Resistance of muskmelons to CYSDV

Abstract

The present invention relates to a Cucumis melo plant, in particular a Cucumis melo plant, which is resistant to CYSDV infection, as a result of the presence in its genotype of a specific combination of QTL on chromosome 1, QTL on chromosome 5 and QTL on chromosome 11, thereby conferring said resistance. The invention also relates to cells, seeds of these plants, as well as various methods and uses to utilize CYSDV resistance.

Description

Technical Field

[0001] This invention relates to resistance and / or tolerance of *Cucumis melo* plants, particularly the subspecies *Cucumis melo*, to criniviruses, including *Cucumis yellow dwarfing disorder virus* (CYSDV) and / or *Cucurbit chlorotic yellows virus* (CCYV). According to the invention, resistance is provided by DNA sequences, particularly QTLs, introgressed from wild melon germplasm at specific loci in the genome of commercial squash plants. Background Technology

[0002] Melon belongs to the Cucurbitaceae family. Cucurbitaceae is a family of about 100 genera and 700 to 900 species (according to the author's count), primarily found in tropical regions. This family includes squash, pumpkin, gourd, watermelon, loofah, and some weeds. Cantaloupe, cucumber, and several types of melon belong to the genus *Cucumis*, which includes about 70 species. Melon encompasses a wide range of cultivated plants, and its center of origin is likely in East Africa.

[0003] Based on the hairiness of the calyx tube, melons are divided into two subspecies: the thick-skinned melon subspecies (Cucumis melo subsp. melo), with long, spreading hairs on the ovary or young fruit, and the common melon subspecies (Cucumis melo subsp. agrestis), with short, appressed hairs. The plant community belonging to the common melon subspecies is found in East Asia, from India to Japan, while the thick-skinned melon subspecies is more commonly found from India to Europe and the New World. Although extraspecific hybridization is sterile, intraspecific hybridization is usually fertile, resulting in a complex range of variations.

[0004] The thick-skinned melon subspecies includes 11 types, such as cantalupensis (cantaloupe), reticulatus (muskmelon), adana, chandalak, ameri, inodorus (winter melon), flexuosus (snake melon), chate, tibish, dudaim, and chito (mango melon, garden melon) (Pitrat et al., 2000).

[0005] Melon is a simple diploid species with 12 pairs of highly differentiated chromosomes. The melon genome comprises more than 375 Mb of sequence and is estimated to contain 27,427 protein-coding genes (Garcia-Mas et al., 2012).

[0006] Many pathogens can affect the productivity of melon plants, including viruses, fungi, bacteria, nematodes, and insects. Melons are particularly susceptible to many viruses, therefore, viral resistance is of major agricultural importance.

[0007] In this respect, cucurbits are susceptible to viruses transmitted by various vectors, such as whiteflies (Bemisia tabaci). Based on the unique particle morphology, length, semi-persistent transmission via hemipteran insect vectors, phloem restriction, cytopathology, genome structure, and expression of viruses, the family Closteroviridae includes a variety of plant viruses. This family includes the genus *Trichovirus* (ssRNA), as well as the genera *Ampelovirus* and *Closterovirus*.

[0008] CYSDV (Cucumber Yellow Dwarf Disorder Virus) and CCYV (Cucumber Chlorosis Virus) are the most common and destructive hairy virus genera in cucurbits.

[0009] From an epidemiological and economic perspective, CYSDV poses the greatest threat to cultivated cucurbits. CYSDV, the cucumber yellow dwarfism virus, spreads in a semi-persistent manner, exhibiting symptoms of interveinal chlorosis (yellowing), commonly observed in cucumber and melon plants. Symptoms of CYSDV first develop in older leaves and resemble those of water shortage. Interveinal chlorosis, where the veins turn yellow, gives the leaves a striped appearance. Eventually, the entire leaf turns yellow except for the veins, which remain green. Sometimes, green spots appear on the leaves. As the plant's internal transport system deteriorates, it begins to shed older leaves in an attempt to protect itself. Without sufficient leaves, the plant weakens and is unable to support or nourish the fruit. Consequently, the fruit is smaller, less sweet, and becomes more difficult to transport or store.

[0010] For example, a representative sequence of CYSDV is the sequence with GenBank accession number FJ492808.

[0011] CCYV also causes yellowing leaf spots and leaf discoloration in various cucurbitaceous plants, including melons and watermelons. Like CYSDV, CCYV is also transmitted in a semi-persistent manner, particularly by whiteflies. The symptoms caused by the two viruses are difficult to distinguish.

[0012] For example, a representative sequence of CCYV is the one with GenBank accession number AB523789.

[0013] Recent studies have shown that CYSDV and CCYV are spreading and causing increasing crop losses year by year, and are spreading to new areas.

[0014] Controlling CCYV and CYSDV is challenging because the primary vector, the whitefly, can develop resistance to most commonly authorized and used pesticides. Furthermore, the virus cycle, acquisition, and transmission are relatively short, meaning even effective authorized pesticides cannot act quickly enough to stop transmission.

[0015] WO2020025631 discloses a modified gene that supposedly provides resistance to CYSDV, namely the modified clathrin assembly protein gene CLAPR1. This modification essentially corresponds to a specific insertion of 9 bp “CAGCAACAA” into the gene on chromosome 5. However, the inventors have detected this insertion in many different strains, but without any association with CYSDV resistance.

[0016] Wild-type TGR-1551 (Pérez-de-Castro et al., 2020) and the SGR material (EP3005862) have been described as resistant to CYSDV. However, the inventors have demonstrated that the first is highly dependent on the isolate and therefore on location, and causes necrotic spots; while the second, conferred by the SGR gene on chromosome 9, is insufficient, primarily masking symptoms due to the "hyperchlorosis" phenotype, but not attenuating viral replication and spread. Symptoms are still observed in the plant. Furthermore, non-dark green leaves are less affected by the "hyperchlorosis" phenotype, and therefore less effective at masking CYSDV and CCYV symptoms.

[0017] Therefore, there is an urgent need to identify the genetic determinants that provide resistance to these viruses and to obtain resistant commercial materials that can also be used to prevent the spread of these viruses.

[0018] This invention provides melon plants exhibiting resistance or tolerance to Cucumber Yellow Dwarfism Virus (CYSDV) and / or Cucurbit Yellowing Virus (CCYV), and methods for producing or identifying melon plants exhibiting resistance and / or tolerance to one or two of these viruses (and possibly other Trichoviruses). The invention also discloses molecular genetic markers, particularly SNPs, associated with the genetic determinants of resistance and / or tolerance to CYSDV and / or CCYV. Summary of the Invention

[0019] The inventors have identified wild melon germplasm with high levels of resistance to CYSDV and have been able to introduce genes from these wild germplasm that confer CYSDV resistance into the genetic background of commercial melons, thereby obtaining resistant melon plants. Furthermore, the inventors emphasize that these genetic determinants also confer CCYV resistance. The resistance of this invention is conferred by newly discovered genetic determinants or QTLs. This resistance is readily transferable to different genetic backgrounds, especially where the inventors have identified genetic markers associated with these genetic determinants. Moreover, this resistance can be transferred without being associated with negative traits, particularly with negative commercial traits of fruit (e.g., fruit size and shape, poor flesh quality, or low sugar content) found in wild germplasm from which these genetic determinants have been introducen. These genetic determinants or QTLs can also be introduce into plants without affecting commercially acceptable fruit quality.

[0020] Therefore, this invention provides introgression sequences or QTLs that confer resistance / tolerance phenotypes to CYSDV and possibly CCYV. This invention also provides melon plants, particularly commercial melon plants, that exhibit high levels of resistance to the genus Trichoviruses, especially to CYSDV and / or CCYV, and methods for producing or identifying melon plants or populations (germplasm) resistant to CYSDV infection, as well as seeds, fruits, and other plant parts (e.g., pollen and ovules) containing resistance-conferring introgression sequences. This invention also discloses molecular genetic markers, particularly SNPs, linked to resistance-conferring introgression sequences.

[0021] Definitions:

[0022] The term "Bryce Brix" (or simply "brix") refers to the amount of soluble solids in an aqueous solution (especially fruit juice), the vast majority of which are sugars. These are primarily determined by a refractometer and measured as Brix. A higher Brix indicates a higher sugar content. Brix measurement is crucial for assessing the taste of melons, as fruits with low Brix and low sugar content are often less popular with consumers.

[0023] The term "resistance," defined by the ISF (International Seed Federation) Vegetable and Ornamental Crop Division, describes a plant's response to pests or pathogens, as well as abiotic stress in the vegetable seed industry. Specifically, resistance refers to the ability of a plant variety to limit the growth and development of a particular pest or pathogen and / or the damage they cause, compared to susceptible plant varieties under similar environmental conditions and pest or pathogen stress. Resistant varieties or plants may exhibit some disease symptoms or damage under severe pest or pathogen stress. Two levels of resistance are defined:

[0024] High resistance (HR): Plants that, compared to susceptible plants, highly limit the growth and / or development of a particular pest and / or the damage it causes under normal pest stress. However, these plants may exhibit some symptoms or damage under severe pest stress.

[0025] Intermediate resistance (IR): Plants that are highly restricted in their growth and / or development and / or the damage caused by a particular pest, but may exhibit a wider range of symptoms or damage compared to highly resistant plants. When grown under similar environmental conditions and / or pest stress, intermediately resistant plants will still exhibit milder symptoms or damage than susceptible plants.

[0026] The term "tolerance" is often used to describe a plant's ability to withstand abiotic stresses without causing serious consequences to its growth, appearance, and yield.

[0027] However, in literature and patents, this term is also used to describe a plant phenotype in which at least some disease symptoms remain absent when the plant is exposed to an infectious dose of the virus, thereby indicating systemic or local infection, viral replication, and, at least under certain culture conditions, the presence of the viral genome sequence and / or its genome integration in the plant cells. Therefore, tolerant plants are resistant to symptom presentation but asymptomatic to virus carriers. Sometimes, the viral sequence may be present or even replicate in the plant without causing disease symptoms. It should be understood that tolerant plants, although infected with the virus, are generally able to at least moderately limit the growth and development of the virus.

[0028] For this reason, plants that are tolerant according to this definition are best characterized as moderately resistant plants.

[0029] Susceptibility: Plant varieties cannot restrict the growth and development of specific pests or pathogens.

[0030] Melon subspecies, such as the commercially available cultivar C. melo subsp. melo Arava, are susceptible to CYSDV.

[0031] Therefore, compared to the Arava variety, the plants according to the invention possess at least enhanced resistance to CYSDV and possibly to CCYV. The resistance of the invention is resistance to CYSDV, particularly to infectious strains causing more severe symptoms, and resistance to infectious infections or high-pressure infections.

[0032] As used herein, the term "offspring" or "progeny" refers to any plant produced as a progeny by asexual or sexual reproduction of one or more parent plants or their progeny. For example, progeny plants can be obtained by cloning or self-pollination of parent plants, or by hybridization of two parent plants, and include self-pollination as well as F1 or F2 or further generations. F1 is the first generation of offspring produced by the parents, where at least one parent is used as the donor for the trait for the first time, while the offspring of the second generation (F2) or subsequent generations (F3, F4, etc.) are samples produced from self-pollination of F1', F2', etc. Thus, F1 can be (and usually is) a hybrid produced by crossing two truly heritable parents (whose true heritability is homozygous for the trait), while F2 can be (and usually is) an offspring produced by self-pollination of said F1 hybrid. Therefore, progeny includes the first generation obtained after one hybridization, and the second generation obtained from the first generation after further hybridization. Preferably, progeny refers to plants obtained from the defined F1 and one or more backcrosses or self-pollinations, preferably less than 10 times. Therefore, the sequences that can be introduced into the genome are limited to those transferred from the first hybridization.

[0033] As used in this article, the terms “hybridization,” “hybrid,” “cross-pollination,” or “hybrid breeding” refer to the process of applying pollen (artificially or naturally) from a flower of one plant to the ovule (stigma) of a flower of another plant.

[0034] As used herein, “genetic determinant” and / or “QTL” refers to any segment of DNA associated with a biological function. QTLs and / or genetic determinants include, but are not limited to, genes, coding sequences, and / or regulatory sequences required for their expression. They may also contain unexpressed segments of DNA, such as recognition sequences that form other proteins. The term “quantitative trait locus (QTL)” more specifically refers to a genomic region that may contain one or more genes or regulatory sequences. For example, a QTL may contain one or more genes whose products confuse genetic resistance or tolerance. Alternatively, a QTL may contain, for example, a regulatory gene or sequence whose products affect the expression of genes at other loci in the plant genome, thereby confusing resistance or tolerance. The QTLs of the present invention can be defined by using one or more molecular genomic markers to indicate their genetic location in the genome of the corresponding pathogen-resistant germplasm. One or more markers then indicate a specific locus. The distance between loci is typically measured by the frequency or crossing over between loci on the same chromosome. The greater the distance between two loci, the more likely crossing over is to occur between them. Conversely, if two loci are close to each other, crossing over is less likely to occur between them. Typically, 1 centimole (cM) equals 1% recombination rate between loci (markers). When a QTL can be indicated by multiple markers, the genetic distance between the terminal markers indicates the size of the QTL.

[0035] As used herein, the term "genotype" refers to the genetic makeup of a single cell, cell culture, tissue, organism (e.g., plant), or population of organisms.

[0036] As used herein, the term “heterozygote” refers to a diploid or polyploid individual cell or plant that has different alleles (in the form of a given gene, genetic sequence, or QTL) present at at least one locus.

[0037] As used in this article, the term "heterozygous" refers to the presence of distinct alleles (in the form of a given gene, genetic sequence, or QTL) at a particular locus.

[0038] As used herein, homologous chromosomes or homologues (or homologs) refer to a set of one maternal chromosome and one paternal chromosome that pair up during meiosis. These copies have the same genes at the same loci and centromere.

[0039] As used in this article, the term "homozygote" refers to an individual cell or plant that has the same alleles at one or more loci on all homologous chromosomes.

[0040] As used in this article, the term "homozygous" means that the same alleles are present at one or more loci in a homologous chromosomal region.

[0041] As used in this article, the term "hybrid" refers to any individual cell, tissue, or plant produced by hybridization between parents that are different in one or more genes.

[0042] As used herein, the term "locus" (plural: "loci") refers to any defined site on a gene. A locus can be a gene, a portion of a gene, or a DNA sequence, and can be occupied by different sequences. A locus can also be defined by an SNP (single nucleotide polymorphism) or multiple SNPs.

[0043] This invention includes plants with different ploidy levels, whether diploid, triploid, tetraploid, etc.

[0044] "Gene infiltration" refers to the infiltration of genes, alleles, or genomic sequences from one species, subspecies, or variety into the gene pool of another species, subspecies, or variety from an initial interspecific hybrid between these species or subspecies.

[0045] "Commercially acceptable fruit quality" refers to edible and marketable fruit that therefore has good size and taste quality, and such fruit preferably has the same shape as fruit that has already been sold (e.g., see illustration). Figure 11 For example, fruits with the same taste qualities as those of commercially marketed varieties. Examples of fruits with commercially acceptable fruit quality could be fruits from the HUGO, ALONSO, ORIGAMI, or VALVEVDE varieties from HM CLAUSE, or the SABROSON variety from Hazela. Wild species typically do not have fruits exhibiting commercially acceptable fruit quality.

[0046] Through association or genetic association, and more specifically genetic linkage, it should be understood that the polymorphism of a genetic marker (e.g., a specific allele of an SNP marker, where an allele can be defined by a nucleotide found on any strand of DNA, i.e., allele A is equivalent to allele T and allele G is equivalent to allele C) and the target phenotype occur simultaneously, i.e., co-inherited, more often than expected to occur randomly, i.e., due to the proximity of the alleles and the genetic sequences responsible for the phenotype in the genome, there is a non-random association between them.

[0047] According to the present invention, CYSDV is a genus of hairy virus that causes the symptoms mentioned in the aforementioned background section, and has a representative sequence FJ492808 (SEQ ID NO: 68), or a sequence having at least 90% sequence identity, preferably at least 95% or at least 98% sequence identity with the representative sequence. Suitable primers for detecting such viruses are disclosed in Example 6 and Abrahamian et al. (2020).

[0048] According to the present invention, CCYV is a genus of hairy virus that causes the symptoms mentioned in the aforementioned background section, and has a representative sequence AB523789 (SEQ ID NO: 69), or a sequence having at least 90% sequence identity, preferably at least 95% or at least 98% sequence identity with the representative sequence. Suitable primers for detecting such viruses are disclosed in Example 6 and Abrahamian et al. (2020). Detailed Implementation

[0049] The inventors have identified genetic determinants, hereinafter also referred to as QTLs, in wild melon germplasm that, when present in specific combinations, confer resistance to trichoviruses (particularly CYSDV and / or CCYV). Therefore, this invention relates to plants, seeds, and cells containing one or more of these genetic determinants, specifically plants, seeds, and cells containing one combination of genetic determinants or QTLs conferring resistance to CYSDV and / or CCYV. The invention also discloses molecular genetic markers, particularly SNPs, linked to resistance loci or genetic determinants. Resistance is preferably resistance to at least CYSDV, at least CCYV, or at least CYSDV and CCYV.

