MELON PLANTS PRODUCING SEEDLESS FRUIT

The present invention is directed to seedless fruit producing melon plants. The present invention also comprises methods for production of said melon plants and the use of nucleic acids encoding homeobox Transcription Factor protein for the production of seedless melon fruits. The gene and protein is referred to as CmBELI herein.

Most commercial seedless fruits have been developed from plants whose fruits normally contain numerous relatively large hard seeds. Seedless fruits are e.g. known for watermelon, tomato, cucumber, eggplant, grapes, banana, citrus fruits, such as orange, lemon and lime. As consumption of seedless fruits is generally easier and more convenient, they are considered valuable.

Fruit development normally begins when one or more egg cells (ovules) in the ovular compartment of the flower are fertilized by sperm nuclei from pollen.

Seedless fruits can result from two different phenomena. In some cases fruit develops without fertilization of the ovule by pollen, a phenomenon known as parthenocarpy. In other cases seedless fruits occur after pollination, when seed (e.g. embryo and/or endosperm) growth is inhibited or the seed dies early, while the remainder of the fruit continues to grow (stenospermocarpy). In contrast to parthenocarpy, stenospermocarpy requires pollination for initiation of fruit growth.

Stenospermocarpy in melon has only been described in WO2015136532A1. Herein a single amino acid substitution in a recessive gene, MEL03C009603, which codes for a Cys2 His2 Zinc Finger (ZF) protein, is described as leading to the ‘superfruiter’ phenotype, i.e. the production of many small, seedless melon fruits upon pollination. MEL03C009603 is located on chromosome 4 of the Cucumis melo genome (cucurbitgenomics.org, DHL92 v3.6.1).

In contrast, herein the loss-of-function of a different recessive gene, on chromosome 9, was found to lead to seedless melon fruits developing. In the mutant plants it was found that a DNA insertion in the allele resulted in a truncated mRNA-transcript and truncated protein being produced, and plants homozygous for the mutant allele produced numerous seedless fruits. The mutant allele is also referred to as a ‘null mutant’ or ‘knock-out mutant’ allele. Similar to the ‘superfruiter’ phenotype, average fruit number was significantly increased in the homozygous mutant plants, while average fruit weight was significantly reduced.

Mapping of the gene lead to the finding that the gene called MEL03C005699 on chromosome 9 was disrupted. However, it was also found that the sequences given in the cucurbitgenomics.org database for this gene did not correspond to the correct sequences. The correct sequences were found by genomic analysis and are provided herein. The null mutant allele had a DNA insertion in the transcribed region of the gene, which lead to a truncated mRNA transcript and truncated protein, whereby the homeodomain-encoding region of the protein was 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 lacked all amino acids starting from Y344 to the end of the protein.

As MEL03C005699 is said to encode a ‘LOW QUALITY PROTEIN: homeobox protein BEL1 homolog’ protein, the gene is herein referred to as CmBELI. However, it should be mentioned that the sequence identity to the Arabidopsis BEL1 protein is only 43.2% and that the name is purely based on the previous annotation given in the database cucurbitgenomics.org. Besides the seedless fruit phenotype in melon plants homozygous for the mutant cmbell, and thus the development of small but numerous seedless fruits, no abnormalities in e.g. the development and/or the morphology of organs, such as flowers, were noticed.

In Arabidopsis thaliana mutations in BEL1 and AP2 disrupt ovule development. In addition to the ovule defects, mutations in BEL1 terminates the inflorescences, changing indeterminate flowering into terminate flowering. Also the developing siliques of the BEL1 mutant sometimes 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 the developing ovules of Arabidopsis, which regulates the transcription of other genes.

Bellaoui et al. 2001 showed that Arabidopsis BEL1 can interact with a specific subset of Arabidopsis KNOX proteins (KNATs) to form heterodimeric complexes. The authors also found that in Arabidopsis BEL1 is expressed in the inflorescence SAM (shoot apical meristem) and suggest that BEL1 has a direct role in maintaining the indeterminate growth of the inflorescence meristem, possibly by inhibiting the floral development program.

Kumar et al. (2007 Plant Cell. 2007 Sep; 19(9): 2719-2735, doi: 10.1105/tpc.106.048769) describe that in Arabidopsis there are 13 members of BEL1-like TALE homeodomain protein (BHL) family, that form heterodimeric complexes with the Class 1 KNOX TALE homeodomain proteins. These BHL proteins are closely related in sequence and the authors study redundancy of two of these BHL proteins (BLH2/SAW1 and BHL4/SAW2). Phylogenetic analysis of the gene family indicates that these two genes are most closely related in sequence to BEL1 .

As mentioned, the inventors have found a recessive gene on chromosome 9, referred to as CmBELI, which, when knocked-out or mutated to encode a non-functional protein, results in melon plants producing seedless fruits. Thus, in one aspect melon plants and plant parts comprising one or two copies of a null mutant allele of the CmBELI gene are one aspect provided herein.

Optionally, plants comprising a CmBELI allele which is knocked-down or mutated to encode a reduced-function protein, may also result in melon plants producing seedless fruits. Such plants and plant parts are also encompassed herein, provided that the seedless fruit phenotype (stenospermocarpy) is seen when the mutant allele is in homozygous form.

Therefore, one aspect herein is a melon plant comprising at least one mutant allele of the CmBELI gene, whereby the mutant allele results in stenospermocarpy when the mutant allele is in homozygous form, due to either a mutant protein being produced which has reduced function or loss-of-function compared to the wild type CmBELI protein, or due to the mutant allele having reduced gene expression or no gene expression compared to the wild type CmBELI allele, resulting in less or no wild type mRNA-transcript or less or no wild type CmBELI protein being made in the plant compared to a control melon plant (lacking a mutant cmbell allele, but comprising two wild type alleles of the CmBELI gene).

A mutant cmbell allele may, thus, comprise one or more amino acids inserted, deleted or replaced compared to the wild type CmBELI protein, or a mutant cmbell allele may comprise one or more mutations in a regulatory region of the protein, such as a promoter or enhancer, resulting in reduced or no functional wild type protein being made from the allele, which thereby results in stenospermocarpy when the mutant allele is in homozygous form.

The CmBELI promoter sequence is located within the 1000 or 2000 bases upstream of the 5’IITR (untranslated region) and is provided herein as SEQ ID NO: 6 or a sequence comprising at least 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 6. In one aspect the CmBELI promoter of SEQ ID NO: 6 or the CmBELI promoter comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6 comprises one or more mutations (insertion, deletion and/or replacement of one or more nucleotides in SEQ ID NO: 6 or in a CmBELI promoter sequence comprising at least 97%, 98% or 99% sequence identity to SEQ ID NO: 6, whereby the transcription of the cmbell gene is abolished completely (no mRNA transcript is generated) or is reduced by at least 80%, 90%, 95%, 96%, 97%, 98% or 99% compared to the wild type CmBELI allele.

A mutant cmbell allele may, in one aspect, comprise one or more nucleotides inserted, deleted or replaced compared to the wild type CmBELI allele, either in the promoter region or in the transcribed region or coding region of the gene, whereby the mutant allele does not produce wild type mRNA-transcript, but produces either e.g. no mRNA transcript or a prematurely terminated mRNA transcript, or an mRNA transcript comprising one or more codons inserted, replaced or deleted compared to the wild type transcript, leading to no functional wild type protein being made from the allele, which thereby results in stenospermocarpy when the mutant allele is in homozygous form.

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 not functional in the plant (e.g. due to a mutation in the allele leading to a non-functional protein).

In one aspect the endogenous allele is disrupted by e.g. a DNA insertion in the gene (e.g. in the promoter or in the transcribed region or in the coding region), e.g. a transposable element (TE) being inserted into the gene, see WO2022/197749 on targeted insertion via transposition of TEs. In one aspect the DNA, e.g. the TE or TE-like element, is inserted into the gene in the promoter region or in the transcribed region or coding region, e.g. in the intron region between exon 2 and exon 3. In one aspect the DNA insert results in premature termination of the mRNA-transcript, e.g. in or after exon 1 , or in or after exon 2, or in or after exon 3. In one aspect 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 in length.

The above mutants, or other mutants in the endogenous CmBELI gene of a plant, can be generated by e.g. random mutagenesis or targeted mutagenesis, such as CRISPR- based methods. A review of targeted gene editing is provided e.g. by Erpen-Dalla Corte et al. in Plants

2019, 8, 601 (doi:10.3390/plants8120601) and by Bed Prakash Bhatta and Subas Malla in Plants

2020, 9, 1360; doi:10.3390/plants9101360. Crispr-based editing has also already been carried out in melon and other cucurbit crops and can thus be used by the skilled person to edit the endogenous CmBELI gene of melon to e.g. generate an endogenous null-allele. For example CRISPR has been used in cucumber to generate mutants in a target gene as described in WO2017098508. Also, in watermelon CRISPR has been successfully used to modify target genes, see e.g. Wang, Y., Wang, J., Guo, S. et al. CRISPR/Cas9-mediated mutagenesis of CIBG1 decreased seed size and promoted seed germination in watermelon. Hortic Res 8, 70 (2021). https://doi.org/10.1038/s41438-021-00506-1. Furthermore, Andrea Giordano et al. (bioRxiv 2022.01.30.478227; doi: https://doi.org/10.1101/ 2022.01.30.478227) published ‘CRISPR/Cas9 gene editing uncovers the role of CTR1 and ROS1 in melon fruit ripening and epigenetic regulation’ wherein melon plants were edited using Crispr/Cas9, generating loss-of- function mutants of two target genes.

Alternatively, mutants in the endogenous CmBELI gene can be generated by targeted insertion via transposition of TEs as described in e.g. WO2022/197749 (incorporated herein by reference).

The wild type, functional CmBELI protein is shown in Figure 1 , whereby the conserved homeodomain is highlighted in the boxed region. The homeodomain is a DNA binding domain involved in transcriptional regulation of other genes in the plant. A mutant CmBELI protein lacking all or part of the homeodomain is, therefore, non-functional in vivo and the mutant allele will, in homozygous form, result in stenospermocarpy.

The homeodomain starts at amino acid W318 and ends at amino acid M379 of SEQ ID NO: 1 , or the equivalent amino acids in a variant sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. In the transcribed region of the gene, the homeodomain starts at nucleotide 1263 of SEQ ID NO: 4 (nucleotide 1263 to 1265 encodes W318) and ends at nucleotide 2427 of SEQ ID NO: 4 (nucleotide 2725 to 2727 encodes M379), or at the equivalent nucleotides in a sequence comprising at least 97%, 98% or 99% sequence identity to SEQ ID NO: 4.

In one aspect a melon plant is provided, comprising a mutant allele of the CmBELI gene, wherein the mutant allele results in one or more amino acids of the homeodomain being deleted, one or more amino acids being inserted into the homeodomain or one or more amino acids of the homeodomain being replaced by different amino acids, rendering the encoded protein nonfunctional in vivo.

Regarding mutations in the homeodomain (or in other parts of the protein), in one aspect especially mutations which lead to amino acid replacements, whereby the properties of the wild type amino acid and the replaced amino acid are different, are one aspect herein, as such different amino acid properties will reduce or abolish the normal function of the protein and/or of the domain. So, for example a replacement of a non-polar amino acid by a polar amino acid (comprising a hydrophilic side chain), or vice versa, or the replacement of an amino acid having a charged side chain with a non-charged 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 lie), Leucine (L or Leu), Methionine (M or Met), Phenylalanine (F or Phe), Proline (P or Pro), Tryptophan (W or Trp), Valine (V or Vai). Polar amino acids are Arginine (R or Arg), Asparagine (N or Asn), Aspartate (D or Asp), Glutamate (E or Glu), Glutamine (Q or Gin), Histidine (H or His), Lysine (K or Lys), Serine (S or Ser), Threonine (T or Thr), Tyrosine (Y or Tyr).

Thus, in one aspect any one (or more) of the non-polar amino acids of the conserved homeodomain are replaced by a polar amino acid and/or any one (or more) of the polar amino acids of a conserved domain are replaced by a non-polar amino acid. The resulting mutant allele can then be tested for its function by generating a plant homozygous for the mutant allele and analysing the phenotype. If the mutant allele results in the plant becoming stenospermocarpic, then the mutant allele is an allele encodes a mutant CmBELI protein having reduced function or no function in vivo. In another aspect the mutant cmbell allele encodes a truncated protein, whereby 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 of the C-terminal end of the wild type CmBELI protein are missing or are optionally replaced by different amino acids, rendering the protein to have a reduced in vivo function or no in vivo function. Examples of mutant alleles encoding a truncated protein are given herein in the Examples, where e.g. the mRNA transcript of the mutant allele terminates after exon 2 and the translated protein comprises only amino acids 1 to 343 of SEQ ID NO: 1 (or 1 to 343 in a variant CmBELI protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1). Also a Y344STOP mutant is described in the Examples. Thus, in these mutant proteins 36 amino acids of the wild type homeodomain, which is 62 amino acids long, are not present, resulting in the loss-of-function. Thus, of the entire BEL1 protein, which is 608 amino acids long, 265 amino acids are missing.

Thus, in one aspect the mutant allele results in a truncated protein, whereby 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 missing (compared to the wild type protein). The truncated protein is preferably truncated at the C-terminal end, whereby 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 function in vivo (or optionally a reduced function in vivo) and confers stenospermocarpy when the mutant allele is in homozygous form.

In another aspect the mutant allele results in a mutant protein, whereby 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 has no function in vivo (or optionally a reduced function in vivo) and confers stenospermocarpy when the mutant allele is in homozygous form.

In yet another aspect the mutant allele results in a mutant protein, whereby at least 1 , 2, 3, 4, 5,

6, 7, 8, 9, 10, or more amino acids are inserted in the homeodomain. The mutant protein preferably has no function in vivo (or optionally a reduced function in vivo) and confers stenospermocarpy when the mutant allele is in homozygous form.

In another aspect the mutant allele results in a mutant protein, whereby at least 1 , 2, 3, 4, 5, 6,

7, 8, 9, 10, or more amino acids are deleted in the homeodomain. The mutant protein preferably has no function in vivo (or optionally a reduced function in vivo) and confers stenospermocarpy when the mutant allele is in homozygous form.

In another aspect the mutant allele results in a truncated protein, whereby at least 50, 60, 70,80, 90, 100, 150, 200, 250, 265 amino acids or more of the C-terminal end of the wild type protein are missing or are replaced by one or more different amino acids (compared to the wild type protein). The mutant protein preferably has no function in vivo (or optionally a reduced function in vivo) and confers stenospermocarpy when the mutant allele is in homozygous form.

These mutant alleles result preferably in a loss-of-function of the wild type protein and, therefore, lead to stenospermocarpy when the mutant allele is in homozygous form in the melon plant. As mentioned, the phenotype can be tested in vivo, by growing the homozygous plant, allowing flowering and pollination, 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 fruits that develop are cut open to inspect whether they are seeded or seedless. Also the average fruit number per plant (per genotype) and/or the average fruit weight per plant (per genotype) can be measured. In one aspect the mutant cmbell allele confers stenospermocarpy and further also a significant increase in the average number of fruits and/or a significant reduction in the average fruit weight compared to the wild type control plant. The control plant is preferably of the same type and same or similar genetic background as the plants comprising the mutant cmbell allele, so that phenotypic effects can be attributed to the mutant allele. In one aspect control plants are isogenic or near isogenic lines or wild type lines which were used as starting material to modify the endogenous CmBELI gene, which differ little or not at all in the genetic background beside the alleles at the CmBELI locus.

Melon plants are diverse for sex expression and may produce different types of flowers, include hermaphrodite (all bisexual flowers), andromonoecious (male flowers and bisexual flowers on the same plant), monoecious (male and female flowers on the same plant), or gynoecious (all female flowers). The most frequent sex type of commercial melon cultivars is andromonoecious, with male and bisexual flowers produced according to a developmental gradient. The flowers develop in the leaf axils or nodes. Pollination herein refers generally to self-pollination of the plant, at least in sex forms where the plant produces flowers that produce pollen (male flowers or bisexual flowers) and female or bisexual flowers on the same plant.

In one aspect the melon CmBELI gene is the gene encoding a CmBELI protein, wherein a CmBELI protein is the protein of SEQ ID NO: 1 or a protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

The melon BEL1 gene may also be referred to as CmBELI, for Cucumis melo BEL1 and the wild type genomic sequence is provided herein in SEQ ID NO: 4, encoding the wild type protein of SEQ ID NO: 1. Other cultivated melons may contain an allelic variant of the CmBELI 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) CmBELI protein having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Such proteins are herein also referred to 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, they should lead to stenospermocarpy when mutated to knock-out gene expression or when mutated to encode a loss-of-function protein or when the gene is deleted. E.g. an allelic variant into which the same mutation is introduced as generated herein in e.g. the Examples, should give the same phenotype (stenospermocarpy) when homozygously present in a diploid plant.

In one aspect of the invention a melon plant or melon plant cell is provided, characterized in that the plant or plant cell has at least one copy of a mutant cmbell allele which causes stenospermocarpy when the mutant allele is in homozygous form, compared to a corresponding wild type plant or plant cell, wherein the CmBELI protein of the wild type plant or plant cell is encoded by nucleic acid molecules selected from the group consisting of: a) nucleic acid molecules, which encodes a protein with the amino acid sequence given under SEQ ID NO: 1 ; b) nucleic acid molecules, which encodes a protein, the sequence of which has an identity of at least 94%, 95%, 96%, 97%, 98% or 99% with the amino acid sequence given under SEQ ID NO: 1 ; c) a nucleic acid molecule of SEQ ID NO: 4 or a sequence comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 and encoding a CmBELI protein.

The mutant cmbell allele may comprise a knock-down or knock-out of the gene expression of the wild type CmBe/1 allele (e.g. through a mutation in the promoter or other regulatory sequence of the wild type BEL1 allele), whereby no wild type mRNA-transcript or reduced wild type mRNA - transcript is made, or through the mutant cmbell allele encoding a loss-of-function or reduced- function CmBell protein (mutant CmBe/1 protein).

In one aspect the plant or plant cell comprises at least one copy of a null mutant allele for the CmBELI gene, i.e. said null allele results in no expression of the wild type gene and therefore no production of any wild type protein, or said null allele encodes a loss-of-function protein, through e.g. an insertion, replacement or deletion of one or more nucleotides in the gene. A null allele or knock-out allele is an allele resulting in absence of wild CmBELI protein being made by that allele in the cell or tissues where the wild type CmBELI protein is normally produced or resulting in a loss-of-function protein being made by that allele in the cell or tissue where the wild type CmBELI protein is normally produced. Thus, when a null allele is present in a plant or plant cell in homozygous form, no functional CmBELI protein is made by the cell or plant homozygous for the null allele. In one aspect the endogenous wild type CmBELI allele is deleted completely, or is deleted partially, or comprises a DNA insert in the allele (e.g. a transposon or a transposon like element being inserted, or a DNA fragment being inserted), whereby the allele is a null allele.

In one aspect the endogenous wild type CmBELI allele is mutated and the mutant cmbell allele encodes a mutant CmBELI protein having decreased function or loss-of-function compared to the wild type protein, e.g. the mutant CmBELI protein comprises one or more amino acids replaced, deleted and/or inserted compared to the wild type protein.

In one aspect the mutant allele encodes a protein which lacks one or more (or all) amino acid of the homeodomain of SEQ ID NO: 1 (which homeodomain starts at amino acid 318 and ends at amino acid 379 of SEQ ID NO: 1 , or the equivalent amino acid of a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% with the amino acid sequence given under SEQ ID NO: 1) or wherein one or more amino acids of the homeodomain are replaced by one or more different amino acid or are missing, e.g. due to a stop codon mutation in a codon preceding the codons of the homeodomain or in a codon of the homeodomain or due to an insertion of one or more nucleotides into the transcribed region of the gene, leading to a reduced function or preferably loss of function CmBELI protein.

