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

BMC Genomics

Lisa Campbell¹  Alan E Pepper¹  Darcie E Ryan² 

[1] Department of Biology, Texas A & M University, College Station, TX 77843, USA;Department of Oceanography, Texas A & M University, College Station, TX 77843, USA

关键词: Comparative transcriptomics;    Cation channel;    Osmoacclimation;    Harmful algal bloom;    De novo transcriptome assembly;   

Others  :  1128468
DOI  :  10.1186/1471-2164-15-888

 received in 2014-02-06, accepted in 2014-09-24,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Ryan et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Steidinger KA, Vargo GA, Tester PA, Tomas CR: Bloom dynamics and physiology of Gymnodinium breve with emphasis on the Gulf of Mexico. NATO ASI Ser G: Ecol Sci 1998, 41:133-154.
• [2]Baden DG: Brevetoxins: unique polyether dinoflagellate toxins. FASEB J 1989, 3(7):1807-1817.
• [3]Dechraoui M-Y, Naar J, Pauillac S, Legrand A-M: Ciguatoxins and brevetoxins, neurotoxic polyether compounds active on sodium channels. Toxicon 1999, 37(1):125-143.
• [4]Huang J, Wu CH, Baden DG: Depolarizing action of a red-tide dinoflagellate brevetoxin on axonal membranes. J Pharmacol 1984, 229(2):615-621.
• [5]Landsberg JH: The effects of harmful algal blooms on aquatic organisms. Rev Fisher Sci 2002, 10(2):113-390.
• [6]Backer LC, Fleming LE, Rowan A, Cheng Y-S, Benson J, Pierce RH, Zaias J, Bean J, Bossart GD, Johnson D: Recreational exposure to aerosolized brevetoxins during Florida red tide events. Harmful Algae 2003, 2(1):19-28.
• [7]Errera R, Campbell L: Correction for Errera and Campbell, Osmotic stress triggers toxin production by the dinoflagellate Karenia brevis. Proc Natl Acad Sci U S A 2012, 109(43):17723-17724.
• [8]Maier Brown AF, Dortch Q, Dolah FMV, Leighfield TA, Morrison W, Thessen AE, Steidinger K, Richardson B, Moncreiff CA, Pennock JR: Effect of salinity on the distribution, growth, and toxicity of Karenia spp. Harmful Algae 2006, 5(2):199-212.
• [9]Errera RM, Bourdelais A, Drennan M, Dodd E, Henrichs D, Campbell L: Variation in brevetoxin and brevenal content among clonal cultures of Karenia brevis may influence bloom toxicity. Toxicon 2010, 55(2):195-203.
• [10]Snyder R, Gibbs P, Palacios A, Abiy L, Dickey R, Lopez J, Rein K: Polyketide synthase genes from marine dinoflagellates. Mar Biotechnol 2003, 5(1):1-12.
• [11]Monroe EA, Van Dolah FM: The toxic dinoflagellate Karenia brevis encodes novel type I-like polyketide synthases containing discrete catalytic domains. Protist 2008, 159(3):471-482.
• [12]Lidie KB, Ryan JC, Barbier M, Van Dolah FM: Gene expression in Florida red tide dinoflagellate Karenia brevis: analysis of an expressed sequence tag library and development of DNA microarray. Mar Biotechnol 2005, 7(5):481-493.
• [13]Lidie KB, Van Dolah FM: Spliced leader RNA‒mediated trans‒splicing in a dinoflagellate, Karenia brevis. J Eukaryot Microbiol 2007, 54(5):427-435.
• [14]Glass AD: Regulation of ion transport. Annu Rev Plant Physiol Plant Mol Biol 1983, 34(1):311-326.
• [15]Agre P, Preston G, Smith B, Jung J, Raina S, Moon C, Guggino WB, Nielsen S: Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol-Renal 1993, 265(4):F463-F476.
• [16]Heymann JB, Engel A: Aquaporins: phylogeny, structure, and physiology of water channels. Physiology 1999, 14(5):187-193.
• [17]Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, Nielsen S: Aquaporin water channels–from atomic structure to clinical medicine. J Physiol 2002, 542(1):3-16.
• [18]Borgnia M, Nielsen S, Engel A, Agre P: Cellular and molecular biology of the aquaporin water channels. Annu Rev Biochem 1999, 68(1):425-458.
• [19]Nelson N, Perzov N, Cohen A, Hagai K, Padler V, Nelson H: The cellular biology of proton-motive force generation by V-ATPases. J Exp Biol 2000, 203(1):89-95.
• [20]Maathuis FJ, Filatov V, Herzyk P, Krijger CG, Axelsen BK, Chen S, Green BJ, Li Y, Madagan KL, Sánchez‒Fernández R: Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress. Plant J 2003, 35(6):675-692.
