Patent Description:
The oomycete Albugo candida (Pers. ) Kuntze is an obligate biotrophic pathogen of the Brassicaceae family, also known as the crucifer family, and causes the widespread disease known as white rust (also known as white blister, white blister rust, staghead, A. cruciferum or A. cruciferatum). This disease is destructive to many vegetable and oilseed crops such as broccoli, cabbage, rape, mustard and radish.

White rust has been observed worldwide, in countries as geographically and environmentally diverse as India, Canada, Europe and Australia. In Australia, white blister rust has spread to Brassicaceae crops in southern parts of the country.

candida is highly specialized, grows between living host cells and causes a range of symptoms that can be the result of local or systemic infection. The genus Albugo includes several species that cause white rust on a range of hosts and within A. candida various host-specialized forms have been reported. One of the characteristics of A. candida is thus its high degree of host specificity.

The growth of A. candida leads to two types of infections, local or systemic. Local infection is characterised by white or creamy-yellow pustules of zoosporangia that form under the epidermis of the host. These usually develop on the lower (abaxial) surface of the leaf and to a lesser extent on the upper (adaxial) surface and occur more commonly on mature leaves. However, they may be localized on any aerial host organ. Initially, the pustules are small and discrete but eventually become large and confluent. Once the pustule is fully developed, the host epidermis ruptures to release a dry, powdery burst of zoosporangia. Subsequently, necrosis of the surrounding leaf tissue may occur.

Localized infection does not usually result in extensive yield loss but would still lead to unmarketable products. Systemic infection can have a severe impact on the productivity of crops grown for seed or floral parts. Systemic infection appears to trigger sexual reproduction of the oomycete and causes distortion, hyperplasia and hypertrophy of the inflorescences, stems and leaves. It can also result in sterility if the flower petals, ovules and pollen grains are malformed as a result of the disease.

candida grows best at moderate temperatures of between <NUM> and <NUM> and in moist conditions. A leaf wetness period of <NUM> hours is sufficient to result in infection, which becomes symptomatic after an incubation period of <NUM> to <NUM> days.

Brassica is a plant genus in the family Brassicaceae (formerly referred to as Cruciferae). The genus Brassica comprises a number of important agricultural and horticultural crops, including rape, cauliflower, broccoli and turnip. Almost all parts of these plants can be used as food. Rape and rape seed are also used for oil, both for consumption and for fuel. Some species with white or purple flowers or distinct colour or shaped leaves are cultivated for ornamental purposes. The Brassicaceae family occurs worldwide and comprises annuals, biennials and perennials. The family also comprises a large number of wild species.

In some crops white rust can cause huge losses in yield. In other Brassica crops white rust affects the appearance of the plant such that the crop is no longer commercially viable because of the cosmetic damage. There is therefore a great need for Brassica crops which are resistant to white rust.

The inheritance of resistance to white blister rust has been shown to vary both between and within host species. Resistance to white rust has been studied in B. napus and A. thaliana and has been found to be controlled either by a single dominant gene, as in B. carinata, B. nigra, or by multiple genes where the resistance is said to be polygenic. Resistance to white rust has also been associated with specific biochemical properties of the host, such as elevated levels of certain sugars, chlorophyll or phenols.

candida is now subdivided into several races that are specialised to infect specific hosts. The current classification of A. candida biological races is based on the response of Brassicaceae hosts to infection by various isolates of A. The different races identified to date are reported in table <NUM>. It is to be noted that for A. candida, the term "race" as used herein refers to a variety of a pathogen that can infect some species of a host genus and not others whereas in most pathosystems it is taken to mean a variety of pathogen that can infect some varieties of a host species but not others.

In the last years, these races of A. candida have been subdivided to acknowledge the existence of different variants within a race. Two broad variants of race <NUM> have been identified, based on their virulence to two varieties of B. rapa, and four variants of race <NUM> have been characterised, based on their virulence to eleven cultivars of B.

candida race <NUM> has spread to B. oleracea var. italica (broccoli) and B. oleracea var. botrytis (cauliflower) crops in southwest Australia, making this pathogen the number one concern for broccoli and cauliflower breeding in Australia. This oomycete pathogen is growing on Broccoli leaves, stem or curds showing white spot symptoms on leaves and transforming buds into white erected pin heads that affect the curd quality and shelf life. The Australian A. candida strain infecting B. oleracea differs from the A. candida race <NUM> generally encountered in other parts of the world, and referred to as European variant; this European variant causes disease essentially on cabbage.

Indeed, the Australian race <NUM> is in some experiments more aggressive towards broccoli than towards cabbage while in Europe, the race <NUM> appears to be more aggressive towards cabbage and Brussels sprouts. It is therefore reported that race <NUM> comprises at least two variants, the European variant, more aggressive towards cabbage and the Australian variant more aggressive towards broccoli (Petkowski et al, Proc. Vth IS on Brassica & XVIth Crucifer Genetics WS: <NUM>-<NUM>; <NUM>); for example Iron CMS and Belstar commercial varieties show resistance to the European variant but are susceptible to the Australian variant, as reported in <CIT>.

The white rust outbreaks caused by A. candida in many broccoli crops have been reported to be very serious. In many situations infection rates have been as high as <NUM>%, with the only recourse being to destroy the entire crop. In neglected fields, complete infection of every part of every plant, from the leaves to the broccoli head, is often observed. The spore load from the mature sporangia is so extensive that a cloud of spores can be seen upon physical disruption of an infected plant.

At the moment few agents are known which can be used to control white rust in Brassicas. Current control methods generally involve a combination of management practices (controlled watering, ventilation, balanced program of nutrition) and a fungicide spray program. However, an increasing number of countries have implemented policy aimed at reducing the use of chemical crop protection agents. If the use of chemical control agents is banned completely, this would result in major problems in the cultivation of Brassica species.

Genetic resistance to A. candida in broccoli (Brassica oleracea var. italica) would be beneficial in the control of white blister rust. Such resistance would not only increase the stability of crop protection, but would also result in a reduced, or eliminated, requirement for environmentally harmful, and not always effective, fungicide applications. Such resistance could also be beneficial to other B. oleracea plants such as cauliflower, Brussel sprouts, white cabbage, savoy cabbage, etc..

<CIT> illustrates B. oleracea plants allegedly containing a monogenic dominant resistance gene to A. candida, but not to the Australian variant of A. candida race <NUM>. <CIT> illustrates B. oleracea plants allegedly containing a monogenic semi-dominant resistance gene to A. candida, present on chromosome <NUM> but not conferring resistance to the Australian variant.

<CIT> illustrates B. oleracea plants containing a monogenic dominant resistance gene to A. candida located on chromosome <NUM>. This gene has been able to confer resistance to some strains of the Australian variant of A. candida race <NUM> but appears to be unable to generally confer resistance to white rust caused by A. candida race <NUM> Australian variant, irrespective of the strain. Similarly, the variety Tyson (<NPL>), although it is disclosed as resistant to the Australian variant of A. candida race <NUM>, appears to be unable to generally confer resistance to white rust caused by A. candida race <NUM> Australian variant, irrespective of the strain, as illustrated in the examples (see Ex. <NUM> and <NUM>).

Therefore, there is still an unfulfilled need for broccoli plants with a resistance to the Australian variant of A. candida race <NUM>, namely a general resistance which is not restricted to some strains.

The present inventors have identified a resistance to the Australian variant of the A. candida race <NUM> pathogen and have been able to introgress this resistance into B. oleracea plants, thus obtaining resistant B. oleracea plants, more specifically broccoli resistant plants. The resistance of the present invention is imparted by the newly discovered sequences conferring the resistance, said resistance being of monogenic and dominant nature and transferable to different B. oleracea genetic backgrounds.

The present invention provides introgressed sequences conferring, when present in homozygous or in heterozygous state, the phenotype of resistance to the Australian variant of the A. candida race <NUM>. The invention also provides B. oleracea plants that display resistance to Australian variant of the A. candida race <NUM>, namely broccoli plants, as well as methods that produce or identify B. oleracea var. italica broccoli plants that display resistance to Australian variant of the A. candida race <NUM> as well as seeds, roots and other plant parts such as pollen and ovules containing the introgressed sequences conferring the resistance. The invention also discloses molecular genetic markers, especially SNPs, linked to the introgressed sequences conferring the resistance, i.e. linked to the resistance locus which is of dominant nature.

oleracea plant resistant to white rust disease caused by the oomycete A. candida race <NUM> Australian variant plant may be selected from the group consisting of B. oleracea convar. Botrytis var. italica (broccoli) or B. oleracea convar. botrytis var. botrytis (cauliflower) and most preferably is broccoli.

candida race <NUM> Australian variant is a strain of A. candida corresponding to the race <NUM> according to the current classification of A. candida biological races established by Pound and William in <NUM> (<NPL>), which is further characterized as being more aggressive towards broccoli than towards cabbage by opposition to the European variant race <NUM> which is more aggressive towards cabbage and Brussels sprouts. The Australian variant is also known as being the variant responsible for the white blister rust outbreaks in Victorian broccoli and cauliflower crops in the summer <NUM>-<NUM> and the subsequent spread of disease to all Australian broccoli and cauliflower cropping areas (<NPL>).

