Melon with extended shelf life

Abstract

The present invention relates to a melon plant, wherein said plant comprises in its genome homozygously a mutant allele of the stay-green (sgr) gene on chromosome 9, wherein said mutant allele of the sgr gene comprises at least one loss-of-function mutation compared to the sequence of the wild type sgr allele (SEQ ID NO: 1), and wherein said mutant allele of the sgr gene confers fruit skin color stability at ripening and / or during the post-harvest period of the fruit of said plant compared to an isogenic non-long shelf life (non-LSL) melon plant not comprising said mutant allele. The present invention also relates to parts, cells and seeds of said plant, as well as related methods and processes.

Description

Technical Field

[0001] This invention relates to melon (C. melono) plants with an extended shelf life compared to existing non-LSL (non-LSL) melon types, while maintaining similar characteristics such as sugar content, cycle length, aroma, or firmness to non-LSL melons. The invention also provides methods for cultivating such plants, as well as methods for detecting and / or selecting such plants. Background Technology

[0002] Melon (Cucumis melo L.) is a globally cultivated cucurbitaceae crop. Most commercial melons produce known sweet fruits, such as Charentais, Cantaloupe, Piel de sapo, Galia, Ananas, and Honeydew. Melon fruit is typically eaten as a dessert fruit.

[0003] Combining long shelf life with desired consumer flavors has always been a challenge for melon breeders. On the one hand, shelf life is a crucial parameter for melon growers and retailers. Fruit with extended shelf life can be stored for longer periods, reducing commercial losses and increasing harvesting and transportation flexibility. Numerous efforts have been made to improve the shelf life of melons. Until the 1980s, the melons sold in the market were primarily of the traditional type, with limited shelf life. Traditional melons are climacteric fruits, their ripening process triggered by the massive production of ethylene and the development of respiration. These events, in turn, trigger many ethylene-dependent processes, such as changes in rind color (typically yellowing), the development of aromas that impart flavor to the melon, and the gradual softening of the fruit. These processes influence the shelf life of the melon.

[0004] In the 1990s, long shelf-life (LSL) melons were introduced and gradually captured a large share of the market. LSL melons are non-climacteric melons, meaning they do not produce the large amounts of ethylene typical of climacteric melons when ripe. Furthermore, LSL melons remain green for a longer period and remain firm after harvest. While the extended shelf life offers significant advantages compared to conventional melons, LSL melons also have a notable drawback: they emit less aroma, leading consumers to often perceive them as having a poorer taste. Candidate mutations have been identified that may contribute to the extended shelf life of LSL melons, particularly in the ACC oxidase gene (Ayub, Ricardo, et al., "Expression of ACC oxidase antisense gene inhibits ripening of cantaloupemelon fruits," Nature Biotechnology 14.7(1996):862-866).

[0005] Recently, medium shelf-life (ISL) melons have been acquired and marketed in an attempt to provide melons with a better flavor than LSL melons while offering an acceptable shelf life. However, this compromise between complex traits presents a challenging task for breeders.

[0006] There is still a need to provide new melon varieties to meet the needs of growers and consumers, combining extended shelf life with good taste and other important commercial characteristics. Summary of the Invention

[0007] The inventors of this application have discovered that inactivating the staygreen (sgr) gene on chromosome 9, for example through splice site mutations, confers increased rind color stability at maturity and post-harvest in non-LSL melon types, whereas no effect on rind color has been observed in LSL melons. This stability is, in turn, associated with extended shelf life. This is surprising because other genes, such as ACC oxidase (Ayub, Ricardo et al., 1996), have previously been involved in controlling melon shelf life, but the sgr gene has not. Surprisingly, the inventors have also found that the sgr mutant possesses many other advantageous traits that are identical or comparable to those in non-LSL melons (such as conventional melons). Growers, retailers, or consumers are interested in these traits: cycle length, firmness, soluble solids content, Brix (i.e., sweetness), or pedicel abscission rate. Therefore, this invention provides a new melon type that combines extended shelf life with commercially valuable non-LSL traits such as cycle length, sweetness, pedicel abscission, and softening at maturity.

[0008] Therefore, in one aspect, the present invention relates to melon plants, wherein the plant contains homozygous sgr gene mutant alleles homozygous in its genome on chromosome 9, wherein the mutant alleles of the sgr gene contain at least one loss-of-function mutation compared to the wild-type sgr allele sequence (SEQ ID NO: 1), wherein the mutant alleles of the sgr gene confer stability of the peel color of the fruit of the plant at ripening and / or during post-harvest period.

[0009] Another object of the present invention relates to cells of melon plants according to the present invention, preferably derived from cells of embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, flowers, cotyledons and / or hypocotyls, wherein said cells homozygous in their genome contain a mutant allele of the 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).

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

[0011] The present invention further relates to melon seeds, which can grow into melon plants according to the present invention.

[0012] In another aspect, the present invention relates to in vitro cell or tissue cultures of regenerative cells of melon plants according to the present invention, wherein the regenerative cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, seeds, flowers, cotyledons and / or hypocotyls.

[0013] This invention also relates to a method for producing melon plants that produce fruits with extended shelf life or are prone to producing fruits with extended shelf life, comprising:

[0014] (a) Obtaining the plant portion of the present invention.

[0015] (b) Asexual reproduction of a portion of the plant to produce a plant from the portion of the plant.

[0016] The present invention also relates to a method for producing melon plants that produce or are prone to producing fruits with extended shelf life, comprising introducing a loss-of-function mutation in the sgr gene (SEQ ID NO:1) on chromosome 9 of a non-LSL melon plant genome, wherein the mutation is introduced by mutagenesis or genome editing, particularly by techniques selected from ethyl methanesulfonate (EMS) mutagenesis, oligonucleotide directed mutagenesis (ODM), zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN), CRISPR / Cas system, engineered wide-range nucleases, reengineered homing endonucleases, and DNA-guided genome editing.

[0017] A method is also provided for identifying, detecting, and / or selecting melon plants that produce or are prone to producing fruits with extended shelf life, the method comprising detecting a mutant allele of the sgr gene on chromosome 9 of the genome of the plant, wherein the mutant allele contains at least one loss-of-function mutation compared to the sequence (SEQ ID NO: 1) of the wild-type sgr allele.

[0018] The present invention also relates to a method for improving the shelf life, marketability and / or yield of melon fruits, wherein the method comprises planting a melon plant according to the invention and harvesting the fruits produced by said plant.

[0019] A method for producing melon fruit is also provided, comprising:

[0020] a) Cultivating the melon plants according to the invention;

[0021] b) Allow the plant to bear fruit; and

[0022] c) Harvest the fruit of the plant, preferably at the early or mature stage.

[0023] Another object of the present invention is the use of the melon plant or its fruit according to the invention in the fresh-cut market or in food processing.

[0024] definition

[0025] Melon types can be divided into three groups based on their post-harvest characteristics: conventional, medium shelf life (ISL), and long shelf life (LSL).

[0026] The term "shelf life" as used herein refers to the period of time a melon fruit can be stored post-harvest before it is deemed unsuitable for sale or consumption. Shelf life is preferably assessed during storage. Shelf life typically takes into account various characteristics of the fruit, such as rind color, flesh color, firmness, aroma, and / or sugar content. Preferably, the extended shelf life of the melons according to the invention is assessed based on improved color stability during harvest and / or post-harvest storage. In particular, the melons according to the invention retain their immature color for a longer period during post-harvest storage compared to melons without the genetic characteristics of the invention.

[0027] Long-Shelf-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. Specifically, LSL melons can be selected from the following types: LSL Charente, LSL Italian netted, Harper, LSL Galia, Yellow Canary, Christmas melon, and Cantaloupe.

[0028] The shelf life of "traditional" melons is typically less than 5 days. Preferably, the shelf life of traditional melons is 2 to 5 days. Generally, traditional melons are climacteric. Specifically, traditional melons can be selected from the following types: traditional Charente melon, traditional Italian netted melon, Western Shipper, Eastern Shipper, traditional Gallia melon, and traditional Mango melon.

[0029] "Intermediate Shelf Life (ISL)" melons refer to melons with a shelf life between that of conventional melons and LSL melons. Preferably, ISL melons have a shelf life of 7 to 14 days. Specifically, ISL melons can be selected from the following types: Charente melons, Italian netted melons, Sandy Beach melons, Oriental melons, Gallia melons, Mango melons, and Cantaloupes.

[0030] As used herein, the term "non-LSL" melon refers to either conventional or ISL melons. Therefore, any reference to non-LSL melons or melon plants should be understood as specifying conventional 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. Specifically, non-LSL melons can be selected from the following types: conventional Charente melon, conventional Italian netted melon, conventional Gallia melon, conventional Mango melon, ISL Charente melon, ISL Italian netted melon, Sandy melon, Oriental melon, ISL Gallia melon, ISL Mango melon, and ISL Hami melon.

