EA041455B1 - MELON PLANTS RESISTANT TO ToLCNDV - Google Patents

Description

The present application relates to melon plants (Cucumis melo) resistant to New Delhi tomato leaf curl virus (ToLCNDV) infection. Resistant melon plants contain an introgression genomic fragment on chromosome 5 that confers dominant resistance to

ToLCNDV. Also described are markers for identifying these fragments, methods for identifying and obtaining melon plants that are resistant to this virus.

New Delhi tomato leaf curl virus (ToLCNDV) is classified as a virus in the Begomovirus genus belonging to the Geminiviridae family. ToLCNDV has a two-component genome consisting of two double-stranded DNA molecules referred to as DNA A and DNA B (Saez et al., 2016, Annals of Applied Biology).

Initially, in 1995, ToLCNDV was found in India to infect tomato (Solarium lycopersicum) plants. It was then found that ToLCNDV also infects other Solanacea plants such as Solarium melongena (eggplant), chili pepper (Capsicum genus) and Solarium tuberosum (potato). In 2012, ToLCNDV-infected gourds (zucchini, Cucurbita rero cultivar giromontiina) were detected in Spain, and in 2015 the virus was identified as the source of the disease in melon, cucumber and zucchini plants in Tunisia. At the same time, the virus has been shown to infect many plants of the genus Curcubitacea, such as Benincasa hispida (wax gourd), Citrullus lanatus (watermelon), Cucumis melo (melon), Cucumis melo, cultivar flexuosus (serpentine melon), Cucumis sativus (cucumber ), Cucurbita moschata (nutmeg pumpkin), Cucurbita pepo (common pumpkin), Cucurbita rero, giromontiina variety (zucchini), Lagenaria siceraria (common gourd), Luffa cylindrica (bast gourd), Momordica charantia (Chinese bitter gourd). Weeds (eg Eclipta prostrata - family Asteraceae) and other cultivated plants such as Hibiscus cannabinus (kenaf - family Malvaceae) and Carica papaya (papaya - family Caricaceae) have also been described. In the Mediterranean region, the disease occurs in various types of crops in Italy (Sicily), Spain and Tunisia. In Asian countries, the infection has been found in various cultures in Bangladesh, India, Indonesia, Pakistan, Philippines, Sri Lanka, Taiwan and Thailand. No additional information is available on the geographic distribution of ToLCNDV, however, based on current observations, it is clear that the virus is further spreading both geographically and to other crops. Symptoms of the disease in general include the phenotypic appearance of a yellow mosaic on the leaves, leaf curl, swelling of the veins, and stunted plants. In melons and gourds, when young plants are infected with ToLCNDV, slow growth and a decrease or decrease in the number of fruits are manifested. Fruits with skin roughness and longitudinal cracking have also been reported. Thus, ToLCNDV causes economically significant yield losses in various important crop species and poses a serious threat. Infection of plants with ToLCNDV occurs continuously through virus transmission by the phloem-feeding whitefly (Bemisia tabaci) (European and Mediterranean Plant Protection Organization, EPPO RS 2015/114, 2016/024, 2016/040, date of entry: 2015-06).

In bast gourd, resistance to ToLCNDV has been shown to be controlled by a single dominant gene (Islam et al., 2010, Euphytica, 174(1):83-89).

Transgenic tomato plants with resistance to ToLCNDV have been obtained by gene silencing of the virus (Varma & Praveen, 2006, ISB News Report).

Methods have been developed to transmit ToLCNDV by mechanical transfer of the sap of an infected zucchini plant to uninfected plants from other genera of cucurbits (Cucumis, Cucurbita, Citrullus, Lagenaria). Five specimens of Cucumis melo have been identified (subsp. agrestis cv. momordica: Mom-KhaInd/ Kharbuja, Mom-PI124Ind/ PI 124112, Mom-PI124Ind/ PI 414723 and subsp. agrestis wt.: Ag-WM9Ind/WM9, Ag-WM7Ind/WM7) , which are resistant to ToLCNDV (Lopez et al., 2015, Euphytica, 204(3), 679-691). Confirmation of the resistance of these accessions to natural whitefly infestation was not performed.

In Saez et al., 2016, p. 214-216 (Proceedings of Cucurbitaeceae, 2016, XI EUCARPIA (European Plant Research Association) Meeting on Genetics and Reproduction of Cucurbitaceae, 24-28 July 2016, Warsaw, Poland) described that the ratio of resistant susceptible plants in the F2 population, obtained from a WM-7 sample of C. melo subsp. agrestis with resistance to ToLCNDV is 3:1, indicating the presence of a single dominant resistance gene present in WM-7. As in Lopez et al., 2015, above, mechanical inoculation was used rather than the natural infection route via the whitefly.

Various species of the genus Cucurbita (C. rero, C. moschata, C. maxima, C. fraternal, C. ficifolia) were screened for resistance to ToLCNDV by screening for mechanical sap transfer. Four specimens of Curcubita moschata (PI 604506, PI 381814, Nigerian local, Kurokawa) were found to show reduced symptoms after mechanical infection with ToLCNDV. With whitefly infestation, however, this result can only be replicated for two accessions (PI 604506, PI 381814), indicating that resistance to ToLCNDV should be tested not only in vitro, but additionally using the natural whitefly infection system (Saez et al ., 2016, Annals of Applied Biology). Thus, it is not clear

- 1 041455 whether specimens identified as resistant to ToLCNDV infection are resistant to natural growing conditions where the virus is carried by whiteflies.

Attempts to control crop ToLCNDV infection include vector control (whitefly) with insecticides and the introduction of cultural practices, including the use of virus-free material (plant transplantation), the allocation of bare fallow and fallow areas, weed control (elimination of virus-infected weeds) and the destruction of infected plants in the field. However, due to the complex epidemiological factors associated with this disease, such attempts are not always effective (Saez et al., 2016, Annals of Applied Biology).

Therefore, there is a need to implement further measures aimed at reducing the incidence of ToLCNDV, preventing further spread of the virus to other geographic areas and spread to other types of crops. To control this disease, breeding ToLCNDV resistant varieties will be of great importance.

The aim of the present invention is to provide measures to control ToLCNDV infection in melon plants.

In accordance with the present invention, plants and plant cells of melon resistant to infections caused by ToLCNDV are described.

It is well known that ToLCNDV infects various plant species of the Curcubitaceae family, including various melon species. It is also well known that ToLCNDV is constantly transmitted from infected plants to non-infected plants via the plant-feeding pest Bemisia tabaci (whitefly). It has been shown that transfer of ToLCNDV from one crop species to other crop species or even from different weed species to crop species is possible. The whitefly can carry ToLCNDV from areas where there are no control measures for the disease, from various plant species to melon plants growing in the area where control measures are taken for this disease. Therefore, control of the vector (whitefly) has some (but limited) effectiveness as a measure to prevent infection with ToLCNDV. ToLCNDV-resistant melon plants have the advantage that they do not become infected with ToLCNDV, and there is no significant yield loss, even if plants growing in adjacent areas are infected with ToLCNDV.

The inventors identified a donor melon specimen with ToLCNDV resistance and mapped resistance in a segregating population by infecting the whitefly with the Spanish ToLCNDV strain to phenotype resistance. A quantitative trait locus (QTL) was identified on chromosome 5 (QTL5) and introgressed from a wild donor sample into an elite cultivated melon line. Seeds of the elite line, including homozygous QTL5 introgression, were entered into the Nunhems B.V. database. under the accession number NCIMB 42585 in accordance with the Budapest Treaty. The donor genotype of single nucleotide polymorphism (SNP) markers (SNP_01-SNP_06) provided in accordance with the present invention is present in these seeds. The donor itself is not homogeneous and has a white fruit pulp with a low solids content. This specimen has no agronomic value. The donor is also no longer in the American seed fund. By identifying and transferring QTL5 from a donor to cultivated melon, it became possible to obtain melon cultivars and varieties of high agronomic value (with uniform characteristics and with fruits of normal market quality, having a high solids content and high keeping quality), with resistance to ToLCNDV; thus, it is possible to grow these melon varieties in ToLCNDV-infested areas without yield loss.

In the context of the present invention, the terms melon plant cells or melon plants, which may also be referred to in the art as cantaloupe plant cells or cantaloupe plants, mean cells derived from plants of the Cucumis melo species or plants belonging to the Cucumis melo species.

The species Cucumis melo can be divided into the following subspecies: C. melo cantalupensis, C. melo inodorous and C. melo reticulatus. C. melo cantalupensis is also called the cantaloupe and is generally round in shape with prominent ribs and almost no reticulation. Most of the fruits have a sweet orange-colored flesh, as a rule, the fruits have a strong, fragrant smell. Unlike the European cantaloupe, the North American cantaloupe is not of this type, but it is a true cantaloupe. C. melo inodorous (or winter melons) are classified into different types such as white melon, pielle de sapo, sugar melon, Japanese melon, etc. C. melo reticulatus is a true muskmelon with a netted skin (net-shell), this subspecies includes Galia melons, Charlene melons, and North American cantaloupe.

The melon and its wild relatives are diploid plants and have 12 pairs of homologous chromosomes, which are numbered from 1 to 12.

In the context of the present invention, the terms resistant or having resistance refer to a plant that is a host species of a specific pathogen and therefore can be infected by this pathogen, however, the plant contains a genetic element (for example,

- 2 041455 fragment of introgression), leading to a decrease in the development and/or spread of the pathogen in the plant after infection compared with a plant susceptible to such a pathogen, in which such a genetic element is absent. In the context of the present invention, the terms resistant or resistant specifically refer to plants or plant cells resistant to ToLCNDV. Resistance is a relative term that can cover a range of (different) reactions caused by a pathogenic infection in a plant or plant cell. The effect of these reactions in a plant or plant cell can be measured in various ways. Typically, the effect is measured by determining the level of symptoms present in the plant or plant cell. Typically, the average symptom severity of several plants in a line (e.g., 10 or more plants) is compared with the average symptom severity of a control line or variety, preferably a control line or variety susceptible to that pathogen. Thus, at a given point in time, at least 10 or more individual plants of a line or variety are evaluated and scored, and an average disease activity index is calculated.

In the context of the present invention, in accordance with phenotypic observations made after infection with ToLCNDV, the following commonly known symptom levels apply.

= Dead plant.

= Extremely severe mosaic and leaf curling, chlorosis and reduced growth. The plant cannot recover.

= Severe mosaic and leaf curling, chlorosis and reduced growth. The plant cannot recover.

= Coagulation and mosaic, chlorosis, little or no growth loss observed. The plant cannot recover.

= Coagulation and mosaic, chlorosis, growth reduction is not observed. There is a slight recovery of the upper part of the plant.

= Slight clotting, mosaic and chlorosis, no growth loss observed. The recovery of the upper and middle parts of the plant is observed.

= Slight clotting, mosaic and chlorosis, no growth loss observed. Symptoms are observed only in the lower part of the plant.

= Weak mosaic.

= No symptoms.

Preferably, young plants are infected with ToLCNDV to determine the level of symptom severity (or disease activity index). Such young plants are preferably plants of first true leaf growth age, preferably about 12-15 days old after sowing. Infection is preferably carried out through the vector of the virus (Bemisia), which eats plants. To do this, the germination and cultivation of plants is carried out under optimal conditions or under conditions close to optimal. The level of symptom severity is preferably determined at least once, for example, 30 days after infection (or later, for example, 31, 32, 33, 34, 35 days after infection). If necessary, to confirm the result, the level of severity of symptoms is determined twice or even three times at different time points after infection, for example, the first assessment is made approximately 15, 20 or 25 days after infection with ToLCNDV, and the second assessment is made approximately 30 days after infection (or later). e.g. 31, 32, 33, 34, 35 days after infection). See also the Examples section. In one aspect, a plant line is said to be resistant to ToLCNDV infection if its mean disease activity index is 5.0 or higher, while a susceptible control line or cultivar, such as cultivar Gandalf (or Gandalf F1, Nunhems B.V.) or Vedantrais has an average disease activity index 2.5 or less, or 2.0 or less when grown under the same conditions and infected in the same way.

It has been observed that introgression of a specific fragment located on chromosome 5 in a wild melon donor plant into cultivated melon plants confers resistance to ToLCNDV infection in melon culture plants or cells derived from them. To confer resistance to ToLCNDV, it is sufficient that the corresponding fragment be present only in the heterozygous state, indicating that the fragment confers dominant resistance to ToLCNDV infection. Single nucleotide polymorphisms (SNPs) have been identified on chromosome 5 that are closely related to the fragment of chromosome 5 conferring resistance to ToLCNDV. In the deposited seeds, the SNP nucleotide of the resistant donor (i.e., the nucleotide of the introgression fragment) is present in the homozygous form, i.e. the donor nucleotide is present in homozygous form for SNP_01-SNP_06 (linked to QTL5). Therefore, the SNP can be used to test for the presence of an introgression fragment containing QTL5 in a cell, tissue, plant part, and/or marker assisted selection (SPM) for QTL transfer to other elite melon lines or cultivars. SNPs can also be used to select for plants containing smaller introgression fragments compared to those present in deposited seeds, such smaller subfragments still containing QTLs. Alternatively, SNPs can be used to identify other donors which include

QTL5, and to introduce these QTLs into cultivated melon.

Thus, the present invention relates to melon plants or plant cells containing an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment contains a donor plant sequence between SNP_01 and SNP_06, preferably a donor plant sequence between SNP_01 and SNP_05, more preferably a donor plant sequence between SNP_01 and SNP_04. The QTL conferring resistance to ToLCNDV is present on the introgression fragment, which can be determined using a resistance assay as described herein.

In a preferred embodiment of the invention, the present invention relates to cultivated plants or melon plant cells containing an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, wherein the introgression fragment contains a donor plant sequence between SNP_02 and SNP_06, preferably the sequence of the donor plant between SNP_02 and SNP_05, more preferably a donor plant sequence between SNP_02 and SNP_04.

