Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 97-107, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-968 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: S. Ma, M. Khayatnezhad, A. Abbas Minaeifar (2021) Genetic diversity and relationships among Hypericum L. species by ISSR Markers: A high value medicinal plant from Northern of Iran. Caryologia 74(1): 97-107. doi: 10.36253/ caryologia-968 Received: June 14, 2020 Accepted: September 24, 2021 Published: July 20, 2021 Copyright: © 2021 S. Ma, M. Khayat- nezhad, A. Abbas Minaeifar. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID SM: 0000-0002-9371-1498 Genetic diversity and relationships among Hypericum L. species by ISSR Markers: A high value medicinal plant from Northern of Iran Shuyan Ma1,*, Majid Khayatnezhad2, Amir Abbas Minaeifar3 1 Tangshan Vocational & Technical College, Department of Agricalture and Forestry Engi- neering, Hebei Tangshan 063000, China 2 Young Researchers Club, Islamic Azad University-Ardabil Branch, Ardabil, Iran 3 Department of Biology. Payame Noor University. P.O. Box19395-3697 Tehran. Iran *Corresponding author. E-mail: ngmashuyan@163.com; aaminaeifar@pnu.ac.ir Abstract. Hypericum L. species are generally known locally in Iran with the names “Hofariqun” which Ebn Sina (or Bo Ali Sina) called it. Plants of the genus Hypericum have traditionally been used as medicinal plants in various parts of the world. Hyperi- cum perforatum L. is the source to one of the most manufactured and used herbal preparations in recent years, especially as a mild antidepressant. Therefore, due to the importance of these plant species, we performed a molecular data for this species. For this study, we used 175 randomly collected plants from 17 species in 9 provinces. Amplification of genomic DNA using 10 primers produced 141 bands, of which 127 were polymorphic (95.78%). The obtained high average PIC and MI values revealed high capacity of ISSR primers to detect polymorphic loci among Hypericum species. The genetic similarities of 17 collections were estimated from 0.617 to 0.911. Accord- ing to Inter-Simple sequence repeats (ISSR) markers analysis, H. androsaemum and H. hirtellum had the lowest similarity and the species of H. perforaturm and H. triquetri- folium had the highest similarity. The aims of present study are: 1) can ISSR markers identify Hypericum species, 2) what is the genetic structure of these taxa in Iran, and 3) to investigate the species inter-relationship? The present study revealed that ISSR markers can identify the species. Keywords: Iran, species identification, structure, Hypericum, ISSR markers. INTRODUCTION Identifying the accurate boundaries of a species is critical to have a bet- ter perspective of any biological studies. Therefore, species delimitation is a subject of extensive part of studies in the framework of biology (Collard & Mackill 2009, Wu et al. 2013). However, defining the criterion which could address the boundaries of species is different and the place of debates (Esfan- dani-Bozchaloyi et al. 2018a, 2018b, 2018c, 2018d). Wild relatives of crops contain genes with the great potential for use in breeding programs and con- stitute a part of their gene pool (Pandey et al. 2008). In addition, the study of 98 Shuyan Ma, Majid Khayatnezhad, Amir Abbas Minaeifar intra-specific levels of genetic variation and investigation of genetic structure of wild populations is crucial for development of effective conservation strategies. The genus Hypericum (Guttiferae, Hypericoideae) is perennial, belonging to the Hypericaceae family, hav- ing 484 species in forms of trees, shrubs, and herbs, dis- tributed in 36 taxonomic sections (Crockett and Robson 2011). The species of the family are distributed worldwide in the temperate zones but are absent in extreme envi- ronmental conditions such as deserts and poles. Iranian species of this genus grow mainly in north, northwest and center of Iran and form floristic elements of Hyrca- nian mountainous areas, Irano-Turanian, Mediterranean