Modified plant architecture in cucumber

Abstract

The present invention is directed to cucumber plants comprising modified plant architecture, such as shorter internodes, more internodes and smaller leaves.

Description

[0001] Modified plant architecture in cucumber

[0002] FIELD

[0003] The present invention is directed to plants and plant parts of the species Cucumis sativus comprising a modified plant architecture through mutation of an endogenous gene, located on chromosome 3, encoding a protein referred to herein as CsAMPl (Cucumis sativus Altered Meristem Program 1), as NCBI BLAST analysis of the protein resulted in the most similar protein being a “probable glutamate carboxypeptidase AMP1”. The functional CsAmpl gene and functional protein appears to be required for normal growth and survival of the plant, as knock-out mutants cannot be found back in homozygous form. The mutant protein identified herein has a single amino acid substitution in the peptidase domain (an L411F substitution), resulting in significantly more and shorter internodes and / or smaller leaves when the mutant allele is in homozygous form, allowing e.g. mutant plants to be grown at a higher density than wild type plants. The mutant plant has significantly higher yield per plant, as more internodes are present e.g. up to the high wire and, therefore, more fruits develop on the stem.

[0004] BACKGROUND

[0005] Although compact cucumber plants already exist, see e.g. US8710303 (B2), there is a need for alternative or improved plants having a compact plant architecture. The compact plant of US8710303 (B2) for example have problems with seed setting, while the herein described plant comprising the csampl mutant allele in homozygous form has normal seed setting.

[0006] So-called ‘compact’ cucumber varieties are commercially available, e.g. Hi-Lisa, Hi-Power, Hi-Light, Hi Pace, Hi Force etc. see Nunhems at nunhems.com / gb / en / Varieties / CUX_cucumber.html.

[0007] WO2017042272 described the causal gene which causes the ‘compact’ phenotype, the gene is the Cullinl gene in cucumber. The gene is found on chromosome 6 of the genome. W02017042270A1 described modifications in the Cullinl gene.

[0008] WO2023152274A1 describes a gene which causes reduced leaf size in cucumber. This DCAF8 gene is found on chromosome 7 of the cucumber genome.

[0009] Xin et al. 2022 (Horticultural Research 9: uhab086; doi.org / 10.1093 / hr / uhab086) edited the ERECTA gene in cucumber, generating plants with shorter internodes. The gene (CsaV3_4G036080) is located in chromosome 4 of the cucumber genome. Xu et al. (2023, Plant Science 327, 111536; doi.org / 10.1016 / j.plantsci.2022.111536) isolated a spontaneous null-mutant in the same gene on chromosome 4, which had smaller leaves, shorter internodes (but same number of internodes), and smaller fruits and seeds. At full maturity of the plant, the above-ground hight was only 37.3 cm.

[0010] Li et al. 2011 (Theor. Appl. Geneti. 123:973-983) describe a recessive compact gene (cp, dwarf) on chromosome 4. The adult plants comprising the mutant in homozygous form had a plant height of less than 50 cm.

[0011] Xin et al. 2012 (African Journal of Biotechnology, Vol. 11(20) pp4493-4498) describe a cucumber superdwarf mutant D0460 whereby the adult plants reach a maximum height (stem length) of less then 50 cm. This gene is located on chromosome 4.

[0012] There is, thus, a need to identify genes which, when modified in cultivated cucumber plants, leads to a different compact plant architecture without negative effects, such as dwarf growth or reduced seed setting.

[0013] The problem is solved by the identification of a mutant plant comprising shorter internode length and concomitantly more (albeit shorter) internodes (thereby not resulting in dwarf plants) and smaller leaves and the identification of the causal gene, which when mutated and present in homozygous form) results in at least shorter average internode length and / or significantly more (albeit shorter) internodes and / or smaller leaves. Due to the significant increase in the number of internodes on the main stem there is also a significant increase in fruits and therefore overall yield per plant. In addition, plants can be grown at a higher density whereby overall yield per area of cultivation is further increased.

[0014] Optionally also one or more of the following characteristics are due to the mutant allele of the CsAMPl gene being present in homozygous form compared to the plant comprising the wild type CsAMP 1 allele in homozygous form: slower growth, shorter fruits (reduction in fruit length). As mentioned, seed setting in the fruits is normal (as in wild type) and not affected by the mutation.

[0015] FIGURES

[0016] Figure 1 - Pairwise protein alignment of the wild type CsAMPl protein of SEQ ID NO: 1 (comprising a Leucine, L, at amino acid 411) and the mutant CsAMPl protein of SEQ ID NO: 4 (comprising a Phenylalanine, F, at amino acid 411). The dashed boxes indicate the M28_PSMA_like domain or peptidase domain of the AMP1 protein. Figure 2 - Left foto: Cucumber plant having shorter internodes and more internodes on the main stem t due to the homozygous mutant allele encoding the mutant protein of SEQ ID NO: 4 (L41 IF mutant). Right foto: wild type plant.

[0017] Figure 3 - Smaller leaf and slightly shorter fruits of cucumber plant comprising the mutant allele encoding the mutant protein of SEQ ID NO: 4 (L41 IF mutant) in homozygous form (left side) and leaves and fruits of wild type cucumber plant (right side).

[0018] GENERAL DEFINITIONS

[0019] 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 plants, etc. Similarly, “a fruit” or “a plant” also refers to a plurality of fruits and plants.

[0020] As used herein, the term “mutant allele of a gene” refers to a mutant allele of a gene, said mutant allele either encodes a protein which, compared to the protein encoded by the wild type allele of the gene, comprises one or more amino acids replaced, deleted and / or inserted, whereby the mutant allele produces a mutant protein which has a “reduced-function” or “loss-of-fimction”, or said mutant allele of the gene has a reduced gene expression or even no expression compared to the gene expression of the wild type (non-mutated) allele of the gene.

[0021] 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, etc.), plant cells, plant protoplasts, plant cell- or tissue- cultures from which whole plants can be regenerated, plant calli, plant cell clumps, plant transplants, seeds from which the plant can be grown and seeds produced by the plant, seedlings, plant cells that are intact in plants, plant clones or micropropagations, or parts of plants, such as plant cuttings, embryos, pollen, ovules, fruits (e.g. harvested tissues or organs), flowers, leaves, 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. As used herein, the term plant includes plant and plant parts comprising one or more of the mutant alleles.

[0022] In one aspect, the term plant part refers to plant cells, or plant tissues or plant organs that comprise one or more of the mutant alleles. In one aspect a plant part (e.g. cell) can grow into a plant and / or live on photosynthesis (i.e. synthesizing carbohydrate and protein from the inorganic substance, such as water, carbon dioxide and mineral salt). In another aspect, a plant part (e.g. cell) cannot grow into a plant and / or live on photosynthesis (i.e. synthesizing carbohydrate and protein from the inorganic substance, such as water, carbon dioxide and mineral salt). Thus, a plant part (e.g. a cell) can be propagating or nonpropagating.

[0023] As used herein, the term “variety” or “cultivar” or “plant variety” means a plant grouping within a single botanical taxon of the lowest known rank, which (irrespective of whether the conditions for the recognition of plant breeder’s rights are fulfilled or not) can be defined on the basis of the expression of characteristics that result from a certain genotype or a combination of genotypes, can be distinguished from any other group of plants by the expression of at least one of those characteristics, and can be regarded as an entity, because it can be multiplied without any change. Therefore, the term “plant variety” cannot be used to denote a group of plants, even if they are of the same kind, if they are all characterized by the presence of one locus or gene or two loci or genes, but which can otherwise differ from one another enormously as regards the other loci or genes.

[0024] The term “allele(s)” means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. 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).

[0025] 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.

[0026] “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.

[0027] “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 3, 4, 5, 6, 7, 8, 9, 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 (e.g. the genetic control) show a statistically significant difference in that characteristic (e.g. the p-value is less than 0.05, p < 0.05, using e.g. ANOVA) from the mean of the control.

[0028] The term “nucleic acid sequence” (or nucleic acid molecule) refers to a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein or protein fragment according to the invention. An “isolated nucleic acid sequence” 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.

[0029] The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein”. An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

[0030] The term “gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA or an RNAi molecule) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3’ non-translated sequence comprising e.g. transcription termination sites. A gene may be an endogenous gene (in the species of origin) or a chimeric gene (e.g. a transgene or cis-gene).

[0031] “Expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA.

[0032] An “active protein” or “functional protein” is a protein which has protein activity as measurable in vivo, e.g. by the phenotype conferred by the protein. A “wild type” protein is a fully functional protein, as present in the wild type plant. A “mutant protein” is herein a protein comprising one or more mutations in the nucleic acid sequence encoding the protein, whereby the mutation(s) results in (the mutant allele encoding) a “reduced-function” or “loss-of-fimction” protein, as e.g. measurable in vivo, e.g. by the phenotype conferred by the mutant allele (e.g. in homozygous form). A “reduced function CsAMP 1 protein” or “reduced activity CsAMP 1 protein” refers to a mutant protein which has a reduced activity and confers at least shorter internode length and / or more internodes and / or smaller leaf size in a cucumber plant comprising such reduced function protein, at least when the allele encoding the mutant protein is present in homozygous form compared to a cucumber plant comprising the wild type allele in homozygous form.

[0033] “Shorter internodes” refers to an average internode length being significantly shorter in the homozygous mutant plant than in the homozygous wild type plant. For example, the average internode length of the mutant plant may be at least 10%, 15%, 20%, 25%, 28%, 30%, 35%, 38%, 40%, 45%, 48% or 50% shorter than the average internode length in the wild type plant. Thus, relative to the wild type having an average internode length set to 100%, the mutant plant comprises an average internode length of equal to or less than 50%, 49%, 48% or 47% of the wild type plant.

[0034] “More internodes” or “more nodes” refers to the average number of nodes or the average number of internodes over a defined main stem length, e.g. 175 cm or 200 cm or 250 cm, being significantly higher in the homozygous mutant plant than in the homozygous wild type plant. For example, the average number of nodes or internodes of the mutant plant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or 80%, 85%, 90%, 100%, 110% (or more) higher than in the wild type plant. E.g. the homozygous mutant plant may develop at least 20 or 25 or more (e.g. 30) internodes on a stem length / height of 175 cm, while the wild type plant develops only 15 or 16 internodes over the same stem length of 175 cm, whereby the average number of internodes of the mutant plant is at least 70% or 75% more, or even 100%, more than that of the wild type. Thus, relative to the wild type plant set to an average number of internodes of 100%, the mutant plant comprises e.g. at least 170%, 175%, 180%, 190%, 200%, 210% internodes on a defined length / height of the main stem.

[0035] “More fruits” refers to the average number of fruits (which develop to harvest maturity) over a defined main stem length / height, e.g. 175 cm or 200 cm or 250 cm, being significantly higher in the homozygous mutant plant than in the homozygous wild type plant. For example, the average number of fruits (which develop to harvest maturity) of the mutant plant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 75% higher than in the wild type plant. Thus, relative to the wild type plant set to an average number of fruits of 100%, the mutant plant comprises e.g. at least 170%, 175%, 180%, 190%, 200%, 210% fruits on a defined length / height of the main stem.

[0036] “Increased fruit yield” refers to refers to the average cucumber fruit yield (which can be expressed in grams of fruit) of harvest-mature fruits over a defined main stem length, e.g. 175 cm or 200 cm or 250 cm, being significantly higher in the homozygous mutant plant than in the homozygous wild type plant. For example, the average fruit yield of the mutant plant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 75% higher than in the wild type plant. Thus, relative to the wild type plant set to an average fruit yield of 100%, the mutant plant comprises e.g. an average yield of at least 110%, 120%, 130%, 140%, 150% yield harvested.

[0037] “Smaller leaf size” refers to an average leaf length and / or average leaf width being significantly shorter in the homozygous mutant plant than in the homozygous wild type plant. For example, the average leaf length and / or leaf width of the mutant plant may be at least 10%, 15%, 20%, 25%, 28%, 30%, 31% or 35% shorter than the average length and / or width in the wild type plant. Alternatively “smaller leaf size” can be measured as leaf surface area, whereby the average leaf surface area of the homozygous mutant plant is equal to or less than 80% of the average leaf surface area of the wild type plant, preferably equal to or less than 70%, 60%, 50%, 40%, 36% or 35% of the wild type leaf surface area. Thus, relative to the wild type plant set to an average leaf area of 100%, the mutant plant comprises e.g. an average leaf surface area of 34%, 35%, 36% or 37% of the average leaf surface area of the wild type plant.

[0038] “Smaller fruit length” or “shorter fruit length” or “shorter fruits” refers to an average fruit length being significantly shorter in the homozygous mutant plant than in the homozygous wild type plant. For example, the average fruit length of the mutant plant may be at least 5%, 6%, 7%, 8%, 9%, 10% or 11% shorter than the average fruit length in the wild type plant. Thus, relative to the wild type plant set to an average fruit length of 100%, the mutant plant comprises e.g. an average fruit length of 86%, 85%, 84% or 83% of the average fruit length of the wild type plant.

[0039] “Dwarf phenotype” refers to plants which at plant maturity develop a main stem length of equal to or less than 60 cm, 55 cm or 50 cm. The plants comprising a mutant csampl allele in homozygous form as described herein are not dwarf plants, as the main stem continues growing to a height of at least 175 cm, 180 cm, 190 cm, 200 cm, or more (until e.g. they are topped).

[0040] "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 genome editing techniques. Induced mutant alleles are thus not natural mutant alleles.

[0041] “Spontaneous mutant” alleles are mutant alleles in which the mutation(s) develop spontaneously. This can occur in cultivated cucumber breeding lines or varieties. “Natural mutant” alleles are mutant alleles in which the mutation(s) have evolved in wild plants or wild relatives of a species or landraces. Such natural mutant alleles can be introgressed into cultivated plants by crossing and selection.

[0042] A “mutation” in a nucleic acid molecule coding for a protein 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. 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.

[0043] A “point mutation” is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide.

[0044] A “nonsense” mutation is a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon is changed into a stop codon. This results in a premature stop codon being present in the mRNA and in a truncated protein. A truncated protein may have reduced function or loss of function.

[0045] 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 reduced function or loss of function.

[0046] 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 reduced function or loss of function.

[0047] 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 reduced function or loss of function.

[0048] A mutation in a regulatory sequence, e.g. in a promoter 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 reduced or no mRNA transcript of the gene being made. In one aspect a mutation in a regulatory sequence of a protein includes a lower level of wild type protein (e.g. due to a lower expression of the allele) or no wild type protein being made (no expression of the allele). Rodriguez-Leal et al., 2017, Cell 171, 470-480 describe for example mutating c / .s-rcgulatory elements to create a continuum of mutant alleles with different expression. 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 or insertion of one or more amino acid residues.

[0049] “Silencing” refers to a down-regulation or complete inhibition of gene expression of the target gene or gene family.

[0050] A “target gene” in gene silencing approaches is the gene or gene family (or one or more specific alleles of the gene) of which the endogenous gene expression is down-regulated or completely inhibited (silenced) when a chimeric silencing gene (or ‘chimeric RNAi gene’) is expressed and for example produces a silencing RNA transcript (e.g. a dsRNA or hairpin RNA capable of silencing the endogenous target gene expression). In mutagenesis or targeted genome editing approaches, a target gene is the endogenous gene which is to be mutated (and / or in which mutations are selected by e.g. TILLING) or edited, leading to a change in (reduction or loss of) gene expression or a change in (reduction or loss of) function of the encoded protein.

[0051] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame so as to produce a “chimeric protein”. A “chimeric protein” or “hybrid protein” is a protein composed of various protein “domains” (or motifs) which is not found as such in nature but which a joined to form a functional protein, which displays the functionality of the joined domains. A chimeric protein may also be a fusion protein of two or more proteins occurring in nature.

