Experience from Lipizzan horse and salmonid species endemic to the Adriatic river system. Examples for the application of molecular markers for preservation of biodiversity and management of animal genetic resources

Management of breeding- and free-living populations, traditionally based on phenotypic traits, relays more and more on availability of reliable information about the basic population genetic parameters as heterozygosity, mean number of alleles per locus, percentage of polymorphic loci, population structuring, genetic distances and others. Therefore, the application of molecular markers, revealing a great deal of phenotypically hidden information, becomes inevitable for population analysis. Conservation geneticists use this information for implementation of appropriate management policies. Application of molecular markers in Lipizzan horse breed, which is an example for a pedigreed breeding population, and in two endangered salmonid fish populations in Slovenia, are presented. In the Lipizzan horse breed, an insight in the population structure, overall heterozygosity, relationship between population parameters and phenotypic traits and reliability of pedigree data was gained by using molecular markers. Marble trout and Adriatic grayling were selected as examples for free-living populations, seriously endangered by human activity in the past. Development of informative molecular markers, their application and suggestions for appropriate conservation actions are described.     

Population structure of species: Eco-geographic units and genetic differentiation between populations

A two-step approach, based on a combined use of environmental, geographic, and genetic data, is suggested for studying population structures of species. First, populations are grouped into eco-geographic units (EGUs) according to the environmental gradients in the studied part of the species range, the types of life strategies, and other non-genetic characteristics that are presumably associated with adaptation and interpopulation gene flows. Second, the selected EGUs are tested for their congruence with genetic data by comparing the genetic differentiation between populations within EGUs to that between populations of different EGUs. Some of the issues discussed are as follows: the relationship of the EGU concept with the concepts of biogeocenosis and evolutionarily significant units (ESUs); designing EGUs in practice; the level of EGUs in a hierarchical population structure; and the weights of genetic and phenotypic markers in estimating population differentiation. The population structure of a salmonid fish, the Sakhalin taimen, in terms of eco-geographic units is considered as an example.     

Cadmium hyperaccumulation and genetic differentiation of Thlaspi caerulescens populations

Although the knowledge on heavy metal hyperaccumulation mechanisms is increasing, the genetic basis of cadmium (Cd) hyperaccumulation remains to be elucidated. Thlaspi caerulescens is an attractive model since Cd accumulation polymorphism observed in this species suggests genetic differences between populations with low versus high Cd hyperaccumulation capacities. In our study, a methodology is proposed to analyse at a regional scale the genetic differentiation of T. caerulescens natural populations in relation to Cd hyperaccumulation capacity while controlling for different environmental, soil, plant parameters and geographic origins of populations. Twenty-two populations were characterised with AFLP markers and cpDNA polymorphism. Over all loci, a partial Mantel test showed no significant genetic structure with regard to the Cd hyperaccumulation capacity. Nevertheless, when comparing the marker variation to a neutral model, seven AFLP fragments (9% of markers) were identified as presenting particularly high genetic differentiation between populations with low and high Cd hyperaccumulation capacity. Using simulations, the number of outlier loci was showed to be significantly higher than expected at random. These loci presented a genetic structure linked to Cd hyperaccumulation capacity independently of the geography, environment, soil parameters and Zn, Pb, Fe and Cu concentrations in plants. Using a canonical correspondence analysis, we identified three of them as particularly related to the Cd hyperaccumulation capacity. This study demonstrates that populations with low and high hyperaccumulation capacities can be significantly distinguished based on molecular data. Further investigations with candidate genes and mapped markers may allow identification and characterization of genomic regions linked to factors involved in Cd hyperaccumulation.     

