Vitis 50 (1), 21–28 (2011)

Recognition and genotyping of minor germplasm of Friuli Venezia Giulia revealed high diversity

M. CRESPAN 1), A. FABBRO2), S. GIANNETTO1), S. MENEGHETTI1), C. PETRUSSI3), F. DEL ZAN2) and P. SIVILOTTI2)

1) C.R.A. Centro di Ricerca per la Viticoltura, CRA-VIT, , 2) ERSA Servizio ricerca e sperimentazione, Pordenone, Italy 3) Viticulturist, , Udine, Italy

Summary We know from the literature that a notable wealth existed in Friuli Venezia Giulia (FVG) in the past, The wealth of vine varieties that used to exist in which has gradually been lost. Market globalization has Friuli Venezia Giulia has been progressively lost. In led to production becoming concentrated on a few well- order to ascertain the current situation regarding vine known varieties, with the consequence of strong competi- germplasm in the region, between 2001 and 2008, a tion for FVG -growers. One possibility for gaining an wide-ranging study was conducted of recovery, conser- extra market share could be the cultivation of some tradi- vation and molecular characterization with microsatel- tional local varieties. This product diversification, which lite markers (SSR) of accessions of minor germplasm is slowly gaining ground, will lead to a strengthening of at risk of erosion or extinction. A total of 178 accessions the bonds between wine and territory through the redis- were analyzed and 93 varieties identified. Of these, 44 covery and appreciation of age-old tastes. With adequate are already registered in the Italian Catalogue, 8 have marketing strategies, this could also allow the opening of been imported and are well-known foreign varieties new commercial outlets, and lesser-known can be an even if not registered in the Italian Catalogue, 38 are effective eno-gastronomic tourist attraction. It should also potentially autochthonous and of these only 15 are de- be emphasized that the survival of the minor varieties over scribed or at least cited in the literature; there are no time has a high selective value, because they are plants hypotheses on the remaining three. In order to obtain adapted to the specific environments where they are found. information on possible genetic similarities, three types This suggests interesting implications regarding the prob- of software were used to process the molecular data, lems of environmental impact linked to plant protection in but only Structure allowed the existence of two prin- vineyards, with the possibility of reducing management cipal groupings to be hypothesized for some of the pre- costs and pesticide use through the selection of genotypes sumed Friuli autochthons: one that gravitates around less susceptible at least to some pests and diseases. ‘’ and the other around ‘Refoscone’. Based on these premises, ERSA and CRA-VIT em- barked on a wide-ranging work of recovery, conservation K e y w o r d s : SSR, microsatellite, minor varieties, en- and molecular characterization with microsatellite markers dangered cultivars, genetic erosion. (SSR) of accessions of minor germplasm found in the ter- ritory of FVG, mainly Centre and East part of the region, and at risk of erosion or extinction. Introduction The aim in the first instance was to identify, preserve and describe the historical, ampelographic, agronomic and Friuli Venezia Giulia (North-East Italy) has a long and enological characteristics of the materials presumed to be consolidated viticultural-enological tradition whose fame native, to understand the extent and characteristics of the extends well beyond the regional and national borders. Nev- varietal heritage currently present in FVG. This paper re- ertheless, in quantitative terms, production is concentrated ports the results relating to the genotyping and the associa- on very few varieties. From the 2007 data, made available tion of at least some of the material with the historically by “Vine card index of Friuli Venezia Giulia region”, we known names. The genetic variability of the material was know that on a total land area of slightly less than 20,000 also evaluated and the existence of any genetic groupings. hectares, 7 varieties cover 78 %, with a prevalence of ‘Pi- not gris’ (24 %) and ‘’ (18 %). Areas of more than 1000 ha are cultivated with ‘Tocai friulano’, ‘’ Material and Methods and ‘Sauvignon’ for the whites, ‘’ and ‘Ca- bernet Sauvignon’ for the reds. Smaller surfaces are culti- V e g e t a l m a t e r i a l : During eight years of vated with ‘Pinot bianco’, ‘ friulano’, ‘Prosecco’, research, from 2001 to 2008, with the aid of technicians ‘Ribolla gialla’, ‘Traminer’, ‘Malvasia istriana’, ‘ and enthusiasts, as well as the existing collections at the italico’, ‘Picolit’, ‘Riesling renano’ and ‘Vitouska’ among University of Udine (Italy) and the “Rinascita” Farm of the the whites, ‘Refosco dal peduncolo rosso’, ‘Pinot nero’, Provincial Administration of Pordenone (Italy), a total of ‘Schioppettino’, ‘Pignolo’, ‘Refosco nostrano’ and ‘Ter- 178 accessions were sampled. In addition to the cited col- rano’ among the reds. lections, the work of retrieval regarded old vineyards, sin-

