Nuclear Restriction Fragment Length Polymorphisms Research Articles

Molecular Support for the Hybrid Origin of the Wild Potato Species Solanum × rechei

Twenty‐seven of the 232 wild potato species (Solanum sect. Petota) have been hypothesized to be of natural hybrid origin. Prior molecular data have failed to support hybrid origins involving two other wild potato species, Solanum raphanifolium Cárdenas and Hawkes and S. chacoense Bitter, and hybrid speciation has never been supported with molecular data in sect. Petota. This study was conducted to test the hybrid origin of Solanum × rechei Hawkes and Hjert. It is a locally common and weedy wild potato species from Argentina, occurring at the extreme southern end of the range of S. microdontum Wittm., and near the northern end of the range of S. kurtzianum Bitter, its two putative parents. Solanum × rechei is diploid (2n = 2x = 24) with triploid (2n = 3x = 36) populations, S. kurtzianum is diploid (2n = 2x = 24), and S. microdontum is diploid (2n = 2x = 24), with triploid populations at its extreme southern range. A prior study supported the hybrid origin of S. × rechei by intermediate morphology of natural and synthetic hybrids, reduced pollen stainability of the natural and synthetic hybrids, and distributional evidence. Our studies of new collections and prior germplasm collections fail to support the morphological intermediacy of S. × rechei, but lack of morphological intermediacy is common for many hybrids. Hybrid origin was instead verified by reduced pollen stainability and additive parent‐specific single‐ to low‐copy nuclear restriction fragment length polymorphisms (RFLPs) in S. × rechei. These data suggest that other wild potato species also may be of hybrid origin, which may help explain some of the taxonomic confusion in the group.

Genetic diversity within and among maize populations: a comparison between isozyme and nuclear RFLP loci

In order to compare the potential of enzyme and DNA markers to investigate genetic diversity within and among populations, ten maize populations were characterized for (1) 20 isozyme loci and (2) restriction fragment length polymorphism (RFLP) for 35 probe-enzyme combinations. Each population was represented by a sample of at least 30 individuals. The average number of alleles detected per locus was clearly higher for RFLPs (6.3) than for isozymes (2.4). Similarly, total diversity was higher for RFLPs (0.60) than for isozymes (0.23). This difference is consistent with observations on inbred-line collections and can be related to the fact that many variations at the DNA level do not change the amino-acid composition or the global charge of proteins. By contrast, the magnitude of population differentiation, relative to the total diversity, was similar for isozymes (23%) and RFLPs (22%). This suggests that the isozyme and RFLP loci considered in this study are subject to similar evolutionary forces, and that both are mostly neutral. However, RFLPs proved clearly superior to isozymes both to (1) identify the origin of a given individual and (2) reveal a relevant genetic structure among populations. The higher polymorphism observed for RFLP loci and the greater number of these loci contributed to the superior discriminative ability of the RFLP data.

The genetic structure of sea beet (Beta vulgaris ssp. maritima) populations. III. Detection of isolation by distance at microsatellite loci

Variation at three microsatellite loci was investigated in five populations of sea beet on the Dorset coast. Genetic variation among the populations was estimated using two different methods. Estimates of FST were obtained using the program FSTAT, and estimates of RST were obtained using the program AMOVA. These different estimates of genetic distance were used to test for isolation by distance with partial matrix correspondence tests and were compared with patterns of variation in FST estimates from isozymes and single-copy nuclear restriction fragment length polymorphisms (RFLPs). FST from isozymes, FST from RFLPs and RST from microsatellites all showed evidence of isolation by distance, whereas FST from microsatellites showed strong similarities within populations that were not related to distance. These results suggest that, compared with isozymes and RFLPs, mutation rates at microsatellite loci are high in these populations. They also suggest that stepwise mutation processes are occurring at these loci. The data provide valuable evidence that all three types of marker can detect similar patterns of population structure. The results are discussed with respect to estimates of genetic distance at microsatellite loci in human populations.

