THE WILD GRAPEVINE IN TUSCANY

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1 BORNICE ET AL., THE WILD GRAPEVINE IN TUSCANY, PAG. 1 THE WILD GRAPEVINE IN TUSCANY Marcello BORNICE*, Giancarlo SCALABRELLI*, Claudio D ONOFRIO*, Franco GIANNETTI** * Department of Agriculture, Food and Environment (DAFE), University of Pisa **CRA, Research Unit for the Viticulture, Arezzo Poster presented at Enoforum 2013, 7-9 May, Arezzo (Italy) Introduction The cultivated vine, Vitis vinifera L. subsp. Sativa belongs from the Vitaceae family (palmate-lobed leaves, berry fruit), subfamily Ampelideae (plants with climbed stem thanks to cauline tendrils), genre Vitis. The latter includes the subgenre Muscadinia (with three spontaneous American species, also known as "muscadinia grapes") and the subgenre Vitis which consists of 108 species (of which 28 fossils) distributed in four groups: - American suitable to temperate climates; - American suitable to warm, tropical and equatorial climates; - Eastern-Asian; - European-Asian suitable to temperate climates. The Vitis vinifera includes 2 subspecies, sylvestris (wild vines) and sativa (cultivated vines). Wild and cultivated vine The wild grape (Fig. 1), Vitis vinifera L. subsp. sylvestris (Gmelin, Hegi) is spontaneous in Europe. It mainly grows in woods or in the thicket where is still possible to identify it, specially in the wetlands of the Mediterranean area and in the Danubian Europe. It is easily recognizable because it is similar to the cultivated vines (according to the most reliable opinions it represents the ancestor) (Fregoni et al., 2005). It differs from these due to substantial ampelographyc differences, the most important of which are: - the presence of inflorescences only male or female (dioecious plant); - the bunches and the berries (frequently black berry) are smaller; - the seeds (Fig. 2) are more rounded with shorter beak (this last character is most important for the classification). These vines are vulgarly know as "Lambruschi" although doesn t always exists a true likeness to the homonymous typical grapevine from the Emilia-Romagna s lowland (Schneider, 2006). The investigations so far carried out have shown a considerable variety of forms among of which, might be present biotypes of botanical or farming interests. The use of molecular analysis s methods, now allow to highlight the degree of relationship with the currently cultivated varieties or with uncommon cultivars, in danger of extinction (Costacurta, 2003; Muñoz-Organero et al., 2001). This possibility, however, would require that these botanical entities had had a common pathway or the same area of distribution, in order to enable pollination. Given the richness of grapevine germplasm in Tuscany (Anzani et al., 1993), this eventuality, even if with a low level of probability, could take shape, knowing better both the genetic profile of wild vines and of autochthonous grape varieties that are present in the area. Researches carried out on archaeological sites (Ciacci et al., Vinum project, Ci.Vin, 2005) have already brought to the identification of molecular profiles of great interest Vignani et al., 2007).The many activities of genotyping of cultivated grapevines through the use of DNA markers, mainly microsatellites (SSR: Simple Sequence Repeat), also allow to carry out a comparative genomics analysis with Vitis vinifera subsp. Sylvestris (Cunha et al., 2007). Today the idea that the vines are exclusively the result of the domestication of wild vines from the near Middle-East seems to falter, while it advances the hypothesis that some of these are derived from the secondary centers of the Western Europe (Arroyo-Garcìa et al., 2006; Forni, 2012). It can not finally to be overlooked as some varieties of V. v. sylvestris are able to ripe grapes of acceptable quality, with a reduced use of pesticides. In some cases giving a wine fairly respectable (Lovicu, 2007) that can be improvable (if not used directly, however, it could help to identify varieties with different levels of diseases s susceptibility).