For example, the mutant allele may encode a truncated protein wherein one or more amino acids of the C-terminal end, including e.g. one or more amino acids of the homeodomain are missing, e.g. the encoded protein may only comprise amino acids 1 to P343 (see also the Examples), due to e.g. DNA insertion between the codon for P343 and Y344 or due to a Y344STOP mutation. In one aspect any codon for amino acid 1 to amino acid 379 may be changed into a premature STOP codon, whereby the truncated protein is rendered non-functional. In another aspect 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 transcribed region of the gene starting at nucleotide 1 and ending at nucleotides 2425, 2426 or 2427 (encoding M379) of SEQ ID NO: 4 is encompassed herein, 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, any nucleotide replacement 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 and ending at nucleotides 2425, 2426 or 2427 (nucleotides 2425-2427 encode M379 of the homeodomain) of SEQ ID NO: 4 is encompassed herein, as the truncated or mutated protein is rendered non-functional. In still another aspect, 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 and ending at nucleotides 2425, 2426 or 2427 (nucleotides 2425-2427 encode M379 of the homeodomain) of SEQ ID NO: 4 is encompassed herein, as the truncated or mutated protein is rendered non-functional. The homeodomain is the domain starting at (and including) amino acid 318 of SEQ ID NO: 1 and ending at (and including) amino acid 379 of SEQ ID NO: 1. The amino acids equivalent to amino acids 318 to 379 of SEQ ID NO: 1 (i.e. , the homeodomain) of a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% with the amino acid sequence given under SEQ ID NO: 1 , can be easily identified by pairwise sequence alignment.

Thus, in one aspect the mutant CmBELI protein comprises one or more amino acids replaced, deleted and/or inserted in the conserved homeodomain of the protein. This insertion, deletion or replacement of one or more amino acids of the homeodomain preferably renders the protein non-functional in vivo, as can be tested by the phenotype when the mutant allele is in homozygous form.

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 an insertion, deletion or replacement of one or more nucleotides into the allele, resulting in a loss-of-function (or decreased function) protein and stenospermocarpy when the allele is in homozygous form (when no wild type allele is present in the diploid plant or plant cell).

Thus, in one aspect the mutant allele comprises an insertion, deletion or replacement of one or more nucleotides in the genomic allele, e.g. into SEQ ID NO: 4 (or a genomic sequence comprising at least 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4), said nucleotide insertion, deletion or replacement preferably resulting in the mutant allele being a null allele. In one aspect one or more nucleotides are inserted, deleted or replaced in the region starting at nucleotide 1 of SEQ ID NO: 4 and ending at nucleotide 2427 of SEQ ID NO: 4 (or the equivalent nucleotides in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4). The insertion, deletion or replacement of one or more nucleotides is in one aspect in a region that is an exon, but can also be in an intron region or in a splice site. Figure 7 shows the intron and exon regions of SEQ ID NO: 4.

In one aspect the insertion, deletion or replacement of one or more nucleotides in the genomic DNA of SEQ ID NO: 4 (or in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4) results in the encoded protein not comprising a wild type, functional homeodomain anymore, e.g. one or more amino acids of the homeodomain are deleted or replaced by one or more other amino acids, or one or more amino acids are inserted into the homeodomain.

In one aspect the insertion, deletion or replacement of one or more nucleotides is in between the codon for W318 and the codon for M379 of SEQ ID NO: 4 (or between codons coding for the equivalent amino acids in a sequence that comprises at least 94% identity to SEQ ID NO: 1); in one aspect the insertion of one or more nucleotides is in between the codon for P343 and the codon for Y344 of SEQ ID NO: 4 (or between codons coding for the equivalent amino acids in a sequence that comprises at least 94% identity to SEQ ID NO: 1). In one aspect the insertion of nucleotides in SEQ ID NO: 4, which renders the allele to encode no wild type protein or a loss- of-function (or reduced function) mutant protein comprises 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.

In another aspect one or more amino acids of the conserved homeodomain are missing, e.g. through a mutation causing a premature STOP codon (e.g. in or prior to the homeodomain, i.e. in or prior to amino acid M379), resulting in a loss-of-function (or decreased function) protein and stenospermocarpy when the allele is in homozygous form (when no wild type allele is present in the diploid plant or plant cell).

In another aspect the mutant protein is truncated, missing 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 of the C-terminal end of the wild type CmBELI protein of SEQ ID NO: 1 (or of a wild type protein comprising at least 94% identity to SEQ ID NO: 1). Optionally the missing wild type amino acids may be replaced by one or more different amino acids, rendering the protein to have no in vivo function (optionally a reduced in vivo function, which still results in the same phenotype).

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

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

In another aspect the insertion, deletion or replacement of one or more nucleotides in the genomic DNA of SEQ ID NO: 4 (or in a sequence comprising 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, rendering the protein non-functional (or having a decreased function) in vivo as seen by the stenospermocarpic phenotype when the mutant allele is in homozygous form.

A loss-of-function of the protein (and optionally a decreased function) is present when the mutant allele changes the in vivo phenotype from the wild type phenotype, i.e. seeded fruits developing after pollination when the wild type allele is present in homozygous form, into stenospermocarpy (seedless fruits developing after pollination) when the mutant allele is in homozygous form in a diploid plant.

The equivalent amino acids in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or more sequence identity to SEQ ID NO: 1 can be identified by pairwise alignment (e.g. using the program Needle) with SEQ ID NO: 1. Likewise the equivalent nucleotide in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or more sequence identity to SEQ ID NO: 4 (or other sequences herein e.g. SEQ ID NO: 2 or 3) can be identified by pairwise alignment (e.g. using the program Needle) with SEQ ID NO: 4 (or with other sequences herein, accordingly, such as SEQ ID NO: 2 or 3).

In one aspect Y344 of the melon protein of SEQ ID NO: 1 (or the equivalent amino acid in a sequence comprising at least 94%, 95%, 96%, 97% or more sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, is deleted or is replaced by a stop codon.

SUMMARY

A cultivated melon plant or plant part is provided comprising at least one copy of a mutant allele of a gene named CmBELI, said mutant allele conferring stenospermocarpy when the mutant allele is in homozygous form.

The mutant allele does not only confer that the fruits that develop on the plant homozygous for the mutant allele are seedless, but in one aspect also confers an increased average number of fruits developing on the plant and/or a reduced average fruit weight of the fruits that develop on the plant homozygous for the mutant allele. In one aspect 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 the control plant (a plant comprising the wild type CmBELI allele in homozygous form), when grown under the same environmental conditions. In another aspect the average weight of the fruits is reduced by at least 5%, 10%, 20%, 30%, 40%, or 50%, or more, compared to the average weight of fruits produced by the control plant (a plant comprising the wild type CmBELI allele in homozygous form), when grown under the same environmental conditions. In one aspect the plant homozygous for the mutant allele produces on average at least 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more fruits on the plant.

In one aspect the CmBELI gene is located on chromosome 9 of the melon genome DHL92, v3.6.1 (cucurbitgenomics.org), especially the gene is located in a region starting at base pair 23597842 and ending at base pair 23600944 of chromosome 9. 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 comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6. In one aspect the promoter which causes mRNA transcription of the CmBELI allele is located upstream of nucleotide 23597097 of chromosome 9, so e.g. in the region starting at nucleotide 23595097 and ending at nucleotide 23597096, or in the region starting at 23596097 and ending at nucleotide 23597096 of chromosome 9.

In one embodiment the melon plant or plant part comprising the mutant allele of the CmBELI gene is diploid. Preferably the mutant allele is present in two copies in a diploid melon plant or plant part.

The melon plant part comprising the mutant allele of the CmBELI gene may be a cell, a flower, a leaf, a stem, a cutting, an ovule, pollen, a root, a rootstock, a fruit, a protoplast, an embryo, an anther.

Also encompassed is a vegetatively propagated melon plant propagated from such a plant part comprising at least one mutant allele of the CmBELI gene, preferably two mutant alleles, so that the plant produces seedless fruits after pollination.

Likewise, a seed from which a plant of the invention can be grown is provided. The seed comprises one or two copies of a mutant allele of the CmBELI gene.

Further, a seedless fruit or fruit part produced by a plant according to the invention is provided. Such a seedless fruit or fruit part comprising two mutant allele of the CmBELI gene in its cells, like the plant on which it is produced.

A method of producing seedless melon fruits is provided, said method comprises growing a diploid plant comprising two copies of a mutant allele of a CmBELI gene and harvesting the fruits produced by said plants. In particular the melon fruits develop after pollination of the flowers (which induces fruit set) and are seedless. The fruit tissue also comprises two copies of the mutant allele.

A method for production of a stenospermocarpic cultivated melon plant is provided, comprising the steps of: a) introducing mutations in a population of melon plants or seeds; or providing a population of mutant melon plants or seeds (e.g. a TILLING population, e.g. M2, M3, M4 or further generation), b) selecting a melon plant producing seedless fruits (after pollination of the flowers); c) optionally verifying if the melon plant selected under b) comprises a mutant allele of a CmBEL 1 gene, especially a null allele; and d) optionally growing the melon plants obtained under c). A method for production of a stenospermocarpic cultivated melon plant is provided comprising the steps of: a) introducing mutations in a cultivated melon plant or seed; or providing a population of mutant melon plants or seeds (e.g. a TILLING population, e.g. M2, M3, M4 or further generation), b) selecting a melon plant comprising a mutant allele of the CmBELI gene; c) optionally selfing the selected plant to generate a melon plant homozygous for the mutant allele of the CmBELI gene, especially a mutant null allele, d) optionally growing the plants, e.g. to allow fruits to develop and confirm that the fruits are seedless.

A melon plant, seed or fruit produced by the method is encompassed herein.

Use of a stenospermocarpic melon plant for producing seedless melon fruits following pollination of the flowers of the plant is also an aspect of the invention.

Use of a mutant cmbell allele of a CmBELI gene as described herein for producing stenospermocarpic melon plants is also an aspect of the invention.

A method for production of a cultivated melon plant producing seedless fruits following pollination is provided, comprising the steps of: a) introducing random or targeted mutations (e.g. using Crispr based methods) into one or more melon plants, plant parts or seeds; or providing a population of mutant plants or seeds (e.g. a TILLING population, e.g. M2, M3, M4 or further generation), b) selecting a plant comprises a mutant allele of a CmBELI gene, e.g. a mutant allele which produces significantly reduced or no wild type CmBELI protein (e.g. a knock-out allele) or which encodes a protein which comprises one or more amino acids deleted, replaced or inserted compared to the wild type protein, especially a null mutant allele or loss-of-function allele, c) optionally removing any transgenic construct (e.g. CRISPR construct) from the plant, and/or d) optionally generating a plant homozygous for the mutant allele and analysing whether seedless fruits develop after pollination.

A method for selecting or identifying melon plants, seeds or plant parts is provided comprising the steps of: a) analysing whether the genomic DNA of the plant or plant part or seed comprises a mutant allele and/or comprises a wild type allele of the CmBELI gene in their genome and optionally b) selecting a plant or plant part or seed comprising one or two copies of a mutant allele of the CmBELI gene in the genome, especially a null allele, wherein the wild type allele of the melon CmBELI gene encodes the protein of SEQ ID NO: 1 (or a wild type protein comprising at least 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1).

Step a) can be carried out in various ways, using e.g. PCR based methods, sequencing based methods, nucleic acid hybridization based methods, gene expression levels, etc. In one aspect for example a KASP assay may be used.

A method for screening (e.g. genotyping) genomic DNA of melon plants, seeds or plant parts is provided comprising the steps of: a) providing a sample (or a plurality of samples) of genomic DNA of a melon plant or of a plurality of plants (e.g. a F2 population, inbred lines, a backcross population, a breeding population, hybrid plants, etc.), b) providing a pair of PCR primers or an oligonucleotide probe, which primers or (oligonucleotide) probe comprise at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 or more consecutive nucleotides of the genomic CmBELI allele of the melon CmBELI gene and can hybridize to the genomic allele and/or amplify part of the genomic allele in a PCR assay, and c) carrying out a PCR assay using the primer pair or a hybridization assay using the probe of step b) on the sample(s) of step a), and optionally d) selecting a plant or plant part or seed comprising one or two copies of an allele (e.g. a wild type allele and/or a mutant allele, especially a null allele) of the melon CmBELI gene in the genome, wherein the wild type allele of the melon CmBELI gene encodes the protein of SEQ ID NO: 1 (or a wild type protein comprising at least 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1).

In step b) a PCR primer pair is at least one forward primer, complementary to one of the DNA strands of the CmBELI allele and one reverse primer complementary to the other DNA strand of the CmBELI allele, which primer pair hybridizes to the denatured genomic DNA and amplifies part of the CmBELI allele in a PCR reaction. Primers can be designed to amplify the wild type or any mutant CmBELI allele using primer design tools. In one aspect two forward primers are used, one designed to amplify the wild type allele and one designed to amplify a mutant allele of the CmBELI gene, and one common reverse primer. These three primers can be used in a KASP-assay to genotype the samples of step a). Thus, in one aspect the assay in step c) is a KASP-assay, but also other genotyping assays can be used, such as those described in world wide web at biosearchtech.com/sectors/agrigenomics/agrigenomics-pcr-qpcr -technologies.

In one aspect the assay discriminates between a wild type allele and a mutant allele of the CmBELI gene (especially a null allele), e.g. between the wild type CmBELI allele and a mutant allele of the Examples, or any mutant allele described elsewhere herein, which mutant allele confers stenospermocarpy when it is in homozygous form.

For analysing the genomic DNA at least crude genomic DNA extraction may be necessary. The presence of a mutant allele and/or a wild type allele in the genomic DNA can be detected directly or indirectly. Directly may for example be by nucleic acid hybridization of e.g. oligonucleotide probes. Indirectly may for example be by nucleic acid amplification using e.g. PCR primers which comprise e.g. a tail sequence attached to the primer and during PCR the allele-specific primer binds to the template DNA and elongates, thereby attaching the tail sequence to the newly synthesized strand and in subsequent PCR rounds a FRET cassette (fluorescent resonant energy transfer cassette) binds to the tail and emits fluorescence. The fluorescent signal can then be detected. This is used e.g. in the KASP-assay.

The mutant allele may differ from the wild type allele in various aspects, e.g. in the promoter sequence or in the protein coding sequence or in the intron/exon splice sites. The mutant allele may have a reduced gene expression or no gene expression or it may result in the production of a protein comprising one or more amino acids deleted, replaced, or inserted or duplicated compared to the wild type protein.

In one aspect the mutant allele is an allele encoding a mutant protein, which protein has a loss- of-function in vivo, e.g. it is truncated and/or lacks all or part of the homeodomain.

In one aspect the mutant allele encodes the mutant protein which is truncated at the C-terminal end and comprising the wild type amino acids corresponding to amino acid 1 to 378, or less (e.g. 1 to 377, 1 to 376, etc.), of the wild type protein, e.g. 1 to 343 or less of the wild type protein of SEQ ID NO: 1 or of a wild type protein comprising at least 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1.

Also, methods of generating and/or selecting melon plants or plant parts comprising at least one mutant allele of the melon CmBELI gene in the genome is provided.

In one aspect also a method for detecting the presence of a wild type allele and/or of a mutant allele of the melon CmBELI gene in the genome is provided. In one aspect a method for detecting whether a melon plant or plant part or seed comprises at least one copy of the wild type allele, e.g. encoding the protein of SEQ ID NO: 1 (or a wild type allele comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1), and/or comprises at least one copy of a mutant allele which encodes a mutant protein comprising e.g. one or more amino acids replaced, inserted and/or deleted with respect to the wild protein, is provided and optionally selecting a plant, plant part or seed comprising at least one copy of a mutant CmBELI allele.

In a further aspect a method for detecting whether a melon plant or plant part or seed comprises at least one copy of the wild type allele comprising the genomic sequence of SEQ ID NO: 4 (or a wild type allele comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4), and/or comprises at least one copy of a mutant allele which comprises one or more nucleotides inserted, deleted and/or replaced with respect to SEQ ID NO: 4 (or with respect to a wild type allele comprising 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 having a reduced-function or a loss-of-function due to e.g. one or more amino acids being replaced, inserted and/or deleted with respect to the functional wild protein, is provided and optionally selecting a plant, plant part or seed comprising at least one copy of a mutant CmBELI allele.

Detection may involve detection of the genomic DNA encoding the mutant or wild type protein, or otherwise detections of the cDNA or mRNA encoding the mutant or wild type protein. In one aspect also detection of the presence of the wild type promoter or mutant promoter is encompassed herein. Thus e.g. the detection of the presence of the wild type sequence or mutant sequence of the wild type sequences of SEQ ID NO: 2 or 3 or detection of a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2 or 3, is encompassed. Also detection of the presence of the wild type or mutant sequence of SEQ ID NO: 6 or detection of a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6 is encompassed herein.

Also a KASP-assay (Kbioscience Kompetitive Allele specific PCR-genotyping Assay) is provided comprising two allele specific forward primers, e.g. a FAM primer and a VIC primer and a Common reverse primer. Obviously, allele specific primers can be developed to detect and/or discriminate between the wild type allele and any mutant allele comprising e.g. one or more amino acids replaced, duplicated, deleted and/or inserted with respect to the wild type protein.

Likewise, isolated sequences or molecules of the (wild type or mutant) genomic sequence, the cDNA or mRNA sequence, protein sequences, or promoter sequence, as well as oligonucleotide primers or probes for detecting a wild type or mutant allele of the melon CmBELI gene are encompassed herein. Also, a method for generating a PCR amplification product and/or an oligonucleotide hybridization product of (a part of the) genomic DNA of melon plants, seeds or plant parts is provided comprising the steps of: a) providing a sample (or a plurality of samples) of genomic DNA of a melon plant or of a plurality of plants (e.g. a F2 population, inbred lines, a backcross population, a breeding population, hybrid plants, etc.), b) providing at least a pair of PCR primers or at least one oligonucleotide probe, which primers or (oligonucleotide) probe comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive nucleotides of the genomic CmBELI allele of the melon CmBELI gene and can hybridize to the genomic allele and/or amplify part of the genomic allele in a PCR assay, and c) carrying out a PCR assay using the primer pair or a hybridization assay using the probe of step b) on the sample(s) of step a) to generate a PCR amplification product and/or an oligonucleotide hybridization product, and optionally d) selecting a plant or plant part or seed comprising one or two copies of an allele (e.g. a wild type allele and/or a mutant allele, especially a null allele) of the CmBELI gene in the genome, wherein the wild type allele of the melon CmBELI gene encodes the protein of SEQ ID NO: 1 (or a wild type protein comprising at least 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1), or wherein the wild type genomic allele of the melon CmBELI gene comprises SEQ ID NO: 4 or a wild type genomic sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4.

Further a method for amplifying and/or hybridizing (a part of the) genomic DNA of melon plants, seeds or plant parts is provided comprising the steps of: a) providing a sample (or a plurality of samples) of genomic DNA of a melon plant or of a plurality of plants (e.g. a F2 population, inbred lines, a backcross population, a breeding population, hybrid plants, etc.), b) providing at least a pair of PCR primers or at least one oligonucleotide probe, which primers or (oligonucleotide) probe comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more consecutive nucleotides of the genomic CmBELI allele of the melon CmBELI gene and can hybridize to the genomic allele and/or amplify part of the genomic allele in a PCR assay, and c) carrying out a PCR assay using the primer pair or a hybridization assay using the probe of step b) on the sample(s) of step a) to generate a PCR amplification product and/or a oligonucleotide hybridization product, and optionally d) selecting a plant or plant part or seed comprising one or two copies of an allele (e.g. a wild type allele and/or a mutant allele, especially a null allele) of the melon CmBELI gene in the genome, wherein the wild type allele of the melon CmBELI gene encodes the protein of SEQ ID NO: 1 (or a wild type protein comprising at least 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1), or wherein the wild type genomic allele of the melon CmBELI gene comprises SEQ ID NO: 4 or a wild type genomic sequence comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4.

Also, a genotyping kit comprising primers and/or probes and reaction components to amplify and/or hybridize part of the genomic DNA of the CmBELI gene is provided.

Primers and probes are preferably labelled or modified by e.g. a tail sequence or label, to be able to detect the amplification or hybridization reaction products.

GENERAL DEFINITION

The verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one", e.g. “a plant” refers also to several cells plants, etc. Similarly, “a fruit” or “a plant” also refers to a plurality of fruits and plants.

As used herein, the term “plant” includes the whole plant or any parts or derivatives thereof, preferably having the same genetic makeup as the plant from which it is obtained, such as plant organs (e.g. harvested or non-harvested fruits, leaves, flowers, anthers, etc.), plant cells, plant protoplasts, plant cell tissue cultures from which whole plants can be regenerated, plant calli, plant cell clumps, plant transplants, seedlings, plant cells that are intact in plants, plant clones or micropropagations, or parts of plants, 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 root stocks) and the like. Also any developmental stage is included, such as seedlings, cuttings prior or after rooting, etc. When “seeds of a plant” are referred to, these either refer to seeds from which the plant can be grown or to seeds produced on the plant, after self-fertilization or cross-fertilization.

