RESULTS: More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis.
CONCLUSIONS: This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.
Methods: First, studies of codon usage in monocots were reviewed. The current information was then extended regarding codon usage, as well as codon-pair context bias, using four completely sequenced non-grass monocot genomes (Musa acuminata, Musa balbisiana, Phoenix dactylifera and Spirodela polyrhiza) for which comparable transcriptome datasets are available. Measurements were taken regarding relative synonymous codon usage, effective number of codons, derived optimal codon and GC content and then the relationships investigated to infer the underlying evolutionary forces.
Key Results: The research identified optimal codons, rare codons and preferred codon-pair context in the non-grass monocot species studied. In contrast to the bimodal distribution of GC3 (GC content in third codon position) in grasses, non-grass monocots showed a unimodal distribution. Disproportionate use of G and C (and of A and T) in two- and four-codon amino acids detected in the analysis rules out the mutational bias hypothesis as an explanation of genomic variation in GC content. There was found to be a positive relationship between CAI (codon adaptation index; predicts the level of expression of a gene) and GC3. In addition, a strong correlation was observed between coding and genomic GC content and negative correlation of GC3 with gene length, indicating a strong impact of GC-biased gene conversion (gBGC) in shaping codon usage and nucleotide composition in non-grass monocots.
Conclusion: Optimal codons in these non-grass monocots show a preference for G/C in the third codon position. These results support the concept that codon usage and nucleotide composition in non-grass monocots are mainly driven by gBGC.