Abstract: Obtaining reliable species identification of the legume genus Caragana has been challenging. Until now, species identification was mostly carried out utilizing diagnostic morphological characteristics, in addition to some successful applications of secondary chemical compounds. This study was designed to establish a DNA barcoding protocol enabling unambiguous identification of 238 accessions belonging to 67 species of Caragana. The performance of four DNA barcoding regions nrITS, trnH-psbA, matK, and rbcL was explored using three analytical approaches, Pairwise Genetic Distance, Sequence Similarity and Phylogenetic Tree method. The chloroplast regions rbcL and matK showed lower discriminatory power compared with the nuclear region internal transcribed spacer (ITS) and the chloroplast region trnH-psbA. The nrITS outperformed the other regions in the resolution rate. The present study brings forth an efficient barcode locus for Caragana. A barcode based either on a single-locus nrITS or the combination of nrITS and trnH-psbA was found to be most suitable for species discrimination with distinctive barcoding gaps. An attempt has also been made to resolve taxonomic issues in the Caragana opulens complex. DNA barcoding tools when complemented with alpha taxonomic evidence can aid in solving complex systematic problems, especially when taxa are characterized by overlapping traits, such as species belonging to the Fabaceae family.

Key words: ITS, molecular taxonomy, species complex, species discrimination, systematics, trnH‐psbA