Hyaloperonospora brassicae resistance gene

Abstract

The invention relates to a gene conferring resistance against Hyaloperonospora brassicae, and a Brassica oleracea plant comprising said gene. The gene comprises a nucleotide sequence encoding a protein having an amino acid sequence, which has at least 70% sequence similarity to SEQ ID No. 3 or a nucleotide sequence comprising a coding sequence which has at least 70% sequence identity to SEQ ID No. 2; or a nucleotide sequence encoding at least the amino acid positions [58] to [146] of SEQ ID No. 3, the amino acid positions [217] to [503] of SEQ ID No. 3 and the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3; or a nucleotide sequence encoding an LRR domain comprising the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3 or encoding an LRR domain which has in order of increased preference at least 95% sequence similarity to the amino acid sequence of the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3. The gene is as comprised in the genome of a Brassica oleracea plant representative seed of which was deposited with the NCIMB under accession number NCIMB 43592.

Description

HYALOPERONOSPORA BRASSICAE RESISTANCE GENEThe present invention relates to a gene conferring resistance against Hyaloperonospora brassicae, and a Brassica oleracea plant comprising said gene. The invention further relates to progeny, seed and part of a Brassica oleracea plant that is resistant to Hyaloperonospora brassicae and to a food product comprising such part of the Brassica oleracea plant. The invention also relates to propagation material suitable for producing the Brassica oleracea plant, to a marker for the identification of a resistant Brassica oleracea plant, to use of the said marker to identify and / or develop a Hyaloperonospora brassicae resistant Brassica plant or other markers, to a method of selecting a Brassica oleracea plant for the resistance and to a method for producing a Hyaloperonospora brassicae resistant Brassica oleracea plant.Brassica is a genus of plants, taxonomically embedded within the Brassicaceae family. It contains many economic important crops that serve as a source of food, but species are also employed in the production of oil. In general, the Brassica genus includes B. napus, B. nigra, B. rapa, B. juncea and B. oleracea. Brassica napus is very important in the production of vegetable oil that is increasingly applied in the fuel industry. Together with Brassica rapa, Brassica nigra and Brassica juncea, Brassica oleracea is a species that plays a very important role in the production of human food. Over time, cultivars originated within B. oleracea amongst which cabbage, Chinese cabbage, cauliflower, collards, broccoli, kohlrabi and Brussels sprouts can be found.The wild cabbage has been bred into a broad range of different horticultural cultivars that remained sexually compatible. However, their appearance does not show a very high level of phylogenetic similarity. The wide range in differences of morphological characteristics within Brassica oleracea has long been of interest and form the foundation of a cultivars’ uniqueness. These include an enlarged inflorescence (cauliflower, broccoli); an enlarged stem (kohlrabi); an enlarged apical bud (cabbage); enlarged lateral buds (Brussels’ sprouts).Breeding of cruciferous vegetables like Brassica oleracea varieties aims at the production of commercial varieties optimally adapted to local growing conditions that allows the grower to maximize the productivity of high quality plants. Many characteristics need to be taken into account during selection which relate to both input as well as output traits. One of the most important input traits in this respect relates to disease resistance, in particular to resistance towards micro-organisms .Brassica oleracea plants are affected by a wide range of pests and diseases. These threats are therefore high priority objects for Brassica oleracea breeders, in order to obtain crops that are vigorous and highly resistant. In absence of resistances in these crops, growers necessarily have toapply agronomic strategies like crop rotation, in order to reduce pest damage in areas where cruciferous vegetables are grown.Downy mildew - in Brassica oleracea plants caused by the oomycete Hyaloperonospora brassicae - is a common, probably the most damaging disease found among cabbages, radishes, turnips and other plants belonging to the Crucifereae family. Downy mildew can be caused by different isolates of Hyaloperonospora brassicae. The obligated parasite is living on Brassica plants and the symptoms of the infection appear primarily on the aerial parts of the plants. Symptoms of the disease such as sporulation and necrotic spots with brown edges can destroy the quality of the leaves and of the plant head.Infection by Hyaloperonospora brassicae occurs worldwide and it has an extensive host range. Cultural practices and / or chemical treatments have been unsuccessful in protecting crops or have proven to be too expensive. Therefore, the development of resistant cultivars is now considered the most economical and efficient method for the control of downy mildew.As for oomycetes like Hyaloperonospora brassicae it is known that they continuously develop the ability to break resistances present in their host plant, new resistance loci are very valuable assets. Therefore, a Brassica oleracea plant having a resistance to Hyaloperonospora brassicae is preferred.Plants can be resistant to Hyaloperonospora brassicae at seedling stage and / or at adult plant stage. If the plants are not resistant to Hyaloperonospora brassicae at seedling stage, the cotyledons or young plants can be infected and show the symptoms of a susceptible plant. The infection at this stage can lead to mature plants that have heads and / or leaves that are not properly developed or it can even lead to the death of the infected plants. The infection at seedling stage thereby leads to reduced yield or to reduced quality of the plant heads or leaves. Therefore, a Brassica oleracea plant resistant to Hyaloperonospora brassicae at all plant stages i.e. at seedling stage and adult plant stage is preferred.Summarizing, there is a need for a reliable resistance towards Hyaloperonospora brassicae at all plant stages. In particular, a resistance towards to multiple isolates of Hyaloperonospora brassicae at all plant stages, is preferred.It is the object of the present invention to obtain Brassica oleracea plants which are resistant to Hyaloperonospora brassicae.In the research that led to the present invention, a novel gene was identified that confers resistance to Hyaloperonospora parasitica when expressed in a Brassica oleracea plant.Thus, the present invention provides a gene, herein referred to as the ‘gene of the invention’, encoding a protein which confers resistance to Hyaloperonospora parasitica when expressed in a Brassica oleracea plant, wherein said gene comprises:a) a nucleotide sequence encoding a protein having an amino acid sequence, which has in order of increased preference at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No. 3; or b) a nucleotide sequence comprising a coding sequence which has in order of increased preference at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 2; or c) a nucleotide sequence encoding at least the amino acid positions

