ASSESSING THE IDENTITY OF GRAPEVINE PLANTS FROM VINEYARDS FROM CRETE AND SAMOS BY MICROSATELLITE PROFILING ÉVALUATION DE L’IDENTITÉ DE PLANTS DE VIGNES ISSUS DES VIGNOBLES DE CRÈTE ET DE SAMOS PAR PROFILAGE À DES LOCI MICROSATELLITES F. LEFORT1,*and K. ROUBELAKIS-ANGELAKIS2 1 : Laboratoire de Biotechnologie et Génétique Appliquée, École d’ingénieurs de Lullier (EIL), Haute École Spécialisée de Suisse Occidentale (HES SO), 150 route de Presinge, 1254 Jussy, Switzerland 2 : Laboratory of Plant Physiology and Biotechnology, Department of Biology, University of Crete, P.O. Box 2208, 71409 Heraklion, Crete, Greece Abstract : Four nuclear microsatellite markers were used for testing 45 plants visually selected from 18 locations in controlled origin wine areas of Crete and Samos, and representing 7 cultivars. Discrepancies between the obtained profile and a profile from a plant of reference of a given cultivar, were found for 26 plants. This sugges- ted that microsatellite profiling at a small number of loci was an efficient procedure in order to detect and remove inappropriate material at an early stage of selection. In a second case, three plants were sampled from a vineyard, located in the controlled origin wine area of Dafnes in Crete, for testing their identity in comparison to cultivars of reference at 15 nuclear microsatellite loci. Only one matched its identity profile of reference. One of them was found to be a continental cultivar, named Fegi and not usually grown in Crete. This showed the resolution of microsatellite profiling for problems where misnaming at an early stage in nursery or trade could have important consequences for growers in controlled origin wine areas. Résumé : Quatre marqueurs microsatellites nucléaires ont été utilisés pour tester l’identité de 45 plantes, sélec- tionnées visuellement pour leurs différences phénotypiques avec les plantes environnantes de 7 cultivars, dans 18 vignes différentes de Crète et de Samos. Des différences entre profils génétiques obtenus et profils génétiques de références pour ces cultivars ont été relevées pour 26 plantes. Cela suggère que le profilage génétique à un nombre réduit de loci microsatellite est une procédure efficace pour détecter et donc éliminer un matériel végétal impropre à une phase précoce d’un processus de sélection à partir de vignes déjà établies. Dans un second cas, l’identité génétique de trois plantes, échantillonnées dans un vignoble privé de l’appellation contrôlée Dafnes en Crète, a été testée avec 15 marqueurs microsatellites nucléaires et les profils génétiques obte- nus ont été comparés avec ceux de cultivars de référence. Une des trois plantes (dite Liatiko Div. Agr.) était iden- tique à son profil de référence. Des deux autres, une (dite Kotsifali Div. Agr.) pouvait être un clone ou un parent de la première. Après comparaison de son profil d’identité génétique avec 298 profils génétiques à 9 loci microsatellites nucléaire de cultivars grecs, la troisième plante (dite Kotsifaloliatiko Div.Agr.) correspondait à un cultivar habituellement non cultivé en Crète mais en Grèce continentale et nommé Fegi. Ces résultats illustrent la résolution du profilage génétique par microsatellite appliqués à des problèmes pratiques d’identification de plantes provenant de vignes, où des erreurs de nom à un stade précoce (pépinière, distribution commerciale) peuvent avoir des conséquences importantes pour le viticulteur, dans les régions d’appelation contrôlée. Key-words: grapevine cultivars, microsatellite profiling, nSSR, Vitis vinifera. Mots-clés : cultivars de vigne, profilage génétique, microsatellite, Vitis vinifera. INTRODUCTION technically a high reproducibility between users of dis- tant laboratories, thus enabling an easy standardization Among the different classes of molecular markers of the sizing of the alleles. Since the characterization available for molecular profiling of genotypes, micro- of the first microsatellite markers in grapevine by satellites (or single sequence repeated, SSR) tend to be THOMAS et al. (1994), more markers have been made the tool of choice, because they are specific, highly available in the past years (BOWERS et al., 1996; DI polymorphic and co-dominant, while they also allow GASPERO et al., 2000; LEFORT et al., 2002a; SEFC J. Int. Sci. Vigne Vin, 2002, 36, n°4, 177-183 *Correspondance : [email protected] - 177 - ©Vigne et Vin Publications Internationales (Bordeaux, France) F. LEFORT et K. ROUBELAKIS-ANGELAKIS et al., 1999), which have been used in a variety of pur- Sampling was carried out by technicians of the Division poses (for a review, see SEFC et al., 2001). They allo- for Agriculture of the Region of Crete and these plants wed in particular for the first time the comparison of are given with the label Div. Agr. along with their sup- molecular genetic profiles of distant gene pools, (SEFC posed name in table II. et al., 2000; SEFC et al., 2002) and the molecular