WO2000006753A1 - Procede de genie genetique servant a obtenir une resistance aux nematodes chez les solanacees - Google Patents

Procede de genie genetique servant a obtenir une resistance aux nematodes chez les solanacees Download PDF

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WO2000006753A1
WO2000006753A1 PCT/NL1998/000445 NL9800445W WO0006753A1 WO 2000006753 A1 WO2000006753 A1 WO 2000006753A1 NL 9800445 W NL9800445 W NL 9800445W WO 0006753 A1 WO0006753 A1 WO 0006753A1
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Prior art keywords
nucleic acid
acid sequence
plant
homologue
seq
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PCT/NL1998/000445
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English (en)
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Voor Plantenveredelings-En Reproductie-Onderzoek Centrum (Cpro-Dlo)
Wageningen Landbouwuniversiteit
Original Assignee
Van Der Vossen, Edwin, Andries, Gerard
Van Der Voort, Jeroen, Nicolaas, Albert, Maria, Rouppe
Lankhorst, Rene, Marcel, Klein
Bakker, Jaap
Stiekema, Wilhelmus, Johannes
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Application filed by Van Der Vossen, Edwin, Andries, Gerard, Van Der Voort, Jeroen, Nicolaas, Albert, Maria, Rouppe, Lankhorst, Rene, Marcel, Klein, Bakker, Jaap, Stiekema, Wilhelmus, Johannes filed Critical Van Der Vossen, Edwin, Andries, Gerard
Priority to AU87518/98A priority Critical patent/AU8751898A/en
Priority to PCT/NL1998/000445 priority patent/WO2000006753A1/fr
Priority to PCT/NL1999/000491 priority patent/WO2000006754A2/fr
Publication of WO2000006753A1 publication Critical patent/WO2000006753A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the Gpa2 resistance gene from potato conferring resistance to phytopathogenic nematodes of the genus Globodera. It further relates to methods and materials employing the gene and processes for identifying related genes. Finally the invention relates to polypeptides encoded by said resistance genes and the use of said polypeptides.
  • Most plants are susceptible to infection by pathogens such as nematodes and develop various undesirable disease symptoms upon infection which cause retarded growth, reduced yield and consequently economical loss to farmers.
  • the plants respond to infection with several defense mechanisms including production of phytoalexins, deposition of lignin-like material, accumulation of cell wall hydroxyproline-rich glycoproteins, expression of pathogenesis related proteins (PR-proteins) and an increase in the activity of several lytic enzymes.
  • PR-proteins pathogenesis related proteins
  • Some of these responses can be induced not only directly by infection but also in some cases by exposure to exogenous chemicals such as ethylene.
  • the full capacity of the defense mechanism of the plant is, however, normally delayed in relation to the onset of infection, and thus, the plant may be severely injured before its defense mechanism reaches its maximum capacity.
  • the defense mechanism of the plant may not in itself be sufficiently strong to effectively combat the infectious organism. This is in particular true for cultivated plants which have often been cultivated with the aim of increasing the yield, decreasing the climate susceptibility, decreasing the nutrient demand etc. Therefore, a normal and necessary procedure is to treat infected plants or plants susceptible to infection with a chemical either as a prophylactic treatment or shortly after infection.
  • a chemical treatment is neither desirable from an ecological nor from an economic point of view.
  • Another procedure to combat the infectious organism is crop rotation. However, this is not able to fully overcome the problem. It would therefore be desirable to be able to enhance the defense of the host plant itself by introducing new and/or improved genes by genetic engineering.
  • the advantageous effect of the latter strategy would be the immediate inhibition of a phytopathogenic attack, leading to a retarded epidemic establishment of the infecting organisms in genetically engineered plant crops and thus an overall reduction in the effect of the infection.
  • PCN potato cyst nematodes
  • the durability of PCN resistance is determined by the extent of variation at (a)virulence loci which occur among the pathogen biotypes and the ability of the pathogen to generate novel specificities at (a)virulence loci.
  • the variation at (a)virulence loci is for the majority determined by the original founders which have been introduced into Europe.
  • PCN are endemic in the Andes region of South-America where they coevolved with their Solanaceous hosts. They are thought to have been introduced into Europe relatively recently, after 1850, together with collections of potato species which were imported for breeding purposes. Only a limited part of the genetic variation present in their centre of origin has been introduced into Europe (Folkertsma 1997).
  • Plant disease resistance genes The majority of plant resistance (R-) genes are located in chromosomal bins containing other disease or insect resistance factors (reviewed in Crute and Pink, 1996). These resistance genes are dominantly inherited, are often involved in resistance processes which are characterized by a hypersensitive response (HR) and are members of multigene families hypothesized to have evolved from common ancestral genes. Most R- loci are characterized by the presence of DNA sequences encoding putative gene products that contain (1) a nucleotide binding site (NBS) and (2) a leucine rich repeat structure (LRR).
  • NBS nucleotide binding site
  • LRR leucine rich repeat structure
  • R-loci clusters Clustering of R-loci in potato has been reported.
  • One of the large R-loci clusters is on the short arm of potato chromosome 5.
  • This cluster comprises at least five R-loci: Rl associated with resistance to Phytophthora infestans (Leonards-Schippers et al. 1992), Nb associated with HR type resistance to potato virus X (de Jong et al. 1997), Rx2 associated with an extreme type of resistance to PVX, and Gpa and Grpl associated with resistance to the PC ⁇ (Kreike et al. 1994; Rouppe van der Voort et al. 1998).
  • PC ⁇ R-locus Gpa5 is also located within the Grpl region (Rouppe van der Voort and Van der Vossen; unpublished data). Additionally, Gpa6 has been mapped to a region on chromosome 9 on which the homologous region in tomato, Sw5, conferring resistance to tomato spotted wilt virus, resides (Rouppe van der Voort and Van der Vossen; unpublished data).
  • the Gpa2 locus in potato has been found to be associated with resistance to G pallida populations D383 and D372 (Arntzen et al. 1994).
  • the presence of a single locus in potato which acts specifically to this small cluster of populations indicates that a gene- for-gene relationship underlies this plant-pathogen interaction (Rouppe van der Voort et al. 1997; Bakker et al. 1993).
  • the Gpa2 locus has previously been mapped on the short arm of chromosome 12 of potato (Rouppe van der Voort et al. 1997a), thusfar no sequence data or precise location were known. The gene was never isolated and no indication as to whether this single sequence would suffice to provide resistance or reduce susceptibility to nematode infection was available.
  • the present invention relates to a nucleic acid sequence providing resistance to infection by a phytopathogenic nematode of the Globodera species when introduced into a host plant, said host plant prior to introduction being susceptible to infection to the phytopathogenic nematode, said introduction occurring in such a way that said nucleic acid sequence is expressed in the host plant. Furthermore the invention relates to sequences which are homologous to the aforementioned sequence and which, when present in a plant, are able to render said plant resistant to infection by Globodera species. More specifically, a sequence according to the invention is preferably that of SEQ ID NO.l or a homologue thereof.
  • the PCN resistance locus Gpa2 when present in a plant such as Solanum spp., is capable of conferring to the plant anti-phytopathogenic activity in the form of resistance to Globodera species which are known to invade and damage the roots of Solanacae.
  • the invention relates to the Gpa2 resistance gene of which the DNA sequence is disclosed herein.
  • the invention also relates to a product encoded by a nucleic acid sequence according to the invention, said product providing nematode resistance activity.
  • the invention relates to genetic constructs, vectors, host cells such as bacterial strains, yeast cells and plant cells comprising a nucleic acid sequence according to the invention.
  • the present invention relates to a genetically transformed plant, preferably of the family Solanacae, especially a genetically transformed potato plant.
  • the expression product of the nucleic acid sequence according to the invention, said expression product providing the anti-nematode activity is produced in an increased amount as compared to the untransformed host cell so as to result in an increased resistance to Globodera species.
  • a process for producing a genetically transformed or transfected nematode resistant plant is additionally provided as is a process for isolating or detecting nucleic acid sequences according to the invention, providing nematode resistance of the aforementioned type.
  • a process for diagnosing whether a plant is resistant to Globodera species and a process for providing resistance to Globodera species to plant material are also disclosed in the present invention.
  • the invention also encompasses a process for producing a polypeptide providing the resistance and a nematocide composition providing said resistance.
  • Antibodies to the polypeptide are also envisaged as embodiments of the invention as is the application thereof in a diagnostic kit for assessing whether a plant is resistant to the aforementioned nematodes.
  • a diagnostic kit according to the invention may also comprise probes and/or primers specific for detection of a nucleic acid sequence providing the resistance.
  • the present invention relates to oligonucleotides corresponding to a part of a sequence according to the invention or being complementary thereto, with which homologous resistance genes can be identified that confer resistance to Globodera species.
  • nucleic acid a double or single stranded DNA or RNA molecule.
  • Oligonucleotide a short single-stranded nucleic acid molecule.
  • primer refers to an oligonucleotide which can prime the synthesis of nucleic acid.
  • Homologous sequence a sequence which has at least 70%, preferably 75%, more preferably 80%, most preferably 85% or even 90% sequence identity with the nucleic acid of the invention, whereby the length of the sequences to be compared for nucleic acids is at least 100 nucleotides, preferably 200 nucleotides and more preferably 300 nucleotides and for polypeptides at least 50 amino acid residues, preferably 75 amino acid residues and more preferably 100 amino acid residues.
