Abstract:
A new and distinct clonal line of oregano named KI-Ov1850 and characterized by elevated levels of thymol and vigorous growth.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. Provisional Patent Application Ser. No. 61/855,068, titled “Oregano Clonal Line Having High Levels of Thymol” which was filed on May 7, 2013, and is incorporated herein in its entirety by this reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates generally to an oregano plant and, more specifically, to a plant of oregano clonal line KI-Ov1850 that produces a high amount of thymol. 
       BACKGROUND OF THE INVENTION 
       [0003]    Oregano ( Orignaum  sp.) has been used for centuries as a traditional herb, spice and also in traditional medicine. Oregano essential oils are used as ingredients in several applications including tooth paste, mouth wash, gums etc. The main active molecules in oregano essential oil are carvacrol and thymol. These molecules have been shown to have potent antimicrobial activity 1  and have been used as feed additives to improve gastrointestinal health in farm animals. Oregano essential oils in animal feed diets have been shown to improve growth and reproductive performance in pigs 2,3  and overall performance in broilers. Oregano oil is also used in several personal care applications. Thymol is also used as a biological control of Varroa mites in bee hives. 
         [0004]    Accumulation of essential oil in oregano and other herbs varies with the developmental stage of the plant 4 . The highest carvacrol and thymol content in  Orignaum onites  was found at the pre-flowering stages 5 . However another study reported highest carvacrol and thymol content during the post flowering and seed formation stages in mid-June 6 . 
         [0005]    The bio active molecules, although controlled genetically, are strongly affected by environmental influences of a particular growing region; and also by agronomic conditions, harvesting time and the type of processing 4 . The age of the plants at the time of harvest seems to affect the oil yield and composition. Total thymol and carvacrol content of oregano oils obtained from younger plants was higher than that of the mature plants 4 . The amount of water received by the plant did not have a significant effect on the thymol and carvacrol content of the oil extracted from Mexican oregano 7 . 
         [0006]    The effects of temperature and duration of day light on the bioactive components of oregano essential oil have been studied. Oregano plants, like many other species, grown under higher temperatures, light intensity and longer day length periods tend to have increased oil content 8 . The harvest time was crucial to maximize the essential oil yield and the concentration of its main components in oregano. Carvacrol and thymol content were generally much higher during summer months 8 . 
         [0007]    While progressive genetic improvement was focused on increasing the essential oils, little or no effort has yet been made in the improvement of oregano for better production of carvacrol, thymol or other antimicrobial or antioxidant molecules. Extraction of thymol from a hyper-accumulating oregano clonal line is crucial for economically viable commercial thymol production. Therefore developing oregano varieties with high levels of thymol combined with high biomass will provide an economically valuable thymol source. 
       SUMMARY OF THE INVENTION 
       [0008]    The invention consists of a plant of oregano named KI-Ov1850 that has elevated thymol levels, excellent vigor and overall agronomic robustness. The variety was selected and propagated from seeds obtained from a commercial source of segregating oregano. A plant with elevated levels of thymol and good growth habits was selected and has been asexually propagated to produce a clonal line of identical plants. 
         [0009]    Plants of the clonal line KI-Ov1850 have not been observed under all possible environmental conditions. The phenotype may vary somewhat with variations in environment and culture such as temperature, light intensity, day length, water status, and/or fertilizer rate or type without, however, any variance in genotype. 
         [0010]    An object of the present invention is a plant with a high level of thymol for use as an antimicrobial or antioxidant in human and animal food, beverages, personal care and household care products. 
         [0011]    Another object of the invention is a variety of oregano that is novel, stable, and uniform and has good agronomic characteristics that permit efficient cultivation of the variety as a crop that produces a high amount of biomass from which thymol can be extracted. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1  is a chart comparing the carvacrol and thymol content in greenhouse-grown and field-grown plants. 
           [0013]      FIG. 2  is a chart of the seasonal variation of the carvacrol/thymol content in selected oregano lines. 
           [0014]      FIG. 3  is a chart of the relative phenotypic vigor scores of selected oregano lines. 
           [0015]      FIG. 4  is a photograph of the length of a leaf of a plant of the variety KI-Ov1850 against a centimeter scale. 
           [0016]      FIG. 5  is a photograph of the width of a leaf of a plant of the variety KI-Ov1850 against a centimeter scale. 
           [0017]      FIG. 6  is a photograph of a lateral branch of the variety KI-Ov1850. 
           [0018]      FIG. 7  is a photograph of the inflorescence of KI-Ov1850. 
           [0019]      FIG. 8  is a chromatogram of prior art Greek Oregano. 
           [0020]      FIG. 9  is a chromatogram of the variety KI-Ov1850. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    We previously identified proprietary clonal lines of oregano that can accumulate either high levels of carvacrol (Hi-C) or high levels of thymol (Hi-T lines) from a large scale genetic screening study under greenhouse conditions. These Hi-C and Hi-T oregano clonal lines were grown under field conditions for carvacrol and thymol accumulation during the growing season as well as general biomass accumulation. The combination of target molecule accumulation, biomass production, and propagation ability has been used to identify the best clonal lines for commercial applications. 
         [0022]    The plants of the present invention were identified from a wild oregano population and have the taxonomic description of being genus  Origanum , unknown species, family Lamiaceae and the common name oregano. Table 2 sets out a description of traits of the plants taken from plants growing in a greenhouse and Table 1 sets out the definitions of the traits of Table 2. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Description of the traits 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Days to first flowering 
                 Number of days from transplanting 
               