[0052] The term “heterozygous” refers to a plant or plant cell having dissimilar pairs of alleles of a gene for any hereditary characteristic. The term “homozygous” or in “homozygous form” refers to a plant or plant cell or plant part (e.g. a fruit) having identical alleles of a gene for any hereditary characteristic, e.g. a diploid cucumber plant or plant part homozygous for the mutant csampl allele comprises two copies of the allele in its genome.

[0053] It is understood that comparisons between different plant lines involves growing a number of plants of a line (e.g. at least 3, 4, 5, 6, 7, 8 or 9 plants, preferably at least 10 plants per line) under the same conditions as the plants of one or more control plant lines (e.g. plants comprising the wild type allele or plants having the same or very similar genetics as the line it is compared with except that the wild type allele is present in homozygous form instead of the mutant allele) and the determination of statistically significant differences between the plant lines when grown under the same environmental conditions and when treated in the same way.

[0054] “Stringent hybridization 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. lOOnt) 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. lOOnt) 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.

[0055] “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 minimises 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, accessible at world wide web under ebi.ac.uk / Tools / emboss / . Alternatively sequence 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 95%, 96%, 97%, 98% or 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 an Blosum62 for proteins). Such sequences are also referred to as ‘variants’ herein, e.g. other allelic variants of the wild type CsAMPl allele and CsAMP 1 protein than the specific nucleic acid and protein sequences disclosed herein can be identified. Mutations in such allelic variants have the same effect on internode length and / or internode number and / or leaf size in plants comprising such variants and cultivated Cucumis sativus plants comprising mutations in such variants are embodiments of the invention.

[0056] “Equivalent position” or “equivalent amino acid or nucleotide” in a variant sequence or in a sequence comprising at least a certain percentage sequence identity with the given sequence (e.g. the equivalent nucleotide / amino acid in a sequence comprising at least 95%, 96%, 97%, 98% or more sequence identity to a certain SEQ ID NO) can be identified by pairwise alignment (e.g. using the program Needle) with the SEQ ID NO.

[0057] “Wild type CsAMPl allele” (WT) refers herein to a version of a gene encoding a fully functional cucumber CsAMPl protein (wild type CsAMPl protein). Such a sequence encoding a fully functional CsAMPl protein of SEQ ID NO: 1 is for example the wild type CsAMPl cDNA (mRNA) sequence depicted in SEQ ID NO: 2, or the wild type CsAMPl genomic sequence of SEQ ID NO: 3. The protein sequence encoded by this wild type CsAMPl mRNA is depicted in SEQ ID NO: 1. It consists of 701 amino acids. Other fully functional CsAMPl protein-encoding alleles (i.e. variant alleles, or allelic variants) may exist in other cucumber plants or wild cucumber or wild relatives of cucumber and may comprise substantial sequence identity with SEQ ID NO: 1, i.e. at least about 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1. Such fully functional wild type CsAMPl proteins are herein referred to as “variants” of SEQ ID NO: 1. Likewise, the genomic DNA sequence encoding a wild type CsAmpl protein may be an allele comprising SEQ ID NO: 3 (encoding the wild type protein of SEQ ID NO: 1) or a variant allele encoding a variant wild type CsAmpl protein, such as a variant genomic DNA comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to the genomic DNA sequence of SEQ ID NO: 3. Also due to the degeneracy of the genetic code a genomic DNA sequence comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 may still encode a wild type protein of SEQ ID NO: 1 or a variant.

[0058] “M28_PMSA_like domain”, or “M28 Zn-peptidase prostate-specific membrane antigen domain”, or also referred to as “peptidase domain” herein, is a conserved zinc peptidase protein domain which e.g. cleaves terminal glutamate from e.g. gamma-linked polyglutamates (carboxypeptidase). It can for example be identified in NCBIs conserved domain database (CDD, see world wide web at ncbi.nlm.nih.gov / cdd / and search for cd08022). The peptidase domain of the CsAMPl protein starts at (and includes) amino acid 322 and ends at (and includes) amino acid 548 of SEQ ID NO: 1 or SEQ ID NO: 4, see Figure 1, dashed boxes. Two other conserved domains of the CsAMP 1 protein are the “Protease Associated (PA) domain” (in CDD named cl28883) which starts at (and includes) amino acid 119 and ends at (and includes) amino acid 292 of SEQ ID NO: 1 or SEQ ID NO: 4 and the “TFR dimer domain” or “Transferrin receptor-like dimerization domain” (pfam04253 or cdd427820) which starts at (and includes) amino acid 581 and ends at (and includes) amino acid 683 of SEQ ID NO: 1 or SEQ ID NO: 4.

[0059] “Cucumber genome” and “physical position on the cucumber genome” and “chromosome 3” refer to the physical genome of cultivated cucumber, for which e.g. reference genomes have been published on the world wide web at / / cucurbitgenomics. org / , e.g. the genome of cultivated cucumber (Chinese Long V2 or V3, or the B 10 V3 genome), and the physical chromosomes and the physical position on the chromosomes.

[0060] “Cucumber plant” or “cultivated cucumber” or “domesticated cucumber” refers to plants of Cucumis sativus var. sativus i.e. varieties, breeding lines or cultivars, cultivated by humans and having good agronomic characteristics, especially producing edible and marketable fruits of good size and quality and uniformity; such plants are not “wild cucumber” or “primitive cucumber” plants , i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and are less uniform genetically and in their physiological and / or morphological characteristics. “Wild plants” of “wild cucumber” include for example ecotypes, landraces or wild accessions or wild relatives of a species.

[0061] “Landrace(s)” refers to primitive cultivars developed in local geographic regions, which often show a high degree of genetic variation in their genome and exhibit a high degree of morphological and / or physiological variation within the landrace (e.g. large variation in fruit size, etc.), i.e. are significantly less uniform than cultivated plants. Landraces are, therefore, herein included in the group “wild” plants, which is distinct from “cultivated” plants.

[0062] “Wild relatives of cucumber” refer to Cucumis sativus var. hardwickii. C. sativus var. sikkimensis. Cucumis sativus var. xishuangbannesis .

[0063] 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, as a result of this selfing, plants of an inbred line are nearly identical to each other in genotype and phenotype. 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.

[0064] “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, the Fl hybrids which are produced from crossing two such inbred lines are highly uniform in their genotypic and phenotypic characteristics and performance. A “recombinant chromosome” refers to a chromosome having a new genetic makeup arising through crossing -over between homologous chromosomes.

[0065] The term “traditional breeding techniques” encompasses herein crossing, backcrossing, selfing, selection, double haploid production, embryo rescue, protoplast fusion, marker assisted selection, mutation breeding etc., all as known to the breeder (i.e. methods other than genetic modification / transformation / transgenic methods), by which, for example, a recombinant chromosome can be obtained, identified and / or transferred.

[0066] “Backcrossing” refers to a breeding method by which a (single) trait, such as a mutant allele, can be transferred from a generally (but not necessarily) inferior genetic background (e.g. a wild plant or wild relative; also referred to as “donor”) into a generally (but not necessarily) superior genetic background (also referred to as “recurrent parent”), e.g. a cultivated plant. An offspring of a cross (e.g. an Fl plant obtained by crossing a donor plant with a e.g. superior genetic background plant; or an F2 plant or F3 plant, etc., obtained from selfing the Fl) is e.g. “backcrossed” to the recurrent parent genetic background, e.g. to the cultivated parent. After repeated backcrossing, the trait of the donor genetic background will have been incorporated into the recurrent parent genetic background.

[0067] “Vegetative propagation”, “vegetative reproduction” or “clonal propagation” are used interchangeably herein and mean the method of taking part of a plant and allowing that plant part to form at least roots where plant part is, e.g., defined as or derived from (e.g. by cutting of) leaf, pollen, embryo, cotyledon, hypocotyl, cells, protoplasts, meristematic cell, root, root tip, pistil, anther, flower, shoot tip, shoot, stem, fruit, petiole, etc. When a whole plant is regenerated by vegetative propagation, it is also referred to as a vegetative propagation or a vegetatively propagated plant. In one aspect propagation by grafting, e.g. a scion onto a rootstock, is included herein.

[0068] “Cell culture” or “tissue culture” refers to the in vitro culture of cells or tissues of a plant.

[0069] “Regeneration” refers to the development of a plant from cell culture or tissue culture or vegetative propagation.

[0070] “Non-regenerable cell” refers to a cell which cannot be regenerated into a whole plant.

[0071] “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. 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.

[0072] “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.

[0073] “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.

[0074] DETAILED DESCRIPTION

[0075] CUCUMBER PLANTS AND PLANT PARTS

[0076] The present invention relates in one aspect to the identification of a gene called CsAMPl which, when mutated, leads to a different growth type, especially shorter internodes and / or more internodes and / or smaller leaves than the wild type plant. Three different mutants were generated and characterized. Two mutants resulted in loss-of-fiinction or near loss of function of the CsAMPl protein (one stop codon mutant, Q448*, and one splice mutant) and it was not possible to generate homozygous plants for the stop codon mutant and plants homozygous for the splice mutant grew not larger than a few centimetres in height. An at least partly functioning CsAMPl protein is, therefore, needed for viability and normal development of the plant. The third mutant was an amino acid substitution mutant at amino acid 411, the codon for Leucine was changed into the codon for Phenylalanine, i.e. L41 IF. When the mutant allele was in homozygous form plants developed normally but had significantly shorter internodes but also more internodes and smaller leaves, see Examples. As internode length was shorter but at the same time the number of internodes was increased, the mutation did not result in a dwarf phenotype (with e.g. a stem length of an adult plant of 50 cm or less) but plants grew up to the wire in the greenhouse. This mutant protein must, therefore, have a reduced function in vivo (but not a loss of function) compared to the wild type protein, as it resulted in viable plants which developed normally but had a modified plant growth (shorter internodes, more internodes per stem length and smaller leaves) when the mutant allele was in homozygous form. Due to the higher number of internodes (and therefore also nodes) per stem length, also the number of fruits that develop at the nodes was significantly higher. Thereby the overall fruit yield per stem length was greatly increased. Thus, not only can more plants (stems) be grown per unit area due to the leaves being smaller, also each stem has a significantly increased number of fruits and thereby overall fruit yield is increased significantly per stem and per unit area in e.g. the greenhouse.

[0077] Further two other mutants, P428S and L538F, were generated and phenotyped when the mutant allele was in homozygous form. However, these two amino acid substitution mutants retained the wild type phenotype. It is, therefore, concluded that these two mutants do not reduce or change the CsAMPl protein function, which is in agreement with the SIFT prediction in Table 1 (‘tolerated’ means that no change in protein function is predicted due to the amino acid substitution).

[0078] The wild type CsAMPl gene provided herein in SEQ ID NO: 3 (wild type genomic DNA, gDNA) was found to be on chromosome 3 of the reference genome Chinese Long V2 (found on cucurbitgenomics.org), starting at nucleotide 30565575 and ending at nucleotide 30572119, albeit not 100% identical to SEQ ID NO: 3 (99.89% identical). The transcript of the gDNA of SEQ ID NO: 3 (provided herein in SEQ ID NO: 2) encodes the wild type CsAMPl protein of SEQ ID NO: 1. The gene on chromosome 3 of the ChineseLong V2 genome encodes the protein Csa3G790960.1, which is 99.7% identical to the wild type CsAMPl protein of SEQ ID NO: 1. The wild type protein Csa3G790960.1 (of the reference genome and also NCBI Ref. XP_004136724.2) comprises a peptidase domain which is 100% identical to the peptidase domain of SEQ ID NO: 1, but comprises two different amino acids in regions outside of the peptidase domain (amino acid 135 is not an Asparagine (Asn, N) but a Tyrosine (Tyr, Y) and amino acid 668 is not a Glutamic acid (Glu, E) but a Valine (Vai, V). Still this CsAMPl protein is a wild type (functional) protein.

[0079] On the Chinese Long V3 reference genome a sequence which is 99.98% identical to SEQ ID NO: 3 is present on chromosome 3 from nucleotide 31587489 to 31594030. This is the same as on the Chinese Long V2 genome. Doing a BLAST against the NCBI database results in a similar protein XP 004136724.3 (Low Quality Protein: probable glutamate carboxypeptidase AMP1 [Cucumis sativus]), which is 99.9% identical to SEQ ID NO: 1, but misses amino acid D293 of SEQ ID NO: 1. No further information on the function is given.

[0080] However, the above sequence variation is likely due to sequence errors. The high-quality cucumber genome of variety BIO (cucumber (B10)V3 genome; Osipowski et al. 2020, Mol Genet Genomics 295, 177-193), which is the highest quality reference genome available for cucumber, contains the CsAMPl gene which is 100% identical to the sequences of the wild type CsAMPl gene and protein provided herein under SEQ ID NO: 1, 2 and 3. On the (B10)V3 genome the wild type CsAmpl gene of SEQ ID NO: 3 is present on chromosome 3 starting at nucleotide 936598 and ending at nucleotide 943140.

[0081] Alignment of the wild type CsAMPl protein provided herein in SEQ ID NO: 1 with the Arabidopsis thaliana AtAMPl protein (At3g54720 or NP_567007.1) showed very little protein sequence identity, the two proteins only had 56.2% sequence identity when aligned pairwise using the program Emboss Needle. AtAMPl has been studied in e.g. Saibo et al. Planta 2007 (Mar;225(4): 831-42) and Fouracre et al. (2020, Development 147, doi: 10.1242 / dev. 186874).

[0082] Analysis of the conserved domains of the protein of SEQ ID NO: 1 showed that three conserved domains are present: a PA-domain, a M28_PMSA_like domain and a TFR dimer domain. The L41 IF amino acid substitution was found in the M28_PMSA_like domain, also referred to as the peptidase domain herein. This domain includes a whole range of active site amino acids.

[0083] The L41 IF mutation is, however, not one of these active site amino acids of the peptidase domain. Still, in the SIFT prediction tool as shown in Table 1 the L411F substitution is predicted to affect protein function, which is in agreement what has been found in vivo.

[0084] Mutants that were generated herein by EMS mutagenesis and that are in the peptidase domain are listed in the Table 1 below and in Table 3. These are encompassed herein if they lead to shorter internodes and / or more internodes and / or smaller leaves when the mutant allele is in homozygous form. Table 1

[0085] In one aspect a cultivated plant or plant part of the species Cucumis sativus is provided comprising at least one copy of a mutant allele of a gene named CsAMPl, said gene encodes a wild type CsAMPl protein of SEQ ID NO: 1 or a wild type protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, wherein the mutant allele encodes a protein having a decreased function compared to the wild type CsAMPl protein. The decreased function is, however, not a loss-of-function, as a loss-of- function is lethal when the mutant allele is in homozygous form. A decreased function of the mutant protein can easily be determined by the phenotype of significantly more internodes and / or significantly shorter internodes and / or significantly smaller leaves of the plant comprising the mutant allele in homozygous form compared to the wild type (control) plant comprising the wild type allele in homozygous form.

[0086] In one aspect a plant or plant part of the species Cucumis sativus is provided comprising at least one copy of a mutant allele of a gene named CsAmpl on chromosome 3, wherein the wild type CsAmpl gene is a gene that encodes a CsAmpl protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, wherein the mutant allele encodes a CsAmpl protein comprising one or more amino acids of the wild type protein are replace by another amino acid (especially in one of the conserved domains, especially the peptidase domain) and / or one or more amino acids are inserted and / or deleted (especially in one of the conserved domains, especially the peptidase domain), and wherein said mutant allele causes a plant which is homozygous for the mutant allele to develop significantly more internodes and / or significantly shorter internodes and / or significantly smaller leaves compared to the plant which is homozygous for the wild type CsAmpl gene.

