Brassica Oleracea Plants Resistant to Diamondback Moth

Abstract

The present invention relates to Brassica oleracea plants resistant to the plant pest diamondback moth Plutella xylostella and, additionally, tolerant to the plant pathogen Xanthomonas campestris pv. campestris. The present invention further relates to methods for identifying the present plants and nucleic acid sequences allowing characterizing, or identifying, the present plants. Specifically, the present invention relates to Brassica oleracea plants resistant to the diamondback moth Plutella xylostella and the plant pathogen Xanthomonas campestris campestris, wherein said resistance to the diamondback moth Plutella xylostella and said tolerance to the plant pathogen Xanthomonas campestris campestris are obtainable from a Brassica oleracea plant deposited under deposit number NCIMB 43822.

Claims

1. Brassica oleracea plant wherein said plant is resistant to the diamondback moth Plutella xylostella, wherein said plant is further tolerant to the plant pathogen Xanthomonas campestris pv. campestris, and wherein said resistance to the diamondback moth Plutella xylostella and said tolerance to the plant pathogen Xanthomonas campestris pv. campestris are obtainable from a Brassica oleracea plant deposited under deposit number NCIMB 43822.

2. The Brassica oleracea plant according to claim 1, wherein said plant further comprises in its genome: a first genomic region located on chromosome 6, said first genomic region is characterized by one or more sequences selected from the group consisting of SEQ ID Nos. 25, 27, 29, and 31.

3. The Brassica oleracea plant according to claim 1, wherein said plant comprises in its genome: a second genomic region located on chromosome 1, said second genomic region is characterized by one or more sequences selected from the group consisting of SEQ ID Nos. 1, 3, 5, and 7.

4. The Brassica oleracea plant according to claim 1, wherein said plant further comprises in its genome: a third genomic region located on chromosome 5, said third genomic region is characterized by one or more sequences selected from the group consisting of SEQ ID Nos. 17, 19, 21, and 23.

5. Brassica oleracea plant according to claim 1, wherein said plant further comprises in its genome: a fourth genomic region located on chromosome 2, said fourth genomic region is characterized by one or more sequences selected from the group consisting of SEQ ID Nos. 9, 11, 13, 15, and 33.

6. (canceled)

7. The Brassica oleracea plant according to claim 1, wherein said plant is obtained, obtainable, or is a Brassica oleracea plant deposited under deposit number NCIMB 43822.

8. The Brassica oleracea plant according to claim 1, wherein said plant is cytoplasmic male sterile (CMS).

9. The Brassica oleracea plant according to claim 1, wherein said plant is a hybrid plant.

10. Seeds, progeny, edible parts, egg cells, callus, suspension culture, somatic embryos, clones, embryos, or plant parts of a Brassica oleracea plant according to claim 1.

11. A method for providing a Brassica oleracea according to claim 1, wherein the method comprises the step of introgressing, either simultaneously or separately: a) one or more sequences selected from the group consisting of SEQ ID Nos. 25, 27, 29, and 31; and/or b) one or more sequences selected from the group consisting of SEQ ID Nos. 1, 3, 5, and 7, and/or c) one or more sequences selected from the group consisting of SEQ ID Nos. 17, 19, 21, and 23, and/or d) one or more sequences selected from the group consisting of SEQ ID Nos. 9, 11, 13, 15, and 33, into a Brassica oleracea plant not comprising the one or more sequences.

12. A method for identifying a Brassica oleracea plant being resistant to Plutella xylostella, the method comprises the step of establishing the presence of a resistance providing genomic region, or resistance providing genomic regions, by determining in the genome of said plant the presence of one or more of the corresponding 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.

13. (canceled)

14. (canceled)

15. The Brassica oleracea plant according to claim 1, wherein said plant is not exclusively obtainable by an essentially biological process.

16. The Brassica oleracea plant according to claim 2, wherein said first genomic region is between positions 21414859 and 23635544 of the Brassica oleracea HDEM assembly.

