【作者】 贺虹；

【导师】 袁锋；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2004， 博士

【摘要】 麦红吸浆虫Sitodiplosis mosellana(Gehin)是一种间歇性大发生的小麦主要害虫, 分布于我国的各主要小麦生产区，在我国历史上曾多次发生麦红吸浆虫对小麦生产造成的灾害，特别是1946-1952年、1984-1991年造成的灾害更为严重。近几年，仍在局部地区爆发成灾，并具有扩散蔓延的趋势，对小麦生产造成潜在的严重威胁。本研究对中国12个地区的麦红吸浆虫种群，选择线粒体DNA细胞色素C氧化酶亚基Ⅱ（COⅡ）和NADH脱氢酶亚基4（ND4）进行序列变异分析，同时结合微卫星引物PCR多态性分析，从分子水平探讨麦红吸浆虫种群传播扩散的途径及其种群的遗传多样性水平，以便为研究该害虫的扩散传播、成灾规律、预测预报及大面积的综合治理提供种群遗传学信息。对线粒体DNA COⅡ和ND4基因片段的序列分析结果表明：1）扩增出的麦红吸浆虫 COII基因片段的长度为550bp左右，在所有11个种群中可用于分析的序列长度为447bp。在得到的11条同源序列中，共检测出7个变异位点（约占核苷酸总数的1.6%），其中转换数为5，颠换数为 2，无碱基的缺失/插入。序列中A+T平均含量为78.6%，G+C平均含量为21.4%。不同序列之间核苷酸替换数最大为6，最小为1，这些变异的多态位点共定义了7个单倍型，群体的单倍型多样度为0.873±0.0079。 2）扩增出的麦红吸浆虫ND4基因片段的序列长度为450bp左右，在所有12个种群中可用于分析的序列长度为327bp。在得到的43条同源序列中，共发现21个变异位点（约占分析位点数的6.4%），其中包括17个转换，4个颠换，无碱基的缺失/插入。序列中A+T平均含量为75.6%，G+C平均含量为24.4%。这些变异的多态位点共定义了22种单倍型，不同单倍型之间的核苷酸替换数最大为8，最小为1，单倍型间的序列差异在0.31%～3.06%之间，平均为1.68%,群体的单倍型多样度为0.8782±0.0017。利用mtDNA COⅡ和ND4基因序列构建的麦红吸浆虫各地理种群单倍型的分子系统树显示，单倍型的系统发育关系与各种群在地理分布上相对应。所有分布在春麦区的麦红吸浆虫地理种群拥有的单倍型聚为一枝；分布于冬麦区的绝大部分单倍型聚为一枝，而其中的陕西长安种群（SC）拥有的单倍型则在两枝中均有出现，成为单倍型分布的一种过渡区域。结合单倍型空间分布的影响因素分析结果，作者认为受限制的基因流动及其在分布区的扩散可能是形成麦红吸浆虫目前单倍型分布模式的主要原因。麦红吸浆虫群体的单倍型频率非配对分布分析表明, 该种群在历史上曾经历两次群体扩张事件, 其中后一次的规模大于前一次。麦红吸浆虫种群遗传分化和基因流水平分析表明，无论在冬麦区还是春麦区，其区域内的各种群之间Fst值均小于0.33，遗传分化很小，在同一类型的耕作区内各种群间存在着较强的基因交流；但在不同类型的耕作区种群之间，其Fst值几乎均大于0.33，遗传分化较大，遗传漂变是影响其分化的主要因素；但陕西长安种群SC和春、冬麦区<WP=6>其他种群的Fst值均小于0.33，这进一步表明陕西长安种群SC所在的区域是麦红吸浆虫在我国冬麦和春麦两种类型耕作区的过渡地带。由此可以认为，麦红吸浆虫的基因流模型应为“距离隔离”模型，即在一个连续分布的群体内，其邻里之间的种群能够发生基因流，且基因流水平与其间的距离成反比。结合麦红吸浆虫生物学特征及其寄主植物的生长发育规律特点，对不同地理种群间的基因流方向和影响基因流动的因素进行分析后，作者认为：随机遗传漂变应是形成冬、春麦区麦红吸浆虫种群分化的最主要原因；其各种群间的基因流主要应是在水流和风的推助作用下，进行种群扩散、传播后完成的（幼虫、蛹或圆茧主要随水流传播，而成虫则借助风力扩散）。日照时间和温湿度应是影响其基因流方向的主要因素，因为各地的日照时间和温湿度差异是影响其寄主植物成熟期形成先后顺序的关键因子，这即造成了成虫的扩散传播只能从东向西、由南向北进行。水流的灌溉作用又是局部地区麦红吸浆虫形成一个随机交配群体的主要原因。利用4个微卫星引物对麦红吸浆虫11个种群的遗传多样性分析表明，冬麦区种群的基因多样度和多态位点率均高于春麦区种群。基于两两种群间相似系数的聚类分析将11个种群聚为3枝，所有春麦区种群聚为一枝，冬麦区的SC、HL、HX、AF种群聚在一起后于春麦区种群合并，最后与种群AS-HN聚在一起,显示出明显的地理分布特点。从mtDNA COⅡ基因序列计算出的麦红吸浆虫11个种群的遗传距离在0.000～0.016之间，由mtDNA ND4基因得到的12个种群的遗传距离在0.0000～0.0180之间，该变异范围属于地理种群一级的变异，因此麦红吸浆虫各种群之间的遗传差异还没有达到种级，仅为种下分化水平。从mtDNA ND4基因估计的麦红吸浆虫12个地理种群内的遗传变异范围在0.0000～0.0145之间，其中安徽宿州种群AS和河北南和种群HN种群内的遗传变异最大，为0.0145, 甘肃武威种群GW和宁夏惠农种群NH种群内的遗传变异最小，为0.0000。 根据不同地理种群间的遗传距离构建出的系统树表明，麦红吸浆虫12个（COⅡ基因构建了11个种群的系统树）不同地理种群明显地聚为春麦区和冬麦区两个大类，其中陕西长安种群（SC）虽处于冬麦区，却与春麦区各种群间的遗传距离?更多还原

