Studies On The Application Of Molecular Biology Techniques To The Identification Of Medicinal Plants Of Fritillaria

Herba Fritillaria, named "Beimu"in Chinese, is one of the most popular herbal medicine with a long history of medicinal use in china, according to the Pharmacopoeia of the People's Republic of China(Edition 2000), Herbal Fritillaria is derived from the bulbs of nine species of Liliaceae belonging to the genus Fritillaria: F.thunbergii Miq, F.ussuriensis Maxim, F.pallidiflora schrenk, F.walujewii Regel, F.hupehensis Hsiao, F.cirrhosa D.Don, F.unibracteata Hsiao etkc.Hsia, F.delavayi Franch, F.przewalskii Maxim.There are more than 50 species of Fritillaria in china. The origin of Fritillaria is much cofused and complex due to it, with the market demands increasing, the confusion is more serious. Many different species of Fritillaria besides above nine species, even some other non- Fritillaria genus,such as:Bolbostemma,Tulipa,ha\e been used as Fritillaria.The identification of Fritillaria is difficult because of the morphological similarities, in order to establish a convenient and precise method for the identification of Fritillaria, also to protect and exploit the resources, we have carried out the studies on Fritillaria and made some progresses as the following list:1 Reviewed the recent research literatures on the identification of the traditional Chinese medicinal plants using molecular markers ,analyzed the history and current applying status of Fritillaria on the base of the resource survey.2 Amplified the sequenced the ITS region(including ITS1,5.8sRNA gene and ITS2)of nuclear ribosomal DNA from 11 Fritillaria species , two outgroup Lilium japonicum and Lilium mackliniae of ITS sequences were downloaded their sequences were used to evaluate the phylogeny ,alignmented with Clustal X and analysed with MEGA 2.0,The UPGMA tree showed that there was high sequence divergence between Fritillaria and Lilium ,and the two genera were monophyletic group Respectively. Four clades could be distinguished in Fritillaria clade as follows:a F.przewalskii, F.unibracteata. F.delavayib F.thunbergii, F.thunbergii var. chekiangensis, F.puqiensis,F.hupehensisc F.pallidiflorad F. ussuriensisaccording to the results of phylogene and genetic distance, it is suggested that F. d elavayi is included in F.przewalskii. the result showed the ITS gene can be a useful molecular marker for the identification of Fritillaria3 In order to simplified the identification method ,PCR-RFLP analysis weretested, PCR products from 5SrRNA spacer gene were digested and identified by restriction enzyme:AclI(Psp 14061) for F. hupehensis, HinfI for F. delavayi ,BcnI for F. przewalskii, .Moreover, PCR products from ITS region were digested and identified by restriction enzyme: SamI for F. cirrhosa ,F. unibracteata ,F. delavayi ,F. przewalskii, EcoSlI for F.pallidiflora.4 Designed 3 pairs of highly specialized diagnostic primers for identifying F.thunbergii Miq based on the sequences of 5S-rDNA spacer region of Fritillaria: FthP1 and FthP2, FthP2 and FthP3, FthPl and FthP4. They were employed to identify F.thunbergii Miq by being amplified with anneal temperature at 60℃,60℃ and 62℃ respectively. Designed 1 pairs of highly specialized diagnostic primers for identifying F.pallidiflora based on the sequences of ITS region of Fritillaria: FpdP2 and FpdP3,. They were employed to identify F.pallidiflora by being amplified with anneal temperature at 67℃.