Resistance to ToLCNDV in melons

Abstract

The present invention is directed to melon plant and seed, namely of C. melo subsp. melo, which are resistant to ToLCNDV, comprising in their genome introgressed sequences from C. melo subsp. agrestis var. acidulous conferring resistance to said virus, when present homozygously. The introgressed sequences are preferably characterized by defined alleles of SNPs on chromosome 11, inter alia allele A of SNP Melon_sbg_14207_58 (SEQ ID No:9). The introgressed sequences can be chosen from those present in the genome of a plant of ToLR1 accession number NCIMB 42506. The invention is also directed to parts of these resistant plants, as well as progeny, to the use of these plants for introgressing the resistance in another genetic background, as well as to different methods for obtaining resistant melon plants or seeds.

Claims

1. A Cucumis melo subsp. melo plant resistant to Tomato leaf curl New Delhi virus (ToLCNDV), having in its genome introgressed sequences from C. melo subsp. agrestis var. acidulous, conferring said resistance only when present homozygously, wherein said introgressed sequences are: (1) located on homologous chromosome 11, within the chromosomal region delimited by (a) SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, and (b) SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12, and (2) the same as the sequence present in the genome of a resistant C. melo seed, deposited as NCIMB accession number NCIMB-42506, on chromosome 11 within the chromosomal region delimited by SNP Melon_sbg_33761_74 and SNP Melon_sbg_22016_36.

2. The plant according to claim 1, wherein the presence of said introgressed sequences on chromosome 11 and conferring said resistance is characterized by at least one marker selected from the group consisting of SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, SNP Melon_sbg_2720_78, the sequence of which is set forth in SEQ ID No:8, SNP Melon_sbg_14207_58, the sequence of which is set forth in SEQ ID No:9, SNP Melon_sbg_22016_27, the sequence of which is set forth in SEQ ID No:10, SNP Melon_sbg_22016_30, the sequence of which is set forth in SEQ ID No:11, and SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12.

3. The plant according to claim 2, wherein the presence of said introgressed sequence is characterized by the presence of allele T of SNP Melon_sbg_33761_74 and/or the presence of allele G of SNP Melon_sbg_2720_78 and/or the presence of allele A of SNP Melon_sbg_14207_58 and/or the presence of allele G of SNP Melon_sbg_22016_27 and/or the presence of allele G of SNP Melon_sbg_22016_30 and/or the presence of allele G of SNP Melon_sbg_22016_36.

4. The plant according to claim 1, wherein the presence of said introgressed sequences on homologous chromosome 11 is characterized by: a) the detection of allele T of SNP Melon_sbg_33761_74 combined with the lack of detection of allele C for said SNP; and/or b) the detection of allele G of SNP Melon_sbg_2720_78 combined with the lack of detection of allele A for said SNP; and/or c) the detection of allele A of SNP Melon_sbg_14207_58 combined with the lack of detection of allele C for said SNP; and/or d) the detection of allele G of SNP Melon_sbg_22016_27 combined with the lack of detection of allele C for said SNP; and/or e) the detection of allele G of SNP Melon_sbg_22016_30 combined with the lack of detection of allele A for said SNP; and/or f) the detection of allele G of SNP Melon_sbg_22016_36 combined with the lack of detection of allele A for said SNP.

5. The plant according to claim 1, wherein said plant is a progeny of, or is derived from a deposited seed under NCIMB accession number 42506.

6. A Hybrid plant of Cucumis melo subsp. melo, obtained by crossing a plant resistant to ToLCNDV according to claim 1 with another C. melo subsp. melo.

7. The plant according to claim 1, wherein said plant can bear marketable melon fruits.

8. A cell of a Cucumis melo subsp. melo plant according to claim 1, comprising in its genome introgressed sequences from C. melo subsp. agrestis var. acidulous on chromosome 11, wherein said introgressed sequences: (1) confer resistance to ToLCNDV only when present homozygously on homologous chromosome 11, within the chromosomal region delimited by (a) SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, and (b) SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12, and (2) are the same as the sequence present in the genome of a resistant C. melo seed, deposited as NCIMB accession number NCIMB-42506, on chromosome 11 within the chromosomal region delimited by SNP Melon_sbg_33761_74 and SNP Melon_sbg_22016_36.

9. A plant part, explant, scion, cutting, seed, fruit, root, rootstock, pollen, ovule, embryo, siliqua, protoplast, leaf, anther, stem or petiole of a C. melo subsp. melo plant having in its genome introgressed sequences conferring resistance to ToLCNDV only when present homozygously, wherein said plant part, explant, scion, cutting, seed, fruit, root, rootstock, pollen, ovule, embryo, siliqua, protoplast, leaf, anther, stem or petiole is obtained from a plant according to claim 1 and comprises cells comprising in their genome introgressed sequences from C. melo subsp. agrestis var. acidulous on chromosome 11, wherein said introgressed sequences: (1) confer resistance to ToLCNDV only when present homozygously on homologous chromosome 11, within the chromosomal region delimited by (a) SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, and (b) SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12, and (2) are the same as the sequence present in the genome of a resistant C. melo seed, deposited as NCIMB accession number NCIMB-42506, on chromosome 11 within the chromosomal region delimited by SNP Melon_sbg_33761_74 and SNP Melon_sbg_22016_36.