17. The Brassica oleracea plant according to claim 3, wherein said second genomic region is between positions 11035789 and 12032392 of the Brassica oleracea HDEM assembly.

18. The Brassica oleracea plant according to claim 4, wherein said third genomic region is between positions 2137498 and 3356008 of the Brassica oleracea HDEM assembly.

19. The Brassica oleracea plant according to claim 5, wherein said fourth genomic region is between positions 1654630 and 3238987 of the Brassica oleracea HDEM assembly.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present invention will be further detailed in the examples below. In the examples, reference is made FIGURES wherein:

[0051] FIG. 1: shows graphs of flow cytometric data wherein the left side represents a haploid (In) plant; the middle represents a diploid or doubled haploid (2n) plant and the right side a tetraploid (4n) plant;

EXAMPLES

Example 1. Crossing Scheme of Plants with Plutella xylostella Resistance Including the Microspore Culture Step

Description of the Invention

Parent 1

TABLE-US-00001 TABLE 1 Breeding scheme of Parent 1 including the Microspore culture step. Name Field number Source Z343079*Z340129 B2836 F1 J5882 Z343079*Z340129 Z343079*Z340129 F1S1 B2836 F1S1 L12399 J5882-1 Z343079*Z340129 F1S1 B2836 F1S1 MC MC17600 L12399-21 MC Z343079*Z340129 MCS1 B2836 MC F1S1 T24527 MC17600-3 Z344539 Z344539

[0052] The first cross was between Z343079 and Z340129. Z343079 is a parent line that has intermediate resistance to Xanthomonas campestris pv. campestris. Z340129 is a Bejo parent line with high resistance against Xanthomonas campestris pv. campestris. The result of this cross was B2836 F1, a Bejo variety that has intermediate resistance against Xanthomonas campestris pv. campestris.

[0053] Variety B2836 F1 was used as a donor to create a new breeding population. This was achieved by a selfing step (F1S1). The F1S1 population was used to screen for resistance against Xanthomonas campestris pv. campestris. The best individuals were selected and used for microspore culture (MC) to obtain doubled haploids (DH).

[0054] The microspore culture resulted in approximately 450 unique progenies. All of the 450 individuals were screened and 32 individuals were selected. One of these individuals became Z344539. See Example 4 for further details regarding the microspore culture.

Parent 2

[0055] The second parent is Z344099. This is a line with intermediate resistance to Xanthomonas campestris pv. campestris.

Final step: Z344539 x Z344099->200234

[0056] The plant 200234 is a plant resistant to Plutella xylostella, which has been deposited as NCIMB 43822. Its parents Z344539 and Z344099 were chosen because of their high intermediate resistance against Xanthomonas campestris pv. campestris. In the process of developing Z344539, microspore culture was employed to generate a homozygous line with increased resistance against Xanthomonas campestris pv. campestris.

[0057] Plant 200234 was developed as a Xanthomonas resistant line. The plant was grown on a field in Guatemala, where Plutella xylostella occurs naturally and it was observed there that plant 200234 is not susceptible to this pest. Thereby, it was coincidently discovered that the line also comprises Plutella xylostella resistance.

Example 2. Field Trial for Assessing Plutella xylostella Resistance and Plants Resistant to Plutella xylostella

[0058] To assess the level of resistance against Plutella present in the newly developed plant 200234, a full field trial was performed. The insect test was carried out under field circumstances in Guatemala at subtropical climate in the wet season. Plutella xylostella is naturally present in this area. The trial was not chemically treated. A susceptible variety was planted as border rows and in rows between the trial to increase pest pressure by allowing the multiplication and infestation of Plutella xylostella.

[0059] In the experiment trail plots of the susceptible variety, the comparator variety and the plant according to the invention were grown in lines, next to each other. The infestation occurred naturally and was not induced. The moment the crop was deemed to have matured, the pest trail was assessed.

