Application of High Resolution Melting combined with DNA-based markers for quantitative analysis of olive oil authenticity and adulteration

Journal Pre-proofs Application of High Resolution Melting combined with DNA-based markers for quantitative analysis of olive oil authenticity and adulteration Elsa Chedid, Myrto Rizou, Panagiotis Kalaitzis PII: DOI: Reference:

S2590-1575(20)30006-7 https://doi.org/10.1016/j.fochx.2020.100082 FOCHX 100082

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Food Chemistry: X

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Please cite this article as: E. Chedid, M. Rizou, P. Kalaitzis, Application of High Resolution Melting combined with DNA-based markers for quantitative analysis of olive oil authenticity and adulteration, Food Chemistry: X (2020), doi: https://doi.org/10.1016/j.fochx.2020.100082

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Application of High Resolution Melting combined with DNA-based markers for

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quantitative analysis of olive oil authenticity and adulteration

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Elsa CHEDID,1 : [email protected]

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Myrto RIZOU,1 : [email protected]

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Panagiotis KALAITZIS1* : [email protected]

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Department of Horticultural Genetics & Biotechnology, Mediterranean Agronomic Institute of Chania, Crete, Greece

*Correspondence: Dr. Panagiotis Kalaitzis, Department of Horticultural Genetics and

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Biotechnology, Mediterranean Agronomic Institute of Chania (MAICh), Alsyllio

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Agrokepiou,

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P.O. Box 85, Chania 73100, Crete, Greece

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E-mail: [email protected]

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Fax: +30-28210-35001

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Abstract

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A plethora of biotechnological methodologies is used to authenticate quality olive

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oils. Among the DNA-based approaches, SNPs and SSRs combined with high

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resolution melting (HRM) provide certain advantages such as speed, simplicity and

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reliability. SNP-HRM and SSR-HRM were used for the detection of monovarietal

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olive oils as well as the quantification of varietal composition in olive oil DNA

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admixtures and olive oil blends of two different cultivars. The SSR-HRM was more

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efficient in distinguishing monovarietal olive oils while the SNP-HRM assay was

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more reliable in discriminating olive oil blends. HRM was also used for the detection

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of adulteration of olive oil with oils of different plant origin by using plastid trnL

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indels and SNPs. The trnL-indels-HRM showed higher discrimination power than the

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trnL-SNP-HRM in determining adulteration in olive oil. These results indicate that

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traceability of adulteration might be more reliable than authentication of the varietal

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origin in olive oil blends.

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Keywords: olive oil, quantitative authentication, high resolution melting, molecular

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markers, SSRs, SNPs, adulteration, sunflower oil, maize oil

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Highlights

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 The varietal composition of olive oil blends was quantified

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 Quantification of olive oil adulteration with oils of different plant origin

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 The quantitative authentication of olive oil blends has limitations

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 The SNP-HRM showed higher authentication ability for oil blends compared to

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SSR-HRM  SNP-HRM indicated a 5% limit of detection for varietal identification of olive oil

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1. Introduction

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Olive oil is a valuable agricultural commodity of the Mediterranean basin and is

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considered the major component of the Mediterranean diet not only due to its health

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benefits but also for its great nutritional value. The olive oil production has a high

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social-economic impact, especially the extra virgin olive oil (EVOO) which is

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considered of premium quality and possess higher prices in the market (Kalaitzis &

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El‐Zein, 2016). The identity of such processed products is practically difficult to be

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determined because is influenced by several factors; olive cultivars, pedoclimatic

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conditions, environment, agricultural practices, fruit maturation and methods of 2

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extraction of olive oil (Avramidou, Doulis, & Petrakis, 2018). Protected designation

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of origin (PDO) and protected geographical indication (PGI) are important labels

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referring to the quality and identity of olive oils. Therefore, authenticity and

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traceability of high quality PDO and PGI monovarietal extra virgin olive oils is a

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major concern for markets and consumers. A major part of authentication efforts

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concentrate on the identification of the varietal origin as well as the adulteration with

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oils of different plant origin.

