EP4236678A1 - Résistance au peronospora dans spinacia oleracea - Google Patents

Résistance au peronospora dans spinacia oleracea

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Publication number
EP4236678A1
EP4236678A1 EP21799297.3A EP21799297A EP4236678A1 EP 4236678 A1 EP4236678 A1 EP 4236678A1 EP 21799297 A EP21799297 A EP 21799297A EP 4236678 A1 EP4236678 A1 EP 4236678A1
Authority
EP
European Patent Office
Prior art keywords
allele
plant
spinach
wolf
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21799297.3A
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German (de)
English (en)
Inventor
Erik DEN BOER
Raoul Jacobus Johannes Maria FRIJTERS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rijk Zwaan Zaadteelt en Zaadhandel BV
Original Assignee
Rijk Zwaan Zaadteelt en Zaadhandel BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US17/085,860 external-priority patent/US11473102B2/en
Application filed by Rijk Zwaan Zaadteelt en Zaadhandel BV filed Critical Rijk Zwaan Zaadteelt en Zaadhandel BV
Publication of EP4236678A1 publication Critical patent/EP4236678A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/02—Amaranthaceae or Chenopodiaceae, e.g. beet or spinach
    • A01H6/028—Spinacia oleracea [spinach]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/1255—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for fungal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • 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
    • C12N15/8282—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 fungal resistance

Definitions

  • the invention relates to a gene capable of conferring resistance to a spinach plant against one or more Peronospora effusa races.
  • the invention also relates to a spinach plant, to propagation material of said spinach plant, to a cell of said spinach plant, and to seed of said spinach plant carrying the gene.
  • the invention further relates to a method of producing a spinach plant carrying the gene and to the use of the gene in breeding to confer resistance against Peronospora effusa.
  • Downy mildew (Peronospora effusa) is a major threat for spinach growers because it directly affects the harvested leaves.
  • downy mildew is caused by the oomycete Peronospora effusa (formerly known as Peronospora farinosa f. sp. spinaciae).
  • Infection makes the leaves unsuitable for sale and consumption, as it manifests itself phenotypically as yellow lesions on the older leaves, and on the abaxial leaf surface a greyish fungal growth can be observed.
  • the infection can spread very rapidly, and it can occur both in glasshouse cultivation and in soil cultivation.
  • the optimal temperature for formation and germination of Peronospora effusa spores is 9 to 12°C, and it is facilitated by a high relative humidity. When spores are deposited on a humid leaf surface they can readily germinate and infect the leaf. Fungal growth is optimal between 8 and 20°C and a relative humidity of >80%, and within 6 and 13 days after infection mycelium growth can be observed. Oospores of P. effusa can survive in the soil for up to 3 years, or as mycelium in seeds or living plants.
  • Pe: 1 to Pe: 19 (Pe: 1 to Pe: 17 were formerly known as Pfs: l to Pfs: 17; Irish et al. Phtypathol. Vol. 98 pg. 894-900, 2008; Plantum NL (Dutch association for breeding, tissue culture, production and trade of seed and young plants) press release, “Benoeming van Pfs: 14, een nieuwe fysio van valse meeldauw in spinazie”, September 19, 2012; Report Jim Correl (Univ.
  • R-genes In commercial spinach varieties resistance against downy mildew is usually caused by so-called R-genes.
  • R-gene mediated resistance is based on the ability of a plant to recognize the invading pathogen. In many cases this recognition occurs after the pathogen has established the first phases of interaction and transferred a so called pathogenicity (or avirulence) factor into the plant cell. These pathogenicity factors interact with host components in order to establish conditions which are favorable for the pathogen to invade the host and thereby cause disease. When a plant is able to recognize the events triggered by the pathogenicity factors a resistance response can be initiated. In many different plant pathogen interaction systems such as the interaction of spinach with different downy mildew strains, the plant initiates these events only after specific recognition of the invading pathogen.
  • R-genes are in spinach still the predominant form of defense against downy mildew. This is mainly due to the fact that it is the only form of defense that gives absolute resistance. So far plant breeders have been very successful in generating downy mildew resistant spinach varieties by making use of resistance genes residing in the wild germplasm of the crop species. Even though R-genes are extensively used in spinach breeding, until now not much is known of these R-genes. Only recently it was discovered that the R-genes officially recognized in spinach are in fact all different alleles of the two tightly linked genes, the alpha- and the beta-WOLF genes.
  • a new allelic variant of the Alpha- WOLF gene as described in WO2018059651 was found.
  • the a/p/ra-WOLF gene encodes a protein that belongs to the CC-NBS-LRR family (Coiled Coil - Nucleotide Binding Site - Leucine-Rich Repeat).
