Tropical Root-Knot Nematode Resistant Carrot Plant

Abstract

The present invention relates to tropical root-knot nematode resistant carrot plants comprising a first tropical root-knot nematode resistance providing genomic fragment. The present invention further relates to methods for identifying tropical root-knot nematode resistant carrot plants, methods for providing tropical root-knot nematode resistant carrot plants and means for identifying tropical root-knot nematode resistant carrot plants.

Claims

1. A carrot plant resistant to tropical root-knot nematodes, comprising on chromosome 4 of said carrot plant a first tropical root-knot nematode resistance providing genomic fragment, wherein said first genomic fragment comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.

2. The carrot plant according to claim 1, further comprising a second tropical root-knot nematode resistance providing genomic fragment on chromosome 8 of the carrot plant, wherein said second genomic fragment comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 25, 27, 29, 31, 33, 35, 37 and 39.

3. The carrot plant according to claim 1 wherein a first tropical root-knot nematode resistance providing genomic fragment is obtained, obtainable, or is from a carrot plant of which representative seed are deposited under deposit number NCIMB 43792.

4. The carrot plant according to claim 1, wherein said carrot plant is cytoplasmic male sterile (CMS).

5. The carrot plant according to claim 1, wherein said carrot plant is a hybrid plant.

6. The carrot plant according to claim 1, wherein said carrot plant is a carrot plant of which representative seed are deposited under deposit number NCIMB 43792.

7. A method for identifying a tropical root-knot nematode resistance providing genomic fragment comprising the steps of: a) isolating or providing a genomic nucleic acid of a carrot plant; b) identifying in said genomic nucleic acid a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 by detecting a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23; and c) optionally, further identifying in said genomic nucleic acid a second tropical root-knot nematode resistance providing genomic fragment on chromosome 8 by detecting a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 25, 27, 29, 31, 33, 35, 37 and 39.

8. A method for providing a tropical root-knot nematode resistant carrot plant comprising the steps of: a) obtaining a carrot plant not comprising a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 of said carrot plant; b) crossing said carrot plant with a carrot plant according to claim 1; c) obtaining a seed from said cross and germinating said seed to obtain a carrot plant; d) isolating genomic DNA from said carrot plant and identifying in the genome of said carrot plant a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23; and e) optionally, further identifying in the genome of said carrot plant a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 25, 27, 29, 31, 33, 35, 37, and 39.

9. A method for providing a tropical root-knot nematode resistant carrot plant, comprising the steps of: a) obtaining a carrot plant not comprising a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 of said carrot plant; and b) introducing in said carrot plant a first tropical root-knot nematode resistance providing genomic fragment as found in a carrot plant of which representative seed has been deposited under deposit number NCIMB 43792, wherein said genomic fragment is not introduced by means of an essentially biological process

10. The method according to claim 9, wherein said method for providing comprises the step of mutagenesis, transformation with Agrobacterium or CRISPR/Cas.

11. A seed of the carrot plant according to claim 1, comprising a first tropical root-knot nematode resistance providing genomic fragment wherein said first genomic fragment comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.

12. The seed according to claim 11, wherein said seed is polished, coated, encrusted, pelleted or primed.

13. A plant part, callus, suspension culture, or clone of a carrot plant according to claim 1.

14. (canceled)

15. (canceled)

16. The seed of claim 11, wherein the seed further comprises a second tropical root-knot nematode resistance providing genomic fragment wherein said second genomic fragment comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 25, 27, 29, 31, 33, 35, 37, and 39.

Description

DESCRIPTION OF THE INVENTION

[0022] Specifically, this object, amongst other objects, is achieved, according to a first aspect, by providing a carrot plant resistant to tropical root-knot nematodes, comprising on chromosome 4 of said carrot plant a first tropical root-knot nematode resistance providing genomic fragment, wherein said first genomic fragment comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No.7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 and SEQ ID No. 23. The indefinite article a or an can be used interchangeably with the phrases one or more or at least one. As such, the word a should be understood to mean one, two, three, four, five, six, or more, or all. Preferably, the first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 comprises SEQ ID No. 9; more preferably SEQ ID No. 9 and SEQ ID No. 11; more preferably SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11; even more preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11; yet even more preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19 and SEQ ID No. 21; and most preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, and SEQ ID No. 23. The first tropical root-knot resistance providing genomic fragment is not comprised of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ, ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, and SEQ ID No. 24.

