Toward a Molecular Genetic Linkage Map for the Apple Maggot Fly (Diptera: Tephritidae): Comparison of Alternative Strategies

Abstract

Apple [Malus pumila (L.)] and hawthorn (Crataegus spp.) infesting populations of Rhagoletis pomonella Walsh are rapidly becoming a model system for the study of host plant specialization and sympatric race formation. Unfortunately, a major impediment to further progress in the Rhagoletis system is the lack of an adequate framework of genetic information. Here we report on the development of a molecular genetic linkage map for the fly and evaluate the relative merits of alternative strategies that we used to build the map. The most efficient method for detecting polymorphism in R. pomonella was based on screening a complementary DNA (cDNA) library for genetic variation. Twenty-four of the 98 cDNA clones that we attempted to analyze from plaques in the library displayed either restriction site (Alu I, Dde I, Sau 3A, or Taq I), fragment length, or single strand conformation polymorphism that could be mapped in R. pomonella and represented 25 different loci. Linkage group relationships were established for all 25 of these loci. Seven cDNA loci mapped to the 3 regions of the R. pomonella genome where the 6 allozyme loci with significant allele frequency differences between the host races are located. Furthermore, 5 of these 7 cDNA loci were in strong linkage disequilibrium with the allozyme loci residing within 2 of these 3 genomic regions, suggesting that the cDNA linkage map will be useful for dissecting the genetics of race formation in R. pomonella. Another successful strategy that we employed to map the locus glucose-6-phosphate dehijdrogenase (G-6-pdh) involved the use of a set of redundant primers that had been produced based on the conserved amino acid sequence of this gene. Surprisingly, a scheme based on screening plasmid libraries of randomly cloned restriction fragments was ineffective at detecting polymorphism, and it had an overall success rate of only 5.9% (3 mapped loci/51 low copy number clones screened for variation). In addition, application of random amplified polymorphic DNAs was unsuccessful in producing useful markers for the linkage map.