Genetic variability and relationship among different accessions of Froriepia subpinata Bail (Gijavash) an endan gered medicinal plant from Iran revealed by ISSR and IRAP markers

The genetic variability of Froriepia subpinata Ledeb. Bail., an endan­ gered Iranian endemic species, has been estimated with a total of 52 accessions using 20 markers including ISSR and IRAP. The results showed the polymorphic band produced by primers was 82.3%. The best mean values of genetic diversi­ ty parameters observed in ISSRs markers, being UBC873, UBC811, and UBC873 the best primers tested. The similarity range among accessions was 34.45% to 93.3%. The cluster analysis classified the accessions into five main groups that in totally, accessions with similarity in region generally were clustered in the same group. Overall, present study could provide elementary information for formulation of conservation strategies and invaluable elementary genetic infor­ mation for next breeding or designing conservation programs.


Introduction
Froriepia subinata Ledeb. Bail. syn= Buplerum subinatum, Froriepia nuda is a biennial medicinal and aromatic plant, locally known as Gijavash, that belong to the Apiaceae family. It is a selfpollinated plant with white flowers and small achene fruits. Gijavash leaves are used in diet people and have antimicrobial, antifungal properties and high antiox idant activity (Salmanian and Sadeghi, 2012). This species is the only endemic threatened one of Froriepia genus in northern Iran. In this genus, somatic chromosome number ranged between 2n= 14 and 2n=16. Some of its phonological characteristics like irregular and delay germina tions and also excessive and improper harvest have exposed it to mortali ty and annihilation (Mozaffarian, 2015).
Nowadays, due to increased human activities and excessive growth in residential area, plants habitats have been destroyed. So, investigation and study on each threatened plant for identifying best way to protect should be considered. Although pastures protection ways as well as molecular finger printing, micropropagation and other biotechnological methods and beneficial techniques (Glover and Abbott, 1995;Sudha et al., 1998) are the most important method for conservation of rare and endangered plants. Therefore, genetic diversity of endangered species is the first step in conservations strategies and selection for domesticating process (Vicente et al., 2011).
Moreover, recently an increasing number of stud ies for plant conservation biology, especially in rare endemic species have demonstrated the value of genetic data (Gaudeul et al., 2000;Bellusci et al., 2008;GonzálezPérez et al., 2009).
One of the most important features for longterm survival and adaptation to environment conditions of population or species is genetic variation within their taxon (Frankham, 2010).Having information on genetic diversity of a plant species is very necessary for its conservation (Höglund, 2009;Frankham, 2010;Laikre, 2010) as losses of genetic diversity are likely to have consequences for plant fitness (Reed and Frankham, 2003;Dostálek et al., 2010). Preserving rare species endangered especially those which have restricted geographic distributions is main concern of scientist because of their habitat destruction and fragmentation. On the other hand, losing allelic rich ness or genetic diversity in fragmented populations due to their genetic drift and inbreeding depression have increased population differentiation (Buza et al., 2000;Tomimatsu and Ohara, 2003).
Thus, an accurate estimate on the level and distri bution of genetic diversity of threatened and endan gered species seems necessary for designing conser vation programs (Smith and Wayne, 1996;Höglund, 2009). In addition, understanding the chance of species survival in the shortterm, formulation of conservation strategy for longterm survival need to population genetic information (Cires et al., 2013). Meanwhile, knowledge of population genetic struc ture can provide important information to under stand the evolution of rare and endangered different species. For example, by identifying populations of greatest evolutionary potential as well as populations best suited for source material for ex situ preserva tion or reintroduction (Furches et al., 2009).
According to the fragmentation distribution and the endangered status of this endemic species, there are no population genetic studies and conservation management plans. As an initial step in developing such plan, we have assessed the genetic variability of 52 natural populations of F. subpinata using inter simple sequence repeat (ISSR) and interretrotrans poson amplified polymorphism (IRAP).These popula tions were naturally grown in their own habitat. To avoid from possibility pollination between different populations, the distance between the populations was considered and tried to elect population which had more difference between each other due to mor phological and environmental characteristics.
ISSR and IRAP were chosen because of their advantages over other DNA polymorphism analysis methods, as they do not require prior sequence knowledge, cloning procedures or characterized probes. It is also generally accepted that they have a comparatively high reproducibility (Jones et al., 1997). Therefore, both techniques have been suc cessfully used in plant population genetic studies, especially for endangered species (Li and Jin, 2007;Gong et al., 2010;Noroozisharaf et al., 2015).
Froriepia subpinata is commonly used in tradition al foods in Iran for its bioactive compounds and antioxidant potential. However, the destruction of its natural habitats by human activity has put a strain on its survival. Therefore, the study of genetic diversity among accessions collected from different areas of Iran would be very useful in the biodiversity manage ment and conservation plans organization.