Rhinanthus minor population genetic structure and subspecies: Potential seed sources of a keystone species in grassland restoration projects

In the last few decades unimproved semi-natural grasslands have been affected by intensification of land use and habitat fragmentation. Because of their biodiversity these species-rich grasslands are of high conservation importance and efforts are under way to restore such habitats. Detailed knowledge of within species diversity will aid deciding on the optimal seed source for such restoration projects, e.g. local genotypes or ecotypes. Rhinanthus minor is a species that is typically found in semi-natural grasslands and is commonly used in grassland restoration projects. This is because R. minor is a hemiparasitic plant that takes minerals and nutrients from its host, which in turn decreases the host's biomass and leads to opportunities for less competitive species in the vegetation. Here, we investigate genetic diversity within and between R. minor populations. This allowed us to test whether the six different subspecies of R. minor that have been described in the UK, based on their morphology, flowering time, and habitat, can be differentiated using molecular markers. We identified moderate levels of genetic differentiation between R. minor populations within the UK. In addition, R. minor individuals from the UK appear to be distinct from R. minor and Rhinanthus angustifolius individuals from other European countries based on microsatellite genotyping and DNA sequencing of cpDNA and rDNA ITS. The molecular markers used in the current study did not separate populations of R. minor based on either their subspecies or habitat. The implication for the use of R. minor in grassland restoration projects seems to be that it is not necessary to use local seeds or seeds from the same subspecies.     

Genetic Diversity of Twelve Switchgrass Populations Using Molecular and Morphological Markers

Switchgrass (Panicum virgatum L.) is a warm season, C₄ perennial grass native to most of North America with numerous applications, including use as a bioenergy feedstock species. To date, no studies on genetic diversity in switchgrass have been conducted that use both molecular and morphological markers. The objectives of this study were to assess genetic diversity and determine differences among and between 12 switchgrass populations grown in New Jersey by examining both morphological and molecular characteristics, and to determine whether morphological, molecular, and/or combined data sets can detect ecotype and/or geographical differences at the population level. Twelve plants from each population were characterized with 16 switchgrass expressed sequence tag-simple sequence repeat markers (EST-SSRs) and seven morphological characters. Data was analyzed using GenAlEx and Unweighted Pair-Group Method of Averages (UPGMA) cluster analysis. Most (64%) of the molecular variation in switchgrass populations exists among individuals within populations, with lesser amounts between populations (36%). Upland and lowland populations were distinguished in all three data sets. Some eastern US and midwestern US populations were distinct in all three data sets. Similarities were observed between all three data sets indicating molecular markers may be useful for identifying morphological differences or other adaptive traits. The combined data set was the most useful in differentiating populations based on geography and found separation between midwestern and eastern upland populations. The results indicate that the combination of morphological and molecular markers may be useful in future applications such as genetic diversity studies, plant variety protection, cultivar identification, and/or identifying geographic origin.     

An assessment of the spatial scale of local adaptation in brown trout (Salmo trutta L.): footprints of selection at microsatellite DNA loci

Local adaptation is considered a paradigm in studies of salmonid fish populations. Yet, little is known about the geographical scale of local adaptation. Is adaptive divergence primarily evident at the scale of regions or individual populations? Also, many salmonid populations are subject to spawning intrusion by farmed conspecifics that experience selection regimes fundamentally different from wild populations. This prompts the question if adaptive differences between wild populations and hatchery strains are more pronounced than between different wild populations? We addressed these issues by analyzing variation at 74 microsatellite loci (including anonymous and expressed sequence tag- and quantitative trait locus-linked markers) in 15 anadromous wild brown trout (Salmo trutta L.) populations, representing five geographical regions, along with two lake populations and two hatchery strains used for stocking some of the populations. F_ST-based outlier tests revealed more outlier loci between different geographical regions separated by 522±228 km (mean±s.d.) than between populations within regions separated by 117±79 km (mean±s.d.). A significant association between geographical distance and number of outliers between regions was evident. There was no evidence for more outliers in comparisons involving hatchery trout, but the loci under putative selection generally were not the same as those found to be outliers between wild populations. Our study supports the notion of local adaption being increasingly important at the scale of regions as compared with individual populations, and suggests that loci involved in adaptation to captive environments are not necessarily the same as those involved in adaptive divergence among wild populations.     

