WO2018146323A1

WO2018146323A1 - Method for altering ripening characteristics of fruit

Info

Publication number: WO2018146323A1
Application number: PCT/EP2018/053482
Authority: WO
Inventors: Liping Gao; René Johannes Maria Hofstede
Original assignee: Keygene N.V.
Priority date: 2017-02-13
Filing date: 2018-02-13
Publication date: 2018-08-16

Abstract

A method for modifying ripening characteristics of fruit of a plant is disclosed, as well as a plant bearing fruit that has such modified fruit ripening characteristics. Further, the invention provides means for modifying ethylene production of fruits from such plants, allowing tailoring of fruit ripening in any type of plant.

Description

Method for altering ripening characteristics of fruit

Field of the invention

The present disclosure is concerned with methods characterizing and/or modifying or modulating fruit ripening characteristics of a plant.

Background of the invention

During the ripening period, fruit undergoes many changes, including a decrease in chlorophyll, an increase in carotenoid, the accumulation of free sugar and organic acid, the production of fragrant components, an increase in breath rate, an increase in the activity of softening enzymes, and the degradation of cell wall components, which lead to the physical property changes and softening phenomena of the fruits. Fruit ripening is achieved through two main mechanisms based on the role of the ethylene hormone in the process: 1 ) climacteric ripening, characterized by an increase in respiration and concomitant ethylene synthesis upon initiation of ripening; and 2) non-climacteric ripening, characterized by a continuous decrease in respiration rate and ethylene production. Climacteric fruits include apple, banana, tomato and avocado, whilst non-climacteric fruits include grape, citrus species, strawberry and pineapple.

In climacteric fruits, the softening process is concomitant with the climacteric ethylene production. This process is very fast, so climacteric fruits usually have a short shelf-life, while in non-climacteric fruits, ethylene is constantly at a low level, and fruits don't show a fast and big change in the physical property. Non-climacteric fruits therefore usually have a long shelf-life.

To prolong the shelf life of climacteric fruit, currently often used techniques include premature harvest, controlled atmosphere storage, and chemically induced ripening to schedule the timing of maturation of the fruit. However, added production, shipping and processing costs are often the consequences of these practices. Specially, premature harvest reduces fruit quality, and for some species or varieties, they become sweet enough only when they are almost ripe, e.g. Cantaloupe melon, so premature harvest is almost not applicable.

There is a need in the art for novel methods for creating plants with modified fruit ripening characteristics. The identification of genes involved in the climacteric ripening process would allow the creation of novel plants altered in their fruit ripening characteristics and/or ethylene production.

Climacteric fruit ripening has been extensively studied in tomato. Most of the enzymes, receptors and other factors involved in ethylene synthesis and signalling have been identified from ethylene response mutants in Arabidopsis, and confirmed in tomato. It has been shown that climacteric ripening represents a combination of both ethylene mediated and developmental control. However, the exact mechanisms to differentiate climacteric from non-climacteric fruit ripening have yet to be elucidated.

The co-existence in melon of both climacteric, intermediate-type and non-climacteric varieties and the availability of a set of genetic and genomic resources make melon a suitable model for genetic studies of fruit ripening. According to a research paper (Vegas et al. Theor Appl Genet. 2013. 126: 1531 ), two QTLs, ETHQB3.5 and ETHQV6.3 on their own, are capable of inducing climatic ripening of the melon fruits. ETHQV6.3 effects being greater than those of ETHQB3.5. They also interact epistatically resulting in a precocity of the fruits, which require less time to mature than in the case of exclusively owning one of the QTLs.

One of these QTLs, ETHQB3.5, is on chromosome 3, between markers CMCTN5 and TJ10 (Argyris ei al. BMC Genomics. 2015. 16: 4) and its physical position is 24975485-27248436. In this region, 146 genes were predicted.

Summary of the invention

Surprisingly, the present inventors identified MELO2C011271 , located on chromosome 3 with a physical position of coding region on 25049667-25050104, as the real gene responsible of

ETHQB3.5 and identified an upstream region, located on chromosome 3 at physical position 25049407, near (closest to) this gene that comprises a molecular marker that correlates with the ripening phenotype.

Based on extensive and detailed expression studies, the inventors found that among the 146 genes predicted in the ETHQB3.5 QTL region, only one single gene, MELO2C01 1271 , positively co- expressed with the 1-aminocyclopropane-1-carboxylate oxidase 1 (AC01 ) gene in non-climacteric types, whilst it was negatively co-expressed with the AC01 gene in the climacteric and intermediate type. The AC01 gene encodes the enzyme (ACC oxidase) catalyzing the last step of ethylene biosynthesis. MELO2C01 1271 encodes a protein annotated as a 14kDa proline rich protein. Further, a 33 bp indel was identified 289 bp upstream of the start codon of this gene that perfectly correlates with the fruit ripening. The 33 bp indel is absent in non-climacteric types, while it is present in climacteric and intermediated types.

The newly identified molecular marker within the domain located about 289 bp upstream of the start codon of the MELO2C01 1271 coding sequence, correlates perfectly with the ripening phenotype. The molecular marker is a 33 bp insertion/deletion polymorphism. The presence of this 33 bp sequence was found in climacteric species, whereas the absence of this sequence was found in non-climacteric species. This indel may play a role in regulation of expression of MELO2C01 1271 encoding the 14kDa proline rich protein, which is the gene closest to the indel. The region comprising this indel is therefore also denominated herein as cis-element of the 14kDa proline rich protein.

Description of the invention

Definitions

In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The disclosures of all publications, patent applications, patents and other references are incorporated herein in their entirety by reference.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to one skilled in the art. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are known to one skilled in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego. The term "comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The scope of the term "comprise" encompasses the scope of the term "consist essentially of" and the scope of the term "consist of." The terms "aligning" and "alignment" refer to the comparison of two or more nucleotide sequences based on the presence of short or long stretches of identical or similar nucleotides. Several methods for alignment of nucleotide sequences are known in the art, as will be further explained below.

"Expression of a gene" refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment). "Ectopic expression" refers to expression in a tissue in which the gene is normally not expressed. "Expression of a protein" is used herein interchangeably with the term expression of a gene. It refers to the process in which a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an mRNA and which is subsequently translated into a protein or peptide (or active peptide fragment). Reduced or increased expression in plant tissue or cells can be assessed by suitable techniques in the art, such as, but not limited to, PCR analysis, sequencing of genomic DNA, sequencing of mRNA transcript, analysing mRNA transcript levels (Northern-blot analysis), analysing copy number (Southern blot analysis), etc. The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g., an mRNA) in a cell, operably linked to suitable regulatory regions (e.g., a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising, e.g., sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3' non-translated sequence comprising, e.g., transcription termination sequence sites.

The term "cDNA" means complementary DNA. Complementary DNA is made by reverse transcribing RNA into a complementary DNA sequence. cDNA sequences thus correspond to RNA sequences that are expressed from genes. As mRNA sequences when expressed from the genome can undergo splicing, i.e., introns are spliced out of the mRNA and exons are joined together, before being translated in the cytoplasm into proteins, it is understood that expression of a cDNA means expression of the mRNA that encodes for the cDNA. The cDNA sequence thus may not be identical to the genomic DNA sequence to which it corresponds as cDNA may encode only the complete open reading frame, consisting of the joined exons, for a protein, whereas the genomic DNA encodes and exons interspersed by intron sequences. Genetically modifying a gene which encodes the cDNA may thus not only relate to modifying the sequences corresponding to the cDNA, but may also involve mutating intronic sequences of the genomic DNA and/or other gene regulatory sequences of that gene, as long as it results in the impairment of gene expression. As used herein, a "locus" is a fixed position on a chromosome and may represent a single nucleotide, a few nucleotides or a large number of nucleotides in a genomic region.