Plant Material
Fresh leaves of Gijavash (F. subpinata) accessions were gathered from 52 different localities of Guilan province, Iran. To accurate estimate the genetic vari ability, based on local people's knowledge and distri bution of the plant, we elected 52 locations through out several cities (Fig. 1). All the accessions are listed in Table 1 with the location and the altitude of each one. Plants were randomly selected from two or three individuals of each site. To reduce the probability duplicate sam pling fresh leaves were taken from each individual separated at least 3 m apart. Samples were immedi ately frozen in liquid nitrogen and kept at 80°C for genomic DNA extraction. In addition, voucher speci mens were collected, dried by pressing in absorbent paper, stored at room temperature, and lodged at the herbarium of the University of Guilan.

DNA extraction
DNA extraction was carried out using the CTAB method described by (Doyle, 1990) with minor modi fications as follow: approximately 5070 mg leaf material was ground in liquid nitrogen, then 600 µL of hot (65°C) extraction buffer 2X (100 mM Tris HCl, pH 8; 20 mM EDTA; 1.4 M NaCl; 2% CTAB; 1% PVP) was added. Subsequently, an equal volume of cold chloroform/Isoamyl alcohol (24:1) was added and mixed by gentile inversion of the tube until a light green single phase emulsion is performed. In the next step, the emulsion centrifuged at 10000 rpm for 10 min. Then, the aqueous phase transferred into a clean tube and addition 100 µL of CTAB solution (10% CTAB, 0.7 M NaCl) and the extraction was repeated. This step may takes several times until no precipitate can be detected at phenol/aqueous layer interface. The aqueous phase is removed and the rest mixed with an equal volume of hot (65°C) CTAB precipita tion buffer (50 mM Tris HCl, pH 8.5, 10 mM EDTA, 1% CTAB). The solution mixed gently and incubated every 35 min at room temperature for 30 minutes. The resulting CTAB/DNA complex is immediately plat ted by centrifugation at 12000 rpm for 10 min. The resulting pellet resuspended in 650 µL high salt buffer (10 mM Tris HCl, pH 8; 1 mM EDTA; 1 M NaCl) and the DNA precipitated by addition 1300 µL of cold 100% ethanol. The precipitation gently mixed and incubated every 35 min on ice for 30 min. then, it centrifuged at 12000 rpm for 10 min. The pellet was washed three times with 1 ml of cold 70% ethanol, and then dried at room temperature. Finally, pellet was resuspended in 100 µL TE buffer (10 mM Tris HCl, pH 8; 1mM EDTA). Extracted DNA was qualified using 1% (w/v) agarose gel electrophoresis. Afterwards, the DNA concentration and contamina tion rate was evaluated by NanoDrop spectropho tometers (Thermo Fisher scientific, 5225 Verona Rd, USA). For PCR reaction, only template of DNA was used which had a purity of 2 in a dilution of 15 ng/ml. PCR amplification PCR reactions were done in 1500 µL reaction vol umes containing 750 µL of sterile double distilled water, 150 µL of Taq polymerase reaction buffer (10×), 1 mM MgCl 2 , 150 µL of dNTPs (10 mM), 100 µL of each primer at 5 mM, 0.5 unit of Taq DNA poly merase, and 200 µL of plant DNA. The planning of thermal cycling was as follows: initial template denaturation at 94°C for 4 min, 35 cycles of denatu ration 94°C for 1 min, annealing at 4250°C (depend ing on primer used) ( Table 2) for 1 min, extension at 72°C for 90s, and final extension at 72°C for 5 min.
The PCR products were loaded on 1.5% (w/v) agarose gel in 1× TAE buffer at voltage of 70 for 90 min. The gel's images were captured using the Biometra gel documentation system (Whatman Biometra, Gottingen, Germany). The produced frag ments size in comparing to size marker was distin guished (GeneRuler 1 kb DNA ladder, SM0241, Fermentase, Ontario, Canada).

