CGMMV RESISTANT CITRULLUS PLANTS

Abstract

The present invention relates to cultivated watermelon plants comprising CGMMV resistance due to introgression of QTLs from a CGMMV resistant donor of the species Citrullus colocynthis.

Claims

1. A cultivated watermelon plant or plant cell comprising an introgression fragment on chromosome 9 and/or on chromosome 7 of a CGMMV resistant donor plant of the species Citrullus colocynthis, wherein the introgression fragment on chromosome 9 comprises a CGMMV resistance QTL from a C. colocynthis donor of which a representative sample has been deposited under accession number NCIMB 42624 and wherein the QTL is in-between marker SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome and marker InDel9.1 at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, and wherein the introgression fragment on chromosome 7 comprises a CGMMV resistance QTL from a C. colocynthis donor of which a representative sample has been deposited under accession number NCIMB 42624 and wherein the QTL is in-between marker InDel7.1 at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome and marker SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome.

2. A cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 9 comprises a Cytosine for marker SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome and/or comprises a Guanine for marker InDel9.1 instead of a Guanine-Cytosine at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to a deletion of the Cytosine at nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, and wherein the introgression fragment on chromosome 7 comprises an Adenine for marker InDel7.1 instead of an Adenine-Thymine at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome and/or comprises a Adenine for marker SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome.

3. The cultivated watermelon plant or plant cell according to claim 1, wherein the QTL on chromosome 9 is in-between SNP22 at nucleotide 51 of SEQ ID NO: 22, corresponding to nucleotide 35,343,850 of chromosome 9, and SNP23 at nucleotide 51 of SEQ ID NO: 23, corresponding to nucleotide 36,923,004 of chromosome 9, and wherein the QTL on chromosome 7 is in-between SNP13 at nucleotide 51 of SEQ ID NO: 13, corresponding to nucleotide 4,784,519 of chromosome 7, and SNP16 at nucleotide 51 of SEQ ID NO: 16, corresponding to nucleotide 11,166,694 of chromosome 7.

4. A cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 7 comprises an Adenine at nucleotide 51 of SEQ ID NO: 14 or an Adenine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 14, and/or a Guanine at nucleotide 51 of SEQ ID NO: 15 or a Guanine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 15.

5. The cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 7 is in homozygous or in heterozygous form and/or wherein the introgression fragment on chromosome 9 is in homozygous form or in heterozygous form.

6. The cultivated watermelon plant or plant cell according to claim 1, wherein the introgression fragment is from a Citrullus colocynthis accession, a representative sample of seeds of the accession having been deposited under accession number NCIMB 42624.

7. The cultivated watermelon plant or plant cell according to claim 1, wherein the plant comprises a CGMMV resistance of scale 3 (mild symptoms) or scale 4 (no symptoms).

8. The cultivated watermelon plant or plant cell according to claim 1, wherein the plant or plant cell is an inbred plant or plant cell, or an F1 hybrid plant or plant cell, or a diploid or tetraploid or triploid plant or plant cell.

9. A seed which grows into a plant according to claim 1.

10. A cultivated watermelon fruit or fruit part comprising plant cells according to claim 1.

11. A cultivated watermelon plant propagation material comprising a cultivated watermelon plant cell according to claim 1.

12. A method for producing triploid hybrid cultivated watermelon seeds comprising the following steps: a) providing a first CGMMV resistant inbred diploid watermelon plant comprising two chromosomes 7, each having an introgression fragment from chromosome 7 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor; b) providing a second CGMMV resistant inbred tetraploid watermelon plant comprising four chromosomes 7 each having an introgression fragment from chromosome 7 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor; c) allowing pollination of the tetraploid watermelon plant provided in step b) with pollen of the diploid watermelon plant provided in step a); and d) collecting seeds from the fruits produced in step c).

13. A method for producing triploid hybrid cultivated watermelon seeds comprising the following steps: a) providing a first CGMMV resistant inbred diploid watermelon plant comprising two chromosomes 9, each having an introgression fragment from chromosome 9 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor; b) providing a second CGMMV resistant inbred tetraploid watermelon plant comprising four chromosomes 9 each having an introgression fragment from chromosome 9 according to claim 1 comprising a CGMMV resistance QTL from a C. colocynthis donor; c) allowing pollination of the tetraploid watermelon plant provided in step b) with pollen of the diploid watermelon plant provided in step a); and d) collecting seeds from the fruits produced in step c).

14. A method of screening plants or plant parts or seeds, or DNA derived therefrom, for the presence of a fragment on chromosome 7 comprising a CGMMV resistance QTL and/or on chromosome 9 comprising a CGMMV resistance QTL conferring CGMMV resistance comprises the steps of: a) screening the genomic DNA for the SNP genotype of one or more or all of SNP13 at nucleotide 51 of SEQ ID NO: 13, InDel7.1 at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome, SNP14 at nucleotide 51 of SEQ ID NO: 14, SNP15 at nucleotide 51 of SEQ ID NO: 15, SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome, SNP16 at nucleotide 51 of SEQ ID NO: 16, SNP17 at nucleotide 51 of SEQ ID NO: 17, SNP18 at nucleotide 51 of SEQ ID NO: 18, linked to the QTL on chromosome 7, and/or for one or more of SNP19 at nucleotide 51 of SEQ ID NO: 19, SNP20 at nucleotide 51 of SEQ ID NO: 20, SNP21 at nucleotide 51 of SEQ ID NO: 21, SNP22 at nucleotide 51 of SEQ ID NO: 22, SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome, InDel9.1 at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, SNP23 at nucleotide 51 of SEQ ID NO: 23 and SNP24 at nucleotide 51 of SEQ ID NO: 25, linked to the QTL on chromosome 9; and b) optionally selecting a plant or plant part which comprises on chromosome 9 a Cytosine for marker SNP FM9.1 at nucleotide 71 of SEQ ID NO: 34 or 35, corresponding to nucleotide 35,675,266 of chromosome 9 of the Watermelon Charleston Grey genome and/or a Guanine for marker InDel9.1 instead of a Guanine-Cytosine at nucleotide 91 of SEQ ID NO: 36 or at nucleotide 91 of SEQ ID NO: 37, corresponding to a deletion of the Cytosine at nucleotide 36,439,966 of chromosome 9 of the Watermelon Charleston Grey genome, or selecting a plant or plant part which comprises on chromosome 7 an Adenine for marker InDel7.1 instead of an Adenine-Thymine at nucleotide 91 of SEQ ID NO: 30 or at nucleotide 91 of SEQ ID NO: 31, corresponding to nucleotide 7,022,773 of chromosome 7 of the Watermelon Charleston Grey genome and/or an Adenine at nucleotide 51 of SEQ ID NO: 14 or an Adenine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 14, and/or a Guanine at nucleotide 51 of SEQ ID NO: 15 or a Guanine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 15, and/or an Adenine for marker SNP FM7.1 at nucleotide 71 of SEQ ID NO: 32 or 33, corresponding to nucleotide 8,733,604 of chromosome 7 of the Watermelon Charleston Grey genome.

15. A cultivated watermelon plant or plant cell according to claim 2, wherein the introgression fragment on chromosome 7 comprises an Adenine at nucleotide 51 of SEQ ID NO: 14 or an Adenine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 14, and/or a Guanine at nucleotide 51 of SEQ ID NO: 15 or a Guanine at the equivalent position in a sequence comprising at least 95% sequence identity to SEQ ID NO: 15.

Description

DESCRIPTION OF FIGURES

[0358] FIG. 1: NCIMB 42624 plant used for production of scions and CGMMV infected Citrullus lanatus cultivar Sugar baby plant (picture taken at 25 dpi) used for production of rootstock (A); NCIMB 42624 scion grafted onto CGMMV infected Citrullus lanatus cultivar Sugar baby rootstock, picture taken after 140 dpi shows no symptoms on scion (Scale 4) (B); CGMMV infected Citrullus lanatus cultivar Sugar baby plant, picture taken after 140 dpi shows severe symptoms (C);

[0359] FIG. 2: Example of Citrullus plants showing severe symptoms, leaf deformation, blistering and clear mosaic after inoculation with CGMMV representing scale 1 according visual evaluation of symptoms described under General Methods.

