Sequencing and Analysis of the Mediterranean Amphioxus (Branchiostoma lanceolatum) Transcriptome

Abstract

BackgroundThe basally divergent phylogenetic position of amphioxus (Cephalochordata), as well as its conserved morphology, development and genetics, make it the best proxy for the chordate ancestor. Particularly, studies using the amphioxus model help our understanding of vertebrate evolution and development. Thus, interest for the amphioxus model led to the characterization of both the transcriptome and complete genome sequence of the American species, Branchiostoma floridae. However, recent technical improvements allowing induction of spawning in the laboratory during the breeding season on a daily basis with the Mediterranean species Branchiostoma lanceolatum have encouraged European Evo-Devo researchers to adopt this species as a model even though no genomic or transcriptomic data have been available. To fill this need we used the pyrosequencing method to characterize the B. lanceolatum transcriptome and then compared our results with the published transcriptome of B. floridae.ResultsStarting with total RNA from nine different developmental stages of B. lanceolatum, a normalized cDNA library was constructed and sequenced on Roche GS FLX (Titanium mode). Around 1.4 million of reads were produced and assembled into 70,530 contigs (average length of 490 bp). Overall 37% of the assembled sequences were annotated by BlastX and their Gene Ontology terms were determined. These results were then compared to genomic and transcriptomic data of B. floridae to assess similarities and specificities of each species.ConclusionWe obtained a high-quality amphioxus (B. lanceolatum) reference transcriptome using a high throughput sequencing approach. We found that 83% of the predicted genes in the B. floridae complete genome sequence are also found in the B. lanceolatum transcriptome, while only 41% were found in the B. floridae transcriptome obtained with traditional Sanger based sequencing. Therefore, given the high degree of sequence conservation between different amphioxus species, this set of ESTs may now be used as the reference transcriptome for the Branchiostoma genus.

Asymmetric Distribution of pl10 and bruno2, New Members of a Conserved Core of Early Germline Determinants in Cephalochordates

“…When B. floridae orthologs had not been previously characterized in the extensive phylogenetic analyses of Kerner et al (2011), we identified putative stem cell markers using BLASTp searches against the genomes of B. floridae and B. belcheri, and confirmed the identity of our B. lanceolatum proteins by comparison with published sequences in other cephalochordates (Supplementary File 4), including transcriptomic data from B. lanceolatum in the NCBI TSA database (Oulion et al, 2012). We generated phylogenies that include, where possible, sequences from more than one amphioxus species to support the identity of these proteins (Supplementary File 5; and see below).…”

Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus

“…An amphioxus (B. lanceolatum) Prdm12 cDNA was amplified by PCR from cDNA generated from total RNA from adults, using primers forward 5′-ATGA-AGCCGACCCTGTTTGATC-3′ and reverse 5′-ACATGGCGACCGAA-CGAACGTC-3′ (Oulion et al, 2012). …”

Expression of Fox genes in the cephalochordate Branchiostoma lanceolatum

“…lanceolatum Fox sequences were recovered from its reference transcriptome (Oulion et al, 2012) by TBLASTN using sequences from B. floridae as queries. Specific primers were then designed for RT-PCR amplification from total RNA.…”

“…We looked for Fox gene sequences in the reference transcriptome of B. lanceolatum (Oulion et al, 2012). The sequences that were recovered were used to conduct a phylogenetic tree reconstruction presented in Figure 1.…”

“…Specific duplications, that occurred in the cephalochordate clade at least in the ancestor of B. floridae and B. lanceolatum, gave rise to three members in the FoxQ2 group (FoxQ2a, FoxQ2b, FoxQ2c), two members in the FoxN1/4 group (FoxN1/4a and FoxN1/4b), and two genes in the FoxE group (FoxEa and FoxEc). We then analyzed the expression pattern during B. lanceolatum embryonic development of 13 of these 28 Fox genes corresponding to those showing a higher expression level in the transcriptome (Oulion et al, 2012).…”