Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli

Fifty-one isolates of Rhizobium leguminosarum biovar phaseoli from various geographic and ecological sources, largely in Mexico, were characterized by the electrophoretic mobilities of 15 metabolic enzymes, and 46 distinctive multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at the enzyme loci. Mean genetic diversity per enzyme locus among the 46 ETs was 0.691, the highest value yet recorded for any species of bacterium. The occurrence of strong nonrandom associations of alleles over loci suggested a basically clonal population structure, reflecting infrequent recombination of chromosomal genes. Multilocus genotypic diversity was unusually high, with the most strongly differentiated pairs of ETs having distinctive alleles at all 15 loci and major clusters of ETs diverging at genetic distances as large as 0.89. This great diversity in the chromosomal genome raises the possibility that R. leguminosarum biovar phaseoli is a polyphyletic assemblage of strains. As other workers have suggested, the inclusion of all strains capable of nodulating beans in a single biovar or species is genetically unrealistic and taxonomically misleading. A biologically meaningful classification of Rhizobium spp. should be based on assessment of variation in the chromosomal genome rather than on phenotypic characters, especially those mediated for the most part or wholly by plasmid-borne genes, such as host relationships.     

Evaluating the utility of microsatellites for investigations of autopolyploid taxa

Autopolyploid taxa present numerous challenges for population genetic analyses due to difficulties determining allele dosage. Dosage ambiguity hinders accurate assessment of allele frequencies, multilocus genotypes (MLGTs), as well as levels and patterns of clonality. The pervasiveness of polyploidy in the evolutionary history of plant taxa makes this a recurring problem. Whereas diploidization of loci may occur over time, duplication of at least some loci is still frequently evident. Fortunately, with high-quality allozyme gels, it is possible to accurately infer allele dosage and, thus, determine exact MLGTs. However, accurately assessing dosage of microsatellite peaks is nearly impossible when studying wild populations with a large number of alleles per locus. Even if precise knowledge of genotypes is not required, for comparable numbers of alleles per locus and loci, the number of "phenotypes" is always lower with microsatellites than allozymes due to the inability to assess allele dosage. Microsatellite loci typically have more alleles per locus relative to allozymes although fewer loci are generally employed. Here, we present a mathematical model for comparing the relative utility of simple sequence repeat (SSR) versus allozyme markers to discriminate MLGTs. For example, the average plant allozyme study (2.6 alleles per locus, 10 polymorphic loci) has better discriminating power than SSR markers with 10 alleles at each of 3 loci, 9 alleles at 4 loci, 6 alleles at 5 loci, 5 alleles at 6 loci, and 4 alleles at 8 loci, demonstrating the value of assessing the relative discriminating power of these markers.     

Genetic relationships within the genus Prevotella analyzed by multilocus enzyme electrophoresis and DNA-DNA hybridization

The genetic diversity and relationships within the genus Prevotella were studied by analyzing twenty-five strains by multilocus enzyme electrophoresis (MLEE) at nine metabolic enzyme loci and DNA-DNA hybridization. MLEE revealed a high genetic diversity with 25 electrophoretic types (ETs) for the 25 strains studied, a mean number of alleles per enzyme locus of 6.8 and a mean genetic diversity per locus of 0.786. The index of association described by Maynard Smith et al. (1993) revealed a clonal structure within the genus Prevotella. A dendrogram generated by cluster analysis of a matrix of ETs showed that species like P. bivia, P. buccae, P. oris, P. oralis, P. nigrescens, and P. denticola form clusters that are consistent with DNA homologies. However, strains identified as P. melaninogenica or P. loescheii by DNA-DNA hybridization did not constitute distinct subpopulations in MLEE. MLEE analysis demonstrated its high power in differentiating closely related strains. It provides an alternative to 16S rRNA analysis for the study of phylogenetic relationships within the genus Prevotella, especially for differentiating strains with high DNA homology or high rRNA homology.     

Genetic characterization of Dirofilaria repens and D. immitis by electrophoretic analysis of gene-enzyme systems

A multilocus genetic analysis based on protein electrophoresis was carried out on 39 adult specimens of Dirofilaria repens and 31 of D. immitis. Seventeen enzymatic loci provided reliable electrophoretic patterns which were compared in the two species. All loci except one were monomorphic. Fixed alternative allozymes were found at 13 loci while only 3 loci shared apparently the same allozyme in the two species. The polymorphic locus, Pgm, also showed alternative allozymes. This remarkably high genetic divergence, which presumably reflects a very old speciation process, allows an easy characterization of D. repens and D. immitis and supports their classification in the subgenera Nochtiella and Dirofilaria, respectively.     

Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.     

Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers

Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.     

Patterns and levels of genetic differentiation in North American populations of the Alaskan wheatgrass complex

Levels and distribution of genetic variation were assessed using six allozymes in 27 populations of Alaskan wheatgrass (Elymus alaskanus) from different locations in Canada, USA, Greenland and Russia to obtain information on the genetic structure of these populations. The enzyme systems were ACO, DIA, GPI, MDH, PGM and SKD. Allozyme variation at the species level was high, with 64.3% (Ps) of the loci being polymorphic, an average number of alleles per locus of 1.9 (As), and an average genetic diversity of 0.17 (Hes). Differentiation was found in the populations studied, with the following findings: (1) statistically significant differences were found in allele frequencies among populations for every polymorphic locus (P < 0.001); (2) 63% of the total allozyme variation at polymorphic loci was partitioned among populations (GST = 0.63); (3) relatively low mean genetic distances between the populations were obtained (mean D = 0.029); (4) the genetic structure of Russian populations are clearly distinct from the other populations, the cluster and principal component analyses revealed the same genetic patterns of relationships among populations. This study also indicates that E. alaskanus contains different levels of allozyme variation in its populations. Furthermore, some banding patterns at the loci Aco-1, Aco-2, Gpi-2, Mdh-1, Skd-1, Skd-2 can be used as markers to identify individual populations.     

Genetic Diversity and Temporal Variation in the E. COLI Population of a Human Host

Electrophoretic techniques were employed to study variation in chromosomal genes encoding enzymes and in the distribution of cryptic plasmids in the E. coli population of a human host over an 11-month period. Thirteen of the 15 enzymes studied were polymorphic, and mean genetic diversity per locus was 0.39. Among 550 clones isolated from fecal samples, protein electrophoresis revealed 53 distinct electrophoretic types (ETs). Most ETs appeared on only one or a few days and were considered transients, but two (ET-12 and ET-13) were observed many times over extended periods and represented residents. Complete turnover in the transient ETs in the population occurred in periods of from two weeks to a month. ETs appearing in one month showed no particular genetic similarity to those of the previous month. — All but 4 of the 53 ETs carried one or more "cryptic" plasmids with molecular weights ranging from 1 to 80 megadaltons. With few exceptions, the plasmid composition of each ET was unique. In the course of the 11-month sampling period, there were changes in the plasmid profiles of the resident strains ET-12 and ET-13, and also in the profile of a recurrent strain, ET-2, which was isolated on four days. Modification of the plasmid profile of ET-12 involved the sequential addition of relatively high molecular weight bands. For ET-2 and ET-13, the changes in the plasmid profiles were radical, suggesting invasions of new cell types rather than merely the addition and deletion of plasmids. — The results of this study provide three lines of evidence that recombination plays a minor role in the generation of genetic diversity in the E. coli population of a single host. (1) Several pairs of loci were in strong linkage disequilibrium; compared to a randomly generated array of genotypes, the sample of ETs contained an excess of pairs differing at one or two loci and too many pairs with highly distinctive combinations of electromorphs. (2) In most cases where pairs of ETs differed at a single locus and, therefore, could reasonably have been generated by phage- or plasmid-mobilized gene transfer, the plasmid profiles of the pair members were radically different and/or the potentially transmitted alleles were not present in other ETs in the population. (3) Although ET-12 was abundant, being represented by 252 of the 550 clones sampled, the electrophoretic type most similar to ET-12 different from it at six loci, and ET-12 carried two unique alleles. We conclude that most of the genetic diversity observed in this human host is a consequence of successive invasions of E. coli genotypes.     

Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers

Abstract.— We examined genetic variation at 21 polymorphic allozyme loci, 15 nuclear DNA loci, and mitochondrial DNA in four spawning populations of sockeye salmon ( Oncorhynchus nerka ) from Cook Inlet, Alaska, to test for differences in the patterns of divergence among different types of markers. We were specifically interested in testing the suggestion that natural selection at allozyme loci compromises the effectiveness of these markers for describing the amount and patterns of gene flow among populations. We found concordance among markers in the amount of genetic variation within and among populations, with the striking exception of one allozyme locus ( sAH ), which exhibited more than three times the amount of among-population differentiation as other loci. A consideration of reports of discordance between allozymes and other loci indicates that these differences usually result from one or two exceptional loci. We conclude that it is important to examine many loci when estimating genetic differentiation to infer historical amounts of gene flow and patterns of genetic exchange among populations. It is less important whether those loci are allozymes or nuclear DNA markers.     

