## 2 Results and Discussion
These data suggest that _DFR_ gene may also be involved in formation and regulation of anthocyanin biosynthesis as well as the significant factor for determination of anthocyanin form in red and green perilla. Based on above results, although we confirmed the subspecies-specific SSR marker for _P. frutescens_ f. _viridis_ from _DFR_ promoter region, there was no significant difference between perilla subspecies. The _P.fLS_, _PfMyb-P1 pro_, and _PfDFRpro_ primers were used to develop a multiplex-PCR assay to distinguish the Korean herbal medicinal plant 'Jasoyeop' (_P. frutescens_ var. _acuta_). The primer specificity, concentration, and melting temperature (Tm) are very crucial in the development of multiplex PCR since its success depends on the capacity of the primers to be relatively annealed with their targets under a single set of PCR conditions [26; 27]. Optimal amplification conditions were determined  
Figure 3: Development of the _PfDFRpro_ marker. (**a**) Identification of a simple sequence repeat (SSR) in the promoter region of the _DFR_ gene. The sequence alignment shows the SSR variation of 5\({}^{\prime}\)-UTR Py-rich stretch in the promoter of _DFR_ among four subspecies of _P. frutescens_. The SSR variations are highlighted in green; (**b**) Application of the _PfDFRpro_ marker in the commercial breeding perilla lines. (M: 100 bp DNA ladder; 1-2: _P. frutescens_ var. _japonica_; 3-5: var. _crispa_; 6–8: var. _acuta_; 9-11:f. _viridis_). by altering the following parameters: annealing time (85-90 s), annealing temperature (58-60 \({}^{\circ}\)C), primer concentration (0.5, 1.0, and 1.0 \(\upmu\)M of each primer). Amplification with this combination of primers yielded distinct DNA fragments with two or three multi-bands that corresponded to the amplicons produced by the individual indel and SSR reactions. The amplicon sizes were used to identify the subspecies. Multiplex PCR with the three primer pairs clearly discriminated the six commercial perilla cultivars: three varieties of _P. frutescens_ var. _japonica_ (Cham-Dlggae, Ip-Dlggae, and Bora-Dlggae), and one variety each of _P. frutescens_ var. _crispa_ (Juneum-soyeop), _acuta_ (Jasoyeop), and f. _viridis_ (Chungsoyeop). The PCR products of the three _P. frutescens_ var. _japonica_ varieties were specific double-band amplicons: Cham-Dlggae had bands at 189 and 316 bp, Ip-Dlggae at 202 and 314 bp, and Bora-Dlggae at 202 and 316 bp, corresponding to _P/DFRpro_ and _P/Myb-P1 pro_, respectively. The PCR amplified three distinct products from the other three subspecies: _P. frutescens_ var. _crispa_ showed bands at 189, 322, and 430 bp; _P. frutescens_ var. _acuta_ showed bands at 189, 330, and 430 bp, and _P. frutescens_ f. _viridis_ had bands at 193, 315, and 430 bp. The bands corresponded to _P/DFRpro_, _P/Myb-P1 pro_, and _P/LS_, respectively (Figure 4, Table 2). Jasoyeop, a mixture of dried leaves and twigs of either _P. frutescens_ var. _acuta_ or _crispa_, is regularly consumed as a tea and herbal supplement in Korea. To validate the utility of multiplex PCR in commercial dried herbal materials, genomic DNA was extracted from leaf samples of 11 Jasoyeop products and amplified using the subspecies-specific primers. Almost all of the samples gave rise to clear triple band patterns consistent with the _P. frutescens_ var. _acuta_ or _crispa_, which are commonly used in _Jasoyeop_ products (Figure 5a). The amplified PCR products were sequenced to confirm the identification of _P. frutescens_ var. _acuta_ (Figure 5b,c). As shown in Figure 5a, the band patterns and sequences revealed that A and B are _P. frutescens_ var. _acuta_, while C-F, H, and J were identified as being _P. frutescens_ var. _crispa_. Although G and I showed similar banding patterns to the other samples, they were determined not to be _crispa_ nor _acuta_ by their mixed size ranges; their _P/DFRpro_ amplicons resembled _P. frutescens_ f. _viridis_ while their _P/Myb-P1 pro_ amplicon resembled _P. frutescens_ var.