Melons with extended shelf life

Abstract

The invention relates to a melon (Cucumis melo) plant comprising in its genome a homozygous mutant allele of the staygreen (sgr) gene on chromosome 9, the mutant allele of the sgr gene comprising at least one loss-of-function mutation compared to the sequence of a wild-type sgr allele (SEQ ID NO: 1), and the mutant allele of the sgr gene confers skin color stability to fruit of the plant at maturity and / or after harvest compared to an isogenic non-extended shelf life (non-LSL) melon plant that does not contain the mutant allele. The invention further relates to parts, cells, and seeds of the plant, as well as related methods and processes.

Description

[Technical Field]

[0001] The present invention relates to a melon (Cucumis melo (C. melo)) plant that extends the shelf life compared to existing non-long-life (non-LSL) melon varieties while retaining characteristics such as sugar content, cycle length, aroma, or firmness similar to non-LSL melons. The present invention also provides a method for producing such a plant, as well as a method for detecting and / or selecting such a plant. [Background technology]

[0002] Melon (Cucumis melo L.) is a globally cultivated crop belonging to the gourd family. Most commercially available melons produce sweet fruit known as Charentais, Cantaloupe, Piel de Sapo, Galia, Ananas, and Honeydew. Melon fruit is typically consumed as a dessert fruit.

[0003] Combining long shelf life with a desirable taste for consumers has always been a challenge for melon growers. Shelf life, in particular, is a crucial parameter for both melon producers and retailers. Fruit with an extended shelf life can be stored for longer periods, reducing commercial losses and increasing flexibility in harvesting and transportation. Much effort has been put into improving the shelf life of melons. Until the 1980s, commercially available melons were primarily traditional varieties with limited shelf life. Traditional melons are climacteric fruits, whose ripening process is triggered by rapid ethylene production accompanied by respiration. These events then trigger many ethylene-dependent processes, such as changes in skin color (generally yellowing), the development of flavorings that give the melon, or the gradual softening of the fruit. These processes affect the shelf life of the melon.

[0004] In the 1990s, long-life (LSL) melon varieties were introduced and gradually came to dominate the market. LSL melons are non-climacteric melons that do not undergo the typical ethylene surge in climacteric melons during maturation. Furthermore, LSL melons maintain their green color for extended periods and retain their firmness after harvest. While the extended shelf life offers significant advantages compared to conventional melons, LSL melons also have a major drawback: they do not develop much aroma, and therefore their flavor is often perceived negatively by consumers. Candidate mutations that may be involved in extending the shelf life of LSL melons, particularly mutations in the ACC oxidase gene, have been identified (Ayub, Ricardo, et al. “Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits.” Nature biotechnology 14.7(1996):862-866).

[0005] Recently, medium-term storage life (ISL) melons have been acquired and marketed as an attempt to provide melons that offer an acceptable storage life while having a better taste than long-term storage life (LSL) melons. However, such compromises between complex characteristics are a difficult task for growers to achieve.

[0006] There remains a need to provide new types of melons that meet the demands of producers and consumers, combining improved shelf life with good taste and other commercially important characteristics. [Overview of the Initiative]

[0007] The inventors of this application have discovered that inactivating the staygreen (sgr) gene on chromosome 9, for example, by splicing site mutation, conferred stability to the rind color at maturity and after harvest compared to non-LSL melon varieties, while observing no effect on rind color in LSL melons. This stability subsequently leads to an increase in fruit storage life. This was surprising because other genes, such as ACC oxidase (Ayub, Ricardo, et al., 1996), had previously been involved in controlling the storage life of melons, but the sgr gene had not. Surprisingly, the inventors also discovered that the sgr mutant possesses many other advantageous properties that remain the same as or equivalent to non-LSL melons, such as conventional melons. These properties are of interest to growers, retailers, or consumers in terms of cycle length, hardness, soluble solids or Brix (i.e., sweetness), or pedicel detachment rate. Therefore, the present invention provides a new type of melon that combines extended shelf life with commercially interesting non-LSL characteristics such as cycle length, sweetness, stalk detachment, and softness at maturity.

[0008] Accordingly, in one embodiment, the present invention relates to a melon (Cucumis melo) plant in which the plant homozygously contains a mutant allele of the staygreen (sgr) gene on chromosome 9 in its genome, the mutant allele of the sgr gene contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1), the mutant allele of the sgr gene does not contain the mutant allele in a homozygous state, and therefore contains a functional sgr gene in a heterozygous or homozygous state, thereby conferring stability of the rind color to the fruit of the plant at maturity and / or after maturity compared to an isogenic non-long-life (non-LSL) melon plant.

[0009] Another object of the present invention relates to cells of a melon plant according to the present invention, preferably cells derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, flowers, cotyledons, and / or hypocotyls, wherein the cells are homozygous for a mutant allele of the staygreen (sgr) gene on chromosome 9 in their genome, and the mutant allele of the sgr gene homozygously contains at least one loss-of-function mutation as compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1).

[0010] The present invention also relates to a plant part of a melon plant comprising at least one cell according to the present invention, preferably an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, and / or hypocotyl, particularly a fruit.

[0011] The present invention further relates to a melon seed that can grow into a melon plant according to the present invention.

[0012] In a further aspect, the present invention relates to an in vitro cell or tissue culture of a renewable cell of a melon plant according to the present invention, wherein the renewable cell is derived from an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, seed, flower, cotyledon, and / or hypocotyl.

[0013] The present invention also relates to a method for producing or facilitating the production of a melon plant that produces fruits with an extended storage life, (a) obtaining a plant part according to the present invention, (b) vegetatively propagating the plant part to generate a plant from the plant part and comprising the method.

[0014] The present invention further relates to a method for producing or producing melon plants that produce fruits with extended shelf life, comprising introducing a loss-of-function mutation into the sgr gene (SEQ ID NO: 1) on chromosome 9 in the genome of a non-LSL melon plant, wherein the mutation is introduced by mutagenesis or genome editing, particularly by techniques selected from ethylmethanesulfonic acid (EMS) mutagenesis, oligonucleotide-directed mutagenesis (ODM), zinc finger nuclease (ZFN) technology, activator-like effector nuclease (TALEN), CRISPR / Cas system, engineered meganuclease, re-engineered homing endonuclease, and DNA-guided genome editing.

[0015] A method is provided for identifying, detecting, and / or selecting melon plants that produce or are likely to produce fruits with extended shelf life, comprising detecting a mutant allele of the sgr gene on chromosome 9 in the genome of the plant, wherein the mutant allele contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1).

[0016] The present invention further relates to a method for improving the storage life of melon fruit, the marketability of melon fruit, and / or the yield of melon production, the method comprising growing a melon plant according to the present invention and harvesting the fruit produced by the plant.

[0017] A method for producing melon fruit, a) Growing melon plants according to the present invention, b) To enable the plant to bear fruit, c) The fruits of the plant are preferably harvested before maturity or at the maturity stage. Methods including the following are also provided.

[0018] Another object of the present invention is the use of the melon plant or its fruit according to the present invention for the fresh cut market or for food processing.

[0019] definition Melon varieties can be classified into three groups based on their post-harvest characteristics: traditional, medium-term storage life (ISL), and long-term storage life (LSL).

[0020] In this specification, the term “storage life” refers to the period after harvest during which a melon fruit can be stored before it is deemed unsuitable for sale or consumption. Storage life is preferably evaluated during storage. Storage life generally takes into account various characteristics of the fruit, such as rind color, flesh color, firmness, aroma, and / or sugar content. Preferably, the increase in storage life of melons according to the present invention is evaluated based on improved color stability at harvest and / or during post-harvest storage. In particular, melons according to the present invention retain their immature color for a longer period during post-harvest storage compared to melons without the genetic characteristics of the present invention.

[0021] Long-life (LSL) melons typically have a shelf life of at least 10 days, preferably at least 14 days. More specifically, LSL melons have a shelf life of 10 to 21 days. LSL melons are non-climacteric. In particular, LSL melons may be selected from the following varieties: LSL Charentais, LSL Italian netted, Harper, LSL Galia, Yellow Canary, Piel de Sapo, and Honey Dew.

[0022] "Traditional" melons typically have a shelf life of less than 5 days. Preferably, traditional melons have a shelf life of 2 to 5 days. Typically, traditional melons are of the climacteric type. In particular, traditional melons may be selected from the following varieties: traditional Charentais, traditional Italian netted, Western Shipper, Eastern Shipper, traditional Galia, and traditional Ananas.

[0023] "Intermediate Storage Life (ISL)" melons are melons that have a storage life between that of traditional melons and that of LSL melons. Preferably, ISL melons have a storage life of 7 to 14 days. In particular, ISL melons may be selected from the following varieties: Charentais, Italian netted, Western Shipper, Eastern Shipper, Galia, Ananas, and Honey Dew.

[0024] As used herein, the term “non-LSL” melon refers to traditional or ISL melons. Therefore, references to non-LSL melons or melon plants should be understood as referring to traditional melons and / or ISL melons or melon plants. Non-LSL melons typically have a shelf life of less than 14 days, preferably less than 10 days. In particular, non-LSL melons may be selected from the following types: traditional Charentais, traditional Italian netted, traditional Galia, traditional Ananas, ISL Charentais, ISL Italian netted, Western Shipper, Eastern Shipper, ISL Galia, ISL Ananas, and ISL Honey Dew.

