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Development of User-Friendly Method to Distinguish Subspecies of the Korean Medicinal Herb Perilla frutescens Using Multiplex-PCR.
Kim Y
,
Kim AY
,
Jo A
,
Choi H
,
Cho SS
,
Choi C
.
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Perilla (Perilla frutescens) is an economically and culturally important plant in East Asia. Plant breeding between cultivars has enhanced the genetic diversity of perilla overall, but means that functionally diverse subspecies are more difficult to identify and distinguish. In this study, we developed gene-based DNA markers to distinguish between the Korean herbal medicinal perilla varieties. We identified informative simple sequence repeat (SSR) regions on the promoter regions of the Myb-P1 and dihydroflavonol 4-reductase (DFR) genes, as well as a large insertion-deletion (indel) region in the limonene synthase (LS) gene, and developed markers to characterize the distinct subspecies differences (PfMyb-P1pro, PfDFRpro, and PfLS, respectively). Using the PfLS primers, a 430-bp region could be amplified from P. frutescens var. acuta, crispa, and f. viridis (known as Jasoyeop, Jureum-soyeop, and Chungsoyeop, respectively), but not from P. frutescens var. japonica (Dlggae). The PfMybpro primers resulted in PCR products of 314 or 316, 330, 322, and 315 bp from Dlggae, Jasoyeop, Jureum-soyeop, and Chungsoyeop, respectively, and the PfDFRpro primers resulted in products of 189 or 202, 187 or 189, 185 or 189, and 193bp, respectively, for the four perilla subspecies. Combining these three reactions into a single multiplex PCR approach resulted in subspecies-specific PCR band patterns for six common types of commercial perilla, distinguishing between three varieties of Dlggae (Cham-Dlggae, Ip-Dlggae, and Bora-Dlggae), as well as identifying Jasoyeop, Jureum-soyeop, and Chungsoyeop. These user-friendly markers will be valuable as a simple and efficient method for identifying the Korean medicinal herb Jasoyeop, as well as distinguishing between other functionally distinct subspecies, which may have broad applications in the Korean herbal industry.
Figure 1. Development of the PfLS marker. (a) LS region specific to PA-type genomes identified in P. frutescens 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. frutescens var. japonica; 3–5:f. viridis; 6–8: var. acuta; 9–11: var. crispa). (c) 430 bp sites of specific target region.
Figure 2. Development of the PfMyb-P1pro 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′-UTR Py-rich stretch and AT-rich regions in the promoter of Myb-P1 among four subspecies of P. frutescens. The SSR variations are highlighted in green; (b) Application of the PfMyb-P1pro marker in the commercial breeding perilla lines. (M: 100bp DNA ladder; 1–2: P. frutescens var. japonica; 3–5: f. viridis; 6–8: var. acuta Kudo; 9–11: var. crispa).
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′-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: 100bp DNA ladder; 1–2: P. frutescens var. japonica; 3–5: var. crispa; 6–8: var. acuta; 9–11:f. viridis).
Figure 4. Multiplex PCR assay using three specific markers for the perilla subspecies in a single reaction. A mixture of three specific markers, PfLS, PfMyb-P1pro, and PfDFRpro, was used for PCR amplification. Lanes on 3% electrophoresis gel: M: 100 bp DNA ladder; 1–3: ‘Cham-Dlggae’, ‘Ip-Dlggae’, and ‘Bora-Dlggae’, respectively, which represent the three cultivar types of P. frutescens var. japonica; 4: ‘Jureum-soyeop’, representing P. frutescens var. crispa; 5: ‘Jasoyeop’, representing P. frutescens var. acuta; 6: ‘Chungsoyeop’, representing P. frutescens f. viridis.
Figure 5. Multiplex PCR identification of 11 commercial dried leaves and twigs of Jasoyeop products. (a) Lanes on 3% electrophoresis gel: M: 100 bp DNA ladder; A–K: purchased commercial dried Jasoyeop products (see Table 3 for full details); (b,c) Sequence analysis of PCR products amplified using the PfMybpro and PfDFRpro marker primers, respectively, aligned using Clustal W2.
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