The present study was carried out to understand the evolutionary dynamics of male based genes using D. pseudoobscura, the second completely sequenced Drosophila genome. About 3500 transcripts were identified with Serial Analysis of Gene Expression (SAGE) in this species. Like in the previous studies with D. melanogaster, the majority of these genes are male based in expression. About 34% of the genes change their expression pattern between the two species. The rate of evolution of these male biased genes is measured in terms of the ratio of non-synonymous substitution (dN) to synonymous substitution (dS). However, due to high divergence between D. melanogaster and D.
pseudoobscura, the synonymous substitutions are saturated between the two species. Hence it is not appropriate to use this method. Since all the genes diverge at the same time, it is reasonable to compare only the non-synonymous substitution rate. We also used codon usage bias and Grantham distance as a measure of rate of evolution. Contrary to the expectation that male biased genes evolve rapidly, the male based genes in D. pseudoobscura do not show accelerated rate of evolution. The genes with male based expression in both species show highest rate of evolution and the genes with male based expression in D. melanogaster show slightly lower rate of evolution. If the accelerated rate of protein evolution is due to sexual selection, our results indicate that there could be different selection regime in D. pseudoobscura. From this analysis, several sex-based genes could not be mapped to specific genes in the study. The majority of these unmapped genes are female biased. There are several possible reasons for this. The inherent disadvantage of the SAGE method is that the tags cannot be mapped if the 3¿ boundaries of the genes are not accurately identified. Alternatively, the unmapped tags might be representing novel genes.
Hence, in the follow up study, 20 male based and 64 female based tags were extended in the 3¿ direction using GLGI method to explore the possible explanations for unmapped SAGE tags. Majority of the SAGE tags were not mapped due to incomplete 3¿UTR annotation. However, among these tags, 5 male biased and 3 female biased tags that represent putatively novel genes. 5¿ RACE analysis indicated that these novel transcripts are very short in length and could contain introns. The excess of novel genes being male based is consistent with the previous analysis in other Drosophila species. In conclusion, the present study shows that there could be different selection regimes acting on male based genes in different species and that several novel male based genes exist in the genome of D. pseudoobscura compared to female based genes.