  • Homology between the sequences may be as defined and determined by the TBLASTN computer programme for nucleic acids or the TBLASTP computer programme for polypeptides, of Altschul et al.
  • a homologous sequence refers to a nucleic acid which can hybridize under stringent conditions to the nucleic acid of the invention.
  • Nucleic acid hybridization is a method for detecting related sequences by hybridization of single- stranded nucleic acid probes with denatured complementary target DNA on supports such as nylon membrane or nitrocellulose filters.
  • Stringent conditions refer to hybridization conditions which allow a nucleic acid sequence of at least 50 nucleotides and preferably about 200 or more nucleotides to hybridize to a particular sequence at about 65 °C in a solution comprising approximately 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65 °C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. These conditions allow the detection of sequences having about 90% or more sequence identity.
  • promoter is intended to mean a short DNA sequence to which RNA polymerase and/or other transcription initiation factors bind prior to transcription of the DNA to which the promoter is functionally connected, allowing transcription to take place.
  • the promoter is usually situated upstream (5') of the coding sequence.
  • promoter includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site. Such regulatory sequences are, e.g., sequences which are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological conditions.
  • the promoter region should be functional in the host cell and preferably corresponds to the natural promoter region of the Gpa2 resistance gene. However, any heterologous promoter region can be used as long as it is functional in the host cell where expression is desired.
  • the heterologous promoter can be either constitutive or regulatable.
  • a constitutive promoter such as the CaMV 35S promoter or T-DNA promoters, all well known to those skilled in the art, is a promoter which is subjected to substantially no regulation such as induction or repression, but which allows for a steady and substantially unchanged transcription of the DNA sequence to which it is functionally bound in all active cells of the organism provided that other requirements for the transcription to take place is fulfilled.
  • a regulatable promoter is a promoter of which the function is regulated by one or more factors. These factors may either be such which by their presence ensure expression of the relevant DNA sequence or may, alternatively, be such which suppress the expression of the DNA sequence so that their absence causes the DNA sequence to be expressed.
  • the promoter and optionally its associated regulatory sequence may be activated by the presence or absence of one or more factors to affect transcription of the DNA sequences of the genetic construct of the invention.
  • Suitable promoter sequences and means for obtaining an increased transcription and expression are known to those skilled in the art.
  • Terminator the transcription terminator serves to terminate the transcription of the DNA into RNA and is preferably selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences and plant virus terminator sequences known to those skilled in the art.
  • - Nematode plant parasitic roundworms of the genus Globodera, i.e. Globodera pallida and G. rostochiensis.
  • nematode resistance denotes the characteristic activity in a plant ascribable to a resistance gene, i.e. the capability of the gene products to reduce or prevent the formation of cysts on the roots of plants in particular of Solanacae like e.g. Solanum spp.
  • the term "gene” is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5 '-upstream or 3 '-downstream region.
  • the 5 '-upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter.
  • the 3 '-downstream region comprises sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3' untranslated region.
  • the term "resistance gene” is a nucleic acid comprising a sequence as depicted in Fig. 3 (SEQ ID NO.3), or part thereof, or any homologous sequence.
  • - Resistance gene product a polypeptide having an amino acid sequence as depicted in Fig. 3 (SEQ ID NO.l) or part thereof, or any homologous sequence exhibiting the characteristic of providing nematode resistance when incorporated and expressed in a plant.
  • the present invention relates to a nucleic acid sequence providing resistance to infection by a phytopathogenic nematode of the genus Globodera when introduced into a host plant, said host plant prior to introduction being susceptible to infection with the phytopathogenic nematode, said introduction occurring in such a way that said nucleic acid sequence is expressed in the host plant. Furthermore the invention relates to resistance sequences which are homologous to the aforementioned sequence and which, when present in a plant, are able to confer to said plant resistance to infection by Globodera species. More specifically, a sequence according to the invention is suitably that of SEQ ID NO.l or a homologue thereof.
  • the PCN resistance locus Gpa2 when present in a plant such as Solanum spp., is capable of conferring, to the plant, anti-phytopathogenic activity in the form of resistance to Globodera species which are known to invade and damage the roots of Solanacae.
  • the invention relates to the Gpa2 resistance gene of which the DNA sequence is disclosed herein.
  • Homologues of the nucleic acid sequence of the abovementioned embodiment of the invention which also provide resistance to Globodera species, said homologues being nucleic acid sequences encoding the amino acid sequence of SEQ ID NO.l, are also within the scope of the invention.
  • a homologue of the nucleic acid sequence according to the invention can suitably also provide the resistance when said homologue is a nucleic acid sequence exhibiting more than 70% homology at nucleic acid level with SEQ ID NO. 1.
  • the homologue is a nucleic acid sequence exhibiting more than 75% homology at nucleic acid level with SEQ ID NO. 1, preferably exhibiting more than 80% homology at nucleic acid level with SEQ ID NO. 1, more preferably exhibiting more than 85% homology at nucleic acid level with SEQ ID NO. 1.
  • a homologue of the nucleic acid sequence according to the invention can also be a nucleic acid sequence exhibiting more than 90% homology at nucleic acid level with SEQ ID NO.l and can even be a nucleic acid sequence exhibiting more than 95% homology at nucleic acid level with SEQ ID NO.l.
  • a homologue also providing the resistance can be a nucleic acid sequence capable of hybridising under normal to stringent conditions to the nucleic acid sequence of SEQ ID NO. 1.
  • Naturally another suitable embodiment of a homologue of the sequence according to the invention, also providing the resistance can be a nucleic acid sequence encoding a deletion, insertion or substitution mutant of the amino acid sequence of SEQ ID NO.l.
  • Such a homologue can be a nucleic acid sequence encoding a deletion, insertion or substitution variant, preferably as occurs in nature, of the amino acid sequence of SEQ ID NO.l.
  • a nucleic acid sequence according to the invention may in addition to any of the embodiments described above or any combinations thereof further comprise at least one intron. Suitable examples of introns and locations thereof are provided in SEQ ID NO.2.
  • a suitable embodiment of the nucleic acid sequence according to the invention is the genomic insert of pBINRGH2 as disclosed in the examples.
  • a nucleic acid sequence according to the invention is suitably identical to that present in the genetic material of a species of the Solanacae family, preferably a species of the genus Solanum.
  • sequences can be found on and are preferably identical to those present in the genome of potato on chromosomes 4, 5, 7, 9, 11 or 12. More specifically, the nucleic acid sequence is identical to that present in the genome of potato at the Gpa2 locus. Obviously, a fragment of any of the above mentioned embodiments exhibiting the characteristic of providing the resistance falls within the scope of the invention.
  • a DNA region comprising the PCN R-locus Gpa2 has been isolated from a potato plant harbouring a wild Solanum genomic introgression segment possessing resistance against nematode infection.
  • This resistance which appears to be very effective in PCN control, is not present in most cultivated potato cultivars. Therefore, one object of the present invention is to provide plants, specifically Solanum spp., which have the features of cultivated plants, with anti- phytopathogenic activity in the form of resistance to Globodera species.
  • the present invention relates to a DNA segment comprising the Gpa2 locus of about 200 kb comprising one or several genes, the gene product or gene products thereof being capable of conferring to the plant resistance to nematodes of the Globodera species.
  • Another aspect of the present invention is a nucleic acid comprising the Gpa2 resistance gene, the nucleic acid having the sequence of all or part of the sequence depicted in Fig. 3 (SEQ ID NO.3) or any homologous sequence, including (where appropriate) both coding and/or noncoding regions and providing nematode resistance upon expression thereof in a plant.
  • the Gpa2 gene comprises the deduced coding sequence provided in Fig. 3 (SEQ ID NO.l) or any homologous sequence, preceded by a promoter region and followed by a terminator sequence.
  • the nucleic acid sequence according to the invention possesses very valuable features with respect to anti-nematode activity.
  • the DNA region comprising the nucleic acid sequence according to the invention encoding a polypeptide conferring/evoking the anti-nematode activity as defined above can be used for the construction of genetically modified hosts having an increased resistance to nematodes as compared to untransformed hosts.
  • the nucleic acid region according to the invention is thus capable of being inserted into the genome of a host plant, which in itself is susceptible to infection by a nematode, in such a way that the nucleic acid sequence is expressed, thereby conferring to the host plant resistance to infection by a phytopathogenic nematode.
  • Another aspect of the present invention relates to a genetic construct consisting of the nucleic acid sequence according to the invention which genetic construct can then be used to genetically transform a host, e.g. a plant such as a cultivated plant, in such a way that it becomes resistant to nematodes.
  • a genetic construct comprising a nucleic acid sequence according to any of the embodiments described above, said sequence being operably linked to a regulatory region for expression, falls within the scope of the invention. Accordingly, the present invention relates to a genetic construct comprising
  • the regulatory region of a genetic construct according to the invention is a Gpa2 regulatory region.
  • a regulatory region can by way of example correspond to that present in the sequence of nucleotides 1-4874 of SEQ ID NO.3.
  • the regulatory region can suitably even correspond to that of nucleotides 1-4874 of SEQ ID NO.3.