[0023] Tropical root-knot nematode is defined as a group consisting of three tropical root-knot nematode species: Meloidogyne incognita, Meloidogyne arenaria and Meloidogyne javanica; preferably, Meloidogyne incognita and Meloidogyne arenaria; most preferably, Meloidogyne incognita.

[0024] Carrot plant means a plant of the species Daucus carota; preferably, a cultivated carrot plant; most preferably, a commercially cultivated carrot plant.

[0025] Alternatively, this object, amongst other objects, is achieved by providing a tropical root-knot nematode resistant carrot plant comprising two tropical root-knot nematode resistance providing genomic fragments, wherein a first tropical root-knot resistance providing genomic fragment is located on chromosome 4 and comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No.7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 and SEQ ID No. 23; and wherein, a second tropical root-knot resistance providing genomic fragment is selected from the group consisting of SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 39. Preferably, the first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 comprises SEQ ID No. 9; more preferably SEQ ID No. 9 and SEQ ID No. 11; more preferably SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11; even more preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11; yet even more preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19 and SEQ ID No. 21; and most preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, and SEQ ID No. 23. The first tropical root-knot resistance providing genomic fragment is not comprised of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ, ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, and SEQ ID No. 24. Preferably, the second tropical root-knot nematode resistance providing genomic fragment on chromosome 8 comprises SEQ ID No. 31, more preferably SEQ ID No. 31 and SEQ ID No. 33; more preferably SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35 and SEQ ID No. 37; even more preferably SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 39; most preferably SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 39.

[0026] Hence, in one preferred embodiment, the tropical root-knot resistant carrot plant comprises SEQ ID No. 9 and SEQ ID No. 31; more preferably SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 31 and SEQ ID No. 33; more preferably SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 31 and SEQ ID No. 33; even more preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35 and SEQ ID No. 37; yet even more preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 39; and most preferably SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37 and SEQ ID No. 39.

[0027] According to a preferred embodiment of the present invention, the first tropical root-knot nematode resistance providing genomic fragment is obtained, obtainable, or is from a carrot plant of which representative seed has been deposited at NCIMB (National Collections of Industrial, Food and Marine Bacteria; 5 NCIMB Limited, Ferguson Building; Craibstone Estate, Bucksburn Aberdeen, Scotland, AB21 9YA United Kingdom) on 9 Jun. 2021 under deposit number NCIMB 43792.

[0028] According to another preferred embodiment, the present carrot plant is cytoplasmic male sterile (CMS).

[0029] According to yet another preferred embodiment, the present carrot plant is a hybrid carrot plant, more preferably a sterile hybrid carrot plant and most preferably a male sterile hybrid carrot plant, e.g., cytoplasmic male sterile.

[0030] The present invention further relates to a method for identifying a tropical root-knot nematode resistance providing genomic fragment as found in the present carrot plant. The method comprises the steps of isolating or providing genomic nucleic acid of a carrot plant and identifying in the genomic nucleic acid a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 by detecting a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 and SEQ ID No. 23.

[0031] Preferably, the present method comprises an optional step of further identifying in the genomic nucleic acid a second tropical root-knot nematode resistance providing genomic fragment on chromosome 8 by detecting a nucleic acid sequence selected from the group consisting of SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37, and SEQ ID No. 39.

[0032] Several common genotyping methods exist for detecting a single nuclear polymorphism (SNP) in a genomic sequence, including PCR-based methods, direct hybridization, fragment analysis, and sequencing. An example of a method suitable for detecting a genomic sequence is isolating DNA from available plant material (e.g., from a piece of a leaf from a plant, or a seed), followed by nucleic acid amplification of isolated DNA (e.g., using PCR) and detecting the presence of said genomic sequence (e.g., by sequencing, measuring fluorescence, or visualizing and analysing PCR amplification using agarose gel electrophoresis).

[0033] Additionally, the present invention relates to a method for providing a carrot plant of the invention comprising the steps of: [0034] a) obtaining a carrot plant not comprising a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 of the carrot plant; [0035] b) crossing the carrot plant with a carrot plant comprising a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 of the carrot plant; obtaining a seed from said cross and germinating the seed to obtain a carrot plant; [0036] c) isolating genomic nucleic acid from the carrot plant and identifying in the genomic nucleic acid a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 of the carrot plant by detecting a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 and SEQ ID No. 23.

[0037] Preferably, the present method comprises an optional step of further identifying in the genomic nucleic acid a second tropical root-knot nematode resistance providing genomic fragment on chromosome 8 of the carrot plant by detecting a nucleic acid sequence selected from the group consisting of SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37, and SEQ ID No. 39.

