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WO2025172110A1 - Architecture végétale modifiée chez le concombre - Google Patents

Architecture végétale modifiée chez le concombre

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Publication number
WO2025172110A1
WO2025172110A1 PCT/EP2025/052805 EP2025052805W WO2025172110A1 WO 2025172110 A1 WO2025172110 A1 WO 2025172110A1 EP 2025052805 W EP2025052805 W EP 2025052805W WO 2025172110 A1 WO2025172110 A1 WO 2025172110A1
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WIPO (PCT)
Prior art keywords
plant
csampl
mutant
allele
protein
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English (en)
Inventor
Lieke MERTENS
Wim VRIEZEN
Frank BEENDERS
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Nunhems Netherlands BV
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Nunhems Netherlands BV
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Publication of WO2025172110A1 publication Critical patent/WO2025172110A1/fr
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Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/121—Plant growth habits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/34—Cucurbitaceae, e.g. bitter melon, cucumber or watermelon 
    • A01H6/346—Cucumis sativus[cucumber]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development

Definitions

  • 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.
  • “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.
  • 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.
  • “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.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • heterozygous refers to a plant or plant cell having dissimilar pairs of alleles of a gene for any hereditary characteristic.
  • 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.
  • 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.
  • a line e.g. at least 3, 4, 5, 6, 7, 8 or 9 plants, preferably at least 10 plants per line
  • 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
  • Stringent conditions for RNA-DNA hybridisations 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 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.
  • sequence identity 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.
  • “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 can be identified by pairwise alignment (e.g. using the program Needle) with the SEQ ID NO.
  • Wild type CsAMPl allele refers herein to a version of a gene encoding a fully functional cucumber CsAMPl protein (wild type CsAMPl protein).
  • 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.
  • 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.
  • 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.
  • PA Protease Associated
  • 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.
  • 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.
  • 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.
  • Wild relatives of cucumber refer to Cucumis sativus var. hardwickii. C. sativus var. sikkimensis. Cucumis sativus var. xishuangbannesis .
  • a “plant line” or “breeding line” refers to a plant and its progeny.
  • 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.
  • 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.
  • “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.
  • 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
  • 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.
  • “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.
  • vegetative propagation it is also referred to as a vegetative propagation or a vegetatively propagated plant.
  • propagation by grafting e.g. a scion onto a rootstock, is included herein.
  • Cell culture or “tissue culture” refers to the in vitro culture of cells or tissues of a plant.
  • Regeneration refers to the development of a plant from cell culture or tissue culture or vegetative propagation.
  • Non-regenerable cell refers to a cell which cannot be regenerated into a whole plant.
  • “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.
  • 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.
  • an oligonucleotide with 19 nucleotides is called a 19-mer.
  • oligonucleotides are designed to base-pair with a strand of DNA or RNA.
  • the most common use for oligonucleotides is as primers for PCR (polymerase chain reaction).
  • Primers are designed with at least part of their sequence complementary to the sequence targeted for amplification.
  • Optimal primer length for a complementary sequence is e.g. 18 to 22 nucleotides.
  • Optimal primer sequences for PCR are usually determined by primer design software.
  • DNA microarrays are arrays which have many microscopic spots of DNA, usually oligonucleotides, bound on a solid support.
  • Assay targets can be DNA, cDNA, or cRNA.
  • 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.
  • SNP single-nucleotide polymorphism
  • 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.
  • CsAMPl 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.
  • 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.
  • 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.
  • 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.
  • the wild type CsAmpl gene of SEQ ID NO: 3 is present on chromosome 3 starting at nucleotide 936598 and ending at nucleotide 943140.
  • AtAMPl 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).
  • 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.
  • 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.
  • 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.
  • amino acid of the conserved peptidase domain of the wild type CsAMP 1 protein that is substituted is selected from: L411, G412 and G530.
  • amino acid that is substituted is selected from L411, G412, and G530.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the plant or plant part comprises the mutant allele in heterozygous or homozygous form.
  • 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.
  • a control plant e.g. the non-mutated plant
  • 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.
  • 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.
  • the extent of the reduction in internode length and/or leaf size may be slightly different in different genetic backgrounds of cultivated cucumber.
  • 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.
  • 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.
  • 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.
  • at least 3.0 or 4.0 stems per square meter can be grown, or even more.
  • yield can be significantly increased.
  • 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.
  • 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.
  • the average internode length can be determined, optionally also the average leaf size, average fruit number, average fruit weight, etc.
  • 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.
  • 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.
  • the mutation preferably occurs in the coding sequence (cds, composed of the exons).