10. A seed of a Cucumis melo subsp. melo plant, giving rise when grown up to a plant according to claim 1.

11. A tissue culture of regenerable cells of the plant according to claim 1, wherein the regenerable cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, siliqua, seeds, flowers, cotyledons, and/or hypocotyls, and contain in their genome introgressed sequences from C. melo subsp. agrestis var. acidulous on chromosome 11 conferring resistance to ToLCNDV only when present homozygously, wherein said introgressed sequences: (1) are located on homologous chromosome 11, within the chromosomal region delimited by (a) SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, and (b) SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12, and (2) are the same as the sequence present in the genome of a resistant C. melo seed, deposited as NCIMB accession number NCIMB-42506, on chromosome 11 within the chromosomal region delimited by SNP Melon_sbg_33761_74 and SNP Melon_sbg_22016_36.

12. The seed or plant part according to claim 9, wherein the presence of said introgressed sequences on chromosome 11 is characterized by: a) the presence of allele T of SNP Melon_sbg_33761_74; and/or b) the presence of allele G of SNP Melon_sbg_2720_78; and/or c) the presence of allele A of SNP Melon_sbg_14207_58; and/or d) the presence of allele G of SNP Melon_sbg_22016_27; and/or e) the presence of allele G of SNP Melon_sbg_22016_30; and/or f) the presence of allele G of SNP Melon_sbg_22016_36.

13. A method for detecting and selecting C. melo subsp. melo plants according to claim 1, comprising detecting at least one of the following markers: allele T of SNP Melon_sbg_33761_74 and/or allele G of SNP Melon_sbg_2720_78 and/or allele A of SNP Melon_sbg_14207_58 and/or allele G of SNP Melon_sbg_22016_27 and/or allele G of SNP Melon_sbg_22016_30 and/or allele G of SNP Melon_sbg_22016_36, in a sample of genetic material from such plants, and selecting plants in which at least one such marker is detected.

14. A method for breeding Cucumis melo subsp. melo plants resistant to ToLCNDV, comprising the steps of crossing a plant grown from the deposited seeds NCIMB 42506 or progeny thereof bearing the sequences conferring the resistance to ToLCNDV, with a Cucumis melo subsp. melo plant susceptible or less resistant to ToLCNDV, wherein said sequences are located on homologous chromosome 11, within the chromosomal region delimited by (a) SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, and (b) SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12, and are introgressed from the deposited seeds NCIMB 42506.

15. A method for the production of Cucumis melo subsp. melo plants resistant to ToLCNDV, comprising the steps of: a) crossing a plant grown from the deposited seeds NCIMB 42506, or progeny thereof bearing the sequences conferring the resistance to ToLCNDV, and a susceptible or less resistant Cucumis melo subsp. melo plant, wherein said sequences are located on homologous chromosome 11, within the chromosomal region delimited by (a) SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, and (b) SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12, and are introgressed from the deposited seeds NCIMB 42506; b) selecting a plant in the progeny thus obtained bearing sequences conferring resistance to ToLCNDV only when present homozygously; c) self-pollinating one or several times the plant obtained at step b) and selecting a resistant plant in the progeny thus obtained.

16. The method of claim 15, wherein SNPs markers are used in steps b) and/or c) for selecting plants bearing sequences conferring resistance to ToLCNDV only when present homozygously and/or plants resistant to ToLCNDV.

17. A method for the production of Cucumis melo subsp. melo plants resistant to ToLCNDV comprising the steps of: a1) crossing a plant grown from the deposited seeds NCIMB 42506 or progeny thereof bearing the sequences conferring the resistance to ToLCNDV, and a susceptible or less resistant C. melo subsp. melo plant, thus generating the F1 population, wherein said sequences are located on homologous chromosome 11, within the chromosomal region delimited by (a) SNP Melon_sbg_33761_74, the sequence of which is set forth in SEQ ID No:7, and (b) SNP Melon_sbg_22016_36, the sequence of which is set forth in SEQ ID No:12, and are introgressed from the deposited seeds NCIMB 42506, a2) advancing the F1 population to create F2 population, b) selecting resistant individuals in the progeny thus obtained; c) optionally self-pollinating one or several times the resistant plant obtained at step b) and selecting a resistant plant in the progeny thus obtained; d) optionally backcrossing the resistant plant selected in step b) or c) with a Cucumis melo subsp. melo plant susceptible to ToLCNDV infection, e) selecting in the progeny a plant bearing sequences linked to the resistance to ToLCNDV, f) self-pollinating the plant obtained at step e) or crossing distinct plants obtained at step e), one or several times, and g) selecting a plant resistant to ToLCNDV.