[0025] "Climacteric" melon varieties are generally characterized by the rapid autocatalytic production of ethylene during maturation, accompanied by increased respiration. Climacteric maturation involves several ethylene-mediated physiological and biochemical events, including softening of the flesh, development of aroma, rapid change in rind color, and detachment from the vine (i.e., detachment of the pedicel). The change in rind color varies depending on the melon variety. In Galia melons, the rind changes from dark green to yellow-orange, while in Charentais melons, the rind changes from green or gray to a creamy yellow. Autocatalytic production of ethylene manifests as an exponentially increasing ethylene concentration in the melon cavity over time, generally progressing from negligible levels to maximums in just a few days. While the absolute magnitude of the peak ethylene levels varies among climachteric melon cultivars, rapid induction of ethylene biosynthesis is characteristic of these varieties.

[0026] Non-climacteric melon varieties do not exhibit such autocatalytic ethylene production, and therefore, they are characterized by reduced or no change in skin color during ripening, as well as reduced hardness during storage and reduced aroma production, which negatively affects the flavor of such melons.

[0027] As used herein, “allele” refers to any of several alternative or variant forms of a genetic unit, such as a gene, that are alternative for inheritance because they are located at the same locus on homologous chromosomes. Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations, or deletions, or of gene regulation caused by, for example, chemical or structural modifications, transcriptional regulation, or post-translational modification / regulation. In diploid cells or organisms, the two alleles of a given gene or genetic element typically occupy the corresponding locus on a pair of homologous chromosomes.

[0028] As used herein, the terms “cross-breeding,” “cross-pollination,” or “cross-breeding” refer to the process by which pollen from one flower of one plant is applied (artificially or naturally) to the ovule (stigma) of another flower of a different plant.

[0029] As used herein, the term “genotype” refers to the genetic structure of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.

[0030] As used herein, the term “heterozygous” refers to a diploid or polyploid individual cell or plant having different alleles (a given gene, gene determinant, or gene sequence form) located at at least one locus.

[0031] As used herein, the term "heterozygous" refers to the presence of different alleles (a given gene, gene determinant, or gene sequence form) at a particular locus.

[0032] As used herein, the terms “homologous chromosome” or “homolog” (or “homologue”) refer to a pair of one maternal chromosome and one paternal chromosome that are paired together during meiosis. These copies have the same genes at the same locus and the same centromere position.

[0033] As used herein, the term “homozygote” refers to an individual cell or plant having the same allele at one or more loci on all homologous chromosomes.

[0034] As used herein, the term “homozygous” means that identical alleles are present at one or more loci in homologous chromosome segments. Therefore, a plant that homozygously contains a mutant allele of the staygreen (sgr) gene on chromosome 9 in its genome contains the mutant allele in all copies of the sgr gene on chromosome 9, for example, in two copies if the plant is diploid and has two homologous sets of chromosome 9.

[0035] As used herein, the term “hybrid” refers to any individual cell, tissue, plant part, or plant resulting from the cross between two parents that differ in one or more genes. An F1 hybrid (HF1) results from the cross between two genetically distinct parent varieties or lines. The hybrid plants according to the present invention are heterozygous for one or more genes in their genome, but homozygous for the sgr gene, i.e., all of their sgr alleles (i.e., two in diploid plants) are loss-of-function mutant alleles. The loss-of-function mutations may or may not be the same for each sgr allele. Example 2 and Figure 1 describe a technique for generating HF1 plants containing homozygous sgr mutant alleles.

[0036] As used herein, two plants are referred to as "isogeneic" if they have the same or essentially the same set of chromosomes and genes, except for one of the sgr genes. Therefore, two isogenic plants contain different alleles of the sgr gene. The effect of allelic variation in the sgr gene can be evaluated by comparing the phenotypes of the two isogenic plants.

[0037] As used herein, “loss-of-function mutation” or “inactivation mutation” is a mutation that results in a gene product with reduced function or no function at all (partially or completely inactivated). When an allele is completely loss-of-function, it is also called a null allele. The phenotype associated with such mutations is generally recessive.

[0038] As used herein, the term “molecular marker” refers to an indicator used in methods for visualizing differences in the characteristics of nucleic acid sequences. Examples of such indicators include restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence characteristic amplified regions (SCARs), truncated amplified polymorphism sequence (CAPS) markers or isozyme markers, or combinations of markers described herein that define specific genetic and chromosomal locations. Mapping molecular markers in the vicinity of alleles is a procedure that can be performed very readily by those skilled in the art using common molecular techniques.

[0039] As used herein, the term “primer” refers to an oligonucleotide that can function as a starting point for DNA synthesis when annealed to an amplification target to bind DNA polymerase, thereby inducing the synthesis of a primer extension product, i.e., in the presence of a polymerization agent such as nucleotides and DNA polymerase, and at a suitable temperature and pH. Primers are preferably single-stranded to maximize amplification efficiency. Preferably, primers are oligodeoxyribonucleotides. Primers must be long enough to initiate the synthesis of the extension product in the presence of a polymerization agent. The exact length of the primer depends on many factors, including temperature and primer composition (A / T and G / C content). A pair of bidirectional primers consists of one forward primer and one reverse primer, as is commonly used in the field of DNA amplification, such as PCR amplification.

[0040] As used herein, a single nucleotide polymorphism (SNP) is a DNA sequence variation that occurs when a single nucleotide (A, T, C, or G) in a genome (or other shared sequence) differs between members of a species or between paired chromosomes in an individual. For example, two sequenced DNA fragments from different individuals contain a single nucleotide difference from AAGCCTA to AAGCTTA. In this case, there are two alleles, C and T.

[0041] As used herein, “marker-based selection,” “marker-assisted selection (MAS),” or “marker-assisted breeding (MAB)” refers to the use of genetic markers to detect one or more nucleic acids from plants, where the nucleic acids are associated with desired traits to identify plants that possess the genes for the desired (or undesirable) traits, and thus those plants can be used (or avoided) in a selective breeding program.

[0042] As used herein, "maturity" refers to the developmental stage of a melon fruit. The aging of the first leaves and fruit tendrils is a common indicator of maturity for both climachteric and non-climacteric melons.

[0043] Additional indicators of maturity in climachteric melons include cracking of the stalk or release of aroma. In non-climacteric melon varieties such as Piel de Sapo or Yellow Canary, maturity is indicated by browning or yellowing of the pistil area and coloration that progresses to the stalk.

[0044] As used herein, the terms “offspring” or “descendant” refer to any plant that arises as an offspring from the vegetative or sexual reproduction of one or more parent plants or their descendants. For example, offspring plants are obtained by cloning or self-pollination of parent plants, or by crossing two parent plants, and may include self-pollination and F1 or F2 or even further generations. F1 is the first generation of offspring produced from at least one parent used for the first time as a trait donor, while the second generation (F2) or subsequent generations (F3, F4, etc.) are specimens produced from self-pollination of F1, F2, etc.

[0045] Therefore, F1 may be a hybrid resulting from a cross between two true breeding parents (true breedings are homozygous for traits) (which is usually the case), and F2 may be an offspring resulting from self-pollination of the F1 hybrid (which is also usually the case).

[0046] As used herein, the term "melon" means any species, variety, or cultivar of the melon (Cucumis melo) species. The present invention encompasses plants of different ploidy levels, including not only diploid plants but also triploid plants, tetraploid plants, and so on.

[0047] As used herein, the term “plant part” refers to any part of a plant, including but not limited to buds, roots, stems, seeds, fruits, leaves, petals, flowers, ovules, branches, petioles, internodes, pollen, stamens, rootstock, scions, etc.

[0048] The term "resistance" is defined by the Vegetable and Ornamental Crops Section of the ISF (International Seed Federation) to describe a plant's response to pests or pathogens and the abiotic stresses of the vegetable seed industry. Specifically, resistance means the ability of a plant variety to limit the growth and development of a particular pest or pathogen, and / or the damage they cause compared to a susceptible plant variety under similar environmental conditions and pressures from the pest or pathogen. Resistant varieties may exhibit some disease symptoms or damage under strong pressures from pests and pathogens.

[0049] As used herein, the term “susceptible” refers to a plant that cannot restrict the growth and development of a particular pest or pathogen.

[0050] As used herein, the terms “inbreed” or “lineage” refer to a relatively true breeding lineage.

[0051] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism (e.g., plant), or group of organisms resulting from the interaction between its individual genetic structure (i.e., genotype) and its environment.

[0052] As used herein, the terms “gene transfer,” “gene transferred,” and “to transfer” refer to the process by which genes from one species, variety, or cultivar are transferred to the genome of another species, variety, or cultivar by crossing those species. Crossing may be natural or artificial. This process can optionally be completed by backcrossing to a repeating parent, in which case gene transfer refers to the intrusion of genes from one species into the gene pool of another species by repeatedly backcrossing an interspecific hybrid with one of its parents. Gene transfer can also be described as heterologous genetic material stably integrated into the genome of the recipient plant.

[0053] In this specification, comparisons between two or more melon plants or fruits, particularly between the melon plant according to the present invention and an isogenic melon that does not contain the mutant allele of the sgr gene on chromosome 9, should be understood as comparisons between plants or fruits grown under the same environmental conditions and at the same maturation stage or post-harvest stage.