Correspondence to: Dr. M. CRESPAN, C.R.A. Centro di Ricerca per la Viticoltura (CRA-VIT), Via Casoni 13/a, 31058 (TV), Italy. Fax: +39-438-738489. E-mail: [email protected] 22 2 M. CRESPAN et al. gle vine rows marking boundaries between holdings, ditch- of the individuals (USEPOPINFO=0). Structure was run es, vegetable gardens and any other places where plants for K values ranging from 1 to 10 and the K value where ln might be identified with characteristics differing from the P(D) was minimum was taken in account. Five independ- principal varieties cultivated in the region. ent runs were performed for the estimated minimum, in After DNA analysis varieties that resulted as being order to valuate the consistency of the datum. original were rescued in a conservation field at Ersagrico- la’s Pantianicco Farm. Over three years data were gathered on the phenology, , composition characteristics of the Results and Discussion berries and sensorial properties of the wines. D N A a n a l y s i s : DNA was extracted from young The molecular analyses of the 178 accessions sampled leaves and genotyping was performed with 11 SSR loci demonstrated the presence of 93 different genotypes. The routinely employed at CRA-VIT for cultivar identifica- SSR profiles obtained were compared with the CRA-VIT tion (VVS2, THOMAS and SCOTT 1993); VVMD5, VVMD7, database and data published in the literature. In Tab. 1 the VVMD27 and VVMD28, BOWERS et al. 1996 and 1999; varieties identified are listed in alphabetic order. The re- VrZAG62 and VrZAG79, (SEFC et al. 1999); ISV2, ISV3, sults of SSR profile comparison are the following from the ISV4 (CRESPAN 2003) and VMCNG4b9 (WELTER et al. 93 different varieties: 2007). The PCR reaction mixture (25 µL final volume) - 44 are registered in the Italian National Catalogue of contained 20 ng total DNA, 10 µl Eppendorf HotMaster- Vine Varieties (RNVV) (marked with an X in Tab. 1). Mix (2.5 x) and 5-10 pmoles of each primer. The PCR was Fifteen out of these are considered as pure FVG: ‘Ci- performed in an AB 9700 thermal cycler with the follow- vidin’, ‘Cjanorie’, ‘Cordenossa’, ‘Forgiarin’, ‘Piccola ing steps: 1 min 30 s at 94 °C; 35 cycles at 94 °C for 30 s, nera’, ‘Picolit’, ‘Piculit neri’, ‘Refosco dal peduncolo 55 °C for 30 s, 65 °C for 30 s; 65 °C for 7 min and a final rosso’, ‘Refoscone’, ‘Ribolla gialla’, ‘Schioppettino’, step of at least 10 min at 8 °C to stop the reaction. On ‘Sciaglin’, ‘Tazzelenghe’, ‘Ucelut’ and ‘Verduzzo friu- the basis of signal intensity on agarose gel, 0.75-1.5 µL of lano’. ‘Cividin’ and ‘Cjanorie’ were registered in 2006 amplified DNA were used for electrophoresis onto a se- and ‘Cordenossa’ even more recently in 2008. The reg- quencing gel (5 % polyacrylamide, 1 x TBE, 7 M urea). istration of these three minor varieties is due to the pas- Amplification products of cultivars with alleles of known sion of some winegrowers in the region aiming the cul- molecular size were used as references for allele sizing. tivation of mainly endangered local varieties. Allele bands were revealed by silver staining and visually - 7 are known foreign varieties and not registered in the scored at least twice, as reported in CRESPAN and MILANI RNVV, i.e. ‘Alicante Bouschet’, ‘Auxerrois’, ‘Hum- (2001). agne’, ‘Jacquez’, ‘Kraljevina’, ‘Tsaousi’ and ‘Veltliner S t a t i s t i c s o n S S R d a t a : SSR markers statis- rot’. They are heterogeneous varieties of highly dispa- tics were computed with Identity 1.0 software, i.e. number rate provenance (VIVC, Vitis International Variety Cat- of alleles/locus, allele frequencies, expected and observed alogue, http://www.vivc.de). Apart from ‘Kraljevina’ heterozygosity, probability of null alleles, and probabil- and ‘Veltliner rot’, which are grown in the neighboring ity of identity (PI). The power of discrimination (PD = 1 regions of and , there is no explication 2 th - ∑gi , where gi is the frequency of the i genotype) was for the presence of the other varieties in FVG. calculated for each locus using the genotype frequencies - 38 are presumed original Friuli varieties (in bold in computed with Excel program. Tab. 1) not registered in the RNVV. Of these, 21 (55 %) G e n o t y p e s c o r r e l a t i o n s : Three differ- have black grapes and 17 (45 %) white. Only 15 have ent methods were used to determine the genetic relations already been described or even just mentioned in the lit- among the genotypes found: 1) the Analysis of Functional erature (CALÒ and COSTACURTA 1991; COSTANTINI et al. Correspondence (AFC) was performed with GENETIX 2007), while the other 22 have new molecular profiles software (BELKHIR et al. 2003), available free at http:// and are not referable to varieties cited in the literature www.univ-montp2.fr/%7Egenetix/genetix/genetix.htm to (at least with that name), so they represent a heritage compare all the genotypes listed in Tab. 1; 2) to focus on that we have just begun to explore. the relationships among all the genotypes found, the dis- - A difficult case is that of ‘Pienel’, which corresponds similarity index calculated as -ln (proportion of shared al- to the ‘Bela ’ found in by ŠTAJNER et al. leles) (DANGL et al. 2001) was used to measure the genetic (2008) but, contrary to what is reported in the VIVC, distance between all pairwise combinations. A dendrogram it is not a synonym of ‘’. Nothing is known of all these genotypes was elaborated with the UPGMA about the geographical origin of this genotype. method and the cophenetic correlation was computed with - One is ‘Siciliana’, whereas two other no named geno- the NTSYS-pc program; 3) to find indications on popula- types could not be identified. They are indicated as un- tion structure. We set the following parameters: length of known G1 and unknown G2. burn-in period 100,000 generations and 106 Markov chain On the basis of our analyses, but unlike what was pre- Monte Carlo replications; we used the admixture model, viously stated (CALÒ and COSTACURTA 1991), ‘Cuneute’ and which estimates the fraction of ancestry from each cluster ‘Vercluna’ are synonymous varieties. In the majority of for each individual (PRITCHARD et al. 2000), and run the cases the identified genotypes grouped between one and analyses with correlated allele frequencies (FALUSH et al. four accessions together, but some varieties were better 2003). No information was given on the presumed origin represented. In particular, 11 samples were identified as Recognition and genotyping of minor germplasm of Friuli Venezia Giulia 23

T a b l e 1

List of the 93 varieties found after analysis of 178 accessions, ordered by identity. In bold those presumed authoctonous of Friuli Venezia Giulia. DB CRA-VIT: SSR database of Centro di Ricerca per la Viticoltura. N: black; B: white, and Rs: pink

