Journal of Applied Botany and Food Quality 93, 186 - 196 (2020), DOI:10.5073/JABFQ.2020.093.023 1Department of Horticultural Science, Faculty of Agricultural Science and Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran 2Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran 3Julius Kühn-Institut, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Berlin, Germany Morphological and phytochemical screening of some Thymus ecotypes (Thymus spp.) native to Iran in order to select elite genotypes Siavash Mohammadi1, Leila Tabrizi1*, Majid Shokrpour1, Javad Hadian2, Hartwig Schulz3, David Riewe3 (Submitted: May 14, 2020; Accepted: September 11, 2020) * Corresponding author Summary Thymus spp. is one of the most important medicinal plants widely used in food, pharmaceutical, and cosmetic industries. In this research, different ecotypes of three Thymus species including T. daenensis, T. kotschyanus and T. lancifolius native to Iran were compared to two commercial cultivars of T. vulgaris (i.e. 'Varico 3' and 'Deutscher Winter') under identical conditions. Based on the results, there was a remarkable diversity among different ecotypes of Thymus species. The highest plant dry weight was found in T. daenensis (Malayer 2), T. kotschyanus (Azerbaijan gharbi), and T. lancifolius (Fars). The highest thymol percentage (> 75%) was obtained by T. daenensis. The ecotype of Ilam belonging to T. daenensis gained highest essential oil percentage (7.83%). In all ecotypes of T. daenensis, thymol was the major constituent in their essential oil. Five chemotypes of citral, carvacrol-thymol, thymol-carvacrol, p-cymene-carvacrol, and geranyl acetate-citral were found in T. kotschyanus ecotypes, while four chemotypes of thymol, α-terpineol-linalool, carvacrol-thymol and thymol- geraniol were identified for T. lancifolius. In addition, in terms of growth, yield, and phytochemical traits, the elite genotypes within ecotypes were selected. Elite ecotypes and genotypes detected during this research could be used in Thymus breeding programs. Keywords: Chemotype, Essential oil, Medicinal plants breeding, Thymol Introduction Thymus species, belonging to the family Lamiaceae, includes more than 215 species in the world, in which 14 species are native to Iran, while 4 species are endemic to Iran including T. persicus, T. carmanicus, T. daenensis, and T. trautvetteri (Jamzad, 2009; Safaei- Ghomi et al., 2009; amiri, 2012). Essential oil of Thymus species has many applications in different industries such as pharmaceutical, food, sanitary, cosmetic and others, due to its antibacterial, antifungal and antioxidant properties, thereby occupying the top position in world trade of medicinal plants (alizadeh et al., 2013; moJab et al., 2008; raSooli and mirmoStafa, 2002). In Thymus species, the major constituents of its essential oil include thymol, carvacrol, and p-cymene (tohidi et al., 2019). In addition to its essential oil, its aerial parts containing tannin, flavonoids, saponin and bitter materials have been received great attention. Thyme is used as an expectorant for curing cough, sore throat, bronchi, and asthma (Petrović et al., 2017; edeoGa et al., 2005). Regarding the application of Thymus in different industries, the breeding and creating thyme genotypes with high qualitative and quantitative yield is imperative. The main breeding objective of Thymus species is to produce plants with higher yield of dry matter, ratio of leaf/shoot, essential oil, thymol content, crop uniformity, freezing tolerance in Winter, upright form of growth, and finally possibility of mechanizing harvesting (Carlen et al., 2010). In this regard, the simplest way for thyme breeding is evaluation of their populations and then selecting the elite genotypes based on their growth and chemotype traits (hadian et al., 2016; heydari et al., 2019). Approximately, 60 to 75% of current medicinal crops have been produced through selecting elite genotypes among wild or landraces populations (bernat, 2000; nemet, 2000). In terms of dry matter yield, essential oil content, and chemical composition, thyme with higher qualitative traits and uniformity, are highly respected. Progress in plant breeding requires an extended genetic pool (rahim malek et al., 2009). Awareness of genetic diversity in crops and their wild relatives is an underlying requirement to improve crop yield (GovindaraJ et al., 2015). There are basic necessities for achieving high amount of bioactive compounds and their homogeneity in pharmaceutically important plants, such as sufficient genetic diversity in populations for creating favorable traits, appropriate genotypes for different cultivation systems, high harvest index, and low sensitivity to diseases and climate perturbations (denG et al., 2019; iSlam et al., 2019; denG et al., 2019). Therefore, progress in breeding being in line with the mentioned requirements depends on genetic diversity created by plant breeders (rahim malek et al., 2009). Thyme usually possesses high genetic diversity in terms of morpho- logical and phytochemical traits (imbrea et al., 2010). According to loapez-puJol et al. (2004), higher genetic diversity was found in different ecotypes of T. loscosil; this was attributed to its polyploidy, resulted naturally from its heterozygosity as well as its biochemical and genetic diversities. Also, the study of morphological diversity of 5 T. glabrescens ecotypes, showed a significant difference in leaf length and width as well as number of leaf secreting trichomes; which are useful traits in thyme breeding programs (daJiC-StevanoviC, 2008). hadian et al. (2016) evaluated morphological and essential oil of 6 populations of T. daenensis on their natural habitats, and found a remarkable morphological diversity between the populations; the content of essential oil in these ecotypes varied from 1.53 to 4.28%, with the highest essential oil content in Ilam ecotype. However, major components of essential oil in different ecotypes of T. kotschy- anus ranged from 1 to 1.41%, and the amount of main components in their essential oil was as follows: thymol (31.2%), carvacrol (19.5%), linalool (2.38-20.06%), alpha-terpineol (0.16-11.64%), and geraniol (0.36-11.37%) (tohidi et al., 2018). Mostly, breeding thyme (especially those native to Iran) has been focused on selecting naturally ecotypes and elite genotypes on the basis of morphological, phytochemical, and genetic traits. Compared to breeding of agricultural crops, there is limited progress in breed- ing of medicinal plants. Therefore, the present study aimed to evalu- ate different ecotypes of three Thymus species (i.e. T. daenensis, Screening of Thymus spp. ecotypes for selection of elite genotypes 187 T. kotschyanus, and T. lancifolius) under field condition and also their comparison with two commercial cultivars of T. vulgaris (i.e. 