Brassica oleracea plants resistant to diamondback moth

Abstract

The present invention relates to a Brassica oleracea plant that is resistant to the plant pest diamondback moth, Plutella xylostella, and further to the plant pathogen Xanthomonas campestris pathotype campestris. The present invention further relates to the plant and a method for identifying the nucleic acid sequence and allowing to characterize or identify the plant. In particular, the present invention relates to a Brassica oleracea plant that is resistant to diamondback moth, Plutella xylostella, and the plant pathogen Xanthomonas campestris campestris, said resistance to diamondback moth, Plutella xylostella, and said resistance to the plant pathogen Xanthomonas campestris campestris can be obtained from a Brassica oleracea plant deposited under the accession number NCIMB43822.

Description

[Technical field]

[0001] statement The present invention relates to a Brassica oleracea plant that is resistant to the plant pest diamondback moth, Plutella xylostella, and further to the plant pathogen Xanthomonas campestris pv. campestris. The present invention further relates to a method for identifying the plant and a nucleic acid sequence that allows the plant to be characterized or identified. [Background technology]

[0002] Brassica oleracea is a member of the family Brassicaceae or cruciferous plants (Cruciferae). The species has many cultivars, which include several edible crops. The most important of these cultivars are B. oleracea convar. capitata var. alba (white cabbage, pointy-head cabbage), B. oleracea convar. capitata var. rubra (red cabbage), B. oleracea convar. botrytis var. botrytis (cauliflower, romanesco, broccoli), B. oleracea convar. botrytis var. asparagoides (sprouting broccoli), B. oleracea convar. oleracea var. gemnifera (cabbage), B. oleracea convar. oleracea var. gemnifera (Brussels sprouts), B. oleracea convar. capitata var. sabauda (Savoy cabbage), B. oleracea convar. acephela var. sabellica (boerekole), B. oleracea convar. acephela var. gongyloides (kohlrabi), and B. oleracea var. tronchuda syn. Costata (Portuguese cabbage, tronchuda).

[0003] Each of these cultivars has been selected and grown for a specific part of the plant: for example, white and red cabbages have prominent leaves and Brussels sprouts are formed by the axial buds of the Brassica plant, while cauliflower is the white inflorescence meristem and broccoli is formed by the flower head of the plant.

[0004] The wild Brassica oleracea plant is native to Southern and Western Europe. It is tolerant to saline conditions and is almost restricted to coastal areas. Wild cabbage is a biennial plant. In the first year, it forms a rosette of leaves. In the second year, generally after a cold period, it produces a flower stalk about 1-2 meters tall with numerous yellow flowers. From this wild species, a variety of different cultivars have been developed over time by a process of selection and breeding.

[0005] Cultivars of B. oleracea convar. capitata, also known as headed cabbage, have a wide phenotypic range. Flat varieties are more prevalent, but round and pointy headed cabbages also exist. The shape can be bred according to the requirements of a given market. The demand for a particular shape of head cabbage may change over time. Previously, the market generally preferred headed cabbages with large heads, whereas now smaller cabbages are in demand.

[0006] Beyond shape, there is relatively large variation among cultivars in the number of growing days before harvest. Early cultivars and varieties mature in 60 days, while late varieties require 150 days of growth. Varieties may also vary in leaf position, waxiness of the head, head size, head shape, and inner core length.

[0007] Head cabbage is consumed worldwide. In many countries, cabbage is a traditional food and is part of many local cuisines. Head cabbage can be eaten raw, cooked, or soured. The souring process can be either due to fermentation or exposure to acetic acid.

[0008] Head cabbage is a rich source of nutrients. The most important are, among others, glucosinolates and glycosides. These compounds contribute to the characteristic taste of head cabbage. The crop also contains large amounts of vitamins B1, B6, folic acid, vitamin C, vitamin K, and to a lesser extent vitamins B2 and B3.

[0009] The relatively long growing season makes head cabbage more vulnerable to various diseases and disorders. Common pathogens causing diseases in head cabbage include Xanthomonas campestris, Fusarium oxysporum, Albugo Candida, Plasmodiophora brassicae, Mycosphaerella brassicae, and Plutella xylostella.