Different strains of A. candida race <NUM> Australian variant co-exist, inter alia during field infection, which may have different virulence on the infected plants.

The term "Resistance" is as defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Section for describing the reaction of plants to pests or pathogens, and abiotic stresses for the Vegetable Seed Industry.

Specifically, by resistance, it is meant the ability of a plant variety to restrict the growth and development of a specified pest or pathogen and/or the damage they cause when compared to susceptible plant varieties under similar environmental conditions and pest or pathogen pressure. Resistant varieties may exhibit some disease symptoms or damage under heavy pest or pathogen pressure.

Susceptibility: The inability of a plant variety to restrict the growth and development of a specified pest or pathogen.

olearacea plant susceptible to white rust, especially caused by A. candida race <NUM> Australian variant, is for example the commercially available plant 'Belstar' or 'Fiesta' from Bejo, the variety 'Rumba' from HM. Clause, the varieties 'Patriot' and 'Green Belt' from Sakata or the variety 'Monaco' from Syngenta. All the commercially available varieties of B. olearacea broccoli are, to date, susceptible to white rust caused by A. candida race <NUM> Australian variant, apart Booster variety of HM. Clause SA and Tyson variety of Syngenta, which are however not resistant to all infections by A. candida race <NUM> Australian variant.

A plant according to the invention has thus at least improved resistance to A. candida race <NUM> Australian variant, and especially with respect to the broccoli plant Booster, and more generally with respect to any commercial variety of broccoli.

As used herein, the term "offspring" or "progeny" refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof. For instance an offspring plant may be obtained by cloning or selfing of a parent plant or by crossing two parents plants and include selfings as well as the F1 or F2 or still further generations. An F1 is a first-generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of second generation (F2) or subsequent generations (F3, F4, etc.) are specimens produced from selfings of F1's, F2's etc. An F1 may thus be (and usually is) a hybrid resulting from a cross between two true breeding parents (true-breeding is homozygous for a trait), while an F2 may be (and usually is) an offspring resulting from self-pollination of said F1 hybrids.

As used herein, the term "cross", "crossing", "cross pollination" or "cross-breeding" refer to the process by which the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.

As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

As used herein, the term "genotype" refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.

As used herein, the term "heterozygote" refers to a diploid or polyploid individual cell or plant having different alleles (forms of a given gene or sequences) present at least at one locus.

As used herein, the term "heterozygous" refers to the presence of different alleles (forms of a given gene or sequences) at a particular locus.

As used herein, the terms "homolog" or "homologue" refer to a nucleic acid or peptide sequence which has a common origin and/or functions similarly to a nucleic acid or peptide sequence from another species.

As used herein, the term "homozygote" refers to an individual cell or plant having the same alleles at one or more loci on all homologous chromosomes.

As used herein, the term "homozygous" refers to the presence of identical alleles at one or more loci in homologous chromosomal segments.

As used herein, the term "hybrid" refers to any individual cell, tissue or plant resulting from a cross between parents that differ in one or more genes.

As used herein, the term "locus" (plural: "loci") refers to any site that has been defined genetically. A locus may be a gene, or part of a gene, or a DNA sequence, and may be occupied by different sequences. A locus may also be defined by a SNP (Single Nucleotide Polymorphism), or by several SNPs.

By introgression, it is meant the infiltration of the genes or of genomic sequences of one species, variant or strain, into the gene pool of another one from an initial hybrid between these species, variants or strains.

The present invention is directed to B. oleracea var. italica broccoli plants that display resistance to Australian variant of A. candida race <NUM>, as well as methods that produce or identify B. oleracea broccoli plants that display resistance to infection by Australian variant of A. candida race <NUM>. The present invention also discloses molecular genetic markers, especially SNPs, linked to the resistance locus. The inventors have also shown that the sequences responsible for the resistance can be introgressed into different genetic backgrounds and still confer the resistance phenotype (as demonstrated inter alia in example <NUM> with different F2 populations and example <NUM> with <NUM> inbred lines).

The seeds and plants according to the invention have been obtained from an initial cross between a Romanesco plant, the introgression partner displaying the phenotype of interest, and an inbred line of broccoli, the recurrent susceptible parent, followed by backcross and a dihaploidization program. A sample of this Brassica oleracea var italica (broccoli) BROCCO-C4 seed has been deposited by HM. Rue Louis Saillant, Z. La Motte, <NUM> Porte les Valence, France pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (the "Budapest treaty") with the National Collection of Industrial, Food and Marine Bacteria (NCIMB), (NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom), on <NUM>th July <NUM>, under accession number NCIMB <NUM>.

The plants grown from these deposited seeds are broccoli plants resistant to white blister rust caused by any Australian variant of A. candida race <NUM> and are commercially acceptable phenotype.

A deposit of this Brassica oleracea varitalica - BROCCO-C4 seed is maintained by HM. Rue Louis Saillant, Z. La Motte, <NUM> Porte les Valence, France.

According to a first aspect, the present invention is thus directed to a B. oleracea var. italica plant or seed, which is resistant to white blister rust caused by the oomycete Albugo candida race <NUM> Australian variant, comprising in its genome introgressed sequences or interval conferring said resistance to white blister rust. Said introgressed sequences confer the resistance to a B. oleracea var. italica plant or seed when present either homozygously, or heterozygously.

The introgressed interval acts as a single dominant allele of a resistance gene responsible for the phenotype (i.e. the resistance trait is monogenic and dominant). Plants homozygous and heterozygous for the introgressed interval both fully exhibit the white rust resistance phenotype. This phenotype can be used to identify progeny that bear the claimed introgressed sequences or interval. The introgressed interval acting as a resistance gene confers the phenotype of interest and is unexpectedly unlinked to negative features incompatible with marketability of the plants, seed or head, such as presence of leaflets on the petioles or "cat-eyes", i.e. the presence of some beads which prematurely break into yellow flowers.

The resistance to A. candida race <NUM> Australian variant infection is advantageously determined by comparison to a susceptible (commercial) line, for example Belstar, Fiesta or Rumba, or to a line resistant to only some strains, for example Booster or Tyson. The resistance may be determined by the pathological test described in example <NUM>, preferably <NUM> days after inoculation. In this resistance scoring, a score of "<NUM>" corresponds to leaves with more than <NUM>% of the surface with sporulation, a score of "<NUM>" corresponds to between <NUM> and <NUM>% of the surface with sporulation, a score of "<NUM>" is <NUM> to <NUM>% of the surface with sporulation, a score of "<NUM>" is <NUM> to <NUM>%, a score of "<NUM>" is <NUM> to <NUM>%, and "<NUM>" corresponds to the absence of sporulation on the leaves. A plant having a score of <NUM>, i.e. without sporulation on the leaf surface, is to be considered as resistant, and more specifically highly resistant.

The introgressed sequences are preferably to be found on chromosome <NUM> in the B. oleracea genome and thus confer resistance to white blister rust caused by the oomycete A. candida race <NUM> Australian variant when they are present on either one or both homologous chromosomes <NUM>. The introgressed sequences conferring the resistance are more preferably located within the chromosomal region of chromosome <NUM> which is delimited on one side by the SNP BO-<NUM> (SEQ ID No:<NUM>) and on the other side by the SNP BN-<NUM> (SEQ ID No:<NUM>), and preferably between SNP BO-<NUM> (SEQ ID No:<NUM>) and SNP BO-<NUM> (SEQ ID No:<NUM>).

The specific polymorphisms corresponding to the SNPs (Single Nucleotide Polymorphism) referred to in this description, as well as the flanking sequences of these SNPs in the B. oleracea genome, are given in the experimental section (see inter alia table <NUM>) and accompanying sequence listing. Their location with respect to the version V2. <NUM> of the Brassica oleracea TO1000 genome, on chromosome <NUM>, is indicated in table <NUM>, and their flanking sequences are also illustrated in table <NUM>.

The introgressed sequences or interval conferring the resistance are preferably chosen from the introgressed sequences present in the genome of a plant of B. oleracea var. Italica -BROCCO-C4, representative seeds of which are deposited at the NCIMB under the accession number NCIMB <NUM>. They are especially chosen from the introgressed sequences present on chromosome <NUM> of said B. oleracea var. Italica -BROCCO-C4. Indeed, the deposited seeds comprise, on one chromosome <NUM> homologue, introgressed sequences conferring the phenotype of interest, wherein said introgressed sequences are also conferring the phenotype in B. oleracea genetic background. A sample of this B. oleracea var. Italica -BROCCO-C4 seed has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB), (NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom), on <NUM>th July <NUM>, under accession number NCIMB <NUM>. The deposited seeds are hybrid seeds, they bear the introgression sequences, conferring the resistance, on only one chromosome <NUM> homologue. The resistance sequences can be easily identified inter alia by the markers disclosed in the application.

