Heredity (2008) 101, 136–144 & 2008 Nature Publishing Group All rights reserved 0018-067X/08 $30.00 www.nature.com/hdy ORIGINAL ARTICLE An integrated approach to the characterization of two autochthonous lentil (Lens culinaris) landraces of Molise (south-central Italy) GS Scippa1, D Trupiano1, M Rocco2, V Viscosi1, M Di Michele3, A D’Andrea1 and D Chiatante4 1Dipartimento di Scienze e Tecnologie per l’Ambiente e il Territorio, University of Molise, Pesche, Italy; 2Dipartimento di Scienze Biologiche e Ambientali, University of Sannio, Benevento, Italy; 3Laboratory of Analytical Techniques and Proteomics, ‘John Paul II’ Center for High Technology, Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy and 4Dipartimento di Scienze Chimiche ed Ambientali, University of Insubria, Como, Italy Plant biodiversity must be safeguarded because it constitu- in the Molise germoplasm bank. The two local landraces tes a resource of genes that may be used, for instance, in were well differentiated from each other, and the Conca breeding programs. Lentil (Lens culinaris Medik.) is one of Casale landrace was separated from the commercial the most ancient crops of the Mediterranean region. varieties at morphological, protein and DNA level. The Extensive differentiation of L. culinaris over millennia has Capracotta landrace, was well separated from the commer- resulted in a myriad of different landraces. However, in more cial varieties, except Castelluccio di Norcia, at DNA level recent times many landraces have disappeared consequent showing a more complex and heterogeneous segregation to environmental and socioeconomic changes. To promote at morphological and biochemical level. The correlation the survival of endangered lentil landraces, we have between morphological, DNA and protein data, illustrates investigated the genetic relationship between two ancient that proteomics is a powerful tool with which to complement landrace cultivated in Capracotta and Conca Casale (Molise, the analysis of biodiversity in ecotypes of a single plant south-central Italy) and widely spread commercial varieties species and to identify physiological and/or environmental using an integrated approach consisting of studies at markers. morphological, DNA and protein level. Seeds of these two Heredity (2008) 101, 136–144; doi:10.1038/hdy.2008.39; landraces were collected from local farmers and conserved published online 14 May 2008 Keywords: biodiversity conservation; plant germoplasm lentil landraces; Lens culinaris M.; proteomic; genetic variation Introduction landraces are on the verge of extinction; others are grown only by elderly farmers, mainly in marginal lands, for Lentil (Lens culinaris Medik.) is an ancient crop that their own consumption and, occasionally, for local originated in the Near East (Zohary, 1999) and subse- markets (Piergiovanni, 2000). Unlike modern cultivars quently spread throughout the Mediterranean Basin and selected for their performance in specific environmental central Asia (Cubero and Malbon, 1984; Lev-Yadun et al., conditions, local landraces have a high genetic varia- 2000). Over time, local constraints produced wide bility. Having evolved adaptive gene complexes con- diversity within the L. culinaris species, resulting in a served by genetic linkage or natural or human selection, myriad of different landraces (Erskine, 1997). However, they are highly adapted to different environmental the cultivation of lentils has progressively decreased in conditions. industrialized countries in favour of more remunerative An understanding of genetic variation within the lentil crops. Consequently, several local populations have germoplasm and of the genetic relationship between disappeared and those still being cultivated are at a landraces is important because: (1) a broad genetic high risk of severe genetic erosion (Ladizinsky, 1993; diversity can accelerate the genetic improvement of Piergiovanni, 2000). crops and (2) the identification of genetic variation is Various local landraces have evolved in several Italian an effective method of stratifying and sampling variation regions thanks to an optimal combination of climate, soil in germoplasm collections and can help to define and moisture, although modern agricultural practises are priorities for conservation programs. eroding the genetic richness of this plant species. Several Thus far, genetic variation of the lentil germoplasm has been evaluated based on morpho-physiological traits (Erskine et al., 1989; Lazaro et al., 2001; Tullu et al., 2001; Correspondence: Professor GS Scippa, Dipartimento di Scienze e Yan’kov et al., 2001), isozymes (De La Rosa and Jouve, Tecnologie per l’Ambiente e il Territorio, University of Molise, C.da Fonte 1992; Rodriguez et al., 