As used herein, the term “variety” or “cultivar” means a plant grouping within a single botanical taxon of the lowest known rank, which can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes. A plant characterized by the presence of alleles of a single gene, e.g. mutant alleles of the CmBELI gene, is not a variety or cultivar, as the remainder of the genome is not characterized.

The term “allele(s)” means any of one or more alternative forms of a gene at a particular locus, e.g. the CmBELI locus (where the CmBELI gene is located; the alleles of the gene may be wild type alleles designated CmBELI, or mutant alleles designated cmbelT), all of which alleles relate to one trait or characteristic at a specific locus (e.g. stenospermocarpy). In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes. A diploid plant species may comprise a large number of different alleles at a particular locus. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous), e.g. two identical copies of the mutant cmbell allele (i.e. cmbell/ cmbell) or one copy of the mutant cmbell allele and one copy of the wild type allele (i.e. cmbel1/CmBEL1).

“CmBELI gene” is a single, recessive gene identified in cultivated melon on chromosome 9, which when mutated results in stenospermocarpy. CmBELI is the wild type (WT), functional allele as present in non-stenospermocarpic cultivated melon plants and cmbell is the mutant allele resulting in stenospermocarpy if the allele is in homozygous form in a diploid (cmbell/ cmbell) melon. In one aspect the CmBELI gene is the gene encoding a protein of SEQ ID NO: 1 or encoding a protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 (melon), when aligned pairwise. In one aspect the CmBELI gene is the genomic allele comprising SEQ ID NO: 4 or a wild type genomic sequence comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4. In one aspect the CmBELI gene is the genomic allele producing a mRNA/cDNA comprising SEQ ID NO: 2 or 3 or a wild type mRNA/cDNA comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2 or 3.

“Stenospermocarpy” is generally understood in the art and also to be understood in connection with the present invention to mean that induction of fruit set and development requires pollination but without the fruits producing mature or viable seeds. Mature or viable seeds are not developed in stenospermocarpic plants due to e.g. arrested seed development or in ovule development or degradation of ovules and/or embryos and/or endosperm or abortion of the ovules and/or embryos and/or endosperm before maturity is reached. “F1 , F2, F3, etc.” refers to the consecutive related generations following a cross between two parent plants or parent lines. The plants grown from the seeds produced by crossing two plants or lines is called the F1 generation. Selfing the F1 plants results in the F2 generation, etc.

“F1 hybrid” plant (or F1 hybrid seed) is the generation obtained from crossing two inbred parent lines. Thus, F1 hybrid seeds are seeds from which F1 hybrid plants grow. F1 hybrids are more vigorous and higher yielding, due to heterosis. Inbred lines are essentially homozygous at most loci in the genome.

A “plant line” or “breeding line” refers to a plant and its progeny. As used herein, the term "inbred line" refers to a plant line which has been repeatedly selfed and is nearly homozygous. Thus, an “inbred line” or “parent line” refers to a plant which has undergone several generations (e.g. at least 5, 6, 7 or more) of inbreeding, resulting in a plant line with a high uniformity.

The term “gene” means a (genomic) DNA sequence comprising a region (transcribed region), which is transcribed into a pre-mRNA, which is processed (by intron splicing) into a messenger RNA molecule (mRNA) in a cell, and an operably linked regulatory region (e.g. a promoter). An example is the CmBELI gene of the invention. Different alleles of a gene are, thus, different alternatives form of the gene, which may be in the form of e.g. differences in one or more nucleotides of the genomic DNA sequence (e.g. in the promoter sequence, the exon sequences, intron sequences, etc.), mRNA and/or amino acid sequence of the encoded protein.

“Mutant cmbell allele” or “bell allele” refers herein to a mutant allele of the CmBELI gene on chromosome 9 in melon, which causes the plant to be stenospermocarpic when the mutant allele is in homozygous form. The mutation in the mutant allele can be any mutation or combination of mutations, including deletions, truncations, insertions, point mutations, non-sense mutations, mis-sense mutations or non-synonymous mutations, splice-site mutations, frame shift mutations and/or mutations in one or more regulatory sequences such as promoter sequence, or enhancer or silencer sequences. In one aspect the mutant cmbell allele is a mutant allele of the CmBELI gene, whereby the CmBELI gene is the gene encoding a protein of SEQ ID NO: 1 or encoding a protein comprising at least 94%, 95%, 96%, 97% or 98% or 99% sequence identity to SEQ ID NO: 1 (when aligned pairwise). In one aspect the mutant cmbell allele is a mutant allele of the wild type genomic allele comprising SEQ ID NO: 4 or of a wild type genomic sequence comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4. In one aspect the mutant cmbell allele is a mutant allele of the wild type genomic allele producing mRNA/cDNA comprising SEQ ID NO: 2 or 3 or of a wild type mRNA/cDNA transcript sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2 or 3. “Wild type CmBELI allele” or “BEL1 allele” refers herein to the functional allele of the CmBELI gene, which causes the plant to have a normal fruit set, requiring normal pollination and fertilization to set fruits which contain seeds. The wild type CmBELI allele is found in any commercial variety of melon (e.g. Nunhems variety Magenta F1 , Kirene F1 , Coliseo F1 and others). In one aspect the wild type CmBELI allele is a wild type allele of the CmBELI gene whereby the CmBELI gene is the gene encoding a protein of SEQ ID NO: 1 or encoding a protein comprising at least 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 1 (when aligned pairwise). In one aspect the wild type genomic CmBELI allele comprises SEQ ID NO: 4 or a wild type genomic sequence comprising 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 produces mRNA/cDNA comprising SEQ ID NO: 2 or 3 or of a wild type mRNA/cDNA transcript sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2 or 3.

The term “locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found. The CmBELI locus is, thus, the location in the genome of melon, where the mutant allele and/or the wild type allele of the CmBELI gene is found. The CmBELI locus is a locus on cultivated melon chromosome 9 (using the chromosome assignment of the published melon genome (Garcia-Mas J et al. 2012, The genome of melon Cucumis melo L. PNAS 109: 11872-11877) found at world wide web at cucurbitgenomics.org under “Genome: melon (DHL92) v3.6.1”, i.e. cmbell was generated in the cultivated melon genome by mutagenesis and the mutant cmbell allele was mapped to a defined region of chromosome 9 of cultivated melon.

"Induced mutant" alleles are mutant alleles in which the mutation(s) is/are/have been induced by human intervention, e.g. by mutagenesis via physical or chemical mutagenesis methods or via e.g. tissue culture (as described in e.g. Zhang et al, Pios 9(5) e96879), including also targeted gene editing techniques (such as Crispr based techniques, TALENS, Base editing, etc.) and also 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).

“Diploid plant” refers to a plant, vegetative plant part(s), or seed from which a diploid plant can be grown, having two sets of chromosome, designated herein as 2n.

A “DH plant” or “doubled-haploid plant” is a diploid plant produced by doubling the haploid genome of the diploid plant using e.g. in vitro techniques. A DH plant is, therefore, homozygous at all loci.

A “seedless fruit” as commonly used in the art and is to be understood in context with the present invention to be a fruit without mature or viable seeds. Sometimes empty seeds or microseeds may be seen. Mature or viable seeds can be germinated in soil under conditions appropriate for the respective plant and grown into plants. This test can be used to determine if a plant produces seedless fruits. Seedless fruits will not produce seed which will germinate and grow into a plant under conditions appropriate for the respective plant.

“Vegetative propagation” or “clonal propagation” refers to propagation of plants from vegetative tissue, e.g. by in vitro propagation or grafting methods (using scions and rootstocks). In vitro propagation involves in vitro cell or tissue culture and regeneration of a whole plant from the in vitro culture. Grafting involves propagation of an original plant by grafting onto a rootstock. Clones (i.e. genetically identical vegetative propagations) of the original plant can thus be generated by either in vitro culture or grafting. “Cell culture” or “tissue culture” refers to the in vitro culture of cells or tissues of a plant. “Regeneration” refers to the development of a plant from cell culture or tissue culture or vegetative propagation. “Non-propagating cell” refers to a cell which cannot be regenerated into a whole plant.

“Recessive” refers to an allele which expresses its phenotype (e.g. stenospermocarpy) when no dominant allele is present in the diploid genome, i.e. when it is homozygous in a diploid. The mutant cmbell allele results in a stenospermocarpic plant when present in two copies in a diploid plant. The dominant allele is herein also referred to as the wild type (WT) allele.

“Melon plant cells” or “melon plants” or “cultivated melon plants or cells” also designated as muskmelon plant cells or muskmelon plants in the art shall be understood in context with the present invention to be plant cells originating from the species Cucumis melo or to be plants belonging to the species Cucumis melo. Cucumis melo, can be classified into: C. melo var. cantalupensis, C. melo var. inodorous and C. melo var. reticulatus. C. melo var. cantalupensis are also referred to as Cantaloupes and are primarily round in shape with prominent ribs and almost no netting. Most have orange, sweet flesh and they are usually very fragrant. In contrast to the European cantaloupe, the North American 'Cantaloupe' is not of this type, but belongs to the true muskmelons. C. melo var. inodorous (or winter melons) can be subdivided into different types, such as Honeydew melon, Piel de Sapo, Sugar melon, Japanese melon, etc. C. melo var. reticulatus is the true muskmelon, with reticulated skin (netted) and includes Galia melons, Sharlyn melons and the North American cantaloupe.

Cultivated melon and the wild relatives of melon is/are diploid and has/have 12 pairs of homologous chromosomes, numbered 1 to 12.

"Cultivated melon plant" refers to plants of Cucumis melo i.e. varieties, breeding lines or cultivars of the species C. melo, cultivated by humans and having good agronomic characteristics, especially producing edible and marketable fruits of good size and quality and uniformity; preferably such plants are not "wild melon plants", i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and e.g. grow naturally in wild populations. "Wild melon plants" include for example ecotypes, PI (Plant Introduction) lines, landraces or wild accessions or wild relatives of a species.

“Pocket melons” or “mini melons” are melon fruits produced by a plant which have an average fruit weight of 1 .0 kg or less, especially 0.9 kg or less, 0.8 kg or less, 0.7kg or less, 0.6kg or less, 0.5 kg or less, 0.4 kg or less.

“Midi melons” or “personal size melons” are melon fruits produced by a plant which have an average fruit weight of 1 .5 to 2.0 kg, or above 1 .0 kg, e.g. 1 .0 to 1.5 kg or 1 .0 to 2.0 kg.

“SNP marker” refers to a Single Nucleotide Polymorphism between e.g. a mutant cmbell allele and a wild type CmBELI allele. Using a SNP marker assay which can distinguish between the mutant and wild type allele of the CmBELI gene (i.e. an allele specific assay) one can screen pants, plant parts or the DNA therefrom for the presence of the mutant allele and/or the wild type allele. For any of the SNP markers a SNP markers assays can be designed based on the sequences provided herein. Such a SNP marker assay can be used to detect the mutant allele, e.g. in Marker Assisted Selection and/or SNP genotyping assays. Thus, using a SNP marker assay which can distinguish between the mutant and wild type allele of the gene (e.g. in an allele specific assay) one can screen pants, plant parts or the DNA therefrom for the presence of the mutant allele.

“INDEL marker” refers to an insertion/deletion polymorphism between e.g. a mutant cmbell allele and a wild type CmBELI allele. Using an INDEL marker assay which can distinguish between the mutant and wild type allele of the gene (e.g. an allele specific assay) one can screen pants, plant parts or the DNA therefrom for the presence of the mutant allele.

“Genotyping” methods are methods whereby the genotype or allelic composition of a plant or plant part or seed can be determined. Bi-allelic genotyping assays, such as KASP-assays, can distinguish between two alleles at a locus.

A “chromosome region comprising the mutant cmbell allele” refers to the genomic region of e.g. chromosome 9 of cultivated melon which region carries the mutant cmbell allele. The presence of the allele can be determined phenotypically and/or by the presence of one or more molecular markers, e.g. SNP markers, INDEL markers or other markers, linked to the mutant cmbell allele or preferably markers distinguishing different cmbell alleles or by the genomic sequence of the allele sequence itself (e.g. sequencing the allele). A marker is “linked to the cmbell allele”, if it is physically coupled to the allele. An “allele specific marker” is a marker which is specific for a particular allele (e.g. a specific mutant allele) and is thus discriminating between e.g. the mutant allele and the 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, e.g. based on a polymorphism between the wild type allele sequence and the mutant allele sequence.

A pair of “flanking markers” refers to two markers, preferably two SNP markers or two sequences comprising the SNP markers, which are linked to the cmbell allele, and/or which are closely linked to the cmbell allele, whereby the cmbell allele is located in-between the two markers or in-between the two sequences comprising the markers.

“Brix” or “degree Brix” or “° brix” refers to the mean total soluble solids content as measured on several mature fruits using a refractometer. Preferably the mean of at least three fruits, each measured between the centre and the rind of the cut-open fruit, is calculated.

“Physical distance” between loci (e.g. between molecular markers and/or between phenotypic markers) on the same chromosome is the actually distance expressed in bases or base pairs (bp), kilo bases or kilo base pairs (kb) or megabases or mega base pairs (Mb).

“Genetic distance” between loci (e.g. between molecular markers and/or between phenotypic markers) on the same chromosome is measured by frequency of crossing-over, or recombination frequency (RF) and is indicated in centimorgans (cM). One cM corresponds to a recombination frequency of about 1 %. If no recombinants can be found, the RF is zero and the loci are either extremely close together physically or they are identical. The further apart two loci are, the higher the RF.

“Uniformity” or “uniform” relates to the genetic and phenotypic characteristics of a plant line or variety. Inbred lines are genetically highly uniform as they are produced by several generations of inbreeding. Likewise, and the F1 hybrids which are produced from such inbred lines are highly uniform in their genotypic and phenotypic characteristics and performance.

A genetic element, an introgression fragment, or a gene or allele conferring a trait (such as stenospermocarpy) is said to be “obtainable from” or can be “obtained from” or “derivable from” or can be “derived from” or “as present in” or “as found in” a plant or seed or tissue or cell if it can be transferred from the plant or seed in which it is present into another plant or seed in which it is not present (such as a non-stenospermocarpic line or variety) using traditional breeding techniques without resulting in a phenotypic change of the recipient plant apart from the addition of the trait conferred by the genetic element, locus, introgression fragment, gene or allele. The terms are used interchangeably and the genetic element, locus, introgression fragment, gene or allele can thus be transferred into any other genetic background lacking the trait. Cultivated melons containing the genetic element, locus, introgression fragment, gene or allele (e.g. a mutant cmbell allele) can be generated de novo, e.g. by mutagenesis (e.g. chemical mutagenesis, CRISPR-Cas induced, etc.) and then e.g. be crossed into other cultivated melons. “Average” or “mean” refers herein to the arithmetic mean and both terms are used interchangeably. The term “average” or “mean” thus refers to the arithmetic mean of several measurements. The skilled person understands that the phenotype of a plant line or variety depends to some extent on growing conditions and that, therefore, arithmetic means of at least 10, 15, 20, 30, 40, 50 or more plants (or plant parts) are measured, preferably in randomized experimental designs with several replicates and suitable 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, when compared to a suitable control show a statistically significant difference in that characteristic (e.g. the p- value is less than 0.05, p < 0.05, using ANOVA) from the (mean of the) control.

The term “traditional breeding techniques” encompasses herein crossing, backcrossing, selfing, selection, double haploid production, chromosome doubling, embryo rescue, protoplast fusion, marker assisted selection, mutation breeding etc., all as known to the breeder (i.e. methods other than genetic modification I transformation I transgenic methods), by which, for example, a chromosome 9 comprising a mutant cmbell allele can be obtained, identified and/or transferred.

“Backcrossing” refers to a breeding method by which a (single) trait, such as the stenospermocarpy trait, can be transferred from one (often an inferior) genetic background (also referred to as “donor”) into another (often a superior) genetic background (also referred to as “recurrent parent”. An offspring of a cross (e.g. an F1 plant obtained by crossing e.g. the donor with the recurrent parent melon, or an F2 plant or F3 plant, etc., obtained from selfing the F1), is “backcrossed” to the parent with e.g. the superior genetic background. After repeated backcrossing, the trait of the one (often inferior) genetic background will have been incorporated into the other (often superior) genetic background.

“Marker assisted selection” or “MAS” is a process of using the presence of molecular markers (such as SNP markers or INDEL markers), which are genetically and physically linked to a particular locus or to a particular chromosome region or allele specific markers, to select plants for the presence of the specific locus or region or allele. For example, a molecular marker genetically and physically linked to the mutant cmbell allele or an allele specific marker, can be used to detect and/or select e.g. melon plants, or plant parts, comprising the cmbell allele. The closer the linkage of the molecular marker to the locus, the less likely it is that the marker is dissociated from the locus through meiotic recombination. Likewise, the closer two markers are linked to each other the less likely it is that the two markers will be separated from one another (and the more likely they will co-segregate as a unit). Allele specific markers are preferred markers, as they select for the allele directly.

A molecular marker (or a sequence comprising a molecular marker) within 5 Mb, 3 Mb, 2.5 Mb,

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

“LOD-score” (logarithm (base 10) of odds) refers to a statistical test often used for linkage analysis in animal and plant populations. The LOD score compares the likelihood of obtaining the test data if the two loci (molecular marker loci and/or a phenotypic trait locus) are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD scores favour the presence of linkage and a LOD score greater than 3.0 is considered evidence for linkage. A LOD score of +3 indicates 1000 to 1 odds that the linkage being observed did not occur by chance.

“Transgene” or “chimeric gene” refers to a genetic locus comprising a DNA sequence, such as a recombinant gene, which has been introduced into the genome of a plant by transformation, such as Agrobacterium mediated transformation. A plant comprising a transgene stably integrated into its genome is referred to as “transgenic plant”.

An “isolated nucleic acid sequence” or “isolated DNA” refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome. When referring to a “sequence” herein, it is understood that the molecule having such a sequence is referred to, e.g. the nucleic acid molecule.

A "host cell" or a "recombinant host cell" or “transformed cell” are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, having been introduced into said cell. The host cell is preferably a plant cell or a bacterial cell. The host cell may contain the nucleic acid as an extra-chromosomally (episomal) replicating molecule, or comprises the nucleic acid integrated in the nuclear or plastid genome of the host cell, or as introduced chromosome, e.g. minichromosome.

“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as "substantially identical” or “essentially similar” when they are optimally aligned by for example the programs GAP or BESTFIT or the Emboss program “Needle” (using default parameters, see below) share at least a certain minimal percentage of sequence identity (as defined further below). These programs use the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimising the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 10 and gap extension penalty = 0.5 (both for nucleotide and protein alignments). For nucleotides the default scoring matrix used is DNAFULL and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignments and scores for percentage sequence identity may for example be determined using computer programs, such as EMBOSS as available on the world wide web under ebi.ac.uk/Tools/psa/emboss_needle/). Alternatively sequence similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences have “substantial sequence identity” if the percentage sequence identity is at least 85%, 90%, 92%, 93%, 94%, 95%, 98%, 99% or more (as determined by Emboss “needle” using default parameters, i.e. gap creation penalty = 10, gap extension penalty = 0.5, using scoring matrix DNAFULL for nucleic acids and Blosum62 for proteins).

When reference is made to a nucleic acid sequence (e.g. DNA or genomic DNA) having “substantial sequence identity to” a reference sequence or having a sequence identity of 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 said nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using stringent hybridisation conditions. In another embodiment, the nucleic acid sequence comprises one or more mutations compared to the given nucleotide sequence but still can be identified using stringent hybridisation conditions.

“Stringent hybridisation conditions” can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g. 100nt) are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions. Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g. 100nt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. “M1 generation” or “M1 plants” in context with the present invention shall refer to the first generation that is produced directly from the mutagenic treatment. A plant grown from seeds treated with a mutagen e.g. is a representative of an M1 generation.

“M2 generation” or “M2 plant” shall refer herein to the generation obtained from self-pollination of the M1 generation. A plant grown from seeds obtained from a self-pollinated M1 plant represents a M2 plant. M3, M4, etc. refers to further generations obtained after self-pollination.

