Background: Orchids are economically important, particularly in horticulture, floristry, pharmaceutical, and fragrance industries. India is a paradise for 1256 orchids, of which 31% (388 species) are endemic. Although Indian orchids are known in floristry, there is still a lot of room to use their bioactive compounds to promote their commercialization. Easy and accurate identification is first step towards conservation and commercial cultivation of endemic and endangered orchid species. This requirement can be accomplished using DNA barcoding technique. Materials and Methods: A total of 62 samples belonging to 35 species, 7 genera were collected for DNA barcoding studies. Evolutionary divergences and barcoding gap were calculated using MEGA-X software to screen the most suitable barcode region among the ITS, matK, rbcL, and trnH-psbA loci. BLAST analysis was used to identify barcoding locus presenting maximum species resolution. Phylogenetic analyses were performed to test harmony between barcoding and taxonomy. Results: We have generated 133 barcoding sequences, out of which, 46 sequences were found unique and new to GenBank database. Evolutionary divergence analysis showed the best result for ITS, where intra-specific and inter-specific divergence ranged between 0.0000-0.0300 and 0.0322-0.3765 correspondingly. It indicated clear barcoding gap, which was sufficient to robustly infer identities for taxa. BLAST-based evaluation concluded that largest number of barcode sequences (94.64%) could be identified using ITS locus followed by rbcL (78.69%) and matK (51.61%). In addition, the optimal phylogenetic tree was established using the ITS locus sequences, which complemented the orchid taxonomy. Conclusion: This study recommends ITS as best single locus barcoding region for identifying the orchids of India.
