Morphological, Chemical, and Genetic Characteristics of Korean Native Thyme Bak-Ri-Hyang (Thymus quinquecostatus Celak.)

Bak-ri-hyang (Thymus quinquecostatus Celak.) is an important medicinal and aromatic plant in Korea. T. quinquecostatus population and is always mixed with other thyme cultivars during cultivation and marketing. Hence, this study aimed to determine the genetic variability and the essential oil composition of three Korean native thyme, T. quinquecostatus cultivars collected from the Wolchul, Jiri, and Odae mountains, in comparison with six commercial thyme cultivars (T. vulgaris), to distinguish Bak-ri-hyang from other thyme cultivars. The composition of essential oils obtained from nine individuals was analyzed by gas chromatography–mass spectrometry (GC–MS). The random amplified polymorphic DNA (RAPD) analysis was accomplished using 16 different primers. The GC–MS analysis revealed that Wolchul, creeping, golden, and orange cultivars belong to the geraniol chemotype. Whereas the Odae, lemon, and silver cultivars belong to the thymol chemotype. Further, linalool was the most abundant component in carpet and Jiri cultivars. The RAPD analysis demonstrated that all thyme cultivars showed characteristic RAPD patterns that allowed their identification. In total, 133 bands were obtained using 16 primers, and 124 bands were polymorphic, corresponding to 93.2% polymorphism. Cluster analysis of RAPD markers established the presence of clear separation from nine thyme cultivars. The highest dissimilarity and similarity coefficient of the RAPD markers were 0.58 and 0.98, respectively. According to the RAPD patterns, the nine thyme cultivars could be divided into two major clusters. Among three Korean cultivars, the Wolchul and Odae cultivars were placed into the same cluster, but they did not show identical clustering with their essential oil compositions. The findings of the present study suggest that RAPD analysis can be a useful tool for marker-assisted identification of T. quinquecostatus from other Thymus species.


Introduction
The genus Thymus (Lamiaceae) consists of approximately 300 species of herbaceous perennials and sub-shrubs, distributed throughout the world and predominantly found in the Mediterranean basin [1,2]. They are widely used as spices, herbal tea, and insecticide in addition to flavor and fragrance materials. Among these, Thymus quinquecostatus (Bak-ri-hyang) is a scrubby subshrub and an important aromatic plant in Korea. Two varieties of T. quinquecostatus such as T. quinquecostatus Celak and T. quinquecostatus var. japonica are found in Korea [3]. In traditional systems of medicine, T. quinquecostatus is used for the treatment of cough, inflammation, preventing excessive intestinal gas, and diaphoresis [3][4][5].
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 ( Figure 1 and Figure 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 (

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.

Essential Oil Extraction
The essential oil from nine thyme samples was isolated by steam distillation, using a Clevengertype 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.

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.

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., Antibiotics 2020, 9, 289 4 of 16 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.

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 nalkanes (C8-C22) and mass spectra from the National Institute of Standards and Technology (NIST, 3.0) library and literature data [22].

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.

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 (C 8 -C 22 ) and mass spectra from the National Institute of Standards and Technology (NIST, 3.0) library and literature data [22].

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.  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 Antibiotics 2020, 9, 289 5 of 16 total DNA, 1× PCR buffer, 3.0 mM MgCl 2 , 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.

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.

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.

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 Antibiotics 2020, 9, 289 6 of 16 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.

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][17][18][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).     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.

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 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.
(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).    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. 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.

