Selection of plant-specific primers

ITS sequences of all the four species were aligned with Clustal W tool using MEGA 5.1 to determine polymorphic nucleotides which were used further to develop species-specific primers.[17] Each site was checked manually to develop primers. In silico tools, OligoCalc and Primer3 softwares were used to test each polymorphic site for its feasibility to design suitable primers on the basis of optimal length, optimum GC content, melting temperature compatibility, hairpin formation, secondary structure, and species specificity through primer blast.[18,19] Bioinformatically tested primers were validated experimentally. ITS sequence comparison revealed absence of any polymorphic site between T. arjuna and T. tomentosa for suitable primer development of T. tomentosa while many polymorphic sites were present between and among other three Terminalia species. One forward and reverse primer were developed for authentication of T. arjuna, and primers for T. bellirica and T. chebula were also developed aiming the detection of the adulterants [Table 1].

Sequence of the primers developed for molecular identification of Terminalia species