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Abstract
BackgroundThe full power of modern genetics has been applied to the study of speciation in only a small handful of genetic model species - all of which speciated allopatrically. Here we report the first large expressed sequence tag (EST) study of a candidate for ecological sympatric speciation, the apple maggot Rhagoletis pomonella, using massively parallel pyrosequencing on the Roche 454-FLX platform. To maximize transcript diversity we created and sequenced separate libraries from larvae, pupae, adult heads, and headless adult bodies.ResultsWe obtained 239,531 sequences which assembled into 24,373 contigs. A total of 6810 unique protein coding genes were identified among the contigs and long singletons, corresponding to 48% of all known Drosophila melanogaster protein-coding genes. Their distribution across GO classes suggests that we have obtained a representative sample of the transcriptome. Among these sequences are many candidates for potential R. pomonella "speciation genes" (or "barrier genes") such as those controlling chemosensory and life-history timing processes. Furthermore, we identified important marker loci including more than 40,000 single nucleotide polymorphisms (SNPs) and over 100 microsatellites. An initial search for SNPs at which the apple and hawthorn host races differ suggested at least 75 loci warranting further work. We also determined that developmental expression differences remained even after normalization; transcripts expected to show different expression levels between larvae and pupae in D. melanogaster also did so in R. pomonella. Preliminary comparative analysis of transcript presences and absences revealed evidence of gene loss in Drosophila and gain in the higher dipteran clade Schizophora.ConclusionsThese data provide a much needed resource for exploring mechanisms of divergence in this important model for sympatric ecological speciation. Our description of ESTs from a substantial portion of the R. pomonella transcriptome will facilitate future functional studies of candidate genes for olfaction and diapause-related life history timing, and will enable large scale expression studies. Similarly, the identification of new SNP and microsatellite markers will facilitate future population and quantitative genetic studies of divergence between the apple and hawthorn-infesting host races.
Highlights
The full power of modern genetics has been applied to the study of speciation in only a small handful of genetic model species - all of which speciated allopatrically
To determine if the transcripts we identified were representative of our expectations for the transcriptome as a whole, we compared the distribution of R. pomonella sequences mapping to the Gene Ontology (GO) sub-categories described above with similar distributions of transcripts from the entire Drosophila melanogaster genome, and the partial transcriptome of the flesh fly Sarcophaga crassipalpis, another higher fly for which a substantial expressed sequence tag (EST) database has recently been developed using 454-pyrosequencing [36]
We found cases in which a single D. melanogaster odorant binding proteins (OBPs) represented the best match to two different R. pomonella ESTs, as is the case in R. suavis [39], indicating either gene loss in D. melanogaster or a duplication in the Rhagoletis lineage after
Summary
The full power of modern genetics has been applied to the study of speciation in only a small handful of genetic model species - all of which speciated allopatrically. We report the first large expressed sequence tag (EST) study of a candidate for ecological sympatric speciation, the apple maggot Rhagoletis pomonella, using massively parallel pyrosequencing on the Roche 454-FLX platform. In only a tiny subset of these organisms has it been possible to apply the full power of modern genetics - the ability to identify, sequence, and experimentally manipulate any gene in the genome - to the study of reproductive isolation [2]. We have applied transcriptome pyrosequencing to an organism proposed to have undergone sympatric, ecological speciation: the apple maggot fly, Rhagoletis pomonella (Walsh). The central premise of sympatric speciation in R. pomonella is that ecological adaptation to apple has resulted in the formation of a new apple-infesting host race of the fly that is partially reproductively isolated from ancestral hawthorn-infesting populations, a critical first step in the process of ecological sympatric speciation
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