Melon plants resistant to scab, aphids and powdery mildew

Abstract

The present invention provides Cucumis melo (C. melo) plants that are resistant to scab, aphids, and powdery mildew (PM) combined with desirable agronomic traits. The present invention also relates to methods for producing such plants, as well as methods for detecting and / or selecting such plants.

Description

[Technical Field]

[0001] The present invention relates to Cucumis melo (C. melo) plants that have resistance to scab, aphids, and powdery mildew (PM) in combination with desirable agronomic traits. The present invention also provides methods for producing said plants, as well as methods for detecting and / or selecting said plants. [Background technology]

[0002] Many pathogens, such as Cladosporium, Podosphaera xantii, or aphids, can colonize melon (Cucumis melo) cultures.

[0003] Cladosporium cucumerinum Ellis and Arthur is a fungus that causes scab disease that attacks the leaves and fruit of several cucurbits, including cucumber and melon, in many parts of the world. Very high levels of single-gene resistance have been systematically introduced into cucumber over the past 50 years, but no resistance has been described in melon. The disease occurs primarily in cool, damp conditions and is widespread in areas such as southwestern France, where growers regularly use fungicides.

[0004] Powdery mildew (PM) is a foliar disease caused by two main pathogens: Golobinomyces sicoracearum and Podosphaera xantii. While Podosphaera xantii predominates in most countries, Golobinomyces sicoracearum can cause the disease in temperate zones. In France, despite the presence of two pathogens, Podosphaera xantii is the most commonly encountered species. Five races of Podosphaera xantii (e.g., races Px-1, Px-2, Px-3, Px-5, and Px-3-5) and two races of Golobinomyces sicoracearum have been described in melon.

[0005] The melon aphid, Aphis gossypii glover, is a widespread pest of economically important host plants, such as cucurbits, and is a major pest of these plants. Colonization of cucurbits by A. gossypii causes stunting and severe leaf curling, which can lead to plant death. A. gossypii is also an efficient virus vector, contributing to the spread of viral diseases. Resistant melon accessions have been described since the 1970s, and the major gene responsible for resistance, the Vat gene, has been identified (Dogimont et al., Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae; Dogimont et al., 2014, The Plant Journal, 80, 993-1004) and introduced into commercial melon lines. However, it has been demonstrated that the Vat gene is associated with the necrotic response (Flaccida necrosis) (Pitrat and Lecoq, 1982, Agronomie, 2:503-508).

[0006] To date, resistance to several races of Podosphaera xantii and the aphid Aphis gossypii currently exists in Charentais cultivars grown in France. Two independent loci, PmV.1 and PmXII.1, conferring resistance to Podosphaera xantii races 1, 2, 3, and 5, were identified by Perchepied et al., 2005, The American Phytopathological Society, Vol. 95(5):556-565, using a RIL population derived from a cross between the resistant melon accession PI 124112 and the susceptible melon 'Vedrantais'. In addition, in Fukuno et al., 2008, Theor. Appl. Genet., 118(1):165-75, using RILs derived from a cross between the resistant AR5 muskmelon breeding line and the susceptible Japanese cultivar Earl's Favourite (Harukei 3), two QTLs conferring PM resistance to Podosphaera xanthiracea races A and B were detected on linkage groups (LG) II (or LG2) and LGXII (LG12). The QTL on LGII in this study was found to be closely linked to markers CMBR8 and CMBR120.

[0007] Fazza et al., 2013, Crop Breeding and Applied Biotechnology, 13:349-355, also disclosed the mapping of a QTL conferring resistance to Podosphaera xantii races 1, 3, and 5 in melon accession PI 414723 to two linked loci on LG2 (LG2). However, the PI 414723 accession is an Indian accession with undesirable agronomic characteristics, including pale, mealy, soft flesh at maturity, large cavities, very yellow / orange skin at maturity, and low sugar levels (6°Brix) (Burger et al., 2010, Horticultural Reviews, 36, 165-198). Fazza et al. also disclosed that LG2 contains other disease resistance genes, such as the Zym gene, which confers resistance to zucchini yellow mosaic virus (ZYMV). However, necrotic streak phenotypes on leaves, stems, and fruits have been observed in Zym / Zym homozygous plants (Pitrat and Lecoq, 1984, Euphytica, 33(1):57-61, US20140059712). Therefore, there is a need to obtain plants that are resistant to PM without the deleterious necrotic effects associated with the presence of the Zym gene.

[0008] Furthermore, the CMBR8 marker identified by Fukino et al. was not mapped by Fazza et al., and the CMBR120 marker was located quite far from the QTL identified in LGII, suggesting that the QTLs identified by Fazza et al. and Fazza et al. are located in different parts of LGII. Due to the low density of genetically mapped markers in the melon genome, it is difficult to determine the exact location of the resistance QTLs. Therefore, the QTLs for resistance to different races of Podosphaera xantii are diverse, and their precise locations are needed to define whether they are potentially combinable and to enable their combination in a single melon plant.

[0009] With the decline in plant protection products used to disinfect floors, an increase in the incidence of all soil-related diseases can be expected. Therefore, the development of multi-resistant varieties, such as those resistant to Cladosporium, Podosphaera xantii, and aphids, is also necessary to have more acceptable and economical means of controlling the impact of pathogens. Summary of the Invention

[0010] The present inventors have been able to introduce quantitative trait loci that confer resistance to scab, aphids, and powdery mildew (PM) in Cucumis melo (C. melo) plants in combination with desirable agronomic traits, and preferably without the necrosis phenotype associated with Zym genes. In a first embodiment, the present invention provides a C. melo plant that is resistant to scab, aphids, and powdery mildew (PM), the plant comprising: - below, (i) at least one QTL that confers resistance to scab, said at least one QTL being present on linkage group 2 (LG2) and / or linkage group 5 (LG5); (ii) at least one QTL that confers resistance to PM, the at least one QTL being present on LG2 and / or LG5 and distinct from the one or more QTLs in (i); and (iii) Vat gene analogues associated with aphid resistance in LG5: and - have commercially acceptable fruit quality;

[0011] Preferably, the plant does not have any necrotic phenotype associated with the Zym gene.

[0012] In some embodiments, the QTL on LG2 that confers resistance to scab is located within the chromosomal region bounded by markers Cm_MU45136_209 (also known as MU45136_209, SEQ ID NO: 1) and Cm_MU45398_32 (also known as MU45398_32, SEQ ID NO: 9).

[0013] In some embodiments, the QTL on LG5 that confers resistance to scab is located within the chromosomal region bounded by marker LG5-M1 (SEQ ID NO: 13) and marker Cm_MU44050_58 (also referred to as MU44050_58, SEQ ID NO: 20).

[0014] In some embodiments, the QTL on LG2 that confers resistance to PM is located within the chromosomal region bounded by marker CMBR120 (which can be identified using primers with SEQ ID NOs: 24 and 25) and marker Cm_MU47536_461 (also referred to as MU47536_461, SEQ ID NO: 30).

[0015] In some embodiments, the QTL on LG5 that confers resistance to PM is located within the chromosomal region bounded by marker Cm_MU45437_855 (also referred to as MU45437_855, SEQ ID NO: 34) and marker LG5-M3 (SEQ ID NO: 42).

[0016] In some embodiments, the scab, aphid, and PM resistant C. melo plant is line MTYVVC721, and a representative seed sample has been deposited with NCIMB under accession number NCIMB 43317.

[0017] Also provided are cells of the Cucumis melo (C. melo) plants described in the present invention.

[0018] Further provided are plant parts obtained from the C. melo plants described herein, hi some embodiments, the plant parts are seeds, fruits, reproductive material, roots, flowers, rootstocks, or scions.

[0019] The present invention also provides seeds of Cucumis melo (C. melo) plants that result when grown into plants according to the present invention.

[0020] Also provided are scab, aphid and powdery mildew (PM) resistant Cucumis melo (C. melo) hybrid plants obtained by crossing a C. melo plant with a resistant plant described in the present invention.

[0021] The present invention also provides a method for detecting and / or selecting Cucumis melo (C. melo) plants that are resistant to scab, aphids and powdery mildew (PM), said method comprising the steps of: a) detecting the presence or absence of: (i) at least one QTL that confers resistance to scab, said at least one QTL being present on linkage group (LG) 2 and / or linkage group 5 (LG5); (ii) at least one QTL conferring resistance to PM, the QTL being present on LG2 and / or LG5 and different from the at least one QTL in (i); and (iii) a Vat gene analogue on LG5 that confers resistance to aphids; b) selecting C. melo plants in which the presence of at least one QTL conferring resistance to scab, at least one QTL conferring resistance to PM, and the Vat gene analogue conferring resistance to aphids has been detected as plants resistant to scab, aphids, and powdery mildew (PM).

[0022] Further provided is the use of one or more markers to detect Cucumis melo (C. melo) plants that are resistant to scab, aphids, and powdery mildew (PM), wherein the one or more markers are located in at least one of the following chromosomal regions: - within the chromosomal region bounded on LG2 by markers Cm_MU45136_209 and Cm_MU45398_32, - within the chromosomal region bounded at LG5 by marker LG5-M1 and marker Cm_MU44050_58, - within the chromosomal region bounded on LG2 by markers CMBR120 and Cm_MU47536_461, - within the chromosomal region bounded on LG5 by marker Cm_MU45437_855 and marker LG5-M3, or - in Vat gene analogs.

[0023] The present invention provides the use of a resistant Cucumis melo (C. melo) plant according to the invention as a breeding partner in a breeding program to obtain C. melo plants that are resistant to scab, aphids and powdery mildew (PM), and preferably do not have any necrosis phenotype associated with the Zym gene.

[0024] Also provided are methods for producing Cucumis melo (C. melo) seeds. In some embodiments, the methods include crossing a Cucumis melo (C. melo) plant described herein with itself or another C. melo plant and harvesting the resulting seeds.

[0025] In environments where scab, aphids and powdery mildew are prevalent, - below, (i) at least one QTL that confers resistance to scab, said at least one QTL being present on linkage group 2 (LG2) and / or linkage group 5 (LG5); (ii) at least one QTL that confers resistance to PM, the at least one QTL being present on LG2 and / or LG5 and distinct from the one or more QTLs in (i); and (iii) Vat gene analogues associated with aphid resistance in LG5: and - have commercially acceptable fruit quality; Further provided is a method of increasing the number of harvestable Cucumis melo (C. melo) plants in said environment, comprising growing C. melo plants that are resistant to scab, aphids, and powdery mildew.

[0026] Preferably, such plants do not have any necrotic phenotypes associated with Zym genes.

[0027] - below, (i) at least one QTL that confers resistance to scab, said at least one QTL being present on linkage group 2 (LG2) and / or linkage group 5 (LG5); (ii) at least one QTL that confers resistance to PM, the at least one QTL being present on LG2 and / or LG5 and distinct from the one or more QTLs in (i); and (iii) Vat gene analogues associated with aphid resistance in LG5: and - have commercially acceptable fruit quality; and - preferably does not have any necrotic phenotype associated with the Zym gene, Also provided is a method for protecting fields from the infestation and / or spread of scab, aphids and powdery mildew (PM), comprising growing C. melo plants that are resistant to scab, aphids and PM.

[0028] - below, (i) at least one QTL that confers resistance to scab, said at least one QTL being present on linkage group 2 (LG2) and / or linkage group 5 (LG5); (ii) at least one QTL that confers resistance to PM, the at least one QTL being present on LG2 and / or LG5 and distinct from the one or more QTLs in (i); and (iii) Vat gene analogues associated with aphid resistance in LG5: and - have commercially acceptable fruit quality; and - preferably does not have any necrotic phenotype associated with the Zym gene, There is also provided the use of scab, aphid and powdery mildew (PM) resistant C. melo plants to control the infestation of scab in a field.

[0029] Further provided is a method for producing scab, aphid and powdery mildew (PM) resistant C. melo plantlets or plants, the method comprising: i. culturing in vitro isolated cells or tissues of a C. melo plant according to the invention to produce scab, aphid and powdery mildew (PM) resistant C. melo microplantlets; and ii. Optionally, subjecting the C. melo microplantlets to further in vivo culture steps to develop scab, aphid, and powdery mildew (PM) resistant C. melo plants. Includes.

[0030] Further provided is a method for improving yield of C. melo plants in environments infested with scab, aphids, and powdery mildew (PM), comprising growing a C. melo plant that is resistant to scab, aphids, and PM, and optionally does not have any necrosis phenotype associated with the Zym gene, wherein the plant comprises in its genome (i) at least one QTL that confers resistance to scab, the at least one QTL being on linkage group (LG) 2 and / or linkage group 5 (LG5), (ii) at least one QTL that confers resistance to PM, the at least one QTL being on LG2 and / or LG5 and different from the at least one QTL in (i), and (iii) a Vat gene analog on LG5 that confers resistance to aphids.

[0031] a. (i) at least one QTL that confers resistance to Cladosporium, wherein the at least one QTL is on linkage group (LG) 2 and / or linkage group 5 (LG5); (ii) at least one QTL conferring resistance to PM, said at least one QTL being present on LG2 and / or LG5 and distinct from the at least one QTL in (i); (iii) a VAT gene analog on LG5 that confers resistance to aphids; Identifying scab, aphid and powdery mildew (PM) resistant C. melo plants comprising in their genomes; and b. Growing the resistant C. melo plants in an environment infested with scab, aphids, and powdery mildew (PM): Also provided is a method for improving yield of C. melo plants in said infested environment, comprising:

[0032] definition

[0033] As used herein, the term "plant part" refers to any part of a plant, including, but not limited to, shoots, roots, stems, seeds, fruits, leaves, petals, flowers, ovules, branches, petioles, internodes, pollen, stamens, rootstocks, scions, etc.

[0034] As used herein, the term "quantitative trait locus (QTL)" refers to a genomic region that may contain one or more genes or regulatory sequences. A QTL may, for example, contain one or more genes whose products confer genetic resistance or tolerance. Alternatively, a QTL may, for example, contain a regulatory gene or sequence whose product affects the expression of genes at other loci in the plant genome, thereby conferring resistance or tolerance. The QTLs of the present invention may be defined by using one or more molecular genomic markers to indicate their genetic location in the genome of each pathogen-resistant accession. The one or more markers indicate specific loci. The distance between loci is usually measured by the frequency or crossover between loci on the same chromosome. The farther apart two loci are, the more likely crossover will occur between them. Conversely, if two loci are close to each other, the less likely crossover will occur between them. As a general rule, 1 centimorgan (cM) is equal to 1% recombination between loci (markers). When a QTL can be indicated by multiple markers, the genetic distance between the endpoint markers indicates the size of the QTL.

[0035] The term "resistance" is as defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Section, which describes the response of plants to pests or pathogens and abiotic stresses in the vegetable seed industry.

[0036] 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 caused by it compared to a susceptible plant variety under similar environmental conditions and influence of the pest or pathogen. A resistant variety may exhibit some disease symptoms or damage under the influence of a strong pest or pathogen.

[0037] "Tolerance" means the ability of a plant variety to withstand biotic and abiotic stresses without serious effects on growth, appearance and yield.

[0038] As used herein, the term "susceptible" refers to a plant that is unable to restrict the growth and development of a particular pest or pathogen.

[0039] As used herein, the term "offspring" or "progeny" refers to any plant resulting from vegetative or sexual propagation of one or more parent plants or their offspring. For example, offspring plants can be obtained by cloning or selfing a parent plant or by crossing two parent plants, including selfing and F1 or F2 or further generations. F1 is the first generation offspring resulting from parents, at least one of which is initially used as a trait donor, and second (F2) or subsequent generations (F3, F4, etc.) offspring are specimens generated from selfing F1, F2, etc. Thus, F1 can be (and typically is) a hybrid resulting from a cross between two true breeding parents (true breeding is homozygous for the trait), while F2 can be (and typically is) a progeny resulting from self-pollination of the F1 hybrid.

[0040] As used herein, the terms "crossing" and "mating" refer to the process by which pollen from one flower of one plant is applied (artificially or naturally) to the ovule (stigma) of a flower of another plant.

[0041] As used herein, the term "heterozygote" refers to a diploid or polyploid cell or plant that has different alleles (forms of a given gene or sequence) present at at least one locus.

[0042] As used herein, the term "heterozygosity" refers to the presence of different alleles (forms of a given gene or sequence) at a particular locus.

[0043] As used herein, the term "homozygote" refers to an individual cell or plant that has the same allele at one or more loci on all homologous chromosomes.

[0044] As used herein, the term "homozygous" refers to the presence of identical alleles at one or more loci on homologous chromosomal segments.

