【作者】 魏晓棠；

【导师】 包振民； 薛芳森；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 大猿叶甲为重要的十字花科蔬菜害虫，从成虫在土中滞育。本研究以江西龙南、江西修水、山东泰、哈尔滨4个地理种群的大猿叶甲为对象，探索了不同群体的滞育变异，并采用几种分子标记技术对其群体遗传多样性和遗传结构进行了研究，同时比较了与其滞育变异之间的相关性。光周期和温度对大猿叶甲不同地理种群滞育诱导的影响在不同的恒温(18-30℃)条件下，测试经历了越冬滞育的大猿叶甲四个地理种群[江西龙南种群(JXL)、江西修水种群(JXX)、山东泰安种群(SDT)、黑龙江哈尔滨种群(HEB)]后代在光照8-16 h下滞育诱导的光周期反应。结果表明，随纬度的增高，光照时间缩短，温度降低，大猿叶甲从南到北呈现明显的地理变异。龙南种群对光周期和温度的反应与山东和哈尔滨种群不同，与修水种群相似。温度在大猿叶甲的滞育诱导中起决定性作用：无论光周期如何，当温度≤18℃时，龙南种群成虫均进入滞育；修水种群在温度≤20℃时，成虫均进入滞育；山东泰安种群在温度＜25℃时，全部个体进入滞育；哈尔滨种群在温度≤28℃时，全部个体进入滞育。光周期对滞育诱导的影响受温度的调节，龙南种群为典型的短日照型昆虫，当温度＞18℃时，随着温度的升高，短日照的滞育抑制作用逐渐增强；修水种群的光周期反应亦表现为典型的短日照类型，当温度＞20℃时，不同短光照条件下诱导的滞育率随温度的升高而明显降低；当温度＞22℃时，不同长光照诱导的滞育率随温度的升高而明显降低；山东泰安种群在28℃时，滞育率在69．23％～90．9l％之间；在30℃时，反应曲线呈不规则波浪状，没有呈现典型的光周期反应。哈尔滨种群没有光周期反应，滞育的诱导完全取决于温度。不同的滞育特性，导致大猿叶甲的生活史不同。遗传多样性研究采用了线粒体DNA COI基因序列、RAPD和PCR-RFLP三种分子标记研究大猿叶甲4个地理种群的遗传结构和群体遗传多样性。1、对长约335bp的mtDNA COI片段进行序列分析，在4个群体64个个体中，共发现37个单倍型，总变异位点35个，多态位点29个，(A+T)含量(63．68％)大于(G+C)含量(36．32％)。4个地理种群的核苷酸多样性指数分别为，0．082(JXL)、0．035(JXX)、0．099(SDT)、0．186(HEB)。Fst分析和中性检验结果显著，显示大猿叶甲群体存在显著的种群遗传结构，mtDNA COI基因不是按照中性理论进化。遗传距离分析表明，哈尔滨种群与江西龙南种群的遗传距离最远，其次是与江西修水种群；山东泰安种群与江西龙南种群的遗传距离最近，江西修水与江西龙南种群的遗传距离大于其与山东泰安种群间的距离。群体遗传距离与其相对地理距离的远近不吻合。2、从80个引物组合中筛选出的11个引物对大猿叶甲4个地理种群进行RAPD分析，共得到65个扩增位点，多态位点53个。多态位点、遗传相似性指数和遗传距离分析表明：4个种群的多态位点的百分率为36．92％～67．69％，Nei遗传多样性指数为0．1049～0．20611，Shannon多样性指数为0．1641～0．3167，江西龙南群体内的遗传变异最低，山东泰安群体内遗传变异最高。遗传距离分析表明，江西龙南种群和江西修水种群间的遗传距离最小，山东泰安种群与哈尔滨种群间的遗传距离次小，哈尔滨种群与江西龙南种群间的遗传距离最大。群体遗传距离与其相对地理距离吻合，与群体的滞育变异及生物学特征相符。3、利用7种限制性内切酶对大猿叶甲COI基因进行PCR-RFLP分析，结果表明，利用AseI、MboI、NlaⅢ、RsaⅠ，在大猿叶甲4个地理群体中均没检测到多态性；利用AluⅠ和DraⅠ只检测到群体间的多态性；利用HaeⅢ既检测到了群体间的多态性，又在山东群体中检测到群体内多态性。根据酶切图谱，共发现4种酶切类型，酶切型的特异性可以作为种群鉴别的标志。根据限制性片段共享度，利用POPGEN3．2计算4个群体的遗似距离，并利用MEGA3．1进行聚类分析，结果表明其遗传变异与其滞育变异的特征不相符。更多还原

【Abstract】 The leaf beetle,Colaphellus bowringi Baly is a serious pest of crucifers,aestivating and hibernating as adults in the soil.Four Populations of C.bowringi fromLongnan,Xiushui of JiangXi provence,Taian of ShanDong provence,Haerbin ofHeiLongJiang provence,were studied to explore the diapause diversity by differenttemprature and photoperiod,and estimate the genetic structure and genetic diversityby different DNA marker technologies,and compaired them with diapause diversity.Temperature and photoperiodic adjustment of diapause in populations of C.bowringiAt differente constant temperature (18-30℃),the photoperiodic response ofdiapause induction of the four populations of C.bowringi from Longnan of JiangXiprovence(JXL),Xiushui of JiangXi provence(JXX),Taian of ShanDong provence(SDT),Haerbin of HeiLongJiang provence(HEB),undergoing winter diapause weretested with daylight of 8-16 h.The result suggest that geographical diversity wasoccurred with the increasing of latitude and depressing of temperature.The responseon photopreiod and temperature is differente between the JXL and SDT or HEB,but itis similar to that of JXX.Temperature play a Crucial role in diapause induction for C.bowringi.Whateverphotoperiod,100% diapause occurred at (?)18℃in JXL,at (?)20℃in JXX,at (?)25℃in SDT,at (?)28℃in HEB.The effect of photoperiod on diapause induction is adjusted by temperature.JXLof C.bowringi is typical short-daylight insects.The effect of diapause control ofshort-daylengths enhance with the increasing