  • CC-NBS-LRR family oiled Coil - Nucleotide Binding Site - Leucine-Rich Repeat.
  • allelic variant or the allelic variants
  • said plant will produce a variant of the WOLF protein that confers a certain resistance profile to pathogenic races of Peronospora effusa.
  • allele or “allelic variant” is used to designate a version of the gene that is linked to a specific phenotype, i.e. resistance profile. It was found that a spinach plant may carry one or two WOLF genes. Each of these two WOLF genes encompasses multiple alleles, each allele conferring a particular resistance profile. In the context of this invention an allele or allelic variant is a nucleic acid.
  • the beta WOLF gene is located on scaffoldl2735 (sequence: GenBank: KQ143339.1), at position 213573-221884. In case the spinach plant also carries or only carries the alpha-WOLF gene, the alpha-WOLF gene is located at approximately the same location as where the beta-WOLF gene is located on scaffoldl2735 in the Viroflay genome assembly.
  • the newly found alpha-WOLF allele provides resistance to at least downy mildew race Pe: 14, Pe: 15 and Pe: 17.
  • a genome assembly for spinach variety Viroflay - which is susceptible to all known pathogenic races of Peronospora effusa - is publicly available (Spinacia oleracea cultivar SynViroflay, whole genome shotgun sequencing project; Bioproject: PRJNA41497; GenBank: AYZV00000000.2; BioSample: SAMN02182572, see also Dohm et al, 2014, Nature 505: 546- 549).
  • the beta- WOLF gene is located on scaffoldl2735 (sequence: GenBank: KQ143339.1), at position 213573-221884.
  • the sequence covered by this interval comprises the entire genomic sequence of the beta- WOLF gene of Viroflay, plus 2000 basepairs sequence upstream from the gene, plus the sequence downstream from the gene, up to the locus of the neighbouring gene that is situated downstream from the WOLF gene.
  • Spinach variety Viroflay only possesses a single WOLF gene, namely a beta- WOLF gene, but most other spinach lines harbor a single alpha-type WOLF gene at the same location in the genome.
  • Other spinach lines harbor two WOLF genes at approximately the same location in the genome. In such cases, the two WOLF genes are positioned adjacent to each other.
  • all WOLF proteins comprise in their amino acid sequence the motif “MAEIGYSVC” (SEQ ID NO: 1) at the N-terminus.
  • all alpha-WOLF proteins comprise the motif “KWMCLR” (SEQ ID NO: 2) in their amino acid sequence
  • all beta- WOLF proteins comprise the motif “HVGCWDR” (SEQ ID NO: 3) in their amino acid sequence.
  • the present invention relates to a new Peronospora effusa resistance conferring allele of the alpha-WOLF gene designated alpha-WOLF 27.
  • the invention relates to a Peronospora effusa resistance conferring allele designated alpha-WOLF 27 wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGYSVC” at its N-terminus; and b) the motif “KWMCLR”; and wherein the LRR domain of the protein has in order of increased preference at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence identity to SEQ ID NO: 10.
  • the alpha-WOLF 27 allele further comprises an additional motif in its amino acid sequence, namely “DQEDEGEDN”.
  • the invention further relates to a Peronospora effusa resistance conferring allele designated alpha-WOLF 27 wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGYSVC” at its N-terminus; and b) the motif “KWMCLR”; and wherein the LRR domain of the protein has in order of increased preference at least 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence similarity to SEQ ID NO: 10.
  • the alpha-WOLF 27 allele further comprises an additional motif in its amino acid sequence, namely “DQEDEGEDN”.
  • the invention also relates to an alpha-WOLF 27 allele having an LRR domain which has a sequence that in order in order of increased preference has at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity to SEQ ID NO: 9.
  • the LRR domain of the protein of the alpha- WOLF 27 allele is defined as the amino acid sequence that in order of increased preference has at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity to SEQ ID NO: 10.
  • the LRR domain of the protein of the alpha- WOLF 27 allele is defined as the amino acid sequence that in order of increased preference has at least 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence similarity to SEQ ID NO: 10.
  • Sequence similarity for an amino acid sequence is calculated using EMBOSS stretcher 6.6.0 (www.ebi.ac.uk/Tools/psa/emboss_stretcher), using the EBLOSUM62 matrix with settings Gap open penalty: 12 and Gap extend penalty: 2.
  • sequence similarity is calculated using the DNA full matrix with settings Gap open penalty: 16 and Gap extend penalty: 4.