and Zagros elements. They generally prefer steep slopes of rocky and calcareous cliffs and margin of mountain- ous forests (Robson 1968; Azadi 1999). Robson (1968) introduced 21 species in the area covered by Flora Irani- ca. Robson (1977) and Assadi (1984) reported H. fursei N. Robson and H. dogonbadanicum Assadi as two endemics of North and South West of Iran. In Flora of Iran, Aza- di (1999) identified 19 species, 4 subspecies arranged in 5 sections (comprising Campylosporus (Spach) R. Keller, Hypericum, Hirtella Stef., Taeniocarpum Jaub. & Spach. and Drosanthe (Spach) Endl.), and two doubtful species including H. heterophyllum Vent. and H. olivieri (Spach) Boiss. Hypericum species are generally known locally in Iran with the names “Hofariqun” which Ebn Sina (or Bo Ali Sina) called it (Rechinger, 1986). St. John’s wort (Hypericum perforatum L.) is the most important medic- inal species of the genus and its main uses in medicine includes treatment of mild and moderate depression, skin wounds and burns (Barnes et al. 2001). The plant con- tains a vast array of secondary metabolites, among which naphthodianthrones (hypericin and pseudohypericin), acylphloroglucinols (hyperforin and adhyperforin) and essential oil can be mentioned (Morshedloo et al. 2012; Radusiene et al. 2005). Molecular markers provide a powerful tool for study- ing the genetic diversity. Among advanced genetic mark- ers, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers have been widely used for diversity analyses (Pharmawati et al. 2004). RAPD technique is quick, easy and requires no pri- or sequence information. The technique detects nucleotide sequence polymorphism using a single primer of arbitrary nucleotide sequence (Moreno et al., 1998). ISSR marker involves PCR amplification of DNA by a single 16-18 bp. long primer composed of a repeated sequence anchored at the 3’ or 5’ end of 2-4 arbitrary nucleotides. The technique is rapid, simple, inexpensive and more reproducible than RAPD (Esfandani-Bozchaloyi et al. 2017a, 2017b, 2017c, 2017d), (Collard & Mackill 2009, Wu et al. 2013). The present investigation has been carried out to evaluate the genetic diversity and relationships among different Hypericum species using new gene-targeted molecular markers, i.e ISSR markers . This is the first study on the use of ISSR markers in Hypericum genus; Therefore, we performed molecular study of 175 collect- ed specimens of 17 Hypericum species. We try to answer the following questions: 1) Is there infra and interspecif- ic genetic diversity among studied species? 2) Is genetic distance among these species correlated with their geo- graphical distance? 3) What is the genetic structure of populations and taxa? 4) Is there any gene exchange between Hypericum species in Iran? MATERIALS AND METHODS Plant materials A total of 175 individuals were sampled representing 17 geographical populations belong 17 Hypericum spe- cies in East Azerbaijan, Lorestan, Kermanshah, Guilan, Mazandaran, Esfahan, Tehran, Hamadan and Kohgi- luyeh and Boyer-Ahmad Provinces of Iran during July- Agust 2016-2019 (Table 1). For ISSR analysis we used 175 plant accessions (Five to twelve samples from each popu- lations) belonging to 17 different populations with dif- ferent eco-geographic characteristics were sampled and stored in -20 till further use. More information about geographical distribution of accessions are in Table 1 and Fig. 1. Morphological studies Five to twelve samples from each species were used for Morphometry. In total 18 morphological (11 quali- tative, 7 quantitative) characters were studied. Data obtained were standardized (Mean= 0, variance = 1) and used to estimate Euclidean distance for clustering and ordination analyses (Podani 2000). Morphological characters studied are: corolla shape, bract shape, calyx shape, calyx length, calyx width, calyx apex, calyx mar- gins, bract length, corolla length, corolla width, corolla apex, leaf length and leaf width, leaf apex, leaf margins, leaf shape, leaf gland and bract