At the start of our work GenBank 27 and CarpBASE 21 together contained 10,615 common carp ESTs, all of which originated from non-gonadal cDNA libraries. We enriched the existing transcriptome dataset for common carp, by generating an additional 6,050 ESTs by random sequencing of clones from five different cDNA libraries derived from differentiating common carp testis (60–100 days post fertilization or dpf; see Additional File 1: Table S1 for details on the libraries). We also added an additional 652 common carp mRNAs extracted from GenBank in order to assist the assembly of ESTs.

Following cleaning and quality control, over 15,000 ESTs {10,283 from GenBank plus CarpBASE and 5,073 from our own data (GenBank: DW719352–DW724424)} were retained and clustered (Fig. 1). The clustered dataset of 8,663 unique sequences (1,643 clusters and 7,020 singletons) contained 2,442 (28.1%) "testis-only" sequences, including clusters with exclusively testis-derived ESTs and singletons isolated from one of the testis cDNA libraries (Fig. 2).

In order to initiate functional annotation of the partial transcriptome of common carp, we identified open reading frames (ORFs) in our clustered EST set using ESTScan 28. An ORF prediction was obtained for 81% of the clusters and 47.5% of the singletons, yielding a total of 4,663 sequences (data not shown). The ORF-containing common carp transcripts were classified into functional categories using protein domain databases (Additional File 2: Table S2; see Materials and Methods for databases used).

In the zebrafish Ensembl annotation (Ensembl_37) genes were annotated using mRNA and proteins from the target species as well as a range of other vertebrates, the closest to zebrafish being Japanese fugu and green spotted pufferfish. We mapped our common carp EST data to the zebrafish genome assembly (v5; 29) according to a multi-step protocol (see Materials and Methods for details and Additional File 8: Figure S1 for flow chart). A total of 1,182 common carp clusters (72% of all clusters) and 3,827 singletons (55% of all singletons) showed sequence similarity to the zebrafish genome with a BLAST E-value cutoff of 1e-04. After stringent filtering – selecting a unique zebrafish genomic location for each mapped common carp cluster (see Materials and Methods for detailed description) and sequence identity of 80% over 70% of the EST length – we assigned 484 clusters (29%) and 1,359 singletons (19%) to the zebrafish genome assembly (from here onwards these sequences will be referred to as "mapped common carp transcripts"). The common carp transcript map coordinates are available from Ensembl version 38 as a DAS track 30.

The 90 percentile of all intron lengths within the zebrafish Ensembl database is 4,657 nucleotides. There were 122 cases, where two common carp clusters/singletons mapped to the zebrafish genome within 4,657 nucleotides. These represent cases where the clusters and/or singletons potentially correspond to the same gene but were partitioned into separate clusters because of the absence of sequence data in the EST database.

Interestingly, there were 84 cases, where at least two clusters and/or singletons overlapped the same zebrafish locus. These represent potential gene family expansions in the common carp relative to zebrafish, but would require experimental validation in the future. These cases provide support for the incorporation of EST sequences from closely related "sequence-poor" species into the analysis pipeline of (nearly) completely sequenced genomes.

Nearly 40% of ESTs obtained from GenBank and those sequenced in our lab are bi-directional due to the EST sequencing protocol used. As a result, the strandedness of the genome-aligned common carp ESTs were obtained using the splice-site orientation as defined in the EST2GENOME algorithm 31. To identify un-annotated regions in the zebrafish genome, we required both plus and minus strands of the zebrafish genome be free of any sequence similarity features to non-common carp cDNA and proteins.

Of the 1,843 common carp transcripts mapped to the zebrafish genome assembly (Ensembl_37), 1,752 overlapped zebrafish cDNAs supported by genes and/or ESTs. The remaining 91 "mapped common carp transcripts" showed sequence identity to regions overlapping zebrafish introns (23), ab-initio predictions (22), non-zebrafish exons (22), intergenic regions (13) and non-zebrafish introns (11) (Additional File 3: Table S3; see Materials and Methods for classification criteria).

