## 2 Results and Discussion
Therefore, we proved that limonene is a chemical major component as well as a subspecies specific marker between PA and PK types at the genetic level. The 1,141-bp sequence of the _Myb-P1_ gene was sequenced from two accessions of _P. frutscens_ var. _japonica_ and nine accessions each of _P. frutscens_ var. _acuta_, _crispa_, and _f. viridis_, then aligned with the previously published sequence (GenBank accession number: AB024053). A 657 bp region of the 5\({}^{\prime}\) upstream promoter of _Myb-P1_ (Myb-P1 pro2 primers) was found to have an informative nuclear SSR in a TA-rich region contained within a 5\({}^{\prime}\)-untranslated region (UTR) Py-rich sequence, a cis-acting element conferring high transcription levels (Table 2). To confirm the best amplification patterns for this polymorphic region, the observed product size including the number of alleles amplified and PIC value for _Myb-P1 pro_ marker used in this study is provided in Table 3. _Myb-P1 pro_ marker produced five alleles for all perilla accessions. As shown in Figure 2, the amplicon sizes of _Myb-P1 pro_ marker were 314 or 316 bp from _P. frutscens_ var. _japonica_, whereas amplicons from _P. frutscens_ var. _acuta_, _crispa_, and _f. viridis_ were 330, 322, and 315 bp respectively (Table 2). The amplicon sizes showed clear differences among the three subspecies, while there was no difference between _P. frutscens_ var. _japonica_ and _f. viridis_. [PERSON] et al. [24] suggested that _Myb-P1_ is a key factor in the regulation of anthocyanin biosynthesis and the main factor responsible for determining anthocyanin formation in red P. frutscens. [PERSON] et al. [17] reported that _Myb-P1_ is a highly upregulated gene associated with the anthocyanin biosynthesis and accumulation from red forms of perilla plants by RNA seq data. Therefore, according to these results, we performed the subspecies-specific SSR region from _Myb-P1_ promoter region, whereas there was no significant difference in coding and encoding region of _Myb-P1_ gene sequence. Figure 2: Development of the _P/Myb-P1 pro_ marker. (**a**) Identification of a simple sequence repeat (SSR) in the promoter region of the _Myb-P1_ gene. The sequence alignment shows the SSR variation of the 5\({}^{\prime}\)-UTR Py-rich stretch and AT-rich regions in the promoter of _Myb-P1_ among four subspecies of _P. frutscens_. The SSR variations are highlighted in green; (**b**) Application of the _P/Myb-P1 pro_ marker in the commercial breeding perilla lines. (M: 100 bp DNA ladder; 1–2: _P. frutscens_ var. _japonica_; 3–5: _f. viridis_; 6–8: var. _acuta_ Kudo; 9–11: var. _crispa_). In the same manner as described above, among total 1,364 bp of DFR sequence, a 745 bp region of the subspecies-specific nuclear SSR (DFRpro1 primers) was identified in aligned region with 5\({}^{\prime}\)-UTR Py-rich stretch between 11 perilla accessions (Table 2). To confirm the best amplification patterns for this polymorphic region, the observed product size including the number of alleles amplified and PIC value for _DFRpro_ marker used in this study is provided in Table 3. _DFRpro_ marker produced seven alleles for all perilla accessions. As shown in Figure 3, the product sizes for the _DFRpro_ primers were as follows: 189 or 202 bp from _P. frutescens_ var. _japonica_, 187 or 189 bp in _acuta_, 185 or 189 bp in _crispa_, and 193 bp in f. _viridis_. Although the amplicon sizes were similar between _P. frutescens_ var. _japonica_, _acuta_, and _crispa_, the _DFRpro_ marker could clearly distinguish these from Chungsoyeop, _P. frutescens_ f. _viridis_ (Table 3). Dihydroflavonol reductase (_DFR_) gene known as one of the vital factor for the formation of anthocyanin, since the mutants lacking this enzyme do not produce anthocyanin [24; 25]. Interaction between _Myb-P1_ and the promoter of gene encoding _DFR_ from P. frutescens was shown that the expression of Myb-P1 was 10-fold abundant in red perilla than in green perilla as well as remarkably induced by light [25].