margins. DNA Extraction and ISSR Assay Fresh leaves were used randomly from one to twelve plants in each of the studied populations. These were dried by silica gel powder. CTAB activated char- 99Genetic diversity and relationships among Hypericum L. species by ISSR Markers coal protocol was used to extract genomic DNA (Esfan- dani-Bozchaloyi et al. 2019). The quality of extracted DNA was examined by running on 0.8% agarose gel. For the ISSR analysis, 22 primers from UBC (Univer- sity of British Columbia) series were tested for DNA amplification. Ten primers were chosen for ISSR anal- ysis of genetic variability, based on band reproducibly (Table 2). PCR reactions were carried in a 25μl volume containing 10 mM Tris-HCl buffer at pH 8; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of a single primer; 20 ng genomic DNA and 3 U of Taq DNA polymerase (Bioron, Ger- many). The amplifications, reactions were performed in Techne thermocycler (Germany) with the following program: 5 min initial denaturation step 94°C, fol- lowed by 40 cycles of 1 min at 94°C; 1 min at 52-57°C and 2 min at 72°C. The reaction was completed by final extension step of 7-10 min at 72°C. The amplification products were observed by running on 1% agarose gel, followed by the ethidium bromide staining. The frag- ment size was estimated by using a 100 bp molecular size ladder (Fermentas, Germany). Table 1. Voucher details of Hypericum species in this study from Iran. No Section Sp. Locality Latitude Longitude Altitude (m) Sp1 Campylosporus (Spach) R. Keller H. dogonbadanicum Assadi Kohgiluyeh and Boyer-Ahmad 38°52’37” 47°23’92” 1144 Sp2 Androsaemum (Duhamel) Godron H. androsaemum L. Mazandaran, Haraz road, Emam Zad-e-Hashem 32°50’03” 51°24’28” 1990 Sp3 Hypericum H. tetrapterum Fries. Guilan, Sangar, Road sid 29°20’07” 51° 52’08” 1610 Sp4 H. perforaturn L. Esfahan:, Ghameshlou, Sanjab 38°52’37” 47°23’92” 1144 Sp5 H. triquetrifolium Turra Lorestan, Oshtorankuh, above Tihun village 33°57’12” 47°57’32” 2500 Sp6 Hirtella Stef. H. lysimachioides Boiss. & Noe in Boiss. Kermanshah, Islamabad 34°52’37” 48°23’92” 2200 Sp7 H. asperulum Jaub. & Spach. Hamedan, Nahavand 38°52’37” 47°23’92” 1144 Sp8 H. scabrum L. Azerbaijan, 78 km from Mianeh to Khalkhl. 35°50’03” 51°24’28” 1700 Sp9 H. hirtellum (Spach) Boiss. Lorestan, Durood 36°14’14” 51°18’07” 1807 Sp10 H. elongaturn Ledeb. Guilan, Lahijan 32°36’93” 51°27’90” 2500 Sp11 H. davisii N. Robson East Azerbaijan, Arasbaran 37°07’02” 49°44’32” 48 Sp12 H. apricum Kar. & Kir. Azarbaiejan, 48 km from Tabriz to Marand 28°57’22” 51°28’31” 430 Sp13 H. helianthemoides (Spach) Boiss. Tehran, Damavand 30°07’24” 53°59’06” 2178 Sp14 H. vermiculare Boiss. & Hausskn Hamedan, Alvand 28°57’22” 51°28’31” 288 Sp15 Taeniocarpium H. hirsutum L. Mazandaran, Nowshahr 34°46’10” 48°30’00” 1870 Sp16 H. linarioides Bosse. Azarbaiejan, West of Tabriz 35°37’77” 46°20’25” 1888 Sp17 H. armennm Jaub. & Spach, Mazandaran, Chalos 33°47’60” 46°07’58” 1250 Figure 1. Map of Iran shows the collection sites and provinces where Hypericum species were obtained for this study; sp1= H. dogonbadanicum; sp2= H. androsaemum; sp3= H. tetrapterum; sp4= H. perforaturm; sp5= H. triquetrifolium; sp 6= H. lysimachioides; sp7= H. asperulum; sp8= H. scabrum; sp9= H. hirtellum; sp10: H. elongaturn ; sp11: H. davisii ; sp12= H. apricum; sp13= H. helian- themoides ; sp14= H. vermiculare; sp15= H. hirsutum; sp16= H. lin- arioides; sp17= H. armennm. 