Molecular and Quantitative Genetic Differentiation in Sitobion avenae Populations from Both Sides of the Qinling Mountains

Quantitative trait differences are often assumed to be correlated with molecular variation, but the relationship is not certain, and empirical evidence is still scarce. To address this issue, we sampled six populations of the cereal aphid Sitobion avenae from areas north and south of the Qinling Mountains, and characterized their molecular variation at seven microsatellite loci and quantitative variation at nine life-history traits. Our results demonstrated that southern populations had slightly longer developmental times of nymphs but much higher lifetime fecundity, compared to northern populations. Of the nine tested quantitative characters, eight differed significantly among populations within regions, as well as between northern and southern regions. Genetic differentiation in neutral markers was likely to have been caused by founder events and drift. Increased subdivision for quantitative characters was found in northern populations, but reduced in southern populations. This phenomenon was not found for molecular characters, suggesting the decoupling between molecular and quantitative variation. The pattern of relationships between F_ST and Q_ST indicated divergent selection and suggested that local adaptation play a role in the differentiation of life-history traits in tested S. avenae populations, particularly in those traits closely related to reproduction. The main role of natural selection over genetic drift was also supported by strong structural differences in G-matrices among S. avenae populations. However, cluster analyses did not result in two groups corresponding to northern and southern regions. Genetic differentiation between northern and southern populations in neutral markers was low, indicating considerable gene flow between them. The relationship between molecular and quantitative variation, as well as its implications for differentiation and evolution of S. avenae populations, was discussed.     

Land use affects flowering time: seasonal and genetic differentiation in the grassland plant Scabiosa columbaria

Management practices like mowing or grazing have a large impact on grassland species. Due to its evolutionary significance, the interaction between the flowering time of plants and land use is of special interest. Asynchronous flowering restricts gene flow between populations and promotes, as a consequence, their differentiation. We selected 12 populations across southern central Europe to study the impact of mowing and grazing on seasonal and genetic differentiation in the grassland species Scabiosa columbaria. We conducted a common garden experiment to analyse floral display between populations and applied molecular markers to assess genetic diversity and genetic differentiation between populations. We demonstrated explicitly that flowering time and genetic differentiation are linked with the type of land use. Populations from mown habitats flowered significantly earlier than populations from grazed sites. Furthermore, genetic differentiation was stronger between populations from sites of different land use than between populations from far away geographic regions. The results of this study indicate that populations of S. columbaria are seasonally adapted to mowing and grazing. Land use is, therefore, an important factor for evolution in grassland species, which promotes the development of seasonal ecotypes and clearly affects intraspecific variation.     

Genetic structure of Pinus henryi and Pinus tabuliformis: Natural landscapes as significant barriers to gene flow among populations

Mountains as natural barriers often have important effects on intraspecific genetic structure through restraining gene flow and enhancing differentiation among populations. While the Qinling and Daba mountains are considered significant geographic barriers, dividing China into temperate and subtropical regions, little is known about how this barrier influences the genetic patterns of sister species represented in distinct habitats. In this study, we analyzed genetic differentiation and the geographic boundary between Pinus henryi and Pinus tabuliformis using chloroplast microsatellite markers. Our data show high levels of among-population differentiation, consistent with the effects of historical demographic bottlenecks, local adaptation and climate effects. Three main geographic boundaries coinciding with mountain systems indicate natural landscapes, such as large rivers, and habitat loss caused by anthropogenic deforestation, are significant barriers to genetic exchange among populations. The divergence between populations in the eastern and western Qinling Mountains populations may possibly be ascribed to fragmentation driven by climate change. The genetic boundary of P. henryi and P. tabuliformis generally coincides with the previous morphological dividing line based on the unweighted pair group method using arithmetic averages and on spatial analysis of molecular variance.     

Ecology, genetic diversity and phylogeography of the Iberian endemic plant Jurinea pinnata (Lag.) DC. (Compositae) on two special edaphic substrates: dolomite and gypsum

Background and aims

Jurinea pinnata is an Iberian vascular plant which only grows on gypsum and dolomite, two types of rocks associated with their exclusive endemic floras. In addition, the plant has an island-like distribution which could affect the differentiation and the genetic variability of wild populations. Thus, the species provides a unique opportunity for comparing (bio)geographical and ecological (edaphic) differentiation by means of molecular markers.

Methods

For our investigation we took 24 soil samples paired with a similar number of foliar samples for nutritional analyses. Our molecular-marker approach (AFLPs) involved 16 populations.

