Nuclear Restriction Fragment Length Polymorphisms Research Articles

Genetic entities and hybridisation within the Acacia microbotrya species complex in Western Australia

Species complexes are often taxonomically challenging because the evolutionary relationships among closely related taxa may not be well reflected by morphological divergence. Molecular data can be a powerful tool in resolving these ambiguities. Using nuclear restriction fragment length polymorphisms (RFLPs), we examined genetic variation among 22 populations of the Acacia microbotrya complex and assessed its congruence with five taxa previously identified using morphological criteria. Four distinct genetic clusters were consistently detected across multiple analytical approaches (Bayesian, UPGMA, maximum likelihood, and principle coordinate). These genetic clusters were strongly congruent with each of four morphologically defined taxa, Acacia amblyophylla, Acacia daphnifolia, A. microbotrya and A. microbotrya (Dandaragan variant). From this, we suggest that the Dandaragan variant warrants formal recognition at the same taxonomic level as the other three taxa. Individuals of the fifth morphologically defined species, Acacia splendens, were not consistently assigned to any one genetic cluster having affinities with A. microbotrya (Dandaragan variant) and A. amblyophylla, although at the population level it showed closest affinity to A. microbotrya (Dandaragan variant). Despite strong genetic and morphological differentiation of these taxa in allopatric regions, we detected a hybrid zone of genetic admixture between A. microbotrya and A. daphnifolia where their geographic ranges overlap. Together, these results highlight a complex evolutionary history of fragmentation, divergence and hybridisation in the A. microbotrya complex. While further work is required to resolve the phylogenetic status of A. splendens, this study provides compelling evidence for the recognition of these four genetic clusters as distinct evolutionary entities in the A. microbotrya complex that should be treated as separate units for conservation, commercial or management purposes.

Rapid genetic identification of brown bullhead (Ameiurus nebulosus), black bullhead (Ameiurus melas) and their hybrids

Hybridization can complicate the effective conservation of cryptic and morphologically similar species. Here we provide a rapid genetic method for the identification of pure-strain brown bullhead (Ameiurus nebulosus) and black bullhead (Ameiurus melas) using nuclear and mitochondrial DNA restriction fragment length polymorphism assays. We confirm earlier reports of introgressive hybridization among A. nebulosus and A. melas in Lake Erie, and reveal the presence of introgressed hybrids in Detroit River. Our method provides a prime example of the utility of internet-based genetic databases for the development of molecular tools in biological and environmental research, and suggests hybridization plays a larger role in the genetic structure of bullheads.

DNA Markers in Eucalyptus with Emphasis on Species Identification

I review the current knowledge on molecular markers for Eucalyptus species, with particular emphasis on individual identification and the prevention of escape of transgenes from transgenic Eucalyptus. Species-specific bands have been found in inter simple sequence repeat (ISSR) markers. Microsatellites are highly polymorphic genetic regions that allow precise discrimination of closely related individuals. Random amplified polymorphic DNA (RAPD) markers are also very useful for individual identification and map construction, although they have limited transferability because they are dominant markers with only two alleles. The amplified fragment length polymorphism (AFLP) technique facilitates the production of high-coverage parental maps in interspecific crosses of eucalypts. Genetic diversity in the continuously distributed Eucalyptus species has been investigated using nuclear restriction fragment length polymorphism (RFLP) loci. Chloroplast DNA, 5S rDNA genes, and ITS regions have proven to be useful in phylogenetic, population, and evolutionary studies of Eucalyptus. Genetic association studies using SNPs are in progress with several Eucalyptus species. Furthermore, microarray technology holds the promise of parallel analysis of thousands of markers in Eucalyptus. Early screening of hybrids with transgenic Eucalyptus using DNA markers will be important to prevent escaping transgenes.

