Clones Identification and Genetic Characterization of Garnacha Grapevine by Means of Different PCR-Derived Marker Systems

This study uses PCR-derived marker systems to investigate the extent and distribution of genetic variability of 53 Garnacha accessions coming from Italy, France and Spain. The samples studied include 28 Italian accessions (named Tocai rosso in Vicenza area; Alicante in Sicily and Elba island; Gamay perugino in Perugia province; Cannonau in Sardinia), 19 Spanish accessions of different types (named Garnacha tinta, Garnacha blanca, Garnacha peluda, Garnacha roja, Garnacha erguida, Garnacha roya) and 6 French accessions (named Grenache and Grenache noir). In order to verify the varietal identity of the samples, analyses based on 14 simple sequence repeat (SSR) loci were performed. The presence of an additional allele at ISV3 locus (151 bp) was found in four Tocai rosso accessions and in a Sardinian Cannonau clone, that are, incidentally, chimeras. In addition to microsatellite analysis, intravarietal variability study was performed using AFLP, SAMPL and M-AFLP molecular markers. AFLPs could discriminate among several Garnacha samples; SAMPLs allowed distinguishing few genotypes on the basis of their geographic origin, whereas M-AFLPs revealed plant-specific markers, differentiating all accessions. Italian samples showed the greatest variability among themselves, especially on the basis of their different provenance, while Spanish samples were the most similar, in spite of their morphological diversity

High-throughput 18K SNP array to assess genetic variability of the main grapevine cultivars from Sicily

The viticulture of Sicily, for its vocation, is one of the most important and ancient forms in Italy. Autochthonous grapevine cultivars, many of which known throughout the world, have always been cultivated in the island from many centuries. With the aim to preserve this large grapevine diversity, previous studies have already started to assess the genetic variability among the Sicilian cultivars by using morphological and microsatellite markers. In this study, simple sequence repeat (SSR) were utilized to verify the true-to-typeness of a large clone collection (101) belonging to 21 biotypes of the most 10 cultivated Sicilian cultivars. Afterwards, 42 Organization Internationale de la Vigne et du Vin (OIV) descriptors and a high-throughput single nucleotide polymorphism (SNP) genotyping array (Vitis18kSNP) were applied to assess genetic variability among cultivars and biotypes of the same cultivar. Ampelographic traits and high-throughput SNP genotyping platforms provided an accuracy estimation of genetic diversity in the Sicilian germplasm, showing the relationships among cultivars by cluster and multivariate analyses. The large SNP panel defined sub-clusters unable to discern among biotypes, previously classified by ampelographic analysis, belonging to each cultivar. These results suggested that a very large number of SNP did not cover the genome regions harboring few morphological traits. Genetic structure of the collection revealed a clear optimum number of groups for K = 3, clustering in the same group a significant portion of family-related genotypes. Parentage analysis highlighted significant relationships among Sicilian grape cultivars and Sangiovese, as already reported, but also the first evidences of the relationships between Nero d’Avola and both Inzolia and Catarratto. Finally, a small panel of highly informative markers (12 SNPs) allowed us to isolate a private profile for each Sicilian cultivar, providing a new tool for cultivar identification

Incidencia de las infecciones víricas en antiguos viñedos de tres variedades de vid autóctonas de Mallorca: consecuencias sobre las estrategias de la selección clonal

Three autochthonous grapevine varieties of Majorca (Spain) were analyzed for the presence of viruses listed by the international certification programs. Enzyme-Linked Immuno-Sorbent Assay (ELISA) screenings were performed in 193 vines from 46 vineyards included in a clonal selection. Virus-free vines were only 6.4%, 9.6% and 11.5%, in Manto Negro, Callet and Moll, respectively. Infections by grapevine leafroll associated viruses (GLRaVs) were ascertained in 71%, 78% and 60% of Manto Negro, Callet and Moll vines, respectively. Each variety was also highly infected by Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV). The percentage of plants displaying multiple infections was 58.4% in Manto Negro, 63.8% in Callet and 42.6% in Moll. Thus, it was very difficult to find virus-free clones with suitable agronomic characteristics to be considered as a reference for the grape market. In order to obtain certified propagation material under such conditions of endemic viral infection, sanitation should be the main focus in clonal selection processes. However, the time and financial requirements for proper sanitation process bring to consideration the need to use, at least temporarily, standard multiplication material while certified clones are achieved.En este estudio se analizó la presencia de las virosis contempladas por las leyes internacionales en tema de certificación en tres variedades de vid autóctonas de Mallorca. Para ello se realizó el test ELISA (enzyme-linked immuno-sorbent assay) sobre 193 cepas procedentes de 46 viñedos incluidos en un programa de selección clonal. Las cepas que resultaron libres de los virus analizados fueron el 6,4% en Manto Negro, el 9,6% en Callet y el 11,5% en Moll. Los porcentajes de cepas infectadas por los virus asociados al síndrome del enrollado de la vid (GLRaVs) fueron 71% en Manto Negro, 78% en Callet y 60% en Moll. Se detectaron también altas tasas de infecciones para el virus del entrenudo corto infeccioso de la vid (GFLV) y el virus del jaspeado de la vid (GFkV) en las tres variedades. Los porcentajes de cepas sujetas a infecciones múltiples fueron 58,4% en Manto Negro, 63,8% en Callet y 42,6% en Moll. Por tanto fue difícil encontrar cepas que cumpliesen tanto los requisitos sanitarios para la certificación como aquellos agronómicos necesarios según las exigencias del mercado vitivinícola. En tales condiciones de infecciones víricas endémicas, el saneamiento debería ser el primer objetivo a seguir con el fin de obtener material de propagación certificado. Sin embargo, el tiempo y los recursos económicos necesarios para llevar a cabo el proceso de saneamiento conllevan la necesidad de considerar el uso temporal de material de propagación estándar hasta que se obtengan clones certificados