[0360] FIG. 3: Example of Citrullus plants showing medium symptoms, mild leaf deformation but clear mosaic after inoculation with CGMMV representing scale 2 according visual evaluation of symptoms described under General Methods.

[0361] FIG. 4: Example of a Citrullus plant showing mild symptoms, very mild mosaic and few clearing spots after inoculation with CGMMV representing scale 3 according visual evaluation of symptoms described under General Methods.

[0362] FIG. 5: Example of fruit showing severe CGMMV symptoms, the fruit being produced by a CGMMV susceptible Citrullus plant inoculated with CGMMV.

[0363] FIG. 6: Example of fruit showing no symptoms, the fruit being produced by a CGMMV resistant Citrullus plant inoculated with CGMMV.

[0364] FIG. 7A: BLAST output between Query (C. colocynthis sequence SEQ ID NO: 30) against Charleston Grey genome of watermelon (Subject sequence provided herein as SEQ ID NO: 31), showing position of InDel7.1 on chromosome 7.

[0365] FIG. 7B: BLAST output between Query (C. colocynthis sequence SEQ ID NO: 32) against Charleston Grey genome of watermelon (Subject sequence provided herein as SEQ ID NO: 33), showing position of SNP FM7.1 on chromosome 7.

[0366] FIG. 8A: BLAST output between Query (C. colocynthis sequence SEQ ID NO: 34) against Charleston Grey genome of watermelon (Subject sequence provided herein as SEQ ID NO: 35), showing position of SNP FM9.1 on chromosome 9.

[0367] FIG. 8B: BLAST output between Query (C. colocynthis sequence SEQ ID NO: 36) against Charleston Grey genome of watermelon (Subject sequence provided herein as SEQ ID NO: 37), showing position of InDel9.1 on chromosome 9.

DESCRIPTION OF SEQUENCES

[0368] SEQ ID NO: 1 to SEQ ID NO:6 refer to sequences of chromosome 1 linked to QTL1.1 from C. colocynthis comprising the donor nucleotide of SNP1 to SNP6 respectively;

[0369] SEQ ID NO: 7 to SEQ ID NO: 9 refer to sequences of chromosome 4 linked to QTL4.1 from C. colocynthis comprising the donor nucleotide of SNP7 to SNP9 respectively;

[0370] SEQ ID NO: 10 to SEQ ID NO: 12 refer to sequences of chromosome 5 linked to QTL5.2 from C. colocynthis comprising the donor nucleotide of SNP10 to SNP12 respectively;

[0371] SEQ ID NO: 13 to SEQ ID NO: 18 refer to sequences of chromosome 7 linked to QTL7.1 from C. colocynthis comprising the donor nucleotide of SNP13 to SNP18 respectively;

[0372] SEQ ID NO: 19 to SEQ ID NO: 24 refer to sequences of chromosome 9 linked to QTL9.1 from C. colocynthis comprising the donor nucleotide of SNP19 to SNP24 respectively;

[0373] SEQ ID NO 25: Reverse primer used in RT PCR for detection of CGMMV RNA in plant tissue.

[0374] SEQ ID NO 26: Forward primer used in RT PCR for detection of CGMMV RNA in plant tissue.

[0375] SEQ ID NO 27: Sense primer used in quantitative RT PCR for quantification of CGMMV RNA in plant tissue.

[0376] SEQ ID NO 28: Antisense primer used in quantitative RT PCR for quantification of CGMMV RNA in plant tissue.

[0377] SEQ ID NO 29: Probe oligonucleotide used in quantitative RT PCR for quantification of CGMMV RNA in plant tissue.

[0378] SEQ ID NO 30: C. colocynthis sequence comprising a deletion of the Thymine following the Adenine at nucleotide 91 (marker InDel7.1) compared to the Citrullus lanatus sequence of SEQ ID NO: 31.

[0379] SEQ ID NO 31: Citrullus lanatus Charleston Grey sequence comprising a Thymine at nucleotide 92, following the Adenine at nucleotide 91 (marker InDel7.1).

[0380] SEQ ID NO 32: C. colocynthis sequence comprising an Adenine (A) at nucleotide 71 (marker SNP FM7.1).

[0381] SEQ ID NO 33: Citrullus lanatus Charleston Grey sequence comprising a Cytosine (C) at nucleotide 71 (marker SNP FM7.1).

[0382] SEQ ID NO 34: C. colocynthis sequence comprising a Cytosine (C) at nucleotide 71 (marker SNP FM9.1).

[0383] SEQ ID NO 35: Citrullus lanatus Charleston Grey sequence comprising an Adenine (A) at nucleotide 71 (marker SNP FM9.1).

[0384] SEQ ID NO 36: C. colocynthis sequence comprising a deletion of the Cytosine (C) following the Guanine at nucleotide 91 (marker InDel9.1) compared to the Citrullus lanatus sequence of SEQ ID NO: 37.

[0385] SEQ ID NO 37: Citrullus lanatus Charleston Grey sequence comprising a Cytosine at nucleotide 92, following the Guanine at nucleotide 91 (marker InDel9.1).

EXAMPLES

Example 1—General Methods

Example: Mechanical Leaf Inoculation to Evaluate CGMMV Resistance

[0386] Item 1: Preparation of CGMMV Inoculum for Plant Infection

[0387] Inoculum for infection of Citrullus plants was produced from CGMMV susceptible cucumber plants (e.g. variety Sheila, Nunhems B.V.) infected with European type strain Ve459 (CNR, Turin). Fresh young leaves showing clear symptoms of CGMMV infection (symptomatic leaves) were taken off the cucumber plants. Per 1 gram of symptomatic cucumber leaves 5 ml of refrigerated (5° C.) 0.03M phosphate buffer were added and per 1 gram of cucumber leaf 0.05 gram active carbon and 0.1 gram diatomaceous earth was added. The leaf material was crushed with a pestle in a mortar kept ice cold during and after crushing.

[0388] For testing if Citrullus plants are resistant to CGMMV it is advisable to use freshly prepared inoculum from freshly multiplied material.

[0389] Item 2: Infection of (Citrullus) Plants with CGMMV

[0390] Leaf inoculation was done by rubbing the inoculum onto the first true leaf of young seedlings. Thus, the seedlings of (Citrullus) plants were inoculated with inoculum, prepared as described under General Methods Item 1, when the first true leaf is expanded (approximately 15-20 days after sowing). Seedlings were inoculated a second time 4 to 5 days after the first inoculation. Control seedlings are inoculated with buffer only (Mock inoculation). The inoculated plants were kept in indirect sunlight and direct sunlight was avoided until the second inoculation. The temperature regime for maintaining the inoculated seedlings was 18° C. at night time and 25° C. at daytime with 12-14 hours of daylight. Inoculate preferably at least 10 plants per genotype (e.g. CGMMV resistant genotype, susceptible control genotype) in at least two replicates. In addition include a number of plants which are mock inoculated.

[0391] Item 3: Assessment of Disease Symptoms

[0392] Plants having been inoculated were analyzed for symptoms and/or presence for CGMMV virus at the following points in time after the first inoculation (dpi, days post inoculation):

[0393] 15 dpi

[0394] 30 dpi

[0395] 45 dpi

[0396] 60 dpi

[0397] In case desired, fruits can be tested for symptoms and/or presence of CGMMV after e.g. about 140 dpi or even later.

[0398] Control Plants

[0399] Controls have to be included into all test series. It is important to include (Citrullus) plants known to be susceptible to CGMMV (e.g. Citrullus lanatus commercial variety Sugar baby) as a positive control to check for efficiency of infection with CGMMV. Preferably at least 95% of the positive control plants should show severe symptoms (scale 1) on leaves after 20 dpi and preferably fruits produced by these plants should show severe symptoms. In addition, negative control plants should be included represented by CGMMV susceptible plants which have been inoculated with phosphate buffer only as Mock inoculation. In addition, non-inoculated plants can be included into the test series. The negative controls should not show symptoms of CGMMV infection at any time in leaves or fruits and CGMMV protein and/or RNA should not be detectable in Mock inoculated plants.

