Gene for parthenogenesis

Abstract

To provide a procedure for inducing apomixis (also called agamospermy) in crops, and a method for producing an apomictic plant and an apomictic seed, and also provide a gene and a protein involved in the process of apomixis, particularly in parthenogenesis.SOLUTION: The invention provides nucleotide sequences and amino acid sequences of the parthenogenesis gene of Taraxacum as well as (functional) homologues, fragments and variants thereof, which provide parthenogenesis as a part of apomixis. Also parthenogenetic plants and methods for making these are provided, as are molecular markers and methods of using these.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly 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 haploid induction. The present invention particularly relates to genes associated with apomixis, as well as the proteins encoded thereby and fragments of both. The present invention relates to methods for suppressing and / or inducing apomixis in plants and crops, particularly in combination with apomixis genes (plural), for the production of apomictic plants capable of doubling their chromosomes to produce doubled haploids, and further relates to the use of genes and / or proteins or fragments thereof.

Background Art

[0002] Apomixis (also called agamospermy) is asexual plant reproduction through seeds. Apomixis has been reported in approximately 400 angiosperm species (Bicknell and Koltunow, 2004). Apomixis in angiosperms occurs in two forms: (1) Gametophytic apomixis, in which the embryo develops from an unreduced unfertilized egg cell by parthenogenesis; (2) Sporophytic apomixis, in which the embryo develops somaticly from a sporophyte cell. Examples of gametophytic apomicts are dandelion (Taraxacum sp.), hawkweed (Hieracium sp.), Kentucky bluegrass (Poa pratensis), and eastern gamagrass (Tripsacum dactyloides). Examples of sporophytic apomixis are citrus (Citrus sp.) and mangosteen (Garcinia mangostana). Gametophytic apomixis involves two developmental processes: (1) Meiotic recombination and avoidance of meiosis (apomeiosis); and (2) Development of egg cells into embryos without fertilization (apomixis) is involved.

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

[0004] F1 fixation by apomixis is a special case of the general property of apomixis, whereby any genotype, regardless of its genetic complexity, gives rise to pure lines in one step. What this means is that it is possible to immediately fix polygenic quantitative traits using apomixis. Note that most yield traits are polygenic. Apomixis can be used for the stacking (or pyramiding) of multiple traits (e.g., various resistances, several transgenes, or multiple quantitative trait loci). Without using apomixis, in order to fix such a series of traits, the loci of each trait need to be made homozygous individually and then combined. As the number of loci involved in a trait increases, making these trait loci homozygous by crossing becomes time-consuming, a difficult task in terms of conducting business, and thereby costly. Furthermore, certain epistatic interactions between alleles are lost by homozygosity. Using apomixis makes it possible to fix this type of non-additive genetic variation. Thus, apomixis, clonal propagation through seeds, 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 immediately fixing any genotype, there are further important agricultural uses of apomixis, whatever its complexity. Sexual interspecific hybrids and autopolyploids often suffer from sterility due to meiotic problems. Since apomixis skips meiosis, using apomixis can solve such sterility problems related to interspecific hybrids and autopolyploids. Since apomixis prevents female hybridization, apomixis combined with male sterility has been proposed for the containment of transgenes in wild relatives of transgenic crops to prevent the introgression of transgenes (Daniell, 2002). In insect-pollinated crops (e.g., Brassica), apomictic seed set should not be limited by insufficient pollinator service. This is becoming more important in light of the increasing health problems in the population of pollinating bees (Varroa mite infestation, African killer bees, etc.). In tuber-propagating crops such as potato, apomixis maintains a clonally superior genotype and also seems to reduce or eliminate the current risk of virus transmission and related 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 may replace labor-intensive and costly tissue culture propagation. In general, apomixis is thought to strongly reduce the costs of cultivar development and plant propagation.

