Melon plants producing seedless fruits

Abstract

The invention relates to a muskmelon producing seedless fruits. The invention also includes methods for producing said plants and methods for producing seedless muskmelons. The plants and plant parts of the invention comprise at least one copy of a mutant allele of a gene named CmBEL1 which confer seed abortion when the mutant allele is in a homozygous form.

Description

[0001] The present invention relates to melon plants that produce seedless fruits. The present invention also includes methods for producing said melon plants and the use of nucleic acids encoding homeobox transcription factor proteins for producing seedless melon fruits. The gene and protein are referred to herein as CmBEL1.

[0002] Most commercial seedless fruits are developed from plants whose fruits typically contain many relatively large and hard seeds. It is known, for example, that watermelon, tomato, cucumber, eggplant, grape, banana, citrus fruits such as orange, lemon and lime have seedless fruits. Since the consumption of seedless fruits is generally easier and more convenient, they are considered valuable.

[0003] Fruit development usually begins when the sperm nucleus of pollen fertilizes one or more egg cells (ovules) in the ovule chamber of a flower.

[0004] Seedless fruits can be caused by two different phenomena. In some cases, fruits develop without pollen fertilizing the ovules, i.e., a phenomenon called parthenocarpy. In other cases, after pollination, when the growth of seeds (e.g., embryo and / or endosperm) is inhibited or the seeds die prematurely while the rest of the fruit continues to grow (seed abortion), seedless fruits appear. In contrast to parthenocarpy, seed abortion requires pollination to initiate fruit growth.

[0005] Only WO2015136532A1 describes seed abortion in melons. Herein, a single amino acid substitution in the recessive gene MELO3C009603 encoding a Cys2His2 zinc finger (ZF) protein is described as producing a "super-fruiting" phenotype, i.e., producing many small seedless melon fruits upon pollination. MELO3C009603 is located on chromosome 4 of the melon (Cucumis melo) genome (cucurbitgenomics.org, DHL92v3.6.1).

[0006] In contrast, herein, it is found that the loss of function of a different recessive gene on chromosome 9 causes seedless melon fruit development. In mutant plants, it is found that a DNA insertion in the allele causes the production of truncated mRNA transcripts and truncated proteins, and plants homozygous for the mutant allele produce many seedless fruits. The mutant allele is also referred to as a "null mutant" or "knockout mutant" allele. Similar to the "super-fruiting" phenotype, the average number of fruits in homozygous mutant plants is significantly increased, while the average fruit weight is significantly decreased.

[0007] Mapping of this gene led to the following discovery: The gene designated MELO3C005699 on chromosome 9 was disrupted. However, it was also found that the sequence of this gene given in the cucurbitgenomics.org database did not correspond to the correct sequence. The correct sequence was found by genomic analysis and is provided herein. The null mutant allele has a DNA insertion in the transcriptional region of the gene, which results in a truncated mRNA transcript and a truncated protein, whereby the coding region of the homologous domain of the protein is disrupted between the codon for amino acid P343 and the codon for amino acid Y344 of the protein (see Figure 1 , black star), whereby the protein lacks all amino acids starting from Y344 to the end of the protein.

[0008] Since MELO3C005699 is thought to encode a "low-quality protein: homeobox protein BEL1 homolog" protein, this gene is referred to herein as CmBEL1. However, it should be noted that the sequence identity with the Arabidopsis BEL1 protein is only 43.2%, and this name is purely based on the previous annotation given in the cucurbitgenomics.org database. Except for the seedless fruit phenotype in melon plants homozygous for the mutant cmbel1 and thus the development of small but numerous seedless fruits, no abnormalities were noticed in the development and / or morphology of organs such as flowers.

[0009] In Arabidopsis, mutations in BEL1 and AP2 disrupt ovule development. In addition to ovule defects, mutations in BEL1 terminate the inflorescence, thereby changing indeterminate flowering to determinate flowering. Developing siliques with BEL1 mutants sometimes also develop carpel-like structures. (Modrusan et al. 1994, The Plant Cell, Vol. 6, 333 - 349 and Bellaoui et al. 2001, The Plant Cell, Vol. 13, 2455 - 2470). In Arabidopsis, BEL1 is a transcription factor protein transcribed in developing ovules of Arabidopsis, regulating the transcription of other genes.

[0010] Bellaoui et al. showed in 2001 that Arabidopsis BEL1 can interact with a specific subset of Arabidopsis KNOX proteins (KNAT) to form a heterodimeric complex. The authors also found that in Arabidopsis, BEL1 is expressed in the shoot apical meristem (SAM) of the inflorescence and showed that BEL1 may have a direct role in maintaining the indeterminate growth of the inflorescence meristem by suppressing the plant development program.

[0011] Kumar et al. (2007 Plant Cell. September 2007; 19(9):2719 - 2735, doi:10.1105 / tpc.106.048769) described the presence in Arabidopsis thaliana of 13 members of the BEL1 - like TALE homeodomain protein (BHL) family that form heterodimeric complexes with class 1 KNOX TALE homeodomain proteins. These BHL proteins are closely related in sequence, and the authors investigated the redundancy of two of these BHL proteins (BLH2 / SAW1 and BHL4 / SAW2). Phylogenetic analysis of the gene family indicated that these two genes are most closely related in sequence to BEL1.

[0012] As mentioned, the present inventors have found that a recessive gene designated CmBEL1 on chromosome 9 causes melon plants to produce seedless fruits when knocked out or mutated to encode a non - functional protein. Thus, in one aspect, melon plants and plant parts containing one or two copies of a null mutant allele of the CmBEL1 gene are provided herein.

[0013] Optionally, plants containing a CmBEL1 allele knocked down or mutated to encode a protein with reduced function can also generate melon plants that produce seedless fruits. Such plants and plant parts are also encompassed herein, provided that a seedless fruit phenotype (seed abortion) is seen when the mutant allele is in homozygous form.

[0014] Thus, one aspect herein is a melon plant containing at least one mutant allele of the CmBEL1 gene, whereby due to the production of a mutant protein with reduced function or loss of function compared to the wild - type CmBEL1 protein, or due to the mutant allele having reduced gene expression or no gene expression compared to the wild - type CmBEL1 allele, there is a reduction or absence of wild - type mRNA transcripts or a reduction or absence of wild - type CmBEL1 protein production in the plant compared to a control melon plant (lacking the mutant CmBEL1 allele but containing two wild - type alleles of the CmBEL1 gene), and thus when the mutant allele is in homozygous form, the mutant allele causes seed abortion.

[0015] Thus, compared to the wild - type CmBEL1 protein, the mutant cmbel1 allele can contain one or more amino acid insertions, deletions, or substitutions, or the mutant cmbel1 allele can contain one or more mutations in the regulatory regions of the protein, such as the promoter or enhancer, resulting in the production of a wild - type protein with reduced function or non - functionality from that allele, thus causing seed abortion when the mutant allele is in homozygous form.

[0016] The CmBEL1 promoter sequence is located within 1000 or 2000 bases upstream of the 5'UTR (untranslated region) and is provided herein as SEQ ID NO: 6 or a sequence having at least 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 6. In one aspect, the CmBEL1 promoter of SEQ ID NO: 6 or a CmBEL1 promoter having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6 contains one or more mutations (insertions, deletions and / or substitutions of one or more nucleotides in SEQ ID NO: 6 or in the CmBEL1 promoter sequence having at least 97%, 98% or 99% sequence identity with SEQ ID NO: 6), whereby compared to the wild-type CmBEL1 allele, transcription of the cmbel1 gene is completely abolished (no mRNA transcript is produced) or reduced by at least 80%, 90%, 95%, 96%, 97%, 98% or 99%.

[0017] In one aspect, compared to the wild-type CmBEL1 allele, the mutant cmbel1 allele may contain one or more nucleotides inserted, deleted or substituted in the promoter region of the gene or in the transcription region or coding region, whereby the mutant allele does not produce a wild-type mRNA transcript but produces, for example, no mRNA transcript or a prematurely terminated mRNA transcript, or an mRNA transcript containing one or more codons inserted, substituted or deleted compared to the wild-type transcript, thus resulting in the failure to generate a functional wild-type protein by the allele, thus leading to seed abortion when the mutant allele is in homozygous form.

[0018] In one aspect, the mutant allele is thus a null allele, i.e., the allele is not expressed (e.g., due to a mutation in the promoter), or the protein product of the allele is non-functional in the plant (e.g., due to a mutation in the allele, thus producing a non-functional protein).

[0019] In one aspect, the endogenous allele is disrupted due to, for example, DNA insertion in the gene (e.g., in the promoter or transcription region or coding region), such as a transposable element (TE) being inserted into the gene. For targeted insertion via TE transposition, see WO2022 / 197749. In one aspect, the DNA, such as a TE or TE-like element, is inserted into the promoter region or transcription region or coding region of the gene, such as in the intron region between exon 2 and exon 3. In one aspect, the DNA insert, for example, causes premature termination of the mRNA transcript within or after exon 1, or within or after exon 2, or within or after exon 3. In one aspect, the length of the DNA insert in the allele is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 nucleotides.

[0020] Other mutants in the endogenous CmBEL1 gene of the above mutants or plants can be generated by, for example, random mutagenesis or targeted mutagenesis, such as CRISPR-based methods. Reviews of targeted gene editing are provided, for example, by Erpen-Dalla Corte et al. in Plants 2019, 8, 601 (doi: 10.3390 / plants8120601) or by Bed Prakash Bhatta and Subas Malla in Plants 2020, 9, 1360; doi: 10.3390 / plants9101360. CRISPR-based editing has also been carried out in melons and other cucurbit crops, and thus those skilled in the art can use it to edit the endogenous CmBEL1 gene of melons to, for example, generate endogenous null alleles. For example, CRISPR has been used in cucumbers to generate mutants in target genes, as described in WO2017098508. In addition, in watermelon, CRISPR has been successfully used to modify target genes, see, for example, Wang, Y., Wang, J., Guo, S. et al. CRISPR / Cas9-mediated mutagenesis of ClBG1 decreased seed size and promoted seed germination in watermelon. Hortic Res 8, 70 (2021). https: / / doi.org / 10.1038 / s41438-021-00506-1. In addition, Andrea Giordano et al. (bioRxiv 2022.01.30.478227; doi: https: / / doi.org / 10.1101 / 2022.01.30.478227) published "CRISPR / Cas9 gene editing reveals the roles of CTR1 and ROS1 in melon fruit ripening and epigenetic regulation", in which Crispr / Cas9 was used to edit melon plants, thereby generating loss-of-function mutants of two target genes.

[0021] Alternatively, mutants in the endogenous CmBEL1 gene can be generated by targeted insertion via TE transposition, as described, for example, in WO2022 / 197749 (incorporated herein by reference).

[0022] The wild-type functional CmBEL1 protein is shown in Figure 1Among them, the conserved homeodomain is highlighted in the boxed region. This homeodomain is a DNA-binding domain that participates in the transcriptional regulation of other genes in plants. Therefore, the mutant CmBEL1 protein lacking all or part of this homeodomain is non-functional in vivo, and the mutant allele in homozygous form will lead to seed abortion.

[0023] This homeodomain starts from amino acid W318 of SEQ ID NO: 1 and ends at amino acid M379, or equivalent amino acids in a variant sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1. In the transcriptional region of this gene, this homeodomain starts from nucleotide 1263 of SEQ ID NO: 4 (nucleotides 1263 to 1265 encode W318) and ends at nucleotide 2427 of SEQ ID NO: 4 (nucleotides 2725 to 2727 encode M379), or ends at equivalent nucleotides in a sequence having at least 97%, 98% or 99% sequence identity with SEQ ID NO: 4.

[0024] In one aspect, there is provided a melon plant comprising a mutant allele of the CmBEL1 gene, wherein the mutant allele results in the deletion of one or more amino acids in the homeodomain, the insertion of one or more amino acids into the homeodomain, or the substitution of one or more amino acids in the homeodomain with different amino acids, such that the encoded protein is non-functional in vivo.

[0025] Regarding mutations in the homeodomain (or other parts of the protein), in one aspect, mutations that particularly result in amino acid substitutions, where the properties of the wild-type amino acid and the substituted amino acid are different, are an aspect herein, because such different amino acid properties will reduce or eliminate the normal function of the protein and / or domain. Thus, for example, substituting a non-polar amino acid (including a hydrophilic side chain) with a polar amino acid, or vice versa, or substituting an amino acid with a charged side chain with an amino acid with an uncharged or differently charged side chain. Non-polar amino acids are alanine (A or Ala), cysteine (C or Cys), glycine (G or Gly), isoleucine (I or Ile), leucine (L or Leu), methionine (M or Met), phenylalanine (F or Phe), proline (P or Pro), tryptophan (W or Trp), valine (V or Val). Polar amino acids are arginine (R or Arg), asparagine (N or Asn), aspartic acid (D or Asp), glutamic acid (E or Glu), glutamine (Q or Gln), histidine (H or His), lysine (K or Lys), serine (S or Ser), threonine (T or Thr), tyrosine (Y or Tyr).

[0026] Thus, in one aspect, any one (or more) of the non-polar amino acids of the conserved homeodomain is replaced with a polar amino acid, and / or any one (or more) of the polar amino acids of the conserved domain is replaced with a non-polar amino acid. Then, the function of the resulting mutant allele can be tested by generating plants homozygous for the mutant allele and analyzing the phenotype. If the mutant allele causes the plant to become seed-aborting, then the mutant allele is an allele encoding a mutant CmBEL1 protein with reduced or no in vivo function.

[0027] In another aspect, the mutant cmbel1 allele encodes a truncated protein in which at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 or more (e.g., at least 230, 240, 250, 255, 260, 265, 270, 280, 300, 400, 500 or 600) amino acids at the C-terminus of the wild-type CmBEL1 protein are deleted or optionally replaced with different amino acids, such that the protein has reduced or no in vivo function. Examples of mutant alleles encoding truncated proteins are given in the examples herein, where, for example, the mRNA transcript of the mutant allele terminates after exon 2, and the translated protein contains only amino acids 1 to 343 of SEQ ID NO: 1 (or 1 to 343 in a variant CmBEL1 protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1). The Y344STOP mutant is also described in the examples. Thus, in these mutant proteins, 36 amino acids of the 62-amino acid wild-type homeodomain are absent, resulting in loss of function. Thus, 265 amino acids are deleted from the entire 608-amino acid BEL1 protein.

[0028] Thus, in one aspect, the mutant allele produces a truncated protein in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 36, 40, 45, 50, 55, 60 or all 62 amino acids of the homeodomain are deleted (compared to the wild-type protein). The truncated protein is preferably truncated at the C-terminus, where the C-terminal truncation includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 36, 40, 45, 50, 55, 60 or all 62 amino acids of the homeodomain. The mutant protein preferably has no in vivo function (or optionally has reduced in vivo function) and confers seed abortion when the mutant allele is in homozygous form.

[0029] In another aspect, the mutant allele produces a mutant protein in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 36, 40, 45, 50, 55, 60 or all 62 amino acids of the homeodomain are replaced by different amino acids (compared to the wild-type protein). The mutant protein preferably does not have an in vivo function (or optionally has a reduced in vivo function), and confers seed abortion when the mutant allele is in the homozygous form.

[0030] In yet another aspect, the mutant allele produces a mutant protein in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are inserted into the homeodomain. The mutant protein preferably does not have an in vivo function (or optionally has a reduced in vivo function), and confers seed abortion when the mutant allele is in the homozygous form.

[0031] In another aspect, the mutant allele produces a mutant protein in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are deleted from the homeodomain. The mutant protein preferably does not have an in vivo function (or optionally has a reduced in vivo function), and confers seed abortion when the mutant allele is in the homozygous form.

[0032] In another aspect, the mutant allele produces a truncated protein in which at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 265 or more amino acids at the C-terminus of the wild-type protein are deleted or replaced by one or more different amino acids (compared to the wild-type protein). The mutant protein preferably does not have an in vivo function (or optionally has a reduced in vivo function), and confers seed abortion when the mutant allele is in the homozygous form.

[0033] These mutant alleles preferably result in a loss of function of the wild-type protein and, thus, when the mutant allele is in homozygous form in a melon plant, result in seed abortion. As mentioned, the phenotype can be tested in vivo by growing the homozygous plants, allowing them to flower and be pollinated, and examining the fruits produced on the plants to see if they are seedless. To assess the phenotype, several plants homozygous for the mutant allele and several plants homozygous for the wild-type allele (control plants) are grown under the same environmental conditions, and the developed fruits are cut open to examine whether they are seeded or seedless. The average fruit number per plant (for each genotype) and / or the average fruit weight per plant (for each genotype) can also be measured. In one aspect, compared to wild-type control plants, the mutant cmbel1 allele confers seed abortion and also confers a significant increase in the average fruit number and / or a significant decrease in the average fruit weight. The control plants preferably have the same type and the same or a similar genetic background as the plants containing the mutant cmbel1 allele so that the phenotypic effect can be attributed to the mutant allele. In one aspect, the control plants are isogenic or near-isogenic lines or wild-type lines that are used as starting materials for modifying the endogenous CmBEL1 gene and have little or no difference in the genetic background except for the allele at the CmBEL1 locus.

[0034] The sex expression of melon plants is different and can produce different types of flowers, including hermaphrodite (all bisexual flowers), andromonoecious (male and bisexual flowers on the same plant), monoecious (male and female flowers on the same plant), or gynoecious (all female flowers). The most common sex type of commercial melon cultivars is andromonoecious, with male and bisexual flowers produced according to a developmental gradient. Flowers develop in the leaf axils or at the stem nodes. Pollination herein generally refers to self-pollination of the plant, at least in the sex forms where the plant produces pollen-producing flowers (male or bisexual flowers) and female or bisexual flowers on the same plant.

[0035] In one aspect, the melon CmBEL1 gene is a gene encoding a CmBEL1 protein, where the CmBEL1 protein is the protein of SEQ ID NO: 1 or a protein having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

[0036] Melon BEL1 may also be referred to as CmBEL1. Regarding Melon BEL1, the wild-type genomic sequence is provided herein as SEQ ID NO: 4 and encodes the wild-type protein of SEQ ID NO: 1. Other cultivated melons may contain allelic variants of the CmBEL1 gene having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to SEQ ID NO: 4 and may encode a wild-type (functional) CmBEL1 protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Such proteins are also referred to herein as functional variants of the protein of SEQ ID NO: 1, and such genes are referred to as allelic variants of the gene of SEQ ID NO: 4. Importantly, when mutated to knockout gene expression or when mutated to encode a protein with loss of function or when the gene is deleted, they should result in seed abortion. For example, when present homozygously in a diploid plant, an allelic variant having the same mutation as the mutation generated in the examples herein should result in the same phenotype (seed abortion).

[0037] In one aspect of the present invention, there is provided a melon plant or a melon plant cell, characterized in that, compared with the corresponding wild-type plant or plant cell, the plant or plant cell has at least one copy of a mutant cmbel1 allele, thereby resulting in seed abortion when the mutant allele is in homozygous form, wherein the CmBEL1 protein of the wild-type plant or plant cell is encoded by a nucleotide molecule selected from the group consisting of:

[0038] a) a nucleic acid molecule encoding a protein having the amino acid sequence given in SEQ ID NO: 1;

[0039] b) a nucleic acid molecule encoding a protein, the sequence of which has at least 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence given in SEQ ID NO: 1;

[0040] c) a nucleic acid molecule having SEQ ID NO: 4 or having at least 90%, 91%, 92%,

[0041] 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 and encoding a CmBEL1 protein.

[0042] The mutant cmbel1 allele may comprise a knockdown or knockout of the gene expression of the wild-type CmBel1 allele (e.g., by a mutation in the promoter or other regulatory sequences of the wild-type BEL1 allele), whereby no wild-type mRNA transcript is produced or a reduced amount of wild-type mRNA transcript is produced, or by a mutant cmbel1 allele encoding a loss-of-function or reduced-function CmBel1 protein (mutant CmBel1 protein).

[0043] In one aspect, the plant or plant cell comprises at least one copy of a null mutant allele of the CmBEL1 gene, i.e., the null allele does not result in the expression of the wild-type gene and thus does not result in the production of any wild-type protein, or the null allele encodes a loss-of-function protein, by, for example, insertion, substitution, or deletion of one or more nucleotides in the gene. A null allele or knockout allele is an allele that results in the absence of the wild-type CmBEL1 protein produced by that allele in a cell or tissue that normally produces the wild-type CmBEL1 protein or results in the production of a loss-of-function protein by that allele in a cell or tissue that normally produces the wild-type CmBEL1 protein. Thus, when the null allele is present in homozygous form in a plant or plant cell, the cell or plant homozygous for the null allele does not produce a functional CmBEL1 protein.

[0044] In one aspect, the endogenous wild-type CmBEL1 allele is completely absent, or partially absent, or contains a DNA insert (e.g., an inserted transposon or transposon-like element, or an inserted DNA fragment) in the allele, whereby the allele is a null allele.

[0045] In one aspect, the endogenous wild-type CmBEL1 allele is mutated, and the mutant cmbel1 allele encodes a mutant CmBEL1 protein with reduced or loss-of-function compared to the wild-type protein, e.g., the mutant CmBEL1 protein contains one or more amino acid substitutions, deletions, and / or insertions compared to the wild-type protein.

[0046] In one aspect, the mutant allele encodes a protein lacking one or more (or all) amino acids of the homeodomain of SEQ ID NO: 1 (the homeodomain starting at amino acid 318 of SEQ ID NO: 1 and ending at amino acid 379, or equivalent amino acids having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence given in SEQ ID NO: 1), or wherein one or more amino acids of the homeodomain are replaced by one or more different amino acids, or deleted, for example due to a stop codon mutation in a codon before or in the codons of the homeodomain, or due to insertion of one or more nucleotides into the transcription region of the gene, resulting in a CmBEL1 protein with reduced function or preferably loss of function.

[0047] For example, the mutant allele can encode a truncated protein in which one or more amino acids at the C-terminus, including for example one or more amino acids of the homeodomain, are deleted, for example due to, for example, DNA insertion between the codon for P343 and the codon for Y344 or due to a Y344STOP mutation, and the encoded protein can contain only amino acids 1 to P343 (see also the Examples). In one aspect, any codon for amino acids 1 to amino acid 379 can be changed to a premature stop codon, whereby the truncated protein is rendered non-functional. In another aspect, covered herein is the insertion of one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 10, 20, 100, 500, 1000 or more) into the transcription region of the gene (encoding M379) starting at nucleotide 1 of SEQ ID NO: 4 and ending at nucleotide 2425, 2426 or 2427, whereby the protein is truncated and lacks at least M379 of the homeodomain, as the truncated protein is rendered non-functional. In yet another aspect, covered herein is any nucleotide substitution of one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 10 or more) in the region of the gene starting at nucleotide 1 of SEQ ID NO: 4 and ending at nucleotide 2425, 2426 or 2427 (nucleotides 2425 - 2427 encode M379 of the homeodomain), as the truncated or mutant protein is rendered non-functional. In still another aspect, covered herein is any nucleotide deletion of one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 10 or more) in the region of the gene starting at nucleotide 1 of SEQ ID NO: 4 and ending at nucleotide 2425, 2426 or 2427 (nucleotides 2425 - 2427 encode M379 of the homeodomain), as the truncated or mutant protein is rendered non-functional.

