Genes for parthenogenesis

Abstract

The present invention provides nucleotide and amino acid sequences of Taraxacum parthenogenetic genes, and (functional) homologs, fragments and variants thereof, which achieve parthenogenesis as part of apomixis. Also provided are parthenogenetic plants and methods for producing them, as well as molecular markers and methods for using them.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, in particular to plant biotechnology, including plant breeding. The present invention particularly relates to the identification and use of genes associated with and useful in, for example, apomixis and monploid induction. The present invention particularly relates to genes associated with parthenogenesis, as well as their encoded proteins and fragments of both. The present invention further relates to methods for inhibiting and / or inducing parthenogenesis in plants and crops, to the use of genes and / or proteins or fragments thereof for apomixis, particularly in combination with apomictic gene(s), or for the production of monploid plants capable of doubling their chromosomes to produce doubled monploids. [Background technology]

[0002] Apomixis (also called agamospermy) is the asexual reproduction of plants through seeds. Apomixis has been reported in approximately 400 flowering plant species (Bicknell and Koltunow, 2004). Apomixis in flowering plants occurs in two forms: (1) Gametophytic apomixis, in which an embryo develops from a nonreduced unfertilized egg cell by parthenogenesis; (2) Sporophytic apomixis, in which the embryo develops somatically from a sporophytic cell. Examples of gametophyte apomicts are dandelion (Taraxacum sp.), hawkweed (Hieracium sp.), Kentucky bluegrass (Poa pratensis) and eastern gamagrass (Tripsacum dactyloides). Examples of sporophyte apomixis are citrus (Citrus sp.) and mangosteen (Garcinia mangostana). Gametophyte apomixis is a developmental process that involves two stages: (1) avoidance of meiotic recombination and reduction (apomeiosis); and (2) The development of an egg cell into an embryo without fertilization (parthenogenesis) But is involved.

[0003] Apomictically produced seeds are genetically identical to the parent plant. It has long been recognized that apomixis can be extremely useful in plant breeding (Asker, 1979; Hermsen, 1980; Asker and Jerling, 1990; Vielle-Calzada et al., 1995). The most obvious advantage of introducing apomixis into crops is the pure breeding of heterosis F1 hybrids. In most crops, F1 hybrids are elite varieties. However, in sexual crops, self-fertilization of F1 hybrids causes loss of heterosis due to recombination in the genome of the F2 progeny plants, so F1 hybrids need to be produced in each generation by mating the homozygous parents of the inbred again. Producing sexual F1 seeds is a complex, costly process that is repeated many times. In contrast, apomictic F1 hybrids are likely to produce pure varieties in perpetuity. In other words, genetic fixation of F1 hybrids through seeds and production of uniform progeny plants are possible.

[0004] F1 fixation by apomixis is a special case of the general property of apomixis, where any genotype, whatever its genetic complexity, will produce purebreds in one step. What this means is that apomixis can be used to instantly fix polygenic quantitative traits. Note that most yield traits are polygenic. Apomixis can be used for stacking (or pyramiding) multiple traits (e.g., various resistances, several transgenes, or multiple quantitative trait loci). Without apomixis, to fix such a set of traits, the loci for each trait must be made homozygous individually and then combined. As the number of loci involved in the traits increases, making these trait loci homozygous by crossing becomes time-consuming, challenging, and therefore expensive. Furthermore, certain epistatic interactions between alleles are lost by homozygosity. Apomixis allows this type of non-additive genetic variation to be fixed. Apomixis, clonal propagation through seeds, therefore has the potential to cause a paradigm shift in plant breeding, commercial seed production, and agriculture (van Dijk et al. 2016; Van Dijk and Schauer 2016).

[0005] In addition to quickly fixing any genotype, whatever its complexity, there are further important agricultural applications of apomixis. Sexual interspecific hybrids and autopolyploids often suffer from sterility due to meiotic problems. Apomixis can be used to solve such sterility problems for interspecific hybrids and autopolyploids, since it skips meiosis. Apomixis combined with male sterility has been proposed for transgene containment, preventing introgression of transgenes in wild relatives of transgenic crops, since apomixis prevents female hybridization (Daniell, 2002). In insect-pollinated crops (e.g., Brassica), apomictic seed set should not be limited by insufficient pollinator service. This becomes even more important in light of growing health problems of pollinating bee populations (Varroa mite infections, African killer bees, etc.). In tuber-propagated crops such as potato, apomixis would maintain clonally superior genotypes but also reduce or eliminate the current risks of virus transmission and associated costs in clean production, containment and certification. Also, the storage costs of apomictic seeds are much lower than those of tubers or other vegetatively propagated plant parts. In ornamentals, apomixis could replace labor-intensive and costly tissue culture propagation. In general, apomixis is considered to strongly reduce the costs of cultivar development and plant propagation.

[0006] Unfortunately, apomixis is not present in any of the major crops. There have been many attempts to introduce apomixis in sexual crops; for example, introgression of apomixis genes, mutation of sexual model species, de novo production of apomixis by hybridization, and cloning of candidate genes. Introgression of apomixis genes from wild apomicts into crop species by far-crossing has so far been unsuccessful (e.g. apomixis from Tripsacum dactyloides to maize - Savidan, Y., 2001; Morgan et al., 1998; WO 97 / 10704). Regarding mutant sexual model species, WO 2007 / 066214 describes the use of an apomixis mutant called Dyad in Arabidopsis. However, Dyad is a recessive mutation with extremely low penetrance. In crop species, this mutation has limited utility. The production of apomixis de novo by hybridization between two sexual ecotypes has not resulted in agronomically interesting apomicts (US Patent Application Publication No. 2004 / 0168216A1 and US Patent Application Publication No. 2005 / 0155111A1). The cloning of candidate apomixis genes by transposon tagging in maize is described in US Patent Application Publication No. 2004 / 0148667. An orthologue of the elongation gene, which is hypothesized to induce apomixis, has been claimed. However, according to Barrell and Grossniklaus (2005), the elongation gene skips meiosis II and therefore does not maintain the maternal genotype, which makes it much less useful.

[0007] In US Patent Application Publication No. 2006 / 0179498, it was described that so-called reverse breeding could be an alternative to apomixis. However, reverse breeding is a technically complex in vitro laboratory procedure, whereas apomixis is an in vivo procedure carried out by the plant itself. Furthermore, when using reverse breeding, crosses still need to be carried out once the parental lines have been reconstituted (doubled gamete homozygotes).

[0008] Apomixis in natural apomicts generally has a genetic basis (reviewed by Ozias-Akins and Van Dijk, 2007). An alternative could therefore be the isolation of apomictic genes from natural apomictic species. However, this is not an easy task, since natural apomicts often have polyploid genomes, and positional cloning in polyploids is extremely difficult. Other complicating factors are the suppression of recombination in chromosomal regions specific for apomixis, repetitive sequences, and segregation distortion during mating. Summary of the Invention

[0009] As described herein, there is a need for a procedure for inducing apomixis in crop plants that is free of at least some of the limitations of the current state of the art. In particular, there is a need for a method for producing apomictic plants and apomictic seeds. There is also a need to provide genes and proteins involved in the process of apomixis, in particular parthenogenesis, that are suitable for use in introducing apomixis into crop plants and that can substantially mimic the apomictic pathway.

[0010] The inventors have now identified and isolated parthenogenetic loci and genes, alleles associated with parthenogenetic phenotypes (herein referred to as parthenogenetic alleles or Par alleles) and non-parthenogenetic phenotypes (herein referred to as sexual or non-parthenogenetic alleles or par alleles), their gene sequences, i.e. promoter or 5'UTR sequences, coding sequences, 3'UTR sequences and encoded protein sequences. Parthenogenesis can be directly introduced into sexual plants, possibly by random or targeted mutagenesis, by transformation or by somatic cell hybridization. By genetically modifying a sexual allele of a parthenogenetic locus of a sexual plant, for example by mutagenesis, transgenesis or by introduction of a double-strand break at a specific site and insertion via homologous recombination, a Par allele may be introduced, enabling the plant and / or its progeny to develop an egg cell into an embryo. [Brief description of the drawings]

[0011] [Figure 1] Figure 1: Multiple alignment of the coding sequence (nucleotides 325-360 of the Par allele coding sequence) and the encoded amino acids of the amplicon from a control plant showing the wild type sequence (SEQ ID NO:23) and from a transgenic plant containing a vector encoding the Cas9 / RNA-1 complex showing modified sequences (SEQ ID NOs:24-27). The gene-specific portions of guide RNA-1 are indicated in boxes. Modifications are indicated in bold and underlined. The wild type sequence includes spacing (-) for alignment reasons. [Diagram 2] Diagram of germination experiment. Top row; A68 control, normal viable black seeds that germinate. Middle row; non-viable, light grey, non-germinated seeds of plant pKG10821-6 carrying a 3 bp deletion in gene 164. Bottom row; all tetraploid, germinated and viable progeny of plant pKG10821-6 pollinated with FCH72 monaploid pollen. Seeds in each petri dish are derived from a single seed head. [Diagram 3]FIG. 1 shows an example of a cleared ovule containing an embryo 75 hours after emasculation of a transgenic lettuce line harboring a Dandelion Par allele driven by the Arabidopsis EC1.1 promoter. When such an embryo was found, it was included in the total observations, as shown in Table 3. [Figure 4] FIG. 13. Examples of polyembryony in cleared ovules 75 hours after emasculation of transgenic lettuce lines harboring the Par allele of Dandelion driven by the Arabidopsis EC1.1 promoter. Each asterisk marks an embryo. [Diagram 5] FIG. 13. Analysis of Par gene expression in APO, PAR and SEX plants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] definition As used herein, the term "locus" (plural: loci) refers to a specific location (or locations) or site on a chromosome where, for example, a gene or genetic marker is found. For example, a "parthenogenetic locus" refers to the location in the genome where a parthenogenetic gene is located, an allele that contributes to the parthenogenetic phenotype, i.e. (parthenogenetic allele or Par allele) and / or its sexual counterpart(s), i.e., non-parthenogenetic gene(s) (non-parthenogenetic allele(s) or par allele(s)). A gene, allele, protein, or nucleic acid that is "functional in parthenogenesis" is understood herein to be one that contributes to the parthenogenetic phenotype and / or that confers to a plant or plant cell the ability to develop an egg cell into an embryo.

[0013] As used herein, the term "allele(s)" refers to any of one or more alternative forms of a gene at a particular locus. In diploid and / or polyploid cells of an organism, alleles of a given gene are located at specific positions or loci on chromosomes, where one allele is present on each chromosome of a set of homologous chromosomes. Diploid and / or polyploid, or plant species can contain a large number of different alleles at a particular locus.

[0014] The term "dominant allele" as used herein refers to the relationship between alleles of a gene in which the effect of one allele on the phenotype (i.e., a dominant allele) masks the contribution of a second allele (i.e., a recessive allele) at the same locus. For genes on autosomes (any chromosome other than the sex chromosomes), the alleles and associated traits of the alleles are autosomal dominant or autosomal recessive. Dominance is an important concept in Mendelian inheritance and classical genetics. For example, dominant alleles can code for functional proteins, whereas recessive alleles do not. In one embodiment, the genes and their fragments or variants taught herein refer to dominant alleles of parthenogenetic genes.

[0015] The term "ovary" (plural: "ovaries") as used herein refers to the enclosure in which spores are formed. A female ovary may consist of a single cell or may be multicellular. All plants, fungi, and many other lineages form an ovary at some point in their life cycle. The ovary can produce spores by mitosis or meiosis. Generally, within each ovary, four monoploid megaspores are produced by meiosis of the megaspore mother cell. In gymnosperms and angiosperms, only one of these four megaspores is functional at maturity, while the other three degenerate. The remaining megaspore divides mitotically and develops into a female gametophyte (megagametophyte), which ultimately produces one egg cell.

[0016] The term "female gamete" as used herein refers to a cell that, under normal (sexual) circumstances, fuses with another ("male") cell during the process of fertilization (conception) in sexually reproducing organisms. In species that produce two morphologically distinct types of gametes, with each individual producing only one type, a female is any individual that produces the larger type of gamete, called an ovule (egg) or egg cell. In plants, female ovules are produced by the ovary of a flower. Upon maturity, the monoploid ovule produces female gametes, which are then ready for fertilization. The male cells are the (mostly monoploid) pollen, which are produced by the anthers.

[0017] The term "genetic marker" or "polymorphic marker" refers to a region on genomic DNA that can be used to "mark" a specific location on a chromosome. If a genetic marker is closely linked to or "on" a gene, it "marks" the DNA where the gene is found and can therefore be used in (molecular) marker assays to select for or against the presence of the gene, for example in marker assisted breeding / selection (MAS) methods. Examples of genetic markers are AFLP (amplified fragment length polymorphism, EP 534858), microsatellites, RFLP (restriction fragment length polymorphism), STS (sequence tagged site), SNP (single nucleotide polymorphism), SFP (single feature polymorphism; see Borevitz et al., 2003), SCAR (sequence characterized amplified region), CAPS markers (truncated amplified polymorphic sequences), etc. The further away a marker is from a gene, the greater the chance that recombination (crossing over) will occur between the marker and the gene, thereby causing loss of linkage (and co-segregation of the marker and gene). Distance between loci is measured in terms of recombination frequency and is given in cM (centimorgan; 1 cM is the meiotic recombination frequency between two markers of 1%). Because genome sizes vary greatly between species, the actual physical distance represented by 1 cM (i.e., the kilobase, kb, between two markers) also varies greatly between species.

[0018] It is understood that when reference is made herein to a marker "linked" to this, this also encompasses a marker "on" the gene itself.

[0019] "MAS" refers to "marker assisted selection", whereby plants are screened for the presence and / or absence of one or more genetic and / or phenotypic markers to accelerate the introgression of a DNA region containing the marker (and optionally lacking flanking regions) into an (elite) breeding line.

[0020] "Molecular marker assay" (or test) refers to a (DNA-based) assay that indicates (directly or indirectly) the presence or absence of an allele, e.g., a Par allele or a par allele, in a plant or plant part. Preferably, the assay allows one to determine whether a particular allele is homozygous or heterozygous at the parthenogenetic locus in any individual plant. For example, in one embodiment, PCR primers are used to amplify nucleic acids linked to the parthenogenetic locus, the amplification products are enzymatically digested, and based on the electrophoretic resolution pattern of the amplification products, it is possible to determine which allele(s) are present in any individual plant and the zygosity of the alleles at the parthenogenetic locus (i.e., the genotype at each locus). Examples include SCAR markers (sequence characterized amplified regions), CAPS markers (truncated amplified polymorphic sequences), and similar marker assays.

[0021] As used herein, the term "heterozygous" refers to a genetic condition that exists when two distinct alleles are present at a particular locus, but are separately located on a corresponding set of homologous chromosomes in a cell. Conversely, as used herein, the term "homozygous" refers to a genetic condition that exists when two identical (or more than two, in the case of polyploids) alleles are present at a particular locus, but are separately located on a corresponding set of homologous chromosomes in a cell.

[0022] "Variety" is used herein in accordance with the UPOV Convention to refer to a grouping of plants within a single plant taxonomic group of the lowest known rank, which grouping can be defined by the expression of characteristics, which can be distinguished from any other plant grouping by the expression of at least one of said characteristics, and which is considered to be a unit of fitness for unaltered (stable) reproduction.

[0023] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule composed of a chain of amino acids, regardless of a particular mode of action, size, three-dimensional structure, or origin. Thus, a "fragment" or "portion" of a protein can still be referred to as a "protein." An "isolated protein" is used to refer to a protein that is no longer in its natural environment, e.g., in vitro or in a recombinant bacterial or plant host cell.

[0024] The term "gene" refers to a DNA sequence comprising a region that is transcribed in a cell into an RNA molecule (e.g., a pre-mRNA that is processed into mRNA) (transcribed region), operably linked to appropriate regulatory regions (e.g., a promoter). Thus, a gene can comprise several operably linked sequences, e.g., a promoter, a 5' leader sequence including, e.g., sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), and a 3' untranslated sequence including, e.g., a transcription termination site.

[0025] A "chimeric gene" (or recombinant gene) refers to any gene not normally found in nature in a species, particularly a gene in which there are one or more portions of nucleotide sequence that are not naturally associated with each other. For example, a promoter is not naturally associated with part or all of the transcribed region or with another regulatory region. The term "chimeric gene" is understood to include expression constructs in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences or to an antisense sequence (the reverse complement of the sense strand) or an inverted repeat sequence (sense and antisense, whereby the RNA transcript forms a double-stranded RNA upon transcription).

[0026] "3'UTR" or "3' untranslated sequence" (often also referred to as 3' untranslated region or 3' end) refers to nucleotide sequences found downstream of the coding sequence of a gene, including, for example, transcription termination sites and (in most but not all eukaryotic mRNAs) a polyadenylation signal (such as, for example, AAUAAA or a variant thereof). After transcription is terminated, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a polyA tail may be added, which is involved in transport of the mRNA into the cytoplasm where translation takes place.

[0027] "5'UTR" or "leader sequence" or "5' untranslated region" is the region of an mRNA transcript, and the corresponding DNA, between the +1 position where mRNA transcription begins and the translation start codon of the coding region (usually AUG on the mRNA or ATG on the DNA). The 5'UTR usually contains sites important for translation, mRNA stability and / or turnover, as well as other regulatory elements.

[0028] "Expression of a gene" refers to the process by which a DNA region operably linked to an appropriate regulatory region, in particular a promoter, is transcribed into RNA that is biologically active, i.e. translatable into a biologically active protein or peptide (or active peptide fragment), or active in itself (e.g. post-transcriptional gene silencing or RNAi). An active protein in certain embodiments refers to a protein that is constitutively active. The coding sequence is preferably in a sense orientation and encodes a desired biologically active protein or peptide, or an active peptide fragment. In the gene silencing approach, the DNA sequence is preferably present in the form of antisense DNA or inverted repeat DNA, and includes a short sequence of the target gene in antisense or in sense and antisense orientation.

[0029] A "transcriptional regulatory sequence" is defined herein as a nucleotide sequence capable of regulating the rate of transcription of a (coding) sequence operably linked to the transcriptional regulatory sequence. Thus, a transcriptional regulatory sequence as defined herein will include all of the sequence elements necessary for initiating transcription (promoter elements), maintaining and regulating transcription, including, for example, attenuators or enhancers. Most refer to transcriptional regulatory sequences upstream (5') of a coding sequence, but regulatory sequences found downstream (3') of a coding sequence are also encompassed by this definition.

[0030] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes by being located upstream in the direction of transcription of the transcription start site of a gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequence, including but not limited to, a transcription factor binding site, a protein binding site for repressors and activators, and any other sequence of nucleotides known to those skilled in the art that act directly or indirectly to regulate the amount of transcription from the promoter. Optionally, the term "promoter" herein also includes the 5'UTR region (e.g., a promoter, as used herein, can include one or more portions upstream (5') of the translation start codon of a gene), because this 5'UTR region can have a role in regulating transcription and / or translation. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically (e.g., by external application of a certain compound) or developmentally regulated. A "tissue-specific" promoter is only active in a particular type of tissue or cell. A "promoter active in plants or plant cells" refers to the general ability of the promoter to drive transcription in plants or plant cells. This does not imply any spatiotemporal activity of the promoter.