100 Shuyan Ma, Majid Khayatnezhad, Amir Abbas Minaeifar Data Analyses Morphological Studies Morphological characters were f irst standard- ized (Mean = 0, Variance = 1) and used to establish Euclidean distance among pairs of taxa (Podani 2000). For grouping of the plant specimens, The UPGMA (Unweighted paired group using average) ordination methods were used (Podani 2000). ANOVA (Analysis of variance) were performed to show morphological differ- ence among the populations while, PCA (Principal com- ponents analysis) biplot was used to identify the most variable morphological characters among the studied populations (Podani 2000). PAST version 2.17 (Hammer et al. 2012) was used for multivariate statistical analyses of morphological data. Molecular Analyses ISSR bands obtained were coded as binary char- acters (presence = 1, absence = 0) and used for genetic diversity analysis. Discriminatory ability of the used primers was evaluated by means of two parameters, polymorphism information content (PIC) and marker index (MI) to characterize the capacity of each primer to detect polymorphic loci among the genotypes (Pow- ell et al. 1996). MI is calculated for each primer as MI = PIC × EMR, where EMR is the product of the number of polymorphic loci per primer (n) and the fraction of polymorphic fragments (β) (Heikrujam et al. 2015). The number of polymorphic bands (NPB) and the effective multiplex ratio (EMR) were calculated for each primer. Parameter like Nei’s gene diversity (H), Shannon infor- mation index (I), number of effective alleles, and per- centage of polymorphism (P% = number of polymorphic loci/number of total loci) were determined (Weising et al, 2005, Freeland et al. 2011). Shannon’s index was cal- culated by the formula: H’ = -Σpiln pi. Rp is defined per primer as: Rp = ∑ Ib, were “Ib” is the band informative- ness, that takes the values of 1-(2x [0.5-p]), being “p” the proportion of each genotype containing the band. The percentage of polymorphic loci, the mean loci by acces- sion and by population, UHe, H’ and PCA were calcu- lated by GenAlEx 6.4 software (Peakall & Smouse 2006). Nei’s genetic distance among populations was used for Neighbor Joining (NJ) clustering and Neighbor-Net net- working (Freeland et al. 2011, Huson & Bryant 2006). Mantel test checked the correlation between geographi- cal and genetic distances of the studied populations (Podani 2000). These analyses were done by PAST ver. 2.17 (Hammer et al. 2012), DARwin ver. 5 (2012) soft- ware. AMOVA (Analysis of molecular variance) test (with 1000 permutations) as implemented in GenAlex 6.4 (Peakall & Smouse 2006) were used to show genetic difference of the populations. Gene flow was determined by (i) Calculating Nm an estimate of gene flow from Gst by PopGene ver. 1.32 (1997) as: Nm = 0.5(1 - Gst)/Gst. This approach considers the equal amount of gene flow among all populations. RESULTS Species identification and inter-relationship Morphometry ANOVA showed significant differences (P <0.01) in quantitative morphological characters among the spe- cies studied. In order to determine the most variable characters among the taxa studied, PCA analysis has been performed. It revealed that the first three factors comprised over 73% of the total variation. In the first PCA axis with 57% of total variation, such characters as corolla shape, calyx shape, calyx length, bract length and leaf shape have shown the highest correlation (>0.7), leaf apex, corolla length, leaf length, leaf width were charac- ters influencing PCA axis 2 and 3 respectively. Differ- ent clustering and ordination methods produced similar results therefore, PCA plot of morphological characters are presented here (Fig. 2). In general, plant samples of each species were grouped together and formed separate groups. This result show that both quantitative and qual- itative morphological characters separated the studied species into distinct groups. In the studied specimens we did not encounter intermediate forms. Species Identification and Genetic Diversity Ten ISSR primers were screened to study genetic relationships among Hypericum species; all the prim- ers produced reproducible polymorphic bands in all 17 Hypericum species. An image of the ISSR amplifica- tion generated by ISSR-5 primer is shown in Figure 3. A total of 127 amplified polymorphic bands were gener- ated across 17 Hypericum species. The size of the ampli- fied fragments ranged from 200 to 3000 bp. The highest and lowest number of polymorphic bands was 18 for ISSR-2 and 7 for ISSR-6, on an average of 12.7 polymor- phic bands per primer. The PIC of the 10 ISSR primers