  • the regulatory region preferably comprises a promoter functionally connected to the nucleic acid sequence as defined in any of the embodiments above or in the examples, said promoter being able to control the transcription of said nucleic acid sequence in a host cell, preferably in a plant cell.
  • the genetic construct may be used in the construction of a genetically modified host in order to produce a host showing an increased anti-nematode activity and thus an increased resistance towards nematodes. It will be understood that a large number of different genetic constructs as defined above may be designed and prepared. Without being an exhaustive list, elements of the genetic constructs which may be varied are the number of copies of each of the nucleic acid sequences of the genetic construct, the specific nucleotide sequence of each of the nucleic acid sequences, the type of promoter and terminator connected to each nucleic acid sequence, and the type of any other associated sequences. Thus, genetic constructs of the present invention may vary within wide limits.
  • the invention also relates to DNA constructs comprising the regulatory sequences, and more preferably the promoter region of the Gpa2 resistance gene in conjunction with a structural gene sequence heterologous to said regulatory sequences.
  • a vector which carries a nucleic acid according to any of the embodiments disclosed above or in the examples or a genetic construct according to any of the embodiments disclosed above or in the examples also falls within the scope of the invention.
  • a vector is capable of replicating in a host organism.
  • the vector may either be one which is capable of autonomous replication, such as a plasmid, or one which is replicated with the host chromosome such as a bacteriophage or integrated into a plant genome.
  • the vector is an expression vector capable of expressing the nucleic acid sequence according to the invention in the organism chosen for the production. Suitable cloning vectors, transformation vectors, expression vectors, etc., are well known to those skilled in the art.
  • a vector according to the invention is constructed to function in a host organism selected from the group consisting of a micro-organism, plant cell, plant, seed, seedling, plant part and protoplast.
  • a host cell capable of resulting in a plant is preferred and suitably the host organism is selected from the group consisting of a plant, plant cell, plant part, seed, seedling and protoplast.
  • the present invention relates to a host organism which carries and which is capable of replicating or expressing an inserted nucleic acid region of the invention.
  • a host organism is preferably selected from the group consisting of a micro-organism, plant cell, plant, seed, seedling, plant part and protoplast, harbouring a vector and/or a genetic construct as defined above.
  • inserted indicates that the nucleic acid region has been inserted into the organism or an ancestor thereof by means of genetic manipulation, in other words, the organism may be one which did not naturally or inherently contain such a nucleic acid region in its genome, or it may be one which naturally or inherently contains such a nucleic acid region, but in a lower number so that the organism with the inserted nucleic acid region has a higher number of such regions than its naturally occurring counterparts.
  • the nucleic acid region carried by the organism may be part of the genome of the organism, or may be carried on a genetic construct or vector as defined above which is harboured in the organism.
  • the nucleic acid region may be present in the genome or expression vector as defined above in frame with one or more second nucleic acid regions encoding a second gene product or part thereof so as to encode a fusion gene product.
  • the organism may be a higher organism such as a plant, or a lower organism such as a micro-organism.
  • a lower organism such as a bacterium, e.g. a gram-negative bacterium such as a bacterium of the genus Escherichia, e.g. E. coli, or a yeast such as of the genus Saccharomyces, is useful for producing a recombinant polypeptide as defined above.
  • the recombinant production may be performed by use of conventional techniques, e.g.
  • the organism may be a cell line, e.g. a plant cell line.
  • the organism is a plant, i.e. a genetically modified plant such as will be discussed in further detail below.
  • the genetic construct is preferably to be used in modifying a plant.
  • the present invention also relates to a genetically transformed plant comprising in its genome a genetic construct as defined above.
  • the genetically transformed plant has an increased anti-nematode activity compared to a plant which does not harbour a genetic construct of the invention, e.g. an untransformed or natural plant or a plant which has been genetically transformed, but not with a genetic construct of the invention.
  • the genetically transformed plant is obtained by introducing the nucleic acid sequence according to the invention within the genome of said plant having a susceptible genotype to nematodes, using standard transformation techniques. It will be apparent from the above disclosure, that the genetically transformed plant according to the invention has an increased resistance to nematodes as compared to plants which have not been genetically transformed according to the invention or as compared to plants which do not harbour the genetic construct as defined above.
  • the present invention relates to seeds, seedlings or plant parts obtained by growing the genetically transformed plant as described above or by genetically transforming a plant cell and generating said part. It will be understood that any plant part or cell derivable from a genetically transformed host of the invention is to be considered within the scope of the present invention.
  • a process for producing a genetically transformed host organism having increased resistance to Globodera species as compared to the host organism prior to the transformation, said process comprising transferring a genetic construct and/or a vector according to any of the embodiments disclosed above and in the examples into the host organism so that it's genetic material comprises the genetic construct and/or vector and subsequently regenerating the host organism into a genetically transformed plant part is also a part of the invention.
  • the host organism may be selected from the group consisting of a plant cell, plant, seed, seedling, plant part and protoplast of the plant type to be rendered resistant and may subsequently be regenerated to a plant.
  • the nematodes against which resistance is provided are selected from the group consisting of Globodera species, more specifically Globodera rostochiensis and Globodera pallida.
  • the host organism which is to be transformed is selected from a plant type of the family Solanacae, preferably a Solanum spp. Plants of the species Solanum tuberosum, comprising commercial potato cultivars, are preferred as this is a particular problem area for the commercial growers of such plants.
  • a genetically transformed plant may be performed by a double transformation event (introducing the genetic construct in two transformation cycles) or may be associated with use of conventional breeding techniques.
  • two genetically modified plants according to the present invention may be the subject of cross breading in order to obtain a plant which contains the genetic construct of each of its parent plants.
  • the present invention also relates to the resistance gene product which is encoded by the nucleic acid sequence according to the invention and which has the deduced amino acid sequence provided in Fig. 3 (SEQ ID NO.l).
  • a polypeptide having an amino acid sequence provided in SEQ ID NO.1 and also a homologue of said amino acid sequence, said homologue being a substitution, insertion or deletion mutant conferring nematode resistance against Globodera species form embodiments of the invention.
  • a polypeptide according to the invention is encoded by a sequence according to any of the embodiments described above or in the examples.
  • a process for producing such polypeptides having an amino acid sequence provided in SEQ ID NO.l, or a homologue of said amino acid sequence, said homologue being a substitution, insertion or deletion mutant possessing resistance to Globodera species said process comprising the expression of the nucleic acid sequence or genetic construct according to any of the embodiments according to the invention and optionally isolating said polypeptide, said expression occurring in a host organism according to the invention, is also covered by the invention.
  • a process comprising an isolation step of the polypeptide in a manner known per se for polypeptide isolation from cell culture or from the host organism itself is also covered.
  • a nematicide composition comprising as active component a polypeptide according to the above or produced according to the process described or a host organism expressing such a polypeptide in a formulation suitable for application as nematicide to a plant and optionally comprising other ingredients required for rendering the polypeptide suitable for application as a nematicide, also falls within the scope of the invention.
  • a nematicide composition comprises the polypeptide in a slow release dosage form. It is quite efficient if such a nematicide composition is formulated and packaged comprising instructions for application as nematicide.
  • Antibodies may be raised against any purified resistance gene product according to the invention by any method known to those skilled in the art (for an overview see “Immunology - 5th Edition” by Roitt, Male: Pub 1998-Mosby Press, London). Such antibodies can be used for the detection of the gene product.
  • nucleic acid sequences comprising at least 16 contiguous nucleotides corresponding to or complementary to the Gpa2 sequence, with the proviso that when such a nucleic acid comprises part or all of the NBS encoding sequence, the nucleic acid also comprises at least one codon of a 5' and/or 3' overhanging portion corresponding to the respective 5' and/or 3' adjacent amino acids of parts of the NBS sequence of the Gpa2, with the following sequence, GGIGKTT or GGLPLA (see Table 4).
  • the Gpa2 sequence is comprised within the sequence of SEQ ID NO.l, 2 or 3.
  • the sequence length is preferably at least 50 nucleotides, preferably more than 100 nucleotides rendering it suitable for use as a probe in a nucleic acid hybridization assay.
  • Oligonucleotides complementary to one strand of the Gpa2 sequence or part thereof can be used as labeled hybridization probes in a Southern hybridization procedure or as primers in an amplification reaction such as the polymerase chain reaction (PCR), for the screening of genomic DNA or cDNA, or constructed libraries thereof, for the identification and isolation of homologous genes.
  • PCR polymerase chain reaction
  • Homologous genes that are identified in this way and which encode a gene product that is involved in conferring reduced susceptibility or resistance to a plant against pathogens, such as nematodes of the genus Globodera, are comprised within the scope of the invention. Suitable embodiments can be selected from any of the sequences SEQ. ID. No.4, 5, 6 and/or 7.
  • a diagnostic kit for assessing the presence of nematode resistance in a plant to infection by a phytopathogenic nematode of the genus Globodera comprising at least one nucleic acid defined above as a probe or primer, for screening of nucleic acid from a plant or plant part to be tested and/or comprising an antibody as defined above, is also comprised within the scope of the invention.
  • the invention also covers a process for isolating or detecting a nucleic acid sequence according to the invention providing nematode resistance as described above and in the examples, said process comprising the screening of genomic nucleic acid of a plant with said nucleic acids or a fragment thereof as probe or primer, said probe or primer being at least 16 nucleotides in length, the identification of positive clones which hybridize to said probe or primer and the isolation of said positive clones and the isolation of the nucleic acid sequence therefrom.
