Restriction Fragment Length Polymorphism Variation Research Articles

Genotypic and Phenotypic Diversity among Induced, stx 2 -Carrying Bacteriophages from Environmental Escherichia coli Strains

Shiga toxin 2 (stx(2)) gene-carrying bacteriophages have been shown to convert Escherichia coli strains to Shiga toxin-producing Escherichia coli (STEC). In this study, 79 E. coli strains belonging to 35 serotypes isolated from wastewaters of both human and animal origin were examined for the presence of stx(2)-carrying bacteriophages in their genomes. The lytic cycle of the bacteriophages was induced by mitomycin, and the bacteriophage fraction was isolated and used for morphological and genetic characterization. The induced bacteriophages showed morphological diversity, as well as restriction fragment length polymorphism variation, in the different strains belonging to different serotypes. The ability to infect new hosts was highly variable, although most of the induced phages infected Shigella sonnei host strain 866. In summary, in spite of carrying either the same or different stx(2) variants and in spite of the fact that they were isolated from strains belonging to the same or different serotypes, the induced bacteriophages were highly variable. The high level of diversity and the great infectious capacity of these phages could enhance the spread of the stx(2) gene and variants of this gene among different bacterial populations in environments to which humans may be exposed.

Comparison of Colletotrichum orbiculare and Several Allied Colletotrichum spp. for mtDNA RFLPs, Intron RFLP and Sequence Variation, Vegetative Compatibility, and Host Specificity

ABSTRACT Based on spore morphology, appressorium development, sequence similarities of the rDNA, and similarities in amplified restriction fragment length polymorphism (AFLP), it has been proposed that Colletotrichum orbiculare, C. trifolii, C. lindemuthianum, and C. malvarum represent a single phylogenetic species, C. orbiculare. In the current study, the phylogenetic relationship among isolates in the C. orbiculare species complex was reassessed. In all, 72 isolates of C. orbiculare from cultivated cucurbit or weed hosts, C. trifolii from alfalfa, C. lindemuthianum from green bean, and C. malvarum from prickly sida (Sida spinosa) were examined for mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs), RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase gene, and vegetative compatibility. In addition, host specificity was examined in foliar inoculations on cucurbit, bean, and alfalfa hosts. Inoculations also were conducted on cucumber fruit. Genetically distinct isolates, based on vegetative compatibility, within the species complex (C. orbiculare, C. trifolii, and C. malvarum) had an identical mtDNA haplotype (haplotype A) when examined with each of three different restriction enzymes. Isolates of C. lindemuthianum had a very similar mtDNA haplotype to haplotype A, with a single polymorphism detected with the enzyme HaeIII. The four species represent a phylogenetically closely related group based on a statistical analysis of the 900- and 200-bp intron sequences. However, distinct RFLPs in the 900-bp intron were consistently associated with each species and could be used to qualitatively and quantitatively distinguish each species. Furthermore, each of the species showed distinct host specificity, with isolates of C. orbiculare (from cucurbits), C. lindemuthianum, and C. trifolii being pathogenic only on cucurbits, green bean, and alfalfa, respectively. Consequently, distinct and fixed nucleotide, or genotypic (intron sequences and RFLPs) and phenotypic (host specificity) characteristics can be used to distinguish C. orbiculare, C. lindemuthianum, and C. trifolii from one another; therefore, they should be recognized as distinct species. This species delineation is consistent with the most current species concepts in fungi. More isolates and further characterization is needed to determine whether C. orbiculare from cocklebur and C. malvarum represent distinct species. RFLPs of the 900-bp intron may represent a relatively inexpensive, reliable, and useful diagnostic tool for general species differentiation in the genus Colletotrichum.

Nucleotide sequence variation does not relate to differences in kinetic properties of neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

Genetic variability in a putative virulence factor, the neutral trehalase ( Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., Km, Vmax, and Kcat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower Kcat/Km. However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.

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.

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.

