**1. Introduction**

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

Tobe, H. & Raven, PH. (1987). Systematic embryology of the Anisophylleaceae. *Annals of the* 

Twyford, AD. & Ennos, RA. (2011). Next generation hybridization and introgression.

Zeng Z-B. (2005). QTL mapping and the genetic basis of adaptation: recent developments. *Genetica*, Vol.123, No.1-2, (February 2005), pp. 25-37, ISSN 0016-6707 Zhang, L-B.; Simmons MP.; ocyan, A. & Renner SS. (2006). Phylogeny of the Cucurbitales

*Missouri botanical garden*, Vol.74, No.1, pp. 1-26, ISSN 0026-6493

*Heredity*, doi: 10.1038/hdy.2011.68., ISSN 0018-067X

Vol.39, No.2, (May 2006), pp. 305-322, ISSN 1055-7903

ISSN 1055-7903

*Molecular Phylogenetics and Evolution*, Vol. 60, No. 3, (September 2011), pp. 428-444,

based on DNA sequences of nine loci from three genomes: Implications for morphological and sexual system evolution. *Molecular Phylogenetics and evolution*,

> Southern Italy has geographical, climatic, historical and traditional peculiarities summarized in a landscape to which olive trees are the backdrop. Olive (*Olea europaea* L. subsp. *europaea*) is cultivated since the third millennium B.C. in the Eastern region of the Mediterranean sea (Loukas & Krimbas, 1983) and spread later around the basin following land and maritime routes to Italy, Spain, North Africa and France (Angiolillo et al., 1999).

> The plant longevity, the cross-pollinating nature of the species and its secular history contributed to determine a wide germplasm biodiversity with more than 1200 cultivars, 9.992 thousand harvested hectares and a total production of 18 million tons (Tab.1; FAO 2009) mostly concentrated in the main olive oil producing countries (Alba et al., 2009a). Europe alone produces the 69% of the total worldwide production (Fig. 1). Italy is one of the main producers and has a rich assortment of olive cultivars, with a total number of 395 registered entries in the national list of *O. europaea* L. Italian cultivars (Official Journal of the Italian Republic, 1994), covering 1,18 million hectares and a total production of 3.6 million tons (ISTAT, 2010). The 80% of the cultivation is mostly concentrated in Southern Italy, where the production touches the 88% of the whole Italian olive production. In particular, Apulia region alone produces 1/3 of the total (about 1,7 milion tons) (Tab. 2).

> Nowadays olive-growing has a crucial commercial role in many Italian regions with a richness of biodiversity spread from North to South in terms of cultivars grown, agronomic practices, processes of transformation of the raw material. Therefore, the preservation and protection of Italian olive biodiversity depends upon the use of genetic research tools like molecular markers for a correct cultivar identification. In countries that belong to the "Union Internationale pour la Protection des Obtentions Végetales" (UPOV) the registration, multiplication, certification, and commercialization of olive varieties rely on the evaluation of morpho-agronomic descriptors. Recently, morphological, biochemical and agronomic traits have been complemented with the large array of DNA molecular marker types

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

2009). \* Data are referred to 2008.

Assessment and Traceability of Processed Products by Means of Molecular Markers 55

Total Production (million tons)

Total olive production (table, oil, double purpose)

thousand ha % on the total area million tons % on the total production

Area Harvested (thousand Ha)

Table 1. Area harvested and total olive production in the main producing countries of the Mediterranean Bacin and in the main worldwide geographical productive areas. (FAO,

> Italian total area allocated to olive

Northern Italy 28,4 2,4 0,49 1,4 Central Itali 205,2 17,3 3,88 10,7 Southern Italy 949,8 80,3 31,75 87,9

Apulia region 377,6 39,8 11,72 36,9 Other Southern regions 572,2 60,2 20,03 63,1

**Total 1183,4 36,11** 

Table 2. Consistency of the Italian olive cultivation (ISTAT, 2010)

Albania 40,0 0,05 Algeria 288,4 0,48 Croatia 15,3 0,03 Cyprus 12,0 0,01 Egypt 110,0 0,50 France 19,0 0,03 Greece\* 800,0 2,31 Israel 16,0 0,03 Italy 1.159,0 3,60 Jordan 60,7 0,14 Libya\* 0,2 0,18 Morocco 550,0 0,77 Portugal 380,7 0,36 Spain 2.500,0 6,20 Syrian 635,7 0,89 Tunisia 2.300,0 0,75 Turkey 727,5 1,29 **World 9.922,8 18,24**  Europe 4.923,2 12,61 Northern Africa 3.248,6 2,68 Western Asia 1.604,0 2,54 Others 146,9 0,42

available, that provide an accurate and unambiguous tool for a correct identification of cultivated olive germplasm. Corrado et al. (2009) report that morpho-agronomic analyses on quantitative data in olive often are in contrast to DNA molecular analyses. Despite this, the authors consider the use of morpho-agronomical descriptors irreplaceable for the description of local or neglected olive cultivars since they are simple to be recorded and focus on important agronomic traits, while an accurate and unambiguous olive cultivars identification cannot be performed without the use of molecular markers.