Registered SSR profile of reference where ampelographic description of Variety Color in the Italian used FVG cultivars may be found Catalogue Aghedene B CRESPAN et al. 2006 CALÒ and COSTACURTA 1991 Aleatico N CRESPAN and MILANI 2001 X Alicante Bouschet N MARTIN et al. 2003 Auxerrois B BOWERS et al. 1999 N THIS et al. 2004 X Berzamino N current paper CALÒ and COSTACURTA 1991 Bianchella B current paper Blanchias B current paper Bontempo N current paper Brambana N current paper CALÒ and COSTACURTA 1991 Cabernet franc N THIS et al. 2004 X Chasselas B SCHNEIDER et al. 2008 X Cjanorie N current paper X Ciavalgian N current paper CALÒ and COSTACURTA 1991 Ciliona N current paper Cilja B current paper COSTANTINI et al. 2007 Cividin B current paper X Codelunghe N current paper CALÒ and COSTACURTA 1991 Corbina N CANCELLIER et al. 2006 X Cordenossa N current paper X Corvinone N CANCELLIER et al. 2006 X Cremin N current paper Croatina N DB CRA-VIT X Cuneute/Vercluna N current paper CALÒ and COSTACURTA 1991 Curvin N current paper COSTANTINI et al. 2007 Duriese B current paper COSTANTINI et al. 2007 Durina B current paper Forgiarin N current paper X Freisa N DB CRA-VIT X Friularo B current paper Fumat N current paper CALÒ and COSTACURTA 1991 Gragnelut N current paper Gran Rap N current paper COSTANTINI et al. 2007 Humagne B. B VOUILLAMOZ et al. 2004 Jacquez N THIS et al. 2004 Kraljevina B-Rs MALETIC et al. 1999 Lambrusco Maestri N DB CRA-VIT X Malvasia bianca lunga B CRESPAN et al. 2007 X Malvasia istriana B DB CRA-VIT X B CRESPAN et al. 2004 X Mocula B current paper Mostosa B DB CRA-VIT X Mueller Thurgau B DB CRA-VIT X Negrat N DB CRA-VIT COSTANTINI et al. 2007 Negretto N DB CRA-VIT X Nerata N current paper Nigrut N current paper Palomba nera N current paper Pelena B current paper Peverina N current paper Piccola nera N current paper X Piciule N current paper Picolit B DB CRA-VIT X Piculit neri N DB CRA-VIT X Pienel B current paper Pignola della Valtellina N CANCELLIER et al. 2009 X Pignolo N CANCELLIER et al. 2009 X Pignoletta N current paper X Portugieser N THIS et al. 2004 X Prosecco lungo B CRESPAN et al. 2006 X Prosecco tondo B CRESPAN et al. 2006 X Raboso veronese N CRESPAN et al. 2004 X Refosco bianco B current paper COSTANTINI et al. 2007 Refosco dal peduncolo rosso N COSTACURTA et al. 2005 X Refosco gentile N COSTACURTA et al. 2005 CALÒ and COSTACURTA 1991 Refoscone N COSTACURTA et al. 2005 X Regina B DB CRA-VIT X Ribolla gialla Rs COSTACURTA et al. 2006 X Ruacit B current paper COSTANTINI et al. 2007 Sagrestana B current paper Sangiovese N CRESPAN and MILANI 2001 X Sbulcisa B current paper Sbulzina N current paper Schiava gentile N DB CRA-VIT X Sciaglin B current paper X 24 4 M. CRESPAN et al.

Tab. 1, continued

Registered SSR profile of reference where ampelographic description of Variety Color in the Italian used FVG cultivars may be found Catalogue Siora B current paper Siciliana N current paper Tazzelenghe N COSTACURTA et al. 2005 X Terrano N COSTACURTA et al. 2005 X Tintoria Lloyd N DB CRA-VIT Trebbiano toscano B DB CRA-VIT X SEFC et al. 2000; Tsaousi B ARADHYA et al. 2003 Ucelut B current paper X CALÒ and COSTACURTA 1991 Veltliner grün B SEFC et al. 2000 X Veltliner rot B THIS et al. 2004 Venere B current paper Verduzzo trevigiano B DB CRA-VIT X Vinoso rosso N current paper Vitouska B CRESPAN et al. 2007 X Vubola B current paper Turca N DB CRA-VIT X unknown G1 N unknown G2 B

