**6. DNA-based markers**

170 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

hydrocarbons, and tocopherols, fatty acids and triglycerides, components may provide basic information on olive cultivars. Minor components, can provide more useful information and have been more widely used to differentiate the botanical origin of *olive oil*s (Montealegre et

There are several parameters, major and minor compositional markers, with varied discriminant power used for *olive oil* traceability according to the variety of olive

To relate the fatty acid composition of *olive oil*s with the cultivar, Mannina et al. (2003) studied *olive oil* in a well- limited geographical region, with no consideration of the pedoclimatic factor (soil characteristics such as temperature and humidity). A relationship between the fatty acid

The *volatile fraction* in *olive oil*s, which represents one of the most important qualitative aspects of this oil, consists of a complex mixture of more than 100 compounds, but the most important substances useful for olive cultivar differentiation are the products of the lipoxygenase pathway (LOX). Only a subset of volatile compounds and a combination among them could provide valuable information for olive cultivar differentiation (Montealegre et al., 2010). Three volatile compounds [hexyl acetate, hexanal, and (E)-hex-2 enal] and the total concentration of ketones have nominated to distinguish the olive varieties (Tena et al., 2007) hich consequently could be used for olive oil, as well. However, it has been found that the level of (E)-hex-2-enal in the analyzed samples showed a variability that suggest an influence of genetic factors on the biosynthesis of this compound. In fact, genetic (Tura et al., 2008; Mahjoub-Haddada et al., 2007) and geographic (Mahjoub-Haddada et al., 2007) factors influence the volatile compound production of the olive fruits and affect the differentiation of *olive oil*s according to their olive variety. The volatile compound contents allowed differentiation among monovarietal *olive oil*s and even identification of the

The color of a virgin *olive oil* is due to the solubilization of the lipophilic chlorophyll and carotenoid pigments present in the fruit. The green-yellowish color is due to various pigments, that is, chlorophylls, pheophytins, and carotenoids (Cichelli & Pertesana, 2004). Chlorophyll a is the major chlorophyll pigment, followed by chlorophyll b. The carotenoid fraction is included lutein, violaxanthin, neoxanthin, β-carotene, β-cryptoxanthin, and

Several researchers reported the same qualitative composition in chlorophyll and carotenoid pigments, independent of the olive variety and the time of picking (Giuffrida et al., 2007 ;

Cerretani et al. (2006) showed that the carotenoid and chlorophyll content determination using UV-vis spectrophotometry was not useful to discriminate oils produced from different olive varieties. Lutein/β-carotene ratio has been reported as a tool to differentiate oils from

Tocopherols and hydrocarbons are the compositional markers less studied to date to differentiate *olive oil*s. An important common aspect is that the content and composition of

composition and some specific cultivars has been observed (Montealegre et al., 2010).

participated in the production of the oil (Arvanitoyannis et al., 2007).

technique used for *olive oil* production (Torres Vaz-Freire et al., 2009).

luteoxanthin (Montealegre et al., 2010).

Roca et al., 2003).

a single cultivar.

al., 2010).

**5.2 Compositional markers** 

Molecular markers are investigated as a diagnostic tool for food authenticity and traceability of variety/type composition of complex food matrices in an increasing number of projects (Palmieri et al., 2004). DNA-based methods make an important contribution to protect highquality *olive oil*s.

Significant amounts of DNA are present in *olive oil* obtained by cold pressing (Consolandi et al., 2008). However, the filtration process lowers DNA concentrations, which tend to disappear due to nuclease degradation (De la Torre et al., 2004; Muzzalupo et al., 2002). On the other hand, the length of storage after milling of the oil can affect the use of DNA as an analyte for molecular traceability. Pafundo et al., 2010 observed a significant decrease of quality of DNA extracted from olive oil, with a consequent loss of information a month later from olive oil production.

