Experimental Review of DNA-Based Methods for Wine Traceability

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Article

Experimental review of DNA-based methods for wine traceability and development of a SNP genotyping assay for quantitative varietal authentication Valentina Catalano, Paula Moreno-Sanz, Silvia Lorenzi, and Maria Stella Grando J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02560 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on September 5, 2016

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Journal of Agricultural and Food Chemistry

Experimental review of DNA-based methods for wine traceability and development of a SNP genotyping assay for quantitative varietal authentication

Valentina Catalano§, Paula Moreno-Sanz§, Silvia Lorenzi, and Maria Stella Grando* Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all’Adige, Trento, Italy

§ these authors contributed equally

* Corresponding Author Phone +39 0461 615197 Fax +39 0461 650956 E-mail [email protected]

ACS Paragon Plus Environment

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Abstract: The genetic varietal authentication of wine was investigated according to

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DNA isolation procedures reported for oenological matrices, and also by testing eleven

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commercial extraction kits and various protocol modifications. Samples were collected

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at different stages of the winemaking process of renowned Italian wines Brunello di

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Montalcino, Lambruschi Modenesi and Trento DOC.

6

Results demonstrated that grape DNA loss is produced not only by the fermentation

7

process, but clarification and stabilization operations also contribute to the reduction of

8

double-stranded DNA content on wine. Despite the presence of inhibitors, downstream

9

PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must

10

samples, whereas no amplification or inconsistent results were obtained at later stages

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of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific

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SNPs was designed which allowed to assess grapevine DNA mixtures. Once the wine-

13

matrix limitations were overcome this sensitive tool may be implemented for the

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relative quantification of cultivars used for blend wines or frauds.

15 16

Keywords: wine, grapevine, genetic traceability, single nucleotide polymorphisms,

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DNA extraction, quantitative PCR.

18 19


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INTRODUCTION

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Wine is one of the most complex and appreciated alcoholic beverages, whose quality is

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highly dependent on different factors among which the grapevine variety is of primary

23

importance. Old World wineries must follow strict rules governing the types of grapes

24

used in their wines. The cultivar used is essential in the case of monovarietal wines or in

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wines identified by an appellation of origin that are produced with more than one

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cultivar in a specific ratio (Regulation EC No. 479/2008). Sometimes, the irregular

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addition of wines derived from other grapevine varieties is used with the aim of

28

enhancing the sensory characteristics of the final product and/or to decrease the

29

production costs. Therefore technical approaches and legislative guidelines have been

30

developed to grape, must, and wine traceability in order to guarantee the product origin

31

and detect fraud (Regulation EC No. 178/2002);1,2. Fingerprinting methods based on the

32

analysis of metabolites such as volatile compounds, amino acids and proteins,

33

polyphenols, anthocyanins, and also minerals, have been developed for the

34

authentication of the geographical origin of wines obtaining promising results,

35

especially in the case of the analysis of the mineral profile and, recently, by the analysis

36

of stable isotopes3,4,5,6. Some of these approaches have also been applied for the varietal

37

identification of wines; however the metabolic composition of grapes and wines

38

depends from environmental conditions and cultural practices, which, instead, do not

39

affect genotype; reason why the varietal control might be more accurate and efficient

40

when DNA based methodologies are used.

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Identification of grapevine varieties from direct plant material - leaves, roots, canes7,8,9 -

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through DNA-based markers is a well-established practise. Among the genetic markers

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available (RFLPs, RAPDs, AFLPs), microsatellites (SSRs) have proved to be the best

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ones for grapevine DNA typing because of their high degree of polymorphism, species-


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specificity, co-dominant Mendelian inheritance, reproducibility and simple data

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interpretation9,10,11. Due to the extensive use of this fingerprinting technology

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worldwide, large international Vitis databases of SSR profiles are now available as

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references for grapevine varietal identification12,13.Wine is a complex matrix where the

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DNA comes not only from the grapes used for its elaboration, but also from the

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spontaneous microbiota, or that inoculated for alcoholic and malolactic fermentations,

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as well as from the additives of biological origin and the concentrated musts that may

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have been used. The potential of a genetic traceability approach in a such heterogeneous

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matrix, indeed, is almost unlimited, since the molecular analysis would allow

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identifying, not only the grape variety from which it has been produced, but also the

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yeasts and bacteria strains used for fermentation and to establish if genetically modified

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organisms (GMO) have been used14,15,16. Thus, the development of daily routine

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methods based on genetic analysis would make possible the traceability of a wine at all

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levels and in all stages of the winemaking process. However winemaking implies

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several processing steps which obviously affect the quantity and quality of grape DNA

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available in wine. On one hand, besides that grape DNA is contaminated with that from

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the existing microbiota, the DNases from the yeasts degrade DNA during fermentation

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generating denatured and fragmented residues. On the other hand, decantation,

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clarification, filtration and other fining treatments contribute to the decrease of the final

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DNA concentration available.

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SSR analysis for grapevine varietal identification can be roughly described in four main

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steps: DNA isolation from plant material, SSR amplification by PCR, analysis of the

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microsatellite fragments produced, usually by a denaturing capillary gel electrophoresis,

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and results interpretation17. This methodology, widely used as routine lab work for

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application to breeding and germplasm management, was first applied for grape juice


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varietal identification by18, and then for varietal wine authentication by19,20, who

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analyzed experimental wines from the start to the end of fermentation. These authors

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performed successful varietal identification by SSR genotyping in musts, but reported

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difficulties for the authentication of the cultivars in finished wines due to the scarce

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DNA isolated. Successive studies have been performed in order to improve DNA

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isolation from wine, but in all cases, although varietal identification of musts was

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possible, reproducibility problems for the systematic authentication of finished

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experimental and/or commercial wines were always reported again due to the extraction

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of low DNA quantity and quality from a wine matrix21,22,23,24,25,26,27,28,29. Rodriguez-

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Plaza et al.30 tried to improve the method by applying a non-denaturing capillary

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electrophoresis, but the reproducibility of the results was even worst. Analysis of other

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marker types, such as chloroplast SSR markers or ISSR markers, has been

82

proposed22,23,24,25,26,27,28,29. Multivarietal must mixtures and blended experimental wines

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have been analysed as well to detect the discrimination power of the SSR technology

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for determining the varieties used in the mixtures20,21,22,26,31. Despite it could be

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discriminated if more than one variety was used, the identification of unknown

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additional cultivars used became impossible, especially when the blends consisted on

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more than two varieties. In this context, the main limiting factor for the development of

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a standard method for wine varietal authentication is the obtainment of grape DNA from

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wine suitable for downstream applications. In order to shed light on the issue, we have

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experimentally reviewed some of the different methods published till now. We focused

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into two main tasks: 1) the optimization of protocols for DNA extraction from wine in

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terms of DNA quantity, purity and functionality in PCR application and 2) the

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development of an alternative genotyping system potentially less affected by the quality

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of DNA and best suited for target quantification purposes.


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MATERIALS AND METHODS

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Plant DNA of reference

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DNA was isolated from 65 mg of fresh grapevine leaves collected at the FEM

98

germplasm collection from true-to-type accessions of the wine varieties involved in this

99

study (Tables 6 and 7). DNeasy Plant mini kit (Qiagen) was applied for plant DNA

100

extraction according to the manufacturers’ protocol. Plant DNA was used to get a

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reference varietal genotype of each cultivar. SSR analysis from leaves’ DNA was

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performed as described by 32.

103 104

Improvement of DNA isolation

105

Must and wine total DNA was isolated as further specified in the different trials

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performed. As in previous experiments with wine

107

extraction was based on the UV absorbance values. Three measurements for each

108

sample were performed using a NanoDrop 8000 UV-vis Spectrophotometer (Thermo

109

Scientific). The origin and quality of the DNA isolates were further validated by PCR

110

amplification of specific grapevine nuclear microsatellite markers (nSSRs) as described

111

below (Trial 2.1).

15, 23, 24, 26, 44

evaluation of DNA

112 113

MUST

114

Trial 1.1: Total DNA extraction from must at three different stages of fermentation.

115

First, 750 mL of Lambrusco Grasparossa must were collected from a commercial wine

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cellar at different stages: pre-fermentation, middle of fermentation and end of

117

fermentation. Samples were stabilized by addition of 2 g/l of potassium metabisulfite

118

(K2S2O5). Before DNA isolation, samples were left decanting overnight for

119

precipitation of solid particles in suspension. Next, 2 mL of the supernatant were


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withdrawn (decanted sample) and then the precipitated solid phase was mixed again

121

with the supernatant in order to take another 2 mL (homogenized sample). Must’s

122

samples were centrifuged at 2500 rpm for 30 min at room temperature and DNA

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extraction was performed from the pellet obtained according to the CTAB-based

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method proposed by 23. In this protocol the pellet was dissolved by briefly vortexing in

125

750 µL of CTAB buffer [25mM EDTA, 1 M Tris-HCl, pH 8, 2 M NaCl and 3% (w/v)

126

CTAB, 0.2% (v/v) 2-mercaptoethanol and 1% (w/v) polyvinilpolypyrrolidone (PVPP)]

127

and left at 65°C for 60 min. Afterwards, a step for proteins’ purification was performed

128

by adding an equal volume of phenol:chloroform:isoamilic alcohol (25:24:1) to the

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sample. The upper layer (DNA-containing), recovered by centrifugation at 13000g for

130

15 min at 4 °C, was transferred into a new 2 mL tube. Subsequently, 0.6 volumes of 2-

131

propanol were added and the sample was left overnight at -20°C for DNA precipitation.

