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WO2025186284A1 - Production accrue du concombre - Google Patents

Production accrue du concombre

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Publication number
WO2025186284A1
WO2025186284A1 PCT/EP2025/055893 EP2025055893W WO2025186284A1 WO 2025186284 A1 WO2025186284 A1 WO 2025186284A1 EP 2025055893 W EP2025055893 W EP 2025055893W WO 2025186284 A1 WO2025186284 A1 WO 2025186284A1
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WO
WIPO (PCT)
Prior art keywords
plant
yieldplus
gene
allele
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/055893
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English (en)
Other versions
WO2025186284A8 (fr
Inventor
Jamila CHAIB
Zahi Paz
Quang-Hien LE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vilmorin SA
Original Assignee
Vilmorin SA
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Filing date
Publication date
Application filed by Vilmorin SA filed Critical Vilmorin SA
Publication of WO2025186284A1 publication Critical patent/WO2025186284A1/fr
Publication of WO2025186284A8 publication Critical patent/WO2025186284A8/fr
Pending legal-status Critical Current
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
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a Cucumis sativus plant having an increased fruit yield.
  • the invention is further related to markers linked to the increased fruit yield phenotype and to the use of such markers to identify or select plants having such phenotype.
  • the invention also relates to the seeds and progeny of such plants and to the propagation material for obtaining such plants.
  • the cucumber genome was the first vegetable genome to be sequenced (Huang et al. 2009, Nature Genetics, Volume 41 , Number 12, p 1275-1283).
  • Fazio et al. 2003 (Theor Appl Genet 107: 864-874) genetically mapped a number of traits, including cumulative fruits per plants over three harvests and morphological traits such as little leaf. Their linkage group 1 appears to correspond to the physical chromosome 6. A locus called fp1 1 .2 (fruits per plant) was consistent in both environments and mapped to the little leaf locus.
  • the present invention relates to a Cucumis sativus plant comprising a mutant allele of a YieldPlus gene on chromosome 3 in its genome, wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele, resulting in an increased fruit yield in comparison to an isogenic plant comprising a wild-type allele of the YieldPlus gene.
  • Embodiment 1 A Cucumis sativus plant comprising a mutant allele of a YieldPlus gene on chromosome 3 in its genome, wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , resulting in an increased fruit yield in comparison to an isogenic plant comprising a wild-type allele of the YieldPlus gene.
  • Embodiment 2 A plant according to embodiment 1 , wherein said mutant allele is present heterozygously in the genome of said plant.
  • Embodiment 3 A plant according to embodiment 1 , wherein said mutant allele is present homozygously in the genome of said plant.
  • Embodiment 4 A plant according to any one of embodiments 1 to 3 wherein said mutant allele comprises a nucleotide sequence having at least 90% identity with the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • Embodiment 5 A plant according to any one of embodiments 1 to 4, wherein said mutation is a loss-of-function mutation and/or a mutation which decreases expression of the YieldPlus protein.
  • Embodiment 6 A plant according to any one of embodiments 1 to 5 wherein said at least one loss-of-function mutation is selected from a missense mutation, a nonsense mutation and a frameshift mutation.
  • Embodiment 7 The plant according to any one of embodiments 1 to 6, wherein said at least one mutation is located in the coding sequence of the YieldPlus gene.
  • Embodiment 8 The plant according to embodiment 7, wherein said at least one mutation results in at least one amino acid change, addition or deletion in the amino acid sequence set forth in SEQ ID NO:3.
  • Embodiment 9 The plant according to embodiment 8, wherein said at least one mutation results in at least one amino acid change, addition or deletion in the C-terminal portion of the protein encoded by the YieldPlus gene, preferably between the amino acid residues in position 150 and 277 of SEQ ID NO:3.
  • Embodiment 10 The plant according to embodiment 9, wherein said at least one mutation results in at least one amino acid change, addition or deletion in position 253 of SEQ ID NO:3.
  • Embodiment 11 The plant according to embodiment 10, wherein said at least one mutation results in a G253S change in the amino acid sequence set forth in SEQ ID NO:3.
  • Embodiment 12 The plant according to embodiment 11 , wherein said at least one mutation is a G757A mutation in SEQ ID NO: 2.
  • Embodiment 13 The plant according to any one of embodiment 1 to 6, wherein said mutation is located in a regulatory sequence selected from a 3’-untranslated region (3 -UTR), a 5’-untranslated region (5 -UTR), a promoter, an enhancer and a polyA signal sequence.
  • a regulatory sequence selected from a 3’-untranslated region (3 -UTR), a 5’-untranslated region (5 -UTR), a promoter, an enhancer and a polyA signal sequence.
  • Embodiment 14 The plant according to any one of embodiments 1 to 13, which produces at least 10 fruits or more, preferably at least 12 fruits or more.
  • Embodiment 15 The plant according to any one of embodiments 1 to 14, which produces at least 10%, preferably at least 20%, still preferably at least 50% more fruits in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • Embodiment 16 The plant according to any one of embodiments 1 to 15, wherein the fruit weight of said plant is at least 10%, preferably at least 20%, still preferably at least 50% higher in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • Embodiment 17 The plant according to any one of embodiments 1 to 16, wherein said plant is a plant from an inbred line or is a hybrid plant.
  • Embodiment 18 The plant according to any one of embodiments 1 to 17, which further comprises one or more traits of agronomical interest selected from resistance to ZYMV (Zucchini Yellow Mosaic Virus), resistance to CVYV (Cucumber Vein Yellowing Virus), resistance to PRSV (Papaya Ringspot Virus), resistance to WMV (Watermelon Mosaic Virus), resistance to CMV (Cucumber Mosaic Virus), resistance to powdery mildew, resistance to potyviruses, resistance to downy mildew, e.g. caused by Pseudoperonospora cubensis, resistance to Fusaria, e.g. caused by Fusariumoxysporum f.sp.
  • ZYMV Ziucchini Yellow Mosaic Virus
  • CVYV Cucumber Vein Yellowing Virus
  • PRSV Papaya Ringspot Virus
  • WMV Watermelon Mosaic Virus
  • CMV Cucumber Mosaic Virus
  • Embodiment 19 A seed for producing a plant of any one of embodiments 1 to 18.
  • Embodiment 20 A cell of a plant according to any one of embodiments 1 to 18, preferably a cell derived from an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, and/or hypocotyl, comprising in its genome a mutant allele of the YieldPlus gene, wherein said mutant allele comprises one or more mutations in the sequence of the gene, or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 .
  • Embodiment 21 A plant part of a C.
  • sativus plant comprising at least one cell according to embodiment 20, preferably an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, and/or hypocotyl, in particular a fruit.
  • Embodiment 22 An isolated polynucleotide, comprising a mutant allele of the YieldPlus gene, wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , wherein said mutant allele confers increased fruit yield to a plant comprising said mutant allele, in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • Embodiment 23 The isolated polynucleotide of embodiment 22, which comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 1 or SEQ ID NO: 2.
  • Embodiment 24 The isolated polynucleotide of embodiment 22, which encodes a polypeptide with at least 95% identity with SEQ ID NO:3.
  • Embodiment 25 An isolated polypeptide, comprising an amino acid sequence with at least 95% identity with SEQ ID NO:3, wherein said polypeptide comprises at least one mutation in the amino acid sequence, in comparison to the sequence of the corresponding wild-type polypeptide as set forth in SEQ ID NO:3, wherein said mutation confers a I oss-of-fu notion phenotype to said polypeptide.
  • Embodiment 26 An in vitro cell or tissue culture of regenerable cells of the C. sativus plant according to any one of embodiments 1 to 18, wherein the regenerable cells are derived from an embryo, protoplast, meristematic cells, callus, pollen, leaf, anther, stem, petiole, root, root tip, seed, flower, cotyledon, and/or hypocotyl.
  • Embodiment 27 A method of producing a C. sativus plant, comprising: a) obtaining a part of a plant according to embodiments 1 to 18, b) vegetatively propagating said plant part to generate a plant from said plant part.
  • Embodiment 28 A method of producing a C. sativus plant, comprising the introduction of at least one mutation in the YieldPlus gene on chromosome 3 in the genome of a C. sativus plant, wherein said mutation is introduced in the sequence of the gene or in a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , resulting in an increased fruit yield in comparison to an isogenic plant comprising a wild-type allele of the YieldPlus gene.
  • Embodiment 29 The method of embodiment 28, wherein said mutation is introduced by mutagenesis or genome editing, in particular by a technique selected from ethyl methanesulfonate (EMS) mutagenesis, oligonucleotide directed mutagenesis (ODM), Zinc finger nuclease (ZFN) technology, Transcription Activator-Like Effector Nucleases (TALENs) the CRISPR/Cas system, the CRISPR/Cpf system engineered meganuclease, re-engineered homing endonucleases and DNA guided genome editing.