[0048] The homeodomain is a domain that starts at (and includes) amino acid 318 of SEQ ID NO: 1 and ends at (and includes) amino acid 379 of SEQ ID NO: 1. Amino acids having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence given in SEQ ID NO: 1 (i.e., the homeodomain), equivalent to amino acids 318 to 379 of SEQ ID NO: 1, can be readily identified by pairwise sequence alignment.

[0049] Thus, in one aspect, the mutant CmBEL1 protein contains one or more amino acids substituted, deleted and / or inserted in the conserved homeodomain of the protein. Such insertion, deletion or substitution of one or more amino acids of the homeodomain preferably renders the protein non-functional in vivo, as can be tested phenotypically when the mutant allele is in homozygous form.

[0050] In one aspect, at least one amino acid of the conserved homeodomain is replaced by another amino acid or by a stop codon, or one or more amino acids of the homeodomain are not transcribed and translated due to insertion, deletion or substitution of one or more nucleotides in the allele, resulting in a loss-of-function (or reduced-function) protein and seed abortion when the allele is in homozygous form (when the wild-type allele is absent in a diploid plant or plant cell).

[0051] Thus, in one aspect, the mutant allele contains an insertion, deletion or substitution of one or more nucleotides in the genomic allele, such as SEQ ID NO: 4 (or a genomic sequence having at least 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 4), and such nucleotide insertion, deletion or substitution preferably results in the mutant allele being a null allele. In one aspect, one or more nucleotides are inserted, deleted or substituted in the region starting at nucleotide 1 of SEQ ID NO: 4 and ending at nucleotide 2427 of SEQ ID NO: 4 (or equivalent nucleotides in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 4). In one aspect, the insertion, deletion or substitution of one or more nucleotides is in a region that is an exon, but can also be in an intron region or at a splice site. Figure 7 The intron regions and exon regions of SEQ ID NO: 4 are shown.

[0052] In one aspect, an insertion, deletion, or substitution of one or more nucleotides in the genomic DNA of SEQ ID NO: 4 (or in a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4) results in the encoded protein no longer containing the wild-type functional homology domain, such as a deletion of one or more amino acids of the homology domain or replacement of one or more amino acids with one or more other amino acids, or insertion of one or more amino acids into the homology domain.

[0053] In one aspect, the insertion, deletion, or substitution of one or more nucleotides is between the codon for W318 of SEQ ID NO: 4 and the codon for M379 (or between codons encoding equivalent amino acids in a sequence having at least 94% identity to SEQ ID NO: 1); in one aspect, the insertion of one or more nucleotides is between the codon for P343 of SEQ ID NO: 4 and the codon for Y344 (or between codons encoding equivalent amino acids in a sequence having at least 94% identity to SEQ ID NO: 1). In one aspect, the insertion of a nucleotide in SEQ ID NO: 4 that results in an allele not encoding a wild-type protein or a loss-of-function (or reduced-function) mutant protein contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000, 1100, 1200, 1210, 1216, or more nucleotides.

[0054] In another aspect, a deletion of one or more amino acids of the conserved homology domain, such as by a mutation that creates a premature stop codon (e.g., within or prior to the homology domain, i.e., within or prior to amino acid M379), results in a loss-of-function (or reduced-function) protein and seed abortion when the allele is in homozygous form (when the wild-type allele is absent in a diploid plant or plant cell).

[0055] In another aspect, the mutant protein is truncated, lacking at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 215, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265 or more amino acids at the C-terminus of the wild-type CmBEL1 protein of SEQ ID NO: 1 (or a wild-type protein having at least 94% identity to SEQ ID NO: 1). Optionally, the deleted wild-type amino acids may be replaced by one or more different amino acids such that the protein does not have in vivo function (optionally, having reduced in vivo function, which still results in the same phenotype).

[0056] In other words, the truncated mutant protein may contain only 500, 450, 400, 380, 370, 360, 350, 345, 344, 343, 342, 341, 340, 330, 320, 318, 317, 300 or fewer N-terminal amino acids corresponding to the wild-type amino acids of SEQ ID NO: 1.

[0057] In one aspect, there is a deletion of Y at position 344 of SEQ ID NO: 1 or at the equivalent position of a protein having at least 94% identity to SEQ ID NO: 1, or replacement by a different amino acid, or replacement by a stop codon. Optionally, all amino acids after Y344 are also deleted, or replaced by one or more different amino acids.

[0058] In another aspect, an insertion, deletion or substitution of one or more nucleotides in the genomic DNA of SEQ ID NO: 4 (or in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4) results in the encoded protein being truncated by at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 260 or 265 C-terminal amino acids, such that the protein is non-functional in vivo (or has reduced function), as visible by the seed abortion phenotype when the mutant allele is in homozygous form.

[0059] There is a loss of function (and optionally a reduction in function) of the protein when the mutant allele changes the in vivo phenotype from the wild-type phenotype, i.e., the seeded fruit that develops after pollination when the wild-type allele is in homozygous form, to seed abortion (seedless fruit that develops after pollination) when the mutant allele is in homozygous form in a diploid plant.

[0060] Equivalent amino acids in a sequence having at least 94%, 95%, 96%, 97%, 98% or higher sequence identity to SEQ ID NO: 1 can be identified by pairwise comparison with SEQ ID NO: 1 (e.g., using the program Needle). Similarly, equivalent nucleotides in a sequence having at least 94%, 95%, 96%, 97%, 98% or higher sequence identity to SEQ ID NO: 4 (or other sequences herein, such as SEQ ID NO: 2 or 3) can be identified by pairwise comparison with SEQ ID NO: 4 (or correspondingly with other sequences herein, such as SEQ ID NO: 2 or 3) (e.g., using the program Needle).

[0061] In one aspect, Y344 of the melon protein of SEQ ID NO: 1 (or an equivalent amino acid in a sequence having at least 94%, 95%, 96%, 97% or higher sequence identity to SEQ ID NO: 1) is substituted with a different amino acid, deleted or substituted with a stop codon. SUMMARY OF THE INVENTION

[0062] There is provided a cultivated melon plant and plant parts thereof, which contain at least one copy of a mutant allele of a gene named CmBEL1, and when the mutant allele is in homozygous form, the mutant allele confers seed abortion.

[0063] The mutant allele not only confers that the fruits developed on plants homozygous for the mutant allele are seedless, but also, in one aspect, confers an increased average number of fruits developed on plants homozygous for the mutant allele and / or a decreased average fruit weight of the fruits developed on plants homozygous for the mutant allele. In one aspect, when grown under the same environmental conditions, the average number of fruits is increased by at least 5%, 10%, 20%, 30%, 40% or 50% or more compared to the average number of fruits produced by control plants (plants containing the wild-type CmBEL1 allele in homozygous form). In another aspect, when grown under the same environmental conditions, the average weight of the fruits is decreased by at least 5%, 10%, 20%, 30%, 40% or 50% or more compared to the average weight of the fruits produced by control plants (plants containing the wild-type CmBEL1 allele in homozygous form). In one aspect, plants homozygous for the mutant allele produce on average at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more fruits per plant.

[0064] In one aspect, the CmBEL1 gene is located on chromosome 9 of the melon genome DHL92, v3.6.1 (cuCurbitgenomics.org), and in particular, this gene is located in the region of chromosome 9 that starts at base pair 23597842 and ends at base pair 23600944. The promoter is in the region 1000 or 2000 bases upstream of base 23597842, so in the region 23596842 to 23597842, or in the region 23595842 to 23597842. In one aspect, the promoter is provided as part of SEQ ID NO: 6 or a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6. In one aspect, the promoter that causes mRNA transcription of the CmBEL1 allele is upstream of nucleotide 23597097 on chromosome 9, so for example in the region that starts at nucleotide 23595097 on chromosome 9 and ends at nucleotide 23597096, or in the region that starts at 23596097 and ends at nucleotide 23597096.

[0065] In one embodiment, a melon plant or plant part comprising a mutant allele of the CmBEL1 gene is diploid. Preferably, the mutant allele is present in two copies in the diploid melon plant or plant part.

[0066] A melon plant part comprising a mutant allele of the CmBEL1 gene can be a cell, flower, leaf, stem, cutting, ovule, pollen, root, rootstock, fruit, protoplast, embryo, anther.

[0067] Also encompassed is a vegetatively propagated melon plant that is propagated from such a plant part comprising at least one mutant allele, preferably two mutant alleles, of the CmBEL1 gene such that the plant produces seedless fruit after pollination.

[0068] Similarly, a seed is provided from which a plant of the present invention can grow. The seed contains one or two copies of a mutant allele of the CmBEL1 gene.

[0069] In addition, a seedless fruit or fruit part is provided that is produced by a plant according to the present invention. Such a seedless fruit or fruit part contains two mutant alleles of the CmBEL1 gene in its cells, as does the plant on which the seedless fruit or fruit part is produced.

[0070] A method for producing a seedless melon fruit is provided, the method comprising growing a diploid plant comprising two copies of a mutant allele of the CmBEL1 gene and harvesting the fruit produced by the plant. In particular, the melon fruit develops after pollination of the flower (which induces fruit set) and is seedless. The fruit tissue also contains two copies of the mutant allele.

[0071] A method for producing a cultivated melon plant with seed abortion is provided, the method comprising the steps of:

[0072] a) introducing a mutation into a melon plant or seed population; or providing a mutant melon plant or seed population (e.g., a TILLING population, e.g., M2, M3, M4 or higher generations),

[0073] b) selecting a melon plant that produces seedless fruit (after pollination of the flower);

[0074] c) optionally verifying whether the melon plant selected in b) contains a mutant allele of the CmBEL1 gene, in particular a null allele; and

[0075] d) optionally growing the melon plant obtained in c).

[0076] A method for producing a cultivated melon plant with seed abortion is provided, the method comprising the steps of:

[0077] a) introducing a mutation into a cultivated melon plant or seed; or providing a mutant melon plant or seed population (e.g., a TILLING population, e.g., M2, M3, M4 or higher generations),

[0078] b) selecting a melon plant that contains a mutant allele of the CmBEL1 gene;

[0079] c) optionally selfing the selected plant to generate a melon plant homozygous for the mutant allele of the CmBEL1 gene, in particular a mutant null allele,

[0080] d) optionally growing the plant, e.g., to allow the fruit to develop and confirm that the fruit is seedless.

[0081] A melon plant, seed or fruit produced by this method is covered herein.

[0082] The use of a seed-aborting melon plant for producing a seedless melon fruit after pollination of the flower of the plant is also an aspect of the present invention.

[0083] The use of the mutant cmbel1 allele of the CmBEL1 gene described herein for producing a seed-aborting melon plant is also an aspect of the present invention.

[0084] A method for generating cultivated melon plants that produce seedless fruits after pollination is provided, the method comprising the following steps:

[0085] a) Introduce a random or targeted mutation (e.g., using a Crispr-based method) into one or more melon plants, plant parts, or seeds; or provide a population of mutant plants or seeds (e.g., a TILLING population, such as M2, M3, M4, or higher generations),

[0086] b) Select mutant alleles that contain the CmBEL1 gene, such as mutant alleles that produce significantly reduced wild-type CmBEL1 protein or do not produce wild-type CmBEL1 protein (e.g., knockout alleles), or mutant alleles that encode a protein containing one or more amino acid deletions, substitutions, or insertions compared to the wild-type protein, especially null mutant alleles or loss-of-function alleles of plants,

[0087] c) Optionally remove any transgenic constructs (e.g., CRISPR constructs) from the plants, and / or

[0088] d) Optionally generate plants homozygous for the mutant alleles and analyze whether seedless fruits develop after pollination.

[0089] A method for selecting or identifying melon plants, seeds, or plant parts is provided, the method comprising the following steps:

[0090] a) Analyze whether the genomic DNA of the plant or plant part or seed contains mutant alleles in its genome and / or contains wild-type alleles of the CmBEL1 gene, and optionally

[0091] b) Select plants or plant parts or seeds that contain mutant alleles of one or two copies of the CmBEL1 gene in the genome, especially null alleles,

[0092] Wherein the wild-type allele of the melon CmBEL1 gene encodes the protein of SEQ ID NO: 1 (or a wild-type protein having at least 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1).

[0093] Step a) can be carried out in a variety of ways, using, for example, PCR-based methods, sequencing-based methods, nucleic acid hybridization-based methods, gene expression levels, etc. In one aspect, for example, the KASP assay can be used.

[0094] A method for screening (e.g., genotyping) genomic DNA of melon plants, seeds or plant parts is provided, the method comprising the following steps:

[0095] a) providing a sample (or samples) of genomic DNA of a melon plant or plants (e.g., F2 population, inbred line, backcross population, breeding population, hybrid plant, etc.);

[0096] b) providing a pair of PCR primers or an oligonucleotide probe, the primer or (oligonucleotide) probe comprising at least 10, 11,

[0097] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive nucleotides of the genomic CmBEL1 allele of the melon CmBEL1 gene, and capable of hybridizing to the genomic allele and / or amplifying a portion of the genomic allele in a PCR assay, and

[0098] c) performing a PCR assay on the sample of step a) using the pair of primers of step b) or a hybridization assay using the probe of step

[0099] b), and optionally

[0100] d) selecting a plant or plant part or seed comprising an allele (e.g., wild-type allele and / or mutant allele, especially null allele) of the melon CmBEL1 gene having one or two copies in the genome;

[0101] wherein the wild-type allele of the melon CmBEL1 gene encodes the protein of SEQ ID NO: 1 (or a wild-type protein having at least 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1).

[0102] In step b), the PCR primer pair is at least one forward primer that is complementary to one DNA strand in the DNA strand of the CmBEL1 allele and one reverse primer that is complementary to the other DNA strand of the CmBEL1 allele. This primer pair hybridizes with the denatured genomic DNA in the PCR reaction and amplifies a portion of the CmBEL1 allele. Primer design tools can be used to design primers to amplify the wild-type or any mutant CmBEL1 allele. In one aspect, two forward primers and a common reverse primer are used. One forward primer is designed to amplify the wild-type allele, and one forward primer is designed to amplify the mutant allele of the CmBEL1 gene. These three primers can be used in a KASP assay to genotype the sample of step a). Thus, in one aspect, the assay in step c) is a KASP assay, but other genotyping assays can also be used, such as those described at the World Wide Web address biosearchtech.com / sectors / agrigenomics / agrigenomics-pcr-qpcr-technologies.

[0103] In one aspect, the assay differentiates between the wild-type allele and the mutant allele (especially the null allele) of the CmBEL1 gene, such as the wild-type CmBEL1 allele and the mutant allele of the Examples, or any mutant allele described elsewhere herein. When the mutant allele is in the homozygous form, it confers seed abortion.

[0104] To analyze the genomic DNA, at least crude genomic DNA extraction may be necessary. The presence of the mutant allele and / or the wild-type allele in the genomic DNA can be detected directly or indirectly. Direct detection can be performed, for example, by nucleic acid hybridization using, for example, an oligonucleotide probe. Indirect detection can be performed, for example, by nucleic acid amplification using, for example, PCR primers that contain, for example, a tail sequence attached to the primer. During PCR, the allele-specific primer binds to the template DNA and extends, thereby attaching the tail sequence to the newly synthesized strand. In subsequent PCR cycles, a FRET cassette (fluorescence resonance energy transfer cassette) binds to the tail and emits fluorescence. Then the fluorescence signal can be detected. This is used, for example, in a KASP assay.

[0105] The mutant allele can differ from the wild-type allele in several aspects, such as differing from the wild-type allele in the promoter sequence, or in the protein-coding sequence, or at the intron / exon splicing site. The mutant allele can have reduced gene expression or no gene expression, or the mutant allele can produce a protein that contains one or more amino acids with deletions, substitutions, insertions, or duplications compared to the wild-type protein.

[0106] In one aspect, a mutant allele is an allele encoding a mutant protein that has a loss of function in vivo, such as being truncated and / or lacking all or part of a homologous domain.

[0107] In one aspect, the mutant allele encodes a mutant protein that is truncated at the C-terminus and comprises amino acids 1 to 378 or fewer (such as 1 to 377, 1 to 376, etc.) corresponding to the wild-type protein, for example, the wild-type protein of SEQ ID NO: 1, or 1 to 343 or fewer wild-type amino acids of a wild-type protein having at least 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 1.

[0108] Furthermore, a method for generating and / or selecting a melon plant or plant part comprising at least one mutant allele of the melon CmBEL1 gene in the genome is provided.

[0109] In one aspect, a method for detecting the presence of the wild-type allele and / or mutant allele of the melon CmBEL1 gene in the genome is also provided.

[0110] In one aspect, a method for detecting whether a melon plant or plant part or seed contains at least one copy of the wild-type allele, such as the wild-type allele encoding the protein of SEQ ID NO: 1 (or a wild-type allele having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1) and / or contains at least one copy of a mutant allele encoding a mutant protein comprising, for example, one or more amino acids with substitutions, insertions and / or deletions relative to the wild-type protein, and optionally selecting a plant, plant part or seed containing at least one copy of the mutant CmBEL1 allele is provided.

[0111] In another aspect, provided is a method for detecting whether a melon plant, plant part, or seed contains at least one copy of a wild-type allele of a genomic sequence comprising SEQ ID NO: 4 (or a wild-type allele having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4) and / or at least one copy of a mutant allele comprising one or more nucleotides having an insertion, deletion, and / or substitution relative to SEQ ID NO: 4 (or relative to a wild-type allele having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4), and wherein said mutant allele is a null allele or encodes a mutant protein with reduced or loss of function due to, for example, substitution, insertion, and / or deletion of one or more amino acids relative to a functional wild-type protein, and optionally selecting a plant, plant part, or seed comprising at least one copy of the mutant CmBEL1 allele.

[0112] Detection can include detecting genomic DNA encoding a mutant or wild-type protein, or otherwise detecting cDNA or mRNA encoding a mutant or wild-type protein. In one aspect, also covered herein is the detection of the presence of a wild-type or mutant promoter. Thus, covered is the detection of the presence of a wild-type sequence such as SEQ ID NO: 2 or 3 or a mutant sequence of a wild-type sequence or a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2 or 3. Also covered herein is the detection of the presence of a wild-type or mutant sequence of SEQ ID NO: 6 or a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6.

[0113] Also provided is a KASP assay (Kbioscience Kompetitive Allele-Specific PCR Genotyping Assay) that includes two allele-specific forward primers, such as a FAM primer and a VIC primer, and a common reverse primer. Obviously, allele-specific primers can be developed to detect and / or distinguish a wild-type allele from any mutant allele that contains, for example, one or more amino acids having a substitution, duplication, deletion, and / or insertion relative to a wild-type protein.

[0114] Similarly, covered herein are isolated sequences or molecules of (wild-type or mutant) genomic sequences, cDNA or mRNA sequences, protein sequences, or promoter sequences, as well as oligonucleotide primers or probes for detecting wild-type or mutant alleles of the melon CmBEL1 gene.

[0115] There is also provided a method for generating PCR amplification products and / or oligonucleotide hybridization products of genomic DNA (a part thereof) of melon plants, seeds or plant parts, the method comprising the following steps:

[0116] a) providing a sample (or samples) of genomic DNA of a melon plant or plants (e.g., an F2 population, an inbred line, a backcross population, a breeding population, a hybrid plant, etc.);

[0117] b) providing at least one pair of PCR primers or at least one oligonucleotide probe, the primer or (oligonucleotide) probe comprising at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive nucleotides of a genomic CmBEL1 allele of the melon CmBEL1 gene and capable of hybridizing to and / or amplifying a part of the genomic allele in a PCR assay, and

[0118] c) performing a PCR assay on the sample of step a) using the primer pair of step b) or a hybridization assay using the probe of step b) to generate a PCR amplification product and / or an oligonucleotide hybridization product, and optionally

[0119] d) selecting a plant or plant part or seed comprising an allele (e.g., a wild-type allele and / or a mutant allele, particularly a null allele) of the CmBEL1 gene having one or two copies in the genome,

[0120] wherein the wild-type allele of the melon CmBEL1 gene encodes the protein of SEQ ID NO: 1 (or a wild-type protein having at least 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1), or

[0121] wherein the wild-type genomic allele of the melon CmBEL1 gene comprises SEQ ID NO: 4 or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4.

[0122] There is also provided a method for amplifying and / or hybridizing genomic DNA (a part thereof) of melon plants, seeds or plant parts, the method comprising the following steps:

[0123] a) providing a sample (or samples) of genomic DNA of a melon plant or plants (e.g., an F2 population, an inbred line, a backcross population, a breeding population, a hybrid plant, etc.);

[0124] b) Provide at least one pair of PCR primers or at least one oligonucleotide probe, the primer or (oligonucleotide) probe comprising at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive nucleotides of the genomic CmBEL1 allele of the melon CmBEL1 gene, and capable of hybridizing to and / or amplifying a portion of the genomic allele in a PCR assay, and

[0125] c) Perform a PCR assay on the sample of step a) using the primer pair of step b) or a hybridization assay using the probe of step b) to generate a PCR amplification product and / or an oligonucleotide hybridization product, and optionally

[0126] d) Select a plant or plant part or seed comprising an allele (e.g., a wild-type allele and / or a mutant allele, especially a null allele) of the melon CmBEL1 gene having one or two copies in the genome,

[0127] wherein the wild-type allele of the melon CmBEL1 gene encodes the protein of SEQ ID NO: 1 (or a wild-type protein having at least 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 1), or

[0128] wherein the wild-type genomic allele of the melon CmBEL1 gene comprises SEQ ID NO: 4 or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEO ID NO: 4.

[0129] Also provided is a genotyping kit, the genotyping kit comprising primers and / or probes for amplifying and / or hybridizing a portion of the genomic DNA of the CmBEL1 gene and reaction components.

[0130] The primers and probes are preferably labeled or modified, for example, by a tail sequence or a label, to enable detection of the amplification or hybridization reaction product.

[0131] General definition

[0132] The verb "comprising" and its inflections are used in their non - limiting sense, meaning including the items following the word, but not excluding items not specifically mentioned. Further, the indefinite article "a" or "an" in reference to an element does not exclude the possibility of more than one element, unless the context clearly requires that there be one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one", for example, "a plant" also refers to several celled plants, etc. Similarly, "a fruit" or "a plant" also refers to several fruits and plants.

[0133] As used herein, the term "plant" includes a whole plant or any part or derivative thereof (preferably having the same genetic constitution as the plant from which it is obtained), such as plant organs (e.g., harvested or unharvested fruits, leaves, flowers, anthers, etc.), plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating a whole plant, plant callus, plant cell aggregates, plant grafts, seedlings, intact plant cells in a plant, plant clones or micropropagations, or plant parts, such as plant cuttings, embryos, pollen, anthers, ovules, fruits (e.g., harvested tissues or organs), flowers, leaves, seeds, clonally propagated plants, roots, stems, root tips, grafts (scions and / or rootstocks), etc. Also included are any developmental stages, such as seedlings, cuttings before or after rooting, etc. When referring to "seeds of a plant", these mean seeds from which a plant can grow or seeds produced on a plant after self - fertilization or cross - fertilization.

[0134] As used herein, the term "variety" or "cultivar" means a grouping of plants within a single botanical taxon of the lowest known rank, which can be defined by the expression of characteristics resulting from a given genotype or combination of genotypes. Plants characterized by the presence of an allele of a single gene, such as a mutant allele of the CmBEL1 gene, are not varieties or cultivars because the remainder of the genome has not been characterized.

