The study of genetic diversity in different plant populations of Artemisia Annua L. native to northern Iran by molecular marker AFLP
Artemisia annua L. is an annual, medicinal and aromatics plant that is widespread in Asia, Europe and North America and has been wide distribution in northern areas of Iran. In this study, AFLP markers were used to investigate the genetic diversity of 15 populations of A. annua. 86 scorable bands were produced by using four primer combinations )E/M( that 58 of them) were polymorphic. The highest number of polymorphic bands (24 bands) using primer combination EcoR11 Mse20 and the lowest number of polymorphic (6 bands) using primer combinations EcoR2 Mse35 were produced. Data matrix using similarity coefficient was converted to similarity matrix (distance matrix) and then using the algorithm (UPGMA) was drawn relevant dendrogram. Dendrogram obtained from UPGMA put 15 populations in four groups. The obtained cluster of cases was consistent with the geographic diversity in some cases. Then correlation coefficient based on the Mantel test was obtained 7.0, indicating good agreement drawn dendrogram with the similarity matrix. The results of principal component analysis and almost coincided with the results of cluster analysis. The results showed that the use of AFLP method to assess molecular diversity in these masses is appropriate. *Corresponding Author: Akbari Farhad Hariri.a.farhad@gmail.com Journal of Biodiversity and Environmental Sciences (JBES) ISSN: 2220 6663 (Print) 2222 3045 (Online) Vol. 9, No. 2, p. 51 59, 2016 http:/www.innspub.net J. Bio. Env. Sci. 2016 52 Reza et al. Introduction Among the 45000 plant species, approximately 15000 are flowering plants and nearly 3000 of these are known to be of medicinal value (Sarwat et al. 2012) All over the world, plant based medicines are gaining popularity due to their almost negligible or no side effects (Hoareau and DaSilva, 1999) These plants are traditional source of quite a big number of biologically active substances used in the pharmaceutical industry. The most valuable phytochemicals are products of the secondary metabolism. Most of them have complicated structure which makes their chemical synthesis very difficult and even impossible (Tasheva and Kosturkova, 2012) Biotechnological tools have potential to overcome the problems of traditional methods of propagation for large scale production of high quality planting material, (Kant and Parmar, 2014) These tools are important for the multiplication and genetic enhancement, for the medicinal plants by adopting techniques such as in vitro regeneration and molecular methods. It could also be harnessed for the production of secondary metabolites using plants as bioreactors (Hoareau and DaSilva, 1999; Siahsar et al. 2011) One of the most applicable methods is, assessment and characterization of the germplasm, can identify novel genotypes and aid future plant improvement programs. There is a need for the assessment and characterization of genetic diversity that is an essential prerequisite for systematic documentation of plant species (Sarwat et al. 2012; Schippmann et al. 2002) Genus Artemisia belongs to the family Asteraceae has more than 444 species in the world (Wright, 2002) that 34 species has wide distribution wildly in Iran in all areas (Asghari and Razban Haghighi, 1999; Verdianrizi, 2008) The great importance of Artemisia species for its application in various fields has caused to have been planted as economic and commercial product widely. Among the most famous commercial species of the genus Artemisia can be pointed to anti malarial species (A. annua) species used in food and cooking (A. dracunculus) insect repellent species (A. vulgris) and ornamental species (A. abrotanum) (Mueller et al. 2004; Tzenkova et al. 2010; A. annua species is an annual, aromatic and medicinal plant. This plant is native to Asia, particularly China and widely distributed in Asia, Europe and North America (Bertea et al. 2006; Majruhi, 2008) The mentioned species in Iran is widely grown in northern strip of country (Sharafi et al. 2006) The major importance of this species in the pharmaceutical industry is due to the presence of a sesquiterpene lactone endoperoxide compound called Artemisinin that has antimalarial activity (Cafferata et al. 2010) Essence of the A. annua species has been used in the cosmetics, health and drug industry (Lari Yazdi et al. 2001; Gupta et al. 2002) and has anti bacterial, antiviral and anti tumor properties (Bhakuni et al. 2001; Santos and Simon, 2002) Essence of this species contains terpenoids (including monoterpenes, sesquiterpene and triterpene) flavonoids and coumarin (Bagchi et al. 2003; Bovard et al. 2014) Compounds which are reported from various geographic regions as a major component in essence of the A. annua are very different. This shows the great influence of environmental conditions on the type and percentage of composition of plant essence. The studies indicate that the essence is distributed in all