brassica napus downy mildew resistance gene
Patent Information
- Application Number
- CN202480086515.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-12-22
- Filing Date
- 2024-12-20
- Publication Date
- 2026-08-28
Smart Images
Figure CN122662736A_ABST
Abstract
Description
[0001] This invention relates to conferring protection against brassinolide (Bacteroides pertussis) Hyaloperonospora brassicae The gene for resistance to ), and cabbage (containing said gene) Brassica oleracea This invention also relates to progeny, seeds, and portions of cabbage plants resistant to Brassica oleracea var. brassicae, and to foods containing portions of such cabbage plants. The invention further relates to propagation materials suitable for producing cabbage plants, to markers for identifying resistant cabbage plants, to the use of said markers for identifying and / or developing cabbage plants resistant to Brassica oleracea var. brassicae or other markers, to methods for selecting cabbage plants with said resistance, and to methods for producing cabbage plants resistant to Brassica oleracea var. brassicae.
[0002] Brassica ( ) Brassica ) is a genus of plants, taxonomically belonging to the Brassicaceae family ( Brassicaceae It contains many important economic crops that serve as food sources, and species are also used for oil production. Generally, the genus *Brassica* includes rapeseed (*Brassica napus*). B. napus ), black mustard ( B. nigra ),turnip( B. rapa ), mustard-type rapeseed ( B. juncea ) and cabbage ( B. oleracea Rapeseed plays a vital role in the production of vegetable oil, which is increasingly used in the fuel industry. Along with turnips, black mustard, and mustard-type rapeseed, cabbage is a species that plays a very important role in human food production. Over time, cultivated varieties originating from cabbage can be found, including head cabbage, Chinese cabbage, cauliflower, kale, broccoli, kohlrabi, and Brussels sprouts.
[0003] Wild cabbage has been bred into a variety of different horticultural cultivars, which still maintain sexual compatibility. However, their appearance does not show a high level of phylogenetic similarity. The wide range of morphological characteristics within cabbage has long been a focus of attention and forms the basis for the uniqueness of cultivars. These characteristics include enlarged inflorescences (cauliflower, broccoli), enlarged stems (Brussels sprouts), enlarged terminal buds (cabbage), and enlarged lateral buds (Brussels sprouts).
[0004] Breeding cruciferous vegetables (such as cabbage varieties) aims to produce commercially viable varieties best suited to local growing conditions, enabling growers to maximize the productivity of high-quality plants. Many characteristics, encompassing both input and output traits, need to be considered during the selection process. In this regard, one of the most important input traits relates to disease resistance, particularly resistance to microorganisms.
[0005] Cabbage plants are susceptible to a variety of pests and diseases. Therefore, these threats are a high priority for cabbage breeders in order to obtain robust and highly resistant crops. If these crops lack resistance, growers must implement agronomic strategies (such as crop rotation) to reduce pest damage in areas where cruciferous vegetables are grown.
[0006] Downy mildew, caused by the oomycete *Brassica oleracea*, is a common and perhaps most destructive disease in cabbage, radish, turnip, and other plants belonging to the Brassicaceae family. Downy mildew can be caused by different isolates of *Brassica oleracea*. This obligate parasite lives on plants of the *Brassica* genus, and symptoms of infection primarily appear on the above-ground parts of the plant. Symptoms such as spore formation and necrotic spots with brown edges can damage the quality of leaves and the top of the plant.
[0007] Downy mildew infection occurs globally and has a wide host range. Cultivational practices and / or chemical treatments have been unsuccessful in protecting crops or have proven too costly. Therefore, developing resistant cultivars is now considered the most cost-effective method for controlling downy mildew.
[0008] For oomycetes like *Brassica oleracea*, they are known to continuously evolve the ability to overcome existing resistance in host plants, making new resistance sites a valuable asset. Therefore, cabbage plants with *Brassica oleracea* resistance are preferred.
[0009] Plants can develop resistance to brassinolide at both the seedling and adult plant stages. If a plant lacks resistance to brassinolide at the seedling stage, the cotyledons or young plants can be infected and exhibit symptoms of susceptible plants. Infection at this stage can lead to the abnormal development of mature plants with heads and / or leaves, and may even result in the death of the infected plant. Infection at the seedling stage thus leads to reduced yield or decreased quality of plant heads or leaves. Therefore, cabbage plants that are resistant to brassinolide at all plant stages (i.e., both seedling and adult stages) are preferred.
[0010] In summary, reliable resistance to *Brassica oleracea* is required at all plant stages. In particular, resistance to multiple isolates of *Brassica oleracea* at all plant stages is preferred.
[0011] The purpose of this invention is to obtain cabbage plants resistant to brassinolide.
