## 2 Results and Discussion  
To identify subspecies-specific genomic regions in the PA and PK perilla varieties, the genome and promoter sequences of three previously identified [17; 18] candidate genes, _LS_, _Myb-P1_, and _DFR_, were downloaded from NCBI. Based on PCR profiling derived from these sequences using 44 primer combinations (Table 2), three genes (_LS_, _Myb-P1_, and _DFR_) were found to contain distinct indel and SSR patterns that varied between the perilla varieties. The primer pairs were developed from nucleotide or promoter sequences containing coding, encoding, 5\({}^{\prime}\) and 3\({}^{\prime}\) UTR regions. These sequences were identified from source sequence Genbank among PA type of perilla. In the 4,315-bp _LS_ sequence of the PA genotype, the four primer pairs (LS1-4) covered an approximately 0.9-1.0-kb region conserved between the PA and PK types (Table 2). Two primer pairs (LS6 and LS7 F/R2) amplified a region with a subspecies-specific difference in sequence length,  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Seen Name** & **Genbank Accession** & **Primer Sequences 5\({}^{\prime}\) to 3\({}^{\prime}\)** & **Tm \({}^{\circ}\)C** & **Size (bp)** & **Location** \\ \hline \multirow{2}{*}{\(\begin{array}{c}\text{{LSI}}^{*}\\ \text{{LSI}}  
amplifying 1012 bp in PA and 582 bp in PK between exon 3 and 3' UTR. Indel DNA fragments were identified by sequencing and aligning the DNA sequences of this region from two accessions of _P. frutscens_ var. _japonica_, as well as three accessions each of _P. frutscens_ var. _acuta_, _crispa_, and _P. frutscens_ f. _viridis_, with the published _LS_ sequence (GenBank accession number: AF241791). This event was identified as a deletion of 430 bp in var. japonica across exon 4 and 3' UTR; therefore, we considered the 430-bp indel site to be a unique identifier of the PA type. The _PfLS_ primer pair generated a single, clear band in the PA-type accessions (Figure 1, Table 3). Perillaldehyde and limonene has been reported as major volatile compounds and marker components in _P. frutscens_ var. _acuta_ grown in East asia. [PERSON] et al. (2016) were reported that the contents of major component in PA chemotype grown in Korea analyzed 57.9% of limonene and 19.6%  
Figure 1: Development of the _PfLS_ marker. (a) _LS_ region specific to PA-type genomes identified in _P. frutscens_ var. _crispa_, _acuta_, and _viridis_. The amplified region covered part of exon 4 and 3′ UTR; (b) Application of the _PfLS_ marker in the commercial breeding perilla lines (1–2: _P. frutscens_ var. _japonica_; 3–5:f. _viridis_; 6–8: var. _acuta_; 9–11: var. _crispa_). (c) 430 bp sites of specific target region. \begin{table}
\begin{tabular}{l l l l l l l l l l} \hline \hline \multirow{2}{*}{**Marker Name**} & \multicolumn{4}{c}{**Marker Sequence**} & \multicolumn{4}{c}{**Product Size (bp)**} & \multirow{2}{*}{**Number of Alleles**} & \multirow{2}{*}{**PIC**} \\ \cline{3-3} \cline{5-10}  & & & **J \({}^{a}\)** & & **A \({}^{b}\)** & & **C \({}^{c}\)** & **V \({}^{d}\)** & \\ \hline \multirow{2}{*}{**PfLS**} & F & GCTATTACCCGAGGCGATTCGAG & & & & & & & \\  & R & TGCTGCCACAGAATCTGGTGC & & N/A & 430 & 430 & 430 & N/A & N/A \\ \hline \multirow{2}{*}{**PfMybre**} & F & GCTATTATTATTACCTTATTACCG & & & & & & & \\  & R & GCTTGATCACAGCTCTCTTCATCAC & & & & & & & \\ \hline \multirow{2}{*}{**PfDFRpro**} & F & GAAGCTGAGTTGATTACCTTAGT & & & & & & & \\  & R & GCAAGAGATTTAGACAGCTGTAGT & & & & & & & \\ \hline \multicolumn{10}{c}{} \\ \end{tabular} \({}^{a}\) P. frutscens_ var. _japonica_; \({}^{b}\) P. frutscens_ var. _acuta_; \({}^{c}\) P. frutscens_ var. _crispa_, \({}^{d}\) P. frutscens_ f. _viridis_.  
\end{table} 
Table 3: Summary of subspecies-specific indel and two SSR markers used in the analysis of the 11 perilla accessions. of perillaldehyde. Based on this result, we obtained the PA type specific indel region from limonene synthase (LS) gene by marker-based approaches.