[0050] According to the first aspect, the present invention therefore relates to a melon plant or seed resistant to the genus Trichophyton (more specifically, resistant to Cucumber Yellow Dwarf Disorder Virus (CYSDV) and / or Cucurbit Yellowing Virus (CCYV)) containing one or more of the following QTLs in its genome or genotype:

[0051] - QTL1 located on chromosome 1;

[0052] - QTL5 located on chromosome 5, and

[0053] - QTL11 located on chromosome 11,

[0054] More specifically, one of the following combinations:

[0055] - Homozygous QTL1, and potentially at least one of QTL5 and QTL11 that are independently homozygous or heterozygous.

[0056] - Heterozygous QTL1, and both QTL5 and QTL11, which are either homozygous or heterozygous, or

[0057] - Both are homozygous QTL5 and QTL11.

[0058] The combination of the QTLs imparts resistance to CYSDV and / or CCYV.

[0059] Hereinafter, the combination of the above-mentioned genetic determinants or QTLs will be referred to as the combination of the present invention, or the combination of the genetic determinants or QTLs of the present invention. A genome or genotype that corresponds only to a homozygous QTL1 will also be referred to as a "combination" because it involves a combination of at least two QTL1s, each of which is homologous on chromosome 1. In a preferred embodiment, the combination according to the present invention comprises at least two different QTLs, i.e., a combination of at least two of the three QTLs, i.e., one of the following combinations:

[0060] - A homozygous QTL1, and at least one of QTL5 and QTL11 that is independently homozygous or heterozygous.

[0061] - Heterozygous QTL1, and both QTL5 and QTL11, which are independently homozygous or heterozygous, and

[0062] - Both are homozygous QTL5 and QTL11.

[0063] The present invention also relates to cells of such plants, seeds or plant parts that contain one of a combination of genetic determinants or QTLs that confer resistance.

[0064] By virtue of resistance or tolerance to CYSDV and / or CCYV, it should be understood that, compared to plants that do not contain one of the combinations of the present invention but potentially contain one of the QTLs according to the present invention, such plants do not exhibit symptoms commonly attributed to CYSDV or CCYV infection, especially leaf spots, yellowing of leaves, etc., of chlorosis, or exhibit fewer or milder symptoms, or have reduced viral replication, thereby reducing viral load. However, plants whose symptoms are masked but still present, such as plants with the SGR phenotype, are not considered resistant.

[0065] Regardless of the plant's growing location, it can exhibit resistance or tolerance according to the invention, although resistance levels are observed to vary with climate. The resistance or tolerance according to the invention is also preferably independent of CYSDV or CCYV isolates.

[0066] Tolerance / resistance phenotypes can be tested and scored as described in the experimental section by natural infection (e.g., using whiteflies) or artificial inoculation (using whiteflies pre-infected with the virus, preferably at the first leaf level). Other protocols are also known to those skilled in the art. Infection can be caused by CYSDV alone, or by both CYSDV and CCYV, as often occurs under natural infection conditions.

[0067] The presence of viral sequences can be detected by ELISA or polymerase chain reaction (PCR), especially quantitative PCR (qPCR). Viral activity can be tested by bioassays on tobacco plants.

[0068] The melon plant, seeds, or cells according to the first aspect of the invention are advantageously commercial plants, seeds, or cells, such as superior varieties. This means that the plant can be cultivated due to its agronomic characteristics, especially fruit quality. When cultivated and pollinated under suitable conditions, this plant is capable of producing marketable fruit, or, when cultivated under suitable conditions, is suitable for pollinating other plants to produce marketable fruit. Preferably, this plant has commercially acceptable fruit quality. Therefore, the melon plant, seeds, or cells according to the invention are not wild germplasm that produces unmarketable fruit as shown in the examples.

[0069] In some embodiments, the plant, cell, or seed is a subspecies of melon, specifically a thick-skinned melon plant, cell, or seed, particularly a melon capable of producing marketable fruit (i.e., having a suitable taste, appropriate size, and being able to be stored and transported).

[0070] The QTLs according to the present invention are indeed not related to negative or harmful factors that cause undesirable phenotypes (e.g., unsellable fruits) to be found in wild germplasm.

[0071] Regarding the QTLs of this invention, they correspond to sequences infiltrated from wild melon germplasm genes.

[0072] QTL1:

[0073] The QTL1 of the present invention corresponds to a gene introgression sequence on chromosome 1, particularly in the chromosomal region flanked by SNP ME-0004372 (SEQ ID NO:1) and SNP ME-0007598 (SEQ ID NO:14), or in a region defined by a boundary less than 5 centimoles, or preferably less than 1 centimole, or alternatively less than 0.1 megabases different from these markers.

[0074] Preferably, the gene introgression sequence is found at the positions corresponding to ME-0006564 (SEQ ID NO:3) and ME-0008111 (SEQ ID NO:6), and even more preferably, within the region defined by ME-0027623 (SEQ ID NO:19) and SNP ME-0002337 (SEQ ID NO:4). In a specific embodiment, the gene introgression sequence is found within the region defined by or flanked by SNP ME-0027365 (SEQ ID NO:20) and SNP ME-0002337 (SEQ ID NO:4). As mentioned above, the boundaries may be different.

[0075] Therefore, the plants, seeds, or cells of the present invention containing this QTL contain a gene introgression sequence derived from wild germplasm on chromosome 1, in the region flanking these markers. When the gene introgression sequence is present in homozygous form, i.e., when it is present homozygous on all chromosomes 1 of the plant, seed, or cell, the gene introgression sequence provides resistance to CYSDV and / or CCYV.

[0076] In the plant, seed or cell of the present invention containing the QTL1, the gene introgression sequence is preferably found in the genome at a genetic distance of less than 20 cM, preferably less than 15 cM, most preferably less than 10 cM, or even more preferably less than 5 cM from the locus corresponding to SNP ME-0027623, ME-0027365 or ME-0002337.

[0077] The specific polymorphisms corresponding to the SNPs (single nucleotide polymorphisms) or markers mentioned in this specification, and the flanking sequences of these SNPs or markers in the melon public genome assembly (DHL92) version 3.6.1 (Garcia-Mas et al., 2012, available at http: / / cucurbitgenomics.org / organism / 18), are given in Table A and the attached sequence listing. Their positions (chromosomes and locations) relative to the melon genome DHL92 and its flanking sequences are also shown in this table.

[0078] In this regard, it should be noted that, by definition, an SNP refers to a single nucleotide in the genome that varies depending on the presence of alleles, while flanking nucleotides are identical. To facilitate clear identification of the locations of different SNPs, their locations are given in the table, identified by SEQ ID numbers by reference to the Melon genome assembly (DHL92) version 3.6.1 and by reference to their flanking sequences. In sequences associated with a specific SNP in this application, such as SEQ ID NO:1 of SNP ME-0004372, only one nucleotide in the sequence actually corresponds to the polymorphism, i.e., nucleotide 26 of SEQ ID NO:1 corresponds to the polymorphic position of SNP ME-0004372, which can be A (or T, depending on the DNA strand) or C (or G, depending on the DNA strand) as shown in Table A, corresponding to M in the sequence list. Flanking sequences are given for locating SNPs in the genome, but they are not part of this polymorphism. The indicative polymorphic nucleotide, A (or T, depending on the DNA strand) or C (or G, depending on the DNA strand), corresponds to position 96324 in the melon genome DHL92 as indicated in the table. It is important to note that the polymorphic alleles at the marker positions in Table A are indicated according to conventional orientation. Therefore, the detection of an SNP marker or its allele refers to the detection of the polymorphic nucleotide on either DNA strand, and it is not necessary for all flanking sequences to be identical.

[0079] Therefore, genomic or chromosomal regions identified by flanking sequences, such as SNP markers, are defined clearly and unambiguously.

[0080] A genomic region defined or flanked by two SNPs X and Y refers to a portion of the genome, more specifically a portion of a chromosome, located between the locations of the two SNPs, preferably including the SNPs, such that the nucleotide sequence of the chromosomal region begins with a nucleotide corresponding to SNP X and ends with a nucleotide corresponding to SNP Y, i.e., according to the invention, the SNPs are contained within the regions they define.

[0081] "A gene introgression sequence from the donor" is present at a given locus in the genome of a melon plant, cell, or seed, and should be understood as the genome sequence found at that locus having the same sequence as the corresponding genome sequence found at the same locus in the donor (i.e., the wild-type gene introgression mate).

[0082] The term "gene introgression sequence from the donor within a designated region defined by or flanked by two SNPs X and Y" in the genome of a melon plant, cell, or seed should be understood as all or part of the genome sequence located between the positions of the two SNPs being a gene introgression sequence from the donor.

[0083] For example, GISH (genetic in situ hybridization) can be used to show the presence of introgression sequences from wild-type donors in the genome of melon plants, seeds, or cells. GISH is indeed a powerful technique for detecting the introgression of chromatin material from one species, subspecies, or germplasm into another. An advantage of GISH is that the introgression process is visualized as a "genomic picture of introgression." This technique can also determine whether a specific genomic region is homozygous or heterozygous, thanks to the use of codominant molecular cytogenetic markers. Furthermore, this technique can identify the chromosome on which the target introgressed gene is present.

[0084] According to a preferred embodiment, the plant, cell, or seed of the present invention contains gene introgression sequences in the region defined by SNPs ME-0004372 and ME-0007598 on chromosome 1. These gene introgression sequences are corresponding sequences at homologous positions in wild-type germplasm donors, and when homozygous, confer resistance to CYSDV and / or CCYV. According to other embodiments, the resistance-conferring gene introgression sequences (when homozygous) are located in the region flanked by markers ME-0006564 and ME-0008111 or markers ME-0027623 and ME-0002337. In a particularly preferred embodiment, the resistance-conferring wild-type germplasm gene introgression sequences are located in the region defined by or flanked by markers ME-0027365 and ME-0002337.

[0085] Preferably, the introgression sequence extends from the position corresponding to marker ME-0027365 to the position corresponding to marker ME-0002337. When homozygous, these introgression sequences confer resistance to CYSDV and / or CCYV.

[0086] According to the implementation method, the first genetic determinant or QTL (corresponding to QTL1) is selected from those present in the seed genome of melon ME22BNGA-F06-52563 / 001. This genetic determinant is indeed present in the genome or genotype of these deposited seeds. Seed samples representing these melon subspecies of thick-skinned melon seeds were deposited on June 7, 2023, at the National Centre for Industrial, Food and Marine Microbiology Collection (NCIMB) (NCIMB, Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK), accession number NCIMB 44156, in accordance with and in compliance with the requirements of the Budapest Treaty.

[0087] Therefore, QTL1s can be obtained, for example, from melon plants grown from seeds of ME22BNGA-F06-52563 / 001 through hybridization and selection, so that genetic determinants or QTL1s conferring resistance to CYSDV and / or CCYV (when homozygous) can be introduced. The genome of ME22BNGA-F06-52563 / 001 seeds does indeed contain homozygous QTL1s. For example, homozygous QTL1s can be obtained through a first hybridization with plants grown from preserved seeds, self-pollination of the F1 generation, and selection of plants containing homozygous QTL1s in the F2 generation.

[0088] Therefore, the presence of introgression sequences on chromosome 1 corresponding to QTL1 or genetic determinants that confer resistance phenotypes can be identified based on phenotype (i.e., CYSDV and / or CCYV resistance) or by using SNP markers associated with introgression sequences.

[0089] In some embodiments, when homozygous, the QTL1 conferring CYSDV and / or CCYV resistance on chromosome 1 is identified or characterized in melon plants, seeds, or cells by detecting one or more of the following markers: ME-0006564, ME-0027363, ME-0027608, ME-0027609, ME-0027622, ​​ME-0027623, ME-0027365, ME-0002337, ME-0006240, ME-0027367, ME-0027328, ME-0027330, ME-0027332, ME-0027334, ME-0027336, and ME-008111, or by any other marker within the chromosomal region defined by ME-0006564 and ME-008111. In some embodiments, the detection of ME-0027623, ME-0027365, and / or ME-0002337 is used to identify the presence of QTL1. A particularly preferred marker is, for example, ME-0027623. In some embodiments, the detection of these markers is performed by amplification, preferably by PCR using specific primers that can be used to amplify the resistance / susceptibility allele for each of these markers, or by sequencing, by hybridization with a suitable probe, or by restriction with a suitable enzyme. Such methods are well known to those skilled in the art.

[0090] Therefore, in the plants, cells, or seeds of the present invention, QTL1 is preferably obtained or available from preserved seeds, i.e., as offspring of said seeds, after 1, 2, 3, 4, or 5 to 10 or more hybridizations, and selected based on at least one of the aforementioned markers or other markers based on distinguishable resistance and susceptibility alleles within a genomic region; this ensures that the QTL1 present in said plants, cells, or seeds is indeed the same as that present in preserved seeds.

[0091] According to a preferred embodiment, the presence of the gene introgression sequence in the melon plant, cell, or seed of the present invention, obtainable from ME22BNGA-F06-52563 / 001 seeds or from another source containing QTL1, can be identified by at least two, preferably at least three, or at least four of the SNP markers described. For example, the presence of a gene introgression sequence conferring CYSDV and / or CCYV resistance from a donor on chromosome 1 can be detected by the presence of a haplotype consisting of at least two SNPs, such as ME-0027365 and ME-0002337, or by at least two SNPs, one of which is ME-0027623.

[0092] For the different SNPs on chromosome 1 disclosed herein, alleles representing QTL1 or introgression sequences that confer resistance to the present invention are reported in Table A. Methods for determining suitable alternative markers are also disclosed in this specification.

[0093] For the above SNP markers, the alleles representing gene introgression into QTL1, thereby allowing the detection of that QTL1, are: allele G of marker ME-0006564, allele G of marker ME-0027363, allele G of marker ME-0027608, allele C of marker ME-0027609, allele C of marker ME-0027622, ​​allele G of marker ME-0027623, and allele A of marker ME-0027365. Allele A of marker ME-0002337, allele G of marker ME-0006240, allele G of marker ME-0027367, allele A of marker ME-0027328, allele G of marker ME-0027330, allele A of marker ME-0027332, allele A of marker ME-0027334, allele A of marker ME-0027336, and allele G of marker ME-008111. The particularly preferred allele is allele G of ME-0027623.

[0094] Therefore, the presence of QTLs in the genome or genotype of melon plants, cells or seeds according to the first aspect of the present invention can be detected or revealed by detecting sequences representing QTL1, more preferably by detecting one or more resistance alleles of the disclosed SNPs, preferably at least two or at least three of these alleles, for example at least five or at least eight.

[0095] In some implementations, the markers are detected by amplification, preferably by PCR, using specific primers that can amplify the resistance / susceptibility alleles of each marker.

[0096] For example, the detection of the marker can be performed using two forward primers, one specifically targeting the resistance allele and the other specifically targeting the susceptibility allele, and a common reverse primer (such as in the KASPAR assay). Alternatively, the marker can be detected using forward and reverse primers flanking the target SNP, and two probes with different fluorescent reporter molecules. The first probe (labeled with one fluorescent dye) detects the resistance allele sequence, while the second probe (labeled with another fluorescent dye) detects the susceptibility allele (such as in the TaqMan assay). This applies to all allele markers disclosed in this specification, or to alternative allele markers that can be readily designed by those skilled in the art based on the teachings of this invention. Other detection methods include, for example, high-resolution melting (HRM), sequencing, hybridization; or other techniques well known to those skilled in the art may also be used.

[0097] The QTL1 or introgression sequence of the present invention, which confers resistance to CYSDV and / or CCYV in the melon plant, cell, or seed when present in homozygosity, can be heterozygous or homozygous. The inventors have experimentally demonstrated that the QTL1 confers resistance to CYSDV and / or CCYV in a homozygous state, and further confers resistance in a heterozygous state when combined with QTL5 and QTL11 as defined according to the present invention. Simultaneous detection of susceptibility and resistance alleles of one or more markers indicates the heterozygous presence of QTL1; detection of a resistance allele alone indicates the homozygous presence of QTL1.

[0098] QTL5:

[0099] The QTL5 of the present invention corresponds to a gene introgression sequence on chromosome 5, particularly within a chromosomal region flanked by SNP ME-0004289 (SEQ ID NO: 27) and SNP ME-0006334 (SEQ ID NO: 40), or within a region defined by a boundary less than 5 centimoles, or preferably less than 1 centimole, or less than 0.1 megabases different from these markers.

[0100] Preferably, the introgression sequence is located within a chromosomal region bounded by markers ME-0027624 (SEQ ID NO:55) and ME-0000109 (SEQ ID NO:35), even more preferably within a region bounded by ME-0004225 (SEQ ID NO:31) and ME-0027388 (SEQ ID NO:60), or within a region bounded by ME-0027624 and ME-0027650 (SEQ ID NO:56), or within a region bounded by ME-0004225 and ME-0027650, or within a region bounded by ME-0003342 (SEQ ID NO:32) and ME-0027650. As mentioned above, the boundaries may be different.