[0135] The term "allele" means any one of one or more alternative forms of a gene at a particular locus, such as the CmBEL1 locus (the locus at which the CmBEL1 gene is located; the allele of this gene can be the wild-type allele named CmBEL1 or the mutant allele named cmbel1), and all alleles are associated with a trait or characteristic (e.g., seed abortion) at a particular locus. In the diploid cells of an organism, the alleles of a given gene are located at a specific position or locus (loci) on a chromosome. One allele is present on each chromosome of a homologous chromosome pair. A diploid plant species can contain a large number of different alleles at a particular locus. These can be the same alleles of a gene (homozygous) or two different alleles (heterozygous), e.g., two identical copies of the mutant cmbel1 allele (i.e., cmbel1 / cmbel1) or one copy of the mutant cmbel1 gene and one copy of the wild-type allele (i.e., cmbel1 / CmBEL1).

[0136] The "CmBEL1" gene is a single recessive gene identified in cultivated melons on chromosome 9, which, when mutated, causes seed abortion. CmBEL1 is the wild-type (WT) functional allele present in cultivated melon plants that do not have seed abortion, and cmbel1 is the mutant allele that causes seed abortion when the allele is in the homozygous form in diploid (cmbel1 / cmbel1) melons. In one aspect, the CmBEL1 gene is a gene encoding the protein of SEQ ID NO: 1 or a protein (melon) that encodes a protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 when compared pairwise. In one aspect, the CmBEL1 gene is a genomic allele comprising SEQ ID NO: 4 or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4. In one aspect, the CmBEL1 gene is a genomic allele that produces an mRNA / cDNA comprising SEQ ID NO: 2 or 3 or a wild-type mRNA / cDNA having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2 or 3.

[0137] "Seed abortion" is generally understood in the art and is also understood in the context of the present invention to mean that pollination is required to induce fruit set and development, but the fruit does not produce mature or viable seeds. Mature or viable seeds do not develop in plants with seed abortion due to, for example, arrested seed development, or ovule development, or degradation of the ovule and / or embryo and / or endosperm, or abortion of the ovule and / or embryo and / or endosperm before reaching maturity.

[0138] "F1, F2, F3, etc." refer to successive related generations after the cross between two parental plants or parental lines. A plant grown from a seed produced by crossing two plants or lines is referred to as the F1 generation. F1 plants self-pollinate to produce the F2 generation, etc.

[0139] An "F1 hybrid" plant (or F1 hybrid seed) is the generation obtained from crossing two inbred parental lines. Thus, an F1 hybrid seed is the seed from which an F1 hybrid plant grows. Due to heterosis, F1 hybrids are stronger and more productive. Inbred lines are substantially homozygous at most loci of the genome.

[0140] A "plant line" or "cultivar" refers to a plant and its progeny. As used herein, the term "inbred line" refers to a plant line that has been repeatedly self-pollinated and is nearly homozygous. Thus, an "inbred line" or "parental line" refers to a plant that has undergone several generations (e.g., at least 5, 6, 7 or more generations) of inbreeding, resulting in a plant line with a high degree of uniformity.

[0141] The term "gene" means a (genomic) DNA sequence that contains a region (transcribed region) that is transcribed into pre-mRNA and processed (by intron splicing) in the cell into a messenger RNA molecule (mRNA), and an operably linked regulatory region (e.g., a promoter). An example is the CmBEL1 gene of the present invention. Thus, different alleles of a gene are different alternative forms of the gene, which can be in the form of differences in one or more nucleotides in, for example, the genomic DNA sequence (e.g., promoter sequence, exon sequence, intron sequence, etc.), the mRNA, and / or the amino acid sequence of the encoded protein.

[0142] "Mutant cmbel1 allele" or "bel1 allele" as used herein refers to a mutant allele of the CmBEL1 gene on chromosome 9 in melon, which, when in homozygous form, causes the plant to be seed abortive. The mutation in the mutant allele can be any mutation or combination of mutations, including deletions, truncations, insertions, point mutations, nonsense mutations, missense mutations or non-synonymous mutations, splice site mutations, frameshift mutations, and / or mutations in one or more regulatory sequences such as promoter sequences or enhancer or silencer sequences. In one aspect, the mutant cmbel1 allele is a mutant allele of the CmBEL1 gene, wherein the CmBEL1 gene is a gene encoding the protein of SEQ ID NO: 1 or a gene encoding a protein having at least 94%, 95%, 96%, 97%, or 98% or 99% sequence identity (when compared pairwise) to SEQ ID NO: 1. In one aspect, the mutant cmbel1 allele is a mutant allele of a wild-type genomic allele comprising SEQ ID NO: 4 or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4. In one aspect, the mutant cmbel1 allele is a mutant allele of a wild-type genomic allele that produces an mRNA / cDNA comprising SEQ ID NO: 2 or 3 or a wild-type mRNA / cDNA transcript sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2 or 3.

[0143] "Wild-type CmBEL1 allele" or "BEL1 allele" as used herein refers to a functional allele of the CmBEL1 gene that enables a plant to have normal fruit set, such that normal pollination and fertilization are required for fruit set, and the fruit is seeded. The wild-type CmBEL1 allele is present in any commercial variety of melon (e.g., Nunhems varieties Magenta F1, Kirene F1, Coliseo F1, etc.). In one aspect, the wild-type CmBEL1 allele is the wild-type allele of the CmBEL1 gene, wherein the CmBEL1 gene is a gene encoding the protein of SEQ ID NO: 1 or a protein encoding a protein having at least 94%, 95%, 96%, 97%, or 98% sequence identity (when pairwise aligned) to SEQ ID NO: 1. In one aspect, the wild-type genomic CmBEL1 allele comprises SEQ ID NO: 4 or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4. In one aspect, the wild-type genomic allele gives rise to an mRNA / cDNA comprising SEQ ID NO: 2 or 3 or a wild-type mRNA / cDNA transcript sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2 or 3.

[0144] The term "locus" (loci) means one or more specific positions or sites on a chromosome, such as where a gene or genetic marker is found. Thus, the CmBEL1 locus is the location in the genome of melon where mutant alleles and / or wild-type alleles of the CmBEL1 gene are present. The CmBEL1 locus is a locus on chromosome 9 of cultivated melon (using the chromosome assignments of the publicly available melon genome (Garcia-Mas J et al. 2012, The genome of melon Cucumis melo L. PNAS 109:11872-11877), available at "Genome: melon (DHL92) v3.6.1" on cucurbitgenomics.org on the world wide web), i.e., cmbel1 was generated in the cultivated melon genome by mutagenesis, and the mutant cmbel1 allele was mapped to a defined region of chromosome 9 of cultivated melon.

[0145] An "induced mutant" allele is a mutant allele where the mutation has been induced by / through human intervention, such as by mutagenesis via physical or chemical mutagenesis methods or via, for example, tissue culture (as described, for example, in Zhang et al., P1os9(5)e96879) (also including targeted gene editing techniques such as Crispr-based techniques, TALENS, base editing, etc.) and insertional mutagenesis techniques such as targeted transposon insertion techniques, etc.) (for a review, see Gao et al. https: / / doi.org / 10.1016 / j.cell.2021.01.005).

[0146] A "diploid plant" refers to a plant, vegetative plant part, or seed having two sets of chromosomes from which a diploid plant can be grown, herein designated 2n.

[0147] A "DH plant" or "doubled haploid plant" is a diploid plant produced by doubling the haploid genome of a diploid plant using, for example, in vitro techniques. Thus, DH plants are homozygous at all loci.

[0148] A "seedless fruit" is commonly used in the art and should be understood in the context of the present invention as a fruit without mature or viable seeds. Sometimes empty seeds or tiny seeds may be seen. Mature or viable seeds can germinate in soil under conditions suitable for the corresponding plant and grow into a plant. Such a test can be used to determine whether a plant produces seedless fruit. A seedless fruit will not produce seeds that will germinate and grow into a plant under conditions suitable for the corresponding plant.

[0149] "Vegetative propagation" or "clonal propagation" refers to the propagation of plants from vegetative tissue, for example, by in vitro propagation or grafting methods (using scions and rootstocks). In vitro propagation involves in vitro cell or tissue culture and the regeneration of an entire plant from in vitro culture. Grafting involves the propagation of the original plant by grafting onto a rootstock. Thus, clones (i.e., genetically identical vegetative propagations) of the original plant can be generated by in vitro culture or grafting. "Cell culture" or "tissue culture" refers to the in vitro culture of plant cells or tissues. "Regeneration" refers to the development of a plant from cell culture or tissue culture or vegetative propagation. "Non-reproductive cells" refer to cells that cannot regenerate into a complete plant.

[0150] "Recessive" refers to an allele that expresses its phenotype (e.g., seed abortion) when the dominant allele is not present in the diploid genome, i.e., when the dominant allele is homozygous in the diploid. When present in two copies in a diploid plant, the mutant cmbel1 allele produces plants with seed abortion. The dominant allele is also referred to herein as the wild-type (WT) allele.

[0151] "Melon plant cell" or "melon plant" or "cultivated melon plant or cell" is also named as muskmelon plant cell or muskmelon plant in the art, and should be understood in the context of the present invention as a plant cell derived from the species Cucumis melo or a plant belonging to the species Cucumis melo. Melons can be classified as: cantaloupe, smooth-skinned melon, and netted melon. Cantaloupe is also known as honeydew melon, and is mainly round, with prominent ribs and almost no netting. Most have orange, sweet flesh, and they are usually very fragrant. Compared with European honeydew melons, the "honeydew melons" in North America are not of this type, but belong to the true muskmelon. Smooth-skinned melons (or winter melons) can be subdivided into different types, such as honey melons, Spanish melons, sugar melons, Japanese melons, etc. Netted melons are true muskmelons, with a netted skin (netted structure), and include Galia melons, Charentais melons, and North American honeydew melons.

[0152] Cultivated melons and wild melon relatives are diploid and have 12 pairs of homologous chromosomes, numbered 1 to 12.

[0153] "Cultivated melon plant" refers to a melon plant, i.e., a variety, breeding line, or cultivar of the species Cucumis melo, cultivated by humans and having good agronomic characteristics, especially producing edible and marketable fruits with good size, quality, and uniformity; preferably, such a plant is not a "wild melon plant", i.e., a plant that generally has a much lower yield and worse agronomic characteristics than cultivated plants and grows naturally, for example, in a wild population. "Wild melon plants" include, for example, ecotypes of a species, Plant Introduction (PI) lines, landraces, or wild accession or wild relatives.

[0154] "Pocket melon" or "mini melon" is a melon fruit produced by a plant with an average fruit weight of 1.0 kg or less, especially 0.9 kg or less, 0.8 kg or less, 0.7 kg or less, 0.6 kg or less, 0.5 kg or less, 0.4 kg or less.

[0155] "Medium-sized melon" or "personal-sized melon" is a melon fruit produced by a plant with an average fruit weight of 1.5 to 2.0 kg or more than 1.0 kg, for example, 1.0 to 1.5 kg or 1.0 to 2.0 kg.

[0156] "SNP marker" refers to, for example, a single nucleotide polymorphism between a mutant cmbel1 allele and a wild-type CmBEL1 allele. One can use an SNP marker assay (i.e., an allele-specific assay) that can distinguish the mutant allele of the CmBEL1 gene from the wild-type allele to screen pants, plant parts, or their DNA for the presence of the mutant allele and / or the wild-type allele. For any SNP marker, an SNP marker assay can be designed based on the sequences provided herein. Such SNP marker assays can be used to detect mutant alleles, for example, in marker-assisted selection and / or SNP genotyping assays. Thus, an SNP marker assay (e.g., in an allele-specific assay) that can distinguish the mutant and wild-type alleles of a gene is used to screen pants, plant parts, or their DNA for the presence of the mutant allele.

[0157] "INDEL marker" refers to, for example, an insertion / deletion polymorphism between a mutant cmbel1 allele and a wild-type CmBELl allele. An INDEL marker assay (e.g., an allele-specific assay) that can distinguish the mutant and wild-type alleles of a gene can be used to screen pants, plant parts, or their DNA for the presence of the mutant allele.

[0158] A "genotyping" method is a method by which the genotype or allelic composition of a plant or plant part or seed can be determined. A bi-allelic genotyping assay (such as a KASP assay) can distinguish the two alleles at a locus.

[0159] "Chromosomal region containing the mutant cmbel1 allele" refers to, for example, a genomic region of chromosome 9 of cultivated melon that carries the mutant cmbel1 allele. The presence of the allele can be determined phenotypically and / or by one or more molecular markers associated with the mutant cmbel1 allele, such as an SNP marker, an INDEL marker, or other markers, or preferably by the presence of markers that distinguish different cmbel1 alleles, or by the genomic sequence of the allele itself (e.g., sequencing the allele). A marker is "associated with the cmbel1 allele" if it is physically coupled to the cmbel1 allele. An "allele-specific marker" is specific for a particular allele (e.g., a particular mutant allele) and thus distinguishes, for example, a mutant allele from a wild-type allele. An allele-specific marker is preferably a marker in the allele itself, i.e., in the promoter region or the transcribed region of the gene, for example, based on the polymorphism between the wild-type allele sequence and the mutant allele sequence.

[0160] A pair of "flanking markers" refers to two markers, preferably two SNP markers or two sequences containing SNP markers, which are associated with and / or in close association with the cmbel1 allele, where the cmbel1 allele is located between the two markers or between the two sequences containing the marker.

[0161] "Brix" refers to the average total soluble solids content measured in several mature fruits using a refractometer. Preferably, the average is calculated for at least three fruits, each measured between the center and the peel of an open fruit.

[0162] The "physical distance" between loci on the same chromosome (e.g., between molecular markers and / or between phenotypic markers) is the actual distance expressed in bases or base pairs (bp), kilobases or kilobase pairs (kb), or megabases or megabase pairs (Mb).

[0163] The "genetic distance" between loci on the same chromosome (e.g., between molecular markers and / or between phenotypic markers) is measured by the frequency of crossing over or recombination frequency (RF) and is indicated in centimorgans (cM). One centimorgan corresponds to approximately 1% recombination frequency. If no recombinants can be found, the RF is zero and the loci are extremely close physically or they are identical. The farther apart two loci are, the higher the RF.

[0164] "Uniformity" or "uniform" relates to the genetic and phenotypic characteristics of a plant line or variety. Inbred lines are highly uniform genetically because they are produced by several generations of close inbreeding. Similarly, F1 hybrids produced from such inbred lines are highly uniform in their genotypic and phenotypic characteristics and performance.

[0165] A genetic element, introgression segment, or gene or allele that confers a trait (such as seed abortion) is said to be "able" or "capable" of being obtained from, or "derived" from, or "present in" or "found in" a plant or seed or tissue or cell if it can be transferred, using traditional breeding techniques, from the plant or seed in which it is present to another plant or seed (such as a non-seed abortion line or variety) in which it is not present, without causing a phenotypic change in the recipient plant other than the addition of the trait conferred by the genetic element, locus, introgression segment, gene, or allele. These terms are used interchangeably, and thus a genetic element, locus, introgression segment, gene, or allele can be transferred into any other genetic background lacking the trait. A cultivated melon containing a genetic element, locus, introgression segment, gene, or allele (such as the mutant cmbel1 allele) can be generated de novo, for example, by mutagenesis (such as chemical mutagenesis, CRISPR-Cas-induced mutagenesis, etc.) and then, for example, by crossing with other cultivated melons.

[0166] "Mean" or "average" as used herein refers to the arithmetic mean, and the two terms are used interchangeably. Thus, the term "mean" or "average" refers to the arithmetic mean of several measurements. One of ordinary skill in the art understands that the phenotype of a plant line or variety depends to some extent on growth conditions, and thus it is preferred to measure the arithmetic mean of at least 10, 15, 20, 30, 40, 50, or more plants (or plant parts) in a randomized experimental design with several replicates and in appropriate control plants grown under the same conditions in the same experiment. "Statistically significant" or "statistically significantly" different or "significantly" different refers to a characteristic of a plant line or variety that shows a statistically significant difference of that characteristic from the control (mean) (e.g., a p-value using ANOVA less than 0.05, p < 0.05) when compared to an appropriate control.

[0167] The term "traditional breeding techniques" as used herein encompasses hybridization, backcrossing, selfing, selection, doubled haploid production, chromosome doubling, embryo rescue, protoplast fusion, marker-assisted selection, mutation breeding, etc., all of which are known to breeders (i.e., methods other than genetic modification / transformation / transgenic methods) by which, for example, a chromosome containing the mutant cmbel1 allele can be obtained, identified, and / or transferred.

[0168] "Backcrossing" refers to a breeding method by which a (single) trait (such as the seed abortion trait) can be transferred from one (usually inferior) genetic background (also referred to as the "donor") to another (usually superior) genetic background (also referred to as the "recurrent parent"). The offspring of the cross (e.g., the F1 plants obtained by crossing, for example, the donor with the recurrent parent melon, or the F2 or F3 plants obtained by selfing the F1) are "backcrossed" to the parent with, for example, the superior genetic background. After repeated backcrossing, the traits of one (usually inferior) genetic background will be incorporated into another (usually superior) genetic background.

[0169] "Marker-assisted selection" or "MAS" is the process of using the presence of molecular markers (such as SNP markers or INDEL markers) to select plants for the presence of a specific genome or region or allele, which are genetically and physically associated with a specific locus or a specific chromosomal region or an allele-specific marker. For example, a molecular marker that is genetically and physically associated with the mutant cmbel1 allele or an allele-specific marker can be used to detect and / or select, for example, melon plants or plant parts that contain the cmbel1 allele. The closer the linkage of the molecular marker to the locus, the lower the probability that the marker will dissociate from the locus by meiotic recombination. Similarly, the closer two markers are associated with each other, the lower the probability that the two markers will separate from each other (and the higher the probability that they will co-segregate as a unit). Allele-specific markers are preferred markers because they directly select for the allele.

[0170] A molecular marker (or a sequence containing a molecular marker) that is 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb, 74 kb, 50 kb, 20 kb, 10 kb, 5 kb, 2 kb, 1 kb or less different from another marker (or a sequence containing a molecular marker) or locus refers to a marker that is physically located within a genomic DNA region 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb, 74 kb, 50 kb, 20 kb, 10 kb, 5 kb, 2 kb, 1 kb or less flanking the marker (i.e., on either side of the marker).

[0171] "LOD score" (logarithm (base 10) of odds) is a statistical test commonly used in linkage analysis in animal and plant populations. The LOD score compares the likelihood of obtaining test data if two loci (a molecular marker locus and / or a phenotypic trait locus) are truly associated with the likelihood of observing the same data purely by chance. A positive LOD score favors the existence of linkage, and a LOD score greater than 3.0 is considered proof of linkage. A LOD score of +3 indicates 1000-to-1 odds that the observed linkage did not occur by chance.

[0172] "Transgene" or "chimeric gene" refers to a genetic locus containing a DNA sequence (such as a recombinant gene) that has been introduced into the genome of a plant by transformation (such as Agrobacterium-mediated transformation). A plant containing a transgene stably integrated into its genome is called a "transgenic plant".

[0173] "Isolated nucleic acid sequence" or "isolated DNA" refers to a nucleic acid sequence that no longer exists in its natural environment from which it was isolated, such as a nucleic acid sequence in a bacterial host cell or in a plant nuclear or plastid genome. When a "sequence" is referred to herein, it is understood that a molecule having such a sequence refers to, for example, a nucleic acid molecule.

[0174] "Host cell" or "recombinant host cell" or "transformed cell" is a term for a new single cell (or organism) that results from the introduction of at least one nucleic acid molecule into the cell. The host cell is preferably a plant cell or a bacterial cell. The host cell may contain the nucleic acid as an extrachromosomal (episomal) repeating molecule, or contain the nucleic acid integrated into the nuclear or plastid genome of the host cell, or the nucleic acid as an introduced chromosome (such as a minichromosome).

[0175] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms. When sequences are optimally aligned by, for example, the programs GAP or BESTFIT or the Emboss program "Needle" (using default parameters, see below), the sequences can be said to be "substantially identical" or "substantially similar", sharing at least a certain minimum percentage of sequence identity (as further defined below). These programs use the Needleman and Wunsch global alignment algorithm to align two sequences over the entire length of the two sequences, maximizing the number of matches and minimizing the number of gaps. Typically, using default parameters, where the gap creation penalty = 10 and the gap extension penalty = 0.5 (for both nucleotide and protein alignments). For nucleotides, the default scoring matrix used is DNAFULL, and for proteins, the default scoring matrix is Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, 10915-10919). Scoring of sequence alignments and sequence identity percentages can be determined, for example, using a computer program such as EMBOSS, which is available on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / . Alternatively, sequence similarity or identity can be determined by searching databases such as FASTA, BLAST, etc., but hits should be retrieved and pairwise aligned to compare sequence identity. Two proteins or two protein domains or two nucleic acid sequences have "substantially sequence identity" (as determined by Emboss "needle" using default parameters (i.e., gap creation penalty = 10, gap extension penalty = 0.5), scoring matrix DNAFULL for nucleic acids and Blosum62 for proteins) if the sequence identity percentage is at least 85%, 90%, 92%, 93%, 94%, 95%, 98%, 99% or more.

[0176] When referring to nucleic acid sequences (e.g., DNA or genomic DNA) having "substantially sequence identity" to a reference sequence or having at least 80%, e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, 99.2%, 99.5%, 99.9% nucleic acid sequence identity to a reference sequence, in one embodiment, the nucleotide sequence is considered to be substantially identical to the given nucleotide sequence and can be identified using stringent hybridization conditions. In another embodiment, the nucleic acid sequence contains one or more mutations compared to the given nucleotide sequence, but can still be identified using stringent hybridization conditions.

[0177] "Stringent hybridization conditions" can be used to identify nucleotide sequences that are substantially identical to a given nucleotide sequence. Stringent conditions depend on the sequence and will vary in different circumstances. Typically, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature at which (at a defined ionic strength and pH) 50% of the target sequence hybridizes to a perfectly matched probe. Stringent conditions are typically selected where the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Decreasing the salt concentration and / or increasing the temperature increases the stringency. Stringent conditions for RNA-DNA hybridization (e.g., Northern blotting using a probe of, for example, 100 nt) are, for example, those that include at least one wash for 20 minutes in 0.2X SSC at 63°C, or equivalent conditions. Stringent conditions for DNA-DNA hybridization (e.g., Southern blotting using a probe of, for example, 100 nt) are, for example, those that include at least one (usually 2) wash for 20 minutes in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, or equivalent conditions.

[0178] In the context of the present invention, "M1 generation" or "M1 plant" refers to the first generation directly resulting from mutagenic treatment. For example, a plant grown from a seed treated with a mutagen is representative of the M1 generation.

[0179] "M2 generation" or "M2 plant" as used herein refers to the generation obtained from self-pollination of the M1 generation. A plant grown from a seed obtained from a self-pollinated M1 plant represents an M2 plant. M3, M4, etc. refer to other generations obtained after self-pollination.

[0180] "mRNA coding sequence" or "mRNA sequence" shall have its ordinary meaning herein. The mRNA coding sequence corresponds to the corresponding DNA coding (cDNA) sequence of the gene / allele, except that thymine (T) is replaced by uracil (U).

