Introduction

Thymus genus which belongs to the family Lamiaceae, includes several hundreds of species distributed over world¹, where Mediterranean basin is considered the main center of this herbal plant ².

Traditionally, most of plants discriminated on morphological-basis; however, these methods still difficult to apply for an accurate discrimination and authentication use³. Thymus genus is usually used for flavoring agents, herbal tea, and medicine and the aerial parts and volatile constituents of thyme are used as a medicinal material². ⁴reported that, Polyploidy and disploidy/aneuploidy within many species in the genus Thymus further complicate the determination of species boundaries and there were have high levels of natural hybridization within and between the species, probably due to the absence of incompatibility and the presence of a dimorphic breeding system, gynodioecy, in which populations comprise female and hermaphroditic individuals.⁵ Reported that, Knowledge of genetic diversity within species is necessary for any improvement of cultivars, and biodiversity maintenance and restoration. DNA-based molecular markers, which are not affected by environmental conditions, have become increasingly important for surveying genetic diversity and genotype identification of medicinal plants⁶. These markers can also be taxonomically useful, i.e. for phylogenetic studies to distinguish plant species and subspecies^7,8,9,10.

The start codon targeted (SCoT) polymorphism is a novel, simple and reliable gene targeted marker technique based on the translation start codon¹¹. Primers for SCoT marker analysis were designed based on the con-served region surrounding the translation initiation codon, ATG. Using a single 18-mer primer as a forward and reverse primer in the PCR, ¹¹designed thirty-six primers that were used successfully for cultivar identification and genetic diversity analysis in many crops. Being characterized by lower recombination levels between its markers and the gene/trait.¹² Conducted that, This ISSR-PCR  technique is rapid, simple, inexpensive and more reproducible than RAPD amplification of DNA.. ISSR used to study the genetic diversity of plants for examples; Nepeta¹³, Thyme¹⁴, Salvia¹⁵, Mentha aquatica L.¹⁶, Satureja¹⁷, Salvia¹⁸, Thymus¹⁹, Phlomis kurdica and Phlomis oppositiflora²⁰ and Ocimum²¹.

Increasingly, the plant breeding approach has taken advantage of de­velopments in molecular biology in order to genotype the species of interest in a way that considerably accelerates their selection and  this types of approach consist of choos­ing desired genotypes on the basis of molecular markers, or having prior knowledge of the genes that determine the formation of a particular trait in a plant²².

The aim of this study is to assess the molecular genetics and morphological assessment among different species of this plant using morphological and SCoT and ISSR markers, with a view toward conservation of this endangered species.

Materials and Methods 

Plant Materials and Growth Conditions

The seedlings of  the five Thymus species were obtained from two countries, Egypt and Kingdome of Saudi Arabia, as a commonly known species showed in Table 1. Thymus  plants were collected during two seasons of 2017/2018 and 2018/2019, at Vegetable and Medicinal and Aromatic Plants Research Departments, Dokki, Giza and Biotechnology Lab. Horticulture Research Institute, Agricultural Research Centre, Egypt. Two months old seedlings of five Thymus Sp.  were obtained from the Experimental Farm of Medicinal and Aromatic Plants Research Department, El-Kanater El-Khairia, Egypt.

Table 1: The Thymus species numbers and the names of the five studied species.

Growth Parameters

Plant height (cm), Number of branches, Leaves fresh weight (g), Leaves dry weight (g) and  Volatile Oil%. 

Molecular Genetic Assessment

DNA Extraction

The DNA extraction of the five species of Thymus was performed as described by²³. DNA quality was checked by means of absorbance ratios A260/A280 through a UV-spectrophotometer where DNA is pure with a ratio A260/A280 from 1.8- 2.0. Moreover, using electrophoresis in 1% agarose gel with ethidium bromide, a qualitative check for DNA samples was done. 

SCoT and ISSR Analysis

Obtaining clear reproducible amplification products require a number of factors were included PCR temperature cycle profile and concentration of each of (template DNA, primer, MgCl₂ and Taq polymerase) which were optimized  according to²⁴ and ²⁵ respectively, in the PCR reaction using 5 SCoT primers and 5 ISSR primers in molecular genetic analysis for the five Thymus species. ISSR primers procured from Bio Basic Company Canada. On the other hand, SCoT primers were designed from consensus sequence derived from the previous studies by²⁶ and ²⁷. ¹¹ and procured from Biobasic Company.

ISSR and SCoT assays were performed as described by ^24,25 and  ²⁸.

Gel Electrophoresis

PCR products were run at 100 V for one 30min. on 1.5 % agarose gel using 100bp Ladder DNA marker to detect polymorphism between five Thymus species  under study.

