1. Meneghetti S.; Costacurta A.; Calò A. EVALUATION OF THE INTRA-VARIETAL VARIABILITY OF GRAPEVINE (II) OIV Organisation Internationale de la Vigne et du Vin Paris, le 18 mars 2009 VIT CENTRO DI RICERCA PER LA VITICOLTURA di Conegliano ITALY

2. GRAPEVINE VARIABILITY The great morphological and genetic variability that characterize the genus Vitis is the principal problem about grapevine cultivars identification. Biotype identification in viticulture have traditionally solved using ampelography, ampelometry and chemical traits analysis. The DNA molecular analyses with ampelography, ampelometry and chemistry, are essential for a sure grapevine identification (SSR) and to investigate the genetic difference among Vitis vinifera clones. SSR (Simple Sequence Repeat) & SSR new generation (3-5 n) SAMPL (Selective Amplification of Microsatellite Polymorphic Loci) M-SAP (Methyl-Sensitive Amplified length Polymorphism) REMAP (REtrotransposon-Microsatellite Amplified Polymorphism) RAPD (Random Amplified Polymorphic DNA) AFLP (Amplified Fragment Length Polymorphism) I-SSR (Inter-microsatellites) SNP (Single Nucleotide Polymorphism) IRAP (Inter-Retrotransposon Amplified Polymorphism) Chloroplast DNA polymorphisms S-SAP (Specific Sequence Amplified Polymorphism) GRAPEVINE & MOLECULAR MARKERS

3. SSR grape identification (DI-NUCLEOTIDE MICROSATELLITES) Silver Staining PAA gel Sequencer (gel) Laser Sequencer (gel) Sequencer (capillary) SSR grape identification 133 bp 155 bp Size VITIS Size standard Sequencer (capillary) CCD camera 5 colors

4. VVS2 6-FAM (151 bp)VVS2 TET (151 bp) 1 amplification product at -2 bp 4 amplification product (-2, -4, -6, +2 bp) 6FAM - blue VIC - green ? size standard 145 155 Allelic standard 123 125 133 131 135 137 129 127 139 145 143 141 147 151 149 153 155 ABOUT DI-NUCLEOTIDE MICROSATELLITES

5. The 19 grapevine chromosomes and the distribution of the new 38 SSR markers TETRA-NUCLEOTIDE MICROSATELLITES

6. 175 bp 222 bp 239 bp Cultivar: CABERNET FRANC Locus SSR: 1a (222-239 bp) Cultivar: BARBERA Locus SSR: 1a (175-222 bp) RAW DATA

7. 218 – 226 bp 222 // bp = 4bp LAMBRUSCO MARANI MALVASIA B. DI CANDIA

8. In order to compare the variability of grapevine cultivated in Italy from a long time with the cultivars of recently introduction we have analyzed Primitivo, Garganega and Merlot genotypes at 1) morphological and 2) molecular level.

9. PRIMITIVO

10. In ancient vineyards sited in typical cultivation areas (Taranto province, Gioia del Colle) of Primitivo new researches have been done to evaluate its genetic variability. Overall based on bunch (shape, size, density) and berry (size, colour of skin) characteristics 46 vines have been identified. Five typologies called A-E have been identified by means of ampelographic, phyllometric and DNA (SSR, AFLP, M-AFLP) analyses. RIF: A. Calò, G. Masi, L. Tarricone, A. Costacurta, S. Meneghetti, M. Crespan, R. Carraro (2008): Ricerche sulla variabilità del Primitivo (V. vinifera L.) in Puglia – Search for Primitivo (V. vinifera L.) variability in Apulia. Riv. Vitic. Enol. 1: p. 3-13.

11. VVS2VVMD5VVMD7VVMD27VVMD28VrZAG 62 133143226236247249179181251261199203 VrZAG 79VMC6E1VMC6F1VMC6G1VMCNG4b9 236258141165139 177 150164 SSR Profile of Primitivo at 11 Loci 1) Six accession of Primitivo with different morphological characteristics were analyzed 3) In order to find intravarietal variability, AFLP, SAMPL and M- AFLP molecular markers were performed 2) For the varietal identification, microsatellite analyses confirmed that these 6 accessions were the same cultivar showed the typical SSR profile of Primitivo

12. Bunches of the five Primitivo typologies A typology

13. Leaves of the five Primitivo typologies

14. Eco+C / Mse+A AFLP polymorphisms among 6 Primitivo genotypes:

15. M-AFLP & ISSR polymorphisms among 6 Primitivo biotypes: 123456123456 All the Primitivo accessions showed a different molecular profile.