[0058] to

[0146] of SEQ ID No. 3, the amino acid positions

[0217] to

[0503] of SEQ ID No. 3 and the amino acid positions

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] of SEQ ID No. 3; or d) a nucleotide sequence encoding an LRR domain comprising the amino acid positions

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] of SEQ ID No. 3 or encoding an LRR domain which has in order of increased preference at least 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to the amino acid sequence of the amino acid positions

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] of SEQ ID No. 3.The gene of the invention is a nucleic acid, in particular a nucleic acid molecule, more in particular an isolated nucleic acid molecule.The gene of the invention comprises a nucleotide sequence encoding a protein having an amino acid sequence, which has in order of increased preference at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity to an amino acid sequence according to SEQ ID No. 3.Preferably, the gene of the invention comprises a nucleotide sequence encoding a protein having an amino acid sequence, which has at least 95% sequence similarity to an amino acid sequence according to SEQ ID No. 3. This nucleotide sequence preferably comprises at least a nucleotide sequence encoding an LRR domain comprising the amino acid positions

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] of SEQ ID No. 3.In a preferred embodiment, the gene of the invention comprises a nucleotide sequence encoding a protein having an amino acid sequence according to SEQ ID No. 3.The gene of the invention comprises a coding sequence which has in order of increased preference at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the coding sequence according to SEQ ID No. 2.Preferably, the gene of the invention comprises a coding sequence which has at least 85% sequence identity to the coding sequence according to SEQ ID No. 2.More preferably, the gene of the invention comprises a coding sequence which has at least 95% sequence identity to the coding sequence according to SEQ ID No. 2. This coding sequence preferably comprises at least a nucleotide sequence encoding an LRR domain comprising the amino acid positions

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] of SEQ ID No. 3.In a preferred embodiment, the gene of the invention comprises a coding sequence according to SEQ ID No. 2.The three main categories of domains that can be recognized in the protein encoded by the gene of the invention are: 1) The coil-coil (CC) domain as defined herein by the amino acids on position 58 to 146 in SEQ ID No. 3. 2) The nucleotide binding site (NBS) domain as defined herein by the amino acids on position 217 to 503 in SEQ ID No. 3. 3) The leucine -rich repeat (LRR) domains as defined herein by the amino acids on position

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] in SEQ ID No. 3.In one embodiment, the gene of the invention comprises a nucleotide sequence encoding a protein, the LRR domain of which comprisespositions

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] of SEQ ID No. 3.In a further embodiment, the gene of the invention comprises a nucleotide sequence encoding a protein comprising at least the amino acid positions 58 to 146 of SEQ ID No. 3, the amino acid positions 217 to 503 of SEQ ID No. 3, and the amino acid positions

