[Show abstract][Hide abstract] ABSTRACT: The ecological consequences of biodiversity have become a prominent public issue. Little is known on the effect of genetic diversity on ecosystem services. Here, a diversity experiment was established with European and North American aspen (Populus tremula, P. tremuloides) planted in plots representing either a single deme only or combinations of two, four and eight demes. The goals of this study were to explore the complex inter- and intraspecific genetic diversity of aspen and to then relate three measures for diversity (deme diversity, genetic diversity determined as Shannon index or as expected heterozygosity) to arthropod abundance. Microsatellite and AFLP markers were used to analyze the genetic variation patterns within and between the aspen demes and deme mixtures. Large differences were observed regarding the genetic diversity within demes. An analysis of molecular variance revealed that most of the total genetic diversity was found within demes, but the genetic differentiation among demes was also high. The complex patterns of genetic diversity and differentiation resulted in large differences of the genetic variation within plots. The average diversity increased from plots with only one deme to plots with two, four, and eight demes, respectively and separated plots with and without American aspen. To test whether intra- and interspecific diversity impacts on ecosystem services, arthropod abundance was determined. Increasing genetic diversity of aspen was related to increasing abundance of arthropods. However, the relationship was mainly driven by the presence of American aspen suggesting that species identity overrode the effect of intraspecific variation of European aspen.
[Show abstract][Hide abstract] ABSTRACT: For investigating genotypic differences in the production potential of Populus tremula L., we grew aspen plants of six full-sib families under optimal water and nutrient conditions and analysed more than 20 physiological and morphological traits with a potential impact on productivity. The six families were produced from controlled crossings of two male and four female trees. Despite genetic distances of 2–28%, the families showed no significant differences in photosynthetic and leaf water status parameters (photosynthetic capacity, leaf water potential and others), even though productivity differed up to twofold between the families. Hence, growth rate was not related to photosynthetic activity but showed a close association with several morphological traits, most closely with the leaf number (L) and total leaf area. Variation in L explained 70% of the growth variation across the six families, and the start of bud burst (BB) correlated with the leaf number (early-starting families produced more leaves). The between-family variation in growth-related morphological traits was much larger than that in physiological traits (coefficient of genetic variation 4–29% vs. 0–4%). Even though the genetic constitution had a significant effect on eight morphological (leaf and root-related) traits, we found no relation between the genetic differences between any two families and the corresponding growth differences. We conclude that the timing of bud burst and the resulting total number of leaves developed are the determinants of growth in P. tremula. Selection programmes should focus on the considerable intraspecific variation in L and BB in order to increase yield.
Biomass and Bioenergy 09/2013; 56:274–283. DOI:10.1016/j.biombioe.2013.05.013 · 3.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In view of the predicted changing environmental conditions, the question arises whether the important tree species European beech (Fagus sylvatica L.) will be adaptable to the future climate in Europe. Only few studies investigated the genetic background of drought stress tolerance in beech. In this study a set of 17 SNP (Single Nucleotide Polymorphism) markers selected from eight candidate genes potentially involved in drought stress response were developed. The polymorphisms of these markers were analysed in 50 adult trees from a population in Germany by using two multiplex sets (SNaPshot® Multiplex Kit (Applied Biosystems)). Observed and expected heterozygosity ranged from 0.060 to 0.520 and from 0.059 to 0.505, respectively. None of the loci showed a significant deviation from Hardy–Weinberg equilibrium.
