Patent Description:
Downy mildew (Peronospora farinosa f. spinaciae) is a major threat for spinach growers because it directly affects the harvested leaves. In spinach, downy mildew is caused by the oomycete Peronospora farinosa f. spinaciae (formerly known as P. 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 P. farinosa f. spinaciae spores is <NUM> to <NUM>, 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 <NUM> and <NUM> and a relative humidity of ≥<NUM>%, and within <NUM> and <NUM> days after infection mycelium growth can be observed. Oospores of P. farinosa can survive in the soil for up to <NUM> years, or as mycelium in seeds or living plants.

To date <NUM> pathogenic races of spinach downy mildew (Pfs) have been officially identified and characterized, and many new candidates are observed in the field. The <NUM> officially recognized races of Peronospora farinosa f. spinaciae, are designated Pfs:<NUM> to Pfs:<NUM> (<NPL>; <NPL>; Report <NPL>; <NPL>; <NPL>; <NPL>). Races <NUM> to <NUM> were identified between <NUM> and <NUM>, while only recently two new Peronospora isolates have been identified, termed UA201519B and US1602, which subsequently have been officially named Pfs:<NUM> and Pfs:<NUM> by the International Working Group on Peronospora (IWGP) (<NPL>; <NPL>. All <NUM> officially recognized Pfs races are publicly available from the Department of Plant Pathology, University of Arkansas, Fayetteville, AR <NUM>, USA, and also from NAK Tuinbouw, Sotaweg <NUM>, <NUM> GD Roelofarendsveen, the Netherlands.

Especially the latest identified Peronospora races can break the resistance of many spinach varieties that are currently used commercially worldwide, and they thus pose a serious threat to the productivity of the spinach industry. Therefore, it is crucial to stay at the forefront of developments in this field, as Peronospora continuously develops the ability to break the resistances that are present in commercial spinach varieties. For this reason new resistance genes against downy mildew are very valuable assets, and they form an important research focus in breeding and particular in spinach and lettuce breeding. One of the main goals of spinach breeders is to rapidly develop spinach varieties with a resistance to as many Peronospora races as possible, including the latest identified races, before these races become wide-spread and pose a threat to the industry.

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.

Co-evolution of plant and pathogen has led to an arms race in which a R-gene mediated resistance is sometimes overcome as a consequence of the capability of the pathogen to interact with and modify alternative host targets or the same targets in a different way, such that the recognition is lost and infection can be established successfully resulting in disease. In order to reestablish resistance in a plant, a new R-gene has to be introduced which is able to recognize the mode of action of an alternative pathogenicity factor.

Despite the fact that the durability of R-genes is relatively low, 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. This was also the first time that R-genes, or better R-alleles were for the first time characterized at the molecular level, i.e. their nucleotide and amino acid sequence was determined. Although this provides the breeder with tools that increase the efficiency of detecting and selecting R-alleles, adequately responding to newly emerging downy mildew races is still crucial for developing commercially successful spinach varieties. Therefore, it is the object of the invention to provide a new resistance allele conferring resistance to a newly emerged downy mildew isolate and to provide molecular biological tools for identifying this new resistance allele.

In the research leading to the present invention, a new allelic variant of the Alpha-WOLF gene as described in <CIT> was found. The alpha-WOLF gene encodes a protein that belongs to the CC-NBS-LRR family (Coiled Coil - Nucleotide Binding Site - Leucine-Rich Repeat). Depending on the allelic variant (or the allelic variants) that is (are) present in a spinach plant, said plant will produce a variant of the WOLF protein that confers a certain resistance profile to pathogenic races of Peronospora farinosa f.

In the context of this invention the term "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 scaffold12735 (sequence: GenBank: KQ143339. <NUM>), at position <NUM>-<NUM>. 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 scaffold12735 in the Viroflay genome assembly.

The newly found alpha-WOLF allele provides at least resistance to downy mildew race Pfs:<NUM>. Alpha-WOLF <NUM> also provides resistance to Pfs:<NUM> and Pfs15.

