COI基因DNA条形码技术在福建省蜱类鉴定中的应用

目的 探讨基于线粒体细胞色素C氧化酶亚基I(COI)基因的DNA条形码技术应用于福建省蜱类鉴定的可行性。方法 蜱标本经形态学鉴定后提取基因组DNA,PCR扩增COI基因片段,并进行测序和比对,用Kimura-2-parameter模型计算种内及种间遗传距离,以邻接法构建系统发育树。结果 经形态学鉴定,97只蜱隶属于5属12种,遗传距离计算结果显示,种间遗传距离(14.65%~26.79%)显著大于种内遗传距离(0~7.14%);BLAST同源性比对发现GenBank数据库中尚无越原血蜱(Haemophysalis Yeni)与基刺硬蜱(Ixodes spinicoxalis)的COI基因序列,褐黄血蜱(H.flava)、台岛血蜱(H.formosensis)、豪猪血蜱(H.hystricis)、粒形硬蜱(I.granulatus)、微小扇头蜱(Rhipicephalus microplus)与血红扇头蜱(R.sanguineus)的形态学鉴定与DNA条形码鉴定结果一致,但是,龟形花蜱(Amblyomma testudinarium)、台湾革蜱(Dermacentor taiwanensis)、中华硬蜱(I.sinensis)的形态学鉴定与COI基因DNA条形码鉴定结果不一致;系统发育树显示,同一物种的不同个体均形成高支持率的单系,种间分支很明显。结论 COI基因DNA条形码测定是一种快速准确的蜱类鉴定新兴技术,但现阶段,COI基因DNA条形码技术尚不能完全脱离传统的形态学鉴定而独立进行,两者应有机结合。

Application of DNA barcoding of COI gene in identification of ticks in Fujian Province,China

To explore the feasibility of mitochondrial cytochrome C oxidase subunit I(COI)gene based DNA barcoding for the identification of ticks in Fujian Province, China. Genomic DNA was extracted from 97 tick specimens belonging to 12 species of 5 genera by morphological identification. COI gene fragments were amplified by PCR, sequenced and aligned. The intra-and inter-species genetic distances were calculated by Kimura-2-parameter model, and a phylogenetic tree was constructed by the neighbor-joining(NJ) method.Results showed that the inter-species genetic distance(14.65%-26.79%) was significantly greater than the intra-species genetic distance(0-7.14%);BLAST homology comparison suggested that there was no COI gene sequence of Haemophysalis Yeni and Ixodes spinicoxalis in Genbank database. Morphological identification of H.flava, H.formosensis, H.hystricis, I.granulatus, Rhipicephalus microplus and R.sanguineus were consistent, while Amblyomma testudinarium, Dermacentor taiwanensis, I.sinensis were inconsistent with DNA barcode identification results. The NJ tree showed that different individuals from the same species formed monophyletic groups with high support value,and inter-species branches were clear.The study has shown that DNA barcoding determination of COI gene as new technique for tick identification is fast and accurate, but at this stage, DNA barcoding of COI gene can not be completely independent from traditional morphological identification, and they should be combined organically.