[Show abstract][Hide abstract] ABSTRACT: European beech (Fagus sylvatica L.) is one of the most important deciduous tree species in Central Europe. The potential of beech to adapt to climate change, higher temperatures, and less precipitation in the summer months is still unknown. Most studies in beech used microsatellite, AFLP (amplified fragment length polymorphism), or isozyme markers, which have only a restricted potential to analyze adaptation. Only few studies investigated genes probably involved in the adaptation to drought stress and bud phenology in beech. In this study, SNP (single nucleotide polymorphisms) markers were developed in order to analyze adaptation and their technical advantages compared to microsatellites and AFLPs were discussed. Partial sequences of ten candidate genes probably involved in drought stress and/or bud phenology were identified at the genomic level, and SNPs and indels (insertions/deletions) in coding and non-coding regions were analyzed. Plant material was sampled along a precipitation gradient in Germany. In total, 8,145 bp were sequenced and analyzed, 4,038 bp were located in exon and 4,107 bp in intron regions. 63 SNPs and 11 indels were detected, which are differently distributed over the studied gene regions. The nucleotide diversity ranged from 0 to 6.62 (π × 10−3) and is comparable to other tree species, whereas the mean nucleotide diversity (2.64) for F. sylvatica is comparatively low. These results will help to investigate the genetic basis of drought stress and bud burst and to conduct association mapping in natural populations. Furthermore, the detected SNPs can also be used for population genetic studies.
European Journal of Forest Research 11/2012; 131(6). DOI:10.1007/s10342-012-0630-9 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the Chernobyl exclusion zone forest trees have to tolerate and to adapt to ionizing radiation, therefore the molecular basis of their adaptive responses is of the utmost interest. Based on SNP analysis and real time PCR nucleotide diversity and expression profiles of gene fragments of catalase (Cat) and glutathione peroxidase (GPx), which are known as radical scavenging genes, were analysed in the needles of irradiated pine trees of the Chernobyl exclusion zone. In acutely and chronically irradiated trees (50 years old) planted before the accident a higher nucleotide diversity of Cat and more somatic mutations were found compared to their control. Chronically irradiated trees (20 years old) planted after the accident showed a similar nucleotide diversity of Cat compared to their control and in both collectives one somatic mutation was found. The nucleotide diversity of GPx was higher in all analysed trees compared to Cat. No somatic mutation events were found in GPx. For both gene fragments, no association between the received dose in a tree and the nucleotide diversity and mutation events was detected. The expression profiles of Cat and GPx in acutely and chronically and in chronically irradiated trees were similar. Compared to their corresponding control collectives, Cat was up-regulated and GPx slightly down-regulated.
[Show abstract][Hide abstract] ABSTRACT: The nuclear accident of Chernobyl caused a drastic change of the environmental conditions for trees growing close to the exploded reactor. Forests in the exclusion zone surrounding the nuclear power plant are dominated by pines (Pinus sylvestris). Pine plantations were either established before the accident or were planted on extremely contaminated soils after the accident. The former experienced high acute followed by chronic radiation while the latter suffer from extremely high levels of chronic radiation. We investigated both types of pine stands, i. e. plantations established before and after the accident, and compared results to stands of the same origin and age in areas with normal levels of background radiation. We observed somatic mutations in different regions of the pine genome by investigating tissue from several twigs with different exposition from the same tree. At microsatellite loci no mutations were observed in control trees and only few mutations in pines close to the reactor. Mutation rates were higher at AFLP (Amplified Fragment Length Polymorphisms); a threefold increase of the mutation rate was observed in trees exposed to high radioactivity in comparison to controls. Comparisons of genetic structures at AFLP revealed significant differences between control populations and plantations established at contaminated sites after the accident. Random fluctuations of gene frequencies were excluded as likely causes of the observed differentiation for approximately six percent of the investigated loci. Selection is likely to act on many of these loci. The adaptive potential of pine trees to high radioactivity is influenced by their genetic constitution, and populations react to high radioactivity by changes of their genetic structures due to selection. The analysis of gene expression patterns in a catalase gene, which is known to be involved in general stress response of many plant species, revealed a strong increase of the gene activity in pines exposed to high radioactivity in comparison to the controls. The opposite tendency was observed for a glutathione peroxidase gene, which is also frequently affected in plants exposed to different types of stress; a slightly higher activity of this gene was observed in control trees. Finally, different methylation patterns were observed between trees exposed to acute and chronic radiation in comparison to their controls. Accordingly, DNA methylation might be another response of pines to environmental change. In conclusion, we observed highly complex reactions of pines to high radioactivity. We observed elevated mutation rates, protection of DNA by increased methylation, modified patterns of gene expression and changes of genetic structures due to selection in response to the high levels of radioactivity experienced by pine trees in the Chernobyl exclusion zone.
