**2.2 AFLP markers**

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

marker class

Olive cultivar Molecular marker class Olive cultivar Molecular

Aglandau AFLP, SSR Manzanilla AFLP, SSR Ascolana Tenera AFLP, SSR Maurino AFLP, SSR Atena AFLP, SSR Mele AFLP, SSR Bella di Cerignola AFLP, SSR Mora AFLP, SSR Bella di Spagna AFLP, SSR Moraiolo AFLP, ISSR, SSR Bouteillan AFLP, SSR Nocellara del Belice AFLP, SSR Canino AFLP, SSR Nocellara Etnea AFLP, SSR Carolea AFLP, SSR Nocellara Messinese AFLP, SSR Cassanese ISSR Nociara AFLP, ISSR, SSR Cazzinicchio AFLP, SSR Nolca AFLP, SSR Cellina di Nardò AFLP, ISSR, SSR Ogliarola barese AFLP, SSR Cerasella AFLP, SSR Ogliarola garganica SSR Cima di Bitonto AFLP, ISSR, SSR Ogliarola salentina ISSR, SSR Cima di Mola SSR Oliastro AFLP, SSR Cima di Melfi AFLP, SSR Oliva rossa SSR Cipressino SSR Pasola AFLP, SSR Conservolia AFLP, SSR Pasola di Andria AFLP, SSR Coratina AFLP, ISSR, SSR Pendolino AFLP, SSR Corniola SSR Peppino AFLP, SSR Dolce di Cassano AFLP, SSR Peranzana AFLP, SSR

Montescaglioso AFLP, SSR Picholine AFLP, SSR Donna Francesca SSR Picual AFLP, SSR Donna Giulietta SSR Primicea AFLP, SSR Frantoio AFLP, SSR S. Agostino AFLP, SSR Gentile di Larino ISSR S. Caterina AFLP, SSR Giarraffa AFLP, SSR S. Francesco AFLP, SSR Gioconda AFLP, SSR Simone AFLP, SSR Grignan AFLP, SSR Selvatic entry AFLP, SSR Grossane AFLP, SSR Sigoise AFLP, SSR Itrana AFLP, ISSR, SSR Tanche AFLP, SSR Kalamata AFLP, SSR Termite di Bitetto AFLP, SSR Leccino AFLP, ISSR, SSR Tonda Iblea AFLP, SSR Leccio del Corno AFLP, SSR Toscanina AFLP, SSR Leo AFLP, SSR Verdale AFLP, SSR Maiatica AFLP, SSR Vergiola AFLP, SSR 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

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

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