  • EMS ethyl methanesulfonate
  • ODM oligonucleotide directed mutagenesis
  • ZFN Zinc finger nuclease
  • TALENs Transcription Activator-Like Effector Nucleases
  • Embodiment 30 The method of embodiment 29, comprising: a) Introducing one or more mutations in cucumber plant(s), seed(s) or plant part(s), b) Optionally, determining if the plant, seed or plant part under a) presents an increased yield compared to a plant not having said at least one mutation; and c) Selecting a plant that comprises a mutant allele of a YieldPlus gene, wherein said mutant allele comprises at least one mutation in the sequence of the gene or in a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , resulting in an increased fruit yield in comparison to an isogenic plant comprising a wild-type allele of the YieldPlus gene.
  • Embodiment 31 The method of embodiment 29 or 30, wherein the produced plant is according to anyone of embodiments 1 to 18.
  • Embodiment 32 A method of producing a C. sativus plant, comprising:
  • step b) optionally self-pollinating and/or backcrossing one or several times the plant selected at step b) and selecting in the progeny thus obtained a plant comprising a mutant allele of the YieldPlus gene.
  • Embodiment 33 A method for detecting and/or selecting a cucumber plant, seed or plant part, comprising the steps of:
  • mutant allele of the YieldPlus gene on chromosome 3 wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 ;
  • Embodiment 34 The method of embodiment 33, wherein step (b) comprises carrying out a genotyping assay, using the DNA samples of a) as template, that discriminates between the wild type YieldPlus allele and the mutant allele, wherein said genotyping assay is based on nucleic acid amplification making use of YieldPlus allele specific oligonucleotide primers, and/or wherein said genotyping assay is based on nucleic acid hybridization making use of YieldPlus allele-specific oligonucleotide probes.
  • Embodiment 35 The method of embodiment 34, wherein said YieldPlus allele specific oligonucleotide primers or said YieldPlus allele-specific oligonucleotide probes comprise at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 or of the complement strand of SEQ ID NO: 1 or SEQ ID NO: 2.
  • Embodiment 36 The method of embodiment 35, wherein said YieldPlus allele specific oligonucleotide primers comprise a sequence selected from the sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 7.
  • Embodiment 37 A method for screening and/or selecting plant, seeds or plant part, or DNA or RNA or protein derived therefrom, for the presence of a mutant allele of the YieldPlus gene on chromosome 3, conferring increased fruit yield in comparison to an isogenic plant comprising a wildtype allele of the YieldPlus gene, comprising one or more of the following steps: a) determining if the level of expression of the YieldPlus gene in the plant, seed or plant part is reduced or abolished in comparison to a plant comprising a wild-type allele of the YieldPlus gene, wherein the sequence of the wild-type YieldPlus gene is set forth in SEQ ID NO: 1 ; b) determining if the amounts of the protein encoded by the YieldPlus gene in the
  • Embodiment 38 A method for improving the yield of cucumber production, wherein said method comprises growing C. sativus plants according to any one of embodiments 1 to 18 and harvesting fruits set by said plants.
  • Embodiment 39 A method of producing cucumber fruit comprising: a) growing a C. sativus plant according to any one of embodiments 1-18. b) allowing said plant to set fruit; and c) harvesting fruit of said plant.
  • Embodiment 40 A method of producing a foodstuff or feedstuff comprising: a) Obtaining fruits of a C. sativus plant according to any one of embodiments 1-18. b) Processing said fruits into a processed food or feedstuff or using said fruit as an ingredient into a foodstuff or feedstuff.
  • Embodiment 41 Use of a C. sativus plant according to any one of embodiments 1-18 or some fruit thereof in the fresh cut market or for food processing.
  • Embodiment 42 A plant according to any one of embodiments 1-18, wherein the average individual fruit weight of fruits of said plant is decreased by less than 50%, in particular less than 40%, more particularly less than 30%, still more particularly less than 20%, even more particularly less than 10%, most particularly less than 5% in comparison to the average individual fruit weight of fruits of an isogenic plant which comprises a wild-type allele of the YieldPlus gene, or is not decreased.
  • Figure 1 shows the mean fruit weight per plant (A) and mean fruit number per plant(B) of progeny plants of the mutant plant which are homozygous for the mutant allele (Homo Mut) and progeny plants which are homozygous for the wild-type allele (Homo WT). Trial conducted for 32 days, 15 picks, 255 mutant fruits, 175 wild-type fruits.
  • Fruit weight: Anova R 2 0.32, P ⁇ 0.001 .
  • Fruit number: Anova R 2 0.24, P ⁇ 0.005.
  • SEQ ID NO: 1 wild-type allele of the YieldPlus gene
  • SEQ ID NO: 2 cds of wild-type YieldPlus gene
  • SEQ ID NO: 3 wild-type YieldPlus protein
  • SEQ ID NO: 4 mutant allele of the YieldPlus gene
  • SEQ ID NO: 5 mutant YieldPlus protein
  • SEQ ID NO: 6 KASP detection of the mutant: primer recognizing the wild-type allele
  • SEQ ID NO: 7 KASP detection of the mutant: primer recognizing the mutant allele
  • SEQ ID NO: 8 KASP detection of the mutant: common primer
  • the term "cucumber” refers to a plant, or any part thereof, of the species Cucumis sativus. This includes, without being limited to, plants commonly referred to as Cucumber American gherkin, Cassabanana, Cuke, Gherkin, Hothouse cucumber, Lemon cucumber, Mandera cucumber, Pickling cucumber, Serpent cucumber, Slicing cucumber, Snake cucumber, and West Indian gherkin.
  • an F1 may thus be (and usually is) a hybrid resulting from a cross between two true breeding parents (true-breeding is homozygous for a trait), while an F2 may be (and usually is) an offspring resulting from self-pollination of said F1 hybrids.
  • crossing As used herein, the terms “cross”, “crossing”, “cross pollination” or “cross-breeding” refer to the process by which the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.
  • the terms “molecular marker” or “marker” refer to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences.
  • indicators are restriction fragment length polymorphism (RFLP) markers, amplification fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location.
  • RFLP restriction fragment length polymorphism
  • AFLP amplification fragment length polymorphism
  • SNPs single nucleotide polymorphisms
  • SSRs single nucleotide polymorphisms
  • SCARs sequence-characterized amplified regions
  • CAS cleaved amplified polymorphic sequence
  • a chromosome can indeed be defined with respect to markers, such as SNPs, insofar as the flanking sequences of said markers are defined in order to unambiguously position them on the genome.
  • markers such as SNPs
  • the present inventors have used SNPs markers, identified by their flanking sequences, present in the cucumber genome, to discriminate between the mutant and wild type allele and to track down the mutation conferring the increased Yield in the cucumber genome.
  • Their location in the cucumber genome refer to the assembly Cucumber (Chinese Long) v3 available at http://cucurbitgenomics.org/v2/organism/19 (based on Li et al, 2019 A chromosome-scale genome assembly of cucumber (Cucumis sativus L.). Gigascience, Volume 8, Issue 6, June 2019, giz072).
  • the term “primer” refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primers extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • a pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.
  • a single nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C, or G — in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes in an individual.
  • SNP single nucleotide polymorphism
  • AAGCCTA to AAGCTTA two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case there are two alleles: C and T.
  • the term “genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.
  • heterozygous refers to the presence of different alleles (forms of a given gene, genetic determinant or sequences) at a particular locus.
  • homozygous refers to the presence of identical alleles at one or more loci in homologous chromosomal segments.
  • homologous chromosomes refer to a set of one maternal and one paternal chromosome that pair up with each other during meiosis. These copies have the same genes in the same loci and the same centromere location.
  • hybrid refers to any individual cell, tissue or plant resulting from a cross between parents that differ in one or more genes.
  • An F1 hybrid results from the cross of two genetically different parent cultivars or lines.
  • two plants are said “isogenic”, when they have the same or essentially the same set of chromosomes and genes, except for one gene, e.g. the YieldPlus gene.
  • the two isogenic plants thus comprise different alleles of the gene. Comparing the phenotype of two isogenic plants allows the evaluation of the effect of an allelic variation of the gene.
  • locus refers to any site that has been defined genetically, this can be a single position (nucleotide) or a chromosomal region.
  • a locus may be a gene, a genetic determinant, or part of a gene, or a DNA sequence, and may be occupied by different sequences (e.g. The YieldPlus locus is, thus, the location in the genome of cucumber, where the mutant allele and/or the wild type allele of the YieldPlus gene is found).
  • a locus may also be defined by a marker, such as a SNP (Single Nucleotide Polymorphism), by several markers (e.g.
  • allele(s) means any of one or more alternative forms of a gene at a particular locus, e.g. the YieldPlus locus.
  • the alleles of the gene may be wild type (e.g. SEQ ID NO: 1), or mutant alleles, which alleles relate to one trait or characteristic at a specific locus (e.g. increased yield).
  • 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.
