WO2023020938A1 - Plante de laitue à montée à graines retardée - Google Patents

Plante de laitue à montée à graines retardée Download PDF

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WO2023020938A1
WO2023020938A1 PCT/EP2022/072583 EP2022072583W WO2023020938A1 WO 2023020938 A1 WO2023020938 A1 WO 2023020938A1 EP 2022072583 W EP2022072583 W EP 2022072583W WO 2023020938 A1 WO2023020938 A1 WO 2023020938A1
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plant
amino acid
seq
mutant
protein
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PCT/EP2022/072583
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English (en)
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Gowtham PRAKASH
Wim VRIEZEN
Lieke MERTENS
Daan SCHREURS
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Nunhems B.V.
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Publication of WO2023020938A1 publication Critical patent/WO2023020938A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/121—Plant growth habits
    • A01H1/1215—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • 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
    • A01H5/12—Leaves
    • 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
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
    • A01H6/1472—Lactuca sativa [lettuce]
    • 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

Definitions

  • the present invention relates to the field of plant breeding.
  • a Lactuca sativa plant having delayed bolting wherein said plant comprises in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene.
  • the present invention provides a seed from which the Lactuca sativa plant according to the present invention can be grown.
  • the present invention further provides a plant cell, tissue or plant part of the plant according to the present invention or of the seed according to the present invention, comprising the mutant allele of the wild type FT gene.
  • the present invention provides a method for identifying and/or selecting a Lactuca sativa plant or plant part comprising determining whether said plant or plant part comprises in its genome at least one copy of a mutant allele of the wild type FT gene. Also a genotyping assay for detecting the mutant allele is provided, which can be used e.g. in marker assisted selection of the mutant allele.
  • the present invention provides a method for generating a Lactuca sativa plant comprising a mutant allele which delays bolting of said plant when present in homozygous form. Further provided is a method of producing a lettuce crop, comprising growing a plant comprising preferably two copies of the mutant allele which delays bolting of the crop and thereby increases the harvest window by e.g.
  • the biomass of the lettuce plants is increased, as the plant does not use its resources to make reproductive organs via flowering, but uses the resources for leaf growth instead, so that at (later) harvest more biomass (e.g. more leaves and/or a more compact head) is present compared to the control.
  • Bolting is an essential process in the growth and development of a lettuce plant (Lactuca sativa L.). Bolting is the switch from vegetative to reproductive stage, when the stem extends to make flowers. Environmental stress, such as an increased temperature, can induce bolting of the lettuce plant, which decreases both the quality and quantity of the harvested plant material. Resistance to early bolting accordingly is an important trait in breeding of lettuce cultivars. A decreased sensitivity to environmental conditions which cause early bolting not only allows the production of lettuce having an improved quality and/or and increased biomass of the lettuce heads but also allows broadening of the harvest window.
  • Lettuce cultivars may vary widely in the number of days necessary from sowing to bolting and flowering. Although there is natural variation in late bolting at the disposal of the breeders to achieve the genetic improvement, complexity of inheritance makes it challenging to breed efficiently. This is partially caused by the fact that the molecular mechanisms regulating the bolting and flowering characteristics in lettuce are largely unknown. Also, as reviewed by Ho et al. 2021 (supra), 167 QTLs have been described in lettuce for bolting, flowering time or both. Bolting and flowering time QTLs are located on all of the 9 chromosomes of the lettuce genome, see Figure 1 of Ho et al.
  • FT Three genes, FT, SOC1 and LFY are so-called ‘floral integrator’ genes, as they connect the floral induction pathways to the floral development pathway (see also the simplified model of Arabidopsis in Figure 2 of Han et al 2021).
  • LsFT encodes a small protein, which is produced in the mature leaves upon induction of gene expression and the wild type protein likely travels via the phloem sap to the shoot apical meristems.
  • Fukuda et al. 2011 J. of Plant Physiology 168, 1602-1607) studied the expression of LsFT and also overexpressed LsFT underthe 35S promoter to see if in transgenic Arabidopsis the function is the same and can be complemented.
  • overexpression of LsFT reduced the number of day to flower, LsFT did not fully complement AtFT in regulating flowering time in Arabidopsis.
  • LsFT knockdown by RNAi (Chen et al. 2018, Front Plant Science 8: 2248, supra), delayed bolting and diminished bolting in response to heat treatment.
  • LsFT knockdown lines also showed reduced gene expression of LsAP1 , LsAP3 and LsLFY, suggesting that these genes function downstream of LsFT in the pathway (see Ho et al. at page 9, left column).
  • Ho et al also mention that further molecular analyses is needed to clarify the regulatory relationship between LsFT and another floral integrator protein, LsSOCI (page 9, right column), which also plays a role in bolting in lettuce, but which appears to be a different role than SOC1 has in other species.
  • the present invention provides in one aspect a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the wild type FT gene encodes a protein of SEQ ID NO: 1 , or a protein comprising at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to (the wild type protein of) SEQ ID NO: 1 , and wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the equivalent amino acid in a wild type protein comprising at least 95% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid residue.
  • the plant may either be heterozygous for the mutant allele, or homozygous for the mutant allele.
  • a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein one amino acid in the Phosphatidylethanolamine Binding domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 , is replaced by a different amino acid, preferably under the proviso that this one amino acid is not an amino acid selected from D71 , Y85, H87, P111 , R112, P113, S114, H118, M120, and wherein the mutant allele delays the start of bolting of the Lactuca sativa plant by at least 7 days when the mutant allele is present in homozygous form, compared to the control plant lacking the mutant allele, and wherein the wild type allele encodes the protein of SEQ ID NO: 1 (or a protein comprising at least 95%, 96%, 97%
  • a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a truncated protein wherein e.g.
  • FT Flowering Locus T
  • mutant allele delays the start of bolting of the Lactuca sativa plant by at least 7 days compared to the control plant lacking the mutant allele and wherein the mutant allele additionally prevents flowering, when the mutant allele is present in homozygous form, and wherein the wild type allele encodes the protein of SEQ ID NO: 1 (or a protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1).
  • the plant may either be heterozygous for the mutant allele, or homozygous for the mutant allele.
  • the present invention provides a seed from which the Lactuca sativa plant according to the present invention can be grown.
  • the present invention further provides a plant cell, tissue or plant part of the Lactuca sativa plant according to the present invention or of the seed according to the present invention, comprising the mutant allele of the wild FT gene, as described herein, in heterozygous or preferably in homozygous form.
  • the present invention provides a method for producing harvested plant material, such as lettuce heads or leaves, having an improved average quality and/or having an increased average mass, said method comprising growing a Lactuca sativa plant or seed according to the present invention, preferably comprising the mutant allele in homozygous form, and harvesting the lettuce head or leaves produced by said Lactuca sativa plant.
  • the present invention provides a method for identifying and/or selecting a Lactuca sativa plant or plant part comprises in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the equivalent amino acid in a wild type protein comprising at least 95% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid residue, said method comprising analysing the genomic DNA for the presence of the mutant allele, e.g. using a PCR method, a SNP genotyping method or sequencing.
  • FT Flowering Locus T
  • the present invention provides a method for identifying and/or selecting a Lactuca sativa plant or plant part comprises in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a truncated protein wherein e.g.
  • FT Flowering Locus T
  • mutant allele delays the start of bolting of the Lactuca sativa plant by at least 7 days compared to the control plant lacking the mutant allele and wherein the mutant allele additionally prevents flowering, when the mutant allele is present in homozygous form, and wherein the wild type allele encodes the protein of SEQ ID NO: 1 (or a protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1), said method comprising analysing the genomic DNA for the presence of the mutant allele, e.g. using a PCR method, a SNP genotyping method or sequencing.
  • the present invention provides a method for producing or generating a Lactuca sativa plant comprising a mutant allele which delays bolting of said plant when present in homozygous form, said method comprising the step(s) of: (i) crossing a first Lactuca sativa plant and a second plant, wherein the first Lactuca sativa plant comprises in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the equivalent amino acid in a wild type protein comprising at least 95% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid residue; and optionally (ii) harvesting seed from the crossing of (i), and optionally (iii) selecting seed comprising said mutant allele in its genome.
  • FT Flowering Locus T
  • the present invention provides a method for producing or generating a Lactuca sativa plant comprising a mutant allele which delays bolting of said plant when present in homozygous form, said method comprising the step(s) of: (i) crossing a first Lactuca sativa plant and a second plant, wherein the first Lactuca sativa plant comprises in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein one amino acid in the Phosphatidylethanolamine Binding domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 , is replaced by a different amino acid, preferably under the proviso that this one amino acid is not an amino acids selected from D71 , Y85, H87, P111, R112, P113, S114, H118, M120, and wherein the mutant allele delays the start of
  • Figure 1 Picture depicting the comparison of shoot apical meristem in transversely cut lettuce head (of the same age and grown under the same conditions) showing the LsFT homozygous P75S mutant on the left and an isogenic wild type control plant on the right.
  • Figure 2 Picture of the LsFT homozygous P75S mutant line on the right (right of the line) and an isogenic azygous control line, comprising the wild type LsFT allele, on the left (left of the line).
  • the age of the plants is 49 das (days after sowing). As can be seen, the control is bolting, while the mutant line is not bolting.
  • FIG. 3 Picture of the W138* mutant homozygous line, which has bolted in the picture, but which has developed whorls of leaves on the stalk (or stem) instead of inflorescences I flowers. The picture is from 115 days after sowing. See Examples, where the W138* mutant starts bolting after 95 das (days after sowing), but never flowers, while the azygous control starts flowering after 95 das. The wild type control is, thus, flowering at this stage, while the W138* mutant developed whorls of leaves on the stem/stalk.
  • Figure 4 Figure which shows the wild type LsFT protein of 175 amino acids, aligned with the conserved, functional Pfam01161 domain of SEQ ID NO: 17 (Phosphatidylethanolamine Binding domain, PB domain). Open triangles pointing at an amino acid represent amino acids in the PB domain which are involved in substrate binding, such as ATP or phosphotidylethanolamine. Boxed amino acids show the mutant proteins generated herein, P75S (leading to delayed bolting and delayed flowering), P136S and P136L leading to no change in bolting or flowering compared to the wild type LsFT protein, and the W138* mutant leading to delayed bolting and no flowering.
  • P75S leading to delayed bolting and delayed flowering
  • P136S and P136L leading to no change in bolting or flowering compared to the wild type LsFT protein
  • W138* mutant leading to delayed bolting and no flowering.
  • FIG. 5 Transverse cuttings of lettuce plants.
  • the left lettuce plant is homozygous for the P136S mutant allele, center one is heterozygous for the P136S mutant allele and right one is azygous (homozygous for the wild type allele).
  • FIG. 6 Transverse cuttings of lettuce plants.
  • the left lettuce plant is homozygous for the P136L mutant allele, center one is heterozygous for the P136L mutant allele and right one is azygous (homozygous for the wild type allele).
  • the term "genome” relates to the genetic material of an organism. It consists of DNA. The genome includes both the genes and the non-coding sequences of the DNA.
  • the Lactuca sativa wild type FT gene appears to be located on Linkage Group 2 in the genome Lsat_1_v8_lg_2 at position 39568061 to 39570403 of the reverse strand, as detected by BLAST analysis against the UC Davies Lactuca sativa V8 genome (world wide web at phytozome-next.jgi.doe.gov/).
  • gene means a (genomic) DNA sequence comprising a region (transcribed region), which is transcribed into a messenger RNA molecule (mRNA) in a cell, and an operably linked regulatory region (also described herein as regulatory sequence, e.g. a promoter).
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3' non-translated sequence comprising e.g. transcription termination sites.
  • Different alleles of a gene are thus different alternative forms of the gene, which may be in the form of e.g.
  • a gene may be an endogenous gene (in the species of origin) or a chimeric gene (e.g. a transgene or cis-gene).
  • the "promoter" of a gene sequence is defined as a region of DNA that initiates transcription of a particular gene. Promoters are located near the genes they transcribe, on the same strand and upstream on the DNA. Promoters can be about 100-1000 base pairs long. In one aspect the promoter is defined as the region of about 1000 base pairs or more e.g. about 1500 or 2000, upstream of the start codon (i.e. ATG) of the protein encoded by the gene.
  • Transgene or “chimeric gene” refers to a genetic locus comprising a DNA sequence, such as a recombinant gene, which has been introduced into the genome of a plant by transformation, such as Agrobacterium mediated transformation.
  • a plant comprising a transgene stably integrated into its genome is referred to as “transgenic plant”.
  • RNA which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide or which is active itself (e.g. in posttranscriptional gene silencing or RNAi).
  • the coding sequence may be in sense-orientation and encodes a desired, biologically active protein or peptide.
  • protein and “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 - dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a "protein”.
  • isolated protein is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • amino acid sequence and “amino acid sequence” refer to the primary amino acid sequence of a protein or polypeptide.
  • locus means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.
  • a QTL quantitative trait locus
  • a hereditary unit (often indicated by one or more molecular genomic markers) that occupies a specific location on a chromosome and that contains the genetic instruction for a particular phenotypic characteristics or trait in a plant.
  • the exact boundaries of a QTL are not known, but can be found without undue burden by a person skilled in the art by using fine mapping techniques well known in the art of genetic mapping and subsequent DNA sequencing routines.
  • the QTL encodes at least one gene of which the expression, alone or in combination with other genes, results in the phenotypic trait being expressed, or that encodes at least one regulatory region that controls the expression of at least one gene the expression of which, alone or in combination with other genes, results in the phenotypic trait being expressed.
  • a QTL may be defined by indicating its genetic location in the genome of the donor of the introgression that contains the QTL using one or more molecular genomic markers. These one or more markers, in turn, indicate a specific locus.
  • centimorgan 1% recombination between loci (markers).
  • markers markers that are linked to the QTL or markers that are in linkage disequilibrium with the QTL.
  • allele(s) means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus.
  • alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome.
  • loci plural locus on a chromosome.
  • One allele is present on each chromosome of the pair of homologous chromosomes.
  • a diploid plant species may comprise a large number of different alleles at a particular locus. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous).
  • allelism test is a test known in the art that can be used to identify whether two genes conferring the same trait are located at the same locus.
  • Wild type allele refers herein to a version of a gene encoding a fully functional protein (wild type protein). Accordingly, the term “wild type FT allele” or “FT allele” or “wild type allele of the Flowering Locus T gene” or “wild type allele of the FT gene” refers to the fully functional allele of the Flowering Locus T gene, which allows the normal bolting and flowering during plant development.
  • a wild type FT allele in the species Lactuca sativa for instance is the wild type genomic DNA which encodes the wild type FT cDNA (mRNA) sequence depicted in SEQ ID NO: 2.
  • SEQ ID NO: 1 The protein sequence encoded by this wild type Lactuca sativa FT cDNA has 175 amino acids and is depicted in SEQ ID NO: 1 , which corresponds to NCBI reference sequence XP_023759400.1 (protein GenBank) derived from the cDNA from mRNA Sequence ID: XM_023903632.1 from gene with ID: 111907824 (LOC111907824) on the Scaffold Reference Lsat_Salinas_v7 Primary Assembly in GenBank.
  • SEQ ID NO: 1 is the protein XP_023759400.1 in GenBank encoded by the coding sequence SEQ ID NO: 2 which has been derived from the cDNA from mRNA sequence XM_023903632.1.
  • the mRNA is from gene with Sequence ID: 111907824 (LOC111907824) on the Scaffold Reference Lsat_Salinas_v7 Primary Assembly in GenBank.
  • the wild type FT allele further comprises functional variants of the wild type genomic DNA which encodes the wild type FT cDNA and amino acid sequences as described herein, such as functional FT proteins comprising at least 95%, 96%, 97%, 98%, 99% or more amino sequence identity to SEQ ID NO: 1. Whether a certain variant of the herein specifically described wild type FT allele represents a functional variant can be determined by using routine methods, including, but not limited to, phenotypic testing for normal bolting behaviour during plant development and in silico prediction of amino acid changes that affect protein function.
  • SIFT Silicon Intolerant From Tolerant
  • a web-based computer program SIFT is a program that predicts whether an amino acid substitution affects protein function; see world wide web at sift.bii.a-star.edu.sg/. Functionally important amino acids will be conserved in the protein family, and so changes at well-conserved positions tend to be predicted as not tolerated or deleterious; see also Ng and Henikoff (2003) Nucleic Acids Res 31(13): 3812-3814. For example, if a position in an alignment of a protein family only contains the amino acid isoleucine, it is presumed that substitution to any other amino acid is selected against and that isoleucine is necessary for protein function.
  • PB domain refers to amino acids 66 to 127 of SEQ ID NO: 1 (or the equivalent domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1).
  • the PB domain is a conserved functional domain, which comprises 9 amino acids (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120) that are involved in substrate binding, see Figure 4.
