Genetic diversity analysis in the Hypericum perforatum populations in the Kashmir valley by using inter-simple sequence repeats ( ISSR ) markers

1 Division of Floriculture Medicinal and Aromatic Plants, Sher-eKashmir University of Agricultural Sciences and Technology, Shalimar, Srinagar, J.K, India. 2 Cancer Genetics Lab, Department of Biochemistry, Maulana Azad Medical College and Associated Hospitals, New Delhi, India. 3 Division of P.B.G, SKAUST, Shalimar, Srinagar J.K, AMU, UP, India. 4 Department of Botany, AMU, UP, India. 5 Dean, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India.


INTRODUCTION
Hypericum perforatum L. (St.John's wort) is one of the most important medicinal plants, which has been used as a medical herb for over 2000 years (Patooka, 2003).H. perforatum (St.John's wort) is a widespread Eurasian perennial plant species with remarkable variation in its morphology, ploidy and breeding system, which ranges from sex to apomixes (Koch et al., 2013).The genus Hypericum L. (St.John's wort, Hypericaceae) includes more than 450 species that occur in temperature or tropical mountain regions of the world.Monographic work on the genus has resulted in the recognition and description of 36 taxonomic sections, delineated by specific combinations of morphological characteristics and biogeographic distribution (Nürk et al., 2011).One of the top-selling medicinal products worldwide is H. perforatum (St.John's Wort).Despite its cosmopolitan distribution and utilization, little is known regarding the relationship of the bioactive compounds in H. perforatum to the plants from which they are purportedly derived and it produces pharmaceutically important metabolites with antidepressive, anticancer and antiviral activities (Zanoli, 2004;Kubin et al., 2005;Percifield et al., 2007).A number of Hypericum species are well known for their therapeutic efficacy and use in traditional medicine.
H. perforatum L. has been used in herbal medicine for the treatment of burns, skin injuries, neuralgia, fibrositis, sciatica and depression (Ghasemi et al., 2013;Barnes et al., 2001).The existence of distinct multilocus genotypes in apomictic H. perforatum populations were reported by Ellstrand and Roose (1987).Also, Noyes and Soltis (1996), reported the existence of different clones in apomictic plant populations.Hısil and Sahin (2005) performed various experimental studies and reported the existence of diverse clones in H. perforatum populations but the source of this genetic diversity remains largely unexplained.Meirmans and Van Tienderen (2004) documented that the clonal diversity within a population reflects the sexual genetic pool from which the clones originated, the frequency of clonal origin, and the somatic mutations that subsequently accumulate in established clones.The diversity arising from the clonal origin is directly dependent on reproductive mode, and hence varying degrees of apomixis between H. perforatum landraces may have a significant impact on the diversity of local populations.The mode of reproduction was also investigated using the FCSS analysis to reconstruct the patterns of seed formation and to infer the sources of genetic variation at the population level (Percifield et al., 2007;Arnholdt-Schmitt, 2000).Earlier studies demonstrated that randomly amplified polymorphic DNA (RAPD) analysis would enable the elucidation of genetic diversity in wild populations and cultivars of H. perforatum.Reproduction modes of H. perforatum, which is facultative apomict with apospory and pseudogamy have been one of the most examined subjects by different molecular markers (Skyba et al., 2010;Arnholdt-Schmitt, 2002) such as RAPD restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) (Mayo and Langridge, 2003) and inter-simple sequence repeats (ISSR).Recently, RAPD and simple sequence repeat (SSR) were performed to estimate correlations between secondary metabolite contents and genetic profile of H. perforatum and other Hypericum species, which demonstrated variable rates of correlation (Smelcerovic et al., 2006;Verma et al., 2008, Joshi et al., 2004).
The H. perforatum clones examined in this study were improved by Ceylan et al. (2005) with clonal selection method from the seeds of H. perforatum accessions collected from eight provinces and 35 locations in the flora of Aegean Region of Turkey.Genetic distance analysis of the AFLP data (Ryan et al., 2007) revealed that the cultivated populations studied share higher genetic identity with the Western and Central European populations (0.925) than with populations from East Europe and Asia (0.828).This could be attributed to the fact that the cultivated varieties used in this study were developed in Germany and Denmark.Crockett et al. (2004) study provides a genetic method for authentication of commercial H. perforatum preparations and allows a preliminary assessment of phylogenetic relationships within the genus, revealing three strongly supported monophyletic clades, plus several secondary monophyletic groupings.Using internal transcribed spacer (ITS) gene sequences, they were able to distinguish H. perforatum from all other species of Hypericum included in this study.Our study demonstrates that there is a great deal of genetic diversity among Hypericum species as well as within H. perforatum, and that this diversity is structured phylogenetically and geographically.These data provide the foundation for future work characterizing the genetic relationships and recent domestication of St. John's wort and its closely related species.The use of ISSR markers gave hints for the occurrence of sexual recombination in H. perforatum plants.In comparison to other molecular *Corresponding author.E-mail: drrashidmamc@gmail.com.Tel: +91-9968584601.