               
                   
                 to when the first spike/inflores- 
               
               
                   
                 cence emerges 
               
               
                 Plant height (cm) 
                 Length of the middle stem at the 
               
               
                   
                 time of flowering 
               
               
                 Length of lateral branches (cm) 
                 Length/Ht of the lateral stems 
               
               
                 Number of lateral branches 
                 Count on the number of lateral 
               
               
                   
                 branches 
               
               
                 Number of nodes on 
                 Count on the number of nodes of 
               
               
                 the middle stem 
                 the middle stem 
               
               
                 Internode length on 
                 Measure the length between 9th and 
               
               
                 the middle stem 
                 10th internode of the middle stem 
               
               
                 Number leaves on 
                 Count the number of spikes per plant 
               
               
                 the middle stem 
               
               
                 Leaf width 
                 Taken from the fully opened leaf 
               
               
                   
                 from 10th node of the middle stem 
               
               
                 Leaf length 
                 Taken from the fully opened leaf 
               
               
                   
                 from 10th node of the middle stem 
               
               
                 Leaf area (cm 2 ) 
               
               
                 Weight of aerial part 
                 Fresh weight during of the biomass 
               
               
                 of single plant (g) 
                 including stems 
               
               
                 Leaf to stem ratio 
                 Leaves separated from stem and 
               
               
                   
                 weighed to obtain ratio 
               
               
                 Thymol content 
                 Top 6 inches of the plant cut and air 
               
               
                   
                 dried for 48 hours for thymol 
               
               
                   
                 quantitation 
               
               
                 Ratio of aerial plant 
                 On fresh weight basis 
               
               
                 parts to roots 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Characteristics of KI-Ov1850 observed in the Greenhouse 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Days to first flowering from date 
                 112 days 
                 109-120 
                 days 
               
               
                 of transplanting rooted cuttings 
               
             
          
           
               
                 Plant height (cm) 
                 83.4 
                 cm 
                 78-88 
                 cm 
               
               
                 Length of lateral branches (cm) 
                 70.8 
                 cm 
                 69-74 
                 cm 
               
             
          
           
               
                 Number of lateral branches 
                 18.4  
                 16-22 
               
               
                 Number of nodes on the middle stem 
                 20.6  
                 18-22 
               
             
          
           
               
                 Internode length on the middle stem 
                 3.48 
                 cm 
                 2.9-3.9 
                 cm 
               
             
          
           
               
                 Number of leaves on the middle stem 
                 449-843 
                 653 
               
             
          
           
               
                 Leaf width 
                 1.92 
                 cm 
                 1.8-2.1 
                 cm 
               
               
                 Leaf length 
                 2.32 
                 cm 
                 2.2-2.5 
                 cm 
               
               
                 Leaf area (cm2) 
                 4.46 
                 cm2 
                 3.96-5.0 
                 cm2 
               
