Vitis 46 (2), 71–76 (2007) Identification and molecular biodiversity of autochthonous grapevine cultivars in the ‘Comarca del Bierzo’, León, Spain F. GONZÁLEZ-ANDRÉS 1) , J. P. MARTÍN 2) , J. YUSTE 3) , J. A. RUBIO 3) , C. ARRANZ 3) and J. M. ORTIZ 2) 1) Dto. Ingeniería Agraria, E.S.T. de Ingeniería Agraria, León, Spain 2) Dto. Biología Vegetal, E.T.S. Ingenieros Agrónomos, Universidad Politécnica de Madrid, Madrid Spain 3) Dto. Viticultura, Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid, Spain Correspondence to: Dr. J. M. ORTIZ, Departamento de Biología Vegetal, E.T.S. Ingenieros Agrónomos. Ciudad Universitaria, 28040 Madrid, Spain. Fax: 913365656. E-mail: jesusmaria.ortizm@upm.es Summary A prospecting work at the ‘Comarca del Bierzo’ in Spain has been carried out in order to evaluate the existing biodiversity and detect the neglected and en- dangered minor grapevine cultivars of the zone. As a result, a total of 79 different accessions were collected and studied using STMS markers for preliminary va- riety identification, that was confirmed on the base of ampelographic observations. Several synonymies and homonymies were detected. A total of 33 different cul- tivars were identified. Comparison of the genotypes, allelic frequencies and allelic sizes are presented. Con- servation of the endangered cultivars is highly recom- mended. K e y w o r d s : ampelography, microsatellites, synony- mies, Vitis vinifera. Introduction The ‘Comarca del Bierzo’, located in the northwest of the León province in Spain is an area of vineyards for obtention of VQPRD (Vin de Qualité Produit dans des Régions Déterminées), and corresponds to the Apellation of Origin ‘D.O. Bierzo’. GARCÍA DE LOS SALMONES (1914) mentioned about 30 white and 30 red cultivars existing in this area, although probably including also a certain number of synonymies. The ‘D.O. Bierzo’, only includes four white, ‘Godello’, ‘Doña Blanca’, ‘Malvasía’ and ‘Pal- omino’, and two red, ‘Mencía’ and ‘Garnacha Tintorera’ cultivars (ORTIZ and VILLASECA 2004). This fact leads to the reduction of the cultivars. As a consequence, an intense erosion of grapevine germplasm exists, with a high risk of extinction of old autochthonous cultivars. During the last years a prospecting study in the search of endangered plant material has been carried out. STMS (sequence-tagged microsatellite site) markers were used in order to identify the collected cultivars, since they have a high level of reproducibility, and are adequate for the genotyping of grapevine cultivars (SEFC et al. 1998, MARTÍN et al. 2003). Complementary ampelographic ob- servations were made to confirm the obtained results. The objective of the present study was to collect mi- nor and endangered cultivars in the ‘D.O. Bierzo’, identify them and analyze their variability. The detection of syno- nymies among the studied cultivars was based on the mo- lecular characterization and ampelographic observations, taking some previous studies into account (MARTÍN et al. 2003, PINTO-CARNIDE et al. 2003). Material and Methods A broad survey of the ‘Comarca del Bierzo’ was car- ried out in order to study the existing variability in the area. The plants selected were either supposed being different cultivars or plants of the same cultivated variety in order to assess their true to typeness. Twenty five plants (‘unknown’ in Tab. 1) were sampled because they were different of the predominant variety in the plot and were not previously identified as being an already known variety. Tab. 1 lists the 79 accessions that were sampled at different plots, with the indication of the berry color as well as the local name of the variety. Samples of young fresh leaves were collected in the field and kept at -80 ºC until analyzed. For the molecular characterization, DNA extraction was carried out by using the MasterPure TM Plant Leaf DNA Purification Kit (Epi- centre Technologies, Madison, USA). The following six STMS loci were used: VVS2 (THOMAS and SCOTT 1993), VVMD5 and VVMD7 (BOWERS et al. 1996), and ssrVr- ZAG47, ssrVrZAG62 and ssrVrZAG79 (SEFC et al. 1999), under the conditions detailed in a previous work (MARTÍN et al. 2003). Polymorphism of the amplified products was detected in an automated DNA sequencer ABI PRISM model 310 (PE Applied Biosystems). As a result of the analysis, genotypes for each cultivar were obtained for the studied loci. Microsatellite genotypes were compared with some of the available databases (SEFC et al. 1998, http://www. genres.de/CF/eccdb/vitis, MARTÍN et al. 2003) by using ref- erence standard cultivars and comparing the relative allele sizes for the different STMS loci. In order to confirm the provisionally identified culti- vars on the base of the molecular results, ampelographic observations were made in the field at different times of the year. The minimal list of descriptors for germplasm collec- tions was used (OIV 1984) (data not presented). For the study of the molecular biodiversity, the allele and genotype frequencies were obtained by counting and direct calculation. The observed heterozygosity H o was calculated as the ratio between heterozygote genotypes and CORE Metadata, citation and similar papers at core.ac.uk Provided by JKI Open Journal Systems (Julius Kühn-Institut)