## 3 Results
### Development of SCAR Markers for Distinguishing Herbal Medicines  
To screen the candidate sequences for locations for species-specific SCAR primers, we analyzed the entire sequence and identified several variable regions for each herbal medicine (Figure S1). We designed several forward primers matching the diverged sequences in ITS1 and one common reverse primer (ITS1 SCAR R) to the conserved 5.8S gene (Figure 1). We verified the specificity of the PCR amplification with the 24 plant samples listed in Table 1. The four SCAR primers, when combined with ITS SCAR R, amplified products with the expected sizes only in their respective samples (Table 3, Figures 1 and 2). The Adenophrae Radix-specific forward primer, AD ITS1 SCAR F, and the reverse primer, ITS1 SCAR R, produced a 231 bp amplicon only with the 12 samples from the three _Adenophora_ species, whereas no PCR products were observed with the other 12 Adenophrae Radix-related plant samples (Figure 2). Therefore, this primer set differentiates Adenophrae Radix from its related herbal medicines. The other three specific forward primers, CL ITS1 ACAR F for _C. lanccolata_, CP ITS1 SCAR F for _C. pilosula_, and GL ITS1 SCAR F for _G. litoralis_, also amplified their expected 267, 167, and 124 bp products only in their respective target species, with no products detected using the other samples (Table 3 and Figure 2). Thus, these three SCAR markers distinguish individual herbal medicines from Adenophrae Radix and identify plant material at the species level. Establishment of a Multiplex SCAR Amplification Method and Monitoring of Commercially-Processed Herbal Medicines  
The four individual SCAR markers produced mutually uniquely-sized amplicons that could be used to differentiate the four herbal medicines in a single gel electrophoresis. Therefore, we used a multiplex-PCR assay to concurrently amplify the SCAR amplicons. The combination of five primers, namely one common reverse primer (ITS1 SCAR R) and four forward primers (AD ITS1 SCAR F, CL ITS1 SCAR F, CP ITS1 SCAR F, and GL ITS1 SCAR F), was used in a single PCR reaction. We optimized the reaction conditions and PCR parameters, obtaining the same SCAR amplicons as those from the individual SCAR primer sets (Figure 3), suggesting that this multiplex-PCR assay can distinguish the four herbal medicines in a single reaction. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Primer Direction** & **Primer Name** & **Primer Sequence (5\({}^{\prime}\)\(\rightarrow\) 3\({}^{\prime}\))** & **Specificity** & **Amplicon Size (bp)** \\ \hline Forward & AD ITS1 SCAR F & ATC GAG CGA AAG CGC GTG AGC T & _Adenophora_ sp. & 231 \\  & CL ITS1 SCAR F & TGG CCC CT CT GCC GAC CT & _C. lanccolata_ & 267 \\  & CP ITS1 SCAR F & AAA ACT TAA CTC AAA GAG CGC GA & _C. pilosula_ & 167 \\  & GL ITS1 SCAR F & GTA CGT CCG TAT CCC GTT AAG G & _G. littoralis_ & 124 \\ \hline Reverse & ITS1 SCAR R & GCA ATT CAC ACC AAG TAT CGC AT & All samples & - \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Sequences of the ITS1-SCAR primers and their specificity, amplicon sequence, and amplicon size. Figure 2: Development of species-specific SCAR markers based on inter-species sequence variability. (**a**) Schematic diagram of primer position and SCAR amplicon sizes; and (**b**) the verification of primer specificity for Adenophrae Radix, Codonopsis Lanceolatae Radix, Codonopsis Pilosulae Radix, and Glehniae Radix. The numbers 1-24 correspond to those listed in Table 1, in the ‘Lane in gel’ column. The precise lengths of DNA fragments are indicated to the right side of the gel images. M represents the 100 bp DNA ladder. To verify the reproducibility and investigate the present state of Adenophorae Radix distribution, we assessed 37 commercial herbal medicines using the multiplex SCAR assay (Figure 4 and Table 4). As shown in Table 4, we purchased 17, nine, eight, and three samples that were listed as Adenophorae Radix, Codonopsis Lanceolatae Radix, Glehniae Radix, and Codonopsis Pilosulae Radix, respectively.