[0045] As used herein, the term "hybrid" refers to any individual cell, tissue, or plant resulting from a cross between one or more genetically distinct parents.

[0046] As used herein, the term "inbred" or "strain" refers to a relatively true-breeding strain.

[0047] As used herein, the term "phenotype" refers to the observable characteristics of an individual cell, cell culture, organism (e.g., plant), or group of organisms that result from the interaction between its individual genetic constitution (i.e., genotype) and the environment.

[0048] As used herein, the terms "introduction," "introduced," and "introducing" refer to the process by which genes from one species, variety, or cultivar are transferred to the genome of another species, variety, or cultivar by crossing the species. Crossing can be natural or artificial. This process can optionally be completed by backcrossing the recurrent parent, in which case introduction refers to the infiltration of genes from one species into the gene pool of another species through repeated backcrossing of an interspecific hybrid with one of its parents. Introduction can also be described as heterogeneous genetic material stably integrated into the genome of a recipient plant.

[0049] As used herein, the term "molecular marker" refers to a marker used in a method for visualizing differences in the characteristics of nucleic acid sequences. Examples of such markers are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characteristic amplified regions (SCARs), cleaved amplified polymorphic sequences (CAPS) markers, or isozyme markers, or a combination of markers described herein that define a specific genetic and chromosomal location. Mapping molecular markers near allele genes is a procedure that can be very easily performed by those skilled in the art using common molecular techniques.

[0050] As used herein, the term "primer" refers to an oligonucleotide that can anneal to an amplification target, allowing DNA polymerase to bind, and serves as a starting point for DNA synthesis when placed under conditions conducive to the synthesis of a primer extension product, i.e., in the presence of a polymerization agent such as nucleotides and DNA polymerase, and at an appropriate temperature and pH. A primer is preferably single-stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of an extension product in the presence of a polymerization agent. The exact length of the primer depends on many factors, including the temperature and composition (A / T and G / C content) of the primer. A pair of bidirectional primers, consisting of one forward primer and one reverse primer, is commonly used in DNA amplification techniques such as PCR amplification.

[0051] "Scab" refers to a fungal disease caused by fungi of the Ascomycete family (indoor and outdoor mold) called Cladosporium. Non-limiting examples of Cladosporium species include Cladosporium elegans, Cladosporium cladosporioides, Cladosporium caligenum, Cladosporium musae, Cladosporium brassicae, Cladosporium cucumerinum, and Cladosporium oxysporum. Preferably, the Cladosporium species is Cladosporium cucumerinum (also known as Cladosporium cucumerinum eris and Arthur). Disease symptoms are seen on all parts of the plant, including leaves, petioles, stems, and fruit. Leaf lesions begin as light green, water-soaked areas that gradually turn gray to white and become angular. Lesions are often surrounded by a yellowish halo and tear the center, leaving irregular holes in the leaves. On fruit, small (1 / 8 inch), gray, sunken, oozing, sticky spots resembling insect "bites" develop. The spots then enlarge and eventually become distinct sunken cavities. The crater-like depressions may develop an irregular, scab-like appearance as the fruit ages and may ooze a sticky substance. (https: / / extension.illinois.edu / hortanswers / detailproblem.cfm?PathogenID=141).

[0052] "Powder mildew" refers to a fungal disease caused by fungi of the order Erysiphalegas. Preferably, the powdery mildew is caused by Golobinomyces sicoracearum (also known as Erysiphe sicoracearum DC) and / or Podosphaera xanti (also known as Sphaerotheca fuliginea or Oidium erysiphoides). More preferably, the powdery mildew is caused by Podosphaera xantiraces Px-1, Px-2, Px-3, Px-5, and / or Px3-5. The disease is characterized by white or pale yellow lesions on stems, petioles, upper and / or lower leaf surfaces, and fruit. As the disease progresses, the lesions become larger and more dense. As the lesions enlarge, conidia are produced from the affected tissue, causing the spots to take on a powdery appearance. Said sporulation of fungi can cause cellular destruction of plant tissues and a loss of photosynthetic efficiency resulting in less energy performance of the plant. (https: / / cuccap.org / disease-management / melon / powdery-mildew / ).

[0053] "Aphids" refers to pests that extensively colonize economically important host plants, such as cucurbits, and are therefore a major pest. Aphid colonization of cucurbits causes stunting and severe leaf curling, which can lead to plant death. Aphids are also efficient virus vectors, contributing to the spread of viral diseases. Preferably, the aphid is a species of Aphis gossypii glover.

[0054] By "Vat gene homolog" is meant the major gene responsible for resistance to aphids, more preferably Aphis gossypii glover, in accession PI 414723, as described in Dogimont et al., 2008, Pitrat M. (ed.), Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae, Avignon (France), May 21-24, 2008, pp. 219-228. The vat gene homolog is located on LG5 (Dogimont et al., 2014, The Plant Journal, 80, 993-1004). In some embodiments, the vat gene analog comprises or consists of a 5897 bp nucleotide sequence that has 99.8% identity to the nucleic acid sequence encoded by the vat gene referenced under GenBank No. KM513660.1 (SEQ ID NO:46), updated on March 24, 2015. In some embodiments, the vat gene analog encodes a polypeptide having an amino acid sequence of 1473 aa that has 99.6% identity to the vat protein referenced under GenBank No. AIU36098.1 (SEQ ID NO:47), updated on October 27, 2014, i.e., the vat gene analog encodes a polypeptide that has 6 different aa compared to the vat protein referenced under GenBank No. AIU36098.1 (SEQ ID NO:47), updated on October 27, 2014. Identification of Vat gene analogs may be carried out as described in patent application FR 2 849 863 using the forward primer Me-VatE-F with the sequence 5'-CTCCACTCAGAATTGGTAGGTGCC-3' (SEQ ID NO: 48) and the reverse primer Me-VatE-R with the sequence 5'-CCTTAGAAGAAGATGAAGTCTCCC-3' (SEQ ID NO: 49). With the above pair of primers, detection of a 1723 bp fragment indicates the presence of a Vat gene analog.

[0055] In the context of the present application, the percentage of identity is calculated using a global alignment (i.e., the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the "needle" program can be used, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) considering the entire length of the two sequences. The Needle program is available, for example, at the ebi.ac.uk World Wide Web site. The percentage of identity described in the present invention is preferably calculated using the EMBOSS::needle(global) program, with a "gap open" parameter equal to 10.0, a "gap extend" parameter equal to 0.5, and a Blosum62 matrix.

[0056] In the context of the present invention, the assignment and orientation of the DNA strand and allele genes for markers LG2-M1, Cm_MU47536_461, LG2-M2, Cm_MU47380_465, LG2-M3, Cm_MU45136_209, LG2-M4, Cm_MU45398_32, Cm_MU46579_322, Cm_MU44050_58, Cm_MU45437_855 and LG5-M3 can be performed according to the TOP / BOT method developed by Illumina. (https: / / www.illumina.com / documents / products / technotes / technote_topbot.pdf).

[0057] The Cm suffix in the names of SNPs may be omitted below, but still refer to the same marker in Cucumis.melo.

[0058] In the context of the present application, a chromosomal region (e.g., SNPs) delimited by two markers X and Y refers to the chromosomal portion located between these two marker positions and including the markers, and thus the nucleotide sequence of this chromosomal region starts with the nucleotide corresponding to marker X and ends with the nucleotide corresponding to marker Y, i.e., the markers are included within the region they delimit.

[0059] The "necrosis phenotype associated with the Zym gene" means the necrosis phenotype as described in Pitrat and Lecoq, 1984, Euphytica, 33(1):57-61, which is homozygous in the presence of the Zym gene. More specifically, the necrosis phenotype corresponds to the appearance of necrosis spots on the leaf epidermis, which gradually spread on the leaf and may completely dry out the leaf or stem.

[0060] "Commercially acceptable fruit quality" means the flesh color from orange to red magenta (i.e., colorimetric values L * c * h measured by a Minolta colorimeter: having a colorimetric ratio of 60 < L < 65, 40 < c < 50, and 66 < h < 75), an average flesh firmness of 4 kg / 0.5 cm measured with a Penefel 2 + / - 2, a Brix level of at least 11°, the ratio of fruit diameter / cavity size found in the Vedrantais variety, and the storage of fruit for more than 8 days at +12 °C, and means fruits of the Charentais, Western Shipper, Harper, or Italian cantaloupe type. Examples of fruits having commercially acceptable fruit quality can be fruits of the HUGO, ALONSO, or FELINO varieties derived from HM-CLAUSE.

[0061] By association, or genetic association, more specifically by genetic linkage, a polymorphism of a gene marker (e.g., a specific allelic gene of a SNP marker) and a phenotype of interest occur simultaneously, i.e., they co-inherit more frequently than expected by chance, i.e., as a result of their genomic proximity, there is a non-random association between the allelic gene and the gene sequence involved in the phenotype.

[0062] Sequence Listing

[0063] SEQ ID NO: 1 shows the flanking sequence of marker Cm_MU45136_209.

[0064] SEQ ID NO: 2 shows the sequence of the forward primer for detecting the susceptibility allele gene of the Cm_MU45136_209 marker.

[0065] SEQ ID NO: 3 shows the sequence of the forward primer for detecting the resistance allele gene of the Cm_MU45136_209 marker.

[0066] SEQ ID NO: 4 shows the sequence of the common reverse primer for detecting the Cm_MU45136_209 marker.

[0067] SEQ ID NO: 5 shows the flanking sequence of marker LG2-M4.

[0068] SEQ ID NO: 6 shows the sequence of the forward primer for detecting the susceptibility allele of the LG2-M4 marker.

[0069] SEQ ID NO: 7 shows the sequence of the forward primer for detecting the resistance allele gene of the LG2-M4 marker.

[0070] SEQ ID NO: 8 shows the sequence of the common reverse primer for detecting the LG2-M4 marker.

[0071] SEQ ID NO: 9 shows the flanking sequence of marker Cm_MU45398_32.

[0072] SEQ ID NO: 10 shows the sequence of the forward primer for detecting the susceptibility allele of the Cm_MU45398_32 marker.

[0073] SEQ ID NO: 11 shows the sequence of the forward primer for detecting the resistance allele gene of the Cm_MU45398_32 marker.

[0074] SEQ ID NO: 12 shows the sequence of the common reverse primer for detecting the Cm_MU45398_32 marker.

[0075] SEQ ID NO: 13 shows the flanking sequence of marker LG5-M1.

[0076] Sequence number 14 shows the sequence of the forward primer for detecting the CMCTN2 marker.

[0077] Sequence number 15 shows the sequence of the reverse primer for detecting the CMCTN2 marker.

[0078] SEQ ID NO: 16 shows the flanking sequence of marker Cm_MU46579_322.

[0079] SEQ ID NO: 17 shows the sequence of the forward primer for detecting the susceptibility allele of the Cm_MU46579_322 marker.

[0080] SEQ ID NO: 18 shows the sequence of the forward primer for detecting the resistance allele gene of the Cm_MU46579_322 marker.

[0081] SEQ ID NO: 19 shows the sequence of the common reverse primer for detecting the Cm_MU46579_322 marker.

[0082] SEQ ID NO: 20 shows the flanking sequence of marker Cm_MU44050_58.

[0083] SEQ ID NO: 21 shows the sequence of the forward primer for detecting the susceptibility allele of the Cm_MU44050_58 marker.

[0084] SEQ ID NO: 22 shows the sequence of the forward primer for detecting the resistance allele gene of the Cm_MU44050_58 marker.

[0085] SEQ ID NO: 23 shows the sequence of the common reverse primer for detecting the Cm_MU44050_58 marker.

[0086] Sequence number 24 shows the sequence of the forward primer for detecting the CMBR120 marker.

[0087] Sequence number 25 shows the sequence of the reverse primer for detecting the CMBR120 marker.

[0088] SEQ ID NO: 26 shows the flanking sequence of marker LG2-M1.

[0089] SEQ ID NO: 27 shows the sequence of the forward primer for detecting the susceptibility allele of the LG2-M1 marker.

[0090] SEQ ID NO: 28 shows the sequence of the forward primer for detecting the resistance allele gene of the LG2-M1 marker.

[0091] SEQ ID NO: 29 shows the sequence of the common reverse primer for detecting the LG2-M1 marker.

[0092] SEQ ID NO: 30 shows the flanking sequence of marker Cm_MU47536_461.

[0093] SEQ ID NO: 31 shows the sequence of the forward primer for detecting the susceptibility allele of the Cm_MU47536_461 marker.

[0094] SEQ ID NO: 32 shows the sequence of the forward primer for detecting the resistance allele gene of the Cm_MU47536_461 marker.

[0095] Sequence number 33 shows the sequence of the reverse primer common to the detection of the Cm_MU47536_461 marker.

[0096] SEQ ID NO: 34 shows the flanking sequence of marker Cm_MU45437_855.

[0097] SEQ ID NO: 35 shows the sequence of the forward primer for detecting the susceptibility allele gene of the Cm_MU45437_855 marker.

[0098] SEQ ID NO: 36 shows the sequence of the forward primer for detecting the resistance allele gene of the Cm_MU45437_855 marker.

[0099] SEQ ID NO: 37 shows the sequence of the common reverse primer for detecting the Cm_MU45437_855 marker.

[0100] SEQ ID NO: 38 shows the sequence of the forward primer for detecting the LG5-M2 marker.

[0101] SEQ ID NO: 39 shows the sequence of the reverse primer for detecting the LG5-M2 marker.

[0102] Sequence number 40 shows the sequence of the forward primer for detecting the CMTAN139 marker.

[0103] SEQ ID NO: 41 shows the sequence of the reverse primer for detecting the CMTAN139 marker.

[0104] SEQ ID NO: 42 shows the flanking sequence of marker LG5-M3.

[0105] SEQ ID NO: 43 shows the sequence of the forward primer for detecting the susceptibility allele of the LG5-M3 marker.

[0106] SEQ ID NO: 44 shows the sequence of the forward primer for detecting the resistance allele of the LG5-M3 marker.

[0107] SEQ ID NO: 45 shows the sequence of the common reverse primer for detecting the LG5-M3 marker.

[0108] SEQ ID NO: 46 represents the genomic sequence of the Vat gene referenced under GenBank number KM513660.1.

[0109] SEQ ID NO: 47 represents the amino acid sequence of the Vat protein referenced in GenBank number AIU36098.1.

[0110] SEQ ID NO: 48 shows the sequence of the forward primer for amplifying the Vat gene analogue.

[0111] SEQ ID NO: 49 shows the sequence of the reverse primer for amplifying the Vat gene analog.

[0112] SEQ ID NO: 50 shows the flanking sequence of marker LG2-M2.

[0113] SEQ ID NO: 51 shows the sequence of the forward primer for detecting the susceptibility allele of the LG2-M2 marker.

[0114] SEQ ID NO: 52 shows the sequence of the forward primer for detecting the resistance allele gene of the LG2-M2 marker.

[0115] SEQ ID NO: 53 shows the sequence of the common reverse primer for detecting the LG2-M2 marker.

[0116] SEQ ID NO: 54 shows the flanking sequence of marker Cm_MU47380_465.

[0117] Sequence number 55 shows the sequence of the forward primer for detecting the susceptibility allele of the Cm_MU47380_465 marker.

[0118] SEQ ID NO: 56 shows the sequence of the forward primer for detecting the resistance allele gene of the Cm_MU47380_465 marker.

[0119] SEQ ID NO: 57 shows the sequence of the common reverse primer for detecting the Cm_MU47380_465 marker.

[0120] SEQ ID NO: 58 shows the flanking sequence of marker LG2-M3.

[0121] SEQ ID NO: 59 shows the sequence of the forward primer for detecting the susceptibility allele of the LG2-M3 marker.

[0122] SEQ ID NO: 60 shows the sequence of the forward primer for detecting the resistance allele gene of the LG2-M3 marker.