  • the Phosphatidylethanolamine Binding Proteins family of proteins is also described under InterPro entry IPR008914 for all organisms (www at ebi.ac.uk/interpro/entry/lnterPro/IPR008914/) and IPR035810 entry for eukaryotes.
  • mutant allele refers herein to an allele that encodes a mutant protein comprising one or more amino acids replaced, inserted or deleted with respect to the wild type protein and resulting in delayed bolting when the mutant allele is in homozygous form.
  • Such a mutant allele accordingly can refer to an allele comprising one or more mutations in the coding sequence (mRNA, cDNA or genomic sequence) compared to the wild type allele.
  • Such mutation(s) e.g. insertion, inversion, deletion and/or replacement of one or more nucleotide(s)
  • Mutant alleles according to the invention can be generated de novo by mutagenesis methods, such as chemical mutagenesis (e.g. using EMS or MNU mutagenesis or mutagenesis by generating reactive oxygen species) or radiation mutagenesis (e.g. using UV radiation or ion beam radiation).
  • mutagenesis methods such as chemical mutagenesis (e.g. using EMS or MNU mutagenesis or mutagenesis by generating reactive oxygen species) or radiation mutagenesis (e.g. using UV radiation or ion beam radiation).
  • mutant Flowering Locus T allele or “ft allele” or “mutant allele of the Flowering Locus T gene” or “mutant allele of the FT gene” refers to an allele of the Flowering Locus T gene (FT gene) comprising one or more mutations leading to one or more amino acids being replaced, deleted or inserted compared to the wild type protein, which delays bolting of the Lactuca sativa plant when the mutant allele is in homozygous form.
  • mutant ft allele refers herein to a mutant ft allele which is not found in plants in the natural population or breeding population, but which is produced by human intervention such as mutagenesis or targeted gene modification (also referred to as targeted gene editing), such as effected through e.g. CRISPR/Cas9, CRISPR/Cpf1 or similar methods.
  • mutagenesis or targeted gene modification (also referred to as targeted gene editing), such as effected through e.g. CRISPR/Cas9, CRISPR/Cpf1 or similar methods.
  • targeted gene editing also referred to as targeted gene editing
  • mutant allele does not encompass mutant alleles which have mutations in regulatory elements (such as promoters or enhancers) and therefore have reduced gene expression or no gene expression.
  • an “active protein” or “functional protein” or “wild type protein” is a protein which has normal (i.e. not reduced) protein activity as measurable in vitro, e.g. by an in vitro activity assay, and/or in vivo, e.g. by the phenotype conferred by the protein.
  • a “wild type” FT protein is a functional protein of SEQ ID NO: 1 or a functional wild type protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 1 (also referred to as a ‘variant’ of SEQ ID NO: 1), or preferably 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7% sequence identity to SEQ ID NO: 1 , when aligned pairwise (e.g. using the program Needle, default parameters).
  • the wild type FT allele is the allele encoding a wild type protein or variant.
  • the wild type FT gene encodes a protein (the “Flowering Locus T protein” or “FT protein”) of SEQ ID NO: 1 or a functional protein comprising at least 95% (96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7%) amino acid sequence identity to SEQ ID NO:1.
  • the protein described by the amino acid sequence SEQ ID NO: 1 represents a wild type FT protein in Lactuca sativa and corresponds to NCBI reference sequence XP_023759400.1 (protein GenBank) and is encoded by the gene Sequence ID: 111907824 (LOC111907824) on the Scaffold Reference Lsat_Salinas_v7 Primary Assembly in GenBank.
  • the wild type FT protein comprises a conserved domain, the Phosphatidyl Ethanolamine-Binding Protein (PB) domain.
  • PB Phosphatidyl Ethanolamine-Binding Protein
  • mutant protein is herein a protein comprising one or more amino acids inserted, deleted or replaced with respect to the wild type protein, due to mutations in the genomic DNA encoding the protein.
  • the mutant protein has an altered function, e.g. measurable in vivo, e.g. by the phenotype conferred by the mutant allele, for example delayed bolting when the mutant allele is in homozygous form compared to a control plant which lacks the mutant allele.
  • mutant proteins comprise “reduced-function” or “loss-of-function” proteins, as e.g. measurable in vivo, e.g. by the phenotype conferred by the mutant allele, or in a phenotypic test to determine the bolting behaviour during plant development.
  • a "Flowering Locus T protein having a W138* mutation” or a “FT protein having a W138* mutation” or a “FT protein having a W138STOP mutation” refers to an Lactuca sativa Flowering Locus T protein of SEQ ID NO: 1 , or variant thereof comprising at least 95%, e.g.
  • an FT protein having a W138* mutation is the Lactuca sativa Flowering Locus T protein of SEQ ID NO: 1 that is truncated due to a mutation of codon 138 into a STOP codon.
  • the truncated W138* protein is shown in SEQ ID NO: 3.
  • the codon at nucleotides 1954 to 1956 of SEQ ID NO: 7 (codon TGG, encoding W, Trp) is mutated to TGA or TAG, which is a STOP codon.
  • This mutant has delayed bolting when in homozygous form (see examples) and does not induce flowering when the mutant allele is in homozygous form, but, surprisingly, produces whorls of leaves on the stem instead of flowers (see Figure 3).
  • a genotyping assay for detecting and/or selecting the mutant allele is provided (see examples).
  • a "Flowering Locus T protein having a P75S mutation” or a “FT protein having a P75S mutation” refers to an Lactuca sativa Flowering Locus T protein of SEQ ID NO: 1 , or variant thereof comprising at least 95%, e.g. at least 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7% amino acid sequence identity to SEQ ID NO: 1 , that has a Serine at amino acid position 75 of SEQ ID NO: 1 , or at the equivalent amino acid position in a wild type FT protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.
  • FT protein that has a replacement (substitution) of amino acid 75, replacing the Proline of the wild type protein (P75) with a Serine.
  • the mutant protein (P75S) is described herein as SEQ ID NO: 5.
  • the codon at nucleotides 340 to 342 of SEQ ID NO: 7 (codon CCA, encoding Proline) is mutated to TCA, which encodes a Serine.
  • This mutant has delayed bolting and delayed flowering when in homozygous form (see Examples).
  • a genotyping assay for detecting and/or selecting the mutant allele is provided (see examples).
  • a "Flowering Locus T protein having a P136S mutation” or a “FT protein having a P136S mutation” refers to an Lactuca sativa Flowering Locus T protein of SEQ ID NO: 1 , or variant thereof comprising at least 95%, e.g. at least 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7% amino acid sequence identity to SEQ ID NO: 1 , that has a Serine at amino acid position 136 of SEQ ID NO: 1 or at the equivalent amino acid position in a wild type FT protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.
  • FT protein that has a replacement (substitution) of amino acid 136, replacing the Proline of the wild type protein (P136) with a Serine.
  • the codon at nucleotides 1948 to 1950 of SEQ ID NO: 7 (codon OCT, encoding Proline) is mutated to TCT, which encodes a Serine.
  • This mutant does not delay bolting and does not delay flowering when in homozygous form, the P136S substitution does, therefore, not affect the protein function.
  • a genotyping assay for detecting and/or selecting the mutant allele is provided (see examples).
  • a "Flowering Locus T protein having a P136L mutation” or a “FT protein having a P136L mutation” refers to an Lactuca sativa Flowering Locus T protein of SEQ ID NO: 1 , or variant thereof comprising at least 95%, e.g. at least 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7% amino acid sequence identity to SEQ ID NO: 1 , that has a Leucine at amino acid position 136 of SEQ ID NO: 1 or at the equivalent amino acid position in a wild type FT protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.
  • FT protein that has a replacement (substitution) of amino acid 136, replacing the Proline of the wild type protein (P136) with a Leucine.
  • the codon at nucleotides 1948 to 1950 of SEQ ID NO: 7 (codon OCT, encoding Proline) is mutated to CTT, which encodes a Leucine.
  • This mutant does not delay bolting and does not delay flowering when in homozygous form, the P136L substitution does, therefore, not affect the protein function.
  • a genotyping assay for detecting and/or selecting the mutant allele is provided (see examples).
  • a "Flowering Locus T protein having a P75X mutation or P136X mutation” or a “FT protein having a P75X mutation or a P136X mutation” refers to an Lactuca sativa Flowering Locus T protein of SEQ ID NO: 1 , or variant thereof comprising at least 95%, e.g.
  • SEQ ID NO: 1 at least 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7% amino acid sequence identity to SEQ ID NO: 1 , that has an amino acid substitution of the Proline at amino acid 75 or at amino acid 136 of SEQ ID NO: 1 , or at the equivalent amino acid position in a wild type FT protein comprising at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , with a different amino acid.
  • the X refers to any other amino acid at this position or at the equivalent position.
  • the equivalent amino acid in a variant protein is referred to.
  • the equivalent amino acid can easily be identified by pairwise alignment of the variant sequence with the sequence of SEQ ID NO: 1. Due to the high percentage of sequence identity, the equivalent amino acid position will be immediately apparent.
  • the Proline at position 75 of SEQ ID NO: 1 may for example be located at position 73, 74, 76 or 77 in a variant protein comprising at least 95% identity to SEQ ID NO: 1.
  • the Proline at position 136 of SEQ ID NO: 1 may for example be located at position 134, 135, 137 or 138 in a variant protein comprising at least 95% identity to SEQ ID NO: 1.
  • Wild type plant refers herein to a Lactuca sativa plant comprising two copies of the wild type FT allele (encoding the wild type protein of SEQ ID NO: 1 or a variant wild type protein comprising at least 95% to SEQ ID NO: 1) showing normal bolting and flowering behaviour. Such plants are for example suitable controls in phenotypic assays.
  • Control plant is a plant genotype comprising two copies of the wild type FT allele, encoding wild type, functional FT protein, which can be suitably used in a phenotyping assay, e.g. to compare the start of bolting and/or flowering (see e.g.
  • control plant examples are between the control plant and a plant homozygous for a mutant ft allele.
  • the control plant is of the same type as the plant comprising the mutant allele in homozygous form (e.g. both are butterhead types or both are Romaine types).
  • the control plant comprising the wild type FT allele in homozygous form is genetically very similar to the plant comprising the mutant ft allele in homozygous form, so that the differences in bolting are compared in the same or similar genetic backgrounds.
  • the control plant may, thus, be e.g. a near isogenic line or isogenic line or a so-called ‘genetic control’ line.
  • a mutant allele may be crossed into a specific lettuce line or variety and the original line or variety, or a backcross line lacking the mutant allele may be used as control.
  • orthologous gene or "ortholog” is defined as genes in different species that have evolved through speciation events. It is generally assumed that orthologs have the same biological functions in different related species. Accordingly, it is particularly preferred that the protein encoded by the ortholog of the wild type Lactuca sativa FT gene in other plants of the genus Lactuca (preferably in wild lettuce) has the same biological function as the wild type Lactuca sativa FT protein. Methods for the identification of orthologs is very well known in the art as it accomplishes two goals: delineating the genealogy of genes to investigate the forces and mechanisms of evolutionary process and creating groups of genes with the same biological functions (Fang G, et al (2010) Getting Started in Gene Orthology and Functional Analysis.
  • orthologs of a specific gene or protein can be identified using sequence alignment or sequence identity of the gene sequence of the protein of interest with gene sequences of other species.
  • Gene alignments or gene sequence identity determinations can be done according to methods known in the art, e.g. by identifying nucleic acid or protein sequences in existing nucleic acid or protein database (e.g. GENBANK, SWISSPROT, TrEMBL) and using standard sequence analysis software, such as sequence similarity search tools (BLASTN, BLASTP, BLASTX, TBLAST, FASTA, etc.).
  • an ortholog of the Lactuca sativa FT protein in other plants of the genus Lactuca has at least 65% (e.g. at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7%) amino acid sequence identity with SEQ ID NO: 1.
  • orthologs of the FT gene product can be found in further plant species of the genus Lactuca, e.g. in wild Lactuca species such as L. serriola, L. altica, L. saligna, L. virosa or L. geogica.
  • the word "trait” in the context of this application refers to the phenotype of the plant.
  • its genome comprises the mutant allele causing the trait of the invention, particularly in the present invention when the mutant allele is in homozygous form.
  • the plant thus, has the genetic determinant of the invention. It is understood that when referring to a plant comprising the trait of the plant of the invention, reference is made to a Lactuca sativa plant comprising the trait of delayed bolting (compared to the control plant having two copies of the wild type FT allele), optionally also delayed flowering or no flowering.
  • Average refers herein to the arithmetic mean.
  • the term “molecular genomic marker” or short “marker” or “DNA marker” refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences, especially different alleles (also referred to as ‘allele specific markers’).
  • indicators are restriction fragment length polymorphism (RFLP) markers, amplified 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 amplified fragment length polymorphism
  • SNPs single nucleotide polymorphisms
  • SSRs single nucleotide polymorphisms
  • SCARs sequence-characterized amplified regions
  • SNPs are for example the C at nucleotide position 340 of SEQ ID NO: 7 and the T at position 340 of SEQ ID NO: 7, whereby the C/T SNP either detects the wild type (encoding a P at amino acid 75) or mutant ft allele (encoding a S at amino acid 75).
  • Another example is the C at nucleotide position 1948 of SEQ ID NO: 7 and the T at nucleotide position 1948 of SEQ ID NO: 7, whereby the C/T SNP either detects the wild type (encoding a P at amino acid 136) or mutant allele (encoding a S at amino acid 136).
  • a further example is the C at nucleotide position 1949 of SEQ ID NO: 7 and the T at nucleotide position 1949 of SEQ ID NO: 7, whereby the C/T SNP either detects the wild type (encoding a P at amino acid 136) or mutant ft allele (encoding a L at amino acid 136).
  • Yet a further example is the G at nucleotide 1955 or 1956 and the A at nucleotide 1955 or 1956, whereby the G/A SNP either detects the wild type (encoding a W, codon TGG), or the mutant encoding a STOP codon (TGA or TAG).
  • a fragment of a mutant protein refers to any subset of the molecule.
  • Variant peptides may be made by direct chemical synthesis, for example, using methods well known in the art.
  • An analogue of a mutant protein refers to a non-natural protein substantially similar to either the entire protein or a fragment thereof.
  • a "mutation" in a nucleic acid molecule is a change of one or more nucleotides compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more nucleotides.
  • a "mutation" in an amino acid molecule making up a protein is a change of one or more amino acids compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more amino acids. Such a protein is then also referred to as a "mutant protein”.
  • a "point mutation” is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide.
  • a "nonsense mutation” is a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon in a nucleic acid molecule is changed into a stop codon. This results in a premature stop codon being present in the mRNA and results in translation of a truncated protein.
  • a truncated protein may have decreased function or loss of function.
  • a "missense or non-synonymous mutation” is a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon is changed to code for a different amino acid.
  • the resulting protein may have decreased function or loss of function.
  • a "splice-site mutation” is a mutation in a nucleic acid sequence encoding a protein, whereby RNA splicing of the pre-mRNA is changed, resulting in an mRNA having a different nucleotide sequence and a protein having a different amino acid sequence than the wild type. The resulting protein may have decreased function or loss of function.
  • a "frame shift mutation” is a mutation in a nucleic acid sequence encoding a protein by which the reading frame of the mRNA is changed, resulting in a different amino acid sequence. The resulting protein may have decreased function or loss of function.
  • a “deletion” in context of the invention shall mean that anywhere in a given nucleic acid sequence at least one nucleotide is missing compared to the nucleic sequence of the corresponding wild type sequence or anywhere in a given amino acid sequence at least one amino acid is missing compared to the amino acid sequence of the corresponding (wild type) sequence.
  • a "truncation” shall be understood to mean that at least one nucleotide at either the 3’-end or the 5’-end of the nucleotide sequence is missing compared to the nucleic sequence of the corresponding wild type sequence or that at least one amino acid at either the N-terminus or the C-terminus of the protein is missing compared to the amino acid sequence of the corresponding wild type protein, whereby in a 3’-end or C-terminal truncation at least the first nucleotide at the 5’-end or the first amino acid at the N-terminus, respectively, is still present and in a 5’-end or N- terminal truncation at least the last nucleotide at the 3’-end or the last amino acid at the C- terminus, respectively, is still present.
  • the 5’-end is determined by the ATG codon used as start codon in translation of a corresponding wild type nucleic acid sequence.
  • Replacement or “substitution” shall mean that at least one nucleotide in a nucleic acid sequence or one amino acid in a protein sequence is different compared to the corresponding wild type nucleic acid sequence or the corresponding wild type amino acid sequence, respectively, due to e.g. an exchange of one or more nucleotides in the coding sequence of the respective protein.
  • “Insertion” shall mean that the nucleic acid sequence or the amino acid sequence of a protein comprises at least one additional nucleotide or amino acid compared to the corresponding wild type nucleic acid sequence or the corresponding wild type amino acid sequence, respectively.