18. The method of claim 17, wherein SNPs markers are used for selecting plants bearing the introgressed sequences linked to resistance to ToLCNDV only when present homozygously or for selecting plants resistant to ToLCNDV.

19. The method according to claim 14, wherein the selection is carried out by detection of at least one of the following alleles: allele T of SNP Melon_sbg_33761_74 , allele G of SNP Melon_sbg_2720_78, allele A of SNP Melon_sbg_14207_58, allele G of SNP Melon_sbg_22016_27, allele G of SNP Melon_sbg_22016_30 and allele G of SNP Melon_sbg_22016_36.

20. The method according to claim 14, wherein the selection of a resistant plant is carried out by the detection of: a) the presence of allele T of SNP Melon_sbg_33761_74 combined with the absence of allele C for said SNP; and/or b) the presence of allele G of SNP Melon_sbg_2720_78 combined with the absence of allele A for said SNP; and/or c) the presence of allele A of SNP Melon_sbg_14207_58 combined with the absence of allele C for said SNP; and/or d) the presence of allele G of SNP Melon_sbg_22016_27 combined with the absence of allele C for said SNP; and/or e) the presence of allele G of SNP Melon_sbg_22016_30 combined with the absence of allele A for said SNP; and/or f) the presence of allele G of SNP Melon_sbg_22016_36 combined with the absence of allele A for said SNP.

Description

LEGEND OF FIGURES

(1) FIG. 1 is the Manhattan plot showing mapping results of RIL population. Vertical axis (y-axis) shows the log 10 (p-value) and horizontal axis (x-axis) represents all SNPs by their positions (in centimorgan CM) by chromosomes along the genetic map.

(2) FIG. 2 illustrates a zooming of FIG. 1 in chromosome 11. Vertical axis shows log 10 (p-value) and horizontal axis represent all SNPs by their position (Mbp) as mapped on the Melon genome.

(3) FIG. 3 is a mosaic plot showing frequencies of phenotypes for each genotypic group determined by Melon_sbg_14207_58. Over all this SNP predicts the phenotype of 81% from RIL. R (resistant) and S (susceptible) proportions are denoted by white and black colors, respectively.

EXAMPLES

Example 1: ToLCNDV Insect Mediated Inoculation and Disease Screening

(4) Tomato leaf curl New Delhi virus (ToLCNDV) is caused by a whitefly transmitted geminivirus. The identification of source of resistance involves the development of a phenotyping system based on the four following aspects that have to be validated for repeatability and reliability: the virus growth and maintenance, the vector growth and maintenance, the inoculation and the scoring methodologies.

(5) Virus Growth and Maintenance

(6) The Tomato leaf curl New Delhi virus (ToLCNDV) was maintained on isolated infected squash plants (Cucurbita pepo) grown in an insect-proof cages, with natural lighting and temperature control (25 C. during the day and 20 C. during the night). Cultures of the ToLCNDV were maintained on susceptible squash plants (Cucurbita pepo) Victoria F1 (HM.Clause) (thus confirming that the ToLCNDV is indeed a strain, giving rise to the severe symptoms described above).

(7) It must be noted that the presence of ToLCNDV was confirmed using PCR as described in Ruiz et al. 2015 and the absence of CVYV and CYSDV confirmed according to Ruiz et al. 2006.

(8) Whitefly Maintenance

(9) Whitefly (Bemisia tabaci, biotype B) colonies were reared on eggplant (Solanum melongena L.) plants grown in muslin-covered cages maintained inside an insect-proof greenhouse with natural lighting and temperature control (25 C. during the day and 20 C. during the night). Adult whiteflies were provided with a 48-h acquisition access period on ToLCNDV infected squash plants (Cucurbita pepo) Victoria F1.

(10) Inoculation of Plants

(11) The acquisition access period was followed by a 72-h inoculation access period on the melon (Cucumis melo) plants to be tested for resistance to ToLCNDV, said plants being at the one true leaf development level. The plant infestation was effective as at least 10 whiteflies per melo plant were observed. Following the inoculation access period, the whiteflies were removed by treating plants with imidacloprid (Confidor; Bayer, Leverkusen, Germany). The plants were transplanted in the greenhouse (insect-proof greenhouse with natural lighting and temperature control (30 C. during the day and 25 C. during the night), during spring and late summer/fall seasons (April and August, respectively), for a six weeks incubation period.

(12) Disease Screening

(13) The development of symptoms (leaves in upper part of plant showing yellowing mosaic, puckering and stunting of the plants) was followed and registered 45 days post inoculation (DPI) using three levels, namely resistant (no symptom), intermediate (moderate yellowing mosaic) or susceptible (symptomatic, with strong yellow mosaic, puckering and stunting).

