Valentina Fanelli1 Wilma Sabetta4 and Valentina di Rienzo12
1Department of Soil Plant and Food Sciences Section of Genetics and Breeding University of Bari Aldo Moro Via Amendola 165A70126 Bari Italy2Spin Off Sinagri srl University of Bari Aldo Moro Via Amendola 165A 70126 Bari Italy3Department of Soil Plant and Food Sciences Section of Food Science Technology University of Bari Aldo Moro Via Amendola 165A70126 Bari Italy4Institute of Biosciences and Bioresources CNR Via Amendola 165A 70126 Bari Italy
Correspondence should be addressed to Cinzia Montemurro cinziamontemurrounibait
Received 29 December 2014 Revised 16 March 2015 Accepted 17 March 2015
Academic Editor Jose A Pereira
Copyright copy 2015 Cinzia Montemurro et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The aim of the research was to verify the applicability of microsatellite (SSR) markers in High Resolution Melting (HRM) analysisfor the identification of the olive cultivars used in the ldquoTerra di Barirdquo PDO extra virgin olive oil A panel of nine cultivarswidespread in Apulia region was tested with seventeen SSR primer pairs and the PCR products were at first analysed with aGenetic Analyzer automatic sequencer An identification key was obtained for the nine cultivars which showed an unambiguousdiscrimination among the varieties constituting the ldquoTerra di Barirdquo PDO extra virgin olive oil Cima di Bitonto Coratina andOgliarola Subsequently an SSR based method was set up with the DCA18 marker coupled with HRM analysis for the distinctionof the Terra di Bari olive oil from non-Terra di Bari olive oil using different mixturesThus this analysis enabled the distinction andidentification of the PDO mixtures Hence this assay provided a flexible cost-effective and closed-tube microsatellite genotypingmethod well suited to varietal identification and authentication analysis in olive oil
1 Introduction
The average worldwide production of olive oil has grownsteadily in the last years mainly due to the recommendationof doctors and nutritionists about the benefit of Mediter-ranean diet in which olive oil is a key element Despitethe fact that Italy remains one of the main producers ofthis sector the total production and consumption of oliveoil have undergone a considerable decrease [1] In thisscenario Apulia is the main oil producing region in Italy[2] Due to their authenticity most of the Apulian olive oilsobtained the quality marks of Protected Denomination ofOrigin (PDO) by the European Community according tothe EC Regulation 208192 This certification implies the useof specific local cultivars and peculiar sensory features ofoil (Official Journal of the European Communities 1992)
Consequently monitoring the origin of raw material andindustrial process becomes of primary importance during theproduction of a high value PDO product
The verification of the cultivars used to produce olive oilis becoming of particular interest in the last decade This factassumes a strong commercial appeal especially in the pro-tection of high-quality olive oils such as PDO which mightbe adulterated with other low-quality oil using minor or lessexpensive cultivars [3] In fact the quality of olive oil strictlydepends on the variety employed for its production and thereis a strong link between the cultivar and the territory of culti-vation Several analytical approaches have been developed tohelp the identification of olive oil cultivars constituents andpossible adulterants The compositional markers monitoredfor traceability purposes are different such as triglyceridessterols fatty acids phenolic compounds volatile compounds
Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 496986 7 pageshttpdxdoiorg1011552015496986
2 Journal of Chemistry
pigments hydrocarbons and tocopherols [4ndash7] However allthese compositional markers can be severely affected by theenvironmental conditions during the plant growth whichmight cause ambiguous or erroneous results [3] Since thechemical analyses are not enough for themselves to verifythe olive oil authenticity or its varietal identification othertraceability markers based on DNA analysis have been usedto identify olive cultivars In fact DNA being less influencedby environment and food processing is the best resource forcomparison of different genetic material [8ndash10]
In this contest molecular markers represent an idealtool to accurately and exclusively characterize olive cultivarsby detection of DNA polymorphisms and the establishmentof an identification key To this purpose microsatellites orSimple Sequence Repeats (SSR) are among the most suitablemolecular markers since they are characterized by a highpolymorphism level due to variations of the repeats numberMoreover SSR analysis is easy to perform just implyingan amplification with species-specific primer pairs and thesubsequent electrophoresis on agarose or acrylamide gelsIn particular microsatellites have been preferred by severalauthors for variability studies germplasm characterizationand varietal fingerprinting of numerous species such aswheat [11] rice [12] grape [13] tomato [14] and olive [15ndash18] SSR markers can allow to determine an unambiguouslyidentification key of the most common Italian monovarietaloils also thanks to their capacity to detect any ldquoalienrdquo allele[9 19]
The extra virgin olive oil ldquoTerra di Barirdquo is one of themostimportant Italian PDO oils whose spread on the nationalmarket is equal to 131 [20] According to the MinisterialDecree of 4 September 1998 the EC Regulation 232597 andthe article 17 of EEC Regulation 208192 the disciplinaryregulations about the production of ldquoTerra di Barirdquo oil withldquoBitontordquo as additional geographical mention expect thatthis oil is obtained from the following olive cultivars in theminimum quantity of 80 alone or mixed together Cimadi Bitonto or Ogliarola Barese and Coratina (UG n 227 of29 September 1998) Thereby the main aim of the presentstudywas to test the applicability ofmicrosatellitemarkers forthe identification of cultivars constituting the ldquoTerra di BarirdquoPDO oil
2 Material and Methods
21 Plant Material and DNA Extraction Nine olive cultivarsdiffused in the Apulia Region (Italy) were sampled at theOlive Pre-multiplication Centre field ldquoConca drsquoOrordquo Pala-giano (Taranto Italy) Cima di Bitonto Ogliarola CoratinaToscanina Maiatica Cellina di Nardo Nociara Cima diMola and Simone These cultivars were previously tested forthe genetic identity and are considered as certified varietiesThe DNAwas extracted both from leaves and 9 monovarietalolive oils The experimental material was enriched with twocommercial PDO olive oils used for DNA extraction andHRM analyses Genomic DNA from leaves was extractedaccording to Li protocol [21] modified as reported by Sabettaet al 2011 [22] starting from 30mg of lyophilized leavesDNA from monovarietal and commercial olive oils was
extracted by means of Gene Elute Plant Kit (Sigma StLouis MO) following the manufacturerrsquos instructions andthe modifications reported in Pasqualone et al [9] Cellularresiduals were obtained by centrifuging 250mL of oil at10000 rpm for 5min The extracted DNA was checked interms of quality and quantity by means both of 08 agarosegel electrophoresis and spectrophotometer (Nanodrop 1000Thermo Scientific Waltham MA USA) at 260 nm DNAfrom lyophilized leaves resulted to have optimal qualityand a concentration of 100 ng120583L whereas DNA extractedfrom oil had lower concentration (5 ng120583L) and was partiallydegraded [23]
22 SSR Markers Analysis and Genetic Relationship Seven-teen microsatellite primer pairs DCA03 DCA04 DCA18DCA05 DCA09 DCA13 DCA14 DCA15 DCA16 DCA17[15] and GAPU103a GAPU71b GAPU101 GAPU45 [24]EMOL EMO90 [25] and UDO43 [26] (Table 1) labelledwith FAM or HEX fluorochromes were used in the anal-ysis and the amplification reactions were carried out in afinal volume of 25 120583L containing the following 50 ng DNA1X PCR buffer 025mM dNTP 025 120583M of each primer25mMMgCl
2 and 006 U Taq Polymerase (Euroclone)The
PCRs were carried out in a C1000 thermal cycler (Bio-Rad)and the conditions were set as follows 5min at 95∘C 35cycles consisting of 30 sec at 95∘C 30 sec at the specificannealing temperature 30 sec at 72∘C 60min at 72∘C forfinal elongation The amplification products were separatedby capillary electrophoresis on an automated sequencer ABIPRISM 3100 Avant Genetic Analyzer (Applied Biosystems)and the obtained electropherograms were analysed by theGeneMapper 37 software (Applied Biosystems)
The amplified fragments were used to get a binarymatrixin which amplicons were marked with 1 for presence and 0for the absence of a fragment to a certain molecular weightGenetic similarity among the olive cultivars was calculated byJaccard coefficient and the Unweighted Pair Group Methodusing Arithmetic Averages (UPGMA) was performed forcluster analysis with NTSYS-PC 20 [27]
23 DNA Mixtures and SSR Genotyping by High Resolu-tion Melting (HRM) Analysis Reference DNAs of cultivarsCoratina Cima di Bitonto and Ogliarola were used aloneor combined in order to get mixtures simulating Terra diBari oil which comprises a 20 of unspecified local varietieswidely spread in the area The local varieties indicated asldquoother cultivarsrdquo were Simone Toscanina Cima di MolaNociara Maiatica and Cellina di Nardo In addition weanalysed two commercial olive oils Terra di Bari and anextra-European monovarietal oil obtained from the cultivarAeleh (Algeria) in order to consider a possible introductionof unlabelled extra community olive oil (Table 2) Progressiveadulteration of a PDO oil sample (monovarietal Coratinaolive oil) was simulated by adding a crescent proportion ofcultivar Aeleh from 10 to 50
We performed a High Resolution Melting (HRM) analy-sis choosing the SSRmarker DCA18 on the basis of its differ-ent allelic profiles in the cultivars Coratina Cima di Bitontoand Ogliarola Barese obtained by capillary electrophoresis
Journal of Chemistry 3
Table 1 Locus name repeat motif annealing temperature primer sequences referred to SSR markers and allelic profiles of the cultivarsincluded in the PDO Terra di Bari olive oil
Locus Repeat motif Primer sequence (51015840-31015840) Annealing temperature Allelic profiles (bp)Cima di Bitonto Ogliarola Barese Coratina
Coratina 100 266 40 40 90 80 70 60 50Ogliarola 100 80 266 40 40Cima di Bitonto 100 80 266 40 40Nociara 80 50Toscanina 80 50Aeleh 80 100 10 20 30 40 50Other cultivarslowastlowast 266 266 266 20 20 20 20 20 20 20lowastOn the bases of PDO ldquoTerra di Barirdquo procedure rules these commercial samples should bemade by 80 of Coratina Ogliarola Barese andor Cima di Bitontoalone or in mixture and a 20 of other cultivarslowastlowastMixture in equal proportion of cultivars Simone Cima di Mola Nociara and Toscanina
4 Journal of Chemistry
HRM reaction was performed in a final volume of 10 120583Lconsisting of 50 ng of genomic DNA 1 times Sso Fast EvaGreenMaster mix (Bio-Rad Hercules CA) and 025 120583M of eachprimer (Sigma-Aldrich St Louis MO) A No TemplateControl (NTC) was included in each run [28]
Amplification and HRM analysis were performed onCFX96 Touch Real Time PCR Detection System (Bio-RadHercules CA) and the cycling program consisted of a touch-down protocol 2min of initial denaturation at 98∘C followedby 5 cycles of denaturation at 98∘C for 8 sec annealing at56∘C for 8 sec (with decrement of 05∘C per cycle) andextension at 72∘C for 12 sec The annealing temperaturewas maintained at 54∘C for the successive 40 steps anddenaturation temperature was decreased to 95∘C acquiringfluorescent data at the end of each cycle The amplificationprotocol was immediately followed by the High ResolutionMelting steps of 95∘C for 10 sec cooling to 58∘C for 30 sec andraising the temperature from 65∘C to 95∘C with increasing of02∘C every 10 sec with fluorescence acquisition
After verification of robust amplification curves themelting curve stage was further analysed by CFX Managersoftware (Bio-Rad Hercules CA) The melt curve was nor-malized along the temperature axis (temperature shifting) topermit easy differentiation of DNA curve
3 Results and Discussion
Among the different types of genetic markers available forvarietal identification purposes the nuclear microsatellite orSSR (Simple Sequence Repeat) is the marker of choice largelyused and the only one accepted for forensic applications [29]This is essentially due to the numerous advantages intrinsic ofsuch marker codominant nature high polymorphism widedistribution across the genome and automated detection AsSSR can be used to distinguish olive varieties when DNA isextracted directly from olive oils and based on our previousworks [30] we identified the most informative and effectivemarkers (DCA04 and DCA18) to genotype the selectedcultivars showing a different allelic profiles (Table 1)
In this work SSR markers were directly applied to DNAextracted fromolive oil which is highly degraded and poor inquantityThebest approach is to select SSRmarkers providinga simple and reproducible pattern whilst they maintain theirinformativeness and efficiency The SSR markers used in thiswork were selected for their high polymorphism and for theclear allelic profile
All 17 SSRmarkers showed a very high value of PD (PowerDiscrimination) [31] with the maximum value of 088 scoredfor DCA18
Figure 1 reports the genetic similarity dendrogramobtained with 17 SSR markers The cultivars Toscanina andNociara showed the highest degree of diversity whereas theothers are separated in two subclusters In the first groupthere are Cima di Mola Ogliarola Maiatica and Simoneand in the second one there are Cellina di Nardo CoratinaCima di Bitonto and Toscanina All the cultivars showedsmall sized fruits characteristic of olive oil attitude cultivarswith Cima di Mola and Ogliarola and Coratina and Cima
Similarity coefficient000 020 040 060 080 100
