The Open Agriculture Journal




ISSN: 1874-3315 ― Volume 13, 2019
RESEARCH ARTICLE

Morphological, Biochemical, and Molecular Analysis of Origanum vulgare L.



Concetta Lotti1, Luigi Ricciardi2, Guglielmo Rainaldi3, Claudia Ruta4, Waed Tarraf4, Giuseppe De Mastro4
1 Department of the Sciences of Agriculture, Food and Environment, University of Foggia, Foggia, Via Napoli, 25-71122, Italy
2 Department of Soil, Plant and Food Sciences, Plant Genetics and Breeding Unit University of Bari, Bari, Italy
3 Department of Biosciences, Biotechnologies and Biopharmaceuticals, University of Bari, Bari, Italy
4 Department of Agriculture and Environmental Science, University of Bari, Bari, Italy

Abstract

Background:

The variation on morphological, biochemical, and genetic characters is very important in germplasm management and conservation strategies.

Objective:

To determine the diversity of 23 accessions from Origanum vulgare L. and a commercial cultivar on the basis of agronomical, biochemical and genetic features.

Methods:

Different characters related to vegetative growth and essential oil production were studied while the genetic relationships between the individuals were evaluated with the use of Amplified Fragment Length Polymorphism.

Results:

Despite the accessions exhibited cymyl- and acyclic-compounds, all the essential oil chemotypes according to the prevalence of essential oil fractions were phenotypically varied. A considerable amount of biomass with maximum values in plant height was achieved by thymol/γ-terpinene chemotype and carvacrol chemotype making them of particular interest for the production of high-quality plant material and further for the mechanical harvest. The AFLP analysis, performed using 10 primer combinations, to obtain a dendrogram of genetic similarity, revealed a genetic variability that could be useful for the selection of the proper genetic groups in future breeding programs.

Conclusion:

We identified two chemotypes thymol/γ-terpinene and carvacrol with their suitability for the production of biomass and essential oil and for the mechanical harvest. The results of the molecular characterization of the species may support and contribute to breeding programmes for agronomic and biochemical traits.

Keywords: Lamiaceae, Origanum vulgare L., Essential oil, Chemical composition, GC-MS, AFLP.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 116
Last Page: 124
Publisher Id: TOASJ-13-116
DOI: 10.2174/1874331501913010116

Article History:

Received Date: 28/06/2019
Revision Received Date: 18/08/2019
Acceptance Date: 19/08/2019
Electronic publication date: 30/09/2019

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© 2019 Lotti et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of the Sciences of Agriculture, Food and Environment, University of Foggia, Foggia, Via Napoli, 25-71122, Italy; Tel/Fax: +39 0881 589347; E-mail: concetta.lotti@unifg.it





1. INTRODUCTION

The genus Origanum belongs to Lamiaceae family, which grows wildly in Mediterranean areas and northern Africa [1Skoula M, Harborne JB. The taxonomy and chemistry of Origanum Oregano: The genera Origanum and Lippia 2002; 67-108.]. It includes various species, subspecies, varieties and hybrids that can be distinguished individually with a high variation in morphological and chemical features [2Azizi A, Wagner C, Honermeier B, Friedt W. Intraspecific diversity and relationship between subspecies of Origanum vulgare revealed by comparative AFLP and SAMPL marker analysis. Plant Syst Evol 2009; 281(1-4): 151-60.
[http://dx.doi.org/10.1007/s00606-009-0197-1]
]. According to the Ietswaart’s classification [3Ietswaart JH. A taxonomic revision of the genus Origanum (Labiatae) 1980.
[http://dx.doi.org/10.1007/978-94-009-9156-9]
], no less than 10 sections, consisting of 49 taxa, had to be recognized on the basis of geographical distribution. Origanum is a monospecific section which includes six subspecies of Origanum vulgare: Gracile (Kock) Ietswaart, glandulosum (Desfontaines) Ietswaart, hirtum (Link) Ietswaart, vulgare L., virens (Hoffmannsegg et Link) Ietswaart, and viride (Boissier) Hayek. They were recognized based on the indumentum, number of sessile glands on leaves, bracts and calyces and the size and colour of bracts and flowers. The southernmost range of O. vulgare in Europe comprises three subspecies: O. vulgare L. subsp. Glandulosum (Desfontaines) Ietswaart, O. vulgare L. subsp. hirtum (Link) Ietswaart, and O. vulgare L. subsp. gracile (Koch) Ietswaart, found to be rich in the EOs while the northern part that includes the other two, O. vulgare L. subsp. virens (Hoffmannsegg et Link) Ietswaart, O. vulgare L. subsp. vulgare L. and O. vulgare L. subsp. viride (Boissier) Hayek, pointed out to be poor in EOs [4Kokkini S. Taxonomy, diversity and distribution of Origanum. Proceedings of the IPGRI International Workshop on Oregano Institute of Plant Genetics and Crop Plant Research 1996; 8-12.].