[0087] In one aspect said wild type protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 but also comprises a peptidase domain which is 100% identical to the peptidase domain of SEQ ID NO: 1, i.e. the sequence variation of the wild type (variant) protein is not in the conserved peptidase domain. The conserved peptidase domain starts at (and includes) amino acid 322 and ends at (and includes) amino acid 548 of SEQ ID NO: 1. Thus, slight variation in the amino acid sequence outside of the peptidase domain may be found in variant wild type CsAMPl proteins. For example the wild type protein Csa3G790960.1 (of the reference genome) comprises a peptidase domain which is 100% identical to the peptidase domain of SEQ ID NO: 1, but comprises two different amino acids in regions outside of the peptidase domain (amino acid 135 is not a asparagine (Asn, N) but a Tyrosine (Tyr, Y) and amino acid 668 is not a glutamic acid (Glu, E) but a Valine (Vai, V). Still this CsAMPl protein is a wild type (functional) protein.

[0088] In one aspect a plant or plant part of the species Cucumis sativus is provided comprising at least one copy of a mutant allele of a gene named CsAmpl on chromosome 3, wherein the wild type CsAmpl gene is a gene that encodes a CsAmpl protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and further comprises a peptidase domain that is 100% identical to the peptidase domain starting at amino acid 322 and ending at amino acid 548 of SEQ ID NO: 1, wherein the mutant allele encodes a CsAmp 1 protein comprising one or more amino acids of the wild type protein are replace by another amino acid (especially in one of the conserved domains, especially the peptidase domain) and / or one or more amino acids are inserted and / or deleted (especially in one of the conserved domains, especially the peptidase domain), and wherein said mutant allele causes a plant which is homozygous for the mutant allele to develop shorter internodes and more internodes over a defined stem length compared to the plant which is homozygous for the wild type CsAmpl gene.

[0089] In a further aspect a plant or plant part of the species Cucumis sativus is provided comprising at least one copy of a mutant allele of a gene named CsAmpl on chromosome 3, wherein the wild type CsAmpl gene is a gene that encodes a wild type CsAmpl protein comprising at least 98% or 99% sequence identity to SEQ ID NO: 1 and further comprises a peptidase domain that is 100% identical to the peptidase domain starting at amino acid 322 and ending at amino acid 548 of SEQ ID NO: 1, wherein the mutant allele encodes a CsAmpl protein comprising a substitution (or replacement) of Leucine 411 of SEQ ID NO: 1, or the equivalent Leucine in the peptidase domain of a wild type CsAmpl protein comprising at least 98% or 99% sequence identity to SEQ ID NO: 1, by another amino acid, preferably by Phenylalanine, and wherein said mutant allele causes a plant which is homozygous for the mutant allele to develop shorter internodes and more internodes over a defined stem length compared to the plant which is homozygous for the wild type CsAmpl gene.

[0090] In yet a further aspect a plant or plant part of the species Cucumis sativus is provided comprising at least one copy of a mutant allele of a gene named CsAmpl on chromosome 3, wherein the wild type CsAmpl gene is a gene that encodes a wild type CsAmpl protein comprising at least 98% or 99% sequence identity to SEQ ID NO: 1, wherein the mutant allele encodes a mutant CsAmpl protein comprising a substitution (or replacement) of Leucine 411 of SEQ ID NO: 1, or the equivalent Leucine in a wild type CsAmpl protein comprising at least 98% or 99% sequence identity to SEQ ID NO: 1, by another amino acid, preferably by Phenylalanine, and wherein said mutant allele causes a plant which is homozygous for the mutant allele to develop shorter internodes and more internodes over a defined stem length compared to the plant which is homozygous for the wild type CsAmpl gene.

[0091] In one aspect a cultivated plant or plant part of the species Cucumis sativus is provided comprising at least one copy of a mutant allele of a gene named CsAMP 1, said gene encodes a CsAMPl protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, wherein the mutant allele encodes a protein comprising an amino acid substitution in the peptidase domain starting at amino acid 322 and ending at amino acid 548 of SEQ ID NO: 1 or in the peptidase domain of said protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

[0092] In one aspect the amino acid substitution in the peptidase domain comprises a substitution of a hydrophobic or non-polar amino acid of the peptidase domain into an amino acid having different physicochemical properties, e.g. into a hydrophilic or polar amino acid or into a ‘very large’ amino acid. For the different physicochemical properties see e.g. world wide web at imgt.org / IMGTeducation / Aide- memoire / _UK / aminoacids / IMGTclasses.html. Without limiting the invention it is believed that such a non-conservative substitution results in a reduced activity of the CsAMPl enzyme. In particular any one of the Leucines (Leu, L) and / or any one of the Glycines (Gly, G) of the peptidase domain is in one aspect changed into a different amino acid having different physicochemical properties.

[0093] In one aspect the amino acid of the conserved peptidase domain of the wild type CsAMP 1 protein that is substituted is selected from any G (Gly) or L (Leu) of the peptidase domain. In one aspect the amino acid of the conserved peptidase domain of the wild type CsAMP 1 protein that is substituted is selected from: L411, G412 and G530. Thus, in one aspect the amino acid that is substituted is selected from L411, G412, and G530. In one aspect the amino acid is substituted by a different amino acid, especially the mutation changes a hydrophobic or non-polar amino acid (e.g. L or G) into a different amino acid, especially into an amino acid having different physicochemical properties, e.g. into a hydrophilic or polar amino acid (e.g. E, S, R) or into a very large amino acid (e.g. F). In one aspect L411 is substituted by F, G412 is substituted by E and G530 is substituted by R.

[0094] In another aspect the plant comprises two copies of the mutant allele (homozygous). In one aspect the mutant allele causes, when in homozygous form, the development of shorter internodes and / or more internodes and / or smaller leaves of the plant. Preferably also fruit number and fruit yield per plant is significantly increased in the plant comprising the mutant allele in homozygous form. In one aspect fruit length may be reduced and / or growth speed may be reduced in the homozygous mutant plant. The phenotypic change caused by the mutant allele is compared to the control plant or wild type plant, which comprises wild type, functional CsAMPl alleles on both of the chromosomes 3 at the locus of the CsAMPl gene. Preferably there is a significant difference in average internode length and / or number of internodes and / or average leaf size between the plant homozygous for the mutant allele and the control plant (homozygous for the wild type allele).

[0095] The cucumber plant or plant part, therefore, comprises at least one copy, preferably two copies, of a mutant allele of the endogenous CsAMPl gene on chromosome 3. The endogenous gene can be mutated by various methods known in the art, e.g. chemical mutagenesis (e.g. as done in the Examples), radiation mutagenesis or targeted gene editing techniques (such as Crispr based gene editing).

[0096] An endogenous cucumber CsAMPl gene (or an CsAMPl allele thereof) is a gene (or allele) encoding a (wild type, functional) CsAMPl protein which comprises at least 95%, 96%, 97%, 98%, 99% or more sequence identity to the cucumber CsAMPl protein of SEQ ID NO: 1 and in one aspect also comprises a peptidase domain as e.g. shown in Figure 1 (dotted line) or as present in SEQ ID NO: 1 from amino acid 322 to 548.

[0097] In the plant or plant part comprising said mutant csampl allele, said mutant csampl allele encodes a protein having a decreased function compared to the wild type CsAmpl protein, i.e. said mutant allele encodes a mutant CsAmpl protein.

[0098] Thus, as a result of the mutation in the endogenous CsAMPl allele a mutant CsAmp 1 protein is produced by the mutant allele, e.g. comprising one or more amino acids replaced, inserted or deleted compared to the functional wild type protein, especially in the conserved peptidase domain (or in the PA domain or TFR dimer domain), thereby leading to a reduced function of the (mutant) CsAmp 1 protein. And consequently, at least when the mutant csampl allele is in homozygous form in the genome of the plant, the plant will develop shorter internodes and / or more internodes and / or smaller leaves compared to the plant comprising two functional / wild type CsAMPl alleles.

[0099] Reduced function and decreased function or reduced activity are used synonymously. Such alleles are known to have reduced function or decreased function or reduced activity when the phenotype (average shorter internodes and / or more internodes and / or smaller leaves) is seen when the mutant allele is in homozygous form. Loss-of-function mutants are deleterious, i.e. it will not be possible to make a homozygous plant for such an allele, thus upon selfing the plant comprising the allele in heterozygous form, no homozygous progeny will be recovered. Therefore, a reduced function of a mutant allele can be identified by generating homozygous plants and determining the phenotype (e.g. the average internode length and / or average number of internodes developing).

[0100] In one embodiment the cucumber plant or plant part comprises a mutant csampl allele, wherein the protein encoded by the mutant csampl allele comprising one or more amino acids replaced and / or inserted and / or deleted compared to the CsAmpl wild type protein of SEQ ID NO: 1 (or a functional variant thereof comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1), especially one or more amino acids replaced and / or inserted and / or deleted in the conserved peptidase domain which is present at amino acid 322 to 548 of SEQ ID NO: 1, or at the equivalent position in a variant protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. Such a protein can have a reduced activity in vivo. Especially one or more of the amino acid L or G of the peptidase domain are replaced by another amino acid, e.g. by a polar or hydrophilic amino acid or by a very large amino acid as described elsewhere herein. In one aspect L is replaced by F. In another aspect G is replaced by E or R. In one aspect L411 is replaced by F. In one aspect G412 is replaced by E or R. In one aspect G530 is replaced by E or R.

[0101] In one embodiment the plant or plant part comprises the mutant allele in heterozygous or homozygous form.

[0102] Whether the replacement and / or insertion and / or deletion of one or more amino acids actually results in shorter average internode length and / or more internodes and / or smaller leaf size in vivo can be tested by growing a plant homozygous for the mutant allele under the same conditions as a control plant (e.g. the non-mutated plant) and comparing or measuring internode length and / or counting the number of internodes over a specific stem length of the plant and / or comparing or measuring leaf size.

[0103] In the plant comprising the L411F mutant allele in homozygous form average internode length was, in one of the Examples, about 8 to 10 cm while in the control plant it was about 14 to 16 cm. Also, the number of internodes counted up to the wire in the greenhouse was 28 in the mutant plant and only 16 in the control plant. The leaves were also significantly smaller with an average leaf length x width of 39 cm x 32 cm compared to the control plant having an average length x width of 52 cm x 46 cm. Average fruit length in the mutant plant was 26 cm while the average fruit length in the control plant was 29 cm. In another one of the Examples a plant homozygous for the L411F mutant allele in a different genetic background had an average internode length of 7.3 cm and 30 internodes up to the wire (at 175 cm), while the wild type control plant had an average internode length of 15 cm and only 15 internodes up to the wire when grown under the same conditions. It is understood that the extent of the reduction in internode length and / or leaf size may be slightly different in different genetic backgrounds of cultivated cucumber. Thus, if the mutant allele is transferred or generated into / in a different genetic background the reduction in internode length and / or leaf size may be slightly different. However, the phenotype(s) will co-segregate with the mutant allele, i.e. when e.g. backcrossed into another genetic background a reduction in internode length and / or leaf size will be seen in the plants of that genetic background which are homozygous for the mutant allele.

[0104] In one aspect the mutant csampl allele results in a cultivated cucumber plant comprising an increased number of shorter internodes over e.g. a defined segment of stem length and smaller leaves compared to the control plant. This more compact growth type allows more plants to be grown per area of cultivation, as plants with smaller leaves can be grown closer together and more stems per area can be grown. For example in high-wire cultivation, where fruits are harvested from the main stem which is lead up to a high wire, at least 3.0 or 4.0 stems per square meter can be grown, or even more. In combination with the significantly increased number of fruits developing due to the increased number of nodes on the main stem, yield can be significantly increased.

[0105] In one aspect the mutant csampl allele in homozygous form results in a cultivated cucumber plant that develops at least (an average of) 25, 26, 27, 28, 29, 30, or more, internodes on the main stem when counted up to a height of e.g. 175 cm. For example, two or three plants comprising the mutant allele in homozygous form, and optionally 1, 2 or 3 wild type control plants, can be grown up to a height of e.g. 175 cm and the number of internodes can be counted. Also, the average internode length can be determined, optionally also the average leaf size, average fruit number, average fruit weight, etc. In one aspect the average internode length of the cucumber plant comprising the mutant allele in homozygous is e.g. about 7 cm (e.g. 7.1 cm, 7.2 cm, 7.3 cm, 7.4 cm or 7.5 cm) or about 8 cm, 8.5 cm or 9 cm.

[0106] In one aspect the mutation in the mutant csampl allele is a missense mutation, especially a missense mutation in a codon of one of the conserved domains, in one aspect the peptidase domain. It is understood that not all missense mutations will lead to the allele having reduced function when in homozygous form in a plant, but it is easy and without burden for the skilled person to generate and select the mutant alleles which do lead to the desired phenotype (shorter internodes, more internodes and smaller leaves than the wild type). The mutation can occur in a DNA sequence comprising the coding sequence of a CsAmpl gene, or in an RNA (which may be depicted as cDNA herein) sequence encoding a CsAMPl protein or it can occur in the amino acid molecule of the CsAMPl protein.

[0107] Concerning a DNA sequence of the CsAMPl allele, the mutation preferably occurs in the coding sequence (cds, composed of the exons). In respect to RNA encoding a CsAmpl protein, the mutation can occur in the pre-mRNA or the mRNA. In one aspect the mutant allele results in the protein having a decrease of function due to one or more amino acids being replaced, inserted or deleted, especially resulting in one or more amino acids being replaced, inserted and / or deleted in the conserved peptidase domain or in one of the other conserved domains. As mentioned and as shown in the examples, its understood that not all mutations in the CsAmpl allele will lead to the allele having reduced function when in homozygous form in a plant, but it is easy and without burden for the skilled person to generate and select the mutant alleles using phenotypic selection which do lead to the expected phenotype similar to the L411F mutant phenotype.

[0108] In one aspect the genomic sequence of the CsAMPl gene comprises a missense mutation in the coding sequence, especially in the coding sequence encoding the peptidase domain of SEQ ID NO: 1. Thus, in one aspect the genomic sequence of SEQ ID NO: 3 or a genomic sequence comprising at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 3 comprises a missense mutation which changes a codon of e.g. the peptidase domain (or one of the other conserved domains) into a codon for a different amino acid. For example the codon CTT (coding for Leucine, L) at nucleotide 3081 to 3083 of SEQ ID NO: 3, or the equivalent codon in a genomic sequence comprising at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 3, may be changed into codon TTT (coding for Phenylalanine, F), whereby the resulting protein comprises a Phenylalanine at amino acid 411. Similarly, the genomic DNA may comprise any other missense mutation which changes a codon coding for an amino acid of e.g. the peptidase domain (or one of the other conserved domains) into a different codon.

[0109] SEQ ID NO: 3 is the wild type genomic sequence which encodes the wild type protein of SEQ ID NO: 1. The cDNA is shown in SEQ ID NO: 2. SEQ ID NO: 6 is a mutant genomic sequence which encodes the mutant protein of SEQ ID NO: 4, comprising a Phenylalanine at amino acid 411. The cDNA is shown in SEQ ID NO: 5. One embodiment therefore concerns cucumber plant cells or plants comprising a mutant allele of a CsAmpl gene, characterized in that the mutant csampl allele comprises or effects one or more of the mutations selected from the group consisting of a) a point mutation or a missense or non-synonymous mutation in the genomic sequence; b) a point mutation or a missense or non-synonymous mutation in the coding sequence; c) a point mutation or missense or non-synonymous mutation in the pre-mRNA or mRNA; and / or d) a replacement, insertion and / or deletion of one or more amino acids in the CsAmpl protein, especially in one of the conserved domains of the protein, such as the peptidase domain.

[0110] In one aspect the mutations result in a replacement (and / or insertion and / or deletion) of one or more amino acids of the peptidase domain of the CsAMPl protein, thereby resulting in a mutant CsAMPl protein. Furthermore, the mutant allele (when in homozygous form) results in the plant producing shorter internodes and / or more internodes and / or smaller leaves compared to the control plant comprising the wild type allele. Especially the mutant allele (when in homozygous form) results in the plant producing shorter internodes and more internodes and smaller leaves compared to the control plant comprising the wild type allele. Also, more fruits and higher fruit yield per plant is preferably seen in the mutant plant, as is higher fruit yield per cultivation area.