Nociara
Cima di Mola
Ogliarola
Maiatica
Simone
Coratina
Cima di Bitonto
Toscanina
oCellina di Nard
Figure 1 Genetic similarity dendrogram of nine olive cultivarsobtained by the analysis of seventeen microsatellite markers
75 76 77 78 79
minus005
000
005
010
015
020
025
80 81 82
Diff
eren
ce R
FU
Temperature
Cima di Bitonto
Coratina
Ogliarola
Figure 2 Normalized difference curve of the three cultivarsCoratina Cima di Bitonto and Ogliarola obtained with the DCA18marker
di Bitonto that are very similar in the morphological traitsOgliarola and Cima di Bitonto are commonly consideredas synonymous referred to the same variety and also thedisciplinary of ldquoTerra di Barirdquo olive oil production induces inan ambiguous appellation of them not clarifying that are twodifferent cultivars The results of our analyses define Cima diBitonto and Ogliarola as two distinct cultivars as confirmedby capillary electrophoresis and HRM analysis (Figure 2)
Among the seventeen markers the DCA18 was the mostpolymorphic SSR able to discriminate all samples and forthis reason it was chosen to realize an identification key(Figure 3) This marker revealed 10 different amplicons thatare combined in 9 unique genotypes and for this reason wasselected for the HRM application In recent years differentauthors reported the use of HRM for the varietal identifica-tion [32 33] genotyping [28 34] and food traceability [35]The advantages of this technique that was originally con-ceived for the human diagnostic [36] such as absenceof manipulation after PCR cost effectiveness closed-tube
Journal of Chemistry 5
Simone
Maiatica
168
Ogliarola
172 Cima di Bitonto
174
Coratina
Cima di Mola
154
158 Nociara
Cellina
168 193
166 Toscanina
Figure 3 Identification key of nine olive cultivars obtained by theanalysis of DCA18 microsatellite markers
analysis and results obtainable in less than 3 hours arenowadays emerging in plant and food sector All the HRMexperiments were realized with the marker DCA18 and inFigure 4 is reported the difference curve of some Terra di Bariexperimental samples compared to commercial Terra di Bariolive oil and one experimental olive oil made up of NociaraandToscaninaTheplotwell established the difference amongthe Terra di Bari group (mixes 3 6 7 9 10 11 12) and non-Terra di Bari group (mix 13) The preparation of differentmixtures (Table 2) is essential to constitute a dataset of melt-ing curve profiles in order to perform a quick preliminaryanalysis and define the belonging of an unknown sample tothe Terra di Bari group specified and declared as well in thelabelThis analysismight be useful as first check of the varietalcomposition of olive oil especially when it is necessary toworkwith large number of samplesThehigh efficiency of dis-crimination of the technique already tested by Vietina et al[37] and Ganopoulos et al [38] in adulteration of olive oilby the addition of cheaper oils obtained from other plants(ie maize sunflower and soybean) is proved also in thevarietal identification if a cultivar is required and specifiedby production disciplinary A further application of HRMmethod is to quantify the presence of adulterants in literatureare reported several studies regarding the identification ofadulterants (ie different species or botanical varieties) andtheir quantification [35 38 39] Figure 5 showed the set-up of quantification of increasing addition of the Algeriancultivar Aeleh to a monovarietal Coratina olive oil It ispossible to appreciate the clear distinction between themonovarietal oil of Coratina and Aeleh showing differentmelting temperature and the placement of the different
76 77 78 79
minus005
minus010
minus015
000
005
Diff
eren
ce R
FU
Shifted temperature
Cima di Bitonto Mix 12
Mix 6Mix 10
Mix 11Mix 7
Mix 9Mix 13
Figure 4 Normalized and temperature-shifted difference curve ofdifferent commercial samples (mixes 11 and 12) Terra di Bari oils(monovarietal Cima di Bitonto oil and mixes 6 7 9 10) and non-Terra di Bari olive oil (mix 13)
770 775 780 785 790
minus002
minus004
minus006
000
004
002
006
008
Diff
eren
ce R
FU
Shifted temperature
Coratina
10 contamination
20 contamination 30 contamination
40 contamination
50 contaminationAeleh
Figure 5 Temperature-shifted difference curve obtained with theaddition of increasing quantity of cultivar Aeleh in Coratina mono-varietal oil
level of adulteration (10ndash20ndash30ndash40ndash50) between themTheobtained results confirmed the possibility to use the HRMtechnique not only for a qualitative application but also fora quantitative detection of the addiction of different amountsof olive oils produced by cultivars not allowed in the PDOdisciplinary production
4 Conclusion
This molecular analysis allowed the distinction of the culti-vars included in the ldquoTerra di Barirdquo PDO disciplinary withrespect to those widely diffused in the Apulian region Inaddition SSR markers were able to provide a specific profilefor Coratina Ogliarola Barese and Cima di Bitonto cultivarsThe production rules of ldquoTerra di Barirdquo olive oil consider thecultivar Cima di Bitonto and Ogliarola as two synonymousreferred to the same genotypes The allelic profiles obtainedwith capillary electrophoresis analysis clarify unambiguouslythat Cima di Bitonto and Ogliarola are two distinct varietiesand this aspect should be taken into account for an accuratereview of the disciplinary
These results should be useful in the agrofood com-partment where the application of molecular techniquescould lead to the identification of raw materials and derivedproducts allowing to trace origin and identity of cultivarsused for the obtainment of typical products
6 Journal of Chemistry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work has been carried out with financial support fromthe University of BarimdashProjects Idea Giovani 201011 andCofin PRIN 2009 coordinated by Dr Cinzia Montemurro
IDPagina7051[3] C Montealegre M L M Alegre and C Garcıa-Ruiz ldquoTrace-
ability markers to the botanical origin in olive oilsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 1 pp 28ndash38 2010
[4] M A Brescia G Alviti V Liuzzi and A Sacco ldquoChemometricsclassification of olive cultivars based on compositional data ofoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 80 no10 pp 945ndash950 2003
[5] T G Diaz I D Meras J S Casas and M F A Franco ldquoChar-acterization of virgin olive oils according to its triglycerides andsterols composition by chemometric methodsrdquo Food Controlvol 16 no 4 pp 339ndash347 2005
[6] M R Alves S C Cunha J S Amaral J A Pereira and M BOliveira ldquoClassification of PDO olive oils on the basis of theirsterol composition by multivariate analysisrdquo Analytica ChimicaActa vol 549 no 1-2 pp 166ndash178 2005
[7] I S Arvanitoyannis and A Vlachos ldquoImplementation ofphysicochemical and sensory analysis in conjunction withmultivariate analysis towards assessing olive oil authentica-tionadulterationrdquo Critical Reviews in Food Science and Nutri-tion vol 47 no 5 pp 441ndash498 2007
[8] S Pafundo C Agrimonti and N Marmiroli ldquoTraceability ofplant contribution in olive oil by amplified fragment lengthpolymorphismsrdquo Journal of Agricultural and Food Chemistryvol 53 no 18 pp 6995ndash7002 2005
[9] A Pasqualone C Montemurro C Summo W Sabetta FCaponio and A Blanco ldquoEffectiveness of microsatellite DNAmarkers in checking the identity of protected designation oforigin extra virgin olive oilrdquo Journal of Agricultural and FoodChemistry vol 55 no 10 pp 3857ndash3862 2007
[10] A Pasqualone V di Rienzo R Nasti A Blanco T Gomes andC Montemurro ldquoTraceability of Italian protected designationof origin (PDO) table olives by means of microsatellite molecu-lar markersrdquo Journal of Agricultural and Food Chemistry vol 61no 12 pp 3068ndash3073 2013
[11] M S Akkaya and E B Buyukunal-Bal ldquoAssessment of geneticvariation of bread wheat varieties using microsatellite markersrdquoEuphytica vol 135 no 2 pp 179ndash185 2004
[12] L Zeng T-R Kwon X Liu C Wilson C M Grieve andG B Gregorio ldquoGenetic diversity analyzed by microsatellitemarkers among rice (Oryza sativa L) genotypes with differentadaptations to saline soilsrdquo Plant Science vol 166 no 5 pp1275ndash1285 2004
[13] G Cipriani A Spadotto I Jurman et al ldquoThe SSR-basedmolecular profile of 1005 grapevine (Vitis vinifera L) accessionsuncovers new synonymy and parentages and reveals a large
admixture amongst varieties of different geographic originrdquoTheoretical and Applied Genetics vol 121 no 8 pp 1569ndash15852010
[14] G M M Bredemeijer R J Cooke M W Ganal et al ldquoCon-struction and testing of a microsatellite database containingmore than 500 tomato varietiesrdquoTheoretical and Applied Genet-ics vol 105 no 6-7 pp 1019ndash1026 2002
[15] K M Sefc M S Lopes D Mendonca M Rodrigues DosSantos M Laimer Da Camara Machado and A Da CamaraMachado ldquoIdentification of microsatellite loci in olive (Oleaeuropaea) and their characterization in Italian and Iberian olivetreesrdquoMolecular Ecology vol 9 no 8 pp 1171ndash1173 2000
[16] C Montemurro R Simeone A Pasqualone E Ferrara andA Blanco ldquoGenetic relationships and cultivar identificationamong 112 olive accessions using AFLP and SSR markersrdquo TheJournal of Horticultural Science and Biotechnology vol 80 no 1pp 105ndash110 2005
[17] M Hosseini-Mazinani R Mariotti B Torkzaban et al ldquoHighgenetic diversity detected in olives beyond the boundaries of theMediterranean seardquo PLoS ONE vol 9 no 4 Article ID e931462014
[18] D Scarano and R Rao ldquoDNA markers for food productsauthenticationrdquo Diversity vol 6 no 3 pp 579ndash596 2014
[19] A Pasqualone C Montemurro F Caponio and A BlancoldquoIdentification of virgin olive oil from different cultivars byanalysis of dnamicrosatellitesrdquo Journal of Agricultural and FoodChemistry vol 52 no 5 pp 1068ndash1071 2004
[20] httpwwwunaprolit[21] J T Li J Yang D C Chen X L Zhang and Z S Tang
ldquoAn optimizedmini-preparationmethod to obtain high-qualitygenomic DNA from mature leaves of sunflowerrdquo Genetics andMolecular Research vol 6 supplement 4 pp 1064ndash1071 2007
[22] W Sabetta V Alba A Blanco and C Montemurro ldquoSunTILLa TILLING resource for gene function analysis in sunflowerrdquoPlant Methods vol 7 article 20 2011
[23] A Pasqualone V di Rienzo A Blanco C Summo F Caponioand C Montemurro ldquoCharacterization of virgin olive oil fromLeucocarpa cultivar by chemical and DNA analysisrdquo FoodResearch International vol 47 no 2 pp 188ndash193 2012
[24] F Carriero G Fontanazza F Cellini and G Giorio ldquoIdentifica-tion of simple sequence repeats (SSRs) in olive (Olea europaeaL)rdquoTheoretical and Applied Genetics vol 104 no 2-3 pp 301ndash307 2002
[25] R de la Rosa C M James and K R Tobutt ldquoIsolation andcharacterization of polymorphic microsatellites in olive (Oleaeuropaea L) and their transferability to other genera in theOleaceaerdquo Molecular Ecology Notes vol 2 no 3 pp 265ndash2672002
[26] G Cipriani M T Marrazzo R Marconi A Cimato andR Testolin ldquoMicrosatellite markers isolated in olive (Oleaeuropaea L) are suitable for individual fingerprinting andreveal polymorphism within ancient cultivarsrdquoTheoretical andApplied Genetics vol 104 no 2-3 pp 223ndash228 2002
[27] F J Rohlf NTSYS-PC Numerical Taxonomy and MultivariateAnalysis System Version 202i Department of Ecology andEvolution State University of New York Setauket NY USA1998
[28] S M Sanzani C Montemurro V di Rienzo M Solfrizzo andA Ippolito ldquoGenetic structure and natural variation associatedwith host of origin in Penicillium expansum strains causing bluemouldrdquo International Journal of Food Microbiology vol 165 no2 pp 111ndash120 2013
Journal of Chemistry 7
[29] M Perez-Jimenez G Besnard G Dorado and P HernandezldquoVarietal tracing of virgin olive oil based on plastid DNAvariation profilingrdquo PLoS ONE vol 8 no 8 Article ID e705072013
[30] V Alba C Montemurro W Sabetta A Pasqualone andA Blanco ldquoSSR-based identification key of cultivars of Oleaeuropaea L diffused in Southern-Italyrdquo Scientia Horticulturaevol 123 no 1 pp 11ndash16 2009
[31] A D Kloosterman B Budowle and P Daselaar ldquoPCR-amplification and detection of the human D1S80 VNTR locusAmplification conditions population genetics and applicationin forensic analysisrdquo International Journal of LegalMedicine vol105 no 5 pp 257ndash264 1993
[32] J F MacKay C D Wright and R G Bonfiglioli ldquoA newapproach to varietal identification in plants by microsatellitehigh resolution melting analysis application to the verificationof grapevine and olive cultivarsrdquo Plant Methods vol 4 article 82008
[33] A Xanthopoulou I Ganopoulos G Koubouris et alldquoMicrosatellite high-resolutionmelting (SSR-HRM) analysis forgenotyping andmolecular characterization of anOlea europaeagermplasm collectionrdquo Plant Genetic Resources vol 12 no 3pp 273ndash277 2014
[34] I Ganopoulos A Argiriou and A Tsaftaris ldquoMicrosatellitehigh resolution melting (SSR-HRM) analysis for authenticitytesting of protected designation of origin (PDO) sweet cherryproductsrdquo Food Control vol 22 no 3-4 pp 532ndash541 2011
[35] I Ganopoulos A Argiriou and A Tsaftaris ldquoAdulterationsin Basmati rice detected quantitatively by combined use ofmicrosatellite and fragrance typing with High Resolution Melt-ing (HRM) analysisrdquo Food Chemistry vol 129 no 2 pp 652ndash659 2011
[36] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquoHumanMutation vol 30 no 6pp 857ndash859 2009
[37] M Vietina C Agrimonti and N Marmiroli ldquoDetection ofplant oil DNA using high resolution melting (HRM) post PCRanalysis a tool for disclosure of olive oil adulterationrdquo FoodChemistry vol 141 no 4 pp 3820ndash3826 2013
[38] I Ganopoulos C Bazakos P Madesis P Kalaitzis and A Tsaf-taris ldquoBarcode DNA high-resolution melting (Bar-HRM) anal-ysis as a novel close-tubed and accurate tool for olive oil forensicuserdquo Journal of the Science of Food and Agriculture vol 93no 9 pp 2281ndash2286 2013