[0031] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter, or rather a transcriptional regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are usually contiguous, and, if necessary, join two protein coding regions that are contiguous and in reading frame to produce a "chimeric protein." A "chimeric protein" or "hybrid protein" is a protein composed of various protein "domains" (or motifs) that are not found in such a form in nature, but are joined to form a functional protein, which exerts the functionality of the domains that are joined. A chimeric protein can also be a fusion protein of two or more naturally occurring proteins. As used herein, the term "domain" refers to any portion(s) or domain(s) of a protein with a particular structure or function that can be transferred to another protein to provide a novel hybrid protein having at least the functional characteristics of the domains.

[0032] The term "targeting peptide" refers to an amino acid sequence that targets a protein or protein fragment to a target intracellular organelle, such as plastids, preferably chloroplasts, mitochondria, or to the extracellular space or apoplast (secretory signal peptide). The nucleotide sequence encoding the targeting peptide may be fused (in frame) to the nucleotide sequence encoding the amino terminus (N-terminus) of the protein or protein fragment, or may be used to replace the native targeting peptide.

[0033] A "nucleic acid construct" or "vector" is understood herein to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and used to deliver foreign DNA into a host cell. The vector backbone can be, for example, a binary or super-binary vector (see, for example, U.S. Pat. No. 5,591,616, U.S. Patent Application Publication No. 2002 / 138879, and WO 95 / 06722), a cointegration vector, or a T-DNA vector, as known in the art and described elsewhere herein, into which a gene or chimeric gene is integrated, or, if appropriate transcriptional regulatory sequences are already present, only the desired nucleotide sequence (e.g., coding sequence, antisense sequence, or inverted repeat sequence) is integrated downstream of the transcriptional regulatory sequence. A vector usually further comprises genetic elements that facilitate the use of the vector in molecular cloning, such as, for example, a selection marker, a multiple cloning site, etc.

[0034] "Recombinant host cell" or "transformed cell" or "transgenic cell" are terms that refer to new individual cells (or organisms) that arise as a result of at least one nucleic acid molecule that contains a nucleotide sequence that, when transcribed, results in an antisense RNA or an inverted repeat RNA (or hairpin RNA) for silencing a target gene / gene family, being introduced into said cell, inter alia. "Isolated nucleic acid" is used to refer to a nucleic acid that is no longer in its natural environment, e.g., in vitro or in a recombinant bacterial or plant host cell.

[0035] A "host cell" is an original cell that is transformed with a transgene to become a recombinant host cell. Preferably, the host cell is a plant cell or a bacterial cell. The recombinant host cell can contain the nucleic acid construct as an extrachromosomally (episomal) replicating molecule, or more preferably, contains a gene or chimeric gene integrated into the nuclear or plastid genome of the host cell.

[0036] A "recombinant plant" or "recombinant plant part" or "transgenic plant" is a plant or plant part (e.g., a seed or fruit or leaf) that contains a recombinant or chimeric gene, even if the gene may not be expressed or may not be expressed in all cells.

[0037] An "elite event" is a recombinant plant that has been selected to contain a recombinant gene at a location in the genome that confers favorable phenotypic and / or agronomic characteristics to the plant. The DNA flanking the integration site can be sequenced to characterize the integration site and to distinguish the event from other transgenic plants that contain the same recombinant gene at other locations in the genome.

[0038] The term "selection marker" is a term well known to those skilled in the art and is used herein to describe any genetic entity that, when expressed, can be used to select a cell or cells that contain the selection marker. The selection marker gene product confers, for example, antibiotic resistance, or more preferably, herbicide resistance or another selectable trait, such as a phenotypic trait (e.g., a change in pigmentation) or an auxotrophy. The term "reporter" is primarily used to refer to a visible marker, such as green fluorescent protein (GFP), eGFP, luciferase, GUS, etc.

[0039] The term "ortholog" of a gene or protein, as used herein, refers to a homologous gene or protein found in another species that has the same function as the gene or protein, but that has (usually) diverged in sequence since the species harboring the gene diverged (i.e., genes that have evolved from a common ancestor by speciation). Thus, orthologs of Dandelion parthenogenetic genes can be identified in other plant species based on both sequence comparisons (e.g., based on the percentage of sequence identity across the entire sequence or across specific domains) and functional analysis.

[0040] The terms "homologous" and "heterologous" refer to the relationship between a nucleic acid or amino acid sequence and its host cell or host organism, particularly in the context of transgenic organisms. Thus, a homologous sequence is naturally found in the host species (e.g., a lettuce plant transformed with a lettuce gene), whereas a heterologous sequence is not naturally found in the host cell (e.g., a lettuce plant transformed with a sequence from a potato plant). Depending on the context, the terms "homolog" or "homologous" may alternatively refer to sequences that are descendants of a common ancestral sequence (e.g., they may be orthologs).

[0041] "Stringent hybridization conditions" can be used to identify nucleotide sequences that are substantially identical to a given nucleotide sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Usually, stringent conditions will be selected 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 the stringency. For RNA-DNA hybridization (e.g., Northern blot using a 100 nt probe), stringent conditions include, for example, at least one wash in 0.2×SSC at 63°C for 20 minutes, or equivalent conditions. Stringent conditions for DNA-DNA hybridization (e.g., Southern blots using 100 nt probes) include, for example, at least one wash (usually two) in 0.2×SSC for 20 minutes at a temperature of at least 50° C., usually about 55° C., or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

[0042] "High stringency" conditions can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6xSSC (20xSSC containing 3.0M NaCl, 0.3M sodium citrate, pH 7.0), 5xDenhardt's (100xDenhardt's containing 2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS) and 20μg / ml denatured carrier DNA (single-stranded fish sperm DNA, with an average length of 120-3000 nucleotides) as a non-specific competitor. Following hybridization, high stringency washes may be performed in several stages, with a final wash (about 30 minutes) in 0.2-0.1xSSC, 0.1% SDS at the hybridization temperature mentioned above.

[0043] "Medium stringency" refers to conditions equivalent to hybridization in the solutions described above, but at about 60-62° C., with the final wash being in 1×SSC, 0.1% SDS at the hybridization temperature.

[0044] "Low stringency" refers to conditions equivalent to hybridization in the above solution at about 50-52° C., with a final wash in 2×SSC, 0.1% SDS at the hybridization temperature. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

[0045] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptide sequences or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman). Sequences can then be called "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (as defined herein) (e.g., when optimally aligned by the programs GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. Global alignment is appropriately used to determine sequence identity when two sequences have similar lengths. Typically, the GAP default parameters are used, with a gap creation penalty of 50 (nucleotides) / 8 (proteins) and a gap extension penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and sequence identity percentage scores may be determined using computer programs such as GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software such as the programs "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) of EmbossWIN version 2.10.0, using the same parameters as GAP above, or using default settings (for both "needle" and "water" and for both protein and DNA alignments, the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; the default scoring matrix is ​​Blosum62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

[0046] Alternatively, the percentage of similarity or identity may be determined by searching against public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as "query sequences" to perform searches against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present invention. BLAST protein searches can be performed using the BLASTx program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0047] As used herein, the term "sexual plant reproduction" refers to a developmental pathway in which a (e.g., diploid) somatic cell called a "megaspore mother cell" undergoes meiosis to produce four meiotic megaspores. One of these megaspores divides by mitosis to form a megagametophyte (also known as an embryo sac) that contains a meiotic egg cell (i.e., a cell with a reduced number of chromosomes compared to the mother) and two meiotic polar nuclei. Fertilization of the egg cell by one sperm cell of the pollen grain gives rise to the (e.g., diploid) embryo, while fertilization of the two polar nuclei by a second sperm cell gives rise to the (e.g., triploid) endosperm (a process called double fertilization).

[0048] The term "megaspore mother cell" or "megasporocyte" as used herein refers to a cell that produces megaspores by reduction, usually meiosis, to create four monoploid megaspores that develop into female gametophytes. In angiosperms (also known as flowering plants), megaspore mother cells produce megaspores that develop into megagametophytes through two distinct processes including megasporogenesis (the formation of megaspores, or megasporangia, in the nucellus) and megagametogenesis (the development of megaspores into megagametophytes).

[0049] The term "asexual plant reproduction" as used herein is the process by which plant reproduction occurs without fertilization and without the fusion of gametes. Asexual reproduction produces new individuals that are genetically identical to the parent plant and to each other, except when mutation or somatic recombination occurs. Plants have two main types of asexual reproduction, including vegetative reproduction (i.e., involving budding, tillering, etc. of vegetative pieces of the original plant) and apomixis.

[0050] The term "apomixis" as used herein refers to the formation of seeds by an asexual process. One form of apomixis is characterized by: 1) apomeiosis, which refers to the formation of a non-reduced embryo sac in the ovary, and 2) parthenogenesis, which refers to the development of a non-reduced egg into an embryo. Hundreds of wild plant species are characterized by apomictic reproduction and reproduce asexually. Apomeiosis is a process that results in the production of non-reduced egg cells with the same chromosome number and identical or highly similar genotype as the somatic tissue of the mother plant. Non-reduced egg cells can be derived from non-reduced megaspores (diplosporous reproduction) or from somatic founder cells (asporous reproduction). In the case of diplosporous reproduction, megasporogenesis replaces mitosis or modified meiosis. The modified meiosis is preferably of the first division restitution type without recombination. Alternatively, the modified meiosis can be of the second division restitution type. In a preferred embodiment, the apomeiosis is of a diplosporous type affecting the first meiotic division. Apomixis is known to occur in various forms, including at least two forms known as gametophytic apomixis and sporophytic apomixis (also called somatic embryogenesis). Examples of plants in which gametophytic apomixis occurs include dandelion (Tandellia spp.), hawkweed (Hexacanthus spp.), Kentucky bluegrass (Luca longa), eastern gamagrass (Trypsacum dactyloides), and others. Examples of plants in which sporophytic apomixis occurs include citrus (Citrus spp.), mangosteen (Garcinia mangostana), and others.

[0051] The term "diploid reproduction" as used herein refers to the situation in which non-meiotic embryo sacs are derived from megaspore mother cells either directly by mitosis or by interrupted meiotic events. Three main types of diploid reproduction have been reported, named after the plants in which they occur: Taraxacum, Ixeris, and Antennaria. In the Taraxacum type, meiotic prophase is initiated, but the process is subsequently interrupted, resulting in two non-meiotic dyads, one of which gives rise to the embryo sac by mitosis. In the Ixeris type, two further mitotic divisions of the nucleus that give rise to the eight-karyon embryo sac follow the equal division after the meiotic prophase. The Taraxacum and Ixeris types are known as meiotic diploid reproduction, since they involve modified meiosis. In contrast, in the Antennaria type, called mitotic diplosporous, the megaspore mother cell does not initiate meiosis but divides directly three times to produce a non-meiotic embryo sac. In diplosporous gametophyte apomixis, a non-meiotic gametophyte is produced from a non-meiotic megaspore, which results from either a mitosis-like division (displory) or a modified meiosis (displory). In both asporous and diplosporous gametophyte apomixis, a non-meiotic egg cell develops parthenogenetically into an embryo. The apomixis in the genus Dandelion is of the diplosporous type, which means that the first female reduction division (meiosis I) is skipped, resulting in two non-meiotic megaspores with the same genotype as the mother plant. One of these megaspores degenerates, and the other, surviving, non-reduced megaspore gives rise to a non-reduced megagametophyte (or embryo sac), which contains a non-reduced egg cell that develops without fertilization into an embryo with the same genotype as the mother plant. The seed resulting from the process of apomixis of the gametophyte is called an apomictic seed.

[0052] The term "diplosporous reproduction capability" refers to the ability to induce diplosporous reproduction in a plant, preferably in the female ovary, preferably in the megaspore mother cell and / or in the female gamete. Thus, a plant into which a diplosporous reproduction capability has been introduced is capable of carrying out the diplosporous reproduction process, i.e., capable of producing non-meiotic gametes via meiosis I restoration.

[0053] The term "diplosporous reproduction as part of gametophyte apomixis" refers to the diplosporous reproduction component of the process of apomixis, i.e., the role that diplosporous reproduction plays in the formation of seeds by an asexual process. In particular, next to the diplosporous reproduction function, the parthenogenetic function is also necessary in establishing the process of apomixis. Thus, the combination of diplosporous and parthenogenetic functions can result in apomixis.

[0054] As used herein, the term "diplosporous plant" refers to a plant that undergoes gametophytic apomixis by diplosporous reproduction, or a plant that has been induced (e.g., by genetic modification) to undergo gametophytic apomixis by diplosporous reproduction. In both cases, the diplosporous plant produces apomictic seeds when combined with a parthenogenetic agent.

[0055] The term "apomictic seed" as used herein refers to a seed obtained from an apomictic plant species or by a plant or crop that has been induced to undergo apomixis, in particular diplosporous gametophytic apomixis. Apomictic seeds are characterized by germinating plants that are clonal and genetically identical to the parent plant, allowing for pure breeding. In the present invention, "apomictic seed" also refers to "clonal apomictic seed".

[0056] The term "apomictic plant(s)" as used herein refers to a plant that reproduces itself asexually, without fertilization. An apomictic plant may be a sexual plant that has been modified to be apomictic, for example a sexual plant that has been genetically modified with one or more of the parthenogenetic genes taught herein to obtain an apomictic plant or a plant that is a descendant of an apomictic plant. In this case, the apomictically produced progeny is genetically identical to the parent plant.

[0057] "Clones" of cells, plants, plant parts, or seeds are characterized in that they are genetically identical to their sibling species and to the parent plant from which they were derived. The genomic DNA sequences of individual clones are nearly identical, however, mutations may cause minor differences.

[0058] As used herein, the term "pure breeding" or "pure breeding organisms" (also known as pure bred organisms) refers to organisms that always transmit certain phenotypic traits unchanged or nearly unchanged to their offspring. An organism can be said to be pure breeding for each trait that the organism applies to, and the term "pure breeding" is also used to describe individual genetic traits.

[0059] The term "F1 hybrid" (or hybrid first generation) as used herein refers to the first hybrid generation of offspring of distinct parent types. The parent types may be inbred or not. F1 hybrids are used in genetics and in selective breeding, where they may appear as F1 crosses. The offspring of distinctly different parent types produce a new uniform phenotype with a combination of characteristics from both parents. F1 hybrids carry distinct advantages such as hybrid vigor and are therefore highly desired in agricultural practice. In embodiments of the present invention, the methods, genes, proteins, variants or fragments thereof taught herein can be used to fix the genotype of F1 hybrids, regardless of their genetic complexity, thereby allowing the creation of organisms that can be purebred in one step.

[0060] The term "pollination" or "pollinating" as used herein refers to the process by which pollen is transferred from the anther (male part) of a plant to the stigma (female part), thereby enabling fertilization and reproduction. Pollination is unique to angiosperms, flowering plants. Each pollen grain is a male monoploid gametophyte that is adapted to be transported to the female gametophyte, where it can carry out fertilization by producing a male gamete (or gametes) in a process of double fertilization. Successful angiosperm pollen grains (gametophytes) containing male gametes are transported to the stigma, where they germinate and their pollen tubes grow down the style into the ovary. The two gametes travel down the tube until the gametophyte(s) containing the female gametes are held within the carpel. One nucleus fuses with the polar body to produce endosperm tissue, and the other nucleus fuses with the ovule to produce the embryo.

[0061] As used herein, the term "parthenogenesis" refers to a form of asexual reproduction in which embryonic growth and development occurs without fertilization. The genes and proteins of the present invention, in combination with diplosporous factors, e.g., genetic or chemical factors, are capable of producing apomictic progeny.

[0062] The term "pyramiding or stacking genes" as used herein refers to the process of combining related or unrelated genes from different parental lines that underlie desired or preferred traits (e.g., disease resistance traits, color, drought resistance, pest resistance, etc.) into one plant. Pyramiding or stacking genes can be performed using traditional breeding methods or can be accelerated by using molecular markers to identify and maintain plants containing the desired allele combinations and discard plants that do not have the desired allele combinations. In one embodiment of the present invention, the parthenogenetic genes taught herein can be advantageously used in gene pyramiding or stacking programs to create apomictic plants or introduce apomixis into sexual crops.

[0063] In this document and the claims, the verb "to comprise" and its conjugations are used in their open-ended sense to mean that the items following this word are included, but not that items not specifically mentioned are excluded. In addition, the reference of an element with the indefinite article "a" or "an" does not exclude the possibility that there is more than one element, unless the context clearly requires one and only one of the elements. Thus, the indefinite article "a" or "an" usually means "at least one". It is further understood that when a "sequence" is referred to herein, it generally refers to an actual physical molecule having a certain sequence of subunits (e.g., amino acids).

[0064] As used herein, the term "plant" includes plant cells, plant tissues or organs, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant cell mass, and intact plant cells in a plant, or plant parts, such as embryos, pollen, ovules, fruits, flowers, leaves (e.g., harvested lettuce crops), seeds, roots, root tips, etc. DETAILED DESCRIPTION OF THE PRESENT EMBODIMENT

[0065] Nucleotide sequences of the invention The present inventors have identified for the first time the genes, coding sequences, promoters, 3'UTRs and proteins responsible for parthenogenesis. The gene sequences, promoter sequences, coding sequences and 3'UTR sequences are located on the Par alleles. The present inventors have also identified the gene sequences, promoter sequences, coding sequences and 3'UTR sequences located on the sexual counterparts of the Par alleles, i.e. on the par alleles. As the sexual counterparts of the dominant alleles that cause parthenogenesis, the presence of the par alleles does not contribute to the parthenogenetic phenotype, but these par alleles are also indicated herein as being associated with parthenogenesis, because the presence of the par alleles may be indicative of a sexual phenotype, i.e. a non-parthenogenetic phenotype. Because the Par alleles may be dominant alleles, confirmation of the sexual phenotype may require evaluation of all alleles of the Par locus as par alleles and / or evaluation of the absence of the Par alleles. In other words, "associated with" is understood herein to refer to a parthenogenetic or non-parthenogenetic phenotype, and optionally to be functional in parthenogenesis. For example, a par allele can be modified to confer a Par allele capable of inducing a parthenogenetic phenotype, by modifying one or more expression control sequences of the par allele, such as a promoter sequence, which results in altered expression of the encoded protein.

[0066] Both the Par and par alleles comprise a gene having a coding sequence encoding a protein referred to herein as a "PAR protein", which contains a zinc finger C2H2-type domain (IPR13087), preferably a zinc finger K2-2-like domain having the consensus sequence C.{2}C.{7}[K / R]A.{2}GH.[R / N].H, which may also be annotated as: CXXCXXXXXXX[K / R]AXXGHX[R / N]XH (SEQ ID NO:37), where X can be any naturally occurring amino acid, [K / R] indicates that the amino acid at position 12 is lysine or arginine, and [R / N] indicates that the amino acid at position 19 is arginine or asparagine (see Englbrecht et al., 2004). In addition to a zinc finger C2H2-type domain, preferably a zinc finger K2-2-like domain as defined herein, the protein comprises an EAR motif having the consensus amino acid sequence DLNXXP (SEQ ID NO: 58) or DLNXP (SEQ ID NO: 59), where X can be any naturally occurring amino acid (see Kagale et al., 2010). Preferably, the protein is at most 400 amino acids, wherein said protein comprises one or two EAR motifs as indicated herein and a zinc finger K2-2-like domain as defined herein. Preferably, the protein is at most 400 amino acids, wherein said protein comprises only one or two EAR motifs as indicated herein and only one zinc finger K2-2-like domain as defined herein, i.e., no further EAR motif as defined herein and no further zinc finger K2-2-like domain as defined herein.In addition to the characteristics of a maximum size of 400 amino acids, only one or two EAR motifs as indicated herein, and a single zinc finger K2-2-like domain as defined, a PAR protein can contain only one additional zinc finger domain having the zinc finger consensus sequence C.{2}C.{12}H.{3}H, which may also be annotated as: CXXCXXXXXXXXXXXXHXXXH (SEQ ID NO: 38), but more preferably does not contain an additional zinc finger domain having the zinc finger consensus sequence C.{2}C.{12}H.{3}H (SEQ ID NO: 38).

