Molecular authentication of medicinal and edible plant Gnaphalium affine (cudweed herb, “Shu-qu-cao”) based on DNA barcode marker ITS2

Cudweed herb is not only a conventional health-protection wild vegetable, but also a traditional Chinese medicine known as “Shu-qu-cao”, refers to Gnaphalium affine D. Don (renamed as Pseudognaphalium affine ), an annual plant in the family Asteraceae. However, due to the similarity of phenotypic characteristics between G. affine and some related species, it is difficult to identify G. affine accurately; besides, “Shu-qu-cao” is usually sold dried, making traditional identification methods difficult, to a certain extent, it caused the species confusion of Gnaphalium medicinal plants, which threatened the safety and effectiveness of cudweed herb in clinical treatment. To more accurately distinguish cudweed herb from its adulterants, DNA barcoding was used in this study. Leaves of not only G. affine , but also the potential adulterant species were also collected. After DNA extraction, PCR amplification, gene sequencing, GeneBank searching and annotation, ITS2 sequences were obtained. 34 ITS2 sequences representing 5 genera (Gnaphalium, Leontopodium, Pulsatilla, Potentilla, Datura) and 13 species ( Gnaphalium affine , Gnaphalium sylvaticum , Gnaphalium norvegicum , Gnaphalium supinum , Gnaphalium tranzschelii , Gnaphalium hypoleucum , Gnaphalium luteoalbum , Gnaphalium adnatum , Gnaphalium japonicum , Leontopodium leontopodioides , Pulsatilla chinensis , Potentilla chinensis , Datura stramonium ) were analyzed by MEGA7.0 software. GC content, intraspecific and interspecific genetic distance of K2P (Kimura 2-parameter) were calculated. The neighbor joining (NJ) method was used to construct the genetic cluster tree, and bootstrap (self expanding support rate 1000 times) was used to test the supporting rate of each branch. The ITS2 secondary structures were predicted by the ITS2 Database. The results showed that a barcoding gap existed among all tested taxa, and the difference in the secondary structures of ITS2 also indicates that cudweed herb and its potential adulterants can be distinguished. Therefore, ITS2 barcode can be used for accurate identification of cudweed herb and its adulterants, and provide the basis for safety and accuracy of rational use of G. affine in clinical application.