摘要
【目的】建立基于线粒体COⅠ和COⅡ基因序列的沙果小食心虫、梨小食心虫分子鉴定方法。【方法】田间采集试虫,利用PCR和基因测序技术,扩增2种食心虫的线粒体COⅠ和COⅡ基因,将获得的COⅠ和COⅡ基因序列在GenBank中进行BLAST比对,并通过GeneDoc软件分析基因序列相似性,利用Mega 5.05软件分别计算基于COⅠ和COⅡ基因序列的遗传距离,并分别构建COⅠ和COⅡ基因的系统发育树。【结果】在分析的2种食心虫样本中,沙果小食心虫COⅠ基因的种内相似度在99.4%以上,梨小食心虫COⅠ基因的种内相似度在99.6%以上,2种食心虫COⅠ基因的种间相似度为95.0%-95.6%;沙果小食心虫COⅡ基因的种内相似度在99.0%以上,梨小食心虫COⅡ基因的种内相似度在99.3%以上,2种食心虫COⅡ基因的种间相似度为94.6%-95.5%;2种食心虫COⅠ基因种间存在30个稳定变异位点,COⅡ基因种间有26个稳定变异位点。2种食心虫种内遗传距离为0.001-0.006（COⅠ）和0.001-0.010（COⅡ）,种间遗传距离为0.046-0.052（COⅠ）和0.047-0.057（COⅡ）,基于COⅠ和COⅡ基因的种间遗传距离均显著大于种内遗传距离。分别构建COⅠ和COⅡ基因单倍型的系统发育树,结果显示,在2个基因的系统发育树上,2种食心虫的基因序列分别位于不同的进化支,且置信度均达到100%,同种食心虫的基因序列位于相同的进化枝。【结论】可以根据本研究所用的COⅠ和COⅡ基因序列的差异性,进行沙果小食心虫和梨小食心虫的分子鉴定。
【Objective】This study aimed to establish a method for identifing Grapholita dimorpha Komai and Grapholita molesta Busck based on mitochondrial COⅠ and COⅡ genes.【Method】PCR andgene sequencing methods were used to obtain COⅠ and COⅡ sequences from the samples of the two species collected from apple orchards.The acquired COⅠ and COⅡ sequences were verified by BLAST program in GenBank.GeneDoc was also used to analyze the similarity between the obtained sequences.Mega5.05 was used to analyze the genetic distances between sequences and to construct the phylogenetic trees of COⅠ and COⅡ gene haplotypes,respectively.【Result】The intraspecific similarities of COⅠ sequences of G.dimorpha and G.molesta were above 99.4% and 99.6%,respectively,whilst the interspecific similarity of COⅠ gene sequences between them was 95.0%-95.6%.The intraspecific similarities of COⅡ sequences of G.dimorphaand G.molesta were above 99.0% and 99.3%,while its interspecific similarity between them was 94.6%-95.5%.A total of 30 variable sites were detected in COⅠ gene sequences from samples of the two species,and 26 were detected in COⅡ sequences.The intraspecific genetic distances were 0.001-0.006（COⅠ）and 0.001-0.010（COⅡ）,whereas the interspecific genetic distances were0.046-0.052（COⅠ）and 0.047-0.057（COⅡ）.The interspecific genetic distance was significantly higher than intraspecific genetic distance.Phylogenetic trees based on COⅠ gene haplotypes and COⅡ gene haplotypes indicated that gene sequences from the same species were clustered in the same branch,whereas gene sequences from different species were clustered in different branches,with the bootstrap value of100%.【Conclusion】The COⅠand COⅡ gene sequences used in this study can be applied in the molecular identification of G.dimorphaand G.molesta.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2016年第2期156-164,共9页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(31071687)
教育部高等学校博士学科点专项科研基金(博导类)项目(20110204110001)
西北农林科技大学人才引进项目
国家科技支撑计划项目(2012BAK11B03)
