基于Cytb基因的穿山甲科物种鉴定研究

Species Identification for Pangolins Based on Cytochrome b Gene

为鉴别混有非穿山甲物种疑似检材的来源物种,基于细胞色素b基因设计了穿山甲科特异的新引物DCW-F1/DCW-R1。试验表明:其特异性良好、灵敏度较高、适用检材类型较广。采用BLAST比对法、遗传距离法和系统发育法分析了4组不同长度Cytb序列,研究序列长度对穿山甲物种鉴定的影响。结果表明,多数样本与相应穿山甲物种同源序列相似度均高于98%,建议将该值作为BLAST比对法的判别标准;除马来穿山甲(211 bp)和树穿山甲(421和211 bp)外,剩余5个穿山甲物种种间最小遗传距离均大于种内最大遗传距离,序列长度的变化和样本量的不平衡会导致该比值不稳定;除马来穿山甲和菲律宾穿山甲,其余物种均为单系,序列长度变短使得系统发育树拓扑结构和分支置信度发生相应变化。

A pair of new specific primers DCW-F1/DCW-R1 were designed based on Cytochrome b(Cytb)gene to identify suspected specimens mixed with non-pangolin species.The results showed that it was good specificity,high sensitivity and could be used for several type samples.Secondly,we used BLAST comparison,genetic distance and constructing phylogenetic tree to analyze the four groups of Cytb sequences with different lengths,in order to estimate the influence of changing sequence length on pangolin identification.The results indicated that the similarity of homologous sequence between most samples and corresponding pangolin was higher than 98%.This suggested that the value should be used as the criterion to identify pangolin species.The minimum inter-specific genetic distance(inter-d)was greater than the maximum Intra-specific genetic distance(intra-d)among five pangolins with the exception of M.javanica(211bp)and P.tricuspis(421 and 211 bp).The variation of sequence length and the imbalance of sample size could cause unstable ratio.Phylogenetic trees showed that most pangolin species except M.javanica and M.culionensis were monophyletic,and their topological structure and confidence for some nodes changed with the sequence being shorter.