Olive Oil DNA Fingerprinting by Multiplex SNP Genotyping on

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Olive oil DNA fingerprinting by multiplex SNPgenotyping on fluorescent microspheres Despina P. Kalogianni, Christos Bazakos, Lemonia M. Boutsika, Mehdi B. Targem, Theodore K. Christopoulos, Panagiotis Kalaitzis, and Penelope C Ioannou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5054657 • Publication Date (Web): 10 Mar 2015 Downloaded from http://pubs.acs.org on March 13, 2015

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Journal of Agricultural and Food Chemistry -1-

Olive oil DNA fingerprinting by multiplex SNP-genotyping on fluorescent microspheres

Despina P. Kalogianni1, Christos Bazakos2,3, Lemonia M. Boutsika1, Mehdi Ben Targem2, Theodore K. Christopoulos1,*, Panagiotis Kalaitzis2, and Penelope C. Ioannou4

1

Department of Chemistry, University of Patras, Patras, Greece 26504.

2

Department of

Horticultural Genetics & Biotechnology, Mediterranean Agronomic Institute, Chania (MAICh), Greece 73100. 3Present address: INRA, UMR 1318, Institut Jean-Pierre Bourgin, RD10, F78000, Versailles, France. 4Department of Chemistry, University of Athens, Athens, Greece 15771.

*Corresponding author. Phone: (+30)2610 962951

E-mail: [email protected]

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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1

ABSTRACT

2

Olive oil cultivar verification is of primary importance for the competitiveness of the

3

product and the protection of consumers and producers from fraudulence. Single

4

nucleotide polymorphisms (SNPs) have emerged as excellent DNA markers for

5

authenticity testing. We report the first multiplex SNP-genotyping assay for olive oil

6

cultivar identification that is performed on a suspension of fluorescence-encoded

7

microspheres. Up to 100 sets of microspheres, with unique ‘fluorescence signatures’,

8

are available. Allele discrimination was accomplished by primer-extension reaction. The

9

reaction products were captured via hybridization on the microspheres and analyzed,

10

within seconds, by a flow-cytometer. The ‘fluorescence signature’ of each microsphere

11

is assigned to a specific allele, whereas the signal from a reporter fluorophore denotes

12

the presence of the allele. As a model, a panel of three SNPs was chosen that enabled

13

identification of five common Greek olive cultivars (Adramytini, Chondrolia Chalkidikis,

14

Kalamon, Koroneiki and Valanolia).

15 16 17 18

KEYWORDS

19

Olea europaea L., Olive oil, varietal origin, genotyping, fluorescence, single-nucleotide

20

polymorphisms, fingerprinting

21


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INTRODUCTION

23

Olive oil is produced from the fruit of Olea europaea L., a tree that is native to the

24

Mediterranean basin.1 Because of its unique taste, flavor and beneficial effects on

25

human health, olive oil is the most expensive edible oil in the market. Olive oil

26

consumption has been constantly growing worldwide. The sensorial characteristics and

27

nutritional properties of olive oil are heavily dependent on the cultivar variety.

28

Monovarietal (single-cultivar) oils of protected designation of origin offer superior quality

29

and therefore have become the center of interest of producers and consumers.2 In fact,

30

the consumers increasingly prefer to purchase certified olive oil from a particular cultivar

31

variety and geographical area.2,3 As a consequence, the verification of the cultivar

32

utilized to produce an olive oil sample is of primary importance in order to enhance the

33

competitiveness of the product and protect both the consumer and the producer by

34

preventing fraudulent practices. The European Union has recognized the differentiation

35

in quality products, such as olive oil, and introduced a new framework for the protection

36

of the geographical origin of the product, according to the EU legislation.4 This

37

legislation permits the labeling of the product as ‘Protected Geographical Indication’

38

(PGI) and ‘Protected Designation of Origin’ (PDO).

39

It should be emphasized that, because of the strong compositional similarities

40

between olive oils obtained from various cultivars, the identification of the cultivar origin

41

is a much more challenging task than the detection of adulteration of olive oil by other

42

vegetable oils.