[0123] SEQ ID NO: 61 shows the sequence of the common reverse primer for detecting the LG2-M3 marker. [Brief explanation of the drawings]

[0124] [Figure 1] This figure includes photographs showing the various melon lines and accessions used in the examples - one photograph of the exterior and one photograph of the interior profile. [Figure 2] This figure shows the genetic map of LG2 as disclosed in Diaz et al., 2011, with the markers of the present invention added, as well as a representation of the corresponding QTL. PM stands for powdery mildew resistance; Scab for resistance to scab, and ZYMV for resistance to zucchini yellow mosaic virus. [Figure 3] This figure shows the genetic map of LG5 as disclosed in Diaz et al., 2011, with the markers of the present invention added, as well as a representation of the corresponding QTL. PM stands for powdery mildew resistance; Scab stands for scab resistance. DETAILED DESCRIPTION OF THE INVENTION

[0125] According to a first embodiment, the present invention relates to a Cucumis melo (C. melo) plant that is resistant to scab, aphids and powdery mildew (PM), said plant comprising: - below, (i) at least one QTL that confers resistance to scab, said at least one QTL being present on linkage group 2 (LG2) and / or linkage group 5 (LG5); (ii) at least one QTL that confers resistance to PM, the at least one QTL being present on LG2 and / or LG5 and distinct from the one or more QTLs in (i); and (iii) vat gene analogues associated with aphid resistance in LG5: and - have commercially acceptable fruit quality;

[0126] According to a preferred embodiment, the plant does not have any necrotic phenotype associated with the Zym gene.

[0127] By "one or more QTLs conferring resistance to scab" it is understood that at least one, two, three, four, five or more QTLs conferring resistance to scab, i.e. at least one, two, three, four, five or more QTLs present on LG2 and / or at least one, two, three, four, five or more QTLs present on LG5.

[0128] It is to be understood that "one or more QTLs conferring resistance to PM" means at least one, two, three, four, five or more QTLs that confer resistance to PM, i.e. at least one, two, three, four, five or more QTLs present on LG2 and / or at least one, two, three, four, five or more QTLs present on LG5, wherein the QTLs that confer resistance to PM on LG2 and / or LG5 are different from the QTLs that confer resistance to scab.

[0129] In some embodiments, the QTL that confers resistance to scab present on LG2 is located within the chromosomal region bounded by markers Cm_MU45136_209 and Cm_MU45398_32.

[0130] In some embodiments, the QTL conferring resistance to present on LG5 is located within the chromosomal region bounded by marker LG5-M1 and marker Cm_MU44050_58.

[0131] In some embodiments, the QTL that confers resistance to PM present on LG2 is located within the chromosomal region bounded by marker CMBR120 and marker Cm_MU47536_461.

[0132] In some embodiments, the QTL that confers resistance to PM present on LG5 is located within the chromosomal region bounded by marker Cm_MU45437_855 and marker LG5-M3.

[0133] Preferably, a C. melo plant according to the invention comprises any combination of QTLs as defined herein, has commercially acceptable fruit quality, and is free of any necrotic phenotype. For example, a C. melo plant according to the invention may comprise the following combination of QTL(s) associated with scab, aphid, and PM resistance:

[0134] (a) A QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, and a Vat gene analogue associated with aphid resistance on LG5;

[0135] (b) a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and the Vat gene associated with aphid resistance on LG5;

[0136] (c) a QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and a Vat gene analogue associated with aphid resistance on LG5;

[0137] (d) a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, and a Vat gene analog associated with aphid resistance on LG5;

[0138] (e) a QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, and a Vat gene analog associated with aphid resistance on LG5;

[0139] (f) a QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and a Vat gene analog associated with aphid resistance on LG5;

[0140] (g) a QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and a Vat gene analog associated with aphid resistance on LG5;

[0141] (h) a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and a Vat gene analog associated with aphid resistance on LG5; or

[0142] (i) A QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and a Vat gene analog associated with aphid resistance on LG5.

[0143] In a particularly preferred embodiment, the C. melo plant according to the invention is - a QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and a Vat gene analogue associated with aphid resistance on LG5, - have commercially acceptable fruit quality; - does not have any necrotic phenotype associated with the Zym gene.

[0144] In some embodiments, the QTL on LG2 that confers resistance to scab is identified by detection of the Cm_MU45136_209, LG2-M4 (SEQ ID NO: 5), and / or Cm_MU45398_32 markers; or other markers within the chromosomal region delimited by markers Cm_MU45136_209 and Cm_MU45398_32. In some embodiments, detection of the Cm_MU45136_209, LG2-M4, and / or Cm_MU45398_32 markers is performed by amplification, preferably PCR, using specific primers that can be used to amplify the resistance / susceptibility alleles of each of the Cm_MU45136_209, LG2-M4, and Cm_MU45398_32 markers.

[0145] In particular, detection of the Cm_MU45136_209 marker on LG2 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which can be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO: 1, or a fragment thereof comprising the [T / C] polymorphism at position 209 of SEQ ID NO: 1. For example, a forward primer for detecting a susceptible allele gene of marker Cm_MU45136_209 may consist of the sequence 5'-ACAAATTTCTTGGAGCTGCAAGACTTA-3' (SEQ ID NO: 2) by amplifying a nucleic acid consisting of SEQ ID NO: 1 or its complementary sequence, a forward primer for detecting a resistant allele gene of marker Cm_MU45136_209 may consist of the sequence 5'-CAAATTTCTTGGAGCTGCAAGACTTG-3' (SEQ ID NO: 3) by amplifying a nucleic acid consisting of SEQ ID NO: 1 or its complementary sequence, and a common reverse primer may consist of the sequence 5'-TATCATCGGTTCTTGTCTCAAGAAGGAAA-3' (SEQ ID NO: 4). When primers consisting of the sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are used, detection of guanine (G) instead of adenine (A) at position 209 of the amplification product consisting of the sequence of SEQ ID NO: 1, or cytosine (C) instead of thymine (T) in the complementary strand, indicates the presence of a QTL on LG2 that confers resistance to scab (see Table 1 below).

[0146] In particular, detection of the LG2-M4 marker on LG2 is carried out using two forward primers, one specific for the resistance allele gene and one specific for the susceptibility allele gene, and one common reverse primer. The two forward primers can be selected so as to be capable of amplifying a nucleic acid comprising or consisting of SEQ ID NO: 5, or a fragment thereof containing the [G / A] polymorphism at position 61 of SEQ ID NO: 5. For example, the forward primer for detecting the susceptibility allele gene of the marker LG2-M4 may consist of the sequence 5'-TTCACACCATTTGTAAGTTTGAACTTTG-3' (SEQ ID NO: 6) by amplifying a nucleic acid consisting of SEQ ID NO: 5, the forward primer for detecting the resistance allele gene of the marker LG2-M4 may consist of the sequence 5'-GTTTTCACACCATTTGTAAGTTTGAACTTTA-3' (SEQ ID NO: 7) by amplifying a nucleic acid consisting of SEQ ID NO: 5, and the common reverse primer may consist of the sequence 5'-GCACGTATGATAACGAGTTCTTTAGTGTT-3' (SEQ ID NO: 8). When primers consisting of the sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 are used, detection of adenine (A) rather than guanine (G) at position 61 of the amplification product consisting of the sequence of SEQ ID NO: 5 indicates the presence of a QTL on LG2 that confers resistance to scab (see Table 1 below).

[0147] In particular, detection of the Cm_MU45398_32 marker on LG2 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which can be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO:9, or a fragment thereof comprising the [T / C] polymorphism at position 32 of SEQ ID NO:9. For example, a forward primer for detecting a susceptibility allele of marker Cm_MU45398_32 may consist of the sequence 5'-CAAAACAGGGTTGTTCCGCTTTACT -3' (SEQ ID NO: 10) by amplifying a nucleic acid consisting of SEQ ID NO: 9, a forward primer for detecting a resistance allele of marker Cm_MU45398_32 may consist of the sequence 5'-AAAACAGGGTTGTTCCGCTTTACC -3' (SEQ ID NO: 11) by amplifying a nucleic acid consisting of SEQ ID NO: 9, and a common reverse primer may consist of the sequence 5'-CGTCTTCTTCTTCTTCTTCTTTGTTGCTA-3' (SEQ ID NO: 12). When primers consisting of the sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 are used, detection of a cytosine (C) rather than a thymine (T) at position 32 of the amplification product consisting of the sequence of SEQ ID NO: 9 indicates the presence of a QTL on LG2 that confers resistance to scab (see Table 1 below).

[0148] In some embodiments, the QTL on LG5 that confers resistance to scab is identified by detection of the LG5-M1, CMCTN2, Cm_MU46579_322, and / or Cm_MU44050_58 markers; or any other marker within the chromosomal region bounded by marker LG5-M1 and marker Cm_MU44050_58. In some embodiments, detection of the LG5-M1, CMCTN2, Cm_MU46579_322, and / or Cm_MU44050_58 markers is performed by amplification, preferably by PCR, using specific primers that can be used to amplify the resistance / susceptibility alleles of each of the LG5-M1, CMCTN2, Cm_MU46579_322, and / or Cm_MU44050_58 markers.

[0149] In particular, detection of the LG5-M1 marker on LG5 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer. The two forward primers can be selected so as to amplify a nucleic acid comprising or consisting of SEQ ID NO: 13, or a fragment thereof containing the [T / C] polymorphism at position 36 of SEQ ID NO: 13 (see Table 1 below). For example, suitable primers that can be used in the KASPar assay can be easily designed by a skilled artisan. Using primers that allow detection of the [T / C] polymorphism at position 36 of SEQ ID NO: 13, detection of guanine (G) instead of adenosine (A), or cytosine (C) instead of thymine (T) in the complementary strand, indicates the presence of a QTL on LG2 that confers resistance to scab at position 36 of the amplification product consisting of the sequence of SEQ ID NO: 13.

[0150] In particular, detection of the CMCTN2 marker on LG5 is carried out by PCR using forward and reverse primers that can be used to amplify the resistance / susceptibility allele of the CMCTN2 marker. In some embodiments, the PCR is followed by digestion of the amplification product with a restriction enzyme or sequencing of the amplification product. In particular, the forward and reverse primers for amplifying the CMCTN2 marker may comprise the sequences 5'-CTGAAAGCAGTTTGTGTCGA-3' (SEQ ID NO: 14) and 5'-AAAGAAGGAAGGGCTGAGA-3' (SEQ ID NO: 15), respectively. Using primers consisting of SEQ ID NO: 14 and SEQ ID NO: 15, detection of a 195-bp amplification product indicates the presence of a QTL on LG5 that confers resistance to scab disease (see Table 2 below).

[0151] In particular, detection of the Cm_MU46579_322 marker on LG5 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which can be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO: 16, or a fragment thereof comprising the [T / C] polymorphism at position 51 of SEQ ID NO: 16. For example, a forward primer for detecting a susceptible allele of marker Cm_MU46579_322 may consist of the sequence 5'-TCCGATCCTCACTGGAACTATCT -3' (SEQ ID NO: 17) by amplifying a nucleic acid consisting of SEQ ID NO: 16, a forward primer for detecting a resistant allele of marker Cm_MU46579_322 may consist of the sequence 5'-CCGATCCTCACTGGAACTATCC -3' (SEQ ID NO: 18) by amplifying a nucleic acid consisting of SEQ ID NO: 16, and a common reverse primer may consist of the sequence 5'-CAGCCTCATCGACTGTGAACTTCAT -3' (SEQ ID NO: 19). When primers consisting of the sequences of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 are used, detection of guanine (G) instead of adenine (A) at position 51 of the amplification product consisting of the sequence of SEQ ID NO: 16, or cytosine (C) instead of thymine (T) in the complementary strand, indicates the presence of a QTL on LG5 that confers resistance to scab (see Table 1 below).

[0152] In particular, detection of the Cm_MU44050_58 marker on LG5 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which can be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO:20, or a fragment thereof comprising the [T / C] polymorphism at position 58 of SEQ ID NO:20. For example, a forward primer for detecting a susceptible allele of marker Cm_MU44050_58 may consist of the sequence 5'-GCGTTGCTTTCATGGCGAGCTTT-3' (SEQ ID NO: 21) by amplifying a nucleic acid consisting of SEQ ID NO: 20, a forward primer for detecting a resistant allele of marker Cm_MU44050_58 may consist of the sequence 5'-CGTTGCTTTCATGGCGAGCTTC-3' (SEQ ID NO: 22) by amplifying a nucleic acid consisting of SEQ ID NO: 20, and a common reverse primer may consist of the sequence 5'-CTGTTGGAACGGAGAAGCTCAAAGAA-3' (SEQ ID NO: 23). When primers consisting of the sequences of SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 are used, detection of guanine (G) instead of adenine (A) at position 58 of the amplification product consisting of the sequence of SEQ ID NO: 20, or cytosine (C) instead of thymine (T) in the complementary strand, indicates the presence of a QTL on LG5 that confers resistance to scab (see Table 1 below).

[0153] In some embodiments, the QTL on LG2 that confers resistance to PM is identified by detection of the CMBR120, LG2-M1 (SEQ ID NO: 26), and / or Cm_MU47536_461 markers; or any other marker within the chromosomal region bounded by markers CMBR120 and Cm_MU47536_461. In some embodiments, detection of the CMBR120, LG2-M1, and / or Cm_MU47536_461 markers is performed by amplification, preferably PCR, using specific primers that can be used to amplify the resistance / susceptibility alleles of each of the CMBR120, LG2-M1, and / or Cm_MU47536_461 markers.

[0154] In particular, detection of the CMBR120 marker on LG2 is carried out by PCR using forward and reverse primers that can be used to amplify the resistance / susceptibility allele of the CMBR120 marker. In some embodiments, the PCR is followed by digesting the amplification product with a restriction enzyme or sequencing the amplification product. In particular, the forward and reverse primers for amplifying the CMBR120 marker may comprise the sequences 5'-CTGGCCCCCTCCTAAACTAA-3' (SEQ ID NO: 24) and 5'-CAAAAAGCATCAAAATGGTTG-3' (SEQ ID NO: 25), respectively. Using primers consisting of SEQ ID NO: 24 and SEQ ID NO: 25, detection of a 165-bp amplification product indicates the presence of a QTL on LG2 that confers resistance to PM (see Table 2 below).

[0155] In particular, detection of the LG2-M1 marker on LG2 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which can be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO:26, or a fragment thereof comprising the [C / T] polymorphism at position 69 of SEQ ID NO:26. For example, a forward primer for detecting a susceptibility allele of marker LG2-M1 may consist of the sequence 5'-CCTCATTTGGGCCCCGGG-3' (SEQ ID NO:27) by amplifying a nucleic acid consisting of SEQ ID NO:26 or its complementary sequence, a forward primer for detecting a resistance allele of marker LG2-M1 may consist of the sequence 5'-AATCCTCATTTGGGCCCCGGA-3' (SEQ ID NO:28) by amplifying a nucleic acid consisting of SEQ ID NO:26 or its complementary sequence, and a common reverse primer may consist of the sequence 5'-TCATGGCTTCTGATACTCGTTCTGATAT-3' (SEQ ID NO:29). When primers consisting of SEQ ID NOs:27, 28, and 29 are used, detection of an adenine (A) rather than a guanine (G) at position 69 of the amplification product consisting of the sequence of SEQ ID NO:26, or a thymine (T) rather than a cytosine (C) in the complementary strand, indicates the presence of a QTL on LG2 that confers resistance to PM (see Table 1 below).

[0156] In particular, detection of the Cm_MU47536_461 marker on LG2 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which may be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO: 30, or a fragment thereof comprising the [A / T] polymorphism at position 51 of SEQ ID NO: 30. For example, a forward primer for detecting a susceptibility allele of marker Cm_MU47536_461 may consist of the sequence 5'-ATGTACAAGATTTTGATAATGTGATTGATACA -3' (SEQ ID NO: 31) by amplifying a nucleic acid consisting of SEQ ID NO: 30, a forward primer for detecting a resistance allele of marker Cm_MU47536_461 may consist of the sequence 5'-ATGTACAAGATTTTGATAATGTGATTGATACT -3' (SEQ ID NO: 32) by amplifying a nucleic acid consisting of SEQ ID NO: 30, and a common reverse primer may consist of the sequence 5'-CGAAGAATATTAGCTGAGCCTTTGATGTT -3' (SEQ ID NO: 33). When primers consisting of the sequences of SEQ ID NOs: 31, 32, and 33 are used, detection of a thymine (T) rather than an adenine (A) at position 51 of the amplification product consisting of the sequence of SEQ ID NO: 30 indicates the presence of a QTL on LG2 that confers resistance to PM (see Table 1 below).

[0157] In some embodiments, the QTL on LG5 that confers resistance to PM is identified by detection of the Cm_MU45437_855, LG5-M2, CMTAN139, and / or LG5-M3 markers; or other markers within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3. In some embodiments, detection of the Cm_MU45437_855, LG5-M2, CMTAN139, and / or LG5-M3 markers is performed by amplification, preferably PCR, using specific primers that can be used to amplify the resistance / susceptibility alleles of each of the Cm_MU45437_855, LG5-M2, CMTAN139, and / or LG5-M3 markers.