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DNA-based approaches are considered complementary to analytical chemistry

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methodologies for olive oil varietal authentication due to their sensitivity, specificity

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and reliability (Agrimonti & Marmiroli, 2019; Avramidou, Doulis, & Petrakis, 2018;

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Lo & Shaw, 2018). In this context, various molecular markers were used for food

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authenticity and traceability but the single nucleotide polymorphisms (SNPs) and the

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microsatellites or single sequence repeats (SSRs) became the markers of choice for

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olive oil traceability purposes (Bazakos, Khanfir, Aoun, Spano, Zein, Chalak, et al.,

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2016; Montemurro, Miazzi, Pasqualone, Fanelli, Sabetta, & di Rienzo, 2015;

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Pasqualone, Montemurro, di Rienzo, Summo, Paradiso, & Caponio, 2016). These

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molecular markers are considered as ideal analytical targets in partially degraded

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DNA preparations such as those of olive oil since they require the amplification of

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short length PCR amplicons. Therefore, many studies have used the SSRs for the

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identification of varietal origin of olive oil with various technological platforms

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(Alba, Sabetta, Blanco, Pasqualone, & Montemurro, 2009; Ben‐Ayed, Kamoun‐Grati,

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& Rebai, 2013; Pasqualone, Montemurro, Summo, Sabetta, Caponio, & Blanco,

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2007).

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Recent reports explored the potential of SNPs in olive oil varietal discrimination by

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using various methodologies (Consolandi, Palmieri, Doveri, Maestri, Marmiroli,

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Reale, et al., 2007; Reale, Doveri, Díaz, Angiolillo, Lucentini, Pilla, et al., 2006) such

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as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

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combined with capillary electrophoresis (CE) (Bazakos, Dulger, Uncu, Spaniolas,

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Spano, & Kalaitzis, 2012; Bazakos, et al., 2016). Moreover, a multiplex SNPs assay

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was also used for the varietal identification of monovarietal olive oils in combination

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with an innovative approach of fluorescence-encoded microspheres (Kalogianni,

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Bazakos, Boutsika, Targem, Christopoulos, Kalaitzis, et al., 2015).

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Plastid markers have also been used for the detection of adulteration of olive oil

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(Besnard, Hernández, Khadari, Dorado, & Savolainen, 2011; Pérez-Jiménez, Besnard,

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Dorado, & Hernandez, 2013). One of the analytical targets of extra interest is the

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plastid trnL (UAA) intron for various reasons. The plastid trnL (UAA) intron has

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been used as an analyte molecule to identify the identity of specific food crops

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(Spaniolas, Bazakos, Spano, Zoghby, & Kalaitzis, 2010). It has been used in the past

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for the identification of plant species (Spaniolas, Bazakos, Spano, Zoghby, &

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Kalaitzis, 2010). This intron shows acceptable discrimination efficiency since it is

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sufficiently variable among species and conserved enough within species (Taberlet,

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Coissac, Pompanon, Gielly, Miquel, Valentini, et al., 2006). Alternatively, a set of 40

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polymorphic loci were identified after sequencing 8 plastid genomes of olive showing

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very low variability in olive tree cultivars (Besnard, Hernandez, Khadari, Dorado &

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Savolainen, 2011). Such loci might be efficiently used for detection of adulteration

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with oils of different plant origin.

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The development of methodologies that require minimum manipulation is mandatory

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when dealing with samples destined for authenticity testing. Therefore, the High

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resolution melting (HRM) technology might be an approach which allows the

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genotyping of varieties in a closed-tube reaction, without further analysis, as long as

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the variants have been previously identified (Druml & Cichna-Markl, 2014). The

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HRM analysis curves can be distinguished on the basis of their shape, due to

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polymorphisms on single nucleotides and/or amplicon length, even though the PCR

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products might have similar Tm values (Ganopoulos, Bosmali, Madesis, & Tsaftaris,

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2012; Pereira, Gomes, Castro, Eiras-Dias, Brazão, Graça, et al., 2017). A barcode-

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HRM approach was established by Madesis, Ganopoulos, Anagnostis, and Tsaftaris

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(2012) on chloroplastic DNA to detect Lupinus adulterants present in Glycine max

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flour. Moreover, the HRM assay combined with molecular markers allowed a quick

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and high-throughput detection of adulteration of monovarietal olive oils (Ganopoulos,

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Bazakos, Madesis, Kalaitzis, & Tsaftaris, 2013). In addition, the SSR-HRM approach

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was reported to efficiently determine the traceability of olive oils (Montemurro,

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Miazzi, Pasqualone, Fanelli, Sabetta, & di Rienzo, 2015) while recently, the SSR-

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HRM methodology was proposed as an efficient, fast, simple and reliable approach to

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authenticate a high number of monovarietal olive oils (Gomes, Breia, Carvalho,

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Carnide, & Martins‐Lopes, 2018).