[0010] The bacterium Xanthomonas campestris pathotype campestris (Xcc) is the main cause of black rot disease in crucifers. Black rot is commonly found in parts of Europe, America, Africa, Asia, Australia, and Oceania. Brassica oleracea is the most economically important host for Xanthomonas, but the bacterium can also infect other crucifers, weeds, and ornamentals.

[0011] Infection with Xanthomonas campestris pathotype campestris generally occurs through natural openings, such as the drainage tissues of the leaf or, in some cases, stomata, or through wounds caused by mechanical damage. After primary infection, the microorganism spreads through the vascular bundles. Infection initially results in small, wilted, V-shaped infected areas that spread inwards from the leaf margins. The bacteria produce a sticky polysaccharide called xanthan, which eventually plugs the vascular tissues in the leaf veins, causing them to disintegrate and turn black. As a result, parts of the leaf wilt, turn yellow, and eventually die.

[0012] Xanthomonas campestris pathotype campestris is a seed-borne disease. It can survive on the seed for many years and infect it during the early stages of plant development. Infection by the microorganism can also occur through infected plant parts, secondary host plants, and irrigation systems.

[0013] Control of the disease through chemicals is not possible. The only means available to combat the disease are the use of disease-free starting material and sanitary measures, such as removal of infected host plants. Disease-free starting material can be obtained by using pathogen-free seeds or by physically treating infected seeds. Genetically resistant plants are highly preferred.

[0014] The diamondback moth (DBM, Plutella xylostella), also known as the cabbage moth, is a pest of Brassica crops. DBM lays eggs on many crops belonging to the Brassicaceae family, including broccoli, cauliflower, Brussels sprouts, Chinese cabbage, kale, and kohlrabi. These plants produce glucosinolates that act as food stimulants for DBM. Other examples of affected crops are rapeseed (Brassica napus) and canola plants (Brassica juncea). However, DBM can also reproduce on weeds.

[0015] This pest occurs naturally in humid subtropical climates. Currently, DBM is present in Japan, China, the Philippines, the United States, Australia, remote areas such as Reunion and New Caledonia, and in colder regions such as Canada. It is particularly problematic in Central and South America, including countries such as Guatemala and Mexico, and South Africa. Due to rising global temperatures as a result of climate change, Plutella xylostella has also spread northward in Europe and can now be found in the Netherlands.

[0016] DBM appears in May or June in the Northern Hemisphere. The moth is grey to brown in colour. It is small, with a wingspan of about 13 mm. It has white to silvery diamond-shaped markings on the wings. The green caterpillar (up to 14 mm long) chews small circular holes in the leaves, often leaving a thin layer of leaves untouched. The caterpillar is very active and mobile. When disturbed in the crop, the moth flies upwards and the caterpillar descends from the leaf on a thin thread.

[0017] DBM is mostly carried between plants by the wind. Although the insects can overwinter, they cannot survive cold winters and must reinvade in the spring in colder regions.

[0018] Eggs may be laid individually or in small groups on stems or along leaf veins, often on the undersides of leaves. When the eggs hatch, the caterpillars begin feeding. In warm, dry weather, the eggs develop into adult moths within 25 days. During moist, cool weather, growth is slower. Females can lay over 150 eggs in a two-week period, and two to five generations are possible per year, so the insects can cause severe problems in a relatively short period of time. Furthermore, Brassica crops are grown worldwide over large areas for year-round production, often as monocultures. Upon infestation, DBM have enough food to multiply and produce large numbers of DBM individuals to infest more plants.

[0019] Infestation by DBM results in the cabbage head formation being disturbed and in the case of cauliflower and broccoli the presence of the larvae in the florets making them unfit for consumption, leading to the discarding of the crop. Frequently, hectares of crops are completely destroyed within a week of infestation. If no action is taken to combat the pest, losses in a given area can easily be 80% and may reach 99% of the crop.