MATERIALS AND METHODS
The study was conducted in the Division of Floriculture, Medicinal and Aromatic Plants (FMAP), Sher-e-Kashmir University of Agricultural Sciences and Technology Kashmir, Srinagar, J and K (India) in collaboration with Medical Phytochemistry Lab, Department of Biochemistry, Maulana Azad Medical College, New Delhi.The plant material from different populations was authenticated in the division of FMAP.

Plant materials
The H. perforatum clones used in the study of genetic diversity and population structure was obtained wild from 20 populations (20 sites), covering 08 geographical locations (Forest Divisions) with altitude ranging 5000-13000 feets from the from the Kashmir valley, India as depicted in Table 1.Each population of H. perforatum consist of about two to three plants with different age groups (1st, 2nd, 3rd and 4th year).Whereas, for analysis of genetic diversity, representative samples of three plants (3 replicates for each population) were used and designated with accession code with respect to the site of collections.The interval between samples was 2-5 m, the pair wise distance between populations was 0.5 -20 Km, whereas, the pair wise distance between forest divisions was 10-200 Km.Morphological feature of each plant sample as well as the environmental factors for each sampling site were also documented.About 5 g of young leaves from each representative plant samples were obtained and placed in a ziplock plastic bag containing silica gel which sped up the drying.The samples were stored at -80°C until use.

DNA extraction from H. perforatum without using liquid nitrogen
There were minor modifications in the protocol already used by Sharma et al. (2003).Approximately, 0.4 g of leaf tissue was collected from healthy plants (H.perforatum) and stored at -80°C.DNA was extracted without using liquid Nitrogen stored in TE buffer at -20°C until use.The quality was checked on 0.8% agarose gel after staining with ethidium bromide (Et-Br, 5 mg/ml) as shown in Figure 1.The DNA was quantified by taking the optical density (OD) at λ 260 with a Thermo Scientific NanoDrop™ 1000 Spectrophotometer.The ratio of absorbance at 260 and 280 nm was used to assess the purity of DNA.A ratio of ~1.8 is generally accepted as "pure" for DNA.If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.The ISSR polymerase chain reaction (PCR) was performed only in those DNA samples in which absorbance were between 1.75 to 1.85.An optical density (or "OD") of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA.

Evaluation of primers
A total of 20 ISSR primers were screened with 10 plant samples.A gradient PCR was performed to figure out what annealing temperatures work best.The primer sequences, GC content and melting temperatures of ISSR primers is depicted in Table 2 and were commercially synthesized by Sigma.Similar ISSR primers were used by Barcaccia et al. (2006).

PCR amplification of ISSR primers in H. perforatum
The PCR cocktail was prepared by using HotStarTaq Master Mix Kit (Qiagen), ready-to-use solution.Twenty five (25 ul) reaction volume was used containing 12 ul of master mix, 50 pmole of 1 ul of primer, 10 ul of molecular grade water and finally added 2 ul of DNA.The thermocycling conditions were initial denaturation for 5 min at 94°C followed by 45 cycles of 1 min at 94°C, 55 s at specific annealing temperature, 45 min at 72°C and a 10 min final extension step at 72°C.The annealing temperature for each primer was determined.
Amplification products were electrophoresed on 2.0% agarose gels run at constant voltage and 1X TBE for approximately 2 h, visualized by staining with ethidium bromide and photographed under ultraviolet light (using Gel Doc, Biorad).Molecular weights were estimated using DNA markers (100 bp) purchased from Gene Aid.Gel-Pro analyzer version 3-1 software was used to score ISSR profile.Each genomic DNA sample was scored for the presence or absence of specific PCR bands generated by a primer as depicted in Figure 2. The presence (1) or absence (0) of the amplified bands was scored in all genotypes for each primer.Faintly stained bands were not considered.