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In this report, a comparative authentication and adulteration of olive oil approach by

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using SSR- and SNP- based HRM was performed at the quantitative level. Initially,

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HRM combined with DNA markers was used for the discrimination and quantitative

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determination of blends of monovarietal olive oils. In this analysis, the discriminatory

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capacity of SNPs compared to SSRs was found to be higher. Moreover, the efficiency

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of HRM was also investigated for the detection of adulteration of olive oil with oils of

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different plant origin. For this approach, plastid trnL (insertions/deletions) indels and

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SNPs were used and it was found that the indels region is more efficient in the

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discrimination of adulterants. Overall, our findings suggest that depending on the

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authentication and adulteration objective, one DNA marker might provide advantages

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over another.

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2. Materials and Methods

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2.1. Plant tissues and oil samples

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The leaf tissue of three Greek olive varieties, Koroneiki, Tsounati and Kalamon, was

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collected from an olive collection maintained at the Mediterranean Agronomic

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Institute of Chania, Crete, Greece and used for DNA extraction. The monovarietal

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olive oil samples were provided by Pamako S.A.and Kolympari SA, Chania, Crete

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and by the Union of Agricultural Cooperatives of Lakonia, Sparta, Peloponnese.

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Commercial maize and sunflower oils were used for the adulteration experiments.

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For the authentication of the olive oil DNA admixtures, genomic DNA was extracted

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from three monovarietal olive oils and diluted to equal concentrations separately.

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Subsequently, the DNA of Koroneiki with Kalamon and Koroneiki with Tsounati

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were blended in ratios of 50-50%, 75-25%, 85-15% and 95-5%. Monovarietal olive

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oil DNA of each variety was also used as positive control. For the authentication of

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olive oil blends, monovarietal olive oils of Koroneiki and Kalamon as well as

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Koroneiki and Tsounati were mixed in ratios of 50-50%, 75-25%, 85-15% and 95-5%

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and then DNA was extracted from the mixtures. In addition, monovarietal olive oils

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of each variety were also used as positive controls. Finally, for the detection of olive

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oil adulteration, two different vegetable oils were used, maize and sunflower.

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Monovarietal Koroneiki olive oil was blended with maize and sunflower oils in ratios

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of 99-1%, 95-5%, 90-10%, 85-15% and 75-25% and subsequently DNA extraction

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was performed in the mixtures. The monovarietal olive oil and the 100% maize and

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sunflower oils were used as positive controls.

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2.2. Isolation of DNA from olive oil

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Genomic DNA was extracted from leaves of the three olive varieties with a standard

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CTAB (cetyltrimethylammonium bromide) protocol as described in Woolley, James,

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and Manning (2001). DNA extraction from olive oil samples was performed using

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two different methods: the CTAB/hexane/chloroform protocol developed by

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Giménez, Pistón, Martín, and Atienza (2010) and Norgen’s Olive Oil DNA

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Isolation Kit (Norgen Biotek co., Canada). DNA quantity and purity were estimated

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on a Nano photometer (Pearl, Implen GmbH Munich Germany)

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2.3. PCR amplification from olive oil DNA

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The amplification of DNA templates comprising the SSR and SNP molecular markers

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(Supplementary Table 1) was performed by PCR reaction in a DYAD thermocycler

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(BIORAD) using 5x Phusion HF Buffer, 4 mM dNTP, 10 μM for each primer, 0.4

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units of Phusion High-Fidelity DNA Polymerase (Applied Biosystems, Foster City,

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CA) and 20 ng of olive DNA in a final volume of 25 μl. The PCR conditions were 98

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°C for 30 seconds, followed by 45 cycles of 98 °C for 10 s, Tm of the primers for 30 s

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and 72 °C for 30s/Kb, with a final extension step at 72 °C for 5 min. The amplicons

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were run on a 1.5% agarose gel and purified with the QIAquick Gel Extraction Kit

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(QİAGEN, Germany) according to the manufacturer’s instructions based on the

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expected lentgh despite the fact that were not visualized.