Data analysis
In all, 20 individual ISSR and IRAP primers with their combinations were used (Table 2). Only repro ducible and well clear bands in the replications were considered as potential polymorphic markers. It was assumed that each band represented the phenotype at a single biallelic locus, because the ISSR and IRAP markers are dominant (Williams et al., 1990). Amplified fragments were scored for presence (1) or absence (0) of homologous bands. According to PCR banding patterns, a data matrix was created for each reaction. Polymorphism information content (PIC), Effective multiplex ratio (EMR) and Marker index (MI) were calculated (Smith and Wayne, 1996).
Effective number of alleles (Ne), Nei's gene diversi ty (Nei, 1972) and Shannon's information index (Shannon and Weaver, 1949) were estimated for total accessions using POPGENE software version 1.31 (Yeh, 1999). Similarity matrix based on simple matching coefficient was constructed from the ISSR and IRAP data. It was used for the cluster analysis and construc tion of dendrogram through unweighted pairgroup method using arithmetic average (UPGMA), per formed by NTSYSPC software (Rohlf, 2000). In order to evaluate fitness between the dendrogram and simi larity matrix, the cophenetic correlation coefficient was calculated. Principal coordinate analysis (PCoA) was accomplished using GenStat (GenStat v12, VSN International Ltd, UK) on a similarity matrix.

Results
Twenty individuals ISSR and IRAP and their combi nations (ISSR+ISSR; ISSR+IRAP) produced 147 distin guishable fragments out of which 121 (82.31%) were polymorphic. The polymorphic rang was from 4 in UBC812 to 9 in UBC811 with an average number of 6.05 polymorphic bands per primer. The products number varied from 5 in UBC812 to 10 in UBC811.
On the whole, among the 20 used primers, maxi mum of the EMR, MI, Ne, H and I recorded in UBC811 (8.1), UBC873 (3.16), UBC825 (1.75), UBC825 (0.41) and UBC825 (0.60), respectively .Also it must be con sidered that the total mean of EMR, MI, Ne, H and I were 5.11, 1.59, 1.49, 028 and 0.43 respectively (Table 3). PIC= polymorphism information content; EMR= effective multiplex ratio; MI= marker index; Ne= effective number of alleles; H= Nei's gene diversity; I= Shannon's information index. In addition, there was a significant correlation at P≤0.01 probability level between most of these indices, so that only between EMR and Ne as well as EMR and H were significant at P≤0.05 level of proba bility (Table 4).
Principal coordinate analysis (PCoA) was con structed based on simple matching coefficient of sim ilarity. The results showed that the first twelve princi pal coordinates account for 70.29% of total variation. The first and second extracted component accounted for 26.43% and 9.11% of the variation, respectively (Fig. 2).
To draw cluster analysis for 52 Gijavash acces sions, the obtaining data from ISSR and IRAP analysis were used. Figure 3 presents the dendrogram of genetic relationships among the accessions as revealed by the UPGMA method. The 52 accessions of Gijavash classified into 5 main groups. The similari ty coefficient range varies from34.45% to 93.27%. The highest similarity was related to G4 and G6 and the lowest similarity observed between G26 and G38 (Fig. 3). Also, high amount of calculated cophenetic correlation coefficient (r=95.2%) showed that UPGMA method was useful in the clustering plant accessions.