43

One approach to the classification of the cultivar origin of olive oil entails the

44

characterization of the metabolic profile, including triacyl glycerols, free fatty acids,



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45

phenols, sterols, various pigments, trace elements etc. Infrared and UV-Vis

46

spectroscopy, various chromatographic methods, calorimetry, mass spectrometry,

47

nuclear magnetic resonance (NMR) and a potentiometric electronic tongue have been

48

employed in combination with powerful chemometric tools for the discrimination of the

49

cultivar origin of olive oil.5-10 However, the metabolic profile of olive oil is affected,

50

heavily, by environmental factors (e.g. soil composition and climate conditions),

51

extraction methods, stage of ripeness, harvesting time and storage period.11 As a

52

consequence, the metabolite analysis is not sufficient to provide verification of the

53

cultivar.11

54

In recent years, it has been shown that, despite the close similarities between the

55

olive cultivars, the diversity of their DNA sequences is sufficient to allow varietal

56

discrimination. A ‘DNA fingerprint’ is an excellent alternative to metabolic profiling,11

57

because DNA markers are unaffected by the aforementioned factors that influence the

58

metabolite composition and, as a result, they enable unambiguous determination of the

59

cultivar variety of the olive oil sample.12 Moreover, it has been shown that the amount of

60

DNA present in olive oil is adequate for carrying out DNA-based authenticity testing.12,13

61

Various DNA markers have been developed for olive cultivar identification. These

62

include genomic microsatellites,14-16 random amplified polymorphic DNA (RAPD),17,18

63

inter simple sequence repeat (ISSR) markers,19,20 sequence-characterized amplified

64

regions (SCAR)21 of RAPD products and amplified fragment length polymorphisms

65

(AFLP).22,23 The above methods are based on the comparison of the patterns of DNA

66

fragments separated by gel electrophoresis or capillary electrophoresis in automated

67

systems. More recently, single-nucleotide polymorphisms (SNPs) have emerged as new


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genetic markers with unique advantages for varietal discrimination.24-27 The progress in

69

automated sequencing technology has facilitated greatly the discovery of characteristic

70

SNPs for olive cultivars.28,29 The main advantages of SNPs over other genetic markers

71

are: 12,29 (a) SNPs enable to pinpoint very similar cultivars because the differentiation is

72

based on a single-nucleotide change rather than a whole DNA sequence. (b) SNP

73

genotyping can be accomplished even with short amplified DNA sequences (e.g. 100

74

bp). This is particularly important in cases where the integrity of the DNA may be

75

affected during the extraction process and storage of olive oil. (c) SNPs constitute the

76

most abundant genetic markers, (d) they are mainly biallelic and stably inherited.

77

Therefore, the trend is that SNP genotyping will replace the classical genetic markers

78

for cultivar identification. The methods reported so far for SNP genotyping of olive oil

79

DNA include capillary electrophoresis of restriction enzyme-digested DNA fragments25,26

80

and DNA-microarray analysis of ligation reaction products.24 It should be noted,

81

however, that microarrays are suitable for parallel genotyping of thousands of SNPs in

82

one sample, i.e., the SNP-throughput per sample is high but the sample-throughput is

83

low.

84

The proposed method for SNP genotyping is based on a suspension of

85

fluorescent microspheres and, in comparison to the above methods, offers the following

86

advantages. i) High sample-throughput, i.e., compared to microarrays, the method is

87

best suited for genotyping of a small number of useful SNPs in a large number of

88

samples. ii) The suspension of microspheres provides flexibility and convenience in

89

modification and/or expansion of the assay by just replacing sets of microspheres or



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90

adding more sets of microspheres in the suspension. iii) The microspheres offer rapid

91

solution kinetics.

92

Previous reports have employed suspension of fluorescent microspheres for the

93

detection and/or quantification of PCR-amplified DNA fragments from food samples.30,31

94

However, to our knowledge, this is the first report of SNP genotyping assays for food

95

authenticity testing using this technological platform.

96

The aim of the present work is the development of a high sample-throughput

97

method for the identification of olive cultivars based on the simultaneous genotyping of

98

single-nucleotide polymorphisms.

99 100

MATERIALS AND METHODS

101

Apparatus and reagents. PCR and primer extension reactions were performed

102

in the MJ Research PTC-0150 thermal cycler (Watertown, MA). The digital camera,

103

Kodak DC 120, and the Gel Analyzer software for DNA documentation were purchased

104

from Kodak (New York, NY). The ultrasonic cleaner from Branson Ultrasonics (Danbury,

105

CT) was used for resuspension of the microspheres. The Luminex 100 IS flow-

106

cytometer (Luminex, Austin, TX) was used for the analysis of the microspheres.

107

Fluorescent carboxylated microspheres (5.6 µm) were purchased from Luminex (Austin,

108

TX). Amplitaq Gold DNA polymerase was from Applied Biosystems (CA, USA) and Vent

109

(exo-) DNA polymerase was from New England Biolabs (Beverly, MA). Ultrapure 2’-

110

deoxyribonucleoside 5’-triphosphates (dNTPs) were purchased from Invitrogen

111

(CarlsBad, CA). KAPA 2G Fast multiplex PCR kit was from Kapa Biosystems (Woburn,

112

MA, USA). Biotin-11-dUTP was from Applichem (Darmstadt, Germany). Streptavidin-R-


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phycoerythrin conjugate (SA-PE) was purchased from Molecular Probes (Eugene, OR).