As used herein, "polymorphism" means the presence of one or more variations of a nucleic acid sequence at one or more loci in a population of one or more individuals. The variation may comprise but is not limited to, one or more base changes, the insertion of one or more nucleotides or the deletion of one or more nucleotides. A polymorphism includes a single nucleotide polymorphism (SNP), repeats such as a perfect or nearly perfect tandem repeat or simple sequence repeat (SSR), a restriction fragment length polymorphism and indels, which are insertions and deletions. A polymorphism may arise from random processes in nucleic acid replication, through mutagenesis, as a result of mobile genomic elements, from copy number variation and during the process of meiosis, such as unequal crossing over, genome duplication and chromosome breaks and fusions. The variation can be commonly found or may exist at low frequency within a population, the former having greater utility in general plant breeding and the later may be associated with rare but important phenotypic variation. As used herein, "marker" means a detectable characteristic that can be used to discriminate between organisms. Examples of such characteristics may include genetic markers, protein composition, protein levels, oil composition, oil levels, carbohydrate composition, carbohydrate levels, fatty acid composition, fatty acid levels, amino acid composition, amino acid levels, biopolymers, pharmaceuticals, starch composition, starch levels, fermentable starch, fermentation yield, fermentation efficiency, energy yield, secondary compounds, metabolites, morphological characteristics, and agronomic characteristics.

As used herein, "genetic marker" means polymorphic nucleic acid sequence or nucleic acid feature. A genetic marker, a gene, a DNA-derived sequence, a RNA-derived sequence, a promoter, a 5' untranslated region of a gene, a 3' untranslated region of a gene, micro RNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, ds mRNA, a transcriptional profile, and a methylation pattern may comprise polymorphisms. "Identity" is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art- recognized meaning and can be calculated using published techniques (see, e.g., COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ). While a number of methods exist to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48: 1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1 ):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403), preferably using default parameters. The percentage sequence identity may preferably be determined over the entire length of the sequence concerned.

As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference polypeptide sequence. Hence, the percentage of identity of a nucleotide sequence to a reference nucleic acid sequence is calculated over the entire length of the reference nucleic acid sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid. Hence, the percentage of identity of an amino acid sequence to a reference amino acid sequence is calculated over the entire length of the reference amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

A "nucleic acid" according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes). The present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxy methylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

A "vector," "expression vector" or "expression construct" refers to a recombinant nucleic acid molecule which is used to deliver exogenous DNA into a host cell. The vector backbone may for example be a binary or superbinary vector (see e.g. U.S. Pat. No. 5,591 ,616, US 2002138879 and WO 95/06722), a co-integrate vector or a T-DNA vector, as known in the art and as described elsewhere herein, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid sequence (e.g. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like. "Promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Optionally the term "promoter" includes also the 5' UTR region (5' Untranslated Region) (e.g. the promoter may herein include one or more parts upstream of the translation initiation codon of a gene, as this region may have a role in regulating transcription and/or translation). A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmental^ regulated. A "tissue specific" promoter is only active in specific types of tissues or cells. A "promoter active in plants or plant cells" refers to the general capability of the promoter to drive transcription within a plant or plant cell. It does not make any implications about the spatio-temporal activity of the promoter.

The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked may mean that the DNA sequences being linked are contiguous.

The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A "fragment" or "portion" of a protein may thus still be referred to as a "protein." An "isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

" ELO3C01 1271 polypeptides" refers to a group of plant proteins characterized by their sequence homology to the protein encoded by the melon MELO3C01 1271 gene (e.g., having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96,%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the polypeptide encoded by the MELO3C01 1271 gene (SEQ ID NO: 1 ), or to the protein encoded by the tomato (LOC101254713) gene (e.g., having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96,%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the SEQ ID NO: 2, as well as their characterisitc in being co-expressed with the AC01 gene, thereby being involved in climacteric fruit ripening. MELO3C01 1271 polypeptides include at least the polypeptide encoded by the MELO3C01 1271 gene (SEQ ID NO: 1 ) and its homolog from tomato (SEQ ID NO: 2). See Table 1 for sequences and their SEQ ID NO.

"MELO3C01 1271 genes" refers to genes encoding the MELO3C01 1271 polypeptides. The MELO3C011271 genes include at least the melon MELO3C01 1271 gene (SEQ ID NO: 3) and its homologs from tomato (SEQ ID NO: 4).

The term "functional" in relation to MELO3C01 1271 proteins or genes refers to the capability of the gene and/or encoded protein to modify fruit ripening characteristics, e.g. from climacteric ripening to non-climacteric ripening type, and/or to modify the AC01 gene expression, e.g., by modifying the expression level of the gene (e.g., silencing or knocking out the expression of the gene), of a plant. For example, the functionality of a MELO3C01 1271 protein obtained from plant species X can be tested by various methods. Preferably, if the protein is a functional MELO3C01 1271 protein, knocking out the gene encoding the protein in plant species X or reducing expression of the gene encoding the protein in a different plant species can lead to modified fruit ripening characteristics, e.g. from non- climacteric ripening characteristics to climacteric ripening characteristics, as can be tested as explained herein in detail. Also, restoring expression of the MELO3C01 1271 protein can delay the ripening phenotype of the wild-type plants. The skilled person will be able to test such functionality. "Plant" refers to either the whole plant or to parts of a plant, such as cells, tissue, callus, explant, or organs (e.g. pollen, seeds, gametes, roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainable from the plant, as well as derivatives of any of these and progenies derived from such a plant by selfing or crossing. "Plant cell(s)" include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., either in isolation or within a tissue, organ or organism. The plant cell, tissue or organ may be a cell, tissue or organ that does not possess the property of photosynthesis.

"Genetically-engineered plant" or "transformed plant" refers herein to a plant or plant cell that has been genetically engineered to introduce for example one or more insertions of a gene expression construct in the genome. A genetically-engineered plant cell may refer to a plant cell in isolation or in tissue culture, or to a plant cell contained in a genetically-engineered plant or in a differentiated organ or tissue, and both possibilities are specifically included herein. Hence, a reference to a plant cell in the description or claims is not meant to refer only to isolated cells or protoplasts in culture, but refers to any plant cell, wherever it may be located or in whatever type of plant tissue or organ it may be present.

A "control plant" as referred to herein is a plant of the same species and preferably same genetic background as the plant having modified fruit ripening characteristics and/or modified MELO3C01 1271 polypeptide expression. The control plant preferably comprises the endogenous MELO3C01 1271 gene, and is preferably a wild type plant. The term "regeneration" as used herein refers the process of growing a plant from a plant cell (e.g.,, plant protoplast, callus or explant).

The term "modulating ripening" or "altering ripening" as used herein may refer to delaying, suppressing, reducing, decreasing, inducing, conferring, restoring, elevating, increasing or otherwise affecting the ripening of a fruit (e.g., changing the fruit ripening characteristics from the climacteric type to the intermediate or non-climacteric type or vice versa) of a plant.

A "non-climacteric phenotype" (also denominated herein as non-climacteric fruit ripening phenotype)is understood herein as a fruit ripening phenotype without a fast and big change in physical property of the fruit during ripening, and wherein the fruits have a relatively long shelf-life. The non-climacteric phenotype may be characterized by a constant and low level of ethylene production. For melon, a non-climacteric phenotype may be a fruit ripening phenotype that is similar to the fruit ripening characteristics of a melon line of the Inodorus group.

A "climacteric phenotype" (also denominated herein as climacteric fruit ripening phenotype) is understood herein as a fruit ripening phenotype that is fast and wherein the fruit have a relatively short shelf-life with a fast and big change in physical property of the fruit during ripening. The climacteric phenotype is characterized by increased ethylene production that is concomitant with the softening process of the fruit during ripening. For melon, a climacteric phenotype may be the fruit ripening phenotype that is similar to the fruit ripening characteristics of a melon line of the Cantalupensis group or Reticulatus group.

An "intermediate phenotype" (also denominated herein as intermediate fruit ripening phenotype) is understood herein as a fruit ripening phenotype that has characteristic that are in between the non- climacteric and climacteric phenotype. For melon, an intermediate phenotype may be the fruit ripening phenotype that is similar to the fruit ripening characteristics of a melon line of Honeydew-TamDew (Tam) and/or PI161375 (SC). "Immediately adjacent" in the sense of the location of a nucleotide sequence is to be understood here as to be directly linked at the 3'-end or 5'-end by a phosphodiester bond within a nucleic acid strand.

