**8. Conclusion**

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

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

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

Several sequences from noncoding spacer region between psbA-trnH and partial coding region of matK of plastid genome provided a good discrimination of pure *olive oil* and its

The plastid based molecular DNA technology has a great potential to be used for rapid

Similar to other woody species, olive is characterized by a long juvenile phase that ranges between 10 and 15 years. This represents a great obstacle to breeding programs and makes the genetic improvement of olive very difficult and expensive. Although seedling-forcing growth protocols have been developed to reduce the length of this phase, the evaluation of the agronomic performance of mature olive plants still requires at least 5 years of experimentation (Santos-Antunes et al. 2005). For this reason, the application of molecular markers both to confirm the parental origins of the progeny and to select early agronomical characteristic-associated markers (Mart´n et al. 2005) can be very useful to reduce the time

With regard the paternity analysis, SSRs are the most suitable to trace the genetic contribution of alleles from the parents to the offspring, being co-dominant and highly polymorphic markers (Mookerjee et al. 2005). The effectiveness of SSRs in the identification of paternity contribution to progeny obtained from olive breeding programs has been demonstrated by several authors (De la Rosa et al. 2004; Diaz et al. 2007). The results demonstrated that SSR analysis is a convenient technique to routinely assess the crosses made in breeding programs and to for check self-incompatibility in olive cultivars (Diaz et al. 2006). These studies have highlighted that no contamination by self-pollen was found, indicating that placing pollination bags well before anthesis is important and that emasculation to avoid selfing is unnecessary (De la Rosa et al. 2004). The analysis also revealed that the main factor affecting the success of crosses seems to be the inter-

authentication purposes when quantification is needed (Gimenez et al.; 2010).

admixture by other vegetable oils such as canola and sunflower.

detection of adulteration easily up to 5% in *olive oil* (Kumar et al., 2011).

and cost of the development of new genotypes (Bracci et al., 2011).

**6.8 Real time-PCR** 

et al.; 2010).

**6.9 DNA barcode** 

**7. Paternity analysis** 

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 consumer

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 POD *olive oils*).

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

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 from degradation and increase in DNA yield, as well.

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 proposed.

Traceability of Origin and Authenticity of *Olive Oil* 179

Arvanitoyannis, I. S. & Vlachos, A. (2007). Implementation of physicochemical and sensory

authentication/adulteration, *Crit. Rev. Food Sci. Nutr*. Vol.47, pp. 441–498 Ayed, R.B.; Grati-Kamoun, N.; Moreau, F. & Rebaï, A. (2009). Comparative study of

Baldoni, L. & Belaj, A. (2009). *Olive*, In: Vollmann J, Rajean I (eds) Oil crops. Handbook of

Baldoni, L.; Tosti, N.; Ricciolini, C.; Belaj, A.; Arcioni, S.; Pannelli, G.; Germana, M.A.; Mulas,

Belaj, A.; Mun˜oz-Diez, C.; Baldoni, L.; Porceddu, A.; Barranco, D. & Satovic, Z. (2007).

Bracci, T.; Busconi, M.; Fogher, C. & Sebastiani, L. (2011). Molecular studies in olive (Olea

Breton, C.; Claux, D.; Metton, I.; Skorski, G. & Bervillè, A. (2004). Comparative Study of

Busconi, M.; Sebastiani, L. & Fogher, C. (2006). Development of SCAR markers for

Busconi, M.;, Foroni, C.; Corradi, M.; Bongiorni, C.; Cattapan, F. & Fogher, C. (2003). DNA

Cantini, C.; Cimato, A. & Sani, G. (1999). Morphological evaluation of olive germplasm

Carriero, F.; Fontanazza, G.; Cellini, F. & Giorio, G. (2002). Identification of simple sequence repeats (SSRs) in olive (*Olea europaea* L.), *Theor Appl Genet*. Vol.104, pp. 301–307 Cerretani, L.; Bendini, A.; Del Caro, A.; Piga, A.; Vacca, V.; Caboni, M. F. & Gallina Toschi,

