本文關(guān)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(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：科學(xué)準(zhǔn)確的鑒定物種是進(jìn)行更深層次生物學(xué)研究的基礎(chǔ)和前提。物種的形態(tài)鑒定需要分類(lèi)學(xué)家投入足夠的時(shí)間和精力,更需要多年經(jīng)驗(yàn)的積累。金小蜂科是一類(lèi)重要的天敵昆蟲(chóng),對(duì)控制害蟲(chóng)種群密度起著重要的作用。但金小蜂個(gè)體微小、形態(tài)多樣,是小蜂總科最難鑒定的類(lèi)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(xué)者認(rèn)可。隨著研究的深入,部分學(xué)者認(rèn)為COI基因本身存在一些問(wèn)題可能會(huì)干擾物種鑒定,如線(xiàn)粒體異質(zhì)性、Wolbachia感染、核線(xiàn)粒體假基因等�；谏鲜鲈�,近年來(lái)有學(xué)者嘗試將核糖體第二轉(zhuǎn)錄間隔區(qū)(ITS2)作為DNA條形碼的補(bǔ)充。因此,本研究選擇COI基因和ITS2作為DNA條形碼,對(duì)2015-2016年于京津冀地區(qū)采集的金小蜂進(jìn)行物種鑒定。經(jīng)過(guò)整理,本次采集共得到568頭金小蜂標(biāo)本。根據(jù)標(biāo)本采集信息和形態(tài)鑒定結(jié)果將標(biāo)本分為325組。隨后提取標(biāo)本基因組DNA,擴(kuò)增COI序列和ITS2序列,得到的序列經(jīng)過(guò)校對(duì)、拼接、對(duì)齊、剪齊后,分別構(gòu)建COI和ITS2的NJ樹(shù),并使用ABGD、j MOTU等方法對(duì)樣本的COI序列劃分分子可操作分類(lèi)單元。最終,COI、ITS2序列分析共涉及288頭京津冀金小蜂標(biāo)本和14頭非京津冀地區(qū)金小蜂標(biāo)本。同時(shí)獲得COI和ITS2序列的標(biāo)本共166頭,結(jié)合ABGD、j MOTU、NJ樹(shù)三種方法的分析,最終將166頭標(biāo)本劃分為54分子可操作分類(lèi)單元。同時(shí)參考劃分結(jié)果中三種方法對(duì)應(yīng)的各項(xiàng)參數(shù),對(duì)只有COI或只有ITS2序列的標(biāo)本進(jìn)行分析,最終將302頭標(biāo)本劃分為96分子可操作分類(lèi)單元。分析還得到以下結(jié)果:(1)獲得COI序列的標(biāo)本227頭被劃分為72分子可操作分類(lèi)單元;獲得ITS2序列的標(biāo)本241頭被劃分為83分子可操作分類(lèi)單元。(2)根據(jù)劃分的分子可操作分類(lèi)單元,部分屬級(jí)形態(tài)鑒定不確定的屬(Arthrolytus、Callimerismus、Chlorocytus、Eurydinota、Sphaeripalpus)屬級(jí)階元不成立;Pteromalus、Gastrancistrus、Trichomalopsis部分屬級(jí)形態(tài)不確定的標(biāo)本的屬級(jí)階元得到肯定;部分形態(tài)鑒定為Cyrtoptyx、Nasonia、Propicrocytus、Pteromalus標(biāo)本的屬級(jí)階元建議更正。(3)根據(jù)劃分的分子可操作分類(lèi)單元,對(duì)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(lèi)建議;成功將9頭無(wú)形態(tài)鑒定結(jié)果的標(biāo)本鑒定至種。(4)新增5中國(guó)新紀(jì)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(duì)98%的標(biāo)本進(jìn)行了物種鑒定。但少數(shù)標(biāo)本的COI序列遺傳距離存在模糊地帶,導(dǎo)致分種不明確。分析過(guò)程中發(fā)現(xiàn)ITS2本身種內(nèi)高度保守,可以對(duì)COI起到非常好的驗(yàn)證和補(bǔ)充作用。此外,實(shí)驗(yàn)中發(fā)現(xiàn)Spalangia的所有標(biāo)本均未能成功擴(kuò)增COI序列,但多頭標(biāo)本成功擴(kuò)增到ITS2序列,因此,將ITS2作為DNA條形碼的補(bǔ)充片段很有必要同時(shí)選用COI基因和ITS2,不但增加了分子鑒定的說(shuō)服力,排除了COI基因單獨(dú)使用可能存在的問(wèn)題,更對(duì)分析過(guò)程中避免了種內(nèi)遺傳距離界定而導(dǎo)致分種不清的情況。此外,目前金小蜂科的大多數(shù)物種尚無(wú)可靠的條形碼,因此物種鑒定很大程度依賴(lài)于形態(tài)鑒定結(jié)果。對(duì)于無(wú)形態(tài)鑒定結(jié)果,且無(wú)條形碼的物種,只能將其與其他物種分開(kāi)而不能確定其為何種�？梢�(jiàn),形態(tài)鑒定與分子鑒定密不可分,相輔相成。同時(shí),構(gòu)建DNA條形碼金小蜂科樣本數(shù)據(jù)庫(kù)非常必要,只有積累更多的物種,才能更好的達(dá)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(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q969

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