Oregano plants are known to possess a special composition of Essential Oil (EO) which is of great interest for pharmaceutical and industrial market [5Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils-a review. Food Chem Toxicol 2008; 46(2): 446-75.
[http://dx.doi.org/10.1016/j.fct.2007.09.106] [PMID: 17996351]
, 6Nieto G. Biological Activities of Three Essential Oils of the Lamiaceae Family. Medicines (Basel) 2017; 4(3): 63.
[http://dx.doi.org/10.3390/medicines4030063] [PMID: 28930277]
]. Furthermore, it was grouped into different chemotypes: Acyclic, cymyl, bicyclic, and/or bornane type compounds depending on the predominance of particular components as (a) linalool, linalyl acetate, β-ocimene and myrcene; (b) p-cymene, γ-terpinene, p-cymene-8-ol, thymol methyl ether, carvacrol methyl ether, thymoquinone, thymol and carvacrol; (c) sabinene, trans-/cis-sabinene hydrate and their acetate; and (d) camphor, borneol, bornyl acetate, respectively [1Skoula M, Harborne JB. The taxonomy and chemistry of Origanum Oregano: The genera Origanum and Lippia 2002; 67-108., 4Kokkini S. Taxonomy, diversity and distribution of Origanum. Proceedings of the IPGRI International Workshop on Oregano Institute of Plant Genetics and Crop Plant Research 1996; 8-12., 7D’Antuono L. Variability of Essential Oil Content and Composition of Origanum vulgare L. Populations from a North Mediterranean Area (Liguria Region, Northern Italy). Ann Bot 2000; 86(3): 471-8.
[http://dx.doi.org/10.1006/anbo.2000.1205]
-10Shafiee-Hajiabad M, Novak J, Honermeier B. Content and composition of essential oil of four Origanum vulgare L. accessions under reduced and normal light intensity conditions. J Appl Bot Food Qual 2016; 89: 126-34.].

In this context, the active compounds of EO are not only responsible for the aroma and flavor of oregano, making O. vulgare the most popular spice for food production, but are also emerged as effective source to provide a vast range of biological activities with antioxidant [11Yan F, Azizi A, Janke S, Schwarz M, Zeller S, Honermeier B. Antioxidant capacity variation in the oregano (Origanum vulgare L.) collection of the German National Genebank. Ind Crops Prod 2016; 92: 19-25.
[http://dx.doi.org/10.1016/j.indcrop.2016.07.038]
], antifungal [12Soković M, Glamočlija J, Marin PD, Brkić D, van Griensven LJ. Antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model. Molecules 2010; 15(11): 7532-46.
[http://dx.doi.org/10.3390/molecules15117532] [PMID: 21030907]
], antibacterial [13De Falco E, Mancini E, Roscigno G, Mignola E, Taglialatela-Scafati O, Senatore F. Chemical composition and biological activity of essential oils of Origanum vulgare L. subsp. vulgare L. under different growth conditions. Molecules 2013; 18(12): 14948-60.
[http://dx.doi.org/10.3390/molecules181214948] [PMID: 24304588]
], anticancer [14Elshafie HS, Armentano MF, Carmosino M, Bufo SA, De Feo V, Camele I. Cytotoxic activity of Origanum vulgare L. on Hepatocellular carcinoma cell line HepG2 and evaluation of its biological activity. Molecules 2017; 22(9): 1435.
[http://dx.doi.org/10.3390/molecules22091435] [PMID: 28867805]
], insecticidal [15Abdelgaleil SAM, Mohamed MIE, Shawir MS, Abou-Taleb HK. Chemical composition, insecticidal and biochemical effects of essential oils of different plant species from Northern Egypt on the rice weevil, Sitophilus oryzae L. J Pest Sci 2016; 89(1): 219-29.
[http://dx.doi.org/10.1007/s10340-015-0665-z]
], herbicidal [16De Mastro G, Fracchiolla M, Verdini L, Montemurro P. Oregano and its potential use as bioherbicide. Acta Hortic 2006; 335-46.
[http://dx.doi.org/10.17660/ActaHortic.2006.723.46]
] and nematicidal [17Barbosa P, Faria JMS, Mendes MD, et al. Bioassays against pinewood nematode: Assessment of a suitable dilution agent and screening for bioactive essential oils. Molecules 2012; 17(10): 12312-29.
[http://dx.doi.org/10.3390/molecules171012312] [PMID: 23085666]
] properties.

The quantitative and qualitative profile of EO is principally influenced by the genotype, the environmental factors, the growing conditions, and the geographical locations [18Johnson CB, Kazantzis A, Skoula M, Mitteregger U, Novak J. Seasonal, populational and ontogenic variation in the volatile oil content and composition of individuals of Origanum vulgare subsp. Hirtum, assessed by GC headspace analysis and by SPME sampling of individual oil glands. Phytochem Anal 2004; 15(5): 286-92.
[http://dx.doi.org/10.1002/pca.780] [PMID: 15508832]
, 21Mastro G, Tarraf W, Verdini L, Brunetti G, Ruta C. Essential oil diversity of Origanum vulgare L. populations from Southern Italy. Food Chem 2017; 235: 1-6.
[http://dx.doi.org/10.1016/j.foodchem.2017.05.019] [PMID: 28554612]
] leading to a noticeable variation between populations and accessions of O. vulgare and within the genus Origanum by itself [22Kokkini S, Vokou D. Carvacrol-rich plants in Greece. Flavour Fragrance J 1989; 4(1): 1-7.
[http://dx.doi.org/10.1002/ffj.2730040102]
-24Kintzios SE. Profile of multifaceted prince of the herbs.Oregano The genera Origanum and Lippia (Medicinal and aromatic plants - industrial profiles) 2002; 3-10.].

