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Background  The  Pythium splendens  is a potentially useful organism for the synthesis of large amounts of eicosapentaenoic acid. Peak biomass and lipid accumulation do not occur at the same time and growth temperature has an effect on the fatty acid composition. Little is known about the pathway or the genes involved in growth, lipid synthesis or temperature resistance in  P. splendens . Analysis of the transcriptome and expression profile data for  P.splendens RBB-5 were used to extend genetic information for this strain and to contribute to a comprehensive understanding of the molecular mechanisms involved in specific biological processes.    Methodology/Principal Findings  This study used transcriptome assembly and gene expression analysis with short-read sequencing technology combined with a tag-based digital gene expression (DGE) system. Assembled sequences were annotated with gene descriptions, such as gene ontology (GO), clusters of orthologous group (COG) terms and KEGG orthology (KO) to generate 23,796 unigenes. In addition, we obtained a larger number of genes at different stages of fermentation (48, 100 and 148 h). The genes related to growth characteristics and lipid biosynthesis were analyzed in detail. Some genes associated with lipid and fatty acid biosynthesis were selected to confirm the digital gene expression (DGE) results by quantitative real-time PCR (qRT-PCR).    Conclusion/Significance  The transcriptome improves our genetic understanding of  P.splendens RBB-5 greatly and makes a large number of gene sequences available for further study. Notably, the transcriptome and DGE profiling data of  P.splendens RBB-5 provide a comprehensive insight into gene expression profiles at different stages of fermentation and lay the foundation for the study of optimizing lipid content and growth speed at the molecular level.

Characterization of Pythium Transcriptome and Gene Expression Analysis at Different Stages of Fermentation