  • the LRR domain of the alpha-WOLF 27 allele as defined herein can be determined by amplifying and sequencing the genomic DNA encoding for the amino acid sequence of LRR domain using specific primers, and subsequently translating the DNA sequence into an amino acid sequence, thereby applying common sense in choosing the correct reading frame.
  • the skilled person is capable of doing this, using freely available online bioinformatics tools such as can be found here: http://web.expasy.org/translate/.
  • the genomic sequence of a LRR domain of an alpha-WOLF gene such as alpha- WOLF 27 can be amplified using a primer pair having a forward primer which is a nucleic acid molecule having the sequence of SEQ ID NO: 4 and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID NO: 5.
  • the invention also relates to a nucleic acid molecule which confers resistance to at least one Peronospora effusa race, wherein the protein encoded by said nucleic acid molecule is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGY SVC” at its N-terminus; and b) the motif “KWMCLR”; and wherein the LRR domain of the protein has in order of increased preference at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence identity to SEQ ID NO: 10.
  • this nucleic acid molecule is an isolated nucleic acid molecule.
  • the invention also relates to a nucleic acid molecule which confers resistance to at least one Peronospora effusa race, wherein the protein encoded by said nucleic acid molecule is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGYSVC” at its N-terminus; and b) the motif “KWMCLR”; and wherein the LRR domain of the protein has in order of increased preference at least 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence similarity to SEQ ID NO: 10.
  • this nucleic acid molecule is an isolated nucleic acid molecule.
  • PCR conditions for amplifying the LRR domain-encoding region of an alpha- WOLF gene using primers having SEQ ID NO: 4 and SEQ ID NO: 5 are, using Platinum Taq enzyme (Thermo Fisher Scientific): 3 minutes at 95 °C (initial denaturing step); 40 amplification cycles, each cycle consisting of: 30 seconds denaturation at 95°C, 30 seconds annealing at 60°C, and 30 seconds extension at 72°C; 2 minutes at 72°C (final extension step).
  • the LRR domain of a beta-WOLF gene e.g. the null allele as present in variety Viroflay, can be amplified using a forward primer which is a nucleic acid molecule having the sequence of SEQ ID NO: 6 and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID NO: 5.
  • PCR conditions for amplifying the LRR domain-encoding region of a beta- WOLF gene using primers having SEQ ID NO: 5 and SEQ ID NO: 6 are as follows, using Platinum Taq enzyme (Thermo Fisher Scientific): 3 minutes at 95°C (initial denaturing step); 40 amplification cycles, each cycle consisting of: 30 seconds denaturation at 95°C, 50 seconds annealing at 58°C and 50 seconds extension at 72°C; 2 minutes at 72°C (final extension step).
  • the invention also relates to a primer pair for amplifying the LRR domain of an alpha-WOLF gene, more in particular for amplifying the LRR domain of an alpha- WOLF 27 allele wherein the forward primer is a nucleic acid molecule having the sequence of SEQ ID NO: 4 and the reverse primer which is a nucleic acid molecule having the sequence of SEQ ID NO: 5.
  • the primers disclosed herein have been specifically designed for selectively amplifying part of a WOLF gene, and not of any other CC-NBS-LRR protein-encoding genes.
  • the invention relates to an alpha-WOLF 27 allele which has a coding sequence that in order of increased preference has at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity to SEQ ID NO: 12.
  • the alpha-WOLF 27 allele encodes for a protein having an amino acid sequence which in order of increased preference has at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity to SEQ ID NO: 13.
  • the alpha-WOLF 27 allele encodes for a protein having an amino acid sequence which in order of increased preference has at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence similarity to SEQ ID NO: 13.
  • the alpha-WOLF 27 allele when present in a spinach plant confers complete resistance to at least one of the 19 officially recognized Peronospora effusa races. In a further embodiment, the alpha-WOLF 27 allele when present in a spinach plant confers complete resistance to at least two of the 19 officially recognized Peronospora effusa races. In a further embodiment, the alpha-WOLF 27 allele when present in a spinach plant confers complete resistance in order of increased preference to at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen of the nineteen officially recognized Peronospora effusa races.
  • the alpha-WOLF 27 allele when homozygously present in a spinach plant confers complete resistance to at least the officially recognized Peronospora effusa races Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 17. More in particular, the alpha-WOLF 27 allele when homozygously present in a spinach plant confers complete resistance to at least the officially recognized Peronospora effusa races Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 17 (see Table 1).
  • the alphaWOLF 27 allele also when homozygously present in a spinach plant confers complete resistance to at least the officially recognized Peronospora effusa races Pe: 16 and Pe: 18. Furthermore, the alphaWOLF 27 allele when homozygously present in a spinach plant confers intermediate resistance to Peronospora effusa race Pfs: 10.
