WO2018146322A1

WO2018146322A1 - Method for altering ripening characteristics of fruit

Info

Publication number: WO2018146322A1
Application number: PCT/EP2018/053480
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, wherein ETHQV6.3 effects are 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, ETHQV6.3, is on chromosome 6, between markers Al_03-B03 and FR14-P22 (Theor Appl Genet. 2013. 126: 1531 ) and its physical position is 21834442-26987092 (Argyris ef al. BMC Genomics. 2015. 16: 4). For ETHQV6.3, based on transcriptome analysis, Vegas (PhD thesis Juan Vegas Renedo, Universitat Automoma de Barcelona 2014, Estudio genetico de la maduracion del fruto en melon en la linea isogenica SC3-5-1 ) defined three candidate genes, namely, two NAC / NAM transcription factor family genes, MELO3C016540 and MELO3C016536, and kinase m13r_c DUFF gene, MELO3C016579. MELO3C016536 was further characterized and a non-synonymous mutation was identified that results in the substitution of a serine in non-climacteric line Piel de Sapo by a proline in the climacteric line SC3-5-1.

However, through fine mapping Rodriguez, from the same research group, (PhD thesis Pablo Rios Rodirguez, Universitat Automoma de Barcelona 2015, Clonaje posicional y validation de un gen candidato eth6.3, un QTL implicado en la maduracion cliaterica del fruto del melon) ruled out MELO3C016536, and concluded that MELO3C016540 was the gene responsible of ETHQV6.3. Two climacteric background mutants from TILLING with mutations in the NAC-domain in MELO3C016540 showed a delay in ripening and the haplotypes within this gene highly conserved among varieties that show the same kind of ripening suggest a central role in its regulation.

Summary of the invention

Surprisingly, the present inventors identified MELO3C016536 on chromosome 6 at physical position 26849432-26851 194 as the real gene responsible of ETHQV6.3 and identified a downstream region localized on chromosome 6 at physical position 26850247-26850317, which is localized near (i.e. closest to) this gene that comprises a molecular marker that highly correlates with the ripening phenotype.

Based on extensive and detailed expression studies, the inventors found that among the 298 genes predicted in the ETHQV6.3 QTL region, only one single gene, MELO3C016536, positively co- expressed with 1-aminocyclopropane-1-carboxylate oxidase 1 (AC01 ) gene in climacteric and intermediate types, whilst it was negatively co-expressed with the ACQ1 gene in the non-climacteric type. The AC01 gene encodes the enzyme (ACC oxidase) catalyzing the last step of ethylene biosynthesis. Additionally, MELO3C016536 was expressed slightly prior to AC01 gene expression in climacteric types, indicating the likelihood of this gene causing the increased expression of the AC01 gene.

The newly identified molecular marker within the domain located about 3 kb downstream of the NAC- TF coding sequence, associates strongly (p-value=2.67E-1 1 ) with the ripening phenotype. The molecular marker is a 36 bp insertion/deletion polymorphism. The indel is a duplication of a sequence that is directly adjacent to the 3'end of said indel. A single copy of said sequence was found in climacteric ripening species, whereas two copies (a nearly perfect tandem repeat) were found in non- climacteric species.

Without wishing to be bound by any theory, the inventors identified an EIN3/EIL (ethylene insensitive transcription factor) binding site (EBS) in the 36 bp fragment. As duplication of such binding site is suggested to severely affect EIN3 binding (Song et al. PLOS ONE, 2015 10: 1 ), the inventors suggest duplication of this fragment to be the genetic cause of the difference in fruit ripening between climacteric and non-climacteric phenotypes, likely via the regulation of expression of MELO3C016536 encoding NAC-TF, which is the gene closest to the indel. The region consisting of the single copy or the nearly perfect tandem repeat, which is located about 3 kb downstream of the NAC-TF coding sequence, is therefore also denominated herein as cis-element of the NAC-TF gene.

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, hydroxymethylated 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 developmentally 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.

"NAC-TF polypeptides" (or "NAC transcription factor polypeptides") refers to a group of plant proteins characterized by their sequence homology to the protein encoded by the melon MELO3C016536 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 MELO3C016536 gene (SEQ ID NO: 1 ), or to the protein encoded by the tomato JA2 (Solyc12g013620) or JA2L (Solyc07g063410) 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 JA2 or JA2L gene (SEQ ID NO: 2 or 3, respectively), as well as their characteristic in being co-expressed with the AC01 gene and being functional as defined herein, thereby being involved in climacteric fruit ripening. NAC-TF polypeptides include at least the MELO3C016536 polypeptide (SEQ ID NO: 1 ) and its homologs from tomato JA2 (SEQ ID NO: 2) and JA2L (SEQ ID NO:3). See Table 1 for sequences and their SEQ ID NO.

"NAC-TF genes" refers to genes encoding the NAC-TF polypeptides. The NAC-TF genes include at least the melon MELO3C016536 gene (SEQ ID NO: 4) and its homologs from tomato (JA2 gene - SEQ ID NO: 5 and JA2L gene - SEQ ID NO: 6). The term "functional" in relation to NAC-TF proteins or genes refers to the capability of the gene and/or encoded protein to modify fruit ripening characteristics, e.g. from non-climacteric ripening to 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 NAC-TF protein obtained from plant species X can be tested by various methods. Preferably, if the protein is a functional NAC-TF 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 climacteric ripening characteristics to non-climacteric ripening characteristics, as can be tested as explained herein in detail. Also, restoring expression of the NAC-TF protein can restore 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 NAC-TF polypeptide expression. The control plant preferably comprises the endogenous NAC-TF 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 (Tarn) and/or P1161375 (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 MELO3C016536, located on chromosome 6 and having the physical position 26849432-26851 194, was the only gene among the 298 genes within the ETHQV6.3 QTL, which is located between markers Al_03-B03 and FR14- P22 (Theor Appl Genet. 2013. 126: 1531 ) that positively co-expressed with the AC01 gene in the climacteric and intermediate type, and negatively in the non-climacteric type in fruit ripening. Besides, this gene expressed is shortly before the AC01 gene in climacteric types, indicating it as a regulator of AC01 expression. By sequencing the MELO3C016536 gene in 126 germplasm lines, a polymorphism (an indel) was identified in a region downstream of the MELO3C016536 gene. Within the 126 melon lines that were sequenced, 94 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 (p-value=2.67E-1 1 ) association was found between the polymorphism within this region and the ripening phenotype.