"A," "an," and "the": these singular form terms include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

As used herein, the term "about" is used to describe and account for small variations. For example, the term can refer to less than or equal to ±10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

"And/or": the term "and/or" refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

Detailed description of the invention

In a largescale and detailed gene expression study, the inventors found that MELO3C01 1271 was the only gene among the 146 genes within the ETHQB3.5 QTL that negatively co-expressed with the AC01 gene in climacteric and intermediate type, and positively with the non-climacteric type in fruit ripening. By sequencing the MELO3C01 1271 gene in 126 germplasm lines, a polymorphism (i.e. an indel) was identified in a region upstream of the MELO3C01 1271 gene. The MELO3C01 1271 gene is within QTL ETHQB 3.5 on chromosome 3, between markers CMCTN5 and TJ10, (Argyris et al. BMC Genomics. 2015, 16: 4), wherein the gene is located at physical position on chromosome 3, 25049667-25050104. 94 of the 126 melon lines that were sequenced, belonged to C. melo subsp. melo. This subspecies is divided into Cantalupensis group, Reticulatus group and Inodorus group, where the former two are in general regarded as climacteric type melons, while the latter one is considered a non-climacteric type. Based on this general categorization, a high association was found between the polymorphism within this region and the ripening phenotype.

The presence of the 33 bp indel (a 36 bp long sequence represented by SEQ ID NO: 5, as compared to a 3 bp long sequence present on this position in the non-climacteric phenotype having the sequence TTT, strongly associates with the climacteric phenotype. The identification of these polymorphisms between the climacteric versus non-climacteric melon genotypes allows for the development of DNA markers that can for instance be used in marker assisted breeding. Use of such markers allows for definitive genotyping of seedlings in a matter of 1-5 days.

Therefore, in a first aspect, provided is a genetic marker for characterizing fruit ripening. The genetic marker is located within the region upstream of the of the MELO3C01 1271 gene as defined herein, preferably about 289 bp upstream, such as between 1 and 1000 bp upstream, or between 100 and 400 bp upstream, or between 200 and 300 bp upstream, of the start cod on of MELO3C01 1271 , within the ETHQB 3.5 QTL sequence that is on chromosome 3 delimited by markers CMCTN5 and TJ10, (Argyris et al. BMC Genomics. 2015, 16: 4), or any orthologous genomic region. The molecular marker is an indel that is located within the region upstream of the MELO3C01 1271 gene as defined above (also see Figure 3). Preferably, the indel has a sequence represented by SEQ ID NO: 5 or has a sequence that has 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. The presence of this indel within the region upstream of MELO3C01 1271 gene as defined herein associates with the climacteric and intermediate phenotype. The absence of this indel associates with the non-climacteric phenotype. Therefore, preferably the marker (also denominated herein as molecular marker or genetic marker) that associates with climacteric and intermediate phenotype has the sequence of SEQ ID NO: 5.

The presence or absence of said indel may be assessed by any technique known in the art such as by sequencing the sequence upstream of the MELO3C01 1271 gene, more preferably the cis-element, which is located at or near the region localised on chromosome 3 at physical position 25049407 or any orthologous position thereof, or a sequence comprising said cis-element, or by using any tagged oligonucleotide that specifically hybridizes to either one of the markers associated with the non- climacteric or climacteric and intermediate phenotype. Alternatively, or in addition, the presence or absence of said indel may be assessed by comparing the length of amplified fragments of the sequence optionally comprising the indel, e.g. the sequence upstream of the ELO3C01 1271 gene, more preferably the sequence comprising the cis-element or a sequence comprising said cis-element. Preferably, such amplified fragments comprise the sequence of about 36 bp starting at position 25049407 on chromosome 3. Examples of such a primers set that can be used to amplify that sequence are represented by SEQ ID NO: 6 and SEQ ID NO: 7. The presence of the indel (SEQ ID NO: 5) associating with the climacteric and intermediate phenotype can be distinguished easily from the absence (i.e. absence of SEQ ID NO: 5, optionally presence of TTT) based on length difference of the amplicons (amplified fragments) prepared using primers such as SEQ ID NO: 6 and SEQ ID NO: 7 in PGR amplification. Further suitable techniques envisioned by the skilled person to detect the above indicated polymorphism for characterizing fruit ripening are encompassed within the invention.

The genetic marker of this aspect may be used in screening, identification and marker-assisted breeding of plants, in particular of plants belonging to the genus Cucumis, particularly Cucumis melo. Current selection methods require confirmation of the genotype through phenotypic analysis of fruit development (i.e., the latest stage of plant development) with confirmation requiring analysis of subsequent progeny. Such phenotypic screening requires considerable growth space and 2-3 months per plant generation cycle. Use of the present markers allows for definitive genotyping of seedlings in a matter of 1-5 days.

In a second aspect, provided is the use of the genetic marker according to the first aspect, alone or in combination, in the field of screening, identifying and/or marker assisted breeding, of plants. Preferably, said screening, identifying and/or breeding is for a genotype that is associated with the climacteric and intermediate or non-climacteric phenotype.

Preferably, the marker is used for a plant(s) that is a climacteric fruit-bearing plant, preferably selected from the group consisting of tomato, nectarine, peach, apricot, avocado, banana, cantaloupe, guava, kiwifruit, mango, papaya, passionfruit, apple, pear, persimmon, plum, date and mulberry. The plant may be Cucumis melo or Solanum lycopersicum. The marker may also be used for a plant(s) that is a non-climacteric fruit-bearing plant, preferably selected from the group consisting of watermelon, strawberry, inodorus melon, grape, pomegranate, pineapple, citrus, coconut, olive, summer squash, blackberry, blackcurrant, blueberry, gooseberry, raspberry, cherry, and fig. The plant may be a monocotyledonous plants or dicotyledonous plants, preferably fruit-bearing plants, preferably of the family Cucurbitaceae or Solanaceae. The plant may be selected from Cucumis, or Solanum (including Lycopersicon), Nicotiana, Capsicum, Petunia and other genera. The plant may also be selected from vegetable species, including tomato (Solanum lycopersicum) such as e.g. cherry tomato, var. cerasiforme or currant tomato, var. pimpinellifolium) or tree tomato (S. betaceum, syn. Cyphomandra betaceae), potato (Solanum tuberosum), eggplant (Solanum melongena), pepino (Solanum muricatum), cocona (Solanum sessiliflorum) and naranjilla (Solanum quitoense), or peppers (Capsicum annuum, Capsicum frutescens, Capsicum baccatum). The plant may be a "crop plant", i.e. plant species which is cultivated and bred by humans. A crop plant may be cultivated for food purposes (e.g. field crops), or for ornamental purposes (e.g. production of flowers for cutting, grasses for lawns, etc.). A crop plant as defined herein also includes plants from which non-food products are harvested, such as oil for fuel, plastic polymers, pharmaceutical products, cork and the like. Preferably, the plant is Cucumis melo.

Specific methods for screening, identifying and marker assisted breeding, wherein the genetic marker according to the first aspect can be used, are provided in the aspects below, more in particular the third, fourth and fifth aspect below. In all these methods, the plant preferably is a plant as defined in this aspect.

In a third aspect, provided is a method for identifying a plant comprising a particular fruit ripening characteristic. For instance, said method may be a method for identifying a plant comprising a genotype that is associated with a non-climacteric phenotype, comprising the steps of:

a) extracting nucleic acid from a plant; and,

b) assessing the absence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype.

Further, said method may be a method for identifying a plant comprising a genotype that is associated with a climacteric and intermediate phenotype, comprising the steps of:

a) extracting nucleic acid from a plant; and,

b) assessing the presence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype in said nucleic acid.

The method of this aspect may further comprise the step of selecting the plant having the genetic marker of step b, optionally from a population of plants. Said selected plant may be used for breeding. In a fourth aspect, provided is a method for screening a plurality of plants comprising a particular fruit ripening characteristics. For instance, said method for screening a plurality of plants for comprising a genotype that is associated with a non-climacteric phenotype comprises the steps of:

a) extracting nucleic acid from said plurality of plants; and,

b) assessing the absence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype in said nucleic acid via a screening technology, preferably a high-throughput screening technology.