TABLE-US-00002 TABLE 2 Results of the field trial for assessing Plutella xylostella resistance (test code: XWV17GU2) Variety/line name Score Botran F1 3 Greenboy F1 3 Alegria F1 3 Escazu F1 = Izalco F1 7 200234 8

[0060] The trial was scored using a scale ranging from 1 to 9, where 1 is fully susceptible and 9 resistant. The variety Greenboy was used as a standard of susceptible variety. Escazu and Izalco are known varieties that show tolerance for Plutella xylostella. Surprisingly, plant 200234 scored 8 and was more resistance against Plutella xylostella than Izalco, which scored 7.

Example 3. Field Tests for Assessing Resistance Against Xanthomonas campestris pv. Campestris

[0061] An inoculum of Xanthomonas campestris pv. campestris was prepared by growing the bacterium on Yeast Dextrose Agar for 3 weeks incubated in dark at 25 C. Bacteria were scraped using water and put in a liter flask. The bacteria were suspended in water with a stirring magnet for 30 minutes. The concentration was measured by measuring absorbance with an UV-VIS spectrophotometer (PerkinElmer) and adjusted to 106 bacteria per ml. Plants were sown on 260-trays at the end of March. The plants were transplanted in the second week of May in a field in the Netherlands. Renton F1 and Morris F1 were used as susceptible control varieties.

[0062] From the second week of June on, the plants were inoculated early in the morning, when guttation takes place, for 10 consecutive weeks. Inoculation is done by spraying the bacterium suspension on the leaves using an ultra-low volume applicator. The inoculation was performed once a week. Roughly 6 weeks after the first inoculation, the first symptoms may appear. Symptoms become more apparent during cultivation. The assessment of disease resistance was carried out when the plants were ready to be harvested.

[0063] If the plant became overmature the assessment was stopped. The scale that was used ranges from 0 (completely susceptible, severe symptoms) to 9 (highly resistant, no symptoms). A score on this scale is referred to as a disease score.

TABLE-US-00003 TABLE 3 Results of the field test against Xanthomonas campestris pv. campestris. Plant Score Escazu F1 2.6 Izalco F1 3.8 Z344539 6.0 Z344099 6.0 Plant 200234 6.6

Example 4. Microspore Culture

[0064] For the purpose of developing a parental line, microspore culture was used to develop doubled haploids, which are 100% homozygous lines. By employing doubled haploid plants obtained by microspore embryogenesis and using only the spontaneously doubled haploids, it was possible to greatly shorten the breeding time required to introduce the desired trait into the plants. For obtaining doubled haploid plants the procedure outlined below was followed: [0065] Breeding lines with high quality and the desired trait were selected by the breeder. These plants were maintained and transferred to the greenhouse, see example 1 for details. [0066] The plants were vernalized at 6 C. and grown until flowering under controlled conditions at a 16-h photoperiod at 18 C. and irrigated daily. [0067] When flowering started, closed buds were collected and checked for the developing stage of the microspores within. [0068] Buds that contained microspores at the late uninucleate stage, which are sensitive for reprogramming, were used for microspore isolation. [0069] Buds were crushed in hormone free NLN media, the microspore suspension that was released, was filtered and collected in 10 ml tubes. [0070] The suspension was washed three times by centrifugation and diluted until a concentration of 40000 msp/ml. [0071] Microspores were given a stress treatment in the dark at 35 C. for 24 hours, to switch from the default developmental pathway toward embryogenesis. [0072] The microspores were kept in the dark at 25 C. and after three weeks the first embryos appeared. [0073] Microspore derived embryos at the late cotyledonary stage (0.50.7 cm) were transferred to solidified media with hormones for further outgrowth and transferred to a culture room with a 16-hour photoperiod at 24 C. [0074] After 7 weeks direct conversion of embryo to plant was determined and the ploidy level of the regenerated plantlets was checked with a Partec Flow Cytometer (FIG. 1). [0075] Plants with a 2n level (doubled haploids) were transferred to fresh media for rooting, while haploid (In), tetraploid (4n) and mixoploid plantlets were discarded. [0076] Rooted DH plants were transferred to the greenhouse for acclimatization and then planted out in the field. [0077] The best performing plants were selected, selfed and crossed, and also checked for their ability as a parental line in an F1 hybrid. [0078] One of those individuals was selected as Z344539, Parent 1 from example 1.