Concordant evolution of mitochondrial and nuclear genomes in the wheat pathogen Phaeosphaeria nodorum

We compared patterns of mitochondrial restriction fragment length polymorphism (RFLP) diversity with patterns of nuclear RFLP diversity to investigate the effects of selection, gene flow, and sexual reproduction on the population genetic structure and evolutionary history of the wheat pathogen Phaeosphaeria nodorum. A total of 315 fungal isolates from Texas, Oregon, and Switzerland were analyzed using seven nuclear RFLP probes that hybridized to discrete loci and purified mitochondrial DNA that hybridized to the entire mtDNA genome. Forty-two different mitochondrial haplotypes and 298 different nuclear haplotypes were detected. The two most frequent mtDNA haplotypes were present in every population and represented 32% of all isolates. High levels of gene flow, low levels of population subdivision, no evidence for either host specificity or cyto-nuclear disequilibrium were inferred from the analysis of both genomes. The concordance in estimates of these population genetic parameters from both genomes suggests that the two genomes experienced similar degrees of migration, genetic drift and selection.

Origin and evolution of Andigena potatoes revealed by chloroplast and nuclear DNA markers

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are important, native-farmer-selected cultivars in the Andes, which form a primary gene pool for improving a worldwide grown potato (S. tuberosum subsp. tuberosum). To elucidate the origin of Andigena, 196 Andigena accessions were compared with 301 accessions of 33 closely related cultivated and wild species using several types of chloroplast DNA (ctDNA) markers and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Fourteen ctDNA types (haplotypes) and 115 RFLP bands were detected in Andigena, of which the main haplotypes and frequent RFLP bands were mostly shared with a cultivated diploid species, S. stenotomum Juz. et Buk. Principal component analysis of nDNA polymorphisms revealed a progressive and continuous variation from Peruvian wild species with C-type ctDNA to a group of wild species having S-type ctDNA in its variation range (S. bukasovii, S. canasense, S. candolleanum, and S. multidissectum), to cultivated diploid potatoes (S. phureja and S. stenotomum), and to cultivated tetraploid potatoes (Andigena and Chilean S. tuberosum subsp. tuberosum). These results suggest that the initial Andigena population arose with multiple origins exclusively from S. stenotomum. The overall evolutionary process toward the present-day Andigena was discussed.

Genetic differentiation among morphological variants of Acacia saligna (Mimosaceae)

This study investigated the pattern of genetic variation in Acacia saligna (Labill.) H.L. Wendl. to facilitate its development as a crop species for dryland salinity management. A. saligna is a morphologically variable species and four main variants are recognized. The genetic structure within A. saligna was investigated in populations across the geographic range of the species using nuclear restriction fragment length polymorphism loci. The analysis identified considerable genetic variation within A. saligna that was genetically structured into three groups. Two of the three groups corresponded to variants recognized in the field study; the third group encompassed the other two variants which, though morphologically different, were not genetically differentiated. The level of genetic differentiation between the groups suggests they may represent different taxa and a taxonomic revision of the species may be required. Identification of different taxa within A. saligna will have implications for the utilization and domestication of the species, as the taxa will need to be evaluated separately to determine their suitability for agroforestry. The high genetic variation between and within groups suggests there is a large genetic base available for breeding improved cultivars.

Genetic Variation and Management of Striped Bass Populations in the Coastal Rivers of South Carolina

Abstract The coastal rivers of South Carolina possess reproducing populations of striped bass Morone saxatilis. Historically, all of these populations have been augmented with hatchery stocks from the Santee–Cooper drainage. Concern has been expressed that locally adapted, reproductively isolated populations may exist in these coastal rivers. Thus, the objective of this effort was to assess the distinctiveness of reproducing striped bass populations in these coastal rivers. Tagging studies indicated that the interriver movement of striped bass was minimal. A population estimate of 406 striped bass longer than 30 cm was obtained for the Combahee River, suggesting a relatively small breeding population. Genetic assays at three nuclear restriction fragment length polymorphism sites showed highly significant allele frequency differences among seven coastal rivers, suggesting reproductive isolation. Partially or fully isolated populations can develop beneficial adaptations to local environmental conditions. Th...