'Varico 3' and 'Deutscher Winter') under identical conditions in order to select elite genotypes and ecotypes qualified with high qualitative and quantitative yield for thyme breeding and cultivation goals. Materials and methods Plant materials The seeds of different ecotypes of Thymus spp., provided by differ- ent places including Research Institute of Forests and Rangelands (RIFR), Medicinal Plants and Drugs Research Institute of Shahid Beheshti University (MDRI-SUB) and Department of Horticultural Science and Landscape Engineering, University of Tehran (HSD- UT) (Tab. 1), were planted in February 2013 in plant growing trays, containing peat moss, grown in the research greenhouse of HSD- UT and irrigated regularly. In May 2013, the seedlings of studied ecotypes of different Thymus spp. such as T. daenensis (12 eco- types), T. kotschyanus (5 ecotypes) and T. lancifolius (5 ecotypes) along with two commercial cultivars of T. vulgaris ('Varico 3' and 'Deutscher Winter') (Tab. 1) were transferred into an experimental field located in Research Station of HSD-UT (located at 35° 46´ N; 50° 55´ E, at an altitude of 1320 m.a.s.l.). The experiment was laid out in Randomized Complete Block Design (RCBD) with three rep- lications. The inter- and intra-planting spaces were 60 and 30 cm, respectively. Each ecotype entailed three blocks containing 16 plants (3×16=48 plants). After transferring transplants into the field, they were irrigated using drip system and weed control was carried out in spring and summer seasons. The average day and night temperatures during growing season were 26.14 °C and 19.92 °C, respectively and precipitation average was 5.47 mm. Physico-chemical characteristics of the soil of field used in this study were: texture: Clay Loam, pH: 8.7, electrical conductivity (EC): 1.3 dS/ m, organic matter: 0.64%, nitrogen (N): 0.07%, Phosphorus (P): 1.03 mg/kg and Potassium (K): 350 mg/kg. Assessment of growth and yield traits As thyme is a perennial herbal plant, its growth and yield perfor- mance is not considerable in first year of cultivation. Therefore, they were solely maintained during first year and then in the second year, their growth and yield traits were evaluated at full flowering stage (in spring and summer of 2014). In our research, morphological traits consisted of plant height, number of lateral shoot per plant, internode length, internode number, main fluorescence’s length, leaf length, leaf width, plant form (from 1, as recumbent to 5, as upright plants), shoot dry weight, leaf dry weight, ratio of leaf/shoot, and percentage of male sterile plants. After measuring growth traits, the plants were harvested at full flowering stage and then dried in shade at room temperature in order to perform phytochemical analysis of essential oil including its content, yield, and constituents. Essential oil extraction and analysis After drying, at room temperature, essential oil was extracted us- ing hydrodistillation method for 3 h with a Clevenger type apparatus based on britiSh pharmaCopoeia (1988) and then, was kept in a vial at 4 °C until to be analysed. Analysis of essential oil was car- ried out using Gas Chromatography/Mass Spectrometry (GC/MS) and Gas Chromatography/Flame Ionization Detector (GC/FID) in Phytochemistry Department of Julius Kühn Institut (JKI), Institute for Ecological Chemistry, Plant Analysis and Stored Product Pro- tection, Berlin, Germany. Afterwards, essential oil of different eco- types was injected to GC/MS (Agilent, MSD 5972/HP) and GC/FID, in which, theGC/MS was equipped with a column of MS-5 HP (30 m × 0.25 mm i.d. and film thickness 0.5 μm). The column temperature was set to start with 60-120 °C at 3 °C/min and then it was held with 220 °C at 8 °C/min and it held for 10 min at this temperature. The temperature of injector was 250 °C. Helium was used as a carrier gas (1.0 mL min-1). Mass Spectrometry operates in electronic ioni- zation mode at 70eV and calculation was carried out using Chem- station software. The detection of Spectrometry was carried out us- ing retention index and resulted data were compared with the indexes found in reference books (adamS, 2017) and information found in computer library. The system of GC/FID (Agilent, N HP6890) was equipped with a column MS-5 HP (30 m × 0.25 mm i.d. and film thickness 0.5 μm). The program of column temperature was set from 60-220 °C at 8 °C/min and then held for 10 min at this temperature. Also, the temperature of injector was 250 °C and helium was used as a carrier gas (1.0 mL min-1). Data analysis The ANOVA was made based on RCBD and the means were com- pared by Duncan’s Multiple Range Test (DMRT) at 5% probability level. The analyses were done using SPSS (Ver. 20) and the plotting of graphs was carried out using Excel 2013. Tab. 1: Plant materials and seed collection origins of thyme samples. Species Ecotype name Origin in Iran Species Ecotype name Origin in Iran (province) (province) 1. Khan-e-miran Markazi 1. Tehran Tehran 2. Lorestan Lorestan 2. Zanjan Zanjan 3. Isfahan 1 Isfahan T. kotschyanus 3. Ghazvin Ghazvin 4. Ilam Ilam 4. Az. Gharbi West Azarbaijan 5. Malayer 1 Hamedan 5. Kordestan Kordestan T. daenensis 6. Arak Markazi 1. Lorestan Lorestan 7. Zagheh Lorestan 2. Kordestan Kordestan 8. Malayer 2 Hamedan T. lancifolius 3. Isfahan Isfahan 9. Fars Fars 4. Fars Fars 10. Isfahan 2 Isfahan 5. Markazi Markazi 11. Markazi 1 Markazi 1. 'Deutscher Winter' Germany 12. Markazi 2 Markazi T. vulgaris 2. Varico 3 Switzerland 3. Isfahan Isfahan 188 S. Mohammadi, L. Tabrizi, M. Shokrpour, J. Hadian, H. Schulz, D. Riewe Results and discussion Yield and morphological traits The Analysis of variance (ANOVA) results of morphological traits in thyme species and ecotypes are shown in Tab. 2. As shown in Tab. 3, the highest plant height was found in T. vul- garis (39.4 cm on average) that was significantly higher than those in other species (i.e. T. daenensis, T. kotschyanus, and T. lancifolius). In T. vulgaris, 'Varico 3' gained the highest plant height (43.6 cm) com- pared to 'Deutscher Winter' and Isfahan ecotype. Carlen et al. (2010) substantiated the superiority of 'Varico 3' over 'Deutscher Winter' in terms of plant height. In other studies, it has been confirmed that the height of T. vulgaris ecotypes were taller than T. daenensis eco- types (Kaveh et al., 2013; MondaK et al., 2014; asKari et al., 2018; MohaMMadi et al., 2019). In T. daenensis, the highest plant height (33 cm) was in Markazi 1 which did not show a significant difference with Markazi 2 and Malayer 1. Likewise, the highest plant height in T. kotschyanus and T. lancifolius was observed in Azerbaijan gharbi (18.6 cm) and Markazi (22.5 cm), respectively (Tab. 4). Our findings are in agreement with those obtained by Kaveh (2013) who con- cluded that Azerbaijan gharbi is the tallest plant among T. kotschya- nus ecotypes. The height of plants is important because of its role in mechanizing and facilitating harvest activities. Also, the highest biomass yield was obtained by 'Varico 3' and Azerbaijan gharbi eco- type, possessing highest plant height. According to results of previ- ous studies carried out on T. pubescens (Yavari et al., 2010) and different Thymus species native to Iran (MondaK et al., 2014), there is a positive relationship between plant height and plant dry weight, and this is in agreement with our findings. In thyme, number of shoots per plant is attributed effectively in yield performance. Among Thymus species evaluated in this research, the highest shoot number (163.2) was observed in T. vulgaris that sig- nificantly higher than T. daenensis (8.5), T. kotschyanus (91.2), and T. lancifolius (85.6) (Tab. 3). Also, between ecotypes of the three last-mentioned Thymus species, no significant difference was found between them in terms of shoot number (Tab 3). The number of shoot in the mentioned species was as follows: T. daenensis (Ilam ecotype, 97), T. kotschyanus (Azerbaijan gharbi, 120), and T. lancifolius (Kurdistan, 95) (Tab. 4). The highest internode length (2.4 cm) belonged to T. daenensis which was significantly higher than that in the other three species. Also, there was no significant difference in terms of internode length between T. vulgaris, T. kotschyanus, and T. lancifolius (Tab. 3). In T. daenensis, the highest (2.93 cm) and lowest (1.27 cm) internode length were observed in Markazi 1 and Ilam, respectively (Tab. 4). While in T. vulgaris, the highest internode length (2.3 cm) belonged to 'Varico 3' which significantly was higher than 'Deutscher Winter' and Isfahan (with no significant difference between the last two ecotypes); (Tab. 4). Also, the highest internode length in T. kotschya- nus (2 cm) and T. lancifolius (1.83 cm) were belonged to Tehran and Isfahan, respectively (Tab. 4). T. vulgaris showed the highest