[0031] Climax melons are characterized by rapid autocatalytic ethylene production during ripening, typically accompanied by increased respiration. Climax ripening is accompanied by several ethylene-mediated physiological and biochemical events, including flesh softening, aroma development, rapid rind color change, and peduncle shedding. The rind color change varies depending on the melon type. In Gallia melons, the rind changes from dark green to yellow-orange, while in Charente melons, the rind changes from green or gray to creamy yellow. The autocatalytic production of ethylene manifests as an exponential increase in ethylene concentration within the melon cavity over time, typically from negligible to maximum within a few days. The absolute magnitude of the peak ethylene levels varies among different climax melon varieties, but rapid induction of ethylene biosynthesis is characteristic of these lines.

[0032] Non-climacteric melons do not exhibit this autocatalytic production of ethylene, and are therefore characterized by reduced or no color change in the peel at ripening, reduced firmness during storage, and reduced aroma production, which negatively impacts the flavor of these melons.

[0033] As used herein, an "allele" refers to any of several alternative or variant forms of a genetic unit, such as a gene, that are genetically substituted because they reside at the same locus on homologous chromosomes. Such alternatives or variant forms can result from single nucleotide polymorphisms, insertions, inversions, translocations, or deletions, or from gene regulation caused by, for example, chemical or structural modifications, transcriptional regulation, or post-translational modifications / regulations. In diploid cells or organisms, the two alleles of a given gene or genetic element typically occupy corresponding loci on a pair of homologous chromosomes.

[0034] As used in this article, the terms “cross,” “crossing,” “cross-pollination,” or “cross-breeding” refer to the process of applying pollen (artificially or naturally) from one flower of one plant to the ovule (stigma) of another flower of a different plant.

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

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

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

[0038] As used in this article, "homologous chromosomes" or "homologues" refers to a pair of maternal and paternal chromosomes that pair up during meiosis. These copies have the same genes at the same loci and centromere.

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

[0040] As used herein, the term "homozygous" refers to the presence of the same allele at one or more loci in a homologous chromosomal segment. Therefore, this plant homozygousally contains the mutant allele of the sgr gene on chromosome 9, the mutant allele in all copies of the sgr gene on chromosome 9 (e.g., two copies if the plant is diploid), and a set of two homologous chromosomes 9.

[0041] As used herein, the term "hybrid" refers to any single cell, tissue, plant part, or plant resulting from hybridization between parents that are different in one or more genes. An F1 hybrid (HF1) is the result of hybridization between two genetically different parent varieties or lines. Hybrid plants according to the invention are heterozygous for one or more genes in their genome, but homozygous for the sgr gene, meaning that all of their sgr alleles (i.e., the two in diploid plants) are loss-of-function alleles. The loss-of-function mutations in each sgr allele may be the same or different. Example 2 and... Figure 1 A technique for producing HF1 plants that homozygousally contain the sgr mutant allele is described.

[0042] As used herein, two plants are referred to as "isogenic" when they have the same or substantially the same set of chromosomes and genes, except for one gene (the sgr gene in this invention). Therefore, these two isogenic plants contain different alleles of the sgr gene. Comparing the phenotypes of the two isogenic plants can assess the impact of allele variation in the sgr gene.

[0043] As used in this article, a "loss-of-function mutation" or "inactivation mutation" is a mutation that results in a reduced or non-functional gene product (partial or complete inactivation). When an allele completely loses its function, it is also called a null allele. The phenotype associated with such mutations is usually recessive.

[0044] As used herein, the term "molecular marker" refers to an indicator used in methods for visualizing characteristic differences in 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), cleaved amplified polymorphic sequences (CAPS) markers, or isoenzyme markers, or combinations of the markers described herein, which define specific genetic and chromosomal locations. Plotting molecular markers near alleles is a procedure readily performed by those skilled in the art using common molecular techniques.

[0045] As used herein, the term "primer" refers to an oligonucleotide capable of annealing to an amplification target to allow DNA polymerase attachment, thus serving as the starting point for DNA synthesis when placed under conditions that induce primer extension product synthesis, i.e., in the presence of nucleotides and a polymerizing agent such as DNA polymerase, and at suitable temperature and pH. To maximize amplification efficiency, primers are preferably single-stranded. Preferably, the primer is an oligodeoxyribonucleotide. Primers must be long enough to initiate the synthesis of extension products in the presence of a polymerizing agent. The exact length of the primer depends on many factors, including temperature and primer composition (A / T and G / C content). A bidirectional primer pair, consisting of a forward primer and a reverse primer, is commonly used in the field of DNA amplification, such as PCR amplification.

[0046] As used in this article, a single nucleotide polymorphism (SNP) is a variation in DNA sequence that occurs when a single nucleotide (A, T, C, or G) in the genome (or other shared sequence) differs between paired chromosomes of members of an organism or individual. For example, two sequenced DNA fragments from different individuals, AAGC... C TA to AAGC T TA represents a difference of a single nucleotide. In this case, there are two alleles: C and T.

[0047] As used herein, “marker-based selection” or “marker-assisted selection (MAS)” or “marker-assisted breeding (MAB)” refers to the use of genetic markers to detect one or more nucleic acids in plants that are associated with a desired trait, in order to identify plants carrying genes for the desired (or unwanted) trait, thereby enabling these plants to be used (or avoided) in selective breeding programs.

[0048] As used in this article, "maturity" is a stage in melon development. The senescence of the first leaf and fruit tendrils is a common indicator of maturity in both climacteric and non-climacteric melons. Other indicators of maturity in climacteric melons are stem-end splitting or aroma release. In non-climacteric melons, such as Christmas melons or Canary melons, maturity is characterized by browning or yellowing of the pistil area and coloration towards the stem end.

[0049] As used herein, the term "offspring" or "progeny" refers to any plant that is a descendant of one or more parent plants or their offspring, produced asexually or sexually. For example, offspring plants can be obtained by cloning or self-pollinating the parent plants or by hybridizing the two parent plants, and include self-pollination as well as F1 or F2 or further generations. F1 refers to the first generation of offspring produced for the first time by at least one parent as a donor of the trait, while the offspring of the second generation (F2) or the offspring of the progeny (F3, F4, etc.) are samples produced by self-pollination of F1, F2, etc. Thus, F1 may be (usually) a hybrid produced by hybridization between two homozygous breeding parents (homozygous breeding is homozygous for the trait), while F2 may be (and usually) a progeny produced by self-pollination of the said F1 hybrid.

[0050] As used herein, the term "melon" refers to any type, variety, or cultivar of melon species. This invention includes plants at different ploidy levels, whether diploid, triploid, tetraploid, etc.

[0051] As used herein, the term "plant part" refers to any part of a plant, including but not limited to branches, roots, stems, seeds, fruits, leaves, petals, flowers, ovules, branches, petioles, internodes, pollen, stamens, rhizomes, scions, etc.

[0052] The term "resistance," defined by the ISF (International Seed Federation) Vegetable and Ornamental Crop Division, describes the response of plants in the vegetable seed industry to pests or pathogens, as well as abiotic stress. Specifically, resistance refers to the ability of a plant variety to limit the growth and development of a particular pest or pathogen and / or the damage they cause, compared to susceptible plant varieties under similar environmental conditions and pest or pathogen stress. Resistant varieties may exhibit some disease symptoms or damage under severe pest or pathogen stress.

[0053] As used in this article, the term "susceptible" refers to a plant that cannot restrict the growth and development of a particular pest or pathogen.

[0054] As used in this article, the terms “inbred line” or “strain” refer to a relatively pure strain.

[0055] As used herein, the term "phenotype" refers to the observable characteristics of an individual cell, cell culture, organism (e.g., plant) or population of organisms resulting from the interaction between an individual's genetic makeup (i.e., genotype) and the environment.

[0056] As used herein, the terms “introgression,” “introgressed,” and “introgressing” refer to the process by which genes from one species, variety, or cultivar enter the genome of another species, variety, or cultivar through hybridization between these species. Hybridization can be natural or artificial. This process can be selectively accomplished through repeated backcrosses with a parent; in this case, introgression refers to the insertion of genes from one species into the gene pool of another species through repeated backcrosses between an interspecific hybrid and one of its parents. Introgression can also be described as heterologous genetic material stably integrated into the genome of the recipient plant.

[0057] In this specification, comparisons between two or more melon plants or fruits, particularly between melon plants according to the invention and allogeneic melons that do not contain a mutant allele of the sgr gene on chromosome 9, are understood to be comparisons between plants or fruits grown under the same environmental conditions at the same ripening stage or at the same stage after harvest.

[0058] sequence list

[0059] SEQ ID NO: 1 shows the sequence of the wild-type sgr gene on chromosome 9.

[0060] SEQ ID NO:2 shows the sequence of the sgr-1 allele of the sgr gene, which contains the G584A mutation.

[0061] SEQ ID NO:3 shows the coding sequence of the wild-type sgr gene on chromosome 9.

[0062] SEQ ID NO:4 shows the amino acid sequence of the wild-type SGR protein.

[0063] SEQ ID NO: 5 shows a neighboring sequence for developing markers around the sgr-1 mutation.

[0064] SEQ ID NO: 6 shows the sequence of the forward primer used to detect the wild-type allele of the sgr gene.

[0065] SEQ ID NO: 7 shows the sequence of the forward primer used to detect the sgr-1 mutant allele of the sgr gene.

