**2. Molecular markers for olive cultivar identification and monovarietal olive oil traceability**

#### **2.1 ISSR markers**

ISSR are DNA based markers that involve amplification via polymerase chain reaction (PCR) of DNA regions situated between adjacent and inversely oriented 16–18 bp long simple sequence repeats, already employed successfully for cultivar identification purposes. ISSR, together with physico-chemical analyses, were used for olive cultivar identification in 2001 by Pasqualone et al.. Here we focus on the molecular aspect of that paper. In particular, 10 olive cultivars were screened with 10 ISSR markers. DNA was extracted from fresh leaves


Table 3. Cultivar identification and olive oil traceability research activities conducted at the Department of Agro-forestry and Environmental Biology and Chemistry, University of Bari, Italy.

and ripe drupes following the protocol proposed by Sharp et al. (1988) with some modifications. Monovarietal olive oil was obtained by mechanical extraction with the method proposed by Catalano & Caponio (1996) and, after centrifugation of 250 ml oil, the cell residue sediment was recovered and used for the DNA extraction with the same protocol of leaves and drupes. DNA extracted from leaves and drupes was rich in PCR interfering substances such as phenolic compounds and lipids, suggesting a further purification achieved by chromatography on a commercial device. On the contrary, the DNA extraction protocol revealed to be inadequate when processing cell residue sediments, yielding degraded or unsuitable for PCR experiments (i.e. all 10 ISSR primers failed to amplify). Three ISSR completely failed to amplify both on leaf and drupe DNA, while 7 ISSR gave distinguishable electrophoretic patterns with appreciable polymorphic content, so that a set-up of an identification key with the lowest number of molecular markers that distinguish among the 10 cultivars was obtained by means of only 2 ISSR. The paper

56 The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribution

Unique sensorial, aromatic, gustatory, nutraceutical and healthy properties of the different Italian extra-virgin olive oil justify the attention devoted to the respect of standard

The skill developed by olive growers after centuries of cultivation has led Italy to become one of the first producing countries of olive oil considering the ratio between quality and quantity. In order to guarantee, preserve and promote the both the olive genetic variability and the technical expertise in cultivating, collecting and transforming processes of olives, Italian producers consortiated and obtained marks of protected designation of origin (PDO) at European level according to EC Regulation no. 510/06. An official production protocol of any PDO extra-virgin olive oil must meet a specific varietal requirement alone or in combination with other varieties eventually allowed by the protocol at well defined percentages. Therefore, it is clear that traceability, of raw and processed materials, specially by means of molecular markers, assumes a crucial role in the certification of products for the

Fig. 1. Percentage distribution of harvested area (A) and total olive production (B) in the

**Europe 50%**

The aim of this paper is to review the research activity conducted in the last decade by the DiBCA in the fields of Southern Italian olive cultivar identification and monovarietal olive oil traceability (tab. 3, tab. 4). To this purpose, only certified material from the Experimental Station "P. Martucci" of the Plant Production Department (D.P.V.) and the DiBCA, from the Nursery "Vivai Giannoccaro" in Bari-Italy, from the Olive Pre-multiplication Centre "Concadoro" in Palagiano - Italy and from the Department of Olive Research, Idleb, Syria

**Northern Africa 15%**

**Western Asia 14%**

**Others 2%**

> **Europe 69%**

**2. Molecular markers for olive cultivar identification and monovarietal olive** 

ISSR are DNA based markers that involve amplification via polymerase chain reaction (PCR) of DNA regions situated between adjacent and inversely oriented 16–18 bp long simple sequence repeats, already employed successfully for cultivar identification purposes. ISSR, together with physico-chemical analyses, were used for olive cultivar identification in 2001 by Pasqualone et al.. Here we focus on the molecular aspect of that paper. In particular, 10 olive cultivars were screened with 10 ISSR markers. DNA was extracted from fresh leaves

main worldwide geographical productive areas. (FAO, 2010).

was analysed.

**Northern Africa 33%**

**oil traceability 2.1 ISSR markers** 

parameters of quality all along the productive chain.

protection of consumers and for fraud prevention.