An “mRNA coding sequence” or “mRNA sequence” shall have the common meaning herein. An mRNA coding sequence corresponds to the respective DNA coding (cDNA) sequence of a gene/allele apart from that thymine (T) is replaced by uracil (II).

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, e.g. by replacement, deletion or insertion of one or more nucleotides. Examples of such a mutation are point mutation, nonsense mutation, missense mutation, splice-site mutation, frame shift mutation or a mutation in a regulatory sequence.

A “nucleic acid molecule” shall have the common understanding in the art. It is composed of nucleotides comprising either of the sugars deoxyribose (DNA) or ribose (RNA).

A “point mutation” is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide.

A “nonsense mutation” is a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon in a nucleic acid molecule is changed into a stop codon. This results in a premature stop codon being present in the mRNA and results in translation of a truncated protein. A truncated protein may have decreased function or loss of function.

A “missense or non-synonymous mutation” is a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon is changed to code for a different amino acid. The resulting protein may have decreased function or loss of function.

A “splice-site mutation” is a mutation in a nucleic acid sequence encoding a protein, whereby RNA splicing of the pre-mRNA is changed, resulting in an mRNA having a different nucleotide sequence and a protein having a different amino acid sequence than the wild type. The resulting protein may have decreased function or loss of function.

A “frame shift mutation” is a mutation in a nucleic acid sequence encoding a protein by which the reading frame of the mRNA is changed, resulting in a different amino acid sequence. The resulting protein may have decreased function or loss of function. A “deletion” in context of the invention shall mean that anywhere in a given nucleic acid sequence at least one nucleotide is missing compared to the nucleic sequence of the corresponding wild type sequence or anywhere in a given amino acid sequence at least one amino acid is missing compared to the amino acid sequence of the corresponding (wild type) sequence.

A “truncation” shall be understood to mean that at least one nucleotide at either the 3’-end or the 5’-end of the nucleotide sequence is missing compared to the nucleic sequence of the corresponding wild type sequence or that at least one amino acid, but preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids, at either the N-terminus or the C-terminus of the protein is missing compared to the amino acid sequence of the corresponding wild type protein. The 5’-end is determined by the ATG codon used as start codon in translation of a corresponding wild type nucleic acid sequence.

“Replacement” shall mean 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 e.g. an exchange of a nucleotide in the coding sequence of the respective protein.

“Insertion” shall mean that the nucleic acid sequence or the amino acid sequence of a protein comprises at least one additional nucleotide or amino acid compared to the corresponding wild type nucleic acid sequence or the corresponding wild type amino acid sequence, respectively.

“Pre-mature stop codon” in context with the present invention means that a stop codon is present in a coding sequence (cds) which is closer to the start codon at the 5’-end compared to the stop codon of a corresponding wild type coding sequence.

A “mutation in a regulatory sequence”, e.g. in a promoter or enhancer of a gene, is a change of one or more nucleotides compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more nucleotides, leading for example to decreased or no mRNA transcript of the gene being made.

A “mutation in a protein” is a change of one or more amino acid residues compared to the wild type sequence, e.g. by replacement, deletion, truncation or insertion of one or more amino acid residues.

“Mutant protein” is herein a protein comprising one or more mutations in the nucleic acid sequence encoding the protein, whereby the mutation results in (the mutant nucleic acid molecule encoding) a "reduced-function" or "loss-of-function" protein, as e.g. measurable in vivo, e.g. by the phenotype conferred by the mutant allele. In context of the present invention, “loss-of-function” of a protein shall mean a loss of the normal in vivo function of a CmBELI protein when compared to a corresponding wild type plant cell or a corresponding wild type plant. “Loss-of-function” can be distinguished from a “reduced- function” or “decreased function” of a protein, whereby the reduced (or decreased) function protein still has some residual in vivo function, albeit less than the wild type protein.

“Null allele” is a non-functional allele. It results in either no wild type gene product being produced, e.g. through mutation in the regulatory elements such as the promoter (e.g. knockout of gene expression), or in the production of a gene product which has lost its function, i.e. a loss-of-function protein. The entire deletion of a gene has phenotypically the same effect as a ‘null allele’ and is also encompassed herein in one aspect.

In context with the present invention, the term "wild type plant cell" or "wild type plant" means that they comprise wild type CmBELI alleles and not mutant cmbell alleles. Thus, the wild type plant or wild type plant cell is a plant or plant cell comprising fully functional CmBELI genes, encoding a fully functional CmBELI proteins (also referred to as wild type CmBELI protein), e.g. regarding melon plants or plant cells a diploid melon plant producing the protein of SEQ ID NO: 1 (or a protein comprising at least 94% sequence identity to SEQ ID NO: 1) and producing fruits with seeds after pollination.

“Knock-out” or “entire knock-out” shall be understood that expression of the respective gene is not detectable anymore.

“Conserved domain” refers to conserved protein domains, such as the homeodomain of SEQ ID NO: 1 , starting at amino acid 318 and ending at amino acid 379 (or the equivalent amino acids in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1). Conserved domains can e.g. be found in the Conserved Domain Database of the NCBI (world wide web at ncbi.nlm.nih.gov/cdd).

'Targeted gene editing” is referred to techniques whereby endogenous target genes can be modified, e.g. one or more nucleotides can be inserted, replaced and/or deleted e.g. in the promoter or coding sequence or transcribed region of a gene. For example CRISPR based techniques, such as Crispr-Cas9 gene editing, Crispr-Cpf/ gene editing, or more recent techniques called ‘base editing’ or ‘primer editing’ can be used to modify endogenous target genes, such as the endogenous wild type CmBELI gene in melon (encoding the protein of SEQ ID NO: 1 , or a wild type protein comprising at least 94% sequence identity to SEQ ID NO: 1). The mutants described herein can, for example, be reproduced by targeted gene editing of the wild type CmBELI gene.

“Oligonucleotides” or “oligos” or “oligonucleotide primers or probes” are short, single-stranded polymers of nucleic acid, e.g. at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more nucleotides in length. Oligos may be unmodified or modified with a variety of chemistries depending on their intended use, for example, the addition of 5' or 3' phosphate groups to enable ligation or block extension, respectively, labelling with radionuclides or fluorophores and/or quenchers for use as probes, the incorporation of thiol, amino, or other reactive moieties to enable the covalent coupling of functional molecules such as enzymes, and extension with other linkers and spacers of diverse functionality. DNA oligos are the most commonly used, but RNA oligos are also available. The length of an oligo is usually designated by adding the suffix -mer. For example, an oligonucleotide with 19 nucleotides (bases) is called a 19-mer. For most uses, oligonucleotides are designed to base-pair with a strand of DNA or RNA. The most common use for oligonucleotides is as primers for PCR (polymerase chain reaction). Primers are designed with at least part of their sequence complementary to the sequence targeted for amplification. Optimal primer length for a complementary sequence is e.g. 18 to 22 nucleotides. Optimal primer sequences for PCR are usually determined by primer design software.

“DNA microarrays” are arrays which have many microscopic spots of DNA, usually oligonucleotides, bound on a solid support. Assay targets can be DNA, cDNA, or cRNA. Depending on the system, the hybridization of targets to specific spots is detected by fluorescence, chemiluminescence, or colloidal silver or gold. Microarrays are used for multiple applications such as simultaneous measurement of the expression of large numbers of genes, enabling genome-wide gene expression analysis, as well as genotyping studies using e.g. single-nucleotide polymorphism (SNP) or InDei analysis.

“Complementary strands” refer to two strands of complementary sequence, and may be referred to as sense (or plus) and anti-sense (or minus) strands for double stranded DNA. The sense I plus strand is, generally, the transcribed sequence of DNA (or the mRNA that was generated in transcription), while the anti-sense I minus strand is the strand that is complementary to the sense sequence. For any of the sequences provided herein only one strand of the sequence is given, but the complementary strand of the given strand is also encompassed herein. The complementary nucleotides of DNA are A complementary to T, and G complementary to C. The complementary nucleotides of RNA are A complementary to II, and G complementary to C.

FIGURES

Figure 1 : shows the wild type CmBELI protein of SEQ ID NO: 1. The vertical black lines delimit the amino acids encoded by exon 1 , exon 2, exon 3 and exon 4 of the transcript. The black star at the second black vertical line (separating amino acids encoded by exon 2 and exon 3) shows the effect of the mutant cmbell allele, which comprises a DNA insert in intron 2 and thereby leads to an mRNA transcript that contains only the transcribed region of exon 1 and exon 2, and thereby results in a truncated protein, comprising only amino acids 1 to 343 of SEQ ID NO: 1. The black box highlights the conserved homeodomain. Figure 2: melon fruits homozygous for the mutant cmbell allele, being seedless.

Figure 3: melon fruits homozygous for the mutant cmbell allele, being seedless and essentially free of empty seed cavity.

Figure 4: melon fruits homozygous for the mutant cmbell allele, being seedless and essentially free of empty seed cavity.

Figure 5: melon fruits (Piel de Sapo) homozygous for the mutant cmbell allele, being seedless and having some empty seed cavity.

Figure 6: RT-PCR with primers that span the cDNA region corresponding to exon 2 and exon 3 results in PCR products in the wild type (seeded) plant, but not in the mutant (seedless) plant, showing that the mRNA transcript in the mutant plant terminates after exon2.

Figure 7: genomic DNA (SEQ ID NO: 4) encoding the wild type CmBELI protein. 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 to1945, 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 the conserved homeodomain. Boxed is codon TGG at nucleotides 1263 to 1265, which encodes the first amino acid of the homeodomain (W318 of SEQ ID NO: 1) and codon ATG at nucleotides 2425 to 2427, which encodes the last amino acid of the homeodomain (M379 of SEQ ID NO: 1). The black vertical line in intron 2 shows the position where the mutant cmbell allele comprises a DNA insert (between nucleotide 1655 and nucleotide 1656), leading to the mRNA transcript lacking the region of exons 3 and 4.

Figure 8: Cantaloupe F2 plants were grown in the field and number of fruits were counted and the fruit weight (grams) was measured in the wild type plants and homozygous mutant plants. The wild type plants had about 3 to 4 fruits of 1600-3000g, while the cmbell mutant plants (comprising the mutant allele in homozygous form) had about 7 to 14 fruits of about 700-1000g.

Figure 9: Normal, seeded Piel de Sapo fruit with wild type CmBELI allele (left) and seedless fruit (comprising the mutant cmbell allele) on the right, whereby the seedless fruit contains microseeds.

DETAILED DESCRIPTION

A first embodiment of the present invention concerns cultivated melon plants, Cucumis melo, comprising at least one copy of a mutant allele of a gene conferring stenospermocarpy when the mutant allele is in homozygous form, and optionally an increased average number of fruits and/or a reduced average fruit weight (compared to the wild type plant comprising the wild type allele of the gene). Thus, in one aspect cultivated melon plants are provided, comprising at least one copy of a mutant allele of a single recessive gene called CmBELI .

The CmBELI gene is an endogenous gene of cultivated melon, which when mutated and in homozygous form results in stenospermocarpy.

A segregating population made by crossing the mutant stenospermocarpic melon plant generated with a melon line enabled mapping of the CmBELI gene to a region on chromosome 9. Further fine-mapping led to the identification of a gene comprising a mutation which led to a premature termination of the mRNA transcript and a truncation of the encoded protein after amino acid P343 of SEQ ID NO: 1 .

As 265 amino acids of the 608 amino acids were missing in the truncated protein (including 36 amino acids of the conserved homeodomain needed for protein function), it was concluded that this truncation of the CmBELI protein led to the protein being non-functional in vivo. As a result, the plant homozygous for this mutant protein (and thus lacking the functional wild type protein) develops seedless fruits. The average fruit weight was significantly reduced to less than half of the weight of the recurrent parent, while the average fruit number was significantly increased, e.g. about doubled compared to the recurrent parent. See Figure 8.

In one aspect a melon plant or plant part is provided comprising at least one copy of a mutant allele of a gene named CmBELI, wherein said mutant allele either a) comprises one or more mutations in a regulatory element, especially in the promoter, resulting in no expression (or alternatively reduced expression) of the allele compared to the wild type allele, and/or b) encodes a mutant protein comprising one or more amino acids replaced, inserted or deleted compared to the wild type protein, especially a non-functional protein, wherein said mutant allele of a) or b) confers stenospermocarpy when the mutant allele is in homozygous form, and wherein the wild type melon allele encodes a protein of SEQ ID NO: 1 or a protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 1.

The wild type functional CmBELI protein of melon is provided in SEQ ID NO: 1. There may however be some amino acid sequence variation within melons and functional CmBELI proteins may comprise e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more amino acids which are different than in SEQ ID NO: 1 provided herein or whereby the protein comprises comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the proteins of SEQ ID NO: 1 (when aligned pairwise using e.g. Emboss-Needle). Therefore, in one aspect functional variants (also referred to as ‘variant CmBELI protein’ herein) of the melon protein of SEQ ID NO: 1 are proteins comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the protein of SEQ ID NO: 1 , when aligned pairwise (using e.g. Needle with default parameters). In one aspect the amino acid sequence variation is found outside the conserved homeodomain. In one aspect the functional proteins, which comprise at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the protein of SEQ ID NO: 1 , therefore comprise 100% identical amino acids to SEQ ID NO: 1 for the homeodomain starting at amino acid 318 of SEQ ID NO: 1 and ending at amino acid 379 of SEQ ID NO: 1 , see also Figure 1 (boxed amino acids).

As the homeodomain is highly conserved within the species, any mutation (deletion, insertion and/or replacement 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 homeodomain is predicted to lead to the mutant CmBELI protein having no function in vivo, thereby leading to the stenospermocarpic phenotype when the mutant allele is in homozygous form in a diploid plant.

Thus, inserting, deleting and/or replacing one or more amino acids in the homeodomain will negatively affect the protein function, especially make the protein non-functional in vivo.

Therefore, in one aspect a melon plant or plant part is provided comprising at least one copy of a mutant allele of a gene named CmBELI , wherein said mutant allele encodes a mutant protein comprising one or more amino acids inserted, deleted or replaced in the homeodomain of the protein starting at amino acid 318 and ending at amino acid 379 of SEQ ID NO: 1 or the equivalent amino acids in a variant CmBELI protein comprising at least 94% sequence identity to SEQ ID NO: 1 and wherein said mutant allele confers stenospermocarpy when the mutant allele is in homozygous form.

The term ‘starting at’ and ‘ending at’ or ‘from’ and ‘to’ includes the first and last amino acid mentioned.

Thus, insertion, deletion and/or replacement of one or more amino acids in the homeodomain may be the insertion, deletion and/or replacement of 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 amino acids of the homeodomain, i.e. of amino acids 318 to 379 of SEQ ID NO: 1 , or the equivalent amino acids in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

In yet another aspect a melon plant or plant part is provided comprising at least one copy of a mutant allele of a gene named CmBELI , wherein said mutant allele encodes a mutant protein comprising 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 inserted, deleted and/or replaced in SEQ ID NO: 1 or in a variant CmBELI protein or a protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , and wherein said mutant allele confers stenospermocarpy when the mutant allele is in homozygous form. The mutant CmBELI protein may, thus, e.g. be truncated at the N-terminal or C-terminal, 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-terminal or C-terminal, 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 may be deleted, replaced or inserted compared to the wild type functional CmBELI protein. As mentioned, the mutant protein is preferably non-functional in vivo, and therefore confers the stenospermocarpy phenotype when the mutant allele encoding the mutant protein is in homozygous form.

In still another aspect a melon plant or plant part is provided comprising at least one copy of a mutant allele of a gene named CmBELI, wherein said mutant allele comprises one or more nucleotides inserted, deleted or replaced in SEQ ID NO: 4 (or a wild type genomic sequence comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4), and wherein said mutant allele confers stenospermocarpy when the mutant allele is in homozygous form. The mutant CmBELI genomic sequence is, in one aspect, a null allele. It may encode a loss-of-function protein which e.g. may be truncated at the N-terminal or C-terminal, 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-terminal or C- terminal, or it may be a mutated protein wherein 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, replaced or inserted compared to the wild type functional CmBELI protein. As mentioned, the mutant allele is preferably a null allele in vivo, and therefore confers the stenospermocarpy phenotype when the mutant allele is in homozygous form.

In yet another aspect a melon plant or plant part is provided comprising at least one copy of a mutant allele of a gene named CmBELI , wherein said mutant allele encodes a mutant protein comprising at least 1 , 2, 3, 4, 5, 6, 7, 8 or 9 or more amino acids inserted, deleted and/or replaced in SEQ ID NO: 1 , or in a variant CmBELI protein or a protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , and wherein said mutant allele confers stenospermocarpy when the mutant allele is in homozygous form. The mutant CmBELI protein may, thus, comprise at least 1 amino acid deleted, replaced and/or inserted compared to the wild type functional CmBELI protein. For example, the amino acid deleted or replaced (e.g. by a stop codon or by a different amino acid) may be P343* or Y344*, or any amino acid of the homeodomain may be replaced by another amino acid or by a STOP codon. As mentioned, the mutant protein is preferably non-functional in vivo, and therefore confers the stenospermocarpy phenotype when the mutant allele encoding the mutant protein is in homozygous form.

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

Any mutant allele which results in an insertion, deletion and/or replacement of one or more amino acids of the wild type, functional protein may result in a mutant protein having reduced function or no function and may, thus, result in the phenotype of stenospermocarpy when the mutant allele is in homozygous form. Plants and plant parts comprising such mutant alleles are one embodiment herein.

The ‘equivalent amino acid’ can easily be determined by amino acid sequence alignment.

In one aspect the amino acid deletion, insertion and/or replacement in the mutant protein is due to a mutation in a codon of the CmBELI gene.

A mutation in the codon may be a (at least one) nucleotide insertion, deletion or replacement in the codon, leading to e.g. a different reading frame or a different codon, e.g. encoding a different amino acid or a STOP codon. Also, the entire codon may be deleted or replaced by a different codon (or optionally a stop codon), resulting in either a deletion of the encoded amino acid, or the replacement thereof.

In one aspect the mutant allele encodes a mutant CmBELI protein comprising an amino acid substitution, deletion or a stop codon of an (or of one or more) amino acid(s) selected from amino acid number 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 the equivalent amino acid in a protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

In one aspect the mutant allele encodes a mutant CmBELI protein which comprises a truncation of 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, 268, 269, 270 amino acids of the C-terminal end of the protein of SEQ ID NO: 1 or of the C-terminal end of a protein comprising at least 94% sequence identity to SEQ ID NO: 1. In one aspect all amino acids starting at (and including) an amino acid selected from amino acid number 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 the equivalent amino acid in a protein comprising at least 94% sequence identity to SEQ ID NO: 1 , are deleted or replaced by one or more different amino acids. “Starting at” means that the mentioned amino acid is deleted or replaced, and that the amino acids following the mentioned amino acid (towards the C- terminal end) are also deleted or replaced.

In a further aspect one or more nucleotides, e.g. 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, are inserted, deleted or replaced in the genomic sequence of SEQ ID NO: 4 (or Figure 7), whereby no functional wild type protein is being made from the mutant allele, e.g. the mRNA transcript is truncated and/or the encoded protein is truncated. In one aspect the one or more nucleotides are a transposable element that is integrated into the genomic sequence of SEQ ID NO: 4. In another aspect the one or more nucleotides are inserted, deleted or replaced by targeted gene editing methods, such as Crispr based methods. In a specific aspect the one or more nucleotides are inserted into, deleted or replaced in an exon region or an intron region of SEQ ID NO: 4, especially into (or in) exon 1 or exon 2 or exon 3, or into (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, e.g. between nucleotide 1655 and 1656 of SEQ ID NO: 4.

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) of the nucleotides of SEQ ID NO: 5.

A transposable element may be a melon transposable element, e.g. described in Morata et al. 2018, Genome Biol. Evol. 10(6): 1584-1595. doi:10.1093/gbe/evy115.