  • Pre-mature stop codon in context with the present invention means that a stop codon is present in a coding sequence (cds) which is closer to the start codon at the 5’-end compared to the stop codon of a corresponding wild type coding sequence.
  • a "mutation in a regulatory sequence” is a change of one or more nucleotides compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more nucleotides, leading for example to decreased or no mRNA transcript of the gene being made.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the nucleic acid sequences being linked are typically contiguous.
  • sequence similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc. Hits are preferably aligned pairwise to compare sequence identity, preferably over the full length of the sequences.
  • hybridisation is used to indicate hybridisation of nucleic acids at appropriate conditions of stringency as would be readily evident to those skilled in the art depending upon the nature of the probe sequence and target sequences.
  • Conditions of hybridisation and washing are well known in the art, and the adjustment of conditions depending upon the desired stringency by varying incubation time, temperature and/or ionic strength of the solution are readily accomplished. See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989.
  • the choice of conditions is dictated by the length of the sequences being hybridised, in particular, the length of the probe sequence, the relative G-C content of the nucleic acids and the amount of mismatches to be permitted. Low stringency conditions are preferred when partial hybridisation between strands that have lesser degrees of complementarity is desired. When perfect or near perfect complementarity is desired, high stringency conditions are preferred.
  • a nucleic acid sequence e.g. DNA or genomic DNA
  • nucleic acid sequence identity to a reference sequence
  • said nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using stringent hybridisation conditions.
  • nucleic acid sequence comprises one or more mutations compared to the given nucleotide sequence but still can be identified using stringent hybridization conditions.
  • Stringent hybridisation conditions can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe.
  • stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency.
  • Stringent conditions for RNA-DNA hybridisations are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.
  • Stringent conditions for DNA-DNA hybridisation are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions.
  • hybridizes to a DNA or RNA molecule is used to indicate that a molecule recognizes and hybridizes to another nucleic acid molecule by base pairing, meaning that there is enough sequence similarity between the two nucleic acid molecules to effect hybridization under appropriate conditions.
  • introgression refers to both a natural and artificial process whereby a genomic fragment of one species, variety or cultivar, termed donor parent, is transduced into the genome of another species, variety or cultivar, termed recipient parent, for example by crossing the donor and recipient parent. The process may optionally be completed by backcrossing the resulting plants to the recipient parent, which is than termed recurrent parent.
  • An introgression fragment is present outside of its natural genomic context, meaning that a plant harbouring an introgression fragment from e.g. Lactuca virosa is not a Lactuca virosa plant.
  • the term “plant” includes the whole plant or any parts or derivatives thereof, such as plant organs (e.g., harvested or non-harvested fruits, leaves, seed, flowers, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, ovaries, fruits (e.g., harvested tissues or organs, such as harvested heads or harvested leaves or parts thereof), flowers, leaves, seeds, clonally propagated plants, roots, root-stocks, stems, root tips and the like. Also any developmental stage is included, such as seedlings, immature and mature, etc.
  • plant organs e.g., harvested or non-harvested fruits, leaves, seed, flowers, etc.
  • plant cells e.g., plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, plant calli, plant cell clump
  • a "plant line” or “breeding line” refers to a plant and its progeny.
  • the term “inbred line” refers to a plant line which has been repeatedly selfed, preferably more than three times, more preferably more than 4, 5 or 6 times.
  • cultivar (or “cultivated” plant) is used herein to denote a plant having a biological status other than a "wild" status, which "wild" status indicates the original non-cultivated, nondomesticated, or natural state of a plant or accession, and the term cultivated does not include such wild, or weedy plants.
  • cultivar does include material with good agronomic characteristics, such as breeding material, research material, breeding lines, elite breeding lines, synthetic population, hybrid, founder stock/base population, inbred lines, cultivars (open pollinated of hybrid cultivar), segregating population, mutant/genetic stock, and advanced/improved cultivar.
  • heirloom varieties or cultivars i.e.
  • cultivar also includes landraces, i.e. plants (or populations) selected and cultivated locally by humans over many years and adapted to a specific geographic environment and sharing a common gene pool.
  • Plant variety is a group of plants within the same botanical taxon of the lowest grade known, which (irrespective of whether the conditions for the recognition of plant breeder’s rights are fulfilled or not) can be defined on the basis of the expression of characteristics that result from a certain genotype or a combination of genotypes, can be distinguished from any other group of plants by the expression of at least one of those characteristics, and can be regarded as an entity, because it can be multiplied without any change.
  • plant variety cannot be used to denote a group of plants, even if they are of the same kind, if they are all characterized by the presence of one locus or gene (or a series of phenotypical characteristics due to this single locus or gene), but which can otherwise differ from one another enormously as regards the other loci or genes.
  • Backcrossing refers to a breeding method by which a (single) trait, such as the delayed bolting trait, can be transferred from one genetic background (also referred to as “donor” generally, but not necessarily, this is an inferior genetic background) into another genetic background (also referred to as “recurrent parent”; generally, but not necessarily, this is a superior genetic background).
  • donor generally, but not necessarily, this is an inferior genetic background
  • recurrent parent also referred to as "recurrent parent”; generally, but not necessarily, this is a superior genetic background.
  • An offspring of a cross e.g.
  • the trait of the donor genetic background e.g. the delayed bolting trait of the present invention, will have been incorporated into the recurrent genetic background.
  • the terms "gene converted” or “conversion plant” or “single locus conversion” in this context refer to plants which are developed by backcrossing wherein essentially all of the desired morphological and/or physiological characteristics of the recurrent parent are recovered in addition to the one or more genes transferred from the donor parent.
  • the plants grown from the seeds produced by backcrossing of the F1 plants with the second parent plant line is referred to as the "BC1 generation”. Plants from the BC1 population may be selfed resulting in the BC1 F2 generation or backcrossed again with the cultivated parent plant line to provide the BC2 generation.
  • M1 population is a plurality of mutagenized seeds I plants of a certain plant line.
  • M2, M3, M4, etc. refers to the consecutive generations obtained following selfing of a first mutagenized seed I plant (M1).
  • “Lettuce” or “cultivated lettuce” or “cultivated Lactuca sativa” refers herein to plants of the species Lactuca sativa L. (or seeds from which the plants can be grown), and parts of such plants, bred by humans for food and having good agronomic characteristics.
  • breeding lines e.g. backcross lines, inbred lines
  • cultivars and varieties of any type Generally heading and non-heading types of lettuce are distinguished. Heading types include for example crisphead, butterhead and Romaine (cos) types, while non-heading types include leaf- types.
  • Cultivated lettuce plants are not “wild lettuce” plants or “wild Lactuca” plants, i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and e.g. grow naturally in wild populations.
  • Wild lettuce or “wild Lactuca” accessions refers to plants of species other than cultivated Lactuca sativa, such as Lactuca virosa, Lactuca serriola, Lactuca saligna, Lactuca perennis, and others.
  • such wild lettuce comprises or consists of Lactuca species which are cross fertile with L. sativa, optionally with the aid of embryo rescue techniques (see Maisonneuve 1987, Agronomique 7: 313-319 and Maisonneuve et al. 1995, Euphytica 85:281-285) and/or chromosome doubling techniques (Thompson and Ryder 1961 , US Dept Agric Tech Bui. 1224), or methods whereby genes can be transferred into L. sativa via a bridge species, such as L. serriola (Eenink et al. 1982, Euphytica 31 , 291-299, //doi.org/10.1007/ BF00021643).
  • the term "food” is any substance consumed to provide nutritional support for the body. It is usually of plant or animal origin, and contains essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals. The substance is ingested by an organism and assimilated by the organism's cells in an effort to produce energy, maintain life, or stimulate growth. The term food includes both substances consumed to provide nutritional support for the human and animal body.
  • Hardvested plant material refers herein to plant parts (e.g., leaves, leaf parts or heads detached from the whole plant) which have been collected for further storage and/or further use.
  • Hardvested seeds refers to seeds harvested from a line or variety, e.g., produced after selffertilization or cross-fertilization and collected.
  • Hardvested leaves or “harvested heads” as used herein refers to lettuce leaves, or leaf parts or heads, i.e., the plant without the root system, for example substantially all (harvested) leaves. Leaves may be whole or cut into parts.
  • Plantative propagation or “clonal propagation” refers to propagation of plants from vegetative tissue, e.g. by propagating plants from cuttings or by in vitro propagation. In vitro propagation involves in vitro cell or tissue culture and regeneration of a whole plant from the in vitro culture. Clones (i.e. genetically identical vegetative propagations) of the original plant can thus be generated by in vitro culture.
  • Cell culture or “tissue culture” refers to the in vitro culture of cells or tissues of a plant.
  • Regeneration refers to the development of a plant from cell culture or tissue culture or vegetative propagation.
  • Non-propagating cell refers to a cell which cannot be regenerated into a whole plant.
  • somatic cells and “reproductive cells” can be distinguished, whereby somatic cells are cells other than gametes (e.g. ovules and pollen), germ cells and gametocytes. Gametes, germ cells and gametocytes are “reproductive cells”.
  • Tissue Culture or “cell culture” refers to an in vitro composition comprising isolated cells of the same or a different type or a collection of such cells organized into plant tissue. Tissue cultures and cell cultures of lettuce, and regeneration of lettuce plants therefrom, is well known and widely published (see, e.g., Teng et al., HortScience. 1992, 27(9): 1030-1032 Teng et al., HortScience. 1993, 28(6): 669-1671 , Zhang et al., Journal of Genetics and Breeding. 1992, 46(3): 287-290).
  • a “plant line” or “breeding line” refers to a plant and its progeny being highly uniform in plant phenotype.
  • inbred line refers to a plant line which has been repeatedly selfed and is nearly homozygous for all alleles.
  • an “inbred line” or “parent line” refers to a plant which has undergone several generations (e.g. at least 3, 4, 5, 6, 7 or more) of inbreeding, resulting in a plant line with a high uniformity.
  • F1 , F2, etc. refer to the consecutive related generations following a cross between two parent plants or parent lines. The plants grown from the seeds produced by crossing two plants or lines is called the F1 generation. Selfing the F1 plants results in the F2 generation, etc.
  • hybrid plant (or hybrid seed) refers to a plant or seed obtained from crossing two inbred parent lines.
  • F1 hybrid plant or “F1 hybrid” seed or “F1 seed” refers to a first- generation plant or seed obtained from crossing two inbred parent lines.
  • Hybrid refers to the seeds harvested from crossing one plant line or variety with another plant line or variety, and the plants or plant parts grown from said seeds.
  • F1 hybrid plant (or F1 hybrid seed) is the generation obtained from crossing two non- isogenic inbred parent lines.
  • F1 hybrid seeds are seeds from which F1 hybrid plants grow.
  • An “interspecific hybrid” refers to a hybrid produced from crossing a plant of one species, e.g. L. sativa, with a plant of another species, e.g. L. virosa.
  • progeny refers to any and all offspring that are derivable from or obtainable from a plant of the invention that comprises at least one copy of a mutant ft allele as described herein and comprises the delayed bolting phenotype as described herein when the mutant ft allele is in homozygous form.
  • Progeny are derived by crossing a plant comprising at least one copy of a mutant / -allele with another lettuce plant and/or selfing a plant comprising at least one copy of a mutant / -allele one or more times, e.g. 2, 3, 4, 5 or more times.
  • Progeny may also be derived by cell culture or by tissue culture, or by producing seeds of a plant.
  • the term progeny may also encompass plants derived from crossing of at least one parent plant with another plant of the same or another variety or (breeding) line.
  • a progeny is directly derived from, obtained from, obtainable from or derivable from the parent plant by, e.g., traditional breeding methods (selfing and/or crossing) or regeneration or transformation.
  • the term "progeny” generally encompasses further generations such as second, third, fourth, fifth, sixth, seventh or more generations, i.e., generations of plants which are derived from, obtained from, obtainable from or derivable from the former generation by, e.g., traditional breeding methods, regeneration or genetic transformation techniques.
  • a second- generation progeny can be produced from a first generation progeny by any of the methods mentioned above.
  • a progeny plant may also be a double haploid plant comprising two copies a mutant ff-allele as described
  • a plant (or plant line or variety or genotype) having a "delayed bolting” relates to a plant (or plant line or variety or genotype, e.g. 5,10,15 or more plants) showing a phenotype wherein the average start of bolting of the plant is delayed by at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days when compared to the average start of bolting of a suitable control plant (or plant line or variety) grown under the same conditions. Plants in which the average start of bolting is delayed by at least 7 or more days will in one aspect also have a delay in the average start of flowering (‘delayed flowering’).
  • the average start of bolting can e.g. be measured by sowing a plurality of seeds of the plant line or genotype and the control line or genotype and e.g. cutting heads transversely at e.g. weekly intervals and inspecting whether the transition from vegetative to reproductive stage has started (as seen by elongation of the shoot apical meristem, see Examples).
  • the average number of days after sowing until the start of bolting is then compared between the plant line or genotype comprising a mutant / -allele in homozygous form and the wild type control line comprising the wild type / -allele in homozygous form.
  • the average start of flowering can e.g. be measured by sowing a plurality of seeds of the plant line or genotype and the control line or genotype and inspecting the plants at e.g. weekly intervals to see whether the shoot apical meristem has formed flowering primordia, see Examples.
  • the average number of days after sowing until the start of flowering is then compared between the plant line or genotype comprising a mutant ff-allele in homozygous form and the wild type control line comprising the wild type / -allele in homozygous form.
  • a plant line or genotype having a "delayed flowering” relates to a plant (or plant line or variety) showing a phenotype wherein the average start of flowering of the plant is delayed by at least ?, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days.
  • a plant (or plant line or variety) having a "delayed bolting" and/or “delayed flowering” does not encompass plants (or plant lines or varieties) that do not flower at all and thus also do not develop seeds.
  • a plant (or plant line or variety) having a delayed bolting does encompass plants which do not flower at all, but which develop whorls of leaves instead, as e.g. shown in Figure 3 for mutant W138*.
  • the plant After germination of the lettuce seed the plant enters the “seedling stage” in which the roots and the seed cotyledons are formed. Typically, the seedling stage lasts 14-60 days, considering different lettuce types and seasons. Following the seedling stage, the lettuce plant enters the “vegetative growth stage” in which the lettuce head is developed, and which starts when the third true leaf is fully developed. Typically, the vegetative growth stage lasts 60-120 days, considering different lettuce types and seasons. In lettuce cultivation for food production, the harvested plant material (i.e. leaves or heads) is collected when the lettuce plant is in the vegetative growth stage, preferably during the second half of the vegetative growth stage.
  • the harvested plant material i.e. leaves or heads
  • the lettuce plant Following the vegetative growth stage, the lettuce plant enters the “bolting stage” which starts when a visibly recognisable stalk has started to develop in the centre of the lettuce head.
  • the lettuce head Prior to bolting, the lettuce head shows a typical compact filling of the leaves, which is also described herein as an “intact shape of the (lettuce) head”. Accordingly, a lettuce head of a plant that is harvested from a lettuce plant that is not yet in bolting stage shows compact filling of the leaves and accordingly an intact shape of the lettuce head.
  • the bolting stage lasts 75-140 days. Following the bolting stage, the lettuce plant enters the “mature reproductive stage” which starts when the first plant reproductive organs become functional.
  • a plant (or plant line or variety) or harvested plant material having an "improved quality” relates to a plant (or plant line or variety) or harvested plant material showing a phenotype wherein the harvested plant material in average shows an “improved quality” when compared to a suitable control plant (or plant line or variety).
  • Such improved quality phenotype may be detected by visual inspection of the harvested plant material.
  • An "improved quality" phenotype accordingly, is a phenotype wherein in average a higher percentage of the harvested plant material shows a high-quality standard when compared to a suitable control plant, preferably an isogenic plant.
  • an improved quality can be measured on the harvested plant material as the percentage of plants (e.g. 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and/or 100% of harvested heads) to have a compact filling of the leaves and/or to have an intact shape of the heads.
  • a plant (or plant line or variety) or harvested plant material having an "increased mass of the harvested lettuce heads and/or harvested lettuce leaves” or “increased biomass” relates to a plant (or plant line or variety) showing a phenotype wherein average weight of the harvested lettuce heads and/or the average weight of the harvested lettuce leaves is increased (e.g. by 1%, 2%, 3%, 4% or more) when compared to a suitable control plant (or plant line or variety).
  • the biomass is increased compared to the control when harvested during the stage when the control is already bolting and/or flowering, while the delayed bolting plant is not bolting yet.
  • comparisons between different plants or plant lines or varieties involves growing a number of plants of a line (or variety) (e.g. at least 5 plants, preferably at least 10 plants per line) under the same conditions as the plants of one or more control plant lines (preferably wild type plants, e.g. isogenic lines or genetic controls) and the determination of differences, preferably statistically significant differences, between the plant lines when grown under the same environmental conditions.
