【作者】 李婷；

【导师】 洪晓月；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2008， 硕士

【摘要】 二斑叶螨（Tetranychus urficae Koch）与朱砂叶螨（T cinnabarinus Boisduval）同属于节肢动物门蛛形纲Arachnida、蜱螨亚纲Acari、真螨目Acariformes、叶螨科Tetranychidae、叶螨属Tetranychus,广泛分布于世界各地,寄主植物广泛,包括果树、花卉、蔬菜、农作物及杂草等多种植物,一般以成螨、若螨群集在叶背面吸食植物汁液,对寄主植物产生机械伤害和毒害作用,引起植物减产和品质下降,给农业生产带来严重的经济损失,这已经成为农业发展中的突出问题。由于两者扩散迅速、世代周期短、发育速度快、繁殖能力强、所以极易在新的环境定殖,形成新的地理种群。本研究利用微卫星标记的方法,揭示中国二斑叶螨和朱砂叶螨的种群遗传结构,为了解其入侵途径和爆发机制,有效地控制其扩散和危害奠定基础。通过比较两者种群遗传结构的差异,为进一步揭示两者的亲缘关系提供可靠的依据。一、实验材料的采集和饲养在全国范围内选择较有代表性的地区分别对二斑叶螨和朱砂叶螨进行采样,共采集了7个二斑叶螨地理种群和6个朱砂叶螨地理种群,采集的生境主要是果园、花圃和蔬菜地。实验室内采用隔水法在菜豆（Phaseolus vulgaris L.）叶片上进行恒温恒湿隔离饲养,饲养条件为25±1℃,L16:D8,相对湿度60%。二、微卫星标记技术在种群遗传结构中的应用微卫星标记是种群遗传结构研究中应用最多的一种分子标记方法。因其具有在基因组中分布广泛、多态信息含量高、共显性标记、无损伤取样、一旦开发易于检测等诸多优点,而被越来越多的应用到分子生态、群体遗传学等研究领域。自Navajas等1998年和2002年在二斑叶螨体内成功开发出微卫星位点以来,越来越多的研究者将微卫星标记用于螨的种群遗传结构的研究。实验方法:首先需进行单位点扩增,成功扩增物种体内已知的微卫星序列;然后进行各个微卫星位点等位基因和基因型检测,本研究使用的方法是银染—非变性聚丙烯酰胺凝胶电泳法;其次利用相关软件进行数据分析,如种群遗传多样性参数统计,包括等位基因频率、基因型频率、等位基因丰富度、杂合度、基因型多样性等;种群之间分化研究,包括遗传分化指数、基因流、遗传距离等;最后基于种群之间遗传距离的远近,采用距离矩阵法（UPGMA）构建系统发育树,更加直观的反应种群之间的亲缘关系,揭示种群遗传结构,为监控害螨种群涨落趋势,及时有效的对其进行防治具有一定的指导意义。三、中国二斑叶螨种群遗传结构的研究本研究所选用的3个微卫星位点在二斑叶螨体内成功扩增,扩增结果显示每个位点平均等位基因数3.7个,平均多态信息含量（PIC）0.5247,当PIC＞0.5时.该位点为高度多态位点,所以这3个位点作为遗传标记进行二斑叶螨种群遗传结构的研究是切实可行的。表征种群遗传多样性的参数指标(N_A、N_AE、H_O和H_E等)在二斑叶螨各个种群中普遍偏低,种群遗传多样性降低。二斑叶螨各个地理种群检测到的私有等位基因占等位基因总数的27.3%,特有基因型占基因型总数的54.5%,单态位点数占检测总数的33%,总样本的基因型多样性远高于各个地理种群的基因型多样性水平,遗传分化指数平均成对F_ST值为0.5695,当F_ST＞0.25时,种群极度分化。以上结果均说明二斑叶螨各个地理种群之间发生了明显的分化。种群之间遗传分化与地理距离呈现一定的相关性（P＜0.05,R²=0.2594）。二斑叶螨种群遗传结构表现出两个明显的特点:种群遗传多样性降低和种群之间极度分化。造成以上结果的原因有生物本身的特性（如生殖系统）、分布状态（如栖境破碎、地理隔离等）、自然选择、迁徙（基因交流）、遗传漂变等多种因素综合作用的影响,其中地理隔离引起的小居群范围内的近亲交配和等位基因遗传漂变很可能起到了决定性的作用。四、中国朱砂叶螨种群遗传结构的研究朱砂叶螨种群遗传结构的研究结果与二斑叶螨有许多类似的地方,如以总体样本为研究对象均能表现出显著的多态性,然而各个地理种群的遗传多样性水平普遍偏低;种群之间极度分化(F_ST＞＞0.25),但是却发现许多种群之间遗传分化与地理距离不成比例的例子;从统计上确定遗传分化程度与地理距离之间的相关关系,结果显示虽然地理距离与种群分化的相关系数很低（二斑叶螨R²=0.2594;朱砂叶螨R²=0.277）,但是仍然表现出显著的相关性（P＜0.05）。所以朱砂叶螨种群遗传结构也表现出种群遗传多样性降低和种群之间极度分化的特点。五、我国二斑叶螨和朱砂叶螨种群遗传结构的差异分析中国二斑叶螨和朱砂叶螨的种群遗传结构经比较差异显著,主要表现在种群遗传多样性水平不同,朱砂叶螨的总体样本的遗传多样性水平明显高于二斑叶螨,而二斑叶螨各个地理种群的遗传多样性水平却比朱砂叶螨高;种群遗传分化程度不同,朱砂叶螨各个种群之间平均成对F_ST值远高于二斑叶螨,说明朱砂叶螨种群遗传分化的程度远高于二斑叶螨;将采自同一个省市的二斑叶螨和朱砂叶螨进行成对比较发现,地理间距缩短并没有降低种群之间的遗传分化水平,说明地理隔离并不是朱砂叶螨和二斑叶螨分化的主要原因,生殖隔离也可能造成同域内种群分化;系统发育分析结果显示7个二斑叶螨地理种群与6个朱砂叶螨地理种群明显聚在不同的分支上。综上所述,本研究利用微卫星标记技术,揭示中国二斑叶螨和朱砂叶螨的种群遗传结构及两者的差异,这将有助于我们了解它们的入侵途径和爆发机制,为有效控制其扩散和危害奠定基础,同时,也有助于了解这两种叶螨的分类地位,为现在具有极大争议的同种或异种学说提供科学依据。更多还原

【Abstract】 Belonging to the Arachinida, Acari, Acariformes, Tetranychidae and Tetranychus, both the two-spotted spider mite Tetranychus urticae Koch and the carmine spider mite T. cinnabarinus （Boisduval） are widely distributed worldwide. The two spider mites have wide range of host plants, including fruit trees, flowers, vegetables, crops and weeds. They live aggregately on the undersurfaces of leaves to suck plant juice, causing damage to host plant and great loss to agriculture. Owing to their rapid development and high reproductive capacities, new geographical populations of these mites are established easily in other areas. In this study, we used the molecular marker of microsatellite to analyze the population genetic structure of T. urticae Koch and T. cinnabarinus （Boisduval） from China. This work will be helpful for understanding their invading approach and outbreak mechanism, laying a solid foundation for controlling their dispersal.1. The collection and rearing of