De novo assembly and characterization of the transcriptome of the toxic dinoflagellate Karenia brevis

Abstract

Background: Karenia brevis is a harmful algal species that blooms in the Gulf of Mexico and produces brevetoxins that cause neurotoxic shellfish poisoning. Elevated brevetoxin levels in K. brevis cells have been measured during laboratory hypo-osmotic stress treatments. To investigate mechanisms underlying K. brevis osmoacclimation and osmoregulation and establish a valuable resource for gene discovery, we assembled reference transcriptomes for three clones: Wilson-CCFWC268, SP3, and SP1 (a low-toxin producing variant). K. brevis transcriptomes were annotated with gene ontology terms and searched for putative transmembrane proteins that may elucidate cellular responses to hypo-osmotic stress. An analysis of single nucleotide polymorphisms among clones was used to characterize genetic divergence. Results: K. brevis reference transcriptomes were assembled with 58.5 (Wilson), 78.0 (SP1), and 51.4 million (SP3) paired reads. Transcriptomes contained 86,580 (Wilson), 93,668 (SP1), and 84,309 (SP3) predicted transcripts. Approximately 40% of the transcripts were homologous to proteins in the BLAST nr database with an E value ≤ 1.00E-6. Greater than 80% of the highly conserved CEGMA core eukaryotic genes were identified in each transcriptome, which supports assembly completeness. Seven putative voltage-gated Na+ or Ca2+ channels, two aquaporin-like proteins, and twelve putative VATPase subunits were discovered in all clones using multiple bioinformatics approaches. Furthermore, 45% (Wilson) and 43% (SP1 and SP3) of the K. brevis putative peptides > 100 amino acids long produced significant hits to a sequence in the NCBI nr protein database. Of these, 77% (Wilson and SP1) and 73% (SP3) were successfully assigned gene ontology functional terms. The predicted single nucleotide polymorphism (SNP) frequencies between clones were 0.0028 (Wilson to SP1), 0.0030 (Wilson to SP3), and 0.0028 (SP1 to SP3). Conclusions: The K. brevis transcriptomes assembled here provide a foundational resource for gene discovery and future RNA-seq experiments. The identification of ion channels, VATPases, and aquaporins in all three transcriptomes indicates that K. brevis regulates cellular ion and water concentrations via transmembrane proteins. Additionally, > 40,000 unannotated loci may include potentially novel K. brevis genes. Ultimately, the SNPs identified among the three ecologically diverse clones with different toxin profiles may help to elucidate variations in K. brevis brevetoxin production.