Marker properties
The ability of the most informative primers to differentiate between the clones was assessed by calculating their resolving power (Rp) according to Prevost and Wilkinson (1999).Polymorphic information content (PIC) or average heterozygosity was calculated using the formula of Roldan-Ruiz et al. (2000).Marker index (MI) was calculated by multiplying the average heterozygosity (Hav) with effective multiplex ratio (EMR) (Powell et al., 1996).EMR and MI were calculated for ISSR analysis to measure the usefulness of the marker system as depicted in Table 3.

Primer resolving power
Primer resolving power (Rp) was calculated according to Prevost and Wilkinson formula: Where, Ib i = 1-(2 × | 0.5 -p i |).'p i ' is the proportion of accessions containing the i th band and Ib i is the informativeness of the i th  band.
According to Prevost and Wilkinson (1999), the resolving power (Rp) of a primer is: Rp = IB where IB (band informativeness) takes the value of: 1-[2x (0.5 -P)] P being the proportion of the n number of genotypes containing the band.

Polymorphism information content (PIC) or diversity index
Diversity index/genetic diversity (DI) is the expected heterozygosity and was calculated according to Weir as: Where, PICi is the polymorphic information content of a marker i; Pij is the frequency of the jth pattern for marker i and the summation extends over n patterns.Where, pi is the frequency of ith allele at the l locus and L is the number of loci.This formula is equivalent to an average of PIC.The PIC obtained for ISSR markers are as depicted in Table 3.

Effective multiplex ratio (EMR)
It is the product of the fraction of polymorphic loci and the number of polymorphic loci for an individual assay.The EMR obtained for ISSR markers are as depicted in Table 3.

Marker index (MI)
MI of each primer was determined according to Powell et al. (1996).Marker index is the product of two functions: Diversity index (DI) and EMR.

DI = 1-Σpi 2
Where, pi is the frequency of ith band; EMR, the product of the fraction of polymorphic loci and the number of polymorphic loci for an individual assay.The MI obtained for ISSR markers are as depicted in Table 3.

HPLC analysis for hypericin content
The air-dried H. perforatum leaves and roots (50 g) were broken into small pieces (2-6 mm) by using a cylindrical crusher, and extracted with ethanol: water solution (80:20) (180 ml) using a Soxhlet apparatus.The mixture was filtered through a paper filter (Whatman, No.1) and evaporated.Crude extract (20 μl) was used for injection into the high performance liquid chromatography (HPLC) system.The residue (5.8 g) was stored in a dark glass bottle for further processing.The resultant residue was dissolved in 10 ml acetonitrile (HPLC grade) and filtered with 0.22 μm durapore membrane filter (Millipore).HPLC analysis was carried out using Nova Pack C18 cartridge column (250 x 4.6 mm) in HPLC system (Water).Acetonitrile: water: methanol (37:58:5) was used as a mobile phase with a flow rate of 1.5 mlmin-1.Hypercin (0.1mg/L Sigma, P-4405) was used as a standard for calculating Hypericin content in the samples on the basis of peak heights.Phytoextracts were prepared by removing solvents under vacuum.