[0650] to

[0679] ,

[0819] to

[0850] ,

[0853] to

[0925] ,

[0941] to

[0972] ,

[0989] to

[1029] ,

[1106] to

[1147] ,

[1180] to

[1210] of SEQ ID No. 3.The gene of the invention may further comprise a nucleotide sequence encoding a protein derived from the protein according to SEQ ID No. 3 by substitution, deletion and / or addition of one or more amino acids.The gene of the invention, when present in a Brassica oleracea plant, confers resistance to Hyaloperonospora parasitica. The Hyaloperonospora parasitica resistance conferred by the gene of the invention inherits in a dominant fashion.As used herein, sequence identity is the percentage of nucleotides or amino acids that is identical between two sequences after proper alignment of those sequences. The person skilled in the art is aware of how to align sequences, for example by using a sequence alignment tool such as BLAST®, which can be used for both nucleotide sequences and protein sequences. To obtain the most significant result, the best possible alignment that gives the highest sequence identity score should be obtained. The percentage sequence identity is calculated through comparison over the length of the shortest sequence in the assessment. In the present case, a nucleotide sequence represents a gene that at least comprises a start codon and a stop codon or encodes an amino acid sequence which comprises a complete protein encoded by such a gene.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.The Brassica oleracea plants of the invention are resistant at all plant stages (i.e. cotyledon stage and adult plant stage) to multiple strains of Hyaloperonospora brassicae.The invention further relates to a Brassica oleracea plant, wherein the plant comprises the gene of the invention in its genome. A Brassica oleracea plant comprising the gene of the invention in its genome is referred to herein as a ‘plant of the invention’.In a further embodiment, the plant of the invention is an agronomically elite plant, preferably an agronomically elite Brassica oleracea plant.In the context of this invention, an agronomically elite plant is a plant having a genotype that, as a result of human intervention, comprises an accumulation of distinguishable and desirable agronomic traits which allow a producer to harvest a product of commercial significance, preferably the agronomically elite plant of the invention 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 Fl hybrid variety.The present invention relates to the species within Brassica oleracea that are affected by infection with Hyaloperonospora brassicae. Brassica oleracea is a plant species that includes many common cultivars. The invention relates to the Brassica oleracea plants of the following cultivars: var. acephala DC. (kales) var. botrytis L. (cauliflower, romanesco) var. capitata L. (red, white, savoy cabbage) var. gemmifera DC. (brussels sprouts) var. gongylodes L. (kohlrabi) var. italica Plenck. (broccoli, calabrese) var. sabauda L. (savoy cabbage) var. sabellica (collards) var. selensia (borecole) var. albiflora Sun [= B. alboglabra] (Chinese kale) var. alboglabra [= B. alboglabra] (Chinese kale) var. chinensis Prain (burma sarson)var. fimbriata Mill, (kitchen kale) var. fruticosa Metz, (thousand-head kale) var. tronchuda L. H. Bailey (tronchuda cabbage) var. costata (Portugese cabbage) var. medullosa (marrow stem kale) var. pamifolia (kale, Jersey kale) var. ramona (thousand-head kale)Preferred are the varieties broccoli, cauliflower, romanesco, red cabbage, white cabbage, savoy cabbage and kohlrabi.Seed of Brassica oleracea convar. botrytis var. italica comprising the gene of the invention was deposited with the NCIMB under accession number NCIMB 43592. The invention thus also relates to plants grown from seed deposited under NCIMB accession numbers NCIMB 43592.Another aspect of the invention relates to a seed capable of growing into a Brassica oleracea plant of the invention wherein said plant comprises the gene of the invention. The invention also relates to use of said seed for the production of a Brassica oleracea plant of the invention, by growing said seed into a Brassica oleracea plant.The invention also relates to propagation material suitable for producing a plant of the invention, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from a microspore, a pollen, an ovary, an ovule, an embryo sac and an egg cell, or is suitable for vegetative reproduction, and is in particular selected from a cutting, a root, a stem a cell, and a protoplast, or is suitable for tissue culture of regenerable cells or protoplasts, and is in particular selected from a leaf, a pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed and a stem, wherein the propagation material comprises the gene of the invention.The invention further relates to a cell of a Hyaloperonospora brassicae resistant Brassica plant of the invention. Such a cell may either be in isolated form, or a part of the complete plant or parts thereof and still forms a cell of the invention because such a cell comprises the gene of the invention. Each cell of a Hyaloperonospora brassicae resistant Brassica plant of the invention carries the gene of the invention. A cell of the invention may also be a regenerable cell that can regenerate into a new plant of the invention.The invention further relates to plant tissue of a plant of the invention, which comprises the gene of the invention. The tissue can be undifferentiated tissue or already differentiated tissue. Undifferentiated tissue is for example a stem tip, an anther, a petal, or pollen, and can be used in micro propagation