A variable minisatellite sequence in the chloroplast genome ofSorbusL. (Rosaceae: Maloideae)

The chloroplast genome is now known to be more variable than was once thought. Reports of RFLP (restriction fragment length polymorphism) and sequence variation, as well as variation in chloroplast microsatellites, are common. Here, data are presented on the variability of a minisatellite sequence in the chloroplast genome of Sorbus species. RFLP analysis of a PCR product comprising the region between the trnM and rbcL genes of nine Sorbus species identified seven size variants. Sequencing revealed the observed size polymorphism to be due to differences in the number of copies of an imperfect 9-bp motif. A more intensive survey of the variability of the minisatellite was undertaken in populations of Sorbus aucuparia. The potential uses of such regions in chloroplast DNA are discussed and a possible mechanism for the evolution of the minisatellite is presented.

Variation in restriction fragment length polymorphisms among serial isolates from patients with Trichophyton rubrum infection.

Molecular genotyping of strains of Trichophyton rubrum and T. mentagrophytes from patients with onychomycosis of the toes was performed to ascertain whether the fungal genotype changes over the course of time as sequential samples were obtained from patients receiving antifungal therapy and during follow-up. Sixty-six serial strains of T. rubrum and 11 strains of T. mentagrophytes were obtained from 20 patients (16 patients with T. rubrum, 4 with T. mentagrophytes) who were treated with oral antifungal therapy and observed over periods of up to 36 months. These strains were screened for genetic variation by hybridization of EcoRI-digested genomic DNAs with a probe amplified from the small-subunit (18S) ribosomal DNA and adjacent internal transcribed spacer regions. A total of five restriction fragment length polymorphism (RFLP) types were observed among 66 strains of T. rubrum. Two major RFLP types, differentiated by one band shift, represented 68% of the samples. None of the patients had a unique genotype. More than one RFLP type was often observed from a single patient (same nail) over a period of 1, 2, or 3 years, even in cases that did not appear cured at any time. Samples taken from different nails of the same patient had either the same or a different genotype. The genotypic variation did not correspond to any detectable phenotypic variation. Furthermore, no correlation was observed between the efficacy of the treatment administered and the genotype observed. While the DNA region studied distinguished among T. rubrum, T. mentagrophytes, and T. tonsurans, intraspecific RFLP variation was observed for T. rubrum and T. mentagrophytes strains. While independent multiple infection and coinhabitation of multiple strains may explain the presence of different genotypes in a nail, microevolutionary events such as rapid substrain shuffling, as seen in studies of repetitive regions in Candida species, may also produce the same result. The recovery of multiple strains during the course of sequential sampling of uncured patients further suggests that the typing system is not able to distinguish between relapse or reinfection, ongoing infection, and de novo infection.

Molecular Variation and F1 Performance among Strains of the Sweet Corn Inbred P39

Some maize (Zea mays L.) inbreds produce morphological variants at unexpectedly high frequencies. In some cases, heterosis has been observed in crosses between variants and the progenitor inbred. Causes of such variants and resulting heterosis are unclear. Unique materials for addressing these questions are strains of the sweet corn inbred P39. Several morphologically distinct strains of P39 have been identified. Molecular variation of P39 strains was analyzed by means of three types of molecular markers, including four probes that hybridize to multiple sequences, 78 single copy restriction fragment length polymorphisms (RFLP), and 671 amplified fragment length polymorphisms (AFLP). All crosses were made among seven P39 strains. Ear weight and plant and ear height of the crosses were measured in 2 yr with one location and eight replications per year. Ten‐ear weight ranged from 0.49 to 0.84 kg. Significant levels of molecular variation were observed among the strains. Summing polymorphisms per inbred pair over the multiple sequence probes resulted in a range among pairwise comparisons of 0 to 18. RFLPs ranged from 6 (7%) to 28 (35%) polymorphisms per inbred pair. AFLPs between inbred pairs ranged from 3 (0.5%) to 145 (22%). Ten‐ear weight was correlated with the number of polymorphisms between pairs of strains for the multiple copy probes. RFLP variation was correlated with plant height and ear height but not ear weight. AFLP variation was correlated with ear weight While initial isolation of some of the P39 strains was due to a mutation at a single gene, the amount of molecular variation was unexpectedly high. The molecular variation and changes in combining ability indicates the occurrence of alterations throughout the genome.