[0006] Unfortunately, apomixis does not exist in any of the major crops. There have been numerous attempts to introduce apomixis into sexual crops. Examples include the gene transfer of apomixis genes, the mutation of sexual model species, the de novo production of apomixis by hybridization, and the cloning of candidate genes. The gene transfer of apomixis genes from wild apomicts to crop species by wide cross has not been successful so far (for example, apomixis from Tripsacum dactyloides to maize - Savidan, Y., 2001; Morgan et al., 1998; International Publication No. 97 / 10704). Regarding mutant sexual model species, International Publication No. 2007 / 066214 describes the use of an apomiotic mutant called Dyad in Arabidopsis. However, Dyad is a recessive mutation with a very low penetrance. In crop species, the use of this mutation is limited. The de novo production of apomixis by hybridization between two sexual ecotypes has not resulted in agriculturally interesting apomicts (U.S. Patent Application Publication No. 2004 / 0168216A1 and U.S. Patent Application Publication No. 2005 / 0155111A1). The cloning of candidate apomixis genes by transposon tagging in maize is described in U.S. Patent Application Publication No. 2004 / 0148667. Orthologs of the elongator gene, which is hypothesized to induce apomixis, are claimed. However, according to Barrell and Grossniklaus (2005), the elongator gene skips meiosis II and thus does not maintain the maternal genotype, which makes the elongator gene of considerably low utility.

[0007] U.S. Patent Application Publication No. 2006 / 0179498 describes that so-called reverse breeding seems to be an alternative to apomixis. However, reverse breeding is a technically complex in vitro laboratory procedure, while apomixis is an in vivo procedure performed by the plant itself. Furthermore, when using reverse breeding, once the parental lines are reconstructed (doubled gamete homozygotes), it is still necessary to perform mating.

[0008] Apomixis in natural apomicts generally has a genetic basis (reviewed by Ozias-Akins and Van Dijk, 2007). Thus, an alternative approach could be the isolation of apomixis genes from natural apomictic species. However, this is not an easy task, as natural apomicts often have polyploid genomes, and positional cloning in polyploids is extremely difficult. Other complicating factors are suppression of recombination in chromosomal regions specific to apomixis, repetitive sequences, and segregation distortion in crosses. SUMMARY OF THE INVENTION

[0009] As described herein, there is a need for procedures for inducing apomixis in crops that are not subject to at least some of the limitations of the current state of the art. In particular, there is a need for methods for producing apomictic plants and apomictic seeds. There is also a need to provide genes and proteins involved in the process of apomixis, particularly parthenogenesis, which are suitable for use in introducing apomixis into crops and can substantially mimic the apomictic pathway.

[0010] In this study, the inventors have identified and isolated the parthenogenesis locus and genes, alleles related to parthenogenesis phenotypes (designated herein as parthenogenesis alleles or Par alleles) and non-parthenogenesis phenotypes (designated herein as sexual or non-parthenogenesis alleles or par alleles), their gene sequences, i.e., promoter or 5'UTR sequences, coding sequences, 3'UTR sequences, and the encoded protein sequences. Parthenogenesis can be directly introduced into sexual plants, presumably by random or targeted mutagenesis, by transformation, or by somatic hybridization. By genetically modifying the sexual alleles of the parthenogenesis locus of sexual plants, for example, by mutagenesis, gene introduction, or introduction of double-strand breaks at specific sites and insertion via homologous recombination, Par alleles may be introduced, and plants and / or their progeny may be capable of developing egg cells into embryos.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] Definition As used herein, the term "locus" (plural: loci) means, for example, a particular one (or more) location or site on a chromosome where a gene or genetic marker is found. For example, the "apomixis locus" refers to the position in the genome where the apomixis gene is located, the allele contributing to the apomictic phenotype, i.e., (the apomictic allele or Par allele) and / or its sexual counterpart(s), i.e., the non-apomictic gene(s) (non-apomictic allele(s) or par allele(s)). A gene, allele, protein or nucleic acid that is "functional in apomixis" is understood herein to be one that contributes to the apomictic phenotype and / or one that converts an egg cell into a plant or plant cell capable of developing into an embryo.

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

[0014] As used herein, the term "dominant allele" refers to the relationship between alleles of a single gene in which the effect of one allele (i.e., the dominant allele) masks the contribution of a second allele (i.e., the recessive allele) at the same locus. For genes on autosomes (any chromosome other than a sex chromosome), the allelic and associated traits are autosomal dominant or autosomal recessive. Dominance is an important concept in Mendelian and classical genetics. For example, a dominant allele can encode a functional protein, while a recessive allele cannot. In one embodiment, the genes and their fragments or variants taught herein refer to the dominant alleles of apomictic genes.