2 BORNICE ET AL., THE WILD GRAPEVINE IN TUSCANY, PAG. 2 Figure 1 - Wild grape. Figure 2 Seed s shape in V. vinifera (on the Left cv. Barsaglina, and V. sylvestris on the right). Save the germplasm The wild grapevine often grows along the rivers by clinging on trees, that regularly are cut, or worse, along ditches continuously cleaned, seriously endangering its natural survival. Our Department is aware about the scientific importance of this plant, for this reason a research project has been undertaken for the safeguard and the ampelographyc, molecular and phylogenetic study of wild vines present in the Tuscan Maremma. We have identified and cataloged some wild vines discovered in different habitats in the provinces of Siena and Grosseto. We have found more than 150 accessions of which about seventy were placed in a collection using a specific training system, the Totem (Fig. 3), on which ampelographyc, qualitative and molecular (nuclear and chloroplast microsatellites) observations are carried out. Figure 3 Training system Totem. Morphological characters Within of the collected material (not everything classified yet) the accessions with male flowers are the 19.5%, while those with female flowers represent more than 49% among of which prevailing the black berry (77%) (Tab. 1). It can be noticed, moreover, as the seeds of V. vinifera subsp. sylvestris are more rounded, with a beak shorter than the V. vinifera subsp. sativa. The shape of the seeds grape was considered particularly important for the classification and to highlight also the phylogenetic relationship (Terral et al., 2010; Rivera et al., 2007). The morphological characteristics of the leaves vary widely, from entire to three-lobed or fivelobed, with petiole sinus open or just open and blistering light or moderate (Fig. 4). Quite different even the pilosity of the lower page, ranging from absent to high, characterized by erect hairs or prostrate hairs. Variable is also the color of the veins and the petiole, from purplish red to green. SEX Un-classified Female Male N % 31,45 49,05 19,5 COLOR White Black N % 23,08 76,92

3 BORNICE ET AL., THE WILD GRAPEVINE IN TUSCANY, PAG. 3 Table 1 Sex and color of the wild vines recovered. Figure 4 Leaves dimorphism. Analysis of microsatellites loci This study has revealed very similar genotypes from the same population, but also quite different genotypes regardless the area of origin. We have also found some accessions becoming from wild sativa or natural hybrids between V. v. subsp. sativa and V. v. subsp. sylvestris. The chloroplast microsatellites analysis have shown that the accessions identify themselves exclusively in chlorotypes A and D: the first is very common in the supposed secondary centers of origin of the Western Europe, while the second predominates in the centers of origin of the Eastern Europe. From our data, the majority of the accessions recovered as V. v. sylvestris have the chlorotype A, while the cultivated vines have in prevalence the chlorotype D (Tab. 2). Moreover, the chlorotype D of certain supposed V. v. sylvestris confirms that these may result from become wild sativa or intraspecific crossbreedings between sylvestris and sativa present in the area of identification. Accessions V. v. sativa V. v. sylvestris Chlorotype A 33,33 73,68 Chlorotype D 66,67 26,32 Table 2 - Chlorotype distribution of the material studied. The anthocyanin profile In the first group there are the cv. Sangiovese, cv. Ciliegiolo and four accessions of sylvestris which have in common the prevalence of malvin respect to peonin and other anthocyanins, with low ratio of anthocyanins trisubstituted/disubstituted and low presence of acetate and paracoumarylated anthocyanins. In the second group together with the cv. Buonamico there are two accessions with a prevalence of malvin, medium ratio of trisubstituted/disubstituted and medium presence of para-coumarylated malvin. The third cluster contains five accessions of sylvestris characterized by prevalence of malvin and greater ratio of trisubstituted/disubstituted with acetate and para-coumarylated anthocyanins. The fourth group includes the cv. Colorino and two sylvestris less homogeneous. However, prevails malvin and above all, is higher the ratio of trisubstituted/disubstituted. Finally, cv. Barbera, Tempranillo (accession n 19, Fig. 5) and the accession n 64 are markedly diversified in several aspects. The latter accession is different for the prevalence of peonin and the very low ratio of trisubstituted/disubstituted (Fig. 5).