114

The primers and probes used in this study were synthesized by VBC-Biotech (Vienna,

115

Austria). The sequences of the oligonucleotides used throughout this work are listed in

116

Table 1. All other common reagents were from Sigma (St. Louis, MO).

117

Experimental Outline. The method includes the following steps: i) DNA

118

isolation, ii) PCR amplification of three DNA segments that contain the 3 SNPs (SNP2,

119

SNP3 and SNP4), iii) multiplex genotyping reaction based on the extension of 6 allele-

120

specific primers, iv) multiplex hybridization assay of the genotyping reaction products on

121

spectrally encoded fluorescent microspheres (Figure 1).

122

Samples and DNA extraction. The Koroneiki and Chondrolia monovarietal extra

123

virgin olive oil samples were provided by Anatoli S.A. Chania, Crete and Makedoniki

124

Elaiourgia S.A. Kalives, Chalkidiki, respectively. The monovarietal olive oil samples of

125

Adramytini, Valanolia and Kalamon were extracted from fruits of the respective cultivars

126

in the pilot olive mill of the Institute of Subtropical Plants and Olive, NAGREF, Chania,

127

Crete. The samples were stored in the dark at room temperature for several months

128

before use. Genomic DNA was extracted from young leaves of five Greek olive varieties

129

(Adramytini, Chondrolia Chalkidikis, Kalamon, Koroneiki and Valanolia) according to the

130

method Saghai-Maroof et al.32 as modified by Angiolillo et al.33 Monovarietal olive oil

131

samples

132

CTAB/hexane/chloroform protocol.34

(500

µL)

were

used

to

extract

genomic

DNA

according

to

the

133

DNA Amplification Protocol A. DNA isolated from olive leaves and oil was

134

subjected to two consecutive rounds of amplification (PCR I and PCR II). PCR I was

135

carried out in a total volume of 50 µL containing 15 mM Tris-HCl, pH 8.0, 50 mM KCl,



Page 8 of 33 -8-

136

2.5 mM MgCl2, 200 µM of each dNTP, 0.3 µM of each primer, 1.25 U of Amplitaq Gold

137

DNA polymerase and 2 µL of genomic DNA or PCR I product. The cycling conditions for

138

PCR I and PCR II for SNPs 2, 3 and 425 were as follows: 10 min at 95 °C followed by 35

139

cycles (40 cycles for PCR I of olive oil) at 95 °C for 30 s, 50 °C (60 °C for PCR I for

140

SNP2) for 30 s and 72 °C for 30 s. At the end of the cycling, the mixture was held at 72

141

°C for 10 min. A 2-µL aliquot from PCR I of olive oil was used directly in PCR II. The

142

products of PCR I from leaves were first diluted 200 times and a 2-µL aliquot was used

143

as DNA template for PCR II. The PCR II primers (nested primers) were as previously

144

reported.25 The cycling conditions for PCR II were exactly the same as PCR I. The PCR

145

II products were confirmed and quantified by 2% agarose gel electrophoresis and

146

ethidium bromide staining. The PCR I products were 501 bp, 224 bp and 239 bp for

147

SNP2, SNP3 and SNP4, respectively, whereas the PCR II products were 112 bp, 105

148

bp and 137 bp for SNP2, SNP3 and SNP4, respectively.

149

DNA Amplification Protocol B (Multiplex PCR). The multiplex PCR was

150

performed in a final volume of 25 µL containing 12.5 µL of Kapa 2G fast multiplex PCR

151

mix, 0.2 µΜ of each of the PCR II primers for the three SNPs and 2 µL of the isolated

152

DNA. The multiplex PCR conditions were: an initial step at 95 0C for 3 min followed by

153

35 cycles of: 95 °C for 15 s, 50 °C for 30 s and 72 °C for 30 s. A final step at 72 °C for

154

10 min was included. An aliquot of 5 µL of the PCR products were then subjected to a

155

multiplex primer extension reaction and an aliquot of 10 µL of the primer extension

156

products were subjected to a multiple hybridization assay (see below).