Evidence for Natural Selection in the Mitochondrial Genome of Mycosphaerella graminicola

ABSTRACT Pathogenicity assays were combined with restriction fragment length polymorphism (RFLP) markers in the mitochondrial and nuclear genomes to compare Mycosphaerella graminicola populations adapted to bread wheat (Triticum aestivum) and durum wheat (T. turgidum) in the Mediterranean Basin. The majority of isolates had unique nuclear DNA fingerprints and multilocus haplotypes. Only six mitochondrial DNA (mtDNA) haplotypes were identified among 108 isolates assayed. There were minor differences in frequencies of alleles at nuclear RFLP loci between the two host-adapted populations, but differences in the frequencies of mtDNA haplotypes were highly significant (P < 0.0001). mtDNA haplotype 1 dominated on the isolates adapted to bread wheat, and its frequency was twice as high as for the isolates adapted to durum wheat. mtDNA haplotype 4, which contained a unique approximately 3-kb insertion, was detected only in isolates showing specificity toward durum wheat and was the dominant haplotype on this species. We propose that the low mitochondrial diversity in this pathogenic fungus is due to a selective sweep and that differences in the frequencies of mtDNA haplotypes between the two host-adapted populations were due to natural selection according to host species.

Nuclear DNA RFLP variation among Atlantic cod in south and south-east Icelandic waters

The aim of this study was to describe the population structure of Atlantic cod, Gadus morhua, in Icelandic waters. A total of 404 cod was collected from five locations in Icelandic waters. The samples were analysed for allelic variation at five restriction fragment length polymorphism (RFLP) (GM862, GM860, GM738, GM777 and GM865) loci. Significant differences between sample sites were found at two loci (GM865 and GM777). The results are in line with other recent findings from this area indicating that the cod in south and south-east Icelandic waters do not belong to one panmictic population but may represent two genetically different reproductive stocks of cod.

The Probable Center of Origin of Fusarium oxysporum f. sp. lycopersici VCG 0033.

Isolates of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici, predominantly from commercial tomato fields in Florida and southwestern Georgia, were characterized using vegetative compatibility grouping (VCG), nuclear restriction fragment length polymorphism (RFLP), and virulence. All field isolates that could be grouped into VCG belonged to VCG 0033. This VCG was first described by Marlatt et al. in 1996 for isolates from northern Florida, Arkansas, and North Carolina. This study demonstrates that VCG 0033 is also widespread in central and southern Florida, in addition to southwestern Georgia, and also was found to be present in Puerto Rico. Population genetic and phylogenetic analyses of 121 isolates indicated that molecular diversity among VCG 0033 isolates was by far the highest in Manatee County, FL, suggesting it to be the probable center of origin of this relatively newly described VCG. Virulence tests with a subset of isolates identified all VCG 0033 isolates as race 3, although differences in aggressiveness were observed among tested isolates, independent of resistance genes in the differential cultivars. The widespread VCG 0030 of F. oxysporum f. sp. lycopersici was not present in our field collections. This was unexpected, as strains from Florida isolated prior to 1990 were predominantly VCG 0030. This would suggest that VCG 0033 has replaced VCG 0030 in recent years in commercial tomato fields of Florida and southwestern Georgia.

Nuclear DNA RFLP variation of Atlantic cod in the North Atlantic Ocean

A total of 551 specimens of Atlantic cod was collected from six locations across the North Atlantic Ocean. The samples were analysed for allelic variation at six nuclear restriction fragment length polymorphism (RFLP) loci scored by five anonymous cDNA clones, and the sequenced and identified synaptophysin ( Syp I). Significant differences in allelic variation were found between the sampling localities. The greatest degree of differentiation was detected between the Barents Sea and Scotian Shelf samples vs. the remaining sample units and a clear differentiation between west and east Atlantic samples was found.

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.