[0054] Sequence List Sequence ID 1 shows the sequence of the wild-type sgr gene on chromosome 9.

[0055] Sequence ID 2 shows the sequence of the sgr-1 allele of the sgr gene, including the G584A mutation.

[0056] Sequence ID 3 shows the coding sequence for the wild-type sgr gene on chromosome 9.

[0057] Sequence ID 4 shows the amino acid sequence of the wild-type SGR protein.

[0058] Sequence ID 5 shows the context sequence for the development of markers around the sgr-1 mutation.

[0059] Sequence ID 6 shows the sequence of a forward primer for detecting the wild-type allele of the sgr gene.

[0060] Sequence ID 7 shows the sequence of a forward primer for detecting the sgr-1 mutant allele of the sgr gene.

[0061] Sequence ID 8 shows the sequences of common reverse primers for detecting the sgr-1 and wild-type mutant alleles of the sgr gene. [Brief explanation of the drawing]

[0062] [Figure 1] Figure 1 shows a breeding scheme for introducing the sgr-1 mutation into HF1 hybrids. [Figure 2] Figure 2 shows photographs of leaves from Charentais, Yellow Canary, and Galia melons containing the wild-type allele or the sgr-1 mutation. [Figure 3] Figure 3 shows photographs of Italian netted melon leaves containing wild-type alleles or sgr-1 mutations under CYSDV pressure. [Figure 4] Figure 4 shows the L*, a*, and b* values ​​for leaf color in varieties V1_Charentais and V2-Yellow Canary, which contain the wild-type allele or the sgr-1 mutation. [Figure 5] Figure 5 shows the change in ΔE* values ​​of leaf color over three dates, where the E* values ​​reflect the difference in leaf color in the CIELAB color space between sgr-1 mutant melons of varieties V1_Charentais and V2-Yellow Canary and their corresponding wild-type (WT) melons. [Figure 6] Figure 6 shows photographs of the peel of the V2-ItalianNet_NLSL variety after 7 days of storage, either with the WT (Panel A) or the sgr-1 mutation (Panel B). [Figure 7]Figure 7 shows the L*, a*, and b* values ​​of the peel color for varieties V1_Charentais_LSL, V2_ItalianNet_NLSL, and V3_ItalianNet_NLSL on the day of harvest (upper panel) or after 7 days of storage (lower panel) (from left to right for each genotype). [Figure 8] Figure 8 shows the ΔE* values ​​for varieties V1_Charentais_LSL, V2_ItalianNet_NLSL, and V3_ItalianNet_NLSL at two different dates (harvest date and 7 days after storage, from left to right). The ΔE value reflects the difference in rind color in the CIELAB color space between the sgr-1 mutant melon and the corresponding wild-type (WT) melon. [Figure 9] Figure 9 shows the L*, a*, and b* values ​​(from left to right for each genotype) of flesh color for varieties V1_Charentais_LSL, V2_ItalianNet_NLSL, and V6_YellowC_LSL containing the wild-type allele or the sgr-1 mutation after 7 days of storage. [Figure 10] Figure 10 shows the evaluation of cycle lengths for different melon varieties (V2_ItalianNet_NLSL, V4_HD_NLSL, and V5_Charentais_NLSL) that contain either the sgr-1 mutation or the wild-type allele. [Figure 11] Figure 11 shows the evaluation of pedicel detachment in different melon genotypes (V2_ItalianNet_NLSL, V4_HD_NLSL, and V5_Charentais_NLSL) containing either the sgr-1 mutation or the wild-type allele. [Figure 12] Figure 12 shows the measured Brix values ​​for different melon genotypes (V2_ItalianNet_NLSL, V4_HD_NLSL, and V5_Charentais_NLSL) that contain either the sgr-1 mutation or the wild-type allele. [Figure 13] Figure 13 shows the hardness measurements of different melons (V2_ItalianNet_NLSL, V4_HD_NLSL, and V5_Charentais_NLSL) containing either the sgr-1 mutation or the wild-type allele. [Modes for carrying out the invention]

[0063] Detailed explanation According to a first aspect, the present invention relates to a melon plant whose genome contains a homozygous mutant allele of the staygreen (sgr) gene on chromosome 9, wherein the mutant allele of the sgr gene contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1), and wherein the mutant allele of the sgr gene confers rind color stability to the fruit of the plant at maturity and / or after harvest compared to isogenic non-long-life (non-LSL) melon plants that do not contain the mutant allele. By containing a homozygous mutant allele (loss-of-function type) of the sgr gene, the mutant allele of the sgr gene is present in all homologs of chromosome 9, but it should be understood that they are not necessarily the same mutant allele, provided that all mutant alleles are actually loss-of-function mutations.

[0064] In one embodiment, the non-LSL melon plant is a traditional melon plant. In such a case, the corresponding isogenic mutant plant is an ISL melon variety or an LSL melon variety. In one embodiment, the non-LSL melon plant is an ISL melon plant. In such a case, the corresponding isogenic mutant plant is an LSL melon variety.

[0065] The melon plant of the present invention is characterized by a homozygous and inactivated sgr gene. The sgr gene is mapped to chromosome 9 of the melon genome (NCBI gene ID 103482692). The sequence of the wild-type allele of the sgr gene is shown in SEQ ID NO: 1. The coding sequence of the wild-type allele of the sgr gene is shown in SEQ ID NO: 3 and deposited in Genbank under accession XM_008438967 (updated June 7, 2016), with the coding sequence located at nucleotides 415-1188. The translated sequence, i.e., the wild-type amino acid sequence of the SGR protein, is deposited in Genbank under accession XP_008437189.1 (updated June 7, 2016) and is as shown in SEQ ID NO: 4.

[0066] In one embodiment, the mutant allele of the sgr gene is a loss-of-function allele, i.e., it contains at least one loss-of-function mutation. The sequence of the mutant allele may differ from the wild-type gene sequence by at least one nucleotide substitution, insertion, or deletion in the sequence. In particular, the mutation may be a single nucleotide polymorphism (SNP). The mutant allele of the sgr gene may also differ from the wild-type sgr gene sequence by one or more insertions or deletions of nucleic acid segments, including a complete gene deletion. The mutation may induce one or more amino acid substitutions in the sequence of the SGR protein, thereby impairing the function of the SGR protein.

[0067] In one embodiment, a loss-of-function mutation in the sgr gene is a null mutation. A null mutation prevents the expression of an active SGR protein. The mutation may be a nonsense mutation that prematurely halts the translation of mRNA into protein, resulting in the expression of a cleaved SGR protein. Alternatively, the mutation may be a framework mutation that causes a framework shift resulting in an abnormal sequence of amino acids. Alternatively, the mutation may be a deletion splicing mutation that causes an error in the splicing of premRNA into mature mRNA. The mutation may be a splicing site mutation, i.e., a mutation located at the splicing site of the gene, or located at any splicing regulatory sequence in an intron or exon.

[0068] In the present invention, nonsense mutations, framework mutations, or deletion splicing mutations have the advantage of resulting in a complete absence of functional protein expression, in contrast to missense mutations (single amino acid substitutions), which are the most frequently expressed and whose activity can be partially preserved.

[0069] Loss-of-function mutations may be located in any exon or intron of the sgr gene. In particular, the mutation may be located in the first, second, or third exon or in one of the first, second, or third introns.

[0070] In one embodiment, the mutation is a nucleotide substitution at the splicing site between a first intron and a second exon. In one embodiment, the mutation consists of substituting guanine with alanine at the last position of the first intron. This guanine is located at position 584 of SEQ ID NO: 1. This splicing site mutation, called sgr-1, was identified by the inventors in EMS mutant plants and translocated into different non-LSL and LSL genotypes. The sequence of the sgr-1 allele is shown in SEQ ID NO: 2.

[0071] Mutant alleles and their corresponding markers can be identified by methods known in the art.

[0072] Mutations in the mutated sgr allele can be induced by methods such as mutagenesis or genetic manipulation. Mutagenesis and genetic manipulation methods are known in the art and are described in more detail below.

[0073] Therefore, the plants according to the present invention may be obtained by different processes and are not exclusively obtained by biological processes.

[0074] The melon fruit according to the present invention is characterized by increased stability of rind color at maturity and after harvest compared to isogenic non-LSL fruit that does not contain the mutant allele of the sgr gene, as defined herein. The stability of rind color can be evaluated by comparing the rind color of mutant melons and isogenic non-mutant melons at different time points from maturity, preferably from the harvest date, and after harvest, preferably 7 to 21 days after harvest, particularly 7 to 14 days after harvest, and most particularly 7 or 14 days after harvest. Preferably, the stability of rind color is evaluated after 7 to 21 days under refrigerated storage conditions at temperatures including 4°C to 15°C. The same parameters can be applied to measuring any property of the melon of the present invention or its isogenic non-mutant counterpart.