The presence of the 36 bp indel in the above indicated position, which strongly associates with the 5 non-climacteric phenotype, results in a 71 bp nearly perfect tandem repeat that is represented herein by SEQ ID NO: 9, which consists of the 36 bp indel (represented herein by SEQ ID NO: 7) and a nearly perfect 35 bp repeat thereof located immediately adjacent to the indel (represented herein by SEQ ID NO: 10). The repeats only differ in that SEQ ID NO: 7 has one additional nucleotide (a cytosine) as compared to SEQ ID NO: 10 inserted between the analogous nucleotide positions 18 and 10 19 of SEQ ID NO: 10. Therefore, preferably the marker (also denominated herein as molecular marker or genetic marker) that associates with the non-climacteric and phenotype has the sequence of SEQ ID NO: 9.

The absence of the indel in the above indicated position, strongly associates with the climacteric and 15 intermediate phenotype. Further, the presence of a single copy of 35 bp nucleotide stretch represented herein by SEQ ID NO: 8 is strongly associated with the climacteric and intermediate phenotype. Therefore, preferably the marker (also denominated herein as molecular marker or genetic marker) that associates with the climacteric and intermediate phenotype has the sequence of SEQ ID NO: 8.

20 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 1-5 days old seedlings.

Therefore, in a first aspect, provided is a genetic marker for characterizing fruit ripening. The genetic 25 marker is located within the region downstream of the of the NAC-TF gene as defined herein, preferably about 3 kb downstream of said gene, such as between 1 and 5 kb downstream of said gene or between 2 and 4 kb downstream of said gene, within the ETHQV6.3 QTL sequence that is on chromosome 6 delimited by markers Al_03-B03 and FR14-P22 (Theor Appl Genet. 2013. 126: 1531 ), or any orthologous genomic region.

30

Preferably, said molecular marker is an indel that is located within the region downstream of the NAC- TF as defined above, which is characterized in that it comprises at least one EIN3 (ethylene insensitive 3) / EIL (EIN3-like) binding site or EBS (see Figure 3). The EBS may have the palindromic sequence ATGCAT. The presence of said indel, resulting in two EBSs within this region separated by 35 a stretch of between 10 and 40 base pairs, between 10 and 35, or between 20 and 35, preferably by a stretch of about 30 base pairs, is associated with the non-climacteric phenotype, whereas the absence of said indel results in a single EBS within this region and is associated with the climacteric and intermediate phenotype.

40 Preferably, the indel has a length of 36 bp and has a sequence represented by SEQ ID NO: 7 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: 7. Preferably, said nucleotide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 comprises an EBS, wherein preferably said EBS has the sequence of ATGCAT. Preferably, said indel is located immediately adjacent and upstream to a nucleotide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10 thereby resulting in the presence of a nearly perfect tandem repeat of about 71 base pairs within the region downstream of the NAC-TF gene as defined herein. Said nearly perfect tandem repeat preferably has a nucleotide sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9, which associates with the non-climacteric phenotype.

In a further embodiment, the molecular marker may be a single copy of a fragment of about 35 bp within said region downstream of the NAC-TF gene, that preferably has a nucleotide sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8, which associates with the climacteric and intermediate phenotype. A single copy is to be understood herein that the regions of similar length (i.e. 35 bp) immediately flanking said copy do not show any sequence similarity, or preferably are do not show more than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1 % sequence identity to SEQ ID NO: 8. This marker associates with the climacteric and intermediate phenotype. The presence or absence of the molecular marker may be assessed by any technique known in the art such as by sequencing the sequence downstream of the NAC-TF gene, more preferably the cis- element which is located at or near the region localized on chromosome 6 at physical position 26850247-26850317 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 the molecular marker may be assessed by comparing the length of amplified fragments of the sequence optionally comprising the indel, e.g. the sequence downstream of the NAC-TF gene, more preferably the sequence comprising the cis-element or a sequence comprising said cis-element. Preferably, such amplified fragments comprise the sequence of position 26850247-26850317 on chromosome 6. An example of a primers set that can be used to amplify that sequence are represented by SEQ ID NO: 1 1 and SEQ ID NO: 12. The presence of the nearly perfect tandem repeat (SEQ ID NO: 9) associating with the non-climacteric phenotype can be distinguished easily from the presence of a single copy (SEQ ID NO: 8) based on length difference of the amplicons (amplified fragments) prepared using primers such as SEQ ID NO: 1 1 and SEQ ID NO: 12 in PCR 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 1-5 days old seedlings. In a second aspect, provided is the use of the genetic marker according to the first aspect 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 fruit ripening 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 presence of the genetic marker according to the first aspect that is associated with the non-climacteric phenotype, and/or 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 and/or assessing the absence of the genetic marker according to the first aspect that is associated with the non- climacteric phenotype.

