DNA markers to study genetic diversity and fingerprinting in Arbutus unedo L. (strawberry tree)

Strawberry tree (Ericaceae) is a species of Mediterranean climates growing in several countries of the Mediterranean basin as well as in Portugal and Ireland. The edible fruits have been used in different ways yet the production of a popular spirit is still the main application. The species is also interesting from an ecological perspective since it is drought tolerant and fire resistant. In spite of its large distribution in Portugal little is known about the genetic diversity of the species and no efforts have been carried out to select the most interesting trees. Material from selected trees (based on fruit production) in a total of 27 was collected all around the country covering the distribution area of the species. In order to find out DNA markers to characterise genetic diversity and to fingerprint Arbutus unedo, random amplified polymorphic DNA (RAPD) and microsatellite markers (SSR) were used. The set of 20 RAPD primers used generated 124 bands, from which 71 (57.3%) were polymorphic and the expected heterozygosity was 0.27±0.014. The cluster analysis (UPGMA) based on the Jaccard similarity index showed a similarity of 71% among the trees of different origins. Only four genotypes could be grouped according to their geographical distribution. Eleven SSR primers designed for Vaccinium (an Ericaceae as A. unedo) were selected according to their polymorphism, number of alleles per locus, cross-amplification, allele scoring quality and the repeat motif, and were tested in A. unedo. Six loci showed to be polymorphic, with 71% mean expected diversity, 10.5 mean number of alleles, and 67% of average polymorphic information content. A mean homozygote excess was found (11%) although not significant. Estimation of null allele frequency was about 9% on average. The linkage disequilibrium test showed that all the loci were independent. The UPGMA clustering based on the Lynch and Ritland coefficient showed that only two genotypes shared about 50% of the alleles, but without a consistent geographic pattern, similarly to the results obtained by RAPD analysis. The Mantel test confirmed that there is no correlation between the genetic and the geographical distance matrices, for both makers. Reduced gene flow, due to fragmentary distribution, together with the type of mating system of this species (pollination by small insects), are plausible explanations for these results. As far as we know our work is the first detailed study to evaluate A. unedo diversity by molecular markers opening new perspectives for fingerprinting analysis and for conservation and improvement purposes