  • control plant lines preferably wild type plants, e.g. isogenic lines or genetic controls
  • the control plants may for example be isogenic or near isogenic to the plants to which they are compared.
  • isogenic plant genotype refers to two plant genotypes which are genetically identical except for the gene of which the effect on the phenotype is to be compared, e.g. the difference in phenotype between a plant genotype comprising the wild type FT allele in homozygous form and a plant comprising a mutant / -allele in homozygous form.
  • near isogenic lines are genetically almost identical in their genetic background, except for the genotype of the FT allele.
  • “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 D, et al. (2014) Tissue Culture- Induced Heritable Genomic Variation in Rice, and Their Phenotypic Implications. PLOS ONE 9(5): e96879), including also targeted gene editing techniques (such as Crispr based techniques, TALENS, etc.).
  • SNP marker refers to a ‘Single Nucleotide Polymorphism’ between e.g. a mutant / -allele, and a wild type FT-allele.
  • SNP marker assay or a SNP genotyping assay, which can distinguish between the mutant and wild type allele of the gene (i.e. an allele specific assay) one can screen pants, plant parts or the DNA therefrom for the presence of the mutant allele and/or the wild type allele, i.e. for their genotype at the FT locus.
  • the SNPs underlying the P75X or the P136X or the W138* codon change are SNP markers that can distinguish between the wild type or mutant allele.
  • a SNP genotyping assay is provided herein for these SNPs, but alternative assays can, off course, be designed and used. For any mutation in the genomic DNA of a mutant ff-allele encoding a mutant protein, a SNP assay can easily be designed.
  • INDEL marker refers to an insertion/deletion polymorphism between e.g. a mutant ft- allele and a wild type FT- allele.
  • an INDEL marker assay or a genotyping assay, which can distinguish between the mutant and wild type allele of the gene (i.e. an allele specific assay) one can screen plants, plant parts or the DNA therefrom for the presence of the mutant allele.
  • “Genotyping” methods or assays are methods whereby the genotype or allelic composition of a plant or plant part or seed can be determined. Bi-allelic genotyping assays, such as KASP- assays, can distinguish between two alleles at a locus, e.g. a wild type FT-allele and a mutant ft- allele.
  • Marker assisted selection is a process of using the presence of molecular markers (such as SNP markers or INDEL markers), which are genetically and physically linked to a particular locus or to a particular chromosome region or allele specific markers, to select plants for the presence of the specific locus or region or allele.
  • molecular markers such as SNP markers or INDEL markers
  • INDEL markers genetically and physically linked to a particular locus or to a particular chromosome region or allele specific markers
  • a molecular marker genetically and physically linked to the mutant ft- allele or an allele specific marker can be used to detect and/or select e.g. lettuce plants, or plant parts, comprising the mutant / -allele. Allele specific markers are preferred markers, as they select for the allele directly.
  • “Targeted gene editing” is referred to techniques whereby endogenous target genes can be modified, e.g. one or more nucleotides can be inserted, replaced and/or deleted e.g. in the promoter or coding sequence.
  • CRISPR based techniques such as Crispr-Cas9 gene editing, Crispr-Cpf/ gene editing, or more recent techniques called ‘base editing’ or ‘primer editing’ can be used to modify endogenous target genes, such as the endogenous wild type FT gene in lettuce (encoding the protein of SEQ ID NO: 1 or a wild type protein comprising at least 95% sequence identity to SEQ ID NO: 1).
  • the mutants described herein can, for example, be reproduced by targeted gene editing of the wild type FT gene.
  • Oligonucleotides or “oligos” or “oligonucleotide primers or probes” are short, singlestranded polymers of nucleic acid, e.g. at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more nucleotides in length.
  • Oligos may be unmodified or modified with a variety of chemistries depending on their intended use, for example, the addition of 5' or 3' phosphate groups to enable ligation or block extension, respectively, labelling with radionucleotides or fluorophores and/or quenchers for use as probes, the incorporation of thiol, amino, or other reactive moieties to enable the covalent coupling of functional molecules such as enzymes, and extension with other linkers and spacers of diverse functionality.
  • DNA oligos are the most commonly used, but RNA oligos are also available. The length of an oligo is usually designated by adding the suffix -mer.
  • an oligonucleotide with 19 nucleotides is called a 19-mer.
  • oligonucleotides are designed to base-pair with a strand of DNA or RNA.
  • the most common use for oligonucleotides is as primers for PCR (polymerase chain reaction).
  • Primers are designed with at least part of their sequence complementary to the sequence targeted for amplification.
  • Optimal primer length for a complementary sequence is e.g. 18 to 22 nucleotides.
  • Optimal primer sequences for PCR are usually determined by primer design software.
  • DNA microarrays are arrays which have many microscopic spots of DNA, usually oligonucleotides, bound on a solid support.
  • Assay targets can be DNA, cDNA, or cRNA.
  • the hybridization of targets to specific spots is detected by fluorescence, chemiluminescence, or colloidal silver or gold.
  • Microarrays are used for multiple applications such as simultaneous measurement of the expression of large numbers of genes, enabling genome-wide gene expression analysis, as well as genotyping studies using e.g. singlenucleotide polymorphism (SNP) or InDei analysis.
  • SNP singlenucleotide polymorphism
  • “Complementary strands” refer to two strands of complementary sequence, and may be referred to as sense (or plus) and anti-sense (or minus) strands for double stranded DNA.
  • the sense I plus strand is, generally, the transcribed sequence of DNA (or the mRNA that was generated in transcription), while the anti-sense I minus strand is the strand that is complementary to the sense sequence.
  • the complementary nucleotides of DNA are A complementary to T, and G complementary to C.
  • the complementary nucleotides of RNA are A complementary to II, and G complementary to C.
  • the present invention provides in one aspect a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the wild type FT gene encodes a protein of SEQ ID NO: 1 , or a protein comprising at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 1 , and wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75) is replaced by a different amino acid residue.
  • FT Flowering Locus T
  • a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the equivalent amino acid in a wild type FT protein comprising at least 95% 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid residue, especially by a Serine.
  • FT Flowering Locus T
  • a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 136 of SEQ ID NO: 1 (amino acid P136), or the equivalent amino acid in a wild type FT protein comprising at least 95% 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid residue, e.g. by a Serine or by a Leucine.
  • FT Flowering Locus T
  • the average start date for the plant line to start bolting was significantly delayed compared to the wild type or isogenic control.
  • the delayed bolting also resulted in delayed flowering, but flowers still did develop and produced seeds.
  • the Proline 75 is replaced by a Serine.
  • the P75S mutant protein is still able to induce bolting (albeit significantly delayed) and subsequent flowering, i.e. the mutant protein was still able to travel in the phloem sap of the plant and to interact with other proteins to e.g. form a ‘florigen activation complex’ in the plant, and to interact in the bolting and flowering pathway. It is speculated that this is on the one hand because the single amino acid replacement is not in an amino acid which binds substrate (see Figure 4), but that probably the conformation of the PB domain is slightly changed. Proline is a small, unique amino acid, which can often be found in very tight turns in protein structures (i.e. where the polypeptide chain must change direction).
  • a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein (at least or only) one amino acid in the Phosphatidylethanolamine Binding domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 , is replaced by a different amino acid, preferably under the proviso that this (at least or only) one amino acid is not an amino acids selected from D71 , Y85, H87, P111 , R112, P113, S114, H118, M120, and wherein the mutant allele delays the start of bolting of the Lactuca sativa plant by at least 7 days when the mutant allele is present in homozygous form, compared to the control plant lacking the mutant allele, and wherein the wild type allele encodes the protein of SEQ ID
  • mutant allele encodes a mutant protein wherein the Proline at position 75 of SEQ ID NO: 1 is replaced by a different amino acid.
  • any of the mutant allele encodes a mutant protein wherein the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 is replaced by a different amino acid.
  • the Lactuca sativa plant comprising any one of the above mutant alleles flowers normally when the mutant allele is in homozygous form, but the start of flowering is delayed as a consequence of the delay in bolting.
  • mutant allele encoding the mutant P75X protein was found to result in an average delay in the start of bolting by at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or more days compared to the control plant grown under the same conditions. Thereby the harvest window or harvest period is significantly extended.
  • the delayed bolting phenotype of the present invention seen e.g. in the P75S homozygous mutant plant, was also found to be associated with an improved quality and/or and increased biomass of the harvested lettuce heads and/or harvested lettuce leaves.
  • biomass compact filling of leaves in the given period crop cycle
  • quality intact shape of the heads
  • the plant of the present invention e.g. the P75S homozygous mutant plant, shows an enhanced field holding capacity, i.e. extended harvest window in e.g. heading lettuce types which should allow the growers to flexible harvest window depending upon the good market prices, adverse weather conditions (decreased heat sensitivity) and shortage of labour or machines for the harvest.
  • enhanced field holding capacity i.e. extended harvest window in e.g. heading lettuce types which should allow the growers to flexible harvest window depending upon the good market prices, adverse weather conditions (decreased heat sensitivity) and shortage of labour or machines for the harvest.
  • the plant of the plants of present invention e.g. the P75S homozygous mutant plant, have the advantage of having smaller flowering primordia (i.e. the hard core inside the lettuce head), which leads to a higher yield of processed leaves.
  • a lettuce plant or lettuce seed comprises at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, especially a mutant allele which encodes a mutant FT protein (comprising one or more amino acids inserted, deleted or replaced), whereby the presence of the mutant allele delays bolting and/or delays flowering when the mutant allele is in homozygous form.
  • the wild type FT gene is a gene which encodes a functional protein (the FT protein) comprising at least 95% amino acid sequence identity to SEQ ID NO: 1 , e.g. 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3% or more preferably 99.7% sequence identity to SEQ ID NO: 1 (as determined using pairwise alignments, e.g. using Needle).
  • the mutant allele encodes a protein in which one (at least one or only one) amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or the equivalent amino acids of a PB domain in a variant sequence comprising at least 95% identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 amino acids of the PB domain that are involved in substrate binding (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120).
  • the mutant allele encodes a mutant protein wherein the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 (or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1) is replaced by a different amino acid, especially by a disfavoured amino acid.
  • the mutant allele of the of the wild type FT gene of the present invention encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or at the equivalent amino acid position in a wild type FT protein comprising at least 95% 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid residue.
  • the substitution of the Proline amino acid residue with a different amino acid residue leads to a reduction of the normal (wild type) protein function of the FT protein, whereby the reduced function is capable of inducing the delayed bolting trait of the present invention.
  • Proline is a non-polar, hydrophobic amino acid which plays an important role in protein structure and replacing Proline with a different amino acid, especially with so-called disfavoured amino acid, may cause a change in structure and/or reduce protein function in vivo.
  • Disfavoured amino acids for replacing Proline are Ala, Lys, Asp, Glu, Gin, Ser, Thr, His, Arg, Asn, Gly, Vai, Met, Leu, Cys, Tyr, lie, Phe and Trp, see world wide web at russelllab.org/aas/Pro.html.
  • the mutant allele of the of the wild type FT gene of the present invention encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or at the equivalent amino acid position in a wild type FT protein comprising at least 95% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid selected from Ala, Lys, Asp, Glu, Gin, Ser, Thr, His, Arg, Asn, Gly, Vai, Met, Leu, ,Cys, Tyr, lie, Phe and Trp.
  • a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein the W (Tryptophan, Trp) amino acid residue at position 138 of SEQ ID NO: 1 (amino acid W138), or the equivalent amino acid in a wild type FT protein comprising at least 95% 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 , is absent and the protein is truncated, i.e. the rest of the wild type protein is missing.
  • FT Flowering Locus T
  • this mutant allele in homozygous form also resulted in a delayed bolting, while it would have been thought that no bolting would occur as the protein was expected to be non-functional. Further, it was surprisingly found that the mutant plant developed whorls of leaves on the extended stem after starting to bolt (see Figure 3). Without being bound by any theory, it appears that the intact PB domain and/or an intact N-terminal of the protein, including the PB domain, (only 38 amino acids at the C-terminal are missing in the W138* mutant) can, thus, still induce bolting (albeit significantly delayed), but the downstream flowering pathway is affected, resulting in a delayed bolting and non-flowering phenotype.
  • the truncated W138* protein induces bolting (the extension of the stem and the initiation of floral primordia), albeit delayed, but the floral primordia proliferate into whorls (or bundles) of leaves, instead of flowers.
  • This is a surprising phenotype, which however is useful in that it increases biomass and leaf tissue, and the leaves can be repeatedly cut off for harvest from the stem (so-called ratooning).
  • mutant alleles which result in truncated LsFT proteins, e.g. mutant proteins missing at least the last 10, 20, 30, 40 or 48 C-terminal amino acids of SEQ ID NO: 1 , show the same or very similar phenotype as the W138* mutant.
  • Amino acids 128 to 175 are the C-terminal amino acids which follow the PB domain. It could be that deletion of all or part of amino acids 128 to 175 (while keeping an intact PB domain or an intact N-terminal including the PB domain, i.e. amino acids 1 to 127) results in the same or similar phenotype as the W138* mutant.
  • Such mutant alleles can easily be generated by e.g.
  • Plants comprising such mutant alleles (encoding a truncated LsFT protein which lacks at least the last 10, 20, 30, 38, 40 or 48 C-terminal amino acids of SEQ ID NO: 1 (but comprise the remaining amino acids, e.g. amino acids 1 to 127) are, in one aspect, also an embodiment of the invention.
  • Such plants have, preferably, delayed bolting and no flowering, but develop whorls of leaves instead of flowers (see Figure 3).
  • the mutant allele of the FT gene may be an “induced mutant allele”, i.e. a mutant allele generated by human interventions such as mutagenesis.
  • Suitable mutagenesis methods comprise chemical mutagenesis (e.g. using EMS or MNU mutagenesis or mutagenesis by generating reactive oxygen species) and radiation mutagenesis (e.g. using UV radiation or ion beam radiation). Such methods are also referred to as random mutagenesis methods, as mutations are induced randomly in the genome. Methods such as TILLING can be used to screen mutagenized seeds or plants for the presence of mutant alleles of the FT gene.
  • the mutant allele of the FT gene may also be generated by targeted mutagenesis methods, such as targeted gene editing methods including, but not limited to, CRISPR/Cas9 -based targeted mutagenesis methods and CRISPR/Cpf1 -based targeted mutagenesis methods. It is noted that the exact same mutant alleles as provided herein or as described herein, e.g. in the Examples, can be generated by the skilled person without undue burden using random or targeted mutagenesis. Selfing of the mutants can then generate plants which are homozygous for the mutant allele and the effect of the homozygous mutant allele on the phenotype (bolting and/or flowering) can be tested as described.
  • targeted mutagenesis methods such as targeted gene editing methods including, but not limited to, CRISPR/Cas9 -based targeted mutagenesis methods and CRISPR/Cpf1 -based targeted mutagenesis methods. It is noted that the exact same mutant alleles as provided herein or as described herein,
  • a mutant / -allele may also be identified by screening of wild lettuce plants (e.g. landraces, PI accession, CGN accessions, etc.) or by screening orthologs of the Lactuca sativa FT gene in wild relatives of Lactuca sativa.
  • Such a “natural mutant allele”, which may be identified in a wild plants and/or in wild relatives of Lactuca sativa, may be introgressed into a cultivated Lactuca sativa plant using standard breeding methods to provide a plant according to the present invention. It is preferred that the mutant allele of the present invention is an induced mutant allele and not a ‘natural mutant allele’.
  • the plant of the present invention preferably is a cultivated Lactuca sativa plant.
  • Introgression of natural mutant alleles from e.g. wild relatives of lettuce has the disadvantage that generally linkage drag, i.e. the additional transfer of undesired traits, occurs. It is also highly questionable whether such ‘natural mutant alleles’ of the FT gene even exist, as they have no advantage to the plant at all, but rather a major disadvantage, namely delayed reproduction or even no reproduction, whereby one would expect that they disappear again as soon as they may have been generated by accident.
  • the mutant allele according to the present invention delays the average start of bolting of the Lactuca sativa plant when present in homozygous form.
  • the mutant allele according to the present invention delays average start of bolting of the Lactuca sativa plant when present in homozygous form by at least 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or more days when compared to a suitable control plant.
  • the mutant allele further also delays the start of flowering, e.g. by about the same time delay as the delay in the start of bolting, but the plant flowers normally.
  • only bolting time is affected and flowering is normal, only its start is delayed as a knock-on effect of the delayed bolting.
  • the mutant allele resulting in delayed bolting and also in delayed flowering is in one aspect a mutant allele in which one (at least one or only one) amino acid in the PB domain is replaced by another amino acid, e.g. P75, with preferably the provision that this one amino acid is not one of the nine amino acids involved in substrate binding (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120 of SEQ ID NO: 1 , or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1).