  • a seedling test is defined as a test wherein spinach plants are planted in trays containing growth medium, fertilized twice a week after seedling emergence. Plants are inoculated at the first true leaf stage with a sporangial suspension having a concentration of approximately 2.5 x 10 5 /ml of one of the pathogenic races of Peronospora effusa or isolates to be tested. Thirty plants per race are tested.
  • the inoculated plants are placed in a dew chamber at 18°C with 100% relative humidity for a 24 h period, and then moved to a growth chamber at 18°C with a 12 h photoperiod for 6 days. After 6 days, the plants are returned to the dew chamber for 24 h to induce sporulation, and subsequently scored for a disease reaction.
  • a plant is completely resistant against a Peronospora effusa race when a plant shows no symptoms in the seedling test described herein.
  • a plant is intermediately resistant against a Peronospora effusa race when a plant shows only symptoms of chlorosis, or sporulation occurring only on the tips of the cotyledons in the seedling test described herein.
  • a plant is susceptible to an isolate of a Peronospora effusa race when a plant shows more than only symptoms of chlorosis, or when sporulation occurs on area larger than only the tips of the cotyledons in the seedling test described herein.
  • Another aspect of the invention relates to a spinach plant, comprising the alpha- WOLF 27 allele of invention, of which a representative sample of seed was deposited with the NCIMB under accession number NCIMB 43668.
  • the plant of the invention which comprises the alpha- WOLF 27 allele is an agronomically elite spinach plant.
  • an agronomically elite spinach plant is a plant having a genotype that results into an accumulation of distinguishable and desirable agronomic traits which allow a producer to harvest a product of commercial significance, preferably the agronomically elite spinach plant comprising the alpha- WOLF 27 allele is a plant of an inbred line or a hybrid.
  • a plant of an inbred line is a plant of a population of plants that is the result of three or more rounds of selfing, or backcrossing; or which plant is a double haploid.
  • An inbred line may e.g. be a parent line used for the production of a commercial hybrid.
  • a hybrid plant is a plant which is the result of a cross between two different plants having different genotypes. More in particular, a hybrid plant is the result of a cross between plants of two different inbred lines, such a hybrid plant may e.g. be a plant of an Fi hybrid variety.
  • a plant carrying the alpha-WOLF 27 allele in heterozygous form may further comprise a beta-WOLF 0 allele as e.g. present in variety Viroflay wherein the beta-WOLF 0 allele does not confer any resistance to downy mildew.
  • a plant heterozygous for the alpha- WOLF 27 allele may further comprise an allele of the alpha/beta-IFOZFgene that does provide resistance to downy mildew.
  • such an allele would complement the alpha-WOLF 27 allele such that the spinach plant will be at least intermediately resistant to one or more other races to which the alpha-WOLF 27 allele does not provide resistance.
  • the other allele of the alpha/beta-JPOLFgene complements the alpha-WOLF 27 allele such that the plant is resistant to Peronospora effusa races Pe: 1 to Pe: 19.
  • such a plant is an agronomically elite plant.
  • the resistance profile of a plant carrying the alpha-WOLF 27 allele is complemented by a resistance conferring allele of a totally different gene.
  • examples of such genes are e.g. DMR1 as described in US8,354,570, DMR6 as described in US9,121,029 and plO as described in US 10,226,016.
  • the invention thus relates to a spinach plant carrying the alpha-WOLF 27 allele and further comprising a genetic determinant resulting in resistance against Peronospora effusa races Pe: 1 to Pe: 19.
  • the genetic determinant can be another resistance conferring alpha/beta- WOLF allele or a resistance conferring allele of a totally different gene.
  • the invention further relates to propagation material comprising the alpha-WOLF 27 allele.
  • the propagation material is suitable for sexual reproduction.
  • Such propagation material comprises for example a microspore, pollen, ovary, ovule, embryo sac and egg cell.
  • the propagation material is suitable for vegetative reproduction.
  • Such propagation material comprises for example a cutting, root, stem, cell, protoplast, and a tissue culture of regenerable cells.
  • a part of the plant that is suitable for preparing tissue cultures is in particular a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root tip, an anther, a flower, a seed and a stem.
  • the invention furthermore relates to a cell of a spinach plant comprising the alpha- WOLF 27 allele.
  • a cell of a spinach plant comprising the alpha- WOLF 27 allele.
  • Such a cell may be either in isolated form or may be part of the complete plant or parts thereof and then still constitutes a cell of the invention because such a cell harbors the alpha- WOLF 27 allele that confers resistance to downy mildew.