A genome assembly for spinach variety Viroflay - which is susceptible to all known pathogenic races of Peronospora farinosa f. spinaciae - is publicly available (Spinacia oleracea cultivar SynViroflay, whole genome shotgun sequencing project; Bioproject: PRJNA41497; GenBank: AYZV00000000. <NUM>; BioSample: SAMN02182572, see also <NPL>). In this genome assembly for Viroflay, the beta-WOLF gene is located on scaffold12735 (sequence: GenBank: KQ143339. <NUM>), at position <NUM>-<NUM>. The sequence covered by this interval comprises the entire genomic sequence of the beta-WOLF gene of Viroflay, plus <NUM> 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. In most spinach lines that harbor two WOLF genes, one of said WOLF genes belongs to the alpha-type, and the other WOLF gene belongs to the beta-type. It was observed that this allelic variation in the WOLF locus is responsible for differences in resistance to pathogenic races of Peronospora farinosa f.

The difference between an allele of an alpha-WOLF gene and an allele of a beta-WOLF gene lies in the presence of specific conserved amino acid motifs in the encoded protein sequence. As mentioned above, all WOLF proteins possess - from N- to C-terminus - the following domains that are generally known in the art: a coiled coil domain (RX-CC-like, cd14798), an NBS domain (also referred to as "NB-ARC domain", pfam00931; <NPL>), and leucine-rich repeats (IPR032675) which encompass the LRR domain. In addition, all WOLF proteins comprise in their amino acid sequence the motif "MAEIGYSVC" at the N-terminus. In addition to this, all alpha-WOLF proteins comprise the motif "KWMCLR" in their amino acid sequence, whereas all beta-WOLF proteins comprise the motif "HVGCVVDR" in their amino acid sequence.

Disclosed herein is a new Peronospora farinosa f. spinaciae resistance conferring allele of the alpha-WOLF gene designated alpha-WOLF <NUM>.

In particular, disclosed herein is a Peronospora farinosa f. spinaciae resistance conferring allele designated alpha-WOLF <NUM> 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 <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>. Optionally, the alpha-WOLF <NUM> allele further comprise an additional motif in their amino acid sequence, namely "DQEDEGEDN".

Further disclosed herein is a Peronospora farinosa f. spinaciae resistance conferring allele designated alpha-WOLF <NUM> 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 <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity to SEQ ID No:<NUM>. Optionally, the alpha-WOLF <NUM> allele further comprise an additional motif in their amino acid sequence, namely "DQEDEGEDN".

An alpha-WOLF <NUM> allele LRR domain in order in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM> is also disclosed herein.

Furthermore, disclosed herein is an alpha-WOLF <NUM> allele having an LRR domain which has a genomic sequence that in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity to SEQ ID No:<NUM>.

For the purpose of this disclosure, the LRR domain of the protein of the alpha-WOLF <NUM> allele is defined as the amino acid sequence that in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>.

For the purpose of this disclosure, the LRR domain of the protein of the alpha-WOLF <NUM> allele is defined as the amino acid sequence that in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity to SEQ ID No:<NUM>.

The skilled person is familiar with methods for the calculation of sequence similarity and sequence identity. Sequence similarity for an amino acid sequence is calculated using EMBOSS stretcher <NUM>. <NUM> (www. uk/Tools/psa/emboss stretcher), using the EBLOSUM62 matrix with settings Gap open: <NUM> and Gap extend: <NUM>. In case of DNA, sequence similarity is calculated using the DNA full matrix with settings Gap open: <NUM> and Gap extend: <NUM>.

The LRR domain of the alpha-WOLF <NUM> 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. org/translate/.

The genomic sequence of a LRR domain of an alpha-WOLF gene such as alpha-WOLF <NUM> can be amplified using a primer pair having a forward primer which is a nucleic acid molecule having the sequence of SEQ ID No:<NUM> and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:<NUM>.