[39] EMader J Ruzicka C Schmiderer and J Novak ldquoQuantitativehigh-resolution melting analysis for detecting adulterationsrdquoAnalytical Biochemistry vol 409 no 1 pp 153ndash155 2011
pigments hydrocarbons and tocopherols [4ndash7] However allthese compositional markers can be severely affected by theenvironmental conditions during the plant growth whichmight cause ambiguous or erroneous results [3] Since thechemical analyses are not enough for themselves to verifythe olive oil authenticity or its varietal identification othertraceability markers based on DNA analysis have been usedto identify olive cultivars In fact DNA being less influencedby environment and food processing is the best resource forcomparison of different genetic material [8ndash10]
In this contest molecular markers represent an idealtool to accurately and exclusively characterize olive cultivarsby detection of DNA polymorphisms and the establishmentof an identification key To this purpose microsatellites orSimple Sequence Repeats (SSR) are among the most suitablemolecular markers since they are characterized by a highpolymorphism level due to variations of the repeats numberMoreover SSR analysis is easy to perform just implyingan amplification with species-specific primer pairs and thesubsequent electrophoresis on agarose or acrylamide gelsIn particular microsatellites have been preferred by severalauthors for variability studies germplasm characterizationand varietal fingerprinting of numerous species such aswheat [11] rice [12] grape [13] tomato [14] and olive [15ndash18] SSR markers can allow to determine an unambiguouslyidentification key of the most common Italian monovarietaloils also thanks to their capacity to detect any ldquoalienrdquo allele[9 19]
The extra virgin olive oil ldquoTerra di Barirdquo is one of themostimportant Italian PDO oils whose spread on the nationalmarket is equal to 131 [20] According to the MinisterialDecree of 4 September 1998 the EC Regulation 232597 andthe article 17 of EEC Regulation 208192 the disciplinaryregulations about the production of ldquoTerra di Barirdquo oil withldquoBitontordquo as additional geographical mention expect thatthis oil is obtained from the following olive cultivars in theminimum quantity of 80 alone or mixed together Cimadi Bitonto or Ogliarola Barese and Coratina (UG n 227 of29 September 1998) Thereby the main aim of the presentstudywas to test the applicability ofmicrosatellitemarkers forthe identification of cultivars constituting the ldquoTerra di BarirdquoPDO oil
2 Material and Methods
21 Plant Material and DNA Extraction Nine olive cultivarsdiffused in the Apulia Region (Italy) were sampled at theOlive Pre-multiplication Centre field ldquoConca drsquoOrordquo Pala-giano (Taranto Italy) Cima di Bitonto Ogliarola CoratinaToscanina Maiatica Cellina di Nardo Nociara Cima diMola and Simone These cultivars were previously tested forthe genetic identity and are considered as certified varietiesThe DNAwas extracted both from leaves and 9 monovarietalolive oils The experimental material was enriched with twocommercial PDO olive oils used for DNA extraction andHRM analyses Genomic DNA from leaves was extractedaccording to Li protocol [21] modified as reported by Sabettaet al 2011 [22] starting from 30mg of lyophilized leavesDNA from monovarietal and commercial olive oils was
extracted by means of Gene Elute Plant Kit (Sigma StLouis MO) following the manufacturerrsquos instructions andthe modifications reported in Pasqualone et al [9] Cellularresiduals were obtained by centrifuging 250mL of oil at10000 rpm for 5min The extracted DNA was checked interms of quality and quantity by means both of 08 agarosegel electrophoresis and spectrophotometer (Nanodrop 1000Thermo Scientific Waltham MA USA) at 260 nm DNAfrom lyophilized leaves resulted to have optimal qualityand a concentration of 100 ng120583L whereas DNA extractedfrom oil had lower concentration (5 ng120583L) and was partiallydegraded [23]
22 SSR Markers Analysis and Genetic Relationship Seven-teen microsatellite primer pairs DCA03 DCA04 DCA18DCA05 DCA09 DCA13 DCA14 DCA15 DCA16 DCA17[15] and GAPU103a GAPU71b GAPU101 GAPU45 [24]EMOL EMO90 [25] and UDO43 [26] (Table 1) labelledwith FAM or HEX fluorochromes were used in the anal-ysis and the amplification reactions were carried out in afinal volume of 25 120583L containing the following 50 ng DNA1X PCR buffer 025mM dNTP 025 120583M of each primer25mMMgCl
2 and 006 U Taq Polymerase (Euroclone)The
PCRs were carried out in a C1000 thermal cycler (Bio-Rad)and the conditions were set as follows 5min at 95∘C 35cycles consisting of 30 sec at 95∘C 30 sec at the specificannealing temperature 30 sec at 72∘C 60min at 72∘C forfinal elongation The amplification products were separatedby capillary electrophoresis on an automated sequencer ABIPRISM 3100 Avant Genetic Analyzer (Applied Biosystems)and the obtained electropherograms were analysed by theGeneMapper 37 software (Applied Biosystems)
The amplified fragments were used to get a binarymatrixin which amplicons were marked with 1 for presence and 0for the absence of a fragment to a certain molecular weightGenetic similarity among the olive cultivars was calculated byJaccard coefficient and the Unweighted Pair Group Methodusing Arithmetic Averages (UPGMA) was performed forcluster analysis with NTSYS-PC 20 [27]
23 DNA Mixtures and SSR Genotyping by High Resolu-tion Melting (HRM) Analysis Reference DNAs of cultivarsCoratina Cima di Bitonto and Ogliarola were used aloneor combined in order to get mixtures simulating Terra diBari oil which comprises a 20 of unspecified local varietieswidely spread in the area The local varieties indicated asldquoother cultivarsrdquo were Simone Toscanina Cima di MolaNociara Maiatica and Cellina di Nardo In addition weanalysed two commercial olive oils Terra di Bari and anextra-European monovarietal oil obtained from the cultivarAeleh (Algeria) in order to consider a possible introductionof unlabelled extra community olive oil (Table 2) Progressiveadulteration of a PDO oil sample (monovarietal Coratinaolive oil) was simulated by adding a crescent proportion ofcultivar Aeleh from 10 to 50
We performed a High Resolution Melting (HRM) analy-sis choosing the SSRmarker DCA18 on the basis of its differ-ent allelic profiles in the cultivars Coratina Cima di Bitontoand Ogliarola Barese obtained by capillary electrophoresis
Journal of Chemistry 3
Table 1 Locus name repeat motif annealing temperature primer sequences referred to SSR markers and allelic profiles of the cultivarsincluded in the PDO Terra di Bari olive oil
Locus Repeat motif Primer sequence (51015840-31015840) Annealing temperature Allelic profiles (bp)Cima di Bitonto Ogliarola Barese Coratina
Coratina 100 266 40 40 90 80 70 60 50Ogliarola 100 80 266 40 40Cima di Bitonto 100 80 266 40 40Nociara 80 50Toscanina 80 50Aeleh 80 100 10 20 30 40 50Other cultivarslowastlowast 266 266 266 20 20 20 20 20 20 20lowastOn the bases of PDO ldquoTerra di Barirdquo procedure rules these commercial samples should bemade by 80 of Coratina Ogliarola Barese andor Cima di Bitontoalone or in mixture and a 20 of other cultivarslowastlowastMixture in equal proportion of cultivars Simone Cima di Mola Nociara and Toscanina
4 Journal of Chemistry
HRM reaction was performed in a final volume of 10 120583Lconsisting of 50 ng of genomic DNA 1 times Sso Fast EvaGreenMaster mix (Bio-Rad Hercules CA) and 025 120583M of eachprimer (Sigma-Aldrich St Louis MO) A No TemplateControl (NTC) was included in each run [28]
Amplification and HRM analysis were performed onCFX96 Touch Real Time PCR Detection System (Bio-RadHercules CA) and the cycling program consisted of a touch-down protocol 2min of initial denaturation at 98∘C followedby 5 cycles of denaturation at 98∘C for 8 sec annealing at56∘C for 8 sec (with decrement of 05∘C per cycle) andextension at 72∘C for 12 sec The annealing temperaturewas maintained at 54∘C for the successive 40 steps anddenaturation temperature was decreased to 95∘C acquiringfluorescent data at the end of each cycle The amplificationprotocol was immediately followed by the High ResolutionMelting steps of 95∘C for 10 sec cooling to 58∘C for 30 sec andraising the temperature from 65∘C to 95∘C with increasing of02∘C every 10 sec with fluorescence acquisition
After verification of robust amplification curves themelting curve stage was further analysed by CFX Managersoftware (Bio-Rad Hercules CA) The melt curve was nor-malized along the temperature axis (temperature shifting) topermit easy differentiation of DNA curve
3 Results and Discussion
Among the different types of genetic markers available forvarietal identification purposes the nuclear microsatellite orSSR (Simple Sequence Repeat) is the marker of choice largelyused and the only one accepted for forensic applications [29]This is essentially due to the numerous advantages intrinsic ofsuch marker codominant nature high polymorphism widedistribution across the genome and automated detection AsSSR can be used to distinguish olive varieties when DNA isextracted directly from olive oils and based on our previousworks [30] we identified the most informative and effectivemarkers (DCA04 and DCA18) to genotype the selectedcultivars showing a different allelic profiles (Table 1)
In this work SSR markers were directly applied to DNAextracted fromolive oil which is highly degraded and poor inquantityThebest approach is to select SSRmarkers providinga simple and reproducible pattern whilst they maintain theirinformativeness and efficiency The SSR markers used in thiswork were selected for their high polymorphism and for theclear allelic profile
All 17 SSRmarkers showed a very high value of PD (PowerDiscrimination) [31] with the maximum value of 088 scoredfor DCA18
Figure 1 reports the genetic similarity dendrogramobtained with 17 SSR markers The cultivars Toscanina andNociara showed the highest degree of diversity whereas theothers are separated in two subclusters In the first groupthere are Cima di Mola Ogliarola Maiatica and Simoneand in the second one there are Cellina di Nardo CoratinaCima di Bitonto and Toscanina All the cultivars showedsmall sized fruits characteristic of olive oil attitude cultivarswith Cima di Mola and Ogliarola and Coratina and Cima
Similarity coefficient000 020 040 060 080 100
Nociara
Cima di Mola
Ogliarola
Maiatica
Simone
Coratina
Cima di Bitonto
Toscanina
oCellina di Nard
Figure 1 Genetic similarity dendrogram of nine olive cultivarsobtained by the analysis of seventeen microsatellite markers
75 76 77 78 79
minus005
000
005
010
015
020
025
80 81 82
Diff
eren
ce R
FU
Temperature
Cima di Bitonto
Coratina
Ogliarola
Figure 2 Normalized difference curve of the three cultivarsCoratina Cima di Bitonto and Ogliarola obtained with the DCA18marker
di Bitonto that are very similar in the morphological traitsOgliarola and Cima di Bitonto are commonly consideredas synonymous referred to the same variety and also thedisciplinary of ldquoTerra di Barirdquo olive oil production induces inan ambiguous appellation of them not clarifying that are twodifferent cultivars The results of our analyses define Cima diBitonto and Ogliarola as two distinct cultivars as confirmedby capillary electrophoresis and HRM analysis (Figure 2)
Among the seventeen markers the DCA18 was the mostpolymorphic SSR able to discriminate all samples and forthis reason it was chosen to realize an identification key(Figure 3) This marker revealed 10 different amplicons thatare combined in 9 unique genotypes and for this reason wasselected for the HRM application In recent years differentauthors reported the use of HRM for the varietal identifica-tion [32 33] genotyping [28 34] and food traceability [35]The advantages of this technique that was originally con-ceived for the human diagnostic [36] such as absenceof manipulation after PCR cost effectiveness closed-tube
Journal of Chemistry 5
Simone
Maiatica
168
Ogliarola
172 Cima di Bitonto
174
Coratina
Cima di Mola
154
158 Nociara
Cellina
168 193
166 Toscanina
Figure 3 Identification key of nine olive cultivars obtained by theanalysis of DCA18 microsatellite markers
analysis and results obtainable in less than 3 hours arenowadays emerging in plant and food sector All the HRMexperiments were realized with the marker DCA18 and inFigure 4 is reported the difference curve of some Terra di Bariexperimental samples compared to commercial Terra di Bariolive oil and one experimental olive oil made up of NociaraandToscaninaTheplotwell established the difference amongthe Terra di Bari group (mixes 3 6 7 9 10 11 12) and non-Terra di Bari group (mix 13) The preparation of differentmixtures (Table 2) is essential to constitute a dataset of melt-ing curve profiles in order to perform a quick preliminaryanalysis and define the belonging of an unknown sample tothe Terra di Bari group specified and declared as well in thelabelThis analysismight be useful as first check of the varietalcomposition of olive oil especially when it is necessary toworkwith large number of samplesThehigh efficiency of dis-crimination of the technique already tested by Vietina et al[37] and Ganopoulos et al [38] in adulteration of olive oilby the addition of cheaper oils obtained from other plants(ie maize sunflower and soybean) is proved also in thevarietal identification if a cultivar is required and specifiedby production disciplinary A further application of HRMmethod is to quantify the presence of adulterants in literatureare reported several studies regarding the identification ofadulterants (ie different species or botanical varieties) andtheir quantification [35 38 39] Figure 5 showed the set-up of quantification of increasing addition of the Algeriancultivar Aeleh to a monovarietal Coratina olive oil It ispossible to appreciate the clear distinction between themonovarietal oil of Coratina and Aeleh showing differentmelting temperature and the placement of the different
