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Molecular Characterization of Bonarda-Type Grapevine (Vitis Vinifera L.) Cultivars from Argentina, Italy, and France

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Molecular Characterization of Bonarda-Type Grapevine (Vitis Vinifera L.) Cultivars from Argentina, Italy, and France Molecular Characterization of Bonarda-type Grapevine (Vitis vinifera L.) Cultivars from Argentina, Italy, and France Liliana Martínez,1* Pablo Cavagnaro,2 Jean-Michel Boursiquot,4 and Cecilia Agüero1,3 Abstract: Bonarda is the second most cultivated red variety in Argentina after Malbec. Morphologically, the Argentine-Bonarda shows strong similarities with three grape varieties from northern Italy, collectively called Bonarda, and the French variety Corbeau. Twelve Argentine-Bonarda accessions, three Bonarda-type cultivars from Italy (Bonarda Piemontese, Croatina, and Uva Rara), and the French variety Corbeau were analyzed at eight microsatellite loci. Overall, the eight SSRs revealed only four distinct genotypes, corresponding to Bonarda Pi- emontese, Croatina, Uva Rara, and Corbeau. All the Argentine-Bonarda accessions were identical in their SSR profiles to the French Corbeau, indicating, with a high confidence level, that they are the same variety. Key words: grapevine, microsatellites, Bonarda, Argentina South American viticulture is relatively recent since At present, the identity of the Bonarda cultivated in Vitis vinifera was first introduced to America in the form Argentina (Argentine-Bonarda) is not clear. Alcalde (1989) of seeds, raisins, and/or wood cuttings in the 15th century described its ampelography for the first time, indicating by the early Spanish conquerors (Dickenson and Unwin remarkable morphological similarities between Argentine- 1992). Later grapevine introductions followed from other Bonarda and Bonarda Piemontese, a variety grown in the European countries, especially France and Italy. Today, Piedmont region of Italy. He suggested that these two germplasm materials derived from these three European denominations could be synonyms for the same variety. origins predominate in the major viticultural regions of However, as cited in Alcalde (1989), ampelographer Paul South America. Truel reported that Argentine-Bonarda corresponds to the In Argentina, Bonarda is the second most important French cultivar Corbeau, based on morphology. According variety for red winemaking, accounting for nearly 10% to others (Rodríguez and Matus 2002, Matus and Rodri- of the total grapevine cultivated area (Instituto Nacio- guez, unpublished data, 2008), Argentine-Bonarda could nal de Vitivinicultura). For many years it was used in be a different variety than the two previously mentioned. bulk together with several local low-quality varieties to In Italy, Bonarda is a name applied to three differ- produce red wine of little enological value. In the last ent grape varieties (Italian Bonarda-type cultivars) used decades, however, Bonarda has gained appreciation from to make red wine, namely Bonarda Piemontese, Croa- viticulturists and consumers and is increasingly used for tina, and Uva Rara. Bonarda Piemontese (also known as high-quality wines characterized by their deep red color Bonarda dell’Astigiano, Bonarda di Chieri, or Bonarda and finely structured qualities. del Monferrato) is grown in Piedmont, whereas Croatina (or Bonarda dell’Oltrepò Pavese) is grown in Lombardy. Uva Rara (or Bonarda Novarese) is grown in the Pied- mont provinces of Novara and Vercelli. The coexistence 1 2 3 Cátedra de Fisiología Vegetal, Cátedra de Horticultura, and Cátedra de of Bonarda Piemontese, Croatina, and Uva Rara in the Viticultura, Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo, Alte Brown 500, M 5528 AHB, Chacras de Coria, Mendoza, Argentina; 4UMR Piedmont region and their morphological similarities 1097 Diversité et Adaptation des Plantes Cultivées, Equipe INRA, Génétique resulted in their misidentification by viticulturists and de la Vigne, 2, place Pierre Viala, 34060 Montpellier, France, and Etablisse- enologists (Schneider and Mannini 1993). Morphological ment National Technique pour l’Amélioration de la Viticulture, Domaine de descriptions of these cultivars have been carried out by l’Espiguette, 30240 Le Grau du roi, France. several ampelographers (Di Rovasenda 1877, Dalmasso *Corresponding author (email: [email protected]; tel: 054-261- 4135000, ext 1236; fax: 054-261-4960469) et al. 1962a, 1962b, 1963, Schneider and Mannini 1993), Acknowledgments: This work was supported by a grant from Secretaría de indicating that it is possible, although not easy, to distin- Ciencia y Técnica, Universidad Nacional de Cuyo. guish them by this methodology. Furthermore, the rela- The authors thank Sandrine Lalet and Mireille Dessup for their kind help tionship among Bonarda Piemontese, Croatina, and Uva collecting and sending the Corbeau samples. The authors also acknowledge Rara remains uncertain. Giuseppe Zatti and Raquel Garcia for providing leaf samples from the Italian Ampelography traditionally has been used for the Bonardas. identification of grape varieties. The analysis of mor- Manuscript submitted December 2007; revised February 2008. Publication costs of this article defrayed in part by page fees. phological characters is a fast and inexpensive method for Copyright © 2008 by the American Society for Enology and Viticulture. All variety identification, especially among distantly related rights reserved. genotypes. However, they are strongly influenced by the 287 Am. J. Enol. Vitic. 