‘Piculit neri’, 9 as ‘Berzamino’, 5 as ‘Prosecco lungo’ and difference between the two values in the case of locus Vr- 5 as ‘Gragnelut’, indicating a wider diffusion of these vari- ZAG79, where the observed heterozygosis is much higher eties in the studied territory. than that expected on the basis of the allelic frequencies. The term “Glera” also gives cause for reflection as The probability of null alleles is less than or very close 4 accessions with this name refer to 4 different varieties: to zero at all loci, excluding ISV4 and VMCNG4b9. The ‘Aghedene’, ‘Mocula’, ‘Prosecco lungo’ and ‘Vitouska’. A three most informative loci, on the basis of both PI and PD, previous study (CRESPAN et al. 2006 a) revealed that the are VVMD28, VMCNG4b9 and VVS2, in that order, while name “Glera”, considered just a synonym of ‘Prosecco’, those least informative are ISV3, ISV4 and VrZAG79. The was more often associated to ‘Prosecco lungo’ and, mar- probability of total identity is 5.72 x 10-11, therefore a high ginally, also to other minor white grape varieties. value, notwithstanding the small number of genotypes be- We also found some peculiarities: the two accessions ing compared. The average PD is also high at 0.9073. of ‘Brambana’ analyzed have three alleles at locus VVS2 The high heterogeneity of the varieties of presumed (135, 151 and 153), whereas the accession of ‘Brambana’ FVG origin thus emerges, but the molecular data are too held in the CRA-VIT collection had alleles 135 and 151. limited to make any suppositions on possible parent/off- The two accessions identified as ‘Sbulzina’ have a small spring relationships. difference at locus VVMD7, accession n. 122 having 239 G e n o t y p e s r e l a t i o n s h i p s : Genetic rela- and 255 alleles and n. 88 having 239 and 257. Lastly, of tions among the 89 unique genotypes (‘Jacquez’, ‘Pigno- the four accessions identified as ‘Pignolo’, two have three letta’, ‘Siciliana’ and unknown G1 were not considered) alleles at VVMD7 (247, 257 and 259). were analysed by Genetix software. It turned out that the Some genotypes, i.e. ‘Jacquez’, ‘Pignoletta’, ‘Sicili- varieties presumed to be native to FVG intermixed with the ana’ and unknown G1, display alleles typical of rootstocks others, with exception of ‘Malvasia istriana’, ‘Fumat’ and or other Vitis species and absent in V. vinifera (CRESPAN ‘Alicante Bouschet’ (data not shown). et al. 2009), which are highlighted in bold in Tab. 2. ‘Jac- The dendrogram of genetic dissimilarity, elaborated quez’ is a known interspecific hybrid (VIVC), while we using all 93 identified genotypes, showed a similar result, would hypothesize that the others are hybrids. Some am- in which the two groups (presumed Friuli vs. non Friuli cv.) pelographic characteristics (colour of the bud and inter- are uniformly mixed and ‘Malvasia istriana’ is confirmed nodes, colour, hairiness and shape of the leaf) reveal signs as being one of most divergent samples (data not shown). of “hybrid blood”, while no phylloxera attacks were found The Structure software was also applied to the same on the leaves, probably because the vineyard is still very 89 genotypes, because it gives an estimate on the possibil- young. ity to find realistic groups according to allele frequencies. R e s u l t s o f t h e e l a b o r a t i o n s w i t h The ln P(D) showed a slight minimum for k = 3 and 4, t h e I d e n t i t y s o f t w a r e : To evaluate the genetic therefore none distinct population was inferred. This re- variability present within the presumed FVG varieties, the sult is in agreement with that obtained by CIPRIANI et al. statistics of the molecular data of the 58 genotypes in bold (2010) analyzing 795 genotypes of Vitis vinifera: Structure in Tab. 1 were elaborated with the Identity software, as failed to divide them in any sub-population. Nevertheless, well as those of ‘Malvasia istriana’, ‘Marzemina bianca’ the 5 repetitions fluctuated very closely around the same and ‘Corbina’, which are also found outside the region. values; moreover, comparing the results of the two bar The results are summarized in Tab. 3. A total of 96 alleles plots, we observed that a more clear separation was ob- were found, with an average of 8.7 alleles per locus. The tained with k = 4. Also with this elaboration the presumed observed heterozygosis was higher than expected at all the FVG varieties were disseminated in all groups individu- loci, excluding ISV4 and VMCNG4b9. There is a very big ated by the software. In an attempt to interpret the group- Recognition and genotyping of minor germplasm of Friuli Venezia Giulia 25 176 158 170 162 172 172 164 176 150 158 176 162 158 164 168 162 162 178 178 158 178 168 172 166 178 166 172 184 178 166 172 158 178 168 172 150 184 176 168 176 150 166 158 158 158 172 166 158 166 158 4B9 VMCNG 150 150 164 158 158 162 138 