[0158] In particular, detection of the Cm_MU45437_855 marker on LG5 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which can be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO: 34, or a fragment thereof comprising the [G / A] polymorphism at position 51 of SEQ ID NO: 34. For example, a forward primer for detecting a susceptibility allele of marker Cm_MU45437_855 may consist of the sequence 5'-AAAGTTTCTGTGTATTAAATCTGAACTCG -3' (SEQ ID NO: 35) by amplifying a nucleic acid consisting of SEQ ID NO: 34, a forward primer for detecting a resistance allele of marker Cm_MU45437_855 may consist of the sequence 5'-AATTAAAGTTTCTGTGTATTAAATCTGAACTCA -3' (SEQ ID NO: 36) by amplifying a nucleic acid consisting of SEQ ID NO: 34, and a common reverse primer may consist of the sequence 5'-CAGAGCACGTTTCGAAGGCACATAT-3' (SEQ ID NO: 37). When primers consisting of the sequences of SEQ ID NOs: 35, 36, and 37 are used, detection of an adenine (A) rather than a guanine (G) at position 51 of the amplification product consisting of the sequence of SEQ ID NO: 34 indicates the presence of a QTL on LG5 that confers resistance to PM (see Table 1 below).

[0159] In particular, detection of the LG5-M2 marker on LG5 is performed by PCR using forward and reverse primers that can be used to amplify the resistance / susceptibility allele of the LG5-M2 marker. In some embodiments, the PCR is followed by digestion of the amplified product with a restriction enzyme or sequencing of the amplified product. In particular, the forward and reverse primers for amplifying the LG5-M2 marker can comprise the sequences 5'-CACTTTCTAAATAGTTTGGAAAAGAG-3' (SEQ ID NO: 38) and 5'-GAGAATGTCTCTTTATCTAC-3' (SEQ ID NO: 39), respectively. Using primers consisting of SEQ ID NO: 38 and SEQ ID NO: 39, detection of a 178-bp amplified product indicates the presence of a QTL on LG5 that confers resistance to PM (see Table 2 below).

[0160] In particular, detection of the CMTAN139 marker on LG5 is performed by PCR using forward and reverse primers that can be used to amplify the resistance / susceptibility allele of the CMTAN139 marker. In some embodiments, the PCR is followed by digestion of the amplification product with a restriction enzyme or sequencing of the amplification product. In particular, the forward and reverse primers for amplifying the CMTAN139 marker can comprise the sequences 5'-CGTAGAAGACACACATAATG-3' (SEQ ID NO: 40) and 5'-GAACTAGAACCACAAATCAC-3' (SEQ ID NO: 41), respectively. Using primers consisting of SEQ ID NO: 40 and SEQ ID NO: 41, detection of a 134-bp amplification product indicates the presence of a QTL on LG5 that confers resistance to PM (see Table 2 below).

[0161] In particular, detection of the LG5-M3 marker on LG5 is carried out using two forward primers, one specific for the resistance allele and one specific for the susceptibility allele, and one common reverse primer, which can be selected to amplify a nucleic acid comprising or consisting of SEQ ID NO: 42, or a fragment thereof comprising the [C / T] polymorphism at position 51 of SEQ ID NO: 42. For example, a forward primer for detecting a susceptible allele gene of marker LG5-M3 may consist of the sequence 5'-CAGTCACAGAATTTGTAGTAGACTTATAG -3' (SEQ ID NO: 43) by amplifying a nucleic acid consisting of SEQ ID NO: 42 or its complementary sequence, a forward primer for detecting a resistant allele gene of marker LG5-M3 may consist of the sequence 5'-CAGTCACAGAATTTGTAGTAGACTTATAA -3' (SEQ ID NO: 44) by amplifying a nucleic acid consisting of SEQ ID NO: 42 or its complementary sequence, and a common reverse primer may consist of the sequence 5'-AGAGTTCTTTCTAACGGGCATTGAGATT-3' (SEQ ID NO: 45). When primers consisting of the sequences of SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 are used, detection of adenine (A) instead of guanine (G) at position 51 of the amplification product consisting of the sequence of SEQ ID NO: 42, or thymine (T) instead of cytosine (C) in the complementary strand, indicates the presence of a QTL on LG5 that confers resistance to PM (see Table 1 below).

[0162] In some embodiments, the Vat gene analog associated with aphid resistance on LG5 is identified by the amplification product of forward primer Me-VatE-F having the sequence 5'-CTCCACTCAGAATTGGTAGGTGCC-3' (SEQ ID NO: 48) and reverse primer Me-VatE-R having the sequence 5'-CCTTAGAAGAAGATGAAGTCCC-3' (SEQ ID NO: 49). Using this pair of primers, detection of a 1723 bp fragment indicates the presence of the Vat gene analog.

[0163] The alleles conferring resistance to scab and powdery mildew (PM) amplified by the markers defined above are listed in Tables 1 and 2.

[0164] [Table 1]

[0165] [Table 2]

[0166] In some embodiments, the QTL(s) conferring resistance to scab, PM and aphids are identified by detecting: - (1) Allele C of Cm_MU45136_209, allele A of LG2-M4, and allele C of Cm_MU45398_32, - (2) Allele C of LG5-M1, allele 195 bp of CMCTN2, allele C of Cm_MU46579_322, and allele C of Cm_MU44050_58, - (3) CMBR120 allele 165 bp, LG2-M1 allele T, and Cm_MU47536_461 allele T, (4) Allele A of Cm_MU45437_855, allele 178 bp of LG5-M2, allele 134 bp of CMTAN139, allele T of LG5-M3, or - (5) Me_VatE allele gene 1723 bp.

[0167] Insofar as the QTLs conferring resistance to scab, PM and aphids can be identified by the specific alleles set forth in Tables 1 and 2, the plants of the present invention preferably comprise any combination of alleles defined herein above, have commercially acceptable fruit quality, and optionally are free of any necrotic phenotype. For example, a C. melo plant according to the present invention may comprise the following combination of alleles associated with resistance to scab, PM and aphids: I) Allele gene combinations (1), (3), and (5); II) allele combinations (2), (4), and (5); III) allele combinations (1), (4), and (5); IV) allele combinations (2), (3) and (5); V) allele combinations (1), (2), (3) and (5); VI) allele combinations (1), (2), (4) and (5); VII) allele combinations (1), (3), (4) and (5); VIII) allele combinations (2), (3), (4), and (5); or IX) allele combinations (1), (2), (3), (4) and (5);

[0168] Thus, in a particularly preferred embodiment, the C. melo plant according to the invention comprises: - containing allele combination IX) as defined above, - have commercially acceptable fruit quality; - optionally, does not have any necrotic phenotype associated with the Zym gene.

[0169] In some embodiments, the one or more QTLs are associated with resistance to Cladosporium, aphids, and powdery mildew and are selected from those present in the genome of a plant of line MTYVVC721, the seeds of which are deposited under NCIMB Accession No. 43317.

[0170] In some embodiments, the one or more QTL associated with resistance to Cladosporium, aphids, and powdery mildew are as found in the genome of the plant corresponding to deposit material MTYVVC721 (NCIMB Accession No. 43317).

[0171] In some embodiments, the C. melo plant described in the present invention is line MTYVVC721, seeds of which are deposited under NCIMB accession number 43317.

[0172] In some embodiments, plants described in the present invention can be progeny or offspring of plants grown from deposited seeds of C. melo line MTYVVC721, deposited with NCIMB under accession number 43317. Plants grown from the deposited seeds are indeed homozygously resistant to scab, aphids, and powdery mildew, have commercially acceptable fruit quality, and do not have the necrosis phenotype associated with the Zym gene, i.e., they have in their genome, in the homozygous state, the QTLs on LG2 and LG5 associated with scab, aphids, and powdery mildew resistance defined above; and do not have any necrosis phenotype associated with the Zym gene. They can be used to transfer the QTLs on LG2 and LG5 in another background by crossing and selfing and / or backcrossing, without transferring any necrosis phenotype associated with the Zym gene. The progeny of plants obtained from the deposited seeds can be identified by one skilled in the art using, for example, the markers Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG5-M1, CMCTN2, Cm_MU46579_322, Cm_MU44050_58, CMBR120, LG2-M1, Cm_MU47536_461, Cm_MU45437_855, LG5-M2, CMTAN139, LG5-M3 and / or Me-VatE. Preferably, said progeny are distinguished by at least two, more preferably at least three, of said markers; according to a preferred embodiment, at least one of the markers is associated with a QTL on LG2 or LG5 associated with resistance to scab and at least one of the markers is associated with a QTL on LG2 or LG5 associated with resistance to powdery mildew; and the third marker may be a marker associated with resistance to aphids, such as Me-VatE.

[0173] Resistance to scab, aphids and powdery mildew is advantageously determined by comparison with susceptible (commercial) lines, such as the Vedrantais line. Resistance to Cladosporium is preferably determined based on inoculation tests of plants at the one-leaf stage, as detailed in Example 1.1. Resistance to PM is preferably determined based on inoculation tests of plants at the one-leaf stage or based on spray tests applied to detached leaves, as detailed in Example 1.2. Resistance to aphids is preferably determined as disclosed in the prior art.

[0174] According to a second aspect, the present invention is directed to a plant part according to the invention.

[0175] In some embodiments, the plant part is a plant cell. Thus, the present invention relates to a cell of a C. melo plant according to the present invention, i.e. (i) at least one QTL that confers resistance to scab, said at least one QTL being present on linkage group 2 (LG2) and / or linkage group 5 (LG5); (ii) at least one QTL that confers resistance to PM, the at least one QTL being present on LG2 and / or LG5 and distinct from the one or more QTLs in (i); and (iii) Vat gene analogues associated with aphid resistance in LG5: The present invention provides a plant cell comprising:

[0176] The distinct characteristics of the QTL on LG2 and LG5 are as defined in relation to the first aspect of the invention and apply mutatis mutandis to this aspect of the invention. The QTL are preferably selected from those present in the genome of the plant corresponding to the deposited material MTYVVC721 (NCIMB accession number 43317). In some embodiments, the QTL on LG2 and LG5 that confer resistance to scab, aphids and powdery mildew are as found in the genome of the plant corresponding to the deposited material MTYVVC721 (NCIMB accession number 43317).

[0177] In some embodiments, the QTL on LG2 and LG5 that confer resistance to scab, aphids and powdery mildew are as defined in the first aspect of the invention.

[0178] In some embodiments, alleles that confer resistance to scab, aphids, and powdery mildew are as set forth in Tables 1 and 2.

[0179] In some embodiments, the plant part according to the invention comprises an allelic gene combination I) to IX) as defined in the first aspect of the invention.

[0180] In some embodiments, the allelic gene combinations described herein above are as found in the genome of the plant corresponding to deposit material MTYVVC721 (NCIMB Accession No. 43317).

[0181] The plant cells of the present invention may have the ability to regenerate into whole plants, which have commercially acceptable fruit quality and are free of any necrotic phenotype.

[0182] Alternatively, the present invention relates to plant cells that are non-regenerative and therefore cannot give rise to a whole plant.

[0183] Preferably, the plant cell according to the invention comprises a QTL conferring resistance to scab located on LG2 within the chromosomal region delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32, a QTL conferring resistance to scab located on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, a QTL conferring resistance to PM located on LG2 within the chromosomal region delimited by marker CMBR120 and marker Cm_MU47536_461, a QTL conferring resistance to PM located on LG5 within the chromosomal region delimited by marker Cm_MU45437_855 and marker LG5-M3, and a Vat gene analogue associated with aphid resistance on LG5.

[0184] According to another embodiment, the plant part may be any other part of a plant according to the invention, in particular a seed, reproductive material, root, flower, fruit, rootstock or scion.

[0185] All embodiments detailed in the previous section in relation to the first aspect of the invention are also preferred embodiments described in this second aspect of the invention.

[0186] The present invention more particularly relates to the seeds of the C. melo plants that result when grown into scab-, aphid-, and powdery mildew-resistant C. melo plants as defined above. Said seeds are "seeds of the plants of the invention," i.e., seeds that give rise to the plants of the invention. The present invention also relates to seeds derived from the plants of the invention, i.e., seeds obtained from said plants after selfing or crossing, provided that the plants obtained from said seeds are resistant to scab, aphids, and powdery mildew due to the QTLs on LG2 and LG5 defined above, and preferably do not have the necrosis phenotype associated with the Zym gene.

[0187] The present invention also relates to tissue cultures of regenerable plant cells as defined above according to the present invention; preferably, the regenerable cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, flowers, cotyledons and / or hypocotyls of the present invention and therefore comprise in their genome the QTLs on LG2 and LG5 that confer resistance to scab, aphids and powdery mildew as described herein above.

[0188] The tissue cultures are preferably capable of regenerating plants having the physiological and morphological characteristics of the aforementioned C. melo plants and are capable of regenerating plants having substantially the same genotype as the aforementioned C. melo plants. The present invention also provides C. melo plants regenerated from the tissue cultures of the present invention.

[0189] The present invention also provides protoplasts of a plant as defined above, or from a tissue culture as defined above, said protoplasts comprising in their genome QTLs on LG2 and LG5 that confer resistance to scab, aphids and powdery mildew as defined herein above.

[0190] All embodiments detailed in the preceding paragraphs in relation to the first aspect of the invention are also embodiments of this second aspect of the invention.

[0191] According to a third aspect, the present invention also relates to the use of a C. melo plant as detailed according to the first aspect of the invention, i.e., a C. melo plant resistant to scab, aphids, and powdery mildew, as a breeding partner in a breeding program to obtain C. melo plants resistant to scab, aphids, and powdery mildew. Indeed, the C. melo plant according to the first aspect carries in its genome the QTLs defined above that confer said resistance, preferably without any necrosis phenotype associated with the Zym gene. By crossing this plant with a susceptible or less resistant plant, it is possible to transfer these QTLs to the offspring, thereby conferring the desired phenotype. The plants described in the present invention can be used as breeding partners for introducing QTLs that confer a desired phenotype to C. melo plants or germplasm (i.e., without introducing any necrosis phenotype associated with the Zym gene). The present invention also relates to similar uses of plants or seeds of MTYVVC721 deposited with NCIMB under accession number 43317. The plants are also suitable as introduction partners in breeding programs aimed at imparting desired phenotypes to C. melo plants or germplasm.

[0192] In said breeding program, selection of progeny exhibiting a desired phenotype, or carrying sequences associated with a desired phenotype, can be advantageously carried out based on the allelic genes of the markers disclosed herein above. The progeny are preferably selected for the presence of one or more of the following specific alleles: C allele of Cm_MU45136_209, A allele of LG2-M4, C allele of Cm_MU45398_32, C allele of LG5-M1, 195bp allele of CMCTN2, Cm_MU46579_322, C allele of Cm_MU44050_58, 165bp allele of CMBR120, T allele of LG2-M1, T allele of Cm_MU47536_461, A allele of Cm_MU45437_855, 178bp allele of LG5-M2, 134bp allele of CMTAN139, T allele of LG5-M3, and 1723 allele of Me_VatE. bp. The offspring are preferably selected for the presence of allelic gene combinations I) to IX) as defined in the first aspect of the invention.

[0193] Selection of progeny with the desired phenotype can also be carried out under pathogen-prevalent conditions, particularly as disclosed in the Examples section on Scab and / or PM tests, or in conjunction with other tests well known to the skilled reader.

[0194] The plants described in the present invention, or the plant deposited under accession number NCIMB 43317, are particularly valuable in marker-assisted selection programs to obtain commercial C. melo lines and varieties that are resistant to scab, aphids and powdery mildew, but preferably do not have any of the necrosis phenotype associated with the Zym gene.

[0195] Any of the embodiments described for the first and second aspects of the invention are also applicable to this aspect of the invention.

[0196] The invention also relates to the use of said plants in programs aimed at identifying, sequencing and / or cloning gene sequences that confer a desired phenotype.

[0197] Any particular embodiment described in the previous aspect of the invention is also applicable to this aspect of the invention, particularly with respect to the characterization of QTLs that confer a phenotype of interest.