[0111] A different embodiment concerns cucumber plant cells, plant parts or plants comprising or synthesising an mRNA encoding a CsAmp 1 protein, wherein the mRNA encoding a CsAmp 1 protein has a missense or non-synonymous mutation.

[0112] In another embodiment plant cells or plants encompassed herein comprise or synthesise an mRNA encoding a CsAmp 1 protein having one or more mutations as described elsewhere, wherein the mRNA is transcribed from a mutant allele of a CsAmp 1 gene. Comprised by these embodiments are plant cells, plant parts or plants comprising or synthesising an mRNA transcribed from a mutant allele of a CsAmpl gene, characterized in that the mRNA comprises a missense or non-synonymous mutation.

[0113] An “mRNA coding sequence” shall have the common meaning herein. An mRNA coding sequence corresponds to the respective DNA coding sequence of a gene / allele apart from that Thymine (T) is replaced by Uracil (U).

[0114] In one aspect the cucumber plant or plant part is homozygous for a mutant csampl allele described herein.

[0115] In one aspect the mutant csampl allele is an induced mutant allele, while in a different aspect the mutant csampl allele is a ‘spontaneous mutant’ allele and in a different aspect the mutant allele is a “natural mutant” allele introgressed into cultivated cucumber by e.g. backcrossing. Mutant alleles can be generated by methods known in the art, such as chemical mutagenesis (e.g. EMS treatment), radiation mutagenesis (UV, gamma rays etc.), targeted mutagenesis, such as Crispr / Cas9 or other Crispr based genome editing techniques or TALENS.

[0116] Suitable chemical mutagens include ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N- ethyl-N-nitrosurea (ENU), trimethylamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N’-nitrosoguanidine (MNNG), nitrosoguanidine, 2- aminopurine, 7,12-dimethylbenz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes, diepoxyoctrane (DEO), diepoxybutane (DEB), 2-methoxy-6-choloro9[3-ethyl- 2-chloro-ethyl]aminopropylamine]acridine dihydrochloride (ICR-170), and formaldehyde. Suitable radiation is UV radiation or radioactive radiation.

[0117] Biotechnological methods for introducing mutations into a desired gene / allele of a plant cell or plant are known in the art. Therefore, mutant alleles of a CsAMPl gene can be produced in plant cells or plants by using these methods. Examples for such technologies are in particular mutagenesis techniques or enzymes which induce double stranded DNA breaks (double stranded DNA break inducing enzyme (DSBI)) in the genome of plants. Known and practised technologies are rare-cleaving endonucleases and custom -tailored rare-cleaving endonucleases including but not limited to homing endonucleases, also called meganucleases, transcription activator-like effectors fused to the catalytic domain of a nuclease (TALENs) and so-called CRISPR systems. CRISPR systems is used broadly herein and does not only encompass the use of the Cas9 nuclease (Crispr / Cas9 system), but also other Crispr systems e.g. using other nucleases, such as Cpf\.. These techniques can also be referred to as targeted genome editing techniques or gene editing techniques or targeted mutagenesis techniques.

[0118] Thus, technologies such as mutagenesis or targeted genome editing techniques are eligible for introducing a mutation into genes in plant cells or plants. Therefore, plant cells and plants having a mutant allele of a CsAmpl gene, wherein the mutation into the mutant allele was introduced by genome editing techniques, e.g. using rare-cleaving endonucleases or custom -tailored rare-cleaving endonucleases, are also an embodiment. Concerning custom-tailored rare-cleaving endonucleases the mutation in the mutant allele of CsAmpl protein has preferably been introduced by a meganuclease, a TALENs or a CRISPR system.

[0119] In cucumber broad virus resistance has been generated by targeted mutagenesis using Crispr / Cas9 by disrupting the function of the recessive eIF4E gene, see Chandrasekaran et al. Molecular Plant Pathology (2016) 17(7), 1140-1153. The transformed T1 plants were selfed and homozygous non-transgenic T3 progeny were selected comprising the mutant eif4e allele in homozygous form, conferring immunity to CVYV and resistance to ZYMV and PRSMV-W. Similarly, Hu etal. (Mol. Plant. 2017; 10: 1575-8) used Crispr-Cas9 to edit the CsWIPl gene in cucumber. Use of the Crispr system in cucumber is, therefore, known to the skilled person.

[0120] Thus, in one aspect the mutant csampl allele is an induced mutant allele, e.g. induced in a breeding line, an inbred line or variety of cultivated cucumber. In one aspect the mutant allele is generated by mutagenesis (e.g. chemical or radiation mutagenesis) or by targeted mutagenesis, especially using the CRISPR system (e.g. Crispr / tU.s'Q or Crispr / Cp / 7 or other nucleases). In one aspect the cultivated cucumber plant comprising the mutant csampl allele is not a transgenic plant, e.g. non transgenic progeny are selected which do not comprise e.g. the CRISPR construct.

[0121] The cultivated cucumber may be of any type, such as pickling cucumbers (e.g. American pickling, European pickling types), slicing cucumbers (e.g. American slicing), long cucumbers, short cucumbers, European greenhouse cucumbers, Beit-Alpha type cucumbers, oriental trellis type cucumbers (also marketed as ‘burpless’), Asian cucumbers, which can be further subdivided into different types, such as Indian Mottled cucumber, Chinese Long cucumber, Korean cucumber and Japanese cucumber types.

[0122] In one aspect a plant or plant part of the species Cucumis sativus var. sativus is provided, comprising a mutant csampl allele on chromosome 3, said mutant allele is of a gene named CsAmpl, said wild type gene encodes a wild type CsAmpl protein of SEQ ID NO: 1 or a wild type protein comprising at least 95% 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1 and further comprising a 100% identical peptidase domain of SEQ ID NO: 1 starting at amino acid 322 of SEQ ID NO: 1 and ending at amino acid 548 of SEQ ID NO: 1, wherein said mutant csampl allele encodes a mutant protein comprising at least one amino acid substitution in e.g. the peptidase domain of the protein. Said amino acid substitution is in one aspect a substitution of a Leucine or of a Glycine by another amino acid. In one aspect said Leucine (L) is replaced by a Phenylalanine (F) and / or said Glycine is replaced by a Glutamine (E) or by an Arginine (R). In one aspect L411 is replaced by another amino acid, e.g. by F. In one aspect G412 or G530 is replaced by another amino acid e.g. E or R.

[0123] In one aspect a plant or plant part of the species Cucumis sativus var. sativus is provided, comprising a mutant csampl allele on chromosome 3, said mutant allele encodes a mutant CsAmpl protein of SEQ ID NO: 4 or a mutant CsAmpl protein comprising at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 4 and further comprising the peptidase domain of SEQ ID NO: 4 starting at amino acid 322 of SEQ ID NO: 4 and ending at amino acid 548 of SEQ ID NO: 4. In one aspect the mutant allele is a natural or spontaneous mutant allele, while in another aspect the mutant allele is an induced mutant allele.

[0124] In one aspect wild or cultivated cucumber plants are screened for the presence of a mutant csampl allele, optionally after a mutation inducing step such as EMS treatment of seeds. This can be done by e.g. analysing (directly or indirectly) the genomic DNA, mRNA (or cDNA) or protein of the CsAmpl gene, using e.g. PCR methods or other molecular methods known in the art, such as sequencing, etc. whereby the presence of a mutant csampl allele can be determined. Wild cucumber plants or wild relatives of cucumber may be CGN accessions, PI accessions (Plant Introductions), or accessions from various seed bank collections. Once an accession is identified to comprise a mutant csampl allele, the accession can be crossed to cultivated cucumber, and the mutant allele can be introgressed into the genome of cultivated cucumber by e.g. backcrossing. The cultivated cucumber introgression line is preferably selfed one or more times to ensure the mutant allele is in homozygous form and the line can then be tested for its phenotype, e.g. internode length, number of internodes and / or leaf size.

[0125] The cultivated cucumber plant and plant parts described herein, comprising either an induced mutant or a natural mutant csampl allele in its genome preferably comprises the mutant allele in homozygous form, as the phenotype is seen when the allele is in homozygous form.

[0126] Also encompassed herein is a method for identifying a plant or plant part comprising a mutant csampl allele. The method involves determining whether a cucumber plant (e.g. a cultivated plant or a wild plant) comprises a mutant allele and optionally transferring the mutant allele into another cucumber by traditional breeding techniques.

[0127] The method comprises obtaining genomic DNA of a wild or cultivated cucumber plant, generating an amplification product (e.g. a PCR product) or hybridization product (e.g. using a nucleic acid probe) of the CsAmpl allele by using primers or probes that amplify or hybridize to part of the allele.

[0128] The primers or probes may be allele-specific primers or probes.

[0129] The identified wild or cultivated cucumber plant which comprises a mutant CsAmpl allele can then be further analyzed for the phenotype (after e.g. selfing) and / or crosses to another cucumber plant (e.g. introduced into other plants by e.g. marker assisted selection). In one embodiment a method for transferring a mutant csampl allele from a cucumber plant into another cucumber plant is provided, comprising a) identifying or providing a cucumber plant comprising a mutant csampl allele as described herein, b) crossing the cucumber plant of a) with another cucumber plant to generate progeny plants comprising the mutant allele, and c) selecting a progeny plant comprising the mutant csampl allele.

[0130] The method may be preceded by a step wherein mutations are induced in e.g. cucumber seeds or plants and then screening for a cucumber plant which comprises a mutant csampl allele in its genome, at least on one chromosome, thereby identifying or providing a cucumber plant comprising a mutant csampl allele. The method may further comprise selecting a cucumber plant comprising a mutant allele which allele confers shorter internodes and / or more internodes and / or smaller leaves when the mutant allele is in homozygous form.

[0131] Step a) and / or c) may involve screening or identification by analyzing the DNA of the plant or of a part of the plant for which csampl alleles are present in the genome, e.g. wild type alleles or a mutant allele. This can be done by e.g. PCR based methods (e.g. SNP genotyping) or hybridization based methods, sequencing, cDNA analysis, etc.

[0132] Step a) and / or c) may involve identification of the plant by phenotype. Optionally there may be a step of selfing the cucumber plant prior to step a) and / or c).

[0133] Thus, step c) may also involve selfing the selected progeny plant to analyze the phenotype and / or step c) may involve analyzing the genomic DNA for the presence of a CsAmpl allele.

[0134] A method of inducing a mutant csampl allele in a cucumber plant or identifying a mutant csampl allele in a cucumber plant is provided herein, comprising: a) inducing mutations in the endogenous wild type CsAmpl allele encoding a protein of SEQ ID NO: 1 (or a functional variant thereof comprising at least 98% or 99% sequence identity to SEQ ID NO: 1), b) screening the cucumber plants for the presence of a mutant csampl allele, and optionally c) selecting a plant comprising a mutant csampl allele, and optionally d) phenotyping the plant comprising the mutant csampl allele in homozygous form and selecting a plant comprising a mutant csampl allele which causes the plant to produce shorter internodes, more internodes and / or smaller leaves compared to the plant comprising the wild type CsAmpl allele in homozygous form. Inducing mutations can be by e.g. random mutagenesis (e.g. chemical mutagenesis) or by targeted mutagenesis, e.g. targeted gene editing. Step b) may be e.g. a PCR based method or a sequencing-based method. Screening may also be for specific mutant alleles, such as e.g. the L41 IF mutant described herein.

[0135] Step a) can also be omitted, whereby a method is provided for identifying a mutant csampl allele in a cucumber plant, comprising: a) screening the cucumber plants for the presence of a mutant csampl allele, and optionally b) selecting a plant comprising a mutant csampl allele, and optionally c) phenotyping the plant comprising the mutant csampl allele in homozygous form and selecting a plant comprising a mutant csampl allele which causes the plant to produce shorter internodes, more internodes and / or smaller leaves compared to the plant comprising the wild type CsAmpl allele in homozygous form.

[0136] Also, a plant generated and / or identified and / or selected by a method described herein is encompassed herein.

[0137] The cultivated cucumber plant comprising a mutant csampl allele may be of any type, e.g. it may be of one of the following cucumber types: pickling cucumbers (e.g. American pickling, European pickling type), slicing cucumbers (e.g. American slicing), long cucumbers, short cucumbers, European greenhouse cucumbers, Beit- Alpha type cucumbers, oriental trellis type cucumbers, Asian cucumbers (e.g. selected from Indian Mottled cucumber, Chinese Long cucumber, Korean cucumber and Japanese cucumber type). In one aspect the cultivated cucumber is an inbred line or a Fl hybrid of a pickling cucumber type, slicing cucumber type, long cucumber type, short cucumber type, European greenhouse cucumbers, Beit-Alpha type cucumbers, oriental trellis type cucumbers, Chinese long cucumber type, Korean cucumber type or Japanese cucumber type. In a specific embodiment the cucumber is an inbred line or an Fl hybrid of a long cucumber, especially a European greenhouse cucumber, or a short cucumber.

[0138] The cultivated cucumber plant may be an inbred line, an OP (open pollinated variety) or an Fl hybrid. In one aspect the Fl hybrid comprises a mutant csampl allele preferably in homozygous form.

[0139] The cultivated cucumber plant preferably has good agronomic and good fruit quality characteristics. The cultivated cucumber plant is in one aspect uniform, both genetically and phenotypically. Especially fruit characteristics are uniform, e.g. regarding shape, skin color, skin thickness, skin ribs, skin toughness, spines (spine color, spine density, etc.), presence / absence of warts, length and diameter at edible and marketable maturity, flavour, etc. Likewise seed characteristics (i.e. characteristics of the seeds from which the plant is grown) are preferably uniform, e.g. seed size, seed color, etc.

[0140] Also, a seed is provided from which a plant or plant part as provided herein can be grown, i.e. comprising a mutant csampl allele on chromosome 3, preferably in homozygous form. In one aspect the plant or the seed is a cultivated cucumber plant or seed comprising the mutant allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution, preferably in homozygous form.

[0141] Further a cucumber fruit produced by a plant as encompassed herein is provided, wherein the fruit comprises the mutant csampl allele, preferably in homozygous form. In one aspect the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L411F substitution, preferably in homozygous form.

[0142] Likewise plant parts of a plant as described herein throughout the description are provided, wherein the plant part is a cell, a flower, a pistil, a leaf, a stem, a petiole, a cutting, a tissue, a seed coat, an ovule, pollen, a root, a rootstock, a scion, a fruit, a cotyledon, a hypocotyl, a protoplast, an embryo, an anther. The plant part comprises in its genome a mutant csampl allele as described elsewhere herein, preferably in homozygous form. In one aspect the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution, preferably in homozygous form. In one aspect the cell is a non-propagating or a non-regenerable cell. In one aspect the non-propagating or non-regenerable cell is part of a tissue or organ of the plant. In a different aspect the non-propagating or non-regenerable cell is in a cell culture or tissue culture.

[0143] Also, a cell culture or a tissue culture of cells or tissues comprising in its genome a mutant csampl allele as disclosed herein is encompassed herein. In one aspect the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L411F substitution, preferably in homozygous form.

[0144] Further a plant regenerated from such a cell or tissue culture is encompassed.

[0145] Further a vegetatively propagated plant propagated from a plant part according to the invention is provided.

[0146] Also, a food or feed product comprising cells or tissues or parts of a plant according to the invention are provided, such as parts of cucumber fruits, e.g. slices or pieces of cucumber fruits, such as salads. In one embodiment a method of cucumber fruit production is provided, said method comprising growing a plant comprising a mutant csampl allele as disclosed herein preferably in homozygous form, said method optionally comprising a reduced spacing of the plants in the greenhouse, and optionally harvesting the fruits produced by said plants.

[0147] Thus, in one embodiment, cucumber plants comprising a mutant csampl allele in homozygous form (and expressing the phenotype caused by the mutant allele) are grown at a stem density of more than 2.2 plants (or stems) per m2preferably at least 2.4, 2.5, 2.6. 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5 plants (or stems) per m2, or more; more preferably about 2.6 - 2.8 stems per m2, especially 3.0 to 4.0 or 3.0 to 4.5 plants (or stems) per m2. In one aspect at least about 20%, preferably at least about 22%, 23% or 24% more stems per m2can be used, in e.g. high-wire cultivation, compared to a cucumber plant which is homozygous for the wild type csampl allele.