157

Allele-Specific Primer Extension Reaction. Each allele-specific primer

158

contained: (a) At the 5’ end, a unique 24-bp oligonucleotide sequence (tag)


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complementary to the anti-tag sequence that was attached to the surface of a specific

160

set of fluorescent microspheres. (b) A 12-bp common spacer, and (c) An allele-specific

161

sequence 17-24 bp (Figure 1). Extension reactions were performed in the presence of a

162

mixture of six primers specific for the six alleles of all three SNPs (SNP2, SNP3 and

163

SNP4). The extension reaction was carried out in a total volume of 20 µL containing 20

164

mM Tris–HCl, 10 mM (NH4)2SO4, 10 mM KCl, 0.1% Triton X-100, pH 8.8, 1.5 U Vent

165

(exo-) DNA polymerase, 400 fmol of amplified DNA, 2 pmol of each primer, 2 mM

166

MgSO4, 10 µM of each dATP, dCTP, dGTP, and 10 µΜ biotin-dUTP. The extension

167

reactions were performed in the thermal cycler as follows: 95 oC for 5 min and 40 cycles

168

of 95 oC for 15 s, 58 oC for 15 s, 72 oC for 15 s.

169

Multiplex Hybridization Assay on Spectrally Distinct Microspheres. The

170

extension reaction products (10 µL) were analyzed by a multianalyte hybridization

171

assay, performed in a total volume of 55 µL (Figure 1). A suspension of six sets of

172

spectrally encoded microspheres was prepared for the assay, each set consisting of

173

5000 identical microspheres. Each microsphere was stained with precise amounts of

174

two fluorescent dyes, thus creating a built-in fluorescent signature. Also, anti-tag

175

oligonucleotides were attached on the surface of each microsphere for molecular

176

recognition of the allele-specific extension products. All the microspheres were

177

suspended in the hybridization buffer (100 mM Tris-HCl, pH 8.0, 200 mM NaCl and 0.8

178

mL/L Triton X-100) and preheated at 37 °C. The extension reaction products were

179

denatured for 3 min at 95 °C, placed immediately on ice for 2 min and subsequently

180

added to the above mixture and incubated for 1 h at 37 °C. The microspheres were then

181

pelleted by centrifugation at 10000 g for 1 min and washed once with 50 µL of



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182

hybridization buffer followed by resuspension in 80 µL of hybridization buffer containing

183

250 ng of streptavidin-phycoerythrin conjugate (SA-PE). The mixture was incubated for

184

20 min at room temperature and the microspheres were analyzed by flow-cytometry

185

without prior washing. During the flow-cytometric detection process, each microsphere

186

was irradiated by two laser beams, 635 nm and 532 nm. The 635-nm beam was used

187

for the excitation of the two fluorescent dyes within the microsphere and the ratio of the

188

fluorescence intensities at 658 and 712 nm provided the classification of the

189

microsphere. The 532-nm laser beam was used for the excitation of the reporter

190

molecule, phycoerythrin, and the fluorescence intensity was related to the presence of a

191

specific allele in the sample. At least 100 microspheres of each set were analyzed by

192

the flow-cytometer and the median fluorescence value was calculated (Figure 1).

193

Genotype assignment. The genotype of a sample, for SNPs 2, 3 and 4, was

194

determined as follows: The fluorescence intensity obtained from each set of

195

microspheres was first normalized to scale of 0-100, i.e., it was expressed as a

196

percentage of the maximum fluorescence value of the set. Then, for each SNP, the

197

allelic fraction R=100FA/(FA+FB) was calculated (as a percentage), where FA and FB are

198

the normalized fluorescence values for the alleles A and B of the SNP, respectively. In

199

the presence of allele A only, the value of R is expected to be close to 100, whereas in

200

the absence of allele A (only allele B present) the value of R would be close to zero. If

201

both alleles, A and B, of the SNP are present, then R is about 50.

202 203

RESULTS AND DISCUSSION


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The olive oil cultivar identification is based on the genotyping of three SNPs, i.e.,

205

SNP2, SNP3 and SNP4, that are located in the plant’s genome giving a unique

206

genotype fingerprint for each variety.25 The SNP database of the 5 Greek olive cultivars

207

is presented in Table 2.25 The principle of the method is illustrated in Figure 1. The

208

method is performed on a suspension of spectrally encoded microspheres in a high

209

sample-throughput and cost-effective format. We used sets of microspheres stained

210

with precise amounts of two spectrally distinct fluorophores, thus providing unique

211

‘fluorescence signatures’ for 100 different sets, which in turn permit the rapid

212

simultaneous genotyping of up to 50 SNPs. Allele discrimination was accomplished

213

through primer-extension reaction. The extension reaction products were captured from

214

the suspension of the microspheres and reacted with a streptavidin-phycoerythrin

215

conjugate. The microspheres were then analyzed, within seconds, by a flow-cytometer.

216

Each microsphere ‘fluorescence signature’ corresponds to a specific allele, whereas the

217

signal of the phycoerythrin reporter denotes the presence of this allele in the sample.

218

The suspension of microspheres allows solution kinetics and great assay flexibility.