As mentioned, the melon plant or seed or plant part may comprise a mutant cmbell allele, wherein the mutant allele is produced by random mutagenesis or targeted mutagenesis, such as CRISPR based methods. Random mutagenesis may for example be chemical induced (e.g. EMS treatment) or radiation induced mutagenesis or other methods, whereby mutations are randomly induced in the genome and then plants or plant parts comprising mutations in the endogenous cmbell gene can be screened for and identified. Targeted mutagenesis are methods whereby mutations are specifically introduced into a target gene, such as the cmbell gene, using e.g. Crispr-Cas9, or Crispr-Cpf/ or other known methods, such as targeted transposon insertion. It is noted that using such methods, the mutant alleles described in e.g. the Examples can be generated without undue burden or other mutant alleles can be made.

When referring herein to a melon plant this encompasses in one aspect a seed from which the plant can be grown, i.e. the embryo in the seed may comprise at least one copy (or two copies) of mutant cmbell allele as described.

In one aspect the plant comprising the mutant allele is not produced exclusively by an essentially biological process, meaning that the mutant allele has at one point been generated by human intervention. If such a human generated mutant allele is transferred from one plant to another by crossing and selection, then the patent covers plants comprising the mutant allele, even if the plant itself has been generated solely by crossing and selection. Preferably the plant is not transgenic, and e.g. any construct used to modify the endogenous gene, in case of e.g. targeted gene editing, has been removed from the genome. Also the plant is preferably not a transgenic plant in that the mutant cmbell allele has not been introduced from the outside and integrated anywhere in the plant genome using plant transformation techniques, but rather the mutant allele is an endogenous, wild type CmBELI allele which has been mutated (using e.g. targeted or random mutagenesis or transposon insertion or DNA insertion) at the locus in the genome where the wild type allele is located.

In one aspect the melon plant is diploid and comprises at least one copy of a mutant cmbell allele as described above, i.e. the plant is heterozygous. As the phenotype is only seen when the mutant allele is in homozygous form, these plants are not stenospermocarpic, but produce normal seeded fruits upon pollination. Selfing of such heterozygous plants will generate a plant which is homozygous and which comprises two copies of the mutant allele. In one aspect the melon plant is diploid and comprises two copies of a mutant cmbell allele as described above, i.e. the plant is homozygous. The plant is, therefore, stenospermocarpic, producing seedless fruits following pollination (which induces fruit set and development).

The plants and plant parts comprising at least one copy of a mutant cmbell allele is preferably a cultivated plant, not a wild plant. So preferably cultivated melon (Cucumis meld). The plant may be an inbred line, a F1 hybrid or a breeding line.

Also seeds from which a plant or plant part as described above can be grown are encompassed herein. In one aspect the seed grows into a plant comprising one copy of the mutant allele. In another aspect the seed grows into a plant comprising two copies of the mutant allele.

Likewise, a fruit produced by a plant described above is encompassed herein, optionally wherein the fruit is seedless, produced by a plant comprising two copies of the mutant allele. As the fruit flesh of the seedless fruit develops from the maternal tissue, the fruit flesh also comprises two copies of the mutant allele. The plant part may be a cell, a flower, a leaf, a stem, a cutting, an ovule, pollen, a root, a rootstock, a scion, a fruit, a protoplast, an embryo, an anther.

Further, a vegetatively propagated plant propagated from a plant part and comprising at least one copy (or two copies) of a mutant cmbell allele in its genome is provided.

In one aspect also a method of producing seedless melon fruits is provided, said method comprising growing a melon plant comprising two copies of a mutant cmbell allele as described, allowing pollination to occur and allowing seedless fruits to develop.

A method for screening or detecting or genotyping plants, seeds, plant parts or DNA therefrom for the presence of a mutant allele of a gene named CmBELI, or for selecting a plant, seed or plant part comprising a mutant allele of a gene named CmBELI, or for generating a plant, seed or plant part comprising a mutant allele of a gene named CmBELI, is provided, wherein said mutant allele either a) comprises one or more mutations in a regulatory element, especially in the promoter, resulting in no expression (or reduced expression) of the allele compared to the wild type allele, and/or b) encodes a mutant protein comprising one or more amino acids replaced, inserted and/or deleted compared to the wild type protein, especially a non-functional protein, wherein the wild type allele encodes a protein of SEQ ID NO: 1 or a protein comprising at least 94% sequence identity to SEQ ID NO: 1 , or wherein the wild type genomic allele comprising SEQ ID NO: 4 or of a wild type genomic sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4.

In one aspect the mutant allele is a null allele.

In one aspect the mutant cmbell allele comprises a mutation in the genomic DNA, resulting in the expression of a mutant CmBELI protein comprising one or more amino acids inserted, deleted or replaced as described above.

In one aspect said mutation is an insertion, deletion and/or replacement of one or more nucleotides into the wild type CmBELI allele, rendering the mutant cmbell allele to be a null allele. For example, the one or more nucleotides inserted, deleted and/or replaced may be in the promoter region preceding SEQ ID NO: 4 (i.e. 5’ to the genomic sequence of SEQ ID NO: 4), e.g. in SEQ ID NO: 6 (or in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6) whereby e.g. the promoter not active anymore. Or the one or more nucleotides inserted, deleted and/or replaced may be in the genomic sequence of SEQ ID NO: 4 (or in a sequence comprising 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 comprising 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 comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2).

In one aspect any null allele which causes stenospermocarpy when in homozygous form is an embodiment of the invention. Different mutant alleles can be generated by the skilled person without undue burden. The skilled person can, for example, generate mutants in the promoter region or in the transcribed region of the CmBELI allele and determine whether the plant is stenospermocarpic when the mutant allele is in homozygous form in a melon plant.

Also a deletion of all or part of the CmBELI gene is encompassed herein, as a deletion will result in the same phenotype as a null allele.

Having identified the nucleotide sequence of the gene, the skilled person can generate melon plants comprising mutants in the CmBELI gene by various methods, e.g. mutagenesis, TILLING or CRISPR-Cas or other methods known in the art. Especially with targeted gene modification technologies such as Crispr-Cas, TALENS and others, targeted mutations can be made in e.g. the promoter or the transcribed sequence or in the coding sequence of the gene by the person skilled in the art. The skilled person can then confirm the phenotype of a plant homozygous for the mutant cmbell allele, i.e. being stenospermocarpic. Therefore, the skilled person is not limited to the specific cmbell mutant generated by the inventors (which the skilled person can also generate), but the skilled person can equally generate other mutations in the cmbell allele of melon and thereby generate other mutants which lead to stenospermocarpy when in homozygous form.

Various mutations can be generated and tested for the resulting phenotype, for example the regulatory elements, especially the promoter, can be mutated to reduce expression (knockdown) or eliminate expression (knock-out) of the allele and thus reduce or eliminate the amount of wild type CmBELI protein present in the cell or plant. Alternatively, mutations which lead to reduced function or loss-of-function of the CmBELI protein can be generated, i.e. mutations (such as missense mutations or frame shift mutations) which lead to one or more amino acids being substituted, inserted and/or deleted, or whereby the protein is truncated through the introduction of a premature stop-codon in e.g. the coding sequence (non-sense mutations). As the CmBELI protein comprises a highly conserved homeodomain it is, in one aspect, encompassed that one or more amino acids are replaced, deleted and/or inserted in this domain, as such mutations will result in a loss of function. Whether the mutation results in the expected phenotype (stenospermocarpy) can then be tested by generating plants homozygous for the mutation through selfing and growing the plant line and allow pollination of the flowers to see if fruits develop which are seedless, and in one aspect produce many more fruits than the wild type plant (increased average fruit number) and/or much smaller fruits (reduced average fruit weight) than the wild type plants (comprising two copies of the wild type CmBELI allele).

In one aspect a melon plant is provided comprising two copies of a mutant cmbell allele as described herein, especially a null allele, wherein said plant produces seedless fruits and produces at least 10%, 15%, 20%, 30%, 40% or 50% more fruits than the plant homozygous for the wild type allele when grown under the same conditions and over 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).

In another aspect a melon plant is provided comprising two copies of a mutant cmbell allele as described herein, especially a null allele, wherein said plant produces at least 10%, 15%, 20%, 30%, 40% or 50% more fruits than the plant homozygous for the wild type allele (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), when grown under the same conditions and over the same period of time and/or wherein the average fruit weight of said fruits is at least 5%, 10%, 15%, 20%, 30%, 40% or 50% lower than the average fruit weight of the plant homozygous for the wild type allele, when grown under the same conditions and over the same period of time. Thus, average fruit number is increased by at least 10%, 15%, 20% or more compared to the control plant comprising the wild type allele in homozygous form and/or the average fruit weight is decreased by at least 5%, 10%, 15%, 20% or more compared to the control plant comprising the wild type allele in homozygous form.

Also provided is a method for production of a stenospermocarpic cultivated melon plant, comprising the steps of: a) introducing mutations in a (population of) melon plant(s) or seed(s), especially a cultivated plant, or providing a (population of) mutated plant(s) or seed or progeny thereof; b) selecting a plant producing seedless fruits after pollination; c) optionally determining if the plant selected under b) comprises a mutant allele of a CmBELI gene; and d) optionally growing the plants obtained under c).

Steps b) and c) can also be switched, so that step b) is selecting a plant comprising a mutant allele of a CmBELI gene and step c) is determining if the plant (or a progeny thereof produced by selfing) producing seedless fruits after pollination. It is understood that the plants of step b must be homozygous, i.e. mutated plants must have been selfed at least once prior to step b.

Step a) can be carried out by e.g. mutagenizing seeds of one or more lines or varieties of melon, for example by treatment with mutagenizing agents such as chemical mutagens, e.g. EMS (ethyl methane sulphonate), or irradiation with UV radiation, X-rays or gamma rays or the like. The population may for example be a TILLING population. Preferably the mutagenized plant population is selfed at least once (e.g. to produce an M2 generation, or M3, M4, etc.) prior to carrying out step b). In step b) relating to phenotyping, plants are grown to allow pollination. Regular visual inspection of flowers, fruit setting and visual inspection of the mature fruits (e.g. presence of viable seeds or seedless) can be carried out to identify mutants which producing seedless fruits. Such plants, or selfing progeny thereof, can be tested for the presence of the mutant CmBELI gene by genotyping the plants for mutations in the CmBELI gene and encoded protein, or expression of the CmBELI gene, sequencing and other methods known to the skilled person. There are, thus, various methods, or combinations of methods, for verifying if a phenotypically selected plant comprises a mutant allele of a CmBELI gene.

If step b) is the selection of plants comprising a mutant allele of the CmBELI gene, the skilled person can also use various methods for detecting the DNA, mRNA or protein of the CmBELI gene in order to identify a plant comprising a mutant cmbell allele. The genomic DNA of the wild type melon CmBELI gene, encoding a functional CmBELI protein (SEQ ID NO: 1 or a variant thereof) is the DNA of SEQ ID NO: 4 (or a variant thereof) and the cDNA (and mRNA) encoding the protein of SEQ ID NO: 1 is given in SEQ ID NO: 2 and 3 (or a variant thereof). The promoter is upstream of this sequence and can e.g. be retrieved by sequencing or from the melon genome database. The promoter is in one aspect provided herein in the sequence of SEQ ID NO: 6, or in a sequence comprising at least 94% sequence identity to SEQ ID NO: 6. As genomic sequences encoding a certain protein may vary slightly (e.g. due to degeneracy of the genetic code or due to variation in intron sequences), the genomic alleles encoding a wild type CmBELI protein may comprise at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 4.

In one aspect the mutant allele of the CmBELI gene is a mutant allele resulting in reduced expression or no expression of the CmBELI gene or is a mutant allele resulting in one or more amino acids of the encoded CmBELI protein being replaced, inserted or deleted, compared to the wild type CmBELI protein.

In one aspect the mutant allele of the CmBELI gene is obtainable by inducing mutations, either targeted or random, into the gene (promoter or other regulatory elements, splice sites, coding region, etc.) and selecting plants, e.g. from the progeny, comprising a mutant cmbell allele. In one aspect an allele comprising a mutation in a codon, especially in a codon of the homeodomain is selected, e.g. a mutation which causes an amino acid replacement, a frame shift or a stopcodon. In one aspect the mutant allele causes a truncation of the encoded melon CmBELI protein. In another aspect an allele comprising a mutation in the genomic sequence of SEQ ID NO: 4, resulting in a non-functional mutant CmBELI protein being produced by the allele, e.g. the genomic sequence comprising an insertion of one or more nucleotides in intron 1 or intron 2, or in exon 1 , exon 2 or exon 3 sequence of SEQ ID NO: 4, whereby the transcript encodes a non-functional protein (e.g. a truncated protein).

In one aspect a SNP marker or INDEL marker is detected in the genome of a melon plant or plant part, or DNA therefrom. This SNP marker or INDEL marker detects the allele comprising a single nucleotide polymorphism or insertion/deletion polymorphism between SEQ ID NO: 4 and a mutant allele of SEQ ID NO: 4, which mutation results in e.g. a non-functional protein being made by the allele. For example, the mutant allele may comprise a STOP codon mutation in a codon, e.g. a codon of the homeodomain, or in a codon of exon 1 , 2 or 3.

For any mutant cmbell allele, SNP markers (or other markers) and SNP genotyping (or other genotyping) assays can easily be designed. Thus, allele specific markers and detection methods are encompassed herein, especially for any mutant allele which results in e.g. an amino acid insertion, deletion or replacement in CmBELI protein of melon, but also other mutant alleles described herein, e.g. null alleles.

The diploid plant heterozygous for the mutant cmbell allele (i.e. cmbell /CmBELI) will be heterozygous for the SNP marker or for the INDEL marker, while a plant homozygous for the mutant cmbell allele (i.e. cmbell /cmbell) will be homozygous for the SNP marker or INDEL marker.

Mutant-allele-specific markers and marker assays can easily be developed for any mutant cmbell allele, as the underlying genomic change, e.g. in a codon, can be used to design a marker assay to detect the genomic change, e.g. underlying the amino acid changes disclosed herein or other genomic changes (e.g. nucleotide insertions in exon or intron sequences of SEQ ID NO: 4) in the mutant cmbell allele compared to the wild type CmBELI allele of SEQ ID NO: 4, or compared to a wild type CmBELI allele comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 4 and encoding a wild type CmBELI protein comprising at least 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1.

Using such allele-specific markers, which detect specific mutant cmbell alleles, genotyping can be carried out to detect the presence and copy number of the allele in plants and plant material (or DNA derived therefrom). PLANTS AND PLANT PARTS

In one embodiment a cultivated melon plant is provided, or a part thereof (such as a cell, a tissue, organ, fruit, etc.), comprising at least one copy of a mutant allele of a gene named CmBELI , said mutant allele conferring stenospermocarpy when the mutant allele is in homozygous form.

In one aspect the mutant allele is a mutant allele of the melon gene which encodes the CmBELI protein of SEQ ID NO: 1 or a protein comprising at least 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 1 (wild type functional protein), whereby the mutant allele has a reduced expression (e.g. 10%, 9%, 8%, 7%, 6% or less of the wild type expression) or no expression, or whereby the mutant allele encodes a mutant CmBELI protein comprising one or more amino acids replaced, inserted and/or deleted compared to the wild type protein, resulting in a nonfunctional protein (optionally a reduced function protein, provided that the stenospermocarpic phenotype is conferred when the allele is in homozygous form).

In one embodiment the one or more amino acid replacements, insertions or deletions comprise or consist of the replacement, insertion or deletion of one or more amino acids in the conserved homeodomain.

The mutant protein has a loss-of-function (or optionally a reduced-function) compared to the wild type protein (and thus compared to a wild type plant comprising the wild type CmBELI gene), preferably the plant cell or plant comprising the mutant allele in homozygous form is stenospermocarpic.

When referring herein to a specific nucleotide or amino acid position, e.g. at amino acid 318 of SEQ ID NO: 1 , “or at amino acid 318 of a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the SEQ ID NO” (or ‘at the equivalent position in a sequence comprising at least 94% ... ’), this means that the nucleotide or amino acid is present in a variant sequence at a nucleotide or amino acid corresponding to the same nucleotide or amino acid (e.g. corresponding to amino acid 318 of SEQ ID NO: 1) in the variant sequence, i.e. in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the mentioned SEQ ID NO. It may for example be that the variant sequence is one or a few nucleotides or amino acids shorter, but when one pairwise aligns the variant sequence with the mentioned SEQ ID NO, 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 for example be amino acid 319 or 317.

The mutant allele is a mutation in an endogenous gene of cultivated melon. The existence of a gene conferring stenospermocarpy enables the skilled person to generate other de novo mutants in the gene, e.g. in any cultivated line or variety. The skilled person can, without undue burden, generate plants according to the invention, e.g. by carrying out a method for generation and/or identification of CmBELI mutants in a mutant population or by targeted gene editing of the CmBELI gene.

As mentioned above, as the CmBELI gene has been identified to be the gene encoding a protein of SEQ ID NO: 1 (wild type melon BEL1 protein) in normal, non-stenospermocarpic melon plants, the same or other mutants than the ones provided in the Examples can be generated de novo.

As natural variation may exist in the wild type, functional CmBELI proteins, the wild type CmBELI protein need not be 100% identical to the protein of SEQ ID NO: 1 but may have less sequence identity to SEQ ID NO: 1 , e.g. at least 94%, 95% 96%, 97%, 98% or 99% when aligned pairwise over the entire length to SEQ ID NO: 1. In one aspect the conserved homeodomain is however 100% identical to that of SEQ ID NO: 1 , so that the variation of at least 94% identity lies outside of the conserved homeodomain.

As mentioned, a mutant allele of a CmBELI gene causes a plant to produce seedless fruits after pollination, when the plant is homozygous for the mutant allele. Concerning the embodiments of the invention, the mutation in the mutant allele of a CmBELI gene can be any mutation, including deletions, truncations, insertions, point mutations, nonsense mutations, missense or non- synonymous mutations, splice-site mutations, frame shift mutations and/or mutations in regulatory sequences, such as the promoter.

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

The mutation can occur in a DNA sequence comprising the transcribed region of the gene or in the coding sequence of a CmBELI gene or in an RNA sequence encoding a CmBELI protein or it can occur in the amino acid of a CmBELI protein. Concerning a DNA sequence of a CmBELI gene the mutation can occur in the coding sequence or it can occur in non-coding sequences like 5’- and 3’-untranslated regions, promoters, enhancers, introns, etc. of a CmBELI gene. In respect to RNA encoding a CmBELI protein the mutation can occur in the pre-mRNA or the mRNA.

In one aspect the mutant allele results in the protein having a loss-of-function (or optionally a decrease of function) due to one or more amino acids being replaced, inserted and/or deleted, for example resulting in one or more amino acids being replaced, inserted and/or deleted at the C-terminal end of the protein and/or in the homeodomain of the protein. For example, truncation of 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 of the C-terminal end of the wild type protein will result in a mutant protein which causes stenospermocarpy, as was shown by the mutant protein of the Examples, which lacks amino acids Y344 and all remaining C-terminal amino acids (i.e. it is truncated at the C-terminal end by 265 amino acids). In particular, this mutant also lacks 36 amino acids (amino acid 344 to 379) of the conserved homeodomain and is, therefore, nonfunctional in vivo, as the homeodomain is required for DNA binding of the protein and transcriptional regulation of other genes. Therefore, any other mutant allele of the CmBELI gene, which encodes a mutant protein that lacks one or more amino acids of the homeodomain (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 homeodomain), or which is truncated at the C-terminal end, whereby 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 homeodomain, is believed to be non-functional in vivo and result in stenospermocarpy.

Thus, in one aspect mutations whereby the homeodomain is deleted completely or in part or whereby one or more amino acids of the homeodomain are replaced by one or more different amino acids, will result in a loss of function of the protein.

For example, a stop codon mutation e.g. in the N-terminal part preceding the homeodomain or a stop codon mutation in the homeodomain results in a truncated protein having a loss of function.

Likewise amino acid insertions, deletions and/or replacements in the N-terminal part preceding the homeodomain or in the homeodomain itself can result in a protein having a loss of function.

Any mutant allele can be analysed for the phenotype when the allele is in homozygous form to see if indeed the plant becomes stenospermocarpic. For phenotyping several plants (e.g. at least 3, 4, 5, 6, 7, 8, 9, 10) of a control plant and a mutant plant (homozygous for the mutant allele) are grown under the same conditions for the same period of time. The mature fruits can then 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.