76 77 78 79
minus005
minus010
minus015
000
005
Diff
eren
ce R
FU
Shifted temperature
Cima di Bitonto Mix 12
Mix 6Mix 10
Mix 11Mix 7
Mix 9Mix 13
Figure 4 Normalized and temperature-shifted difference curve ofdifferent commercial samples (mixes 11 and 12) Terra di Bari oils(monovarietal Cima di Bitonto oil and mixes 6 7 9 10) and non-Terra di Bari olive oil (mix 13)
770 775 780 785 790
minus002
minus004
minus006
000
004
002
006
008
Diff
eren
ce R
FU
Shifted temperature
Coratina
10 contamination
20 contamination 30 contamination
40 contamination
50 contaminationAeleh
Figure 5 Temperature-shifted difference curve obtained with theaddition of increasing quantity of cultivar Aeleh in Coratina mono-varietal oil
level of adulteration (10ndash20ndash30ndash40ndash50) between themTheobtained results confirmed the possibility to use the HRMtechnique not only for a qualitative application but also fora quantitative detection of the addiction of different amountsof olive oils produced by cultivars not allowed in the PDOdisciplinary production
4 Conclusion
This molecular analysis allowed the distinction of the culti-vars included in the ldquoTerra di Barirdquo PDO disciplinary withrespect to those widely diffused in the Apulian region Inaddition SSR markers were able to provide a specific profilefor Coratina Ogliarola Barese and Cima di Bitonto cultivarsThe production rules of ldquoTerra di Barirdquo olive oil consider thecultivar Cima di Bitonto and Ogliarola as two synonymousreferred to the same genotypes The allelic profiles obtainedwith capillary electrophoresis analysis clarify unambiguouslythat Cima di Bitonto and Ogliarola are two distinct varietiesand this aspect should be taken into account for an accuratereview of the disciplinary
These results should be useful in the agrofood com-partment where the application of molecular techniquescould lead to the identification of raw materials and derivedproducts allowing to trace origin and identity of cultivarsused for the obtainment of typical products
6 Journal of Chemistry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work has been carried out with financial support fromthe University of BarimdashProjects Idea Giovani 201011 andCofin PRIN 2009 coordinated by Dr Cinzia Montemurro
IDPagina7051[3] C Montealegre M L M Alegre and C Garcıa-Ruiz ldquoTrace-
ability markers to the botanical origin in olive oilsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 1 pp 28ndash38 2010
[4] M A Brescia G Alviti V Liuzzi and A Sacco ldquoChemometricsclassification of olive cultivars based on compositional data ofoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 80 no10 pp 945ndash950 2003
[5] T G Diaz I D Meras J S Casas and M F A Franco ldquoChar-acterization of virgin olive oils according to its triglycerides andsterols composition by chemometric methodsrdquo Food Controlvol 16 no 4 pp 339ndash347 2005
[6] M R Alves S C Cunha J S Amaral J A Pereira and M BOliveira ldquoClassification of PDO olive oils on the basis of theirsterol composition by multivariate analysisrdquo Analytica ChimicaActa vol 549 no 1-2 pp 166ndash178 2005
[7] I S Arvanitoyannis and A Vlachos ldquoImplementation ofphysicochemical and sensory analysis in conjunction withmultivariate analysis towards assessing olive oil authentica-tionadulterationrdquo Critical Reviews in Food Science and Nutri-tion vol 47 no 5 pp 441ndash498 2007
[8] S Pafundo C Agrimonti and N Marmiroli ldquoTraceability ofplant contribution in olive oil by amplified fragment lengthpolymorphismsrdquo Journal of Agricultural and Food Chemistryvol 53 no 18 pp 6995ndash7002 2005
[9] A Pasqualone C Montemurro C Summo W Sabetta FCaponio and A Blanco ldquoEffectiveness of microsatellite DNAmarkers in checking the identity of protected designation oforigin extra virgin olive oilrdquo Journal of Agricultural and FoodChemistry vol 55 no 10 pp 3857ndash3862 2007
[10] A Pasqualone V di Rienzo R Nasti A Blanco T Gomes andC Montemurro ldquoTraceability of Italian protected designationof origin (PDO) table olives by means of microsatellite molecu-lar markersrdquo Journal of Agricultural and Food Chemistry vol 61no 12 pp 3068ndash3073 2013
[11] M S Akkaya and E B Buyukunal-Bal ldquoAssessment of geneticvariation of bread wheat varieties using microsatellite markersrdquoEuphytica vol 135 no 2 pp 179ndash185 2004
[12] L Zeng T-R Kwon X Liu C Wilson C M Grieve andG B Gregorio ldquoGenetic diversity analyzed by microsatellitemarkers among rice (Oryza sativa L) genotypes with differentadaptations to saline soilsrdquo Plant Science vol 166 no 5 pp1275ndash1285 2004
[13] G Cipriani A Spadotto I Jurman et al ldquoThe SSR-basedmolecular profile of 1005 grapevine (Vitis vinifera L) accessionsuncovers new synonymy and parentages and reveals a large
admixture amongst varieties of different geographic originrdquoTheoretical and Applied Genetics vol 121 no 8 pp 1569ndash15852010
[14] G M M Bredemeijer R J Cooke M W Ganal et al ldquoCon-struction and testing of a microsatellite database containingmore than 500 tomato varietiesrdquoTheoretical and Applied Genet-ics vol 105 no 6-7 pp 1019ndash1026 2002
[15] K M Sefc M S Lopes D Mendonca M Rodrigues DosSantos M Laimer Da Camara Machado and A Da CamaraMachado ldquoIdentification of microsatellite loci in olive (Oleaeuropaea) and their characterization in Italian and Iberian olivetreesrdquoMolecular Ecology vol 9 no 8 pp 1171ndash1173 2000
[16] C Montemurro R Simeone A Pasqualone E Ferrara andA Blanco ldquoGenetic relationships and cultivar identificationamong 112 olive accessions using AFLP and SSR markersrdquo TheJournal of Horticultural Science and Biotechnology vol 80 no 1pp 105ndash110 2005
[17] M Hosseini-Mazinani R Mariotti B Torkzaban et al ldquoHighgenetic diversity detected in olives beyond the boundaries of theMediterranean seardquo PLoS ONE vol 9 no 4 Article ID e931462014
[18] D Scarano and R Rao ldquoDNA markers for food productsauthenticationrdquo Diversity vol 6 no 3 pp 579ndash596 2014
[19] A Pasqualone C Montemurro F Caponio and A BlancoldquoIdentification of virgin olive oil from different cultivars byanalysis of dnamicrosatellitesrdquo Journal of Agricultural and FoodChemistry vol 52 no 5 pp 1068ndash1071 2004
[20] httpwwwunaprolit[21] J T Li J Yang D C Chen X L Zhang and Z S Tang
ldquoAn optimizedmini-preparationmethod to obtain high-qualitygenomic DNA from mature leaves of sunflowerrdquo Genetics andMolecular Research vol 6 supplement 4 pp 1064ndash1071 2007
[22] W Sabetta V Alba A Blanco and C Montemurro ldquoSunTILLa TILLING resource for gene function analysis in sunflowerrdquoPlant Methods vol 7 article 20 2011
[23] A Pasqualone V di Rienzo A Blanco C Summo F Caponioand C Montemurro ldquoCharacterization of virgin olive oil fromLeucocarpa cultivar by chemical and DNA analysisrdquo FoodResearch International vol 47 no 2 pp 188ndash193 2012
[24] F Carriero G Fontanazza F Cellini and G Giorio ldquoIdentifica-tion of simple sequence repeats (SSRs) in olive (Olea europaeaL)rdquoTheoretical and Applied Genetics vol 104 no 2-3 pp 301ndash307 2002
[25] R de la Rosa C M James and K R Tobutt ldquoIsolation andcharacterization of polymorphic microsatellites in olive (Oleaeuropaea L) and their transferability to other genera in theOleaceaerdquo Molecular Ecology Notes vol 2 no 3 pp 265ndash2672002
[26] G Cipriani M T Marrazzo R Marconi A Cimato andR Testolin ldquoMicrosatellite markers isolated in olive (Oleaeuropaea L) are suitable for individual fingerprinting andreveal polymorphism within ancient cultivarsrdquoTheoretical andApplied Genetics vol 104 no 2-3 pp 223ndash228 2002
[27] F J Rohlf NTSYS-PC Numerical Taxonomy and MultivariateAnalysis System Version 202i Department of Ecology andEvolution State University of New York Setauket NY USA1998
[28] S M Sanzani C Montemurro V di Rienzo M Solfrizzo andA Ippolito ldquoGenetic structure and natural variation associatedwith host of origin in Penicillium expansum strains causing bluemouldrdquo International Journal of Food Microbiology vol 165 no2 pp 111ndash120 2013
Journal of Chemistry 7
[29] M Perez-Jimenez G Besnard G Dorado and P HernandezldquoVarietal tracing of virgin olive oil based on plastid DNAvariation profilingrdquo PLoS ONE vol 8 no 8 Article ID e705072013
[30] V Alba C Montemurro W Sabetta A Pasqualone andA Blanco ldquoSSR-based identification key of cultivars of Oleaeuropaea L diffused in Southern-Italyrdquo Scientia Horticulturaevol 123 no 1 pp 11ndash16 2009
[31] A D Kloosterman B Budowle and P Daselaar ldquoPCR-amplification and detection of the human D1S80 VNTR locusAmplification conditions population genetics and applicationin forensic analysisrdquo International Journal of LegalMedicine vol105 no 5 pp 257ndash264 1993
[32] J F MacKay C D Wright and R G Bonfiglioli ldquoA newapproach to varietal identification in plants by microsatellitehigh resolution melting analysis application to the verificationof grapevine and olive cultivarsrdquo Plant Methods vol 4 article 82008
[33] A Xanthopoulou I Ganopoulos G Koubouris et alldquoMicrosatellite high-resolutionmelting (SSR-HRM) analysis forgenotyping andmolecular characterization of anOlea europaeagermplasm collectionrdquo Plant Genetic Resources vol 12 no 3pp 273ndash277 2014
[34] I Ganopoulos A Argiriou and A Tsaftaris ldquoMicrosatellitehigh resolution melting (SSR-HRM) analysis for authenticitytesting of protected designation of origin (PDO) sweet cherryproductsrdquo Food Control vol 22 no 3-4 pp 532ndash541 2011
[35] I Ganopoulos A Argiriou and A Tsaftaris ldquoAdulterationsin Basmati rice detected quantitatively by combined use ofmicrosatellite and fragrance typing with High Resolution Melt-ing (HRM) analysisrdquo Food Chemistry vol 129 no 2 pp 652ndash659 2011
[36] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquoHumanMutation vol 30 no 6pp 857ndash859 2009
[37] M Vietina C Agrimonti and N Marmiroli ldquoDetection ofplant oil DNA using high resolution melting (HRM) post PCRanalysis a tool for disclosure of olive oil adulterationrdquo FoodChemistry vol 141 no 4 pp 3820ndash3826 2013
[38] I Ganopoulos C Bazakos P Madesis P Kalaitzis and A Tsaf-taris ldquoBarcode DNA high-resolution melting (Bar-HRM) anal-ysis as a novel close-tubed and accurate tool for olive oil forensicuserdquo Journal of the Science of Food and Agriculture vol 93no 9 pp 2281ndash2286 2013
[39] EMader J Ruzicka C Schmiderer and J Novak ldquoQuantitativehigh-resolution melting analysis for detecting adulterationsrdquoAnalytical Biochemistry vol 409 no 1 pp 153ndash155 2011
Table 1 Locus name repeat motif annealing temperature primer sequences referred to SSR markers and allelic profiles of the cultivarsincluded in the PDO Terra di Bari olive oil
Locus Repeat motif Primer sequence (51015840-31015840) Annealing temperature Allelic profiles (bp)Cima di Bitonto Ogliarola Barese Coratina
Coratina 100 266 40 40 90 80 70 60 50Ogliarola 100 80 266 40 40Cima di Bitonto 100 80 266 40 40Nociara 80 50Toscanina 80 50Aeleh 80 100 10 20 30 40 50Other cultivarslowastlowast 266 266 266 20 20 20 20 20 20 20lowastOn the bases of PDO ldquoTerra di Barirdquo procedure rules these commercial samples should bemade by 80 of Coratina Ogliarola Barese andor Cima di Bitontoalone or in mixture and a 20 of other cultivarslowastlowastMixture in equal proportion of cultivars Simone Cima di Mola Nociara and Toscanina
4 Journal of Chemistry
HRM reaction was performed in a final volume of 10 120583Lconsisting of 50 ng of genomic DNA 1 times Sso Fast EvaGreenMaster mix (Bio-Rad Hercules CA) and 025 120583M of eachprimer (Sigma-Aldrich St Louis MO) A No TemplateControl (NTC) was included in each run [28]
Amplification and HRM analysis were performed onCFX96 Touch Real Time PCR Detection System (Bio-RadHercules CA) and the cycling program consisted of a touch-down protocol 2min of initial denaturation at 98∘C followedby 5 cycles of denaturation at 98∘C for 8 sec annealing at56∘C for 8 sec (with decrement of 05∘C per cycle) andextension at 72∘C for 12 sec The annealing temperaturewas maintained at 54∘C for the successive 40 steps anddenaturation temperature was decreased to 95∘C acquiringfluorescent data at the end of each cycle The amplificationprotocol was immediately followed by the High ResolutionMelting steps of 95∘C for 10 sec cooling to 58∘C for 30 sec andraising the temperature from 65∘C to 95∘C with increasing of02∘C every 10 sec with fluorescence acquisition
After verification of robust amplification curves themelting curve stage was further analysed by CFX Managersoftware (Bio-Rad Hercules CA) The melt curve was nor-malized along the temperature axis (temperature shifting) topermit easy differentiation of DNA curve
3 Results and Discussion
Among the different types of genetic markers available forvarietal identification purposes the nuclear microsatellite orSSR (Simple Sequence Repeat) is the marker of choice largelyused and the only one accepted for forensic applications [29]This is essentially due to the numerous advantages intrinsic ofsuch marker codominant nature high polymorphism widedistribution across the genome and automated detection AsSSR can be used to distinguish olive varieties when DNA isextracted directly from olive oils and based on our previousworks [30] we identified the most informative and effectivemarkers (DCA04 and DCA18) to genotype the selectedcultivars showing a different allelic profiles (Table 1)
In this work SSR markers were directly applied to DNAextracted fromolive oil which is highly degraded and poor inquantityThebest approach is to select SSRmarkers providinga simple and reproducible pattern whilst they maintain theirinformativeness and efficiency The SSR markers used in thiswork were selected for their high polymorphism and for theclear allelic profile