The method of this aspect may further comprise the step of selecting the plant comprising 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 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 presence of the genetic marker according to the first aspect that is associated with the non-climacteric phenotype, and/or 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, and/or assessing the absence of the genetic marker according to the first aspect that is associated with the non-climacteric 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 the plant(s) comprising the genotype associated with non-climacteric phenotype or climacteric and intermediate phenotype, 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: determining the presence the genetic marker according to the first aspect that is associated with the non-climacteric phenotype and/or determining the absence the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype;

selfing or crossing the plant in which the marker associated with a non-climacteric phenotype is present and/or in which the marker associated with a climacteric and intermediate phenotype is absent; and

optionally, determining the presence of the genetic marker according to the first aspect that is associated with the non-climacteric phenotype, and/or determining the absence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate 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 and/or determining the absence the genetic marker according to the first aspect that is associated with the non-climacteric phenotype; b) selfing or crossing the plant in which the marker associated with a climacteric and intermediate phenotype is present and/or in which the marker associated with a non- climacteric phenotype is absent; and

optionally, determining the presence of the genetic marker according to the first aspect that is associated with the climacteric and intermediate phenotype, and/or determining the absence of the genetic marker according to the first aspect that is associated with the non-climacteric 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 lacking the marker associated with a non-climacteric phenotype and/or comprising the marker associated with a climacteric and intermediate phenotype. Optionally, the plant in step b) is heterozygous for the marker associated with a non-climacteric phenotype. The method of the invention may be aiming for progeny that is homozygous for the marker associated with a non-climacteric 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 and/or comprising the marker associated with a non-climacteric 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 or absence of the genetic marker 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 downstream of the NAC-TF gene as defined in the first aspect herein, or preferably the modification of the cis-element and/or modification of NAC-TF expression.

Said method may comprise the steps of altering the nucleotide sequence of the region downstream of the NAC-TF gene, preferably of the cis-element of the NAC-TF gene as defined herein. Preferably said sequence comprises at least one EIN3 (ethylene insensitive 3) / EIL (EIN3-like) binding site (EBS). The EBS may have the sequence ATGCAT.

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;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in the deletion of an EBS downstream of the NAC-TF 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 comprising two EBSs within the cis- element as defined herein, or may comprise an indel as defined herein downstream of the NAC-TF coding sequence, optionally in a heterozygous or homozygous. Said plant may have a non-climacteric phenotype. The plant regenerated in step c) may be a transgenic plant. Said EBS preferably has the sequence ATGCAT. Deleting an EBS may encompass the deletion of a nucleotide sequence comprising an EBS or by altering a nucleotide sequence (e.g. by deletion, insertion and/or substitution of one or more nucleotides) thereby resulting in the disruption of an EBS. 5 Optionally, the sequence to be deleted or modified has a length of 6 base pairs and has the sequence of an EBS, preferably ATGCAT and is comprised within a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9.

Preferably, this sequence to be deleted is directly adjacent to, preferably at the 5'-end, of SEQ ID NO: 10 10, or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, or at the 3'-end of SEQ ID NO: 7, or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7, of the non-climacteric plant. Preferably, said nucleotide sequence 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 15 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. Preferably, said nucleotide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 10 comprises an EBS, wherein preferably said EBS has the sequence of ATGCAT.

20

Optionally, said method further comprises the step of assessing the non-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 climacteric phenotype of the plant after deleting 25 the indicated nucleotide sequence, e.g. by assessing the deletion 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 30 plant;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in the insertion of an EBS downstream of the NAC-TF 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.

35

The plant cell or protoplast in step a) may be derived from a plant having a single EBS within the cis- element as defined herein, or lack the indel as defined herein downstream of the NAC-TF coding sequence, optionally in a heterozygous or homozygous manner. Said plant may have a climacteric or intermediate phenotype. The plant regenerated in step c) may be a transgenic plant.

40 Said EBS preferably has the sequence ATGCAT. Inserting an EBS may encompass the insertion of a nucleotide sequence comprising an EBS or by altering a nucleotide sequence (e.g. by deletion, insertion and/or substitution of one or more nucleotides) thereby resulting in the formation of an EBS. Preferably this sequence to be inserted is located 10-40 base pairs or 15-35 base pairs, or 20, 21 , 22, 5 23, 24, 25, 26, 27, 28, 29 or 30 base pairs from a further EBS located downstream of the NAC-TF coding sequence, preferably within the cis-element as defined herein. Preferably, this sequence to be inserted is directly adjacent to, either at the 5'- or 3'-end, SEQ ID NO: 8, or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8, of the climacteric plant. Optionally, this sequence is inserted directly adjacent to, either at the 5'-end,

10 SEQ ID NO: 8 of the climacteric plant. Preferably, said nucleotide sequence 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 to be inserted may have at least 50%, 60%, 70%,

15 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7. Preferably, said nucleotide sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7 comprises an EBS, wherein preferably said EBS has the sequence of ATGCAT. Optionally, the sequence to be inserted has a length of 6 base pairs and has the sequence of an EBS, preferably ATGCAT. Optionally, said method further comprises the step

20 of assessing the 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 non-climacteric phenotype of the plant after inserting the indicated nucleotide sequence, e.g. by assessing the presence of the insertion of step b.

25

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 30 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 downstream sequence of the NAC-TF coding sequence has been modified, NAC-TF expression and/or activity may be altered. Inducing an indel, thereby 35 inducing an EBS, may result in a decrease in NAC-TF expression and/or activity, preferably during fruit ripening. Deleting the indel, thereby deleting an EBS, may result in an increase in NAC-TF expression and/or activity, preferably during fruit ripening. Therefore, the present invention also provides for methods for altering NAC-TF expression and/or activity, preferably during fruit ripening.

40 The method of this aspect may also or alternatively comprise the step of modifying expression and/or activity of a NAC transcription factor (NAC-TF) polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 , 2 or 3, 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 , 2, or 3 in said plant. Expression and/or activity of the NAC-TF 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 reducing or abolishing expression and/or activity of a NAC transcription factor (NAC-TF) polypeptide comprising or consisting the amino acid sequence of SEQ ID NO: 1 , 2 or 3, 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 , 2, or 3, preferably over the entire length, in said plant, wherein preferably said reducing or abolishing expression and/or activity of a NAC transcription factor (NAC-TF) is compared to the expression and/or activity of the NAC transcription factor (NAC- TF) of a control plant.