Hypericin content
Among the different locations of Baramulla, the highest hypericin contents were obtained from clones of H. perforatum (leaves) collected from Tangmarg (1.37% on dry weight basis).Also, the highest hypericin contents were obtained from clones of H. perforatum collected from Kupwara division (1.22% on dry weight basis).In the different locations of Ganderbal division, the highest hypericin contents were obtained from clones of H. perforatum collected from Sonmarg (0.96% on dry weight basis).Among the different locations of Srinagar, the highest hypericin contents were obtained from clones of H. perforatum collected from Hazaratbal (0.87% on dry weight basis).
The hypericin contents obtained among the different locations of Anathnag was comparatively more (0.89 to 0.97% on dry weight basis) than that of the samples collected from other forest divisions of Badgam, Shopian and Bandipore.The hypericin content was correlated with altitude and genetic diversity among the H. perforatum populations.Similar results were reported by Aziz et al. (2002).The Hypericin contents obtained among the H. perforatum populations with respect to altitude by HPLC are as depicted in Table 4.

ISSR polymorphism
The number of scored bands, number of polymorphic bands, percentage of polymorphism and total fragments amplified were shown in Table 5.A total of 20 ISSR primers were screened with 10 plant samples.Ten pairs of ISSR primers were used to screen 60 randomly selected plant samples from 20 sites (populations) covering eight geographical locations (regions).A total of 68 ISSR loci were detected.Out of the 98 loci surveyed, 71 were polymorphic (74%).The amplified PCR fragment size ranged from 150 to 1650 bp with an average of 9.8 bands per primer.Out of these 10 primers, ISSR1 to ISSR 07 revealed 24 monomorphic loci existed in all of the 20 populations.Among the divisions by ISSR markers, the numbers of polymorphic bands varied in between 23 (sumbalhill) and a maximum of 74 (Shalimar).Also polymorphism differed substantially and was found to be between minimum 46% and a maximum of 69.66%.Out of the 98 loci surveyed, 71 were polymorphic (74%).The total number of polymorphic alleles were 71, thereby giving an estimate of profound (>74%) polymorphism.The high reproducibility of ISSR markers may be due to the use of longer primers and higher annealing temperature.
The ISSR primers possess the highest Rp values (>10) ISSR1 to ISSR 10 except ISSR7 and ISSR8.These primers are able to distinguish between all the 20 populations of P. hexandrum collected from the different locations.A particular band would have optimal discriminating power when it is scored in the 50% of the genotypes.The band discriminating powers and a primer's discriminating power increases with the increasing number of bands of that primer.The high reproducibility of ISSR markers may be due to the use of longer primers and higher annealing temperature than those used for RAPD.The highest DI or PIC (0.51) was found in ISSR6 primer and lowest DI (0.23) was found in ISSR5.The ability of different assay to infer genetic relationship was also observed.The PIC value of ISSR primers varied from 0.23 to 0.51 with an average of 0.38.
The primer ISSR-6 showed the highest value of average Rp and PIC.The suitability of each ISSR system was examined in terms of number of loci revealed EMR and the amount of polymorphism detected (DI).For the ISSR markers used in this study, the EMR ranged from 2.00 to 11.Based on EMR, ISSR markers used were efficient tools for H. perforatum accessions.vThe MI was used to evaluate the overall utility of each marker system.The high MI is the reflection of efficiency of ISSR markers to simultaneously analyze a large number of bands, rather than level of polymorphism detected.The ISSR resulted in the highest marker indices ranged from 0.60 to 4.40 (average 2.31) as depicted in Table 6.

Phylogenetic analysis
Genetic distance based on the Jaccard's coefficient was calculated by making a pairwise comparison between 20 H. perforatum clones by ISSR marker using the NTSYSpc software version 2.01e.Based on the Jaccard similarity coefficient between individuals, a cluster analysis was carried out and a dendrogram was generated that represented the genetic relationship among 20 populations or 60 individuals as depicted in the Figure 3.In the dendrogram, all the individuals in each population clustered together.Based on ISSR markers, the similarity index values ranged from 0.54 to 0.84 as depicted in Table 7.These values were used to construct a dendrogram using UPGMA.Based on the presence or absence of the amplification of alleles, the pair-wise genetic similarity according to Jaccard's coefficient was analyzed ranging from 0.54 to 0.84.
The cluster analysis of ISSR data based on similarity matrix among the populations with respect to their geographical location generated a dendrogram with 5 clusters.Cluster I represents Ganderbalhill, Arhomahill, Khilanmarg, Pombai,Yusmarg, Tangmarg, Darogabagh

Genetic diversity and hypercin content
There was a significant correlation between hypericin contents and ISSR data of the clones.In the dendrogram (Figure 3), clones 1, 3, 11, 15, 12, 16, 17 and 18 were grouped in same subcluster.A close relationship among these clones could be seen in both genetic and hypericin data.
Furthermore, close relationship among clones 4, 5, 8 and 7 could be seen in both dendrograms constructed from genetic and hypericin data.
Clone-12 and clone-14 had the highest hypericin content (0.137 and 0.98%, respectively) with respect to the mean hypericin content as shown in Table 8.