For better management of oregano genetic resources, extensive efforts to improve cultivars and restore endangered varieties [25Karpa K, Bhat KV, Ayad WG, Hodgkin T. Molecular Tools in Plant Genetic Resources Conservation: A Guide to the Tecnologies 1997.] with a higher content of bioactive compounds are urgently needed. Moreover, extending the research about the morphological and chemical diversity of oregano populations is very important. Furthermore, oriented exploitation of its germplasm in Italy could enable plant breeders of selecting the interesting parental sources for breeding programs [26Kokkini S, Karousou R, Vokou D. Pattern of geographic variations of Origanum vulgare trichomes and essential oil content in Greece. Biochem Syst Ecol 1994; 22(5): 517-28.
[http://dx.doi.org/10.1016/0305-1978(94)90046-9]
].

The safeguard of genetic resources implies not just the collection and the storage of material but also the evaluation and the characterization through morphological, biochemical and molecular analysis. All this should be done to prevent the conservation of redundant genetic materials that do not induce important variation and that contribute to increasing the biodiversity management cost [27Ricciardi L, Filippetti A. L’erosione genetica di specie agrarie in ambito mediterraneo: Rilevanza del problema e strategie di intervento. Options Méditerranéennes 2000; 53(53): 191-223., 28Govindaraj M, Vetriventhan M, Srinivasan M. Importance of genetic diversity assessment in crop plants and its recent advances: An overview of its analytical perspectives. Genet Res Int 2015; 1-14.
[http://dx.doi.org/10.1155/2015/431487] [PMID: 25874132]
]. Moreover, the assessment of genetic diversity within collections becomes significant to identify genotypes of agronomic interest, useful in crop-improvement programs.

The molecular analysis, able to evaluate DNA polymorphism to univocally characterize and estimate the genetic distances among plant materials, was performed using AFLP (Amplified Fragment Length Polymorphism) markers on DNA isolated from leaves. The choice of AFLPs is due to the potential capability of this technique to detect a large level of polymorphism, particular when combined with the fluorescence detection system. AFLP is used for different applications, such as analysis of population polymorphism, phylogenetic relationship, genetic diversity assessment, identification of loci linked to economically valuable traits, genetic maps design and varietal fingerprinting [29Renganayaki K, Read JC, Fritz AK. Genetic diversity among Texas bluegrass genotypes (Poa arachnifera Torr.) revealed by AFLP and RAPD markers. Theor Appl Genet 2001; 102(6-7): 1037-45.
[http://dx.doi.org/10.1007/s001220000521]
-32Paun O, Schönswetter P. Amplified fragment length polymorphism: An invaluable fingerprinting technique for genomic, transcriptomic, and epigenetic studies. Methods Mol Biol 2012; 862: 75-87.
[http://dx.doi.org/10.1007/978-1-61779-609-8_7] [PMID: 22419490]
]. This method also can be used by breeders to preliminary assess the initial genetic material to plan the strategy of crosses, to identify the best combinations of genotypes, and for general selection [33Lanteri S, Acquadro A, Saba E, Portis E. Molecular fingerprinting and evaluation of genetic distances among selected clones of globe artichoke (Cynara cardunculus L. var. scolymus L.). J Hortic Sci Biotechnol 2004; 79(6): 863-70.
[http://dx.doi.org/10.1080/14620316.2004.11511858]
, 34Tuberosa R, Frison E, Graner A. Genomics of Plant Genetic Resources 2014.].

The aim of the current study was to determine the morphological and the phytochemical variability within different O. vulgare accessions collected from various geographical areas of Southern Italy and to show the usefulness of AFLP technique in the assessment and genetic characterisation, making possible the identification of promising material to use in future breeding programs besides evaluating the chemical essential oil composition in order to identify genotypes with higher content in carvacrol, thymol and γ-terpinene.

2. MATERIALS AND METHODS

2.1. Plant Material and Experimental Field

A total of 24 accessions of O. vulgare were investigated, 23 wild populations collected, according to Good Agricultural Practices (GAP) [35World Health Organization. WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants. World Health 2003; 99: 67-73.], from different locations of Southern Italy (Apulia, Basilicata and Calabria) along with one commercial cultivar “Sais” (S.A.I.S., S.p.a., Cesena, Italy), belonging to the genus Origanum. Additional information related to the coordinates (i.e latitude, longitude) plus the altitude were provided in Table 1. All oregano plants were identified and then cuttings of each accession were grown at Enrico Pantanelli farm of the University of Bari Aldo Moro (Policoro, Italy). The soil of this experimental farm is loamy (sand 398 g/kg, silt 374 g/kg clay 228 g/kg). The chemical characteristics of soil as described by Pontonio et al. [36Pontonio E, Di Cagno R, Tarraf W, Filannino P, De Mastro G, Gobbetti M. Dynamic and assembly of epiphyte and endophyte lactic acid bacteria during the life cycle of Origanum vulgare L. Front Microbiol 2018; 9: 1372.
[http://dx.doi.org/10.3389/fmicb.2018.01372] [PMID: 29997592]
]: pH 7.7; 2.3% organic matter, 1.7 g/kg total N (Kjeldahl method) and 27.6 mg/kg available P2O5 (Olsen method). The experimental design was a randomized complete block with 3 replications following GAP for medicinal plants [35World Health Organization. WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants. World Health 2003; 99: 67-73.]. The planting distance was 0.75 m between rows and 0.35 m within rows.