               
                 Weight of aerial part of single plant (g) 
                 331.6 
                 g 
                 280-460 
                 g 
               
             
          
           
               
                 Leaf to stem ratio 
                  1.50 
                 1.39-1.61 
               
               
                 Thymol content (mg/g) during maturity 
                 34.6  
                 23.6-51.6 
               
               
                 Ratio of aerial plant parts to roots 
                  1.407 
                  1.14-1.589 
               
               
                   
               
             
          
         
       
     
         [0023]    In an embodiment, the plant KI-Ov1850 produces thymol comprising 4% on a dry matter basis during the active growing season (May to September) 
         [0024]    The present invention is related to the development of a novel, stable, uniform vigorously growing robust plant KI-Ov1850 of oregano. This plant is unique and clearly distinct from all other existing varieties of oregano. 
       Example 1 
     Selection and Development of Clonal Line 
     Materials and Methods 
       [0025]    Proprietary Oregano Clonal Lines. 
         [0026]    Seventeen oregano selections identified from the previous genetic screening were chosen to be planted under field conditions at a field site located close to Indianola, Iowa. The carvacrol content of the 14 high-carvacrol (Hi-C) oregano clonal lines and the thymol content of the three high-thymol (Hi-T) clonal lines grown under greenhouse conditions are given in Table 3. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Carvacrol and thymol content of the selected 
               
               
                 oregano clonal lines grown in the greenhouse 
               
             
          
           
               
                   
                 Clonal line* 
                 Carvacrol % 
                 Thymol % 
               
               
                   
                   
               
               
                   
                 KI-Ov1750 
                 5.140 
                 0.006 
               
               
                   
                 KI-Ov1817 
                 4.401 
                 0.039 
               
               
                   
                 KI-Ov1835 
                 4.408 
                 — 
               
               
                   
                 KI-Ov1680 
                 4.412 
                 0.010 
               
               
                   
                 KI-Ov1689 
                 4.326 
                 0.041 
               
               
                   
                 KI-Ov1786 
                 4.309 
                 0.025 
               
               
                   
                 KI-Ov1781 
                 4.282 
                 0.006 
               
               
                   
                 KI-Ov1826 
                 4.258 
                 0.007 
               
               
                   
                 KI-Ov1843 
                 4.137 
                 — 
               
               
                   
                 KI-Ov1676 
                 4.156 
                 0.038 
               
               
                   
                 KI-Ov1796 
                 4.115 
                 — 
               
               
                   
                 KI-Ov1765 
                 4.081 
                 0.020 
               
               
                   
                 KI-Ov1721 
                 4.013 
                 — 
               
               
                   
                 KI-Ov1791 
                 4.019 
                 0.004 
               
               
                   
                 KI-Ov1855 
                 0.385 
                 3.852 
               
               
                   
                 KI-Ov1850 
                 0.281 
                 3.767 
               
               
                   
                 KI-Ov2098 
                 0.063 
                 3.135 
               
               
                   
                   
               
               
                   
                 — means undetectable levels 
               
             
          
         
       