temperature at＞18℃;The population ofXiushui are also short-daylight insects.The diapause rate induced under differenteshort-light decreases with increasing temperature at＞20℃,and decreases underdifferent long-daylengths with increasing temperature distinctly at＞22℃;For SDT, the diapause rate is 69.23%～90.91% at 28℃.But its photoperiodic responses curve isanomalous wavilness at 30℃,showing the photoperiodic responses didn’t occured.HEB don’t responce on photoperiod,the diapause induction was decided bytemperature completely.More,The examination showed the different diapausecharacters results in the different life-cycles in the four population of C.bowringi.Genetic diversity analyse of four populations of C.bowringi by mtDNA COIsequence,RAPD and PCR-RFLP1.A total of 30 mutation loci,25 polymorphic loci,37 haplotypes,weredetected among four populations,64 individuals by analyzing the sequences datafrom mtDNA COI gene about 335bp.The content of A+T (63.68%) is obviouslyhigher than that of G+C (36.32%).The nucleotide diversity index were 0.0056(JXL)、0.0023 (JXX)、0.0093 (SDT)、0.0151 (HEB),respectively.The result ofpairwise Fst and Neutrality tests revealed significant genetic differentiation amongfour populations,and evolution of mtDNA COI gene incompliance with the Neutralitytheory。The genetic distance was the biggest between HEB and JXL,and was thesmallest between SDT and JXL among the 4 populations.While the genetic distancebetween JXL and JXX is bigger than that between SDT and JXX.The geneticdistance ofpopulaions dismatch with their geographic distance.2.Four geographical populatios of C.bowringi were amplified using 11 primersselected from 80 random primers.There were 53 polymorphic loci based on 65amplification loci obtained by RAPD analysis.Polymorphisms,genetic similarityindices and genetic diatance were calculated.The results suggesting:the percent ofpolymorphisms was ranging 36.92%～67.69%.Nei genetic diversity was ranging from0.1049～0.20611.And Shannon diversity was ranging from 0.1641～0.3167.Tthegenetic variation within SDT was the highest,while that within JXL is the lowest.Thegenetic distance between JXL and JXX was the smallest,that between JXL and HEBwas the biggest.While the genetic distance between HEB and SDT was smaller thanthat between HEB and JXX.The genetic distances of C.bowringi populations matchtheir geographic distances between the sources of each population,and werecorrelated with their biologic comparability.3.Seven endonucleases were used to analyze the amplified products of COI gene.The result showed that AseⅠ,MboⅠ,NlaⅢ,RsaⅠdidn’t produce restrictionfragment-length polymorphism among C.bowringi populations.The polymorphismwas detected among the 4 populations with the enzymes AluⅠand DraⅠ.Whereas,the restriction fragment-length polymorphism was found eihter among 4 populationsor within SDT with HaeⅢ.Four restriction types were found in all individuals.Obviously,the populations could be distinguished with unique restriction type.According to the porportion of restriction fragments shared,the genetic distancesamong the 4 populations were calculated with popgene3.2,and a cladogram wasproduced with MEGA3.1.The result showed that the genetic variance dismatch withtheir diapause variance.更多还原