  • the term “gene” means a (genomic) DNA sequence comprising a region (transcribed region), which is transcribed into a messenger RNA molecule (mRNA) in a cell, and operably linked regulatory regions (e.g. a promoter, enhancer, 3’UTR region, 5’-UTR region, polyA sequence or intron).
  • mRNA messenger RNA molecule
  • operably linked regulatory regions e.g. a promoter, enhancer, 3’UTR region, 5’-UTR region, polyA sequence or intron.
  • mRNA messenger RNA molecule
  • allele refers to any of several alternative or variant forms of a genetic unit, such as a gene, which are alternative in inheritance because they are positioned at the same locus in homologous chromosomes.
  • Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused, for example, by chemical or structural modification, transcription regulation or post- translational modification/regulation.
  • the two alleles of a given gene or genetic element typically occupy corresponding loci on a pair of homologous chromosomes.
  • wild-type allele such as in “wild type YieldPlus allele” refers herein to the functional allele of the gene, which causes the plant to develop a normal phenotype, e.g. a normal fruit yield.
  • mutant allele such as in “mutant YieldPlus allele” refers herein to an allele of a gene comprising one or more mutations in comparison to a wild-type allele, which causes the cucumber plant to exhibit a modified phenotype, e.g. an increased fruit yield.
  • the mutation(s) in the mutant allele can be any mutation or combination of mutations, including deletions, substitutions, truncations, insertions, point mutations, non-sense mutations, mis-sense mutations, frame shift mutations and/or mutations in the coding sequence and/or in one or more regulatory sequences such as a promoter sequence, a 3’UTR sequence, a 5’-UTR sequence, an enhancer sequence, a silencer sequence, a polyA sequence and an intron sequence.
  • the term "Induced mutant alleles” are mutant alleles in which the mutation(s) is/are/have been induced by human intervention, e.g. by mutagenesis via physical or chemical mutagenesis methods or via e.g. tissue culture (as described in e.g. Zhang et al, Pios 9(5) e96879), including also targeted gene editing techniques (such as CRISPR based techniques, TALENS, etc.).
  • Backcrossing is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first - generation hybrid F1 , with one of the parental genotypes of the F1 , hybrid.
  • the term “Marker assisted selection” or “MAS” is a process of using the presence of molecular markers (such as SNP markers), which are genetically and physically linked to a particular locus or to a particular chromosome region or allele specific markers, to select plants for the presence of the specific locus or region or allele.
  • molecular markers such as SNP markers
  • a molecular marker genetically and physically linked to the mutant YieldPlus allele or an allele specific marker can be used to detect and/or select e.g. cucumber plants, or plant parts, comprising the mutant YieldPlus allele. Allele specific markers are preferred markers, as they select for the allele directly.
  • M2 generation or “M2 plant” shall refer herein to the generation obtained from self-pollination of the Ml generation.
  • a plant grown from seeds obtained from a selfpollinated Ml plant represents a M2 plant.
  • M3, M4, etc. refers to further generations obtained after self- pollination.
  • An “mRNA coding sequence” shall have the common meaning herein.
  • An mRNA coding sequence corresponds to the respective DNA coding (cDNA) sequence of a gene/allele apart from that thymine (T) is replaced by uracil (U).
  • mutation in a nucleic acid molecule (DNA or RNA) refers to a change of one or more nucleotides compared to the corresponding wild-type sequence, e.g. by substitution, deletion and/or insertion of one or more nucleotides. Examples of such a mutation are point mutation, nonsense mutation, missense mutation, frame shift mutation or a mutation in a regulatory sequence.
  • mutant protein is herein a protein comprising one or more mutations in the nucleic acid sequence encoding the protein, whereby the mutation results in (the mutant nucleic acid molecule encoding) a "loss-of- function" protein, as e.g. measurable in vivo, e.g. by the phenotype conferred by the mutant allele.
  • mutation in an amino acid sequence (e.g. polypeptide or protein sequence) refers to a change of one or more amino acids compared to the corresponding wild-type amino acid sequence, e.g. by substitution, deletion and/or insertion of one or more amino acids.
  • An amino acid substitution may be conservative or non-conservative.
  • Consservative amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to induce an immune response when administered to a subject.
  • conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.
  • deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some embodiments less than 1 %) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.
  • Non-conservative substitutions are generally those that reduce an activity or function of a given protein, here the YieldPlus protein, such as the ability to induce an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.
  • point mutation refers to the replacement, insertion or deletion of a single nucleotide.
  • nonsense mutation refers to a mutation within a gene encoding a protein, that changes a sense codon which corresponds to an amino acid residue in a protein, into a stop codon which ends synthesis of the protein at a premature position.
  • a nonsense mutation shortens the length of the protein.
  • a truncated protein may have loss of function.
  • missense mutation refers to a mutation within a gene encoding a protein, that substitutes an amino acid of the protein by a different amino acid.
  • a missense mutation is also called a nonsynonymous mutation.
  • the resulting protein has a modified amino acid sequence and may have loss of function.
  • frame shift mutation refers to a mutation within a gene encoding a protein by which the reading frame of the mRNA transcribed from the gene is shifted, i.e. changed, resulting in a different amino acid sequence.
  • the resulting protein may have loss of function.
  • substitution is when at least one nucleotide in the gene sequence is replaced by another nucleotide compared to the corresponding wild type nucleic acid sequence, or at least one amino acid in a protein sequence is different compared to the corresponding wild type amino acid sequence.
  • the term “insertion” or “addition” shall mean that the nucleic acid sequence or the amino acid sequence of a protein comprises at least one additional nucleotide or amino acid compared to the corresponding wild type nucleic acid sequence or the corresponding wild type amino acid sequence, respectively.
  • premature stop codon in context with the present invention means that a stop codon is present in a coding sequence (cds) which is closer to the start codon at the 5 ’-end compared to the stop codon of a corresponding wild type coding sequence.
  • truncation shall be understood to mean that at least one nucleotide at either the 3’-end or the 5’-end of a nucleotide sequence is missing compared to the corresponding wild type sequence.
  • truncation shall be understood to mean that at least one amino acid, at either the N-terminus or the C -terminus of the amino acid sequence e.g. polypeptide or protein sequence, is missing compared to the amino acid sequence of the corresponding wild type protein.
  • knock-out refers to the ablation of a gene expression (i.e., expression of the respective gene is not detectable anymore).
  • knock-down refers to the decrease of a gene expression (i.e., expression of the respective gene is still detectable).
  • a “loss-of -function mutation” is a mutation which results in the gene product having a reduced function or no function at all (being partially or wholly inactivated). The degree to which the function is lost can vary. When the mutation induces a complete loss of function, it is also called a null mutation. Phenotypes associated with such mutations are generally recessive. Is also possible that some function may remain, but not at the level of the wild type allele. These are called leaky mutations. As a result of a I oss-of-fu notion mutation, the plant may produce a phenotypic change (e.g., increased fruit yield). As mentioned, it was found that the YieldPlus protein function directly reflects (and causes) a modification in fruit yield. The I oss-of-fu notion protein can thus be determined phenotypically.
  • a “decreased activity” of a protein shall mean a decrease in activity of a protein, e.g. the YieldPlus protein, when compared to a corresponding wild type protein.
  • a “decreased activity” shall in one aspect comprises a decreased or abolished gene expression (e.g., knock-down or knock-out of the YieldPlus gene), in fine, leading to the apparition of the phenotype of interest (i.e., increased fruit yield).
  • a “decreased activity” may refer to a loss-of- function YieldPlus protein (e.g., a mutant YieldPlus protein may have lost function compared to the wild type, functional YieldPlus protein). A loss-of-function of the protein is present when the mutant allele changes the phenotype from the wild type phenotype, i.e. normal yield when the wild type allele is present, into an increased fruit yield when the mutant allele is present.
  • the term “Complementary strands” refers to two strands of complementary sequence and may be referred to as sense (or plus) and anti-sense (or minus) strands for double stranded DNA.
  • the sense / plus strand is, generally, the transcribed sequence of DNA (or the mRNA that was generated in transcription), while the anti-sense / minus strand is the strand that is complementary to the sense sequence.
  • the complementary nucleotides of DNA are A complementary to T, and G complementary to C.
  • the complementary nucleotides of RNA are A complementary to U, and G complementary to C.
  • yield or “fruit yield” or “average yield” refers to the average number of fruits per plant (FrPP) and/or the average fruit weight (grams) per plant (GrPP) at a single harvest time-point.
  • the single harvest time-point is in line with growers practice and chosen to maximize the number of fruits for each plant.
  • growers will identify the standard size (length and diameter) that need to be reached at each single harvesting time point. For example, for a European long type, standard size is defined as 34 to 38 cm long and 4cm to 4.5cm diameter. Thus, in one aspect all fruits per plant are harvested and only the ones fitting the standard size (length and diameter) are chosen.