[0066] SEQ ID NO: 8 shows the sequence of a universal reverse primer for detecting the sgr-1 and wild-type mutant alleles of the sgr gene. Attached Figure Description

[0067] Figure 1 The breeding scheme for introducing the sgr-1 mutation into the HF1 hybrid is shown.

[0068] Figure 2 Images of leaves of Charente melon, Canary melon, and Gallia melon, which are wild-type alleles or contain the sgr-1 mutation, are shown.

[0069] Figure 3 Images show leaves of Italian netted melons under CYSDV stress, either wild-type alleles or containing the sgr-1 mutation.

[0070] Figure 4 The L*, a*, and b* values ​​of leaf color are shown for wild-type alleles or V1-Charente melon and V2-Canary melon varieties containing the sgr-1 mutation.

[0071] Figure 5 The evolution of leaf color ΔE* values ​​over three dates is shown, where ΔE* values ​​reflect the differences in leaf color in the CIELAB color space between the sgr-1 mutant melons of the V1_Charente and V2-Canary varieties and their corresponding wild-type (WT) melons.

[0072] Figure 6 The image shows a photograph of the rind of the WT (Image A) or V2_Italian netted melon_NLSL variety containing the sgr-1 mutation (Image B) after 7 days of storage.

[0073] Figure 7 The L*, a*, and b* values ​​of the peel color of the V1_Charente Melon_LSL, V2_Italian Netted Melon_NLSL, and V3_Italian Netted Melon_NLSL varieties are shown (from left to right) on the day of harvest (top) or after 7 days of storage (bottom).

[0074] Figure 8 The values ​​of ΔE* for the varieties V1_Charente Melon_LSL, V2_Italian Netted Melon_NLSL, and V3_Italian Netted Melon_NLSL are given on two dates (from left to right: the day of harvest and 7 days after storage). The ΔE values ​​reflect the difference in skin color between the sgr-1 mutant melons and their corresponding wild-type (WT) melons in the CIELAB color space.

[0075] Figure 9The L*, a*, and b* values ​​of flesh color (from left to right) are shown for wild-type alleles or varieties containing the sgr-1 mutation, namely V1_Charente Melon_LSL, V2_Italian Netted Melon_NLSL, and V6_Canary Melon_LSL.

[0076] Figure 10 The cycle lengths of different melon varieties (V2_Italian Netted Melon_NLSL, V4_HD_NLSL, and V5_Charentes Melon_NLSL) containing the sgr-1 mutation or wild-type allele are shown.

[0077] Figure 11 The assessment of peduncle drop was shown for different melon genotypes (V2_Italian netted melon_NLSL, V4_HD_NLSL and V5_Charentes melon_NLSL) containing the sgr-1 mutant or wild-type allele.

[0078] Figure 12 The Brix levels of different melon genotypes (V2_Italian netted melon_NLSL, V4_HD_NLSL, and V5_Charentes melon_NLSL) containing the sgr-1 mutant or wild-type allele are shown.

[0079] Figure 13 The firmness measurements of different melons (V2_Italian netted melon_NLSL, V4_HD_NLSL and V5_Charentes melon_NLSL) containing the sgr-1 mutation or wild-type allele are shown. Detailed Implementation

[0080] According to a first aspect, the present invention relates to melon plants, wherein the plants homozygously contain a mutant allele of the sgr gene on chromosome 9 in their genome, 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 pericarp color stability of the fruit of the plant during ripening and / or post-harvest, compared to isogenetic non-LSL melon plants that do not contain the mutant allele. By homozygously containing the mutant allele of the sgr gene (loss-of-function), it should be understood that the mutant allele of the sgr gene is present on every homologue of chromosome 9, but not necessarily the same mutant allele, as long as all mutant alleles are indeed loss-of-function mutations.

[0081] In one embodiment, the non-LSL melon plant is a conventional melon plant. In this case, the corresponding isogenetic mutant plant is either an ISL melon type or an LSL melon type. In one embodiment, the non-LSL melon plant is an ISL melon plant. In this case, the corresponding isogenetic mutant plant is an LSL melon type.

[0082] The muskmelon plant of this invention is characterized by a homozygous inactivated sgr gene. The sgr gene has been mapped to chromosome 9 of the muskmelon 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 is deposited in GenBank, accession number XM_008438967 (updated June 7, 2016), wherein the coding sequence is located between nucleotides 415 and 1188. The translated sequence, i.e., the wild-type amino acid sequence of the SGR protein, is deposited in GenBank, accession number XP_008437189.1 (updated June 7, 2016), and is shown in SEQ ID NO: 4.

[0083] In one embodiment, the mutant allele of the sgr gene is a loss-of-function allele, meaning it contains at least one loss-of-function mutation. The sequence of the mutant allele can differ from the wild-type sequence of the gene by substitution, insertion, or deletion of at least one nucleotide in the sequence. In particular, the mutation can be a single nucleotide polymorphism (SNP). The mutant allele of the sgr gene can also differ from the wild-type sequence of the sgr gene by insertion or deletion of one or more nucleic acid segments (including deletion of the entire gene). This mutation can induce one or more amino acid substitutions in the SGR protein sequence and impair the function of the SGR protein.

[0084] In one implementation, the loss-of-function mutation in the sgr gene is a null mutation. A null mutation prevents the expression of the active SGR protein. The mutation can be a nonsense mutation, causing premature cessation of mRNA translation into protein, resulting in the expression of a truncated form of the SGR protein. Alternatively, the mutation can be a skeletal mutation, causing a skeletal shift that leads to the translation of an abnormal amino acid string. Alternatively, the mutation can be a defective splicing mutation, causing splicing errors from precursor mRNA to mature mRNA. The mutation can be a splice site mutation, i.e., a mutation located at a gene splice site, or it can be located in an intron or exon of any splicing regulatory sequence.

[0085] In this invention, the advantage of nonsense, structural, or defective splicing mutations is that they typically result in a complete loss of expression of the functional protein, as opposed to missense mutations (single amino acid substitutions), in which the protein is most frequently expressed and its activity may be partially preserved.

[0086] Loss-of-function mutations can be located in any exon or intron of the sgr gene. In particular, the mutation may be located in one of the first, second, or third exons or one of the first, second, or third introns.

[0087] According to one aspect, the mutation is a nucleotide substitution at the splice site between the first intron and the second exon. In one embodiment, the mutation involves the replacement of guanine with alanine at the last position of the first intron. This guanine is located at position 584 of SEQ ID NO: 1. This splice site mutation, designated sgr-1, has been identified by the inventors in EMS mutant plants and has infiltrated into different non-LSL and LSL genotypes. The sequence of the sgr-1 allele is shown in SEQ ID NO: 2.

[0088] Mutant alleles and corresponding markers can be identified using methods known in the art.

[0089] Mutations in the sgr allele can be induced by methods such as mutagenesis or by genetic engineering. Mutagenesis methods and genetic engineering methods are known in the art and are described in more detail below.

[0090] Therefore, the plants according to the invention can be obtained by different methods and not entirely by fundamentally biological methods.

[0091] Compared to isogenetic non-LSL fruits that do not contain mutant alleles of the sgr gene as defined herein, the melon fruits according to the invention are characterized by increased rind color stability at maturity and during the post-harvest period. The stability of rind color can be assessed by comparing the rind color of mutant and isogenetic non-mutant melons at different time points (from maturity, preferably the day of harvest, to post-harvest, preferably 7 to 21 days post-harvest, particularly 7 to 14 days post-harvest, and most particularly 7 or 14 days post-harvest). Preferably, the stability of rind color is assessed after 7 to 21 days under refrigeration at a temperature of 4°C to 15°C. The same parameters apply to the measurement of any characteristic of the melons of the invention or their isogenetic non-mutant counterparts.

[0092] In some implementations, the color of the melon rind is evaluated using colorimetry with a colorimeter such as the Konica Minolta CR400 or 2D image analysis of a picture of the fruit. Color measurements can be expressed in the CIELAB color space (also known as the CIE L*a*b*). The CIELAB color space was defined by the International Commission on Illumination (CIE) in 1976. It represents color as three values: L* represents the brightness from black (0) to white (100), a* represents the measurement from green (-) to red (+), and b* represents the measurement from blue (-) to yellow (+). CIELAB is designed such that equal numbers of numerical changes in these values ​​roughly correspond to equal numbers of visually perceived changes. In this color space, melons that appear greener have a lower a* value, while melons that appear yellower have a higher b* value.

[0093] In one embodiment, the melon fruit according to the invention is characterized by having a lower a* value and / or a lower b* value at maturity and / or during the post-harvest period compared to isogenetic non-LSL melon fruit containing the mutant allele of the sgr gene. In one embodiment, the difference in a* and / or b* values ​​between the melon fruit according to the invention and isogenetic non-LSL melon fruit containing the mutant allele of the sgr gene is statistically significant. In one embodiment, the a* and / or b* values ​​of the melon fruit according to the invention are respectively at least 10%, preferably 20%, and more preferably 30% lower than the a* and / or b* values ​​of isogenetic non-LSL melon fruit containing the mutant allele of the sgr gene.