internode number (15.9) that signifi- cantly differs from that in the other three species, although the differ- ence between these three thyme species was not significant (Tab. 3). The highest internode number (10.7) in T. daenensis was allocated to Ilam, being significantly higher than other ecotypes belonged to this species. Likewise, the highest internode number (16.5) was found in 'Deutscher Winter' that significantly longer than 'Varico 3' (14.7), but did not significant difference with Isfahan (Tab. 4). Azerbaijan gharbi (9.6) and Kurdistan (7.6) gained the highest internode number in T. kotschyanus and T. lancifolius ecotypes, respectively (Tab. 4). Of all Thymus species investigated in this research, the highest flo- rescence length (3.6 cm) was found in T. daenensis which higher than T. kotschyanus and T. lancifolius, whereas it did not have a signifi- cant difference with T. vulgaris (Tab. 3). In T. daenensis, the longest florescence (5.5 cm) was in Markazi 1 which did not have significant difference with Markazi 2 (Tab. 4). Tab. 2: Analysis of variance (ANOVA) of morphological traits in thyme species and ecotypes. Sources df ANOVA of variance Mean of the squares (MS) (S.O.V) Traits Plant Lateral Internode Internode Inflores- Leaf Leaf Plant Shoot Leaf to Male height shoot length number cence length width form dry weight shoot sterile number length ratio plants Block 2 12.058* 102.01ns 0.033ns 0.333ns 0.029ns 0.030* 0.0002ns 0.599* 648.6* 8.093ns 46.45ns Species 3 1331.6** 583.8** 0.411** 179.5** 14.45** 4.61** 0.061** 7.28** 43912.6** 1153.3** 6990.4** Ecotypes 21 36.3** 15535.5** 3.33** 3.74** 0.86** 0.065** 0.0067** 0.585** 1724.6** 52.82** 606.1** within species Error 50 172.2 312.4 0.026 0.316 0.114 0.007 0.0009 0.088 42.49 2.87 22.67 Coefficient of 7.6 18.4 8.1 6.5 11.7 6.3 6.7 8.4 8.2 2.9 13.3 variation (CV) (%) * and ** indicate statistical significance at 95% and 99%; ns show statistical insignificance. Tab. 3: Morphological traits in different Thymus species Species Plant Lateral Internode Internode Inflores- Leaf Leaf Plant Shoot Leaf to Male height shoot length number cence length width form dry weight shoot ratio sterile (cm) number (cm) length (cm) (cm) (cm) (g) (%) plants (%) T. daenensis 26.9 b 85.5 b 2.4 a 8.0 b 3.9 a 1.8 a 0.44 a 3.4 b 68.4 b 62.1 a 25.7 b T. vulgaris 39.4 a 163.2 a 1.9 b 15.9 a 3.8 a 0.6 c 0.32 b 5.0 a 192.4 a 46.2 b 61.2 a T. kotschyanus 15.1 c 91.2 b 1.6 b 7.6 b 2.2 b 1.0 b 0.49 a 3.3 b 65.9 b 49.0 b 27.3 b T. lancifolius 18.9 c 85.6 b 1.6 b 7.1 b 2.4 b 1.3 b 0.46 a 3.3 b 53.9 b 63.0 a 59.2 a Any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. Screening of Thymus spp. ecotypes for selection of elite genotypes 189 According to Tab. 3, the highest and lowest leaf length were found in T. daenensis (1.8 cm) and T. vulgaris (0.6 cm), respectively. In T. daenensis, the highest leaf length was seen in Khane-e-miran and Lorestan (1.9 cm) that, except for Isfahan 2, Markazi 1 and 2, did not have a significant difference with other ecotypes belonging to this species. In terms of leaf length, the ecotypes of T. vulgaris (with leaf length of 0.6 cm on average), did not show a significant differ- ence with each other. Also, the highest leaf length in T. kotschyanus and T. lancifolius was allocated to Tehran and Markazi, respectively (Tab. 4). Among the four investigated Thymus species, the highest leaf width (0.49 cm) was seen in T. kotschyanus which did not have a significant difference with T. daenensis and T. kotschyanus species. The lowest leaf width was observed in T. vulgaris (0.32 cm) (Tab. 3). The ecotypes of Zagheh (0.53 cm) (T. daenensis), Azerbaijan gharbi (0.60 cm) (T. kotschyanus), and Fars (0.51 cm) (T. lancifolius) were superior in leaf width among ecotypes of the three mentioned spe- cies. Moreover, the leaf width in ecotypes of T. vulgaris was insig- nificant (Tab. 4). In terms of mechanizing harvest, plants’ form plays a significant role in breeding program of Thymus spp. (Carlen et al., 2010). As stated before, the plants’ form is a qualitative trait and ratified by digits in this research as follows: 5 signifies completely upright plant, 3: semi- upright plants and 1: completely recumbent plant. In this regard, T. vulgaris significantly ranked 5 and other thymus species occupied other positions, although difference between plants’ form of other species (i.e. T. daenensis. T. kotschyanus and T. lancifolius) was not statistically significant and they were qualified with semi-upright plants (Tab. 3). In this regard, Ilam (4.8 on average) remarkably demonstrated more upright form in comparison to other ecotypes of T. daenensis. In T. kotschyanus and T. lancifolius, the most upright plants belonged to Azerbaijan gharbi and Fars, respectively (Tab. 4). The results of our research revealed that shoot dry weight of T. vul- garis (192.4 g) was significantly higher than other Thymus species. Also, there was no significant difference between shoot dry weight of T. daenensis (68.5 g), T. lancifolius (65.9 g), and T. kotschyanus (53.9 g) (Tab. 3). Our findings are in agreement with those found by kaveh et al., 2013; mondak et al., 2014; aSkari et al., 2018; mohammadi et al., 2019 who stated that shoot dry weight of T. vul- garis is higher than in the other three mentioned Thymus species. The highest shoot dry weight in T. vulgaris belonged to 'Varico 3' (213 g) that was significantly higher than in 'Deutscher Winter' and Isfahan (Tab. 4). It is necessary to mention that 'Varico 3' is a hy- brid produced from T. vulgaris in the Agroscope Research Institute, Switzerland. The results of Carlen et al. (2010) also proved the superiority of 'Varico 3' over 'Deutscher Winter' in terms of shoot dry weight. According to Tab. 4, the highest shoot dry weight was in Malayer 2 (85 g) (T. daenensis), Azerbaijan gharbi (152 g) (T. lanci- folius) and Fars (61 g) (T. kotschyanus). The highest leaf/shoot ratio (63%) was found in T. lancifolius which did not have a significant difference with T. daenensis (62.2%), and the lowest (42.2%) was obtained by T. vulgaris (Tab. 3). In T. dae- nensis, the highest ratio of leaf/shoot was observed in Zagheh (69%) which did not show a significant difference with Ilam (66%), al- though it had a significant difference with other ecotypes of T. dae- T. d ae ne ns is T. la nc ifo liu s T. k ot sc hy an us T. v ul ga ri s Tab. 4: Morphological traits in different Thymus ecotypes Species Ecotype Plant Lateral Internode Internode Inflorescence Leaf Leaf Plant Shoot Leaf to Male height shoot length number length length width form dry weight shoot ratio sterile (cm) number (cm) (cm) (cm) (cm) (g) (%) plants (%) Khane miran 28.4 bc 96 a 2.63 ab 8.5 b 3.5 de 1.90 a 0.45 bc 3.5 bcd 83.8 a 63.0 bc 27.7 cd Lorestan 26.0 cd 94 a 2.17 c 8.7 b 3.4 de 1.90 a 0.45 bc 2.9 d 62.2 de 61.0 c 0.0 h Esfahan1 26.3 cd 95 a 2.27 bc 8.3 b 4.0 bcd 1.87 a 0.36 d 3.1 d 68.3 cd 60.7 c 31.0 bc Ilam 23.5 d 97 a 1.27 d 10.7 a 4.4 bc 1.83 a 0.46 bc 4.6 a 48.5 f 66.3 ab 7.7 gh Malayer 1 29.4 abc 86 ab 2.63 ab 7.8 bc 3.6 cde 1.83 a 0.42 bcd 4.0 ab 77.1 abc 60.3 c 21.7 def Arak 26.8 cd 67 bc 2.67 ab 7.0 cd 3.8 cd 1.80 ab 0.45 bc 3.1 d 59.1 e 62.3 c 38.7 b Zagheh 18.2 e 55 c 2.00 c 6.2 d 2.8 e 1.80 ab 0.53 a 3.3 bcd 47.1 f 69.0 a 28.0 cd Malayer 2 26.9 cd 69 bc 2.63 ab 6.1 d 3.7 cde 1.77 ab 0.48 ab 3.9 bc 84.6 a 60.0 c 55.3 a Fars 28.6 bc 94 a 2.70 a 7.9 bc 3.9 bcd 1.73 ab 0.39 cd 3.2 d 62.6 de 62.7 bc 26.0 cde Esfahan 2 23.7 d 84 ab 2.17 c 7.6 bc 3.4 de 1.60 bc 0.43 bcd 2.9 d 70.4 bcd 60.7 c 38.0 gh Markazi 1 33.0 a 94 a 2.93 a 8.5 b 5.5 a 1.60 bc 0.42 bcd 3.1 d 78.7 ab 60.7 c 16.0 fg Markazi 2 32.2 ab 96 a 2.80 a 8.4 b 4.8 ab 1.43 c 0.46 abc 3.3 cd 79.3 ab 59.3 c 18.3 ef 'Deutscher Winter' 37.2 b 155 b 1.8 b 16.5 a 3.73 a 0.57 a 0.31 a 5.0 a 183.1 b 52.3 a 58.7 b Esfahan 37.3 b 156 b 1.7 b 16.3 a 3.67 a 0.53 a 0.31 a 5.0 a 182.6 b 52.0 a 57.3 b 'Varico 3' 43.6 a 179 a 2.3 a 14.7 b 3.87 a 0.57 a 0.33 a 5.0 a 212.6 a 34.0 b 67.7 a Tehran 14.4 b 88 b 2.0 a 6.4 c 2.4 a 1.1 a 0.49 b 3.2 bc 49.3 bc 48.3 a 18.0 b Zanjan 15.4 b 82 b 1.7 ab 8.1 ab 2.3 a 1.0 ab 0.49 b 3.5 ab 33.2 d 48.3 a 28.7 ab Ghazvin 13.6 b 89 b 1.7 ab 6.6 bc 2.2 a 1.0 a 0.42 b 2.9 c 54.5 b 50.0 a 41.7 a Az. Gharbi 18.6 a 120 a 1.4 b 9.6 a 2.1 a 0.8 c 0.60 a 3.9 a 152.1 a 48.0 a 28.7 ab Kordestan 13.7 b 77 b 1.3 b 7.2 bc 2.1 a 0.9 bc 0.46 b 2.9 c 40.4 cd 50.3 a 19.7 b Lorestan 14.6 c 89 a 1.5 a 6.8 a 2.2 a 1.0 c 0.48 a 2.8 b 49.1 bc 56.7 d 33.3 c Kordestan 17.6 bc 95 a 1.5 a 7.6 a 2.5 a 1.1 c 0.47 ab 3.1 ab 46.2 c 63.7 bc 48.3 bc Esfahan 20.4 ab 89 a 1.8 a 6.7 a 2.7 a 1.3 b 0.39 b 3.4 ab 54.7 abc 67.3 a 46.0 bc Fars 19.2 ab 86 a 1.6 a 7.4 a 2.7 a 1.4 b 0.51 a 3.7 a 61.3 a 65.0 b 52.7 c Markazi 22.5 a 69 a 1.8 a 6.8 a 2.1 a 1.6 a 0.43 ab 3.5 a 58.1 ab 62.3 c 84.3 a Any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. 