Genetics of adaptive radiation in Hawaiian and Cook Islands species of Tetramolopium (Asteraceae; Astereae). I. Nuclear RFLP marker diversity

Thirty-three nuclear RFLP (restriction fragment length polymorphism) probes were used to study genetic diversity in Hawaiian and Cook Islands species of Tetramolopium for comparison with previous morphological and isozyme studies and to provide greater resolution of the events associated with adaptive radiation in the genus. Levels of RFLP diversity are greater than those reported for isozymes, yet are still low in comparison to continental species. Genetic differentiation is greatest among species in sections rather than among sections and is concordant with the hypothesis of phyletic sorting of initial variability as suggested for morphological traits. Hypothesized introgression between T. lepidotum and T. filiforme is supported, but the evidence suggests bidirectional gene flow. Systematic relationships derived from the data agree with hypotheses based on morphology in the placement of populations within their respective species and the recognition of three main lineages within Hawaii. Inclusion of the Cook Islands species, however, renders section Tetramolopium paraphyletic, contradicting morphological, ecological, and crossing evidence. Interpreting these results in light of evidence from previous studies, the genetic diversity and relationships seen among species and sections of Hawaiian and Cook Islands Tetramolopium reflect the recent and rapid evolution of this group, limited addition of new variability, and phyletic sorting.

Predominant Paternal Transmission of the Mitochondrial Genome in Cucumber

Segregation analyses of restriction fragment length polymorphisms (RFLPs) in cucumber revealed one paternally inherited Pstl genomic clone, possibly from either the mitochondrial (mt) or chloroplast (cp) genome. Polymorphisms in the mtDNA were identified with clones apocytochrome B and atp9. No polymorphisms were detected in the cpDNA using restriction enzymes that recognize 6 and 4 bp sequences. Two pairs of reciprocal crosses were made between individual plants of the breeding line Gy14 and cultivar Wisconsin SMR18 with USDA plant introduction 432860. A nuclear RFLP was biparentally inherited and established the hybrid origin of all progenies. For 68 of 70 progenies (97%), the mt genome of cucumber showed paternal transmission. Two hybrid plants possessed both parental fragments for one of the two polymorphic probe enzyme combinations, likely due to mitochondrial heterogeneity in the pollen parent.

Gene Flow and Sexual Reproduction in the Wheat Glume Blotch Pathogen Phaeosphaeria nodorum (Anamorph Stagonospora nodorum)

ABSTRACT Restriction fragment length polymorphisms (RFLPs) were used to characterize the genetic structures of three field populations of Phaeosphaeria nodorum from Texas, Oregon, and Switzerland. Data from seven nuclear RFLP loci were used to estimate gene diversity and genetic distances and to make indirect measures of gene flow between populations. Three of the seven RFLP loci differed significantly in allele frequencies across populations. On average, 96% of the total gene diversity was found within populations. There was little evidence for population subdivision, suggesting that gene flow was not restricted among populations. Based on an average population differentiation of 0.04, we estimated that the exchange of 11 migrants among populations per generation would be needed to account for the present level of population subdivision. Genotype diversity based on DNA fingerprints was at a maximum for the Swiss population, whereas populations in Texas and Oregon had lower genotype diversities. Many multilocus haplotypes were found in each population. Ninety-five percent of RFLP allele pairs were in gametic equilibrium. The data were consistent with random mating within each population.

RFLP and Cytogenetic Evidence on the Origin and Evolution of Allotetraploid Domesticated Peanut, Arachis hypogaea (Leguminosae)

Nuclear restriction fragment length polymorphism (RFLP) analysis was used to determine the wild diploid Arachis species that hybridized to form tetraploid domesticated peanut. Results using 20 previously mapped cDNA clones strongly indicated A. duranensis as the progenitor of the A genome of domesticated peanut and A. ipaensis as the B genome parent. A large amount of RFLP variability was found among the various accessions of A. duranensis, and accessions most similar to the A genome of cultivated peanut were identified. Chloroplast DNA RFLP analysis determined that A. duranensis was the female parent of the original hybridization event. Domesticated peanut is known to have one genome with a distinctly smaller pair of chromosomes (“A”), and one genome that lacks this pair. Cytogenetic analysis demonstrated that A. duranensis has a pair of “A” chromosomes, and A. ipaensis does not. The cytogenetic evidence is thus consistent with the RFLP evidence concerning the identity of the progenitors. RFLP and cytogenetic evidence indicate a single origin for domesticated peanut in Northern Argentina or Southern Bolivia, followed by diversification under the influence of cultivation.

