Amplified fragment length polymorphisms reveal genetic differentiation among strains of Xanthomonas albilineans

Xanthomonas albilineans, the causative agent of leaf scald disease (LSD), colonizes the vascular system of sugarcane (Saccharum spp. hybrids). In this study X. albilineans strains from 28 countries were differentiated by using two methods of amplified fragment length polymorphism (AFLP). In the manual procedure, AFLP reactions were performed on 57 X. albilineans strains and after selective amplification using radiolabelled primers, the resulting products were separated using polyacrylamide gel electrophoresis. The autoradiographs were analyzed using GelCompar version 4.1 software (Applied Maths) to construct dendograms from similarity matrices. Fluorescent AFLP (FAFLP) was also employed on 52 X. albilineans strains using three fluorescently labelled primer combinations (automated AFLP). The FAFLP data was converted to a binary format using the Genemapper Software 3.7 (Applied Biosystems). Thereafter, dendograms were generated using the NTSYSpc. Software (USA). Distinct AFLP profiles were produced for the majority of the strains and were found to be useful in differentiating X. albilineans strains from various geographical locations. Fingerprints unique to each strain were reproducibly obtained and may be used to create a database for use in the identification of the various X. albilineans strains. It can be also concluded from the results obtained that the FAFLP has considerable technical advantages compared with the manual AFLP and also that the FAFLP is more sensitive than AFLP using radiolabelled primers in differentiating X. albilineans.     

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     

Genetic monitoring of laboratory rat strains by restriction fragment length polymorphisms of mitochondrial DNA

Restriction fragment length polymorphisms of rat mitochondrial DNA (mtDNA) were examined using various kinds of laboratory rat strains in Japan. The results show that mtDNA of laboratory rats, Rattus norvegicus, are highly polymorphic; at least 7 types, Aa, Ba, Bb, Cb, Dc, Eb, and Fa, were found with the use of 6 restriction enzymes, EcoR I, Hind II, Hha I, Hpa II, Taq I, and Hinf I. Types Aa, Ba, and Eb were distributed widely in several strains, whereas types Ba, Cb, Dc and Fa were limited to some specific strains. These results indicate that restriction fragment length polymorphisms of mtDNA can be applied to genetic monitoring of laboratory rat strains.     

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     

Use of restriction fragment length polymorphisms to identify sea turtle eggs and cooked meats to species

One of the many threats to sea turtlepopulations is the take of turtles and theireggs for consumption and sale. Improved speciesidentification methods for sea turtle eggs andcooked meats would facilitate prosecution ofthose involved. Fatty acid-based methods toidentify eggs cannot resolve loggerheads andthe two ridley species. Protein-based methodsare not applicable to eggs or cooked meat. Wepresent methods to extract DNA from turtle eggand cooked meat and to produce diagnosticrestriction fragment length polymorphismpatterns in the cytochrome b region of themitochondrial DNA. This method works on DNAfrom any tissue, and provides wildlife lawenforcement another tool to combat illegal takeof endangered species.     

Use of restriction fragment length polymorphisms for genetic counseling: Population genetic considerations

Two-locus population genetic models are analyzed to evaluate the utility of restriction fragment length polymorphisms for purposes of genetic counseling. It is shown that the linkage disequilibrium between a neutral marker and a tightly linked overdominant mutant will increase rapidly as the mutant moves to its polymorphic equilibrium. The linkage disequilibrium decays for deleterious recessive mutants. Two measures involving the linkage disequilibrium are investigated to determine how much information the transmission of the neutral marker provides about the transmission of the selected gene. In certain kinds of matings, where the parental two-locus genotypes and linkage phases are known, it is possible to determine whether or not a progeny is homozygous for the selected gene on the basis of the fetal genotype at the marker locus. A quantity of primary interest is the fraction of matings between individuals heterozygous for the selected gene in which exact diagnosis can be made in this way. The expected proportion of such matings, taken over all two-locus matings involving heterozygotes at the selected locus, is calculated as a function of the gene frequencies at the two loci and the linkage disequilibrium between them. This expected value is maximized when the linkage disequilibrium is at its maximum in absolute value. Fewer than half of all matings are informative if the linkage disequilibrium is small in magnitude or if the gene frequencies at the two loci are quite different. Consideration is also given to various conditional measures of association that may be useful when the parental two-locus genotypes are unknown. The results suggest that the utility of tightly linked neutral marker genes in predicting the transmission of a selected gene is generally less when selection acts against a recessive gene than for overdominant selection.     

BglII restriction fragment length polymorphisms at the human p53 gene locus

Summary A new restriction fragment length polymorphism detected with the restriction endonuclease BglII (AGATCT) is presented.     