Discussion
The identification of the Thymus species is extremely difficult because of the high levels of diversity within the genus. This genus contains several commercially important aromatic species. For this purpose, the relationship among the chemical composition of essential oils and molecular analysis was carried out for different Thymus species [20,23]. In this context, the essential oil composition and molecular analysis of nine thyme cultivars were investigated in this study, to distinguish between commercial thyme cultivars and Korean native thyme cultivars. In the morphological study, the T. quinquecostatus and T. vulgaris cultivars exhibited a significant level of variability in recorded parameters. In the qualitative traits, a considerable variability was observed in stem type, stem color, length and number of stem branches, leaf shape, and trichome position, among and within T. quinquecostatus and T. vulgaris cultivars.
The present study showed a high chemical diversity among nine thyme cultivars. Results revealed that essential oils from Korean cultivars (T. quinquecostatus) belonged to the geraniol, thymol, and linalool chemotypes. Essential oils from the commercial thyme cultivars (T. vulgaris) such as creeping, golden, and orange belonged to the geraniol chemotype and lemon, and the silver cultivars belonged to the thymol chemotype. Further, carpet cultivar belonged to the linalool chemotype. In particular, these essential oils were dominated by monoterpenes. 1-Octen-3-ol, γterpinene, linalool, borneol, α-terpineol, nerol, geraniol, thymol, β-cubebene, β-elemene, caryophyllene, β-bisabolene, butylated hydroxytoluene, β-sesquiphellandrene, and caryophyllene oxide were detected in all six essential oils from the commercial cultivars. With regards to the chemical composition of T. vugaris essential oils, seven different chemotypes such as thymol,