[0101] Therefore, the plants, seeds, or cells of the present invention containing this QTL contain, on chromosome 5, in the regions flanking these markers, introgression sequences derived from wild germplasm. These introgression sequences participate in resistance to CYSDV and / or CCYV, conferring resistance when combined with QTL1 and QTL11, or when homozygous in combination with homozygous QTL11; these sequences, when homozygous, also enhance the resistance provided by homozygous QTL1 (see [link to original text]). Figure 5 ).

[0102] According to a preferred embodiment, the plant, cell, or seed of the present invention containing the QTL5 includes a gene introgression sequence in the region defined by SNPs ME-0004289 and ME-0006334 on chromosome 5. These gene introgression sequences are corresponding sequences at homologous positions in wild-type germplasm donors and, when combined with QTL1 and QTL11, or when present homozygous with a homozygous QTL11, confer resistance to CYSDV and / or CCYV. According to other embodiments, the gene introgression sequence involved in resistance is found in a region flanked by markers ME-0027624 and ME-0000109, or ME-0004225 and ME0027388, or ME-0027624 and ME-0027650, or ME-004225 and ME-0027650, or ME-0003342 and ME-0027650.

[0103] Preferably, the introgression sequence extends from the position corresponding to marker ME-0004225 to the position corresponding to marker ME-0000109, or from ME-0027624 to ME-0000109, or from ME-0004225 to ME-0027388, or from ME-0027624 to ME-0027650, or from ME-004225 to ME-0027650, or from ME-0003342 to ME-0027650. When these introgression sequences are combined with QTL1 and QTL11, or when they are homozygous with homozygous QTL11, these introgression sequences confer CYSDV and / or CCYV resistance; when they are homozygous, they also enhance the resistance conferred by homozygous QTL1.

[0104] In the plant, seed, or cell of the present invention containing the QTL5, the introgression sequence is preferably found in the genome at a genetic distance of less than 20 cM, preferably less than 15 cM, most preferably less than 10 cM, and even more preferably less than 5 cM from the locus corresponding to SNP ME-0004225, ME-0003342, ME-0027650, ME-0027384, ME-0027385, ME-0027387, ME-0027388, or ME-0006334.

[0105] According to the implementation method, the genetic determinant or QTL (corresponding to QTL5) is selected from those present in the seed genome of melon ME22BNGA-F06-52563 / 001. This genetic determinant is indeed present in the genome or genotype of these preserved seeds.

[0106] Therefore, QTL5s can be obtained, for example, from melon plants grown from seeds of the melon variety ME22BNGA-F06-52563 / 001 through hybridization and selection, so that genes can be introduced into the genetic determinant or QTL5, thereby conferring resistance to CYSDV and / or CCYV. The genome of the ME22BNGA-F06-52563 / 001 seed does indeed contain homozygous QTL5s. For example, homozygous QTL5s can be obtained through a first hybridization with a plant grown from preserved seeds, self-pollination of the F1 generation, and selection of plants containing homozygous QTL5s in the F2 generation. Heterozygous QTL5s can be obtained, for example, through a first hybridization with a plant grown from preserved seeds.

[0107] Therefore, when combined with QTL1 and QTL11, or when it is homozygous with a homozygous QTL1 or homozygous with a homozygous QTL11, the presence of introgression sequences corresponding to QTL5 or genetic determinants on chromosome 5 that participate in the resistance phenotype can be identified based on the phenotype (i.e., CYSDV and / or CCYV resistance) or by using SNP markers associated with the introgression sequences.

[0108] In some embodiments, the QTL5 on chromosome 5 involved in CYSDV and / or CCYV resistance can be identified or characterized in melon plants by detecting one or more of the following markers: ME-0027624, ME-0004225, ME-0003342, ME-0027650, ME-0009162, ME-0009163, ME-0027384, ME-0027385, ME-0027387, ME-0027388, ME-0007780 and / or SNPs. ME-0000109, or by detecting one or more of the markers ME-0027624, ME-0004225, ME-0003342, ME-0027650, ME-0009163, ME-0027384, ME-0027385, ME-0027387, ME-0027388, ME-0007780 and / or SNP ME-0000109, or by any other marker within the chromosomal region defined by ME-00004289 and ME-0006334, in melon plants. In some embodiments, detection of these markers is performed by amplification, preferably by PCR, using specific primers suitable for amplifying the resistance / susceptibility alleles in each of these markers, or by sequencing, by hybridization with a suitable probe, or by restriction with a suitable enzyme. This method is well known to those skilled in the art.

[0109] Therefore, in the plants, cells, or seeds of the present invention, QTL5 is preferably obtained or available from preserved seeds, i.e., as offspring of said seeds, after 1, 2, 3, 4, or 5 to 10 or more hybridizations, and selected based on at least one of the aforementioned markers or other markers based on distinguishable resistance and susceptibility alleles within a genomic region; this ensures that the QTL5 present in said plants, cells, or seeds is indeed the same as that present in preserved seeds.

[0110] According to a preferred embodiment, the presence of the introgression sequence in the melon plant, cell, or seed of the present invention, obtainable from ME22BNGA-F06-52563 / 001 seeds or from another source containing QTL1, can be identified by at least two, preferably at least three, or at least four of the SNP markers described. For example, the presence of a donor-derived introgression sequence on chromosome 5 involved in CYSDV and / or CCYV resistance can be detected by the presence of a haplotype consisting of at least two SNPs.

[0111] For the different SNPs on chromosome 5 disclosed, the alleles of these molecular markers, representing QTL5 or gene introgression sequences that confer resistance to the present invention, are reported in Table A.

[0112] For the above SNP markers, the alleles representing gene introgression into QTL5 and thus allowing the detection of that QTL5 are allele G of marker ME-0027624, allele A of marker ME-0004225, allele A of marker ME-0003342, allele C of marker ME-0027650, allele A of marker ME-0009162, allele A of marker ME-0009163, allele G of marker ME-0027384, allele G of marker ME-0027385, allele G of marker ME-0027387, allele G of marker ME-0027388, allele G of marker ME-0007780, and allele G of marker ME-0000109; preferably, marker ME-00276 Allele G of marker ME-0004225, allele A of marker ME-0003342, allele C of marker ME-0027650, allele A of marker ME-0009163, allele G of marker ME-0027384, allele G of marker ME-0027385, and allele of marker ME-0027387. G, the allele G of marker ME-0027388, the allele G of marker ME-0007780, and the allele G of marker ME-000010; even more preferably, the allele G of marker ME-0027624, the allele A of marker ME-0004225, the allele A of marker ME-0003342, and the allele C of marker ME-0027650.

[0113] Therefore, the presence of QTLs in the genome or genotype of melon plants, cells or seeds according to the present invention can be detected or revealed by detecting sequences representing QTL5, more preferably by detecting one or more resistance alleles of the SNPs disclosed above, preferably at least two or at least three of these alleles, for example at least five or at least eight.

[0114] For example, the detection of the marker can use two forward primers, one specifically targeting the resistance allele and the other specifically targeting the susceptibility allele, and a common reverse primer (such as in the KASPAR assay). Alternatively, the detection of the marker can use forward and reverse primers flanking the target SNP, and two probes with different fluorescent reporter molecules. The first probe (labeled with one fluorescent dye) detects the resistance allele sequence, while the second probe (labeled with another fluorescent dye) detects the susceptibility allele (such as in the TaqMan assay). This applies to all allele markers disclosed in this specification, or to alternative allele markers that can be readily designed by those skilled in the art based on the teachings of this invention. Other detection methods include, for example, high-resolution melting (HRM), sequencing, and hybridization; or other techniques well known to those skilled in the art may also be used.

[0115] The melon plants, cells, or seeds of this invention can be heterozygous or homozygous for the QTL5 or gene introgression sequence of this invention. The inventors have indeed demonstrated in the experimental section that when QTL5 is combined with QTL1 and QTL11, or when it is homozygous in combination with homozygous QTL11, said QTL5 can confer resistance to CYSDV and / or CCYV; when QTL5 is homozygous, it can also enhance the resistance to CYSDV and / or CCYV conferred by homozygous QTL1. Simultaneous detection of susceptibility and resistance alleles of one or more markers indicates the heterozygous presence of QTL5; detection of a resistance allele alone indicates the homozygous presence of QTL5.

[0116] QTL11:

[0117] The QTL11 of the present invention corresponds to a gene introgression sequence on chromosome 11, particularly in the chromosomal region flanked by SNP ME-0005874 (SEQ ID NO:41) and SNP ME-0000595 (SEQ ID NO:44), or in a region defined by a boundary less than 5 centimoles, or preferably less than 1 centimole, or alternatively less than 0.1 megabases different from these markers.

[0118] Preferably, the gene introgression sequence is located within the chromosomal region defined by markers ME-0005874 (SEQ ID NO:41) and ME-0019064 (SEQ ID NO:43), more preferably within the region defined by SNP ME-0027670 (SEQ ID NO:52) and SNP ME-00027671 (SEQ ID NO:53). As mentioned above, the boundaries may be different.

[0119] Therefore, the plants, seeds, or cells of the present invention containing this QTL contain introgression sequences derived from wild germplasm on chromosome 11, in the regions flanking these markers. These introgression sequences are involved in resistance to CYSDV and / or CCYV, i.e., they confer resistance when combined with QTL1 and QTL5, or when homozygous for their combination with homozygous QTL5; these sequences, when homozygous, also enhance the resistance provided by homozygous QTL1 (see [link to original text]). Figure 5 ).

[0120] In the plant, seed, or cell of the present invention containing the QTL11, the gene introgression sequence is preferably found in the genome at a genetic distance of less than 20 cM, preferably less than 15 cM, most preferably less than 10 cM, and even more preferably less than 5 cM from the locus corresponding to SNP ME-0005874, ME-0019064, ME-0027670, or ME-00027671.

[0121] According to a preferred embodiment, the plant, cell, or seed of the present invention containing the QTL includes a gene introgression sequence in the region on chromosome 11 defined by SNPs ME-0005874 and ME-0000595. These gene introgression sequences are corresponding sequences at homologous positions in wild-type germplasm donors that, when combined with QTL1 and QTL5, or when homozygous with a homozygous QTL5, confer resistance to CYSDV and / or CCYV, or improve resistance provided by QTL1. According to other embodiments, the gene introgression sequence involved in resistance is found in a region flanked by markers ME-0005874 and ME-0019064, or markers ME-0027670 and ME-0027671.

[0122] Preferably, the gene introgression sequence extends from the position corresponding to marker ME-0005874 to the position corresponding to marker ME-0000595. When combined with QTL1 and QTL5, or when homozygous in combination with homozygous QTL5, the gene introgression sequence confers resistance to CYSDV and / or CCYV; when homozygous in combination, they also enhance the resistance conferred by homozygous QTL1.

[0123] According to the implementation method, the genetic determinant or QTL (corresponding to QTL11) is selected from those present in the seed genome of ME22BNGA-F06-52563 / 001. This genetic determinant is indeed present in the genome or genotype of these preserved seeds.

[0124] Therefore, QTL11 can be obtained from melon plants grown from seeds of melon ME22BNGA-F06-52563 / 001, for example, through hybridization and selection, so that genes can be introduced into the genetic determinants of CYSDV and / or CCYV resistance, or QTL11. The genome of the ME22BNGA-F06-52563 / 001 seed does indeed contain a homozygous QTL11. For example, a homozygous QTL11 can be obtained through a first hybridization with a plant grown from a preserved seed, self-pollination of the F1 generation, and selection of plants containing a homozygous QTL11 in the F2 generation. For example, a heterozygous QTL11 can be obtained through a first hybridization with a plant grown from a preserved seed.

[0125] Therefore, when combined with QTL1 and QTL5, or when homozygous QTL1 is combined with homozygous QTL5, the presence of introgression sequences corresponding to QTL11 or genetic determinants on chromosome 11 that participate in the resistance phenotype can be identified based on the phenotype (i.e., CYSDV and / or CCYV resistance) or by using SNP markers associated with the introgression sequences.

[0126] In some embodiments, the QTL11 on chromosome 11 involved in CYSDV and / or CCYV resistance can be identified or characterized in melon plants by detecting one or more of the following markers: SNPs ME-0005874, ME-0027651, ME-0027655, ME-0027656, ME-0027659, ME-0027664, ME-027667, ME-027668, ME-0027670, ME-0027671, ME-0027675, ME-0007096, ME-0019064, and ME-0000595, or by any other marker within the chromosomal region defined by ME-0005874 and ME-0000595. In some embodiments, the detection of ME-0027670, ME-0027671, and / or ME-0007096 is used to identify the presence of QTL11. A particularly preferred marker is, for example, ME-0027670. In some embodiments, the detection of these markers is performed by amplification, preferably by PCR, using specific primers suitable for amplifying the resistance / susceptibility allele in each of these markers, or by sequencing, by hybridization with a suitable probe, or by restriction with a suitable enzyme. Such methods are well known to those skilled in the art.

[0127] Therefore, in the plants, cells, or seeds of the present invention, QTL11 is preferably obtained or available from preserved seeds, i.e., as offspring of said seeds, after 1, 2, 3, 4, or 5 to 10 or more hybridizations, and selected based on at least one of the aforementioned markers or other markers based on distinguishable resistance and susceptibility alleles within a genomic region; this ensures that the QTL11 present in said plants, cells, or seeds is indeed the same as that present in preserved seeds.

[0128] According to a preferred embodiment, the presence of the introgression sequence in the melon plant, cell, or seed of the present invention, obtained from ME22BNGA-F06-52563 / 001 seeds or from another source containing QTL11, can be identified by at least two, preferably at least three, or at least four of the SNP markers. For example, the presence of a donor-derived introgression sequence on chromosome 11 involved in CYSDV and / or CCYV resistance can be detected by the presence of a haplotype consisting of at least two SNPs, such as ME-0027670 and ME-27671.

[0129] For the different SNPs on chromosome 11 disclosed, alleles representing these molecular markers, such as QTL11 or introgression sequences, conferring resistance to the present invention are reported in Table A.

[0130] For the above SNP markers, the alleles representing the introgression of genes into QTL11, thereby allowing the detection of this QTL11, are allele C of marker ME-0005874, allele A of marker ME-0007096, allele A of marker ME-0019064, allele A of marker ME-0027651, allele C of marker ME-0027655, and allele C of marker ME-0027656. The alleles G of marker ME-0027659, A of marker ME-0027664, A of marker ME-0027667, G of marker ME-027668, G of marker ME-0027670, A of marker ME-0027671, A of marker ME-0027675, and C of marker ME-0000595 are particularly preferred. The allele G of ME-0027670 is especially preferred.

[0131] Therefore, the presence of QTLs in the genome or genotype of melon plants, cells or seeds according to the present invention can be detected or revealed by detecting sequences representing QTL11, more preferably by detecting one or more resistance alleles of the SNPs disclosed above, preferably at least two or at least three of these alleles, for example at least five or at least eight.

[0132] In some implementations, the markers are detected by amplification, preferably by PCR, using specific primers that can amplify the resistance / susceptibility alleles of each marker.

[0133] For example, the detection of the marker can be performed using two forward primers, one specifically targeting the resistance allele and the other specifically targeting the susceptibility allele, and a common reverse primer (such as the KASPAR assay). Alternatively, the detection of the marker can be performed using forward and reverse primers flanking the target SNP, and two probes with different fluorescent reporter molecules. The first probe (labeled with one fluorescent dye) detects the resistance allele sequence, while the second probe (labeled with another fluorescent dye) detects the susceptibility allele (such as the TaqMan assay). This applies to all allele markers disclosed in this specification, or to alternative allele markers that can be easily designed by those skilled in the art based on the teachings of this invention. Other detection methods include, for example, high-resolution melting (HRM), sequencing, and hybridization; or other techniques well known to those skilled in the art may also be used.

[0134] The melon plants, cells, or seeds of this invention can be heterozygous or homozygous for the QTL11 or gene introgression sequence of this invention. The inventors have indeed demonstrated in the experimental section that when QTL11 is combined with QTL1 and QTL5, or when it is homozygous in combination with also homozygous QTL5, said QTL11 can confer resistance to CYSDV and / or CCYV; when QTL11 is homozygous, it can also enhance the resistance to CYSDV and / or CCYV conferred by homozygous QTL1. Simultaneous detection of susceptibility and resistance alleles of one or more markers indicates the heterozygous presence of QTL11; detection of a resistance allele alone indicates the homozygous presence of QTL11.

