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  • 1
    ISSN: 1617-4623
    Keywords: Potato ; Solanum tuberosum ; Phytophthora infestans ; RFLP ; R genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F1 populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F1 populations analysed, whereas the RI allele segregated with a 1:1 ratio as expected in five F1 populations. The mode of inheritance of the R10 allele could not be deduced as only very few F1 hybrids bearing R10 were obtained. Linkage analysis in two F1 populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromdsome XI.
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  • 2
    ISSN: 1573-8469
    Keywords: transposon tagging ; potato ; T-DNA IPCR ; RFLP mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A general strategy for the isolation of disease resistance genes is presented, employing a two-step approach of transposon targeting near genes of interest followed by transposon tagging. A library of transposon (Ac/Ds) transformants in a self fertile potato diploid are being mapped by deriving genomic DNA probes flanking the transposon containing T-DNA insertions with the inverse polymerase chain reaction and using these probes for RFLP analysis. We have produced a large number of transposon (Ac/Ds) transformants in a self fertile potato diploid. Genomic DNA probes, flanking the transposon containing T-DNA insertions, are produced by the inverse polymerase chain reaction (IPCR) and mapped by restriction fragment length polymorphism (RFLP) analysis in a segragating potato location. A transposon mapped close to a resistance gene can be recombined cis to the gene and used for efficient transposon targeting due to preferential transposition to linked sites.
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  • 3
    ISSN: 1573-8469
    Keywords: antibacterial ; antimicrobial ; genetic engineering ; thionin ; toxicity assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic forClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker on tomato,C. m. subsp.sepedonicus, the causal agent of ring rot on potato, andXanthomonas campestris pv.vesicatoria, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.
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  • 4
    ISSN: 1573-8469
    Keywords: Monoclonal antibodies ; single chain antibodies ; scFv ; potato cyst nematodes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests. Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (VL) and heavy chain (VH) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.
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  • 5
    ISSN: 1573-5028
    Keywords: Antimicrobial ; ion channel ; phosphoinositides ; plant defence ; thionins ; toxins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Thionins are low-molecular-weight proteins (M r ca. 5000) occurring in seeds, stems, roots and leaves of a number of plant species. The different members of this family of plant proteins show both sequence and structural homology, and are toxic to bacteria, fungi, yeasts and various naked cells in vitro. Toxicity requires an electrostatic interaction of the positively charged thionin with the negatively charged phospholipids making up the membrane, followed by either pore formation or a specific interaction with a certain lipid domain. This domain might be composed of phosphoinositides, which mediate transduction of environmental signals in eukaryotes. Their in vitro toxicity to plant pathogenic bacteria and fungi could reflect a direct role in plant defence, although, in view of the many divergent activities displayed by thionins both in vitro and in vivo, a biological role other than inhibition of microbial growth is equally plausible.
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  • 6
    ISSN: 1617-4623
    Keywords: Heterodera schachtii ; Beta vulgaris ; Monosomic additions ; Molecular markers ; Disease resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In cultivated beet no useful level of resistance of the beet cyst nematode (BCN) Heterodera schachtii Schm. has been found, unlike the situation in wild species of the section Procumbentes. Stable introgression of resistance genes from the wild species into Beta vulgaris has not been achieved, but resistant monosomic additions (2n =18 + 1), diploids of B. vulgaris with an extra alien chromosome carrying the resistance locus, have been obtained. Here we describe a new series of resistant monosomic fragment addition material of B. patellaris chromosome 1 (pat-1). We further describe the cloning of a single-copy DNA marker that specifically hybridizes with a monosomic addition fragment of approximately 8 Mb (AN5-90) carrying the BCN resistance locus. This marker and another fragment-specific, single-copy DNA marker probably flank the BCN locus on the addition fragment present in the AN5-203 material, which is approximately 19 Mb in size. Furthermore, several specific repetitive DNA markers have been isolated, one of which hybridizes to AN5-90 and also to DNA from a smaller DNA segment of Beta procumbens, present in line B883, carrying a BCN resistance locus introgressed into the B. vulgaris genome. This suggests that the specific repetitive marker is closely linked to the BCN locus.