[0012] In the research leading to this invention, a novel gene was identified that, when expressed in cabbage plants, confers resistance to downy mildew (…). Hyaloperonospora parasitica Resistance to ).
[0013] Therefore, this invention provides a gene, referred to herein as "the gene of this invention," which encodes a protein that, when expressed in cabbage plants, confers resistance to parasitic downy mildew, wherein said gene comprises:
[0014] a) A nucleotide sequence encoding a protein having an amino acid sequence that, in ascending order of priority, has at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID No. 3; or
[0015] b) A nucleotide sequence containing a coding sequence that, in ascending order of priority, has at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID No. 2; or
[0016] c) A nucleotide sequence that encodes at least amino acids
[58] to
[146] ,
[217] to
[503] , and
[650] to
[679] ,
[819] to
[850] ,
[853] to
[925] ,
[941] to
[972] ,
[989] to
[1029] ,
[1106] to
[1147] , and
[1180] to
[1210] amino acids of SEQ ID No. 3; or
[0017] d) A nucleotide sequence encoding an LRR domain comprising amino acids
[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 in ascending order of priority as specified in SEQ ID No. 3. The amino acid sequences of amino acids
[650] to
[679] ,
[819] to
[850] ,
[853] to
[925] ,
[941] to
[972] ,
[989] to
[1029] ,
[1106] to
[1147] ,
[1180] to
[1210] of 3 have LRR domains with at least 95%, 96%, 97%, 98%, 99%, and 100% sequence similarity.
[0018] The genes of this invention are nucleic acids, particularly nucleic acid molecules, and even more particularly isolated nucleic acid molecules.
[0019] The gene of the present invention comprises a nucleotide sequence encoding a protein having an amino acid sequence having at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence similarity to SEQ ID No. 3 in ascending order of priority.
[0020] Preferably, the gene of the present invention comprises a nucleotide sequence encoding a protein having an amino acid sequence having at least 95% sequence similarity to the amino acid sequence according to SEQ ID No. 3. The nucleotide sequence preferably comprises at least a nucleotide sequence encoding an LRR domain comprising amino acids
[650] to
[679] ,
[819] to
[850] ,
[853] to
[925] ,
[941] to
[972] ,
[989] to
[1029] ,
[1106] to
[1147] , and
[1180] to
[1210] of SEQ ID No. 3.
[0021] In a preferred embodiment, the gene of the present invention comprises a nucleotide sequence encoding a protein having the amino acid sequence according to SEQ ID No. 3.
[0022] The gene of the present invention comprises a coding sequence that, in ascending order of priority, has at least 70%, 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the coding sequence according to SEQ ID No. 2.
[0023] Preferably, the gene of the present invention comprises a coding sequence having at least 85% sequence identity with the coding sequence according to SEQ ID No. 2.
[0024] More preferably, the gene of the present invention comprises a coding sequence having at least 95% sequence identity with the coding sequence according to SEQ ID No. 2. The coding sequence preferably comprises at least a nucleotide sequence encoding an LRR domain, the LRR domain comprising amino acids
[650] to
[679] ,
[819] to
[850] ,
[853] to
[925] ,
[941] to
[972] ,
[989] to
[1029] ,
[1106] to
[1147] , and
[1180] to
[1210] of SEQ ID No. 3.
[0025] In a preferred embodiment, the gene of the present invention comprises the coding sequence according to SEQ ID No. 2.
[0026] The three main identifiable domain categories in the protein encoded by the gene of this invention are: 1) coiled-coil (CC) domain, defined herein as amino acids 58 to 146 of SEQ ID No. 3; 2) nucleotide binding site (NBS) domain, defined herein as amino acids 217 to 503 of SEQ ID No. 3; and 3) leucine-rich repeat (LRR) domain, defined herein as amino acids
[650] to
[679] ,
[819] to
[850] ,
[853] to
[925] ,
[941] to
[972] ,
[989] to
[1029] ,
[1106] to
[1147] , and
[1180] to
[1210] of SEQ ID No. 3.
[0027] In one embodiment, the gene of the present invention comprises a nucleotide sequence encoding a protein whose LRR domain comprises amino acids
[650] to
[679] ,
[819] to
[850] ,
[853] to
[925] ,
[941] to
[972] ,
[989] to
[1029] ,
[1106] to
[1147] , and
[1180] to
[1210] of SEQ ID No. 3.
[0028] In a further embodiment, the gene of the present invention comprises a nucleotide sequence encoding a protein that comprises at least amino acids 58 to 146 of SEQ ID No. 3, amino acids 217 to 503 of SEQ ID No. 3, and amino acids
[650] to
[679] ,
[819] to
[850] ,
[853] to
[925] ,
[941] to
[972] ,
[989] to
[1029] ,
[1106] to
[1147] , and
[1180] to
[1210] of SEQ ID No. 3.