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Next, DNA was recovered by centrifugation at 13000g for 30 min at 10°C, washed in

133

70% ethanol for 30 min under the same conditions and, once dried, DNA was

134

resuspended in 100 µL of bidistilled water. Finally, an additional treatment with 1 µL of

135

RNase A at 37°C for 60 min was performed to the isolates, which were then kept until

136

use at -20°C. Two replicates per sample were analysed.

137 138

Trial 1.2: DNA extraction at intermediate steps of the winemaking chain.

139

Isolation of DNA from Lambrusco (L. Sorbara and L. Grasparossa) and Trento DOC

140

(Chardonnay and Pinot Noir) musts was performed at intermediate steps of the

141

winemaking process (Tables 1-2). Musts came from different commercial wine cellars.

142

Musts were left decanting overnight in order to withdraw decanted and homogenized

143

samples. DNA extraction was performed as in Trial 1 according to

144

replicates per sample were analyzed.


23

. Two-three

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Trial 1.3: Introduction of modifications to the total DNA extraction protocol from musts

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DNA was isolated from L. Sorbara and L. Grasparossa musts at different fermentation

148

stages (Table 3) with the original CTAB protocol of 23 , completely described in trial 1.1

149

(SM method), as well as with a modified one by adding 100 µL of proteinase K (20

150

mg/ml) to the CTAB extraction buffer before incubation at 65°C for 60 min (SM-K

151

method). The performance of an additional DNA purification step was also evaluated by

152

testing the NucleoSpin CleanUp commercial kit (Macherey-Nagel), applied according

153

to the manufacturer’s instructions. Three replicates per sample were analyzed.

154 155

WINE

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Analyzed Sangiovese experimental wines were supplied by the experimental wine cellar

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of the Fondazione Edmund Mach (FEM). Grape harvest was performed in Montalcino

158

area (Tuscany, Italy) at oenological maturity. Grapes were mechanically pressed being

159

the global parameters of the initial must obtained as follows: 23ºBrix, pH of 3.4 and

160

titratable acidity of 4.8 g/l tartaric acid. Must was stabilized by addition of 15 mg/l of

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SO2. For alcoholic fermentation 50 g/hl of Saccharomyces cerevisiae Blastosel (Kappa

162

Perdomini Spa) were added. Temperature during fermentation was 28ºC. Maceration

163

lasted seven days with two manual cap punching per day. In the end of the alcoholic

164

fermentation 30 mg/l of sulphur dioxide (SO2) were added. For malolactic fermentation

165

the bacteria Oenoccocus oeni Viniflora oenos (CHR Hansen) was added. Finally, wine

166

samples were filtered and bottled. With regard to the commercial wines, monovarietal

167

wines of Sangiovese were provided by the Consorzio del Vino Brunello di Montalcino,

168

while, in the case of Lambrusco wines, they were supplied by the Consorzio Marchio

169

Storico dei Lambruschi Modenesi. Finally sparkling base wines were provided by


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Consorzio Trento DOC. Before withdrawing the wine aliquots for DNA isolation, wine

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was previously homogenized by inverting the bottle several times.

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Trial 1.4: Comparison of wine DNA extraction methods.

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DNA extraction from Sangiovese experimental wines from 2010 vintage was performed

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by using different methods: Savazzini and Martinelli

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Bigliazzi 27. Savazzini and Martinelli modified extraction method (SM-K) was the same

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described in trial 1.3, but the initial volume of wine was of 10 ml and wine samples

178

were concentrated before DNA extraction by 2-propanol precipitation (0.7 v/v) at -20°C

179

for two weeks. DNA extraction was then performed from the pellet obtained by

180

centrifugation at 2500 rpm for 30 min at room temperature. In addition, three

181

commercial wines (Brunello 2008, Trento DOC and Lambrusco) were analyzed with

182

Pereira and Bigliazzi methods

183

following the manufacturer’s specifications. Plant genomic DNA miniprep kit (Sigma),

184

Dneasy Plant mini kit (Qiagen), High Pure PCR template preparation kit (Roche),

185

Power Plant DNA isolation kit (MO-BIO), Power Soil DNA isolation kit (MO-BIO),

186

FastDNA spin kit for soil (MP Biomedicals), NucleoSpin Food (Macherey-Nagel),

187

Genomic DNA from food (Macherey-Nagel), QIAamp DNA Stool for human DNA

188

analysis (Qiagen) and Dneasy mericon Food kit (Qiagen) were based on resin columns

189

that selectively bind nucleic acids; while DNA Purification system for food (Promega)

190

was based on magnetic bead-based nucleic acid isolation technology. Sangiovese

191

experimental wine from vintage 2010 was analyzed with all kits and for some of them

192

other varietal wine types were tested additionally: Cabernet Sauvignon 2010, Merlot

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2010 and Teroldego 2010 (from the FEM wine cellar), Brunello 2008, Trento DOC and

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Lambrusco Grasparossa (Table 4). Wine samples were subjected to a concentration

23

modified, Pereira

26

and

26,27

. Several commercial extraction kits were also tested


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treatment with 2-isopropanol (see trial 1.5) before DNA isolation with each kit.

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Modifications were introduced to those kits with the best DNA isolation performance

197

consisting on the addition of α-amylase (100 µL and incubation for 30 min at 80ºC)

198

and/or proteinase K treatment (40 µL and incubation for 30 min at 55ºC). The

199

treatments were introduced after the first kit buffer solution was added.

200 201

Trial 1.5: Effect of concentration and purification treatments before and after DNA

202

isolation from wine, respectively.

203

Several treatments for sample concentration were performed before DNA isolation from

204

Sangiovese 2010 experimental wine: i) addition of 0.6 volumes of 2-isopropanol to 30

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mL of wine and incubation at -20°C for two weeks, then centrifugation for 15’ at 8000

206

rpm, the supernatant was discarded while the pellet was eluted in 1 mL of Milli-Q water

207

and concentrated again by centrifugation, ii) precipitation of 45 mL of wine with 5 mL

208

of a NaCl solution 1.2 M, incubation at -20°C for one week and centrifugation, iii)

209

precipitation of 43 mL of wine with 7 mL of a solution of Na acetate 3 M, pH 5.2,

210

incubation at -20°C for two weeks and centrifugation, iv) filtration of 30 mL of wine by

211

using a Millipore membrane of 10 or 3 kDa, aliquots of the filtered solution in 1.5 mL

212

tubes were performed and centrifuged for concentration, v) precipitation of the wine

213

sample in 5 volumes of absolute ethanol by overnight incubation at 2 – 4°C, the pellet

214

was then recovered by centrifugation at 8000 rpm for 15’, washed twice with 10 mL of

215

ethanol and recovered again by centrifugation with the same conditions, then the pellet

216

was dried at room temperature, resuspended in 1 mL Milli-Q water and concentrated by

217

centrifugation and vi) filtration of 500 mL of wine with Sep-Pak C18 Classic Cartridge,

218

previously cleaned with 30 mL Milli-Q water and activated with 30 mL of methanol.

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The fraction recovered was dried for concentration.


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After each treatment, DNA extraction was performed according to the protocols

221

described by 26 and 23 modified. In the case of samples extracted with Pereira method 26,

222

a further purification step was carried out with NucleoSpin Clean Up kit or following

223

the purification procedure described by 33.

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Trial 1.6: Effect of wine aging on DNA isolation

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Sangiovese experimental wines from vintages 2008, 2009 and 2010, and pre-

227

commercial Brunello wines from vintages 2008 and 2010 were analyzed. DNA was

228

extracted according to Pereira CTAB method

229

analyzed.

26

. Three replicates per sample were

230 231

Trial 1.7: Introduction of modifications into Pereira et al. (2011) extraction method to

232

improve its performance.

233

Pereira DNA extraction method 26 consisted on:

234

Step 1: Precipitation of the wine samples by adding 0.7 volumes of 2-isopropanol at

235

-20°C for two weeks and recovery of the precipitated DNA by centrifugation (4000g for

236

30 min at room temperature).

237

Step 2: Resuspension of the obtained pellet in 750 µL of preheated extraction buffer

238

[20mM of EDTA, 10mM of Tris-HCl pH 8.0, 1.4M NaCl and 2% (w/v) of CTAB, 1%

239

(v/v) of 2-mercaptoethanol, 2% (w/v) of PVPP and proteinase K (20 mg/ml)] and

240

incubation for 60 min at 65°C.

241

Step 3: An extraction by adding an equal volume of chloroform:isoamyl alcohol (CIA)

242

(24:1) (v/v). Recovery of the DNA layer by centrifugation at 13000 g for 15 min at 4°C.

243

Step 4: RNase treatment (10 mg/ml) at 37°C for 30 min.


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Step 5: DNA precipitation by overnight incubation at -20°C with 0.6 volumes of cold 2-

245

isopropanol. Recovery of the precipitated DNA by centrifugation at 10000 g for 15 min

246

at 4°C.

247

Step 6: Dilution of the DNA in 300 µL TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

248

Step 7: Extraction by adding an equal volume of neutral phenol and recovery of the

249

DNA layer by centrifugation at 13000 g for 15 min at 4°C.

250

Step 8: Repeat step 5

251

Step 9: Wash the pellet with a buffer of 76% ethanol and 10 mM NH4 Acetate for 5

252

min.

253

Step 10: Dry the pellet at room temperature and elute in 50 µL of TE. Keep sample at -

254

20°C until use.

255 256

The following modifications to the protocol were introduced:

257

- Modification 1: Introduction of an extra extraction with CIA (step 3) between steps 7

258

and 8.

