Isolation and characterisation of microsatellite DNA in olive (Olea europaea L.)

We have isolated and sequenced 52 microsatellites or SSR from nearly sixty positive clones obtained from a "Frantoio" olive genomic library enriched in (AC/GT) and (AG/CT) repeats. The repeat-containing fragments obtained from genomic DNA restricted with Tsp5091 were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs, which gave correct re-amplification in the source genome, were used to assay the polymorphism of 28 olive genotypes, representing five Italian cultivars ("Coratina", "Frantoio", "Leccino", "Maurino", "Pendolino") and eight local cultivars grown in the lake Garda area ("Casaliva", "Favarol", "Fort", "Grignan", "Less", "Raza", "Rossanel", "Trep"). The local cultivars were each represented by 2 to 4 long-living individuals. The analysis was carried out using P-33-labelled primers and 6 % acrylamide sequencing gels. All microsatellites except two showed polymorphism, the number of alleles varying from 1 to 7. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individuals of the same variety. This was the case of "Casaliva", "Fort", "Raza", and "Rossaanel". We were able to solve the identity of "Casaliva", which is probably a sport mutation of "Frantoio"; whereas "Leccino" which is sometimes said to be similar to the local variety "Less", showed patterns largely different from those of the local cultivar. Interestingly, several primer pairs (17 %) of the 30 assayed amplified more than one locus, resulting in patterns of three of more alleles in numerous individuals. This would agree with the hypothesis that domesticated olive (Olea europaea L.) is an ancient polyploid, as postulated since 1954.