[0020] Insecticides have been used to combat DBM, but they have various drawbacks. In the last century, the moth has developed resistance to various insecticides, such as pyrethroids. It can be expected that the moth will become resistant to compounds to which it is currently sensitive. Another drawback of using insecticides is that these compounds are toxic to beneficial insects, specifically pollinators. Due to stricter regulations on the use of insecticides, the number of chemicals approved for use in agriculture is becoming more and more limited. Furthermore, insecticides need to be actively applied to the crops, which is labor-intensive and expensive, especially since this treatment needs to be repeated every 5 days. Furthermore, the use of chemical insecticides is not permitted for organic food production. There is a high demand for organic food from consumers, and its market share is steadily increasing, making organic production more and more meaningful.

[0021] Currently, there are no commercial cultivars known to have absolute resistance to Plutella xylostella. However, a few commercial cultivars have some degree of resistance to Plutella xylostella. This includes the cabbage cultivar Izalco and related cultivars, such as Escazu. Thus, a need exists for Brassica plants that have natural, genetically encoded resistance to DBM.

[0022] Doubled haploids (DH) can be obtained by androgenesis, the development of an embryo from only male germ cells, or by gynogenesis, where the embryo genome is exclusively derived from female origin. In the case of Brassica, DHs are typically obtained by androgenesis, plants that originate from a single pollen grain and are doubled naturally or artificially to form homozygous diploids. Doubled haploids (DH) allow homozygous plants to be obtained in a single generation. In contrast, conventional inbreeding is slow, taking an average of 6-8 generations to obtain a nearly perfect homozygous plant. Another disadvantage of conventional breeding is that selection in early generations is inefficient due to heterozygosity. With androgenesis, homozygosity can be achieved in one generation, allowing for evaluation and selection of better crosses in a shorter period of time.

[0023] There are three ways by which DH plants can be obtained: elimination of the female genome after fertilization, callus formation from mitotic cells, and microspore embryogenesis.

[0024] Microspore embryogenesis is a technique used to generate pure 100% homozygous lines in a short period of time. During microspore embryogenesis, pollen grains or microspores are reprogrammed and diverted from their original pathway towards embryogenesis. Vacuolated microspores and young bicellular pollen are reprogrammed from the natural gametophyte pathway towards embryogenesis. Most microspores die immediately and some continue to follow a pollen-like development. Only a few microspores are effectively induced to divide and form microspore-derived embryos as a result of the treatment.

[0025] During successful microspore embryogenesis, the microspores swell significantly, their nuclei move towards the cell centre, and the vacuoles break into smaller fragments. The first division of the microspores is symmetrical rather than asymmetrical, similar to that of somatic cells. Spontaneous doubling of the haploid genome can occur at any time during culture, but most frequently occurs during the first division of the embryogenic embryo. Spontaneous doubling is preferred over artificial doubling, e.g. using colchicine, but this depends on the crop and genotype. After the first division, the development of the proembryo forms a complete embryo in about 3-5 weeks, which can be transferred to fresh medium and grown into a plantlet. If the pollen parents are heterozygous, each doubled haploid plant produced is genetically distinct. Because DH plants are 100% homozygous, microspore culture is a powerful tool for generating pure lines that can be used as parental lines in F1 hybrids.

[0026] Molecular markers are used by researchers as tools to introduce desired genes into cultivars. For example, the presence or absence of genes providing resistance to plant pests, such as Plutella xylostella, can be detected using molecular markers. This technique saves time, since laborious insect tests are not necessary to identify resistant plants. Tests with makers can also be performed at all stages of plant development, which is not always the case in reliable disease tests. Furthermore, markers are not dependent on field conditions, where pest pressure may vary from year to year and are therefore generally less susceptible to experimental variations compared to disease tests.

[0027] Developing informative markers can require a significant effort. The nature of the resistance is a factor, including whether the resistance is monogenic or polygenic, dominant or recessive. The availability and quality of insect tests, the size of the test population, and the quality of the reference genome used can also influence how difficult it is to develop a marker for a particular resistance.

[0028] Complex genomes can make it difficult to identify informative markers, such as single nucleotide polymorphism (SNP) markers. Markers used in plant breeding should be closely linked to the locus of interest, preferably at a genetic distance of less than 5 cM, even more preferably adjacent to the gene of interest, and ideally intragenic.