The invention relates to a nucleic acid molecule which confers complete resistance to at least Peronospora farinosa f. spinacea race Pfs:<NUM>, Pfs15 and Pfs:<NUM> and does not confer resistance to Pfs:<NUM>, 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 <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>. Optionally this nucleic acid molecule is an isolated nucleic acid molecule.

Another embodiment of the present invention is the nucleic acid molecule as defined herein, wherein the genomic DNA sequence of the LRR domain in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>.

Disclosed herein is a nucleic acid molecule which confers resistance to at least one Peronospora farinosa f. spinacea 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 <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity to SEQ ID No:<NUM>. Optionally this nucleic acid molecule is an isolated nucleic acid molecule.

The allele shows a segregation pattern that is consistent with that of a dominant inheritance for the resistance it confers to downy mildew races Pfs8, Pfs15 and Pfs16.

PCR conditions for amplifying the LRR domain-encoding region of an alpha- WOLF gene using primers having SEQ ID No:<NUM> and SEQ ID No:<NUM> are, using Platinum Taq enzyme (Thermo Fisher Scientific): <NUM> minutes at <NUM> (initial denaturing step); <NUM> amplification cycles, each cycle consisting of: <NUM> seconds denaturation at <NUM>, <NUM> seconds annealing at <NUM>, and <NUM> seconds extension at <NUM>; <NUM> minutes at <NUM> (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:<NUM> and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:<NUM>.

PCR conditions for amplifying the LRR domain-encoding region of a beta- WOLF gene using primers having SEQ ID No:<NUM> and SEQ ID No:<NUM> are as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):- <NUM> minutes at <NUM> (initial denaturing step); <NUM> amplification cycles, each cycle consisting of: <NUM> seconds denaturation at <NUM>, <NUM> seconds annealing at <NUM> and <NUM> seconds extension at <NUM>; <NUM> minutes at <NUM> (final extension step).

Also disclosed herein is 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 <NUM> allele wherein the forward primer is a nucleic acid molecule having the sequence of SEQ ID No:<NUM> and the reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:<NUM>. 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.

Further disclosed herein is an alpha-WOLF <NUM> allele which has a coding sequence that in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%,<NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>.

In addition, disclosed herein is an alpha-WOLF <NUM> allele which has a coding sequence that in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%,<NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity to SEQ ID No:<NUM>.

The alpha-WOLF <NUM> allele may encode for a protein having an amino acid sequence which in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>.

The alpha-WOLF <NUM> allele encoding for a protein having an amino acid sequence which in order of increased preference has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity to SEQ ID No:<NUM> is also disclosed herein.

The alpha-WOLF <NUM> allele when present in a spinach plant confers complete resistance to at least one of the <NUM> officially recognized Peronospora farinosa f. spinacea races. In a further embodiment, the alpha-WOLF <NUM> allele when present in a spinach plant confers complete resistance to at least two of the <NUM> officially recognized Peronospora farinosa f. spinacea races. In a further embodiment, the alpha-WOLF <NUM> 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 all of the seventeen officially recognized Peronospora farinosa f. spinacea races.

The alpha-WOLF <NUM> allele when heterozygously or homozygously present in a spinach plant confers at least complete resistance to the officially recognized Peronospora farinosa f. spinacea races Pfs:<NUM>, Pfs15 and Pfs:<NUM>, and does not confer resistance to downy mildew race Pfs:<NUM> (see Table <NUM>).

The resistance of a spinach plant against one or more races of Peronospora farinosa f. spinaciae can be determined using a seedling test. Herein, a seedling test is defined as a test wherein spinach plants are planted in trays containing growth medium, optionally fertilized twice a week after seedling emergence. Plants were inoculated at the first true leaf stage with a sporangial suspension having a concentration of approximately <NUM> × <NUM><NUM>/ml of one of the pathogenic races of Peronospora farinosa f. spinaciae or isolates to be tested. The inoculated plants are placed in a dew chamber at <NUM> with <NUM>% relative humidity for a <NUM> period, and then moved to a growth chamber at <NUM> with a <NUM> photoperiod for <NUM> days. After <NUM> days, the plants are returned to the dew chamber for <NUM> to induce sporulation, and subsequently scored for a disease reaction. Preferably, <NUM> plants per race are tested.

