10were firstly clustered wit h HD95 , ( ) GT95 , XX96 and GT96 pop ulatio ns , and mo st C Y96 hi ndividuals were clustered wit h ( ) GT96 gpop ulatio n . These result s suggested t hat C Y pop ulatio ns had hi gh gene flow wit h GT , HD , XX and D F pop ulatio ns , especially wit h bot h GT pop ulatio ns. As t here were no overwi nter pop ulatio ns i n t he no rt heast of Chi na , such as Liao ni ng Provi nce , t he o rigi n of ( ) cot to n bullwo r m i n t his area has been discussed fo r a lo ng ti me Zhu et al . 1997. Our re2 sult s suggested t hat t he C Y pop ulatio ns might co me f ro m t he so ut h place to Liao ni ng Provi nce , mo stly f ro m Hebei , Shando ng and Henan Provi nces. This mi ght be a result of t he relatively f requent so ut h to no rt h wi nd i n t he no rt her n part of Chi na . The average bands shari ng bet ween C Y95 and C Y96 , GT95 and GT96 were lower ( ) 0138 , 0142t han t ho se bet ween ot hers. It i ndicated t hat genetic variatio n of samples f ro m t he same locatio n but f ro m different years differed significantly. These result s suggested t hat t here was f requently migratio n amo ng cot to n bollwo r m pop ulatio ns. Recent st udies o n t he p hysiology and behavio r of Hel icover pa a r m i gera demo nst rated t hat it has ver y st ro ng ( ) flyi ng abilit y Guo 1997, however , whet her effective i nter2pop ulatio n migratio n do occur o r t here is significant i mpact o n t he genetic st ruct ure and pop ulatio n dynamics are not known. Our result s suggested high rate of gene flow amo ng local pop ulatio ns , and t hus , suppo rted t he mi gratio n hypot hesis. The co nclusio n of hi gh genetic variatio n and high gene flow wo uld have great i mpo rtance fo r explai ni ng t he pesticide resistance pat ter n , ho st adap2 tatio n and o ut break of t he pest . Ackno wledgement Mr . L I Shi2peng p rovided assistance i n data anal ysis. Ref erences Avise , J . C. 1994 Molecular Mar ker , Nat ural History and Evolutio n. New Yor k : Chap man & Hall . pp . 9542956 . Chen , X. F. , X. Zhao , S. P. Li et al . 1997 Co mpariso n of t he RA PD DNA polymorp hism in nat ural and ( ) laboratory pop ulatio ns of China . I n : Progress in Ento mology Research eds Yang , K. X. and Wu H. . Beijing : China Forest ry Press , pp . 43246 . Chen , X. F. , J . Chen , S. P. Li et al . 1996 Pop ulatio n genetic variatio n of cot to n bollworm Hel icover pa ( a r m i gera detected by RA PD2PCR. 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M . and Y. Y. Guo 1996 Ecological form of Hel icover pa a r m i gera in China . I n : Progress o n Plant () Protectio n Research eds The Chinese Society of Plant Protectio n. Beijing : China Science and Technology Press , pp . 4082414 . Wu , K. M . and Y. Y. Guo 1997 Investigatio n o n t he fit ness of cot to n bollworm Hel icover pa a r m i gera. ( ) A ct a Entom ol . S i n . 40 supplement:7212 . Zhu , M . L . , G. Q . Xu , J . Q . Zhao . et al . 1997 Investigatio n o n t he incidence of cot to n bollworm in ( ) nort heast China . A ct a Entom ol . S i n . 40 supplement:2102213 . 棉铃虫种群遗传变化的研究 陈晓峰 谭声江 刘仁义 王 瑛 李典谟 农业虫鼠害综合治理国家重点实验室 , 中国科学院动物研究所 , 北京 100080 利用重复序列引物扩增 DNA 多态性方法研究了几个产棉区的棉铃虫的种群遗传结构变化 。结果 明 :实验室种群比自然种群的遗传变化低 ,自然种群间的遗传差异不显著 ,同一地点不同年间种群间存在 ( ) 较大遗传差异 ,表明棉铃虫的迁飞扩散现象比较明显 ;聚类分析结果表明 ,来自辽宁的朝阳种群 C Y与来 ( ) () 自山东高唐 GT、江苏大丰 D F的种群间的有较强的基因流 ,为我国东北地区棉铃虫主要来自河北 、山东 等地的假说提供了一定的理论依据 。本研究所选用的遗传标记能灵敏地检测种群的遗传结构变化 。 棉铃虫 遗传变化 简单重复序列引物 关键词