16. DENDROGRAM of Primitivo Primitivo biotype B Primitivo biotype D Primitivo biotype A Primitivo biotype C Primitivo biotype E Primitivo biotype RIF 0.80 0.900.850.951.00 Dice’s Genetic Similarity Coefficient All the Primitivo genotypes were diversified by cluster analysis using AFLP-derived markers.

17. GARGANEGA

18. VVS2VVMD5VVMD7VVMD27VVMD28VrZAG 62 181 226232249253179194239251199 VrZAG 79VMC6E1VMC6F1VMC6G1VMCNG4b9 250 141165133139177187176178 SSR Profile of Garganega at 11 Loci 1) Nine accession of Garganega with different morphological characteristics were analyzed 3) In order to find intravarietal variability, AFLP, SAMPL and M- AFLP molecular markers were performed 2) For the varietal identification, microsatellite analyses confirmed that these 9 accessions were the same cultivar showed the typical SSR profile of Garganega Garganega is cultivated for centuries ago in Veneto region according to Pier de Crescenzi in "Opus ruralium commodorum" at the end of 1200 (citation).

19. Bunches of Garganega

20. AFLP: Pst + AT / Mse+ AAG high genetic variability

21. DENDROGRAM of Garganega All the Garganega genotypes were diversified by cluster analysis using AFLP-derived markers. 0.80 0.900.850.951.00 Garganega 1 Garganega 8 Garganega 9 Garganega 4 Garganega 7 Garganega 3 Garganega 6 Garganega 5 Garganega 2 Dice’s Genetic Similarity Coefficient

22. MERLOT

23. VVS2VVMD5VVMD7VVMD27VVMD28VrZAG 62 139151226236239247189191231237193 VrZAG 79VMC6E1VMC6F1VMC6G1VMCNG4b9 258 151 133139169191162172 SSR Profile of Merlot at 11 Loci 1) Fifteen accession of Merlot were analyzed. 3) In order to find intravarietal variability, AFLP, SAMPL and M- AFLP molecular markers were performed. 2) For the varietal identification, microsatellite analyses confirmed that these 15 accessions were the same cultivar showed the typical SSR profile of Merlot. The Merlot is a cultivar of recent cultivation in Italy according to S. Mondini on “I Vitigni Stranieri da vino coltivati in Italia, 1903” - The foreign grapevines for wine cultivated in Italy: “Merlot is present in the collections of marquis Incisa in Piedmont region (Rocchetta Tanaro) on 1850” (citation).

24. ISVFV2 ISVFV5 ISVFV6 Bunches of Merlot

25. Five Merlot molecular profiles were diversified by cluster analysis using AFLP-derived markers. DENDROGRAM of Merlot Merlot 6 Merlot 1 Merlot 2 Merlot 3 Merlot 4 Merlot 7 Merlot 8 Merlot 9 Merlot 14 Merlot 15 Merlot 11 Merlot 13 Merlot 12 Merlot 10 Merlot 5 0.80 0.900.850.951.00 Dice’s Genetic Similarity Coefficient 1 2 3 4 5

26. CONCLUSIONS

27. M-AFLP: MD27f / Mse+ AGG 9/9=100% 9 GARGANEGA15 MERLOT6 PRIMITIVO 9 profiles 123456789 5/15=33% 5 profiles 122231222341522 6/6=100% 6 profiles 123456

28. G P M DENDROGRAM (G = Garganega; P = Primitivo; M = Merlot) There are 9 different genotypes of Garganega (9/9) GS = 0.812 There are 5 different genotypes of Primitivo (5/5) GS = 0.853 Dice’s Genetic Similarity Coefficient There are 5 different genotypes of Merlot (5/15) GS = 0.913

29. Analyses showed: 1) high morphological variability within the Primitivo accessions 2) high morphological variability within the Garganega accessions 3) low morphological variability within the Merlot accessions 4) The high morphological variability of cultivars corresponds to elevated intra-varietal genetic variability using molecular markers (Garganega and Primitivo from Italy) 5) The low morphological variability of Merlot accessions also corresponds to low intra-varietal genetic variability shown by the molecular markers Correspondence between morphological and genetic variability the grapevine cultivated for a long time (in Italy) show great variability (M&G) of those with more recent introduction/cultivation

30. THANKS