The Molecular Identification Of Angelica Sinensis And Its Adulterants

Objective:To establish a new method for identification of Angelica sinensis from its adulterants by analyzing trnL-F and ropC1 sequences;To establish the inter-simple sequence repeat(ISSR)DNA fingerprinting database of A.sinensis,and to construct corresponding specific sequence characterized amplified region(SCAR)marker,for identification and analysis of the genetic relationship among A.sinensis and its adulterants;Preliminary explore the applicability of the SCAR marker in A.sinensis by its proprietary Chinese medicine;Methods:The plastid trnL-F and ropC1 sequences of 25 samples of A.sinensisand its adulterants were amplified,sequenced and analyzed.The K-2-P distances of A.sinensis and its adulterants were calculated,and phylogenetic trees were constructed;We used ISSR(inter-simple sequence repeats)within A.sinensis and its closely related species,detect specific bands in A.sinensis.The bands were separated,extracted,cloned and sequenced.A pair of unique primers SCAR marker was designed by this specific sequences;Four kinds of DNA extraction:CTAB,SDS,Magnetic bead,modified CTAB,were compared in proprietary Chinese medicine;Results:TrnL-F and ropC1 sequence of A.sinensis and its adulterants were successful amplified and sequenced,there were significant differences between A.sinensis and its adulterants based on trnL-F sequences,phylogeny tree reconstructed using MP analysis based on trnL-F sequences could effectively distinguish A.sinensis and its adulterants,however,analysis of the ropC1 sequences could not identify A.sinensis and its adulterants;Through 14 ISSR primers,a total of amplification of 135 bands,the polymorphism proportion is as high as 90.37%,there are four specific bands of A.sinensis;Based on SCAR markers,for 6 A.sinensis were positive results,for the rest of were negative result;Four methods to extract DNA were not showed there as a result,but after amplification by the trnL-F and SCAR markers,there is an obvious bands by modified CTAB method,no amplification bands on the rest of the way;Conclusions: The ropC1 sequence has poor capability for identification of A.sinensis and its adulterants,the trnL-F sequences could be used as an efficient molecular marker for authenticating A.sinensis and its adulterants;The SCAR markers can be successful to identification of A.sinensis and its adulterants,it has a great of significance for identification of A.sinensis;The modified CTAB method opened a new road through the molecular identification of proprietary Chinese medicine,and has significant effect on the application of SCAR marker in A.sinensis proprietary Chinese medicine.