## 2 Results
### Transcriptome Sequencing, Assembly, and Annotation of the Fresh Samples  
We also identified the metabolites in fresh roots (Figure S2 and Table S5). There were 852, 756, 751, 835, 789, 762, and 822 metabolites detected in the fresh roots of BcR, BcR_W, BsR_W, BmsR, BmR, and BmR, respectively. In the fresh samples, PC1, PC2, and PC3 accounted for 32.66%, 20.06%, and 16%, respectively. We also screened out different metabolites between BmsR and other species. Compared with the other four species/varieties, the contents of hydrocarbons, ketones, and esters in BmsR were relatively up-regulated (Figure S3 and Table S6). To investigate the expression differences of the key genes involved in the biosynthesis of the related components of volatile oil in different _Bupleurum_ species/varieties, 21 RNA-Seq libraries (5 different species/varieties, 3 biological repeats) were constructed. After removing the repeats with the low correlation values (<0.6) with other repeats, 16 RNA-Seq libraries were finally obtained (three repeats for _B. scorzonerifolium_ Willd. (wild) and _B. marginatum_ var. _stenophyllum_, respectively; two repeats for other species/varieties). After sequencing and filtering the raw reads, 19.05-25.49 million clean reads were obtained, with the GC contents ranging from 42.55% to 43.27%, respectively (Table S7). The Q30 value of each sample was all higher than 92.09%. A total of 99,960 unigenes were assembled with the N50 value of 2105 bp (Table S8). Finally, 21,863, 56,228, 46,059, 38,866, 52,190, 45,450, 65,470, 52,819, and 71,495 assembled unigenes were annotated by the GO, COG, KEGG, Pfam, Swissprot, KOG, eggnog, TrEMBL, and Nr databases, respectively (Table S9). The RNA-Seq data were available on NCBI (accession number PRJNA981284). In line with the metabolomic results of fresh samples, when using BcR as control, BmsR showed the highest number of DEGs at 28,996, while BsR_W was the closest to BcR, with only 533 DEGs. The number of DEGs between BsR, BcR_W, and BcR was 4625 and 10,613, respectively, suggesting the relatively high similarity between BcR and BsR (Figure 5A and Table S10). Accordingly, as shown in the PCA results, BmsR was significantly separated from all the samples; _B. chinense_ DC. and _B. scorzonerifolium_ Willd. exhibited cross similarity between their wild and cultivated samples (Figure 5B). Figure 5: The bioinformatic analysis of the transcriptome data of the fresh samples of the five species/varieties of the genus _Bupleurum_. (**A**) The volcano plot of the differentially expressed genes in each fresh sample of the 6 species/varieties. Fresh samples of _B. chinense_ was used as control. (**B**) The PCA analysis is based on transcriptome data of the five species/varieties. DEGs Involved in the Synthesis or Metabolism of Volatile Substances in B. marginatum var. stenophyllum  
Next, we conducted transcriptome sequencing to explore the possible molecular mechanism that contributes to the different levels of related compounds among the seven samples. Generally, the biosynthetic pathways of volatile compounds can be divided into three pathways: terpenoid pathway, amino acid pathway, and fatty acid pathway. We found that many differential metabolites belong to terpenoids. Based on the metabolomics results, we focused on the genes involved in the biosynthesis or metabolism of terpenoids, ketones, and esters. Numerous key enzyme genes of triterpene saponin biosynthesis have been reported in _B. chinense_; many of key enzyme genes, such as TPS, AACT, DXS, GPS, GGPS, etc., exhibited significant differential expressions in this study (Figure 6). Only TPSs, which have pivotal roles in the biosynthesis of triterpenes and monoterepenes, showed relatively consistent up-regulation expression in all samples; the differentially expressed TPSs in _B. scorzonerifolium_ Wild were all up-regulated. However, another large group of TPSs were only differentially expressed in _B. marginatum_ var. _stenophyllum_ and exhibited down-regulation.