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

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

[0017] The term "gene 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 gene marker is closely linked to a gene or is "on" the gene, the gene marker "marks" the DNA where the gene is found, and thus the gene marker can be used in a (molecular) marker assay to select for the presence of the gene or against the presence of the gene, for example in marker-assisted breeding / selection (MAS) methods. Examples of gene markers are AFLP (amplified fragment length polymorphism, European Patent No. 534858), microsatellite, 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 marker (cleaved amplified polymorphic sequence), etc. The further a marker is from a gene, the more likely recombination (crossing over) will occur between the marker and the gene, thereby increasing the likelihood of losing linkage (and co-segregation of the marker and the gene). The distance between loci is measured in terms of recombination frequency and is given in cM units (centimorgan; 1 cM is the meiotic recombination frequency between two markers of 1%). Since the genome size varies greatly between species, the actual physical distance represented by 1 cM (i.e., the kilobases, kb, between two markers) also varies greatly between species.

[0018] When referring to "linked" markers in this specification, it is understood that this also encompasses markers "on" the gene itself.

[0019] "MAS" refers to "marker-assisted selection", by which plants are screened for the presence and / or absence of one or more genetic markers and / or phenotypic markers in order to accelerate the transfer of DNA regions containing the markers (and optionally lacking adjacent regions) into (elite) breeding lines.

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

[0021] As used herein, the term "heterozygous" means a genetic state that exists when two different alleles are present at a particular locus but are individually arranged on the corresponding sets of homologous chromosomes in a cell. Conversely, as used herein, the term "homozygous" means a genetic state that exists when two identical (or more than two in the case of polyploids) alleles are present at a particular locus but are individually arranged on the corresponding sets of homologous chromosomes in a cell.

[0022] "Variety" as used herein in accordance with the UPOV Convention refers to a grouping of plants within the lowest known rank of a single plant taxon, which grouping can be defined by the expression of characteristics, and can be distinguished from any other grouping of plants by the expression of at least one of said characteristics, and is considered to be a unit with respect to the suitability for propagation unchanged (stable).

[0023] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule composed of an amino acid chain, 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". The term "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example, in vitro or in recombinant bacterial cells or plant host cells.

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

[0025] A "chimeric gene" (or recombinant gene) refers to any gene that is not normally found in nature in a species, particularly a gene in which one or more portions of nucleotide sequences that do not naturally associate with each other are present. For example, a promoter is not naturally associated with a part or all of the transcribed region or another regulatory region. The term "chimeric gene" is understood to include an expression construct 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 double-stranded RNA during transcription).

[0026] "3' UTR" or "3' untranslated sequence" (often also called the 3' untranslated region or 3' end) refers to the nucleotide sequence found downstream of the coding sequence of a gene, which includes, for example, a transcription termination site and (in most but not all eukaryotic mRNAs) a polyadenylation signal (such as AAUAAA or its variants, etc.). After transcription termination, the mRNA transcript may be cleaved downstream of the polyadenylation signal, and a polyA tail that is involved in the transport of the mRNA to the cytoplasm (where translation occurs) may be added.

[0027] "5' UTR" or "leader sequence" or "5' untranslated region" refers to the region of the 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 that are important for translation, mRNA stability and / or turnover, and other regulatory elements.

[0028] "Gene expression" refers to the process by which a DNA region operably linked to a suitable regulatory region, particularly a promoter, is transcribed into RNA that is biologically active, i.e., translatable into a biologically active protein or peptide (or an active peptide fragment), or is itself active (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 the sense orientation and encodes a desired biologically active protein or peptide, or an active peptide fragment. In a gene silencing approach, the DNA sequence preferably exists in the form of antisense DNA or inverted repeat DNA and contains a short sequence of the target gene in the antisense or in both the sense and antisense orientations.

