title: Barcoding the  major Mediterranean leguminous crops by combining universal chloroplast and  nuclear DNA sequence targets.
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 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.
authors.affiliation: Institute of Agrobiotechnology, Centre for Research and Technology Hellas, Thessaloniki, Greece.
authors.forename: P
authors.initials: P
authors.lastname: Madesis
authors.forename: I
authors.initials: I
authors.lastname: Ganopoulos
authors.forename: P
authors.initials: P
authors.lastname: Ralli
authors.forename: A
authors.initials: A
authors.lastname: Tsaftaris
mesh_terms: D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics*; D018556:Crops, Agricultural / Q000235:genetics*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D018742:DNA, Chloroplast / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D007887:Fabaceae / Q000145:classification* / Q000235:genetics*; D019083:Mediterranean Region; D008969:Molecular Sequence Data; D010802:Phylogeny; D013045:Species Specificity
chemical_list: D018742:DNA, Chloroplast; D018744:DNA, Plant