Morphological and Molecular Genetic Assessment Of Some Thymus Species

1Department of Biological Sciences, Faculty of Science and Humanities, Shaqra University, P.O. Box 1040,Ad-Dawadimi, 11911, Saudi Arabia. 2Biology Department, Faculty of Arts and Science, Northern Border University, Rafha, 91911, Saudi Arabia. 3Vegetable and Medicinal and Aromatic Plants Research Departments, Dokki, Giza and Biotechnology Lab. Horticulture Research Institute, Agricultural Research Centre, 12619, Egypt.

Thymus genus which belongs to the family Lamiaceae, includes several hundreds of species distributed over world 1 , 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 use 3 .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 material 2,4 .reported 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. 5Reported 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 plants 6 .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, 11 designed 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. 12onducted 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; Nepeta 13 , Thyme 14 , Salvia 15 , Mentha aquatica L. 16 , Satureja 17 , Salvia 18 , Thymus 19 , Phlomis kurdica and Phlomis oppositiflora 20 and Ocimum 21 .
Increasingly, the plant breeding approach has taken advantage of de velopments 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 choos ing 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 plant 22 .
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.

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

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 by 26 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 to 29 .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.

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

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.

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

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.
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  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.
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 Nepeta 13 , Thyme 14 , Mentha aquatica L. 16 , Satureja 17 , Salvia 18 and Thymus 19 .dendrogram analysis of Combination Between sCot and issr analysis 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, Salvia 18 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.

Fig. 1 .
Fig. 1.SCoT-PCR Profile for five species of Thymus amplified with five primers for each analysis

Fig. 2 .
Fig. 2. ISSR-PCR Profile for five species of Thymusamplified with five primers for each analysis

table 3 .table 4 .
Molecular genetic data produced from amplified banding patterns of SCoT technique Molecular genetic data produced from amplified banding patterns of ISSR technique

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).

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

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.
cuts were as follow(23.74and27.51gm) 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(11.13and11.07gm).

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

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