     
         [0027]    Establishment of Field Plots. 
         [0028]    Selected oregano clonal lines were asexually propagated as rooted cuttings at a propagation greenhouse in Texas for transplanting in late May, 2011. Approximately 38,000 plants, representing 17 selections were transplanted into field plots totaling 3.1 acres during May-June 2011, near Indianola, Iowa. Multiple strategies of weed control were employed including, mechanical tillage, hand wicking with Glyphosate, hand hoeing, broadcast of selective grass herbicide (Poast), and finally broadcast spraying of a tank mix for grasses, sedges, broadleaves and weed seed germination (Poast/Basagran/Sinbar). 
         [0029]    Tissue Sampling. 
         [0030]    Leaf tissue was collected from each of the selections once during September, 2011 after full establishment, for carvacrol and thymol analysis. During the growing period in 2012, leaf tissue from each of the clonal lines was collected once every two weeks from April to September 2012. The top 4″-6″ of leaf tissue including stems was collected from each clonal line; air dried for 48 hours and sent for analysis. A rapid method developed and validated for the determination of carvacrol and thymol in oregano dried leaf tissue was used to quantitate carvacrol and thymol content in these lines. Three replicate extractions were carried out from each bulk leaf tissue sample representing each clonal line. Carvacrol and thymol contents were determined on a per unit dry weight basis and means were calculated for statistical comparison. 
         [0031]    Phenotype Vigor Scores. 
         [0032]    All the clonal lines were scored for their overall vigor and growth on the basis of a visual assessment ranging from 1-5. A score of 1 was given for oregano lines with very poor vigor and growth; while a score of 5 was given to oregano lines with the best vigor and growth. 
         [0033]    Data Analysis. 
         [0034]    Statistical analysis was performed on the data obtained from the 17 clonal lines, using Stat Centurion Graphics in order to determine differences among clonal lines for carvacrol and thymol accumulation. Carvacrol and thymol content were analyzed using a general linear model. Fisher&#39;s least significant difference (LSD) was used to discriminate the means and was computed using multiple range tests. 
       Results 
       [0035]    Variation for Carvacrol and Thymol. 
         [0036]    There was a significant variation among clonal lines for both carvacrol and thymol content (Table 4). There was also a significant variation in sampling time for carvacrol and thymol content for each clonal line. A significant genotype (clonal line)×date of sampling was also observed (Table 4). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Analysis of variance (ANOVA) for carvacrol and thymol 
               
               
                 content among genotypes sampled at different dates 
               
             
          
           
               
                   
                 Carvacrol 
                 Thymol 
               
             
          
           
               
                 Source 
                 Df 
                 F-Ratio 
                 P-Value 
                 Df 
                 F-Ratio 
                 P-Value 
               
               
                   
               
             
          
           
               
                 Clonal lines 
                 13 
                 1077.33 
                 0.0000 
                 2 
                 54.62 
                 0.0028 
               
               
                 Replicate 
                 2 
                 0.93 
                 0.3941 
                 2 
                 0.12 
                 0.8874 
               
               
                 Date of sampling 
                 10 
                 5248.78 
                 0.0000 
                 10 
                 248.42 
                 0.0000 
               
               
                 Clonal lines*Date 
                 130 
                 162.16 
                 0.0000 
                 20 
                 76.83 
                 0.0004 
               
               
                 of sampling 
               
               
                   
               
             
          
         
       
     
         [0037]    Carvacrol and thymol content during Year 1. 
         [0038]    The field plots of the clonal lines were transplanted during May, 2011 and were established by August, 2011. Leaf tissue samples collected from the 1 st  year growth showed reasonable carvacrol/thymol content despite experiencing transplanting stress, heavy rain and waterlogged soils ( FIG. 1 ). 
         [0039]    Carvacrol and Thymol Content During Year 2. 
         [0040]    The carvacrol and thymol content of clonal lines were monitored throughout the growing season in Year 2. Carvacrol and thymol content from the leaf tissue collected semi-monthly (or bi-weekly) from April to September is shown in Table 5. The carvacrol and thymol content of the clonal lines varied with growing stages throughout the growing season. During the spring regrowth period in April, the carvacrol and thymol content, irrespective of the clonal line, was at the lowest level. A gradual increase in carvacrol or thymol content was observed in all the clonal lines starting from April onwards and reached maximum levels in June. 
         [0041]    Across clonal lines and sampling time, the highest carvarol content was around 7% in clonal line KI-Ov1750. This clonal line consistently accumulated the highest levels of carvacrol from May to September; followed by KI-Ov1791, KI-Ov1843, KI-Ov1721, and KI-Ov1835. Amongst the three Hi-T lines planted, clonal line KI-Ov1855 did not survive the winter very well; and clonal line KI-Ov1850 showed the highest levels of thymol accumulation at &gt;5% on a dry matter basis. Thymol hyper-accumulating clonal lines of oregano were much rarer and more difficult to find than the carvacrol hyper-accumulating lines. 
         [0042]    All lines were harvested twice in 2012; the first harvest during the first week of June and second harvest during the first week of September. Five Hi-C lines KI-Ov1750, KI-Ov1721, KI-Ov1791, KI-Ov1843, and KI-Ov1835 showed the highest carvacrol content with KI-Ov1750, being the highest at the time of harvest. Again during the 2 nd  harvest in September, a similar trend was observed. At the time of the 2 nd  harvest, KI-Ov1750 had significantly higher levels of carvacrol than other Hi-C clonal lines. On the other hand, maximum accumulation of thymol was observed only in June in clonal line KI-Ov1850 (5.16%) and the content had declined by 50% at the time of second harvest in September. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Carvacrol % and thymol % from clonal lines collected from April to September §   
               
