Nuclear male sterility in watermelons

Abstract

The invention relates to a modified male reduced mother cell death 1-like (MMD1-like) gene, the wild type of which comprises a nucleotide sequence of SEQ ID NO.2, the modified male reduced mother cell death 1-like gene encodes an MMD 1-like protein comprising one or more modifications in the wild-type amino acid sequence of SEQ ID NO. 1 or in an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 1. The invention further relates to watermelon plants comprising said modified MMD1-like gene and to methods for producing and identifying watermelon plants comprising said modified MMD1-like gene. Finally, the invention relates to a method for conferring nuclear male sterility to watermelon plants, comprising introducing a modification to the mmdl-like gene.

Description

TECHNICAL FIELD

[0001] The present invention relates to a modified gene conferring genic male sterility to the nucleus of a plant, in particular a watermelon plant. The invention further relates to plants comprising said modified gene, markers capable of detecting said modified gene, and to methods and uses of said modified gene and said markers. BACKGROUND

[0002] In commercial plant breeding, the production of hybrid seed is of great importance. Plants grown from hybrid seed are often very uniform and they benefit from heterosis / hybrid vigour which can result in a significant increase in yield and / or performance compared to the parental lines of the hybrid or to outcrossed (open pollinated) lines. Typically, the parental lines used for hybrid seed production are inbred, which means that their genome is essentially homozygous. If the two parental lines are genetically unrelated or not closely related, the combination of two essentially homozygous genomes into a hybrid results in a high degree of heterozygosity.

[0003] In plant species that are capable of self-pollination, effective hybrid seed production requires appropriate measures to prevent self-pollination of the plants on which the hybrid seed is produced. A number of strategies have been developed to achieve this and to obtain an effective hybrid seed production system. However, the complexity and the amount of labour required for each of these strategies varies greatly.

[0004] One strategy that is naturally present in certain plant species is the physical separation of male and female reproductive organs in separate flowers, either on separate plants (dioecious species) or on the same plant (monoecious species). This system naturally promotes outcrossing and it can easily be applied to hybrid seed production.

[0005] Another natural strategy is self-incompatibility, which has been extensively studied in, for example, Brassica species. In this case, pollen is physically unable to fertilise an egg cell from the same plant. The precise mechanism of the incompatibility interaction can vary. Either pollen hydration or germination is prevented, or the female tissue inhibits the growth of the pollen tube through the style, or the pollen tube is not attracted to the mature ovule, or the sperm nucleus cannot fuse with the egg nucleus to form a viable zygote. Again, this naturally occurring system is very effective and useful for preventing self-pollination and promoting outcrossing.

[0006] Another method to prevent selfing is to mechanically remove all male flowers (emasculation), a method commonly used in e.g. maize. Only female flowers remain on the plant and these flowers can be hand pollinated with pollen from a selected paternal line to obtain ears that are completely hybrid.

[0007] In plant species with hermaphrodite flowers (both ovules and pollen grains are produced in the same flower), a common strategy to prevent selfing is emasculation by mechanical removal of anthers and / or pollen before flowering. Anthers are mechanically removed before pollen grains are released from the anther chamber and / or before the filaments extend enough to match the height of the stigma to effectively prevent selfing. Subsequently, the female reproductive parts of the emasculated flower are allowed to mature normally, after which pollen grains from a selected paternal plant can be applied to the stigma to obtain pure hybrid seeds from a cross. However, especially for commercial scale applications, this method is very labor intensive and not 100% reliable: if the anthers are removed slightly later in development, or if one anther is accidentally not removed, this can result in a mixed seed set consisting of hybrid seeds and maternal seeds. This results in a non-uniformity of commercial seed batches, which is undesirable for customers expecting uniform and consistently good seeds, and it brings inbred maternal lines of hybrid varieties to the market, which is undesirable for breeding companies. Therefore, a 100% reliable hybridization system is desired.

[0008] Male sterility occurs in plants when the anthers or pollen of the plant are not functional. This condition manifests itself in a severe reduction or absence of pollen, malformation or absence of flowers or stamens, or pollen that does not dehisce. Induction of male sterility can be achieved chemically. This so-called maleicide method can be achieved by treatment with e.g. gibberellins (in rice and maize), sodium methanearsonate (in rice) or maleic acid (in wheat and onions). A disadvantage of this method is that this male sterility is not heritable, as it is not caused by a genetic determinant present in the plant genome, and chemical treatment is labor intensive and not 100% reliable.

[0009] Another class of mechanisms that can prevent selfing is called genetic male sterility. Here three different approaches can be distinguished: genetically engineered male sterility (transgenic MS), cytoplasmic male sterility (CMS) and genic male sterility (GMS). Transgenic MS includes all approaches that use transgenes to ensure that pollen grains are unable to fertilize ovules, either by causing the pollen grains to die before flowering or by causing the pollen grains to be dysfunctional at the time of flowering. One well-known example is the reversible Barnase / Barstar system, in which the Barnase enzyme is transgenically expressed in the tapetum, causing pollen sterility. However, when the Barstar protein is co-expressed in the tapetum, it blocks the Barnase activity and restores pollen fertility.

[0010] CMS is a type of sterility that is controlled by extranuclear cytoplasmic factors, more precisely plasmid origin. Usually, mutations in the mitochondrial genome are the basis for CMS, and they are usually inherited in a maternal way. Conventional hybrid seed production using CMS lines requires the use of maintainer lines and restorer lines, which complicates the production process and increases the costs and time required for commercial hybrid seed production.

[0011] GMS includes nuclear effects on male fertility, in contrast to cytoplasmic effects caused by organelle factors. Due to mutations in e.g. nuclear genes, a plant is unable to produce viable and / or functional pollen grains or male spores, and / or is unable to disperse its pollen due to e.g. its anthers not dehiscing.

[0012] Watermelon belongs to the genus Citrullus, which is part of the family Cucurbitaceae. The modern cultivated watermelon is known as Citrullus lanatus var. lanatus (Thunb.) Matsum. & Nakai. Watermelon is widely cultivated in the tropics and subtropics of the world, mainly for its delicious flesh to be eaten. The southern United States, China, the Middle East, Africa, India, Japan and southern Europe are the most important watermelon producing regions.