Further, said method for screening a plurality of plants for comprising a genotype that is associated with a climacteric and intermediate phenotype comprises the steps of:

a) extracting nucleic acid from said plurality of plants; and,

b) assessing the presence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype in said nucleic acid via a screening technology, preferably a high-throughput screening technology.

The method of this aspect may further comprise the step of selecting plant(s) lacking or comprising said marker, respectively, optionally for use in breeding.

In a fifth aspect, provided is a method for obtaining a plant with particular fruit ripening characteristics. Said method may be a method for obtaining a plant having a non-climacteric phenotype or increased non-climacteric phenotype, comprising the steps of:

a) determining the absence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype:

b) selfing or crossing the plant in which said marker is absent; and

c) optionally, determining the absence the marker according to the first aspect that is associated with the non-climacteric phenotype in the progeny of the selfing or crossing step.

Further, said method may be a method for obtaining a plant having a climacteric or intermediate phenotype or increased climacteric phenotype, comprising the steps of:

a) determining the presence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype;

b) selfing or crossing the plant in which said marker is present; and

c) optionally, determining the presence of the marker according to the first aspect that is associated with the climacteric and intermediate phenotype in the progeny of the selfing or crossing step.

In case the method of this aspect is to obtain a plant having a non-climacteric phenotype or increased non-climacteric phenotype, the plant is step b) may be crossed with a plant comprising the marker associated with a climacteric and intermediate phenotype. Optionally, the plant in step b) is heterozygous for lacking the marker associated with a climacteric and intermediate phenotype. The method of the invention may be aiming for progeny that is homozygous for the lacking the marker associated with a climacteric and intermediate phenotype, for instance by selfing the heterozygous plant in step b).

In case the method of this aspect is to obtain a plant having a climacteric phenotype or increased climacteric and intermediate phenotype, the plant is step b) may be crossed with a plant lacking the marker associated with a climacteric and intermediate phenotype. Optionally, the plant in step b) is heterozygous for the marker associated with a climacteric and intermediate phenotype. The method of the invention may be aiming for progeny that is homozygous for the marker associated with a climacteric and intermediate phenotype, for instance by selfing the heterozygous plant in step b).

Determining the presence of the genetic maker in step a of the method of this aspect may be in a single plant or plant cell or in a plurality of plants or plant cells and/or in a genomic library of a plurality of plants. Preferably said plant (optionally within said plurality of plants) is a plant as defined in the second aspect herein.

Determining the presence or absence of the genetic marker in step a) and c) of the method of the invention may encompass assessing the zygosity of this marker within the genome of the plant and/or progeny. The method may comprise, a subsequent step of producing seeds from the plant obtained in step b) and/or assessed in step c). The method can further comprise, for example, growing the seeds into plants having modulated or modified fruit ripening phenotype.

The method may comprise, a subsequent step of producing progenies of the plant obtained in step b) and/or assessed in step c), or plant protoplast or plant cell derived therefrom, and selecting one or more progenies that have particular fruit ripening characteristics, i.e. climacteric or non-climacteric.

In a sixth aspect, provided is a method for altering fruit ripening characteristics in a plant, and/or a method for producing a plant having altered fruit ripening characteristics, via genetic modification. Said modification may be the modification of the sequence upstream of the MELO3C01 1271 gene as defined in the first aspect herein, or preferably the modification of the cis-element and/or modification of MELO3C01 1271 expression.

Said method may comprise the steps of altering the nucleotide sequence of the region upstream of the MELO3C01 1271 gene, preferably of the cis-element of the MELO3C01 1271 gene as defined herein.

Said method may be a method for producing a plant having a climacteric or intermediate phenotype or an increased climacteric phenotype that comprises the step of:

a) providing a plant cell or protoplast, wherein preferably said cell or protoplast is derived from a plant; and b) genetically engineering said plant cell or protoplast, wherein said engineering results in the insertion of an indel upstream of the ELO3C01 1271 coding sequence; and,

c) regenerating a plant from said plant cell, wherein the fruit ripening of said plant is accelerated or wherein the fruit ripening of the plant is climacteric or intermediate.

The plant cell or protoplast in step a) may be derived from a plant not comprising the indel as defined herein upstream of the MELO3C01 1271 , optionally in a heterozygous or homozygous manner. Said plant may have a non-climacteric phenotype. The plant regenerated in step c) may be a transgenic plant.

Preferably this sequence to be inserted (which may result of insertion of a sequence and/or by alteration of a nucleic acid sequence, i.e. deletion, insertion and/or substitution of one or more nucleotides) in step b) is located 289 bp upstream of the MELO3C01 1271 coding sequence, preferably in the cis-element as defined herein. Preferably, this replaces the sequence represented by TTT on position 25049408-25049410 of the non-climacteric plant as indicated in Figure 3. Preferably, said nucleotide sequence to be inserted or replaced has a length of at least 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16. 17, 18, 19, 20, 21 , 22, 23, 24, 25. 26, 27, 28, 29, 30. 31. 32, 33, 34, 35 or 36 base pairs and preferably has a length of at most 80. 70. 60, 50, 49. 48, 47, 46, 45, 44. 43. 42, 41 , 40, 39, 38, 37, 36 base pairs, or has a length of any one of the combinations of said indicated lower and upper limit. Said nucleotide sequence may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. Optionally, said method further comprises the step of assessing the non-climacteric phenotype of the plant before inserting the indicated nucleotide sequence, e.g. by assessing the presence of a genetic marker according to the first aspect that is associated with the non-climacteric phenotype. Optionally, said method further comprises the step of assessing the climacteric phenotype of the plant after inserting the indel in step b), e.g. by assessing the presence of the indel of step b).

Further, said method may be a method for producing a plant having a non-climacteric phenotype or an increased non-climacteric phenotype that comprises the step of:

a) providing a plant cell or protoplast, wherein preferably said cell or protoplast is derived from a plant; and

b) genetically engineering said plant cell or protoplast, wherein said engineering results in the deletion of an indel upstream of the MELO3C011271 coding sequence; and,

c) regenerating a plant from said plant cell, wherein the fruit ripening of said plant is delayed or wherein the fruit ripening of the plant is non-climacteric.

The plant cell or protoplast in step a) may be derived from a plant comprising the indel as defined herein upstream of the MELO3C01 1271 , optionally in a heterozygous or homozygous manner. Said plant may have a climacteric phenotype. The plant regenerated in step c) may be a transgenic plant. Preferably this sequence to be deleted (which may result of deletion of a sequence and/or by alteration of a nucleic acid sequence, i.e. deletion, insertion and/or substitution of one or more nucleotides) is located 289 bp upstream of the MELO3C01 1271 coding sequence, preferably in the cis-element as defined herein. Preferably, this sequence in a climacteric plant is replaced by the sequence represented by TTT. Preferably, said nucleotide sequence to be deleted has a length of at least 6, 7. 8, 9, 10, 1 1 , 12, 13. 14. 15, 16, 17, 18, 19. 20, 21. 22, 23. 24, 25. 26. 27. 28. 29, 30. 31. 32, 33, 34, 35 or 36 base pairs and preferably has a length of at most 80, 70. 60, 50, 49, 48, 47. 46. 45, 44, 43, 42. 41 , 40, 39, 38. 37. 36 base pairs, or has a length of any one of the combinations of said indicated lower and upper limit. Said nucleotide sequence may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. Optionally, said method further comprises the step of assessing the climacteric phenotype of the plant before deleting the indicated nucleotide sequence, e.g. by assessing the presence of a genetic marker according to the first aspect that is associated with the climacteric phenotype. Optionally, said method further comprises the step of assessing the non-climacteric phenotype of the plant after deleting the indicated nucleotide sequence, e.g. by assessing the absence of the indel of step b).

The incorporation, deletion or altering of any one of the nucleotide sequences as indicated above may be achieved using routine targeted mutagenesis methods, which include, without limitation, those employing zinc finger nucleases, CRISPR-nucleases (e.g. Cas9-like, Cas9/crRNA/tracrRNA, Cas9/gRNA or Cpf1 CRISPR systems), or targeted mutagenesis methods employing mutagenic oligonucleotides (e.g., KeyBase® or TALENs). Altering may be inserting, deleting and/or replacing at least one nucleotide.