[0198] In another aspect, the present invention also relates to a method for producing C. melo plants, particularly commercial plants, that are resistant to scab, aphids and powdery mildew. The method or process for producing plants with these characteristics comprises the following steps: a) crossing a plant according to the first aspect of the present invention (e.g., a plant corresponding to the deposited seed (NCIMB 43317)) with a susceptible or less resistant C. melo plant to introduce or improve a desired phenotype; b) selecting one plant that is resistant to scab, aphids and powdery mildew but preferably does not have any necrotic phenotype associated with the Zym gene in the resulting progeny, or one plant that has a QTL associated with resistance to scab, aphids and powdery mildew but preferably does not have any necrotic phenotype associated with the Zym gene; c) optionally self-pollinating the resistant plants obtained in step b) once or several times to select for plants of the resulting progeny that are resistant to scab, aphids and powdery mildew, but preferably do not have any necrotic phenotype associated with the Zym gene; d) backcrossing the resistant plants selected in step b) or c) with susceptible C. melo plants (i.e., susceptible to scab, aphids and / or powdery mildew); and e) Selecting plants that are resistant to scab, aphids and powdery mildew, but preferably do not have any necrotic phenotypes associated with the Zym gene.

[0199] Alternatively, the method or process may include the following steps: a1) Crossing a plant according to the first aspect of the present invention (e.g., a plant corresponding to the deposited seeds (NCIMB 43317)) with a C. melo plant having low susceptibility or resistance to introduce or improve a desired phenotype and generate an F1 population; a2) Self-pollinating the F1 population to create an F2 population; b) Selecting, in the obtained progeny, resistant individuals and preferably individuals having no necrotic phenotype associated with the Zym gene; c) Optionally, self-pollinating the resistant plants obtained in step b) one or several times, preferably selecting resistant plants having no necrotic phenotype associated with the Zym gene in the obtained progeny; d) Backcrossing the resistant plants selected in step b) or c) with a susceptible C. melo plant (i.e., a plant susceptible to scab, aphids and / or powdery mildew); e) Selecting a plant resistant to scab, aphids and powdery mildew and preferably having no necrotic phenotype associated with the Zym gene.

[0200] In some embodiments, plants that are resistant to scab, aphids and powdery mildew but have no necrotic phenotype associated with the Zym gene can be selected in steps b), c) and e).

[0201] The plant selected in step e) is preferably a commercial plant, particularly a fruit of the Charentais, Western Shipper, Harp, or Italian Cantaloupe type having a flesh color from orange to red magenta (i.e., a colorimetric value measured by a Minolta colorimeter: 60 < L < 65, 40 < c < 50 and 66 < h < 75), an average flesh firmness of 4 kg / 0.5 cm 2 + / - 2 measured with a Penefel, a Brix level of at least 11°, a fruit diameter / cavity size ratio found in the Vedrantais variety, and fruit storage at +12 °C for more than 8 days.

[0202] Preferably, steps d) and e) are repeated at least two times, and preferably three times, but not necessarily with the same susceptible C. melo plant. Preferably, said susceptible C. melo plant is a breeding line.

[0203] The self-pollination and backcrossing steps may be performed and interspersed in any order, for example, a backcross may be performed before or after one or more self-pollinations, and a self-pollination may be envisaged before or after one or more backcrosses.

[0204] In some embodiments, the method is advantageously carried out by using markers as described herein for one or more of the selection steps carried out in steps b), c) and / or e) to select plants that are resistant to scab, aphids and powdery mildew, and preferably do not have any necrotic phenotype associated with the Zym gene.

[0205] In some embodiments, markers for selecting plants that are resistant to scab, aphids and powdery mildew, and preferably do not have any necrotic phenotypes associated with the Zym gene, are as follows: - one or more of the markers Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG2-M1, Cm_MU47536_461, CMBR120 and Me-VatE, or all of the markers Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG2-M1, Cm_MU47536_461, CMBR120 and Me-VatE, - one or more of the markers LG5-M1, Cm_MU46579_322, Cm_MU44050_58, CMCTN2, Cm_MU45437_855, LG5-M3, LG5-M2, CMTAN139 and Me-VatE, or all of the markers LG5-M1, Cm_MU46579_322, Cm_MU44050_58, CMCTN2, Cm_MU45437_855, LG5-M3, LG5-M2, CMTAN139 and Me-VatE, - one or more markers of Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG2-M1, Cm_MU47536_461, CMBR120, LG5-M1, Cm_MU46579_322, Cm_MU44050_58, CMCTN2, Cm_MU45437_855, LG5-M3, LG5-M2, CMTAN139, and Me-VatE, or Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG2-M1, Cm_MU47536_461, CMBR120, LG5-M1, Cm_MU46579_322, Cm_MU44050_58, CMCTN2, Cm_MU45437_855, LG5-M3, LG5-M2, CMTAN139, and all markers in Me-VatE.

[0206] In some embodiments, the plant selected in any one of steps b), c) and / or e) is preferably selected in the presence of one of the allele combinations I) to IX) defined in the first aspect of the present invention.

[0207] Selection of progeny with the desired phenotype can also be performed under conditions of pathogen infestation, particularly as disclosed in the Scab test and / or PM test sections of the Examples, or with other tests well known to the skilled reader.

[0208] The method used for allelic detection can be based on any technique that allows the differentiation of two different allelic markers on a particular chromosome.

[0209] The present invention also relates to a C. melo plant obtained or obtainable by said method, said plant being a scab-, aphid- and powdery mildew-resistant C. melo plant according to the first aspect of the invention.

[0210] According to a further aspect, the present invention also relates to a C.melo plant obtained by crossing a resistant plant according to the first aspect of the present invention, for example, the plant MTYVVC721 which is a representative sample of the seeds deposited under NCIMB accession number 43317, or a resistant plant obtained by the method disclosed above, with a plant susceptible to, for example, scab, aphids and powdery mildew infections, or a plant having different levels of resistance to scab, aphids and powdery mildew infections. Particularly preferred hybrid C.melo plants are plants that exhibit any agronomically beneficial trait or phenotype.

[0211] The present invention also relates to a method for obtaining a commercially available C.melo plant resistant to scab, aphids and powdery mildew, said method comprising the following steps: - Backcrossing a plant obtained by germinating the deposited seeds MTYVVC721 (NCIMB accession number 43317) or a C.melo plant according to the first aspect of the present invention with, for example, a C.melo plant susceptible to scab, aphids and powdery mildew; - Selecting a plant resistant to scab, aphids and powdery mildew and preferably not having a necrotic phenotype associated with the Zym gene.

[0212] The selection in the second step is preferably carried out as detailed above for the other methods of the present invention. Said selection is preferably carried out in the presence of one or more of the specific allelic genes of the markers and Vat gene analogues described herein, as found in the line MTYVVC721.

[0213] The plant is preferably a commercial plant, particularly having flesh color from orange to red magenta (i.e., colorimetric values measured by a Minolta colorimeter: 60 < L < 65, 40 < c < 50 and 66 < h < 75), and an average value of 4 kg / 0.5 cm measured with a Pennefeer. 2Fruit of the Charentais, Western Sipper, Harper or Italian cantaloupe type, with a flesh firmness of + / -2, a Brix level of at least 11°, a fruit diameter / cavity size ratio found in the Vedrantais variety, and a fruit storage period of more than 8 days at +12°C.

[0214] Also provided are methods for producing seeds of C. melo plants, in some embodiments, the methods comprise crossing a C. melo plant described herein with itself or another C. melo plant and harvesting the resulting seeds.

[0215] In addition to introducing QTL associated with scab, aphid, and powdery mildew resistance, as detailed in the methods of the present invention, the sequences can also be introduced into a C. melo background by genetic engineering to obtain commercial C. melo plants that are resistant to scab, aphid, and powdery mildew. It is routine for one skilled in the art to identify and clone introduced QTL from C. melo, particularly from a deposit, that confer a desired phenotype.

[0216] According to a further aspect, the present invention provides a plant obtained or obtainable by one of the methods described above, said plant being in fact a C. melo plant having the desired phenotype according to the first aspect of the invention, namely resistance to scab, aphids and powdery mildew, and preferably being free of any necrotic phenotype associated with the Zym gene.

[0217] It should be noted that the seeds or plants of the invention may be obtained by different processes, in particular technical processes such as UV mutagenesis or genetic engineering such as induced recombination, and are not exclusively obtained by essentially biological processes.

[0218] According to this aspect, the invention relates to a C. melo plant or seed, preferably a non-naturally occurring C. melo plant or seed, which may contain one or more mutations in its genome that provide the mutant plant with resistance to scab, aphids and / or powdery mildew, for example, the mutations present in the genome of a plant a representative sample of which has been deposited with NCIMB under accession number NCIMB 43317.

[0219] Preferably, the mutation is the incorporation of (i) at least one QTL that confers resistance to scab, said QTL being present on linkage group (LG) 2 and / or linkage group 5 (LG5), (ii) at least one QTL that confers resistance to PM, said QTL being present on LG2 and / or LG5 and different from one or more QTLs in (i), and (iii) a vat gene analog associated with aphid resistance on LG5 in place of the homologous sequence of a C. melo plant. Even more preferably, the mutation is (i) a replacement of the homologous sequence on LG2 in the genome of a plant a representative sample of which has been deposited at NCIMB under accession number NCIMB 43317 with the sequence delimited by marker Cm_MU45136_209 and marker Cm_MU45398_32 on LG2 of the C. melo genome, or a fragment thereof; (ii) a replacement of the homologous sequence on LG5 in the genome of a plant a representative sample of which has been deposited at NCIMB under accession number NCIMB 43317 with the sequence delimited by marker LG5-M1 and marker Cm_MU44050_58 on LG5 of the C. melo genome, or a fragment thereof; or (iii) a replacement of the homologous sequence on LG2 in the genome of a plant a representative sample of which has been deposited at NCIMB under accession number NCIMB 43317 with the sequence delimited by marker CMBR120 and marker Cm_MU44050_58 on LG2 of the C. melo genome, or a fragment thereof. (iv) replacement of a homologous sequence on LG5 in the genome of a plant of which a representative sample has been deposited with NCIMB under accession number NCIMB 43317 with a sequence or a fragment thereof delimited by marker Cm_MU45437_855 and marker LG5-M3 on LG5 of the C. melo genome; and (v) the vat gene analogue, which sequence or fragment confers resistance to scab, aphids and PM in combination.

[0220] In one embodiment, the present invention relates to a method for carrying a C. melo plant or seed having one or more mutations in its genome, providing the plant with resistance to scab, aphids and PM. The method is exemplified in Example 6 and may include: a) treating M0 seeds of the C. melo plant to be modified with a mutagenic agent to obtain M1 seeds; b) growing plants from the M1 seeds thus obtained to obtain M1 plants; c) producing M2 seeds by self-pollination of the M1 plants; and d) Optionally repeating steps b) and c) n times to obtain M2+n seeds.

[0221] The M2+n seeds are grown into plants and exposed to scab, aphid, and PM infection. Surviving plants or plants with mild scab, aphid, and PM infection are propagated for one or more generations as long as selection for resistance to scab, aphid, and PM continues. In this method, the M1 seeds of step a) can be obtained through chemical mutagenesis, such as EMS mutagenesis. Other chemical mutagenic agents include, but are not limited to, diethyl sulfate (des), ethyleneimine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (enu), and sodium azide. Alternatively, mutations can be induced by irradiation, e.g., X-rays, fast neutrons, or UV radiation.

[0222] In another embodiment of the invention, mutations are induced by genetic engineering, including the incorporation of sequences that confer scab, aphid and PM resistance, as well as the replacement of existing sequences with alternative sequences that confer scab, aphid and PM resistance.

[0223] Available genetic engineering tools include the use of all techniques known as novel breeding techniques, which are a variety of novel techniques developed and / or used to create new traits in plants through genetic variation, with the goal of targeted mutagenesis, targeted introduction of new genes, or gene silencing (RdDM). Examples of novel breeding techniques include targeted sequence alterations facilitated through the use of zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2, and ZFN-3, see U.S. Patent No. 9,145,565, incorporated by reference in its entirety), oligonucleotide-directed mutagenesis (ODM), cisgenesis and intragenesis, RNA-dependent DNA methylation (RdDM, which does not necessarily alter the nucleotide sequence but can alter the biological activity of the sequence), grafting (on GM rootstocks), reverse breeding, agroinfiltration (agroinfiltration "sensu stricto," agroinoculation, floral dip), transcription activator-like effector nucleases (TDM), and the like. ALENs, see U.S. Patent Nos. 8,586,363 and 9,181,535, which are incorporated by reference in their entireties), CRISPR / Cas systems (see U.S. Patent Nos. 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, which are incorporated by reference in their entireties), engineered meganucleases, redesigned homing endonucleases, DNA-guided genome editing (Gao (E. et al., Nature Biotechnology (2016), doi: 10.1038 / nbt.3547, incorporated by reference in its entirety), and Synthetic 5 Genomics). The majority of targeted genome editing (another designation for novel breeding techniques) today involves the induction of DNA split breaks (DSBs) at selected locations within the genome where modification is intended. Directed repair of DSBs enables targeted genome editing.These applications can be used to generate mutations (e.g., targeted mutations or precise natural gene editing) and precisely insert genes (e.g., cis-genes, intragenes, or transgenes). Mutation-inducing applications are often identified as site-specific nuclease (SDN) technologies, such as SDN1, SDN2, and SDN3. In the case of SDN1, targeted, nonspecific gene deletion mutations are generated: the location of the DNA DSB is precisely selected, but DNA repair by the host cell is random, resulting in the deletion, addition, or substitution of small nucleotides. In the case of SDN2, SDN is used to generate a targeted DSB, and then repair the DSB using a DNA repair template (a short DNA sequence identical to the target DSB DNA sequence except for one to a few nucleotide changes): this results in a targeted, predetermined point mutation in a desired gene of interest. In the case of SDN3, SDN is used with a DNA repair template containing a new DNA sequence (e.g., a gene). The outcome of this technology can be the integration of that DNA sequence into the plant genome. The most likely use of SDN3 is to insert cisgenic, intragenic, or transgenic expression cassettes into selected genomic locations. A complete description of each of these techniques is provided in a report entitled "New plant breeding techniques - State-of-the-art and prospects for commercial development," prepared by the Joint Research Center (JRC) Institute for Prospective Technological Studies of the European Commission in 2011, which is incorporated herein by reference.

[0224] The present invention also provides a method for detecting and / or selecting C. melo plants that are resistant to scab, aphids and powdery mildew (PM), and optionally do not have any necrotic phenotype associated with the Zym gene, said method comprising the steps of: (i) detecting the presence of at least one QTL conferring resistance to scab, wherein said at least one QTL is present on linkage group (LG) 2 and / or linkage group 5 (LG5); (ii) at least one QTL that confers resistance to PM, the at least one QTL being present on LG2 and / or LG5 and different from the at least one QTL in (i); and (iii) detecting the presence of a Vat gene analog associated with aphid resistance on LG5; and (iv) optionally detecting the absence of a necrosis phenotype associated with the Zym gene.

[0225] Preferably, the QTL present on LG2 that confers resistance to scab is located within the chromosomal region bounded by marker Cm_MU45136_209 and marker Cm_MU45398_32. In some embodiments, the QTL present on LG2 can be identified by amplifying any one of the following markers: Cm_MU45136_209, LG2-M4, and Cm_MU45398_32; or any other marker within the chromosomal region bounded by marker Cm_MU45136_209 and marker Cm_MU45398_32.

[0226] Preferably, the QTL present on LG5 that confers resistance to scab is located within the chromosomal region bounded by marker LG5-M1 and marker Cm_MU44050_58. In some embodiments, the QTL present on LG5 can be identified by amplifying any one of the following markers: LG5-M1, CMCTN2, Cm_MU46579_322, and Cm_MU44050_58; or any other marker within the chromosomal region bounded by marker LG5-M1 and marker Cm_MU44050_58.

[0227] Preferably, the QTL present on LG2 that confers resistance to PM is located within the chromosomal region bounded by marker CMBR120 and marker Cm_MU47536_461. In some embodiments, the QTL present on LG2 can be identified by amplifying any one of the following markers: CMBR120, LG2-M1, and Cm_MU47536_461; or any other marker within the chromosomal region bounded by marker CMBR120 and marker Cm_MU47536_461.

[0228] Preferably, the QTL present on LG5 that confers resistance to PM is located within the chromosomal region bounded by marker Cm_MU45437_855 and marker LG5-M3. In some embodiments, the QTL present on LG5 can be identified by amplifying any one of the following markers: Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3; or any other marker within the chromosomal region bounded by marker Cm_MU45437_855 and marker LG5-M3.

[0229] According to a preferred embodiment, the plants according to the invention do not homozygously contain a Zym gene on LG2. However, plants of the invention may heterozygously contain said gene that confers resistance to potyviruses, in particular ZYMV. Therefore, the methods or processes of the invention preferably also include a step of detecting whether a Zym gene is present.