Further, said method may be a method for producing a plant having a climacteric or intermediate phenotype or an increased climacteric phenotype, or a method for accelerating fruit ripening, wherein said method comprises the step of increasing expression and/or activity of or overexpressing a NAC transcription factor (NAC-TF) polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , 2 or 3, 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 , 2, or 3, preferably over the entire length, in said plant, wherein preferably said increasing expression and/or activity of a NAC transcription factor (NAC-TF) is compared to the expression and/or activity of the NAC transcription factor (NAC-TF) of a control plant. The NAC-TF 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 NAC-TF 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: 4, 5, or 6.

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 NAC-TF 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 reducing or abolishing NAC-TF expression and/or activity; and,

5 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 downstream of the NAC-TF coding sequence, optionally in a homozygous or heterozygous 10 manner. Said plant may have a climacteric or intermediate phenotype. The plant regenerated in step c) may be a transgenic plant.

Preferably, the NAC-TF polypeptide comprises the amino acid sequence of SEQ ID NO: 1 , 2 or 3, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 15 99% sequence identity with any of SEQ ID NOs: 1 , 2, or 3, preferably over the entire length. The plant obtained in step c) may show a reduction in NAC-TF 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).

20 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:

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 25 increasing NAC-TF 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 climacteric or intermediate.

The plant cell or protoplast in step a) may be derived from a plant not comprising the indel as defined 30 herein downstream of the NAC-TF coding sequence, optionally in a homozygous or heterozygous manner. Said plant may be a non-climacteric plant. The plant regenerated in step c) may be a transgenic plant.

Preferably, the NAC-TF polypeptide comprises the amino acid sequence of SEQ ID NO: 1 , 2 or 3, or 35 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 , 2, or 3, preferably over the entire length. The plant obtained in step c) may show an increase in NACT-TF 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).

40 Decreasing or abolishing of the expression and/or activity of the NAC-TF polypeptide can be achieved, for example, by T-DNA insertion in the gene encoding said polypeptide, or by silencing NAC-TF polypeptide expression and/or activity, e.g. using an expression vector comprising an antisense NAC-TF gene as taught herein. Increasing expression and/or activity of the NAC-TF 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 NAC-TF polypeptide. The additional copy may be a copy of endogenous NAC-TF or may involve ectopic expression or the novo expression of NAC- TF that is normally not expressed in the plant or plant cell to be transformed, e.g. heterogeneous NAC-TF. Further ways are modulating promoter and/or further regulating sequences that are operably linked to the NAC-TF polypeptide-encoding sequence and resulting in reduced, abolished, or increased expression and/or activity of the NAC-TF 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 NAC-TF sequence, or a sense NAC-TF sequence. An antisense NAC-TF 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 NAC-TF gene, either by activating enzymatic breakdown and/or by steric blocking. The antisense sequence may be complementary to a portion to the entire NAC-TF 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 NAC-TF sequence is to be understood herein a nucleic acid sequence that encodes mRNA encoding NAC-TF.

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 yAgrofciacier/t/m-mediated 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 altered (increased, reduced or abolished) expression and/or activity of the NAC-TF polypeptide. Methods for testing the expression level of the NAC-TF 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 NAC-TF 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 NAC-TF 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 NAC-TF sequence as defined herein can be used for accelerating fruit ripening or inducing climacteric fruit ripening characteristics. A nucleic acid construct, expression cassette or vector comprising an antisense NAC-TF sequence as defined herein can be used for delaying fruit ripening or inducing non-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 decreased in a plant by knocking out expression of the NAC-TF polypeptide taught herein, e.g., by T-DNA insertion in the gene encoding said polypeptide, or by silencing NAC-TF polypeptide expression, e.g. using an expression vector comprising an antisense NAC-TF gene as taught herein. Alternatively, the sequence downstream of the NAC-TF 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 NAC-TF protein and/or altered sequence downstream the NAC-TF encoding sequence, e.g. via the introduction of an early stop codon (such as by using a CRISPR system) in the NAC-TF encoding sequence and/or via the introduction of an EBS in the sequence downstream of the NAC-TF 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 downstream of the NAC-TF 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 NAC-TF 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 downstream of the NAC-TF gene, modified cis-element, modified NAC-TF coding sequence and /or modified expression and/or activity of a NAC-TF 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:4, 5, or 6, 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:4, 5, or 6, 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:4, 5, or 6, 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:4, 5, or 6. Further, the plant, plant cell, seed or fruit, may be characterized in that a NAC- TF polypeptide expression and/or activity of as taught herein is upregulated, e.g., by overexpression of said NAC-TF 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 NAC-TF polypeptide and/or sequence encoding for heterogeneous NAC-TF.

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, 5 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 NAC transcription factor (NAC-TF) polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , 2 or 3, or an amino acid sequence comprising at least 50%, 60%, 70%, 80%, 85%, 10 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 1 , 2, or 3, 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 NAC transcription factor (NAC-TF) polypeptide encoded by a nucleic acid sequence 15 having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 4, 5, or 6, 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 NAC transcription factor (NAC-TF) polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , 20 2 or 3, 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 , 2, or 3, 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 25 a NAC transcription factor (NAC-TF) 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: 4, 5, or 6, particularly when compared to a control plant.

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

30

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, 35 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 NAC-TF polypeptide expression and/or activity, and/or modified region downstream of the NAC-TF gene and/or cis-element as defined herein, as compared to products derived from a control plant.

