Detection of genetic diversity among some species of Anthemis L . ( Asteraceae ) in Saudi Arabia by using RAPD-PCR analysis

The aim of the present study was to determine the unique molecular markers among, three species of the genus Anthemis and the construction of phylogenetic tree using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique. Genetic diversity was analyzed among 15 populations of three species of Anthemis (Anthemis melampodina, Anthemis, pseudocotula and Anthemis, bornmuelleri), collected from different locations at Saudi Arabia by using RAPD primers. Pairwise genetic distance was calculated based on Nei and Li coefficient. Unweighted pair group method with an average (UPGMA) was used for construction of dendrogram, based on the similarity matrix data. Results showed wide variations among A. bornmuelleri and other two species. A wide close genetic relation was observed between A. melampodina and A. pseudocotula. RAPD-PCR technique was shown to be an accurate tool, in other to ascertain plant relationships among species of genus Anthemis.


INTRODUCTION
Anthemis L. is one of the largest genus of family Asteraceae, including more than 210 described species (Oberprieler et al., 2007).They are widely distributed across Europe extending into extreme southern Arabia and tropical east Africa and other parts of the world (Oberprieler, 2001).
The main center of biological diversity or species diversity is located in Mediterranean region (Lo Presti, 2010) and southwestern Asia with 150 to 210 species, including all of the presently accepted subgenera and sections (Kilica et al., 2011).Some species inhabit northern America and southern hemisphere as well (Oberprieler, 2001).Anthemis is a diverse group that can be easily distinguished by the paleaceous receptacle of E-mail: sqarinet@gmail.com.Tel: +966555504086.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License its mostly radiate capitula and the achene morphology.Some species are widely used in pharmaceutical, cosmetics, and food industry.They are also used as herbal tea for treatment of anxiety, flatulence, stomach disorders, insomnia and toothache (Vaverkova et al., 2001).Saudi Arabia contains a large number of wild plants, both in arid and high lands.The greatest plant diversity was recorded in Asir, Hijaz and the western area, bordering the Red Sea (Collenette, 1998).Many previous studies showed that landscape of an area and the climatic influences are the main factors affecting the degree of species diversity (El-Kady et al., 1995;Shaltout et al., 1997).Some species of Anthemis are distributed depending on the chemical nature of bedrock and climate of geographic area.Four species of Anthemis were illustrated by Migahid (1996), 12 species by (Ghafoor and Al Turki, 1999) and 19 species by (Ghafoor, 2010).
Molecular markers such as Random Amplified Polymorphic DNA (RAPD), Variable Number of Tandem Repeats (VNTR) and Restriction Fragment Length Polymorphism (RFLP) have been established as useful markers, for discovering genetic diversity of floras (Wang, et al., 1996).RAPD is a PCR-based technique developed by (Williams et al., 1990) and (Welsh and McClelland, 1990) which employs decamer random primers, for amplifying random DNA fragments from genomic DNA without any prior knowledge of the genomic sequence of any organism.Recently, several studies have used this tool to measure the levels and patterns of variations within different plants (Abd El-Ghani and El-Sawaf, 2004;Sarwat et al., 2008;Thendral et al., 2010;Hammadi and Qari, 2012;Ismail et al., 2016;Lu et al., 2016;Patil et al., 2016).
The present study aimed to use DNA (RAPD) markers to investigate genetic diversity, genetic relationships and polymorphism in natural populations of three species of Anthemis in Saudi Arabia.

Plant materials
During the period of March to May 2015, 15 plant samples of three species of Anthemis were collected from different geographic regions in Saudi Arabia through Jizan, Makkah, Hail and Asir (Figure 1

DNA extraction
Total genomic DNA extraction of plant leaves was performed using a modified CTAB method according to the protocol of (Doyle and Doyle, 1990).Quantitative estimation of total genomic DNA in each sample was confirmed spectrophotometericaly at 260 and 280 nm, Whereas, quality was checked by running samples on 1.2% agarose electrophoresis with DNA ladder and visualized under UV light in gel documentation system.