The sequences conferring the resistance are present in the genome of all deposited seeds; as such the genome of these deposited seeds thus represent a reservoir of introgressed sequences in the B. oleracea genome conferring resistance to white rust caused by A. candida race <NUM> Australian variant according to the invention. A plant or seed of the invention comprises in its genome introgressed sequences which are chosen from this reservoir.

Whereas all deposited seeds possess an introgressed fragment at the same locus on one chromosome <NUM>, and conferring the phenotype according to the invention, this introgressed fragment may slightly vary in length between the seeds.

The present invention is thus also directed to a B. oleracea var. italica seed or plant, preferably a cultivated B. oleracea var. italica broccoli seed or plant, having in its genome introgressed sequences conferring resistance to white rust caused by A. candida race <NUM> Australian variant, wherein said introgressed sequences conferring the resistance are chosen from the introgressed sequences present in the genome of a seed of B. oleracea var. Italica -BROCCO-C4 corresponding to NCIMB <NUM> deposit.

By "introgressed sequences or intervals at a given locus" or "introgressed sequences or intervals present/found at a given locus", it is to be understood that the genomic interval found at this given locus has not the same sequence as the corresponding genomic interval found in the B. oleracea broccoli germplasm before introgression, but has, at this locus, the sequence found in the corresponding genomic interval of B. oleracea var. Italica -BROCCO-C4 (NCIMB <NUM>) at the same locus; the introgressed sequences are thus exogenous with respect to a plant of the invention. By having the "same sequence", it means that the two sequences to be compared are identical to the exception of potential point mutations which may occur during transmission of the genomic interval to progeny, i.e. preferably at least <NUM>% identical on a length of <NUM> kbase. It can be deduced that a genomic interval under test has the same sequence, in the sense of the invention, as the corresponding genomic interval found in the B. oleracea var. Italica - BROCCO-C4 at the same locus, if said genomic interval under test is also capable of conferring resistance to white rust caused by A. candida race <NUM> Australian variant.

The presence of introgressed sequences into the genome of a B. oleracea plant, seed or cell may for example be shown by GISH (genetic in situ hybridization). GISH is indeed a powerful technique for detection of the introgression of chromatin material from one species or strain onto another species or strain. The advantage of GISH is that the introgression process is visualized by means of 'pictures of the introgressed genome'. With this technique, it is also possible to establish if a particular region of the genome is homozygous or heterozygous, thanks to the use of molecular cytogenetic markers which are co-dominant. By this technique, it is also possible to determine in which chromosome an introgressed gene of interest is present.

According to a preferred embodiment, the introgressed sequences conferring the resistance phenotype of the invention, i.e. resistance to white blister rust caused by the oomycete Albugo candida race <NUM> Australian variant, are introgressed from a romanesco B. oleracea plant.

According to a preferred embodiment, the introgressed sequences conferring the resistance, and which are also to be found in the genome of the deposited seeds, are introgressed on chromosome <NUM> of a B. oleracea plant according to the invention, preferably broccoli, they are more precisely within the chromosomal region of chromosome <NUM> which is delimited on one side by the SNP BO-<NUM> (SEQ ID No:<NUM>) and on the other side by the SNP BN-<NUM> (SEQ ID No:<NUM>), and more preferably between SNP BO-<NUM> (SEQ ID No:<NUM>) and SNP BO-<NUM> (SEQ ID No:<NUM>). In other words, in the genome of a B. oleracea plant or seed of the invention, the section on chromosome <NUM> within the region delimited on one side by the SNP BO-<NUM> (SEQ ID No:<NUM>) and on the other side by the SNP BN-<NUM> (SEQ ID No:<NUM>), comprises sequences which are responsible for the resistance to white rust caused by A. candida race <NUM> Australian variant, in B. oleracea genetic background, and more preferably between SNP BO-<NUM> (SEQ ID No:<NUM>) and SNP BO-<NUM> (SEQ ID No:<NUM>). Said introgressed sequences are preferably from a romanesco B. oleracea plant.

According to a preferred embodiment, the introgressed sequences present in the genome of a plant or seed of the invention are to be found at one or more of the following loci:.

The locus encompassing SNP BO-<NUM> is highly preferred.

It is noted in this respect that specific positions in a chromosome can indeed be defined with respect to single nucleotide polymorphism, insofar as the flanking sequences of said SNPs are defined. The present inventors have used SNPs, identified by their flanking sequences, present in all B. oleracea genomes, especially B. oleracea var. Italica and Romanesco B. oleracea, to discriminate between introgressed and endogenously residing sequences and to track down the introgressed sequences from Romanesco in the B. oleracea broccoli genome.

The present inventors have identified that introgressed sequences essential for the phenotype of interest, i.e. white rust caused by A. candida race <NUM> Australian variant, are to be found in the vicinity of the SNP BN-<NUM> (SEQ ID No:<NUM>) or preferably in the vicinity of SNP BO-<NUM> (SEQ ID No:<NUM>), or in the vicinity of both SNPs. Preferably the introgressed sequences are to be found in a locus encompassing the position of SNP BO-<NUM>. It is to be noted that the locus of SNP BN-<NUM> and SNP BO-<NUM> are located within the chromosomal region defined above, i.e. delimited on one side by the SNP BO-<NUM> and on the other side by the SNP BN-<NUM> on B. oleracea chromosome <NUM>.

When the introgressed sequences conferring the resistance are found in a locus encompassing SNP BN-<NUM> then the allele of SNP BN-<NUM> is the allele of SNP BN-<NUM> found in the Romanesco introgression partner, and also in the deposited resistant B. oleracea var. Italica -BROCCO-C4, and demonstrated in the experimental section as genetically linked to the resistance, i.e. allele T of SNP BN-<NUM>. The <NUM>' flanking region of SNP BN-<NUM>, or the <NUM>' flanking region of SNP BN-<NUM>, or both regions, are also identical B. oleracea var. Italica -BROCCO-C4 sequences in this region. Therefore, the SNP BN-<NUM> may form part of the <NUM>' border or <NUM>' border of the introgressed interval, or may be within the introgressed interval conferring the desired phenotype.

When the introgressed sequences conferring the resistance are found in a locus encompassing SNP BO-<NUM>, then the allele of SNP BO-<NUM> is the allele of SNP BO-<NUM> found in the Romanesco introgression partner, and also in the deposited resistant BROCCO-C4, and demonstrated in the experimental section as genetically linked to the resistance i.e. allele C of SNP BO-<NUM>. The <NUM>' flanking region of SNP BO-<NUM>, or the <NUM>' flanking region of SNP BO-<NUM>, or both regions, are also identical to B. oleracea var. Italica -BROCCO-C4 sequences in this region. Therefore, the SNP BO-<NUM> may form part of the <NUM>' border or <NUM>' border of the introgressed interval, or may be within the introgressed interval conferring the desired phenotype.

The presence of the introgressed sequences of interest can indeed be revealed by the presence of specific alleles of given SNPs, wherein said alleles are characteristic of the introgression partner, thus associated with the resistance, and distinct from the allele of the recurrent B. oleracea broccoli parent for these SNPs, associated with the susceptibility. The alleles of given SNPs can thus reflect the presence of the introgression sequences of the invention. Examples of appropriate SNPs are all or part of the list consisting of SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BO-<NUM> (SEQ ID No:<NUM>), SNP BN-<NUM> (SEQ ID No:<NUM>) and SNP BN-<NUM> (SEQ ID No:<NUM>). These alleles, associated with the resistance are inter alia allele G of SNP BO-<NUM> (SEQ ID No:<NUM>); allele C of SNP BO-<NUM> (SEQ ID No:<NUM>); allele G of SNP BO-<NUM> (SEQ ID No:<NUM>); allele A of SNP BO-<NUM> (SEQ ID No:<NUM>); allele C of SNP BO-<NUM> (SEQ ID No:<NUM>); allele A of SNP BO-<NUM> (SEQ ID No:<NUM>); allele G of SNP BO-<NUM> (SEQ ID No:<NUM>); allele C of SNP BO-<NUM> (SEQ ID No:<NUM>); allele C of SNP BO-<NUM> (SEQ ID No:<NUM>); allele T of SNP BN-<NUM> (SEQ ID No:<NUM>) and allele G of SNP BN-<NUM> (SEQ ID No:<NUM>); these alleles correspond to the alleles of these SNPs found in the deposited seeds NCIMB <NUM>. Table <NUM> discloses for the <NUM> SNPs of the invention the allele which is associated with the resistance phenotype, i.e. the allele which co-segregates with the resistance phenotype, and the allele which is associated with the susceptibility.

