›› 2017, Vol. 60 ›› Issue (6): 681-690.doi: 10.16380/j.kcxb.2017.06.008

• 研究论文 • 上一篇    下一篇


陈珊, 周琼*, 李纲   

  1. (湖南师范大学生命科学学院, 长沙 410081)
  • 出版日期:2017-06-20 发布日期:2017-06-20

DNA barcoding of various developmental stages of Endoclita davidi (Lepidoptera: Hepialidae) based on mtDNA COI gene sequence

CHEN Shan, ZHOU Qiong*, LI Gang   

  1. (College of Life Sciences, Hunan Normal University, Changsha 410081, China)
  • Online:2017-06-20 Published:2017-06-20

摘要: 【目的】巨疖蝙蛾Endoclita davidi (Poujade, 1886)是雪峰虫草菌Ophiocordyceps xuefengensis的重要寄主。本研究分析了该虫线粒体DNA COI 基因条形码,以在不同虫态及被虫草菌寄生的僵虫状态下能快速准确鉴定寄主种类。【方法】PCR扩增巨疖蝙蛾成虫(足一对,胸肌,翅基部)、卵(单粒)、幼虫(胸足一对)、蛹(少量体壁组织)及虫草僵虫(胸足或体壁)组织共计21个样品的COI基因片段(约658 bp)并进行测序和比对;以MEGA6.0软件的K2-P双参数模型计算种内及种间遗传距离;以邻接法(neighbor-joining, NJ)和最大简约法(maximum parsimony, MP)两种方法构建系统发育树。【结果】聚类分析与形态学鉴定结果一致,所纳入分析的20个样品均为同一物种,节点支持率可达99%。种内遗传距离为0.00%~1.08%,平均0.54%。与蝙蝠蛾科其他6种昆虫种间遗传距离为5.36%~13.31%,平均10.47%;种间遗传距离为种内遗传距离的19.39倍(10.47% vs 0.54%),而且种内与种间遗传距离没有重叠区域。【结论】基于mtDNA COI基因DNA条形码技术可以用于巨疖蝙蛾的快速准确鉴定,并对蝙蝠蛾科(Hepialidae)昆虫的分子系统发育分析有重要意义。

关键词: 巨疖蝙蛾, DNA 条形码, 线粒体DNA COI基因, 物种鉴定, 分子系统发育

【Aim】 Endoclita davidi is the primary host of the entomogenous fungus Ophiocordyceps xuefengensis. In order to identify the host species in different developmental stages and its caterpillar infected by the Cordyceps fungi, the gene sequence of mtDNA cytochrome oxidase subunit I (COI) of E. davidi was analyzed for DNA barcoding. 【Methods】 The partial fragments (about 658 bp) of the mtDNA COI gene were amplified from 21 samples of tissues of adults (thoracic legs, pectorals, wing base), egg, larva (thoracic legs), pupa (body wall), caterpillar fungus (thoracic legs, body wall) of E. davidi by PCR, and the obtained COI gene fragments were sequenced and aligned. The intra-and inter-species genetic distances were calculated with MEGA version 6.0 software using the Kimura 2-parameter model. The phylogenetic trees were constructed among the closely-related species with neighbor-joining (NJ) and maximum parsimony (MP) methods. 【Results】 The identification results based on the phylogenetic tree were consistent with those based on the morphological analysis of the 20 samples, which were all verified to be originated from the same host species. The bootstrap values of the original divergence among different haplotypes within the same species were over 99%. The average inter-species genetic distance between  E. davidi and other six hepialid species was 19.39 times as high as the average intra-species genetic distance of the 20 samples of E. davidi (10.47% vs 0.54%). There was no overlap between intra-and inter-species genetic distances. 【Conclusion】 The results indicate that DNA barcoding based on the partial sequence of mtDNA COI gene provides rapid and accurate identification of E. davidi. The technology has important significance for molecular phylogenetic studies of Hepialidae.

Key words: Endoclita davidi; DNA barcoding, mtDNA COI gene, species identification, molecular phylogeny