2.2. Agronomical Evaluation/Collection Data

The plants were collected during the full flowering between the end of June and the middle of July. Different characters related to vegetative growth and leaves production including days’ number of early flowering, plant height, biomass, and leaf area, were measured with 15 individuals per each population.

2.3. Extraction of Essential Oils

The aerial parts of all the investigated accessions were dried (air drying oven, 35 °C) and then a fraction of each (20 g) was submitted, according to European Pharmacopoeia [37European Pharmacopoeia. Ph Eur 60 2007.], to hydro-distillation (Clevenger apparatus, 4h). The obtained EOs were dried over anhydrous sodium sulfate and stored at 4 °C, in amber vials, until further analysis. The oil content was calculated as v/w on the basis of the dry matter of the initial material.

Table 1
Collection sites and proportions (values of peak area percentages) of metabolic pathway-compounds of studied populations of Origanum vulgare L.


2.4. Analysis of Essential Oils Using Gas Chromatography-Mass Spectrometry

The GC-MS analysis was done by Agilent 6890N gas chromatograph coupled to an Agilent mass spectrometer 5973N (Agilent Technologies, Cernusco sul Naviglio, MI, Italy). The separation of the volatile compounds was carried out on a capillary column HP-5MS (30m ×0.25mm × 0.25μm film thicknesses). The carrier gas was Helium (flow rate, 1.1 ml min-1), the temperature of the injector and transfer line were set to 250 and 300, respectively. The initial column temperature was 60 °C, then ramped to 110 °C at the rate of 2 °C min-1 and again to 220 °C at 10 °C min-1. 1.0 μl of the sample was submitted to GC–MS using the split mode (split ratio 1:50). A mixture of aliphatic hydrocarbons (C8−C30; Sigma, IT-Milan) in n-hexane was directly injected into the GC under the same analytical conditions in order to calculate the Retention Indices (RIs) of peaks in the chromatogram. All the mass spectra were acquired using the Electron-Impact (EI) mode with an ionization voltage of 70 eV [38Tirillini B, Pagiotti R, Angelini P, Pintore G, Chessa M, Menghini L. Chemical composition and fungicidal activity of the essential oil of Laserpitium garganicum from Italy. Chem Nat Compd 2009; 45(1): 103-5.
[http://dx.doi.org/10.1007/s10600-009-9237-x]
].

2.5. Identification of EO Composition

The constituents of the essential oils were identified based on the retention index, mass spectra obtained from Wiley [39Wiley Registry of Mass Spectral Data 6th ed. 1995.], NIST [40NIST. Mass Spectral Library (NIST/EPA/NIH) National Institute of Standards and Technology 1994.] and Adams [41Adams RP. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy 2001.] libraries and those reported in the literature. The content of each component corresponded to the relative percentage of the total peak area without the use of correction factors [42Marriot PJ, Shellie R, Cornwell C. Gas chromatographic technologies for the analysis of essential oils. J Chromatogr A 2001; 936(1-2): 1-22.
[http://dx.doi.org/10.1016/S0021-9673(01)01314-0] [PMID: 11760992]
].

2.6. DNA Extraction and AFLP Analysis

DNA was extracted from tissue leaf by mean of a commercial kit (GenEluteTMPlant Genomic DNA Kit -Sigma-Aldrich Chemie, Steinheim, Germany). Quality and quantity of genomic DNA were evaluated both on 0.8% agarose gel electrophoresis, comparing band intensity of DNA samples to Lambda DNA standard and using the Nano Drop 2000 UV-Vis Spectrophotometer (ThermoScientific, Waltham, Massachusetts, US). AFLP analyses were performed as reported by Vos et al. [43Vos P, Hogers R, Bleeker M, et al. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res 1995; 23(21): 4407-14.
[http://dx.doi.org/10.1093/nar/23.21.4407] [PMID: 7501463]
]. DNA (50 ng) was digested with two restriction enzymes: the six-cutter EcoRI and the four-cutter MseI. Restriction fragments size were assessed on 1.5% agarose gel. Adapters were ligated to fragments and ligation products were diluted 1:10 in TE (Tris-HCl 100mM, pH 8.0, EDTA 0.01 mM). Pre-selective and selective PCR was performed as reported in the AFLP-TM Plant Mapping Kit Manual (Applied Biosystems Corp., Norwalk, Connecticut, U.S.) In Tables 2 and 3 sequences of adapters and primers are reported. Labeled fragments obtained by using FAM, NED and JOE EcoRI primers were analyzed on an ABI PRISM 310 Automated DNA Sequencer (Applied Biosystems Corp., Norwalk, Connecticut, U.S.).

Table 2
Adapter and non-selective primer sequences used in the AFLP reactions.


Table 3
Selective primers used in the AFLP experiments and polymorphic level detected on the 24 Origanum genotypes.