Relationships between populations
To assess the overall distribution of diversity within and among these populations, analysis of molecular variance (AMOVA) was completed.Highly significant (P < 0.000 2) genetic differentiation was observed among the 20 populations.The summary of nested analysis of molecular variance (AMOVA) based on ISSR genotypes obtained in H. perforatum is depicted in Table 9.The levels of significance were based on 1000 interation steps.
Analysis of molecular variance indicated that over half of the total variation in the studied populations (58%) could be accounted for by differences among the 8 divisions, with a further 42% being accounted for by the variation among populations within a division (Table 9).All components of molecular variance were significant (P < 0.001).This is helpful in making strategy for germplasm collection and evaluation.

Population genetic structure
However, with the hypervariable nature of ISSR, asymmetrical patterns of gene flow are easily detected.Low frequency markers are sometimes more useful in detecting these patterns than species-typical markers.
After the identification of particular marker alleles, the frequency of occurrence of the markers in the popula- lation of interest is measured, and the hypothesis of gene flow is evaluated based on the patterns detected.The data for observed number of alleles, effective number of alleles, Nei's genetic diversity, Shannon's information index, for all the twenty populations were analysed using ten ISSR markers and their respective values were found as 1.775, 1.421, 0.265 and 0.399 as depicted in Table 10.The value for total genotype diversity among population (Ht) was 0.253 while within population diversity (Hs) was found to be 0.157.Mean coefficient of gene differentiation (Gst) value 0.451.Estimate of gene flow in the population was found as 0.30432.A close relationship among the clones in dendrogram generated by ISSR markers, that there is a stronger correlation between hypericin contents and genetic structure in H. perforatum.The existing variation in hypericin content among the H. perforatum populations was proved to be coupled with geographical altitude but rarely with genetic variations by ISSR as shown in Figure 4.The genetic relationship of different populations in H. perforatum studied by ISSR markers is equally important.Based on this, we suggest that in situ conservation be an important and practical measure for maintaining the genetic diversity of this species.

