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.     

The suitability of restriction fragment length polymorphisms as genetic markers in maize

Summary Strain identification in Zea mays by restriction fragment length polymorphism should be feasible due to the high degree of polymorphism found at many loci. The polymorphism in maize is apparently higher than that currently known for any other organism. Five randomly selected maize inbred lines were examined by Southern filter hybridization with probes of cloned low copy sequences. Typically, several alleles could be distinguished among the inbred lines with any one probe and an appropriately selected restriction enzyme. Despite considerable polymorphism at the DNA level, 16 RFLP markers in three inbred lines of maize were examined for six to 11 generations and found be stable. Mapping of RFLP markers in maize can be accelerated by the use of B-A translocation stocks, which enable localization of a marker to chromosome arm in one generation. The use of recombinant inbred lines in further refinement of the map is discussed.     

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.     

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 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.     

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.     

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.     

Genetic difference in somatic embryogenesis from seeds in melon (Cucumis melo L.)

Significant differences in somatic embryogenesis from melon seeds were observed among 18 cultivars; especially, cultivars Earl's Favorite and Barnett which produced a large number of somatic embryos. F₁ seeds were obtained by reciprocal crosses between cultivars. Some lines produced a large number of somatic embryos whereas others showed no or poor embryogenic response. Most of the F₁ seeds formed somatic embryos. The frequency of somatic embryogenesis decreased as compared to the parents with the highest potential. Transfer of the frequency of somatic embryogenesis from superior responding cultivars to inferior cultivars was proved. It was difficult to determine the mode of inheritance of somatic embryogenesis because there was a large variation in the range of somatic embryogenesis from F₂ seeds, and cytoplasmic effect was recognized in certain combinations.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Rapid investigation of French sourdough microbiota by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region

The aim of the present research is to identify rapidly the lactic acid bacteria (LAB) microflora of four natural French sourdoughs (GO, BF, VB and RF), applying a biphasic (restriction length polymorphism (RFLP) and sequencing) approach for bacterial identification. For this purpose, a database with the RFLP patterns of 30 lactobacilli type strains was created. So-developed ISR-RFLP algorithm was further applied for the differentiation and identification of 134 sourdough isolates. The 16S-23S rDNA intergenic spacer region was amplified by primers t^Ala and 23S/10, and then digested by HindIII, HinfI and α-TaqI enzymes. Nucleotide sequences of the cloned 16S-23S intergenic spacer region (ISR) were determined by the dideoxynucleotide chain termination method. The T7Prom and M13rev primers flanking the multiple cloning site of pCR2.1 DNA were used to sequence both DNA strands. The RFLP profile obtained upon digestion with HindIII, HinfI and α-TaqI enzymes can be used to discriminate Lactobacillus sanfranciscensis (66%), Lactobacillus panis (17%), Lactobacillus nantensis (11%) and Lactobacillus hammesii(6%) in sourdough GO, Lactobacillus sanfranciscensis (80%), Lactobacillus spicheri (14%) and Lactobacillus pontis(6%) in sourdoughs BF. In sourdoughs VB, which differed in the process temperature, we can differentiate Lactobacillus sanfranciscensis (89%) and Leuconostoc mesenteroidessubsp. mesenteroides (11%). Lactobacillus frumenti(47%), Lactobacillus hammesii (8%), and Lactobacillus paralimentarius (45%) were differentiated in sourdough RF.     

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.     

A linkage map ofBrassica rapa (syn.campestris) based on restriction fragment length polymorphism loci

Summary A detailed linkage map ofB. rapa (syn.campestris) was constructed based on segregation of 280 restriction fragment length polymorphism loci, detected by using 188 genomic DNA clones as probes on DNAs from a F₂ population of Chinese cabbage MichihilF×Spring broccoli. These genetic markers covered 1,850 centiMorgans (cM) and defined ten linkage groups, which equals the haploid chromosome number of this species. Extensive sequence duplication was evident by the detection of two or more segregating loci with each of 69 clones (36.7% of the total). Although some duplicated loci were randomly distributed throughout the genome, many had linkage arrangements that were conserved on different linkage groups, suggesting that large chromosome fragments were present in multiple copies. However, conservation in the linkage arrangement of duplicate loci throughout entire pairs of linkage groups was not observed. Single-copy loci were often found to be located within conserved duplicated regions, and linkage distances between some loci having conserved duplicated arrangements were substantially different between the duplicated regions. Structural rearrangements, such as insertions, deletions, and inversions or combinations of these events, seemed to be related to the alternations of map distances between duplicated loci and to the dispersal of duplicated chromosome fragments. These results suggest thatB. rapa has evolved in part by duplication of chromosomes or large chromosome fragments with subsequent structural rearrangements.     

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.     

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.     

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.     

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.     

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.     

Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis

Summary A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.     

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

Summary Preliminary analysis using nuclear RFLPs provided evidence that subspecies within Brassica rapa originated from two different centers. One center is in Europe, represented by turnip and turnip rape from which the oilseed sarson was derived. A second center is in South China containing a variety of Chinese vegetables of which pak choi and narinosa seem to be the most ancient forms. Based on RFLP data, the accessions of B. oleracea examined could be divided into three distinct groups, represented by thousand head kale, broccoli and cabbage. Thousand head kale and Chinese kale appear to be the primitive types. Observations of parallel variation among subspecies of both species are discussed.     

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.     

Intraspecific classification of melons (Cucumis melo L.) in view of their phenotypic and molecular variation

Cucumis melo L. (melon) genotypes differ widely in morphological and biochemical traits. Intraspecific classification of such variability has been difficult, and most taxonomists still rely on the work of Naudin (1859). A collection of 54 accessions representing diverse genotypes from 23 countries was surveyed. Morphological traits related to the vegetative and flowering stages and mature fruit morphology and quality parameters, e.g., taste, aroma, sugar composition and pH, were scored. These were used to construct a botanical-morphological dendrogram that generally reflected the classification ofCucumis melo into several horticultural varieties. DNA polymorphism among the accessions was assessed using the Inter-SSR-PCR and RAPD techniques that detected abundant DNA polymorphism among melon genotypes. Cluster analysis indicated that the largest divergence was between North American and Europeancantalupensis andinodorus cultivars as one group, and the more exotic varieties:conomon, chito, dudaim, agrestis andmomordica, as a second group. The molecular phylogeny agreed, broadly, with the classification of melon into two subspecies, and did not contradict the division into horticultural varieties. It was apparent, however, that the infra-specific division is rather loose, molecular variation being distributed continuously between and within cultivar groups. We suggest that despite the morphological diversity, separation between varietal-groups may be based on a too small number of genes to enable unambiguous infra-specific classification based on DNA diversity.