RFLP AND CYTOGENETIC EVIDENCE ON THE ORIGIN AND EVOLUTION OF ALLOTETRAPLOID DOMESTICATED peanut,Arachis hypogaea(Leguminosae)

Nuclear restriction fragment length polymorphism (RFLP) analysis was used to determine the wild diploidArachisspecies that hybridized to form tetraploid domesticated peanut. Results using 20 previously mapped cDNA clones strongly indicatedA. duranensisas the progenitor of the A genome of domesticated peanut andA. ipaensisas the B genome parent. A large amount of RFLP variability was found among the various accessions ofA. duranensis, and accessions most similar to the A genome of cultivated peanut were identified. Chloroplast DNA RFLP analysis determined thatA. duranensiswas the female parent of the original hybridization event. Domesticated peanut is known to have one genome with a distinctly smaller pair of chromosomes (“A”), and one genome that lacks this pair. Cytogenetic analysis demonstrated thatA. duranensishas a pair of “A” chromosomes, andA. ipaensisdoes not. The cytogenetic evidence is thus consistent with the RFLP evidence concerning the identity of the progenitors. RFLP and cytogenetic evidence indicate a single origin for domesticated peanut in Northern Argentina or Southern Bolivia, followed by diversification under the influence of cultivation.

Genetic structure of the global population of banana black leaf streak fungus, Mycosphaerella fijiensis

The genetic structure of Mycosphaerella fijiensis populations around the world was examined using DNA restriction fragment length polymorphism (RFLP) markers. Allele frequencies at 19 nuclear RFLP loci were estimated in a sample of 136 M. fijiensis isolates from five geographical populations representative of banana‐producing areas (South‐East Asia including the Philippines and Papua New Guinea, Africa, Latin America and Pacific Islands). Within each population, gametic disequilibrium tests between the 19 nuclear RFLP loci were mainly non significant (P > 0.05), indicating that random sexual reproduction occurred in these populations. All M. fijiensis populations had a high level of genotypic and allelic diversity (H, gene diversity: 0.25–0.59). The highest levels of gene diversity were found in the two South‐East Asian populations (H: 0.57 and 0.59). Most of the alleles (> 88%) detected in Africa, Latin America and Pacific Islands populations were also detected in South‐East Asian populations. Furthermore, a high and significant (P < 0.05) level of genetic differentiation was observed among M. fijiensis geographical populations (overall estimate of Fst: 0.32). These results were consistent with the hypothesis that M. fijiensis originated in South‐East Asia and spread recently to other parts of the world. The level of allelic diversity in M. fijiensis populations from regions other than South‐East Asia was drastically reduced, indicating founder effects. The data also suggested rare occurrence of migration of M. fijiensis between continents.

Genetic structure of the global population of banana black leaf streak fungus, Mycosphaerella fijiensis

AbstractThe genetic structure of Mycosphaerella fijiensis populations around the world was examined using DNA restriction fragment length polymorphism (RFLP) markers. Allele frequencies at 19 nuclear RFLP loci were estimated in a sample of 136 M. fijiensis isolates from five geographical populations representative of banana‐producing areas (South‐East Asia including the Philippines and Papua New Guinea, Africa, Latin America and Pacific Islands). Within each population, gametic disequilibrium tests between the 19 nuclear RFLP loci were mainly non significant (P > 0.05), indicating that random sexual reproduction occurred in these populations. All M. fijiensis populations had a high level of genotypic and allelic diversity (H, gene diversity: 0.25–0.59). The highest levels of gene diversity were found in the two South‐East Asian populations (H: 0.57 and 0.59). Most of the alleles (> 88%) detected in Africa, Latin America and Pacific Islands populations were also detected in South‐East Asian populations. Furthermore, a high and significant (P < 0.05) level of genetic differentiation was observed among M. fijiensis geographical populations (overall estimate of Fst: 0.32). These results were consistent with the hypothesis that M. fijiensis originated in South‐East Asia and spread recently to other parts of the world. The level of allelic diversity in M. fijiensis populations from regions other than South‐East Asia was drastically reduced, indicating founder effects. The data also suggested rare occurrence of migration of M. fijiensis between continents.