of plant organs but the major part of it is concentrated in the leaves and flowers, and tiny amounts are in the main stem, branches and roots (Charles et al. 1991; Bovard et al. 2014) The objective of this study was to estimate the genetic diversity among the 15 native populations of the medicinal plant Artemisia annua from North of Iran with using the Amplified Fragment Length Polymorphism (AFLP) Materials and methods Plant material and collecting population of Artemisia annua 15 native populations were collected from different cities from North of Iran. And cultivated in order to perform tests and take samples in the Research farm of Institute of Medicinal Plant, Karaj, Iran. J. Bio. Env. Sci. 2016 53 Reza et al. In order to achieve accurate and comprehensive information regarding to north Iranian Populations of A. annua, we have attempted that collected native populations to be appropriate for the geographic distribution throughout the region (Table 1) Cultivation and production of plant material First the seeds were collected from plants of natural habitats. The seeds were put in Petri dishes on wet filter paper and transferred to plastic pots after germination in order to grow these plants to a height of 10 to 15 cm and also leaves grow enough. After three weeks, fresh leaves from each pot were picked for genomic DNA extraction. Genome extraction and purification Total genomic DNA was extracted and the quality of the extracted DNA was tested on 1% agarose gel electrophoresis and quantity was measured with a spectrophotometer (Hariri Akbari et al. 2012) The fresh and young leaves of each sample produced in a mortar were ground to a fine powder in liquid nitrogen, and transferred to the tubes 1/5 ml. 900 ml of SDS 4% extraction buffer was added per tube and placed for 45 Min in water bath at 65 deg C. and then added 300 ml of potassium acetate per tube for 25 Min under ice container. The tubes were centrifuged at a temperature of 3 deg C for 15 Min and at 12000rpm. 750 ml of the upper liquid transferred to a new tube and 750 ml of cold isopropanol added to it and placed 5 Min at laboratory temperature (25 deg C) Then the tubes were centrifuged at a temperature of 3 deg C for 15 Min and at 12000 rpm and after removing supernatant and tubes containing plates were incubated at 37 deg C for 20 to 30 Min. In order to purify, 700 ml of autoclaved deionized distilled water (DDW) was added to each tube that has been kept for 1 h at 4 deg C. and then added the chloroform isoAmyl alcohol solution (Powell et al. 1996) volume of 700 ml and shaken well until a uniform emulsion was obtained. Centrifugation was performed for 10 Min at 13000 rpm, the lower phase is chloroform Isoamyl alcohol and upper phase is solution containing DNA. The upper phase are transferred to a new 2 ml tube and was added 0/1 volume of sodium acetate supernatant liquid (3 M, pH =8) Absolute alcohol (100% that is 2/5 times volume of supernatant liquid was added and then dehydrated after 5 Min. centrifuge was performed for 10 Min at 13000 rpm and discarded the supernatant liquid and added 100 ml 70% alcohol to the sediment. The alcohol was removed and the samples incubated at 37 deg C for 20 to 30 Min to dry completely and then added TE buffer (100 ml) or deionized distilled water (DDW) The quality of the extracted DNA was tested on 1% agarose gel (Fig. 1) electrophoresis and quantity was measured with a spectrophotometer (Hariri Akbari et al. 2012) Statistical analysis The similarity matrix and cluster analysis was performed using NTSYS pc analytical software v.2. and dendrogram was constructed based on DICE similarity coefficient and the accessions were grouped by cluster analysis using the UPGMA algorithm method. To determine the correlation between similarity matrices and cophenetic, Mantel test was used that MAXCOMP coefficient obtained via COPH and SM coefficients was applied for the Mantel test (Powell et al. 1996) Results DNA extraction Extracted samples by Hariri et al.(2012) method on 1% agarose gel showed sharp bands and no fracture (Fig. 1) The DNA fracture on agarose gel can be investigated by this method. Broken DNA in the form of spots or smears can be seen as pieces with low molecular weight (Charles et al. 1991; Hariri Akbari et al. 2012) AFLP reaction Molecular markers, in particular DNA based markers provide reliable genetic information because of the in dependence of the confounding effects of environmental factors (Powell et al. 1996) J. Bio. Env. Sci. 2016 54 Reza et al. Table 1. Province, city and coordinate for 15 populations of North of Iran. Row Province City Coordinate 1 Golestan Aliabad 36deg54'30''N 54deg52'08''E 2 Golestan Azadshahr 37deg05'13''N 55deg10'26''E 3 Golestan Gonbad e Kavus 37deg15'00''N 55deg10'02''E 4 Golestan Gomishan 37deg04'18''N 54deg04'36''E 5 Golestan Maraveh Tappeh 37deg54'15''N 55deg57'21''E 6 North Khorasan Ashkhaneh 37deg33'41''N 56deg55'16''E 7 Mazandaran Sari 36deg33'48''N 53deg03'36''E 8 Gilan Asalem 37deg43'50''N 48deg57'13''E 9 Gilan Lowshan 3