Characterization of North American Armillaria species by nuclear DNA content and RFLP analysis

Twenty-six isolates representing nine North American Armillaria species were investigated with flow cytometry and RFLP (restriction fragment length polymorphism) analyses to determine their nuclear DNA content and RFLP profile. Three putatively diploid isolates of A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, and North American Biological Species (NABS) X were analyzed, and two putatively diploid isolates of NABS XI also were analyzed. Nuclear DNA contents of Armillaria species were 0.11–0.17 pg per nucleus (55–84 X 106 bp/C), depending on species. Among the nine North American Armillaria species tested, A. ostoyae, A. gemina, and A. mellea possessed relatively small nuclear DNA contents (0.11–0.12 pg per nucleus), whereas A. gallica possessed a relatively large nuclear DNA content (0.17 pg per nucleus). A. nabsnona has a slightly larger nuclear DNA content (0.13 pg per nucleus) than A. ostoyae, A. gemina, and A. mellea. Other species (A. calvescens, A. sinapina, NABS X, and NABS XI) possessed moderate nuclear DNA contents (ca 0.15 pg per nucleus). Polymerase chain reaction (PCR) and RFLP of the intergenic spacer region-1 (IGS-1) generated banding patterns for nine Armillaria species. In addition to previously reported banding patterns, new banding patterns are presented for A. gemina, A. calvescens, A. mellea, and A. gallica.

Regional Population Diversity of Pyricularia grisea in Arkansas and the Influence of Host Selection

MGR586 DNA fingerprinting has been widely used to characterize population diversity of the rice blast pathogen, Pyricularia grisea. However, the frequency and distribution of particular haplotypes (individuals) within MGR-delimited lineages has not been examined in the United States. MGR586 DNA fingerprinting, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLPs), and virulence phenotyping were used to examine genetic diversity of P. grisea in Arkansas. A total of 470 monoconidial isolates were recovered from eight rice cultivars in 18 commercial fields in nine counties in Arkansas. All isolates were examined for nuclear DNA RFLPs with the MGR586 DNA fingerprint probe, and both the MGR lineage (isolates with >80% similarity) and the haplotype frequencies were determined. Four distinct MGR586 DNA fingerprint lineages (designated A, B, C, and D) were identified among the 470 field isolates. All four lineages were found in 9 of the 18 locations. Three lineages were found in four locations, two lineages in three locations, and only a single lineage was found at two locations. In all, 10, 19, 16, and 13 haplotypes (isolates which had MGR586 DNA fingerprints which differed by 1 to 20%) were identified within lineages A, B, C, and D, respectively, among the 470 isolates examined. Within each lineage, a single haplotype (clone) predominated, representing 51 to 71% of the isolates collected for each of the four lineages. Overall, 60% of the 470 isolates belonged to one of only four haplotypes (A1, B1, C1, and D1) and these four predominant haplotypes were recovered from between 7 and 14 of the 18 locations sampled, indicating a widespread distribution of these four clones. These data indicate an exceptionally low level of genetic diversity in the regional rice blast pathogen population in Arkansas relative to several other populations of P. grisea examined from tropical environments. In addition, no mtDNA RFLPs were detected among representative haplotypes within each of the lineages, indicating a single mtDNA haplotype was present in the population. Examination of virulence indicated that two races predominated in the regional collection. All 30 isolates in lineages A and C tested had an IB-49 virulence phenotype. Out of 30 isolates in lineages B and D, 29 had an IC-17 virulence phenotype. One isolate in lineage B, isolated from a highly susceptible cultivar (L201), had an IG-1 virulence phenotype. The frequencies of the four lineages varied among the locations sampled and may have been due, in part, to the cultivar from which isolates were recovered. A single lineage was recovered from two cultivars, Mars and Millie. Although only a single field of each of these cultivars was sampled, the data indicate that certain cultivars grown in Arkansas may serve as a "bottleneck", selecting out specific lineages in the regional population. To test this hypothesis, an additional 283 isolates were recovered from replicated plots of cvs. M204 and Mars located within commercial rice fields at two locations during two seasons. All four MGR586 lineages were recovered from each location. However, there was a strong bias for lineage B on cv. M204 (79% of all isolates) and a strong bias for lineage A on cv. Mars (95% of all isolates), indicating some cultivars were effective in excluding certain lineages.

Implications of Sexual Reproduction for Phytophthora infestans in the United States: Generation of an Aggressive Lineage.

