利用COI基因序列鉴别赛加羚羊角与其代用品

Discrimination of Saiga Antelope Horn from Substitutes in “Lingyangjiao” Markets by Genetic Identification Technology

目的：用COI序列作为分子标记鉴定赛加羚羊角与其代用品山羊Capra hircus、绵羊Ovis aries、藏原羚Procapra picticaudata、蒙原羚P. gutturosa、鹅喉羚Gazella subgutturosa和藏羚Pantholops hodgsonii的角，为监管羚羊角市场贸易和控制赛加羚羊角走私提供技术支持。方法：从3种不同类型的赛加羚羊角以及藏原羚、蒙原羚、鹅喉羚样品中提取基因组DNA，设计巢式PCR体系，扩增349 bp COI片段，结合从GenBank中下载的序列构建赛加羚羊及其代用品的系统发育树。结果：保存期1～2年的新鲜赛加羚羊角中提取出的基因组DNA浓度比陈旧羚羊角、羚羊角片的高，而陈旧羚羊角不同位置提取出的基因组DNA浓度差别较小。NJ树上，7个物种分别单独聚群，形成独立的分枝，支持率都达到了99%以上。结论：DNA分子鉴定技术结合针对低浓度基因组DNA模板的巢式PCR反应体系客观准确地区分了赛加羚羊角和其易混代用品。本研究不仅为赛加羚羊角的分子鉴定技术建立了操作方法，而且提供了参考序列，简化了实际应用中的鉴定流程，对赛加羚羊贸易监管以及走私监控具有实践参考意义。

Objective： Employ genetic species identification technology to distinguish Saiga antelope horus from the substitutes in Traditional Chinese Medicine （TCM） markets, including horus of goat （Capra hircus）, sheep （ Ovis aries ） , Tibetan gazelle （ Procapra picticaudata ）, Mongolia gazelle （ Procapra gutturosa ）, Goitere d gazelle （Gazella subgutturosa） and Tibetan antelope （Pantholops hodgsonii）. Those substitutes were morphologically similar to Saiga horn, especially when they were processed, for instance, when the horus were grinded into powder, chopped into slides and segments. The final aim of this study was to facilitate the management of TCM markets of ＂Lingyangjiao＂ and control Saiga antelope or other endangered Chinese gazelles smuggling. Methods： Genomic DNA was extracted from three Saiga horn samples and samples of Goitered gazelle, Tibetan gazelle and Mongolia gazelle. Two newly designed primer sets were used to amplify COI fragments of 349 bp with nest-PCR systems. Phylogenetic NJ tree of saiga antelope and six substituted species was constructed using the sequences of this study and those retrieved from GenBank. Results： Genomic DNA extracted from different parts of the timeworn Saiga horn, such as the tip, middle part and the base, were low, as well as the processed horn block, whereas concentration of DNA from the new Saiga antelope horn was much higher. In NJ tree, all the seven ＂Lingyangjiao＂ species formed independent clades with 〉 99% support values. Conclusion： Combining with nest-PCR systems, molecular diagnosis technology using COI gene as markers proved to be a powerful tool to discriminate Saiga antelope horus from the other ＂Lingyangjiao＂ species, especially when dealing with degraded samples from processed or timeworn horus. With the diagnostic protocol and reference sequences provided in this study, identification of biological origins of ＂Lingyangjiao＂ sold in TCM could be simplified. The technology might contribute to monitoring the trade of Saiga antelope and controlling Saiga antelope and the other endangered Chinese gazelle smuggling.