Five of the 13 common carp transcripts that map to intergenic regions are located less than 1 kb from the 5' end of the nearest neighbouring gene. Considering their close proximity to an annotated gene, these common carp transcripts represent potential untranslated regions (UTRs). In fact, the five neighbouring genes are annotated as developmental genes (data not shown). Developmental genes are highly conserved among species and very often the sequence conservation extends to their regulatory regions 1314. Furthermore, each of the common carp transcripts mapped to the zebrafish genome have sequence identity in excess of 80%, suggesting that the use of a lower threshold for common carp EST mapping might retrieve many more UTR sequences that could be subjected to similar UTR analyses as described in the Materials and Methods. The remaining eight common carp transcripts that map to intergenic regions are located between 5 and 150 kb away from the nearest zebrafish locus, suggesting the presence of novel gene loci that require experimental verification in the future.

Forty-two of the 91 mapped common carp transcripts have not been identified in the zebrafish and fathead minnow EST collections so far, therefore they represented novel cyprinid sequences. Another 16 of the 91 mapped common carp transcripts showed significant sequence similarity to the zebrafish and fathead minnow UniGene collection (build 91). (This indicated that the overlapping zebrafish transcripts might not have been available at the time of annotating the zebrafish genome version 5.). The remaining 33 common carp transcripts shared very weak sequence similarity (<40% identity) with either zebrafish or fathead minnow, thus might point to genes that diverged from their orthologs. Alternatively, these transcripts could represent sequences orthologous to zebrafish UTRs that are yet to be assigned to the annotated zebrafish genome.

The above cases illustrate the potential advantages of utilizing partial transcriptomes from related species in order to provide information on the functional properties of (a) un-annotated parts of genomes to be assembled as well as (b) regions annotated with distantly related species.

EST-based analysis of alternative splicing has been performed earlier in mammals; the results suggest that 40–60% of the genes produce alternatively spliced transcripts 32. Only a few studies have been performed on fish sequences (see e.g. 3334) resulting in a limited amount of data on splicing from teleosts.

Interestingly, out of 1,752 common carp transcripts that map to coding regions, there were 26 cases where the exon structure showed evidence for a missing exon compared to the overlapping zebrafish Ensembl gene (example: Fig. 3A; full list: Additional File 4: Table S4). Similar comparisons yielded 16 cases where an exon that was present in the overlapping common carp ESTs was missing from the zebrafish transcript (example: Fig. 3B; full list: Additional File 4: Table S4). There are four possibilities to explain such differences: i) the exon in question is missing from one of the two genomes; ii) exclusive usage of different splice products in the two related species; iii) different preferences of alternative splice products; and iv) virtual difference due to partial transcriptomes.

At the time of submission GenBank contained over 1.3 million ESTs for zebrafish, fathead minnow and common carp. We propose that the broad mRNA diversity contained in teleost EST resources could be leveraged to understand the extent of alternative splicing within this diverse group of teleosts using analyses similar to those reported for human ESTs 35.

The UniGene collection (build 91) contains datasets for two cyprinid species namely the zebrafish and fathead minnow. The common carp EST data reported in this study, sampled by nearly 9,000 unique transcripts, represent an additional cyprinid species that will be included in subsequent UniGene releases. The new EST data for common carp has also provided an opportunity to examine the value of tissue-specific sequencing on the existing gene collections. The common carp EST data were compared to the zebrafish UniGene collection (build 91) and subsequently to the fathead minnow data set using a BLAST E-value <1e-04 and sequence identity over 40% of the sequence length.

A total of 932 testis-derived common carp singletons and 42 clusters containing exclusively testis-derived common carp ESTs (Additional File 5: Table S5) did not overlap any of the zebrafish and fathead minnow UniGene transcripts. This dataset added 974 potentially novel sequences to the combined testis transcriptome of cyprinid teleosts (a fraction of these might represent UTR or coding sequences that are derived from fast-evolving genes).

