## 2 Results
### Unigene Functional Annotation and Overview of Unigene Expression  
Among the 135,968 unigenes, 102,154 genes (75.13%) were annotated in public databases, including 70.86%, 51.42%, 53.76%, 55.01%, 56.03%, 53.73%, and 37.67% in NCBI nonredundant protein sequences (NR), NCBI nucleotide sequences (NT), a manually annotated and reviewed protein sequence database (SwissProt), Kyoto Encyclopedia of Genes and Genomes (KEGG), clusters of euKaryotic Orthologous Groups (KOG), Pfam, and gene ontology (GO), respectively (Table 1). According to Venn diagram analysis, 49,785 (36.62%) unigenes were co-annotated in five databases (Supplementary Figure S3A). Additionally, 96,353 unigenes were annotated in the NR database. Just over half (55.48%) of the annotated unigenes were mapped to Sesamumo indicum, 20.10% were mapped to Erythranthe guttata, 19.76% were mapped to Dorcoceras hygrometricum, and 4.65% were mapped to others (Supplementary Figure S3B). Moreover, we conducted an annotation of unigenes in C. chinense using the MAPMAN software. The most unigenes were enriched in the categories of \"protein\", \"RNA\", \"signaling\", \"miscellaneous function (misc)\", \"transport\", and \"stress\" (Supplementary Figure S4). Based on the unigenes with fragments per kilobase of transcript per million mapped reads (FPKM) \(>\) 1, an overview of metabolic and secondary metabolic pathways was developed using MAPMAN analysis. In the metabolic pathway, the most unigenes were mapped to \"lipids metabolism\" and \"secondary metabolism\". In secondary metabolic pathways, the most unigenes were enriched in the \"phenylpropanoids\", \"flavonoids\", and \"lignin and lignans\" pathways (Supplementary Figures S5 and S6). The annotated unigenes were described using GO terms which were divided into three classes: biological process, cellular component, and molecular function; 51,220 unigenes were matched with one or more GO terms comprising of 55 subcategories. We focused on the biological process and molecular function categories in this study. The most abundant categories under molecular function were cellular process, metabolic process, and transporter activity (24,757, 24,210, and 2761 unigenes, respectively). The most abundant categories under biological processes were cellular process, metabolic process, and biological regulation (17,210, 16,877, and 4937 unigenes, respectively) (Supplementary Figure S7). The expression values of transcripts in each tissue were calculated based on FPKM \(>\) 1. The numbers of expressed unigenes were 46,136, 61,118, and 50,304 in leaf, stem, and root tissues, respectively (Figure 1A). We observed that the overall expression level of unigenes in roots was lower than in leaves or stems (Figure 1B). Identification of Candidate Genes Involved in Triterpenoid Saponin Biosynthesis by KEGG Pathway Analysis  
To determine the main biological processes in C. chinense, 74,791 unigenes were annotated in the KEGG database; these were classified into five categories (cellular process, genetic information processing, metabolism, organismal systems, and environmental information processing) (Supplementary Figure S8) and distributed to 136 KEGG pathways (Supplementary Table S1). Seven pathways were assigned \begin{table}
\begin{tabular}{l l l} \hline \hline
**Database** & **Number Annotated** & **Annotated Unigene Ratio (\%)** \\ \hline NR & 96,353 & 70.86 \\ NT & 69,913 & 51.42 \\ SwissProt & 73,100 & 53.76 \\ KOG & 76,187 & 56.03 \\ KEGG & 74,791 & 55.01 \\ Pfam & 73,053 & 53.73 \\ GO & 51,220 & 37.67 \\ Overall & 102,154 & 75.13 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Summary statistics of annotations for C. chinense unigenes via seven public databases. Figure 1: Overall expression profiles in C. chinense leaf, stem and root tissues. (**A**) Distribution of number of expressed unigenes (FPKM \(>\) 1) in tissues. (**B**) Boxplot of unigene expression in tissues. In (**B**), the x-axis represents tissue type and the y-axis represents log2 (FPKM + 1) values.