[0013] Watermelon is a monoecious species, i.e. both male and female flowers are present on the same plant. To produce high purity hybrid watermelon seeds, controlled pollination is necessary. Controlled pollination requires artificial pollination of the female flowers of the female parent with pollen of a selected male parent. This method prevents undesired self-pollination from the male flowers of the female parent or unwanted cross-pollination, but this method is time-consuming and labor-intensive, all of which significantly increase the production costs. SUMMARY

[0014] In view of current practices, it is an object of the present invention to provide a solution to this expensive and laborious system by eliminating the need for controlled pollination in the production of watermelon hybrids.

[0015] In the research leading to the present invention, a new type of cytoplasmic male sterile watermelon plant was developed. The cytoplasmic male sterile watermelon plant comprises flowers with non-viable pollen or pollen-deficient flowers, or flowers that completely eliminate the production of pollen, thus producing an ideal female parent. If male sterile plants are used, artificial pollination can be removed from the production process. Conversely, when male sterile (female) plants are planted together with the desired male fertile plants, insect pollination can be used. Surprisingly, it was found that the GMS trait is caused by a modification of the Male Meiocyte Death 1-like (MMD1-like) gene. The MMD1-like gene encodes a protein comprising a domain belonging to the conserved protein domain family PHD_MMD1_like (CDD entry: cd15556). Proteins belonging to the PHD_MMD1_like domain family are plant homeodomain (PHD) zinc finger proteins, which are expressed in male meiosis. PHD zinc fingers are often found in transcriptional regulatory proteins and proteins associated with chromatin-remodeling complexes, suggesting that MMD1-like proteins can play a role in male meiosis as transcriptional regulators.

[0016] The GMS trait of the present invention is controlled by a modification of the MMD1-like gene, which is inherited in a manner consistent with the inheritance of a monogenic recessive trait. In the context of the present application, the term "recessive trait" means that the fully realized trait is observed when the modified MMD1-like gene is present in homozygous form in the genome, i.e. both alleles of the MMD1-like gene comprise the modification. When the modified MMD1-like gene is present in heterozygous form in the genome, only one allele of the MMD1-like gene is modified, thus not conferring GMS. Since the inheritance of this trait is comparable to the inheritance of a monogenic trait, it has the advantage that it can be easily integrated into a variety of plant types of a given plant species.

[0017] In the context of the present application, a "gene" comprises an exon sequence and regulatory sequences, such as a promoter sequence, and optionally an intron sequence. In the present application, the term "modification" or "modified" refers to an alteration of a wild-type MMD1 -like gene sequence resulting in a variant version of the wild-type gene. An alteration or modification of the coding sequence (CDS) of a gene results in an alteration of the amino acid sequence of the encoded protein, whereas an alteration or modification of a regulatory sequence of a gene results in an alteration of the transcription of the gene. The resulting modified MMD1 -like protein has reduced levels, reduced activity or is completely missing the encoded MMD1 -like protein. As used herein, "wild-type" refers to the typical or most common, unmutated or unmodified form of an organism, line, gene, protein, characteristic or trait, as opposed to, for example, a mutated or modified form. In the context of the present application, a wild-type MMD1 -like gene does not confer GMS.

[0018] The modified MMD1 -like gene of the present application encodes a modified protein comprising one or more modifications in the wild-type amino acid sequence of SEQ ID NO: 1. In another aspect of the present application, the modified MMD1 -like gene of the present application encodes a modified protein comprising one or more modifications in an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. The modifications are not included in the calculation of the sequence identity difference, as this modification is always present. The skilled person is familiar with methods for calculating sequence identity. Suitably, the sequence identity is calculated using the Sequence Identity and Similarity (SIAS) tool, which is accessible at imed.med.ucm.es / Tools / sias.html. SIAS calculates the percentage of pairwise sequence identity and similarity between each pair of sequences from a multiple sequence alignment. The calculation of sequence identity uses a method that takes gaps into account; the calculation of sequence similarity is based on groupings of amino acids with similar properties. The default settings of SIM percentage, grouping of similar amino acids, sequence length, normalized similarity score, matrix and gap penalty are used in the calculation.

[0019] Thus, the present application relates to a modified MMD1 -like gene, whose wild-type encodes a protein comprising the amino acid sequence as set forth in SEQ ID NO: 1.

[0020] The wild-type MMD1 -like gene of the application comprises the nucleotide sequence of SEQ ID NO: 2. In the publicly available Citrullus lanatus cv. 97103 genome assembly (version 1, see Guo et al., 2013, the draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nature Genetics 45(1 ):51 -58), the wild-type of the modified MMD1 -like gene of the application is located at position 1185039-1187019 (+) on chromosome 10. The term "wild-type MMD1 -like gene of the application" also includes genes having a nucleotide sequence with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity (in order of increasing preference) to the nucleotide sequence of SEQ ID NO: 2.

[0021] The wild-type MMD1 -like gene of the application encodes a protein comprising the amino acid sequence of SEQ ID NO: 1. This wild-type MMD1 -like protein comprises the following conserved domain: PHD_MMD1_like domain (amino acids 551 -595 of SEQ ID NO: 1 ). The term "wild-type MMD1 gene of the application" also includes genes encoding a protein having an amino acid sequence with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity (in order of increasing preference) to the amino acid sequence of SEQ ID NO: 1.

[0022] In one embodiment, the modified MMD1 -like gene of the application is a nucleic acid, in particular a nucleic acid molecule, more particularly an isolated nucleic acid molecule.

[0023] The DNA sequence of a gene can be altered in a variety of ways and can have altered effects, depending on where the modifications are present and whether they change the function of the encoded protein. Examples of such modifications include amino acid substitutions, premature stop codons, insertions, deletions, or frameshift mutations.

[0024] Insertions alter the number of DNA bases by adding one or more base pairs. Deletions alter the number of DNA bases by removing one or several base pairs or even entire genes or adjacent genes. These types of modifications can change the function of the encoded protein.

[0025] A frameshift mutation is caused by the insertion or deletion of one or more base pairs in the DNA sequence encoding a protein. When the number of base pairs inserted or deleted at a certain position is not a multiple of 3, a triplet codon for a single amino acid in the sequence of the encoded protein is shifted relative to the original open reading frame, and then the sequence of the encoded protein is significantly changed. Protein translation results in an amino acid sequence that is different from the amino acid sequence of the original encoded protein, and frameshifts can often result in a premature stop codon in the open reading frame. The overall result is that the encoded protein no longer has the same biological function as the original encoded protein.

