## 4 Discussion
triphylla_ in a recent reconsideration of the taxonomic system for _Adenophora_ [PERSON], although the original plant material for Adenophorae Radix was described as the roots of _A. tetraphylla_ and _A. stricta_ in Chinese pharmacopoeia [6; 7]. Therefore, we considered _A. tetraphylla_ as _A. triphylla_ in our taxonomic identification (Table 1). Since DNA is usually degraded in commercial herbal medicines, genetic analysis must be amenable to short DNA fragments [33]. We considered this point for developing SCAR markers and designed diverse SCAR primers that amplify variable ranges of DNA fragments. We designed SCAR markers in the ITS1 region based on aligned nrDNA-ITS sequences. Sequence substitutions and indels were more abundant in ITS1 than in ITS2 (Figure S1). In fact, differences between _C. lanccolata_ and _C. pilosula_ were rarely identified in the ITS2 region. The SCAR primers were, therefore, designed only for the ITS1 region and selected for different amplicon sizes depending on the herbal medicine, allowing for optical discriminability and stable amplificability by the multiplexed SCAR assay (Figure 1). To assess and increase the reproducibility of single and multiplexed SCAR markers, we optimized the PCR conditions by adjusting the annealing temperature, time, and concentration of primers and templates as described in Materials and Methods. Finally, we confirmed the discriminability and amplificability of our assay by assessing the adulteration of commercially-processed Adenophorae Radix, Codonopsis Lanccolatae Radix, Glehniae Radix, and Codonopsis Pilosulae Radix samples. We successfully assessed 37 commercially-processed herbal medicines and identified eight cases of adulteration (Figure 4 and Table 4). To confirm the botanical origin and sequence identity of the PCR amplicons obtained using the multiplexed SCAR assay, PCR products were sub-cloned into the pGEM-T Easy Vector and sequenced. The resulting sequences were aligned with the entire nrDNA-ITS sequences obtained from control plant materials listed in Table 1 and were confirmed against GenBank data using BLAST searches. All the sequences were identical to the corresponding sequences of control plants listed in Table 1. These results demonstrate that our multiplexed SCAR marker assay is a useful genetic tool for differentiating between Adenophorae Radix and its commercial adulterants. In this study, we could not identify the species used in Adenophorae Radix because the genus _Adenophora_ cannot be well resolved at the species-level using universal DNA barcodes (Figures S1 and S2). In a previous study, we analyzed the nrDNA-ITS regions and the chloroplast \(mat\)K and \(rb\)CL gene sequences using several _Adenophora_ species, but could not identify useful species-specific variability [34]. In addition, another study using the ITS2 sequence was unable to discriminate between species of the genus _Adenophora_[3]. We also could not differentiate three _Adenophora_ species using nrDNA-ITS sequences in this study. These results confirm the low level of ribosomal sequence variation among species of the genus _Adenophora_. Therefore, to verify the accurate botanical origins of the roots of _Adenophora_ species, further studies are needed to develop genetic markers for use in genomic fingerprinting analyses, such as RAPD, and comparative analyses of organellar or whole genome sequences are needed to obtain intra-species sequence information. The following are available online at www.mdpi.com/2076-3417/7/7/660/s1: Figure S1, Comparative analysis of nrDNA-ITS sequences used in this study; Data S1: FASTA file of nucleotide sequences of nrDNA-ITS used in this study; Figure S2: Phylogenetic relationships of medicinal plant species based on the nrDNA-ITS sequences. We thank the 'Classification and Identification Committee of the KIOM' for critical identification and the Korean Herbarium of Standard Herbal Resources (IH code KIOM) for providing plant materials.