Establishing the pangolin mitochondrial D-loop sequences from the confiscated scales.
Pangolin scales are encountered in traditional East Asian medicines (TEAM) and the ever increasing demand for these scales has escalated the decline in the numbers of these mammals. The identification of protected pangolin species is necessary to enforce international and national legislation as well as assist with conservation measures. There is limited morphological feature on a pangolin scale thus requiring DNA analysis as a means of identification. We report on the isolation of DNA from pangolin scales and a strategy for obtaining the full length of the mitochondrial D-loop, being 1159 bp. Primer sets creating five overlapping amplicons were designed to amplify sections of this mitochondrial DNA locus. DNA from the blood stain of nineteen Formosan pangolins (Manis pentadactyla pentadactyla) along with 145 scale samples that were suspected to have come from pangolins, was amplified and sequenced; leading to a total of 91 D-loop sequences being obtained. The 19 Formosan pangolin sequences produced 5 haplotypes and 72 of the 145 seized scales provided useable sequence classified as a further 38 haplotypes. The D-loop sequences from those scales suspected to be from a pangolin had a higher similarity to any of the 19 samples taken from M. p. pentadactyla compared to a D-loop sequence from Manis tetradactyla (the only pangolin D-loop sequence in GenBank, NC_004027). These 43 haplotypes were used to establish a local database for the D-loop sequence of pangolins and add to the data of Manis sp. held on GenBank. The PCR amplification strategy development in this study could be used in forensic DNA identification of scales suspected to be from protected pangolin species.
Hsieh
Hsing-Mei
HM
Department of Forensic Science, Central Police University, 56 Shu-Jen Road, Kwei-San, Taoyuan 33334, Taiwan, ROC.
Lee
James Chun-I
JC
Wu
Jane-Hong
JH
Chen
Chao-An
CA
Chen
Yen-Jean
YJ
Wang
Guan-Bang
GB
Chin
Shih-Chien
SC
Wang
Lih-Chiann
LC
Linacre
Adrian
A
Tsai
Li-Chin
LC
D000818:Animals; D003247:Conservation of Natural Resources; D004272:DNA, Mitochondrial / Q000235:genetics*; D030561:Databases, Nucleic Acid; D006239:Haplotypes; D008322:Mammals / Q000235:genetics*; D008518:Medicine, East Asian Traditional; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D004272:DNA, Mitochondrial