## 2 Results and Discussion
### Identification of Genes Related to Steroidal Backbone Biosynthesis  
Steroidal saponins are biosynthesized from C5 units, isopentenyl diphosphate (IPP), which is derived either from the cytosolic mevalonate pathway (MVA pathway) or from the plastidic methylerythitol phosphate pathway (MEP pathway). In the \(D\). _zingiberensis_ transcriptome of this study, eleven unigenes putatively code for the MVA pathway enzymes (Table 2), including three for AACT, one for HMGS, one for HMGR, two for MK, one for PMK, one for MVD, and two for IDI; seven unigenes were identified as the putative genes in the MEP pathway (Table 2), which included two for  
\begin{table}
\begin{tabular}{c c c c c c c} \hline
**Item** & **Sample** & **Number** & **Q20(\%)** & **Q30(\%)** & **GC(\%)** & **N50(bp)** \\ \hline Raw read & Aug\_L & 55,798,190 & ND & ND & ND & ND \\  & Aug\_R & 45,367,360 & ND & ND & ND & ND \\  & Nov\_R & 42,564,054 & ND & ND & ND & ND \\ Clean read & Aug\_L & 53,330,426 & 97.68 & 94.42 & 49.52 & ND \\  & Aug\_R & 43,234,636 & 97.68 & 94.44 & 47.54 & ND \\  & Nov\_R & 40,686,226 & 97.72 & 94.51 & 47.87 & ND \\ Total & Unigenes & 143,245 & ND & ND & ND & 814 \\ \hline \end{tabular}
\end{table}
Table 1: Summary of the sequencing data.  
DXS, one for DXR, one for CMK, one for MECPS, one for HDS and one for HDR. Among the MVA pathway unigenes, the transcripts of the c46893_g1 and c102447_g1 coding for AACT, the c76083_g1 for MK, the c61824_g1 for HMGS, and the c74391_g1 for IDI were specifically expressed in the rhizomes (Aug_R) but not in the leaves (Aug_L), which is in accordance with the rhizome-specificity for dioscin biosynthesis in _D. zingiberensis_. On the other hand, all the MEP pathway backbone unigenes were expressed at much higher levels in the leaves (Aug_L) than in the rhizomes (Aug_R), and their expressions in both rhizome tissues (Aug_R and Nov_R) were essentially similar (Table 2), which does not match the metabolite accumulation pattern (Figure 2C). These results indicate that dioscin biosynthesis is likely to be mainly from the MVA pathway.  
In the _Solanaceae_ plant family, especially those producing the steroidal glycoalkaloids (SGAs), there are two separate sets of pathway enzymes responsible for the biosynthesis of cholesterol and phytosterols [11]. However, based on the bioinformatics analysis here, we proposed the same set of enzymes probably occurring in the cholesterol and phytosterol pathways in _D. zingiberensis_. A similar case was also previously suggested for another diosgenin-producing plant _Trigonella fonium-gracum_[17]. The unigenes encoding all the known pathway enzymes from IPP to cholesterol were discovered in the _D. zingiberensis_ transcriptome (Table 2), including two for GPPS, one for FPPS, one for SS, one for SE, one for CAS, two for SSR, one for SMO1, one for CYP51, one for C14-R, one for 8,7 SI, one for SMO2, one for C5-SD, and one for 7-DR. Except for the unigenes putatively coding for GPPS, FPPS, CAS, CPI, and 8,7 SI, all the other unigenes in the pathway were more highly expressed in the rhizomes (Aug_R) than in the leaves (Aug_L) and their transcript abundances were much higher in the younger rhizomes (Aug_R) than in the mature rhizomes (Nov_R) (Table 2). Taken together, except for the MEP pathway genes displaying higher expression in the leaves (Aug_L), most of the unigenes involved in the steroidal saponin backbone biosynthesis showed higher expression in the Aug_R, followed by the Nov_R, and Aug_L(Table 2), which parallels to the accumulation pattern of diosgenin or dioscin in the plants (Figure 2C). This data implicates that the biosynthesis of the steroidal metabolites in _D. zingiberensis_ is highly controlled by the transcriptional regulation of steroidal backbone biosynthesis genes.