Example 5. Genetic Mapping Plutella xylostella Resistance

Population

[0079] For the genetic mapping of the Plutella xylostella (DBM) resistance, an FIS1 mapping population was made. The population is build up from the DBM resistant line A and susceptible line B. The F1 cross A*B is selfed which formed the FIS1 population. A population of 432 plants has been tested in a DBM field trial for determining the resistance levels.

Genetic Map

[0080] For the genetic mapping informative SNP markers between line A and line B are selected. These SNP markers are developed on the DNA sequences of the respective lines A and B. The genetic map is constructed with 150 SNP markers.

QTL Mapping

[0081] The genetic map and DBM scores are together used for the QTL mapping of the DBM resistance trait. Four QTLs with a LOD value higher than the threshold were found in the population.

[0082] In Table 4 below informative SNPs are identified for the detection of the DBM resistance QTLs. In Table 5, sequences of the SNPs from Table 4 are provided. The markers have been validated on the deposit NCIMB 43822 (Brassica oleracea 200234) and they were found to be informative.

TABLE-US-00004 TABLE 4 SNPs for the detection of DBM resistance in cabbage. Physical position Resistant Alternative SNP No. KSNP Chromosome HDEM (bp)* allele allele 1 1009-2163.1 C1 11035789 A G 2 1009-2161.1 C1 11066385 A G 3 1009-2159.1 C1 11134129 A C 4 1009-1500.1 C1 12032392 T G 5 C2 16546306 T C 6 C2 17852976 A G 7 C2 20270792 A G 8 C2 29962502 T C 9 1009-0319.1 C2 32389878 A G 10 1009-7049.1 C5 2137498 T C 11 1009-0498.1 C5 2145769 T A 12 1009-7050.1 C5 2562211 A G 13 1009-0627.1 C5 3356008 C A 14 1009-7060.1 C6 21414859 A C 15 1009-7062.1 C6 22636135 A C 16 1009-7063.1 C6 23182069 A G 17 1009-7065.1 C6 23635544 C T *The Brassica oleracea HDEM assembly was used according to Belser et al,, Nature Plants Vol. 4 (2018): 879-887.