Results

The edaphic parameters revealed significant dissimilarities between dolomitic and gypsum soils. These differences are also found in the mineral composition of the leaves. However, molecular data revealed that the differentiation between populations correlates better with geographical isolation than with the substrate character.

Conclusions

The populations showing the greatest genetic diversity are those of the East Baetic territory where the species grow on both substrates and its populations are closer together. The plant tolerance to gypsum and dolomite can be explained either as a result of common adaptive mechanisms or of a more general adaptation to arid environments.     

Genetic and allelopathic differences between populations of daisy fleabane Erigeron annuus (L.) Pers. (Asteraceae) from disturbed and stable habitats

The interactions between invasive plants and their habitats may vary at different phases of the invasion process and depend on the phenotypic plasticity or local adaptations of each species. In this study, we investigated whether habitat changes during the invasion process are related to variations in the physiological traits (allelopathic properties) and genetic differentiation of daisy fleabane (Erigeron annuus (L.) Pers.). E. annuus is a winter annual invasive species that originated in North America and is now distributed throughout Europe. Genetic and genotypic diversity analyses were performed for 37 populations of E. annuus based on inter simple sequence repeat (ISSR) polymorphisms. In total, 684 plants were analyzed; 342 were from stable habitats and 342 were from disturbed habitats. The genetic differences among the populations from the different habitats were studied using a Bayesian cluster analysis and an analysis of molecular variance (AMOVA) and by calculating the genetic and genotypic diversity parameters. A germination test using the juglone index was employed to examine the potential allelopathic properties of the plants from the different habitats. Bayesian cluster analysis, AMOVA and allelopathic effects evaluation revealed differences in the allelopathic potential and genetic structure of the E. annuus populations from the disturbed and stable habitats. This differentiation of populations could be associated with founder effects or with different selection pressures among habitats.     

A morphometric analysis and taxonomic study of Panax bipinnatifidus Seem. (Araliaceae) species complex from Sikkim Himalaya, India

Cattleya elongata is a rupicolous orchid species spread throughout and endemic to outcrop islands in campo rupestre vegetation of the Chapada Diamantina, northeastern Brazil. We scored nine natural populations of C. elongata for morphological and genetic variability, covering the whole distribution area of the species, using allozymes and ISSR markers and morphometric multivariate analyses. Genetic variability in allozimes was relatively high (H _e?=?0.12?C0.25), and unexpectedly higher than the values based on ISSR (H _e?=?0.16?C0.19). The populations present moderate structuring (allozymes, ??_PT?=?0.14; ISSR, ??_PT?=?0.18) and low inbreeding (allozymes, F _IS?=?0.06). Genetic similarity among the populations was high in both markers, in spite of the discontinuity of the outcrops of the Chapada Diamantina. We found no particular biogeographical pattern to the distribution of the genetic and morphologic similarity among the populations of C. elongata. We found high morphological variability with moderate differentiation among the populations. We did not find any correlation among genetic, morphological, and geographical distances, and among the variability found in the morphological and genetic markers. The differences observed between the two genetic markers and the various morphological markers examined here indicated that the isolated use of any single parameter of these different populations for conservation planning or management would not consider all of the variability to be found in the species, as found in other Brazilian campos rupestres plants.     

Glacial History Affected Phenotypic Differentiation in the Alpine Plant,Campanula thyrsoides