259

- Modification 2: The neutral phenol extraction was skipped, so after step 5 protocol

260

continued directly with step 9.

261

- Modification 3: Addition of chloroform after Step 7. Different volumes of chloroform

262

were tested (50 µL, 100 µL, and 1 volume).

263 264

Development of an authentication system for varietal traceability of wine:

265

Trial 2.1: Varietal identification by SSR markers’ genotyping

266

Nuclear (nSSR) and chloroplast (cpSSR) microsatellite markers were tested for wine

267

varietal fingerprinting.


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Ten nSSR markers were analyzed: VVS2 9, VVMD5 and VVMD7

10

269

VVMD27, VVMD28 and VVMD32 34, VrZAG62 and VrZAG79 11, VMC1B1135 . The

270

forward primer of each pair was labelled with a fluorophore among 6-FAM, HEX or

271

NED. Single amplification reactions were carried out in a final volume of 50 µL

272

containing 10 ng of DNA template, 0.2 µM of each primer, 200 µM of each dNTP, 2.5

273

mM of MgCl2, 1x of reaction buffer and 2 Units of FastStart Taq DNA Polymerase

274

(Roche). Reactions were performed in a 96 wells Veriti® Thermal Cycler (Applied

275

Biosystems) using a hot start cycle of 4 min at 95°C, followed by 35 cycles of 30 sec at

276

95°C, 30 sec at 54°C, 40 sec at 72°C, and a final extension step of 10 min at 72°C. Two

277

other DNA Taq Polymerases were tested: Kapa2G Robust HotStart (Kapa Biosystems)

278

and AmpliTaq Gold (Applied Biosystems).

279

Eight mononucleotide microsatellite markers from chloroplasts were analyzed: NTCP8,

280

NTCP12, ccmp2, ccmp3, ccmp4, ccmp6, ccmp10 and ccSSR14

281

primer of each pair was labelled with a fluorophore. Single PCRs for each locus were

282

performed in 50 µL of total volume containing 1.5mM of MgCl2, 200 µM of each

283

dNTP, 0.5 µM of each primer, 1x of reaction buffer, 2 U of FastStart Taq DNA

284

Polymerase (Roche) and 5 µL of DNA (5-10 ng). Single amplification reactions were

285

carried out in a 96 wells Veriti® Thermal Cycler (Applied Biosystems) according to the

286

conditions described by 39, although the final extension cycle was prolonged for a total

287

of 60 minutes.

288

Genotyping analysis: The SSR amplification products were mixed with 9.5 µL of

289

HIDITM formamide and 0.2 µL of GENESCANTM500ROX size standard and run in an

290

ABIPRISM 3130xl Genetic Analyzer (Applied Biosystems). The microsatellite

291

fragments were sized using Genemapper® v.4.0 software (Applied Biosystems).

, VVMD25,

36,37,38

. The forward

292


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Trial 2.2: Varietal authentication by TaqMan SNP genotyping

294

SNP selection: Specific target SNPs for Sangiovese – the only permissible grape variety

295

for the Brunello wines - and for the cultivars allowed in the Lambruschi Modenesi

296

appellation (L. Sorbara, L. Salamino, Ancellotta, Fortana, L. Grasparossa and Malbo

297

Gentile) were identified within a dataset of 384 Illumina BeadArray SNPs previously

298

genotyped in 730 V. vinifera sativa and other 350 accessions of V. v. sylvestris and

299

grape hybrids of the FEM germplasm collection

300

chromosomal regions containing Sangiovese SNPs were designed with PrimerExpress®

301

software v.3.0 (Applied Biosystems) for further targeted resequencing (Table 9). The

302

correct design of the primers’ pairs was confirmed by PCR amplification of DNA from

303

Sangiovese young leaves, which was performed in 25 µl final reaction volume

304

containing 1x of reaction buffer, 200 µM of each dNTP, 0.2 µM of each primer, 1 U of

305

FastStart DNA Taq Polymerase (Roche) and 2 µl DNA template (5-10 ng). The

306

amplification programme consisted on one cycle at 95°C for 4 min, 35 cycles at 95°C

307

for 30 sec, 55°C for 30 sec and 72°C for 1 min, and a final cycle at 72°C for 7 min.

308

Results were visualized in a 1% agarose gel using FastRuler Middle Range DNA ladder

309

(Fermentas).

310

Resequencing: Amplifications using the forward (fw) and reverse (rw) primers

311

separately were performed. Since 6.5 ng of DNA was needed for each 200 bp to be

312

resequenced, PCR products were quantified by agarose gel electrophoresis and further

313

purified with ExoSap-IT (Affymetrix) following the manufacturers’ specifications, just

314

modifying the incubation time at 37°C to 45 min. The sequencing reaction was

315

performed in 10 µl of final volume containing 5.5 µl of purified and quantified PCR

316

product and 4.5 µl of sequencing mix (1µl of primer - fw or rw, 3.2 µM -, 2 µl of 5x

317

sequencing buffer, 1 µl of BigDye terminator v.3.1 and Milli-Q water till reaching the

32

. Primers for amplification of the


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318

final volume). The reaction was performed in a 96 wells Veriti® Thermal Cycler

319

(Applied Biosystems) according to the following conditions: one cycle of 2 min at 96°C

320

and 40 cycles of 10 sec at 96°C, 5sec at 50°C and 4 min at 60°C. After the addition of

321

16 µl of Milli-Q water and 64 µl of 96% chilled ethanol (-20°C) to each resequencing

322

reaction, they were incubated for 15 min at room temperature and then centrifuged at

323

4000 rpm for 30 min at 4°C. The supernatant was discarded. Next, the plate was

324

inverted and short spin at 400 rpm for 10 sec at 4°C to eliminate the remaining ethanol.

325

Another purification step was performed, but in this case 180 µl of 70% cold ethanol

326

(4°C) were added to the samples followed by centrifugation at 4000 rpm for 20 min at

327

4°C. The supernatant was discarded and the remaining ethanol was eliminated again as

328

described above. The plate was then kept for 2 hours at room temperature. Once the

329

samples were completely dry, DNA was resuspended in 20 µL of Milli-Q water

330

(incubation for 1-2-hours at room temperature) and shacked for 5 min. Finally 7 µl of

331

HIFITM formamide were added to 7 µl of the purified sequencing reaction, denatured for

332

5 min at 95°C, cooled on ice, spin and charged in an ABIPRISM 3130xl Genetic

333

Analyzer (Applied Biosystems). BioEdit software

334

uniqueness of the Illumina BeadArray data in Sangiovese cultivar. Then, for each

335

confirmed SNP, TaqMan probes and nested primers flanking the probes were designed

336

(Table 9).

337

TaqMan SNP genotyping assay. SNPs 98 and 222 were further analyzed by Real-Time

338

PCR using the specific TaqMan probes designed. The amplification reaction was

339

performed in a final volume of 15 µl containing 0.67 µM of each nested primer, 10 µl of

340

Master mix and 0.5 µl of each probe (100 nM). RT-PCR was performed in a

341

LightCycler 480 system (Roche) using a touch-down PCR program consisting on one

342

denaturation cycle (95°C for 10 min), 55 annealing cycles [95°C for 10 sec, 63°C for 15

40,41

was used to confirm the


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343

sec (-0.5°C per cycle till 60°C), 72°C for 5 sec], and one final cooling cycle (40°C for

344

30 sec). Experimental mixtures of genomic DNA of Sangiovese were performed (100%

345

of Sangiovese gDNA, 95%, 90%, 80%, 70%, 50%, 30%, 20%, 10%, 5%, 0%

346

Sangiovese and 100% other variety) in order to evaluate the discrimination power of the

347

method for quantification purposes. Three replicates per sample were always analyzed.

348

Results were subjected to endpoint analysis.

349 350

RESULTS AND DISCUSSION

351

Improvement of DNA isolation

352

Genetic varietal authentication of wine through DNA fingerprinting, unlike analysis

353

from raw plant material, presents some limitations due to the difficulty of extracting

354

high quality grape DNA when analyzing a complex and heterogeneous matrix such as

355

wine

356

different published DNA extraction protocols from must and wine were evaluated, some

357

modifications were also introduced. As wine is the fully processed final product, in

358

order to detect the steps in the winemaking process where the biggest DNA loss is

359

produced, we first evaluated the ratios of absorbance 260/280 and 230/280 of DNA

360

extracts obtained from musts at three points of the fermentation process (trial 1.1), then

361

from musts at intermediate steps in the winemaking process (trial 1.2 and 1.3) and

362

finally from finished wines (trials 1.4 – 1.7).

19-26

. With the aim of improving this limiting step for genetic wine traceability,

363 364

DNA extraction from must

365

Different protocols have been previously optimized and successfully applied for DNA

366

isolation from must

367

reduced. Because of that, and in order to develop a standard final method that will allow

18-20

but, when applied to wine, the efficiency was drastically


16

Page 17 of 47


368

the wine genetic traceability at all stages of the winemaking process, we chose the first

369

CTAB-based method optimized for wine DNA isolation by

370

extracting nucleic acids also from must. In all trials, samples from decanted and

371

homogenized must were analyzed. Based on absorbance values at 260 nm, DNA was

372

mostly extracted from the homogenized samples (containing solids in suspension),

373

while almost no DNA was isolated from the must aqueous phase (decanted samples;

374

Fig. 1; Tables 1-2). These results agree with those obtained by

375

the solid part, mainly constituted by cellular traces, is the main source of DNA in musts.

23

as the starting point for

19-20

and evidenced that

376 377

Trial 1.1: Total DNA extraction from must at three different stages of fermentation.