TABLE-US-00005 TABLE5 SequencesoftheSNPsusedtodetecttheDBMresistanceincabbage. Physical SeqID positionin Sequence No. SNP Chromosome HDEM(bp) (SNPnucleotideisboldandinbrackets) 1 1 C1 11035789 AGGTATGTCKTCCTCCATATTCACCGATCC AGCTAGATTCTGGCTGTGTYCATATGGAGA CGTGTAAGAGTCTTCTKCTCCAGTTTCATC ATCGGTTGTC[A]TGTCCGTAGCTGTTTGCA TTTGTCTAGGAGTCTCGTCTTCCTCGTAAG ACTTTGTATATGAAGTTTCTCTGCTYGTGG ATTCTGAACACGTTCTCTGCAA 2 1 C1 11035789 AGGTATGTCKTCCTCCATATTCACCGATCC AGCTAGATTCTGGCTGTGTYCATATGGAGA CGTGTAAGAGTCTTCTKCTCCAGTTTCATC ATCGGTTGTC[G]TGTCCGTAGCTGTTTGCA TTTGTCTAGGAGTCTCGTCTTCCTCGTAAG ACTTTGTATATGAAGTTTCTCTGCTYGTGG ATTCTGAACACGTTCTCTGCAA 3 2 C1 11066385 ATAACATGAAATTCTAAGAGAATTGTAAAC CATGTGATAATCTTTTTACAAATTGATCAG [A]AATAAGAAAAAAGAGAGGTGGAATATC TTCTAYATCTCTATACTCTTTCTTTCCCTT GTG 4 2 C1 11066385 ATAACATGAAATTCTAAGAGAATTGTAAAC CATGTGATAATCTTTTTACAAATTGATCAG [G]AATAAGAAAAAAGAGAGGTGGAATATC TTCTAYATCTCTATACTCTTTCTTTCCCTT GTG 5 3 C1 11134129 AATCAACATAAAATTCCTGTGTTTGACCAC TTGCAAGGACTCTATCACAGTCTTATACAT [A]TGATAAGAGAAATTCAAGTGAACATAT TAAAAACCAACAATTAGTGTTTGCTCACAG AAG 6 3 C1 11134129 AATCAACATAAAATTCCTGTGTTTGACCAC TTGCAAGGACTCTATCACAGTCTTATACAT [C]TGATAAGAGAAATTCAAGTGAACATAT TAAAAACCAACAATTAGTGTTTGCTCACAG AAG 7 4 C1 12032392 AGCGGTTTGTCTGACGCCTCTCCGTCTTCTT TAATCCACGGATGTTTTAGTACTTCACTCG CTGTTAGCCGGTCTTTAGGGTCATATGTCA ACATTTTCTTCACAAGATCTTTGGCACCGTT GGATACA[T]CAGGCCATGGATCAGCTGAA AAATCAAGCTTTCCTTCTAGAATGGCATCG AAGATCCCTGTCTCATTTTCTCCCCAGAAA GGTGGGACACCACTGAGAAGGATGTACAG AATCACACCGGCACTCCAAATATCAGCCTC TGGTCCATAGTTCCGTTTTAAAACTTCCGG GGCAACATAGTATGCACTTCCAACAAGATC C 8 4 C1 12032392 AGCGGTTTGTCTGACGCCTCTCCGTCTTCTT TAATCCACGGATGTTTTAGTACTTCACTCG CTGTTAGCCGGTCTTTAGGGTCATATGTCA ACATTTTCTTCACAAGATCTTTGGCACCGTT GGATACA[G]CAGGCCATGGATCAGCTGAA AAATCAAGCTTTCCTTCTAGAATGGCATCG AAGATCCCTGTCTCATTTTCTCCCCAGAAA GGTGGGACACCACTGAGAAGGATGTACAG AATCACACCGGCACTCCAAATATCAGCCTC TGGTCCATAGTTCCGTTTTAAAACTTCCGG GGCAACATAGTATGCACTTCCAACAAGATC C 9 5 C2 16546306 CAGCAAGCTTCAGATAAATCCATTGTGATA AGTGAAATTCTGGACTCAAGAACAAAGAAC [T]TTGTCTCTGTCTCCAGAATAAGTGATT ACGTACTATCCAACGAGCTAGTTAGTATGG CTC 10 5 