Discussion and Conclusions
Using dominant molecular markers for assessing genetic diversity is usually similar and directly compa rable (Nybom, 2004). So that, these dominant mark ers widely have been used for earning genetic infor mation in large number of endemic and endangered species from different plant families (Jeong et al., 2010;Brütting et al., 2012;Cires et al., 2013;Noroozisharaf et al., 2015) and also for formulation and implementation conservation strategies, along with testing genetic relationships between species (GonzálezPérez et al., 2009). We applied 20 ISSR and IRAP primers to examine the genetic diversity of 52 accessions from the natural distribution of wild F. subpinata. The results showed that a high genetic diversity has been achieved in this species (H= 0.28, I= 0.43) in comparison with the corresponding genet ic coefficients of other endangered species (Hamrick and Godt, 1996;Nybom, 2004;Zheng et al., 2008). Accessions codes are identified in Table 1. The results of ISSR and IRAP markers demonstrat ed similar overall trends for genetic diversity. Nevertheless, the genetic diversity indices from IRAP approximately are lower than those from ISSR due to IRAP tending to produce somewhat low estimates of withinpopulation variation (Nybom, 2004).
According to the attributes of F. subpinata acces sions (i.e. fragmented, endemic) it could expected that there should be low genetic diversity, but in gen eral, it seems that the total genetic diversity based on ISSR and IRAP markers is similar to, or slightly higher, than most of those used by different authors in other plants like Primula heterochroma, Bupleurum rotundifolium, Changium smyrnioide, Cycas guizhouensis. Nei's genetic diversity accounts in other ISSR and IRAP studies ranged from 0.100.28 (Qiu et al., 2004;Wu et al., 2004;Xiao et al., 2004;Shao et al., 2009;Jeong et al., 2010;Brütting et al., 2012;Noroozisharaf et al., 2015). Base on this result and high polymorphism rate (82.31%), our research has manifested the potential of ISSR and IRAP mark ers, reproducible and useful methods for classifying different accessions.
Principal coordinate (PCoA) showed that acces sions were divided into two groups, (i) the first group of accessions who collected from East of Guilan Province and (ii) the second group belong to West of Guilan Province origination. Many biological factors can influence both the species genetic diversity and its distribution among populations. Among these, the geographic distribution has been considered as one of the most important (Hamrick and Godt, 1990).
In contrast, in another study the geographical range had no significant influence on genetic diversi ty (Nybom, 2004). Our finding may be related to self pollination character of this plant, that cause, acces sions with less distance from each other had more genetic similarity.
The result of cluster analysis (Fig. 2) also showed that accessions with same region had more similarity to each other, so that maximum of similarity (93.27%) was between G4 and G6, and also the low est of similarity (34.45%) was between G26 and G38. The G26 and G38 accessions originated from the east (Rudsar city) and west (Fuman city) of Guilan province, respectively (Fig. 1) and it could confirm the relative between genetic similarity and geograph ic distance in this research.
Overall, present study could provide invaluable elementary genetic information for next breeding plan.
Genetic diversity of different Gijavash accessions was analyzed using ISSR and IRAP molecular markers for the first time.
Results revealed that using of ISSR and IRAP mark ers had high efficiency for differentiating among the various accessions. Among all used primers, the high est PIC value, EMR and MI was belonging to UBC873, UBC811 and UBC873, respectively. The maximum of Ne, H and I observed in UBC825. With respect to these findings the UBC873, UBC811, UBC873 and specially UBC825 were the most informative primers which could be used to determine the diversity of Gijavash accessions.