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2.4. High Resolution Melting Analysis

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The purified PCR amplicons and the sets of primers used for their amplification were

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also used for the subsequent HRM assays. HRM analysis was conducted in a 96-well

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plate using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad).

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Duplicates of each sample were prepared in a final volume of 10 μL containing 2x

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Precision Melt Supermix (Bio-Rad), 0.2 μM of each primer and 20 ng/μL of amplified

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and purified template DNA. A negative control was included in each assay. An initial

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2 min step at 95 °C was followed by 40 cycles of 95 °C for 10 s, Tm of the primers

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for 30s and 72 °C for 30 s. The melting curve was obtained in continuous, performed

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as follow: 95 °C for 30 s, 65 °C for 1min rising 0.2 °C/s, 95 °C for 10 s. During the

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incremental melting step, fluorescence data were continuously acquired. Precision

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Melt Analysis™ Software (Bio-Rad) was used to analyze the data. After

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normalization and temperature shift determination, the different melting curves of the

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several plots were generated. Three biological replicates were performed for each

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HRM assay.

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3. Results

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3.1. Detection of monovarietal olive oils using SSR- and SNP-HRM analysis

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The major bottleneck of the DNA-based approaches for the identification of the

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varietal origin of olive oil as well as the detection of adulteration with oils of different

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plant origin is the isolation of adequate quality DNA for PCR amplification from the

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oil samples. Therefore, two different methodologies were initially tested, the

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CTAB/hexane/chloroform protocol (Giménez, Pistón, Martín, & Atienza, 2010) and

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the Norgen kit for olive oil (Norgen Biotek, Thorold, Canada) using 600 µl (Bazakos,

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et al., 2016) and 500 µl of olive oil sample, respectively. The Norgen DNA extraction

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procedure required less time compared to the CTAB protocol and was considered

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more reliable in providing olive oil DNA isolates which were more consistently

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leading to successful PCR amplifications. Therefore, the Norgen kit was used for the

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isolation of DNA from the monovarietal olive oils as well as their blends in order to

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be PCR amplified and purified after fractionation in agarose gel electrophoresis.

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Although the visualization of the PCR amplicons was not possible in the agarose gel,

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the PCR products were purified as gel fragments according to their expected length

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(Supplementary Figure 1). These purified PCR products were successfully re-

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amplified and used for HRM analysis (Supplementary Figure 1). The same set of

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primers was used for the initial and the additional PCR amplification for the HRM

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analysis.

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The olive SSR loci ssrOe-DCA3, ssrOe-DCA9, ssrOe-DCA16 and ssrOe-DCA18

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(Sefc, Lopes, Mendonça, Santos, Machado, & Machado, 2000) were selected among

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several others such as ssrDCA5, ssrEMO90 and ssrGAPU71 (Carriero, Fontanazza,

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Cellini, & Giorio, 2002) because they were more consistent in the PCR amplification

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of the SSR marker fragments. The nucleotide sequence of the SSR amplicons was

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determined in order to validate amplification of the selected SSR loci. Representative

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sequences of ssrOe-DCA3 and ssrOe-DCA18 which were amplified from the

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Kalamon, Koroneiki and Tsounati are shown in Supplementary Figure 2. The HRM

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difference plot with the ssrOe-DCA18 showed three different melting profiles of

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various shapes, one for every variety, allowing their discrimination (Figure 1A).

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Moreover, the HRM assay with the ssrOe-DCA3 generated two different melting

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profiles of similar shape distinguishing the Kalamon (Tm=70.40 ˚C) from Koroneiki

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and Tsounati both with a Tm of 70.20˚C (Figure 1B). Leaf DNA of Koroneiki was

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used as a positive control template (Figure 1A, B).

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The SNP1, SNP2 and SNP3 were successfully used for the determination of the

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varietal origin of Kalamon, Koroneiki and Tsounati as was previously reported after

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validation by sequencing of the PCR amplicons comprising these SNPs (Bazakos et

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al., 2012; Bazakos et al., 2016).