219

Biotin-dUTP was incorporated during the extension, and hence the reaction

220

products were labeled with biotin. The products were heat-denatured and allowed to

221

hybridize with a mixture of six sets of microspheres, each set carrying a characteristic

222

anti-tag sequence. The hybrids were detected with a streptavidin-phycoerythrin

223

conjugate. The fluorescence of the microspheres was finally measured by a flow-

224

cytometer.

225 226

As a model, the proposed method was applied to the authentication of 5 olive varieties: Adramytini, Chondrolia Chalkidikis, Kalamon, Koroneiki and Valanolia.



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227

The PCR amplification yielded fragments with sizes 112 bp, 105 bp and 137 bp

228

for SNP2, SNP3 and SNP4, respectively. The PCR products were subjected to the

229

extension reaction in the presence of six allele-specific primers (two primers per SNP).

230

Several parameters were evaluated for the optimization of the allele-specific

231

primer extension reaction. The concentration of primers was tested in the range of 0.05

232

and 0.1 µΜ. The concentration of dATP, dGTP and dCTP was studied in the range of

233

2.5 to 25 µM of each and the concentration of biotin-dUTP was tested in the range of

234

1.25 to 10 µM. The annealing temperature of the reaction was performed at 50 to 58 oC,

235

as well as the reaction was carried out for 20 to 40 cycles. The optimized conditions are

236

reported in the Materials and Methods section. Finally, the effect of the PCR product

237

purification prior to the PEXT reaction was tested. Several PCR products were treated

238

with exonuclease I and alkaline phosphatase, which hydrolyse the primers and the

239

dNTPs respectively. The treated PCR products were subjected to extension reaction in

240

parallel with untreated products. The untreated products performed the same with

241

treated ones. Optimization studies for hybridization assays on fluorescent microspheres

242

included the effect of the number of microspheres (500-10000) on the signal of the

243

assay, the hybridization time (30 to 120 min), the amount of streptavidin-phycoerythrin

244

conjugate (100-500 ng) and the incubation time for the conjugate (10-30 min). Finally,

245

the volume of the extension reaction mixture used for the multiplex hybridization assay

246

was optimized and 10 µL of the product were sufficient to obtain high fluorescence

247

signal.

248

Synthetic DNA Controls for Olive Varieties. The method was initially evaluated

249

by using synthetic DNA controls representative of each variety. A synthetic control was


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a mixture of single-stranded DNA target sequences (60-mers), each corresponding to

251

an allele (two targets per SNP). The sequences of the synthetic targets are listed in

252

Table 1. Mixtures of the synthetic targets of the three SNPs were prepared, according to

253

the known genotype of each olive variety. Given that the two alleles of each SNP are

254

denoted with the letters A and B, the known genotypes of the five olive varieties for

255

SNP2-SNP3-SNP4 are as follows:25 Adramytini (AD): AA-AB-AA; Chondrolia (CH): AB-

256

AB-AA; Kalamon (KA): AA-AA-AA; Koroneiki (KO): AB-AB-AB; Valanolia (VA): AB-AA-

257

AA. Thus, the combination of the three genotypes provided a unique varietal code.

258

We produced synthetic controls that represent the five olive varieties: Adramytini,

259

Chondrolia, Kalamon, Koroneiki, and Valanolia. The synthetic controls contained 200

260

fmol/µL of each allele in the case of a ‘homozygote’ control of the specific SNP, and 100

261

fmol/µL of each allele in the case of a ‘heterozygote’ control. A 1-µL aliquot of each

262

synthetic control was subjected to a multiple extension reaction using a mixture of 6

263

extension primers, specific for the SNP2, SNP3 and SNP4 alleles. The extension

264

products were subjected to a multiplex hybridization assay with the six sets of

265

microspheres. The genotyping results for the synthetic controls are presented as plots

266

of the allelic fraction R versus the olive variety that corresponds to each synthetic

267

control (Figure 2). A color-code presentation of the SNP2, SNP3 and SNP4 genotypes

268

for the five olive varieties is shown in Figure 2. It is demonstrated that the combination

269

of the three genotypes gives a unique identification code for each variety.

270

Genotyping of Olive Leaves and Olive Oil. The proposed method was applied

271

to the genotyping of DNA samples isolated from leaves and oil of the 5 olive varieties.