The present invention also relates to melon plants or plant cells containing an introgression fragment from chromosome 5 of a ToLCNDV-resistant donor plant, wherein the introgression fragment contains a ToLCNDV-resistant donor plant sequence between SNP_03 and SNP_06, preferably a plant- a donor with ToLCNDV resistance between SNP_03 and SNP_05.

Most preferably, the present invention relates to melon plants or plant cells comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment contains a ToLCNDV resistant melon donor plant sequence between SNP_03 and SNP_04.

In one aspect, the expression an introgression fragment containing a sequence between two SNPs (single nucleotide polymorphisms) means that one or both of the two SNPs also come from a resistant donor, i.e. have a donor nucleotide at the SNP position. In another aspect, the two SNPs are derived from a recipient, such as a non-resistance melon plant, and only the region between the two SNPs is derived from a resistant donor and confers resistance to ToLCNDV, i.e. the donor fragment conferring resistance is located between two SNP markers. For example, the plant may contain an introgression fragment containing the sequence of a ToLCNDV resistant melon donor plant between SNP_03 and SNP_04, in one aspect, this plant contains thymine (T) at nucleotide 68 of SEQ ID NO: 3 and/or adenine (A ) at nucleotide 227 of SEQ ID NO: 4, i. donor nucleotides. In another aspect, only the region (all or part thereof) between these two SNPs is from the donor, while SNP_03 and SNP_04 are from the recipient, for example, with cytosine (C) at nucleotide 68 of SEQ ID NO: 3 and nucleotide guanine (G) at nucleotide 227 of SEQ ID NO: 4.

Thus, with respect to QTLs on chromosome 5, SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06 may all have a resistant donor genotype. Or only SNP_01 and SNP_02 may have the genotype of a resistant donor; or only SNP_02 and SNP_03 may have a stable donor genotype; or only SNP_03 and SNP_04 can have the genotype of a resistant donor, etc. Or just one SNP, i.e. only SNP_01, or only SNP_02, or only SNP_03, or only SNP_04, or only SNP_05, or only SNP_06 has the genotype of a resistant donor. Thus, SNPs that do not have the genotype of the resistant donor have a different genotype, the genotype of the recipient. The recipient genotype for the SNP can be any of the other 3 nucleotides, i.e. for SNP_01, the recipient's genotype may be adenine, guanine, or thymine. Thus, for example, when it is indicated that the introgression fragment is located between SNP_03 and SNP_04 in relation to the QTL on chromosome 5, both SNP_03 and SNP_04 may have a resistant donor genotype. Or just one SNP, i.e. only SNP_03 or only SNP_04 may have a resistant donor genotype, or neither SNP_03 nor SNP_04 may have a resistant donor genotype, with the sequence between them containing QTL5.

The reason that not all SNPs according to the present invention need to be of the resistant donor genotype is that the introgression fragment containing the QTL from the donor may be smaller than the chromosome fragment introgressed into e.g. deposited seeds, but it is a fragment still contains QTL5. However, it can be determined that a plant contains an introgression fragment (containing QTL5) by phenotype and/or by transferring the fragment into a susceptible plant and hence transferring the ToLCNDV resistance phenotype or by sequencing the region between SNP markers to identify the donor fragment or by other means. methods known to those skilled in the art, such as saturating the region with more SNP markers, testing for allelism, identifying the causative gene, and so on.

Thus, a combination of methods can be used to demonstrate that QTL5 is present in a plant or plant cell, even if not all associated SNPs have a donor SNP genotype. The average ToLCNDV resistance index conferred on QTL5 is at least 5.0 when transferred to a line or cultivar that is not resistant, provided that the trait is dominant.

In a preferred embodiment of the invention, an introgression fragment from chromosome 5 of a donor plant between SNP_01 and SNP_06, between SNP_01 and SNP_05, between SNP_01 and SNP_04, between SNP_02 and SNP_06, between SNP_02 and SNP_05, between SNP_02 and SNP_04, between SNP_03 and SNP_06, between SNP_03 and SNP_05, preferably between SNP_03 and SNP_04 confers resistance to ToLCNDV to the crop plants or melon plant cells of the present invention.

Preferably, the melon plant cell of the present invention is derived from a melon crop plant, or the melon plant of the present invention is a melon crop plant.

In accordance with one aspect, the present invention relates to cultivated plants (or plant parts) or melon plant cells containing an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, wherein the introgression fragment confers resistance to ToLCNDV and the introgression fragment can be identified by donor plant SNP genotype (comprises the donor plant SNP genotype) for one or more (or all) of the following SNPs: SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06 and optionally any SNP between SNP_01 and SNP_06.

In one aspect, the plant, plant part, or plant cell contains QTL5 and contains the donor SNP genotype for at least SNP_03, as this SNP is most strongly associated with QTL5 resistance to ToLCNDV. In another aspect, the plant, plant part, or plant cell contains the donor SNP genotype for at least SNP_03 and SNP_04, or at least SNP_03 and SNP_02. Optionally, the plant, plant part or plant cell contains QTL5 and contains the donor SNP genotype for at least SNP_01, SNP_02 and SNP_03; or for SNP_02, SNP_03 and SNP_04.

Therefore, an introgression fragment may contain a donor SNP genotype for all QTL5 associated SNP markers (as in seeds deposited according to the present invention) or a smaller fragment resulting in the absence of one or more SNP markers. As described below, all donor SNP markers or all but one donor SNP marker may be absent, yet QTL5 will still be present on the introgression fragment.

The nucleotide sequences (SEQ ID NOs: 1-6) containing the SNPs of the present invention are those of a stable donor, i. they contain the donor nucleotide SNP. Therefore, in accordance with one aspect, the present invention relates to cultivated plants (or plant parts) or melon plant cells, containing an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, wherein the introgression fragment confers resistance to ToLCNDV, and the introgression fragment may be identified by (comprises) SEQ ID NO: 1 or identified by cytosine (comprises cytosine) at nucleotide 101 of SEQ ID NO: 1 or identified by cytosine (comprises cytosine) at an equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO: 1 and/or according to SEQ ID NO: 2, or according to thymine at nucleotide 945 of SEQ ID NO: 2, or according to thymine in the equivalent nucleotide of a sequence having significant sequence identity with SEQ ID NO: 2, and/or according to SEQ ID NO : 3, or at the thymine at nucleotide 68 of SEQ ID NO: 3, or at the thymine at the equivalent nucleotide of a sequence having significant identity b sequences with SEQ ID NO: 3, and/or at SEQ ID NO: 4, or at adenine at nucleotide 227 of SEQ ID NO: 4, or at adenine at an equivalent nucleotide of a sequence having significant sequence identity to SEQ ID NO: 4, and /or by SEQ ID NO: 5, or by cytosine at nucleotide 839 of SEQ ID NO: 5, or by cytosine in an equivalent nucleotide of a sequence having significant sequence identity with SEQ ID NO: 5, and/or by SEQ ID NO: 6, or adenine at nucleotide 445 of SEQ ID NO: 6, or an adenine at an equivalent nucleotide of a sequence having significant sequence identity to SEQ ID NO: 6. A QTL conferring resistance to ToLCNDV is present on the introgression fragment.

In the context of the present invention, the term donor plant cell or donor plant means a melon cell or melon plant that is resistant to ToLCNDV. Similarly, the term DNA fragment or introgression fragment from a donor plant or donor plant cell means a fragment of chromosome 5 of a melon plant that is resistant to ToLCNDV, whereby this fragment confers resistance to TOLCNDV when transferred to a melon plant that is not resistant to ToCNDV. In a preferred embodiment of the invention, the donor plant is a wild species or wild melon specimen. According to a particularly preferred embodiment of the invention, the DNA fragments or introgression fragments from donor plants or cells of donor plants are donor fragments obtained from plants grown from seeds deposited under No. NCLMB 42585, or from progeny obtained from plants grown from seed deposited under NCLMB 42585 or from plants obtained by crossing with plants grown from seeds deposited under NCLMB 42585.

Melon donor plants can be obtained from a variety of sources. Those skilled in the art are aware of methods for determining from which sources melon donor plants with ToLCNDV resistance were obtained. To identify such sources of ToLCNDV resistant donor plants, in general, melon plants can be infected with ToLCNDV, mechanically as described in Lopez et al. (2015, Euphytica, 204(3), 679-691) or by virus transmission from the whitefly. In the context of the present invention, the infection is preferably via the whitefly. Plants that exhibit reduced levels of symptoms compared to non-resistance control plants can then be selected and such plants can be used as a source of genome fragments or sequences conferring resistance to ToLCNDV. The preferred method for infecting melon plants with ToLCNDV and methods for determining the severity of symptoms of infected plants are given in this document in the General Methods section.

In the context of the present invention, the donor plants preferably have an average symptom score equal to or greater than 5.0, more preferably equal to or greater than 6.0, more preferably equal to or greater than 7.0, even more preferably equal to or greater than 8.0, and most preferably equal to or greater than 9.0. In one aspect, the donor plant contains an SNP donor genotype for one or more or all of SNP_01, SNP_2, SNP_3, SNP_04, SNP_05, and SNP_06, as shown in Table 1. 2. Preferably, the donor SNP genotype is homozygous. In accordance with the present invention, the term donor, for example, refers to a wild melon that has no agronomic value, such as a white-fleshed, low solids plant, etc.

In accordance with the present invention, the terms recurrent plant cell or recurrent plant or recipient plant means a melon plant or plant cell that is susceptible (synonymous with susceptible) or that does not have resistance to ToLCNDV infection. Whether a plant is susceptible or resistant to ToLCNDV can be determined by observing symptom levels following infection with ToLCNDV. The recurrent plant preferably has an average symptom score below 3.0, more preferably at or below 2.5, or at or below 2.0. Symptom levels and methods of infecting melon plants with ToLCNDV are described throughout this document and are applied herein accordingly. In a preferred embodiment of the invention, the cell of the recurrent melon plant of the present invention is derived from a cultivated melon plant, or the recurrent melon plant of the present invention is a cultivated melon plant. Preferably it is an elite line, breeding line or variety.

The term introgression fragment refers to a fragment of a chromosome, a portion or region of a chromosome that has been introduced into another plant of the same or related species by crossing or conventional breeding methods. As a result of the introgression of a fragment from a donor plant into a recurrent plant, the progeny obtained by crossing the donor plant and the recurrent plant acquires a phenotype that is absent in the recurrent plant. In the context of the present invention, the phenotype transferred from the donor plant to the recurrent plant is resistance to ToLCNDV, eg the average disease activity index in such a plant is 5.0. For the introgression of a fragment into a particular breeding line or variety, the first crossing step may, for example, be followed by one or more backcrosses with the intended breeding line or variety. As used throughout this document, the term introgression may refer to the first crossing of a donor plant resistant to ToLCNDV with a recurrent plant not resistant to ToLCNDV, and then backcrossing one or more plants resistant to ToLCNDV resulting from the first crossing, with recipient plants that should acquire resistance to ToLCNDV. In such a case, the introgressed fragment results from the application of breeding methods for which the term introgression is used (such as backcrossing) to the recipient variety or breeding line. Thus, the introgression of the ToLCNDV resistance trait into a recurrent plant is a technical anthropogenic process. In particular, as used herein, the term introgression refers to an artificial breeding process or method. Such a process may use one or more or all of the molecular markers (SNP markers) according to the present invention. The resulting plant, i.e. a cultural line or variety that contains one introgression fragment (on chromosome 5) from a donor, i.e. contain recombinant chromosome 5, is also artificial and does not exist in nature.

The introgression fragment may be large, e.g. it may be half a chromosome, but is preferably smaller, e.g., about 15 Mb or less, e.g., about 10 Mb or less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less than, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2 Mb or less, about 1 Mb (which is equal to 1,000,000 bases or less), or about 0.8 Mb (which is equal to 800,000 base pairs) or less.

- 6 041455

The introgression fragment may originate from a wild melon plant or wild melon specimen or wild melon relatives or landraces (donor). Wild melon plants or wild melon accessions or wild plant relatives or landraces of melon can be used to introgress the donor genome fragments into the genome of the cultivated melon Cucumis melo to create crossbreeding lines or varieties with good agronomic characteristics. Thus, such a cultivated melon plant has the genome of the cultivated plant C. melo, but contains in its genome a donor fragment, for example, an introgression fragment of a related wild genome of Cucumis genome, for example, Cucumis melo ssp. agrestis, C. melo ssp. melo, C. melo ssp. a cidulous, C. callosus, C. trigonus, C. picrocarpus or other wild melon or wild melon relative. Obviously, the term introgression fragment never includes the whole chromosome, but only part of the chromosome. Therefore, the introgression-bearing chromosomes also include a part or parts of a recurrent melon plant (recipient plant) and, in addition, parts of a melon donor plant.

When cultivated melon chromosome 5 contains an introgression fragment, this therefore means that the cultivated melon plant contains recombinant chromosome 5, whereby the introgressed fragment includes a QTL conferring resistance to ToLCNDV. As described herein, an introgression fragment from a donor may contain one or more or all SNP donor nucleotides (for SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06) or one or more or all sequences containing SNP donor nucleotides (SEQ ID NO: 1, 2, 3, 4, 5 and/or SEQ ID NO: 6).

Thus, for example, an introgression fragment (in homozygous or heterozygous form) may contain and be identified by one or more or all of the following SNP genotypes: CC or CT genotype for SNP_01 in SEQ ID NO: 1, and/or TT or TG genotype for SNP_02 in SEQ ID NO: 2, and/or TT or TC genotype for SNP_03 in SEQ ID NO: 3, and/or AA or AG genotype for SNP_04 in SEQ ID NO: 4, and/or CC or CT genotype for SNP_05 in SEQ ID NO: 5, and/or AA or AG genotype for SNP_06 in SEQ ID NO: 6.