All 17 SSRmarkers showed a very high value of PD (PowerDiscrimination) [31] with the maximum value of 088 scoredfor DCA18
Figure 1 reports the genetic similarity dendrogramobtained with 17 SSR markers The cultivars Toscanina andNociara showed the highest degree of diversity whereas theothers are separated in two subclusters In the first groupthere are Cima di Mola Ogliarola Maiatica and Simoneand in the second one there are Cellina di Nardo CoratinaCima di Bitonto and Toscanina All the cultivars showedsmall sized fruits characteristic of olive oil attitude cultivarswith Cima di Mola and Ogliarola and Coratina and Cima
Similarity coefficient000 020 040 060 080 100
Nociara
Cima di Mola
Ogliarola
Maiatica
Simone
Coratina
Cima di Bitonto
Toscanina
oCellina di Nard
Figure 1 Genetic similarity dendrogram of nine olive cultivarsobtained by the analysis of seventeen microsatellite markers
75 76 77 78 79
minus005
000
005
010
015
020
025
80 81 82
Diff
eren
ce R
FU
Temperature
Cima di Bitonto
Coratina
Ogliarola
Figure 2 Normalized difference curve of the three cultivarsCoratina Cima di Bitonto and Ogliarola obtained with the DCA18marker
di Bitonto that are very similar in the morphological traitsOgliarola and Cima di Bitonto are commonly consideredas synonymous referred to the same variety and also thedisciplinary of ldquoTerra di Barirdquo olive oil production induces inan ambiguous appellation of them not clarifying that are twodifferent cultivars The results of our analyses define Cima diBitonto and Ogliarola as two distinct cultivars as confirmedby capillary electrophoresis and HRM analysis (Figure 2)
Among the seventeen markers the DCA18 was the mostpolymorphic SSR able to discriminate all samples and forthis reason it was chosen to realize an identification key(Figure 3) This marker revealed 10 different amplicons thatare combined in 9 unique genotypes and for this reason wasselected for the HRM application In recent years differentauthors reported the use of HRM for the varietal identifica-tion [32 33] genotyping [28 34] and food traceability [35]The advantages of this technique that was originally con-ceived for the human diagnostic [36] such as absenceof manipulation after PCR cost effectiveness closed-tube
Journal of Chemistry 5
Simone
Maiatica
168
Ogliarola
172 Cima di Bitonto
174
Coratina
Cima di Mola
154
158 Nociara
Cellina
168 193
166 Toscanina
Figure 3 Identification key of nine olive cultivars obtained by theanalysis of DCA18 microsatellite markers
analysis and results obtainable in less than 3 hours arenowadays emerging in plant and food sector All the HRMexperiments were realized with the marker DCA18 and inFigure 4 is reported the difference curve of some Terra di Bariexperimental samples compared to commercial Terra di Bariolive oil and one experimental olive oil made up of NociaraandToscaninaTheplotwell established the difference amongthe Terra di Bari group (mixes 3 6 7 9 10 11 12) and non-Terra di Bari group (mix 13) The preparation of differentmixtures (Table 2) is essential to constitute a dataset of melt-ing curve profiles in order to perform a quick preliminaryanalysis and define the belonging of an unknown sample tothe Terra di Bari group specified and declared as well in thelabelThis analysismight be useful as first check of the varietalcomposition of olive oil especially when it is necessary toworkwith large number of samplesThehigh efficiency of dis-crimination of the technique already tested by Vietina et al[37] and Ganopoulos et al [38] in adulteration of olive oilby the addition of cheaper oils obtained from other plants(ie maize sunflower and soybean) is proved also in thevarietal identification if a cultivar is required and specifiedby production disciplinary A further application of HRMmethod is to quantify the presence of adulterants in literatureare reported several studies regarding the identification ofadulterants (ie different species or botanical varieties) andtheir quantification [35 38 39] Figure 5 showed the set-up of quantification of increasing addition of the Algeriancultivar Aeleh to a monovarietal Coratina olive oil It ispossible to appreciate the clear distinction between themonovarietal oil of Coratina and Aeleh showing differentmelting temperature and the placement of the different
76 77 78 79
minus005
minus010
minus015
000
005
Diff
eren
ce R
FU
Shifted temperature
Cima di Bitonto Mix 12
Mix 6Mix 10
Mix 11Mix 7
Mix 9Mix 13
Figure 4 Normalized and temperature-shifted difference curve ofdifferent commercial samples (mixes 11 and 12) Terra di Bari oils(monovarietal Cima di Bitonto oil and mixes 6 7 9 10) and non-Terra di Bari olive oil (mix 13)
770 775 780 785 790
minus002
minus004
minus006
000
004
002
006
008
Diff
eren
ce R
FU
Shifted temperature
Coratina
10 contamination
20 contamination 30 contamination
40 contamination
50 contaminationAeleh
Figure 5 Temperature-shifted difference curve obtained with theaddition of increasing quantity of cultivar Aeleh in Coratina mono-varietal oil
level of adulteration (10ndash20ndash30ndash40ndash50) between themTheobtained results confirmed the possibility to use the HRMtechnique not only for a qualitative application but also fora quantitative detection of the addiction of different amountsof olive oils produced by cultivars not allowed in the PDOdisciplinary production
4 Conclusion
This molecular analysis allowed the distinction of the culti-vars included in the ldquoTerra di Barirdquo PDO disciplinary withrespect to those widely diffused in the Apulian region Inaddition SSR markers were able to provide a specific profilefor Coratina Ogliarola Barese and Cima di Bitonto cultivarsThe production rules of ldquoTerra di Barirdquo olive oil consider thecultivar Cima di Bitonto and Ogliarola as two synonymousreferred to the same genotypes The allelic profiles obtainedwith capillary electrophoresis analysis clarify unambiguouslythat Cima di Bitonto and Ogliarola are two distinct varietiesand this aspect should be taken into account for an accuratereview of the disciplinary
These results should be useful in the agrofood com-partment where the application of molecular techniquescould lead to the identification of raw materials and derivedproducts allowing to trace origin and identity of cultivarsused for the obtainment of typical products
6 Journal of Chemistry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work has been carried out with financial support fromthe University of BarimdashProjects Idea Giovani 201011 andCofin PRIN 2009 coordinated by Dr Cinzia Montemurro
IDPagina7051[3] C Montealegre M L M Alegre and C Garcıa-Ruiz ldquoTrace-
ability markers to the botanical origin in olive oilsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 1 pp 28ndash38 2010
[4] M A Brescia G Alviti V Liuzzi and A Sacco ldquoChemometricsclassification of olive cultivars based on compositional data ofoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 80 no10 pp 945ndash950 2003
[5] T G Diaz I D Meras J S Casas and M F A Franco ldquoChar-acterization of virgin olive oils according to its triglycerides andsterols composition by chemometric methodsrdquo Food Controlvol 16 no 4 pp 339ndash347 2005
[6] M R Alves S C Cunha J S Amaral J A Pereira and M BOliveira ldquoClassification of PDO olive oils on the basis of theirsterol composition by multivariate analysisrdquo Analytica ChimicaActa vol 549 no 1-2 pp 166ndash178 2005
[7] I S Arvanitoyannis and A Vlachos ldquoImplementation ofphysicochemical and sensory analysis in conjunction withmultivariate analysis towards assessing olive oil authentica-tionadulterationrdquo Critical Reviews in Food Science and Nutri-tion vol 47 no 5 pp 441ndash498 2007
[8] S Pafundo C Agrimonti and N Marmiroli ldquoTraceability ofplant contribution in olive oil by amplified fragment lengthpolymorphismsrdquo Journal of Agricultural and Food Chemistryvol 53 no 18 pp 6995ndash7002 2005
[9] A Pasqualone C Montemurro C Summo W Sabetta FCaponio and A Blanco ldquoEffectiveness of microsatellite DNAmarkers in checking the identity of protected designation oforigin extra virgin olive oilrdquo Journal of Agricultural and FoodChemistry vol 55 no 10 pp 3857ndash3862 2007
[10] A Pasqualone V di Rienzo R Nasti A Blanco T Gomes andC Montemurro ldquoTraceability of Italian protected designationof origin (PDO) table olives by means of microsatellite molecu-lar markersrdquo Journal of Agricultural and Food Chemistry vol 61no 12 pp 3068ndash3073 2013
[11] M S Akkaya and E B Buyukunal-Bal ldquoAssessment of geneticvariation of bread wheat varieties using microsatellite markersrdquoEuphytica vol 135 no 2 pp 179ndash185 2004
[12] L Zeng T-R Kwon X Liu C Wilson C M Grieve andG B Gregorio ldquoGenetic diversity analyzed by microsatellitemarkers among rice (Oryza sativa L) genotypes with differentadaptations to saline soilsrdquo Plant Science vol 166 no 5 pp1275ndash1285 2004
[13] G Cipriani A Spadotto I Jurman et al ldquoThe SSR-basedmolecular profile of 1005 grapevine (Vitis vinifera L) accessionsuncovers new synonymy and parentages and reveals a large
admixture amongst varieties of different geographic originrdquoTheoretical and Applied Genetics vol 121 no 8 pp 1569ndash15852010
[14] G M M Bredemeijer R J Cooke M W Ganal et al ldquoCon-struction and testing of a microsatellite database containingmore than 500 tomato varietiesrdquoTheoretical and Applied Genet-ics vol 105 no 6-7 pp 1019ndash1026 2002
[15] K M Sefc M S Lopes D Mendonca M Rodrigues DosSantos M Laimer Da Camara Machado and A Da CamaraMachado ldquoIdentification of microsatellite loci in olive (Oleaeuropaea) and their characterization in Italian and Iberian olivetreesrdquoMolecular Ecology vol 9 no 8 pp 1171ndash1173 2000
[16] C Montemurro R Simeone A Pasqualone E Ferrara andA Blanco ldquoGenetic relationships and cultivar identificationamong 112 olive accessions using AFLP and SSR markersrdquo TheJournal of Horticultural Science and Biotechnology vol 80 no 1pp 105ndash110 2005
[17] M Hosseini-Mazinani R Mariotti B Torkzaban et al ldquoHighgenetic diversity detected in olives beyond the boundaries of theMediterranean seardquo PLoS ONE vol 9 no 4 Article ID e931462014
[18] D Scarano and R Rao ldquoDNA markers for food productsauthenticationrdquo Diversity vol 6 no 3 pp 579ndash596 2014
[19] A Pasqualone C Montemurro F Caponio and A BlancoldquoIdentification of virgin olive oil from different cultivars byanalysis of dnamicrosatellitesrdquo Journal of Agricultural and FoodChemistry vol 52 no 5 pp 1068ndash1071 2004
[20] httpwwwunaprolit[21] J T Li J Yang D C Chen X L Zhang and Z S Tang
ldquoAn optimizedmini-preparationmethod to obtain high-qualitygenomic DNA from mature leaves of sunflowerrdquo Genetics andMolecular Research vol 6 supplement 4 pp 1064ndash1071 2007
[22] W Sabetta V Alba A Blanco and C Montemurro ldquoSunTILLa TILLING resource for gene function analysis in sunflowerrdquoPlant Methods vol 7 article 20 2011
[23] A Pasqualone V di Rienzo A Blanco C Summo F Caponioand C Montemurro ldquoCharacterization of virgin olive oil fromLeucocarpa cultivar by chemical and DNA analysisrdquo FoodResearch International vol 47 no 2 pp 188ndash193 2012
[24] F Carriero G Fontanazza F Cellini and G Giorio ldquoIdentifica-tion of simple sequence repeats (SSRs) in olive (Olea europaeaL)rdquoTheoretical and Applied Genetics vol 104 no 2-3 pp 301ndash307 2002
[25] R de la Rosa C M James and K R Tobutt ldquoIsolation andcharacterization of polymorphic microsatellites in olive (Oleaeuropaea L) and their transferability to other genera in theOleaceaerdquo Molecular Ecology Notes vol 2 no 3 pp 265ndash2672002
[26] G Cipriani M T Marrazzo R Marconi A Cimato andR Testolin ldquoMicrosatellite markers isolated in olive (Oleaeuropaea L) are suitable for individual fingerprinting andreveal polymorphism within ancient cultivarsrdquoTheoretical andApplied Genetics vol 104 no 2-3 pp 223ndash228 2002
[27] F J Rohlf NTSYS-PC Numerical Taxonomy and MultivariateAnalysis System Version 202i Department of Ecology andEvolution State University of New York Setauket NY USA1998
[28] S M Sanzani C Montemurro V di Rienzo M Solfrizzo andA Ippolito ldquoGenetic structure and natural variation associatedwith host of origin in Penicillium expansum strains causing bluemouldrdquo International Journal of Food Microbiology vol 165 no2 pp 111ndash120 2013
Journal of Chemistry 7
[29] M Perez-Jimenez G Besnard G Dorado and P HernandezldquoVarietal tracing of virgin olive oil based on plastid DNAvariation profilingrdquo PLoS ONE vol 8 no 8 Article ID e705072013
[30] V Alba C Montemurro W Sabetta A Pasqualone andA Blanco ldquoSSR-based identification key of cultivars of Oleaeuropaea L diffused in Southern-Italyrdquo Scientia Horticulturaevol 123 no 1 pp 11ndash16 2009
[31] A D Kloosterman B Budowle and P Daselaar ldquoPCR-amplification and detection of the human D1S80 VNTR locusAmplification conditions population genetics and applicationin forensic analysisrdquo International Journal of LegalMedicine vol105 no 5 pp 257ndash264 1993
[32] J F MacKay C D Wright and R G Bonfiglioli ldquoA newapproach to varietal identification in plants by microsatellitehigh resolution melting analysis application to the verificationof grapevine and olive cultivarsrdquo Plant Methods vol 4 article 82008
[33] A Xanthopoulou I Ganopoulos G Koubouris et alldquoMicrosatellite high-resolutionmelting (SSR-HRM) analysis forgenotyping andmolecular characterization of anOlea europaeagermplasm collectionrdquo Plant Genetic Resources vol 12 no 3pp 273ndash277 2014
[34] I Ganopoulos A Argiriou and A Tsaftaris ldquoMicrosatellitehigh resolution melting (SSR-HRM) analysis for authenticitytesting of protected designation of origin (PDO) sweet cherryproductsrdquo Food Control vol 22 no 3-4 pp 532ndash541 2011
