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Transciptome analysis based on next generation sequencing allows quantitative comparisons of gene expression across diverse species. Using Illumina sequencing, we generated a total of 35.7 and 30.1 million paired-end reads with lengths of 100 bp from Paphiopedilum concolor tessellated leaves and Paphiopedilum hirsutissimum green leaves, respectively. De novo assembly yielded 68,602 and 54,273 unigenes with average lengths of 844 and 874 bp for each species leaves, respectively. Based on BLAST searches with known protein sequences, 46.6% unigenes from P. concolor and 48.6% unigenes from P. hirsutissimum were annotated. Gene ontology, cluster of orthologous groups and kyoto encyclopedia of genes and genomes annotations revealed that the functions of the transcripts from the two Paphiopedilum species leaves covered a similarly broad set of molecular functions, biological processes and metabolic pathways. Gene expression profiles analyses between the two Paphiopedilum species leaves revealed that a total of 1,544 genes were obviously differentially expressed. To confirm the differential expression results, the expression profiles of 8 selected genes were analyzed by quantitative real-time PCR. Both transcript differences analysis and leaf internal morphology observation between the two Paphiopedilum species leaves demonstrated that chloroplast, cytoplasm, thylakoid membrane, extracellular region, and nucleus related genes probably played crucial roles in the two Paphiopedilum species leaves formation during evolutional processes. Finally, 8,523 potential EST-SSRs were identified, and 7,864 primer pairs for 6,210 SSRs were obtained. This study provides a valuable clue to understand the mechanisms of Paphiopedilum leaves formation during evolutional adaptation, and supplies us with a large leaf sequence resource for novel gene discovery and marker-assisted studies in Paphiopedilum plants.
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