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The use of SNPs for HRM analysis generated two melting plots per SNP, as expected.

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Specifically, the SNP1 and SNP3 (Bazakos, et al., 2016) discriminated Tsounati from

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Koroneiki and Kalamon and Kalamon from Koroneiki and Tsounati, respectively

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(Figure 1C, D).

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3.2. Authentication of Olive Oil DNA admixtures using SSR- and SNP-HRM

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analysis

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Genomic DNA was extracted from three monovarietal olive oils and diluted to equal

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concentrations for admixtures preparations as described in materials and methods.

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The admixtures and the positive control samples were used for PCR amplification in

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order to generate the templates for HRM analysis.

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Three SSRs and three SNPs markers were used for the discrimination of the two sets

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of admixtures in a preliminary, comparative study to determine which DNA marker

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might be more suitable for authentication of olive oil admixtures.

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The ssrOe-DCA18 and ssrOe-DCA16 exhibited 5 different HRM melting profiles out

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of 6 samples for the DNA mixtures of Koroneiki-Tsounati (Figure 2A, B). The

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melting curves of 95-5% and 85-15% ratios showed overlapping profiles and could

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not be discriminated (Figure 2A, B). Although, curves of variable shapes were

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generated for some ratios compared to others, the discrimination of all ratios was not

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possible (Figure 2A, B). The SNP1 and SNP2 (Bazakos, et al., 2016) generated six

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different melting profiles distinguishing the four ratios and the two monovarietal

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samples generating curves of similar shape (Figure 2C, D).

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The ssrOe-DCA9 and ssrOe-DCA18 showed 5 different HRM melting profiles out of

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6 samples for the Koroneiki-Kalamon blends (Figure 3). The melting curves of 95-5%

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and 85-15% ratios also showed overlapping curves and could not be discriminated

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(Figure 3A, B). However, the SNP2 and SNP3 (Bazakos, et al., 2016) resulted in six

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different melting profiles distinguishing the four ratios and the two monovarietal

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samples generating curves of similar, though, distinct shapes (Figure 3).

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These results indicate higher discriminatory capacity of SNPs compared to SSRs in

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DNA admixtures. Although the monovarietal DNA samples exhibited melting curves

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of similar shape, they were distinguished based on the Tm differences (Figure 2,

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Figure 3). However, the discrimination of the ratios was mainly based on the shape of

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the visualized melting profiles (Figure 2, Figure 3).

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Moreover, the limit of detection for both DNA markers was determined at the levels

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of 5% since the 95-5% ratios of both blends were discriminated efficiently and

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reliably (Figure 2, Figure 3).

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3.3. Authentication of Olive oil blends

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Olive oil blends were prepared and DNA extraction was performed as described in

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detail in Materials & Methods. The two-cultivar blends at four different ratios and

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their two monovarietal oils were used for SSR- and SNP-HRM analysis.

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Initially the ssrOe-DCA16, ssrOe-DCA18 and ssrOe-DCA9, ssrOe-DCA18 pair of

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markers were used with blends of Koroneiki, Tsounati and Koroneiki, Kalamon,

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respectively (Figure 4, Figure 5). The blends of 95-5% and 85-15% and those of 75-

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25% and 50-50% showed overlapping melting profiles and therefore could not be

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discriminated (Figure 4, Figure 5). These results indicate that blends with ratios in the

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range between 95 to 85% and between 75 to 50% could not be distinguished by using

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these specific pairs of SSR markers (Figure 4, Figure 5).

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Moreover, the SNP1, SNP2 and the SNP2, SNP3 pair of markers were used with

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blends of Koroneiki, Tsounati and Koroneiki, Kalamon, respectively (Figure 4, Figure

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5). The blends of 95-5% and 50-50% showed distinct melting profiles and therefore

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could be discriminated by these combinations of SNP markers (Figure 4, Figure 5).

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However, the blends of 85-15% and 75-25% showed overlapping melting profiles and

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therefore could not be discriminated (Figure 4, Figure 5). These results indicate that

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blends with ratios in the range between 85 to 75% could not be distinguished with

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these specific pair of SNP markers (Figure 4). Overall, the SNPs showed higher

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capacity to distinguish olive oil blends compared to the SSR markers.