378

DNA extraction was performed from L. Grasparossa must at pre-, middle- and end of

379

fermentation stages with the aim of study the evolution of total DNA concentration

380

during the process. Sample collected at the end of fermentation was also subjected to

381

further fining treatments before analysis. Total DNA concentration increased until the

382

middle of the fermentation and then decreased dramatically at the end of the process

383

(Fig. 1). During fermentation yeasts multiply, so, on one hand the increase of total DNA

384

it is likely due to yeast DNA increase. On the other hand, at the end of fermentation the

385

DNA concentration was even lower than that at the beginning of the process. Grape

386

DNA is degraded by yeast DNases during fermentation, and it also occurs the death of

387

the yeasts (and therefore their DNA degradation as well) due to the presence of the

388

waste toxic products generated by themselves (e.g. ethanol), so at the end of the process

389

it only remains residual DNA.

390

concentration during the fermentation process. These authors demonstrated that the

391

must solid fraction also decreased during fermentation because of structural changes of

19

observed the same kinetic regarding total DNA


17


Page 18 of 47

392

the tissue integrity. In fact, no big differences regarding DNA concentration were

393

observed between decanted and homogenized samples at the end of the process (Fig. 1).

394

DNA quantification by UV spectrometry may be confounded by various contamination

395

and may overestimate DNA concentration. In this study the quantity of DNA derived

396

from absorbance at 260 nm was intended as a rough indicator for comparing the effect

397

of different procedures on the same or similar samples. Likewise, the quality/purity of

398

DNA extracts was based on commonly used absorbance ratios. For samples of good

399

quality 260/280 and 260/230 values are expected to lie in the range of 1.8 – 2 and 2 –

400

2.2 respectively.

401

For homogenized samples, acceptable 260/280 values were obtained at pre- and middle

402

of fermentation, while they were a bit low at the end of the process which may indicate

403

protein contamination, traces of phenol or alcohols in the sample. In the case of 260/230

404

ratio in all three stages the values determined were much lower than expected which

405

may indicate contamination of polysaccharides, salts and/or organic compounds,

406

especially at the end of fermentation. In this last step, the low absorbance’s ratios were

407

probably mainly due to the loss of DNA quantity, rather than for an increment of

408

contaminants. The addition of fining agents after fermentation might also be one of the

409

causes of the reduction of DNA content.

410

The presence of grape DNA in the total DNA extracted from the homogenized samples

411

was confirmed by amplification of VrZAG79 marker, which is one of the microsatellite

412

markers used for grapevine varietal identification13. Expected allelic sizes (see Table 6

413

for reference genetic profiles) were obtained for all samples at pre- and middle of

414

fermentation stages, while for samples collected after fermentation usually erratic peaks

415

were observed in addition to the true alleles, making confusing the interpretation of the


18

Page 19 of 47


19

416

results.

also found that amplification at the end of the fermentation process was

417

weaker and, in some cases, not successful, because of scarce DNA extraction.

418 419

Trial 1.2: DNA extraction at intermediate steps of the winemaking chain.

420

With the aim of determining the effect on DNA isolation efficiency and SSR

421

genotyping analysis of the different treatments carried out in a wine cellar during the

422

winemaking process - such as addition SO2, clarification and/or other fining treatments -

423

DNA extraction was further evaluated in samples collected at intermediate steps of the

424

chain in Lambruschi Modenesi DOP and Trento DOC (Tables 1-2).

425

Concerning DNA isolation efficiency, no clear effect of floatation was observed, since it

426

produced a decrease of DNA concentration in L. Sorbara must, while no decrease was

427

observed in L. Grasparossa one after the treatment. Draining of L. Sorbara must before

428

fermentation greatly reduced the concentration of DNA. Fining treatments after the end

429

of fermentation such as centrifugation, filtration with perlites and cold stabilization

430

dramatically reduced the DNA yield and, therefore, the quality ratios of absorbance

431

became worst. However, the absence of skin contact before fermentation did not

432

negatively influence the DNA extraction yield. This is particularly important in the case

433

of vinification of white wines, for which maceration with grapes’ skin is not performed,

434

or is very reduced. Transport of L. Grasparossa must for about 30 min. in a tanker, and

435

then refrigerated at destination while waiting for fermentation, also reduced the total

436

DNA available in the must, which suggests that DNA degradation occurred (Table 1).

437

With regard to SSR genotyping analysis, all nSSR markers successfully amplified in the

438

analyzed commercial must samples of Lambrusco (L. Grasparossa and L. Sorbara)

439

before fermentation, even if stabilised with K2S2O5. In all cases the SSR genotypes

440

obtained in musts perfectly matched with the reference ones. These results agree with


19


Page 20 of 47

441

those obtained for must in previous works 19,20,31. However, amplification did not occur

442

in all replicates and for some markers (VVS2, VVMD5, VVMD27 and VVMD7) in

443

Lambrusco musts subjected to a floatation treatment before the start of fermentation,

444

and for the markers for which amplification was obtained, wrong allele sizes compared

445

to the expected ones reported in Table 6 or allele dropout phenomenon (one allele is

446

missing) were observed. After must draining no reproducible and reliable results were

447

obtained among replicates neither. For Lambrusco musts analyzed at the end of the

448

fermentation process no amplification was observed.

449

In Trento DOC musts, clarification, filtration and decantation drastically reduced the

450

extracted DNA yield and, consequently, the absorbance ratios as well. However, in the

451

case of Chardonnay samples CR1272 and CR1273 (Table 2) a moderate DNA

452

concentration was still available after clarification. Pressing did not have a clear effect

453

(Table 2). Complete nSSR genotypes matching the reference plant DNA were obtained

454

when analyzing grape juices, sulphited musts, or must after draining, but for must

455

samples after treatments such as filtration, clarification or decantation, which drastically

456

reduced the amount of DNA available, no reliable and reproducible results were

457

obtained since amplification did not work or there were allelic dropouts or additional

458

peaks of wrong sizes. Only reproducible and reliable results were obtained for the

459

clarified Chardonnay must for which a moderate concentration of total DNA could be

460

isolated (samples CR1273). These results contrast with those obtained by

461

analyzed Moscato d’Asti musts after static clarification or floatation getting the

462

successful profile for the 9 nSSRs analyzed, even if samples presented low DNA

463

concentration (5 – 10 ng/µl).

464

Must and wine stabilization by addition of SO2 under the form of K2S2O5 is a common

465

practice in wineries to avoid or stop the fermentation of samples collected at certain


28

, who

20

Page 21 of 47


466

steps of the process in order to perform additional chemical analysis. According to our

467

results, musts’ stabilization by K2S2O5 did not have any negative effect on DNA

468

isolation (Tables 1-2) and did not interfere with the DNA polymerase used for SSR

469

amplification by PCR. This result confirms what observed first by

470

stabilized samples by addition of SO2 obtaining successful SSR amplification results,

471

and later by 21, who studied this effect by analyzing stabilized samples with K2S2O5 and

472

not stabilized controls, observing no qualitative and quantitative differences of the

473

extracted DNA and successful SSR genotyping in both cases. This result is particularly

474

interesting, since it allows the oenologist to stabilize the samples taken at certain steps

475

of fermentation not only for performing chemical controls, but also to store and keep

476

them for further genetic analysis for authentication purposes.

19,20

, who analyzed

477 478

Trial 1.3: Introduction of modifications to the total DNA extraction protocol from musts

479

A treatment with proteinase K was introduced to the original protocol of 23 with the aim

480

of better purifying DNA from proteins. This enzyme is active in the presence of

481

chemicals that denature proteins (SDS, urea) and of chelating agents such as EDTA,

482

and, besides degrading proteins, it also effectively inactivates DNases

483

to the results obtained (Table 3), in general a greater DNA extraction yield was

484

achieved with proteinase K treatment, but no big effect was observed in the 260/280

485

ratio, while for 260/230 ratio ues were obtained for SM-K method. Another

486

modification introduced in order to improve the DNA purity was the performance of a

487

subsequent purification with NucleoSpin CleanUp kit (Macherey-Nagel). In this case a

488

great DNA loss was observed, and DNA purity was not improved (Table 3). PCR

489

amplification of purified samples was not always successful or results were not

490

consistent with the expected varietal SSR profiles.


42,43

. According

21


Page 22 of 47

491 492

DNA extraction from wine

493

Trial 1.4: Comparison of wine DNA extraction methods.

494

26

495

commercial kit - DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and other five

496

already published methods

497

one

498

(absorbance 260/280 and 260/230 ratios around 1.7). Later,

499

methods for wine varietal DNA extraction: TEPC (Tris-EDTA-CTAB-PVPP) and

500

QDEK (modified Qiagen DNeasy Kit), and evaluated them, through the RT-PCR

501

quantification method developed by 23, obtaining more grape DNA with the first one. In

502

this study we tested all three methods, Pereira 26 TEPC and QDEK 27 , together with 23,

503

to evaluate the one with a best performance. The results obtained are summarized in

504

Table 4. Among the two methods proposed by 27, a higher total DNA concentration was

505

obtained with QDEK one. Anyway, despite 260/280 was usually better, DNA quantity

506

isolated from both QDEK and TEPC was very scarce, if not absent, compared to that

507

obtained with 26 and 23 (Table 4; Supporting Information, Fig. S1). It is remarkable that

508

in the case of Lambrusco commercial wines, the pellet was absent when samples were

509

concentrated before DNA extraction, while a big pellet was observed for Brunello 2008

510

commercial wine. This could be due to the differences in the vinification process of

511

both types of wine. For elaboration of Lambrusco wine, maceration of the must with

512

grapes’ skin usually occurs before the fermentation process for a reduced period of time,

513

while in the case of Brunello wines, grapes’ skin is in contact with must during the

514

whole fermentation process such that the content on solids in suspension in the wine

developed a new protocol for DNA isolation from wine and compared it with a

26

22-24,30,44

. Among the traditional extraction methods, Pereira

showed the best performance regarding DNA yield and quality values


27

published two other

22

Page 23 of 47


515

will be higher. In fact, a much higher DNA concentration was obtained from Brunello

516

than from Lambrusco wine (Table 4).