Molecular characterization of the freshwater snail Lymnaea natalensis (Gastropoda: Lymnaeidae) on Madagascar with an observation of an unusual polymorphism in ribosomal small subunit genes

AbstractRecent characterization of nuclear ribosomal small subunit (SSU) genes has shown that variant nucleotides within this region could be useful for species and species group identification within the genus Lymnaea (Gastropoda: Lymnaeidae). This study aimed to characterize a range of populations of Lymnaea natalensis Krauss, 1848 on Madagascar, and addressed two related questions. First, is there any evidence of intraspecific variation of the SSU and, if so, what might be its significance? Secondly, might this variation jeopardize the use of SSU for lymnaeid taxonomy and phylogeny?Lymnaea natalensis (n= 212) was collected from 17 sampling localities, spanning the northern and southern ends of the island. Variation within a selected region of the SSU known to vary between species, the V1 and V2, was assayed by polymerase chain reaction (PCR) linked restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) analysis. The PCR‐RFLP profiles indicated a striking dimorphism across populations at two restriction site loci (CfoI & MspI) within the E10‐1 helix of the V2 region. The observed RFLP variation was confirmed by direct sequencing and by genomic digestion with subsequent hybridization. Putative heterozygotes were also encountered and in these individuals the SSU arrays composed of two distinct types approximately 1% divergent. A severe departure from Hardy–Weinberg equilibrium with a highly statistically significant (P < 10−5) heterozygote deficiency was found and genetic variation among populations was highly structured (Fst= 0.53). The geographic distribution of the variants was mapped, revealing that one variant was restricted to higher, predominately colder environments and was thought to be an adaptation. The molecular basis of the SSU variation was caused by single nucleotide polymorphisms (SNPs). To test for the possibility of cryptic taxa, an analysis of individuals representative of the SSU variant types with isoenzyme analysis (ISA), randomly amplified polymorphic DNA (RAPDs) and PCR‐RFLP analysis of the ribosomal Internal Transcribed Spacer (ITS) was performed. Little variation was revealed and none that correlated to the groups suggested by SSU, confirming that the SSU variation was intraspecific. The levels of intraspecific divergence of the V1 and V2 within Lymnaea were not appreciably different (1%) from interspecific and would therefore question the validity of these data for lymnaeid taxonomy and phylogeny.

Molecular characterization of the freshwater snail Lymnaea natalensis (Gastropoda: Lymnaeidae) on Madagascar with an observation of an unusual polymorphism in ribosomal small subunit genes

Recent characterization of nuclear ribosomal small subunit (SSU) genes has shown that variant nucleotides within this region could be useful for species and species group identification within the genus Lymnaea (Gastropoda: Lymnaeidae). This study aimed to characterize a range of populations of Lymnaea natalensis Krauss, 1848 on Madagascar, and addressed two related questions. First, is there any evidence of intraspecific variation of the SSU and, if so, what might be its significance? Secondly, might this variation jeopardize the use of SSU for lymnaeid taxonomy and phylogeny?Lymnaea natalensis (n= 212) was collected from 17 sampling localities, spanning the northern and southern ends of the island. Variation within a selected region of the SSU known to vary between species, the V1 and V2, was assayed by polymerase chain reaction (PCR) linked restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) analysis. The PCR-RFLP profiles indicated a striking dimorphism across populations at two restriction site loci (CfoI & MspI) within the E10-1 helix of the V2 region. The observed RFLP variation was confirmed by direct sequencing and by genomic digestion with subsequent hybridization. Putative heterozygotes were also encountered and in these individuals the SSU arrays composed of two distinct types approximately 1% divergent. A severe departure from Hardy–Weinberg equilibrium with a highly statistically significant (P < 10−5) heterozygote deficiency was found and genetic variation among populations was highly structured (Fst= 0.53). The geographic distribution of the variants was mapped, revealing that one variant was restricted to higher, predominately colder environments and was thought to be an adaptation. The molecular basis of the SSU variation was caused by single nucleotide polymorphisms (SNPs). To test for the possibility of cryptic taxa, an analysis of individuals representative of the SSU variant types with isoenzyme analysis (ISA), randomly amplified polymorphic DNA (RAPDs) and PCR-RFLP analysis of the ribosomal Internal Transcribed Spacer (ITS) was performed. Little variation was revealed and none that correlated to the groups suggested by SSU, confirming that the SSU variation was intraspecific. The levels of intraspecific divergence of the V1 and V2 within Lymnaea were not appreciably different (1%) from interspecific and would therefore question the validity of these data for lymnaeid taxonomy and phylogeny.

Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment length polymorphism (RFLP) markers.

A total of 72 hypovirus-infected isolates of the chestnut blight fungus Cryphonectria parasitica were sampled from nine European countries between 1975 and 1997. The double-stranded RNA of the Cryphonectria hypoviruses (CHV1) was isolated and reverse transcription (RT)-PCR products were obtained for two different regions of the viral genome (ORF A and ORF B) using primer sequences of the type species CHV1-EP713. Both PCR products of each viral isolate were digested with four restriction endonucleases recognizing sequences of four nucleotides. The restriction fragment length polymorphism (RFLP) analysis revealed 41 genetically distinct RFLP types of CHV1 with 10 types occurring more than once. Identical RFLP types were detected nine times among viruses collected in the same location. Cluster analysis based on the RFLP banding patterns separated the viral isolates into five CHV1 clusters or subtypes. Most viral isolates (64 out of 72) grouped into one large cluster which comprised all viruses from Italy (including CHV1-EP747), Switzerland, Crotia, Bosnia, Hungary, Greece, and the French island Corsica, as well as five out of 11 isolates from continental France. Two additional subtypes of CHV1 were found in France (one related to CHV1-EP713) and one each in Spain and Germany. The Swiss samples collected over a period of 20 years showed that very little RFLP variation has evolved during this time. The results of this study are consistent with the hypothesis of multiple introductions of CHV1 into Europe.

Genetic diversity among Paspalum spp. as determined by RFLPs

The genus Paspalum is characterized by over 400 species that are indigenous to a wide range of stressful habitats and marginal environments. Fifty-one accessions representing 29 Paspalum species were analyzed for DNA restriction fragment length polymorphisms (RFLPs). Fifteen random genomic probes were used in combination with restriction enzyme EcoRI to detect RFLPs, and data were analyzed phenetically. Hybridization with the 15 selected clones resulted in the detection of 261 RFLPs. Among the 261 restriction fragments scored, 204 (78.2%) were phenetically informative. Extensive RFLP variation was found between the species studied. Species affinities based on RFLP data were found to be in close agreement with previously determined relationships based on both morphological and cytological characteristics.

Variation in Mitochondrial DNA in Populations of Peromyscus eremicus from the Chihuahuan and Sonoran Deserts

A 1,439-bp region of the mitochondrial DNA was analyzed for restriction-fragment-length polymorphisms (RFLPs) within and among populations of cactus mice ( Peromyscus eremicus ) from the Chihuahuan and Sonoran deserts. Although five mtDNA haplotypes were detected, none of these was shared between Sonoran and Chihuahuan populations. All analyses of the RFLP variation and concordant morphological, biochemical, and biogeographic data are consistent with the hypothesis that the P. eremicus from the Sonoran and Chihuahuan deserts represent recently diverged species. Pliocene or late Pleistocene pluvial-interpluvial climatic fluctuations are implicated as the primary mechanism in the diversification of the cactus mice from the Sonoran and Chihuahuan deserts.

Resolution of recent radiations within three evolutionary lineages of felidae using mitochondrial restriction fragment length polymorphism variation

Patterns of mitochondrial restriction fragment length polymorphism (RFLP) variation were used to resolve more recent relationships among the species of the Felidae ocelot lineage, domestic cat lineage, and pantherine lineage. Twenty-five of 28 restriction enzymes revealed site variation in at least 1 of 21 cat species. The ocelot lineage was resolved into three separate sistertaxa groups: Geoffroy's cat (Oncifelis geoffroyi) and kodkod (O. guigna), ocelot (Leopardus pardalis) and margay (L. wiedii), and pampas cat (Lynchailurus colocolo) and most of the tigrina samples (Leopardus tigrina). Within the domestic cat lineage, domestic cat (Felis catus), European wild cat (F. silvestris), and African wild cat (F. libyca) formed a monophyletic trichotomy, which was joined with sand cat (F. margarita) to a common ancestor. Jungle cat (F. chaus) and black-footed cat (F. nigripes) mtDNAs diverged earlier than those of the other domestic cat lineage species and are less closely related. Within the pantherine lineage, phylogenetic analysis identified two distinct groups, uniting lion (P. leo) with leopard (P. pardus) and tiger (P. tigris) with snow leopard (P. uncia).