  • average individual fruit weight refers to the total fruit weight (grams) of a plant divided by the number of fruits of the plant, i.e. reflecting the average fruit weight (grams) per fruit in a particular plant.
  • the term “increased fruit yield” refers to a cucumber plant, typically mutant, having a statistically significantly higher average number of fruits per plant (FrPP) and/or a significantly higher average fruit weight per plant (GrPP) compared to a reference plant, typically wild-type, at the same stage of development and when grown under the same environmental conditions.
  • the reference plant is preferably an isogenic plant homozygously comprising a wild-type allele of the YieldPlus gene.
  • Fruit yield comparisons are made in the same growth conditions, wherein the fruits are harvested at a same harvest time point.
  • field trials are carried out in several replicates (2, 3, or more) in several locations (2, 3, or more), with sufficient plants (e.g. at least 10, 15, 20, 30, 40, or more plants per line) comprising a mutant allele (e.g., SEQ ID NO: 4) and the wild-type allele (e.g. SEQ ID NO: 1).
  • plant part refers to any part of a plant including but not limited to the shoot, root, stem, seeds, fruits, leaves, petals, flowers, ovules, branches, petioles, internodes, pollen, stamen, rootstock, scion and the like.
  • resistance is as defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Section for describing the reaction of plants to pests or pathogens, and abiotic stresses for the Vegetable Seed Industry. Specifically, by resistance, it is meant the ability of a plant variety to restrict the growth and development of a specified pest or pathogen and/or the damage they cause when compared to susceptible plant varieties under similar environmental conditions and pest or pathogen pressure. Resistant varieties may exhibit some disease symptoms or damage under heavy pest or pathogen pressure.
  • the term “susceptible” refers to a plant that is unable to restrict the growth and development of a specified pest or pathogen.
  • inbred or “line” refers to a relatively true-breeding strain.
  • phenotype refers to the observable characters of an individual cell, cell culture, organism (e.g. a plant), or group of organisms which results from the interaction between that individual genetic makeup (i.e. genotype) and the environment.
  • introgression refers to the process whereby genes of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species.
  • the crossing may be natural or artificial.
  • the process may optionally be completed by backcrossing to the recurrent parent, in which case introgression refers to infiltration of the genes of one species into the gene pool of another through repeated backcrossing of an interspecific hybrid with one of its parents.
  • An introgression may be also described as a heterologous genetic material stably integrated in the genome of a recipient plant.
  • a comparison between two or more plants or fruits in particular a comparison between a cucumber plant according to the invention with an isogenic plant not comprising a mutant allele of the Yieldplus gene, is understood to be a comparison between plants or fruits at the same stage of maturity or at the same stage post-harvest, grown in the same environmental conditions.
  • the present inventors have identified that the presence of a mutant allele of a gene named YieldPlus on chromosome 3 in the genome of Cucumis sativus plants, provides to these plants an increased fruit yield.
  • the present invention thus provides cultivated cucumber plant or plant part that displays an increased fruit yield as well as a method for producing a cucumber plant that exhibits the increased yield.
  • the present invention also discloses a method for screening and/or selecting a plant having an increased fruit yield.
  • the present invention also discloses molecular genetic markers, especially KASP markers, linked the phenotype of interest (i.e., increased fruit yield). Plants obtained through the uses of such molecular markers are also provided. Methods for identifying further molecular markers linked to the phenotype of interest are also provided.
  • Said increased fruit yield is moreover easily transferable to different genetic backgrounds and the invention also extends to different methods allowing the transfer or introgression of the mutated allele conferring the phenotype as well as method for introducing the mutated allele in a cucumber plant.
  • Said increased fruit yield is characterized by increased total fruit weight per plant, increased fruit number per plant, and/or increased individual fruit weight per fruit of the plant.
  • the invention also provides methods for improving the yield of cucumber production and methods for producing cucumber fruits.
  • the invention relates to a Cucumis sativus plant comprising a mutant allele of a YieldPlus gene on chromosome 3 in its genome, wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele.
  • the mutation results in an increased fruit yield, particularly in an increased fruit yield in comparison to an isogenic plant comprising a wild-type allele of the YieldPlus gene.
  • the plant according to the invention is preferably a cultivated cucumber, namely a cultivated C. sativus var. sativus plant.
  • the YieldPlus gene has been mapped to chromosome 3 of the genome (nucleotide 31790724 to nucleotide 31792269 of reference genome Cucumis sativus (Cucumber 9930 (Chinese Long) v3.0).
  • a sequence of a wild-type allele of the YieldPlus gene is set forth in SEQ ID NO: 1 .
  • a wild-type coding sequence is set forth in SEQ ID NO: 2. Wild-type YieldPlus alleles also encompass any nucleotide sequence which retains the activity and expression level of the YieldPlus gene as encoded by SEQ ID NO: 1.
  • the wild-type YieldPlus allele comprises a nucleotide sequence with at least 80%, preferably at least 90%, still preferably at least 95%, more preferably at least 98%, most preferably at least 99% identity with SEQ ID NO: 1.
  • a translated sequence of the wild-type YieldPlus gene i.e. the wild-type amino acid sequence of the YieldPlus protein is set forth in SEQ ID NO:3.
  • the YieldPlus protein (SEQ ID NO:3) is a zinc finger protein 8-like transcription factor. The YieldPlus protein has been annotated and reported to be involved in the cell’s response to gibberellin, trichome differentiation and trichome morphogenesis.
  • I oss-of-fu notion alleles of the YieldPlus gene can be identified by assessing a C. sativus plant or cell’s response to a gibberellin stimulus and/or trichome morphogenesis and/or trichome differentiation in a cell, and comparing said response to the response of a plant or cell comprising a wild-type allele of the YieldPlus gene to a gibberellin stimulus and/or trichome morphogenesis and/or trichome differentiation.
  • Loss-of-fu notion alleles of the YieldPlus are also those which have the same effect, or a similar effect, as a reference loss-of-function allele, e.g.
  • an allele comprising a YieldPlus gene sequence encoding a protein comprising a non-conservative substitution within the zinc finger domain, as located from amino acids 85 to 120 of the amino acid sequence of the YieldPlus protein, as set forth in SEQ ID NO:3.
  • the reference loss-of-function allele comprises a YieldPlus gene encoding a protein comprising a non-conservative substitution within the motif QAALGH in positions 99 to 104 of SEQ ID NO: 3.
  • the same effect, or a similar effect refers, in particular to a same or similar effect on any phenotype distinguishing a plant or a cell with the reference allele and a wild-type plant or cell, in particular an effect on cell’s response to a gibberellin stimulus and/or trichome morphogenesis and/or trichome differentiation, and/or an effect on fruit yield as defined in the present specification.
  • a same or similar effect may be an effect of identical nature (e.g. the presence or absence or a decrease or increase of a given phenotype), with the same intensity or a higher or lower intensity than the reference plant.
  • Loss-of-function of the YieldPlus gene can also be assessed by a decrease or loss of the YieldPlus protein’s ability to bind target DNA.
  • Wild-type YieldPlus protein also encompass any amino acid sequence which retains the activity and expression level of the YieldPlus protein as encoded by SEQ ID NO: 3.
  • the wild-type YieldPlus protein comprises an amino acid sequence with at least 90%, preferably at least 95%, still preferably at least 98%, more preferably at least 99% identity with SEQ ID NO:3.
  • a decreased activity of the YieldPlus protein causes a phenotype of interest, i.e. an increased fruit yield.
  • a modification i.e., mutation
  • leading to a decreased or abolished activity of the YieldPlus protein, or a decreased or abolished expression of the YieldPlus protein will cause the phenotype of interest.
  • the presence of a mutant YieldPlus allele may result into a decreased or abolished activity or expression of the YieldPlus protein.
  • the mutant allele of the YieldPlus gene is a loss-of-function allele, i.e. it comprises at least one loss-of-function mutation.
  • the degree to which the function is lost can vary.
  • the sequence of the mutant allele can differ from the wild-type sequence of the gene by at least one nucleotide substitution, insertion or deletion in said sequence.
  • the mutation can be a point mutation. More particular, the mutation can be a single nucleotide polymorphism (SNP).
  • the mutant allele of the YieldPlus gene can also differ from the wild-type allele by the insertion or the deletion of one or more nucleic acid segments, including the deletion of a part or the totality of the gene or a regulatory sequence of the gene, e.g. a promoter, enhancer, 3’-UTR, 5’-UTR and/or polyA sequence.
  • the loss-of-function mutation is a null mutation.
  • a null mutation prevents expression of an active YieldPlus protein, i.e. the function is fully lost.
  • the mutant allele of the YieldPlus gene may be a null allele or knockout allele.
  • the mutation is a nonsense mutation.
  • a nonsense mutation causes a premature stop in the translation of the mRNA into a protein, resulting into the expression of a truncated form of the YieldPlus protein.
  • the mutation is a missense mutation.