190 S. Mohammadi, L. Tabrizi, M. Shokrpour, J. Hadian, H. Schulz, D. Riewe nensis (Tab. 4). In T. vulgaris, 'Varico 3' significantly gained the lowest ratio of leaf/shoot (34%) which was lower than 'Deutscher Winter' and Isfahan (Tab. 4). Also, there was no significant differ- ence between ecotypes of T. kotschyanus in terms of ratio of leaf/ shoot (Tab. 4). In T. lancifolius, the highest ratio of leaf/shoot (67%) was shown by Isfahan that significantly was higher than that in other ecotypes belonging to this species (Tab. 4). The ratio of leaf/shoot (called as a harvest index) is of important traits suitable for breeding of Thymus species. Regarding higher essential oil content in leaf and flower compared to shoot and also high density of trichrome in leaf and flower secreting essential oil (hadian et al. 2016), the yield of essential oil in Thymus species with high ratio of leaf/shoot is higher than the other species with low ratio of leaf/shoot. In our research, the ratio of leaf/shoot in T. vulgaris was lower than the other spe- cies investigated here. Although 'Varico 3' showed highest leaf dry weight (34%), its leaf/shoot ratio was lower than 'Deutscher Winter' (52%). Because of depressing leaf/shoot ratio of 'Varico 3' compared to 'Deutscher Winter', high harvest index (or ratio of leaf/shoot) is one of the most important advantages in Thymus species native to Iran such as T. daenensis. Ratio of leaf/shoot is directly related to leaf number and essential oil yield; accordingly, it plays a crucial role in cultivation and breeding of Thymus species. Compared to T. vulgaris, the three-mentioned species also possess higher shoot dry weight and ratio of leaf/shoot, thereby having higher leaf number and essential oil yield. Percent of male sterile plant The percent of male sterile plant among different ecotypes of T. daenensis is one of most important traits useful in thyme breed- ing via hybridization. Among the four investigated Thymus species, the highest percentage of male sterile plant (61.2%) was seen in T. vulgaris that did not show a significant difference with T. lancifo- lius (59.2%) (Tab. 3). The highest percentage of male sterile plant in T. vulgaris was found in 'Varico 3' (68%) that showed a significant difference with 'Deutscher Winter' and Isfahan (Tab. 4). In T. dae- nensis, the highest percentage of male sterile plant was obtained by Malayer 2 (55%) that was significantly higher than that in other eco- types of this species. Also, in Lorestan ecotype, even one male sterile plant was not found (Tab. 4). The highest percentage of male sterile plant (42%) was found in Qazvin ecotype belonged to T. kotschyanus which did not show a significant difference with Azerbaijan gharbi and Zanjan ecotypes of this species (Tab. 4). Among ecotypes be- longing to T. lancifolius, the highest percentage of male sterile plant (84%) was obtained by Markazi which significantly differed to other T. lancifolius ecotypes (Tab. 4). As described earlier, male sterility plays an important role in thyme breeding. In general, emasculation of thyme flowers is difficult, due to flower smallness and high density of flower on a fluorescence as well as non-simultaneity in time of opening flowers (rey, 1993; meweS and pank, 2006). Accordingly, identifying male sterile plant in thyme populations is important for hybridization activities. The variety 'Varico 3' is produced from T. vulgaris via hybridization using male sterile plant (Carlen et al., 2010). Also, the percentage of male sterile plant in 'Varico 3' was higher than that in 'Deutscher Winter', and this may be due to exploit- ing male sterile plant in hybridization as maternal parents (Carlen et al., 2010). Moreover, the type of male sterility in thyme is cytoplas- mic; accordingly, male sterility modulation is a complex process and controlled by at least 5 genes (belhaSSen et al., 1991). Cluster analysis of Thymus ecotypes In our research, growth and yield traits were subjected to cluster analysis using WARD method. The results showed that all ecotypes were categorized into four groups (Fig. 1). The first group consisted of 11 ecotypes classified into two subclusters. The first sub-cluster consisted of 9 ecotypes of T. daenensis (Khan-e-miran, Malayer 1 and 2, Markazi 1 and 2, Lorstan, Isfahan 1, Arak and Isfahan 2). In the second sub-cluster, only Ilam belonging to T. daenensis was placed (Fig. 1). In is noteworthy that 11 ecotypes of T. daenensis in this group were qualified with high phytochemical attributes (such as thymol) and ordinary growth and yield. In the second group, 10 ecotypes were found in forms of two sub-clusters; at first sub-cluster, 5 ecotypes of T. lancifolius including Lorstan, Kurdistan, Isfahan, and Fars and 1 ecotype of T. daenensis including Zagheh were ob- served, and in second sub-cluster, just 4 ecotypes of T. kotschyna- nus including Tehran, Zanjan, Kurdistan, and Qazvin were placed (Fig. 1). This group was qualified with high phytochemical (espe- cially essential oil content and thymol) and low growth and yield. Zagheh, belonging to T. daenensis, solely occupied this place, be- cause of its unique characteristics such as low essential oil percent- age and low chemical compounds such as thymol, growth, and yield, Fig. 1: Clustering different ecotypes and species of Thymus based on morphological traits using WARD method Screening of Thymus spp. ecotypes for selection of elite genotypes 191 in comparison to the other T. daenensis’s ecotypes. In the third group, only Azerbaijan gharbi, belonging T. kotschyanus and quali- fied with relatively high growth and yield, but low phytochemicals (especially essential oil and thymol) was placed. In the fourth group, 3 ecotypes of T. vulgaris were placed in forms of two sub-clusters: Isfahan and 'Deutscher Winter' in the first sub-cluster and 'Varico 3' in the second sub-cluster (Fig. 1). This group is characterized by high growth and yield and a moderate rate of essential oil and thymol. In general, it can be concluded that the cluster analysis categorized different ecotypes of Thymus species into four groups, in such a way that 11 groups belonged to the first group, 10 ecotypes to the sec- ond, one ecotype to the third, and finally three ecotypes to the fourth group (Fig. 1). In a research carried out by mondak et al. (2014), 22 ecotypes of Thymus native to Iran were placed into two main groups in terms of morphologic and essential oil traits. The first group included T. daenensis ecotypes and the second group belonged to T. lancifolius and T. kotschynanus ecotypes. Phytochemical traits Essential oil content Among four Thymus species investigated in this research, T. dae- nensis gained the highest essential oil content (5.76%) and its con- tent in other three species was as follows: T. vulgaris (3.59%), T. kotschyanus (2.43%), and T. lancifolius (3.96%) (Fig. 2). In T. dae- nensis, the highest essential oil content was observed in Ilam being significantly higher when compared to other ecotypes of this species (Fig. 2). In T. vulgaris, 'Varico 3' showed the highest essential oil content (4.42%) which was significantly higher than in 'Deutscher Winter' (3.21%) and Isfahan (3.16%) (Fig. 2). Azerbaijan gharbi, be- longing to T. kotschyanus, gained the highest essential oil content (2.79%), although it did not have a significant difference with that in Zanjan and Qazvin belonging to this species (Fig. 2). Among ecotypes belonging to T. lancifolius, Fars significantly obtained the highest essential oil (5.38%) in comparison to the other ecotypes be- longing to this species (Fig. 2). As presented in Fig. 2, there was a relatively high diversity among ecotypes and species of Thymus in this regard. The highest essential oil content (7.83%) was found in T. daenensis (Ilam). Also, essential oil in T. daenensis ecotypes varied from 2.5 to 5%. Moreover, khorShidi et al. (2018) reported a remarkable diversity among T. daenensis ecotypes (2.5-5%). Based on ruStaiee et al. (2010), the content of essential oil among T. dae- nensis ecotypes, naturally growing in Iran, was 1.8 to 3.83%, and the highest essential oil and thymol content were found in Arak, while the lowest was observed in Daenensis ecotypes. In another study, the content of essential oil in T. daenensis ecotypes, gathered from Hamadan regions (niCaver et al., 2005), was about 2.4%, and its content in ecotypes gathered from 5 regions of Isfahan province, Iran, was about 3.3%. The previous studies showed a higher essen- tial oil content in T. daenensis ecotypes, as compared to T. vulgaris (mondak et al., 2014; aSkari et al., 2018; mohammadi et al., 2019). According to our findings, the essential oil content in 'Varico 3' was significantly higher than in 'Deutscher Winter' (Fig. 2), and this is in agreement with that obtained by Carlen et al. (2010), who recorded a superiority of 'Varico 3' over 'Deutscher Winter' in terms of essential oil content. In our research, the lowest essential oil content was ob- served in T. kotschyanus (Fig. 2). Also, kaveh et al. (2013) reported various levels of essential oil in T. kotschyanus, from 0.42 to 2.17%. In other research, the amount of essential oil in this species was 1.1 to 2.5% (khoShSokhan et al., 2014). Also, in this research, essential oil content in T. lancifolius, depending on the individual ecotype, varied from 2.93 to 5.38%. Furthermore, the essential oil content in T. lancifolius under greenhouse condition was reported to have 0.93 to 2.32% (mondak et al., 2014). Essential oil yield As shown in Fig. 3, the highest essential oil yield was found in T. vul- garis (3.08 mL per plant), although it did not have a significant differ- ence with that in T. daenensis (2.47 mL per plant) and conversely had a significant difference with T. kotschyanus (0.8 mL per plant) and T. lancifolius (1.38 mL per plant) (Fig. 3). The highest yield of essen- tial oil (3.19 mL per plant) was obtained by 'Varico 3', although it did not show a significant difference with 'Deutscher Winter' (3.07 mL per plant) (Fig. 3). Among ecotypes of T. kotschyanus, Azerbaijan gharbi gained the highest yield of essential oil (2.04 mL per plant) that was significantly higher than other ecotypes belonging to this species (Fig. 3). In T. lancifolius, the highest yield of essential oil was in Fars (2.14 mL per plant) that was significantly higher than other ecotypes of T. lancifolius species (Fig. 3). In Thymus species, essential oil yield is of the most important traits gained through mul- tiplying essential oil and leaf dry weight. As shown in Fig. 3, the highest yield of essential oil was obtained by T. vulgaris which did not show a significant difference with T. daenensis. Although dry weight of T. vulgaris was higher than T. daenensis and, conversely, ratio of leaf/shoot in T. daenensis was higher than T. vulgaris, but the essential oil yield in T. vulgaris and T. daenensis was the same. Also, in our research, the yield of essential oil in 'Varico 3' and 'Deutscher Winter' did not show a significant difference (Fig. 3). Although the rate of essential oil and shoot dry weight in 'Varico 3' was higher than b d c a bc b f b e de b c b b a a a b a b c c b a b 0 1 2 3 4 5 6 7 8 K ha ne m ir an Lo re st an E sf ah an 1 Ila m M al ay er 1 A ra k Za gh eh M al ay er 2 Fa rs E sf ah an 2 M ar ka zi 1 M ar ka zi 2 D . W in te r E sf ah an V ar ic o 3 Te hr an Za nj an G ha zv in A z. G ha rb i K or de st an Lo re st an K or de st an E sf ah an Fa rs M ar ka zi T. daenensis T. vulgaris T. kotschianus T. lancifolius Es se n ti al o il co n te n t (% ) 5.76 A 3.59 BC 2.34 C 3.96 B Fig. 2: Mean essential oil content in different Thymus ecotypes and species 192 S. Mohammadi, L. Tabrizi, M. Shokrpour, J. Hadian, H. Schulz, D. Riewe 'Deutscher Winter', the ratio of leaf/shoot in 'Deutscher Winter' (52%) remarkably was higher than 'Varico 3' (34%); So, leaf dry weight in 'Deutscher Winter' was higher than 'Varico 3', and this leads to in- significant difference in both cultivars in terms of essential oil yield (Fig. 3). Major essential oil constitutes of T. daenensis’s ecotypes According to Tab. 5, the main components found in different eco- types of T. daenensis include thymol (47.1-82%), carvacrol (0.77- 24.39%), p-cymene (2.76-5.37%) and γ-terpinen (1.65-4.07%). Thy- mol was the major constituent widely found in all ecotypes of T. daenensis; all ecotypes, except for ecotype Zagheh (47.08%), con- tained more than 75% thymol. In contrast, Zagheh showed highest rate of carvacrol (24.39%) in comparison to the other T. daenensis ecotypes which contained lower than 5% of carvacrol. In different studies, thymol has been found as a main constituent of T. daenensis ecotypes (niCkavar et al., 2005; mondak et al., 2014; khorShidi et al., 2018, haydari et al., 2019). Also, the rate of carvacrol in Zagheh was 24.4%, while carvacrol in other ecotypes of T. daenensis was lower than 5% (Tab. 5). In a study carried out by khorShidi et al. (2018), Zagheh was shown to have higher carvacrol rather than the other ecotypes of T. daenensis. Also, among 12 ecotypes of T. daenensis, 11 ecotypes were introduced as thymol chemotypes and one of them (i.e. Zagheh) as carvacrol-thymol chemotype. Major essential oil constitutes of T. vulgaris In T. vulgaris, the main components of essential oil included thy- mol (38.33-42.23%), p-cymene (22.06-24.48%), γ-terpinene (9.97- 14.37%), and carvacrol (2.83-2.94%) (Tab. 6). In this respect, 'Varico 3' gained the highest thymol (42.23%). In terms of carvacrol, there was not found a remarkable diversity between ecotypes of T. vul- garis, and its rate in this species was reported 2.9% (Tab. 6). Carlen et al. (2010) recorded high essential oil content in 'Varico 3' com- pared to 'Deutscher Winter'. In this regard, mondak et al. (2014) a gh def cde bc efg i a h fgh ab cd a a a b c b a c c c b a b 0 1 2 3 4 K ha ne m ir an Lo re st an E sf ah an 1 Ila m M al ay er 1 A ra k Za gh eh M al ay er 2 Fa rs E sf ah an 2 M ar ka zi 1 M ar ka zi 2 D . W in te r E sf ah an V ar ic o 3 Te hr an Za nj an G ha zv in A z. G ha rb i K or de st an Lo re st an K or de st an E sf ah an Fa rs M ar ka zi T. daenensis T. vulgaris T. kotschianus T. lancifolius Es se n ti al o il yi el d ( m L p er p la n t) 2.47 A 3.08 A 0.79 B 1.38 B Fig. 3: Mean essential oil yield in different Thymus ecotypes and species Tab. 5: Major constituents in essential oil of different ecotypes of T. daenensis Ecotype p-Cymene (%) γ-Terpinene (%) Thymol (%) Carvacrol (%) Chemotype Khane miran 2.83 b 3.61 a 78.93 a 1.38 b Thymol Lorestan 5.37 a 4.07 a 75.98 a 3.52 b Thymol Isfahan 1 3.32 b 2.94 a 79.48 a 3.38 b Thymol Ilam 