Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species

A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.     

Evaluation of restriction fragment length polymorphism in Cucumis melo

Summary The objectives of this study were to assess the degree of restriction fragment length polymorphism (RFLP) in Cucumis melo and to determine interrelationships among cultivated varieties. Initial screening of a genomic PstI library revealed that approximately 40% of the clones were repetitive. A total of 162 unique and low-copy sequence clones were hybridized to seven diverse accesions of C. melo and a C. sativus cultivar Pacer to evaluate RFLP variation. Of these, 130 probes (80%) detected a polymorphism between C. melo accessions and C. sativus, and the majority were polymorphic with more than one enzyme digest. In contrast, only 53 probes (33%) were useful in differentiating at least one of the seven accessions. Of those, only 9% were informative with more than one enzyme digest. This indicates that within C. melo, the differences among accessions are due to infrequent base substitutions, whereas between the two species, differences are mainly due to genome rearrangements such as insertions and deletions or numerous base substitutions. Of the informative probes, 34 were used in analyzing 44 C. melo lines to establish a data base of RFLP hybridization patterns. Percent similarity based on RFLP profiles was computed among lines and analyzed by principal component analysis, to visualize relationships among lines. There were clear demarcations among, but not within, muskmelon and honeydew groups.     

Plasmid profiles and restriction fragment length polymorphism of Rhizobium leguminosarum bv. viciae in field populations

Abstract 56 isolates of Rhizobium leguminosarum biovar viciae from one field were characterized by analysis of plasmid profile, total DNA restriction pattern and restriction fragment length polymorphism (RFLP) of 2 chromosomal regions and of symbiotic (Sym) plasmid. Different levels of similarity exist in patterns generated by the different techniques. At the level of partial similarity these techniques give comparable results for more than 80% of the isolates, with the exception of RFLP profiling with the Sym probe. Analysis at this level allows the grouping of the isolates that have most of their non-Sym genome similarly organized. At the level of total similarity, the techniques are no more equivalent and provide complementary information on possible evolution of the different elements of the genome identified by each specific technique. The non-Sym plasmids defining classes were strongly associated with specific chromosomal backgrounds. In contrast, variations in Sym plasmids were not related with variations in the remaining genome. Host range towards chromosomes was variable among the Sym plasmids, which may reflect plasmid transfer between strains.     

In vitro propagation, restriction fragment length polymorphism, and random amplified polymorphic DNA analyses of Angelica plants

Angelica acutiloba, a medicinal plant used as a natural medicine Touki, was clonally propagated through axillary buds in vitro. No substantial differences were found in the random amplified polymorphic DNA (RAPD) pattern between the original A. acutiloba and the plant propagated in vitro, suggesting no changes in the DNA sequences and structure during in vitro propagation. The genetic similarities of several Angelica plants were investigated by restriction fragment length polymorphism (RFLP) and RAPD analyses. The RFLP and RAPD patterns of A. sinensis Diels were substantially different from those of A. acutiloba. Using ten different restriction enzymes, no RFLP was observed in the varieties of A. acutiloba. By RAPD analysis, A. acutiloba varieties can be classified into two major subgroups, i.e., A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyamae Hikino. The varieties of A. acutiloba Kitagawa in Japan and Angelica spp. in northeast China exhibited a very close genetic relationship. Received: 13 March 1998 / Revision received: 28 July 1998 / Accepted: 21 August 1998     

Linkage arrangement of restriction fragment length polymorphism loci in Brassica oleracea