Example 2: ToLCNDV Mechanical Inoculation

(14) In addition to the insect mediated inoculation described in example 1, the present inventors also developed an artificial test based on mechanical inoculation. Naturally squash plants infected with ToLCNDV were collected in Almeria and the virus was isolated. Infected leaves were kept in a plastic bag at 80 C. up to their use. 1 g of infected leaves is grinded with 4 ml of a 0.03M Na2HPO4 buffer (pH9) containing 0.2% sodium diethyldithiocarbamate with carborundum (7.5%) and activated carbon (10%). Seedlings with first leaf not yet emerged are inoculated on both cotyledons by gentle rubbing and plants are rinsed after inoculation. Plants are then maintained in growth chamber with a day temperature of 25 C. for 14 hours and a night temperature of 20 C. for 10 hours. A second inoculation can be performed one week after the initial one in order to insure infection.

(15) A first scoring is done 3 weeks after inoculation, when the susceptible check show clear symptoms, a second one being done one week later to confirm the reactions. The rating is on a 1 to 9 scale where 1 is for plants having severe symptoms (leaf cupping, plant stunting); 3 is for plants with obvious symptoms such as vein banding or vein clearing, and beginning of lead cupping, 5 if for plants having mild symptoms such as yellow mosaic, 7 is for plants having only slight symptoms, i.e. light yellowing or light leaf curling and 9 is for symptomless plants.

Example 3: Identification of a Source of Resistance to ToLCNDV Through Insect Mediated Inoculation

(16) As a starting point of the realization of the invention, the present inventors have conducted several experiments to screen for ToLCNDV resistance among several Cucumis melo plants, both from Cucumis melo subsp. agrestis and from Cucumis melo subsp. melo. It must be noted that, to date, Cucumis melo subsp. agrestis var. acidulous has not been identified as a possible source of resistance to ToLCNDV.

(17) A panel of 27 commercial Cucumis melo subsp. melo hybrids as well as 8 Cucumis melo subsp. agrestis and Cucumis melo subsp. melo were screened according to the process described in Example 1. A susceptible check was added as control.

(18) TABLE-US-00001 TABLE 1 Plants tested for resistance Nb of Nb of Nb of Total Plant identifier and Resistant Intermediate Susceptible Plant type of plants tested plants plants plants nb H-MLCND-1: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-2: C. melo subsp. 0 0 5 5 melo var. cantalupensis Galia H-MLCND-3: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-4: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-5: C. melo subsp. 0 0 5 5 melo var. cantalupensis Galia H-MLCND-6: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-7: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-8: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-9: C. melo subsp. 0 0 5 5 melo var. cantalupensis Galia H-MLCND-10: C. melo subsp. 0 0 7 7 melo var. cantalupensis Galia H-MLCND-11: C. melo subsp. 0 0 7 7 melo var. cantalupensis Galia H-MLCND-12: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-13: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-14: C. melo subsp. 0 3 6 9 melo var. cantalupensis Galia H-MLCND-15: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-16: C. melo subsp. 0 0 8 8 melo var. cantalupensis Galia H-MLCND-17: C. melo subsp. 0 0 8 8 melo var. cantalupensis Galia H-MLCND-18: C. melo subsp. 0 0 8 8 melo var. cantalupensis Galia H-MLCND-19: C. melo subsp. 0 0 5 5 melo var. cantalupensis Galia H-MLCND-20: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-21: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-22: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-23: C. melo subsp. 0 1 9 10 melo var. cantalupensis Galia H-MLCND-24: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-25: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-26: C. melo subsp. 0 0 10 10 melo var. cantalupensis Galia H-MLCND-27: C. melo subsp. 0 0 10 10 melo var. ameri ananas H-MLCND-28: C. melo subsp. 0 0 10 10 melo var. ameri ananas H-MLCND-31: C. melo subsp. 0 7 2 9 agrestis var. chinemsis H-MLCND-32: C. melo subsp. 4 2 1 7 agrestis var. acidulous H-MLCND-33: Not defined 0 6 0 6 H-MLCND-34: Not defined 0 0 10 10 H-MLCND-35: Not defined 4 5 1 10 H-MLCND-36: Not defined 0 7 0 7

(19) Two resistant leads were identified, namely H-MLCND-32 and H-MLCND-35. Lead H-MLCND-32 was chosen by the present inventors as a possible source of resistance to ToLCNDV. It is a Cucumis melo subsp. agrestis var. acidulous, with white green, firm and crisp flesh with low sugar content (a brix level of 5.5) and no aroma, all such characteristics that would render any melon fruit unmarketable.

Example 4: Phenotyping of a RIL Mapping Population

(20) For a genetic mapping experiment, 82 Recombinant Inbred Lines (RIL) were selected. These are F6-F8 lines developed by Single Seed Descent from a cross between C. melo subsp agrestis var. acidulous H-MLCND-32, with a susceptible line C. melo subsp. melo Vedrantais. All lines were infected as described in the example 1 and then transplanted in the greenhouse and screened for resistance against the ToLCNDV according to the disease screening notation described in example 1. It is to be noted that none of tested plants were scored as intermediate. The experiment was conducted in complete blocks design with 4 replicates of single plants. In addition, one block with un-infected plants were grown in the greenhouse to enable comparison between infected and non-infected plants of each line.