2.7. Statistical Analysis

The results obtained from growth and yield characteristics are expressed as mean±standard deviation while the EO components data are presented as mean. All scored bands in AFLP analysis were processed using the Genotyper software (A.B. Corp., Norwalk, Connecticut, U.S.) obtaining a binary matrix based on the presence (1) or absence (0) of each polymorphic DNA fragment detected on the 24 genotypes. The software GenAlex 6.5 [44Peakall R, Smouse P E. GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 2012; 28: 2537e2539.] was used to calculate allele frequencies, Nei distance matrix and to perform PCo analysis (PCoA). A dendrogram of genetic distance was constructed using the Unweigth Pair Group Method with Aritmetic averages (UPGMA) implemented in the Mega software 6.0 [45Kumar S, Nei M, Dudley J, Tamura K. MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 2008; 9(4): 299-306.
[http://dx.doi.org/10.1093/bib/bbn017] [PMID: 18417537]
].

3. RESULTS

The research led to identifying 23 accessions belonging to different sites located in Apulia, Basilicata and Calabria regions- Southern Italy, from which 23 accessions of oregano were collected. All populations were classified as Origanum vulgare L. For a clear presentation of the data, the collected plants were grouped into cymyl-compounds and acyclic-compounds according to the metabolic pathway EO fractions (Table 1). OR13, OR14, OR15 were regarded as acyclic pathway (sum of linalool, linalyl acetate, β-ocimene and myrcene peak area percentage) while all the other populations were exhibited cymyl pathway (sum of p-cymene, γ-terpinene, thymol methyl ether, carvacrol methyl ether, thymol and carvacrol peak area percentage) except OR24 was classified into mixed chemotype including the sabinyl pathway (sum of sabinene, trans-sabinene hydrate and cis-sabinene hydrate peak area percentage) with a presence of sesquiterpenes compounds.

3.1. Growth and Yield Characteristics

Mean and standard deviation values for the studied measured characters are reported in Table 4. Eight chemotypes according to the prevalence of EOs compounds were phenotypically diverse. Plant height was the lowest (52.01cm) in carvacrol methyl ether/γ-terpinene chemotype while it was the highest in oregano populations belonging to carvacrol (76.70cm) and thymol/γ-terpinene (75.20cm) chemotypes. Carvacrol chemotype also reported the maximum values in early flowering days (151.33) compared to 139.67 in linalyl acetate chemotype. The leaf area ranged from 1.01cm2 leaf-1 (chemotype of carvacrol methyl ether/γ-terpinene) to 2.74 cm2 leaf-1 (carvacrol chemotype). In addition, the biomass (g plant-1) harvested from carvacrol chemotype (248.80) was comparable with the other chemotypes (113.33-185.33) but less than thymol/γ-terpinene chemotype (257.83).

3.2. Content of Essential Oil Among Oregano Populations

It was evident that the presence of variation in EO content among all the oregano populations (Table 2), carvacrol group was the richest (4.02) compared to γ-terpinene group. It seems that the potential yield of accessions contained γ-terpinene in combination with carvacrol methyl ether was decreased (1.01%) to be less than of OR2, OR5, OR7, OR8, OR12, OR18 (1.28%) where γ-terpinene was the main component. However, the populations of linalyl acetate, thymol, thymol/ γ-terpinene, thymol/γ-terpinene/p-cymene and sabinyl-compounds+sesquiterpenes (β-caryophyllene) characterized by a high essential oil content (2%) than the other two populations γ-terpinene and carvacrol methyl ether/γ-terpinene (~1%).

3.3. Chemical Analysis of Essential Oil

A total of 43 compounds were identified in the 23 studied populations and single cultivar which further classified as the following: thirteen are monoterpenes hydrocarbons (11.14- 61.11%), twelve of oxygenated monoterpenes (1.48-68.89%), two of phenolic monoterpenes (4.30-70.24%), twelve of sesquiterpene hydrocarbons (5.69-44.93%), three oxygenated sesquiterpene (0.22-6.83%) and one is uncategorized compound. Only the main EO constituents obtained from 24 oregano accessions are presented (Table 5) where is the variation in the chemical composition is quite remarkable (84.94-94.69%). In OR1, OR4 and OR9 the main constituent found to be carvacrol with a total of 72.20%, 70.03 and 69.03%, respectively (carvacrol chemotype) while the other phenolic monoterpene (thymol) was the major compound in OR10, OR11, OR16, OR17, OR19 having a higher percentage from 32.85% to 52.03% (thymol chemotype). Linalyl acetate was the first main oxygenated monoterpenes with 59.36, 59.59 and 66.30% respectively in OR13, OR14 and OR15 (linalyl acetate chemotype) while carvacrol methyl ether was interestingly the highest in OR22 population (25.65%). Among the monoterpenes hydrocarbons, γ-terpinene appeared to be the most dominant in the essential oils from twelve accessions tested, ranging from 18.08% to 34.95% followed by p-cymene (18.67%) which detected only in OR23 or sabinene (36.70%) in OR24.

3.4. AFLP Fingerprinting and Genetic Similarity

The 10 AFLP primer combinations yielded a total of 3315 scorable fragments, of which 1179 were polymorphic. The number of polymorphic bands generated by each AFLP primer combination (Table 3) varied from 225 (E-ACG/M-CAT) to 557 (E-ACA/M-CAG). As shown in Table 3, the 10 primer combinations detected 35.8% of polymorphism.