As used herein, a plant is completely resistant against a Peronospora farinosa f. spinaciae race when a plant shows no symptoms in the seedling test described herein.

As used herein, a plant is intermediately resistant against a Peronospora farinosa f. spinaciae 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.

As used herein, a plant is susceptible to an isolate of a Peronospora farinosa f. spinaciae 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.

Further disclosed herein is a a spinach plant, comprising the alpha-WOLF <NUM> allele of invention, of which a representative sample of seed was deposited with the NCIMB under NCIMB accession number <NUM>.

The plant which comprises the alpha-WOLF <NUM> allele may be an agronomically elite spinach plant. In the context of this invention 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 <NUM> allele is a plant of an inbred line or a hybrid.

As used herein, 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.

As used herein, 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 F<NUM> hybrid variety.

A plant carrying the alpha-WOLF <NUM> allele in heterozygous form may further comprise a beta-WOLF <NUM> allele as e.g. present in variety Viroflay wherein the beta-WOLF <NUM> allele does not confer any resistance to downy mildew. However, a plant heterozygous for the alpha-WOLF <NUM> allele may further comprise an allele of the alpha/beta-WOLF gene that does provide resistance to downy mildew. Preferably, such an allele would complement the alpha-WOLF <NUM> allele such that the spinach plant will be at least intermediately resistant to one or more other races to which the alpha-WOLF <NUM> allele does not provide resistance. Most preferably the other allele of the alpha/beta-WOLF gene complements the alpha-WOLF <NUM> allele such that the plant is resistant to Peronospora farinosa f. spinaciae races Pfs:<NUM> to Pfs:<NUM>. In one embodiment such a plant is an agronomically elite plant.

Alternatively, the resistance profile of a plant carrying the alpha-WOLF 25allele is complemented by a resistance conferring allele of a totally different gene. Examples of such genes are e.g. DMR1 as described in <CIT>, DMR6 as described in <CIT> and p10 as described in <CIT>.

Thus, disclosed herein is a spinach plant carrying the alpha-WOLF <NUM> allele and further comprising a genetic determinant resulting in resistance against Peronospora farinosa f. spinacea races Pfs:<NUM> to Pfs:<NUM>. The genetic determinant can be another resistance conferring alpha/beta-WOLF allele or a resistance conferring allele of a totally different gene.

Also disclosed herein is propagation material comprising the alpha-WOLF <NUM> allele. In one embodiment, the propagation material is suitable for sexual reproduction. Such propagation material comprises for example a microspore, pollen, ovary, ovule, embryo sac and egg cell. In another embodiment, 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.

In addition, disclosed herein is a cell of a spinach plant comprising the alpha-WOLF <NUM> allele. Such a cell may be either in isolated form or may be part of the complete plant or parts thereof and harbors the alpha-WOLF <NUM> allele that confers resistance to downy mildew. Each cell of a plant carrying the alpha-WOLF <NUM> allele of the invention carries the genetic information that confers resistance to Peronospora farinosa f. Such a cell may also be a regenerable cell that may be used to regenerate a new plant comprising the allele of the invention.

Yet further disclosed herein is 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 <NUM> allele. In particular embodiment, the first and/or second parent plant is a plant of an inbred line as defined herein.

Furthermore, disclosed herein is 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 <NUM> 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, sequence-specific 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 <CIT>, and in <NPL>) followed by sequencing, etcetera.

In one embodiment the invention relates to a method for identifying a plant carrying the alpha-WOLF <NUM> allele comprises determining the DNA sequence coding for the LRR domain as defined herein.

In a further embodiment of the method the LRR domain of the alpha-WOLF <NUM> 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:<NUM> and the reverse primer is preferably a nucleic acid molecule having the sequence of SEQ ID No:<NUM>.