[0075] In some embodiments, the color of the melon rind is evaluated by colorimetric analysis using a colorimeter such as a Konica Minolta CR400 or 2D image analysis from photographs of the fruit. The color measurements are in the CIELAB color space (CIE L). * a * b * It can be expressed as (also known as). The CIELAB color space is a color space defined by the International Commission on Illumination (CIE) in 1976. It is a color space for lightness from black (0) to white (100). * a from green (-) to red (+) * , and b from blue (-) to yellow (+)* represents color with three values. CIELAB is designed such that approximately the same amount of numerical change in these values corresponds to a visually recognized change. In this color space, a melon fruit that is visually recognized as greener has a lower a * value, and a melon that is visually recognized as yellower has a higher b * value.

[0076] In one embodiment, a melon fruit according to the present invention has a lower a * value and / or b * value at maturity and / or after harvest as compared to a homozygous non-LSL melon fruit that does not contain a mutant allele of the sgr gene. In one embodiment, the difference in the a * value and / or b * value between a melon fruit according to the present invention and a homozygous non-LSL melon fruit that does not contain a mutant allele of the sgr gene is statistically significant. In one embodiment, the a* value and / or b * value of the melon fruit of the present invention is at least 10%, preferably 20%, more preferably 30% lower, respectively, than the a * value and / or b * value of a homozygous non-LSL melon fruit that does not contain a mutant allele of the sgr gene.

[0077] The color difference in the CIELAB color space can be evaluated by the formula

Equation

[0078] In one embodiment, the difference in skin color between melon fruit according to the present invention and isogenic non-LSL melon fruit that does not contain the mutant allele of the sgr gene is statistically significant.

[0079] The color difference in the rind of non-mutant melons can also be evaluated visually using color evaluation tools.

[0080] The melon according to the present invention is also characterized by the fact that several characteristics of non-LSL melons, such as Brix, firmness, stalk detachment rate and aroma, or flesh color, remain unchanged or substantially unchanged. These non-LSL-like characteristics are particularly advantageous to producers and consumers and therefore have commercial value.

[0081] In one embodiment, the Brix degree of the fruit of the melon plant according to the present invention at maturity and / or after harvest is substantially unchanged compared to the fruit of a non-LSL isogenic plant grown at the same maturation stage and under the same environmental conditions, wherein the isogenic plant does not contain the mutant allele of the sgr gene in a homozygous state in its genome.

[0082] In particular, the Brix degree of the melon plant fruit according to the present invention varies by less than 20%, preferably less than 10%, and more preferably less than 5%, compared to the fruit of an isogenic non-mutant plant.

[0083] The term "Brix degree" or "Brix" refers to the soluble solids content of an aqueous solution of fruit juice, the majority of which is sugar. These are primarily estimated by a refractometer and measured as Brix degree. A higher degree indicates a higher sugar content. Brix measurement is important for evaluating the taste of melons, as fruits with low Brix, and therefore low sugar content, are not appreciated by consumers. Brix can be measured with a Brixmeter, also known as a refractometer, as is known to those skilled in the art.

[0084] The melons according to the present invention have a longer shelf life while retaining the same or substantially the same Brix as non-LSL isogenic melons that do not contain the sgr mutation, in order to accumulate the sweetness of non-LSL melons, particularly traditional melons. Therefore, the melons of the present invention avoid the typical drawbacks of LSL melons, where increased shelf life is generally associated with a lack of flavor.

[0085] In one embodiment, the hardness of the fruit of the melon plant according to the present invention at maturity and / or after harvest is substantially the same as that of the fruit of an isogenic plant grown at the same maturation stage and under the same environmental conditions, and the isogenic plant does not contain the mutant allele of the sgr gene in a homozygous state in its genome.

[0086] In particular, the hardness of the fruit of the melon plant according to the present invention differs by less than 20%, preferably less than 10%, compared to the fruit of an isogenic plant. Hardness can be measured by a penetrometer, as is known to those skilled in the art.

[0087] Non-LSL melons gradually lose their firmness during maturation through an ethylene-dependent process. The melons of the present invention exhibit firmness characteristics similar to or essentially similar to non-LSL melons, and therefore tend to soften in a similar manner to non-LSL melons during maturation.

[0088] In one embodiment, the degree of pedicel detachment of the fruit of the melon plant according to the present invention during maturation and / or after harvest is substantially the same as that of the fruit of an isogenic plant grown at the same maturation stage and under the same environmental conditions, and the isogenic plant does not contain the mutant allele of the sgr gene in a homozygous state in its genome.

[0089] In particular, the degree of detachment of the fruit stalk of the melon plant according to the present invention varies by less than 20%, preferably less than 10%, compared to the fruit of the isogenic plant.

[0090] Detachment of the fruit stalk is a good indicator of maturity. In commercial maturity, non-LSL melon varieties generally form a detachment layer at the attachment point of the fruit stalk, while LSL melon varieties do not. For this reason, LSL melons are also called non-slip melon fruits because they need to be cut from the vine for harvesting. Therefore, the presence of a detachment layer on the fruit stalk is particularly useful for growers in determining when the melons can be harvested. The melons of this invention have the advantage of having a visible detachment layer and are similar to, or essentially similar to, isogenic non-mutant non-LSL plants in terms of appearance and development.

[0091] The stage of detachment of the fruit stalk can be visually evaluated on a scale from 1 to 9, where 1 means it has fallen off and 9 means it has not fallen off.

[0092] In one embodiment, the length of the fruit cycle of the melon plant according to the present invention at maturity is substantially the same as that of the fruit of an isogenic plant grown under the same environmental conditions, and the isogenic plant does not contain the mutant allele of the sgr gene in a homozygous state in its genome.

[0093] In particular, the length of the fruit cycle of the plant according to the present invention varies by less than 20%, preferably less than 10%, and more preferably less than 5%, compared to the fruit of the isogenic plant. The length of the cycle corresponds to a period, for example, the number of days from planting to harvest. Non-LSL melons become ready for harvest earlier than LSL melons, meaning their cycle length is shorter compared to LSL melons, which leads to an increase in yield. Therefore, it is desirable to maintain a cycle length similar to that of non-LSL melon varieties, and thus the same harvest period.

[0094] The SGR mutation in melon plants according to the present invention can also affect the leaves, more specifically, the leaf color. In particular, the plants of the present invention show a reduction in leaf yellowing and necrosis.

[0095] In one embodiment, the flesh color of the melon plant fruit according to the present invention at maturity and / or after harvest is substantially unchanged compared to the fruit of an isogenic plant grown at the same maturation stage and under the same environmental conditions, and the isogenic plant does not contain the mutant allele of the sgr gene in a homozygous state in its genome. In particular, the flesh color of the melon plant fruit according to the present invention * value and / or b * The value varies by less than 20%, preferably less than 10%, compared to the fruit of the isogenic plant. The color difference of the flesh is also expressed by formula ΔE * This can be evaluated in the CIELAB color space. In one embodiment, the ΔE of the flesh color between a melon fruit according to the present invention and an isogenic non-LSL melon fruit that does not homozygously contain the mutant allele of the sgr gene in its genome. * The value is less than 50, preferably less than 10, more preferably less than 2, and more preferably less than 1.

[0096] In one embodiment, the leaves of the melon plant according to the present invention show reduced yellowing compared to the isogenic non-LSL plant, and the isogenic plant does not contain the mutant allele of the sgr gene in a homozygous state in its genome.

[0097] Leaf color can be evaluated visually or by colorimetric analysis using a colorimeter. The CIElab color system, in particular, can be used.

[0098] In one embodiment, the leaf color of the melon plant according to the present invention is characterized by lower a* and / or lower b* values ​​compared to an isogenic non-LSL plant that does not contain the mutant allele of the sgr gene.

[0099] Therefore, leaf color evaluation can be used as a substitute for identifying non-LSL plants that exhibit the desired phenotype, i.e., stability of pericarp color at maturity and / or after harvest.

[0100] The reduction in leaf yellowing exhibited by the plants of the present invention is also reflected as resistance, and more specifically, as partial resistance of the plants of the present invention to yellowing diseases such as CYSDV (cucurbit yellow stunting disorder virus).

[0101] Therefore, in some embodiments, the melon plants according to the present invention are resistant to CYSDV (Cucurbit Yellow Developmental Disorder Virus), and such resistance is provided by allelic mutations in the sgr gene. In particular, the resistance is partial resistance.

[0102] CYSDV is a crossterovirus transmitted in nature by the tobacco whitefly (Bemisia tabaci). CYSDV induces yellowing spots between the veins of mature leaves, which can expand and eventually fuse, causing the entire leaf to yellow except for the veins that remain green. The yellowing symptoms result in a significant decrease in fruit yield and quality, and therefore, this virus is of high economic importance.

[0103] The sgr mutation in melon plants according to the present invention can reduce damage caused by CYSDV by masking specific symptoms of CYSDV in infected plants, particularly leaf yellowing. Resistance to CYSDV is advantageously determined by comparison with susceptible (commercially available) lines.

[0104] In one embodiment, the melon plant according to the present invention is a plant derived from an inbreeding melon line.

[0105] In a preferred embodiment, the melon plant according to the present invention is an F1 hybrid melon plant.

[0106] The present invention also relates to a group of melon plants according to the present invention, wherein the group comprises at least five plants, in particular at least ten plants, more particularly at least 20 plants, even more particularly at least 50 or 100 plants, or more particularly at least 1000 plants.

[0107] The present invention also covers further embodiments, which are detailed below. All embodiments detailed in the above section relating to the first aspect of the present invention are also embodiments according to these further aspects of the present invention.