(21) TABLE-US-00002 TABLE 2 level of resistance of the 82 RILs and of the 2 recurrent parents. RIL Phenotype RIL Phenotype RIL Phenotype RIL Phenotype RIL-1 R RIL-2 S RIL-3 S RIL-4 S RIL-5 S RIL-6 S RIL-7 R RIL-8 S RIL-9 R RIL-10 S RIL-11 S RIL-12 R RIL-13 R RIL-14 S RIL-15 S RIL-16 R RIL-17 S RIL-18 R RIL-19 R RIL-20 S RIL-21 S RIL-22 R RIL-23 S RIL-24 S RIL-25 S RIL-26 R RIL-27 S RIL-28 R RIL-29 R RIL-30 R RIL-31 S RIL-32 S RIL-33 R RIL-34 R RIL-35 R RIL-36 S RIL-37 S RIL-38 S RIL-39 R RIL-40 R RIL-41 R RIL-42 S RIL-43 R RIL-44 R RIL-45 S RIL-46 S RIL-47 R RIL-48 R RIL-49 S RIL-50 R RIL-51 S RIL-52 R RIL-53 S RIL-54 S RIL-55 S RIL-56 R RIL-57 R RIL-58 S RIL-59 R RIL-60 S RIL-61 S RIL-62 R RIL-63 R RIL-64 S RIL-65 R RIL-66 R RIL-67 S RIL-68 R RlL-69 R RIL-70 S RIL-71 S RIL-72 S RIL-73 R RIL-74 R RIL-75 R RIL-76 S RIL-77 S RIL-78 R RIL-79 R RIL-80 S RIL-81 S RIL-82 R HMLCND-32 R Vendrantais S R stands for resistant, S stands for susceptible.

Example 5: Confirmation of the Resistance

(22) A plant derived from the same C. melo subsp. agrestis var. acidulous H-MLCND-32 was used in a ToLCNDV mechanical inoculation pathology test according to example 2 and resulted in the confirmation of the resistance.

Example 6: Genetic Determinism of the Resistance

(23) A test was performed to identify the genetic determinism of the resistance identified in C. melo subsp. agrestis var. acidulous H-MLCND-32: The plant of example 5 was crossed with a susceptible melon proprietary line and the F1 progenies plants thereof were inoculated and scored according to the protocol of example 2, whereby scoring of 1 or 3 will be considered as susceptible plants, 5 would be intermediate resistance plants having mild symptoms and 7 and 9 would be for resistant plants. The resistance was preliminary identified as a recessive resistance as all F1 plants scored susceptible.

(24) The scoring was repeated on F2 plants that confirmed the preliminary identification as a monogenic recessive inheritance of the resistance ( of resistant plants and of susceptible ones).

(25) TABLE-US-00003 TABLE 3 scoring of F2 plants. Nb of Observed Expected p- Plant plants R IR S ratio ratio value Susceptible 26 0 0 26 0:26 0:26 control Susceptible 10 0 0 10 0:10 0:10 parent Resistant 10 9 1 0 9:1 1:0 0.3049 parent F1 plant 10 0 0 10 0:10 0:10 F2 plant 206 39 9 158 39:167 52:154 0.1226 R = resistant; S = susceptible, IR = intermediate resistant

(26) Each plant was scored and analyses were done using JMP software (SAS) (statistical software). Those qualitative data were analyzed according non parametric statistic tests (Khi.sup.2). The p value shows that the differences between the expected ratio and the observed ratio for a recessive gene are not significatively different, from a statistical point of view.

Example 7: Development of Genetic Data and Molecular Markers

(27) SNP Genotyping (Sequence-Based Genotyping)

(28) The sequencing procedure was started with an in silico analysis to select the best Primer Combination (PC), i.e. to choose a PC that generates the least fragments in organelles. Next, DNA of RILs and the two parents were used to generate one library based on the High Throughput AFLP-based polymorphism detection method developed by Keygene (see WO2007/073165 and WO2008/007951). In the present case, the DNA was digested by EcoRI and MseI and adapters were added. These adapters further contain selective bases allowing to reduce the genome to be sequenced. Accordingly to the nomenclature described by Keygene, the library is an EcoRI/MseI+0/+2, meaning that no selective base was added to the 3 end of the primer complementary to the restriction site made by the EcoRI digestion while 2 selective bases have been added to the 3 end of the primer complementary to the restriction site made by the MseI. The library was prepared for Illumina single-end sequencing on the HiSeq system. This library was used for SNP discovery and genotyping. Illumina reads were filtered on quality, presence of sample identification tags and the restriction site motif. From the remaining reads, sample identification tags were removed. Secondly a reference was created: reads from all samples that passed the pre-processing were combined and grouped. Ideally, each cluster represents a single restriction fragment and each restriction fragment is represented by a single cluster. A single sequence was selected from each cluster as representative of the corresponding restriction fragment. Together, these sequences form the reference set. Subsequently filtered reads were mapped to the reference using the BWA software and genotyped for SNPs using the GATK UnifiedGenotyper software. Further information on Sequence-Based Genotyping as well as kits for performing such processes, such as for example the SBG 100-Kit can be ordered from Keygene B.V.