[0135] Therefore, the plants, seeds, or cells of the present invention contain in their genome a combination of defined genetic determinants, namely, a resistance QTL (homozygous or heterozygous) on chromosome 1, potentially with a QTL on chromosome 5 and / or a QTL on chromosome 11, or both QTL5 and QTL11, said combination providing CYSDV and / or CCYV resistance. The plants, seeds, or cells according to the present invention preferably contain one of the following combinations of genetic determinants:

[0136] a) The resistance QTL (QTL1) on homozygous chromosome 1,

[0137] b) Homozygous QTL1 and heterozygous resistance QTL (QTL5) on chromosome 5,

[0138] c) Homozygous QTL1 and heterozygous QTL5;

[0139] d) Homozygous QTL1 and homozygous resistance QTL on chromosome 11 (QTL11)

[0140] e) Homozygous QTL1 and heterozygous QTL11;

[0141] f) Homozygous QTL1, heterozygous QTL5, and heterozygous QTL11,

[0142] g) Homozygous QTL1, homozygous QTL5, and heterozygous QTL11,

[0143] h) Homozygous QTL1, heterozygous QTL5, and homozygous QTL11,

[0144] i) Homozygous QTL1, homozygous QTL5, and homozygous QTL11,

[0145] j) Heterozygous QTL1, homozygous QTL5, and heterozygous QTL11,

[0146] k) Heterozygous QTL1, heterozygous QTL5, and homozygous QTL11,

[0147] l) Heterozygous QTL1, homozygous QTL5, and homozygous QTL11,

[0148] m) Heterogeneous QTL1, heterogeneous QTL5, and heterogeneous QTL11,

[0149] n) Homozygous QTL5 and homozygous QTL11.

[0150] According to the specific implementation, the combination is selected from the combination a), j), k) and n) detailed above; or j), k) and n).

[0151] According to the invention, such plants, seeds, or cells, comprising combinations corresponding to homozygous QTL1, or combinations of QTL1, QTL5, and QTL11, or combinations of QTL5 and QTL11, can be obtained from the seeds of melon ME22BNGA-F06-52563 / 001, deposited under accession number NCIMB 44156. The deposited seeds do indeed include the three defined QTLs; therefore, QTLs can be introduced through hybridization and selection, particularly from this source. Selection can be based on the disclosed markers for each QTL; these markers do indeed allow for the detection of the presence of homozygous or heterozygous QTLs in offspring produced by hybridization with plants corresponding to the deposited seeds.

[0152] Specifically, the presence of QTL1 can be identified by detecting one or more of the following markers: ME-0006564, ME-0027363, ME-0027608, ME-0027609, ME-0027622, ​​ME-0027623, ME-0027365, ME-0002337, ME-0006240, ME-0027367, ME-0027328, ME-0027330, ME-0027332, ME-0027334, ME-0027336, and ME-000811, preferably ME-0027623, ME-0027365, and / or ME-0002337. It is preferred to detect the presence of QTL1 using at least two, preferably three or more, of these markers.

[0153] That is, the presence of the resistance allele corresponding to QTL1 can be identified by detecting one or more of the following alleles: allele G of marker ME-0006564, allele G of marker ME-0027363, allele G of marker ME-0027608, allele C of marker ME-0027609, allele C of marker ME-0027622, ​​allele G of marker ME-0027623, and allele of marker ME-0027365. A. Allele A of marker ME-0002337, allele G of marker ME-0006240, allele G of marker ME-0027367, allele A of marker ME-0027328, allele G of marker ME-0027330, allele A of marker ME-0027332, allele A of marker ME-0027334, allele A of marker ME-0027336, and allele G of marker ME-00811.

[0154] The presence of QTL5 can be identified by detecting one or more of the following markers: ME-0027624, ME-0004225, ME-0003342, ME-0027650, ME-0009162, ME-0009163, ME-0027384, ME-0027385, ME-0027387, ME-0027388, ME-0007780 and / or SNP. ME-0000109, and preferably identified by one or more of the following markers: ME0027624, ME-0004225, ME-0003342, ME-0027650, ME-0009163, ME-0027384, ME-0027385, ME-0027387, ME-0027388, ME-0007780 and / or SNP ME-0000109. The presence of QTL5 is preferably detected by at least two or more of these markers.

[0155] Specifically, the presence of QTL5 can be identified by detecting one or more of the following alleles: allele G of marker ME-0027624, allele A of marker ME-0004225, allele A of marker ME-0003342, allele C of marker ME-0027650, allele A of marker ME-0009162, allele A of marker ME-0009163, allele G of marker ME-0027384, allele G of marker ME-0027385, allele G of marker ME-0027387, allele G of marker ME-0027388, allele G of marker ME-0007780, and allele G of marker ME-0000109.

[0156] The presence of QTL11 can be identified by detecting one or more of the following markers: ME-0005874, ME-0027651, ME-0027655, ME-0027656, ME-0027659, ME-0027664, ME-0027667, ME-027668, ME-0027670, ME-0027671, ME-0027675, ME-0007096, ME-0019064 and / or ME-0000595, preferably ME-0027670, ME-0027671 and / or ME-0007096. It is preferred that the presence of QTL11 be detected by at least two, preferably three or more of these markers.

[0157] Specifically, the presence of QTL11 can be identified by detecting one or more of the following alleles: allele C of marker ME-0005874, allele A of marker ME-0007096, allele A of marker ME-0019064, allele A of marker ME-0027651, allele C of marker ME-0027655, allele C of marker ME-0027656, and marker... Allele G of ME-0027659, allele A of marker ME-0027664, allele A of marker ME-0027667, allele G of marker ME-027668, allele G of marker ME-0027670, allele A of marker ME-0027671, allele A of marker ME-0027675, and allele C of marker ME-0000595.

[0158] When cultivated under suitable conditions, the plants of the present invention produce fruit of commercially acceptable quality. Furthermore, the number of fruits per plant is substantially unaffected by the presence of combinations of the genetic determinants of the present invention; that is, the yield of plants according to the present invention differs by no more than 20%, preferably no more than 10%, from that of plants with the same genotype but without the aforementioned combinations.

[0159] As described above, the present invention relates to melon plants that exhibit enhanced CYSDV and / or CCYV resistance due to a combination of genetic determinants or QTLs, as well as the seeds that produce those plants, and the cells or other plant parts of those plants or seeds that contain the combination in their genome or genotype, and the offspring of such plants of the present invention that contain the combination of genetic determinants in their genome or genotype.

[0160] Offspring include first, second, and all other progeny resulting from hybridization with the plant according to the invention, wherein hybridization includes self-pollination (crossing with itself) or backcrossing with another plant. The plant or seed according to the invention can be the offspring or progeny of a plant grown from seed ME22BNGA-F06-52563 / 001, deposited in NCIMB with accession number NCIMB 44156. Plants grown from these deposited seeds do indeed contain the QTLs according to the invention. They can be used to transfer one or more QTLs to another background through hybridization and self-pollination and / or backcrossing to obtain a plant containing one of the combinations of QTLs according to the invention, which provides CYSDV and / or CCYV resistance.

[0161] Regarding the preserved seeds NCIMB 44156, it should be noted that these seeds do not correspond to plant varieties and are not homozygous for most genes except for the QTLs on chromosomes 1, 5, and 11; therefore, their phenotypes are not fixed during reproduction except for the CYSDV and / or CCYV resistance / tolerance QTLs; and most of their phenotypic traits segregate during reproduction except for the QTLs of this invention.

[0162] The aforementioned offspring or descendants refer to offspring selected according to the present invention in response to the presence of a combination of genetic determinants.

[0163] According to a preferred embodiment, the present invention relates to seeds as described above, which develop into plants according to the first aspect of the invention, thereby possessing resistance to CYSDV and / or CCYV infection due to the presence of a combination of genetic determinants as described above, particularly one of the combinations of a) to n), or one of the combinations of b) to n), and preferably one of the combinations of a), j), k), and n), or one of the combinations of j), k), and n).

[0164] The present invention also relates to cells of melon plants, such that the cells contain in their genome a combination of the genetic determinants of the present invention that confer resistance to CYSDV and / or CCYV in melon plants, i.e., plant cells comprising:

[0165] - QTL1 located on chromosome 1, within the chromosomal region flanked by SNPs ME-0004372 and ME-0007598.

[0166] - QTL5 is located on chromosome 5, within the chromosomal region flanked by SNPs ME-0004289 and ME-0006334, and

[0167] - QTL11 is located on chromosome 11, within the chromosomal region flanked by SNPs ME-0005874 and ME-0000595.

[0168] The combination includes:

[0169] - A homozygous QTL1, and at least one of potentially homozygous or heterozygous QTL5 and QTL11,

[0170] - Heterogeneous QTL1, and both homozygous or heterozygous QTL5 and QTL11, or

[0171] - Both are homozygous QTL5 and QTL11, and

[0172] The combination of QTLs confers resistance to CYSDV and / or CCYV.

[0173] The cells were derived from melon plants with commercially acceptable fruit quality, such as the thick-skinned melon subspecies with commercially acceptable fruit quality.

[0174] The combination of genetic determinants is a combination already defined within the framework of this invention, preferably a combination of a) to n), or b) to n), preferably a combination of a), j), k), or n); or a combination of j), k), or n); characterized by the same features and preferred embodiments disclosed with respect to plants and seeds according to the foregoing aspects of this invention. The presence of genetic determinants responsible for the target phenotype can be revealed by techniques disclosed above and well known to those skilled in the art.

[0175] The cells according to the invention can be any type of melon cell, especially isolated cells and / or cells capable of regenerating into complete melon plants, carrying a combination of the genetic determinants of the invention. The cell can be a regenerable or non-regenerable cell.

[0176] The present invention also relates to tissue cultures of non-regenerative or regenerative plant cells as defined above according to the present invention; preferably, the regenerative cells are derived from the embryo, protoplast, meristematic cells, callus, pollen, leaf, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, and / or hypocotyl of the present invention, and the cells contain a combination of the genetic determinants of the present invention in their genome, which confer resistance to CYSDV and / or CCYV. Preferably, the tissue culture is an in vitro cell culture or an in vitro tissue culture.

[0177] The tissue culture is preferably capable of regenerating plants with the physiological and morphological characteristics of the aforementioned melon plants, and is also capable of regenerating plants with substantially the same genotype as the aforementioned melon plants. The present invention also provides melon plants regenerated from the tissue culture of the present invention.

[0178] The present invention also relates to a plant part of a plant according to the invention, comprising cells as defined above, and comprising a combination of the QTLs of the invention. According to embodiments of the invention, a plant part is any part of a plant according to the invention, which may in particular be a seed, reproductive material, propagation material, root, flower, fruit, rootstock, or scion. It comprises the defined cells.

[0179] All embodiments detailed in the preceding sections in conjunction with the previous aspects of the invention are also preferred embodiments according to this aspect of the invention.

[0180] The present invention also provides protoplasts of plants as defined above, or protoplasts from tissue cultures as defined above, wherein the protoplasts contain a combination of the genetic determinants of the present invention, conferring the defined phenotype.

[0181] The present invention also relates to tissues of the plant of the present invention; such tissues may be undifferentiated or differentiated tissues. These tissues comprise one or more cells containing combinations of the genetic elements of the present invention.

[0182] The present invention also relates to propagation material capable of producing resistant melon plants (particularly cucurbitaceous plants) according to the invention, comprising a combination of genetic determinants or elements as defined above, and having commercially acceptable fruit quality. Particularly preferred propagation material is seeds. According to embodiments, the present invention relates to seeds of melon plants that develop into plants according to the invention, or derived from plants according to the invention, and which contain a combination of disclosed genetic determinants in their genome.

[0183] The present invention also relates to a container comprising a melon plant resistant to CYSDV and / or CCYV as defined above due to the combination of QTLs of the present invention, or the defined plant portion, or the seeds as defined in the context of the present invention. This plant has commercially acceptable fruit quality.

[0184] According to another aspect, the invention also relates to melon hybrids obtained by crossing melon plants with resistant plants according to the invention, which contain homozygous QTL1s as defined. The resulting hybrids may or may not be resistant to CYSDV and / or CCYV; and contain at least the defined QTL1s in their genome, thus allowing them to be combined with QTL5 and QTL11.

[0185] According to other aspects, the present invention also relates to the use of plants, seeds, parts thereof, or progeny deposited in NCIMB with accession number NCIMB 44156 at NCIMB address ME22BNGA-F06-52563 / 001 as breeding partners in a breeding procedure to obtain plants having the resistance phenotype of the present invention (i.e., CYSDV and / or CCYV resistant plants, especially the melon subspecies *Melon spp.*, thick-skinned melon, preferably commercial melon plants), said plants, seeds, parts thereof, or progeny carrying QTL1, QTL5, and QTL11 according to the present invention, said breeding procedure being intended to infiltrate or transfer these genetic determinants or QTL genes into melon plants. In embodiments, the breeding procedure is used to confer resistance to CYSDV and / or CCYV on melon plants susceptible to CYSDV and CCYV, particularly on plants with commercially acceptable fruit quality.

[0186] This invention also relates not only to the preserved seeds and plants of this invention, but also to plants or seeds containing three QTLs in one of the combinations of this invention, namely, containing QTL1, QTL5, and QTL11, or containing only QTL1, or containing both QTL5 and QTL11 (for use as breeding couples or gene introgression couples in a breeding process to obtain other melon plants with the resistance phenotype of this invention). The combination of genetic determinants will advantageously introduce other desired genetic traits into varieties containing other desirable traits, such as disease resistance, early fruit ripening, drought tolerance, fruit shape, etc., and commercially acceptable fruit quality. Preferably, the QTLs are similarly present in the breeding couple.

[0187] In such a breeding program, the selection of offspring that exhibit the desired phenotype or carry a combination of genetic determinants associated with the desired phenotype can be advantageously based on the alleles of the SNP markers of QTL1, QTL5 and / or QTL11 mentioned above.

[0188] In practice, this selection can be based on the presence of any one or a combination of resistance alleles of the SNP linked to the genetic determinant providing the target phenotype. Preferred selection of offspring is based on the presence of one or more of the following specific alleles: allele G of marker ME-0027623, allele A of marker ME-0027365, allele A of marker ME-0002337, allele A of marker ME-0004225, allele A of marker ME-0003342, allele A of marker ME-0009163, allele G of marker ME-0007780, allele G of marker ME-0000109; allele G of marker ME-0027670, and allele A of marker ME-0027671.

[0189] This selection will be made when the target allele is present in a sample of the genetic material of the plant to be selected. The presence of this or these alleles confirms the presence of a QTL at the locus defined by the SNP on chromosomes 1, 5, and / or 11. However, following a point mutation or recombination event, it is conceivable that at least one or two of these alleles may be lost, but the remaining chromosomal segments carrying the target QTL1, QTL5, and / or QTL11 may still confer the target phenotype.

[0190] Therefore, plants according to the invention, or plants grown from seeds of the invention, are particularly valuable in using marker-assisted selection to obtain commercial melon lines and varieties with the resistance phenotype of the invention.

[0191] The present invention also relates to the use of the plant in procedures aimed at identifying, sequencing and / or cloning gene sequences that confer a desired phenotype.

[0192] Any specific embodiments described in the foregoing aspects of the present invention are also applicable to this aspect of the present invention, particularly with respect to the features of QTLs that impart a target phenotype.

[0193] In its embodiments, the present invention also relates to a method for cultivating melon plants resistant to CYSDV and / or CCYV, comprising at least the following steps:

[0194] - Hybridize CYSDV-susceptible initial melon plants with plants grown from preserved seeds NCIMB 44156, their progeny, or other sources, wherein the plants, their progeny, or other sources carry QTL1 on chromosome 1, preferably also carrying QTL5 on chromosome 5 and / or QTL11 on chromosome 11, and

[0195] - Select plants that contain a combination of QTLs according to claim 1.

[0196] Specifically, QTL1, QTL5, and QTL11 are present in the genome of the seeds of plant ME22BNGA-F06-52563 / 001, NCIMB accession number 44156. For QTL1, it can be identified by allele G of ME-0027623, allele A of ME-0027365, or allele A of ME-0002337. For QTL5, it can be identified by allele A of ME-0004225, allele A of ME-0003342, allele A of ME-0009163, allele G of ME-0007780, or allele G of ME-0000109. For QTL11, it can be identified by allele A of ME-0007096, allele G of ME-0027670, or allele A of ME-0027671.

[0197] According to another aspect, the invention also relates to methods or processes for producing or breeding melon plants, particularly commercial plants, and particularly hybrid and inbred parent lines, with desired phenotypes of resistance to CYSDC and / or CCYV. The invention also relates to transferring combinations of the genetic determinants of the invention that confer resistance to other melon plants, particularly other melon varieties, or other species or inbred parent lines of the melon subspecies *Melon thunbergii*, and can be used to produce new types and varieties of melons, particularly *Melon thunbergii*.

[0198] According to some embodiments, the present invention therefore relates to a method or process for producing plants resistant to CYSDV and / or CCYV or for conferring resistance to CYSDV and / or CCYV on melon plants, comprising the following steps:

[0199] a) A hybridization was performed between plants grown from the preserved seed NCIMB 44156, or their progeny or other sources (containing QTL1 on chromosome 1, and preferably also QTL5 on chromosome 5 and / or QTL11 on chromosome 11) and the initial melon plants susceptible to CYSDV and CCYV.

[0200] b) Select plants from the resulting offspring that carry the combination of QTLs according to the invention (which confer resistance to CYSDV and / or CCYV);

[0201] c) Optionally, the plants obtained in step b) are subjected to multiple self-pollinations, hybridizations, or backcrosses, and the resulting offspring are selected for plants exhibiting CYSDV and / or CCYV resistance, thus resulting in the following plants:

[0202] - A homozygous QTL1, and at least one of potentially homozygous or heterozygous QTL5 and QTL11.