517

All the classical DNA extraction methods discussed above are long and laborious;

518

therefore, with the aim of reducing the DNA isolation time, 11 commercial kits were

519

tested following the manufacturers’ indications. Practically no DNA was isolated with

520

none of the tested kits (DNA concentrations lower than 12 ng/µl - data not shown for all

521

kits) and absorbance ratios were very low (Table 4). Acceptable 260/280 ratios were

522

generally obtained only for High Pure PCR template preparation kit (Roche) and Power

523

Soil DNA isolation kit (MO-BIO), so additional steps were introduced into these kits’

524

protocol with the aim of improving their performance. Modifications consisted on the

525

addition of α-amylase for Roche kit – enzyme which breaks down long-chain

526

polysaccharides - and addition of both α-amylase and proteinase K treatments for MO-

527

BIO kit. These modifications carried to a higher concentration of isolated DNA less

528

contaminated by proteins but likely contaminated by polysaccharides and organic

529

compounds based on absorbance ratios.

530

with DNeasy Plant Mini Kit.

531

Amplification of VrZAG79 marker was performed to confirm the presence of varietal

532

DNA. For DNA samples obtained by

533

same PCR conditions for SSR amplification used by these authors. These authors

534

reported good amplification results for four of the seven wines they analyzed for this

535

SSR marker, among them Sangiovese wine for the TEPC method, but not for the QDEK

536

one. In our study, unlike 27,45, amplification products could be observed sometimes only

537

for QDEK method, but not for the exact expected sizes. In addition, the fluorescence

538

intensity was very low, which made more difficult the interpretation of the results. Low

539

reproducibility was also observed and usually no amplification was obtained with the

27

26

got no results neither when extracting DNA

methods - QDEK and TEPC - we applied the


23


Page 24 of 47

540

other nSSRs. Sangiovese 2010 experimental wine and Brunello 2008 and Lambrusco

541

commercial wines, extracted with Pereira method

542

marker. Alleles of the expected size were observed in some cases, but results were not

543

reproducible, usually no amplification was observed. This result contrasts with the

544

results obtained by

545

amplification for commercial wines. When the rest of nSSR markers were analyzed no

546

amplification was observed. Amplification of grape DNA was also tested on samples

547

extracted with Roche and MO-BIO kits, but no amplification was obtained.

548

26

549

contamination of the DNA extracts with this solvent (which absorbs at 270 nm) was

550

overcome by further purification with 2-propanol and a later washing with 76% ethanol

551

supplemented with 10mM NH4 acetate. However, in all repetitions performed in this

552

study, with this same extraction method, the phenol contamination was always present.

553

In fact, DNA absorbance peak at 260 nm was hidden each time by a more intense

554

absorbance peak at 270 nm. None of the methods tested overcame this contamination

555

problem (Supporting Information, Fig. S1).

556

We chose the UV-Spectrophotometer NanoDrop 8000 method for total DNA

557

quantification because it allowed us to evaluate the purity of the extracts through the

558

absorbance ratios. Nevertheless, we further analyzed several samples with a

559

Fluorospectrometer NanoDrop 3300, which selectively determines double-stranded

560

DNA by using PicoGreen® dye. Though we report a poor relationship between DNA

561

amounts estimated by the two methods, a proportion of dsDNA can be consistently

562

derived in preparations from modified commercial kits (Supporting Information, Tab.

563

S1). Grapevine DNA quantification by RT-PCR using SYBRGreen dye was also tested,

564

but no reliable results were obtained (data not shown).

26

, were also analyzed for this same

26

, while it is in agreement with

21

, who obtained no SSR

reported that, in spite of using phenol in their protocol (a PCR inhibitor),


24

Page 25 of 47


565

Trial 1.5: Effect of concentration and purification treatments before and after DNA

566

isolation from wine, respectively.

567

19

568

extraction performance itself. In this study, Savazzini and Martinelli modified DNA

569

isolation method was performed applying as concentration treatments salts precipitation

570

- as described in these authors’ protocol

571

Concentration with 3kDa and 10kDa Millipore filters were also tested, but the DNA

572

isolates were brown pigmented in all repetitions and quantification with UV-

573

Spectrophotometer NanoDrop 8000 was not possible since negative values of the

574

260/230 ratio were always obtained, despite the samples were highly diluted. It might

575

be due to an excess of contaminants or may be some of these pigmented compounds’

576

absorbance overlaps giving no reliable quantification results (data not shown). Anyway,

577

the best performance for this extraction method was observed for precipitation with

578

NaCl treatment, although the best purity ratios were given by ethanol precipitation. No

579

big differences were observed regarding DNA yield between Na acetate precipitation,

580

Sep-Pak and ethanol precipitation (Fig. 2A). Sample concentration with NaCl, Na

581

acetate and 2-propanol treatments had already been tested before by

582

observed big differences among them; these authors only reported that salt precipitation

583

gave higher efficiency as number of samples extracted and quantified, while 2-propanol

584

precipitation gave higher DNA concentration. As our main interest was to isolate

585

enough DNA from wine, 2-propanol precipitation treatment was included together with

586

Millipore filters, ethanol precipitation and Sep-Pak sample concentration approaches,

587

but in this case in combination with Pereira wine DNA extraction method

588

case, except Sep-Pak, all other treatments applied allowed the isolation of moderate

589

concentrations of total wine DNA. In overall, higher DNA yield was observed for

hypothesized that concentration treatments before DNA extraction could improve the

23

-, ethanol precipitation and Sep-Pak.


23

, who did not

26

. In this

25


Page 26 of 47

590

26

591

samples tested with ethanol precipitation presented again less contamination, although

592

the DNA purity was not optimal in any case (Fig. 2A, 2B).

593

for concentrate the wine samples before DNA isolation (concentration by centrifugation

594

of the wine solid phase, 2-propanol precipitation, dialysis, concentration under low

595

pressure and by lyophilisation) but they concluded that these treatments did not improve

596

the SSR analysis from wine, likely because these methods were not selective and

597

concentrate also contaminants. Other authors used lyophilisation as wine concentration

598

method or precipitation with 2-propanol supplemented with 0.3 M of Na acetate 24,25,27.

599

We also tested lyophilisation of the pellet obtained after just wine sample centrifugation

600

or after centrifugation after a concentration treatment, but no differences were observed

601

on DNA yield extraction between lyophilised or just centrifuged samples (data not

602

shown).

603

Further purification performed with NucleoSpin CleanUp kit after wine DNA extraction

604

according to

605

the absorbance ratios (260/280 and 260/230). 20 obtained also a very low DNA yield (10

606

– 25 ng/µl in musts and < 1 ng/µl in wines) after purifying the DNA extracts with

607

DNeasy Plant Mini kit. The purification method described by

608

carried to a ten-fold loss of DNA and its purity did not improve anyway, since phenol is

609

again used in this purification method increasing the contamination risk.

method than for the previous one. Concerning its purity and according to the ratios,

26

21

applied some treatments

supposed the almost completely lost of DNA without improvement of

33

was also tested. It

610 611

Trial 1.6: Effect of wine aging on DNA isolation.

612

Wine is a dynamic matrix, that is to say that the compounds that compose it, such as

613

sugars, acids, polyphenols, do not remain stationary over time, but can experience

614

chemical changes that will alter the aroma, colour, mouth feel and taste of a wine,


26

Page 27 of 47


615

making it more balanced, finesse and rounded. Due to this dynamicity, some types of

616

wines are subjected to aging, since this process can potentially improve their quality.

617

However, during aging further degradation of the residual DNA present in wine also

618

occurs, as it was appreciated by

619

Chardonnay wines of 2002 than from Chardonnay vintages 2004 and 2005. (25) also

620

observed that in experimental wines the concentration of DNA - extracted after 6, 12

621

and 18 months after alcoholic fermentation - decreased, being practically absent in

622

commercial aged wines (yields < 4 ng/µL and < 2 ng/µL in 12 and 18 months aged

623

wines, respectively). The results obtained in this study from the analysis of Sangiovese

624

experimental wines and Brunello from different vintages also illustrate this fact

625

(Supporting Information, Fig. S2).

24

, which isolated lower amount of DNA from

626 627

Trial 1.7: Introduction of modifications into Pereira et al. (2011) extraction method to

628

improve its performance.

629

Modifications introduced to Pereira DNA extraction protocol

630

purpose of reducing the phenol contamination, since the presence of this compound,

631

inhibitor of the PCR amplification, was the main problem that we reported when using

632

this method (Supporting Information, Fig. S1). According to the results obtained (Table

633

5), the step with phenol extraction cannot be eliminated because it drastically reduced

634

the amount of DNA isolated, as pointed by

635

extraction step was introduced after phenol extraction (modification 1), which should

636

have eliminated residual phenol. However, results showed that CIA purification did not

637

improve the amount of DNA isolated nor its purity, moreover, the introduction of the

638

CIA step drastically reduced the DNA yield, even more than when phenol step was

639

eliminated. Finally, the use of chloroform (modification 3) should have removed any

26

were applied with the

26

. A chloroform: isoamilic alcohol (CIA)


27


Page 28 of 47

46

640

lingering traces of phenol

, but this treatment was not successful; in fact the use of

641

high volumes of chloroform decreased the yield.