Diversity among genotypes was assessed by the construction of a UPGMA dendrogram (Fig. 1), showing a wide variability among genotypes. Three main clusters are valuable: the first grouped five genotypes: from OR8 to OR4; the second seven genotypes from OR7 to OR11; the third genotypes from OR23 and OR17.

PCoA analysis was also performed (Fig. 2), supporting the evidence of wide variability among genotypes, in which only OR8 genotype is not grouped with the remaining ones.

Table 4
Collected data (mean±SD) from 24 oregano populations classified according to main chemotypes of EOs.


Fig. (1)
Dendrogram of genetic similarity of 24 genotypes of oregano obtained using MEGA software.


Fig. (2)
Principal component analysis (PCoA) performed on 24 genotypes of oregano using GenAlex 6.5.


Table 5
Mean (%) of the main 15 essential oil compounds in the studied populations of O. vulgare L.



4. DISCUSSION

4.1. Agronomic Characteristics

The focus of the current study was to investigate the variability among different wild-growing O. vulgare accessions. Populations belonging to thymol/γ-terpinene chemotype (4 accessions) and carvacrol chemotype (3 accessions) have produced a considerable amount of the biomass, making them of particular interest for biomass-production purposes and further for the production of essential oils [46Tuttolomondo T, La Bella S, Licata M, et al. Biomolecular characterization of wild sicilian oregano: Phytochemical screening of essential oils and extracts, and evaluation of their antioxidant activities. Chem Biodivers 2013; 10(3): 411-33.
[http://dx.doi.org/10.1002/cbdv.201200219] [PMID: 23495158]
]. In addition to their individuals reached maximum values in plant height which is of great importance in medicinal plant breeding for the mechanical harvest [47Khadivi-Khub A, Karimi E, Hadian J. Population genetic structure and trait associations in forest savory using molecular, morphological and phytochemical markers. Gene 2014; 546(2): 297-308.
[http://dx.doi.org/10.1016/j.gene.2014.05.062] [PMID: 24878369]
]. For most of the evaluated characters, a noticeable heterogeneity between populations were observed in the present study. Our results are in agreement with the available literature, where a substantial variable in plant height and dry mass have been underlined in a germplasm collection of Origanum vulgare L. from Europe [19Azizi A, Hadian J, Gholami M, Friedt W, Honermeier B. Correlations between genetic, morphological, and chemical diversities in a germplasm collection of the medicinal plant Origanum vulgare L. Chem Biodivers 2012; 9(12): 2784-801.
[http://dx.doi.org/10.1002/cbdv.201200125] [PMID: 23255448]
] as well as wild Tunisian oregano [48Mechergui K, Coelho JA, Serra MC, Lamine SB, Boukhchina S, Khouja ML. Essential oils of Origanum vulgare L. subsp. glandulosum (Desf.) Ietswaart from Tunisia: Chemical composition and antioxidant activity. J Sci Food Agric 2010; 90(10): 1745-9.
[http://dx.doi.org/10.1002/jsfa.4011] [PMID: 20564436]
]. Also, the Hungarian Origanum vulgare populations showed a high degree of variability in response to altitude, soil type and humus content [49Cserháti B, Juhos K, Begyik A, Radácsi P, Németh É, Szabó K. In situ morphological variability of wild marjoram (Origanum vulgare L.) populations in Hungary. Acta Aliment 2012; 41(Suppl. 1): 12-23.
[http://dx.doi.org/10.1556/AAlim.41.2012.Suppl.2]
]. This indicates the importance of location features on oregano growth which can further illustrate the high variation in plant performance under different environments [50Kassahun BM, Tilahun S, Zigene ZD, Teferi Z, Mekonnen M, Melka B. Morpho-agronomic characteristics, essential oil content and essential oil yield of oregano (Origanum vulgare L.) in Ethiopia. Sch J Agric Sci 2014; 4(12): 565-71.].

