Meneghetti S.; Costacurta A.; Calò A.

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

VITCENTRO DI RICERCA PER LA VITICOLTURA di Conegliano

ITALY

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

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 camera5 colors

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

129127

139

145143

141

147

151

149153 155

ABOUT DI-NUCLEOTIDE MICROSATELLITES

The 19 grapevine chromosomes and the distribution of the new 38 SSR markers

TETRA-NUCLEOTIDE MICROSATELLITES

TETRA-NUCLEOTIDE MICROSATELLITES

175 bp

222 bp

222 bp 239 bp

Cultivar: CABERNET FRANC

Locus SSR: 1a (222-239 bp)

Cultivar: BARBERA

Locus SSR: 1a (175-222 bp)

RAW DATA

218 – 226 bp

222 // bp = 4bp

LAMBRUSCO MARANI

MALVASIA B.DI CANDIA

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.

PRIMITIVO

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.

VVS2 VVMD5 VVMD7 VVMD27 VVMD28 VrZAG 62

133 143 226 236 247 249 179 181 251 261 199 203

VrZAG 79 VMC6E1 VMC6F1 VMC6G1 VMCNG4b9

236 258 141 165 139 139 177 177 150 164

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

Bunches of the five Primitivo typologies

A typology

Leaves of the five Primitivo typologies

Eco+C / Mse+A

AFLP polymorphisms among 6 Primitivo genotypes:

M-AFLP & ISSR polymorphisms among 6 Primitivo biotypes:

1 2 3 4 5 61 2 3 4 5 6

All the Primitivo accessions showed a different molecular profile.

DENDROGRAM of Primitivo6 tipologieprimitivo

Coefficient0.80 0.85 0.90 0.95 1.00

1

1

4

6

2

3

5

Primitivo biotype B

Primitivo biotype D

Primitivo biotype A

Primitivo biotype C

Primitivo biotype E

Primitivo biotype RIF

6 tipologieprimitivo

Coefficient0.80 0.85 0.90 0.95 1.00

1

1

4

6

2

3

5

0.80 0.900.85 0.95 1.00

Dice’s Genetic Similarity Coefficient

All the Primitivo genotypes were diversified by cluster analysis using AFLP-derived markers.

GARGANEGA

VVS2 VVMD5 VVMD7 VVMD27 VVMD28 VrZAG 62

181 181 226 232 249 253 179 194 239 251 199 199

VrZAG 79 VMC6E1 VMC6F1 VMC6G1 VMCNG4b9

250 250 141 165 133 139 177 187 176 178

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).

Bunches of Garganega

AFLP: Pst + AT / Mse+ AAG

high genetic variability

DENDROGRAM of Garganega

All the Garganega genotypes were diversified by cluster analysis using AFLP-derived markers.

6 tipologieprimitivo

Coefficient0.80 0.85 0.90 0.95 1.00

1

1

4

6

2

3

5

0.80 0.900.85 0.95 1.00

Garganega 1

Garganega 8

Garganega 9

Garganega 4

Garganega 7

Garganega 3

Garganega 6

Garganega 5

Garganega 2

Dice’s Genetic Similarity Coefficient

MERLOT

VVS2 VVMD5 VVMD7 VVMD27 VVMD28 VrZAG 62

139 151 226 236 239 247 189 191 231 237 193 193

VrZAG 79 VMC6E1 VMC6F1 VMC6G1 VMCNG4b9

258 258 151 151 133 139 169 191 162 172

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).

ISVFV2 ISVFV5 ISVFV6

Bunches of Merlot

Five Merlot molecular profiles were diversified by cluster analysis using AFLP-derived markers.

DENDROGRAM of Merlot

Merlot 6

Merlot 1Merlot 2Merlot 3Merlot 4

Merlot 7Merlot 8

Merlot 9Merlot 14

Merlot 15Merlot 11Merlot 13Merlot 12

Merlot 10Merlot 5

6 tipologieprimitivo

Coefficient0.80 0.85 0.90 0.95 1.00

1

1

4

6

2

3

5

0.80 0.900.85 0.95 1.00Dice’s Genetic Similarity Coefficient

1

2

3

4

5

CONCLUSIONS

M-AFLP: MD27f / Mse+ AGG

9/9=100%

9 GARGANEGA 15 MERLOT 6 PRIMITIVO

9 profiles9 profiles

1 2 3 4 5 6 7 8 9

5/15=33%

5 profiles5 profiles

1 2 2 2 3 1 2 2 2 3 4 1 5 2 2

6/6=100%

6 profiles6 profiles

1 2 3 4 5 6

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

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

THANKS