Numerous widespread Alpine plant species show molecular differentiation among populations from distinct regions. This has been explained as the result of genetic drift during glacial survival in isolated refugia along the border of the European Alps. Since genetic drift may affect molecular markers and phenotypic traits alike, we asked whether phenotypic differentiation mirrors molecular patterns among Alpine plant populations from different regions. Phenotypic traits can be under selection, so we additionally investigated whether part of the phenotypic differentiation can be explained by past selection and/or current adaptation. Using the monocarpic Campanula thyrsoides as our study species, a common garden experiment with plants from 21 populations from four phylogeographic groups located in regions across the Alps and the Jura Mountains was performed to test for differentiation in morphological and phenological traits. Past selection was investigated by comparing phenotypic differentiation among and within regions with molecular differentiation among and within regions. The common garden results indicated regional differentiation among populations for all investigated phenotypic traits, particularly in phenology. Delayed flowering in plants from the South-eastern Alps suggested adaptation to long sub-mediterranean summers and contrasted with earlier flowering of plants experiencing shorter growing seasons in regions with higher elevation to the West. Comparisons between molecular and phenotypic differentiation revealed diversifying selection among regions in height and biomass, which is consistent with adaptation to environmental conditions in glacial refugia. Within regions, past selection acted against strong diversification for most phenotypic traits, causing restricted postglacial adaptation. Evidence consistent with post-glacial adaptation was also given by negative correlation coefficients between several phenotypic traits and elevation of the population''s origin. In conclusion, our study suggests that, irrespective of adaptation of plants to their current environment, glacial history can have a strong and long-lasting influence on the phenotypic evolution of Alpine plants.     

Analysis of Genetic Variation in Natural Populations of Medicago truncatula of Southern Tunisian Ecological Areas, Using Morphological Traits and SSR Markers

We used 19 quantitative traits and 14 microsatellite markers (SSRs) to analyze the genetic variation in four natural populations of the model legume Medicago truncatula sampled in southern Tunisia. The greatest genetic variation of quantitative traits and molecular markers occurred within populations (>71%). In contrast to quantitative population differentiation (Q _ST ?=?0.09), a high level of molecular differentiation (F _ST ?=?0.23) was found among populations. The majority of quantitative traits exhibited Q _ST values significantly less than F _ST values, suggesting that selection may be acting to suppress differentiation for these traits. There was no significant correlation between genetic variation of quantitative traits and molecular markers within populations. On the other hand, significant correlations were found between measured quantitative characters and the site-of-origin environmental factors. The eco-geographical factors with the greatest influence on the variation of measured traits among populations were altitude, followed by soil texture, assimilated phosphorus (P₂O₅) and organic matter. Nevertheless, there were no consistent patterns of associations between gene diversity (He) and eco-geographical factors.     

Molecular differentiation within and among island populations of the endemic plant Scalesia affinis (Asteraceae) from the Galápagos Islands

Molecular variance was estimated in seven populations of the endemic species Scalesia affinis within and among islands of the Galapagos. The analysis, based on 157 polymorphic AFLP markers, revealed a high differentiation among populations, of which most was partitioned among islands. In addition, the information content of AFLP markers was tested with sets of discriminant analyses based on different numbers of AFLP markers. This indicated that the markers were highly informative in discriminating the populations. Although one of four populations from the island Isabela was sampled from a volcano 100 km away from the remaining populations, this population resembled the others on Isabela. The partitioning of molecular variance (AFLP) resulted in two unities, one consisting of populations from Isabela and one of populations from Santa Cruz and Floreana. The differentiation in two chloroplast microsatellites was higher than for AFLP markers and equally partitioned among populations within islands as among islands. Thus, gene flow via fruits within islands is as limited as among islands. The lower differentiation within islands in the nuclear AFLP markers may thus indicate that gene flow within islands is mostly accounted for by pollen transfer. S. affinis is the only species in the genus that is not listed in 2000 IUCN Red List of Threatened Species. However, due to prominent grazing and land exploitation, some populations have recently been reduced markedly, which was reflected in lower diversity. As inbreeding depression is present in the species, the rapid bottlenecks are threats to the populations.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

High Genetic Differentiation of Hippophae rhamnoides ssp. yunnanensis (Elaeagnaceae), a Plant Endemic to the Qinghai-Tibet Plateau

RAPD markers were used to detect genetic diversity and population genetic differentiation of Hippophae rhamnoides ssp. yunnanensis, a sea buckthorn endemic to the Qinghai-Tibet plateau. The genetic parameters of percentage of polymorphic bands (92.86%), Nei’s gene diversity (h, 0.255), and Shannon’s index (I, 0.397) indicated high genetic diversity in this subspecies. The subpopulation differentiation suggested that 45.9% of genetic variation was among populations. High genetic differentiation among populations was also detected using AMOVA (47.02%). The main factors responsible for high genetic differentiation are probably related to natural geographic barriers among populations, gene drift, and limited gene flow caused by restricted pollen flow and seed flow. A Mantel test indicated that geographic distances were significantly correlated with genetic distances. The UPGMA phenogram based on Nei’s unbiased genetic distances and the result of three-dimensional model plots performed by principal coordinate analysis also supported the correlation. Altitude, however, did not have any clear effect on genetic differentiation.     