[Show abstract][Hide abstract] ABSTRACT: For the first time in sessile oak [Quercus petraea (Matt.) Liebl.], the isolation and characterisation of a full-length dehydrin gene and its promoter region, as well as its allelic variation in natural populations, is reported. Dehydrins (Dhn) are stress-related genes important for the survival of perennial plants in a seasonal climate. A full-length dehydrin gene (Dhn3) was characterised at the nucleotide level and the protein structure was modelled. Additionally, the allelic variation was analysed in five natural populations of Quercus petraea (Matt.) Liebl. sampled along an altitudinal gradient in the French Pyrenees. The analysed sequences contain typical domains of the K(n) class of dehydrins in the coding region. Also, the 5'untranslated region (promoter) of the gene was amplified, which shows typical motifs essential for drought- and cold-responsive gene expression. Single nucleotide substitutions and indels (insertions/deletions) within the coding region determine large biochemical differences at the protein level. However, only low levels of genetic differentiation between populations from different altitudes were detectable.
[Show abstract][Hide abstract] ABSTRACT: Knowledge on phenological, morphometric, and phytochemical variation of local progenies of European aspen (Populus tremula, L.) is limited. The goal of this study was to characterize variation in growth and ecologically important leaf properties
in aspen full-sib families in relation to interacting organisms (mycorrhiza, endophytes, and insects) and to determine whether
these interactions were affected by soil application of a systemic fungicide. In local progenies, within-family variation
of neutral molecular genetic markers (nuclear microsatellites) was higher than between families. Significant variation in
growth, production of phenolic defensive compounds and other phytochemical leaf traits was found between families. Phenolic
compounds showed clear negative correlation with generalist herbivores, but did not result in negative trade-off with biomass
production. Differences in mycorrhizal colonization were not found among full-sib families and application of a systemic fungicide
suppressed neither mycorrhizal colonization nor infestation with insects. However, a strong suppression of endophytes occurred,
whose long-term consequences may require attention when fungicides are used in agroforestry plantations.
KeywordsAgroforestry–Molecular marker–Nitrogen–Nutrition–Phenolic compounds–
European Journal of Forest Research 09/2011; 130(5):707-716. DOI:10.1007/s10342-010-0460-6 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ionizing radiation is a strong mutagenic factor and, accordingly, elevated mutation rates would be expected in plants exposed to high chronic or acute radiation after the Chernobyl accident in 1986. Somatic mutations were analyzed in pines (Pinus sylvestris L.) planted before and after the Chernobyl accident and in control material of the same origin planted in sites with natural radiation. Microsatellites (SSRs) and amplified fragment-length polymorphisms (AFLPs) were investigated. The mutation rates for microsatellites were estimated as 2.8 × 10(-4)-7.1 × 10(-4) per locus for different irradiated tree populations; no mutations were detected in the controls. In the case of AFLPs, the observed mutation rates were 3.74 × 10(-3) -3.99 × 10(-3) and 1.06 × 10(-3) per locus for contaminated and control areas, respectively. Thus a statistically highly significant three-fold increase in number of mutations was found by the use of AFLP markers, indicating that ionizing radiation causes strong DNA damage across the entire genome and that AFLPs may be the appropriate marker system for this kind of analysis.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2011; 725(1-2):29-35. DOI:10.1016/j.mrgentox.2011.07.003 · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ackee (Blighia sapida) is a native multipurpose species important for the livelihoods of the rural populations in Benin. Trees are found in natural
forests or are managed by farmers in different traditional agroforestry systems. Genetic variation at amplified fragment length
polymorphism (AFLP) markers, four nuclear microsatellites (nSSRs) and one chloroplast microsatellite (cpSSR) were investigated
in 279 individuals from six wild and eight cultivated populations from Benin. The AFLP data revealed moderate levels of diversity
of ackee in Benin (mean diversity values are proportion of polymorphic loci=52.8% and Nei’s gene diversity=0.157, for
375 AFLP fragments). The mean diversity values based on nSSR-markers are expected heterozygosity=0.286, allelic richness=2.77.