1999), seed storage proteins (De La Lappone, Pesche (IS) 86090, Italy. E-mail: [email protected] Rosa and Jouve, 1992, Echeverrigaray et al., 1998, Received 15 January 2008; revised 26 March 2008; accepted 4 April Piergiovanni and Taranto, 2005) or such molecular 2008; published online 14 May 2008 markers as random amplified polymorphic DNA (Sharma Characterization of two autochthonous lentil landraces GS Scippa et al 137 et al., 1995) and ISSR (inter-simple sequence repeat) basic data matrix as follows: the variables were trans- (Alvarez et al., 1997; Sonnante and Pignone, 2001). More formed to obtain a mean of zero and a variance of one. A recently, proteomics, an innovative approach involving standardized matrix of 18 populations using eight the comparison of several hundred to several thousand morphological variables was subjected to principal gene products revealed by two-dimensional gel electro- component analysis (PCA) and cluster analysis, which phoresis of protein extracts, has been successfully was performed using the unweighted pair group method applied to investigations of natural variations within with arithmetic (UPGMA) mean method (Sokal and plant species populations. Indeed, protein spots resolved Minchener, 1958). by two-dimensional gel electrophoresis are de-facto genetic and physiological markers (Damerval et al., 1994; De Vienne et al., 1996) that can be used to assess Protein analysis genetic variability and to establish genetic distances and Total protein was extracted according to the method of phylogenetic relationships between lines, species and Rabilloud (2000) with minor modifications. Independent genus (Thiellement et al., 1999). samples (2.0 g of dry seeds) were finely powdered in Different methods sample genetic variation at different liquid N2 using a mortar, and the resulting powder was levels, and have different powers of genetic resolution: suspended in 10 ml of buffer A (10% trichloroacetic acid ‘neutral’ DNA markers like microsatellites, widely used or tricarboxylic acid, 0.07% b-mercaptoethanol in cold to study genetic diversity among populations, describe acetone at À20 1C) and then filtered through Miracloth. relations between species in terms of time divergence Proteins were precipitated at À20 1C for 4 h, centrifuged (‘molecular clock’) (Thiellement et al., 1999), whereas at 35 000 g for 15 min and rinsed twice with cold acetone, phenotypic markers like morpho-physiological traits can 0.07% b-mercaptoethanol for 4 h at À20 1C. The pellet provide information about adaptive responses to macro- was recovered by centrifuging at 35 000 g for 15 min, environmental conditions (David et al., 1997). Obviously, dried under vacuum and solubilized in 300 mlofL2 a study involving all these techniques would provide buffer (urea 7 M, thiourea 2 M, Chaps 4%, Triton X-100 more information about genetic variation than each 1%, Tris 20 mM, dithiothreitol (DTT) 1% and ampholine technique used alone. 3–10 0.2% and 5–7 0.15%). Protein concentration was This study had two main goals: (1) to investigate the estimated according to Peterson (Lowry’s method genetic relationship between two local lentil landraces modified) using bovine serum albumin as standard severely threatened by genetic erosion, using an inte- (Peterson, 1977). grated approach and (2) to explore the potential of For two-dimensional gel electrophoresis, protein sam- proteomics as a tool with which to investigate natural ples were focused on immobilized pII gradient (IPG) variation within and between lentil populations. strips and then subjected to SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis. IPG strips (17 cm pH 4–7, Bio-Rad ReadyStrip; Bio-Rad, Hercules, CA, Materials and methods USA) were rehydrated overnight with 300 ml of isoelec- trofocusing (IEF) buffer containing 600 mg of total Plant materials proteins. Proteins were focused using a Protean IEF Cell In this study, we compared Molise lentil landraces (six (Bio-Rad) at 12 1C, applying 250 V (90 min), 500 V from Conca Casale and seven from Capracotta—two (90 min), 1000 V (180 min) and 8000 V for a total of small villages in the Molise region of central-south Italy) 55 kVh. After focusing, proteins were reduced by with five others: the widely marketed Turkish red lentils incubating the IPG strips with 1% w/v DTT for 15 min, and Canadian lentils, the Castelluccio da Norcia ecotype, and alkylated with 2.5% w/v iodoacetamide in 10 ml of which is the only one in Italy to be awarded the IGP 50 mM Tris-HCl pH 8.8, 6 M urea, 30% w/v glycerol, 2% (geographically protected brand), and with two other w/v SDS and a dash of bromophenol blue, for 15 min. autochthonous landraces from the central Appenine Two-dimensional electrophoresis was carried out using a area,