SSRs

RAPDs AFLPs

RFLPs ISOENZIMI

Experimental basic steps

 DNA residues purification

 Qalitative PCR amplification at 15 SSRs loci

 Allele sizing by MEGABace 500 (GE Healthcare)

NERETO GRANOIR

ABROSTINE SAN COLOMBANO

ABRUSCO VERMENTINO BIANCO

ANSONICA MAZZESE

B45BB GIACCHE'

BARSAGLINA ALEATICO

BIV1-420 MONTEPULCIANO D'ABRUZZO

BRONTOLO NEGROTINO

C2 GRAND NOIR

CANAIOLO NERO PETIT VERDOT

CILIEGIOLO MERLOT

FOGLIA FRASTAGLIATA GAMAY

FOGLIA TONDA GRANE’

M235 SYRAH

M238 CABERNET FRANC

MAMMOLO CABERNET SAUVIGNON

POLLERA TEROLDEGO

PRUGNOLO GENTILE ANCELLOTTA

PUGNITELLO TINTURIER

SANGIOVESE APSG2 REFOSCO DAL PEDUNCOLO ROSSO

SANGIOVESE R24 MORONE

SANGOVESE F9 COLORINO

VERDELLO BIOT.PITIGL. RASPO ROSSO

VERDELLO VERMENTINO NERO

WINE AND DNA?

WINE TRACK AUSTRALIA

BRL HARDY COMPANY

BOTTLE WITH SEAL IN WHICH THERE IS A CODE WHERE DNA EXTRACTED FROM GRAPEVINE IS MIXED WITH INORGANIC CHEMICALS AND USED AS AUTHENTICITY LABEL MATRIX TM

AND THE 'COMBINED WITH INK, MARCATORE, RESINS ETC.

LABELS ARE READ BY AN AN ELECTRONIC SCANNER THAT ENSURES TRACEABILITY OF BOTTLES

WINE TRACK

EFSA (European Food Safety Authority) STABLE ISOTOPE ANALYSIS (C,H,O)

ISOTOPE RELATIVE RATIO DEPENDS ON GEOGRAPHICAL PROVENIENCE, CLIMATE CONDITIONS

Chemometric anthocyanines wine profiling (Iztok Joz et al. Analytica Chimica ActaVolume 513, 18 June 2004, Pages 277-282 ) • Merlot, Cabernet sauvignon, Pinot noir • Environment effects

DNA and consumer confidence (EMBO reports 4, 3, 232ﾐ234 (2003)) In forensics DNA testing is fundamental for individual identification PCR is the key technique, which allows to analyse even DNA highly degraded (fossil specimens) or present in traces (a few or even one hairbulb, biological specimens present in crime scenes)

• Identify potential suspects whose DNA may match evidence left at crime scenes • Identify crime and catastrophe victims • Establish paternity and other family relationships

IN MEDICINA FORENSE SI ESTRAE DNA DA CELLULE RESIDUE, OSSA,TESSUTI,CAPELLI, RESIDUI DI LIQUIDI ORGANICI E SANGUE ECC.

DNA purification from WINE

DNA purification by use of linear acrylamide (Dràbek et al., Eur Food Res Technol 2008) or other chelating agents (Argylla technologies, Tucson, AZ, USA)

Siliceous matrix-based kits (Qiagen)

Chemical extraction (SDS, CTAB) after an initial massive precipitation step (Siret et al., J. Agric. Food Chem. 2002; Savazzini et al. Analytica Chimica Acta 2006)

• Siret et al, 2000

Centrifugazione

sovranatante Pellet

2 X CTAB

2 X clorof.-isomilico +ppt in alcool e sale

DNA crudo

DNA crudo

Proteinasi K

1 x Estrazione fenol-clorof.

Ppt 2 vol alcool + sale

Purificazione mediante colonne

Qiagen

2° prelievo

1° eluizione

2° eluizione

• Left: DNA fractions extracted from wine (Wang, personal communication). Right: DNA extracted following a modification of the Siret protocol (Siret et al.,2000). DNA presence is evidenced with coloured ovals. Lambda HindIII is used as molecular weight standard (asterisk)

• Residual grapevine DNA is estimated to be 2 picograms-4000picograms/ml

* *

ZAG47

ZAG79 D14

D21 Sangiovese 243/249

D31 Sangiovese 212

D25 Sangiovese 241

Sangiovese and more?..

SANGIOVESE genotype wine 179-185 179-185-189

175 175 175 175

159-161 159-161 135 135-137

260-262 260-262-254-256 156-162 156-162-166

240 240

Quantitative assay by Real time-PCR

A new set of three- and tetra- nucleotidic markers under development (in collaboration with VIGNA project)

Apply molecular genotyping to the vineyards to establish a detailed program of analytical traceability for each producer (from field to bottle.. to meet real genetic

diversity population)

Department of Environmental Science at the University of Siena

“The Grapevine group” Monica Scali, PhD, Proteomics and

molecular biology Elisa Paolucci, Jacopo Bigliazzi,

genotyping Thilo Nagel in vitro transformation

assays, ELISA test, micropropagation