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

[0030] As used herein, the term "promoter" refers to a nucleic acid fragment that is located upstream of the transcription start site of a gene in the direction of transcription and functions to control the transcription of one or more genes, and includes, but is not limited to, transcription factor binding sites, protein binding sites for repressors and activators, and any other arbitrary sequences of nucleotides known to those skilled in the art that act directly or indirectly to regulate the amount of transcription from the promoter, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other arbitrary DNA sequence. Optionally, herein the term "promoter" also includes the 5'UTR region (for example, the promoter can include one or more portions upstream (5') of the translation start codon of the gene herein), 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 regulated physiologically (e.g., by the external application of a particular compound) or developmentally. A "tissue-specific" promoter is active only in a particular type of tissue or cell. A "promoter active in a plant or plant cell" refers to the general ability of a promoter to drive transcription within a plant or plant cell. This does not imply anything about the spatio-temporal activity of the promoter.

[0031] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. Nucleic acids are "operably linked" when placed in a functional relationship with another nucleotide sequence. By way of example, a promoter, or rather a transcriptional regulatory sequence, is operably linked to a coding sequence when it affects the transcription of the coding sequence. Being operably linked means that the DNA sequences being linked are usually adjacent, and optionally, connect two protein-coding regions that are adjacent and in the same 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 connected to form a functional protein but are not found in nature in such a form, and this exhibits the functionality of the connected domains. A chimeric protein can also be a fusion protein of two or more naturally occurring proteins. As used herein, the term "domain" means any part(s) or domain(s) of a protein having a specific structure or function that can be transferred to another protein to provide a novel hybrid protein having at least the functional characteristics of the domain.

[0032] The term "targeting peptide" refers to an amino acid sequence that directs a protein or protein fragment into an intracellular organelle that is a target, such as a plastid, preferably a chloroplast, mitochondrion, etc., or into 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] As used herein, the term "nucleic acid construct" or "vector" is understood to mean an artificial nucleic acid molecule obtained from the use of recombinant DNA technology and used for the delivery of foreign DNA into a host cell. The vector backbone can be, for example, a binary or super-binary vector (see, for example, U.S. Patent No. 5,591,616, U.S. Patent Application Publication No. 2002 / 138879, and International Publication No. 95 / 06722), a co-integrate 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., a coding sequence, an antisense sequence or an inverted repeat sequence) is integrated downstream of the transcriptional regulatory sequences. A vector usually further contains genetic elements that facilitate the use of the vector in molecular cloning, such as, for example, a selectable marker, a multiple cloning site, etc.

[0034] The term "recombinant host cell" or "transformed cell" or "transgenic cell" refers specifically to a new individual cell (or organism) resulting from the introduction into said cell of at least one nucleic acid molecule containing a gene or chimeric gene encoding a desired protein, or a nucleotide sequence that gives rise to an antisense RNA or an inverted repeat RNA (or hairpin RNA) that silences a target gene / gene family when transcribed. The term "isolated nucleic acid" is used to refer to a nucleic acid that is no longer in its natural environment, for example, in vitro or in a recombinant bacterial cell or a plant host cell.

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

[0036] The term "recombinant plant" or "recombinant plant part" or "transgenic plant" refers to a plant or plant part (e.g., seed or fruit or leaf) that contains a recombinant gene 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 selected to contain a recombinant gene at a position in the genome that confers good phenotypic and / or agronomic characteristics in the plant. Sequencing of the adjacent DNA at the integration site can be used to characterize the integration site and to distinguish the event from other transgenic plants containing the same recombinant gene at other positions in the genome.

[0038] The term "selectable marker" is 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 one or more cells containing the selectable marker. The selectable 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 auxotrophy. The term "reporter" is used primarily to refer to visible markers such as green fluorescent protein (GFP), eGFP, luciferase, GUS, etc.