Spaniolas et al. (2008b) has used lambda DNA as a marker to monitor the length of DNA fragments in olive oil during storage time when determined the varietal origin of olive oil. Lambda DNA is a linear molecule of approximately 50 kb, a length that probably resembles that of olive DNA present in olive oil. Based on the fact, PCR-based fingerprinting techniques, which require templates longer than 100 bp, might not be able to successfully amplify the target sequences from olive oil samples low in DNA content and stored for several months, they conduct their analyses. They have deduced the detection of polymorphic markers requiring DNA templates shorter than 100 bp might have a wider range of applications in DNA fingerprinting of olive oil.

In *olive oil*, once the barrier of DNA extraction has been overcome, several markers could be used to identify olive cultivars that made up a certain *olive oil*. (Consolandi et al., 2008)

DNA recovery methods from *olive oil* have been developed by many authors (Busconi et al. 2003; Doveri et al. 2006; Pasqualone et al. 2007; Consolandi et al. 2008). Several commercial kits, providing adapted protocols, were used in different works (Martins-Lopes et al. 2008; Spaniolas et al. 2008a; Ayed et al. 2009; Pafundo et al. 2010). All of these studies confirmed that the DNA of the cultivars is recoverable from extra virgin *olive oil*, but it has low quantity and quality. The first researches, carried out using genomic DNA extracted from drupes. That DNA had a good potential to amplify correctly using RAPDs markers (Cresti et al. 1997). By means of SCAR and AFLP markers, Busconi et al. (2003) were able to show that DNA recovered from *olive oil* had both organellar and nuclear origin. Pafundo et al. (2005) traced the cultivar composition of monovarietal *olive oil*s by AFLPs, suggesting that DNA extraction is the most critical step affecting the procedure. Pafundo et al. (2007), performed amplification of DNA isolated from *olive oil* using AFLPs. They have also developed some SCARs to amplify successfully the DNA extracted from *olive oil*. Using SSR analysis, Pasqualone et al. (2007) demonstrated that microsatellites are useful in checking the presence of a specific cultivar in a PDO oil, thus verifying the identity of the product. However, they obtained only the marker profile of the main cultivar in the oil: no signal was detected for the secondary varieties. Montemurro et al. (2008) analyzed ten virgin monovarietal *olive oil*s prepared in the laboratory by AFLP markers. They were able to

Traceability of Origin and Authenticity of *Olive Oil* 173

The applicability to the traceability of cultivars in the *olive oil* by means of RAPDs was evaluated by Muzzalupo & Perri (2002). This kind of markers was used, together with other DNA-based markers, in the construction of the first olive linkage maps (De la Rosa et al.

AFLPs have been widely used for DNA fingerprinting of cultivars (Angiolillo et al. 1999; Owen et al. 2005), to analyse the relationships between wild and cultivated olive (Baldoni et al. 2006), for the construction of linkage maps (de la Rosa et al. 2003) and for cultivar

SCARs have been used for cultivar identification (Busconi et al. 2006) and in *olive oil* traceability (De la Torre et al. 2004; Pafundo et al. 2007). Putative associations of several SCAR markers with fruit characteristics (Mekuria et al. 2002) and resistance to pathogenic fungi (Herna´ndez et al. 2001) were found, suggesting the applicability of this type of

Many authors have reported on SSR development in olive and several of them are currently available for DNA analysis (Cipriani et al. 2002; De la Rosa et al. 2002; Rabiei & Tahmasebi Enferadi 2009; Sabino Gil et al. 2006; Sefc et al. 2000). This technique benefits from the use of microsatellites which are short stretches (1-6-bp long) of DNA, tandemly repeated several times. The number of repeats can vary from one individual to another. Besides, they are abundant in eucaryotic genomes. A combination of several SSRs loci allows virtually discrimination of individuals originated through sexual reproduction, especially in out-

In Olea europaea, these markers have been used for different applications such as cultivar discrimination (Sarri et al. 2006; Fendri et al. 2010), study of relationships between wild and cultivated olive tree (Belaj et al. 2007), construction of association maps (De la Rosa et al. 2003), paternity analysis (Mookerjee et al. 2005) and identification of *olive oil* varietal composition (Alba et al. 2009; Ayed et al. 2009; Rabiei et al. 2010). A list of recommended SSR markers and protocols for olive genotyping has been provided with the aim to develop a robust method to

Table 4. List of microsatellites isolated in olive which their related primers are available in the literature, most of them have been used in case of *olive oil* traceability (Bracci et al., 2011)

track the origin of olive cultivars (Doveri et al. 2008; Baldoni et al. 2009) (table 4).