[0400] Visual Evaluation of CGMMV Symptoms on Leaves

[0401] Visually evaluated symptoms on leaves are classified according to the following scale:

[0402] Scale 1: Severe symptoms: leaf deformation, blistering and clear mosaic (exemplified in FIG. 2)

[0403] Scale 2: Medium symptoms: mild leaf deformation but clear mosaic (exemplified in FIG. 3)

[0404] Scale 3: Mild symptoms: very mild mosaic and few clearing spots (exemplified in FIG. 4)

[0405] Scale 4: No symptoms (exemplified in FIG. 1B)

[0406] Virus detection in inoculated plant tissue: ELISA

[0407] For demonstrating presence or absence of CGMMV in plants an ELISA assay was performed after the second evaluation (30 dpi) for all plants classified as scale 4 by visual evaluation of symptoms. Depending on the results obtained by the ELISA assay, the plants tested were classified according to the following scale:

[0408] Negative by ELISA

[0409] Low positive if Absorbance is <0.5

[0410] Positive by ELISA if Absorbance is >0.5

[0411] Presence or absence of CGMMV in plant material can also be evaluated by RT-PCR or quantified by RT-qPCR detection (see General Methods item 4) and/or by the Tissue print or DotBlot (General methods item 5).

[0412] Item 4: Virus Detection in Inoculated Plant Tissue: PCR Detection of CGMMV in Plant Tissue

[0413] Presence of CGMMV in plant material was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and for quantification of CGMMV in plant material reverse transcriptase quantitative polymerase chain reaction (RT-qPCR, TaqMan) was used. See also Hongyun et al., 2008, J Virological Methods 149, 326-329, incorporated by reference.

[0414] a) Extraction of RNA from Plant Material

[0415] Leaf tissue (200 mg) was ground to a fine powder in liquid nitrogen with a pestle in a mortar and transferred into a sterile microcentrifuge tube. Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's instruction. The resulting RNA pellet was resuspended in 30 μl DEPC-treated water and stored at −80° C. RNA concentration was determined by ND-1000 Spectrophotometer (NanoDrop Technologies).

[0416] b) Reverse Transcriptase PCR (RT-PCR)

[0417] The oligonucleotides used for RT-PCR were designed based on the CGMMV coat protein gene and 3′ noncoding region (NCR) nucleotide sequence available from the EMBL database. The first strand of cDNA was synthesized by the SuperScript II reverse transcriptase (Invitrogen) primed with reverse primer: 5′-TTG CAT GCT GGG CCC CTA CCC GGG GAA AG-3′ (SphI; SEQ ID NO 25). The sequence of forward primer used for PCR is: 5′-CCG AAT TCA TGG CTT ACA ATC CGA TCA C-3′ (EcoRI; SEQ ID NO 26). The thermal cycling scheme was: 3 minutes at 94° C., 35 cycles of 30 seconds at 94° C., 45 seconds at 53° C., 1 minute at 72° C. followed by a final incubation of 7 minutes at 72° C. Expected PCR products of 700 base pairs were cloned into pGEM-3Zf plasmid and positive clones were sequenced in both directions with an automated ABI 3730 DNA sequencer to confirm the amplified sequence to be obtained from CGMMV virus RNA.

[0418] c) Rt-Qpcr

[0419] The primers and probe for the TaqMan assay were designed with the Primer Express software (Applied Biosystems, ver. 2.0), following the procedure as described in the instruction supplied by manufacturer. CGMMV specific primers were 5′-GCA TAG TGC TTT CCC GTT CAC-3′ (sense; SEQ ID NO 27) and 5′-TGC AGA ATT ACT GCC CAT AGA AAC-3′ (antisense; SEQ ID NO 28). The probe was 5′-CGG TTT GCT CAT TGG TTT GCG GA-3′ (SEQ ID NO 29), labelled with 6-carboxyfluorescein (FAM) and 3′ end was labelled with N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA).

[0420] All reactions were carried out in a final volume of 50 μl containing 25 μl 2× Master Mix without UNG, 1.25 μl 40× MultiScribe and RNase Inhibitor Mix, 1 μl forward primer (20 μM), 2 μl reverse primer (20 μM), 0.5 μl probe (20 μM), 1 μl total RNA or 5 μl RNA transcripts, and the volume of DEPC-water was adjusted with template. Thermo-cycling was run on LightCycler® 96 (Roche LifeScience) as follows: 30 minutes at 48° C., 10 minutes at 95° C., 40 cycles of 15 seconds at 95° C., 1° min at 60° C. Conversion of microgram of single stranded RNA to picomole and the number of RNA molecules was performed as described by Olmos et al. (2005, J Virol Methods 128(1-2), 151-155). Tenfold serial dilutions of the transcripts were prepared from 5×1012 to 50 copies per 5 μl, aliquoted and stored at −80° C. Dilutions from 5×108 to 5×100 were amplified by the real time RT-PCR assay. The stable amplification could be observed as low as 50 copies of RNA transcripts undertaking three repetitions of assay.

[0421] Item 5: Detection of CGMMV by Hybridization (Tissue Print or DotBlot)

[0422] Membrane Preparation for Tissue Print

[0423] Wearing gloves, prepare the nylon membrane by cutting to the required size with sterilized scissors, and place a micro-plate grid template on top (actual size depends on the type of sample being used). Make a transversal cut across the leaf stalk with a clean, sterile scalpel, and lightly press into one of the grids until all sap is absorbed. Make a fresh cut for each subsequent print. For purified DNA or RNA samples, 1 μl of sample is sufficient.

[0424] Include healthy negative controls and known infected positive controls of a respective same plant species. Purified virus or an amplified PCR product thereof should also be included as a positive control. Wait until the membrane is totally dry, and fix the nucleic acids to the membrane using UV light at 0.120 J cm-2 (crosslink). Store the dry membranes at room temperature, sealed in plastic bags.

[0425] Membrane Preparation for DotBlot

[0426] Sample tissue is weighed and ground in the appropriate extraction buffer at the appropriate dilution. Take 10-20 μl of extract with a micropipette, and carefully remove the tip, ensuring the liquid inside does not rise and become air-locked. Lightly touch (swiftly) the pipette tip against the membrane, using the micro-plate grid as a guide and apply the extract onto the membrane (printing). The resulting dots are about 250 nl in volume, and 4 can be placed in each grid position. Use 2-4 dots per sample, depending on the purpose of the membrane and quality of hybridization probe.

[0427] Print controls, crosslink and store as described above for Tissue Print membranes.

[0428] You may print duplicate membranes as back-up in case problems during processing should arise.

[0429] Prehybridization of Membranes

[0430] Place the membrane prepared as described above for tissue prints or DotBlots into a hybridization tube using flat tweezers. Add pre-hybridization solution (DIG Easy plus 1% blocking agent) at room temperature (sufficient to cover the membrane; e.g. 20 ml for 8×12 cm membrane, 10 ml for 8×6 cm). Incubate for aprox. 2 hours at 54° C. rotating in an hybridization oven. Ensure that the membrane does not dry out from now on.

[0431] Hybridization of Membranes

[0432] Prepare fresh hybridization solution (DIG Easy solution plus 1% blocking agent), using frozen stock probe solutions to obtain 80-120 ng probe per 8×12 cm membrane. Pre-heat the hybridization solution for 10 minutes at 100° C. (for DNA probes) or 65° C. for RNA probes. Discard the pre-hybridization solution from the membranes and add the diluted probe solution to the membranes. Incubate overnight at 54° C. rotating in the hybridization oven.

[0433] Washing of Membranes

[0434] Discard hybridization solution from the membranes and carry out the following steps in the hybridization tube with rotation in the hybridization oven:

[0435] 1. Wash with 2× hybridization volume in 6×SSC+1% SDS for 40 minutes at hybridization temperature.

[0436] 2. Discard liquid and repeat wash.

[0437] 3. Transfer membrane to a detection tray+Solution 1 (avoid drying out of the membrane).