[0029] Advances in molecular genetics are making genome sequencing cheaper, more accessible, and faster. Concomitantly, the increasing availability of high-quality genomic data has led to the development of more informative markers, including SNP markers. For example, AFLP or RAMP makers that were commonly used 15 years ago would not be satisfactory for modern plant breeding companies today. Developing new markers to track traits is far from obvious today, as each new gene-encoded resistance requires a custom approach. [Prior art documents] [Non-patent literature]

[0030] [Non-Patent Document 1] Belser et al., Nature Plants, Volume 4 (2018): 879-887, link: https: / / www.genoscope.cns.fr / exteme / plants / Summary of the Invention [Problem to be solved by the invention]

[0031] It is an object of the present invention to provide, among other objects, a Brassica oleracea plant that is resistant to the plant pest diamondback moth, Plutella xylostella, and additionally to the plant pathogen Xanthomonas campestris pathovar campestris, and to provide, among other objects, means for identifying or characterizing the plant. [Means for solving the problem]

[0032] The above objects, among others, are met by the invention as outlined in the appended claims.

[0033] Specifically, among other objects, the above objects are met by the present invention by providing a Brassica oleracea plant which is resistant to the diamondback moth, Plutella xylostella, and further resistant to the plant pathogen Xanthomonas campestris pathoval campestris, wherein the resistance to the diamondback moth, Plutella xylostella, and the resistance to the plant pathogen Xanthomonas campestris pathoval campestris are obtainable from a Brassica oleracea plant deposited under accession number NCIMB43822 (NCIMB Limited, Craibstone Estate, 35 Ferguson Building, Bucksbum, Aberdeen AB21 9YA, United Kingdom).

[0034] The present inventors have surprisingly identified a genetic source that provides resistance or tolerance to two major pathogens of Brassica oleracea, which not only provides combined resistance or tolerance, but which is stronger or higher than previously observed.

[0035] According to the invention, the Brassica oleracea plant preferably comprises in its genome: - a first genomic region located on chromosome 6 and characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 25, 27, 29 and 31, preferably comprised in the region corresponding to the region between positions 21414859 and 23635544 of the Brassica oleracea HDEM assembly, and / or - a second genomic region located on chromosome 1 and characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5 and 7, preferably comprised in the region corresponding to the region between positions 11035789 and 12032392 of the Brassica oleracea HDEM assembly, and / or - a third genomic region located on chromosome 5 and characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 17, 19, 21 and 23, preferably comprised in the region corresponding to the region between positions 2137498 and 3356008 of the Brassica oleracea HDEM assembly, and / or - a fourth genomic region located on chromosome 2 and characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 9, 11, 13, 15 and 33, preferably included in the region corresponding to the region between positions 1654630 and 3238987 of the Brassica oleracea HDEM assembly; Includes.

[0036] The Brassica oleracea plant as defined above is preferably a Brassica oleracea plant obtained, obtainable or deposited under the accession number NCIMB 43822. Furthermore, the Brassica oleracea plant is preferably cytoplasmic male sterile (CMS) and / or a hybrid.

[0037] Considering the beneficial properties of the Brassica oleracea plant, the present invention also relates to seeds, progeny, edible parts, egg cells, callus, suspension cultures, somatic embryos, clones, embryos or plant parts of the Brassica oleracea plant as defined above.

[0038] The present invention also relates to a method for providing Brassica oleracea as defined above, comprising the steps of: a) a first genomic region as defined above, and / or b) a second genomic region as defined above, and / or c) a third genomic region as defined above, and / or d) the fourth genomic region as defined above; The present invention further relates to a method comprising the steps of introgressing the first, second, third, and / or fourth genomic regions, either simultaneously or separately, into a Brassica oleracea plant that does not contain the first, second, third, and / or fourth genomic regions, preferably by using microspore culture to provide a doubled haploid.

[0039] The Brassica oleracea plant can be readily identified by a method for establishing the presence of a resistance-conferring genomic fragment or resistance-conferring genomic fragment as defined above, the method comprising determining the presence of a nucleic acid sequence or a plurality of sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 in the genome of the Brassica oleracea plant.