[0026] When a mutation of one or more base pairs in the coding sequence results in a change in the triplet codon, which usually encodes a different amino acid, an amino acid substitution occurs in the sequence of the encoded protein. Due to the redundancy of the genetic code, not all point mutations result in an amino acid change. Such mutations are referred to as "silent mutations". Some amino acid changes are "conservative", i.e. they result in the replacement of one amino acid by another amino acid with similar properties, so that the mutation is unlikely to significantly change the folding of the mature protein or affect its function. Conservative amino acid substitutions can be made based on chemical properties, e.g. similarity of residues in polarity, charge, solubility, hydrophobicity, hydrophilicity or amphipathicity, in which case the resulting protein can still function normally. Other amino acid changes are non-silent, non-conservative amino acid changes in domains that play a role in substrate recognition, active sites of enzymes, interaction domains or major domains such as transmembrane helices, which can partially or completely destroy the function of the encoded protein without necessarily affecting the expression level of the encoding gene. Whether an amino acid substitution is conservative or non-conservative can be predicted based on chemical properties, e.g. similarity of amino acids in polarity, charge, solubility, hydrophobicity, hydrophilicity or amphipathicity.

[0027] Deletions, insertions, frameshift mutations and / or amino acid substitutions can result in nonsense mutations. A nonsense mutation is a mutation in a nucleic acid molecule encoding a protein in which a codon is changed to a premature stop codon. The conversion of an amino acid to a premature stop codon results in a truncated protein. How much of the protein is missing determines whether the protein still has a function. Especially when the truncated protein is missing all or part of a conserved functional domain, the protein function is likely to be affected. A premature stop codon can also result in nonsense-mediated decay, in which mRNA transcribed from the allele carrying the nonsense mutation is eliminated, resulting in low RNA expression levels and no or very little protein.

[0028] Deletions, insertions, frameshift mutations and / or amino acid substitutions can result in null mutations or knock-out mutations. A null mutation or knock-out mutation is a mutation that eliminates the function of the affected gene. For example, a null mutation in a gene that normally encodes a particular enzyme can result in the production of an enzyme that has no functionality or in the complete absence of the enzyme.

[0029] The wild type of the MMD1-like gene of the application encodes a protein comprising the amino acid sequence of SEQ ID NO: 1. The wild type MMD1-like protein comprises the following conserved domain: PHD MMD1-like domain (amino acids 551-595 of SEQ ID NO: 1). The term "wild type of the MMD1-like gene of the application" also encompasses a gene encoding a protein having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity (in order of increasing preference) with the amino acid sequence of SEQ ID NO: 1.

[0030] The modified MMD1-like gene of the application comprises one or more replaced, inserted and / or deleted nucleotides relative to the wild type gene sequence, and said one or more replaced, inserted and / or deleted nucleotides result in a lack of functional MMD1-like protein.

[0031] In the context of the present application, the term "lack of functional MMD1-like protein" means either that no MMD1-like protein is expressed, or that the MMD1-like protein expressed is non-functional and does not have MMD1-like activity. The modification of the MMD1-like gene can result in a lack of MMD-1-like RNA or a significant reduction of MMD-1-like RNA levels, resulting in a lack of MMD1-like protein. Alternatively, the modified MMD-1-like protein is expressed, but is non-functional: the lack of one or more functional domains of the MMD-1-like protein results in the modified MMD1-like protein being unable to perform its function in the transcriptional regulation of the male mother cell.

[0032] In one embodiment, the modified MMD1-like gene of the application comprises a premature stop codon resulting in a lack of functional MMD1-like protein. In another embodiment, the modified MMD1-like gene of the application comprises a premature stop codon resulting in a lack of PHD MMD1-like domain in the encoded modified MMD1-like protein. In a preferred embodiment, the one or more nucleotides replaced, inserted and / or deleted in the modified MMD1-like gene of the application relative to the wild type are located at positions 1 to 1815 of SEQ ID NO: 2, resulting in a premature stop codon resulting in a lack of functional protein. In a most preferred embodiment, the modified MMD1-like gene comprises a substitution from guanine to adenine at position 12 of SEQ ID NO: 2 (i.e. G12A).

[0033] The modified MMD1-like gene of the application confers GMS to a plant when present in homozygous form.

[0034] In one embodiment, the modified MMD1-like gene of the application is a nucleic acid, in particular a nucleic acid molecule, more particularly an isolated nucleic acid molecule.

[0035] The application relates to a watermelon plant comprising a modified MMD1-like gene of the application, wherein the modified MMD1-like gene present in homozygous form confers GMS to the plant. The plant can comprise the modified MMD1-like gene of the application in heterozygous form, in which case the plant is not cytoplasmically male sterile, but the plant can be used to transfer the modified MMD1-like gene of the application to another plant.

[0036] Cytoplasmically male sterile watermelon plants comprise both male and female flowers. The female flowers of cytoplasmically male sterile watermelon plants have a normal flower morphology. The male flowers can have a normal flower morphology or the male flowers can be abortive. The main difference between the male flowers of cytoplasmically male sterile watermelon plants and the male flowers of wild type (fertile) watermelon plants is that the male flowers of the cytoplasmically male sterile plants either have non-viable pollen grains or produce no pollen at all (see Figure 2 A, 2B, 2C, comparative Figure 2 D, 2E, 2F). As described herein, the lack of viable pollen grains is attributed to the modified MMD1-like gene of the application.

[0037] The term "watermelon plant of the application" or "plant of the application" as used herein means a watermelon (Citrullus lanatus var. lanatus) plant comprising a modified MMD1-like gene of the application.

[0038] The watermelon plant of the application can be a watermelon plant of any type, of any fruit morphology or fruit color, and is preferably an agronomically elite watermelon plant. In the context of the present application, an agronomically elite plant is a plant having a genotype that, as a result of directed crosses and selection by human intervention, comprises an accumulation of distinguishable and desirable agronomic traits that allow the producer to harvest a product of commercial interest.