[0438] Detection

[0439] Transfer the membrane to a tray slightly larger than the membrane. From here on, the process is at room temperature and with gentle agitation. Adjust the solution volumes to the tray and membrane size (e.g. 600 μl/cm2 or 50 ml for 8×12 cm membrane). It is important that the membrane does not dry out during the following steps:

[0440] Wash the membrane with the following solutions: [0441] 1. Solution I, 1 min [0442] 2. Wash solution, 5 min [0443] 3. Solution I, 1 min [0444] 4. Solution II, 30 min (prepare 2× volume in measuring cylinder) [0445] 5. Solution II (from previous step)+Anti-DIG (1:20000), 30 min [0446] 6. Wash solution, 15 min [0447] 7. Wash solution, 15 min [0448] 8. Solution I, 1 min [0449] 9. Solution III, 5 min minimum

[0450] Dilute alkaline-phosphatase chemiluminescent substrate (CDP-Star (1:100) in 600 μl of Solution III. Place the membrane between two sheets of acetate and distribute the substrate evenly over the membrane using a micropipette (1 drop=4 small quadrants). Mix by lifting and lowering the top acetate several times, taking care not to introduce air bubbles. Incubate 5 minutes in the dark (e.g. cover with aluminum foil). Remove excess substrate by pressing with filter paper and heat-seal edges of acetate.

[0451] The following reagents are needed for preparing the solutions mentioned herein:

[0452] Reagents:

[0453] Blocking agent (Roche)

[0454] Maleic Acid.

[0455] NaCl

[0456] Tris-HCL

[0457] Tween 20

[0458] Anti-Dig (Roche).

[0459] CDP-Star (Roche).

[0460] Solutions:

[0461] Solution I: 0.1 M Maleic acid, 0.15 M NaCl, pH 7.5

[0462] Solution II: 1% blocking agent in Solution I.

[0463] Solution III: 0.1 M Tris-HCl pH 9.5, 0.1 M NaCl

[0464] Wash Solution: 0.3% Tween 20 in Solution I

[0465] Digital Imaging

[0466] Place membrane in Digital Imaging chamber, and take one photograph with ambient lighting (Nikon Control Pro2, 50 mm objective, manual control with remote shooting enabled, ISO 0.3 step over 6400, long-exposure noise reduction box is checked, camera pre-chilled 30 minutes minimum, <10° C.).

[0467] Close the chamber, change shutter speed to Bulb and Aperture to 1.4. Using DSLR_Bot, Long Exposure function, set exposure time to 5 minutes 03 seconds. Ensure infrared cable is fully inserted, output level is at maximum and press Start (a “click” should be audible). Camera takes one image with aperture open, and a second with aperture closed for noise reduction—total time of 10 minutes 06 seconds for each 5 minute 03 seconds photo.

[0468] One photo of 5 min exposure is roughly equivalent to a conventional image of 2-4 hours exposure. If stronger signal is required, a 10 minutes digital exposure (equivalent to overnight conventional image) can be done. For external diagnostics or low signal, a conventional overnight exposure is recommended.

[0469] Item 6: Grafting of Citrullus Plants

[0470] Rootstocks were prepared from 15 days old CGMMV inoculated Citrullus lanatus cultivar Sugar baby plants or cultivar Ercole (Nunhems B.V.). Scions were prepared from 20 days old NCIMB 42624 plants. Scions were grafted onto the rootstock by making a slant cut on the stem of the rootstock and on the shoot of the scion and joining these so that the vascular system is joined. After healing at high humidity, grafted plants were transferred to the greenhouse.

Example 2—Results

[0471] Results Item 1: Identification of CGMMV Resistant Citrullus Plants

[0472] A group of various different Citrullus species accessions was grown in a greenhouse or climate chamber infected with CGMMV according to the method described herein under General Methods. Inoculum for CGMMV infection was produced according to General Methods Item 1 and inoculated plants were evaluated visually for symptoms as described under General Methods Item 3. One single Citrullus colocynthis plant being free of CGMMV induced symptoms (scale 4) was identified. The plant was selfed for a number of times and a selected CGMMV resistant plant was multiplied to obtain seeds for seed deposit under the Budapest treaty, designated as having accession number NCIMB 42624.

[0473] Results Item 2: Production of CGMMV Resistant Plants and Analysis Thereof

[0474] Citrullus lanatus Plants

[0475] Pollen from NCIMB 42624 was used to pollinate elite lines of Citrullus lanatus subspecies vulgaris. F1 plants were grown from seeds produced by the crosses for further analysis in respect to CGMMV resistance.

[0476] Also F2 plants were made by selfing the F1 plants. It has to be noted, that it was complicated to obtain F2 seeds and backcrosses from F1 plants (NCIMB 42624×elite lines of Citrullus lanatus subspecies vulgaris). Only few fruits did develop and in those fruits most of the seeds were empty.

[0477] The F1 plants were tested for CGMMV resistance and presence of CGMMV virus using the mechanical leaf inoculation method described in General Method Item 1-3 and RT-qPCR described in General Method Item 4.

[0478] The results were as follows. The Mock inoculated plants showed no symptoms. The positive control, Sugar baby, plants showed CGMMV symptoms of Scale 1. The F1 plants were CGMMV resistant and showed no symptoms (Scale 4).

TABLE-US-00002 TABLE 2 Quantitative analysis of CGMMV RNA in the plants was performed 15 dpi, 30 dpi and 60 dpi for each set of plants: Average Concentration (ng/μl) Plant 15 dpi 35 dpi 60 dpi Mock Sugar Baby — — — Mock Fl — — — Sugar baby inoculated_rep 1 1.4 3.35 × 10.sup.−2 1.26 Sugar baby inoculated_rep 2 2.1 1.53 × 10.sup.−2 0.41 Average of Sugar baby  1.75 2.44 × 10.sup.−2 0.83 inoculated F1 inoculated_rep 1 1.2 × 10.sup.−4 — 1.17 × 10.sup.−5 F1 inoculated_rep 2 — 1.09 × 10.sup.−4 3.68 × 10.sup.−5 F1 inoculated_rep 3 5.3 × 10.sup.−5 — 1.86 × 10.sup.−5 F1 inoculated_rep 4 7.2 × 10.sup.−6 1.81 × 10.sup.−6 1.03 × 10.sup.−5 Average of F1 inoculated 4.5 × 10.sup.−5 2.7 × 10.sup.−5 1.93 × 10.sup.−5

[0479] Results of RT-qPCR above shows the amount of CGMMV RNA in plant material. “Mock” indicates plants inoculated with buffer but without CGMMV. “inoculated” indicates plants having been inoculated with CGMMV as described in the mechanical leaf inoculation test in General Methods Item 1-3. “Sugar” indicates susceptible Citrullus lanatus cultivar Sugar baby plants. “F1” indicates plants obtained from crosses between NCIMB 42624×Citrullus lanatus subspecies vulgaris elite lines. “rep” indicates different plants, wherein each rep consists of a group of 6 plants.

[0480] RT-qPCR is a highly sensitive method for detecting CGMMV RNA molecules in plant samples (Hongyun et al., 2008, J Virological Methods 149, 326-329). As can be seen from the Table 2 above using the highly sensitive RT-qPCR method CGMMV RNA can be detected in all plants analysed at nearly all points in time (15 dpi, 30 dpi and 60 dpi). It is clearly derivable from the Table 2 that the amount of CGMMV RNA in F1 plants obtained from a cross between NCIMB 42624×Citrullus lanatus subspecies vulgaris elite lines is orders of magnitude lower than the amount of CGMMV RNA in CGMMV susceptible Citrullus lanatus cultivar Sugar baby. The amount was at all time points below 5.0×10-5 ng/μl on average of the four replicates and progressively lower at later time points. The susceptible control had an average of at least 0.02 ng/μl (at 35 dpi) but even much higher levels at 15 dpi and 60 dpi, above 1.7 ng/μl and above 0.8 ng/μl respectively.

[0481] From the visual evaluation of symptoms it is clear that F1 plants are resistant to CGMMV as they produce no symptoms after inoculation with CGMMV (scale 4). Citrullus lanatus cultivar Sugar baby is known to be susceptible to CGMMV and does show severe symptoms (scale 1) after inoculation with CGMMV. Therefore, the very low amounts of CGMMV in F1 plants shown in the Table above clearly indicates that CGMMV resistance is correlated with a significant decrease of CGMMV RNA in respective plant material. The F2 plants were also without CGMMV symptoms (scale 4) when tested in the mechanical leaf inoculation method described in the General Method items 1-3. Also the fruits produced on the F2 plants showed no symptoms on the outside or inside. F2 plants which were without symptoms (scale 4) also had an extremely low amount of virus titer in the leaves (RT-qPCR data not shown), significantly reduced compared to the susceptible control.