  • Each cell of a plant of the invention carries the genetic information that confers resistance to Peronospora effusa.
  • Such a cell of the invention may also be a regenerable cell that may be used to regenerate a new plant comprising the allele of the invention.
  • Yet another aspect of the invention relates to a method for making a hybrid spinach seed comprising crossing a first parent spinach plant with a second parent spinach plant and harvesting the resultant hybrid spinach seed, wherein said first and/or second parent spinach plant comprises the alpha-WOLF 27 allele.
  • the first and/or second parent plant is a plant of an inbred line as defined herein.
  • the invention further relates to a hybrid spinach plant grown from seed produced by crossing a first parent spinach plant with a second parent spinach plant and harvesting the resultant hybrid spinach seed, wherein said first and/or second parent spinach plant comprises the alpha-WOLF 27 allele.
  • Determining the genomic DNA or coding DNA sequence of at least part of a WOLF gene in the genome of a spinach plant may be performed using any suitable molecular biological method known in the art, including but not limited to (genomic) PCR amplification followed by Sanger sequencing, whole-genome-sequencing, transcriptome sequencing, sequencespecific target capture followed by next-generation sequencing (using, for example, the xGen® target capture system of Integrated DNA Technologies), specific amplification of LRR- domain-comprising gene sequences (using, for example, the RenSeq methodology, as described in US patent application 14/627116, and in Jupe et al., 2013, Plant J. 76: 530-544) followed by sequencing, etcetera.
  • any suitable molecular biological method known in the art, including but not limited to (genomic) PCR amplification followed by Sanger sequencing, whole-genome-sequencing, transcriptome sequencing, sequencespecific target capture followed by next-generation sequencing (using, for example, the xGen® target
  • the invention relates to a method for identifying a plant carrying the alpha-WOLF 27 allele comprises determining the DNA sequence coding for the LRR domain as defined herein.
  • the LRR domain of the alpha-WOLF 27 allele is determined by using a primer pair to amplify the genomic DNA region of the LRR domain.
  • the forward primer is preferably a nucleic acid molecule having the sequence of SEQ ID NO: 4 and the reverse primer is preferably a nucleic acid molecule having the sequence of SEQ ID NO: 5.
  • Another aspect of the invention relates to a method for producing a spinach plant comprising resistance to Peronospora effusa comprising: (a) crossing a plant comprising the alpha- WOLF 27 allele, with another plant; (b) optionally performing one or more rounds of selfing and/or crossing; (c) optionally selecting after each round of selfing or crossing for a plant that comprises the alpha-WOLF 27 allele.
  • Selecting a plant comprising the alpha-WOLF 27 allele can be done by determining the presence of the DNA sequence of the NBS-LRR domain of the allele having in order of increased preference 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity to SEQ ID NO: 9.
  • selecting a plant comprising the alpha-WOLF 27 allele can be done by determining the presence the coding sequence of the entire allele.
  • the presence of the alpha-WOLF 27 allele can be determined phenotypically by assaying a plant in a disease test, for example the test as described herein.
  • the invention further relates to the use of a spinach plant carrying the alpha- WOLF 27 allele in breeding to confer resistance against Peronospora effusa.
  • the invention also relates to a breeding method for the development of spinach plants carrying the alpha-WOLF 27 allele of the invention wherein germplasm which comprises said allele is used. Seed capable of growing into a plant comprising the allele of the invention and being representative for the germplasm was deposited with the NCIMB under accession number NCIMB 43668.
  • the invention in another aspect, relates to a method for the production of a spinach plant which comprises alpha-WOLF 27 allele, which method comprises: (a) crossing a plant comprising the allele with another plant; (b) optionally selecting for plants comprising said allele in the F 1 ; (c) optionally backcrossing the resulting F 1 with the preferred parent and selecting for plants that have the said allele in the BC1F1; (d) optionally performing one or more additional rounds of selfing, crossing, and/or backcrossing, and subsequently selecting for a plant which comprises the said allele or shows the resistance profile corresponding to said allele.
  • the invention also encompasses a spinach plant produced by this method.
  • the invention also relates to a harvested leaf of a spinach plant of the invention, to a food product which comprises a harvested leaf of a spinach plant of the invention, either in natural or in processed form.
  • Spinach leaves are sold in packaged form, including without limitation as prepackaged spinach leaves or as processed in a salad comprising said leaves. Mention of such a package is e.g. made in US Patent No. 5,523,136, which provides packaging film, and packages from such packaging film, including such packaging containing leafy produce, and methods for making and using such packaging film and packages, which are suitable for use with the spinach leaves of the invention.