(29) Identifying Markers Significantly Linked to Resistance to ToLCNDV

(30) Phenotypic data: Phenotypic data was collected as described in example 4.

(31) Linkage analysis: The genotyping information described in the SNP genotyping section and the phenotypic measurements were used as input to linkage analysis via Chi-Square test, (JMP), i.e., for each marker the frequencies of resistant and susceptible lines were compared between genotypic groups.

(32) Results

(33) Linkage analysis identified a single major QTL on chromosome 11 which is defined by set of markers significantly linked to resistance to ToLCNDV. FIG. 1 showing the mapping results of the RIL population illustrates this point. FIG. 2, specific for chromosome 11, illustrates the specific portion of chromosome 11 bearing this major QTL.

(34) The list of associated markers and their significance are summarized in table 4. The SNPs were physically mapped to the public available melon genomic map (https://melonomics.net/files/Genome/Melon_genome_v3.5.1/).

Example 8: Definition of the Introgressed Fragment Conferring Resistance Unlinked to Unmarketable Characteristics

(35) The p-value obtained for each SNP marker, and the phenotype of the different RIL harboring the SNP marker allele linked to the resistance, have been taken into consideration in order to define the introgressed fragment, conferring the resistance to ToLCNDV when present homozygously without conferring unmarketable characteristics. The three lines RIL-30, RIL-69 and RIL-82, having fruits displaying the phenotype of marketable melons have been retained. Their genotype with respect to the SNP identified in the previous examples is reported in table 5.