A plant or seeds of the invention is preferably characterized by allele C of BO-<NUM>, preferably in combination with at least one, preferably at least two, <NUM>, <NUM> or all of allele G of BO-<NUM>, allele G of BO-<NUM>, allele A of BO-<NUM>, allele C of BO-<NUM>, allele A of BO-<NUM>, allele G of BO-<NUM>, allele C of BO-<NUM>, allele C of BO-<NUM>, allele T of BN-<NUM> and allele G of BN-<NUM>. As disclosed in example <NUM>, allele C of BO-<NUM> appears very highly associated with the phenotype and is thus mostly preferred according to the invention.

According to another embodiment, the introgressed sequences present in the genome of a plant or seed of the invention are to be found at one or more of the following loci:.

in addition or in place of the introgressed sequences found at the locus encompassing SNP BN-<NUM> and/or preferably SNP BO-<NUM> according to the previous embodiment, preferably in all these <NUM> loci.

According to a preferred embodiment, introgressed sequences are to be found at the loci encompassing SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM> and SNP BO-<NUM>.

Consequently, a resistant plant or seed of the invention may also be characterized by the allele T of SNP BN-<NUM>, or preferably by allele C of SNP BO-<NUM>, or both. They may also be characterized in addition to allele C of BO-<NUM>, by at least one of the following alleles: allele G of SNP BO-<NUM>; allele G of SNP BO-<NUM>; allele A of SNP BO-<NUM>; allele C of SNP BO-<NUM>; allele A of SNP BO-<NUM>; allele G of SNP BO-<NUM>; allele C of SNP BO-<NUM>; allele C of SNP BO-<NUM>; allele T of SNP BN-<NUM> and allele G of SNP BN-<NUM>.

The presence of the introgressed sequences can also be revealed by genic amplification of sequences in the proximity of the SNPs defined in the present invention, especially SNP BO-<NUM> and comparison with the sequence of the respective amplification fragment, obtainable by carrying out the amplification on seeds deposited at the NCIMB under accession number NCIMB <NUM>. Primers for the genic amplification can be defined by use of the flanking sequences disclosed in the present invention, potentially in combination with the available B. oleracea genome assembly.

Preferably, the introgressed sequences conferring the phenotype of interest, are in linkage disequilibrium with the allele of SNP BO-<NUM>. According to a preferred embodiment, the allele of SNP BO-<NUM> is "C". According to another embodiment, the allele 'T' of SNP BN-<NUM> and allele C of SNP BO-<NUM> are in linkage disequilibrium with the introgressed sequences conferring the resistance. Linkage disequilibrium indeed is used to describe common inheritance of genomic sequences in a cross population analysis when no linkage exists. Linkage describes common inheritance of genomic sequences in a population structure pending on the frequency of recombination.

The linkage disequilibrium score may be any positive score, meaning that the association of SNP BO-<NUM> with the introgressed sequences is not random.

In a plant or seed of the invention, thus comprising in its genome introgressed sequences, said introgressed sequences are preferably to be found in the genome at a genetic distance of less than <NUM>, preferably less than <NUM>, most preferably less than <NUM>, and even preferably less than <NUM> from the locus corresponding to SNP BO-<NUM>.

In another embodiment, the introgressed sequences are to be found in the genome of a plant or seed of the invention in the vicinity of both SNPs BO-<NUM> and BN-<NUM>, preferably at less than <NUM> from both SNPs, or preferably less than <NUM> from both; for example the introgressed sequences are located at less than <NUM> from the locus corresponding to SNP BO-<NUM> and at <NUM> or less from SNP BN-<NUM>.

According to an embodiment, a seed or plant of the invention, preferably a broccoli seed or plant, is characterized by the presence of allele C of SNP BO-<NUM> on chromosome <NUM>, optionally in combination with absence of allele A of said SNP. Indeed, presence of allele C of SNP BO-<NUM> confirms the presence of introgressed sequences at the locus of SNP BO-<NUM> and thus of the resistance; moreover the absence of allele A indicates that the introgressed sequences are homozygously present, i.e. present on all the homologues of chromosome <NUM>. Alternatively, a seed or plant of the invention, preferably a broccoli seed or plant, is characterized by the presence of both alleles C and A of SNP BO-<NUM> on chromosome <NUM>, confirming the heterozygous presence of the introgressed sequences.

According to a preferred embodiment of the present invention, the introgressed sequences or interval present in the genome of a broccoli seed or plant and conferring the resistance to white rust caused by A. candida race <NUM> Australian variant, are at least <NUM> kilobases long, and preferably at least <NUM>, <NUM> or <NUM> kb long.

Preferably, the introgressed sequences or intervals are however not too long in order to avoid introgression of non-commercial features associated with Romanesco introgression partner. It is thus preferred according to the invention that the introgressed sequences mentioned above are less than <NUM> in length, preferably less than <NUM> or less than <NUM>. According to more preferred embodiments, the introgressed sequences are less than <NUM> or even less than <NUM> in length and most preferably less than <NUM> in order to avoid or limit linkage drag.

According to a preferred embodiment, said introgressed sequences are minimized to contain as few as possible sequences unrelated to the desired phenotype.

The resistance according to the invention is a resistance to white rust caused by A. candida race <NUM> Australian variant infection irrespective of the strain of Australian variant, contrary to the plant Booster, referred to in Petkowski et al and described in <CIT> or the plant Tyson, referred to in Minchinton et al. In this respect, it is to be noted that, in fields, white rust infection is generally due to a plurality of different strains of A. candida race <NUM> Australian variant, stressing the importance of a resistance irrespective of the strain of Australian variant.

The seed or plant according to this aspect of the invention is preferably highly resistant to A. candida race <NUM> Australian variant, inter alia it remains free of symptoms till the end of the season when grown under natural infection conditions. In any case, a seed or plant of the invention, which is resistant to A. candida race <NUM> Australian variant has less than <NUM>% of the leave surface with sporulation, and preferably less than <NUM>% according to the pathology test described in example <NUM>, and even preferably no sporulation on the leaf surface.

Plants or seeds of the invention, resistant to A. candida race <NUM> Australian variant, are also preferably resistant or tolerant to another pest, especially to pest of major agricultural importance.

Plants or seeds of the invention preferably also comprise other white rust resistance genes, preferably the monogenic dominant gene on chromosome <NUM> as disclosed in <CIT>, more preferably said plants or seeds comprise in their genome SEQ ID NO:<NUM> and/or SEQ ID NO:<NUM> as defined in <CIT>.

As detailed above, the invention is directed to B. oleracea broccoli plants, resistant to A. candida race <NUM> Australian variant infection, as well as to seeds giving rise to those plants.

oleracea var. italica broccoli plant according to the invention may be a commercial plant or line or variety, preferably cultivated for its head. Such a commercial plant or line gives rise to head which are marketable, when grown in suitable conditions.

The plant according to invention may be an inbred or a dihaploid plant or a hybrid, and is a broccoli. It is preferably a cultivated B. oleracea broccoli.

The plant according to the invention may also be cytoplasmic male sterile, for example having the Ogura mitochondrial sterility.

Plants or seeds according to the invention are Broccoli plants (B. oleracea var. Alternatively, in a non-claimed embodiment, the description also concerns other B. oleracea plants, which are known as host for A. candida race <NUM> Australian variant, namely cauliflower (B. oleracea convar. Botrytis var. botrytis), Brussels sprouts (B. oleracea convar. Oleracea var. gemnifera), white cabbage, oxheart cabbage (B. oleracea convar. Capitata var. alba) and savoy cabbage (B. oleracea convar. Capitata var. The introgressed sequences, conferring the resistance, can be transferred from a resistant broccoli, inter alia BROCCO-C4, to another B. oleracea plants, by a breeding program using the markers exemplified in the examples. A plant or seed of the invention is advantageously not a Romanesco plant or seed.

In this respect, it is to be noted that the inventors have, for the first time, identified sequences responsible for the resistance, have identified SNP markers associated with these sequences, and have proved that said sequences, initially present in a Romanesco plant can be transferred to another B. oleracea plants, without loss of the resistance phenotype, and without causing any other detrimental phenotype. Once the inventors have demonstrated that such a resistance gene exists, can be transferred from one genetic background to another one, while conserving its ability to confer resistance (penetration) without imparting negative features and once they have identified genetic markers linked to this resistance, it is then possible by a breeding program to transfer said sequences to another B. oleracea plants, and thus to cauliflower, Brussel sprouts, white cabbage and savoy cabbage.

A plant or seed according to the invention may be a progeny or offspring of a plant grown from the deposited seeds of B. oleracea var. Italica-BROCCO-C4, NCIMB <NUM>. Plants grown from the deposited seeds are indeed resistant to A. candida race <NUM> Australian variant, they thus bear in their genome the introgressed sequences conferring resistance. They can be used, in a non-claimed aspect, to transfer these sequences in another background by crossing and selfing and / or backcrossing, and selecting the plants of interest.