[0482] Grafted Plants

[0483] Scions from NCIMB 42624 were grafted onto CGMMV infected rootstocks prepared from commercial Citrullus lanatus subspecies vulgaris plants (cultivar Sugar Baby) or from a Curcubita maxima×Curcubita moshata cultivar Ercole hybrid (Nunhems B.V.) according to the method described herein under General Methods.

[0484] Analysis of the Plants

[0485] Greenhouse grown F1 plants (NCIMB 42624×Citrullus lanatus subspecies vulgaris elite lines), grafted plants composed of a scion from NCIMB 42624 and an infected rootstock from Citrullus lanatus subspecies vulgaris cultivar Sugar Baby (“NCIMB42624—Sugar baby”) and grafted plants composed of a scion from NCIMB 42624 and an infected rootstock from a Curcubita maxima×Curcubita moshata cultivar Ercole hybrid (“NCIMB42624—Ercole”) were analysed. The non-grafted plants (NCIMB42624, F1, Sugar Baby) were inoculated with CGMMV using the mechanical leaf inoculation. The grafted plants were made using the rootstock of a CGMMV infected Sugar Baby plant or Ercole plant as described in the General Methods.

[0486] In none of the plants symptoms on leaves caused by CGMMV could be detected by visual analysis. All plants were determined to be scale 4 by the visual evaluation of symptoms as described herein under General Methods item 3.

[0487] Furthermore, all plants did produce fruits appearing normal from outside and showing no symptoms of deteriorated or water soaked pulp on the inside. A representative picture of a fruit produced by such plants is given in FIG. 6.

[0488] Nearly all non-grafted Citrullus lanatus subspecies vulgaris cultivar Sugar Baby plants inoculated with CGMMV did show clear symptoms and have been determined to be scale 1 or 2 by the visual evaluation of symptoms as described herein under General Methods Item 3. Fruits produced by these plants did show severe pulp deterioration. A representative picture of a fruit produced by such plants is given in FIG. 5.

[0489] Citrullus lanatus F1 plants and grafted plants (“NCIMB42624—Sugar Baby” and “NCIMB42624—Ercole”) were further analysed for the presence of CGMMV Virus RNA in the leaves by means of dot-blot hybridization analyses (*) as described herein under General Methods items 5 and RT-PCR analysis (**) as described under General Methods Item 4 b). Results obtained are given in the following Table.

TABLE-US-00003 TABLE 3 dpi Host 14(*) 30(**) 40(*) 60(**) 90(*) NCIMB 42624 +/− +/− — — — F1 +/− Not — — — (NCIMB 42624 × elite lines) tested Graft — Not — Not Not (“NCIMB 42624 - Ercole”) tested tested tested Graft — Not — +/− — (“NCIMB 42624 - Sugar baby”) tested Sugar baby (Positive control) + + + + +

[0490] The above Table 3 shows the results of the detection of CGMMV RNA in leaves of CGMMV resistant Citrullus colocynthis plants (NCIMB 42624), F1 plants obtained from crossing NCIMB 42624×Citrullus lanatus subspecies vulgaris and grafted plants (“NCIMB 42624—Ercole” and “NCIMB 42624—Sugar baby”) after infection with CGMMV by mechanical inoculation of the leaves or by grafting a non-infected scion to an infected root-stock. Plants from the CGMMV susceptible commercial variety Sugar Baby, were used as positive control. +/− indicates a very weak signal, − indicates no signal, + indicates a strong positive signal; dpi: days post infection. (*) Dot Blot method, (**) RT-PCR method.

[0491] For CGMMV susceptible control plants, the virus was detected throughout development of the plants from early (dpi 14) to late stage (dpi 90). Very weak signals for CGMMV RNA were detected in NCIMB42624 at early stages (dpi 14 and 30), but those signals disappeared at later stages (from dpi 40 on). A similar result was obtained for F1 plants (NCIMB 42624×elite lines). For grafted plants no CGMMV RNA was detected in the leaves at any stage of development (from dpi 14 to dpi 90) with the exception that a weak signal was detected for the “NCIMB 42624—Sugar baby” graft at dpi 60. As can be seen from the following Table 4, further analysis of “NCIMB 42624—Sugar baby” grafts showed that a weak signal indicating presence of low amounts of CGMMV RNA was only due to virus being found in a single plant out of eight plants at early stage (dpi 14). At later stage (dpi 60) the signal in the same plant was neither detectable by dot-blot nor by PCR anymore.

TABLE-US-00004 TABLE 4 Graft “NCIMB 42624 - Dot-Blot Dot-Blot PCR Sugar baby” 14 dpi 60 dpi 60 dpi 1 — — — 2 — — — 3 — — — 4 — — — 5 — — No tested 6 −/+ — No tested 7 — — No tested 8 — — No tested

[0492] The above Table 4 shows the results of the detection of CGMMV RNA in grafted plants (“NCIMB 42624—Sugar baby”) after grafting to a CGMMV infected rootstock. +/− indicates a weak signal, − indicates no signal.

[0493] The above, non-quantitative but only qualitative, results of Table 3 and 4 are in conformity with the earlier quantitative results in Table 2, in that the very low level of virus at the early stages of resistant plants becomes progressively lower at later stages and is then only detectible using quantitative methods, i.e. only extremely low virus titers are found at 40 dpi or later at 60 dpi or thereafter. In addition, in the resistant scions no CGMMV virus could be detected using these qualitative methods. Further, also RT-qPCT was carried out on leaves obtained from scion-rootstock combinations, where the rootstock was infected with CGMMV as described in General Methods Item 6. The rootstock used was Sugar Baby.

[0494] Average Ct-values at 140-150 dpi of leaf samples were found to be as follows. Each replicate consists of 6 plants. Note that a low Ct-value indicates a high virus titer, while a high Ct-value indicates a low amount of virus.

TABLE-US-00005 TABLE 5 Average Ct-value Plant at 140-150 dpi Symptoms Leaf Sugar baby without CGMMV 26.22 No symptoms (mock) Leaf of Sugar Baby infected with 9.15 Severe CGMVV (rep 1) symptoms (scale 1) Leaf of Sugar Baby infected with 8.92 Severe CGMVV (rep2) symptoms (scale 1) Leaf of scion of graft: 27.36 No symptoms “NCIMB42624 - Sugar Baby” (rep 1) (scale 4) Leaf of scion of graft: 26.49 No symptoms “NCIMB42624 - Sugar Baby” (rep2) (scale 4)

[0495] The above clearly demonstrated that NCIMB 42624 plants and plants of crosses between NCIMB 42624×Citrullus lanatus subspecies vulgaris elite lines did not show any symptoms of CGMMV infection and contained very low, almost undetectable levels of virus in the upper leaves; and grafted plants comprising an NCIMB42624 scion grafted onto a CGMMV infected rootstock (“NCIMB42624—Sugar Baby” and “NCIMB42624—Ercole”) do not show any symptoms of CGMMV infection on the scion. Thus, NCIMB 42624 plants are resistant to CGMMV, the resistance to CGMMV is transferable to other Citrullus species including Citrullus lanatus subspecies vulgaris elite lines and the resistance is maintained when CGMMV resistant scions are grafted onto CGMMV infected rootstocks.

[0496] Results item 3—QTL mapping of CGMMV resistance found in NCIMB 42624 and marker development Three mapping populations were generated by crossing NCIMB 42624 with an elite watermelon line which is susceptible to CGMMV. One BC1 populations (backcross) and two F2 populations were made. Plants were mechanically inoculated with CGMMV as described and symptoms were evaluated using the visual scale described and also using ELISA tests to evaluate virus titers. Mapping was done using mostly symptoms assessed at 30 dpi or later.

[0497] In total seven QTLs were mapped (two on chromosome 1, one on chromosome 4, two on chromosome 5 and one on chromosome 7 and one on chromosome 9), of which 5 QTLs (QTL.1.1 on chromosome 1, QTL4.1 on chromosome 4, QTL5.2 on chromosome 5, QTL7.1 on chromosome 7 and QTL9.1 on chromosome 9, see Table 6 below, were used in further backcrossing and for these five QTLs SNP markers linked to these QTLs are provided below.