[35] I Ganopoulos A Argiriou and A Tsaftaris ldquoAdulterationsin Basmati rice detected quantitatively by combined use ofmicrosatellite and fragrance typing with High Resolution Melt-ing (HRM) analysisrdquo Food Chemistry vol 129 no 2 pp 652ndash659 2011
[36] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquoHumanMutation vol 30 no 6pp 857ndash859 2009
[37] M Vietina C Agrimonti and N Marmiroli ldquoDetection ofplant oil DNA using high resolution melting (HRM) post PCRanalysis a tool for disclosure of olive oil adulterationrdquo FoodChemistry vol 141 no 4 pp 3820ndash3826 2013
[38] I Ganopoulos C Bazakos P Madesis P Kalaitzis and A Tsaf-taris ldquoBarcode DNA high-resolution melting (Bar-HRM) anal-ysis as a novel close-tubed and accurate tool for olive oil forensicuserdquo Journal of the Science of Food and Agriculture vol 93no 9 pp 2281ndash2286 2013
[39] EMader J Ruzicka C Schmiderer and J Novak ldquoQuantitativehigh-resolution melting analysis for detecting adulterationsrdquoAnalytical Biochemistry vol 409 no 1 pp 153ndash155 2011
HRM reaction was performed in a final volume of 10 120583Lconsisting of 50 ng of genomic DNA 1 times Sso Fast EvaGreenMaster mix (Bio-Rad Hercules CA) and 025 120583M of eachprimer (Sigma-Aldrich St Louis MO) A No TemplateControl (NTC) was included in each run [28]
Amplification and HRM analysis were performed onCFX96 Touch Real Time PCR Detection System (Bio-RadHercules CA) and the cycling program consisted of a touch-down protocol 2min of initial denaturation at 98∘C followedby 5 cycles of denaturation at 98∘C for 8 sec annealing at56∘C for 8 sec (with decrement of 05∘C per cycle) andextension at 72∘C for 12 sec The annealing temperaturewas maintained at 54∘C for the successive 40 steps anddenaturation temperature was decreased to 95∘C acquiringfluorescent data at the end of each cycle The amplificationprotocol was immediately followed by the High ResolutionMelting steps of 95∘C for 10 sec cooling to 58∘C for 30 sec andraising the temperature from 65∘C to 95∘C with increasing of02∘C every 10 sec with fluorescence acquisition
After verification of robust amplification curves themelting curve stage was further analysed by CFX Managersoftware (Bio-Rad Hercules CA) The melt curve was nor-malized along the temperature axis (temperature shifting) topermit easy differentiation of DNA curve
3 Results and Discussion
Among the different types of genetic markers available forvarietal identification purposes the nuclear microsatellite orSSR (Simple Sequence Repeat) is the marker of choice largelyused and the only one accepted for forensic applications [29]This is essentially due to the numerous advantages intrinsic ofsuch marker codominant nature high polymorphism widedistribution across the genome and automated detection AsSSR can be used to distinguish olive varieties when DNA isextracted directly from olive oils and based on our previousworks [30] we identified the most informative and effectivemarkers (DCA04 and DCA18) to genotype the selectedcultivars showing a different allelic profiles (Table 1)
In this work SSR markers were directly applied to DNAextracted fromolive oil which is highly degraded and poor inquantityThebest approach is to select SSRmarkers providinga simple and reproducible pattern whilst they maintain theirinformativeness and efficiency The SSR markers used in thiswork were selected for their high polymorphism and for theclear allelic profile
All 17 SSRmarkers showed a very high value of PD (PowerDiscrimination) [31] with the maximum value of 088 scoredfor DCA18
Figure 1 reports the genetic similarity dendrogramobtained with 17 SSR markers The cultivars Toscanina andNociara showed the highest degree of diversity whereas theothers are separated in two subclusters In the first groupthere are Cima di Mola Ogliarola Maiatica and Simoneand in the second one there are Cellina di Nardo CoratinaCima di Bitonto and Toscanina All the cultivars showedsmall sized fruits characteristic of olive oil attitude cultivarswith Cima di Mola and Ogliarola and Coratina and Cima
Similarity coefficient000 020 040 060 080 100
Nociara
Cima di Mola
Ogliarola
Maiatica
Simone
Coratina
Cima di Bitonto
Toscanina
oCellina di Nard
Figure 1 Genetic similarity dendrogram of nine olive cultivarsobtained by the analysis of seventeen microsatellite markers
75 76 77 78 79
minus005
000
005
010
015
020
025
80 81 82
Diff
eren
ce R
FU
Temperature
Cima di Bitonto
Coratina
Ogliarola
Figure 2 Normalized difference curve of the three cultivarsCoratina Cima di Bitonto and Ogliarola obtained with the DCA18marker
di Bitonto that are very similar in the morphological traitsOgliarola and Cima di Bitonto are commonly consideredas synonymous referred to the same variety and also thedisciplinary of ldquoTerra di Barirdquo olive oil production induces inan ambiguous appellation of them not clarifying that are twodifferent cultivars The results of our analyses define Cima diBitonto and Ogliarola as two distinct cultivars as confirmedby capillary electrophoresis and HRM analysis (Figure 2)
Among the seventeen markers the DCA18 was the mostpolymorphic SSR able to discriminate all samples and forthis reason it was chosen to realize an identification key(Figure 3) This marker revealed 10 different amplicons thatare combined in 9 unique genotypes and for this reason wasselected for the HRM application In recent years differentauthors reported the use of HRM for the varietal identifica-tion [32 33] genotyping [28 34] and food traceability [35]The advantages of this technique that was originally con-ceived for the human diagnostic [36] such as absenceof manipulation after PCR cost effectiveness closed-tube
Journal of Chemistry 5
Simone
Maiatica
168
Ogliarola
172 Cima di Bitonto
174
Coratina
Cima di Mola
154
158 Nociara
Cellina
168 193
166 Toscanina
Figure 3 Identification key of nine olive cultivars obtained by theanalysis of DCA18 microsatellite markers
analysis and results obtainable in less than 3 hours arenowadays emerging in plant and food sector All the HRMexperiments were realized with the marker DCA18 and inFigure 4 is reported the difference curve of some Terra di Bariexperimental samples compared to commercial Terra di Bariolive oil and one experimental olive oil made up of NociaraandToscaninaTheplotwell established the difference amongthe Terra di Bari group (mixes 3 6 7 9 10 11 12) and non-Terra di Bari group (mix 13) The preparation of differentmixtures (Table 2) is essential to constitute a dataset of melt-ing curve profiles in order to perform a quick preliminaryanalysis and define the belonging of an unknown sample tothe Terra di Bari group specified and declared as well in thelabelThis analysismight be useful as first check of the varietalcomposition of olive oil especially when it is necessary toworkwith large number of samplesThehigh efficiency of dis-crimination of the technique already tested by Vietina et al[37] and Ganopoulos et al [38] in adulteration of olive oilby the addition of cheaper oils obtained from other plants(ie maize sunflower and soybean) is proved also in thevarietal identification if a cultivar is required and specifiedby production disciplinary A further application of HRMmethod is to quantify the presence of adulterants in literatureare reported several studies regarding the identification ofadulterants (ie different species or botanical varieties) andtheir quantification [35 38 39] Figure 5 showed the set-up of quantification of increasing addition of the Algeriancultivar Aeleh to a monovarietal Coratina olive oil It ispossible to appreciate the clear distinction between themonovarietal oil of Coratina and Aeleh showing differentmelting temperature and the placement of the different
76 77 78 79
minus005
minus010
minus015
000
005
Diff
eren
ce R
FU
Shifted temperature
Cima di Bitonto Mix 12
Mix 6Mix 10
Mix 11Mix 7
Mix 9Mix 13
Figure 4 Normalized and temperature-shifted difference curve ofdifferent commercial samples (mixes 11 and 12) Terra di Bari oils(monovarietal Cima di Bitonto oil and mixes 6 7 9 10) and non-Terra di Bari olive oil (mix 13)
770 775 780 785 790
minus002
minus004
minus006
000
004
002
006
008
Diff
eren
ce R
FU
Shifted temperature
Coratina
10 contamination
20 contamination 30 contamination
40 contamination
50 contaminationAeleh
Figure 5 Temperature-shifted difference curve obtained with theaddition of increasing quantity of cultivar Aeleh in Coratina mono-varietal oil
level of adulteration (10ndash20ndash30ndash40ndash50) between themTheobtained results confirmed the possibility to use the HRMtechnique not only for a qualitative application but also fora quantitative detection of the addiction of different amountsof olive oils produced by cultivars not allowed in the PDOdisciplinary production
4 Conclusion
This molecular analysis allowed the distinction of the culti-vars included in the ldquoTerra di Barirdquo PDO disciplinary withrespect to those widely diffused in the Apulian region Inaddition SSR markers were able to provide a specific profilefor Coratina Ogliarola Barese and Cima di Bitonto cultivarsThe production rules of ldquoTerra di Barirdquo olive oil consider thecultivar Cima di Bitonto and Ogliarola as two synonymousreferred to the same genotypes The allelic profiles obtainedwith capillary electrophoresis analysis clarify unambiguouslythat Cima di Bitonto and Ogliarola are two distinct varietiesand this aspect should be taken into account for an accuratereview of the disciplinary
These results should be useful in the agrofood com-partment where the application of molecular techniquescould lead to the identification of raw materials and derivedproducts allowing to trace origin and identity of cultivarsused for the obtainment of typical products
6 Journal of Chemistry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work has been carried out with financial support fromthe University of BarimdashProjects Idea Giovani 201011 andCofin PRIN 2009 coordinated by Dr Cinzia Montemurro
IDPagina7051[3] C Montealegre M L M Alegre and C Garcıa-Ruiz ldquoTrace-
ability markers to the botanical origin in olive oilsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 1 pp 28ndash38 2010
[4] M A Brescia G Alviti V Liuzzi and A Sacco ldquoChemometricsclassification of olive cultivars based on compositional data ofoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 80 no10 pp 945ndash950 2003
[5] T G Diaz I D Meras J S Casas and M F A Franco ldquoChar-acterization of virgin olive oils according to its triglycerides andsterols composition by chemometric methodsrdquo Food Controlvol 16 no 4 pp 339ndash347 2005
[6] M R Alves S C Cunha J S Amaral J A Pereira and M BOliveira ldquoClassification of PDO olive oils on the basis of theirsterol composition by multivariate analysisrdquo Analytica ChimicaActa vol 549 no 1-2 pp 166ndash178 2005
[7] I S Arvanitoyannis and A Vlachos ldquoImplementation ofphysicochemical and sensory analysis in conjunction withmultivariate analysis towards assessing olive oil authentica-tionadulterationrdquo Critical Reviews in Food Science and Nutri-tion vol 47 no 5 pp 441ndash498 2007
[8] S Pafundo C Agrimonti and N Marmiroli ldquoTraceability ofplant contribution in olive oil by amplified fragment lengthpolymorphismsrdquo Journal of Agricultural and Food Chemistryvol 53 no 18 pp 6995ndash7002 2005
[9] A Pasqualone C Montemurro C Summo W Sabetta FCaponio and A Blanco ldquoEffectiveness of microsatellite DNAmarkers in checking the identity of protected designation oforigin extra virgin olive oilrdquo Journal of Agricultural and FoodChemistry vol 55 no 10 pp 3857ndash3862 2007
[10] A Pasqualone V di Rienzo R Nasti A Blanco T Gomes andC Montemurro ldquoTraceability of Italian protected designationof origin (PDO) table olives by means of microsatellite molecu-lar markersrdquo Journal of Agricultural and Food Chemistry vol 61no 12 pp 3068ndash3073 2013
[11] M S Akkaya and E B Buyukunal-Bal ldquoAssessment of geneticvariation of bread wheat varieties using microsatellite markersrdquoEuphytica vol 135 no 2 pp 179ndash185 2004
[12] L Zeng T-R Kwon X Liu C Wilson C M Grieve andG B Gregorio ldquoGenetic diversity analyzed by microsatellitemarkers among rice (Oryza sativa L) genotypes with differentadaptations to saline soilsrdquo Plant Science vol 166 no 5 pp1275ndash1285 2004
[13] G Cipriani A Spadotto I Jurman et al ldquoThe SSR-basedmolecular profile of 1005 grapevine (Vitis vinifera L) accessionsuncovers new synonymy and parentages and reveals a large
admixture amongst varieties of different geographic originrdquoTheoretical and Applied Genetics vol 121 no 8 pp 1569ndash15852010
[14] G M M Bredemeijer R J Cooke M W Ganal et al ldquoCon-struction and testing of a microsatellite database containingmore than 500 tomato varietiesrdquoTheoretical and Applied Genet-ics vol 105 no 6-7 pp 1019ndash1026 2002
[15] K M Sefc M S Lopes D Mendonca M Rodrigues DosSantos M Laimer Da Camara Machado and A Da CamaraMachado ldquoIdentification of microsatellite loci in olive (Oleaeuropaea) and their characterization in Italian and Iberian olivetreesrdquoMolecular Ecology vol 9 no 8 pp 1171ndash1173 2000
[16] C Montemurro R Simeone A Pasqualone E Ferrara andA Blanco ldquoGenetic relationships and cultivar identificationamong 112 olive accessions using AFLP and SSR markersrdquo TheJournal of Horticultural Science and Biotechnology vol 80 no 1pp 105ndash110 2005
[17] M Hosseini-Mazinani R Mariotti B Torkzaban et al ldquoHighgenetic diversity detected in olives beyond the boundaries of theMediterranean seardquo PLoS ONE vol 9 no 4 Article ID e931462014
[18] D Scarano and R Rao ldquoDNA markers for food productsauthenticationrdquo Diversity vol 6 no 3 pp 579ndash596 2014
[19] A Pasqualone C Montemurro F Caponio and A BlancoldquoIdentification of virgin olive oil from different cultivars byanalysis of dnamicrosatellitesrdquo Journal of Agricultural and FoodChemistry vol 52 no 5 pp 1068ndash1071 2004