[0029] The gene of the present invention may also 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.
[0030] When present in cabbage plants, the genes of this invention confer resistance to parasitic downy mildew. The parasitic downy mildew resistance conferred by the genes of this invention is inherited in a dominant manner.
[0031] As used herein, sequence identity refers to the percentage of identical nucleotides or amino acids between two sequences after proper alignment. Those skilled in the art understand how to align sequences, for example, using sequence alignment tools such as BLAST®, which can be used for both nucleotide and protein sequences. To obtain the most significant results, the best possible alignment that gives the highest sequence identity score should be obtained. The percentage of sequence identity is calculated by comparing the shortest sequences based on their lengths. In this context, the nucleotide sequence represents a gene containing at least a start codon and a stop codon, or an amino acid sequence encoding a complete protein encoded by such a gene.
[0032] The sequence similarity of amino acid sequences was calculated using EMBOSS stretcher 6.6.0 (www.ebi.ac.uk / Tools / psa / emboss_stretcher) with an EBLOSUM62 matrix, setting the vacancy opening penalty to 12 and the vacancy extension penalty to 2.
[0033] The cabbage plants of this invention are resistant to multiple strains of *Brassica napus* at all plant stages (i.e., cotyledon stage and mature plant stage).
[0034] This invention also relates to cabbage plants, wherein the plants contain the genes of this invention in their genomes. Cabbage plants containing the genes of this invention in their genomes are referred to herein as "plants of this invention".
[0035] In a further embodiment, the plant of the present invention is an agronomically superior plant, preferably a cabbage plant.
[0036] In the context of this invention, an agronomically superior plant refers to a plant with a genotype that has accumulated distinguishable and desirable agronomic traits due to human intervention, enabling producers to harvest commercially significant products. Preferably, the agronomically superior plants of this invention are inbred lines or hybrids.
[0037] As used herein, an inbred line refers to a plant in a population resulting from three or more rounds of self-pollination or backcrossing; or the plant is a double haploid plant. An inbred line can be, for example, a parent line used for the production of commercial hybrids.
[0038] As used herein, a hybrid plant is the result of crossing two different plants with different genotypes. More specifically, a hybrid plant is the result of crossing two different inbred lines of plants. Such a hybrid plant can be, for example, an F1 hybrid variety.
[0039] This invention relates to species of cabbage affected by Brassica downy mildew infection. Cabbage is a plant species comprising many common cultivars. This invention relates to the following cultivars of cabbage:
[0040] var. headless DC. (Kale)
[0041] var. botrytis L. (cauliflower, Romanesco broccoli)
[0042] var. capita L. (Red cabbage, white cabbage, wrinkled cabbage)
[0043] var. gemmifera DC. (Brussels sprouts)
[0044] var. gongylodes L. (Brassica oleracea var. rubra)
[0045] var. italic Plenck. (broccoli, calabrese)
[0046] var. sabauda L. (wrinkled cabbage)
[0047] var. sabellic (Kale)
[0048] var. Selensia (Borecole)
[0049] var. albiflora Sun [= B. alboglabra] (Chinese kale)
[0050] var. white-haired [= B. alboglabra] (Chinese kale)
[0051] var. Chinese Prain (Burmese wild rapeseed (burma sarson))
[0052] var. fimbriated Mill. (Kitchen kale)
[0053] var. fruit-bearing Metz. (Thousand-headed kale)
[0054] var. trunk LH Bailey (tronchuda cabbage)
[0055] var. rib (Portuguese cabbage)
[0056] var. medullary (Pithed kale)
[0057] var. palm leaf (Kale, Jersey kale)
[0058] var. ramona (Thousand-headed kale)
[0059] The preferred varieties are broccoli, cauliflower, romaine cauliflower, red cabbage, white cabbage, curly cabbage, and kohlrabi.
[0060] Genes containing the present invention Brassica oleracea convar . botrytis var. italica The seeds are deposited in NCIMB with accession number NCIMB 43592. Therefore, the present invention also relates to plants grown from seeds deposited with NCIMB accession number NCIMB 43592.
[0061] Another aspect of the invention relates to seeds capable of growing into the cabbage plant of the invention, wherein said plant contains the genes of the invention. The invention also relates to the use of said seeds for producing the cabbage plant of the invention by cultivating said seeds into a cabbage plant.