642 643

Development of an authentication system for varietal traceability of wine

644

Due to their economical importance, Lambrusco and Sangiovese Italian wines were

645

used for the application and development of genetic methods for wine authentication.

646

“Lambruschi” are the most famous sparkling red wines in the world. Their production is

647

subjected to the rules of DOP (Protected Designation of Origin) which covers

648

agricultural products and foodstuffs that are produced, processed and prepared in a

649

given geographical area using recognised know-how. Lambrusco di Sorbara, L.

650

Salamino, L. Grasparossa, Ancellotta, Fortana and Malbo gentile are the grapevine

651

cultivars from which the Lambruschi Modenesi DOP wines can be obtained

652

case of Sangiovese, wines endorsed by Brunello di Montalcino DOCG must be

653

elaborated 100% with this variety according to the designation regulations

654

obtained from the grapevines Sauvignon blanc, Riesling Renano, Chardonnay, Petit

655

Verdot, Teroldego, Tannat, Cannaiolo, Montepulciano, Ciliegiolo, Primitivo, Cabernet

656

Sauvignon, Merlot and Tempranillo were also included in our study as control cultivars

657

for the Sangiovese wine authentication system.

47

. In the

48

. DNAs

658 659

Trial 2.1: Varietal identification by SSR markers’ genotyping

660

Since fingerprinting by SSR markers has been widely used for varietal identification in

661

grapevine 13,17, this approach was tested for wine samples. Both nuclear and chloroplast

662

microsatellite markers were used.

663

Nuclear SSRs. Microsatellite profiles of reference were obtained from leaf DNA of

664

true-to-type accessions of each cultivar and were used as controls for comparison with


28

Page 29 of 47


665

the genotyping results obtained from must and wine (Table 6). SSR genotyping protocol

666

was optimized for the analysis of pre-fermented musts, musts at intermediate stages of

667

the fermentation process and wines. From all the Taq DNA polymerase enzymes tested,

668

the best performance was obtained with the FastStart Taq DNA polymerase (Roche).

669

When nSSR markers were combined in multiplex PCRs not reproducible and reliable

670

results were obtained (data not shown), so each locus was individually analyzed. The

671

marker with the best performance and highest reproducibility was VrZAG79, whose

672

amplification occurred even when none of the other markers did. Because of that this

673

marker was selected to confirm the presence of grape DNA in wine DNA isolates. For

674

markers VVS2, VVMD5 and VVMD27 usually no amplification was observed. Results

675

of the nSSR genotyping have already been discussed for each trial (see above).

676

Chloroplast SSR markers. Chloroplast DNA (cpDNA) presents some advantages in

677

relation to the nuclear genome (nDNA) for traceability purposes: i) it is more abundant,

678

since thousands of copies are present per cell, ii) it is more resistant to exonucleases due

679

to its circular form and iii) it presents more resistance against degradation because it is

680

contained in organelles with a double membrane. However, cpDNA has a low mutation

681

rate and is inherited from a single parent, thus it is not adapted for the discrimination of

682

all grapevine cultivars. Since residual varietal DNA in wine is quite degraded,

683

chloroplast SSR loci (cpSSR) were proposed as an alternative to nSSRs for wine

684

authentication because of their higher stability 22. We analyzed eight cpSSRs in order to

685

check if wine authentication could be better achieved by using these type of markers.

686

DNA from young leaves was first analyzed in order to get the microsatellite profile and

687

haplotypes of the different varieties investigated (Table 7). Four cpSSRs showed no

688

polymorphism (ccmp2, ccmp4, ccmp6 and NTcp12); while ccmp10 and ccSSR14 were

689

the most polymorphic ones with three alleles each one. These results agree with


29


28

Page 30 of 47

690

previous works

. Four haplotypes (chlorotypes) were discriminated, all of them

691

present in the set of varieties applied for Sangiovese authentication, while only

692

haplotypes A and D were found among Lambrusco cultivars. Only Teroldego cultivar

693

presented the haplotype B.

694

28

695

ccmp10, NTcp8, NTcp12 and ccSSR14) obtaining a unique haplotype for Moscato

696

bianco respect to the other nine possible varieties used as fraudulent contaminants for

697

the elaboration of Asti Spumante wines. These authors could amplify all cpSSRs in

698

musts, musts after floatation before fermentation and in commercial wines. In our case,

699

for Lambrusco wine, the set of cpSSRs used did not allow discrimination among

700

Lambrusco cultivars and no haplotypes specific of Lambruschi were detected neither

701

(Table 7). L. Grasparossa and L. Sorbara musts were analyzed with the four

702

polymorphic cpSSRs, as well as a commercial wine of L. Grasparossa and musts of this

703

same cultivar collected in different stages of the fermentation process (after floatation

704

before fermentation, in the middle of the process, at the end of fermentation and at the

705

end of fermentation after further fining treatments). Successful amplification of the

706

expected allele sizes was obtained for musts before fermentation, musts after floatation

707

before fermentation and also must collected in the middle of fermentation. However,

708

unlike 28, no reliable and conclusive results were obtained in the case of musts collected

709

at the end of fermentation or in the commercial wine, for which amplification did not

710

occur for all markers and/or wrong fragment sizes were observed.

711

In the case of Sangiovese, experimental and commercial wines were analyzed.

712

Amplification was successful on the experimental Sangiovese wine for the four cpSSRs

713

analyzed, although for ccSSR14 an additional unspecific peak could be observed. With

714

regard to the commercial wine of Brunello di Montalcino DOCG, amplification not

applied a set of 7 cpSSR loci (five of them in common with this study: ccmp3,


30

Page 31 of 47


715

always occurred and in some cases the results interpretation was difficult because weak

716

signal and extra peaks (Supporting Information, Fig. S3). In any case, the most reliable

717

cpSSR was always ccmp3, which showed the best performance.

718

Although better amplification results were obtained for cpSSRs than for nSSRs, their

719

use as a standardize method for authentication purposes is not feasible because of their

720

low level of polymorphism. Only rare cpSSR haplotypes may be useful as they can help

721

to efficiently discriminate some varieties.

722 723

Trial 2.2: Varietal authentication by TaqMan SNP genotyping

724

Single nucleotide polymorphism markers (SNPs) were chosen to design an alternative

725

method to SSR genotyping for wine authentication because, first, they can be detected

726

in low quality fragmented DNA and, second, they can be potentially applied for the

727

relative quantification of the several cultivars that may have been used for blended

728

wines or frauds. As the method is based on the analysis of cultivar specific SNPs, the

729

technique has to be optimized for each variety target for authentication. We optimized

730

this method for the authentication of 100% Sangiovese wines from Brunello di

731

Montalcino DOCG.

732

Three candidate SNPs unique of Sangiovese were selected among 1080 diverse

733

genotypes constituting the FEM grapevine germplasm collection previously

734

characterized for a set of 384 SNPs

735

each SNP further confirmed their uniqueness for Sangiovese among the set of cultivars

736

compared (Table 8). Sangiovese presented allele C in homozygosis for all three SNPs,

737

while the other cultivars were homozygous for the alternative allele or heterozygous;

738

they could be detected in eventual fraud by targeting the specific allele. Further

739

experimental verification was performed for SNPs 98 and 222. The TaqMan SNP

32

. The resequenced genomic regions containing


31


Page 32 of 47

740

genotyping assay was first tested with plant genomic DNA (gDNA) from true-to-type

741

accessions. The allele specific TaqMan probes allowed discrimination of Sangiovese

742

cultivar among all the other ones for both SNPs (Fig. 3). This method also allowed a

743

good discrimination among leaf DNA extracts mixtures in which the gDNA proportion

744

differed of at least 10%, and in some cases of 5% (Fig. 4).

745

limit of 10% (v/v) when leaf DNA extracts were mixed, not being possible the detection

746

through SSR genotyping of a variety mixed in a proportion below. VIC dye channel

747

showed the decrease of Sangiovese DNA proportion in each solution, while 6-FAM dye

748

channel showed the increment of the other cultivar (Supporting Information, Figure S4).

749

This method is more sensitive and precise for quantitative purposes than the methods

750

reported till now for relative quantification of each variety in a mixture 22,31 , because is

751

based on specific allele probes. The other methods, instead, were based on the

752

relationship between the proportion of each variety and the signal intensity of the SSR

753

alleles given by fluorescence or by densitometry, respectively, but the presence of

754

overlapping SSR alleles of the varieties mixed was a limitation of these approaches. In

755

addition, they are also influenced by the amplification efficiency of each allele and

756

when the proportion of one of the varieties is low, one of the alleles could be not

757

amplified.

758

(alleles) with the relative proportion of each variety.

759

When the method was applied to Sangiovese 2010 wine samples, no successful results

760

were obtained because there was no DNA amplification due to scarce yield and quality

761

of the DNA isolated from wine matrices (Fig. 5).

762

The SNPs detected by the Illumina BeadArray tecnology as unique for each Lambrusco

763

cultivar are shown in Supporting Information, Tab. S2. They will be experimentally

764

confirmed and tested for Lambrusco varietal authentication in a further work.