4 BORNICE ET AL., THE WILD GRAPEVINE IN TUSCANY, PAG. 4 Figure 5 - Dendrogram obtained from the anthocyanin profile in some Tuscan grapes, in accessions of V. v.. sylvestris (Sil_Mar_and number) and other samples recovered in Maremma (operational numerical symbols). (Accession n 19 Malv_n. Br represent the cv. Tempranillo) Delfinin Cianin Petunin Peonin Malvin Trisost./Disost. Percentage (%) Barbera Syl 2 Syl 5 Buonamico Syl 10 Syl 28 Ciliegiolo Syl 37 Syl 41 Colorino Syl 61 Syl 63 Sangiovese Syl 64 Syl 65 Tempranillo Syl 96 Figure 6 - Profile of anthocyanins in a series of Vitis sylvestris and in some Tuscan s vines. Conclusions The material retrieved, has large morphological and genetic variability, in addition presents very particular anthocyanins profiles of black grapes. The nuclear microsatellites analysis has showed that certain supposed accessions of Vitis vinifera subsp. sylvestris retrieved in Tuscany seem to derive from the vines already cultivated that become wild, while others accessions would be intraspecific crossbreedings sativa-sylvestris.

5 BORNICE ET AL., THE WILD GRAPEVINE IN TUSCANY, PAG. 5 The retrieval of V. v. sylvestris continues on increasing the number of sites, subjecting the accessions to ampelographyc anthocyanin profile of black grapes observations. Further genotyping through other types of molecular DNA markers are in progress. Acknowledgements Project financed by the foundation Bertarelli Montecucco. We wish also thank the Ente Parco Regionale della Maremma for having hosted the surveys. References - Anzani R., Failla O., Scienza A., De Micheli L. Individuazione e conservazione del germoplasma di vite selvatica (Vitis vinifera sylvestris) in Italia. Vignevini 1993, 6, pp.51-61; - R. Arroyo-Garcìa, L. Ruiz-Garcìa, L. Bolling, R. Ocete, M. A. Lòpez, C. Arnold, A. Ergul. Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms Molecular Ecology (2006) 15, ; - A. Costacurta. Le metodologie per l identificazione e la caratterizzazione dei vitigni. Istituto Sperimentale per la Viticoltura Conegliano; - J. Cunha, M. Baleiras-Couto, J. P. Cunha, J. Banza, A. Soveral, L. C. Carneiro, J. E. Eiras-Dias. Characterization of Portuguese populations of Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi. Genet Resour Crop Evol (2007) 54: ; - J. Cunha, M. Teixeira Santos, L. C. Carneiro, P. Fevereiro, J. E. Eiras-Dias. Portuguese traditional grapevine cultivars and wild vines (Vitis vinifera L.) share morphological and genetic traits. Genet Resour Crop Evol (2009) 56: ; - Fregoni M. Viticoltura di Qualità, Phytoline, 2005; - Schneider A. Aspetti genetici nello studio dei vitigni del territorio. Quad. Vitic. Enol. Univ. Torino, 28, ; - Lovicu G. La Sardegna della vite è selvatica, antica, biodiversa. Darwin, quaderno n 3, 2007; - G. Muñoz-Organero, F. Cabello, A. Benito, S. García-Muñoz, L. Gaforio, C. Rubio, M.A. López, R. Ocete. Caracterizaciòn de vides silvestres (Vitis vinifera subsp. sylvestris) en España. Am. J. Enol. Vitic., Vol. 50, No. 4, 1999; - D. Rivera, B. Miralles, C. Obòn, E. Carreno, J. A. Palazòn. Multivariate analysis of Vitis subgenus Vitis seed morphology. Vitis 46 (4), (2007); - J. F. Terral, E. Tabard, L. Bouby, S. Ivorra, T. Pastor, I. Figueiral, S. Picq, J. B. Chevance, C. Jung, et al. Evolution and history of grapevine (Vitis vinifera) under domestication: new morphometric perspectives to understand seed domestication syndrome and reveal origins of ancient European cultivars. Annals of Botany 105: , 2010.