**4.2 Amplified Fragment Length Polymorphism (AFLP)**

The principle of amplified fragment length polymorphism (AFLP) (Vos et al., 1995) technique is basically simple and its procedure consists of three main steps: (a) template preparation, (b) fragment amplification, and (c) gel analysis. The fingerprinting patterns are obtained by detection of genomic restriction fragments by PCR amplification. This technique has been widely employed. Because of its effectiveness, reliability and efficiency in genetic diversity studies (Ercisli et al., 2009), the AFLP technique has been widely used in olive Spanish cultivars considering intra-varietal diversity (Sanz-Cortés et al., 2003), and to assess genetic inter-relationships among cultivated cultivars in the Eastern Mediterranean Basin (Owen et al., 2005). The results showed significant genetic distance between Greek and Turkish cultivars, and a clear separation of most of the Spanish and Italian clones, suggesting that an East-West divergence of olive cultivars occurred. Using the AFLP markers Angiolillo et al. (1999) have shown that wild olives from the Western Mediterranean and cultivated cultivars did not cluster together, and were relatively distant. However, a few oleasters clustered with the cultivars suggesting a common origin.

The first linkage map of the olive genome was constructed using a combination of molecular markers (e.g., RAPD, AFLP, RFLP and SSR) (De la Rosa et al., 2003). Maps can be used to select important traits and to study genes that control expression of polygenic traits. Molecular marker linkage maps are widely recognized as essential tools for genetic research and breeding in many species.

### **4.3 Microsatellites (SSR)**

The simple sequence repeat (SSR) (Tautz et al., 1986; Litt & Luty, 1989) consists of short (1-6 base pair long) stretches of DNA tandem repeated several times, occurring in the genomes of many higher organisms (Rafalski & Tingey, 1993; Wu & Tanksley, 1993). The simple sequence repeat or microsatellites, as one of the most popular marker system, are widely used in plant genetic research for diversity studies, namely in olive tree and to test the breeding success as they are transferable, highly polymorphic, ideal for genetic map development, linkage analysis, marker-assisted selection and fingerprinting studies (Bracci et al., 2009; Cipriani et al., 2002; De la Rosa el al., 2004; Gomes et al., 2009; Karp et al., 1996; Muzzalupo et al., 2009; Rallo et al., 2002; Sefc et al., 2000). When compared with RAPD or AFLP markers, the SSR have the advantage of their codominant nature, as two alleles may be identified at each locus. The main constrain of SSR markers is the development requires previous DNA sequencing for primer designing.

The microsatellites loci have been isolated from olive tree (Carriero et al., 2002; Cipriani et al., 2002; De la Rosa et al., 2002; Rallo et al., 2000; Sefc et al., 2000) and are used either alone or in combination with other molecular markers to characterize olive cultivars (Belaj et al., 2004; Gomes et al., 2009; Khadari et al., 2003; Wu & Sedgley, 2004). This methodology has

(2000) used the same methodology for olive geographic location and confirmed the hypothesis of autochthonic origin of most olive-tree cultivars. Nowadays, obtaining specific markers, such as sequence characterized amplified region (SCAR) and sequence-tagged-site (STS), from RAPD markers could be a way to overcome the lack of reproducibility, proper of

The principle of amplified fragment length polymorphism (AFLP) (Vos et al., 1995) technique is basically simple and its procedure consists of three main steps: (a) template preparation, (b) fragment amplification, and (c) gel analysis. The fingerprinting patterns are obtained by detection of genomic restriction fragments by PCR amplification. This technique has been widely employed. Because of its effectiveness, reliability and efficiency in genetic diversity studies (Ercisli et al., 2009), the AFLP technique has been widely used in olive Spanish cultivars considering intra-varietal diversity (Sanz-Cortés et al., 2003), and to assess genetic inter-relationships among cultivated cultivars in the Eastern Mediterranean Basin (Owen et al., 2005). The results showed significant genetic distance between Greek and Turkish cultivars, and a clear separation of most of the Spanish and Italian clones, suggesting that an East-West divergence of olive cultivars occurred. Using the AFLP markers Angiolillo et al. (1999) have shown that wild olives from the Western Mediterranean and cultivated cultivars did not cluster together, and were relatively distant.

However, a few oleasters clustered with the cultivars suggesting a common origin.

The first linkage map of the olive genome was constructed using a combination of molecular markers (e.g., RAPD, AFLP, RFLP and SSR) (De la Rosa et al., 2003). Maps can be used to select important traits and to study genes that control expression of polygenic traits. Molecular marker linkage maps are widely recognized as essential tools for genetic research

The simple sequence repeat (SSR) (Tautz et al., 1986; Litt & Luty, 1989) consists of short (1-6 base pair long) stretches of DNA tandem repeated several times, occurring in the genomes of many higher organisms (Rafalski & Tingey, 1993; Wu & Tanksley, 1993). The simple sequence repeat or microsatellites, as one of the most popular marker system, are widely used in plant genetic research for diversity studies, namely in olive tree and to test the breeding success as they are transferable, highly polymorphic, ideal for genetic map development, linkage analysis, marker-assisted selection and fingerprinting studies (Bracci et al., 2009; Cipriani et al., 2002; De la Rosa el al., 2004; Gomes et al., 2009; Karp et al., 1996; Muzzalupo et al., 2009; Rallo et al., 2002; Sefc et al., 2000). When compared with RAPD or AFLP markers, the SSR have the advantage of their codominant nature, as two alleles may be identified at each locus. The main constrain of SSR markers is the development requires

The microsatellites loci have been isolated from olive tree (Carriero et al., 2002; Cipriani et al., 2002; De la Rosa et al., 2002; Rallo et al., 2000; Sefc et al., 2000) and are used either alone or in combination with other molecular markers to characterize olive cultivars (Belaj et al., 2004; Gomes et al., 2009; Khadari et al., 2003; Wu & Sedgley, 2004). This methodology has

**4.2 Amplified Fragment Length Polymorphism (AFLP)** 

RAPD markers.

and breeding in many species.

previous DNA sequencing for primer designing.