In any of the methods above, where the upstream sequence of the MELO3C01 1271 coding sequence has been modified, MELO3C01 1271 expression and/or activity may be altered. Inserting the indel may result in a decrease in MELO3C01 1271 expression and/or activity, preferably during fruit ripening. Deleting the indel may result in an increase in MELO3C01 1271 expression and/or activity, preferably during fruit ripening. Therefore, the present invention also provides for methods for altering MELO3C01 1271 expression and/or activity, preferably during fruit ripening.

The method of this aspect may also or alternatively comprise the step of modifying expression and/or activity of a MELO3C01 1271 encoded polypeptide (a 14kDa proline rich protein) comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 1 or 2 in said plant.

Expression and/or activity of the MELO3C01 1271 polypeptide may be either downregulated or upregulated, depending on the desired fruit ripening characteristics. Said method may be a method for producing a plant having a non-climacteric phenotype or an increased non-climacteric phenotype, or a method for delaying fruit ripening, wherein said method comprises the step of increasing expression and/or activity of a MELO3C01 1271 encoded polypeptide comprising or consisting the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 1 or 2, preferably over the entire length, in said plant, wherein preferably said reducing or abolishing expression and/or activity of a MELO3C01 1271 is compared to the expression and/or activity of the MELO3C01 1271 of a control plant.

Further, said method may be a method for producing a plant having a climacteric phenotype or an increased climacteric phenotype, or a method for accelerating fruit ripening, wherein said method comprises the step of decreasing or abolishing expression and/or activity of or overexpressing a MELO3C01 1271 encoded polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 1 or 2, preferably over the entire length, in said plant. The MELO3C01 1271 polypeptide may be heterogeneous, i.e. originating from a different species, or homogeneous, i.e. of the same species, to the plant to be transformed, and therefore to the control plant.

The MELO3C011271 polypeptide can be encoded by, for example, a nucleic acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of SEQ ID NOs: 3 or 4.

The methods of this aspect can comprise, for example, genetically engineering a plant, plant protoplast or plant cell to modulate expression and/or activity of the MELO3C01 1271 polypeptide. Such genetically engineering may be achieved through transiently or stably incorporating a vector, such as an expression vector, a silencing vector, or other construct into a plant, plant cell, or plant protoplast.

Therefore, provided is a method for producing a plant having a non-climacteric phenotype or an increased non-climacteric phenotype that comprises the step of:

a) providing a plant cell or protoplast, wherein preferably said cell or protoplast is derived from a plant;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in increasing MELO3C01 1271 expression and/or activity; and,

c) regenerating a plant from said plant cell, wherein the fruit ripening of said plant is delayed or wherein the fruit ripening phenotype of the plant is non-climacteric.

The plant cell or protoplast in step a) may be derived from a plant comprising the indel as defined herein upstream of the MELO3C01 1271 , optionally in a heterozygous or homozygous manner. Said plant may be a climacteric plant. The plant regenerated in step c) may be a transgenic plant. Preferably, the MELO3C01 1271 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 1 or 2, preferably over the entire length. The plant obtained in step c) may show an increase in MELO3C01 1271 expression and/or activity of at least 50%, 60%, 70%, 80%, 90% or 100% as compared to the plant used as starting material for the plant cell or protoplast used in step a).

Also provided is a method for producing a plant having a climacteric or intermediate phenotype or an increased climacteric phenotype that comprises the step of:

b) genetically engineering said plant cell or protoplast, wherein said engineering results in reducing or abolishing MELO3C01 1271 expression and/or activity; and,

c) regenerating a plant from said plant cell, wherein the fruit ripening of said plant is accelerated or wherein the fruit ripening phenotype of the plant is climacteric or intermediate.

Preferably, the MELO3C01 1271 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 1 or 2, preferably over the entire length. The plant obtained in step c) may show a reduction in MELO3C01 1271 expression and/or activity of at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to the plant used as starting material for the plant cell or protoplast used in step a).

Decreasing or abolishing of the expression and/or activity of the MELO3C01 1271 polypeptide can be achieved, for example, by T-DNA insertion in the gene encoding said polypeptide, or by silencing MELO3C01 1271 polypeptide expression and/or activity, e.g. using an expression vector comprising an antisense MELO3C01 1271 gene as taught herein. Increasing expression and/or activity of the MELO3C01 1271 polypeptide can be achieved by inserting into a plant, plant protoplast or plant cell at least one additional copy of a nucleic acid molecule encoding the MELO3C01 1271 polypeptide. The additional copy may be a copy of endogenous MELO3C011271 or may involve ectopic expression or the novo expression of MELO3C01 1271 that is normally not expressed in the plant or plant cell to be transformed, e.g. heterogeneous MELO3C01 1271. Further ways are modulating promoter and/or further regulating sequences that are operably linked to the MELO3C01 1271 polypeptide-encoding sequence and resulting in reduced, abolished, or increased expression and/or activity of the MELO3C01 1271 polypeptide. The method can comprise, for example, transforming a plant protoplast or plant cell with a vector or expression construct comprising a recombinant nucleic acid comprising an antisense ELO3C01 1271 sequence, or a sense MELO3C01 1271 sequence. An antisense MELO3C01 1271 sequence is to be understood as a nucleic acid or DNA sequence that results in a transcript which is complementary to and therefore binds and inactivates or silences the mRNA produced by the MELO3C01 1271 gene, either by activating enzymatic breakdown and/or by steric blocking. The antisense sequence may be complementary to a portion to the entire MELO3C01 1271 coding strand or to only a portion thereof. The antisense sequence may be at least six nucleotides in length, but may be about 8, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Sense MELO3C011271 sequence is to be understood herein a nucleic acid sequence that encodes mRNA encoding MELO3C01 1271. A sense ELO3C01 1271 sequence

Both constitutive promoters (such as CMV) and inducible or developmental^ regulated promoters (such as that for the polygalacturonase gene, or abcission-zone-specific or flower-specific promoters) may be used in an expression construct or vector used in the method of the invention as circumstances require. Use of a ripening-specific promoter may be preferred. Thus, ethylene production may, for example, only be inhibited during fruit ripening and not in other stages of development. A non-limiting example of a ripening-specific inducible promoter that could be used is the E8 promoter (Diekman & Fischer, EMBO Journal 7, 3315-3320, 1988).

The degree of production of antisense RNA in the plant cells can be controlled by suitable choice of promoter sequences, or by selecting the number of copies, or the site of integration, of the DNA sequences that are introduced into the plant genome. In this way, for example, it may prove possible to delay softening of fruits for a greater or lesser period after ripening.

The constructs may be used to transform cells of both monocotyledonous and dicotyledonous plants in various ways known to the art. In many cases such plant cells (particularly when they are cells of dicotyledonous plants) may be cultured to regenerate whole plants, which can subsequently reproduce to give successive generations of genetically modified plants. Preferred plants according to the present invention are those bearing climacteric fruit, including, as well as tomatoes, fruits such as mangoes, peaches, apples, pears, bananas and melons.

The method may comprise ^grobacfer/um-med iated transformation (e.g., contacting the plant protoplast or plant cell with an Agrobacterium strain comprising the vector or expression construct to introduce the recombinant nucleic acid into the plant protoplast or plant cell).

The method may further comprise regenerating the plant protoplast or plant cell into a plant.

The method may comprise a subsequent step of producing seeds from the plant obtained in step c) having modulated or modified phenotype as discussed above, e.g., modulated or modified fruit ripening phenotype as compared to a control plant. The method can further comprise, for example, growing the seeds into plants having modulated or modified fruit ripening phenotype.

The method may comprise a subsequent step of testing the plant obtained in step c), or plant protoplast or plant cell derived therefrom, for reduced or abolished expression and/or activity of the MELO3C01 1271 polypeptide. Methods for testing the expression level of the MELO3C01 1271 polypeptide include, without limitation, PCR analysis, sequencing of genomic DNA, sequencing of mRNA transcript, analysing mRNA transcript levels (Northern-blot analysis), analysing copy number (Southern blot analysis), etc.