Series Number Institute References ssrOeUA-DCA 15 Boku, Vienna, Austria Sefc et al 2000 IAS-oli 5 CSIC, Córdoba, Spagna Rallo et al 2000 GAPU 20 Agrobios, Matera, Italia Carriero et al 2002 UDO 30 Università di Udine, Italia Cipriani et al 2002 EMO 7 ETSIAM, Córdoba, Spagna De La Rosa et al 2002

**6.1 Random Amplified Polymorphic DNA (RAPDs)** 

**6.2 Amplified Fragment Length Polymorphism (AFLPs)** 

traceability of *olive oil* (Busconi et al. 2003; Pafundo et al. 2005).

marker for marker-assisted breeding programs (Bracci et al., 2011).

crossing species, where the level of heterozygosity is high.

**6.4 Simple Sequence Repeats (SSRs)** 

*Totale 77*

**6.3 Development of sequence-characterized amplified regions (SCARs)** 

2004; Wu et al. 2004) as well.

distinguish all the *olive oil*s examined, even if only a partial correspondence with the AFLP profile obtained from the leaves was obtained. Martins-Lopes et al. (2008) evaluated the efficiency of RAPD, ISSR and SSR molecular markers for *olive oil* varietal identification and their possible use in certification purposes (Bracci et al., 2011).

Consolandi et al. (2008) reported the development of a semi-automated SNP genotyping assay to verify the origin and the authenticity of extra-virgin *olive oil*s. The authors developed a Ligation Detection Reaction (LDR)/Universal Array (UA) platform by using several olive SNPs. They found that 13 accurately chosen SNPs were sufficient to unequivocally discriminate a panel of 49 different cultivars (Bracci et al., 2011).

Doveri et al. (2006) published a cautionary note on the use of DNA markers for provenance testing. Their observations were based on non-concordance between the genetic profiles of *olive oil* and fruit. They suggested that this could be due to the contribution of pollen donors in DNA extracted from the paste obtained by crushing whole fruits. They concluded that care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues (Martins-Lopes et al., 2008). It is to note that, the possible presence of additional alleles due to paternal contribution in oils extracted from entire drupes, should be taken into consideration for variety traceability purposes when comparing the amplification profiles of leaves with the corresponding oils for (Alba et al., 2009)

In a recent study, Pafundo et al. (2010) investigated the effect of the storage time on the degradation of the DNA purified from the oil; a negative correlation between storage time and quality–quantity of recovered DNA has been observed. The authors showed that 1 month after the production of the oil the degradation increases making harder traceability goals (Bracci et al., 2011). Table 3 presents a brief report on the application of DNA-based molecular markers for cultivar traceability in *olive oil*.


Table 3. Applications of DNA-based molecular markers for cultivar traceability in *olive oil*

distinguish all the *olive oil*s examined, even if only a partial correspondence with the AFLP profile obtained from the leaves was obtained. Martins-Lopes et al. (2008) evaluated the efficiency of RAPD, ISSR and SSR molecular markers for *olive oil* varietal identification and

Consolandi et al. (2008) reported the development of a semi-automated SNP genotyping assay to verify the origin and the authenticity of extra-virgin *olive oil*s. The authors developed a Ligation Detection Reaction (LDR)/Universal Array (UA) platform by using several olive SNPs. They found that 13 accurately chosen SNPs were sufficient to

Doveri et al. (2006) published a cautionary note on the use of DNA markers for provenance testing. Their observations were based on non-concordance between the genetic profiles of *olive oil* and fruit. They suggested that this could be due to the contribution of pollen donors in DNA extracted from the paste obtained by crushing whole fruits. They concluded that care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues (Martins-Lopes et al., 2008). It is to note that, the possible presence of additional alleles due to paternal contribution in oils extracted from entire drupes, should be taken into consideration for variety traceability purposes when comparing the amplification profiles of leaves with the corresponding oils

