Genetic variation and cultivar identification in Cymbidium ensifolium

As a popular flowering species with many cultivars, Cymbidium ensifolium (L.) is commercially important in horticulture. However, so far little has been known about genetic diversity and conservation genetics of this species. Understanding of the genetic variation and relationships in cultivars of C.?ensifolium is a prerequisite for development of future germplasm conservation and cultivar improvement. Here we report assessment of genetic variations in C.?ensifolium cultivars using the DNA fingerprinting technique of inter-simple sequence repeats (ISSR). A total of 239 ISSR loci were identified and used for evaluation of genetic variation with a selection of 19 ISSR primers. Among these ISSR loci, 99.16% were polymorphic with wide genetic variation as shown by Nei??s gene diversity (H?=?0.2431) among 85 tested cultivars. ISSR fingerprinting profiles showed that each cultivar had its characteristic DNA pattern, indicating unequivocal cultivar identification at molecular level. Eighteen cultivar-specific ISSR markers were identified in seven cultivars. The cultivar Sijiwenhan was confirmed as hybrid by four ISSR primers. Several cultivars with same name but different geographical origins were distinguished based on their ISSR profiles. A dendrogram generated with ISSR markers could group 73 of 85 cultivars into four major clusters. Further analysis of ISSR variation revealed that about 69% of total genetic variation in this species is due to genetic divergence inside geographical groups. Our results suggest that both germplasm collection and in?situ conservation are important for future planning of C.?ensifolium species conservation.     

Polyploidization as a retraction force in plant genome evolution: sequence rearrangements in triticale

Background

Polyploidization is a major evolutionary process in plants where hybridization and chromosome doubling induce enormous genomic stress and can generate genetic and epigenetic modifications. However, proper evaluation of DNA sequence restructuring events and the precise characterization of sequences involved are still sparse.

Methodology/Principal Findings

Inter Retrotransposons Amplified Polymorphism (IRAP), Retrotransposons Microsatellite Amplified Polymorphism (REMAP) and Inter Simple Sequence Repeat (ISSR) largely confirmed the absence of any intraspecific variation in wheat, rye and triticale. The comparative analysis of banding profiles between wheat and rye inbred lines revealed 34% of monomorphic (common to both parental species) bands for the ten different primer combinations used. The analysis of triticale plants uncovered nearly 51% of rearranged bands in the polyploid, being the majority of these modifications, due to the loss of rye bands (83%). Sequence analysis of rye fragments absent in triticale revealed for instance homology with hydroxyproline-rich glycoproteins (HRGP), a protein that belongs to a major family of inducible defence response proteins. Conversely, a wheat-specific band absent in triticale comprises a nested structure of copia-like retrotransposons elements, namely Claudia and Barbara. Sequencing of a polyploid-specific band (absent in both parents) revealed a microsatellite related sequence. Cytological studies using Fluorescent In Situ Hybridization (FISH) with REMAP products revealed a widespread distribution of retrotransposon and/or microsatellite flanking sequences on rye chromosomes, with a preferential accumulation in heterochromatic sub-telomeric domains.

Conclusions/Significance

Here, we used PCR-based molecular marker techniques involving retrotransposons and microsatellites to uncover polyploidization induced genetic restructuring in triticale. Sequence analysis of rearranged genomic fragments either from rye or wheat origin showed these to be retrotransposon-related as well as coding sequences. Further FISH analysis revealed possible chromosome hotspots for sequence rearrangements. The role of chromatin condensation on the origin of genomic rearrangements mediated by polyploidization in triticale is also discussed.     