[0038] The present invention also relates to a method of producing a carrot plant of the invention comprising the steps of obtaining a carrot plant not comprising a first tropical root-knot nematode resistance providing genomic fragment on chromosome 4 of said carrot plant; introducing in said carrot plant a first tropical root-knot nematode resistance providing genomic fragment as found in a carrot plant of the present invention, wherein said genomic fragment is not introduced by means of an essentially biological process.

[0039] Mutagenesis, transformation with Agrobacterium or CRISPR/Cas can be used to introduce a first tropical root-knot nematode resistance providing genomic fragment by means of a non-essentially biological process.

[0040] Suitable mutagenesis methods comprise chemical mutagenesis (e.g., using ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), sodium azide (NaN3), methylnitrosoguanidine (MNNG), diethyl sulfonate (DES), TILLING, or mutagenesis by generating reactive oxygen species) and radiation mutagenesis (e.g., using UV radiation or ion beam radiation). Mutagenesis can lead to one or more mutations located in the coding sequence (mRNA, cDNA or genomic sequence) or in the associated non-coding sequence and/or regulatory sequence regulating the level of expression of the coding sequence. The presence of one or more mutations (e.g., insertion, inversion, deletion and/or replacement of one or more nucleotide(s)) may lead to the encoded protein having a new or altered functionality (gain of function), reduced functionality (reduced function) or no functionality (loss-of-function), e.g., due to the protein being truncated or having an amino acid sequence wherein one or more amino acids are deleted, inserted or replaced. Such changes may lead to the protein having a different 3D structure or conformation, being targeted to a different sub-cellular compartment, having one or more modified catalytic domains, having a modified binding activity to nucleic acids or proteins, etcetera.

[0041] Alternatively, the resistance according to the invention can be introduced in a plant cell by transformation (e.g., using Agrobacterium tumefaciens). Genomic fragments can be amplified by long-range PCR amplifications, de novo synthesized, or isolated from gels or columns (e.g., after restriction digestion). The resulting fragments can be reassembled (e.g., in yeast) or introduced in an expression vector, subsequently transformed into carrot plant cells and allowed to integrate or recombine with the carrot plant genome. The fragment may be introduced in a single step or in a series of transformations ultimately resulting in a carrot plant comprising the resistance of the present invention.

[0042] Alternatively, the CRISPR/Cas9 system can be used to introduce tropical root-knot nematode resistance in a carrot plant by enabling robust and precise targeted genome modifications, as well as CRISPR-based screening, e.g., using pooled gRNA libraries with complete coverage and distribution over the carrot genome, and whole genome mutagenesis.

[0043] According to a second aspect, the present invention also relates to a seed capable of providing a plant of the present invention

[0044] Seeds can be coated, coloured, washed, polished, encrusted, pelleted, primed or undergo a combination of treatments. Coated seeds are covered by a relatively thin layer of polymer supplied to the seed; to this polymer fungicides or insecticides can be added to protect the seed against soil borne pathogens and insect damage. Additionally, a dye can be added. This added colour gives the farmer the opportunity to check for correct drilling of the seeds. Alternatively, also other beneficial compounds can be added as micronutrients or beneficial micro-organisms promoting the growth of the young seedlings. Encrusted seeds are not only covered by a polymer with or without extra substances, as described above, but the seeds are provided with a smooth surface as well. This makes drilling easier, and the added weight enables a more precise direct drilling of the seeds. Polishing removes the outermost layer of the seed, so that the seed assumes a more rounded form. Polishing and washing promotes germination of the seed. With pelleting the seeds are covered with more material, e.g., polymer bound clay, to produce a regularly shaped, round pellet. This pellet, besides having protecting substances described above, can be constructed in such a way that it will melt or split after water uptake. Priming or pre-germination is a treatment in which seeds are given enough moisture to initiate germination of the embryo inside the seed. This results in a faster emergence of the seedling, a higher emergence rate and better growth. It is believed that priming leads to a better root system and faster growth.

[0045] According to a third aspect, the present invention further relates to a plant cell, a protoplast, a plant organ, plant tissue, edible parts, pollen, microspores, ovaries, ovules, egg cells, callus, suspension culture, somatic embryos, embryos, or plant parts of the present plants resistant to tropical root-knot nematodes. Plant parts include, but are not limited to, the shoot, the stalk, the stem, leaves, blossoms, inflorescence, roots, fruits, and cuttings.