4.2. EO Content

The essential oil of O. vulgare samples has been analysed and its chemotypes were identified [51Skoula M, Gotsiou P, Naxakis G, Johnson CB. A chemosystematic investigation on the mono- and sesquiterpenoids in the genus Origanum (Labiatae). Phytochemistry 1999; 52(4): 649-57.
[http://dx.doi.org/10.1016/S0031-9422(99)00268-X]
]. Essential oil contents from leaves and flowers, ranged between carvacrol chemotype, as the most productive group to reach the lowest content in carvacrol methyl ether/γ-terpinene chemotype. The correlation between the oil yield and carvacrol content was strongly reported in populations featured by a very high amount of carvacrol and EO content as well [52Baser KH, Özek T, Kürkçüoglu M, Tümen G. The essential oil of Origanum vulgare subsp. hirtum of Turkish Origin. J Essent Oil Res 1994; 6(1): 31-6.
[http://dx.doi.org/10.1080/10412905.1994.9698321]
, 53Baser KHC. The Turkish Origanum species.Oregano The Genera Origanum and Lippia 2002; 109-26.]. Therefore, best EO content in OR1, OR4, OR9 (carvacrol chemotype) accompanied by the maximum presence of carvacrol up to 72.20% [54Morshedloo MR, Salami SA, Nazeri V, Maggi F, Craker L. Essential oil profile of oregano (Origanum vulgare L.) populations grown under similar soil and climate conditions. Ind Crops Prod 2018; 119: 183-90.
[http://dx.doi.org/10.1016/j.indcrop.2018.03.049]
]. The other chemotypes of O. vulgare accessions were poor in EO content (<2%) as OR22, OR2, OR5, OR7, OR8, OR12 and OR18 in which the main constituents were γ-terpinene, carvacrol methyl ether/γ-terpinene [55Afsharypour S, Sajjadi SE, Erfan-Manesh M. Volatile constituents of Origanum vulgare ssp. viride (syn. O. heracleoticum) from Iran. Planta Med 1997; 63(2): 179-80.
[http://dx.doi.org/10.1055/s-2006-957640] [PMID: 17252342]
]. In detail, the monoterpene fraction of the above essential oil-poor accessions comprised a higher cymyl-compounds (γ-terpinene, carvacrol methyl ether) whereas the acyclic compounds were minimized or even deactivated. Such an observation led to exceptional chemotypes with ‘low quality’ plant material poor in phenolic monoterpene which is less important for commercial purposes [9Lukas B, Schmiderer C, Novak J. Essential oil diversity of European Origanum vulgare L. (Lamiaceae). Phytochemistry 2015; 119: 32-40.
[http://dx.doi.org/10.1016/j.phytochem.2015.09.008] [PMID: 26454793]
]. This confirms the difficulties to ensure large scale production, from a wild collection of native populations, with the better quality required by consumers. Generally, the diversity in the accumulation of EO constituents and commonly the carvacrol content is one of the most important breeding goals, which in turn has led to improve cultivars of great importance in oregano species [51Skoula M, Gotsiou P, Naxakis G, Johnson CB. A chemosystematic investigation on the mono- and sesquiterpenoids in the genus Origanum (Labiatae). Phytochemistry 1999; 52(4): 649-57.
[http://dx.doi.org/10.1016/S0031-9422(99)00268-X]
].

4.3. EO Composition and Main Chemotypes

Over forty mono- and sesquiterpenes have been reported from the complex and highly variable essential oil of Origanum vulgare plants. Almost 60% of these compounds were monoterpenes from acyclic or cymyl-pathway, including carvacrol, thymol, γ-terpinene, and p-cymene while sabinyl-compounds with the presence of sesquiterpenes (mainly β-caryophyllene and Germacrene D) was only reported in few cases. Comparing the current data with results from other studies demonstrated considerable variability in the chemical profile and the identified chemotypes of oregano EO either for the single compounds or even for the biosynthetic pathway. Most of the papers described the essential oil composition of O. vulgare from different geographic areas. Sicilian oregano was found to be rich in thymol (phenolic monoterpenes) with a variable percentage of γ-terpinene, p-cymene, and carvacrol [56Napoli EM, Curcuruto G, Ruberto G. Screening of the essential oil composition of wild Sicilian rosemary. Biochem Syst Ecol 2010; 38(4): 659-70.
[http://dx.doi.org/10.1016/j.bse.2010.04.001]
] or thymol (phenolic monoterpenes) and γ-terpinene (monoterpenes hydrocarbons) followed by p-cymene, carvacrol and thymol methyl ethers [57Bonfanti C, Iannì R, Mazzaglia A, Lanza CM, Napoli EM, Ruberto G. Emerging cultivation of oregano in Sicily: Sensory evaluation of plants and chemical composition of essential oils. Ind Crops Prod 2012; 35(1): 160-5.
[http://dx.doi.org/10.1016/j.indcrop.2011.06.029]
]. Recently, native populations of this species from Calabria region showed the predominate of carvacrol, thymol and γ-terpinene as well as linalyl acetate (oxygenated monoterpenes) which it resulted from the active acyclic pathway [21Mastro G, Tarraf W, Verdini L, Brunetti G, Ruta C. Essential oil diversity of Origanum vulgare L. populations from Southern Italy. Food Chem 2017; 235: 1-6.
[http://dx.doi.org/10.1016/j.foodchem.2017.05.019] [PMID: 28554612]
]. More chemotypes have been determined also in Calabria on the basis of the phenolic content, i.e., thymol, carvacrol, thymol/carvacrol, and carvacrol/thymol chemotypes [58Russo M, Galletti GC, Bocchini P, Carnacini A. Essential oil chemical composition of wild populations of Italian oregano spice (Origanum vulgare ssp. hirtum (Link) Ietswaart): A preliminary evaluation of their use in chemotaxonomy by cluster analysis. 1. Inflorescences. J Agric Food Chem 1998; 46(9): 3741-6.
[http://dx.doi.org/10.1021/jf980087w]
]. It was also underlined the differentiation in EOs composition based on the metabolic pathway, sesquiterpenes rich type and partly sabinyl compounds were the characteristic of O. vulgare individuals native to northern Italy [9Lukas B, Schmiderer C, Novak J. Essential oil diversity of European Origanum vulgare L. (Lamiaceae). Phytochemistry 2015; 119: 32-40.
[http://dx.doi.org/10.1016/j.phytochem.2015.09.008] [PMID: 26454793]
]. In an earlier investigation on oregano EOs from Liguria and Emilia regions, various groups have been identified: the first was rich in carvacrol/thymol content, the second had a high prevalence of linalool with sesquiterpenes and lastly, the third was characterized by the abundance of sesquiterpenes [7D’Antuono L. Variability of Essential Oil Content and Composition of Origanum vulgare L. Populations from a North Mediterranean Area (Liguria Region, Northern Italy). Ann Bot 2000; 86(3): 471-8.
[http://dx.doi.org/10.1006/anbo.2000.1205]
]. Likewise, the relationship between chemical composition and biotypes and/or chemotypes was stated in three chemotypes from Campania region: one rich in carvacrol/thymol while the other two were described for the first time as thymol/α-terpineol and linalyl acetate/linalool [59De Martino L, De Feo V, Formisano C, Mignola E, Senatore F. Chemical composition and antimicrobial activity of the essential oils from three chemotypes of Origanum vulgare L. ssp. hirtum (Link) Ietswaart growing wild in Campania (Southern Italy). Molecules 2009; 14(8): 2735-46.
[http://dx.doi.org/10.3390/molecules14082735] [PMID: 19701120]
]. Despite the fact that numerous data reported wide differences in monoterpenes accumulation depending on geographical areas, the findings obtained from the present research possibly correlated to the different growing conditions, in which the analyzed plants were collected [58Russo M, Galletti GC, Bocchini P, Carnacini A. Essential oil chemical composition of wild populations of Italian oregano spice (Origanum vulgare ssp. hirtum (Link) Ietswaart): A preliminary evaluation of their use in chemotaxonomy by cluster analysis. 1. Inflorescences. J Agric Food Chem 1998; 46(9): 3741-6.
[http://dx.doi.org/10.1021/jf980087w]
]. Another point probably indicates the dissimilarity of EO chemotypes in specific area, is that the pollination in O. vulgare plants is mostly cross [60Kheyr-Pour A. Wide nucleo-cytoplasmic polymorphism for male sterility in Origanum vulgare L. J Hered 1981; 72(1): 45-51.
[http://dx.doi.org/10.1093/oxfordjournals.jhered.a109424]
], which can cause a wide variation in the levels of carvacrol, thymol, p-cymene, and γ-terpinene in the essential oils as a result of sexual polymorphism or genetic mechanism [61Gershenzon J, Croteau R. Regulation of Monoterpene Biosynthesis in Higher PlantsBiochemistry of the Mevalonic Acid Pathway to Terpenoids 1990; 99-160.
[http://dx.doi.org/10.1007/978-1-4684-8789-3_3]
].