Genetic variation of wild and cultivated populations did not differ markedly. AMOVA revealed that only 7.3 and 5.2% of the
variation was partitioned among populations for nSSR- and AFLP-markers, respectively. A Mantel test based on these both marker-types
revealed significant correlations between population pairwise geographic distance and genetic differentiation. Differentiation
among cultivated populations was higher than among wild populations. The only polymorphic chloroplast microsatellite marker
(ccmp7) showed three haplotypes. Cultivated populations from northeastern Benin were fixed on one haplotype which was not
observed elsewhere indicating a different origin of these populations possibly from neighboring Nigeria. Farmer-led domestication
had an impact on the spatial distribution of genetic variation but did not result in significant losses of diversity within
populations. Measures to conserve genetic resources of ackee in each of the three main bioclimatic zones in Benin are proposed.
KeywordsBlighia sapida–AFLP–Nuclear and chloroplast microsatellites–Genetic structure–Domestication–Human impact–Agroforestry
[Show abstract][Hide abstract] ABSTRACT: There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size ( approximately 23.8 Gb/C).
A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates).
This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.
PLoS ONE 06/2010; 5(6):e11034. DOI:10.1371/journal.pone.0011034 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the increasing availability of sequence information at putatively important genes or regulatory regions, the characterization of adaptive genetic diversity and their association with phenotypic trait variation becomes feasible for many non-model organisms such as forest trees. Especially in predominantly outcrossing forest tree populations with large effective size, a high genetic variation in relevant genes is maintained, that is the raw material for the adaptation to changing and variable environments, and likewise for plant breeding. Oaks (Quercus spp.) are excellent model species to study the adaptation of forest trees to changing environments. They show a wide geographic distribution in Europe as dominant tree species in many forests and grow under a wide range of climatic and edaphic conditions. With the availability of a growing amount of functional and expressional candidate genes, we are now able to test the functional importance of single nucleotide polymorphisms (SNPs) by associating nucleotide variation in these genes with phenotypic variation in adaptive traits in segregating or natural populations. Here, we report on quantitative trait locus (QTL), candidate gene and association mapping approaches that are applicable to characterize gene markers and SNPs associated with variation in adaptive traits, such as bud burst, drought resistance and other traits showing selective responses to environmental change and stress. Because genome-wide association mapping studies are not feasible because of the enormous amount of SNP markers required in outcrossing trees with high recombination rates, the success of such an approach depends largely on the reasonable selection of candidate genes.
Physiologia Plantarum 07/2009; 137(4):509-19. DOI:10.1111/j.1399-3054.2009.01263.x · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Black poplar (Populus nigra L.) is a keystone species for riparian ecosystems in Europe. We analysed the structure of genetic diversity of 17 populations from 11 river valleys that are part of seven catchment systems (Danube, Ebro, Elbe, Po, Rhine, Rhone, and Usk) in Europe, in relation to geography and river management. In total, 1069 trees were genotyped using AFLP and microsatellite markers.The trees had an observed heterozygosity of 0.74 (range 0.59–0.82 across microsatellite loci). The majority (72.6–90.8%, depending on the marker system) of the genetic variation was present within populations. Most pairs of populations along a river were relatively similar (pairwise Fst 0.042–0.135 based on AFLP, 0.002–0.037 based on microsatellites). Overall population differentiation among rivers was considerable (Fst among populations was 0.268 based on AFLP, and 0.081 based on microsatellites). An analysis using the program Structure indicated that all populations recruited plants from several clusters. Geographically close populations tended to draw from the same Structure clusters, including populations from adjacent catchments. The Danube and Inn populations in Austria were genetically more similar to the Vltava population (Elbe catchment) in Czech Republic than the geographically more distant populations along the Tisa and Prut rivers of the Danube catchment in Ukraine. This indicates that gene flow and dispersal takes place across fairly large distances and between river catchments. Consistent with this result, a principal coordinate analysis of genetic distances among individual trees based on AFLP bands showed large overlap of populations, although the French and Spanish samples formed distinct clusters, and the samples from the Ticino (Italy) were at an intermediate position.The extent of clonal duplication was highest along regulated rivers, with e.g., 41% clonal duplication along the Rhine in The Netherlands (up to 32 trees for one genet). The Usk contained a man-made population (two genotypes along the entire river, one genet present as 70 trees out of 72 trees sampled). No clonal duplication was found along dynamic rivers, such as the Ebro (Spain), the Drome (France), and the Tisa and Prut (Ukraine).It is concluded that the restoration of the natural habitat and the re-creation of the natural dynamics of the floodplain, in combination with sufficiently sized and spaced natural populations as seed sources, are the most important measures for sufficient natural regeneration and conservation of this species in the future.