[0108] According to a second aspect, the present invention relates to a cell of a melon plant according to the present invention, wherein the cell contains a mutant allele of the staygreen (sgr) gene on chromosome 9 in its genome, and the mutant allele of the sgr gene contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1).

[0109] The plant cells of the present invention may have the ability to regenerate throughout the entire plant.

[0110] Alternatively, the present invention also relates to plant cells that are not regenerative and therefore cannot produce an entire plant.

[0111] According to one embodiment, the cells are derived from an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, and / or hypocotyl.

[0112] In one embodiment, the present invention relates to the plant portion of a melon plant. The present invention also relates to the plant portion of a melon plant comprising at least one cell according to the present invention.

[0113] According to one embodiment, the plant part is an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, and / or hypocotyl. In one embodiment, the plant part is the fruit of a melon plant according to the present invention.

[0114] Another aspect of the present invention relates to melon seeds that can be grown into melon plants according to the present invention. Thus, such seeds are the seeds of the plant of the present invention, i.e., seeds that give rise to the plant of the present invention. The present invention also relates to seeds derived from the plant of the present invention, i.e., seeds obtained from such plants after self-pollination or cross-pollination, provided that the plants obtained from such seeds contain, in a homozygous manner, a loss-of-function mutant allele of the sgr gene that confers rind color stability to the fruit of the plant at maturity and / or after harvest, compared to isogenic non-LSL melon plants that do not contain the mutant allele.

[0115] The present invention also relates to a group of melon seeds according to the present invention, wherein the group comprises at least two seeds, in particular at least ten seeds, in particular at least 100 seeds, and even more particularly at least 1000 seeds.

[0116] Another aspect of the present invention is an in vitro cell or tissue culture of regenerative cells of a melon plant according to the present invention. Preferably, the regenerative cells are derived from an embryo, protoplast, meristem cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, seed, flower, cotyledon, and / or hypocotyl. The regenerative cells contain a loss-of-function mutant allele of the sgr gene described above in their genome.

[0117] The tissue culture can preferably regenerate plants having the physiological and morphological characteristics of the aforementioned melon plants, and can regenerate plants having substantially the same genotype as the aforementioned melon plants. The present invention also provides melon plants regenerated from the tissue culture of the present invention.

[0118] The present invention also provides a protoplast of a plant as defined above or a protoplast derived from tissue culture as defined above, which contains a loss-of-function mutant allele of the sgr gene in its genome.

[0119] In another aspect, the present invention also relates to the use of melon plants detailed in accordance with the present invention as breeding partners in a breeding program for obtaining melon plants with increased shelf life, and in particular increased stability of rind color at maturity and / or after harvest. In fact, such a melon plant according to the first aspect possesses in its genome a loss-of-function allele of the sgr gene defined above, which confers rind color stability at maturity and / or after harvest. By crossing this plant with a plant that does not contain the mutation, it is possible to transmit this allele conferring the desired phenotype to the offspring. Thus, the plant according to the present invention can be used as a breeding partner for gene transfer of a mutant allele conferring the desired phenotype into a melon plant or germplasm.

[0120] In such breeding programs, the selection of offspring exhibiting a desired phenotype or having sequences associated with a desired phenotype can be advantageously carried out based on the alleles and corresponding markers disclosed above herein.

[0121] The present invention also relates to the use of the plant in a program aimed at identifying, sequencing, and / or cloning gene sequences that confer a desired phenotype.

[0122] In another aspect, the present invention also relates to a method for producing melon plants, particularly commercial plants, with increased shelf life. A method or process for producing plants having these features is as follows: (a1) A step of crossing a melon plant according to the present invention, which contains a mutant allele of the sgr gene in a homozygous state (wherein the sequence of the mutant allele of the sgr gene contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1)), with a second melon plant that does not contain the mutant allele in a homozygous state, thereby generating an F1 population. (a2) A step of advancing the F1 group to create the F2 group, (b) The step of selecting one plant from the offspring thus obtained that contains the mutant allele in a homozygous state, (c) Optionally, the plants obtained in step b) are self-pollinated once or several times, (d) Optionally, the plant selected in step b) or c) is backcrossed with a melon plant that does not homozygously contain the mutant allele, (e) A step of selecting a plant that contains the mutant allele in a homozygous manner, wherein the plant produces fruit with increased shelf life, (f) The optional step of crossing the selected plant with a different melon plant that contains the mutant sgr allele in a homozygous state, thereby producing a hybrid melon plant that contains the mutant sgr allele in a homozygous state. including

[0123] The plant selected in step (e) or produced in step (f) is preferably a commercial variety, cultivar, or type of melon. In some embodiments, the selected plant is derived from one of the following species: Charentais, Italian netted, Western Shipper, Eastern Shipper, Galia, Ananas, and Honey Dew.

[0124] Preferably, steps c) and / or d) are repeated at least twice, preferably three times, but it is not necessary to use the same melon plant that does not homozygously contain the mutant allele. The melon plant that does not homozygously contain the mutant allele is preferably a breeding line.

[0125] The steps of self-pollination and backcrossing may be performed in any order and may be inserted in between. For example, backcrossing may be performed before and after one or more self-pollinations, and self-pollination may be performed before and after one or more backcrosses.

[0126] In some embodiments, such a method is advantageously carried out by using nucleic acid markers for one or more selections performed in step b) or e) to select plants containing a mutant allele of the sgr gene in a homozygous state.

[0127] The selection made in step b) or e) can be made using any type of genetic marker, in particular restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), simple sequence length polymorphism (SSLP), single nucleotide polymorphism (SNP), insertion / deletion polymorphism (indel), variable number tandem repeat (VNTR), and randomly amplified polymorphic DNA (RAPD), isozymes, as well as other markers known to those skilled in the art.

[0128] Methods used for detecting markers and alleles can be based on any technique that allows for the distinction between two different alleles of a marker on a particular chromosome. Polymorphism detection can be performed by gel electrophoresis, including single-strand conformational polymorphism (Orita, et al. (1989) Genomics, 8(2), 271-278), denaturing gradient gel electrophoresis (Myers (1985) EPO0273085), or cleavage length polymorphism (Life Technologies, Inc., Gaithersburg, MD), but with the widespread availability of DNA sequencing, it is often easier to simply and directly sequence amplification products. If differences in polymorphic sequences are known, rapid assays for detecting polymorphisms can be designed for progeny testing, generally including several forms of PCR amplification of a specific allele (PASA; Sommer, et al. (1992) Biotechniques 12(1), 82-87) or PCR amplification of multiple specific alleles (PAMSA; Dutton and Sommer (1991) Biotechniques, 11(6), 700-7002). In certain examples, PCR detection and quantification are performed using two labeled fluorescent oligonucleotide forward primers and an unlabeled common reverse primer, e.g., KASPar® (KBiosciences). Polymorphism detection can also be performed by electrophoretic techniques, including single-strand conformational polymorphism (Orita, et al. (1989) Genomics, 8(2), 271-278), denaturing gradient gel electrophoresis (Myers (1985) EPO0273085), or cleavage length polymorphism (Life Technologies, Inc., Gaithersburg, Md.). With the widespread availability of DNA sequencing, it has also become possible to directly sequence amplification products.

[0129] The present invention also relates to melon plants that can be obtained or obtained by the methods described herein. Such plants are, in fact, melon plants having the characteristics described in a first aspect of the present invention.

[0130] The plant is preferably a commercial variety, cultivar, or type of melon. The plant is preferably an F1 hybrid melon plant. In some embodiments, the plant is one of the following types: Charentais, Italian netted, Western Shipper, Eastern Shipper, Galia, Ananas, and Honey Dew.

[0131] Methods for producing seeds of melon plants are also provided. In some embodiments, the method includes crossing the melon plant according to the present invention with itself or another melon plant, and harvesting the resulting seeds.

[0132] In addition to the gene transfer of the mutant allele of the sgr gene detailed in the method of the present invention, the sequence can be introduced into the melon background by genetic engineering to obtain a particularly advantageous feature of the present invention, namely, a commercially viable melon plant with increased shelf life. Identification and cloning of the gene-transferred mutant allele conferring the desired phenotype is routine for those skilled in the art.

[0133] It should be noted that the seeds or plants of the present invention may be obtained by different processes, particularly by technical processes such as mutagenesis, for example, chemical mutagenesis or UV mutagenesis, or by genetic manipulation such as guided recombination or genome editing, and are not exclusively obtained by essentially biological processes.

[0134] In one embodiment, the present invention relates to a method for producing or producing melon plants that produce fruits with increased shelf life, comprising introducing a loss-of-function mutation into the sgr gene on chromosome 9 in the genome of a non-LSL melon plant, wherein the mutation is introduced by mutagenesis or genome editing, particularly by techniques selected from ethylmethanesulfonic acid (EMS) mutagenesis, oligonucleotide-specific mutagenesis (ODM), zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN), CRISPR / Cas system, engineered meganuclease, reengineered homing endonuclease, and DNA-guided genome editing. Preferably, the loss-of-function mutation is introduced into all copies of the sgr gene present on chromosome 9.