ranged from 0.23 (ISSR-3) to 0.44 (ISSR-6) with an aver- 101Genetic diversity and relationships among Hypericum L. species by ISSR Markers age of 0.36 per primer. MI of the primers ranged from 1.37 (ISSR-9) to 4.47 (ISSR-1) with an average of 3.8 per primer. EMR of the ISSR primers ranged from 4.60 (ISSR-6) to 11.11 (ISSR-9) with an average of 8.9 per primer (Table 2). The primers with the high EMR values were considered to be more informative in distinguish- ing the genotypes. The genetic parameters were calculated for all the 17 Hypericum species amplified with ISSR primers (Table 3). Unbiased expected heterozygosity (H) ranged from 0.10 (H. hirsutum) to 0.31 (H. elongaturn), with a mean of 0.21. A similar pattern was observed for Shan- non’s information index (I), with the highest value of 0.33 observed in H. elongaturn and the lowest value of 0.13 observed in H. hirsutum with a mean of 0.23. The observed number of alleles (Na) ranged from 0.23 in H. linarioides to 0.56 in H. apricum. The effective number of alleles (Ne) ranged from 1.01 (H. scabrum) to 1.38 (H. elongaturn). AMOVA test showed significant genetic difference (P = 0.001) among studied species. It revealed that 63% of total variation was among species and 37% was with- in species (Table 4) Moreover, genetic differentiation of these species was demonstrated by significant Nei’s GST (0.31, P = 0.001) and D_est values (0.167, P = 0.001). These results revealed a higher distribution of genetic diversity among Hypericum species compared to within species. Different clustering and ordination methods pro- duced similar results therefore, UPGMA clustering are presented here (Figure 4). In general, plant samples of each species belong to a distinct section, were grouped together and formed separate cluster. This result show Figure 2. PCA plots of morphological characters revealing species delimitation in th Hypericum species; sp1= H. dogonbadanicum; sp2= H. androsaemum; sp3= H. tetrapterum; sp4= H. perforaturm; sp5= H. triquetrifolium; sp 6= H. lysimachioides; sp7= H. asperulum; sp8= H. sca- brum; sp9= H. hirtellum; sp10: H. elongaturn ; sp11: H. davisii ; sp12= H. apricum; sp13= H. helianthemoides ; sp14= H. vermiculare; sp15= H. hirsutum; sp16= H. linarioides; sp17= H. armennm. Figure 3. Electrophoresis gel of studied ecotypes from DNA frag- ments produced by ISSR-7. sp1= H. dogonbadanicum; sp2= H. androsaemum; sp3= H. tetrapterum; sp4= H. perforaturm; sp5= H. triquetrifolium; sp 6= H. lysimachioides; sp7= H. asperulum; sp8= H. scabrum; sp9= H. hirtellum; sp10: H. elongaturn ; sp11: H. davisii ; sp12= H. apricum; sp13= H. helianthemoides ; sp14= H. vermicu- lare; sp15= H. hirsutum; sp16= H. linarioides; sp17= H. armennm. 102 Shuyan Ma, Majid Khayatnezhad, Amir Abbas Minaeifar that molecular characters studied can delimit Hyperi- cum species in two different major clusters or groups. In the studied specimens we did not encounter intermedi- ate forms. In general, two major clusters were formed in UPGMA tree (Figure. 4), Populations of H. dogonbadan- icum (sect. Campylosporus) and H. vermiculare (sect. Hirtella) were placed in the first major cluster and were placed with great distance from the other species. The second major cluster included two sub-clusters. Plants of H. perforaturm and H. triquetrifolium (sect. Hypericum) and H. androsaemum (sect. Androsaemum) comprised the first sub-cluster, while plants of H. lysimachioides; H. asperulum; H. scabrum; H. hirtellum; H. elongaturn; H. davisii; H. apricum; H. helianthemoides (sect. Hirtella) formed the second sub-cluster. In general, relationships obtained from ISSR data agrees well with species relationship obtained from morphological. This is in agreement with AMOVA and Table 3. Genetic diversity parameters in the studied Hypericum species. SP N Na Ne I He UHe %P H. dogonbadanicum 8.000 0.499 1.067 0.18 0.171 0.14 49.26% H. androsaemum 9.000 0.261 1.024 0.192 0.23 0.23 43.15% H. tetrapterum 6.000 0.555 1.021 0.29 0.25 0.18 43.53% H. perforaturn 10.000 0.431 1.088 