(36) TABLE-US-00004 TABLE4 SNPslinkedtoToLCNDVresistance,locationandflankingsequences: Loc:Locationonchromosome11-NtR:Nucleotideinresistantbulk,correspondingtoalleles ofH-MLCND-32.NtS:Nucleotideinsusceptiblebulk.Itmustbeborneinmindthat,outside theintrogressionfragmentoftheinvention,delimitedbySNPMelon_sbg_617_42andMelon_sbg_ 16835_17,preferablybyMelon_sbg_33761_74andMelon_sbg_22016_36,theassociationwith susceptible/resistancephenotypeisverylow. Left Right SNP SNP SequencesurroundingtheSNP border border position P-Value NtR NtS SeqID Melon_sbg_ TCACGCCGTGTTTTCAACGCACAAATTGAATT 28,024,900 28,024,988 28,024,985 0.002715 T/T C/C 17 2869_86 TCCGACACCCTTATATTATTATCACCTCCACT CCCCCACCGCCGCCGCCTCTC[C/T]CGC Melon_sbg_ ACCG[G/T]CGACTCATTGGTCCGGCGACAGA 28,404,869 28,404,957 28,404,873 0.005816 T/T G/G 18 3859_5 GGAGAGAGAGAGAGATTGATGCTCAAGCGTG CGAGCATGCGAGGGGAAGGGGTTCGCACGC Melon_sbg_ ACCG[G/T]CGACTCATTGGTCCGGCGACAGA 28,404,869 28,404,959 28,404,873 0.007771 T/T G/G 19 21303_5 GGAGAGAGAGAGATTGATGCTCAAGCGTGCG AGCATGCGAGGGGAAGGGGTTCGCACGCGC Melon_sbg_ GAGGCAGTCTTCTCCCCATGTAGACTTCATTC 28,468,747 28,468,835 28,468,780 0.003917 T/T A/A 20 15226_34 T[T/A]CCCGTGGAATAGATCGAATACGTAACC GATGATCCCGCCGGAACGAAGGTTCCGT Melon_sbg_ GCACCCGATTGATCTTCAAGACGTGTTGTTAC 28,469,424 28,469,512 28,469,463 0.005557 A/A T/T 21 40291_40 GTCTTAC[T/A]TTTGATAATATTTGTGGTTTGG CTTTTGGAAAAGATCCAATGACTTGTG Melon_sbg_ ACCGGGGCGG[C/T]GGCTTTCAGGACGTTGA 28,524,917 28,525,005 28,524,927 0.001933 C/C T/T 22 8091_11 AGGAAAATGAATGGACCAAACGAGAACGACC AAAGTGGGTCGACGTCCAAATCAGCATTTC Melon_sbg_ GGAGCAATGCGACGACGCCGTCACCGTCTG 28,541,411 28,541,499 28,541,475 0.000741 G/G C/C 23 29917_65 ACGTGGAGGAGTTGGATTATGTGGAAGACGA TGA[C/G]GACGATGAGGAGGAGGAGGACGGC Melon_sbg_ CACGGCTGCATTCCTAGCACCAT[T/A]GGCCA 28,547,660 28,547,748 28,547,683 0.001933 T/T A/A 24 1548_24 AATGTCTAACCTCAACCAAATTCTCTTTCTCG GCAACAAGCTCGGTGGTTGTTTCCCACC Melon_sbg_ TTGTTTGGAGAGTCTGGCTTGCGGCGTTCCA 28,815,310 28,815,398 28,815,381 0.000347 A/A G/G 25 22966_49 ACCGTGGCGTTTCCACA[G/A]TGGTCCGATCA AGCGACCAACACTAAGATCATTCAGGACT Melon_sbg_ TTGTTTGGAGAGTCTGGCTTGCGGCGTTCCA 28,815,310 28,815,398 28,815,358 0.000347 G/G C/C 26 22966_72 ACCGTGGCGTTTCCACAGTGGTCCGATCAAG CGACCAACA[C/G]TAAGATCATTCAGGACT Melon_sbg_ AACCAAAGTACCTGTTGGGGCTCTTTCCCGT 28,922,005 28,922,093 28,922,046 0.000867 T/T G/G 1 617_42 CAATCCGGTC[G/T]GGCTCCGCATGTGCCACA GCTACAATGTGGCGTCTGGCCAAACCGGT Melon_sbg_ AACCAAAGTACCTGTTGGGGCTCTTTCCCGT 28,922,005 28,922,093 28,922,088 0,000867 G/G A/A 2 617_84 CAATCCGGTCGGGCTCCGCATGTGCCACAGC TACAATGTGGCGTCTGGCCAA[A/G]CCGGT Melon_sbg_ CTTTGTCGAAAGCGTATGAACGCCGTGCGAG 29,092,886 29,092,974 29,092,967 0.000418 A/A T/T 3 20578_63 TGAAATTCAATTTATGAAACCAATAAATTCA[A/T] AGAAGAATAATAAACATGCTTCTCAT Melon_sbg_ CTTTGTCGAAAGCGTATGAACGCCGTGCGAG 29,092,886 29,092,974 29,092,948 0.000418 C/C A/A 4 20578_82 TGAAATTCAATTTATGAAACCAATAAATTCAAA GAAGAATAATAAACATG[C/A]TTCTCAT Melon_sbg_ GACAACCAACTTTACC[G/A]ATAACCTTCCCG 29,307,652 29,307,740 29,307,668 0.000878 A/A G/G 5 55680_17 ATCTGCTTGGAGGGCACAACGATTCTGAAAA GCACGTCGTGGGCCTTCGCTTGTCTTTTA Melon_sbg_ TATCCCTCCACTCGTCTCATTTATGATGTCCT 29,618,689 29,618,777 29,618,762 8.671E-05 A/A G/G 6 60684_74 CCTTTGGGTGTTTGGGTGCACAACCTATGTTC TTAGCCATG[G/A]TCCTAACCAAACTAA Melon_sbg_ CTCAGATAATCAACCTCTGGTACGACTGGCC 30,171,021 30,171,109 30,171,094 3.580E-07 T/T C/C 7 33761_74_ TAATGGTCGGAACTTCAACATCATCCAACACC AGCTCGGCAA[C/T]GTTCTCTACGACCTC Melon_sbg_ AACCAACACTGTTTCTTGTGAAAATTGTTTGG 30,328,018 30,328,106 30,328,095 7.282E-07 G/G A/A 8 2720_78 TTGTTATATCTGTATGCAGAGGCCTATTATGT GGTGCTTTGATGG[G/A]TGGTTTAGAAT Melon_sbg_ GGCCTAAAAAAATCGTAGCATTATAGAGAAAT 30,335,962 30,336,050 30,336,019 2.523E-07 A/A C/C 9 14207_58 GCAAACAAGGGTAGAAGGGTAGAAG[C/A]GC TTGCCTTGTACAAGAACTCCGCATAGTTA Melon_sbg_ CCACCTGAAGACGTGGAGATCCAACG[G/C]TC 30,391,940 30,392,028 30,391,966 3.437E-06 G/G C/C 10 22016_27 GAGATCGAACCGTAACGGCCTCGCCAAAATC CAACGGCAAAAAACTAGCGAAAGTGGAAG Melon_sbg_ CCACCTGAAGACGTGGAGATCCAACGGTC[G/A] 30,391,940 30,392,028 30,391,969 3.437E-06 G/G A/A 11 22016_30 AGATCGAACCGTAACGGCCTCGCCAAAATC CAACGGCAAAAAACTAGCGAAAGTGGAAG Melon_sbg_ CCACCTGAAGACGTGGAGATCCAACGGTCGA 30,391,940 30,392,028 30,391,975 3.437E-06 G/G A/A 12 22016_36 GATC[G/A]AACCGTAACGGCCTCGCCAAAATC CAACGGCAAAAAACTAGCGAAAGTGGAAG Melon_sbg_ TCGAGCCAAAGA[C/T]TGAAGATCACCATCTT 31,136,530 31,136,618 31,136,542 8.788E-05 C/C T/T 13 11556_13 CACCCATCTGATCTTGATAATCAACTGGGTTC AGAAAGCTATTTGTCATTTCGTTACAAC Melon_sbg_ TGGTGGGATTTGAATCCAAGATGTCTGTCAGT 31,159,064 31,159,152 31,159,108 8.788E-05 C/C G/G 14 24259_45 GCTTCCTCTTCA[C/G]ACCGCAGGGATTGGAC AAAAGCCGGACAGCGCGGTGATGCGCCG Melon_sbg_ CCAG[C/T]GGTCTTCGTCGGCGGCGGCGATT 31,209,200 31,209,288 31,209,204 8.788E-05 T/T C/C 15 16835_5 GAGTCTACACTCCGGTTTTCAACTCCGACGC AGGGCTGCAAATTTGATTATCGAGGGAAAA Melon_sbg_ CCAGCGGTOTTCGTCG[G/T]CGGCGGCGATT 31,209,200 31,209,288 31,209,216 5.811E-05 T/T G/G 16 16835_17 GAGTCTACACTCCGGTTTTCAACTCCGACGC AGGGCTGCAAATTTGATTATCGAGGGAAAA