[0148] The method comprises e.g. growing plants or seeds of a cultivated cucumber plant comprising a mutant CsAmpl allele in homozygous form (e.g. the L41 IF mutant allele) in a greenhouse in a high-wire system at a stem density of at least 2.9, 3.0, 3.5, 4.0 stems per square meter area, leading the stem up to the high wire, harvesting the fruits on the stem.

[0149] In one aspect the cucumber fruits are seedless. In one aspect the cucumber plant is of the European long cucumber type. Seedless means that no viable seeds develop in the fruits.

[0150] As mentioned previously, a mutant csampl allele can be induced, i.e. it can be generated by mutating the endogenous CsAmpl allele in cultivated cucumber seeds or plants or plant parts (or optionally in wild plants) and / or by selecting induced mutants e.g. tissue culture induced mutants or TILLING mutants.

[0151] Therefore, methods for producing and / or selecting plants having a mutant csampl allele, resulting in a mutant allele encoding a mutant protein having a decreased function (but not a loss-of-function) compared to the wild type protein are encompassed herein. To generate such mutant alleles conventional mutagenic agents, like chemicals or high energy radiation (e.g. x-rays, neutron radiation, gamma radiation or UV radiation) may be used. It is also possible to generate mutant alleles by means of biotechnology methods as described above (e.g. targeted gene editing technology).

[0152] In one aspect a method is provided for generating a cucumber plant comprising a mutant allele of a gene named CsAMPl, said gene encodes a CsAmpl protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, comprising inducing random or targeted mutations in a population of cucumber plants or seeds and thereby inducing random or targeted mutations in the endogenous CsAmpl allele, and selecting a plant from the mutant cucumber plants, or progeny thereof obtained by selfing, comprising a mutant csampl allele. Therefore, in one aspect a method is provided for generating a cucumber plant comprising a mutant csampl allele using e.g. a targeted genome editing technique, such as a Crispr system (e.g. Crispr / Cas9). In one aspect the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution or any other mutant allele disclosed herein.

[0153] In one aspect a method is provided for identifying a cucumber plant comprising a mutant allele of a gene named CsAMPl, said gene encodes a CsAmpl protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, comprising identifying and optionally selecting a plant in a mutant cucumber population, or progeny thereof obtained by selfing, comprising a mutant csampl allele. In one aspect the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution.

[0154] A ‘mutant cucumber population’ or ‘population of mutant cucumber plants’ refers in one aspect to a plurality of cucumber seeds or plants or plant parts which have been treated with a conventional mutagenic agents, like chemicals or high energy radiation (e.g. x-rays, neutron radiation, gamma radiation or UV radiation) or progeny thereof obtained by selfing, to ensure that mutations are in homozygous form. These can be plants or seeds or plant parts of a cultivated cucumber breeding line, variety, inbred line or any plurality of cultivated cucumber plants or seeds. Alternatively, these may be wild cucumber plants or wild relatives of cucumber.

[0155] Cucumber plants as provided herein, comprising shorter internodes and / or more internodes and / or smaller leaves, can be produced by introducing one or more mutations into an allele of a CsAmpl gene.

[0156] An embodiment, therefore, concerns a method for production of a cucumber plant comprising the steps of a) providing a population of mutant cucumber plants or seeds, b) optionally selecting a plant which comprising shorter internodes and / or more internodes and / or smaller leaves than a non-mutated plant, c) determining if a plant of the mutant population of a) or selected under b) has a mutation in an allele of a CsAmpl gene, optionally d) growing / cultivating the plants obtained under c).

[0157] In one aspect is a method for production of a cucumber plant comprising the steps of a) introducing mutations in a population of cucumber plants or seeds (and optionally selfing the plants), b) optionally selecting a plant which comprising shorter internodes and / or more internodes and / or smaller leaves than a non-mutated plant, c) determining if the plant selected under b) has a mutation in an allele of a CsAmpl gene and selecting a plant comprising such a mutation, and optionally d) growing / cultivating the plants obtained under c).

[0158] However, in one aspect the order of the steps can also be different, comprising: a) providing a population of mutant cucumber plants or seeds, b) determining if a plant of the mutant population of a) has a mutation in an allele of a CsAmpl gene, optionally c) selecting a plant comprising a mutation in an allele of a CsAmpl gene, and optionally d) selfing the plant of b) or c) to generate a plant comprising the mutant allele in homozygous form, and optionally e) determining if the plant of step c) or d) comprising shorter internodes and / or more internodes and / or smaller leaves than a non-mutated plant.

[0159] Or the steps may comprise: a) introducing mutations in a population of cucumber plants or seeds (and optionally selfing the plants), b) determining if a plant of a) has a mutation in an allele of a CsAmpl gene and optionally c) selecting a plant comprising such a mutation, and optionally d) selfing the plant of b) or c) to generate a plant comprising the mutant allele in homozygous form, and optionally e) determining if the plant of step c) or d) comprising shorter internodes and / or more internodes and / or smaller leaves than a non-mutated plant.

[0160] A non-mutated plant may be e.g. a control plant, such as a plant comprising wild type, functional CsAmpl allele in homozygous form.

[0161] In one aspect the plant selected in one of the above methods comprises the mutant allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution.

[0162] Optionally, the methods comprise selecting a plant comprising at least one copy of a mutant allele of a gene encoding a CsAmpl protein. The selected plants are also encompassed herein. In one aspect the plant selected comprises the mutant allele which encodes a L411 amino acid substitution, e.g. an L411F substitution. Chemical substances, which can be used to produce chemically induced mutations, and the mutations resulting from the effect of the corresponding mutagens are, for example described in Ehrenberg and Husain, 1981, (Mutation Research 86, 1-113), Muller, 1972 (Biologisches Zentralblatt 91 (1), 31-48). The production of rice mutants using gamma radiation, ethyl methane sulphonate (EMS), N-methyl-N- nitrosurea or sodium azide (NaNs) is described, for example, in Jauhar and Siddiq (1999, Indian Journal of Genetics, 59 (1), 23-28), in Rao (1977, Cytologica 42, 443-450), Gupta and Sharma (1990, Oryza 27, 217-219) and Satoh and Omura (1981, Japanese Journal of Breeding 31 (3), 316-326). The production of wheat mutants using NaNs or maleic hydrazide is described in Arora et al. (1992, Annals of Biology 8 (1), 65-69). An overview of the production of wheat mutants using different types of energy-rich radiation and chemical substances is presented in Scarascia-Mugnozza et al. (1993, Mutation Breeding Review 10, 1- 28). Svec et al. (1998, Cereal Research Communications 26 (4), 391-396) describes the use ofN-ethyl-N- nitrosurea for producing mutations in triticale. The use of MMS (methyl methane sulphonic acid) and gamma radiation for the production of millet mutants is described in Shashidhara et al. (1990, Journal of Maharashtra Agricultural Universities 15 (1), 20-23).

[0163] All these methods are basically suitable in the method for production of a plant according to the invention for producing mutant alleles in genes encoding a CsAmpl protein.

[0164] The plants generated and / or selected by these methods are also an embodiment herein. These plants can be used to make breeding lines and varieties comprising the mutant alleles.

[0165] Selecting plants having shorter internodes and / or more internodes and / or smaller leaves can be done visually and / or by taking measurements. As the phenotype is at least seen in homozygous condition, selfing of the plant or the population of mutagenized plants is preferred before phenotyping.

[0166] Determining the presence of a mutant allele of the CsAmpl gene (and selecting a plant comprising such a mutant allele) can be done with the help of methods known to the person skilled in the art. In particular, analyses based on hybridisations with probes (Southern Blot), amplification by means of polymerase chain reaction (PCR), sequencing of related genomic sequences and the search for individual nucleotide exchanges can be used for this purpose. Methods, which allow several plants to be investigated for mutations in certain genes in a short time, are particularly suitable. Such a method, so-called TILLING (Targeting Induced Local Lesions IN Genomes), has been described by McCallum et al. (2000, Plant Physiology 123, 439-442).

[0167] Other methods for identifying if a plant cell or plant part or a plant or a seed comprises a mutant allele of a CsAmpl gene comprise sequencing of the respective alleles and SNP marker analyses with methods common in the art and e.g. discussed in Thomson (2014, Plant Breeding and Biotechnology 2,195-212). Also analysis of CsAmpl mRNA / cDNA sequence can be done to determine whether the allele is a mutant allele or a wild type allele and which mutation is present in a mutant allele.

[0168] These methods are basically suitable for identifying plant cells and plants having a mutant allele of a CsAmpl gene.

[0169] In one aspect, a method for identifying and / or selecting a cucumber plant or plant part or cell comprising in its genome at least one copy of a mutant allele of the CsAmpl gene is provided, said method comprising determining whether the plant or plant part or cell comprises in its genome at least one mutant csampl allele (e.g. any mutant allele, such as mutant alleles described elsewhere herein, especially mutant alleles which in homozygous form result in shorter internodes and / or more internodes and / or smaller leaves as described).

[0170] In one aspect, a method for identifying and / or selecting a cucumber plant or plant part or cell comprising in its genome at least one copy of a mutant allele of the CsAmpl gene is provided, said method comprising determining whether the plant or plant part or cell comprises in its genome at least one mutant csampl allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution, or determining whether the plant or plant part or cell comprises in its genome one or two copies of a mutant csampl allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution,

[0171] As mentioned previously, determining the presence of a mutant csampl allele can be done phenotypically and / or using molecular methods, such as PCR based methods or hybridization-based methods or sequencing based methods. Such methods may comprise a step of nucleic acid isolation from plant material, especially genomic DNA.

[0172] The cucumber plant or plant part may be a cultivated plant or a wild cucumber plant or wild relative of cucumber.

[0173] This method may involve analysing (directly or indirectly) the genomic nucleotide sequence of the csampl allele, or the mRNA / cDNA nucleotide sequence of the csampl allele, or the protein sequence of the CsAmpl protein, e.g. to determine if the encoded protein comprises one or more amino acid replacements, insertions or deletions compared to the wild type CsAmpl protein, especially in the peptidase domain as described. One method for analysing the presence of a mutant csampl allele, is for example to assay the presence of a Single Nucleotide Polymorphism (SNP) between the genomic sequence of the mutant csampl allele and the wild type CsAmpl allele, by, for example, designing primers for the SNP and genotyping plants or plant parts for the genotype of that particular SNP. For example a KASP assay can be used for detecting a SNP and thereby the mutant allele.

[0174] For example, the SNP underlying the L411F replacement is found at nucleotide 3081 of SEQ ID NO: 3 and of SEQ ID NO: 6. In SEQ ID NO: 3 (wild type genomic sequence) a C (Cytosine) is present while in SEQ ID NO: 6 (mutant genomic sequence) a Thymine is present at nucleotide 3081. A SNP assay for this SNP (C / T SNP at nucleotide 3081 of SEQ ID NO: 3 and 6) can be used to detect the presence of the wild type or the mutant csampl allele. In the ChineseLongV2 genome the nucleotide of the SNP position is at nucleotide 30568657. See Table 3.

[0175] Thus, in one aspect a SNP assay (or SNP detection assay) is provided to detect whether e.g. a cucumber chromosome 3 comprises nucleotide C (Cytosine) or a T (Thymine) in the csampl allele in the codon for amino acid 411 at e.g. nucleotide 30568657 of chromosome 3 of the ChineseLong V2 genome or at the equivalent position in the genome. By this SNP, the codon CTT (encoding Leucine at amino acid 411 of the protein, L411) is changed to TTT (encoding Phenylalanine at amino acid 411 of the protein). See Table 3 herein.

[0176] For any other SNP in the genomic DNA, especially any SNP in a codon of e.g. the peptidase domain of the CsAMPl protein (e.g. any SNP of Table 3 or any other SNP that changes a codon), a SNP assay can easily be developed. The peptidase domain is shown in e.g. Table 3. It is encoded in the genomic DNA od SEQ ID NO: 3 starting at nucleotide 2474 in Exon 2 and ending at nucleotide 5159 of SEQ ID NO: 3 in Exon 7. In the reference genome Chinese Long V2 (cucurbitgenomics.org) this corresponds to nucleotides 30568048 to 30570735 of chromosome 3 of the ChineseLong V2 genome. Therefore, in one aspect a cucumber plant or plant part is provided herein which comprises a mutant csampl allele which comprises a mutation which changes at least one amino acid of the peptidase domain into another amino acid and a method for generating said mutant allele (e.g. by chemical mutagenesis or targeted gene editing) and / or a method for detecting said mutant allele and / or transferring the mutant allele into another cucumber plant.

[0177] Thus, for example, the G412E mutant has codon GGA (encoding Glycine) in the wild type genomic DNA of SEQ ID NO: 3 at nucleotide 3084 to 3086 of SEQ ID NO: 3 and in the mutant the codon is GAA (encoding Glutamine, E). Thus, in one aspect a SNP assay is provided to detect whether a cucumber chromosome 3 comprises nucleotide G (Guanine) at nucleotide 30568661 of chromosome 3 or an A (Adenine) at nucleotide 30568661 of chromosome 3. See Table 3 herein.

[0178] For example, the P428S mutant has codon CCA (encoding Proline, P) in the wild type genomic DNA of SEQ ID NO: 3 at nucleotide 3132 to 3134 of SEQ ID NO: 3 and in the mutant the codon is TCA (encoding Serine, S). Thus, in one aspect a SNP assay is provided to detect whether a cucumber chromosome 3 comprises nucleotide C (Cytosine) at nucleotide 30568708 of chromosome 3 or a T (Thymine) at nucleotide 30568708 of chromosome 3. See Table 3 herein.

[0179] For example, the G530R mutant has codon 5103 to 5105 (GGT encoding Glycine, G) in the wild type genomic DNA of SEQ ID NO: 3 and in the mutant the codon is CGT (encoding Arginine, R). Thus, in one aspect a SNP assay is provided to detect whether a cucumber chromosome 3 comprises nucleotide G (Guanine) at nucleotide 30570679 of chromosome 3 or a C (Cytosine) at nucleotide 30570679 of chromosome 3. See Table 3 herein.

[0180] Further, the L538F mutant has codon 5127 to 5129 (CTT encoding Leucine, L) in the wild type genomic DNA of SEQ ID NO: 3 and in the mutant the codon is TTT (encoding Phenylalanine, F). Thus, in one aspect a SNP assay is provided to detect whether a cucumber chromosome 3 comprises nucleotide C (Cytosine) at nucleotide 30570703 of chromosome 3 or a T (Thymine) at nucleotide 30570703 of chromosome 3. See Table 3 herein.

[0181] Thus, for any of the single amino acid substitutions in the CsAMPl protein (e.g. the ones mentioned in Table 1 and Table 3 and any others), especially in the peptidase domain of the protein, a SNP assay can easily be developed to discriminate between the wild type allele and the mutant allele being present on chromosome 3 at the CsAmpl gene locus.

[0182] Thus, primers can be designed for allele specific DNA amplification, e.g. distinguishing a wild type allele from a mutant allele. Such PCR primers can be designed based on the differences between the wild type and the mutant allele. So, if the mutant allele comprises a substitution of a nucleotide, e.g. in a codon, the primers can be designed to differentiate between the presence of the mutant allele and the wild type allele. The skilled person can easily develop a molecular assay to detect a mutant allele.

[0183] So, one aspect comprises a method for determining whether a cucumber plant, plant part or plant cell comprises one or more copies of a mutant csampl allele by a method selected from analysing one or more nucleotides of the genomic csampl allele, analysing the mRNA (or cDNA) expressed by the csampl allele or analysing the CsAmpl protein amino acid sequence. Thus, a method for determining whether a cucumber plant or plant part comprises at least one copy of a mutant allele of a gene named CsAmpl is provided, said gene encodes a CsAmpl protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, said method comprising analysing the CsAmpl DNA, RNA or protein of the plant or plant part.