59:3 (2008) 288 – Martínez et al. environment and their interpretation is sometimes subjec- assayed: VVMD5, VVMD7 (Bowers et al. 1996), VVS2 tive, thus reliable discriminations among morphologically (Thomas and Scott 1993), VVMD27, VVMD31, VVMD32 similar germplasm are difficult. This source of error fre- (Bowers et al. 1999), VrZAG62, and VrZAG79 (Sefc et quently leads to mislabeling of individuals, giving rise to al. 1999). cultivar homonyms and/or synonyms. PCR reactions were carried out in a final volume of Molecular DNA markers, on the other hand, are not 25 μL, containing 100 ng of genomic DNA, 1 unit of Taq influenced by the environment and their interpretation is polymerase (Go Taq Promega, Madison, WI), 1 x Taq more objective. Therefore, they are a valuable alternative polymerase buffer (Promega), 0.2 mM of each dNTP (In- for fingerprinting closely related genotypes. Among them, vitrogen, Carlsbad, CA), and 0.128 mM of each primer. random amplified polymorphic DNA (RAPD) (Grando et Amplification reactions were carried out on an Ep- al. 1995, Moreno et al. 1995), restriction fragment length pendorf Mastercycler Gradient thermocycler (Hamburg, polyphorphism (RFLP) (Bowers and Meredith 1996), am- Germany) with the following conditions for all markers: plified fragment length polymorphism (AFLP) (Sensi et 94°C for 2 min followed by 40 cycles at 92°C for 1 min, al. 1996, Cervera et al. 1998), and simple sequence re- 52 to 56°C for 1 min, and 72°C for 2 min, and a final peat (SSR) (Bowers et al. 1996, Sefc et al. 2000) have extension step at 72°C for 7 min. been used for cultivar identification and genetic diversity Aliquots of the amplification products were run in 2% studies in grapevine. SSRs represent codominant, hyper- agarose gel electrophoresis and visualized with ethidium variable loci that are suitable for discriminating among bromide. The remaining PCR products were separated by closely related taxa. They have become the DNA marker polyacrylamide gel (6% polyacrylamide, 7 M urea) elec- of preference for a number of grapevine genetic studies trophoresis and visualized by silver staining as described (Bowers et al. 1996, Bowers and Meredith 1997, Meredith by a Promega Silver Staining kit. Fragments sizes were et al. 1999, Sefc et al. 2000, Hinrichsen et al. 2001, Mar- estimated by using a 100 bp DNA Ladder (Invitrogen) tín et al. 2002, Agüero et al. 2003, González Techera et and by comparison to reference cultivars that were run al. 2004, Martínez et al. 2006a, 2006b). in adjacent lanes in the same gel. The purpose of this work was to characterize these The absence or presence of bands was visually scored. cultivars using microsatellite markers and to clarify the A microsatellite binary matrix was created and analyzed identity of Argentine-Bonarda. To accomplish this, we using Numerical Taxonomy System Software (NTSYS- characterized SSR profiles from Argentine and Italian pc version 1.80; Rohlf 1993). Genetic similarity values Bonarda-type cultivars and French Corbeau and estab- among pairs of genotypes were calculated using the lished genetic relationships among them. simple matching coefficient (Sneath and Sokal 1973) and used to generate a dendrogram based on the unweight- Materials and Methods ed pair-group method of arithmetic averages (UPGMA; Plant material. A total of 16 Bonarda-type grapevine Sokal and Michener 1958). accessions were analyzed in this study. Plant materials from 11 Argentine-Bonarda accessions were provided by Results and Discussion Bodegas y Viñedos Trapiche (Santa Rosa, Mendoza, Ar- Microsatellite analysis resulted in four distinct SSR gentina), and materials were selected with the purpose genotypes that corresponded to the three Italian Bonarda- of capturing potential intracultivar variation. Thus, the type varieties (Bonarda Piemontese, Croatina, and Uva Argentine-Bonarda plants sampled had distinctive phe- Rara) and to the French Corbeau (Table 1). DNA profiles notypes for one or more morphological traits. An acces- of all accessions of Argentine-Bonarda were identical to sion of Argentine-Bonarda was also obtained from the that of Corbeau. The UPGMA dendrogram based on the germplasm collection of the Estación Experimental Agro- analysis of eight SSR loci showed that these four variet- pecuaria
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