[0203] - Heterozygous QTL1, and homozygous or heterozygous QTL5 and QTL11,

[0204] The QTLs on chromosomes 1, 5, and 11 are present in the genome of a plant seed, ME22BNGA-F06-52563 / 001, NCIMB accession number 44156.

[0205] Alternatively, the method or process may include the following steps instead of step a):

[0206] a1) A plant or its progeny (containing QTL1 on chromosome 1, and preferably also QTL5 on chromosome 5 and / or QTL11 on chromosome 11) grown from the preserved seed NCIMB 44156 is crossed with an initial melon plant susceptible to CYSDV and CCYV to produce an F1 hybrid.

[0207] a2) Propagate the F1 hybrid through self-pollination to generate the F2 population.

[0208] According to other embodiments, the present invention also relates to a method or process for producing plants with resistance to CYSDV and / or CCYV, or for conferring resistance to CYSDV and / or CCYV on melon plants, comprising the following steps:

[0209] a) A hybridization was performed between a plant grown from a preserved seed NCIMB 44156, or its progeny or other sources (containing QTL5 on chromosome 5 and QTL11 on chromosome 11, preferably both homozygous) and a primary melon plant susceptible to CYSDV and CCYV.

[0210] b) Select plants from the resulting offspring that carry the QTL combination according to the invention, which confers resistance to CYSDV and / or CCYV;

[0211] c) Optionally, the plants obtained in step b) are subjected to one or more self-pollination, hybridization, or backcrossing, and the resulting offspring are selected to have plants with CYSDV and / or CCYV resistance, thereby possessing:

[0212] - Both QTL5 and QTL11 are homozygous.

[0213] Alternatively, the method or process may include the following steps instead of step a):

[0214] a1) A plant or its progeny (containing QTL5 on chromosome 5 and QTL11 on chromosome 11, preferably both homozygous) grown from the preserved seed NCIMB 44156 is crossed with an initial melon plant susceptible to CYSDV and CCYV to produce an F1 hybrid.

[0215] a2) Propagate the F1 hybrid through self-pollination to generate the F2 population.

[0216] As an alternative to or supplement to self-crossing in step a2), the method may also include backcrossing, or self-crossing and backcrossing.

[0217] The method or process defined above may advantageously include a backcrossing step d) with one or more other superior lines, preferably after step c), in order to obtain a plant with all the characteristic features of a commercial melon plant, particularly the melon subspecies *Melon thunbergii*.

[0218] The method or process may also include step e), the step of selecting plants resistant to CYSDV and / or CCYV.

[0219] The plant used in step a), i.e., the plant corresponding to the preserved seed, can be a plant grown from the preserved seed; according to other embodiments, it can alternatively be any plant according to the first aspect of the invention, carrying a phenotype-conferring QTL1, preferably carrying homozygous sequences of these sequences. Alternatively, the plant used in step a) can be a plant containing QTL5 and QTL11, but not necessarily containing QTL1. The initial melon plant is preferably a plant that does not contain said QTL.

[0220] The plants selected in step c) or e) are preferably commercial plants, especially those with commercially acceptable fruit quality.

[0221] Preferably, steps d) and e) are repeated at least twice, preferably three times, and the same susceptible melon plant is not necessarily used. The susceptible melon plant is preferably a breeding strain.

[0222] Self-pollination and backcrossing can be performed in any order and can be interspersed. For example, backcrossing can be performed before or after one or more self-pollinations, and self-pollination can be performed before or after one or more backcrosses.

[0223] In the above method or process, it is preferred to use SNP markers in steps b), c) and / or e) to select resistant plants by selecting plants having one of the combinations of the present invention’s QTLs that confer resistance to CYSDV and / or CCYV.

[0224] The SNP markers are preferably those disclosed in conjunction with the foregoing aspects of the present invention.

[0225] When selecting plants based on alleles of one or more SNPs, it should be understood that the plant is selected when the allele of the SNP corresponds to the “resistance” allele of the SNP as defined in Table A. This selection can also be based on any other marker linked to genetic determinants or QTLs, and the presence of these genetic determinants is represented by their sequence being the reverse of the inherent sequence of the susceptible parent.

[0226] In some implementations, the markers used to select plants resistant to CYSDV and / or CCYV are:

[0227] - For QTL1, it is one or more of the following markers: ME-0006564, ME-0027363, ME-0027608, ME-0027609, ME-0027622, ​​ME-0027623, ME-0027365, ME-0002337, ME-0006240, ME-0027367, ME-0027328, ME-0027330, ME-0027332, ME-0027334, ME-0027336, and ME-0008111, preferably ME-0027623, ME-0027365, and / or ME-0002337.

[0228] - For QTL5, it is one or more of the following markers: ME-0027624, ME-0004225, ME-0003342, ME-0027650, ME-0009162, ME-0009163, ME-0027384, ME-0027385, ME-0027387, ME-0027388, ME-0007780 and / or SNP ME-0000109.

[0229] - For QTL11, it is one or more or all of the following markers: ME-0005874, ME-0027651, ME-0027655, ME-0027656, ME-0027659, ME-0027664, ME-027667, ME-027668, ME-0027670, ME-0027671, ME-0027675, ME-0007096, ME-0019064 and / or ME-0000595, preferably ME-0027670, ME-0027671 and / or ME-0007096.

[0230] According to other preferred embodiments, QTL1, QTL5, and QTL11 can be identified as follows: For QTL1, it can be identified by allele G of ME-0027623, allele A of ME-0027365, or allele A of ME-0002337; For QTL5, it can be identified by allele A of ME-0004225, allele A of ME-0003342, allele A of ME-0009163, allele G of ME-0007780, or allele G of ME-0000109; For QTL11, it can be identified by allele A of ME-0007096, allele G of ME-0027670, or allele A of ME-0027671.

[0231] In some embodiments, the plant selected in any of steps b), c), and / or e) is preferably selected based on the presence of one of the allele combinations a) to n) or b) to n) as defined in the first aspect of the invention, for example, combinations a), j), k), or n); or j), k), or n).

[0232] The selection of offspring with the desired phenotype can also be carried out under pathogen infection conditions, especially as disclosed in the examples or other tests known to those skilled in the art.

[0233] Methods for allele detection can be based on any technology that allows two different alleles of a marker to be distinguished or identified on a specific chromosome.

[0234] The present invention also relates to melon plants obtained or obtainable by this method, particularly the melon subspecies *Melon thunbergii*, preferably plants with commercially acceptable fruit quality. Such plants are, in fact, melon plants resistant to CYSDV and / or CCYV according to the first aspect of the present invention.

[0235] The present invention also relates to a method for obtaining commercial melon plants resistant to CYSDV and / or CCYV, the method comprising the following steps:

[0236] - Plants obtained by germinating preserved seeds ME22BNGA-F06-52563 / 001 (NCIMB accession number 44156) or melon plants according to the first aspect of the invention are backcrossed with melon plants (e.g., melon plants susceptible to CYSDV and / or CCYV).

[0237] - Select plants resistant to CYSDV and / or CCYV.

[0238] The selection in the second step is preferably performed as detailed above with respect to other methods of the present invention. The selection is preferably performed in the presence of one or more specific alleles of the marker, as described above, such as those found in the ME22BNGA-F06-52563 / 001 strain.

[0239] The selected plants are preferably commercial plants, especially those with commercially acceptable fruit quality.

[0240] A method for producing melon plant seeds is also provided. In some embodiments, the method includes hybridizing the melon plant according to the invention with itself or with another melon plant and harvesting the resulting seeds.

[0241] In addition to the introgression of QTLs associated with CYSDV and / or CCYV resistance as detailed in the method of this invention, the sequences can also be introduced into a melon background via genetic engineering to obtain commercially viable melon plants resistant to CYSDV and / or CCYV, particularly those with commercially acceptable fruit quality. For those skilled in the art, the identification and cloning of introgression QTLs from melons conferred with the desired phenotype, especially from preserved seeds, is routine.

[0242] Therefore, the present invention also relates to a method for conferring resistance to CYSDV and / or CCYV on melon plants, the method comprising genetically modifying the plant to heterozygous or homozygous introduce QTL1 on chromosome 1 and potentially introduce QTL5 on chromosome 5 or QTL11 on chromosome 11, or both QTL5 on chromosome 5 and QTL11 on chromosome 11, conferring the resistance, or introducing homozygous QTL5 and QTL11, wherein the QTL1 on chromosome 1, the QTL5 on chromosome 5, and the QTL11 on chromosome 11 are as previously defined. Therefore, they exist in the seed genome of plant ME22BNGA-F06-52563 / 001, with representative seeds deposited in NCIMB with accession number 44156. For QTL1, they can be identified by allele G of ME-0027623, allele A of ME-0027365, or allele A of ME-0002337; for QTL5, they can be identified by allele A of ME-0004225, allele A of ME-0003342, allele A of ME-0009163, allele G of ME-000780, or allele G of ME-0000109; and for QTL11, they can be identified by allele A of ME-0007096, allele G of ME-0027670, or allele A of ME-0027671. According to a preferred embodiment, a genetically modified melon plant (which is advantageously a thick-skinned melon plant), for example, one of the combinations a) to n) or b) to n) as described above, is introduced into its genome or genotype, for example, combinations a), j), k) or n).

[0243] It should be noted that the seeds or plants of the present invention can be obtained by different methods, not just by inherently biological methods. In some embodiments, the plants and seeds are not obtained solely by inherently biological methods.

[0244] Therefore, the present invention also relates to seeds and plants specifically obtained through non-biological processes. In some embodiments, the present invention relates to plants and seeds obtained, or specifically obtained through techniques such as targeted mutagenesis, cisgenesis, including intragenesis, and most preferably cisgenesis. Cisgenesis involves inserting genetic material from a sexually compatible donor into a recipient organism, whether or not it has been modified / rearranged.

[0245] According to these embodiments, the plants or seeds of the present invention are obtained by inserting the QTL of the present invention, whether or not the endogenous sequence is replaced, using at least one of site-specific nuclease, oligonucleotide directed mutagenesis, chemical mutagenesis, or tilling.

[0246] According to this aspect, the present invention relates to melon plants or seeds, preferably non-naturally occurring melon plants or seeds, which may contain one or more mutations in their genome that confer resistance to CYSDV and / or CCYV, the mutations corresponding to resistance QTLs on chromosome 1, 5 and / or 11, in combinations defined according to the invention, particularly one of a) to n) or b) to n), such as a), j), k) or n).

[0247] In another embodiment, the present invention relates to a method for obtaining a melon plant or seed carrying one or more mutations in its genome, which provides the plant with resistance to CYSDV and / or CCYV as defined according to the invention. This method is illustrated in the following experimental section and may include:

[0248] a) Treat M0 seeds of melon or thick-skinned melon plants to be modified with a mutagen to obtain M1 seeds;

[0249] b) Grow plants from the M1 seeds thus obtained to obtain M1 plants;

[0250] c) Producing M2 seeds through self-pollination of M1 plants; and

[0251] d) Optionally, repeat steps b) and c) n times to obtain the M1+n seed.

[0252] M1+n seeds grow into plants and are infected with CYSDV and / or CCYV. The surviving plants, or those with milder symptoms of CYSDV and / or CCYV infection, reproduce for one or more generations while continuing to select for resistance to CYSDV.

[0253] In this method, the M1 seed of step a) can be obtained by chemical mutagenesis (e.g., EMS mutagenesis). Other chemical mutagens include, but are not limited to, diethyl sulfate (des), ethyleneimine (ei), propanesulfonate lactone, N-methyl-N-nitrosocarbamate (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (enu), and sodium azide.

[0254] Alternatively, mutations can be induced by radiation, such as radiation selected from X-rays, fast neutrons, or ultraviolet radiation.

[0255] In another embodiment of the invention, the mutation is induced by genetic engineering. This mutation also includes the integration of sequences conferring resistance to CYSDV and / or CCYV, and the replacement of the resident sequence with an alternative sequence conferring resistance to CYSDV and / or CCYV. Preferably, as described above, the mutation is the integration of one of the resistance QTLs and potential QTL5 and QTL11 on chromosome 1 to replace a homologous sequence in the melon plant. According to one embodiment, the mutation is the replacement of sequences or fragments thereof (e.g., sequences contained in SNP ME-0004372 and SNP ME-0007598 on chromosome 1 of the melon genome) with homologous sequences present on chromosome 1 of the plant genome, a representative sample of which is deposited in NCIMB with accession number NCIMB 44156, wherein the sequences or fragments thereof, when homozygous, confer resistance to CYSDV and / or CCYV.

[0256] According to another embodiment, the mutation is the replacement of a sequence or fragment thereof (e.g., a sequence contained in SNP ME-0004289 and SNP ME-0006334 on chromosome 5 of the melon genome) with a homologous sequence present on chromosome 5 of the plant genome, a representative sample of which is deposited in NCIMB with accession number NCIMB 44156, wherein the sequence or fragment thereof participates in resistance to CYSDV and / or CCYV, i.e., conferring resistance to CYSDV and / or CCYV when combined with QTL1 and QTL11, or enhancing the resistance to CYSDV and / or CCYV provided by homozygous QTL1 when homozygous is present.

[0257] In this embodiment, the mutation is the replacement of a sequence or fragment thereof (e.g., a sequence contained in SNPs ME-0005874 and ME-0000595 on chromosome 11 of the melon genome) with a homologous sequence present on chromosome 11 of the plant genome, with a representative sample of the plant deposited in NCIMB with accession number NCIMB 44156. The sequence or fragment thereof participates in resistance to CYSDV and / or CCYV, i.e., conferring resistance to CYSDV and / or CCYV when combined with QTL1 and QTL5, or enhancing the resistance to CYSDV and / or CCYV provided by homozygous QTL1 when homozygous is present.

[0258] Available genetic engineering techniques include the use of all techniques known as New Breeding Techniques, which are various new techniques developed and / or used to create new traits in plants through genetic variation with the aim of targeted mutagenesis, targeted introduction of new genes, or gene silencing (RdDM). Examples of such New Breeding Techniques are those that promote targeted sequence changes through the use of techniques such as: zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2, and ZFN-3, see US Patent No. 9,145,565), oligonucleotide directed mutagenesis (ODM), cisgenesis and intragenesis, grafting (in GM rhizomes), reverse breeding, and Agrobacterium-infiltration. "Strictly speaking," Agrobacterium inoculation (flower dipping method), transcription activator-like effector nucleases (TALENs, see US patents 8,586,363 and 9,181,535), CRISPR / Cas systems (see US patents 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641), engineered meganucleases, reprogrammed homing endonucleases, DNA-guided genome editing (Gao et al., 2016), and synthetic genomics. A key component of targeted genome editing, also known as a new breeding technique, is the application of inducing DNA double-strand breaks (DSBs) at selected sites in the genome where the intended modification is desired. Such applications can be used to generate mutations (e.g., targeted mutations or precise natural gene editing) and to precisely insert genes (e.g., homologous, endogenous, or heterologous genes). A complete description of each of these techniques can be found in the 2011 report entitled "New Plant Breeding Techniques – Existing Technologies and Prospects for Commercial Development" published by the Joint Research Centre for Advanced Technologies (JRC) of the European Commission.

[0259] The present invention also provides a method for detecting and / or selecting melon plants resistant to CYSDV and / or CCYV, wherein the method comprises the following steps:

[0260] (i) Detecting the presence of QTL1 on chromosome 1 as defined according to the invention, and

[0261] (ii) Potentially detecting the presence of QTL5 on chromosome 5 as defined according to the invention, and

[0262] (iii) Potentially detect the presence of QTL11 on chromosome 11 as defined in the present invention.

[0263] If a homozygous QTL1 is detected, steps (ii) and (iii) are optional.

[0264] The present invention also provides a method for detecting melon plants resistant to CYSDV and / or CCYV, wherein the method comprises the following steps:

[0265] (i) Detecting the presence of at least one genetic marker linked to QTL1 on chromosome 1 as described in claim 1, and potentially,

[0266] (ii) Detecting the presence of at least one genetic marker linked to QTL5 on chromosome 5 and / or at least one genetic marker linked to QTL11 on chromosome 11, as described in claim 1.

[0267] The plant is resistant to CYSDV and / or CCYV if the marker linked to QTL1 is homozygous, or if the markers linked to QTL1, QTL5 and QTL11 are detected simultaneously.

[0268] The present invention also provides a method for detecting and / or selecting melon plants resistant to CYSDV and / or CCYV, wherein the method comprises the following steps:

[0269] (i) Detection of homozygous QTL5 on chromosome 5 as defined according to the present invention, and

[0270] (ii) Detecting homozygous QTL11 on chromosome 11 as defined in the present invention.

[0271] In some embodiments, a plant is selected if any of the allele combinations a) to n) as defined in the first aspect of the invention are detected in a sample of the genetic material of the plant to be selected, for example, combinations a), j), k) or n).