[0067] Thus, the present invention provides a nucleic acid associated with parthenogenesis in plants, wherein the nucleic acid comprises a nucleotide sequence encoding a PAR protein as defined herein. The present invention also provides a promoter sequence and a 3'UTR operably linked to the nucleotide sequence encoding the PAR protein. Taraxacum officinale comprises one dominant Par allele capable of inducing parthenogenesis and two sexual counterparts, namely par allele-1 and par allele-2, encoding PAR proteins having the amino acid sequence of SEQ ID NO: 1, 6, or 11, respectively. The Par allele comprises a gene having the nucleotide sequence of SEQ ID NO: 5, par allele-1 comprises a par gene having the nucleotide sequence of SEQ ID NO: 10, and par allele-2 comprises a par gene having the nucleotide sequence of SEQ ID NO: 15. The Par gene comprises a promoter sequence having SEQ ID NO: 2, a coding sequence having SEQ ID NO: 3, and a 3'UTR having SEQ ID NO: 4. The par gene-1 comprises a promoter sequence having SEQ ID NO: 7, a coding sequence having SEQ ID NO: 8, and a 3'UTR having SEQ ID NO: 9. The par gene-2 ​​comprises a promoter sequence having SEQ ID NO: 12, a coding sequence having SEQ ID NO: 13, and a 3'UTR having SEQ ID NO: 14. Thus, the present invention provides a) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 1, 6 or 11; b) a promoter having the nucleotide sequence of SEQ ID NO: 2, 7 or 12; c) a coding sequence having the nucleotide sequence of SEQ ID NO: 3, 8 or 13; d) a 3'UTR having the nucleotide sequence of SEQ ID NO: 4, 9 or 14; e) a gene having the nucleotide sequence of SEQ ID NO: 5, 10 or 15; f) any one of the variants a) to e); and g) A fragment of any one of a) to f) The present invention provides nucleic acids associated with parthenogenesis in plants, comprising at least one of:

[0068] Table 1 provides an overview of all SEQ ID NOs used herein.

[0069] Preferably, the nucleic acid is functional in parthenogenesis. In one embodiment, the nucleic acid of the invention is: a) a gene encoding a protein having the amino acid sequence of SEQ ID NO:1; b) a promoter having the nucleotide sequence of SEQ ID NO:2; c) a coding sequence having the nucleotide sequence of SEQ ID NO:3; d) a 3'UTR having the nucleotide sequence of SEQ ID NO:4; e) a gene having the nucleotide sequence of SEQ ID NO:5; f) any one of the variants a) to e); and g) A fragment of any one of a) to f). The present invention includes or consists of at least one of the following:

[0070] Preferably, the nucleic acid of this embodiment and / or a product derived therefrom, such as its RNA transcript or its encoded protein, directs parthenogenesis, e.g. a plant comprising said nucleic acid directs said plant to exhibit parthenogenesis, meaning that said plant has the ability to develop an embryo from a meiotic or non-meiotic egg cell. Preferably, said nucleic acid and / or a product derived therefrom, such as its RNA transcript or its encoded protein, is functional in parthenogenesis, preferably when present in a plant or plant cell, and even more preferably induces or is capable of inducing parthenogenesis.

[0071] In another embodiment, the nucleic acid of the invention is: a) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 6 or 11; b) a promoter having the nucleotide sequence of SEQ ID NO: 7 or 12; c) a coding sequence having the nucleotide sequence of SEQ ID NO: 8 or 13; d) a 3'UTR having the nucleotide sequence of SEQ ID NO: 9 or 14; e) a gene having the nucleotide sequence of SEQ ID NO: 10 or 15; f) any one of the variants a) to e); and g) A fragment of any one of a) to f) The present invention includes or consists of at least one of the following:

[0072] Preferably, said nucleic acid of this embodiment and / or products derived therefrom, such as its RNA transcript or encoded protein, preferably when present in a homozygous state in a plant or plant cell, does not or is incapable of inducing parthenogenesis. In other words, the presence of the nucleic acid of this embodiment may be indicative of a non-parthenogenetic or sexual phenotype, e.g. a plant comprising said nucleic acid is indicative of said plant being of a sexual phenotype, i.e. incapable of developing an embryo from an egg cell.

[0073] The Par allele may be a dominant allele. If the Par allele is dominant, then all alleles at the Par locus of the plant need to be evaluated as par alleles to confirm that the plant is of a non-parthenogenetic phenotype, and the presence of a single Par allele is sufficient to indicate that the plant is capable of parthenogenesis.

[0074] The nucleic acids of the invention can be used for screening and / or genotyping. Optionally, the functionality in parthenogenesis of the putative nucleic acid or gene and / or its derivatives, or the ability of the putative nucleic acid and / or its derivatives to induce parthenogenesis, can be assessed by reducing expression, silencing or knocking out said nucleic acid or gene in parthenogenetic plants, for example by introducing a premature stop in the coding sequence of said gene. Subsequent loss of parthenogenetic phenotype means that the putative nucleic acid and / or its derivatives are capable of inducing parthenogenesis. The ability to induce parthenogenesis can also be assessed by complementing a loss-of-function apomictic plant with the putative nucleic acid and / or its derivatives (mRNA or protein). Such a loss-of-function apomictic plant can be Taraxacum officinale isolate A68 modified to lose the apomictic phenotype by reducing expression of a functional Par allele (e.g. by deletion or knockout). Such a loss-of-function apomictic plant may be Taraxacum officinale isolate A68, comprising a Par allele, in which SEQ ID NO: 23, as defined herein, has been modified to any one of SEQ ID NOs: 24-27 (see Table 2). Such loss of functional apomictic plants of Taraxacum officinale isolate A68 may be obtained by targeted genome editing using a CRISPR-Cas9 / guide RNA complex, in which, as exemplified herein, said guide RNA (also designated herein as gRNA) comprises the target-specific sequence of SEQ ID NO: 19. Deletion of the Par allele of Taraxacum officinale isolate A68 results in a loss of parthenogenesis, and thus a loss of apomixis. If said putative nucleic acid or a derivative thereof is capable of inducing parthenogenesis, then introduction of said nucleic acid or derivative into said isolate, for example by transfecting said isolate with a vector comprising said nucleic acid and / or encoding said derivative, will restore (or rescue) the apomictic phenotype. Such vectors preferably contain sequences suitable for driving expression of the encoded derivative in the isolate.By way of example, a putative nucleic acid possibly encoding a PAR protein of the invention may be operably linked in said vector to a promoter as defined herein by SEQ ID NO: 2 and, optionally, to a 3'UTR as defined herein by SEQ ID NO: 4. In the case of Taraxacum officinale isolate A68, high seed set in the absence of cross-pollination is a clear indication of apomixis. Self-pollination in this isolate can be excluded as an alternative explanation, since sexually reproduced egg cells and pollen grains have very low fertility due to unbalanced triploid male and female meiosis.

[0075] Preferably, the variant nucleic acid as defined herein is a homologue or orthologue of the Par allele or gene, promoter, coding sequence and / or 3'UTR of the par allele of Taraxacum officinale isolate A68 as defined herein. Preferably, said variant nucleic acid and / or product derived therefrom, such as its RNA transcript or encoded protein, when present preferably in a plant or plant cell, is associated with and optionally induces or is capable of inducing parthenogenesis as defined herein. Said variant preferably encodes a PAR protein as defined herein or is operably linked to a sequence encoding a PAR protein. Orthologues of the Par genes and par genes identified in Taraxacum officinale isolate A68 in other plant species can be identified based on the characteristics of the PAR proteins as defined herein. Such a gene may encode any one of PAR proteins selected from the group consisting of, but not limited to, a PAR protein from Ananas comosus (e.g., UniProtKB: A0A199URK4), a PAR protein from Apostasia shenzhenica (e.g., UniProtKB: A0A2I0AZW3), a PAR protein from Arabidopsis thaliana (e.g., UniProtKB: Q8GXP9, A0A178V2S4, O81793, A0A178V1Q3, A0MFC1, O81801), a PAR protein from Arabidopsis lyrata subsp. Lyrata (e.g., UniProtKB: D7MC52 or D7MCE8), a PAR protein from Arachis ipaensis (e.g., UniProtKB: D7MC53 or D7MCE8), a PAR protein from Arachis spp. (e.g., UniProtKB: D7MC54 or D7MCE8), a PAR protein from Arachis spp. (e.g., UniProtKB: D7MC55 or D7MCE8), a PAR protein from Arachis spp. (e.g., UniProtKB: D7MC56 or D7MCE8), a PAR protein from Arachis spp. (e.g., UniProtKB: D7MC57 or D7MCE8), a PAR protein from Arachis spp. (e.g., UniProtKB: D7MC59 ...7 or D7MCE8), a PAR protein from Arachis spp. (e.g., UniProtKB: D7MC5 ipaensis (e.g., SEQ ID NO: 45 or SEQ ID NO: 49), Brachypodium distachyon (e.g., UniProtKB: I1J0D9), Brassica oleracea var.oleracea PAR proteins (e.g. UniProtKB: A0A0D3A1Q6 or A0A0D3A1Q3), Brassica campestris PAR proteins (e.g. UniProtKB: A0A398AHT1), Brassica rapa PAR proteins (e.g. SEQ ID NO: 47), Brassica rapa subsp. Pekinensis PAR proteins (e.g. UniProtKB: M4D574 or M4D571), Brassica oleracea PAR proteins (e.g. UniProtKB: A0A3P6ESB1 or A0A3P6F726), Brassica campestris PAR proteins (e.g. UniProtKB: A0A3P5ZMM3 or A0A3P5Z1M1), Cajanus cajan PAR protein (e.g. SEQ ID NO: 46), Capsella rubella PAR protein (e.g. UniProtKB: R0H2J1 or R0H0C2), Cephalotus follicularis PAR protein (e.g. UniProtKB: A0A1Q3CSK1), Cicer arietinum PAR protein (e.g. UniProtKB: A0A3Q7YBZ1, A0A1S2YZL9, A0A3Q7Y0Z6 or A0A1S2YZM6; or SEQ ID NO: 55, 56 or 57), Cichorium endivia PAR protein (e.g. SEQ ID NO: 39), Cucumis sativus PAR protein (e.g. UniProtKB: A0A0A0KGW4 or A0A0A0L0X7), Cucumis melo (e.g., UniProtKB: A0A1S3BLF2 or A0A1S3B298), cucumber (e.g., UniProtKB: A0A0A0KAW8), Japanese pumpkin (Cucurbita moschata) PAR protein (e.g., SEQ ID NO: 43), American dodder (Cuscutacampestris PAR proteins (e.g. UniProtKB: A0A484MGR1), Dendrobium catenatum PAR proteins (e.g. UniProtKB: A0A2I0V7N9, A0A2I0X2T2 or A0A2I0W0Q8), Dorcoceras hygrometricum PAR proteins (e.g. UniProtKB: A0A2Z7D3Y1), Eutrema salsugineum PAR proteins (e.g. UniProtKB: V4LSH0; or SEQ ID NO: 44), Fagus sylvatica PAR proteins (e.g. UniProtKB: A0A2N9E5Y5, A0A2N9HAB9, or A0A2N9H993), Genlisea aurea PAR proteins (e.g. UniProtKB: A0A2N9E5Y5, A0A2N9HAB9, or A0A2N9H993), aurea PAR protein (e.g. UniProtKB: S8E1M6), soybean (Glycine max) PAR protein (e.g. SEQ ID NO: 51, 52, 53 or 54), upland cotton (Gossypium hirsutum) PAR protein (e.g. UniProtKB: A0A1U8LDU9), sunflower (Helianthus annuus) PAR protein (e.g. SEQ ID NO: 21), rubber tree (Hevea brasiliensis) PAR protein (e.g. SEQ ID NO: 42), dandelion (Hieracium aurantiacum) PAR protein (e.g. SEQ ID NO: 40), Persian walnut (Juglans regia) PAR protein (e.g. UniProtKB: A0A2I4E6B1), lettuce (Lactuca sativa) PAR protein (e.g. UniProtKB: A0A2J6KZF7; or SEQ ID NO: 22), bottle gourd (Lagenaria siceraria PAR protein (e.g., SEQ ID NO: 48), Medicago truncatula PAR protein (e.g., UniProtKB: G7K024), Morusnotabilis PAR proteins (e.g. UniProtKB: W9SMY3 or W9SMQ7), Mucuna pruriens PAR proteins (e.g. UniProtKB: A0A371ELJ8), Nicotiana attenuata PAR proteins (e.g. UniProtKB: A0A1J6IQI6), Nicotiana sylvestris PAR proteins (e.g. UniProtKB: A0A1U7VXJ0), Nicotiana tabacum PAR proteins (e.g. UniProtKB: A0A1S4A651 or A0A1S3YHQ2), Oryza sativa subsp. Japonica PAR proteins (e.g. UniProtKB: B9FGH8), Oryza balsii PAR proteins (e.g. UniProtKB: B9FGH9), barthii PAR proteins (e.g. UniProtKB: A0A0D3FWX3), Panicum miliaceum PAR proteins (e.g. UniProtKB: A0A3L6Q010 or A0A3L6T1D6), Parasponia andersonii PAR proteins (e.g. UniProtKB: A0A2P5BMI5), Populus alba PAR proteins (e.g. UniProtKB: A0A4U5PSY9), Populus trichocarpa PAR proteins (e.g. UniProtKB: B9H661), Punica granatum PAR proteins (e.g. UniProtKB: A0A2I0IBB9, A0A218XB85 or A0A218W102), Senecio cambrensis PAR proteins (e.g. UniProtKB: A0A2I0IBB9, A0A218XB85 or A0A218W102), cambrensis PAR protein (e.g., SEQ ID NO: 41), peach (Prunus persica PAR protein (e.g., SEQ ID NO: 50), Trema orientale PAR protein (e.g., UniProtKB: A0A2P5EB04), red clover (Trifoliumpratense (e.g. UniProtKB: A0A2K3N851), Trifolium subterraneum (e.g. UniProtKB: A0A2Z6MYD3 or A0A2Z6MDR7), red clover (e.g. UniProtKB: A0A2K3PR44), Vitis vinifera (e.g. UniProtKB: A0A438C778, A0A438ESC4 or A0A438DBR4) and Zea mays (e.g. UniProtKB: A0A1D6HF46, B6UAC5, A0A3L6F4S1, A0A3L6EMC6, A0A3L6EMC6, K7UHQ6 or A0A1D6KHZ4). Such genes may also encode a PAR protein selected from the group consisting of: a PAR protein from Actinidia chinensis (UniProtKB: A0A2R6S2S9), a PAR protein from sugar beet (Beta vulgaris) (UniProtKB: XP_010690656.1), a PAR protein from potato (Solanum tuberosum) (UniProtKB: XP_015159151.1), a PAR protein from tomato (Solanum lycopersicum) (UniProtKB: A0A3Q7GXB3), a PAR protein from yellow pepper (Capsicum baccatum) (UniProtKB: A0A2G2WJR7), a PAR protein from eggplant (Solanum melongena) (UniProtKB: AVC18974.1), a PAR protein from wild soybean (Glycine max) (UniProtKB: AVC18974.1), a PAR protein from cereals (Citrus moniliforme ... soja (GenBank accession: XP_028201014.1, XP_006596577.1 or UniprotKB: A0A445M3M6), peanut (Arachis hypogaea) (UniProtKB: A0A444WUX5), kidney bean (Phaseolus vulgaris) (UniProtKB: V7CIF6), carrot (Daucuscarota (GenBank accession: XP_017245413.1), bread wheat (Triticum aestivum) (UniProtKB: A0A3B6RP64), rice subsp. indica (Oryza sativa subsp. indica) (UniProtKB: A2YH63), rice subsp. japonica (UniProtKB: Q5Z7P5) and cacao (Theobroma cacao) PAR protein (UniProtKB: A0A061DL63). The present invention relates to these orthologous genes, their promoters, and It includes the driver sequence, the coding sequence (including the cDNA and mRNA sequences) and the 3'UTR.

[0076] The nucleic acid of the present invention may be DNA, such as, but not limited to, genomic DNA, cDNA, or RNA, such as mRNA.Preferably, the nucleic acid of the present invention is an isolated nucleic acid.Preferably, the variant nucleic acid as defined herein preferably comprises at least about 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more nucleotide sequence identity with any one of the sequences of SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14 and 15, and / or any one of the sequences encoding SEQ ID NOs: 1, 6 and 11, respectively, or their complements, when pairwise aligned, for example, using the Needleman and Wunsch algorithm (global sequence alignment) with default parameters. For example, a variant of the coding sequence of SEQ ID NO:3 preferably comprises at least 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more nucleotide sequence identity to SEQ ID NO:3; a variant of the coding sequence of SEQ ID NO:5 preferably comprises at least about 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more nucleotide sequence identity to SEQ ID NO:5, etc.

[0077] Preferably, the variant differs from any one of the sequences encoding SEQ ID NO: 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14 and 15 and SEQ ID NO: 1, 6 and 11 or their complements by one or more nucleotide deletions, insertions and / or substitutions, including natural and / or synthetic / artificial variants. A "natural variant" is a variant found in nature, for example in other Taraxacum species or in other plants. Preferably, the variant is a nucleotide sequence (gene, promoter sequence or coding sequence) from a different plant species, for example from a Taraxacum species different from Taraxacum officinale, for example from a different cultivar, accession or breeding line. The variant can also be found and / or isolated from plants other than those belonging to the Taraxacum genus.

[0078] As indicated herein, the nucleic acid of the invention also encompasses a fragment of a gene, promoter or coding sequence of a Par allele or a par allele definition as defined herein, or any variant thereof. A "fragment" comprises or consists of at least about 10, 12, 15, 18, 20, 30, 50, 100, 150, 200, 250, 300, 500, 1000, 2000 or more contiguous nucleotides of any one of SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14 and 15 and / or any one of SEQ ID NOs: 1, 6 and 11 coding sequences, or variants thereof, or complements thereof, preferably capable of hybridizing to said sequences. In one embodiment, such a fragment may be functional in parthenogenesis as defined herein (preferably capable of inducing parthenogenesis). In another embodiment, such fragments may not be functional in parthenogenesis, but may be associated with parthenogenesis, for example because they can hybridize to sequences that are functional in parthenogenesis and thus direct parthenogenesis. Such fragments may be useful, for example, as PCR primers or hybridization probes, and thus can be used as genetic markers for use in mapping or molecular assays and / or to identify and / or isolate Par alleles or par alleles from other plants.