0.23 0.22 0.23 57.53% H. triquetrifolium 3.000 0.255 1.021 0.15 0.18 0.12 42.15% H. lysimachioides 3.000 0.288 1.024 0.23 0.25 0.27 64.30% H. asperulum 9.000 0.352 1.083 0.23 0.22 0.14 45.05% H. scabrum 8.000 0.333 1.016 0.192 0.12 0.22 48.23% H. hirtellum 12.000 0.247 1.199 0.271 0.184 0.192 55.91% H. elongaturn 5.000 0.358 1.380 0.334 0.30 0.31 66.50% I. davisii 6.000 0.299 1.029 0.231 0.18 0.23 44.38% H. apricum 3.000 0.567 1.062 0.24 0.224 0.213 44.73% H. helianthemoides 8.000 0.499 1.067 0.14 0.181 0.14 49.26% H. vermiculare 9.000 0.261 1.034 0.142 0.13 0.13 33.15% H. hirsutum 6.000 0.545 1.021 0.13 0.10 0.10 23.53% H. linarioides 6.000 0.234 1.032 0.26 0.23 0.18 45.53% H. armennm 8.000 0.499 1.067 0.19 0.191 0.14 39.26% Abbreviations: (N = number of samples, Na= number of different alleles; Ne = number of effective alleles, I= Shannon’s information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism, populations). Table 2. ISSR primers used for this study and the extent of polymorphism. Primer name Primer sequence (5’-3’) TNB NPB PPB PIC PI EMR MI ISSR-1 DBDACACACACACACACA 12 12 100.00% 0.26 5.86 8.55 2.45 ISSR-2 GGATGGATGGATGGAT 10 9 84.99% 0.23 2.91 7.43 3.85 ISSR-3 GACAGACAGACAGACA 15 15 100.00% 0.44 3.34 10.55 2.44 ISSR-4 AGAGAGAGAGAGAGAGYT 10 10 100.00% 0.37 3.88 6.56 1.85 ISSR-5 ACACACACACACACACC 18 18 100.00% 0.35 5.23 6.23 4.47 ISSR-6 GAGAGAGAGAGAGAGARC 15 14 93.74% 0.37 4.66 5.56 3.67 ISSR-7 CTCTCTCTCTCTCTCTG 13 12 92.31% 0.34 4.21 4.60 3.55 ISSR-8 CACACACACACACACAG 13 13 100.00% 0.27 3.32 9.55 3.45 ISSR-9 GTGTGTGTGTGTGTGTYG 11 7 82.89% 0.43 5.56 6.34 3.11 ISSR-10 CACACACACACACACARG 17 17 100.00% 0.29 3.25 11.11 1.37 Mean 14.1 12.7 95.78% 0.36 4.8 8.9 3.8 Total 141 127 Note: TNB - the number of total bands, NPB: the number of polymorphic bands, PPB (%): the percentage of polymorphic bands, PI: poly- morphism index, EMR, effective multiplex ratio; MI, marker index; PIC, polymorphism information content for each of CBDP primers 103Genetic diversity and relationships among Hypericum L. species by ISSR Markers genetic diversity parameters presented before. The spe- cies are genetically well differentiated from each other. These results indicate that ISSR molecular markers can be used in Hypericum species taxonomy. The Nm analy- sis by Popgene software also produced mean Nm= 0.123, that is considered very low value of gene flow among the studied species. Mantel test with 5000 permutations showed a sig- nificant correlation (r = 0.23, p=0.0002) between genetic distance and geographical distance, so isolation by dis- tance (IBD) occurred among the Hypericum species studied. Nei’s genetic identity and the genetic distance deter- mined among the studied species (Table 5). The results showed that the highest degree of genetic similarity Figure 4. UPGMA tree of ISSR data revealing species delimitation in the Hypericum. Table 4. Analysis of molecular variance (AMOVA) of the studied species. Source df SS MS Est. Var. % ΦPT Among Pops 28 1901.364 73.789 12.154 63% 63% Within Pops 129 234.443 3.805 2.888 37% Total 144 1955.807 13.060 100% df: degree of freedom; SS: sum of squared observations; MS: mean of squared observations; EV: estimated variance; ΦPT: proportion of the total genetic variance among individuals within an accession, (P < 0.001). Table 5. The matrix of Nei genetic similarity (Gs) estimates using ISSR molecular markers among 17 Hypericum species.sp1= H. dogon- badanicum; sp2= H. androsaemum; sp3= H. tetrapterum; sp4= H. perforaturm; sp5= H. triquetrifolium; sp 6= H. lysimachioides; sp7= H. asperulum; sp8= H. scabrum; sp9= H. hirtellum; sp10: H. elongaturn ; sp11: H. davisii ; sp12= H. apricum; sp13= H. helianthemoides ; sp14= H. vermiculare; sp15= H. hirsutum; sp16= H. linarioides; sp17= H. armennm. sp1 sp2 sp3 sp4 sp5 sp6 sp7 sp8 sp9 sp10 sp11 sp12 sp13 sp14 sp15 sp16 sp17 sp1 1.000 sp2 0.742 1.000 sp3 0.786 0.833 1.000 sp4 0.767 0.836 0.842 1.000 sp5 0.823 0.823 0.786 0.911 1.000 sp6 0.781 0.766 0.767 0.757 0.793 1.000 sp7 0.749 0.683 0.823 0.759 0.836 