[0039] The term "ortholog" of a gene or protein refers herein to a homologous gene or protein found in another species that has the same function as the gene or protein but that has diverged in sequence (usually) since the time the species containing the gene diverged (i.e., a gene that has evolved from a common ancestor by speciation). Thus, orthologs of the parthenogenesis gene in the genus Taraxacum can be identified in other plant species based on both sequence comparison (e.g., based on the percentage of sequence identity across the entire sequence or across a specific domain) 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 organism, particularly in the context of transgenic organisms. Thus, a homologous sequence is found naturally in the host species (e.g., a lettuce plant transformed with a lettuce gene), while a heterologous sequence is not found naturally 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 sequence from a common ancestor (e.g., they may be orthologs).

[0041] Using "stringent hybridization conditions", nucleotide sequences that are substantially identical to a given nucleotide sequence can be identified. Stringent conditions are sequence-dependent and will vary in different circumstances. In general, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (under defined ionic strength and pH). Typically, stringent conditions will be selected where the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60 °C. Stringency increases with a decrease in salt concentration and / or an increase in temperature. For RNA-DNA hybridization (e.g., Northern blot using a 100 nt probe), stringent conditions include, for example, conditions that include at least one wash in 0.2× SSC at 63 °C for 20 minutes, or equivalent conditions. For DNA-DNA hybridization (e.g., Southern blot using a 100 nt probe), stringent conditions include, for example, conditions that include at least one wash (usually two washes) 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] For the "high stringency" condition, it can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6×SSC (20×SSC containing 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0), 5×Denhardt's (100×Denhardt's containing 2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS) and 20 μg / ml denatured carrier DNA (single-stranded salmon sperm DNA having an average length of 120 - 3000 nucleotides) as a non-specific competitor. Following hybridization, high stringency washing may be performed in several steps under the conditions of final washing (for about 30 minutes) at the hybridization temperature in 0.2 - 0.1×SSC, 0.1% SDS.

[0043] "Medium stringency" refers to conditions equivalent to hybridization in the solution described above but at about 60 - 62°C. In that case, the final washing is performed at the hybridization temperature in 1×SSC, 0.1% SDS.

[0044] "Low stringency" refers to conditions equivalent to hybridization in the solution described above but at about 50 - 52°C. In that case, the final washing is performed at the hybridization temperature in 2×SSC, 0.1% SDS. 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 lengths 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). The sequences can then be referred to as "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (as defined herein), when optimally aligned (e.g., by the programs GAP or BESTFIT using default parameters). GAP uses the global alignment algorithm of Needleman and Wunsch 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. Generally, GAP default parameters are used with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and a gap extension penalty = 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).Array alignment and percentage sequence identity scores may be determined using a computer program such as GCG Wisconsin Package, version 10.3, obtainable from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752, USA, or an open source software such as the program "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 open 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 the sequences have substantially different full lengths, local alignment, such as that using the Smith Waterman algorithm, is preferred.

[0046] Alternatively, the percentage of similarity or identity may be determined by searching a public database using an algorithm such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can be further used as a "query sequence" to perform a search against a public database, for example, to 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. The BLAST nucleotide search 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. The BLAST protein search 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 an alignment with gaps for comparison purposes, Gapped BLAST can be utilized as described in Altschul, et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (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 the 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 the embryo sac) containing a meiotic egg cell (i.e., a cell with a reduced chromosome number compared to the mother) and two meiotic polar nuclei. Fertilization of the egg cell by one sperm cell of the pollen grain results in a (e.g., diploid) embryo, while fertilization of the two polar nuclei by the second sperm cell results in a (e.g., triploid) endosperm (a process called double fertilization).

[0048] As used herein, the term "megaspore mother cell" or "megasporocyte" refers to a cell that produces megaspores by meiosis, typically meiosis, to create four haploid megaspores that develop into the female gametophyte. In angiosperms (also known as flowering plants), the megaspore mother cell produces megaspores that develop into the female gametophyte through two different processes including megasporogenesis (formation of megaspores in the nucellus, or megasporangium) and megagametogenesis (development of megaspores into the megagametophyte).

[0049] As used herein, the term "asexual plant reproduction" is a process by which plants reproduce 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 mutations or somatic recombination occur. Plants have two main types of asexual reproduction, including vegetative reproduction (i.e., budding, fragmentation, etc. of vegetative parts of the original plant) and apomixis.

