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Meneghetti S.; Costacurta A.; Calò A.

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

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Meneghetti S.; Costacurta A.; Calò A.

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

  2. SSR (Simple Sequence Repeat) & SSR new generation (3-5 n) I-SSR (Inter-microsatellites) RAPD (Random Amplified Polymorphic DNA) AFLP (Amplified Fragment Length Polymorphism) SAMPL (Selective Amplification of Microsatellite Polymorphic Loci) SNP (Single Nucleotide Polymorphism) S-SAP(Specific Sequence Amplified Polymorphism) M-SAP (Methyl-Sensitive Amplified length Polymorphism) REMAP (REtrotransposon-Microsatellite Amplified Polymorphism) IRAP (Inter-Retrotransposon Amplified Polymorphism) Chloroplast DNA polymorphisms 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 viniferaclones. GRAPEVINE & MOLECULAR MARKERS

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

  4. 155 VVS26-FAM (151 bp) VVS2 TET (151 bp) 145 Allelic standard 6FAM - blue 133 139 123 125 147 135 151 137 ? 131 143 153 145 VIC - green 155 149 129 141 127 1 amplification product at -2 bp 4 amplification product (-2, -4, -6, +2 bp) size standard ABOUT DI-NUCLEOTIDE MICROSATELLITES

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

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

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

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

  12. Bunches of the five Primitivo typologies A typology

  13. Leaves of the five Primitivo typologies

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

  15. 1 2 3 4 5 6 1 2 3 4 5 6 M-AFLP & ISSR polymorphisms among 6 Primitivo biotypes: All the Primitivo accessions showed a different molecular profile.

  16. Primitivo biotype B Primitivo biotype D Primitivo biotype A Primitivo biotype C Primitivo biotype E Primitivo biotype RIF 0.80 0.85 0.90 0.95 1.00 Dice’s Genetic Similarity Coefficient DENDROGRAM of Primitivo All the Primitivo genotypes were diversified by cluster analysis using AFLP-derived markers.

  17. GARGANEGA

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

  19. Bunches of Garganega

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

  21. Garganega 1 Garganega 8 Garganega 9 Garganega 4 Garganega 7 Garganega 3 Garganega 6 Garganega 5 Garganega 2 0.80 0.85 0.90 0.95 1.00 Dice’s Genetic Similarity Coefficient DENDROGRAM of Garganega All the Garganega genotypes were diversified by cluster analysis using AFLP-derived markers.

  22. MERLOT

  23. 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). 1) Fifteen accession of Merlot were analyzed. 2) For the varietal identification, microsatelliteanalyses confirmed that these 15 accessions were the same cultivar showed the typical SSR profile of Merlot. SSR Profile of Merlot at 11 Loci 3) In order to find intravarietal variability, AFLP, SAMPL and M-AFLP molecular markers were performed.

  24. ISVFV2 ISVFV6 ISVFV5 Bunches of Merlot

  25. 1 Merlot 6 Merlot 1 Merlot 2 Merlot 3 Merlot 4 2 Merlot 7 Merlot 8 Merlot 9 Merlot 14 Merlot 15 3 Merlot 11 Merlot 13 4 Merlot 12 Merlot 10 5 Merlot 5 0.80 0.85 0.90 0.95 1.00 Dice’s Genetic Similarity Coefficient DENDROGRAM of Merlot Five Merlot molecular profiles were diversified by cluster analysis using AFLP-derived markers.

  26. CONCLUSIONS

  27. 9 profiles 5 profiles 6 profiles 1 2 3 4 5 6 7 8 9 1 2 2 2 3 1 2 2 2 3 4 1 5 2 2 1 2 3 4 5 6 9 GARGANEGA 15 MERLOT 6 PRIMITIVO 5/15=33% 6/6=100% M-AFLP: MD27f / Mse+ AGG 9/9=100%

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

  29. 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 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

  30. THANKS

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