Jordan Journal of Agricultural Sciences, Volume 11, No.4 2015

Assessment of Genetic Diversity of () 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 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 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.

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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

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Table 2. Primer sequences used for ISSR analysis in performed using the numerical 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

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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.

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The PIC values, a reflection of allele diversity and bupleuroide, S. chlorifolia, S. noctiflora and S. coronaria. frequency among the varieties, obtained high for all the The second subcluster manifested of S. aucheriana, S. ISSR loci tested. The PIC value ranged from 0.74 (UBC araratica, S. parjumanensis, S. spergulifolia and S. 835) to 0.95 (UBC 826) with a mean of 0.87.The indepransa. Cluster ІI consisted of S. latifolia and S. similarity coefficients based on 207 ISSR markers conoidea. S. vulgaris and S. dichothoma were included in ranged from 0.15 to 0.38 (Table 4). S. vulgaris and S. cluster III. Within cluster I, S. araratica and S. spergulifolia showed the lowest genetic similarity (0.15), parjumanensis appeared to be closer to each other. and S. araratica and S. parjumanensis appeared to have Correlation coefficient of similarity matrix and ISSR the highest similarity (0.38). dendrogram was calculated (0.73) which shows good The Complete Linkage method split the 13 species into relationship between the results of similarity matrix and three main clusters (Figure 3). Cluster І was divided into cluster analysis. two sub clusters. The first sub cluster comprised of S.

Table 4. Jaccard,s similarity coefficient between13 Silene species based on ISSR molecular marker.

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Figure 3. Dendrogram obtained by complete linkage cluster analysis based on ISSR data.

DISCUSSION difference in average band production could be due to Silene has a complicated species relationship due to differences in the studied species and populations. the complex nature of the genus as frequent Thirteen ISSR primers, used in this study, generated hybridization occurs among its species and also due to high levels of polymorphism that varied from 89.5 by rapid species/subspecies radiation in the genus (Rettig et UBC 847 to 100% by many other primers. The obtained al. 1992; Sheidai et al. 2010). polymorphism was similar to report of Sheidai (2012) In this study, 13 ISSR primers generated a total of studying 3 species of Silene at inert-population level 204 distinct polymorphic bands. DNA fragments (80.8 to100 %). The number of polymorphic bands were amplified by ISSR markers are associated to both coding variable from 9 (UBC 835 and UBC 868) to 27 (UBC and non-coding genome sections (Roldan-Ruiz et al. 826). Compared to other studies on genetic variation e.g, 2001). Three out of 207 bands, scored for ISSR primers, Sepehry (2012) on Salvia and Muthusamy (2008) on rice were monomorphic in all species and 204 bands showed bean, UBC 826 primer produced more polymorphic polymorphism with an average of 15.9 fragments per bands in Silene. Genetic diversity assessment based on primer. These observations were in accordance with the RAPD molecular marker detected 98% polymorphism results of Sheidai (2012) on genetic variability of three level in Iranian Auriculatae sect. Silene (Sheidai et al. populations of Silene species using ISSR molecular 2010) and Oxelman detected 64% polymorphism in marker with an average number of 15.6.The obtained Silene sectioidea (Caryophyllaceae) (Oxelman, 2005). value appeared to be 14.3 for Dianthus accessions (Xiao Polymorphic information content (PIC), as a et al. 2008) and 18.6 for Eremochloa ophiuroides diagnostic capacity of the marker, is associated not only (Milla-Lewis et al. 2012). The reason behind the to polymorphic alleles, but also to polymorphic allele

-1043- Assessment of Genetic… Tahereh Aghaee Bargish and Fatemeh Rahmani frequencies (Botstein et al. 1980). In ISSR analysis, the too. S. noctiflora and S. coronaria belong to Sect. mean PIC value was obtained 0.87 (0.74 to 0.95) with Melandriformes (Boiss, 1867) and Sect. Lychnidiformes the lowest owned by UBC 835 primer and highest (Melzh, 1988), respectively. Morphologically, they are belonging to the UBC 826 primer. In fact, PIC value the closest relatives. Sect. Lychnidiformes differs in estimate discriminatory power of a marker and, marker number of styles, calyx veins and indumentum from with higher PIC value possess higher identification Sect. Melandriformes (Edalatiyan et al. 2010). Our ISSR power (Botstein et al. 1980). Therefore, obtained high analysis also placed S. noctiflora and S. coronaria in PIC values in our study, demonstrate high efficiency of the same subcluster together. used primers in the isolation of Silene samples which According to our molecular marker investigation, S. can be proposed for similar studies. In a similar study, araratica, S. parjumanensis, S. spergulifolia and S. the mean PIC value reported 0.28 on average with a indepransa were grouped in cluster I. S. spergulifolia range of 0.18 to 0.34 in Eremochloa ophiuroides plant belongs to Sect. Spergulifoliae (Boiss, 1867) and S. (Milla-Lewis et al. 2012). araratica, S. parjumanensis and S. indeprensa belong to In our present study, Jaccard's similarity varied from Sect. Auriculatae (Boiss, 1867). These two sections are 0.15 to 0.38 for ISSR marker. Therefore, ISSR could detect morphologically close with difference in flower type and high diversity between Silene species. This capability could inflorescence (Edalatiyan et al. 2010). The ISSR data put be attributed to the different nature of this marker for target S. araratica and S. parjumanensis closest to each other, regions of amplification. Jolivet and Bernasconi (2007) also grouped in cluster I, too. studied genetic and morphological diversity in six In conclusion, genetic variation evaluation using populations of S. latifolia and reported significant these markers showed that diversity between species is molecular and genetic differentiation. sufficient. We hope that this research could be a good For accurate detection of genetic diversity between introduction to start working on other species of this species, cluster analysis was performed which revealed genus with taxonomical and medicinal values. three clusters. In general, ISSR clustering was in accordance with morphological classification. Based on ACKNOWLEDGMENT morphological classification, S. bupleuroides and This research was financially supported by the S.chlorifolia belong to Sect. Sclerocalycinae (Boiss, Ministry of Sciences and carried out in the 1867). Clustering based on ISSR, placed the S. Biotechnology Research Center of the Urmia University. bupleuroides and S.chlorifolia in one cluster (cluster I),