A total of 214 out of 974 testis-only transcripts contained an ORF. Among these 214 transcripts, three testis-derived clusters contained an interleukin-8-like domain (IPR001811). The absence of significant sequence identity to zebrafish and fathead minnow at the nucleotide level is partly due to cytokines representing rapidly diverging genes involved in regulation of the immune system. Another domain, tissue inhibitor of metalloproteinase (IPR001820), which was identified in a common carp testis-derived transcript was present in two zebrafish UniGene clusters (Dr.240 and Dr.31907), but not sampled by any gonad-derived zebrafish sequences. The remaining 210 common carp transcripts do not show the presence of any characterized domains. These unique testis-derived transcripts could provide starting material for the isolation of their zebrafish orthologs, if any, and their potential application as markers for functional studies on gonad differentiation.

The potential homologs of 474 common carp clusters with at least one testis-derived EST were identified in the zebrafish UniGene data collection (Additional File 6: Table S6). When compared to the fathead minnow EST collection, six of these 474 common carp clusters showed sequence identity to adult testis-derived ESTs only (Additional File 6: Table S6). The common carp data correspond to differentiating testis (60–100 dpf), whereas the testis-derived zebrafish and fathead minnow clones presently found in the public databases are all from an adult organ. Therefore our results have complemented the previously available knowledge about the expression of these genes with experimental data on their activity during testis differentiation, providing indications on potentially conserved aspects of cyprinid gonad development. Moreover, the fact that common carp transcripts help the identification or confirmation of these coding regions in zebrafish exemplifies the usefulness of sequences from closely related species for the annotation of model genomes.

There is an average 82% sequence identity between the coding region of homologous gene pairs in zebrafish and common carp, whereas the same value for their 5' and 3' UTRs is only 61% and 58%, respectively (see Materials and Methods for details). We set out to explore the extent to which common carp and zebrafish retained similarity in the 5'UTR regions of their orthologous genes as this can reveal aspects of regulatory roles of these regions in both species. This task was difficult for two reasons: i) the fact that only limited sequence information is available from common carp dramatically decreased our ability to identify large number of orthologs between these species; and ii) the usual approaches to evaluate similarity based on local alignments are not really suitable for the similarity assessment of regulatory regions as demonstrated by Blanco and colleagues 36.

By analyzing 48 pairs of orthologous sequences and an additional six paralogs, which contained at least 50 bp at their 5' UTR (see Additional File 7: Table S7 for the complete list) we identified motif families shared in the 5' UTR of common carp and zebrafish mRNAs. Analyzing each of the orthologous pairs individually (plus the paralogs, whenever applicable), we determined the order of a maximum of 10 shared motifs between common carp and zebrafish.

The distribution of coverage for all orthologous pairs relative to the number of motifs in these arrangements is represented in Additional File 9: Figure S2. About two-third of the orthologous 5'UTR pairs tested shared 4–6 motifs in the conserved positional arrangement, whereas most of the rest shared 7–10. The distribution of identified motifs together with the conserved arrangement in the zebrafish caudal type homeobox transcription factor 4 (cdx4) (RefSeq:NM_131109) and its common carp ortholog, cdx1 (Genbank:X80668) are shown in Figure 4 as an example.

A detailed UTR analysis is not within the scope of the present manuscript, therefore we propose a large-scale analysis to find out whether 5'UTR regions from different orthologous pairs share motifs from the same family. The presence of such shared motif families would suggest the existence of regulatory components common to both species suitable for further evaluation.

Androgenetic common carp "supermales" (YY; 37) have been crossed with wild type females (XX) to give rise to an all-male offspring population. (This approach allows for testis isolation without the need for sexing the fish.) The gonad has been isolated from a minimum of 6 individuals at 59/60 (a mixture of 59 and 60 days-old individuals), 70, 80 and 100 dpf, respectively. One of the two gonads from each individual has been processed for histological analyses (data not shown), while the other one has been stored in RNAlater (Ambion) for the use of RNA isolation.

Total RNA was isolated from the testis of 59/60, 80 and 100 days-old individuals, respectively. Full-length cDNA was synthesized using Creator Smart Library Construction Kit (Clontech) according to the manufacturer's instruction. After SfiI restriction enzyme digestion the adaptors and short cDNAs were removed by ChromaSpin 400 column (Clontech). The size fractionated cDNA pool was then cloned into a pBluescript based vector (detailed map is available on request) and transformed into E. coli XL10-Gold cells. Clones were picked into thirty, twenty and ten 96-well plates from the libraries generated from testes collected at 60, 80 and 100 dpf, respectively, and their insert was sequenced using M13 forward primer as described in 26.