[0181] A "mutation" in a nucleic acid molecule (DNA or RNA) is a change of one or more nucleotides compared to the corresponding wild-type sequence, for example, by substitution, deletion, or insertion of one or more nucleotides. Examples of such mutations are point mutations, nonsense mutations, missense mutations, splice site mutations, frameshift mutations, or mutations in regulatory sequences.

[0182] "Nucleic acid molecule" has its ordinary understanding in the art. It is composed of nucleotides containing either deoxyribose (DNA) or ribose (RNA).

[0183] A "point mutation" is a substitution of a single nucleotide, or an insertion or deletion of a single nucleotide.

[0184] "Nonsense mutation" is a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon in the nucleic acid molecule is changed to a stop codon. This results in the presence of a premature stop codon in the mRNA and leads to the translation of a truncated protein. The truncated protein can have reduced function or loss of function.

[0185] "Missense or non-synonymous mutation" is a (point) mutation in a nucleic acid sequence encoding a protein, whereby the codon is changed to encode a different amino acid. The resulting protein can have reduced function or loss of function.

[0186] "Splice site mutation" is a mutation in a nucleic acid sequence encoding a protein, whereby the RNA splicing of the pre-mRNA is altered, resulting in an mRNA with a nucleotide sequence different from the wild type and a protein with an amino acid sequence different from the wild type. The resulting protein can have reduced function or loss of function.

[0187] "Frameshift mutation" is a mutation in a nucleic acid sequence encoding a protein, by which the reading frame of the mRNA is altered, resulting in a different amino acid sequence. The resulting protein can have reduced function or loss of function.

[0188] "Deletion" in the context of the present invention means the deletion of at least one nucleotide anywhere in a given nucleic acid sequence compared to the nucleic acid sequence of the corresponding wild type sequence, or the deletion of at least one amino acid anywhere in a given amino acid sequence compared to the amino acid sequence of the corresponding (wild type) sequence.

[0189] "Truncated" should be understood to mean the deletion of at least one nucleotide at the 3' or 5' end of the nucleotide sequence compared to the nucleic acid sequence of the corresponding wild type sequence, or the deletion of at least one amino acid at the N-terminus or C-terminus of the protein compared to the amino acid sequence of the corresponding wild type protein, but preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids. The 5' end is determined by the ATG codon that serves as the start codon in the translation of the corresponding wild type nucleic acid sequence.

[0190] "Substitution" means that at least one nucleotide in a nucleic acid sequence or one amino acid in a protein sequence is different compared to the corresponding wild type nucleic acid sequence or the corresponding wild type amino acid sequence, respectively, due to, for example, the exchange of nucleotides in the coding sequence of the corresponding protein.

[0191] "Insertion" means that a nucleic acid sequence or the amino acid sequence of a protein contains at least one additional nucleotide or amino acid, respectively, compared to the corresponding wild type nucleic acid sequence or the corresponding wild type amino acid sequence.

[0192] An "early termination codon" in the context of the present invention means that a termination codon is present in the coding sequence (cds) closer to the 5' - terminal start codon compared to the termination codon of the corresponding wild - type coding sequence.

[0193] A "mutation in a regulatory sequence", such as a mutation in the promoter or enhancer of a gene, is an alteration of one or more nucleotides compared to the wild - type sequence, for example, by substitution, deletion, or insertion of one or more nucleotides, resulting in, for example, reduced or no production of the gene's mRNA transcript.

[0194] A "mutation in a protein" is an alteration of one or more amino acid residues compared to the wild - type sequence, for example, by substitution, deletion, truncation, or insertion of one or more amino acid residues.

[0195] A "mutant protein" herein is a protein that contains one or more mutations in the nucleic acid sequence encoding the protein, whereby the mutation results in a protein with "reduced function" or "loss of function", such as a protein with a measurable "reduced function" or "loss of function" in vivo, for example, through the phenotype conferred by the mutant allele (the encoded mutant nucleic acid molecule).

[0196] In the context of the present invention, "loss of function" of a protein means the loss of the normal in - vivo function of the CmBEL1 protein when compared to the corresponding wild - type plant cell or the corresponding wild - type plant. "Loss of function" can be distinguished from "reduced function" or "decreased function" of a protein, where a protein with reduced (or decreased) function still has some residual in - vivo function, although less than the wild - type protein.

[0197] A "null allele" is a non - functional allele. It results in no production of the wild - type gene product, for example, through a mutation in a regulatory element such as a promoter (e.g., gene expression knockout), or produces a gene product that has lost its function, i.e., a loss - of - function protein. A complete deletion of a gene has the same phenotypic effect as a "null allele" and is also encompassed in one aspect herein.

[0198] In the context of the present invention, the terms "wild - type plant cell" or "wild - type plant" mean that they contain the wild - type CmBEL1 allele and not the mutant cmbel1 allele. Thus, a wild - type plant or wild - type plant cell is a plant or plant cell that contains a fully functional CmBEL1 gene, which encodes a fully functional CmBEL1 protein (also called the wild - type CmBEL1 protein). For example, in the case of a melon plant or plant cell, a diploid melon plant produces the protein of SEQ ID NO: 1 (or a protein having at least 94% sequence identity with SEQ ID NO: 1) after pollination and produces seeded fruits.

[0199] "Knockout" or "complete knockout" should be understood as the expression of the corresponding gene being no longer detectable.

[0200] "Conserved domain" refers to a conserved protein domain, such as the homologous domain of SEQ ID NO: 1, which starts at amino acid 318 and ends at amino acid 379 (or equivalent amino acids in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1). Conserved domains can be found, for example, in the Conserved Domain Database of NCBI (ncbi.nlm.nih.gov / cdd on the World Wide Web).

[0201] "Targeted gene editing" refers to techniques by which endogenous target genes can be modified, for example, one or more nucleotides can be inserted, replaced and / or deleted in, for example, the promoter or coding sequence or transcription region of a gene. For example, CRISPR-based techniques, such as Crispr-Cas9 gene editing, Crispr-CpfI gene editing or more recent techniques known as "base editing" or "prime editing", can be used to modify endogenous target genes, such as the endogenous wild-type CmBEL1 gene in melon (encoding the protein of SEQ ID NO: 1 or a wild-type protein having at least 94% sequence identity with SEQ ID NO: 1). The mutants described herein can be replicated, for example, by targeted gene editing of the wild-type CmBEL1 gene.

[0202] "Oligonucleotides (oligonucleotides / oligos)" or "oligonucleotide primers or probes" are short single-stranded nucleic acid polymers, such as at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more nucleotides in length. Oligonucleotides can be unmodified or chemically modified in various ways, depending on their intended use, such as adding 5′ or 3′ phosphate groups to effect ligation or block extension respectively, labeling with radionuclides or fluorophores and / or quenchers for use as probes, incorporating thiols, amines or other reactive moieties for covalent coupling of functional molecules such as enzymes, and extending with other linkers and spacers of different functionality. DNA oligonucleotides are the most commonly used, but RNA oligonucleotides are also available. The length of an oligonucleotide is usually named by adding the suffix -mer. For example, an oligonucleotide with 19 nucleotides (bases) is called a 19-mer. For most applications, oligonucleotides are designed to base pair with DNA or RNA strands. Oligonucleotides are most commonly used as primers for PCR (polymerase chain reaction). Primers are designed so that at least a portion of their sequence is complementary to the sequence targeted for amplification. The optimal primer length for the complementary sequence is, for example, 18 to 22 nucleotides. The optimal primer sequence for PCR is usually determined by primer design software.

[0203] A "DNA microarray" is an array of microscopic spots with many DNAs (usually oligonucleotides) bound to a solid support. The assay target can be DNA, cDNA or cRNA. Depending on the system, hybridization of the target to a specific spot is detected by fluorescence, chemiluminescence or colloidal silver or gold. Microarrays are used for a variety of applications, such as the simultaneous measurement of the expression of large numbers of genes, enabling whole-genome gene expression analysis, and genotyping studies using, for example, single nucleotide polymorphisms (SNPs) or InDel analysis.

[0204] A "complementary strand" refers to the two strands of a complementary sequence and can be referred to as the sense (or positive) and antisense (or negative) strands for double-stranded DNA. The sense / positive strand is usually the transcribed sequence of DNA (or the mRNA generated during transcription), while the antisense / negative strand is the strand complementary to the sense sequence. For any sequence provided herein, only one strand of the sequence is given, but the complementary strand of the given strand is also covered herein. The complementary nucleotides of DNA are A that is complementary to T and G that is complementary to C. The complementary nucleotides of RNA are A that is complementary to U and G that is complementary to C. BRIEF DESCRIPTION OF THE DRAWINGS

[0205] Figure 1: Displays the wild-type CmBEL1 protein of SEQ ID NO: 1. The vertical black lines demarcate the amino acids encoded by exon 1, exon 2, exon 3, and exon 4 of the transcript. The black star at the second vertical black line (separating the amino acids encoded by exon 2 and exon 3) shows the effect of the mutant cmbel1 allele, which contains a DNA insert in intron 2, resulting in an mRNA transcript with a transcription region containing only exons 1 and 2, and thus producing a truncated protein containing only amino acids 1 to 343 of SEQ ID NO: 1. The black box highlights the conserved homology domain.

[0206] Figure 2 : Seedless melon fruits homozygous for the mutant cmbel1 allele.

[0207] Figure 3 : Seedless and substantially empty seed cavity-free melon fruits homozygous for the mutant cmbel1 allele.

[0208] Figure 4 : Seedless and substantially empty seed cavity-free melon fruits homozygous for the mutant cmbel1 allele.

[0209] Figure 5 : Seedless and having some empty seed cavities (Spanish melons) melon fruits homozygous for the mutant cmbel1 allele.

[0210] Figure 6 : RT-PCR performed with primers spanning the cDNA region corresponding to exons 2 and 3 produced a PCR product in wild-type (seed-bearing) plants but not in mutant (seedless) plants, indicating that the mRNA transcript in mutant plants terminates after exon 2.

[0211] Figure 7: Genomic DNA encoding wild-type CmBEL1 protein (SEQ ID NO: 4). Exons are underlined (there are 4 exons, exon 1 from nucleotide 1 to nucleotide 645, exon 2 from 957 to 1340, exon 3 from 1886 to 1945, exon 4 from 2380 to 3117; there are three introns, intron 1 from nucleotide 646 to 956, intron 2 from 1341 to 1885, intron 3 from 1946 to 2379). Bold codons encode conserved homologous domains. The boxed codons are TGG at nucleotides 1263 to 1265, which encodes the first amino acid of the homologous domain (W318 of SEQ ID NO: 1); and ATG at nucleotides 2425 to 2427, which encodes the last amino acid of the homologous domain (M379 of SEQ ID NO: 1). The vertical black line in intron 2 shows the position where the mutant cmbel1 allele contains a DNA insert (between nucleotides 1655 and 1656), resulting in an mRNA transcript lacking the regions of exons 3 and 4.

[0212] Figure 8 : Cantaloupe F2 plants were grown in the field, and the number of fruits and the fruit weight (grams) were counted and measured in wild-type plants and homozygous mutant plants. Wild-type plants had approximately 3 to 4 fruits weighing 1600 - 3000 g, while cmbel1 mutant plants (containing the mutant allele in homozygous form) had approximately 7 to 14 fruits weighing approximately 700 - 1000 g.

[0213] Figure 9 : Normal-seeded cantaloupe fruits (left) with wild-type CmBEL1 allele and seedless fruits (containing mutant cmbel1 allele) (right), where the seedless fruits contain tiny seeds. Detailed Description of the Invention

[0214] A first embodiment of the present invention relates to a cultivated melon plant, Cucumis melo, which contains at least one copy of a mutant allele of a gene that, when in homozygous form, confers seed abortion, and the cultivated melon plant optionally has an increased average number of fruits and / or a decreased average fruit weight (compared to wild-type plants containing the wild-type allele of the gene). Thus, in one aspect, there is provided a cultivated melon plant that contains at least one copy of a mutant allele of a single recessive gene called CmBEL1.

[0215] The CmBEL1 gene is an endogenous gene of cultivated melons, and when mutated and in homozygous form, it causes seed abortion.

[0216] The segregating population created by crossing the generated mutant seed-aborting melon plants with a melon line enabled the mapping of the CmBEL1 gene to a region on chromosome 9. Further fine mapping led to the identification of the gene containing the mutation that causes premature termination of the mRNA transcript and truncation of the protein encoded after amino acid P343 of SEQ ID NO: 1.

[0217] Since 265 of the 608 amino acids are missing in the truncated protein (including 36 amino acids of the conserved homeodomain required for protein function), it was concluded that this truncation of the CmBEL1 protein results in the protein being non-functional in vivo. Thus, plants homozygous for this mutant protein (and thus lacking the functional wild-type protein) develop seedless fruits. The average fruit weight is significantly reduced to less than half the weight of the recurrent parent, while the average fruit number is significantly increased, for example, by about two-fold compared to the recurrent parent. See Figure 8 。

[0218] In one aspect, there is provided a melon plant or plant part that contains at least one copy of a mutant allele of a gene named CmBEL1, wherein the mutant allele

[0219] a) contains one or more mutations in regulatory elements, particularly in a promoter, resulting in non-expression (or alternatively, reduced expression) of the allele compared to the wild-type allele, and / or

[0220] b) encodes a mutant protein containing one or more amino acid substitutions, insertions, or deletions compared to the wild-type protein, particularly a non-functional protein,

[0221] wherein when the mutant allele of a) or b) is in homozygous form, the mutant allele confers seed abortion, and wherein the wild-type melon allele encodes the protein of SEQ ID NO: 1 or a protein having at least 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with SEQ ID NO: 1.

[0222] The wild-type functional CmBEL1 protein of melon is provided in SEQ ID NO: 1. However, there may be some amino acid sequence variants within melon, and the functional CmBEL1 protein can contain, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids different from SEQ ID NO: 1 provided herein, or wherein the protein has at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein of SEQ ID NO: 1 (when using pairwise alignment such as Emboss-Needle).

[0223] Accordingly, in one aspect, a functional variant of the melon protein of SEQ ID NO: 1 (also referred to herein as "variant CmBEL1 protein") is a protein that has at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein of SEQ ID NO: 1 when subjected to pairwise alignment (using, for example, Needle with default parameters). In one aspect, the amino acid sequence variants are outside of the conserved homologous domain. Accordingly, in one aspect, a functional protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein of SEQ ID NO: 1 contains amino acids that are 100% identical to the homologous domain of SEQ ID NO: 1, which homologous domain starts at amino acid 318 of SEQ ID NO: 1 and ends at amino acid 379 of SEQ ID NO: 1, also see Figure 1 (boxed amino acids).

[0224] Since the homologous domain is highly conserved within a species, any mutations (deletions, insertions and / or substitutions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 36 or more amino acids) in the homologous domain are predicted to result in the mutant CmBEL1 protein being non-functional in vivo, and thus, when the mutant allele is in homozygous form in a diploid plant, results in a seed abortion phenotype.

[0225] Accordingly, inserting, deleting and / or substituting one or more amino acids in the homologous domain will have a negative impact on protein function, especially rendering the protein non-functional in vivo.

[0226] Thus, in one aspect, there is provided a melon plant or plant part comprising at least one copy of a mutant allele of a gene named CmBEL1, wherein the mutant allele encodes a mutant protein as follows: one or more amino acids containing an insertion, deletion or substitution in the homeodomain of a protein starting at amino acid 318 of SEQ ID NO: 1 and ending at amino acid 379, or equivalent amino acids in a variant CmBEL1 protein having at least 94% sequence identity with SEQ ID NO: 1, and wherein when the mutant allele is in homozygous form, the mutant allele confers seed abortion.

[0227] The terms "starting at" and "ending at" or "from" and "to" include the first and last amino acids mentioned.

[0228] Thus, the insertion, deletion and / or substitution of one or more amino acids in the homeodomain can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40 or more amino acids or more of the homeodomain, i.e., the insertion, deletion and / or substitution of amino acids 318 to 379 of SEQ ID NO: 1, or equivalent amino acids in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1.

[0229] In yet another aspect, there is provided a melon plant or plant part comprising at least one copy of a mutant allele of the gene named CmBEL1, wherein the mutant allele encodes a mutant protein as follows: comprising an insertion, deletion, and / or substitution of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 260, 264, 265, 266 or more amino acids in SEQ ID NO: 1 or in a variant CmBEL1 protein or a protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, and wherein when the mutant allele is in homozygous form, the mutant allele confers seed abortion. Thus, the mutant CmBEL1 protein can be truncated, for example, at the N-terminus or the C-terminus, thereby lacking said at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 260, 264, 265, 266 or more amino acids at the N-terminus or the C-terminus, or any other at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 260, 264, 265, 266 amino acids can be deleted, substituted or inserted compared to the wild-type functional CmBEL1 protein. As mentioned, the mutant protein is preferably non-functional in vivo and thus confers a seed abortion phenotype when the mutant allele encoding the mutant protein is in homozygous form.

[0230] In yet another aspect, a melon plant or plant part is provided that comprises at least one copy of a mutant allele of a gene named CmBEL1, wherein the mutant allele comprises one or more nucleotides inserted, deleted or substituted in SEQ ID NO: 4 (or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 4), and wherein when the mutant allele is in homozygous form, the mutant allele confers seed abortion. In one aspect, the mutant CmBEL1 genomic sequence is a null allele. It may encode a loss-of-function protein, which may be truncated, for example, at the N-terminus or C-terminus, thereby lacking said at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 260, 264, 265, 266 or more amino acids at the N-terminus or C-terminus, or it may be a mutant protein, wherein compared to the wild-type functional CmBEL1 protein, any at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 260, 264, 265, 266 amino acids may be deleted, substituted or inserted. As mentioned, the mutant allele is preferably a null allele in vivo and thus confers a seed abortion phenotype when the mutant allele is in homozygous form.

[0231] In yet another aspect, there is provided a melon plant or plant part that comprises at least one copy of a mutant allele of a gene named CmBEL1, wherein the mutant allele encodes a mutant protein as follows: at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 or more amino acids comprising insertions, deletions and / or substitutions in SEQ ID NO: 1 or in a variant CmBEL1 protein or a protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, and wherein when the mutant allele is in homozygous form, the mutant allele confers seed abortion. Thus, compared to the wild-type functional CmBEL1 protein, the mutant CmBEL1 protein can comprise at least 1 amino acid of deletion, substitution and / or insertion. For example, the amino acid of deletion or substitution (e.g., by a stop codon or by a different amino acid) can be P343* or Y344*, or any amino acid of the homologous domain can be substituted by another amino acid or by a stop codon. As mentioned, the mutant protein is preferably non-functional in vivo, and thus when the mutant allele encoding the mutant protein is in homozygous form, it confers a seed abortion phenotype.

[0232] Mutant alleles can be generated by various techniques such as random mutagenesis or targeted gene editing, and then the phenotype of the mutant allele can be analyzed in plants homozygous for the mutant allele.

[0233] Any mutant allele that results in an insertion, deletion and / or substitution of one or more amino acids of the wild-type functional protein can produce a mutant protein with reduced or no function, and thus can result in a seed abortion phenotype when the mutant allele is in homozygous form. Plants and plant parts comprising such mutant alleles are an embodiment herein.

[0234] "Equivalent amino acids" can be readily determined by amino acid sequence alignment.

[0235] In one aspect, the amino acid deletions, insertions and / or substitutions in the mutant protein are attributed to mutations in the codons of the CmBEL1 gene.

[0236] The mutation in the codon can be an (at least one) nucleotide insertion, deletion or substitution in the codon, resulting in, for example, a different reading frame or a different codon, thus, for example, encoding a different amino acid or a stop codon. An entire codon can also be deleted or replaced by a different codon (or optionally a stop codon), resulting in a deletion or substitution of the encoded amino acid.

[0237] In one aspect, the mutant allele encodes a mutant CmBEL1 protein that comprises one (or more) amino acids selected from amino acid numbers W318, R319, P320, Q321, R322, G323, L324, P325, E326, R327, S328, V329, S330, V331, L332, R333, A334, W335, L336, F337, E338, H339, F340, L341, H342, P343, Y344, P345, S346, D347, V348, D349, K350, H351, I352, L353, A354, R355, Q356, T357, G358, L359, S360, R361, S362, Q363, V364, S365, N366, W367, F368, I369, N370, A371, R372, V373, R374, L375, W376, K377, P378 or M379 of SEQ ID NO: 1, or an amino acid substitution, deletion or stop codon of an equivalent amino acid in a protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0238] In one aspect, the mutant allele encodes a mutant CmBEL1 protein that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 213, 225, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267,

[0241] truncation of 268, 269, 270 amino acids of the C-terminus of the protein of SEQ ID NO: 1 or the C-terminus of a protein having at least 94% sequence identity to SEQ ID NO: 1.

[0242] In one aspect, all amino acid deletions or substitutions with one or more different amino acids start from (and include) an amino acid selected from amino acid numbers W318, R319, P320, Q321, R322, G323, L324, P325, E326, R327, S328, V329, S330, V331, L332, R333, A334, W335, L336, F337, E338, H339, F340, L341, H342, P343, Y344, P345, S346, D347, V348, D349, K350, H351, I352, L353, A354, R355, Q356, T357, G358, L359, S360, R361, S362, Q363, V364, S365, N366, W367, F368, I369, N370, A371, R372, V373, R374, L375, W376, K377, P378 or M379 of SEQ ID NO: 1, or equivalent amino acids in a protein having at least 94% sequence identity with SEQ ID NO: 1. "Starting from" means that the mentioned amino acid is deleted or substituted, and the amino acids after the mentioned amino acid (towards the C-terminus) are also deleted or substituted.

[0243] In another aspect, one or more, for example at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or more nucleotides are inserted, deleted or substituted in the genomic sequence of SEQ ID NO: 4 (or Figure 7 ), where a functional wild-type protein is not produced by the mutant allele, for example the mRNA transcript is truncated and / or the encoded protein is truncated. In one aspect, the one or more nucleotides are transposable elements integrated into the genomic sequence of SEQ ID NO: 4. In another aspect, the one or more nucleotides are inserted, deleted or substituted by a targeted gene editing method, such as a Crispr-based method. In a specific aspect, the one or more nucleotides are inserted, deleted or substituted in the exon region or intron region of SEQ ID NO: 4, especially in exon 1 or exon 2 or exon 3, or in intron 1, intron 2 or intron 3 (see Figure 7 ). In one aspect, the one or more nucleotides are inserted into intron 2, for example between nucleotides 1655 and 1656 of SEQ ID NO: 4.

[0244] In one aspect, the one or more nucleotides inserted into SEQ ID NO: 4 comprise at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or more (e.g., all) nucleotides of SEQ ID NO: 5.

[0245] The transposable element can be a melon transposable element, such as that described in Morata et al 2018, Genome Biol. Evol. 10(6):1584-1595. doi:10.1093 / gbe / evy115.