In accordance with the present invention, in one aspect, the lower part of chromosome 5 of cultivated melon below the CMGAAN144 marker on chromosome 5 on page 3 according to Diaz et al., 2015 (Mol. Breeding, 35:188) or between the CMGAAN144 marker and the AI_13-H12 marker contains QTL5 from a donor.

In one aspect, the donor plant of the invention is not a plant from the five specimens of Cucumis melo subsp. agrestis (subsp. agrestis var. momordica: Mom-Khalnd/Kharbuja, MomPI124Ind/PI124112, Mom-PI124Ind/PI414723 and subsp. agrestis wild types: Ag- WM9Ind/WM9, AgWM7Ind/WM7) that are resistant to ToLCNDV identified by Lopez et al., 2015, Euphytica, 204(3), 679-691.

The term breeding in this document includes crossing, backcrossing, selfing, breeding, double haploid production, embryonic rescue, protoplast fusion, marker-assisted selection, mutational selection, and other methods known to the breeder (i.e., other methods that do not include genetic modification), by which, for example, the production, identification and/or transfer of recombinant chromosome 5 can be carried out.

In a preferred embodiment of the present invention, the introgression fragment is from a wild Cucumis plant or a wild Cucumis specimen, most preferably the introgression fragment is from a wild Cucumis melo ssp. melo, with small fruits (no more than 6 cm long), with white flesh of the fruit with a sour taste. This donor was used in the present invention, however, one skilled in the art will be able to identify other donors that include, for example, the same SNP genotype for SNP_01-SNP_06 and contain QTLs in the same region of chromosome 6.

In one aspect, a suitable donor is a wild plant or C. melo specimen having an average disease activity index

ToLCNDV of at least 7.0 or at least 7.1, 7.2, 7.3 or 7.4 or 7.5 or 7.6 on a scale of 1=dead plant to 9=asymptomatic that includes one or more or all of the following SNP genotypes: CC or CT genotype for SNP_01 in SEQ ID NO: 1, and/or TT or TG genotype for SNP_02 in SEQ ID NO: 2, and/or TT or TC genotype for SNP_03 in SEQ ID NO: 3, and/or AA or AG genotype for SNP_04 in SEQ ID NO: 4 and/or CC or CT genotype for SNP_05 in SEQ ID NO: 5 and/or AA or AG genotype for SNP_06 in SEQ ID NO: 6.

A plant variety is a group of plants within a single botanical taxon of the lowest known class which (whether or not the conditions for recognizing the right to protection of the created plant variety are met) can be determined based on the expression of characteristics that result from a particular genotype or combination of genotypes. , which can be distinguished from any other group of plants on the basis of the expression of at least one of these characteristics and which can be considered as a whole, because when such plants are propagated, no changes occur. Thus, the term plant variety cannot be used to refer to a group of plants, even if they are of the same genus, if they are all characterized by the presence of one or two loci or genes (or phenotypic characteristics in connection with such specific

- 7 041455 loci or genes), but otherwise may be extremely different from each other relative to other loci and genes.

F1, F2, F3, etc. refer to successive, related generations after crossing two parent plants or parental lines. Plants grown from seeds obtained by crossing two plants or lines are called the F1 generation. As a result of self-pollination of F1 plants, the F2 generation is obtained, and so on.

An F1 hybrid plant or (F1 hybrid seed) is a generation obtained by crossing two inbred parent lines. Thus, F1 hybrid seeds are seeds from which F1 hybrid plants are grown. F1 hybrids have more power, they give more yield due to heterosis. Inbred lines are predominantly homozygous at most loci in the genome.

The term plant line or cross line refers to a plant and its progeny. As used throughout this document, the term inbred line refers to a plant line that has been produced by repeated self-pollination and is substantially homozygous. Thus, the terms inbred line or parental line refer to a plant that has been inbred for several generations (eg, at least 5, 6, 7 or more generations) resulting in a plant line with high uniformity.

The term uniformity or homogeneous refers to the genetic and phenotypic characteristics of a plant line or variety. Inbred lines are genetically highly homogeneous, as they are obtained in several generations as a result of inbreeding. Similarly, F1 hybrids that are derived from such inbred lines are highly uniform in their genotypic and phenotypic characteristics and properties.

In a preferred particular embodiment of the invention, the plants and plant cells of the present invention are characterized in that the introgression fragment conferring resistance to ToLCNDV is obtained from seeds deposited under NCIMB 42585 or from their progeny.

In yet another preferred embodiment of the invention, the melon plant cell of the present invention is derived from a cultivated melon plant, or the melon plant of the present invention is a cultivated melon plant and the introgression fragment is obtained from a wild Cucumis plant or from a wild Cucumis specimen or from donor plants described herein as the preferred donor plants, or derived from seeds deposited under NCIMB 42585, or from their progeny.

In the context of the present invention, chromosome 5 of the melon plant means scaffolds, fragments, regions, markers and nucleotide sequences that, according to ICuGI (International Cucurbite Genome Initiative), belong to chromosome 5 of the melon genome.

The term orthologous chromosome 5 refers to chromosome 5 of wild melon relatives, parts of which can be introgressed into chromosome 5 of cultivated melon.

The term recombinant chromosome refers to a chromosome with a new genetic structure resulting from crossing between homologous chromosomes, for example recombinant chromosome 5 means chromosome 5 that is not present in any of the parent plants and resulted from a rare crossing over event between homologous chromosomes of chromosome pair 5. For example, according to the present invention, a recombinant melon chromosome 5 containing a QTL conferring resistance to ToLCNDV is provided. Thus, recombinant chromosome 5 is a cultivated melon chromosome with an introgression fragment from a wild donor, resulting in the introgression fragment including a QTL conferring resistance to ToLCNDV.

In this document, the term ICuGI refers to data on Cucumis melo published by the International Cucurbit Genome Initiative, which publishes genetic maps such as Cucumis melo (http://www.icugi.org/cgi-bin/cmap/mapset_info?speciesacc= CM). The current version of the C. melo genome map is CM_3.5.1 on March 4, 2012, and the chromosome 5 map is called ICuGI_V (or LG_V, or Linkage Group V). Additional information, including additional marker information and mapping information in addition to ICuCI data, can be found in Diaz et al. (2015, Mol. Breeding., 35, 188), as well as in additional data included in the online version of the corresponding article.

The term melon cultivated plant means plants of the species Cucumis melo, ie. varieties, crossing lines or cultivars of the species C. melo which are cultivated by humans and have high agronomic characteristics, in particular plants producing edible fruits of normal market quality, of good size and quality and of sufficient uniformity; such plants are not wild-type plants, ie. plants that generally have significantly lower yields and agronomic characteristics compared to cultivated plants, and which, for example, grow naturally in wild populations. Wild-type plants include, for example, ecotypes, plant introduction lines, landraces, wild specimens, or wild relatives of a species.

In the context of the present invention, the term SNP (=single nucleotide polymorphism)

- 8 041455 means a variation in a single nucleotide that occurs at a specific position in the genome. A SNP is a single nucleotide variation at a specific position in the genome of two plants. If a wild melon plant with resistance to ToLCNDV (donor plant) has a different nucleotide in the corresponding sequence at a certain single position than the corresponding nucleotide in the same position of the cultivated melon plant, this position defines the SNP between wild and cultivated melon. If the donor plant has one of the four possible nucleotides (A, C, T, or G) at a particular position, a SNP occurs if the crop plant has one of the remaining three possible nucleotides at the same corresponding position in the sequence. Therefore, in a cultivated melon plant containing an introgression fragment from a donor, it can be easily determined whether a single SNP nucleotide was obtained from a donor or from a cultivated melon (recipient).

The term SNP nucleotide refers to a single nucleotide and the term SNP genotype refers to a pair of nucleotides in a diploid plant cell. Thus, for SNP_01, the nucleotide of the ToLCNDV-resistant SNP donor SNP is cytosine (C) for nucleotide 101 of SEQ ID NO: 1, while the genotype of a plant or cell SNP containing SEQ ID NO: 1 can be CC (cytosine on both chromosomes) or CT (cytosine on one chromosome and thymine on the other chromosome), resulting in the nucleotide SNP (cytosine) of the ToLCNDV-resistant donor and therefore SEQ ID NO: 1 (or a sequence with a significant degree of identity to SEQ ID NO: 1) is homozygous or heterozygous . The term donor SNP genotype refers to a donor SNP nucleotide present in homozygous or heterozygous form, i. for SNP_01, the donor SNP genotype is CC or CT.

In the context of the present invention, the term SNP_01, which is alternatively referred to as mME11320_k, means the SNP at position 101 in SEQ ID NO: 1. According to ICuGI, SEQ ID NO: 1 or a sequence with a significant degree of identity to SEQ ID NO: 1 can be found on chromosome 5 The relative position of SNP_01 according to the markers published by the ICuGI can be inferred from Table 1. 1. Preferably, the nucleotide sequence containing SNP_01 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO: 1, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO: 1, most preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO: 1, even more preferably at least 97% identity with the nucleotide sequence shown under SEQ ID NO: 1, still most preferably at least 98 % identity with the nucleotide sequence shown under SEQ ID NO: 1, in particular preferably at least 99% identity with the nucleotide sequence shown under SEQ ID NO: 1, or more particularly preferably at least 99.5% identity with the nucleotide sequence, shown under SEQ ID NO: 1, provided that in each case tea, the nucleotide at position 101 in SEQ ID NO: 1 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1 are referred to as sequences with significant sequence identity to SEQ ID NO: 1.

ToLCNDV resistant donor plants according to the invention have C (cytosine) at position 101 in SEQ ID NO: 1 or in a sequence having significant sequence identity with SEQ ID NO: 1. According to a preferred embodiment of the invention SNP_01 characterized in that the recurrent plant has an A, G, or T at position 101 in SEQ ID NO: 1. In accordance with one aspect of the invention, the recurrent plant has a T at position 101 in SEQ ID NO: 1.

In accordance with one embodiment of the invention, SNP_01 is characterized in that the donor plant has C at position 101 in SEQ ID NO: 1 (or an equivalent position in a sequence having significant sequence identity with SEQ ID NO: 1) and the recurrent plant has T at position 101 in SEQ ID NO: 1.

In the context of the present invention, the term SNP_02, which is alternatively referred to as mME43070_k, means the SNP at position 945 in SEQ ID NO: 2. According to ICuGI, SEQ ID NO: 2 or a sequence with a significant degree of identity to SEQ ID NO: 2 can be found on chromosome 5 The relative position of SNP_02 according to the markers published by the ICuGI can be inferred from Table 1. 1. Preferably, the nucleotide sequence containing SNP_02 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO: 2, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO: 2, most preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO: 2, even more preferably at least 97% identity with the nucleotide sequence shown under SEQ ID NO: 2, still most preferably at least 98 % identity with the nucleotide sequence shown under SEQ ID NO: 2, particularly preferably at least 99% identity with the nucleotide sequence shown under SEQ ID NO: 2, or more particularly preferably at least 99.5% identity with the nucleotide- 9 041455 sequence shown under SEQ ID NO: 2, provided that in in each case, the nucleotide at position 945 in SEQ ID NO: 2 is different from the corresponding nucleotide at the same position in the recurrent plant. Such sequences having at least 85, 90, 95, 97, 98, 99 or more sequence identities to SEQ ID NO: 2 are considered to have significant sequence identity to SEQ ID NO: 2.

Donor plants with resistance to ToLCNDV according to the invention have T (thymine) at position 945 in SEQ ID NO: 2 or in a sequence having significant sequence identity with SEQ ID NO: 2.

In accordance with one embodiment of the invention, SNP_04 is characterized in that the recurrent plant has A, C, or G at position 945 in SEQ ID NO: 2, or in a sequence having significant sequence identity with SEQ ID NO: 2.

In accordance with one aspect of the invention, the recurrent plant has G (guanine) at position 945 in SEQ ID NO: 2, or in a sequence having significant sequence identity with SEQ ID NO: 2.

In accordance with a particular embodiment of the invention, SNP_02 is characterized in that the donor plant has a T at position 945 in SEQ ID NO: 2 (or in a sequence having a significant sequence identity with SEQ ID NO: 2) and the recurrent plant has a G at position 945 in SEQ ID NO: 2 (or in a sequence having significant sequence identity with SEQ ID NO: 2).

In the context of the present invention, the term SNP03, which is alternatively referred to as mME10621_k, means the SNP at position 68 in SEQ ID NO: 3. According to ICuGI, SEQ ID NO: 3 or a sequence with a significant degree of identity to SEQ ID NO: 3 can be found on chromosome 5 The relative position of SNP_03 according to markers published by ICuGI can be inferred from Table 1. 1. Preferably, the nucleotide sequence containing SNP_03 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO: 3, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO: 3, most preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO: 3, even more preferably at least 97% identity with the nucleotide sequence shown under SEQ ID NO: 3, still most preferably at least 98 % identity with the nucleotide sequence shown under SEQ ID NO: 3, particularly preferably at least 99% identity with the nucleotide sequence shown under SEQ ID NO: 3, or more particularly preferably at least 99.5% identity with the nucleotide sequence, shown under SEQ ID NO: 3, provided that in each case ae nucleotide at position 68 in SEQ ID NO: 3 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 3 are considered to have significant sequence identity to SEQ ID NO: 3.

Donor plants with resistance to ToLCNDV according to the invention have T (thymine) at position 68 in SEQ ID NO: 3 (or in a sequence having significant sequence identity with SEQ ID NO: 3). In accordance with one embodiment of the invention, SNP_03 is characterized in that the recurrent plant has A, C or G at position 68 in SEQ ID NO: 3 (or in a sequence having significant sequence identity with SEQ ID NO: 3). In accordance with one aspect of the invention, the recurrent plant has C at position 68 in SEQ ID NO: 3 (or in a sequence having significant sequence identity with SEQ ID NO: 3).

In accordance with one embodiment of the invention, SNP_03 is characterized in that the donor plant has a T at position 68 in SEQ ID NO: 3 (or in a sequence having a significant sequence identity with SEQ ID NO: 3) and the recurrent plant has a C at position 68 in SEQ ID NO: 3 (or in a sequence having significant sequence identity with SEQ ID NO: 3).