云南松居群遗传学研究的等位酶分析方法

针对１５个云南松Ｐｉｎｕｓｙｕｎｎａｎｅｎｓｉｓ居群,开展了１４种酶系统的水平切片淀粉凝胶电泳实验,在谱带遗传分析的基础上确定了３３个等位酶位点及其等位基因。其中有３２个等位酶位点是多态的（有２个以上的等位基因）,只有一个单态位点Ｄｉａ－４。有３个等位基因的位点有Ｌａｐ－１、Ｌａｐ－２、Ａａ－３、Ｓｋｄ－１、Ｓｋｄ－２、Ａｄｈ－１、Ａｄｈ－３、Ｇｄｈ、Ｐｇｄ－１、Ｐｇｍ－１、Ｐｇｍ－３、Ｐｇｉ－１、Ｐｇｉ－３、Ｍｄｈ－１、Ｍｅ、Ｇ６ｐｄ、Ｄｉａ－１、Ｔｐｉ－１、Ｔｐｉ－２、Ｔｐｉ－３和Ｔｐｉ－４,有４个等位基因的位点有Ｓｋｄ－３、Ａｄｈ－２、Ｐｇｄ－２、Ｍｄｈ－２、Ｍｄｈ－３、Ｍｄｈ－４和Ｄｉａ－２,有５个等位基因的位点有Ａａｔ－１和Ｄｉａ－３。云南松居群的等位基因平均数Ａ＝２１,在松属中居于中上水平。本研究揭示了云南松居群酶位点及其等位基因带谱的变异式样,为松属植物的遗传多样性研究提供了一批酶位点及其等位基因的参考图谱     

Genetic Variation and Relationships Within Taxus and Between the Genus and Pseudotaxus in China

In the present paper, genetic diversity of 12 populations of four Taxus taxa and one species of monotypic genus Pseudotaxus in China were studied by horizontal starch gel electrophoresis. Seven enzymes encoded by nine loci were assessed. Genetic diversity within 5 taxa were relatively high with the proportion of polymorphic loci ( P ) ranging from 44.4 % to 77.8 %, average number of alleles per locus (A) from 1.6 to 2.1; mean expected and observed heterozygosity ( He, Ho) per locus from 0. 065 to 0.152 and 0. 068 to 0.111, respectively. Combined with the analysis of other characters, the results from allozyme analysis suggest that there be very close relationships between the three species and one variety in Taxus, with high genetic identities (I) between them(from 0. 727 to 0. 995) except those between T. yunnanensis and other taxa in Taxus (0. 727～ 0. 804). Consequently, we propose that these taxa in Taxus be treated as different geographical races within a single species. Also the taxonomic position of Pseudotaxus as a distinct genus is supported by ourallozyme data.     

Microsatellite polymorphism and genetic differentiation in three Norwegian populations of Elymus alaskanus (Poaceae)

 Variation at seven microsatellite loci was investigated in three local E. alaskanus populations from Norway and microsatellite variation was compared with allozyme variation. The percentage of polymorphic loci was 81%, the mean number of alleles per polymorphic locus was 5.7 and expected heterozygosity was 0.37. An F-statistic analysis revealed an overall 48% deficit of heterozygotes over Hardy-Weinberg expectations. Gene diversity is mainly explained by the within population component. The averaged between population differentiation coefficient, F _st , over 7 loci is only 0.13, which accounts for only 13% of the whole diversity and was contrary to allozyme analysis. The mean genetic distance between populations was 0.12. However, a χ² -test showed that allele frequencies were different (p < 0.05) among the populations at 5 of the 7 loci. In comparison with the genetic variation detected by allozymes, microsatellite loci showed higher levels of genetic variation. Microsatellite analysis revealed that population H10576 possesses the lowest genetic variation among the tested three populations, which concur with allozyme analysis. The dendrogram generated by microsatellites agreed very well with allozymic data. Our results suggest that natural selection may be an important factor in shaping the genetic diversity in these three local E. alaskanus populations. Possible explanations for deficit heterozygosity and incongruence between microsatellites and allozymes are discussed. Received November 6, 2001; accepted April 24, 2002 Published online: November 14, 2002 Addresses of the authors: Genlou Sun (e-mail: Genlou.sun@STMARYS.CA), Biology Department, Saint Mary's University, Halifax. Nova Scotia, B3H 3C3, Canada. B. Salomon, R. von Bothmer, Department of Crop Science, The Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53, Alnarp, Sweden.     