138 150 158 162 158 158 162 158 150 158 158 162 158 158 150 166 162 158 162 150 164 150 150 168 158 176 150 154 150 158 150 138 166 138 158 138 158 158 166 162 150 150 150 187 169 187 197 177 177 191 177 187 191 191 177 169 187 191 191 197 177 169 187 187 177 187 197 169 177 177 187 197 169 191 169 177 187 187 177 177 177 197 177 177 187 197 169 177 177 169 169 197 169 ISV4 (VMC6G1) 177 169 187 169 169 169 177 177 177 169 187 169 169 169 169 169 169 169 169 169 169 169 187 169 169 169 169 187 187 169 191 169 169 169 175 169 169 177 191 169 177 177 169 169 177 169 169 169 177 169 133 139 139 145 133 141 139 139 145 141 139 139 137 139 141 139 139 141 141 141 141 141 145 139 141 139 145 141 139 139 133 139 141 139 143 139 145 139 139 139 133 139 139 139 139 141 141 139 133 141 ISV3 (VMC6F1) 133 131 135 139 133 139 137 139 139 139 139 133 137 139 139 131 133 139 139 139 139 139 139 133 133 121 139 133 135 131 133 133 133 133 139 133 133 133 133 133 133 133 133 139 133 133 139 133 133 139 151 143 165 165 151 169 143 143 169 161 165 165 161 169 165 143 151 165 165 161 165 151 165 165 161 165 165 165 161 151 165 151 165 165 151 169 169 165 165 165 165 169 161 151 165 169 165 151 165 165 6E1) 141 143 151 151 143 165 141 141 143 151 151 141 151 165 151 141 141 165 151 141 151 151 141 141 151 141 151 151 151 141 161 151 141 151 141 143 151 143 161 141 151 141 151 141 151 169 151 151 151 143 ISV2 (VMC 248 254 256 244 258 258 250 250 250 250 258 258 250 250 258 258 248 258 250 258 250 250 244 250 258 250 250 258 250 258 260 250 250 246 250 250 250 250 254 250 258 256 244 250 248 258 250 256 258 258 V. vinifera V. VRZAG 79 242 248 242 242 242 250 242 242 236 242 246 250 250 242 250 242 242 250 242 250 250 250 244 242 258 248 236 250 250 250 242 244 242 238 248 250 244 242 236 248 248 250 242 250 244 258 242 250 242 250 195 195 187 195 199 195 203 203 189 203 203 203 203 199 193 201 199 199 195 193 203 193 195 203 195 203 203 203 199 195 199 199 193 199 197 195 201 199 187 199 203 195 193 203 203 199 195 195 195 199 193 185 187 187 191 193 191 199 189 195 193 193 193 187 193 195 187 193 191 187 187 193 185 193 193 193 187 193 193 193 193 187 191 187 185 193 191 195 187 193 195 187 193 195 193 187 193 193 187 193 VRZAG 62 251 249 263 239 263 271 251 249 261 271 239 271 261 249 261 261 251 261 267 267 261 247 239 237 261 237 263 267 271 271 271 251 267 261 239 237 267 257 281 239 251 261 247 239 239 249 239 251 257 267 VVMD28 247 239 247 231 237 249 239 239 251 261 231 221 247 237 239 247 237 251 239 251 237 239 237 231 239 237 251 247 247 239 231 231 247 247 233 231 237 251 257 239 247 237 237 231 237 237 239 239 247 221 T a b l e 2 T 194 194 194 189 189 185 189 189 194 189 189 189 194 185 185 185 194 181 189 189 185 191 194 185 181 189 194 189 189 189 191 189 189 189 189 183 194 179 179 194 194 191 181 189 189 181 189 194 179 189 VVMD27 185 179 181 181 185 181 181 181 179 185 181 185 179 181 181 181 185 179 185 181 179 189 189 185 181 185 185 181 185 179 185 185 185 185 179 179 191 179 179 185 179 189 181 185 183 179 185 189 179 179 247 249 243 243 253 263 247 249 249 257 263 247 263 249 263 247 249 263 257 263 239 263 249 263 257 257 247 257 249 247 261 249 249 253 239 247 257 253 239 253 247 247 257 257 257 249 247 247 239 261 VVMD7 239 239 239 239 249 257 239 247 249 247 239 239 247 239 239 247 239 249 249 239 233 239 247 247 247 247 243 247 247 247 249 239 247 243 237 239 253 239 239 239 239 243 247 247 247 239 239 247 239 239 SSR profi les. In bold are the alleles found in rootstocks and not 246 228 238 238 226 240 228 234 228 236 240 236 228 232 228 238 228 228 232 228 240 236 236 236 236 228 234 240 232 232 236 238 232 236 244 240 232 240 240 238 234 246 228 232 232 236 228 232 246 226 VVMD5 226 226 226 234 226 232 226 226 226 232 226 228 226 226 226 236 226 226 232 226 232 226 236 232 232 228 226 232 226 226 228 226 226 226 228 234 228 226 222 226 226 226 226 232 226 236 226 226 240 226 155 135 145 151 135 155 153 153 145 151 147 143 151 155 151 143 155 151 155 155 151 143 151 155 151 151 143 141 155 135 151 151 155 151 143 143 155 145 143 133 155 145 151 135 155 151 155 143 145 151 VVS2 135 133 133 133 133 151 151 133 143 135 139 133 139 133 135 141 133 133 135 135 135 133 139 143 135 133 143 133 133 135 133 139 155 133 139 135 135 145 143 133 153 133 143 133 133 135 155 135 133 143 Variety Aghedene Aleatico Alicante Bouschet Auxerrois Barbera Berzamino Bianchella Blanchias Bontempo Brambana* Cabernet franc Chasselas Ciavalgian Ciliona Cilsa Cividin Cjanorie Codelunghe Corbina Cordenossa Corvinone Cremin Croatina Curvin Duriese Durina Freisa Friularo Fumat Gragnelut Gran Rap Humagne B. Jacquez Kraljevina Lambrusco Maestri Malvasia bianca lunga Malvasia istriana Marzemina bianca Mocula Mostosa Negrat Negretto Nerata Nigrut Palomba nera Pelena Peverina Cuneute/Vercluna Forgiarin Thurgau