  • the missense mutation may consist in the substitution of one amino acid of the YieldPlus protein by another. In some embodiments, more than one amino acid are substituted.
  • a missense mutation preferably results in a protein with decreased activity or no activity at all.
  • a preferred missense mutation is a mutation inducing the substitution of the glycine residue in position 253 of SEQ ID NO: 3. In particular, the glycine residue in position 253 of SEQ ID NO: 3 is substituted by a serine. More particularly, this missense mutation has been identified by the inventors in a mutant plant and introgressed into a different genotype. The amino acid sequence of the corresponding mutant allele is set forth in SEQ ID NO: 5.
  • the at least one mutation is a G757A mutation in the coding sequence of the Yieldplus gene, as set forth in SEQ ID NO: 2. In some embodiments the at least one mutation is a G1077A mutation in the sequence set forth in SEQ ID NO: 1.
  • the mutation is situated within the zinc finger domain of the YieldPlus protein, in particular from amino acid residues 85 to 120 of SEQ ID NO:3. In some embodiments, the mutation is located outside of the zinc finger domain of the YieldPlus, wherein said zinger domain is located from residues 85 to 120 of the amino acid sequence of the YieldPlus protein, as set forth in SEQ ID NO:3. In particular, the mutation is located at a position from amino acid residues 1 to 84 and/or from amino acid residues 121 to 277 C-terminally of the zinc finger domain of the amino acid sequence of the YieldPlus protein, as set forth in SEQ ID NO:3. More particularly, the mutation is located within the C-terminal disordered region of the YieldPlus protein. Preferably, the mutation is located at a position from amino acid residues 150 to 277 of the YieldPlus protein.
  • the substitution is a non-conservative substitution.
  • non-conservative substitutions may be defined by substitutions between or outside the groups of amino acids reflected as follows:
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1 .3); proline (-1 .6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the at least one mutation is in the coding sequence of the YieldPlus gene. In other aspects, the at least one mutation is a mutation in a regulatory sequence of the YieldPlus gene. Regulatory sequences are sequences which control a gene expression. Mutations within such sequences are known to affect the expression of the gene that they control. In some aspects, the mutation is in a regulatory sequence selected from a promoter, an enhancer, a 3’-UTR sequence, a 5’- UTR sequence and a polyA sequence.
  • the mutation can be of any type.
  • the mutation is selected from a substitution, a deletion or an addition of one or more nucleotides.
  • 1 to 100 nucleotides in particular from 1 to 50 nucleotides, more particularly from 1 to 20 nucleotides, most particularly from 1 to 10 nucleotides are mutated.
  • 1 , 2, 3, 4, 5 ,6, 7, 8, 9 or 10 nucleotides are mutated.
  • the mutation can affect one or more codons encoding the YieldPlus protein.
  • the mutant allele comprises at least one codon inserted or duplicated and/or at least one codon changed into a different codon (e.g., through a single nucleotide change), at least one codon changed into a stop codon, or at least one codon deleted.
  • from 1 to 30 codons in particular from 1 to 20 codons, more particularly from 1 to 10 codons, most particularly from 1 to 5 codons are mutated.
  • 1 , 2, 3, 4, 5 ,6, 7, 8, 9 or 10 codons are mutated.
  • the mutation affects the amino acid sequence of the YieldPlus protein
  • from 1 to 30 amino acids in particular from 1 to 20 amino acids, more particularly from 1 to 10 amino acids, most particularly from 1 to 5 amino acids may be mutated.
  • 1 , 2, 3, 4, 5 ,6, 7, 8, 9 or 10 amino acids are mutated.
  • Mutant alleles and corresponding markers can be identified by methods known in the art.
  • the mutation in the mutant YieldPlus allele can be induced via methods such as mutagenesis or by means of genetic engineering. Mutagenesis methods and methods of genetic engineering are known in the art and are described below in more details. Accordingly, the plants according to the invention may be obtained by different processes and are preferably not exclusively obtained by means of an essentially biological process.
  • said mutant allele is introgressed in the genome of said Cucumis sativus plant. [0150] In some embodiments, said mutant allele is in homozygous form. In alternative embodiments, said mutant allele is in a heterozygous form.
  • Said mutant allele confers the plant of the invention with an increased fruit yield.
  • the fruit yield is increased in comparison to an isogenic plant comprising a wild-type allele of the YieldPlus gene.
  • the plant of the invention produces at least 10%, preferably at least 20%, still preferably at least 50% more fruits in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • said Cucumis sativus plant produces at least 1 more fruit, in particular at least 2 more fruits, more particularly at least 5 more fruits, still more particularly at least 10 more fruits in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • said Cucumis sativus plant produces at least 10 fruits or more, preferably at least 12 fruits or more.
  • the fruit weight of said plant is at least 10%, preferably at least 20%, still preferably at least 50% higher in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • the average individual fruit weight of the fruits of said plant is decreased by less than 50%, in particular less than 40%, more particularly less than 30%, still more particularly less than 20%, even more particularly less than 10%, most particularly by less than 5%, in comparison to the average individual fruit weight of the fruits of an isogenic plant which comprises a wild-type allele of the YieldPlus gene, or is not decreased.
  • the average individual fruit weight of the fruits of said plant is increased by at least 10%, in particular at least 20%, more particularly at least 30%, still more particularly at least 40%, even more particularly at least 50% in comparison to the average individual fruit weight of the fruits of an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • the plant according to the invention comprises an increased average number of fruits, in particular commercial-sized fruits, per node, in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • the average number of fruits per node, in particular commercial-sized fruits is increased by at least 5%, in particular at least 10%, more particularly at least 20%, still more particularly at least 30%, even more particularly at least 40%, most particularly at least 50%, in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • the average number of fruits per node is increased by 5- 100%, in particular 5-75%, more particularly 5-50% in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • the average number of fruits per node, in particular commercialsized fruits is measured at a single harvest time-point, at a commercial stage, which can be chosen in line with growers practice, as defined in the present specification in relation the measurement of yield parameters.
  • the plant according to the invention has a faster fruit filling than an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • Faster fruit filling is assessed by measuring the time elapsed from anthesis to harvest.
  • the time elapsed from anthesis to harvest of a plant according to the invention is decreased by at least 5%, in particular at least 10%, more particularly at least 20%, still more particularly at least 30%, even more particularly at least 40%, most particularly at least 50%, in comparison to an isogenic plant which comprises a wildtype allele of the YieldPlus gene.
  • the time elapsed from anthesis to harvest of a plant according to the invention is decreased by 5-75%, in particular 5-50%, more particularly 5-30% in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • the time of harvest is a single harvest time-point, at a commercial stage, chosen in line with growers practice, as defined in the present specification in relation to the measurement yield parameters.
  • said cucumber plant is not exclusively obtained by means of an essentially biological process.
  • a C. sativus plant according to the invention is a cultivated plant or line, more preferably a commercial plant or line or hybrid.
  • a plant or line thus has generally 4 primary branches or less, generally less than 3 and more preferably has a single primary branch.
  • Such a commercial plant or line preferably also exhibits resistance to viruses.
  • a commercial plant is resistant to ZYMV (Zucchini Yellow Mosaic Virus) and/or to CVYV (Cucumber Vein Yellowing Virus). Resistances to PRSV (Papaya Ringspot Virus) and WMV (Watermelon Mosaic Virus) and CMV (Cucumber Mosaic Virus) are also generally found in commercial plants.
  • a plant of the invention is thus advantageously resistant at least to powdery mildew and to potyviruses.
  • resistances or tolerances are also envisaged according to the invention, inter alia resistance to downy mildew caused Pseudoperonospora cubensis, resistance to Fusaria caused by Fusariumoxysporum f.sp. cucumerinum or by Fusarium oxysporum f.sp. radicis cucumerinum, resistance to scab caused by Cladosporium cucumerinum, resistance to WMV, to CYSDV and to CCYV, resistance to angular leaf sport and, resistance to anthracnose.
  • the present invention is directed to one or more plant parts of a plant according to the invention.
  • Such plant part comprises a mutant allele of the YieldPlus gene.
  • the different features of said YieldPlus gene that have been defined in relation with the above aspects of the invention apply mutatis mutandis to this aspect of the invention.
  • the plant part is a seed, explant, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole or flower.
  • a particularly preferred plant part according to the invention is a seed produced by the plant of the invention.
  • the seed comprises homozygously the mutant alleles of the YieldPlus gene.
  • the plant part is a plant cell.
  • one aspect of the invention relates to a cell of a C. sativus plant according to the invention, i.e. a plant cell comprising a mutant allele of the YieldPlus gene.
  • the different features of said YieldPlus gene that have been defined in relation with the above aspects of the invention apply mutatis mutandis to this aspect of the invention.
  • a plant cell of the invention may have the capacity to be regenerated into a whole plant.
  • a plant cell according to the invention is derived from a seed, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole or flower.
  • the invention further relates to a seed from a plant of the invention.