  • Such a process comprises screening genomic nucleic acid of a plant, preferably such a process comprises the screening of a genomic library of a plant with a nucleic acid sequence according to SEQ ID NO 3 or a fragment thereof as probe or primer, said probe being at least 16 nucleotides in length.
  • a process comprises the screening of a cDNA library of a plant with the coding portion of a nucleic acid sequence according to the invention providing the nematode resistance, or a fragment thereof as probe or primer, said probe or primer being at least 16 nucleotides in length.
  • the coding portion of a nucleic acid according to SEQ ID NO.l or a fragment thereof is used as probe or primer.
  • the probe or primer can be comprised within the sequence of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3.
  • the above processes can use a nucleic acid amplification reaction such as PCR in conjunction with at least one primer corresponding to or being complementary to the nucleic acid sequence according to the invention providing the nematode resistance, or a fragment thereof, said primer being at least 16 nucleotides in length.
  • the primer can be complementary to the nucleic acid sequence of SEQ ID NO.l, SEQ ID NO.2 or SEQ ID NO.3 or a fragment thereof, said primer being at least 16 nucleotides in length.
  • a probe or primer in such a process comprises a nucleic acid sequence encoding the amino acid sequence of a part or all of the NBS sequence of Gpa2.
  • Suitable examples of primers comprising a nucleic acid sequence encoding the amino acid sequence of a specific part or all of the NBS sequence of Gpa2 are given below (see Table 4).
  • the process can comprise application of a primer comprising at least part of the NBS sequence of Gpa2 and at least one codon of a 5' and/or 3' overhanging portion corresponding to the respective 5' and/or 3' adjacent amino acids of the previously specified NBS sequence of Gpa2.
  • a primer comprises the specified part of the NBS sequence of Gpa2 and at least one codon of a 5' and/or 3' overhanging portion corresponding to the respective 5' and/or 3' adjacent amino acids of the NBS sequence of Gpa2 of SEQ ID NO.l.
  • said primers correspond to a sequence selected from SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and/or SEQ ID NO.7.
  • a process for diagnosing whether a plant is resistant to a phytopathogenic Globodera species comprising the detection of the presence of a nucleic acid sequence providing nematode resistance as defined in any of the embodiments according to the invention, the presence of a genetic construct according to any of the embodiments according to the invention, the presence of a vector according to any of the embodiments according to the invention or the presence of a polypeptide as defmed according to the invention, in the genetic material of plant material of a plant to be tested falls within the scope of the invention. Combinations of detection of the various elements are also covered.
  • the nucleic acid sequence and the polypeptide being detected can be naturally present in the plant to be tested or can have been introduced via genetic engineering.
  • a process for diagnosis according to the invention can comprise any of the nucleic acid sequence detection processes already described above as embodiments of the invention. More specifically the process can comprise applications of the diagnostic kit described according to the invention in an analogous manner to application of other nucleic acid assay kits comprising probes or primers or antibody known in the art. Suitably such a kit according to the invention will be provided with the appropriate instructions for application thereof. Amplification reactions of nucleic acid , use of probes in Southern analysis and use of antibodies in immunoassays are suitable examples of applications known in the art.
  • Another process within the scope of the invention is a process for providing resistance to a phytopathogenic Globodera species to plant material, said process comprising the introduction into the plant genome of a nucleic acid sequence providing nematode resistance as defmed in any of the embodiments according to the invention, a genetic construct according to any of the embodiments according to the invention, a vector according to any of the embodiments according to the invention in the genetic material of plant material of a plant to be tested and thereby producing a transformed plant cell, plant propagating material, plant part or plant.
  • Such introduction of genetic material should result in a transformed host with the introduced genetic material stably present in the host such that replication of said host is possible with said genetic material.
  • Such a process may further comprise regenerating the resulting transformed or transfected plant cell, plant propagating material or plant part.
  • the process of introduction of the genetic material can occur as commonly described in the art for introduction of genetic material into the appropriate host type.
  • nucleic acid sequence comprising the resistance as provided by the present invention has numerous applications of which some are described herein but which are not limiting to the scope of the invention.
  • the present invention is further described in detail below, whereby the map-based cloning strategy used to isolate the Gpa2 resistance gene of the invention is explained.
  • the strategy to isolate the Gpa2 gene was as follows: 1) genetic fine mapping of the Gpa2 locus; 2) construction of a B AC contig spanning the Gpa2 locus;
  • the Gpa2 locus was initially mapped on chromosome 12 using information on the genomic positions of 733 known AFLP markers (Rouppe van der Voort et al, 1997a and 1997b). By use of RFLP probes, Gpa2 was mapped more precisely between markers GP34 and CT79 on the distal end of chromosome 12 (Rouppe van der Voort et al, 1997a), a 6 cM genetic interval that was previously shown to harbour the potato virus X (PVX) resistance gene Rx ⁇ (Fig. 1; Bendahmane et al, 1997).
  • PVX potato virus X
  • Fine mapping of the Gpa2 locus was subsequently carried out using cleaved amplified polymorphic sequence (CAPS; Konieczny and Ausubel, 1993) markers derived from the IPM3-IPM5 interval, all of which were initially developed for the cloning of Rx ⁇ (Fig. 1). 2,788 Sl-Cara genotypes were assayed for recombination events in the IPM3-IPM5 region. In addition, 598 FISHxRH genotypes were subjected to a GP34/IPM5 marker screening as marker IPM3 was not informative in population FISHxRH.
  • CAPS cleaved amplified polymorphic sequence
  • BAC clones BACH, BAC45, BAC221 and BAC111, which map to the 0.06 cM IPM4c-l llR genetic interval harbouring the Gpa2 locus, were isolated from a BAC library prepared from a progeny of a selfed cv. Cara (Fig. IC). However these four BAC clones did not completely cover the Gpa2 interval. Screening of the Cara BAC library with CAPS markers 77R and 45L (Fig. IB) did not lead to the identification of Cara BAC clones that spanned the region between markers 77R and 45L. A second BAC library was constructed from the diploid potato genotype SH83-92-488 (SH83).
  • Gpa2 resistance gene by complementation analysis, BAC clones SHBAC43, BAC45, BAC221a and BAC111 were analysed for the presence of R-gene homologous sequences.
  • R-gene homologous sequences Despite the general lack in DNA sequence conservation between R-genes, there are a few conserved protein motifs in the NBS region present in many of these genes. Leister et al (1996) has shown that it is possible to amplify resistance gene like sequences from potato using degenerate primers based on these homologous regions.
  • Eggs and second stage juveniles are obtained by crushing cysts which have been soaked in tap water for one week.
  • the egg/J 2 suspension is poured through a 100 ⁇ m sieve to remove debris and cystwalls.
  • three to four week old potato stem cuttings are transferred from a peat mixture to 900 gram pots containing a mixture of silversand and a sandy loam fertilized with Osmocote (N-P-K granulates).
  • plants are inoculated with nematodes to a final density of 5 egg/J 2 per gram soil.
  • nematodes are inoculated.
  • Resistant standards are Solanum tuberosum cv. Multa (resistant to G pallida D383), S. vernei hybrid 58.1642/4 (resistant to G rostochiensis line Ro,-19) and S. vernei hybrid 62-33-3 (resistant to both D383 and R ⁇ j -19).
  • the susceptible standard is S. tuberosum cv.Eigenheimer. Plants are arranged in a randomized block design and grown in a greenhouse with 15°C and 25°C as minimum and maximum temperature, respectively.
  • cysts are recovered from the soil with a Fenwick can (Fenwick 1940) and the size of the root systems is judged on a scale of 0 to 3. Resistance data of a genotype are only recorded when at least two well-rooted plants of this genotype are available. The mean cyst numbers developed per genotype are standardized using a log 10 ( + 1) transformation and then subjected to SAS Ward's minimum variance cluster analysis (SAS Institute Inc., Cary NC, USA). On the basis of this analysis the plants are devided into a resistant, an unassigned or a susceptible class.
  • the resistance assay is carried out on sterile tissue culture plants in agar. Two or three nodia from each in vitro grown (transgenic) potato plant are grown on solidified B5 medium (Gamborg et al. 1968) with 0.5% PhytagelTM (Sigma) and 2% sucrose for one week (25 °C and 16 hr light regime). Each new root tip (on average 2 per nodium) is then inoculated with 15 sterilized second stage juveniles. Preparation of inoculum is substantially as described by Heismes et al. (1995) with slight modifications.
  • Cysts are collected in a modified 20 ml syringe with a 22 ⁇ m nylon mesh and surface sterilized in 90% ethanol for 15 sec followed by an 8 min wash in 1.3% (w/v) commercial bleach. To remove excess bleach, the cysts are washed three times in sterile tap water for 5 min and incubated in sterile tap water for 3 days at 20°C in the dark. Cysts are then transferred to filter sterile potato root differentiate (PRD) and left to hatch for 5 days at 20°C in the dark.
  • PRD sterile potato root differentiate
  • Second stage juveniles are subsequently transferred to a 5 ⁇ m sieve- syringe and incubated first in 0.5% (w/v) streptomycine-penicilline G solution for 20 min, then in 0.1% (w/v) ampicillin-gentamycin solution for 20 min and finally in 0.1% chlorhexidin solution for 3 min.