to obtain new plantlets that are grown into new plants of the invention. The tissue can also be grown from a cell of the invention.The invention further relates to a method for the production of a plant comprising the gene of the invention, which plant is resistant to Hyaloperonospora brassicae, by using tissue culture or by using vegetative propagation.The invention moreover relates to progeny of a plant, a cell, a tissue, or a seed of the invention, which progeny comprises the gene of the invention. Such progeny can in itself be a plant, a cell, a tissue, or a seed. The progeny can in particular be progeny of a plant of the invention, representative seeds of which were deposited under NCIMB number 43592. As used herein, progeny comprises the first and all further descendants from a cross with a plant of the invention, wherein a cross comprises a cross with itself or a cross with another plant, and wherein a descendant that is determined to be progeny comprises the gene of the invention. Descendants can be obtained through selfing and / or further crossing of the deposit. Progeny also encompasses material that is obtained by vegetative propagation or another form of multiplication.The invention further relates to the germplasm of plants of the invention. The germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the resistance trait of the invention. The germplasm can be used in a breeding program for the development of Brassica oleracea plants that show resistance to Hyaloperonospora brassicae. The use of germplasm that comprises the gene of the invention in breeding is also part of the present invention.The invention also relates to the use of the gene of the invention for producing a Brassica oleracea plant that is resistant to Hyaloperonospora brassicae. The plant is preferably a plant that belongs to the group of broccoli, cauliflower, romanesco, red cabbage, white cabbage, savoy cabbage and kohlrabi. Said use does not mean that Brassica oleracea plants thus produced are obtained by crossing and selecting the gene of the invention.The invention also relates to a marker for the identification of Hyaloperonospora brassicae resistance in a Brassica oleracea plant, which marker comprises a single nucleotide polymorphism (SNP). The use of a marker for identification of Hyaloperonospora brassicae resistance in a Brassica oleracea plant is also part of the invention. Any of these markers can also be used to develop other markers for the identification of the gene of the invention when present in a Brassica oleracea plant, the use of said marker is also part of the present invention.The invention further relates to the molecular marker and to the use of the molecular marker detected by the method for the identification of molecular markers as described herein.The current invention also relates to the use of a plant of the invention as a crop, as a source of seed or as a source of propagation material.The present invention further relates to a method for identifying a Brassica oleracea plant resistant to Hyaloperonospora brassicae, wherein the method comprises the following steps: a) detecting in the genome of a plant the gene of the invention, and / orb) testing of the plant comprising the gene of the invention for exhibiting resistance to Hyaloperonospora brassicae. Optionally, this method for identifying a Brassica oleracea plant resistant to Hyaloperonospora brassicae comprises a step of detecting an unique polymorphism in the gene of the invention.The invention further relates to a method for seed production comprising growing a Brassica oleracea plant from a seed of the invention that comprises the gene of the invention homozygously, allowing the Brassica oleracea plant to produce seed and harvesting the seed. Production of the seed is suitably done by selfing or by crossing with another plant that is optionally also a plant of the invention. The plant grown from the seed produced as described herein is resistant to Hyaloperonospora brassicae.The invention also relates to a method for producing hybrid seed, comprising crossing a first Brassica oleracea parent plant with a second Brassica oleracea parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and / or the second parent plant is a plant of the invention comprising the gene of the invention. Preferably, at least one of the parent plants comprises the gene of the invention homozygously.The invention also relates to the hybrid seed produced by the method described herein and a hybrid plant grown from said hybrid seed.The invention further relates to a method for growing a Brassica oleracea plant that is resistant to Hyaloperonospora brassicae, comprising the step of planting a seed comprising the gene of the invention, a representative sample of which seed was deposited with the NCIMB under accession number NCIMB 43592.Transgenic techniques used for transferring nucleotide sequences between plants that are sexually incompatible can also be used to produce a plant of the invention, by transferring the gene of the invention from one species to another. Techniques that can suitably be used comprise general plant transformation techniques known to the skilled person, such as the use of an Agrobacterium-mediated transformation method. Genome editing methods such as the use of a CRISPR / Cas system might also be employed to obtain a plant of the invention. A Brassica oleracea plant of the deposit or a descendant thereof is a suitable source of the modified gene.Introduction of the gene of the invention can also be done through introgression from a Brassica oleracea plant comprising said gene, for