The invention is also directed to the deposited seeds B. oleracea var. Italica -BROCCO-C4 (NCIMB <NUM>) and to plants grown from one of these seeds. These seeds contain heterozygously the introgressed sequence conferring the phenotype of interest.

The presence of the introgressed sequences according to the invention can be revealed by the sequencing of the SNP identified by the inventors, and more specifically by allele C of SNP BO-<NUM>. The heterozygous status of the genome of a plant, seed or plant part with respect to the introgressed sequences of interest can be brought to light by the simultaneous presence of alleles C and A of SNP BO-<NUM>. The homozygous status of the genome of a plant, seed or plant part with respect to the introgressed sequences of interest can be brought to light by the presence of only allele C of SNP BO-<NUM>.

The invention also concerns any broccoli plant likely to be obtained from seed or plants of the invention as described above, and also plant parts of such a plant, and most preferably explant, scion, cutting, seed, root, rootstock, pollen, ovule, embryo, siliqua, protoplast, leaf, anther, stem, petiole, head and any other plants part, wherein said plant, explant, scion, cutting, seed, root, rootstock, pollen, ovule, embryo, siliqua, protoplast, leaf, anther, stem, petiole, head and/or plant part is obtainable from a seed or plant according to the first aspect of the invention, i.e. bearing the introgressed sequences of interest in their genome either homozygously or heterozygously. These plant parts, inter alia explant, scion, cutting, seed, root, rootstock, pollen, ovule, embryo, siliqua, protoplast, leaf, anther, stem, petiole or head, comprise in their genome the introgressed sequences conferring the resistance to white rust caused by A. candida race <NUM> Australian variant. The introgressed sequences referred to in this aspect of the invention are those defined above in the context of plants of the invention. The different features of the introgressed sequences defined in relation with the first aspect of the invention apply mutatis mutandis to this aspect of the invention. The introgressed sequences are thus preferably chosen from those present in the genome of a plant corresponding to the deposited material B. oleracea var. Italica -BROCCO-C4 (NCIMB accession number <NUM>) and conferring the resistance. They are advantageously characterized by the presence of allele T of SNP BN-<NUM> (SEQ ID No:<NUM>) and/or allele C for SNP BO-<NUM> (SEQ ID No:<NUM>), preferably allele C for SNP BO-<NUM>. The invention is also directed to cells of B. oleracea var. Italica cells, such that these cells comprise, in their genome, introgressed sequences conferring the phenotype of interest. The introgressed sequences are those already defined in the frame of the present invention, they are characterized by the same features and preferred embodiments already disclosed with respect to the plants and seeds according to the preceding embodiments of the invention. The presence of these introgressed sequences can be revealed by the techniques disclosed above and well known to the skilled reader. It can inter alia be determined whether the introgressed sequences are present homozygously or heterozygously in the genome of such a cell of the invention. They are advantageously characterized by the presence of allele T for SNP BN-<NUM> (SEQ ID No:<NUM>) and/or allele C for SNP BO-<NUM> (SEQ ID No:<NUM>), preferably allele C for SNP BO-<NUM>. A plant, explant, scion, cutting, seed, root, rootstock, pollen, ovule, embryo, siliqua, protoplast, leaf, anther, stem, petiole, head and/or plant part as defined above preferably comprises at least one cell of the invention, preferably the majority of the cells thereof are cells according to the invention, more preferably all cells of the plant part as defined are cells of the invention.

Cells according to the invention can be any type of B. oleracea var. Italica cell, inter alia a cell capable of regenerating a whole B. oleracea plant, bearing the introgressed sequences. Alternatively, the cell according to the invention can be a cell specifically not capable of regenerating a whole B. oleracea plant.

The cell according to the invention may be an isolated cell.

The present invention is also directed to a tissue culture of regenerable cells of the plant as defined above according to the present invention; preferably, the regenerable cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, siliqua, seeds, flowers, cotyledons, and/or hypocotyls, and contain in their genome introgressed sequences on chromosome <NUM> conferring resistance to white rust caused by A. candida race <NUM> Australian variant.

The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing broccoli plant, and of regenerating plants having substantially the same genotype as the foregoing broccoli plant. The present invention also provides broccoli plants regenerated from the tissue cultures of the invention.

The invention also provides a protoplast of the plant defined above, or from the tissue culture defined above, said protoplast containing said introgressed sequences conferring resistance to A. candida race <NUM> Australian variant.

According to another non claimed aspect, the present invention is also directed to the use of a B. oleracea broccoli plant as detailed according to the first aspect of the invention, i.e. resistant, as a breeding partner in a breeding program for obtaining B. oleracea plants resistant to white rust caused by A. candida race <NUM> Australian variant. Indeed, such a plant according to the first aspect harbors in its genome introgressed sequences conferring the phenotype of interest, i.e. resistance. By crossing this plant with susceptible or less resistant plants, it is thus possible to transfer these sequences, conferring the desired phenotype, to the progeny as the phenotype is a monogenic trait. Preferably, a plant to be used as a breeding partner is a plant homozygous with respect to the introgressed sequence conferring the resistance, such that all hybrid plants resulting from the crossing of this plant with another B. oleracea will also bear the introgressed sequence conferring the resistance phenotype. Alternatively, a heterozygous plant according to the first aspect of the invention can also be used; in this case a selection step will advantageously be added, for example based on the presence of the SNP markers of the invention, in view of the segregation of the phenotype.

A plant according to the invention can thus be used as a breeding partner for introgressing sequences conferring the desired phenotype into a B. oleracea plant or germplasm.

The introgressed sequences of interest will advantageously be introduced into plant or varieties that contain other desirable genetic traits such as resistance to other diseases.

The introgressed sequences of interest will advantageously be introduced into plant or varieties that contain other white rust resistance genes such as the monogenic dominant gene on chromosome <NUM> as disclosed in <CIT>; more preferably they comprise in their genome SEQ ID NO:<NUM> and/or SEQ ID NO:<NUM> as defined in <CIT>.

The description is also directed, in a non-claimed embodiment, to the same use with plants or seed of B. oleracea var. Italica -BROCCO-C4, deposited at the NCIMB under the accession number NCIMB <NUM>, or progeny thereof bearing the introgressed sequences of the invention conferring the resistance to white blister rust, especially progeny thereof having allele C of SNP BO-<NUM>. Said plants are also suitable as introgression partners in a breeding program aiming at conferring the desired phenotype to B. oleracea plant or germplasm, although it is heterozygous for the introgression sequences of interest, i.e. for obtaining B. oleracea plants resistant to white blister rust caused by A. candida race <NUM> Australian variant.

In such a breeding program, the selection of the progeny displaying the desired phenotype, or bearing sequences linked to the desired phenotype, can advantageously be carried out on the basis of the alleles of the SNP markers. The progeny is preferably selected on the presence of allele C of SNP BO-<NUM><NUM> on chromosome <NUM>. The selection can alternatively be made on the basis of the simultaneous presence of allele T of SNP BN-<NUM> and allele C of SNP BO-<NUM>.

Alternatively, the other SNPs of the invention, namely SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM> and SNP BN-<NUM> can also be used as detailed above.

The selection of the progeny having the desired phenotype can also be made on conditions of A. candida race <NUM> Australian variant infection, as disclosed inter alia in example <NUM> (pathology test).

A plant according to the invention, or grown from a seed as deposited under accession number NCIMB <NUM>, is thus particularly valuable in a marker assisted selection for obtaining commercial broccoli lines and varieties resistant to white rust caused by A. candida race <NUM> Australian variant, as well as progeny thereof bearing the introgressed sequences of the invention conferring the resistance to white blister rust, especially progeny thereof having allele C of SNP BO-<NUM>.

The invention is also directed to the use of said plants in a program aiming at identifying, sequencing and / or cloning the genes conferring the desired phenotype, i.e. resistance to white blister rust caused by the oomycete Albugo candida race <NUM> Australian variant.

Any specific embodiment described for the preceding aspects of the invention is also applicable to this aspect of the invention, especially with regard to the features of the introgressed sequences conferring the resistance.

According to still another non-claimed aspect, the invention also concerns methods or processes for the production of B. oleracea plants, preferably Brassica oleracea var. italica broccoli plants, having the desired phenotype, especially commercial plants.

The present invention is indeed also directed to transferring the introgressed sequences conferring the resistance, from the broccoli plant to other B. oleracea varieties and species, especially to other broccoli species or to cauliflower (B. oleracea convar. Botrytis var. botrytis), Brussels sprouts (B. oleracea convar. Oleracea var. gemnifera), white cabbage, oxheart cabbage (B. oleracea convar. Capitata var. alba) and savoy cabbage (B. oleracea convar. Capitata var. sabauda); the invention is useful for producing new types and varieties of A. candida race <NUM> Australian variant resistant broccoli.

