Jordan Journal of Agricultural Sciences, Volume 11, No.4 2015 Assessment of Genetic Diversity of Silene (Caryophyllaceae) Species Using ISSR Molecular Marker Tahereh Aghaee Bargish1 and Fatemeh Rahmani2 ABSTRACT In the present work, we employed Inter-Simple Sequence Repeat (ISSR), a polymerase chain reaction (PCR)- based system to evaluate the genetic diversity of thirteen species of Silene in Iran. Thirteen ISSR primers amplified a total of 207 DNA fragments, of which 204 (98.85%) were polymorphic. The obtained mean polymorphism information content (PIC) value was 0.88. Jaccard’s similarity coefficient ranged from 0.15 to 0.38 according to ISSR. Cluster analysis data revealed three main clusters. ISSR detected high polymorphism level and represented high genetic variation at interspecies level in Silene genus. Keywords: Genetic variation; Silene; Polymorphism; ISSR. INTRODUCTION Jurgens et al. 2002, a; Moore et al. 2003; Tero et al. 2003; Bernasconi et al. 2009). Most of the Silene species are Silene L. is one of the largest genera of the world’s flora diploid having 2n = 2x = 24, or 2n = 2x = 20, some others with 700 species worldwide (Greuter, 1995). The genus is are tetraploid (4x = 48) and hexaploid (6x = 72) with a few widespread and most abundant in the Mediterranean species showing higher polyploidy level, 2n =96, 120 and (Oxelman et al. 1997). In Iran, Silene includes 110 species 192 (Bari, 1973). which 35 are endemic (Melzheimer, 1988). It contains Different molecular markers have been used in several important weeds, horticultural plants and some systematic and genetic diversity studies (Tonguc and medicinal species (Swank, 1932; Vestal, 1952). Silene often Griffiths, 2004; Hale and Farnham, 2006; Louarn et al. 2007; serves as model system for manifold scientific questions Lu et al. 2009; Lingling et al. 2012). Knowledge of genetic due to its enormous diversity of ecological and diversity among individuals provides an important data such morphological characters, reproductive systems and as genetic structure and gene flow (Al-Fawaeer et al. 2011). parasite host interactions (Alexander and Antonovics 1988; Molecular analyses provide an effectively scientific Delph and Meagher 1995; Desfeux and Lejeune 1996; basis for parent’s selection; development of the right strategy for germplasm collection, evaluation, conservation, 1 MSc, Department of Biology and Institute of and foreign genes introgression or exchange. The Inter- Biotechnology, Urmia University, Urmia, Iran 2 PhD, Assistant of Professor, Department of Biology Simple Sequence Repeat (ISSR) marker involves and Institute of Biotechnology, Urmia University, polymerase chain reaction (PCR) amplifications of DNA Urmia, Iran using a primer composed of a microsatellite sequence [email protected] Received on 14/7/2014 and Accepted for Publication on (Zietkiewicz et al. 1994). ISSRs have been successfully 30/11/2014. used in genetic diversity studies of large number of plants (Al-Maarri et al. 2014). © 2015 DAR Publishers/The University of Jordan. All Rights Reserved. -1037- Assessment of Genetic… Tahereh Aghaee Bargish and Fatemeh Rahmani A few molecular studies have been performed on Silene MATERIALS AND METHODS in the world and there are not much systematic studies on Plant materials. Species of Silene genus were the genetic relationship among the species of Silene based collected from their natural growing regions of West on molecular markers in Iran. In the present study; we Azerbijan and Khorasan area (Figure 1).The details on systematically investigate the genetic relationship between sample collection are given in Table 1. Fresh young thirteen species of Silene genus in Iran using ISSR marker. leaves of 5 to 6 plants were collected from the field- grown plants and stored at -80°C prior to DNA extraction. Plant leaves of the same species were mixed prior to DNA extraction. Table 1. Location of Silene species used in the present study. Species Location 1 S. bupleuroides Urmia, Salmas road, Ghoshchi 1800 m. 2 S. vulgaris Maku Between GarehKhaj and Dibak, 1850 m. 3 S. aucheriana Maku Between GarehKhaj and Torkan 1850 m. 4 S. dichotoma Urmia, Jade Sero, Nazlu, 1273 m. 5 S. latifolia Urmia, Jade Emamzade, 1273 m. 6 S. chlorifolia Urmia, Ghoshchi, koheSomakh, 1800 m. 7 S. araratica Maku Between GarehKhaj and Dibak, 1850 m. 8 S. conoidea Khoy, Psak Village,1700 m. 9 S. spergulifolia Urmia,45 km Oshnaviyeh, 1300 m. 10 S. parjumanensis Western Khorasan, W-Ghaeen, Tajan mountains, 2000 m. 11 S. noctiflora North Khorasan, W-S Bojnord, Rien, 1700 m. 12 S. indeprensa Khorasan, Daregaz, International Park Tounduoreh, 2400 m. 13 S. coronaria Goulestan, E-N International Park Goulestan, 1200 m. -1038- Jordan Journal of Agricultural Sciences, Volume 11, No.4 2015 Figure 1.Geographical