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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Applied microbiology and biotechnology 38 (1992), S. 70-76 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·μl−1 and high MgCl2 levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.
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  • 2
    ISSN: 1432-2048
    Keywords: Feedback regulation (nodulation) ; Medicago (nodulation) ; Nodulation (spontaneous) ; Rhizobium ; Symbiosis (legume-Rhizobium)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.
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  • 3
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Co-inoculation ; Medicago ; Mutant (Rhizobium) ; Nodulation ; Rhizobium ; Root nodule initiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·103 bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (103 cells/plant) were co-inoculated with 106 cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular “signals” that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.
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  • 4
    ISSN: 1432-2048
    Keywords: Medicago ; Mutant (Rhizobium) ; Rhizobium (host-specificity mutants) ; Root nodule initiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pairs of Rhizobium meliloti nod mutants were co-inoculated onto alfalfa (Medicago saliva L.) roots to determine whether one nod mutant could correct, in situ, for defects in nodule initiation of another nod mutant. None of the Tn5 or nod deletion mutants were able to help each other form nodules when co-inoculated together in the absence of the wild-type. However, as previously observed, individual nod mutants significantly increased nodule initiation by low dosages of co-inoculated wild-type cells. Thus, nod mutants do produce certain signal substances or other factors which overcome limits to nodule initiation by the wild-type. When pairs of nod mutants were co-inoculated together with the wild-type, the stimulation of nodulation provided by individual nodABC mutants was not additive. However, clearly additive or synergistic stimulation was observed between pairs of mutants with a defective host-specificity gene (nodE, nodF, or nodH). Each pair of host-specificity mutants stimulated first nodule formation to nearly the maximum levels obtainable with high dosages of the wild-type. Mutant bacteria were recovered from only about 10% of these nodules, whereas the co-inoculated wild-type was present in all these nodules and substantially outnumbered mutant bacteria in nodules occupied by both. Thus, these mutant co-inoculants appeared to help their parent in situ even though they could not help each other. Sterile culture filtrates from wild-type cells stimulated nodule initiation by low dosages of the wild-type, but only when a host-specificity mutant was also present. The results from our studies seem consistent with the possibility that pairs of host-specificity mutants are able to help the wild-type initiate nodule formation by sustained production of complementary signals required for induction of symbiotic host responses.
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  • 5
    ISSN: 1617-4623
    Keywords: DNA amplification fingerprinting (DAF) ; PCR ; Primer-template interactions ; Single oligonucleotide primer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5′ terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3′ terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3′-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.
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  • 6
    ISSN: 1617-4623
    Keywords: Amplification fragment length polymorphism ; Arbitrary primers ; DNA amplification fingerprinting (DAF) ; Genetic polymorphism ; Nodulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F2 families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.
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  • 7
    ISSN: 1573-5060
    Keywords: ASAP ; bermudagrass ; cultivar identification ; Cynodon ; DAF analysis ; genetic stability ; mini-hairpin primers ; off-types
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract DNA amplification fingerprinting (DAF) and arbitrary signatures from amplification profiles (ASAP) were used to evaluate the genetic stability of two important bermudagrass (Cynodon) cultivars, the interspecific cross Tifgreen and its somatic mutant Tifdwarf, and study genetic diversity and origin of derived bermudagrass off-types that exhibit patches of contrasting morphology and performance. Mini-hairpin primers produced complex and reproducible DAF and ASAP profiles with high levels of polymorphic DNA, and established genetic relationships between 11 Tifgreen and 8 Tifdwarf turf plot accessions and 16 off-types. DAF analysis revealed an average 14.1 ± 5.6 (SE) polymorphic bands/primer within cultivar accessions. In contrast, the higher resolving power of ASAP detected 24.5 ± 2.1 polymorphic bands/primer. Similarly, DAF and ASAP produced 13.0 ± 5.5 (SE) and 20 ± 2.8 polymorphic bands/primer within off-types, respectively. Phenetic analysis using cluster (UPGMA) and ordination (PCO) techniques showed that both Tifdwarf and Tifgreen were genetically unstable. The analysis showed that almost all cultivar accessions and one-half of the off-types studied were genetically distinct, but very close to each other. In this case, genetic variation was probably the result of somatic mutations. The other off-types and some Tifgreen accessions represented a genetically distant and diverse bermudagrass group of interspecific hybrid (n=27) origin. Off-types were probably the result of sod contamination. Results complement a previous study that established that the interspecific Tifway bermudagrass was genetically stable whereas derived off-types were contaminants rather than somatic mutants. Tifgreen and Tifdwarf showed genetic instabilities that were readily detected by DNA amplification with mini-hairpin primers. The present study offers a direct molecular alternative capable of evaluating the genetic stability of selected cultivars.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Plant and soil 148 (1993), S. 265-270 