In the context of the present invention, the term SNP_04, which is alternatively referred to as mME50729_k, means the SNP at position 227 in SEQ ID NO: 4. According to ICuGI SEQ ID NO: 4 or a sequence with a significant degree of identity to SEQ ID NO: 4 can be found on chromosome 5 The relative position of SNP_04 according to the markers published by the ICuGI can be inferred from Table 1. 1. Preferably, the nucleotide sequence containing SNP_04 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO: 4, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO: 4, most preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO: 4, even more preferably at least 97% identity with the nucleotide sequence shown under SEQ ID NO: 4, still most preferably at least 98 %

- 10 041455 identity with the nucleotide sequence shown under SEQ ID NO: 4, in particular preferably at least 99% identity with the nucleotide sequence shown under SEQ ID NO: 4 or more particularly preferably at least 99.5% identity with the nucleotide the sequence shown under SEQ ID NO: 4, provided that in each case the nucleotide at position 227 in SEQ ID NO: 4 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 4 are considered to have significant sequence identity to SEQ ID NO: 4.

Donor plants with resistance to ToLCNDV according to the invention have A (adenine) at position 227 in SEQ ID NO: 4 (or in a sequence having significant sequence identity with SEQ ID NO: 4). In accordance with one embodiment of the invention, SNP_04 is characterized in that the recurrent plant has C, T or G at position 227 in SEQ ID NO: 4 (or in a sequence having significant sequence identity with SEQ ID NO: 4). In accordance with one aspect of the invention, the recurrent plant has a G at position 227 in SEQ ID NO: 4 (or in a sequence having significant sequence identity with SEQ ID NO: 4).

In accordance with one embodiment of the invention, SNP_04 is characterized in that the ToLCNDV resistant donor plant has A at position 227 in SEQ ID NO: 4 (or in a sequence having significant sequence identity to SEQ ID NO: 4), and the recurrent plant has a G at position 227 in SEQ ID NO: 4 (or in a sequence having significant sequence identity with SEQ ID NO: 4).

In the context of the present invention, the term SNP_05, which is alternatively referred to as mME32395_k, means the SNP at position 839 in SEQ ID NO: 5. According to ICuGI SEQ ID NO: 5 or a sequence with a significant degree of identity to SEQ ID NO: 5 can be found on chromosome 5 The relative position of SNP_05 according to markers published by the ICuGI can be inferred from Table 1. 1. Preferably, the nucleotide sequence containing SNP_05 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO: 5, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO: 5, most preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO: 5, even more preferably at least 97% identity with the nucleotide sequence shown under SEQ ID NO: 5, still most preferably at least 98 % identity with the nucleotide sequence shown under SEQ ID NO: 5, particularly preferably at least 99% identity with the nucleotide sequence shown under SEQ ID NO: 5 or more particularly preferably at least 99.5% identity with the nucleotide sequence shown under SEQ ID NO: 5, provided that in each case tea, the nucleotide at position 839 in SEQ ID NO: 5 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 5 are considered to have significant sequence identity to SEQ ID NO: 5.

Donor plants with resistance to ToLCNDV according to the invention have C (cytosine) at position 839 in SEQ ID NO: 5 (or in a sequence having significant sequence identity with SEQ III NO: 5). In accordance with one embodiment of the invention, SNP_05 is characterized in that the recurrent plant has A, T or G at position 839 in SEQ ID NO: 5 (or in a sequence having significant sequence identity with SEQ ID NO: 5). In accordance with one aspect of the invention, the recurrent plant has a T at position 839 in SEQ ID NO: 5 (or in a sequence having significant sequence identity with SEQ ID NO: 5).

According to one embodiment of the invention, SNP_05 is characterized in that the ToLCNDV resistant donor plant has C at position 839 in SEQ ID NO: 5 (or in a sequence having significant sequence identity to SEQ ID NO: 5), and the recurrent plant has a T at position 839 in SEQ ID NO: 5 (or in a sequence having significant sequence identity with SEQ ID NO: 5).

In the context of the present invention, the term SNP_06, which is alternatively referred to as mME49184_k, means the SNP at position 445 in SEQ ID NO: 6. According to ICuGI SEQ ID NO: 6 or a sequence with a significant degree of identity to SEQ ID NO: 6 can be found on chromosome 5 The relative position of SNP_06 according to markers published by the ICuGI can be inferred from Table 1. 1. Preferably, the nucleotide sequence containing SNP_06 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO: 6, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO: 6, most preferably at least 95% identity with the nucleotide sequence shown

- 11 041455 under SEQ ID NO: 6, even more preferably at least 97% identity with the nucleotide sequence shown under SEQ ID NO: 6, even more preferably at least 98% identity with the nucleotide sequence shown under SEQ ID NO: 6, particularly preferably at least 99% identity with the nucleotide sequence shown under SEQ ID NO: 6, or more particularly preferably at least 99.5% identity with the nucleotide sequence shown under SEQ ID NO: 6, provided that in in each case, the nucleotide at position 445 in SEQ ID NO: 6 is different from the corresponding nucleotide at the same position in the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 are considered to have significant sequence identity to SEQ ID NO: 6.

Donor plants with resistance to ToLCNDV according to the invention have A (adenine) at position 445 in SEQ ID NO: 6 (or in a sequence having significant sequence identity with SEQ ID NO: 6). In accordance with one embodiment of the invention, SNP_06 is characterized in that the recurrent plant has C, T or G at position 445 in SEQ ID NO: 6 (or in a sequence having significant sequence identity with SEQ ID NO: 6). In accordance with one aspect of the invention, the recurrent plant has a G at position 445 in SEQ ID NO: 6 (or in a sequence having significant sequence identity with SEQ ID NO: 6).

In accordance with a particular embodiment of the invention, SNP_06 is characterized in that the ToLCNDV resistant donor plant has A at position 445 in SEQ ID NO: 6 (or in a sequence having significant sequence identity with SEQ ID NO: 6) and is recurrent the plant has a G at position 445 in SEQ ID NO: 6 (or in a sequence having significant sequence identity with SEQ ID NO: 6).

Table 1

Name marker Type mark pa lame ohm Pseudomolecule coordinates cM GarciaMas et al. (2012) cM Argyris et al. (2015) LGICuGI cM ICuGI Scaffold v3.5.1 (address in Internet: melonomics.net) Scaffold coordinates CMPSNP460 SNP 5 12.966.671 57.3 CM3.5.1_scaffold00009 1.816.109 SNP_06 SNP 5 CM3.5.1_scaffold00009 2.607.437 CMPSNP682 SNP 5 15.854.444 57.3 50.4 CM3.5.1_scaffold00009 4.703.882 CMN62_05 SSR 5 16.570.623 CM3.5.1_scaffold00009 5.420.061 CMPSNP1005 SNP 5 17.239.621 57.3 50.4 CM3.5.1_scaffold00009 6.089.059 CMN61_15 SSR 5 17.453.772 CM3.5.1_scaffold00009 6.303.210 CMPSNP39 SNP 5 17.651.074 57.3 CM3.5.1_scaffold00009 6.500.512 PS03-B08 SNP 5 18.119.560 57.3 50.4 CM3.5.1_scaffold00009 6.968.998 ECM203 SSR 5 18.459.732 CM3.5.1_scaffold00009 7.309.170 CMPSNP2005 SNP 5 18.833.037 50.4 CM3.5.1_scafFold00009 7.682.475 CMN01_49 SSR 5 19.305.580 CM3.5.1_scaffold00003 26.051 ECM142 SSR 5 19.743.282 5 54 CM3.5.1_scaffold00003 463.753 CMGAN3 SSR 5 19.871.767 5 56 CM3.5.1_scaffold00003 592.238 CMPSNP1018 SNP 5 20.088.198 50.9 50.7 CM3.5.1_scaffold00003 808.669 CMPSNP613 SNP 5 20.406.864 CM3.5.1_scaffold00003 1.127.335 CMN05_89 SSR 5 21.514.552 CM3.5.1_scaffold00003 2.235.023 CMN23_06 SSR 5 21.949.502 CM3.5.1_scaffold00003 2.669.973

- 12 041455

GCM295 SSR 5 23.318.454 CM3.5.1_scaffold00003 4.038.925 CMPSNP741 SNP 5 23.620.132 CM3.5.1 _scaffold00003 4.340.603 ECM115 SSR 5 23.786.719 CM3.5.1 _scaffold00003 4.507.190 CMPSNP1136 SNP 5 24.100.004 67.0 67.7 CM3.5.1 _scaffold00003 4.820.475 ECM206 SSR 5 24.175.232 CM3.5.1 _scaffold00003 4.895.703 CMCTN2 SSR 5 24.313.118 5 73 CM3.5.1 _scaffold00003 5.033.589 CMGAAN144 SSR 5 24.403.405 5 73 CM3.5.1 _scaffold00003 5.123.876 SNP_05 SNP 5 CM3.5.1 _scaffold00003 5.202.092 3J84-496 SNP 5 24.884.331 CM3.5.1 _scaffold00003 5.604.802 3J84-19 SNP 5 24.884.834 CM3.5.1 _scaffold00003 5.605.305 60k53-404 SNP 5 25.036.462 CM3.5.1 _scaffold00003 5.756.933 60k53-147 SNP 5 25.036.692 CM3.5.1 _scaffold00003 5.757.163 60k53-80 SNP 5 25.036.780 CM3.5.1 _scaffold00003 5.757.251 60k49-351 SNP 5 25.040.592 CM3.5.1 _scaffold00003 5.761.063 60k49-307 SNP 5 25.040.689 CM3.5.1 _scaffold00003 5.761.160 60k49-182 SNP 5 25.040.770 CM3.5.1 _scaffold00003 5.761.241 60k45.389 SNP 5 25.043.452 73.4 CM3.5.1 _scaffold00003 5.763.923 60k45.288 SNP 5 25.043.521 CM3.5.1 _scaffold00003 5.763.992 60k45.213 SNP 5 25.043.624 CM3.5.1 _scaffold00003 5.764.095 60k45.14 SNP 5 25.043.807 73.4 CM3.5.1 _scaffold00003 5.764.278 60k42-490 SNP 5 25.045.798 CM3.5.1 _scaffold00003 5.766.269 60k42-411 SNP 5 25.045.907 CM3.5.1 _scaffold00003 5.766.378 60k42-126 SNP 5 25.046.183 76.6 CM3.5.1 _scaffold00003 5.766.654 60k42-29 SNP 5 25.046.269 CM3.5.1 _scaffold00003 5.766.740 60k41.333 SNP 5 25.047.374 CM3.5.1 _scaffold00003 5.767.845 60k41.243 SNP 5 25.047.449 73.4 CM3.5.1 _scaffold00003 5.767.920 60k41.49 SNP 5 25.047.654 CM3.5.1 _scaffold00003 5.768.125 SNP_04 SNP 5 CM3.5.1 _scaffold00003 5.785.550 CMTAAN128 SSR 5 25.081.134 5 72 CM3.5.1 _scaffold00003 5.801.605 CMTAN138 SSR 5 25.081.134 5 70 CM3.5.1 _scaffold00003 5.801.605 CMTAN139 SSR 5 25.081.134 5 70 CM3.5.1 _scaffold00003 5.801.605 CMPSNP588 SNP 5 25.271.831 75.0 78.3 CM3.5.1 _scaffold00003 5.992.302 CMPSNP464 SNP 5 25.639.164 75.8 CM3.5.1 _scaffold00003 6.359.635 SNP_03 SNP 5 CM3.5.1 _scaffold00003 6.553.350 CMPSNP 1155 SNP 5 26.124.693 79.8 CM3.5.1 _scaffold00003 6.845.164 SNP_02 SNP 5 CM3.5.1 _scaffold00003 6.950.286 CMPSNP690 SNP 5 26.340.629 79.8 90.0 CM3.5.1 _scaffold00003 7.061.100 CMTCN227 SSR 5 26.453.003 5 61 CM3.5.1 _scaffold00003 7.173.474 CMPSNP1115 SNP 5 26.639.938 84.6 CM3.5.1 _scaffold00003 7.360.409 ECM213 SSR 5 26.907.003 CM3.5.1 _scaffold00003 7.627.474 SNP_01 SNP 5 CM3.5.1 _scaffold00003 7.824.960 AI13-H12 SNP 5 27.276.249 89.4 CM3.5.1 _scaffold00003 7.996.720

The molecular markers described herein can be detected by standard methods. For example, SNP markers can be detected by KASP analysis (see www.kpbioscience.co.uk) or other types of analysis. For the SNPs described herein, a KASP analysis was developed. For more information, see the Examples section. The sequences used in the respective KASP assays are shown in the sequence listing. Two allele-specific forward primers and one allele-specific reverse primer have been developed to develop KASP assays for SNPs in accordance with public knowledge in the art (see, e.g., Allen et al., 2011, Plant Biotechnology J., 9 , 1086-1099, in particular p097-098 for KASP analysis).

- 13 041455

Sequence identity and sequence similarity can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms. Thus, sequences may be referred to as substantially identical or substantially identical when they have a certain overall minimum percentage of sequence identity (as defined below) when optimally aligned using the GAP or BESTFIT software or the Needle program (Emboss package) with parameters according to default, see below. These programs use the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of deletions. Typically default parameters are used, deletion penalty = 10, deletion extension penalty = 0.5 (for both nucleotide sequence alignment and protein sequence alignment). For nucleotides, the default substitution matrix is DNAFULL, and for proteins Blosum62 (Henikoff & Henikoff, 1992, PNAS, 89, 10915-10919). Sequence alignments and percent sequence identity scores, for example, can be determined using computer programs such as EMBOSS, which is available at: ebi.ac.uk/Tools/psa/emboss_needle/). Alternatively, percent similarity or identity can be determined by searching databases such as FASTA, BLAST, etc., however, to compare sequence identities, matches must be obtained and matched pairwise. Two proteins or two protein domains or two nucleotide sequences have significant sequence identity if the percent sequence identity is at least 85%, 90%, 95%, 98%, 99% or more (e.g., at least 99.1, 99.2, 99, 3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or more (as defined for the Needle program (Emboss package) using default parameters, i.e. fine per deletion = 10, penalty per deletion = 0.5 using DNAFULL substitution matrix for nucleic acids and Blosum62 for proteins).