[0135] In particular, one embodiment of the present invention is a method for producing fruit with an increased shelf life or for obtaining a melon plant or its seeds that are easy to produce such fruit with, a) Treating M0 seeds of a modified melon plant, preferably a non-LSL melon plant, with a mutagenic agent to obtain M1 seeds, b) To grow plants from the M1 seeds obtained in this way and obtain M1 plants, c) Producing M2 seeds through self-fertilization of M1 plants, d) Optionally, repeat steps b) and c) n times to obtain M2+n seeds. This includes methods.

[0136] In this method, the M1 seeds of step a) can be obtained by chemical mutagenesis such as EMS mutagenesis, or by any other chemical mutagenerator including, but not limited to, diethyl sulfate (des), ethyleneimine (ei), propanesultone, N-methyl-N-nitrosulfurethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (enu), and sodium azide. Alternatively, the mutation can be induced by irradiation, for example, selected from X-rays, fast neutrons, or UV rays.

[0137] In another embodiment of the present invention, mutations are induced by genetic engineering. Such mutations include the incorporation of sequences that confer the phenotype of the mutant plant according to the present invention, particularly stability of the pericarp color, and the substitution of constitutive sequences with alternative sequences that confer the phenotype of the mutant plant according to the present invention, particularly stability of the pericarp color.

[0138] The genetic engineering manipulation methods that can be used include the use of all such techniques called novel breeding techniques, which are various new techniques developed and / or used to create new traits in plants by genetic mutation for the purpose of targeted mutagenesis, targeted introduction of new genes, or gene silencing (RdDM). Examples of such novel breeding techniques include zinc finger nuclease (ZFN) techniques (ZFN-1, ZFN-2, and ZFN-3, see U.S. Patent No. 9,145,565 (the whole is incorporated by reference)), oligonucleotide-specific mutagenesis (ODM), cisgenesis and intragenesis, RNA-dependent DNA methylation (RdDM, which does not necessarily alter the nucleotide sequence but can alter the biological activity of the sequence), grafting (on genetically modified (GM) rootstock), reverse breeding, agroinfiltration (agroinfiltration "narrowly defined", agroinoculation, floral dip), and transcription activator-like effector nucleases. Aase (TALEN, see U.S. Patents 8,586,363 and 9,181,535 (all incorporated herein by reference)), CRISPR / Cas systems (see U.S. Patents 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233 and 8,999,641 (all incorporated herein by reference)), manipulated meganucleases, remanufactured homing endonucleases, DNA-guided genome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038 / nbt.3547 (the entire article is incorporated by reference), and targeted sequence changes facilitated by the use of synthetic 5 genomics. Most targeted genome editing today (another name for new breeding techniques) is an application that induces DNA double-strand breaks (DSBs) at selected sites in the genome where modification is intended. Directed repair of DSBs enables targeted genome editing.Such applications can be used to induce mutations (such as targeted mutations or precise native gene editing) and precise gene insertions (such as cisgenesis, intragenesis, and transgenesis). Applications that induce mutations are often identified as site-directed nuclease (SDN) techniques, e.g., SDN1, SDN2, and SDN3. In the case of SDN1, the result is a targeted, nonspecific gene deletion mutation. The site of the DNA DSB is precisely selected, but DNA repair by the host cell is random, resulting in small nucleotide deletions, additions, or substitutions. In the case of SDN2, SDN is used to induce a targeted DSB, and a DNA repair template (a short DNA sequence identical to the targeted DSB DNA sequence except for one or more nucleotide changes) is used to repair the DSB, thereby resulting in a targeted point mutation in the desired gene of interest. With regard to SDN3, SDN is used in conjunction with a DNA repair template containing a new DNA sequence (such as a gene). The result of this technique is the integration of that DNA sequence into the plant genome. The most likely application illustrating the use of SDN3 is the insertion of cisgenic, intragenic, or transgenic expression cassettes at selected genomic locations. A complete description of each of these techniques can be found in the report (in its entirety by reference) prepared in 2011 by the Institute for Predictive Technologies of the Joint Research Centre (JRC) of the European Commission, entitled "New plant breeding techniques—State-of-the-art and prospects for commercial development."

[0139] DNA editing technology has been successfully used in melons to inactivate targeted genes at specific locations (Hooghvorst, et al. “Efficient knockout of phytoene desaturase gene using CRISPR / Cas9 in melon.” Scientific reports 9.1(2019):1-7).

[0140] The present invention also provides a method for identifying, detecting, and / or selecting melon plants that produce or are more likely to produce fruits with increased shelf life, comprising detecting a mutant allele of the sgr gene on chromosome 9 in the genome of the plant, wherein the mutant allele contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1).

[0141] In one embodiment, the loss-of-function allele is selected from nonsense mutations, indel mutations, framework mutations, or deletion splicing mutations, particularly splicing site mutations.

[0142] In one embodiment, the method includes the detection of a guanine substitution at position 584 of SEQ ID NO: 1, i.e., the allele sgr-1 whose sequence is shown in SEQ ID NO: 2.

[0143] In some embodiments, detection of mutant alleles of the sgr gene is performed by amplification by PCR using, for example, KASPar® (KBiosciences) technology, with, for each marker, one forward primer that can be used to amplify the resistance allele, one forward primer that can be used to amplify the sensitivity allele, and one common reverse primer. In particular, the primers for amplifying each of the markers may have the sequences listed in Table 1.

[0144] In a preferred embodiment, amplification is performed using a two-step touchdown method in which the extension and annealing steps are combined into a single step. The temperature used in the annealing step determines the specificity of the reaction and, therefore, the ability of the primers to anneal to the DNA template. Touchdown PCR includes a first step of Taq polymerase activation, followed by a second step called the touchdown step, which includes a high annealing temperature and gradually decreases the annealing temperature in each PCR cycle, and a third step of DNA amplification. The higher annealing temperature in the initial touchdown cycle ensures that only highly specific base pairing occurs between the DNA and primers, and therefore the first sequence amplified is most likely to be the sequence of interest. Gradually decreasing the annealing temperature increases the efficiency of the reaction. The region initially amplified during the highly specific initial touchdown cycle is further amplified, outcompeting any nonspecific amplification that may occur at lower temperatures.

[0145] In another embodiment, amplification of the SNP marker is recommended in the KASPar assay and is carried out as illustrated in the example (see Example 1).

[0146] In a further embodiment, the present invention also provides one or more molecular markers for identifying melon plants that produce or are prone to producing fruits with increased shelf life, wherein the molecular markers detect loss-of-function mutations in the sgr gene on chromosome 9.

[0147] The use of one or more molecular markers for detecting melon plants that produce or are prone to producing fruits with increased shelf life, wherein the molecular markers detect loss-of-function mutations in the sgr gene on chromosome 9, is further provided.

[0148] According to these embodiments of the present invention, the molecular marker may be located in the sgr gene or in a chromosomal region genetically related to the sgr gene. In one embodiment, the molecular marker identifies a guanine substitution by alanine at position 584 of SEQ ID NO: 1. In one embodiment, the molecular marker is located within the sequence shown in SEQ ID NO: 5.

[0149] The present invention also provides a method for identifying molecular markers suitable for detecting melon plants that produce or readily produce fruits with increased shelf life, (a) Identifying molecular markers in the sgr gene or chromosomal regions genetically related to the sgr gene, (b) To determine whether the molecular marker is associated with or related to a phenotype of increased shelf life of melon fruit, particularly increased stability of rind color at maturity and / or after harvest. This applies to methods that include [specific methods].

[0150] In a further embodiment, the present invention relates to a method for producing melon plantlets or plants that produce fruit with increased shelf life or that facilitate the production of such fruit, i. To produce melon microplants by culturing isolated cells or tissues of a melon plant in vitro according to the present invention, ii. To produce melon microplants with increased shelf life or to develop them into melon plants that are more likely to produce such fruits by using them during the in vivo cultivation period. This applies to methods that include [specific methods].

[0151] The isolated cells or tissues used to produce microplants are explants obtained under sterile conditions from the melon parent plant of the present invention to be propagated. The explants include, for example, cotyledons, hypocotyls, stem tissue, leaves, embryos, meristems, node buds, shoot apices, or protoplasts. The explants can be surface-sterilized before being placed in a culture medium for micropropagation.

[0152] Conditions and culture media suitable for micropropagation of plants are well known to those skilled in the art of plant cultivation, and are described, for example, in "Plant Propagation by Tissue Culture, Handbook and Directory of Commercial Laboratories, eds. Edwin F George and Paul D Sherrington, Exegetics Ltd, 1984".

[0153] Microproliferation typically includes: 1. Axillary bud formation: Axillary bud proliferation is induced by adding cytokinin to the bud culture medium, preferably with minimal callus formation, to produce buds; ii. Generation of adventitious buds: Root formation is induced by adding auxin to the culture medium to produce small plants that can be transplanted into the soil. Alternatively, root formation can be induced directly in the soil.

[0154] The small plants can be further subjected to cultivation in soil under laboratory conditions, followed by an in vivo cultivation period with progressive adaptation to the natural climate, and grown into melon plants according to the present invention.

[0155] The reduction in leaf yellowing exhibited by the melons of the present invention makes it possible to reduce yield losses caused by various physiological or pathological conditions, such as leaf yellowing due to aging or the presence of yellowing diseases such as CYSDV infection. Accordingly, the present invention also relates to a method for improving the yield of melon plants or a method for increasing the number of harvestable melon plants or fruits, comprising growing melon plants according to the present invention that homozygously contain a mutant allele of the sgr gene on chromosome 9, wherein the mutant allele contains at least one loss-of-function mutation, resulting in a reduction in leaf yellowing. In one embodiment, melon plants according to the present invention are grown in an environment infected with CYSDV.