The method may comprise a subsequent step of testing the plant obtained in step c), or particularly its fruits, for modified fruit ripening characteristics.

The method may comprise a subsequent step of producing progenies of the plant obtained in step c), or plant protoplast or plant cell derived therefrom, and selecting one or more progenies that have modified (e.g., increased, reduced or abolished) expression of the MELO3C01 1271 polypeptide.

The method may further comprise producing progenies of the plant obtained in step c), or plant protoplast or plant cell derived therefrom and selecting one or more progenies plants that have modified fruit ripening phenotype.

The plant, plant protoplast or plant cell used as starting material, i.e. in step a) is preferably a plant as defined in the second aspect herein. The invention further relates to the use of a nucleic acid construct, expression cassette or vector comprising a sense or antisense MELO3C01 1271 sequence as defined herein for modulating fruit ripening characteristics in a plant, plant protoplast or plant cell. As can be understood from the disclosure, a nucleic acid construct, expression cassette or vector comprising a sense MELO3C01 1271 sequence as defined herein can be used for delaying fruit ripening or inducing non- climacteric fruit ripening characteristics. A nucleic acid construct, expression cassette or vector comprising an antisense MELO3C01 1271 sequence as defined herein can be used for accelerating fruit ripening or inducing climacteric fruit ripening characteristics. Said plant, protoplast or plant cell to be modified may be any plant as defined herein. The genetic marker and any of the methods of the aspects defined herein above can be used for development of hybrid lines with slow or delayed ripening and/or long-shelf-life characteristics. Such new lines are relevant for instance in order to accelerate ripening of important early season crops, and/or controlled or delayed ripening of crops permitting longer shipping handling, storage and post- retail shelf-life. Effects on ethylene production which could be effected with the present disclosure include either decreased or increased production of ethylene, whichever is desired. Changes in production of ethylene may affect fruit ripening, organ abscission, seed or pollen dehiscence/shattering, tissue senescence, and disease resistance.

This invention can thus be used to control fruit ripening and softening, as well as plant growth and flower and fruit development of many flowering plants. Reduction in ethylene levels in plants will delay such phenomena, particularly fruit development and fruit softening, including rate of pigment formation as well as the induction of cell wall changes. The effect of these changes will be prolonged ripening time and storage life of fruit. Over-ripening as seen in many fruits (including, for example, tomatoes, mangoes, peaches, apples, pears, bananas and melons) may be prevented or delayed. It is expected that leaf senescence could be delayed, allowing the creation of leaf vegetables (e.g. lettuce, cabbage, spinach) that will stay green longer. It is further expected that flower petal senescence and abscission will be delayed. This could find use in the horticultural industry, leading to cut flowers (roses, chrysanthemums, carnations, tulips, daffodils, etc.) and pot plants having a longer shelf life.

The methods taught herein may be used to realize any of the above-mentioned phenotypic effects in potentially any plant species, but of course fruit ripening effects can be expected only in fruit-bearing plant species. For example, fruit ripening or ethylene production could be accelerated in a plant by knocking out expression of the MELO3C01 1271 polypeptide taught herein, e.g., by T-DNA insertion in the gene encoding said polypeptide, or by silencing MELO3C01 1271 polypeptide expression, e.g. using an expression vector comprising an antisense MELO3C01 1271 gene as taught herein. Alternatively, the sequence upstream of the ELO3C01 1271 encoding sequence may be manipulated, such as by inserting an indel as further specified herein. Alternative methods in the art are random or targeted mutagenesis resulting in a dysfunctional MELO3C01 1271 protein and/or altered sequence upstream the MELO3C01 1271 encoding sequence, e.g. via the introduction of an early stop codon (such as by using a CRISPR system) in the MELO3C01 1271 encoding sequence and/or via the introduction of an indel in the sequence upstream of the MELO3C01 1271 encoding sequence, respectively, for instance by insertion, deletion or substitution of at least one nucleotide. Mutagenesis may be performed by techniques known in the art such as by treatment with radiation or with ethyl methanesulfonate

Any one of the methods of the aspects as defined herein may comprise the step of assessing climacteric or non-climacteric fruit ripening in a phenotypic assay. Such assay may be based on the detection of ethylene production during fruit ripening. The assay may further comprise the assessment shelf-life of the fruit after ripening.

In a seventh aspect, provided is a plant, plant cell, seed or fruit with modified fruit ripening characteristics. Said plant, plant cell, seed or fruit is can be obtained or is obtainable by any of the methods of the fifth and sixth aspect described herein. Said plant, plant cell, seed or fruit may be characterized in that it comprises a modified or introgressed region upstream of the MELO3C01 1271 gene and/or the cis-element as define herein, preferably as compared to control plant. Said plant, plant cell, seed or fruit may be characterized in that it comprises an altered nucleotide sequence in the region encoding MELO3C01 1271 as define herein, preferably as compared to control plant. Said plant, plant cell, seed or fruit, may be characterized in that it has an altered ethylene production, modified region upstream of the MELO3C01 1271 gene, modified cis-element, modified MELO3C01 1271 coding sequence and /or modified expression and/or activity of a MELO3C01 1271 polypeptide (up or down-regulated or silenced) as taught herein, particularly when compared to a control plant. Preferably the plant is a plant as defined in the second aspect herein. The pant, seed or fruit may be characterized by comprising a silencing vector comprising a nucleotide sequence that, when expressed, binds to complementary target mRNA molecules comprising a nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3 or 4, thereby preventing translation of the mRNA molecule. Further, the pant, seed or fruit may be characterized by comprising a vector comprising a nucleotide sequence of SEQ ID NO: 3 or 4, or a nucleotide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3 or 4. Further, the plant, plant cell, seed or fruit, may be characterized in that a MELO3C01 1271 polypeptide expression of as taught herein is upregulated, e.g., by overexpression of said MELO3C01 1271 polypeptide, particularly when compared to a control plant. Also or alternatively, the plant, plant cell, seed or fruit, may be characterized in that it comprises a heterogeneous MELO3C01 1271 polypeptide and/or sequence encoding for heterogeneous MELO3C01 1271.

The plant, plant cell, seed or fruit may be any plant, plant cell, seed or fruit, or may be derived from any plant, such as monocotyledonous plants or dicotyledonous plants, preferably fruit-bearing plants, preferably of the family Cucurbitaceae or Solanaceae. In some embodiments, the plant is selected from Cucumis, or Solanum (including Lycopersicon), Nicotiana, Capsicum, Petunia and other genera. In some embodiments, the plant is selected from vegetable species, including tomato (Solanum lycopersicum) such as e.g. cherry tomato, var. cerasiforme or currant tomato, var. pimpinellifolium) or tree tomato (S. betaceum, syn. Cyphomandra betaceae), potato (Solanum tuberosum), eggplant (Solanum melongena), pepino (Solanum muricatum), cocona (Solanum sessiliflorum) and naranjilla (Solanum quitoense), or peppers (Capsicum annuum, Capsicum frutescens, Capsicum baccatum).

In some embodiments, the plant is selected from "crop plants", i.e. plant species which is cultivated and bred by humans. A crop plant may be cultivated for food purposes (e.g. field crops), or for ornamental purposes (e.g. production of flowers for cutting, grasses for lawns, etc.). A crop plant as defined herein also includes plants from which non-food products are harvested, such as oil for fuel, plastic polymers, pharmaceutical products, cork and the like.

In an embodiment, the plant, plant cell, seed or fruit has reduced or abolished expression and/or activity of a MELO3C01 1271 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs:1 or 2, particularly when compared to a control plant.

In some embodiments, the plant, plant cell, seed or fruit has reduced or abolished expression and/or activity of a MELO3C01 1271 polypeptide encoded by a nucleic acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 3 or 4, particularly when compared to a control plant.

In another embodiment, the plant, plant cell, seed or fruit has increased expression and/or activity of a MELO3C01 1271 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence comprising at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 1 or 2, particularly when compared to a control plant.

In a further embodiment, the plant, plant cell, seed or fruit has increased expression and/or activity of a MELO3C01 1271 polypeptide encoded by a nucleic acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 3 or 4, particularly when compared to a control plant.