  • the seed may be produced by such a plant after selfing or crossing.
  • said seed is capable of germinating into a plant with increased fruit yield.
  • the invention also provides C. sativus plants grown from the seeds of the invention.
  • the invention further relates to a seed for producing a plant according to the invention.
  • Said seed can be grown into a plant of the invention.
  • said plant, seed, cell or part comprises a mutant allele of a YieldPlus gene on chromosome 3 in its genome, wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , resulting in an increased fruit yield of a plant grown from said seed, in comparison to an isogenic plant comprising a wild-type allele of the YieldPlus gene.
  • the invention also relates to a population of C. sativus seeds according to the invention, wherein said population comprises at least 2 seeds, especially at least 10 seeds, particularly at least 100 seeds, even more particularly at least 10 5 or 10 6 seeds.
  • the present invention is also directed to an in vitro cell or tissue culture of regenerable cells of the plant as defined above according to the present invention.
  • the regenerable cells are derived from a seed, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole or flower of a plant of the invention, and comprise in their genome a mutant allele of the YieldPlus gene as described here above.
  • the tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing C. sativus plant, and of regenerating plants having substantially the same genotype as the foregoing C. sativus plant.
  • the present invention also provides C. sativus plants regenerated from the tissue cultures of the invention.
  • the invention also provides a protoplast of the plant defined above, or from the tissue culture defined above, said protoplast comprising in its genome a mutant allele of the YieldPlus gene, as described here above.
  • the present invention also encompasses asexual processes of propagation. Accordingly, one aspect of the invention relates to a method of producing C. sativus plant with an increased fruit yield, comprising: (a) obtaining a part of a plant according to the invention, such as a cutting; and
  • the generated C. sativus plant comprises in its genome a mutant allele of the YieldPlus gene, wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , resulting in an increased fruit yield.
  • the invention described above encompasses a plant, seed, plant part and cell of any ploidy levels. It encompasses inter alia diploid, triploid, tetrapioid and/or allopolyploid Cucumis sativus plant, plant part, cell or seed.
  • the invention further relates to an isolated polynucleotide comprising a mutant allele of the YieldPlus gene, wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , wherein said mutant allele confers increased fruit yield to a plant comprising said mutant allele, in comparison to an isogenic plant which comprises a wild-type allele of the YieldPlus gene.
  • said polynucleotide is isolated or susceptible to be isolated from a plant according to the invention.
  • the invention relates to a plant, plant part or plant cell comprising a polynucleotide according to the invention.
  • said polynucleotide comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 2.
  • said polynucleotide comprises a nucleotide sequence having at least 85%, more particularly at least 90%, even more particularly at least 95%, at least 96%, at least 97%, at least 98% or at least 99% with SEQ ID NO: 2.
  • said polynucleotide comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 1.
  • said polynucleotide comprises a nucleotide sequence having at least 85%, more particularly at least 90%, even more particularly at least 95%, at least 96%, at least 97%, at least 98% or at least 99% with SEQ ID NO: 1 .
  • said polynucleotide encodes a polypeptide having at least 80% sequence identity with SEQ ID NO: 3.
  • said polynucleotide encodes a polypeptide having at least 85%, more particularly at least 90%, even more particularly at least 95%, at least 96%, at least 97%, at least 98% or at least 99% with SEQ ID NO: 3.
  • said polypeptide comprises the sequence of SEQ ID NO: 3 in which at least 1 %, in particular at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of said sequence has been modified.
  • said polypeptide comprises the sequence of SEQ ID NO: 3 in which 1-10%, 10-20%, 20- 30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of said sequence has been modified.
  • said polypeptide comprises the sequence of SEQ ID NO: 3, or a sequence with at least 95%, in particular at least 98%, more particularly at least 99% identity with SEQ ID NO:3, in which at least 1 %, in particular at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of said sequence has been deleted and/or truncated, and/or in which 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-80%, 80-90% or 90-100% of said sequence has been deleted and/or truncated.
  • said polypeptide comprises the sequence of SEQ ID NO: 3, or a sequence with at least 95%, in particular at least 98%, more particularly at least 99% identity with SEQ ID NO:3, in which at least 1 %, in particular at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the C-terminal domain of the YieldPlus protein, preferably of the fragment from amino acid residues 150 to 277 of the YieldPlus protein, has been deleted and/or truncated, and/or in which 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the C-terminal domain of the YieldPlus protein has been deleted and/or truncated.
  • said polynucleotide comprises the sequence of SEQ ID NO:4.
  • said polynucleotide encodes the sequence of SEQ ID NO:5.
  • the invention also relates to a polypeptide encoded by a polynucleotide of the invention.
  • the invention relates to an isolated polypeptide comprising an amino acid sequence with at least 95%, in particular at least 98%, more particularly at least 99% identity with SEQ ID NO:3, wherein said polypeptide comprises at least one mutation in the amino acid sequence, in comparison to the sequence of the corresponding wild-type polypeptide as set forth in SEQ ID NO:3, wherein said mutation confers a loss-of-function phenotype to said polypeptide.
  • said polypeptide is as defined in the present specification.
  • the PILEUP and BLAST algorithms can also be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul, 1993, J Mol Evol 36:290-300; Altschul ef al, 1990, J Mol Biol 215:403-10, the disclosures of which are incorporated herein by reference).
  • said polypeptide has the sequence of SEQ ID NO:5.
  • the invention is also directed to the use of the information provided herewith by the present inventors, namely a mutant YieldPlus allele conferring an improved fruit yield to C. sativus plants compared to the wild type allele, as set forth in SEQ ID NO: 1 .
  • This knowledge can be used inter alia to generate new mutant plants comprising a mutated YieldPlus allele and expressing the phenotype of interest (i.e., increased fruit yield).
  • new mutant can be generated de novo by e.g. targeted gene editing techniques, such as CRISPR based techniques or by mutagenesis, such as radiation induced mutagenesis or chemically induced mutagenesis.
  • the skilled person can for example generate a plant with a mutant YieldPlus gene and determine whether it results in an increased fruit yield compared to the same plant with the wild type YieldPlus gene, in particular in homozygous form (i.e. isogenic plants).
  • a plant homozygous for the mutant allele can be generated by selfing the plant and then growing the homozygous plant to determine whether the yield is higher in the homozygous mutant plant in comparison to a wild-type control.
  • the invention is thus also directed to a method for generating or producing cucumber plant comprising a mutant allele of the YieldPlus gene.
  • a method for generating or producing cucumber plant comprising a mutant allele of the YieldPlus gene.
  • such method may comprise mutagenizing one or more Cucumis sativus seed, plant or plant part and screening/selecting the M1 or M2 generation for YieldPlus mutant alleles.
  • the present invention also relates to a method for producing a C. sativus plant with an increased fruit yield, comprising the introduction of at least one mutation in the YieldPlus gene on chromosome 3 in the genome of a C. sativus plant, wherein said mutation is introduced in the sequence of the gene or in a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 , resulting in an increased fruit yield.
  • the present invention also relates to a method for producing a C. sativus plant with an increased fruit yield, comprising: a) Introducing one or more mutations in cucumber plant(s), seed(s) or plant part(s), b) Optionally, determining if the plant, seed or plant part under a) presents an increased yield compared to a plant not having said at least one mutation; and c) Selecting a plant that comprises a mutant allele of a YieldPlus gene, wherein said mutant allele comprises at least one mutation in the sequence of the gene or in a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele, as set forth in SEQ ID NO: 1 , resulting in an increased fruit yield.
  • said mutation is introduced by mutagenesis or genome editing.
  • said mutation is introduced by a technique selected from ethyl methanesulfonate (EMS) mutagenesis, oligonucleotide directed mutagenesis (ODM), Zinc finger nuclease (ZFN) technology, Transcription Activator-Like Effector Nucleases (TALENs) the CRISPR/Cas system, the CRISPR/Cpf system engineered meganuclease, re-engineered homing endonucleases and DNA guided genome editing.
  • EMS ethyl methanesulfonate
  • EMS ethyl methanesulfonate
  • said mutation is introduced by an endonuclease-mediated technique selected from the Zinc finger nuclease (ZFN) technology, the TALEN technology, the CRISPR/Cas system and the CRISPR/Cpf system, preferably the CRISPR/Cas system, most preferably the CRISPR/Cas9 system.
  • ZFN Zinc finger nuclease
  • TALEN Zinc finger nuclease
  • CRISPR/Cas system preferably the CRISPR/Cas system, most preferably the CRISPR/Cas9 system.
  • Mutagenesis methods include chemical mutagenesis using ethyl methanesulfonate (EMS).
  • Other chemical mutagenic agents include but are not limited to, diethyl sulfate (des), ethyleneimine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea(enu), and sodium azide.
  • the mutations can be induced by means of irradiation, which is for example selected from x-rays, fast neutrons, UV radiation.