A non-claimed method or process for the production of a plant having these features, i. resistant to A. candida race <NUM> Australian variant, may comprise the following steps:.

wherein SNPs markers are preferably used in steps b) and c), for selecting plants bearing sequences conferring resistance to white rust caused by A. candida race <NUM> Australian variant. The SNP markers are preferably one or more of the <NUM> SNP markers of the invention, and preferably SNP BO-<NUM>. According to a preferred embodiment, the selection is at least partly carried out by detecting the alleles of SNP BN-<NUM> and SNP BO-<NUM>. Alternatively, the selection can be made on the detection of the allele of at least <NUM> SNPs chosen amongst SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM> and SNP BN-<NUM>, preferably at least <NUM> SNPs, for example at least <NUM>, <NUM> or <NUM>, one of them being SNP BO-<NUM>. The selection can also be made on the detection of the alleles of all these <NUM> SNPs. The plant, which is selected at the selection step disclosed above, is preferably selected on the presence of allele C of SNP BO-<NUM>.

Preferably, the susceptible or less resistant B. oleracea plant of step a) is an elite line, used in order to introduce commercially desired traits or desired horticultural traits. It is preferably a broccoli plant.

A method or process for the production of a plant having these features may also comprise the following additional steps:.

The plant selected at step b), c) or g) of the preceding method may be a commercial plant.

Steps d), e) and / f) may be repeated twice or three times or more, not necessarily with the same susceptible B. oleracea plant. Said susceptible B. oleracea plant is preferably a breeding line. This plant is preferably an elite line, used in order to introduce commercially desired traits or desired horticultural traits. It is preferably a broccoli plant.

The self-pollination/crossing and backcrossing steps may be carried out in any order and can be intercalated.

Moreover, these non-claimed methods are advantageously carried out by using SNP markers for one or more of the selection steps for selecting plants bearing the introgressed sequences linked to the resistance, or for selecting plants having the phenotype of interest.

A method for the production of resistant B. oleracea broccoli plants thus advantageously comprises the steps of:.

wherein SNPs markers are used in steps b), c) and/or d) for selecting plants resistant to white rust, as disclosed herein.

The SNP markers are preferably one or more of the <NUM> SNP markers of the invention, and preferably SNP BO-<NUM>. According to a preferred embodiment, the selection is at least partly made on the basis of the allele of SNP BO-<NUM> on chromosome <NUM>. The selection is for example carried out by detecting the alleles carrying/characterized by SNP BN-<NUM> and SNP BO-<NUM>.

The plant selected at the selection step disclosed above is preferably selected on the presence of allele C of SNP BO-<NUM>. In order to identify plants bearing homozygously the introgressed sequences responsible for the resistance, the presence of allele C of SNP BO-<NUM> is detected in combination with the absence of allele A of SNP BO-<NUM>. Alternatively, the other SNPs of the invention can also advantageously be used, namely SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BN-<NUM> and SNP BN-<NUM>, in place or advantageously in addition to SNP BO-<NUM>.

The selection of the progeny having the desired phenotype can also be made on conditions of A. candida race <NUM> Australian variant infection, as disclosed inter alia in example <NUM>.

The method used for allele detection can be based on any technique allowing the distinction between two different alleles of a SNP, on a specific chromosome locus.

Preferably, the plant according to the first aspect of the invention used in step a of the methods is a plant obtained by germinating a deposited seed BROCCO-C4 NCIMB accession number <NUM> or progeny thereof bearing the sequences conferring the resistance to white blister rust, especially progeny thereof having allele C of SNP BO-<NUM>.

According to another non-claimed embodiment, the invention also provides a method for the production of B. oleracea plants, preferably broccoli, resistant to white rust caused by the oomycete Albugo candida race <NUM> Australian variant, comprising the steps of:.

The description also provides, in a non-claimed embodiment, a method for obtaining commercial broccoli plants, resistant to white blister rust caused by A. candida race <NUM> Australian variant, comprising the steps of:.

According to a preferred embodiment of the methods, the selection of a resistant plant is carried out by detection of at least one of the following alleles: allele G of BO-<NUM>, allele C of BO-<NUM>, allele G of BO-<NUM>, allele A of BO-<NUM>, allele C of BO-<NUM>, allele A of BO-<NUM>, allele G of BO-<NUM>, allele C of BO-<NUM>, allele C of BO-<NUM>, allele T of BN-<NUM> and allele G of BN-<NUM>, preferably by detecting allele C of BO-<NUM>. Preferably, the plant according to the first aspect of the invention used in step a1) or a) of these methods is a plant obtained by germinating a deposited seed BROCCO-C4 NCIMB accession number <NUM>, or progeny thereof bearing the sequences conferring the resistance to white blister rust, especially progeny thereof having allele C of SNP BO-<NUM>.

The present invention also concerns a B. oleracea var. Italica broccoli plant obtained or obtainable by one of the methods described above. Such a plant is indeed a B. oleracea var. italica plant having the desired phenotype according to the first aspect of the invention, i.e. resistant to white rust caused by A. candida race <NUM> Australian variant.

The invention is moreover directed to a method for detecting and/or selecting B. oleracea var. italica broccoli plants having introgressed sequences conferring resistance to white rust caused by A. candida race <NUM> Australian variant, on the basis of the allele detection. Such a method is based on the detection of the allele(s) of at least one of the <NUM> SNPs of the invention, especially based on the detection of the allele which is associated with the resistance, for at least one of the SNPs of the invention. In this respect, it is noted that, for the <NUM> SNPs of the invention, detection of the allele associated with the resistance is, on its own, indicative of the resistance phenotype, irrespective of whether the allele associated with the susceptibility is also detected, whereas the absence of detection of the allele associated with the resistance is indicative of susceptibility. The detection of the "susceptible" allele is not informative on its own. The method thus advantageously comprises the detection of whether at least one of the following alleles is present in the plant under test: allele G of BO-<NUM>, allele C of BO-<NUM>, allele G of BO-<NUM>, allele A of BO-<NUM>, allele C of BO-<NUM>, allele A of BO-<NUM>, allele G of BO-<NUM>, allele C of BO-<NUM>, allele C of BO-<NUM>, allele T of BN-<NUM>, and allele G of BN-<NUM>, the detection of at least one of these alleles allowing to conclude that the plant under test is resistant to white rust caused by A. candida race <NUM> Australian variant. Preferably, the method comprises the detection of the alleles of at least one SNP chosen amongst SNP BN-<NUM> and SNP BO-<NUM> on chromosome <NUM>, preferably SNP BO-<NUM>, whereas detection of allele C of BO-<NUM> is indicative of resistance, and/or allele T of SNP BN-<NUM>. The method can be carried out on B. oleracea plants resistant to white rust; the method thus can be used to confirm that such plants comprise in their genome the introgressed sequences according to this invention, and thus have been obtained according to the present invention.

Preferably, plants bearing the introgressed sequences are selected if allele C of SNP BO-<NUM> is detected in a genetic material sample of the plant to be selected. The allele of interest can be present homozygously or heterozygously in the selected plant.

According to a particularly preferred embodiment, the selection is thus made on the simultaneous presence of allele T of SNP BN-<NUM> and allele C of SNP BO-<NUM>. For selection of plants displaying homozygously the phenotype of resistance, the selection is preferably made on the simultaneous detection of allele T of SNP BN-<NUM> and allele C of SNP BO-<NUM> in combination with no detection of any other allele for these SNPs, and especially no detection of allele C of SNP BN-<NUM> or allele A of SNP BO-<NUM>. Conversely, the detection of both alleles of a SNP is indicative of a heterozygous plant with respect to the resistance sequences.

Such a combination of alleles is to be found in plants grown up from to the deposited seeds.

The method of the invention allows selection of plants bearing the resistance sequences homozygously, by detecting plants only displaying the allele associated with the resistance for a given SNP of the invention, and also of plants bearing these sequences heterozygously, by detecting plants displaying both alleles associated with the resistance and with the susceptibility. The method also allows to make a distinction between these two types of resistant plants, those bearing the sequences heterozygously being characterized by the simultaneous presence of both the alleles associated with the resistance and with the susceptibility, for at least one of the SNPs of the invention.

The detection or selection may also comprise the identification of the alleles of at least one of SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BN-<NUM> and SNP BN-<NUM>, or at least <NUM>, <NUM>, or all <NUM> SNPs, or the identification of the alleles of at least one of SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, SNP BO-<NUM>, preferably in addition to SNP BO-<NUM>.

The method of the invention is thus suitable for detecting and/or selecting B. oleracea, especially broccoli plants, having introgressed sequences conferring resistance to white blister rust caused by the oomycete A. candida race <NUM> Australian variant, e.g. as found in the genome of a plant of BROCCO-C4 (NCIMB accession number <NUM>), comprising detection of at least one of the following alleles: allele G of BO-<NUM>, allele C of BO-<NUM>, allele G of BO-<NUM>, allele A of BO-<NUM>, allele C of BO-<NUM>, allele A of BO-<NUM>, allele G of BO-<NUM>, allele C of BO-<NUM>, allele C of BO-<NUM>, allele T of BN-<NUM> and allele G of BN-<NUM>, in a genetic material sample of the plant to be selected and/or detected. Detection of allele C of BO-<NUM> is highly preferred.