[0062] The present invention also relates to propagation materials suitable for producing the plants of the present invention, wherein the propagation materials are suitable for sexual reproduction, particularly selected from microspores, pollen, ovary, ovule, embryo sac and egg cell, or suitable for vegetative reproduction, particularly selected from cuttings, roots, stem cells and protoplasts, or suitable for tissue culture of regenerable cells or protoplasts, particularly selected from leaves, pollen, embryo, cotyledons, hypocotyl, meristematic cells, roots, root tips, anthers, flowers, seeds and stems, wherein the propagation materials contain the genes of the present invention.
[0063] This invention also relates to the cells of Brassica plants resistant to Brassica downy mildew according to the present invention. These cells may be in isolated form or part of a whole plant or its components, and still constitute the cells of the present invention because they contain the genes of the present invention. Each cell of the Brassica plant resistant to Brassica downy mildew according to the present invention carries the genes of the present invention. The cells of the present invention may also be regenerative cells capable of regenerating new plants of the present invention.
[0064] This invention also relates to plant tissues of the plant of the invention that contain the genes of the invention. This tissue can be undifferentiated or differentiated. Undifferentiated tissues are, for example, shoot tips, anthers, petals, or pollen, and can be used for micropropagation to obtain new plants that grow into new plants of the invention. This tissue can also be formed from cells of the invention.
[0065] The present invention also relates to a method for producing plants containing the genes of the present invention by means of tissue culture or by means of vegetative propagation, the plants being resistant to brassinolide.
[0066] Furthermore, this invention relates to the offspring of the plants, cells, tissues, or seeds of the present invention, which contain the genes of the present invention. Such offspring can themselves be plants, cells, tissues, or seeds. In particular, the offspring can be offspring of the plants of the present invention, whose representative seeds are deposited under NCIMB number 43592. As used herein, offspring include the first generation hybridized with the plants of the present invention and all further offspring, wherein hybridization includes hybridization with itself or with another plant, and wherein the offspring identified as offspring contain the genes of the present invention. Offspring can be obtained through self-pollination and / or further hybridization of the deposit. Offspring also encompass material obtained through vegetative propagation or other forms of proliferation.
[0067] This invention also relates to germplasm of the plants of this invention. Germplasm comprises all the genetic characteristics of an organism and, according to the invention, at least covers the resistance traits of this invention. This germplasm can be used in breeding programs to develop cabbage plants exhibiting resistance to Brassica downy mildew. The use of germplasm containing genes of this invention in breeding is also part of this invention.
[0068] This invention also relates to the use of the genes of this invention for producing cabbage plants resistant to Brassica downy mildew. The plant is preferably a plant belonging to the group consisting of broccoli, cauliflower, romaine broccoli, red cabbage, white cabbage, curly cabbage, and kohlrabi. This use does not imply that the resulting cabbage plants are obtained through hybridization and selection of the genes of this invention.
[0069] This invention also relates to markers for identifying brassinolide resistance in cabbage plants, the markers comprising single nucleotide polymorphisms (SNPs). The use of markers for identifying brassinolide resistance in cabbage plants is also part of this invention. Any of these markers may also be used to develop other markers to identify genes of this invention present in cabbage plants, the use of which is also part of this invention.
[0070] The present invention also relates to molecular markers and the use of molecular markers detected by the identification methods for molecular markers described herein.
[0071] The present invention also relates to the use of the plant of the present invention as a crop, as a source of seeds, or as a source of propagation material.
[0072] The present invention also relates to a method for identifying cabbage plants resistant to brassinolide, wherein the method comprises the following steps:
[0073] a) Detecting the gene of the present invention in the plant genome, and / or
[0074] b) Testing whether plants containing the genes of the present invention exhibit resistance to brassinolide. Optionally, the method for identifying cabbage plants resistant to brassinolide includes the step of detecting unique polymorphisms in the genes of the present invention.
[0075] This invention also relates to a method of seed production, comprising growing a cabbage plant from seeds of the invention that contain the genes of the invention in a homozygous state, causing the cabbage plant to produce seeds, and harvesting the seeds. Seed production is suitably accomplished by self-pollination or by hybridization with another plant (optionally also a plant of the invention). As described herein, plants grown from the produced seeds are resistant to Brassica downy mildew.
[0076] The present invention also relates to a method for producing hybrid seeds, comprising crossing a first cabbage parent plant with a second cabbage parent plant and harvesting the resulting hybrid seeds, wherein the first parent plant and / or the second parent plant are plants of the present invention containing the genes of the present invention. Preferably, at least one parent plant contains the genes of the present invention in a homozygous state.
[0077] The present invention also relates to hybrid seeds produced by the methods described herein and hybrid plants grown from said hybrid seeds.
[0078] The present invention also relates to a method for growing cabbage plants resistant to brassinolide, comprising the step of planting seeds containing the gene of the present invention, a representative sample of which is deposited in NCIMB with accession number NCIMB 43592.
