Interspecific mitochondrial DNA restriction fragment length polymorphisms in Aspergillus section Flavi

Mitochondrial DNA restriction fragment length polymorphisms (RFLPs) are described for 64 isolates, representing 11 species of Aspergillus section Flavi. Mitochondrial DNA haplotypes were identified following digestion of total cellular DNA with the restriction enzymes HaeIII, AseI, or DraI. In general, isolates of the same species possessed identical mitochondrial DNA haplotypes. Three haplotypes were found in multiple, closely related species: one in A. flavus, A. oryzae, and A. subolivaceus; a second in A. parasiticus and A. sojae; and a third in A. tamarii and A. flavofurcatis. Four distinct haplotypes were each associated with a single species: A. nomius, A. avenaceus, A. leporis, and A. zonatus. Mitochondrial DNA haplotypes complement traditional morphological and growth criteria in making taxonomic decisions within Aspergillus section Flavi.     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

Some combinatorial problems of DNA restriction fragment length polymorphisms

Recombinant DNA techniques provide a means of defining new polymorphisms at the DNA sequence level. Polymorphisms arise when individuals differ in the location and number of sites where restriction endonucleases can cleave their DNA. Each such site exhibits two possible states: one for the presence of a specific endonuclease recognition sequence, the other for its absence. The states of a system of adjacent sites can be revealed experimentally by cleaving a person's DNA into a set of fragments. For experimentally well-understood systems of sites, we consider problems of counting numbers of possible fragments, haplotypes, genotypes, and phenotypes, and the means of resolving phenotype-genotype ambiguities. The degree of polymorphism generated by such systems and the importance to gene mapping are discussed.     

Chromosome 13 restriction fragment length polymorphisms

Summary The gene locus for hereditary retinoblastoma is on human chromosome 13, band q14. With this gene localization in mind, we cloned DNA fragments from this chromosome. Three of the fragments identify restriction fragment length polymorphisms. These three fragments are from the region 13q12–13q22, the chromosome region which contains the retinoblastoma locus. We expect that these restriction fragment length polymorphisms will be linked to the retinoblastoma locus, and that they will serve in certain retinoblastoma families as predictors of retinoblastoma gene carriers.They will also be useful in studies of other gene loci thought to be on chromosome 13.This research was supported by grants from the National Institutes of Health HD04807, CA29883, and EY04543, by a grant from Fight for Sight, Inc., New York City, and by the Anna Fuller Fund     

A rapid protocol for the purification of mitochondrial DNA suitable for studying restriction fragment length polymorphisms

When analyzing mitochondrial DNA (mtDNA) from various normal and malignant human tissues, it became necessary to enhance mtDNA isolation for improved yields and quality. The method described here consists of rapid and simple-to-perform steps, avoiding complicated instrumentation. It was designed for preparation of undegraded mtDNA and is highly useful when limited amounts of tissues, cells and unique biopsies of tumors (fresh or frozen) are available. The resulting mtDNA is sufficiently pure for restriction analysis, subcloning, labeling and various types of hybridization. Using Sau3A and MspI, restriction analysis revealed new restriction-fragment length polymorphisms for Caucasians, independent of the DNA source, and hence excluding tissue-specific DNA modifications.     

Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

Analysis of mitochondrial DNA restriction fragment length polymorphisms for examining genetic variability among isolates of Phellinus linteus

 Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNAs (mtDNAs) from nine Japanese wild isolates of Phellinus linteus was carried out to examine their genetic variability. BamHI and EcoRI digests of mtDNAs from these isolates produced four and five distinct RFLP patterns, respectively. By combining the RFLP patterns obtained with the two endonucleases, mtDNAs from the nine isolates could be assigned to five different genotypes, but no mtDNA variation was detected among the isolates collected from a small area. Distance values calculated among all pairs of mtDNA genotypes, based on the presence or absence of comigrating restriction fragments, were clearly smaller than those among the mtDNA genotypes of Lentinula edodes and Pleurotus ostreatus samples collected worldwide, suggesting the necessity of collecting P. linteus wild isolates for genetic resources from geographically wider areas. Received: June 27, 2002 / Accepted: August 19, 2002 Correspondence to:T. Nakamura     