RFLP variation and genetic relationships in cultivated cucumber

Two sets of cucumber (Cucumis sativus L.) germplasm were used to determine the potential use of restriction fragment length polymorphisms (RFLPs) for estimating genetic relationships. Sixteen accessions [15 domesticated variety sativus and one feral variety hardwickii (PI 183967)] of diverse origin were used to assess RFLP variation in cucumber, and to determine if genetic relationships based on RFLPs were similar to those obtained by isozyme analysis. Additionally, 35 commercial lines or cultivars were surveyed to determine genetic relationships among and within common cucumber types (narrow genetic base). The 16 accessions were surveyed with 440 low copy clones from two libraries (Pst I partial genomic and cDNA) using two restriction enzymes. Data from a subset of 104 random (mapped and unmapped) and a set of 30 mapped RFLPs were used to estimate genetic relationships among the 16 cultigens. Variability was low among RFLPs (33% of all probes) and putative alleles (∼ 2.2 polymorphic fragments/probe). RFLP variation between sativus lines and hardwickii (21±4%) was greater than among sativus lines (12±2%). RFLPs among the 16 accessions revealed genetic relationships which agree with those obtained using isozymes. Genetic relationships estimated using mapped and unmapped RFLPs were similar. The 35 elite lines were surveyed using a set of 40 RFLPs from 3 libraries (Pst I and EcoR I partial genomic and cDNA) to evaluate the discriminatory value of RFLPs among and between commercial cucumber types. The RFLP-derived genetic relationships among this germplasm were in agreement with predictions based on fruit type and pedigree information. Thus, RFLPs are a useful addition to the morphological characters and isozyme loci currently used for taxonomic classification and plant variety protection of cucumber.

Influence of genetic variability in the nondeletion LDL-receptor allele on phenotypic variation in French-Canadian familial hypercholesterolemia heterozygotes sharing a ‘null’ LDL-receptor gene defect

We investigated the associations between low density lipoprotein (LDL)-receptor gene haplotypes and lipid and lipoprotein levels in French-Canadian individuals with familial hypercholesterolemia (FH). The 112 unrelated patients studied shared the same > 10 Kb deletion in the 5′ region of the LDL-receptor gene, leading to a null allele. Support for the hypothesis that the deletion is carried on only one LDL-receptor restriction fragment length polymorphism (RFLP) haplotype in this sample has previously been demonstrated [1]. We studied associations of genetic variability in DNA polymorphisms of the nondeletion LDL-receptor allele with variation in plasma lipid levels in these patients heterozygous for the deletion. All analyses were done separately in males and females. The traits were adjusted for variation in the concomitants age, height and weight, and for variation in apolipoprotein (apo) E phenotype, and then the association between variability in haplotypes defined by two RFLP loci and variation in trait levels were tested. The results indicated that in this sample of French-Canadian >10 Kb deletion FH heterozygotes, variability at the LDL-receptor gene contributes to quantitative variation in measures of lipid metabolism and that the effects are different in males and females. The results indicated that variability at the LDL-receptor gene defined by two RFLP loci contributes to quantitative variation in high density lipoprotein (HDL)-cholesterol and LDL-cholesterol concentrations in French-Canadian FH women heterozygous for the >10 Kb deletion and not in men.

PCR and RFLP variation of conserved region of small subunit ribosomal DNA among Acanthamoeba isolates assigned to either A. castellanii or A. polyphaga.

Twelve isolates of Acanthamoeba spp. assigned to either A. castellanii or A. polyphaga, and type strains of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were examined by restriction fragment length polymorphism (RFLP) of a conserved region of small subunit ribosomal RNA gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellaii was 9.8% whereas that among the isolates assigned to A. polyphaga 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. castellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. polyphaga was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. castellanii and A. polyphaga (2.6%) which appeared between the Castellani (or CCAP 1501/2 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. astronyxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphaga. It is suggested that taxonomic validity of the isolates assigned to either A. castellanii or A. polyphaga should be reevaluated.