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Representative amplification plots for the ssrOe-DCA18 and the SNP2 indicate

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differences in the amplification efficiency but not in the shape of the plot which might

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be attributed to the quality of the DNA template of the olive oil samples and their

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blends considering that the same primers and similar HRM reaction conditions were

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used for each SSR and SNP marker (Supplementary Figure 3).

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3.4 Detection of maize and sunflower oil adulteration in extra virgin olive oil

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Two different polymorphic molecular markers of the same chloroplastic trnL intron

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region were used for the detection of adulteration (Spaniolas, Bazakos, Spano,

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Zoghby, & Kalaitzis, 2010). The trnL-indels and the trnL-SNPs primers were

287

designed to amplify two different regions of this intron which can discriminate

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between different species based on length polymorphisms and single nucleotide

289

polymorphisms, respectively (Supplementary Table 1). The trnL-SNPs amplicon was

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comprising two SNPs (Spaniolas, Bazakos, Spano, Zoghby, & Kalaitzis, 2010).

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DNA was extracted from the five blends for each adulterant and from the three

292

different plant species oil, and then used as a template for the amplification and the

293

HRM analysis of the two DNA markers, trnL-indels and trnL-SNPs.

294

The HRM-trnL-indels difference plots of Koroneiki with maize and sunflower

295

adulteration mixtures exhibited discrimination of all seven samples, four ratios and

296

three 100% oil samples (Figure 6). The shape of the melting profiles was similar in all

297

blends regardless of the botanical origin of the adulterant and the percentage of

298

adulteration (Figure 6 A, B).

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However, the HRM- trnL-SNPs difference plots of the same samples detected five out

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of seven samples (Figure 6C, D). The ratios of 99-1% with 95-5% and of 90-10%

301

with 85-15% showed overlapping melting profiles indicating the higher

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discriminatory capacity of the trnL-indels marker compared to the trnL-SNPs (Figure

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6C, D). The shape of the melting curves was different in the maize adulterant plot

304

compared to the sunflower plot (Figure 6 C, D). Moreover, in the sunflower

305

difference plot the profiles of 100% and 25% sunflower oil exhibited different shape

306

compared to the profiles of the other samples (Figure 6 C, D).

307

According to these results, the limit of detection for the HRM-trnL-indels analysis

308

was determined at the levels of 1%. For the HRM-trnL-SNPs limit of detection was

309

set at the range of 1-5% due to the overlapping curves of 99-1% and 95-5% blends

310

(Figure 6).

311

Representative amplification plots indicate differences in the efficiency of

312

amplification but not in the shape of the plots which might be explained by

313

differences in the quality of the DNA templates (Supplementary Figure 3).

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4. Discussion

316

Three monovarietal olive oils were selected for varietal authentication by using SSR-

317

HRM and SNP-HRM analysis not only at the monovarietal level but also at the

318

admixtures level of various ratios between two varieties each time. Moreover, this

319

comparative study between SSRs and SNPs suggested possible advantages of one

320

DNA marker over the other depending on the authentication objective.

321

Many studies revealed the efficiency of SSR-HRM analysis for the identification and

322

differentiation of cultivars and closely related species (Bosmali, Ganopoulos,

323

Madesis, & Tsaftaris, 2012; Distefano, Caruso, La Malfa, Gentile, & Wu, 2012;

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324

Gomes, Breia, Carvalho, Carnide, & Martins‐Lopes, 2018; Li, Xiong, He, Lu, Zhang,

325

Chen, et al., 2018; Mackay, Wright, & Bonfiglioli, 2008). In this study, one SSR

326

locus was adequate to discriminate the three olive varieties while two SNP loci were

327

required to distinguish among them. Usually more SNP loci are required in order to

328

acquire the same level of discriminatory power compared to SSRs (Avramidou,

329

Doulis, & Petrakis, 2018). Therefore, the SSR-HRM might be considered the

330

molecular marker of choice combined with HRM analysis if the objective is to

331

distinguish a higher number of monovarietal olive oils.