[0039] In one embodiment, the mature fruit of the watermelon plant of the application has red, orange or yellow flesh. In another embodiment, the mature fruit of the plant has a soluble solid content of the flesh of at least 5.0 °Brix, 6.0 °Brix, 7.0 °Brix, 8.0 °Brix, 9.0 °Brix, 9.5 °Brix, 10.0 °Brix, 10.5 °Brix, 11.0 °Brix, 11.5 °Brix, 12.0 °Brix, 12.5 °Brix, 13.0 °Brix, 13.5 °Brix, 14.0 °Brix, 14.5 °Brix, 15.0 °Brix, 15.5 °Brix, 16.0 °Brix or 17.0 °Brix (in order of increasing preference). Preferably, the mature fruit of the plant has a soluble solid content of no more than 18 °Brix. In another embodiment, the watermelon plant of the application is an inbred line plant or a hybrid plant.

[0040] In the present application, all watermelon plants referred to are diploid. If a plant referred to in the present application is not diploid, it is indicated as being triploid or tetraploid. The plants of the present application can be diploid, tetraploid or triploid watermelon plants. Triploid watermelon plants of the present application can be generated by treating a diploid watermelon line comprising a modified MMD1-like gene in heterozygous form with colchicine to obtain a tetraploid watermelon plant, which comprises the MMD1-like gene in homozygous form after successive selfing and which is cytoplasmically male sterile. The cytoplasmically male sterile tetraploid plant can be successively crossed with a diploid watermelon plant which does not comprise a modified MMD1-like gene and which is therefore not cytoplasmically male sterile, to generate a triploid watermelon plant which produces triploid seedless watermelon fruits.

[0041] The plants of the present application can be inbred line plants, hybrid plants, double haploid plants or plants of an isolated population. As used herein, an inbred line plant is a plant of a population resulting from three or more rounds of selfing or backcrossing; or the plant is a double haploid plant. For example, the inbred line can be a parental line used to generate a commercial hybrid.

[0042] As used herein, a hybrid plant is a plant resulting from a cross between two different plants having different genotypes. More particularly, a hybrid plant is a plant resulting from a cross between two different inbred lines, such a hybrid plant can for example be a plant of an Fl hybrid variety.

[0043] The present application also encompasses watermelon seeds comprising a modified MMD1-like gene of the present application, wherein a plant grown from the seed is cytoplasmically male sterile because the modified MMD1-like gene is present in homozygous form.

[0044] The present invention further relates to a part of the watermelon plant of the present invention, said part comprising a fruit of the plant of the present invention or a seed of the plant of the present invention, wherein said plant part comprises the modified MMD1-like gene of the present invention.

[0045] The present invention further relates to a watermelon fruit produced by the watermelon plant of the present invention. Said watermelon fruit is a fruit of the present invention.

[0046] Furthermore, the present invention also relates to a food product or a processed food product comprising a fruit or part thereof of the present invention. Said food product can have been subjected to one or more processing steps. Such processing steps can include, but are not limited to, any one or a combination of the following treatments: peeling, cutting, washing, juicing, cooking, cooling or preparing a salad mix comprising the fruit of the present invention. The obtained processed form is also part of the present invention as it comprises DNA where the modified MMD1-like gene is present.

[0047] The present invention further relates to a cell of the plant of the present invention, said cell comprising the modified MMD1-like gene of the present invention. Such a cell can be in isolated form or part of a whole plant or part thereof and still constitutes a cell of the present invention as such a cell comprises the genetic information conferring GMS. Each cell of the plant of the present invention carries the genetic information leading to GMS. The cell of the present invention can also be a regenerable cell, which is capable of regenerating into a new plant of the present invention. The presence of genetic information as used herein is the presence of the modified MMD1-like gene of the present invention.

[0048] The present invention further relates to a plant tissue of the plant of the present invention, said tissue comprising the modified MMD1-like gene of the present invention. Said tissue can be undifferentiated tissue or differentiated tissue. Undifferentiated tissue is for example a meristem, an anther, a petal or pollen and can be used in micropropagation to obtain new plantlets, which grow into new plants of the present invention. Said tissue can also grow from a cell of the present invention.

[0049] The present invention furthermore relates to a progeny of the plant, cell, tissue or seed of the present invention, said progeny comprising the modified MMD1-like gene of the present invention. Such a progeny can itself be a plant, cell, tissue or seed. As used herein, "progeny" means the first generation descendants from a cross with a plant of the present invention and all further generations, wherein crossing includes crossing with itself or with another plant and wherein the progeny that is determined to be a descendant comprises the modified MMD1-like gene of the present invention. Progeny also includes material obtained by vegetative reproduction or another form of propagation.

[0050] The parent can also be a progeny plant from a seed, or a progeny plant from a seed that has been identified by other methods to have (or to have acquired) the trait of the invention. In one embodiment, the present invention relates to a Citrullus lanatus var. lanatus plant carrying the trait of the invention, as well as to a Citrullus lanatus var. lanatus plant that has acquired the trait by introducing the genetic information responsible for the trait from a suitable source, either by conventional breeding, or by genetic modification, in particular by cis- or trans-genesis. Cis-genesis is the genetic modification of a plant using a natural gene that encodes an (agro) trait from the plant itself or from a sexually compatible donor plant. Trans-genesis is the genetic modification of a plant using a gene from a non-crossable species or using a synthetic gene.

[0051] The present invention also relates to propagation material capable of developing into or derived from a plant of the invention, wherein said propagation material comprises a modified MMD1-like gene of the invention. The propagation material is selected from the group consisting of microspores, pollen, ovary, ovule, embryo, embryo sac, egg cell, cutting, root, root tip, hypocotyl, cotyledon, stem, leaf, flower, anther, seed, meristematic cell, protoplast and a cell or tissue culture thereof.

[0052] The present invention further relates to the use of a modified MMD1-like gene of the invention for the production of a cytoplasmic male sterile plant. The cytoplasmic male sterile plant can be produced by introducing a modified MMD1-like gene into its genome, in particular by mutagenesis, introgression, close transgenesis or distant transgenesis or a combination thereof.

[0053] The present invention further relates to a marker for identifying a modified MMD1-like gene, wherein said marker comprises any modification in a MMD1-like gene as described herein, such that the modification can be identified. Such a marker for identification comprises a nucleotide sequence comprising a specific polymorphism in its sequence compared to the same sequence segment in a wild type MMD1-like gene, which polymorphism results in a modification in the encoded protein sequence that alters the function or activity of the MMD1-like protein. The marker of the invention is in particular a marker comprising an oligonucleotide that detects a single nucleotide polymorphism (SNP) at position 12 of SEQ ID NO: 2 from guanine to adenine (i.e. G12A). A nucleotide sequence comprising said polymorphism suitable for identifying the polymorphism in SEQ NO. 2 is set forth in SEQ ID NO. 5 or SEQ ID NO. 6 or a portion thereof. Optionally, the sequence used as a marker can be extended on either side of the modification to ensure that the sequence is unique on the genome and maps to the MMD1-like gene.