When referring in the text to a nucleotide sequence (e.g., DNA or genomic DNA) that has significant sequence identity with a reference sequence or that has at least 80% sequence identity, such as at least 85, 90, 95, 98, 99, 99.2, 99.5, 99.9% Nucleotide Sequence Identity with a Reference Sequence, according to one embodiment of the invention, said nucleotide sequence is considered to have significant sequence identity with a particular nucleotide sequence and can be identified using stringent hybridization conditions. In yet another embodiment of the invention, the nucleotide sequence contains one or more mutations compared to a certain nucleotide sequence, however, it can still be identified using stringent hybridization conditions.

Cultivated melon plants containing a ToLCNDV resistance-conferring fragment introgressed into chromosome 5 show reduced symptoms when infected with ToLCNDV, while control susceptible plants (without the introgression fragment) show the expected severe symptoms under the same conditions.

In a preferred embodiment of the invention, the plants or plant cells of the present invention are characterized in that after they are infected with ToLCNDV, the average symptom score is at least 4.0, more preferably at least 5.0. As stated earlier, QTL5 confers resistance when the QTL is in homozygous or heterozygous form. From a ToLCNDV susceptible plant into which an introgression fragment containing QTL5 is introduced, by e.g. , SNP_05 and SNP_06 in homozygous form), a plant is obtained that is resistant to ToLCNDV infection and has an average disease activity index of at least 4.0, preferably at least 5.0, preferably at least 6.0, or preferably at least 7.0. The symptom severity levels that occur after infection with ToLCNDV, between 1 and 9, where 1 is defined as the most severe symptom level and 9 is defined as the highest level of resistance, have been described herein above and are applied accordingly. The preferred test for determining symptom levels is listed below in the General Methods section.

In yet another embodiment of the invention, the plants or plant cells of the present invention are characterized in that, after being infected with ToLCNDV, the severity of symptoms is in the range of 4-6, more preferably the severity of symptoms is in the range of 5-6, while susceptible control plants exhibit a symptom severity score of 2 or less.

ToLCNDV resistance conferred by an introgression fragment is expressed in a dominant manner, thus it can be observed when only one chromosome 5 contains an introgression fragment containing a donor plant sequence between the chromosome 5 markers described herein.

- 14 041455

Thus, other particular embodiments of the invention relate to melon plants or melon plant cells of the present invention, in which an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, containing the sequence of the donor plant between SNP_01 and SNP_06, between SNP_01 and SNP_05 , between SNP_01 and SNP_04, between SNP_02 and SNP_06, between SNP_02 and SNP_05, between SNP_02 and SNP_04, between SNP_03 and SNP_06, between SNP_03 and SNP_05, or preferably between SNP_03 and SNP_04 is present in a heterozygous state. Accordingly, it is sufficient that at least one chromosome 5 in the cells of the plants according to the invention or in the plants according to the invention contains the described introgression fragment. However, it is clear that chromosome 5 with respect to the described introgression fragment can also be present in a homozygous state without reducing the degree of resistance due to the dominant resistance to ToLCNDV, which is conferred by the described introgression fragment. Thus, the present invention encompasses the plants or plant cells of the present invention containing the described introgression fragment in a heterozygous or homozygous state.

In table. Figure 2 shows the SNP genotype of plants or cells containing ToLCNDV-resistant donor SNPs in homozygous or heterozygous form, as well as the SNP genotype of the recurrent parent plant lacking the introgression fragment.

table 2

SNP and nucleotide position (nt) in the sequence SNP genotype in melon plant containing donor fragment in homozygous form SNP genotype in a melon plant containing a donor fragment in heterozygous form SNP genotype of the recurrent parent in which the introgression fragment is absent SNP_01 (nt 101 SEQ GO NO: 1) SS ST TT SNP_02 (nt 945 SEQ ID NO: 2) TT TG GG SNP_03 (nt 68 SEQ ID NO: 3) TT TS SS SNP_04 (nt 227 SEQ ID NO: 4) AA AG GG SNP_05 (nt 839 SEQ ID NO: 5) SS ST TT SNP_06 (nt 445 SEQ ID NO: 6) AA AG GG

In one embodiment, a melon plant (sp. C. melo) is provided that contains a recombinant chromosome 5, whereby the recombinant chromosome 5 contains an introgression fragment that confers resistance to ToLCNDV to the melon plant if it is present in homozygous or heterozygous form, wherein the introgression fragment was obtained from a wild donor of the species C. melo. In accordance with one aspect, the introgression fragment contains an SNP donor genotype for one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06. In accordance with one embodiment of the invention, the introgression fragment contains an SNP donor genotype for at least SNP_03 and/or SNP_04. The donor SNP genotype may be present in homozygous form (if the introgression fragment is in homozygous form) or in heterozygous form (if the introgression fragment is in heterozygous form). Thus, for example, a plant, plant cell, or plant part may contain the TT or TC genotype for SNP_03 at nucleotide 68 of SEQ ID NO: 3. Thus, a plant, plant cell, or plant part may contain SEQ ID NO: 3, or a sequence including at least 95, 97 or 98% sequence identity with SEQ ID NO: 3, in homozygous or heterozygous form.

In accordance with one embodiment of the invention, melon crop plants or cells of these plants are provided that contain an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers resistance to ToLCNDV, such introgression fragment being between SNP_01 and SNP_06 (or between SEQ ID NO: 1 and SEQ ID NO: 6, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity with SEQ ID NO: 1 or with SEQ ID NO : 6); or wherein the introgression fragment is between SNP_01 and SNP_05 (or between SEQ ID NO: 1 and SEQ ID NO: 5, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 5).

In accordance with one aspect, melon crop plants or cells of these plants contain

- 15 041455 fragment of an introgression from a wild donor on chromosome 5, and this fragment of introgression confers resistance to ToLCNDV, while such an introgression fragment is located between SNP_01 and SNP_06 (or between SEQ ID NO: 1 and SEQ ID NO: 6, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 6), or the introgression fragment is between SNP_01 and SNP_05 (or between SEQ ID NO: 1 and SEQ ID NO: 5, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity with SEQ ID NO: 1 or with SEQ ID NO: 5), and the introgression fragment contains thymine (T) at nucleotide 68 of SEQ ID NO: 3 or an equivalent nucleotide of a sequence having significant sequence identity with SEQ ID NO: 3.

According to yet another aspect, the introgression fragment optionally contains adenine (A) at nucleotide 227 of SEQ ID NO: 4 or an equivalent nucleotide of a sequence having significant sequence identity to SEQ ID NO: 4.

In accordance with another aspect, the introgression fragment, if necessary, contains the donor nucleotide SNP_02 and/or SNP_05. Thus, an introgression fragment may contain a donor genotype for SNP_03, SNP_04 and SNP_05; or for SNP_03 and SNP_02; or for SNP_03 and SNP_02 and SNP_04; or for SNP_03, SNP_02, SNP_04 and SNP_05.

According to another aspect, the introgression fragment optionally contains a donor nucleotide SNP_02 and/or SNP_01. Thus, an introgression fragment may contain a donor genotype for SNP_03 and SNP_02; or for SNP_03, SNP_02 and SNP_01; and if necessary also for SNP_04, and further if necessary also for SNP_05, and further if necessary also for SNP_06.

In yet another aspect, the introgression fragment optionally contains a donor nucleotide SNP_05 and/or SNP_06.

Thus, in accordance with one aspect, melon crop plants or cells of these plants contain an introgression fragment from a wild donor on chromosome 5, and this introgression fragment confers resistance to ToLCNDV, while such an introgression fragment is located between SNP_01 and SNP_06 (or between SEQ ID NO : 1 and SEQ ID NO: 6, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 6), and wherein the introgression fragment contains an SNP genotype with resistance to ToLCNDV for one or more or all SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06. In accordance with one aspect, the fragment contains an SNP donor genotype for SNP_03 and/or SNP_04.

Thus, in accordance with one aspect, melon crop plants or cells of these plants contain an introgression fragment from a wild donor on chromosome 5, and this introgression fragment confers resistance to ToLCNDV, while such an introgression fragment is located between SNP_01 and SNP_05 (or between SEQ ID NO : 1 and SEQ ID NO: 6, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 5), and wherein the introgression fragment contains an SNP genotype with resistance to ToLCNDV for one or more or all SNP_01, SNP_02, SNP_03, SNP_04 and SNP_05. In accordance with one aspect, the fragment contains an SNP donor genotype for SNP_03 and/or SNP_04. If necessary, the SNP resistance genotype is also present for SNP_06.

Thus, in accordance with one aspect, melon crop plants or cells of these plants contain an introgression fragment from a wild donor on chromosome 5, and this introgression fragment confers resistance to ToLCNDV, while such an introgression fragment is located between SNP_03 and SNP_04 (or between SEQ ID NO : 1 and SEQ ID NO: 6, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4), and wherein the introgression fragment optionally contains the ToLCNDV resistant SNP genotype for SNP_03 and/or SNP_04. Optionally, the donor genotype is also present for one or more or all of the SNP markers selected from SNP_01, SNP_02, SNP_05 and SNP_06.

In another aspect, melon crop plants or cells of these plants contain an introgression fragment from a wild donor on chromosome 5, this introgression fragment conferring resistance to ToLCNDV, such introgression fragment being between SNP_02 and SNP_04 (or between SEQ ID NO: 2 and SEQ ID NO: 4, or a sequence containing at least 90%, or at least 95%, or at least 97 or 98% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4), while the introgression fragment optionally contains the ToLCNDV resistant SNP genotype for SNP_02 and/or SNP_03 and/or SNP_04. Optionally, the donor genotype is also present for one or more or all of the SNP markers selected from SNP_01, SNP_05 and SNP_06.

Plants containing plant cells according to the invention are another embodiment of the present invention.

- 16 041455

The melon plant of the present invention may be an inbred line, a free pollinated variety (COS), or an F1 hybrid. In one aspect, the F1 hybrid contains the introgression fragment in heterozygous form, i.e. an introgression fragment obtained by crossing two inbred parental lines, one of which contains an introgression fragment (preferably, but not necessarily in homozygous form) and collecting seeds of the F1 hybrid resulting from such crossing. The F1 hybrid may also contain an introgression fragment in a homozygous form, i.e. obtained by crossing two inbred parental lines, each of which contains an introgression fragment in a homozygous or heterozygous form.

The melon plant of the present invention may be of any type. Preferably, such a plant has good agronomic characteristics and good fruit quality characteristics, such as, for example, large average fruit size (at least 500, 600, 700, 800, 900, 1000 g or more), high solids content (for example, medium refractometer solids percentage of total soluble solids is at least 10%, 12%, 14%, 16%, 18% or more), large number of fruits per plant, dense fruit pulp, etc.

Other types of resistance can also be introduced into the melon plants of the invention, such as resistance to one or more of the following diseases: bacterial wilt, root rot, crown wilt, melon rust, powdery mildew, verticillium wilt, sulfur blight, scab, watermelon mosaic , downy mildew, Fusarium oxysporum specialized form melonis (FOM) race 0, Fusarium oxysporum specialized form melonis (FOM) race 1, Fusarium oxysporum specialized form melonis (FOM) race 2, Fusarium oxysporum specialized form melonis (FOM) race 1.2, Fusarium R2 wilt, root wart (nematode), anthracnose, cucumber mosaic and squash mosaic, and/or resistance to one or more of the following pests: aphid, moth, dark beetle, ground beetle, bordered flea beetle, mites, eleven-spotted flea beetle, melon grasshopper , melon worm, Acalymma trivittata or melon leaf miner. Other genes for resistance to pathogenic viruses, fungi, bacteria or pests can also be introduced.

A specific aspect of the invention relates to plants or plant cells containing an introgression fragment of the invention, wherein such an introgression fragment can be obtained from seeds deposited under NCIMB 42585 or from their progeny. The deposited seeds are BC4S4 generation melon crops containing the introgression fragment in homozygous form, with the donor nucleotide present in homozygous form for SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06. It is most likely that the ToLCNDV resistance region lies between SNP_03 and SNP_04, which means that the size of the donor introgression region can be reduced by selecting recombinants with smaller introgression fragments. Thus, plants containing subfragments of an introgression fragment (and said subfragments still confer resistance to ToLCNDV) containing a donor SNP for SNP_03 and/or SNP_04, but having the recurrent parent's SNP genotype for one or more or all of the other SNPs, can be produced by methods known to the skilled person.

Whether a plant has acquired a ToLCNDV resistance site from deposited seeds (same size introgression fragment or subfragment) can be determined by various methods such as sequencing and sequence comparison of introgression fragments and recombination sites.

Melon plants and plant parts (such as leaves, stems, roots, fruits, pollen, flowers, etc.) containing melon plant cells of the invention are also an embodiment of the present invention. Similarly, the present invention includes seeds from which such plants can be grown, as well as parts of such seeds (for example, cells or tissues of the seeds, such as the seed coat, germ, etc.).

Another aspect of the present invention relates to melon seeds containing an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment contains the sequence of a ToLCNDV resistant melon donor plant, between SNP_01 and SNP_06, between SNP_01 and SNP_05 , between SNP_01 and SNP_04, between SNP_02 and SNP_06, between SNP_02 and SNP_05, between SNP_02 and SNP_04, SNP_03 and SNP_06, between SNP_03 and SNP_05, or between SNP_03 and SNP_04. In a preferred embodiment, the seed contains an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment contains a ToLCNDV resistant donor plant sequence between SNP_01 and SNP_06, more preferably between SNP_01 and SNP_05, even more preferably between SNP_02 and SNP_05, moreover, preferably between SNP_02 and SNP_04, and most preferably between SNP_03 and SNP_04.

