Abstract
Cartilaginous fishes are the most ancient group of living jawed vertebrates (gnathostomes) and are, therefore, an important reference group for understanding the evolution of vertebrates. The elephant shark (Callorhinchus milii), a holocephalan cartilaginous fish, has been identified as a model cartilaginous fish genome because of its compact genome (∼910 Mb) and a genome project has been initiated to obtain its whole genome sequence. In this study, we have generated and sequenced full-length enriched cDNA libraries of the elephant shark using the ‘oligo-capping’ method and Sanger sequencing. A total of 6,778 full-length protein-coding cDNA and 10,701 full-length noncoding cDNA were sequenced from six tissues (gills, intestine, kidney, liver, spleen, and testis) of the elephant shark. Analysis of their polyadenylation signals showed that polyadenylation usage in elephant shark is similar to that in mammals. Furthermore, both coding and noncoding transcripts of the elephant shark use the same proportion of canonical polyadenylation sites. Besides BLASTX searches, protein-coding transcripts were annotated by Gene Ontology, InterPro domain, and KEGG pathway analyses. By comparing elephant shark genes to bony vertebrate genes, we identified several ancient genes present in elephant shark but differentially lost in tetrapods or teleosts. Only ∼6% of elephant shark noncoding cDNA showed similarity to known noncoding RNAs (ncRNAs). The rest are either highly divergent ncRNAs or novel ncRNAs. In addition to full-length transcripts, 30,375 5′-ESTs and 41,317 3′-ESTs were sequenced and annotated. The clones and transcripts generated in this study are valuable resources for annotating transcription start sites, exon-intron boundaries, and UTRs of genes in the elephant shark genome, and for the functional characterization of protein sequences. These resources will also be useful for annotating genes in other cartilaginous fishes whose genomes have been targeted for whole genome sequencing.
Highlights
Cartilaginous fishes are the most basal phylogenetic group of living jawed vertebrates. They shared a common ancestor with bony vertebrates approximately 450 million years ago (Mya) [1]
Total RNA was isolated from various tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. 150 mg of total RNA from each tissue was treated with 100 U of DNase I (Roche) and 80 U RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) for 30 min at 37uC and purified by RNeasy Mini Kit (Qiagen)
CDNA library construction DNAse treated total RNA was used for making libraries enriched for full-length cDNA by the ‘oligo-capping’ method developed by Suzuki and Sugano [21]
Summary
Cartilaginous fishes are the most basal phylogenetic group of living jawed vertebrates (gnathostomes). The elephant shark (Callorhinchus milii), a holocephalan chimaera, was identified as a model cartilaginous fish genome because of its relatively small genome size (,910 Mb) [5] and sequenced to 1.46 coverage by Sanger sequencing [6]. 107,231, 103, 996 and 92,334 ESTs were sequenced from the embryos of the small-spotted catshark (stage 24–30), the little skate (stage 20–29) and the elephant shark (stage 32), respectively [8]. In addition to facilitating the annotation of proteincoding sequences, exon-intron boundaries, alternative exons, transcription start sites and UTRs, full-length cDNA clones are valuable for expressing proteins and generating mutant clones that can shed light on the function of proteins. The cDNA clones and sequences generated in this study are useful resources for annotating genes in elephant shark and other cartilaginous fish genomes and for functional studies of elephant shark genes
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