Statistical Analysis

A randomized complete block design was adopted for the present investigation data were statistically analyzed by the standard methods according to²⁹. The new L.S.D. test was used for comparison between means. The DNA bands generated by each primer were counted and their molecular sizes were compared with those of the DNA markers. The bands scored from DNA profiles generated by each primer were pooled together. Then the presence or absence of each DNA band was treated as a binary character in a data matrix (coded 1 and 0, respectively) to calculate genetic similarity and to construct dendrogram tree among the studied five Thymus Species. Calculation was achieved using Dice similarity coefficients³⁰ as implemented in the computer program SPSS-10.

Results and Discussion

Growth Composition Diameter

The growth parameters results were including Plant height (cm), number of branches/plant and fresh and dry weights of leaves/plant (g), of (Thymus species) seedlings in both two seasons are shown in Table (2).

Plant Height

Table (2) represented that plant height, Origanum vulgare was the highest plant in first season in the two cuts were as follow (31.39 and 35.37cm) and increased in the second season was cuts as follows (35.90 and 32.31cm) and this followed by Thymus citriodorus, Thymus vulgaris and Origanum syricum. While, the lowest in the Thymus Sp.  in plant height was Thymus 3 which results in first season in the two cuts were as follows (6.82 and 8.91cm) and in the second season data in the tow cuts were as follows (9.62 and 9.19cm), respectively.

Branch Number

The data in Table (2) revealed that,  the number of branches it was clear from that the greatest branches number were revealed by Thym.1in the first season: in two cuts were as follow (16 and 39) and in the second season: in the two cuts were as follow (39 and 42.67) and this results were followed by Thymus argenteus, Thymus citriodorus and Origanum syricum. While, the lowest number of branches were recorded in Origanum vulgare which were in the first season: in the two cuts as follow (3.67 and 4) and in the second season: in the two cuts ere as follow (4.33 and 5.33), respectively.

Fresh and Dry Weight of Leaves /Plant

Results of fresh weight of leaves /plant and dry weight of leaves /plant in the five sp. of Thymus in the two seasons data were revealed in Table (2), Thymus argenteus results were recorded as the highest data in all Thymus sp. under study and results were as follow, in the first season : fresh weight of leaves/plant in the two cuts were as follow (422.4 and 519 gm) and in dry weight of leaves/plant were as follow  in the two cuts (49.25 and 53.98gm). While, in the second season: fresh weight of leaves/plant in the two cuts were as follow (522.14 and 659.99 gm) and in dry weight of leaves/plant were as follow  in the two cuts (52.34 and 65.20gm) and this results were followed by Origanum vulgare, Thymus vulgaris and Thymus citriodorus, respectively. On the other hand, Origanum syricum was the lowest in both fresh and dry weight of leaves/plant in the two seasons and the results were as follow, the first season: fresh weight of leaves/plant in the two cuts were as follow (23.74 and 27.51 gm) and in dry weight of leaves/plant were as follow  in the two cuts (8.41 and 9.57gm). While, in the second season: fresh weight of leaves/plant in the two cuts were as follow (29.36 and 30.40 gm) and in dry weight of leaves/plant were as follow  in the two cuts (11.13 and 11.07gm).

Volatile Oil %

The results were observed in Table (2) illustrated the largest amount of volatile oil % was in Origanum vulgare in both two seasons as follow, the first season: results in the two cut were as follow (0.27 and 0.29%) and in the second season: results in the two cuts were as follow: (0.31 and 0.33%) respectively, and this results were followed by Thymus citriodorus, Thymus vulgaris and Thymus argenteus. While, the lowest amount of volatile oil % was observed in Origanum syricum in both two seasons and the results as follow, the first season: the two cut were as follow (0.05 and 0.06%) and in the second season: results in the two cuts were as follow: (0.07 and 0.12%), respectively.

Table 2: Vegetative parameters, plant height, branches number, fresh weight, dry weight and volatile oil % of five Thymus species through two cuts and two seasons.

Molecular  Genetics Assessment

This results of  the genetic variability in five species of Thymus using SCoT-PCR and ISSR-PCR analysis. Where five SCoT  primers out of ten tested primers were succeeded on the five different Thymus Species, and  five ISSR primers out of fourteen tested primers generated reproducible amplified bands.