[0094] Color difference in the ClELAB color space can also be evaluated using the following formula: The color difference is represented by a non-empty ΔE*, as assessed by a pairwise comparison statistical tool. In one embodiment, the ΔE* value of the peel color between the melon fruit according to the invention and the allogeneic non-LSL melon fruit that does not contain the mutant allele of the sgr gene is greater than 1, preferably greater than 2, more preferably greater than 10, and even more preferably greater than 50.

[0095] In one embodiment, the difference in peel color between the melon fruit according to the present invention and the allogeneic non-LSL melon fruit that does not contain the mutant allele of the sgr gene is statistically significant.

[0096] The color differences in the rind of non-mutant melons can also be assessed visually, for example, by using color assessment tools.

[0097] The melons according to the invention are further characterized by retaining several characteristics of non-LSL melons unchanged or substantially unchanged, such as their Brix content, firmness, pedicel abscission rate and aroma, or their flesh color. These non-LSL characteristics are particularly advantageous to growers and consumers, and therefore have commercial value.

[0098] In one embodiment, the Brix content of the melon plant according to the invention remains substantially unchanged at maturity and / or during post-harvest compared to the fruit of a non-LSL isogenetic plant at the same maturity stage and grown under the same environmental conditions, wherein the isogenetic plant contains, heterozygous, the mutant allele of the sgr gene in its genome.

[0099] In particular, compared with the fruit of isogenetic non-mutant plants, the Brix content of the melon plant according to the present invention varies by less than 20%, preferably less than 10%, and more preferably less than 5%.

[0100] The term "Bryce Brix" or simply "Bryce Brix" refers to the soluble solids content of an aqueous solution, especially fruit juice, of which the vast majority is sugar. These are primarily estimated by a refractometer and measured as Brix. The higher the number, the greater the sugar content. Brix measurement is important for assessing the taste of melons because fruits with low Brix and therefore low sugar content are not popular with customers. Brix can be measured using a Brix meter, also known as a refractometer, as is known to those skilled in the art.

[0101] Maintaining the same or substantially the same Brix level as non-LSL melons that do not contain the sgr mutation is particularly advantageous, because the melons according to the invention accumulate the sweetness of non-LSL melons, especially traditional melons with longer shelf lives. Therefore, the melons of the present invention avoid the typical disadvantages of LSL melons, in which extended shelf life is usually associated with a lack of flavor.

[0102] In one embodiment, the fruits of the melon plants according to the invention exhibit substantially no change in firmness at maturity and / or during the post-harvest period compared to the fruits of syngeneic plants at the same maturity stage and grown under the same environmental conditions, wherein the syngeneic plants contain, heterozygous, the mutant allele of the sgr gene in their genome.

[0103] In particular, compared with the fruits of the isogenetic plants, the fruit firmness of the melon plants according to the present invention varies by less than 20%, preferably less than 10%.

[0104] Hardness can be measured using a hardness tester known to technicians.

[0105] Non-LSL melons gradually lose firmness as they ripen through an ethylene-dependent process. The melons of this invention exhibit firmness characteristics similar to or substantially similar to those of non-LSL melons, and therefore tend to soften as they ripen in a similar manner to non-LSL melons.

[0106] In one embodiment, the degree of pedicel abscission in the fruits of the melon plants according to the invention is substantially unchanged at maturity and / or during post-harvest, compared to the fruits of syngeneic plants at the same stage of maturity and grown under the same environmental conditions, wherein the syngeneic plants contain heterozygous mutant alleles of the sgr gene in their genome.

[0107] In particular, compared with the fruits of the isogenetic plants, the degree of pedicel abscission of the melon fruits according to the present invention varies by less than 20%, preferably less than 10%.

[0108] Abscission of the pedicel is a good indicator of maturity. At commercial maturity, non-LSL melon types generally form an abscission layer at the pedicel attachment point, while LSL melon types do not. For this reason, LSL melons are also known as abscission-resistant melons, as they need to be cut from the vine for harvest. Therefore, the presence of the abscission layer is particularly helpful for growers in determining when the melons are ready for harvest. The melons of this invention are similar to or substantially similar to isogenetic non-mutant non-LSL plants in various characteristics and developmental advantages, possessing a visible abscission layer.

[0109] The stages of flower stalk shedding can be visually assessed on a scale of 1 to 9, where 1 = completely shed and 9 = not shed.

[0110] In one embodiment, the fruit of the melon plant according to the invention exhibits essentially no change in the cycle length at maturity compared to the fruit of an isogenetic plant grown under the same environmental conditions, wherein the isogenetic plant contains, heterozygous, a mutant allele of the sgr gene in its genome.

[0111] Specifically, compared to the fruits of the isogenetic plants described above, the variation in cycle length of the fruits according to the invention is less than 20%, preferably less than 10%, and more preferably less than 5%. Cycle length corresponds to a time period, such as the number of days between the sowing date and the harvest date. Maintaining a similar cycle length to non-LSL type melons and thus maintaining the same harvest window is desirable, since non-LSL melons can be harvested earlier than LSL melons, i.e., their cycle length is shorter than LSL melons, thereby increasing yield.

[0112] The sgr mutation in the 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 exhibit reduced leaf yellowing and necrosis.

[0113] In one embodiment, the flesh color of the melon plant according to the invention remains substantially unchanged during ripening and / or post-harvest compared to the fruit of an isogenetic plant at the same maturity stage and grown under the same environmental conditions, wherein the isogenetic plant heterozygously contains the mutant allele of the sgr gene in its genome. Specifically, the a* and / or b* values ​​of the flesh color of the melon plant fruit according to the invention change by less than 20%, preferably less than 10%, compared to the fruit of the isogenetic plant. The difference in flesh color can also be assessed using the formula ΔE* in the CIELAB color space. In one embodiment, the ΔE* value of the flesh color between the melon fruit according to the invention and an isogenetic non-LSL melon fruit heterozygously containing the mutant allele of the sgr gene in its genome is less than 50, preferably less than 10, even more preferably less than 2, and more preferably less than 1.

[0114] In one embodiment, the leaves of the melon plant according to the invention exhibit reduced yellowing compared to isogenetic non-LSL plants, wherein the isogenetic plants contain, heterozygous, the mutant allele of the sgr gene in their genome.

[0115] The color of the leaves can be evaluated by visual inspection or by colorimetry using a colorimeter. In particular, the CIEIab color system can be used.

[0116] In one embodiment, the leaf color of the melon plant according to the invention is characterized by a lower a* value and / or a lower b* value compared to an isogenetic non-LSL plant that does not contain a mutant allele of the sgr gene.

[0117] Therefore, assessment of leaf color can be used as an alternative for identifying non-LSL plants that exhibit the phenotype of interest (i.e., pericarp color stability at maturity and / or post-harvest).

[0118] The reduced leaf yellowing exhibited by the plants of this invention also reflects resistance, more specifically, partial resistance of the plants of this invention to yellowing diseases, such as CYSDV (Cucurbit Yellow Developmental Disorder Virus).

[0119] Therefore, in some embodiments, the melon plants according to the invention are resistant to CYSDV (Calabash Yellow Developmental Disorder Virus), wherein this resistance is provided by an allelic mutant of the sgr gene. In particular, the resistance is partial resistance.

[0120] CYSDV is a clostridium fungus spread naturally by the whitefly (Bemisia tabaci). CYSDV induces interveinal chlorosis spots in mature leaves, which may enlarge and eventually merge, causing the entire leaf to turn yellow except for the veins, which remain green. This yellowing symptom is accompanied by a significant decline in fruit yield and quality, making the virus of high economic importance.

[0121] The sgr mutation in melon plants according to the invention can reduce the damage caused by CYSDV by hiding certain symptoms of CYSDV in infected plants, particularly leaf yellowing. Resistance to CYSDV is advantageously determined by comparison with susceptible (commercial) lines.

[0122] In one embodiment, the melon plant according to the invention is a plant derived from an inbred melon strain.

[0123] In a preferred embodiment, the melon plant according to the invention is an F1 hybrid melon plant.

[0124] The present invention also relates to a melon plant population according to the invention, wherein the population comprises at least 5 plants, particularly at least 10 plants, more particularly at least 20 plants, even more particularly at least 50 or 100 plants, or more particularly at least 1000 plants.

[0125] The present invention also relates to further aspects, which are detailed below. All embodiments described in detail in the foregoing section in conjunction with the first aspect of the invention are also embodiments according to these further aspects of the invention.

[0126] According to a second aspect, the present invention relates to cells of melon plants according to the invention, wherein the cells contain a mutant allele of the sgr gene on chromosome 9 in their genome, 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).

[0127] The plant cells of this invention can regenerate into complete plants.

[0128] Alternatively, the invention also relates to non-renewable plant cells that cannot grow into complete plants.

[0129] According to one embodiment, the cells are derived from the embryo, protoplast, meristematic cells, callus, pollen, leaf, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon and / or hypocotyl.

[0130] In one aspect, the present invention relates to plant parts of melon plants according to the present invention. The present invention also relates to plant parts of melon plants comprising at least one cell according to the present invention.