C2 16546306 CAGCAAGCTTCAGATAAATCCATTGTGATA AGTGAAATTCTGGACTCAAGAACAAAGAAC [C]TTGTCTCTGTCTCCAGAATAAGTGATT ACGTACTATCCAACGAGCTAGTTAGTATGG CTC 11 6 C2 17852976 GAGACACTCTATCCTTATACATTGCCATCA CCTCCTCGGCTGTTAGCAGCGTCCTAATAC [A]AGAAGACCGTGGTGAACAGAAACATAT TTTCTACACCAGCAAGCGGATGATGGAACC GGA 12 6 C2 17852976 GAGACACTCTATCCTTATACATTGCCATCA CCTCCTCGGCTGTTAGCAGCGTCCTAATAC [G]AGAAGACCGTGGTGAACAGAAACATAT TTTCTACACCAGCAAGCGGATGATGGAACC GGA 13 7 C2 20270792 TTGTTCTCCGCTTTCTTCGCAAATTAGCACT CCGCTTACTGCGTGTTTGGAGGTTGCCAC[A] TACAAAAAGAGGGTCTCTCCTATGATTGGT TTGACAGAATGGGCGGCTCGCTGATATATG 14 7 C2 20270792 TTGTTCTCCGCTTTCTTCGCAAATTAGCACT CCGCTTACTGCGTGTTTGGAGGTTGCCAC[G] TACAAAAAGAGGGTCTCTCCTATGATTGGT TTGACAGAATGGGCGGCTCGCTGATATATG 15 8 C2 29962502 GATAAAATAAAATTGAATTGTTTTCAACAT AGATGAGTGAAAGCAGTGAATCATGATATT [T]TTTGGTCTAGGTTTTGACAACATATGT TATAGTATTGTATGTATTCTTAGGGTTAGA TTT 16 8 C2 29962502 GATAAAATAAAATTGAATTGTTTTCAACAT AGATGAGTGAAAGCAGTGAATCATGATAT T[C] TTTGGTCTAGGTTTTGACAACATATGTTAT AGTATTGTATGTATTCTTAGGGTTAGATTT 33 9 C2 32389878 GGCAACTTGTGGCGCTGTACTTCTTGTCTCT TTGGTTTCGTCTTTGGGAATGGCCTTTTT[A] TCAAAGGCAGCTAAAGCTTGCTGAAAACA TAACATTCTCTATCTGTTAGACTTTCATTTA 34 9 C2 32389878 GGCAACTTGTGGCGCTGTACTTCTTGTCTCT TTGGTTTCGTCTTTGGGAATGGCCTTTTT[G] TCAATGGCAGCTAAAGCTTGCTGAAAACAT AACATTCTCTATCTGTTAGACTTTCATTTA 17 10 C5 2137498 CTAAATCCTGTGTGGATTTGGCTGGCAGCT TTTGTCTAAAATAAAATCTAGATAACCACA ACATTCTTCAACGCTGTTACTCTGCCCTCAT TATGTATGG[T]ATGCCTATGATATTGCGCT TGTCTTGTCTTGATGACGTTTTCATTTGTTT TTTCTGCTTCAGATGAAGTGGTGTCCTGCC CCAGGATGTGAGAATGCAAT 18 10 C5 2137498 CTAAATCCTGTGTGGATTTGGCTGGCAGCT TTTGTCTAAAATAAAATCTAGATAACCACA ACATTCTTCAACGCTGTTACTCTGCCCTCAT TATGTATGG[C]ATGCCTATGATATTGCGCT TGTCTTGTCTTGATGACGTTTTCATTTGTTT TTTCTGCTTCAGATGAAGTGGTGTCCTGCC CCAGGATGTGAGAATGCAAT 19 11 C5 2145769 TCTTGCAAGATGTTGATCTCATTGTGAAAA ATGCCAAGGCCTACAATGGAGATGATTAC GCTGGAGCGAGAATTGTCAGTAGAGCCTA CGAGCTTCGGGATGTAGTGCATGGGATGCT GGCGCAGATGGACCCAGCACTGCTAACAT ATTGTGACAAAATCGC[T]GCTGAAGGTGGT CCTTCACAGATACCAGATGATCTGAGTGAA TCTATCCTTGGTTTAGCTCCTGTGGTGCAG ATGGGGACTGTTACTAGAGCAAGTGCCCG GCTTCGAAACATGCAGCCAGAGGTTAATAT