  • the second stage juveniles are suspended in the required volume (sterile tap water) for inoculation.
  • the petridishes with the inoculated root tips are incubated in the dark at 20°C.
  • the level of infection is determined by counting the number of female nematodes formed on the roots.
  • EXAMPLE 2 COSEGREGATION OF Gpal NEMATODE RESISTANCE AND Rx ⁇ VIRUS RESISTANCE.
  • the Gpa2 locus was initially mapped to chromosome 12 using information on the genomic positions of 733 known AFLP markers (Rouppe van der Voort et al, 1997 'a and 1997b). By use of RFLP probes, Gpa2 was mapped more precisely between markers GP34 and CT79 on the distal end of chromosome 12 (Fig. 2 A; Rouppe van der Voort et al, 1997a), a 6 cM genetic interval that was previously shown to harbour the potato virus X (PVX) resistance gene Rx ⁇ (Bendahmane et al, 1997).
  • PVX potato virus X
  • the resistance tests showed a clear reduction in the number of cysts of G pallida population D383 on genotypes which were resistant to PVX CP4 .
  • the number of cysts developed on the resistant Sl-Cara genotypes appeared to be slightly higher than the number of cysts found on the resistant genotypes of population FISHxRH.
  • a considerable reduction in size of these cysts was observed as compared to the cysts developed on a susceptible genotype. This observation was corroborated after comparing the number of eggs per cyst developed on resistant and susceptible genotypes. Average cyst contents were determined from at least 30 cysts (if possible) and subjected to a t- test.
  • EXAMPLE 3 ISOLATION OF CARA BAC CLONES AND PRODUCTION OF CAPS MARKERS DERIVED FROM THE RxllGpal LOCUS (according to the unpublished article in preparation of Kanyuka, K., Bendahmane, A., Rouppe van der Voort, J.N.A.M., van der Vossen, E.A.G. and Baulcombe, D.C. Mapping of intra-locus duplications and introgressed DNA: aids to map-based cloning of genes from complex genomes illustrated by analysis of the Rx locus in tetraploid potato).
  • a BAC library of 160,000 clones was prepared from plant SC-781 which is a progeny of selfed cv Cara carrying Rxl in the duplex condition (Rx,Rx,rx,rx).
  • High molecular weight DNA was prepared in agarose plugs from potato protoplasts essentially as described in Bendahmane et al. (1997). The agarose plugs were dialysed three times for 30 min against TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA), once at room temperature and twice at 4°C.
  • the plugs were then equilibrated in Hindlll buffer (10 mM Tris-HCl, 10 mM MgCl 2 , 50 mM NaCl, 1 mM DTT, pH 7.9) twice on ice for 1 h.
  • Half of each plug ( ⁇ 5 ⁇ g of DNA) was transferred to a test tube containing 360 ⁇ l of H dIII buffer and 10-15 units of HbzdIII restriction enzyme. The enzyme was allowed to diffuse into a plug at 4°C for 1 h and the digestion was carried out at 37°C for 30 min.
  • the reaction was stopped by adding 1 ml of 0.5 M EDTA and plugs were immediately loaded into a 1% low melting point agarose gel and subjected to contour-clamped homogeneous electric fields (CHEF; Chu, 1989) electrophoresis in a CHEF DR II system (Bio-Rad Laboratories, USA) in 0.5 X TBE buffer (45 mM Tris- borate pH 8.0, 1 mM EDTA) at 150 volts for 10 h at 4°C and constant pulse time of 5 sec or 8 sec. Compression zones containing the DNA fragments of 100 kb or 150 kb were excised from the gel and dialysed against 30 ml TE in a 15 cm Petri dish for 2 h at 4°C.
  • CHEF contour-clamped homogeneous electric fields
  • Dialysed agarose slices were then transferred to a 1.5 ml test tube, melted at 70°C for 10 min and digested with 1 unit of GELASE (Epicentre Technologies, USA) per 100 mg of agarose gel for 1 h at 45°C.
  • the size selected potato DNA (25-50 ng) was ligated to 25-50 ng of H dIII-digested and dephosphorylated pBeloBACl l (Shizuya et al, 1992) using 400 to 800 units of T4 DNA LIGASE (New England BioLabs, USA) at 16°C for 24 hours in a total volume of 50 ⁇ l.
  • the ligation products were dialysed against 1 X TE using 0.025 ⁇ m MF- MILLIPORE MEMBRANE FILTER (Millipore, UK) at 4°C for 2 h and 30 min at room temperature using the "drop dialysis" method of Maruzyk and Sergeant (1980).
  • E. coli D ⁇ 10B cells were carried out by electroporation using a BRL CEM1-PORATOR SYSTEM (Life Technologies Ltd, UK). To 20 ⁇ l of electro- competent cells, 0.5-3 ⁇ l of ligation mixture was added. After electroporation, E. coli cells were mixed with 1 ml SOC medium (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM glucose) and incubated at 37°C for 1 h with gentle shaking (80 rpm).
  • SOC medium 0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM glucose
  • the cells were spread on Luria broth (LB) agar plates containing chloramphenicol (12.5 lg/ml), 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactoside (Xgal) (40 lg/ml) and isopropyl- 1-thio- ⁇ -D-galactoside (IPTG) (0.12 mg/ml). Plates were incubated at 37°C for 24 hours. DNA from the compression zones of 100 and 150 kb led to clones with an average insert size of 100 kb and a transformation efficiency of approximately 1000 and 150 white colonies per 11 ligation mixture, respectively.
  • LB Luria broth
  • Another 100 bacterial pools containing -500- 1000 white colonies each (these pools also contained approximately 500-1500 blue bacterial colonies with an empty pBeloBACl l) were prepared by scraping the colonies from agar plates into the LB medium containing 18% glycerol and 12.5 ⁇ g/ml chloramphenicol using a sterile glass spreader. These pools were also stored at -80°C.
  • the Cara BAC library was initially screened with CAPS markers IPM3, IPM4 and IPM5 corresponding to the AFLP markers PM3, PM4 and PM5 flanking the Rxl locus (Bendahmane et al, 1997). This was carried out as follows. For the first part of the library of 92,160 clones stored in 384 well microtiter plates the plasmid DNA was isolated using the standard alkaline lysis protocol (Hilor et al, 1997) from pooled bacteria of each plate to produce 240 plate pools. Aliquots of these plate pools were combined to prepare 26 'superpools' containing DNA from 9 plate pools, and one superpool containing DNA from 6 plate pools.
  • plasmid DNA was isolated from each pool of clones using the standard alkaline lysis protocol and PCR was carried out to identify positive pools. Bacteria corresponding to positive pools were diluted, plated on LB agar plates and subsequently colony hybridisation was carried out as described in Sambrook et al. (1989) using 2 P-labelled DNA probes corresponding to the CAPS markers. PCR with the corresponding CAPS primers was used to distinguish between hybridising colonies carrying the markers previously mapped to homologues located elsewhere in the genome and those derived from the Rxl locus.
  • Positive BAC clones were analysed by isolating plasmid DNA from 5 ml overnight cultures (LB medium supplemented with 12.5 mg/ml chloramphenicol) using the standard alkaline lysis miniprep protocol (Engebrecht et al, 1997) and resuspended in 50 ⁇ l TE. Plasmid DNA (10 ⁇ l) was digested with Notl for 3 h at 37°C to free the genomic D ⁇ A from the pBeloBACl l vector.
  • the digested D ⁇ A was separated by CHEF electrophoresis in a 1% agarose gel in 0.5 X TBE at 4°C using a BIO-RAD CHEF DR II system (Bio-Rad Laboratories, USA) at 150 volts with a constant pulse time of 14 sec for 16 h.
  • Ligations were carried out using 5-50 ng of digested D ⁇ A and 5-10 units of T4 D ⁇ A LIGASE (Boehringer Mannheim, Germany) in a total volume of 100 ⁇ l.
  • PCR amplification of the recircularised D ⁇ A was carried out using 3 ⁇ l of self-ligated D ⁇ A as the template.
  • AB1 (5'- C C T A A A T A G C T T G G C G T A A T C A T G - 3 ' ) a n d A B 2 ( 5 ' - TGACACTATAGAATACTCAAGCTT-3') primers were used for PCR amplification of the left end sequence of insert D ⁇ A.
  • AB3 (5'-CGACCTGCAGGCATGCAAGCTT-3') and AB4 (5'-ACTCTAGAGGATCCCCGGGTAC-3') primers were used for PCR amplification of the right end sequence of insert D ⁇ A.
  • PCR conditions were as follows: 94°C for 15 sec, 60°C for 15 sec, 72°C for 90 sec - for 35 cycles.
  • PCR products were digested simultaneously with Hindlll and the restriction enzyme used in the preparation of IPCR DNA template. The released insert ends were gel purified and cloned into pGEM-3Z(f+) (Promega, USA).
  • 117L and IPM3 markers were separated from Rxl by two and three recombination events respectively whereas the GP34 marker, present in BAC 167, was separated from Rxl by thirteen recombinations (Fig. IB).
  • the BAC library did not contain additional BACs extending further towards Rxl from the 191L marker.