4.4. AFLP Fingerprinting and Genetic Similarity

AFLP analysis is a powerful tool to analyze germplasm investigating the whole genome. The choice of this class of markers was also due to a low efficiency showed by using RAPD markers on the 24 oregano genotypes (data not shown), revealing not reproducible data and, for some genotypes, lack of amplification patterns.

Furthermore, polymorphisms were detected using a fluorescence system, allowing to improve the efficiency of the classical AFLP procedure, based on radioactive detection of bands after electrophoresis on polyacrylamide gels. In fact, AFLP analysis was also performed on oregano by other Authors, detecting a lower number of total and polymorphic bands [1Skoula M, Harborne JB. The taxonomy and chemistry of Origanum Oregano: The genera Origanum and Lippia 2002; 67-108., 19Azizi A, Hadian J, Gholami M, Friedt W, Honermeier B. Correlations between genetic, morphological, and chemical diversities in a germplasm collection of the medicinal plant Origanum vulgare L. Chem Biodivers 2012; 9(12): 2784-801.
[http://dx.doi.org/10.1002/cbdv.201200125] [PMID: 23255448]
, 62Azizi A, Ardalani H, Honermeier B. Statistical analysis of the associations between phenolic monoterpenes and molecular markers, AFLPs and SAMPLs in the spice plant Oregano. Herba Pol 2016; 62(2): 42-56.
[http://dx.doi.org/10.1515/hepo-2016-0010]
].

Polymorphism level detected on the 24 oregano genotypes was lower in respect to data reported by Azizi et al. [1Skoula M, Harborne JB. The taxonomy and chemistry of Origanum Oregano: The genera Origanum and Lippia 2002; 67-108.], who analyzed a wide germplasm collection from different Countries.

CONCLUSION

The wild O. vulgare accessions collected from different locations of Southern Italy have been evaluated in terms of morphological, biochemical and molecular properties. Considerable variability in the EO profile among the oregano populations was underlined, leading to identify 4 accessions belong to thymol/γ-terpinene chemotype and other 3 accessions from carvacrol chemotype. Such accessions are of great interest for biomass and EO production besides the suitability for the mechanical harvest since their individuals reached maximum values in plant height.

Molecular characterization, performed by means of AFLPs, showed a wide genetic variability within oregano germplasm. Due to the scant information about the molecular characterization of the species results obtained in the present work could support and contribute to breeding programmes for agronomic and biochemical traits.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

RESEARCH INVOLVING PLANTS

The reported experiments were in accordance with the United Nations (UN, 1992) convention on biological diversity.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

All individuals listed as authors were contributed substantially to the design, performance, analysis, and reporting of the work.

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"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

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Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
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Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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