  • the mutant allele encodes a mutant protein wherein the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 (or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1) is replaced by a different amino acid.
  • the mutant ff-allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75) of SEQ ID NO: 1 , or the Proline amino acid residue at the equivalent amino acid position in a wild type (variant) FT protein comprising at least 95% sequence identity to SEQ ID NO: 1 , is replaced by a different amino acid residue.
  • the Proline amino acid residue at position 75 of SEQ ID NO: 1 may be replaced by an Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamine, Glutamic acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Serine, Threonine, Tryptophan, Tyrosine, or Valine amino acid residue.
  • the mutant allele according to the present invention encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the Proline amino acid residue at the corresponding amino acid position, is replaced by a Leucine, Alanine, Arginine, Threonine, Glutamine or Serine amino acid residue.
  • the mutant allele according to the present invention encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the Proline amino acid residue at the equivalent position in a variant FT protein comprising at least 95% identity to SEQ ID NO: 1 , is replaced by a Serine amino acid residue.
  • the plant according to the present invention comprises at least one copy of the mutant allele as provided herewith.
  • Such a plant may, thus, be heterozygous for the mutant allele of the present invention.
  • Such a heterozygous plant comprises (at least) one copy of the wild type allele and (at least) one copy of the mutant allele of the present invention.
  • As the gene is recessive such a heterozygous plant shows a normal bolting phenotype, i.e. the same as wild type plants (i.e. plants homozygous for the wild type FT allele).
  • the present invention thus, is also directed to plants comprising the mutant ft allele of the present invention in heterozygous form.
  • Such heterozygous plants can also be advantageously used in accordance with the present invention for breeding to generate offspring that is homozygous for the mutant ft allele as further described herein.
  • the Lactuca sativa plant according to the present invention is homozygous for the mutant allele.
  • Such a plant is inter alia characterized in that it shows an average delayed bolting and, optionally, further also an average delayed flowering of the plant, as further described herein.
  • the Lactuca sativa plant according to the present invention is homozygous for the mutant allele which results in a truncated LsFT protein and the plant is inter alia characterized in that it shows an average delayed bolting and no flowering of the plant but instead whorls of leaves developing on the stem, as further described herein.
  • the plant is heterozygous for the mutant allele and can flower normally, especially to be able to generate progeny seeds from the heterozygous plant, which seeds comprise the mutant allele in homozygous form or heterozygous form.
  • the Lactuca sativa plant comprising one or two copies of the mutant / -allele may be an inbred plant, a dihaploid (or double haploid) plant or a hybrid plant.
  • lettuce is a self pollinating crop, it is preferably an inbred plant line or variety comprising one or two copies of the mutant ft- allele.
  • the plants of the present invention may be any plant of the species Lactuca sativa as described herein, comprising in its genome at least one copy of a mutant allele of the wild type FT gene.
  • the Lactuca sativa plant according to the present invention is an inbred plant, a dihaploid plant or a hybrid plant.
  • the present invention provides that the plant of the present invention is an inbred plant.
  • Such an inbred plant is highly homozygous, for instance by repeated selfing or self pollination steps.
  • the disclosure provides for haploid plants and/or dihaploid (double haploid) plants of plant of the invention are encompassed herein, which comprise the mutant ft allele as described herein.
  • Haploid and dihaploid plants can for example be produced by anther or microspore culture and regeneration into a whole plant.
  • chromosome doubling may be induced using known methods, such as colchicine treatment or the like. So, in one aspect a Lactuca sativa plant is provided, comprising a delayed bolting phenotype as described, wherein the plant is a dihaploid plant.
  • the plants provided by the present invention may be used to produce food.
  • the present invention thus, provides the use of a plant of the species Lactuca sativa as provided herein as a crop for consumption.
  • the harvested leaves and/or harvested heads produced by the plants of the present invention can be advantageously used as a crop for consumption since these harvested leaves and/or harvested heads have an improved quality and/or and increased mass of the harvested heads and/or harvested leaves.
  • the Lactuca sativa plant according to the invention may be of any type, such as heading and non-heading types of lettuce.
  • the Lactuca sativa plant according to the present invention is of the head lettuce type or of the teen leaf type, loose leaf type or baby leaf type.
  • the plants provided by the present invention may be used to produce propagation material.
  • Such propagation material comprises propagation material suitable for and/or resulting from sexual reproduction, such as pollen and seeds.
  • Such propagation material comprises propagation material suitable for and/or resulting from asexual or vegetative reproduction including, but not limited to cuttings, grafts, tubers, cell culture and tissue culture.
  • the present invention thus, further provides the use of a Lactuca sativa plant as provided herein as a source of propagation material.
  • the present invention provides seed from which the Lactuca sativa plant according to the present invention can be grown. Furthermore, the invention provides a plurality of such seed.
  • a seed of the invention can be distinguished from other seeds due to the presence of the mutant allele of the wild type FT gene as described herein, either phenotypically (based on plants having the delayed bolting phenotype of the present invention) and/or using molecular methods to detect the mutant allele in the cells or tissues, such as molecular genotyping methods to detect the mutant allele of the present invention or sequencing.
  • the present invention provides seed from which any plant according to the invention can be grown, wherein said seed comprises in its genome at least one copy of a mutant allele of the wild type FT gene wherein said mutant allele encodes a protein in which one amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 amino acids involved in substrate binding of the PB domain (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120 of SEQ ID NO: 1 , or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1).
  • the present invention provides seed from which any plant according to the invention can be grown, wherein said seed comprises in its genome at least one copy of a mutant allele of the wild type FT gene wherein said mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the Proline amino acid residue at the equivalent amino acid position in a variant sequence comprising at least 95% identity to SEQ ID NO: 1 , is replaced by a different amino acid residue.
  • the present invention provides seed from which any plant according to the invention can be grown, wherein said seed comprises in its genome at least one copy of a mutant allele of the wild type FT gene wherein said mutant allele encodes a truncated protein at least 10, 20, 30, 38, 40, or 48 of the C-terminal amino acids are missing, e.g. the 38 C-terminal amino acids of SEQ ID NO: 1 (or the equivalent amino acids in a variant wild type FT protein comprising at least 95% sequence identity to SEQ ID NO: 1) are missing.
  • seed are provided from which any plant according to the invention can be grown, wherein said seed comprises in its genome at least one copy of a mutant allele of the wild type FT gene wherein said mutant allele encodes a mutant protein wherein the W (Tryptophan, Trp) amino acid residue at position 138 of SEQ ID NO: 1 (amino acid W138), or the W (Tryptophan, Trp) amino acid residue at the equivalent amino acid position in a variant sequence comprising at least 95% identity to SEQ ID NO: 1 , is absent and the protein is truncated and misses all further amino acids.
  • the truncated protein thus, only comprises amino acids 1 to 137 of the wild type protein.
  • the plant homozygous for this mutant allele does not flower and does not produce seeds, the plant is maintained in heterozygous form for sexual reproduction.
  • a heterozygous plant ft/FT
  • Seeds or seedlings then can be genotyped and optionally sorted, in order to provide seeds or seedlings of a certain genotype, e.g. seeds or seedlings homozygous for the mutant / -allele. For example non-destructive seed genotyping methods can be used.
  • Seeds include, for example, seeds produced by a plant of the invention which is heterozygous for the mutant allele after self-pollination and optionally selection of those seeds which comprise one or two copies of the mutant allele (e.g. by non-destructive seed sampling methods and analysis of the presence of the mutant ft allele), or seed produced after crosspollination, e.g. pollination of a plant of the invention with pollen from another Lactuca plant, preferably from another Lactuca sativa plant, or pollination of another Lactuca sativa plant with pollen of a plant of the invention.
  • the present invention provides pollen or seed produced by the plant according to the present invention, or seed from which a plant of the invention can be grown, wherein said plant is a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type FT gene, wherein the wild type FT gene encodes a protein of SEQ ID NO: 1 or a protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 1 , e.g.
  • mutant allele encodes a mutant protein in which one amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 amino acids involved in substrate binding (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120 of SEQ ID NO: 1 , or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1).
  • Reference to ‘one amino acid’ of the PB domain refers to either ‘at least one’ or, preferably, ‘only one’ of the amino acids of the PB domain, as the domain needs to keep in vivo functionality.
  • the plant By only changing one amino acid (and preferably not one of the 9 amino acids involved in substrate binding), the plant will be able to bolt and flower, albeit delayed. Changing more than one amino acid may have a too severe effect on in vivo functionality of the domain and the protein, but it may also be possible to change e.g. two or three amino acids.
  • the ‘one amino acid’ of the PB domain that is replaced by a different amino acid is selected from: the Proline at position 72, the Proline at position 75, the Proline at position 77, the Proline at position 80, the Proline at position 94 of SEQ ID NO: 1 (or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1).
  • the present invention provides pollen or seed produced by the plant according to the present invention, or seed from which a plant of the invention can be grown, wherein said plant is a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the wild type FT gene, wherein the wild type FT gene encodes a protein of SEQ ID NO: 1 or a protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 1 , e.g.
  • mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the Proline amino acid residue at the equivalent amino acid position in a variant, is replaced by a different amino acid residue or wherein the mutant allele encodes a truncated protein comprising only amino acids 1 to 137 (i.e. wherein the codon for W138, or the equivalent amino acid in a variant protein, is changed into a STOP codon).
  • the present invention provides pollen or seed produced by the plant according to the present invention, or seed from which a plant of the invention can be grown, wherein the pollen or seed comprises the mutant allele of the wild type FT gene as defined as defined herein that is capable of causing the delayed bolting phenotype when the mutant allele is in homozygous form.
  • the present invention provides seed from which the plant of the present invention can be grown.
  • the present invention further provides seeds obtained from the methods of producing plants as described herein. Also provided is a method of genotyping the DNA of plants or of seeds or plant parts for the presence of a mutant allele of the ff-gene and optionally selection of a plant or seed or plant part or a plurality plants or seeds or plant parts for a particular genotype, e.g. homozygous for the mutant ft-allele.
  • a KASP-assay may be used for genotyping, as e.g. provided in the Examples.
  • other genotyping assays may be used, such as Taqman, etc.
  • a plurality of seed is packaged into a container (e.g. a bag, a carton, a can etc.).
  • Containers may be any size.
  • the seeds may be pelleted prior to packing (to form pills or pellets) and/or treated with various compounds, including seed coatings.
  • a plant part obtained from (obtainable from) a plant of the invention is provided herein, and a container or a package comprising said plant part.
  • the present invention provides a plant cell, tissue or plant part of the Lactuca sativa plant according to present invention or of the seed from which the Lactuca sativa plant according to the present invention can be grown, comprising the mutant allele of the wild type FT gene as defined herein.
  • the plant part is a lettuce head, a leaf, a leaf part or a plurality of leaves comprising in their genome one or two copies of a mutant / -allele as described.
  • the head, leaf or leaf part is harvested and optionally packaged in bags, cartons or the like. In another aspect it is still connected to the roots, e.g. the plants may be in trays or pots.
  • the plant part is a plant cell.
  • the plant part is a non-regenerable cell or a regenerable cell.
  • the plant cell is a non-propagating cell.
  • the plant cell is a somatic cell.
  • a non-regenerable cell or a non-propagating cell is a cell which cannot be regenerated into a whole plant through in vitro culture.
  • the non-regenerable cell may be in a plant or plant part (e.g. leaves) of the invention.
  • the non-regenerable cell may be a cell in a seed, or in the seed- coat of said seed.
  • the plant cell is a reproductive cell, such as an ovule or a cell which is part of a pollen.
  • the pollen cell is the vegetative (non-reproductive) cell, or the sperm cell (Tiezzi, Electron Microsc. Review, 1991).
  • a reproductive cell is haploid. When it is regenerated into whole a plant, it comprises the haploid genome of the starting plant. If chromosome doubling occurs (e.g. through chemical treatment), a double haploid plant can be regenerated.
  • the plant of the invention comprising the mutant allele of the wild type FT gene as described herein is a haploid or a double haploid Lactuca sativa plant according to the present invention.
  • an in vitro cell culture or tissue culture of the Lactuca sativa plant of the invention in which the cell- or tissue culture is derived from a plant part described herein, such as, for example and without limitation, a leaf, a pollen, an embryo, cotyledon, hypocotyls, callus, a root, a root tip, an anther, a flower, a seed or a stem, or a part of any of them, or a meristematic cell, a somatic cell, or a reproductive cell.
  • a plant part described herein such as, for example and without limitation, a leaf, a pollen, an embryo, cotyledon, hypocotyls, callus, a root, a root tip, an anther, a flower, a seed or a stem, or a part of any of them, or a meristematic cell, a somatic cell, or a reproductive cell.
  • the present invention further provides a vegetatively propagated plant, wherein said plant is propagated from a plant part according to the present invention.
  • isolated cells in vitro cell cultures and tissue cultures, protoplast cultures, plant parts, harvested material (e.g. harvested plant material), pollen, ovaries, flowers, seeds, stamen, flower parts, etc. comprising in each cell at least one copy of the the mutant allele of the wild type FT gene as described herein are provided.
  • the plant comprises mutant allele of the wild type FT gene as described herein capable of conferring the delayed bolting phenotype of the present invention.
  • an in vitro cell culture and/or tissue culture of cells or tissues of plants of the invention is provided.
  • the cell or tissue culture can be treated with shooting and/or rooting media to regenerate a Lactuca sativa plant.
  • vegetative or clonal propagation of plants according to the invention is encompassed herein.
  • obtaining a part of a plant of the invention e.g. cells or tissues, e.g. cuttings
  • a method for vegetatively reproducing a Lactuca sativa plant of the invention comprising the mutant allele of the wild type FT gene as described herein is provided.
  • a vegetatively produced Lactuca sativa plant comprising the mutant allele of the wild type FT gene as described herein is provided.
  • a Lactuca sativa plant according to the invention, comprising the mutant allele of the wild type FT gene as described herein is propagated by somatic embryogenesis techniques.
  • Lactuca sativa plant regenerated from any of the above-described plant parts, or regenerated from the above-described cell or tissue cultures, said regenerated plant comprising in its genome the mutant allele of the wild type FT gene as described herein.
  • This plant can also be referred to as a vegetative propagation of plants of the invention.
  • the invention also relates to a food or feed product comprising or consisting of a plant part described herein.
  • the food or feed product may be fresh or processed, e.g., canned, steamed, boiled, fried, blanched and/or frozen etc. Examples are sandwiches, salads, juices, sauces, plant pastes or other food products comprising a plant or a part of a plant of the invention.
  • the present invention provides a plant part, obtained from (obtainable from) a plant of the invention, and a container or a package comprising said plant part.
  • the present invention provides a part from the plant of the present invention, wherein the part comprises in its genome at least one copy of the mutant allele of the wild type FT gene as described herein, preferably wherein the part is selected from the group consisting of a leaf, anther, pistil, stem, petiole, root, ovule, pollen, protoplast, tissue, seed, flower, cotyledon, hypocotyl and embryo.
  • the present invention provides a part of the plant according to the present invention, wherein said plant part is a leaf, anther, pistil, stem, petiole, root, ovule, pollen, protoplast, tissue, seed, flower, cotyledon, hypocotyl or embryo and wherein said part comprises in its genome at least one copy of the mutant allele of the wild type FT gene as described herein.
  • the part of the plant according to the present invention comprising in its genome at least one copy of the mutant allele of the wild type FT gene preferably is homozygous for the mutant ff-allele as described herein.
  • the various stages of development of aforementioned plant parts are comprised, as are parts thereof (e.g. parts of leaves, seeds, etc.).
  • harvested plant material such as harvested leaves or harvested heads produced by the plant of the present invention.
  • Plants according to the present invention may be homozygous or heterozygous for the mutant ff-allele as described herein.
  • the harvested plant material is homozygous for the mutant ff-allele as described herein.
  • the harvested plant material produced by the plant of the present invention can be distinguished from the harvested plant material according to the prior art by the presence of the mutant allele.
  • the harvested material has an improved (average) quality and/or and increased (average) biomass of the harvested lettuce heads and/or harvested lettuce leaves compared to the control of the same age and grown under the same conditions.
  • the present invention provides a method for producing harvested plant material having an increased percentage of compact filling of the leaves and/or having an intact shape of the heads when compared to isogenic plants not comprising the mutant allele of the present invention and grown in the same way (and being the same age), wherein said method comprises growing a Lactuca sativa plant according to present invention and harvesting plant material produced by said Lactuca sativa plant.
  • the present invention provides a method for producing harvested plant material having an improved average quality and/or having an increased average mass, said method comprising growing a Lactuca sativa plant according to present invention and harvesting plant material produced by said Lactuca sativa.
  • the harvesting may be done at a stage when the control plants are already bolting, i.e. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days after the control plant (homozygous for the wild type FT-allele) would start or starts to bolt.