[0046] Additionally, the present invention relates to use of a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 and SEQ ID No. 23 for identifying or providing a first tropical root-knot nematode resistance providing genomic fragment as found in a carrot plant of which representative seed has been deposited under deposit number NCIMB 43792. The indefinite article a or an can also here be used interchangeably with the phrases one or more or at least one. As such, the word a should be understood to mean one, two, three, four, five, six, or more, or all. The nucleic acid sequences are closely linked to the first tropical root-knot nematode resistance providing genomic fragment and can be used as molecular markers, e.g., in molecular marker-assisted breeding. Molecular marker-assisted breeding greatly increases efficiency and precision of plant breeding, and includes several modern breeding strategies, including marker-assisted selection, marker-assisted backcrossing, marker-assisted recurrent selection, and genome-wide selection or genomic selection.

[0047] The present invention also relates to nucleic acid sequences which co-segregate with a first tropical root-knot nematode providing genomic fragment as present in deposit number NCIMB 43792, which nucleic acid sequences are selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, and SEQ ID No. 23.

[0048] The present invention will be further detailed in the following examples.

EXAMPLES

Example 1: Mutagenesis of Carrot Plants to Introduce Tropical Root-Knot Nematode Resistance

[0049] Random mutagenesis followed by forward screening is a useful method for identifying mutant carrot plants with resistance to tropical root-knot nematodes. A mutagenized library can be generated by subjecting seeds to a step of mutagenesis, preferably random mutagenesis. Such a step may comprise, but is not limited to, the treatment of a pool of 100.000 to 200.000 seeds with a chemical mutagen, or a mixture of chemical mutagens, e.g., 0.25% EMS for 16 hours at room temperature; alternatively, radiation can be used (e.g., gamma-radiation from a radioactive Cobalt-60 source). Preferably, only a mildly mutagenized library (fewer than 1% of all genes contain a mutation in a coding region) is generated. Nevertheless, the mutagenesis step will lead to the loss of germination in some seeds. In contrast to irradiation, which can lead to mutations varying from single base substitutions or deletions to large deletions, EMS produces predominantly random point mutations by nucleotide substitution, particularly by guanine alkylation.

[0050] The mutagenized seeds can be sown and propagated in a field using standard practices. The mutagenized seed will generate plants that each have a particular set of mutations. Plants can be harvested in pools and viable seeds can be sown again (F1 population). To obtain an F1S1 population seeds can be collected from the F1 plants after selfing. As the mutations will segregate in an FIS1 population, the resistance in this population can be evaluated and used to map the resistance against tropical root-knot nematodes.

Example 2. General Protocol for Assessing Resistance

[0051] Four-week-old carrot plants are transplanted into pots filled with fine white sand. One week after transplanting, plants are tested for tropical root-knot nematode resistance by injecting 1 ml inoculation medium containing 2000-2500 tropical root-knot nematode eggs/ml next to the root system at a depth of 0.5 cm. Plants are not watered for 48 h after inoculation to prevent flushing of tropical root-knot nematode eggs.

[0052] Eight to ten weeks after inoculation plants are assessed for severity of tropical root-knot nematode infection. Plants are uprooted and the root system is flushed with tap water to remove the potting soil and fine white sand. After washing, plants are placed in 10% carmine staining solution for five minutes. The number of galls is subsequently counted under a microscope. The maximum number of galls counted per plant is 50. It is carefully checked that the susceptible control plants indeed show the expected symptoms of tropical root-knot nematode infection and that the resistant controls show no infection. Per accession the average number of galls is calculated (AVG), as well as the standard deviation (STD), the percentage of plants with only 2 galls or less (% ?2) or 5 galls or less (% ?5), which are all indicative values for the level of resistance.

Example 3. Results of Assessment for Resistance Against Tropical Root-Knot Nematode Resistance

[0053]

TABLE-US-00002 TABLE 2 Several sources of resistance were tested with different populations, including sources described in literature. Mi-WU-1 is a Meloidogyne incognita population obtained from the nematology group at Wageningen University. MAU is a Meloidogyne arenaria population originating from North America. A minimum of 10 plants were tested per condition. Population AVG STD % ?2 % ?5 Sus 1 Mi-WU-1 50.0 0.0 0% 0% Sus 2 Mi-WU-1 42.1 13.8 0% 0% Res Mi-WU-1 2.4 2.8 50% 88% SFF Mi-WU-1 7.3 7.6 32% 56% Homs Mi-WU-1 12.5 11.4 15% 32% Sus 1 MAU 16.0 8.5 0% 13% Sus 2 MAU 35.6 11.2 0% 0% Res MAU 0.2 0.7 94% 100% SFF MAU 0.6 0.7 100% 100% Homs MAU 0.2 0.4 100% 100%

[0054] Explanation of abbreviations used: AVG is the average number of galls counted per plant under a microscope; STD is the standard deviation of the number of galls counted per plant under a microscope; % ?2 is the percentage of plants that had 2 or fewer galls; % ?5 is the percentage of plants that had 5 or fewer galls.