Discussion
The identification of the Thymus species is extremely difficult because of the high levels of diversity within the genus. This genus contains several commercially important aromatic species. For this purpose, the relationship among the chemical composition of essential oils and molecular analysis was carried out for different Thymus species [20,23]. In this context, the essential oil composition and molecular analysis of nine thyme cultivars were investigated in this study, to distinguish between commercial thyme cultivars and Korean native thyme cultivars. In the morphological study, the T. quinquecostatus and T. vulgaris cultivars exhibited a significant level of variability in recorded parameters. In the qualitative traits, a considerable variability was observed in stem type, stem color, length and number of stem branches, leaf shape, and trichome position, among and within T. quinquecostatus and T. vulgaris cultivars.
Hudaib and Aburjai [24] determined variations in the composition of essential oils from cultivated and wild-growing plants of T. vulgaris grown in Jordan. Higher oil yields were obtained in plants growing wild, when compared to the cultivated plants. Among the four different samples, thymol (0.8-63.8%) and carvacrol (6.9-86.1%) were the most abundant components in the T. vulgaris essential oils. A study indicated that the essential oil composition of T. vulgaris highly varied both qualitatively and quantitatively during the vegetative cycle [25]. The variations in the yield and composition of essential oils could be influenced by various factors, such as the geographical region of the plant, plant's maturity, cultivation practices, and weather parameters (temperature, humidity, sunlight duration, and rainfall) [26][27][28]. In addition, the genetic constitution of the cultivars also played a considerable role in the essential oil composition [1,25].
According to previous reports, it is difficult to distinguish Thymus species and cultivars by analyzing the essential oil profile alone. Hence, the combined analysis of chemical composition and molecular techniques was used for the correct identification of the different plant species. In recent decades, the correlation between the chemical composition and molecular analysis of different Thymus species were investigated by various researchers [1,13,20,21]. Previous studies showed that both essential oil composition and RAPD analysis could be used to distinguish different thyme cultivars, and especially, to determine their relationships [1]. In addition, RAPD analysis revealed high polymorphisms even when using closely related genotypes. Even though the essential oil composition of plants was different from one another, RAPD analysis clustered these plants together, owing to their similar genetic background [15].
In the present study, 16 primers were used to amplify segments of DNA of the genome of three Korean thyme cultivars and six commercial thyme cultivars, to investigate the genetic variations. A total of 133 bands were obtained and the average percentage of the polymorphic bands was 93.23%. Based on the RAPD data, the similarity of the cultivars, estimated by the Jaccard's coefficient, is depicted in Figure 5. The nine cultivars of thyme fell into two clusters. Cluster 1 was formed by six cultivars (lemon, golden, creeping, silver, carpet, and Jiri) and cluster 2 by three cultivars (orange, Wolchul, and Odae). This emphasized the obvious variation between the Korean cultivars (except Jiri cultivar) and the commercial cultivars. The dendrogram indicated a clear separation of T. quinquecostatus from T. vulgaris, with the exception of the Jiri cultivar. According to the RAPD similarity matrix, it was observed that the Wolchul and Odae cultivars were closely related. Nevertheless, there was no significant relationship between the essential oil composition and RAPD data. The ability to discriminate all studied cultivars using RAPD bands indicated that RAPD analysis can provide a rapid and inexpensive technique to identify phenotypically similar thyme cultivars.
Based on previous reports, a high correlation between genetic and chemical relationships was attained in several plants. These data indicated that the composition of the essential oil is regulated by a number of genes that are extensively distributed throughout the plant genome [1,29,30]. Khalil et al. [31] used RAPD analysis to determine the genetic relationship between T. vulgaris populations collected in Syria. In their study, 13 individuals were analyzed using 27 primers, which generated 180 polymorphic bands from 198 bands. The authors found a significant correlation between T. vulgaris populations and their geographic areas. The present study also proved that the geographic distribution had a significant influence on genetic variation. Comparing the groups formed by the cluster analysis based on RAPD data ( Figure 5) and chemotype, based on essential oil composition, we can observe that the groups formed in both cases were not identical.
In another study, the composition of essential oils and genetic relationships between six commercial cultivars of T. vulgaris were analyzed. A total of 104 were polymorphic RAPD bands (63.8%) were obtained using 15 primers. Among 15 primers, the highest percentage of polymorphism was obtained by the OPA-05 primer (90.9%). Similar to the essential oil composition, the six T. vulgaris cultivars fell into two major clusters, according to the RAPD patterns, with a correlation coefficient of −0.779 [1].
The chemical and genetic variations of 20 taxa from four Hungarian Thymus species (T. glabrescens, T. pannonicus, T. praecox, and T. pulegioides) were studied by Pluhár et al. [23]. In the molecular analysis, 114 polymorphic RAPD bands (80.8%) were obtained using 13 primers. The results revealed that partial correlation was found between the essential oil and RAPD analyses. The essential oil composition and genetic variation in six micropropagated genotypes (in vitro and in vivo) of T. saturejoides were investigated by Nordine et al. [32]. RAPD results and the essential oil composition grouped these six genotypes into three clusters exhibiting significant intraspecific chemical and genetic differences. Furthermore, a significant correlation was observed between RAPD and essential oil composition obtained from the in vitro genotypes.
Similar to our report, several studies also reported that the combined use of RAPD and essential oil analyses were not significantly correlated. For example, the genetic and chemical relationships among 31 individuals of T. caespititius collected from the islands of Pico, Sao Jorge, and Terceira (Azores) were determined. In the RAPD analysis, 187 polymorphic bands were obtained using 17 primers. However, there was no close relationship between the collection site, the essential oil composition, and RAPD analysis [15]. Rustaiee et al. [20] also studied the essential oil composition and genetic variability between some Thymus species such as T. daenensis (two populations), T. fallax, T. fedtschenkoi, T. migricus, and T. vulgaris, using GC-MS and RAPD. Although the RAPD markers allowed a perfect distinction among different Thymus species according to their characteristic genetic background, there was no identical clustering with the essential oil composition. In addition, Masi et al. [33] found that the essential oil compositions did not match with the results achieved from agronomic and genetic analyses in Ocimum basilicum. In another study, there was no correlation between RAPD and the essential oil obtained from the in vivo genotypes of T. saturejoides [32]. Based on the previous and present studies, marker-assisted RAPD technique had a high advantage for the assessment of the genetic differences of plant species without prior molecular knowledge.
Results of the present study revealed that there was a significant correlation between the genetic and geographic distances of the Korean thyme cultivars (Wolchul and Odae cultivars), compared to the commercial thyme cultivars. However, the chemical polymorphism of these thyme cultivars is not well-understood. Hence, other molecular techniques should be investigated in order to understand this question in T. quinquecostatus and other Thymus cultivars.

Conclusions
The present study emphasized that RAPD analysis allowed a perfect distinction between the Korean thyme cultivars (Wolchul and Odae) and commercial thyme cultivars, based on their unique genetic background. However, the chemical composition of the Wolchul and Odae cultivars was not identical. Furthermore, there was no significant relationship between the RAPD data and essential oil composition of both T. quinquecostatus and T. vulgaris cultivars. The chemical composition and molecular data obtained in this study delivered a good starting point for future investigations. It could be concluded that the RAPD markers proved to be an effective tool for discriminating different Thymus species. The sample collection must be done from different geographical regions in Korea to understand the genetic and chemical variability of the T. quinquecostatus cultivars.