[0079] Preferably, the nucleic acid of the invention comprises or consists of a regulatory sequence, preferably a promoter sequence, of a gene encoding a PAR protein as defined herein, wherein said regulatory sequence, preferably a promoter sequence, comprises a nucleic acid insert, preferably a double-stranded DNA insert, wherein said insert has a length of between 50 and 2000 bp, between 100 and 1900 bp, between 200 and 1800 bp, between 300 and 1700 bp, between 400 and 1600 bp, between 500 and 1500 bp, between 600 and 1400 bp, between 1000 and 1400, between 1200 and 1400, or between 1300 and 1400 bp. Even more preferably, said insert has a length of about 1300 bp. Preferably, the insert is associated with a parthenogenetic phenotype as defined herein, and optionally functional in the parthenogenetic phenotype. Preferably, the insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding the PAR protein, such that the distance between the 3' end of the insert and the start codon of the sequence encoding the PAR protein is between 50 and 200 bp, preferably about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 bp, most preferably about 102 bp. Preferably, the insert is located such that the 3' terminal nucleotide of the insert is at a position that is homologous to nucleotide 1798 of SEQ ID NO:2 and / or nucleotide 1798 of SEQ ID NO:5. Preferably, the insert is devoid of an open reading frame. Even more preferably, the insert is a miniature inverted repeat transposable element (MITE) or MITE-like sequence, which is a non-autonomous element characterized in that it contains an internal sequence with no open reading frame, which is flanked by terminal inverted repeats (TIRs) that are in turn flanked by small direct repeats (target site overlaps). For details on MITEs, TIRs, and sequences, see Guo et al., Scientific Reports. 2017 Jun. 1;7(1):2634, which is incorporated herein by reference.Said insert, preferably said MITE or MITE-like sequence, may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, said insert is associated with and optionally functional in a parthenogenetic phenotype as defined herein. In a further preferred embodiment, the nucleic acid of the invention comprises or consists of a regulatory sequence, preferably a promoter sequence, and includes said insert at a position as defined herein above. Preferably, the nucleic acid of the invention comprises or consists of a sequence encoding a PAR protein as defined herein, operably linked to said promoter sequence, wherein preferably said promoter sequence is located directly upstream of the sequence encoding a PAR protein. Optionally, said nucleic acid of the invention may comprise one or more further transcriptional regulatory sequences.

[0080] In one embodiment, the nucleic acid of the invention can be derived from the Taraxacum family (eg, Taraxacum officinale sensu lato) or from other species.

[0081] In one embodiment, the nucleic acid of the invention is derived from a source other than the genus Taraxacum or Taraxacum officinale semblée.

[0082] In one embodiment, the invention encompasses homologous or orthologous Par alleles from plants where parthenogenesis exists, such as wild or cultivated plants, and / or from other plants. Such homologs or orthologs can be easily isolated by using the provided nucleotide sequences or parts thereof as primers or probes. For example, moderate or stringent nucleic acid hybridization methods can be used, for example, using fragments of the nucleotide sequences defined herein, or their complements. Variants can also be isolated from other wild or cultivated apomictic or non-apomictic plants (and / or from other plants using known methods such as PCR, stringent hybridization methods, etc.). Thus, variants of any one of SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14 and 15, and / or the sequences encoding SEQ ID NOs: 1, 6 and 11, also include nucleic acids that are found naturally (or in nature) in other Dandelion plants, strains or cultivars, and / or that are found naturally in other plants.

[0083] For optimal expression in a host or host cell, the coding sequences taught herein can be codon-optimized by adapting the codon usage (e.g., more suitable for expression in the plant of interest) to those most preferred in plant genes, particularly genes native to the plant genus or species of interest (Bennetzen and Hall, 1982, J. Biol. Chem. 257, 3026-3031; Itakura et al., 1977 Science 198, 1056-1063) using available codon usage tables. Codon usage tables for various plant species are published, for example, by Ikemura (1993, in Plant Molecular Biology Labfax, edited by Croy, Bios Scientific Publishers Ltd.) and Nakamura et al. (2000, Nucl. Acids Res. 28, 292.) and in major DNA sequence databases (e.g., EMBL, Heidelberg, Germany). Thus, a synthetic DNA sequence can be constructed so that the same or substantially the same protein can be produced using said synthetic DNA sequence. Several techniques for modifying codon usage to that preferred by the host cell can be found in the patent and scientific literature. The exact method of modifying codon usage is not critical to the present invention.

[0084] Minor modifications to any one of the sequences encoding SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, and 15, and / or SEQ ID NOs: 1, 6, and 11, or variants thereof, can be routinely made, i.e., by random or targeted mutagenesis (e.g., by chemical mutagenesis or CRISPR-endonuclease mediated mutagenesis). More significant modifications to the sequences taught herein can be routinely made by de novo DNA synthesis of the desired sequence using available techniques.

[0085] In one embodiment, the nucleic acid of the invention can be modified so that the N-terminus of the protein of the invention encoded by said nucleic acid has an optimal translation initiation situation by adding or deleting one or more amino acids at the N-terminus of the protein. In many cases, it is preferred that the protein of the invention expressed in plant cells starts with a Met-Asp or Met-Ala dipeptide for optimal translation initiation. Thus, an Asp or Ala codon may be inserted following an existing Met, or the second codon Val can be replaced with an Asp (GAT or GAC) or Ala (GOT, GCC, GCA, or GCG) codon. The nucleotide sequence can also be modified to remove illegitimate splice sites.

[0086] In one embodiment, the nucleic acid of the invention may have a (genetically) dominant function that is preferably provided by (over)expressing a functional protein having the amino acid sequence of SEQ ID NO:1, or a variant or functional fragment thereof, such as an orthologue or fragment thereof found in another plant (i.e. other than Taraxacum or Taraxacum officinale sect.).

[0087] Preferably, the nucleic acid of the invention encodes a protein or functional fragment(s) thereof that is functional when produced in a plant and induces and / or enhances parthenogenesis. For example, when a nucleic acid comprising SEQ ID NO: 3 or 5, or a variant or fragment thereof, is expressed (transcribed and translated) and an appropriate amount of a protein of the invention is produced in an appropriate plant tissue, the parthenogenetic effect is significantly enhanced compared to a plant that differs only in that it lacks said nucleic acid. Functionality can also be easily tested by (over)expressing the nucleic acid of the invention in a suitable host plant, such as a non-parthenogenetic Taraxacum line, and analyzing the parthenogenetic effect of the transformant in a bioassay, for example as described in Example 2. The functionality of said nucleic acids is preferably assessed by comparing a test plant in which one or more of these nucleic acids are (over)expressed with a control plant that differs from the test plant only in that the control plant lacks (over)expression of said nucleic acid. Alternatively, silencing or disruption of the nucleic acid of the invention associated with parthenogenesis can lead to a loss of function, i.e. reduced parthenogenesis.

[0088] The nucleic acids of the present invention can be used to create vectors or plasmids for expressing the proteins of the present invention in suitable host cells or for silencing one or more endogenous parthenogenetic genes or gene families. Thus, constructs, vectors and / or plasmids comprising the nucleic acids of the present invention, and / or silencing constructs are also encompassed by the present invention.

[0089] Amino acid sequences according to the invention The present invention provides a PAR protein as defined herein. The present invention also provides a protein associated with parthenogenesis in plants, wherein said protein is: a) encoded by a nucleic acid of the invention; b) having the amino acid sequence of SEQ ID NO: 1, 6 or 11; c) a variant of a) and / or b); and / or d) A fragment of any one of a) to c); Preferably, herein, said protein is functional in parthenogenesis. In one embodiment, the protein of the invention comprises: a) encoded by any one of the nucleic acids of SEQ ID NO: 3, 8, or 13; b) having the amino acid sequence of SEQ ID NO: 1, 6 or 11; c) a variant of a) and / or b); and / or d) A fragment of any one of a) to c); Here, preferably, the protein of the invention is suitable for inducing parthenogenesis. In one embodiment, the protein of the invention comprises: a) encoded by a nucleic acid of SEQ ID NO: 3 or 5; b) having the amino acid sequence of SEQ ID NO:1; c) a variant of a) and / or b); and / or d) A fragment of any one of a) to c); Preferably, the protein of the invention herein is suitable for inducing parthenogenesis. The variant is preferably a PAR protein as defined herein. Preferably, the protein or protein fragment is encoded by a nucleic acid of SEQ ID NO: 3 or 5, or a variant and / or fragment thereof, or such protein comprises SEQ ID NO: 1, or a variant and / or fragment thereof. Preferably, said variant comprises or consists of an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 1, 6 or 11, respectively, preferably when pairwise aligned using, for example, the Needleman and Wunsch algorithm (global sequence alignment) with default parameters. Variants differ from the provided sequence by deletion, insertion and / or substitution of one or more amino acid residues, said variants including natural and / or synthetic / artificial variants. A variant of a protein having an amino acid sequence encoded by a nucleic acid of the present invention, preferably a variant of a protein encoded by any one of SEQ ID NOs: 3, 5, 8, 10, 13, 15, or a variant of a protein having an amino acid sequence of any one of SEQ ID NOs: 1, 6 or 11, may be a homolog or ortholog. Such orthologous proteins encompassed by the present invention may be, but are not limited to, any one of the PAR proteins selected from the group consisting of: a PAR protein from Ananas sativa (e.g., UniProtKB: A0A199URK4), a PAR protein from Apostasia siensis (e.g., UniProtKB: A0A2I0AZW3), a PAR protein from Arabidopsis thaliana (e.g., UniProtKB: Q8GXP9, A0A178V2S4, O81793, A0A178V1Q3, A0MFC1, O81801), a PAR protein from Acanthus subsp. lyrata (e.g., UniProtKB: D7MC52 or D7MCE8), a PAR protein from Arachis ipaensis (e.g., SEQ ID NO: 45 or SEQ ID NO: 49),PAR proteins from Brassica campestris (e.g. UniProtKB: A0A398AHT1), PAR proteins from Brassica rapa (e.g. SEQ ID NO: 47), PAR proteins from Brassica rapa subsp. pekinensis (e.g. UniProtKB: M4D574 or M4D571), PAR proteins from Brassica napus (e.g. UniProtKB: I1J0D9), PAR proteins from Brassica napus var. orracea (e.g. UniProtKB: A0A0D3A1Q6 or A0A0D3A1Q3), PAR proteins from Brassica campestris (e.g. UniProtKB: A0A398AHT1), PAR proteins from Brassica rapa (e.g. SEQ ID NO: 47), PAR proteins from Brassica rapa subsp. pekinensis (e.g. UniProtKB: M4D574 or M4D571), PAR proteins from Brassica napus (e.g. UniProtKB: I1J0D9), PAR protein (e.g. UniProtKB: A0A3P6ESB1 or A0A3P6F726), PAR protein from Brassica campestris (e.g. UniProtKB: A0A3P5ZMM3 or A0A3P5Z1M1), PAR protein from pigeonpea (e.g. SEQ ID NO: 46), PAR protein from Rubera herbacea (e.g. UniProtKB: R0H2J1 or R0H0C2), PAR protein from Saxifrage (e.g. UniProtKB: A0A1Q3CSK1), PAR protein from chickpea ... or SEQ ID NO:55, 56 or 57), a PAR protein from endive (e.g., SEQ ID NO:39), a PAR protein from cucumber (e.g., UniProtKB:A0A0A0KGW4 or A0A0A0L0X7), a PAR protein from muskmelon (e.g., UniProtKB:A0A1S3BLF2 or A0A1S3B298), a PAR protein from cucumber (e.g., UniProtKB:A0A0A0KAW8), a PAR protein from Cucurbita gracilis (e.g., SEQ ID NO: 43), a PAR protein from Cucurbita dodder (e.g., UniProtKB: A0A484MGR1), a PAR protein from Dendrobium nigricans (e.g., UniProtKB: A0A2I0V7N9, A0A2I0X2T2, or A0A2I0W0Q8), a PAR protein from Dorcoceras hygrometricum (e.g., UniProtKB: A0A2Z7D3Y1), a PAR protein from Eutrema sarsuginaeum (e.g., UniProtKB: V4LSH0; or SEQ ID NO: 44),PAR proteins from Fagus crucian carp (e.g., UniProtKB: A0A2N9E5Y5, A0A2N9HAB9, or A0A2N9H993), PAR proteins from Germplasm aurea (e.g., UniProtKB: S8E1M6), PAR proteins from Soybean (e.g., SEQ ID NO: 51, 52, 53, or 54), PAR proteins from Upland Cotton (e.g., UniProtKB: A0A1U8LDU9), PAR proteins from Sunflower (e.g., SEQ ID NO: 21), PAR proteins from Hevea brasiliensis (e.g., SEQ ID NO: 42), PAR protein from Dandelion (e.g., SEQ ID NO: 40), PAR protein from Persian walnut (e.g., UniProtKB: A0A2I4E6B1), PAR protein from lettuce (e.g., UniProtKB: A0A2J6KZF7; or SEQ ID NO: 22), PAR protein from bottle gourd (e.g., SEQ ID NO: 48), PAR protein from Medicago sativa (e.g., UniProtKB: G7K024), PAR protein from Marbagus vulgare (e.g., UniProtKB: W9SMY3 or W9SMQ7), PAR protein from Vitis vinifera ... PAR proteins from Nicotiana attenuata (e.g. UniProtKB: A0A1J6IQI6), PAR proteins from Nicotiana sylvestris (e.g. UniProtKB: A0A1U7VXJ0), PAR proteins from Nicotiana tabacum (e.g. UniProtKB: A0A1S4A651 or A0A1S3YHQ2), PAR proteins from Oryza sativa subsp. japonica (e.g. UniProtKB: B9FGH8), PAR proteins from Oryza balsii (e.g. UniProtKB: U niProtKB:A0A0D3FWX3), PAR proteins from millet (e.g. UniProtKB:A0A3L6Q010 or A0A3L6T1D6), PAR proteins from Parasponia andersonii (e.g. UniProtKB:A0A2P5BMI5), PAR proteins from poplar trees (e.g. UniProtKB:A0A4U5PSY9), PAR proteins from black cottonwood (e.g. UniProtKB:B9H661), PAR proteins from pomegranate (e.g. UniProtKB:A0A2I0IBB9,A0A218XB85 or A0A218W102), a PAR protein from Senecio cambrensis (e.g. SEQ ID NO: 41), a PAR protein from peach (e.g. SEQ ID NO: 50), a PAR protein from Enoki mushroom (e.g. UniProtKB: A0A2P5EB04), a PAR protein from Red clover (e.g. UniProtKB: A0A2K3N851), a PAR protein from Japanese clover (e.g. UniProtKB: A0A2Z6MYD3 or A0A2Z 6MDR7), PAR proteins from red clover (e.g. UniProtKB: A0A2K3PR44), PAR proteins from European grape (e.g. UniProtKB: A0A438C778, A0A438ESC4 or A0A438DBR4) and PAR proteins from maize (e.g. UniProtKB: A0A1D6HF46, B6UAC5, A0A3L6F4S1, A0A3L6EMC6, A0A3L6EMC6, K7UHQ6 or A0A1D6KHZ4). Such an orthologous protein may also be a PAR protein selected from the group consisting of: a PAR protein from Actinidia chinensis (UniProtKB: A0A2R6S2S9), a PAR protein from sugar beet (UniProtKB: XP_010690656.1), a PAR protein from potato (UniProtKB: XP_015159151.1), a PAR protein from tomato (UniProtKB: A0A3Q7GXB3), a PAR protein from melon pepper (UniProtKB: A0A2G2WJR7), a PAR protein from eggplant (UniProtKB: A0A2G2WJR7), a PAR protein from thyme ... protein (UniProtKB: AVC18974.1), PAR protein from wild soybean (GenBank accession: XP_028201014.1, XP_006596577.1 or UniprotKB: A0A445M3M6), PAR protein from peanut (UniProtKB: A0A444WUX5), PAR protein from common bean (UniProtKB: V7CIF6), PAR protein from carrot (GenBank accession: XP_017245413.1), PAR protein from bread wheat (UniProtKB: A0A3B6RP64),PAR protein from rice subspecies indica (UniProtKB: A2YH63), PAR protein from rice subspecies japonica (UniProtKB: Q5Z7P5), and PAR protein from cacao (UniProtKB: A0A061DL63).

[0090] Thus, variants of the protein of SEQ ID NO:1 encompassed by the present invention may be, but are not limited to, any one of the orthologous PAR proteins defined herein.

[0091] The PAR protein of the invention and / or variants of proteins with SEQ ID NO: 1, 6 or 11 may be capable of inducing parthenogenesis when present in a plant or plant cell. The variants of proteins may be endogenous or non-endogenous proteins of said plant or plant cell. Optionally, the PAR protein of the invention and / or variants of proteins with SEQ ID NO: 1, 6 or 11 may be capable of inducing parthenogenesis when the expression of said protein is altered, preferably increased. Preferably, such altered, preferably increased, expression is in an egg cell. The altered or increased expression may be de novo expression of said protein in the plant or plant cell or may be increased expression of an endogenous protein in the plant or plant cell. The skilled person knows how to increase the expression of a protein. De novo expression of the protein in a plant or plant cell may be induced, for example, by transfecting a construct or vector encoding the protein into the plant or plant cell, by introgressing a gene encoding the protein into the progeny of the plant or plant cell, and / or by modifying an endogenous sequence to provide a sequence encoding the protein, for example by genetic modification. Optionally, such a construct or vector comprises a sequence encoding a PAR protein operably linked to an egg cell promoter. Those skilled in the art are aware of egg cell promoters. Exemplary egg cell promoters capable of driving expression in plant egg cells include, but are not limited to, the promoters of the egg cell-specific genes ECl.1, ECl.2, ECl.3, ECl.4, or ECl.5 (see, e.g., Sprunck et al. Science, 338:1093-1097 (2012); AT2G21740; Steffen et al., Plant Journal 51:281-292 (2007)), the Arabidopsis DD45 promoter (Ohnishi et al. Plant Physiology 165:1533-1543 (2014)).Preferably, the construct or vector of the present invention comprises a sequence encoding a PAR protein operably linked to a regulatory sequence, preferably a promoter sequence, and comprises a nucleic acid insert, preferably a double-stranded DNA insert, wherein said insert has a length of between 50 and 2000 bp, between 100 and 1900 bp, between 200 and 1800 bp, between 300 and 1700 bp, between 400 and 1600 bp, between 500 and 1500 bp, between 600 and 1400 bp, between 1000 and 1400, between 1200 and 1400, or between 1300 and 1400 bp. Even more preferably, said insert has a length of about 1300 bp. Preferably, the insert is associated with a parthenogenetic phenotype as defined herein, and optionally is functional in the parthenogenetic phenotype. Preferably, the insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding the PAR protein, such that the distance between the 3' end of the insert and the start codon of the sequence encoding the PAR protein is between 50 and 200 bp, preferably about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 bp, most preferably about 102 bp. Preferably, the insert is located such that the 3' terminal nucleotide of the insert is at a position that is homologous to nucleotide 1798 of SEQ ID NO:2 and / or nucleotide 1798 of SEQ ID NO:5. Preferably, the insert is devoid of an open reading frame. Even more preferably, the insert is a miniature inverted repeat transposable element (MITE) or MITE-like sequence, which is a non-autonomous element characterized in that it contains an internal sequence with no open reading frame, which is flanked by terminal inverted repeats (TIRs) that are in turn flanked by small direct repeats (target site overlaps). For details on MITEs, TIRs, and sequences, see Guo et al., Scientific Reports. 2017 Jun. 1;7(1):2634, which is incorporated herein by reference.Said insert, preferably said MITE or MITE-like sequence, may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, said insert is associated with and optionally functional in a parthenogenetic phenotype as defined herein. In a further preferred embodiment, the construct or vector of the invention comprises or consists of a regulatory sequence, preferably a promoter sequence, and includes said insert in a position as defined herein above. Preferably, the construct or vector comprises or consists of a sequence encoding a PAR protein as defined herein, operably linked to said promoter sequence, wherein preferably said promoter sequence is located directly upstream of the sequence encoding the PAR protein. Optionally, said construct or vector of the invention may comprise one or more further transcriptional regulatory sequences.