[0054] The use of markers for identifying and / or selecting a nucleare male sterile watermelon plant is also part of the present application.

[0055] The present application further relates to primers for detecting a modification in a MMD1-like gene as described herein. The primers are suitable as PCR primers and are complementary to the start and end of the DNA sequence comprising the polymorphism leading to the modification of the present application. In one embodiment, the primers are complementary to both ends of SEQ ID NO. 5 or SEQ ID NO. 6.

[0056] The present application also relates to probes for detecting a modification in a MMD1-like gene as described herein. A probe is an oligonucleotide capable of hybridizing to a DNA sequence comprising the polymorphism leading to the modification of the present application. The sequence of the probe is thus complementary to the part of the MMD1-like gene comprising the polymorphism. In one embodiment, the probe is complementary to SEQ ID NO. 5 or SEQ ID NO. 6.

[0057] The present application further relates to a method for identifying and / or selecting a nucleare male sterile watermelon plant, the method comprising determining whether the plant has one or more modifications in a MMD1-like gene, identifying and / or selecting a plant comprising said modification in homozygous form as a nucleare male sterile plant, and optionally, verifying whether the plant is nucleare male sterile. Identifying whether a modification is present in a MMD1-like gene can be performed by using a marker as defined above.

[0058] The present application further relates to a method for producing a nucleare male sterile watermelon plant, the method comprising modifying a wild type of a MMD1-like gene of the present application, wherein the modification leads to a lack of functional MMD1-like protein, and the lack of functional MMD1 protein leads to a nucleare male sterile plant. The wild type of a MMD1-like gene of the present application is a gene having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity (in order of increasing preference) to SEQ ID NO: 2.

[0059] The present application relates to a method for producing a nucleare male sterile watermelon plant, the method comprising:

[0060] a) crossing a plant comprising a modified MMD1-like gene of the present application with a plant not comprising said modified MMD1-like gene;

[0061] b) optionally, subjecting the plants resulting from step a) to one or more rounds of selfing and / or crossing to obtain a subsequent generation population;

[0062] c) selecting from the population plants comprising in homozygous form the modified MMD1-like gene which generates a cytoplasmic male sterile plant.

[0063] The present invention relates to a method for producing hybrid watermelon seeds, said method comprising:

[0064] a) crossing a plant comprising a modified MMD1-like gene of the present invention with a plant not comprising said modified MMD1-like gene or comprising this gene in heterozygous form;

[0065] b) harvesting the resulting hybrid seeds.

[0066] The presence of a modified MMD1-like gene leading to GMS can be detected using routine methods known to the skilled person, such as RT-PCR, PCR, antibody-based assays, sequencing and genotyping assays, or combinations thereof. Such methods can be used to determine in plant material or plant parts or DNA or RNA or proteins derived therefrom, for example, a reduction in expression of a wild-type MMD1-like gene, a reduction in expression of a wild-type MMD1-like protein, the presence of a modified mRNA, cDNA or genomic DNA encoding a modified MMD1-like protein, or the presence of a modified MMD1-like protein.

[0067] An example of a routinely used genotyping assay is KASP (Competitive Allele-Specific PCR), which is based on allele-specific oligonucleotide extension. The person skilled in the art is familiar with designing and performing KASP assays for the detection of genetic variations in plants. In the present invention, KASP markers were designed based on SEQ ID NO. 5 and SEQ ID NO. 6 to detect the G to A SNP mutation. The ‘A’ haplotype means that a plant comprises in homozygous form the wild-type MMD1-like gene, the ‘B’ haplotype means that a plant comprises in homozygous form the mutant MMD1-like gene, and the ‘H’ haplotype means that a plant comprises in heterozygous form the mutant MMD1-like gene. Other genotyping techniques can also be used to detect the SNP associated with the trait of the present invention.

[0068] Modifications or mutations of the wild-type MMD1-like gene can be introduced randomly by means of one or more chemical compounds, such as ethyl methanesulfonate (EMS), nitrosomethylurea, hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine, N-ethyl-N-nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethyl sulfate, ethyleneimine, sodium azide, formaldehyde, ethyl carbamate, phenol and oxirane, and / or by physical means, such as UV-irradiation, fast neutron exposure, X-rays, gamma radiation, and / or by insertion of genetic elements, such as transposons, T-DNA, retroviral elements.

[0069] Mutagenesis also includes more specific targeted introduction of at least one modification by homologous recombination, oligonucleotide-based mutagenesis introduction, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) system.

[0070] Modifications to the wild-type MMD1 -like gene can also include a step of targeted genome editing, wherein the sequence of the wild-type MMD1 -like gene is modified, or wherein the wild-type MMD1 -like gene is replaced with another modified MMD1 -like gene. This can be achieved by any method known in the art for modifying DNA in the genome of a plant, or by methods of gene replacement. Such methods include genome editing techniques and homologous recombination.

[0071] Homologous recombination allows for targeted insertion of a nucleic acid construct into the genome, this targeting being based on unique sequences flanking the integration site that is targeted. For example, a wild-type site of a MMD1 -like gene can be replaced by a nucleic acid construct comprising a modified MMD1 -like gene.

[0072] Modifying the wild-type MMD1 -like gene can involve the use of a double-strand break induced in the DNA using a zinc finger nuclease (ZFN), a TAL (transcription activator-like) effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeat / CRISPR-associated nuclease (CRISPR / Cas nuclease), or a homing endonuclease that has been engineered to create a double-strand break at a specific recognition sequence in the genome of a plant, another organism, or a host cell.

[0073] TAL effector nucleases (TALENs) can be used to create a double-strand break at a specific recognition sequence in the genome of a plant, thereby allowing for gene modification or gene replacement by homologous recombination. TAL effector nucleases are a class of sequence-specific nucleases that can be used to create a double-strand break at a specific target sequence in the genome of a plant or other organism. TAL effector nucleases are created by fusing a natural or engineered transcription activator-like (TAL) effector (or a functional part thereof) to the catalytic domain of an endonuclease, such as Fok I. The unique, modular TAL effector DNA-binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA-binding domain of a TAL effector nuclease can be engineered to recognize a specific DNA target site, and thereby be used to create a double-strand break at the desired target sequence.