[0050] As used herein, the term "apomixis" refers to seed formation by an asexual process. One form of apomixis is characterized by the following: 1) apomeiosis, which refers to the formation of an unreduced embryo sac in the ovary, and 2) parthenogenesis, which refers to the development of an unreduced 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 unreduced egg cells that have the same chromosome number and the same or highly similar genotype as the somatic tissue of the mother plant. Unreduced egg cells can be derived from unreduced megaspores (diplospory) or from somatic progenitor cells (apospory). In the case of diplospory, megasporogenesis is replaced by mitosis or by a modified meiosis. The modified meiosis is preferably of the first division restitution type without recombination. Alternatively, the modified meiosis may be of the second division restitution type. In a preferred embodiment, apomeiosis is of the diplosporous type that affects the first meiosis. Apomixis occurs in various forms, including at least two forms known as gametophytic apomixis and sporophytic apomixis (also called adventitious embryony). Examples of plants in which gametophytic apomixis occurs include dandelion (species of Taraxacum), hawkweed (species of Hieracium), Kentucky bluegrass (Poa pratensis), eastern gamagrass (Tripsacum dactyloides), and others. Examples of plants in which sporophytic apomixis occurs include citrus (species of Citrus), mangosteen (Garcinia mangostana), and others.

[0051] As used herein, the term "diplospory" refers to the situation where the unreduced embryo sac is directly derived from the megaspore mother cell either by mitosis or by a meiotic event that is interrupted. Three main types of diplospory have been reported, and these types are named after the plants in which they occur, which are the genus Taraxacum, the genus Ixeris, and the genus Antennaria. In the Taraxacum type, meiosis prophase is initiated, but the process is then interrupted, resulting in two unreduced dyad chromosomes, one of which gives rise to the embryo sac by mitosis. In the Ixeris type, two additional mitotic divisions of the nuclei that give rise to the 8-nucleate embryo sac follow an equational division after meiosis prophase. The Taraxacum type and the Ixeris type are known as meiotic diplospory because they involve modifications of meiosis. In contrast, in the Antennaria type, which is called mitotic diplospory, the megaspore mother cell does not initiate meiosis and divides directly three times to produce an unreduced embryo sac. In gametophytic apomixis by diplospory, an unreduced gametophyte is produced from an unreduced megaspore. This unreduced megaspore results from either a mitosis-like division (displory) or a modified meiosis (meiotic modification). In both gametophytic apomixis by apospory and gametophytic apomixis by diplospory, the unreduced egg cell develops parthenogenetically into an embryo. The apomixis of the genus Taraxacum is of the diplosporous type, which means that the first female reduction division (meiosis I) is skipped, resulting in two unreduced megaspores with the same genotype as the mother plant. One of these megaspores degenerates, and the surviving unreduced megaspore gives rise to an unreduced megagametophyte (or embryo sac), which contains an unreduced egg cell. This unreduced egg cell develops into an embryo with the same genotype as the mother plant without fertilization. The seeds resulting from the process of gametophytic apomixis are called apomictic seeds.

[0052] The term "diplospory function" preferably refers to the ability to induce diplospory in plants, preferably within the female ovary, preferably within the megaspore mother cell and / or within the female gamete. Thus, a plant into which the diplospory function has been introduced is capable of performing the diplospory process, i.e., producing apomeiotic gametes via restitution meiosis I.

[0053] The term "diplospory as part of gametophytic apomixis" refers to the diplospory component of the apomixis process, i.e., the role that diplospory plays in seed formation by an asexual process. In particular, following the diplospory function, the parthenogenesis function is likewise necessary for establishing the apomixis process. Thus, the combination of the diplospory function and the parthenogenesis function can result in apomixis.

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

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

[0056] As used herein, the term "apomictic plant(s)" refers to a plant that reproduces asexually by itself 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 a plant that is an apomictic plant or a descendant of an apomictic plant. In that case, the apomictically produced offspring are 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 as well as to the parent plant from which they are derived. The genomic DNA sequences of individual clones are mostly identical, although mutations may cause minor differences.