distribution of selected Silene spp. used in this study. The collection area has been highliighted with grrey color. DNA extraction. The genomic DNA was extracted (1cycle); 2) Denaturation at 95˚С for 30 s, 54˚С for 45 s following the modified CTAB method (Liu et al. 2003). using Auto Delta program with reduction of one degree in The concentration of each DNA sample was determined every cycle, extension at 72˚С for 2 min (35 cycles); 3) spectrophotometrically at 260 nm (Biophotometer 6131; final extension at 72˚С for 10 min (1 cycles). The PCR Eppendorf, Hamburg, Germany). DNA samples having products were electrophoresed (Bio-Rad) on 1.5% agarose good degree of purity (1.8-2) were considered for PCR in 0.5X TBE buffer. The gels stained with ethidium amplifications. The integrity of genomic DNA was bromide (10μg /ml) and phhotographed under ultraviolet determined by electrophoresis on1.0% (w/v) agarose gel (UV) light in a gel documentation system (Carestream and then diluted to 10 ng /µl for PCR reactions. 212 Pro Imaging System, USA). Primers which gave ISSR-PCR amplification. Thirteen ISSR primers were reproducible fingerprints (DNA bands) were considered purchased from Cinnagen; Tehran, Iran (Table 2). DNA for data analysis. Size of DNA fragments were estimated amplifications were performed in a 96-well Applied by comparison with the Gene Ruler DNA size marker Biosystem using Touchdown program with the following 1Kb (Fermentas). cycle profile: 1) Initial denaturation at 95˚С for 3 min -1039- Assessment of Genetic… Tahereh Aghaee Bargish and Fatemeh Rahmani Table 2. Primer sequences used for ISSR analysis in performed using the numerical taxonomy multivariate this study. analysis system Software package (NTSYS-pc); version Annealing 2.02 (Yap and Nelson, 1996; Rohlf, 2002). ISSR primers (5’ -3’) temperature Polymorphism information content (PIC) values were UBC807 (AG)8T 48˚C estimated according to the formula: PIC= 1 – Σ (Pij) 2, UBC816 (CA)8T 44˚C Where, Pij is the frequency of the ith pattern revealed UBC826 (AC)8C 44˚C by the jth primer summed across all patterns revealed by UBC835 (AG)8TC 44˚C the primers (Botstein et al. 1980). Dendrogram was UBC836 (AG)8YA 44˚C constructed based on the genetic similarity matrix using UBC841 (GA)8YC 42˚C Complete Linkage method. The representativeness of the UBC842 (GA)8YG 44˚C dendrogram was evaluated by estimating cophenetic UBC847 (CA)8RC 49˚C correlation coefficient and comparing it with the UBC868 (GAA)6 62˚C similarity matrix using Mantel matrix correspondence UBC888 BDB(CA)7 52˚C test (Mantel, 1967). UBC889 DBD(AC)7 50˚C UBC890 VHV(GT)7 52˚C RESULTS UBC891 HVH(GT)7 44˚C ISSR analysis. Thirteen ISSR primers amplified a R = (A, G); Y = (C, T); B = (C, G, T); D = (A, G, T); H total of 207scorable bands (Table 3) of which 204 were = (A, C, T); V = (A, C, G) polymorphic (99%). High level of molecular polymorphism was detected ranging from 89.47% Data analyses. ISSR amplifications were repeated (primer UBC 847) to 100% (most of the other primers). twice and only clear bands considered for scoring. Faint Typical result obtained with the UBC 888 primer is or unclear bands were not considered .The amplified shown in Figure 2. The number of scorable bands DNA fragments were recorded as either (1) or (0), produced per primer ranged from 9 (UBC 835& UBC representing the presence or absence of the band, 868) to 27(UBC 826), with an average of 15.9 fragments respectively. Data analysis was conducted using per primer. The size of the amplified products ranged polymorphic bands. Amplified fragments were scored to from 190 bp to 2500 bp. create binary data matrices. Data analyses were -1040- Jordan Journal of Agricultural Sciences, Volume 11, No.4 2015 Table 3. Genetic diversity of Silene species revealed by ISSR. Total number Number of Number of Percentage of PIC Primers of bands polymorphic bands monomorphic bands polymorphic bands (PPB) value UBC807 17 17 0 100 0.91 UBC816 13 13 0 100 0.86 UBC826 27 27 0 100 0.95 UBC835 9 9 0 100 0.74 UBC836 13 13 0 100 0.91 UBC841 12 12 0 100 0.94 UBC842 23 22 1 95.6 0.92 UBC847 19 17 2 89.47 0.85 UBC868 9 9 0 100 0.78 UBC888 14 14 0 100 0.87 UBC889 11 11 0 100 0.86 UBC890 23 23 0 100 0.91 UBC891 17 17 0 100 0.91 Total 207 204 0 - - Mean 15.9 15.6 - 98.85 0.87 Figure 2.Amplified results of Silene species using primer UBC 888. The numbers from left to right are representative of 1) S.bupleuroides; 2 ) S.vulgaris; 3) S.aucheriana; 4) S.dichotoma; 5) S.latifolia; 6) S.chlorifolia; 7) S.araratica; 8) S. conoidea; 9) S.spergulifolia; 10) S.parjumanensis; 11) S.noctiflora; 12) S.indeprensa; 13) S.coronaria. L represents 1Kb DNA ladder. -1041- Assessment of Genetic… Tahereh Aghaee Bargish and Fatemeh Rahmani The PIC values, a reflection of allele diversity and bupleuroide, S.