  • the invention comprehends the use of and methods for making and using the leaves of the spinach plant of the invention, as well as leaves of spinach plants derived from the invention.
  • the invention further relates to a container which comprises one or more plants of the invention, or one or more spinach plants derived from a plant of the invention, in a growth substrate for harvest of leaves from the plant, in a domestic environment. This way the consumer may pick very fresh leaves for use in salads, when the plant is in a ready-to-harvest condition.
  • the invention also relates to the use of a spinach plant, of which representative seed was deposited with the NCIMB under accession number NCIMB 43668, in the production of a spinach plant comprising the alpha-WOLF 27 allele.
  • the said spinach plant is a hybrid, doubled haploid, or inbred spinach plant.
  • the spinach plant of the invention may comprise the alpha-WOLF 27 allele heterozygously or homozygously.
  • Another aspect of the invention is the use of a cell comprising the alpha-WOLF 27 allele for the production of a spinach plant showing resistance to Peronospora effusa.
  • the invention relates to an allele designated alpha-WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 17.
  • the alpha-WOLF 27 allele when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 17.
  • the alpha-WOLF 27 allele when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10.
  • the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGY SVC” at its N-terminus; and b) the motif “KWMCLR”; and wherein the LRR domain of the protein has in order of increased preference at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% sequence identity to SEQ ID NO: 10.
  • the invention further relates to an allele designated alpha-WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at ⁇ Qas Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid
  • the invention further relates to an allele designated alpha-WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at ⁇ Qas Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid
  • the invention further relates to an allele designated alpha-WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a
  • the invention also relates to a spinach plant comprising an allele designated alpha- WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 17.
  • alpha-WOLF 27 allele when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 17.
  • the alpha-WOLF 27 allele when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10.
  • the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGY SVC” at its N-terminus; and b) the motif “KWMCLR”; and wherein the LRR domain of the protein has in order of increased preference at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence identity to SEQ ID NO: 10.
  • this spinach plant is an agronomically elite spinach plant.
  • the invention also relates to a spinach plant comprising an allele designated alpha- WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at ⁇ Qas Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10, wherein the protein encoded by said allele is a CC-NBS-LRR
  • the invention also relates to a spinach plant comprising an allele designated alpha- WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at ⁇ Qas Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10, wherein the protein encoded by said allele is a CC-NBS-LRR
  • the invention also relates to a spinach plant comprising an allele designated alpha- WOLF 27 which when present in a spinach plant homozygously confers complete resistance to at least Peronospora effusa race Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at least Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15 and Pe: 17; or confers complete resistance to at ⁇ Qas Peronospora effusa race Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 16, Pe: 17 and Pe: 18 and intermediate resistance to Peronospora effusa race Pe: 10, wherein the protein encoded by said allele is a CC-NBS-LRR
  • An agronomically elite spinach plant comprising an allele which confers resistance to at least one Peronospora effusa race when present in a spinach plant and encodes a protein that in order of increased preference has at least 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence identity to a protein comprising an amino acid sequence SEQ ID NO: 13; wherein said protein comprises in its amino acid sequence: a) SEQ ID NO: 1, b) SEQ ID NO: 2, and wherein the LRR domain of the protein has in order of increased preference at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99
  • An agronomically elite spinach plant comprising an allele which when homozygously present in a spinach plant encodes a protein that confers complete resistance to at least Peronospora effusa races Pe: 1, Pe: 2, Pe: 3, Pe: 4, Pe: 5, Pe: 6, Pe: 7, Pe: 8, Pe: 9, Pe: 11, Pe: 12, Pe: 13, Pe: 14, Pe: 15, Pe: 17, wherein the allele has a nucleotide sequence which has in order of increased preference at least 95%, 95.3%, 95.5%, 95.8%, 96%, 96.3%, 96.5%, 96.8%, 97%, 97.3%, 97.5%, 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence identity to SEQ ID NO: 12.
  • a method of producing an Fl hybrid spinach seed comprising crossing a first parent spinach plant with a second parent spinach plant and harvesting the resultant hybrid spinach seed, wherein said first parent spinach plant and/or said second parent spinach plant is the agronomically elite spinach plant of any of the paragraphs 1 to 7.
  • a method for producing a spinach plant showing resistance to Peronospora effusa comprising: (a) crossing the agronomically elite spinach plant of any of the paragraphs 1 to 7 with another spinach plant; (b) optionally performing one or more rounds of selfing and/or crossing; (c) optionally selecting after the crossing or the one or more rounds of selfing and/or crossing for a plant that comprises said allele.