Relevant genetic differentiation among Brazilian populations of Anastrepha fraterculus (Diptera,Tephritidae)

We used a population genetic approach to detect the presence of genetic diversity among six populations of Anastrepha fraterculus across Brazil. To this aim, we used Simple Sequence Repeat (SSR) markers, which may capture the presence of differentiative processes across the genome in distinct populations. Spatial analyses of molecular variance were used to identify groups of populations that are both genetically and geographically homogeneous while also being maximally differentiated from each other. The spatial analysis of genetic diversity indicates that the levels of diversity among the six populations vary significantly on an eco-geographical basis. Particularly, altitude seems to represent a differentiating adaptation, as the main genetic differentiation is detected between the two populations present at higher altitudes and the other four populations at sea level. The data, together with the outcomes from different cluster analyses, identify a genetic diversity pattern that overlaps with the distribution of the known morphotypes in the Brazilian area.     

The use of nuclear DNA molecular markers for studying speciation and systematics as exemplified by the “Lacerta agilis complex” (Sauria: Lacertidae)

Four types of nuclear DNA markers identified by the taxonprint, RAPD, and IMP (Inter-MIR-PCR) methods, and the nucleotide sequences of satellite DNA monomers have been used to analyze the molecular genetic similarity between some populations, subspecies, and species of lizards combined into the group Lacerta s. str., as well as representatives of some other genera. The notions on the systematics and phylogeny of this group based on morphological and zoogeographic criteria have been compared to the conclusions based on molecular genetic data. The genus and species subdivisions of populations based on nuclear molecular markers and morphological characters generally agree with each other, the degree of genetic differences being correlated with the taxonomy suggested by zoomorphologists. The degree of differences between the subspecies of one of the species studied, Lacerta agilis, varies depending on the molecular markers used: according to the results of RAPD analysis, all subspecies substantially differ from one another, the variation within populations being small; with respect to other markers, the differences are smaller and not equivalent. The existence of the so-called eastern and western clades of this species earlier assumed by other researchers on the basis of mtDNA and morphological data has been confirmed. There are no distinct gradations exceeding individual variation in 14 populations of L. agilis exigua (the eastern clade) with respect to IMP markers, although these populations inhabit a vast area from the Ural Mountains to the Kabardino-Balkar Republic (the Caucasus). These data suggest that the subspecies has been rapidly spreading northwards since the Pleistocene glaciation (about 15,000 years ago).     

Genetic diversity and population structure of Butea monosperma (Lam.) Taub.- a potential medicinal legume tree

Three molecular marker systems, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) were employed to investigate the genetic structure and diversity among the 14 natural populations of Butea monosperma collected from different geographical regions of India. Detected by 17 RAPD, 15 ISSR and 11 SRAP primer combinations, the proportions of polymorphic bands were 84.2 %, 77.2 % and 91.9 %, respectively, and the mean Nei’s genetic distances among the populations were 0.13, 0.10 and 0.13, respectively. Partitioning of genetic variability by Analysis of molecular variance (AMOVA) revealed that the high genetic diversity was distributed within the populations. AMOVA also revealed that the coefficient of gene differentiation among populations based on F_ST was very high irrespective of markers used. The overall gene flow among populations (Nm) was very low. Cophenetic correlation coefficients of Nei’s distance values and clustering pattern by Mental test were statistically significant for all three marker systems used but poor fit for ISSR data than for RAPD, SRAP and combined data set of all three markers. For all markers, a high similarity in dendrogram topologies was obtained, although some differences were observed with ISSR. The dendrogram obtained by RAPD, SRAP and combined data set of all three markers reflect relationship of most of the populations according to their geographic distribution.