[20] httpwwwunaprolit[21] J T Li J Yang D C Chen X L Zhang and Z S Tang
ldquoAn optimizedmini-preparationmethod to obtain high-qualitygenomic DNA from mature leaves of sunflowerrdquo Genetics andMolecular Research vol 6 supplement 4 pp 1064ndash1071 2007
[22] W Sabetta V Alba A Blanco and C Montemurro ldquoSunTILLa TILLING resource for gene function analysis in sunflowerrdquoPlant Methods vol 7 article 20 2011
[23] A Pasqualone V di Rienzo A Blanco C Summo F Caponioand C Montemurro ldquoCharacterization of virgin olive oil fromLeucocarpa cultivar by chemical and DNA analysisrdquo FoodResearch International vol 47 no 2 pp 188ndash193 2012
[24] F Carriero G Fontanazza F Cellini and G Giorio ldquoIdentifica-tion of simple sequence repeats (SSRs) in olive (Olea europaeaL)rdquoTheoretical and Applied Genetics vol 104 no 2-3 pp 301ndash307 2002
[25] R de la Rosa C M James and K R Tobutt ldquoIsolation andcharacterization of polymorphic microsatellites in olive (Oleaeuropaea L) and their transferability to other genera in theOleaceaerdquo Molecular Ecology Notes vol 2 no 3 pp 265ndash2672002
[26] G Cipriani M T Marrazzo R Marconi A Cimato andR Testolin ldquoMicrosatellite markers isolated in olive (Oleaeuropaea L) are suitable for individual fingerprinting andreveal polymorphism within ancient cultivarsrdquoTheoretical andApplied Genetics vol 104 no 2-3 pp 223ndash228 2002
[27] F J Rohlf NTSYS-PC Numerical Taxonomy and MultivariateAnalysis System Version 202i Department of Ecology andEvolution State University of New York Setauket NY USA1998
[28] S M Sanzani C Montemurro V di Rienzo M Solfrizzo andA Ippolito ldquoGenetic structure and natural variation associatedwith host of origin in Penicillium expansum strains causing bluemouldrdquo International Journal of Food Microbiology vol 165 no2 pp 111ndash120 2013
Journal of Chemistry 7
[29] M Perez-Jimenez G Besnard G Dorado and P HernandezldquoVarietal tracing of virgin olive oil based on plastid DNAvariation profilingrdquo PLoS ONE vol 8 no 8 Article ID e705072013
[30] V Alba C Montemurro W Sabetta A Pasqualone andA Blanco ldquoSSR-based identification key of cultivars of Oleaeuropaea L diffused in Southern-Italyrdquo Scientia Horticulturaevol 123 no 1 pp 11ndash16 2009
[31] A D Kloosterman B Budowle and P Daselaar ldquoPCR-amplification and detection of the human D1S80 VNTR locusAmplification conditions population genetics and applicationin forensic analysisrdquo International Journal of LegalMedicine vol105 no 5 pp 257ndash264 1993
[32] J F MacKay C D Wright and R G Bonfiglioli ldquoA newapproach to varietal identification in plants by microsatellitehigh resolution melting analysis application to the verificationof grapevine and olive cultivarsrdquo Plant Methods vol 4 article 82008
[33] A Xanthopoulou I Ganopoulos G Koubouris et alldquoMicrosatellite high-resolutionmelting (SSR-HRM) analysis forgenotyping andmolecular characterization of anOlea europaeagermplasm collectionrdquo Plant Genetic Resources vol 12 no 3pp 273ndash277 2014
[34] I Ganopoulos A Argiriou and A Tsaftaris ldquoMicrosatellitehigh resolution melting (SSR-HRM) analysis for authenticitytesting of protected designation of origin (PDO) sweet cherryproductsrdquo Food Control vol 22 no 3-4 pp 532ndash541 2011
[35] I Ganopoulos A Argiriou and A Tsaftaris ldquoAdulterationsin Basmati rice detected quantitatively by combined use ofmicrosatellite and fragrance typing with High Resolution Melt-ing (HRM) analysisrdquo Food Chemistry vol 129 no 2 pp 652ndash659 2011
[36] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquoHumanMutation vol 30 no 6pp 857ndash859 2009
[37] M Vietina C Agrimonti and N Marmiroli ldquoDetection ofplant oil DNA using high resolution melting (HRM) post PCRanalysis a tool for disclosure of olive oil adulterationrdquo FoodChemistry vol 141 no 4 pp 3820ndash3826 2013
[38] I Ganopoulos C Bazakos P Madesis P Kalaitzis and A Tsaf-taris ldquoBarcode DNA high-resolution melting (Bar-HRM) anal-ysis as a novel close-tubed and accurate tool for olive oil forensicuserdquo Journal of the Science of Food and Agriculture vol 93no 9 pp 2281ndash2286 2013
[39] EMader J Ruzicka C Schmiderer and J Novak ldquoQuantitativehigh-resolution melting analysis for detecting adulterationsrdquoAnalytical Biochemistry vol 409 no 1 pp 153ndash155 2011
Figure 3 Identification key of nine olive cultivars obtained by theanalysis of DCA18 microsatellite markers
analysis and results obtainable in less than 3 hours arenowadays emerging in plant and food sector All the HRMexperiments were realized with the marker DCA18 and inFigure 4 is reported the difference curve of some Terra di Bariexperimental samples compared to commercial Terra di Bariolive oil and one experimental olive oil made up of NociaraandToscaninaTheplotwell established the difference amongthe Terra di Bari group (mixes 3 6 7 9 10 11 12) and non-Terra di Bari group (mix 13) The preparation of differentmixtures (Table 2) is essential to constitute a dataset of melt-ing curve profiles in order to perform a quick preliminaryanalysis and define the belonging of an unknown sample tothe Terra di Bari group specified and declared as well in thelabelThis analysismight be useful as first check of the varietalcomposition of olive oil especially when it is necessary toworkwith large number of samplesThehigh efficiency of dis-crimination of the technique already tested by Vietina et al[37] and Ganopoulos et al [38] in adulteration of olive oilby the addition of cheaper oils obtained from other plants(ie maize sunflower and soybean) is proved also in thevarietal identification if a cultivar is required and specifiedby production disciplinary A further application of HRMmethod is to quantify the presence of adulterants in literatureare reported several studies regarding the identification ofadulterants (ie different species or botanical varieties) andtheir quantification [35 38 39] Figure 5 showed the set-up of quantification of increasing addition of the Algeriancultivar Aeleh to a monovarietal Coratina olive oil It ispossible to appreciate the clear distinction between themonovarietal oil of Coratina and Aeleh showing differentmelting temperature and the placement of the different
76 77 78 79
minus005
minus010
minus015
000
005
Diff
eren
ce R
FU
Shifted temperature
Cima di Bitonto Mix 12
Mix 6Mix 10
Mix 11Mix 7
Mix 9Mix 13
Figure 4 Normalized and temperature-shifted difference curve ofdifferent commercial samples (mixes 11 and 12) Terra di Bari oils(monovarietal Cima di Bitonto oil and mixes 6 7 9 10) and non-Terra di Bari olive oil (mix 13)
770 775 780 785 790
minus002
minus004
minus006
000
004
002
006
008
Diff
eren
ce R
FU
Shifted temperature
Coratina
10 contamination
20 contamination 30 contamination
40 contamination
50 contaminationAeleh
Figure 5 Temperature-shifted difference curve obtained with theaddition of increasing quantity of cultivar Aeleh in Coratina mono-varietal oil
level of adulteration (10ndash20ndash30ndash40ndash50) between themTheobtained results confirmed the possibility to use the HRMtechnique not only for a qualitative application but also fora quantitative detection of the addiction of different amountsof olive oils produced by cultivars not allowed in the PDOdisciplinary production
4 Conclusion
This molecular analysis allowed the distinction of the culti-vars included in the ldquoTerra di Barirdquo PDO disciplinary withrespect to those widely diffused in the Apulian region Inaddition SSR markers were able to provide a specific profilefor Coratina Ogliarola Barese and Cima di Bitonto cultivarsThe production rules of ldquoTerra di Barirdquo olive oil consider thecultivar Cima di Bitonto and Ogliarola as two synonymousreferred to the same genotypes The allelic profiles obtainedwith capillary electrophoresis analysis clarify unambiguouslythat Cima di Bitonto and Ogliarola are two distinct varietiesand this aspect should be taken into account for an accuratereview of the disciplinary
These results should be useful in the agrofood com-partment where the application of molecular techniquescould lead to the identification of raw materials and derivedproducts allowing to trace origin and identity of cultivarsused for the obtainment of typical products
6 Journal of Chemistry
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work has been carried out with financial support fromthe University of BarimdashProjects Idea Giovani 201011 andCofin PRIN 2009 coordinated by Dr Cinzia Montemurro
IDPagina7051[3] C Montealegre M L M Alegre and C Garcıa-Ruiz ldquoTrace-
ability markers to the botanical origin in olive oilsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 1 pp 28ndash38 2010
[4] M A Brescia G Alviti V Liuzzi and A Sacco ldquoChemometricsclassification of olive cultivars based on compositional data ofoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 80 no10 pp 945ndash950 2003
[5] T G Diaz I D Meras J S Casas and M F A Franco ldquoChar-acterization of virgin olive oils according to its triglycerides andsterols composition by chemometric methodsrdquo Food Controlvol 16 no 4 pp 339ndash347 2005
[6] M R Alves S C Cunha J S Amaral J A Pereira and M BOliveira ldquoClassification of PDO olive oils on the basis of theirsterol composition by multivariate analysisrdquo Analytica ChimicaActa vol 549 no 1-2 pp 166ndash178 2005
[7] I S Arvanitoyannis and A Vlachos ldquoImplementation ofphysicochemical and sensory analysis in conjunction withmultivariate analysis towards assessing olive oil authentica-tionadulterationrdquo Critical Reviews in Food Science and Nutri-tion vol 47 no 5 pp 441ndash498 2007
[8] S Pafundo C Agrimonti and N Marmiroli ldquoTraceability ofplant contribution in olive oil by amplified fragment lengthpolymorphismsrdquo Journal of Agricultural and Food Chemistryvol 53 no 18 pp 6995ndash7002 2005
[9] A Pasqualone C Montemurro C Summo W Sabetta FCaponio and A Blanco ldquoEffectiveness of microsatellite DNAmarkers in checking the identity of protected designation oforigin extra virgin olive oilrdquo Journal of Agricultural and FoodChemistry vol 55 no 10 pp 3857ndash3862 2007
[10] A Pasqualone V di Rienzo R Nasti A Blanco T Gomes andC Montemurro ldquoTraceability of Italian protected designationof origin (PDO) table olives by means of microsatellite molecu-lar markersrdquo Journal of Agricultural and Food Chemistry vol 61no 12 pp 3068ndash3073 2013
[11] M S Akkaya and E B Buyukunal-Bal ldquoAssessment of geneticvariation of bread wheat varieties using microsatellite markersrdquoEuphytica vol 135 no 2 pp 179ndash185 2004
[12] L Zeng T-R Kwon X Liu C Wilson C M Grieve andG B Gregorio ldquoGenetic diversity analyzed by microsatellitemarkers among rice (Oryza sativa L) genotypes with differentadaptations to saline soilsrdquo Plant Science vol 166 no 5 pp1275ndash1285 2004
[13] G Cipriani A Spadotto I Jurman et al ldquoThe SSR-basedmolecular profile of 1005 grapevine (Vitis vinifera L) accessionsuncovers new synonymy and parentages and reveals a large
admixture amongst varieties of different geographic originrdquoTheoretical and Applied Genetics vol 121 no 8 pp 1569ndash15852010
[14] G M M Bredemeijer R J Cooke M W Ganal et al ldquoCon-struction and testing of a microsatellite database containingmore than 500 tomato varietiesrdquoTheoretical and Applied Genet-ics vol 105 no 6-7 pp 1019ndash1026 2002
[15] K M Sefc M S Lopes D Mendonca M Rodrigues DosSantos M Laimer Da Camara Machado and A Da CamaraMachado ldquoIdentification of microsatellite loci in olive (Oleaeuropaea) and their characterization in Italian and Iberian olivetreesrdquoMolecular Ecology vol 9 no 8 pp 1171ndash1173 2000
[16] C Montemurro R Simeone A Pasqualone E Ferrara andA Blanco ldquoGenetic relationships and cultivar identificationamong 112 olive accessions using AFLP and SSR markersrdquo TheJournal of Horticultural Science and Biotechnology vol 80 no 1pp 105ndash110 2005
[17] M Hosseini-Mazinani R Mariotti B Torkzaban et al ldquoHighgenetic diversity detected in olives beyond the boundaries of theMediterranean seardquo PLoS ONE vol 9 no 4 Article ID e931462014
[18] D Scarano and R Rao ldquoDNA markers for food productsauthenticationrdquo Diversity vol 6 no 3 pp 579ndash596 2014
[19] A Pasqualone C Montemurro F Caponio and A BlancoldquoIdentification of virgin olive oil from different cultivars byanalysis of dnamicrosatellitesrdquo Journal of Agricultural and FoodChemistry vol 52 no 5 pp 1068ndash1071 2004
[20] httpwwwunaprolit[21] J T Li J Yang D C Chen X L Zhang and Z S Tang
ldquoAn optimizedmini-preparationmethod to obtain high-qualitygenomic DNA from mature leaves of sunflowerrdquo Genetics andMolecular Research vol 6 supplement 4 pp 1064ndash1071 2007
[22] W Sabetta V Alba A Blanco and C Montemurro ldquoSunTILLa TILLING resource for gene function analysis in sunflowerrdquoPlant Methods vol 7 article 20 2011
[23] A Pasqualone V di Rienzo A Blanco C Summo F Caponioand C Montemurro ldquoCharacterization of virgin olive oil fromLeucocarpa cultivar by chemical and DNA analysisrdquo FoodResearch International vol 47 no 2 pp 188ndash193 2012
[24] F Carriero G Fontanazza F Cellini and G Giorio ldquoIdentifica-tion of simple sequence repeats (SSRs) in olive (Olea europaeaL)rdquoTheoretical and Applied Genetics vol 104 no 2-3 pp 301ndash307 2002
[25] R de la Rosa C M James and K R Tobutt ldquoIsolation andcharacterization of polymorphic microsatellites in olive (Oleaeuropaea L) and their transferability to other genera in theOleaceaerdquo Molecular Ecology Notes vol 2 no 3 pp 265ndash2672002
[26] G Cipriani M T Marrazzo R Marconi A Cimato andR Testolin ldquoMicrosatellite markers isolated in olive (Oleaeuropaea L) are suitable for individual fingerprinting andreveal polymorphism within ancient cultivarsrdquoTheoretical andApplied Genetics vol 104 no 2-3 pp 223ndash228 2002