Genetic diversity of Indian jujube cultivars using SCoT,ISSR, and rDNA markers

Genetic variation and relationships among 37 cultivars of Ziziphus mauritiana (Lamk.) native of India were analyzed using start codon targeted (SCoT), inter-simple sequence repeats (ISSR), and ribosomal DNA (rDNA) markers. High level of polymorphism among SCoT (61.6%) and ISSR (61%) primers with higher PIC values ranging from 63.1 to 90.4% of SCoT and 47.3 to 88.8% of ISSR primers was recorded. SCoT and ISSR dendrograms revealed similarity coefficients ranging from 0.80 to 0.92 and 0.79 to 0.96, respectively, and clearly delineated all the cultivars of Z. mauritiana into well-supported distinct clusters. Greater Gst signifies higher amount of differentiation observed over multiple loci among seven Z. mauritiana populations. On the other hand, higher gene flow demonstrating a very high migration rate between Z. mauritiana populations indicated higher rates of transfer of alleles or genes from one population to another. The genetic diversity of population 1 (Rajasthan) was the richest among all the seven populations. The largest genetic distance was measured between Maharashtra and West Bengal and the least between Rajasthan and Punjab cultivars. Most of the genetic diversity exists within population rather than among populations. Substantial variation in the ITS-1 region signifies its phylogenetic utility specifically in assessing genetic diversity in Z. mauritiana. The clustering patterns using three molecular marker systems vis-à-vis place of origin exhibited no consistency in grouping of Z. mauritiana cultivars as cultivars from the same place of origin were genetically cataloged into different SCoT, ISSR, and ITS phylogram clusters indicating wide genetic diversity and distribution across agro-climatic zones validating the robustness of marker systems tested.     

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.     

Genetic,Epigenetic, and HPLC Fingerprint Differentiation between Natural and Ex Situ Populations of Rhodiola sachalinensis from Changbai Mountain,China

Rhodiola sachalinensis is an endangered species with important medicinal value. We used inter-simple sequence repeat (ISSR) and methylation-sensitive amplified polymorphism (MSAP) markers to analyze genetic and epigenetic differentiation in different populations of R. sachalinensis, including three natural populations and an ex situ population. Chromatographic fingerprint was used to reveal HPLC fingerprint differentiation. According to our results, the ex situ population of R. sachalinensis has higher level genetic diversity and greater HPLC fingerprint variation than natural populations, but shows lower epigenetic diversity. Most genetic variation (54.88%) was found to be distributed within populations, and epigenetic variation was primarily distributed among populations (63.87%). UPGMA cluster analysis of ISSR and MSAP data showed identical results, with individuals from each given population grouping together. The results of UPGMA cluster analysis of HPLC fingerprint patterns was significantly different from results obtained from ISSR and MSAP data. Correlation analysis revealed close relationships among altitude, genetic structure, epigenetic structure, and HPLC fingerprint patterns (R² = 0.98 for genetic and epigenetic distance; R² = 0.90 for DNA methylation level and altitude; R² = –0.95 for HPLC fingerprint and altitude). Taken together, our results indicate that ex situ population of R. sachalinensis show significantly different genetic and epigenetic population structures and HPLC fingerprint patterns. Along with other potential explanations, these findings suggest that the ex situ environmental factors caused by different altitude play an important role in keeping hereditary characteristic of R. sachalinensis.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

Assessment of somaclonal variation during sugarcane micropropagation in temporary immersion bioreactors by intersimple sequence repeat (ISSR) markers

Somaclonal variation refers to the genetic and epigenetic changes in plants regenerated from plant tissue culture. In this study, using intersimple sequence repeat (ISSR) molecular markers, the somaclonal variation during micropropagation of sugarcane using temporary immersion bioreactors (TIBs) was evaluated. Apices of the cultivar Mex 69-290 were established and multiplied by ten subcultures in TIBs. After 30 d in each subculture, the number and length of shoots per explant were recorded. For the molecular analysis, ten plants were taken per subculture, and a total of 109 bands from ten ISSR primers were obtained. For each subculture, the polymorphism (%) was calculated. A dendrogram of genetic distances between subcultures and the donor plant was obtained using a matrix of Nei’s genetic distances and the unweighted pair group method with arithmetic mean (UPGMA). The results showed that the production of sugarcane shoots tends to increase until subculture 8, while shoot length decreases. ISSR markers showed the existence of somaclonal variation during micropropagation of sugarcane. The subcultures with the highest percentage of polymorphism (%) and genetic distances (GD) were the 1°, 9°, and 10° (with 10.1, 15.6, and 10.1% and 0.0222, 0.0181, and 0.0181 GD, respectively). The molecular and statistical analysis showed that in vitro establishment and the number of subcultures are both factors that affected the frequency of somaclonal variation during the micropropagation of sugarcane using TIBs. Thus, it is important to determine the optimal number of subcultures that can be made from an explant for each species to be micropropagated.     