example from a Brassica oleracea plant, representative seed of which was deposited as NCIMB 43592, or from progeny thereof, or from another plant that is resistant to Hyaloperonospora brassicae and in which the gene of the invention was identified. Breeding methods such as crossing and selection, backcrossing, recombinant selection, or other breeding methods that result in the transfer of a genetic sequence from a resistant plant to a susceptible plant can be used. A resistant plant can be of the same species or of a different and / or wild species. Difficulties in crossing between species can beovercome through techniques known in the art such as embryo rescue, or cisgenesis can be applied. Progeny of a deposit can be sexual or vegetative descendants of that deposit, which can be selfed and / or crossed, and can be of an Fl, F2, or further generation as long as the descendants of the deposit still comprise the modified gene the invention as present in seed of that deposit. A Brassica oleracea plant produced by such method is also a part of the invention.The invention also relates to a method for the production of a Brassica oleracea plant exhibiting resistance against Hyaloperonospora brassicae, comprising the steps of: a) crossing a first parent plant comprising the gene of the invention with a second parent plant to obtain an Fl population; b) optionally performing one or more rounds of selfing and / or crossing with a plant from the Fl population to obtain a further generation; c) selecting from the further generation a plant that comprises the gene as a resistant plant. The invention also relates to a method for the production of a Brassica oleracea plant which is resistant to Hyaloperonospora brassicae, said method comprising: a) crossing a first parent plant comprising the gene of the invention with a second parent plant not comprising the gene of the invention; b) backcrossing the plant resulting from step a) with the second parent plant for at least three generations; c) selecting from the third or higher backcross population a plant that comprises at least the gene of the invention of the first parent plant of step a) as the plant which is resistant to Hyaloperonospora brassicae.The invention additionally provides for a method of introducing another desired trait into a Brassica oleracea plant that is resistant to Hyaloperonospora brassicae, comprising: a) crossing a plant comprising the gene of the invention with a second plant that comprises the other desired trait to produce Fl progeny; b) optionally selecting in the Fl for a plant that comprises the resistance and the other desired trait; c) crossing the optionally selected Fl progeny with one of the parents for at least three generations, to produce backcross progeny; d) selecting backcross progeny comprising the resistance and the other desired trait; and e) optionally repeating steps c) and d) one or more times in succession to produce selected fourth or higher backcross progeny that comprises the resistance and the other desired trait.Optionally, selfing steps are performed after any of the crossing or backcrossing steps in above-described methods. Selection of a Brassica oleracea plant comprising the Hyaloperonospora brassicae resistance and the other desired trait can alternatively be done following any crossing or selfing step of the method. The other desired trait can be selected from,but is not limited to, the following group: resistance to bacterial, fungal or viral diseases, insect or pest resistance, improved germination, plant size, plant type, improved shelf-life, water stress and heat stress tolerance, and male sterility.The invention relates to the use of a Brassica oleracea plant of the invention, for transferring the resistance to Hyaloperonospora brassicae into another Brassica plant.The invention relates to the use of seeds that were deposited with the NCIMB under accession number NCIMB 43592 on, for transferring the resistance to Hyaloperonospora brassicae into another Brassica oleracea plant.The invention also relates to harvested parts of Brassica oleracea plants of the invention or parts thereof, to food products comprising harvested parts of Brassica oleracea plants of the invention or parts thereof, either in natural or optionally in processed form.The harvested part or food product can be or comprises a cabbage head, a curd, a stem, a leaf, a root, a sprout, a seed, or any other part of a Brassica oleracea plant. The harvested part may also be used for the production of bio-fuel. The food product or harvested part, may have undergone one or more processing steps. Such a processing step might comprise but is not limited to any one of the following treatments or combinations thereof: cutting, washing, cooking, steaming, baking, frying, pasteurizing, freezing, grinding, extracting oil, pickling, or fermenting. The processed form that is obtained is also part of this invention.The invention additionally relates to the use of a plant of the invention in plant breeding. The invention thus also relates to a breeding method for the development of a cultivated Brassica plant that is resistant to Hyaloperonospora brassicae, wherein a plant comprising the gene of the invention, for conferring said resistance to another Brassica oleracea plant is used. Seed being representative for a Brassica oleracea plant that can be used in plant breeding to develop another Brassica oleracea plant with Hyaloperonospora brassicae resistance was deposited with the NCIMB under accession number NCIMB 43592.As used herein, “resistance to Hyaloperonospora brassicae”, “resistance against