[0272] According to another aspect, the present invention also relates to a method for selecting melon plants having QTLs conferring resistance to CYSDV and / or CCYV, the method comprising:

[0273] a) Determine whether a melon plant has at least one first genetic marker that is genetically linked to a QTL1 as defined in this invention;

[0274] b) Potentially, determining whether the plant contains at least one second genetic marker genetically linked to a QTL5 as defined according to the present invention.

[0275] c) Potentially, determining whether the plant has at least one third genetic marker genetically linked to QTL11 as defined in the present invention;

[0276] d) Select plants containing three genetic markers and QTL1, QTL5 and QTL11 conferring resistance to CYSDV and / or CCYV, or plants containing the first genetic marker and a homozygous QTL1.

[0277] Alternatively, methods for selecting melon plants with QTLs that confer resistance to CYSDV or CCYV include:

[0278] a) Determine whether a melon plant has at least one first genetic marker that is genetically linked to a QTL5 as defined in this invention;

[0279] b) Determine whether the plant contains at least one second genetic marker genetically linked to QTL11 as defined in this invention;

[0280] c) Select plants containing two genetic markers and homozygous QTL5 and QTL11 conferring resistance to CYSDV and / or CCYV.

[0281] QTL1, QTL5, and QTL11 are defined as follows: QTL1 and the genetic marker are found in the genomic region on chromosome 1 defined by SNPs ME-0004372 and ME-0007598, preferably in ME-0006564 and ME-0008111; QTL5 and the genetic marker are found in the genomic region on chromosome 5 defined by SNPs ME-0004289 and ME-0006334, preferably in ME-0027624 and ME-0000109; and QTL11 and the genetic marker are found in the genomic region on chromosome 11 defined by SNPs ME-0005874 and ME-000595, preferably in ME-0005874 and ME-0010064.

[0282] If the marker linked to QTL1 is homozygous, or if the marker linked to QTL1, QTL5, and QTL11 is detected simultaneously, or if the marker linked to QTL5 and QTL11 is homozygous, then the plant is detected or selected as resistant to CYSDV and / or CCYV.

[0283] This method is preferably performed when at least two genetic markers are present for each QTL, for example, two different genetic markers, or three or more different genetic markers. For QTL1, at least one marker is ME-0027623. For QTL11, at least one marker is ME-0027670.

[0284] In another aspect, the present invention relates to a method for producing melon seedlings or plants resistant to CYSDV and / or CCYV, the method comprising:

[0285] i. In vitro culture of isolated cells or tissues of melon plants according to the present invention to produce melon microplants resistant to CYSDV and / or CCYV, and

[0286] ii. Optionally, the melon microplants may be further cultured in vivo to develop into melon plants resistant to CYSDV and / or CCYV.

[0287] The isolated cells or tissues used for producing microplants are explants obtained under sterile conditions from the melon parent plant of the present invention to be propagated. The explants comprise, for example, cotyledons, hypocotyls, stem tissues, leaves, embryos, meristems, nodular buds, shoot tips, or protoplasts, or are composed of these. The explants may be surface-sterilized before being placed on a culture medium for micropropagation.

[0288] The conditions and culture media suitable for plant micropropagation are well known to those skilled in the field of plant culture, and are described, for example, in "Plant Propagation by TissueCulture, Handbook and Directory of Commercial Laboratories" (eds. Edwin F. George and Paul D. Sherrington, Exegetics Ltd, 1984).

[0289] Micropropagation typically involves:

[0290] Micropropagation typically involves:

[0291] i. Axillary bud production: Axillary bud proliferation is induced by adding cytokinins to the bud culture medium to produce buds with minimal callus formation;

[0292] ii. Adventitious bud production: Adding auxin to the culture medium induces root formation to produce small plantlets that can be transferred to the soil. Alternatively, root formation can be induced directly in the soil.

[0293] The small plants can then undergo an in vivo culture stage, by being cultured in soil under laboratory conditions and then gradually adapted to the natural climate, to develop into melon plants resistant to CYSDV and / or CCYV, especially the thick-skinned melon subspecies, and with commercially acceptable fruit quality.

[0294] Given the ability of the resistant plants of the present invention to limit damage caused by CYSDV and / or CCYV, they are advantageously grown in environments that have been or may be infected by viruses transmitted by these whiteflies, or that have been or may be infected by whiteflies carrying or potentially carrying these viruses; under these conditions, the resistant plants of the present invention produce more marketable fruit than susceptible plants (including seedless fruit), particularly thick-skinned melons. Therefore, the present invention also relates to a method for increasing the yield of melon plants or increasing the number of harvestable melon plants or fruits, particularly the number of thick-skinned melon subspecies plants in environments infected with CYSDV and / or CCYV transmitted by whiteflies, the method comprising growing melon plants resistant to CCYV and / or CYSDV as defined in said environment, i.e., containing, on chromosome 1, and potentially on chromosomes 5 and 11, or on chromosomes 5 and 11, a QTL or sequence according to the present invention, which confers resistance to CYSDV and / or CCYV to said plant. The present invention also relates to a method for increasing the number of harvestable seedless melon fruits, particularly the melon subspecies *Melon thunbergii*, in an environment infected with CYSDV and / or CCYV transmitted by whiteflies, the method comprising growing melon plants resistant to CCYV and / or CYSDV as defined in the environment, i.e., containing the QTL or sequence according to the invention on chromosome 1, and potentially on chromosomes 5 and 11, or on chromosomes 5 and 11, which confers resistance to CYSDV and / or CCYV, and pollinating with melon plants of different ploidy levels, particularly triploid parents, so that the fruits are seedless.

[0295] Preferably, the method includes a first step: selecting or screening melon plants containing the target sequence or QTL combination, the target sequence or QTL combination conferring resistance to CYSDV and / or CCYV, preferably combinations a) to n), or b) to n), preferably one of a), j), k), or n), or j), k), or n). The method can also be defined as a method for improving the productivity of melon fields, tunnels, or greenhouses, or a method for reducing the intensity or frequency of chemical or fungicide application in melon, especially seedless melon production.

[0296] The present invention also relates to a method for reducing melon production losses under conditions of CYSDV and / or CCYV infection, comprising planting melon plants as described above. The method may advantageously include a step of pollinating with plants of different ploidy levels to obtain seedless fruit.

[0297] The resistant plants of the present invention can also limit the viral replication or reproduction of CYSDV and / or CCYV within the plant, thereby limiting infection by other insects, and consequently limiting infection by other plants and viral proliferation. Therefore, the present invention also relates to a method for protecting fields, tunnels, greenhouses, or any other type of cultivation from infection by whitefly-borne CYSDV and / or CCYV, or at least limiting the level of infection or limiting the spread of whitefly-borne CYSDV and / or CCYV. Such a method preferably includes the step of growing the resistant plants of the present invention, i.e., plants containing, on chromosome 1, and potentially on chromosomes 5 and 11, or on chromosomes 5 and 11, one of combinations of sequences or QTLs conferring resistance to CYSDV and / or CCYV (a) to n).

[0298] The present invention also relates to the use of the CYSDV and / or CCYV resistant melon plants according to the present invention for controlling CYSDV and / or CCYV infection in fields, tunnels or greenhouses or other cultivation, or for reducing losses of melons under conditions of CYSDV and / or CCYV infection or contamination.

[0299] All preferred features of the QTL, as defined in conjunction with other aspects of the invention, are preferably present in the seed ME22BNGA-F06-52563 / 001 (NCIMB accession number 44156) and can be identified by a mark defined according to the invention.

[0300] This invention also relates to a method for increasing melon plant yield in an environment infected with CYSDV or CCYV transmitted by whiteflies, comprising:

[0301] (a) Identifying melon plants resistant to CYSDV and / or CCYV, said melon plant containing in its genome: a combination of QTL1 located on chromosome 1 within a chromosomal region defined by SNP ME-0004372 and SNP ME-0007598, QTL5 located on chromosome 5 within a chromosomal region defined by SNP ME-0004289 and SNP ME-0006334, and / or QTL11 located on chromosome 11 within a chromosomal region defined by SNP ME-0005874 and SNP ME-0000595, wherein said combination confers CYSDV resistance and comprises:

[0302] - At least one of homozygous QTL1 and potentially homozygous or heterozygous QTL5 and QTL11, or

[0303] - Both heterozygous QTL1 and homozygous or heterozygous QTL5 and / or QTL11, and

[0304] (b) Cultivate the resistant melon plants in the infected environment.

[0305] In this way, if the melon plant population increases, more marketable thick-skinned melons can be harvested, more commercially viable thick-skinned melons can be produced, or more seeds can be obtained.

[0306] In another aspect, the present invention also relates to a method for producing thick-skinned melons, comprising:

[0307] a) Growing the melon plant of the present invention, as described above;

[0308] b) To cause the plant to bear fruit; and

[0309] c) Harvest the fruit of the plant, preferably at maturity and / or before maturity.

[0310] All preferred embodiments of the melon plant have been disclosed in the context of the prior art.

[0311] The method may advantageously include further steps of processing the melon into processed food products.

[0312] According to another aspect, the present invention also relates to a combination of molecular markers for detecting melon plants, particularly the thick-skinned melon subspecies, resistant to CYSDV and / or CCYV infection, wherein the markers are located in at least one of the following chromosomal regions:

[0313] - On chromosome 1, within the chromosomal region defined by ME-0004372 and SNP ME-0007598,

[0314] - On chromosome 5, within the chromosomal region defined by ME-0004289 and SNP ME-0006334, and

[0315] - On chromosome 11, within the chromosomal region defined by ME-0005874 and SNP ME-0000595.

[0316] The combination comprises at least one marker on chromosome 1 in the region and at least one marker on chromosome 5 or 11 in the region; or comprises at least one marker on chromosome 5 in the region and at least one marker on chromosome 11 in the region.

[0317] Preferably, the combination of markers according to the invention includes at least one marker on chromosome 1 in the region, at least one marker on chromosome 5 in the region, and at least one marker on chromosome 11 in the region. In an embodiment, the combination includes:

[0318] - At least one of the following markers: ME-0006564, ME-0027363, ME-0027608, ME-0027609, ME-0027622, ​​ME-0027623, ME-0027365, ME-0002337, ME-0006240, ME-0027367, ME-0027328, ME-0027330, ME-0027332, ME-0027334, ME-0027336, and ME-0008111, preferably ME-0027623, ME-0027365, or ME-0002337; preferably at least two or three such markers.

[0319] - At least one marker selected from ME-0027624, ME-0004225, ME-0003342, ME-0027650, ME-0009162, ME-0009163, ME-0027384, ME-0027385, ME-0027387, ME-0027388, ME-0007780 and SNP ME-0000109; preferably at least two or three such markers, and

[0320] - At least one of the following markers: ME-0005874, ME-0027651, ME-0027655, ME-0027656, ME-0027659, ME-0027664, ME-0027667, ME-027668, ME-0027670, ME-0027671, ME-0027675, ME-0007096, ME-0019064 and ME-0000595, preferably ME-0027670, ME-0027671 or ME-0007096, and preferably at least two or three such markers.

[0321] This invention also relates to methods for identifying other or alternative markers to supplement or replace those described herein, and methods for identifying or selecting plants having the QTLs of this invention. Having knowledge of the markers of this invention and being able to obtain plants according to this invention, this invention also relates to the use of these markers and their genomic locations for identifying alternative markers. The method may include screening for resistant plants and susceptible materials according to this invention using SNP arrays, or using KASPar markers, targeted sequencing screening, or any other suitable potential genotyping technique. The method may also include tracking the genetic status of potential markers, and tracking the genetic status of one or more markers of this invention in a population derived from plants according to this invention. For QTL1, the potential marker is found in the region defined by SNP ME-0004372 and SNP ME-0007598; for QTL5, in the region defined by SNP ME-0004289 and SNP ME-0006334; and for QTL11, in the region defined by ME-0005874 and SNP ME-0000595.

[0322] Once resistant material and QTL-defining markers are available, alternative markers can also be designed through sequencing of resistant plants. Sequencing methods can include, for example, short-read sequencing, long-read sequencing, or Sanger sequencing. It can target one or more of QTL1, QTL5, and QTL11, or genes found within or within any genomic region of a QTL. It can include untargeted sequencing, generating a large number of reads distributed along the genome. It can include DNA or transcriptome sequencing. Sequence analysis, including comparison of resistant sequences with sequences found in susceptible samples, identifies variations that can be used to design markers.

[0323] Throughout this application, the term "comprising" should be interpreted to encompass all specifically mentioned features as well as optional, additional, or unspecified features. As used herein, the use of the term "comprising" also discloses implementations that do not include any other features besides those specifically mentioned (i.e., "composed of").

[0324] Table A The list of markers mentioned in this specification includes their SEQ ID numbers (first column), their names (second column), chromosomes and their positions on the chromosome from which DHL92_3.6.1 is assembled according to the genome (third and fourth columns, respectively), their flanking sequences (fifth column), and the resistance and susceptibility alleles of the markers (sixth and seventh columns, respectively).

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331] Seed preservation:

[0332] A sample of melon seed ME22BNGA-F06-52563 / 001 was deposited on June 7, 2023, at the National Centre for the Preservation of Microorganisms for Industrial, Food and Marine Use (NCIMB, Ltd, Wellheads Place, Aberdeen, Dyce, AB21 7GB, Scotland, United Kingdom) (NCIMB, Ltd, Wellheads Place, Aberdeen, Dyce, AB21 7GB, United Kingdom) by Mazera Seeds Ltd. (Berurim, MP Shikmim 79837, Israel), in accordance with and satisfying the requirements of the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Proceedings (“Budapest Treaty”), with accession number NCIMB 44156. This melon seed deposit is maintained by Hazera Seeds Ltd. (Berurim, MP Shikmim 79837, Israel). Attached Figure Description

[0333] Figure 1 Phenotypic distribution (1=S, 9=R). A. Viridian (Viridis) selection (resistance source from the thick-skinned melon subspecies). B. F2 population (Viridis × GAL).

[0334] Figure 2 F2 QTL mapping results of the 12 chromosomes of melon (controlled infection).

[0335] Figure 3: Controlled Infection

[0336] Figure 3A - Phenotypic differences between genetic populations identified in Chr1 QTL (ME-0002337, R2=0.2), corresponding to QTL1. Partial dominance effects were observed.

[0337] Figure 3B - Phenotypic distribution of F2 plants carrying homozygous QTL1 (i.e., carrying the R / R allele of chr1 QTL, corresponding to the A / A allele of QTL1 (marker ME-0002337)). These plants had a mean CYSDV score of 7.9. All were resistant plants (scores 6–9).

[0338] Figure 3C - Interaction analysis and CYSDV prediction in F2 generation. Chr1 represents QTL1, Chr5 represents QTL5, and Chr11 represents QTL11. The marker for QTL1 is ME-0002337, the marker for QTL5 is ME-0009162 (SEQ ID NO: 61), A is the resistance allele and G is the susceptibility allele, and the marker for QTL11 is ME-0007096.

[0339] Figure 4. Controlled Infection

[0340] Figure 4A The main function of QTL1. The average rating of CYSDV in F3 generation.

[0341] Figure 4B QTL combination effects. CYSDV resistance levels observed in F3 individuals carried different QTL combinations. Different letters (AE) showed significant differences.

[0342] Figure 5 QTL combinations affecting different melon types. For example, in the BC1F3 strain, the resistance levels of Ananas, WS, YC, and Galia-CYSDV melons carried different QTL combinations. Different letters (AD) showed significant differences.

[0343] Figure 6 Location of QTL1 on chromosome 1.

[0344] Figure 7 Fine mapping of QTL1 on chromosome 1. CYSDV score of recombinant lines (after recombinant screening within the major identified QTL peaks (ME-0006564 to ME-0008111)). Alleles of resistance source are dark gray (e.g., allele A / A in ME-0002337). Alleles of susceptible parents are light gray (e.g., allele G / G in ME-0002337). QTL intervals are narrowed to 30 kbp between markers ME-0027623 and ME-0002337. Chr1 action refers to QTL1 action.

[0345] Figure 8 Location of QTL5 on chromosome 5.

[0346] Figure 9 Location of QTL11 on chromosome 11.

[0347] Figure 10Fine mapping of QTL 11 on chromosome 11. CYSDV score of recombinant lines (after recombination screening within the major identified QTL peaks (ME-0005874 to ME-0000595)). Alleles of resistance origin are dark gray (e.g., allele A / A in ME-0027671). Alleles of susceptible parents are light gray (e.g., allele G / G in ME-0027671). QTL interval narrowed to 70 kbp between markers ME-0027670 and ME-0027671.

[0348] Figure 11 Photographs of mature plants and fruits. A: Viridis, resistance source, with slender, non-restricted fruits. B: Susceptible parental line (Western shipper), with round, reticulated fruits. C. Parental line, in which the resistance QTL has been incorporated: round, reticulated fruits, similar to the original (susceptible) parental line.

[0349] Figure 12 CYSDV scoring under controlled infection conditions during the Fall 2018 trials. A. Susceptible control. B. Identified source of resistance, Viridis. C. Alternative sources of candidates: TGR 1551,90625 (also known as PI 313970) and SGR material.