experimental materialSamples of T. urticae Koch and T. cinnabarinus （Boisduval） used in this study were collected from seven and six geographical localities, respectively. Their collections cover an extensive geographical scale representing different regions and environments in China. Mites were then reared separately in the laboratory on leaves of Phaseolus vulgaris L., placed on a water-saturated tissue in a glass dish. All populations were kept in an illumination culture tank at 25±1℃, 60% R.H. and under L16:D8 conditions.2. The application of microsatellite markers in the studies of population genetic structure.Microsatellite, as a molecular marker, is an important tool in the study of population genetic structure. It has a lot of advantages such as extensive in eukaryotic genome, richness of polymorphism information content, codominant genetic marker, easily to be investigated. Because of many advantages, it has often been used in the fields of molecular ecology, population genetics, and so on. Microsatellite has been used in the population genetic structure of the mite by more and more researchers after successfully exploiting microsatellite locus in Tetranychus urticae Koch by Navajas in 1998 and 2002.The method of this experiment was as follows: （1） to amplify known loci successfully; （2） to investigate alleles and allelic genotype at every locus in every sample; （3） PCR products were separated on polyacrylamide gel and silver staining to showing bands; （4） different softwares were used to analyze parameters of population genetic diversity such as allelic frequency, genotypic frequency, allelic richness, heterozygosity, genotypic diversity, and so on. The study of population genetic differentiation includes genetic differentiation index, gene flow, genetic distance, and so on. Finally, the phylogenetic tree based on genetic distances between populations was established. Cluster analysis was performed using the UPGMA algorithm.3. The population genetic structure of Tetranychus urticae Koch from ChinaThe three microsatellite loci selected in this research were amplified successfully. Amplification results showed that a mean number 3.7 of alleles per locus, the average polymorphism information content （PIC） 0.5247. When the value of PIC is bigger than 0.5, the site is highly polymorphic. So these three loci as the genetic marker for the study of T. urticae Koch population genetic structure are practicable. Parameters standing for population genetic diversity is generally low in various geographical populations, these results illustrate the low level genetic diversity of T. urticae Koch populations. The rates of private allele, special genotype and monomorphic locus detected were 27.3%, 54.5% and 33%, respectively. The genotypic diversity of total samples was much higher than respective population. Average pairwise F_ST was 0.5695, when F_ST>0.25, population was divided extremely. The above results show that extreme division has happened to T. urticae Koch. Genetic differentiation between populations had a direct bearing on the geographical distance.The result of population genetic structure of T. urticae Koch showed two distinct characteristics: the lower level of population genetic diversity and extreme differentiation among populations. These results caused by intrinsic biological feature （such as mating system）, distribution （such as habitat fragmentation, geographical isolation etc）, natural selection, migration （gene flow）, genetic drift, etc. Therefore, both inbreeding within populations and genetic drift of alleles have probably had a decisive effect on the population genetic structure of T. urticae Koch.4. The population genetic structure of T. cinnabarinus （Boisduval） from ChinaThe population genetic structure of T. cinnabarinus （Boisduval） displayed many similarities to T. urticae Koch. The overall sample study showed significant polymorphism, but all the geographical populations had generally lower levels of genetic diversity. There existed an extreme differentiation between populations (F_ST>> 0.25). However, the genetic differentiations among populations were disproportionate with geographical distance. The correlation analysis between the genetic differentiation and the geographical distance from the point view of statistics showed a low correlation coefficient between the geographic distance and the population division （R² = 0.277; R² = 0.2594）, and a significant correlation （P<0.05）. Therefore, the population genetic structure of T. cinnabarinus （Boisduval） also showed a lower level of genetic diversity and the extreme polarization characteristics between populations.5. The differences of population genetic structure between Tetranychus urticae Koch and T. cinnabarinus （Boisduval） from China.By comparing the population genetic structures of T. urticae Koch and T. cinnabarinus （Boisduval） from China, we found significant differences between them. Firstly, they had different levels of genetic diversity of populations in these two species. The overall level of genetic diversity of T. cinnabarinus （Boisduval） was significantly higher than that of T. urticae Koch, but the various geographical levels of genetic diversity in T. cinnabarinus （Boisduval） were oppositely lower than that of T. urticae Koch. Secondly, the degree of population differentiation between the two spider species was different. Average pairwise F_ST between geographical populations of T. cinnabarinus （Boisduval） was far higher than that of T. urticae Koch. This result demonstrated the level of population genetic differentiation of T. cinnabarinus （Boisduval） was much higher than that of T. urticae Koch. Thirdly, by comparing T. cinnabarinus （Boisduval） and T. urticae Koch collected in the same area; we could not find the reduction of the level of genetic differentiation due to the shortening geographical distances between different populations. Geographic isolation between populations wasn’t the main reason for genetic differentiation; reproductive isolation could also result in differentiation among populations in the same area. Finally, phylogenetic analysis revealed that seven geographic populations of T. urticae Koch and six geographic populations of T. cinnabarinus （Boisduval） were gathered in different branches.In conclusion, by using microsatellite marker, we revealed population genetic stricture of T. urticae Koch and T. cinnabarinus （Boisduval） and differences between them. It will help us understand their ways of invasion and outbreak mechanism, and lay foundations for effectively controlling them. Additionally, these results will also be helpful for understanding their classification status, and offering the scientific evidence to homogeneous or heterogeneous theory.更多还原