332

Blends of specific varieties grown in certain regions are considered premium olive

333

oils of higher value. Therefore there is a pressing need for reliable identification of the

334

genetic identity of premium quality olive oils (Kalaitzis & El‐Zein, 2016). In the

335

current study, the limit of detection of artificial DNA admixtures of two olive oil

336

cultivars was determined down to 5% for the SNP-HRM assays while for the SSR-

337

HRM assays the limit of detection ranged between 5 and 15%. This is an

338

improvement considering that Bazakos, et al. (2016) determined the limit of detection

339

of olive oil blends at the levels of 10% by using a SNP-based PCR-RFLP approach.

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Significant differences in the discrimination accuracy of HRM analysis was detected

341

between DNA mixtures and monovarietal olive oil mixtures. The SSR-HRM assay of

342

olive oil admixtures of Koroneiki-Kalamon and Koroneiki-Tsounati revealed similar

343

results; the HRM profiles of blends of 5% and 15% ratios were overlapping as well as

344

the blends of 25% and 50% ratios indicating that there are limits in the discriminatory

345

power of the SSR markers. However, the SNP-HRM assay distinguished all ratios

346

except the 5% and 15% indicating that this analytical approach might be more

347

efficient in olive oil traceability and authenticity efforts with strong commercial

348

application potential as was previously described by Reed and Wittwer (2004). 14

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The greatest challenges one faces while using DNA technology is the low quality and

350

highly degraded DNA recovered from the fatty matrices and the impact of oil

351

extraction processing on the size of the recovered DNA (Enferadi & Rabiei, 2013).

352

The critical steps of the extraction process that affects the most the DNA, are the

353

malaxation and separation steps where oil might be exposed to high temperatures

354

causing higher degradation of DNA. This can result in variable quality DNA isolates.

355

In these cases, a blend comprised of differentially processed monovarietal olive oils

356

might result in DNA isolates which might not be representative of the blend ratios.

357

Consequently, false estimation of DNA isolate for each variety from the blend might

358

be obtained. Thus, this might be a limiting factor for olive oil authenticity at the

359

quantitative level potentially leading to inconclusive results.

360

Various qualities of isolated DNA from monovarietal olive oils or blends might lead

361

to significant variation in the amplification plots in the HRM reactions. In addition,

362

there is the possibility that the presence of contaminants might interfere with the PCR

363

amplification leading to a reduction in the amplification efficiency (Scollo, Egea,

364

Gentile, La Malfa, Dorado & Hernandez, 2016). However, the difference plots

365

provided by the HRM software represent the melting kinetics of each PCR amplicon

366

which is affected by the SNP- or SSR-based polymorphic nature of the sequence of

367

the amplicon and is not related to the amplification plots which are affected by the

368

DNA templates per se.

369

The low quality and partially degraded DNA in olive oil samples make the

370

traceability more challenging and less accurate due to the mostly short DNA

371

templates present in the extracts. In this context, the amplicon length is important in

372

determining the outcome of PCR-based amplification of DNA templates. Therefore,

373

the shorter the amplicon is, the higher the probability of successful amplification

15

374

(Spaniolas, Bazakos, Ntourou, Bihmidine, Georgousakis, & Kalaitzis, 2008).

375

Therefore, it is suggested that the SNP-HRM might be considered the molecular

376

marker of choice compared to the SSR-HRM for authentication of olive and vegetable

377

oil blends due to the requirement for relatively shorter DNA templates.

378

DNA mixtures extracted from adequately high quality olive oil DNA samples might

379

result in the detection of the ratios of two varieties and distinguish among 5%, 15%,

380

25% and 50% by using SNP-HRM assays. However, it was suggested that CE

381

electrophoretograms are more suitable for large data sets due to many samples

382

compared to SNP-HRM because it is easier to digitalize and quantify peaks than

383

melting curves (Lian & Zeng, 2017).

384

It was demonstrated that the discrimination of various percentages of olive oil

385

mixtures is possible at limits of detection set at 5% by using this efficient, closed tube

386

HRM approach. However, mixtures which differ in the range of 10 to 25% might not

387

be possible to be discriminated regardless of high or low ratio percentages neither by

388

SSR-HRM nor by SNPs-HRM.