Schistosoma mansoni: use of a cloned ribosomal RNA gene probe to detect restriction fragment length polymorphisms in the intermediate host Biomphalaria glabrata.

Adult susceptibility of Biomphalaria glabrata to Schistosoma mansoni infection is controlled by simple Mendelian genetics. In this study a molecular approach was used to determine the degree of genetic variation between well-defined lines of B. glabrata which are either resistant (10-R2) or susceptible (M-line) to S. mansoni infection. A cloned probe pSM389, which contains part of the S. mansoni small ribosomal RNA gene and a portion of the nontranscribed spacer was found to cross-hybridize with B. glabrata DNA and was used in Southern hybridizations to detect restriction fragment length polymorphisms (RFLPs) between the above snail stocks. Polymorphisms were noted with a variety of restriction enzymes, namely Bg/II, BamHI, AccI, AvaII, ClaI, EcoRI, EcoRV, KpnI, PvuII, and NcoI. Although most RFLPs were relatively minor, a significant difference was observed with EcoRV. Further analysis of the EcoRV RFLPs among other isolates of the resistant stock demonstrated that a high frequency of genetic variation exists even among isolates of the same origin, but maintained in separate laboratories. Interestingly, RFLPs in the EcoRV site were detected in DNA isolated from a single generation of selfed progeny of a single 10-R2 parent. RFLPs associated with this site were found to occur between B. glabrata and B. tenagophila, B. straminea, and B. schrammi, indicating that Southern blot analysis using ribosomal gene probes may be useful for the molecular differentiation of B. glabrata from other intermediate hosts and from morphologically similar species that are refractory to infection.     

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.     

Use of restriction fragment length polymorphism to detect dihydropteroate synthase gene mutations in strains of Pneumocystis jirovecii isolated in Poland

Point mutation of the dihydropteroate synthase gene causes Pneumocystis jirovecii resistance to sulfa drugs. The RFLP method was used to search for DHPS polymorphism in P. jirovecii strains isolated in Poland. Positive results of DHPS gene fragment amplification using nested PCR were achieved in 25 out of the 30 examined Pneumocystis DNA samples. Two (8%) of the 25 isolates had point mutation at codon 55 (Thr55Ala). The mutation-positive strains were obtained from HIV positive (1/15) and HIV negative (1/10) patients. The observed DHPS gene polymorphism may point to the appearance of P. jirovecii strains resistant to sulfa drugs in Poland.     

Relationship of restriction fragment length polymorphisms to single-cross hybrid performance of maize

Summary Isozymes and restriction fragment length polymorphisms (RFLPs) have been proposed for use in varietal identification and selection for agronomic traits. Although the use of isozymes for these purposes has been well documented, evaluation of the efficacy of RFLP technology as applied to crop improvement is far from complete. This investigation was conducted to study the relationship between RFLP-derived genotypes and heterotic patterns of a group of maize (Zea mays L.) inbred lines. A total of 22 inbreds was crossed to four testers (B73, B76, Mo17, and Va26) in combinations that minimized crossing within heterotic groups. Forty-seven single-cross progeny were subsequently evaluated for several agronomic traits (including grain yield and moisture, ear height, and root lodging) over 2–4 consecutive years at two to four Iowa locations in a randomized complete-block design. The inbred lines were subjected to RFLP analysis, which involved 47 genomic clones and the restriction enzymes EcoRI and HindIII. Hybrid RFLP patterns were predicted from their inbred parents. Modified Roger's distances were computed to estimate genetic distance among the inbred lines. Principal component analysis facilitated ascertainment of relative dispersion of the inbreds based on the frequency of variants at specific RFLP loci. Evident associations of variants with genes affecting agronomic traits were identified by principal component regression analysis, in which adjusted hybrid means were regressed on the matrix of hybrid variants frequencies. The hybrid means were adjusted by removing environmental effects, using residuals as dependent variables in the regression analysis. Results from this study suggest that RFLP analysis may be of value in allocating maize inbreds to heterotic groups, but no relationship between RFLP-based genetic distance and hybrid performance was apparent. Principal component regression identified variants potentially linked to genes that control specific agronomic traits.Joint contribution: USDA-ARS and Journal Paper No. J-13590 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA. Projects No. 2818 and 2778     

Linkage of restriction fragment length polymorphisms and isozymes in Citrus

Summary Genetic linkage analysis was performed using two segregating populations of citrus. One population arose from an intergeneric backcross of Citrus grandis (L.) Osb. cv Thong Dee and Poncirus trifoliata (L.) Raf. cv Pomeroy, using the former as the recurrent (female) parent. The other population came from an interspecific backcross of C. reticulata Blanco cv Clementine and C. x paradisi Macf. cv Duncan, using the former as the recurrent (male) parent. A total of 11 isozyme and 58 restriction fragment length polymorphisms were found to segregate in a monogenic fashion in one or both populations. Linkage analysis revealed that 62 of the loci examined mapped to 11 linkage groups, while 7 loci segregated independently from all other markers. Gene order was highly conserved between the maps generated from the two divergent segregating populations. Possible applications of the use of such maps in tree fruit breeding are discussed.     