0.862 1.000 sp8 0.681 0.776 0.727 0.728 0.834 0.750 0.799 1.000 sp9 0.817 0.610 0.746 0.796 0.768 0.675 0.727 0.728 1.000 sp10 0.715 0.884 0.800 0.709 0.720 0.681 0.746 0.796 0.680 1.000 sp11 0.645 0.754 0.785 0.676 0.829 0.733 0.800 0.709 0.820 0.721 1.000 sp12 0.745 0.757 0.741 0.758 0.816 0.740 0.785 0.676 0.725 0.635 0.839 1.000 sp13 0.666 0.737 0.890 0.722 0.719 0.853 0.741 0.758 0.834 0.750 0.799 0.642 1.000 sp14 0.649 0.807 0.799 0.755 0.812 0.774 0.990 0.722 0.768 0.675 0.727 0.728 0.684 1.000 sp15 0.617 0.782 0.744 0.636 0.834 0.750 0.799 0.755 0.720 0.681 0.746 0.796 0.676 0.722 1.000 sp16 0.778 0.702 0.757 0.703 0.778 0.691 0.744 0.636 0.829 0.733 0.800 0.709 0.770 0.754 0.770 1.000 sp17 0.641 0.814 0.800 0.681 0.710 0.688 0.757 0.703 0.816 0.740 0.785 0.676 0.699 0.756 0.735 0.778 1.000 104 Shuyan Ma, Majid Khayatnezhad, Amir Abbas Minaeifar (0.91) occurred between H. perforaturm and H. triquetri- folium. The lowest degree of genetic similarity occurred between H. androsaemum and H. hirtellum (0.61). The low Nm value (0.123) indicates limited gene f low or ancestrally shared alleles between the species studied and indicating high genetic differentiation among and within Hypericum species. DISCUSSION Genetic diversity is an important role in biology of long-term evolution of a taxon or a population. The basis of existence, growth, and evolution of taxon. Thus, the study of genetic diversity of taxon is fundamental to recognize the taxonomy, origin, and evolution of taxon. Moreover, such research will provide a theoretical basis for the germplasm resource conservation, development, utilization, and breeding (Lubbers et al., 1991). The present research, revealed interesting data about its genetic variability, genetic stratification and mor- phological divergence in north and west part of Iran. Degree of genetic variability within a species is highly correlated with its reproductive mode, the higher degree of open pollination/ cross breeding brings about higher level of genetic variability in the studied taxon (Meu- sel et al., 1965). PIC and MI characteristics of a primer help in determining its effectiveness in genetic diver- sity analysis. Sivaprakash et al. (2004) suggested that the ability of a marker technique to resolve genetic variabil- ity may be more directly related to the degree of poly- morphism. Generally, PIC value between zero to 0.25 suggest a very low genetic diversity among genotypes, between 0.25 to 0.50 shows a mid-level of genetic diver- sity and value ≥0.50 suggests a high level of genetic diver- sity (Tams et al., 2005). In this research, the ISSR prim- ers’ PIC values ranged from 0.23 to 0.44, with a mean value of 0.36, which indicated a mid-level ability of ISSR primers in determining genetic diversity among the spe- cies of Hypericum. All of 10 primer pairs showed a good polymorphism in taxon of Hypericum. A total 141 alleles were recognized for the studied species. Total number of bands per primers ranged from 7 to 18 polymorphic bands and the mean of the allele number in loci was 12.7. In most studies, population size is limited to sev- eral vegetative accession (Meusel et al., 1965; Uotila, 1996). This population could be showed genetic drift, whose effect are observed in the high level of FIS and low level of genetic diversity. The isolation of the popula- tion and absence the gene flow led to fragmentation of the Hypericum populations. Between genetic diversity parameters and population size were showing positive correlations that confirmed various studies (Leimu et al. 2006). There are two reasons for the positive correlation between genetic diversity and population size (Leimu et al., 2006). 