[Show abstract][Hide abstract] ABSTRACT: Obwohl die molekulare Analyse von Pflanzenteilen nicht zu forensischen Standardverfahren zählt, gibt es zahlreiche Anwendungen für den Einsatz molekulargenetischer Marker bei Pflanzen zur Klärung gerichtsrelevanter Sachverhalte. Für forensische Fragestellungen geeignete DNA-Marker sind insbesondere anonyme Fingerprint-Methoden (RAPDs, AFLPs) sowie artspezifische Marker, insbesondere Kernmikrosatelliten [SSRs; (SSR ist der in der Botanik gebräuchliche Begriff für STR)] und Sequenzen von DNAFragmenten. Auch durch eine Analyse von DNA aus uniparental vererbten Chloroplasten können spezifische Fragestellungen geklärt werden. Die molekulare Untersuchung von Pflanzenteilen an Tatorten oder an der Kleidung von Tatverdächtigen und ihr Vergleich zu Referenzproben, etwa einer am Tatort wachsenden Pflanze, kann zur Aufklärung von Kapitalverbrechen beitragen. Die Zugehörigkeit von Pflanzenteilen, die Schäden verursacht haben (z. B. ein herabgefallener Ast) zu einer bestimmten Pflanze kann mit molekularen Methoden überprüft werden. Eine molekulargenetische Analyse von illegal gehandelten Pflanzen und Produkten aus Pflanzen, insbesondere von Rauschmitteln, kann für die Klärung von Straftaten bedeutsam sein. Molekulare Marker können genutzt werden, um durch Erkennung des Ursprungs von Pflanzenteilen, beispielsweise Holz, Beiträge zur Klärung von Fällen von Umweltkriminalität zu erbringen. Schließlich können molekulare Methoden genutzt werden, um besondere Rechtsvorschriften, die sich auf die Produktion und den Handel mit beispielsweise gentechnisch veränderten Pflanzen und Pflanzenprodukten befassen, zu überprüfen. Aufgrund der großen Vielfalt von Fragestellungen, Untersuchungsmaterialien und Methoden stößt die Festlegung verbindlicher Standards für die molekulare Untersuchung von Pflanzenteilen auf große Schwierigkeiten. Eine für gerichtliche Entscheidungen erforderliche, sehr hohe und exakt quantifizierbare Sicherheit von Aussagen kann durch Untersuchung pflanzlicher DNA seltener erreicht werden als bei der Analyse von menschlicher DNA, obwohl die methodischen Ansätze ähnlich sind. Es bedarf einer auf den jeweiligen konkreten Fall bezogenen Prüfung, ob entsprechende Untersuchungen an Pflanzenresten aussichtsreich sind.
