【作者】 李菁；

【导师】 王振营；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 亚洲玉米螟Ostrinia furnacalis (Guenée)是威胁我国玉米生产的主要害虫,除为害玉米外,还为害多种寄主植物,其在我国的地理分布跨越了很大的经纬度区域,其所处的不同分布区内自然生态环境具有很大的差异,亚洲玉米螟种群自北向南一年可发生1~7代。据此推测我国亚洲玉米螟不同地理种群之间已经产生了一定程度的遗传分化。本研究采用微卫星与线粒体DNA分子标记技术相结合的方法,对分布在我国及邻国朝鲜的亚洲玉米螟不同地理种群的遗传结构进行了分析,并对不同种群间的遗传分化程度与基因流情况进行了检测;同时对所采集的亚洲玉米螟地理种群Wolbachia感染情况进行了研究,明确了各种群中Wolbachia的感染率和感染类群,并对亚洲玉米螟的两种寄生性天敌——腰带长体茧蜂Macrocentrus cingulum Brischke和玉米螟厉寄蝇Lydella grisescens Robineau- Desvoidy若干种群进行了Wolabchia检测,以揭示亚洲玉米螟与这两种寄生性天敌之间是否存在Wolbachia的水平传播现象。全文获得了以下主要研究结果:1.对亚洲玉米螟32个不同地理种群的4个线粒体基因COI、COII、ND1以及ND5全长或片段进行了PCR扩增并测序,序列分析结果发现,亚洲玉米螟群体中线粒体基因多态性比较高,共检出COI基因单倍型108种,COII基因单倍型53种,ND1基因单倍型72种,ND5基因单倍型37种。2.建立了适合亚洲玉米螟种群分子遗传学分析的微卫星体系,选用6个微卫星座位进行PCR扩增并应用GeneScan测序技术测定了各位点片段的长度多态性。在这些微卫星座位中,有两个位点偏离了Hardy-Weinberg平衡(P < 0.05),其余四个位点均符合Hardy-Weinberg平衡,经检验本研究所用的各位点间不存在连锁不平衡现象。不同微卫星座位的观测等位基因数范围在12~26之间,各种群平均Shannon信息指数为1.566,平均观测杂合度为0.572,平均期望杂合度为0.734,平均等位基因丰富度为7.929,平均多态信息含量为0.692。3.亚洲玉米螟种群间的系统发育树显示,朝鲜平壤种群与我国的玉米螟种群之间存在明显的遗传分化。我国的31个亚洲玉米螟种群在系统聚类树上呈现出较混乱的分布格局,各种群所处的地理位置与系统树上的种群间的聚类结构不一致。成对种群间的遗传距离Fst / (1- Fst)与地理距离之间具有较低的相关性(r = 0.135, P = 0.0025)。4.分子方差分析(AMOVA)结果显示亚洲玉米螟群体中分子遗传变异的主要方差组分来自于种群内的个体之间,其次少部分来自区域内种群间,只有极少部分来自于区域种群之间。说明我国亚洲玉米螟种群在不同玉米产区的之间没有明显的分化。不同玉米产区内种群间的遗传分化与基因交流程度不同。黄淮海平原夏播玉米区内种群之间没有产生显著的遗传分化,种群之间基因交流非常频繁,而在北方春播玉米区和南方丘陵玉米区内的种群之间产生了显著的遗传分化,基因交流程度很低。5.通过4个线粒体基因的单倍型歧点分布及中性检验,显示出我国亚洲玉米螟群体单倍型歧点分布情况为双峰或多峰模式,Tajima’s D与Fu’s Fs中性检验值总体均不显著,表明我国亚洲玉米螟群体在过去较近的历史时期内没有经历种群扩张事件,群体大小保持相对稳定。6.亚洲玉米螟44个种群中Wolbachia的感染率在0~71.1%不等,平均感染率为10.5%。在wsp基因片段序列的基础上,将亚洲玉米螟中检测到感染的Wolbahcia分为9个品系,分别命名为wFur1-wFur9 (GU166588-GU166596),品系wFur1-wFur7属于A组,wFur8-wFur9属于B组。其中wFur1、wFur2、wFur7和wFur9是亚洲玉米螟中较为常见的感染品系,而其余5个品系在亚洲玉米螟种群中均比较罕见。亚洲玉米螟中普遍存在Wolbachia的超感染现象。各地理种群中的Wolbachia感染频率和感染类群不具有明显的空间分布格局。7.腰带长体茧蜂中Wolbachia的感染率非常低,平均感染率为2.22%,克隆得到的两种Wolbachia品系分别命名为wCin1和wCin2 (GU166602-GU166603)。玉米螟厉寄蝇中Wolbachia的感染率非常高,平均感染率为95.7%,鉴定出的3个Wolbachia品系分别命名为wGri1、wGri2和wGri3 (GU166604-GU166606)。在这两种玉米螟寄生性天敌中均发现了Wolbachia的超感染现象。8.经亚洲玉米螟、腰带长体茧蜂和玉米螟厉寄蝇中感染的Wolbachia中wsp序列进行系统发育分析,结果发现亚洲玉米螟与这两种寄生性天敌之间均存在Wolbachia的水平传播现象。更多还原

【Abstract】 Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae) is one of the dominant pests in maize, Zea mays (L.) in China, which injures many other host plants except for maize, and its spatial distribution covers vast geographic distances throughout the country. There exist significant natural and geographical environment discrepancies among different corn regions. Due to high variety of postdiapause development period, voltinism diversity in the Chinese O. furnacalis populations from north to south is present from 1 to 7 generations per year. Based on these properties, it is assumed that the genetic divergency has already happened among different O. furnacalis geographic populations in China.Investigations of the O. furnacalis population genetic structure from China and North Korea using both microsatellite and mitochondrial DNA markers were performed in this research. Genetic differentiation and gene flow among geographic populations were detected. In this study we also investigated prevalence and diversity of Wolbachia infection in O. furnacalis populations and two larval parasitoids: Macrocentrus cingulum Brischke and Lydella grisescens Robineau-Desvoidy to explore the potential horizontal transimissions of Wolbachia between Asian corn borer and its parasitoids. The primary achievements of this dissertation are listed as follows:1. Four mitochondrial genes including COI, COII, ND1 and ND5 full length or fragments were amplified and analyzed with the nucleotide sequences. The results showed high genetic diversity in mtDNA genes of O. furnacalis populations. 