The SNPs markers of the invention can also be used to map the resistance locus, such that the smallest possible introgression conferring the phenotype can be identified and/or selected.

In addition to introgression of the sequences conferring resistance, as detailed in the non-claimed methods of the description, these sequences of the invention can also be introduced into B. oleracea background by genetic engineering in order to obtain a commercial B. oleracea plant resistant to white rust caused by A. candida race <NUM> Australian variant. The identification and cloning of the introgressed sequences conferring the desired phenotype, inter alia from the deposit NCIMB <NUM>, can be carried out by the skilled person, on the basis of the sequence information given in the present application and deposited material. The description also concerns the genetic manipulations aiming at cloning the introgressed sequences conferring the phenotype, and/or integrating them into a B. oleracea plant, as well as the plants thus obtained, preferably broccoli plants.

According to a further aspect, the present invention is also directed to hybrid broccoli plants of B. oleracea, obtainable by crossing a resistant plant according to the first aspect of the invention, or a resistant plant obtainable by the methods disclosed above, with a plant of B. oleracea, for example a plant susceptible to white rust caused by A. candida race <NUM> Australian variant, or a plant with a different level of resistance to white rust caused by A. candida race <NUM> Australian variant. The resistant plant is preferably homozygous for the sequences conferring the resistance. If the resistant plant is heterozygous for the sequences conferring the resistance, only half of the progeny will be resistant; a selection step of the plants bearing the sequence conferring the resistance is thus preferably to be added.

A particularly preferred hybrid B. oleracea plant, is a plant which displays male sterility, or any other trait or phenotype of agronomical interest.

The invention also provides a method of protecting a field of B. oleracea plants, namely broccoli plants, against infection by A. candida race <NUM> Australian variant, said method comprising planting seeds according to the invention, or growing plants according to the invention, exhibiting the resistance phenotype.

In a field trial held in Brittany, France, a romanesco Brassica oleracea plant showed resistance under high natural Albugo candida pressure.

The present inventors then decided to try this very same romanesco B. oleracea plant in a field trial in Australia so to check whether the resistance phenotype seen in Brittany could sustain the Albugo candida Australian variant pressure, although romanesco Brassica oleracea plants are not known to be the preferred host of A. candida race <NUM> Australian variant.

The results were positive and it was thought that the resistance determinism to both European and Australian variants was possibly driven by a unique genetic sequence.

Preliminary mapping work together with pathology tests did however show otherwise, as the mapping work on segregating populations identified two different localizations, on two different chromosomes. The genetic sequences conferring the resistance to the Albugo candida Australian variant of race <NUM> did therefore not confer resistance to the European variant. The initially tested romanesco plants probably possessed both sequences.

The present inventors crossed the original romanesco B. oleracea plant with a broccoli inbred line to obtain a first generation hybrid, that was further backcrossed twice to the broccoli inbred line. The plants obtained thereof have been screened through the pathology test described hereunder, with an European A. candida race <NUM> strain and a symptomless plant was kept for the next steps backcross steps. Next generation plants were again tested in pathology test, this time with an Australian A. candida race <NUM> variant.

One of the resistant plants was used in a dihaploid program to produce a resistant inbred line homozygous for the resistance to the Australian A. candida race <NUM> variant. This plant has been further crossed to a susceptible broccoli plant and the resulting hybrid progeny plants BROCCO-C4, harbouring the resistance in a heterozygous way are resistant to the Australian A. candida race <NUM> variant. Seeds thereof were deposited at the NCIMB on <NUM>th July <NUM> under accession number NCIMB <NUM>.

In order to better identify the nature of the resistance to the Australian variant as well as to map the genetic location of this new resistance, a test was set up, made of <NUM> different F2 populations; indeed the original romanesco resistant plants were crossed to <NUM> different susceptible broccoli plants and the resulting F1 plants have been self-pollinated to produce F2 seeds.

An Australian variant A. candida strain has been collected in Templestowe, Australia and is kept in the freezer at -<NUM>. Every <NUM>-<NUM> months, it is periodically multiplied on susceptible varieties : the frozen cotyledons containing the pustules are washed with demineralized water to suspend the spores and the suspension is then vaporized on susceptible plants (cotyledon stage, <NUM> week after sowing) placed on growing chambers (<NUM> during the night, <NUM> during the day, with <NUM> hours for each condition).

The cotyledon with the fresh, new pustules are collected into an Erlenmeyer flask together with <NUM> of distilled water and the solution adjusted at <NUM><NUM> spores/ml in <NUM> of distilled water, thereby creating the inoculum.

The plant material is composed of <NUM> plants per genotype to be tested (i.e. <NUM> plants per F2 population), together with one susceptible and one resistant controls : seeds are sown and placed in greenhouses for about <NUM> weeks, up to the <NUM>-<NUM> leaves stage. Then, the leaves are gently rubbed to remove the leaf wax and the inoculum is sprayed on them. The reading is carried out <NUM> days after the spray, the plants being cultivated in growing chambers (<NUM> during the night, <NUM> during the day, with <NUM> hours for each condition).

The reading is carried out with a <NUM> to <NUM> scale, where "<NUM>" corresponds to leaves with more than <NUM>% of the surface with sporulation, "<NUM>" <NUM>-<NUM>%, "<NUM>" is <NUM> to <NUM>% of sporulation, "<NUM>" is <NUM> to <NUM>%, "<NUM>" is <NUM> to <NUM>% and "<NUM>" is no sporulation. The results are presented in table <NUM>.

Some seeds did not germinate so that the exact number of plants per population was slightly lower than <NUM>.

All results are in favour of a <NUM>:<NUM> segregation ratio demonstrating that the resistance to the A. candida Australian variant acts as a dominant one. The population n°<NUM> shows a slight deviation at <NUM>%, but not at <NUM>% (significance assessed by a Chi-Square test).

A chi-square test was performed using was performed using R statistical software (http://www. org/) to confirm the hypothesis of one dominant resistance gene. The results are presented in table <NUM>.

Each F2 plant was sampled individually and DNA was isolated, using magnetic beads (NucleoMag® <NUM> Plant), according to the protocol of the manufacturer of the beads, Macherey-Nagel.

For each of the <NUM> F2 populations that best followed the <NUM>:<NUM> segregation ratio, i.e. populations n°<NUM>, <NUM>, <NUM> and <NUM>, <NUM> F2 plants were genotyped with <NUM> SNP markers well spread over the B. oleracea genome, using Illumina Veracode Golden Gate technology. For each of the <NUM> populations, among the <NUM> plants, <NUM> of them were randomly taken among the plants scored <NUM>, and <NUM> of them were taken among the plants scored <NUM>. If, for a given population, less than <NUM> plants had been scored <NUM>, all the plants scored <NUM> were genotyped and additional plants were taken among the plants scored <NUM> to make a total of <NUM> susceptible plants genotyped.

A QTL detection was performed across the <NUM> populations (<NUM> x <NUM> = <NUM> individuals in total) using a Kruskal-Wallis test (R statistical software http://www. Results showed that resistance locus was located on chromosome <NUM> (C4) between position <NUM><NUM><NUM> and <NUM><NUM><NUM>, such physical position on the genome being based on the version V2. <NUM> of the Brassica oleracea TO1000 genome published by the European Nucleotide Archive and EnsemblPLants - http://plants. org/Brassica_oleracea/Info/Index.

The results are presented in table <NUM>. The physical position (of the polymorphic nucleotide within the marker) is given with respect to the version V2. <NUM> of the Brassica oleracea TO1000 genome, on chromosome <NUM>. The sequences of the markers are detailed in table <NUM>.

The most tightly linked markers identified were tested on all the plants of four populations. Additional markers, located in the same genome region but initially not in the set of <NUM> SNP used for discovery were also genotyped. F2 population n°<NUM> was not tested in this first phase. A QTL detection was performed for each populations separately using a Kruskal-Wallis test (R statistical software http://www. Table <NUM> shows the -log10 (p-value) value for each marker in one or more populations. NT means 'not tested' in table <NUM>. Results showed that marker BN-<NUM> located at position C4: <NUM><NUM><NUM> was the marker linked the more closely with the resistance, in view of the results obtained for F2 population n°<NUM>.

A second Genotyping experiment was done using KASP™ technology.

<NUM> SNP located between positions C4: <NUM><NUM><NUM> and C4: <NUM><NUM><NUM> were screened for polymorphism among the two parental lines of F2 population n°<NUM>. <NUM> of them showed polymorphism and were further genotyped on the whole F2 population n°<NUM>. F2 population n°<NUM> was thus tested in this <NUM>nd phase. One marker did not show the expected segregation and has thus been discarded from the analysis. Association test was performed using a Kruskal-Wallis test (R statistical software http://www. The results are presented in table <NUM>.