The plant may be a transformed or genetically-engineered plant.

In an embodiment, the invention also provides for products derived from the plant as defined herein above, e.g. plant fats, plant oils, plant starch, and plant protein fractions, either crushed, milled or still intact, mixed with other materials, dried, frozen, and so on. These products may be non-propagating. Preferably, these products comprise at least a fraction of or whole recombinant nucleic acid or protein, introgressed nucleic acid or protein, or heterogeneous nucleic acid or protein as defined herein, which allows to assess that the plant product is derived from a plant of the invention, e.g. having modified MELO3C01 1271 polypeptide expression and/or activity, and/or modified region upstream of the MELO3C01 1271 gene and/or cis-element as defined herein, as compared to products derived from a control plant.

Brief description of the Figures

Figure 1 shows co-expression of the MELO3C01 1271 gene with AC01 gene in climacteric Vedrantais ("Ved_a", "Ved_b"), Noy Yizre'el ("NY_a", "NY_b") and Dulce ("Dul_a", "Dul_b"), intermediate type Honeydew-TamDew ("Tarn") and PI 161375 ( "SC"),and non-climacteric type Piel de Sapo ("PS") melon.

Figure 2 shows the scheme of resequencing of MELO3C01 1271 and at about 289 bp upstream of the start codon of MELO3C01 1271 , there was a 33 bp insertion/deletion polymorphism. Figure 3 shows the nucleic acid sequence of the 33 bp insertion/deletion polymorphism. The presence and absence of the 33 bp indel correlate to the climacteric (CL) and non-climacteric (NC) phenotype, respectively. Examples

Genes in QTL region

According to a research paper (Vegas et al. Theor Appl Genet. 2013. 126: 1531 ), two QTLs, ETHQB3.5 and ETHQV6.3, are capable of inducing, individually, the climatic ripening of the melon fruits. ETHQV6.3 effects being greater than those of ETHQB3.5. They also interact epistatically resulting in a precocity of the fruits, which require less time to mature than in the case of exclusively owning one of the QTLs. QTL ETHQB 3.5 is on chr. 3, between markers CMCTN5 and TJ10, and according to melon WGS (Argyris et al. BMC Genomics. 2015. 16: 4), its physical position is 24975485-27248436. In this region, 146 genes were predicted.

RNASeq and gene expression

Six melon (Cucumis melo L.) genotypes, including three climacteric type, Vedrantais (Ved), Noy Yizre'el (NY) and Dulce (Dul); one non-climacteric type, Piel de Sapo (PS); and two intermediate types, Honeydew-TamDew (Tarn) and P1161375 (SC); were grown under KeyGene greenhouse conditions (day/night temperature: 18/22 C, light: 6:00-21 :00). Flowers were hand pollinated and fruit (one per plant) age was decided as days after pollination (DAP). Fruits were harvested at different time points since 24-30 DAP when fruit size stopped increasing until peduncle abscission layer formed and slip, except for PS, which didn't form abscission layer until 60 DAP. At each sampling time, flesh (mesocarp) was collected from the middle of the fruit, avoiding rind, seed and jelly tissue, immediately frozen in liquid nitrogen and stored at -80 °C until RNA isolation. For genotypes Ved, NY and Dul, there were two biological repeats (a and b), and for the rest three genotypes, there was no biological repeat.

RNA was isolated and reverse transcripted into cDNA using QIAGEN kits. Libraries were made and sequenced on HISeq sequenser. Reads were mapped to DHL 92 genome sequence and reads per gene were summed up using CLCBio software. Log2(RPKM+1 ) (RPKM stands for Reads Per Kilobase of transcript per Million mapped reads) was used to measure gene expression level.

1-aminocyclopropane-1-carboxylate oxidase 1 (AC01 ) gene, encodes the enzyme (ACC oxdase) catalyzing the last step of ethylene biosynthesis. The RNASeq data showed that melon AC01 gene (MELO3C014437) expression increased considerably (ca. 60-1000 folds) in the three climacteric genotypes, increased intermediately in the two intermediate types (less than 60 folds) and stayed low in non-climacteric PS (< 4 fold) during fruit ripening. Among those 146 genes in the QTL region, there was only one gene, MELO3C011271 , turned out to be coexpressed with AC01 gene negatively in climacteric and intermediate types, and positively in non-climacteric type (see Figure 1 ). This gene is annotated as 14 kDa proline-rich protein, physical position of coding region is on chr_3, 25049667- 25050104. Resequence in a germplasm panel

A germplasm panel consists of 126 melon lines were grown in greenhouse and young leaves were collected for DNA isolation. Gene specific primers with pacbio adapters (see below) were designed to amplify and sequence MELO3C011271 using Pacbio sequencer.

AP1 F: /5AmMC6/gcagtcgaacatgtagctgactcaggtcacGCTAAGAGGTTTGATGCTTTTCA (SEQ ID NO: 8)

AP1 R: /5AmMC6/tggatcacttgtgcaagcatcacatcgtagTGTGGTAGCTTCATGTTTTCAGA (SEQ ID NO: 9) AP2F: /5AmMC6/gcagtcgaacatgtagctgactcaggtcacGTGGGGGACTTAGTTTGGTTATC (SEQ ID NO: 10)

AP2R: /5AmMC6/tggatcacttgtgcaagcatcacatcgtagCCCTTCCTTGTTTGAGTTTTCTT (SEQ ID NO: 11 ) The resequencing results showed that at about 289 bp upstream of the start codon of MELO3C011271 , there was a 33 bp insertion/deletion polymorphism, the physical position is chr_3, 25049407 (see Figure 2 and 3).

In the 126 melon lines, there were 94 lines were from C. melo subsp. Melo. This subspecies is divided into Cantalupensis group, Reticulatus group and Inodorus group, where the former two are in general regarded as climacteric type melons, and the later one, non-climacteric type. Based on this classification, absence of this 33 bp-fragment correlates perfectly with the non-climacteric ripening phenotype.

Combine this indel with the indel found in the 3' of MELO3C016536 can predict the time to ripe of the fruit, which is a very valuable trait for improving shelf life of climacteric melon types.

Screening EMS population for MELO3C011271 mutants

Two EMS mutation populations from 'Noy Yizre'el', a Galia' melon-type, and 'Cezanne', a 'Charentais' type melon were produced by Agricultural Research Orgnization in Israel (Tadmor et al. 2007; Galpaz et al. 2013), and KeyGene bought them. These two populations were used to screen for mutants.

Reference

Juan Vegas, Jordi Garcia-Mas, Antonio Jose Monforte. Interaction between QTLs induces an advance in ethylene biosynthesis during melon fruit ripening. Theoretical and Applied Genetics. 2013, 126(6): 1531-1544

Saladie M, Cafiizares J, Phillips MA, Rodriguez-Concepcion M, Larrigaudiere C, Gibon Y, Stitt M, Lunn JE, Garcia-Mas J. Comparative transcriptional profiling analysis of developing melon (Cucumis melo L.) fruit from climacteric and non-climacteric varieties. BMC Genomics. 2015; 16(1 ): 440

Argyris JM, Ruiz-Herrera A, Madriz-Masis P, Sanseverino W, Morata J, Pujol M, Ramos-Onsins SE, Garcia-Mas J. Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly. BMC Genomics. 2015; 16(1 ): 4. Galpaz N, Burger Y, Lavee T, Tzuri G, Sherman A, elamed T, Eshed R, Meir A, Portnoy V, Bar E, Shimoni-Shor E, Feder A, Saar Y, Saar U, Baumkoler F, Lewinsohn E, Schaffer AA, Katzir N, Tadmor Y. Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene. 2013, 539 (2): 1 17-125

Tadmor Y, Katzir N, Meir A, Yaniv-Yaakov A, Sa'ar U, Baumkoler F, Lavee T, Lewinsohn E, Schaffer A, and Burger J. Induced mutagenesis to augment the natural genetic variability of melon (Cucumis melo L). 2007, 55 (2): 159-169