【Abstract】 The wheat midge, Sitodiplosis mosellana (Gehin), is a serious pest of wheat, outbreaking sporadically and distributing in the main wheat-growing region of China. It outbroke and plagued many times in history and made great losses in the wheat production. Forties and eighties of 20th century were its main outbroke periods. Recently about ten years, it still outbreaks and plagues at certain wheat fields and has a trend to extend their habit, having the potential threat to the wheat production of China. In this paper, the genetic diversity of 12 geographic populations of wheat midges was studied through sequence analysis of mtDNA COⅡ and ND4 gene and the PCR technique with repeat sequence primer, in order to analyze its population variation, dispersal and migration patterns and the level of genetic diversity in molecular level. This investigation will provide some essential information for understanding possible local adaptation and migration patterns of the pest, and might provide useful data for the pest forecasting and control in a large area.The results about mtDNA COⅡ and ND4 sequence analysis showed that: 1) 550bp DNA fragment was amplified from COⅡ gene and only 447bp can be reliably read for all 11 geographic populations. Among those analyzed sequences, there are 7 variable sites including 5 transitions, 2 transversions and no any delete and insert. Mean A%+T% content for the sequences was 78.6%, while mean G%+C% content was 21.4%. Through comparing pairwise differences among sequences, it was found that the maximum of nucleotide substitions was 6 and minimum was 1, defining 7 haplotypes designated as H1-H7. The haplotype diversity (Hd) of the wheat midge was 0.8782±0.0017. 2) 450bp DNA fragment was amplified from ND4 gene and only 327bp can be reliably read for all 43 individuals from 12 geographic populations. Among those analyzed sequences, there were 21 variable sites including 17 transitions and 4 transversions and no any delete and insert. Mean A%+T% content for the sequences was 75.6%, while G%+C% content was 24.4%. Through comparing pairwise differences among sequences, it was found that the maximum of nucleotide substitions was 8 and minimum was 1, defining 22 haplotypes designated as Hap1-Hap22. The sequence differences among haplotypes was 0.31%～3.06% (mean was 1.68%), the haplotype diversity (Hd) was 0.8782±0.0017.The phylogenetic tree of haplotype constructed by mtDNA COⅡ and ND4 gene showed that the phylogeny among haplotypes was closely related with geographical distribution. The haplotypes from the spring wheat region were clustered into one group, and most ones from <WP=9>the winter wheat region were clustered into another group, but the haplotypes in SC population in winter wheat region were found synchronously in each group, so distribution area of SC population become a transition place of haplotype distribution. The main factors that may influence the haplotype distribution were analyzed. It was concluded that the restricted gene flow and distributing pervasion were the main reasons that can effect the haplotype distribution of the wheat midge. The analysis of mismatch distribution of haplotype frequence indicated that the population of the wheat midge had undergone two large population expanding in history.The analysis results of genetic differentiation and gene flow about different geographic populations showed that Fst values within populations in winter wheat region and spring wheat region respectively were less than 0.33, so their genetic differentiations were very small and there existed more gene flow. But Fst values between winter wheat region populations and spring wheat region populations were larger than 0.33. This maybe was caused mainly by genetic drift. The Fst values between SC population and other populations were all less than 0.33. Those imply that there existed more gene flows between SC population and other populations. The gene flow model among different geographic populations of wheat midge should belong to “distance-isolation” model. That means gene f更多还原