[0230] As detailed in the Experimental Section of the present application, in particular in Example 5, the presence of the Zym gene confers resistance and can be detected by SNP markers LG2-M2 (SEQ ID NO: 50), Cm-MU47380_465 (also known as MU47380_465, SEQ ID NO: 54), and / or LG2-M3 (SEQ ID NO: 58). Preferably, the QTL or gene conferring resistance to ZYMV present on LG2 is located within the chromosomal region bounded by markers LG2-M2 and LG2-M3. In some embodiments, the QTL present on LG2 can be identified by amplifying any one of the following markers: LG2-M2, Cm-MU47380_465, and LG2-M3; or any other marker within the chromosomal region bounded by markers LG2-M2 and LG2-M3. According to the present invention, plants are selected if they contain at least one of the following alleles: LG2-M2 allele C, LG2-M3 allele G, or MU47380_465 allele A. These alleles represent the absence of QTLs or genes that actually confer resistance to potyviruses, and detection of one or more of these alleles indicates the homozygous absence of the Zym gene, thus ensuring the absence of the necrosis phenotype associated with the Zym gene.

[0231] Markers and alleles associated with resistance to ZYMV are listed in Table D. Potential primers that can be used to amplify marker sequences and distinguish between different alleles of the SNP are reported in Table G.

[0232] In some embodiments, a plant is selected if any one of allele combinations I) to IX) defined in the first aspect of the invention is detected in a genetic material sample of the selected plant. Preferably, a plant is selected if allele combination IX) defined in the first aspect of the invention is detected in a genetic material sample of the selected plant.

[0233] Preferably, the plant is detected when, in addition to the allele combinations I) to IX) defined in the first aspect of the present invention, one of the alleles representing the absence of the Zym gene is detected.

[0234] In some embodiments, detection of markers Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG5-M1, Cm_MU46579_322, Cm_MU44050_58, LG2-M1, Cm_MU47536_461, Cm_MU45437_855, and / or LG5-M3 is carried out by amplifying, e.g., by PCR, one forward primer that can be used to amplify the resistance allele gene, one forward primer that can be used to amplify the susceptibility allele gene, and one common reverse primer for each marker. In particular, the primers for amplifying each of said markers may have sequences as described in the first aspect of the invention and detailed in Table G. The same applies to markers related to the Zym gene disclosed above; potential primers are disclosed in Table G.

[0235] In a preferred embodiment, amplification is as described in the Examples. In a more preferred embodiment, amplification is performed using a two-step touchdown method, in which the extension and annealing steps are combined into a single step. The temperature used in the annealing step determines the specificity of the reaction and the ability of the primers to anneal to the DNA template. Touchdown PCR involves a first step of Taq polymerase activation, followed by a second step, called the touchdown step, which involves a high annealing temperature and gradually decreasing the annealing temperature with each PCR cycle, and a third step of DNA amplification. The high annealing temperature in the early cycles of touchdown ensures that only highly specific base pairing occurs between the DNA and the primers, making the initially amplified sequence most likely to be the desired sequence. The annealing temperature is gradually decreased, increasing the efficiency of the reaction. The region initially amplified during the highly specific initial touchdown cycles is further amplified, overcoming nonspecific amplification that may occur at lower temperatures.

[0236] In another embodiment, amplification of SNP markers is performed by PCR cycling as recommended for the KASPar assay and illustrated in the Examples (see Example 5), including an initial denaturation step at 94°C for about 15 minutes, 10 cycles at 94°C for about 20 seconds followed by about 60 seconds at a decreasing temperature (65°C in the first cycle to 57°C in the last cycle), and about 35 cycles at 94°C for about 20 seconds followed by about 60 seconds at 57°C. This protocol can be easily adapted by the skilled artisan depending on the type of primers used.

[0237] In some embodiments, detection of markers LG5-M2, CMTAN139, CMCTN2, CMBR120, and / or Me_VatE is carried out by amplification, e.g., by PCR, using one forward and one reverse primer for each marker. In particular, the primers for amplifying each of said markers may have the sequences described in the first aspect of the invention.

[0238] The present invention also relates to hybrid C. melo plants obtained or obtainable by crossing a C. melo plant according to the first aspect of the invention, or a resistant plant obtained or obtainable by the method disclosed above, with a C. melo plant that is susceptible to scab, aphids and PM, or a C. melo plant that has a different level of resistance to scab, aphids and PM.

[0239] According to a further aspect, the present invention also provides molecular markers associated with QTLs on LG2 and / or LG5 as defined above that confer resistance to scab, aphids and / or powdery mildew (PM).

[0240] In some embodiments, the molecular marker associated with a QTL conferring resistance to scab on LG2 is one or more of markers Cm_MU45136_209, LG2-M4, and Cm_MU45398_32, or all of markers Cm_MU45136_209, LG2-M4, and Cm_MU45398_32, or a combination of markers Cm_MU45136_209, LG2-M4, and Cm_MU45398_32, or any other marker within the chromosomal region delimited by markers Cm_MU45136_209 and Cm_MU45398_32.

[0241] In some embodiments, the molecular marker associated with a QTL conferring resistance to scab on LG5 is one or more of markers LG5-M1, CMCTN2, Cm_MU46579_322, and Cm_MU44050_58, or all of markers LG5-M1, CMCTN2, Cm_MU46579_322, and Cm_MU44050_58, or a combination of markers LG5-M1, CMCTN2, Cm_MU46579_322, and Cm_MU44050_58, or any other marker within the chromosomal region bounded by marker LG5-M1 and marker Cm_MU44050_58.

[0242] In some embodiments, the molecular marker associated with a QTL conferring resistance to PM on LG2 is one or more of markers CMBR120, LG2-M1, and Cm_MU47536_461, or all of markers CMBR120, LG2-M1, and Cm_MU47536_461, or a combination of markers CMBR120, LG2-M1, and Cm_MU47536_461, or any other marker within the chromosomal region bounded by marker CMBR120 and marker Cm_MU47536_461.

[0243] In some embodiments, the molecular marker associated with a QTL conferring resistance to PM on LG5 is one or more of markers Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3, or all of markers Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3, or a combination of markers Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3, or any other marker within the chromosomal region bounded by marker Cm_MU45437_855 and marker LG5-M3.

[0244] The sequences of the above-mentioned markers are set out in Tables 1 and 2 and in the Examples.

[0245] one or more molecular markers Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG5-M1, CMCTN2, Cm_MU46579_322, Cm_MU44050_58, CMBR120, LG2-M1, Cm_MU47536_461, Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3, or markers All of Cm_MU45136_209, LG2-M4, Cm_MU45398_32, LG5-M1, CMCTN2, Cm_MU46579_322, Cm_MU44050_58, CMBR120, LG2-M1, Cm_MU47536_461, Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3, or markers Cm_MU45136_209, LG2-M4, Cm_MU Further provided is the use of a combination of 45398_32, LG5-M1, CMCTN2, Cm_MU46579_322, Cm_MU44050_58, CMBR120, LG2-M1, Cm_MU47536_461, Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3 to detect C. melo plants that are resistant to scab, aphids, and / or powdery mildew (PM). The use may also further include the use of one or all of the SNP markers LG2-M2, Cm-MU47380_ and LG2-M3 to detect C. melo plants that do not exhibit the necrosis phenotype associated with the Zym gene.

[0246] The present invention also relates to the use of at least one of the markers of the list Cm_MU45136_209, LG2-M4, Cm_MU45398_32, CMBR120, LG2-M1, Cm_MU47536_461, LG5-M1, CMCTN2, Cm_MU46579_322, Cm_MU44050_58, Cm_MU45437_855, LG5-M2, CMTAN139 and LG5-M3 associated with a QTL on LG2 (SNPs 1 to 6 of the list) and LG5 (SNPs 7 to 14 of the list) conferring resistance to scab, aphids and / or powdery mildew (PM) according to the invention for identifying a surrogate molecular marker associated with said QTL, wherein said surrogate molecular marker is: within the chromosomal region delimited on LG2 by markers Cm_MU45136_209 and Cm_MU45398_32, or markers CMBR120 and Cm_MU47536_461, on LG5 within the chromosomal region delimited by marker LG5-M1 and marker Cm_MU44050_58, or marker Cm_MU45437_855 and marker LG5-M3, less than 2 megabases from the locus of 14 markers of the invention, namely Cm_MU45136_209, LG2-M4, Cm_MU45398_32, CMBR120, LG2-M1, Cm_MU47536_461, LG5-M1, CMCTN2, Cm_MU46579_322, Cm_MU44050_58, Cm_MU45437_855, LG5-M2, CMTAN139 and LG5-M3 is.

[0247] The surrogate molecular markers are preferably associated with said QTL(s) with a p-value of 0.05 or less, preferably less than 0.01. The QTL are contained within the deposited seed NCIMB 43317.

[0248] The present invention also relates to a method for identifying molecular markers associated with QTLs conferring resistance to scab, aphids and / or powdery mildew (PM), when the molecular markers are present heterozygously or homozygously, said method comprising: - identifying molecular markers in the following chromosomal regions: within the chromosomal region bounded on LG2 by markers Cm_MU45136_209 and Cm_MU45398_32, or markers CMBR120 and Cm_MU47536_461, within the chromosomal region bounded at LG5 by marker LG5-M1 and marker Cm_MU44050_58, or by marker Cm_MU45437_855 and marker LG5-M3, less than 2 megabases from the 15 SNP markers of the present invention, namely, Cm_MU45136_209, LG2-M4, Cm_MU45398_32, CMBR120, LG2-M1, Cm_MU47536_461, LG5-M1, CMCTN2, Cm_MU46579_322, Cm_MU44050_58, Cm_MU45437_855, LG5-M2, CMTAN139 and LG5-M3 loci, and - determining whether said molecular markers are associated with or related to resistance to scab, aphids and / or powdery mildew (PM) in segregating populations generated from said resistant plants.

[0249] The population is preferably generated from plants grown from deposited seed NCIMB 43317 or its progeny, and exhibits resistance to scab, aphids and / or powdery mildew (PM) as described in the present invention.

[0250] The QTLs on LG2 and LG5 described above that confer resistance to scab, aphids and / or powdery mildew (PM) according to the present invention are QTLs present in MTYVVC721 (NCIMB 43317).

[0251] Genetic association or linkage is as described above. Preferably, the association or linkage has a p-value of less than 0.05, most preferably less than 0.01 or less.

[0252] The molecular marker and the resistance phenotype are preferably co-inherited in more than 90% of meiotic divisions, preferably more than 95%.

[0253] In a further aspect, the present invention relates to a method for producing scab, aphid and / or powdery mildew (PM) resistant C. melo plantlets or plants, the method comprising: i. culturing isolated cells or tissues of a C. melo plant in vitro according to the present invention to produce scab, aphid and powdery mildew (PM) resistant C. melo microplantlets; and ii. Optionally, subjecting the C. melo microplantlets to further in vivo culture steps to develop scab, aphid and powdery mildew (PM) resistant C. melo plants; Includes.

[0254] The isolated cells or tissues used to produce microplants are explants obtained under sterile conditions from the C. melo parent plant of the invention to be propagated. The explants may comprise or consist of, for example, cotyledons, hypocotyls, stem tissue, leaves, embryos, meristems, nodal buds, shoot tips, or protoplasts. The explants may be surface sterilized before being placed on culture media for tissue culture.

[0255] Conditions and media that can be suitably used for plant tissue culture are well known to those skilled in the art of plant cultivation, and are described, for example, in "Plant Propagation by Tissue Culture, Handbook and Directory of Commercial Laboratories, eds. Edwin F George and Paul D Sherrington, Exegetics Ltd, 1984."

[0256] Tissue culture typically involves: i. Axillary Shoot Proliferation: Axillary shoot proliferation is induced by adding cytokinin to the shoot culture medium, preferably producing shoots with minimal callus formation. ii. Adventitious shoot propagation: Root formation is induced by the addition of auxin to the medium to produce plantlets that can be transferred to soil. Alternatively, root formation can be induced directly in soil.

[0257] The plantlets can be further subjected to an in vivo culture stage by culturing them in soil under laboratory conditions and then gradually adapting them to natural climate, resulting in C. melo plants that are resistant to scab, aphids and powdery mildew (PM).

[0258] In view of the ability of the resistant plants of the present invention to limit damage caused by scab, aphids, and PM, they are advantageously grown in environments that are infested with or likely to be infected by aphids, Cladosporium, Podosphaera xantii, and / or Golobinomyces sicoracearum; under these conditions, the resistant plants of the present invention produce more marketable melons than susceptible plants. The present invention also relates to a method for improving the yield of C. melo plants or increasing the number of harvestable C. melo plants or fruits, comprising growing a scab, aphid, and PM-resistant C. melo plant in an environment infested with scab, aphids, and powdery mildew (PM), the plant comprising a QTL or sequence described in the present invention on LG2 and / or LG5 that confers resistance to scab, aphids, and powdery mildew (PM) on the plant.

[0259] Preferably, the method comprises a first step of screening or selecting a C. melo plant that comprises said sequence of interest, which confers resistance to scab, aphids and PM on said plant, and preferably does not comprise any necrosis phenotype associated with the Zym gene. The method may also be defined as a method for increasing the productivity of C. melo fields, tunnels or greenhouses, or for reducing the intensity or number of chemical or fungicide sprays in melon production.

[0260] The present invention also relates to a method for reducing the loss of C. melo production under scab, aphid and PM infestation or infection conditions, comprising growing a C. melo plant as defined above.

[0261] Resistant plants of the invention are also able to limit the proliferation of the pathogens causing scab and PM, as well as aphids, limiting further plant infection and the proliferation of pathogens and aphids. The invention also relates to a method for protecting fields, tunnels or greenhouses, or any other type of cultivation area, from the infestation of scab, aphids and PM, or at least limiting the level of infestation or limiting the infestation of scab, aphids and PM. The method preferably comprises the step of growing a resistant or tolerant plant of the invention, i.e. a plant comprising a sequence on LG2 and / or LG5 that confers resistance to scab, aphids and PM, preferably without any necrotic phenotype linked to the Zym gene.

[0262] The present invention also relates to the use of scab-, aphid- and PM-resistant C. melo plants according to the invention for controlling scab, aphid and PM infestations in fields, tunnels or greenhouses or any other growing area.

[0263] All preferred characteristics of the QTL are as defined in relation to other aspects of the present invention, i.e. are preferably present in seeds of MTYVVC721 (NCIMB accession number 43317) and are identifiable by markers defined according to the present invention.

[0264] The present invention also relates to a method for improving the yield of C. melo plants in environments infested with scab, aphids and powdery mildew (PM), comprising the steps of: a. Identifying scab, aphid and PM resistant C. melo plants that contain in their genome (i) at least one QTL that confers resistance to Cladosporium, said QTL being present on linkage group (LG) 2 and / or linkage group 5 (LG5), (ii) at least one QTL that confers resistance to PM, said QTL being present on LG2 and / or LG5 and different from the at least one QTL in (i), and (iii) a VAT gene analog on LG5 that confers resistance to aphids; b. Growing resistant C. melo plants in said infested environment.

[0265] This method increases the yield of C. melo plants, resulting in, among other things, more marketable melons being harvested, more commercial melons being produced, or more seeds being obtained.

[0266] In a still further aspect, the present invention also relates to a method for producing a melon comprising the steps of: a) cultivating a C. melo plant of the present invention as defined above; b) allowing the plant to produce fruit; and c) harvesting the fruits of said plants, preferably at and / or before maturity.

[0267] All preferred embodiments relating to the C. melo plant are as already disclosed in the foregoing aspects of the invention.

[0268] The method may beneficially include the further step of processing said melon into a processed food product.

[0269] Throughout this application, the term "comprising" should be interpreted as embracing all specifically mentioned features as well as any additional, unspecified features. As used herein, use of the term "comprising" also discloses embodiments in which no features are present other than those specifically mentioned (i.e., "consisting of").

[0270] Seed deposit

[0271] A representative sample of seeds from the C. melo plant described in the present invention (i.e., seeds from the MTYVVC721 plant) was deposited by HM-Clause, SA, Rue Louis Saillant, ZI La Motte, BP83, 26802 Portes-les-Valence cedex, France, on December 13, 2018, under accession number 43317, in accordance with and in compliance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (the "Budapest Treaty") with the National Collection of Industrial, Food and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB21 9YA, United Kingdom.