108, 53, 72 and 37 haplotypes were observed in COI, COII, ND1 and ND5 respectively.2. Practical microsatellite system for O. furnacalis population studies was developed based on microsatellite loci reported in European corn borer, O. nubilalis. Six polymorphic SSR loci were estimated by GeneScan sequencing technology. Two loci showed deviation from Hardy-Weinberg equilibrium (P-value < 0.05). The genotypic data showed no significant linkage disequilibrium between pairs of loci (P > 0.05). The number of alleles ranged from 12 to 26 at different loci across each population. The mean Shannon’s information index was 1.566, and the average observed (HO) and expected (HE) heterozygosity in all populations was 0.572 and 0.734 respectively. The mean allelic richness (AR) was 7.929 and the mean polymorphism information content (PIC) was 0.692. 3. Phylogenic trees reconstructed from the microsatellite data indicated that the Pyongyangpopulation from North Korea was significantly differentiated from the 31 geographic populations collected in China. Topological structure of the phylogenic tree demonstrated no geographic clustering was observed. There was low correlation between Fst / (1 - Fst) and geographical distance (r = 0.135, P = 0.0025).4. Analysis of molecular variance (AMOVA) results revealed that most of the genetic variation was within populations; less variation was detected among populations within each corn region and even lesser variation among the five corn regions, which suggested no obvious genetic differentiation among these corn regions. The degree of genetic divergence and gene flow varied within different corn regions. The relatively lowest Fst and highest degree of gene flow (Nm) was among populations in Huanghuaihai Summer Corn Region, while there detected significant genetic differentiation and relatively low Nm among populations in both North Spring Corn Region and Southeast Hilly Corn Region.5. Mismatch distribution analysis showed multimodal in O. furnacalis populations. In addition, results of Tajima’s D and Fu’s Fs neutrality tests (not significant, p > 0.05) implied that there might not be a population expansion in recent time and the Asian corn borer populations was at demographic equilibrium.6. Infection frequency of Wolbachia in populations of O. furnacalis ranged from 0~71.1% with an average infection rate at 10.5%. Nine strains of Wolbachia (wFur1-wFur9) were identified based on wsp sequences isolated in this insect species (GU166588-GU166596). Among these variants, wFur1-wFur7 belonged to Group-A and wFur8-wFur9 belonged to Group-B. wFur1, wFur2, wFur7 and wFur9 were prevalent types, and the rest strains were uncommon among O. furnacalis populations. Superinfection commonly appeared in individuals of O. furnacalis and the coinfection patterns were very complex. There was no obvious geographical pattern for the prevalence and distribution of the nine Wolbachia strains.7. Wolbachia infection rate was extremely low in M. cingulum populations (average infection frequency = 2.2%). Two variants isolated from M. cingulum were designated as wCin1 and wCin2 separately (GU166602 & GU166603). Whereas infection frequencies of Wolbachia in L. grisescens were remarkably high (average infection frequency = 95.7%). Three distinct strains were identified in L. grisescens, named as wGri1, wGri2 and wGri3 (GU166604-GU166606), separately. Superinfection was also a common infection pattern observed in the two parasitoid species.8. Phylogenetic evidences based on wsp gene strongly suggested that horizontal transmission events occurred between O. furnacalis and its larval parasitoids.更多还原