  • TILLING Targeting Induced Local Lesions IN Genomes
  • TILLING 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.
  • TILLING uses traditional chemical mutagenesis (e.g. EMS or MNU mutagenesis) or other mutagenesis methods (e.g.
  • S1 nucleases such as CEL1 or ENDO1
  • electrophoresis such as a Ll- 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 cucumber (Fraenkel et al., BMC Res Notes. 2014 Nov 26;7:846. doi: 10.1186/1756-0500-7-846).
  • the mutation(s) is(are) induced by means of genetic engineering.
  • the genetic engineering means which can be used include the use of all such techniques called New Breeding Techniques which are various new technologies developed and/or used to create new characteristics in plants through genetic variation, the aim being targeted mutagenesis, targeted introduction of new genes or gene silencing (RdDM).
  • New Breeding Techniques which are various new technologies developed and/or used to create new characteristics in plants through genetic variation, the aim being targeted mutagenesis, targeted introduction of new genes or gene silencing (RdDM).
  • Example of such new breeding techniques are targeted sequence changes facilitated through the use of Zinc finger nuclease (ZFN) technology (ZFN- 1 , ZFN-2 and ZFN-3, see U.S. Pat. No.
  • Oligonucleotide directed mutagenesis ODM
  • Cisgenesis RNA-dependent DNA methylation
  • RdDM RNA-dependent DNA methylation
  • Grafting on GM rootstock
  • Transcription Activator-Like Effector Nucleases TALENs, see U.S. Pat. Nos. 8,586,363 and 9,181 ,535, incorporated by reference in their entireties
  • the CRISPR/Cas system see U.S. Pat. Nos.
  • Such applications can be utilized to generate mutations (e.g., targeted mutations or precise native gene editing) as well as precise insertion of genes (e.g., cisgenes, intragenes, or transgenes).
  • the applications leading to mutations are often identified as site-directed nuclease (SDN) technology, such as SDN1 , SDN2 and SDN3.
  • SDN site-directed nuclease
  • the outcome is a targeted, non-specific genetic deletion mutation: the position of the DNA DSB is precisely selected, but the DNA repair by the host cell is random and results in small nucleotide deletions, additions or substitutions.
  • a SDN is used to generate a targeted DSB and a DNA repair template (a short DNA sequence identical to the targeted DSB DNA sequence except for one or a few nucleotide changes) is used to repair the DSB: this results in a targeted and predetermined point mutation in the desired gene of interest.
  • the SDN3 is used along with a DNA repair template that contains new DNA sequence (e.g. gene). The outcome of the technology would be the integration of that DNA sequence into the plant genome.
  • the invention relates to a method for obtaining a C. sativus plant or seed carrying one or more mutations in its genome.
  • a method for obtaining a C. sativus plant or seed carrying one or more mutations in its genome Such a method is illustrated in the Examples and may comprise: a) treating M0 seeds of a C. sativus plant to be modified with a mutagenic agent to obtain M1 seeds; b) growing plants from the thus obtained M1 seeds to obtain M1 plants; c) producing M2 seeds by self-fertilisation of M1 plants; and d) optionally repeating step b) and c) n times to obtain M2+n seeds.
  • the M1 seeds of step a) can be obtained via chemical mutagenesis such as EMS mutagenesis.
  • chemical mutagenic agents include but are not limited to, diethyl sulfate (des), ethyleneimine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea(enu), and sodium azide.
  • the mutations are induced by means of irradiation, which is for example selected from x-rays, fast neutrons, UV radiation.
  • the mutation(s) is(are) induced by means of genetic engineering.
  • Such mutations also include the integration of sequences, as well as the substitution of residing sequences by alternative sequences.
  • a further aspect of the invention relates to the use of a C. sativus plant or seed according to the invention, as a breeding partner in a breeding program for conferring increased fruit yield to progeny C. sativus plants. By crossing a C. sativus plant of the invention with another plant, e.g. having a different genotype, for instance a susceptible or less resistant plant, it is possible to transfer the mutant YieldPlus allele, conferring the desired phenotype, to the progeny.
  • the selection of the progeny displaying the desired phenotype, or bearing sequences linked to the desired phenotype can advantageously be carried out with markers, e.g. the markers disclosed in the present specification.
  • the selection of the progeny having the desired phenotype can also be made by assessing the phenotype of the progeny plants.
  • the invention is also directed to the use of said plants in a program aiming at identifying, sequencing and/or cloning the genetic sequences conferring the desired phenotype.
  • the invention also concerns methods for the production of a C. sativus plant having increased fruit yield, comprising the following steps:
  • step b) optionally self-pollinating and/or backcrossing one or several times the plant selected at step b) and selecting in the progeny thus obtained a plant a mutant allele of the YieldPlus gene.
  • the self-pollination and backcrossing steps may be carried out in any order and can be intercalated, for example a backcross can be carried out before and after one or several self-pollinations, and self-pollinations can be performed before and after one or several backcrosses.
  • the selection of the progeny can advantageously be carried out with markers, e.g. the markers disclosed in the present specification.
  • the selection of the progeny having the desired phenotype can also be made by assessing the phenotype of the progeny plants.
  • the inventors have been able to develop a genotyping assay to distinguish between the wild type allele and a mutant allele of the YieldPlus gene. Thus, is it now possible to detect the presence of a mutant allele of the YieldPlus gene.
  • SEQ ID NO: 6 Three nucleotide sequences were designed, two forward and one reverse primers (SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8). These three sequences were designed based on the following sequences: SEQ ID NO: 1 or 2 (i.e., wild type allele) and SEQ ID NO:4 (i.e., mutant allele). These are sequences of the reverse strand (- strand) of the alleles.
  • the invention is also directed to a method for detection and/or selecting a cucumber plant, seed or, plant part having an increased fruit yield, comprising the steps of:
  • mutant allele of the YieldPlus gene on chromosome 3 wherein said mutant allele comprises at least one mutation in the sequence of the gene or a regulatory sequence thereof, in comparison to the sequence of the corresponding wild-type allele as set forth in SEQ ID NO: 1 ;
  • step (b) comprises carrying out a genotyping assay, using the DNA samples of a) as template, that discriminates between the wild type YieldPlus allele and the mutant allele, wherein said genotyping assay is based on nucleic acid amplification making use of oligonucleotide primers and probes.
  • said genotyping assay uses at least one pair of oligonucleotide primers specific for the mutant YieldPlus allele. Said genotyping assay may also use at least one pair of oligonucleotide primers specific for the wild-type YieldPlus allele. In other embodiments, said genotyping assay is based on nucleic acid hybridization making use of YieldPlus allele-specific oligonucleotide probes.
  • said oligonucleotide primers comprise at least 10 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 or of the complement strand of SEQ ID NO: 1 or SEQ ID NO: 2.
  • said plant selected at step (c) comprises one ortwo copies of said mutant allele.
  • step a) comprises the isolation of genomic DNA from the plant, seeds, plant part, cell or tissue to be analyzed in the genotyping assay. Crude DNA extractions methods can be used as known in the art.
  • the plant may be mutagenized using any mutagenesis technique as described herein, e.g. chemical radiation, mutagens or gene editing techniques.
  • the method may comprise, prior to step a), a step of mutagenizing a Cucumis sativus plant to induce one or more mutations in the YieldPlus gene or regulatory sequence thereof.
  • step b) the genotyping assay is carrying out using the DNA samples of a) as template.
  • Various genotyping assays can be used, as long as they can detect the at least one mutation present in the mutant allele of the YieldPlus gene, e.g. an insertion, deletion or substitution, and can differentiate between the wild type allele of SEQ ID NO: 1 being present in the genomic DNA sample (at the YieldPlus locus on chromosome 3) or a mutant allele of the YieldPlus gene being present in the genomic DNA sample.
  • 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 BHQPIus probes (see also biosearchtech.com).
  • genotyping assays are the KASP-assay (by LGC, see at LGCgenomics.com and also 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 at LGCgenomics.com and also at biosearchtech.com/products/pcr-kits-and-reagents/genotyping-assays/kasp-genotyping- chemistry
  • the KASP-assay is also described in He C, Holme J, Anthony J. ‘SNP genotyping: the KASP assay. Methods Mol Biol. 2014;1145:75-86’ and EP1726664B1 or US7615620 B2, incorporated by reference.
  • the KASP genotyping assay utilizes a unique form of competitive allele-specific PCR combined with a novel, homogeneous, fluorescence-based reporting system for the identification and measurement of genetic variation occurring at the nucleotide level to detect single nucleotide polymorphisms (SNPs) or inserts and deletions (InDeis).
  • the KASP technology is suitable for use on a variety of equipment platforms and provides flexibility in terms of the number of SNPs and the number of samples able to be analyzed.
  • the KASP chemistry functions equally well in 96-, 384-, and 1 ,536-well microtiter plate formats and has been utilized over many years in large and small laboratories by users across the fields of human, animal, and plant genetics.