[0040] In a further embodiment, the present invention relates to the use of one or more of the genomic sequences comprising one or more DNA sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 for identifying or providing Brassica oleracea plants that are resistant to Plutella xylostella.

[0041] The present invention also relates to the use of one or more of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 for identifying or providing Brassica oleracea plants that are resistant to Plutella xylostella, as well as to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.

[0042] The invention will now be described in more detail in the following examples, in which reference is made to the figures. [Brief description of the drawings]

[0043] [Figure 1] FIG. 1 shows graphs of flow cytometry data; the left side represents haploid (1n) plants, the middle represents diploid or doubled haploid (2n) plants, and the right side represents tetraploid (4n) plants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0044] Working Example Example 1 Breeding scheme for plants with Plutella xylostella resistance including microspore culture step.

[0045] Parent 1

[0046] [Table 1]

[0047] The first cross was between Z343079 and Z340129. Z343079 is a parent line with moderate resistance to Xanthomonas campestris pathovr. campestris. Z340129 is a Bejo parent line with high resistance to Xanthomonas campestris pathovr. campestris. The result of this cross was B2836 F1, a Bejo cultivar with moderate resistance to Xanthomonas campestris pathovr. campestris.

[0048] Cultivar B2836 F1 was used as a donor to create a new breeding population. This was achieved by a self-pollination process (F1S1). The F1S1 population was used to screen for resistance to Xanthomonas campestris pathovar campestris. The best individuals were selected and used for microspore culture (MC) to obtain doubled haploids (DH).

[0049] Microspore culture yielded approximately 450 unique progeny. All 450 individuals were screened and 32 individuals were selected. One of these individuals became Z344539. For further details regarding microspore culture, see Example 4.

[0050] Parent 2 The second parent is Z344099, a line with intermediate resistance to Xanthomonas campestris pathovar campestris. Final process: Z344539×Z344099→200234

[0051] Plant 200234 is a Plutella xylostella resistant plant and has been deposited as NCIMB43822. Its parents Z344539 and Z344099 were selected for their high intermediate resistance to Xanthomonas campestris pathoval campestris. In the process of developing Z344539, microspore culture was used to generate homozygous lines with increased resistance to Xanthomonas campestris pathoval campestris.

[0052] Plant 200234 was developed as a Xanthomonas resistance line. The plants were grown in a field in Guatemala where Plutella xylostella naturally occurs, and it was observed that plant 200234 was not susceptible to this pest. This led to the serendipitous discovery that this line also contained Plutella xylostella resistance.

[0053] Example 2 Field trials to evaluate Plutella xylostella resistance and plants resistant to Plutella xylostella. A full field trial was conducted to evaluate the level of resistance to Plutella present in the newly developed plant 200234. Insect trials were conducted during the rainy season under field conditions in Guatemala, which has a subtropical climate. Plutella xylostella occurs naturally in the region. Trials were not chemically treated. Susceptible varieties were planted as border rows and in rows between trials to increase pest pressure by allowing proliferation and infestation of Plutella xylostella.

[0054] In the experiment, trail plots of susceptible varieties, comparison varieties, and plants according to the invention were grown adjacent to each other in rows. Infestation was natural and not induced. Pest trails were assessed at the moment the crop was considered mature.

[0055] [Table 2]

[0056] The tests were scored using a scale ranging from 1 to 9, with 1 being fully susceptible and 9 being resistant. The Green Boy cultivar was used as a standard for susceptible cultivars. Escazú and Izalco are cultivars known to be resistant to Plutella xylostella. Surprisingly, plant 200234 had a score of 8, making it more resistant to Plutella xylostella than Izalco, which had a score of 7.

[0057] Example 3 Field trials to evaluate resistance to Xanthomonas campestris pathovar campestris. An inoculum of Xanthomonas campestris pathovar campestris was prepared by growing the bacteria on yeast dextrose agar for 3 weeks by incubation at 25°C in the dark. The bacteria were scraped using water and placed in a 1-liter flask. The bacteria were suspended in water for 30 min using a stirring magnet. Concentrations were determined by measuring absorbance with a UV-VIS spectrophotometer (PerkinElmer) and were diluted to 10. 6The bacteria was adjusted to 1000 bacteria / ml. At the end of March, plants were sown on 260 trays. In the second week of May, plants were transplanted to a field in the Netherlands. Renton F1 and Morris F1 were used as susceptible control varieties.