[0092] Additionally or alternatively, such constructs or vectors comprise a sequence encoding a PAR protein operably linked to the promoter of SEQ ID NO:2. The altered or increased expression of the endogenous protein may be induced by modifying one or more regulatory sequences operably linked to the coding sequence. For example, the promoter sequence operably linked to the protein coding sequence may be modified, for example by genetic modification. In a preferred embodiment, an insert as defined herein above is introduced into the promoter sequence, preferably at a position as defined herein above. Such functionality capable of inducing parthenogenesis may be evaluated by using a test suitable for the functionality in parthenogenesis of the nucleic acid encoding said variant, as described herein. The protein of the invention may be an isolated protein.

[0093] "Natural variants" are those found in nature, e.g., in cultivated or wild lettuce plants and / or other plants. Also included are fragments, i.e., non-full-length peptides, preferably functional fragments, of the proteins of the invention, i.e., the fragments are capable of inducing parthenogenesis when expressed in a suitable host plant. Fragments of the proteins taught herein include peptides comprising or consisting of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250 or more consecutive amino acid sequences encoded by the nucleic acids of the invention, in particular peptides comprising or consisting of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250 or more consecutive amino acids of SEQ ID NO: 1, 6, or 11, or variants thereof (as defined herein). Sequences found in nature are also referred to herein as "wild type".

[0094] The proteins of the invention may be isolated from natural sources, synthesized de novo by chemical synthesis (e.g., using a peptide synthesizer such as those supplied by Applied Biosystems), or produced by a recombinant host cell by expressing a nucleotide sequence as taught herein that encodes a protein of the invention. The proteins of the invention may also be produced by expression from a nucleic acid of the invention as defined herein.

[0095] Protein variants can include conservative amino acid substitutions within the categories of basic (e.g., Arg, His, Lys), acidic (e.g., Asp, Glu), non-polar (e.g., Ala, Val, Trp, Leu, Ile, Pro, Met, Phe, Trp) or polar (e.g., Gly, Ser, Thr, Tyr, Cys, Asn, Gln). In addition, non-conservative amino acid substitutions are also encompassed within the scope of the invention.

[0096] Also encompassed herein are chimeric proteins, such as proteins composed of domains from different sources, such as the N-terminus of a protein of SEQ ID NO: 1, 6 or 11 (e.g., obtained from Taxaracum or plant species X) and the middle and / or C-terminal domain of a variant of SEQ ID NO: 1, 6 or 11 (e.g., obtained from Taxaracum or plant species Y or another plant species). Preferably, a chimeric protein is composed of domains from at least two orthologous proteins. Such chimeric proteins can have improved functionality, for example, in the sense that they can confer parthenogenesis more efficiently than the native protein when expressed in a plant host.

[0097] Also encompassed by the present invention are all nucleotide sequences (RNA, cDNA, genomic DNA, etc.) that code for the proteins, protein variants or protein fragments of the present invention. Due to the degeneracy of the genetic code, various nucleotide sequences can code for the same amino acid sequence.

[0098] Parthenogenetic plants and methods for producing them In a further aspect, the present invention relates to plants (including, for example, plant cells, organs, seeds and plant parts) and methods for producing plants exhibiting modified parthenogenesis, optionally transgenic plants with modified, preferably induced, parthenogenesis compared to natural or unmodified plants. Such plants can be produced using various methods, for example as further described herein. Preferably, the plants of the present invention are obtained by technical means, preferably by the methods described herein. Such technical means are well known to those skilled in the art and include, for example, genetic modifications such as at least one of random mutagenesis, targeted mutagenesis, and nucleic acid insertion.

[0099] Preferably, the plant of the present invention is not obtained by an essentially biological process. Preferably, the plant of the present invention is not obtained exclusively by an essentially biological process. Preferably, the plant of the present invention is not obtained, preferably not directly obtained, by an essentially biological process of introducing parthenogenesis into a plant. Preferably, the plant of the present invention is not obtained exclusively by an essentially biological process of introducing parthenogenesis into a plant. Preferably, the plant of the present invention is not a plant of natural origin, i.e. is not a plant occurring in nature. In particular, the present invention provides a method for producing a parthenogenetic plant, comprising the steps of: a) introducing into one or more plant cells a nucleic acid of the invention and / or a derivative thereof capable of inducing parthenogenesis and / or functional in parthenogenesis; b) optionally selecting plant cells containing said nucleic acid, preferably wherein said nucleic acid is integrated into the genome of said plant cells; c) regenerating a plant from said plant cell. Including, Preferably, said nucleic acid of the invention herein encodes or is operably linked to a sequence encoding a PAR protein as defined herein that is functional in parthenogenesis and / or is any one of SEQ ID NOs: 2 to 5, or encodes the protein of SEQ ID NO: 1, or is a variant or fragment thereof. The present invention further provides a method of producing an apomictic plant, comprising the steps of: a) introducing into one or more plant cells capable of apomyosis a nucleic acid of the invention and / or a derivative thereof capable of inducing parthenogenesis; b) optionally selecting plant cells containing said nucleic acid, preferably wherein said nucleic acid is integrated into the genome of said plant cells; c) regenerating a plant from said plant cell. Including, Preferably, said nucleic acid of the invention herein encodes or is operably linked to a sequence encoding a PAR protein as defined herein that is functional in parthenogenesis and / or is any one of SEQ ID NOs: 2-5, or encodes a protein of SEQ ID NO: 1, or is a variant or fragment thereof. A plant cell capable of apomeiosis may be obtained by a step of introducing a nucleic acid capable of conferring apomeiosis. Optionally, said nucleic acid is introduced into the plant cell before, together with or after the introduction of a nucleic acid of the invention.

[0100] The nucleic acids of the invention can be introduced into one or more plant cells by transformation, gene transfer, somatic cell hybridization and / or protoplast fusion. Such nucleic acids may be exogenous nucleic acids, i.e. nucleic acids that are not naturally present in said plant cells.

[0101] The nucleic acid of the invention can be introduced into one or more plant cells by modifying an endogenous nucleic acid to obtain the nucleic acid of the invention. The modification of the endogenous gene preferably includes random or targeted mutation of one or more nucleotides in the coding sequence and / or in the regulatory and / or promoter sequences, or insertion or deletion of short or larger sequences, for example by homologous recombination, to alter the expression of the endogenous protein. Such methods preferably result in the modification of one or more endogenous par alleles to Par alleles as defined herein. Random mutagenesis may be, but is not limited to, chemical mutagenesis and gamma radiation. Non-limiting examples of chemical mutagenesis include, but are not limited to, EMS (ethyl methanesulfonate), MMS (methyl methanesulfonate), NaN3 (sodium azide) D), ENU (N-ethyl-N-nitrosourea), AzaC (azacytidine) and NQO (4-nitroquinoline 1-oxide). Optionally, a mutagenesis system such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al., 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporated herein by reference) may be used to generate plant lines with modified genes as defined herein. TILLING uses traditional chemical mutagenesis (e.g., EMS mutagenesis) followed by high-throughput screening for mutations. Thus, plants, seeds and tissues containing genes with one or more desired mutations may be obtained using TILLING. Targeted mutagenesis is mutagenesis that can be designed to alter specific nucleotides or nucleic acid sequences, such as, but not limited to, oligo-directed mutagenesis, RNA-guided endonucleases (e.g., CRISPR technology), TALEN or zinc finger technology.

[0102] Preferably, the modification is a modification in the promoter sequence of a gene encoding a PAR protein as defined herein. Preferably, the modification introduces or increases the expression of a PAR protein as defined herein. Preferably, the modification introduces or increases the expression of a PAR protein as defined herein in an egg cell. Thus, the method of the present invention comprises the following steps: a) modifying in one or more plant cells a nucleic acid which is or is operably linked to a sequence encoding a protein associated with and / or functional in parthenogenesis, preferably said nucleic acid being within the genome of said one or more plant cells; b) optionally selecting plant cells containing the modified nucleic acid; c) regenerating a plant from said plant cell. may include Preferably herein, said protein associated with and / or functional in parthenogenesis has an amino acid sequence according to the invention as described herein above. Preferably, the nucleic acid to be modified in step a) is an endogenous nucleic acid, preferably comprising or consisting of a nucleotide sequence which is or is operably linked to a PAR protein as defined herein and / or a protein having the amino acid sequence of SEQ ID NO: 1, 6 or 11, or a variant or fragment thereof.

[0103] In a particular preferred embodiment, said nucleic acid is a (5'UTR) promoter sequence of a gene encoding a protein associated with parthenogenesis as defined herein. Preferably, said modification is the introduction of a nucleic acid insert, preferably a double-stranded DNA insert, wherein said insert has a length of between 50 and 2000 bp, between 100 and 1900 bp, between 200 and 1800 bp, between 300 and 1700 bp, between 400 and 1600 bp, between 500 and 1500 bp, between 600 and 1400 bp, between 1000 and 1400, between 1200 and 1400, or between 1300 and 1400 bp. Even more preferably, said insert has a length of about 1300 bp. Preferably, the insert is associated with a parthenogenesis phenotype as defined herein, and optionally is functional in the parthenogenesis phenotype. Preferably, the insert is introduced into a promoter sequence located immediately upstream (3') of the sequence encoding a PAR protein, such that the distance between the 3' end of the insert and the start codon of the sequence encoding a PAR protein is between 50 and 200 bp, preferably about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 bp, most preferably about 102 bp. Preferably, the insert is introduced such that the 3' terminal nucleotide of the insert is at a position homologous to nucleotide 1798 of SEQ ID NO:2 and / or nucleotide 1798 of SEQ ID NO:5. Preferably, the insert is devoid of an open reading frame. Even more preferably, the insert is a miniature inverted repeat transposable element (MITE) or MITE-like sequence, which is a non-autonomous element characterized in that it contains an internal sequence with no open reading frame, which is flanked by terminal inverted repeats (TIRs) that are in turn flanked by small direct repeats (target site overlaps). For details on MITEs, TIRs, and sequences, see Guo et al., Scientific Reports. 2017 Jun. 1;7(1):2634, which is incorporated herein by reference.The insert, preferably the MITE or MITE-like sequence, may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO: 60. Preferably, the insert is associated with, and optionally functional in, a parthenogenetic phenotype as defined herein.

[0104] Preferably, modification of the nucleotide sequence results in the introduced or increased expression of said protein, preferably in a plant egg cell regenerated from the plant cell. Preferably, the modified promoter sequence comprises a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2. Further, the method of the present invention comprises the steps of: a) modifying in one or more plant cells capable of apomeiosis a nucleic acid which is or is operably linked to a sequence encoding a protein associated with and / or functional in parthenogenesis, preferably said nucleic acid being within the genome of said one or more plant cells; b) optionally selecting plant cells containing the modified or altered nucleic acid; c) regenerating a plant from said plant cell. may include Preferably herein, said protein associated with and / or functional in parthenogenesis has an amino acid sequence according to the protein of the invention as described herein above. Preferably, the nucleic acid modified in step a) is an endogenous nucleic acid, preferably comprising or consisting of a nucleotide sequence which is or is operably linked to a PAR protein as defined herein and / or a protein having the amino acid sequence of SEQ ID NO: 1, 6 or 11, or a variant or fragment thereof. Preferably, the nucleic acid modified in step a) is an endogenous nucleic acid.

[0105] In a particular preferred embodiment, said nucleic acid is a promoter sequence of a gene encoding a protein associated with and / or functional in parthenogenesis as defined herein. Preferably, modification of the nucleotide sequence results in an introduced or increased expression of said protein, preferably in an egg cell of a plant regenerated from said plant cell. Preferably, the modified promoter sequence is a promoter sequence operably linked to a coding sequence of a PAR protein as defined herein. Preferably, said modified promoter sequence is modified to contain an insert as defined herein above, preferably in a position as defined herein above.

[0106] Preferably, the modified promoter sequence comprises a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2. The present invention also provides a method for producing apomictic hybrid seeds, comprising the steps of: a) cross-fertilizing a sexually reproduced first plant with pollen from a second plant to produce F1 hybrid seed; b) optionally selecting seeds containing an apomictic phenotype from said F1 seeds; Including, wherein said first plant and / or said second plant are capable of apomyosis, said second plant comprises a nucleic acid of the present invention, and preferably said selecting step is performed by genotyping. Preferably, said second plant comprises a nucleic acid of the present invention, which is any one of SEQ ID NOs: 2 to 5 or encodes a protein of SEQ ID NO: 1, or a variant or fragment thereof.

[0107] The nucleic acid of the invention may be comprised in a chimeric gene, a gene construct or a nucleic acid vector. In one embodiment of the invention, the nucleic acid of the invention may be used to generate a chimeric gene and / or a vector comprising the nucleic acid for the transfer of the nucleic acid into a host cell and the production of a functional (preferably parthenogenetically inducible) protein encoded by said nucleic acid in the host cell. Vectors intended for the production of such proteins (or protein fragments or variants) in plant cells are herein referred to as "expression vectors". The host cell is preferably a plant cell.

[0108] The construction of chimeric genes, constructs and / or vectors for the optional transient, but preferably stable, introduction of nucleotide sequences encoding proteins into the genome of a host cell is generally known in the art. To create chimeric genes for inducing parthenogenesis and / or improving functionality in parthenogenesis, the nucleotide sequences encoding the proteins of SEQ ID NO: 1, 6 or 11, or functional variants and / or functional fragments thereof, may be operably linked to a promoter sequence suitable for expression in a host cell using standard molecular biology techniques. The promoter sequence may already be present in the vector, so that the nucleotide sequence encoding the protein can simply be inserted downstream of the promoter sequence into the vector. The vector may then be used to transform a host cell, and the nucleic acid and / or chimeric gene of the present invention may be inserted into the nuclear genome or into the genome of plastids, mitochondria or chloroplasts, and expressed in the host cell using a suitable promoter (e.g., Mc Bride et al., 1995; U.S. Patent No. 5,693,507). In one embodiment, the nucleic acid and / or chimeric gene of the invention can comprise a promoter suitable for expression in a plant cell or a microbial cell (e.g., a bacterium) operably linked to a nucleotide sequence encoding the protein of the invention, optionally followed by a 3' untranslated nucleotide sequence. The coding sequence is optionally preceded by a 5' UTR sequence. The promoter, 3' UTR and / or 5' UTR can be, for example, from a naturally occurring parthenogenetic gene, or alternatively from other sources.

[0109] A nucleic acid as taught herein encoding a protein capable of inducing parthenogenesis as taught herein can be stably inserted into the nuclear genome of a single plant cell, and the plant cell so transformed can be used to generate a transformed plant having an altered phenotype due to the presence of the protein in a particular cell at a particular time. In a non-limiting example, a T-DNA vector containing a nucleic acid as taught herein encoding a protein functional in parthenogenesis as taught herein can be used to transform a plant cell in Agrobacterium tumefaciens, and the transformed plant can then be regenerated from the transformed plant cell using procedures described, for example, in EP 0116718, EP 0270822, PCT Publication WO 84 / 02913, and EP 0242246, as well as in Gould et al. (1991). Construction of T-DNA vectors for Agrobacterium-mediated plant transformation is well known in the art. The T-DNA vector may be either a binary vector as described in EP 0120561 and EP 0120515, or a cointegrate vector that can be integrated into an Agrobacterium Ti plasmid by homologous recombination as described in EP 0116718. Lettuce transformation protocols are described, for example, in Michelmore et al. (1987) and Chupeau et al. (1989).

[0110] A preferred T-DNA vector contains a promoter operably linked to a nucleotide sequence encoding a protein of the invention; for example, the promoter is operably linked to a nucleotide sequence of SEQ ID NO: 3 or a variant or functional fragment thereof between the T-DNA border sequences or at least located to the left of the right border sequence. Preferably, said promoter is a promoter comprising a nucleic acid insert, preferably a double-stranded DNA insert, wherein said insert has a length of between 50 and 2000 bp, between 100 and 1900 bp, between 200 and 1800 bp, between 300 and 1700 bp, between 400 and 1600 bp, between 500 and 1500 bp, between 600 and 1400 bp, between 1000 and 1400, between 1200 and 1400, or between 1300 and 1400 bp. Even more preferably, said insert has a length of about 1300 bp. Preferably, the insert is associated with a parthenogenetic phenotype as defined herein, and optionally functional in the parthenogenetic phenotype. Preferably, the insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding a PAR protein, such that the distance between the 3' end of the insert and the start codon of the sequence encoding a PAR protein is between 50 and 200 bp, preferably about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 bp, most preferably about 102 bp. Preferably, the insert is located such that the 3' terminal nucleotide of the insert is at a position that is homologous to the position of nucleotide 1798 of SEQ ID NO:2 and / or nucleotide 1798 of SEQ ID NO:5. Preferably, the insert is devoid of an open reading frame. Even more preferably, the insert is a miniature inverted repeat transposable element (MITE) or MITE-like sequence, which is a non-autonomous element characterized in that it contains an internal sequence without an open reading frame, which internal sequence is flanked by terminal inverted repeats (TIRs) which are in turn flanked by small direct repeat sequences (target site overlap).For details of MITEs, TIRs and sequences, see Guo et al., Scientific Reports. 2017 Jun 1;7(1):2634, incorporated herein by reference. The insert, preferably the MITE or MITE-like sequence, may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO: 60. Preferably, the insert is associated with and optionally functional in a parthenogenetic phenotype as defined herein. In a further preferred embodiment, the T-DNA vector comprises or consists of a regulatory sequence, preferably a promoter sequence, and includes the insert at a position as defined herein above. Preferably, the T-DNA vector comprises or consists of a sequence encoding a PAR protein as defined herein, operably linked to the promoter sequence, wherein preferably, the promoter sequence is located directly upstream of the sequence encoding the PAR protein. Optionally, the T-DNA vector can include one or more additional transcriptional regulatory sequences.

[0111] Border sequences are described in Gielen et al., 1984. Of course, other types of vectors can be used to transform plant cells using procedures such as direct gene transfer (e.g., as described in EP 0223247), pollen-mediated transformation (e.g., as described in EP 0270356 and WO 85 / 01856), protoplast transformation as described, for example, in U.S. Pat. No. 4,684,611, plant RNA virus-mediated transformation (e.g., as described in EP 0067553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (e.g., as described in U.S. Pat. No. 4,536,475), and other methods.

[0112] In a further embodiment, the nucleic acid of the present invention may be introduced by somatic cell hybridization, which may be performed by protoplast fusion (see, for example, Holmes, 2018).

[0113] The nucleic acid of the present invention can also be integrated into the genome, for example, by using one or more specific endonucleases (such as CRISPR-endonuclease / guide RNA complexes) to introduce double-strand breaks at appropriate sites in the genome and a donor construct comprising the nucleic acid of the present invention to be integrated into the genome.Those skilled in the art know how to design such CRISPR-endonuclease / guide RNA complexes to introduce double-strand breaks and donor constructs suitable for integration (for review, see Bortesi and Fischer, 2015).