Example 4: Molecular Characterization of Genomic DNA and Mapping of the Resistance Genes

[0055] Applying the genetic resource for resistance, here referred to as Res, an F1S1 population was made by crossing the source of resistance to the sus 1 carrot line, after which the resulting F1 plant was self-pollinated.

[0056] At least 2000 seeds were harvested from the F1S1 generation of a cross between the distinctive source of resistance and the sus 1 carrot line. To perform a QTL mapping, 240 plants of the cross were tested with the Mi-WU-1 isolate. From each individual plant, leaf material was used for DNA isolation and successive marker analysis.

[0057] Using SNP markers covering the entire genome, a QTL was found on chromosome 4 (C4). This QTL is defined by the SNP markers listed in the table below.

TABLE-US-00003 TABLE 3 SNPs for the detection of the tropical root-knot nematode resistance providing genomic fragment on chromosome 4. The genomic position of each SNP was determined using the published genome assembly PRJNA268187 Daucus carota Ver2. Position on Allele linked Alternative SNP ID Chromosome chromosome (bp) to resistance allele 1 9847 4 10231839 G A 2 6624 4 10367643 C A 3 9848 4 10375226 A G 4 9849 4 10503519 C T 5 6873 4 10507269 C T 6 9850 4 10627267 A G 7 6524 4 10667421 A G 8 8738 4 10674185 T C 9 2281 4 10705926 T G 10 8363 4 10734365 C T 11 9851 4 10753940 T G 12 7147 4 10957339 A G