[0079] The plants of the present invention can also be produced using transgenic techniques for transferring nucleotide sequences between sex-incompatible plants by transferring the genes of the present invention from one species to another. Suitable techniques include general plant transformation techniques known to those skilled in the art, such as those using Agrobacterium (…). Agrobacterium Transformation methods mediated by [the genome editing process] can also be used. Genome editing methods, such as those using the CRISPR / Cas system, can also be employed to obtain the plants of this invention. Preserved cabbage plants or their progeny are suitable sources of the modified genes.
[0080] The introduction of the gene of the present invention can also be achieved through introgression from a cabbage plant containing the gene (e.g., from a cabbage plant preserved with representative seeds under NCIMB 43592, or from its progeny, or from another pair of plants resistant to Brassica oleracea and in which the gene of the present invention has been identified). Breeding methods such as hybridization and selection, backcrossing, recombination selection, or other breeding methods that result in the transfer of the genetic sequence from a resistant plant to a susceptible plant can be used. The resistant plant can be the same species or different species and / or wild species. Difficulties in interspecific hybridization can be overcome by techniques known in the art, such as embryo rescue or by applying homologous transformation. The progeny of the preserved plant can be its sexual or asexual offspring, which can be self-pollinated and / or hybridized, and can be F1, F2, or further generations, as long as the progeny of the preserved plant still contains the modified gene of the present invention as present in the preserved seed. Cabbage plants produced by this method are also part of the present invention.
[0081] The present invention also relates to a method for producing cabbage plants exhibiting resistance to brassinolide, comprising the following steps:
[0082] a) Cross the first parent plant containing the gene of this invention with the second parent plant to obtain the F1 population;
[0083] b) Optionally, one or more rounds of self-pollination and / or hybridization with plants from the F1 population may be carried out to obtain further generations;
[0084] c) Select plants containing the gene from further generations as resistant plants.
[0085] The present invention also relates to a method for producing cabbage plants resistant to brassinolide, the method comprising:
[0086] a) Hybridize a first parent plant containing the gene of this invention with a second parent plant that does not contain the gene of this invention;
[0087] b) Backcross the plants produced in step a) with the second parent plant for at least three generations;
[0088] c) Select from the third backcross population or higher backcross populations a plant that contains at least the gene of the present invention of the first parent plant from step a) as a plant resistant to Brassica downy mildew.
[0089] The present invention further provides a method for introducing another desired trait into cabbage plants resistant to brassinolide, comprising:
[0090] a) Crossing a plant containing the gene of the present invention with a second plant containing other desired traits to produce F1 offspring;
[0091] b) Optionally select plants in F1 that contain resistance and other desired traits;
[0092] c) Cross the randomly selected F1 offspring with one of the parents for at least three generations to produce backcross offspring;
[0093] d) Select backcross progeny that contain resistance and other desired traits; and
[0094] e) Optionally repeat steps c) and d) once or more to produce selected fourth-generation backcross progeny or higher backcross progeny containing resistance and other desired traits.
[0095] Optionally, a self-pollination step may be performed after any hybridization or backcrossing step of the methods described above. Selection of cabbage plants containing brassica downy mildew resistance and other desired traits may alternatively be performed after any hybridization or self-pollination step of the methods described above. Other desired traits may be selected from, but are 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; tolerance to water stress and heat stress; and male sterility.
[0096] This invention relates to the use of the cabbage plant of this invention for transferring Brassica downy mildew resistance to another Brassica plant.
[0097] This invention relates to the use of seeds deposited in NCIMB with accession number NCIMB 43592 for transferring resistance to brassinolide to another cabbage plant.
[0098] The present invention also relates to the harvested portion or part thereof of the cabbage plant of the present invention, and to food products (whether in natural form or optionally processed form) comprising the harvested portion or part thereof of the cabbage plant of the present invention.
[0099] The harvested portion or food can be or includes the cabbage head, flower head, stem, leaves, roots, buds, seeds, or any other part of the cabbage plant. The harvested portion can also be used to produce biofuel. The food or harvested portion may have undergone one or more processing steps. Such processing steps may include, but are not limited to, any one or a combination of the following treatments: cutting, washing, cooking, steaming, baking, frying, pasteurizing, freezing, grinding, oil extraction, pickling, or fermentation. The resulting processed form is also part of this invention.
[0100] This invention also relates to the use of the plants of this invention in plant breeding. Therefore, this invention also relates to a breeding method for developing cultivated Brassica plants resistant to Brassica downy mildew, wherein the resistance is conferred on another Brassica plant using a plant containing the genes of this invention. Representative seeds of the Brassica plant that can be used for plant breeding to develop another Brassica plant with Brassica downy mildew resistance are deposited in NCIMB with accession number NCIMB 43592.