             
          
           
               
                   
                 April 
                 April 
                 May 
                 May 
                 June 
                 July 
                 July 
                 August 
                 August 
                 August 
                 Sept 
               
               
                 Sample 
                 9 th   
                 27 th   
                 8 th   
                 22 nd   
                 4 th   
                 5th 
                 20 th   
                 3 rd   
                 17 th   
                 31 th   
                 7 th   
               
               
                   
               
             
          
           
               
                 1750 
                 3.03 c   
                 3.78 c   
                 5.76 
                 6.35 
                 6.97 a   
                 6.17 a   
                 5.09 e   
                 5.16 c   
                 6.21 a   
                 6.62 a   
                 5.54 a   
               
               
                 1817 
                 2.71 d   
                 2.98 h   
                 4.35 
                 6.15 
                 6.94 a   
                 4.66 e   
                 6.27 a   
                 4.00 l   
                 4.99 f   
                 4.40 fg   
                 3.18 h   
               
               
                 1826 
                 3.19 b   
                 4.12 b   
                 5.85 
                 6.47 
                 6.32 b   
                 5.39 b   
                 4.45 
                 4.42 g   
                 5.55 d   
                 4.24 g   
                 3.04 i   
               
               
                 1835 
                 3.90 a   
                 4.89 a   
                 5.92 
                 6.91 
                 6.30 b   
                 4.72 e   
                 4.71 f   
                 4.50 fg   
                 6.02 b   
                 5.58 c   
                 4.59 c   
               
               
                 1689 
                 2.40 f   
                 3.63 de   
                 3.57 
                 5.16 
                 6.24 bc   
                 4.31 f   
                 4.36 g   
                 4.64 f   
                 4.87 fg   
                 4.46 f   
                 3.08 hi   
               
               
                 1791 
                 3.17 b   
                 4.21 b   
                 4.93 
                 6.81 
                 6.22 bc   
                 5.04 cd   
                 5.72 c   
                 5.16 c   
                 4.71 g   
                 4.19 g   
                 2.77 j   
               
               
                 1796 
                 2.99 c   
                 3.79 c   
                 5.94 
                 6.46 
                 6.13 c   
                 5.04 cd   
                 5.05 e   
                 3.57 j   
                 5.23 e   
                 4.96 e   
                 2.76 j   
               
               
                 1765 
                 2.70 d   
                 3.43 f   
                 5.00 
                 6.20 
                 5.95 d   
                 4.06 g   
                 4.67 f   
                 4.85 de   
                 5.01 f   
                 4.28 g   
                 4.22 e   
               
               
                 1721 
                 3.15 b   
                 4.09 b   
                 4.82 
                 5.73 
                 5.93 d   
                 5.19 c   
                 5.09 e   
                 5.22 b   
                 5.48 d   
                 5.02 e   
                 2.75 j   
               
               
                 1843 
                 3.87 a   
                 3.58 e   
                 4.54 
                 5.80 
                 5.78 de   
                 4.03 h   
                 5.31 d   
                 5.06 bc   
                 5.27 e   
                 5.53 c   
                 4.47 cd   
               
               
                 1676 
                 2.75 d   
                 3.30 fg   
                 4.43 
                 5.77 
                 5.42 e   
                 4.05 h   
                 4.50 g   
                 4.69 f   
                 5.17 e   
                 5.30 d   
                 4.41 d   
               
               
                 1786 
                 2.42 f   
                 3.74 cd   
                 3.79 
                 5.86 
                 5.18 f   
                 3.94 h   
                 3.87 h   
                 4.21 h   
                 4.78 g   
                 4.20 g   
                 3.77 g   
               