[0184] In one aspect the protein which comprises at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 comprises a peptidase domain which is 100% identical to the peptidase domain of SEQ ID NO: 1. See also Examples and Table 3.

[0185] In one aspect, especially in respect of the European Patent Convention, the cucumber plant is “not obtained exclusively by an essentially biological process”, or in one aspect the mutant csampl 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 cucumber plant comprising a mutant allele (e.g. a commercial variety of the applicant) to cross the mutant allele into a different background of cucumber 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.

[0186] The plants which comprise a mutant csampl allele, and thereby comprise a modified architecture, such as shorter internodes and / or more internodes and / or smaller leaves, can be grown in methods for fruit production, whereby in one aspect a higher density of plants (i.e. stems) is grown per area.

[0187] In one aspect a plant or plant part of the species Cucumis sativus is provided comprising at least one copy of a mutant allele of a gene named CsAmpl, said wild type gene encodes a CsAmpl protein of SEQ ID NO: 1 or a wild type protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, wherein said mutant allele encodes a protein having a decreased function compared to the wild type CsAmpl protein. In one aspect the wild type protein which comprises at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 also comprises a peptidase domain which is 100% identical to the peptidase domain of SEQ ID NO: 1, i.e. the variation of the wild type variant protein is outside of the peptidase domain.

[0188] The plant or plant part according to the above embodiment, wherein said mutant allele confers shorter internode length and / or more internodes and / or smaller leaves when the mutant allele is in homozygous form compared to a plant or plant part homozygous for the wild type allele of the CsAmpl allele. The plant or plant part according to any one of the embodiments described herein, wherein said protein comprising one or more amino acids replaced and / or inserted and / or deleted compared to the CsAmp 1 wild type protein, especially one or more amino acids replaced and / or inserted and / or deleted in the conserved Peptidase Domain at amino acid 322 to 548 of SEQ ID NO: 1.

[0189] The plant or plant part according to any one of the embodiments described herein, wherein said plant or plant part is a cucumber plant or plant part and wherein said mutant allele is an induced allele or a natural allele.

[0190] The plant or plant part according to any one of the embodiments herein is in one aspect a cucumber plant or plant part and wherein said mutant allele is not a loss-of-function allele.

[0191] The plant or plant part according to any one of the embodiments herein is preferably homozygous for the mutant allele.

[0192] Also provided is a seed from which a plant or plant part according to any of the embodiments described herein can be grown.

[0193] Further provided is a fruit produced by a plant according to any one of the embodiments described herein, wherein the fruit comprises the mutant csampl allele preferably in homozygous form. The fruit is preferably seedless.

[0194] The plant part according to any one of the embodiments described herein is e.g. a cell, a flower, a pistil, a leaf, a stem, a petiole, a cutting, a tissue, a seed coat, an ovule, pollen, a root, a rootstock, a scion, a fruit, a cotyledon, a hypocotyl, a protoplast, an embryo, an anther.

[0195] Also provided is a vegetatively propagated plant propagated from a plant part according to e.g. the above embodiment.

[0196] In one aspect a method of cucumber fruit production is provided, said method comprises growing a plant according to any one of the embodiments comprising the mutant csampl allele preferably in homozygous form, said method optionally comprises a reduced plant density during cultivation and optionally harvesting the fruits produced by said plants from e.g. the main stem

[0197] The method encompasses harvesting a significantly higher fruit yield of fruits comprising the mutant allele in homozygous form compared to plants comprising the wild type allele in homozygous form. Thus, the method comprises growing the mutant plants at a higher density and / or harvesting a higher number and higher fruit yield of harvest mature fruits from the plants comprising the mutant csampl allele in homozygous form.

[0198] A method for generating and / or identifying a cucumber plant comprising a mutant allele of a gene named CsAmplis provided, said gene encodes a CsAmpl protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, comprising identifying or selecting a plant in a mutant cucumber population, or progeny thereof obtained by selfing, comprising a mutant csampl allele, or generating a cucumber plant comprising a mutant csampl allele using a genome editing technique. In one aspect the mutant allele generated comprises one or more amino acids replaced and / or inserted and / or deleted with respect to the wild type protein, preferably one or more amino acids replaced, inserted and / or deleted in the peptidase domain, preferably any of the mutant alleles are generated and / or identified as described elsewhere herein, e.g. the allele where L411 of SEQ ID NO: 1 or the equivalent amino acid in a variant sequence is replaced by a different amino acid, preferably by Phenylalanine (F).

[0199] Thus, in one aspect, a method for generating a cucumber plant comprising a mutant allele of a gene named CsAmpl is provided, wherein the wild type CsAmpl gene is a gene on chromosome 3 that encodes a CsAmpl protein comprising at least 98% or 99% sequence identity to SEQ ID NO: 1, said method comprising using a targeted genome editing technique (such as Crispr-based editing) or random techniques mutagenesis (such as radiation or chemical mutagenesis) to generate a mutant allele of the CsAmpl gene. The method may further comprising identifying or selecting a plant comprising a mutant csampl allele. The method may also further comprise analysing the phenotype of a plant comprising the mutant allele in homozygous form.

[0200] In the method the mutant allele generated may in one aspect encode a CsAmpl protein comprising a substitution of Leucine 411 of SEQ ID NO: 1, or the equivalent Leucine in a CsAmpl protein comprising at least 98% or 99% sequence identity to SEQ ID NO: 1, by another amino acid, preferably by Phenylalanine. Or in the method any other mutant allele may be generated as described elsewhere herein.

[0201] Furthermore, a method is provided for determining whether a cucumber plant or plant part comprises at least one copy of a mutant allele of a gene named CsAmpl, said gene encodes a CsAmpl protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, said method comprising analysing the CsAmpl DNA, RNA or protein of the plant or plant part.

[0202] In the method for determining whether a cucumber plant or plant part (e.g. a leaf, fruit, fruit part, etc.) comprises at least one copy of a mutant allele of the CsAmpl gene the mutant allele detected may in one aspect encode a CsAmpl protein comprising a substitution of Leucine 411 of SEQ ID NO: 1, or the equivalent Leucine in a CsAmpl protein comprising at least 99% sequence identity to SEQ ID NO: 1, by another amino acid, preferably by Phenylalanine. Or in the method any other mutant allele may be detected as described elsewhere herein.

[0203] In order to determine whether a plant or plant part comprises at least one copy of a mutant csampl allele in its genome at least one of the following methods may be used: a PCR - assay, a SNP -genotyping assay such as a KASP-assay or a TaqMan-assay, a High Resolution Melting (HRM) assay, or DNA sequencing. The assay may be designed for detecting the specific mutant allele (so the assay may be allele-specific) and may also differentiate between the presence of one copy or two copies of the mutant allele.

[0204] The cucumber plant or plant part (or seed from which such a plant is grown) may, in one aspect, be subjected to a mutation-inducing step prior to determining whether the cucumber plant or plant part comprises a mutant allele of the CsAmpl gene. Such a mutation inducing step may comprise e.g. chemical mutagenesis, such as the use of ethyl methanesulfonate (EMS) as mutagenic agent, or radiation mutagaenesis, such as e.g. UV- or gamma radiation.

[0205] Further a method for producing a cucumber plant comprising a mutant allele which confers shorter internodes and / or more internodes and / or smaller leaves when present in homozygous form is provided, said method comprising the step(s) of:

[0206] (i) crossing a first cucumber plant and a second cucumber plant, wherein the first cucumber plant comprises in its genome at least one copy of a mutant allele of the CsAmpl gene as described elsewhere herein,

[0207] (ii) optionally harvesting seeds from the crossing of (i) and

[0208] (iii) optionally selecting seeds comprising said mutant allele in its genome.

[0209] In one aspect the first cucumber plant is homozygous for the mutant allele and, therefore, has the desired phenotype.

[0210] As the phenotype is only seen when the mutant allele is in homozygous form the method may further comprise one or more selfing steps of progeny of the cross to ensure homozygosity. So the seeds selected under iii) may for example be grown into plants and selfed one or more times. Further methods of producing or of identifying and / or selecting a plant, seed or plant part

[0211] Further provided are methods wherein a Cucumis sativus plant, as described herein, comprising at least one copy of a mutant allele of the CsAmpl gene, is used and / or obtained.

[0212] In one aspect, a method for identifying and / or selecting a Cucumis sativus plant or plant part or seed is provided, comprising determining whether said Cucumis sativus plant or plant part or seed comprises in its genome at least one copy of a mutant allele of the CsAmpl gene as described herein, wherein the mutant allele encodes a mutant protein comprising e.g. at least one amino acid substitution, preferably in the peptidase domain, compared to the wild type CsAmpl protein (resulting in reduced function protein as described).

[0213] The method may comprise steps like obtaining genomic DNA from the plant or plant part or seed or seed part, generating a PCR product or a nucleic acid hybridization product for a specific mutant CsAmpl - allele as described herein or generating sequence information of the mutant CsAmpl -allele, optionally selecting a plant, plant part or seed comprising a specific mutant CsAmpl -allele as described herein and as detected in the previous step.

[0214] Herein the wild type allele is encoded by nucleic acid molecules selected from the group consisting of: a) nucleic acid molecules, which encode a protein with the amino acid sequence given under SEQ ID NO: 1; b) nucleic acid molecules, which encode a protein, the sequence of which has an identity of at least 95%, 96%, 97%, 98% or 99% with the amino acid sequence given under SEQ ID NO: 1; c) nucleic acid molecules, which comprise the nucleotide sequence shown under SEQ ID NO: 2 or 3 or a complimentary sequence of either of these; d) nucleic acid molecules, which have an identity of at least 95%, 96%, 97%, 98% or 99% with the nucleic acid sequences described under c); e) nucleic acid molecules, which hybridize with at least one strand of the nucleic acid molecules described under a), b), c), or d) under stringent conditions of at least one wash in 0.2X SSC at a temperature of at least 50°C for 20 min; and f) nucleic acid molecules, the nucleotide sequence of which deviates from the sequence of the nucleic acid molecules identified under a) or b) due to the degeneration of the genetic code.

[0215] In one aspect a method is provided for identifying and / or selecting a cucumber plant or plant part or seed comprising in its genome at least one copy of a mutant allele of the CsAmpl gene, wherein the mutant allele encodes a mutant protein wherein at least one or more amino acids of the wild type protein are replaced and / or inserted and / or deleted by another amino acid, especially in the peptidase domain, e.g. amino acid L411 is replaced by a different amino acid, said method comprising analysing whether the genomic DNA of said cucumber plant or plant part or seed comprises the mutant allele.

[0216] The method above may comprise steps like obtaining genomic DNA from the plant or plant part or seed or seed part, generating a PCR product or a nucleic acid hybridization product for a specific mutant CsAmpl - allele as described herein or generating sequence information of the mutant CsAmpl -allele, optionally selecting a plant, plant part or seed comprising a specific mutant CsAmpl -allele as described herein and as detected in the previous step.

[0217] The presence of any mutant allele described herein can be detected using known methods or any of the above methods or any methods described throughout the description.

[0218] So for example a method for identifying and / or selecting a cucumber (Cucumis sativus) plant or plant part or seed is provided comprising determining whether said plant or plant part or seed comprises in its genome at least one copy of a mutant allele of the CsAmpl gene as described herein, e.g. the mutant allele the codon for Leucine at amino acid 411 of SEQ ID NO: 1 (amino acid L411), or the equivalent amino acid in a wild type CsAmpl protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, is changed into a different amino acid. The same applies for other mutant alleles described herein.

[0219] Preferably, the mutant allele of the CsAmpl gene as comprised in the Cucumis sativus plant or plant part or seed as used in the identification and / or selection method results in e.g. at least shorter internodes and more internodes of the Cucumis sativus plant when the mutant allele is present in homozygous form compared to the wild type / control plant.

[0220] 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. 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 or plant part or cell or seed or seed part 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 70 base pairs upstream and 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, PlantBiotechnology J. 9, 1086-1099, especially p097-1098 for KASP- assay method. Examples are e.g. forward and reverse primers, which are allele specific and can be used in the detection or genotyping of the mutant allele, e.g. encoding the L41 IF mutation, or any mutant allele described herein. Obviously, various different primers can be designed for detection of the same or different mutations in the genomic DNA. E.g. the primers may be slightly different, e.g. longer or shorter or degenerate with respect of the template DNA. The primers may be designed based on the forward strand or based on the reverse (complementary) strand of the genomic sequence.

[0221] 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.

[0222] Apart from in-gene markers (like the SNP markers in the CsAmpl -allele provided or described herein), also other molecular markers may be used to aid in the identification of the plants (or plant parts or nucleic acids obtained therefrom) containing a mutant CsAmpl allele. For example, one can develop one or more suitable molecular markers which are closely genetically (and preferably also physically) linked to the mutant CsAmpl allele.

[0223] In one aspect, the method for identifying and / or selecting a Cucumis sativus plant or plant part further comprises a step wherein the plant or plant part is subjected to a mutation inducing step prior to determining whether the plant or plant part comprises a mutant allele of the CsAmpl gene.

[0224] A mutagenizing step may involve the use of ethyl methanesulfonate (EMS) as mutagenic agent. Preferably, the seed, plant or plant part is subjected to a mutation inducing step prior to determining whether the seed, plant or plant part comprises a mutant allele of the CsAmpl gene. In one aspect ethyl methanesulfonate (EMS) is used as mutagenic agent.

[0225] Accordingly, said mutation inducing step may comprise contacting said seed, plant or plant part with a mutagen. Preferably, the seed or plant or plant part that is contacted with the mutagen comprises a wild type CsAmpl allele in homozygous form.

[0226] Said mutation inducing step may alternatively involve targeted mutagenesis techniques that depend on e.g. the site-specific induction of a double strand break in the genomic DNA of a host plant cell. Inducing such a double strand break may comprise contacting a plant or plant part (e.g. a plant cell) with an engineered nuclease upon which said double strand break may be repaired by the cell’s endogenous DNA double stranded break repair mechanisms (e.g. the homology directed repair mechanism), which allows a site-specific deletion or inversion of DNA in a target cell. Engineered nucleases useful in genome editing methods include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases. Genome editing methods particularly useful in the context of the present invention include, but are not limited to, CRISPR / Cas9 -based targeted mutagenesis methods and CRISPR / Casl2 (or a subtype of Casl2 such as Casl2a, also known as CRISPR / Cpfl)-based targeted mutagenesis methods; see e.g. Brooks et al. (2014) Plant Physiol 166, 1292-1297 and W02016 / 205711 Al. See also e.g. Front. Plant Sci., 02 November 2020, Sec. Plant Biotechnology, Volume 11 - 2020, / / doi.org / 10.3389 / fpls.2020.584151 for Crispr based systems for plant gene editing, especially for using Crispr-Casl2a as editing system.

[0227] The mutation-inducing step subsequently causes a mutation in the CsAmpl allele to provide a mutant CsAmpl allele that results in the mutant phenotype (shorter internodes and / or more internodes and / or smaller leaves). The specific mutant alleles provided or described herein can be reproduced or generated de novo by any of the above mutagenesis techniques.

[0228] Accordingly provided is a method of producing a Cucumis sativus plant comprising the steps of:

[0229] (a) obtaining or providing seeds or plant material of a Cucumis sativus plant;

[0230] (b) treating said seed or plant material with a mutagen to create mutagenized seed or plant material;

[0231] (c) analyzing said mutagenized seed or plant material to identify a plant having at least one mutation in the CsAmpl gene as defined herein, wherein the wild type CsAmpl gene encodes a protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 1, and wherein the mutant allele is a mutant allele as described anywhere herein, e.g. comprising a mutation in the transcribed region or coding region whereby the mutant protein comprises one or more amino acids inserted and / or deleted and / or replaced compared to the wild type protein.