[0058] Starting from the second week of June, plants were inoculated early in the morning when drainage occurs for 10 consecutive weeks. Inoculation is performed by spraying the leaves with a bacterial suspension using an ultra-low volume applicator. Inoculation was performed once a week. The first symptoms can appear about 6 weeks after the first inoculation. Symptoms become more evident during cultivation. When the plants were ready for harvest, evaluation of disease resistance was performed.

[0059] When the plants became overripe, the evaluation was stopped. The scale used ranged from 0 (fully susceptible, severe symptoms) to 9 (highly resistant, no symptoms). The score on this scale is called the disease score.

[0060] [Table 3]

[0061] Example 4 Microspore culture. For the purpose of developing parental lines, doubled haploids were developed using microspore culture and are 100% homozygous lines. Using doubled haploid plants obtained by microspore embryogenesis, it was possible to greatly reduce the breeding time required to introduce the desired traits into the plants by using only naturally occurring doubled haploids. To obtain doubled haploid plants, the procedure outlined below was followed: Breeding lines with high quality and desired traits were selected by the breeders. These plants were maintained and transferred to a greenhouse. See Example 1 for details. · Plants were vernalized at 6°C and grown under controlled conditions, a 16-h photoperiod at 18°C ​​with daily irrigation, until flowering. Once flowering had commenced, closed buds were collected and examined for the developmental stage of microspores within them. Buds that contained late uninucleate stage microspores that were susceptible to reprogramming were used for microspore isolation. Buds were crushed in hormone-free NLN medium and the released microspore suspension was filtered and collected in a 10 ml tube. The suspension was washed three times by centrifugation and diluted to a concentration of 40000msp / ml. Microspores were stressed for 24 hours at 35°C in the dark to switch from the default developmental pathway towards embryogenesis. The microspores were kept in the dark at 25°C and the first embryos appeared after 3 weeks. · Embryos (0.5–0.7 cm) derived from microspores at the late cotyledon stage were transferred to solidified medium containing hormones for further elongation and transferred to a 24°C incubation room with a 16-hour photoperiod. After 7 weeks, direct embryo-to-plant conversion was determined and the ploidy level of the regenerated plantlets was examined using a Partec flow cytometer (Figure 1). · Plants with 2n level (doubled haploid) were transferred to fresh medium for rooting, while haploid (1n), tetraploid (4n) and mixoploid plantlets were discarded. · Rooted DH plants were transferred to a greenhouse for acclimatization and then planted in the field. The best performing plants were selected, selfed, crossed and also examined for their ability as parents in F1 hybrids. One of these individuals was selected as Z344539, parent 1 for Example 1.

[0062] Example 5 Genetic mapping of Plutella xylostella resistance. Group For genetic mapping of Plutella xylostella (DBM) resistance, an F1S1 mapping population was created. The population was constructed from DBM-resistant line A and susceptible line B. F1 cross A * B is allowed to self-pollinated, thus forming the F1S1 population. A population of 432 plants is being tested in a DBM field trial to determine resistance levels.

[0063] Gene Map For genetic mapping, informative SNP markers between line A and line B are selected. These SNP markers are developed for the DNA sequences of line A and B, respectively. A genetic map is constructed using 150 SNP markers.

[0064] QTL mapping The genetic map and DBM score are used together for QTL mapping of the DBM resistance trait. Four QTLs with LOD values ​​higher than the threshold were found in the population.

[0065] In Table 4 below, informative SNPs have been identified for detection of DBM resistance QTL. In Table 5, the sequences of the SNPs from Table 4 are provided. Markers have been validated on accession NCIMB43822 (Brassica oleracea 200234) and these were found to be informative.