[0101] As used herein, "resistance to Brassica downy mildew," "resistance against Brassica downy mildew," or "resistance of the present invention" is defined as the ability of a plant to resist infection by one or more strains of Brassica downy mildew at all plant stages. Brassica downy mildew-resistant plants are identified by visual phenotype. Resistance is appropriately graded according to the ratings shown in Table 2, with plants of the present invention preferably rated from 1 to 3 for resistance to Brassica downy mildew. In bioassays, resistant Brassica plants exhibit clean cotyledons, optionally with a few or many necrotic spots, but without any spore formation on the cotyledons.
[0102] As used herein, “increased resistance” in a cabbage plant refers to a lower score, as shown in Table 2, than that of a non-resistant cabbage plant compared to it. Non-resistant plants are plants that do not contain the genes of the present invention. Plants with increased resistance exhibit fewer necrotic spots and / or spore formation compared to their comparison plants, and preferably none at all.
[0103] As used herein, the marker is genetically linked to the gene of the present invention, and therefore can be used to identify the gene when the marker and trait co-segregate in a segregating population resulting from a cross between a plant containing the gene of the present invention and a plant not containing the gene.
[0104] As used herein, "offspring" is intended to refer to the first generation and all further offspring of hybridization with plants of the present invention. "Offspring" also encompasses cabbage plants carrying the genes of the present invention and possessing the traits of the present invention, as well as cabbage plants obtained through vegetative propagation or vegetative proliferation from other plants of the present invention or the offspring of plants of the present invention. Offspring of the present invention contain the genes of the present invention and exhibit resistance to downy mildew.
[0105] As used herein, the term "trait" is intended to refer to the resistance phenotype of cultivated cabbage plants. In particular, the term "trait" refers to the trait of the present invention, and more specifically, resistance to *Persistentia brassicae*. When a cultivated cabbage plant exhibits the trait of the present invention, its genome contains the genes of the present invention that lead to the trait of the present invention. Therefore, the cultivated cabbage plant contains the genes of the present invention. Therefore, as used herein, "trait of the present invention" or "phenotype of the present invention" is intended to refer to the trait of resistance to *Persistentia brassicae*.
[0106] As used herein, "plant of the present invention" is defined as a cabbage plant resistant to Brassica oleracea var. brassicae. The plant of the present invention contains the genes of the present invention that confer resistance to Brassica oleracea var. brassicae. In the context of this invention, when referring to cabbage plants, unless otherwise stated, it means cultivated cabbage plants.
[0107] As used herein, “wild-type plant” is defined as a cabbage plant that does not contain the genes of the present invention that confer resistance to brassinolide and is therefore susceptible to brassinolide.
[0108] The phrase “exists in” can also mean “found in”, “contains in”, or “available from” the genome of a plant grown from a preserved seed or the preserved seed itself. These phrases are intended to indicate that the gene of the present invention is substantially the same as or identical to the gene in the genome of the preserved seed. The gene does not need to be exactly the same and may contain polymorphisms (i.e., sequence variations) compared to the gene of the present invention, but these polymorphisms do not affect the function of the gene in inducing downy mildew resistance in brassica.
[0109] The present invention will be further illustrated by the following embodiments, which are for illustrative purposes only. These embodiments are not intended to limit the invention in any way.
[0110] preservation
[0111] Broccoli containing a modified gene located on chromosome 2 that confers resistance to brassica downy mildew ( Brassica oleracea var. italic Seeds of the plant *Plenck* were deposited on April 7, 2020, at NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA) under accession number NCIMB 43592. All deposited seeds contain the modified gene on chromosome 2 in a homozygous state. Plants grown from these seeds therefore possess resistance to *Brassica napus* downy mildew.
[0112] The preserved seeds do not meet the DUS criteria required for obtaining plant variety protection and therefore cannot be considered as plant varieties. Attached Figure Description
[0113] Figure 1 :
[0114] This figure shows the QTL mapping results for *Bacillus brassicae* as described in Example 2. Each group represents the nine chromosomes present in the cabbage. The X-axis represents the relative position (cM) on the respective chromosomes of the population, and the Y-axis represents the LOD score. The figure clearly shows that the mapping results yielded a QTL located on chromosome 2.