1- A positive correlation could imply the pres- ence of an extinction vortex, where the drop-in popula- tion size lowers genetic diversity, which leads to inbreed- ing depression. The second reason is the fact that plant fitness differentiates populations based on variations in habitat quality (Vergeer et al., 2003). According to Booy et al. (2000) the low levels of genetic diversity could reduce plant fitness and restrict a population’s ability to respond to changing environmen- tal conditions through selection and adaptation. Genetic diversity (37%) was obtained within populations, where- as 63% of genetic variation obtained between the evalu- ated populations. One of the key factors determining the distribution of genetic variation is the breeding system in plant species (Duminil, 2007). Couvet (Booy et al., 2000) revealed that one migrant per generation cannot be existed to guarantee long-term survival of small pop- ulations and that the number of migrants is demonstrate through life history characters and population genetic (Vergeer et al., 2003). Genetic variances between the three groups were very similar, but statistically important. There are two hypotheses for the absence of differences between iso- lated populations. The first hypothesis explained that genetic diversity within and between populations demon- strate gene flow processes, which led to the fragmentation of larger populations (Dostálek et al., 2010). The second hypothesis presented that geographically proximate pop- ulations are more efficiently connected through gene flow than populations separated by greater distance. A high level of variation among H. perforatum populations was also reported by Percifield et al. (2007) which confirms results of the present study. Simi- lar results have been reported on this species using the RAPD markers by Hazler Pilepic et al. (2008). The high genetic diversity of H. perforatum populations is as a result of its mating systems. In fact, propagation method(s) of plant species is considered as one of the most important factors determining their levels of genet- ic diversity (Hamrick 1982; Hamrick and Godt 1989). Self-incompatibility is a wide spread phenomenon in the genus Hypericum (Robson 1981), resulting in the high levels of genetic variability (Borba et al. 2001). Further- more, this perennial plant produces a great number of seeds every year in favor of the high amounts of diver- sity in this species (Zhao et al. 2007). Since widespread species may possess the higher lev- els of genetic diversity than narrowly distributed plants (Hamrick and Godt 1996; Singh et al. 1998), the wide 105Genetic diversity and relationships among Hypericum L. species by ISSR Markers range of H. perforatum distribution is an important fac- tor in this respect. Considering the low level of gene flow rate among studied wild populations of H. perforatum, therefore, genetic drift might be inevitable. In H. perforatum, the low rate of gene flow may be due to factors such as prevailing apomixes and short dis- tance of seed dispersal as stated by Hazler Pilepic et al. (2008). Molecular markers have been used to investigate the genetic diversity, population structure, and repro- ductive biology of H. perforatum (Arnholdt-Schmitt, 2000; Haluŝková and Koŝuth, 2003; Barcaccia et al., 2006; Percifield et al., 2007). However, due to the lack of a specific marker system for these plants, most of the studies used marker systems such as RAPD and ISSR. In the present work, we took advantage of the ubiquity and abundance of ISSR method in plant genomes and their role in genomic diversification to develop and apply ret- rotransposon markers based on the ISSR method for the first time to Hypericum. High among-population variation was previously reported in Hypericum species by Percifield et al. (2007), Pilepić et al. (2008), and Farooq et al. (2014). High dif- ferentiation among populations is mostly coupled with limited gene flow among them. The low gene flow and the high differentiation among populations has been explained mainly by founder events such as time since colonization (Jacquemyn et al., 2004). In conclusion, the results of this study showed that to evaluate the genetic diversity of the Hypericum genus, the primers derived from ISSR were more effective than the other molecular markers. 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