[27] F J Rohlf NTSYS-PC Numerical Taxonomy and MultivariateAnalysis System Version 202i Department of Ecology andEvolution State University of New York Setauket NY USA1998
[28] S M Sanzani C Montemurro V di Rienzo M Solfrizzo andA Ippolito ldquoGenetic structure and natural variation associatedwith host of origin in Penicillium expansum strains causing bluemouldrdquo International Journal of Food Microbiology vol 165 no2 pp 111ndash120 2013
Journal of Chemistry 7
[29] M Perez-Jimenez G Besnard G Dorado and P HernandezldquoVarietal tracing of virgin olive oil based on plastid DNAvariation profilingrdquo PLoS ONE vol 8 no 8 Article ID e705072013
[30] V Alba C Montemurro W Sabetta A Pasqualone andA Blanco ldquoSSR-based identification key of cultivars of Oleaeuropaea L diffused in Southern-Italyrdquo Scientia Horticulturaevol 123 no 1 pp 11ndash16 2009
[31] A D Kloosterman B Budowle and P Daselaar ldquoPCR-amplification and detection of the human D1S80 VNTR locusAmplification conditions population genetics and applicationin forensic analysisrdquo International Journal of LegalMedicine vol105 no 5 pp 257ndash264 1993
[32] J F MacKay C D Wright and R G Bonfiglioli ldquoA newapproach to varietal identification in plants by microsatellitehigh resolution melting analysis application to the verificationof grapevine and olive cultivarsrdquo Plant Methods vol 4 article 82008
[33] A Xanthopoulou I Ganopoulos G Koubouris et alldquoMicrosatellite high-resolutionmelting (SSR-HRM) analysis forgenotyping andmolecular characterization of anOlea europaeagermplasm collectionrdquo Plant Genetic Resources vol 12 no 3pp 273ndash277 2014
[34] I Ganopoulos A Argiriou and A Tsaftaris ldquoMicrosatellitehigh resolution melting (SSR-HRM) analysis for authenticitytesting of protected designation of origin (PDO) sweet cherryproductsrdquo Food Control vol 22 no 3-4 pp 532ndash541 2011
[35] I Ganopoulos A Argiriou and A Tsaftaris ldquoAdulterationsin Basmati rice detected quantitatively by combined use ofmicrosatellite and fragrance typing with High Resolution Melt-ing (HRM) analysisrdquo Food Chemistry vol 129 no 2 pp 652ndash659 2011
[36] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquoHumanMutation vol 30 no 6pp 857ndash859 2009
[37] M Vietina C Agrimonti and N Marmiroli ldquoDetection ofplant oil DNA using high resolution melting (HRM) post PCRanalysis a tool for disclosure of olive oil adulterationrdquo FoodChemistry vol 141 no 4 pp 3820ndash3826 2013
[38] I Ganopoulos C Bazakos P Madesis P Kalaitzis and A Tsaf-taris ldquoBarcode DNA high-resolution melting (Bar-HRM) anal-ysis as a novel close-tubed and accurate tool for olive oil forensicuserdquo Journal of the Science of Food and Agriculture vol 93no 9 pp 2281ndash2286 2013
[39] EMader J Ruzicka C Schmiderer and J Novak ldquoQuantitativehigh-resolution melting analysis for detecting adulterationsrdquoAnalytical Biochemistry vol 409 no 1 pp 153ndash155 2011
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work has been carried out with financial support fromthe University of BarimdashProjects Idea Giovani 201011 andCofin PRIN 2009 coordinated by Dr Cinzia Montemurro
IDPagina7051[3] C Montealegre M L M Alegre and C Garcıa-Ruiz ldquoTrace-
ability markers to the botanical origin in olive oilsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 1 pp 28ndash38 2010
[4] M A Brescia G Alviti V Liuzzi and A Sacco ldquoChemometricsclassification of olive cultivars based on compositional data ofoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 80 no10 pp 945ndash950 2003
[5] T G Diaz I D Meras J S Casas and M F A Franco ldquoChar-acterization of virgin olive oils according to its triglycerides andsterols composition by chemometric methodsrdquo Food Controlvol 16 no 4 pp 339ndash347 2005
[6] M R Alves S C Cunha J S Amaral J A Pereira and M BOliveira ldquoClassification of PDO olive oils on the basis of theirsterol composition by multivariate analysisrdquo Analytica ChimicaActa vol 549 no 1-2 pp 166ndash178 2005
[7] I S Arvanitoyannis and A Vlachos ldquoImplementation ofphysicochemical and sensory analysis in conjunction withmultivariate analysis towards assessing olive oil authentica-tionadulterationrdquo Critical Reviews in Food Science and Nutri-tion vol 47 no 5 pp 441ndash498 2007
[8] S Pafundo C Agrimonti and N Marmiroli ldquoTraceability ofplant contribution in olive oil by amplified fragment lengthpolymorphismsrdquo Journal of Agricultural and Food Chemistryvol 53 no 18 pp 6995ndash7002 2005
[9] A Pasqualone C Montemurro C Summo W Sabetta FCaponio and A Blanco ldquoEffectiveness of microsatellite DNAmarkers in checking the identity of protected designation oforigin extra virgin olive oilrdquo Journal of Agricultural and FoodChemistry vol 55 no 10 pp 3857ndash3862 2007
[10] A Pasqualone V di Rienzo R Nasti A Blanco T Gomes andC Montemurro ldquoTraceability of Italian protected designationof origin (PDO) table olives by means of microsatellite molecu-lar markersrdquo Journal of Agricultural and Food Chemistry vol 61no 12 pp 3068ndash3073 2013
[11] M S Akkaya and E B Buyukunal-Bal ldquoAssessment of geneticvariation of bread wheat varieties using microsatellite markersrdquoEuphytica vol 135 no 2 pp 179ndash185 2004
[12] L Zeng T-R Kwon X Liu C Wilson C M Grieve andG B Gregorio ldquoGenetic diversity analyzed by microsatellitemarkers among rice (Oryza sativa L) genotypes with differentadaptations to saline soilsrdquo Plant Science vol 166 no 5 pp1275ndash1285 2004
[13] G Cipriani A Spadotto I Jurman et al ldquoThe SSR-basedmolecular profile of 1005 grapevine (Vitis vinifera L) accessionsuncovers new synonymy and parentages and reveals a large
admixture amongst varieties of different geographic originrdquoTheoretical and Applied Genetics vol 121 no 8 pp 1569ndash15852010
[14] G M M Bredemeijer R J Cooke M W Ganal et al ldquoCon-struction and testing of a microsatellite database containingmore than 500 tomato varietiesrdquoTheoretical and Applied Genet-ics vol 105 no 6-7 pp 1019ndash1026 2002
[15] K M Sefc M S Lopes D Mendonca M Rodrigues DosSantos M Laimer Da Camara Machado and A Da CamaraMachado ldquoIdentification of microsatellite loci in olive (Oleaeuropaea) and their characterization in Italian and Iberian olivetreesrdquoMolecular Ecology vol 9 no 8 pp 1171ndash1173 2000
[16] C Montemurro R Simeone A Pasqualone E Ferrara andA Blanco ldquoGenetic relationships and cultivar identificationamong 112 olive accessions using AFLP and SSR markersrdquo TheJournal of Horticultural Science and Biotechnology vol 80 no 1pp 105ndash110 2005
[17] M Hosseini-Mazinani R Mariotti B Torkzaban et al ldquoHighgenetic diversity detected in olives beyond the boundaries of theMediterranean seardquo PLoS ONE vol 9 no 4 Article ID e931462014
[18] D Scarano and R Rao ldquoDNA markers for food productsauthenticationrdquo Diversity vol 6 no 3 pp 579ndash596 2014
[19] A Pasqualone C Montemurro F Caponio and A BlancoldquoIdentification of virgin olive oil from different cultivars byanalysis of dnamicrosatellitesrdquo Journal of Agricultural and FoodChemistry vol 52 no 5 pp 1068ndash1071 2004
[20] httpwwwunaprolit[21] J T Li J Yang D C Chen X L Zhang and Z S Tang
ldquoAn optimizedmini-preparationmethod to obtain high-qualitygenomic DNA from mature leaves of sunflowerrdquo Genetics andMolecular Research vol 6 supplement 4 pp 1064ndash1071 2007
[22] W Sabetta V Alba A Blanco and C Montemurro ldquoSunTILLa TILLING resource for gene function analysis in sunflowerrdquoPlant Methods vol 7 article 20 2011
[23] A Pasqualone V di Rienzo A Blanco C Summo F Caponioand C Montemurro ldquoCharacterization of virgin olive oil fromLeucocarpa cultivar by chemical and DNA analysisrdquo FoodResearch International vol 47 no 2 pp 188ndash193 2012
[24] F Carriero G Fontanazza F Cellini and G Giorio ldquoIdentifica-tion of simple sequence repeats (SSRs) in olive (Olea europaeaL)rdquoTheoretical and Applied Genetics vol 104 no 2-3 pp 301ndash307 2002
[25] R de la Rosa C M James and K R Tobutt ldquoIsolation andcharacterization of polymorphic microsatellites in olive (Oleaeuropaea L) and their transferability to other genera in theOleaceaerdquo Molecular Ecology Notes vol 2 no 3 pp 265ndash2672002
[26] G Cipriani M T Marrazzo R Marconi A Cimato andR Testolin ldquoMicrosatellite markers isolated in olive (Oleaeuropaea L) are suitable for individual fingerprinting andreveal polymorphism within ancient cultivarsrdquoTheoretical andApplied Genetics vol 104 no 2-3 pp 223ndash228 2002
[27] F J Rohlf NTSYS-PC Numerical Taxonomy and MultivariateAnalysis System Version 202i Department of Ecology andEvolution State University of New York Setauket NY USA1998
[28] S M Sanzani C Montemurro V di Rienzo M Solfrizzo andA Ippolito ldquoGenetic structure and natural variation associatedwith host of origin in Penicillium expansum strains causing bluemouldrdquo International Journal of Food Microbiology vol 165 no2 pp 111ndash120 2013
Journal of Chemistry 7
[29] M Perez-Jimenez G Besnard G Dorado and P HernandezldquoVarietal tracing of virgin olive oil based on plastid DNAvariation profilingrdquo PLoS ONE vol 8 no 8 Article ID e705072013
[30] V Alba C Montemurro W Sabetta A Pasqualone andA Blanco ldquoSSR-based identification key of cultivars of Oleaeuropaea L diffused in Southern-Italyrdquo Scientia Horticulturaevol 123 no 1 pp 11ndash16 2009
[31] A D Kloosterman B Budowle and P Daselaar ldquoPCR-amplification and detection of the human D1S80 VNTR locusAmplification conditions population genetics and applicationin forensic analysisrdquo International Journal of LegalMedicine vol105 no 5 pp 257ndash264 1993
[32] J F MacKay C D Wright and R G Bonfiglioli ldquoA newapproach to varietal identification in plants by microsatellitehigh resolution melting analysis application to the verificationof grapevine and olive cultivarsrdquo Plant Methods vol 4 article 82008
[33] A Xanthopoulou I Ganopoulos G Koubouris et alldquoMicrosatellite high-resolutionmelting (SSR-HRM) analysis forgenotyping andmolecular characterization of anOlea europaeagermplasm collectionrdquo Plant Genetic Resources vol 12 no 3pp 273ndash277 2014
[34] I Ganopoulos A Argiriou and A Tsaftaris ldquoMicrosatellitehigh resolution melting (SSR-HRM) analysis for authenticitytesting of protected designation of origin (PDO) sweet cherryproductsrdquo Food Control vol 22 no 3-4 pp 532ndash541 2011
[35] I Ganopoulos A Argiriou and A Tsaftaris ldquoAdulterationsin Basmati rice detected quantitatively by combined use ofmicrosatellite and fragrance typing with High Resolution Melt-ing (HRM) analysisrdquo Food Chemistry vol 129 no 2 pp 652ndash659 2011
[36] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquoHumanMutation vol 30 no 6pp 857ndash859 2009
[37] M Vietina C Agrimonti and N Marmiroli ldquoDetection ofplant oil DNA using high resolution melting (HRM) post PCRanalysis a tool for disclosure of olive oil adulterationrdquo FoodChemistry vol 141 no 4 pp 3820ndash3826 2013
[38] I Ganopoulos C Bazakos P Madesis P Kalaitzis and A Tsaf-taris ldquoBarcode DNA high-resolution melting (Bar-HRM) anal-ysis as a novel close-tubed and accurate tool for olive oil forensicuserdquo Journal of the Science of Food and Agriculture vol 93no 9 pp 2281ndash2286 2013
[39] EMader J Ruzicka C Schmiderer and J Novak ldquoQuantitativehigh-resolution melting analysis for detecting adulterationsrdquoAnalytical Biochemistry vol 409 no 1 pp 153ndash155 2011
[29] M Perez-Jimenez G Besnard G Dorado and P HernandezldquoVarietal tracing of virgin olive oil based on plastid DNAvariation profilingrdquo PLoS ONE vol 8 no 8 Article ID e705072013
[30] V Alba C Montemurro W Sabetta A Pasqualone andA Blanco ldquoSSR-based identification key of cultivars of Oleaeuropaea L diffused in Southern-Italyrdquo Scientia Horticulturaevol 123 no 1 pp 11ndash16 2009
[31] A D Kloosterman B Budowle and P Daselaar ldquoPCR-amplification and detection of the human D1S80 VNTR locusAmplification conditions population genetics and applicationin forensic analysisrdquo International Journal of LegalMedicine vol105 no 5 pp 257ndash264 1993
[32] J F MacKay C D Wright and R G Bonfiglioli ldquoA newapproach to varietal identification in plants by microsatellitehigh resolution melting analysis application to the verificationof grapevine and olive cultivarsrdquo Plant Methods vol 4 article 82008
[33] A Xanthopoulou I Ganopoulos G Koubouris et alldquoMicrosatellite high-resolutionmelting (SSR-HRM) analysis forgenotyping andmolecular characterization of anOlea europaeagermplasm collectionrdquo Plant Genetic Resources vol 12 no 3pp 273ndash277 2014
[34] I Ganopoulos A Argiriou and A Tsaftaris ldquoMicrosatellitehigh resolution melting (SSR-HRM) analysis for authenticitytesting of protected designation of origin (PDO) sweet cherryproductsrdquo Food Control vol 22 no 3-4 pp 532ndash541 2011
[35] I Ganopoulos A Argiriou and A Tsaftaris ldquoAdulterationsin Basmati rice detected quantitatively by combined use ofmicrosatellite and fragrance typing with High Resolution Melt-ing (HRM) analysisrdquo Food Chemistry vol 129 no 2 pp 652ndash659 2011
[36] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquoHumanMutation vol 30 no 6pp 857ndash859 2009
[37] M Vietina C Agrimonti and N Marmiroli ldquoDetection ofplant oil DNA using high resolution melting (HRM) post PCRanalysis a tool for disclosure of olive oil adulterationrdquo FoodChemistry vol 141 no 4 pp 3820ndash3826 2013
[38] I Ganopoulos C Bazakos P Madesis P Kalaitzis and A Tsaf-taris ldquoBarcode DNA high-resolution melting (Bar-HRM) anal-ysis as a novel close-tubed and accurate tool for olive oil forensicuserdquo Journal of the Science of Food and Agriculture vol 93no 9 pp 2281ndash2286 2013
[39] EMader J Ruzicka C Schmiderer and J Novak ldquoQuantitativehigh-resolution melting analysis for detecting adulterationsrdquoAnalytical Biochemistry vol 409 no 1 pp 153ndash155 2011