Another embodiment of the present invention relates to melon seeds obtained or obtained from plants according to the invention, or seeds containing plant cells according to the present invention.

Another aspect of the present invention relates to melon fruits containing an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, in which the introgression fragment contains the sequence of a melon donor plant with resistance to

- 17 041455

ToLCNDV, between SNP_01 and SNP_06, between SNP_01 and SNP_05, between SNP_01 and SNP_04, between SNP_02 and SNP_06, between SNP_02 and SNP_05, between SNP_02 and SNP_04, SNP_03 and SNP_06, between SNP_03 and SNP_05, or between SNP_03 and SNP_04. In a preferred embodiment, the seed contains an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment contains a ToLCNDV resistant donor plant sequence between SNP_01 and SNP_06, more preferably between SNP_01 and SNP_05, even more preferably between SNP_02 and SNP_05, moreover, preferably between SNP_02 and SNP_04, and most preferably between SNP_03 and SNP_04.

Another embodiment of the present invention relates to melon fruits obtained or obtained from plants according to the invention, or to fruits containing plant cells according to the present invention.

Preferably, the melon fruits of the present invention are characterized in that they contain an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, containing the sequence of the donor plant between SNP_01 and SNP_06, between SNP_01 and SNP_05, between SNP_01 and SNP_04, between SNP_02 and SNP_06 , between SNP_02 and SNP_05, between SNP_02 and SNP_04, between SNP_03 and SNP_06, between SNP_03 and SNP_05, or preferably between SNP_03 and SNP_04 in a heterozygous or homozygous state.

The preferred embodiments and other embodiments described herein for melon plants or melon plant cells of the invention apply to the preferred and other embodiments relating to fruits of melon plants, respectively.

Another aspect of the present invention relates to a melon plant propagation material comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment contains a ToLCNDV resistant donor plant sequence between SNP_01 and SNP_06, between SNP_01 and SNP_05, between SNP_01 and SNP_04, between SNP_02 and SNP_06, between SNP_02 and SNP_05, between SNP_02 and SNP_04, SNP_03 and SNP_06, between SNP_03 and SNP_05, or between SNP_03 and SNP_04. In a preferred embodiment, the propagation material comprises an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment contains a ToLCNDV resistant donor plant sequence between SNP_01 and SNP_06, more preferably between SNP_01 and SNP_05, even more preferably between SNP_02 and SNP_05 is further preferably between SNP_02 and SNP_04, and most preferably between SNP_03 and SNP_04.

Another embodiment of the present invention relates to a melon plant propagation material which is/can be obtained from the plants of the invention, or to a melon plant propagation material comprising the plant cells of the present invention.

The preferred embodiments and other embodiments described herein for the plants or plant cells of the invention apply, respectively, to the preferred and other embodiments relating to melon plant propagation material.

The term propagating material includes those plant components that are suitable for vegetative (asexual) or generative (sexual) procreation. For vegetative propagation, for example, cuttings, in vitro tissue cultures, cells, protoplasts, embryos or calli, micropropagative material, rhizomes or tubers can be used. Other propagation material includes, for example, fruits, seeds, seedlings that are homozygous or heterozygous for the chromosome 5 introgression fragment that confers resistance to ToLCNDV, etc. In one aspect, the propagation material is cuttings that are propagated by grafting them onto another rhizome or in vitro tissue culture material, in particular germ cultures. Particularly preferred is the propagation material in the form of in vitro tissue culture material, in particular in vitro germ cultures.

In accordance with one aspect, non-reproducing plant cells are provided that contain the recombinant chromosome 5 described herein. In one aspect, however, such non-reproducing plant cells may be part of a melon plant.

Another embodiment of the invention relates to a method for producing a melon plant with resistance to ToLCNDV, comprising the following steps:

a) selecting a donor plant that is resistant to ToLCNDV;

b) crossing the donor plant selected in step a) with a recurrent plant lacking resistance to ToLCNDV;

c) obtaining seeds from plants obtained by crossing in step b), and if necessary;

d) checking whether the plants grown from the seeds obtained in step c) are ToLCNDV resistant and/or contain one or more SNPs from the donor plant which are selected from the group SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.

- 18 041455

In step a) of the method for producing a ToLCNDV-resistant melon plant according to the invention, a ToLCNDV-resistant plant donor can be selected by infecting melon plants with ToLCNDV and determining the severity of symptoms of ToLCNDV infection in the melon plant as described herein. The same applies to checking in steps b) and c) of the method for producing melon plants having ToLCNDV resistance of the present invention to confirm whether the plant has ToLCNDV resistance or not, respectively.

In a preferred embodiment of the method for producing a ToLCNDV resistant melon plant according to the present invention, the ToLCNDV resistant donor plant in step a) contains a fragment on chromosome 5 conferring ToLCNDV resistance, which fragment contains the plant sequence - between SNP_01 and SNP_06, between SNP_01 and SNP_05, between SNP_01 and SNP_04, between SNP_02 and SNP_06, between SNP_02 and SNP_05, between SNP_02 and SNP_04, between SNP_03 and SNP_06, between SNP_03 and SNP_05, or between SNP_03 and SNP_04. Most preferably, the fragment on chromosome 5 conferring resistance to ToLCNDV contains a sequence between SNP_03 and SNP_04. In a particularly preferred embodiment of the invention, in the process for producing a ToLCNDV-resistant melon plant according to the invention, the ToLCNDV-resistant donor plant in step a) contains a chromosome 5 fragment conferring ToLCNDV resistance, comprising a sequence between SNP_01 and SNP_06, between SNP_01 and SNP_05, between SNP_01 and SNP_04, between SNP_02 and SNP_06, between SNP_02 and SNP_05, between SNP_02 and SNP_04, between SNP_03 and SNP_06, between SNP_03 and SNP_05, or preferably between SNP_03 and SNP_04, as found in seeds stored under NCIMB 42585.

In a preferred embodiment of the invention, the method of obtaining a plant with resistance to ToLCNDV, according to the present invention is used to obtain a plant according to the present invention. The preferred embodiments and other embodiments described herein for the plants of the invention are applicable, respectively, to the method for producing a ToLCNDV resistant melon plant of the present invention.

The plants obtained or obtained by the method for producing ToLCNDV resistant melon plants of the present invention are also an embodiment of the invention.

Another embodiment of the invention relates to a method for obtaining melon seeds, comprising the following steps:

a) growing a melon plant containing at least one chromosome 5 with an introgression fragment from chromosome 5 of a donor plant having resistance to ToLCNDV, wherein the introgression fragment contains the sequence of the donor plant between SNP_03 and SNP_04;

b) harvesting the fruit of the melon plants grown in step a);

c) collecting seeds from fruits obtained in step b).

In a preferred embodiment, the melon plants in step a) of the inventive melon seed production process have the specific characteristics described herein as preferred and additional embodiments for the inventive plants. The preferred embodiments and other embodiments described herein for the plants of the invention apply accordingly to the method for producing the hybrid melon seed of the present invention.

Seeds that can be obtained by the melon seed production method of the present invention are also an embodiment of the invention.

Another embodiment of the invention relates to a method for producing hybrid melon seeds, which includes the following steps:

a) obtaining a first inbred melon plant containing at least one chromosome 5 with an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, wherein the introgression fragment contains the sequence of the donor plant between SNP_03 and SNP_04;

b) obtaining a second inbred melon plant containing or not containing chromosome 5 with an introgression fragment from chromosome 5 of a donor plant with resistance to ToLCNDV, while the introgression fragment contains the sequence of the donor plant between SNP_03 and SNP_04;

c) crossing the plant obtained in step a) with the plant obtained in step b);

d) selection of seeds resulting from the cross in step c).

In the context of the present invention, the term inbred plant or inbred line means plants that have been produced by self-pollination over several generations and are highly uniform in their genetic structure and phenotypic appearance.

In a preferred embodiment of the invention, the inbred lines in steps a) and b) of the method for producing hybrid melon seeds of the invention have the specific characteristics described herein as preferred and additional embodiments for the plants of the present invention. Preferred Embodiments and Other Options

- 19 041455 embodiments described herein for plants according to the invention are applicable accordingly to the method for producing a hybrid melon seed according to the present invention.

Hybrid seeds obtained or obtained by the method for producing hybrid melon seeds of the present invention are also an embodiment of the invention.

Another embodiment of the present invention relates to a method for obtaining a melon fruit, which includes the following steps:

a) growing a plant containing at least one chromosome 5 with an introgression fragment from chromosome 5 of a donor plant having resistance to ToLCNDV, wherein the introgression fragment contains the sequence of the donor plant between SNP_03 and SNP_04;

b) harvesting the fruits of the plants grown in step a).

The term fruit in its botanical meaning usually means the seed-containing structure that develops from the ovary of angiosperm flowers.

Melon fruits obtained or obtained by the method for producing melon fruits of the present invention are also an embodiment of the invention.

ToLCNDV resistant melon donor plants can be identified using SNP markers, in particular one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06, which are described herein. Thus, the present invention provides for the first time an opportunity for a person skilled in the art to identify donor plants from which an introgression fragment conferring ToLCNDV resistance in melon plants can be transferred to recurrent melon plants.

Thus, another embodiment of the invention relates to the use of one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 or SNP_06 to identify a melon plant with resistance to ToLCNDV, or parts thereof (such as cells, fruits, leaves). Preferably, the application relates to the identification of ToLCNDV resistant melon donor plants and/or recurrent melon plants, as well as the identification of cross lines, varieties or varieties containing recombinant chromosome 5, for example recombinant chromosome 5, obtained from seeds deposited in accordance with this document. , which optionally includes a subfragment of the introgression fragment present in the deposited seeds.

Another embodiment of the invention relates to the use of one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 or SNP_06 to introgress a ToLCNDV resistance characteristic into a melon plant lacking ToLCNDV resistance, in particular a melon cultivar or cultivar.

In addition, another embodiment of the invention relates to the use of one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 or SNP_06 to breed ToLCNDV resistant melon plants.

Also provided is a method for screening plants or plant material or DNA derived therefrom for the presence of a fragment on chromosome 5 conferring resistance to ToLCNDV. This method includes the following steps:

screening the genomic DNA for the SNP genotype of one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06; and optionally selecting plants or plant material that contain the stable donor genotype of one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.

Also provided is a method for obtaining a cultivated plant C. melo containing an introgression fragment on chromosome 5, while the specified introgression fragment contains QTL ToLCNDV, while this method includes the following steps:

crossing a first cultivated melon plant susceptible to ToLCNDV with a second wild melon plant resistant to ToLCNDV, said second melon plant having the CC or CT genotype for SNP_01 in SEQ: ID NO: 1, and/or the TT or TG genotype for SNP_02 in SEQ ID NO: 2, and/or TT or TC genotype for SNP_03 in SEQ ID NO: 3, and/or AA or AG genotype for SNP_04 in SEQ ID NO: 4, and/or CC or CT genotype for SNP_05 in SEQ ID NO: 5, and/or AA or AG genotype for SNP_06 in SEQ ID NO: 6;

harvesting the seeds of the F1 plant resulting from said cross and backcrossing the F1 plant with the first melon plant to produce a backcross population (BC1) or selfing said F1 plants one or more times to produce an F2 or F3 population and, if necessary, selfing the backcross population to obtain a BC1S1 population, wherein said F2, F3, BC1 or BC1S1 plant contains the CC or CT genotype for SNP_01 in SEQ: ID NO: 1, and/or the TT or TG genotype for SNP_02 in SEQ ID NO: 2, and/or TT or TC genotype for SNP_03 in SEQ ID NO: 3, and/or AA or AG genotype for SNP_04 in SEQ ID NO: 4, and/or CC or CT genotype for SNP_05 in SEQ ID NO: 5, and/or AA or AG genotype for SNP_06 in SEQ ID NO: 6.

Also provided is a method for identifying or defining a C. melo crop containing an introgression fragment on chromosome 5, said introgression fragment containing a ToLCNDV resistant allele, said method comprising the following steps:

screening a Cucumis melo plant using a molecular marker assay that can detect at least one of the SNP markers selected from the group consisting of: SNP_01 in SEQ ID NO: 1, SNP_02 in SEQ ID NO: 2, SNP_03 in SEQ ID NO: 3

SNP_04 in SEQ ID NO: 4, SNP_05 in SEQ ID NO: 5 and/or SNP_06 in SEQ ID NO: 6; and identifying and/or selecting a plant containing the CC or CT genotype for SNP_01 in SEQ: ID NO: 1, and/or the TT or TG genotype for SNP_02 in SEQ ID NO: 2, and/or the TT or TC genotype for SNP_03 in SEQ ID NO: 3, and/or AA or AG genotype for SNP_04 in SEQ ID NO: 4, and/or CC or CT genotype for SNP_05 in SEQ ID NO: 5, and/or AA or AG genotype for SNP_06 in SEQ ID NO : 6.

A method is provided for producing C. melo F1 hybrid plants containing a ToLCNDV resistance phenotype, said method comprising the following steps:

crossing a first inbred melon plant containing at least one recombinant chromosome 5, wherein the recombinant chromosome 5 contains an introgression fragment that confers resistance to ToLCNDV to the first inbred melon plant when it is present in homozygous or heterozygous form, and said introgression fragment is obtained from a wild plant of the species Cucumis melo, with a second inbred melon plant, which may or may not contain said at least one recombinant chromosome 5, and collecting F1 hybrid seeds resulting from said crossing.