Also disclosed herein is a method for producing a spinach plant comprising resistance to Peronospora farinosa f. spinaciae comprising: (a) crossing a plant comprising the alpha-WOLF <NUM> 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 <NUM> allele.

Selecting a plant comprising the alpha-WOLF <NUM> allele can be done genotypically by determining the presence of the genomic DNA sequence of the NBS-LRR domain of the allele having in order of increased preference <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>, or <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence identity to SEQ ID No:<NUM>.

In another embodiment, selecting a plant comprising the alpha-WOLF <NUM> allele can be done genotypically by determining the presence the coding sequence of the entire allele.

Alternatively, the presence of the alpha-WOLF <NUM> allele can be determined phenotypically by assaying a plant in a disease test, for example the test as described herein.

Also disclosed herein is the use of a spinach plant carrying the alpha-WOLF <NUM> allele in breeding to confer resistance against Peronospora farinosa f.

Furthermore, disclosed herein is a breeding method for the development of spinach plants carrying the alpha-WOLF <NUM> 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 deposit number NCIMB <NUM>.

Further disclosed herein is a method for the production of a spinach plant which comprises alpha-WOLF <NUM> allele, which method comprises: (a) crossing a plant comprising the allele with another plant; (b) optionally selecting for plants comprising said allele in the F1; (c) optionally backcrossing the resulting F1 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. A spinach plant produced by this method is also disclosed herein.

In addition, disclosed herein is a harvested leaf of a spinach plant carrying the alpha-WOLF <NUM> allele of the invention, to a food product which comprises a harvested leaf of a spinach plant carrying the alpha-WOLF <NUM> allele 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 <CIT>, 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. Disclosed herein is also the use of and methods for making and using the leaves of the spinach plant carrying the alpha-WOLF <NUM> allele of the invention, as well as leaves of spinach plants derived from the invention.

Also disclosed herein is a container which comprises one or more plants carrying the alpha-WOLF <NUM> allele of the invention, or one or more spinach plants derived from a plant carrying the alpha-WOLF <NUM> allele 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.

Furthermore, disclosed herein is the use of a spinach plant, of which representative seed was deposited with the NCIMB under accession number NCIMB <NUM>, in the production of a spinach plant comprising the alpha-WOLF <NUM> allele.

In a further embodiment the said spinach plant is a hybrid, doubled haploid, or inbred spinach plant.

Additionally, disclosed herein is the use of a cell comprising the alpha-WOLF <NUM> allele for the production of a spinach plant showing resistance to Peronospora farinosa f.

Further disclosed herein is the use of a tissue culture comprising the alpha-WOLF <NUM> allele for the production of a spinach plant showing resistance to Peronospora farinosa f.

Seeds of a plant that comprises the alpha-WOLF <NUM> allele of the invention in its genome were deposited with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK, on <NUM> October <NUM> , under deposit accession number NCIMB <NUM>. The deposit was made pursuant to the terms of the Budapest Treaty. Upon issuance of a patent, all restrictions upon the deposit will be removed, and the deposit is intended to meet the requirements of <NUM> CFR § <NUM>-<NUM>. The deposit will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposit will be maintained in the depository for a period of <NUM> years, or <NUM> years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.

The present invention will be further clarified in the Examples that follow and that are given for illustration purposes only and are not intended to limit the invention in any way.

The resistance to downy mildew infection was assayed as described by <NPL>), using a differential set. Spinach plants of the invention were sown along with spinach plants from different other genotypes (see Table <NUM>) in trays containing Scotts Redi-Earth medium, and fertilized twice a week after seedling emergence with Osmocote Peter's (<NUM>-<NUM>-<NUM>) fertilizer (Scotts). Plants were inoculated with a sporangial suspension (<NUM> × <NUM><NUM>/ml) of a pathogenic race of Peronospora farinosa f. spinaciae at the first true leaf stage. In this manner, <NUM> officially recognized pathogenic race were tested.

