**3. Results**

#### *3.1. Morphological Characteristics of the Thyme Cultivars*

The morphological characteristics of six commercial and three Korean native thyme cultivars are presented in Table 2. In these, all three *T. quinquecostatus* cultivars had a creeping type of stem. On the other hand, lemon, golden, orange, and silver cultivars possessed an erect stem type. The length of the stem branch varied among different cultivars. In the creeping stem type, the length of the stem branch ranged from 2 to 8 cm, whereas the length of the stem branch in the erect type ranged from 3 to 11 cm. Carpet cultivar possessed a higher number of stem branches than the other cultivars. The shape of the leaves was mainly oval, followed by oblanceolate. In the case of the Bak-ri-hyang cultivars, the Odae and Jiri cultivars had an oval shape of leaves. The leaf shape of the Wolchul cultivar was oblanceolate. Furthermore, the Bak-ri-hyang cultivars possessed a higher number of auxiliary leaves when compared with the commercial thyme cultivars. The trichome position was mainly observed at the leaf petiole. In the Wolchul cultivar, the trichome position was observed at the leaf margin.

#### *3.2. The Chemical Composition of Essential Oils*

The yield and chemical composition of essentials oils obtained from the nine thyme cultivars are presented in Tables 3 and 4. The essential oil components and their concentration produced by thyme cultivars were very diverse. The essential oil yields ranged between 0.12% and 0.43% (v/w) for the *T. quinquecostatus* cultivars. The highest yield was obtained from the Odae cultivar (0.43%). For commercial cultivars of *T. vulgaris*, the essential oil yields ranged from 0.23% to 0.33%. In these, the highest yields were obtained from the lemon and silver cultivars (0.34% and 0.33%, respectively), and the lowest yield was obtained from the carpet cultivar (0.23%). The color profile of the essential oils obtained from the *T. quinquecostatus* cultivars was measured. The *L*\* value of the essential oils of the Wolchul, Odae, and Jiri cultivars was 92.48, 92.54, and 92.39, respectively. Wolchul and Jiri cultivars possessed similar *a*\* value (0.18). With regards to the *b*\* value, the Wolchul cultivar showed the highest value (2.29) and the Odae cultivar showed the lowest value (1.89). The total number of components in the analyzed essential oils ranged between 32 (creeping cultivar) and 43 (lemon cultivar). In these nine samples, twelve compounds were detected in all essential oil samples and these oils were dominated by monoterpenes, accounting for 79.95–92.16% with 0.03–46.47% of monoterpene hydrocarbons and 43.86–88.46% of oxygenated monoterpenes. Whereas sesquiterpenes achieved 6.50–29.17% with 5.83–15.07% of sesquiterpene hydrocarbons and 0.32%–17.23% of oxygenated sesquiterpenes.

Geraniol, geranyl acetate, linalool, phenylethyl alcohol, γ-terpinene, and thymol were detected as the most abundant components, which comprised more than 20% in at least one essential oil (Figure 3). Results of the essential oil composition revealed that all three *T. quinquecostatus* cultivars tested belonged to di fferent chemotypes (Supplementary Figure S1). Cultivars of Wolchul, Odae, and Jiri were mainly composed of geraniol (42.94%), thymol (30.54%), and linalool (47.89%), respectively. In the case of the commercial cultivars of *T. vulgaris*, the lemon and silver cultivars belonged to the thymol chemotype (43.91% and 66.24%, respectively). On the other hand, the creeping, golden, and orange cultivars belonged to the geraniol chemotype (29.57%, 65.99%, and 44.70%, respectively). With regards to the carpet cultivar, linalool (48.16%) was recorded as the most abundant component. Furthermore, geranyl acetate was detected as a major component in the Wolchul (26.49%) and silver (29.86%) cultivars. γ-Terpinene (23.92%) and *p*-cymene (11.13%) were major components in the Odae cultivar. In the creeping cultivar, neral (11.75%) and geranial (18.21%) were also recorded as major components. Other important compounds detected in all essential oils were caryophyllene (2.87%–7.02%), borneol (0.41%–5.91%), β-bisabolene (0.23%–3.86%), and 1-octen-3-ol (0.39%–3.61%). α-Elemol (11.62% and 8.52%, respectively) was also recorded as a major component in the carpet and creeping cultivars.

## *3.3. RAPD Analysis*

The molecular analysis revealed that the RAPD primers produced clear and reproducible polymorphic bands (Figure 4) among 9 thyme cultivars, and generated a total of 133 amplicons from 16 primers. The number of bands per primer varied from 4 (OPA-12, OPA-14, and OPB-04) to 16 (OPA-19), with an average of 8.31 bands per primer. In these, 124 amplicons were polymorphic, corresponding to 93.23% polymorphism (Table 5). Eight primers gave the highest percentage of polymorphism (100%), while the lowest percentage of polymorphism (75%) was obtained by OPA-12 and OPB-04 primers (Table 5).


**Table 2.** Morphological characteristics of six commercial *Thymus vulgaris* cultivars and three Korean native *Thymus quinquecostatus* cultivars.

**Table 3.** The yield and color of the essential oils isolated from the six commercial *Thymus vulgaris* cultivars and the three Korean native *Thymus quinquecostatus* cultivars.


**Table 4.** The chemical composition of essential oils isolated from the six commercial *Thymus vulgaris* cultivars and the three Korean native *Thymus quinquecostatus* cultivars.



138

**Table 4.** *Cont.*

*Antibiotics* **2020**, *9*, 289


**Table 4.** *Cont.*

mean of the three replicate determinations ± standard deviation.

#### *Antibiotics* **2020**, *9*, 289

**Figure 3.** Structure of the major components identified in the essential oils of commercial *Thymus vulgaris* cultivars and Korean native *Thymus quinquecostatus* cultivars. Geraniol, geranyl acetate, linalool, phenylethyl alcohol, γ-terpinene, and thymol were identified as the major components, comprising>20% in at least one of the essential oil obtained from the different cultivars.

**Figure 4.** The example of a pattern among the three Korean native *Thymus quinquecostatus* cultivars and the six commercial *Thymus vulgaris* cultivars using OPB-01 (top) and OPA-11 (bottom) primers separated in 1.2% agarose gel electrophoresis. M, PCR marker; 1–6, commercial *Thymus vulgaris* cultivars: (1, lemon; 2, golden; 3, carpet; 4, orange; 5, silver; and 6, creeping); 7–9, Korean native *Thymus quinquecostatus* cultivars (7, Odae Mt; 8, Wolchul; 9, Jiri).


**Table 5.** Bands and polymorphism revealed by the RAPD primers among the 9 *Thymus* cultivars.

The dendrogram realized from the RAPD markers grouped the 9 thyme cultivars into two major clusters and showed a clear separation (Figure 5). Levels of genetic similarity indices ranged from

0.58 to 0.98. Cluster 1 consisted of lemon, golden, creeping, silver, carpet, and Jiri. Whereas cluster 2 consisted of orange, Wolchul, and Odae.

**Figure 5.** Clustering tree of the three *Thymus quinquecostatus* cultivars and the six commercial *Thymus vulgaris* cultivars, based on the unweighted pair-group method with the arithmetic average (UPGMA), using 16 RAPD markers.