  • a method of growing a lettuce plant comprising a mutant ff-allele in homozygous form comprises sowing seeds of the lettuce plant and harvesting the leaves or heads, optionally harvesting at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days after the control plant (homozygous for the wild type FT-allele) would start or starts to bolt.
  • plants, seeds, plant parts and cells according to the present invention are obtained by a technical method as described herein.
  • plants, plant parts and cells according to the present invention are not exclusively obtained by means of an essentially biological process, e.g. as defined by Rule 28(2) EPC.
  • a process for the production of plants or animals is essentially biological if it consists entirely of natural phenomena such as crossing or selection e.g. as defined by Rule 26(5) EPC.
  • the plants, plant parts or seeds are non-GMO (i.e. they do not contain genetic construct inserted into the genome through transformation).
  • the mutant alleles are generated by random mutagenesis (e.g. chemical or radiation mutagenesis) or by targeted mutagenesis, especially using the CRISPR system (e.g. Crispr/Cas9 or Crispr/Cpf/ or other nucleases).
  • the cultivated plant comprising the mutant ft- allele is not a transgenic plant, i.e. non-transgenic progeny are selected which do not comprise any vector construct or parts thereof e.g. a CRISPR construct.
  • the mutant allele of the LsFT gene comprises a human induced mutation, i.e. a mutation introduced by random mutagenesis techniques, such as chemical mutagenesis or radiation mutagenesis, or targeted mutagenesis techniques, such as Crispr based techniques.
  • a method for targeted mutagenesis of the endogenous FT gene in lettuce is provided herein, using any targeted gene modification method, such as CRISPR based methods (e.g. Crispr/Cas9 or Crispr/Cpfl), TALENS, Zinc Fingers or other methods.
  • CRISPR based methods e.g. Crispr/Cas9 or Crispr/Cpfl
  • TALENS Zinc Fingers or other methods.
  • the present invention further provides methods wherein a Lactuca sativa plant as described herein comprising at least one copy of a mutant allele of the wild type FT gene of the present invention is used and/or obtained.
  • the present invention provides a method for identifying and/or selecting a Lactuca sativa plant or plant part or seed comprising determining whether said Lactuca sativa plant or plant part comprises in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene as described herein, wherein the mutant allele encodes a mutant protein in which one amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 substrate binding site amino acids of the PB domain.
  • FT Flowering Locus T
  • the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1 can be easily identified by pairwise alignment of SEQ ID NO: 1 with the variant sequence, using e.g. Emboss Needle.
  • SEQ ID NO: 17 can be aligned with the variant sequence in a pairwise alignment to identify the PB domain in the variant sequence.
  • mutant allele in the above method encodes a mutant protein wherein the Proline at position 75 of SEQ ID NO: 1 is replaced by a different amino acid.
  • any of the mutant allele in the above method encodes a mutant protein wherein the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 is replaced by a different amino acid.
  • the method may comprise steps like obtaining genomic DNA from the plant or plant part or seed, generating a PCR product or a nucleic acid hybridization product for a specific mutant / -allele as described herein or generating sequence information of the mutant ff-allele, optionally selecting a plant, plant part or seed comprising a specific mutant / -allele as described herein and as detected in the previous step.
  • the present invention provides a method for identifying and/or selecting a Lactuca sativa plant or plant part comprising determining whether said Lactuca sativa plant or plant part comprises in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene as described herein, e.g. wherein the mutant allele encodes a mutant protein, wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75) (or the equivalent amino acid in a variant of SEQ ID NO: 1) is replaced by a different amino acid residue; or e.g.
  • FT Flowering Locus T
  • the wild type FT gene encodes a protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 1.
  • the wild type FT gene according to the present invention encodes a protein (the FT protein) comprising at least 95% amino acid sequence identity to SEQ ID NO: 1 , e.g.
  • the mutant allele of the FT gene as comprised in the Lactuca sativa plant or plant part as used in the identification and/or selection method of the present invention delays bolting of the Lactuca sativa plant when present in homozygous form.
  • the method comprises screening at the DNA, RNA (or cDNA) or protein level using known methods, in order to detect the presence of the mutant allele according to the present invention.
  • the SNP can easily be detected using a KASP-assay (see world wide web at kpbioscience.co.uk) or other SNP genotyping assays.
  • a KASP-assay for example 70 base pairs upstream and 70 base pairs downstream of the SNP can be selected and two allele-specific forward primers and one allele specific reverse primer can be designed. See e.g. Allen et al. 2011 , Plant Biotechnology J. 9, 1086-1099, especially p097-1098 for KASP-assay method.
  • forward and reverse primers which are allele specific and can be used in the detection or genotyping of the mutant allele encoding the P75S mutation, or the P136S or P136L mutation, or the P138* mutation are provided in the Examples, or any mutant allele described herein.
  • other primers can be designed for detection of the same or different mutations in the genomic DNA. E.g.
  • the primers may be slightly different, e.g. longer or shorter than the ones provided in the Examples or degenerate with respect of the template DNA.
  • the primers may be designed based on the forward strand or based on the reverse (complementary) strand of the genomic sequence.
  • genotyping assays can be used.
  • a TaqMan SNP genotyping assay a High Resolution Melting (HRM) assay
  • HRM High Resolution Melting
  • SNP- genotyping arrays e.g. Fluidigm, Illumina, etc.
  • DNA sequencing may equally be used.
  • in-gene markers like the SNP markers in the / -allele provided or described herein
  • other molecular markers may be used to aid in the identification of the plants (or plant parts or nucleic acids obtained therefrom) containing a mutant ft allele of the present invention.
  • a segregating population e.g. F2, F3 or backcross population
  • the segregating population can then be phenotyped for delayed bolting and genotyped using e.g. molecular markers such as SNPs (Single Nucleotide Polymorphisms), AFLPs (Amplified Fragment Length Polymorphisms; see, e.g., EP 534858), or others, and by software analysis molecular markers which co-segregate with the delayed bolting trait in the segregating population can be identified and their order and genetic distance (centimorgan distance, cM) to the FT gene (or locus) can be identified.
  • cM centimorgan distance
  • markers at a 5, 3, 2 or 1 cM distance or less can then be used in detecting and/or selecting plants (e.g. plants of the invention or progeny of a plant of the invention) or plant parts comprising or retaining the introgression fragment comprising the mutant ft allele.
  • Such closely linked molecular markers can replace phenotypic selection (or be used in addition to phenotypic selection) in breeding programs, i.e. in Marker Assisted Selection (MAS).
  • linked markers are used in MAS. More preferably, flanking markers are used in MAS, i.e. one marker on either side of the locus of the mutant ft allele.
  • the method for identifying and/or selecting a Lactuca sativa plant or plant part further comprises a step wherein the Lactuca sativa plant or plant part is subjected to a mutation inducing step prior to determining whether the Lactuca sativa plant or plant part comprises a mutant allele of the FT gene.
  • a mutagenizing step may involve the use of ethyl methanesulfonate (EMS) as mutagenic agent.
  • EMS ethyl methanesulfonate
  • the seed, plant or plant part is subjected to a mutation inducing step prior to determining whether the seed, plant or plant part comprises a mutant allele of the FT gene.
  • EMS ethyl methanesulfonate
  • said mutation inducing step may comprise contacting said seed, plant or plant part with a mutagen.
  • the seed or plant or plant part that is contacted with the mutagen comprises a wild type FT allele in homozygous form.
  • Said mutation inducing step may alternatively involve targeted mutagenesis techniques that depend on the site-specific induction of a double strand break in the genomic DNA of a host plant cell.
  • Inducing such a double strand break may comprise contacting a plant or plant part (e.g. a plant cell) with an engineered nuclease upon which said double strand break may be repaired by the cell’s endogenous DNA double stranded break repair mechanisms (e.g. the homology directed repair mechanism), which allows a site-specific deletion or inversion of DNA in a target cell.
  • Engineered nucleases useful in genome editing methods include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases.
  • Genome editing methods particularly useful in the context of the present invention include, but are not limited to, CRISPR/Cas9 -based targeted mutagenesis methods and CRISPR/Cas12 (also known as CRISPR/Cpf1)-based targeted mutagenesis methods; see e.g. Brooks et al. (2014) Plant Physiol 166, 1292-1297 and W02016/205711 A1.
  • the mutation inducing step subsequently causes a mutation in the wild type FT allele to provide a mutant ft allele that is capable of inducing the delayed bolting trait of the present invention.
  • the specific mutant alleles provided or described herein can be reproduced or generated de novo by any of the above mutagenesis techniques.
  • transgenic plants can be made, e.g. by using the mutant ft nucleotide sequences of the invention using known plant transformation and regeneration techniques in the art.
  • An "elite event” can be selected, which is a transformation event having the chimeric gene (comprising a promoter operably linked to a nucleotide sequence encoding mutant allele of the present invention) inserted in a particular location in the genome, which results in good expression of the desired phenotype.
  • transgenic plants can be made comprising a construct which reduces or abolishes the expression of the endogenous (wild type) FT gene, such as an RNAi construct, as described in further detail herein below.
  • the present invention accordingly provides a method of producing a Lactuca sativa plant comprising the steps of:
  • mutant allele in the above method encodes a mutant protein wherein the Proline at position 75 of SEQ ID NO: 1 is replaced by a different amino acid.
  • mutant allele in the above method encodes a mutant protein wherein the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 is replaced by a different amino acid.
  • the present invention also provides a method of producing a Lactuca sativa plant comprising the steps of:
  • 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 mutagenesis by generating reactive oxygen species) or other mutagenesis methods (e.g. by radiation mutagenesis using e.g.
  • S1 nucleases such as CEL1 or ENDO1
  • electrophoresis such as a LI-COR gel analyzer system, see e.g. Henikoff et al. Plant Physiology 2004, 135: 630-636.
  • TILLING has been applied in many plant species, including Lactuca sativa plants. (see http://tilling.ucdavis.edu/index.php/Tomato_Tilling ), rice (Till et al.
  • nucleic acid sequences encoding such mutant ft protein comprise one or more non-sense and/or missense mutations, e.g. transitions (replacement of purine with another purine (A ⁇ - G) or pyrimidine with another pyrimidine (C ⁇ - T) or transversions (replacement of purine with pyrimidine, or vice versa (C/T A/G).
  • non-sense and/or missense mutations e.g. transitions (replacement of purine with another purine (A ⁇ - G) or pyrimidine with another pyrimidine (C ⁇ - T) or transversions (replacement of purine with pyrimidine, or vice versa (C/T A/G).
  • the mutation in the FT gene results in the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75) being replaced by a different amino acid residue.
  • the mutation in the FT gene results in the Proline amino acid residue at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 being replaced by a different amino acid.
  • the mutation in the FT gene results in a truncated ft protein, wherein at least the 10, 20, 30, 38, 40 or 48 C-terminal amino acids are missing, while the amino acids 1 to 127 are present.
  • the mutation in the FT gene results in the codon for the Tryptophan (W) amino acid residue at position 138 of SEQ ID NO: 1 (amino acid W138) being replaced by a STOP codon.
  • a FT gene nucleotide sequence comprising one or more non-sense and/or missense mutations in one of the exon- encoding sequence are provided, as well as a plant comprising such a mutant allele resulting in a plant capable of producing plants having a delayed bolting phenotype when said mutant allele is present in homozygous form.
  • the plant or plant part is identified and/or selected from a TILLING population that was obtained by subjecting seeds, plants or plant parts to a mutagen as described in further detail herein below.
  • a method for producing a Lactuca sativa plant comprising the steps of:
  • mutant allele either i) encodes a mutant protein in which one amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 amino acids involved in substrate binding (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120 of SEQ ID NO: 1 , or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1); or ii) encodes a truncated protein, wherein the final at least 10, 20, 30, 38, 40 or 48 C
  • the mutant allele in step c) i) encodes a mutant protein wherein the Proline at position 75 of SEQ ID NO: 1 is replaced by a different amino acid.
  • the mutant allele in step c) i) encodes a mutant protein wherein the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 (or the equivalent amino acid in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1) is replaced by a different amino acid.
  • the mutant allele in step c) ii) encodes a mutant protein wherein the codon for W138 is changed into a STOP codon.
  • the mutant allele delays bolting of the Lactuca sativa plant when present in homozygous form.
  • a method for producing a Lactuca sativa plant comprising the steps of:
  • mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75), or the Proline at the equivalent position in a variant comprising at least 95% identity to SEQ ID NO: 1 , is replaced by a different amino acid residue; or where the codon for the Tryptophan (W) amino acid residue at position 138 of SEQ ID NO: 1 or the W at the equivalent position in a variant comprising at least 95% identity to SEQ ID NO: 1 (amino acid W138) is replaced by a STOP codon.
  • the mutant allele delays bolting of the Lactuca sativa plant when present in homozygous form.
  • Mutant plants (M1) are preferably selfed one or more times to generate for example M2 populations or preferably M3 or M4 populations for phenotyping.
  • M2 populations the mutant allele is present in a ratio of 1 (homozygous for mutant allele) : 2 (heterozygous for mutant allele) : 1 (homozygous for wild type allele).
  • the present invention provides a method for producing a Lactuca sativa plant comprising a mutant allele which delays bolting of said plant when present in homozygous form, said method comprising the step(s) of: a) crossing a first Lactuca sativa plant and a second plant, wherein the first Lactuca sativa plant comprises in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene, wherein said mutant allele either i) encodes a mutant protein in which one amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 amino acids involved in substrate binding (D71 , Y85, H87, P111 , R11
  • the present invention provides a method for producing a Lactuca sativa plant comprising a mutant allele which delays bolting of said plant when present in homozygous form, said method comprising the step(s) of: i) crossing a first Lactuca sativa plant and a second plant, wherein the first Lactuca sativa plant comprises in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75) or the Proline at the equivalent position in a variant comprising at least 95% identity to SEQ ID NO: 1 is replaced by a different amino acid residue, ii) optionally harvesting seed from the crossing of (i) and selecting seed comprising said mutant allele in its genome.
  • FT Flowering Locus T
  • the present invention provides a method for producing a Lactuca sativa plant capable of producing harvested plant material having an improved average quality and/or having an increased average mass, said method comprising the step(s) of: i) crossing a first Lactuca sativa plant and a second plant, wherein the first Lactuca sativa plant comprises in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene, wherein said mutant allele encodes a mutant protein in which one amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 amino acids involved in substrate binding (D71 , Y85, H87, P111 , R112, P113, S114, H
  • the present invention provides a method for producing a Lactuca sativa plant capable of producing harvested plant material having an improved average quality and/or having an increased average mass, said method comprising the step(s) of: a) crossing a first Lactuca sativa plant and a second plant, wherein the first Lactuca sativa plant comprises in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene, wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75) or the Proline at the equivalent position in a variant comprising at least 95% identity to SEQ ID NO: 1 is replaced by a different amino acid residue, b) optionally harvesting seed from the crossing of (i) and selecting seed comprising said mutant allele in its genome.
  • FT Flowering Locus T
  • both the first Lactuca sativa plant and the second Lactuca sativa plant in step (i) of the method of producing the Lactuca sativa plant capable of producing harvested plant material having an improved average quality and/or having an increased average mass as provided herein are plants according to the present invention comprising comprises a mutant allele of the wild type FT gene as described herein in their genome.
  • the present invention further provides a plant grown from seeds obtained by the method of identifying and/or selecting a Lactuca sativa plant or plant part comprising a mutant allele of the FT gene as described herein.
  • the present invention further provides a plant grown from seeds obtained by the method of producing a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the FT gene as defined herein.
  • the present invention further provides a method for the production of a Lactuca sativa plant capable of producing harvested plant material having delayed bolting by growing a seed according to the present invention, wherein said plant is homozygous for the mutant allele.
  • the herein-below described cell or cells is a non-regenerable cell as defined herein above.
  • the herein-below described cell or cells is a non-propagating cell.
  • non-propagating plant cell is a plant cell which is unable to maintain its life by synthesizing carbohydrate and protein from the inorganic substance, such as water, carbon dioxide and mineral salt and so on through photosynthesis.
  • the present invention provides a cell of the Lactuca sativa plant as further described herein. Accordingly, the present invention provides a cell of a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the Flowering Locus T (FT) gene, wherein said mutant allele either i) encodes a mutant protein in which one amino acid of the PB domain, starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1), is replaced by a different amino acid, preferably under the proviso that this one amino acid is not one of the 9 amino acids involved in substrate binding (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120 of SEQ ID NO: 1 , or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID
  • the present invention provides a cell of a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the FT gene, wherein the mutant allele encodes a mutant protein wherein the Proline amino acid residue at position 75 of SEQ ID NO: 1 (amino acid P75) or the Proline at the equivalent position in a variant comprising at least 95% identity to SEQ ID NO: 1 is replaced by a different amino acid residue.