In these last years the Department of Agro-forestry and Environmental Biology and Chemistry of the University of Bari (DiBCA) employed molecular markers both for olive cultivar identification and olive oil traceability. Amplified fragment Polymorphism (AFLP), in absence of sequence information about the olive genome, have been largely employed for simultaneous screening of a large number of loci with high efficiency in revealing inter and intra-cultivar variability (Angiolillo et al., 1999; Belaj et al., 2003; Grati-Kamoun et al., 2006; Montemurro et al., 2005; Montemurro et al., 2008; Sensi et al., 2003). Also Inter Simple Sequence Repeats (ISSR) have been efficiently employed for cultivar identification of olive (Pasqualone et al., 2001). As sequences of genomic DNA in olive became available, Simple Sequence Repeats (SSR) have become the markers of choice for varietal identification studies as they are transferable, hypervariable, highly polymorphic, multiallelic, polymerase chain reaction (PCR)-based co-dominant markers. They are relatively simple to interpret and show a high information content (Ganino et al., 2006). In fact, SSR result less laborious and cheaper than AFLP and do not require special equipment and manipulation of radiolabelled substances, making possible to transfer SSR protocols for routine analyses. Recently, several olive SSRs have been published (Carriero et al., 2002; Cipriani et al., 2002; De la Rosa et al., 2002; Diaz et al., 2006; Sefc et al., 2000) and screened on a huge number of cultivars. The most part of these SSR have been evaluated by Baldoni et al. (2009) on a set of 77 cultivars from different geographical origins. The authors provided details on reproducibility, power of discrimination and number of amplified loci/alleles and precise band size alleles, indicating the most suitable SSR markers for olive cultivar identification. The allele sequencing makes it possible to have a reference point when screening olive cultivars with SSR, since the right size dimension of SSR alleles represents a bottleneck when phenomena of stuttering or genotyping errors occurr. Further information on olive germplasm, cultivar names, homonyms and synonims, SSR data and Expressed Sequence Tag (EST) are available at Oleadb database (www.Oleadb.it).

The advantages enfisized for SSR are much more justified by their applicability on processed material since significant amounts of DNA, suitable for PCR analysis, are present in residues from olive oil mechanic extraction (Alba et al., 2009b; Breton et al., 2004; Consolandi et al., 2008; de la Torre et al., 2004, Montemurro et al., 2008; Pasqualone et al., 2004a; 2004b).

Extra-virgin olive oil represent the pride of the Italian olive production and is only obtained by mechanical or physical extraction with any chemical change. It must fufil a series of physicchemical quality parameters , such as a free acidity below 0.8 g/100 g expressed in free oleic acid content (Commission Regulation EU No 61/2011). For its fatty acid composition, with a high monounsaturated – to - polyunsaturated fatty acid ratio and the presence of minor compounds having anti-oxidant activity, extra-virgin olive oil is especially appreciated for its high resistance to oxidative deterioration, playng a crucial role in the so called Mediterranean diet (Bendini et al., 2007; Harwood and Yaqoob, 2002; Visioli and Galli, 2002).


Olive (*Olea Europaea* L.): Southern-Italian Biodiversity Assessment and Traceability of Processed Products by Means of Molecular Markers 55

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

available, that provide an accurate and unambiguous tool for a correct identification of cultivated olive germplasm. Corrado et al. (2009) report that morpho-agronomic analyses on quantitative data in olive often are in contrast to DNA molecular analyses. Despite this, the authors consider the use of morpho-agronomical descriptors irreplaceable for the description of local or neglected olive cultivars since they are simple to be recorded and focus on important agronomic traits, while an accurate and unambiguous olive cultivars