[0272] The MTYVVC721 seed deposit is maintained by HM-Clause, SA, Rue Louis Saillant, ZI La Motte, BP83, 26802 Portes-les-Valence cedex, France. [Example]

[0273] The aim of the following experiments was to obtain a new disease resistance package useful for breeding lines and commercial hybrids, accumulating, by molecular marker analysis, two resistance QTLs (one scab resistance QTL and one PM / vat resistance QTL) derived from two wild accessions on linkage group 5, and two resistance QTLs on LG2 (one PM resistance QTL derived from a wild accession and one scab resistance QTL, identified in Charentais lines), thus accumulating a total of four resistance QTLs on the two linkage groups.

[0274] By using markers, tests including PM artificial tests, many selfing and backcrossing cycles (eight breeding cycles), and a large number of plants over several cycles (24 to over 100), the inventors were able to introduce two small chromosomal regions from wild accession genomes and successfully limit the occurrence of linkage drag associated with fruit shape and skin color.

[0275] To introduce Scab resistance into the elite breeding line, we used a wild landrace and had to discard all undesirable traits associated with this landrace. This melon, in fact, exhibits a pale flesh color and an undesirable shape with large cavities (see Figure 1D). Its Brix level is also quite low, approximately 10–11°B at full maturity, and the flesh becomes very soft after the climacteric crisis. We were able to recover internal quality, shelf life potential, and fruit appearance and shape from the Charentais elite line. Furthermore, the use of flanking markers linked to two identified QTLs on two linkage group (LG) carriers (LG2 and LG5) and random markers on non-carrier LGs, as well as artificial pathology testing of backcross plants, allowed for the recovery of the Charentais background.

[0276] For each backcross or selfing cycle, we manage 100-300 plants, using these different tools, plus fruit evaluation to discard undesirable ones (elongated shape, soft, pale flesh, gray / green skin that turns orange at maturity, etc.), and retain those with the most desirable Charentais elite lineage characteristics.

[0277] Using flanking markers and a large number of plants screened with Cladosporium tests and marker analysis, the inventors introduced small genomic fragments from wild accessions and avoided undesirable traits from these melon accessions (such as cavity size, flesh firmness, flesh color) and necrotic symptoms that have been described in the art as being associated with PM and Scab resistance.

[0278] Materials and Methods

[0279] Cladosporium test (or Scab test)

[0280] The Cladosporium cucumerium strain used in the study was stored at -80°C. The inoculum is prepared by culturing the fungus for 14 days before inoculation.

[0281] For each line or genotype tested, 20 different seeds / plants are tested, as well as two susceptible and two resistant controls.

[0282] The test is performed on plants at the one-leaf stage of growth (i.e., approximately 10-14 days after sowing). For inoculation, conidia are suspended in water and filtered to reach a concentration of 10-10 conidia per ml. Inoculation is performed by spraying the inoculum onto the leaves of the plants to be tested. The plants are then cultured for the first few days under saturated humidity conditions, equivalent to 18°C ​​night / 22°C day with 14 hours of sunlight.

[0283] The Scab test results are read on the seventh day after inoculation (first reading), and the second reading is taken five days after the first reading on a 1-9 scoring scale of symptoms, as follows:

[0284] [Table 3]

[0285] The disease index (DI) of a plant population is calculated based on the resistance scores of individual plants as follows:

[0286] DI = [(0 × number of plants with a resistance score of 1) + (3 × number of plants with a resistance score of 3) + (5 × number of plants with a resistance score of 5) + (7 × number of plants with a resistance score of 7) + (9 × number of plants with a resistance score of 9)] / (9 × total number of plants).

[0287] If DI = 1, all plants are resistant.

[0288] If DI = 0, all plants are susceptible.

[0289] 1.2. Powdery mildew test

[0290] First test: Leaf disc assay:

[0291] Various fungi can cause powdery mildew on melon, including Podosphaera xantii (Px) and Golobinomyces sicoracearum var. sicoracearum (Gc). To the extent that these fungi are obligate fungi, they have been maintained on susceptible zucchini and melon plants, namely, the Tosca and Edisto cultivars, respectively.

[0292] The test is carried out on plants at the one-leaf stage of growth, when the second leaf has emerged (ie, approximately 11-13 days after sowing).

[0293] For each line or genotype tested, 24 different seeds / plants are tested as well as eight different controls ranging from more susceptible to more resistant: Vedrantais, Nantais oblong, PMR 45, WMR 29, Edisto 47, PMR 5, PI 124112, MR1 and PM1.

[0294] For inoculation, conidia from zucchini plants are suspended in water to reach a concentration of 104-105 conidia per ml. Inoculation is performed by spraying the inoculum onto the leaves of the plants to be tested. A second inoculation is also performed three days after the first inoculation by dusting the leaves with the inoculum.

[0295] Result reading: The reading is taken 10 days after inoculation. The second reading is taken 5-6 days after the first reading.

[0296] Symptoms are graded as follows (resistance score):

[0297] [Table 4]

[0298] Improved Test Cut Leaf Test:

[0299] To improve the speed of the PM test, a second test was designed and applied to detached leaves. In this test, the progeny of 15 different plants under test were tested simultaneously, with two replicates per plant (R1 and R2) and two lectures per plant (Lect 1 and Lect 2). One or two control plants were tested, with two replicates per plant (R1 and R2) and two lectures per plant (Lect 1 and Lect 2). Isolated Podosphaera xantii progeny (Px1, Px2, Px3, Px5, Px3-5) and Goloivinomyces sicolacerum progeny 1 (GC) were inoculated by spraying each progeny onto leaf discs spread on agar plates. The occurrence of sporulation in each disc was measured twice: Read 1 and Read 2 (9 and 11 days after inoculation, respectively). The scoring scale (susceptibility scoring scale) is as follows:

[0300] 0: no sporulation (resistant), 1: light sporulation in <10% of leaf discs (resistant), 3: sporulation in <30% of leaf discs (intermediate), 5: sporulation in <60% of leaf discs (intermediate), 7: sporulation in >60% of leaf discs (susceptible), 9: sporulation in the entire disc (susceptible).

[0301] Of note, the susceptibility scores determined according to this test are adjusted to fit the following relationship:

[0302] "Susceptibility score" = 9 - "Resistance score"

[0303] 1.3. Necrosis Test:

[0304] The incidence of necrosis was tested in a humid greenhouse and plants were classified as showing necrosis or not.

[0305] "Zym gene-associated necrotic phenotype" refers to a necrotic phenotype as described in Pitrat and Lecoq, 1984, Euphytica, 33(1):57-61. More specifically, such a necrotic phenotype corresponds to the appearance of necrotic spots in the leaf epidermis, which gradually spread on the leaf and may cause the leaf or stem to dry out completely.

[0306] Virus isolation and maintenance:

[0307] The necrosis phenotype was assessed by inoculating melon plants with an isolate of Zucchini Yellow Mosaic Potyvirus (ZYMV) and storing them in plastic bags at −80°C or on dry infected leaves at +4°C. The inoculum is propagated on melon plantlets following a mechanical inoculation process.

[0308] Mechanical inoculation:

[0309] The inoculum was prepared by crushing 1 g of infected leaves with 4 ml of 0.03 M NaHPO buffer containing 0.2% sodium diethyldithiocarbamate, carborundum (7.5%), and activated charcoal (10%). Seedlings with the first expanded leaves were inoculated on both cotyledons.

[0310] The trials were conducted in a growth chamber under a photoperiod of 14 hours at 24°C during the day and 10 hours at 20°C at night.

[0311] Symptom evaluation

[0312] Evaluation is performed when susceptible controls show symptoms (approximately 10-14 days after inoculation).

[0313] One "necrotic" control (with the Fn gene); one "mosaic" control (without the gene) and a resistant control are included in the test to validate the test and check the virulence of the virus.

[0314] The rating is made on the leaves according to an ordinal scale: 1: death of the plant, complete wilting of the plant; 3: obvious clearing of the veins; 5: mild mosaic; 7: light chlorosis spots on young leaves; 9: no symptoms at all.

[0315] A second scoring can be done a few days later to monitor the progression of symptoms.

[0316] 1.4. Melon accessions and lines

[0317] Charentais elite line C (Figure 1B) belongs to the species Cucumis melo L. subsp. melo. This elite line is susceptible to P. xanthii races Px-1, Px-2, Px-3, Px-5, and Px3-5 and has deep orange, firm flesh at maturity, dark green sutures, a smooth yellow skin, good shelf life, and a high sugar content (15–16° Brix).

[0318] Charentais Elite Line A (Figure 1A) belongs to the species Cucumis melo L. subsp. melo. This elite line is susceptible to C. cucumerinum and resistant to powdery mildew, and has orange, firm flesh at maturity, dark green sutures, little or no yellow, thinly reticulated skin, good shelf life, and high sugar levels (15-16° Brix).

[0319] Charentais Hybrid B belongs to the species Cucumis melo L. subsp. melo. This hybrid has high internal quality (bright orange flesh with a high Brix (15-16°)), a denser mesh, early maturity, and a good storage life.

[0320] Melon accession PM1 (Figure 1E) belongs to the species Cucumis melo sp. Momordica. This accession is resistant to P. xanthii races Px-1, Px-2, Px-3, Px-5, and Px3-5 and has pale, soft flesh at maturity, large cavities, a very yellow / orange skin at maturity, and a low sugar content (10°Brix).

[0321] Melon accession SC1 (Figure 1D) belongs to the species Cucumis melo L. subsp. melo. This landrace is resistant to C. cucumerinum and, at maturity, has pale-orange color and soft flesh, a large cavity, a ribbed shape, a very yellow skin, and a low sugar content (10°Brix).

[0322] Charentais elite line F (Figure 1C) belongs to the species Cucumis melo L. subsp. melo. This elite line is susceptible to SCAB and PM. It is a hermaphrodite line, rounded, with a net-like structure and bright, dark green sutures. It has medium internal quality, orange flesh, medium firmness, an average BRIX of 12–14°C, and is classified as an early-ripening line with yellow skin.

[0323] 1.5. DNA Extraction and Genotyping Protocol

[0324] DNA purification

[0325] Automated genomic DNA isolation was performed on primary young leaf tissue using the NucleoMag® 96 plant kit (Macherey Nagel) according to the manufacturer's instructions.

[0326] SSR genotyping was performed according to methods well known to those skilled in the art, and suitable primers are disclosed in Table E below.

[0327] SNP genotyping was also performed according to well-known methods, in particular allele determination by KASPar assay (KBioscience competitive allele-specific polymerase chain reaction assay). This SNP genotyping assay from KBioscience is based on competitive allele-specific PCR (one primer per allele) and FRET (fluorescence resonance energy transfer), which allows for the detection of SNPs without the need for a separation step.

[0328] For each SNP, a KASPar mix containing two competitive allele-specific forward primers and one common reverse primer is prepared. The KASP assay mix is ​​specific to the target SNP. The KASP assay mix is ​​mixed with the DNA sample to be tested and a buffer solution optimized for the KASP Maser mix® containing a universal FRET cassette, ROX™ passive reference dye, Taq polymerase, free nucleotides, and MgCl2.

[0329] The PCR cycle was as follows: Step 1: Denaturation: 94°C for 15 minutes Step 2: 94 °C for 20 s, 10 cycles of 65–57 °C (−0.8 °C per cycle) for 60 s Step 3: 35 cycles of 94°C for 20 seconds and 57°C for 60 seconds Step 4: 15°C

[0330] Allele fluorescence was detected with a Pherastar Plate Reader, and parsing into FAM / VIC alleles was performed with KlusterCaller software with predefined cluster assignment parameters.

[0331] 2. Marker-assisted introduction of powdery mildew resistance with the PM1 accession in Charentais melon

[0332] A breeding program using QTLs was initiated to develop melon lines with high levels of resistance to different races of P. xanthii. To avoid genetic drift in the melon line by introducing a QTL from melon accession PM1 (Figure 1E), molecular markers were used to introduce the resistance QTL and break linkage to undesirable agronomic traits known to be associated with the QTL conferring powdery mildew (PM) resistance (particularly pale, soft flesh at maturity, large cavities, yellow / orange skin at maturity, and low sugar content (<10°Brix)).

[0333] Charentais elite line C (Figure 1B) was crossed with melon accession PM1, a Cucumis melo sp. Momordica plant known to be resistant but possessing undesirable agronomic traits. The resulting F1 seeds were germinated, plants were grown from the germinated seeds, and the resulting plants were self-cultivated to produce F2 seeds / plants for further selection and breeding. 300 F2 plants were submitted for PM leaf disc assays using the local isolate Px3-5. Among the resistant plants, F2 plants were selected using a set of approximately 50 marker microsatellites (SSRs). These markers, centered on LG2 and LG5, were known in the literature to harbor powdery mildew resistance QTLs in Cucumis melo sp. Momordica. F2 plants that retained QTLs on LG2 and LG5 potentially associated with PM resistance at the homozygous stage were selected. Six F2 plants out of 300 were selected and backcrossed with Charentais elite line C to obtain BC1 seeds / plants. At this heterozygosity level, no PM pressure was performed. Only phenotypic evaluation of the BC1 plants was performed. The fruits of the BC1 plants were observed internally and externally. Plants with clear skin color, as regularly reticulated as possible, round or not extremely oblong, and firm, colorful, sweet, and opaque fruits were retained after storage at 12°C for 5 days and at room temperature for 2 days after opening.

[0334] A similar approach has been adopted in most of the following selection stages, where plants are selected primarily on the basis of their agronomic characteristics, and plants that exhibit necrotrophic factors are eliminated.

[0335] Furthermore, genotyping with approximately 50 microsatellites (SSRs) allowed the calculation of the percentage of the repeat genome on these BC1s and the size of the introduced fragments. A population of BC1 plants was selected for its smaller introduced fragments, distinct bark and bright, dark green lines, deep orange flesh, firm, sweet taste, and slightly elongated shape. BC1 plants from this population were selfed to produce F2BC1 seeds / plants. 236 F2BC1 plants were submitted to a PM test using the local isolate Px 3-5. Next, 67 resistant plants were screened with SSRs to select plants with shorter QTL / introgressions from PM1 on LG2 and LG5 at the homozygous or heterozygous stage. Five F2BC1 plants were selected from the 236 F2BC1 plants and selfed to produce F3BC1 seeds / plants or backcrossed with Charentais elite line C to obtain BC2 seeds / plants. The F3BC1 progeny were then subjected to PM individual races Px1 / 2 / 3 / 5 / 3-5 by leaf disc assay to verify the broad spectrum of the selected plants. All plants tested had good levels of resistance to all these races.

[0336] To retain the shorter PM1 fragment on the carrier linkage group (LG) and the more highly repeated genome on the non-carrier linkage group, 170 BC2 plants were screened with the same SSRs. From the 170 BC2 plants, 15 BC2 plants were selected and selfed to produce F2BC2 seeds / plants. The F2BC2 plants were selfed to produce F3BC2 seeds / plants that were backcrossed with a Charentais elite line to produce BC3 seeds / plants. The BC3 plants were selfed to produce F2BC3 seeds / plants that were selfed to produce F3BC3 seeds / plants. The F3BC3 seeds / plants were again selfed to produce F4BC3 seeds / plants.

[0337] After artificial PM leaf disc assays using individual races Px1 / 2 / 3 / 5 / 3-5, 33 F4BC3 progeny were analyzed for three SSRs on each carrier LG (CMAG36, NR39, and CMBR120 for LG2, and CNTAAN128, NR2, and LG5-M2 for LG5). The analysis demonstrated that the PM1-derived resistance QTL was indeed located on LG2 and LG5 and had been fixed by a breeding scheme based essentially on phenotypic selection. F4BC3 plants were selected and crossed with line F (Figure 1C) to produce BC4 seeds / plants. The BC4 plants were selfed four times to produce F5 plants that could be used as donor lines for the PM resistance QTL on LG2 and LG5. However, at this stage, linkage to the necrotic factor on LG2 still exists, making the plant susceptible to excessive necrosis due to contact with water droplets.

[0338] 3. Marker-assisted introduction of scab resistance from the SC1 landrace of Charentais melon

[0339] To develop melon lines highly resistant to C. cucumerinum, we initiated a breeding program using QTLs present in wild species. To avoid genetic drift in the wild melon lines by introducing the QTL from melon accession SC1 (Figure 1D), we used molecular markers to introduce the resistance QTL and break linkage to undesirable agronomic traits associated with resistance, particularly pale orange and soft flesh at maturity, large cavities, yellow / orange skin at maturity, and low sugar content (10°Brix).