[0114] Alternatively, the plant may be transformed by modifying the endogenous nucleotide sequence, thereby, for example, converting one or more par alleles contained in the plant into one or more Par alleles, for example by random or targeted mutagenesis. Said mutagenesis may include mutagenesis of coding sequences, but also of regulatory sequences such as promoter sequences, 5'UTR and / or 3'UTR. Said endogenous 5'UTR promoter nucleotide sequence of a par allele may be modified to include an insert as defined herein above, preferably at a position as defined herein above.

[0115] Similarly, the selection and regeneration of transformed plants from transformed cells is well known in the art. Clearly, protocols have been specifically designed to regenerate transformants at high frequency for a variety of species, and even for a variety of varieties or cultivars of a single species. The present invention also encompasses the progeny of transformed plants that exhibit parthenogenesis and contain the nucleic acids and / or proteins of the present invention.

[0116] In addition to the transformation of the nuclear genome, the transformation of the plastid genome, preferably the chloroplast genome, is also included in the present invention. One advantage of the transformation of the plastid genome is that it can reduce the risk of the spread of the introduced gene(s). The transformation of the plastid genome can be carried out as known in the art, see for example Sidorov et al. (1999) or Lutz et al. (2004).

[0117] The resulting transformed plants can be used in conventional plant breeding schemes to generate more transformed plants containing the transgene. Single copy transformants can be selected, for example, using Southern blot analysis or PCR-based methods or Invader® Technology Assay (Third Wave Technologies, Inc.). Transformed cells and plants can be easily distinguished from non-transformed ones by the presence of the nucleic acid or protein and / or chimeric gene of the present invention. Sequences of plant DNA adjacent to the insertion site of the transgene can also be sequenced, allowing the development of "event-specific" detection methods for routine use. See, for example, WO 0141558, which describes elite event detection kits (such as PCR detection kits) based on integrated and adjacent (genomic) sequences.

[0118] The nucleic acid of the invention may be inserted into the plant cell genome such that the inserted coding sequence(s) is downstream (i.e., 3') of and under the control of a promoter capable of directing expression in the plant cell. This is preferably achieved by inserting a chimeric gene containing such elements into the plant cell genome, particularly the nuclear or plastid (e.g., chloroplast) genome.

[0119] The promoter that can be operably linked to SEQ ID NO:3, or a variant or fragment thereof, may be, for example, a constitutively active promoter, such as, for example: the strong constitutive 35S promoter or enhanced 35S promoter ("35S promoter") of the cauliflower mosaic virus (CaMV) of isolates CM1841 (Gardner et al., 1981), CabbB-2 (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the 35S promoter described by Odell et al. (1985) or in U.S. Pat. No. 5,164,316, promoters from the ubiquitin family (e.g., Christensen et al., 1992; the maize ubiquitin promoter in EP 0 342 926; see also Cornejo et al., 1993), the gos2 promoter (de Arabidopsis actin promoters such as the promoter described by Pater et al., 1992), the emu promoter (Last et al., 1990), the promoter described by An et al. (1996), rice actin promoters such as the promoter described by Zhang et al. (1991) and the promoter described in U.S. Pat. No. 5,641,876 or the rice actin 2 promoter described in WO 070067; the promoter of Cassava Vein Mosaic Virus (WO 97 / 48819, Verdaguer et al. 1998), Subterranean Clover dwarf virus (Clover dwarf virus) and the like. pPLEX series promoters from Stunt virus (WO 96 / 06932, especially the S7 promoter), alcohol dehydrogenase promoters such as pAdh1S (GenBank Accession Nos. X04049, X00581), and the TR1' and TR2' promoters (the "TR1' promoter" and "TR2' promoter", respectively) driving expression of the 1' and 2' genes of the T-DNA (Velten et al., 1984), the Figwort Mosaic Virus promoter described in U.S. Pat. No. 6,051,753 and EP 426641, histone gene promoters such as the Ph4a748 promoter from Arabidopsis (PMB 8:179-191), or others.

[0120] Alternatively, a promoter that is not constitutive, but rather specific to one or more tissues or organs of the plant (tissue-preferential / tissue-specific, including developmentally regulated promoters), such as an egg cell-specific promoter, can be utilized, whereby the protein of the invention is expressed only or preferentially in cells of a specific tissue(s) or organ(s) and / or only during certain developmental stages.

[0121] Since constitutive production of the protein of the invention may be costly to plant fitness, in one embodiment it is preferred to use a promoter whose activity is inducible. An example of an inducible promoter is a wound-inducible promoter, such as the MPI promoter described by Cordera et al. (1994), which is wound-induced (e.g., caused by insects or physical wounds), or the COMPTII promoter (WO 0056897) or the RP1 promoter described in US Pat. No. 6,031,151. Alternatively, the promoter may be inducible by chemicals such as dexamethasone, as described by Aoyama and Chua (1997) and in US Pat. No. 6,063,985, or by tetracycline (TOPFREE or TOP10 promoters, see Gatz, 1997 and Love et al., 2000).

[0122] The term "inducible" does not necessarily require that the promoter is completely inactive in the absence of inducer stimulation. Low levels of non-specific activity may be present as long as this does not result in severe yield or quality penalties for the plant. Thus, inducible preferably refers to an increase in the activity of the promoter, resulting in increased transcription of the downstream coding region encoding the protein of the invention following contact with an inducer.

[0123] In one embodiment, a promoter of a naturally occurring parthenogenetic gene is used. For example, the promoter of the Dandelion Par allele or the par allele may be isolated and operably linked to the coding region encoding the protein according to the invention. In one embodiment, the promoter (upstream transcriptional regulatory region, e.g., within about 2000 bp upstream of the translation start codon and / or the transcription start codon) can be isolated from apomictic plants and / or other plants using known methods such as TAIL-PCR (Liu et al., 1995; Liu et al., 2005), linker-PCR, or inverse PCR (IPCR).

[0124] In one embodiment, a promoter of a naturally occurring parthenogenetic gene or a promoter derived therefrom is used. For example, a promoter derived from SEQ ID NO: 2 or a variant or fragment thereof may be used. Preferably, said promoter is a promoter comprising a nucleic acid insert, preferably a double-stranded DNA insert, wherein said insert has a length of between 50 and 2000 bp, between 100 and 1900 bp, between 200 and 1800 bp, between 300 and 1700 bp, between 400 and 1600 bp, between 500 and 1500 bp, between 600 and 1400 bp, between 1000 and 1400, between 1200 and 1400, or between 1300 and 1400 bp. Even more preferably, said insert has a length of about 1300 bp. Preferably, the insert is associated with a parthenogenetic phenotype as defined herein, and optionally functional in the parthenogenetic phenotype. Preferably, the insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding the PAR protein, such that the distance between the 3' end of the insert and the start codon of the sequence encoding the PAR protein is between 50 and 200 bp, preferably about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 bp, most preferably about 102 bp. Preferably, the insert is located such that the 3' terminal nucleotide of the insert is at a position that is homologous to nucleotide 1798 of SEQ ID NO:2 and / or nucleotide 1798 of SEQ ID NO:5. Preferably, the insert is devoid of an open reading frame. Even more preferably, the insert is a miniature inverted repeat transposable element (MITE) or MITE-like sequence, which is a non-autonomous element characterized in that it contains an internal sequence with no open reading frame, which is flanked by terminal inverted repeats (TIRs) that are in turn flanked by small direct repeats (target site overlaps). For details on MITEs, TIRs, and sequences, see Guo et al., Scientific Reports. 2017 Jun. 1;7(1):2634, which is incorporated herein by reference.The insert, preferably the MITE or MITE-like sequence, may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, the insert is associated with a parthenogenetic phenotype as defined herein, and optionally is functional in the parthenogenetic phenotype. The promoter may have the nucleotide sequence of SEQ ID NO: 2. Longer sequences than those described herein may also be used. The region up to about 2000 bp upstream of the translation start codon of the coding region may contain transcriptional regulatory elements (i.e., promoters). Thus, in one embodiment, nucleotide sequences 2000 bp, 1500 bp, 1000 bp, 800 bp, 500 bp, 300 bp or less upstream of the translation start codon of the sequence encoding the protein of the invention may be isolated and tested for promoter activity, and if functional, the sequence may be operably linked to the sequence encoding the protein of the invention as taught herein. The promoter activity of the entire sequence and fragments thereof can be tested, for example, by deletion analysis, whereby the 5' and / or 3' portions are deleted and the promoter activity is tested using known methods (e.g., operably linking the promoter or fragment to a reporter gene).

[0125] It is preferred to insert the coding sequence taught herein into the plant genome such that said coding sequence is upstream (i.e., 5') of a suitable 3'-end untranslated region ("3' end" or 3'UTR). Suitable 3' ends include the 3' end of the CaMV 35S gene ("3'35S"), the nopaline synthase gene ("3'nos") (Depicker et al., 1982), the octopine synthase gene ("3'ocs") (Gielen et al., 1984) and the T-DNA gene 7 ("3'gene 7") (Velten and Schell, 1985), which act as 3'-untranslated DNA sequences in transformed plant cells and the like. In one embodiment, the 3'UTR of a naturally occurring parthenogenetic gene, or a 3'UTR derived therefrom, is used. For example, any 3'UTR derived from SEQ ID NO:4, or a variant or fragment thereof, may be used. The 3'UTR may have the nucleotide sequence of SEQ ID NO:4.

[0126] In one embodiment, a promoter having a nucleotide sequence of SEQ ID NO: 2, or variants and / or fragments thereof, may be operably linked to a nucleic acid encoding a protein of the invention, preferably a nucleotide sequence encoding a protein capable of inducing parthenogenesis as taught herein, more preferably having an amino acid sequence of SEQ ID NO: 1, or variants and / or fragments thereof. Preferably, said promoter and coding sequence is further operably linked to a 3'UTR of SEQ ID NO: 4, or variants and / or fragments thereof.

[0127] Introduction of the T-DNA vector into Agrobacterium can be carried out using known methods such as electroporation or triparental mating.

[0128] The coding sequences taught herein can optionally be inserted into a plant genome as hybrid gene sequences whereby the coding sequences are linked in frame to a gene encoding a selectable or scorable marker (U.S. Pat. No. 5,254,799; Vaeck et al., 1987), such as the neo (or nptII) gene encoding kanamycin resistance (European Patent No. 0242236), such that the plant expresses a fusion protein that is easily detectable.

[0129] All or part of the sequence coding for the protein of the invention can be used to transform microorganisms, such as bacteria (e.g. Escherichia coli, Pseudomonas, Agrobacterium, Bacillus, etc.), fungi, or algae or insects, or to generate recombinant viruses. This is particularly suitable for the production and subsequent purification of proteins, preferably isolated proteins. Transformation of bacteria with all or part of the coding sequence taught herein, incorporated in a suitable cloning vehicle, can be carried out in conventional manner, preferably using conventional electroporation techniques as described in Maillon et al. (1989) and WO 90 / 06999. For expression in prokaryotic host cells, the codon usage of the nucleic acid sequence may be optimized accordingly (as described herein for plants). Intron sequences may need to be removed and other adaptations for optimal expression may be made as known. Such prokaryotic host cells containing the nucleic acids and / or expressing the proteins of the invention are encompassed by the present invention. Such host cells may be used to produce the proteins and / or nucleic acids of the invention.

[0130] The DNA sequences of the nucleic acids of the invention can be further altered in a translationally neutral manner to remove potentially inhibitory DNA sequences present in gene portions and / or by introducing changes in the codon usage, e.g. by adapting the codon usage to the most preferred codon usage in plants, e.g. in the particular relevant plant genus described herein as the host plant.

[0131] According to one embodiment of the invention, the protein of the invention is targeted to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or is secreted from the cell, thus potentially optimizing protein stability and / or expression. Similarly, the protein may be targeted to the vacuole. To this end, in one embodiment of the invention, the chimeric gene of the invention comprises a coding region encoding a signal or targeting peptide linked to a region encoding the protein of the invention. Particularly preferred peptides to be included in the protein of the invention are chloroplast or other plastid-directed transit peptides, which target overlapping transit peptide regions, especially from plant genes whose gene products are targeted to plastids, the optimized transit peptide of Capellades et al. (U.S. Pat. No. 5,635,618), the transit peptide of ferredoxin-NADP+ oxidoreductase from spinach (Oelmuller et al., 1993), the transit peptide described in Wong et al. (1992, Plant Molec. Biol. 20, 81-93), and the targeting peptides of published PCT patent application WO 00 / 26371. Also preferred are peptides that signal the secretion of proteins linked to them extracellularly, such as the secretion signal of potato proteinase inhibitor II (Keil et al., 1986), the secretion signal of the rice alpha-amylase 3 gene (Sutliff et al., 1991) and the secretion signal of the tobacco PR1 protein (Cornelissen et al., 1986). Particularly useful signal peptides according to the invention include chloroplast transit peptides (e.g., Van Den Broeck et al., 1985) or the optimized chloroplast transit peptides of U.S. Pat. Nos. 5,510,471 and 5,635,618 which cause transport of the protein into the chloroplast, secretion signal peptides or peptides which target the protein to other plastids, mitochondria, the ER or another organelle.Signal sequences for targeting to intracellular organelles or for secretion outside the plant cell or into the cell wall are found in naturally targeted or secreted proteins, preferably those described by Klosgen et al. (1989), Klosgen and Weil (1991), Neuhaus & Rogers (1998), Bih et al. (1999), Morris et al. (1999), Hesse et al. (1989), Tavladoraki et al. (1998), Terashima et al. (1999), Park et al. (1997), Shcherban et al. (1995).

[0132] In one embodiment, the inventive proteins taught herein are co-expressed, optionally under the control of various promoters, with other proteins that regulate, preferably enhance or induce, parthenogenesis, apomyosis or apomixis in a single host. Such other genes may be, for example, genes for conferring apomyosis, such as diplosporous reproduction, as described in WO 2017 / 039452 A1, which is incorporated herein by reference.

[0133] In another embodiment, the proteins of the invention are introgressed into germplasm that preferably contains other genes of interest, such as genes for conferring apomeiosis (e.g., genes for diplosporic reproduction).Through breeding and selection, hybrids are produced that are capable of stacking several genes of interest.

[0134] Co-expressing host plants can be easily obtained by transforming plants already expressing the protein of the invention or by crossing plants transformed with different nucleic acids of the invention. It is understood that the different proteins can be expressed in the same plant or can be expressed in a single plant and then combined in the same plant by crossing the single plants with each other. For example, in hybrid seed production, each parent plant can express each of the proteins that are desired to be co-expressed. When the parent plants are crossed to produce a hybrid, both proteins are combined in the hybrid plant. Such hybrids or their progeny that contain both genes and / or express both proteins are encompassed by the present invention.

[0135] Preferably, for selection purposes as well as for weed control options, the transgenic plants of the invention are also transformed with DNA encoding a protein that confers resistance to herbicides, for example broad-spectrum herbicides, such as herbicides based on glufosinate ammonium (e.g. Liberty® or BASTA; resistance conferred by the PAT or bar genes; see EP 0242236 and EP 0242246) or glyphosate (e.g. RoundUp®; resistance conferred by the EPSPS gene; see EP 0508909 and EP 0507698) as active ingredient. The use of herbicide resistance genes (or other genes conferring the desired phenotype) as selection markers has the additional advantage that the introduction of antibiotic resistance genes can be avoided.

[0136] Alternatively or additionally, other selectable marker genes such as antibiotic resistance genes may be used. Since it is generally not permitted to carry antibiotic resistance genes in transformed host plants, these genes can be removed again following selection of transformants. Various techniques exist for the removal of the transgene. One way to achieve this is by flanking the transgene with lox sites and, following selection, crossing the transformed plant with a CRE recombinase expressing plant (see, for example, EP506763B1). Site-specific recombination leads to excision of the marker gene. Another site-specific recombination system is the FLP / FRT system described in EP686191 and US Pat. No. 5,527,695. Site-specific recombination systems such as CRE / LOX and FLP / FRT can also be used for gene stacking purposes. Additionally, one-component excision systems have been described, see, for example, WO97 / 37012 or WO95 / 00555.

[0137] Preferably, the nucleic acid of the invention is used to generate transgenic plant cells, plants, plant seeds, etc., and any derivatives / progeny thereof, with enhanced parthenogenesis phenotype. Transgenic plants with enhanced parthenogenesis can be generated by transforming a plant host cell with a nucleic acid of the invention, or a variant and / or fragment thereof, preferably encoding a protein having the amino acid sequence of SEQ ID NO: 1, under the control of a suitable promoter, as described herein, and regenerating a transgenic plant from said cell. Preferably, the transgenic plant of the invention comprises enhanced parthenogenesis compared to a non-transformed or empty vector control. As a result, for example, a transgenic lettuce plant is provided that comprises enhanced parthenogenesis. Thus, a transformed plant expressing a protein according to the invention exhibits enhanced parthenogenesis when it exhibits a marked increase in parthenogenesis compared to a non-transformed or empty vector transformed control. Enhancement of the parthenogenesis phenotype can be fine-tuned by expressing an appropriate amount of a protein of the invention capable of inducing parthenogenesis at an appropriate time and / or location. Such fine tuning may be achieved by determining the most suitable promoter and / or by selecting the transgenic "event" that exhibits the desired expression level.

[0138] Transformants, hybrids or inbreds expressing the desired level of the protein of the invention and / or containing the desired or desired level of the nucleic acid of the invention are selected, for example, by analyzing copy number (Southern blot analysis), mRNA transcription levels (e.g., RT-PCR using primer pairs or flanking primers capable of amplifying the protein of the invention), or by analyzing the presence and levels of the unitary reproductive protein in various tissues (e.g., SDS-PAGE; ELISA assays, etc.). For regulatory reasons, for example, single copy transformants may be selected, but the sequences flanking the insertion site of the transgene are analyzed, preferably sequenced, to characterize the "event". Transgenic events resulting in high or moderate expression of the protein of the invention are selected for further development until high-performing elite events carrying stable transgenes are obtained.

[0139] The transformants expressing the proteins of the invention and / or containing the nucleic acids of the invention can also contain other transgenes, for example other transgenes that confer disease resistance or resistance to other biotic and / or abiotic stresses or that confer diplosporous reproduction. To obtain such plants with "stacked" transgenes, other transgenes can either be introduced into the transformant, or the transformant can be subsequently transformed with one or more other genes, or alternatively several chimeric genes can be used to transform a plant line or variety. For example, several transgenes can be present on a single vector or on different vectors that are co-transformed.

[0140] In one embodiment, the following genes are combined with the nucleic acid of the invention: known disease resistance genes, particularly genes that confer enhanced resistance to necrotrophic pathogens, virus resistance genes, insect resistance genes, abiotic stress resistance genes (e.g. drought, salt, heat or cold tolerance, etc.), herbicide resistance genes, etc. Thus, the stacked transformants can have a broader range of biotic and / or abiotic stress tolerance, such as pathogen resistance, insect resistance, nematode resistance, salinity, cold stress, heat stress, water stress, etc. Silencing approaches can also be combined with expression approaches in a single plant, for example silencing of a Par allele can be combined with expression of a par allele, or vice versa. Optionally, the nucleic acid of the invention may be used to suppress parthenogenesis, for example by silencing, knocking down or reducing the expression of parthenogenetic genes on one or more Par alleles in a plant or plant cell. This may be done by modifying the coding sequence or one or more regulatory sequences (e.g., promoter sequences) of the Par allele(s) present in said plant or plant cell, or by introducing an RNAi targeting the transcript of the Par allele(s). Thus, the invention also provides a method for reducing or eliminating parthenogenesis in a plant or plant cell, comprising the steps of: a) reducing or eliminating expression of a nucleic acid capable of inducing parthenogenesis and / or functional in parthenogenesis, as defined herein, in one or more plant cells; b) selecting plant cells in which said expression is reduced or absent; c) regenerating a plant from said plant cell. Includes.

