【作者】 赵广宇；

【导师】 彩万志；

【作者基本信息】 中国农业大学 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 DNA条形码技术因其快速、客观、易操作等优点自产生经过这短短不到十年的时间,已经受到越来越多学者的重视,它已经成为分类鉴定的一种常规手段。昆虫线粒体基因组基因组成稳定,易扩增,普遍为母系遗传,极少发生重组,基因进化速率较核基因快,被广泛用于系统发育、种群遗传结构和谱系地理学等方面的研究。本研究测得812条猎蝽科昆虫COI条形码序列,代表形态鉴定得出的9亚科,75属,150种,对其进行DNA条形码分析,探讨COI条形码在猎蝽科的鉴定效力。针对条形码结果与形态学鉴定相悖的盗猎蝽属三个近缘种污黑盗猎蝽Peirates turpis Walker、茶褐盗猎蝽Peirates fulvescens Lindberg和黄纹盗猎蝽Peirates atromaculatus (Stal),我们又引入了ITS2基因和线粒体基因组方面的数据,一方面为了解决此分类问题,另一方面对于线粒体基因组被用做分子分类的可行性进行研究。结论如下：(1)在本研究中,大部分的物种能够明显的区分开,将近70%的种内差异没有超过之前普遍应用的界限2%,证明COI基因作为条形码区分猎蝽科物种的可行性。这些序列差异在某种程度上可以作为一个参考,不同的情况还是需要我们结合形态学、生态学方面的数据来分析。(2)基于COI基因的DNA条形码研究有助于猎蝽科物种鉴定,有助于发现可能存在的分类问题,比如物种的同物异名现象和隐存种。(3)ITS2数据帮助我们进一步确定COI的鉴定结果,在本研究中,COI和ITS2对于猎蝽科种级分类鉴定显示出非常有效且一致的鉴定效力。ITS2在种内具有高度保守性,其长度具有物种特异性,可作为猎蝽科条形码备选基因与COI结合使用。(4)盗猎蝽属昆虫线粒体基因组大小范围为15,702bp-16,314bp,线粒体基因组大小的差异主要源于非编码区的数量和长度特别是控制区的长度。所有线粒体基因组中,37个基因按与果蝇D. yakuba相同的方式排列。碱基组成都具有明显的AT偏向性。rRNA基因在序列和二级结构上均非常保守。除tRNASer(AGN)的DHU臂由7个不配对的核苷酸组成而无法形成典型的茎环结构外,所有的22个tRNA基因都可以折叠为典型的三叶草结构。(5)基于COI条形码序列、ITS2序列以及比较线粒体基因组学数据,本研究认为长久以来根据前翅色斑型区分的近缘种污黑盗猎蝽、茶褐盗猎蝽和黄纹盗猎蝽应被视为一个种。(6)本研究证实了线粒体基因组可被用作较低阶元种级阶元的分类研究,鉴于高通量测序技术使得高效且成本有效地测定大量样本的线粒体全基因组用于研究种级分类成为可能,线粒体基因组序列未来有可能成为每个种用于分类的标准序列,就像现在COI条形码这样。更多还原

【Abstract】 DNA barcoding has gained more and more attention of scholars because of its rapid, objective and easy-to-operate advantages and became a routine method of classification and identification. Insect mitochondrial genome has been widely applied in the study|of phylogenetic, population structure and phylogeographic studies due to its stable gene content, uniparental inheritance, lack of extensive recombination and high evolve rate.In order to evaluate the effectiveness of barcodes in the identification of assassin bugs, we recovered812barcode records from specimens representing150species belonging to75genera in9subfamilies. Furthermore, we introduced the ITS2gene and mitochondrial genomics data to understand the species’status of three closely related species Peirates turpis Walker, Peirates fulvescens Lindberg and Peirates atromaculatus (Stal), due to the fact that DNA barcoding results opposed the current taxonomic status of these three species. On the other hand, we tested the utility of mitogenomic data in species identification. Conclusions were drawn as follows:(1)In this study, most species can be distinguished obviously, analysis of the COI gene revealed less than2%intra-specific divergence in nearly70%of the taxa examined, these results verified the utility of COI gene as DNA barcode in the discrimination of Reduviidae. These sequence divergences can be used as a reference to some extent, although we need to combine morphological and ecological data in different situations.(2) Our results indicated that DNA barcodes will aid the identification of Reduviidae, the discovery of taxonomic problems such as synonym and cryptic species that may exist.(3) ITS2data showed similar results with DNA barcoding analysis. The identification of Reduviidae based on COI and ITS2data showed effective and consistent performance. ITS2sequences were highly conserved within species, its lengths was species-specific. These advantages made ITS2a candidate barcode of Reduviidae.(4) Peirates mitochondrial genomes ranged in size from15702bp to16314bp, with length variations mainly in non-coding regions especially in control regions. The mitogenomes all harboured a typical set of37genes and an identical gene order to the mitogenome of D. yakuba. The nucleotide compositions were biased toward adenine and thymine. In addition, comparative studies of ribosomal RNAs showed high conservation at both primary sequence level and secondary structures level among these six mitogenomes. Among all tRNA genes, only trnSl did not exhibit the classic clover-leaf secondary structure, due to the deficiency of the dihydrouridine (DHU) arm.(5) P. atromaculatus, P. fulvescens and P. turpis should be treated as members of a single species inferred from COI, ITS2and mitochondrial genome data. (6) The utility of mitogenomic data in species identification has been testifie in this study, Furthermore, the generation of next-generation sequencing (NGS) technologies makes it possible to efficiently and cost-effectively obtain entire mt genome from large number of samples for resolving relationships at the level of species. Therefore, mitogenome sequences in future may be obtained for each species and become the standard for taxonomic sequencing, much like the COI barcodes today.更多还原