  • the present invention also provides a cell of a Lactuca sativa plant comprising in its genome at least one copy of a mutant allele of the FT gene, wherein the mutant allele encodes a mutant protein which is truncated at the W138 of SEQ IS NO: 1 or at the W at the equivalent position in a variant comprising at least 95% identity to SEQ ID NO: 1.
  • the mutant allele as comprised in the cell of the Lactuca sativa plant according to the present invention preferably delays bolting of the Lactuca sativa plant when present in homozygous form.
  • the cell of the Lactuca sativa plant of the present invention preferably is homozygous for the mutant allele.
  • the cell provided by the present invention preferably is of a Lactuca sativa plant that is an inbred plant, a dihaploid plant or a hybrid plant.
  • the cell provided by the present invention may be of any type, preferably it is of a head lettuce type plant or of a teen-leaf type, a loose-leaf type or a baby leaf type plant.
  • a method of reproducing a lettuce plant comprising a mutant / -allele, wherein the codon for W138 is mutated into a STOP codon (or wherein the codon for the W at the equivalent position of a wild type FT protein comprising at least 95% sequence identity to SEQ ID NO: 1 is mutated into a STOP codon), or wherein a codon for any one of amino acid 127 to 166 is mutated into a STOP codon (resulting in a truncated protein lacking at least 10, 20, 30, 38, 40 or 48 C- terminal amino acids, as described elsewhere), via seeds.
  • the method comprises providing a plant which is heterozygous for the mutant ff-allele, selfing the plant to obtain seeds which segregate for the genotype of the mutant ff-allele, and optionally sorting the seeds or seedlings grown from the seeds by genotyping the genomic DNA for the presence of the mutant / -allele and/or wild type FT-allele.
  • seeds or seedlings comprising the mutant / -allele in homozygous form are selected and e.g. sold for sowing or planting.
  • a KASP assay may be used in the genotyping of the seeds or seedlings.
  • the primers provided in the Examples may be used in the KASP assay, but also other primers may be developed for allele specific genotyping.
  • the DNA sampling is non-destructive to the seeds or seedlings, i.e. they survive the DNA sampling.
  • a genotyping assay for genotyping lettuce plants, seeds, plant parts, cells or tissues, comprising the steps: a) providing genomic DNA of one or more lettuce plants or a population of plants, and b) carrying out a genotyping assay which detects the presence of the wild type allele of SEQ ID NO: 7 (or the complement strand thereof) and/or the presence of a mutant allele, wherein the mutant allele comprises one or more nucleotides inserted, deleted, replaced or duplicated with respect of SEQ ID NO: 7, resulting in a mutant protein or truncated protein as described elsewhere herein, and optionally c) selecting a plant, seed, plant part, cell or tissue comprising either two copies of the wild type allele, or one copy of the wild type allele and one copy of a mutant allele, or two copies of a mutant allele.
  • Step c) may also be selecting a plant, seed, plant part, cell or tissue comprising at least one copy of a mutant allele.
  • a genotyping assay genotyping lettuce plants, plant parts, cells or tissues comprising the steps: a) providing genomic DNA of one or more lettuce plants or a population of plants (e.g. breeding population, F2 population, backcross population etc.), and b) carrying out a genotyping assay which detects the presence of the wild type allele encoding the protein of SEQ ID NO: 1 and/or the presence of a mutant allele, wherein the mutant allele comprises one or more amino acids inserted, deleted, replaced or duplicated with respect of SEQ ID NO: 1 , especially as described elsewhere herein, and optionally c) selecting a plant, seed, plant part, cell or tissue comprising either two copies of the wild type allele, or one copy of the wild type allele and one copy of a mutant allele, or two copies of a mutant allele.
  • Step c) may also be selecting a plant, seed, plant part, cell or tissue comprising at least one copy of a mutant allele.
  • Step a) may comprise isolation of genomic DNA from the plant, seeds, plant part, cell or tissue to be analyzed in the genotyping assay. Often crude DNA extractions methods can be used, as known in the art.
  • Step b) preferably comprises a bi-allelic genotyping assay, which makes use of allelespecific primers and/or allele-specific probes.
  • the genotyping assay in step b) discriminates between the wild type FT-allele, encoding a protein of SEQ ID NO: 1 or a protein comprising at least 95% identity to SEQ ID NO: 1 , and one of the mutant alleles provided or described herein, selected from e.g. a mutant allele encoding a protein wherein one amino acid in the PB domain is replaced by another amino acid (as described elsewhere); or a mutant allele encoding a protein encoding a P75X amino acid substitution, e.g. P75S, i.e.
  • W138* truncation
  • Genotyping assays can be used, as long as they can detect INDELs and SNPs and can differentiate between e.g. the wild type allele of SEQ ID NO: 7 being present in the genomic DNA (at the FT locus) or a mutant allele of the FT gene being present in the genomic DNA. Genotyping assays may also discriminate between different mutant alleles.
  • 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 www at LGCgenomics.com and also www at biosearchtech.com/products/ pcr-kits-and-reagents/ genotyping-assays/ kasp-genotyping-chemistry), based on competitive allele-specific PCR and end-point fluorescent detection, the TaqMan-assay (Applied Biosytstems), which is also PCR based, HRM assays (High Resolution Melting Assay), wherein allele-specific probes are detected using real time PCR, or the rhAmp assay, based on Rnase H2-dependent PCR, BHQplus genotyping, BHQplex CoPrimer genotyping and many others.
  • KASP-assay by LGC, see www at LGCgenomics.com and also www at biosearchtech.com/products/ pcr-kits-and-reagents/ genotyping-assays/ kasp-genotyping-
  • the KASP-assay is also described in He C, Holme J, Anthony J. ‘SNP genotyping: the KASP assay. Methods Mol Biol. 2014;1145:75-86’ and EP1726664B1 or US7615620 B2, incorporated by reference.
  • the KASP genotyping assay utilizes a unique form of competitive allele-specific PCR combined with a novel, homogeneous, fluorescence-based reporting system for the identification and measurement of genetic variation occurring at the nucleotide level to detect single nucleotide polymorphisms (SNPs) or inserts and deletions (InDeis).
  • 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.
  • TaqMan genotyping assays is also described in Woodward J. ‘Bi-allelic SNP genotyping using the TaqMan® assay.’ Methods Mol Biol. 2014;1145:67-74, US5210015 and US5487972, incorporated herein by reference. With TaqMan(®) technology allele-specific probes are utilized for quick and reliable genotyping of known polymorphic sites. TaqMan assays are robust in genotyping multiple variant types, including single nucleotide polymorphisms, insertions/deletions, and presence/absence variants.
  • two TaqMan probes labelled with distinct fluorophores are designed such that they hybridize to different alleles during PCR-based amplification of a surrounding target region.
  • the 5'-3' exonuclease activity of Taq polymerase cleaves and releases the fluorophores from bound probes.
  • the emission intensity of each fluorophore is measured and allele determination at the queried site can be made.
  • Various genotyping assays can, therefore, be used, which can differentiate between the presence of e.g. the wild type allele of the FT gene, encoding the protein of SEQ ID NO: 1 , or a mutant allele of the FT gene; or between different mutant alleles of the FT gene.
  • Various mutant alleles of the FT gene can be detected. So, not only the mutant allele encoding the protein comprising a P75X or P136X amino acid substitution or an amino acid substitution in the PB domain or a truncation at the C-terminal end, such as the W138* truncation, but the assay can be designed to detect any other mutant allele of the FT gene, e.g. any mutant allele described herein.
  • 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, 21 , or 22 nucleotides complementary to SEQ ID NO: 7 (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 e.g. the wild type sequence from the mutant sequence of the allele.
  • the two forward primers thereby have different binding specificity (or preference) to either the wild type allele or to the mutant allele.
  • a KASP- assay can easily be designed to differentiate between the wild type allele of SEQ ID NO: 7 and any mutant allele of the FT gene which differs from the wild type allele in one or more nucleotides being inserted, deleted or replaced, so e.g. the assay can be designed for any SNP or INDEL that differentiates two alleles.
  • the amino acid change P75S is due to codon 340 to 342 of SEQ ID NO: 7 being mutated from CCA to TCA.
  • the mutated nucleotide is thus nucleotide 340 of SEQ ID NO: 7 (mutated from C to T).
  • the two forward KASP primers of SEQ ID NO: 8 and SEQ ID NO: 9 comprise a stretch of nucleotides complementary to the sequence preceding the mutated nucleotide plus either the wild type nucleotide (C) or the mutant nucleotide (T). Together with the reverse common primer of SEQ ID NO: 10, they amplify either the wild type allele or the mutant allele in the KASP assay.
  • the genotype is thereby determined for the SNP, either being homozygous wild type, homozygous mutant or heterozygous for mutant and wild type.
  • the SNP (C/T) at nucleotide 340 of SEQ ID NO: 7 is detected using a genotyping assay, such as a KASP assay, e.g. using the two forward primers and the reverse primer of SEQ ID NO: 8, 9 and 10), or other primers can be developed.
  • a genotyping assay such as a KASP assay, e.g. using the two forward primers and the reverse primer of SEQ ID NO: 8, 9 and 10
  • the SNP (C/T) at nucleotide 1948 of SEQ ID NO: 7 is detected using a genotyping assay, such as a KASP assay, e.g. using the two forward primers and the reverse primer of SEQ ID NO: 14, 15 and 16), or other primers can be developed.
  • a genotyping assay such as a KASP assay, e.g. using the two forward primers and the reverse primer of SEQ ID NO: 14, 15 and 16
  • the SNP (G/A) at nucleotide 1956 of SEQ ID NO: 7 is detected using a genotyping assay, such as a KASP assay, e.g. using the two forward primers and the reverse primer of SEQ ID NO: 11 , 12 and 13), or other primers can be developed.
  • a genotyping assay such as a KASP assay, e.g. using the two forward primers and the reverse primer of SEQ ID NO: 11 , 12 and 13
  • the SNP (C/T) at nucleotide 1949 of SEQ ID NO: 7 is detected using a genotyping assay, such as a KASP assay, e.g. using the two forward primers and the reverse primer.
  • a genotyping assay such as a KASP assay
  • mutant allele of the FT gene encodes a protein comprising one or more amino acids inserted, duplicated, replaced or deleted with respect of the wild type protein of SEQ ID NO: 1.
  • the mutant allele of the FT gene encodes a protein comprising one amino acid replaced in the PB domain of the protein, which is the domain starting at amino acid 66 of SEQ ID NO: 1 and ending at amino acid 127 of SEQ ID NO: 1 (or of the equivalent PB domain in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1 , preferably under the proviso that this one amino acid is not one of the 9 amino acids involved in substrate binding (D71 , Y85, H87, P111 , R112, P113, S114, H118, M120 of SEQ ID NO: 1 , or the equivalent amino acid in a variant sequence comprising at least 95% identity to SEQ ID NO: 1.
  • mutant alleles result in delayed bolting and delayed flowering when in homozygous form, as described for the P75S mutant allele, and plants and seeds comprising one or two copies of any of these mutant alleles are an embodiment herein.
  • the mutant allele comprises a Proline amino acid of the PB domain replaced by another amino acid, e.g. by a Serine, selected from: the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 (or the equivalent amino acid in a variant sequence comprising at least 95% sequence identity to SEQ ID NO: 1) is replaced by a different amino acid.
  • a Serine selected from: the Proline at position 72, or the Proline at position 75, or the Proline at position 77, or the Proline at position 80, or the Proline at position 94 of SEQ ID NO: 1 (or the equivalent amino acid in a variant sequence comprising at least 95% sequence identity to SEQ
  • the mutant allele of the FT gene encodes a protein which is truncated in comparison to the protein of SEQ ID NO: 1 , e.g. at least 10, 15, 20, 30, 37, 38, 39, 40, 48, amino acids are missing at the C-terminal end.
  • the C-terminal end has R175 as last amino acid, and when referring to a certain number of amino acids missing at the C-terminal end, it is understood that e.g. at least the final 10, 15, 20, etc. amino acids are missing, which are counted from amino acid R175.
  • the remaining part of the truncated protein is not modified compared to the wild type FT protein sequence of SEQ ID NO: 1 (or a functional variant thereof).
  • the N-terminal region starting from amino acid 1 and ending at amino acid 127, and especially the PB domain starting from amino acid 66 and ending at amino acid 127, is thus preferably identical to the wild type FT protein in truncated proteins.
  • These mutant alleles result in delayed bolting and no flowering (but instead whorls of leaves forming from the floral primordia), as described for the W138* mutant allele, and plants and seeds comprising one or two copies of any of these mutant alleles are an embodiment herein.
  • the mutant allele of the FT gene encodes a protein which comprises one or more amino acids deleted or replaced in comparison to the protein of SEQ ID NO: 1 , e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are deleted or replaced by one or more different amino acids.
  • the mutant allele of the FT gene encodes a protein which comprises one or more amino acids inserted or duplicated in comparison to the protein of SEQ ID NO: 1 , e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more amino acids are inserted or duplicated.
  • a method for detecting, and optionally selecting, a lettuce plant, seed or plant part comprising at least one copy of a wild type allele and/or of a mutant allele of a gene name FT gene comprising: a) providing genomic DNA of a lettuce plant or of a plurality of plants (e.g. a breeding population, F2, backcross, etc.), b) carrying out an assay (e.g. a bi-allelic genotyping assay) that discriminates or can discriminate between the presence of alleles in the genomic DNA of a), based on nucleic acid amplification (e.g.
  • the mutant allele comprises one or more nucleotides inserted, duplicated, deleted or replaced with respect to the sequence of SEQ ID NO: 7 (or the mutant allele encodes a protein comprising one or more amino acids inserted, duplicated, deleted or replaced with respect to the wild type protein of SEQ ID NO: 1 or a variant comprising at least 95% identity to SEQ ID NO: 1), and optionally c) selecting a plant, seed or plant part comprising one or two copies of the mutant allele
  • the mutant allele detected and optionally selected in any of the above methods is preferably any of the mutant alleles described herein, e.g. resulting in delayed bolting and delayed flowering when in homozygous form, as e.g. the P75S mutant allele, or resulting in delayed bolting and no flowering when in homozygous form, as e.g. the W138* mutant allele.
  • the genotyping assay discriminates between the wild type and the mutant alleles based on nucleic acid (especially DNA) amplification reactions making use of e.g. oligonucleotide primers, such as PCR (Polymerase Chain Reaction) and PCR primers, preferably allele-specific primers, and/or nucleic acid hybridization making use of as oligonucleotide probes, preferably allele-specific probes.
  • oligonucleotide primers such as PCR (Polymerase Chain Reaction) and PCR primers, preferably allele-specific primers, and/or nucleic acid hybridization making use of as oligonucleotide probes, preferably allele-specific probes.
  • the assay uses one or more FT allele specific primers or one or more FT allele specific probes.
  • genomic sequences which encode the protein of SEQ ID NO: 1 or a variant thereof (e.g. a genomic sequence comprising at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 7) or the genomic sequence of a mutant allele which encodes e.g.
  • PCR primers and nucleic acid probes can be designed using known methods or software programs for oligonucleotide design.
  • Primers and probes may for example be at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more nucleotides (bases) in length and anneal to (or hybridize to) the template DNA sequence, i.e. they preferably have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the target sequence.
  • the primer or probe specificity to a wild type allele or a mutant allele is due to at least 1 , 2, 3 or more nucleotides of the primer or probe being specific for either allele.
  • the primers or probes are thus designed around the polymorphism (e.g. the SNP or InDei) between the two alleles of the target gene, so that they discriminate between these.
  • the assay is a bi-allelic genotyping assay selected from e.g. a KASP-assay, a TaqMan-assay, a BHQplus probe assay or any other bi-allelic genotyping assay.
  • the mutant allele comprises at least one codon inserted or duplicated in the coding region of the allele, or at least one codon changed into another codon (e.g. through a single nucleotide change), or at least one codon deleted or changed into a STOP codon.
  • the mutant allele comprises a nucleotide replaced in codon 340 to 342 of SEQ ID NO:7 (e.g. nucleotide 340 is replaced, e.g. from Cytosine to Thymine), leading to the P75X amino acid replacement in the protein.
  • the mutant allele comprises a nucleotide replaced in codon 1948 to 1950 of SEQ ID NO:7 (e.g. nucleotide 1948 or nucleotide 1949 is replaced, e.g. from Cytosine to Thymine), leading to the P136X amino acid replacement in the protein.
  • the mutant allele comprises a nucleotide replaced in codon 1954 to 1956 of SEQ ID NO: 7 (e.g. nucleotide 1956 is replaced, e.g. from Guanine to Adenine, or nucleotide 1955 is replaced, e.g. from Guanine to Adenine), leading to the W138* in the protein.