[0141] The nucleic acid is preferably a nucleic acid comprising or consisting of any one of SEQ ID NOs: 2 to 5, and variants and / or fragments thereof, and / or a nucleic acid encoding the protein of SEQ ID NO: 1 and / or a variant or fragment thereof.

[0142] Encompassed herein are whole plants, plant parts (e.g., seeds, cells, tissues), and plant products (e.g., fruits) and progeny of any of the transformed plants described herein. The whole plants, plant parts, and plant products and progeny can be identified by the presence of the transgene, for example, by PCR analysis using total genomic DNA as a template and a PCR primer pair specific for the parthenogenetic gene, and / or by using genomic mutation analysis, such as, but not limited to, sequence-based genotyping (SBG) or KeyGene® SNPSelect analysis. Also, "event-specific" PCR diagnostics can be developed, where the PCR primers are based on plant DNA flanking the inserted transgene, see U.S. Patent No. 6,563,026. Similarly, event-specific AFLP or RFLP fingerprints can be developed to identify the transgenic plant or any plant, seed, tissue, or cell derived therefrom.

[0143] It is understood that transgenic plants according to the invention preferably do not exhibit undesirable phenotypes such as reduced yield, increased susceptibility to disease (especially to necrotrophs), or undesirable structural changes (dwarfism, deformation), and that if such phenotypes are present in the primary transformants, they can be removed by conventional methods. Any of the transgenic plants described herein may be heterozygous, homozygous, or hemizygous for the transgene.

[0144] The present invention also relates to plants, seeds, plant parts (e.g. plant cells) and plant products, preferably comprising a protein of the invention, a nucleic acid of the invention and / or a construct of the invention, obtained or obtainable by the methods detailed herein. Preferably, said protein, nucleic acid and / or construct is capable of inducing parthenogenesis and / or is functional in parthenogenesis, as detailed herein. The plants of the invention are preferably of the species listed herein as suitable host plants. Such methods include the introgression of a nucleic acid of the invention from a plant to progeny, and / or the transformation of a plant cell with a nucleic acid of the invention as a transgene, and the subsequent regeneration of a plant from said plant cell. Preferably, the plant, plant part and / or plant product is not of the Taraxacum species broadly defined but comprises the nucleic acid of the invention, wherein said plant or plant cell is preferably of a species listed herein as a suitable host plant, preferably from a family selected from the group consisting of Brassicaceae, Cucurbitaceae, Fabaceae, Gramineae, Solanaceae and Asteraceae (Compositae).

[0145] Preferably, the plant, plant part and / or plant product comprises the nucleic acid of the invention by genetic modification or by introgression, wherein preferably said nucleic acid is integrated into the genome of the plant, plant part and / or plant product. Preferably, said plant, plant part and / or plant product is capable of parthenogenesis and / or functional in parthenogenesis. Even more preferably, said plant, plant part and / or plant product is further capable of apomeiosis. The invention provides a seed, plant part or plant product of the plant of the invention, or a plant cell.

[0146] The present invention also relates to plant parts and plant products derived from the plants of the invention, where the plant parts and / or plant products comprise the proteins of the invention as defined herein, the nucleic acids of the invention as defined herein, and / or the constructs of the invention as defined herein, which may be fragments as defined herein, that allow the assessment of the presence of such proteins, nucleic acids or constructs in the plant from which the plant parts of the plant products are derived. Such parts and / or products may be seeds or fruits and / or products derived therefrom (e.g. sugars or proteins). Such parts, products and / or products derived therefrom may be non-growing material.

[0147] Any plant can be a suitable host, but most preferably the host plant species is required to be a plant species that would benefit from increased or reduced parthenogenesis. Suitable hosts include any plant species. In particular, cultivars or breeding lines that have otherwise good agronomic properties are preferred. Those skilled in the art know how to test whether the nucleic acid and / or protein taught herein, and / or their variants or fragments, can confer the required increased or reduced parthenogenesis on the host plant by producing transgenic plants and evaluating parthenogenesis together with suitable control plants.

[0148] Suitable host plants include, for example, hosts belonging to the families Brassicaceae, Cucurbitaceae, Fabaceae, Gramineae, Solanaceae, Asteraceae, Rosaceae, and Poaceae.

[0149] In a preferred embodiment, the host plant may be a plant species selected from the group consisting of Dandelion, Lactuca, Pisum, Capsicum, Solanum, Cucumis, Zea, Gossypium, Glycine, Triticum, Oryza and Sorghum.

[0150] In a preferred embodiment, the plant, plant part, plant cell, or seed taught herein is selected from the group consisting of genus Dandelion, Lactuca, Citrus, Capsicum, Solanum, Cucumber, Maize, Cotton, Glycine, Wheat, Oryza, Allium, Brassica, Helianthus, Beta, Cichorium, Chrysanthemum, Pennisetum, Lamium, Lactuca sativa, Lactic acid ... The plant is derived from a plant species selected from the group consisting of Secale, Hordeum, Medicago, Phaseolus, Rosa, Lilium, Coffea, Linum, Canabis, Cassava, Daucus, Cucurbita, Citrullus, and Sorghum.

[0151] Suitable host plants include, for example, maize / corn (Zea mays spp.), wheat (Triticum spp.), barley (e.g. Hordeum vulgare), oats (e.g. Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp., e.g. Glycine soja), cotton (Gossypium species, e.g. upland cotton, G. barbadense), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B. rapa, etc.), sunflower (Helianthus annus), safflower, yams, cassava, alfalfa (Medicago sativa), rice (Oryza species, e.g., O. sativa indica or japonica cultivars), forage grasses, pearl millet (Pennisetum spp., e.g., P. glaucum),glaucum), tree species (Pinus, poplar, fir, plantain, etc.), tea plant, coffee plant, oil palm, coconut, vegetable species such as pea, zucchini, beans (e.g. Phaseolus spp.), pepper, cucumber, artichoke, asparagus, eggplant, broccoli, garlic, leek, lettuce, onion, radish, turnip, tomato, potato, Brussels sprout, carrot, cauliflower, chicory, celery, spinach, endive, fennel, beet, fruits with flesh (grapes, peaches, pralines, etc.), Fruits and vegetables include, for example, citrus, strawberry, mango, apple, plum, cherry, apricot, banana, blackberry, blueberry, citrus, kiwi, fig, lemon, lime, nectarine, raspberry, watermelon, orange, grapefruit, etc.), ornamental species (e.g., rose, petunia, chrysanthemum, lily, Gerbera species), herbs (mint, parsley, basil, thyme, etc.), woody trees (e.g., species of the genera Populus, Salix, Quercus, Eucalyptus), fiber species such as flax (Linum usitatissimum) and cannabis (Cannabis sativa).

[0152] Marker-assisted selection and introgression or combination of one or more Par alleles The nucleic acids of the invention can be used as genetic markers for marker-assisted selection of Par alleles or par alleles in Dandelion species and / or other plant species, as well as for the introgression of different or identical Par alleles or par alleles and / or combinations thereof into / in plants of interest and / or into plants which can be used to create intra- or inter-specific hybrids with plants in which the Par alleles or par alleles (or variants) are found.

[0153] Based on these sequences, many different marker assays can be developed. The development of marker assays generally involves the identification of polymorphisms between Par and par alleles, such that the polymorphisms are genetic markers that "mark" the particular allele. The polymorphism(s) are then used in the marker assay. For example, the sequences of the Par alleles taught herein are correlated with the presence or enhancement of parthenogenesis. This is done, for example, by screening parthenogenetic and / or non-parthenogenetic plant material for (part of) the nucleotide sequences of the Par or par alleles taught herein to correlate the particular allele with parthenogenesis or non-parthenogenesis. Thus, PCR primers or probes may be created that detect such nucleotide sequences in samples (e.g. RNA, cDNA or genomic DNA samples) obtained from (non-)parthenogenetic plant material. The sequences or parts thereof are compared and polymorphic markers that correlate with parthenogenesis are identified. Polymorphic markers, such as SNP markers linked to Par or par alleles, can then be developed into rapid molecular assays to screen plant material for the presence or absence of parthenogenetic alleles. Thus, the presence or absence of these "genetic markers" indicates the presence of the Par or par allele linked to the marker, and detection of the Par or par allele can be replaced by detection of the genetic markers.

[0154] Preferably, a simple and rapid marker assay is used that allows the rapid detection of a Par allele or a par allele, or a combination of alleles, in a sample (e.g., a DNA sample).Thus, in one embodiment, the use of the nucleic acid of the invention in a molecular assay for determining the presence or absence of a Par allele or a par allele in a sample and / or for determining the homozygosity or heterozygosity of said alleles is provided herein. Such an assay can, for example, include the following steps: (a) providing parthenogenetic and non-parthenogenetic plant material and / or nucleic acid samples thereof; (b) determining the nucleotide sequence of all or part of the nucleic acid of the invention in said material of (a).

[0155] In one aspect, PCR primers and / or probes, molecular markers and kits are provided for detecting the nucleic acid of the present invention or related or derived RNA sequences (such as transcripts).Degenerate or specific PCR primer pairs for amplifying the nucleic acid of the present invention from a sample can be synthesized based on the nucleotide sequences taught herein or their variants, as known in the art (see Dieffenbach and Dveksler, 1995; and McPherson et al., 2000).For example, any stretch of 9, 10, 11, 12, 13, 14, 15, 16, 18 or more consecutive nucleotides of these sequences (or complementary strands) can be used as primers or probes.

[0156] Similarly, DNA fragments containing the sequences of Par alleles or par alleles taught herein, or their complements, can be used as hybridization probes. Detection kits provided herein include either Par(allele-) specific primers and / or Par(allele-) specific probes, and associated protocols, to use said primers or probes to detect the nucleic acids of the invention in a sample. Such detection kits can be used, for example, to determine whether a plant has been transformed with a nucleic acid of the invention, or to screen dandelion germplasm and / or germplasm of other plant species for the presence of Par alleles and, optionally, zygosity determination. Thus, in one embodiment, there is provided a method for detecting the presence or absence of a nucleotide sequence encoding a protein of the invention in a plant tissue, such as a Dandelion tissue, or a nucleic acid sample thereof. a) obtaining a plant tissue sample or a nucleic acid sample thereof from one or more plants; b) analyzing the nucleic acid sample for the presence or absence of one or more markers linked to the Par allele using a molecular marker assay, the marker assay detecting the presence of a nucleic acid of the invention associated with parthenogenesis, and optionally c) selecting plants containing one or more of said markers for further use; may include. Alternatively or additionally, the method of the invention comprises: a) obtaining a plant tissue sample or a nucleic acid sample thereof from one or more plants; b) analyzing the nucleic acid sample for the presence or absence of one or more markers linked to the par allele using a molecular marker assay, the marker assay detecting the presence of the nucleic acid of the invention associated with non-parthenogenesis, and optionally c) selecting plants containing one or more of said markers for further use; may include.

[0157] Preferably, the plant or plants used in any of these methods are suitable as host plants, as further defined herein.

[0158] Applications of parthenogenesis The nucleic acids and / or proteins of the invention may be used for screening (e.g., one or more parthenogenetic loci in a plant or plant cell), genotyping, conferring parthenogenesis, for conferring apomixis to increase ploidy, and / or for the production of doubled haploids. Preferably, said uses are in plant biotechnology and / or breeding, i.e. in / on a plant or plant cell.

[0159] Parthenogenesis is an element of apomixis, and parthenogenesis genes can be used in combination with apomeiosis (e.g. diplosporous) genes to create apomixis, so that apomixis can be preferably used for the applications listed herein. These genes can be introduced into sexual crops by transformation, introgression, or by modifying endogenous appropriate genes, thereby converting parthenogenesis genes into apomeiotic (or diplosporous) genes. Knowledge of the structure and function of apomixis genes can also be used to modify endogenous sexual reproduction genes so that they become apomixis genes. A preferred use would be to place apomixis genes under an inducible promoter so that apomixis can be switched off when sexual reproduction creates new genotypes, and switched on when apomixis is needed to propagate elite genotypes.

[0160] Nucleic acids or derivatives thereof can be used as components of apomixis. Both apomeiosis and parthenogenesis are necessary for apomixis in a functional gametophyte. Apomeiosis can be achieved by a combination of mutations affecting meiosis (Crismani et al., 2013), with the result that chromosome reduction does not occur in the megaspore, i.e. mitosis rather than meiosis. Somatic cells that take on a gametophytic fate through epigenetic changes (Grimanelli, 2012) also result in non-meiotic spore-like cells that may be able to give rise to non-meiotic gametes (egg cells). In another embodiment, apomeiosis is achieved by transgenic or non-transgenic expression of native apomeiosis genes. By whatever means, non-meiotic egg cells are formed, and by appropriate temporal and spatial expression of the nucleic acids of the invention capable of inducing parthenogenesis, egg cells can be induced to behave as zygotes and divide in the absence of fertilization.

[0161] Parthenogenetic genes can be used in entirely new ways, for example, not directly as a tool for apomixis. For example, in apomixis both parthenogenesis and apomeiosis are combined in a single plant, whereas using apomeiosis in one generation and parthenogenesis in the next would combine the sexual gene pool of the crop at the diploid and polyploid levels, with apomeiosis increasing the ploidy level and parthenogenesis decreasing the ploidy level. This is very useful, because polyploid populations can be better for mutation induction, since they can tolerate more mutations. Polyploid plants can also be more robust. However, diploid populations are better for selection, and crossing diploids is better for genetic mapping, building BAC libraries, etc. Parthenogenesis of polyploids can produce uniploids that can cross with diploids. Diploid diplosporogenesis produces non-reduced 2n egg cells that can be fertilized by polyploid-derived pollen to produce polyploid offspring. Thus, the alternation of apomeiosis and parthenogenesis in different breeding generations combines diploid and polyploid gene pools.

[0162] Another use of nucleic acids and their derivatives (transcripts or encoded proteins) without apomeiosis is the production of monad progeny, which can be used for the production of monads and by genome doubling of doubled monads (DH) (e.g. spontaneous genome doubling, colchicine, sodium azide or other chemicals). Doubled monads can be used as parents to produce sexual F1 hybrids. Doubled monads are the fastest way to make plants homozygous. Doubled monads can make plants homozygous, while the second fastest method, self-pollination, takes 5-7 generations to reach significantly high levels of homozygosity in diploid plants. There are several ways to produce doubled monads. In some plant species, monads can be produced by microspore culture. Other methods are the production of monploid embryos (gynogenesis) by pollination with irradiated pollen (melon) or with specific pollinator stocks (corn, potato). These methods have limitations such as cost, genotypic refractoriness, labor intensity, etc. For some crops, methods for monploid production do not exist (e.g. tomato). Dominant alleles of parthenogenetic genes could significantly increase the frequency of gynogenesis and reduce the cost of monploid production.

[0163] The following non-limiting examples illustrate various embodiments of the invention. Unless otherwise stated in the examples, all recombinant DNA techniques are performed according to standard protocols as described in Sambrook et al. (1989), and Sambrook and Russell (2001); and Ausubel et al. (1994), volumes 1 and 2. Standard materials and methods for plant molecular research are described in Plant Molecular Biology Labfax by RD D Croy (1993), co-published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

[0164] [Table 1] TIFF0007674266000002.tif206149 TIFF0007674266000003.tif37149

[0165] [Table 2] EXAMPLES

[0166] Example 1 Materials and Methods plant material The wild-type apomictic triploid Taraxacum officinale A68 and the sexual diploid Taraxacum officinale FCH72.

[0167] DNA constructs A binary vector was constructed with the following components encoded on the T-DNA region; a parsley ubiquitin promoter (SEQ ID NO: 16) driving a Cas9 gene (SEQ ID NO: 17) with a 35S terminator, and a tomato U6 promoter (SEQ ID NO: 18, Nekrasov et al. 2013) driving guide RNA-1 (having a target specific sequence of SEQ ID NO: 19) with a TTTTTT terminator sequence, and a glufosinate resistance gene for selection. A similar binary vector was constructed in which the sequence of guide RNA-1 was replaced by the sequence of guide RNA-2 (having a target specific sequence of SEQ ID NO: 20). Suitable technologies for generating such binary vectors are Gateway®, Golden Gate, or Gibson Assembly® (see, for example, Ma et al., 2015). A vector encoding 35S-GUS on the T-DNA region was used as a control construct.

[0168] Plant transformation methods Agrobacterium transformation was performed according to a modified version of the protocol by Oscarsson (Oscarsson, Lotta. "Production of rubber from dandelion-a proof of concept for a new method of cultivation." 2015). Dandelion A68 explants obtained from subcultured in vitro propagated seed-derived plants grown on half-strength MS20 medium containing 0.8% agar served as starting material for plant transformation. 50 ml of overnight culture in LB medium of Agrobacterium tumefaciens (Rhizobium radiobacter) such as strain C58C1 carrying the binary vector was used at a 10× dilution (resuspended and diluted in liquid MS20) for co-cultivation. Explants were cut to approximately 0.5 cm. 2 The explants were cut into small pieces and co-cultivated for 2-3 days. The explants were then transferred to callus induction medium (CIM; sucrose 20g l-1, MS with micro- and macro-nutrients 4.4g l-1, agar 8g l-1, BAP 1mg l-1, IAA 0.2mg l-1, glufosinate 3mg l-1 for plant selection, vancomycin 100mg l-1 and cefotaxime 100mg l-1, pH 5.8). The explants were transferred to fresh CIM every week. When calli appeared, they were transferred to shoot induction medium (SIM; sucrose 20gl-1, MS with micro- and macro-nutrients 4.4gl-1, agar 8gl-1, zeatin 2mgl-1, IAA 0.1mgl-1, GA3 0.05mgl-1, glufosinate 3mgl-1 for plant selection, vancomycin 100mgl-1 and cefotaxime 100mgl-1, pH 5.8). Finally, the formed shoots, several centimeters in diameter, were rooted on rooting medium (RM; sucrose 20gl-1, MS with micro- and macro-nutrients 2.2gl-1, agar 8gl-1, vancomycin 100mgl-1 and cefotaxime 100mgl-1, pH 5.8). The rooted shoots were transferred to potting soil in the greenhouse.

[0169] result Rooted plants obtained from Agrobacterium transformation were genotyped for the presence of Cas9 and the respective T-DNA encoding guide RNA-1 or guide RNA-2 in the plant genome by PCR. Plants that were positive in this test (herein referred to as transgenic plants) were grown to seed. Individual transgenic plants derived from individual calli containing any one of these constructs had normal viable dark black-gray seeds, and some of these plants had abnormal light gray seeds (see Table 2). These light gray seeds were found to be empty, lacking embryos, found to be non-viable, and did not germinate. Control plants (negative for T-DNA or transformed with 35S-GUS control construct) never had similar abnormal light gray seeds, and all control plants had normal seed heads with fertile black-gray seeds. All transgenic plants were then genotyped by amplicon sequencing of the guide RNA-1 target genomic DNA region on an Illumina MiSeq System. All transgenic plants that showed abnormal light gray seeds were found to have small deletions or small insertions in the parthenogenetic gene, more specifically within the stretch of DNA targeted by gRNA-1. A68 is a triploid plant. The sequences of this gene on the other two alleles were identified and are represented herein by SEQ ID NOs: 10 and 15. The sequences of these two alleles lack the PAM sequence required for Cas9 / guide RNA to induce DSBs.

