**2. Methods**

#### *2.1. Plant Materials*

First, a survey of the native thyme species (*T. quinquecostatus*) grown in Korea was carried out. We also interviewed experts who had ethnobotanical knowledge on Korean native thyme. According to their ethnobotanical information, we collected *T. quinquecostatus* from three accessions, such as Odae Mt, Wolchul Mt, and Jiri Mt in Korea, during April 2018 (Figures 1 and 2). In addition, fresh plants of six *T. vulgaris* cultivars (lemon, golden lemon (golden), carpet, orange, silver, and creeping) were purchased from Daerim Horticulture, Gwachon, Happy Horticulture, Goyang and Nature Horticulture, Yangju, Republic of Korea (Figure 2).

The plants were authenticated and deposited in the Herbarium, Daejin University, Pocheon, Gyeonggi-do, Republic of Korea, with voucher numbers: lemon—DJU20180713, golden—DJU20180712, carpet—DJU20180717, orange—DJU20180715, silver—DJU20180714, creeping—DJU20180716, Odae Mt.—DJU20180718, Wolchul Mt.—DJU20180719, and Jiri Mt.—DJU20180720. The collected samples were kept at –20 ◦C for the essential oil analysis and –80 ◦C for the molecular analysis.

**Figure 1.** The map showing the collection sites of three accessions (Wolchul, Odae, and Jiri mountains) of *Thymus quinquecostatus* in South Korea.

## *2.2. Morphological Characteristics*

The morphological parameters such as stem type, stem branch, stem color, leaf shape, number of auxiliary leaves, and trichome position were observed for the six commercial and three Korean native thyme cultivars.

#### *2.3. Essential Oil Extraction*

The essential oil from nine thyme samples was isolated by steam distillation, using a Clevenger-type apparatus. The steam distillation was performed at 100 ◦C for 90 min. The essential oil isolation was carried out in triplicates and the yield (%) was calculated as volume (mL) of the isolated oil per 100 g of the fresh plant material. The isolated essential oil was dried using anhydrous sodium sulfate and stored at 4 ◦C, until tested. The color of essential oils obtained from the three Korean native *T. quinquecostatus* cultivars was measured, using the Chromameter CT-300 (Mintola Camera Co. Ltd., Japan). The intensity of the color was expressed in terms of *L*\* lightness, *a*\* greenness, and *b*\* yellowness. The color values of *L*\*, *a*\*, and *b*\* were taken in triplicates for each sample.

**Figure 2.** The morphology of six commercial *Thymus vulgaris* cultivars and three Korean native *Thymus quinquecostatus* cultivars. (1) Lemon; (2) golden; (3) carpet; (4) orange; (5) silver; (6) creeping; (7) Odae Mt.; (8) Wolchul Mt.; and (9) Jiri Mt.

#### *2.4. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis*

The identification of the essential oil components from different thyme cultivars was performed using a Varian CP3800 gas chromatograph coupled with a Varian 1200 L mass detector (Varian, CA, USA). The GC–MS was equipped with a VF-5MS polydimethylsiloxane capillary column (30 m × 0.25 mm × 0.25 μm). The oven temperature was programmed from 50 ◦C to 250 ◦C, at a rate of 5 ◦C/min. The injector temperature was 250 ◦C and the ionization detector temperature was 200 ◦C. Helium was the carrier gas (1 mL/min) and the injected volume of the sample was 2 μL, with a split ratio of 10:1. For mass spectra, an electron ionization system with ionization energy of 70 eV was used. The mass range was 50–500 m/z. The determination of the percentage composition of each component was based on the normalization of the GC peak areas. The identification of the essential oil components was based on the comparison of their retention indices (RIs), relative to a homologous series of *n*-alkanes (C8–C22) and mass spectra from the National Institute of Standards and Technology (NIST, 3.0) library and literature data [22].

#### *2.5. DNA Extraction*

The total genomic DNA was isolated from one gram of young leaves of plants, according to the CTAB (cetyl trimethylammonium bromide) extraction method [15]. DNA pellets were dissolved in TE (Tris–EDTA) buffer and RNA was removed by digestion with DNase-free RNase A. The purified total DNA was quantified and its quality was verified using a spectrophotometer, and a diluted solution with the same concentration (10 ng/μL) was prepared by adding TE buffer and was stored at 4 ◦C.

#### *2.6. Randomly Amplified Polymorphic DNA (RAPD) Analysis*

A total of 16 primers (OPA-09, OPA-10, OPA-11, OPA-12, OPA-13, OPA-14, OPA-15, OPA-16, OPA-17, OPA-18, OPA-19, OPA-20, OPB-01, OPB-02, OPB-03, and OPB-04) were used for the RAPD analysis (Table 1). The selection of primers was based on high polymorphisms and good reproducibility of the fragments generated. RAPD amplification was performed in a volume of 25 μL containing 10 ng total DNA, 1× PCR buffer, 3.0 mM MgCl2, 200 μM deoxynucleotide triphosphates (dNTPs), 1 μM primer, 1 μg/mL (w/v) Bovine Serum Albumin (BSA), and 1 unit *Taq* DNA polymerase (Invitrogen). The amplification reactions were performed in a thermocycler and consisted of an initial 5 min denaturation step at 95 ◦C, followed by 40 cycles of 20 s at 95 ◦C, 40 s at 35 ◦C, and 60 s at 72 ◦C. A final extension of 5 min at 72 ◦C completed the amplification. The PCR products were separated in 1.2% agarose gels 1× TAE buffer (Tris–Acetate). The gels were stained with ethidium bromide, visualized with a UV transilluminator.

**Table 1.** The names and sequences of the primers used for random amplified polymorphic DNA (RAPD) analysis.


#### *2.7. Statistical Analysis*

To calculate RAPD polymorphism, the RAPD markers were scored for the presence (1) or absence (0) of amplified bands for 9 thyme cultivars. Genetic similarity was estimated using the Jaccard's coefficients. Cluster analysis was performed using the unweighted pair group method with an arithmetic mean (UPGMA), and dendrograms were drawn using NTSYS software version 2.02.