Comparative electrophoretic polymorphism of esterases and other enzymes in Escherichia coli

The electrophoretic polymorphism of esterases was compared with that of other enzymes in Escherichia coli populations by investigating allozyme distribution of four esterases (A, B, C and I) within both the subspecific groups I, II and III and the new groups A, B1, B2, C, D and E, which have been distinguished by electrophoretic analysis of 11 and 35 enzymes respectively in the 72 reference strains of the ECOR collection. Electrophoretic distribution of esterases was distinct for each of the three subspecific groups as indicated by distributions of allozymes and electrophoretic types (distinctive combination of allozyme for the four esterases). Esterase polymorphisms of the three subspecific groups appeared to have similar features to those of three previously studied natural populations of strains obtained from human and animal gastro-intestinal tracts and extra-intestinal infections in humans. Multiple correspondence analyses using data obtained from the four esterases and the 11 other enzymes also distinguished the groups A, B1, B2, C, D and E. All strains of group B2 showed the B2 electrophoretic pattern of esterase B, which appeared to be a marker of a distinct cluster of strains frequently implicated in extra-intestinal infections.     

Inheritance of restriction fragment length polymorphisms and random amplified polymorphic DNAs in coastal Douglas-fir

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29 RAPD loci was demonstrated based on single-locus segregation in a sample of F₂ progeny. One RFLP locus, PtIFG2025, showed segregation distortion. Probe pPtIFG2025 is a loblolly pine cDNA probe encoding for rbcS. The 16 RFLP loci and 23 allozyme loci were also assayed in a sample of 16 Douglas-fir seed-orchard clones. Allelism was determined at 11 of the 16 RFLP loci. RFLPs were able to detect slightly more variation (4.0 alleles per locus) than allozymes (3.1 alleles per locus). The inheritance of an additional 80 RAPD loci was determined based on haploid segregation analysis of megagametophytes from parent tree 013-1. Once 200–300 markers are identified and placed on a genetic map, quantitative trait loci affecting bud phenology will be mapped.     

Evolutionary relationships among 12 species belonging to three genera of the family Microhylidae in Papua New Guinea revealed by allozyme analysis

To elucidate the potential of electrophoretic analysis for understanding relationships among microhylid frogs in Papua New Guinea, an allozyme analysis was conducted. A total of 119 individuals from nine species of Cophixalus, two species of Sphenophryne and one species of Barygenys, all of which belong to the family Microhylidae, were studied. Fourteen enzymes extracted from skeletal muscles and livers were analyzed by starch-gel electrophoresis. These enzymes were encoded by genes at 20 loci. There were 2-15 phenotypes produced by 2-12 alleles at these loci. The mean proportion of heterozygous loci per individual, mean proportion of polymorphic loci per population, and mean number of alleles per locus in 12 species were 6.1%, 17.1% and 1.17a on average, respectively. The NJ and ML trees constructed from Nei's genetic distances showed that the genus Sphenophryne can be distinguished biochemically from Cophixalus and Barygenys, and that the species groups of Cophixalus, which are similar in external morphology, can be divided biochemically into several species.     

The Study of Genic Variation by Electrophoretic and Heat Denaturation Techniques at the Octanol Dehydrogenase Locus in Members of the DROSOPHILA VIRILIS Group

Heat denaturation studies of three different electrophoretic allozymes of octanol dehydrogenase were performed from 10 species of the Drosophila virilis group. A total of 18 alleles were discovered, and in 3 species in which electrophoretic studies indicated that the locus was monomorphic, heat denaturation showed that the locus was polymorphic. We show that electrophoretic studies underestimate the number of alleles at this locus by a factor of 2.6 in these species. The results are discussed in the light of the continuing controversy over selection and neutral theories of genetic variation.     

Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, and outer membrane protein pattern.

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 genes encoding enzymes in 650 isolates of eight serogroups (A, B, C, W135, X, Y, Z, and 29E) and 38 nonserogroupable isolates. A total of 331 distinctive multilocus genotypes (electrophoretic types, ETs) was identified, among which mean genetic diversity per locus (H = 0.547) was greater than in Escherichia coli and other bacterial species thus far studied. The intercontinental distribution of some ETs and the recovery of organisms of identical genotype over periods of many years strongly suggest that the genetic structure of N. meningitidis is basically clonal as a consequence of low rates of recombination of chromosomal genes. Variation among strains in serogroup, serotype, and the electrophoretic pattern of the major outer membrane proteins has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at genetic distances greater than 0.50. Genetic diversity among ETs of isolates of the same serogroup was, on average, 84% of that in the total sample. Clones of serogroup A were unusual in being genotypically less heterogeneous than those of other serogroups and in forming a single phylogenetic group. Isolates of the same serotype or outer membrane protein pattern were also highly heterogeneous; on average, 87 and 97%, respectively, of the total species diversity was represented by ETs of the same serotype or outer membrane protein.     