In a recent study, Pafundo et al. (2010) investigated the effect of the storage time on the degradation of the DNA purified from the oil; a negative correlation between storage time and quality–quantity of recovered DNA has been observed. The authors showed that 1 month after the production of the oil the degradation increases making harder traceability goals (Bracci et al., 2011). Table 3 presents a brief report on the application of DNA-based

Molecular marker Developers References

Paran & Michelmore (1993)

Morgante & Olivieri (1993)

Table 3. Applications of DNA-based molecular markers for cultivar traceability in *olive oil*

Williams et al. (1990) Muzzalupo and Perri (2002),

Zietkiewicz et al. (1994) Pasqualone et al. (2001),

Botstein et al. (1980) Intrieri et al. (2007)

Vos et al. (1995) Busconi et al. 2003, Pafundo et

Martins-Lopes et al. (2008)

al. 2005

De la Torre et al. (2004), Pafundo et al. (2007)

Martins-Lopes et al. (2008), Alba et al. (2009), Rabiei et al. (2010)

Martins-Lopes et al. (2008)

unequivocally discriminate a panel of 49 different cultivars (Bracci et al., 2011).

their possible use in certification purposes (Bracci et al., 2011).

molecular markers for cultivar traceability in *olive oil*.

for (Alba et al., 2009)

RAPD (Random Amplified Polymorphic DNA)

AFLP (Amplified Fragment Length Polymorphism)

SCAR (Sequence Characterized Amplified Region)

SSRs (Simple Sequence Repeats)

ISSR (Inter Simple sequence Repeats)

Chloroplast and mitochondrial (Direct sequencing)

## **6.1 Random Amplified Polymorphic DNA (RAPDs)**

The applicability to the traceability of cultivars in the *olive oil* by means of RAPDs was evaluated by Muzzalupo & Perri (2002). This kind of markers was used, together with other DNA-based markers, in the construction of the first olive linkage maps (De la Rosa et al. 2004; Wu et al. 2004) as well.

#### **6.2 Amplified Fragment Length Polymorphism (AFLPs)**

AFLPs have been widely used for DNA fingerprinting of cultivars (Angiolillo et al. 1999; Owen et al. 2005), to analyse the relationships between wild and cultivated olive (Baldoni et al. 2006), for the construction of linkage maps (de la Rosa et al. 2003) and for cultivar traceability of *olive oil* (Busconi et al. 2003; Pafundo et al. 2005).

#### **6.3 Development of sequence-characterized amplified regions (SCARs)**

SCARs have been used for cultivar identification (Busconi et al. 2006) and in *olive oil* traceability (De la Torre et al. 2004; Pafundo et al. 2007). Putative associations of several SCAR markers with fruit characteristics (Mekuria et al. 2002) and resistance to pathogenic fungi (Herna´ndez et al. 2001) were found, suggesting the applicability of this type of marker for marker-assisted breeding programs (Bracci et al., 2011).

#### **6.4 Simple Sequence Repeats (SSRs)**

Many authors have reported on SSR development in olive and several of them are currently available for DNA analysis (Cipriani et al. 2002; De la Rosa et al. 2002; Rabiei & Tahmasebi Enferadi 2009; Sabino Gil et al. 2006; Sefc et al. 2000). This technique benefits from the use of microsatellites which are short stretches (1-6-bp long) of DNA, tandemly repeated several times. The number of repeats can vary from one individual to another. Besides, they are abundant in eucaryotic genomes. A combination of several SSRs loci allows virtually discrimination of individuals originated through sexual reproduction, especially in outcrossing species, where the level of heterozygosity is high.