3.9 b 3.69 a 78.47 a 3.83 b Thymol Malayer 1 3.11 b 3.13 a 80.25 a 2.37 b Thymol Arak 4.73 ab 1.065 b 78.15 a 2.67 b Thymol Zagheh 5.08 a 3.32 a 47.08 b 24.39 a Thymol/Carvacrol Malayer 2 2.76 b 2.33 a 82.01 a 0.77 b Thymol Fars 3.57 b 2.36 a 76.26 a 4.01 b Thymol Isfahan 2 3.94 b 2.12 ab 77.27 a 3.15 b Thymol Markazi 1 3.08 b 2.58 a 78.19 a 3.17 b Thymol Markazi 2 3.05 b 2.32 a 76.69 a 2.63 b Thymol Any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. Tab. 6: Major constituents in essential oil of T. vulgaris Cultivar p-Cymene (%) γ-Terpinene (%) Thymol (%) Carvacrol (%) β-caryophyllene (%) Chemotype 'Deutscher Winter' 24.68 a 14.37 a 38.33 b 2.94 a 0.85 a Thymol Isfahan 24.16 a 14.3 a 39.1 b 2.9 a 0.79 a Thymol 'Varico 3' 22.06 b 9.97 b 47.23 a 2.83 a 1.0 a Thymol Any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. Screening of Thymus spp. ecotypes for selection of elite genotypes 193 also reported that thymol was the major constituent of essential oil in T. vulgaris. Also, it was introduced thymol (51.2%) as a major con- stitute of monoterpene index in essential oil of T. vulgaris; however, with low rate of carvacrol (4%) in this species. In general, thymol was introduced as a major constituent in essential oil of T. vulgaris (Carlen et al., 2010). Major essential oil constitutes of T. kotschyanus’s ecotypes In T. kotschyanus, the main components of its essential oil were dif- ferent, due to not only type of ecotype but also type and content of main components of essential oil (Tab. 7). The main essential oil components of T. kotschyanus were as follows: citral (0.48-42.09%), thymol (7.32-34.07), carvacrol (2.34-19.37%), geranial astat (0.95- 24.81%), p-cymene (0.43-18.98%) and gamma terpinene (0.24- 11.23%) (Tab. 7). According to type of ecotype, the main essential oil components of T. kotschyanus’s ecotypes were consisted of Tehran (citral, carvacrol, thymol, and geranial astat), Zanjan (thymol, carva- crol, geranyol, p-cymene), Qazvin (carvacrol, thymol, p-cymene, and beta-Terpineol), Azerbaijan gharbi (p-cymene, carvacrol, gamma terpinen and thymol), and Kurdistan (geranial astat, citral, thymol, and linalool) (Tab. 7). However, for niCkavar et al. (2006), the main components of T. kotschyanus were thymol (38.6%) and carvacrol (33.9%). In another study, the main components found in this spe- cies were as follows: polygon (18.7%), isomenton (17.8%), thymol (14.3%), Eucalyptol (9%), piperiton (6.3%) and carvacrol (5.5%) (Semnani et al., 2006). In this regard, it has also been reported that the major constituents found in essential oil of T. kotschyanus eco- types were different and included thymol (31.2%), carvacrol (19.5%), p-cymene (11.2%), and γ-terpinene (8.4%) (kiliC and Özdemir, 2017). According to tohidi et al. (2018), major constituents of es- sential oil in different ecotypes of T. kotschyanus varied from 1 to 1.41% and main components in its essential oil were consisted of thy- mol (31.2%), carvacrol (19.5%), linalool (2.38-20.06%), α-terpineol (0.16-11.64%), and geraniol (0.36-11.37%). Also, among 43 chemi- cals found in essential oil of T. kotschyanus, SaJadi and khatamSaz (2003) reported that thymol, carvacrol, and p-cymene were major constituents. Major essential oil constituents of T. lancifolius’s ecotypes As shown in Tab. 8, the main components of essential oil in T. lan- cifolius were largely different, depending on type of ecotype. The main components of essential oil in ecotypes of T. lancifolius were as follows: thymol (13.81-68.67), carvacrol (3.64-48.74), geraniol (0.57-25.57), α-terpineol (4.19-14.81), linalool (3.56-12.15), citral (7.88-10.08), geranil astat (1.06-7.33), α-terpinel astat (3.72-6.75), p- cymene (2.10-5.12), and borneol (1.47-4.08) (Tab. 8). Also, the main components of essential oil in all ecotypes of T. lancifolius were as follows: Lorstan (carvacrol, thymol and p-cymene), Kurdistan (alpha terpineol, thymol, linalool, carvacrol and citral), Isfahan (thymol, linalool, and alpha terpineol), Fars (thymol, geraniol, citral, and geranil astat), and Markazi (thymol and alpha terpineol astat) (Tab. 8). Furthermore, mondak et al. (2014) found thymol, carvacrol, and geraniol as major constituents of essential oil in T. lancifolius. Elite genotypes By evaluating different ecotypes of Thymus species in terms of growth, yield, and phytochemical traits, some genotypes were se- lected within ecotypes qualified with interesting traits. The prelimi- nary selection of elite genotypes was carried out on the basis of some visual traits such as plant height, growth form, shoot dry weight, and ratio of leaf/shoot; subsequently the qualified genotypes were evaluated in terms of phytochemical traits such as content and yield of essential oil as well as major constituents of essential oil such as thymol, and eventually three genotypes (numbered as 1, 2, and 3), as elite genotypes, were identified. The results showed that all three selected genotypes belong to T. daenensis and their characteristics a presented in Tab. 9. These genotypes were qualified with some important traits such as plant height, plant form, number of lateral shoots, dry and fresh weight of shoot, ratio of leaf/shoot, the percent and yield of essential oil, and rate of thymol. The form of plant is important because of its role in mechanizing crop harvest. In our re- search, the elite genotypes (1, 2, and 3) were in upright form and suit- able for harvesting mechanically (Tab. 9). Shoot dry weight in elite genotypes remarkably was higher than that in T. daenensis and even in T. vulgaris. The ratio of leaf/shoot in elite genotypes was similar to Tab. 7: Major constituents in essential oil of different ecotypes of T. kotschyanus Ecotype p-Cymene γ-Terpinene beta-terpineol Linalool Citral Geraniol Thymol Carvacrol Geranyl Chemotype (%) (%) (%) (%) (%) (%) (%) (%) acetate (%) Tehran 3.37 c 0.81 c 0.17 b 3.5 b 42.09 a 0.46 bc 7.32 c 19.37 a 5.1 b Citral Zanjan 8.74 b 2.68 b 0.09 b 1.3 b 0.11 e 9.08 a 34.07 a 18.74 a 0.95 c Thymol/Carvacrol Ghazvin 7.00 b 4.44 b 6.79 a 0.25 c 5.49 c 0.12 c 18.62 b 19.35 a 0.16 d Thymol/Carvacrol Az. Gharbi 18.98 a 11.23 a 0.21 b 2.27 b 0.48 d 0.18 c 11.09 c 17.31 a 0.12 d p-Cymene /Carvacrol Kordestan 0.43 d 0.24 c 0.08 b 8.25 a 22.41 b 2.13 b 14.79 bc 2.34 b 24.81 a Geranyl acetate/Citral Any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. Tab. 8: Major constitutes in essential oil of different ecotype of T. lancifolius Ecotype Linalool Borneol α-terpineol Citral Geraniol Thymol Carvacrol α-terpineol Geranyl Chemotype (%) (%) (%) (%) (%) (%) (%) acetate (%) acetate (%) Lorestan 3.56 c 1.84 b 0.09 c 0.22 c 1.12 b 19.46 d 48.74 a 0.20 c 1.06 c Carvacrol Kordestan 12.15 a 4.08 a 14.81 a 7.88 b 0.57 b 13.81 e 8.4 b 0.16 c 2.38 b α-terpineol Isfahan 6.63 b 0.11 c 4.19 b 0.15 c 0.10 c 60.14 b 4 c 3.72 b 0.12 d Thymol Fars 0.21 d 1.47 b 0.13 c 10.08 a 25.57 a 28.69 c 4.5 c 0.11 c 7.33 a Thymol/Geraniol Markazi 0.14 d 0.18 c 0.11 c 0.18 c 0.18 c 68.67 a 3.64 c 6.75 a 0.14 d Thymol Any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. 