DISCUSSION
Research reports on medicinal and aromatic plants are increasing day by day because of the search for new active molecules, for improvement of the plant production systems and new species or molecules for the herbal pharmaceutical industries (phytotherapy or allopathy).However, one of the most important problems related to medicinal plants is that there is an extreme variability in the phytochemical content, mostly resulting from environmental factors and other contaminations during cropping.It has been reported that one obstacle facing breeders, horticulturists, researchers, and oversight agencies working with medicinals is the inability to genetically determine the source of plant material.Markers generated in this study may aid in overcoming this obstacle.Collectively, the 10 unique H. perforatum markers may aid breeders in determining genetic identity and source, can be employed as a tool by producers to accurately diagnose the identity of individual plant lots, and could be useful to agencies or consumer groups as a means to evaluate end-user H. perforatum "St.John's wort" preparations.Additionally, this molecular marker study provides the foundation for future work focused on developing speciesspecific primers that could be used to identify material purported to be H. perforatum with a single PCR reaction.
The present study utilized ISSR markers for analysis that was successful in detecting genetic diversity and relationships among the H. perforatum clones.A moderate level to high level genetic diversity was found in the clones as was expected since the H. perforatum clones subjected to this study originated from only eight provinces of Kashmir valley of India.As a result of genetic variation studied in this by ISSR markers, 98 loci were reported, 71 were polymorphic (74%) and 29 were monomorphic.The numbers of polymorphic bands varied in between 23 (Sumbalhill) and a maximum of 74 (Shalimar).Also, polymorphism differed substantially and was found to be in between minimum of 46% and a maximum of 69.66%.The total number of polymorphic alleles were 71, thereby giving an estimate of profound (>74%) polymorphism.Ellstrand and Roose (1987), reported the existence of distinct multilocus genotypes in apomictic H. perforatum populations.Also, Noyes and Soltis (1996), reported the existence of different clones in apomictic plant populations.Similarly, Chapman et al. (2000) documented the genetic diversity in H. perforatum.Similar results were reported by Hısil et al. (2005) who performed various experimental studies and reported the existence of diverse clones in H. perforatum populations but the source of this genetic diversity remains largely unexplained.Meirmans and Van Tienderen (2004) documented that the clonal diversity within a population reflects the sexual genetic pool from which the clones originated, the frequency of clonal origin, and the somatic mutations that subsequently accumulate in established clones.The diversity arising from the clonal origin is directly dependent on reproductive mode, and hence varying degrees of apomixis between H. perforatum landraces may have a significant impact on the diversity of local populations.The mode of reproduction was also investigated using the FCSS analysis to reconstruct the patterns of seed formation and to infer the sources of genetic variation at the population level.The EMR and MI were calculated for RAPD analysis to measure the usefulness of the marker system according to Powell et al. (1996).
The PIC or average heterozygosity (Hav) is estimated by taking the average of PIC values obtained for all the markers.MI was calculated by multiplying the average heterozygosity with EMR (Powell et al., 1996).MI and Rp were two different methods used to measure the ability of primers or techniques to distinguish between genotypes (Prevost and Wilkinson, 1999;Fatma et al., 2011).In comparison to the work of Sarwat et al. (2008), the marker index in the present study was higher (4.4) and reported a marker index for molecular markers of 3.3 in Tribulus terrestris.This difference may be explained by higher average heterozygosity and EMR values, which were measured as 0.34 and 12 in the study, respectively.The resolving power values of ISSR primers in the present study (9.2 to 19) were found to be higher than that of Fernandez et al. (2002) (1.38-8.88)but lower than that of Prakash and Van Staden (2007) (9.4-26.8).
ISSR analysis was successful in detecting genetic diversity and relationships among the H. perforatum clones.A low level of genetic diversity was found in the clones of Ganderbal division as was expected since the H. perforatum clones used originated from six sites of only one district of Kashmir Valley (59.47%).High level of genetic similarity in H. perforatum accessions collected from different locations in India was previously reported by Verma et al. (2008).According to similarity coefficients of cluster analysis of the H. perforatum clones and variety Topaz, expected results were found.Clustering or similarity coefficient showed that there was a partial relation-ship on a regional basis of the clones.Hence from our observations, the clones of H. perforatum collected from Ganderbal, Arhomahill, Khilanmarg, Pombai were more phylogenetically related with that of clones obtained from Yusmarg, Tangmarg, Darogabagh and Aharbal and were clustered together in Cluster V. Similarly, plants collected from Tangmarg, Yusmarg were similar in genetic makeup and were clustered together.The genotypes of H. perforatum collected from Pranghill, Mansbal, Kokarnag and Gulmarg were similar in genetic makeup and were clustered together in Cluster V.The clones of H. perforatum collected from Yousmarg were more phylogenetically related with that of clones obtained from and Tangmarg.Clusters obtained from ISSR marker were less overlapping indicating comparatively good extent of genetic diversity assessment by ISSR marker.The distribution pattern of clones of diverse origin in cluster I indicated that genetic diversity observed within H. perforatum clones was not related to geographical origin.In general, the results of ISSR analysis confirmed the efficacy of ISSR markers for detecting DNA polymorphism among H. perforatum clones.
Similarly, several studies have exploited RAPD and ISSR markers to characterize H. perforatum accessions and populations (Arnholdt-Schmitt, 2000;Arnholdt-Schmitt, 2002;Halušková and Košuth, 2003;Barcaccia et al., 2006;Verma et al., 2008) and confirmed effectiveness of molecular markers.Significant similarities were observed between the dendrograms constructed from hypericin and ISSR data.A close relationship among the clones in both dendrograms demonstrated that there is a stronger correlation between hypericin contents and genetic structure in H. perforatum.Contrary to our results, Verma et al. (2008) found a partial correlation between secondary metabolite contents and molecular data in H. perforatum accessions.This result could be explained by differences in chemical composition and genetic profile among the accessions of one species, which show less significance compared to different species.
AMOVA revealed that there was significant variation arising from habitat-correlated genetic difference (58%) suggesting that, besides the effects of gene flow and genetic drifts, local ecological conditions (altitude, distance, temperature, rainfall, humidity, soil, pH among others) also played an important role in the variation of the genetic structure in the study populations of H. perforatum.Considering the high genetic differentiation among the wild populations of H. perforatum, presservation of only a few populations may not adequately protect the genetic variation within the species in Himalayan region.AMOVA indicated that over half of the total variation in the studied populations (58%) could be accounted for by differences among the eight divisions, with a further 42% being accounted for by the variation among populations within a division.All components of molecular variance were significant (P < 0.001).This is helpful in making strategy for germplasm collection and evaluation.
The hypericin contents and genetic structures of superior H. perforatum clones were identified by HPLC or spectrophotometric analysis and ISSR markers.Cluster analyses of hypericin contents and ISSR markers grouped the clones in five major clusters and significant correlations were observed between them.The combined analysis of ISSR markers and hypericin content is an optimal approach for further progress and breeding programs.There was a significant correlation between hypericin contents and ISSR data of the clones.Clone-12 and clone-14 had the highest hypericin content (0.137% and 0.98%, respectively) with respect to the mean hypericin content.The observed increase in genetic diversity as well as hypericin contents with increase in altitude is an interesting phenomenon that requires further research.