DNA restriction fragment length polymorphisms and heterozygosity in the human genome

Summary A list is presented of published reports of DNA polymorphisms found in the human genome by restriction enzyme analysis. While the list indicates the large number of restriction fragment length polymorphisms (RFLPs) detected to date, the information collated is insufficient to permit an estimate of heterozygosity for the genome as a whole. Data from our laboratory are therefore also presented on RFLPs detected using a random sample of cloned DNA segments. Such an analysis has permitted a first unbiassed estimate of heterozygosity for the human genome. Since this figure is an order of magnitude higher than previous estimates derived from protein data, the majority of polymorphic variation present in the human genome must, by implication, occur in noncoding sequences. In addition it was confirmed that enzymes containing the dinucleotide CpG in their recognition sequences detect more polymorphic variation than those that do not contain a CpG. Also presented are the clinical applications of DNA polymorphisms in the diagnosis of human genetic disease.Supported by the Deutsche Forschungsgemeinschaft     

DNA restriction fragment length polymorphisms in the rDNA repeat unit of Entomophaga

A heterologous rDNA probe was used to detect restriction fragment length polymorphisms in Entomophaga rDNA sequences. Six Canadian strains of Entomophaga aulicae, isolated from the spruce bud worm or hemlock looper in Ontario or Newfoundland, showed no detectable rDNA variation at 10 different restriction enzyme loci: BamHI, DraI, EcoRI, EcoRV, HindIII, HinfI, MspI, PstI, RsaI, or TaqI. A total of 14 isolates of E. aulicae representative of several different geographic and host origins were compared at the DraI and HindIII rDNA loci and two different banding patterns were detected. Of these, 12 showed the same patterns and were designated E. aulicae type 1. The two members which differed were designated E. aulicae type 2. The variations in rDNA restriction sites did not appear to be geographically dependent. Entomophaga maimaiga, a recently reclassified species from the E. aulicae complex, displayed an rDNA banding pattern clearly distinguishable from the E. aulicae patterns with DraI, EcoRI, EcoRV, or HindIII. Members of the E. grylli species complex exhibited patterns which clearly differed from the patterns seen with either E. aulicae or E. maimaiga isolates. However, members of the E. grylli species complex appeared to be more heterogeneous than those in the E. aulicae complex. Among four E. grylli members, three different rDNA banding patterns were detected with either HindIII or DraI. These were designated as E. grylli type 1, type 2, and type 3. An undesignated Entomophaga isolate from a dipteran host displayed rDNA polymorphisms not previously noted in either the lepidopteran or orthopteran isolates. Our results suggest that RFLPs in rDNA are useful in the delineation of genera and species within the Entomophthorales, but may not be as useful at lower taxonomic levels. These and other RFLPs can however provide useful information regarding the epidemiology of Entomophaga epizootics.     

Characterization of highly virulent Escherichia coli strains by ribosomal DNA restriction fragment length polymorphism

Patterns of ribosomal DNA polymorphism were examined to compare carboxylesterase B type B1 strains and B2 strains of Escherichia coli isolated from extra-intestinal infections. DNA from 14 type B2 strains showing the presence of alpha-haemolysin and mannose-resistant haemagglutinin and lethality to mice and 14 type B1 strains lacking these characteristics, was digested with HindIII, EcoRI, BamHI or BglII restriction enzymes and analysed by Southern blotting. The obtained ribotypes clearly differentiated the B2 strains from the B1 strains. These results indicate that genotypes of the highly virulent B2 strains are different from that of the less virulent B1 strains.     