The inoculated plants were placed in a dew chamber at <NUM> with <NUM>% relative humidity for a <NUM> period, and then moved to a growth chamber at <NUM> with a <NUM> photoperiod for <NUM> days. After <NUM> days, the plants were returned to the dew chamber for <NUM> 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 <NPL>). 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 <NUM> shows the resistance of a plant carrying the alpha-WOLF <NUM> allele to each one of these pathogenic races. Table <NUM> 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). A weak resistance response is indicated as "(-)", which in practice means a slightly reduced 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 <NUM> allele, of which a representative sample of seed was deposited with the NCIMB under NCIMB accession number <NUM> was used in polymerase chain reactions (PCR), using forward primer ACAAGTGGATGTGTCTTAGG (SEQ ID No:<NUM>) and reverse primer TTCGCCCTCATCTTCCTGG (SEQ ID No:<NUM>). The primer pair amplifies the LRR domain-encoding 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:<NUM> and SEQ ID No:<NUM> were as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):.

The isolated genomic DNA of a spinach plant of variety Viroflay comprising the beta-WOLF <NUM> allele was used in polymerase chain reactions (PCR), using forward primer TCACGTGGGTTGTGTTGT (SEQ ID No:<NUM>) and reverse primer TTCGCCCTCATCTTCCTGG (SEQ ID No:<NUM>). 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:<NUM> and SEQ ID No:<NUM> were as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):.

The 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 sequence of the LRR domain of the alpha-WOLF <NUM> allele amplified by primers having SEQ ID No:<NUM> and SEQ ID No:<NUM> is provided in Table <NUM> under SEQ ID No:<NUM>.

The sequence of the LRR domain of the beta-WOLF <NUM> allele amplified by primers having SEQ ID No:<NUM> and SEQ ID No:<NUM> is provided in Table <NUM> under SEQ ID No:<NUM>.

Finally, the obtained sequences were translated into the corresponding amino acid sequence of the LRR domain having SEQ ID No:<NUM> and SEQ ID No:<NUM> for the alpha-WOLF <NUM> allele and the beta-WOLF <NUM>, respectively (See also Table <NUM>).

If PCR products were to be sequenced using SMRT sequencing (Pacific Biosciences), PCR primers and PCR conditions were different.

To the above-mentioned forward primers the following standard amplification sequence was added: GCAGTCGAACATGTAGCTGACTCAGGTCAC.

To the reverse primer, the following standard amplification sequence was added: TGGATCACTTGTGCAAGCATCACATCGTAG.

A spinach plant comprising the alpha-WOLF <NUM> allele, of which a representative sample of seed was deposited with the NCIMB under NCIMB accession number <NUM> was crossed with a plant of variety Viroflay carrying the beta-WOLF <NUM> allele to obtain a F1 generation. Subsequently, a F1 plant was selfed to obtain a F2 population.

Plants of the F2 population were assayed as described in Example <NUM> for resistance to Peronospora farinosa f. spinaciae Pfs: <NUM>. Approximately <NUM>% of the plants scored completely resistant in the assay. This segregation pattern is consistent with that of a dominant inheritance.

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 <NUM>.

The PCR products were visualized on agarose gel (not shown), this demonstrated that approximately <NUM>% of the plants contained an alpha-WOLF fragment, and that the remaining approximately <NUM>% of the plants only contained a beta-WOLF fragment. The plants containing the alpha-WOLF fragment completely correlated with the plants that scored resistant for Pfs: <NUM>. The plants only comprising the beta-WOLF fragment completely correlated with the plants that scored susceptible for Pfs: <NUM>.

Claim 1:
Nucleic acid molecule designated alpha-WOLF <NUM> which confers complete resistance to at least Peronospora farinosa f. sp. spinacea race Pfs:<NUM>, Pfs15 and Pfs:<NUM> and does not confer resistance to Pfs:<NUM>, 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 <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% sequence similarity to SEQ ID No:<NUM>.