[0131] According to one embodiment, the plant part includes the embryo, protoplast, meristematic cells, 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.

[0132] Another aspect of the invention relates to melon seeds that can grow into melon plants according to the invention. Such seeds are therefore “seeds of the plants of the invention,” i.e., seeds that produce the plants of the invention. The invention also relates to seeds derived from the plants of the invention, i.e., seeds obtained from such plants through self-pollination or hybridization, provided that the plants obtained from said seeds homozygously contain a loss-of-function mutant allele of the sgr gene that confers stability of the pericarp color of the fruit at maturity and / or during post-harvest, compared to isogenetic non-LSL melon plants that do not contain said mutant alleles.

[0133] The present invention also relates to a melon seed population according to the invention, wherein the population comprises at least 2 seeds, particularly at least 10 seeds, especially at least 100 seeds, and even more particularly at least 1000 seeds.

[0134] Another aspect of the invention is an in vitro cell or tissue culture of regenerative cells from a melon plant according to the invention. Preferably, the regenerative cells are derived from the embryo, protoplast, meristematic cells, callus, pollen, leaf, anther, stem, petiole, root, root tip, seed, flower, cotyledon, and / or hypocotyl. The regenerative cells contain, in their genome, a loss-of-function mutant allele of the sgr gene as described above.

[0135] The tissue culture will preferably be able to regenerate plants having the aforementioned physiological and morphological characteristics of melon plants, and will be able to regenerate plants having a genotype substantially the same as the aforementioned melon plants. The present invention also provides melon plants regenerated from the tissue cultures of the present invention.

[0136] The present invention also provides protoplasts from plants or tissue cultures as defined above, wherein the protoplasts contain loss-of-function mutant alleles of the sgr gene as described above in their genome.

[0137] According to another aspect, the present invention also relates to the use of melon plants, as detailed herein, as breeding partners in breeding programs for obtaining melon plants with extended shelf life, particularly with increased pericarp color stability during maturity and / or post-harvest. In fact, such melon plants according to the first aspect possess a loss-of-function allele of the sgr gene in their genome, as defined above, conferring pericarp color stability during maturity and / or post-harvest. By crossing this plant with a plant that does not contain the mutation, this allele can thus be transferred to offspring, thereby conferring the desired phenotype. Therefore, the plants according to the invention can be used as breeding partners for introgression of mutant alleles conferring the desired phenotype into melon plants or germplasm.

[0138] In such breeding programs, the selection of offspring that exhibit the desired phenotype or carry sequences linked to the desired phenotype can be advantageously based on alleles and corresponding markers as disclosed above.

[0139] The present invention also relates to the use of the plant in procedures aimed at identifying, sequencing and / or cloning genetic sequences that confer a desired phenotype.

[0140] According to another aspect, the present invention also relates to a method for producing melon plants, particularly commercial plants, with extended shelf life. The method or process for producing plants having these characteristics includes the following steps:

[0141] (a1) A melon plant according to the invention, homozygous for the mutant allele of the sgr gene, is crossed with a second melon plant heterozygous for the mutant allele, thereby producing an F1 population, wherein the sequence of the mutant allele of the sgr gene, compared with the sequence of the wild-type sgr allele (SEQ ID NO: 1), includes at least one loss-of-function mutation.

[0142] (a2) Promote the generation of the F2 population from the F1 population.

[0143] (b) Select a plant that homozygousally contains the mutant allele in the offspring obtained therefrom;

[0144] (c) Optionally, the plants obtained in step b) are self-pollinated once or several times;

[0145] (d) Optionally, backcross the plant selected in step b) or c) with a melon plant that is not homozygous to contain the said mutant allele, and

[0146] (e) Select plants that homozygous for the mutant allele, wherein the plants produce fruit with an extended shelf life.

[0147] (f) Optionally, the selected plant is crossed with different melon plants that are homozygous to contain the mutant sgr allele, thereby producing a hybrid melon plant that is homozygous to contain the mutant sgr allele.

[0148] 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 from one of the following types: Charente melon, Italian netted melon, Sandy melon, Oriental melon, Gallia melon, Mango melon, and Cantaloupe.

[0149] Preferably, steps c) and / or d) are repeated at least twice and preferably three times, and it is not necessary to use the same melon plant that heterozygously contains the mutant allele. The melon plant that heterozygously contains the mutant allele is preferably a breeding line.

[0150] Self-pollination and backcrossing can be performed in any order and can be inserted; for example, backcrossing can be performed before and after one or more self-pollinations, and self-pollination can be conceived before and after one or more backcrosses.

[0151] In some embodiments, such a method is advantageously carried out by using one or more nucleic acid markers for selection in step b) or e) to select plants that homozygousally contain mutant alleles of the sgr gene.

[0152] The selection made in step b) or e) can be made using any type of genetic marker, particularly 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 random amplified polymorphic DNA (RAPD), isoenzymes and other markers known to those skilled in the art.

[0153] Methods for marker and allele detection can be based on any technique that allows the differentiation of two distinct alleles of a marker on a specific chromosome. Polymorphism can be detected by electrophoretic techniques, including single-strand conformation polymorphism (Orita et al., (1989) Genomics, 8(2), 271-278), denaturing gradient gel electrophoresis (Myers (1985) EPO0273085), or fragment length polymorphism (Life Technologies, Inc., Gaithersburg, Md.), but the broad applicability of DNA sequencing often makes it easier to directly sequence the amplified products. Once the polymorphic sequence differences are known, rapid assays for detecting polymorphisms can be designed for offspring testing. These typically involve some form 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 specific examples, PCR detection and quantification are performed using two labeled fluorescent oligonucleotide forward primers and one unlabeled universal reverse primer, such as KASPar (KBiosciences). Polymorphisms can also be detected using electrophoresis techniques, including single-strand conformation polymorphism (Orita et al., (1989) Genomics, 8(2), 271-278), denaturing gradient gel electrophoresis (Myers (1985) EPO0273085), or fragment length polymorphism (Life Technologies, Inc., Gaithersburg, Md.). The widespread application of DNA sequencing also often allows for direct sequencing of amplified products.

[0154] The present invention also relates to melon plants that are obtained or obtainable by the methods described herein. Such plants are indeed melon plants possessing the characteristics described in the first aspect of the invention.

[0155] The plant is preferably a commercial variety, cultivar, or melon type. The plant is preferably an F1 hybrid melon plant. In some embodiments, the plant is one of the following types: Charente melon, Italian netted melon, Sandwich melon, Oriental melon, Gallia melon, Mango melon, and Cantaloupe.

[0156] A method for producing melon plant seeds is also provided. In some embodiments, the method includes hybridizing the melon plant according to the invention with itself or with another melon plant and harvesting the resulting seeds.

[0157] In addition to the introgression of mutant alleles of the sgr gene, as detailed in the method of this invention, the sequence can also be introduced into a melon background via genetic engineering to obtain commercial melon plants with the advantageous characteristics of this invention, particularly extended shelf life. Identification and cloning of the introgressed mutant alleles conferring the desired phenotype are routine for those skilled in the art.

[0158] It should be noted that the seeds or plants of the present invention can be obtained through different processes, particularly technical methods such as mutagenesis (e.g., chemical mutagenesis or UV mutagenesis) or genetic engineering (e.g., guided recombination or genome editing), and are not entirely obtained through inherently biological processes.

[0159] In one embodiment, the present invention relates to a method for producing melon plants that produce or are prone to producing melons with extended shelf life, comprising introducing a loss-of-function mutation in the sgr gene on chromosome 9 of a non-LSL melon plant genome, wherein the mutation is introduced by mutagenesis or genome editing, particularly by techniques selected from ethyl methanesulfonate (EMS) mutagenesis, oligonucleotide directed mutagenesis (ODM), zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN), CRISPR / Cas systems, engineered wide-ranging nucleases, reengineered homing endonucleases, and DNA-guided genome editing. Preferably, the loss-of-function mutation is introduced in all copies of the sgr gene present on chromosome 9.

[0160] In particular, one embodiment of the present invention relates to a method for obtaining a melon plant or its seeds that produce fruits with extended shelf life or are prone to producing fruits with extended shelf life, the method comprising:

[0161] a) Treat the melon plants to be modified with a mutagen, preferably M0 seeds from non-LSL melon plants, to obtain M1 seeds;

[0162] b) Plant plants from the M1 seeds obtained therefrom to obtain M1 plants;

[0163] c) Producing M2 seeds through self-pollination of M1 plants; and

[0164] d) Optionally repeat steps b) and c) n times to obtain M2+n seed.

[0165] In this method, the M1 seed in step a) can be obtained by chemical mutagenesis such as EMS mutagenesis, or by any other chemical mutagen, including but not limited to diethyl sulfate (DES), ethylene imine (EI), propanesulfonate lactone, N-methyl-N-nitrosourea (MNU), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (ENU), and sodium azide. Alternatively, the mutation can be induced by radiation, such as radiation selected from X-rays, fast neutrons, and UV radiation.

