## 4 Plant Materials and Methods  
### Plant Materials  
The medicinal _Bupleurum_ species / varieties that are mainly distributed in China were selected in this study, including _B. chinense_ DC. (BcR), _B. scorzomerifolium_ Wild. (BsR), _B. marginatum_ var. _stenophyllum_ (Wolff) [PERSON]. [PERSON] (BmsR), _B. marginatum_ Wall. ex DC. (BmR), and _B. smithii_ Wolff (BsmR), among which _B. chinense_ DC. and _B. scorzomerifolium_ Wild. are listed in the _Chinese Pharmacopoeia_, and _B. marginatum_ Wall. ex DC. and _B. smithii_ Wolff are included in different local standards in China. According to our review [1], all the sampling locations for the above species (BcR, BsR, BmR, BsmR)/varieties (BmsR is the varieties of BmR) are currently their optimal production areas. For both _B. chinense_ DC. and _B. scorzomerifolium_ Wild., their cultivated germplasm and the wild germplasm were collected from the same areas to avoid additional errors caused by geographical factors. The resources of the other three species/varieties were all cultivated germplasm. The detailed information of location, altitude, latitude, longitude, and growth years (all two years) were shown in Table 2, the soil type of all the sampling locations was sandy soil. All the plant materials were harvested in early October. After harvesting, the fresh roots were \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Species** & **Abbreviation** & **Type** & **Producing Area** & \begin{tabular}{c} **Latitude and** \\ **Longitude** \\ \end{tabular} & **Altitude** \\ \hline _B. chinense_ DC. & BcR & Cultivated & \begin{tabular}{c} Chencang District, Baoji City, \\ Shanxi Province, China \\ \end{tabular} & \begin{tabular}{c} 107°37° E 34°35° N \\ \end{tabular} & 700 m \\  & BcR\_W & Wild & \begin{tabular}{c} Chencang District, Baoji City, \\ Shanxi Province, China \\ \end{tabular} & \begin{tabular}{c} 107°37° E 34°35° N \\ \end{tabular} & 700 m \\ _B. scorzomerifolium_ & BsR & Cultivated & \begin{tabular}{c} Lindian County, Daqing City, \\ Heilongjiang Province, China \\ \end{tabular} & \begin{tabular}{c} 124°87° E 47°17° N \\ \end{tabular} & 150 m \\  & BsR\_W & Wild & \begin{tabular}{c} Lindian County, Daqing City, \\ Heilongjiang Province, China \\ \end{tabular} & \begin{tabular}{c} 124°87° E 47°17° N \\ \end{tabular} & 150 m \\ _B. marginatum_ var. & & & & & \\ _stenophyllum_ (Wolff) & BmsR & Cultivated & \begin{tabular}{c} Lintao County, Dingxi City, \\ Gansu Province, China \\ \end{tabular} & \begin{tabular}{c} 103°86° E 35°40° N \\ \end{tabular} & 1800 m \\ _B. marginatum_ Wall. ex DC. & BmR & Cultivated & \begin{tabular}{c} Rong County, Zigong City, \\ Sichuan Province, China \\ \end{tabular} & \begin{tabular}{c} 104°42° E 29°45° N \\ \end{tabular} & 350 m \\ _B. smithii_ Wolff & BsmR & Cultivated & \
\begin{tabular}{c} Pinshun County, Changzhi \\ City, Shanxi Province, China \\ \end{tabular} &
\begin{tabular}{c} 113°44° E 36°20° N \\ \end{tabular} & 1200 m \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The sampling information of all the plant materials. immediately frozen in liquid nitrogen as fresh samples; the remaining roots were dried in the shade to obtain the dried sample. For the dried samples that were used for e-nose analysis, metabolomics analysis, and the hydrochloric acid phloroglucinol staining, the fully shaped, non-moldy, and disease-free dried roots were used. For _Bupleurum marginatum_ var. _stenophyllum_ ([PERSON] et Y. [PERSON] and the other four species/varieties (including the 2 planting types for _B. clinense_ and _B. scorzorifolium_, respectively), there were 7 groups in total. For each group, three dried roots were randomly selected and mixed as one sample, each group had three samples as three replicates; the biological replications for the dried samples that used for e-nose analysis was five. For the fresh samples that were used for DNA bar-coding analysis, metabolomics analysis, transcriptome analysis, PCR analysis, and safranine and fast green staining, the fully shaped, healthy fresh roots were used.