Phytophthora infestans isolates (n = 26) collected in the Columbia Basin of Oregon and Washington in 1993, which had been characterized previously for mating type, metalaxyl sensitivity, and alleles at the glucose-6-phosphate isomerase locus, were analyzed for nuclear restriction fragment length polymorphism (RFLP) bands detected by probe RG57 and mitochondrial haplotype. Analyses involving the larger set of markers indicated that this group of isolates satisfied expectations of a sexual progeny: they contained much greater genetic diversity than has been reported for most other epidemic populations of P. infestans in the United States and Canada (16 unique multilocus genotypes); both mating types were present in proximity; all possible combinations of alleles occurred at many pairs of polymorphic loci; and two distinct mitochondrial haplotypes were distributed among the isolates. An in vitro laboratory cross involving the putative parents (US-6 and US-7) as parental strains produced progeny with the same general characteristics as the field isolates. Among the field progeny were two genotypes, US-11 and US-16, that had been described previously but from subsequent and largely clonal collections. Isolates obtained from tomatoes (n = 40) and potatoes (n = 7) in 24 counties in California in 1998 were analyzed as described above, and all except one US-8 isolate from potatoes were of the US-11 clonal lineage, consistent with the hypothesis that the US-11 lineage is an especially fit clonal lineage that has survived over time and can dominate pathogen populations over a large area. We conclude that the 1993 Columbia Basin collection represents a sexual progeny that generated the US-11 lineage, and that this lineage is particularly fit when tomatoes are part of the agroecosystem.

Nuclear restriction fragment length polymorphism analysis of genetic diversity in western redcedar

Variation at 41 putative genetic loci, uncovered by 29 single or low-copy nuclear probes of restriction fragment length polymorphism, was examined in western redcedar (Thuja plicata Donn. ex D. Don). Only 8 of the 29 probes produced polymorphisms when HindIII-digested DNA samples from 18 trees from each of five geographic regions (90 trees in total) covering the entire geographic range of the species were analysed. The eight polymorphic probes were used against an additional sample of 160 trees (for a total of 250 trees or 50 per region) to permit higher resolving power of differentiation between the regions. Species-level expected heterozygosity (or total gene diversity, HT) was estimated at 0.070, a low value that is quite similar to that at isozyme loci. Nei's genetic distances between the regions were quite small and DST, the average gene diversity between populations, was only 0.0042, indicating a low degree of differentiation among geographic regions. No private alleles were found, and low-frequency alleles were widespread across the species range. These results suggest that the present range of western redcedar is occupied by descendants of a small number of lineages from a single refugial population that survived the drastic changes in climatic conditions associated with the last ice age.

SINE Insertions as Clade Markers for Wild Crucifer Species

Short interspersed nuclear elements (SINEs) are small noncoding transposable elements that are widespread in most eukaryotic genomes. Plant S1 SINEs are present in crucifers, especially in species of the Brassiceae tribe, and were generated by several waves of amplification of different evolutionary ages, suggesting that S1 insertional variability (presence/absence of an S1 at a given genomic site) could be used as a classification criterion to evaluate phylogenetic relationships. We applied this strategy on closely related species from the Brassica oleracea species complex using 21 independent S1 genomic sites. The microsatellite-like variation of S1 39 poly(A) tails was also used as a complementary classification criterion to obtain internal resolution in two different clades. The phylogenetic tree obtained by this approach is in general agreement with the classification made from chloroplast DNA restriction site polymorphisms and differs significantly from two other (nonequivalent) classifications made using nuclear restriction fragment length polymorphisms. Brassica incana, Brassica montana, and Brassica hilarionis are confirmed as the closest relatives of B. oleracea. From our data, we suggest that Brassica drepanensis emerged recently from a B. incana/Brassica villosa hybrid (with B. villosa as the maternal parent) following backcrosses to B. incana. We also detected several introgressions, confirming that these highly related species are capable of genetic exchange in their natural habitat. S1 markers are therefore very useful in understanding the detailed evolutionary history of wild Brassica species and could also be used to identify potential gene flow between cultivated (including transgenic) Brassica and their wild relatives.