RAPD analysis
Polymorphic primers were identified by screening fifteen random decamer primers (Table 2) with DNA of Anthemis sp.Out of 15 primers, only 5 gave precise and stable PCR-production for RAPD analysis (Table 2).DNA amplification was performed in a Perkin ElmerCetus 480 DNA Thermal Cycler programmed for 45 cycles as follows: 1st cycle of 3.5 min at 92°C, 1 min at 35°C, 2 min at 72°C; followed by 44 cycles each of 1 min at 92°C, 1 min at 35°C, 2 min at 72°C followed by one final extension cycle of 7 min at 72°C.The amplification products were resolved by agarose gels electrophoresis.Controls lacking template DNA were included.Amplified PCR products were resolved on 1% agarose gel electrophoresis, visualized under UV light and photographed with gel documentation system.Each band was considered as RAPD marker.All the reactions were repeated for at least twice.

Data analysis
A binary matrix was prepared by manually scoring of photographed bands on gels, where 1 or 0 represent.The data were used for similaritybased analysis using the software program NTSYS (2.20).RAPD analyses were analyzed using the Nei genetic similarity index (Nei and Li, 1979).On the basis of the similarity matrix data, a dendrogram was constructed by unweighted pair group method with average (UPGMA) cluster analysis (Figure 2).

RESULTS AND DISCUSSION
Different approaches in genetic diversity analyses, reveal the different level of polymorphism (Porter and Smith, 1982) and also, DNA markers are independence of environmental or localities factors which show a greater level of polymorphism (Heywood, 2002).Therefore, they    assessment of the genetic differences between genotypes (Williams et al., 1990).Meanwhile, polymorphism detected by RAPD markers has proven to be useful for discrimination of genetic diversity and relationships in several plant species.The present work is a preliminary study, aim to use DNA (RAPD) markers to investigate genetic diversity, genetic relationships and polymorphism in natural populations of only three species of this genus (A.melampodina, A. pseudocotula and A. bornmuelleri) collected from Saudi Arabia.In this study, a total of 15 RAPD-PCR primers were used to test 15 samples (Table 1).Out of these, only 5 primers showed reproducible results and they were chosen to amplify the whole 15 samples (Figure 3).RAPD-PCR assay had been positively used in several taxonomical and genetic diversity studies (Hammadi and Qari, 2012;Alam et al., 2009).The percentages of polymorphism and monomorphism of the obtained bands are presented in (Table 3).A total of 63 bands were produced for 15 samples, 51 bands of them were polymorphic and present one or more but not all of them.Mehetre et al., (2004) reported that monomorphic bands should present in all producible bands, and the unique ones should present in at least one producible band, not in any others.The mean percentage of polymorphic bands was 69.32% with molecular sizes ranged from 263 up to 2791 bp; approximately, 18 bands of the 63 were commonly detected in all the samples, so it could be the recognized genus bands of Anthemis.
The results of the total fragments amplified (TFA), amplified fragment (AF) specific markers (SM) for each species of Anthemis: (A. melampodina, A. pseudocotula and A. bornmuelleri) are presented in Table 4 and the genetic distance matrix for indices of the studied samples are presented in Table 5. Welsh and McClelland (1990) found that, reproducible fingerprints of genomes could be generated using arbitrary primers with PCR technique.Species-specific markers varied between three types of Anthemis species (TSM=29 markers) and are clearly shown in (Table 4). A. melampodina revealed 13 specific bands, while A. pseudocotula and A. bornmuelleri showed 10 and 6 specific bands (SM), respectively.
The plants were identified by the Plants Taxonomist at the Herbarium of the Faculty of Science, Umm Al-Qura University, Makkah, Saudi Arabia.Young leaves were harvested and preserved in sealed bags with suitable label.Leaves were used immediately for DNA extraction, while excess leaves were stored in -80 °C for subsequent use.

Table 1 .
Locations of plant collection.
A. bornmuelleri Malaki Dam15 km east of Abu Arish, Jizan Region are considered as valuable tools for determining genetic relationships.Among various molecular markers, Random amplified polymorphic DNA (RAPD) markers have proved to be a very useful tool, providing a convenient and rapid

Table 3 .
RAPD primers data and the percentages of polymorphic bands.

Table 4 .
Number of amplified fragments and specific markers of the three species of Anthemis (A. melampodina, A. pseudocotula and A. bornmuelleri) using RAPD analysis with five primers.