  • the method includes performing the optional selection, and the selection of the plant comprising the allele expressing the protein comprises determining the presence of the allele according to a method comprising any or more of; determining the presence of a genomic nucleotide sequence in the genome of a plant, wherein said sequence in order of increased preference at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% sequence identity to SEQ ID NO: 11, or determining the presence of a nucleotide sequence in a plant, wherein said sequence has in order of increased preference at least 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence identity to SEQ ID NO: 12, or determining the presence of a LRR
  • the method includes performing the optional one or more rounds of selfing and/or crossing and the optional selection, and the selection of the plant comprising the allele expressing the protein comprises determining the presence of the allele according to a method comprising any or more of: determining the presence of a genomic nucleotide sequence in the genome of a plant, wherein said sequence in order of increased preference at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% sequence identity to SEQ ID NO: 11, or determining the presence of a nucleotide sequence in a plant, wherein said sequence has in order of increased preference at least 97.8%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3%, 99.5%, 99.8%, 100% sequence identity to
  • a method of producing an Fl hybrid spinach seed comprising crossing a first parent spinach plant with a second parent spinach plant and harvesting the resultant hybrid spinach seed, wherein said first parent spinach plant and/or said second parent spinach plant is the agronomically elite spinach plant of any of the paragraphs 1 to 7.
  • Resistance profile conferred by the alpha-WOLF 27 allele when homozygously present in a spinach plant A means complete resistance against a particular downy mildew race; means intermediate resistance against a particular downy mildew race; “+” means that the allele confers no resistance and would cause a plant only carrying the alpha-WOLF 27 allele to be fully susceptible for that particular downy mildew race; “nt” means that it has not been tested against that isolate.
  • the inoculated plants were placed in a dew chamber at 18°C with 100% relative humidity for a 24 h period, and then moved to a growth chamber at 18°C with a 12 h photoperiod for 6 days. After 6 days, the plants were returned to the dew chamber for 24 h to induce sporulation, and they were scored for disease reaction.
  • Plants for this specific test were scored as resistant, intermediately resistant, or susceptible based on symptoms of chlorosis and signs of pathogen sporulation on the cotyledons and true leaves, as described by Irish et al. (2007; Plant Dis. 91: 1392-1396). Plants exhibiting no evidence of chlorosis and sporulation were in this specific test considered as resistant. Resistant plants were re-inoculated to assess whether plants initially scored as resistant had escaped infection, or whether they were truly resistant. Plants that showed only symptoms of chlorosis, or sporulation occurring only on the tips of the cotyledons were scored as intermediately resistant. Plants showing more than these symptoms of downy mildew infection were scored as being susceptible.
  • Table 1 shows the resistance of a plant carrying the alpha- WOLF 27 allele to each one of these pathogenic races.
  • Table 3 shows the differential set of spinach downy mildew races and the resistance of various spinach varieties (hybrids) to each one of these pathogenic races.
  • a susceptible reaction is scored as “+” (indicating a successful infection by the fungus, with sporulation occurring on the entire cotyledon), and resistance is depicted as (absence of sporulation on the cotyledons).
  • An intermediate resistance response is indicated as “(-)”, which in practice means a slightly level of infection (with only symptoms of chlorosis, or sporulation only occurring on the tips of the cotyledons in the differential seedling test).
  • the isolated genomic DNA of a spinach plant comprising the alpha-WOLF 27 allele, of which a representative sample of seed was deposited with the NCIMB under accession number NCIMB 43668 was used in polymerase chain reactions (PCR), using forward primer ACAAGTGGATGTGTCTTAGG (SEQ ID NO: 4) and reverse primer TTCGCCCTCATCTTCCTGG (SEQ ID NO: 5).
  • PCR polymerase chain reactions
  • the primer pair amplifies the LRR domainencoding region of an alpha- WOLF gene, and has been designed for selectively amplifying part of a WOLF gene, and not of other CC-NBS-LRR protein-encoding genes.
  • PCR conditions for amplifying the LRR domain-encoding region of an alpha- WOLF gene using primers having SEQ ID NO: 4 and SEQ ID NO: 5 were as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):
  • each cycle consisting of: 30 seconds denaturation at 95°C, 30 seconds annealing at 60°C, and 30 seconds extension at 72°C
  • the isolated genomic DNA of a spinach plant of variety Viroflay comprising the beta-WOLF 0 allele was used in polymerase chain reactions (PCR), using forward primer TCACGTGGGTTGTGTTGT (SEQ ID NO: 6) and reverse primer TTCGCCCTCATCTTCCTGG (SEQ ID NO: 5).
