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Background  The genome of the pico-eukaryotic (bacterial-sized) prasinophyte green alga  Ostreococcus lucimarinus  has one of the highest gene densities known in eukaryotes, yet it contains many introns. Phylogenetic studies suggest this unusually compact genome (13.2 Mb) is an evolutionarily derived state among prasinophytes. The presence of introns in the highly reduced  O. lucimarinus  genome appears to be in opposition to simple explanations of genome evolution based on unidirectional tendencies, either neutral or selective. Therefore, patterns of intron retention in this species can potentially provide insights into the forces governing intron evolution.    Methodology/Principal Findings  Here we studied intron features and levels of expression in  O. lucimarinus  using expressed sequence tags (ESTs) to annotate the current genome assembly. ESTs were assembled into unigene clusters that were mapped back to the  O. lucimarinus  Build 2.0 assembly using BLAST and the level of gene expression was inferred from the number of ESTs in each cluster. We find a positive correlation between expression levels and both intron number (R = +0.0893,  p  = <0.0005) and intron density (number of introns/kb of CDS; R = +0.0753,  p  = <0.005).    Conclusions/Significance  In a species with a genome that has been recently subjected to a great reduction of non-coding DNA, these results imply the existence of selective/functional roles for introns that are principally detectable in highly expressed genes. In these cases, introns are likely maintained by balancing the selective forces favoring their maintenance with other mutational and/or selective forces acting on genome size.

EST Analysis of Ostreococcus lucimarinus, the Most Compact Eukaryotic Genome, Shows an Excess of Introns in Highly Expressed Genes