## 2 Results and Discussion
### Identification of CYP450 Unigenes Putatively Involved in Diosgenin Biosynthesis  
It has been suggested that diosgenin is biosynthesized from the steroidal skeleton compound cholesterol by a series of oxidative reactions at the C-22, C-26, and C-16 positions [18] (Figure 1). The hydroxylation of cholesterol is likely catalyzed by cytochrome P450 (CYP) enzymes. Through annotation using different databases, a total of 485 CYP-encoding unigenes were discovered in _D. zingiberensis_ transcriptome (Table S5), among which 165 CYP unigenes showed the highest expression level in Aug_R, followed by Nov_R and Aug_L (Table S6), coincident with the accumulation pattern of diosgenin in those tissue samples. These CYP unigenes exhibiting correlating expression pattern with diosgenin accumulation were considered as the potential candidates to be involved in diosgenin biosynthesis. Among them, there were 22 CYP unigenes putatively being presented at full-length cDNAs (Table S6), out of which the unigene c56203_g1 was annotated as a sterol 14\(\alpha\)-demethylase functions in the pathway steps to demethylase. It is well known that the sterol 14\(\alpha\)-demethylase functions in the pathway steps to cholesterol formation [11]. Cholesterol was proposed to be the precursor for diosgeninbiosynthesis, and thus identification of sterol 14\(\alpha\)- demethylase annotated unigene within our list of the potential P450 candidates indicates that some of the other CYP candidates might play an important role in diosgenin biosynthesis. We performed phylogenetic analysis for these full-length CYP candidates together with the well characterized CYPs from various metabolic pathways, including the ones that are relevant to the biosynthesis of triterpenoids and steroidal saponins. The 2-oxoglutarate-dependent dioxygenenses from potato (St16 DOX) and tomato (Sl16 DOX), which oxidize hydroxycholesterol at the C-16 position [19], were included in the alignments as the outgroup. As shown in Figure 3, the unigene c65998_g1, which belongs to the CYP90B subfamily, grouped together with AtCYP90B1 from _A. thaliana_[20], and OSCYP90B2 and OsCYP724B1 from rice [21] that were all characterized to be a steroid C-22 hydroxylase in brassinosteroid pathway, strongly suggesting that the unigene c65998_g1 might be the CYP candidate responsible for the cholesterol C-22 oxidation in diosgenin biosynthesis. It should be noted that a CYP enzyme StCYP72A188 from potato, which also catalyzes a sterol C-22 hydroxylation in steroidal glycoalkaloid (SGA) biosynthesis [22], was in the clade distinctly different from the above mentioned sterol C-22 hydroxylases (Figure 3). Also, among the full-length CYP candidates matching the accumulation of diosgenin (Table S6), no members of the CYP72A group were found. These data suggest that the C-22 hydroxylase in SGA pathway might evolve independently from the CYPs for diosgenin or brassinosteroid pathway. The unigene c47099_g1, which belongs to CYP505 family, showed a relatively closer relationship to the sterol C-26 hydroxyles that include SICYP73A47 from _Solanum Lyopersicum_[23], OsCYP73A1 from rice [24] ([PERSON] et al., 2011), and StCYP72A208 from potato [22], suggesting that the unigene c47099_g1 might encode the C-26 hydroxylase in diosgenin biosynthesis. CYP enzymes displaying the sterol C-16 hydroxylation activity have not been identified yet, however, a 2-oxoglutarate-dependent dioxygenase (designated St16 DOX) has recently been reported to play a role in SGA biosynthesis in potato by catalyzing the C-16 oxidation on hydroxycholesterols [19]. Its equivalent in tomato, designated Sl16 DOX, has essentially the same biochemical activity as does St16 DOX [19]. From the _D. zingiberensis_ transcriptome data, we searched for close homologs of St16 DOX. However, the closest homolog unigene c51094_g1 displayed only 36% amino acid identity while showing a 77% identity to Oryza sativa _Japonica_ 2-oxoglutarate-Fe-dioxygenase. The unigene c51094_g1 was specifically found in the _D.