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A. Belaj, G. Cipriani, R. Testolin, L. Rallo and I. Trujillo

Nine simple-sequence-repeat (SSR) primer pairs were assayed in 35 Spanish and Italian olive cultivars of commercial interest. All microsatellites were polymorphic, showing 5 to 13 alleles per locus (7.5 alleles per locus on average). The frequency of each alleles was generally low, with most of the alleles present at one or two cultivars. Heterozigosity ranged from 0.15 to 0.95; the discrimination power (PD) ranged from 0.30 to 0.93 (mean 0.79). The set of microsatellites analyzed discriminated all cultivars investigated. The combination of only three SSR primer pairs—UDO99-009+UDO99-043+UDO99-14—made possible the identification of all cultivars included in the study. Cluster analysis did not find differences between Spanish and Italian cultivars, but most of the cultivars from southern and central Spain grouped together. Hence, microsatellites markers are recommended for olive fingerprinting to generate a database for olive cultivar identification.

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A. Belaj, L. Rallo, I. Trujillo and L. Baldoni

Eight and seven clones, respectively selected within the olive cultivars `Arbequina' and `Manzanilla de Sevilla', were studied by means of randomly amplified polymorphic DNA (RAPD) and amplified fragment-length polymorphism (AFLP) markers. Two clones of `Arbequina', C3 and C12, showed polymorphism with respect to the standard cultivar by means of both markers. In fact, about 33.6% RAPD bands and 9.2% AFLP bands were polymorphic for these clones. This high level of polymorphism and the presence of a high percentage of bands absent in `Arbequina' suggest their possible origin as `Arbequina' seedlings. The dendrogram obtained by both molecular markers also supports the hypothesis of a seedling origin of these clones as they clustered separately from the original cultivar and the rest of monomorphic clones at low values of similarity. Also within the `Manzanilla de Sevilla' group, two clones (31 and 44) showed diversity with respect to the standard cultivar; 4.5% RAPD and 6.3% AFLP markers were polymorphic for these genotypes while all the other clones didn't show any difference with the standard `Manzanilla de Sevilla'. RAPD and AFLP markers effectively revealed intracultivar variability due to gametic or multiple mutational events, while the detection of other kind of differences such as eventual single mutations remains uncertain and requires further investigation.

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A. Belaj, I. Trujillo, D. Barranco and L. Rallo

Thirteen randomly amplified polymorphic DNA (RAPD) primers were assayed in 82 Spanish olive cultivars of economical interest. A total of 82 bands were scored giving an average of 6.3 bands per primer. A total of 4 (OPA-01) to 10 bands (OPA-19) was amplified, while the number of polymorphic fragments ranged from 2 (OPK-07) to 9 (OPA-19) with a mean of 5.7 polymorphic bands per primer. A total of 89% of the amplification products (73 bands) were polymorphic. The 13 primers yielded 184 banding patterns (14.9 per primer). The number of banding patterns per primer ranged from 4 (OPK-07) to 39 (OPA-19). Fifty-three unique banding patterns were found, the majority of them resulted from different combinations of polymorphic bands. The combination of only five primers OPA-19, OPF-06, OPX-01, OPX-03, and OPI-12, allowed identification of all the cultivars. Seventy-four cultivars (90%) were identified only by the combination of the first four primers. The addition of the fifth primer (OPI-12) was necessary for the identification of the eight remaining cultivars (10%). The ordination of the primers according to their practical discriminating capacity in this study was: OPA-19 > OPF-06 > OPX-01 > OPX-03 > OPI-12. Hence RAPD markers are recommended for olive fingerprinting in order to generate a database for olive cultivar identification.

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A. Belaj, Z. Satovic, I. Trujillo and L. Rallo

Eighty-two Spanish olive cultivars from the World Germplasm Bank of the Centro de Investigación y Formación Agraria (CIFA) Alameda del Obispo in Cordoba (Spain) were analysed by RAPD markers to assess their genetic relatedness and to study patterns of genetic variation. The dendrogram based on unweighted pair group cluster analysis using Jaccard's index included two major groups that consisted mostly of cultivars from the southern and central part of Spain. Clustering together of cultivars from the Levante zone was also observed. The pattern of genetic variation among olive cultivars from three different Spanish zones (Levante, central and Andalusia) was analysed by means of the analysis of molecular variance (AMOVA). Although most of the genetic variability was attributable to differences of cultivars within each zone (95.88%), significant φ-values among zones (φst = 0.041; p < 0.001) suggested the existence of phenotypic differentiation. These results are consistent with the predominantly allogamous nature of Olea europaea L. species. Significant values of φst for the pair Andalusia/Levante indicate the presence of differentiation. The negative value of φst observed in the case of the Andalusia/central pair suggests that some varieties from central Spain are more similar to the Andalusian ones than to the varieties of their own geographic area, and vice versa.

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A. Belaj, Z. Satovic, L. Rallo and I. Trujillo

The aim of this work was to study in depth the resolving power of RAPD markers for rapid and reliable identification of olive cultivars in germplasm collections. The D parameter (the probability that two randomly chosen cultivars have different banding patterns), used for that purpose, showed high values for most of the 21 primers tested and its values ranged from 0.6114 (OPI-13) to 0.9762 (OPK-16) with a mean value of 0.8566. This parameter was used to select the five most discriminating primers: OPK-16, OPA-19, OPX-09, OPF-06 and OPZ-11. The joint confusion probability and the statistical number of indistinguishable pairs of cultivars were estimated for these primers (under independence hypothesis). The combination of three primers (OPK-16, OPA-19 and OPX-09) was found optimal for rapid discrimination of 103 cultivars with a very low value of cumulative confusion probability (1.72 × 10-5), leaving 0.09 pairs of cultivars indistinguishable. This fact, together with the efficiency of the most discriminating primers combination on an increasing number of cultivars, evidenced the utility of RAPD markers for discrimination of olive cultivars in collections and in nurseries.

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A. Belaj, I. Trujillo, R. de la Rosa, L. Rallo and M.J. Giménez

Random amplified polymorphic DNA (RAPD) analysis was performed on the main Mediterranean cultivars of olive (Olea europaea L.) from the Germplasm Bank of the Centro de Investigación y Formación Agraria “Alameda del Obispo” in Cordoba, Spain. One hundred and ninety reproducible amplification fragments were identified using 46 random primers followed by agarose gel electrophoresis. Some 63.2% of the amplification products were polymorphic, with an average of 2.6 RAPD markers obtained for each primer. The combination of polymorphic markers resulted in 244 banding patterns. The high degree of polymorphism detected made identification of all the cultivars (51) possible by combining the RAPD banding patterns of just only four primers: OPA-01, OPK-08, OPX-01, and OPX-03. Cultivar-specific RAPD markers and banding patterns were also found. A dendrogram based on unweighted pair-group method cluster analysis was constructed using a similarity matrix derived from the RAPD amplification products generated by the 46 primers. Three major groups of cultivars could be distinguished by RAPD analysis: 1) cultivars from east and northeast Spain, 2) Turkish, Syrian, and Tunisian cultivars, and 3) the majority of common olive cultivars in Spain. The dendrogram thus showed a good correlation between the banding patterns of olive cultivars and their geographic origin. A higher level of polymorphism was observed when polyacrylamide gel electrophoresis was used to separate the amplification products. Thus, adequate use of RAPD technology offers a valuable tool to distinguish between olive cultivars.