## 2 Results and Discussion  
### Sequence Generation and Function Analysis  
A half plate 454 run yielded 88.4 MB bases from 374,444 reads with an average 236 bp. The sequences were deposited at NCBI under the accession number SRX128593. After trimming the adapter sequences and removing low quality sequences, a total of 341,170 clean reads were assembled. The assembly was carried out using CAP3; assembly of the trimmed, size-selected sequences generated a total of 58,822 unique sequences including 21,504 contigs and 37,318 singletons. The average contig length was 397 bases with an average of 14.1 reads assembled per contig. To date, only a small number of rehmannia ESTs have been deposited in GenBank, alignment analysis showed that the majority of ESTs can be found in our 454 sequence collection. The annotation was based on sequence similarity searches against the known databases, _i.e._, the NCBI non-redundant protein (Nr) database, the Universal Protein Resource (UniProt) database, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the Clusters of Orthologous Groups of proteins (COG) database and the NCBI non-redundant nucleotide (Nt) database, respectively, with an e-value cutoff at e-5. Due to lack of genome and EST information, only 49.0% of the unique sequences were annotated. The annotation summary is shown in Table 1. In order to distinguish redundant sequences from homologous sequences, \"unigene\" was used in this study to minimize redundancy, each unique sequence was assigned a unigene ID according to the accession number of the best-hit homologue in the Nr database; finally 23,953 unigenes were obtained. Based on Gene Ontology (GO) classifications, 25,468 sequences represented by 46 functional groups were classified into three main GO categories (Figure 2). Among cellular components, cell (40.6%) and organelle (18.7%) categories represented the most dominant groups. Within the molecular function category, binding represented the most abundant category (59.6%) followed by catalytic activity (39.8%) and transport activity (5.9%). A total of 794 unique sequences (4.4%) were designated as transcriptional regulators, with the majority (480) being subcategorized as \"transcription factor activity\". Transcription factors play a significant role in the regulation of secondary metabolite biosynthesis by controlling gene expression [25]. In _C. roseus_, overexpression of the jasmonate-responsive transcription factor ORCA3 can enhance TIA production and activate the expression of iridoid biosynthesis related genes [26]. Interestingly, an ORCA3 homologue (contig1996) was also found in the sequence collection. For biological processes, the majority of sequences were grouped into metabolic (56.9%) and cellular process (52.0%). Compared with the sweet potato tuberous root, in which only 30.46% genes were classified into metabolic process [27], the tuberous root of _R. glutinosa_ contains a higher percentage of metabolic genes, indicating that more metabolic activities occur in _R. glutinosa_. KEGG analysis provides an alternative functional annotation of genes. Of the unique sequences, 25,258 (42.9%) sequences have sequence similarities to KEGG database. Among them, 4,178 unique sequences having enzyme commission (EC) were assigned to metabolic pathways. As shown in Figure 3A, the metabolic pathways were well represented by carbohydrate metabolism, amino acid metabolism, energy metabolism, and nucleotide metabolism, showing that primary metabolism is vital to the tuberous root growth and development. Within the secondary metabolism category, terpenoid backbone biosynthesis, phenylpropanol biosynthesis and isoquinoline alkaloid biosynthesis were prominently represented; the genes involved in terpenoid backbone biosynthesis constitute the largest group, accounting for 42.6% of the total genes assigned to this category, showing that the terpenoid biosynthesis occupies a distinguished position among the secondary metabolic activities in _R. glutinosa_ (Figure 3B).