We developed a new set of functional microsatellite markers for genetic studies in olive (Olea europaea L.). A total of 98,924 ESTs from developing olive fruit were examined for the presence of SSRs, and 984 SSR motifs from 923 SSR containing ESTs were identified, among which dinucleotide SSRs were the most abundant, followed by trinucleotides. The motif GA was the most common among dinucleotides, while GAA was the most abundant trinucleotide SSR motif. A total of 110 EST-SSRs with annotated genes was selected for primer designing, of which 46 EST-SSRs finally showed polymorphism. The sequencing and alignment of the olive EST-SSR alleles confirmed the amplification of predicted microsatellite motifs and revealed a high degree of conservation of the SSRs flanking regions. New EST-SSRs possess genetic diversity properties comparable to previously developed genomic SSRs. The average number of alleles per locus, observed heterozygosity, expected heterozygosity, and polymorphic information content were 4.5, 0.649, 0.604 and 0.539, respectively. Cluster analysis based on EST-SSR or SSR genotyping data and Jaccard"s similarity coefficient clearly distinguished 24 olive varieties, but intra-cultivar variability in "Ascolana tenera" and "Santa Caterina" was well resolved only by genomic SSRs. Seven new EST-SSR loci with high polymorphic information content were used for paternity analysis. Paternity testing with three computer programs (FAMOZ, CERVUS, PARENTE) showed that "Istrska belica" is cross-compatible with other olive varieties. The index of self-incompatibility was between 0.2 and 1.0 and "Istrska belica" variety was classified as partly self-incompatible.
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