Απομόνωση μικροδορυφόρων από το δάκο της ελιάς bactrocera oleae και χρησιμοποίησή τους στην ανάλυση φυσικών πληθυσμών του είδους

Abstract

The olive fruit fly, Bactrocera oleae, is the main pest of the olive fruit. Because of its great economic importance, especially for the Mediterranean countries, there is a need for a more effective control method. The application of an integrated, environmental friendly, management of this pest requires a better knowledge of its biology and of the genetic structure of its natural populations. The aim of the present study was the development of DNA microsatellite markers for the analysis of the natural populations of the olive fruit fly. These markers are abundant in the genome of any species studied so far and highly polymorphic. Three different strategies were used for the isolation of microsatellite markers. The first was the construction and screening of genomic libraries of the insect, the second was the construction of genomic libraries, enriched for microsatellites and the third was the use of primer pairs that were designed for the amplification of microsatellite markers in the closely related species Bactrocera oleae and Ceratitis capitata(cross-species amplification). A total of 69 microsatellite containing clones were isolated from libraries. The next step was the design of primer pairs in the microsatellite flanking sequences. A total of 42 primer pairs was designed and tested for their abillty to amplify the expected product. Test was performed through PCR and analysis of the PCR products through electrophoresis on agarose gel. Twenty primer pairs designed for the amplification of Bactrocera tryoni’ s microsatellites and 42 primer pairs designed for the amplification of Ceratitis capitata microsatellites were also tested. All three strategies gave 67 primer pairs that amplified the expected product. The degree of polymorphism of these primer pairs and their ability to amplify easily resolvable alleles was tested through PCR with DNA template of nine individuals. PCR products were analysed through polyacrylamide gel electrophoresis. Twenty eight out of the 49 primer pairs tested produced clear bands and twenty five of them were polymorphic. A small scale population analysis was then performed, using tventy four of the markers available. The main purpose of this analysis was to demonstate the quality of the markers and lead to the exclusion of six markers: one of them was monomorphic, another didn’t show reproducible results and four more showed deviations from H-W equilibrium, probably because of the presence of null alleles. Twelve of the remaining loci were used in a large scale analysis of B. oleae’s populations in the European part of the Mediterranean basin. Nineteen samples, varying from nine to fifty individuals, were analysed. These samples were collected from six different countries (Greece, Cyprus, Turkey, Italy, Spain and Portugal). The analysis revealed relatively low genetic distances, which, however, demonsrated a statistically important differentiation of the samples in three subpopulations. The first consisted of the samples from Cyprus, the second of the samples from Greece, Turkey and Italy and the third of the samples from the Iberian Peninsula. The statistical analyses performed showed the statistically important contribution of geographic distance to the generation of genetic distance. These three groups of samples also demonstrate a clear loss of polymorphism towards West. This loss is statistically important and, if we take into account the hypothesis that the colonization process of a species is followed by a loss in polymorphism, it suggests a colonization process of the olive fruit fly towards West in the European part of the Mediterranean basin, with a first expansion area in the Eastern part of the Mediterranean basin. Cross-species amplification experiments indicate close phylogenetic relationships among the species studied, mainly between B. tryoni-B. oleae and B. oleae-C. capitata. These results support the usefulness of the markers isolated in phylogenetic studies in these species, as well as in other, closely related species. The identification of a high percentage if conserved microsatellites in species that have been well separated for millions of years is in agreement with the hypothesis that microsatellites are not useless genomic regions (junj DNA) but they perform specific functions in the genome. Polymorphism analysis in the crosses of individuals from laboratory strains was very encouraging. The high degree of polymorpism showes that they can be used in genetic mapping of the species