  • the amplification may be carried out by PCR cycles, comprising a first denaturation step at 94°C during around 15 minutes, at least 10 cycles of around 20 seconds at 94°C followed by around 60 second at a decreasing temperature from 65°C for the 1st cycle to 57°C for the last cycle, and around 35 cycles of around 20 seconds at 94°C followed by around 60 seconds at 57°C.
  • This protocol can easily be adapted by a skilled person, depending on the type of primers used.
  • Various genotyping assays can, therefore, be used, which can differentiate between the presence of the wild type allele of the YieldPlus gene, encoding the protein of SEQ ID NO: 3, and a mutant allele of the YieldPlus gene.
  • a bi-allelic genotyping assay 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.
  • a bi-allelic genotyping assay 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.
  • the genotyping assay in step b) of the methods above is a KASP-assay.
  • a competitive PCR is carried out using two forward primers and one common reverse primer.
  • the two forward primers comprise at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides complementary to SEQ ID NO: 1 or SEQ ID NO:2 (or the complement strand thereof).
  • 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 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.
  • the Fam- primer comprises 17 nucleotides of the wild type sequence and 1 nucleotide specific for the insertion allele
  • the VIC -primer comprises 18 nucleotides of the wild type allele and 1 nucleotide specific to the ‘deletion’ allele.
  • a KASP-assay can easily be designed to differentiate between the wild type allele of SEQ ID NO: 1 and any mutant allele of the YieldPlus gene which differs from the wild type allele in one or more nucleotides being inserted, deleted or substituted, so e.g. the assay can be designed for any SNP or INDEL that differentiates two alleles.
  • the invention relates to a method for the production of C. sativus plantlets or plants, which method comprises:
  • the isolated cell or tissue used to produce a micro-plantlet is an explant obtained under sterile conditions from a C. sativus parent plant of the invention to be propagated.
  • the explant comprises or consists, for instance, of a cotyledon, hypocotyl, stem tissue, leaf, embryo, meristem, node bud, shoot apice, or protoplast.
  • the explant can be surface sterilized before being placed on a culture medium for micropropagation. Conditions and culture media that can be suitably used in plant micropropagation are well known to those skilled in the art of plant cultivation and are described, for example, in "Plant Propagation by Tissue Culture, Handbook and Directory of Commercial Laboratories, eds. Edwin F George and Paul D Sherrington, Exegetics Ltd, 1984".
  • Micropropagation typically involves: axillary shoot production: axillary shoot proliferation is induced by adding cytokinin to the shoot culture medium, to produce shoots preferably with minimum callus formation; adventitious shoot production: addition of auxin to the medium induces root formation, in order to produce plantlets that are able to be transferred into the soil. Alternatively, root formation can be induced directly into the soil.
  • Plantlets can be further subjected to an in vivo culture phase, by culture into the soil under lab conditions, and then progressive adaptation to natural climate, to develop into C. sativus plants.
  • the invention is thus also directed to a method for improving the yield of C. sativus fruits and/or for increasing the number of harvestable C. sativus fruits comprising growing C. sativus according to the invention.
  • the invention is also directed to the use of the C. sativus plants of the invention for improving the yield of C. sativus plants and/or fruits, and/or for increasing the number of harvestable cucumber fruits.
  • the invention is also directed to a method of increasing the productivity of a C. sativus field, tunnel or glasshouse.
  • the method comprises a first step of choosing or selecting a C. sativus plant comprising a mutant allele of the YieldPlus gene.
  • the method comprises:
  • the yield of cucumber production is increased, inter alia more marketable cucumbers can be harvested, and/or more seeds are obtained.
  • the invention relates to a method of producing cucumber fruits comprising:
  • the method may comprise a step of processing said cucumber fruit into a processed food and/or a step of mixing the cucumber fruits or part thereof with one or more food ingredients.
  • the present invention also relates to a method of producing a food product, comprising mixing a cucumber fruit of the invention, or part thereof, with one or more food ingredients.
  • the method comprises cooking and/or processing the cucumber fruit of the invention, alone or in mixture with the one or more food ingredients.
  • the present invention also relates to a food product made of a cucumber fruit of the invention or parts thereof, optionally in processed form.
  • the invention relates to the use of a C. sativus plant according to the invention or a fruit thereof in the fresh cut market or for food processing.
  • Example 1 Generation of an EMS population.
  • a cucumber (Cucumis sativus) mutant TILLING population was developed from a cucumber pickling line using an EMS (Ethyl methanesulfonate) treatment, in order to introduce random point mutations in the genome.
  • EMS Ethyl methanesulfonate
  • the YieldPlus gene encodes a zinc finger protein 8-like transcription factor.
  • this gene is located on the chromosome 3 (negative strand) from 31 ,790,724 nucleotide to 31 ,792,269 nucleotide (Cucumis. sativus L. var. sativus var. 9930 v3; http://cucurbitgenomics.org/feature/gene/CsaV3_3G038590).
  • Table 1 Description of the mutant plant generated. Nucleotide mutated position refers to the nucleotide number in the corresponding sequence number, i.e., SEQ ID NO.
  • SEQ ID NO The genome position mentioned with respect to the cucumber as published by Li et al, 2019 A chromosome-scale genome assembly of cucumber (Cucumis sativus L.). Gigascience, Volume 8, Issue 6, June 2019, giz072).
  • a KASP marker-assay was developed to distinguish between the wild type allele of the gene, shown in SEQ ID NO: 1 , and the mutant allele of the YieldPlus gene comprising a mutation, in the present case a substitution, shown in SEQ ID NO: 4.
  • Table 2 markers and primers for mutation detection on minus DNA strand
  • DNA sequences for the KASP assay were designed on the reverse DNA strand (minus strand) but can be equally designed based on the plus strand of the allele.
  • Plus and minus strands are complementary strands of the double stranded DNA.
  • Nucleotide G corresponds to a C in the complementary strand and nucleotide A corresponds to a T in the complementary strand.
  • Example 2 Introgression of the mutant allele
  • mutant family was selfed to get cucumber plants bearing the mutant allele homozygously (MutantHOM).
  • the BC2F2 conversions of the mutant family show a significant difference in fruit weight per plant (Figure 1A) and fruit number per plant (Figure 1 B) between the homozygote conversion (A:A) and the one that does not have the mutation (G:G).
  • the mutant allele of the YieldPlus gene (SEQ ID NO: 4) is responsible forthe increased yield of the cucumber plants comprising the mutant allele in homozygous form.
  • the BC2F2 conversions of the mutant family do show a difference below 10%, even below 5%, in average individual fruit weight between the plants with the homozygote conversion (A:A) and the ones that do not have the mutation (G:G). Plants having the mutant allele demonstrate an average individual fruit weight of 136g whereas plants having the wild-type allele demonstrate an average individual fruit weight of 142g.
  • the point mutation in the mutant corresponds to a substitution of a G by a A at position 757 of SEQ ID NO: 2.
  • This substitution corresponds to a change of a glycine into a serine in position 253 of the protein sequence (SEQ ID NO:3).
  • This amino acid residue is located in a C-terminal disordered portion of the protein and not in the zinc finger domain.
  • This mutation is predicted to have a neutral impact using the Provean (1.528) and SIFT (0.551) scores.
  • Cas9/sgRNA constructs were designed to target sequences in the coding sequence of the YieldPlus gene (SEQ ID NO: 1) or in 3’-UTR or 5’-UTR sequences.
  • the Cucumis sativus TO-mutant plant are cross-pollinated with a wild type cucumber (Beit-Alpha parthenocarpy cucumber).
  • the mutant alleles are followed by molecular marking, evidencing stable transmission of the mutation.
  • the T1 mutant plants are grown and crosspollinated to produce mutant plants, with a homozygous mutation in the YieldPlus gene.
  • T2 and T3 plants are further generated.
  • the edited plants are evaluated for their fruit yield phenotype. Plants showing increased fruit yield with respect to wild-type control plants are selected for further introgression of the edited allele in cucumber genotypes of agricultural interest.
  • Another mechanism investigated as possibly responsible for increased yield is faster fruit filling.
  • a fruit When a fruit reaches commercial size and is picked, it contributes to increased yield.
  • the time elapsed from anthesis to harvest at a commercial stage is measured.
  • the time elapsed from anthesis to harvest at a commercial stage is measured in a mutant line according to the invention, as compared to a reference line which does not comprise the mutant allele, e.g. an isogenic wild-type line.
  • the line with the favorable allele e.g. the mutant allele

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Abstract

La présente invention concerne une plante Cucumis sativus ayant une production de fruits accrue. L'invention concerne en outre des marqueurs liés au phénotype de production de fruits accrue et l'utilisation de tels marqueurs pour identifier ou sélectionner des plantes ayant un tel phénotype. L'invention concerne également les graines et la descendance de telles plantes et le matériel de propagation pour obtenir de telles plantes.
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