[0498] Two QTLs, QTL7.1 and QTL9.1, were dominant and resulted in plants having a symptom level of scale 3 or scale 4. They also had a significant effect on virus titer, which was significantly reduced in cultivated watermelons into which these QTLs were backcrossed.

[0499] In the Table below (Table 6), the chromosome of each of the five QTLs is indicated, as is the base pair (bp) position of the SNP in the genome of cultivated watermelon (see cucurbitgenomics.org, “Watermelon (Chareleston Gray) genome”). The genotype of the SNP is indicated for the resistant parent (i.e. NCIMB 42624) and the Susceptible parent (elite watermelon line). The seed deposit NCIMB 42625 is homozygous for the SNP genotype and contains all five QTLs in homozygous form.

TABLE-US-00006 TABLE 6 Nucleotide position of the SNP Cultivated in the SNP watermelon watermelon genotype of SNP genotype chromosome genome (base SNP marker linked to QTL (most susceptible of CGMMV (Chr) number); closely linked marker is indicated) parent resistant donor Chrl  6,555,391 SNP1 refers to nucleotide 51 in TT CC (QTL1.1) SEQ ID NO: 1 Chrl 16,631,610 SNP2 refers to nucleotide 51 in AA GG (QTL1.1) SEQ ID NO: 2 Chrl 26,238,488 SNP3 refers to nucleotide 51 in TT CC (QTL1.1) SEQ ID NO: 3 Chrl 34,609,471 SNP4 refers to nucleotide 51 in TT CC (QTL1.1) SEQ ID NO: 4 Chrl 35,238,267 SNP5 refers to nucleotide 51 in AA GG (QTL1.1) SEQ ID NO: 5 (most closely linked) Chrl 35,766,439 SNP6 refers to nucleotide 51 in CC TT (QTL1.1) SEQ ID NO: 6 Chr4  4,230,711 SNP7 refers to nucleotide 51 in AA TT (QTL4.1) SEQ ID NO: 7 Chr4 24,321,690 SNP8 refers to nucleotide 51 in AA CC (QTL4.1) SEQ ID NO: 8 (most closely linked) Chr4 25,526,846 SNP9 refers to nucleotide 51 in GG AA (QTL4.1) SEQ ID NO: 9 Chr5 30,786,503 SNP10 refers to nucleotide 51 in CC TT (QTL5.2) SEQ ID NO: 10 Chr5 32,788,817 SNP11 refers to nucleotide 51 in GG AA (QTL5.2) SEQ ID NO: 11 (most closely linked) Chr5 36,842,501 SNP12 refers to nucleotide 51 in CC TT (QTL5.2) SEQ ID NO: 12 Chr7  4,784,519 SNP13 refers to nucleotide 51 in GG AA (QTL7.1) SEQ ID NO: 13 Chr7  7,848,633 SNP14 refers to nucleotide 51 in GG AA (QTL7.1) SEQ ID NO: 14 (most closely linked) Chr7  8,334,624 SNP15 refers to nucleotide 51 in AA GG (QTL7.1) SEQ ID NO: 15 Chr7 11,166,694 SNP16 refers to nucleotide 51 in TT GG (QTL7.1) SEQ ID NO: 16 Chr7 18,775,728 SNP17 refers to nucleotide 51 in CC TT (QTL7.1) SEQ ID NO: 17 Chr7 21,389,410 SNP18 refers to nucleotide 51 in GG AA (QTL7.1) SEQ ID NO: 18 Chr9 31,394,464 SNP19 refers to nucleotide 51 in TT CC (QTL9.1) SEQ ID NO: 19 Chr9 32,499,549 SNP20 refers to nucleotide 51 in CC GG (QTL9.1) SEQ ID NO: 20 Chr9 34,607,108 SNP21 refers to nucleotide 51 in AA GG (QTL9.1) SEQ ID NO: 21 Chr9 35,343,850 5NP22 refers to nucleotide 51 in TT CC (QTL9.1) SEQ ID NO: 22 (most closely linked) Chr9 36,923,004 5NP23 refers to nucleotide 51 in TT CC (QTL9.1) SEQ ID NO: 23 Chr9 38,321,162 SNP24 refers to nucleotide 51 in TT GG (QTL9.1) SEQ ID NO: 24

[0500] For each SNP the sequence is provided in the sequence listing and herein and the sequence has the same number as the SNP. The sequence contains the SNP nucleotide of the resistant donor. So for SNP1 the sequence comprising the resistant parent SNP (a ‘C’ at position 51) is provided as SEQ ID NO: 1, etc. See also Table 1.

[0501] BC3 and F2 lines have been generated containing different combinations of QTLs and single QTLs. These will be evaluated for their CGMMV resistance and virus titers following inoculation. Also the virus titers will be evaluated and whether the very low virus titers result in transmissibility of the virus to susceptible watermelon plants or to other hosts, such as cucumber. Likewise, transmissibility of the virus to seeds produced on CGMMV resistant plants will be studied. In addition, fine mapping will be done. “Fine-mapping” refers to methods by which the position of a QTL can be narrowed down. For example a large population segregating for the trait can be analysed for segregation of the trait and the SNP markers and plants comprising recombination events in the region between the markers can be selected, in order to determine between which pair of SNP markers the QTL is located. One can also search for additional markers near or flanking the most linked marker to narrow down the interval in which the QTL is located.

Example 3: Virus Transmission from NCIMB42624 Plants (C. colocynthis Accession) to Cucumber and Susceptible Watermelon Plants

[0502] Leaves of a plant of NCIMB42624, which was mechanically inoculated with CGMMV virus European type VE459 (as described above) but showed no symptoms (scale 4), was used to make inoculum and the inoculum was used to mechanically inoculate 215 plants of the cucumber variety Sheila and 193 plants of watermelon variety Sugar Baby. After about 30 dpi the plants were visually assessed for CGMMV symptoms and also a PCR assay was carried out to detect virus in the leaves.

[0503] The results are shown below in Table 7:

TABLE-US-00007 Cucumber variety Sheila Nr. of Nr. of Nr of % of plants plants plants plants wherein with without positive in virus was Symptoms symptoms CGMMV PCR transmitted 1 214 1 0.46% Watermelon variety Sugar Baby 0 193 0 0.00%

[0504] The results showed that only a single cucumber plant was infected by the inoculum (a single plant showed symptoms and was positive in the PCR test), while none of the watermelon plants were infected by the inoculum. This means that the resistant C. colocynthis accession deposited under NCIMB 42624 does not allow CGMMV transmission to watermelon plants and only very low levels of transmission to cucumber plants (less than 1% of plants become infected).

Example 4: Seed Transmissibility of CGMMV in Watermelon Lines Containing Various Combinations of QTLs from the C. colocynthis Donor (NCIMB 42624)

[0505] Various F3 families which contained one or more QTLs from the C. colocynthis donor and which had survived mechanical CGMMV inoculation were allowed to set fruits and the seeds produced in these fruits were analyzed for CGMMV virus using quantitative PCR (RT-qPCR) as described above. The aim was to determine whether the QTLs could prevent seed infection and thus seed transmissibility of the virus. qPCR was carried out at 210 dpi. A low Ct value indicates a high virus titre and a high Ct value (above 30) indicates a low virus titre.

[0506] Results are shown in Table 8:

TABLE-US-00008 Fruit qPCR on seeds of Interpretation of seed Line symptoms fruits: Ct value infection of the virus X16_4305-15 NO 37.09 Negative X16_4310-17 NO 36.93 Negative X16_4311-11 NO 34.08 Negative X16_4311-8 NO 35.46 Negative Sugar baby Mock NO nd Negative X16_4306-1 NO 33.70 Negative X16_4310-9 NO 34.28 Negative X16_4310-35 NO 35.49 Negative X16_4310-8 NO 36.00 Negative X16_4307-24 NO 38.01 Negative X16_4311-6 NO 32.50 Negative X16_4305-20 NO 32.25 Negative X16_4312-4 NO 38.66 Negative X16_4312-2 NO 22.86 Positive X16_4307-22 NO 22.99 Positive X16_4312-6 NO 32.40 Negative X16_4311-3 NO 32.85 Negative X16_4305-8 NO 34.55 Negative X16_4305-17 NO 18.56 Positive X16_4310-15 NO 25.22 Positive X16_4312-16 NO 24.98 Positive X16_4307-9 NO 24.45 Positive X16_4312-10 NO 19.28 Positive X16_4311-1 NO 17.44 Positive Sugar baby YES 19.70 Positive X16_4305-10 NO 20.16 Positive X16_4307-12 NO 17.46 Positive

[0507] The results showed that all lines which contained at least QTL 9.1 and/or QTL 7.1 were negative for CGMMV virus transmission to the seeds. Especially presence of at least QTL9.1 seemed to prevent seed transmission of the virus.