194 S. Mohammadi, L. Tabrizi, M. Shokrpour, J. Hadian, H. Schulz, D. Riewe that in T. daenensis, but larger than T. vulgaris. Essential oil content in elite genotypes of 1, 2, and 3 (8.22%, 7.80%, and 9.07%, respec- tively) was remarkably higher than those in T. vulgaris (3.56%) and T. daenensis (5.76%). The essential oil yield, as an important traits in Thymus species, in elite genotypes of 1, 2, and 3 (11.89, 10.27, and 17.55 mL per plant, respectively), was remarkably higher than essential oil yield in T. daenensis (2.47 mL per plant) and T. vulgaris (3.18 mL per plant). In terms of thymol, elite genotypes contained 73.61, 73.91 and 75.19%, being similar to those in T. daenensis (75.73%), and conversely larger than that in T. vulgaris (41.55%) (Tab. 9). The results of our research revealed a remarkable diversity between and within Thymus species in terms of morphological and phyto- chemical traits. Among four Thymus species investigated in this re- search, the highest shoot dry weight was found in T. vulgaris that was significantly higher than that in the other three Thymus species. An average yield of dry matter in the mentioned three species, com- pared to T. vulgaris, was relatively low. Depending on the individual ecotype, the rate of dry matter among Thymus ecotypes was differ- ent and Khan-e-miran and Malayer 2 belonging to T. daenensis and Azerbaijan gharbi belonging to T. kotschyanus and Fars of T. lan- cifolius had higher dry matter in comparison to the other ecotypes. Moreover, among the four Thymus species, the highest essential oil content was found in T. daenensis which was remarkably higher than in the others. In T. daenensis, Ilam (7.83%) gained highest essential oil content and its essential oil yield did not show a significant dif- ference to T. vulgaris; also, the ecotypes of Khan-E-miran, Malayer 2 and Markazi 1 had higher yield in comparison to the other Thymus species, especially T. vulgaris. According to the results, the essential oil content and major constitu- ents of essential oil were clearly dependent on the individual species and ecotype. In this regard, our findings exhibited high diversity in the Thymus species and their ecotypes. In a nutshell, thymol was found to be a major component in the essential oils of all T. daenensis ecotypes, and all the ecotypes contained approximately 75% thymol, except for Zagheh ecotype. Also, thymol was a major constituent in essential oil of T. vulgaris (43% on average). In addition to thymol, other main components including p-cymene and γ-terpinene were found in T. kotschyanus and T. lancifolius. It is necessary to mention that the type and content of essential oil in the Thymus species was different, dependent on type of ecotype. In our research, five chemo- types (citral, carvacrol-thymol, thymol-carvacrol, p-cymene-carva- crol and geranial astat) and four chemotypes (thymol, alpha terpin- eol-linalool, carvacrol-thymol, thymol-geraniol) were identified in different ecotypes of T. kotschyanus and T. lancifolius, respectively. Therefore, such high diversity in type and content of thyme essential oil is suitable for employing in different industries. Finally, the three elite genotypes were recognized based on their morphological and phytochemical traits. So, after propagating elite genotypes and examining their yield, they can be used as candidate parents for thyme breeding and cultivation. Acknowledgments Hereby we want to appreciate Department of Horticultural Science of University of Tehran and Julius Kühn-Institut (JKI), Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, for supporting this research. We also thank Dr. Torsten Meiners for his detailed comments and suggestion which highly improved this Project. Conflict of interest No potential conflict of interest was reported by the authors. References adamS, R.P., 2017: Identification of essential oil components by gas chro- matography/mass spectrometry. ed. 4.1. Allured Publishing Corporation. alizadeh, a., alizadeh, o., amari, G., zare, m., 2013: Essential oil com- position, total phenolic content, antioxidant activity and antifungal proper- ties of Iranian Thymus daenensis subsp. daenensis Celak. as in influenced by ontogenetical variation. J. Essent. Oil-Bear. Plants 16, 59-70. DOI: 10.1080/0972060X.2013.764190 amiri, h., 2012: Essential oils composition and antioxidant properties of three thymus species. Evid.-Based Complementary Altern. Med. DOI: 10.1155/2012/728065 aSkary, m., behdani, m.a., parSa, S., mahmoodi, S., Jamialahmadi, m., 2018: Water stress and manure application affect the quantity and quality of essential oil of Thymus daenensis and Thymus vulgaris. Ind. Crops Prod. 111, 336-344. DOI: 10.1016/j.indcrop.2017.09.056 bernath, J., 2002: Strategies and recent achievements in selection of me- dicinal and aromatic plants. Acta Hortic.. DOI: 10.17660/ActaHortic.2002.576.19 Carlen, C., SChaller, m., Carron, C.a., vouillamoz, J.f., baroffio, C.a., 2010: The new Thymus vulgaris L. hybrid cultivar (Varico 3) com- pared to five established cultivars from Germany, France and Switzerland. Acta Hortic. 860, 161-166. DOI: 10.17660/ActaHortic.2010.860.23 Dajić-Stevanović, Z., ŠoŠtarić, i., Marin, P.D., Stojanović, D., riStić, m., 2008: Population variability in Thymus glabrescens Willd. from Serbia: morphology, anatomy and essential oil composition. Arch. Biol. Sci. 60, 475-483. DOI: 10.2298/ABS0803475D denG, b., fanG, S., ShanG, X., fu, X., yanG, w., 2019: Influence of geno- types and environmental factors on leaf triterpenoid content and growth of Cyclocarya paliurus. J. For. Res. 30, 789-798. DOI: 10.1007/s11676-018-0680-z denG, y., huanG, l., zhanG, C., Xie, p., ChenG, J., wanG, X., 2019: Physicochemical and functional properties of water soluble gum from Tab. 9: Morphological and phytochemical traits of selected elite genotypes in comparison to different species. Species Internode Inter- Inflores- Leaf Leaf Plant Shoot Plant Shoot Shoot Leaf Essential Essential Thymol length node cence length width height number growth fresh dry to shoot oil content oil yield (%) (cm) number length (cm) (cm) (cm) (cm) form weight (g) weight (g) ratio (%) (ml per plant) T. daenensis 2.4 a 8.0 b 3.9 a 1.8 a 0.44 a 26.9 b 85.5 b 3.4 b 134.3 b 68.5 b 62.1 a 5.76 a 2.47 a 75.73 a T. vulgaris 1.9 b 15.9 a 3.8 a 0.6 c 0.32 b 39.4 a 163.2 a 5.0 a 405.7 a 192.4 a 46.2 b 3.59 bc 3.18 a 41.55 b T. kotschyanus 1.6 b 7.6 b 2.2 b 1.0 b 0.49 a 15.1 c 91.2 b 3.3 b 153.4 b 65.9 b 49 b 2.34 c 0.80 b 17.18 c T. lancifolius 1.6 b 7.1 b 2.4 b 1.3 b 0.46 a 18.9 c 85.6 b 3.3 b 150.8 b 53.9 b 63 a 3.96 b 1.38 b 38.15 b Genotype 1 1.5 11 10 2 0.5 38 250 5 576.5 226.5 63 8.33 11.89 73.61 Genotype 2 2.2 8 5 2.1 0.5 32 190 4 536.5 219.5 60 7.8 10.27 73.91 Genotype 3 1.7 11 6 2 0.5 32 300 4 765.5 322.5 60 9.07 17.55 75.19 Any two means within a column followed by same letters are not significantly different at p ≤ 0.05, n = 3. http://dx.doi.org/10.1080/0972060X.2013.764190 http://dx.doi.org/10.1155/2012/728065 http://dx.doi.org/10.1016/j.indcrop.2017.09.056 http://dx.doi.org/10.17660/ActaHortic.2002.576.19 http://dx.doi.org/10.17660/ActaHortic.2010.860.23 http://dx.doi.org/10.2298/ABS0803475D http://dx.doi.org/10.1007/s11676-018-0680-z Screening of Thymus spp. ecotypes for selection of elite genotypes 195 Wrinkle Floweringquince (Chaenomeles Speciosa) seeds. J. Bioresour. Bioprod. 4, 222-230. DOI: 10.12162/jbb.v4i4.012 edeoGa, h.o., okwu, d.e., mbaebie, b.o., 2005: Phytochemical constitu- ents of some Nigerian medicinal plants. Afr. J. Biotechnol. 