Molecular Characterization of Bonarda-type Grapevine ( L.) Cultivars from Argentina, Italy, and

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 . Morphologically, the Argentine-Bonarda shows strong similarities with three varieties from northern Italy, collectively called Bonarda, and the French variety Corbeau. Twelve Argentine-Bonarda accessions, three Bonarda-type cultivars from Italy (, , 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 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 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to others (Rodríguez and Matus 2002, Matus and Rodri- of the total grapevine cultivated area (Instituto Nacio- guez, unpublished data, 2008), Argentine-Bonarda could nal de Vitivinicultura).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 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 , 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 INTA-Luján de Cuyo, Mendoza. Leaf tissues of ies have less than 40% of the SSR alleles in common French Corbeau were obtained from the germplasm col- (Figure 1). lection of INRA, Domaine de Vassal, Marseillan, France. Despite their ampelographic differences, Italian Bon- Leaves samples from three Italian Bonarda-type cultivars arda-type varieties, Bonarda Piemontese, Croatina, and (Bonarda Piemontese, Uva Rara, and Croatina) grown in Uva Rara are often not differentiated and are simply the Piedmont and Liguria regions of Italy were provided called Bonarda in Italy (Schneider and Mannini 1993). by Dr. Giuseppe Zatti. Our results, from the SSR analysis, showed a low degree DNA extraction and PCR amplification. Genomic of similarity among these varieties and no direct genetic DNA was isolated from young leaves of individual vines relationships could be established among them. This is using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) the first report indicating such low level of relatedness and quantified either by visual comparison with λ-DNA (at the DNA level) among Italian Bonarda. molecular marker on ethidium bromide stained agarose Argentine-Bonarda samples used in this study, coming gels or by spectrophotometry using a Pharmacia Gene from a common vineyard, displayed apparent phenotyp- Quant Spectrophotometer (Pharmacia, Biotech, Cam- ic differences among them; however, they had the same bridge, UK). A total of eight microsatellite markers were SSRs profiles as the Argentine-Bonarda from the INTA

Am. J. Enol. Vitic. 59:3 (2008) Molecular Characterization of Bonarda-type Grapevine – 289 germplasm collection and Corbeau. This sample set of Ar- (Alcalde 1989) using ampelographic traits suggesting that gentine-Bonarda, collected from a commercial vineyard, Argentine-Bonarda and the French Corbeau are the same included plants that were originally obtained from differ- variety. ent nurseries or grapegrowers during the last 40 years. Interestingly, in California, Corbeau is grown under Therefore, phenotypic variations observed among them the name of Charbono, another variety from Piedmont, It- in the field could be due to differences in plant age and aly. All Charbono accessions at Foundation Plant Services physiological status rather than genetic variation. Interest- (FPS), University of California, Davis, fully matched the ingly, the fact that these materials had different origins DNA of Corbeau (Meredith et al. 1999). DNA profiles of within Argentina (e.g., different nurseries or growers) and synonymous varieties of Corbeau (Courbu, Courbu noir, still had identical SSR profiles suggests genetic homoge- and Petit Courbu) from Montpellier vines also matched neity for this variety in Argentina. This suggestion is con- Charbono. sistent with Corbeau being introduced once, followed by vegetative propagation through- out the region, as opposed to many different introductions throughout history. However, a broader SSR-fingerprinting analysis includ- ing more Argentine-Bonarda plants from several viticultural regions of Argentina would provide a more conclusive answer. The probability that the Argentine-Bonarda SSR genotype could occur by chance in another variety can be estimated from the frequen- cies of the individual alleles in the general population of Vitis vinifera cultivars. Based on allele frequencies obtained from a survey of 644 to 694 cultivars (C.P. Meredith, per- sonal communication, 2001) the probability that Argentine-Bonarda is not Corbeau is 5.0 x 10 -12. This indicates, with a very high level of confidence, that Argentine-Bonarda and Corbeau are the same genotype. These re- Figure 1 Dendrogram of Bonarda-type varieties using SSR data (AB: Argentine- sults agree with those reported by Paul Truel Bonarda).

Table 1 Comparison of Argentine-Bonarda (AB), Corbeau, Bonarda Piemontese, Croatina, and Uva Rara alleles at eight SSR loci. Cultivars VVMD5 VVMD7 VVS2 VVMD27 VVMD31 VVMD32 VrZAG62 VrZAG79 AB 1 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 2 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 3 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 4 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 5 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 6 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 7 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 8 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 9 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 10 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB 11 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 AB INTA 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 Corbeau 228-238 249-263 151-151 189-189 204-212 241-273 195-201 251-259 Bonarda Piemontese 246-246 249-255 143-155 185-185 204-210 241-273 201-205 237-251 Croatina 236-236 247-247 141-153 185-185 216-216 257-273 187-197 245-245 Uva Rara 236-240 247-247 133-143 185-189 212-212 257-273 197-201 243-243

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In France, Corbeau is considered an old variety that important reasons to preserve the name Argentine-Bonar- traditionally was grown mostly in . Corbeau produc- da on the label. Nevertheless, our results provide further tion in France was widespread in the second part of the evidence that the real identity of Argentine-Bonarda is 19th century (Mas and Pulliat 1874–1879), but its impor- the French Corbeau. tance decreased drastically during the 20th century, with 957 ha grown in 1958 (IVCC: Cadastre Viticole 1958), 576 Literature Cited ha in 1968 (IVCC: Cadastre Viticole 1968), and 55 ha in Agüero, C.B., J.G. Rodríguez, L.E. Martínez, G.S. Dangl, and C.P. 1979 (Ministère de l’Agriculture SCEES–INSEE: Recen- Meredith. 2003. Identity and parentage of Torrontés cultivars in sement Général Agricole 1979). To date the total surface Argentina. Am. J. Enol. Vitic. 54:318-321. cultivated with Corbeau in France is ~2 ha (J.M. Boursi- Alcalde, A.J. 1989. Cultivares vitícolas argentinas. Asociación Co quot, personal estimation, 2007). Its origin is supposed to operadora de la Estación Experimental Agropecuaria, INTA, be Savoie or north Italy (Viala and Vermorel 1902–1910). Mendoza, Argentina. In the Domaine de Vassal grapevine repository, sever- Bowers, J.E., and C.P. Meredith. 1996. Genetic similarities al accessions of this variety have been introduced from among wine grape cultivars revealed by restriction fragment different locations and with distinct names (synonyms) length polymorphism (RFLP) analysis. J. Am. Soc. Hortic. Sci. such us Douce noire de Savoie, Picot rouge, Promèche 121:620-624. rose, Côte rouge (eastern France), Alcantino (northeastern Bowers, J.E., and C.P. Meredith. 1997. The parentage of a classic France), and Turca (Italy). wines of Corbeau were wine grape . Nat. Genet. 16:84-87. considered of moderate quality and not really adapted for Bowers, J.E., G.S. Dangl, R. Vignani, and C.P. Meredith. 1996. Iso- aging. 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