[0156] Preferably, the method includes a first step of selecting or choosing melon plants containing the mutant allele. The method may also be defined as a method for increasing the productivity of melon fields, tunnels, or greenhouses, or as a method for reducing the intensity or frequency of the use of chemicals or fungicides in melon production.

[0157] The present invention also relates to a method for reducing the loss of melon production, comprising growing the melon plants as defined above. In particular, the method involves growing the melon plants in a state of CYSDV infection.

[0158] In another embodiment, the present invention relates to a method for protecting melon fields, tunnels, or greenhouses, or any other type of planting, from yellowing diseases such as CYSDV infection, or at least a method for limiting the level of infection or limiting the spread of the disease. Such a method preferably comprises the step of growing a yellowing disease-resistant plant of the present invention, i.e., a plant having a mutant allele of the sgr gene on chromosome 9, wherein the mutant allele contains at least one loss-of-function mutation.

[0159] The present invention also relates to the use of melon plants resistant, particularly partially resistant, to yellowing diseases such as CYSDV, according to the present invention, in fields, tunnels, greenhouses, or other plantings.

[0160] The present invention also provides a method for improving the yield of melon plants in an environment infected with CYSDV, (a) Identifying a melon plant resistant to CYSDV, wherein the plant contains a mutant allele of the sgr gene on chromosome 9 in a homozygous state, and the mutant allele contains at least one loss-of-function mutation. (b) Growing the resistant melon plants in the infected environment This applies to methods that include [specific methods].

[0161] This method increases the yield of melon plants, in particular allowing for the harvesting of more marketable melons, or the production of more commercially viable melons, or the acquisition of more seeds.

[0162] The present invention further relates to a method for improving the shelf life of melon fruit, the marketability of melon fruit, and / or the yield of melon production, comprising growing melon plants according to the present invention and harvesting the fruit produced by the plants. Due to the increased shelf life, melons according to the present invention can be harvested less frequently than existing non-LSL melons, particularly 2 to 4 times a week. Accordingly, the present invention also relates to a method for increasing the flexibility of melon harvesting, comprising growing melon plants according to the present invention and harvesting the fruit produced by the plants.

[0163] In one embodiment of these methods, the fruit is stored for at least 7 days, preferably 7 to 21 days, after harvesting.

[0164] In yet another embodiment, the present invention also relates to a method for producing melon fruit, (a) To grow the melon plant of the present invention as defined above, (b) To enable the plant to bear fruit, (c) The fruits of the plant are preferably harvested when they are mature and / or before they are mature. This includes methods.

[0165] All preferred embodiments relating to melon plants have already been disclosed in the context of earlier aspects of the present invention.

[0166] The method may, advantageously, include a further step of processing the melon plant into a processed food.

[0167] In another aspect, the present invention relates to the use of the melon plant or its fruit according to the present invention in the fresh cut market or for food processing.

[0168] Throughout this application, the term “including” should be interpreted as encompassing all features specifically mentioned, as well as any additional, unspecified features of any choice. As used herein, the use of the term “including” also discloses embodiments that consist of no features other than those specifically mentioned (i.e., “consisting of”). [Examples]

[0169] Example 1: Generation and identification of mutant melons by EMS mutagenesis Mutagenesis was induced by soaking mature seeds from climachteric Charentais cultivars in 1%–3% ethyl methanesulfonate (EMS) for 16 hours, followed by washing with 0.1M Na2SO3. The seeds were then rinsed and sown in soil. M2 seeds were collected from M1 plants. Genomic DNA was extracted from M2 plants, and a SNP in the sgr gene on chromosome 9 was identified, which was a G→A substitution at the splicing site at the end of the first intron. This mutant allele is designated sgr-1. Its sequence is shown in Sequence ID No. 2.

[0170] Sgr-1 mutations can be identified using the KASPar® (KBiosciences) assay with two labeled fluorescent oligonucleotide forward primers and a common unlabeled reverse primer (Table 1).

[0171] [Table 1]

[0172] Example 2: Gene transfer of sgr-1 mutation The sgr-1 mutation was introduced into different elite genotypes. The sgr-1 mutation from the EMS population is recessive, and its effect is only present when the mutation is homozygous. Various parent lines were transformed to produce HF1 hybrids exhibiting this effect. After the initial cross between the parent line and the sgr-1 source, the sgr-1 mutation was backcrossed several times in the parent line (Figure 1). The two resulting transformed lines were crossed together to produce homozygous HF1 hybrids for the sgr-1 mutation, which are nearly isogeneic lines (NIL) with respect to HF1 lacking the sgr-1 mutation.

[0173] Several genotypes, including the following orange-fleshed varieties: Charentais, Italian netted, Western Shipper, and Eastern Shipper, as well as the following white and green-fleshed varieties: Yellow Canary, Piel de Sapo, Galia, and Honey Dew, were transformed with the sgr-1 mutation.

[0174] Example 3: Effects of sgr-1 mutation on leaves To evaluate the effect of the sgr-1 mutation on leaf yellowing and necrosis, the inventors evaluated the leaf color of different melon genotypes with and without the sgr-1 mutation. Leaf color is evaluated at various stages during plant growth, typically in the early stages before fruit set (date 1), during fruit maturation (date 2), and in the later stages during or immediately after fruit harvest (date 3). Several plants were evaluated for each genotype (5-10 plants), and leaf color could be evaluated visually and using a colorimeter. For example, leaf color could be measured using a Konica Minolta CR400 colorimeter. After obtaining two measurements in the plant for a given date, the three coordinates (L) in the CIELAB color space were used. * a * , and b * The average plant value is obtained by averaging over ). The leaves measured are selected to represent the plant (not too young, not too old). Then, the average of the values ​​from all plants is used to obtain the average L at the genotype level for a given date.* a * , and b * It is possible to calculate this.

[0175] A significant and visible effect on leaf color was observed in the V1_Charentais and V2_Yellow Canary lines (Figure 2).

[0176] To more accurately evaluate color progression and color difference, leaf color was measured using a colorimeter. Lower L values ​​were observed for the sgr-1 genotype. * a * , and b * The values ​​reflect a darker, greener leaf color. Furthermore, the reduced variance in the sgr-1 data indicates greater leaf color stability and a decrease in leaf yellowing due to the sgr-1 mutation compared to the initial genotype (Figure 4).

[0177] When comparing the mean of the sgr-1 converted line with the original line using the ANOVA test, a significant effect was observed in three coordinates (L). * a * , b * It is recorded in at least one of the following (Table 2). In particular, a clear conclusion can be drawn about the significant effect of the sgr-1 mutation on leaf color at the fruit maturation and late stages.

[0178] [Table 2]

[0179] The color difference ΔE is calculated using the following formula. * The calculation supports this conclusion.

number

[0180] In addition to the visible effects of sgr-1 on leaves under normal conditions, a strong effect of sgr was also observed under CYSDV pressure (natural infection in areas severely affected by the virus). While the virus was still present in the leaves of plants with the sgr-1 mutant allele, visible symptoms disappeared, and plants with the sgr-1 mutant allele showed less yellowing than plants with the corresponding wild-type allele. The sgr-1 mutation provides interesting partial resistance to the yellowing symptoms of CYSDV (Figure 3).

[0181] Example 4: Effect of sgr-1 mutation on peel color During the conversion of the strains, the inventors observed the effect of the sgr-1 mutation on the rind color of melons of different varieties. The fruits tended to remain greener. The inventors evaluated this effect by visual and colorimetric analysis both on the day of harvest and 7 days after storage. Observations and measurements were performed on orange-fleshed material from different melon genotypes, more specifically from the following three varieties: V1_Charentais_LSL, V2_ItalianNet_NLSL, and V3_ItalianNet_NLSL.

[0182] In orange-fleshed fruit material, a clear color difference was observed between the V2_ItalianNet_NLSL wild type and the sgr-1 converted variety at harvest. The V2_Italian netted variety is non-LSL and turns yellow when mature. However, the peel of the sgr-1 form remains green.

[0183] After 7 days of storage, the fruits of wild-type (WT) V2_ItalianNet_NLSL increasingly change to a yellowish-orange color, while the fruits of V2_ItalianNet_NLSL sgr-1 do not appear to change and retain their green peel color (Figure 6). The color version of Figure 6 clearly shows the effect of sgr-1 on the peel color. Color versions of all figures in this application, including Figure 6, are submitted with this application and are available upon request.

[0184] We also observed V1_Charentais_LSL, an LSL genotype that does not change to yellow when mature. At harvest, no color difference was observed between the sgr-1 V1_Charentais_LSL and the wild type. This indicates that the sgr-1 genotype does not affect the skin color of the LSL genotype of melon, or only slightly.

[0185] Furthermore, the recessive effect of the sgr mutation was also confirmed in terms of pericarp color. No differences were observed between the wild-type V3_ItalianNet_NLSL and the sgr-1 heterozygous morphology.

