Research Note Molecular Correlation of Zinfandel (Primitivo) with Austrian, Croatian, and Hungarian Cultivars and Kratošija, an Additional Synonym

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1 Research Note Molecular Correlation of Zinfandel () with Austrian, Croatian, and Hungarian Cultivars and, an Additional Synonym Antonio Calò, 1 * Angelo Costacurta, 1 Vesna Maraš, 2 Stefano Meneghetti, 1 and Manna Crespan 1 Abstract: Results of comparative DNA analysis of Zinfandel with Austrian, Croatian, and Hungarian grape varieties by means of 13 SSR nuclear markers are reported. Twenty-six additional cultivars, representing italian and foreign grapevines, were used as an outgroup in the search for clues that might indicate possible links with Zinfandel. Zinfandel clustered with the following cultivars: its offspring Plavac mali and Plavina, Dobričić, the other parent of Plavac mali, three Malvasias known to have a historical presence along Dalmatian coast, the Croatian cultivars Jarbola and Hrvatica, the Austrian cultivars Franconia and Veltliner frührot, and the Hungarian cultivars Furmint, Harzevelu, and Kadarka. All these varieties are localized in a well-defined geographic area that includes Austria, Dalmatia, and Hungary. The possible first-degree relationship of Zinfandel with Plavac mali and Plavina has been corroborated by results from 21 other SSR loci. an additional synonym for Zinfandel has been identified:, an old variety growing in Montenegro. Key words: Plavina, Verdeca, pedigree, SSR Interest in the origins of Zinfandel and its diffusion has stimulated several research projects aimed at verifying variety homonyms and synonyms in different areas, particularly in Croatia. Similarity was hypothesized between Zinfandel and the grape varieties Plavac and Plavina (Calò 1987) on the basis of an earlier report (Ramey 1977). It is now recognized that Zinfandel is a synonym for a Croatian grape variety in the Spalato area, Crljenak Kastelanski, and that Plavac mali is a progeny of Zinfandel and Dobričić (Maletić et al. 2004). Plavac mali was planted widely in the districts of Chin, Sebenico, and Zara on the Quarnero Islands; it was known as Brajda or Brajdica on the islands of Pago and Arbe (Bulić 1949). Plavina was reportedly the most common grape variety along the Yugoslavian coast (Bulić 1949) and was known as Pagadebiti in Curcola, as Crnac or Kasteljanac in Trau, as Viska in Zara, and as Crljenak in Spalato, which is now assimilated to Zinfandel. Previous research on Plavina after it was added to the ampelographic collection of the Istituto Sperimentale per la Viticoltura (Conegliano, Italy) at the end of the 1980s is again of interest with the onset of DNA analysis with SSR markers. Analysis of historical documents, ampelographic literature, and botanical paintings has shown that Zinfandel was present in the Carpathians, in Hungary, Slovakia, Moravia, Bohemia, and Slovenia under many different names (Jung 2007). Given this history, the possible relationships of Zinfandel with Hungarian and Croatian varieties have been investigated by comparison of SSR profiles. Additional cultivars that may be related to Zinfandel based on morphological similarities (Dolcetto, Pignola, and Veltliner frührot) have also been considered, together with 24 Austrian, Montenegrin, northeastern Italian, and outgroup varieties. 1 C.R.A. Centro di Ricerca per la Viticoltura, Viale 28 Aprile, 26, Conegliano (TV), Italy; and 2 Plantaže A.D. U.I. Radomira Ivanovića br Podgorica, Crna Gora, Montenegro. *Corresponding author ( antonio.calo@entecra.it) Acknowledgments: The authors thank Ivan Pejić and Edi Maletić for Crljenak Kaštelanski and Dobričić samples and Barbara Sladonja for Plavac mali, Plavina, Jarbola bijela, and Hrvatica samples. This research was supported by Trattato Internazionale FAO, Progetto Risorse Genetiche Vegetali, funded by the Italian Ministero delle Politiche Agricole, Alimentari e Forestali. Manuscript submitted April 2007; revised November Publication costs of the article defrayed in part by page fees. Copyright 2008 by the American Society for Enology and Viticulture. All rights reserved. 205 Materials and Methods Plant material. Accessions from different sources belonging to the following grape varieties were compared: Crljenak Kaštelanski, Dobričić, Dolcetto, Franconia, Furmint, Harzevelu, Hrvatica, Jarbola bijela, Kadarka,, Pignola, Plavac mali, Plavina,, Riesling italico, Veltliner frührot, Vranac, and Zinfandel (Table 1). Samples came from the collections of the Centro di ricerca per la Viticoltura at Susegana (Italy), the Vivai Cooperativi di Rauscedo (Pordenone, Italy), the 13 Jul Plantaže a.d. company vineyards in Podgorica (Montenegro), and the Institute of Agriculture and Tourism of Poreč (Croatia).