Total RNA was isolated from the testis of 70 and 100 day old individuals, respectively. Two sets of subtractive hybridizations were performed: 70 dpf male gonad (driver) from 100 dpf testis (tester), and 100 dpf testis (d) from 70 dpf male gonad (t). The PCR-Select™ cDNA subtraction kit (Clontech) was used to enrich for developmental stage-specific fragments from the SMART cDNA template according to the recommendations of the manufacturer. The selectively amplified cDNA fragments (in average 400–800 bp in length) were ligated into pGEM-T (Promega) cloning vector. In total 2,500 clones have been picked from the two libraries and their insert was sequenced using M13 forward primer.

A total of 10,620 common carp ESTs, sequenced from a range of tissues other than gonad, were downloaded from GenBank (26 April 2005); 9,303 of those sequences are also available from CarpBASE 21 (see Additional File 1: Table S1 for details of clone origins). They were combined with 652 mRNAs from GenBank and with 6,050 gonad-derived common carp ESTs generated in our labs within the framework of this project (Fig. 1). Low quality regions were trimmed at the 3' end of ESTs prior to masking against libraries of repeats, mitochondrial and ribosomal sequences using RepeatMasker 38. Sequences that comprised at least 70% unmasked nucleotides (10,283 GenBank and CarpBASE ESTs and mRNAs, 5,073 TLL ESTs) were retained for further analysis. (The processed TLL ESTs were submitted to GenBank and can be found under the following IDs: DW719352-DW724424.) The combined EST data set was clustered using the STACKPACK clustering tools 3940 on HPCompaq Alpha ES40 architecture.

Common carp transcripts (clusters and singletons) were partitioned into ORF- and nonORF-containing sequences using ESTScan 28. The ORF-containing transcripts were annotated for protein domains and functional sites by matching them against the PFAM, PROSITE and PRINTS databases 414243 using hmmpfam, a program within the HMMER package that uses hidden Markov models to do sensitive searching of a protein database 44. The protein domains were mapped to gene ontology categories using GO tables 45.

In order to further categorize the common carp transcripts they were searched against the zebrafish genome assembly (version 5). The possibility of multigene families within EST clusters allow for common carp clusters to map to multiple zebrafish genomic locations. A single high quality zebrafish genomic location was identified for each mapped common cluster in order to screen for novel genes and potential alternative splice variants.

Transcripts that map to the zebrafish genome with BLAST E-value of at least 1e-04 where passed through a set of stringent filters as defined in Additional File 8: Figure S1 in order to identify a single zebrafish genomic location for each of the mapped common carp clusters. The best zebrafish chromosome location for each EST in a common carp cluster was considered: the zebrafish chromosome locus shared by all ESTs within a cluster was chosen as the mapped genomic locus for the corresponding common carp cluster. EST clusters that represented best hits to different chromosome locations for constituent ESTs were screened for a common zebrafish chromosome hit by considering the top five best hits for each EST in a cluster. A common zebrafish chromosome hit identified in the top five best hits was assigned as the unique map location for the common carp cluster. Mapped common carp clusters were not considered if there was not at least one zebrafish chromosome hit shared among all the ESTs in a cluster. All common carp transcripts that passed these filtering criteria were aligned to the specific segment of the overlapping zebrafish genome using EST2GENOME 31.

Exon-intron boundaries were extracted from the EST2GENOME results and served as a DAS track on the ensembl browser 30.

Genome-aligned common carp ESTs were classified according to one of five criteria that were satisfied on the plus or minus strand. Common carp ESTs overlapped (1) zebrafish coding regions: exons corresponding to an Ensembl gene or zebrafish EST; or (2) zebrafish introns: the entire genome-aligned common carp EST was contained within the intronic region(s) of a zebrafish gene; or (3) non-zebrafish exons: common carp ESTs mapped to regions of the zebrafish genome that overlapped non-zebrafish cDNA; (4) non-zebrafish introns: the non-coding portions of cDNA or proteins aligned to the zebrafish genome; (5) intergenic: regions of the zebrafish genome void of any annotations; (6) ab initio predictions: common carp ESTs mapped to regions of the zebrafish genome with an in silico gene prediction only.