40 Brief description of the Figures

Figure 1 shows co-expression of the MELO3C016536 gene with AC01 gene in A) climacteric Vedrantais ("Ved_a", "Ved_b");

B) Noy Yizre'el ("NY_a", "NY_b"),

C) Dulce ("Dul_a", "Dul_b"),

D) intermediate type Honeydew-TamDew ("Tarn"),

E) PI 161375 ("SC"),and

F) non-climacteric type Piel de Sapo ("PS") melon.

Figure 2 shows the scheme of resequencing of MELO3C016536 and at about 3 kb downstream of the stop codon of MELO3C016536, there was a 36 bp insertion/deletion polymorphism.

Figure 3 shows the nucleic acid sequence of the 36 bp insertion/deletion polymorphism. In boldface a binding site (ATGCAT) of EIN3 (Ethylene Insensitive transcription factor) is depicted. The single and double copy presence of the 36 bp indel correlate to the climacteric (CL) and non-climacteric (NC) phenotype, respectively.

Figure 4 shows the blue histochemical staining of GUS activity in melon flesh five days after injection.

Figure 5 shows the qPCR results (Cp values) of actin as internal reference gene and ACS5 in melon fruits injected with GUS gene (control) and MELO3C016536 (MEL036).

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 ETHQV6.3 is on chr. 6, between markers Al_03-B03 and FR14-P22, and according to melon WGS (Argyris et al. BMC Genomics. 2015. 16: 4), its physical position is 21834442-26987092. In this region, 298 genes were predicted.

RNASeq

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 abscised 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.

Candidate gene selection

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 fold) in the three climacteric genotypes, increased intermediately in the two intermediate types (less than 60 fold) and stayed low in non-climacteric PS (< 4 fold) during fruit ripening. Among those 298 genes in the QTL region, there was only one gene, MELO3C016536, turned out to be coexpressed with AC01 gene positively in climacteric and intermediate types, and negatively in non-climacteric type (see Figure 1 ). Besides, this gene expressed a bit earlier than AC01 gene in climacteric types, indicating this gene might be the cause, instead of consequence, of AC01 gene. Based on the protein sequence, this gene is annotated as a gene from NAC transcription factor family, a family where another two members have been shown to be involved in fruit ripening in tomato (SINAC3, patent CN102787124A. 2012; NOR gene, patent US6762347B1 . 2004). Because of its location (in QTL), expression and annotation, MELO3C016536 was selected as the candidate causal gene for climacteric ripening.

Resequence candidate gene 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 MELO3C016536 using Pacbio sequencer.

AP1_F_26856766: acactgacgacatggttctacaTCATTTTGAGGTATTGGACATTTTT (SEQ ID NO: 13) AP1_R_26853733: tacggtagcagagacttggtctGGCGTTACTGTATTCCTTACTTGAA (SEQ ID NO: 14) AP2_F_26853007: /5AmMC6/gcagtcgaacatgtagctgactcaggtcacGGATTGCTTGTAAAAAGAGGAAAA (SEQ ID NO: 15)

AP2_R_26850771 : /5AmMC6/tggatcacttgtgcaagcatcacatcgtagCCCACCATCCTATAAGTGAGAGAT (SEQ ID NO: 16)

AP3_F_26851221 : /5AmMC6/gcagtcgaacatgtagctgactcaggtcacTTTTAACCGTGCAAGTTATTGCT (SEQ ID NO: 17)

AP3_R_26849061 : /5AmMC6/tggatcacttgtgcaagcatcacatcgtagACGCCATCGTTACTTGAGATAAA (SEQ ID NO: 18)

The resequencing results showed that at about 3 kb downstream of the stop codon of MELO3C016536, chr. 6, 26850247-26850317, there was a 36 bp insertion/deletion polymorphism (see Figure 2 and 3). Very interesting, there was a binding site (ATGCAT) of EIN3 (Ethylene Insensitive transcription factor) lies in it, and when it is an insertion, there is another copy of this fragment adjacent to it.

MELO3C016536 is the only gene closest to this indel.

In the 126 melon lines, there were 94 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, while the later one, non-climacteric type. Based on this general categorization, a high (p- value=2.67E-1 1 ) association was found between the 36 bp indel and the climacteric phenotype.

Using primer pair F: TTTTGAGATTACCTTTTTCTAGTTTGA (SEQ ID NO: 1 1 ) and R: ACGAGTGTTGGGTTTATAATAGGA (SEQ ID NO: 12), the indel can be easily genotyped by PCR. Combine the genotype of this indel with the indel in MELO3C01 1271 , they can predict the time to ripe of the fruit, which is a very valuable trait for improving shelf life of climacteric melons.

Transient expression of MELO3C016536 in melon fruit

To validate the function of gene MELO3C016536 in inducing ethylene production, MELO3C016536 was transiently over expressed in young fruits of Vedrantais and Piel de Sapo to test if genes on ethylene biosynthesis path way were switched on.

First, gene MELO3C016536 driven by 35S promoter was cloned to a binary vector and transferred to

Agrobacteria strain EHA105. A Plasmid with a GUS gene (β-qlucuronidase) was used as a control. Then for each fruit, 100 μΙ_ liquid Agrobacteria cultures with the MELO3C016536 or the GUS gene were injected using syringe with a needle at two sites on the equator region of the fruit, respectively.

Three fruits (23-26 DAP) from Vedrantais and one fruit (32 DAP) from Piel de Sapo were used. Five days after injection, flesh tissues with injection were sampled for RNA isolation and qPCR.

MELO3C010779 (ACS5) is the 1-aminocyclopropane-1-carboxylate synthase gene on ethylene biosynthesis pathway during melon fruit ripening (Stitt et al., 2015). Primers used to detect this gene expression by qPCR were:

ELO3C010779_qPCR_F: CGCTATAGCCAATGCAATCC (SEQ ID NO: 19)

MELO3C010779_qPCR_R: GTTCGAAACTTTGGGTGCTC (SEQ ID NO: 20)

Actin gene is used as an internal reference gene, and the primers used were:

Cm_ACT_F: TTG C AG ACAG G ATG AG C AAG (SEQ ID NO: 21 )

Cm_ACT_R: ACCCTCCAATCCAAACACTG (SEQ ID NO: 22)

At the same time, extra samples from injection of GUS gene were made for GUS assay (Jefferson et al., 1987).