[0246] As mentioned, a melon plant or seed or plant part can comprise a mutant cmbel1 allele, where the mutant allele is generated by random mutagenesis or targeted mutagenesis, such as a CRISPR-based method. Random mutagenesis can be, for example, chemically induced (e.g., EMS treatment) or radiation-induced mutagenesis or other methods by which mutations are randomly induced in the genome and then plants or plant parts containing mutations in the endogenous cmbel1 gene can be screened and identified. Targeted mutagenesis is a method in which mutations are specifically introduced into a target gene, such as the cmbel1 gene, using, for example, Crispr-Cas9 or Crispr-CpfI or other known methods, such as targeted transposon insertion. It should be noted that using such methods, mutant alleles such as those described in the examples can be made without undue burden, or other mutant alleles can be made.

[0247] When a melon plant is referred to herein, in one aspect, this encompasses a seed from which a plant can grow, i.e., the embryo in the seed can comprise at least one copy (or two copies) of the described mutant cmbel1 allele.

[0248] In one aspect, a plant containing a mutant allele is not produced solely by substantially biological processes, which means that the mutant allele is generated at a certain point by human intervention. If such a human-generated mutant allele is transferred from one plant to another by crossing and selection, the patent covers the plant containing the mutant allele, even if the plant itself is generated only by crossing and selection. Preferably, the plant is not transgenic, and in the case of, for example, targeted gene editing, any construct used to modify the endogenous gene has been removed from the genome. Additionally, the plant is preferably not a transgenic plant because the mutant cmbel1 allele has not been introduced from the outside and is not integrated anywhere in the plant genome using plant transformation techniques, but the mutant allele is the endogenous wild-type CmBEL1 allele that has been mutated at the locus in the genome where the wild-type allele is located (using, for example, targeted or random mutagenesis or transposon insertion or DNA insertion).

[0249] In one aspect, the melon plant is diploid and contains at least one copy of the mutant cmbel1 allele described above, i.e., the plant is heterozygous. Since the phenotype is only seen when the mutant allele is in the homozygous form, these plants are not seed abortive but produce normal-seeded fruits after pollination. Self-crossing of such heterozygous plants will generate plants that are homozygous and contain two copies of the mutant allele. In one aspect, the melon plant is diploid and contains two copies of the mutant cmbel1 allele described above, i.e., the plant is homozygous. Therefore, the plant is seed abortive and produces seedless fruits after pollination (which induces fruit set and development).

[0250] Plants and plant parts containing at least one copy of the mutant cmbel1 allele are preferably cultivated plants, not wild plants. Thus, preferably, cultivated melons (Cucumis melo). The plant can be an inbred line, an F1 hybrid, or a breeding line.

[0251] Also covered herein are seeds that can grow the above-described plants or plant parts. In one aspect, the seeds grow into plants containing one copy of the mutant allele. In another aspect, the seeds grow into plants containing two copies of the mutant allele.

[0252] Similarly, covered herein are fruits produced by the plants described above, optionally wherein the fruit is seedless and is produced by a plant containing two copies of the mutant allele. Since the pulp of the seedless fruit develops from maternal tissue, the pulp also contains two copies of the mutant allele.

[0253] The plant part can be a cell, flower, leaf, stem, cutting, ovule, pollen, root, rootstock, scion, fruit, protoplast, embryo, anther.

[0254] In addition, a vegetatively propagated plant is provided that is propagated from a plant part and contains at least one copy (or two copies) of a mutant cmbel1 allele in its genome.

[0255] In one aspect, a method of producing seedless melon fruit is also provided, the method comprising growing a melon plant containing two copies of the described mutant cmbel1 allele, thereby allowing pollination to occur and allowing seedless fruit to develop.

[0256] A method is provided for screening or detecting or genotyping plants, seeds, plant parts or their DNA for the presence of a mutant allele of the gene named CmBEL1, or for selecting plants, seeds or plant parts containing a mutant allele of the gene named CmBEL1, or for generating plants, seeds or plant parts containing a mutant allele of the gene named CmBEL1, wherein the mutant allele

[0257] a) contains one or more mutations in a regulatory element, particularly in a promoter, resulting in non-expression of the allele (or reduced allele expression) compared to the wild-type allele, and / or

[0258] b) encodes a mutant protein containing one or more amino acid substitutions, insertions and / or deletions compared to the wild-type protein, particularly a non-functional protein,

[0259] wherein the wild-type allele encodes the protein of SEQ ID NO: 1 or a protein having at least 94% sequence identity with SEQ ID NO: 1, or

[0260] wherein the wild-type genomic allele contains SEQ ID NO: 4 or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4.

[0261] In one aspect, the mutant allele is a null allele.

[0262] In one aspect, the mutant cmbel1 allele contains a mutation in the genomic DNA, resulting in the expression of a mutant CmBEL1 protein containing one or more amino acids with the insertions, deletions or substitutions described above.

[0263] In one aspect, the mutation is an insertion, deletion, and / or substitution of one or more nucleotides in the wild-type CmBEL1 allele such that the mutant cmbel1 allele is a null allele. For example, the one or more nucleotides inserted, deleted, and / or substituted can be in the promoter region (i.e., 5′ of the genomic sequence of SEQ ID NO: 4) before SEQ ID NO: 4, such as in SEQ ID NO: 6 (or in a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6), where for example the promoter is no longer active. Alternatively, the one or more nucleotides inserted, deleted, and / or substituted can be in the genomic sequence of SEQ ID NO: 4 (or in a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4), and / or in the mRNA of SEQ ID NO: 3 (or in a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3), and / or in the cDNA of SEQ ID NO: 2 (or in a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2).

[0264] In one aspect, any null allele that results in seed abortion when in homozygous form is an embodiment of the present invention. Different mutant alleles can be generated by a person skilled in the art without undue burden. The person skilled in the art can generate mutants, for example, in the promoter region or the transcribed region of the CmBEL1 allele, and determine whether the plant is seed abortive when the mutant allele is in homozygous form in a melon plant.

[0265] Also covered herein is a deletion of all or part of the CmBEL1 gene, as the deletion will produce the same phenotype as the null allele.

[0266] After identifying the nucleotide sequence of the gene, a technician can generate melon plants containing CmBEL1 gene mutants by various methods, such as mutagenesis, TILLING or CRISPR-Cas or other methods known in the art. In particular, when using targeted gene modification techniques, such as Crispr-Cas, TALENS, etc., a person skilled in the art can introduce targeted mutations in, for example, the promoter or transcriptional sequence or coding sequence of the gene. Then, the technician can confirm the phenotype of the plants that are homozygous for the mutant cmbel1 allele, i.e., plants with seed abortion. Thus, the technician is not limited to the specific cmbel1 mutants generated by the inventors (the technician can also generate them), but the technician can equally introduce other mutations in the cmbel1 allele of melon and thus generate other mutants that result in seed abortion when in homozygous form.

[0267] Various mutations can be generated and the resulting phenotypes can be tested. For example, regulatory elements, especially promoters, can be mutated to reduce the expression of the allele (knockdown) or eliminate the expression of the allele (knockout), and thus reduce or eliminate the amount of wild-type CmBEL1 protein present in the cell or plant. Alternatively, mutations can be generated that result in a decrease or loss of function of the CmBEL1 protein, i.e., mutations that result in the substitution, insertion, and / or deletion of one or more amino acids (such as missense mutations or frameshift mutations), or truncation of the protein by introducing a premature stop codon (nonsense mutation) in, for example, the coding sequence. Since the CmBEL1 protein contains a highly conserved homologous domain, in one aspect, substitutions, deletions, and / or insertions of one or more amino acids in this domain are encompassed, as such mutations will result in loss of function. Then, plants that are homozygous for the mutation can be generated by self-crossing and the plant lines can be grown to test whether the mutation results in the expected phenotype (seed abortion), and the flowers can be allowed to be pollinated to see if seedless fruits develop, and in one aspect produce significantly more fruits (increased average fruit number) and / or significantly smaller fruits (decreased average fruit weight) than wild-type plants (plants containing two copies of the wild-type CmBEL1 allele).

[0268] In one aspect, there is provided a melon plant that contains two copies of the mutant cmbel1 allele described herein, especially a null allele, wherein the plant produces seedless fruits and produces at least 10%, 15%, 20%, 30%, 40% or 50% more fruits than plants that are homozygous for the wild-type allele when grown under the same conditions and for the same period of time. For example, the plant produces at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more seedless fruits (average number of fruits produced by the plant).

[0269] On the other hand, a melon plant is provided that comprises two copies of the mutant cmbel1 allele, particularly a null allele, described herein, wherein when grown under the same conditions and for the same period of time, the plant produces at least 10%, 15%, 20%, 30%, 40% or 50% more fruit (e.g., the plant produces at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more seedless fruits; the average number of fruits produced by the plant) than a plant homozygous for the wild-type allele, and / or wherein when grown under the same conditions and for the same period of time, the average fruit weight of the fruit is at least 5%, 10%, 15%, 20%, 30%, 40% or 50% lower than the average fruit weight of a plant homozygous for the wild-type allele. Thus, compared to a control plant comprising the wild-type allele in homozygous form, the average fruit number is increased by at least 10%, 15%, 20% or more, and / or compared to a control plant comprising the wild-type allele in homozygous form, the average fruit weight is decreased by at least 5%, 10%, 15%, 20% or more.

[0270] Also provided is a method for producing a cultivated melon plant with seed abortion, the method comprising the following steps:

[0271] a) introducing a mutation into a melon plant or seed, particularly a cultivated plant (population), or providing a mutant plant or seed or their progeny (population);

[0272] b) selecting a plant that produces seedless fruits after pollination;

[0273] c) optionally determining whether the plant selected in b) contains a mutant allele of the CmBEL1 gene; and

[0274] d) optionally growing the plant obtained in c).

[0275] Steps b) and c) can also be exchanged such that step b) is to select a plant containing a mutant allele of the CmBEL1 gene, and step c) is to determine whether the plant (or its progeny produced by selfing) produces seedless fruits after pollination.

[0276] It should be understood that the plant in step b) must be homozygous, i.e., the mutant plant must have been selfed at least once before step b).

[0277] Step a) can be carried out, for example, by mutagenizing the seeds of one or more lines or varieties of melons, for example, by treatment with a mutagen (such as a chemical mutagen, for example EMS (ethyl methane sulfonate)), or by irradiation with UV radiation, X-rays or γ-rays, etc. For example, the population can be a TILLING population. Preferably, before performing step b), the mutagenized plant population is self-crossed at least once (for example, to produce the M2 generation, or M3, M4, etc.). In step b) related to phenotyping, the plants are grown to allow pollination. Regular visual inspection of the flowers, fruit set and visual inspection of the mature fruits (for example, the presence of viable seeds or seedless) can be carried out to identify mutants that produce seedless fruits. Such plants or their self-crossed offspring can be tested for the presence of the mutant CmBEL1 gene by genotyping the plants for mutations in the CmBEL1 gene and the encoded protein, sequencing, and other methods known to the person skilled in the art. Thus, there are various methods or combinations of methods for verifying whether the plants selected based on the phenotype contain mutant alleles of the CmBEL1 gene.

[0278] If step b) is the selection of plants containing mutant alleles of the CmBEL1 gene, the person skilled in the art can also use various methods for detecting the DNA, mRNA or protein of the CmBEL1 gene in order to identify plants containing mutant cmbel1 alleles. The genomic DNA of the wild-type melon CmBEL1 gene encoding the functional CmBEL1 protein (SEQ ID NO: 1 or its variant) is the DNA of SEQ ID NO: 4 (or its variant), and the cDNA (and mRNA) encoding the protein of SEQ ID NO: 1 is given in SEQ ID NO: 2 and 3 (or its variant). The promoter is located upstream of this sequence and can be retrieved, for example, by sequencing or from the melon genome database. In one aspect, a promoter in the sequence of SEQ ID NO: 6 or in a sequence having at least 94% sequence identity with SEQ ID NO: 6 is provided herein. Since the genomic sequences encoding a particular protein may vary slightly (for example, due to the degeneracy of the genetic code or due to variants in the intron sequences), the genomic alleles encoding the wild-type CmBEL1 protein can have at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 4.

[0279] In one aspect, the mutant alleles of the CmBEL1 gene are mutant alleles that result in reduced or no expression of the CmBEL1 gene, or mutant alleles that result in the substitution, insertion or deletion of one or more amino acids of the encoded CmBEL1 protein compared to the wild-type CmBEL1 protein.

[0280] In one aspect, mutant alleles of the CmBEL1 gene can be obtained by inducing targeted or random mutations (promoters or other regulatory elements, splice sites, coding regions, etc.) in the gene and selecting, for example, plants containing the mutant cmbel1 allele from the progeny. In one aspect, alleles are selected that contain mutations in codons, particularly in codons of the homeodomain, such as mutations that cause amino acid substitutions, frameshifts, or stop codons. In one aspect, the mutant allele results in truncation of the encoded melon CmBEL1 protein. In another aspect, the allele contains a mutation in the genomic sequence of SEQ ID NO: 4, resulting in a non-functional mutant CmBEL1 protein produced by the allele, such as a genomic sequence that contains an insertion of one or more nucleotides in intron 1 or intron 2 of SEQ ID NO: 4, or in the exon 1, exon 2, or exon 3 sequences, whereby the transcript encodes a non-functional protein (such as a truncated protein).

[0281] In one aspect, SNP markers or INDEL markers are detected in the genome of a melon plant or plant part, or their DNA. Such SNP marker or INDEL marker detection is for alleles that contain a single nucleotide polymorphism or insertion / deletion polymorphism between SEQ ID NO: 4 and the mutant allele of SEQ ID NO: 4, which mutation results in, for example, a non-functional protein being made by the allele. For example, the mutant allele can contain a stop codon mutation in a codon, such as a codon of the homeodomain or in the codons of exons 1, 2, or 3.

[0282] For any mutant cmbel1 allele, SNP markers (or other markers) and SNP genotyping (or other genotyping) assays can be readily designed. Accordingly, allele-specific markers and detection methods are covered herein, particularly for any mutant allele that results in, for example, an amino acid insertion, deletion, or substitution in the CmBEL1 protein of melon, and other mutant alleles described herein, such as null alleles.

[0283] Diploid plants that are heterozygous for the mutant cmbel1 allele (i.e., cmbel1 / CmBEL1) will be heterozygous for the SNP marker or for the INDEL marker, while plants that are homozygous for the mutant cmbel1 allele (i.e., cmbel1 / cmbel1) will be homozygous for the SNP marker or INDEL marker.

[0284] Mutant allele-specific markers and marker assays can be readily developed for any mutant cmbel1 allele because, for example, potential genomic changes in codons can be used to design marker assays to detect, for example, amino acid changes disclosed herein in the mutant cmbel1 allele, or other genomic changes (such as nucleotide insertions in the exon or intron sequences of SEQ ID NO: 4), compared to the wild-type CmBEL1 allele of SEQ ID NO: 4, or compared to a wild-type CmBEL1 allele having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 4 and encoding a wild-type CmBEL1 protein having at least 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1.

[0285] Using such allele-specific markers that detect a specific mutant cmbel1 allele, genotyping can be performed to detect the presence and copy number of the allele (or DNA derived therefrom) in plants and plant materials.

[0286] Plants and plant parts

[0287] In one embodiment, there is provided a cultivated melon plant or a part thereof (such as a cell, tissue, organ, fruit, etc.) that comprises at least one copy of a mutant allele of a gene named CmBEL1, which, when the mutant allele is in homozygous form, confers seed abortion.

[0288] In one aspect, the mutant allele is a mutant allele of a melon gene encoding the CmBEL1 protein of SEQ ID NO: 1 or a protein having at least 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 1 (wild-type functional protein), wherein the mutant allele has reduced expression (such as 10%, 9%, 8%, 7%, 6% or less of wild-type expression) or no expression, or wherein the mutant allele encodes a mutant CmBEL1 protein that comprises one or more amino acid substitutions, insertions and / or deletions compared to the wild-type protein, thereby resulting in a non-functional protein (optionally a protein with reduced function, provided that it confers a seed abortion phenotype when the allele is in homozygous form).

[0289] In one embodiment, the one or more amino acid substitutions, insertions or deletions comprise or consist of one or more amino acid substitutions, insertions or deletions in a conserved homologous domain.

[0290] Compared with the wild-type protein (and thus with wild-type plants containing the wild-type CmBEL1 gene), the mutant protein has a loss of function (or optionally a reduced function), and preferably, a plant cell or plant containing the mutant allele in homozygous form is seed abortive.

[0291] When referring herein to a specific nucleotide or amino acid position, such as at amino acid 318 of SEQ ID NO: 1, "or at amino acid 318 of a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the SEQ ID NO" (or "at the equivalent position in a sequence having at least 94%..."), this means that the nucleotide or amino acid is present at the nucleotide or amino acid in the variant sequence corresponding to the same nucleotide or amino acid in the variant sequence (e.g., corresponding to amino acid 318 of SEQ ID NO: 1), i.e., in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the SEQ ID NO mentioned. For example, the variant sequence may be one or several nucleotides or amino acids shorter, but when one makes a pairwise comparison of the variant sequence with the SEQ ID NO mentioned, one can see which nucleotide or amino acid of the variant sequence corresponds to the same nucleotide or amino acid. In the variant sequence, amino acid 318 may be, for example, amino acid 319 or 317.

[0292] The mutant allele is a mutation in an endogenous gene of cultivated melon. The presence of the gene conferring seed abortion enables a person skilled in the art to generate other re-mutants in this gene, for example, in any cultivated line or variety.

[0293] A person skilled in the art can generate the plants according to the invention without undue burden, for example, by performing methods for generating and / or identifying CmBEL1 mutants in a mutant population or by targeted gene editing of the CmBEL1 gene.

[0294] As mentioned above, since the CmBEL1 gene was identified as the gene encoding the protein of SEQ ID NO: 1 (wild-type melon BEL1 protein) in normal non-seed abortive melon plants, mutants identical to those provided in the examples or mutants other than those provided in the examples can be regenerated.

[0295] Since natural variants may exist in the wild-type functional CmBEL1 protein, the wild-type CmBEL1 protein need not be 100% identical to the protein of SEQ ID NO: 1, but may have a lower, e.g., at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 when pairwise aligned over its entire length. However, in one aspect, the conserved homologous domain is 100% identical to the homologous domain of SEQ ID NO: 1 such that variants with at least 94% identity are outside the conserved homologous domain.

[0296] As mentioned, when a plant is homozygous for a mutant allele, the mutant allele of the CmBEL1 gene causes the plant to produce seedless fruits after pollination. With respect to embodiments of the present invention, the mutation in the mutant allele of the CmBEL1 gene can be any mutation, including deletions, truncations, insertions, point mutations, nonsense mutations, missense or non-synonymous mutations, splice site mutations, frameshift mutations, and / or regulatory sequences, such as mutations in promoters.

[0297] In one aspect, the mutation in the mutant allele of the CmBEL1 gene is a point mutation. In another aspect, the mutation in the mutant allele of the CmBEL1 gene is an insertion of one or more nucleotides, e.g., an INDEL or a transposon or transposable element insertion.

[0298] The mutation can occur in the DNA sequence including the transcriptional region of the gene, or in the coding sequence of the CmBEL1 gene, or in the RNA sequence encoding the CmBEL1 protein, or it can occur in the amino acids of the CmBEL1 protein. With respect to the DNA sequence of the CmBEL1 gene, the mutation can occur in the coding sequence of the CmBEL1 gene, or it can occur in non-coding sequences such as the 5'-untranslated region and 3'-untranslated region, promoter, enhancer, intron, etc. With respect to the RNA encoding the CmBEL1 protein, the mutation can occur in the pre-mRNA or mRNA.

[0299] In one aspect, a mutant allele causes a protein to lose function (or optionally have reduced function) due to one or more amino acid substitutions, insertions, and / or deletions, such as causing substitution, insertion, and / or deletion of one or more amino acids at the C-terminus of the protein and / or in the homologous domain of the protein. For example, truncating the protein to cause deletion of at least 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 230, 240, 250, 260, 265 or more amino acids at the C-terminus of the wild-type protein will produce a mutant protein that causes seed abortion, as shown by the mutant proteins of the examples, which lack amino acid Y344 and all remaining C-terminal amino acids (i.e., it is truncated by 265 amino acids at the C-terminus). In particular, such mutants also lack 36 amino acids of the conserved homologous domain (amino acids 344 to 379) and are thus non-functional in vivo because the homologous domain is required for DNA binding of the protein and transcriptional repression of other genes. Thus, it is believed that mutant proteins encoding the deletion of one or more amino acids of the homologous domain (at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 36 amino acids of the conserved homologous domain) or any other mutant allele of the CmBEL1 gene truncated at the C-terminus is non-functional in vivo and causes seed abortion, where the truncation includes the absence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 36 amino acids of the conserved homologous domain.

[0300] Thus, in one aspect, mutations resulting in complete or partial deletion of the homologous domain or substitution of one or more amino acids of the homologous domain with one or more different amino acids will cause loss of function of the protein.

[0301] For example, a stop codon mutation in the N-terminal portion before the homologous domain or a stop codon mutation in the homologous domain results in loss of function of the truncated protein.

[0302] Similarly, amino acid insertions, deletions, and / or substitutions in the N-terminal portion before the homologous domain or in the homologous domain itself may cause loss of function of the protein.

[0303] When the allele is in homozygous form, the phenotype of any mutant allele can be analyzed to see if the plant has indeed become seed-aborting. For phenotyping, several plants (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 plants) of the control plant and the mutant plant (homozygous for the mutant allele) are grown for the same period of time under the same conditions. Then, the mature fruits can be cut open to see if they are seeded or seedless. Optionally, the number of fruits can be counted and / or the fruit weight can be measured.

[0304] Accordingly, one embodiment of the invention relates to a plant cell or a plant according to the invention, which plant cell or plant comprises a mutant allele of the gene encoding the CmBEL1 protein, and which plant cell or plant is characterized in that the mutant allele comprises or effects one or more mutations selected from the group consisting of:

[0305] a) deletions, truncations, insertions, point mutations, nonsense mutations, missense or non-synonymous mutations, splice site mutations, frameshift mutations in the genomic sequence;

[0306] b) mutations in one or more regulatory sequences;

[0307] c) deletions, truncations, insertions, point mutations, nonsense mutations, missense or non-synonymous mutations, splice site mutations, frameshift mutations in the transcriptional sequence or the coding sequence;

[0308] d) deletions, truncations, insertions, point mutations, nonsense mutations, missense or non-synonymous mutations, splice site mutations, frameshift mutations in the pre-mRNA or mRNA; and / or

[0309] e) deletions, truncations, insertions or substitutions of one or more amino acids in the CmBEL1 protein.

[0310] In one aspect, the mutant allele results in reduced or particularly no expression of the CmBEL1 gene, or the mutant allele encodes a protein with reduced function or particularly loss of function.