One embodiment of the invention, therefore, concerns plant cells or plants according to the invention comprising a mutant allele of a CmBELI protein-encoding gene characterized in that the mutant allele comprises or effects one or more of the mutations selected from the group consisting of a) a deletion, truncation, insertion, point mutation, nonsense mutation, missense or non- synonymous mutation, splice-site mutation, frame shift mutation in the genomic sequence; b) a mutation in one or more regulatory sequences; c) a deletion, truncation, insertion, point mutation, nonsense mutation, missense or non- synonymous mutation, splice-site mutation, frame shift mutation in the transcribed sequence or coding sequence; d) a deletion, truncation, insertion, point mutation, nonsense mutation, missense or non- synonymous mutation, splice-site mutation, frame shift mutation in the pre-mRNA or mRNA; and/or e) a deletion, truncation, insertion or replacement of one or more amino acids in the CmBELI protein.

In one aspect the mutant allele results in reduced expression or especially in no expression of the CmBELI gene or the mutant allele encodes a protein having a decreased function or especially a loss-of-function.

Reduced expression or no expression means that there is a mutation in a regulatory region of the CmBELI gene, such as the promoter, whereby significantly reduced mRNA transcript (e.g. 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less, of the wild type allele transcript) or no mRNA transcript of the CmBELI allele is being made, compared to plants and plant parts comprising a wild type CmBELI allele. The decrease in the expression can, for example, be determined by measuring the quantity of mRNA transcripts encoding CmBELI protein, e.g. using Northern blot analysis or RT-PCR. Here, a significant reduction preferably means a reduction in the amount of mRNA transcripts by at least 50%, in particular by at least 70%, optionally by at least 85% or by at least 95%, 96%, 97%, 98%, 99% or even by 100% (no expression) compared to the plant or plant part comprising a wild type CmBELI gene. Expression can be analysed e.g. in leaf tissue or ovary tissue. Various techniques can be used to study gene expression, e.g. quantitative real-time Polymerase Chain Reaction, northern blots, RNA sequencing, microarrays, etc. Thus, the mRNA transcript levels encoding the wild type CmBELI protein of SEQ ID NO: 1 or a protein comprising at least 94% sequence identity to SEQ ID NO: 1 can be analyzed. The cDNA (which corresponds to the mRNA, except that Uracil is shown as Thymine in the cDNA) is provided in SEQ ID NO: 2 and in SEQ ID NO: 3 (wherein also UTR’s are included), or in a variant of these sequences. Thus, in one aspect the level of mRNA transcript (or the corresponding cDNA) is reduced or not produced in planta due to one or more mutations in the promoter sequence. The promoter is provided in SEQ ID NO: 6 or in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6. The promoter is also found in the genome of melon (database cucurbitgenomics.org genome DHL v. 3.6.1) 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. In one aspect the melon plant or plant part comprises one or more mutations in the promoter of the CmBELI allele, whereby no mRNA transcript (or significantly reduced mRNA transcript, see above) of the wild type CmBELI allele is produced, as determined for example by the absence of cDNA encoding the wild type CmBELI protein of SEQ ID NO: 1 or a wild type protein comprising at least 94% sequence identity to SEQ ID NO: 1 (or the significant reduction of cDNA encoding the wild type CmBELI protein of SEQ ID NO: 1 or a wild type protein comprising at least 94% sequence identity to SEQ ID NO: 1).

In one aspect the protein comprising one or more amino acids replaced, inserted or deleted compared to the wild type protein. Thus, for melon, one or more amino acids are inserted, deleted or replaced compared to the wild type CmBELI protein of SEQ ID NO: 1 or a wild type CmBELI protein comprising at least 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 1 ; whereby the mutant protein has a loss of function (optionally a reduced function) compared to the wild type protein and, thus, results in stenospermocarpy when the mutant allele is present in homozygous form in a diploid plant.

In one aspect the wild type CmBELI protein comprises the conserved homeodomain. Thus, in one aspect the mutant allele is a mutant allele of the gene CmBELI , which gene encodes a wild type protein of SEQ ID NO: 1 or a wild type protein comprising at least 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 1 , and whereby the wild type protein comprises the conserved homeodomain of amino acids 318 to 379 of SEQ ID NO: 1.

In one aspect the wild type CmBELI protein comprises the conserved homeodomain, i.e. any variation of the functional wild type protein is outside the conserved homeodomain.

The mutant alleles of the above wild type alleles are in one aspect mutant alleles having reduced expression or no expression (through e.g. mutations in the promoter or enhancer elements) or producing a mutant protein which comprises one or more amino acids inserted, deleted or replaced compared to the wild type protein, whereby the mutant protein has no function in vivo (or optionally a reduced function), as can be determined when the mutant allele is in homozygous form in a plant and by analysing whether the plant produces seedless fruits.

If the mutant allele causes stenospermocarpy in vivo, while the control plant comprising only the wild type CmBELI alleles is not stenospermocarpic, then the mutant protein has no function (or optionally a severely reduced function) compared to the wild type protein. The same phenotypic analysis can be done for a mutant allele having no gene expression (or optionally severely reduced gene expression, e.g. a reduction in the amount of RNA transcript by 95%, 96%, 97%, 98% or 99%). Thus, any mutant allele can be made homozygous in the plant and the phenotype can be compared to the control plant comprising the original, non-mutated allele in homozygous form. The homeodomain was found to be a highly conserved protein domain, which will be 100% identical in other wild type, functional CmBELI variants also, as they will be required for proper functioning of the protein in the plant. Therefore, mutating the homeodomain by inserting, deleting and/or replacing one or more of its amino acids will abolish the CmBELI protein function in vivo.

In one aspect, therefore, a plant provided herein comprises a mutant CmBELI allele which encodes a CmBELI protein comprising one or more amino acids inserted, deleted and/or replaced in the homeodomain.

The wild type, functional CmBELI protein which is mutated to comprise one or more amino acids inserted, replaced and/or deleted is, in one aspect, selected from CmBELI of SEQ ID NO: 1 or a protein comprising at least 94% identity to SEQ ID NO: 1 , whereby the wild type protein e.g. comprises the homeodomain of SEQ ID NO: 1 or alternatively a wild type homeodomain comprising at least 98% or 99% sequence identity to the homeodomain of SEQ ID NO: 1 .

A mutant protein comprising a frame shift leading to a change of one or more amino acids in the homeodomain or a mutant protein comprising a truncation leading to the deletion of one or more amino acids of the homeodomain is hereby encompassed as being a mutant protein comprising no function in vivo.

In one aspect, therefore, a mutant CmBELI allele is provided encoding a mutant protein wherein the Y344 of SEQ ID NO: 1 (or the equivalent amino acid in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1), is replaced by another amino acid or is deleted, e.g. the codon being replaced by a STOP codon.

In one aspect, therefore, a mutant CmBELI allele is provided encoding a mutant protein wherein at least one, or more, amino acids of the homeodomain, i.e. selected from W318 to M379 of SEQ ID NO: 1 (or the equivalent amino acid in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1), are replaced by another amino acid or are deleted, e.g. the codon being replaced by a STOP codon.

When amino acids ‘from one amino acid to another amino acid’ are mentioned herein this includes the start/first and end/last amino acid mentioned.

When referring to an amino acid being ‘deleted’, this includes a mutation whereby the codon is changed into a stop codon, or the codon is deleted, or a mutation whereby there is a frameshift, resulting in the amino acid not being encoded, or a mutation in the transcribed region whereby the mRNA transcript becomes truncated and translation results in a truncated protein. Equally, when referring to an amino acid being ‘replaced’, this includes a mutation whereby the codon encodes a different amino acid, or a codon is inserted, or a mutation whereby there is a frameshift resulting in a different amino acid being encoded.

In one aspect the mutant CmBELI allele is heterozygous in a diploid melon plant cell or plant. In another aspect the mutant CmBELI allele is homozygous in a diploid melon plant cell or plant.

The plant cells and plants are preferably cultivated plants, such as elite breeding lines or varieties, and not wild plants. Melon may be any type of melon, such as Piel de Sapo, Charantais, Galia, Honey Dew, Yellow Canary, etc.

A diploid melon plant may thus have the genotype cmbel1/CmBEL1 (heterozygous for the mutant allele) or cmbel1/cmbel1 (homozygous for the mutant allele). In one aspect the diploid melon plant comprising the cmbell allele in homozygous form is a double haploid plant (DH), e.g. a double haploid melon, plant or plant cell or plant part. DH plants can be made by chromosome doubling (e.g. through colchicine treatment) of haploid cells.

In one aspect the melon plant is homozygous for cmbell, in another aspect it is heterozygous for cmbell. In one aspect it is an inbred line or a variety. In a further aspect it is an F1 hybrid.

In one aspect the melon plant is a diploid plant (e.g. an inbred line), comprising at least one mutant copy of cmbell, preferably two mutant copies (i.e. is homozygous for cmbell). After pollination of the flowers (which is required for fruit set and development), the diploid plant homozygous for cmbell will produce fruits which are seedless. Mature or viable seeds are not developed in stenospermocarpic plants due to arrested development or interruption of the normal seed development process. Thus, when diploid plants homozygous for a mutant cmbell allele (cmbell /cmbell are self-pollinated or pollinated by pollen from another plant, they produced seedless, diploid fruits.

A mutant allele of cmbell causes a plant to be male fertile but the plant produces seedless fruits, when the plant is homozygous for the mutant allele. The mutation in the CmBELI gene can be any mutation, including deletions, truncations, insertions, point mutations, nonsense mutations, missense or non-synonymous mutations, splice-site mutations, frame shift mutations and/or mutations in regulatory sequences. The mutation can occur in the genomic DNA sequence in e.g. the coding region (exons) or the non-coding region e.g. introns of the CmBELI gene and/or in the RNA sequence encoding a CmBELI protein or it can occur in the amino acid of the CmBELI protein. Concerning a genomic DNA sequence of the CmBELI gene the mutation can occur in the coding sequence (cds, composed of the exons) or it can occur in non-coding sequences like 5’- and 3’-untranslated regions, introns, promoters, enhancers etc. In respect to RNA encoding a CmBELI protein the mutation can occur in the pre-mRNA or the mRNA. In one aspect the invention, therefore, relates to a melon plant or plant part comprising at least one copy of the mutant cmbell allele, preferably two copies.

Seeds from which such a diploid plant can be grown are also encompassed herein, as are parts of such a plant, such as diploid seedless fruits, flowers, leaves, stems, roots, vegetative propagations, cells, cuttings, seed propagations (e.g. selfings) and also in vitro cell- or tissue cultures, as well as pollen, ovaries, etc. are encompassed herein. Thus, in one embodiment the diploid plant, or seeds from which the plant can be grown, or tissue or parts of the plant (pollen, anthers, ovules) comprises a mutant cmbell allele as described elsewhere herein.

In one aspect the diploid plant or seed comprises one or two copies of the mutant cmbell allele which encodes a non-functional (loss-of-function) protein (optionally a reduced-function protein) which comprises one or more amino acids replaced, inserted and/or deleted with respect to the wild type functional protein of SEQ ID NO: 1 , or with respect to a functional variant comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

In one aspect the diploid plant or seed comprises one or two copies of the mutant cmbell allele which encodes a non-functional (loss-of-function) protein (optionally a reduced-function protein) which comprises one or more amino acids replaced, inserted and/or deleted in the homeodomain of SEQ ID NO: 1 or in the homeodomain of a functional variant comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

In one aspect the diploid plant or seed comprises one or two copies of the mutant cmbell allele which encodes a non-functional (loss-of-function) protein (optionally a reduced-function protein) which is truncated at the C-terminal end and said truncation starts in or prior to the homeodomain of SEQ ID NO: 1 (or in or prior to the homeodomain of a functional variant comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1), whereby 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 homeodomain) of the homeodomain are missing in the truncated protein. Thus, the truncation leads to a mutant protein comprising at most amino acids 1 to 378 of SEQ ID NO: 1 (or amino acids 1 to 378 of a functional variant comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1).

When referring to ‘truncation’ of the wild type, functional protein, this encompasses that the missing amino acids may be replaced by one or more different amino acids (i.e. amino acids having a different sequence than the wild type sequence), e.g. as occurs when a frame shift occurs which lead to the reading frame being changed.

In one aspect the diploid plant or seed comprises one or two copies of the mutant cmbell allele which encodes the truncated protein comprising only amino acids 1 to 343 of SEQ ID NO: 1 , or which encodes a truncated protein comprising the equivalent amino acids of amino acids 1 to 343 of SEQ ID NO: 1 in a sequence comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

The underlying cause of the truncation of the protein that is made may be various, e.g. change of an amino acid codon into a STOP codon, insertion of one or more nucleotides which e.g. terminates transcription and/or translation, a frame shift mutation, a splice site mutation, etc.

In one aspect, the truncation is caused by a DNA insertion in intron 2 of the gene, leading to only the codons of exon 1 and exon 2 being transcribed from the gene. Thus, in one aspect the mRNA transcript comprises only the codons of exon 1 and exon 2. The translated protein, therefore, comprises only amino acids 1 to 343 of SEQ ID NO: 1.

In one aspect the DNA insertion, in e.g. 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 the DNA insert, in e.g. intron 2, comprises the nucleotides of SEQ ID NO: 5.

The melon plant comprising one or preferably two copies of a mutant cmbell allele may be an inbred line, an open pollinated variety (OP) or an F1 hybrid plant.

A mutant cmbell allele can be generated in any background melon type, or can be introduced by crossing from one background (e.g. in which it was generated) into another background, e.g. Piel de Sapo, honeydew, Galia, Charantais, etc. Thus, any melon type can be made which has the stenospermocarpy trait. On one aspect, the melon plant produces mini melons or midi melons, so that the mini melons or midi melons are seedless. In such small sized fruits, the seedless phenotype has the advantage that seeds do not have to be removed prior to eating or processing and the fruits can be consumed or processed directly. Some midi size varieties are for example the variety Kukino F1 (a Piel de Sapo variety of Nunhems). The stenospermocarpy trait can, thus, be introduced into e.g. Kukino F1 , to make the fruits seedless. In one aspect, the mutant cmbell allele, when present in homozygous form, reduces average fruit weight and/or increases average fruit number. So, when introduced into a variety which produces seeded fruits of an average weight of 1.5 kg to 2.0 kg, the mutant allele may result in significantly lower average fruit weight and/or significantly higher fruit number.

In the Examples various of the melon fruits did not show empty seed cavities in the seedless fruit. In one aspect, thus, the seedless melon fruits are free of (or essentially free of) empty seed cavities.

In one aspect the F1 hybrid comprises the mutant cmbell allele in homozygous form (cmbell /cmbell) and is grown from F1 hybrid seed that comprises the mutant cmbell allele in homozygous form. As the F1 hybrid plant which is grown from seeds comprising the cmbell allele in homozygous form will produce seedless fruits, the F1 hybrid plant may be propagated vegetatively. Thus, in one aspect a vegetatively propagated F1 hybrid melon plant is provided comprising the mutant cmbell allele in homozygous form. The plant will produce seedless fruits, e.g. many small fruits, which are seedless.

Alternatively, the F1 hybrid comprises the mutant cmbell allele in homozygous form (cmbell /cmbell) may be produced by crossing a cmbel1/CmBEL1 plant (heterozygous for the mutant allele) with a cmbel1/cmbel1 plant (homozygous for the mutant allele), whereby the resulting seeds will be about 50% cmbel1/cmbel1 and 50% cmbel1/CmBEL1 genetically. Optionally the seeds or seedlings comprising the mutant allele in homozygous form may be selected from the mixed seeds. Thus, in one aspect a seed mix is provided comprising seeds which are about 50% cmbel1/cmbel1 and about 50% cmbel1/CmBEL1 genotypically.

Another method for producing the F1 hybrid comprises the mutant cmbell allele in homozygous form (cmbell /cmbell is by crossing a cmbel1/CmBEL1 plant (heterozygous for the mutant allele) with a cmbel1/CmBEL1 plant (heterozygous for the mutant allele). The resulting seeds will be about 25% cmbell /cmbell and 50% cmbel1/CmBEL1 and 25% CmBEL1/CmBEL1 genetically. Optionally the seeds or seedlings comprising the mutant allele in homozygous form may be selected from the mixed seeds. Thus, in one aspect a seed mix is provided comprising seeds which are about 25% cmbell /cmbell and about 50% cmbel1/CmBEL1 and about 25% CmBEL1/CmBEL1 genotypically.

Yet another method for producing the plant comprises the mutant cmbell allele in homozygous form (cmbell /cmbell) is by selfing a cmbel1/CmBEL1 plant (heterozygous for the mutant allele). The resulting seeds will be about 25% cmbel1/cmbel1 and 50% cmbel1/CmBEL1 and 25% CmBEL1/CmBEL1 genetically. Optionally the seeds or seedlings comprising the mutant allele in homozygous form may be selected from the mixed seeds. Thus, in one aspect a seed mix is provided comprising seeds which are about 25% cmbel1/cmbel1 and about 50% cmbel1/CmBEL1 and about 25% CmBEL1/CmBEL1 genotypically.

In one embodiment of the invention a non-destructive seed based genotyping method is used to determine the presence of one or more mutant cmbell alleles in the embryo, especially a nondestructive genotyping of single plant seeds, whereby the viability of the seed is not affected, but the genotype of the plant that grows from the seed can be determined. Various nondestructive 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 nondestructive genotyping system from a single seed for marker-assisted selection in watermelon”) describes such a method. In another aspect the seeds, e.g. of a seed mix, may be germinated to select plants (e.g. seedlings) which are homozygous for the cmbell mutant allele. Plants, e.g. seedlings, can be selected by taking a tissue or DNA sample and genotyping the DNA to determine whether the mutant cmbell allele is present and whether one or two copies are present. Plants, e.g. seedlings, comprising two copies of the mutant cmbell allele can then be selected and grown to produce seedless melon fruits.

Thus, in one aspect a method for selecting a plant which comprises the mutant cmbell allele in homozygous form is provided, comprising selecting plants, e.g. seedlings, comprising two mutant cmbell alleles.

This method may comprise the steps of providing a plurality of seeds, e.g. seeds comprising different genotypes for the mutant (cmbell) and wild type (CmBELI) allele, e.g. seeds comprising mixtures of genotypes, such as cmbel1/CmBEL1 (heterozygous for the mutant allele) and cmbel1/cmbel1 (homozygous for the mutant allele), and germinating the seeds so that the plants can be analyzed for their genotype.

Thus, in one embodiment of the invention, a population of seeds is provided comprising a mixture of genotypes of CmBELI alleles, wherein said population is a population of seeds comprising about 50% of seeds wherein the mutant cmbell allele is in heterozygous form (cmbell /CmBELI) and about 50% of seeds wherein the mutant cmbell allele is in homozygous form (cmbell/ cmbell). A population of seeds which segregates in a 50% cmbell /CmBELI to 50% cmbell/ cmbell ratio can be generated by crossing a parent line A (preferably as female parent), which is heterozygous for the mutant cmbell allele, with a parent line B (preferably as male parent), which is homozygous for the mutant cmbell allele. Seeds harvested from this cross will segregate in the approximate 50:50 ratio mentioned and from this population of seeds the approximately 50% homozygous mutant cmbell allele seeds can be selected as described. It is understood that the cross of parent line A with parent line B can be carried out between many plants to obtain a large amount of F1 seeds which segregate in the approximate 50:50 ratio of CmBEL1/cmbe/7 : cmbell/ cmbell seeds.

The actual step of crossing line A with line B is, in one aspect of the invention, also part of the method. Thus, in one aspect a method of generating and/or selecting seeds which are homozygous for the mutant cmbell allele is provided, comprising a) Crossing a parent line A, which is heterozygous for a mutant cmbell allele with parent line B, which is homozygous for a mutant cmbell, b) Harvesting the F1 seeds of the cross, c) Germinating the F1 seeds, d) Selecting plants of step c) which comprise the cmbell allele in homozygous form, and optionally e) Allowing the selected plants to produce seedless fruits (after pollination of the flowers), and optionally f) Harvesting the seedless fruits.

As mentioned, parent line A is the female parent line and parent line B is the male parent line in this method, because the line homozygous for the mutant cmbell allele cannot be used to produce seeds, as it will produce seedless fruits upon pollination of the flowers.