26,45

19

also reported a detection

reported the impossibility of associating the intensity of the peaks


32

Page 33 of 47


Acknowledgement We are grateful to Federica Camin, Roberto Larcher, Fulvio Mattivi, Giorgio Nicolini, Panagiotis Arapitsas and Tomas Roman Villegas for providing must and wine samples. We also like to thank Maddalena Sordo and Francesco Emanuelli for valuable technical suggestions and Anna Schneider for grapevine cultivars identification.

Supporting Information: Absorbance plots of DNA extracts obtained from wine using different DNA isolation methods; Residual wine DNA concentration obtained from wines of three vintage years; Wine DNA quantification based on UV absorbance and fluorescent (PicoGreen) measurements; Electropherograms of chloroplast DNA markers from Sangiovese wines and leaves; RT-PCR plot of Sangiovese (CC) and Canaiolo (GG) with SNP98 TaqMan probes; SNPs unique for each grapevine cultivar allowed in the Lambruschi Modenesi appellation.

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10471.

Financial support of the work: This study was funded by Consorzio del Vino Brunello di Montalcino and by AGER Agroalimentare e Ricerca Contract N. 2011-0285.

FIGURE CAPTIONS

Figure 1. Absorbance-based DNA concentration and purity ratios of total DNA extracted from 2 mL of Lambrusco Grasparossa musts at three different fermentation stages.

Figure 2. Absorbance-based DNA concentration and purity ratios of samples obtained by using five different concentration treatments before DNA extraction and two different purification methods afterwards. A) DNA isolated using the method Savazzini and Martinelli (2006); B) DNA isolated using the method Pereira et al (2011).

Figure 3. Endpoint genotyping results. Scatter plot (A) and Fluorescence emitted by the TaqMan probe tagged with VIC dye (allele C) for SNP 98 (B). Two replicates per sample are shown.

Figure 4. Fluorescence emitted for SNP 98 (A) and SNP 222 (B ) by the TaqMan probe tagged with VIC dye for samples containing different proportions of Sangiovese genomic DNA. Two replicates per sample are shown.

Figure 5. RT-PCR results when performing TaqMan SNP genotyping for SNP 222 with wine samples. Genomic DNA of Sangiovese (positive control) in green color.


38

Page 39 of 47


TABLES Table 1. Absorbance-based DNA concentration and purity ratios of Lambrusco samples collected at different stages of the winemaking process. decanted sample

homogenized sample

varietya LS

ng/µl

A260/A280

A260/A230

ng/µl

A260/A280

A260/A230

CM1a

first silo of Lambrusco Sorbara grape juice, added with SO2

129,97

1,99

1,42

-

-

-

CM1b

LS

second silo of Lambrusco Sorbara grape juice, added with SO2

601,83

1,68

0,82

-

-

-

CM2ab

LS

mixing of CM1a and CM1b samples, after flotation process and starting fermentation

38,95

2,2

2,71

-

-

-

CM3ab

LS

sample code

step in the winemaking process

fermented juice

233,9

2,33

2,68

-

-

-

CM4ab

LS

fermented juice after centrifugation

46,28

1,87

0,88

103,94

1,94

1,21

CM5ab

LS

sample after low temperature stabilization, with addition of PAG_CM1c

11,4

1,17

0,26

18,3

1,53

0,47

PB1a

LS

grape juice coming from the first silo after filtration

PB1b

LS

grape juice coming from the second silo after filtration

PB2ab b PB3ab

LS

mixing of products PB1a and PB1b at the end of the fermentation process

4,02

0,55

0,07

144,4

10,33

0,99

0,21

151,43

1,92

0,95

5,13

0,7

0,09

180,7

2,03

1,89

1,48

0,86

LS

fermented sample after SO2 addition

CM1d

LG

first silo of Lambrusco Grasparossa grape juice, added with SO2

CM1e

LG

second silo of Lambrusco Grasparossa grape juice, added with SO2

103,4

2,08

1,41

CM2de

LG

mixing of CM1d and CM1e samples, after flotation process and starting fermentation

223,1

2,12

1,62

CM3de b CM4cde

LG

fermented juice

62,87

1,75

0,79

1986,5

LG


41,82

1,19

0,53

19,81

1,3

0,4

CM1f

LG

third silo of Lambrusco Grasparossa grape juice, added with SO2

158,7

2,16

1,71

728,8

2,13

2,14

CM1g

LG

3,03

0,52

0,07

6,33

0,08

0,13

75,64

1,93

1,09

2450,5

2,04

1,71

670,1

2,1

1,96

1920,5

2,14

1,94

1,77

0,8

fourth silo of Lambrusco Grasparossa grape juice, added with SO2

122,65

2,1

1,46

1467,5

2,14

2,23

CM2fg

LG

CM1f + CM1g without skins at start of fermentation

237,05

2,07

1,64

1892,5

2,09

2,11

CM3fg

LG

fermented juice

-

-

-

110,48

1,81

1,09

CM4fg

LG


26,57

1,29

0,51

20,64

1,46

0,41

CM5cdefg

LG

Must 1

Must 2

CM4cde + CM4fg

22,71

1,31

0,4

48,09

1,74

0,68

LG

must before filtration

205,25

2,14

1,86

1692,5

2,2

2,41

LG

must after filtration with perlites

9,06

1,11

0,22

11,47

1,28

0,25

LG

end of fermentation and after centrifugation

6,78

1,04

0,18

15,09

1,3

0,28

LG


6,54

1,05

0,17

88,6

1,91

1,14

LG

Must 3


10,83

1,09

0,28

LG


10,25

1,06

0,25

8,35

1,14

0,24

LG


254

2,1

1,41

1205

2,16

2,12

LG

a


6,57

0,92

6,49

1,11

0,21

0,09

5,44

0,86

0,09

LG

must to be fermented

101,86

1,86

0,75

169,65

2,09

1,15

LG

end of fermentation, during cold stabilization

172,43

1,51

0,78

37,61

1,85

0,57

LG


7,03

1

0,15

13,04

0,77

0,28

LS = Lambrusco di Sorbara; LG = Lambrusco Grasparossa

b

Not stabilized with addition of

c

concentrated must obtained from Sorbara grapes

Table 2. Absorbance-based DNA concentration and purity ratios of Trento DOC samples collected at different stages of the winemaking process. decanted sample varietya

homogenized sample A260/A280

A260/A230

CR1272

CH

grape juice

30,71

1,49

0,35

255

1,77

0,95

CR1273

CH

clarified must

13,25

1,49

0,62

101,56

1,59

0,75

CR1274

CH

grape juice

13,78

1,78

0,62

238,55

1,7

0,83

CR1275

CH

sulphited must

17,13

1,36

0,36

249,1

1,87

1,11

CR1276

CH

sulphited and filtered must

51,09

1,21

0,44

14,27

1,18

0,24

CR1277

CH

grape juice

32,11

1,49

0,53

500,05

1,8

1,04

CR1278

CH

sulphited grape juice

8,34

0,77

0,11

167,45

1,83

0,97

CR1279

CH

sulphited and clarified must

5,58

0,69

0,1

32,73

1,43

0,43

CR1280

PN

drained must

12,83

1,65

0,39

340,25

1,85

1,11

CR1281

PN

clarified must

17,18

1,38

0,42

40,71

1,59

0,5

CR1282

CH

drained must

74,87

1,52

0,88

318,6

1,71

0,93

CR1283

CH

clarified must

8,39

1,28

0,26

37,9

1,81

0,7

CR1284

CH

drained must

6,23

1,11

0,18

241,9

1,79

1,05

CR1285

CH

clarified must

32,31

1,41

0,51

31

1,43

0,39

CR1286

CH

decanted must

2,83

0,81

0,1

8,03

1,22

0,23

CR1287

CH

inoculated must

6,67

1,11

0,21

130,8

2,04

1,48

CR1288

PN

must w ithout SO2

12,17

1,4

0,37

395,9

1,82

1,01

CR1289

PN

must after pressing

13,3

1,3

0,33

147,6

1,66

0,71

CR1290

CH

must w ithout SO2

5,74

1,2

0,19

150,35

1,75

0,79

CR1291

CH

must after pressing

37,57

1,46

0,68

199,95

1,78

0,87

sam ple code

a

sam ple type

ng/µl

A260/A280

A260/A230

ng/µl

CH= Chardonnay ; PN= Pinot Noir


39


Page 40 of 47

Table 3. Absorbance-based DNA concentration and purity ratios obtained for the modified extraction methods compared to the original one. sample code varietya

sample type

stage

method

ng/µl

A260/A280

decanted

must

SM

77.4

1.34

0.35

CM1a

decanted

must

SM-K

144

1.21

0.32

CM1a

decanted

must

SM +CleanUp

10.77

0.97

0.03

CM1a

decanted

must

SM-K + CleanUp

38.73

1.25

0.1

LS

homogenized

must

SM + CleanUp

46.68

1.72

0.18

decanted

must

SM + CleanUp

3.86

1.81

0.02

LS

homogenized

start of fermentation

SM + CleanUp

51.88

2.03

0.14

decanted


SM + CleanUp

30.12

2.03

0.07

CM1a

CM1b

LS

CM1b CM2ab CM2ab

A260/A230

CM3ab

LS

decanted

end of fermentation

SM + CleanUp

73.86

1.99

0.2

CM2 c

LG

decanted


SM

1839

2.17

2.21

decanted


SM-K

3392

2.1

1.72

decanted

end of fermentation

SM

5244

1.98

1.94

SM-K

4044

2.05

1.73

CM2 c CM3cb

LG

CM3cb decanted end of fermentation LS = Lambrusco di Sorbara; LG = Lambrusco Grasparossa b Sample not stabilized with potassium metabisulphite.