Results showed that blue histochemical staining of GUS activity could be seen after incubation in staining buffer with X-Gluc over night at 37°C (Figure 4). The qPCR results showed that ACS5 gene expression with MELO3C016536 injection increased 2.8-14.2 folds compared to control with injection of GUS gene in the four fruits tested (Figure 5).

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, Canizares 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, Melamed 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

Jefferson RA, Kavanagh TA, and Bevan MW (1987) GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907.

Table 1 Identification of SEQ ID NOs

SEQ Name Sequence

ID NO

1 MELO3C016536 protein MDPLTQLSLPPGFRFFPTDEELLVQYLCRKVAGHHFSLQLI

AEIDLYKFDPWVLPGKALFGEKEWYFFSPRDRKYPNGSR

PNRVAGSGYWKATGTDKIISSEGKNVGIKKALVFYVGKAP

KGTKTNWIMHEYRLITSSRKTGSSKLDDWVLCRIYKKNSS

CQKPTGSISSKEYSNASPSSSIDEVIESLPETGDDFFAYPK

TTLQHNDIMNKFNFEIPADSVHSDWASLAGLYSVPELAPV

DHSGTFDFNNNNNTIADLYVPSVTSSFCQVDYPPASAFRY

STQQRDGGGVFGFSQ

2 JA2 protein MGVQEKDPLLQLSLPPGFRFYPTDEELLVQYLCKKVAGHD

FPLQIIGEIDLYKFDPWVLPSKATFGEKEWYFFSPRDRKYP

NGSRPNRVAGSGYWKATGTDKVITSQGRKVGIKKALVFY

VGKAPKGSKTNWIMHEYRLFESSRKNNGSSKLDEWVLCR

IYKKNSSGPKPLMSGLHSSNEYSHGSSTSSSSQFDDMLES

LPEMDDRFSNLPRLNSLKAEKFNLDRLDSANFDWAILAGL

KPMPELGPANQAPGVQGQAQGHVNNHIHSDNNNMNFLN

DVYAHPPNFRGNTKVESINLDEEVESGKRNQRIDQSSYFQ

QSLNGFSQAYTNNVDQFGIQCPNQTLNLGFKQ

3 JA2L protein MGVQEMDPLTQLSLPPGFRFYPTDEELLVQYLCRKVAGH

DFSLQIIAEIDLYKFDPWVLPSKAIFGEKEWYFFSPRDRKY

PNGSRPNRVAGSGYWKATGTDKVITTDGRKVGIKKALVFY

IGKAPKGTKTNWIMHEYRLSEPTTKTGSSRLDDWVLCRIY

KKNSGGQKSSCSDLQNKDISHASSSSSSSQFDDMLESLPA

IEDRYFSLPRVNSIRNFQQNDKINLQQLSSGNFDWAAMAG

LNSFPELRTGNQVPTPGNQTPVLINTNQYHNHNDNLNNFN

EFFANSTALNFHGEVKFEGGVDQEVESSVRAQRLNSVNP

GFFQENSTGFSSSYTNSVPDPFGIRYPTQTVNMGFTG

4 MELO3C016536 encoding AAAAAAAAAAAGAAAGAAAGAAAGAAAGGGAAAGAAAAA sequence AAGAAAAATCTCAAATTTCGTCTCTCCTTTCTCCTCCTTA

CAATCTCCTCTATACAATG G ACCCACTG ACG CAG CTTAG CTTACCG CCG G G ATTC AG ATTTTTTCCG ACCG ATG AAG A GCTTTTAGTTCAATATCTTTGCCGGAAAGTGGCCGGCC ACCA I I I I AG CTTG CAACTC ATCG CTG AG ATTG ACTTGT ACAAATTCG ATCCATG G GTTTTACCTG G AAAG G CTTTAT TCG G G G AAAAG G AATG GTACTTTTTC AGCCC AAG AG AC CGGAAATATCCAAACGGGTCTAGACCAAACCGGGTAGC CG GTTCG G GTTACTG G AAG G CG ACCG GTACG G ATAAAA TAATCTCGTCG G AAG G G AAG AACGTCG G AATTAAAAAG G CTCTG GTTTTCTACGTCG G AAAAG CTCCTAAG G G AAC

AAAAACGAATTGGATTATGCATGAGTATCGCCTTATTAC TTCCTCC AG AAAAACAG G AAG CTCC AAG CTGG ACG ATT GGGTTTTATGTCGGATTTATAAGAAGAATTCGAGTTGTC AAAAGCCGACGGGGAGTA I I I CAAGTAAGGAATACAGT AACGCCTCACCCTCGTCGTCAATCGACGAAGTCATCGA ATCCCTACCAG AAACG G G CG ACG ATTTCTTTG CATACCC AAAAACAACATTACAACATAACGACATTATGAATAAATTC AAC I I I G AAATTCCG G CG G ACTCTGTACATTCCG ATTG G GCGAGTTTGGCCGGGCTTTACTCAGTGCCGGAACTCGC TCCCGTCGACCATTCGGGGACATTCGA I I I CAACAACAA CAATAACACGATCGCTGATCTGTATGTTCCTTCAGTTAC ATCGTCG I I I I GCCAGGTGGATTATCCGCCGGCGTCGG CGTTCCGTTACTCGACGCAACAAAGGGACGGCGGCGG AGTGTTCG G ATTCAG CC AATG AG ATG AATAC ATATG GC A TTTG G ATTTAC AAACG G AGTGTAAATAG AATTG G ATTCT G AAAATG G AAG AAAAAG G AAAAA I I I I GAAGGGAAAAA GAGAATGAAGAAAGAAAAAGTGAAAAACAGAGCATACC CTGTTTTTGACCATAAATTTTTAATTCTCTAACAAGTTTA ATGTCCCATTCTATTAGACATTAGATTAAACAATGGAATA CAGTTGTACAAATTATTG I I I AG G ATTG G AACTTC ATTAA TTAGTTTAGTGTGTTAGTTTTTTTTGGAAATTATTTTTCTT TTCTCTGCCCATCCTTAATAAATTATTATGATTGTTATGA G I N ATG G ATTGCTTGTAAAAAG AG G AAAAG AAAAAG AA AAAGGAAATTGTTATTGCC

