Barcoding The Major Mediterranean Leguminous Crops By Combining Universal Chloroplast And Nuclear Dna Sequence Targets

Barcoding the major Mediterranean leguminous crops by combining universal chloroplast and nuclear DNA sequence targets
P. Madesis1*, I. Ganopoulos2*, P. Ralli3 and A. Tsaftaris1,2 Institute of Agrobiotechnology, Centre for Research and Technology Hellas, Thessaloniki, Greece 2 Department of Genetics and Plant Breeding, School of Agriculture, Aristotle University of Thessaloniki, Thessaloniki,Greece 3 Greek Gene Bank, Agricultural Research Centre of Northern Greece, National Agricultural Research Foundation, Thessaloniki, Greece
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*These authors contributed equally to this study. Corresponding author: A. Tsaftaris E-mail: tsaft@certh.gr Genet. Mol. Res. 11 (3): 2548-2558 (2012) Received October 27, 2011 Accepted May 5, 2012 Published July 10, 2012 DOIhttp://dx.doi.org/10.4238/2012.July.10.10

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 evaluatedthe 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 withthe 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
Genetics and Molecular Research 11 (3): 2548-2558 (2012) ©FUNPEC-RP www.funpecrp.com.br

DNA barcoding of leguminous crops

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nuclear region ITS2. PCR products were sequenced and the sequences were aligned usingCLUSTAL 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). Fourtree-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. TheDNA 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 wereboth 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. Key words: Barcoding; trnL; rpoC1; ITS2; Legumes; Mediterranean

INTRODUCTION
Leguminous crops belong tothe Fabaceae family and are second only to cereals in their importance for human nutrition. The Mediterranean basin is a center of origin for legumes (Perrino, 1988). They have played an important role in human nutrition in the region since ancient times, increasing not only the quantity but also the quality of cereal-based food for the population. Legumes constitute the main protein component...