[0311] Reduced or no expression means that there is a mutation in the regulatory region of the CmBEL1 gene, such as in the promoter, thereby producing significantly reduced mRNA transcripts of the CmBEL1 allele (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the wild-type allele transcript) or no mRNA transcripts compared to plants and plant parts containing the wild-type CmBEL1 allele. The reduction in expression can be determined, for example, by measuring the amount of mRNA transcripts encoding the CmBEL1 protein, such as using Northern blot analysis or RT-PCR. Here, a significant reduction preferably means that the amount of mRNA transcripts is reduced by at least 50%, particularly by at least 70%, optionally by at least 85% or by at least 95%, 96%, 97%, 98%, 99% or even 100% (no expression) compared to plants or plant parts containing the wild-type CmBEL1 gene. Expression can be analyzed, for example, in leaf tissue or ovary tissue. Various techniques can be used, such as quantitative real-time polymerase chain reaction, RNA blotting, RNA sequencing, microarrays, etc. to study gene expression. Thus, the mRNA transcript levels of the wild-type CmBEL1 protein encoded by SEQ ID NO: 1 or a protein having at least 94% sequence identity with SEQ ID NO: 1 can be analyzed. cDNA (which corresponds to mRNA, except that uracil is shown as thymine in cDNA) is provided in SEQ ID NO: 2 and in SEQ ID NO: 3 (which also includes UTR’s) or in variants of these sequences. Thus, in one aspect, due to one or more mutations in the promoter sequence, in botany, the level of mRNA transcripts (or corresponding cDNA) is reduced or not produced. The promoter is provided in SEQ ID NO: 6 or in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6. The promoter is also present in the melon genome on chromosome 9 in the region starting at nucleotide 23595097 and ending at nucleotide 23597096 or in the region starting at nucleotide 23596097 and ending at nucleotide 23597096 (database cucurbitgenomics.org genomeDHL v.3.6.1).

[0312] In one aspect, the melon plant or plant part contains one or more mutations in the promoter of the CmBEL1 allele, whereby no mRNA transcript of the wild-type CmBEL1 allele is produced (or a significantly reduced mRNA transcript is produced, see above), as determined, for example, by the absence (or a significant reduction) of the cDNA encoding the wild-type CmBEL1 protein of SEQ ID NO: 1 or a wild-type protein having at least 94% sequence identity with SEQ ID NO: 1.

[0313] In one aspect, the protein contains one or more amino acid substitutions, insertions or deletions as compared to the wild-type protein. Thus, for melon, one or more amino acids are inserted, deleted or substituted as compared to the wild-type CmBEL1 protein of SEQ ID NO: 1 or a wild-type CmBEL1 protein having at least 94%, 95%, 96%, 97% or 98% sequence identity with SEQ ID NO: 1; whereby the mutant protein has a loss of function (optionally a reduced function) as compared to the wild-type protein, and thus when the mutant allele is present in homozygous form in a diploid plant, it results in seed abortion.

[0314] In one aspect, the wild-type CmBEL1 protein contains a conserved homologous domain. Thus, in one aspect, the mutant allele is a mutant allele of the gene CmBEL1, which encodes the wild-type protein of SEQ ID NO: 1 or a wild-type protein having at least 94%, 95%, 96%, 97% or 98% sequence identity with SEQ ID NO: 1, and wherein the wild-type protein contains the conserved homologous domain of amino acids 318 to 379 of SEQ ID NO: 1.

[0315] In one aspect, the wild-type CmBEL1 protein contains a conserved homologous domain, i.e., any variant of the functional wild-type protein is outside the conserved homologous domain.

[0316] In one aspect, the mutant allele of the above wild-type allele is a mutant allele with reduced expression or no expression (by mutations in, for example, promoter or enhancer elements) or a mutant allele that produces a mutant protein containing one or more amino acid insertions, deletions or substitutions as compared to the wild-type protein, whereby the mutant protein does not have an in vivo function (or optionally a reduced function), as determined when the mutant allele is in homozygous form in a plant and by analyzing whether the plant produces seedless fruits.

[0317] If the mutant allele results in seed abortion in vivo, while control plants containing only the wild-type CmBEL1 allele are not seed abortive, then the mutant protein does not have a function (or optionally has a severely reduced function) compared to the wild-type protein. The same phenotypic analysis can be performed on mutant alleles that do not have gene expression (or optionally have severely reduced gene expression, e.g., the amount of RNA transcript is reduced by 95%, 96%, 97%, 98%, or 99%). Thus, any mutant allele can be made homozygous in a plant, and the phenotype can be compared to a control plant containing the original non-mutant allele in homozygous form.

[0318] It was found that the homeodomain is a highly conserved protein domain and would be 100% identical in other wild-type functional CmBEL1 variants, as they would be required for the proper functioning of the protein in the plant. Thus, mutating the homeodomain by inserting, deleting, and / or substituting one or more of its amino acids will abolish the in vivo function of the CmBEL1 protein.

[0319] Thus, in one aspect, the plants provided herein contain a mutant CmBEL1 allele encoding a CmBEL1 protein containing one or more amino acids inserted, deleted, and / or substituted in the homeodomain.

[0320] In one aspect, the wild-type functional CmBEL1 protein that has been mutated to contain one or more amino acids inserted, substituted, and / or deleted is selected from CmBEL1 of SEQ ID NO: 1 or a protein having at least 94% identity to SEQ ID NO: 1, wherein the wild-type protein contains, for example, the homeodomain of SEQ ID NO: 1 or alternatively a wild-type homeodomain having at least 98% or 99% sequence identity to the homeodomain of SEQ ID NO: 1.

[0321] Mutant proteins containing a frameshift that results in one or more amino acid changes in the homeodomain or truncated mutant proteins containing one or more amino acid deletions in the homeodomain are herein encompassed as mutant proteins that do not contain in vivo function.

[0322] Thus, in one aspect, a mutant CmBEL1 allele encoding a mutant protein is provided, wherein Y344 of SEQ ID NO: 1 (or the equivalent amino acid in a sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1) is substituted or deleted with another amino acid, e.g., the codon is substituted with a stop codon.

[0323] Thus, in one aspect, provided are mutant CmBEL1 alleles encoding mutant proteins, wherein at least one or more amino acids of the homeodomain (i.e., W318 to M379 selected from SEQ ID NO: 1) (or equivalent amino acids in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1) are replaced or deleted by another amino acid, such as a codon being replaced by a stop codon.

[0324] When referring to an amino acid "from one amino acid to another" herein, this includes the starting / first amino acid and the ending / last amino acid mentioned.

[0325] When referring to an amino acid "deletion", this includes mutations where a codon is changed to a stop codon or a codon is deleted, or mutations where there is a frameshift resulting in an amino acid not being encoded, or mutations in the transcript region where the mRNA transcript becomes truncated and translation produces a truncated protein. Similarly, when referring to an amino acid being "replaced", this includes mutations where a codon encodes a different amino acid or a codon is inserted, or mutations where there is a frameshift resulting in a different amino acid being encoded.

[0326] In one aspect, the mutant CmBEL1 allele is heterozygous in a diploid melon plant cell or plant. In another aspect, the mutant CmBEL1 allele is homozygous in a diploid melon plant cell or plant.

[0327] The plant cell and plant are preferably cultivated plants, such as elite breeding lines or varieties, rather than wild plants. The melon can be any type of melon, such as Spanish melon, Charantais melon, Galia melon, Honey Dew melon, Yellow Canary melon, etc.

[0328] Thus, a diploid melon plant can have the genotype cmbel1 / CmBEL1 (heterozygous for the mutant allele) or cmbel1 / cmbel1 (homozygous for the mutant allele). In one aspect, a diploid melon plant containing the cmbel1 allele in homozygous form is a doubled haploid (DH) (e.g., doubled haploid melon) plant or plant cell or plant part. DH plants can be produced by chromosome doubling of haploid cells (e.g., by colchicine treatment).

[0329] In one aspect, the melon plant is homozygous for cmbel1, and in another aspect, it is heterozygous for cmbel1. In one aspect, it is an inbred line or variety. In another aspect, it is an F1 hybrid.

[0330] In one aspect, the melon plant is a diploid plant (e.g., an inbred line) that contains at least one mutant copy, preferably two mutant copies, of cmbel1 (i.e., homozygous for cmbel1). After the flower is pollinated (which is required for fruit set and development), the diploid plant that is homozygous for cmbel1 will produce seedless fruits. Due to developmental arrest or disruption of the normal seed development process, mature or viable seeds do not develop in plants with seed abortion. Thus, when a diploid plant that is homozygous for the mutant cmbel1 allele (cmbel1 / cmbel1) self-pollinates or is pollinated with pollen from another plant, the diploid plant produces seedless diploid fruits.

[0331] The mutant allele of cmbel1 renders the plant male-fertile, but when the plant is homozygous for the mutant allele, the plant produces seedless fruits. The mutation in the CmBEL1 gene can be any mutation, including deletions, truncations, insertions, point mutations, nonsense mutations, missense or non-synonymous mutations, splice site mutations, frameshift mutations, and / or mutations in regulatory sequences. The mutation can occur, for example, in the genomic DNA sequence in the coding region (exons) or non-coding region (e.g., introns) of the CmBEL1 gene and / or in the RNA sequence encoding the CmBEL1 protein, or it can occur in the amino acids of the CmBEL1 protein. With respect to the genomic DNA sequence of the CmBEL1 gene, the mutation can occur in the coding sequence (cds, composed of exons), or it can occur in non-coding sequences such as the 5′-untranslated region and 3′-untranslated region, introns, promoters, enhancers, etc. With respect to the RNA encoding the CmBEL1 protein, the mutation can occur in the pre-mRNA or mRNA.

[0332] Thus, in one aspect, the invention relates to a melon plant or plant part that contains at least one copy, preferably two copies, of the mutant cmbel1 allele.

[0333] Also covered herein are the seeds from which such diploid plants can grow, just as plant parts such as diploid seedless fruits, flowers, leaves, stems, roots, vegetative propagules, cells, cuttings, seed propagules (e.g., selfing), and in vitro cell or tissue cultures, as well as pollen, ovaries, etc. are covered herein. Thus, in one embodiment, the diploid plant or the seeds from which the plant can grow or the tissue or parts of the plant (pollen, anthers, ovules) contain the mutant cmbel1 allele as described elsewhere herein.

[0334] In one aspect, a diploid plant or seed comprises one or two copies of a mutant cmbel1 allele encoding a non-functional (loss-of-function) protein (optionally a protein with reduced function), wherein the non-functional protein comprises one or more amino acid substitutions, insertions, and / or deletions relative to the wild-type functional protein of SEQ ID NO: 1 or relative to a functional variant having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0335] In one aspect, a diploid plant or seed comprises one or two copies of a mutant cmbel1 allele encoding a non-functional (loss-of-function) protein (optionally a protein with reduced function), wherein the non-functional protein comprises one or more amino acid substitutions, insertions, and / or deletions in the homologous domain of SEQ ID NO: 1 or in the homologous domain of a functional variant having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0336] In one aspect, a diploid plant or seed comprises one or two copies of a mutant cmbel1 allele encoding a non-functional (loss-of-function) protein (optionally a protein with reduced function), wherein the non-functional protein is truncated at the C-terminus and the truncation starts within or before the homologous domain of SEQ ID NO: 1 (or within or before the homologous domain of a functional variant having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1), and wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 36, 40, 45, 50, 55, 60, 61, or all 62 amino acids (i.e., the complete homologous domain) of the homologous domain are missing from the truncated protein. Thus, the truncation results in a mutant protein comprising up to amino acids 1 to 378 of SEQ ID NO: 1 (or amino acids 1 to 378 of a functional variant having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1).

[0337] When referring to a "truncation" of the wild-type functional protein, this encompasses that the missing amino acids can be replaced by one or more different amino acids (i.e., amino acids having a sequence different from the wild-type sequence), for example, when a frameshift that results in a change in the reading frame occurs.

[0338] In one aspect, a diploid plant or seed contains one or two copies of a mutant cmbel1 allele encoding a truncated protein comprising only amino acids 1 to 343 of SEQ ID NO: 1 or encoding a truncated protein comprising equivalent amino acids of amino acids 1 to 343 of SEQ ID NO: 1 in a sequence having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0339] The underlying cause of the truncation of the protein produced can be of various kinds, such as an amino acid codon becoming a stop codon, insertion of one or more nucleotides to, for example, terminate transcription and / or translation, frameshift mutation, splice site mutation, etc.

[0340] In one aspect, the truncation is caused by a DNA insertion in intron 2 of the gene, such that only the codons of exons 1 and 2 are transcribed from the gene. Thus, in one aspect, the mRNA transcript contains only the codons of exons 1 and 2. Accordingly, the translated protein contains only amino acids 1 to 343 of SEQ ID NO: 1.

[0341] In one aspect, for example, the DNA insertion in intron 2 is an insert of at least 10, 20, 30, 40, 50, 60, 100, 200, 500, 600, 700, 800, 900, 1000 or more nucleotides. In one aspect, the DNA insertion comprises at least 10, 20, 30, 40, 50, 60, 100, 200, 500, 600, 700, 800, 900, 1000 or more nucleotides of SEQ ID NO: 5. In another aspect, for example, the DNA insertion in intron 2 comprises the nucleotides of SEQ ID NO: 5.

[0342] A melon plant containing one or preferably two copies of the mutant cmbel1 allele can be an inbred line, an open-pollinated variety (OP) or an F1 hybrid plant.

[0343] Mutant cmbel1 alleles can be generated in any background melon type or can be introduced by crossing from one background (e.g., where it was generated) to another background (e.g., Spanish melons, honeydews, Galia melons, Charentais melons, etc.). Thus, any melon type with the seed abortion trait can be produced. In one aspect, the melon plant produces small or medium-sized melons such that the small or medium-sized melons are seedless. In such small-sized fruits, the seedless phenotype has the advantage that seeds do not have to be removed before eating or processing, and the fruit can be eaten or processed directly. Some medium-sized varieties are, for example, variety Kukino F1 (a Spanish melon variety from Nunhems). Thus, the seed abortion trait can be introduced into, for example, Kukino F1 to make the fruit seedless. In one aspect, when present in homozygous form, the mutant cmbel1 allele reduces the average fruit weight and / or increases the average fruit number. Thus, when introduced into a variety that produces seeded fruits with an average weight of 1.5 kg to 2.0 kg, the mutant allele can cause a significantly lower average fruit weight and / or a significantly higher fruit number.

[0344] In the examples, various melon fruits did not show empty seed cavities in the seedless fruits. Thus, in one aspect, the seedless melon fruits do not contain (or substantially do not contain) empty seed cavities.

[0345] In one aspect, the F1 hybrid contains the mutant cmbel1 allele in homozygous form (cmbel1 / cmbel1) and grows from F1 hybrid seeds containing the mutant cmbel1 allele in homozygous form. Since the F1 hybrid plants grown from seeds containing the cmbel1 allele in homozygous form will produce seedless fruits, the F1 hybrid plants can be vegetatively propagated. Thus, in one aspect, there is provided a vegetatively propagated F1 hybrid melon plant that contains the mutant cmbel1 allele in homozygous form. The plant will produce seedless fruits, such as many small seedless fruits.

[0346] Alternatively, F1 hybrids containing the mutant cmbel1 allele in homozygous form (cmbel1 / cmbel1) can be produced by crossing cmbel1 / CmBEL1 plants (heterozygous for the mutant allele) with cmbel1 / cmbel1 plants (homozygous for the mutant allele), whereby the resulting seeds will be genetically approximately 50% cmbel1 / cmbel1 and 50% cmbel1 / CmBEL1. Optionally, seeds or seedlings containing the mutant allele in homozygous form can be selected from the mixed seeds. Thus, in one aspect, a seed mixture is provided that contains seeds that are genetically approximately 50% cmbel1 / cmbel1 and 50% cmbel1 / CmBEL1.

[0347] Another method for producing F1 hybrids containing the mutant cmbel1 allele in homozygous form (cmbel1 / cmbel1) is by crossing cmbel1 / CmBEL1 plants (heterozygous for the mutant allele) with cmbel1 / CmBEL1 plants (heterozygous for the mutant allele). The resulting seeds will be genetically approximately 25% cmbel1 / cmbel1, 50% cmbel1 / CmBEL1, and 25% CmBEL1 / CmBEL1. Optionally, seeds or seedlings containing the mutant allele in homozygous form can be selected from the mixed seeds. Thus, in one aspect, a seed mixture is provided that contains seeds that are genetically approximately 25% cmbel1 / cmbel1, 50% cmbel1 / CmBEL1, and approximately 25% CmBEL1 / CmBEL1.

[0348] Yet another method for producing a plant containing the mutant cmbel1 allele in homozygous form (cmbel1 / cmbel1) is by selfing cmbel1 / CmBEL1 plants (heterozygous for the mutant allele). The resulting seeds will be genetically approximately 25% cmbel1 / cmbel1, 50% cmbel1 / CmBEL1, and 25% CmBEL1 / CmBEL1. Optionally, seeds or seedlings containing the mutant allele in homozygous form can be selected from the mixed seeds. Thus, in one aspect, a seed mixture is provided that contains seeds that are genetically approximately 25% cmbel1 / cmbel1, 50% cmbel1 / CmBEL1, and approximately 25% CmBEL1 / CmBEL1.

[0349] In one embodiment of the present invention, a non-destructive seed-based genotyping method is used to determine the presence of one or more mutant cmbel1 alleles in an embryo, particularly non-destructive genotyping of individual plant seeds, whereby the viability of the seeds is not affected, but the genotype of the plants grown from the seeds can be determined. A variety of non-destructive single-seed genotyping methods have been developed and can be used. For example, Meru et al. (2013, Genetics and Molecular Research 12(1): 702-709, "A non-destructive genotyping system from a single seed for marker-assisted selection in watermelon") describe such methods.

[0350] In another aspect, seeds such as a seed mixture can be germinated to select plants (e.g., seedlings) that are homozygous for the cmbel1 mutant allele. Plants, such as seedlings, can be selected by taking a tissue or DNA sample and genotyping the DNA to determine the presence of the mutant cmbel1 allele and whether one or two copies are present. Then, plants, such as seedlings, that contain two copies of the mutant cmbel1 allele can be selected and grown to produce seedless melon fruits.

[0351] Thus, in one aspect, there is provided a method for selecting a plant that contains a mutant cmbel1 allele in homozygous form, the method comprising

[0352] - selecting a plant, such as a seedling, that contains two mutant cmbel1 alleles.

[0353] This method may comprise the steps of:

[0354] - providing a plurality of seeds, such as seeds of different genotypes that contain a mutant (cmbel1) allele and a wild-type

[0355] (CmBEL1) allele, such as seeds that contain a genotype mixture, such as cmbel1 / CmBEL1 (heterozygous for the mutant allele) and

[0356] cmbel1 / cmbel1 (homozygous for the mutant allele) seeds, and

[0357] - germinating the seeds so that the genotype of the plants can be analyzed.

[0358] Accordingly, in one embodiment of the present invention, there is provided a seed population that comprises a genotype mixture of the CmBEL1 allele, wherein said population is a seed population that comprises approximately 50% seeds in which the mutant cmbel1 allele is in heterozygous form (cmbel1 / CmBEL1) and approximately 50% seeds in which the mutant cmbel1 allele is in homozygous form (cmbel1 / cmbel1). A seed population segregating in a ratio of 50% cmbel1 / CmBEL1 to 50% cmbel1 / cmbel1 can be generated by crossing a parental line A that is heterozygous for the mutant cmbel1 allele (preferably as the female parent) with a parental line B that is homozygous for the mutant cmbel1 allele (preferably as the male parent). Seeds harvested from this cross will segregate in the aforementioned approximate 50:50 ratio, and approximately 50% homozygous mutant cmbel1 allele seeds can be selected from this seed population as described. It should be understood that crosses between parental line A and parental line B can be made among many plants to obtain a large number of F1 seeds that segregate in an approximate 50:50 ratio of CmBEL1 / cmbel1:cmbel1 / cmbel1 seeds.

[0359] In one aspect of the present invention, the actual steps of crossing line A with line B are also part of the method. Accordingly, in one aspect, there is provided a method for generating and / or selecting seeds that are homozygous for the mutant cmbel1 allele, the method comprising

[0360] a) crossing a parental line A that is heterozygous for the mutant cmbel1 allele with a parental

[0361] line B that is homozygous for mutant cmbel1,

[0362] b) harvesting the F1 seeds of the cross,

[0363] c) germinating the F1 seeds,

[0364] d) selecting the plants of step c) that comprise the cmbel1 allele in homozygous form, and optionally

[0365] e) causing the selected plants to produce seedless fruits (after the flowers are pollinated), and optionally

[0366] f) harvesting the seedless fruits.

[0367] As mentioned, in this method, parental line A is the female parental line and parental line B is the male parental line because a line that is homozygous for the mutant cmbel1 allele cannot be used to produce seeds because it will produce seedless fruits after the flowers are pollinated.

[0368] Another aspect of the present invention provides a seed population that comprises a genotype mixture of the CmBEL1 allele, wherein the seed population is a population of seeds comprising approximately 50% seeds in which the mutant cmbel1 allele is in heterozygous form (cmbel1 / CmBEL1), approximately 25% seeds in which the mutant cmbel1 allele is in homozygous form (cmbel1 / cmbel1), and approximately 25% seeds in which the wild-type CmBEL1 allele is in homozygous form (CmBEL1 / CmBEL1). A seed population separated at a ratio of 50% cmbel1 / CmBEL1 to 25% cmbel1 / cmbel1 to 25% CmBEL1 / CmBEL1 can be generated by crossing parental line A that is heterozygous for the mutant cmbel1 allele with parental line B that is also heterozygous for the mutant cmbel1 allele, or by selfing a plant that is heterozygous for the mutant cmbel1 allele. Seeds harvested from this cross will segregate at the approximate ratio of 50 (CmBEL1 / cmbel1):25 cmbel1 / cmbel1):25 (CmBEL1 / CmBEL1) mentioned, and approximately 25% homozygous mutant cmbel1 allele seeds can be selected from this seed population as described.

[0369] In one aspect of the present invention, the actual steps of crossing heterozygous line A with heterozygous line B or selfing a line that is heterozygous for the mutant cmbel1 allele are also part of the method. Thus, in one aspect, there is provided a method for generating and / or selecting seeds that are homozygous for the mutant cmbel1 allele, the method comprising

[0370] a) crossing parental line A that is heterozygous for the mutant cmbel1 allele of the present invention with parental line B that is heterozygous for the mutant cmbel1 allele of the present invention, or selfing a line that is heterozygous for the mutant cmbel1 allele of the present invention,

[0371] b) harvesting the F1 seeds of the cross,

[0372] c) germinating the F1 seeds,

[0373] d) selecting the plants of step c) that comprise the cmbel1 allele in homozygous form, and optionally

[0374] e) causing the selected plants to produce seedless fruits (after pollination of the flowers), and optionally

[0375] f) harvesting the seedless fruits.

[0376] There is also provided a method for producing fruits with seed abortion, the method comprising growing a plant comprising a mutant cmbel1 allele in homozygous form (as described throughout the specification), and optionally harvesting the seedless fruits produced by said plant.

[0377] Other aspects of the invention are a method for selecting a plant or plant part capable of forming fruits with seed abortion, the method comprising selecting a plant or plant part comprising at least one copy (preferably two copies) of the mutant cmbel1 allele, wherein when the allele is in homozygous form, the mutant allele causes the formation of fruits with seed abortion. The selection of the plant or plant part can include direct and / or indirect methods, such as DNA analysis of the endogenous CmBEL1 allele (e.g., sequence analysis, allele-specific genotyping methods, etc.), RNA analysis of the CmBEL1 allele (e.g., mRNA expression), CmBEL1 protein analysis, etc. Thus, in the above method, any phenotypic selection step of germinated seedlings can be replaced by a selection based on the detection of the mutant cmbel1 allele in ungerminated seeds (i.e., omitting the seed germination step) and / or in seedlings or plants (or their tissues, such as leaf discs) with germinated seeds.