In Olea europaea, these markers have been used for different applications such as cultivar discrimination (Sarri et al. 2006; Fendri et al. 2010), study of relationships between wild and cultivated olive tree (Belaj et al. 2007), construction of association maps (De la Rosa et al. 2003), paternity analysis (Mookerjee et al. 2005) and identification of *olive oil* varietal composition (Alba et al. 2009; Ayed et al. 2009; Rabiei et al. 2010). A list of recommended SSR markers and protocols for olive genotyping has been provided with the aim to develop a robust method to track the origin of olive cultivars (Doveri et al. 2008; Baldoni et al. 2009) (table 4).


Table 4. List of microsatellites isolated in olive which their related primers are available in the literature, most of them have been used in case of *olive oil* traceability (Bracci et al., 2011) For studying the informative potential of the microsatellites, the observed (*Ho*) and expected (*He*) heterozygosities generally are calculated using the software POPGENE ver. 1.31 (Yeh et al., 1999, as cited in Alba et al., 2009). *He* values were estimated using the formula proposed by Nei et al. (1973):

$$H\_e = 1 - \sum p\_i^2 \tag{1}$$

Traceability of Origin and Authenticity of *Olive Oil* 175

Fig. 1. Electropheregram of PCR products separated by capillary electrophoresis of

intensity of amplified allele detection (Rabiei et al., 2010).

and characteristics of olive fruit (Bracci et al., 2011).

et al. 1994, as cited in Bracci et al., 2011) (Table 1).

**6.7 Expressed Sequence Tags (ESTs)** 

microsatellite loci UDO008 and GAPU 101, obtained from DNA extracted from Carolea and Frantoio leaves and oils. Allele sizes are below the x axis. The scores on Y-axis are the

Understanding the function of genes and other parts of the genome is known as functional genomics. In olive, efforts to improve the identification and annotation of genes are prevalently based on EST identification, which are predominantly related to pollen allergens

The first nucleotide sequences isolated in 1994 in olive coded for allergenic proteins (Villalba

where *pi* is the frequency of the *ith* allele. The power of discrimination (PD) [21] of microsatellite primer pairs are also calculated as reported by Cipriani et al. (2002), where the allele frequency of the *He* formula is replaced by the genotype frequency.(Alba et al., 2009)

A research carried on the use of SSRs as a tool to identify the genetic background of *olive oil* which was involved the analysis of DNA sequences using a panel of seven simple sequence repeats (SSRs) to provide genotype-specific allelic profiles (Rabiei et al., 2010). The amplified SSR fragments and the DNA profiles from the monovarietal oil corresponded to the profiles from the leaves of the same cultivar. The most reliable SSR in providing correct allele sizing in distinguishing either single cultivar *olive oil* samples or the different ratios of their blends are DCA3, DCA4, DCA16, DCA17, and GAPU101, while DCA9, GAPU59 produced less concordance against data obtained by the genetic analysis of leaf samples. Desalted PCR product has been analyzed on a MegaBACE 500 capillary sequencer (Amersham Biosciences) using Genetic Profiler v2.0 software to estimate allele sizes (figure 1).

Rabiei et al., 2010 concluded PCR product purification and selection of a set of markers with a highly robust amplification pattern is needed to have reproducible results in certify the genetic background of *olive oil*.

### **6.5 Inter simple sequence repeat (ISSR) polymorphisms**

ISSRs are DNA fragments of about 100–3,000 bp located between adjacent, oppositely oriented microsatellite regions. These markers were used with success to distinguish 10 Italian varieties, by analysing genomic DNA extracted from the olive fruit (Pasqualone et al. 2001), and for the study of cultivar traceability in *olive oil* (Pasqualone et al. 2001, Martins-Lopes et al. 2008).

