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.     

Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera L.

Forty-six accessions of grapevine (V. vinifera L.) were compared by restriction fragment length polmorphism (RFLP) analysis, and 111 informative or unique restriction fragments were found that revealed an important level of polymorphism. RFLP patterns were compared in two ways: by calculating electrophoretic similarity degree values further analyzed by principal component analysis and by studying the distribution of rare restriction fragments. Six taxonomic groups could be defined, which partially confirmed relationships derived from ampelographical data. Our data support the existence of ecogeographical groups.     

Restriction fragment length polymorphism and allozyme linkage map of Cuphea lanceolata

Summary Cuphea lanceolata Ait. has had a significant role in the domestication of Cuphea and is a useful experimental organism for investigating how medium-chain lipids are synthesized in developing seeds. To expand the genetics of this species, a linkage map of the C. lanceolata genome was constructed using five allozyme and 32 restriction-fragment-length-polymorphism (RFLP) marker loci. These loci were assigned to six linkage groups that correspond to the six chromosomes of this species. Map length is 288 cM. Levels of polymorphism were estimated for three inbred lines of C. lanceolata and an inbred line of C. viscosissima using 84 random genomic clones and two restriction enzymes, EcoRI and HindIII. Of the probes 29% detected RFLPs between C. lanceolata and C. viscosissima lines. Crosses between these species can be exploited to expand the map.     

Restriction fragment length polymorphism differences among Illinois long-term selection oil strains

Restriction fragment length polymorphism (RFLP) analysis was used to characterize variability in the Illinois Long-Term Selection Experiment oil strains. Considerable polymorphism was detected within each oil strain and among oil strains. Fifty-two individual plants from each of the Illinois High Oil (IHO), Illinois Low Oil (ILO), Reverse High Oil (RHO) and Reverse Low Oil (RLO) strains were sampled to determine RFLP allele/variant frequencies. Generation 90 was sampled for IHO, RHO, and RLO whereas generation 87 was sampled for ILO. Forty-nine RFLP probes distributed throughout the maize genome were used. Chi-square analysis was performed to determine if RFLP genotypes at each of the 49 RFLP loci were significantly different among strains. Oil strains that have been separated for 90 generations showed high levels of significantly-different RFLP genotypic frequencies. The comparison of ILO vs RHO gave only significant chi-square values while the comparisons of IHO vs RLO and RHO vs RLO had 111 ratios of significant to non-significant chi-square values. Strains that have been separated for only 42 generations showed a lower level of significantly-different RFLP genotypic frequencies. The comparisons of IHO vs RHO and ILO vs RLO both had only a 32 ratio of significant to non-significant chi-squares values. Detection of multiple RFLP alleles/variants among the oil strains was common with 59% of the RFLP loci examined exhibiting multiple variants. A number of RFLP loci in RHO (3) and RLO (11) were associated with a trend in RFLP allele/variant frequencies consistent with a response to reverse selection for oil concentration.     

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.     

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.     

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.     

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.     

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.     

Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species

Abstract DNA from several species of fast growing mycobacteria displayed a characteristic restriction fragment length polymorphism (RFLP) pattern when hybridizated to a Mycobacterium fortuitum 16S rRNA gene fragment. The resulting patterns were identical when comparing different strains belonging to the same species. The RFLP results were consistent with those obtained by DNA-DNA hybridization studies. Using this approach, we have been able to identify the number of copies for 16S rRNA genes in several fast-growing mycobacteria.     

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.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

Molecular marker analysis of Helianthus annuus L. 1. Restriction fragment length polymorphism between inbred lines of cultivated sunflower

cDNA and PstI genomic clones have been used to assess levels of restriction fragment length polymorphism (RFLP) in Helianthus annuus and to determine the inter-relationships between a diverse set of 24 inbred lines. Of the cDNA clones screened 45% were useful as RFLP probes, compared to less than 20% from the PstI library, which showed high levels of redundancy for high copy sequences. Fifty-seven low-copy DNA probes (23 PstI and 34 cDNA clones) were used to fingerprint 12 maintainer (B) lines and 12 restorer (R) lines. The average number of RFLP variants per probe was found to be 3.2, with a mean polymorphic index of 0.49, indicating that high levels of nuclear DNA polymorphism are to be found in cultivated sunflower. Cluster and principal coordinate analysis of the fingerprinting data clearly separated the maintainer and restorer lines, but there was a degree of association between 2 unbranched R-lines and the B-line germ plasm pool.     