Genetic evidence for the existence of cryptic species in theAnopheles albitarsis complex in Brazil: Allozymes and mitochondrial DNA restriction fragment length polymorphisms

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species ofAn. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes.An. marajoara form CM had a higher variability (mean heterozygosity,H=0.22, and percentage of polymorphic loci,P=66.7) than did form IG and form MA (H=0.08 in both, andP=25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates ofF statistics, and genetic similarities, we propose thatAn. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1)An. deaneorum (currently one taxon) and (2) theAn. marajoara species complex, which further consists of at least three cryptic forms,marajoara form MA,marajoara form IG, andmarajoara form CM. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense of the United States. This research was conducted when the senior author was on the staff of the USDA-ARS Laboratory in Gainesville, Florida.     

Chloroplast DNA restriction fragment length polymorphisms (RFLPs) in Musa species

Summary Taxonomic and phylogenetic determinations within the genus Musa are established using a numerical, morphology-based scoring system. However, within this system, the classification and relationships of some types are disputed. The application of chloroplast DNA (cpDNA) restriction fragment length polymorphism (RFLP) analysis to Musa taxonomy provided valuable, supplemental information about the classification of, and relationships between, Musa species and subspecies. Whole-cell DNA was extracted from lyophilized Musa leaf-blade tissue and digested with various restriction enzymes, Southern blotted onto nylon membranes, and probed using radioactively labeled heterologous orchid cpDNA fragments. Phylogenies were inferred from cpDNA RFLP patterns using PAUP software. The relationships between most species examined were as expected; however, some species (M. beccarii and M. basjoo) did not conform to the conventional morphology-based phylogeny.     

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs Triton and Tower. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.     

Typing of Legionella pneumophila serogroups 2–14 strains by analysis of restriction fragment length polymorphisms

A typing method based on analysis of restriction fragment length polymorphisms has previously been developed for Legionella pneumophila serogroup 1. Here data are presented demonstrating the utility of this method for typing strains of all other L. pneumophila serogroups described to date. The method, which is highly discriminatory, should be of considerable value in epidemiological investigations of legionella infections.     

Genetic analysis of tolerance to low-phosphorus stress in maize using restriction fragment length polymorphisms

Summary An understanding of the genetic nature underlying tolerance to low-phosphorus (low-P) stress could aid in the efficient development of tolerant plant strains. The objective of this study was to identify the number of loci in a maize (Zea mays L.) population segregating for tolerance to low-P stress, their approximate location, and the magnitude of their effect.Seventy-seven restriction fragment length polymorphisms (RFLPs) were identified and scored in a maize F₂ population derived from a cross between line NY821 and line H99. The F₂ individuals were self-pollinated to produce F₃ families. Ninety F₃ families were grown in a sand-alumina system, which simulated diffusion-limited, low-P soil conditions. The F₃ families were evaluated for vegetative growth in a controlled-environment experiment. To identify quantitative trait loci (QTLs) underlying tolerance to low-P stress, the mean phenotypic performances of the F₃ families were contrasted based on genotypic classification at each of 77 RFLP marker loci.Six RFLP marker loci were significantly associated with performance under low-P stress (P<0.01). One marker locus accounted for 25% of the total phenotypic variation. Additive gene action was predominant for all of the QTLs identified. Significant marker loci were located on four separate chromosomes representing five unlinked genomic regions. Two marker loci were associated with an additive by additive epistatic interaction. A multiple regression model including three marker loci and the significant epistatic interaction accounted for 46% of the total phenotypic variation. Heterozygosity per se was not predictive of phenotypic performance.     

Genetic analyses of restriction fragment length polymorphisms using high molecular weight DNA from sperm or lymphocytes embedded in agarose

A simple and efficient method for determining restriction fragment length polymorphism types on large numbers of individuals using small samples of peripheral blood or sperm cells is described. Whole cells embedded in low gelling/melting temperature agarose were treated with a series of enzyme, detergent, and washing steps to release high molecular weight DNA that was then digested with standard restriction enzymes such as EcoRI and PstI, electrophoresed, blotted, and probed as in normal Southern analyses. The technique should be readily adaptable to any application requiring DNA from small numbers of cells for Southern analyses or pulsed field gel electrophoresis.