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.)

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.     

Hérédité et épigénétique: un rôle inattendu pour l’ARN

Although Mendel’s first laws explain the transmission of most characteristics, there has recently been a renewed interest in the notion that DNA is not the sole determinant of our inherited phenotype. Human epidemiology studies and animal and plant genetic studies have provided evidence that epigenetic information (“epigenetic” describes an inherited effect on chromosome or gene function that is not accompanied by any alteration of the nucleotide sequence) can be inherited from parents to offspring. Most of the mechanisms involved in epigenetic “memory” are paramutation events, which are heritable epigenetic changes in the phenotype of a “paramutable” allele. Initially demonstrated in plants, paramutation is defined as an interaction between two alleles of a single locus that results in heritable changes of one allele that is induced by the other. The authors describe an unexpected example of paramutation in the mouse revealed by a recent analysis of an epigenetic variation modulating expression of theKit locus. The progeny of hétérozygote intercrosses (carrying one mutant and one wild-type allele) showed persistence of the white patches (characteristic of hétérozygotes) in the homozygous Kit^+/+ progeny. The DNA sequences of the two wild-type alleles were structurally normal, revealing an epigenetic modification. Further investigations showed that RNA and microRNA, released by sperm, mediate this epigenetic inheritance. The molecular mechanisms involved in this unexpected mode of inheritance and the role of RNA molecules in the spermatozoon head as possible vectors for the hereditary transfer of such modifications — implying that paternal inheritance is not limited to just one haploid copy of the genome — are still a matter of debate. Paramutations may be considered to be one possibility of epigenetic modification in the case of familial disease predispositions not fully explained by Mendelian analysis.     

Epigenetic Variation in Mangrove Plants Occurring in Contrasting Natural Environment

Background

Epigenetic modifications, such as cytosine methylation, are inherited in plant species and may occur in response to biotic or abiotic stress, affecting gene expression without changing genome sequence. Laguncularia racemosa, a mangrove species, occurs in naturally contrasting habitats where it is subjected daily to salinity and nutrient variations leading to morphological differences. This work aims at unraveling how CpG-methylation variation is distributed among individuals from two nearby habitats, at a riverside (RS) or near a salt marsh (SM), with different environmental pressures and how this variation is correlated with the observed morphological variation.

Principal Findings

Significant differences were observed in morphological traits such as tree height, tree diameter, leaf width and leaf area between plants from RS and SM locations, resulting in smaller plants and smaller leaf size in SM plants. Methyl-Sensitive Amplified Polymorphism (MSAP) was used to assess genetic and epigenetic (CpG-methylation) variation in L. racemosa genomes from these populations. SM plants were hypomethylated (14.6% of loci had methylated samples) in comparison to RS (32.1% of loci had methylated samples). Within-population diversity was significantly greater for epigenetic than genetic data in both locations, but SM also had less epigenetic diversity than RS. Frequency-based (G_ST) and multivariate (β_ST) methods that estimate population structure showed significantly greater differentiation among locations for epigenetic than genetic data. Co-Inertia analysis, exploring jointly the genetic and epigenetic data, showed that individuals with similar genetic profiles presented divergent epigenetic profiles that were characteristic of the population in a particular environment, suggesting that CpG-methylation changes may be associated with environmental heterogeneity.