Mueller 266 M. CRESPAN et al. 176 176 168 162 176 178 158 158 158 158 176 166 162 168 168 168 158 172 176 164 168 176 158 178 162 170 178 178 158 158 176 138 176 176 168 178 176 150 168 158 162 176 176 4B9 VMCNG 158 166 164 158 176 162 158 150 138 150 166 158 158 158 150 162 150 158 138 164 158 138 150 158 150 151 158 162 150 158 162 138 162 158 160 158 158 138 164 150 162 166 162 197 197 177 187 177 177 197 177 177 197 197 169 169 177 169 169 191 197 197 177 197 197 197 187 169 187 197 169 169 191 187 197 169 177 181 191 183 197 177 169 169 197 187 ISV4 (VMC6G1) 187 169 169 169 169 169 185 177 169 169 169 169 169 169 169 169 177 191 191 177 177 187 177 169 169 177 169 169 169 169 177 177 169 177 177 177 169 187 169 169 169 177 177 139 141 133 141 139 139 139 133 139 139 139 139 139 139 139 139 139 139 139 139 139 139 135 145 133 139 139 139 141 139 139 133 145 139 139 139 145 139 139 141 139 133 139 ISV3 (VMC6F1) 133 139 131 133 139 135 139 131 135 133 133 133 133 133 133 133 139 133 139 131 139 139 133 141 131 139 133 133 139 131 133 131 133 133 137 133 133 133 133 133 133 133 133 141 151 161 145 151 165 151 167 151 167 151 165 165 165 165 165 167 169 159 169 165 165 169 151 161 165 165 165 143 165 161 171 151 159 151 151 161 151 169 167 165 165 167 ISV2 (VMC 6E1) 141 141 141 141 143 159 133 141 141 141 141 151 151 141 143 165 143 165 141 141 143 141 141 151 141 133 141 151 141 151 141 143 141 151 151 143 141 143 141 159 151 151 143 258 258 258 250 258 258 258 258 258 258 258 250 258 258 258 250 250 250 258 258 258 258 258 258 258 258 258 250 250 250 250 248 258 254 260 250 248 250 258 258 250 248 248 VRZAG 79 248 250 238 248 250 250 244 250 248 248 248 242 250 250 250 242 242 236 248 250 242 242 250 238 250 242 248 242 238 244 244 246 250 250 242 246 244 250 238 250 242 242 242 195 195 203 195 195 193 187 195 203 193 203 195 199 193 203 199 187 199 203 191 195 195 203 199 193 192 199 199 193 195 199 203 199 199 193 201 203 195 203 203 203 203 195 187 195 191 193 187 193 187 193 187 187 187 193 195 193 193 193 185 195 191 187 193 187 193 191 191 187 195 195 191 193 193 187 193 193 193 199 193 191 193 193 199 195 187 VRZAG 62 251 267 237 267 257 239 263 247 263 247 247 239 271 239 247 271 261 237 249 249 247 251 263 257 237 245 239 261 237 237 251 261 247 237 261 251 251 271 239 237 267 257 251 VVMD28 239 251 231 239 239 237 255 239 231 239 239 237 247 237 237 237 237 231 237 231 237 239 249 239 231 231 237 237 221 231 247 261 231 237 251 237 237 249 231 231 237 239 247 194 189 185 194 179 189 194 191 194 194 194 185 191 189 185 185 185 185 194 191 185 181 194 185 179 201 181 185 189 194 183 183 189 189 186 183 194 194 185 189 191 194 194 VVMD27 181 181 181 179 179 181 185 179 181 179 179 185 185 181 179 185 185 179 181 181 179 181 181 181 179 181 181 185 189 189 179 179 189 179 181 179 189 183 181 179 185 179 185 239 257 247 263 239 263 243 257 255 247 247 257 257 247 263 263 249 249 247 247 263 239 257 249 263 243 249 263 249 247 253 249 263 263 251 249 257 253 263 263 249 247 257 VVMD7 239 239 247 239 239 253 239 247 243 239 239 239 249 239 247 249 239 239 247 239 239 239 239 247 247 243 239 249 247 239 249 247 249 249 247 249 247 239 247 247 247 239 239 246 232 238 246 246 236 244 232 232 246 246 238 232 232 226 232 232 234 226 232 236 246 226 238 232 264 232 232 228 238 232 238 238 236 244 236 232 246 232 228 226 240 228 VVMD5 228 228 232 232 226 232 226 226 226 226 226 232 228 226 226 226 226 232 226 226 226 234 226 236 228 234 228 228 226 236 226 228 228 228 236 226 232 240 226 226 226 226 226 135 155 139 151 145 155 133 155 151 143 143 135 151 133 155 155 135 151 151 151 133 153 143 155 135 143 139 153 155 151 143 151 151 143 147 151 151 133 151 151 155 145 143 VVS2 133 133 135 135 143 155 125 133 143 133 133 133 135 133 133 151 133 143 143 139 133 135 143 139 133 133 133 151 135 133 133 135 151 133 133 133 133 131 135 143 155 133 135 Valtellina della Variety Piccola nera Piciule Picolit Piculit neri Pienel Pignola Pignoletta Pignolo** Portugieser Prosecco lungo Prosecco tondo Raboso veronese Refosco bianco Refosco dal peduncolo rosso Refosco gentile Refoscone Regina Ribolla gialla Ruacit Sagrestana Sangiovese Sbulcisa Sbulzina*** Schiava gentile Sciaglin Siciliana Siora Ucelut unknown G1 unknown G2 Tazzelenghe Terrano Lloyd Tintoria toscano Trebbiano Tsaousi Turca grün Veltliner rot Veltliner Venere trevigiano Verduzzo rosso Vinoso Vitouska Vubola *The two accessions analyzed have three alleles at VVS2 locus: 135, 151 and 153. *The two accessions analyzed have three alleles at VVMD7: 247, 257 and 259. accessions out of the four analyzed have three alleles at **Two VVMD7, one having 239-255 and the other 88 239-257. at ***The two accessions analyzed showed a difference Tab. 2, continued Tab. Recognition and genotyping of minor germplasm of Friuli Venezia Giulia 27