Christopoulou, E.; Lazakari, M.; Komaitis, M. & Kaselimis, K. (2004). Effectiveness of

present in Tuscany region, *Euphytica*. Vol.109, pp. 173– 181

cultivars in Sardinia, *Eur. Food Res. Technol.* Vol.222, pp. 354–361

*olive oil*s with vegetable oils, *Food Chem*. Vol.84, pp. 463–474

genome analysis, *Plant Cell Rep*. DOI 10.1007/s00299-010-0991-9

Mediterranean assessed by SSR markers, *Ann Bot*. Vol.100, pp. 449–458 Botstein, D.; White, R.L.; Skolnick, M. & Davis, R.W. (1980). Construction of a genetic map in

central Mediterranean basin, *Ann Bot.* Vol.98, pp. 935–942 Bartolini, G. (2008). *Olea databases*, Available at: http://www.oleadb.it

Vol.108, pp. 374–383

*Eur Food Res Technol.* Vol. 229, pp. 757–762

10.1007/978-0-387-77594-4\_13

*Chem,*Vol.52, No.3, pp. 531-537

*Food Chem.;* Voh.83, No.1, pp. 127-134

314–331

59–68

(2008). Geographical origin classification of *olive oil*s by PTR-MS, *Food Chem*.

analysis in conjunction with multivariate analysis towards assessing *olive oil*

microsatellite proWles of DNA from oil and leaves of two Tunisian olive cultivars,

plant breeding, vol 4. Springer Science Business Media, New York, pp. 397–421. doi

M. & Porceddu, A. (2006). Genetic structure of wild and cultivated olives in the

Genetic diversity and population structure of wild olives from the North-western

man using restriction fragment length polymorphisms, *Am J Hum Genet*. Vol.32, pp.

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

Methods for DNA Preparation from *Olive oil* Samples to Identify Cultivar SSR Alleles in Commercial Oil Samples: Possible Forensic Applications, *J. Agric. Food* 

germplasm characterisation in olive tree (Olea europaea L.), *Mol Breed*. Vol.17, pp.

extraction from *olive oil* and its use in the identification of the production cultivar,

T. (2006). Preliminary characterisation of virgin *olive oil*s obtained from different

determination of fatty acids and triglycerides for the detection of adulteration of

Several authors recommended sequences of DNA that show polymorphism at low hierarchical level are therefore suitable for distinguishing between individuals within the same species. They clearly pointed non-coding nuclear DNA sequences could be the best choice. Among those sequences, the microsatellites are likely the most suitable ones. However SNPs that require shorter than 100 bp DNA templates, considered to be successfully used for a wider range of olive oil identification.

In some cases of using microsatellite, the microsatellite profiles obtained with the monovarietal oil-derived DNA were generally consistent with the cultivar used, although some ambiguities were recorded likely due to contamination in monovarietal oils by other cultivars grown in the same block or contaminations occurred at the mill. Moreover, in some cases the lack of matching in leaf and oil profiles has been reported that was due to the presence of embryos in berry seeds that brought the alleles of pollinators. Other cases of mis-amplification were recorded as a missing allele, due either to the preferential amplification of one of the two alleles in oil-derived DNA templates, or to the excess of degradation of the DNA template of the miss allele, that limited the production of a sufficient number of copies of that allele to be detected. In such a case, real-time PCR assay could possibly solve this kind of problems.

To trace out the adulteration in *olive oil* using combined approach of molecular biology and bioinformatics based on unique SNPs present in conserved DNA sequence of plastid genomes of sunflower, canola and olive has been already performed. In general, plastid/chloroplasts are miniature organelles (approx. 5 X 3 µm in size) enclosed in double layer membranes. They are present in abundance (10–100 per cell, and each plastid contains about 100 copies of circular plastid genomes, average size 150 kb) and there is probability that most of the plastid organelles may be left intact due to miniature size when cold pressed to extract the oil from seeds of olive, canola and sunflower.

Moreover, plastid DNA present in extracted oil could be safe from nucleases activities due to double layer membranes and present in large number of copies in comparison to 1–2 nuclear genomic DNA which may be more prone to degradation.

In addition, a new chloroplast marker represents a valuable tool to assess the level of olive intercultivar plastome variation for use in population genetic analysis, phylogenesis, cultivar characterisation and DNA food tracking is recommended.

In summary, molecular biological techniques have become an every-day tool to solve a number of problems and questions in the section of varietal/species identification, fraud, traceability and paternity analysis.