TABLE-US-00004 TABLE4 Sequencesforthedetectionoftheresistance againsttropicalroot-knotnematodeson chromosome4.Thegenomicpositionofeach SNPwasdeterminedusingthepublishedgenome assemblyPRJNA268187DaucuscarotaVer2. Nucleotidebasecodesareaccordingtothe InternationalUnionofPureandApplied Chemistry(IUPAC)code(seeTable5). SEQ Genomic ID position No. SNP ofSNP* Nucleicacidsequence 1 1 CHR4_ TGTTTGAGACTCWACTCTTCTCCTTTACCAACG 10231839 AAAGTATTTATTTGTGA[G]CAATCTTTCATGTCA AACCTAGCAAGTACCTTCTCAATATGGCTCTTTT A 2 1 CHR4_ TGTTTGAGACTCWACTCTTCTCCTTTACCAACG 10231839 AAAGTATTTATTTGTGA[A]CAATCTTTCATGTCA AACCTAGCAAGTACCTTCTCAATATGGCTCTTTT A 3 2 CHR4_ TCCTATCTCTCTTCCTCCTCACAATCCTTCCCATT 10367643 CT[C]CACTCCTTACCAGCCATACCAGACCCAAC CACA 4 2 CHR4_ TCCTATCTCTCTTCCTCCTCACAATCCTTCCCATT 10367643 CT[A]CACTCCTTACCAGCCATACCAGACCCAAC CACA 5 3 CHR4_ ATTATACTTATACTTGTTTAAATATGTGGTAACT 10375226 TTGGGTGCCAATCACT[A]GAGCAAATTTTTATG TAAAAGTTGTCAAAAATACAAACAAAGRGAGA GAA 6 3 CHR4_ ATTATACTTATACTTGTTTAAATATGTGGTAACT 10375226 TTGGGTGCCAATCACT[G]GAGCAAATTTTTATG TAAAAGTTGTCAAAAATACAAACAAAGRGAGA GAA 7 4 CHR4_ CTTCACAACTATTTACHAAGTAATTTAAAACTTC 10503519 TTTAATCTGTGAAGCT[C]GATATCTTATTGCTTT AACTCCTATAACCCGACWTTCTCAACATATTTC T 8 4 CHR4_ CTTCACAACTATTTACHAAGTAATTTAAAACTTC 10503519 TTTAATCTGTGAAGCT[T]GATATCTTATTGCTTT AACTCCTATAACCCGACWTTCTCAACATATTTC T 9 5 CHR4_ CGACCAATGATATCAGTTTTTTCGACGATAATA 10507269 ATCT[C]GGGTCGTTAGAATTGGATTCTCTCTGGA CTTGA 10 5 CHR4_ CGACCAATGATATCAGTTTTTTCGACGATAATA 10507269 ATCT[T]GGGTCGTTAGAATTGGATTCTCTCTGGA CTTGA 11 6 CHR4_ TTATATACTGTATAAAACAATGATTAGTGCAAC 10627267 AAAAATTAGTGATAGTC[A]TAGTTATAAATACT GGTAAGGAAACTAGTTTTTAGTTTGTATTTAAG AGT 12 6 CHR4_ TTATATACTGTATAAAACAATGATTAGTGCAAC 10627267 AAAAATTAGTGATAGTC[G]TAGTTATAAATACT GGTAAGGAAACTAGTTTTTAGTTTGTATTTAAG AGT 13 7 CHR4_ ATCCGTCAATTAGAAAAAGTTAAAAAACACTCT 10667421 CTCT[A]GACACCCATTCTCATTTGAAGCAACAC GTGATG 14 7 CHR4_ ATCCGTCAATTAGAAAAAGTTAAAAAACACTCT 10667421 CTCT[G]GACACCCATTCTCATTTGAAGCAACAC GTGATG 15 8 CHR4_ GCTACAACTTTTATCTTTTTCAGTCTTTACATGA 10674185 AGTAMTAGCCGTAAAT[T]TTKCAAGCCAAAGGT GCAACACATTGTAGACTCGACAATATCTTTCAC TC 16 8 CHR4_ GCTACAACTTTTATCTTTTTCAGTCTTTACATGA 10674185 AGTAMTAGCCGTAAAT[C]TTKCAAGCCAAAGGT GCAACACATTGTAGACTCGACAATATCTTTCAC TC 17 9 CHR4_ CACTTGTCCTGAAGAATGTCAAGGCAAATATTC 10705926 CCATACTGGTCAACATT[T]GGATGGAAGCACAT TGTCTCAAACTTCACTTGGGGAGGCTTGAAAGG ATA 18 9 CHR4_ CACTTGTCCTGAAGAATGTCAAGGCAAATATTC 10705926 CCATACTGGTCAACATT[G]GGATGGAAGCACAT TGTCTCAAACTTCACTTGGGGAGGCTTGAAAGG ATA 19 10 CHR4_ TAAGGCTTGCRTTGGMTTAAARAGTTGCAGCAT 10734365 CGACGTATCAGTGTCAA[C]TTTTGGGAATCCGT GTAGAGGAGTTACAAAGAGTTTAGCAGTAGAA GCWT 20 10 CHR4_ TAAGGCTTGCRTTGGMTTAAARAGTTGCAGCAT 10734365 CGACGTATCAGTGTCAA[T]TTTTGGGAATCCGT GTAGAGGAGTTACAAAGAGTTTAGCAGTAGAA GCWT 21 11 CHR4_ ATGACACCAACACGGTGAAAGTCAGTGTATCTG 10753940 GCCTTTTGGAGGTGGAT[T]TGAAGGTGACTCCA ATAAAAGAAAAGGAAAACAAGGTGCACAACTA CCAG 22 11 CHR4_ ATGACACCAACACGGTGAAAGTCAGTGTATCTG 10753940 GCCTTTTGGAGGTGGAT[G]TGAAGGTGACTCCA ATAAAAGAAAAGGAAAACAAGGTGCACAACTA CCAG 23 12 CHR4_ CCATACCCATTCTTTTGAAATGAAAATGCAAAA 10957339 CAGA[A]GTCTTCCAGTAGCTCTCTACCTGCATTT GACAA 24 12 CHR4_ CCATACCCATTCTTTTGAAATGAAAATGCAAAA 10957339 CAGA[G]GTCTTCCAGTAGCTCTCTACCTGCATTT GACAA *CHR4 =chromosome 4

TABLE-US-00005 TABLE 5 Nucleotide base codes according to the International Union of Pure and Applied Chemistry (IUPAC) code. 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 Nor C B Not A

Example 5: QTL on C4 is Not Allelic to Resistance on Chromosome 4 Previously Discovered in SFF

[0058] Several individual plants from the SPF line were used to make crosses with the sus 1 carrot line. These individual plants were also self-pollinated. The progeny was tested for resistance. The selfings of the individual plants from SFF still show a level of resistance, while all hybrids are fully susceptible.