#### **6.6 Chloroplast genome sequencing**

A very important results, recently published, in *Olea europaea* L. genomic studies have been the DNA sequencing of the entire plastome of the Italian cultivar 'Frantoio' (Mariotti et al. 2010). This sequence has a length of 155,889 bp and showed an organization and gene order that is conserved among numerous Angiosperms. The olive chloroplast contains 130 genes and 644 repetitive sequences (among which 633 mono-nucleotide SSRs, 6 di-, 3 tetra- 2 penta-nucleotide SSRs were identified) (Bracci et al., 2011)

The annotated sequence was used to evaluate the content of coding genes, the extent, and distribution of repeated and long dispersed sequences and the nucleotide composition pattern. These analyses provided essential information for structural, functional and comparative genomic studies in olive plastids. Furthermore, the alignment of the olive plastome sequence to those of other varieties and species identified 30 new organellar polymorphisms within the cultivated olive. chloroplast DNA polymorphisms has been used as molecular markers to identify cultivars of *Olea europaea* L. (Intrieri et al. 2007).

For studying the informative potential of the microsatellites, the observed (*Ho*) and expected (*He*) heterozygosities generally are calculated using the software POPGENE ver. 1.31 (Yeh et al., 1999, as cited in Alba et al., 2009). *He* values were estimated using the formula proposed

where *pi* is the frequency of the *ith* allele. The power of discrimination (PD) [21] of microsatellite primer pairs are also calculated as reported by Cipriani et al. (2002), where the allele frequency of the *He* formula is replaced by the genotype frequency.(Alba et al., 2009) A research carried on the use of SSRs as a tool to identify the genetic background of *olive oil* which was involved the analysis of DNA sequences using a panel of seven simple sequence repeats (SSRs) to provide genotype-specific allelic profiles (Rabiei et al., 2010). The amplified SSR fragments and the DNA profiles from the monovarietal oil corresponded to the profiles from the leaves of the same cultivar. The most reliable SSR in providing correct allele sizing in distinguishing either single cultivar *olive oil* samples or the different ratios of their blends are DCA3, DCA4, DCA16, DCA17, and GAPU101, while DCA9, GAPU59 produced less concordance against data obtained by the genetic analysis of leaf samples. Desalted PCR product has been analyzed on a MegaBACE 500 capillary sequencer (Amersham

Biosciences) using Genetic Profiler v2.0 software to estimate allele sizes (figure 1).

**6.5 Inter simple sequence repeat (ISSR) polymorphisms** 

penta-nucleotide SSRs were identified) (Bracci et al., 2011)

Rabiei et al., 2010 concluded PCR product purification and selection of a set of markers with a highly robust amplification pattern is needed to have reproducible results in certify the

ISSRs are DNA fragments of about 100–3,000 bp located between adjacent, oppositely oriented microsatellite regions. These markers were used with success to distinguish 10 Italian varieties, by analysing genomic DNA extracted from the olive fruit (Pasqualone et al. 2001), and for the study of cultivar traceability in *olive oil* (Pasqualone et al. 2001, Martins-Lopes et al. 2008).

A very important results, recently published, in *Olea europaea* L. genomic studies have been the DNA sequencing of the entire plastome of the Italian cultivar 'Frantoio' (Mariotti et al. 2010). This sequence has a length of 155,889 bp and showed an organization and gene order that is conserved among numerous Angiosperms. The olive chloroplast contains 130 genes and 644 repetitive sequences (among which 633 mono-nucleotide SSRs, 6 di-, 3 tetra- 2

The annotated sequence was used to evaluate the content of coding genes, the extent, and distribution of repeated and long dispersed sequences and the nucleotide composition pattern. These analyses provided essential information for structural, functional and comparative genomic studies in olive plastids. Furthermore, the alignment of the olive plastome sequence to those of other varieties and species identified 30 new organellar polymorphisms within the cultivated olive. chloroplast DNA polymorphisms has been used

as molecular markers to identify cultivars of *Olea europaea* L. (Intrieri et al. 2007).

<sup>2</sup> <sup>1</sup> *H p e i* (1)

by Nei et al. (1973):

genetic background of *olive oil*.

**6.6 Chloroplast genome sequencing** 

Fig. 1. Electropheregram of PCR products separated by capillary electrophoresis of microsatellite loci UDO008 and GAPU 101, obtained from DNA extracted from Carolea and Frantoio leaves and oils. Allele sizes are below the x axis. The scores on Y-axis are the intensity of amplified allele detection (Rabiei et al., 2010).