Another aspect of the invention a population of seeds is provided comprising a mixture of genotypes of CmBELI alleles wherein said population of seeds is a population of seeds comprising about 50% of seeds wherein the mutant cmbell allele is in heterozygous form (cmbell /CmBELI) and about 25% of seeds wherein the mutant cmbell allele is in homozygous form (cmbell/ cmbell) and about 25% wherein the wild type CmBELI allele is in homozygous form (CmBEL1/CmBEL1). A population of seeds which segregates in a 50% cmbell /CmBELI to 25% cmbell/ cmbell to 25% CmBEL1/CmBEL1 ratio can be generated by crossing a parent line A, which is heterozygous for the mutant cmbell allele, with a parent line B, which is also heterozygous for the mutant cmbell allele, or by selfing a plant which is heterozygous for the mutant cmbell allele. Seeds harvested from this cross will segregate in the approximate 50 (CmBELI/ cmbell) : 25 cmbell/ cmbell) : 25 (CmBEL1/CmBEL1) ratio mentioned and from this population of seeds the approximately 25% homozygous mutant cmbell allele seeds can be selected as described.

The actual step of crossing heterozygous line A with heterozygous line B, or selfing a line which is heterozygous for the mutant cmbell allele is, in one aspect of the invention, also part of the method. Thus, in one aspect a method of generating and/or selecting seeds which are homozygous for the mutant cmbell allele is provided, comprising a) Crossing a parent line A, which is heterozygous for a mutant cmbell allele of the invention with parent line B, which is heterozygous for a mutant cmbell allele of the invention, or selfing a line which is heterozygous for a mutant cmbell allele of the invention, b) Harvesting the F1 seeds of the cross, c) Germinating the F1 seeds, d) Selecting plants of step c) which comprise the cmbell allele in homozygous form, and optionally e) Allowing the selected plants to produce seedless fruits (after pollination of the flowers), and optionally f) Harvesting the seedless fruits.

Also provided is a method for producing stenospermocarpic fruits, said method comprising growing a plant comprising a mutant cmbell allele (as described throughout the specification) in homozygous form and optionally harvesting the seedless fruits produced by said plant.

Further aspects of the invention are a method for selecting a plant or plant part capable of stenospermocarpic fruit formation, said method comprising selecting a plant or plant part comprising at least one copy of a mutant cmbell allele (preferably two copies), wherein the mutant allele causes stenospermocarpic fruit formation when the allele is in homozygous form. The selection of the plant or plant part may involve direct and/or indirect methods, such as DNA analysis of the endogenous CmBELI alleles (e.g. sequence analysis, allele specific genotyping methods, etc.), RNA analysis of the CmBELI alleles (e.g. mRNA expression), CmBELI protein analysis, etc. Thus, in the above methods any phenotypic selection step on germinated seedlings can be replaced by selection based on the detection of the mutant cmbell allele in either the non-germinated seeds (i.e. omitting the germination of seeds step) and/or in seedlings or plants (or tissue thereof, such as leaf discs) of germinated seeds.

In a further aspect of the invention a method for generating a plant or plant part capable of stenospermocarpic fruit formation, said method comprising contacting a plant or plant part with a mutagen and subsequently (optionally after one or more selfings of the mutagenized plant) selecting a plant or plant part comprising at least one copy of a mutant cmbell allele, wherein the mutant allele causes stenospermocarpic fruit formation when the allele is in homozygous form.

Also provided is a method for producing a plant capable of stenospermocarpic fruit formation, comprising a) Crossing a plant comprising in its genome at least one mutant cmbell allele as described (which mutant cmbell allele causes stenospermocarpic fruit formation when in homozygous form) with another plant, and optionally b) Harvesting seeds from said cross, and optionally c) Selecting seeds, or plants grown from the seeds (e.g. seedlings), comprising at least one copy of the mutant cmbell allele, preferably selecting seeds comprising two copies of the mutant cmbell allele. In a preferred aspect, a plant which is heterozygous for the mutant cmbell allele is used as female parent and is crossed with a male parent plant which is homozygous for the mutant cmbell allele.

The selection in step c) can be based on method which detect the mutant cmbell allele directly (e.g. using a genotyping assay) or based on the phenotype.

Also the seeds and plants selected by the method are encompassed herein, as are plants grown from the seeds and seedless fruits produced by the plants grown from the seeds homozygous for the mutant cmbell allele.

In one aspect, a method for identifying a plant or plant part or cell comprising in its genome at least one copy of a mutant allele of cmbell gene is provided, said method comprising determining whether the plant or plant part or cell comprises in its genome at least one mutant cmbell allele.

This method may involve analysing (directly or indirectly) the gene expression of the cmbell allele, and/or the genomic nucleotide sequence of the cmbell allele, or the mRNA nucleotide sequence of the cmbell allele, or the protein sequence of the CmBELI protein, or the protein amounts of the CmBELI protein of the plant or plant part or plant cell, to determine if the gene expression is knocked down or knocked out compared to the wild type plant or plant part or plant cell, or if the mRNA is truncated or comprises one or more nucleotides inserted, deleted or replaced compared to the wild type mRNA, or if the encoded protein comprises one or more amino acid insertions, deletions or replacements compared to the wild type CmBELI protein.

One method for analysing the presence of a mutant cmbell allele, is for example to assay the presence of a Single Nucleotide Polymorphism (SNP) or INDEL in the genomic sequence of the cmbell allele, by, for example, designing primers for the SNP or INDEL and genotyping plants or plant parts for the genotype of that particular SNP or INDEL.

So one aspect of the invention comprises a method for determining whether a plant, plant part or plant cell comprises one or more copies of a mutant cmbell allele by a method selected from analysing one or more nucleotides of the genomic cmbell allele in a genotyping assay, analysing the mRNA (or cDNA) expressed by the cmbell allele or analysing the CmBELI protein amount and/or amino acid sequence (using e.g, antibody based detection).

In one aspect, especially in respect of the European Patent Convention, the plant according to the invention is “not obtained exclusively by an essentially biological process”, or in one aspect the mutant cmbell allele is not a natural mutant allele. If such a disclaimer is present in the claim of the European patent, it should be noted that using a plant comprising a mutant allele (e.g. a commercial variety of the applicant) to cross the mutant allele into a different background will still be seen as falling under the claim, even though an exclusively essentially biological process (only crossing and selection) may have been used to transfer the allele into a different background.

In one aspect the mutant cmbell allele is an induced mutant allele.

In one aspect melon plant is provided, comprising two copies of a mutant cmbell allele, which in homozygous form leads to stenospermocarpy, wherein the melon plant is a vegetative propagation, e.g. produced by cuttings which are induced to develop roots and shoots, and upon flowering and pollination will produce seedless fruits. Vegetative propagation may be done using methods well-known in the art, for example in vitro plant tissue culture, e.g. rooting cuttings. In one embodiment, a method of vegetatively propagating a plant comprising one or two copies of a mutant cmbell allele in its genome comprises: a) collecting tissue of a plant comprising one or two copies of a mutant cmbell allele as described herein; b) cultivating said tissue to obtain proliferated shoots; c) rooting said proliferated shoots to obtain rooted plantlets; and d) growing plants from said rooted plantlets. Cuttings according to this aspect of the present invention may include roots, stems, leaves, cotyledons, flowers, fruit, embryos and pollen.

In one aspect a screening method for identifying and/or selecting seeds, plants or plant parts or DNA from such seeds, plants or plant parts comprising in their genome a mutant cmbell allele is provided.

The method comprises screening at the DNA, RNA (or cDNA) or protein level using known methods, in order to detect the presence of the mutant allele. There are many methods to detect the presence of a mutant allele of a gene.

Thus, a method for screening and/or selecting plants, seeds or plant material or plant parts, or DNA or RNA or protein derived therefrom, for the presence of a mutant cmbell allele is provided comprising one or more of the following steps: a) determining if the gene expression of the endogenous CmBELI gene is reduced or abolished; b) determining if the amount of wild type CmBELI protein is reduced or abolished; c) determining if a mutant mRNA, cDNA or genomic DNA encoding a mutant CmBELI protein is present; d) determining if a mutant CmBELI protein is present; e) determining if plants or progeny thereof are stenospermocarpic.

Routine methods can be used, such as RT-PCR, PCR, antibody based assays, sequencing, genotyping assays (e.g. allele-specific genotyping), phenotyping, etc. The plants or plant material or plant parts may be melon plants or plant materials or plant parts, such as leaves, leaf parts, cells, fruits, fruit parts, ovaries, stem, hypocotyl, seed, parts of seeds, seed coat, embryo, etc.

For example if there is a single nucleotide difference (single nucleotide polymorphism, SNP) between the wild type and the mutant allele, a SNP genotyping assay can be used to detect whether a plant, seed or plant part or cell comprises the wild type nucleotide or the mutant nucleotide in its genome. For example the SNP can easily be detected using a KASP-assay (see world wide web at kpbioscience.co.uk) or other SNP genotyping assays. For developing 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 e.g. Allen et al. 2011 , Plant Biotechnology J. 9, 1086-1099, especially p097-1098 for KASP-assay method.

Equally other genotyping assays can be used. For example, a TaqMan SNP genotyping assay, a High Resolution Melting (HRM) assay, SNP- genotyping arrays (e.g. Fluidigm, Illumina, etc.) or DNA sequencing may equally be used.

Based on the difference between the genomic sequence of the wild type allele and the mutant allele, the skilled person can easily develop a genotyping assay which can be used to detect specific alleles.

Also provided herein is a method for identifying a melon plant (or plant part) comprising a mutant cmbell allele, the method comprising detecting in the plant (or plant part) the presence of a mutant cmbell allele, wherein the presence is detected by at least one marker (or nucleotide difference) within the cmbell allele or by detecting the protein encoded by the cmbell allele. The method for detecting the mutant cmbell allele is selected from the group consisting of PCR amplification, nucleic acid sequencing, nucleic acid hybridization and an antibody-based assay (e.g. immunoassay) for detecting the cmbell protein encoded by the allele.

Also provided herein is a method for identifying a melon plant (or plant part) comprising a mutant cmbell allele comprising a mutation in a regulatory element, the method comprising detecting in the plant (or plant part) the reduced gene expression or absence of gene expression of the mutant cmbell allele, wherein the presence is detected by mRNA levels (cDNA) of the wild type CmBELI allele or by detecting the protein levels of the wild type CmBELI protein. The method for detecting the mutant cmbell allele is selected from the group consisting of PCR amplification (e.g. RT-PCR), nucleic acid sequencing, western blotting and an antibody based assay (e.g. immunoassay) for detecting the CmBELI protein encoded by the allele.

Also provided is a method for determining, or detecting or assaying, whether a cell of a melon plant or plant part comprises a mutant allele of a gene named CmBELI encoding a protein of SEQ ID NO: 1 , or a protein comprising at least 94%, 95%, 96%, 97% or 98% sequence identity to SEQ ID NO: 1 , is provided herein. In one aspect the method comprises determining the expression of the allele, and/or determining the coding sequence of the allele and/or determining part of the coding sequence of the allele (e.g. a SNP genotype of the allele), and/or determining the amino acid sequence of the protein produced and/or the amount of protein produced.

Various method can be used to determine whether a plant or part thereof comprises a mutant cmbell allele of the invention. As mentioned, the mRNA (or cDNA) level of the wild type allele may be determined, or the wild type protein level may be determined, to see if there is a reduced expression or no expression of the wild type allele. Also, the transcribed sequence or the coding sequence or part thereof may be analysed, for example if one already knows which mutant allele may be present, an assay can be developed to detect the mutation, e.g. a SNP genotyping assay can e.g. distinguish between the presence of the mutant allele and the wild type allele.

A method for selection of a plant or seed comprising the steps of: a) identifying a plant or seed which has a mutation in an allele of a gene encoding a CmBELI protein, wherein the wild type allele of the gene encodes a CmBELI protein comprising at least 94%, 95%, 96%, 97% or 98% or 99% sequence identity to SEQ ID NO:1 , and optionally b) determining whether the plant, or a progeny plant produced by self-fertilization, is stenospermocarpic and optionally c) selecting a plant or seed comprising at least on copy of the mutant allele of step a).

A method for production of a melon plant, comprising the steps of: a) introducing mutations in a population of plants or seeds, b) selecting a plant producing seedless fruits after pollination and/or selecting a plant or seed comprising a mutant cmbell allele in its genome, c) optionally verifying if the plant selected under b) has a mutation in an allele encoding a CmBELI protein, and optionally d) growing or cultivating the plant or seed obtained under c), wherein the wild type allele of the gene encodes a CmBELI protein comprising at least 94% sequence identity to any one of the proteins selected from the group of: SEQ ID NO:1.

In another aspect a melon plant, seed and plant part is provided, comprising a mutation in the endogenous CmBELI gene, e.g. an induced mutation generated e.g. by random mutagenesis or by targeted mutagenesis, whereby either the gene expression is reduced or abolished or the expressed gene encodes a reduced function or loss of function CmBELI protein compared to the wild type protein.

Also provided herein is a method for screening melon plants, seeds, plant parts, or DNA therefrom, for the presence of a mutant allele of a gene named CmBELI , or for selecting a melon plant, seed or plant part comprising a mutant allele of a gene named CmBELI , comprising the steps: a) analysing whether the genomic DNA comprises a wild type CmBELI allele which encodes a protein of SEQ ID NO: 1 (or a wild type protein comprising at least 94% identity to SEQ ID NO: 1), and/or a mutant cmbell allele which encodes a mutant protein comprising one or more amino acids replaced, inserted or deleted compared to the wild type CmBELI protein, or a mutant allele that is not expressed or has reduced expression, and optionally b) selecting a plant, seed or plant part comprising e.g. 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.

In one aspect the method step a) comprises a method selected from: i) amplification of at least part of the CmBELI allele using one or more oligonucleotide primers which hybridize to the DNA of the CmBELI allele, ii) hybridization of one or more oligonucleotide probes to at least part of the DNA of the CmBELI allele, iii) sequencing the DNA, mRNA or cDNA of the CmBELI allele.

So, for example a DNA sample can be obtained from a plant, seed or plant part, and a PCR reaction can be carried out to amplify part of the wild type CmBELI allele and/or part of the mutant cmbell allele.

Competitive PCR methods, for example, can be used (such as a KASP assay) to generate amplification products of the alleles present at the CmBELI locus in the genomic DNA. Similarly, oligonucleotide probes can generate hybridization products of the alleles present at the CmBELI locus in the genomic DNA. Primers or probes may be designed to be specific to a particular cmbell allele, e.g. to differentiate between the wild type allele and a mutant allele.

In one aspect, a genotyping assay is provided for genotyping melon plants, seeds, plant parts, cells or tissues, comprising the steps: a) providing genomic DNA of one or more melon plants or a population of plants, and b) carrying out a genotyping assay which detects the presence of the wild type allele encoding the protein of SEQ ID NO: 1 or a wild type allele encoding a protein comprising at least 94% sequence identity to SEQ I D NO: 1 and/or the presence of a mutant allele (or two different mutant alleles), wherein the mutant allele encodes a mutant protein which comprises one or more amino acids inserted, deleted or replaced compared to the wild type protein of SEQ ID NO: 1 or compared to a wild type protein comprising at least 94% sequence identity to SEQ ID NO: 1 , or a mutant allele that is not expressed or has reduced expression, and optionally c) selecting a plant, seed, plant part, cell or tissue comprising e.g. either two copies of the wild type allele, or one copy of the wild type allele and one copy of a mutant allele, or two copies of a mutant allele.

In step b) the mutation in the mutant allele preferably causes one or more amino acids to be inserted, deleted or replaced with respect to the wild type protein, e.g. the mutant allele encodes one of the mutant CmBELI proteins described herein. Alternatively in step b) the promoter is mutated leading to reduced or no expression of the allele.

Obviously, also the presence of one or two mutant alleles can be detected in the above assay, e.g. one or two copies of a specific mutant allele or of two different mutant alleles. In the method above the assay can detect the genotype of any CmBELI allele, be it a wild type allele and/or one or more mutant alleles.

The wild type alleles are for example the genomic DNA at the CmBELI locus on chromosome 9. For example SEQ ID NO: 4 provides herein the genomic DNA encoding a wild type CmBELI protein, but likewise genomic sequences comprising at least 90%, 91 %, 92%, 93% 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 may be genomic DNA sequences encoding wild type CmBELI proteins.

In one aspect, therefore, one or more of the following alleles are detected in step b of the method above: a wild type CmBELI allele encoding a protein SEQ ID NO: 1 or a wild type protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 ; a mutant cmbell allele encoding a CmBELI mutant protein comprising one or more amino acids inserted, replaced or deleted with respect to the wild type CmBELI allele encoding a protein SEQ ID NO: 1 or a wild type protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 (see also elsewhere herein); a mutant cmbell allele encoding a mutant CmBELI protein comprising one or more amino acids replaced, inserted or deleted in the homeodomain of SEQ ID NO: 1 or in the homeodomain of a wild type protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 ; a mutant cmbell allele encoding a mutant CmBELI protein which is truncated at the C- terminal end of SEQ ID NO: 1 or at the C-terminal end of a wild type protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , preferably wherein the truncation starts in or prior to the homeodomain; a mutant allele wherein the promoter is mutated so that the allele has reduced expression or no expression.

Step a) may comprise isolation of genomic DNA from the plant, seeds, plant part, cell or tissue to be analyzed in the genotyping assay. Often crude DNA extractions methods can be used, as known in the art.

Step b) preferably comprises a bi-allelic genotyping assay, which makes use of allele-specific oligonucleotide primers and/or allele-specific probes, i.e. primers or probes which discriminate between e.g. the wild type allele and the mutant allele or between two mutant alleles.

The plants of step a) may be mutagenized using e.g. chemical or radiation mutagens or gene editing techniques. Thus prior to step a) there may be a step of treating the plants, seeds or plant parts with a mutagenic agent or induce targeted mutations in the CmBELI allele.

Various genotyping assays can be used, as long as they can detect INDELs and/or SNPs and can differentiate between e.g. the wild type allele being present in the genomic DNA (at the CmBELI locus on chromosome 9) and/or one or more mutant alleles of the CmBELI gene being present in the genomic DNA.

Genotyping assays are generally based on allele-specific primers used in PCR or thermal cycling reactions (polymerase chain reaction) to amplify either the wild type or mutant allele and detect the amplification product or on allele-specific oligonucleotide probes, which hybridize to either the wild type allele or the mutant allele, or both. For example genotyping with BHQplus probes uses two allele specific probes and two primers that flank the region of the polymorphism, and during thermal cycling the polymerase encounters the allele-specific probes bound to the DNA and releases a fluorescent signal. Allele discrimination involves competitive binding of the two allele-specific BHQPIus probes (see also biosearchtech.com).

Examples of genotyping assays are the KASP-assay (by LGC, see www at LGCgenomics.com and also www at biosearchtech.com/products/ pcr-kits-and-reagents/ genotyping-assays/ kasp- genotyping-chemistry), based on competitive allele-specific PCR and end-point fluorescent detection, the Taq Man-assay (Applied Biosytstems), which is also PCR based, HRM assays (High Resolution Melting Assay), wherein allele-specific probes are detected using real time PCR, or the rhAmp assay, based on Rnase H2-dependent PCR, BHQplus genotyping, BHQplex CoPrimer genotyping and many others.

The KASP-assay is also described in He C, Holme J, Anthony J. ‘SNP genotyping: the KASP assay. Methods Mol Biol. 2014;1145:75-86’ and EP1726664B1 or US7615620 B2, incorporated by reference. The KASP genotyping assay utilizes a unique form of competitive allele-specific PCR combined with a novel, homogeneous, fluorescence-based reporting system for the identification and measurement of genetic variation occurring at the nucleotide level to detect single nucleotide polymorphisms (SNPs) or inserts and deletions (InDeis). The KASP technology is suitable for use on a variety of equipment platforms and provides flexibility in terms of the number of SNPs and the number of samples able to be analyzed. The KASP chemistry functions equally well in 96-, 384-, and 1 ,536-well microtiter plate formats and has been utilized over many years in large and small laboratories by users across the fields of human, animal, and plant genetics.

The TaqMan genotyping assays is also described in Woodward J. ‘Bi-allelic SNP genotyping using the TaqMan® assay.’ Methods Mol Biol. 2014;1145:67-74, US5210015 and US5487972, incorporated herin by reference. With TaqMan(®) technology allele-specific probes are utilized for quick and reliable genotyping of known polymorphic sites. TaqMan assays are robust in genotyping multiple variant types, including single nucleotide polymorphisms, insertions/deletions, and presence/absence variants. To query a single bi-allelic polymorphism, two TaqMan probes labeled with distinct fluorophores are designed such that they hybridize to different alleles during PCR-based amplification of a surrounding target region. During the primer extension phase of PCR, the 5'-3' exonuclease activity of Taq polymerase cleaves and releases the fluorophores from bound probes. At the end of PCR, the emission intensity of each fluorophore is measured and allele determination at the queried site can be made.