T a b l e 3

Statistics on the 11 SSR markers applied on the 58 genotypes presumed to be autochthonous of Friuli Venezia Giulia region. He: expected heterozygosis, Ho: observed heterozygosis, Δ = Ho-He, r: probability of null alleles, PI: probability of identity; PD: power of discrimination

Locus no of alleles He Ho Δ r PI PD VVMD28 13 0,881 0,931 0,050 -0,026 0,047 0,9625 VMCNG4b9 11 0,869 0,844 -0,025 0,013 0,055 0,9548 VVS2 9 0,842 0,844 0,002 -0,001 0,084 0,9435 VVMD27 6 0,809 0,810 0,001 0,000 0,120 0,9239 VrZAG62 8 0,816 0,896 0,080 -0,044 0,108 0,9227 ISV2 9 0,807 0,879 0,072 -0,039 0,117 0,9197 VVMD7 8 0,780 0,810 0,030 -0,016 0,147 0,9174 VVMD5 9 0,791 0,844 0,053 -0,029 0,124 0,9132 VrZAG79 10 0,754 0,913 0,159 -0,091 0,168 0,8596 ISV4 5 0,682 0,603 -0,079 0,047 0,228 0,8489 ISV3 8 0,689 0,810 0,121 -0,071 0,249 0,8145 mean 8,7 0,793 0,835 5,72 E-11* 0,9073 *PI over all 11 SSR loci was computed as the product of PI at each locus. ing proposed, we set an arbitrary limit on the recognition hypothesize on its spread from east to west or vice versa, of origin and imposed a threshold equal to 85 % or above nevertheless its similarity to the other varieties of FVG for a genotype to be included in one of the four groups. would suggest the former possibility. Based on the Struc- With this limit, two groups encompassing respectively 21 ture results, therefore, at least two principal groups can be and 17 genotypes resulted “anonymous”, because none va- identified among the presumed varieties from FVG, one riety could be picked out. A third group included 13 varie- referring to ‘Prosecco’ and the other to ‘Refoscone’. The ties, 10 of them being above the threshold limit, mainly elaboration of further comparisons between these materi- white grapes; they were, in decreasing order: ‘Pelena’, als, when their ampelographic, phenological and chemical ‘Vitouska’, ‘Pienel’, ‘Prosecco’, ‘Piccola nera’, ‘Malvasia characterization has been completed, will allow the valid- bianca lunga’, ‘Sbulcisa’, ‘Mocula’, ‘Prosecco lungo’ and ity of these preliminary findings to be verified. ‘Aghedene’. The presence of ‘Malvasia bianca lunga’ in this group is no surprise, it being the parent, with ‘Pros- ecco’, of ‘Vitouska’ (Crespan et al. 2007); almost all the Conclusions others are presumed to be autochthonous (Tab. 1). Lastly, in the fourth group of 38 varieties, 12 overcame the thresh- The meticulous work in the field, flanked by literature old limit: ‘Corbina’, ‘Refoscone’, ‘Nigrut’, ‘Vinoso rosso’, searches and molecular analyses of the materials sampled ‘Refosco bianco’, ‘Negrat’, ‘Curvin’, ‘Tazzelenghe’, ‘Cil- over six years of research on the FVG territory, has allowed ja’, ‘Raboso veronese’, ‘Gran rap’ and ‘Brambana’. In this much of the autochthonous material of the region to be res- case they are mainly red grape and are all minor varieties of cued, to characterize it genetically, identify the original FVG, except ‘Raboso veronese’. The presence of ‘Raboso materials and evidence the synonymies. The elaboration of veronese’ suggests the existence of a link between the molecular data has also highlighted the presence of two and FVG as it is a spontaneous hybrid between ‘Raboso groups of varieties that might have a common origin. Piave’ and ‘Marzemina bianca’ (CRESPAN et al. 2006): the The results of this research are a prerequisite for mov- cultivation area of the former is confined to Veneto, mainly ing on to the successive stages of the project of requalifi- in the province of , while the latter has a more ex- cation of the ampelographic heritage of FVG, which are tensive distribution area that also includes FVG, as proved underway and involve the ampelographic, ampelometric, by the 4 accessions identified as ‘Marzemina bianca’ in agronomic and oenological characterization of all the “po- this work. Nevertheless, ‘Marzemina bianca’ is related to tentially original” accessions. A third phase will consist in ‘’, another centuries-old variety of Veneto, with a more detailed evaluation in different environments of the which it has a parent-offspring relationship (CRESPAN et al. varieties considered potentially more interesting, aimed at 2008); it was attributed by Structure to the previous group reintroducing cultivation of the best ones, with registration with a percentage of 65 %, much lower than the threshold. in the National Catalogue of Italian Vine Varieties. We therefore re-processed the data with Structure, adding ‘Raboso Piave’ and imposing k = 4: the variety was clas- sified in the fourth group, with a presumed recognition of Acknowledgements origin of 91 %, demonstrating a close similarity to those varieties, previously unsuspected, and which explains the This research was carried out as part of the programme position of ‘Raboso veronese’ in the same group. ‘Pignola’ “Characterization of ancient Friuli varieties” and was financed is instead typical of the province of Sondrio (North Lom- by the Regional Agency for Agricultural Development of Friuli bardy), but it has also been found in Veneto, under name of Venezia Giulia, with funds made available by the FVG Regional ‘Groppello di ’ (CANCELLIER et al. 2009), and now Law 22 April 2002, n. 11 “Preservation of autochthonous genetic also in FVG. With the current information it is difficult to resources of agricultural and forestry interest”. The authors thank 28 22 M. CRESPAN et al.