**4.3 Microsatellites (SSR)** 

been used to analyze the genetic variability of the somatic embryogenesis induction process in *Olea europaea* L. and *Olea europaea* var. *maderensis*. The authors reported the maintenance of the genomic integrities between species suggesting the absence of somaclonal variation (Lopes et al., 2009). New insights about genetic diversity and gene flow between the wild (oleaster) and the cultivated form, using SSR marker was reported (Breton et al., 2006). A database containing a consensus list of SSR profiles for true-to-type olive genotyping has been constructed. This platform will allow results' comparison among laboratories, in order to establish a common olive database (Baldoni et al., 2009).

During many years the agarose gel electrophoresis has been used as the common detection method for SSR analysis. The agarose gel is efficient when the alleles are long enough, that is, more than 200-300 base pair and the differences among alleles are also significant to be visualized (i.e., more than 10-20 base pair). The high resolution polyacrylamide gels have been used when small differences between alleles, less than 1-10 base pair, must be identified. Nowadays, the separation of SSR markers using sequencing apparatus revealed to be very suitable, since the detection of alleles is performed automatically. The major advantages of automated detection are: (a) faster in obtaining results, (b) automated data analysis, (c) multiplex analysis, (d) high reproducibility, and (e) exclusion of silver-staining procedure. However, between different apparatus there may be found a shift among allele size, which has to be undertaken when comparing results among laboratories.

#### **4.4 Inter Simple Sequence Repeats (ISSR)**

In order to resolve some of the inconveniences associated with RAPD (low reproducibility), the high AFLP cost, and the need to know the flanking sequences in order to developed primers for SSR polymorphism, ISSR were developed (Terzopoulos et al., 2005; Zietkiewicz et al., 1994). ISSR markers are based on the amplification of regions (200-2000 base pair) between inversely oriented closely spaced microsatellites. The ISSR show the specificity of microsatellite markers, but need no sequence information for primer synthesis. The ISSR alone or in combination with other marker systems, have been widely used to analyze clonal variation and genetic variability in olive cultivars (Gemas et al., 2004; Gomes et al., 2008; Martins-Lopes et al., 2007, 2009; Terzopoulos et al., 2005).

Previous studies have concluded that ISSR markers are efficient in assessing phylogenetic relationships in the *O. europaea* complex (Gemas et al., 2004; Hess et al., 2000) and for olive fruits and leaves identification (Pasqualone et al., 2001). The simultaneous use of ISSR with other markers such as RAPD has made possible the discrimination between 30 Portuguese and 8 foreign olive cultivars (Martins-Lopes et al., 2007).

#### **4.5 Sequence Characterized Amplified Region (SCAR)**

Since PCR-based molecular markers have been developed, several PCR-based markers modifications have emerged. Due to the certification process of orchards and regions, crucial for protected denomination of origin (PDO), there is an urgent need for early and efficient methods able to discriminate and indentify olive cultivars. The development of cultivar-specific DNA markers can also be useful in olive industry in order to avoid olive oil adulteration that affects the oil quality (Marieschi et al., 2011; Pafundo et al., 2007).

The sequence characterized amplified region (SCAR) have been widely developed for plant breeding studies in several species such as wheat (Hernández et al., 1999), grapevine (Vidal et al., 2000), tomato (Zhang & Stommel, 2001), and pear (Lee et al., 2004; Marieschi et al., 2011). In olive, this type of marker has also been applied for olive germplasm evaluation and mapping (Bautista et al., 2003; Busconi et al., 2006; Hernández et al., 2001a), and for analysis of complex agro-food matrixes (olive oil traceability) (Pafundo et al., 2007).

The development of sequence characterized amplified region (SCAR) involves cloning of the amplified product, and then sequencing the two ends of the cloned product that appeared to be specific. The SCAR has the advantage of being inherited in a codominant fashion in contrast to RAPD which are inherited in a dominant manner (Mohan et al., 1997). Bautista et al. (2003) used this technology to develop specific markers useful for olive cultivar identification and mapping. They demonstrated that the use of SCAR markers is enough to provide a simple, cheap, and reliable procedure to identify geographically related olive cultivars. The development of SCAR markers by directly sequencing olive RAPD bands was reported by Hernández et al. (2001a) and they demonstrated that the generated markers were useful for the marker assisted selection of the high flesh/stone ratio. This type of marker has also been applied for olive germplasm evaluation and mapping (Bautista et al., 2003; Busconi et al., 2006). Wu et al. (2004) combined RAPD, SCAR and SSR markers to construct a linkage map from a cross-pollinated F1 population of 'Frantoio' × 'Kalamata' olive cultivars.

#### **4.6 Single Nucleotide Polymorphisms (SNP)**

The single nucleotide polymorphisms are a marker system that can differentiate individuals based on variations detected at the level of a single nucleotide base in the genome. Such variations are present in large abundance in the genomes of higher organisms including plants (Agarwal et al., 2008). The SNP-based markers have been used in many plant species.

In olive, due to olive unknown genome, this technique has not been widely applied. Reale et al. (2006) used SNP markers to genotype 65 olive samples obtained from Europe and Australia, and observed that 77% of the cultivars were clearly discriminate. However, the authors developed SNP markers from olive gene sequences available in the GenBank database and from arbitrary sampling using the sequence-related amplification polymorphism (SRAP) method.
