Barcoding the major Mediterranean leguminous crops by combining universal chloroplast and nuclear DNA sequence targets

Abstract

ABSTRACT. The ability to discriminate all species is the ultimate target in barcoding. The Mediterranean basin is a center of origin for legumes and thus they have played a key role in feeding the Mediterranean population. It is also a region with important protected designation of origin and protected geographical indication legumes that provide income in rural areas. We evaluated the use of two chloroplast regions, trnL and rpoC1, and a nuclear internal transcriber region, ITS2, for their efficiency to barcode the main Mediterranean leguminous crops. Twenty-five legume species were studied. Plant material of pasture and legumes was obtained from the Greek GenBank and the Fodder Crops and Pastures Institute (National Agricultural Research Foundation). DNA was extracted with the Qiagen DNeasy plant mini-kit and PCR amplification was performed using the Kapa Taq DNA polymerase using primers amplifying the chloroplast trnL and rpoC1 regions or the ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 11 (3): 2548-2558 (2012) DNA barcoding of leguminous crops 2549 nuclear region ITS2. PCR products were sequenced and the sequences were aligned using CLUSTAL W. Species identification based on the sequence similarity approach was performed using the GenBank database. In order to evaluate intraspecific and interspecific divergence in legumes we used Molecular Evolutionary Genetics Analysis 5 and for pairwise Kimura 2-parameter distance calculations for all 3 DNA regions (2 chloroplast regions, trnL and rpoC1, and the nuclear region ITS2). Four tree-based methods (neighbor joining and maximum parsimony, maximum likelihood, and Bayesian inference analyses) were used to exhibit the molecular identification results to represent differences as an uprooted dendrogram. Additionally, the sequence character-based method was used with DnaSP and the information from each site was treated as a character to distinguish the species from one another. The DNA regions trnL and ITS2 successfully (100%) discriminated the Mediterranean crop legume species used, while rpoC1 identified only 72% of them. Furthermore, the use of the trnL region enabled the discrimination of even very closely related species, like Phaseolus lunatus and P. coccineus or Vicia faba subsp major with V. faba subsp minor, which are so closely related that even in NCBI they were both referred as Phaseolus vulgaris and V. faba, respectively. We conclude that trnL and ITS2 are efficient DNA barcoding target regions in order to discriminate Mediterranean leguminous crops and provide a reliable and efficient tool for the scientific, agricultural and industrial community.

DNA Barcoding the Canadian Arctic Flora: Core Plastid Barcodes (rbcL + matK) for 490 Vascular Plant Species

“…Studies that sample multiple closely related species generally have the lowest species resolution among barcoding studies, whereas studies that do not sample multiple close relatives generally have the highest species discrimination [51]. The latter includes studies of non-taxonomic groups such as medicinal plants [57,88,89], crop species [90,91], and invasive species [92,93]. Another subset of non-taxonomic studies has focused on barcoding the floras or subsets of the floras of restricted geographical areas, in which diversity is lower and fewer closely related species are expected compared with floras of broader geographical areas.…”

Cadherin Characterization and Cytochrome Oxidase (COI) HRM Analysis in Different Geographical Populations of the Mediterranean Corn Borer, Sesamia nonagrioides

“…Barcoding in combination with High Resolution Melting (HRM) analysis has been increasingly used lately in order to conduct rapid genotyping of animal and plant species (Brechon et al, 2013;Madesis et al, 2012a). HRM curve analysis is a quick, low cost, post PCR method which permits the rapid detection of genetic variation between species.…”

Evaluation of cpDNA Barcodes in Selected Medicinal Plants of Mt. Arayat National Park, Pampanga, the Philippines

“…Though trnL-F is not considered as a core barcode by CBOL-PWG (2009), the marker has been used for the identification not only for angiosperms but also of trees and leguminous crops. [27][28] In fact, trnL-F has been recommended as an efficient tool to identify field gametophytes 29 and as a good barcode for the identification of wild, ethnomedicinal plants. 30 While the choice of rbcL and matK as core barcodes by CBOL-PWG (2009) are based on its recoverability, sequence quality and level of species discrimination.…”