               
                 1781 
                 2.55 e   
                 3.26 g   
                 4.06 
                 4.98 
                 5.01 f   
                 5.16 c   
                 5.99 b   
                 5.86 a   
                 5.89 c   
                 5.74 b   
                 4.76 b   
               
               
                 1680 
                 2.04 g   
                 2.59 i   
                 2.86 
                 3.98 
                 4.17 g   
                 3.47 i   
                 3.98 h   
                 4.93 d   
                 4.26 h   
                 4.22 g   
                 3.93 f   
               
               
                 LSD 
                 0.10 
                 0.14 
                 0.186 
                 0.178 
                 0.205 
                 0.175 
                 0.165 
                 0.146 
                 0.162 
                 0.164 
                 0.154 
               
               
                 1850 £   
                 1.86 
                 2.35 
                 4.16 
                 4.51 
                 5.16 
                 4.09 
                 2.69 
                 2.36 
                 2.87 
                 2.77 
                 2.53 
               
               
                 2098 £   
                 1.63 
                 1.83 
                 2.25 
                 3.32 
                 3.23 
                 2.43 
                 2.45 
                 2.17 
                 2.95 
                 3.15 
                 2.47 
               
               
                   
               
               
                   £ KI-Ov1850 and KI-Ov2098 represents Hi-T lines 
               
               
                   § Means bearing different superscripts are significantly different (p &lt; 0.05). 
               
             
          
         
       
