## 2 Results and Discussion
### Identification of Putative Genes Related to Iridoid Biosynthesis  
Iridoids are derived from ispentenyl diphosphate (IPP) and its allylic isomer dimethylally diphosphate (DMAPP) through multiple steps. In higher plants, IPP itself can be formed through either  
Figure 3: Pathway assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG). (**A**) Classification based on metabolism categories; (**B**) Classification based on secondary metabolism categories. Figure 2: Gene Ontology analysis of genes in the 454 sequence collection of _R. glutinosa_. the plastitial 2-C-methyl-d-erythnitol-4-phosphate (MEP) pathway or the cytosolic mevalonic acid (MVA) pathway. Although experiments indicated that there exists a crosstalk between the MVA and MEP pathways in the terpenoid biosynthetic network in some species, it is well established that MEP pathway mainly leads to monoterpenoids, triterpenoids, the prenyl side chains of chlorophyls and carotenoids, as well as to the phytohormones abscisic acid, cytokinin and gibberellins, and the MVA pathway predominantly gives rise to sterols, sesquiterpenoids and ubiquionones [28]. In TIA producing plants, through precursor feeding experiments, it had been confirmed that the secoiridoids are derived directly from the MEP pathway rather than MVA pathway [29, 30, 31]. The crucial role of the MEP pathway in TIA biosynthesis have also been demonstrated at molecular level, the expressions of the three MEP pathway genes, _1-Deoxy-D-xylulose-5-phosphate synthase_ (_DXS_), _1-Deoxy-D-xylulose-5-phosphate reductoisomerase_ (_DXR_) and _2-C-methyl-D-erythnitol 2,4-cyclodiphosphate synthase_ (_MECS_) in _C. roseus_ are consistent with the TIA production [17, 18, 23, 24]. Based on the gene annotation information, we identified all MEP pathway genes. Among them, the transcripts of DXS and DXR were represented by many 454 reads, indicating that they are highly expressed in the tuberous roots of _R. glutinosa_. Interestingly, genes encoding all enzymes in MVA pathway were also found in the 454 dataset, especially the transcripts of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) were represented by more than one hundred 454 reads, which seemed to show that the MVA pathway plays important roles in tuberous roots (Table 2). Present data cannot exclude the participation of the MVA pathway to the biosynthesis of route II iridoids, as evidences from _C. roseus_ showed that the MVA pathway may be implicated in the regulation of MEP and secoiridoid pathway genes through protein prenylation [32]. Interestingly, a set of genes encoding putative farnesyltransferasees were found in our sequence dataset (data not shown). However, the MEP pathway will be the major source of precursors (IPP and DMAPP) for the iridoid biosynthesis as it is in other monoterpenoid producing plants. The presence of high amount of MVA pathway gene transcripts is most likely to show that other terpenoids exist, derived from this pathway, for example, the biosynthesis of sterols and other triterpenoids that are generally derived from the MVA pathway. A relatively large amount of genes involved in the biosynthesis of other terpenoids, including steroid biosynthetic genes, had also been identified in the datasets (Supplementary File 1). Most interestingly, putative genes encoding enzymes of the secoiridoid pathway including GES, G10H, CPR and 10 HGO were found in the 454 dataset. Among these enzymes, G10H and 10 HGO have only been identified in TIA producing plants. The alignment results showed that these genes share significant sequence similarities with the secoiridoid pathway genes (Supplementary File 3). The predicted amino acid sequences of the putative _R. glutinosa G10H_ gene (contig14037) are 80% identical to those of CrG10H (Supplementary File 4), which implied that it may catalyze the hydroxylation of geraniol as CrG10H does in _C. roseus_. It is known that the early steps of secoiridoid pathway involve GPP, geraniol, 10-hydroxygeraniol and 10-oxygenenia, enzymes catalyzing above steps have been characterized (Figure 1).