Identification of ungulates used in a traditional Chinese medicine with DNA barcoding technology.

Jing Chen,Shaopeng Cui,Hongjun Chu,Zhigang Jiang,Xiaoge Ping,Songhua Tang,Binwan Liu,Chunlin Li

doi:10.1002/ece3.1457

Abstract

Horns of Saiga antelope (Saiga tatarica) have always been an ingredient of “Lingyangjiao”, a traditional Chinese medicine (TCM). Persistent hunting for Saiga antelope has already threatened the survival of critical endangered populations in wild. To control the growing pressure, CITES and Chinese government have legislated for monitoring the trade of Saiga horns. However, similar ungulate horns are difficult to identify by their morphological characteristics, which has impeded the law enforcement. Besides Saiga antelope, other seven ungulate species which have similar horns are also sold and marked as “Lingyangjiao” in TCM markets to offset shortage of Saiga antelope horns. Such species are Gazella subgutturosa, Pantholops hodgsonii, Procapra picticaudata, Procapra gutturosa, Procapra przewalskii, Capra hircus, and Ovis aries. Our study aimed at implementing DNA barcoding technology to diagnose Saiga horns and the substitutes. We successfully extracted genomic DNA from horn samples. We recovered COI sequences of 644 bp with specific primers and 349 bp with nested PCR primers designed for degraded horn samples. The mean interspecific genetic distance of data set of the 644-bp full barcodes and the 349-bp mini-barcodes was 14.96% and 15.38%, respectively, and the mean intraspecific distance was 0.24% and 0.20%, respectively. Each species formed independent clades in neighbor-joining (NJ) phylogenetic tree of the two data sets with >99% supporting values, except P. gutturosa and P. przewalskii. The deep genetic distances gap and clear species clades in NJ tree of either full barcodes or mini-barcodes suggest that barcoding technology is an effective tool to diagnose Saiga horns and their substitutes. Barcoding diagnosis protocol developed here will simplify diagnosis of “Lingyangjiao” species and will facilitate conservation of endangered ungulates involved in TCM “Lingyangjiao” markets, especially the Saiga antelope.

Highlights

Introduction“Lingyangjiao”, a traditional Chinese medicine (TCM), has been used for almost 2,000 years in Chinese communities (Chan et al 1995)
Genomic DNA of “Lingyangjiao” from traditional Chinese medicine (TCM) markets was likely to be degraded because they were sometimes processed into slice, block, or powder before sale and stored at room temperature and high humidity for a long period
DNA yields of old or processed horn samples of S. tatarica and C. hircus were so poor that the final concentrations were only 0.8 to 2.5 ng/lL

Summary

Introduction

“Lingyangjiao”, a traditional Chinese medicine (TCM), has been used for almost 2,000 years in Chinese communities (Chan et al 1995). Horns of goitered gazelle (Gazella subgutturosa), Tibetan antelope (Pantholops hodgsonii), Tibetan gazelle (Procapra picticaudata), Mongolian gazelle (Procapra gutturosa), and Przewalski’s gazelle (Procapra przewalskii) are marked and sold as “Lingyangjiao” in TCM markets without declaration of the real ingredients (Liu 1982; Chan et al 1995). These wild ungulates are all listed as national key protected wild animals of whose trade is forbidden (Table 1) (Jiang et al 1996). We expected to build an effective protocol for “Lingyangjiao” samples and provide a series of reference sequences for quick diagnosis

Materials and Methods

Results and Discussion

Conclusions