Transcriptome resources for the frogs Lithobates clamitans and Pseudacris regilla, emphasizing antimicrobial peptides and conserved loci for phylogenetics

Laura S. Robertson, Robert S. Cornman

We developed genetic resources for two North American frogs, Lithobates clamitans and Pseudacris regilla, widespread native amphibians that are potential indicator species of environmental health. For both species, mRNA from multiple tissues was sequenced using 454 technology. De novo assemblies with Mira3 resulted in 50 238 contigs (N50 = 687 bp) and 48 213 contigs (N50 = 686 bp) for L. clamitans and P. regilla, respectively, after clustering with CD-Hit-EST and purging contigs below 200 bp. We performed BLASTX similarity searches against the Xenopus tropicalis proteome and, for predicted ORFs, HMMER similarity searches against the Pfam-A database. Because there is broad interest in amphibian immune factors, we manually annotated putative antimicrobial peptides. To identify conserved regions suitable for amplicon resequencing across a broad taxonomic range, we performed an additional assembly of public short-read transcriptome data derived from two species of the genus Rana and identified reciprocal best TBLASTX matches among all assemblies. Although P. regilla, a hylid frog, is substantially more diverged from the ranid species, we identified 56 genes that were sufficiently conserved to allow nondegenerate primer design with Primer3. In addition to providing a foundation for comparative genomics and quantitative gene expression analysis, our results enable quick development of nuclear sequence-based markers for phylogenetics or population genetics.