    ISSN: 1573-5036
    Keywords: autoregulation ; Bradyrhizobium japonicum ; Glycine max ; nodulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The distribution of nodules of soybean (Glycine max (L.) Merr.) cultivar Bragg and the supernodulating mutant derivative nts382 was examined on the primary root relative to the first emerging lateral root, and on laterals relative to the base of the roots of plants grown in sand-vermiculite. Mutant nts382 nodulates profusely even in the presence of nitrate and appears defective in a systemic autoregulatory response that regulates nodule number in soybean. Nodules were clustered on primary roots about the first 4 cm down from the first emerging lateral root in both genotypes. Nodulation profiles showed reduced nodulation in younger and older regions of the primary root. Similarly, nodules appeared clustered close to the base of the lateral roots. Decreasing inoculum dose shifted nodule emergence to younger regions of the primary root and to lateral roots emerging in younger portions of the primary root. Our results indicate that the supernodulating mutant is able to regulate nodule number in both primary and lateral roots in the particulate matrix.
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  • 9
    ISSN: 1573-5036
    Keywords: bacterial adsorption ; calcium ; magnesium ; Medicago sativa ; pH ; Rhizobium meliloti ; root surface
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Adsorption ofRhizobium meliloti L5-30 in low numbers to alfalfa (Medicago sativa L.) roots was dependent on the presence of divalent cations, and required neutral pH. Adsorption was proportional to Ca and/or Mg concentrations up to 1.5 mM. Ca was not substituted by Sr, Ba or Mn. Adsorption was abolished and viability decreased at pH≤6. When lowering pH, higher Ca concentrations were required to attain similar adsorption levels, indicating a marked interactive effect between Ca and H ions. Pretreatment of the roots with Ca and low pH did not affect subsequent adsorption of the bacteria. However, Ca pretreatment ofR. meliloti sustained further adsorption at low Ca levels and low pH substantially affected their ability to adsorb. Low pH appears to affect the stability of binding causing desorption of the previously bound bacteria. The presence of saturating concentrations of heterologousR. leguminosarum bv.trifolii A118, did not prevent the expression of divalent cations and pH requirements, as well as their interaction. Our results suggest that rhizobial binding to the root surface already shows the Ca and pH dependence of alfalfa nodulation, which was generally associated to some event prior to rhizobial penetration of root hairs.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Crop science 38 (1998), S. 1415-1424 
    ISSN: 1435-0653
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Turfgrasses constitute an heterogeneous group of species differing widely in reproductive strategy, genome organization, and evolutionary history. Cultivars derived from vegetative propagation or from apomictic or self-pollinated species can be homogeneous at the genetic level, exhibiting little genetic variation. Alternatively, cultivars with an outcrossing breeding system can be genetically heterogeneous, such as those from open-pollinated seeded species. A careful study of the degree and distribution of turfgrass genetic variation is therefore essential for the efficient selection of superior plant material for breeding, an adequate management of genetic resources, and the effective preservation of biodiversity. An array of molecular techniques have targeted nucleic acids to evaluate molecular diversity at the species, population, and within-population levels. These techniques have only recently been applied to the breeding and management of turfgrass germplasm. Using the hybridization and amplification of nucleic acids, DNA profiling techniques have established patterns of genetic variation at the species level in grass systematics, and at the subspecies level in the study of natural populations, breeding lines, cultivars, and accessions. In some cases, chromosome analysis by flow cytometry and genomic in situ hybridization have notably complemented the use of nucleic acid markers. DNA profiling techniques have also assessed the genetic stability of cultivars and the appearance of offtypes, and have provided molecular estimates of turfgrass evolution. The review of current efforts to evaluate turfgrass genetic diversity clearly indicates that the application of the tools of genome analysis to the study of germplasm diversity may finally unlock the genetic potential of wild and cultivated turfgrass resources.
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