  • the BAC library was also screened with the IPM5 CAPS marker which is on the same side of Rxl as IPM4, but further from Rxl (Bendahmane et al, 1997). It was anticipated that BACs containing IPM5 would orientate the 11 IL and 73 L markers relative to Rxl. These analyses identified BAC218, carrying an allele of IPM5 identified by Pstl digestion, as being linked in cis to Rxl (Fig. IC). The end sequences of BAC218 insert DNA were converted into the CAPS markers, 218L and 218R, and mapped genetically to the recombination events between GP34 and IPM5.
  • Marker 218L was positioned 0.48 cM (recombination fraction: 8/ 1720) from Rxl, between IPM5 and CT129.
  • the 218R marker was positioned between IPM4 and IPM5, 0.30 cM (recombination fraction: 5/1720) from Rxl.
  • a single BAC pool #29 was also identified which contains three markers: 218R, 73L and 111R. CAPS analysis revealed that each of these markers in the BAC pool #29 is represented by the allele linked in cis to Rxl.
  • BAC pool #29 contains a single BAC clone, BAC29, with DNA insert linked in cis to Rxl. Therefore, BAC29 provided a link between BAC218 and the IPM4 BAC contig and orientated the markers from the IPM4 contig in the following order: Rxl, 111L, IPM4, 73L (Fig. IB).
  • BAC221 By screening the BAC library with 111L allele-specific primers BAC221 was identified which carries an insert DNA of 40 kb and is linked in cis to Rxl. The left end sequence of BAC221 is located inside of BACH 1 whereas the right end sequence of BAC221 extends further towards Rxl (Fig. IC). However the marker 221R co-segregated with IPM4 in the Sl-Cara mapping population and was separated from Rxl by the recombination event in plant SI -761 (Fig. IB).
  • BAC45 which has an insert DNA of 40 kb and is linked in cis to Rxl.
  • the right end sequence of BAC45 is located inside of BAC221, whereas the left end sequence of BAC45, 45L, extends further towards Rxl (Fig. IC).
  • BAC45 does not contain Rxl as the CAPS marker 45L is genetically separated from Rxl by the recombination event in plant SI -761 (Fig. IB). Additional PCR screening of the BAC library with the 45L marker failed to identify any new BAC clones therefore leaving a gap between the IPM3 and IPM4 BAC contigs (Fig. IC).
  • Primer annealing temperatures in PCR reactions was 5 to 10°C lower than in conditions originally developed for each CAPS primer pair (Table 1) so that amplification of related sequences, in addition to the original marker, could take place.
  • the PCR products obtained with a number of tested CAPS primer pairs were the same size as the products produced under high stringency conditions.
  • digestion of these low stringency PCR products with either Taql, Alul or Ddel restriction enzymes revealed several new DNA fragments that were not identified previously. These included fragments that were nonpolymorphic as well as fragments polymorphic between the R and S pools. Digestion of the low stringency IPM4 products from the R pool with Taql identified the original IPM4 locus (designated IPM4a) in BAC111.
  • IPM4 restriction fragments There were also new IPM4 restriction fragments that had not been detected previously.
  • IPM4b was nonpolymorphic in the R and S pools. This fragment originated from BAC221 as the Taql restriction fragment of similar size was also detectable after digestion of the IPM4b allele derived from this BAC (Fig. IB).
  • a second new DNA fragment was polymorphic between R and S pools and was not detected after digestion of either IMP4a or IPM4b alleles derived from BACl l l and BAC221, respectively.
  • This fragment cosegregated with Rxl in all the plants of the Sl-Cara mapping population, including plant SI -761 and others with recombination events between GP34 and IPM5. This new IPM4 marker allele was designated IPM4c (see Fig. IB).
  • a total of 2,788 Sl-Cara genotypes were assayed for recombination events in the IPM3-IPM5 interval.
  • 598 FISHxRH genotypes were subjected to a GP34/IPM5 marker screening as marker IPM3 is not informative in population FISHxRH.
  • the GP34 CAPS marker is derived from a sequenced insert of RFLP clone GP34.
  • the CAPS marker screening provided a total of 20 recombinants in the Sl-Cara population and 9 recombinants in the FISHxRH population. These recombinants were subsequently tested for the presence of markers 77L, IPM4c, 77R, 45L, 221R, IPM4a, 111R, 73L and 218R, all of which are derived from the PM3-IPM5 interval (see Fig. 2B), as well as for Gpa2 resistance.
  • the Gpa2 resistance test was carried out using G pallida population D383 (Rouppe van der Voort et al. 1997a).
  • the nematode resistance assays were performed on plants derived from in vitro stocks, stem cuttings or tubers.
  • Gpa2 is located between markers IPM4c and 111R (Fig. 2B).
  • Fig. 2B markers IPM4c and 111R
  • SI -761 markers IPM4c and 111R
  • genotype S1-B811 identified marker 111R as a flanking marker for the Gpa2 interval.
  • Marker orders deduced from the analysis of FISHxRH corresponded to those found in population Sl-Cara. Estimates of recombination frequencies and their standard errors were calculated with the aid of the program Linkage- 1 (Suiter et al. 1983) by choosing the appropriate genetic model for each cross. Data for the non-recombinant class of genotypes were set for either a 3:1 segregation ratio for population Sl-Cara or a 1 :1 segregation ratio for population FISHxRH since only strongly skewed segregation ratios will influence estimates of recombination frequencies notably (Sail and Nilsson 1994; Manly 1994). A chi-square test was used to test for differences in recombination frequencies between the marker intervals.
  • the chi-square test criterion was determined from the recombinant and non-recombinant classes for each marker interval. Differences (rejection of the null hypothesis) were significant when the test criterion was greater than the X 2 [0. os ] value. Estimates of recombination frequencies deduced from both populations were merged to obtain an estimate of the average recombination value for each marker interval.
  • the graphical genotypes Young and Tanksley, 1992) shown in Fig. 1 display the boundaries of the Gpa2 interval.
  • Example 3 describes the preparation of a Cara BAC library from a progeny of a selfed cv. Cara and the identification and isolation of BAC clones BAC77, BAC45, BAC221 and BACl l l, which map to the 0.06 cM IPM4c-l l lR genetic interval harbouring the Gpa2 locus (Fig. IC). Additional PCR screening of the Cara BAC library with markers 45L and 77R failed to identify any BAC clones that spanned the region between BAC77 and BAC45.
  • a second BAC library was constructed from the diploid potato genotype SH83-92-488.
  • High molecular weight potato DNA was prepared in agarose plugs from potato nuclei as described in Liu et al. (1994) with the following modifications.
  • Plant nuclei were isolated by grinding leaf tissue (10 g) in liquid nitrogen, suspending the powder in 100 ml nuclei isolation buffer (10 mM Tris-HCl pH 9.5, 10 mM EDTA, 100 mM KC1, 0.5 M sucrose, 4 mM spermidine 1.0 mM spermine, 0.1% mercaptoethanol) and sequential filtering through one layer each of 280, 88, 55 and 20 ⁇ m nylon mesh.
  • nuclei isolation buffer (10 mM Tris-HCl pH 9.5, 10 mM EDTA, 100 mM KC1, 0.5 M sucrose, 4 mM spermidine 1.0 mM spermine, 0.1% mercaptoethanol) and sequential filtering through one layer each of 280, 88, 55 and 20 ⁇ m nylon mesh.
  • isolation buffer 10 mM Tris-HCl pH 9.5, 10 mM EDTA, 100 mM KC1, 0.5 M sucrose, 4 mM spermidine 1.0
  • the nuclei were pelleted at 4°C (in 50 ml screwcap tubes) at 2200 rpm for 10 min and resuspended with isolation buffer to a final volume of 1 ml.
  • FMC low-melting point inCert agarose
  • the agarose plugs were treated with lysis buffer (1% sarkosyl, 0.4 M EDTA pH 8.5, 0.2 mg/ml proteinase K and 3.8 mg/ml sodiumdisulfite) at 50 °C for 2 days with one change of lysis buffer. Proteinase K activity was inhibited by incubating the agarose plugs 12 hours at 50°C in T 10 E 10 buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA) supplemented with 40 ⁇ g/ml PMSF.
  • lysis buffer 1% sarkosyl, 0.4 M EDTA pH 8.5, 0.2 mg/ml proteinase K and 3.8 mg/ml sodiumdisulfite
  • the lanes containing the lambda DNA ladder were removed and stained with ethidium bromide to locate the region of the gel containing potato DNA fragments ranging from 100 to 150 kb in size.
  • This region was excised from the gel using a glass coverslip and subjected to a second size selection step in a 1% SeaPlaque (low-melting point) agarose gel (FMC).
  • FMC SeaPlaque (low-melting point) agarose gel
  • CHEF electrophoresis was carried out for 10 hr at 4°C using a field strength of 4 V/cm and a constant pulse time of 5 sec.
  • the compression zone containing DNA fragments of 100 kb was excised from the gel as described above and dialysed against 50 ml TE for 2 hr at 4°C.
  • Dialysed agarose slices were then transferred to a 1.5 ml Eppendorf tube, melted at 70°C for 5 min and digested with 1 unit of GELASE (Epicentre Technologies, USA) per 100 mg of agarose gel for 1 hr at 45°C.
  • S ⁇ BAC43 By screening the S ⁇ BAC library, as described in Example 3, with CAPS markers 77R and 45L BAC clone S ⁇ BAC43 was identified (see Fig. 2C).