[0059] In contrast, when similar crosses were made with Res plants, the progeny of the selfings showed a high level of resistance. Also, hybrids made by crossing with the susceptible sus 1 and sus 2 carrot lines still showed a level of resistance.

[0060] From these tests, it is clear that the previously described QTL on C4 derived from SFF does not provide any level of resistance when present in a heterozygous form, in contrast to the QTL derived from Res.

TABLE-US-00006 TABLE 6 Several selfings of SFF and Res as well as crosses with susceptible carrot lines were tested with a Meloidogyne incognita population obtained from the nematology group at Wageningen University (Mi-WU-1). Name # plants AVG STD % ?2 % ?5 Res*Res 6 1.8 0.8 83% 100% Sus 1*Sus 1 8 50.0 0.0 0% 0% Sus 2*Sus 2 8 42.1 13.8 0% 0% Sus 1*Res (C4) 235 15.1 9.6 2% 15% SFF*SFF 10 8.0 4.1 10% 20% Sus 1*SFF 14 50.0 0.0 0% 0%
Explanation of abbreviations used: #plants is the number of plants tested, AVG is the average number of galls counted per plant under a microscope; STD is the standard deviation of the number of galls counted per plant under a microscope; % ?2 is the percentage of plants that had 2 or fewer galls; % ?5 is the percentage of plants that had 5 or fewer galls.

Example 6: QTL on C4 can be Combined With QTL on C8 to Further Enhance Resistance

[0061] Several resistances against Meloidogyne javanica and Meloidogyne incognita have been previously described in literature (e.g., Parsons et al. 2015). As the QTL on chromosome 8 (C8), referred to as Mj-1, has been described as a monogenic dominant trait that imparts resistance to Meloidogyne javanica and a partial resistance, plants carrying a tropical root-knot nematode resistance providing genomic fragment on C4 and C8 were combined and tested for resistance to determine whether this provided an even more robust resistance to tropical root-knot nematodes.

[0062] The tests showed that the resistance against tropical root-knot nematodes was further improved by combining the QTLs on C4 and C8.

TABLE-US-00007 TABLE 7 Carrot plants with a QTL on C4, C4 and C8 and only C8 were tested for root-knot nematode resistance. Mi-WU-1 is a Meloidogyne incognita population obtained from the nematology group at Wageningen University. MAU is a Meloidogyne arenaria population originating from North America. ID Locus Isolate #plants AVG STD % ? 2 % ? 5 Y9380 C4 Mi-WU-1 89 0.8 2.9 93 99 Y9381 C4, C8 Mi-WU-1 24 0.1 0.4 100 100 Y9383 C8 Mi-WU-1 176 23.0 16.1 4 10 Y9380 C4 MAU 68 1.9 3.7 74 97 Y9381 C4, C8 MAU 18 0.2 0.7 95 100 Y9383 C8 MAU 176 7.0 9.3 42 61
Explanation of abbreviations used: #plants is the number of plants tested, AVG is the average number of galls counted per plant under a microscope; STD is the standard deviation of the number of galls counted per plant under a microscope; % ?2 is the percentage of plants that had 2 or fewer galls; % ?5 is the percentage of plants that had 5 or fewer galls

[0063] SNP markers were used to detect the tropical root-knot resistance providing genomic fragment on C8 and to identify and provide plants carrying this resistance providing fragment.

TABLE-US-00008 TABLE 8 SNPs for the detection of the tropical root-knot nematode resistance providing genomic fragment on chromosome 8. The genomic position of each SNP was determined using the published genome assembly PRJNA268187 Daucus carota Ver2. Position on Allele linked Alternative SNP ID Chromosome chromosome (bp) to resistance allele 13 7136 8 17676345 C G 14 6692 8 17781631 G A 15 4421 8 17968674 C A 16 6419 8 18836406 G C 17 5095 8 21101599 T C 18 4255 8 21235813 A G 19 5020 8 21254357 T C 20 4441 8 21422127 C T