Phase <NUM> of the markers validation allowed refining the right boundary position of the resistance (i. e, downstream boundary of the interval according to coordinates along the chromosome), located on BN-<NUM> at position C4: <NUM><NUM><NUM>.

Phase <NUM> of the markers validation allowed refining the left boundary position of the resistance (i. e, upstream boundary of the interval according to coordinates along the chromosome), located on marker BN-<NUM> at position C4:<NUM><NUM><NUM>.

It is noted that whereas phase <NUM> has been carried out on different populations, phase <NUM> has been carried out on a single population, namely F2 population n°<NUM>.

In order to determine the smallest introgression fragment, imparting the resistance, it must first be determined the highest -log p value, in a given population, and then to set the boundary of the introgression fragment to the nearest SNP showing a decrease in this value, preferably confirmed by results on another population.

The interval containing the resistance is thus spanning a <NUM><NUM><NUM> bp region between positions <NUM><NUM><NUM> and <NUM><NUM><NUM> on chromosome C4.

Genotyping was done using KASP™ technology.

To further fine map the resistance locus, <NUM> SNPs located between positions C4: <NUM><NUM><NUM> and C4: <NUM><NUM><NUM> were screened for polymorphism among the parental lines of the <NUM> F2 populations. Only one of them (marker BO-<NUM> located at position C4: <NUM><NUM><NUM>) was polymorphic (in population n°<NUM>) and was further tested in the whole population n°<NUM>. Association test was then performed for marker BO-<NUM> on population n°<NUM> using a Kruskal-Wallis test (R statistical software http://www. The results are presented in table <NUM>.

Thanks to the study of populations, and the overlap of confidence intervals, the resistance interval boundaries could be set up to: left boundary: C4: <NUM><NUM><NUM>, corresponding to marker BO-<NUM> and right boundary: C4: <NUM><NUM><NUM>, corresponding to marker BN-<NUM>. The interval containing the resistance is thus spanning a <NUM><NUM> bp region between positions <NUM><NUM><NUM> and <NUM><NUM><NUM> on chromosome C4.

The interval containing the resistance is spanning a <NUM><NUM> bp region between positions <NUM><NUM><NUM> and <NUM><NUM><NUM> on chromosome C4. Looking at the reference genome sequence mentioned herebefore, <NUM> genes were identified in this region. Polymorphisms within these genes were studied more closely, after amplification by PCR and sequencing the amplified region. The most promising amplified region was generated by using the following primers:.

PCR amplification was performed on a Applied Biosystems <NUM> Thermal Cycler using the following program:.

Amplification was performed on parental lines of the <NUM> populations (including the resistance source) and one recombinant plant of population n°<NUM>. This plant was scored <NUM> in the pathology test, has a susceptible parent profile upstream the resistance region and a heterozygous profile downstream the resistance region. As it is phenotypically susceptible, the plant is expected to carry the susceptible allele at the resistance gene location.

Sequencing of the amplicons was performed on both strands by GATC Biotech using Sanger sequencing technology. Sequences analysis was performed using BioEdit software before merging both strands sequences. No polymorphism could be identified among the <NUM> susceptible parents and the susceptible recombinant plant. Several nucleotide polymorphisms between the consensus susceptible sequence and the resistant source sequence were identified in the amplified fragment; out of them <NUM> SNPs were selected as particularly robust at distinguishing the resistant parent sequence from the susceptible parents consensus sequence and the reference sequence. The alleles of these markers are reported in table <NUM>.

Genotyping was done using KASP™ technology. Some of the markers were genotyped in the F2 segregating populations n°<NUM> and <NUM>.

Association test was performed on each population separately using a Kruskal-Wallis test (R statistical software http://www. The results are presented in table <NUM>.

The interval containing the resistance is thus with a very high probability in a region between positions <NUM><NUM><NUM> and <NUM><NUM><NUM> on chromosome C4.

Genotyping was done using KASP™ technology. <NUM> different genotypes were sampled and DNA was isolated, using magnetic beads (NucleoMag® <NUM> Plant), according to the protocol of the manufacturer of the beads, Macherey-Nagel. The <NUM> genotypes are constituted by:.

Association test was performed for the marker BO-<NUM> (SEQ ID N°<NUM>) using a Kruskal-Wallis test (R statistical software http://www. A -log10 (p-value) of <NUM> (K statistic = <NUM>) was found. A pearson correlation test was also performed (R statistical software http://www. A correlation coefficient of <NUM> was observed at <NUM>% confidence interval (t statistic = <NUM>, p-value = <NUM>).

These tests have shown a correlation of <NUM>% between the genotype and the phenotype, i.e. all the resistant plants (Initial Romanesco, BROCCO-C4 and the <NUM> resistant lines) exhibit the presence of allele C of BO-<NUM>, whereas all the other plants, susceptible to A. candida race <NUM> Australian variant do not exhibit the presence of this allele, but only allele T of BO-<NUM>.

Marker BO-<NUM> was confirmed as the most predictive SNP for identifying and selecting broccoli plants resistant to A. candida race <NUM> Australian variant.

As detailed in example <NUM>, a resistant plant obtained from an initial cross between a Romanesco B. oleracea plant and an inbred broccoli line, was used in a dihaploid program to produce a resistant inbred line homozygous for the resistance to the Australian A. candida race <NUM> variant. This plant has been further crossed to a susceptible broccoli plant and the resulting hybrid progeny plants BROCCO-C4, harbouring the resistance in a heterozygous way, are resistant to the Australian A. candida race <NUM> variant. Seeds of BROCO-C4 plants, giving rise to hybrid plant bearing the introgressed sequences linked to the resistance were filed at the NCIMB on <NUM>th July <NUM>, under accession number NCIMB <NUM>.

Plants of BROCCO-C4 were genotyped for the markers identified in examples <NUM>-<NUM>. It was confirmed that BROCCO-C4 plants/seeds exhibit all the alleles of the SNPs shown above as linked to the resistance, as detailed in table <NUM>.

These results thus confirm that BROCCO-C4 plants are plants bearing the introgression fragment conferring the resistance to white rust caused by the A. candida race <NUM>, Australian variant.

Different plants, corresponding to <NUM> different genotypes, were grown and inoculated as described in example <NUM>. The tested plants are as follows:.

The resistance assay was repeated <NUM> times, with <NUM> plants for each genotype A-F and in each repetition. The results are reported in table <NUM> below.

As can be deduced from these results, only the plants bearing the introgression fragment of BROCCO C4 on chromosome <NUM> (genotypes C, E and F) display resistance to Albugo candida race <NUM> Australian variant, namely high resistance.

In order to confirm that the plant Tyson, referred to in Minchinton et al, does not possess the introgression fragment of the present invention, the inventors have moreover determined the allele of SNP BO-<NUM> present in Tyson. The plant Tyson is homozygous for the allele A of BO-<NUM>, contrary to the plants of the invention, bearing allele C of this SNP.

Different hybrids, derived from the deposited seeds were obtained and tested in field trials, with other commercial hybrids. The field trials were carried out in autumn of two consecutive years, in Australia. Tables <NUM> (autumn of year N) and <NUM> (autumn of year N+<NUM>) report, for every plants, the total number of plants grown, the number of infected plants with white rust and the percentage of plants infected. Of note, a particularly stringent test of resistance was adopted in these field trials, a plant was indeed considered as being "infected by white rust" when it has a score of strictly less than <NUM> in the pathology test of example <NUM> and only the plants having no spores at all, i.e. highly resistant, were considered as uninfected.

As can be deduced from these tables, the <NUM> hybrids according to the invention are the sole resistant plants, with <NUM>% of the tested plants being uninfected. The tests have been reproduced in different localizations in Australia, for one given hybrid according to the invention: white rust has never been observed for this hybrid, confirming that the resistance to white rust caused by the oomycete Albugo candida race <NUM> Australian variant is indeed a general resistance, which is not strain-dependent.

Claim 1:
A Brassica oleracea var. italica broccoli plant resistant to white blister rust caused by the oomycete Albugo candida race <NUM> Australian variant, having in its genome introgressed sequences conferring the resistance,
wherein said introgressed sequences are located on chromosome <NUM>, within the chromosomal region delimited by SNP BO-<NUM> (SEQ ID No:<NUM>) and SNP BN-<NUM> (SEQ ID No:<NUM>), preferably within the region delimited by SNP BO-<NUM> (SEQ ID No:<NUM>) and SNP BO-<NUM> (SEQ ID N°<NUM>) and are chosen from the introgressed sequences present in the genome of a seed of B. oleracea BROCCO-C4, deposited at NCIMB accession number NCIMB-<NUM>, on chromosome <NUM> and conferring the resistance.