a

Table 4. Absorbance-based DNA concentration and purity ratios obtained for the different methods tested for wine DNA extraction. ng/µl

A260/A280

A260/A230

kit Roche (modified)

extraction method Sangiovese 2010

wine

69,55

1,88

0,28

kit Roche (modified)

Brunello 2008

197,80

1,39

0,41

Kit Roche (modified)

Trento DOC

71,63

2,06

0,32

Kit Roche (modified)

L. Grasparossa 2010

71,96

2,01

0,32

MO-BIO soil

Cabernet Sauvignon 2010

4,75

2,66

0,52

MO-BIO soil

Merlot 2010

3,23

2,59

1,01

MO-BIO soil

Teroldego 2010

4,56

2,13

0,36

MO-BIO Soil (modified)

Sangiovese 2010

32,96

1,85

0,17


Brunello 2008

13,81

1,99

0,09


Trento DOC

16,22

2,01

0,10


L. Grasparossa 2010

27,78

1,56

0,15

Pereira et al., 2011

Sangiovese 2010


Brunello 2008


266,10

1,45

0,54

1430,00

1,02

0,25

Trento DOC

58,61

1,02

0,12


L. Grasparossa 2010

208,70

1,05

0,26

PowerPlant Pro DNA isolation kit (MO-BIO)

Sangiovese 2010

2,58

0,96

0,21

QDEK - Bigliazzi et al., 2012

Sangiovese 2010

12,62

1,79

0,09


Brunello 2008

24,18

1,36

0,11


Trento DOC

8,34

2,52

0,05


L. Grasparossa 2010

7,74

2,62

0,05

QIAamp DNA Stool for human DNA analysis

Sangiovese 2010

11,77

1,23

0,32


Cabernet Sauvignon 2010

4,70

1,52

0,17


Merlot 2010

0,65

1,12

0,26


Teroldego 2010

3,50

1,71

0,28

QIAamp DNA Stool for human DNA analysis a

Sangiovese 2010

8,77

1,27

0,20

QIAamp DNA Stool for human DNA analysis b

Sangiovese 2010

1,53

1,97

0,18

Savazzini and Martinelli 2006

Sangiovese 2010

208,78

1,15

0,58

TEPC - Bigliazzi et al., 2012

Sangiovese 2010

n.d

n.d

n.d


Brunello 2008

4,05

1,52

0,14


L. Grasparossa 2010

n.d

n.d

n.d

a

ethanol precipitation for 24h; b Sep-Pak

n.d = no DNA was isolated.


40

Page 41 of 47


Table 5. Results obtained with the modifications introduced to Pereira et al. (2011) method wine

method modification

ng/µl

A260/A280

A260/A230

original

174,1

1,43

0,58

modification 1

0,49

0,55

0,05

modification 2

29,92

0,83

0,1

original

133,4

1,3

0,36

Sangiovese 2010

Sangiovese 2009 Sangiovese 2008 Brunello 2010

modification 2

53,73

0,84

0,1

original

164,55

1,41

0,48

modification 2

51,21

0,9

0,11

original

266,1

1,45

0,54

modification 3 (100 ul)

121

1,37

0,46

modification 3 ( 1 volume)

93,33

1,29

0,35 1,48

Lambrusco Grasparossa 2010

original

603,4

1,84

modification 3 (50 ul)

133,7

1,31

0,39

modification 3 (100 uL)

87,86

1,33

0,53

modification 3 (1 volume)

48,57

1,33

0,52

Table 6. Nuclear microsatellite profiles of the grapevine cultivars related to this study VvMD5

VvMD7

VvMD25

VvMD27

VvMD28

VvMD32

VvS2

VrZAG62

VrZAG79

VMC1B11

Fortana

225

231

247

253

241

257

179

189

236

236

251

271

132

150

191

203

245

259

167

Lambrusco Sorbara

226

226

239

247

250

256

179

187

230

236

271

271

132

150

193

195

251

29

169

185 185

Lambrusco Salamino

227

231

233

263

251

257

183

187

238

260

241

251

150

150

193

203

247

251

167

167

Ancellotta

231

231

239

263

242

256

183

187

236

246

241

271

130

154

193

193

245

247

167

167

Lambrusco Grasparossa

231

231

239

249

243

257

183

187

220

248

241

263

130

130

193

199

245

245

167

183

Malbo gentile

231

245

247

253

243

243

179

193

236

236

253

259

144

154

193

199

251

259

183

185

Sangiovese

224

234

239

263

242

242

177

183

235

245

253

257

130

130

193

195

243

259

167

167

Cabernet Sauvignon

230

238

239

239

240

250

173

187

235

237

241

241

136

148

187

193

247

247

185

185

Merlot

224

234

239

247

240

250

187

189

229

235

241

241

136

148

193

193

259

259

173

185

Pinot Noir

226

236

239

243

240

250

183

187

219

237

241

271

134

150

187

193

239

245

167

173

Chardonnay

232

236

239

243

240

256

181

187

219

229

241

271

134

140

187

195

243

245

167

185

Table 7. Chloroplast microsatellite markers of the grapevine varieties included in this study Variety Montepulciano Canaiolo Chardonnay Ciliegiolo Cabernet Sauvignon Merlot Pinot Noir Primitivo Petit Verdot Riesling Renano Sangiovese Sauvignon Blanc Tannat Tempranillo Teroldego Ancellotta Fortana L. Grasparossa L. Salamino L. Sorbara Malbo Gentile

NTcp12 NTcp8

167 167 167 167 167 167 167 167 167 167 167 167 167 167 167 167 167 167 167 167 167

250 249 249 250 250 249 249 249 249 249 250 250 250 249 249 249 250 250 249 250 250

ccmp10

110 109 111 110 110 111 109 111 109 109 110 110 110 109 110 109 110 110 109 110 110

ccmp3

104 103 103 104 104 103 103 103 103 103 104 104 104 103 104 103 104 104 103 104 104

ccmp2 ccmp4 ccmp6 ccSSR14 Chlorotype

206 206 206 206 206 206 206 206 206 206 206 206 206 206 206 206 206 206 206 206 206


126 126 126 126 126 126 126 126 126 126 126 126 126 126 126 126 126 126 126 126 126

106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106 106

204 203 205 204 204 205 203 205 203 203 204 204 204 203 204 203 204 204 203 204 204

D A C D D C A C A A D D D A B A D D A D D

41


Page 42 of 47

Table 8. SNP genotypes confirmed by targeted resequencing of the grapevine cultivars Variety Canaiolo Chardonnay Ciliegiolo Cabernet Sauvignon Merlot Montepulciano Pinot Noir Primitivo Petit Verdot Riesling Renano Sangiovese Sauvignon Blanc Tannat Tempranillo Teroldego

SNP 222

SNP 244

SNP 98

TT CT CT TT TT TT CT TT TT TT CC CT TT CT CT

AA AA AA AA AA AC AA AC AA AA CC AA AA AA AA

GG CG CG GG CG CG CG CG CG CG CC CG CG GG CG

Table 9. Primers designed for amplification and analysis of the unique SNPs for the Sangiovese cultivar

SNP

amplicon primer

222

Fw-5’AGACT GACTT T T GAAACACC3’ Rw-5’T T CCT GGAT T GGGT AT G3’

98

889 bp

nested product size

RwNest-5’CCAGGCAAGT AACACAAG3’

790 bp

128 bp

RwNest-5’T T T CCT AAT CCT T GTT GG3’

RwNest-5’CCCAGT T CCAT TCCTACACC3’


VIC-AGCAAT GT GGGCC GA 6FAM-T AGGAT TT AT GAAGGGAAG

136 bp

FwNest-5’AAT CCCCAT CCCGAAGTG3’ 721 bp

TaqMan probe 6FAM-AGCAAT GT GGGCTGA

FwNest-5’GTT AGT GT AAGGT GAT GCC3’

Fw-5’AAACGCAGGAGAAT GT C3’ Rw-5’T T CAACCT GAT GCCT AAC3’

nested primer FwNest-5’AAGACACCCACCAAGTT C3’

Fw-5’T T CAAAGCGAAGAACCAG3’ Rw-5’ACCCT T CAACAAACCAAC3’

244

amplicon size

VIC-T AGGAT T T AT GAAGGCAAG Probe1-CCT TT CT GGGT T GAACA

136 bp

Probe2- CCT TT CT GGGT T GCACA

42

Page 43 of 47


FIGURES

Fig. 1


43


Page 44 of 47

350.0

1.60

300.0

1.40

250.0

1.20 1.00

200.0

0.80 150.0

0.60

100.0

0.40

50.0

0.20

0.0

absorbance ratio

DNA ng/µl

Fig. 2 A

0.00 Ethanol prec.

Sep-Pak

NaCl

Na Acetate

Treatment DNA ng/µl

260/280 ratio

260/230 ratio

Fig. 2 B


44

Page 45 of 47


Fig. 3 (A, B)

A

B

Green: homozygous CC, Sangiovese Red: heterozygous CG, Chardonnay, Ciliegiolo, Merlot, Montepulciano, Petit Verdot, Pinot Noir, Primitivo, Sauvignon Blanc and Tannat Blue: homozygous GG, Cabernet Sauvignon and Canaiolo


45


Page 46 of 47

Fig. 4 (A, B)

Fig. 5


46

Page 47 of 47


Graphic for table of contents

500

2.50

400

2.00

300

1.50

200

1.00

100

0.50

0

Absorbance ratio

DNA (ng/µl)

(Original graphic)

0.00 Pre-fermentation

Middle of fermentation

End of fermentation


47

Experimental Review of DNA-Based Methods for Wine Traceability

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