DAN条形码在小食心虫属鉴定中的应用研究

发布时间：2018-03-01 03:30

本文关键词： 小食心虫 DNA条形码 COI ITS2 分子鉴定系统发育　出处：《山西农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：小食心虫属(鳞翅目：卷蛾科)的一些物种是我国北方果树的重要害虫,其幼虫蛀果危害,严重影响植物的生长和结实,常给农林生产造成很大的损失。为了更好的防治这类害虫,准确的分类鉴定尤为必要。目前,经典的形态学鉴定是最常用的方法,但需要由专业人员鉴定,且近缘种的鉴定较难掌握,因此需要寻求一种可以对物种进行简单、快速、准确的辅助鉴定方法。随着分子生物学技术在昆虫分类中的广泛应用,DNABarcoding为快速准确地鉴定小食心虫提供了新思路。本论文以小食心虫属部分种类作为研究对象,以线粒体COI基因片段作为主要分子标记,核糖体内转录间隔区ITS2序列为辅助分子标记,对不同性别、不同虫态、不同地域的梨小食心虫、李小食心虫、麻小食心虫和沙果小食心虫线粒体COI基因片段进行了测序,结合GenBank[中已递交的小食心虫序列,对主要小食心虫进行系统发育分析；对梨小食心虫、李小食心虫、麻小食心虫和沙果小食心虫部分个体的ITS2部分序列进行了初步研究,结果如下：1.四种小食心虫经过传统形态学鉴定,扩增不同地理种群的COI和ITS2序列情况如下：梨小食心虫COI序列35条,有5个单倍体基因型,ITS2序列13条;沙果小食心虫COI序列1条,ITS2序列1条；李小食心虫COI序列4条,有4个单倍体基因型,ITS2序列4条；麻小食心虫COI序列14条,有3个单倍体基因型,ITS2序列4条;共获得小食心虫属COI序列54条,ITS2序列22条。进一步丰富了小食心虫属DNA条形码序列信息库。2.对不同性别,不同虫态,不同地域,不同物种小食心虫COI基因序列片段分析发现：COI基因序列无性别、虫态差异,也无明显地理差异(李小食心虫除外),不同物种有固定的核酸位点,表明COI基因片段作为分子标记,对种内不同地理种群、不同性别、不同虫态的识别效力有限。3.对四种小食心虫属昆虫与外群苹果蠹蛾大量样本线粒体部分COI基因序列研究发现,考虑到地理因素和近缘种因素后,结合种间-种内遗传距离分析,并运用邻接法(NJ)和最大简约法(MP)法建树分析,发现小食心虫属内麻小食心虫与外群苹果蠹蛾的亲缘关系较与属内昆虫的亲缘关系更近,表明COI序列单独作为小食心虫分类DNA条形码标记基因只具有一定的识别效力。4.对小食心虫属(四种)大量样本核基因ITS2研究发现,考虑到地理因素和近缘种因素后,并结合种间-种内遗传距离分析和NJ、MP法建树分析,表明ITS2序列适合作为鳞翅目理想的辅助分子标记。5.利用COI和ITS2基因探讨小食心虫的亲缘关系：梨小食心虫与沙果小食心虫亲缘关系最近,其次是和李小食心虫,和麻小食心虫的亲缘关系最远。
[Abstract]:Some species of the genus Lepidoptera (Lepidoptera: Crixidae) are important pests of fruit trees in northern China, and their larvae are harmful to fruit borer, which seriously affects the growth and fruiting of plants. It often causes great losses to agricultural and forestry production. In order to better control such pests, accurate classification and identification is particularly necessary. At present, classical morphological identification is the most commonly used method, but it needs to be identified by professionals. And the identification of relative species is difficult to master, so we need to find a simple and fast way to identify the species. With the wide application of molecular biological techniques in insect taxonomy, DNA Barcoding provides a new idea for rapid and accurate identification of minnow. The mitochondrial COI gene fragment was used as the main molecular marker, and the ITS2 sequence of ribosomal intertranscription spacer was used as the auxiliary molecular marker. The mitochondrial COI gene fragments were sequenced and phylogenetic analysis was carried out for the main minisculids in combination with the previously submitted GenBank sequences. The ITS2 partial sequences of some individuals were studied. The results were as follows: 1. The four species were identified by traditional morphology. The COI and ITS2 sequences of different geographic populations were amplified as follows: 35 COI sequences, 13 haploid genotypes, 1 COI sequence, 1 ITS2 sequence and 4 COI sequences, respectively. There were 4 haploid genotypes, 4 ITS2 sequences, 14 COI sequences and 4 haploid genotypes. A total of 54 COI sequences and 22 ITS2 sequences were obtained, which further enriched the information base of DNA barcode sequences. 2. The sequence analysis of COI gene in different species showed that there was no sex, no difference in insect status, and no significant geographical difference (except for C. prunculus, different species had fixed nucleic acid loci, indicating that COI gene fragment was used as a molecular marker. For different geographical populations, different genders and different insect species, the recognition efficiency of different species was limited. 3. The mitochondrial partial COI gene sequences of a large number of samples of four species of the genus Coccidioctonia and the outer coterworm moth were studied, and the results were as follows: 1. Taking into account geographical factors and relative species, combined with the analysis of interspecific and intra-specific genetic distance, and using the method of NJ) and the maximum minimization method (MPP) to construct the tree analysis, It was found that the phylogenetic relationship between the inner eater and the outer coda was closer to that of the intra genus. The results showed that the COI sequence alone as the DNA barcode marker gene in the classification of hearth only had a certain recognition effect. 4. For a large number of nuclear gene ITS2 of the genus (four species), it was found that, considering geographical factors and relative species factors, Based on the analysis of genetic distance between species and intraspecies and NJMP method, The results showed that the ITS2 sequence was suitable as an ideal auxiliary molecular marker for Lepidoptera. 5. Using COI and ITS2 genes to explore the genetic relationship of minnow, the relationship between Pear fruit-eater and fruit-eater was the most recent, followed by Phellodendron prickly. It is the furthest related to the orifices.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433

【相似文献】