Hyaloperonospora brassicae” or “resistance of the invention” is defined as the capacity of a plant to resist infection by one or more of Hyaloperonospora brassicae strains, in all plant stages. The plants are visually phenotyped to identify a Hyaloperonospora brassicae resistant plant. Resistance is suitably scored on a scale as presented in Table 2, plants of the invention that are resistant to Hyaloperonospora brassicae have preferably a score of 1 to 3. In a bio-assay the resistant Brassica plant shows clean cotyledons, optionally a few necrotic spots or many necrotic spots on the cotyledons, but do not show any sporulation on the cotyledons.As used herein, “increased resistance” of a Brassica oleracea plant is intended to mean the plant has a score as presented in Table 2 that is lower than the non-resistant Brassica oleracea plant to which it is compared. The non-resistant plant is a plant not comprising the gene of theinvention. The plant that has increased resistance shows less necrotic spots and / or sporulation than the plant to which it is compared, preferably no necrotic spots and / or sporulation.As used herein a marker is genetically linked to the gene of the invention, and can therefore be used for the identification thereof, when the marker and the trait co-segregate in a segregating population resulting from a cross between a plant comprising the gene of the invention and a plant lacking the gene.As used herein “progeny” is intended to mean the first and all further descendants from a cross with a plant of the invention. “Progeny” also encompasses Brassica oleracea plants that carry the gene of the invention and have the trait of the invention, and are obtained from other plants, or progeny of plants, of the invention by vegetative propagation or multiplication. Progeny of the invention comprise the gene of the invention and show resistance to Hyaloperonospora brassicae.As used herein “trait” is intended to mean the resistance phenotype of the cultivated Brassica oleracea plant. In particular, the word “trait” refers to the trait of the invention, more in particular to the resistance to Hyaloperonospora brassicae. When a cultivated Brassica oleracea plant exhibits the trait of the invention, its genome comprises the gene of the invention causing the trait of the invention. The cultivated Brassica oleracea plant thus comprises the gene of the invention. Hence, the “trait of the invention” or “phenotype of the invention” as used herein is intended to refer to the trait of resistance to Hyaloperonospora brassicae.As used herein, “plant of the invention” is defined as a Brassica oleracea plant, that is resistant to Hyaloperonospora brassicae. The plant of the invention comprises the gene of the invention which confers resistance to the Hyaloperonospora brassicae. In the context of this invention, when referring to a Brassica oleracea plant, unless otherwise specified, is a cultivated Brassica oleracea plant.As used herein “wild type plant” is defined as a Brassica oleracea plant that does not comprise the gene of the invention that confers resistance to Hyaloperonospora brassicae and is therefore susceptible to Hyaloperonospora brassicae.The phrase “present in” may also mean “found in” or “contained in” or “obtainable from” (the genome of) plants grown from seeds of the deposit or the deposited seeds themselves. These phrases are intended to indicate that the gene of the invention is essentially the same or the same as the gene in the genome of the deposited material. The gene needs not be identical and may comprise polymorphisms (i.e. variation in sequence) as compared to the gene of the invention, but these polymorphisms do not have any bearing on the function of the gene in causing the resistance to Hyaloperonospora brassicae.The present invention will be further illustrated in the Examples that follow and that are for illustration purposes only. The Examples are not intended to limit the invention in any way.DEPOSITSeeds of a broccoli (Brassica oleracea var. italica Plenck) plant comprising the modified gene located on chromosome 2 which confers resistance to Hyaloperonospora brassicae were deposited under accession number NCIMB 43592 on April, 7th, 2020 with NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9 YA). All seeds of the deposit comprise the modified gene on chromosome 2 homozygously. Plants grown from these seeds are thus resistant against Hyaloperonospora brassicae.The deposited seeds do not meet the DUS criteria which are required for obtaining plant variety protection and can therefore not be considered to be plant varieties.FIGURESFigure 1 :This figure shows the results from QTL-mapping for Hyaloperonospora brassicae as described in example 2. The groups represent the 9 chromosomes that are present in Brassica oleracea. On the X-axis, the relative position in cM on the respective chromosome of the described population is indicated and on the Y-axis the LOD score is indicated. This figure clearly shows that the mapping resulted in one QTL, located on chromosome 2.SEQUENCE INFORMATIONTable 1: sequencesEXAMPLESEXAMPLE 1Hyaloperonospora brassicae resistance testing of Brassica oleracea plantsTo test whether a Brassica oleracea plant is resistant to Hyaloperonospora brassicae, a bio-assay was performed. About 20 seeds per plot were sown and one row between two plots was left empty. Brassica plants were grown under standard Dutch greenhouse conditions at a temperature regime of 15°C / 15°C night / day. At 10 days after sowing the cotyledons were sprayed with the sporangial suspension of an Hyaloperonospora brassicae