[0350] Figure 13 QTL5 decreasing interval.

[0351] Reduce the QTL5 region on chromosome 5. CYSDV score of recombinant lines (after recombination screening within the major identified QTL peaks). Alleles of resistance origin are dark gray. Alleles of susceptible parents are light gray. Chr5 action refers to QTL5 action.

[0352] Figure 14 Phenotypic comparison between Viridis and materials derived from SGR.

[0353] CYSDV scoring was performed under controlled infection (Almeria) conditions during the fall 2018 trial. Virdis showed highly stable resistance, while symptoms were observed on SGR material.

[0354] Examples:

[0355] Example 1: Materials and Methods

[0356] A. Infection

[0357] CYSDV infection was carried out via its natural vector, the whitefly (Bemisia tabaci) (whitefly, WF), in cages covered with insect-proof netting (50 mesh). CYSDV-infected Cucurbita plants (Victoria F1, HM. CLAUSE) were used as inoculum. Virus-free WF were placed on the infected plants. The WF population acquired the virus and multiplied until it reached a sufficient number. At this stage, melon seedlings (at the 1-true-leaf stage) were inserted into the cages for three days to allow for effective plant infection (an average of 10 WF / melon plant was observed). After three days, an insecticide was applied to remove the WF, and the infected melon seedlings were then transplanted into a greenhouse.

[0358] B. Phenotypic Disease Index:

[0359] Phenotypic analysis was performed on all plants using the following levels:

[0360] 9 – Resistance: The lower part of the plant shows no symptoms or only a few symptoms.

[0361] 7 – Resistance (-): Only 1-3 lower leaves show a small number of symptoms.

[0362] 5 – Moderate: Symptoms begin at the bottom and extend upwards to 5 leaves from the bottom.

[0363] 3 – Susceptible (+): Symptoms develop from the bottom and reach 3 / 4 of the height (from the bottom).

[0364] 1 – Susceptible: The plant shows yellowing symptoms completely (or almost completely).

[0365] Example 2: Identification of potential sources of resistance.

[0366] The inventors conducted multiple rounds of CYSDV infection and resistant individual selection on multiple wild species at different locations to identify reliable sources of resistance.

[0367] According to the scheme disclosed in Example 1, or through natural infection, wild species TGR-1551, described as resistant to CYSDV (Pérez-de-Castro et al., 2020), and SGR material (EP3005862) were tested in different locations, namely Spain (Almeria), Arizona (Yuma), and Israel.

[0368] Phenotypic resistance scores for these wild or cultivated species varied considerably depending on the location of TGR-1551, with TGR-1551 scoring very low (around 3 in some locations). Regarding SGR materials, the phenotypic disease index was more consistent, but still 7 or lower; however, the inventors have found that these plants primarily masked symptoms due to the "hyperchlorosis" phenotype, without inhibiting viral replication and spread. Symptoms were still observed in the plants, allowing the virus to multiply and spread. SGR materials can be identified, in particular, by the marker NCMEL009102569 on chromosome 9, as described in EP3005862.

[0369] Among other wild species tested, the inventors identified a melon subspecies, Thick-skinned Melon, with resistance scores ranging from 8 to 9, consistent across different locations. Therefore, a wild germplasm source named Viridis, bearing non-commercially viable fruit, was selected for further steps (see [link to previous section]). Figure 11 ). Figure 12 The results of the CYSDV phenotype observed in a controlled infection trial conducted in Israel in the fall of 2018 are shown.

[0370] Example 3: Identification of DNA markers associated with resistance.

[0371] 3.1. Mapping population - phenotypic analysis

[0372] Twenty-two F2 plants (derived from Viridis × GAL, Galia type with long shelf life, round shape, no fruit drop, yellow skin at maturity, reticulate pattern, green flesh, Brix range of 10° to 15°, fruit weight approximately 1 kg, susceptible to CYSDV) were infected according to the method described in Example 1 and then transplanted into a netted greenhouse. The experiment was conducted in Israel during the fall of 2019, using a complete block design with 12 blocks (6 rows, each row containing north and south blocks). Phenotypic analysis of all plants was performed as described in Example 1.

[0373] Figure 1 Phenotypic disease indices from Viridis and F2 generations are shown.

[0374] Resistance was observed to segregate in the F2 generation population (see [link]). Figure 1 B).

[0375] 3.2. Mapping population - QTL identification

[0376] An SNP array covering approximately 1700 informative SNPs, distributed along all melon chromosomes, was used to test for linkage with resistance in the F2 generation population. Major QTLs (QTL1) were identified on chromosome 1, and minor QTLs (QTL5 and QTL11) were identified on chromosomes 5 and 11, respectively.

[0377] The result is Figure 2 As shown in the image.

[0378] Genetic mechanism studies were conducted based on the F2 generation. Figure 3A The differences between genetic populations identified in QTL1 based on the ME-0002337 allele (R²=0.2) are shown. Furthermore, plants carrying homozygous QTL1s were all rated as resistant, with a mean CYSDV score of 7.9.

[0379] It can be inferred that there is a partial dominant effect of QTL1. Figure 3B The phenotypic distribution of F2 individuals fixed with the ME-0002337 allele (A / A) is shown, i.e., containing homozygous alleles corresponding to QTL1.

[0380] Figure 3C Interactive analysis and CYSDV predictions are shown.

[0381] (A) Combinations of QTL1(- / -), QTL5(- / -), and QTL11(- / -). CYSDV prediction range is 2–5.7, with an average of 3.85.

[0382] (B) Combinations of QTL1(+ / -), QTL5, and QTL11(+ / -). The CYSDV prediction range is 5.7–7.4, with an average of 6.55.

[0383] (C) Combinations of QTL1(+ / -), QTL5(+ / -), and QTL11(+ / +). CYSDV prediction range is 6.85–8.9, with an average of 7.9.

[0384] (D) Combinations of QTL1(+ / -), QTL5(+ / +), and QTL11(+ / -). The CYSDV prediction range is 6.89–9, with a mean of 8.

[0385] Combining a heterozygous QTL1 with a heterozygous QTL5 and a homozygous QTL11 yields a resistance level similar to that obtained by combining a heterozygous QTL1 with a homozygous QTL5 and a heterozygous QTL11.

[0386] 3.3. QTL validation - F3 generation family

[0387] In the spring of 2020, the inventors verified the main role of QTL1 in F3 family pedigrees for QTL1 based on SNP ME-000237 (A=resistance allele, G=susceptibility allele), QTL5 based on ME-0009163 (A=resistance allele, G=susceptibility allele), and QTL11 based on ME-0007096 (A=resistance allele, G=susceptibility allele).

[0388] The results are shown in Figure 4.

[0389] 3.4 QTL validation - different melon types (Ananas, Yellow-Canary, Western-Shiper) Figure 5

[0390] In the fall of 2022, the inventors tested the effects of the QTL combination on different melon types (BC1F3).

[0391] The result is Figure 6 The results show that homozygous QTL1 already conferred a significant level of resistance, which could be enhanced by the presence of QTL5 and QTL11. These results confirm the importance of QTL1 as the dominant QTL, which can be further enhanced by the presence of QTL5 and QTL11.

[0392] Example 4: DNA markers associated with resistance

[0393] QTL1.

[0394] QTL1 was located based on the p-values ​​of different SNP markers along the chromosome. Figure 7 The evolution along the QTL1 genomic region - Log10 (p value) is shown.

[0395] The main QTL spanning 2.6 Mbps was identified. The flanking regions were markers ME-0004372 and ME-0007598. In particular, the QTL peak was between ME-0006564 and ME-0008111, spanning 0.6 Mbps.

[0396] Table A lists the sequences of markers in this region, along with detailed information on alleles linked to resistance and susceptibility QTLs, namely ME-0004372, ME-0008627, ME-0006564, ME-0002337, ME-0006240, ME-0008111, ME-0011885, ME-0011886, ME-0006851, ME-0006850, ME-0006008, ME-0008213, ME-0007599, and ME-0007598.

[0397] Then, fine-grained localization was performed to define, as precisely as possible, the minimum length of the QTL on chromosome 1 necessary to provide the desired effect. The results are shown in... Figure 8 The sequence and location of additional SNPs are also listed in Table A.

[0398] QTL5.

[0399] QTL5 was located based on the p-values ​​of different SNP markers along the chromosome. Figure 13 The evolution along the QTL genomic region - Log10 (p value) is shown.

[0400] The dominant QTL spanning 13.4 Mbp was identified. The flanking regions were markers ME-0004289 and ME-0006334. In particular, the QTL peak was located between markers ME-0004225 and ME-0000109, spanning 8.7 Mbp.

[0401] Table A lists the sequences of markers in this region, along with detailed information on alleles linked to resistance and susceptibility QTLs, namely ME-0004289, ME-0017469, ME-0008946, ME-0001758, ME-0004225, ME-0003342, ME-0009163, ME-0007780, ME-0000109, ME-0000155, ME-0000160, ME-0000170, ME-0006335, and ME-0006334.

[0402] Sequence data from “Viridis”, the GAL parental line, and R and S individuals were analyzed to identify additional SNPs in the QTL regions. Table A lists the sequences of the markers, along with details of the alleles linked to resistance and susceptibility QTLs, namely ME-0027624, ME-0027650, ME-0027384, ME-0027385, ME-0027386, ME-0027387, and ME-0027388.

[0403] Then, additional mapping was performed on different strains to limit the potential reduction in length of QTLs on chromosome 5 that could provide an effect, even to a small extent. The results are presented in... Figure 9 The reduced QTLs were identified. The sequences and locations of the additional SNPs are also given in Table A.

[0404] QTL11.

[0405] QTL11 was located based on the p-values ​​of different SNP markers along the chromosome. Figure 10 The changes along the chromosome -Log10 (p value) are shown.

[0406] The main QTL spanning 0.8 Mbp was identified. The flanks of the interval are markers ME-0005874 and ME-0000595. In particular, the optimal connection marker is ME-0007096.

[0407] Table A lists the sequences of markers in this region, along with detailed information on alleles linked to resistance and susceptibility QTLs, namely ME-0005874, ME-0007096, ME-0019064, and ME-0000595.

[0408] Then, fine-grained localization was performed to define, as precisely as possible, the minimum length of the QTL on chromosome 11 necessary to provide the desired effect. The results are shown in... References middle.

[0409] Table A above shows the sequences of markers in QTLs, as well as detailed information on alleles linked to resistance QTLs and susceptibility alleles.

[0410] Example 5: Resistance study based on WO2020025631.

[0411] The gene described in patent application WO2020025631 is CLAPR1. The sequence of the melon gene described in this document is identified on chromosome 5.

[0412] This document describes a specific 9bp insert, “CAGCAACAA,” that led to an resistance response.

[0413] Specific insertion sequences were identified in 38 samples (sequence data: Demirci, Sevgin et al. "Chasing breeding footprints through structural variations in Cucumis melo and wild relatives" G3 11.1 (2021):jkaa038), including the Dulce strain.

[0414] Some of these melons have been tested, yet they are not resistant to CYSDV. DULCE, for example, is a susceptible strain of CYSDV (as reported by McCreight, JD, et al.). Therefore, the insert disclosed in WO2020025631 is not the cause of resistance.

[0415] Example 6: CCYV resistance test.

[0416] Trials were conducted in an open area in Yuma, Arizona. Resistance to CYSDV and CCYV was assessed in naturally infected plants in the fall of 2022. CYSDV and CCYV are actually frequently co-circulated in the same area.

[0417] RT-PCR was used to detect the spread of the virus in the field, as described by Gyoutoku et al. (2009) and Abrahamian et al. (2020). The primers used are as follows:

[0418]

[0419] The presence of two viruses was confirmed using these primers.

[0420] Plant scores are: -1 = S (susceptibility) and 9 = R (resistance), for the two types of infection, namely CCYV and CYSDV, respectively.

[0421] Each strain has two replicates, each containing 20 plants.

[0422] The table below reports resistance scores to both CYSDV and CCYV viruses for plants from parental sources (Viridis), recurrent susceptible parents (GAL), and plants with different combinations of QTL1, QTL5, and QTL11 according to the present invention. In the second column regarding the genotype of the tested plants, R represents a homozygous QTL resistance allele, S represents a homozygous susceptible allele, and the three letters correspond to the three QTLs, QTL1, QTL5, and QTL11, respectively.

[0423] In the third column, the resistance score is a comprehensive resistance score for both CCYV and CYSDV.

[0424] This value is the average of two distinct repeats.

[0425]

[0426] These results indicate that QTLs on chromosomes 1, 5, and 11, namely QTL1, QTL5, and QTL11 identified in the foregoing examples, provide enhanced resistance to both CYSDV and CCYV infection in the aforementioned combination.

[0427] Example 7: Genetic modification of melon seeds by ethyl methanesulfonate (EMS)

[0428] Melon seeds were treated with EMS by immersing approximately 2,000 seeds in an aerated solution of 1% (w / v) or 2% EMS for 24 hours at room temperature.

[0429] Each EMS dose, approximately 1500 treated seeds, is used to germinate and the resulting plants are grown, preferably in a greenhouse, for example, from March to September, to produce seeds.

[0430] Upon maturity, M2 generation seeds were harvested and expanded in one pool for each variety per treatment. The resulting M2 seed pools were used as starting material to identify individual M2 seeds and plants resistant to CYSDV and / or CCYV.

[0431] Example 8: Comparison of Virdis and SGR derivatives.

[0432] Virdis and SGR materials (from the commercial variety SV5133 of Semnis, containing the SGR gene disclosed in EP3005862; the presence of the SGR allele was detected using the marker NCMEL00910256) were tested in Almeria in the fall of 18.

[0433] As previously described (high viral pressure), CYSDV infection was performed. Although all Virdis plants (18 strains) were asymptomatic and highly resistant (score = 9), for the SGR material, 15 out of the 18 plants scored 7, meaning that symptoms were masked by the chlorosis effect but were still observable on the plants.

[0434] ​

[0435] Abrahamian et al., 2013

[0436] Garcia-Mas et al., 2012, PNAS 109(29):11872-11877.

[0437] Gyoutoku, H., et al. 2009. Jpn. J. Phytopathol. 75:109. Kirkbride, JH, Jr. 1993. Biosystematic monograph of thegenus Cucumis (Cucurbitaceae). 84.

[0438] McCreight, JD, et al. "Recessive resistance to CYSDV in melon TGR 1551." V International Symposium on Cucurbits 1151. 2015

[0439] Pérez-de-Castro et al., 2020, “Melon Genome Regions Associated with TGR-1551-Derived Resistance to Cucurbit Yellow stunting disorder virus”. Int. J. Mol. Sci. 2020, 21(17), 5970.

[0440] Pitrat, MP Hanelt and K. Hammer. 2000. Some comments on intraspecific classification of cultivars of melon. Acta Hort. 510:29-36.

[0441] EP3005862

[0442] WO2020025631

Claims

1. A melon (Cucumis melo) resistant to cucumber yellow dwarfism disorder (CYSDV) and possessing commercially acceptable fruit quality, containing a combination of quantitative trait loci (QTLs) in its genome. in, The QTL is selected from: - QTL1 located on chromosome 1, flanked by SNP ME-0004372 (SEQ ID NO:1) and SNP ME-0007598 (SEQ ID NO:14), - QTL5 is located on chromosome 5, flanked by SNP ME-0004289 (SEQ ID NO: 27) and SNP ME-0006334 (SEQ ID NO: 40), and - QTL11 is located on chromosome 11, flanked by SNP ME-0005874 (SEQ ID NO: 41) and SNP ME-0000595 (SEQ ID NO: 44). The combination includes: - A homozygous QTL1, and at least one of potentially homozygous or heterozygous QTL5 and QTL11. - Heterozygous QTL1, and both homozygous or heterozygous QTL5 and QTL11, or - Both are homozygous QTL5 and QTL11, and The combination of QTLs imparts resistance to CYSDV.

2. The melon plant according to claim 1, wherein, The QTL1, QTL5, and QTL11 are present in the genome of the seed of Cucumis melo subsp. melo ME22BNGA-F06-52563 / 001, which is deposited with accession number NCIMB 44156, and are available from the deposited seed, for example, by hybridization with a plant grown from the deposited seed.

3. The melon plant according to any one of claims 1-2, wherein: - The QTL1 on chromosome 1 is identified by detecting one or more of the following markers: ME-0006564, ME-0027363, ME-0027608, ME-0027609, ME-0027622, ​​ME-0027623, ME-0027365, ME-0002337, ME-0006240, ME-0027367, ME-0027328, ME-0027330, ME-0027332, ME-0027334, ME-0027336 and ME-000811, preferably ME-0027623, ME-0027365 and / or ME-0002337; - The QTL5 on chromosome 5 is identified by detecting one or more of the following markers: ME-0027624, ME-0004225, ME-0003342, ME-0027650, ME-0009162, ME-0009163, ME-0027384, ME-0027385, ME-0027387, ME-0027388, ME-0007780 and / or SNP ME-0000109; - The QTL11 on chromosome 11 is identified by detecting one or more of the following markers: ME-0005874, ME-0027651, ME-0027655, ME-0027656, ME-0027659, ME-0027664, ME-027667, ME-027668, ME-0027670, ME-0027671, ME-0027675, ME-0007096, ME-0019064 and / or ME-0000595, preferably ME-0027670, ME-0027671 and / or ME-0007096.

