  Citation: Bosmali, I.; Lagiotis, G.; Haider, N.; Osathanunkul, M.; Biliaderis, C.; Madesis, P. DNA-Based Identification of Eurasian Vicia Species Using Chloroplast and Nuclear DNA Barcodes. Plants 2022, 11, 947. https://doi.org/10.3390/ plants11070947 Academic Editors: Fouad S. Maalouf and Dil Thavarajah Received: 8 February 2022 Accepted: 28 March 2022 Published: 31 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article DNA-Based Identification of Eurasian Vicia Species Using Chloroplast and Nuclear DNA Barcodes Irene Bosmali 1,2 , Georgios Lagiotis 1 , Nadia Haider 3 , Maslin Osathanunkul 4,5 , Costas Biliaderis 2 and Panagiotis Madesis 1,6, * 1 Centre for Research and Technology Hellas, Institute of Applied Biosciences, 57001 Thessaloniki, Greece; eirinimposmali@certh.gr (I.B.); glagiotis@certh.gr (G.L.) 2 Laboratory of Food Chemistry and Biochemistry, Department of Food Science and Technology, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; biliader@agro.auth.gr 3 The Department of Molecular Biology and Biotechnology, Atomic Energy Commission (AEC), Damascus 6091, Syria; ascientific1@aec.org.sy 4 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand; maslin.cmu@gmail.com 5 Research Center in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 6 Laboratory of Molecular Biology of Plants, School of Agricultural Sciences, University of Thessaly, 38446 Volos, Greece * Correspondence: pmadesis@uth.gr Abstract: Many legume species of the Vicia L. genus (Fabaceae Lindl.) are key components of the Mediterranean diet and have an integral role in sustainable agriculture. Given the importance of the Vicia species for Eurasian culture, it is necessary to implement methodologies, such as DNA barcoding, that can enable the effective authentication and identification of species in the genus. In this study, we analysed the chloroplast trnL and rpoC1, as well as the nuclear ITS2 DNA barcoding regions, to identify 71 Vicia specimens of Eurasian descent. Both the trnL and ITS2 regions were highly effective in discriminating the analysed taxa, while the more conserved rpoC1 region could not identify all of the selected species due to high sequence conservation or non-annotated or absent rpoC1 species sequences in GenBank. A dendrographic representation of the generated trnL data showed sufficient clustering for most of the analysed taxa, although some topological discrepancies were observed. ITS2 and rpoC1 reconstructions were also used for resolving the topological discrepancies observed in the trnL tree. Our analysis suggests that a combination of DNA barcoding regions is essential for accurate species discrimination within the Vicia genus, while single-locus analyses do not provide the necessary resolution. Keywords: DNA barcoding; ITS2; rpoC1; trnL; Vicia 1. Introduction Legumes are the second most important crop after cereals in terms of agricultural production, human nutrition, and animal feed [1]. Many leguminous species have been an integral part of the human diet since ancient times due to their low fat and high protein content, richness in fibre and other nutrients, as well as their anticarcinogenic and prebiotic potential [1–5]. Furthermore, legumes are a nutrient-rich feed for domesticated animals. Apart from the use of legumes for human or animal consumption, many species are also utilized in agricultural practices, such as crop rotation, given many species form symbiotic relationships with nitrogen-fixing bacteria, thus replenishing soil nitrogen levels. Finally, legumes are also an invaluable source of nutraceuticals and pharmaceuticals [4,6,7], and are important in the production of other industrial products [8]. Legume seeds can also be fractionated into high protein-, fibre-, and starch-based raw ingredients for the food, Plants 2022, 11, 947. https://doi.org/10.3390/plants11070947 https://www.mdpi.com/journal/plants