Testis-only transcripts for common carp were defined as clusters or singletons represented by ESTs obtained exclusively from common carp testis cDNA libraries. Gonad derived genes for zebrafish were sampled from the UniGene zebrafish collection (build 91) where UniGene clusters contained ESTs that were sampled from zebrafish testis or ovary cDNA libraries. Common carp testis-derived transcripts were searched against the zebrafish gonad-derived UniGene dataset using BLASTN with (i) an E-value < 1e-04; and (ii) sequence overlap where 40% of the query sequence overlapped the matching database sequence. The common carp transcripts without identity to zebrafish gonad derived sequences were searched against the remainder of the zebrafish UniGene build 91 using an E-value < 1e-04 but without the requirement for 50% of the query sequence overlapping the database sequence. This relaxed criteria resulted in the identification of fewer common carp ESTs without homologous zebrafish ESTs in UniGene (build 91). However, these common carp ESTs provide a minimum dataset of testis-derived sequences not sampled by the zebrafish EST collection. The resulting "unique" common carp transcripts were searched against the fathead minnow UniGene (build 91) EST data using the same criteria as used for zebrafish.

A total 652 common carp mRNA sequences were downloaded from GenBank. About 292 mRNAs represented partial mRNA sequences and were removed. The sequences corresponding to the remaining 360 mRNA records in GenBank were searched against NCBI's non-redundant database using protein-protein BLAST (blastp; 46). The BLAST results were filtered for a significant sequence match to zebrafish (E-value < 1e-05) and matching zebrafish mRNAs that were partial sequences was filtered. The remaining 183 common carp and zebrafish homologous pairs were screened manually for orthologous relationships using cross-linked information including publications, curated annotations and filtering for redundant GenBank records. Eventually 120 pairs of orthologous genes were selected for sequence comparison between coding and non-coding regions (Additional File 7: Table S7) and a subset containing 48 pairs, plus six additional paralogs (all with at least 50 nucleotides upstream of the first protein coding exon) was used for motif searches (highlighted sequences in Additional File 7: Table S7).

First we analyzed the sequence similarity among the coding regions and the UTRs for the orthologous gene set. At the nucleotide level, sequence conservation was observed more often in the CDS regions, followed by the 5' UTR and 3' UTR regions, respectively (Additional File 10: Figure S3). Specifically, 75% of the orthologous pairs are captured when we set a sequence identity threshold of 80% at the CDS and protein levels. In comparison, only 25% of the 5' UTR sequences are captured under the same conditions (Additional File 10: Fig. S3). The threshold of 80% sequence identity was implemented for subsequent BLAST searches of common carp ESTs against the zebrafish genome assembly.

For the identification of motifs in 5'UTR regions, we compared the efficiency of Dragon Motif Builder system 47 with a local alignment method, ClustalW 48. With the Dragon Motif Builder we searched for any motif with the length between 6 bp and 10 bp, used the matrix score threshold of 0.9, and searched for up to 10 motifs in the two sequences of a given orthologous pair (in some cases the 5'UTR regions were very short providing not enough sequence length to harbour all 10 motifs). For ClustalW the common motifs were manually identified and restricted to the same criteria as those used by Dragon Motif Builder (motifs of length 6 to 10 bp). A significant difference was observed showing that ClustalW was not able to identify sufficient similarity between the ortholog sequences in the 5'UTR regions (Additional File 9: Figure S2) as the segments that contain similar arrangement of common motifs between the two species were not residing at similar genomic locations.

Once the motifs were identified, we analyzed the motif arrangements. We selected the group of motifs that contained the largest number of common motifs, but retained the same positional arrangement in the two species (see Fig. 4 for a specific example). Thus each of the ortholog pairs was screened for such a representative motif arrangement. We used the number of motifs in the representative arrangements as a possible measure of similarity between the 5'UTR regions. In most cases, the regions where this arrangement had been spotted, was found at significantly different distances from the starting codon.