TABLE-US-00009 TABLE9 Sequencesforthedetectionoftheresistance againsttropicalroot-knotnematodeson chromosome8.Thegenomicpositionofeach SNPwasdeterminedusingthepublishedgenome assemblyPRJNA268187DaucuscarotaVer2. Nucleotidebasecodesareaccordingtothe InternationalUnionofPureandApplied Chemistry(IUPAC)code(seeTable5). SEQ Genomic ID position No. SNP ofSNP* Nucleicacidsequence 25 13 CHR8_ AAACATTAAACTTGGCGGATGATGATATATATA 17676345 ATGC[C]GTGCTACTTGTTGCAGCTGCTTATAATC ACTTT 26 13 CHR8_ AAACATTAAACTTGGCGGATGATGATATATATA 17676345 ATGC[G]GTGCTACTTGTTGCAGCTGCTTATAATC ACTTT 27 14 CHR8_ GATCGAAGGGGGTGCGGCCAGAGGTCTTTGCCC 17781631 GAGC[G]TATGCCCGTGTCTTCTCCTCGTTACTTG TCCTT 28 14 CHR8_ GATCGAAGGGGGTGCGGCCAGAGGTCTTTGCCC 17781631 GAGC[A]TATGCCCGTGTCTTCTCCTCGTTACTTG TCCTT 29 15 CHR8_ CTCTCGGRACCCAACCAATTACCCTACGAACTT 17968674 TCTC[C]TCGAAGAGTACTACACATGTTTTTGCTG CCTCA 30 15 CHR8_ CTCTCGGRACCCAACCAATTACCCTACGAACTT 17968674 TCTC[A]TCGAAGAGTACTACACATGTTTTTGCTG CCTCA 31 16 CHR8_ GAGAATCTGCCGTACGGCGTGTTCAAGCCTGAC 18836406 GAAT[G]TTCGGCACCTCGACCTGGCGTTGCTAT CGGRGA 32 16 CHR8_ GAGAATCTGCCGTACGGCGTGTTCAAGCCTGAC 18836406 GAAT[C]TTCGGCACCTCGACCTGGCGTTGCTAT CGGRGA 33 17 CHR8_ GCCAAAGGCTACTACAAAGTGGTTGCAAGTTTC 21101599 AAAA[T]TGATGCACAAGGGTGCTGTGTCAAGTG AAGATC 34 17 CHR8_ GCCAAAGGCTACTACAAAGTGGTTGCAAGTTTC 21101599 AAAA[C]TGATGCACAAGGGTGCTGTGTCAAGTG AAGATC 35 18 CHR8_ GAAAATATCATCGCCACGAGATAATTTTTGCTG 21235813 ATCA[A]CAAAGACTTGAGAGTAACATTCGACTT CCCGTG 36 18 CHR8_ GAAAATATCATCGCCACGAGATAATTTTTGCTG 21235813 ATCA[G]CAAAGACTTGAGAGTAACATTCGACTT CCCGTG 37 19 CHR8_ TTTTACATAGTTACAATTAGAAATTCAGATCCC 21254357 ACCT[T]TTAGACTCCAACCTCTCCGGCTAACTTG AAAGG 38 19 CHR8_ TTTTACATAGTTACAATTAGAAATTCAGATCCC 21254357 ACCT[C]TTAGACTCCAACCTCTCCGGCTAACTTG AAAGG 39 20 CHR8_ TACAACTCGATGCTTATATATCACGAAAAGAAC 21422127 TCTT[C]AAGGCAGAAGCAAAATATTGGAATGAA ATAATA 40 20 CHR8_ TACAACTCGATGCTTATATATCACGAAAAGAAC 21422127 TCTT[T]AAGGCAGAAGCAAAATATTGGAATGAA ATAATA *CHR8 =chromosome 8

Example 7: Introduction of Tropical Root-Knot Resistance Into a Carrot Plant Using Agrobacterium

[0064] Transformation of plants using the Agrobacterium tumefaciens system is commonly used to generate plants resistant to pathogens by introducing resistance genes in plants. The Agrobacterium tumefaciens system can also be used to introduce a tropical root-knot resistance providing genomic fragment according to the present invention. Such a fragment is isolated from gels or columns after restriction digestion of a carrot plant comprising a first tropical root-knot nematode resistance providing genomic fragment. The digested fragment can be introduced in a single step or in a series of transformations using Agrobacterium tumefaciens into carrot plant cells. After transformation, stable transformants comprising the resistance of the present invention will be selected by performing a disease test or by determining the presence of the resistance providing genomic fragment using molecular markers.

Deposit Information

[0065] Seed samples of the sources of resistance mentioned above have been deposited at the NCIMB (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, Scotland, AB21 9YA) on 9 Jun. 2021 as NCIMB 43792.