AGATCGAGATCATGAAGGTTTTAAAAAGC CTAAGAAAACAGCTGATGCTGCCTGTACAG ATTCAGCTGC 20 11 C5 2145769 TCTTGCAAGATGTTGATCTCATTGTGAAAA ATGCCAAGGCCTACAATGGAGATGATTAC GCTGGAGCGAGAATTGTCAGTAGAGCCTA CGAGCTTCGGGATGTAGTGCATGGGATGCT GGCGCAGATGGACCCAGCACTGCTAACAT ATTGTGACAAAATCGC[A]GCTGAAGGTGGT CCTTCACAGATACCAGATGATCTGAGTGAA TCTATCCTTGGTTTAGCTCCTGTGGTGCAG ATGGGGACTGTTACTAGAGCAAGTGCCCG GCTTCGAAACATGCAGCCAGAGGTTAATAT AGATCGAGATCATGAAGGTTTTAAAAAGC CTAAGAAAACAGCTGATGCTGCCTGTACAG ATTCAGCTGC 21 12 C5 2562211 AACAACACACGGTATAAATGAAAATACCT GAGTGTGAAGATGTCAACGACGCAGCTGA ATCCCTTAGTCTCCTTACACTCTCAGCAGC ATCCAAAGTTTC[A]GAATACTCCTCTGATG TTGGTTCAATCAGACTATCCCTCTTAAGGC CCAGCAGCGCATTTTCGGAGGCCTAGGTCA ACGTCAGAACAATGTCATATACCG 22 12 C5 2562211 AACAACACACGGTATAAATGAAAATACCT GAGTGTGAAGATGTCAACGACGCAGCTGA ATCCCTTAGTCTCCTTACACTCTCAGCAGC ATCCAAAGTTTC[G]GAATACTCCTCTGATG TTGGTTCAATCAGACTATCCCTCTTAAGGC CCAGCAGCGCATTTTCGGAGGCCTAGGTCA ACGTCAGAACAATGTCATATACCG 23 13 C5 3356008 ACAGGAACAGCGTTTTGCTCAACCATCTGT TTCTGTCTGCCGCGAACAACCTCTGGCTGC CAAACGTTCAAAGCTACTGATGCTTGCCTG ACTCTCTGCAGATTTGAGAACTCAACAGAA GCAGCCTGCAACCTCTCTGCCTCCTCWGAT AACCTCCTCTCCAATTCACGAATCCTCCTG CCCCACTCCGAAATCTCGCT[C]TCCTTCC TCAGCAGAGATCTTCTATGAGCAGCTAACC TCCTCTCCTTCTCCCTCTCAAACCTTGTCC TGTAGATAAACTTGGTCGGAGATTCAGAAG AGGAAGAAGAAGAAGTAGAGTCGGTGTGGG TGGGGAAATCAATGGCGAGCAGAAAGTTCC AGAGGCAATCTTCGGAGGATAGTCGGCAG AGACGGCGAGAGGA 24 13 C5 3356008 ACAGGAACAGCGTTTTGCTCAACCATCTGT TTCTGTCTGCCGCGAACAACCTCTGGCTGC CAAACGTTCAAAGCTACTGATGCTTGCCTG ACTCTCTGCAGATTTGAGAACTCAACAGAA GCAGCCTGCAACCTCTCTGCCTCCTCWGAT AACCTCCTCTCCAATTCACGAATCCTCCTG CCCCACTCCGAAATCTCGCT[A]TCCTTCC TCAGCAGAGATCTTCTATGAGCAGCTAACC TCCTCTCCTTCTCCCTCTCAAACCTTGTCC TGTAGATAAACTTGGTCGGAGATTCAGAAG AGGAAGAAGAAGAAGTAGAGTCGGTGTGGG TGGGGAAATCAATGGCGAGCAGAAAGTTCC AGAGGCAATCTTCGGAGGATAGTCGGCAG AGACGGCGAGAGGA 25 14 C6 21414859 CTGTTACTTAGAAACTTCATTTTTTTTTATA GGTTTTTGCTAACTGTTAGATACAAAGTATT ATTATTTGAAGTTTTTTTCATTGTTGTCAAA TCTCGTT[A]TCTTCAGTTACAAAGCATTCT CCAATAAATCTCTTTGTCTCTTTTTTACCCA AAAAACAAAATCTATCTGTCTTTGTTGTTA AATTTTTCATGGGGTCTT 26 14 C6 21414859 CTGTTACTTAGAAACTTCATTTTTTTTTATA