Restriction Fragment Length Polymorphisms Reveal Considerable Nuclear Divergence within a Well-Supported Maternal Clade in Allium Section Cepa (Alliaceae)

Maternal phylogenies estimated by restriction fragment length polymorphisms (RFLPs) in the chloroplast DNA (cpDNA) delineated a well-supported clade containing Allium altaicum, A. cepa (bulb onion), A. fistulosum (Japanese bunching onion), A. galanthum, and A. vavilovii. Few polymorphic restriction-enzyme sites were detected among the wild and cultivated species within this clade, and relationships could not be confidently estimated. Random nuclear RFLPs revealed considerable variation among these species and three distinct groups were identified (Altaicum, Cepa, and Galanthum). Relationships were estimated using principal components and cluster analyses of the Jaccard's similarity matrix. For five out of six analyses, nuclear phylogenies estimated by UPGMA and neighbor joining of the Jaccard's similarity matrix produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, and A. galanthum, disagreeing with maternal phylogenies that produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, A. cepa, and A. vavilovii. Allium oschaninii was closely related to A. galanthum and these two species may represent the progenitor types. Overall, restriction-enzyme analyses of the nuclear and cpDNA produced few synapomorphies among closely related species in Allium section Cepa.

Mitochondrial DNA diversity in wild and cultivated sorghum.

Cultivated sorghum (Sorghum bicolor ssp. bicolor) is classified into five main races on the basis of spikelet morphology. Isozyme analyses provided new insight into the genetic diversity of sorghum and revealed marked geographic grouping, while nuclear restriction fragment length polymorphisms showed racial differentiation and intraguinea race differentiation. Wild sorghum is diploid or tetraploid and African sorghum (S. bicolor ssp. arundinaceum) is classified into four races, that are considered to be progenitors of cultivated sorghum. We performed mitochondrial DNA analyses to compare the diversity of wild and cultivated sorghum and to study the genetic origin of guinea margaritiferum. The same overall patterns were obtained with the different phenogram construction techniques. Our results confirmed the specificity of guinea margaritiferum and demonstrated the presence of two genetic entities within this subrace. Another guinea group was also noted, which corresponded to Asian guinea roxburghii. In wild sorghum, the arundinaceum race appeared to be homogenous, while the verticilliflorum race was separated into two groups, one of which was associated with the arundinaceum race. The diversity observed in cultivated forms was found to be encompassed within the wild pool, except for one guinea margaritiferum group. There did not seem to be any particular relationship between wild races and cultivated races.

Genetic Variation and Mapping of the Carrot Nuclear and Chloroplast Genomes

Studies of genetic variation at the DNA level in the genus Dacus have been very limited. Molecular markers based on restriction fragment length polymorphism (RFLP) have been highly useful and efficient gene markers in other plant species. We have evaluated the chloroplast genome of 19 Dacus species and inbreds cut with 10 restriction enzymes and probed with 14 petunia chloroplast clones. A phylogenetic tree generated to date shows three major groupings of these accessions as seen with data generated by an earlier study using nuclear RFLPs. However, D. glochidiatus and D. guttatus are in different groups. Details of the chloroplast study will be discussed. Also, a genetic linkage map of carrot will be constructed. The map will be used to determine the genomic regions conditioning traits such as pigmentation root and core diameter, and root length.

Similarities and relationships among populations of the bulb onion as estimated by nuclear RFLPs

Random nuclear restriction fragment length polymorphisms (RFLPs) were used to assess similarities and relationships among open-pollinated (OP) populations of the cultivated bulb onion (Allium cepa). Seventeen OP populations and 2 inbreds of contrasting daylength response [termed by convention as long (LD) and short (SD) day], 1 shallot (A. cepa var. ascalonicum), and one cultivar of bunching onion (Allium fistulosum) were examined with 104 cDNA clones and two to four restriction enzymes. Sixty (58%) clones detected at least 1 polymorphic fragment scorable among the OP populations and were used for analyses. The average number of polymorphic fragments per polymorphic probe-enzyme combination was 1.9, reflecting that numerous monomorphic fragments were usually present. Similarities were estimated as the proportion of polymorphic fragments shared by 2 populations. Average similarity values among LD, among SD, and between LD and SD OP populations were 0.79, 0.67, and 0.68, respectively. Relationships among the OP populations were estimated by parsimony, cluster analysis of similarities using the unweighted-pair-group method (UPGMA), and multivariate analysis using principle components. Parsimony analysis generated a strict consensus tree that grouped all but 1 LD onion with unresolved relationships to the SD OP populations. The UPGMA analysis placed together the LD storage OP populations. Principal component analysis grouped all but 2 LD onions; the other OP populations were dispersed. The results suggest that LD and SD onions do not represent distinct germ plasm, but that LD storage onions represent a derived group selected for production at higher latitudes. If it is assumed that the sampled populations are representative of all onion OP populations, the lower similarities among SD OP populations indicate that their collection and maintenance in germ plasm collections is important for the preservation of genetic diversity.