[0378] In another aspect of the invention, there is provided a method for generating a plant or plant part capable of forming fruits with seed abortion, the method comprising contacting a plant or plant part with a mutagen, and subsequently (optionally after one or more self-pollinations of the mutagenized plant) selecting a plant or plant part comprising at least one copy of the mutant cmbel1 allele, wherein when the allele is in homozygous form, the mutant allele causes the formation of fruits with seed abortion.

[0379] There is also provided a method for producing a plant capable of forming fruits with seed abortion, the method comprising

[0380] a) crossing a plant comprising at least one of the described mutant cmbel1 alleles in its genome (the mutant cmbel1 allele causing the formation of fruits with seed abortion when in homozygous form) with another plant, and optionally

[0381] b) harvesting the seeds from said cross, and optionally

[0382] c) selecting seeds or plants (e.g., seedlings) grown from the seeds comprising at least one copy of the mutant cmbel1 allele, preferably selecting seeds comprising two copies of the mutant cmbel1 allele.

[0383] In a preferred aspect, plants heterozygous for the mutant cmbel1 allele are used as the female parent and crossed with male parent plants homozygous for the mutant cmbel1 allele.

[0384] The selection in step c) can be based on methods for directly detecting the mutant cmbel1 allele (e.g., using genotyping assays) or on the phenotype.

[0385] Also covered herein are seeds and plants selected by this method, just as plants grown from seeds homozygous for the mutant cmbel1 allele and seedless fruits produced by the plants grown from such seeds are covered herein.

[0386] In one aspect, a method for identifying a plant or plant part or cell that contains at least one copy of the mutant allele of the cmbel1 gene in its genome is provided, the method comprising

[0387] - determining whether the plant or plant part or cell contains at least one mutant cmbel1 allele in its genome.

[0388] This method can include (directly or indirectly) analyzing the gene expression of the cmbel1 allele of the plant or plant part or plant cell, and / or the genomic nucleotide sequence of the cmbel1 allele, or the mRNA nucleotide sequence of the cmbel1 allele, or the protein sequence of the CmBEL1 protein, or the amount of the CmBEL1 protein to determine whether the gene expression is knocked down or knocked out compared to a wild-type plant or plant part or plant cell, or whether the mRNA is truncated or contains one or more inserted, deleted, or substituted nucleotides compared to the wild-type mRNA, or whether the encoded protein contains one or more amino acid insertions, deletions, or substitutions compared to the wild-type CmBEL1 protein.

[0389] One method for analyzing the presence of the mutant cmbel1 allele is, for example, to assay for the presence of single nucleotide polymorphisms (SNPs) or insertions and deletions (INDELs) in the genomic sequence of the cmbel1 allele by, for example, designing primers for SNPs or INDELs and genotyping the plant or plant part for the genotype of a specific SNP or INDEL.

[0390] Accordingly, one aspect of the invention includes a method for determining whether a plant, plant part or plant cell contains one or more copies of a mutant cmbel1 allele, the method being carried out by a method selected from: analyzing one or more nucleotides of the genomic cmbel1 allele in a genotyping assay, analyzing the mRNA (or cDNA) expressed by the cmbel1 allele or analyzing the amount and / or amino acid sequence of the CmBEL1 protein (using, for example, antibody-based detection).

[0391] In one aspect, particularly with respect to the European Patent Convention, a plant according to the invention "is not obtained only by essentially biological processes", or in one aspect, the mutant cmbel1 allele is not a naturally occurring mutant allele. If such a disclaimer is present in the claims of a European patent, it should be noted that using a plant containing the mutant allele (such as the applicant's commercial variety) to cross the mutant allele into a different background will still be considered to fall within the claims, even if specialized essentially biological processes (only crossing and selection) may have been used to transfer the allele into the different background.

[0392] In one aspect, the mutant cmbel1 allele is an induced mutant allele.

[0393] In one aspect, there is provided a melon plant that contains two copies of the mutant cmbel1 allele in homozygous form, resulting in seed abortion, wherein the melon plant is vegetatively propagated, for example, produced from cuttings induced to develop roots and shoots, and will produce seedless fruits after flowering and pollination. Vegetative propagation can be carried out using methods well known in the art, such as in vitro plant tissue culture, for example, rooting cuttings. In one embodiment, a method of vegetatively propagating a plant that contains one or two copies of the mutant cmbel1 allele described herein in its genome includes: a) collecting tissue from a plant that contains one or two copies of the mutant cmbel1 allele described herein; b) culturing the tissue to obtain proliferating shoots; c) rooting the proliferating shoots to obtain rooted plantlets; and d) growing a plant from the rooted plantlets. Cuttings according to this aspect of the invention can include roots, stems, leaves, cotyledons, flowers, fruits, embryos and pollen.

[0394] In one aspect, there is provided a screening method for identifying and / or selecting seeds, plants or plant parts or DNA from such seeds, plants or plant parts, wherein the seeds, plants or plant parts or DNA contain a mutant cmbel1 allele in their genome.

[0395] The method includes screening at the DNA, RNA (or cDNA), or protein level using known methods in order to detect the presence of mutant alleles. There are many methods for detecting the presence of mutant alleles of a gene.

[0396] Accordingly, provided is a method for screening and / or selecting plants, seeds, or plant materials or plant parts, or DNA or RNA or protein derived therefrom, for the presence of mutant cmbel1 alleles, the method including one or more of the following steps:

[0397] a) determining whether the gene expression of the endogenous CmBEL1 gene is reduced or eliminated;

[0398] b) determining whether the amount of wild-type CmBEL1 protein is reduced or eliminated;

[0399] c) determining whether there is mutant mRNA, cDNA, or genomic DNA encoding a mutant CmBEL1 protein;

[0400] d) determining whether there is a mutant CmBEL1 protein;

[0401] e) determining whether the plant or its progeny is seed-aborted.

[0402] Conventional methods can be used, such as RT-PCR, PCR, antibody-based assays, sequencing, genotyping assays (e.g., allele-specific genotyping), phenotyping, etc.

[0403] The plant or plant material or plant part can be a melon plant or plant material or plant part, such as a leaf, leaf part, cell, fruit, fruit part, ovary, stem, hypocotyl, seed, seed part, seed coat, embryo, etc.

[0404] For example, if there is a single nucleotide difference (single nucleotide polymorphism, SNP) between the wild-type allele and the mutant allele, then SNP genotyping assays can be used to detect whether a plant, seed, or plant part or cell contains a wild-type nucleotide or a mutant nucleotide in its genome. For example, the KASP assay (see kpbioscience.co.uk on the World Wide Web) or other SNP genotyping assays can be readily used to detect SNPs. To develop a KASP assay, for example, 50, 60, or 70 base pairs upstream and 50, 60, or 70 base pairs downstream of the SNP can be selected, and two allele-specific forward primers and one allele-specific reverse primer can be designed. See, for example, Allen et al. 2011, Plant Biotechnology J. 9, 1086-1099, especially pages 1097-1098 regarding the KASP assay method.

[0405] Other genotyping assays can also be used. For example, TaqMan SNP genotyping assays, high-resolution melting (HRM) assays, SNP genotyping arrays (e.g., Fluidigm, Illumina, etc.), or DNA sequencing can also be used.

[0406] Based on the differences between the genomic sequences of the wild-type allele and the mutant allele, a person skilled in the art can easily develop a genotyping assay that can be used to detect specific alleles.

[0407] The present invention also provides a method for identifying a melon plant (or plant part) comprising a mutant cmbel1 allele, the method comprising detecting the presence of the mutant cmbel1 allele in the plant (or plant part), wherein the presence is detected by at least one marker (or nucleotide difference) within the cmbel1 allele or by detecting the protein encoded by the cmbel1 allele. The methods for detecting the mutant cmbel1 allele are selected from the group consisting of: PCR amplification, nucleic acid sequencing, nucleic acid hybridization, and antibody-based assays (e.g., immunoassays) for detecting the CmBEL1 protein encoded by the allele.

[0408] The present invention also provides a method for identifying a melon plant (or plant part) comprising a mutant cmbel1 allele containing a mutation in a regulatory element, the method comprising detecting a decrease or absence of gene expression of the mutant cmbel1 allele in the plant (or plant part), wherein the presence is detected by the mRNA level (cDNA) of the wild-type CmBEL1 allele or by detecting the protein level of the wild-type CmBEL1 protein. The methods for detecting the mutant cmbel1 allele are selected from the group consisting of: PCR amplification (e.g., RT-PCR), nucleic acid sequencing, Western blotting, and antibody-based assays (e.g., immunoassays) for detecting the CmBEL1 protein encoded by the allele.

[0409] The present invention also provides a method for determining, or detecting, or assaying whether a cell of a melon plant or plant part contains a mutant allele of a gene named CmBEL1 encoding a protein of SEQ ID NO: 1 or a protein having at least 94%, 95%, 96%, 97%, or 98% sequence identity to SEQ ID NO: 1. In one aspect, the method comprises determining the expression of the allele, and / or determining the coding sequence of the allele, and / or determining a portion of the coding sequence of the allele (e.g., the SNP genotype of the allele), and / or determining the amino acid sequence of the protein produced and / or the amount of the protein produced.

[0410] Multiple methods can be used to determine whether a plant or a part thereof contains the mutant cmbel1 allele of the present invention. As mentioned, the mRNA (or cDNA) level of the wild-type allele can be determined, or the wild-type protein level can be determined, to observe whether there is a decrease in the expression of the wild-type allele or no expression. The transcribed sequence or the coding sequence or a part thereof can also be analyzed. For example, if one already knows which mutant alleles might be present, assays can be developed to detect the mutations. For example, SNP genotyping assays can distinguish, for example, the presence of a mutant allele from the presence of a wild-type allele.

[0411] A method for selecting a plant or a seed, the method comprising the steps of:

[0412] a) identifying a plant or a seed having a mutation in an allele of the gene encoding the CmBEL1 protein, wherein the wild-type allele of the gene encodes a CmBEL1 protein having at least 94%, 95%, 96%, 97%, or 98% or 99% sequence identity with SEQ ID NO: 1, and optionally

[0413] b) determining whether the plant or the progeny plant produced by self-fertilization is seed-abortive, and optionally

[0414] c) selecting a plant or a seed comprising at least one copy of the mutant allele of step a).

[0415] A method for producing a melon plant, the method comprising the steps of:

[0416] a) introducing a mutation into a plant or a seed population,

[0417] b) selecting a plant that produces seedless fruits after pollination and / or selecting a plant or a seed that contains the mutant cmbel1 allele in its genome,

[0418] c) optionally verifying whether the plant selected in b) has a mutation in the allele encoding the CmBEL1 protein, and optionally

[0419] d) growing or cultivating the plant or the seed obtained in c),

[0420] wherein the wild-type allele of the gene encodes a CmBEL1 protein having at least 94% sequence identity with any one of the proteins selected from the group consisting of SEQ ID NO: 1.

[0421] In another aspect, a melon plant, seed, and plant part are provided, which contain a mutation in the endogenous CmBEL1 gene, for example, an induced mutation generated by random mutagenesis or by targeted mutagenesis, wherein compared to the wild-type protein, gene expression is reduced or eliminated, or the expressed gene encodes a CmBEL1 protein with reduced function or loss of function.

[0422] Also provided herein is a method for screening a melon plant, seed, plant part, or their DNA for the presence of a mutant allele of a gene named CmBEL1, or for selecting a melon plant, seed, or plant part containing a mutant allele of a gene named CmBEL1, the method comprising the following steps:

[0423] a) Analyzing whether the genomic DNA contains a wild-type CmBEL1 allele encoding the protein of SEQ ID NO: 1 (or a wild-type protein having at least 94% identity with SEQ ID NO: 1), and / or a mutant cmbel1 allele encoding a mutant protein containing one or more amino acid substitutions, insertions, or deletions compared to the wild-type CmBEL1 protein, or a mutant allele that is not expressed or has reduced expression, and optionally

[0424] b) Selecting a plant, seed, or plant part containing, for example, two copies of the wild-type allele, or two copies of the mutant allele, or one copy of the wild-type allele and one copy of the mutant allele.

[0425] In one aspect, method step a) includes methods selected from the following:

[0426] i) Amplifying at least a portion of the CmBEL1 allele using one or more oligonucleotide primers that hybridize to DNA having the CmBEL1 allele,

[0427] ii) Hybridizing one or more oligonucleotide probes to at least a portion of the DNA having the CmBEL1 allele,

[0428] iii) Sequencing the DNA, mRNA, or cDNA having the CmBEL1 allele.

[0429] Thus, for example, a DNA sample can be obtained from a plant, seed, or plant part, and a PCR reaction can be performed to amplify a portion of the wild-type CmBEL1 allele and / or a portion of the mutant cmbel1 allele.

[0430] For example, a competitive PCR method such as KASP assay can be used to generate amplification products of alleles present at the CmBEL1 locus in genomic DNA. Similarly, oligonucleotide probes can generate hybridization products of alleles present at the CmBEL1 locus in genomic DNA. Primers or probes can be designed to be specific for a particular cmbel1 allele, for example to distinguish a wild-type allele from a mutant allele.

[0431] In one aspect, there is provided a genotyping assay for genotyping a melon plant, seed, plant part, cell or tissue, the assay comprising the steps of:

[0432] a) providing genomic DNA of one or more melon plants or plant populations, and

[0433] b) performing a genotyping assay that detects: the presence of a wild-type allele encoding the protein of SEQ ID NO: 1 or a wild-type allele encoding a protein having at least 94% sequence identity with SEQ ID NO: 1; and / or the presence of a mutant allele (or two different mutant alleles), wherein the mutant allele encodes a mutant protein comprising one or more amino acid insertions, deletions or substitutions compared to the wild-type protein of SEQ ID NO: 1 or compared to a wild-type protein having at least 94% sequence identity with SEQ ID NO: 1; or a mutant allele that is not expressed or has reduced expression, and optionally

[0434] c) selecting a plant, seed, plant part, cell or tissue comprising, for example, two copies of the wild-type allele or one copy of the wild-type allele and one copy of the mutant allele or two copies of the mutant allele.

[0435] In step b), the mutation in the mutant allele preferably results in the insertion, deletion or substitution of one or more amino acids relative to the wild-type protein, for example the mutant allele encodes one of the mutant CmBEL1 proteins described herein. Alternatively, in step b), the promoter is mutated, resulting in reduced or no expression of the allele.

[0436] Obviously, the presence of one or two mutant alleles can also be detected in the above assay, for example one or two copies of a specific mutant allele or two different mutant alleles. In the above method, the assay can detect the genotype of any CmBEL1 allele, whether it is a wild-type allele and / or one or more mutant alleles.

[0437] The wild-type allele is genomic DNA at the CmBEL1 locus on chromosome 9, for example. For example, SEQ ID NO: 4 provides herein the genomic DNA encoding the wild-type CmBEL1 protein, but equally, genomic sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 can also be genomic DNA sequences encoding the wild-type CmBEL1 protein.

[0438] Thus, in one aspect, one or more of the following alleles are detected in step b of the above method:

[0439] - the wild-type CmBEL1 allele encoding the protein SEQ ID NO: 1 or a wild-type protein having at least 94%, 95%,

[0440] 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1;

[0441] - the mutant cmbel1 allele encoding the CmBEL1 mutant protein, relative to the wild-type CmBEL1 allele encoding the protein SEQ ID NO: 1 or a wild-type protein having at least 94%, 95%,

[0442] 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, the mutant cmbel1 allele contains one or more amino acids inserted, substituted or deleted (see also elsewhere herein);

[0443] - the mutant cmbel1 allele encoding the mutant CmBEL1 protein, the mutant CmBEL1 protein contains one or more amino acids inserted, substituted or deleted in the homologous domain of SEQ ID NO: 1 or in the homologous domain of a wild-type protein having at least 94%, 95%,

[0444] 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1;

[0445] - the mutant cmbel1 allele encoding the mutant CmBEL1 protein, the mutant CmBEL1 protein is truncated at the C-terminus of SEQ ID NO: 1 or at the C-terminus of a wild-type protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, preferably wherein the truncation starts within or before the homologous domain;

[0446] - A mutant allele in which the promoter is mutated such that the allele has reduced expression or no expression.

[0447] Step a) may include isolating genomic DNA from a plant, seed, plant part, cell or tissue to be analyzed in a genotyping assay. As is known in the art, crude DNA extraction methods can generally be used.

[0448] Step b) preferably includes a biallelic genotyping assay that utilizes allele-specific oligonucleotide primers and / or allele-specific probes, i.e., primers or probes that distinguish, for example, a wild-type allele from a mutant allele or two mutant alleles.

[0449] A mutagen such as a chemical or radiation mutagen or a gene editing technique can be used to mutagenize the plant of step a). Thus, prior to step a), there may be a step of treating the plant, seed or plant part with a mutagen or inducing a targeted mutation in the CmBEL1 allele.

[0450] Various genotyping assays can be used as long as they can detect INDELs and / or SNPs and can distinguish, for example, a wild-type allele present in genomic DNA (at the CmBEL1 locus on chromosome 9) and / or one or more mutant alleles of the CmBEL1 gene present in genomic DNA.

[0451] Genotyping assays are generally based on allele-specific primers used in a PCR or thermal cycling reaction (polymerase chain reaction) to amplify a wild-type or mutant allele and detect the amplification product, or on allele-specific oligonucleotide probes that hybridize to a wild-type allele or a mutant allele or both. For example, genotyping using BHQplus probes uses two allele-specific probes and two primers flanking the polymorphic region, and during thermal cycling, the polymerase encounters an allele-specific probe bound to the DNA and releases a fluorescent signal. Allele discrimination involves the competitive binding of two allele-specific BHQPlus probes (see also biosearchtech.com).

[0452] Examples of genotyping assays are the KASP assay (performed by LGC, see www.LGCgenomics.com and www.biosearchtech.com / products / pcr-kits-and-reagents / genotyping-assays / kasp-genotyping-chemistry), which is based on competitive allele-specific PCR and end-point fluorescence detection, the TaqMan assay (Applied Biosytstems) which is also PCR-based, the HRM assay (high-resolution melting assay) in which allele-specific probes are detected using real-time PCR, or the rhAmp assay, BHQplus genotyping, BHQplexCoPrimer genotyping, etc., which are based on RNase H2-dependent PCR.

[0453] The KASP assay is also described in He C, Holme J, Anthony J. “SNP genotyping: the KASP assay. Methods Mol Bio1. 2014;1145:75-86” and EP1726664B1 or US7615620B2, which are incorporated herein by reference. The KASP genotyping assay uses a unique form of competitive allele-specific PCR combined with a new, homogeneous, fluorescence-based reporting system to identify and measure genetic variation occurring at the nucleotide level to detect single nucleotide polymorphisms (SNPs) or insertions and deletions (InDels). The KASP technology is applicable to a variety of device platforms and offers flexibility in terms of the number of SNPs and the number of samples that can be analyzed. The KASP chemistry works equally well in 96-well, 384-well, and 1,536-well microtiter plate formats and has been used by users in large and small laboratories in the fields of human, animal, and plant genetics for many years.

[0454] The TaqMan genotyping assay is also described in Woodward J. “Bi-allelic SNP genotyping using the assay.” Methods Mol Bio1. 2014;1145:67-74, US5210015, and US5487972, which are incorporated herein by reference. By utilizing TaqMan Techniques, allele-specific probes are used for rapid and reliable genotyping of known polymorphic sites. TaqMan assays are robust for genotyping a variety of variant types, including single nucleotide polymorphisms, insertions / deletions, and presence / absence variants. To interrogate a single diallelic polymorphism, two TaqMan probes labeled with different fluorophores are designed such that they hybridize to different alleles during PCR-based amplification of the surrounding target region. During the primer extension phase of PCR, the 5′-3′ exonuclease activity of Taq polymerase cleaves and releases the fluorophore from the bound probe. At the end of PCR, the emission intensity of each fluorophore is measured, and allele determination can be made at the interrogated site.

[0455] Thus, various genotyping assays can be used that can distinguish the presence and / or the presence of one or more mutant alleles of the CmBEL1 gene encoding the protein of SEQ ID NO: 1 or a protein having at least 94% identity to SEQ ID NO: 1, for example. Various mutant alleles of the CmBEL1 gene can be detected.

[0456] As mentioned, it is preferred to use a diallelic genotyping assay, such as the KASP assay, TaqMan assay, BHQplus assay, PACE genotyping (see world wide web idtdna.com / pages / products / qpcr-and-pcr / genotyping / pace-snp-genotyping-assays), or any other diallelic genotyping assay.

[0457] In one aspect, the genotyping assay in step b) of the above method is a KASP assay. Thus, in step b), competitive PCR is carried out using two forward primers and a common reverse primer. The two forward primers contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides complementary to the genomic sequence (or its complementary strand). Additionally, the two forward primers contain 1, 2, 3 or more nucleotides (preferably at the 3′ end of the primer), which provide specificity for, for example, differentiating an SNP or INDEL that differentiates, for example, the wild-type sequence of an allele from, for example, a mutant sequence or differentiates two mutant alleles. Thus, the two forward primers have different binding specificities (or preferences) for, for example, the wild-type allele and / or for, for example, the mutant allele. For example, the Fam- primer may contain, for example, 17 nucleotides of the wild-type sequence and 1 nucleotide specific for the nucleotides of the mutant allele, while the VIC- primer may contain 18 nucleotides of the wild-type allele and 1 nucleotide specific for the nucleotides of the wild-type allele. A KASP assay can be easily designed to differentiate, for example, the wild-type allele of the CmBEL1 gene and / or any mutant allele (which differs from the wild-type allele in one or more nucleotides by insertion, deletion or substitution) or to differentiate different mutant alleles of the gene, and thus, for example, the assay can be designed to detect any SNP or INDEL that differentiates any two CmBEL1 alleles.

[0458] In one aspect, the mutant allele of the CmBEL1 gene encodes a protein that contains one or more amino acids inserted, substituted or deleted relative to the wild-type protein of SEQ ID NO: 1.

[0459] Thus, in one embodiment, a method is provided for detecting and optionally selecting a melon plant, seed or plant part that contains at least one copy of the wild-type allele and / or mutant allele of a gene named CmBEL1, the method comprising:

[0460] a) providing genomic DNA of a melon plant or plants (e.g., breeding population, F2, backcross, etc.),

[0461] b) performing an assay that differentiates or can differentiate the presence of alleles in the genomic DNA of a) based on nucleic acid amplification (e.g., including using allele - specific oligonucleotide primers) and / or nucleic acid hybridization (e.g., including using allele - specific oligonucleotide probes), such as a diploid genotyping assay, to detect the presence of the wild - type allele of the gene and / or one or more mutant alleles of the gene, wherein the wild - type allele encodes the protein of SEQ ID NO: 1 (or a wild - type CmBEL1 protein having at least 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1), and the mutant allele encodes a protein having one or more amino acids inserted, deleted or substituted relative to the wild - type protein of SEQ ID NO: 1 (or relative to the wild - type CmBEL1 protein having at least 94%,

[0462] 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1); or the mutant allele contains a mutation in the promoter that results in reduced or no expression of the allele;

[0463] and optionally

[0464] c) selecting a plant, seed or plant part that contains one or two copies of the mutant allele.

