本文關鍵詞： DNA條形碼 金小蜂科 物種鑒定 京津冀地區(qū)　出處：《河北大學》2017年碩士論文　論文類型：學位論文

【摘要】：科學準確的鑒定物種是進行更深層次生物學研究的基礎和前提。物種的形態(tài)鑒定需要分類學家投入足夠的時間和精力,更需要多年經驗的積累。金小蜂科是一類重要的天敵昆蟲,對控制害蟲種群密度起著重要的作用。但金小蜂個體微小、形態(tài)多樣,是小蜂總科最難鑒定的類群。近幾年DNA條形碼作為鑒定物種的有效手段得到了廣大學者認可。隨著研究的深入,部分學者認為COI基因本身存在一些問題可能會干擾物種鑒定,如線粒體異質性、Wolbachia感染、核線粒體假基因等�；谏鲜鲈�,近年來有學者嘗試將核糖體第二轉錄間隔區(qū)(ITS2)作為DNA條形碼的補充。因此,本研究選擇COI基因和ITS2作為DNA條形碼,對2015-2016年于京津冀地區(qū)采集的金小蜂進行物種鑒定。經過整理,本次采集共得到568頭金小蜂標本。根據標本采集信息和形態(tài)鑒定結果將標本分為325組。隨后提取標本基因組DNA,擴增COI序列和ITS2序列,得到的序列經過校對、拼接、對齊、剪齊后,分別構建COI和ITS2的NJ樹,并使用ABGD、j MOTU等方法對樣本的COI序列劃分分子可操作分類單元。最終,COI、ITS2序列分析共涉及288頭京津冀金小蜂標本和14頭非京津冀地區(qū)金小蜂標本。同時獲得COI和ITS2序列的標本共166頭,結合ABGD、j MOTU、NJ樹三種方法的分析,最終將166頭標本劃分為54分子可操作分類單元。同時參考劃分結果中三種方法對應的各項參數,對只有COI或只有ITS2序列的標本進行分析,最終將302頭標本劃分為96分子可操作分類單元。分析還得到以下結果:(1)獲得COI序列的標本227頭被劃分為72分子可操作分類單元;獲得ITS2序列的標本241頭被劃分為83分子可操作分類單元。(2)根據劃分的分子可操作分類單元,部分屬級形態(tài)鑒定不確定的屬(Arthrolytus、Callimerismus、Chlorocytus、Eurydinota、Sphaeripalpus)屬級階元不成立;Pteromalus、Gastrancistrus、Trichomalopsis部分屬級形態(tài)不確定的標本的屬級階元得到肯定;部分形態(tài)鑒定為Cyrtoptyx、Nasonia、Propicrocytus、Pteromalus標本的屬級階元建議更正。(3)根據劃分的分子可操作分類單元,對部分形態(tài)鑒定不確定的或形態(tài)鑒定有誤的種提出了分類建議;成功將9頭無形態(tài)鑒定結果的標本鑒定至種。(4)新增5中國新紀錄屬,分別為Dinarmus、Dinotoides、Heteroprymna、Sympotomus、Synedrus;京津冀新增記錄8屬,包括Ablaxia、Agiommatus、Ammeia、Cerocephala、Gastrancistrus、Hyperimerus、Norbanus、Zdenekiana。綜上,COI和ITS2鑒定物種非常有效,本研究成功對98%的標本進行了物種鑒定。但少數標本的COI序列遺傳距離存在模糊地帶,導致分種不明確。分析過程中發(fā)現(xiàn)ITS2本身種內高度保守,可以對COI起到非常好的驗證和補充作用。此外,實驗中發(fā)現(xiàn)Spalangia的所有標本均未能成功擴增COI序列,但多頭標本成功擴增到ITS2序列,因此,將ITS2作為DNA條形碼的補充片段很有必要同時選用COI基因和ITS2,不但增加了分子鑒定的說服力,排除了COI基因單獨使用可能存在的問題,更對分析過程中避免了種內遺傳距離界定而導致分種不清的情況。此外,目前金小蜂科的大多數物種尚無可靠的條形碼,因此物種鑒定很大程度依賴于形態(tài)鑒定結果。對于無形態(tài)鑒定結果,且無條形碼的物種,只能將其與其他物種分開而不能確定其為何種。可見,形態(tài)鑒定與分子鑒定密不可分,相輔相成。同時,構建DNA條形碼金小蜂科樣本數據庫非常必要,只有積累更多的物種,才能更好的達到物種鑒定的目的。
[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.

【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q969

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