Towards the ictalurid catfish transcriptome: generation and analysis of 31,215 catfish ESTs

Abstract

Background: EST sequencing is one of the most efficient means for gene discovery and molecular marker development, and can be additionally utilized in both comparative genome analysis and evaluation of gene duplications. While much progress has been made in catfish genomics, large-scale EST resources have been lacking. The objectives of this project were to construct primary cDNA libraries, to conduct initial EST sequencing to generate catfish EST resources, and to obtain baseline information about highly expressed genes in various catfish organs to provide a guide for the production of normalized and subtracted cDNA libraries for large-scale transcriptome analysis in catfish.

Results: A total of 17 cDNA libraries were constructed including 12 from channel catfish (Ictalurus punctatus) and 5 from blue catfish (I. furcatus). A total of 31,215 ESTs, with average length of 778 bp, were generated including 20,451 from the channel catfish and 10,764 from blue catfish. Cluster analysis indicated that 73% of channel catfish and 67% of blue catfish ESTs were unique within the project. Over 53% and 50% of the channel catfish and blue catfish ESTs, respectively, had significant similarities to known genes. All ESTs have been deposited in GenBank. Evaluation of the catfish EST resources demonstrated their potential for molecular marker development, comparative genome analysis, and evaluation of ancient and recent gene duplications. Subtraction of abundantly expressed genes in a variety of catfish tissues, identified here, will allow the production of low-redundancy libraries for in-depth sequencing.

Conclusion: The sequencing of 31,215 ESTs from channel catfish and blue catfish has significantly increased the EST resources in catfish. The EST resources should provide the potential for microarray development, polymorphic marker identification, mapping, and comparative genome analysis.

Figure 1

Bar graph of distribution of Tetraodon gene hits on catfish ESTs. For example, 575 catfish ESTs were each hit by three Tetraodon queries. A logarithmic scale was used for the Y-axis to better show the wide range of values.

Figure 2

Selected examples of the ability to differentiate between catfish allelic variants and gene duplicates (paralogues) using both blue catfish and channel catfish sequences. Highly similar channel catfish sequences (Channel) and at least one blue catfish sequence (Blue) sharing the same BLAST identity were subjected to phylogenetic analysis. The topological stability of the neighbor joining trees was evaluated by 1000 bootstrapping replications, and the bootstrapping values are indicated by numbers at the nodes. Channel catfish and blue catfish genes placed into the same clade indicate that the additional, related channel catfish sequence is likely a paralogue rather than an allelic variant.