Example 5: Comparison of C. colocynthis Accessions for CGMMV Resistance

[0508] Ten seeds of various C. colocynthis accessions were germinated and the plants were mechanically inoculated as described above, in order to compare the CGMMV resistance to the resistance of the accession deposited under NCIMB 42624. Each plant was phenotyped using the scale 1 (severe symptoms), 2 (medium symptoms), 3 (mild symptoms) and 4 (no symptoms). It is noted that PI accessions are known to be heterogenous.

[0509] Many PI accessions did not germinate and could therefore not be assessed.

[0510] The following PI accessions could be assessed: PI537300, PI542616, PI549161, GRIF14201, PI220778, PI386015, PI432337, PI388770 and PI386018. Three of these accessions (PI537300, PI386015, PI388770) were part of the USDA core collection and have been described to have potential CGMMV tolerance (CucCAP, Second Annual CucCAP Team Meeting, Mar. 27-28 2017, Charleston, page 26 and 27, ‘Evaluation of the watermelon PI collection for resistance to Cucumber green mottle mosaic virus CGMMM and conducting genome-wide association mapping to identify SNPs associated with CGMMV resistance’, in combination with the C. colocynthis accessions of the USDA core collection as indicated in the GRIN collection in which only 4 C. colocynthis accessions are part of the ‘core subset’; three of the 4 core subsets were included in this test, but PI525082 could unfortunately not be included in this test).

[0511] However, in our test all tested accessions, including the three accessions of the USDA core collection, had an average symptom score of 3 or less (average of the individual plants), with not a single plant in the entire test having a score of 4 (no symptoms), except NCIMB 42624 of the invention (all of which plants had a score of 4). Of all the other C. colocynthis accessions PI386015 had the best CGMMV tolerance, with all plants having a score of 3 (mild symptoms). PI537300 had 6 plants with a score of 3 (mild symptoms) and 4 plants with a score of 2 (medium symptoms). PI388770 had 6 plants with a score of 1 (severe symptoms), three plants with a score of 2 (medium symptoms) and one plant with a score of 3 (mild symptoms).

[0512] The test confirmed that the CGMMV resistance in the selected C. colocynthis deposited under NCIMB42624 is of a higher resistance level than any of the other C. colocynthis accessions tested, which at most have tolerance (score 3, mild symptoms).

Example 6—Fine Mapping

[0513] QTL7.1 and QTL9.1 were fine mapped, in order to further narrow down the location of the QTLs on the chromosomes.

[0514] QTL7.1 was fine-mapped to be located in-between the INDEL (Insertion/deletion) marker at nucleotide 7,022,773, referred to as marker “InDel7.1”, and the SNP marker at nucleotide 8,733,604 of chromosome 7 of the reference genome (Charleston Grey genome in the world wide web at cucurbitgenomics.org), referred to as “SNP FM7.1”. These two markers, therefore, flank the QTL7.1, which is located in the region in-between them.

[0515] The C. colocynthis donor comprises a deletion of the (T) Thymine found at nucleotide 7,022,773 of chromosome 7 of the reference genome, while the recurrent parent and the reference genome comprise a Thymine (T) at nucleotide 7,022,773 of chromosome 7. The marker InDel7.1 can also be written as ‘AT’ at nucleotides 7,022,772 and 7,022,773 of chromosome 7 (in the susceptible parent), versus ‘A’ at 7,022,772 of chromosome 7, with the T at nucleotide 7,022,773 being absent (in the CGMMV resistant parent). The InDel7.1 marker, thus, differentiates between ‘A-’ and ‘AT’ at nucleotide position 7,022,772 and 7,022,773 of chromosome 7.

[0516] See also FIG. 7A, showing an alignment of the Query sequence of SEQ ID NO: 30 from C. colocynthis and the Subject (added as SEQ ID NO: 31 herein) of the reference genome Charleston Grey. The marker InDel7.1 is highlighted with a box. The alignment was generated as output of a BLAST analysis of the Query sequence of SEQ ID NO: 30 against the “Charleston Grey (Watermelon)” genome on the website cucurbitgenomics.org. As there are some nucleotide ambiguities in the query sequence (e.g. Y=C or T, R=A or G, K=G or T, etc. see IUPAC nucleotide code at e.g. world wide web at bioinformatics.org/sms/iupac.html) the two sequences have a sequence identity of 94.51%.

[0517] The InDel7.1 marker, thus, differentiates between ‘A’ and ‘AT’ at nucleotide position 91 in SEQ ID NO: 30 or at nucleotide 91 (and 92) in SEQ ID NO: 31, or at the equivalent position in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30 or 31. When referring to an ‘A’ instead of an ‘AT’ at nucleotide 91 of SEQ ID NO: 30 or of SEQ ID NO: 31 (or an A at the equivalent position in a sequence comprising at least 94% identity to SEQ ID NO: 30 or 31), it is understood that the InDel7.1 marker of the resistant donor (linked to QTL7.1) is referred to, e.g. the sequence comprising the deletion of the T at the position 7022773 on chromosome 7.

[0518] The C. colocynthis donor further comprises an Adenine (A) at nucleotide 8,733,604 of chromosome 7 of the reference genome, while the recurrent parent and the reference genome comprise a Cytosine (C) at nucleotide 8,733,604 of chromosome 7, referred to as marker SNP FM7.1.

[0519] Marker SNP FM7.1 is shown in FIG. 7B, showing an alignment of the Query sequence of SEQ ID NO: 32 from C. colocynthis and the Subject (added as SEQ ID NO: 33 herein) of the reference genome Charleston Grey. The SNP marker is highlighted with a box. The alignment was generated as output of a BLAST analysis of the Query sequence against the “Charleston Grey (Watermelon)” genome on the website cucurbitgenomics.org. As there are some nucleotide ambiguities in the query sequence (e.g. Y=C or T, R=A or G, K=G or T, etc. see IUPAC nucleotide code at e.g. world wide web at bioinformatics.org/sms/iupac.html) the two sequences have a sequence identity of 95.74%.

[0520] The SNP FM7.1 marker, thus, differentiates between ‘A’ and ‘C’ at nucleotide position 71 in SEQ ID NO: 32 and SEQ ID NO: 33, or at the equivalent position in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32 or 33. When referring to an ‘A’ instead of an ‘C’ at nucleotide 71 of SEQ ID NO: 32 or of SEQ ID NO: 33 (or an A at the equivalent position in a sequence comprising at least 94% identity to SEQ ID NO: 32 or 33), it is understood that the SNP FM7.1 marker of the resistant donor (linked to QTL7.1) is referred to, e.g. the sequence comprising the A at the position 8, 733,604 on chromosome 7.

TABLE-US-00009 TABLE 7 fine mapping op QTL7.1, with new markers flanking the QTL7.1 shaded Nucleotide position of the SNP Cultivated in the SNP watermelon watermelon genotype of SNP genotype of chromosome genome (base susceptible CGMMV resistant (Chr) number); SNP marker linked to QTL parent donor Chr7 7,022,773 INDEL (InsertionDeletion) II DD (QTL7.1) marker named “InDe17.1” (Insertion/ (Deletion/Deletion) (see FIG. 7A) Insertion) (A/A) (AT/AT) or (A-/A-) Chr7 7,848,633 SNP14 refers to nucleotide 51 in GG AA (QTL7.1) SEQ ID NO: 14 (most closely linked) Chr7 8,334,624 SNP15 refers to nucleotide 51 in AA GG (QTL7.1) SEQ ID NO: 15 Chr7 8,733,604 SNP marker named “SNP FM7.1” CC AA (QTL7.1) (See FIG. 7B)

[0521] The introgression fragment comprising QTL7.1 comprises, thus, the sequence of the donor in-between marker “InDel7.1” and the SNP marker “SNP FM7.1”. Optionally the introgression fragment comprises the QTL and the resistant donor nucleotide for SNP14 and/or SNP15. Optionally the introgression fragment comprises the QTL and the resistant donor haplotype or genotype for InDel7.1 and/or SNP FM7.1. In one aspect the introgression fragment comprises the QTL7.1 and comprises the donor marker haplotype or donor genotype for one or more or all of InDel7.1, SNP14, SNP15 and SNP FM7.1.