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ﺗﻘﻳﻳم اﻟﺗﻧوع اﻟﺟﻳﻧﻲ ﻟﺻﻧف ﻧﺑﺎت اﻟﺳﻳﻠﻳﻧﺔ ﻣن اﻟﻌﺎﺋﻠﺔ (اﻟﻘرﻧﻔﻠﻳﺔ ﺑﺎﺳﺗﺧدام اﻟواﺳﻣﺎت اﻟﺟزﻳﺋﻳﺔ ISSR )

1 1 ط ﺎ ﻫ رﻩ آﻗﺎﻳﯽ و ﻓﺎطﻣﻪ رﺣﻣﺎﻧﯽ

ﻣﻠﺧـص

ﻓﻲ اﻟﻌﻣﻝ اﻟﺣﺎﻟﻲ، ﻗﻣﻧﺎ ﺑﺗوظﻳف اﻟواﺳم اﻟﺟزﻳﺋﻲ ﻟﻠﺳﻠﺳﺔ اﻟﺑﺳﻳطﺔ اﻟﻣﺗداﺧﻠﺔ اﻟﻣﺗ ﻛررة (ISSR)، وﺗﻔﺎﻋﻝ ا ﻟ ﺑ ﻠ ﻣ رة اﻟﻣﺗﺳﻠﺳﻝ (PCR)ﻟﺗﻘﻳﻳم اﻟﺗﻧوع اﻟﺟﻳﻧﻲ ﻟﺛﻼﺛﺔ ﻋﺷر ﻧوﻋﺎ ﻣن ﻧﺑﺎت ﺳﻳﻠﻳﻧﺔ ﻓﻲ إﻳران. ﺗﺿﺧﻣت ﺛﻼﺛﺔ ﻋﺷر ﻣن ﺑﺎدﺋﺎت ISSR ﻟﻠﺣﻣض ا ﻟ ﻧ ووي ﻣﺎ ﻣﺟﻣوﻋﻪ 207 ﻣن ﺷظﺎﻳﺎ اﻟﺣﻣض اﻟﻧووي، ﻣﻧﻬﺎ 204 (98.85٪) ﻣن ﻣﺗﻌدد اﻷﺷﻛﺎﻝ. وﻛﺎﻧت اﻟﻘﻳﻣﺔ اﻟﺗﻲ ﺗم اﻟﺣﺻوﻝ ﻋﻠﻳﻬﺎ (PIC) 0.88. را وح ﻣﻌﺎﻣﻝ اﻟﺗﺷﺎﺑﻪ Jaccard ﻣن 0.15-38.0 ﺗﺑﻌﺎ ﻝ ISSR. ﻛﺷﻔت ﺑﻳﺎﻧﺎت اﻟﺗﺣﻠﻳﻝ اﻟﻌﻧﻘودي ﺛﻼث ﻣﺟﻣوﻋﺎت رﺋﻳﺳﻳﺔ و ﻛﺷف. ISSR ، ﻋن ﻣ ﺳ ﺗ وى ﻋﺎﻝ ﻟﺗﻌدد اﻷﺷﻛﺎﻝ وﺗﻣﺛﻝ اﻻﺧﺗﻼف اﻟﺟﻳﻧﻲ اﻟﻌﺎﻟﻲ ﺑﻳن اﻷﻧواع اﻟﻣﺗداﺧﻠﺔ ﻋﻠﻰ ﻣ ﺳ ﺗ وى ﺟﻧس ﻧﺑﺎت ﺳﻳﻠﻳﻧﺔ. اﻟﻛﻠﻣﺎت اﻟداﻟﺔ: اﻟﺗﺑﺎﻳن اﻟوراﺛﻲ. ﺳﻳﻠﻳﻧﺔ، ﺗﻌدد اﻷﺷﻛﺎﻝ، ISSR ..

1 ﻗﺳم ﻋﻠوم اﻷﺣﻳﺎء، ﺟﺎﻣﻌﺔ ّأورﻣﻳﺎ، اﻳران. [email protected] ﺗﺎرﻳﺦ اﺳﺗﻼم اﻟﺑﺣث 14/7/2014 وﺗﺎرﻳﺦ ﻗﺑوﻟﻪ 2014/11/30.

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