[0186] Even when the effect of sgr-1 is clearly visible, it can also be shown using colorimetric analysis data. Due to the presence of netting on the surface of the peel, colorimetric tools were not suitable for such measurements. Instead, image analysis was used to extract the color of the peel and L * a * , and b * The value can be accessed.

[0187] L * Value and b * Compared to the wild type, a significant statistical effect was observed in the sgr-1 mutation of the non-LSL genotype in variety 2 regarding the value (Figure 7). * a * b * The values ​​reflect the fact that the wild type has a more yellow peel color compared to the greener sgr-1 morph. No difference was observed between sgr-1 and the wild type in variety 1, which is LSL. No significant difference was recorded between the heterozygous sgr-1 morph and the wild type in non-LSL variety 3. To clarify the effect on peel color, the mutation needs to be homozygous and in a non-LSL background.

[0188] ΔE between sgr-1 conversion mode and WT corresponding system *The color difference can be calculated using the formula. The effect of the sgr-1 mutation on variety 2 is evident at J0 (harvest day), and increases slightly at J7 (7 days of storage), with ΔE * While the ΔE of other varieties is greater than 10 (26.1 and 30.7 respectively), * It is less than 10 (Figure 8).

[0189] Example 5: Effect of sgr-1 mutation on fruit pulp color To control the potential impact on the quality characteristics of this fruit, the inventors further evaluated the effect of the sgr-1 mutation on the color of melon flesh.

[0190] The flesh color of melons of different genotypes was visually observed and measured by colorimetric analysis after storage in a 14°C refrigerator for 7 days following harvest. For two categories of melons: wild type (WT) and sgr-1 form, the orange flesh material of varieties V1_Charentais_LSL and V2_ItalianNet_NLSL, and the white flesh material of variety V6_YellowC_LSL were evaluated.

[0191] The flesh color of 10 fruits was evaluated for each genotype. A colorimeter was used to measure the equatorial slice of the fruit. Two measurements were obtained for each fruit, opposite in the diametrical direction, and the average was then calculated to obtain the color at the fruit level.

[0192] Using pairwise comparisons with Tukey's test, color differences were evaluated, and no significant differences were recorded between the sgr-1 line and its wild type in both orange-fleshed and white-fleshed materials (Figure 9).

[0193] The same results are observed with green pulp material.

[0194] Example 6: Detailed phenotypic analysis of SGR-1 converted varieties We performed detailed phenotypic analyses of several traits in three varieties containing the WT allele, V2_ItalianNet_NLSL, V4_HD_NLSL, and V5_Charentais_NLSL, as well as in their three corresponding converted varieties for the sgr-1 mutation. A total of 14 fruits were harvested for each genotype, and phenotypic analyses were performed to evaluate the impact of the sgr-1 mutation on other important traits associated with non-LSL varieties.

[0195] The cycle length corresponds to the number of days calculated from planting to harvest. Non-LSL material is known to have the shortest cycle, with fruit being harvested 55 days after planting, while LSL material has a longer cycle, with fruit being harvested approximately 90 days after planting. In the experiment, all plants were planted in the field on the same day (approximately 20 days after sowing), and the harvested fruit was recorded along with the harvest date for calculation. Among the different genotypes observed, there was no significant difference in cycle length between the sgr-1 converted varieties and their corresponding WT varieties (Figure 10). The staygreen mutation does not significantly delay the harvest time.

[0196] Pedicel detachment is an important indicator of maturity, along with the progression of peel color or the senescence of the first leaves and tendrils. Fruit is harvested when one or more of these indicators change. Therefore, pedicel detachment was observed on the fruit harvest date and evaluated on a scale of 1 to 9 (1 = completely detached, 9 = not detached). The sgr-1 mutation did not significantly affect the maturity indicator of pedicel detachment, and no significant difference was found using pairwise comparisons with Tukey's test (Figure 11). The detached material remained detached, facilitating harvesting by growers.

[0197] Brix measurements performed on harvest day at the equatorial slice of each melon using an electronic refractometer did not show a significant difference in Brix levels between the sgr-1 mutant and the wild type. Compared to the corresponding melon with the WT allele, the sgr-1 mutation did not affect sugar levels and therefore sweetness (Figure 12).

[0198] Hardness was also measured in each equatorial slice of all harvested fruit. Using a penetrometer, measurements were taken at two opposite points in the diametrical direction of the equatorial slice. The average of the two measurements was then calculated. No statistical differences were observed between the sgr-1 genotype and the WT genotype (Figure 13).

[0199] In conclusion, observations made with different sgr-1 converted varieties compared to the original variety (WT) classified as a non-LSL genotype indicate that the sgr-1 mutation has resulted in a new ideal type of melon where storage life is extended due to the stability of rind color progression, but without affecting indicators of fruit quality and maturity. In other words, it provides growers with better field maintenance and flexibility in fruit harvesting without extending the harvest time. The lack of rind color progression during storage provides greater flexibility for retailers. For the end consumer, the initial fruit quality of the produce is maintained.

[0200] We evaluated other climacheric fruits from different melon types, and the same conclusion was reached.

Claims

1. A melon (Cucumis melo) plant, wherein the plant contains a homozygous mutant allele of the staygreen (sgr) gene on chromosome 9 in its genome, the mutant allele of the sgr gene contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1), the mutant allele of the sgr gene confers rind color stability to the fruit of the plant at maturity and after harvest compared to an isogenic non-LSL melon plant that does not contain the mutant allele, and the rind color stability is evaluated by comparing the rind color of the mutant and the rind color of an isogenic non-mutant melon at different time points from maturity to 7 to 21 days after harvest.

2. The plant according to claim 1, wherein the at least one loss-of-function mutation is selected from a nonsense mutation, a framework mutation, and a defective splicing mutation.

3. The plant according to claim 2, wherein the at least one loss-of-function mutation is the G584A mutation of Sequence ID No.

1.

4. The plant according to any one of claims 1 to 3, wherein the hardness of the fruit of the plant at maturity, the degree of detachment of the fruit stalk at maturity, and / or the length of the cycle differ by less than 20% compared to the fruit of an isogenic plant grown at the same maturation stage and under the same environmental conditions, and the isogenic plant does not contain a homozygous mutant allele of the sgr gene in its genome.

5. The plant according to any one of claims 1 to 4, wherein the plant exhibits reduced leaf yellowing and necrosis compared to an isogenic non-long-storage-life (non-LSL) melon (Cucumis melo) plant that does not contain the mutant allele.

6. The plant according to any one of claims 1 to 5, wherein the plant is resistant to cucumber yellow stunt virus (CYSDV), and the resistance is provided by a mutant allele of the sgr gene.

7. The plant according to claim 6, wherein the resistance is partial resistance.

8. The plant according to any one of claims 1 to 7, wherein the melon plant is a plant derived from an inbreeding melon line or an F1 hybrid melon plant.

9. A cell of a melon plant according to any one of claims 1 to 8, wherein the cell contains a mutant allele of the staygreen (sgr) gene on chromosome 9 in its genome, and the mutant allele of the sgr gene contains at least one loss-of-function mutation in a homozygous manner, compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1).

10. A plant portion of a melon plant comprising at least one cell as described in claim 9.

11. Melon seeds that can grow into a melon plant according to any one of claims 1 to 8.

12. In vitro cells or tissue cultures of regenerative cells of a melon plant according to any one of claims 1 to 8, wherein the regenerative cells are derived from an embryo, protoplast, meristem cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, seed, flower, cotyledon, and / or hypocotyl.

13. A method for producing melon plants that produce fruit with increased shelf life, (a) To obtain the part of the plant described in claims 1 to 8, (b) Propagating the plant parts vegetatively to produce plants from the plant parts Methods that include...

14. A method for producing a melon plant that produces fruit with an extended shelf life, comprising introducing a loss-of-function mutation into the sgr gene on chromosome 9 in the genome of a non-LSL melon plant, wherein the mutation is introduced by mutagenesis or genome editing, and the mutation is introduced in all copies of the sgr gene on chromosome 9.

15. The method according to claim 14, wherein the mutation is introduced by a technique selected from ethylmethanesulfonic acid (EMS) mutagenesis, oligonucleotide-directed mutagenesis (ODM), zinc finger nuclease (ZFN) technology, activator-like effector nuclease (TALEN), CRISPR / Cas system, engineered meganuclease, re-engineered homing endonucleases, and DNA-guided genome editing.

16. A method for identifying, detecting, and / or selecting melon plants that produce fruit with extended shelf life, comprising detecting homozygous mutant alleles of the sgr gene on chromosome 9 in the genome of non-LSL melon plants, A method wherein the mutant allele contains at least one loss-of-function mutation compared to the sequence of the wild-type sgr allele (SEQ ID NO: 1).

17. The method according to claim 16, wherein the at least one loss-of-function mutation is selected from nonsense mutations, indel mutations, framework mutations, and defective splicing mutations.

18. The method according to claim 16 or 17, comprising detecting the sequence shown in Sequence ID No.

2.

19. A method for improving the storage life of melon fruit, the marketability of melon fruit, and / or melon production yield, comprising growing a melon plant according to any one of claims 1 to 8, and harvesting fruit produced by the plant.

20. A method for producing melon fruit, d) Growing a melon plant according to any one of claims 1 to 8, e) To enable the aforementioned plants to bear fruit, f) Harvesting the fruit of the aforementioned plant Methods that include...

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