Genetic population structure and gene flow in the Atlantic cod Gadus morhua: a comparison of allozyme and nuclear RFLP loci.

High levels of gene flow have been implicated in producing uniform patterns of allozyme variation among populations of many marine fish species. We have examined whether gene flow is responsible for the limited population structure in the Atlantic cod, Gadus morhua L., by comparing the previously published patterns of variation at 10 allozyme loci to 17 nuclear restriction fragment length polymorphism (RFLP) loci scored by 11 anonymous cDNA clones. Unlike the allozyme loci, highly significant differences were observed among all populations at the DNA markers in a pattern consistent with an isolation-by-distance model of population structure. The magnitude of allele frequency variation at the nuclear RFLP loci significantly exceeded that observed at the protein loci (chi 2 = 24.6, d.f. = 5, P < 0.001). Estimates of gene flow from the private alleles method were similar for the allozymes and nuclear RFLPs. From the infinite island model, however, estimates of gene flow from the DNA markers were fivefold lower than indicated by the proteins. The discrepancy between gene flow estimates, combined with the observation of a large excess of rare RFLP alleles, suggests that the Atlantic cod has undergone a recent expansion in population size and that populations are significantly displaced from equilibrium. Because gene flow is a process that affects all loci equally, the heterogeneity observed among populations at the DNA level eliminates gene flow as the explanation for the homogeneous allozyme patterns. Our results suggest that a recent origin of cod populations has acted to constrain the extent of population differentiation observed at weakly polymorphic loci and implicate a role for selection in affecting the distribution of protein variation among natural populations in this species.

DNA science and conservation

A wide array of DNA-based genetic techniques are now available for the study of many problems in conservation biology. Either directly or indirectly, such techniques are becoming increasingly available to scientists and managers alike. Although these technologies are generally known to conservationists, there is a need to clearly outline the principal characteristics of such genetic tools and to detail how they can most appropriately be used in the management of wildlife species. The essential characteristics of mitochondrial and chloroplast restriction fragment analyses are detailed, together with discussions of single locus nuclear restriction fragment length polymorphisms (RFLPs), multilocus DNA fingerprinting, microsatellite DNA, randomly amplified polymorphic DNAs (RAPDs), and DNA sequence variation. We also give relevant information about the development times for these techniques, their relative costs, and the quality of tissue required. In addition, we discuss which conservation problems are appropriate to each of these methods and give examples of their application and potential use in relation to New Zealand organisms. The following problems are considered: sex assignment; parentage and kinship; migration; species, population and strain identification; forensic applications; genetic effects of population bottlenecks; disease identification; feeding preferences; philopatry; pest control; and understanding population extinction. Finally, we suggest that both microsatellite and minisatellite DNA techniques have particular advantages over many other currently-available techniques and conclude that these two approaches are applicable to a wide range of the conservation problems.

A study of genetic variation and evolution of Phyllostachys (Bambusoideae: Poaceae) using nuclear restriction fragment length polymorphisms

Phylogenetic and taxonomic difficulties are common within the woody bamboos, due to their unique life cycle, which severely limits the availability of floral characters. To addresss some of these problems, 20 species of woody bamboos in the genus Phyllostachys were analyzed using nuclear restriction fragment length polymorphisms (RFLPs). The RFLP data were used to generate genetic distances between all pairs of taxa and to examine the degree of genetic variation within and among bamboo species. The genetic distances were also used to create dendrograms of accessions and species. These trees supported the current division of the genus into two sections and provided some information on the thorny taxonomic problems in this group. We show that RFLPs can be used for species identification and the delineation of species limits.

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.

Allozyme and RFLP heterozygosities as correlates of growth rate in the scallop Placopecten magellanicus: a test of the associative overdominance hypothesis.