2 206 Calò et al. DNA analysis with SSR markers and data elaboration. In order to identify the accessions, SSR profiles were produced using the following 13 microsatellite loci: VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, and VrZAG79 (the internationally adopted reference set) (This et al. 2004); VVMD28, VVMD32, and VVMD36 (Bowers et al. 1999); and ISV2, ISV3, ISV4, and VMC NG 4b9 (Crespan 2003). The analysis was extended to other microsatellite loci, for a total of 34, to integrate the SSR data relating possible parent-progeny relationships of Zinfandel with Plavac mali and Plavina: VVS1 (Thomas and Scott 1993); VVS29 (M.R. Thomas, CSIRO, personal communication, 1998); VVMD8 (Bowers et al. 1996); VVMD17, VVMD21, VVMD24, VVMD25, VVMD26, and VVMD31 (Bowers et al. 1999); VrZAG21, VrZAG64, and VrZAG83 (Sefc et al. 1999); scu05 (Scott et al. 2000); VMC4g6 and VMC4h6 (Di Gaspero et al. 2000); VMC6e10 (Arroyo-García and Martínez-Zapater Accession Crljenak Kaštelanski VV-102 Dobričić Dolcetto R3 Dolcetto Franconia Furmint Harzevelu Hrvatica Jarbola bijela Kadarka I II IV VI Pignola Plavac mali Plavac n. 1 Plavac n. 2 Plavina Plavina n. 1 Plavina n. 2 Table 1 Varieties and related accessions analyzed and the associated supplier. Supplier University of Zagreb, Croatia University of Zagreb, Croatia Repository ISV of Arezzo, Italy Institut of Puly, Svizzera Unknown Institute for Agricolture and Tourism of Poreč, Croatia Institute for Agricolture and Tourism of Poreč, Croatia Unknown Plantaže, Montenegro Gluhi do Crmnica, Montenegro Beri, Montenegro Ispettorato Provinciale Agrario of Sondrio, Italy Institut for Agricolture and Tourism of Poreč, Croatia Institut for Agricolture and Tourism of Poreč, Croatia Crispiano (Taranto), Italy Copertino (Lecce), Italy Sava (Taranto), Italy Gioia del Colle (Bari), Italy Riesling italico University of Milan (clone RI 12 V 18) Veltliner frührot Vranac Zinfandel n. 2 Zinfandel n. 6 Vivai Mario Pinat, Perteole (Udine), Italy Plantaže, Podgorica, Crna Gora, Montenegro University of California, Davis University of California, Davis 2004); and VMC3d7, VMC2h9, VMC1e12, VMC2g2, and VMC2h4 from the Vitis Microsatellite Consortium. PCR reactions were multiplexed and allele lengths were determined using reference varieties as previously described (Crespan et al. 2006). The dissimilarity index calculated as ln (proportion of shared alleles) was used to measure the genetic distance between all pair-wise combinations of cultivars (Table 1). This method was suggested for its ability to find correlations without preliminary assumptions about the population under study or the frequency of alleles within that population; it is well suited for use with highly variable SSR loci and unnatural populations such as grape (Dangl et al. 2001). Twenty-four other varieties were added, using molecular data from the SSR database of Centro di Ricerca per la Viticoltura, Conegliano. These included Malvasia grapevines with a long history along the east side of the Adriatic Sea (Malvasia bianca lunga, Malvasia delle Lipari, and Malvasia istriana), Friuli and Veneto cultivars (Corvina, Garganega, Marzemina bianca, Piculit neri, Prosecco tondo, Raboso Piave, Refosco dal peduncolo rosso, Refoscone, Ribolla gialla, Tazzelenghe, and Terrano), and some outgroup varieties (Cabernet Sauvignon, Ciliegiolo, Croatina, Merlot, Montepulciano, Moscato bianco, Pinot nero, Riesling renano, Sangiovese, and Sultanina). A dendrogram of the 39 cultivars was elaborated with the UPGMA method and the cophenetic correlation was calculated using NTSYS (Exeter Software, Setauket, NY). Results and Discussion SSR profiles for the first 13 markers are presented (Table 2), and the three reference varieties are listed separately for easier data comparison (Table 3). As expected, the same molecular profile was obtained for accessions of Zinfandel,, and Crljenak Kaštelanski, which are recognized synonyms (Calò 1987, Maletić et al. 2004). Surprisingly, a new synonym for Zinfandel was identified:, an old Montenegrin grape variety that, according to the literature, existed in these regions before Vranac (Ulićević 1966). Vranac today predominates in Montenegro and is often cultivated together with. Our data suggest a probable first-degree relationship between these two cultivars, as indicated previously (Maletić et al. 2004). The population consists of extremely heterogeneous plants, indicating that its age-old presence in the area has led to differentiation of many morphotypes, which have also been studied (Pejović 1988, Maraš et al. 2003, 2004).