  • the primer pair amplifies the LRR domain-encoding region of a beta-WOLF gene, and has been designed for selectively amplifying part of a WOLF gene, and not of other CC- NBS-LRR protein-encoding genes.
  • PCR conditions for amplifying the LRR domain-encoding region of a beta- WOLF gene using primers having SEQ ID NO: 5 and SEQ ID NO: 6 were as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):
  • each cycle consisting of: 30 seconds denaturation at 95°C, 50 seconds annealing at 58°C and 50 seconds extension at 72°C
  • PCR products were visualized on agarose gel (not shown), and DNA was purified from the PCR reaction. Subsequently the sequence of the PCR products was determined using methods well known in the art.
  • the DNA sequence of the LRR domain of the alpha-WOLF 27 allele amplified by primers having SEQ ID NO: 4 and SEQ ID NO: 5 is provided in Table 2 under SEQ ID NO: 9.
  • the DNA sequence of the LRR domain of the beta-WOLF 0 allele amplified by primers having SEQ ID NO: 5 and SEQ ID NO: 6 is provided in Table 2 under SEQ ID NO: 7.
  • PCR primers and PCR conditions were different.
  • a spinach plant comprising the alpha-WOLF 27 allele, of which a representative sample of seed was deposited with the NCIMB under accession number NCIMB 43668 was crossed with a plant of variety Viroflay carrying the beta-WOLF 0 allele to obtain a Fl generation. Subsequently, a Fl plant was selfed to obtain a F2 population.
  • Plants of the F2 population were assayed as described in Example 1 for resistance to Peronospora effusa Pe: 14, Pe: 15 and Pe: 17.
  • Genomic DNA of each plant of the same F2 population was isolated and used in two different polymerase chain reactions (PCR). The first PCR reaction was done using primers for amplifying the LRR domain of an alpha-WOLF allele and the second PCR reaction was done using primers for amplifying the LRR domain of a beta-WOLF allele, both as described in Example 2.
  • DNA from the PCR reaction was purified, and subsequently the sequence of the PCR products was determined.
  • the alpha-WOLF PCR products gave a sequence that corresponded to the sequence of SEQ ID NO: 9, the genomic sequence of the LRR domain of the alpha-WOLF 27 allele.
  • the beta-WOLF PCR products gave a sequence that corresponded to the sequence of SEQ ID NO: 7 the genomic sequence of the LRR domain of the beta-WOLF 0 allele.

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Abstract

La présente invention concerne un allèle désigné alpha-WOLF 27 qui confère une résistance à au moins une race de Peronospora effusa, la protéine codée par ledit allèle étant une protéine CC-NBS-LRR qui comprend dans sa séquence d'acides aminés : a) le motif "MAEIGY SVC" à son extrémité N-terminale ; et b) le motif "KWMCLR" ; le domaine LRR de la protéine ayant dans l'ordre de préférence croissant au moins least 95 %, 95,5 %, 96 %, 96,5 %, 97 %, 97,5 %, 98 %, 98,5 %, 99 %, 99,5 %, 100 % de similarité de séquence avec la SEQ ID No : 10. L'allèle, lorsqu'il est présent dans une plante d'épinard, confère de façon homozygote une résistance complète à au moins la race de Peronospora effusa Pe : 7, Pe : 8, Pe : 9, Pe : 11, Pe : 12, Pe : 13, Pe : 14, Pe : 15, Pe : 17
EP21799297.3A 2020-10-30 2021-11-01 Résistance au peronospora dans spinacia oleracea Pending EP4236678A1 (fr)

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US5523136A (en) 1993-04-30 1996-06-04 Cypress Packaging Packaging film, packages and methods for using them
WO2007051483A1 (fr) 2005-11-01 2007-05-10 Universiteit Utrecht Holding B.V. Plantes résistantes aux maladies
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WO2015036469A1 (fr) * 2013-09-13 2015-03-19 Nunhems B.V. Plantes d'épinard résistant au mildiou
WO2015036378A1 (fr) * 2013-09-13 2015-03-19 Nunhems B.V. Épinards résistants au mildiou
AU2014221305B2 (en) * 2013-09-13 2019-09-19 Nunhems B.V. Spinach plants that are resistant to downy mildew
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US9402363B1 (en) * 2015-11-20 2016-08-02 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Peronospora resistance in spinacia oleracea
US10226016B2 (en) 2016-05-13 2019-03-12 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Non R-gene mediated resistance
WO2018059651A1 (fr) 2016-09-30 2018-04-05 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Procédé destiné à modifier le profil de résistance de spinacia oleracea au mildiou

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