Also, the present invention includes a method for producing a melon plant that includes resistance to ToLCNDV on chromosome 5, comprising the following steps:

a) screening one or more wild melon accessions using a molecular marker assay that can detect at least one of the SNP markers selected from the group consisting of SNP_01 in SEQ ID NO: 1, SNP_02 in SEQ ID NO: 2 , SNP_03 in SEQ ID NO: 3, SNP_04 in SEQ ID NO: 4, SNP_05 in SEQ ID NO: 5 and/or SNP_06 in SEQ ID NO: 6;

b) identifying and/or selecting a wild melon plant containing the CC or CT genotype for SNP_01 in SEQ: ID NO: 1, and/or the TT or TG genotype for SNP_02 in SEQ ID NO: 2, and/or the TT or TC genotype for SNP_03 in SEQ ID NO: 3, and/or AA or AG genotype for SNP_04 in SEQ ID NO: 4, and/or CC or CT genotype for SNP_05 in SEQ ID NO: 5, and/or AA or AG genotype for SNP_06 in SEQ ID NO: 6;

c) if necessary, confirm the presence of ToLCNDV resistance characterization by resistance testing; And

d) if necessary, introducing said resistance to ToLCNDV from said wild specimen into a cultivated melon plant.

Provided is a Cucumis melo plant or a portion thereof comprising a recombinant chromosome 5, wherein such recombinant chromosome 5 contains an introgression fragment that confers resistance to ToLCNDV to the Cucumis melo plant when present in homozygous or heterozygous form, said introgression fragment containing one or more , or all single-nuclear polymorphism (SNP) markers from the following group: CC or CT genotype for SNP_01 in SEQ ID NO: 1, TT or TG genotype for SNP_02 in SEQ ID NO: 2, TT or TC genotype for SNP_03 in SEQ ID NO: 3, AA or AG genotype for SNP_04 in SEQ ID NO: 4, CC or CT genotype for SNP_05 in SEQ ID NO: 5, and/or AA or AG genotype for SNP_06 in SEQ ID NO: 6, wherein said introgression fragment is obtained from a wild plant of the species Cucumis melo, said wild plant having a mean ToLCNDV disease activity index of at least 7.0 on a scale from 1 = dead plant to 9 = no symptoms.

In one aspect, the melon crop plant contains at least the TT or TC genotype for SNP_03 in SEQ ID NO: 3 and/or the AA or AG genotype for SNP_04 in SEQ ID NO: 4.

In one aspect, a QTL conferring resistance to ToLCNDV, or an introgression fragment containing a QTL, can be derived from/is present in those seeds whose representative sample was deposited under accession number NCIMB42585 or in their progeny (therefore, the progeny retains the ToLCNDV resistance trait) .

Information about the introduction of seeds into the database.

A representative sample of cultivated melon seed, named Cucumis melo TOLCHR5, containing an introgression fragment conferring resistance to ToLCNDV that is introgressed on chromosome 5 (backcross 4, self-pollination generation 4, BC4S4), was deposited by Nunhems B.V. June 6, 2016 at NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn Aberdeen, Scotland, AB21 9YA, UK) under the Budapest Treaty based on expert judgment (EPC 2000, Rule 32(1)). Seeds were assigned the following deposit numbers: NCIMB 42585.

At the request of the applicant, samples of biological material and any material derived from it may be released only to an authorized examiner in accordance with EBU Rule 32(1) or in accordance with applicable national laws or treaties that contain similar provisions and rules, until release is indicated. patent, or until the expiration of 20 years from the date of filing of the application, if the application was rejected, withdrawn or considered withdrawn.

During the period of consideration of this application, access to the designs entered in the database will be provided by persons designated by the authorized director of the United States Patent Office,

- 21 041455 on request. Subject to the provisions of Title 37 of the US Code of Federal Regulations, § 1.808(b), any restrictions imposed by the contributor on material entered into the database are irrevocably removed upon grant of the patent. The material entered into the database is retained for 30 years or for 5 years from the date of the last request or for the duration of the patent, whichever ends later; if during this period the virus becomes non-viable, the material must be replaced. Applicant does not waive any rights granted under this patent under this application or under the Breeders' Rights Protection Act (Title 7 USC 2321 et seq.).

Description of sequences.

Symbols other than G (guanine), A (adenine), T (thymine), and C (cytosine) in the sequences shown in the sequence listing have the following meanings.

R: G or A.

Y: T or S.

M: A or C.

K: G or T.

S: G or S.

W: A or T.

N: A, or C, or T.

B: G or T or C.

V: G or C or A.

D: G, or A, or T.

N: G or A or T or C.

In SEQ ID NO: 1-6, the SNP nucleotide of the resistant donor is indicated in bold and underlined.

SEQ ID NO: 1: ToLCNDV resistant donor plant sequence containing SNP_01.

SEQ ID NO: 1:

agctggtgca aagctggcat tcaaatcgaa tgaagaaata gcagtacaag tgaagtcaat 60 tccactagat gaagtaatcc cggattcaga acgagtgctt ctaataaaaa tcgatgttca 120

SEQ ID NO: 2: ToLCNDV resistant donor plant sequence containing SNP_02.

SEQ ID NO: 2:

ttgtaatcat ggccattgcc tgatytgcag aatgtggcgt cttgattggg cacggtctgg 60 atgaagttac tccaaagctt gtggaagtta atgggttctt ctcttcaccr tctgrtctcc 120 ttcccatcga caatgtggtt tcttcttcaa acacaaaaga aacccctgtt gaagctgtag 180 aagaaagctg tgagttcttt tctccactca gttctaggtt tagtttttta tttgtttttt 240 ttttcttttt tcctttttac ttgctttgag ctgtaggaca caacacaaaa tgataagaac 300 aaaaatcaat ctatgttatt gttgtcattg tctcgcctgc ctcttcaagg taactaggct 360 ataacactga tccaaaatgt ataatcttta ttgctaagta acttatattg caaagattgg 420 gatggaatag gagcataggt tgcatctaat agttttgtcc aactttggtt atagtttcag 480 ccagtttgtt cagatgaaat tcttagtgat tatcactaat ggaattgttt ttataaatgt 540 tgtaggtgtt gaaagaagtg aagaatatga aaaggaaagc aggggaaccg agaaggctga 600 aatcttaccg acaaaagcaa catctgaagc aggttctgaa gtccaacctg tttccagtga 660 ttctgctcag atggtaccca atatgttgga gctcggtgat gcttataagc tagctgtagg 720 tgctagagga ggaagacaat tgtctggcaa gcttttggaa caatggatyg ggaaggaatc 780 ttcaaaagtt agtgaagatc tgaagcttct cttgacacaa ctctcattta atcgtttgaa 840 tgaccaatca cgggagatga gtccaaggct gtccgtaaat ggagacgagg tgaggaactt 900

- 22 041455 tgattacttg agygctgttg ggatgcaaat gctacaaaaa aggwtttcgc ttgaaagaaa 960 tgagtccggt gtagaatctt tagatggaag cataattagt gaaatcgatg gggaaaacat 1020 ggctgatagg ttgaaacgac agattgagta tgataagaag gttmtgartt ctttatacaa 1080 ggaattggag gaagaaagaa atgcatccgc aattgctgca aatcaggcaa tggccatgat 1140 tacaa 1145

SEQ ID NO: 3: ToLCNDV resistant donor plant sequence containing SNP_03.

SEQ ID NO: 3:

agacggcgta catgcagcat tctgttaccc cgcgatgccc agcgatgaaa caactccttt 60

SEQ ID NO: 4: ToLCNDV resistant donor plant sequence containing SNP_04.

SEQ ID NO: 4:

ggagkcttca tccgtcttta ccttcctcac ttttttacct aaccaagtta cgtcttgtag 60 gttgcaagat aacaaatttg gatttcttag aaacaattgt ttatgttgcc ccttcrttga 120 aagagttgga cttgtccgaa aacaactttt gtagamtacc ctcrtgtatt attaatttta 180 aatccctgaa atatctttat acaatggatt gtgagttgct ygaagaaatt tcaaaggttc 240 cagaaggtgt aatttgtacg agtgccgcag gatgcaaatc attggctaga tttcccgaca 300 acttagctga tttcatatct tgtggtaatt ctgcggtgcg taccatatct ctttctcatg 360 acttcaccat tatctctagc tcatgtatat ttaatttcat tcatataata tatattactt 420 ataactattt actgatctca tggtgcagga atgttgtaaa ggtggatg 468

SEQ ID NO: 5: ToLCNDV resistant donor plant sequence containing SNP_05.

SEQ ID NO: 5:

tgcaggtagt caaaccaatt gataatattt gctcaccatc aaaattggta accatagaca 60 tagaytgacc aagcccaggc aaaacggcat catctttttg ttttctatca ttactattct 120 gattacccaa aagaaatctt ctttttgtac ttggagagaa ataatccccg tctgcacatt 180 gcatggaagg tgtgctgtca tgagcatcag cattacacac accagaatta gatttcttgc 240 tagccaagat ttcacaacca ccaagagaag caggacctac gcctggggtc tctgcctyag 300 aaagccaags atcaaagcac acttgtgaag gacctgaaac tgtctcagat ccagggattt 360 ctcctacatt gggtaagcta gcctgttcaa agccatcaca cacatctaca agcggcagtc 420 taccatacat ggaactatta gaataagcag gcttattttc ataaaacaca ttaacgcttg 480 catttggatt agttaaacat ggcatcaaat gggaagaatc ccgaaatctc aagctgttct 540 cattggaccg cagcactcca gaattcaagt ctcttgacca tacttttttt tgcctgctat 600 caacagtaaa attttgagct ttaccatatc cacagtcaag tggctcattt cgagagttac 660 gcttcctcct cattaaatca cktacagaac ggccttccct ggagttatgt gtgcctgtac 720 ctgcatttcg taatgtaaca tccacctgcc ctccatcttc acttgaaaag caactaatga 780 actcagaatc tttggtcgaa cttgtccata tgtcaggcct gcaagaattg acacacgtcg 840 aatctgtatt cactttctca gcctcatgaa aaggtaaaga tccccaaaat gatttctctt 900 tcttgtgact gcaagaacct gaatcaatgc catgcaataa tattgcaagt ttggaagatc 960 tttcattctc aacattaaga tctctatcag gtgagttttc agtcgaccct ccagaagaag 1020 agaacatatc atctgcagt 1acgcagt

SEQ ID NO: 6: ToLCNDV resistant donor plant sequence containing SNP_06.

- 23 041455

SEQ ID NO: 6:

gcgtacmtgc aggcaaagaa tggcacagta cagtaatggt agctcatctg ttcatcaagg 60 agaatcaagc tgcctcagcc attcagtatc ggttccaccc ctgatcatct cctacaatga 120 tcgcattcgt cctctccttg atgctgttga caagcttcgt cacctcatga tcatgagaga 180 aggcatccaa ctgcctacca tagttgttgt tggtgatcag tcawccggta agtcaagtgt 240 cctcgagtcg ttggctggga tcagcctacc tcgaggtcag ggcatctgca ccagggtccc 300 tctgataatg aggctccaaa accatcctga tcccgaaccc gagcttgttt tggagtacaa 360 tgggaaaaag atccacaccg acgaatcctt cattgctgaa gacatctgta cagctacaga 420 ggagattgct ggcagtggca aaggaatatc gaaagcgcca ttgactttga ttgtgaagaa 480 aaatggtgtt cctgatctta caatggttga tctccctgga attacragag tgcctgttaa 540 agatcagcct gaagacattt atgaccaaat aaaagatata atcatggaac atatcaagcc 600 agaagagagc atcatcttga atgtcttgtc tgcgacggtt gattttccaa cttgtgaatc 660 gatacggatg tctcaaagtg tcgacaagac gggaatgaga acgttggcag ttgtgactaa 720 gtctgacaag gcaccagaag gcctacacga gaaggtcacc rcggatgatg tcagtatcgg 780 ccttggttat gtttgcgtta ggaaccgaat tggcaatgag acatatgagg aagctcgggt 840 tgcagaagcc aaattgtttt caactcatcc tcttctctcc aaaattgaca aatctgttgt 900 gggcattcca gtcttggctc agaagttggt gcaaattcaa gcaggtaccc aaactaattc 960 ctgactcaaa agctaggttc cgttagataa ccattttgtt ttagaaaatc aagtttattt 1020 tctctaaatm gtgtaccatg attttcatct ttcttaaata aaaaagttgm attcttwact 1080 aaattttaaa agcaaaaaca agttttaata cttttttt 1118

SEQ ID NO: 7: Allele specific PCR (KASP) primer for the FAM SNP_01 allele.

SEQ ID NO: 8: Primer for VIC allele SNP_01 KASP analysis.

SEQ ID NO: 9: General primer for SNP_01 KASP analysis.

SEQ ID NO: 10: primer for FAM allele SNP_02 KASP analysis.

SEQ ID NO: 11: Primer for VIC allele SNP_02 KASP analysis.

SEQ ID NO: 12: General primer for SNP_02 KASP analysis.

SEQ ID NO: 13: Primer for FAM allele SNP_03 KASP analysis.

SEQ ID NO: 14: Primer for VIC allele SNP_03 KASP analysis.

SEQ ID NO: 15: General primer for SNP_03 KASP analysis.

SEQ ID NO: 16: Primer for FAM allele SNP_04 KASP analysis.

SEQ ID NO: 17: Primer for VIC allele SNP_04 KASP analysis.

SEQ ID NO: 18: General primer for SNP_04 KASP analysis.

SEQ ID NO: 19: Primer for FAM allele SNP_05 KASP analysis.

SEQ ID NO: 20: Primer for VIC allele SNP_05 KASP analysis.

SEQ ID NO: 21: General primer for SNP_05 KASP analysis.

SEQ ID NO: 22: Primer for FAM allele SNP_06 KASP analysis.

SEQ ID NO: 23: Primer for VIC allele SNP_06 KASP analysis.

SEQ ID NO: 24: General primer for SNP_06 KASP analysis.

Description of drawings

Fig. 1. Shown is a recurrent plant sensitive to ToLCNDV (upper figure) and a recurrent plant that has been rendered resistant to ToLCNDV (lower figure) by introgression of a fragment containing a donor sequence between SNP_01 and SNP_06. The picture was taken 25 days after infection (dpz) ToLCNDV with whiteflies.