[0066] [Table 4]

[0067] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9]

[0068] Abbreviations follow the IUPAC nucleotide code:

[0069] [Table 6]

[0070] Information regarding accession NCIMB43822. The plant according to the present invention, Brassica oleracea 200234, accession NCIMB43822, was deposited on 26 July 2021 to NCIMB Limited, Craibstone Estate, 35 Ferguson Building, Bucksbum, Aberdeen AB21 9YA, United Kingdom. [Accession number]

[0071] NCIMB43822

Claims

1. 1. A Brassica oleracea plant, said plant being resistant to the diamondback moth, Plutella xylostella, said plant being further resistant to the plant pathogen Xanthomonas campestris pathoval campestris, said resistance to the diamondback moth, Plutella xylostella and said resistance to the plant pathogen Xanthomonas campestris pathoval campestris being obtainable from a Brassica oleracea plant deposited under accession number NCIMB43822, preferably said plant being not exclusively obtainable by an essentially biological process.

2. The plant has in its genome: - a first genomic region located on chromosome 6, said first genomic region being characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 25, 27, 29 and 31, preferably said first genomic region being included in the region corresponding to the region between positions 21414859 and 23635544 of the Brassica oleracea HDEM assembly, 2. The Brassica oleracea plant of claim 1.

3. The plant has in its genome: - a second genomic region located on chromosome 1, said second genomic region being characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5 and 7, preferably said second genomic region being comprised in the region corresponding to the region between positions 11035789 and 12032392 of the Brassica oleracea HDEM assembly, 3. A Brassica oleracea plant according to claim 1 or claim 2.

4. The plant has in its genome: - a third genomic region located on chromosome 5, said third genomic region being characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 17, 19, 21 and 23, preferably said third genomic region being comprised in the region corresponding to the region between positions 2137498 and 3356008 of the Brassica oleracea HDEM assembly, 4. A Brassica oleracea plant according to any one of claims 1 to 3.

5. The plant has in its genome: - a fourth genomic region located on chromosome 2, said fourth genomic region being characterized by one or more sequences selected from the group consisting of SEQ ID NOs: 9, 11, 13, 15 and 33, preferably said fourth genomic region being comprised in the region corresponding to the region between positions 1654630 and 3238987 of the Brassica oleracea HDEM assembly, 5. A Brassica oleracea plant according to any one of claims 1 to 4.

6. 1. A Brassica oleracea plant according to claims 1 and 2, or claims 1, 2 and 3, or claims 1, 2 and 4, or claims 1, 2, 3 and 4, or claims 1, 2 and 5, or claims 1, 2, 3 and 5, or claims 1, 2, 4 and 5, or claims 1, 2, 3, 4 and 5.

7. 7. The Brassica oleracea plant according to any one of claims 1 to 6, wherein the plant is obtained, obtainable or is a Brassica oleracea plant deposited under accession number NCIMB43822.

8. 8. The Brassica oleracea plant of claim 1, wherein the plant is cytoplasmic male sterile (CMS).

9. 9. The Brassica oleracea plant of claim 1, wherein the plant is a hybrid plant.

10. 10. A seed, progeny, edible part, egg cell, callus, suspension culture, somatic embryo, clone, embryo or plant part of a Brassica oleracea plant according to any one of claims 1 to 9.

11. 10. A method for providing a Brassica oleracea according to any one of claims 1 to 9, said method comprising: a) a first genomic region as defined in claim 2, and / or b) a second genomic region as defined in claim 3, and / or c) a third genomic region as defined in claim 4, and / or d) a fourth genomic region as defined in claim 5. either simultaneously or separately, into a Brassica oleracea plant not containing said first, said second, said third and / or said fourth genomic region, preferably wherein said method comprises providing a doubled haploid, preferably by using microspore culture.

12. 10. A method for identifying a Brassica oleracea plant that is resistant to Plutella xylostella, the method comprising the step of establishing the presence of a genomic region or a plurality of genomic regions that provide resistance as defined in any one of claims 2 to 6 by determining the presence, in the genome of the plant, of one or more of the corresponding nucleic acid sequences or sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.

13. Use of one or more of the genomic or nucleic acid sequences comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 for identifying or providing a Brassica oleracea plant that is resistant to Plutella xylostella.

14. A nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.

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