Regional subdivision in wild barley allozyme variation: adaptive or neutral?

We examined the adaptive importance of allozyme variation in wild barley (Hordeum spontaneum). The test involved a nested sampling design with four population groups, each representing a different environment, and a comparison of observed allozyme variation with that expected under the assumption that allozymes are not neutral. Measurements of plant fitness in indigenous and alien environments in reciprocal introductions of seeds and seedlings in the four environments provided a guideline for the expected pattern of allozyme variation. The results showed considerable variation in both the degree of regional and population subdivision and the pattern of the subdivision among loci. The observed pattern of variation was ambiguous. Although two alleles exhibited a pattern of distribution that cannot be explained by genetic drift as a function of geographic distance, we failed to detect either a significant relationship between genetic distance and environmental similarity or any favored epistatic allele combinations across the four environments. Our results suggest that interpretation of allozyme variation in wild barley as adaptive and directly related to local environment still needs justification. Although we could not reject the null hypothesis, a proposed methodology seeking a concordance between observed and "adaptive" (i.e., expected under hypothesis that allozymes are not neutral) allozyme variation may prove to be effective in resolving the neutralist-selectionist debate when applied to other species.     

Genetic structure of an endangered plant, Antirrhinum microphyllum (Scrophulariaceae): allozyme and RAPD analysis

Thirteen allozyme loci and 68 random amplified polymorphic DNA (RAPD) markers were analyzed to assess the genetic diversity and population structure of threatened Antirrhinum microphyllum (Scrophulariaceae), a narrow endemic of central Spain known from only four populations. According to allozyme data, species genetic diversity (p = 46.15%, A = 2.61, and H(e) = 0.218), as well as within-population genetic diversity (p = 44.23%, A = 2.10, and H(e) = 0.204), were high when compared to average estimates for other narrowly distributed plant species. Ninety-four percent of species genetic diversity corresponded to within-population genetic diversity. Nevertheless, significant differences were found among populations in allele frequencies of four of the six polymorphic loci, and three private alleles were detected. Inbreeding coefficients (F(IS)) suggest that populations are structured in genetic neighborhoods. The RAPDs also showed high levels of genetic diversity (p = 89.71% and H(e) = 0.188 at the species level, and p = 67.65% and H(e) = 0.171 at the population level). Nei's genetic distances estimated both from allozymes and RAPDs indicated low differentiation among populations. In spite of this, the low frequencies of certain alleles and the presence of private alleles indicate that efforts should be made to conserve all four remaining populations.     

Comparison of allozyme,RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen

We examined genetic variation in allozyme loci, nuclear DNA restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs) in 130 trembling aspen (Populus tremuloides) and 105 bigtooth aspen (P. grandidentata) trees. In trembling aspen 10 out of 13 allozyme loci assayed (77%) were polymorphic (P), with 2.8 alleles per locus (A) and an expected heterozygosity (H_e) of 0.25. In contrast, bigtooth aspen had a much lower allozyme genetic variability (P=29%; A=1.4; H_e=0.08). The two species could be distinguished by mutually exclusive alleles at Idh-1, and bigtooth aspen has what appears to be a duplicate 6PG locus not present in trembling aspen. We used 138 random aspen genomic probes to reveal RFLPs in HindIII digests of aspen DNA. The majority of the probes were from sequences of low copy number. RFLP results were consistent with those of the allozyme analyses, with trembling aspen displaying higher genetic variation than bigtooth aspen (P=71%, A=2.7, and H_e=0.25 for trembling aspen; P=65%, A=1.8, and H_e=0.13 for bigtooth aspen). The two species could be distinguished by RFLPs revealed by 21 probes (15% of total probes assayed). RAPD patterns in both species were studied using four arbitrary decamer primers that revealed a total of 61 different amplified DNA fragments in trembling aspen and 56 in bigtooth aspen. Assuming a Hardy-Weinberg equilibrium, estimates of P=100%, A=2, and H_e=0.30 in trembling aspen and P=88%, A=1.9, and H_e=0.31 in bigtooth aspen were obtained from the RAPD data. Five amplified DNA fragments were species diagnostic. All individuals within both species, except for 2 that likely belong to the same clone, could be distinguished by comparing their RAPD patterns. These results indicate that (1) RFLPs and allozymes reveal comparable patterns of genetic variation in the two species, (2) trembling aspen is more genetically variable than bigtooth aspen at both the allozyme and DNA levels, (3) one can generate more polymorphic and species-specific loci with DNA markers than with allozymes in aspen, and (4) RAPDs provide a very powerful tool for fingerprinting aspen individuals.