272

The olive leaves and fruits used for olive oil production of Adramytini, Valanolia and



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273

Kalamon were collected from the same olive trees. Adramytini and Valanolia samples

274

were harvested from olive groves in the Lesvos island and Kalamon samples from an

275

olive grove in Chalkidiki. The Koroneiki leaf sample was harvested from the

276

experimental olive grove at MAICh. The Chondrolia leaf and fruit samples were

277

harvested from an olive grove in the Chalkidiki area. The fruits were then transferred for

278

olive oil extraction in the Makedoniki Elaiourgia Company (Greece). The leaf and olive

279

oil DNA samples of all cultivars were characterized accordingly.25 Following PCR, an

280

amount of 400 fmol of amplified DNA was subjected to the multiplex primer extension

281

reaction in the presence of the six allele-specific primers. A volume of 10 µL of the

282

extension products, after heat denaturation for 3 min at 95 oC, was hybridized to a

283

mixture of the six sets of microspheres. The results of the genotyping of the leaf

284

samples are presented in Figure 3. Similarly, the proposed method was applied to the

285

genotyping of DNA isolated from various olive oil samples and the results are presented

286

in Figure 4. We observe that, for each olive variety, the genotypes obtained from the

287

leaf samples and from olive oil samples coincide with those of the synthetic controls.

288

An All-Multiplex Method. An all-multiplex method was developed comprising (a)

289

multiplex PCR amplification, (b) multiplex extension reaction and (c) multiplex

290

hybridization assay. The multiplex PCR of all three SNP segments in a single round of

291

amplification was accomplished by using the Kapa 2G fast DNA polymerase. The

292

genotyping results, presented in Figure 5, show the agreement of the all-multiplex

293

protocol with the single-PCR approach.

294

Reproducibility. The reproducibility of the method was assessed by analyzing

295

DNA samples from Valanolia, Kalamon and Koroneiki. The analyses were performed in


Page 15 of 33


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triplicate and the mean allelic fractions along with their coefficient of variation (%CV)

297

were calculated for each SNP. The results are presented in Table 3. In all cases the

298

reproducibility of the allelic fraction was very good for all three SNPs and genotypes.

299 300

The proposed method can be easily expanded to include more olive varieties

301

since there are 100 sets of commercially available and spectrally distinguishable

302

fluorescent microspheres carrying anti-tag sequences. Given that each set corresponds

303

to an allele the potential of the present method extends to the multiplex genotyping of

304

50 biallelic SNPs. Furthermore, the flexibility of the proposed method arises from the

305

fact that the suspension of microspheres is not a fixed array (as in the case of planar

306

arrays) but it can be easily adapted to specific laboratory needs, since it is convenient to

307

select and mix only those microsphere sets that are relevant to the SNP panel that is

308

used for the interrogation of a particular olive oil sample. Moreover, the PCR products

309

are introduced directly (without prior purification) to a single-tube multiplex primer-

310

extension reaction followed by a single-tube multiplex hybridization of all extension

311

products on the microspheres and flow-cytometric analysis. The flow-cytometric

312

detection is completed within 30 seconds. The method is accurate since the results

313

obtained from the five olive cultivars are in full agreement with previous studies25 that

314

established the genotypes of the 5 olive varieties for SNPs 2, 3 & 4. Moreover, for each

315

cultivar, DNA isolated from leaf samples gave identical genotypes with DNA isolated

316

from olive oil.

317

In our opinion, the proposed high sample-throughput assay will contribute

318

significantly to the traceability and authenticity of olive oil samples. It is expected to be



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319

particularly useful in an industrial context and in laboratories involved in official control,

320

i.e., laboratories that require methods offering high sample-throughput.

321


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REFERENCES

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(1) Loukas, M.; Krimbas, C.B. History of olive cultivars based on genetic distances. J.

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Hortic. Sci. 1983, 58, 121-127. (2) Stefanoudaki, E.; Kotsifaki, F.; Koutsaftakis, A. Sensory and chemical profiles of

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three

European olive

varieties

(Olea europea

L); an approach for the

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characterisation and authentication of the extracted oils. Sci. Food. Agric. 2000, 80,

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381-389.

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(3) Casale, M.; Oliveri, P.; Casolino, C.; Sinelli, N.; Zunin, P.; Armanino, C.; Forina, M.;

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Lanteri, S. Characterisation of PDO olive oil Chianti Classico by non-selective (UV-

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visible, NIR and MIR spectroscopy) and selective (fatty acid composition). Anal.

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Chim. Acta 2012, 712, 56−63.

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(4) European Council Regulation No 510/2006 of 20 Mar 2006.

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spectroscopy. Food Res. Int. 2010, 43, 2126–2131.

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tongue. Food Chem. 2014, 160, 321–329.

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(Olea europaea L.): overview on DNA markers applications and recent advances in

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Effectiveness of microsatellite DNA markers in checking the identity of protected

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designation of origin extra virgin olive oil. J. Agric. Food Chem. 2007, 55, 3857–

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markers for identification of drupes from different Olea europaea L. cultivars. Eur.

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diversity in the Olive tree (Olea europaea L. subsp. europaea) cultivated in Portugal

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revealed by RAPD and ISSR markers. Genet. Resour. Crop Ev. 2004, 51, 501-511.