     
         [0043]    The gradual increase in carvacrol content in the five Hi-C clonal lines (1750, 1721, 1791, 1835 and 1843); and thymol content in clonal line KI-Ov1850 is shown in the  FIG. 2 , below. All the Hi-C clonal lines showed the highest level of carvacrol content during the first week of June and again during the last week of August. The Hi-T clonal line KI-Ov1850, showed the highest thymol content in June with no second peak later in the summer. 
         [0044]    HPLC was performed on prior art Greek Oregano ( FIG. 8 ) and the present invention ( FIG. 9 ), demonstrating the high thymol and low carvacrol content of the present subject line. 
         [0045]    Phenotype Vigor Scores. 
         [0046]    All the clonal lines were scored for the overall plant vigor and growth during the growing period. Clonal lines (1843, 1791, 1835 and 1750) consistently exhibited vigorous growth throughout the growing period ( FIG. 3 ). Of all the lines, KI-Ov1843 flowered much earlier than the other lines. 
       DISCUSSION 
       [0047]    Previously, we had identified several proprietary Hi-C (14 clonal lines with carvacrol content &gt;4%) and Hi-T (3 clonal lines with thymol content &gt;3%) oregano clonal lines under the greenhouse conditions. These lines were grown in Iowa to monitor the accumulation of carvacrol and thymol under field conditions during 2011. Understanding the fluctuations of carvacrol and thymol content during the growing season is essential in order to harvest biomass in time to maximize the yield of carvacrol and thymol. The first year was mainly focused on establishing these clonal lines in the field. The carvacrol and thymol accumulation was not at the maximum during the first year of growth. 
         [0048]    During the 2 nd  year of growth in 2012, carvacrol and thymol content was monitored in these lines throughout the growing season from April to September. Clonal line KI-Ov1750 consistently accumulated the highest levels of carvacrol throughout the growing season. Other clonal lines (KI-Ov1791, KI-Ov1843, KI-Ov1721, and KI-Ov1835) also showed higher levels of carvacrol (&gt;5%) relative to the rest of the clonal lines tested. Amongst the three Hi-T lines planted, KI-Ov1855 did not survive the winter, while KI-Ov1850 showed the highest levels of thymol accumulation in Year 2. The data from Year 1 was not correlated to Year 2 since Year 1 was mostly considered as the establishment year. Clonal line KI-Ov1750 had the highest content of carvacrol at up to 7% on a dry matter basis at the time of harvest in June; and 6.7% at the time of harvest in September. Clonal line KI-Ov1850 had the highest content of thymol at up to 5.2% on a dry matter basis at the time of harvest in June. The lack of a second peak accumulation later in the summer may have been a function of the very hot and dry conditions experienced during 2012. In general, the most common oregano grown commercially is Greek oregano ( Origanum vulgare , sub species  hirtum ). Greek oregano has also been reported to have unusually high levels of carvacrol and thymol. Assessment of carvacrol and thymol content in Greek oregano and common oregano (cv. Hot &amp; Spicy) was shown by us to be &lt;3% on a dry matter basis. Carvacrol and thymol hyper-accumulation &gt;5% on a dry matter basis has not previously been reported to our knowledge. This makes clonal lines 1750, 1721, 1791, 1843 and 1850 unique and patentable as commercially viable sources of carvacrol and thymol, respectively. Clonal lines (1850, 1843, 1791, 1835 and 1750) consistently exhibited vigorous growth throughout the growing period despite the fact that these plots underwent water stress due to lack of rain during the summer of 2012. KI-Ov1843 flowered much earlier than the other lines during Year 2. 
         [0049]    The essential oil and their contents were reported to be at the highest during the pre-flowering stage 5 . All the clonal lines were flowering at the time of harvest during the first week of June and yet the carvacrol and thymol content were found to be at the maximum levels. Hence oregano can be harvested either pre-flowering or at flowering in order to maximize the carvacrol and thymol yield. Under suitable growing conditions, with adequate rainfall, these lines could be harvested twice during the growing season; once during late May and again during the last week of August. Hi-C clonal lines KI-Ov1750 and KI-Ov1721; and Hi-T clonal line, KI-Ov1850 will be advanced to large scale commercial field planting. 
       Evidence of Uniformity and Stability 
       [0050]    No variants of any kind have been observed since the clonal line KI-Ov1850 was identified, indicating the stability and uniformity of the genotype. It is clear from these results that the KI-Ov1850 clonal line is stable and reproduces true to type in successive generations of asexual reproduction. 
       Statement of Distinction 
       [0051]    KI-Ov1850 is more vigorous and generates more biomass per acre than any other oregano clonal line we have observed or have knowledge of. Due to vigorous vegetative growth this genotype can be harvested multiple times in a season and has the potential of growing in any temperate climate. 
         [0052]    DNA sample from KI-Ov1850 extracted from tissue was sequenced by Data2Bio, LLC (Ames, Iowa) in two Ilumina HiSeq 2000 paired-end (PE) lanes (lanes 5 and 6). Each genomic DNA sample was prepared using the Illumina protocol outlined in “TruSeq DNA Sample Preparation Guide” (Catalog# PE-940-2001). First, gDNA was fragmented (Covaris Sheraing duration time 120 sec) to produce 300-400 bp inserts. The DNA fragment ends were repaired and phosphorylated using Klenow, T4 DNA polymerase and T4 polynucleotide kinase. Next, an “A” base was added to the 3′ end of the blunted fragments, followed by ligation of Illumina adapters via T-A mediated ligation. The ligated products were size selected by AMPure XP Beads and then PCR amplified using Illumina primers. The library size and concentration were determined using an Agilent Bioanalyzer 1000 chip. Raw reads from both lanes were combined into a single archive and summarized in Table 6. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Summary of Raw Sequence Reads 
               
             
          
           
               
                   
                 RAW READS 
               
             
          
           
               
                 File Name 
                 No. Reads 
                 Base Pairs 
                 Read Length (BP) 
               
               
                   
               
               
                 KI-1850-1-1_1 
                 488,327,668 
                 49,321,094,468 
                 101 
               
               
                 KI-1850-1-1_2 
                 488,327,668 
                 49,321,094,468 
                 101 
               
               
                 KI-1850 Total 
                 976,655,336 
                 98,642,188,936 
                 101 
               
               
                   
               
             
          
         
       
     
         [0053]    The nucleotides of each raw read were scanned for low quality. Bases with PHRED quality value &lt;15 (out of 40) (Ewing, B. and P. Green, 1998 Base-calling of automated sequencer traces using phred. II. Error probabilities.  Genome Res.  8(3): 186-194), i.e., error rates of &lt;0.03%, were removed by the trimming pipeline. Each read was examined in two phases. In the first phase reads were scanned starting at each end and nucleotides with quality values lower than the threshold were removed. The remaining nucleotides were then scanned using overlapping windows of 10 bp and sequences beyond the last window with average quality value less than the specified threshold were truncated. The trimming parameters were referred to the trimming software, Lucy (Chou, R H., G. Sutton, A, Glodek and J, Scott, 1998 Lucy—A Sequence Cleanup Program, pp. in  Proceedings of the Tenth Annual Genome Sequencing and Annotation Conference  ( GSAC X ), Miami, Fla.). A statistical summary of raw reads is shown in Table 7. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Summary of Raw Reads Trimming 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 RAW READS 
               