Several studies have reported positive correlations between the degree of enzyme heterozygosity and fitness-related traits. Notable among these are the correlations between heterozygosity and growth rate in marine bivalves. Whether the correlation is the result of intrinsic functional differences between enzyme variants at the electrophoretic loci scored or arises from non-random genotypic associations between these loci and others segregating for deleterious recessive genes (the associative overdominance hypothesis) is a matter of continuing debate. A prediction of the associative overdominance hypothesis, not shared by explanations that treat the enzyme loci as causative agents of the correlation, is that the correlation is not specific to the type of genetic marker used. We have tested this prediction by scoring heterozygosity at single locus nuclear restriction fragment length polymorphisms (RFLPs) in a cohort of juvenile scallops (Placopecten magellanicus) in which growth rate was known to be positively correlated with an individual's degree of allozyme heterozygosity. A total of 222 individuals were scored for their genotypes at seven allozyme loci, two nonspecific protein loci of unknown function and eight nuclear RFLPs detected by anonymous cDNA probes. In contrast to the enzyme loci, no correlation was observed between growth rate and the degree of heterozygosity at the DNA markers. Furthermore, there was no relationship between the magnitude of heterozygote deficiency at a locus and its effect on the correlation. The differences observed between the effects of allozyme and RFLP heterozygosity on growth rate provide evidence against the associative overdominance hypothesis, but a strong case against this explanation must await corroboration from similar studies in different species.

A Reexamination of Species Boundaries and Hypotheses of Hybridization Concerning Solanum megistacrolobum and S. toralapanum (Solanum sect. Petota, series Megistacroloba): Molecular Data

Solanum megistacrolobum and S. toralapanum are two phenetically similar wild potato (Solanum sect. Petota) species, classified in series Megistacroloba, that together are distributed from southern Peru to northwestern Argentina. They have variously been synonymized, recognized as varieties of S. megistacrolobum, or recognized as distinct species. We used 22 singleto low-copy random genomic DNA probes from potato, hybridized to total DNA digested with Dral, EcoRI, EcoRV, and HindIII, to investigate their taxonomic status. We also investigated the hybrid origin of S. raphanifolium and the hybrid origin of S. acaule subsp. aemulans. Our results are concordant with a separate morphological study, showing weak differentiation between S. megistacrolobum and S. toralapanum, but possible only with multivariate methods. These combined morphological and molecular results most closely fit the contemporary treatment of S. megistacrolobum and S. toralapanum at the varietal level. We propose the new combination S. megistacrolobum subsp. toralapanum in order to bring consistency to taxa within sect. Petota, where different authors recognize identical taxa as varieties or subspecies. Our results also show possible hybridization between S. megistacrolobum and S. acaule subsp. aemulans in Argentina, discount the hybridization hypothesis of S. raphanifolium, and show that the species-specific bands of S. albicans relative to S. acaule are shared with many other species in sect. Petota. Recent advances in molecular biology have provided new tools for systematic questions. At the species level or below, restriction fragment length polymorphisms (RFLP's) have provided powerful new data to assess phenetic similarity. Combined with morphological data, RFLP studies are enabling researchers to answer questions not possible with previous methods. Beckman and Soller (1986a, 1986b), Tanksley (1983), and Whitkus et al. (in press) have described many of the useful properties of RFLP's. These include ubiquity, stability, Mendelian codominant inheritance, the presence of multiple alleles for a given locus, and freedom from pleiotropic effects. Additionally, RFLP's are detectable in all tissues at all developmental stages. This, coupled with the relatively long shelf life of DNA, makes RFLP analysis a convenient method for assessing genetic variation and lower-rank systematic questions. This study employs single-to low-copy nuclear DNA probes to generate nuclear restriction fragment length polymorphisms (nRFLP's) to investigate hypotheses of species boundaries and hybridization involving Solanum megistacrolobum Bitter and S. toralapanum Card. and Hawkes. These are closely related and phenetically similar wild potato taxa (Solanum L. sect. Petota Dumort.) distributed from southern Peru to northwestern Argentina. Both are classified in series Megistacroloba, a group of diploid (2n = 24), self-incompatible, and largely interfertile species (Buck 1966; Hawkes 1990; Ochoa 1990; Pandey 1960). There is much intra-and interspecific morphological variability between S. megistacrolobum and S. toralapanum. These two taxa have been taxonomically treated as conspecific (Ochoa 1984), varieties of a single species (Ochoa 1990), or as distinct species (Correll 1962; Gorbatenko 1989; Hawkes 1990). Previous studies have shown that nRFLP's can result from both structural changes (i.e., insertions, deletions, inversions) and site mutations. Data distinguishing these alternatives have come from inferences based on comparisons of numbers of nRFLP patterns and length of areas being probed, or on recurrent patterns of polymorphisms using the same probe with different enzymes (Neuhausen 1992; Wang and Tanksley 1989; Wang et al. 1992). The only way