Conclusions

In spite of significant morphological dissimilarities, individuals of L. racemosa from salt marsh and riverside presented little genetic but abundant DNA methylation differentiation, suggesting that epigenetic variation in natural plant populations has an important role in helping individuals to cope with different environments.     

Century-scale Methylome Stability in a Recently Diverged Arabidopsis thaliana Lineage

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.     

Epigenetic variation associated with responses to different habitats in the context of genetic divergence in Phragmites australis

The mechanisms underlying heritable phenotypic divergence associated with adaptation in response to environmental stresses may involve both genetic and epigenetic variations. Several prior studies have revealed even higher levels of epigenetic variation than genetic variation. However, few population‐level studies have explored the effects of epigenetic variation on species with high levels of genetic diversity distributed across different habitats. Using AFLP and methylation‐sensitive AFLP markers, we tested the hypothesis that epigenetic variation may contribute to differences in plants occupying different habitats when genetic variation alone cannot fully explain adaptation. As a cosmopolitan invasive species, Phragmites australis (common reed) together with high genetic diversity and remarkable adaptability has been suggested as a model for responses to global change and indicators of environmental fluctuations. We found high levels of genetic and epigenetic diversity and significant genetic/epigenetic structure within each of 12 studied populations sampled from four natural habitats of P. australis. Possible adaptive epigenetic variation was suggested by significant correlations between DNA methylation‐based epigenetic differentiation and adaptive genetic divergence in populations across the habitats. Meanwhile, various AMOVAs indicated that some epigenetic differences may respond to various local habitats. A partial Mantel test was used to tease out the correlations between genetic/epigenetic variation and habitat after controlling for the correlation between genetic and epigenetic variations. We found that epigenetic diversity was affected mostly by soil nutrient availability, suggesting that at least some epigenetic differentiation occurred independently of genetic variation. We also found stronger correlations between epigenetic variation and phenotypic traits than between genetic variation and such traits. Overall, our findings indicate that genetically based differentiation correlates with heterogeneous habitats, while epigenetic variation plays an important role in ecological differentiation in natural populations of P. australis. In addition, our results suggest that when assessing global change responses of plant species, intraspecific variation needs to be considered.     

Epigenetic changes and transposon reactivation in Thai rice hybrids

Inter- or intraspecific hybridization is the first step in transferring exogenous traits to the germplasm of a recipient crop. One of the complicating factors is the occurrence of epigenetic modifications of the hybrids, which in turn can change their gene expression and phenotype. In this study we present an analysis of epigenome changes in rice hybrids that were obtained by crossing rice cultivars, most of them of indica type and Thai origin. Comparing amplified fragment length polymorphism (AFLP) fingerprints of twenty-four cultivars, we calculated Nei’s indexes for measuring genetic relationships. Epigenetic changes in their hybrids were established using methylation-sensitive AFLP fingerprinting and transposon display of the rice transposable elements (TEs) Stowaway Os-1 and Mashu, leading to the question whether the relationship between parental genomes is a predictor of epigenome changes, TE reactivation and changes in TE methylation. Our study now reveals that the genetic relationship between the parents and DNA methylation changes in their hybrids is not significantly correlated. Moreover, genetic distance correlates only weakly with Mashu reactivation, whereas it does not correlate with Stowaway Os-1 reactivation. Our observations also suggest that epigenome changes in the hybrids are localized events affecting specific chromosomal regions and transposons rather than affecting the genomic methylation landscape as a whole. The weak correlation between genetic distance and Mashu methylation and reactivation points at only limited influence of genetic background on the epigenetic status of the transposon. Our study further demonstrates that hybridizations between and among specific japonica and indica cultivars induce both genomic DNA methylation and reactivation/methylation change in the Stowaway Os-1 and Mashu transposons. The observed epigenetic changes seem to affect the transposons in a clear manner, partly driven by stochastic processes, which may account for a broader phenotypic plasticity of the hybrids. A better understanding of the epigenome changes leading to such transposon activation can lead to the development of novel tools for more variability in future rice breeding.     

Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.     