#### **6.7 Expressed Sequence Tags (ESTs)**

Understanding the function of genes and other parts of the genome is known as functional genomics. In olive, efforts to improve the identification and annotation of genes are prevalently based on EST identification, which are predominantly related to pollen allergens and characteristics of olive fruit (Bracci et al., 2011).

The first nucleotide sequences isolated in 1994 in olive coded for allergenic proteins (Villalba et al. 1994, as cited in Bracci et al., 2011) (Table 1).

Traceability of Origin and Authenticity of *Olive Oil* 177

compatibility among the parental cultivars, since this significantly influences the rate of contamination from external pollen donors. These results indicate that knowledge of crosscompatibility among cultivars is necessary to plan efficient olive breeding crosses (Diaz et

The possibility of associating genetic characteristics and DNA-based molecular markers is very important to select the progeny showing interesting agronomical traits and even specific organoleptic characteristics at the first stages of development which may use as a marker for future *olive oil* identification. However, this technique, called marker-assisted selection (MAS), requires some knowledge on the co-segregation of molecular markers and

For the inefficiency of analytical parameters in showing variability among samples of the same cultivar/blend due to the environmental conditions and pressing technologies, Several DNA-based technologies and traceability analysis has been used to reveal the different origin of lots that have contributed to the olive oil blend. In this regard, DNA-based methods make an important contribution to protect high quality brand names and in turn

The knowledge of genome nucleotide sequences also could be useful to identify new sequence polymorphisms, which will be very useful in the development of many new cultivar-specific molecular markers (e.g.; Single Nucleotide Polymorphisms, SNPs) and in the implementation of more efficient protocols for tracking and protect *olive oil* origin (in

The greatest challenges one faces while using DNA technology is the low quality and highly degraded DNA recovered from the fatty matrices and the impact of oil extraction processing on the size of the recovered DNA. DNA of low, difficult to determine content and of unknown, variable quality would potentially lead to inconsistent and consequently inconclusive results. Although, the concentration of DNA did not appear to be limiting; rather, successful PCR amplification likely depended on the ability of the DNA extraction

It is to be considered if the DNA is damaged, it could be not properly accessible to the DNA polymerase, which stalls at the sites of damage and the reaction may be interrupted; this being able to influence the length and significance of the synthesized amplicons. The use of proteinase K during extraction process has recommended for a better protection of DNA

Identification of molecular markers suitable for tracing the genetic identity of olive cultivars from which oil is produced, on the other hand, has a great importance**.** For making decision, which molecular markers will be more useful in obtaining reliable results through the numerous molecular markers existing in the literature, many of them have been practically examined (including RAPDs, AFLPs, SCARs, SSRs, ISSR, SNPs, …). A combination of molecular markers (RAPD, ISSR, and SSR) to establish a relationship between small-scaleproduced monovarietal and commercial *olive oil* samples for certification purposes has been

method to free DNA from inhibitors of PCR present in the *olive oil*.

from degradation and increase in DNA yield, as well.

genetic characteristics in the progeny. (Bracci et al., 2011)

al. 2007).

**8. Conclusion** 

the consumer

POD *olive oils*).

proposed.

#### **6.8 Real time-PCR**

The detection of frauds, either due to the mixtures with oils of other species such as hazelnut, or to the certification of PDOs would need quantitative tools. At its best, conventional PCR remains a semi-quantitative technique, and therefore, it is not optimal for authentication purposes when quantification is needed (Gimenez et al.; 2010).

The use of real-time chemistries allows for the detection of PCR amplification during the early phases of the reaction, providing a distinct advantage over detection of amplification at the final phase or end-point of the PCR reaction. qRT-PCR is a useful tool in the development of molecular markers for *olive oil* authentication since it allows inspecting the PCR efficiency. Besides qRT-PCR should be used for the optimisation of the amplicon size and the DNA isolation procedure which are critical aspects in *olive oil* authentication. The potential of cpDNA for *olive oil* authentication should be addressed in the future (Gimenez et al.; 2010).
