Background

Smuggling and illegal trade of pangolins and their scales has drastically reduced the wild population of pangolins. Accurate species identification is currently in urgent need as a powerful weapon for combating pangolin smuggling and trade and conserving the already endangered pangolin species.

Aim of the study

To develop an efficient method based on DNA mini-barcodes for accurate pangolin species identification and authentication of processed pangolin scales against the non-target species.

Materials and methods

The primers for amplifying the DNA mini-barcodes were designed based on cytochrome C oxidase subunit I (COI) gene fragments. The mini-barcodes were compared with the two universal barcodes (COI and Cytb) for performance in pangolin species identification by calculating the Kimura-2-parameter (K2P) distance, assessing the clustering dendrogram, and analyzing the BLAST similarity and barcoding gap. The accuracy of the three barcodes was also compared for authentication of pangolin scales against non-target species.

Results

Comparison of the three barcodes showed that the mini-barcode form COI had the highest amplification success rate (100%) and high variable sites (40.0%), with the ratio of mean inter- to intraspecific distance ratio was 25 and a distinct DNA barcoding gap. In the neighbor-joining (NJ) tree constructed based on the mini-barcode regions, each species of the pangolin family formed an obvious clade respectively, and the clades were all separated from those of the non-target species, indicating that the genetic information in the mini-barcode was sufficient for species identification.

Conclusion

The DNA mini-barcodes based on COI gene fragments provide an effective and accurate method for identification of pangolin species and authentication of pangolin scale products.

中文翻译：

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DNA迷你条形码，保护和打击穿山甲非法贸易的潜在武器

背景

穿山甲及其鳞片的走私和非法贸易大大减少了穿山甲的野生种群。目前迫切需要准确的物种识别，作为打击穿山甲走私和贸易以及保护已经濒临灭绝的穿山甲物种的有力武器。

研究目的

开发一种基于 DNA 迷你条形码的有效方法，用于准确识别穿山甲物种，并针对非目标物种对加工后的穿山甲鳞片进行认证。

材料和方法

基于细胞色素 C 氧化酶亚基 I (COI) 基因片段设计用于扩增 DNA 迷你条形码的引物。通过计算 Kimura-2 参数 (K2P) 距离、评估聚类树状图以及分析 BLAST 相似性和条形码差距，将迷你条形码与两种通用条形码（COI 和 Cytb）在穿山甲物种鉴定中的性能进行了比较。还比较了三种条形码的准确性，以验证穿山甲鳞片对非目标物种的验证。

结果

三种条码的比较表明，COI 形式的迷你条码具有最高的扩增成功率（100%）和高可变位点（40.0%），平均种间距离比为 25，DNA 条码清晰差距。在基于迷你条码区域构建的邻居连接（NJ）树中，穿山甲科的每个物种分别形成了一个明显的进化枝，进化枝与非目标物种的进化枝全部分离，表明遗传信息在迷你条形码中足以识别物种。

结论

基于COI基因片段的DNA迷你条码为穿山甲物种的鉴定和穿山甲鳞片产品的鉴定提供了一种有效、准确的方法。