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.     

Restriction fragment length polymorphism analysis of loci associated with disease resistance genes and developmental traits in Pisum sativum L.

An F₂ population of pea (Pisum sativum L.) consisting of 174 plants was analysed by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. Ascochyta pisi race C resistance, plant height, flowering earliness and number of nodes were measured in order to map the genes responsible for their variation. We have constructed a partial linkage map including 3 morphological character genes, 4 disease resistance genes, 56 RFLP loci, 4 microsatellite loci and 2 RAPD loci. Molecular markers linked to each resistance gene were found: Fusarium wilt (6 cM from Fw), powdery mildew (11 cM from er) and pea common Mosaic virus (15 cM from mo). QTLs (quantitative traits loci) for Ascochyta pisi race C resistance were mapped, with most of the variation explained by only three chromosomal regions. The QTL with the largest effect, on chromosome 4, was also mapped using a qualitative, Mendelian approach. Another QTL displayed a transgressive segregation, i.e. the parental line that was susceptible to Ascochyta blight had a resistance allele at this QTL. Analysis of correlations between developmental traits in terms of QTL effects and positions suggested a common genetic control of the number of nodes and earliness, and a loose relationship between these traits and height.     

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.     

Single-enzyme amplified fragment length polymorphism and its applicability for Salmonella epidemiology

Amplified fragment length polymorphism (AFLP) is a PCR-based DNA fingerprinting technique whereby restriction fragments may be visualized without prior knowledge of nucleotide sequences. In AFLP analysis, bacterial genomic DNA is digested with a restriction enzyme and ligated to adapter oligonucleotides. A subset of DNA fragments are then amplified using primers which contain adapter-defined sequences. Selective amplification is achieved by the use of primers containing adapter-defined sequences with one additional arbitrary nucleotide. We used four primers complementary to the adapter sequence, but each differing in the final 3' base that extended into the fragment DNA. The usefulness of these primers for fingerprinting Salmonella enterica was assessed in a hierarchical manner. Using a single-enzyme approach (SAFLP) we have used this method to fingerprint 30 strains of S. enterica, belonging to 14 different serotypes. SAFLP profiles derived from Hind III fragments differentiated between the serotypes. In addition, SAFLP profiles for each serotype differentiated between the phage types and individual strains. The technique is significantly faster to perform than other DNA-based methods and has given reproducible and discriminatory results. This hierarchical SAFLP technique may provide a valuable addition to existing methods for the DNA fingerprinting of S. enterica for epidemiological studies.     

Mapped DNA probes from loblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm. Thirty complementary DNA and two genomic DNA probes from loblolly pine were hybridized to Southern blots containing DNA from five species of Pinus (P. elliottii, P. lambertiana, P. radiata, P. sylvestris, and P. taeda), one species from each of four other genera of Pinaceae (Abies concolor, Larix laricina, Picea abies, and Pseudotsuga menziesii), one species from each of three other families of Coniferales [Sequoia sempervirens (Taxodiaceae), Torreya californica (Taxaceae) and Calocedrus decurrens (Cupressaceae)], and to one angiosperm species (Populus nigra). Results showed that mapped DNA probes from lobolly pine will cross-hybridize to genomic DNA of other species of Pinus and some other genera of the Pinaceae. Only a small proportion of the probes hybridized to genomic DNA from three other families of the Coniferales and the one angiosperm examined. This study demonstrates that mapped DNA probes from loblolly pine can be used to construct RFLP maps for related species, thus enabling the opportunity for comparative genome mapping in conifers.     

Restriction fragment length polymorphisms in the genusActinidia (Actinidiaceae)

A series of chloroplast and nuclear probes were used to examine restriction fragment length polymorphisms (RFLPs) in kiwifruit (Actinidia deliciosa) and three of its closest relatives. The four species fell into two pairs, withA. chinensis andA. deliciosa closely related but some distance away from the other two species,A. latifolia andA. eriantha. The results are consistent with the hypothesis that the diploid species,A. chinensis, is a precursor ofA. deliciosa, which is hexaploid.