**Others 1%**

**A B** 

**Western Asia 16%**

Olive (*Olea Europaea* L.): Southern-Italian Biodiversity

researchers to overcome the problem.

**2.2 AFLP markers** 

**2.3 SSR markers** 

Assessment and Traceability of Processed Products by Means of Molecular Markers 59

method for extracting DNA from olive oil is a prerequisite for developing a platform of marker-based assessment of olive oil cultivar composition (Consolandi et al, 2008). Today, many commercial kits to extract DNA from processed food are available and help

AFLP markers (Vos et al., 1995) allow multi-locus screening of a genome in absence of preliminary sequence knowledge. The use of AFLP for olive cultivar identification and oil traceability has been described before. In particular, Montemurro et al. (2005) tested 3 AFLP primer combinations and 27 SSR (discussed later) on a set of 60 olive cultivars, some of them present in replicates and coming from different germplasm collections, for a total of 112 samples, from the Mediterranean basin. DNA was extracted from lyophilized leaves with the commercial Gene Elute Plant kit and a biodiversity study was conducted. A dendrogram of genetic similarity based on Jaccard index (1901) was calculated with NTSYS-PC (Rohlf, 1992) using AFLP and SSR data. All the cultivars resulted distinguished and clustered according to their type of use: oil, table, and dual purpose cultivars. Differences between the two techniques were detected. The ratio of polymorphic bands/total bands ranged from a maximum of 100% for SSRs to 42% for AFLP, in line with other authors (Belaj et al., 2003; Powell et al., 1996; Russell et al., 1997) that confirmed the higher efficiency of SSR to distinguish among olive cultivars, but revealed the ability of AFLP to distinguish intravarietal clones respect to SSR. Therefore, AFLP represent the best choice for studies into

Based on this experience, the authors hypothesized and wanted to verify the effectiveness of AFLP in distinguishing among oils from different cultivars. To this purpose, Montemurro et al. (2008) tested the efficiency of AFLP on 10 different virgin oils. Oils were obtained with a laboratory-scale pilot plant consisting of a SK1 hammer-crusher (Retsch, Haan, Germany) and 200 ml were centrifuged to recover residues. In this case, in order to overcome problems of DNA of low quality encountered in the past, the commercial Gene Elute Plant kit (Sigma) to extract DNA from cell residue sediments and lyophilized leaves was adopted. All the 6 AFLP primer combination yielded polymorphic electrophoretic patterns for all the 10 cultivars both for leaf and oil DNA. The level of degradation, the low DNA concentration extractable from oil and the template needed by the restriction enzymes for the initial digestion (100 ng) in the AFLP method suggested a standard protocol implementation in case of DNA from oil (Montemurro et al., 2005). Specifically, no dilution of both restricted/ligated and preamplified products were performed before selective amplifications, on the contrary 30 μl of restricted/ligated product, much higher than usual, was used for ensuring good amounts of DNA template. Differences about the band intensity emerged for the amplicons of DNA from oil, reducing the number of scorable bands after

genetic relationship or for accurate evaluation of intra-cultivar variability.

electrophoresis, but showing sufficient information useful for olive oil traceability.

As discussed before, SSR efficiency compared with other DNA-based markers in olive was demonstrated and nowadays they are preferred by several authors for biodiversity studies, germplasm characterization and varietal fingerprinting. Moreover, the recent introduction of fluorescent labeled primers that avoid radioactive labeled ones for PCR-based markers let the use of automatic sequencers, less time consuming and with minor health implications for operators. Besides, automatic sequencers are more effective, because capillary


Table 4. List of the olive cultivars screened by means of AFLP, ISSR and SSR at the DiBCA

concluded that ISSR represent a powerful tool to distinguish among olive cultivars but revealed an important aspect in relation to the establishment of more accurate protocols to be used for DNA extraction from cell residue sediments of olive oil. A robust and efficient method for extracting DNA from olive oil is a prerequisite for developing a platform of marker-based assessment of olive oil cultivar composition (Consolandi et al, 2008). Today, many commercial kits to extract DNA from processed food are available and help researchers to overcome the problem.