  • plasmid DNA was isolated from 5 ml overnight cultures (LB medium supplemented with 12.5 mg/ml chloramphenicol) using the standard alkaline lysis miniprep protocol (Engebrecht et al, 1997) and resuspended in 50 ⁇ l TE. Plasmid DNA (10 ⁇ l) was digested with Notl for 3 h at 37°C to release the insert D ⁇ A from the pBeloBACl l vector, and subsequently analysed by CHEF electrophoresis. Comparison of the electrophoretic mobility of the SHBAC43 insert with that of the lambda concatemer ladder (BioRad) lead to the conclusion that SHBAC43 contains a BAC insert of approximately 110 kb.
  • the DNA sequence of BAC clones SHBAC43, BAC221a and BAC111 was determined by shotgun sequence analysis. For each BAC clone a set of random subclones with an average insert size of 2 kb was generated. 10 ⁇ g of CsCl purified DNA was sheared for 6 seconds on ice at 6 amplitude microns in 200 ⁇ l T ⁇ using a MS ⁇ soniprep 150 sonicator.
  • the fraction with a size of 1.5-2.5 kb was excised from the gel and dialysed against 50 ml TE for 2 hr at 4°C. Dialysed agarose slices were then transferred to a 1.5 ml Eppendorf tube, melted at 70°C for 5 min, digested with 1 unit of GELASE (Epicentre Technologies, USA) per 100 mg of agarose gel for 1 hr at 45°C, and the DNA was subsequentty precipitated. The 1.5-2.5 kb fragments were ligated at 16°C in a EcoRV restricted and dephosphorylated pBluescript SK + vector (Stratagene Inc.). The ligation mixture was subsequently used to transform ⁇ lectroMAX E.
  • coli DH10B competent cells (Life Technologies, UK) by electroporation using the BioRad Gene Pulser. Settings on the BioRad Gene Pulser were as recommended for E. coli by the manufacturer.
  • the cells were spread on Luria broth (LB) agar plates containing ampicillin (100 ⁇ g/ml), 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside (Xgal) (64 ⁇ g/ml) and isopropyl- 1-thio- ⁇ -D-galactoside (IPTG) (32 ⁇ g/ml). Plates were incubated at 37°C for 24 hours. Individual white colonies were grown in 96-well flat-bottom blocks (1.5 ml Terrific Broth medium containing 100 ⁇ g/ml ampicillin).
  • Plasmid DNA was isolated using the QIAprep 96 Turbo Miniprep system in conjunction with the BioRobotTM 9600 (QIAG ⁇ N) according to the manufacturers instructions.
  • the ABI PRISM dye terminator cycle sequencing ready kit was used to perform sequencing reactions in a PTC-200 Peltier Thermal Cycler (MJ Research).
  • the DNA sequences of the clones were determined using standard Ml 3 forward and reverse primers. Sequence assembly was done using the 1994 version of the STAD ⁇ N sequence analysis programme (Dear and Staden, 1991).
  • tumefaciens by the manufacturer. The cells were spread on Luria broth (LB) agar plates containing kanamycin (100 mg/L) and rifampicin (50 mg/L). Plates were incubated at 28°C for 48 hours. Small-scale cultures from selected colonies were grown in LB medium containing kanamycin (100 mg/1) and rifampicin (50 mg/1). Plasmid DNA was isolated as described previously and the integrity of the plasmids was verified by restriction analysis upon reisolation from A. tumefaciens and subsequent transformation to E. coli. A tumefaciens cultures harbouring a plasmid with the correct DNA pattern were used to transform a susceptible potato genotype.
  • Transformation of the susceptible potato genotype, clone V was essentially performed as described by Visser (1991) and is described briefly below.
  • Stem explants (1 cm long internodes) were prepared from 5 week old tissue culture plants and precultured for 24 hours (25°C, 16 hour light regime) in Petri dishes containing 2 sterile filter papers saturated with PACM (feeding layers: MS30 medium supplemented with 2 g/1 caseinehydrolysate, 1 mg/1 2,4 D and 0.5 mg/1 kinetine, pH 5.8) which were placed on R3B medium (MS30 medium supplemented with 2 mg/1 NAA and 1 mg/1 BAP, pH 5.8). The explants were then infected for 10 minutes with an overnight culture of A.
  • tumefaciens strain AGLO containing either pBINRGHl, pBINRGH2, pBINRGH3 or the pBINPLUS vector, blotted dry on sterile filter paper and cocultured for 48 hours on the original feeder layer plates. Culture conditions were as described above. Overnight __. tumefaciens cultures were pelleted and resuspended in liquid MS20 medium prior to infection. Following cocultivation, the explants were transferred to MS20 medium (pH 5.8) supplemented with 1 mg/1 zeatin, 200 mg/1 cefotaxime, 200 mg/1 vancomycin and 100 mg/1 kanamycin and cultured under the culture conditions described above. The explants were transferred to fresh medium every 3 weeks. Emerging shoots were isolated and transferred to glass jars with selective medium lacking zeatin for root induction. Only transgenic shoots were able to root on the kanamycin containing medium. EXAMPLE 8: COMPLEMENTATION ANALYSIS
  • In vitro grown transgenic (P ⁇ ,) plants were initially subjected to the in vitro resistance assay as described in Example IB whereby sterilized second stage juveniles of G pallida popultion D383 were used as inoculum.
  • Three nodes from four independent primary transformants of the 4 different transformations were assayed; R o (RGHl), R o (RGH2) and Ro(RGH3) transgenic plants contain the candidate genes RGH1, RGH2 and RGH3, respectively, and R 0 (BINPLUS) transgenic plants are without insert DNA and function as control plants.
  • Fig. 3 The sequence of the 10.3 kb Xbal-Xbal insert of pBINRGH2 is provided in Fig. 3 (SEQ ID NO.3).
  • the computer programme GENSCAN predicted the presence of one single gene harbouring two introns in the 3 '-end of the gene. Comparison of the genomic sequence of RGH2 with the sequence of isolated RGH2 cDNAs confirms the position of these two introns.
  • the Gpa2 encoding nucleic acid sequence (RGH2) provided in Fig. 3 (SEQ ID NO.l), codes for a putative polypeptide sequence of 939 amino acids, the sequence of which is also provided in Fig. 3 (SEQ ID NO.l).
  • EXAMPLE 9 IDENTIFICATION AND MAPPING OF HOMOLOGOUS GENES.
  • primers were designed for specific amplification of nucleic acid sequences cognate to the NBS of RGH1-4 (primers RG3 and RG4; see Tables 3 and 4).
  • the position of primer RG3 corresponds to nucleotides 514-533 of SEQ ID NO.l (Fig. 3).
  • Primer RG4 is complementary to nucleotides 985-1002 of SEQ ID NO.l (Fig. 3).
  • These primers differ from RGl and RG2 and those designed by Leister et al. (1996) in that the 3' terminal nucleotides are designed on the basis of amino acid residues that exceed the conserved residues used for the design of the former primers (see Table 4).
  • Primer sequences RG5 and RG6 were designed on the basis of sequences outside of the NBS of RGH 1-4.
  • the position of primer RG5 corresponds to nucleotides 199-221 of SEQ ID NO.2 (Fig. 3).
  • Primer RG6 is complementary to nucleotides 2681-2701 of SEQ ID NO.2 (Fig. 3).
  • Mapping of the Gpa2 homologues identified with primers RG3 and RG4 is carried out by developing CAPS markers designed on the end sequences of each BAC insert. These CAPS markers are used to screen 136 genotypes of population FISHxRH. The data on marker segregation are scored and the respective loci are mapped on the SH83 genome by use of the computer package JoinMap2.0 (Stam, 1993). It is likely that one or more of these homologues map to regions of the potato genome harbouring mono- or polygenic resistance loci that confer resistance to other G. pallida or G. rostochiensis populations; such as HI (Pineda et al. 1993; Gebhardt et al. 1993), Gpa (Kreike et al.
  • Antisense primers are written in opposite orientation to the peptide sequence
  • EXAMPLE 10 A MARKER ASSISTED SELECTION ASSAY FOR Gpa2
  • the Gpa2 locus is hypothesized to be introgressed from S. tuberosum spp. andigena CPC1673 into European cultivars. Flanking markers tightly linked to Gpa2 are likely to be diagnostic for the presence of Gpa2 in these cultivars. Therefore, Gpa2 linked CAPS markers were used to screen two clones (abbreviated as CPC1673-a and CPC1673-b) of the wild species Solanum tuberosum spp. andigena CPC 1673 (hereafter referred to as CPC1673) as well as nine potato cultivars harbouring introgressions from CPC 1673.
  • CPC1673-a and CPC1673-b the wild species Solanum tuberosum spp. andigena CPC 1673
  • the CAPS marker profiles were highly similar for the selfed CPC 1673 genotypes and the analyzed potato cultivars harboring introgressions from CPC 1673.
  • the CAPS marker alleles linked to Gpa2 were only identified in regions which appeared to be of CPC 1673 origin.
  • five differences in the size of an introgressed region of 0.9 cM were observed.
  • Fig. 1 High resolution map of the Rx locus (not drawn to scale).