• [21]Oren-Shamir M, Pick U, Avron M: Involvement of the plasma membrane ATPase in the osmoregulatory mechanism of the alga Dunaliella salina. Plant Physiol 1989, 89(4):1258-1263.
• [22]Kim YS, Martin DF: Effects of salinity on synthesis of DNA, acidic polysaccharide, and ichthyotoxin in Gymnodinium breve. Phytochemistry 1974, 13(3):533-538.
• [23]Rizzo P, Jones M, Ray S: Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve (Davis). J Eukaryot Microbiol 1982, 29(2):217-222.
• [24]Sigee DC: Structural DNA and genetically active DNA in dinoflagellate chromosomes. Biosystems 1984, 16(3):203-210.
• [25]Kamykowski D, Milligan EJ, Reed RE: Biochemical relationships with the orientation of the autotrophic dinoflagellate Gymnodinium breve under nutrient replete conditions. Mar Ecol-Prog Ser 1998, 167:105-117.
• [26]Van Dolah FM, Lidie KB, Monroe EA, Bhattacharya D, Campbell L, Doucette GJ, Kamykowski D: The Florida red tide dinoflagellate Karenia brevis: new insights into cellular and molecular processes underlying bloom dynamics. Harmful Algae 2009, 8(4):562-572.
• [27]Zhang H, Hou Y, Miranda L, Campbell DA, Sturm NR, Gaasterland T, Lin S: Spliced leader RNA trans-splicing in dinoflagellates. Proc Natl Acad Sci 2007, 104:4618-4623.
• [28]Lin S: Genomic understanding of dinoflagellates. Res Microbiol 2011, 162(6):551-569.
• [29]Rizzo PJ: Those amazing dinoflagellate chromosomes. Cell Res 2003, 13(4):215-217.
• [30]Schulz MH, Zerbino DR, Vingron M, Birney E: Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics 2012, 28(8):1086-1092.
• [31]Zerbino DR, Birney E: Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008, 18(5):821-829.
• [32]Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol İ: ABySS: a parallel assembler for short read sequence data. Genome Res 2009, 19(6):1117-1123.
• [33]Van Bel M, Proost S, Van Neste C, Deforce D, Van de Peer Y, Vandepoele K: TRAPID: an efficient online tool for the functional and comparative analysis of de novo RNA-Seq transcriptomes. Genome Biol 2013, 14(12):R134. BioMed Central Full Text
• [34]Parra G, Bradnam K, Korf I: CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 2007, 23(9):1061-1067.
• [35]Xu L, Chen H, Hu X, Zhang R, Zhang Z, Luo Z: Average gene length is highly conserved in prokaryotes and eukaryotes and diverges only between the two kingdoms. Mol Biol Evol 2006, 23(6):1107-1108.
• [36]Van de Peer Y, De Wachter R: Evolutionary relationships among the eukaryotic crown taxa taking into account site-to-site rate variation in 18S rRNA. J Mol Evol 1997, 45:619-630.
• [37]Wegmann K: Osmoregulation in eukaryotic algae. FEMS Microbiol Lett 1986, 39(1):37-43.
• [38]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5(7):621-628.
• [39]Guillard R, Hargraves P: Stichochrysis immobilis is a diatom, not a chrysophyte. Phycologia 1993, 32(3):234-236.
• [40]Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Genome Acids Res 1997, 25(17):3389-3402.
• [41]Bachvaroff TR, Gornik SG, Concepcion GT, Waller RF, Mendez GS, Lippmeier JC, Delwiche CF: Dinoflagellate phylogeny revisited: Using ribosomal proteins to resolve deep branching dinoflagellate clades. Mol Phylogenet Evol 2014, 70:314-322.
• [42]Kortschak D: longorf.pl [perlscript]. Available at https://github.com/bioperl/bioperl-live/blob/master/examples/longorf.pl webcite (Accessed 12 December 2013)
• [43]Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21(18):3674-3676.
• [44]Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011, 7:539.
• [45]Krogh A, Larsson B, Von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001, 305(3):567-580.
• [46]Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR: CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 2011, 39(suppl 1):D225-D229.
• [47]Marchler-Bauer A, Bryant SH: CD-Search: protein domain annotations on the fly. Nucleic Acids Res 2004, 32(suppl 2):W327-W331.
• [48]Forgac M: Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev 1989, 69(3):765-796.
• [49]Marban E, Yamagishi T, Tomaselli GF: Structure and function of voltage-gated sodium channels. J Physiol 1998, 508(3):647-657.