isolate. The isolate of Hyaloperonospora brassicae was maintained on living Hyaloperonospora brassicae susceptible Brassica oleracea plants. The inoculated cotyledons were incubated under controlled conditions being a 12°C / 14°C night / day regime. Each plant was visually scored according to Table 2 at 7 days and 14 days after inoculation to phenotypically identify Hyaloperonospora brassicae resistant Brassica oleracea plants. This screen identified a number of Hyaloperonospora brassicae resistant Brassica oleracea plants, of which one was selected for further research. This was a broccoli (Brassica oleracea var. italicd) plant.Table 2Overview of infection symptoms and classification stadia of Hyaloperonospora brassicae infected Brassica plants (scale 1-9) with grey colorationThe selected plant was tested in a combined screen performed with 15 different Hyaloperonospora brassicae isolates collected in different locations of the world. The selected plant of the invention scored resistant against 8 isolates out of the 15 isolates tested. Considering that the isolates were collected in very different countries and locations, these results show that the plant of the invention has a sufficient resistance phenotype.When comparing several lines for their phenotypes against two important isolates from the test described above, the plant of the invention was shown to be resistant to both isolates (line 2 in Table 3 below). The other lines showed at least resistance to one of the isolates.Table 3.Phenotypes against two different Hyaloperonospora brassicae isolatesEXAMPLE 2QTL mappingInput data comprised genetic marker data and phenotypic measurements on resistance / susceptibility for downy mildew, Hyaloperonospora brassicae. Data was obtained on a population of a cross between two broccoli parents (one susceptible and one resistant to Hyaloperonospora brassicae).In total 162 markers (out of 786 analyzed) were informative for mapping, and a linkage analysis was performed to construct a population-specific map. QTL mapping was performed with MapQTL 6.0. First interval mapping was performed, followed by cofactor selection and MQM- mapping. A single QTL is detected on chromosome 2 explaining at maximum 71.6% of the trait variance at approximately 34 cM.Mapping of resistance to Hyaloperonospora parasitica at this position is supported by the results of an earlier cross between broccoli and cauliflower (data not shown), where the best linked marker was found to be positioned at 32 cM.Taken together, these data support the monogenic control of the trait, said gene being located on chromosome 2.EXAMPLE 3Identification of the gene conferring resistance to Hyaloperonospora parasiticaBecause of the monogenic dominant nature of the resistance as demonstrated above, it was hypothesized that the presence of a so-called R-gene could explain the nature of the resistance. QTL analysis and marker development did show that the QTL was located on chromosome 2. All R-genes present on chromosome 2 of seven different genome assemblies were analysed and compared. The genome assemblies were:1. the resistant broccoli (Brassica oleracea var. italica) line comprising the gene of the invention2. a proprietary susceptible broccoli (Brassica oleracea var. italica) line3. TolOOO a susceptible Brassica oleracea plant for which the genome is publicly available.4. a proprietary susceptible, cauliflower (Brassica oleracea var. botrytis) line5. a proprietary susceptible, head cabbage (Brassica oleracea var. capitata) line6. a proprietary susceptible, kohlrabi (Brassica oleracea var. gongylodes) line7. a proprietary susceptible, head cabbage (Brassica oleracea var. capitata) lineA NLRome (pangenome approach) analysis was performed using all 7 assemblies mentioned as input, in order to determine whether the candidate gene was indeed unique. Plant pan-NLRomes aim to fully capture intraspecific diversity of the highly variable NLR immune receptors, enabling systematic analyses of NLR genes and alleles and their roles in disease resistance. The analysis highlighted that the candidate gene landed in a cluster with 12 genes. In this cluster, one or more genes of 6 of the 7 assemblies also landed. The first column refers to the assemblies indicated above.Table 4Overview of genesBecause the percentages are all low, it was concluded that the candidate gene was indeed unique. It was also concluded that the gene was indeed expressed.EXAMPLE 4Transferring the Hyaloperonospora brassicae resistance gene into susceptible Brassica oleracea plantsFurthermore, also the segregation phenotype of the resistance was studied. The resistant Brassica oleracea var. italica (broccoli) line identified above was crossed with susceptible Brassica oleracea lines. These were proprietary cauliflower, kohlrabi and broccoli lines. Data of F2 populations and populations made out of an inbred of a back-cross were gathered. The observed distribution for all crosses shown below in Table 5 did not significantly differ from the expected distribution according to a chi squared test (data not shown). The table shows the susceptible Brassica oleracea type.The results below show that the Hyaloperonospora brassicae resistance gene of the invention was successfully transferred into susceptible Brassica oleracea plants. It also shows that when introgressing the gene of the invention in whichever background, the observed segregation phenotype is the same for all crosses, being 3:1. This confirms that the trait of the invention is monogenic and dominant.Table 5. Segregation of the resistance phenotype when transferring the gene of the invention