Various genotyping assays can, therefore, be used, which can differentiate between the presence of the e.g. one or more wild type alleles of the CmBELI gene, encoding the protein of SEQ ID NO: 1 or a protein comprising at least 94% identity to SEQ ID NO: 1 , and/or one or more mutant alleles of the CmBELI gene. Various mutant alleles of the CmBELI gene can be detected.

As mentioned preferably a bi-allelic genotyping assay is used, e.g. a KASP-assay, a TaqMan assay, a BHQplus assay, PACE genotyping (see world wide web at idtdna.com/pages/products/qpcr-and-pcr/genotyping/pace-snp-g enotyping-assays) or any other bi-allelic genotyping assay.

In one aspect the genotyping assay in step b) of the methods above is a KASP-assay. Thus in step b) a competitive PCR is carried out using two forward primers and one common reverse primer. The two forward primers comprise at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides complementary to the genomic sequence (or the complement strand thereof). In addition the two forward primers comprise 1 , 2, 3 or more nucleotides (preferably at the 3’end of the primers) which provide specificity to e.g. the SNP or INDEL which differentiates e.g. the wild type sequence from e.g. the mutant sequence of the allele or which differentiates the sequences of two mutant alleles. The two forward primers thereby have different binding specificity (or preference) to e.g. either the wild type allele and/or e.g. to the mutant allele. For example the Fam-primer may comprise e.g. 17 nucleotides of the wild type sequence and 1 nucleotide specific for the nucleotide of the mutant allele, and the VIC-primer may comprise 18 nucleotides of the wild type allele and 1 nucleotide specific to the nucleotide of the wild type allele. A KASP- assay can easily be designed to differentiate between e.g. the wild type allele and/or any mutant allele of the CmBELI gene (which differs from the wild type allele in one or more nucleotides being inserted, deleted or replaced) or which differentiates between different mutant alleles of the gene., so e.g. the assay can be designed for e.g. any SNP or INDEL that differentiates any two CmBELI alleles.

In one aspect the mutant allele of the CmBELI gene encodes a protein comprising one or more amino acids inserted, replaced or deleted with respect of the wild type protein of SEQ ID NO: 1 , as already described elsewhere herein.

Therefore, in one embodiment a method is provided for detecting, and optionally selecting, a melon plant, seed or plant part comprising at least one copy of a wild type allele and/or of a mutant allele of a gene named CmBELI , comprising: a) providing genomic DNA of a melon plant or of a plurality of plants (e.g. a breeding population, F2, backcross, etc.), b) carrying out an assay (e.g. a bi-allelic genotyping assay) that discriminates or can discriminate between the presence of alleles in the genomic DNA of a), based on nucleic acid amplification (e.g. comprising the use of allele specific oligonucleotide primers) and/or nucleic acid hybridization (e.g. comprising the use of allele-specific oligonucleotide probes), to detect the presence of a wild type allele of the gene and/or one or more mutant alleles of the gene, wherein the wild type allele encodes a protein of SEQ ID NO: 1 (or a wild type CmBELI protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1), and the mutant allele encodes a protein comprising one or more amino acids inserted, deleted or replaced with respect to the wild type protein of SEQ ID NO: 1 (or with respect to a wild type CmBELI protein comprising at least 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1); or the mutant allele comprises a mutation in the promoter resulting in reduced expression or no expression of the allele; and optionally c) selecting a plant, seed or plant part comprising one or two copies of the mutant allele.

Under step b) the genotyping assay discriminates between e.g. the wild type and/or the one or more mutant alleles based on nucleic acid (especially DNA) amplification reactions making use of e.g. oligonucleotide primers, such as PCR (Polymerase Chain Reaction) and PCR primers, preferably allele-specific primers, and/or nucleic acid hybridization making use of as oligonucleotide probes, preferably allele-specific probes.

The primers or probes are preferably modified to comprise a label, e.g. a fluorescent label, or to comprise a tail sequence or other modification.

In one aspect, in any of the above methods the assay uses one or more CmBELI allele specific primers or one or more CmBELI allele specific probes.

As mentioned, based on the genomic sequence of SEQ ID NO: 4, or other (e.g. degenerate) genomic sequences which encode the protein of SEQ ID NO: 1 , or the genomic sequence of a mutant allele which encodes e.g. a protein comprising one or more amino acids inserted, deleted or replaced in comparison to SEQ ID NO: 1 , PCR primers and nucleic acid probes can be designed using known methods or software programs for oligonucleotide design. Primers and probes may for example be at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more nucleotides (bases) in length and anneal to (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 primer or probe specificity to e.g. a wild type allele or a mutant allele (or to two or more mutant alleles) is due to at least 1 , 2, 3 or more nucleotides of the primer or probe being specific for either allele. The primers or probes are thus designed around the polymorphism (e.g. the SNP or InDei) between the two (or more) alleles of the target gene, so that they discriminate between these. In one aspect the assay is a bi-allelic genotyping assay selected from e.g. a KASP-assay, a TaqMan-assay, a BHQplus probe assay or any other bi- allelic genotyping assay.

In one aspect the mutant allele is a null allele.

In one aspect, the mutant allele comprises at least one codon inserted or duplicated in the coding region of the allele, or at least one codon changed into another codon (e.g. through a single nucleotide change), or at least one codon deleted or changed into a STOP codon.

In any of the methods above, in one aspect the mutant allele encodes a protein which is truncated, e.g. which lacks one or more amino acids at the C-terminal end, especially which lacks at least one (or more) amino acid of the homeodomain and all C-terminal amino acids thereafter. Thus, in one aspect the methods can be used to discriminate between plants, seeds or plant parts comprising two copies of the wild type CmBELI allele encoding the protein of SEQ ID NO: 1 , two copies of the mutant cmbell allele encoding e.g. a truncated CmBELI protein, or one copy of each allele (heterozygous). In another aspect the methods can be used to discriminate between plants, seeds or plant parts comprising one or two copies of any one or more mutant CmBELI alleles encoding e.g. a truncated CmBELI protein. Optionally plants, plant parts or seeds comprising any of these genotypes may be selected for e.g. further breeding or for use in melon fruit production, especially seedless fruit production.

Although any DNA genotyping assay may be used in the above methods, be it PCR based (using PCR primers) and/or hybridization based (using probes), in one aspect a KASP-assay is used to discriminate between the wild type and the mutant allele. The assay can be used in a high throughput way, e.g. in 96 well plates or more well plates (e.g. 384 well plates).

In one aspect the assay discriminates between the wild type allele of SEQ ID NO: 4 and the mutant allele comprising a DNA insert in intron 2, e.g. between nucleotide 1655 and nucleotide 1656 of SEQ ID NO: 4, resulting in the transcript comprising only exonl and exon2.

In another aspect the assay discriminates between the wild type allele of SEQ ID NO: 4 and the mutant allele comprising a STOP codon in the homeodomain or prior to the homeodomain. For example, the codon for Y344 (TAC at nucleotide 1886-1888 of SEQ ID NO: 4) may be changed into a STOP codon (TAG or TAA).

Depending on differences (e.g. the SNP or INDEL) between the wild type and/or mutant CmBELI allele, various allele-specific primers and probes can be designed for use in the assays.

In one aspect two forward primers (e.g. one for the wild type allele and one for the mutant allele) and one common reverse primer (e.g. for both the wild type and the mutant allele) are used in the 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 genomic CmBELI sequence or of the complement sequence thereof. The forward primers further comprise at least 1 , 2, or 3 nucleotides (preferably at the 3’end of the primer) which confer specificity (or preference) to either amplification of e.g. the wild type allele or amplification of the mutant allele; or which confer specificity to different mutant alleles. Each forward primer forms a primer pair with the common reverse primer to amplify the DNA sequence of the target allele in between the primer pair, during thermal cycling. Standard components for thermal cycling are used and standard components for KASP-assays.

In another embodiment a method is provided for producing a hybridization product or an amplification product of e.g. a wild type allele and/or of a (or one or two or more) mutant alleles of a gene named CmBELI , comprising: a) providing genomic DNA of a melon plant or of a plurality of plants (e.g. a breeding population, F2, backcross, etc.), b) carrying out an assay (e.g. a bi-allelic genotyping assay) that discriminates or can discriminate between the presence of alleles in the genomic DNA of a), which assay generates a nucleic acid amplification product (e.g. through the use of allele specific oligonucleotide primers to generate the product) and/or which assay generates a nucleic acid hybridization product (e.g. through the use of allele-specific oligonucleotide probes to generate the hybridization product), whereby the amplification product or hybridization product indicates the presence of a wild type allele of the gene and/or a 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 comprising at least 94% sequence identity to SEQ ID NO: 1) and the mutant allele encodes a protein comprising one or more amino acids inserted, deleted or replaced with respect to the wild type protein of SEQ ID NO: 1 or the wild type protein comprising at least 94% sequence identity to SEQ ID NO: 1), or the mutant allele comprises a mutation in the promoter, leading to reduced expression or no expression, and optionally c) selecting a plant, seed or plant part comprising one or two copies of the mutant allele.

Also a method of amplifying all or part of a mutant and/or wild type CmBELI allele from a genomic DNA sample derived from a melon plant, plant part or seed is provided, comprising contacting genomic DNA with a primer pair which amplifies all or part of a mutant cmbell allele and/or of a wild type CmBELI allele in the sample, and detecting the amplification products.

Also a method of hybridizing a probe to a mutant and/or wild type CmBELI allele in a genomic DNA sample derived from a melon plant, plant part or seed is provided, comprising contacting genomic DNA with a oligonucleotide probe which hybridizes to a mutant cmbell allele and/or a wild type CmBELI allele in the sample, and detecting the hybridization products.

All embodiments described above and elsewhere herein also apply to these embodiments. The amplification product may thus be a PCR amplification product, e.g. competitive PCR amplification product generated in e.g. a KASP assay or other assay, to detect the mutant allele (or one or two or more mutant alleles) and/or a wild type allele in the DNA sample. The hybridization product may thus be a hybridization product of an oligonucleotide probe which hybridizes to the nucleic acid in the DNA sample, to detect e.g. the mutant and/or wild type allele in the DNA sample. The primer pairs or probes preferably are allele specific, and the products are thus distinguishable as being e.g. either two copies of the wild type allele, two copies of the mutant allele or one copy of each being present in the genomic DNA of the melon plant, plant part or seed.

The primers or probes are preferably modified, e.g. labelled by a tail sequence or fluorescent label or otherwise modified with respect to the wild type sequence which they amplify or hybridize.

As the described methods require detection of a mutant and/or wild type allele in the genomic DNA of the plant, plant part or seed, the genomic DNA needs to be accessible for detection, e.g. it may be extracted from the plant cells using DNA extraction methods or at least eluted from the damaged cells into a solution (e.g. a buffer solution).

The above assays can be used for marker assisted selection (MAS) of plants in e.g. a breeding program to select plants comprising a certain genotype, e.g. homozygous for the wild type allele of the CmBELI gene, homozygous or heterozygous for a mutant allele of the cmbell allele.

Therefore, also a method of breeding melon plants is provided herein, said method comprising genotyping one or more seeds or plants for the allele composition at the CmBELI locus in the genome and optionally selecting one or more seeds or plants having a specific genotype at the CmBELI locus. In one aspect also genotyping-by-sequencing may be done for the CmBELI gene.

As mentioned, optionally the plants or seeds which comprise two copies of a mutant cmbell allele can be grown and phenotyped for stenospermocarpy. The mutant allele is, in one aspect, a mutant allele which, in homozygous form, confers stenospermocarpy, especially a null allele.

In a different aspect a melon plant, seed or plant part is provided comprising at least one copy of a mutant allele of a gene named CmBELI in melon, wherein said mutant allele either a) comprises one or more mutations in the promoter, resulting in no expression or optionally reduced expression of the allele compared to the wild type allele, and/or b) encodes a mutant protein comprising one or more amino acids replaced, inserted, or deleted compared to the wild type protein, especially wherein the protein is non-functional, wherein said mutant allele of a) or b) confers stenospermocarpy when the mutant allele is in homozygous form (compared to the plant comprising the wild type allele in homozygous form), and wherein the wild type melon CmBELI allele encodes a protein of SEQ ID NO: 1 or a protein comprising at least 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1 , or wherein the wild type genomic CmBELI allele comprising SEQ ID NO: 4 or of a wild type genomic sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 4. Further a method of crossing a plant comprising at least one mutant cmbell allele as described herein with a plant, e.g. lacking a mutant cmbell allele, is provided and selecting progeny comprising at least one copy of the mutant cmbell allele is provided.

Thus, in one aspect a method for generating a melon plant is provided comprising the steps of: a) Providing a melon plant comprising at least one copy of a mutant cmbell allele, as described; b) Crossing said melon plant with another melon plant to produce F1 seeds; c) Selecting an F1 seed comprising at least 1 copy of the mutant allele.

Optionally selection or detection of the presence of the mutant cmbell allele in the method can be done using molecular methods, such as SNP or INDEL genotyping, sequencing and the like.

Preferably the allele in step a) is a mutant allele which confers stenospermocarpy when in homozygous form. In one aspect the plant in step a) is a melon plant comprising a mutant allele as described herein, e.g. encoding a truncated protein, either in heterozygous or homozygous form.

Also provided is a method for production of a melon plant comprising the steps of: a) introducing mutations in a population of melon plants or providing a population of mutagenized melon plants, e.g. a TILLING population of the M2, M3 or further generation, b) identifying a plant which has a mutation in an allele encoding a CmBELI protein wherein the wild type allele of the gene encodes a CmBELI protein comprising at least 94% sequence identity to the protein of SEQ ID NO 1.

The method may further comprise one or both steps of: selecting a plant comprising at least two copies of the mutant allele of step b), determining if the plant produces seedless fruits after pollination.

Further any sequences and molecules of the sequences are encompassed, as are sequences comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% sequence identity to the provided sequences. Also, any fragments and/or modified sequences (e.g. primers or probes comprising at least 10, 15, 16, 17, 18, 19, 20 or more nucleotides of the sequence or the complement sequence) and their use in breeding (e.g. MAS) or in detecting or selecting plants or plant parts is provided.

When a mutant protein is described, it is clear that the genomic sequence and mRNA or cDNA sequence encoding the mutation leading to the mutation in the protein is encompassed herein and can be used to detect an allele in the genome comprising the mutation leading to the amino acid change, and to e.g. carry out a genotyping assay directed at the mutant allele.

SEQUENCE DESCRIPTION

SEQ ID NO 1 : wild type CmBELI protein

SEQ ID NO 2: cDNA encoding the wild type CmBELI protein

SEQ ID NO 3: mRNA encoding the wild type CmBELI protein, including the 5’UTR (nucleotides 1 to 630), the translation start codon at nucleotide 631 to 633, the translation stop codon at nucleotide 2455 to 2457, and the 3’UTR (nucleotides 2458 to 2814)

SEQ ID NO 4: genomic DNA encoding the wild type CmBELI protein, which is transcribed into pre-mRNA (containing introns and exons) and the pre-mRNA is processed to mRNA (intron splicing), see Figure 7

SEQ ID NO 5: DNA insert

SEQ ID NO 6: Promoter sequence of the wild type CmBELI allele

EXAMPLES

Example 1

Fruits of an in-house population of cultivated melon, grown in the field, were cut open and analyzed. One plant was identified which did not contain seeds in the fruits.

The seedless mutant was fine-mapped in an F2 population derived from the cross H99 (seeded/recurrent) x H11X.7616-K (seedless).

The inheritance of the seedless trait was evaluated, and it was mapped as a recessive locus on chromosome 9.

As pollination was necessary to induce fruit set in plants homozygous for the mutant allele, it was concluded that the seedless trait is due to stenospermocarpy.

The mutant allele conferring the seedless trait was crossed into Honeydew, Cantaloupe and Piel de Sapo backgrounds. Long reads analysis identified the gene to be located at the locus designated MEL03C005699, annotated as a ‘LOW QUALITY PROTEIN: homeobox protein BEL1 homolog’ in the cucurbitgenomics.org database.

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, black line indicating DNA insertion location after nucleotide1655).

The sequence analysis revealed that the sequence information in the cucurbitgenomics database is incorrect. The correct sequences are provided herein. Table 1 The gene was named CmBELI , purely based on the name of the locus designated MEL03C005699.

The gene has four exons and three introns, see Figure 7, exons are underlined. The DNA insert was present in the second intron (between exon 2 and exon 3), between nucleotide 1655 and 1656 of the genomic DNA (SEQ ID NO: 4). The amino acids encoded by the four exons is also indicated in Figure 1 , with three vertical black lines delimiting the amino acids encoded by the four exons. The vertical black line with the black star is the location between exon 2 and exon 3.

RT-PCR results showed that the mutant CmBELI allele generated a truncated mRNA transcript, which only contained exonl and exon2 and lacked exon 3 and exon 4.

RT-PCR with primers based on the cDNA corresponding to exon 1 and 2 produced an amplicon with cDNA from both wild type and mutant melon. However, RT-PCR with primers that span the cDNA region corresponding to exon 2 to 3 only produced an amplification product in the wild type, not in the mutant plant (Figure 6). This shows that only the wild type CmBELI allele produced the full mRNA transcript, having exon 2 and exon 3, while in the mutant allele the mRNA transcript was terminated after exon 2. The mutant CmBELI allele thus produces a truncated mRNA transcript.

It can be concluded that the mutant allele generates a truncated CmBELI protein, which lacks the amino acids of exon 3 and exon 4.

As the truncated protein lacks most of the homeodomain, which is functional in DNA binding and the role of the protein as a transcription factor, it is concluded that the truncated protein is nonfunctional, at least its homeodomain is non-functional and cannot exert its role in DNA binding and regulation of gene expression of other genes.

Example 2 - Phenotype of homozygous mutants

Segregating populations comprising the mutant CmBELI allele of Example 1 were generated in Piel De Sapo, Honeydew and Cantaloupe backgrounds and analyzed in the field.

In all backgrounds the plants homozygous for the mutant CmBELI allele produced seedless fruits, see Figures 2, 3, 4 and 5. Furthermore, the plants homozygous for the mutant CmBELI allele produced many more fruits, but with a lower fruit weight on average, although quite a range of fruit sizes were seen. See for example Figure 8, which shows the mutant and wild type fruit weight (grams) and number of fruits in a Cantaloupe background.

There was generally a strong negative correlation (e.g. r = - 0.46) between average fruit weight and fruit number. Empty seed cavity was seen in some genotypes, but not in others. Also, some genotypes showed microseeds, which are small, non-viable seeds (they may also be referred to as ‘empty seeds’). See e.g. Figure 9 with a seeded (wild type) Piel de Sapo fruit on the left and a seedless fruit (comprising the mutant cmbell allele) on the right, whereby the seedless fruit contains microseeds. Microseeds could be seen in some genotypes with either empty seed cavity or with non-empty cavity (filled with placenta tissue).

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

Constructs are made which result in a TE (transposable element) to insert into the endogenous allele of the CmBELI gene of SEQ ID NO: 4.

The TE insert results in premature termination of the gene transcript after exon 2 and a truncated protein is being translated from the transcript.

As the TE insert is in intron 2, the protein lacks the amino acids encoded by exon 3 and exon 4.

The plant is selfed to produce a plant which comprises the mutant cmbell allele in homozygous form.

Plants are grown and the developed melon fruits are cut open at maturity. The fruits are seedless due to the lack of a functional CmBELI protein being made, whereby normal seed development cannot take place.

Example 4 - TILLING

A plant comprising a mutant cmbell allele is identified from a melon TILLING population (generated by EMS mutagenesis). In the cmbell allele present in the plant the codon for Y344 (TAC at nucleotide 1886-1888 of SEQ ID NO: 4) is changed into a STOP codon (TAG or TAA).

The plant is selfed to produce a plant which comprises the Y344STOP mutant cmbell allele in homozygous form.

Plants are grown and the developed melon fruits are cut open at maturity. The fruits are seedless due to the lack of a functional CmBELI protein being made, whereby normal seed development cannot take place.