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to Food Genomics: Olive Oil Traceability. J. Agric. Food Chem. 2007, 55, 6052–

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molecular markers to identify virgin olive oils from single Italian cultivars. Eur. Food

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analysis. Eur. Food Res. Technol. 2008, 229, 375–382.

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virgin olive oils based on plastid DNA variation profiling. PLoS One 2013, 8, e70507.

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hybridization assay performed on spectrally encoded microspheres. Anal. Chem.

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(32) Saghai-Maroof, M.A.; Soliman, K.M.; Jorgensen, R.A.; Allard, R.W. Ribosomal

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DNA spacer length polymorphisms in barley: Mendelian inheritance, chromosomal

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419 420



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421

FIGURE CAPTIONS

422

Figure 1

423

(A) Allele-specific primer extension reaction. Each allele-specific primer consists of a

424

segment complementary to the target sequence, a tag sequence at the 5’ end and a 12-

425

nt spacer. The two allele-specific primers differ only at the 3’ end nucleotide. Extension

426

by DNA polymerase occurs only when the primer anneals to the corresponding allele.

427

(B) Hybridization assay on spectrally-encoded fluorescent microspheres. The extension

428

reaction products are captured on the surface of the microspheres through hybridization

429

with the immobilized anti-tag sequences. The added streptavidin-phycoerythrin

430

conjugate binds only to the extended primers that contain biotin moieties. The

431

fluorescence of phycoerythrin of each microsphere is measured by flow-cytometry.

432

(C) Classification of the fluorescent microspheres by flow-cytometry. The classification

433

is based on the fluorescence intensities of the two dyes used for the staining of the

434

microspheres. Each cluster represents a set of microspheres. The dots inside the

435

cluster are data from at least 100 microspheres.

436 437

Figure 2

438

(A) Genotyping results for synthetic controls that represent the five Greek olive

439

varieties, that is, Adramytini (AD), Chondrolia Chalkidikis (CH), Kalamon (KA), Koroneiki

440

(KO) and Valanolia (VA). For each SNP, an allelic fraction R (expressed as a

441

percentage) close to 100 corresponds to the presence of the allele A only, whereas an

442

R value close to 50 indicates the presence of both alleles A and B. ‘R 2A’: Allelic


Page 23 of 33


443

fraction (percentage) of allele A of SNP2. ‘R 3A’: Allelic fraction (percentage) of allele A

444

of SNP3. ‘R 4A’: Allelic fraction (percentage) of allele A of SNP4.

445

(B) Color-code for the genotypes of the five olive varieties as assigned by the

446

combination of the three SNPs. For each SNP, the green circle shows the presence of

447

the allele A only, whereas the yellow circle indicates the presence of both alleles (A and

448

B).

449 450

Figure 3

451

Genotyping results obtained from leaf samples of the five olive varieties: Adramytini

452

(AD), Chondrolia Chalkidikis (CH), Kalamon (KA), Koroneiki (KO) and Valanolia (VA).

453

For each SNP, an allelic fraction R close to 100 corresponds to the presence of the

454

allele A only, whereas an R value close to 50 indicates the presence of both alleles A

455

and B.

456 457

Figure 4

458

Genotyping results obtained from olive oil samples of the five cultivars: Adramytini (AD),

459

Chondrolia Chalkidikis (CH), Kalamon (KA), Koroneiki (KO) and Valanolia (VA). For

460

each SNP, an allelic fraction R close to 100 corresponds to the presence of the allele A

461

only, whereas an R value close to 50 indicates the presence of both alleles A and B.

462 463

Figure 5

464

Genotyping results of the all-multiplex method for the five cultivars: Adramytini (AD),

465

Chondrolia Chalkidikis (CH), Kalamon (KA), Koroneiki (KO) and Valanolia (VA). For



Page 24 of 33 -24-

466

each SNP, an allelic fraction R close to 100 corresponds to the presence of the allele A

467

only, whereas an R value close to 50 indicates the presence of both alleles A and B.