3 Molecular Correlation of Zinfandel 207 Table 2 SSR profiles of 15 individual varieties (allele lengths in bp). Veltliner frührot Vranac Riesling italico Plavac mali Plavina Jarbola bijela Kadarka Pignola Crljenak Kaštelanski Zinfandel Dobričić Dolcetto Franconia Furmint Harzevelu Hrvatica SSR loci VVS VVMD VVMD VVMD VVMD VVMD VVMD VrZAG VrZAG ISV (VMC 6e1) ISV (VMC 6f1) ISV (VMC 6g1) VMCNG4b Table 3 SSR profiles of three reference varieties (allele lengths in bp). SSR loci Moscato bianco Sultanina Pinot noir VVS VVMD VVMD VVMD VVMD VVMD VVMD VrZAG VrZAG ISV (VMC 6e1) ISV (VMC 6f1) ISV4 (VMC 6g1) VMCNG4b the various accessions of Plavac mali and Plavina were confirmed as belonging to the respective grape variety. Plavina was also confirmed as a different variety than Zinfandel. the Dolcetto SSR profile corresponds to that reported elsewhere for other specimens (Sefc et al. 2000); in particular, the clone R3 presents a third, 249 bp allele at the locus VVMD7. these small mutations are rare but have been documented previously in the literature (Franks et al. 2002, Riaz et al. 2002, Crespan 2004). Their presence does not compromise the validity of the SSR profile for varietal identification. Our data exclude a parent-progeny link of Zinfandel with Dolcetto, Pignola, and Veltliner frührot. The proposed parentoffspring relationship of Zinfandel with Plavac mali, and perhaps with Plavina and Vranac (Maletić et al. 2004), is corroborated by our data using different SSR loci (Table 2, Table 4). Moreover, others have suggested that Plavina could be an offspring of Zinfandel and Verdeca, a white cultivar grown in southern Italy, particularly in Apulia, where Zinfandel is largely cultivated (Lacombe et al. 2007).

4 208 Calò et al. Table 4 Additional SSR data for pedigree analysis. SSR loci Plavac mali Zinfandel Plavina VVS VVS VVMD VVMD VVMD VVMD VVMD VVMD VVMD VrZAG VrZAG VMC 4g VMC 6e VMC 3d VMC 2h VMC 1e VMC 2g VrZAG VMC 2h VMC 4h scu The dendrogram based on cluster analysis of genetic dissimilarity shows four main groups (Figure 1): Zinfandel is included in the higher branch, A, together with its progeny, Plavac mali and Plavina. Dobričić, the other parent of Plavac, groups tightly with them. In the same cluster are three Malvasias known to have a historical presence along Dalmatian coast: Malvasia delle Lipari alias Malvasia dubrovačka (Crespan et al. 2006), Malvasia istriana (Galet 2000), and Malvasia bianca lunga alias Maraština (S. Šimon et al., unpublished data, 2007). Others in this cluster include the Croatian Jarbola and Hrvatica (Sladonja et al. 2007), the Austrian Franconia and Veltliner frührot, and the Hungarian Furmint, Harzevelu, and Kadarka. Vranac is included in another branch; its parent-progeny relationship with Zinfandel needs to be confirmed with additional molecular data, but it has been Figure 1 Dendrogram of 39 cultivars based on cluster analysis of genetic dissimilarity (cophenetic correlation value: ). noted that an offspring may cluster far from one parent, particularly when the cross is wide (Dangl et al. 2001). A close similarity has also emerged between Furmint and Harzevelu, the classic grape varieties that are the ancestors of Tokai. They share at least one allele at each SSR locus analyzed, so a parent-progeny relationship can be confidently hypothesized. What seems to be more relevant is that group A encompasses varieties grown in a well-defined geographic area, from Austria to Dalmatia and Hungary. Despite the observation about the spread of Zinfandel in Europe (Jung 2007), we found that none of the available SSR profiles of Hungarian cultivars from different repositories match that of Zinfandel (Halász et al. 2005, Jahnke et al. 2007). Such information is also absent in an analysis of Bulgarian samples (Hrvaleva et al. 2004). Zinfandel was grown in Germany, since it was found in some very old vineyards near Heidelberg (Jung and Maul 2004). The potential kinships emerging from molecular data comparison among Zinfandel, Plavina, Verdeca, and Vranac should be examined more closely to better delineate the pedigree relations and therefore the evolution of current grapevine germplasm. Conclusions Given the interest in Zinfandel () origin and spread, the present research focused on the possible relationships among this cultivar and those of different countries by means of SSR molecular markers. An additional synonym was found:, an old Montenegrin

5 Molecular Correlation of Zinfandel 209 variety encompassing many heterogeneous morphotypes. Thus, new information was added that enlarges the ancient growing areas of Zinfandel. First-degree relationships among Zinfandel () and some northern Italy cultivars could be excluded, especially with the highly morphologically similar Dolcetto. The genetic dissimilarity dendrogram showed that Zinfandel () is close not only to its ascertained offsprings but also to other Austrian, Croatian, and Hungarian cultivars, including three Malvasias known to exist for a long time along Dalmatian coasts. In addition to Zinfandel relationships, molecular data suggested a parent-progeny relationship between Furmint and Harzevelu, the typical white varieties of the Tokaj area in Hungary. Literature Cited Arroyo-García, R., and J.M. Martínez-Zapater Development and characterization of new microsatellite markers for grape. Vitis 43: Bowers, J.E., G.S. Dangl, R. Vignani, and C.P. 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