目的 利用DNA特征序列（DNA signature sequence，DSS）标记，建立一种快速高效的伊犁贝母特异性DSS标记分子鉴定方法。方法 通过植物叶绿体基因组综合数据库（CGIR）获取贝母属植物全部叶绿体基因组数据，利用DSS鉴定软件IdenDSS进行序列分析、DSS标记筛选和特异性引物对设计，获得伊犁贝母候选DSS标记及相应的引物，通过特异性引物扩增、DNA测序及序列比对验证筛选的DSS标记，检测待测样品中是否含有目标物种伊犁贝母的DSS或其反向互补序列进行基原鉴定。结果 通过比对验证5组DSS标记与伊犁贝母、川贝母6种正品来源以及《中国药典》中收载的其他4种药用贝母平贝母、浙贝母、湖北贝母、新疆贝母序列一致性，最终确定5组DSS标记均可用于伊犁贝母快速鉴定，将上述序列上传至中药分子鉴定平台（www.herbsdna.com）标准数据库中，结合数据库DSS鉴定模块BLAST功能，可实现对伊犁贝母快速、准确鉴定。结论 筛选的5组DSS标记均可用于伊犁贝母快速基原鉴定，可有效解决伊犁贝母替代川贝母使用难题。

Objective To establish a rapid and efficient DSS markers molecular identification method for Fritillaria pallidiflora using DNA signature sequence tags (DSS). Methods The complete chloroplast genome sequences of Fritillaria was download from the Chloroplast Genome Information Resource (CGIR) database, the IdenDSS software was used to perform sequence analysis, DSS marker screening, and specific primer design to obtain candidate DSS markers and corresponding primers for F. pallidiflora. The screened DSS markers were validated through specific primer amplification, DNA sequencing, and sequence alignment. The target species’ DSS or its reverse complementary sequence of F. pallidiflora was detected in the test sample for motif identification. Results By comparing and verifying the consistency of five pairs of DSS markers with six authentic sources of F. pallidiflora and F. cirrhosae, as well as the other four medicinal Fritillaria listed in the Chinese Pharmacopoeia, including F. ussuriensis, F. thunbergii, F. hupehensis, and F. walujewii, it was ultimately determined that all five sets of DSS markers can be used for rapid identification of F. pallidiflora. The aforementioned sequences were added to the Traditional Chinese Medicine Molecular Identification Platform’s standard database (www.herbsdna.com). When paired with the database DSS identification module’s BLAST function, F. pallidiflora could be quickly and accurately identified. Conclusion The all five selected DSS markers could be used for rapid identification of F. pallidiflora, which can effectively solve the problem of using F. pallidiflora as a substitute for F. cirrhosae.

R286.12

国家自然科学基金项目（82474075）