[0115] Sequence information
[0116] Table 1: Sequences
[0117]
[0118]
[0119]
[0120]
[0121]
[0122] Example
[0123] Example 1
[0124] Brassica napus downy mildew resistance test
[0125] Bioassays were conducted to test the resistance of cabbage plants to brassinolide. Approximately 20 seeds were sown per plot, with one empty row between plots. Cabbage plants were grown under standard Dutch greenhouse conditions with a temperature regime of 15°C / 15°C (night / day). Ten days after sowing, cotyledons were sprayed with a suspension of sporangia from brassinolide isolates. The brassinolide isolates were maintained on live brassinolide-susceptible cabbage plants. The inoculated cotyledons were cultured under controlled conditions, i.e., a 12°C / 14°C (night / day) regime. At 7 and 14 days post-inoculation, each plant was visually scored according to Table 2 to phenotypically identify resistant cabbage plants to brassinolide. This screening identified several resistant cabbage plants to brassinolide, one of which was selected for further study. This is broccoli (… Brassica oleracea (var. italica) plant.
[0126] Table 2. Overview of infection symptoms and classification stages of Brassica oleracea plants infected with downy mildew (grades 1-9, grayed out)
[0127]
[0128] The selected plants were tested using a combined screening of 15 different *Brassica napus* isolates collected from different locations around the world. The plants selected for this invention were rated as resistant to 8 of the 15 isolates tested. Considering that these isolates were collected from very different countries and regions, these results indicate that the plants of this invention possess a sufficient resistance phenotype.
[0129] When comparing the phenotypes of several strains against the two important isolates in the above tests, the plants of the present invention were shown to be resistant to both isolates (strain 2 in Table 3 below). Other strains showed resistance to at least one of the isolates.
[0130] Table 3
[0131] Phenotypic analysis of two different brassinomenium isolates
[0132]
[0133] Example 2
[0134] QTL plotting
[0135] Input data included genetic marker data and phenotypic measurements of resistance / susceptibility to downy mildew (Brassica napus). Data were derived from populations resulting from crosses between two broccoli parents (one susceptible, one resistant to Brassica napus).
[0136] A total of 162 markers (out of 786 analyzed) were informative for trait mapping, and linkage analysis was performed to construct population-specific maps. QTL mapping was performed using MapQTL 6.0. First, interval mapping was performed, followed by auxiliary factor selection and MQM mapping. A single QTL was detected at approximately 34 cM on chromosome 2, explaining up to 71.6% of the trait variance.
[0137] The location of resistance to parasitic downy mildew is supported by previous results of broccoli and cauliflower hybridization (data not shown), in which the optimal linkage marker is located at 32 cM.
[0138] In summary, these data support the single-gene control of this trait, located on chromosome 2.
[0139] Example 3
[0140] Identification of genes conferring resistance to parasitic downy mildew
[0141] Since the above demonstrates the single-gene dominant nature of resistance, it was hypothesized that the existence of so-called R genes could explain the nature of resistance. QTL analysis and marker development indeed showed that the QTL is located on chromosome 2. All R genes present on chromosome 2 in seven different genome assemblies were analyzed and compared. These genome assemblies were as follows:
[0142] 1. Resistant broccoli containing the gene of the present invention ( Brassica oleracea var. italic )strain
[0143] 2. Proprietary susceptible broccoli ( Brassica oleracea var. italic )strain
[0144] 3. To1000, susceptible cabbage plants with publicly available genomes
[0145] 4. Proprietary susceptible cauliflower ( Brassica oleracea var. botrytis )strain
[0146] 5. Specific susceptible cabbage heads ( Brassica oleracea var. capita )strain
[0147] 6. Proprietary susceptible bulb broccoli ( Brassica oleracea var. gongylodes )strain
[0148] 7. Specific susceptible cabbage heads ( Brassica oleracea var. capita )strain
[0149] NLRome (pan-genome approach) analysis was performed using all seven assemblies mentioned above as input to determine whether candidate genes were truly unique. The plant pan-NLRome aims to fully capture the intraspecific diversity of the highly variable NLR immune receptor, enabling a systematic analysis of NLR genes and alleles and their roles in disease resistance. The analysis emphasized candidate genes falling into clusters containing 12 genes. Within these clusters, one or more genes from six of the seven assemblies also fell into the clusters. The first column refers to the aforementioned assemblies.
[0150] Table 4
[0151] Gene Overview
[0152]
[0153] Because the percentages were all low, it was concluded that the candidate gene was indeed unique. It was also concluded that the gene was indeed expressed.
[0154] Example 4
[0155] Transferring downy mildew resistance genes from brassica plants to susceptible cabbage plants
[0156] Furthermore, the segregation phenotype of resistance was studied. The resistance identified above was... Brassica oleracea var. italic Broccoli lines were crossed with susceptible cabbage lines. These are proprietary cauliflower, kohlrabi, and broccoli lines. Data were collected from the F2 population and from populations composed of backcrossed inbred lines. As shown in Table 5 below, the observed distributions of all hybrids did not differ significantly from the expected distributions based on the chi-square test (data not shown). The table shows the types of susceptible cabbage.