M. GIUST and E. PETERLUNGER for access to the “Rinascita” Farm 2006. Acta Hortic. 827, 217-220. and S. Osvaldo collections, respectively. CRESPAN, M.; CANCELLIER, S.; CHIES, R.; GIANNETTO, S.; MENEGHETTI, S.; 2006 b: Individuati i genitori del Raboso veronese: una nuova ipo- tesi sulla sua origine (The parents of Raboso veronese were discov- ered: a new hypothesis on its origin). Riv. Vit. Enol. 1, 3-12. References CRESPAN, M.; CRESPAN, G.; GIANNETTO, S.; MENEGHETTI, S.; COSTACURTA, A.; 2007: ‘Vitouska’ is the progeny of ‘Prosecco tondo’ and ‘Malvasia ARADHYA, M. K.; DANGL, G. S.; PRINS, B. H.; BOURSIQUOT, J. M.; WALKER, bianca lunga’. Vitis 46, 192-194. M. A.; MEREDITH, C.; SIMON, C. J;. 2003: Genetic structure and dif- CRESPAN, M.; MENEGHETTI, S.; CANCELLIER, S.; 2009: Identification and ferentiation in cultivated grape, Vitis vinifera L. Genet. Res. 81, genetic relationship of the principal rootstocks cultivated in Italy. 179-192. Am. J. Enol. Vitic. 60, 349-356. BELKHIR, K.; BORSA, P.; CHIKHI, L.; RAUFASTE, N.; BONHOMME, F.; 2003: DANGL, G. S.; MENDUM, M. L.; PRINS, B. H.; WALKER, M. A.; MEREDITH, C. GENTIX 4.04, Logiciel sous Windows TM pour la Génétique des P.; SIMON, C. J.; 2001: Simple sequence repeat analysis of a clonally Populations. Laboratoire Génome, Populations, Interactions, Adap- propagated species: a tool for managing a grape germplasm collec- tations, UMR 5171. Univ. Montpellier 2, Montpellier, . tion. Genome 44, 432-438. BOWERS, J. E.; DANGL, G. S.; VIGNANI, R.; MEREDITH, C. P.; 1996: Isolation FALUSH, D.; STEPHENS, M.; PRITCHARD, J. K.; 2003. Inference of population and characterisation of the new polymorphic simple sequence repeat structure: Extensions to linked loci and correlated allele frequencies. loci in grape (Vitis vinifera L.). Genome 39, 628-633. Genetics 164, 1567-1587. BOWERS, J. E.; DANGL, G. S.; MEREDITH, C. P.; 1999: Development and MALETIĆ, E.; SEFC, M. K.; STEINKELLNER, H.; KAROGLAN KONTIĆ, J.; characterisation of additional microsatellite DNA markers for grape. PEJIĆ, I.; 1999: Genetic characterization of Croatian grapevine culti- Am. J. Enol. Vitic. 50, 243-246. vars and detection of synonymous cultivars in neighboring regions. CALÒ, A.; COSTACURTA, A.; 1991: Delle viti in Friuli. Ed. Arti Grafiche Vitis 38, 79-83. Friulane. MARTÍN, J. P.; BORREGO, J.; CABELLO, F.; ORTIZ, J. M.; 2003: Characterization CANCELLIER, S.; GIANNETTO, S.; MENEGHETTI, S.; MILANI, N.; CRESPAN, of Spanish grapevine cultivar diversity using sequence-tagged mic- M.; 2006: Approfondimenti sulle popolazioni delle Corbine e delle rosatellite site markers. Genome 46, 10-18. Corvine presenti nel Veneto. Proceedings of the National Congress PRITCHARD, J. K; STEPHENS, M; DONNELLY, P.; 2000. Inference of population “I varietà autoctoni minori: aspetti tecnici, normativi e commercia- structure from multi-locus genotype data. Genetics 155, 945-959. th st li”, Villa Gualino, Torino 30 November - 1 December 2006. Testo SCHNEIDER, A.; TORELLO MARINONI, D.; CRESPAN, M.; 2008. Genetics and negli Atti su CD, sessione 2, 57-63. trace the origin of Muscat fleur d’oranger. Am. J. CANCELLIER, S.; GIANNETTO, S.; CRESPAN, M.; 2009: Groppello di Breganze Enol. Vitic. 59, 200-204. e Pignola sono lo stesso varietà. Groppello di Breganze and Pignola SEFC, K. M.; REGNER, F.; TURETSCHEK, E.; GLÖSSL, J.; STEINKELLNER, H.; are the same cultivar. Riv. Vit. Enol. 2-3, 3-9. 1999: Identification of microsatellite sequences in Vitis riparia and CIPRIANI, G.; SPADOTTO, A.; JURMAN, I.; DI GASPERO, G.; CRESPAN, M.; their applicability for genotyping of different Vitis species. Genome MENEGHETTI, S.; FRARE, E.; VIGNANI, R.; CRESTI, M.; MORGANTE, M.; 42, 367-373. PEZZOTTI, M.; PÈ, E.; POLICRITI, A.; TESTOLIN R.; 2010: The SSR- SEFC, M. K.; LOPES, M. S.; LEFORT, F.; BOTTA, R.; ROUBELAKIS-ANGELAKIS, based molecular profile of 1005 grapevine (Vitis vinifera L.) acces- K. A.; IBÁÑEZ, J.; PEJÍC, I.; WAGNER, H. W.; GLÖSSL, J.; STEINKELLNER, sions uncovers new synonymy and parentages, and reveals a large H.; 2000: Microsatellite variability in grapevine cultivars from dif- admixture among varieties of different geographic origin. Theor. ferent European regions and evaluation of assignment testing to Appl. Genet. (DOI: 10.1007/s00122-010-1411-9, in press). assess the geographic origin of cultivars. Theor. Appl. Genet. 100, COSTACURTA, A.; CALÒ, A.; CARRARO, R.; GIUST, M.; AGGIO, L.; BORSA, 498-505. D.; DI STEFANO, R.; OF THE ZAN, F.; FABBRO, A.; CRESPAN, M.; 2005: ŠTAJNER, N.; KOROŠEC-KORUZA, Z.; RUSJAN, D.; JAVORNIK, B.; 2008. L’identificazione e la caratterizzazione dei Refoschi, 25-45. In: Microsatellite genotyping of old Slovenian grapevine varieties (Vitis Proc. Conference “dei Refoschi”. Ed. Lloyd of San Dorligo della vinifera L.) of the Primorje (coastal) winegrowing region. Vitis 47, Valle, Trieste. 201-204. COSTACURTA, A.; GIANNETTO, S.; MENEGHETTI, S.; CRESPAN, M.; 2006: THIS, P.; JUNG, A.; BOCCACCI, P.; BORREGO, J.; BOTTA, R.; COSTANTINI, L.; Does it exist a Greek ampelographical heredity in ? SSR CRESPAN, M.; DANGL, G.S.; EISENHELD, C.; FERREIRA-MONTEIRO, F.; profiles comparison of cultivars growing in both Countries, 9-14. GRANDO, S.; IBÁÑEZ, J.; LACOMBE, T.; LAUCOU, V.; MAGALHÃES, M.; Proc. “Ampelos 2006” 2nd Int. Symp. Evaluation and Exploitation MEREDITH, C.P.; MILANI, N.; PETERLUNGER, E.; REGNER, F.; ZULINI, of Grapes of Corresponding Terroir through Winemaking and L.; MAUL, E.; 2004: Development of a standard set of microsatellite Commercialization of Wines, Santorini, Greece, 1-3 June 2006. reference alleles for identification of grape cultivars. Theor. Appl. COSTANTINI, E.; MATTALONI, C.; PETRUSSI, C.; 2007: La vite nella storia e Genetics 109, 1448-1458. nella cultura del Friuli. Ed. Forum, Udine. THOMAS, M. R.; SCOTT, N. S.; 1993: Microsatellite repeats in grapevine re- CRESPAN, M.; MILANI, N.; 2001: The Muscats: a molecular analysis of syn- veal DNA polymorphisms when analysed as sequence-tagged sites onyms, homonyms and genetic relationships within a large family of (STSs). Theor. Appl. Genet. 86, 985-990. grapevine cultivars. Vitis 40, 23 - 30. VOUILLAMOZ, J. F.; MAGRE, D.; MEREDITH, C. P.; 2004: Identity and parent- CRESPAN, M.; 2003. The parentage of Muscat of Hamburg. Vitis 42, age of two alpine grape cultivars from (Vitis vinifera L. 193-197. Lafnetscha and Himbertscha). Vitis 43, 81-87. CRESPAN, M.; CALÒ, A.; GIANNETTO, S.; SPARACIO, A.; STORCHI, P.; COSTA- WELTER, L. J.; GÖKTÜRK-BAYDAR, N.; AKKURT, M.; MAUL, E.; EIBACH, R.; CURTA, A.; 2008: ‘Sangiovese’ and ‘Garganega’ are two key varieties TÖPFER, R.; ZYPRIAN, E. M.; 2007: Genetic mapping and localiza- of the Italian grapevine assortment evolution. Vitis 47, 97-104. tion of quantitative trait loci affecting fungal disease resistance and CRESPAN, M.; CANCELLIER, S.; CHIES, R.; GIANETTO, S.; MENEGHETTI, S.; leaf morphology in grapevine (Vitis vinifera L.). Mol. Breed. 20, COSTACURTA, A.; 2006a. Molecular contribution to the knowledge 359-374. of two ancient varietal populations: Rabosi and Glere. Proc. 9th Int. Conference on Grape Genetics and Breeding. Udine, 2-6 luglio Received May 4, 2010