Primer sets developed to amplify conserved genes from filamentous ascomycetes are useful in differentiating fusarium species associated with conifers

We examined the usefulness of primer sets designed to amplify introns within conserved genes in filamentous ascomycetes to differentiate 35 isolates representing six different species of Fusarium commonly found in association with conifer seedlings. We analyzed restriction fragment length polymorphisms (RFLP) in five amplified PCR products from each Fusarium isolate. The primers used in this study were constructed on the basis of sequence information from the H3, H4, and (beta)-tubulin genes in Neurospora crassa. Primers previously developed for the intergenic transcribed spacer region of the ribosomal DNA were also used. The degree of interspecific polymorphism observed in the PCR products from the six Fusarium species allowed differentiation by a limited number of amplifications and restriction endonuclease digestions. The level of intraspecific RFLP variation in the five PCR products was low in both Fusarium proliferatum and F. avenaceum but was high in a population sample of F. oxysporum isolates. Clustering of the 35 isolates by statistical analyses gave similar dendrograms for H3, H4, and (beta)-tubulin RFLP analysis, but a dendrogram produced by intergenic transcribed spacer analysis varied in the placement of some F. oxysporum isolates.

Reevaluating the origin of domesticated cotton (Gossypium hirsutum; Malvaceae) using nuclear restriction fragment length polymorphisms (RFLPs)

The origin of domestication in Mesoamerican G. hirsutum populations is obscured by several factors, including the absence of a clearly identified wild progenitor, a complex population genetic structure, and many centuries of human‐mediated dispersal and gene flow. Phenetic and phylogenetic analyses of allelic variation at 205 restriction fragment length polymorphism (RFLP) loci were conducted in an effort to unravel this complicated history. The RFLP data, in conjunction with previously published molecular, morphological, and anthropological information, suggest that coastal Yucatan populations are truly wild rather than reestablished feral derivatives. The geographical proximity of these wild coastal populations to agronomically primitive forms of G. hirsutum implicates the Yucatan peninsula as the primary site for the earliest stages of domestication. Agronomically advanced cultigens developed in southern Mexico and Guatemala appear to have been derived from introduced Yucatan peninsular forms, thereby creating the secondary center of diversity that has traditionally been interpreted as the geographical point of origin of domesticated G. hirsutum. The gene pool of modern, improved (Upland) cultivars derives from Mexican highland populations that, in turn, trace their origins to southern Mexico and Guatemala. Gossypium hirsutum is the first tetraploid perennial surveyed for RFLP variation. Levels of RFLP variation in G. hirsutum (HT = 0.048, A = 1.24, and P = 22%) are low relative to other plant taxa, and, in contrast to the few comparable studies, levels of allozyme variation are higher than levels of RFLP variation. Despite assaying 205 loci, only six of the 23 Upland cultivars were found to have unique multilocus genotypes.

RFLP variation and genealogical distance, multivariate distance, heterosis, and genetic variance in oats

Patterns of restriction fragment length polymorphisms (RFLPs) have been proposed as estimators of genetic diversity among breeding lines and as predictors of heterosis and genetic variance. We evaluated these proposals by using a set of nine elite oat lines crossed in a diallel mating design without reciprocals. RFLP analysis was conducted using HindIII-digested DNA and a total of 107 probes from three different sources: 14 heterologous wheat cDNA clones, 17 oat genomic clones, and 76 oat cDNA clones. Of the 77 probes that produced high-quality autoradiographs, 26 detected polymorphisms among this set of lines, with an average of 2.6 variants per probe. RFLP-based genetic distance (FD) was calculated from these data by using Nei and Li's measure of genetic similarity, and was compared with two other measures of genetic divergence. Genealogical distance (GD (*)) was obtained from the coefficients of parentage based on known parental pedigrees, and multivariate distance (DI) was calculated by using the first five principal components of the parental correlation matrix for 12 agronomic traits. FD was significantly correlated with GD (*) (r=0.63, P<0.01), but not with DI (r=-0.05). Cluster analysis based on these three distance estimates did not produce equivalent groupings, but the FD and GD (*) clusters were more similar to each other than to the DI clusters. These results indicate that: (1) sufficient variation exists for further application of RFLP technologyto oats, (2) RFLPs could provide accurate estimates of genetic divergence among elite oat lines, and (3) it is unlikely that dispersed markers can predict heterosis or population genetic variance in oats. Further investigations will require more parental lines, a larger set of markers, and more information on the linkage relationships between RFLP markers and loci controlling the trait of interest.