Moused-amino-acid oxidase gene: Restriction fragment length polymorphism among mouse strains

Summary Genomic DNA was extracted from mice of 15 strains (A/J, AKR, BALB/c, C3H/He, C57BL/6, CBA/J, CD-1, CF#1, DBA/2, ddY/DAO⁺, ddY/DAO^–, ICR, NC, NZB and NZW) for the examination of the difference in the structure of thed-amino-acid oxidase gene among the mouse strains. The DNAs were digested with restriction endonucleases and analyzed by Southern hybridization usingd-amino-acid oxidase cDNA as a probe. The 15 strains showed the same hybridization patterns in theEcoRV,BamHI orBglII digestion. In theEcoRI digestion, the DBA/2 strain showed a different hybridization pattern from the other 14 strains. In thePvuII andXbaI digestion, C3H/He, CBA/J, ddY/DAO⁺ and NC strains were different from the other 11 strains. In thePstI andHindIII digestion, restriction fragment length polymorphisms were observed, and the 15 strains were classified into four groups according to their hybridization patterns. These results indicate that the 15 strains of mice carry a structurally similard-amino-acid oxidase gene, but there is a variation in its inside sequence among the groups of the strains.     

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.     

Analysis of three restriction fragment length polymorphisms in the human type II procollagen gene.

Cloned genomic DNA sequences corresponding to various regions of the human type II procollagen gene were used to analyze the DNA from 78 normal volunteers. Southern hybridization experiments detected polymorphic HindIII, BamHI, and EcoRI sites. The presence of the polymorphic HindIII site results in a 7.0-kilobase (kb) band, and the absence of this site results in a 14.0-kb band. When present, the BamH1 polymorphic site yields a 4.8-kb band, and when absent, yields a 7.2-kb band. The presence of the EcoRI polymorphic site results in a 3.7-kb band, and its absence results in a 7.0-kb band. Each polymorphic site was mapped. Analyses of the data demonstrated that the sites are present in overall gene frequencies of .39 for HindIII, .04 for BamHI, and .02 for EcoRI. Gene frequencies of the polymorphic sites were also studied with respect to race. The polymorphic sites are present in a Hardy-Weinberg distribution in the study population. Study of an extended family demonstrated that the segregation of the HindIII polymorphic site is consistent with Mendelian inheritance.     

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.     

Association analysis of lipid levels and apolipoprotein restriction fragment length polymorphisms

Summary We have analyzed the correlation between restriction site variants (RFLPs; restriction fragment length polymorphisms) of the apolipoprotein AI, AII, B, CI and CII genes and serum lipid levels in a sample of male Norwegians. We find no significant association between any of the RFLPs and lipid levels.     

The use of restriction fragment length polymorphisms in paternity analysis.

This paper examines the utility of restriction fragment length polymorphisms (RFLPs) for paternity analysis. While, on the average, 99% of falsely accused males can be excluded with the standard battery of blood group antigens, red cell enzymes, serum proteins, and HLA antigens, there are still mother-child pairs for whom the exclusion probability is not high. It has been suggested that additional resolution would be available with RFLPs. We have examined the strategic aspects of using RFLPs for paternity analysis, comparing the efficacy and cost of a multimarker haplotypic set with those of a comparable set of unlinked RFLPs, using published frequencies for the beta-globin complex, the serum albumin region, and the growth hormone region. There are four major findings. (1) Greater resolution is obtained with a carefully chosen set of tightly linked RFLPs producing chromosomal haplotypes than with a comparable set (same allele frequencies) of unlinked markers, but only if it is possible to establish linkage phase unambiguously. (2) Assay of linked sets is cheaper than is the assay of unlinked markers, but the cost advantage is optimized with sets of no more than two or three linked markers. (3) Also, with more than two or three tightly linked markers, the haplotypic frequencies are too poorly estimated to provide a reliable measure of the probability of paternity for unexcluded males, given the sample sizes likely to be available in the near future. (4) Optimal resolution, minimal cost, and acceptable accuracy are obtained with several independent sets of no more than two or three tightly linked RFLP markers each. With current technology, RFLP analysis is more expensive for the same level of genetic resolution than is the standard battery, but gradual replacement of the latter can be anticipated as economies of scale reduce the cost of the DNA technology.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.