基于线粒体COⅠ和COⅡ基因的米象与玉米象的分子鉴定

Molecular Identification of Sitophilus Oryzea(Linnaeus) and Sitophilus Zeamais Motschulsky Based on Mitochondrial COⅠ and COⅡ Gene

为建立基于线粒体COI和COⅡ基因序列的米象和玉米象的分子鉴定方法,对采自全国6个地理种群的米象和2个地理种群的玉米象,PCR扩增试虫线粒体COI和COⅡ基因,将获得的基因序列在GenBank中进行BLAST比对,分析基因序列相似性,分别计算基于COI和COⅡ基因序列的遗传距离,并构建系统发育树。分析结果表明,米象COI基因的种内相似度在98.17%以上,玉米象COI基因的种内相似度100%,米象和玉米象COI基因的种间相似度为85.84%~86.92%;米象COⅡ基因的种内相似度在98.37%以上,玉米象COⅡ基因的种内相似度在97.77%以上,2种象虫COⅡ基因的种间相似度为86.27%~88.31%。米象的种内遗传距离为0.002~0.020(COI)和0.011~0.015(COⅡ),玉米象的种内遗传距离为0.001~0.023(COⅡ)。两种象虫的种间遗传距离为0.145~0.185(COI)和0.128~0.154(COⅡ),基于COI和COⅡ基因的种间遗传距离显著高于种内遗传距离。系统发育树结果显示,在2个基因的系统发育树上,米象和玉米象的基因序列分别位于不同的进化支,同种象虫的基因序列位于相同的进化枝。根据本研究所用的线粒体COI和COⅡ基因序列的差异性可以进行米象和玉米象的分子鉴定。

This study aimed to establish a molecular identification method for S. oryzea and S. zeamais based on mitochondrial COⅠ and COⅡ gene sequences. Six field populations of S. oryzea and two field populations of S. zeamais were collected from different cities in China. PCR and gene sequencing methods were used to obtain COⅠ and COⅡ sequences from the samples. The acquired COⅠ and COⅡ sequences have been verified by BLAST program in GenBank and analyzed the similarity of these gene sequences. Then, the acquired sequences were analyzed the genetic distances and to construct the phylogenetic trees of COⅠ and COⅡ gene haplotype, respectively. Result showed that the intraspecific similarity of COⅠ sequences of S. oryzea and S. zeamais were over 98.17% and 100%, the interspecific similarity of COⅠ sequences between S. oryzea and S. zeamais was 85.84%~86.92%. The intraspecific similarity of COⅡ sequences of S. oryzea and S. zeamais were over 98.37% and 97.77%, its interspecific similarity between them was 86.27%~88.31%. The intraspecific genetic distance of S. oryzea was 0.002~0.020(COⅠ)and 0.011~0.023(COⅡ), The intraspecific genetic distance of S. zeamais was 0.001~0.023(COⅡ);and the interspecific genetic distance were 0.145~0.185(COⅠ)and 0.128~0.154(COⅡ). Also, the interspecific genetic distance was significantly higher than the interspecific genetic distance. Phylogenetic trees showed that the gene sequences were located in different evolutionary branches, and the gene sequences of the same species were located in the same evolutionary branches. Mitochondrial COⅠ and COⅡ gene sequences used in this study can be applied in Molecular identification of S. oryzea and S. zeamais.