## 2 Results and Discussion  
### Generation of D. Zingiberensis Transcriptomes from its Leaf and Rhizome Tissues  
It is well known that rhizome tissue is the richest source for dioscin production in \(D\). _zingiberensis_. We confirmed this tissue-specificity for dioscin as well as its aglycone diosgenin by measuring their contents in its leaf and newly-formed rhizome tissues, which we collected at a relatively earlier stage on 28 August 2015 (Figure 2A). Rhizomes (five months old) were shown to produce higher amounts of dioscin and diosgenin (3.50 mg g\({}^{-1}\) for dioscin and 5.84 mg g\({}^{-1}\) for diosgenin) while in leaf tissue  
Figure 2: Phytochemical analysis of the leaf and rhizome tissues of \(D\). _zingiberensis_. (**A**,**B**) representative pictures are shown for the \(D\). _zingiberensis_ plants collected in August (**A**) and November (**B**) of 2015; (**C**), the content of diosgenin and dioscin in the different plant parts, including the leaves harvested in August (Aug_L), the rhizomes harvested in August (Aug_R), and the rhizomes harvested in November (Nov_R). Two biological replicates were included for this experiment. Different letters on the bar indicate a significant difference at \(p<0.05\) based on generalized linear models with Bonferroni multicomparison tests.  
(five months old) much less of them were detected (0.12 mg g\({}^{-1}\) for dioscin and 0.20 mg g\({}^{-1}\) for diosgenin) (Figure 2C). At the later stage (2 November 2015), most of leaves faded and fell off from the plants (Figure 2B), and in the rhizome tissue (eight months old) of this stage the biosynthesis of either dioscin or diosgenin largely decreased in comparison with that in the younger rhizomes (1.23 mg g\({}^{-1}\) of dioscin and 2.00 mg g\({}^{-1}\) of diosgenin were detected from the rhizomes of this later stage) (Figure 2C).  
Based on the metabolite accumulation pattern described above, the leaf (described as Aug_L) and newly-formed rhizomes (Aug_R) harvested at the younger stage, and the newly-formed rhizomes (Nov_R) harvested at the later stage were chosen for comparative transcriptome analysis to understand the molecular mechanism underlying dioscin biosynthesis. Total RNA from each tissue sample was prepared and was sent to Novogene Company (Beijing, China) for the cDNA library construction and the RNA-Seq analysis. The number of the resulting raw reads, clean reads, Q20, Q30, GC content of each sample, and N50 is shown in Table 1. All the raw data have been submitted to the NCBI sequence read archive (SRA; [[http://www.ncbi.nlm.nih.gov/sra](http://www.ncbi.nlm.nih.gov/sra)]([http://www.ncbi.nlm.nih.gov/sra](http://www.ncbi.nlm.nih.gov/sra))) under the accession number of SRR6281651 for Aug_L, SRR6281650 for Aug_R, and SRR6281649 for Nov_R, respectively. The _de novo_ assembly of all the clean reads was performed using Trinity software. A total of 176,406 transcripts were assembled with a mean length of 715 bp in a range of 201 to 16,773 bp. Transcripts were further assembled into 143,245 unigenes and the assembled unigenes were then searched against several public databases including NCBI non-redundant protein database (Nr), NCBI nucleotide sequences database (Nt), Swiss-Prot protein database, Pfam database, Clusters of Orthologous Groups of protein databases (COG) and Gene ontology (GO). In total, 74,908 (52.29%) unigenes were successfully annotated in at least one of the above public databases (Table S1). According to Nr annotation, 8229 (16.2%) unigenes had the most hits from _Elaesis__sinensis_, followed by _Phoenix dactylifera_ (6857, 13.5%), _Musa acuminate_ (2692, 5.3%), _Hordeum vulgare_ (2387, 4.7%), and _Vitis vinifera_ (1117, 2.2%).