Figure 1: SCoT-PCR Profile for five species of Thymus amplified with five primers for each analysis. Click here to view figure

Figure 2: ISSR-PCR Profile for five species of Thymus amplified with five primers for each analysis. Click here to view figure

SCoT and ISSR Analysis Assessment

Molecular genetic data produced by SCoT and ISSR analysis were shown in Figs (1 and 2) and Tables (3 and 4). These  data showed that, in SCoT results, primer (SCoT-6) was resulted in the highest number of amplified bands and primer (SCoT-4) was represented the lowest number of amplified bands compared with  other SCoT primers. On the other hand, in ISSR data,  primer 44B resulted in the highest number of amplified bands and primer (HB-14) showed the lowest number of amplified bands in all ISSR primers.

On the  other hand,  SCoT primers except SCoT 4 and SCoT-8 generated 10 unique bands  out of 39 amplified bands and ISSR primers except (44B, HB-10 and HB-14) generated 4 unique bands out of 23 amplified bands, May be these unique bands were useful as unique markers as explained by³¹ in cymbopogon; ³² in canolla; ³³ in tomato and ³⁴ in pumpkin.

Table 3: Molecular genetic data produced from amplified banding patterns of  SCoT technique.

Table 4: Molecular genetic data produced from amplified banding patterns of ISSR technique.

Table 5: Polymorphic, Monomorphic, Unique bands and Polymorphic percentage generated by the (ISSR and SCoT) analysis.

Also, Table 5 showed that five species of Thymus, (Thymus vulgaris, Origanum vulgare, Thymus argenteus, Thymus citriodorus and Origanum syricum) characterized by five SCoT primers and five ISSR primers data, 23 polymorphic bands from 38 amplified bands were produced by SCoT primers with polymorphic average 60.52%. While, 14 polymorphic bands from 23 amplified bands with polymorphic average 60.86% were generated by ISSR primers. On the other hand, in the combined results there were 37  polymorphic bands from total 61 amplified bands with total polymorphic average 60.65%. These obtained data indicates that SCoT-PCR and ISSR-PCR techniques were succeeded in differentiate between five Thymus species studied.

Molecular Distance of Combination of SCoT and ISSR Analysis

On the other hand, Table (6) illustrated that, results of molecular distance (MD) matrix between all five species of Thymus studied based on SCoT  and ISSRs combined results.

Molecular distances (MD) based on SCoT markers data were ranged from 0.633 (between Thymus vulgaris and Thymus citriodorus species) to 0.843 (between Thymus vulgaris and Origanum vulgare  species) was lower than range of MD based on ISSR ranged from 0.603 (between Thymus vulgaris and Thymus citriodorus species) to 0.942 (between Thymus vulgaris and Origanum vulgare  species). While in combined data were ranged from 0.164 to 0.404 among the same genotypes were defined by SCoT technique.

Table 6: Molecular distances (MD) between five Thymus Species based on Dice dissimilarity index for SCoT & ISSR and combined data.

Previously data represented the important of SCoT-PCR technique in  molecular genetic assessment in Thymus species in comparison with ISSR-PCR technique. These results were in agreement with Nepeta¹³, Thyme¹⁴, Mentha aquatica L.¹⁶, Satureja ¹⁷, Salvia¹⁸ and Thymus¹⁹.

Dendrogram Analysis of Combination Between SCoT and ISSR Analysis 

Fig. 3 represented dendrogram from UPGMA method using Dice-dissimilarity index Dendrogram data were divided the five Thymus Species into two main clusters: The first cluster contained two Thymus sp. (Thymus argenteus and Thymus citriodorus) and the second cluster was divided into two sub-clusters: the first sub-cluster included Origanum syricum only. While the second sub-cluster included the two other species (Thymus vulgaris and Origanum vulgare).

This results were conducted that SCoT and ISSR analysis could be useful as tools for identifying Thymes species in breeding programs  and this combination data of SCoT and ISSR analysis were suitable for evaluating the genetic relationships among  five species of Thymus and this results were  in agreement with genetic analysis has been conducted by^35,36,37, Salvia¹⁸ and Thymus ¹⁹ Phlomis kurdica and Phlomis oppositiflora²⁰ and Ocimum^21,38. Revealed that, by using of ISSR-PCR technique of some accessions of Thymus daenensis, was obtained two geographically diverse groups were generated by dendrogram.

Figure 3: Dendrogram derived by UPGMA method using Dice-dissimilarity coefficient for combined binary data Click here to view figure

Acknowledgment

The authors appreciate the constant help and work support provided by Shaqra University & Northern Border University, Saudi Arabia and Agricultural Research Centre, Egypt.

Funding Source

This work is self-funding and the authors did not get any fund from any organization or persons.

Conflict of Interest

Authors declare that we have no conflict of interests.

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