[0166] In another embodiment of the invention, the mutation is induced by genetic engineering. Such mutations also include the integration of sequences that confer phenotypic characteristics, particularly pericarp color stability, to the mutant plants according to the invention, and the replacement of existing sequences with alternative sequences that confer phenotypic characteristics, particularly pericarp color stability, to the mutant plants according to the invention.

[0167] The genetic engineering techniques that can be used include all such techniques known as novel breeding techniques, which are various new techniques developed and / or used to create new traits in plants through genetic variation with the aim of directed mutagenesis, directed introduction of new genes, or gene silencing (RdDM). Examples of such novel breeding techniques include targeted sequence alteration facilitated by zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2, and ZFN-3, see U.S. Patent No. 9,145,565, incorporated herein by reference in its entirety), oligonucleotide directed mutagenesis (ODM), cisgenesis and intragenesis, RNA-dependent DNA methylation (RdDM, which does not necessarily alter the nucleotide sequence but can alter the biological activity of the sequence), grafting (on the primary root of a transgenic plant), reverse breeding, agro-osmosis (agro-osmosis "strictly speaking," agricultural inoculation, flower immersion), transcription activator-like effector nucleases (… TALEN (see U.S. Patent Nos. 8,586,363 and 9,181,535, all incorporated herein by reference), CRISPR / Cas systems (see U.S. Patent Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, all incorporated herein by reference), engineered large-scale nucleases and reengineered homing endonucleases, DNA-guided genome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038 / nbt.3547, all incorporated herein by reference), and synthetic genomics. A major part of today's targeted genome editing, also known as a new breeding technique, is the application of inducing DNA double-strand breaks (DSBs) at selected sites in the genome where modifications are intended. Directed repair of DSBs allows for targeted genome editing. Such applications can be used to generate mutations (e.g., targeted mutations or precise natural gene editing) and to precisely insert genes (e.g., homologous transgenes, intragenes, or transgenes). Applications that result in mutations are typically identified as site-directed nuclease (SDN) techniques, such as SDN1, SDN2, and SDN3. For SDN1, the result is a targeted, non-specific gene deletion mutation: the location of the DNA DSB is precisely chosen, but the DNA repair in the host cell is random, resulting in small nucleotide deletions, additions, or substitutions.For SDN2, SDN is used to generate a targeted DSB and a DNA repair template (a short DNA sequence identical to the targeted DSB DNA sequence, except for one or a few nucleotide variations) for DSB repair: this results in a targeted and predetermined point mutation in the desired gene of interest. As for SDN3, SDN is used in conjunction with a DNA repair template containing a novel DNA sequence (e.g., a gene). The outcome of this technology will be the integration of this DNA sequence into the plant genome. The most likely application of SDN3 is the insertion of homologous transgenes, intragenetic or transgene expression cassettes at selected genomic locations. Each technology is fully described in the 2011 report entitled "New plant breeding techniques - State-of-the-art and prospects for commercial development," published by the Joint Research Centre of the European Commission (JRC) Institute for Forward-Looking Technologies, which is incorporated herein by reference in its entirety.

[0168] DNA editing technology has been successfully used to inactivate target genes at specific locations in melons (Hooghvorst et al., "Efficient knockout of phytoene desaturase gene using CRISPR / Cas9 in melon." Scientific Reports, 9.1(2019):1-7).

[0169] The present invention also provides a method for identifying, detecting and / or selecting melon plants that produce or are prone to producing fruits with extended shelf life, the method comprising detecting a mutant allele of the sgr gene on chromosome 9 of 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).

[0170] In one embodiment, the loss-of-function allele is selected from nonsense mutations, insertion / deletion mutations, frame mutations, or defective splicing mutations, particularly splicing site mutations.

[0171] In one embodiment, the method includes detecting a substitution of guanine at position 584 of SEQ ID NO: 1, namely the allele sgr-1, the sequence of which is shown in SEQ ID NO: 2.

[0172] In some implementations, the detection of mutant alleles of the sgr gene is performed by amplification, such as by PCR, using a forward primer for amplifying the resistance allele, a forward primer for amplifying the susceptibility allele, and a universal reverse primer, such as KASPar, for each marker. TM (KBiosciences) technology. In particular, the primers used to amplify each of the markers may have sequences as described in Table 1.

[0173] In a preferred embodiment, amplification is performed using a two-step touchdown method, where the extension and annealing steps are combined into one step. The temperature used in the annealing phase determines the specificity of the reaction, thereby determining the ability of the primers to anneal with the DNA template. Touchdown PCR involves a first step of Taq polymerase activation, followed by a second step called the touchdown step, which involves a high annealing temperature that is gradually decreased in each PCR cycle, and a third step of DNA amplification. The higher annealing temperature in the early touchdown cycles ensures that only very specific base pairing occurs between the DNA and the primers, so the first sequence to be amplified is most likely the sequence of interest. The annealing temperature is gradually decreased to increase the efficiency of the reaction. The region initially amplified during the highly specific early touchdown cycles will be further amplified and outperform any non-specific amplification that might occur at lower temperatures.

[0174] In another embodiment, the amplification of SNP markers was performed according to the recommendations in the KASPar assay and the descriptions in the examples (see Example 1).

[0175] According to another aspect, the present invention also provides one or more molecular markers for identifying melon plants that produce fruits with extended shelf life or are prone to producing fruits with extended shelf life, wherein the molecular markers detect loss-of-function mutations in the sgr gene on chromosome 9.

[0176] It also provides the use of one or more molecular markers for detecting melon plants that produce or are prone to producing fruits with extended shelf life, wherein the molecular markers detect loss-of-function mutations in the sgr gene on chromosome 9.

[0177] According to these aspects of the invention, the molecular marker may be located in the sgr gene or in a chromosomal region genetically linked to the sgr gene. In one embodiment, the molecular marker recognizes the substitution of guanine for alanine at position 584 of SEQ ID NO: 1. In another embodiment, the molecular marker is located within the sequence shown in SEQ ID NO: 5.

[0178] This invention also relates to a method for identifying molecular markers suitable for detecting melon plants that produce or are prone to producing fruits with extended shelf life, comprising:

[0179] (a) Identify molecular markers in or in chromosomal regions genetically linked to the sgr gene.

[0180] (b) Determine whether the molecular markers are associated with or related to a phenotype of extended shelf life, particularly increased pericarp color stability in melon fruits during ripening and / or post-harvest.

[0181] In a further aspect, the present invention relates to a method for producing melon seedlings or plants that produce or are prone to producing fruits with extended shelf life, comprising:

[0182] i. To culture isolated cells or tissues of the melon plant according to the invention in vitro to produce melon micro-plantlets, and

[0183] ii. Optionally, the melon microplants may be further subjected to an in vivo culture stage to develop into melon plants that produce or readily produce fruits with extended shelf life.

[0184] The isolated cells or tissues used to generate microplants are explants obtained under sterile conditions from the melon parent plant of the present invention to be propagated. The explants comprise or consist of, for example, cotyledons, hypocotyls, stem tissue, leaves, embryos, meristems, nodular buds, shoot tips, or protoplasts. The explants may be surface-sterilized before being placed on a culture medium for micropropagation.

[0185] The conditions and culture media suitable for plant micropropagation 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”, by Edwin F George and Paul D Sherrington, Exegetics Ltd, 1984.

[0186] Micropropagation typically involves:

[0187] i. Axillary bud production: Axillary bud proliferation is induced by adding cytokinin to the bud culture medium to produce buds with minimal callus formation.

[0188] ii. Adventitious bud production: Adding auxin to the culture medium induces root formation to produce small plantlets that can be transferred to the soil. Alternatively, root formation can be induced directly in the soil.

[0189] The small plant can then undergo an in vivo culture stage, by culturing it in soil under laboratory conditions and then gradually adapting it to the natural climate, in order to develop into a melon plant according to the present invention.

[0190] The reduced leaf yellowing exhibited by the melons of the present invention allows for reduced yield loss caused by leaf yellowing under various physiological or pathological conditions, such as senescence or the occurrence of yellowing diseases, such as CYSDV infection. Therefore, the present invention also relates to a method for increasing melon plant yield or increasing the number of harvestable melon plants or fruits, comprising planting melon plants according to the present invention, which homozygously contains a mutant allele of the sgr gene on chromosome 9, wherein said mutant allele contains at least one loss-of-function mutation and confers reduced leaf yellowing. In one embodiment, the melon plants according to the present invention are grown in an environment infected with CYSDV.

[0191] Preferably, the method includes a first step of screening or selecting melon plants containing the mutant allele. The method can also be defined as a method for increasing the productivity of melon fields, tunnels, or greenhouses, or a method for reducing the intensity or amount of chemical or fungicide application in melon production.

[0192] The present invention also relates to a method for reducing melon production losses, comprising planting melon plants as defined above, particularly melon plants grown under CYSDV infection conditions.

[0193] In another embodiment, the present invention relates to a method for protecting melon fields, tunnels, greenhouses, or any other type of plantation from yellowing disease, such as CYSDV infection, or at least limiting infection levels or disease spread. The method preferably includes the step of planting yellowing-resistant plants of the present invention, i.e., plants containing a mutant allele of the sgr gene on chromosome 9, wherein the mutant allele contains at least one loss-of-function mutation.