Conclusion
This is the first report on the genetic profile of H. perforatum originating from Kashmir valley of India.There is a stronger correlation between hypericin contents and genetic structure in H. perforatum.The existing variation in hypericin content among the H. perforatum populations was proved to be coupled with geographical altitude but rarely with genetic variations by ISSR.Based on this, we suggest that in situ conservation be an important and practical measure for maintaining the genetic diversity of this species.Especially, this knowledge should be considered in the breeding of superior hybrid genotypes for adaptation to different environments and utilization systems.

Figure 3 .
Figure 3. Dendrogram illustrating genetic relationships among 20 H.perforatum populations in population diversity study generated by UPGMA cluster analysis calculated from 1090 ISSR bands produced by 10 primers.

Figure 4 .
Figure 4. Diagrammatical representation of genetic variation and polymorphic features estimated using ISSR markers among the H. perforatum populations with respect to their divisions.

Table 1 .
Twenty populations of H. perforatum collected from different sites at different altitudes covering eight divisions in Kashmir valley.

Table 3 .
Total number (n) and number of polymorphic bands (np), percentage of polymorphic bands (%P), main values of proportion of clones containing band (mp), main values of band informativeness (mIb), resolving power (Rp) and polymorphic information content (PIC) of ISSR primers among the clones of H. perforatum.Marker Resolving power Effective multiplex ratio (EMR) Marker Index PIC or diversity indexPIC, Polymorphism information content.

Table 4 .
Hypericin contents among the H. perforatum populations with respect to altitude by HPLC.

Table 5 .
Total number of scored bands, number of polymorphic bands, percentage of polymorphism, and total fragments amplified by using ISSR primers among the clones of H. perforatum.

Table 6 .
Average value of total number of scored bands, number of polymorphic bands, percentage of polymorphism, and total fragments amplified by using ISSR primers among the clones of H. perforatum.

Table 7 .
Distance matrix for H. perforatum clones by Jaccard's coefficient based on ISSR bands.

Table 8 .
Genetic variation and polymorphic features estimated using ISSR markers among the H perforatum populations.

Table 9 .
Summary of nested analysis of molecular variance (AMOVA) based on ISSR genotypes of Hypericum perforatum.
d.f, Degree of freedom; SSD, sum of square deviation; P-value, probability of null distribution.

Table 10 .
Genetic variation and polymorphic features estimated using ISSR markers among the H. perforatum populations.Observed number of alleles; Ne, effective number of alleles; H, Nei's genetic diversity; I, Shannon's information index; Ht, total gene diversity.