Claims

CLAIMS1. A gene encoding a protein which confers resistance to Hyaloperonospora parasitica when expressed in a Brassica oleracea plant, wherein said gene comprises: a) a nucleotide sequence encoding a protein having an amino acid sequence, which has at least 70% sequence similarity to SEQ ID No. 3; or b) a nucleotide sequence comprising a coding sequence which has at least 70% sequence identity to SEQ ID No. 2; or c) a nucleotide sequence encoding at least the amino acid positions [58] to [146] of SEQ ID No. 3, the amino acid positions [217] to [503] of SEQ ID No. 3 and the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3; or d) a nucleotide sequence encoding an LRR domain comprising the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3 or encoding an LRR domain which has in order of increased preference at least 95% sequence similarity to the amino acid sequence of the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3.

2. The gene according to claim 1 , wherein a) the nucleotide sequence encoding a protein comprises an amino acid sequence, which has in order of increased preference at least 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No. 3; or b) the coding sequence of the gene has in order of increased preference at least 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to SEQ ID No. 2; or c) the nucleotide sequence encoding the LRR domain of the gene comprising the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3 has in order of increased preference at least 96%, 97%, 98%, 99%, 100% sequence similarity to the amino acid sequence of the amino acid positions [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], [1180] to [1210] of SEQ ID No. 3.

3. A protein encoded by the gene as claimed in claims 1 or 2.

4. Use of the gene as claimed in claim 1 or 2 for producing a Brassica oleracea plant that is resistant against Hyaloperonospora parasitica.

5. Method for identifying a Brassica oleracea plant resistant to Hyalopersonospora parasitica, wherein the method comprises the following steps: a) detecting in the genome of a Brassica oleracea plant the gene as claimed in claim 1 or 2, and / or b) testing of the Brassica oleracea plant comprising the gene as claimed in claim 1 or 2 for exhibiting resistance to Hyaloperonospora parasitica6. Method according to claim 5, wherein the method between step a) and step b) comprises a step of detecting an unique polymorphism in the gene as claimed in claim 1 or 2.

7. A Brassica oleracea plant, comprising the gene as claimed in claim 1 or 2, wherein the plant is resistant to Hyaloperonospora parasitica.

8. The Brassica oleracea plant of claim 7, wherein the plant is an agronomically elite plant, in particular a hybrid variety or an inbred line.

9. The Brassica oleracea plant as claimed in any one of the claims 7 and 8, wherein the plant is a broccoli, cauliflower, romanesco, red cabbage, white cabbage, savoy cabbage or kohlrabi plant.

10. A seed capable of growing into a plant as claimed in any one of the claims 7-9.

11. Propagation material suitable for producing a plant as claimed in any one of the claims 7 to 9, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from a microspore, pollen, ovary, ovule, embryo sac and egg cell, or is suitable for vegetative reproduction, and is in particular selected from a cutting, root, stem cell, and protoplast, or is suitable for tissue culture of regenerable cells or protoplasts, which regenerable cells or protoplasts are in particular selected from a leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, anther, flower and stem, and wherein the propagation material comprises the gene as claimed in claim 1 or 2.

12. A method for selecting a Brassica oleracea plant resistant to Hyaloperonospora parasitica, comprising identifying the presence of the gene as claimed in claim 1 or 2, optionallytesting the Brassica oleracea plant for resistance against Hyaloperonospora parasitica, and selecting a plant comprising said gene, wherein the presence of said gene in said plant confers resistance to Hyaloperonospora parasitica.

13. A method for producing a Brassica oleracea plant exhibiting resistance against Hyaloperonospora parasitica comprising the steps of: a) crossing a first parent plant comprising the gene as claimed in claim 1 or 2 with a second parent plant to obtain an Fl population; b) optionally performing one or more rounds of selfing and / or crossing with a plant from the Fl population to obtain a further generation population; c) selecting from the further generation population a plant that comprises the gene as claimed in claim 1 or 2 as a resistant plant.

14. A method for producing hybrid seed resistant to Hyaloperonospora parasitica, comprising the steps of crossing a first parent plant with a second parent plant, wherein one or both parent plants are homozygous for the gene as described in claim 1 or 2 and harvesting the hybrid seed.

15. The hybrid seed produced by the method of claim 14.

16. A plant grown from the hybrid seed of claim 15.

17. A marker for the identification of the gene as claimed in claim 1 or 2, wherein the marker comprises a SNP.

18. Use of the marker as claimed in claim 17 for identification of a plant comprising the gene as claimed in claim 1 or 2.

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