5 JA2 encoding sequence AGTTTTTTATTAATTAAAAAAAAAATTATGGGTGTTCAAG

AAAAAG ATCCACTTTTG CAATTAAGTTTACCACCAG GAT

TTAGATTTTATCCAACTGATGAAGAGCTTTTAGTTCAATA

TTTATGTAAGAAAGTTGCTGGACATGATTTTCCTCTACA

AATTATTGGTGAAATTGATTTATACAAATTTGATCCTTGG

GTTTTACCTAGTAAGGCGACATTTGGTGAAAAAGAATGG

TA I I I CTTCAGTCCGAGGGATAGGAAGTATCCGAATGG

ATCTAGACCGAATCGAGTAGCAGGTTCGGGTTATTGGA

AAGCAACGGGGACGGATAAGGTGATAACTTCGCAAGGA

AG AAAAGTTG G AATTAAG AAAG CTCTTGTG I I I I ATGTG

GGTAAAGCTCCAAAAGGATCCAAGACGAATTGGATTAT

GCATGAATATAGACTTTTTGAATCTTCAAGGAAAAATAAT

GGAAGTTCAAAGCTAGATGAATGGGTGC I I I GTCGAAT

TTATAAGAAGAATTCAAGTGGACCAAAACCTCTGATGTC

TGG I I I ACACAGCAGTAATGAATACAGCCATGGTTCGTC GACTTCGTCATCATCCCAATTCGATGATATGCTCGAATC

ATTACCAGAAATGGACGATCGATTCTCCAA 1 1 I ACCGAG

GTTGAACTCTCTCAAGGCCGAGAAATTCAACCTCGATC

GTCTGGATTCAGCCAA 1 1 1 CGATTGGGCAATTCTCGCTG

G GCTC AAACC AATG CCGG AATTG G G CCC AGC AAATC AA

GCTCCAGGCGTTCAGGGTCAGGCTCAGGGGCATGTCA

ATAACCAC ATTCAC AG CG ACAACAAC AATATG AA 1 1 1 1 CT

CAACGATGTTTACGCCCATCCTCCAAATTTCCGAGGCAA

CACAAAGGTTGAAAGTATTAATCTAGACGAAGAAGTTGA

AAGCGGGAAAAGAAATCAACGGATTGACCAATCGAGTT

ACTTCCAACAGAGTCTCAATGGA 1 1 1 1 CCCAAGCGTATA

CAAACAATGTGGATCAATTCGGGATCCAATGTCCGAATC

AGACGTTAAATCTCGGGTTCAAGCAGTAGAGGATACTG

GAAAGTAAGAAAAGTTGTAGCTCGAAATCTGAATGCCA

GTATACTGTGTATATATACAGAAGGCCTGGA 1 1 I AGATT

TCCTTGGCCCAACGGATTCGAAGAAAGAGCAAGTACAG

TATCATGCCAAGGAAATTAAGTCCTTCGAGGGCACGTCT

GGGCAGACGTCGTGGACGTTA 1 1 1 GCAAGTTCTTGCAA

TTGTGTCTGAAGTCATTAGGCTGAGTGGCACTTCTTATT

TGCAAGTTGTG 1 1 1 GAAGTCATTAGGCCGAAGGCACGT

CTGCGTGGACGATAAAATAATGTACACAAACAGGAGATA

TATGCAGGGGCGTGTATATTC 1 1 1 1 AGGAAACTGGGCTT

TTAGTTTGTTTTATTTTATGATACATTTCCAGATTTGGAT

GTATTTTGTAATTTTTACATTTCTGTGTAGCAAGTTAGAA

GATATATAGAAAGATACTTTTAAAATACTTTTTATAAAAA

AAAAAAAAAAAAAA

JA2L encoding sequence AAATTTCAAAAAAAAATCTATATTTTTCTTCTCTCTCTACA

1 1 1 1 GCTTACTTGTGAGAAGGAAAAAAAATGGGTGTTCA AG AAATG G ATCCTCTTACACAG CTAAG CTTACCACCCG G GTTCCGGTTTTACCCGACTGATGAAGAACTTTTAGTTCA ATATTTATGCCGTAAAGTTGCTGGTCATGATTTTTCTCT G CAAATTATTG CTG AAATTG A 1 1 1 GTACAAATTCGATCCA TGGGTTCTTCCAAGTAAGGCGA 1 1 1 1 CGGAGAAAAAGA ATGGTA 1 1 1 CTTCAGTCCAAGAGATCGGAAGTATCCGAA TG G ATCTAG ACC AAAC AG AGTAG CTG G GTCTG GTTATT G G AAAG CAACTG G AACTG ATAAAGTTATTACTACAG ACG GTAGAAAAGTCGGAATCAAAAAGGCTTTAGTGTTTTACA TTGGTAAAGCACCTAAAGGAACTAAAACAAATTGGATTA TGCACGAATACAGGCTCAGTGAACCTACAACGAAAACT GGAAGTTCAAGGCTCGACGATTGGGTTCTATGTAGGAT TTACAAG AAG AATTCAG GTG G ACAAAAATCG AGTTG CTC TGA I I I ACAG AACAAG G ATATAAGTC ATG CTTC ATC ATC GTCATCGTCATCTCAG I I I G ATG AT ATG CTG G AATCTCT ACCGGCAATTGAAGATCGTTA I I I CTCATTGCCGAGGGT GAATTCTATAAGGAA I I I I CAACAAAATGACAAGATCAAT CTTCAACAATTG AG CTCTG G G AACTTCG ATTGG G CTG C TATGGCGGGATTAAACTCATTCCCGGAATTACGTACCG GAAATCAAGTTCCAACGCCGGGAAATCAAACTCCGGTG CTGATAAACACCAATCAGTATCACAATCACAACGACAAT TTGAATAATTTCAACGAATTTTTCGCCAATTCAACGGCG TTAAATTTTCACGGTGAAGTTAAGTTTGAAGGAGGAGTT GATCAAGAAGTAGAAAGCAGTGTTAGAGCTCAACGACT TAACAGCGTTAACCCGGG I I I CTTCCAAGAGAACTCAAC CGGG I I I I CAAGTTCTTATACAAACTCGGTACCCGACCC A I I I GGGATTCGGTACCCGACCCAAACAGTAAATATGG G I I I I ACTG G GTAAATAGTG G AAG G GTAAAATGTG AATG CTCATAAATTAACTTGAAGACAAA I I I GGAAAAATTGTCA TG GTTATTG AG GTTG AAAG G G CATTTTATTCTTTG GTTG G G G ATTAAAATAG CAAAAAAAAAAAAACAAG A I I I I GTT CATG C AATTATG C ACATATAC AATGTA I I I I GTTGTACTA AAATTTATAG CTTTG GTAATGTACTTATTATAG G AATG CA TATTG GAG G ATTTTAG CTTATTATATTTTGTAAATTTTG A TCTTCAAAAAAAAAA

Indel in non-climacteric AAACTCG ATATG C ATAAACTC AAAACACAAG CTATC phenotype

Single copy in climacteric AAACTCG ATATG C ATAACTC AAAACACAAG CTATC type

Nearly perfect tandem repeat AAACTCG ATATG CATAAACTCAAAAC ACAAG CTATC AAA in non-climacteric phenotype CTCGATATGCATAAATCAAAACACAAGCTATC

Copy of indel in non- AAACTCGATATGCATAAATCAAAACACAAGCTATC climacteric phenotype

Forward primer TTTTGAGATTACCTTTTTCTAGTTTGA