[0157] The following results show that the downy mildew resistance gene of the present invention has been successfully transferred into susceptible cabbage plants. It also shows that when the gene of the present invention is introgressed into any background, the observed segregating phenotypes in all hybrids are identical, i.e., 3:1. This confirms that the trait of the present invention is a single gene and dominant.
[0158] Table 5. Segregation of resistance phenotypes during gene transfer of the present invention
[0159] PCT / RO / 134 form
Claims
1. A gene encoding a protein, said protein being found in cabbage ( Brassica oleracea When expressed in plants, it confers resistance to parasitic downy mildew ( Hyaloperonospora parasitica Resistance to ), wherein the gene comprises: a) A nucleotide sequence encoding a protein having an amino acid sequence that has at least 70% sequence similarity to SEQ ID No. 3; or b) A nucleotide sequence containing a coding sequence having at least 70% sequence identity with SEQ ID No. 2; or c) A nucleotide sequence that encodes at least amino acids [58] to [146], [217] to [503], and [650] to [679], [819] to [850], [853] to [925], [941] to [972], [989] to [1029], [1106] to [1147], and [1180] to [1210] amino acids of SEQ ID No. 3; or d) A nucleotide sequence encoding an LRR domain comprising amino acids [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 having at least 95% sequence similarity to amino acids [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 in ascending order of priority.
2. The gene according to claim 1, wherein a) The nucleotide sequence encodes a protein comprising an amino acid sequence, wherein the amino acid sequence has at least 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID No. 3 in ascending order of priority; or b) The coding sequence of the gene has at least 75%, 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID No. 2 in ascending order of priority; or c) The nucleotide sequence of the gene, wherein the LRR domain encoding amino acids [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 at least 96%, 97%, 98%, 99%, and 100% sequence similarity, in ascending order, to the amino acid sequence of amino acids [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 according to claim 1 or 2.
4. Use of the gene described in claim 1 or 2 for producing cabbage plants resistant to parasitic downy mildew.
5. A method for identifying cabbage plants resistant to downy mildew, wherein the method comprises the following steps: a) Detecting the gene described in claim 1 or 2 in the genome of a cabbage plant, and / or b) Test whether the cabbage plant containing the gene of claim 1 or 2 exhibits resistance to parasitic downy mildew.
6. The method of claim 5, wherein the method includes, between step a) and step b), a step of detecting a unique polymorphism in the gene of claim 1 or 2.
7. A cabbage plant comprising the gene of claim 1 or 2, wherein the plant is resistant to parasitic downy mildew.
8. The cabbage plant according to claim 7, wherein the plant is an agronomically superior plant, particularly a hybrid variety or inbred line.
9. The cabbage plant according to any one of claims 7 and 8, wherein the plant is broccoli, cauliflower, Romanesco broccoli, red cabbage, white cabbage, curly cabbage or kohlrabi.
10. A seed capable of growing into the plant described in any one of claims 7-9.
11. Reproductive material suitable for producing the plant of any one of claims 7 to 9, wherein the reproductive material is suitable for sexual reproduction, particularly selected from microspores, pollen, ovary, ovule, embryo sac and egg cell, or suitable for vegetative reproduction, particularly selected from cuttings, roots, stem cells and protoplasts, or suitable for tissue culture of regenerable cells or protoplasts, wherein the regenerable cells or protoplasts are particularly selected from leaves, pollen, embryo, cotyledons, hypocotyl, meristematic cells, roots, root tips, anthers, flowers and stems, and wherein the reproductive material contains the gene of claim 1 or 2.
12. A method for selecting cabbage plants resistant to downy mildew, comprising identifying the presence of the gene of claim 1 or 2, optionally testing the resistance of the cabbage plant to downy mildew, and selecting plants containing the gene, wherein the presence of the gene in the plant confers resistance to downy mildew.
13. A method for producing cabbage plants exhibiting resistance to parasitic downy mildew, comprising the steps of: a) Crossing a first parent plant containing the gene described in claim 1 or 2 with a second parent plant to obtain an F1 population; b) Optionally, one or more rounds of self-pollination and / or hybridization with plants in the F1 population may be performed to obtain populations for further generations; c) Select plants containing the gene described in claim 1 or 2 from the population of the further generations as resistant plants.
14. A method for producing hybrid seeds resistant to downy mildew, comprising the steps of hybridizing a first parent plant with a second parent plant, wherein one or both parent plants are homozygous for the gene described in claim 1 or 2, and harvesting the hybrid seeds.
15. Hybrid seeds produced by the method of claim 14.
16. A plant grown from the hybrid seed of claim 15.
17. A marker for identifying the gene of claim 1 or 2, wherein the marker comprises an SNP.
18. The use of the marker of claim 17 for identifying plants containing the gene of claim 1 or 2.