  • the mutation can occur in the pre-mRNA or the mRNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • more fruits and higher fruit yield per plant is preferably seen in the mutant plant, as is higher fruit yield per cultivation area.
  • 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.
  • 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.
  • 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.
  • 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).
  • the cucumber plant or plant part is homozygous for a mutant csampl allele described herein.
  • 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.
  • 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-ethy
  • 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.
  • DSBI double stranded DNA break inducing enzyme
  • 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.
  • 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.
  • the mutant csampl allele is an induced mutant allele, e.g. induced in a breeding line, an inbred line or variety of cultivated cucumber.
  • 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).
  • 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.
  • 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.
  • pickling cucumbers e.g. American pickling, European pickling types
  • slicing cucumbers e.g. American slicing
  • long cucumbers short cucumbers
  • European greenhouse cucumbers European greenhouse cucumbers
  • Beit-Alpha type cucumbers oriental trellis 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.
  • a plant or plant part of the species Cucumis sativus var. sativus 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.
  • Said amino acid substitution is in one aspect a substitution of a Leucine or of a Glycine by another amino acid.
  • said Leucine (L) is replaced by a Phenylalanine (F) and/or said Glycine is replaced by a Glutamine (E) or by an Arginine (R).
  • L411 is replaced by another amino acid, e.g. by F.
  • G412 or G530 is replaced by another amino acid e.g. E or R.
  • a plant or plant part of the species Cucumis sativus var. sativus 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.
  • the mutant allele is a natural or spontaneous mutant allele, while in another aspect the mutant allele is an induced mutant allele.
  • 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.
  • 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.
  • 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.
  • a cucumber plant e.g. a cultivated plant or a wild plant
  • 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.
  • an amplification product e.g. a PCR product
  • hybridization product e.g. using a nucleic acid probe
  • the primers or probes may be allele-specific primers or probes.
  • 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).
  • a method for transferring a mutant csampl allele from a cucumber plant into another cucumber plant 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.
  • 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.
  • 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.
  • PCR based methods e.g. SNP genotyping
  • hybridization based methods sequencing, cDNA analysis, etc.
  • Step a) and/or c) may involve identification of the plant by phenotype.
  • 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.
  • a method of inducing a mutant csampl allele in a cucumber plant or identifying a mutant csampl allele in a cucumber plant 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.
  • 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.
  • 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).
  • pickling cucumbers e.g. American pickling, European pickling type
  • slicing cucumbers e.g. American slicing
  • long cucumbers short cucumbers
  • European greenhouse 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.
  • 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.
  • the cucumber is an inbred line or an Fl hybrid of a long cucumber, especially a European greenhouse cucumber, or a short cucumber.
  • the cultivated cucumber plant may be an inbred line, an OP (open pollinated variety) or an Fl hybrid.
  • the Fl hybrid comprises a mutant csampl allele preferably in homozygous form.
  • 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.
  • 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.
  • seed characteristics i.e. characteristics of the seeds from which the plant is grown
  • seed characteristics are preferably uniform, e.g. seed size, seed color, etc.
  • 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.
  • 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.
  • the fruit comprises the mutant csampl allele, preferably in homozygous form.
  • the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L411F substitution, preferably in homozygous form.
  • 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.
  • the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g.
  • the cell is a non-propagating or a non-regenerable cell.
  • 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.
  • mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L411F substitution, preferably in homozygous form.
  • a food or feed product comprising cells or tissues or parts of a plant according to the invention, such as parts of cucumber fruits, e.g. slices or pieces of cucumber fruits, such as salads.
  • a method of cucumber fruit production 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.
  • 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 m 2 preferably 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 m 2 , or more; more preferably about 2.6 - 2.8 stems per m 2 , especially 3.0 to 4.0 or 3.0 to 4.5 plants (or stems) per m 2 .
  • at least about 20%, preferably at least about 22%, 23% or 24% more stems per m 2 can be used, in e.g. high-wire cultivation, compared to a cucumber plant which is homozygous for the wild type csampl allele.
  • 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.
  • a mutant CsAmpl allele in homozygous form e.g. the L41 IF mutant allele
  • 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.
  • mutant alleles are encompassed herein.
  • 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).
  • a method 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.
  • a method 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).
  • a targeted genome editing technique such as a Crispr system (e.g. Crispr/Cas9).
  • 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.
  • a method 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.
  • the mutant csampl allele is the mutant allele which encodes a L411 amino acid substitution, e.g. an L41 IF substitution.
  • 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.
  • 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.
  • these may be wild cucumber plants or wild relatives of cucumber.
  • 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.
  • 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).
  • 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).