Reversed primer ACG AGTGTTG G GTTTATAATAG G A

AP1_F_26856766 acactgacgacatggttctacaTCATTTTGAGGTATTGGACATTTT

T

AP1_R_26853733 tacggtagcagagacttggtctGGCGTTACTGTATTCCTTACTTGA A

AP2_F_26853007: gcagtcgaacatgtagctgactcaggtcacGGATTGCTTGTAAAAAGA /5AmMC6/ GGAAAA

AP2_R_26850771 : tggatcacttgtgcaagcatcacatcgtagCCCACCATCCTATAAGTG /5AmMC6/ AGAGAT

AP3_F_26851221 : gcagtcgaacatgtagctgactcaggtcacTTTTAACCGTGCAAGTTA /5AmMC6/ TTGCT

AP3_R_26849061 : tggatcacttgtgcaagcatcacatcgtagACGCCATCGTTACTTGAG /5AmMC6/ ATAAA

MELO3C010779_qPCR_F CGCTATAGCCAATGCAATCC

MELO3C010779_qPCR_R GTTCGAAACTTTGGGTGCTC

Cm_ACT_F TTGCAGACAGGATGAGCAAG

Cm_ACT_R ACCCTCCAATCCAAACACTG

Claims

1. A genetic marker for characterizing fruit ripening, wherein said marker is localized in a region downstream of the NAC-TF gene.

2. A genetic marker according to claim 1 , wherein said marker has a sequence that is at least 80% identical to the sequence of SEQ ID NO: 9.

3. A genetic marker according to claim 2, wherein the presence of said marker associates with a non- climacteric fruit ripening phenotype, and wherein the absence of said marker associates with a climacteric and intermediate 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 presence of the genetic marker of any one of claims 1-3 that is associated with the non-climacteric fruit ripening phenotype.

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

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

b) assessing the absence of the genetic marker of any one of claims 1-3 that is associated with the non-climacteric 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 presence of the genetic marker of any one of claims 1-3 that is associated with the non-climacteric 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 absence of the genetic marker of any one of claims 1-3 that is associated with the non-climacteric 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 presence of the genetic marker of any one of claims 1-3 that is associated with the non-climacteric 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 5 associated with the non-climacteric fruit ripening phenotype in the progeny of the selfing or crossing step.

10. A method for obtaining a plant having a climacteric or 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 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 fruit ripening phenotype in the progeny of the selfing or crossing step.

15

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

a) providing a plant cell or protoplast;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in the 20 deletion of an ethylene insensitive transcription factor binding site downstream of the NAC-TF 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.

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;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in the insertion of an ethylene insensitive transcription factor binding site downstream of the NAC-TF coding

30 sequence; and,

13. A method for producing a plant having a non-climacteric fruit ripening phenotype or an increased 35 non-climacteric fruit ripening phenotype that comprises the step of:

a) providing a plant cell or protoplast;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in reducing or abolishing NAC-TF expression; and,

c) regenerating a plant from said plant cell, wherein the fruit ripening of said plant is delayed or 40 wherein the fruit ripening of the plant is non-climacteric.

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;

b) genetically engineering said plant cell or protoplast, wherein said engineering results in increasing NAC-TF expression; and,

15. A plant, plant cell, seed or fruit obtained or obtainable by any of the methods claims 9-14, 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.