Population Genetic Analysis Corroborates Dispersal of Fusarium oxysporum f. sp. radicis-lycopersici from Florida to Europe

ABSTRACT Fusarium oxysporum isolates from tomato plants displaying crown and root rot symptoms were collected in central and southern Florida and analyzed using vegetative compatibility grouping (VCG) and nuclear restriction fragment length polymorphism (RFLP) data. VCG 0094 of F. oxysporum f. sp. radicis-lycopersici, previously known only from northwestern Europe, was predominant among 387 isolates assessed. In addition, two newly described VCGs (0098 and 0099) were detected at low frequencies. Floridian VCG 0094 isolates displayed a continuum of compatibilities, which is in contrast to the three distinct subgroups previously identified among European VCG 0094 isolates. RFLP haplotypes were constructed using one repetitive and three low-copy probes. Population subdivision of VCG 0094 from various Floridian counties and from northwestern Europe (Belgium, the Netherlands, and the United Kingdom) was evaluated by analysis of molecular variance. A "natural" population structure was revealed, differentiating populations from the east and west coasts of Florida. In addition, isolates from Europe were statistically indistinguishable from the Palm Beach County, FL, population. Furthermore, gene diversity among Palm Beach County VCG 0094 isolates was more than five times greater than among European isolates. Results from both VCG and RFLP analyses strongly support the inference that the European VCG 0094 constitutes a founder population that resulted from intercontinental migration of a few isolates from Palm Beach County, FL.

Genetic Relationships and Diversity in Carrot and other Daucus Taxa Based on Nuclear Restriction Fragment Length Polymorphisms

Current classifications of the genus Daucus are based on morphological and anatomical characteristics. We have used single to low copy nuclear restriction fragment length polymorphisms (nRFLPs) to describe the phylogeny and relationships of eight Daucus species including cultivated carrot (D. carota L.). Parsimony analysis of 247 characters (DNA fragments from 58 probe-enzyme combinations) yielded a tree in which accessions were grouped into three major clades and phenetic analysis using Jaccard's coefficient yielded two major clusters. The phylogenetic relationships from the nuclear RFLP data generally agreed with an earlier morphological classification. Resolution and placement of D. guttatus and D. muricatus were not consistent with the morphological classifications. Molecular variation among carrot inbreds was large.

Variability for Restriction Fragment-length Polymorphisms (RFLPs) and Relationships among Elite Commercial Inbred and Virtual Hybrid Onion Populations

Nuclear RFLPs were used to estimate relationships among 14 elite commercial inbreds of bulb onion (Allium cepa) from Holland, Japan, and the United States. Variability for known alleles at 75 RFLP loci and 194 polymorphic fragments revealed by 69 anonymous cDNA probes and a clone of alliinase were scored to yield genetically characterized and uncharacterized data sets, respectively. The inbred onion populations possessed more than two alleles at 20 of 43 (46%) codominant RFLP loci. Relationships among the inbreds were estimated by cluster analysis of simple-matching (genetically characterized data) and Jaccard (genetically uncharacterized data) coefficients using the unweighted pair group method and agreed with known pedigrees. RFLPs confidently distinguished among elite inbreds within and between specific market classes. RFLP profiles for virtual hybrids were computer-generated by combining gametic arrays among inbreds of the same market class and analyzed as described above. Allelic and genetically uncharacterized RFLPs confidently distinguished among these hybrids, even though heterozygosity for many markers produced a majority of monomorphic fragments. We randomly sampled decreasing numbers of RFLPs from the complete data sets and calculated simple-matching and Jaccard distances, noting the numbers of probes that were unable to distinguish any two inbreds or hybrids. As few as 10 polymorphic probe-enzyme combinations distinguished among all the inbreds and samples of 20 genetically characterized or 10 genetically uncharacterized clones distinguished all the virtual hybrids. This study demonstrated that the previously reported few RFLPs observed among open-pollinated (OP) onion populations were due to the highly heterozygous nature of the OP population.