  • the methods can be used to discriminate between plants, seeds or plant parts comprising two copies of the wild type FT allele encoding the protein of SEQ ID NO:
  • mutant ft- allele selected from e.g. a mutant ft-allele encoding a protein of SEQ ID NO: 1 in which one amino acid of the PB domain is replaced by another amino acid (as described elsewhere), such as e.g. a mutant / -allele encoding the protein of SEQ ID NO: 1 in which amino acid Proline 75 is changed into another amino acid, or a mutant / -allele in which a codon coding for any one of amino acids 128 to 166 is changed into a STOP codon resulting in a truncated protein (as described elsewhere herein), such as e.g. the codon for W (Trp) at position 138 is changed into a STOP codon, or one copy of each the wild type and a mutant / -allele (heterozygous).
  • plants, plant parts or seeds comprising any of these genotypes may be selected for e.g. further breeding or for use in lettuce production.
  • any DNA genotyping assay may be used in the above methods, be it PCR based (using PCR primers) and/or hybridization based (using probes), in one aspect a KASP-assay is used to discriminate between the wild type and the mutant allele.
  • the assay can be used in a high throughput way, e.g. in 96 well plates or more well plates (e.g. 384 well plates).
  • two forward primers (one for the wild type allele and one for the mutant allele) and one common reverse primer (for both the wild type and the mutant allele) are used in the KASP-assay.
  • the two forward primers and the reverse primer comprise at least 10,
  • the forward primers further comprise at least 1 , 2, or 3 nucleotides (preferably at the 3’end of the primer) which confer specificity (or preference) to either amplification of the wild type allele or amplification of the mutant allele.
  • Each forward primer forms a primer pair with the common reverse primer to amplify the DNA sequence of the target allele in between the primer pair, during thermal cycling. Standard components for thermal cycling are used and standard components for KASP-assays.
  • the KASP-assay discriminates between the SNP found between the wild type and mutant ft- allele, i.e. the KASP-assay can discriminate between the presence in the genomic DNA of SEQ ID NO: 7 in homozygous form (FT wild type, normal bolting allele), and the presence of e.g. one of the mutant ff-alleles described herein in homozygous form (mutant ff-allele, resulting in delayed bolting).
  • Different forward and reverse primers can be designed to achieve allele discrimination in the assay.
  • the forward primers comprise the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 9, or the complement sequence of either of these.
  • the common primer optionally comprises the sequence of SEQ ID NO: 10 or the complement sequence thereof.
  • the primers comprise one or more of SEQ ID NO: 8 (forward primer), SEQ ID NO: 9 (forward primer), and SEQ ID NO: 10 (common primer), or a sequence comprising at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 8, SQ ID NO: 9 or SEQ ID NO: 10 or a complementary sequence of any one of these sequences.
  • the forward primers comprise the sequence of SEQ ID NO: 11 and/or SEQ ID NO: 12, or the complement sequence of either of these.
  • the common primer optionally comprises the sequence of SEQ ID NO: 13 or the complement sequence thereof.
  • the primers comprise one or more of SEQ ID NO: 11 (forward primer), SEQ ID NO: 12 (forward primer), and SEQ ID NO: 13 (common primer), or a sequence comprising at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 11 , SQ ID NO: 12 or SEQ ID NO: 13 or a complementary sequence of any one of these sequences.
  • the forward primers comprise the sequence of SEQ ID NO: 14 and/or SEQ ID NO: 15, or the complement sequence of either of these.
  • the common primer optionally comprises the sequence of SEQ ID NO: 16 or the complement sequence thereof.
  • the primers comprise one or more of SEQ ID NO: 14 (forward primer), SEQ ID NO: 15 (forward primer), and SEQ ID NO: 16 (common primer), or a sequence comprising at least 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 14, SQ ID NO: 15 or SEQ ID NO: 16 or a complementary sequence of any one of these sequences.
  • Such a genotyping assay can be used for marker assisted selection (MAS) of plants in e.g. a breeding program to select plants comprising a certain genotype, e.g. homozygous for the wild type allele of the FT gene (having normal bolting), homozygous or heterozygous for a mutant allele of the FT-gene.
  • MAS marker assisted selection
  • a method of breeding lettuce plants comprising genotyping one or more plants for the allele composition at the FT locus in the genome and optionally selecting one or more plants having a specific genotype at the FT locus.
  • genotyping-by-sequencing may be done for the FT gene.
  • the plants or seeds which comprise two copies of a mutant ft- allele can be grown and phenotyped for the delayed bolting phenotype.
  • the mutant allele is in one aspect a mutant allele which, in homozygous form, confers delayed bolting.
  • the mutant allele encodes a protein comprising an amino acid replacement for an amino acid of the PB domain of SEQ ID NO: 1 (or the equivalent PB domain in a protein comprising at least 95% identity to SEQ ID NO: 1).
  • the mutant allele encodes a protein comprising an amino acid replacement for the Proline 75 of SEQ ID NO: 1 (or the equivalent amino acids in a protein comprising at least 95% identity to SEQ ID NO: 1).
  • the mutant allele encodes a protein comprising a truncation of at least 10, 20, 30, 38, 40 or 48 amino acids at the C-terminal end of SEQ ID NO: 1 (or the truncation in a protein comprising at least 95% identity to SEQ ID NO: 1).
  • the mutant allele encodes a protein comprising a change of the codon encoding the W138 of SEQ ID NO: 1 (or the equivalent amino acid in a protein comprising at least 95% identity to SEQ ID NO: 1) into a STOP codon.
  • a lettuce especially Lactuca sativa, plant, seed or plant part
  • said mutant allele encodes a mutant protein comprising one or more amino acids replaced, inserted, duplicated or deleted compared to the wild type protein, wherein said mutant allele confers delayed bolting when the mutant allele is in homozygous form (compared to the plant comprising the wild type allele in homozygous form), and wherein the wild type FT allele encodes a protein of SEQ ID NO: 1 or a protein comprising at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1.
  • SEQ ID NO : 7 genomic sequence encoding wild type FT protein, the codon CCA at nucleotides 340-342 codes for Proline , P75 )
  • SEQ ID NO : 8 FAM primer for KASP assay (wild type allele of codon at nucleotide 340-342 of SEQ ID NO : 7 ) gaaggtgacc aagttcatgc tgtgatggtc gatcctgatg etc
  • SEQ ID NO : 11 to SEQ ID NO : 16 are primers for KASP assays , as shown in Example 3
  • SEQ ID NO : 17 is the conserved Pfam01161 sequence of Figure 4
  • a highly homozygous inbred line used in commercial lettuce breeding can be used for mutagenesis treatment with the following protocol.
  • EMS is rinsed out by washing 5 times, 5 minutes, in 100ml fresh demineralized water.
  • seeds are directly sown in the greenhouse. Out of the seeds that germinate, a sufficient number of plantlets are transplanted in the field. From these plantlets, seeds are harvested from the surviving and seed bearing plants. For instance, from each remaining M1 mutant plant, M2 seeds are isolated. From the obtained M2 seed family, specific seed lots may be excluded from the population due to low seed set.
  • DNA is extracted from a pool of 10 seeds originating from each M2 seed lot. Per mutant line, 10 seeds are pooled in a Micronic® deepwell tube; http://www.micronic.com from a 96 deepwell plate, 2 stainless balls are added to each tube. The tubes and seeds are frozen in liquid nitrogen for 1 minute and seeds are immediately ground to a fine powder in a Deepwell shaker (Vaskon 96 grinder, Belgium; http://www.vaskon.com) for 2 minutes at 16,8 Hz (80% of the maximum speed).
  • a Micronic® deepwell tube http://www.micronic.com from a 96 deepwell plate
  • 2 stainless balls are added to each tube.
  • the tubes and seeds are frozen in liquid nitrogen for 1 minute and seeds are immediately ground to a fine powder in a Deepwell shaker (Vaskon 96 grinder, Belgium; http://www.vaskon.com) for 2 minutes at 16,8 Hz (80% of the maximum speed).
  • 300 pl Agowa® Lysis buffer P from the AGOWA® Plant DNA Isolation Kit http://www.agowa.de is added to the sample plate and the powder is suspended in solution by shaking 1 minute at 16,8 Hz in the Deepwell shaker. Plates are centrifuged for 10 minutes at 4000 rpm. 75 pl of the supernatant is pipetted out to a 96 Kingfisher plate using a Janus MDT® (Perkin Elmer, USA; http://www.perkinelmer.com) platform (96 head). The following steps are performed using a Perkin Elmer Janus® liquid handler robot and a 96 Kingfisher® (Thermo labsystems, Finland; http://www.thermo.com).
  • the supernatant containing the DNA is diluted with binding buffer (150 pl) and magnetic beads (20 pl). Once DNA is bound to the beads, two successive washing steps are carried out (Wash buffer 1 : Agowa wash buffer 1 1/3, ethanol 1/3, isopropanol 1/3; Wash buffer 2: 70% ethanol, 30% Agowa wash buffer 2) and finally eluted in elution buffer (100 pl MQ, 0,025 pl Tween).
  • HRM High Resolution Melt curve analysis
  • the double stranded PCR product starts to melt, releasing the dye.
  • the release of dye results in decreased fluorescence that is recorded as a melting curve by the fluorescence detector. Pools containing a mutation form hetero duplexes in the post-PCR fragment mix. These are identified as differential melting temperature curves in comparison to homo duplexes.
  • Primers useful to amplify gene fragments for HRM are designed using a computer program (Primer3, http://primer3.sourceforge.net/). The length of the amplification product is limited between 200 and 400 base pairs. Quality of the primers is determined by a test PCR reaction that should yield a single product.
  • PCR Polymerase Chain Reaction
  • 4pl reaction buffer 5x Reaction Buffer
  • 2pl 10xLC dye ((LCGreen+ dye, Idaho Technology Inc., UT, USA)
  • 5pmole of forward and reverse primers each 4nmole dNTPs (Life Technologies, NY, USA)
  • 1 unit DNA polymerase Hot Start II DNA Polymerase
  • Reaction conditions were: 30s 98°C, then 40 cycles of 10s. 98°C, 15s 60°C, 25s of 72°C and finally 60s at 72°C.
  • SIFT Send Intolerant From Tolerant
  • SIFT is a program that identifies the region(s) of a user-selected gene and of its coding sequence where the anticipated point mutations are most likely to result in deleterious effects on the gene's function based on protein sequence conserveness as described in more detail above.
  • P75S codon for Proline at amino acid 75
  • Genotyping can be used for e.g. marker assisted selection in breeding or for sorting seeds in non-destructive seed sorting methods.
  • KASP assays use two allele specific forward PCR primers (hereunder the FAM and VIC primers) and one common reverse PCR primer to amplify the wild type and/or mutant alleles of a genomic DNA sample in a competitive allele-specific PCR.
  • the allele discrimination plot thereafter distinguishes between the genotype of the plant, which is either homozygous for the wild type allele, homozygous for the mutant allele or heterozygous (one copy of the mutant allele and one copy of the wild type allele).
  • Target-specific genome editing using engineered nucleases has become widespread in various fields. Single-base substitutions can be performed by homologous recombination (HR). Calli from plants can be mutagenized by co-transformation with a plasmid and donor fragment through particle bombardment, as described in e.g. Okamoto et al. (2019) Scientific Reports 9:4811 //doi.org/10.1038/s41598-019-41121-4 . Such plasmid would harbour cassettes expressing CAS9 and two guideRNAs (gRNAs) and a donor fragment as template for homology- directed repair (HDR). Expression of the Cas9 gene and gRNA are driven by a strong promoter such as a ubiquitin promoter. The gRNAs are be designed at opposite strands of the of the two targeting sites.
  • HR homologous recombination
  • the donor fragment contains the desired mutation in the middle of a fragment of, for example, 476 bp that corresponds to the sequence of the target gene (except for the mutation).
  • additional synonymous mutations that do not change amino acid residues in the donor fragment, would prevent Cas9 from cutting the donor fragment again, once HDR is successfully achieved.
  • the fragment is flanked with two gRNA target sequences including the PAM motifs, respectively, so that the donor DNA can be released by Cas9/gRNAs from the plasmid; see e.g. Sun et al. (2016) Molecular Plant 9, 628-631 DOI: 10.1016/j.molp.2016.01.001.
  • additional free DNA donor fragment can be co-introduced in the plant calli by particle bombardment. After calli bombardment, regenerated shoots selected based on plasmid encoded antibiotics resistance, are grown and analysed for the presence of mutations. This could be done by primers to amplify a target gene sequence from DNA by PCR. Primer are designed so that they cannot amplify a fragment from the plasmid. The amplified product can be sequenced to validate the presence of the mutation.
  • Plants can be regenerated from plant material comprising the desired mutation, such as calli or cultured plant cells, using standard methods.
  • P136L Another mutant allele had the codon for Proline at amino acid 136 (P136) changed into a codon for Leucine. The mutant is referred to as P136L.
  • the trial included 1 mutant plant homozygous for the P136S allele, 5 plants which were heterozygous for the P136S allele and 11 azygous control plants.
  • the trial further included 6 mutant plants which were homozygous for the P136L allele, 15 plants which were heterozygous for the P136L allele and 28 azygous control plants.
  • the wild type LsFT protein was analyzed using various bioinformatics software tools, such as the NCBI conserved domain database (CDD) tool, and it was found that the P75S substitution lies in the Pfam01161 domain, near D71 , which is one of 9 amino acids that are involved in substrate binding.
  • CDD NCBI conserved domain database
  • Substrate binding sites were indicated as D71 , Y85, H87, P111 , R112, P113, S114, H118 and M120.
  • SEQ ID NO: 17 The alignment with the Pfam01161 domain (herein SEQ ID NO: 17) is shown in Figure 4.
  • PEBPs PhosphatidylEthanolamine- Binding Proteins
  • a number of biological roles for members of the PEBP family include serine protease inhibition, membrane biogenesis, regulation of flowering, plant stem architecture, and Raf-1 kinase inhibition.
  • the members of the PEBP family bind very different substrates including phospholipids, opioids, and hydrophobic odorant molecules as well as having different oligomerization states (monomer/dimer/tetramer).
  • BLAST of the LsFT protein against the PDB shows that the LsFT protein aligns to Chain A of the rice ‘Florigen Activation Complex’ (FAC), which consists of 6 proteins.
  • the 6 proteins are a heterohexamer, which is composed of two molecules each of Hd3a (florigen), GF14c and OsFD1.
  • LsFT aligns with one of the rice Hd3a proteins.
  • the database entry is PDB ID: 3AXY_A and is described in Taoka, Ki., Ohki, I., Tsuji, H. et al. “14-3-3 proteins act as intracellular receptors for rice Hd3a florigen”, Nature 476, 332-335 (2011 ; //doi.org/10.1038/nature10272).
  • Figure 4g of this article shows a model of FAC formation: Once Hd3a florigen enters a shoot apical cell, it initially binds 14-3-3 proteins in the cytoplasm. When the Hd3a-14-3-3 complex enters the nucleus, it forms a complex with OsFD1 , which is retained in the nucleus and activates OsMADSI 5 transcription, leading to floral induction.
  • LsFT acts as a ‘florigen’ signal that is produced in leaves and is transported to the shoot apical meristem via the phloem, similar to the rice florigen signal, and may form part of a similar ‘Florigen Activation Complex’ (FAC), which translocates to the nucleus of apical shoot cells, where it induces transcription of downstream genes to initiate flowering.
  • FAC Florigen Activation Complex
  • the mutant still initiates bolting, albeit delayed compared to the wild type LsFT protein, showing that bolting is not prevented by the absence of the final 38 amino acids.
  • the P75S mutant has a delay in bolting and a delay in flowering which is about the same time delay, and it is though that the P75S mutant can, thus, still form an active FAC to induce floral induction, but that there may be a conformational change to the protein which delays bolting.
  • the delay in flowering is thought to be a knock-on effect of the delay in bolting.

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Abstract

La présente invention concerne une plante Lactuca sativa ayant une montée à graines retardée, ladite plante comprenant dans son génome au moins une copie d'un allèle mutant du gène Flowering Locus T (FT) de type sauvage. La présente invention concerne une graine à partir de laquelle la plante Lactuca sativa selon la présente invention peut être cultivée. La présente invention concerne en outre une cellule végétale, un tissu ou partie de plante de la plante selon la présente invention ou de la graine selon la présente invention comprenant l'allèle mutant du gène FT de type sauvage. La présente invention concerne un procédé d'identification et/ou de sélection d'une plante ou d'une partie de plante Lactuca sativa consistant à déterminer si ladite plante ou partie de plante comprend dans son génome au moins une copie d'un allèle mutant du gène FT de type sauvage. La présente invention concerne un procédé de production d'une plante Lactuca sativa comprenant un allèle mutant qui retarde la montée à graines de ladite plante lorsqu'il est présent sous une forme homozygote.
PCT/EP2022/072583 2021-08-16 2022-08-11 Plante de laitue à montée à graines retardée WO2023020938A1 (fr)

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