  • 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.
  • a non-mutated plant may be e.g. a control plant, such as a plant comprising wild type, functional CsAmpl allele in homozygous form.
  • 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.
  • 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.
  • 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 Monblatt 91 (1), 31-48).
  • 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.
  • 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.
  • 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.
  • Determining the presence of a mutant allele of the CsAmpl gene can be done with the help of methods known to the person skilled in the art.
  • analyses based on hybridisations with probes Southern Blot
  • PCR polymerase chain reaction
  • 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.
  • TILLING Targeting Induced Local Lesions IN Genomes
  • These methods are basically suitable for identifying plant cells and plants having a mutant allele of a CsAmpl gene.
  • 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 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).
  • 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.
  • 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 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,
  • 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.
  • the cucumber plant or plant part may be a cultivated plant or a wild cucumber plant or wild relative of cucumber.
  • 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.
  • SNP Single Nucleotide Polymorphism
  • KASP assay can be used for detecting a SNP and thereby the mutant allele.
  • the SNP underlying the L411F replacement is found at nucleotide 3081 of SEQ ID NO: 3 and of SEQ ID NO: 6.
  • SEQ ID NO: 3 wild type genomic sequence
  • a C Cytosine
  • 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.
  • the nucleotide of the SNP position is at nucleotide 30568657. See Table 3.
  • 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.
  • the codon CTT encoding Leucine at amino acid 411 of the protein, L411
  • TTT encoding Phenylalanine at amino acid 411 of the protein
  • 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.
  • Chinese Long V2 (cucurbitgenomics.org) this corresponds to nucleotides 30568048 to 30570735 of chromosome 3 of the ChineseLong V2 genome.
  • a cucumber plant or plant part 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.
  • 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).
  • C Cytosine
  • T Thymine
  • 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.
  • a mutant allele e.g. a commercial variety of the applicant
  • a plant or plant part of the species Cucumis sativus 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the plant or plant part according to any one of the embodiments herein is preferably homozygous for the mutant allele.
  • the fruit is preferably seedless.
  • 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.
  • 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.
  • 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.
  • the cucumber plant or plant part 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.
  • 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.
  • EMS ethyl methanesulfonate
  • radiation mutagaenesis such as e.g. UV- or gamma radiation.
  • the first cucumber plant is homozygous for the mutant allele and, therefore, has the desired phenotype.
  • 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.
  • 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.
  • 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.
  • 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.
  • said mutation inducing step may comprise contacting said seed, plant or plant part with a mutagen.
  • the seed or plant or plant part that is contacted with the mutagen comprises a wild type CsAmpl allele in homozygous form.
  • 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.
  • 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.
  • a method of producing a Cucumis sativus plant comprising the steps of:
  • 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.
  • TILLING Targeting Induced Local Lesions IN Genomes
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • a method for producing a Cucumis sativus plant comprising the steps of:
  • 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).
  • 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.
  • a genotyping assay genotyping cucumber plants, 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 (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
  • Step c) may also be selecting a plant, seed, plant part, cell or tissue comprising at least one copy of a mutant allele.
  • 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.
  • 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.
  • 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) .
  • 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.
  • KASP-assay by LGC, see www at LGCgenomics.com and also www at biosearchtech.com/products/ pcr-kits-and-reagents/ genotyping-assays/ kasp-genotyping-
  • 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).
  • SNPs single nucleotide polymorphisms
  • InDeis inserts and deletions
  • 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
  • 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.
  • 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.
  • the 5'-3' exonuclease activity of Taq polymerase cleaves and releases the fluorophores from bound probes.
  • the emission intensity of each fluorophore is measured and allele determination at the queried site can be made.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • plants, plant parts or seeds comprising any of these genotypes may be selected for e.g. further breeding or for use in cucumber production.
  • 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).
  • 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.
  • MAS marker assisted selection
  • 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.
  • SEQ ID NO: 3 depicts the genomic DNA encoding the protein of SEQ ID NO: 1.
  • SEQ ID NO: 5 depicts the CsAmpl cDNA encoding the mutant protein of SEQ ID NO: 4.
  • SEQ ID NO: 6 depicts the genomic DNA encoding the protein of SEQ ID NO: 4.
  • a cucumber TILLING population (containing EMS induced mutations) was screened for mutants in a target gene (Csa3G790960.1 on chromosome 3).
  • 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.
  • 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.

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Abstract

La présente invention concerne des plants de concombre présentant une architecture végétale modifiée, comme des entre-noeuds plus courts, une plus grande quantité d'entre-noeuds et des feuilles plus petites.
PCT/EP2025/052805 2024-02-16 2025-02-04 Architecture végétale modifiée chez le concombre Pending WO2025172110A1 (fr)

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