(37) Seeds of RILs 69 and 82 have been deposited by Hazera Seeds Ltd, Berurim, M. P. Shikmim 79837, Israel, with the NCIMB (NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom), on 23 Dec. 2015, under the name ToLR1 and accession number NCIMB 42506. Some of their characteristics are studied in example 10. They are also characterized by the following features: the flower female distribute along the shoots, the shoots are not thin and long as in the wild-types. The RILs set at internode 8-15 similar to cultural plants, there is no strong branching.

(38) TABLE-US-00005 TABLE 5 SNP RIL-30 RIL-69 RIL-82 H-MLCND-32 Melon_sbg_2869_86 T/T T/T T/T T/T Melon_sbg_3859_5 G/G T/T T/T T/T Melon_sbg_21303_5 G/G T/T T/T T/T Melon_sbg_15226_34 A/A T/T T/T T/T Melon_sbg_40291_40 NA A/A A/A A/A Melon_sbg_8091_11 T/T C/C C/C C/C Melon_sbg_29917_65 C/C G/G G/G G/G Melon_sbg_1548_24 A/A T/T T/T T/T Melon_sbg_22966_49 NA A/A NA A/A Melon_sbg_22966_72 NA G/G NA G/G Melon_sbg_617_42 G/G T/T T/T T/T Melon_sbg_617_84 A/A G/G G/G G/G Melon_sbg_20578_63 T/T A/A A/A A/A Melon_sbg_20578_82 A/A C/C C/C C/C Melon_sbg_55680_17 G/G A/A A/A A/A Melon_sbg_60684_74 NA A/A A/A A/A Melon_sbg_33761_74 T/T T/T T/T T/T Melon_sbg_2720_78 G/G G/G G/G G/G Melon_sbg_14207_58 A/A A/A A/A A/A Melon_sbg_22016_27 G/G G/G G/G G/G Melon_sbg_22016_30 G/G G/G G/G G/G Melon_sbg_22016_36 G/G G/G G/G G/G Melon_sbg_11556_13 C/C C/C C/C C/C Melon_sbg_24259_45 C/C C/C C/C C/C Melon_sbg_16835_5 T/T T/T T/T T/T Melon_sbg_16835_17 T/T T/T T/T T/T

Example 9: Marker Prediction Value

(39) FIG. 3 is a mosaic plot showing frequencies of phenotypes for each genotypic group determined by Melon_sbg_14207_58 (SEQ ID No:9). Over all this SNP predicts the phenotype of 81% from RIL.

Example 10: Cucumis melo Subsp melo Plants Resistant to ToLCNDV

(40) Once the resistance has been identified, the present inventors have been able to obtain Cucumis melo subsp melo plants resistant to ToLCNDV that do not harbor the phenotypic characteristics of the original Cucumis melo subsp agrestis var. acidulous plant, especially in terms of brix and flesh color.

(41) Table 6 illustrates the phenotypic trait of these plants.

(42) TABLE-US-00006 TABLE 6 Resistance to Plant ToLCNDV Brix Fruit shape Flesh color RIL-30 Resistant 12 Oval OR1 RIL-66 Resistant 9 Oval White green RIL 69 Resistant 9 Round-Oval OR1 RIL 82 Resistant 10 Oval OR-Crm Vedrantais Susceptible 12 Round OR1-OR2 H-MLCND-32 Resistant 5.5 Oval White green

(43) In table 6, OR1 standard orange is the standard color of orange flesh in commercial melon.

(44) OR-Crm means that a mix color is in the flesh (patches of cream color).

(45) The scale of orangefrom light to dark:

(46) OR0light orange; OR1standard orange; OR2deep orange; OR3very deep orange

(47) A genetic analysis demonstrates that the resistant plants harbor the introgressed sequences containing the resistance as evidenced by the presence of the SNP identified by the present inventors.

(48) TABLE-US-00007 Plant Melon_sbg_22016_27 Melon_sbg_14207_58 Melon_sbg_2720_78 RIL 30 G/G A/A G/G RIL 66 G/G A/A G/G RIL69 G/G A/A G/G RIL82 G/G A/A G/G Vedrantais C/C C/C A/A H-MLCND-32 G/G A/A G/G

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