[0232] In one aspect a method of producing a Cucumis sativus plant is provided comprising the steps of:

[0233] (a) obtaining or providing seeds or plant material of a Cucumis sativus plant;

[0234] (b) treating said seed or plant material with a mutagen to create mutagenized seed or plant material;

[0235] (c) analyzing said mutagenized seed or plant material to identify a plant having at least one mutation in the CsAmpl gene as defined herein, wherein the wild type CsAmpl gene encodes a protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 1, and wherein the mutant allele which encodes a mutant protein comprising an amino acid replacement and / or insertion and / or deletion, preferably in the peptidase domain, e.g. amino acid L411 of SEQ ID NO: 1 or G412 of SEQ ID NO: 1 or G530 of SEQ ID NO: 1 (or the equivalent amino acid in a sequence comprising at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 1) is replaced by a different amino acid.

[0236] Also TILLING (Targeting Induced Local Lesions IN Genomes) is a general reverse genetics technique that uses traditional chemical mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high throughput screens for the discovery of mutations. TILLING combines chemical mutagenesis with mutation screens of pooled PCR products, resulting in the isolation of missense and non-sense mutant alleles of the targeted genes. Thus, TILLING uses traditional chemical mutagenesis (e.g. EMS or MNU mutagenesis or mutagenesis by generating reactive oxygen species) or other mutagenesis methods (e.g. by radiation mutagenesis using e.g. UV radiation or ion beam radiation) followed by high-throughput screening for mutations in specific target genes, such as the CsAmpl gene.

[0237] SI nucleases, such as CEL1 or ENDO1, are used to cleave heteroduplexes of mutant and wildtype target DNA and detection of cleavage products using e.g. electrophoresis such as a LI-COR gel analyzer system, see e.g. Henikoff et al. Plant Physiology 2004, 135: 630-636. TILLING has been applied in many plant species, including Lactuca sativa plants, tomato, rice (Till et al. 2007, BMC Plant Biol 7: 19), Arabidopsis (Till et al. 2006, Methods Mol Biol 323: 127-35), Brassica, maize (Till et al. 2004, BMC Plant Biol 4: 12), etc. Also EcoTILLING, whereby mutants in natural populations are detected, has been widely used, see Till et al. 2006 (Nat Protoc 1: 2465-77) and Comai et al. 2004 (Plant J 37: 778-86).

[0238] In one embodiment (cDNA or genomic) nucleic acid sequences encoding such mutant CsAmp 1 protein comprise one or more non-sense and / or missense mutations, e.g. transitions (replacement of purine with another purine (A «-> G) or pyrimidine with another pyrimidine (C «-> T) or transversions (replacement of purine with pyrimidine, or vice versa (C / T «-> A / G).

[0239] In one embodiment a CsAmpl gene nucleotide sequence comprising one or more non-sense and / or missense mutations in one of the exon- encoding sequence are provided, as well as a plant comprising such a mutant allele resulting in a plant capable of producing more and / or shorter internodes and / or smaller leaves when said mutant allele is present in homozygous form.

[0240] In one aspect, accordingly, the plant or plant part is identified and / or selected from a TILLING population that was obtained by subjecting seeds, plants or plant parts to a mutagen as described in further detail herein below. Thus, in one aspect a method for producing a Cucumis sativus plant is provided comprising the steps of:

[0241] (a) providing a TILLING population of a Cucumis sativus plant species,

[0242] (b) screening said TILLING population for mutants in the CsAmpl gene as described herein, and

[0243] (c) selecting from the mutant plants of (b) those plants (or progeny of those plants) which comprise in their genome at least one copy of a mutant allele of the CsAmpl gene, and wherein the mutant allele is a mutant allele as described anywhere herein, e.g. comprising a mutation in the coding region or transcribed region, whereby the mutant protein comprises one or more amino acids inserted and / or deleted and / or replaced compared to the wild type protein (especially in the peptidase domain).

[0244] Therefore, in one aspect a method for producing a Cucumis sativus plant is provided comprising the steps of:

[0245] (a) providing a TILLING population of a Cucumis sativus plant species,

[0246] (b) screening said TILLING population for mutants in the CsAmpl gene as described herein, and

[0247] (c) selecting from the mutant plants of (b) those plants (or progeny of those plants) which comprise in their genome at least one copy of a mutant allele of the CsAmpl gene, wherein said mutant allele encodes a mutant protein comprising an amino acid replacement and / or insertion and / or deletion, preferably in the peptidase domain, e.g. amino acid L411 of SEQ ID NO: 1 or G412 of SEQ ID NO: 1 or G530 of SEQ ID NO: 1 (or the equivalent amino acid in a sequence comprising at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 1) is replaced by a different amino acid.

[0248] Mutant plants (Ml) are preferably selfed one or more times to generate for example M2 populations or preferably M3 or M4 populations for phenotyping. In M2 populations the mutant allele is present in a ratio of 1 (homozygous for mutant allele) : 2 (heterozygous for mutant allele) : 1 (homozygous for wild type allele).

[0249] In one aspect a method for producing a Cucumis sativus plant comprising a mutant allele which causes the plant to develop significantly shorter but significantly more internodes when the mutant allele is present in homozygous form is provided, said method comprising the step(s) of: a) crossing a first Cucumis sativus plant and a second Cucumis sativus plant, wherein the first Cucumis sativus plant comprises in its genome at least one copy of a mutant allele of the CsAmpl gene, and wherein the mutant allele is a mutant allele as described anywhere herein, e.g. comprising a mutation in the coding region or transcribed region, whereby the mutant protein comprises one or more amino acids inserted and / or deleted and / or replaced compared to the wild type protein and optionally b) harvesting seed from said cross of step a) and selecting seed comprising said mutant allele. Therefore, in one aspect, a method for producing a Cucumis sativus plant comprising a mutant allele which causes the plant to produce more internodes and / or shorter internodes and / or smaller leaves when the mutant allele is present in homozygous form is provided, said method comprising the step(s) of: a) crossing a first Cucumis sativus plant and a second Cucumis sativus plant, wherein the first Cucumis sativus plant comprises in its genome at least one copy of a mutant allele of the CsAmpl gene, wherein said mutant allele encodes a mutant protein comprising an amino acid replacement and / or insertion and / or deletion, preferably in the peptidase domain, e.g. amino acid L411 of SEQ ID NO: 1 or G412 of SEQ ID NO: 1 or G530 of SEQ ID NO: 1 (or the equivalent amino acid in a sequence comprising at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 1) is replaced by a different amino acid.

[0250] In one aspect a method for producing a Cucumis sativus plant comprising a mutant allele which causes the plant to develop more internodes and / or shorter internodes and / or smaller leaves when the mutant allele is present in homozygous form is provided, said method comprising the step(s) of: i) crossing a first Cucumis sativus plant and a second Cucumis sativus plant, wherein the first Cucumis sativus plant comprises in its genome at least one copy of a mutant allele of the CsAmp 1 gene, wherein the mutant allele encodes a mutant protein wherein the L amino acid residue at position 411 of SEQ ID NO: 1 (amino acid L411) or the L at the equivalent position in a variant comprising at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 is replaced by a different amino acid residue, ii) optionally harvesting seed from the crossing of (i) and selecting seed comprising said mutant allele in its genome.

[0251] Likewise, the above method is provided wherein the mutant allele under step i) encodes a mutant protein as described elsewhere herein.

[0252] More preferably, both the first Cucumis sativus plant and the second Cucumis sativus plant in any one of the methods above of producing the Cucumis sativus plant are plants comprises a mutant allele of the CsAmpl gene as described herein in their genome.

[0253] In one aspect also a plant grown from seeds, or a seed, obtained e.g. by any of the methods of identifying and / or selecting a Cucumis sativus plant or plant part is provided, wherein said plant or seed comprises a mutant allele of the CsAmpl gene as described herein.

[0254] Further provided is a method for the growing of a Cucumis sativus plant or producing harvested plant material (e.g. fruits) of said plant by growing a seed or plant, wherein said seed or plant is homozygous for the mutant allele. In one aspect growing comprises growing in a greenhouse or glasshouse whereby the main stem is lead up to a high wire at a height of e.g. at least 150 cm, 160 cm, 170 cm, 175 cm, 180 cm, 190 cm or 200 cm. In one aspect the fruits are harvested from the main stem.

[0255] To compare the phenotypes the seeds or plants comprising the mutant CsAmpl allele in homozygous form are grown under the same conditions as a plant comprising the wild type CsAmpl allele in homozygous form. The number of internodes on the main stem and / or the average length of internodes on the main stem e.g. over a defined stem length, such as at least 1 meter, 1.5 meters, 1.75 meters, 1.8 meters, 1.9 meters or 2.0 meters can be measured on both the mutant and wild type control plants. Also leaf size can be measured, fruit size and / or fruit weight per plant, etc. In this way the phenotype of any mutant allele of the CsAmpl gene can be determined and a mutant allele (or a plant or seed comprising a mutant allele) can be selected for further use in breeding or to generate commercial varieties having the phenotype. Mutant alleles of the CsAmpl gene (or a plant or seed comprising such mutant alleles) which do not result in the desired phenotype (shorter internodes and / or more internodes and / or smaller leaves), or which result in the wild type phenotype can be discarded. It is an undue burden for the skilled person to generate and / or select mutant CsAmpl alleles which result in the desired phenotype as described herein, e.g. for the L41 IF mutant allele. It is, therefore, also an undue burden to generate and / or select a mutant allele which has a reduced function in vivo, as only alleles with a reduced function will show the phenotype of shorter internodes and more internodes and smaller leaf size. Loss-of-fimction mutants are deleterious and mutants that do not reduce function of the CsAmpl protein result in a wild type phenotype.

[0256] In one aspect the seeds or plants are grown in a greenhouse or glasshouse or other controlled environment (e.g. tunnels). The plants are, in one aspect, suitable for so-called high-tech cultivation in high-tech glasshouses, either for the traditional cucumber cultivation system (e.g. umbrella cultivation) or the high- wire cultivation system. As the leaves are smaller the plants may, thus, be grown ‘vertically’ up to a wire and a higher plant density can be reached used compared to the wild type plants with larger leaves, especially in high-wire cultivation. For example, 3 to 4 stems per square meter, or even more (e.g. 4.4, 4.5, 4.6 stems per square meter) can be grown.

[0257] In one aspect the Cucumis sativus seed or plant comprising the mutant CsAmpl allele in homozygous form is of the long cucumber type, but the mutant allele may also be used in other types, such as slicers, short cucumber types, mini-types, etc. or in field grown types (horizontally grown), such as gherkins.

[0258] In one aspect a genotyping assay is provided for genotyping cucumber plants, seeds, plant parts, cells or tissues, comprising the steps: a) providing genomic DNA of one or more cucumber plants or a population of plants or seeds, and b) carrying out a genotyping assay which detects the presence of the wild type allele of SEQ ID NO: 3 (or the complement strand thereof) or a wild type allele comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 and / or the presence of a mutant allele, wherein the mutant allele comprises one or more nucleotides inserted, deleted and / or replaced with respect of SEQ ID NO: 3 or with respect of the wild type allele comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3, resulting in said mutant allele encoding a mutant protein as described elsewhere herein, 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.

[0259] Step c) may also be selecting a plant, seed, plant part, cell or tissue comprising at least one copy of a mutant allele.

[0260] In one aspect the assay distinguishes between the presence of SEQ ID NO: 3 and SEQ ID NO: 6 in the genome.

[0261] In one aspect, a genotyping assay genotyping cucumber plants, plant parts, cells or tissues, comprising the steps is provided, comprising the steps: a) providing genomic DNA of one or more cucumber plants or a population of plants or seeds (e.g. breeding population, F2 population, backcross population etc.), 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 comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1) and / or the presence of a mutant allele, wherein the mutant allele encodes a protein which comprises one or more amino acids inserted and / or deleted and / or replaced with respect of SEQ ID NO: 1 (or with respect of a wild type allele comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1), especially as described elsewhere herein, and optionally c) selecting a plant, seed, plant part, cell or tissue comprising 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.

[0262] Step c) may also be selecting a plant, seed, plant part, cell or tissue comprising at least one copy of a mutant allele.

[0263] In one aspect the assay detects the allele encoding SEQ ID NO: 4. 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 primers and / or allele-specific probes.

[0264] In one aspect the genotyping assay in step b) discriminates between the wild type CsAmpl -allele, encoding a protein of SEQ ID NO: 1 or a wild type protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, and one of the mutant alleles provided or described elsewhere herein, e.g. a mutant allele with one or more amino acids inserted and / or deleted and / or replaced in the peptidase domain.

[0265] Various genotyping assays can be used, as long as they can detect INDELs and SNPs and can differentiate between e.g. the wild type allele of SEQ ID NO: 3 (or a wild type allele comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3) being present in the genomic DNA (at the CsAmpl locus) or a mutant allele of the CsAmpl gene being present in the genomic DNA, such as e.g. SEQ ID NO: 6. Genotyping assays may also discriminate between different mutant alleles.

[0266] 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 BHQPlus probes (see also biosearchtech.com) .

[0267] 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 TaqMan- 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.

[0268] 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.

[0269] 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 herein 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 labelled 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.

[0270] Various genotyping assays can, therefore, be used, which can differentiate between the presence of e.g. the wild type allele of the CsAmpl gene, encoding the protein of SEQ ID NO: 1 (or a wild type protein comprising at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1), or a mutant allele of the CsAmpl gene; or between different mutant alleles of the CsAmpl gene. Various mutant alleles of the CsAmpl gene can be detected. So, not only the mutant allele encoding the protein comprising an amino acid substitution in e.g. the peptidase domain, such as the L41 IF mutant, but the assay can be designed to detect any other mutant allele of the CsAmpl gene, e.g. any mutant allele described herein.

[0271] 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-genotyping-assays) or any other bi-allelic genotyping assay.

[0272] 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, 21, or 22 nucleotides complementary to SEQ ID NO: 3 or complementary to a wild type sequence comprising at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 3 (or the complement strand of any of these). 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 the SNP or INDEL which differentiates e.g. the wild type sequence from the mutant sequence of the allele. The two forward primers thereby have different binding specificity (or preference) to either the wild type allele or to the mutant allele. A KASP-assay can easily be designed to differentiate between the wild type allele of SEQ ID NO: 3 (or a wild type sequence comprising at least 95% sequence identity to SEQ ID NO: 3) and any mutant allele of the CsAmpl gene (such as SEQ ID NO: 6) which differs from the wild type allele in one or more nucleotides being inserted and / or deleted and / or replaced, so e.g. the assay can be designed for any SNP or INDEL that differentiates two alleles.

[0273] For example, the amino acid change L41 IF is due to codon at nucleotides 3081 to 3083 of SEQ ID NO: 3 being mutated from CTT to TTT. The mutated nucleotide is thus nucleotide 3081 of SEQ ID NO: 3, see Table 3 herein. The two forward KASP primers can then comprise a stretch of nucleotides complementary to the sequence preceding the mutated nucleotide plus either the wild type nucleotide (C) or the mutant nucleotide (T). Together with the reverse common primer, they amplify either the wild type allele or the mutant allele in the KASP assay. The genotype is, thereby, determined for the SNP, either being homozygous wild type, homozygous mutant or heterozygous for mutant and wild type.

[0274] Thus, in one aspect the SNP (C / T) at nucleotide 3081 of SEQ ID NO: 3 is detected using a genotyping assay, such as a KASP assay, e.g. using two forward primers and a reverse primer. The same applies for the other mutant alleles of Table 3 or any other mutant allele.

[0275] In one aspect the mutant allele of the CsAmpl gene encodes a protein comprising one or more amino acids inserted and / or replaced and / or deleted with respect of the wild type protein of SEQ ID NO: 1 (or a wild type protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1).

[0276] In one aspect the mutant allele of the CsAmpl gene encodes a protein which comprises one or more amino acids replaced and / or inserted and / or deleted in comparison to the protein of SEQ ID NO: 1, e.g. at least 1, 2, 3 or more amino acids are replaced by one or more different amino acids (or are inserted or deleted), especially one or more amino acids of the peptidase domain as described elsewhere herein.