In these last years the Department of Agro-forestry and Environmental Biology and Chemistry of the University of Bari (DiBCA) employed molecular markers both for olive cultivar identification and olive oil traceability. Amplified fragment Polymorphism (AFLP), in absence of sequence information about the olive genome, have been largely employed for simultaneous screening of a large number of loci with high efficiency in revealing inter and intra-cultivar variability (Angiolillo et al., 1999; Belaj et al., 2003; Grati-Kamoun et al., 2006; Montemurro et al., 2005; Montemurro et al., 2008; Sensi et al., 2003). Also Inter Simple Sequence Repeats (ISSR) have been efficiently employed for cultivar identification of olive (Pasqualone et al., 2001). As sequences of genomic DNA in olive became available, Simple Sequence Repeats (SSR) have become the markers of choice for varietal identification studies as they are transferable, hypervariable, highly polymorphic, multiallelic, polymerase chain reaction (PCR)-based co-dominant markers. They are relatively simple to interpret and show a high information content (Ganino et al., 2006). In fact, SSR result less laborious and cheaper than AFLP and do not require special equipment and manipulation of radiolabelled substances, making possible to transfer SSR protocols for routine analyses. Recently, several olive SSRs have been published (Carriero et al., 2002; Cipriani et al., 2002; De la Rosa et al., 2002; Diaz et al., 2006; Sefc et al., 2000) and screened on a huge number of cultivars. The most part of these SSR have been evaluated by Baldoni et al. (2009) on a set of 77 cultivars from different geographical origins. The authors provided details on reproducibility, power of discrimination and number of amplified loci/alleles and precise band size alleles, indicating the most suitable SSR markers for olive cultivar identification. The allele sequencing makes it possible to have a reference point when screening olive cultivars with SSR, since the right size dimension of SSR alleles represents a bottleneck when phenomena of stuttering or genotyping errors occurr. Further information on olive germplasm, cultivar names, homonyms and synonims, SSR data and Expressed Sequence Tag (EST) are available

The advantages enfisized for SSR are much more justified by their applicability on processed material since significant amounts of DNA, suitable for PCR analysis, are present in residues from olive oil mechanic extraction (Alba et al., 2009b; Breton et al., 2004; Consolandi et al., 2008; de la Torre et al., 2004, Montemurro et al., 2008; Pasqualone et al.,

Extra-virgin olive oil represent the pride of the Italian olive production and is only obtained by mechanical or physical extraction with any chemical change. It must fufil a series of physicchemical quality parameters , such as a free acidity below 0.8 g/100 g expressed in free oleic acid content (Commission Regulation EU No 61/2011). For its fatty acid composition, with a high monounsaturated – to - polyunsaturated fatty acid ratio and the presence of minor compounds having anti-oxidant activity, extra-virgin olive oil is especially appreciated for its high resistance to oxidative deterioration, playng a crucial role in the so called Mediterranean

diet (Bendini et al., 2007; Harwood and Yaqoob, 2002; Visioli and Galli, 2002).

identification cannot be performed without the use of molecular markers.

at Oleadb database (www.Oleadb.it).

2004a; 2004b).

Table 1. Area harvested and total olive production in the main producing countries of the Mediterranean Bacin and in the main worldwide geographical productive areas. (FAO, 2009). \* Data are referred to 2008.


Table 2. Consistency of the Italian olive cultivation (ISTAT, 2010)

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

Matrix for DNA extraction

Leaves Drupes Virgin oil

Leaves Drupes Virgin oil

SSR Leaves <sup>3</sup>

AFLP Leaves <sup>27</sup>

SSR Leaves

SSR Leaves

SSR Leaves

AFLP Leaves

SSR Leaves

Molecular marker class

ISSR

SSR

AFLP

SSR

Italy.

Assessment and Traceability of Processed Products by Means of Molecular Markers 57

N° Olive cultivars Minimum n° of markers distinguishing cultivars

10 10 2 Pasqualone et

7 10 3 Pasqualone et

27 60 - Montemurro

3 37 - Montemurro

Virgin oil 10 5 2 Pasqualone et

Virgin oil 4 2 1 Pasqualone et

Virgin oil 1 75 - Pasqualone et

Virgin oil 6 10 1 Montemurro

Virgin oil 10 7 1 Alba et al.,

SSR Virgin oil 7 7 - Pasqualone et

SSR Leaves 4 9 - Alba et al.,

SSR Leaves 16 30 2 Alba et al.,

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,

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

Source

al., 2001

al., 2003

al., 2004a

al., 2004b

et al., 2005

al., 2007a

al., 2007b

2008

et al., 2008a

et al., 2008b

2009a

2009b

N° Markers tested

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 parameters of quality all along the productive chain.

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 protection of consumers and for fraud prevention.

Fig. 1. Percentage distribution of harvested area (A) and total olive production (B) in the main worldwide geographical productive areas. (FAO, 2010).

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 was analysed.