Fig. 2. Symptom scores are shown 35 days post-infection (dps) with ToLCNDV by whitefly transmission of a ToLCNDV resistant donor plant (wild donor), a recurrent plant (recurrent plant), and a plant obtained after introgression of a ToLCNDV resistant fragment. recurrent plant from a donor plant (introgression). Symptom levels were determined as described in this document under General Methods.

General Methods

1. Determination of the severity of symptoms in plants infected with ToLCNDV.

1.1. Plants and pathogens (virus).

To infect melon plants, the ToLCNDV strain infecting melon plants (Cu-24 041455 cumis melo) is used. In the present invention, the ToLCNDV strain isolated in Murcia, Spain was used as the inoculum.

1.2. Distribution of ToLCNDV.

The source of infection of ToLCNDV persists on living infected melon plants. It must be ensured that pure virus isolates are used and that neither the virus source nor the whiteflies are infected with other diseases, in particular other viruses (eg CGMMV, CYSDV, CYVY SqMV). To prepropagate the ToLCNDV inoculum, whiteflies (Bemisia tabaci) are fed ToLCNDV-susceptible melon plants in an insect cage. Prior to infection of test plants, ToLCNDV-infected plants were placed in an insect cage, whiteflies were placed in the same cage and allowed to feed on ToLCNDV-infected plants for about 3 days.

1.3. Inoculation of the studied plants.

For each melon plant genotype analyzed, 14 plants were grown until the first true leaf appeared (typically 12-15 days after sowing), 12 of which were infected and 2 plants were falsely infected. Also included were 12 susceptible cultivars, in this experiment cultivar Gandalf F1 (Hild Samen) and cultivar Vedantrais. The 12 plants for each genotype to be tested for resistance to ToLCNDV were placed in an insect cage, then infected whiteflies were placed in the insect cage as described in 1.2 above. It must be ensured that there are at least 5-10 whiteflies in the cage for each plant studied. Whiteflies and test plants are caged for approximately 48 hours, after which the whiteflies are killed with an appropriate insecticide. Also, two plants per genotype were falsely infected, i.e. they were kept under the same conditions as the test plants, except that the whiteflies used for infection were not infected with ToLCNDV.

1.4. Cultivation of infected test plants.

Infected test plants, obtained as described in 1.3, were transplanted into larger pots and transferred to a greenhouse with cooling equipment. Plants were grown at a temperature of approximately 18°C at night and approximately 25°C during the day with a photoperiod of 14-16 hours. Infected plants for each infected genotype were grown in two replicate experiments in two different plots, each of which hosted 6 plants infected with ToLCNDV and one falsely infected plant. Plots were randomized in terms of growing area.

1.5. Assessing the severity of symptoms of ToLCNDV infection Assessing the severity of symptoms can be performed as early as about 15 days after infection (dc) with ToLCNDV, but is preferably done at or after about 30 days after infection (dc) with ToLCNDV. In the event that there are plants showing recovery from the viral infection, a further symptom assessment is carried out approximately 45 days post-infection (dpz) with ToLCNDV.

- 25 041455

Use the following levels of symptom severity according to the phenotypes listed below.

Level Observed phenotype severity of symptoms 1 dead plant 2 Extremely severe mosaic and leaf curling, chlorosis and reduced growth. The plant cannot recover. 3 Strong mosaic and leaf curling, chlorosis and reduced growth. The plant cannot recover. 4 Coagulation and mosaic, chlorosis, there is a slight decrease in growth, or no decrease in growth is observed. The plant cannot recover. 5 Coagulation and mosaic, chlorosis, decrease in growth is not observed. There is a slight recovery of the upper part of the plant 6 Slight coagulation, mosaic and chlorosis, growth reduction is not observed. There is a recovery of the upper and middle parts of the plant 7 Slight coagulation, mosaic and chlorosis, growth reduction is not observed. Symptoms are observed only in the lower part of the plant 8 Weak mosaic 9 No symptoms

1.6. Additional research as needed.

It is recommended to use at least one highly ToLCNDV resistant genotype (symptom level 8-9) and one highly susceptible ToLCNDV genotype (symptom level 1) in each experimental model. In addition, it is recommended that each experimental model also include a genotype that is intermediately resistant to ToLCNDV infection. The best results are achieved when the mentioned genotypes are included and the symptom levels of each genotype are evaluated relative to the results obtained for high resistance, high susceptibility and intermediate resistance genotypes. These genotypes also provide a clear indication of the extent of ToLCNDV infestation of melon plants by whiteflies.

In addition, it is recommended to test for infection and spread of ToLCNDV on infected and control plants. This can be done by checking for the presence and amount of viral DNA in the upper parts of the plants. A suitable way to test for the presence and amount of ToLCNDV DNA in the upper parts of the plant is to hybridize the plant material with a probe that hybridizes to the DNA of the ToLCNDV strain used. Various hybridization techniques are well known to those skilled in the art. To obtain valuable results, a simple, so-called point, analysis is sufficient. PCR or quantitative PCR methods can also be used.

Examples

1. Selection of a donor plant with resistance to ToLCNDV.

A study was conducted on the level of symptom severity for wild samples of melon tested for resistance to ToLCNDV in accordance with the study described in the General Methods section. A wild donor plant was identified that is highly resistant to ToLCNDV and has a resistance level of about 7 (as will be shown below, the average disease activity index is 7.4, while the susceptible plant has an average disease activity index of 2.0).

2. Identification of the genomic location of resistance to ToLCNDV.

Three mapping populations were developed, including using the donor plant obtained in Example 1 to map the position of the ToLCNDV resistance-conferring (QTL) fragment in the genome of the melon donor plant.

Analysis in these mapped populations revealed one major resistance-associated QTL located on chromosome 5 and showing dominant inheritance patterns.

The size of the detected QTL fragment and the observed patterns of inheritance suggest the presence of a single dominant gene locus. BC (backcrossing) lines have been developed from sustainable material by the inventors for accurate mapping and further investigation of donor-derived resistance. Genotypic results in 10 advanced BC families developed through phenotypic selection showed >93% agreement with individual phenotypes.

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Claims (11)

Markers identified during fine mapping and their respective positions according to public knowledge from Diaz et al. (2015, Mol. Breeding., 35, 188) are presented in the following table. Identificational Position Coordinates SNP marker number AI13-H12 CM3.5_scaffold00003 SNP 7996720 pseudomolecules 27276249 SNP_01 mME11320_k CM3.5_scaffold00003 7824960 SNP_02 mME43070_k CM3.5_scaffold00003 6950286 SNP_03 mME10621_k CM3.5_scaffold00003 6553350 SNP_04 mME50729_k CM3.5_scaffold00003 5785550 SNP_05 mME32395_k CMGAAN144 CMPSNP682 CM3.5_scaffold00003 CM3.5_scaffold00003 CM3.5_scaffold00009 5202092 5123876 4703882 24403405 15854444 SNP_06 mME49184_k CMPSNP460 CM3.5_scaffold00009 CM3.5_scaffold00009 2607437 1816109 12966671 3. Development of KASP-analysis. To identify SNPs flanking QTLs, a KASP analysis was developed. QTL associated SNPs can be determined using the following primers in a KASP assay. SNP FAM allele VIC allele General Primer SNP_01 SEQ ID NO: 7 SEQ ID NO: 8 SEQ W NO: 9 SNP_02 SEQ ID NO: 10 SEQ W NO: 11 SEQ W NO: 12 SNP_03 SEQ ID NO: 13 SEQ W NO: 14 SEQ ID NO: 15 SNP_04 SEQ W NO: 16 SEQ W NO: 17 SEQ ID NO: 18 SNP_05 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SNP_06 SEQ ID NO: 22 SEQ W NO: 23 SEQ ID NO: 24 4. Introgression of ToLCNDV into a cultivated melon plant. Carried out backcrossing with cultivated melon plants and received recurrent melon plants with increased resistance to ToLCNDV. The presence of QTLs in these lines was determined using the KASP assay sequences shown in Example 3. A representative result for the ToLCNDV symptom score obtained for these plants is presented in the following table and in FIG. 2. Symptom scores were determined 35 days post infection (dps) according to the method described in this document under General Methods. SZDIG SO wild donor 7.40 2.07 recurrent plant 2.00 0.00 introgression 5.11 1.45 SPDIG: average value for the studied genotype; CO: mean deviation. Additional backcrossing is done to introduce resistance into the elite varieties Piel de Sapo, Galia, Cantaloupe and Charente. Genotypic results in 10 advanced BC families developed through phenotypic selection show >93% agreement with individual phenotypes. CLAIM 1. A melon plant cell containing an introgression fragment on chromosome 5 from a donor plant with resistance to New Delhi tomato leaf curl virus (ToLCNDV), characterized in that the introgression fragment confers resistance to ToLCNDV and contains thymine at nucleotide 68 of SEQ ID NO : 3 and/or adenine at nucleotide 227 of SEQ ID NO: 4, wherein the introgression fragment is obtained from seed deposited under accession number NCIMB 42585. 2. A melon plant having resistance to ToLCNDV, containing melon plant cells according to claim 1. 3. Melon seed containing plant cells according to claim 1. 4. Melon fruit containing plant cells according to claim 1. 5. A melon plant propagation material comprising the plant cells of claim 1, wherein the propagation material is selected from cuttings or in vitro tissue culture material. 6. A method for producing a melon plant with resistance to ToLCNDV, comprising the following steps: - 27 041455 a) selection of a donor plant with resistance to ToLCNDV, containing a fragment on chromosome 5, where the fragment confers resistance to ToLCNDV and contains thymine at nucleotide 68 SEQ ID NO: 3 and/or adenine at nucleotide 227 of SEQ ID NO: 4 and where the fragment is obtained from seeds deposited under accession number NCIMB 42585; b) crossing the donor plant selected in step a) with a plant lacking resistance to ToLCNDV; c) obtaining seeds from plants obtained by crossing in step b); And d) checking whether the plants grown from the seeds obtained in step c) are ToLCNDV resistant and contain thymine at nucleotide 68 of SEQ ID NO: 3 and/or adenine at nucleotide 227 of SEQ ID NO: 4. 7. A method for obtaining melon seeds, including the following steps: a) growing a melon plant containing at least one chromosome 5 with an introgression fragment on chromosome 5 from a donor plant having resistance to ToLCNDV, the introgression fragment conferring resistance to ToLCNDV and containing thymine at nucleotide 68 of SEQ ID NO: 3 and/or adenine at nucleotide 227 of SEQ ID NO: 4, where the introgression fragment is obtained from seeds deposited under accession number NCIMB 42585; b) harvesting the fruit of the melon plants grown in step a); And c) collecting seeds from fruits obtained in step b). 8. A method for producing hybrid melon seeds, comprising the following steps: a) obtaining a first inbred melon plant containing at least one chromosome 5 with an introgression fragment on chromosome 5 from a donor plant having resistance to ToLCNDV, the introgression fragment conferring resistance to ToLCNDV and containing thymine at nucleotide 68 of SEQ ID NO: 3 and /or adenine at nucleotide 227 of SEQ ID NO: 4, where the introgression fragment is obtained from seeds deposited under accession number NCIMB 42585; b) obtaining a second inbred melon plant containing or not containing chromosome 5 with an introgression fragment on chromosome 5 from a donor plant with resistance to ToLCNDV, the introgression fragment conferring resistance to ToLCNDV and containing thymine at nucleotide 68 of SEQ ID NO: 3 and/ or adenine at nucleotide 227 of SEQ ID NO: 4, where the introgression fragment is obtained from seeds deposited under accession number NCIMB 42585; c) crossing the plant obtained in step a) with the plant obtained in step b); And d) selection of seeds resulting from the cross in step c). 9. A method for obtaining a melon fruit, including the following steps: a) growing a plant containing at least one chromosome 5 with an introgression fragment on chromosome 5 from a donor plant with resistance to ToLCNDV, wherein the introgression fragment confers resistance to ToLCNDV and contains thymine at nucleotide 68 of SEQ ID NO: 3 and/or adenine at nucleotide 227 of SEQ ID NO: 4, where the introgression fragment is obtained from seeds deposited under accession number NCIMB 42585; b) harvesting the fruits of the plants grown in step a). 10. Method for screening plants or plant material or DNA derived from them for the presence of an introgression fragment on chromosome 5 conferring resistance to ToLCNDV, the introgression fragment conferring resistance to ToLCNDV and containing thymine at nucleotide 68 of SEQ ID NO: 3 and/or adenine at nucleotide 227 of SEQ ID NO: 4, wherein the introgression fragment is obtained from seeds deposited under accession number NCIMB 42585, the method comprising the following steps: screening the genomic DNA for the SNP genotype of one, or more, or all of SNP_03 at nucleotide 68 of SEQ ID NO: 3 and SNP_04 at nucleotide 227 of SEQ ID NO: 4; and selecting plants or plant material that contains the TT or TC genotype for SNP_03 in SEQ ID NO: 3 and/or the AA or AG genotype for SNP_04 in SEQ ID NO: 4. 11. A method for identifying or determining a cultivated plant C. melo containing an introgression fragment on chromosome 5, where the specified introgression fragment contains an allele resistant to ToLCNDV, and the introgression fragment confers resistance to ToLCNDV and contains thymine at nucleotide 68 of SEQ ID NO: 3 and/ or adenine at nucleotide 227 of SEQ ID NO: 4, wherein the introgression fragment is obtained from seeds deposited under accession number NCIMB 42585, the method comprising the following steps: screening a Cucumis melo plant using a molecular marker assay detecting at least one of the SNP markers selected from the group consisting of SNP_03 in SEQ ID NO: 3 and SNP_04 in SEQ ID NO: 4; and identifying and/or selecting a plant containing the TT or TC genotype for SNP_03 in SEQ ID NO: 3 and/or the AA or AG genotype for SNP_04 in SEQ ID NO: 4. -