[0170] All transgenic plants with normal black seeds had no alterations in the sequence of the gene. Table 2 summarizes the small deletions or insertions observed and their effect on the translation of the coding sequence into protein sequence, and Figure 1 shows a multiple alignment of the amplicons.

[0171] The fruit set observed for transgenic plants carrying a small deletion in the gene of SEQ ID NO:5 was interpreted as an indication of loss of the apomictic phenotype (referred to herein as Loss of Apomixis or LoA) and also of loss of the parthenogenetic phenotype (Loss of Parthenogenesis or LoP). Apomictic plants always have a dominant Par allele.

[0172] High seed set in triploid dandelions in the absence of cross-pollination is a clear indication of apomixis. Self-pollination can be excluded as an alternative explanation, since sexually reproduced egg cells and pollen grains would have extremely low fertility due to unbalanced triploid male and female meiosis. Deletion of the Par allele results in LoP and therefore LoA. However, LoA can also be caused by disturbances in other developmental processes. Thus, LoP plants are a subset of LoA plants, and further testing is required to identify the observed phenotype as a LoP deletion phenotype.

[0173] To further investigate the nature of the observed light grey seed phenotype, crosses were performed. LoP in triploid transgenic plants was detected by cross-pollinating triploid transgenic A68 plants with monoploid pollen from sexual FCH72 diploid plants. Seeds of these crosses were collected and sown, and the ploidy levels of the progeny were measured by flow cytometry. Uniformly tetraploid progeny were found, indicating that LoA plants were diplosporous and capable of seed propagation but lacked parthenogenesis.

[0174] As a control, seeds of apomixis triploid A68 plants were sown and were found to be all triploid. At the same sowing, seeds from various plants carrying T-DNA containing guide RNA-1, which showed a light gray phenotype, were also taken, but these seeds never showed germination (Figure 2). Similar germination test results after crossing with FHC72 would be expected for plants carrying T-DNA containing guide RNA-2, which showed a penny phenotype (germination experiments were not performed). In summary, it was concluded that Dandelion A86 has a dominant Par allele with the sequence of SEQ ID NO:5, which is essential for parthenogenesis, and two recessive sexual alleles with the sequences of SEQ ID NO:10 and SEQ ID NO:15, respectively.

[0175] Example 2 Genes essential for parthenogenesis can be used to transfer parthenogenetic traits to plants without apomixis or without parthenogenesis. Either the coding sequence of the gene essential for parthenogenesis or the gene with SEQ ID NO:5 or a homologous gene can be used to transfer such parthenogenetic traits. A binary vector is prepared with a T-DNA carrying at least the gene with SEQ ID NO:5 or a homologous gene, driven by its native promoter or a female gamete-specific promoter. This genetic construct is transformed into a plant without parthenogenesis, such as lettuce or Arabidopsis, by Agrobacterium-mediated transformation. Plants that test positive for the presence of the transgene are evaluated for the occurrence of parthenogenesis. Since the trait is dominant, the test is performed on the primary transformed plants (T0). Parthenogenesis can be detected in non-apomictic plants microscopically by Nomarski Differential Interference Microscopy (DIC) of ovules permeabilized with methyl salicylate (Van Baarlen et al. 2002). In the absence of cross- or self-fertilization, the parthenogenetic egg cell develops into an embryo, at least some of which are found in plants harboring the T-DNA described above.

[0176] plant material In this experiment, wild-type lettuce: Iceberg type, Legacy, Takii Seed Co., Ltd. and Red Romaine type, Baker Creek Heirloom Seeds were used.

[0177] DNA constructs A binary vector was constructed with the following components encoded on the T-DNA region: the Arabidopsis EC1.1 promoter (as in Sprunk et al. 2012) driving the expression of the Taraxacum officinale Par allele CDS sequence (SEQ ID NO: 3), followed by the first 250 bases of the 3'UTR (the first 250 bases of SEQ ID NO: 4), followed by the 35S terminator and the neomycin phosphotransferase gene (nptII) for selection. Suitable technologies for generating such binary vectors are Gateway®, Golden Gate, or Gibson Assembly® (see, for example, Ma et al. 2015). The transgenic line harboring this T-DNA was numbered with the code pKG10824.

[0178] Plant transformation methods Agrobacterium transformation was performed by genotype-independent transformation of lettuce using Agrobacterium tumefaciens. Such methods are well known in the art and are taught, for example, in Curtis et al. Any other method suitable for genetic transformation of lettuce, such as those described in Michelmore et al. (1987) or Chupeau et al. (1989), may be used to generate plants harboring the desired T-DNA.

[0179] result Plants that tested positive for the presence of the transgene were evaluated for the occurrence of parthenogenesis as described under the "DNA construct" section above. Testing was performed on primary transformed plants (T0) since the trait is dominant. In the absence of cross- or self-fertilization, parthenogenetic egg cells develop into embryos. To prevent fertilization of any plants harboring the transgene, plants were grown in a greenhouse and all flowers were manually emasculated prior to microscopic observation. Emasculation was performed by clipping the involucre before the corolla developed. Parthenogenesis can be detected in non-apomictic plants microscopically by Nomarski Differential Interference Microscopy (DIC) of permeabilized ovules. Here, a permeabilization method using chloral hydrate was applied, which is a method commonly used to permeabilize plant ovules for microscopic imaging (e.g. Franks et al. 2016). 75 hours after emasculation, the flower buds were harvested and the ovules were permeabilized with chloral hydrate. In all seven transgenic lines evaluated, multiple embryos were observed in these permeabilized ovules (see Table 3, which shows data for five of these lines). Figure 3 shows examples of such observed embryos. In some single ovules, multiple embryos were observed (polyembryony). Figure 4 shows examples of observed polyembryony. However, polyembryony was observed much less frequently than single embryos. In non-emasculated transgenic lines, embryos could already be observed before the completion of male gametogenesis, and therefore before fertilization. Also, in some rare cases, polyembryony was observed in these non-emasculated transgenic plants. In non-transformed control plants, which were emasculated and imaged in the same way, no embryos were observed at all.

[0180] [Table 3]

[0181] These results demonstrate that the gene of the Par allele of Dandelion is sufficient by itself to induce embryogenesis in lettuce. This is a clear example of the gene of the Par allele of Dandelion inducing parthenogenesis in lettuce, as an egg cell develops into an embryo in the absence of cross- or self-fertilization. Similar results are expected when the lettuce homolog (SEQ ID NO: 22) is used to transform plants in the same way, for example when transforming the lettuce plant with a vector containing a T-DNA region containing the Arabidopsis EC1.1 promoter (as in Sprunk et al. 2012) driving the expression of a sequence encoding the lettuce homolog (SEQ ID NO: 22), together with a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection.

[0182] Example 3 The gene of SEQ ID NO:5 has homologs in parthenogenetic and non-parthenogenetic plant species. All such sequences, including 5' and 3' regulatory sequences, were compared by multiple alignment and variant calling to determine which differences are exclusively represented in the parthenogenetic plant species version of the gene of SEQ ID NO:5.

[0183] The inventors have identified a 1335 bp miniature inverted repeat transposable element (MITE) sequence or MITE-like (as defined herein by SEQ ID NO: 60) in the promoter sequence of the Par allele (SEQ ID NO: 2) at a distance of 102 bp upstream (3') of the start codon, which is absent in the sexual counterpart (SEQ ID NOs: 7 and 12). This MITE or MITE-like sequence is expected to direct and may be responsible for the parthenogenetic phenotype, for example by being responsible for altered expression levels of the encoded protein.

[0184] These specific polymorphisms, insertions or deletions of parthenogenetic alleles can be introduced into non-parthenogenetic plants by chemical mutagenesis or targeted gene editing of sexual alleles homologous to the parthenogenetic genes of the present invention. For example, the promoter sequence of the PAR gene can be replaced by the promoter of the Taraxacum allele, i.e. SEQ ID NO: 2, and a MITE sequence can be introduced into the PAR gene of a non-parthenogenetic plant at a position homologous to the MITE sequence of the Taraxacum Par allele, as shown above. The introduction of these specific polymorphisms, insertions or deletions of parthenogenetic alleles results in the plant acquiring parthenogenetic traits. Parthenogenesis can be detected microscopically in non-parthenogenetic plants by Nomarski Differential Interference Microscopy (DIC) of ovules permeabilized with methyl salicylate (Van Baarlen et al. 2002). In the absence of cross- or self-fertilization, parthenogenetic egg cells develop into embryos. At least some of such embryos are found in plants harboring the specific polymorphism, insertion or deletion described above.

[0185] Example 4 Triploid and tetraploid Taraxacum apomicts were crossed with diploid Taraxacum koksaghyz plants as pollen donors. The pollen donors themselves were obtained by crossing sexual Taraxacum koksaghyz with apomictic Taraxacum brevicorniculatum pollen donors. Thus, the apomixis genes were derived from Taraxacum brevicorniculatum (Kirschner et al. 2012). Triploid progeny plants were tested for the presence of Par and diplosporous (Dip) alleles (see WO 2017 / 039452 A1) using PCR markers and for apomictic seed production. Apomictic seed set was defined as the production of viable seeds on triploid plants without cross-pollination.

[0186] Primers DIP_F (SEQ ID NO: 33) and DIP_R (SEQ ID NO: 34) were designed on the diplosporic gene VPS13 to specifically amplify the Dip allele. Using these primers, the presence of the Dip allele resulted in a PCR product of 829 bp, whereas the absence of this allele resulted in no PCR product.

[0187] Primers PAR_F (SEQ ID NO: 35) and PAR_R (SEQ ID NO: 36) were designed on SEQ ID NO: 2 and SEQ ID NO: 4 to amplify any one of the Par, par1, and par2 alleles. As shown in Table 4, the presence of Par alleles could be distinguished by the length of the PCR products.

[0188] [Table 4]

[0189] As shown in Table 5 reported herein below, 56 progeny plants were tested and a 100% correlation was observed between the presence of the Par allele and parthenogenesis. No plants were observed that produced apomictic seeds and were negative for DIP and PAR markers.

[0190] [Table 5]

[0191] It can therefore be concluded that markers developed from the Par locus in Dandelion also identified the presence of parthenogenesis in a different species, Dandelion rubber, which is further evidence that the Par alleles cause parthenogenesis.

[0192] Example 5 Construction of a gamma-irradiated deletion population of apomictic A68 Approximately 3 x 2000 seeds from clone A68 were gamma irradiated with three different doses: one third at 250 Gy, one third at 300 Gy, and one third at 400 Gy. A total of 3075 plants from the irradiated seeds were grown in pots in a greenhouse. After a vernalization period of 2 months at <10°C, the plants were grown again in a heated greenhouse. More than 90% of the plants flowered and produced seeds. Plants were classified as showing or not showing a loss of apomixis phenotype (LoA). Apomixis A68 plants spontaneously produced seeds and formed large white seedheads with a dark brown center, where the seeds (achene: a fruit with one seed) were attached to the receptacle. In the loss of apomixis phenotype, the seedheads had a lighter center and often the seedheads were reduced in diameter. Finally, 102 plants were identified as having a loss-of-apomixis phenotype.

[0193] Single-dose dominant markers can be mapped in autopolyploid plants using the method of Wu et al. (1992). To find AFLP markers (Vos et al. 1995) linked to the Par locus, a segregation analysis approach was used (Michelmore et al. 1991). Two contrasting DNA pools were constructed, pool A with DNA from 10 triploid PAR plants and pool B with DNA from 10 triploid non-PAR plants, all progeny from a cross TJX3-20 (diploid sexual) × A68. Non-Par plants were carefully phenotyped for the absence of parthenogenesis using a Nomarski DIC microscope (Van Baarlen et al. 2002). For the Par pool, apomictic plants were used. 147 AFLP primer combinations (Vos et al. 1995) were screened for the presence of the fragment in pool A and for the absence of the fragment in pool B. The contrasting fragments in both pools were verified in individuals from both pools. Seventeen AFLP markers were used to construct a genetic map of the Par-locus chromosomal region based on the TJX3-20 × A68 cross (76 plants). Fourteen of the 17 AFLP markers strictly cosegregated with the Par phenotype, an indication of suppression of recombination near the Par locus.

[0194] Partial deletion of one of the three homologous chromosomes results in the loss of the single dose AFLP marker located in the deletion region. AFLP analysis of LoA plants showed that some LoA plants had lost one or more AFLP markers genetically linked to the Par locus. After crossing with diploid pollen donors, LoA plants that lacked Par-linked AFLP markers produced tetraploid progeny. This indicated that these LoA plants had lost the apomixis phenotype, but were still diplosporous and produced non-meiotic egg cells. These LoA plants could be ranked based on the number of Par genetically linked AFLP markers that they lost. The number of lost AFLP markers is an indicator of the size of the deletion. The AFLP markers that were most frequently lost in LoA plants were considered to be closest to the Par locus. Plant i34 had lost the fewest PAR-linked AFLP markers and was therefore considered to have the smallest deletion.

[0195] Example 6 Genotype- and allele-specific expression of Par genes in megagametophytes in apomictic plants versus Par-deficient and sexual plants Cells and tissues from different developmental stages of the gametophyte were isolated by laser-assisted microdissection (LAM) using the SL μCut instrument (2001, Medical Micro Instruments, Glattbrugg, Switzerland), which uses a solid-state UV-A laser (wavelength approximately 350 nm) to cut the tissue, as described by Wuest et al. (2010) and Florez-Rueda et al. (2020). Transcriptome analysis was then performed. RNA was extracted using the PicoPure™ RNA Isolation Kit according to the manufacturer's instructions (Thermo Fisher Scientific). After reverse transcription to DNA, to maintain the initial expression differences between samples, mRNA was linearly amplified using the CEL-seq and CEL-seq2 protocols, as described by Hashimshony et al. (2012) and Hashimshony et al. (2016).

[0196] Three plant lines were compared: 1. the triploid apomict A68 (abbreviation: APO) originating from the Netherlands, 2. the tetraploid PAR deletion progeny (abbreviation: DEL) from a cross between the triploid deletion line i34 (a PAR deletion line derived from A68, see Example 5 above) and the diploid pollen donor FCH72, and 3. the diploid sexual plant FCH72 (abbreviation: SEX) originating from France.

[0197] Five different developmental stages / tissue types were sampled per plant line (Table 6). For very young stages, single samples were analyzed. From mature embryo sacs, the central cell and oocyte apparatus (egg and synergid cells) were sampled in triplicate. Together, these represent nine samples per plant line (Table 6).

[0198] [Table 6]

[0199] Linear amplified DNA was sequenced on an Illumina Hiseq platform. Individual reads were mapped to sequences of Par genes (Figure 5). Expression of Par genes was not detected in either PAR deletion or SEX plants (all stages and tissues). In APO lines, Par gene-specific reads were found in all samples of mature gametophytes, both in the egg cell apparatus and in the central cell. Some transcriptional reads were also detected in one of the younger developmental stages of the apomict. Due to the 3'-end amplification bias of this method, most reads were mapped to the 3'-end of the coding sequence and the 3'-UTR of the gene.

[0200] Thus, the Par gene is expressed in seven samples of apomicts but not in seven samples of deletion or sexual lines at comparable developmental states, further emphasizing that ectopic expression of genes in the central cell and egg apparatus is responsible for the loss of egg arrest and, consequently, parthenogenetic development of the embryo.

[0201] As shown in Example 3, the expression of the Par genes in the apomicts of these cells cannot be repressed as in the sexual case, possibly due to the influence of MITE sequences in the promoter region, which may physically interfere with the binding of transcription factors to the Par genes.

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Claims

1. A nucleic acid associated with parthenogenesis in plants, a) a gene encoding a protein having the amino acid sequence of SEQ ID NO:1; b) a coding sequence of the gene of a), said coding sequence having the nucleotide sequence of SEQ ID NO:3; and c) the gene according to a), which has the nucleotide sequence of SEQ ID NO:5; A nucleic acid comprising at least one of:

2. The nucleic acid of claim 1, which contributes to a parthenogenetic phenotype.

3. 3. The nucleic acid of claim 1 or 2, comprised in a chimeric gene, a gene construct or a nucleic acid vector.

4. A protein associated with parthenogenesis in plants, a) encoded by a nucleic acid according to claim 1; and / or b) A protein having the amino acid sequence of SEQ ID NO:

1.

5. The protein of claim 4 which contributes to a parthenogenetic phenotype.

6. 10. A plant or plant cell comprising a nucleic acid according to claim 1 and / or a protein according to claim 4, said plant or plant cell being not of the species Taraxacum officinale in the broad sense.

7. 7. The plant or plant cell of claim 6, which is from a family selected from the group consisting of Brassicaceae, Cucurbitaceae, Fabaceae, Gramineae, Solanaceae, Asteraceae (Compositae), Rosaceae and Poaceae.

8. 7. A plant or plant cell as claimed in claim 6, comprising a nucleic acid as claimed in claim 1 by genetic modification or by gene transfer.

9. The plant or plant cell of claim 8, wherein the nucleic acid is integrated into the genome of the plant or plant cell.

10. The plant or plant cell according to any one of claims 6 to 9, which is capable of parthenogenesis.

11. A plant or plant cell according to any one of claims 6 to 10, further capable of apomeiosis.

12. 12. A plant or plant cell according to claim 11, which is capable of apomixis.

13. A seed, plant part or fruit of a plant or plant cell according to any one of claims 6 to 12.

14. i) introducing a nucleic acid into one or more plant cells to obtain a parthenogenetic gene, said nucleic acid comprising a) a gene encoding a protein having the amino acid sequence of SEQ ID NO:1; b) a promoter having the nucleotide sequence of SEQ ID NO:2; c) a coding sequence having the nucleotide sequence of SEQ ID NO:3; and d) the gene according to a), which has the nucleotide sequence of SEQ ID NO:5; and at least one of the steps of: ii) selecting a plant cell containing said nucleic acid; iii) regenerating a plant from said plant cell, said regenerated plant being a parthenogenetic plant; A method for producing a parthenogenetic plant, comprising:

15. The method of claim 14, wherein in step iii) the nucleic acid is integrated into the genome of the selected plant cell.

16. A method for producing an apomictic plant, comprising steps i) to iii) according to claim 14 or 15, wherein the one or more plant cells of step i) are capable of apomicsis.

17. a) cross-fertilizing a sexually reproduced first plant with pollen from a second plant to produce F1 hybrid seeds, said second plant comprising the nucleic acid of claim 1, said first plant and / or said second plant being capable of apomeiosis. A method for producing apomictic F1 hybrid seeds, comprising:

18. b) selecting from said F1 seeds containing an apomictic phenotype 20. The method of claim 17, further comprising:

19. The method of claim 18, wherein said selecting is performed by genotyping.

20. The method comprises the steps according to any one of claims 17 to 19, c) growing at least one F1 plant from said F1 hybrid seed. The method of producing an apomictic hybrid plant further comprises:

21. A plant, seed, plant part or fruit obtainable by the method according to any one of claims 14 to 20.

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