[0194] 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 plantations.

[0195] This invention also relates to a method for increasing the yield of melon plants in an environment infected with CYSDV, comprising:

[0196] (a) Identification of melon plants resistant to CYSDV, wherein the plants homozygous contain a mutant allele of the sgr gene on chromosome 9, the mutant allele comprising at least one loss-of-function mutation, and

[0197] (b) Cultivate the tolerant melon plants in the contaminated environment.

[0198] This method increases the yield of melon plants, especially by harvesting more marketable melons, producing more commercial melons, or obtaining more seeds.

[0199] The present invention also relates to a method for improving the shelf life, marketability, and / or yield of melon fruits, wherein the method comprises planting a melon plant according to the invention and harvesting the fruits produced by said plant. Due to its extended shelf life, melons according to the invention can be harvested at a lower frequency, particularly 2 to 4 times per week, compared to existing non-LSL melons. Therefore, the present invention also relates to a method for increasing the flexibility of melon harvesting, wherein the method comprises planting a melon plant according to the invention and harvesting the fruits produced by said plant.

[0200] In one embodiment of these methods, the fruit is stored for at least 7 days after harvest, preferably 7 to 21 days.

[0201] Furthermore, the present invention also relates to a method for producing melon fruit, comprising:

[0202] (a) Cultivating the melon plants of the present invention as defined above;

[0203] (b) allow the plant to bear fruit; and

[0204] (c) Harvest the fruit of the plant, preferably at maturity and / or before maturity.

[0205] All preferred embodiments of the melon plant have been disclosed in the context of the foregoing aspects of the invention.

[0206] The method may advantageously include a further step of processing the melon plant into a processed food product.

[0207] On the other hand, the present invention relates to the use of melon plants or their fruits according to the present invention in the fresh-cut market or in food processing.

[0208] Throughout this application, the term "comprising" should be interpreted as encompassing all specifically mentioned features as well as optional, additional, or unspecified features. As used herein, the use of the term "comprising" also discloses an implementation in which no other features besides those specifically mentioned exist (i.e., "consisting of").

[0209] Example

[0210] Example 1: Generating and identifying mutant melons via EMS mutagenesis

[0211] Mature seeds of the climacteric Charente variety were mutagenized by soaking in 1% to 3% ethyl methanesulfonate (EMS) for 16 hours, followed by washing with 0.1 M 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 on the sgr gene on chromosome 9 was identified, showing a G->A substitution at the splice site at the end of the first intron. This mutant allele was named sgr-1, and its sequence is given in SEQ ID NO: 2.

[0212] The sgr-1 mutation can be identified using the KASPar (KBiosciences) assay, which uses two labeled fluorescent oligonucleotide forward primers and one unlabeled universal reverse primer (Table 1).

[0213]

[0214] Table 1: PCR primers used for detecting sgr-1

[0215] Example 2: Introgression of sgr-1 mutant genes

[0216] The sgr-1 mutation infiltrates into different desirable genotypes. The sgr-1 mutation from the EMS population is recessive, and its effect exists only when the variant is homozygous. To produce HF1 hybrids exhibiting this effect, various parental lines were converted. After the first hybridization between the parental lines and the sgr-1 source, the sgr-1 mutation was backcrossed multiple times in the parental lines. Figure 1 The two transformed lines were crossed together to produce HF1 hybrids homozygous for the sgr-1 mutation, which are near-isogenic lines (NILs) for HF1 lacking the sgr-1 mutation.

[0217] Several genotypes were converted through the sgr-1 mutation, including the following orange-fleshed types: Charente melon, Italian netted melon, beach melon, and Oriental melon, as well as the following white and green-fleshed types: Canary melon, Christmas melon, Gallia melon, and Hami melon.

[0218] Example 3: Effects of sgr-1 mutation on leaves

[0219] To assess the effects 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.

[0220] Leaf color is assessed at different times during plant growth, typically in the early pre-fruiting stage (date 1), during fruit ripening (date 2), and during or shortly after fruit harvest (date 3). Several plants (5 to 10 plants) are assessed for each genotype, and leaf color can be assessed visually and using a colorimeter. For example, a Konica Minolta R400 colorimeter can be used to measure leaf color. For a given date, the plant is measured twice before averaging to obtain the average plant values ​​for the three coordinates (L*, a*, and b*) in the ClELAB color space. Leaves are selected to represent the plant (neither too young nor too old). The average L*, a*, and b* values ​​at the genotype level for a given date can then be calculated using the average of all plant values.

[0221] A significant effect on leaf color was clearly observed in the V1_Charente melon and V2_Canary melon varieties. Figure 2 ).

[0222] To more accurately assess color evolution and variation, leaf color was measured using a colorimeter. Lower L*, a*, and b* values ​​observed for the sgr-1 genotype reflected a deeper and greener leaf color. Furthermore, the reduced dispersion of the sgr-1 data compared to the initial genotype indicates greater stability of leaf color and suggests reduced leaf yellowing in the sgr-1 mutation. Figure 4 ).

[0223] When using ANOVA to compare the means between the sgr-1 transformed lines and the original lines, significant effects were recorded on at least one of the three coordinates (L*, a*, b*) (Table 2). We can definitively conclude that the sgr-1 variation has a significant effect on leaf color, especially during and later stages of fruit ripening.

[0224]

[0225] Table 2: Effect of SGR-1 on leaf color

[0226] The color difference ΔE* calculated using the following formula supports this conclusion.

[0227]

[0228] ΔE* is low in the early stages, meaning that the leaf color is very similar between sgr-1 and the corresponding wild-type (WT) line. Then, ΔE* increases over time, showing the evolution of color differences, which is more pronounced in later stages of plant development. Figure 5 ).

[0229] In addition to the effects of sgr-1 on leaves visible under normal conditions, a strong sgr effect is also observed under CYSDV stress (natural infection in areas severely affected by the virus). The virus is still present on the leaves of plants carrying the sgr-1 mutant allele, but visible symptoms are masked, and plants carrying the sgr-1 mutant allele exhibit less yellowing than those with the wild-type allele. The sgr-1 mutation provides a partial resistance of interest to CYSDV yellowing. Figure 3 ).

[0230] Example 4: Effect of sgr-1 mutation on fruit peel color

[0231] During the varietal conversion process, the inventors observed the effect of the sgr-1 mutation on the rind color based on melon type. The fruit tended to be greener. The inventors evaluated this effect visually and colorimetrically on the day of harvest and 7 days after storage. Observations and measurements were conducted on different melon genotypes, specifically targeting the following three varieties of orange-fleshed materials: V1_Charentes Melon_LSL, V2_Italian Netted Melon_NLSL, and V3_Italian Netted Melon_NLSL.

[0232] In orange-fleshed material, a clear color difference was observed at harvest between the V2_Italian Netted Melon_NLSL WT type and the sgr-1 transgenic variety. The V2_Italian Netted Melon variety is non-LSL and turns yellow upon ripening. However, the sgr-1 type retains its green rind.

[0233] After 7 days of storage, the wild-type (WT) V2_Italian netted melon_NLSL fruit became increasingly yellow-orange, while the V2_Italian netted melon_NLSL sgr-1 fruit showed no external evolution and retained its green rind color. Figure 6 ). Figure 6 The color version clearly shows the effect of SGR-1 on the pericarp color. Color versions of all figures in this application, including... Figure 6 It shall be submitted with this application and may be provided upon request.

[0234] The V1_Charentes Melon_LSL genotype, which does not turn yellow at maturity, was also observed. At harvest, no color difference was observed between the sgr-1 and WT types of V1_Charentes Melon_LSL. This indicates that the sgr-1 genotype does not affect, or only slightly affects, the rind color of the melon LSL genotype.

[0235] Furthermore, the recessive effect of the sgr variant on pericarp color was confirmed. No differences were observed in the WT type and the heterozygous sgr-1 type of V3_Italian netted melon_NLSL.

[0236] Even if the Sgr-1 effect is very pronounced, it can be demonstrated using colorimetric data. Because of the network-like structure on the peel surface, colorimeter tools are not suitable for this type of measurement. Instead, image analysis can be used to extract the peel color and obtain L*, a*, and b* values.

[0237] Compared with the L* and b* values ​​of the WT type, a significant statistical effect of the sgr-1 variation was observed on variety 2 and the non-LSL genotype. Figure 7 The higher L*a*b* values ​​compared to the greener sgr-1 type reflect a more yellow peel color in the WT type. No difference was observed between the sgr-1 and WT types (i.e., LSL) in variety 1. Furthermore, no significant difference was recorded between the heterozygous sgr-1 and WT types in the non-LSL variety 3. The mutation needs to be homozygous and in a non-LSL background to reveal its effect on peel color.

[0238] The color difference can be calculated using the ΔE* formula between the Sgr-1 convert and the corresponding WT strain. The effect of the Sgr-1 variation on Variety 2 is significant at J0 (harvest date), increases slightly at J7 (7 days of storage), and has a ΔE* higher than 10 (26.1 and 30.7 respectively), while the ΔE* of other varieties is lower than 10. Figure 8 ).

