京津冀金小蜂科DNA条形码

发布时间：2018-01-24 20:20

本文关键词： DNA条形码金小蜂科物种鉴定京津冀地区　出处：《河北大学》2017年硕士论文　论文类型：学位论文

【摘要】：科学准确的鉴定物种是进行更深层次生物学研究的基础和前提。物种的形态鉴定需要分类学家投入足够的时间和精力,更需要多年经验的积累。金小蜂科是一类重要的天敌昆虫,对控制害虫种群密度起着重要的作用。但金小蜂个体微小、形态多样,是小蜂总科最难鉴定的类群。近几年DNA条形码作为鉴定物种的有效手段得到了广大学者认可。随着研究的深入,部分学者认为COI基因本身存在一些问题可能会干扰物种鉴定,如线粒体异质性、Wolbachia感染、核线粒体假基因等。基于上述原因,近年来有学者尝试将核糖体第二转录间隔区(ITS2)作为DNA条形码的补充。因此,本研究选择COI基因和ITS2作为DNA条形码,对2015-2016年于京津冀地区采集的金小蜂进行物种鉴定。经过整理,本次采集共得到568头金小蜂标本。根据标本采集信息和形态鉴定结果将标本分为325组。随后提取标本基因组DNA,扩增COI序列和ITS2序列,得到的序列经过校对、拼接、对齐、剪齐后,分别构建COI和ITS2的NJ树,并使用ABGD、j MOTU等方法对样本的COI序列划分分子可操作分类单元。最终,COI、ITS2序列分析共涉及288头京津冀金小蜂标本和14头非京津冀地区金小蜂标本。同时获得COI和ITS2序列的标本共166头,结合ABGD、j MOTU、NJ树三种方法的分析,最终将166头标本划分为54分子可操作分类单元。同时参考划分结果中三种方法对应的各项参数,对只有COI或只有ITS2序列的标本进行分析,最终将302头标本划分为96分子可操作分类单元。分析还得到以下结果:(1)获得COI序列的标本227头被划分为72分子可操作分类单元;获得ITS2序列的标本241头被划分为83分子可操作分类单元。(2)根据划分的分子可操作分类单元,部分属级形态鉴定不确定的属(Arthrolytus、Callimerismus、Chlorocytus、Eurydinota、Sphaeripalpus)属级阶元不成立;Pteromalus、Gastrancistrus、Trichomalopsis部分属级形态不确定的标本的属级阶元得到肯定;部分形态鉴定为Cyrtoptyx、Nasonia、Propicrocytus、Pteromalus标本的属级阶元建议更正。(3)根据划分的分子可操作分类单元,对部分形态鉴定不确定的或形态鉴定有误的种提出了分类建议;成功将9头无形态鉴定结果的标本鉴定至种。(4)新增5中国新纪录属,分别为Dinarmus、Dinotoides、Heteroprymna、Sympotomus、Synedrus;京津冀新增记录8属,包括Ablaxia、Agiommatus、Ammeia、Cerocephala、Gastrancistrus、Hyperimerus、Norbanus、Zdenekiana。综上,COI和ITS2鉴定物种非常有效,本研究成功对98%的标本进行了物种鉴定。但少数标本的COI序列遗传距离存在模糊地带,导致分种不明确。分析过程中发现ITS2本身种内高度保守,可以对COI起到非常好的验证和补充作用。此外,实验中发现Spalangia的所有标本均未能成功扩增COI序列,但多头标本成功扩增到ITS2序列,因此,将ITS2作为DNA条形码的补充片段很有必要同时选用COI基因和ITS2,不但增加了分子鉴定的说服力,排除了COI基因单独使用可能存在的问题,更对分析过程中避免了种内遗传距离界定而导致分种不清的情况。此外,目前金小蜂科的大多数物种尚无可靠的条形码,因此物种鉴定很大程度依赖于形态鉴定结果。对于无形态鉴定结果,且无条形码的物种,只能将其与其他物种分开而不能确定其为何种。可见,形态鉴定与分子鉴定密不可分,相辅相成。同时,构建DNA条形码金小蜂科样本数据库非常必要,只有积累更多的物种,才能更好的达到物种鉴定的目的。
[Abstract]:The identification of the species accurately is the premise and basis for further biological research. Species identification of taxonomists need to devote enough time and energy to the accumulation of years of experience. Pteromalidae is one of the important natural enemies of insects, pest control, population density plays an important role. But p.puparum individual small, diverse forms, is the most difficult to identify groups of Chalcidoidea. In recent years, DNA bar code as an effective means of species identification by the majority of scholars. With the deepening of research, some scholars believe that the COI gene itself has some problems that may interfere with species identification, such as mitochondrial heterogeneity, Wolbachia infection, nuclear pseudogene. Based on the above reasons, in recent years, scholars have tried to ribosomal internal transcribed spacer second (ITS2) as a supplement to the DNA barcode. Therefore, this study selected COI gene and ITS2 DNA bar code, species identification of 2015-2016 on the acquisition of the Beijing Tianjin Hebei region p.puparum. After finishing, the acquisition of a total of 568 head p.puparum specimens. According to specimen collection information and morphological identification results of samples were divided into 325 groups. Then extract genomic DNA, amplification of COI sequence and ITS2 sequence, the sequence after proofreading, stitching, alignment, trim, COI and ITS2 respectively to construct the NJ tree, and use ABGD, J MOTU and other methods of sample COI sequence molecular operational taxonomic unit. Finally, COI ITS2 sequence analysis, involving a total of 