  • A Simplified genetic map of potato chromosome XII (denoted by a horizontal line) in which the area left of the arrow is reversed between the potato and tomato genetic maps (Tanksley et al, 1992). Vertical lines indicate positions of previously mapped RFLP markers (Bendahmane et al, 1997; Tanksley et al, 1992). The filled rectangle denotes a genetic interval between markers GP34 and 218L, which is magnified in panels B and C.
  • B Genetic map of the GP34-218L interval (denoted by a horizontal line).
  • Positions of the RFLP marker GP34 and the AFLP markers IPM3, IPM4a and IPM5 are indicated by vertical lines.
  • the positions of BAC end-derived markers and low-stringency PCR markers are indicated by vertical arrows.
  • the symbols L and R denote the BAC ends that were mapped relative to Rxl.
  • the numbers in brackets below the bar indicate the numbers of Sl-Cara individuals containing recombination events in each marker interval, identified in the initial Sl-Cara mapping population of 1720 individuals.
  • the predicted position of Rxl, delimited by the cross-over events in plants Sl-1146 and SI -761, is indicated by the horizontal arrow.
  • C Positions of Cara BAC clones in the GP34-218L interval. Each open rectangle represents one BAC insert DNA. Inside of each rectangle is the name of the BAC clone, the estimated insert size in kb (except for the BAC29).
  • Fig. 2 High resolution genetic and physical map of the Gpa2 locus.
  • A Relative position of the Gpa2 locus on chromosome 12 of potato. Vertical lines indicate positions of previously mapped RFLP markers. The filled rectangle denotes the Gpa2 locus between markers IPM3 and IPM5 which is magnified in panel B.
  • B High resolution genetic map and graphical genotypes of the IPM3-IPM5 interval, indicating differences in the size of Solanum tuberosum spp. andigena CPC 1673 derived segments in different potato genotypes. The relative positions of CAPS markers used to fine-map Gpa2 are indicated by vertical bars. The presented genotypes border the Gpa2 interval. Introgression segments are indicated by thick bars.
  • R for resistant
  • S for susceptible
  • C High resolution physical map of the Gpa2 locus. The relative positions of CAPS markers are indicated by vertical bars.
  • the open rectangles represent BAC clones isolated from the Cara BAC library.
  • the shaded rectangle represents a BAC clone isolated from the SH83 BAC library.
  • the name of each BAC clone is depicted within the rectangle and the estimated insert size is in given in kb.
  • the predicted position of Gpa2 is indicated by the horizontal anow.
  • Recombinant Sl-Cara genotypes SI -761 and S1-B811 delimit the Gpa2 genetic interval.
  • D Relative positions of four resistance gene homologues (RGH1-4) identified within the IPM4c-l llR Gpa2 interval.
  • Fig. 3 Nucleic and amino acid sequence of the Gpa2 gene.
  • A Coding nucleic acid and deduced amino acid sequence of the Gpa2 gene.
  • B Coding sequence of the Gpa2 gene including introns. The positions of the introns (intron 1 position 2691-2947; intron 2 position 3067-3178) are indicated by boxes.
  • C Sequence of the 10.3 kb Xbal-Xbal genomic DNA fragment inserted in pBINRGH2, harbouring the Gpa2 gene. The initiation codon (ATG position 4875-4877) and the termination codon
  • TAG position 8058-8060 are underlined.
  • the positions of the introns are indicated by boxes.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Gpa2 coding and non coding sequence of S. tuberosum
  • CTTCTCTCCA AAATCAGTAA AACATTGGAT GAGTGGCAAA ATGTTGCGGA GAATGTACGT 1080
  • GAAGCTGTTC CCTCATCAAT AATAGACATT CCTCTATCGA TATCAAGCCT ATGCTATCTG 1800
  • AAAGTTCTTG ATGCTGTCAT TGTGATTGAT TCGAATCCTT CCAATATTGT GTAACTTGTT 2880
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Xbal -Xbal pBINRGH2 fragment containing Gpa2 promoter, coding and non coding sequence of S. tuberosum
  • CTCTCTCTCA GATATTTTCT TTAGCAGATT GAGCTTTGAG ACATACTCGA GAGGTAGCGG 1980
  • TTCTCGTATT TCTACTTCTC TATCGTTGTG GTTGGGTTAG GCTGACGTGT CTGGTGGGAA 2220
  • CGCATCTCTA ATTAATCTCG TAAAGGGATT AAGGGGCCAA TTTCAAAGAA TTAGGCGATT 3660
  • TTATTTTGTG CAACTTATAT GGAACCCTTC GTAGGGAGTT AGTCTCACAC TTTTTAGAGT 3780
  • TTTTTTTAGC ACGGAATGTG GGGAAAAGAA GCAGGGCTAT GTGGGGGATT TTTTTCGTCT 5160
  • CTTTGAGTTA CCATCACTTG CCTTCTCACC TAAAACCGTG TTTTCTGTAT TTTGCAATTT 6060
  • TTCAACTATC ACACAATGCC TTCAAAGGCG AGGAGTGGGA AGTAGTTGAG GAAGGGTTTC 7320
  • ATCACTTTCC ATACCTTGAA CGACTTTTTC TTAGCGATTG CTTTTATTTG GATTCAATCC 7440 CTCGAGATTT TGCAGATATA ACCACACTAG CTCTTATTGA TATATTTCGC TGCCAACAAT 7500
  • TAACTCATCA TCATAGTAAA CTCGATAATA ATCTGGATAA TAGCTTTAGT AAGTCAAATT 7680
  • CAACTTTATA CAAGTTTATG TGCATACTTG TGCATACCCA AAGTTGAATA ACATAAACAT 8460
  • ATCTTTAGCA TAATATCTGA TTATATTATT TTGATATACT TTCTCTATCC CTAATTACTT 8700
  • RPS2 of Arabidopsis thaliana A leucine-rich repeat class of plant disease resistance genes. Science 265: 1856-1860.
  • L6 gene for flax rust resistance is related to the Arabidopsis bacterial resistance gene RPS2 and the tobacco viral resistance gene N Plant Cell 7:1195-1206.
  • Mindrinos M., Katagiri, F., Yu, G. L., and Ausubel, F. M. (1994).
  • the A. thaliana disease resistance gene RPS2 encodes a protein a nucleotide-binding site and leucine -rich repeats. Cell 78, 1089-1099.
  • Amplification of flanking sequences by inverse PCR. In PCR Protocols (Irmis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., eds).
  • pBIBPLUS an improved plant transformation vector based on pBIN19.

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Abstract

L'invention concerne le gène de résistance Gpa2 de la pomme de terre conférant une résistance contre les nématodes phytopathogènes du genre Globodera. Elle concerne également des procédés et des matériaux mettant ce gène en application, ainsi que des procédés servant à identifier des gènes apparentés. Elle concerne enfin des polypeptides codés par lesdits gènes de résistance et l'utilisation de ces polypeptides.
PCT/NL1998/000445 1998-07-31 1998-07-31 Procede de genie genetique servant a obtenir une resistance aux nematodes chez les solanacees WO2000006753A1 (fr)

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AU87518/98A AU8751898A (en) 1998-07-31 1998-07-31 Engineering nematode resistance in solanacae
PCT/NL1998/000445 WO2000006753A1 (fr) 1998-07-31 1998-07-31 Procede de genie genetique servant a obtenir une resistance aux nematodes chez les solanacees
PCT/NL1999/000491 WO2000006754A2 (fr) 1998-07-31 1999-07-30 Resistance aux nematodes obtenue par genie genetique dans les solanacees

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2003080838A1 (fr) * 2002-03-27 2003-10-02 University Of Tsukuba Gene resistant au genre meloidogyne, et utilisation

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EP1922409B1 (fr) 2005-08-08 2017-11-08 Bayer CropScience NV Cotonniers tolérants aux herbicides et leurs procédés d'identification

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WO1996022372A2 (fr) * 1995-01-17 1996-07-25 Landbouwuniversiteit Wageningen Anticorps utilisables dans la lutte contre les nematodes kystiques, et plantes transgeniques les exprimant

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BENDAHMANE, A., ET AL.: "high-resolution genetical and physical mapping of the Rx gene for extreme resistance to potato virus X in tetraploid potato", THEORETICAL AND APPLIED GENETICS, vol. 95, 1997, pages 153 - 162, XP002098294 *
KREIKE, C.M., ET AL.: "quantitatively-inherited resistance to Globodera pallida is dominated by one major locus in Solanum spegazzinii", THEORETICAL AND APPLIED GENETICS, vol. 88, 1994, pages 764 - 769, XP002098295 *
ROUPPE VAN DER VOORT,J., ET AL.: "mapping of the cyst nematode resistance locus Gpa2 in potato using a strategy based on comigrating AFLP markers", THEORETICAL AND APPLIED GENETICS, vol. 95, 1997, pages 874 - 880, XP002098292 *

Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2003080838A1 (fr) * 2002-03-27 2003-10-02 University Of Tsukuba Gene resistant au genre meloidogyne, et utilisation
CN1311078C (zh) * 2002-03-27 2007-04-18 国立大学法人筑波大学 根结线虫(Meloidogyne)抗性基因及其用途
AU2002349585B2 (en) * 2002-03-27 2007-05-31 University Of Tsukuba Novel meloidogyne-resistance gene and utilization thereof
US8304609B2 (en) 2002-03-27 2012-11-06 University Of Tsukuba Root-knot nematode-resistance gene and application thereof

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