mar- kers-assisted management of genetic resources (MALE- II - DNA EXTRACTION TIC et al., 1999 ; LOPES et al., 1999). In some cases they were also useful for discriminating clonal lines DNA was extracted from young leaves with a (VIGNANI et al., 1996). Greek genetic resources have micro-method of DNA isolation (LEFORT and been evaluated with previously characterized micro- DOUGLAS, 1999) modified for grapevine (LEFORT satellite markers and genetic profiles were made avai- and ROUBELAKIS-ANGELAKIS, 2001). lable in combination with the last advances in information technology (LEFORT and ROUBELA- III - MICROSATELLITE PCR AND MICROSA- KIS-ANGELAKIS, 2000; LEFORT and ROUBE- TELLITE PROFILE ANALYSIS LAKIS- Amplification primer sequences for nuclear micro- ANGELAKIS, 2001; LEFORT, 2002; LEFORT et al., satellite loci (nSSRs) from Vitis riparia ssrVrZAG21, 2002b). Markers of the same type but targeting the ssrVrZAG47, ssrVrZAG 62, ssrVrZAG64, ssrVrZAG67, chloroplast genome instead of the nuclear genome can ssrVrZAG79, ssrVrZAG83, ssrVrZAG112 (SEFC et also complete the genetic information provided by nuclear microsatellites and their potential had also been al., 1999) and from Vitis vinifera, VVS1, VVS2, VVS3, assessed in Greek cultivars of grapevine (ARROYO VVS4, VVS5 (THOMAS et al., 1994), ssrVvUCH11, et al., 2002; LEFORT et al., 2000) ssrVvUCH29 (LEFORT et al., 2002a) were used for DNA amplification. Four nSSR loci were used for Following this initial step of characterization of screening plants selected from vineyards in Crete and genetic resources of reference conserved in ampelo- Samos and 15 loci were used to check identities of graphic collections in Greece, genetic profiling with 3 plants from a private vineyard in Crete. microsatellite markers can now be used for checking the identity of plants selected from the vineyard in selec- PCR amplifications were carried out in 96-well pro- tion programs, or more generally in the future for pylene plates in 20 µl final volume reaction mixtures controlling the conformity of cultivars and rootstocks in a PTC-100 thermal cycler (M.J. Research Inc., provided by private nurseries, in order to avoid mis- Watertown, MA). PCR reactions were as follows: 1 µM takes which could have harsh consequences for the of each primer, 100 µM of each dNTPs (Biofinex, wine grower. Praroman, Switzerland), 1.5 mM MgCl2 in the buf- fer 75 mm Tris-HCl (pH 9.0), 50 mM KCl, 20 mM In order to illustrate the power of resolution of mole- (NH )2 SO , 0.5 units Taq polymerase (Biotools, cular profiling for agricultural purposes, we report here 4 4 such a kind of a procedure applied to plants of diffe- Madrid, Spain) and 50 ng DNA template. The forward rent cultivars, selected from different vineyards in primer in each case was labeled with the Licor IR800 controlled origin wine areas of Crete and Samos, as fluorochrome. The following thermal cycling proto- well as a case of identification of plant material from col was applied for all loci: 95 °C for 5 min, 10 cycles a private vineyard from Crete. of 15 s at 50 °C, 15 s at 94 °C, followed by 23 cycles of 15 s at 50 °C, 15 s at 89 °C and terminated imme- MATERIAL AND METHODS diately at 4 °C; except for ssrVrZAG64 which had an optimal annealing temperature of 58 °C. The ampli- I - PLANT MATERIAL fication success was checked by electrophoresis in 2 % agarose gel in 1xTBE buffer (0.09 M Tris-borate, pH In the frame of a project of clone selection for a few cultivars from Crete and Samos, 45 grapevine plants 8.3, 2 mM EDTA, pH 8.0) and ethidium bromide stai- were sampled from 18 private vineyards located in ning. PCR products analysis was carried out on 8 % controlled origin wine areas of Crete and Samos polyacrylamide, 7M urea, 1xTBE sequencing gels in (table I), on the basis of phenotypic differences com- a Licor 2400 DNA Sequencer (Licor, Lincoln, NE), paratively to surrounding plants. Cuttings were culti- and alleles were sized with the software Gene Profiler vated in pots in the green house of the Laboratory of v3.54 (Scanalytics, Fairfax, VA). PCR products for Plant Physiology of the University of Crete. In the plants described in table I were run on the same gel. second case described, three plants were sampled in So was it in the second case, where PCR products of a private vineyard in Agia Barbara, a location of the the 3 investigated plants were run on the same gel along controlled origin wine area of Dafnes in Crete. with PCR products from reference plants (table II). J. Int. Sci. Vigne Vin, 2002, 36, n°4, 177-183 ©Vigne et Vin Publications Internationales (Bordeaux, France) - 178 - Identity of grapevine plants by microsatellite profiling Table I - Microsatellite profiles of 47 grapevine plants from Crete and Samos at 4 microsatellite loci. Expected allele sizes are those of plants of reference for a given cultivar and are according the Greek Vitis data- base (http://www.biology.uoc.gr/gvd). Allele sizes are in base pairs (bp).