[0074] ZFNs can be used to create double-strand breaks at specific recognition sequences in the plant genome, thereby allowing for gene modification or gene replacement by homologous recombination. Zinc finger nucleases (ZFNs) are fusion proteins that comprise a portion of the Fokl restriction endonuclease protein responsible for DNA cleavage fused to a zinc finger protein that recognizes specific, designed genomic sequences and cleaves double-stranded DNA at these sequences, thereby creating free DNA ends (Urnov et al., 2010, Nat. Rev. Genet. 11 :636-46; Carroll, 2011, Genetics 188:773-82).

[0075] The CRISPR / Cas nuclease system can also be used to create double-stranded breaks at specific recognition sequences in the genome of a plant, thereby allowing for gene modification or gene replacement by homologous recombination. The CRISPR / Cas nuclease system is an RNA-guided DNA endonuclease system that makes sequence-specific double-stranded breaks in DNA fragments homologous to the designed RNA. The specificity of the sequence can be designed (Jinek et al., 2012, Science 337:816-821; Cho et al., 2013, Nat. Biotechnol. 31 :230-232; Cong et al., 2013, Science 339:819-823; Mali et al., 2013, Science 339:823-826; Feng et al., 2013, Cell Res. 23:1229-1232). Cas9 is an RNA-guided endonuclease with the ability to create double-stranded breaks in DNA, both in vitro and in vivo (as well as in eukaryotic cells). Cas9 is part of an RNA-mediated adaptive defense system named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in bacteria and archaea. Cas9 gets sequence specificity when it is bound to a guide RNA molecule that can target a sequence present in the DNA of an organism based on their sequence. Cas9 requires the presence of a protospacer adjacent motif (PAM) immediately after the DNA sequence targeted by the guide RNA. The Cas9 enzyme was originally isolated from Streptococcus pyogenes (SpCas9), but functional homologues from many other bacterial species have been reported, such as Neisseria meningitides, Treponema denticola, Streptococcus thermophilus, Francisella novicida, Staphylococcus aureus, and others. For SpCas9, the PAM sequence is 5'-NGG-3', while various Cas9 proteins from other bacteria have been shown to recognize different PAM sequences. In nature, the guide RNA is a duplex between crRNA and tracrRNA, but single guide RNA (sgRNA) molecules containing both crRNA and tracrRNA have been shown to be equally effective (Jinek et al., 2012, Science 337:816-821). The advantage of using sgRNA is that it reduces the complexity of the CRISPR-Cas9 system to two components, rather than three. This is an important simplification for use in experimental systems (either in vitro or in vivo).

[0076] One alternative to Cas9 is, for example, Cpfl, which does not require a tracrRNA to function, recognizes a different PAM sequence and makes a sticky end cut in DNA, whereas Cas9 makes a blunt end. In one aspect, gene modification techniques can be applied to express site-specific nucleases, such as RNA-guided endonucleases and / or guide RNAs, in eukaryotic cells. One or more DNA constructs encoding an RNA-guided endonuclease and at least one guide RNA can be introduced into a cell or organism by stable transformation, in which the DNA construct is integrated into the genome, or by transient expression, in which the DNA construct is not integrated into the genome, but the RNA-guided endonuclease and at least one guide RNA are expressed in a transient manner. This approach requires the use of a transformation vector and a suitable promoter for expression in the cell or organism. Organisms that have been introduced with foreign DNA are considered genetically modified organisms (GMOs), which also apply to their derived cells and the offspring of these organisms. In an important part of the world food market, genetically modified food is not allowed for human consumption and is not accepted by the public. However, there is also an alternative, "DNA-free" delivery method that delivers CRISPR-Cas components into intact plants without involving the introduction of DNA constructs into cells or organisms.

[0077] For example, after in vitro transcription of the mRNA from a DNA construct encoding an RNA-guided endonuclease together with at least one guide RNA, the mRNA encoding Cas9 has been described to be introduced into a cell or organism. This approach does not require the use of a transformation vector and a suitable promoter for expression in the cell or organism.

[0078] Another known method is to assemble ribonucleoprotein (RNP) complexes in vitro, which comprise an RNA-guided endonuclease protein (e.g. Cas9) and at least one guide RNA, and subsequently introduce the RNP complex into a cell or organism. In plants, the use of RNPs has been demonstrated in protoplasts, for example using polyethylene glycol (PEG) transfection (Woo et al. 2015, Nat. Biotech. 33: 1162-1164). After the modification of the genomic sequence has taken place, protoplasts or cells can be used to generate plants carrying the modification in their genome using any plant regeneration method known in the art, such as in vitro tissue culture.

[0079] The use of site-specific nucleases, for example the nucleases described above, to break DNA can increase the rate of homologous recombination within the region of the break. Thus, the coupling of the above-mentioned effectors to nucleases can enable targeted alterations in the genome, including additions, deletions, and other modifications.

[0080] Recently developed genome editing technologies include base editing and prime editing, which enable highly precise and targeted alterations to the genome. In brief, base editing is a genome editing technology that creates precise point mutations in genomic DNA or in cellular RNA without creating double-strand breaks, thereby avoiding random insertions and deletions associated with DNA breaks. Prime editing is another genome editing technology that can make targeted small fragment insertions, deletions, and substitutions in a precise targeted manner and also functions without the need for double-strand breaks.

[0081] In the present application, the two words "modification" and "mutation" are used interchangeably. It is understood that a mutation can be a type of modification, and a modification can also be denoted as a mutation.

[0082] The present application will be further illustrated in the following examples, which are for illustrative purposes only. The examples are not intended to limit the present application in any way. In the examples and throughout the present application, reference is made to the following figures: BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1A - Wild-type MMD-1-like protein sequence (SEQ ID NO. 1) and wild-type MMD1-like gene CDS (SEQ ID NO. 2); mutant MMD-1-like protein sequence (SEQ ID NO. 3) and mutant MMD1-like gene CDS (SEQ ID NO. 4).

[0084] underlined The underlined and bolded amino acids or nucleotides indicate the position of the corresponding amino acid or nucleotide affected by the modification. * indicates protein truncation due to a premature stop codon in the corresponding nucleotide sequence.