[0522] Thus, in one aspect the haplotype of the plant comprising one copy of the introgression fragment (and comprising QTL7.1) is the donor haplotype for one or more markers selected from: [0523] deletion of T at nucleotide 7,022,773 of chromosome 7 for marker “InDel7.1”, or an A instead of an AT at nucleotide 91 of SEQ ID NO: 30 or 31 or an A (instead of an AT) at the equivalent nucleotide in a sequence comprising at least 94%, 95%, 96%, 97%, 98% identity to SEQ ID NO: 30 or 31; [0524] Adenine for SNP14 at nucleotide 51 of SEQ ID NO: 14 (or an Adenine at the equivalent nucleotide in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or more sequence identity to SEQ ID NO: 14), [0525] Guanine for SNP15 at nucleotide 51 of SEQ ID NO: 15 (or a Guanine at the equivalent nucleotide in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or more sequence identity to SEQ ID NO: 15), [0526] Adenine for “SNP FM7.1” at nucleotide 71 of SEQ ID NO: 32 or 33 (or an Adenine at the equivalent nucleotide in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or more sequence identity to SEQ ID NO: 32 or 33), e.g. an Adenine at nucleotide 8,733,604 of chromosome 7.

[0527] QTL9.1 was fine-mapped to be located in-between the SNP marker at nucleotide 35,675,266 (called “FM9.1”) and the INDEL marker at nucleotide 36,439,966 of chromosome 9 (referred to as “InDel9.1”) of the reference genome (Charleston Grey genome in the world wide web at cucurbitgenomics.org). These two markers, therefore, are flanking markers of QTL9.1, i.e. the QTL9.1 is located between the two markers on the C. colocynthis introgression fragment.

[0528] The C. colocynthis donor comprises a (C) Cytosine for SNP FM9.1 found at nucleotide 35,675,266 of chromosome 9 of the reference genome, while the recurrent parent and the reference genome comprise an Adenine (A) at nucleotide 35,675,266 of chromosome 9 for SNP FM9.1.

[0529] See also FIG. 8A, showing an alignment of the Query sequence of SEQ ID NO: 34 from C. colocynthis and the Subject (added as SEQ ID NO: 35 herein) of the reference genome Charleston Grey. The SNP FM9.1 marker is highlighted with a box. The alignment was generated as output of a BLAST analysis of the Query sequence against the “Charleston Grey (Watermelon)” genome on the website cucurbitgenomics.org. As there are some nucleotide ambiguities in the query sequence (e.g. Y=C or T, R=A or G, K=G or T, etc. see IUPAC nucleotide code at e.g. world wide web at bioinformatics.org/sms/iupac.html) the two sequences have a sequence identity of 98.57%.

[0530] The SNP FM9.1 marker, thus, differentiates between ‘C’ and ‘A’ at nucleotide position 71 in SEQ ID NO: 34 and SEQ ID NO: 35, or at the equivalent position in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 34 or 35. When referring to an ‘C’ instead of an ‘A’ at nucleotide 71 of SEQ ID NO: 34 or of SEQ ID NO: 35 (or an C at the equivalent position in a sequence comprising at least 94% identity to SEQ ID NO: 34 or 35), it is understood that the SNP FM9.1 marker of the resistant donor (linked to QTL9.1) is referred to, e.g. the sequence comprising the C at the position 35,675,266 on chromosome 9.

[0531] The C. colocynthis donor comprises a deletion of the (C) Cytosine found at nucleotide 36,439,966 of chromosome 9 of the reference genome, while the recurrent parent and the reference genome comprise a Cytosine (C) at nucleotide 36,439,966 of chromosome 9. The marker InDel9.1 can also be written as ‘GC’ at nucleotides 36,439,965 and 36,439,966 of chromosome 9 (in the susceptible parent), versus ‘G’ at 36,439,965 of chromosome 9, with the C at nucleotide 36,439,966 being absent (in the CGMMV resistant parent). The InDel9.1 marker, thus, differentiates between ‘G-’ and ‘GC’ at nucleotide position 36,439,965 and 36,439,966 of chromosome 9.

[0532] See also FIG. 8B, showing an alignment of the Query sequence of SEQ ID NO: 36 from C. colocynthis and the Subject (added as SEQ ID NO: 37 herein) of the reference genome Charleston Grey. The marker InDel9.1 is highlighted with a box. The alignment was generated as output of a BLAST analysis of the Query sequence of SEQ ID NO: 36 against the “Charleston Grey (Watermelon)” genome on the website cucurbitgenomics.org. As there are some nucleotide ambiguities in the query sequence (e.g. Y=C or T, R=A or G, K=G or T, etc. see IUPAC nucleotide code at e.g. world wide web at bioinformatics.org/sms/iupac.html) the two sequences have a sequence identity of 93.85%.

[0533] The InDel9.1 marker, thus, differentiates between ‘G’ and ‘GC’ at nucleotide position 91 in SEQ ID NO: 36 or at nucleotide 91 (and 92) in SEQ ID NO: 37, or at the equivalent position in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 36 or 37. When referring to an ‘G’ instead of an ‘GC’ at nucleotide 91 of SEQ ID NO: 36 or of SEQ ID NO: 37 (or a G at the equivalent position in a sequence comprising at least 94% identity to SEQ ID NO: 36 or 37), it is understood that the InDel9.1 marker of the resistant donor (linked to QTL9.1) is referred to, e.g. the sequence comprising the deletion of the C at the position 36,439,966 on chromosome 9.

TABLE-US-00010 TABLE 8 fine mapping op QTL9.1, with new markers flanking the QTL9.1 shaded Nucleotide position of the SNP Cultivated in the SNP watermelon watermelon genotype of SNP genotype chromosome genome (base susceptible of CGMMV (Chr) number); SNP marker linked to QTL parent resistant donor Chr9 35,343,850 SNP22 refers to nucleotide 51 in TT CC (QTL9.1) SEQ ID NO: 22 Chr9 35,675,266 SNP marker named “SNP FM9.1” AA CC (QTL9.1) See FIG. 8A Chr9 QTL9.1 (QTL9.1) Chr9 36,439,966 INDEL (InsertionDeletion) marker I/I DID (QTL9.1) named “InDe19.1” (Insertion/ (Deletion/ (See FIG. 8B) Insertion) Deletion) GC/GC GIG or G-/G- Chr9 36,923,004 5NP23 refers to nucleotide 51 in TT CC (QTL9.1) SEQ ID NO: 23

[0534] The introgression fragment comprising QTL9.1 comprises thus the sequence of the donor in-between INDEL marker “InDel9.1” and “SNP FM9.1”. Optionally the introgression fragment comprises the resistant donor nucleotide for SNP22 and/or SNP23.

[0535] Optionally the introgression fragment comprises the QTL and the resistant donor haplotype or genotype for InDel9.1 and/or SNP FM9.1. In one aspect the introgression fragment, thus, comprises the QTL9.1 and comprises the donor marker haplotype or donor genotype for one or more or all of InDel9.1 and/or SNP FM9.1.

[0536] Thus, in one aspect the haplotype of the plant comprising one copy of the introgression fragment (and comprising QTL9.1) is the donor haplotype for one or more markers selected from: [0537] deletion of C (Cytosine) at nucleotide 36,439,966 of chromosome 9 for marker “InDel9.1”, or a Guanine (G) instead of a GC (Guanine-Cytosine) at nucleotide 91 of SEQ ID NO: 36 or 37 or a G (instead of an GC) at the equivalent nucleotide in a sequence comprising at least 94%, 95%, 96%, 97%, 98% identity to SEQ ID NO: 36 or 37; [0538] Cytosine for “SNP FM9.1” at nucleotide 71 of SEQ ID NO: 34 or 35 (or a Cytosine at the equivalent nucleotide in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or more sequence identity to SEQ ID NO: 34 or 35), e.g. a Cytosine at nucleotide 35,675,266 of chromosome 9.