Summary A detailed genetic linkage map of Brassica oleracea was constructed based on the segregation of 258 restriction fragment length polymorphism loci in a broccoli × cabbage F₂ population. The genetic markers defined nine linkage groups, covering 820 recombination units. A majority of the informative genomic DNA probes hybridized to more than two restriction fragments in the F₂ population. Duplicate sequences having restriction fragment length polymorphism were generally found to be unlinked for any given probe. Many of these duplicated loci were clustered non-randomly on certain pairs of linkage groups, and conservation of the relative linkage arrangement of the loci between linkage groups was observed. While these data support previous cytological evidence for the existence of duplicated regions and the evolution of B. oleracea from a lower chromosome number progenitor, no evidence was provided for the current existence of blocks of homoeology spanning entire pairs of linkage groups. The arrangement of the analyzed duplicated loci suggests that a fairly high degree of genetic rearrangement has occurred in the evolution of B. oleracea. Several probes used in this study were useful in detecting rearrangements between the B. oleracea accessions used as parents, indicating that genetic rearrangements have occurred in the relatively recent evolution of this species.     

PCR-based restriction fragment length polymorphism (RFLP) analysis and serotyping of Campylobacter jejuni isolates from diarrheic patients in China and Japan

Abstract A molecular typing approach for Campylobacter jejuni was applied with restriction fragment length polymorphism (RFLP) analysis of a 702-bp PCR-amplified portion of the flagellin-A ( flaA ) gene. We analyzed a total of 179 strains, including 69 independent clinical isolates from diarrheic patients in Japan, 85 isolates in China, and 25 heat-stable (HS) serotype strains by Penner and Hennessy ((1980) J. Clin. Microbiol. 12, 732–737). Six Afa I, seven Mbo I, and five Hae III RFLPs were found in the 702-bp flaA segment from the 179 strains. Using a combination of these three enzymes, 25 separate RFLP groups were recognized. While 59 of 154 (38.3%) strains obtained in Japan and China were nontypeable by the HS antigenic scheme, all but two of 154 (98.7%) could be typed by RFLP typing. All 11 isolates of HS-19 strains, which are frequently isolated from Guillain-Barré syndrome (GBS) patients, showed an identical RFLP pattern (Cj-1), and Cj-1 consisted only of HS-19 strains. This suggests that the HS-19:Cj-l strain is distinct among C. jejuni strains. This molecular typing method provides a rapid and reliable typing scheme for epidemiological studies of C. jejuni , and may also be useful for the analysis of C. jejuni subtypes from GBS patients.     

Detection of linkage between quantitative trait loci and restriction fragment length polymorphisms using inbred lines

Summary In segregating populations, large numbers of individuals are needed to detect linkage between markers, such as restriction fragment length polymorphisms (RFLPs), and quantitative trait loci (QTL), limiting the potential use of such markers for detecting linkage. Fewer individuals from inbred lines are needed to detect linkage. Simulation data were used to test the utility of two methods to detect linkage: maximum likelihood and comparison of marker genotype means. When there is tight linkage, the two methods have similar power, but when there is loose linkage, maximum likelihood is much more powerful. Once inbred lines have been established, they can be screened rapidly to detect QTL for several traits simultaneously. If there is sufficient coverage of the genome with RFLPs, several QTL for each trait may be detected.     

Characterisation of the lpdA gene from Neisseria meningitidis by polymerase chain reaction, restriction fragment length polymorphism and sequencing

P64k protein from Neisseria meningitidis is well recognised in sera from individuals convalescent from meningococcal disease or vaccinated with the Cuban antimeningococcal vaccine VA-MENGOC-BC. The presence of the protein in more than 80 meningococcal strains has also been verified. It is immunogenic in animal models and the antibodies elicited show bactericidal activity against meningococci. To further investigate at the molecular level whether lpdA, the gene coding for P64k protein, is conserved among different N. meningitidis strains, a total of 20 strains isolated from different geographic areas were differentiated on the basis of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the lpdA gene and restriction endonuclease digestion with HpaII. Although a total of five different PCR-RFLP patterns were present, nucleotide sequence determination showed that identity levels were as high as 93-99% among the N. meningitidis strains analysed.     

Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysis

AIMS: The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. METHODS AND RESULTS: Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37 degrees C) treatment of high-strength [10 g chemical oxygen demand (COD) l(-1)] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum- and Methanococcus-like organisms and uncultured Crenarchaeota in R1. CONCLUSIONS: The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities.     

Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

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).To whom correspondence should be addressed.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary RFLPs were used to study genome evolution and phylogeny in Brassica and related genera. Thirtyeight accessions, including 10 accessions of B. rapa (syn. campestris), 9 cultivated types of B. oleracea, 13 nine-chromosome wild brassicas related to B. oleracea, and 6 other species in Brassica and allied genera, were examined with more then 30 random genomic DNA probes, which identified RFLPs mapping to nine different linkage groups of the B. rapa genome. Based on the RFLP data, phylogenetic trees were constructed using the PAUP microcomputer program. Within B. rapa, accessions of pak choi, narinosa, and Chinese cabbage from East Asia constituted a group distinct from turnip and wild European populations, consistent with the hypothesis that B. rapa had two centers of domestication. A wild B. rapa accession from India was positioned in the tree between European types and East Asian types, suggesting an evolutionary pathway from Europe to India, then to South China. Cultivated B. oleracea morphotypes showed monophyletic origin with wild B. oleracea or B. alboglabra as possible ancestors. Various kales constitute a highly diverse group, and represent the primitive morphotypes of cultivated B. oleracea from which cabbage, broccoli, cauliflower, etc. probably have evolved. Cauliflower was found to be closely related to broccoli, whereas cabbage was closely related to leafy kales. A great diversity existed among the 13 collections of nine-chromosome wild brassicas related to B. oleracea, representing various taxonomic states from subspecies to species. Results from these studies suggested that two basic evolutionary pathways exist for the diploid species examined. One pathway gave rise to B. fruticulosa, B. nigra, and Sinapis arvensis, with B. adpressa or a close relative as the initial ancestor. Another pathway gave rise to B. oleracea and B. rapa, with Diplotaxis erucoides or a close relative as the initial ancestor. Raphanus sativus and Eruca sativus represented intermediate types between the two lineages, and might have been derived from introgression or hybridization between species belonging to different lineages. Molecular evidence for an ascending order of chromosome numbers in the evolution of Brassica and allied genera was obtained on the basis of RFLP data and phylogenetic analysis.     

Restriction fragment length polymorphism species-specific patterns in the identification of white truffles

A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.     

Sheep linkage mapping: restriction fragment length polymorphism detection with heterologous cDNA probes

Summary. A selection of cattle, human and sheep cDNA probes were screened against sheep genomic DNA, cut with 10 different restriction enzymes, to assess the usefulness of these probes for restriction fragment length polymorphism (RFLP) linkage studies in sheep. Two-thirds of the cattle cDNA probes showed moderate to strong homology with sheep DNA samples, compared with less than half of the human cDNA probes at the final washing stringency chosen for the experiments. The set of probes tested detected a useful frequency of RFLPs. Fifty-seven per cent of probes showing moderate to strong homology identified RFLPs with one or more restriction enzymes. Restriction enzymes that detected RFLPs most frequently in sheep were Taq I and Msp I. The results show that sheep and cattle cDNA probes, including candidate genes for production traits, identified a high frequency of RFLPs suitable for genetic mapping in sheep.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

Restriction fragment length polymorphism diversity in soybean

Summary Fifty-eight soybean accessions from the genus Glycine, subgenus Soja, were surveyed with 17 restriction fragment length polymorphism (RFLP) genetic markers to assess the level of molecular diversity and to evaluate the usefulness of previously identified RFLP markers. In general, only low levels of molecular diversity were observed: 2 of the 17 markers exhibited three alleles per locus, whereas all others had only two alleles. Thirty-five percent of the markers had rare alleles present in only 1 or 2 of the 58 accessions. Molecular diversity was least among cultivated soybeans and greatest between accessions of different soybean species such as Glycine max (L.) Merr. and G. soja Sieb. and Zucc. Principal component analysis was useful in reducing the multidimensional genotype data set and identifying genetic relationships.     

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