[0085] Figure 1B - Nucleotide sequence of the MMD1-like gene used for the development of the marker (SEQ ID NO. 5 and SEQ ID NO. 6). Haplotypes of wild-type and mutant alleles are annotated. underlined The underlined and bolded nucleotides indicate the position of the corresponding nucleotide affected by the modification.

[0086] Figure 2 - Representative photographs of a male flower (A) obtained from a plant comprising a homozygous mutant MMD1-like gene, anthers from a male flower (B) and a male flower lacking viable pollen grains (C); representative photographs of a male flower (D) obtained from a plant comprising a wild-type MMD1-like gene, anthers from a male flower (E) and a male flower with viable pollen grains (F).

[0087] Figure 3- F1 progeny seeds and their associated genetic relationship to the GMS maternal and non-GMS paternal lines. DETAILED DESCRIPTION

[0088] Example 1

[0089] Genetic modification of watermelon seeds by ethyl methanesulfonate (EMS)

[0090] Watermelon (Citrullus lanatus) seeds from an inbred diploid watermelon line were treated by mutagenic EMS treatment. Approximately 10,000 seeds were swelled in 1% (w / v) EMS solution at a temperature of 28°C under agitation (60 rpm) for 16 hours at 1 ml / seed (150 ml per dose). After treatment, the EMS solution was discarded and the seeds were washed / incubated gently in water for 15 minutes. The seed washing process was repeated at least an additional seven times.

[0091] The treated seeds were germinated in a nursery and the resulting Ml plants (approximately 5,000 plants) were transplanted, grown and self-pollinated in a greenhouse to produce seeds. The resulting seeds were used as starting material M2 to identify individual plants that exhibited mutations.

[0092] Example 2

[0093] Identification of a cell-nuclear male sterile watermelon plant

[0094] Upon TILLING screening of the M2 plants produced in Example 1, a mutant watermelon plant was identified that had a mutant version of the MMD1-like gene. DNA sequencing revealed that in one M2 plant, a guanine (G) to adenine (A) substitution occurred at position 12 of the nucleotide sequence (SEQ ID NO: 4) (i.e., G12A). This substitution or mutation replaces a tryptophan (SEQ ID NO. 1) with a premature stop codon at position 4 of the wild-type protein sequence, resulting in a truncated version of the MMD1-like protein (SEQ ID NO: 3).

[0095] GMS phenotype was assessed on watermelon plants from generation M3 and genotypes were assessed for the presence of a mutated MMD1-like gene by KASP genotyping assays. KASP (Competitive Allele-Specific PCR) is based on allele-specific oligonucleotide extension. Single nucleotide polymorphisms (SNPs) occur when a single nucleotide in a DNA sequence differs between samples. The person skilled in the art is familiar with designing and performing KASP assays for the detection of genetic variations in plants. For the detection of a G to A SNP mutation, KASP markers were designed based on SEQ ID NO: 5 and SEQ ID NO: 6. The‘A’ haplotype means that the plant comprises the wild type MMD1-like gene in homozygous form, the‘B’ haplotype means that the plant comprises the mutated MMD1-like gene in homozygous form, and the‘H’ haplotype means that the plant comprises the mutated MMD1-like gene in heterozygous form. Other genotyping techniques can also be used to detect SNPs.

[0096] Several plants were identified that comprise the mutated MMD1-like gene in homozygous form and scored‘B’. The male flowers of these mutant watermelon plants appeared normal in morphology and development (see Figure 2 A), but produced no pollen at all (see Figure 2 B, 2C). Homozygous mutant plants did not set seed when self-pollinated.

[0097] The seven mutant plants were grafted together with several plants that lack the mutated MMD1-like gene (e.g. wild type MMD1-like gene, scored‘A’) into an enclosed greenhouse. The mutant plants were used as the female parent in a cross. Three weeks after grafting, male flowers were assessed from all plants. Mature male flowers that contained pollen (see Figure 2 D) were only present in the wild type paternal line (i.e. male fertile plants that lack the mutated MMD1-like gene) (see Figure 2 E, 2F). Pollination was allowed using honey bees. 75 days after grafting, mature fruits were collected from the female lines. Cross seeds were removed from the fruits, washed and sown. The genetic profile of the cross seeds was verified using 83 markers covering the entire watermelon genome. Based on the genetic relationships of this map (see Figure 3 ), it was verified that no inbred seeds were obtained from the fruits grown on the female lines, further demonstrating that the female lines are cytoplasmic male sterile and that the female fertility of plants comprising the mutated MMD1-like gene in homozygous form is not impaired.

[0098] Additional experiments were performed on M3 generation watermelon plants between 2019 and 2021. In addition to the self-pollination experiments on mutant plants comprising the MMD1-like gene in homozygous form (results as shown in Table 1), the following additional observations were made: T19R.155-4 plants comprising the mutant MMD1-like gene in heterozygous form were self-pollinated, produced fruits with seeds, and the mutant plants comprising the MMD1-like gene in homozygous form grown from these seeds were male sterile; T20R.401 - pollination of wild-type female flowers with pollen from male flowers of a mutant plant comprising the MMD1-like gene in homozygous form did not result in fruits, indicating that the male flowers were cytoplasmically male sterile; pollination of female flowers of a mutant plant comprising the MMD1-like gene in homozygous form with wild-type male flowers resulted in fruits with seeds, indicating that the female flowers were fertile; T20R.417 and T21 R.406: pollen viability was assessed on 4 plants comprising the MMD1-like gene in homozygous form and 5 plants comprising the MMD1-like gene in heterozygous form, on a medium that allows observation of pollen tubes, which are an indication of pollen viability; male flowers of plants comprising the MMD1-like gene in homozygous form had normal flower development, but did not contain pollen, compared to wild-type male flowers; male flowers of plants comprising the MMD1-like gene in heterozygous form had normal flower development, and viable pollen was observed, compared to wild-type male flowers.