[Show abstract][Hide abstract] ABSTRACT: Eleven laboratories have collaborated to study chloroplast DNA (cpDNA) variation in black poplar (Populus nigra L.) across Europe in order to improve our understanding of the location of glacial refugia and the subsequent postglacial routes of recolonisation. A common analysis based on the restricted fragments produced by five primer pairs was used to determine the cpDNA haplotype of 637 samples obtained from genebank collections established in nine European countries. Haplotype 2 was particularly common and was found in 46% of the non-hybrid samples. A total of 81 non-hybrid chloroplast variants were detected. Three haplotypes (from four trees believed to originate from Eastern Europe) clustered together and were very different from the rest of the samples. The remaining samples were divided into two groups, one of which had a largely eastern distribution and samples from the other group were mostly located in the west. This, along with the fact that Spain in the southwest and Austria and Italy in the southeast had high diversity, suggest that there were ice age refugia of black poplar in both southwestern (Spain) and southeastern Europe (Italy and/or Balkan). Results also indicate that the Pyrenees formed a significant barrier, since only 7 of the 45 haplotypes in Spain exist elsewhere in Europe.
[Show abstract][Hide abstract] ABSTRACT: Microsatellites (SSRs) were surveyed to detect somatic mutations caused by chronic irradiation in Pinus sylvestris from Chernobyl (Ukraine). The analysed trees grow close to the exploded nuclear power plant. They survived the accident, but show morphological anomalies and reduced growth and vitality. Ionising radiation is a known mutagen which causes double-strand breaks of DNA in all living organisms. Using six SSR markers, one tree was found in which a mutation (deletion) was detected at the gene locus SPAG 7.14.
European Journal of Forest Research 10/2004; 123(3):245-248. DOI:10.1007/s10342-004-0026-6 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The spatial distribution of alleles is described in a naturally regenerated, isolated pure beech (Fagus sylvaticaL.) stand consisting of 99 adult trees. After testing nine microsatellite loci originally developed for F. crenata, each tree was genotyped at four well-scorable microsatellite loci. Specific primers were developed for one locus of F. sylvaticaL. For the characterization of spatial genetic structures, two different statistics were used. One method is based on the mean genetic distance between trees in different spatial distance classes, and the other one is Moran's index I. The results show the same tendency of a strong family structure in the distance classes up to 30m in comparison with that expected for a spatially non-systematic distribution of genotypes. In general, microsatellites are more useful to detect spatial genetic structures than allozymes. Spatial genetic structures are influenced by unpredictable factors such as wind direction at anthesis and can therefore vary from year to year. We recommend that seed collections should cover large areas in order to prevent a preponderance of few families and a reduction of the adaptive potential of the next generation.
[Show abstract][Hide abstract] ABSTRACT: Populus nigra L. is a pioneer tree species of riparian ecosystems that is threatened with extinction because of the loss of its natural habitat. To evaluate the existing genetic diversity of P. nigra within ex-situ collections, we analyzed 675 P. nigra L. accessions from nine European gene banks with three amplified fragment length polymorphism (AFLP) and five microsatellite [or simple sequence repeat (SSR)] primer combinations, and 11 isozyme systems. With isozyme analysis, hybrids could be detected, and only 3% were found in the gene bank collection. AFLP and SSR analyses revealed effectively that 26% of the accessions were duplicated and that the level of clonal duplication varied from 0% in the French gene bank collection up to 78% in the Belgian gene bank collection. SSR analysis was preferred because AFLP was technically more demanding and more prone to scoring errors. To assess the genetic diversity, we grouped material from the gene banks according to topography of the location from which the accessions were originally collected (river system or regions separated by mountains). Genetic diversity was expressed in terms of the following parameters: percentage of polymorphic loci, observed and effective number of alleles, and Nei's expected heterozygosity or gene diversity (for AFLP). Genetic diversity varied from region to region and depended, to some extent, on the marker system used. The most unique alleles were identified in the Danube region (Austria), the Rhône region (France), Italy, the Rijn region (The Netherlands), and the Ebro region (Spain). In general, the diversity was largest in the material collected from the regions in Southern Europe. Dendrograms and principal component analysis resulted in a clustering according to topography. Material from the same river systems, but from different countries, clustered together. The genetic differentiation among the regions (F(st)/G(st)) was moderate.