[0465] In step b), the genotyping assay is based on nucleic acid (especially DNA) amplification reactions using, for example, oligonucleotide primers (such as PCR (polymerase chain reaction) and PCR primers, preferably allele - specific primers) and / or nucleic acid hybridization using probes that are oligonucleotides (preferably allele - specific probes) to differentiate, for example, the wild - type allele and / or one or more mutant alleles.

[0466] The primers or probes are preferably modified to contain a label, such as a fluorescent label, or contain a tail sequence or other modifications.

[0467] In one aspect, in any of the above methods, the assay uses one or more CmBEL1 allele - specific primers or one or more CmBEL1 allele - specific probes.

[0468]

[0469] ​As mentioned, PCR primers and nucleic acid probes can be designed using known methods or software programs for oligonucleotide design based on the genomic sequence of SEQ ID NO: 4 or other (e.g., degenerate) genomic sequences encoding the protein of SEQ ID NO: 1 or genomic sequences encoding mutant alleles of proteins that contain, for example, insertions, deletions, or substitutions of one or more amino acids compared to SEQ ID NO: 1. The length of the primers and probes can be, for example, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more nucleotides (bases) and anneal (or hybridize) to the template DNA sequence, i.e., they preferably have at least 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the target sequence. The specificity of the primer or probe pair for, for example, a wild-type allele or a mutant allele (or for two or more mutant alleles) is attributed to at least 1, 2, 3, or more nucleotides of the primer or probe being specific for either allele. Thus, primers or probes are designed around the polymorphism (e.g., SNP or InDel) between two (or more) alleles of the target gene such that they distinguish these alleles. In one aspect, the assay is a biallelic genotyping assay selected from, for example, a KASP assay, a TaqMan assay, a BHQplus probe assay, or any other biallelic genotyping assay.

[0470] In one aspect, the mutant allele is a null allele.

[0471] In one aspect, the mutant allele contains at least one codon inserted or duplicated in the coding region of the allele, or at least one codon changed to another codon (e.g., by a single nucleotide change), or at least one codon deleted or changed to a stop codon.

[0472] In any of the above methods, in one aspect, the mutant allele encodes a truncated protein that, for example, lacks one or more amino acids at the C-terminus, particularly at least one (or more) amino acids of the homeodomain and all subsequent C-terminal amino acids. Thus, in one aspect, the method can be used to distinguish plants, seeds, or plant parts that contain two copies of the wild-type CmBEL1 allele encoding the protein of SEQ ID NO: 1, two copies of the mutant cmbel1 allele encoding, for example, a truncated CmBEL1 protein, or one copy of each allele (heterozygous). In another aspect, the method can be used to distinguish plants, seeds, or plant parts that contain any one or more mutant CmBEL1 alleles encoding one or two copies of, for example, a truncated CmBEL1 protein. Optionally, plants, plant parts, or seeds containing any of these genotypes can be selected for, for example, further breeding or for melon fruit production, particularly seedless fruit production.

[0473] Although any DNA genotyping assay based on PCR (using PCR primers) and / or based on hybridization (using probes) can be used in the above methods, in one aspect, the KASP assay is used to distinguish wild-type alleles and mutant alleles. The assay can be used in a high-throughput manner, for example, in 96-well plates or more (e.g., 384-well plates).

[0474] In one aspect, the assay distinguishes the wild-type allele of SEQ ID NO: 4 from a mutant allele that contains a DNA insert in intron 2 between, for example, nucleotide 1655 and nucleotide 1656 of SEQ ID NO: 4, resulting in a transcript that contains only exon 1 and exon 2.

[0475] In another aspect, the assay distinguishes the wild-type allele of SEQ ID NO: 4 from a mutant allele that contains a stop codon within or before the homeodomain. For example, the codon for Y344 (TAC at nucleotides 1886 - 1888 of SEQ ID NO: 4) can be changed to a stop codon (TAG or TAA).

[0476] Depending on the differences (e.g., SNPs or INDELs) between the wild-type and / or mutant CmBEL1 alleles, various allele-specific primers and probes can be designed for use in the assay.

[0477] In one aspect, two forward primers (e.g., one for the wild-type allele and one for the mutant allele) and a common reverse primer (e.g., for both the wild-type allele and the mutant allele) are used in a KASP assay. In one aspect, the two forward primers and the reverse primer comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more nucleotides of the genomic CmBEL1 sequence or its complementary sequence. The forward primers further comprise at least 1, 2 or 3 nucleotides (preferably at the 3′ end of the primer) that confer specificity (or preference) for the amplification of, for example, the wild-type allele or the mutant allele; or confer specificity for different mutant alleles. Each forward primer forms a primer pair with the common reverse primer to amplify the DNA sequence of the target allele between the primer pairs during thermal cycling. Standard components for thermal cycling and standard components for the KASP assay are used.

[0478] In another embodiment, a method is provided for generating a hybridization product or an amplification product of, for example, one wild-type allele and / or one (or one or two or more) mutant alleles of a gene named CmBEL1, the method comprising:

[0479] a) providing genomic DNA of a melon plant or plants (e.g., a breeding population, F2, backcross, etc.),

[0480] b) performing an assay that differentiates or can differentiate the presence of alleles in the genomic DNA of a) (e.g., a diallelic genotyping assay) that generates a nucleic acid amplification product (e.g., by using allele-specific oligonucleotide primers to generate the product)

[0481] and / or the assay generates a nucleic acid hybridization product (e.g., by using allele-specific oligonucleotide probes to generate the hybridization product), whereby the amplification product or the hybridization product indicates the presence of the wild-type allele and / or the mutant allele of the gene in the DNA, wherein the wild-type allele encodes the protein of SEQ ID NO: 1 or a wild-type protein having at least 94% sequence identity with SEQ ID NO: 1), and the mutant allele encodes a protein that contains one or more amino acid insertions, deletions or substitutions relative to the wild-type protein of SEQ ID NO: 1 or a wild-type protein having at least 94% sequence identity with SEQ ID NO: 1),

[0482] or the mutant allele contains a mutation in the promoter that results in reduced expression or non-expression,

[0483] and optionally

[0484] c) Select plants, seeds or plant parts comprising a mutant allele with one or two copies.

[0485] Also provided is a method for amplifying all or part of a mutant and / or wild-type CmBEL1 allele from a genomic DNA sample derived from a melon plant, plant part or seed, the method comprising contacting the genomic DNA with a primer pair that amplifies all or part of the mutant cmbel1 allele and / or wild-type CmBEL1 allele in the sample, and detecting the amplification product.

[0486] Also provided is a method for hybridizing a probe to a mutant and / or wild-type CmBEL1 allele in a genomic DNA sample derived from a melon plant, plant part or seed, the method comprising contacting the genomic DNA with an oligonucleotide probe that hybridizes to the mutant cmbel1 allele and / or wild-type CmBEL1 allele in the sample, and detecting the hybridization product.

[0487] All embodiments described above and elsewhere herein also apply to these embodiments. Thus, the amplification product can be a PCR amplification product, such as a competitive PCR amplification product generated in, for example, a KASP assay or other assay, to detect a mutant allele (or one or two or more mutant alleles) and / or wild-type allele in a DNA sample. Thus, the hybridization product can be a hybridization product of an oligonucleotide probe that hybridizes to a nucleic acid in the DNA sample to detect, for example, a mutant and / or wild-type allele in the DNA sample. The primer pair or probe is preferably allele-specific, and thus the products can be distinguished as, for example, two copies of the wild-type allele, two copies of the mutant allele, or one copy of the wild-type allele and one copy of the mutant allele present in the genomic DNA of a melon plant, plant part or seed.

[0488] Preferably, the primer or probe is modified, for example, by labeling with a tail sequence or a fluorescent label, or otherwise modified relative to the wild-type sequence to which the primer or probe amplifies or hybridizes.

[0489] Since the method requires detecting a mutant and / or wild-type allele in the genomic DNA of a plant, plant part or seed, the genomic DNA needs to be easily detectable, for example, it can be extracted from plant cells using a DNA extraction method or at least eluted into a solution (such as a buffer solution) from damaged cells.

[0490] The above determination can be used, for example, in marker-assisted selection (MAS) of plants in breeding programs to select plants containing a specific genotype, such as plants homozygous for the wild-type allele of the CmBEL1 gene, plants homozygous or heterozygous for the mutant allele of the cmbel1 allele.

[0491] Accordingly, the present invention also provides a method for cultivating melon plants, the method comprising genotyping one or more seeds or plants for the allelic composition at the CmBEL1 locus in the genome, and optionally selecting one or more seeds or plants having a specific genotype at the CmBEL1 locus. In one aspect, genotyping of the CmBEL1 gene can also be performed by sequencing.

[0492] As mentioned, optionally, plants or seeds containing two copies of the mutant cmbel1 allele can be grown and phenotyped for seed abortion. In one aspect, the mutant allele is a mutant allele in homozygous form that confers seed abortion, especially a null allele.

[0493] In a different aspect, there is provided a melon plant, seed or plant part that contains at least one copy of a mutant allele of the gene named CmBEL1 in melons, wherein the mutant allele

[0494] a) contains one or more mutations in the promoter, resulting in non-expression or optionally reduced expression of the allele compared to the wild-type allele, and / or

[0495] b) encodes a mutant protein that contains one or more amino acid substitutions, insertions or deletions compared to the wild-type protein (especially where the protein is non-functional),

[0496] wherein the mutant allele of a) or b) confers seed abortion when the mutant allele is in homozygous form (compared to a plant containing the wild-type allele in homozygous form), and wherein the wild-type melon CmBEL1 allele encodes the protein of SEQ ID NO: 1 or a protein having at least 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 1, or wherein the wild-type genomic CmBEL1 allele contains SEQ ID NO: 4 or a wild-type genomic sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4.

[0497]

[0498] ​In addition, a method of crossing a plant comprising at least one of the mutant cmbel1 alleles described herein with a plant lacking, for example, the mutant cmbel1 allele is provided, and a method of selecting progeny comprising at least one copy of the mutant cmbel1 allele is provided.

[0499] Thus, in one aspect, a method for producing a melon plant is provided, the method comprising the steps of:

[0500] a) providing a melon plant comprising at least one copy of the described mutant cmbel1 allele;

[0501] b) crossing the melon plant with another melon plant to produce F1 seeds;

[0502] c) selecting F1 seeds comprising at least 1 copy of the mutant allele.

[0503] Optionally, selection or detection of the presence of the mutant cmbel1 allele in the method can be carried out using molecular methods such as SNP or INDEL genotyping, sequencing, etc.

[0504] Preferably, the allele in step a) is a mutant allele that, when in homozygous form, confers seed abortion. In one aspect, the plant in step a) is a melon plant comprising the mutant allele described herein, such as a truncated protein-encoding allele, in heterozygous or homozygous form.

[0505] A method for producing a melon plant is also provided, the method comprising the steps of:

[0506] a) introducing a mutation in a population of melon plants or providing a mutagenized population of melon plants, such as

[0507] a TILLING population of M2, M3 or higher generations,

[0508] b) identifying a plant having a mutation in the allele encoding the CmBEL1 protein, wherein the wild-type allele of the gene encodes a CmBEL1 protein having at least 94%

[0509] sequence identity to the protein of SEQ ID NO 1.

[0510] The method may further comprise one or both of the following steps:

[0511] selecting a plant comprising at least two copies of the mutant allele of step b),

[0512] determining whether the plant produces seedless fruits after pollination.

[0513] In addition, any sequence and sequence molecule is covered, just as sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% sequence identity to the provided sequence are covered. In addition, any fragment and / or modified sequence (e.g., a primer or probe comprising at least 10, 15, 16, 17, 18, 19, 20 or more nucleotides of the sequence or the complementary sequence) and their use for breeding (e.g., MAS) or for detecting or selecting plants or plant parts are provided.

[0514] When describing mutant proteins, obviously, genomic sequences and mRNA or cDNA sequences encoding the mutations that result in mutations in the proteins are covered herein, and these sequences can be used to detect alleles in the genome containing mutations that result in amino acid changes, and for example, for performing genotyping assays for mutant alleles.

[0515] Sequence description

[0516] SEQ ID NO 1: Wild-type CmBEL1 protein

[0517] SEQ ID NO 2: cDNA encoding wild-type CmBEL1 protein

[0518] SEQ ID NO 3: mRNA encoding wild-type CmBEL1 protein, which includes 5'UTR (nucleotides 1 to 630), a translation start codon at nucleotides 631 to 633, a translation stop codon at nucleotides 2455 to 2457, and 3'UTR (nucleotides 2458 to 2814)

[0519] SEQ ID NO 4: Genomic DNA encoding wild-type CmBEL1 protein, which is transcribed into pre-mRNA (containing introns and exons), and the pre-mRNA is processed into mRNA (intron splicing), see Figure 7

[0520] SEQ ID NO 5: DNA insert

[0521] SEQ ID NO 6: Promoter sequence of wild-type CmBEL1 allele

[0522] Examples

[0523] Example 1

[0524] Fruits of an in vitro cultivated melon population grown in the field were cut open and analyzed. A plant with seedless fruits was identified.

[0525] The seedless mutant was finely mapped in the F2 population derived from the cross H99 (seeded / recurrent) × H11X.7616-K (seedless).

[0526] The inheritance of the seedless trait was evaluated and it was mapped to a recessive locus on chromosome 9.

[0527] Since pollination was necessary to induce fruit set in plants homozygous for the mutant allele, it was concluded that the seedless trait was attributed to seed abortion.

[0528] The mutant allele conferring the seedless trait was introgressed into melon, cantaloupe, and Spanish melon backgrounds.

[0529] Long-read analysis identified a gene annotated as "low-quality protein: homeobox protein BEL1 homolog" to be located at the locus named MELO3C005699 in the cucurbitgenomics.org database.

[0530] Sequencing identified the wild-type genomic sequence (Seq ID No: 4) and the mutant genomic sequence (corresponding to SEQ ID NO: 4 but with a DNA insert in the second intron, see Figure 7 , the black line indicates the DNA insertion position after nucleotide 1655).

[0531] Sequence analysis revealed that the sequence information in the cucurbitgenomics database was incorrect. The correct sequence is provided herein.

[0532] Table 1

[0533]

[0534]

[0535] This gene was named CmBEL1 purely based on the name of the locus named MELO3C005699.

[0536] This gene has four exons and three introns, see Figure 7 , the exons are underlined. The DNA insert is present in the second intron (between exon 2 and exon 3) between nucleotides 1655 and 1656 of the genomic DNA (SEQ ID NO: 4). The amino acids encoded by the four exons are also indicated in Figure 1 , where three vertical black lines demarcate the amino acids encoded by the four exons. The vertical black line with a black star is the position between exon 2 and exon 3.

[0537] RT-PCR results showed that the mutant CmBEL1 allele generated truncated mRNA transcripts containing only exons 1 and 2 and lacking exons 3 and 4.

[0538] RT-PCR performed with primers corresponding to the cDNA of exons 1 and 2 produced amplicons with cDNA from both wild-type and mutant melons. However, RT-PCR performed with primers spanning the cDNA region corresponding to exons 2 to 3 produced amplification products only in wild-type plants and not in mutant plants ( Figure 6 ). This indicates that only the wild-type CmBEL1 allele produced full-length mRNA transcripts with exons 2 and 3, while in the mutant allele, the mRNA transcript terminated after exon 2. Thus, the mutant CmBEL1 allele produced truncated mRNA transcripts.

[0539] It can be concluded that the mutant allele generated a truncated CmBEL1 protein lacking the amino acids of exons 3 and 4.

[0540] Since the truncated protein lacks most of the homologous domains that are functional in DNA binding and the role of the protein as a transcription factor, it is concluded that the truncated protein is non-functional, at least its homologous domains are non-functional and cannot play its role in DNA binding and the regulation of gene expression of other genes.

[0541] Example 2 - Phenotype of homozygous mutants

[0542] Segregating populations containing the mutant CmBEL1 allele of Example 1 were generated in Spanish melon, cantaloupe, and honeydew backgrounds and analyzed in the field.

[0543] In all backgrounds, plants homozygous for the mutant CmBEL1 allele produced seedless fruits, see Figure 2 , Figure 3 , Figure 4 and Figure 5 . In addition, plants homozygous for the mutant CmBEL1 allele produced many more fruits, but with a lower average fruit weight, despite seeing a fairly wide range of fruit sizes. See, for example, Figure 8 , which shows the mutant and wild-type fruit weights (grams) and fruit numbers in a honeydew background.

[0544] There was generally a strong negative correlation between average fruit weight and fruit number (e.g., r = -0.46).

[0545] Empty seed cavities are seen in some genotypes but not in others. In addition, some genotypes show minute seeds, which are small, non-viable seeds (they can also be referred to as "empty seeds"). See, for example Figure 9 , where on the left is a seeded (wild-type) Spanish melon, while on the right is a seedless fruit (containing the mutant cmbel1 allele), and the seedless fruit contains minute seeds. Minute seeds with empty seed cavities or with non-empty cavities (filled with placental tissue) can be seen in some genotypes.

[0546] Example 3 - Targeted transposition (e.g., as described in WO2022 / 197749)

[0547] Constructs are made such that a TE (transposable element) is inserted into the endogenous allele of the CmBEL1 gene of SEQ ID NO: 4.

[0548] The TE insertion results in premature termination of the gene transcript after exon 2, and a truncated protein is translated from this transcript.

[0549] Since the TE insertion is in intron 2, the protein lacks the amino acids encoded by exons 3 and 4.

[0550] Plants are self-crossed to produce plants containing the mutant cmbel1 allele in homozygous form.

[0551] The plants are grown, and the developing melon fruits are cut open at maturity. Due to the lack of production of functional CmBEL1 protein, the fruits are seedless, and thus normal seed development cannot occur.

[0552] Example 4 - TILLING

[0553] Plants containing the mutant cmbel1 allele are identified from a melon TILLING population (generated by EMS mutagenesis). In the cmbel1 allele present in the plants, the codon for Y344 (TAC at nucleotides 1886 - 1888 of SEQ ID NO: 4) is changed to a stop codon (TAG or TAA).

[0554] Plants are self-crossed to produce plants containing the Y344STOP mutant cmbel1 allele in homozygous form.

[0555] The plants are grown, and the developing melon fruits are cut open at maturity. Due to the lack of production of functional CmBEL1 protein, the fruits are seedless, and thus normal seed development cannot occur.

Claims

1. A melon plant or plant part of the species melon, said melon plant or plant part comprising at least one copy of a mutant allele of a gene named CmBEL1, wherein said mutant allele a) comprises one or more mutations in the promoter of said CmBEL1 allele, resulting in no wild-type CmBEL1 mRNA transcript being produced, or b) encodes a loss-of-function protein comprising one or more amino acids with substitutions, insertions or deletions compared to the wild-type protein, wherein when the mutant allele of a) or b) is in homozygous form, said mutant allele confers seed abortion, and wherein the wild-type CmBEL1 allele encodes the protein of SEQ ID NO: 1 or a protein having at least 94% sequence identity with SEQ ID NO:

1.

2. The melon plant or plant part according to claim 1, wherein said mutant allele encodes a loss-of-function protein lacking the amino acids encoded by exon 4 and optionally one or more amino acids encoded by exon 3.

3. The melon plant or plant part according to claim 1, wherein said mutant allele encodes a loss-of-function protein lacking one or more amino acids of the homeodomain, said homeodomain starting at amino acid 318 of SEQ ID NO: 1 and ending at amino acid 379.

4. The melon plant or plant part according to any one of the preceding claims, wherein said mutant allele comprises a mutation resulting in a truncated protein, wherein said truncation starts within or before said homeodomain, said homeodomain starting at amino acid 318 of SEQ ID NO: 1 and ending at amino acid 379, such that at least amino acid M379 of said homeodomain is missing.

5. The melon plant or plant part according to any one of the preceding claims, wherein said mutant allele comprises a DNA insertion in the genomic sequence between exon 2 and exon 3 of the genomic sequence of SEQ ID NO:

4.

6. The melon plant or plant part according to any one of the preceding claims, wherein said mutant allele comprises a premature stop codon or a DNA insertion, resulting in a truncated loss-of-function protein.

7. The melon plant or plant part according to any one of the preceding claims, wherein said mutant allele is produced by random mutagenesis, or targeted mutagenesis such as a CRISPR-based method, or targeted transposition.

8. The melon plant or plant part according to any one of the preceding claims, wherein the plant is homozygous for said mutant allele.

9. A seed from which a melon plant according to any one of the preceding claims can grow.

10. A fruit produced by the melon plant according to claim 8, wherein the fruit is seedless.

11. A melon plant according to any one of claims 1 to 8, wherein the plant is homozygous for the mutant allele and produces at least 30% more fruit when grown under the same conditions than the plant that is homozygous for the wild-type allele.

12. A melon plant according to any one of claims 1 to 8, wherein the plant is homozygous for the mutant allele and produces at least 7, 8, 9, or 10 seedless fruits.

13. A melon plant part according to any one of claims 1 to 8, the melon plant part comprising at least one mutant allele according to any one of claims 1 to 7, wherein the plant part is a cell, flower, leaf, stem, cutting, ovule, pollen, root, fruit, protoplast, embryo, anther.

14. A melon plant according to any one of claims 1 to 8, wherein the plant is a vegetatively propagated plant.

15. A method of producing a seedless melon fruit, the method comprising growing a melon plant according to claim 8, whereby pollination of the flower induces fruit set, and wherein the fruit is seedless.

16. A method for screening a melon plant, a seed plant part, or their DNA for the presence of a mutant allele of a gene named CmBEL1, or for selecting a melon plant, seed, or plant part comprising a mutant allele of a gene named CmBEL1, the method comprising the steps of: a) analyzing whether the genomic DNA contains a wild-type CmBEL1 allele encoding a protein of SEQ ID NO: 1 or a protein having at least 94% sequence identity with SEQ ID NO: 1 and / or a mutant CmBEL1 allele encoding a mutant protein comprising one or more amino acid substitutions, insertions, or deletions compared to the wild-type CmBEL1 protein, and optionally b) selecting a plant, seed, or plant part comprising two copies of the wild-type allele, two copies of the mutant allele, or one copy of the wild-type allele and one copy of the mutant allele.

17. The method according to claim 16, wherein step a) comprises a method selected from the following: i) amplifying at least a portion of the CmBEL1 allele using one or more oligonucleotide primers that hybridize to the DNA having the CmBEL1 allele, ii) hybridizing one or more oligonucleotide probes to at least a portion of the DNA having the CmBEL1 allele, iii) sequencing the genomic DNA, mRNA, or cDNA having the CmBEL1 allele, wherein the CmBEL1 allele is an allele encoding a protein of SEQ ID NO: 1 or a protein having at least 94% sequence identity with SEQ ID NO:

1.

18. A method for screening and / or selecting melon plants, seeds or plant materials or plant parts, or DNA or RNA or proteins derived therefrom for the presence of a mutant CmBEL1 allele, the method comprising one or more of the following steps: a) determining whether the gene expression of the endogenous CmBEL1 allele is reduced or eliminated; b) determining whether the amount of the wild-type CmBEL1 protein is reduced or eliminated; c) determining whether there is mutant mRNA, cDNA or genomic DNA encoding a mutant CmBEL1 protein; d) determining whether there is a mutant CmBEL1 protein; wherein the endogenous CmBEL1 allele is an allele encoding the wild-type CmBEL1 protein of SEQ ID NO: 1 or a protein having at least 94% sequence identity with SEQ ID NO: 1.

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