GGTTTTTGCTAACTGTTAGATACAAAGTAT TATTATTTGAAGTTTTTTTCATTGTTGTCAA ATCTCGTT[C]TCTTCAGTTACAAAGCATTCT CCAATAAATCTCTTTGTCTCTTTTTTACCCA AAAAACAAAATCTATCTGTCTTTGTTGTTA AATTTTTCATGGGGTCTT 27 15 C6 22636135 ACTGGCTGAGTGTCCTCTATTATTTCCTCTA CAGGCTGAATGTCACCATGCTTCTCTTCCA CCACCTGAGCAACTTGCTCAACTGGTTCAG TGTTATCCA[A]CATTTCCTCAATCAGTTGTG TCTCATCTCCTACTTCCGTAACAAGAATGT CATCGTCAGCATGCTTCTCCTCCACCAATT GTGCCTCCTGAGCTACTTCT 28 15 C6 22636135 ACTGGCTGAGTGTCCTCTATTATTTCCTCTA CAGGCTGAATGTCACCATGCTTCTCTTCCA CCACCTGAGCAACTTGCTCAACTGGTTCAG TGTTATCCA[C]CATTTCCTCAATCAGTTGTG TCTCATCTCCTACTTCCGTAACAAGAATGT CATCGTCAGCATGCTTCTCCTCCACCAATT GTGCCTCCTGAGCTACTTCT 29 16 C6 23182069 TTATTAGATATGAGGAAGGAGAATGTCAA ACCTAGCATAAGAGGAATTCTGGAGAGAT AGTAGAACCAGATTAAGCTTACAAAATATT TGTATGGTTAAT[A]AGTTTTGAAGTTCAAA ATCACAATTCAGTGGTCACTACAAGAAAAC ACATGGTTAACGACGAAAATTAACGAGGA AAAACAATCCTCGTAAATTTGCGTC 30 16 C6 23182069 TTATTAGATATGAGGAAGGAGAATGTCAA ACCTAGCATAAGAGGAATTCTGGAGAGAT AGTAGAACCAGATTAAGCTTACAAAATATT TGTATGGTTAAT[G]AGTTTTGAAGTTCAAA ATCACAATTCAGTGGTCACTACAAGAAAAC ACATGGTTAACGACGAAAATTAACGAGGA AAAACAATCCTCGTAAATTTGCGTC 31 17 C6 23635544 TCCATGCTTCCTTCATCAGTTCCTGGGTAA ACAAGGATTTTACCATGGCCAGCAATTCCC GGTCCAGCTCTAACCGCAATCGGTTTACCC CAACCAAAAT[C]GTTTCCAAACACATTGAA CCGTGGAGAGCTAGCAACAACGAGCGAAT TATTCACCATCAAACCATTCGGTATTTTAG GGTTCTTCACCCAATTTTCAGCA 32 17 C6 23635544 TCCATGCTTCCTTCATCAGTTCCTGGGTAA ACAAGGATTTTACCATGGCCAGCAATTCCC GGTCCAGCTCTAACCGCAATCGGTTTACCC CAACCAAAAT[T]GTTTCCAAACACATTGAA CCGTGGAGAGCTAGCAACAACGAGCGAAT TATTCACCATCAAACCATTCGGTATTTTAG GGTTCTTCACCCAATTTTCAGCA

[0083] The abbreviations are according to IUPAC nucleotide code:

TABLE-US-00006 Symbol Nucleotide Base A Adenine C Cytosine G Guanine T Thymine N A or C or G or T M A or C R A or G W A or T S C or G Y C or T K G or T V Not T H Not G D Not C B Not A
Information about the Deposit NCIMB 43822.

[0084] A deposit of a plant according to the invention, Brassica oleracea 200234, deposit NCIMB 43822 was deposited at NCIMB Limited, Craibetone Estate, 35 Ferguson Building, Bucksburn, Aberdeen AB21 9YA, United Kingdom on Jul. 26, 2021.