Isolation and characterization of microsatellite markers from the olive fly, Bactrocera oleae, and their cross-species amplification in the Tephritidae family

Abstract

Background: The Tephritidae family of insects includes the most important agricultural pests of fruits and vegetables, belonging mainly to four genera (Bactrocera, Ceratitis, Anastrepha and Rhagoletis). The olive fruit fly, Bactrocera oleae, is the major pest of the olive fruit. Currently, its control is based on chemical insecticides. Environmentally friendlier methods have been attempted in the past (Sterile Insect Technique), albeit with limited success. This was mainly attributed to the lack of knowledge on the insect's behaviour, ecology and genetic structure of natural populations. The development of molecular markers could facilitate the access in the genome and contribute to the solution of the aforementioned problems. We chose to focus on microsatellite markers due to their abundance in the genome, high degree of polymorphism and easiness of isolation.

Results: Fifty-eight microsatellite-containing clones were isolated from the olive fly, Bactrocera oleae, bearing a total of sixty-two discrete microsatellite motifs. Forty-two primer pairs were designed on the unique sequences flanking the microsatellite motif and thirty-one of them amplified a PCR product of the expected size. The level of polymorphism was evaluated against wild and laboratory flies and the majority of the markers (93.5%) proved highly polymorphic. Thirteen of them presented a unique position on the olive fly polytene chromosomes by in situ hybridization, which can serve as anchors to correlate future genetic and cytological maps of the species, as well as entry points to the genome. Cross-species amplification of these markers to eleven Tephritidae species and sequencing of thirty-one of the amplified products revealed a varying degree of conservation that declines outside the Bactrocera genus.

Conclusion: Microsatellite markers are very powerful tools for genetic and population analyses, particularly in species deprived of any other means of genetic analysis. The presented set of microsatellite markers possesses all features that would render them useful in such analyses. This could also prove helpful for species where SIT is a desired outcome, since the development of effective SIT can be aided by detailed knowledge at the genetic and molecular level. Furthermore, their presented efficacy in several other species of the Tephritidae family not only makes them useful for their analysis but also provides tools for phylogenetic comparisons among them.

Figure 1

Schematic representation of the in situ localization of microsatellite markers on the polytene chromosomes of Bactrocera oleae. Arrows that originate from numbers in bold stand for the Boms microsatellite markers. Underlined numbers refer to microsatellite markers that give multiple signals. All other arrows refer to previously mapped loci [Zambetaki et al 1999].

Figure 2

In situ hybridization of several microsatellite probes on the salivary gland polytene chromosomes of Bactrocera oleae. a: Boms23; b: Boms2; c: Boms17; d: Boms31; e: Boms24; f: Boms34. Arrows indicate the hybridization signals.

Figure 3

Cross-species amplification of Bactrocera oleae microsatellites in four Tephritidae genera. 1: Percentage of cross-amplified primer pairs. 2: Percentage of primer pairs that produced a fragment of the expected size.