[0277] In one aspect the mutant allele comprises a missense mutation in the wild type allele of SEQ ID NO: 3, especially in the exons encoding the peptidase domain. Therefore, in one embodiment a method is provided for detecting, and optionally selecting, a cucumber plant, seed or plant part comprising at least one copy of a wild type allele and / or of a mutant allele of a gene name CsAmpl gene, comprising: a) providing genomic DNA of a cucumber plant or of a plurality of plants (e.g. a breeding population, F2, backcross, etc.) or seeds or plant parts, b) carrying out an assay (e.g. abi-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 CsAmp 1 gene and / or a mutant allele of the CsAmp 1 gene, wherein the wild type allele comprises the sequence of SEQ ID NO: 3 (or wherein the wild type allele encodes the protein of SEQ ID NO: 1 or a variant comprising at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1) and the mutant allele comprises one or more nucleotides inserted and / or deleted and / or replaced with respect to the sequence of SEQ ID NO: 3 (or the mutant allele encodes a protein comprising one or more amino acids inserted and / or deleted and / or replaced with respect to the wild type protein of SEQ ID NO: 1 or a variant comprising at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1), and optionally c) selecting a plant, seed or plant part comprising one or two copies of the mutant allele.

[0278] The mutant allele detected and optionally selected in any of the above methods is preferably any of the mutant alleles described herein, e.g. resulting in more internodes and / or shorter internodes and / or smaller leaves when in homozygous form, as e.g. the L41 IF mutant allele.

[0279] Under step b) the genotyping assay discriminates between the wild type and the 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.

[0280] In one aspect, in any of the above methods the assay uses one or more CsAmpl allele-specific primers or one or more CsAmpl allele-specific probes. As mentioned, based on the genomic sequence of SEQ ID NO: 3 or other (e.g. degenerate) genomic sequences which encode the protein of SEQ ID NO: 1 or a variant thereof (e.g. a genomic sequence comprising at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 3) or the genomic sequence of a mutant allele which encodes e.g. a protein comprising one or more amino acids inserted and / or deleted and / 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 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the target sequence. The primer or probe specificity to a wild type allele or a mutant allele 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 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.

[0281] In one aspect, the mutant allele comprises at least one codon inserted 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.

[0282] In any of the methods above, in one aspect the mutant allele comprises a nucleotide replaced in codon 3081 to 3083 of SEQ ID NO:3 (e.g. nucleotide 3081 is replaced, e.g. from Cytosine to Thymine), leading to the codon for L411 being a different codon. In any of the methods above, in one aspect the mutant allele comprises a nucleotide replaced in codon of SEQ ID NO:3 mentioned e.g. in Table 3 or any other codon, e.g. any codon of the peptidase domain, which is encoded by the genomic sequence starting at nucleotide 2474 of SEQ ID NO: 3 and ending at nucleotide 5159 of SEQ ID NO: 3.

[0283] Thus, in one aspect the methods can be used to discriminate between plants, seeds or plant parts comprising two copies of the wild type CsAmpl allele, two copies of a mutant CsAmpl allele or one copy of each.

[0284] Optionally plants, plant parts or seeds comprising any of these genotypes may be selected for e.g. further breeding or for use in cucumber production.

[0285] 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).

[0286] In one aspect the KASP-assay discriminates between the SNP found between the wild type and mutant allele, i.e. the KASP-assay can discriminate between the presence in the genomic DNA of SEQ ID NO: 3 in homozygous form (CsAmpl wild type allele), and the presence of e.g. one of the mutant alleles described herein in homozygous form (mutant allele, resulting in shorter internodes and / or more internodes and / or smaller leaves). Different forward and reverse primers can be designed to achieve allele discrimination in the assay.

[0287] Such a genotyping assay can be used for marker assisted selection (MAS) of plants in e.g. a breeding program to select plants or seeds or plant parts comprising a certain genotype, e.g. homozygous for the wild type allele, homozygous or heterozygous for a mutant allele.

[0288] Therefore, also a method of breeding cucumber plants is provided herein, said method comprising genotyping one or more plants for the allele composition at the CsAmpl locus in the genome and optionally selecting one or more plants having a specific genotype at the CsAmpl locus. In one aspect also genotyping -by-sequencing may be done for the CsAmpl gene.

[0289] As mentioned, optionally the plants or seeds which comprise two copies of a mutant CsAmpl allele can be grown and phenotyped. The mutant allele is in one aspect a mutant allele which, in homozygous form, confers shorter internodes and / or more internodes per stem length and / or smaller leaves.

[0290] SEQUENCE DESCRIPTION

[0291] SEQ ID NO: 1 depicts a functional, wild type CsAmpl protein.

[0292] MVQSPLKQLA TICTSRPAPL PTFFFVIIIC VLGFYTFHFS TSSSFSVTSS PRNSVRFQQL 60

[0293] LLSSGSNYTV ASYLRSLTLH PHLAGTEPSS ETVRYVESHF RDLGLETHSI QYDALLSYPK 120

[0294] STSLSVLLSN GTWNI PLSE NVEGWQPYH AYSPSGTAYG PAVFVNYGRD EDYRELAKMG 180

[0295] VTWGCIAVA RKGEFPRGW VAKAEANGAK GVLLYVEGDG FRQGFERGTV MRGIGDPLSP 240

[0296] GWAAVDGAER LNLNDSEVLK RFPKI PSMPL SAESAEIILS SLDTASVPPE WRDKKANLGS 300

[0297] AAVGPGGPI F INFTYQGERK VATIRNVIAV IKGLEEPDRF VLMGNHRDAW SFGAVDPNSG 360

[0298] TAALLDIARR FSLLRRLGWN PRRTILLCSW DAEEFGMIGS TEWVEQNIVN LGTKAVAYLN 420

[0299] VDCAVQGPGF FAGATPQLDD LLHDVTAQVQ DPDVKGATVH DTWTAENGIG NIERLGAVNS 480

[0300] DFAAFVQHAG VPSVDVYYGR DFPVYHTAFD TYDWMANYGD PLFHRHVTVG SIWGLLALRL 540

[0301] SDDLILPFSY ISYANQLQAY KDTLNHLLDG SVSLHSLSSS IEELKFAAQE IENEAKRLRE 600

[0302] QEASSDVALF QKRALNDRLM LAERGFLDVD GLRGHPWFKH LVYGPLSNYE SALVYFPGIA 660

[0303] DAVSESKEIN KRELEELIQH EIWRVARAIR RAAAALKGEL S 701

[0304] SEQ ID NO: 2 depicts the CsAmpl cDNA encoding the protein of SEQ ID NO: 1.

[0305] SEQ ID NO: 3 depicts the genomic DNA encoding the protein of SEQ ID NO: 1.

[0306] SEQ ID NO: 4 depicts a mutant CsAmpl protein comprising a L41 IF amino acid substitution.

[0307] MVQSPLKQLA TICTSRPAPL PTFFFVIIIC VLGFYTFHFS TSSSFSVTSS PRNSVRFQQL 60

[0308] LLSSGSNYTV ASYLRSLTLH PHLAGTEPSS ETVRYVESHF RDLGLETHSI QYDALLSYPK 120

[0309] STSLSVLLSN GTWNI PLSE NVEGWQPYH AYSPSGTAYG PAVFVNYGRD EDYRELAKMG 180

[0310] VTWGCIAVA RKGEFPRGW VAKAEANGAK GVLLYVEGDG FRQGFERGTV MRGIGDPLSP 240

[0311] GWAAVDGAER LNLNDSEVLK RFPKI PSMPL SAESAEIILS SLDTASVPPE WRDKKANLGS 300

[0312] AAVGPGGPI F INFTYQGERK VATIRNVIAV IKGLEEPDRF VLMGNHRDAW SFGAVDPNSG 360

[0313] TAALLDIARR FSLLRRLGWN PRRTILLCSW DAEEFGMIGS TEWVEQNIVN FGTKAVAYLN 420 VDCAVQGPGF FAGATPQLDD LLHDVTAQVQ DPDVKGATVH DTWTAENGIG NIERLGAVNS 480

[0314] DFAAFVQHAG VPSVDVYYGR DFPVYHTAFD TYDWMANYGD PLFHRHVTVG SIWGLLALRL 540

[0315] SDDLILPFSY ISYANQLQAY KDTLNHLLDG SVSLHSLSSS IEELKFAAQE IENEAKRLRE 600

[0316] QEASSDVALF QKRALNDRLM LAERGFLDVD GLRGHPWFKH LVYGPLSNYE SALVYFPGIA 660

[0317] DAVSESKEIN KRELEELIQH EIWRVARAIR RAAAALKGEL S 701

[0318] SEQ ID NO: 5 depicts the CsAmpl cDNA encoding the mutant protein of SEQ ID NO: 4.

[0319] SEQ ID NO: 6 depicts the genomic DNA encoding the protein of SEQ ID NO: 4.

[0320] Non-limiting examples are provided herein below.

[0321] EXAMPLES

[0322] Example 1

[0323] A cucumber TILLING population (containing EMS induced mutations) was screened for mutants in a target gene (Csa3G790960.1 on chromosome 3).

[0324] A stop codon mutant (Q448*) and a splice mutation were found. However, plants with the stopcodon in homozygous form could not be found and plants homozygous for the splice mutation had short hypocotyls and did not grow for more than a few centimetres above ground. A functioning gene and functional protein is, therefore, necessary for survival and growth of the plant.

[0325] One missense mutation was classified as ‘not tolerated’ (i.e. predicted to affect protein function) in a SIFT analysis. Plants with this mutation (L411F) homozygously showed a modified plant architecture in the greenhouse. See Figures 2 and 3.

[0326] Table 2 Other mutants were also identified and will be phenotyped after the plants are selfed and the mutation is in homozygous form, see Table 1 and Table 3. The L538F mutation was found twice in the population screening. Sanger sequencing was carried out to determine the genomic sequence of the wild type and the mutant plant. Table 3 shows the missense mutations found in the cucumber plants. All mutations were in the peptidase domain of the gene.

[0327] Table 3

[0328] The genomic DNA is transcribed into a pre-mRNA comprising 10 exons and 9 introns. The conserved peptidase domain starts at the beginning of Exon 2 and ends in Exon 7. Example 2

[0329] The mutant L411F allele was crossed into an elite cucumber line. The cucumber plant comprising the L41 IF mutation in homozygous form was characterized in the greenhouse in comparison to the recurrent parent comprising the wild type (non-mutated) CsAMPl gene. Table 4 Table 4 continued

[0330] Example 3 Cucumber plants comprising mutant P428S in homozygous form or mutant L538F in homozygous form were grown in the greenhouse and did not show a modified phenotype compared to the wild type. Around 15 or 16 internodes were present in these mutants up to the high-wire, the same as in the wild type plant comprising the wild type CsAmpl gene.

Claims

CLAIMS1. A plant or seed or plant part of the species Cucumis sativus comprising at least one copy of a mutant allele of a gene named CsAmpl on chromosome 3, wherein the wild type CsAmpl gene is a gene that encodes a CsAmpl protein comprising at least 99% sequence identity to SEQ ID NO: 1, wherein the mutant allele encodes a CsAmpl protein comprising a substitution of Leucine 411 of SEQ ID NO: 1, or the equivalent Leucine in a CsAmpl protein comprising at least 99% sequence identity to SEQ ID NO: 1, by another amino acid, preferably by Phenylalanine, and wherein said mutant allele causes a plant which is homozygous for the mutant allele to develop significantly shorter internodes and significantly more internodes on a stem length of 175 cm compared to the plant which is homozygous for the wild type CsAmpl gene.

2. The plant or seed or plant part according to claim 1, wherein said mutant allele is present in homozygous form.

3. The plant or seed or plant part according to any one of claims 1 or 2, wherein said mutant allele further causes a plant which is homozygous for the mutant allele to have significantly smaller leaves than a plant homozygous for the wild type allele.

4. The plant or seed or plant part according to any one of claims 1 to 3, wherein said mutant allele is present in homozygous form and wherein at least 70% more internodes develop on the stem compared to a plant comprising the wild type CsAmpl gene.

5. The plant or seed according to any one of the preceding claims, wherein the plant or seed is homozygous for the mutant csampl allele and said plant develops at least 25, 26, 27, or more, internodes on said stem length.

6. The plant or seed or plant part according to any one of the preceding claims, wherein the mutant csampl allele is a mutant allele generated by chemical mutagenesis, radiation mutagenesis, tissue culture or targeted genome editing techniques, such as Crispr based techniques.

7. A seed according to any one of the preceding claims, wherein said seed comprises two copies of said mutant csampl allele.

8. A fruit produced by a plant according to any one of the preceding claims, wherein the fruit comprises the mutant csampl allele in homozygous form.

9. The plant part according to any one of claims 1 to 6, wherein the plant part is a cell, a flower, a pistil, a leaf, a stem, a petiole, a cutting, a node, an internode, a tissue, a seed coat, an ovule, pollen, a root, a rootstock, a scion, a fruit, a cotyledon, a hypocotyl, a protoplast, an embryo, an anther or a part of any of these.

10. A vegetatively propagated plant propagated from a plant part according to claim 9.

11. A method of cucumber fruit production, said method comprises growing a plant according to any one of claims 1 to 6 or 10 comprising said mutant csampl allele in homozygous form at a stem density of at least 3.0 or 4.0 plants (or stems) per m2.

12. A method for generating a cucumber plant comprising a mutant allele of a gene named CsAmpl, wherein the wild type CsAmpl gene is a gene on chromosome 3 that encodes a wild type CsAmpl protein comprising at least 99% sequence identity to SEQ ID NO: 1, comprising using a targeted genome editing technique or random mutagenesis techniques to generate a mutant allele of the endogenous CsAmpl gene.

13. The method according to claim 12 further comprising identifying or selecting a plant comprising a mutant csampl allele using primers or probes which amplify or which hybridize to at least part of said mutant csampl allele or sequencing.

14. The method according to claim 13, wherein said mutant allele encodes a CsAmpl protein comprising a substitution of Leucine 411 of SEQ ID NO: 1, or the equivalent Leucine in a CsAmpl protein comprising at least 99% sequence identity to SEQ ID NO: 1, by another amino acid, preferably by Phenylalanine.

15. A method for determining whether a cucumber plant or plant part comprises at least one copy of a mutant allele of a gene named CsAmpl, wherein the wild type CsAmpl gene is a gene that encodes a CsAmpl protein comprising at least 99% sequence identity to SEQ ID NO: 1, said method comprising analysing the CsAmpl DNA, RNA or protein of the plant or plant part.

16. The method according to claim 15, wherein the mutant allele encodes a CsAmpl protein comprising a substitution of Leucine 411 of SEQ ID NO: 1, or the equivalent Leucine in a CsAmpl protein comprising at least 99% sequence identity to SEQ ID NO: 1, by another amino acid, preferably by Phenylalanine.

17. The method according to claim 15 or 16, wherein at least one of the following methods is used to determine the presence of said mutant csampl allele in the genome: a PCR - assay, a SNP -genotyping assay such as a KASP-assay or a TaqMan-assay, a High-Resolution Melting (HRM) assay, or DNA sequencing.

18. The method according to any one of claims 15 to 17, wherein the cucumber plant or plant part is subjected to a mutation-inducing step prior to determining whether the cucumber plant or plant part comprises a mutant allele of the CsAmpl gene.

19. The method according to claim 18, wherein the mutation inducing step comprises chemical mutagenesis, preferably using ethyl methanesulfonate (EMS) as mutagenic agent.

20. A method for producing a cucumber plant comprising a mutant allele which confers significantly shorter internodes and significantly more internodes when present in homozygous form, said method comprising the step(s) of:(i) crossing a first cucumber plant and a second cucumber plant, wherein the first cucumber plant comprises in its genome at least one copy of a mutant allele according to claim 1,(ii) optionally harvesting seed from the crossing of (i) and(iii) optionally selecting seed comprising said mutant allele in its genome.