4, 685-688. DOI: 10.5897/AJB2005.000-3127 GovindaraJ, m., vetriventhan, m., SrinivaSan, m., 2015: Importance of genetic diversity assessment in crop plants and its recent advances: an overview of its analytical perspectives. Genet. Res. Int. DOI: 10.1155/2015/431487 hadian, J., karimi, e., Shuryabi, m., nadJafi, f., kanani, m.r., 2016: Evaluation of morphological variation and path coefficient analysis of oil content of Thymus daenensis Celak populations. Plant Production Technology 8, 41-56. In Persian. heydari, a., hadian, J., eSmaeili, h., kanani, m.r., mirJalili, m.h., SarkhoSh, a., 2019: Introduction of Thymus daenensis into cultivation: Analysis of agro-morphological, phytochemical and genetic diversity of cultivated clones. Ind. Crops Prod. 131, 14-24. DOI: 10.1016/j.indcrop.2019.01.033 HMSO, 1988: British pharmacopoeia. London, HMSO. imbrea, i., prodan, m., niColin, a., butnariu, m., imbrea, f., 2010: Valorising Thymus glabrescens Willd. from the Aninei mountains. Res. J. Agric. Sci. 42, 260-263. iSlam, m.m., barman, a., kundu, G.k., kabir, m.a., paul, b., 2019: Vulnerability of inland and coastal aquaculture to climate change: Evidence from a developing country. Aquac. Fish 4, 183-189. DOI: 10.1016/j.aaf.2019.02.007 Jamzad, z., 2009: Iranian Thymus and Sourings. Research Institute of Forests and Rangelands. kaveh, S., zeinali, h., Safaei, l., arefi, h.m., aflakian, S., 2013: Com- parison of morphological and phytochemical traits in different popula- tions of Thymus kotschyanus Boiss. & Hohen. with Thymus vulgaris L. genotypes. Iranian Journal of Medicinal and Aromatic Plants 29 (1). In Persian. khorShidi, J., Shokrpour, m., nazeri, v., 2018: Effect of water deficit stress on yield, essential oil and some of physiological traits in different ecotypes of Thymus daenensis subsp. daenensis in Karaj region. Iranian Journal of Horticultural Science 49 (3), 613-624. In Persian. khoShSokhan, f., poormeidani, a., babalar, m., moGhadam, m.f., 2014: Analysis of the essential oils of Thymus kotschyanus L. (10 popu- lations) from Iran. Cercetari Agronomice in Moldova 47 (2), 49-59. DOI: 10.2478/cerce-2014-0016 lopez-puJol, J.o.r.d.i., boSCh, m., Simon, J., blanChé, C., 2004: Allo- zyme diversity in the tetraploid endemic Thymus loscosii (Lamiaceae). Ann. Bot. 93, 323-332. DOI: 10.1093/aob/mch039 meweS, S., pank, f., 2006: Inheritance of the gynodioecy of thyme (Thymus vulgaris L.). In: I International Symposium on the Labiatae: Advances in Production, Biotechnology and Utilisation 723, 79-84. DOI: 10.17660/ActaHortic.2006.723.6 mohammadi, h., amirikia, f., Ghorbanpour, m., fatehi, f., haShempour, h., 2019: Salicylic acid induced changes in physiological traits and es- sential oil constituents in different ecotypes of Thymus kotschyanus and Thymus vulgaris under well-watered and water stress conditions. Ind. Crops Prod. 129, 561-574. DOI: 10.1016/j.indcrop.2018.12.046 moJab, f., pourSaeed, m., mehrGan, h., pakdaman, S., 2008: Antibacterial activity of Thymus daenensis methanolic extract. Pak. J. Pharm. Sci. 21, 210-213. mondak, b., mohammadi, v., hadian, J., zeinali, h., 2014: Evaluation of yield and essential oil content in iranian Thymus populations. 3 rd. National Congress on Medicinal Plants. 14, 15 May 2014. Mashhad- Iran. németh, e., Szekely, G., 2000: Floral biology of medicinal plants II. Lamiaceae species. Int. J. Hortic. Sci. 6, 137-140. DOI: 10.31421/IJHS/6/3/124 niCkavar, b., moJab, f., dolat-abadi, r., 2005: Analysis of the essential oils of two Thymus species from Iran. Food Chem. 90, 609-611. DOI: 10.1016/j.foodchem.2004.04.020 Petrović, S., UŠjak, L., MiLenković, M., arSenijević, j., Drobac, M., DrnDarević, a., niketić, M., 2017: Thymus dacicus as a new source of antioxidant and antimicrobial metabolites. J. Funct. Foods 28, 114-121. DOI: 10.1016/j.jff.2016.11.007 rahimmalek, m., bahreinineJad, b., khorrami, m., tabatabaei, b.e.S., 2009: Genetic variability and geographic differentiation in Thymus dae- nensis subsp. daenensis, an endangered medicinal plant, as revealed by Inter Simple Sequence Repeat (ISSR) markers. Biochem. Genet. 47, 11-12. DOI: 10.1007/s10528-009-9281-z rahimmalek, m., tabatabaei, b.e.S., arzani, a., etemadi, n., 2009: Assessment of genetic diversity among and within Achillea species us- ing amplified fragment length polymorphism (AFLP). Biochem. Syst. Ecol. 37, 354-361. DOI: 10.1016/j.bse.2009.06.002 raSooli, i., mirmoStafa, S.a., 2002: Antibacterial properties of Thymus pubescens and Thymus serpyllum essential oils. Fitoterapia 73, 244-250. DOI: 10.1016/S0367-326X(02)00064-3 rey, C.h., 1993: Selection of thyme (Thymus vulgaris L.). In: International Symposium on Medicinal and Aromatic Plants 344, 404-410. DOI: 10.17660/ActaHortic.1993.344.46 ruStaiee, a.r., khorShidi, J., tabatabaei, m.f., omidbaiGi, r., Sefidkon, f., 2010: Essential oil composition of Thymus daenensis Celak. during its phenological cycle. J. Essent. Oil-Bear. Plants 13, 556-560. DOI: 10.1080/0972060X.2010.10643862 Safaei-Ghomi, J., ebrahimabadi, a.h., dJafari-bidGoli, z., batooli, h., 2009: GC/MS analysis and in vitro antioxidant activity of essential oil and methanol extracts of Thymus caramanicus Jalas and its main con- stituent carvacrol. Food Chem. 115, 1524-1528. DOI: 10.1016/j.foodchem.2009.01.051 SaJJadi, S.e., khatamSaz, m., 2003: Composition of the essential oil of Thymus lancifolius. J. Essent. Oil Res. 15, 34-45. DOI: 10.1080/10412905.2003.9712257 Semnani, k., roStami, b., akbarzadeh, m., 2006: Essential oil composi- tion of Thymus kotschyanus and Thymus pubescens from Iran. J. Essent. Oil Res. 18, 272-274. DOI: 10.1080/10412905.2006.9699085 tohidi, b., rahimmalek, m., arzani, a., 2018: Variations in chemical composition and bioactive compounds of Thymus kotschyanus Boiss. & Hohen populations originated from different collection sites. J. Essent. Oil-Bear. Plants 21, 1272-1283. DOI: 10.1080/0972060X.2018.1533435 tohidi, b., rahimmalek, m., trindade, h., 2019: Review on essential oil, extracts composition, molecular and phytochemical properties of Thymus species in Iran. Ind. Crops Prod. 134, 89-99. DOI: 10.1016/j.indcrop.2019.02.038 yavari, a.r., nazeri, v., Sefidkon f., haSSani. m.e., 2010: Study on some ecological factors, morphological traits, ploidy levels and essential oil composition of Thymus pubescens Boiss. & Kotschy ex Celak in two nat- ural regions of East Azerbaijan province, Iranian Journal of Medicinal and Aromatic Plants 26, 500-512. In Persian. ORCID Siavash Mohammadi https://orcid.org/0000-0001-7718-2648 Leila Tabrizi https://orcid.org/0000-0002-8878-7412 Majid Shokrpour https://orcid.org/0000-0001-9377-554X Javad Hadian https://orcid.org/0000-0002-5033-8857 Hartwig Schulz https://orcid.org/0000-0002-7551-3906 David Riewe https://orcid.org/0000-0002-9095-5518 Addresses of the authors: Siavash Mohammadi, Department of Horticultural Science, Faculty of Agricultural Science and Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, 31587, Iran Leila Tabrizi, Department of Horticultural Science, Faculty of Agricultural Science and Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, 31587, Iran E-mail: l.tabrizi@ut.ac.ir Majid Shokrpour, Department of Horticultural Science, Faculty of Agri- cultural Science and Engineering, College of Agriculture and Natural http://dx.doi.org/10.12162/jbb.v4i4.012 http://dx.doi.org/10.5897/AJB2005.000-3127 http://dx.doi.org/10.1155/2015/431487 http://dx.doi.org/10.1016/j.indcrop.2019.01.033 http://dx.doi.org/10.1016/j.aaf.2019.02.007 http://dx.doi.org/10.2478/cerce-2014-0016 http://dx.doi.org/10.1093/aob/mch039 http://dx.doi.org/10.17660/ActaHortic.2006.723.6 http://dx.doi.org/10.1016/j.indcrop.2018.12.046 http://dx.doi.org/10.31421/IJHS/6/3/124 http://dx.doi.org/10.1016/j.foodchem.2004.04.020 http://dx.doi.org/10.1016/j.jff.2016.11.007 http://dx.doi.org/10.1007/s10528-009-9281-z http://dx.doi.org/10.1016/j.bse.2009.06.002 http://dx.doi.org/10.1016/S0367-326X(02)00064-3 http://dx.doi.org/10.17660/ActaHortic.1993.344.46 http://dx.doi.org/10.1080/0972060X.2010.10643862 http://dx.doi.org/10.1016/j.foodchem.2009.01.051 http://dx.doi.org/10.1080/10412905.2003.9712257 http://dx.doi.org/10.1080/10412905.2006.9699085 http://dx.doi.org/10.1080/0972060X.2018.1533435 http://dx.doi.org/10.1016/j.indcrop.2019.02.038 196 S. Mohammadi, L. Tabrizi, M. Shokrpour, J. Hadian, H. Schulz, D. Riewe Resources, University of Tehran, Karaj, 31587, Iran Javad Hadian, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, 1983969411 Tehran, Iran Hartwig Schulz, Julius Kühn-Institut, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Königin-Luise-Strasse 19, 14195 Berlin, Germany David Riewe, Julius Kühn-Institut, Institute for Ecological Chemistry, Plant © The Author(s) 2020. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en). Analysis and Stored Product Protection, Königin-Luise-Strasse 19, 14195 Berlin, Germany