4. The melon plant according to any one of claims 1-3, wherein: - The QTL1 on chromosome 1 is identified by detecting one or more of the following alleles: allele G of marker ME-0006564, allele G of marker ME-0027363, allele G of marker ME-0027608, allele C of marker ME-0027609, allele C of marker ME-0027622, ​​allele G of marker ME-0027623, allele A of marker ME-0027365, and marker... Allele A of marker ME-0002337, allele G of marker ME-0006240, allele G of marker ME-0027367, allele A of marker ME-0027328, allele G of marker ME-0027330, allele A of marker ME-0027332, allele A of marker ME-0027334, allele A of marker ME-0027336, and allele G of marker ME-008111; - The QTL5 on chromosome 5 is identified by detecting one or more of the following alleles: allele G of marker ME-0027624, allele A of marker ME-0004225, allele A of marker ME-0003342, allele C of marker ME-0027650, allele A of marker ME-0009162, allele A of marker ME-0009163, allele G of marker ME-0027384, allele G of marker ME-0027385, allele G of marker ME-0027387, allele G of marker ME-0027388, allele G of marker ME-0007780, and allele G of marker ME-0000109; and / or - QTL 11 on chromosome 11 is identified by detecting one or more of the following alleles: allele C of marker ME-0005874, allele A of marker ME-0007096, allele A of marker ME-0019064, allele A of marker ME-0027651, allele C of marker ME-0027655, allele C of marker ME-0027656, and so on. The allele G of marker ME-0027659, the allele A of marker ME-0027664, the allele A of marker ME-0027667, the allele G of marker ME-027668, the allele G of marker ME-0027670, the allele A of marker ME-0027671, the allele A of marker ME-0027675, and the allele C of marker ME-0000595.

5. The thick-skinned melon subspecies according to any one of claims 1-4, wherein, The plant in question is a descendant of the ME22BNGA-F06-52563 / 001 strain (NCIMB accession number 44156).

6. The thick-skinned melon subspecies according to any one of claims 1-5, wherein, The plant contains: - Purely synthetic QTL1, - Homozygous QTL1 and heterozygous QTL5, - Homozygous QTL1 and homozygous QTL5, - Homozygous QTL1 and heterozygous QTL11, - Homozygous QTL1 and homozygous QTL11, - Homozygous QTL1, heterozygous QTL5, and heterozygous QTL11, - Homozygous QTL1, homozygous QTL5, and heterozygous QTL11, - Homozygous QTL1, heterozygous QTL5, and homozygous QTL11, - Homozygous QTL1, homozygous QTL5, and homozygous QTL11, - Heterozygous QTL1, homozygous QTL5, and heterozygous QTL11, - Heterozygous QTL1, heterozygous QTL5, and homozygous QTL11, - Heterozygous QTL1, homozygous QTL5, and homozygous QTL11, - Heterogeneous QTL1, heterogeneous QTL5, and heterogeneous QTL11, and / or - Homozygous QTL5 and heterozygous QTL11.

7. The thick-skinned melon subspecies according to any one of claims 1 to 6, wherein, The plant in question is a cultivated or commercial plant that produces fruit that can be sold.

8. The thick-skinned melon subspecies according to any one of claims 1 to 7, wherein, The QTL1 on chromosome 1 is found in the chromosomal region defined by markers ME-0006564 (SEQ ID NO:3) and ME-0008111 (SEQ ID NO:6), preferably in the region defined by ME-0027623 (SEQ ID NO:19) and SNP ME-0002337 (SEQ ID NO:4), and even more preferably in the region defined by SNP ME-0027365 (SEQ ID NO:20) and SNP ME-0002337 (SEQ ID NO:4).

9. The thick-skinned melon subspecies according to any one of claims 1 to 7, wherein, The QTL5 on chromosome 5 is found in the chromosomal region defined by markers ME-0004225 (SEQ ID NO:31) and ME-0000109 (SEQ ID NO:35), preferably in the region defined by ME-0027624 (SEQ ID NO:55) and SNP ME-0000109 (SEQ ID NO:35), and even more preferably in the region defined by SNP ME-0004225 (SEQ ID NO:31) and SNP ME-0027388 (SEQ ID NO:60).

10. The thick-skinned melon subspecies according to any one of claims 1 to 7, wherein, The QTL11 on chromosome 11 is found in the chromosomal region defined by markers ME-0005874 (SEQ ID NO:41) and ME-0019064 (SEQ ID NO:43), preferably in the region defined by SNP ME-0027670 (SEQ ID NO:52) and SNP ME-00027671 (SEQ ID NO:53).

11. The melon plant according to any one of claims 1 to 10, wherein, The plant is also resistant to cucurbit chlorosis virus (CCYV).

12. A cell of a melon plant according to any one of claims 1 to 11, comprising a combination of the quantitative trait loci in its genome.

13. The cell according to claim 12, wherein, The cells may be regenerable or non-regenerable.

14. An in vitro cell or tissue culture of plant cells according to any one of claims 1 to 11, wherein, The cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, seeds, flowers, cotyledons, and / or hypocotyls.

15. A plant part obtained from the melon plant according to any one of claims 1-11, comprising the cells according to claim 12.

16. The plant portion according to claim 15, wherein, The plant parts are seeds, fruits, reproductive materials, propagation materials, roots, flowers, rootstocks, or scions.

17. A seed of a melon plant, which, upon growth and development, produces a plant according to any one of claims 1 to 11.

18. A melon hybrid plant, obtained by crossing a melon plant with a resistant plant according to any one of claims 1 to 11, comprising a homozygous QTL1.

19. A melon plant, or seed, or part thereof, or progeny thereof carrying homozygous QTL1, QTL5 and QTL11 as described in claim 1, for use as a breeding mate in a breeding program to confer resistance to CYSDV on a melon plant, wherein the melon seed is deposited in NCIMB with accession number NCIMB 44156, and the melon plant is preferably a melon plant susceptible to CYSDV.

20. The use of the melon plant or seeds according to any one of claims 1-11 or 17 as a breeding mate in a breeding process for conferring resistance to CYSDV on melon plants, wherein the melon plant is preferably a melon plant susceptible to CYSDV.

21. A method for detecting melon plants resistant to CYSDV, wherein, The method includes the following steps: - Detect the presence of at least one genetic marker linked to QTL1 on chromosome 1 as described in claim 1, and potentially, - Detect the presence of at least one genetic marker linked to QTL5 on chromosome 5 and / or at least one genetic marker linked to QTL11 on chromosome 11, as described in claim 1. The plant is resistant to CYSDV if the marker linked to QTL1 is homozygous, or if the markers linked to QTL1, QTL5, and QTL11 are detected simultaneously.

22. A method for selecting melon plants with QTLs conferring CYSDV resistance, the method comprising: a) Determine whether a melon plant contains at least one first genetic marker genetically linked to QTL1 as described in claim 1; b) Potentially, determining whether the plant contains at least one second genetic marker genetically linked to the QTL5 as described in claim 1. c) Potentially, determining whether the plant has at least one third genetic marker genetically linked to QTL11 as described in claim 1; d) Select plants containing three genetic markers and QTL1, QTL5, and QTL11 conferring CYSDV resistance; or plants containing the first genetic marker and homozygous QTL1; or plants containing the second genetic marker, the third genetic marker, and homozygous QTL5 and QTL11. QTL1 and the genetic markers are found on chromosome 1 in the genomic region defined by SNPs ME-0004372 and ME-0007598, preferably in ME-0006564 and ME-0008111. QTL5 and the genetic markers are found on chromosome 5 within the genomic region defined by SNPs ME-0004289 and ME-0006334, preferably within ME-0004225 and ME-0000109. QTL11 and the genetic marker are found on chromosome 11 in the genomic region defined by SNP ME-0005874 and SNP ME-000595, preferably in ME-0005874 and ME-0010064.

23. A method for selecting melon plants with QTLs conferring resistance to CYSDV and CCYV, the method comprising: a) Determine whether a melon plant contains at least one first genetic marker genetically linked to QTL1 as described in claim 1; b) Potentially, determining whether the plant contains at least one second genetic marker genetically linked to the QTL5 as described in claim 1. c) Potentially, determining whether the plant has at least one third genetic marker genetically linked to QTL11 as described in claim 1; d) Select plants containing three genetic markers and QTL1, QTL5, and QTL11 conferring resistance to CYSDV and CCYV, or plants containing the first genetic marker and a homozygous QTL1, or plants containing the second genetic marker, the third genetic marker, and homozygous QTL5 and QTL11. QTL1 and the genetic markers are found on chromosome 1 in the genomic region defined by SNPs ME-0004372 and ME-0007598, preferably in ME-0006564 and ME-0008111. QTL5 and the genetic markers are found on chromosome 5 within the genomic region defined by SNPs ME-0004289 and ME-0006334, preferably within ME-0004225 and ME-0000109. QTL11 and the genetic marker are found on chromosome 11 in the genomic region defined by SNP ME-0005874 and SNP ME-000595, preferably in ME-0005874 and ME-0010064.

24. A method for cultivating melon plants resistant to CYSDV, comprising at least the following steps: - Initial melon plants susceptible to CYSDV will be crossed with plants grown from the preserved seed NCIMB 44156 or their progeny, wherein the plants grown from the preserved seed NCIMB 44156 or their progeny carry QTL1 on chromosome 1, preferably also carrying QTL5 on chromosome 5 and / or QTL11 on chromosome 11, and - Select plants that contain a combination of QTLs as described in claim 1. Specifically, QTL1, QTL5, and QTL11 are present in the genome of the seed of plant ME22BNGA-F06-52563 / 001, whose NCIMB accession number is 44156. For QTL1, it can be identified by allele G of ME-0027623, allele A of ME-0027365, or allele A of ME-0002337; for QTL5, it can be identified by... Allele A of ME-0004225, allele A of ME-0003342, allele A of ME-0009163, allele G of ME-0007780, or allele G of ME-0000109 can be used for identification; for QTL11, it can be identified by allele A of ME-0007096, allele G of ME-0027670, or allele A of ME-0027671.

25. A method for conferring resistance to CYSDV in a melon plant, comprising genetically modifying the plant to introduce a heterozygous or homozygous QTL1 on chromosome 1, and potentially introducing QTL5 on chromosome 5 or QTL11 on chromosome 11, or both, to confer the resistance, wherein... QTL1 on chromosome 1, QTL5 on chromosome 5, and QTL11 on chromosome 11 are present in the genome of the seed of plant ME22BNGA-F06-52563 / 001, whose NCIMB accession number is 44156. Furthermore, QTL1 can be identified by allele G of ME-0027623, allele A of ME-0027365, or allele A of ME-0002337. For QTL5, it can be identified by allele A of ME-0004225, allele A of ME-0003342, allele A of ME-0009163, allele G of ME-000780, or allele G of ME-0000109; and for QTL11, it can be identified by allele A of ME-0007096, allele G of ME-0027670, or allele A of ME-0027671.

26. A method for conferring resistance to CYSDV and CCYV in melon plants, comprising genetically modifying said plant to introduce a heterozygous or homozygous QTL1 on chromosome 1, and potentially introducing QTL5 on chromosome 5, or QTL11 on chromosome 11, or both QTL5 and QTL11 on chromosome 11, to confer said resistance, wherein, QTL1 on chromosome 1, QTL5 on chromosome 5, and QTL11 on chromosome 11 are present in the genome of the seed of plant ME22BNGA-F06-52563 / 001, which has NCIMB accession number 44156. Furthermore, QTL1 can be identified by allele G of ME-0027623, allele A of ME-0027365, or allele A of ME-0002337. For QTL5, it can be identified by allele A of ME-0004225, allele A of ME-0003342, allele A of ME-0009163, allele G of ME-000780, or allele G of ME-0000109; and for QTL11, it can be identified by allele A of ME-0007096, allele G of ME-0027670, or allele A of ME-0027671.

27. A method for conferring resistance to CYSDV on melon plants, comprising the following steps: (a) A plant grown from a preserved seed NCIMB 44156 or its progeny is crossed with an initial melon plant, wherein the plant grown from the preserved seed NCIMB 44156 or its progeny carries QTL1 on chromosome 1 and potentially QTL5 on chromosome 5 and / or QTL11 on chromosome 11, wherein the initial melon plant preferably does not contain the QTLs. (b) Select plants from the resulting offspring that carry at least QTL1; (c) Optionally, the plants obtained in step b) are self-pollinated once or multiple times, and plants resistant to CYSDV and having the following characteristics are selected from the resulting offspring: - A homozygous QTL1, and at least one of potentially homozygous or heterozygous QTL5 and QTL11. - Heterozygous QTL1, and homozygous or heterozygous QTL5 and QTL11, The QTLs on chromosomes 1, 5, and 11 are present in the genome of the seed of plant ME22BNGA-F06-52563 / 001, which has the NCIMB accession number 44156.

28. A method for conferring resistance to CYSDV on melon plants, comprising the following steps: (a1) A plant or its progeny grown from the preserved seed NCIMB 44156 is crossed with an initial melon plant, the plant or its progeny grown from the preserved seed NCIMB 44156 carrying QTL1 on chromosome 1 and potentially QTL5 on chromosome 5 and / or QTL11 on chromosome 11, the initial melon plant preferably not containing the QTLs, thereby producing an F1 hybrid; (a2) Self-cross or backcross the F1 hybrids to produce the F2 population. (b) Select individuals from the resulting offspring that possess the following characteristics: - A homozygous QTL1, and at least one of potentially homozygous or heterozygous QTL5 and QTL11. - Heterozygous QTL1, and homozygous or heterozygous QTL5 and / or QTL11, The QTLs on chromosomes 1, 5, and 11 are present in the genome of the seed of plant ME22BNGA-F06-52563 / 001, which has the NCIMB accession number 44156.

29. The method according to claim 27 or 28, wherein, In steps b) and / or c), SNP markers are used to select plants carrying QTL1 and potential QTL5 and / or QTL11 that confer resistance to CYSDV.

30. A melon plant obtainable by the method according to any one of claims 25 to 29.

31. A method for increasing the harvestable number of melons from a melon subspecies *Melon thunbergii* plant grown in an environment infected by whiteflies and transmitted by CYSDV, comprising planting a CYSDV-resistant melon subspecies *Melon thunbergii* plant as described in any one of claims 1 to 11 in said environment and allowing it to bear fruit.

32. A method for protecting fields, greenhouses or tunnels from infection and / or spread of CYSDV transmitted by whiteflies, comprising planting a thick-skinned melon subspecies resistant to CYSDV as described in any one of claims 1 to 11 in the fields, greenhouses or tunnels.

33. The use of CYSDV-resistant melon plants as described in any one of claims 1 to 11 for controlling CYSDV infection in fields, greenhouses or tunnels.

34. A method for increasing the yield of melon plants in an environment infected with CYSDV transmitted by whiteflies, comprising: - Identify a melon plant resistant to CYSDV, said melon plant containing in its genome a combination of: QTL1 located on chromosome 1 within a chromosomal region defined by SNP ME-0004372 and SNP ME-0007598, QTL5 located on chromosome 5 within a chromosomal region defined by SNP ME-0004289 and SNP ME-0006334, and / or QTL11 located on chromosome 11 within a chromosomal region defined by SNP ME-0005874 and SNP ME-0000595, wherein said combination confers CYSDV resistance and comprises: - At least one of homozygous QTL1 and potentially homozygous or heterozygous QTL5 and QTL11, or - Both heterozygous QTL1 and homozygous or heterozygous QTL5 and QTL11, and - The resistant melon plants are grown in the infected environment.

35. A combination of molecular markers for detecting resistance to CYSDV infection in melon plants, wherein, The marker is located in at least one of the following chromosome regions: - On chromosome 1, within the chromosomal region defined by SNP ME-0004372 and SNP ME-0007598, - On chromosome 5, within the chromosomal region defined by SNP ME-0004289 and SNP ME-0006334, and - On chromosome 11, within the chromosomal region defined by SNP ME-0005874 and SNP ME-0000595, The combination includes at least one marker on chromosome 1 in the region and at least one marker on chromosome 5 or chromosome 11 in the region.

36. The combination of markers according to claim 35, comprising at least one marker on chromosome 1 in the region, at least one marker on chromosome 5 in the region, and at least one marker on chromosome 11 in the region.

37. A container comprising a melon plant according to any one of claims 1 to 11, a plant portion according to claim 15 or 16, or a seed according to claim 17.

38. A method for producing melon saplings or plants resistant to CYSDV, the method comprising: i. In vitro culture of the isolated cells or tissues according to claim 12 to produce melon microplants resistant to CYSDV, and ii. Optionally, the melon microplants are further cultured in vivo to develop into melon plants resistant to CYSDV.

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