Standardization of a set of microsatellite markers for use in cultivar identification studies in olive (Olea europaea L.)

Abstract The comparability of eight olive microsatellite profiles in 17 cultivars generated by four laboratories using different DNA genotyping platforms was tested. In total, 54 alleles were identified, from a minimum of 3 alleles (DCA15) to a maximum of 12 (DCA9), averaging 6.75 alleles per marker. A representative sample of the olive genetic variability can be obtained by selecting a relatively low number of sufficiently different cultivars. Initial comparison of the data generated, revealed the presence of a few discrepancies between the laboratories, most of them due to easily identifiable rounding errors. However, 94.9% of the genotypes were in agreement between at least two laboratories after the harmonisation of the results and only in seven cases it was not possible to determine the genotype. No discrepancies between the four laboratories were observed at all in 106 genotypes (77.9%), while 18 (13.2%) showed discrepancies at one allele and 12 (8.8%) at two alleles. Most of the differences (73.8%) were due to results obtained by only one different laboratory each time. Markers DCA3, DCA8, DCA11, DCA13, DCA14 and DCA15 showed the highest concordance percentage between datapoints scored from all partners, while DCA4 and DCA9 produced less concordant results. Forty-three percent of the discrepancies were due to heterozygous/homozygous misreadings, that is often related to the presence of stutter peaks. The determining factors for obtaining reproducible results seem to be the utilization of unique sources of plant material, the employment of the same reference cultivars by all the laboratories, the standardization of PCR conditions and the selection of the markers with the most robust amplification pattern.

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