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  • 7
    ISSN: 1432-2242
    Keywords: Potato cyst-nematode ; Quantitative trait loci RFLP ; Distorted segregation ; Solanum spegazzinii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A high level of resistance toGlobodera pallida pathotypes Pa2 and Pa3 exists inSolanum spegazzinii, a wild relative of potato (S. tuberosum ssp.tuberosum). Here we report the mapping of loci involved in quantitatively-inherited nematode resistance with the use of RFLPs. One major locus,Gpa, was mapped on chromosome 5 and two minor loci on chromosomes 4 and 7 ofS. spegazzinii. Additionally, the contribution of the susceptible parent to nematode resistance was determined. TheGpa locus was solely responsible for the high resistance level found in the segregating population. However, the RFLP marker closely linked to this resistance locus showed a distorted segregation, with a shortage of plants having the resistance linked allele. Our results indicate that a prediction of the genetic constitution of a quantitative trait based solely on phenotypic observations can lead to erroneous conclusions.
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  • 8
    ISSN: 1432-2242
    Keywords: Morphological markers ; Isozymes ; Non-inbred species ; Combined map ; JoinMap
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A genetic map of potato (Solanum tuberosum L.) integrating molecular markers with morphological and isozyme markers was constructed using a backcross population of 67 diploid potato plants. A general method for map construction is described that differs from previous methods employed in potato and other outbreeding plants. First, separate maps for the female and male parents were constructed. The female map contained 132 markers, whereas the male map contained 138 markers. Second, on the basis of the markers in common the two integrated parental maps were combined into one with the computer programme JoinMap. This combined map consisted of 175 molecular markers, 10 morphological markers and 8 isozyme markers. Ninety-two of the molecular markers were derived from DNA sequences flanking either T-DNA inserts in potato or reintegrated maize transposable elements originating from these T-DNA constructs. Clusters of distorted segregation were found on chromosomes 1,2,8 and 11 for the male parent and chromosome 5 for both parents. The total length of the combined map is 1120 cM.
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  • 9
    ISSN: 1432-2242
    Keywords: Key words  RFLPs ; Solanum spegazzinii ; Globodera sp. ; Interlocus interaction ; Intralocus interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract   A backcross population, derived from the cross (S. tuberosum×S. spegazzinii)×S. tuberosum was used to map QTLs involved in nematode resistance, tuber yield and root development. Complete linkage maps were available for the interspecific hybrid parent as well as the S. tuberosum parent, and interval mapping for all traits was performed for both. Additionally, the intra- and inter-locus interactions of the QTLs were examined. The Gro1.2 locus, involved in resistance to G. rostochiensis pathotype Ro1, that was previously mapped in the S. tuberosum×S. spegazzinii F1 population, was located more precisely on chromosome 10. A new resistance locus, Gro1.4, also conferring resistance to G. rostochiensis pathotype Ro1, was found on chromosome 3. Different alleles of this locus originating from both parents contributed to the resistant phenotype, indicating multiallelism at this locus. No interlocus interactions were observed between these two resistance loci. For resistance to G. pallida no QTLs were detected. One minor QTL involved in tuber yield was located on chromosome 4. Two QTLs involved in root development and having large effects were mapped on chromosomes 2 and 6 and an epistatic interaction was found between these two loci.
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  • 10
    ISSN: 1432-2242
    Keywords: RFLPs ; Solanum spegazzinii ; Globodera sp ; Interlocus interaction ; Intralocus interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A backcross population, derived from the cross (S. tuberosumxS. spegazzinii)xS. tuberosum was used to map QTLs involved in nematode resistance, tuber yield and root development. Complete linkage maps were available for the interspecific hybrid parent as well as the S. tuberosum parent, and interval mapping for all traits was performed for both. Additionally, the intra- and inter-locus interactions of the QTLs were examined. The Gro1.2 locus, involved in resistance to G. rostochiensis pathotype Ro1, that was previously mapped in the S. tuberosumxS. spegazzinii F1 population, was located more precisely on chromosome 10. A new resistance locus, Gro1.4, also conferring resistance to G. rostochiensis pathotype Ro1, was found on chromosome 3. Different alleles of this locus originating from both parents contributed to the resistant phenotype, indicating multiallelism at this locus. No interlocus interactions were observed between these two resistance loci. For resistance to G. pallida no QTLs were detected. One minor QTL involved in tuber yield was located on chromosome 4. Two QTLs involved in root development and having large effects were mapped on chromosomes 2 and 6 and an epistatic interaction was found between these two loci.
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