389

The use of plastidial trnL (UAA) intron as an analytical target to discriminate among

390

oils of different botanical origin by using HRM analysis proved to be an accurate and

391

effective approach. The various polymorphisms in this plastid DNA region among

392

different plant oils were previously reported by Spaniolas, Bazakos, Awad, and

393

Kalaitzis (2008) and Spaniolas, Bazakos, Spano, Zoghby, and Kalaitzis (2010) using a

394

CE assay indicating thus the potential use of this intron in the detection of adulteration

395

of olive oil.

396

Moreover, the HRM assay with trnL-indels locus and trnL-SNPs locus revealed a

397

limit of detection of 1% (v/v) of plant oil adulterant in olive oil. Similar results were

398

obtained by Ganopoulos, Bazakos, Madesis, Kalaitzis, and Tsaftaris (2013) which

16

399

reported a barcode-HRM analysis with an rbcL marker determining the limit of

400

detection at 1% of canola oil in olive oil. In addition, they were able to distinguish

401

different ratio admixtures of olive oil and canola.

402

In this preliminary, comparative report it was demonstrated that the trnL-indels locus

403

revealed better discrimination power than the trnL-SNPs locus, distinguishing all

404

mixture ratios, even though it requires a longer DNA template.

405

The fact that HRM analysis of DNA mixtures is more accurate compared to olive oil

406

mixtures indicates that the quantitative determination of the varietal origin of olive

407

oils has limitations which are directly related to the olive oil production process

408

and/or storage conditions. However, the SNP-based HRM approach might be

409

considered of higher discriminatory potential compared to SSR-based approaches for

410

quantitative authentication purposes.

411

The use of trnL intel-HRM analysis showed higher discrimination power than the

412

trnL SNP-HRM determining all mixture ratios of adulteration with maize oil as well

413

as sunflower oil indicating that traceability of adulteration might be more reliable

414

compared to authentication of the varietal origin of olive oil blends.

415 416

5. Conclusion

417

The authentication, traceability and adulteration of olive oil are of major importance

418

in order to monitor for fraudulent practices. The SSR-HRM and SNP-HRM assays

419

were both efficiently determined the varietal origin of monovarietal olive oils. In two

420

cultivar admixtures, SNP-HRM showed higher efficiency to discriminate olive oil

421

blends of various ratios except those of 95-5% and 85-15%. Moreover, the limit of

422

detection was determined at the level of 5% only for the SNP-HRM assay. The HRM-

423

based comparative quantitative determination of adulteration of olive oil with oils of

17

424

different plant origin indicated that the indels within the trnL region were more

425

efficient compared to trnL SNPs. The limit of detection for adulteration was

426

determined at the level of 1% in accordance with previous reports. These results

427

indicate that the quantitative validation of the varietal composition of olive oil blends

428

might be possible, though not always reliable due to variations in the processing

429

and/or storage of olive oil samples. However, the quantitative detection of

430

adulteration might be considered more efficient despite the heavily processed nature

431

of oils of plant origin.

432 433

Acknowledgements

434

This research has been financed by Greek national funds through the Public

435

Investments Program (PIP) of General Secretariat for Research & Technology

436

(GSRT), under the Emblematic Αction "The Οlive Road" (project code:

437

2018ΣΕ01300000)

438 439

Conflict of Interest Statement

440

The authors declare that there is no conflict of interest.

441 442

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574 575 576

Elsa Chedid: Investigation, Validation, Writing - Original Draft

577

Myrto Rizou: Investigation, Validation, Writing - Original Draft, Writing - Review

578

& Editing,

579

Panagiotis Kalaitzis: Conceptualization, Methodology, Supervision, Writing -

580

Original Draft, Writing - Review & Editing,

581 582 583 584

Declaration of interests

585 586 587

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

588 589 590 591

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

592 593 594 24

595 596 597

Highlights

598

 The varietal composition of olive oil blends was quantified

599

 Quantification of olive oil adulteration with oils of different plant origin

600

 The quantitative authentication of olive oil blends has limitations

601

 The SNP-HRM showed higher authentication ability for oil blends compared to

602 603

SSR-HRM  SNP-HRM indicated a 5% limit of detection for varietal identification of olive oil

604 605

25