468 469


Page 25 of 33


TABLES Table 1. Oligonucleotide Sequences Oligonucleotide Name

Sequence (5΄ → 3΄)

PCR primers SNP2 Forward 2 PCR I Reverse 2 PCR I Forward 2 PCR II Reverse 2 PCR II

GCAACTCAAATGAATGAATCATGAT CTAACTCGATGGCCGTTTTCTAA GAAAATAACCTGCATTTTCG TATTGCTTGTTGGGTAGAAG

SNP3 Forward 3 PCR I Reverse 3 PCR I Forward 3 PCR II Reverse 3 PCR II

TTCACAGGTAAAATTCTTCTTTCC AACCCTCAGCTGTGCAATCTG TCATTCATGGCATTTATTCTTC CTGGGCAATCCTCTAACACC

SNP4 Forward 4 PCR I Reverse 4 PCR I Forward 4 PCR II Reverse 4 PCR II

TGTTGGTGAACCACTGGATG CAGCAACAAAATCCAGGAAG AGGCGATTGTACGATGCTG TCCCTTTCCGAAAACAACAG

Extension primers tag2A tag2B tag3A tag3B tag4A tag4B

TTACCTTTATACCTTTCTTTTTACCCGTAACTCATTAAGACATATAGCACGTATACCT TACACTTTCTTTCTTTCTTTCTTTCCGTAACTCATTAAGACATATAGCACGTATACCG CTTTATCAATACATACTACAATCACCGTAACTCATTTTCTTCTATTCTCAGTTTTTCTAG CAATTTCATCATTCATTCATTTCACCGTAACTCATTTTCTTCTATTCTCAGTTTTTCTAA TCATCAATCAATCTTTTTCACTTTCCGTAACTCATTCAACCAAACTAATCAAATTAAAAG ATACTACATCATAATCAAACATCACCGTAACTCATTCAACCAAACTAATCAAATTAAAAA

Anti-tags anti-tag2A anti-tag2B anti-tag3A anti-tag3B anti-tag4A anti-tag4B

GTAAAAAGAAAGGTATAAAGGTAA AAAGAAAGAAAGAAAGAAAGTGTA TGATTGTAGTATGTATTGATAAAG TGAAATGAATGAATGATGAAATTG AAAGTGAAAAAGATTGATTGATGA TGATGTTTGATTATGATGTAGTAT

Synthetic Targets Target2A Target2B Target3A Target3B Target4A Target4B

CTGCATTTTCGGGCCATCTTCGGCGACCCAGAAGTAATCAGGTATACGTGCTATATGTCTT CTGCATTTTCGGGCCATCTTCGGCGACCCAGAAGTAATCCGGTATACGTGCTATATGTCTT ACAAAAAGGAAATTGTAAACCAATTAACATGGAAGTCTAGAAAAACTGAGAATAGAAGAA ACAAAAAGGAAATTGTAAACCAATTAACATGGAAGTTTAGAAAAACTGAGAATAGAAGAA ATGCTGTTAATGTCATCCTTTCATTACAGGTATTGAAGCTTTTAATTTGATTAGTTTGGTTG ATGCTGTTAATGTCATCCTTTCATTACAGGTATTGAAGTTTTTAATTTGATTAGTTTGGTTG



Page 26 of 33 -26-

Table 2. SNP Database of the 5 Greek Olive Cultivars. A=Adenine; C=Cytosine; G=Guanine

SNPs SNP2 SNP3 SNP4

Adramytini A/A A/G C/C

Chondrolia Kalamon A/C A/A A/G G/G C/C C/C

Koroneiki A/C A/G C/T


Valanolia A/C G/G C/C

Page 27 of 33


Table 3. Reproducibility Study.

SNPs

SNP2 SNP3 SNP4

Valanolia Allelic Genotype fraction (Mean) AB 51.9 AA 98.7 AA 99.8

%CV (n=3) 5.2 0.4 0.1

Kalamon Allelic Genotype fraction (Mean) AA 94.2 AA 99.8 AA 99.7

%CV (n=3) 2.5 0.4 0.1


Koroneiki Allelic Genotype fraction (Mean) AB 48.5 AB 52.3 AB 49.8

%CV (n=3) 4.4 4.0 1.4


Page 28 of 33 -28-

FIGURES


Page 29 of 33


100

R 4A

A

75

B

50

Genotypes for SNPs 2, 3 & 4

25

2 3 4

0

Adramytini (AD)

100

R 3A

75

Chondrolia (CH)

50 25

Kalamon (KA)

0 100

Koroneiki (KO)

R 2A

75 50

Valanolia (VA)

25 0

AD

CH

KA

KO

VA

Variety

Figure 2



Page 30 of 33 -30-

100

R 4A

75 50 25 0 100

R 3A

75 50 25 0 100

R 2A

75 50 25 0

AD

CH

KA

KO

VA

Variety

Figure 3


Page 31 of 33


100

R 4A

75 50 25 0 100

R 3A

75 50 25 0 100

R 2A

75 50 25 0

AD

CH

KA

KO

VA

Variety

Figure 4



Page 32 of 33 -32-

100

R 4A

75 50 25 0 100

R 3A

75 50 25 0 100

R 2A

75 50 25 0

AD

CH

KA

KO

VA

Variety

Figure 5


Page 33 of 33


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Olive Oil DNA Fingerprinting by Multiplex SNP Genotyping on

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