Table 1. SEQ ID NO identification

SEQ ID Name Sequence

NO

1 MELO2C011271 MASKSLSLPTILLLSLLLCSALATPCDQPKTKPAPSVPKKKPAIP

(protein) KIIRPPAASSPAVQSSYCPKDTLKLGVCADILGIGSVVIGSPVSN

ECCALLSGLTDVEAAACLCTAIKANVLGINLDIPVSLSLLISSCQ KTLPAGYQCK

2 Tomato orthologues MASKKFFSTLALLLSLNLIFFTFVSSCNTCPKPKPKPKPSCPPP

(protein) PYVPKYKTCPIDTLKLGVCANVLGLVNVVVGSPPVTPCCSLISG

LADVEAALCLCTALKANVLGINLNVPISLSLLLNVCSKKVPNGF QCPN

3 MELO2C011271 ATGGCTTCCAAATCTCTTTCTTTACCAACCATTCTGTTGCTC

(encoding sequence) TCTCTTCTCCTTTG CTCAG CTCTGG CC ACCCCATG CG ACC A

ACCCAAAACCAAGCCAGCACCGTCAGTGCCCAAGAAAAAAC

CCGCCATCCCCAAAATAATCCGGCCACCGGCGGCATCTTC

TCCGGCAGTACAATCGAGTTACTGTCCAAAAGACACCTTGA

AGTTGGGCGTTTGTGCCGATATTCTTGGAATTGGGAGTGTC

GTAATTGGGAGCCCTGTTTCGAACGAGTGCTGTGCTCTTCT

TAGTGGGCTGACAGATGTTGAAGCTGCTGCTTGTCTTTGTA

CCG CCATTAAAG CCAATGTTCTTG G AATTAACTTGG ATATC

CCTGTTTCACTCAGTTTGCTCATCAGCTCTTGCCAAAAAACT

CTTCCCGCTGGCTACCAGTGTAAATGA

4 Tomato orthologues ATGGCTTCCAAGAAGTTTTTCTCTACTCTTGCTCTATTACTT

(encoding sequence) TCCCTTAACCTAA I I I I CTTCACATTTGTTTCAAGTTGTAACA

CTTGCCCTAAACCTAAACCTAAACCAAAGCCATCATGTCCA

CCACCACCATATGTACCAAAATACAAGACTTGTCCAATTGAT

ACCTTAAAATTAGGTGTTTGTGCAAATGTTCTTGGATTAGTT

AATGTTGTTGTTGGTTCACCACCAGTCACACCTTGTTGTAG

TCTTATTTCTGGACTTGCTGATGTTGAAGCTGCCCTTTGTCT

TTGTACTGCTCTTAAGGCTAATGTCTTAGGGATTAACCTTAA

TGTCCCTATTTCATTGAGCTTGCTTCTTAATGTTTGTTCCAA

GAAAGTTCCTAATGGATTCCAATGTCCTAATTAG

5 Marker non- GTTTCGCAGTATAAACTGATCAGAGTTTGAGTTACC

climacteric type

6 forward primer tgtggagcttaagaagttacgg

7 reversed primer ttgtgggggaaactgtaagg AP1 F: /5AmMC6/ gcagtcgaacatgtagctgactcaggtcacGCTAAGAGGTTTGATGCTTT TCA

AP1 R: /5AmMC6/ tggatcacttgtgcaagcatcacatcgtagTGTGGTAGCTTCATGTTTTCA

GA

AP2F: /5AmMC6/ gcagtcgaacatgtagctgactcaggtcacGTGGGGGACTTAGTTTGGTT

ATC

AP2R: /5AmMC6/ tggatcacttgtg caag catcacatcgtag CCCTTCCTTGTTTGAGTTTTC

TT

Claims

1. A genetic marker for characterizing fruit ripening, wherein said marker is localized in a region upstream of the MELO3C01 1271 gene.

2. A genetic marker according to claim 1 , wherein said marker has a sequence that is at least 80% identical to SEQ ID NO: 5.

3. A genetic marker according to claim 1 , wherein the presence of said marker is associated with a climacteric and intermediate fruit ripening phenotype, and wherein the absence of said marker is associated with a non-climacteric fruit ripening phenotype.

4. Use of a genetic marker according to any one of claims 1-3 for screening, identifying and/or marker assisted breeding of plants.

5. A method for identifying a plant comprising a genotype that is associated with a non-climacteric fruit ripening phenotype, comprising the steps of:

a) extracting a nucleic acid from a plant; and,

b) assessing the absence of a genetic marker of any one of claims 1-3 that is associated with the climacteric and intermediate fruit ripening phenotype.

6. A method for identifying a plant comprising a genotype that is associated with a climacteric and intermediate fruit ripening phenotype, comprising the steps of:

a) extracting a nucleic acid from a plant ; and,

b) assessing the presence of a genetic marker of any one of claims 1-3 that is associated with the climacteric and intermediate fruit ripening phenotype.

7. A method for screening a plurality of plants for comprising a genotype that is associated with a non- climacteric fruit ripening phenotype comprises the steps of:

a) extracting nucleic acids from said plurality of plants; and,

b) assessing the absence of a genetic marker of any one of claims 1-3 that is associated with the climacteric and intermediate fruit ripening phenotype in said nucleic acid via a screening technology.

8. A method for screening a plurality of plants for comprising a genotype that is associated with a climacteric and intermediate fruit ripening phenotype comprises the steps of:

a) extracting nucleic acids from said plurality of plants; and,

b) assessing the presence of a marker of any one of claims 1-3 that is associated with the climacteric and intermediate fruit ripening phenotype in said nucleic acid via a screening technology.

9. A method for obtaining a plant having a non-climacteric fruit ripening phenotype, comprising the steps of: a) determining the absence of the genetic marker of any one of claims 1-3 that is associated with the climacteric and intermediate fruit ripening phenotype;

b) selfing or crossing the plant in which said marker is absent; and

c) optionally, determining the absence of the genetic marker of any one of claims 1-3 that is 5 associated with the climacteric and intermediate fruit ripening phenotype in the progeny of the selfing or crossing step.

10. A method for obtaining a plant having a climacteric and intermediate fruit ripening phenotype, comprising the steps of:

10 a) determining the presence of the genetic marker of any one of claims 1-3 that is associated with the climacteric and intermediate fruit ripening phenotype;

b) selfing or crossing the plant in which said marker is present; and

c) optionally, determining the presence of the genetic marker of any one of claims 1-3 that is associated with the climacteric and intermediate fruit ripening phenotype in the progeny of the selfing

15 or crossing step.

11. A method for producing a plant having a climacteric or intermediate fruit ripening phenotype or an increased climacteric fruit ripening phenotype that comprises the step of:

a) providing a plant cell or protoplast; and

20 b) genetically engineering said plant cell or protoplast, wherein said engineering results in the insertion of an indel upstream of the MELO3C01 1271 coding sequence; and,

25 12. A method for producing a plant having a non-climacteric fruit ripening phenotype or an increased non-climacteric fruit ripening phenotype that comprises the step of:

a) providing a plant cell or protoplast; and

b) genetically engineering said plant cell or protoplast, wherein said engineering results in the deletion of an indel upstream of the MELO3C01 1271 coding sequence; and,

30 c) regenerating a plant from said plant cell, wherein the fruit ripening of said plant is delayed or wherein the fruit ripening of the plant is non-climacteric.

13. A method for producing a plant having a non-climacteric fruit ripening phenotype or an increased non-climacteric fruit ripening phenotype that comprises the step of:

35 a) providing a plant cell or protoplast;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in increasing MELO3C01 1271 expression; and,

40

14. A method for producing a plant having a climacteric or intermediate fruit ripening phenotype or an increased climacteric fruit ripening phenotype that comprises the step of:

a) providing a plant cell or protoplast; and

b) genetically engineering said plant cell or protoplast, wherein said engineering results in reducing or abolishing MELO3C01 1271 expression; and,

15. A plant, plant cell, seed or fruit obtained or obtainable by any of the methods claims 11-16, wherein said plant, plant cell, seed or fruit has modified fruit ripening characteristics as compared to a control plant.

16. A plant product obtained or obtainable from a plant, plant cell, seed or fruit of claim 15.