RYS1, a foldback transposon, is activated by tissue culture and shows preferential insertion points into the rye genome

The study of two variable amplicons of rye indicates that RYS1, a mobile element, is activated during tissue culture. We propose that RYS1 could be a foldback (FB) transposon. The FB transposons have been rarely reported in plants; RYS1 is the first described in rye and also the first active plant FB transposon reported. Preferential integration points in the rye genome exist, because the new insertions seem to be located, in all studied cases, in the same genome positions. We assume that RYS1 became active in rye very recently, as different plants from in vivo-growing cultivars showed that these elements were present or absent in the same genomic position in which the in vitro-activated element was found. This high rate of modification in these particular loci, both in the in vivo and in vitro populations, could indicate that probably the mechanisms promoting genetic variability in nature are the same that induce variation in vitro, and the modifications induced by somaclonal variation could be already present in vivo populations     

DNA fingerprints of living fossil Ginkgo biloba by using ISSR and improved RAPD analysis

In this study, we have aimed to genetically characterize Ginkgo biloba. Nine G. biloba samples from different places of China were collected, and DNA was extracted from the leaves of these samples for inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) analysis. ISSR analysis showed high genetic variation among the nine varieties of G. biloba; the polymorphism and similarity coefficients were 87% and 0.40–0.84, respectively. RAPD analysis also showed 93% polymorphism, and the similarity coefficients ranged from 0.44 to 0.87. Persistent genetic isolation that developed for millions of years might influence the genetic variability between the samples of G. biloba. This study generates a genetic map of G. biloba, and reports the highly variable intra-species genetic characteristics of this living fossil among different geographical locations of China. Our study also suggests that ISSR and the improved RAPD markers are useful molecular tools for the genetic characterization of plants.     

Genetic homogeneity of in vitro raised plants of grapevine cv. Crimson Seedless revealed by ISSR and microsatellite markers

The present study was conducted to test the clonal homogeneity of six month old tissue culture raised plants of grapevine cv. Crimson Seedless using Inter Simple Sequence Repeat (ISSR) and Simple Sequence Repeat (SSR) markers. Visible assessment of these in vitro raised plants maintained in polyhouse did not show any morphological differences among themselves. However, to test the genetic homogeneity of these plants, we screened 50 ISSR primers out of which, 22 primers produced scorable and repeatable bands. These 22 primers were used further for assessing genetic homogeneity of in vitro raised plants of Crimson Seedless. These 22 ISSR primers generated 134 distinct band classes with a total of 3216 scorable bands. All the primers showed uniform banding pattern for all the in vitro raised plants and the mother plant. In case of 5 SSR primers (VS1, VVMD5, VVS2, VMCNG4c8 and VVMD31) used, a total of 288 scorable bands were obtained. The allele sizes ranged from 98 to 254 bp. Allelic composition of 23 in vitro raised plants and the mother plant at 5 SSR loci did not show any polymorphism. The results of the two marker systems in the present study revealed the genetic uniformity among the in vitro raised plants demonstrating the reliability of in vitro propagation system used for the cultivar.     

Genotypic diversity and structure of Satureja mutica revealed by inter-simple sequence repeat markers

Satureja mutica (Lamiaceae) is an herbaceous medicinal plant which grows in Iran. The objective of the study was to obtain an overview of the genetic relatedness among and within seven populations of this species using inter-simple sequence repeat (ISSR). Fourteen ISSR primers amplified a total of 197 DNA fragments of which 176 (88.91%) were polymorphic. All ISSR primers were highly effective in discriminating among the populations. Genetic similarity coefficients ranged from 0.45 to 0.94, indicating considerable distance and diversity in the germplasm and were confirmed by clustering analysis. The dendrogram showed a clear clustering pattern of plants indicating a significant association between genetic similarity and geographical distance. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (75%) rather than among populations (25%). The study indicated that ISSR markers were effective and reliable for assessing the degree of genetic variation of S. mutica. These findings can support future research on the selection of S. mutica for breeding and medicinal plant development.