[0340] Charentais elite line A (Figure 1A) was crossed with melon accession SC1. The resulting F1 seeds were germinated, plants were grown from the germinated seeds, and the resulting plants were backcrossed with Charentais elite line A to produce BC1 seeds / plants for further selection and breeding. Ninety-six BC1 plants were deposited in the Scab study (see Example 1.1). Among the resistant plants, a set of 36 molecular markers spanning the genome and capable of distinguishing genomic sequences from both sources was used to select BC1 plants with the highest elite genome ratio, i.e., plants with the least amount of genomic sequence introduced from SC1. Of the dozen selected BC1 plants, the best three were selected based on fruit evaluation and selfed to produce BC1l1 seeds / plants. Twenty-four BC1l1 plants were deposited in the Scab study, and the resulting data, related to phenotypic evaluation of the BC1 plants and fruit, allowed for the selection of the best two to three BC1 plants and their successor BC2 progeny. The best two to three BC1 plants were backcrossed with Charentais elite line A to produce BC2 seeds / plants. Ninety-six BC2 plants were submitted for Scab testing. Among the resistant plants, the BC2 plants with the highest elite genome ratio were selected using the same set of 36 molecular markers. The selected BC2 plants were selfed to produce BC2l1 seeds / plants. The process continued with one additional backcross, with significant selection pressure applied to agronomic traits of plants selected for further steps, particularly fruit morphology, flesh color and firmness, cavity size, and bark color at maturity. Furthermore, in a third backcross, alternate hybrid lines were tested as recurrent parents to promote diversification of Scab resistance while also introducing earliness, netting, and internal flesh quality. Two additional selfings were performed, maintaining selection pressure for scab resistance and agronomic traits. One particular line was maintained for combination with material carrying the introduced QTL from PM1 that confers powdery mildew resistance selected in Example 2.

[0341] 4. Combination of powdery mildew resistance by the PM1 accession and scab resistance by the SC1 accession in Charentais melon

[0342] F2 plants were generated from a cross between one line from the PM1 accession breeding scheme (see Example 2) and one line from the SC1 breeding scheme (see Example 3). 400 F2 plants were deposited in the SCAB trial and analyzed using markers on LG5 to identify potential recombination events that could potentially break the linkage to the sequence presumed to be responsible for the poor fruit quality found on LG5. Nine F2 plants were retained in the SCAB trial based on potential recombination events and their behavior. Selection continued in the F3 progeny, and 200 F3 plants / progeny were deposited through the SCAB trial. Approximately 50% of the plants were identified as resistant using markers on LG5 and LG2. 13-16 F3 plants per progeny were retained. Within each family, each F3 plant was evaluated for external and internal fruit quality, necrotic behavior, and PM levels in the greenhouse. The F4 progeny of the retained F3 plants were tested in race-to-race PM and ZYMV tests. The results obtained by the present inventors showed that the necrotic response of F3 PM-resistant plants is closely linked to the ZYMV locus. F3 plants with low necrotic activity are more susceptible to ZYMV or segregation. Conversely, all ZYMV-resistant plants are actually necrotic.

[0343] The selection scheme continued with two specific F4 progeny (one susceptible to ZYMV (progeny 1607 / 003) and one susceptible to segregation (progeny 1607 / 007) respectively). PM testing revealed the same necrosis pattern, i.e., slightly higher levels in the second test (see Table A) with the absence of a necrosis pattern. F5 progeny no. 570 (issued from F4 1607 / 007) was selected for further testing for Scab resistance. Specifically, 20 plants of F5 progeny no. 570 (derived from F4 1607 / 003) were tested, as well as two resistant and two susceptible controls.

[0344] The results are reported in Table B below and show very low disease indices for these plants in the SCAB test.

[0345] Seeds of this F5 progeny, designated MTYVVC721, obtained by self-pollination and resistant to PM and SCAB, were deposited at NCIMB on December 13, 2018, under accession number NCIMB 43317. These plants (see example plant in Figure 1F) have essentially identical phenotypic characteristics to those of commercial melon lines (see example plants in Figures 1A, 1B, and 1C).

[0346] [Table 5]

[0347] The susceptibility scores obtained by the PM detached leaf test (see Example 1.2) were applied to 15 plants of two progenies, 1607 / 003 and 16 / 007, as well as to resistant controls, i.e., PM1 transgenic partners, PMR5 (two plants, controls for several strains of Podosphaera xantii), and susceptible controls, i.e., Vedrantais and Nantais oblongata. Plants known to be resistant to at least one strain were also tested (Edisto 47, PMR45). ND=Undetermined.

[0348] [Table 6]

[0349] The disease index is calculated as follows: DI = [(0 × number of plants with a resistance score of 1) + (3 × number of plants with a resistance score of 3) + (5 × number of plants with a resistance score of 5) + (7 × number of plants with a resistance score of 7) + (9 × number of plants with a resistance score of 9)] / (9 × total number of plants).

[0350] These results confirm that the seeds deposited at NCIMB are resistant to PM and SCAB. Resistance to aphids provided by the Vat gene is also confirmed. These plants also have commercially acceptable fruit quality and morphology (see Figure 1F) and do not have the necrotic phenotype associated with the Zym gene.

[0351] 5. Development of specific markers

[0352] Using a genome-scanning approach, over 4,500 SNP markers, evenly distributed along the genome and fully covering 12 melon chromosomes (corresponding to 12 linkage groups), were genotyped on a line panel. This panel included ZYMV-resistant, PM-resistant, SCAB-resistant, and susceptible lines, as well as the initial SCAB and PM resistance donors, PM1 and SC1, the first susceptible lines in the breeding process, elite lines A and C, and the final breeding line (MTYVVC721 and its progeny) that pyramids the SCAB and PM QTLs without affecting fruit quality. Data analysis allowed the identification of specific marker haplotypes for the SCAB resistance QTLs on LG2 and LG5, and the PM resistance QTLs on LG2 and LG5.

[0353] For the SCAB resistance QTL on LG2 and LG5 and the PM resistance QTL on LG2 and LG5, the flanking markers and additional markers within the region bounded by these flanking markers are reported in Table C. Furthermore, the positions of the markers on the melon (DHL92) version 3.6.1 (Garcia-Mas et al., 2012) genome assembly available at the following address: http: / / cucurbitgenomics.org / organism / 18 are also reported in Table C. The positions of the markers relative to some of the markers disclosed in Diaz et al., 2011 are also illustrated in Figure 2 (LG2) and Figure 3 (LG5).

[0354] Furthermore, by tracing the ZYMV gene, known to be near the PM resistance QTL on LG2, the inventors confirmed that this gene was absent in plants with genomes corresponding to the deposited seeds. Thus, the inventors confirmed at the molecular level that the recombination event observed at the phenotypic level, i.e., loss of the ZYMV gene through recombination, results in plants without necrotic symptoms. The markers used to detect the presence or absence of the ZYMV gene, as well as their physical locations on the same version of the melon genome, are also shown in Table C. SSR and SNP genotyping was performed as detailed in Example 1.

[0355] Table D details, for the SNP markers in Table C, the polymorphisms themselves, the alleles associated with the presence of the resistance QTL, the sequences surrounding the informative polymorphisms (i.e., the polymorphic nucleotide and the 3' and 5' flanking sequences), and the corresponding sequence numbers in the accompanying sequence listing.

[0356] Table E reports the forward and reverse primers used for the amplification of the microsatellites and the length of the amplified fragments for the SSR markers in Table C, as well as the presence of resistance QTLs (PM or SCAB resistance QTLs).

[0357] Table F reports the alleles found in the genotyped pedigree panel for the markers in Tables D and E.

[0358] Table G lists the primers used by the inventors to detect alleles of the SNPs disclosed in this invention.

[0359] Table C: List of markers. The columns indicate the marker name, the type of marker (SSR or SNP), the type of resistance marked, the linkage group, and the position on the melon genome version 3.6.1 on this LG of each marker (corresponding to the position of the polymorphic nucleotide of the SNP; in the case of microsatellites, the position given is relative to the sequence of the primer used for amplification), and whether the marker is a flanking marker of a resistance QTL.

[0360] [Table 7]

[0361] Table D: SNP markers. This table reports the various SNP markers (including polymorphic nucleotides) for which marker sequences were identified for different resistances. The column entitled "Polym R / S" indicates the two alleles of the polymorphic nucleotide, with the allele associated with resistance ("resistance allele") listed first and the allele representing susceptibility listed second.

[0362] [Table 8-1]

[0363] [Table 8-2]

[0364] Table E: SSR Markers This table reports the sequences of two primers that can be used to amplify the microsatellite region for the different resistances, as well as the length of the amplified fragment, an indication of the presence of a resistance QTL, and the various SSR markers identified.

[0365] [Table 9]

[0366] Table F: Genotyping of parental lines and deposited plants with respect to identified markers

[0367] [Table 10]

[0368] Table G: Primers used by the inventors for detection of SNP alleles using KASpar technology and corresponding SEQ ID NOs.

[0369] [Table 11]

[0370] 6. Genetic modification of C. melo seeds with ethyl methanesulfonate (EMS)

[0371] Seeds of C. melo plants are treated with EMS by submerging approximately 2000 seeds in an aerated solution of 0.5% (w / v) or 0.7% EMS for 24 hours at room temperature.

[0372] Approximately 1500 treated seeds per EMS dose are germinated and the resulting plants are grown, preferably in a greenhouse, for example from March to September, to produce seed.

[0373] After maturity, M2 seeds are harvested and pooled, one per variety per treatment. The resulting M2 seed pools are used as starting material to identify individual M2 seeds and plants resistant to scab, aphids, and powdery mildew (PM).

[0374] References

[0375] Burger et al., 2010, Horticultural Reviews, 36, 165-198

[0376] Diaz et al., 2011, “A consensus linkage map for molecular markers and Quantitative Trait Loci associated with economically important traits in melon (Cucumis melo L.)”. BMC Plant Biology 2011 11:111.

[0377] Dogimont et al., Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae;

[0378] Dogimont et al., 2014, The Plant Journal, 80, 993-1004

[0379] Fazza et al., 2013, Crop Breeding and Applied Biotechnology, 13:349-355

[0380] Fukino et al., 2008, Theor. Appl. Genet., 118(1):165-75

[0381] Garcia-Mas et al, 2012, PNAS 109(29):11872-11877.

[0382] Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453)

[0383] Perchepied et al., 2005, The American Phytopathological Society, Vol.95(5):556-565

[0384] Pitrat and Lecoq, 1982, Agronomie, 2:503-508

[0385] Pitrat and Lecoq, 1984, Euphytica, 33(1):57-61, US20140059712

Claims

1. 1. A Cucumis melo (C. melo) plant that is resistant to scab, aphids, and powdery mildew, comprising: - below, (i) at least one QTL conferring resistance to scab, which is identified on linkage group 2 (LG2) by the detection of markers Cm_MU45136_209, LG2-M4, and Cm_MU45398_32, and / or on linkage group 5 (LG5) by the detection of markers LG5-M1, CMCTN2, Cm_MU46579_322, and Cm_MU44050_58; (ii) at least one QTL conferring resistance to PM, identified on LG2 by detection of markers CMBR120, LG2-M1, and Cm_MU47536_461, and / or identified on LG5 by detection of markers Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3; and (iii) Vat gene analogs associated with aphid resistance on LG5, identified by a forward primer having SEQ ID NO: 48 and a reverse primer having SEQ ID NO: 49: and - have commercially acceptable fruit quality; The plant.

2. The C. melo plant of claim 1 , wherein the plant does not have any necrotic phenotype associated with the Zym gene.

3. (i) the QTL on LG2 that confers resistance to scab is located within the chromosomal region bounded by marker Cm_MU45136_209 and marker Cm_MU45398_32; and (ii) the QTL conferring resistance to scab present on LG5 is located within a chromosomal region bounded by marker LG5-M1 and marker Cm_MU44050_58; (iii) the QTL on LG2 that confers resistance to PM is located within a chromosomal region bounded by marker CMBR120 and marker Cm_MU47536_461; and (iv) the QTL conferring resistance to PM present on LG5 is located within a chromosomal region bounded by marker Cm_MU45437_855 and marker LG5-M3; The C. melo plant of claim 1.

4. 4. The C. melo plant of claim 1, wherein the one or more QTL associated with resistance to scab, aphids and powdery mildew are selected from those present in the genome of a plant of line MTYVVC721 (NCIMB accession number 43317).

5. 5. The C. melo plant of any one of claims 1 to 4, wherein the plant is a descendant of a plant of line MTYVVC721 (NCIMB accession number 43317).

6. 6. The C. melo plant of claim 1, wherein the scab disease is caused by Cladosporium cucumerinum.

7. 7. The C. melo plant of claim 1, wherein the powdery mildew is caused by Podosphaera xantii.

8. A cell of a C. melo plant according to any one of claims 1 to 7.

9. A plant part obtainable from a C. melo plant as defined in any one of claims 1 to 7.

10. 10. The plant part of claim 9, wherein the plant part is a seed, fruit, reproductive material, root, flower, rootstock or scion.

11. A seed of a C. melo plant which when grown into a plant according to any one of claims 1 to 7 results.

12. A hybrid plant of C. melo obtained by crossing a C. melo plant with a resistant plant according to any one of claims 1 to 7.

13. 1. A method for detecting and / or selecting C. melo plants that are resistant to scab, aphids and powdery mildew, comprising the steps of: (i) detecting the presence of at least one QTL conferring resistance to scab, wherein said at least one QTL is identified by detection of markers Cm_MU45136_209, LG2-M4, and Cm_MU45398_32 on linkage group 2 (LG2) and / or by detection of markers LG5-M1, CMCTN2, Cm_MU46579_322, and Cm_MU44050_58 on linkage group 5 (LG5); (ii) detecting the presence of at least one QTL conferring resistance to PM, wherein said at least one QTL is identified on LG2 by detection of markers CMBR120, LG2-M1, and Cm_MU47536_461, and / or on LG5 by detection of markers Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3; and (iii) detecting the presence of a Vat gene analog associated with aphid resistance on LG5, wherein the Vat gene analog is identified by a forward primer having SEQ ID NO: 48 and a reverse primer having SEQ ID NO: 49; and (iv) optionally detecting the absence of a necrosis phenotype associated with the Zym gene. The method comprising:

14. Use of a resistant Cucumis melo (C. melo) plant according to any one of claims 1 to 7 as a breeding partner in a breeding program to obtain C. melo plants resistant to scab, aphids and powdery mildew (PM).

15. 8. A method for increasing the number of harvestable Cucumis melo (C. melo) plants in an environment infested with scab, aphids and powdery mildew (PM), comprising growing scab, aphid and powdery mildew (PM) resistant C. melo plants as defined in any one of claims 1 to 7 in said environment.

16. 8. A method for protecting fields from the infestation and / or spread of scab, aphids and powdery mildew (PM), comprising growing a scab, aphid and powdery mildew (PM) resistant C. melo plant as defined in any one of claims 1 to 7 in an environment infested with scab, aphids and PM.

17. 8. Use of a scab, aphid and powdery mildew (PM) resistant C. melo plant as defined in any one of claims 1 to 7 for controlling field infestation by scab, aphids and PM.

18. 1. A method for improving yield of C. melo plants in environments infested with scab, aphids and powdery mildew (PM), comprising: a. (i) at least one QTL conferring resistance to Cladosporium, which is identified on linkage group 2 (LG2) by the detection of markers Cm_MU45136_209, LG2-M4, and Cm_MU45398_32, and / or on linkage group 5 (LG5) by the detection of markers LG5-M1, CMCTN2, Cm_MU46579_322, and Cm_MU44050_58; (ii) at least one QTL conferring resistance to PM, identified on LG2 by detection of markers CMBR120, LG2-M1, and Cm_MU47536_461, and / or identified on LG5 by detection of markers Cm_MU45437_855, LG5-M2, CMTAN139, and LG5-M3; and (iii) a VAT gene analogue that confers resistance to aphids on LG5, which VAT gene analogue is identified by a forward primer having SEQ ID NO: 48 and a reverse primer having SEQ ID NO: 49; Identifying scab, aphid and PM resistant C. melo plants comprising in their genomes; and b. Growing the resistant C. melo plants in the infested environment: The method comprising:

19. A container containing a C. melo plant as defined in any one of claims 1 to 7, a plant part as defined in claim 9 or 10, a seed as defined in claim 11, or a hybrid plant as defined in claim 12.

20. 1. A method for producing scab, aphid, and powdery mildew (PM) resistant C. melo plantlets or plants, comprising: i. Culturing isolated cells or tissues of the C. melo plant of any one of claims 1 to 7 in vitro to produce scab, aphid and PM resistant C. melo microplantlets; ii. Optionally, subjecting the C. melo microplantlets to further in vivo culture steps to develop scab, aphid, and PM resistant C. melo plants. The method comprising:

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