DNA barcoding of authentic and substitute samples of herb of the family Asparagaceae and Asclepiadaceae based on the ITS2 region.

ABSTRACT

Background: Herbal drugs used to treat illness according to Ayurveda are often misidentifi ed or adulterated with similar plant materials. Objective: To aid taxonomical identifi cation, we used DNA barcoding to evaluate authentic and substitute samples of herb and phylogenetic relationship of four medicinal plants of family Asparagaceace and Asclepiadaceae. Materials and Methods: DNA extracted from dry root samples of two authentic and two substitutes of four specimens belonging to four species were subjected to polymerase chain reaction (PCR) and DNA sequencing. Primers for nuclear DNA (nu ITS2) and plastid DNA (matK and rpoC1) were used for PCR and sequence analysis was performed by Clustal W. The intraspecifi c variation and interspecifi c divergence were calculated using MEGA V 4.0. Statistical Analysis: Kimura’s two parameter model, neighbor joining and bootstrapping methods were used in this work. Results: The result indicates the effi ciency of amplifi cation for ITS2 candidate DNA barcodes was 100% for four species tested. The average interspecifi c divergence is 0.12 and intraspecifi c variation was 0.232 in the case of two Asparagaceae species. In two Asclepiadaceae species, average interspecifi c divergence and intraspecifi c variation were 0.178 and 0.004 respectively. Conclusions: Our fi ndings show that the ITS2 region can effectively discriminate Asparagus racemosus and Hemidesmus indicus from its substitute samples and hence can resolve species admixtures in raw samples. The ITS2 region may be used as one of the standard DNA barcodes to identify closely related species of family Asclepiadaceae but was noninformative for Asparagaceae species suggesting a need for the development of new markers for each family. More detailed studies involving more species and substitutes are warranted.