[0239] Example 5: Effect of sgr-1 mutation on pulp color

[0240] The inventors further evaluated the effect of the sgr-1 mutation on the flesh color of melons to control for potential impacts on this fruit quality trait.

[0241] The flesh color of different melon genotypes was determined by visual observation and colorimetric methods after harvest, following storage at 14°C for 7 days in a cold storage room. Two types of melons were evaluated: orange-fleshed materials of varieties V1_Charente Melon_LSL and V2_Italian Netted Melon_NLSL, and white-fleshed materials of variety V6_Canary Melon_LSL, as well as their respective versions, wild-type (WT) and sgr-1 type.

[0242] The flesh color of 10 fruits for each genotype was evaluated. These measurements were performed using a colorimeter on equatorial slices of the fruit. Two measurements of opposite diameters were performed for each fruit, and the average was calculated to obtain the fruit-level color.

[0243] Paired comparisons using the Tukey test were used to assess color differences, and no significant differences were recorded between the sgr-1 line and their WT types in both orange and white flesh materials. Figure 9 ).

[0244] The same results were observed in green pulp material.

[0245] Example 6: Fine phenotyping of sgr-1 transformed varieties

[0246] Fine phenotyping of several traits was performed on three varieties, including the WT allele, V2_Italian netted melon_NLSL, V4_HD_NLSL, and V5_Charentes melon_NLSL, and their corresponding three sgr-1 variants. Fourteen fruits were harvested for each genotype and phenotypic analysis was conducted to assess the impact of the sgr-1 mutation on other important traits associated with the non-LSL type.

[0247] Cycle length corresponds to the number of days calculated from the transplanting date to the harvest date. Non-LSL materials are considered to have the shortest cycle, with fruits harvestable from 55 days after transplanting, while LSL materials have a longer cycle, with fruits harvestable approximately 90 days after transplanting. In the experiment, all plants were transplanted to the field on the same day (approximately 20 days after sowing), and the harvest date for each harvested fruit was recorded for calculation. Among the observed genotypes, there was no significant difference in cycle length between the sgr-1 transformant and its corresponding WT variety. Figure 10 The lag mutation does not significantly delay harvest time.

[0248] Flower stalk abscission is an important indicator of maturity, whether it's due to changes in skin color or the senescence of the first leaf and tendrils. Fruit is harvested when one or more of these indicators are evolving. Therefore, flower stalk abscission is observed on the day of fruit harvest and evaluated on a scale of 1 to 9, where 1 = complete abscission and 9 = no abscission. The sgr-1 mutation does not significantly affect the maturity indicator (i.e., flower stalk abscission), and no significant difference was found in pairwise comparisons using the Tukey test. Figure 11 The shed material will continue to fall off, which will reduce the grower's harvest.

[0249] Brix measurements of equatorial slices of each melon taken at harvest time using an electron refractometer also showed no significant difference in Brix levels between the sgr-1 mutant and the WT type. Compared to the corresponding melons carrying the WT allele, the sgr-1 mutation does not affect sugar levels and therefore does not affect sweetness. Figure 12 ).

[0250] The hardness of each equatorial slice of all harvested fruit was also measured. Measurements were taken using a translucency meter at two points opposite each other on the equatorial slice. The average of the two measurements was then calculated. No statistically significant difference was observed between the sgr-1 and WT genotypes. Figure 13 )

[0251] In summary, observations of different sgr-1 transformants compared to the original variety (WT) classified as non-LSL genotypes indicate that the sgr-1 mutation introduces a new ideal plant type for melons, extending shelf life due to the stability of rind color evolution, without affecting fruit quality or maturity indicators. In other words, it will provide growers with better field holding power and harvest flexibility without extending the harvest window. The lack of rind color evolution during storage provides retailers with greater flexibility. For the end consumer, the initial fruit quality of the product is maintained.

[0252] Other climacteric fruits from different melon types were evaluated, and the same conclusion was reached.

Claims

1. A non-regenerative cell of a melon plant (Cucumis melo), wherein, The plant contains homozygous mutant alleles of the sgr gene on chromosome 9 in its genome, wherein the mutant alleles of the sgr gene are G584A mutations in the wild-type sgr allele sequence as shown in SEQ ID NO: 1, and wherein the mutant alleles of the sgr gene confer stability of the peel color of the fruit of the plant at maturity and / or during the post-harvest period, compared to isogenetic non-long-shelf-life melon plants that do not contain the mutant alleles.

2. The cell according to claim 1, wherein, The melon plant is a plant derived from an inbred melon strain or an F1 hybrid melon plant.

3. A method for producing melon plants that produce fruits with extended shelf life or are prone to producing fruits with extended shelf life, comprising: (a) Obtaining a portion of a melon plant, wherein the plant homozygously contains a mutant allele of the sgr gene on chromosome 9 in its genome, wherein the mutant allele of the sgr gene is a G584A mutation in the wild-type sgr allele sequence as shown in SEQ ID NO: 1, and wherein, compared to an isogenetic non-long-shelf-life melon plant that does not contain the mutant allele, the mutant allele of the sgr gene confers rind color stability of the fruit of the plant at maturity and / or during the post-harvest period. (b) Asexual reproduction of parts of the plant to produce plants from the parts of the plant.

4. A method for producing melon plants that produce fruits with extended shelf life or are prone to producing fruits with extended shelf life, comprising: (a) Using melon plants as breeding mates to introduce a mutant allele of the sgr gene into progeny melon plants, wherein the plant homozygously contains a mutant allele of the sgr gene on chromosome 9 in its genome, wherein the mutant allele of the sgr gene is a G584A mutation in the wild-type sgr allele sequence as shown in SEQ ID NO: 1, and wherein the mutant allele of the sgr gene confers rind color stability of the fruit at maturity and / or during the post-harvest period, compared to isogenetic non-long-shelf-life melon plants that do not contain the mutant allele; or (b) Introducing a loss-of-function mutation in the sgr gene on chromosome 9 into the genome of a melon plant by genome editing, wherein the plant homozygously contains a mutant allele of the sgr gene on chromosome 9 in its genome, wherein the mutant allele of the sgr gene is a G584A mutation in the wild-type sgr allele sequence as shown in SEQ ID NO: 1, and wherein the mutant allele of the sgr gene confers rind color stability of the fruit of the plant at maturity and / or during the post-harvest period, compared to an isogenetic non-long-shelf-life melon plant that does not contain the mutant allele.

5. The method according to claim 4, wherein, Using the melon plant as a breeding mate to introduce the mutant allele of the sgr gene into offspring melon plants includes: (a1) The melon plant is crossed with a second melon plant that is not homozygous for a mutant allele of the sgr gene, thereby producing an F1 population. (a2) Promote the generation of the F2 population from the F1 population. (b) Select a plant that homozygousally contains the mutant allele in the offspring obtained therefrom.

6. The method according to claim 5 further includes self-pollinating and / or backcrossing the plant obtained in step b) once or several times.

7. The method according to claim 6, wherein, The step of introducing a loss-of-function mutation in the sgr gene on chromosome 9 into the genome of a melon plant via genome editing is performed using a technique selected from the following: zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN), CRISPR / Cas system, engineered large-scale nucleases, reengineered homing endonucleases, and DNA-guided genome editing.

8. A method for producing melon plants with extended shelf life, comprising the following steps: (a1) A melon plant homozygous for containing the mutant allele of the sgr gene according to any one of claims 1 to 3 is crossed with a second melon plant heterozygous for containing the mutant allele, thereby producing an F1 population, wherein the mutant allele of the sgr gene, compared to the wild-type sgr allele sequence shown in SEQ ID NO: 1, is a G584A mutation in the wild-type sgr allele sequence. (a2) Promote the generation of the F2 population from the F1 population. (b) Select a plant that homozygousally contains the mutant allele in the offspring obtained therefrom.

9. The method according to claim 8, further comprising: (c) Self-pollinate the plant selected in step b) once or several times; and / or (d) Backcross the plant selected in step b) or c) with a melon plant that is heterozygous to contain the mutant allele; and (e) Select plants that homozygously contain the mutant allele, wherein the plants produce fruits with extended shelf life.

10. The method according to claim 8 or 9, further comprising: The selected plant is crossed with different melon plants that homozygously contain the mutant allele of the sgr gene, thereby producing a hybrid melon plant that homozygously contains the mutant allele of the sgr gene.

11. A method for identifying, detecting, and / or selecting melon plants that produce or are prone to producing fruits with extended shelf life, said method comprising detecting mutations in the sgr gene on chromosome 9 of the plant's genome, wherein, The mutation is a G584A mutation performed in the wild-type sgr allele sequence as shown in SEQ ID NO: 1, compared to the wild-type sgr allele sequence.

12. A method for improving the shelf life, marketability, and / or yield of melons, wherein, The method includes planting melon plants produced according to any one of claims 3 to 10 and harvesting the fruits produced by the plants.

13. A method for producing melon fruit, comprising: a) Cultivating melon plants produced by the method according to any one of claims 3 to 10; b) The plant is permitted to bear fruit; and c) Harvest the fruit of the plant.

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