First report of Grapevine leafroll-associated virus 4 (GLRaV-4) in Spain

During phytosanitary certification of three grapevine clones (one of Gorgollosa and two of Mances de Tibus) received at IMIDA from the Balearic Islands (Spain), we observed discrepancies between symptoms on Cabernet Franc indicator plants and the results of ELISA testing for viruses associated with grapevine leafroll.According to DAS-ELISA analysis (Bioreba, Nyon, Switzerland), the three clones were free from GLRaV-1 and GLRaV-3 but positive for GLRaV-2. However, in four consecutive seasons on indicator rootstocks, symptom severity led to a suspicion that other leafroll viruses might be present; in our experience, symptoms produced by GLRaV-2 infection alone are rarely as severe as those that were observed. Testing of the clones with real-time RT-PCR using TaqMan ® probes (Osman et al., 2007) detected Grapevine leafroll-associated virus 4.Using similar real-time PCR methods we confirmed the presence of GLRaV-2 but we did not detect either GLRaV-1 or GLRaV-3. Moreover we could also detect Grapevine fanleaf virus

Nuevas prospecciones y recuperación de variedades antiguas de vid en España

8 Pags.- 2 Tabls.- 1 Fig.El patrimonio varietal de vid en España, ha sufrido un proceso importante de erosión y cambio durante los últimos cien años por diversas razones fitopatológicas, comerciales y legislativas. Desde hace algunos años, expertos en viticultura de toda España están prospectando, para su recuperación y conservación, gran parte de estas variedades que habían dejado de cultivarse. Actualmente organismos de las 17 Comunidades Autónomas, están trabajando en colaboración en un proyecto común para la identificación, conservación y documentación de material de variedades abandonadas o minoritarias. La metodología que se está utilizando para la identificación del material es la suma de 35 caracteres morfológicos y 8 marcadores microsatélites con alto poder discriminante. Se han analizado 1.825 muestras y los resultados moleculares se han comparado con bases de datos nacionales e internacionales. Entre el material encontrado aparecen 299 genotipos nuevos de los que cerca de 100 se han encontrado en varias parcelas de una o más Comunidades Autónomas, por lo que se trata sin duda de material cultivado en el pasado y que es importante conservar y evaluar desde el punto de vista agronómico y enológico. Paralelamente, con la ayuda del Centro Nacional de Recursos Fitogenéticos del Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria (CRFINIA), responsable de la documentación de las colecciones de la red del Programa Nacional de Recursos Fitogenéticos, están armonizando los datos de pasaporte de todas las colecciones de vid en España. Esta información está disponible en el Inventario Nacional.Agradecemos al Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) y a los fondos FEDER la financiación de este trabajo a través del proyecto RF2012-00027- C5-00. También a la Oficina Española de Variedades Vegetales el apoyo técnico prestado. La dedicación de la Dra. J Bota ha sido también financiada, en el marco del proyecto PD/027/2013, por la Conselleria de Educación, Cultura y Universidades del Govern de les IIles Balears y por el Fondo Social Europeo a través del programa operativo del FSE de las lIIes Balears para el periodo 2013-2017. 32Peer reviewe

Nuevas prospecciones y recuperación de variedades antiguas de vid en España

1 Pag.Actualmente organismos de las 17 Comunidades Autónomas, están trabajando en colaboración en un proyecto común para la identificación, conservación y documentación de material de variedades abandonadas o minoritarias. La metodología que se está utilizando para la identificación del material es la suma de 35 caracteres morfológicos y 8 marcadores microsatélites con alto poder discriminante.Peer reviewe

Assessment of the uniformity and stability of grapevine cultivars using a set of microsatellite markers

Solidity of microsatellite markers is a key issue for varietal identification, especially when they are used for legal purposes, what includes their probable future use in the distinctness, uniformity and stability testing of new varieties needed for the granting of Plant Breeders' Rights. Nine grapevine microsatellites (VVS2, VVMD5, VVMD27, VVMD28, ssrVrZAG29, ssrVrZAG62, ssrVrZAG67, ssrVrZAG83 and ssrVrZAG112), which had previously demonstrated its capacity to discriminate any grapevine variety, have been assessed to evaluate its uniformity and stability. 19 varieties were selected, representative of a high diversity for morphological, agronomical, cultural and historical aspects, as well as for microsatellite allele variability. Then, for each variety, uniformity and stability were evaluated through the analysis of 50 plants from each of three different plots, and five plants from each of seven additional plots. Material from 4,137 plants of 229 plots of the 19 varieties was sampled in seven countries. Of 3,654 plants analyzed with the set of nine microsatellites, 3,299 were of the right variety and used for the survey. An average of 172 individual values was studied for each allele of each microsatellite of each variety, and none differences were detected that could not be explained as technical variations, with the exception of several putative chimeras in two varieties. Of the total of 171 variety x microsatellite combinations, only in one combination ('Merlot' x VVMD27) the number of off-types exceeded the threshold allowed. The remaining 170 combinations have been found uniform and stable according to internationally accepted rules. © 2012 Springer Science+Business Media B.V.This study was financially supported by the project VIN01-025 (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Agriculture Ministry of Spain).Peer Reviewe

Recovery of minor old grape varieties in Spain

2 Pags.Recent works by experts in viticullure and oenology in Spain under the same projecl, in collaboralion with the Spanish Planl Variety Office and Ihe Plant Genetic Resources Center, have allowed Ihe identificalion of old vine genotypes in relictic areas. Molecular and ampelographic methods have been used to identify the plant material, combining 8 microsatellites with high discriminant power and 67 morphological traits including those required by the Community Plant Variety Office.Peer reviewe