[0099] Table 1:

[0100]

[0101] Example 3

[0102] Production of triploid watermelon hybrids using GMS mutants

[0103] To use GMS mutant plants to produce triploid watermelon hybrids, a tetraploid mutant MMD1-like gene female line is first generated. The tetraploid mutant MMD1-like gene female line is generated by first selecting a diploid watermelon plant that has the MMD1-like gene in heterozygous form (determined by KASP marker assay as described in Example 2). This plant is then treated with colchicine to allow chromosome doubling to occur. After colchicine treatment, the plant is selfed for several generations and the resulting seeds are grown after checking that the level of ploidy is maintained. Only plants that contain the MMD1-like gene in homozygous form (determined by KASP marker assay as described in Example 2) are selected and used as the female line. The resulting GMS tetraploid female line is then crossed with an inbred diploid non-GMS male line (i.e. containing wild type MMD1-like gene) to produce triploid hybrid plants that produce triploid seedless watermelon fruits. In this way, the GMS mutant of the application eases the laborious control pollination process in triploid watermelon hybrid production.

Claims

1. A modified male meiotic failure 1-like (MMD1-like) gene whose wild type comprises the nucleotide sequence of SEQ ID NO. 2, the modified MMD1-like gene encoding a MMD1-like protein comprising one or more modifications in the wild type amino acid sequence of SEQ ID NO. 1 or in an amino acid sequence having at least 90% sequence identity to SEQ ID NO.

1.

2. The modified MMD1-like gene of claim 1, wherein the modified MMD1-like gene encodes an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO.

1.

3. The modified MMD1-like gene of claim 1 or 2, wherein the modification is a premature stop codon that results in a truncated protein.

4. The modified MMD1-like gene of any one of claims 1 to 3, wherein the premature stop codon is the result of a nucleotide substitution from guanine to adenine at position 12 of SEQ ID NO.

2.

5. The modified MMD1-like gene of any one of claims 1 to 4, wherein the MMD1-like protein encoded by the modified MMD1-like gene is non-functional.

6. The modified MMD1-like gene of any one of claims 1 to 5, wherein the MMD1-like protein encoded by the modified MMD1-like gene comprises SEQ ID NO.

3.

7. The modified MMD1-like gene of any one of claims 1 to 6, wherein the modified protein resulting from the one or more modifications confers cytoplasmic male sterility when present in homozygous form in a plant.

8. A watermelon plant (Citrullus lanatus var. lanatus) comprising the modified MMD1-like gene of any one of claims 1 to 7.

9. The watermelon plant of claim 8, wherein the modified MMD1-like gene encoding the modified MMD-1 protein confers cytoplasmic male sterility when present in homozygous form in the genome of the plant.

10. A watermelon seed comprising the modified MMD1-like gene of any one of claims 1 to 7, wherein a plant grown from the seed is the plant of any one of claims 8 or 9.

11. The watermelon plant of claim 8 or 9 or a progeny plant from a plant grown from the watermelon seed of claim 10, wherein the progeny plant comprises the modified MMD1-like gene of any one of claims 1 to 7.

12. A fruit harvested from the watermelon plant of claim 8 or 9 or a watermelon plant grown from the watermelon seed of claim 10, wherein the fruit comprises the modified MMD1-like gene of any one of claims 1 to 7.

13. Propagation material capable to develop into or derived from a plant according to claim 8 or 9, wherein said propagation material comprises a modified MMD1-like gene according to any one of claims 1 to 7, and wherein said propagation material is selected from the group consisting of microspores, pollen, ovary, ovule, embryo, embryo sac, egg cell, cutting, root, root tip, hypocotyl, cotyledon, stem, leaf, flower, anther, seed, meristem cell, protoplast and cell or tissue culture thereof.

14. Use of a modified MMD1-like gene according to any one of claims 1 to 7 for the production of a cytoplasmic male sterile plant.

15. Use according to claim 14, wherein said cytoplasmic male sterile plant is produced by introducing said modified MMD1-like gene into its genome, in particular by mutagenesis, introgression, cis-transgenesis, transgenesis or a combination thereof.

16. A marker for detecting a modified MMD1-like gene, wherein said marker comprises an oligonucleotide detecting a nucleotide substitution from guanine to adenine at position 12 of SEQ ID NO.

2.

17. Marker according to claim 16, comprising the sequence of SEQ ID NO. 5 or SEQ ID NO. 6, or a portion thereof.

18. Use of a marker according to claim 16 or 17 for identifying and / or selecting a Watermelon plant comprising a modified MMD1-like gene.

19. A method for conferring cytoplasmic male sterility in a Watermelon plant, said method comprising introducing a modification in a MMD1-like gene, wherein said modification results in a MMD1-like gene as defined in any one of claims 1 to 7.

20. A method for producing a cytoplasmic male sterile Watermelon plant, said method comprising introducing a modification in a MMD1-like gene comprising SEQ ID NO.

1.

21. Method according to claim 21, wherein said modification results in a MMD1-like gene as defined in any one of claims 1 to 7.

22. A method for producing a cytoplasmic male sterile Watermelon plant, said method comprising: (a) crossing a plant according to any one of claims 8 or 9 with another plant; (b) optionally, subjecting the plants resulting from step a) to one or more rounds of selfing and / or crossing to obtain a next generation population; (c) selecting from the population resulting from step a) or from the next generation population of step b) a plant comprising a modified MMD1-like gene as defined in any one of claims 1 to 7 in homozygous form.

23. A method of producing hybrid seed, said method comprising crossing a first parent plant with a second parent plant and harvesting the resulting hybrid seed, wherein said first parent plant is a plant according to any one of claims 8 or 9, and said second parent plant is a plant not comprising or comprising said modified MMD1-like gene in heterozygous form.

24. Hybrid seed produced by the method of claim 24.

25. A method for identifying and / or selecting a watermelon plant comprising a modified MMD1-like gene of any one of claims 1 to 7, the method comprising: (a) assaying a nucleic acid of a plant for the presence of one or more modifications in the MMD1-like gene; (b) identifying and / or selecting the plant as a cytoplasmic male sterile plant if one or more modifications in the MMD1-like gene are present; and (c) optionally, verifying whether the plant is cytoplasmic male sterile.

26. A method for identifying and / or selecting a watermelon plant comprising a modified MMD1-like gene of any one of claims 1 to 7, the method comprising: (a) assaying a nucleic acid of a plant for the presence of one or more modifications in the MMD1-like gene; (b) identifying and / or selecting the plant as a cytoplasmic male sterile plant if one or more modifications in the MMD1-like gene are present; and (c) optionally, verifying whether the plant is cytoplasmic male sterile.

27. A method for identifying and / or selecting a watermelon plant comprising a modified MMD1-like gene of any one of claims 1 to 7, the method comprising: (a) assaying a nucleic acid

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