             
          
           
               
                 File Name 
                 No. Reads 
                 Base Pairs 
                 Read Length (BP) 
               
               
                   
               
               
                 KI-1850-1-1_1 
                 488,327,668 
                 49,321,094,468 
                 101 
               
               
                 KI-1850-1-1_2 
                 488,327,668 
                 49,321,094,468 
                 101 
               
               
                 KI-1850 Total 
                 976,655,336 
                 98,642,188,936 
                 101 
               
               
                   
               
             
          
           
               
                   
                 TRIMMED READS 
               
             
          
           
               
                   
                 No. Reads 
                 Base Pairs 
                   
               
               
                 File Name 
                 (%, trimmed/raw) 
                 (%, trimmed/raw) 
                 Read Length (BP) 
               
               
                   
               
               
                 KI-1850-1-1_1 
                 486,718,410 (99.7%) 
                 47,795,202,034 (96.9%) 
                 99 
               
               
                 KI-1850-1-1_2 
                 477,176,905 (97.7%) 
                 46,318,741,222 (93.9%) 
                 98 
               
               
                 KI-1850 Total 
                 963,895,315 (98.7%) 
                 94,113,943,256 (95.4%) 
                 98 
               
               
                   
               
             
          
         
       
     
         [0054]    The raw sequences have been deposited and uploaded to the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, Bethesda, Md., and those sequences are incorporated herein in their entirety by this reference. The submission accession assigned by NCBI is SRX220840 and it was released to the public on May 7, 2013. Those skilled in the art can analyze the deposited raw sequence information to determine the genetic sequence of KI-Ov1850. 
         [0055]    The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art that have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention. 
       REFERENCES 
       [0000]    
       
         1. Bendahou M, Muselli A, Grignon-Dubois M, Benyoucef M, Desjobert J M, Bernardini A F, Costa J 2008 Antimicrobial activity and chemical composition of  Origanum glandulosum  Desf. essential oil and extract obtained by microwave extraction: Comparison with hydrodistillation. Food Chemistry. 106:132-139 . SA -10-02422 
         2. Manzanilla E G, Perez J F, Martin M, Kamel C, Baucells F and Casa J 2004 Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 82, 3210-3218.  SA -08-03666 
         3. Allan P and G. Bilkei. 2005 Oregano improves reproductive performance of sows. Theriogenology 63, 716-721 . SA -10-00360 
         4. Sangwan, N. S, Farooqi, A. H. A, Shabih F, and Sangwan R. S. 2001 Regulation of essential oil production in plants. Plant Growth Regulation. 34. 3-21  SA -12-02838 
         5. Toncer O, Karaman S, Kizil S and Diraz E. 2009 Changes in essential oil composition of oregano ( O. onites  L.) due to diurnal variations at different developmental stages. Notulae Botanicae Hord Agrobotanici 37:177-181  SA -12-02837 
         6. Yaldiz G, Sekeroglu N, Ozguven M and Kirpik M 2005. Seasonal and diurnal variability of essential oil and its components in  Origanum onites  L. grown in the ecological conditions of Cukurova. Grasas. Y. Aceites. 56: 254-258  SA -12-02839 
         7. Dunford N T and Vazquez R. S. 2005 Effect of water stress on plant growth and thymol and carvacrol concentrations in Mexican oregano grown under controlled conditions. Journal of Applied Horticulture. 7:20-22 . SA -12-02863 
         8. Putievsky E, Dudai N, and Ravid U. 2005 Cultivation, selection and conservation of Oregano species in Israel conditions. Proceedings of the IPGRI International Workshop on Oregano 8-12 May 1996, CIHEAM, Valenzano, Bari, Italy  SA -12-02864