288 head of Beijing Tianjin Hebei p.puparum specimens and 14 non Beijing Tianjin Hebei gold wasp specimens. Simultaneously obtained COI and ITS2 sequences were 166, with ABGD, J MOTU, NJ tree analysis of the three methods, the final 166 heads were divided into 54 molecular operational taxonomic units. At the same time reference division results corresponding to three kinds of methods in the The parameters of only COI or only ITS2 sequences were analyzed, the final 302 heads were divided into 96 molecular operational taxonomic unit. The analysis results are as follows: (1) sequence of COI samples from 227 head was divided into 72 molecular operational taxonomic unit; ITS2 sequence is divided 241 head specimens 83 molecular operational taxonomic unit. (2) operational taxonomic units according to the division of the molecule, belonging to the class of uncertain morphological identification of genus (Arthrolytus, Callimerismus, Chlorocytus, Eurydinota, Sphaeripalpus) level is not established; Pteromalus, Gastrancistrus, Trichomalopsis were uncertain of the genus form belongs to the order yuan to be sure; the form was identified as Cyrtoptyx, Nasonia, Propicrocytus, Pteromalus specimens of the genus level. (3) suggest corrections can be operated according to the division of the molecular classification unit, the uncertainty on the part of morphological identification The wrong kind or morphological identification is presented; the success of the 9 head no results of morphological identification were identified to species. (4) Chinese added 5 new record genus, respectively Dinarmus, Dinotoides, Heteroprymna, Sympotomus, Synedrus; Tianjin new records of 8 genera, including Ablaxia, Agiommatus, Ammeia, Cerocephala Gastrancistrus, Hyperimerus, Norbanus, Zdenekiana., COI and ITS2 in the identification of the species is very effective, this study successfully carried out samples of 98% species identified. But the genetic distance of COI sequence and a few specimens are fuzzy zone, cause minutes is not clear. In the process of analysis showed that ITS2 itself in the highly conservative, can be very good to verify and supplement of COI. In addition, the experiment found that all samples of Spalangia were not successful amplification of COI sequences, but the bulls were successfully amplified to ITS2 sequence, therefore, ITS2 as a complement of DNA barcode It is necessary to charge at the same time using the COI gene fragment and ITS2, not only increase the molecular identification and persuasion, ruled out the use of COI gene alone may exist, more of the analysis process to avoid intraspecific genetic distance to define minutes is unclear. In addition, most species of bee in gold and there is no reliable the bar code, so the identification of species depends largely on the results of morphological identification. For the morphological identification, and no barcode species can only be separated from other species and can not determine what. Obviously, the morphological and molecular identification of closely related each other. At the same time, it is necessary to build DNA barcode Pteromalidae sample database only, the accumulation of more species, in order to better achieve the purpose of species identification.

【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q969

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