[0058] As used herein, the term "pure breeding" or "pure breeding organism" (also known as purebred) refers to an organism that always transmits a particular phenotypic trait that is unchanged or almost unchanged to its offspring. An organism is said to be pure breeding for each trait to which the organism applies, and the term "pure breeding" is also used to describe individual genetic traits.

[0059] As used herein, the term "F1 hybrid" (or first filial generation hybrid) refers to the first hybrid generation of offspring of clearly different parent types. The parent types may be inbred lines, but they may not be. F1 hybrids are used in genetics and in selective breeding, in which case they may appear as F1 crosses. Offspring with clearly different parent types produce a new uniform phenotype with a combination of characteristics from both parents. F1 hybrids have distinct advantages such as hybrid vigor and are therefore highly desirable in agricultural practice. In embodiments of the present invention, the genotypes of F1 hybrids can be fixed, regardless of their genetic complexity, using the methods, genes, proteins, their variants or fragments taught herein, thereby enabling the production of organisms that can be purebred in one step.

[0060] As used herein, the term "pollination" or "pollinating" 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, plants that bear flowers. Each pollen grain is a male haploid gametophyte, configured to be transported to the female gametophyte, where the male haploid gametophyte can effect fertilization by producing male gametes (or gametes) in the process of double fertilization. A successful angiosperm pollen grain (gametophyte) containing male gametes is transported to the stigma, where it germinates and its pollen tube grows down the style into the ovary. The two gametes travel down the tube until they reach the gametophyte(s) containing the female gametes, which are held within the carpel. One nucleus fuses with the polar bodies to produce endosperm tissue, and the other nucleus fuses with the ovule to produce an embryo.

[0061] As used herein, the term "apomixis" refers to a form of asexual reproduction in which embryo growth and development occur without fertilization. The genes and proteins of the present invention can, in combination with apomictic sporogenesis factors, such as genes or chemical factors, produce apomictic progeny.

[0062] As used herein, the term "pyramiding gene or stacking gene" refers to the process of combining related or unrelated genes from different parental lines underlying desirable or preferred traits (e.g., disease resistance traits, color, drought tolerance, pest resistance, etc.) into one plant. Pyramiding genes or stacking genes can be carried out using conventional breeding methods or can be accelerated by using molecular markers to identify and maintain plants containing the desired combination of alleles and discard plants that do not have the desired combination of alleles. In one embodiment of the present invention, the apomixis genes taught herein are advantageously used in a gene pyramiding program or stacking program to produce apomictic plants or to introduce apomixis into sexual crops.

[0063] In this specification and the claims thereof, the verb "to comprise" and its inflected forms are used in their non-limiting sense so as to mean that the items following this word are included, without excluding items not particularly recited. In addition, unless the context clearly requires otherwise, the mention of an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present. Thus, the indefinite article "a" or "an" generally means "at least one". When reference is made herein to an "array", it is further understood that this generally refers to an actual physical molecule having a particular array 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 from which plants can be regenerated, plant calli, plant cell aggregates, and intact plant cells or parts of plants in plants, such as embryos, pollen, ovules, fruits, flowers, leaves (e.g., harvested lettuce crops), seeds, roots, root tips, etc. Detailed Description of the Invention

[0065] Nucleotide sequence of the present invention The inventors have for the first time identified 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 allele. The inventors have also identified gene sequences, promoter sequences, coding sequences, and 3'UTR sequences located on the sexual counterpart of the Par allele, i.e., on the par allele. As the sexual counterpart of the dominant allele that causes parthenogenesis, the presence of the par allele does not contribute to the parthenogenetic phenotype, but these par alleles are also indicated herein as being related to parthenogenesis, because the presence of the par allele can result in a sexual phenotype, i.e., a non-parthenogenetic phenotype. Since the Par allele may be a dominant allele, the confirmation of the sexual phenotype may require the evaluation of all alleles at the Par locus as the par allele and / or the evaluation of the absence of the Par allele. In other words, "associated with" is to be understood herein as indicating a parthenogenetic or non-parthenogenetic phenotype and, optionally, as being functional in parthenogenesis. By way of example, it is possible to confer the par allele to a Par allele capable of inducing a parthenogenetic phenotype by modifying one or more expression regulatory sequences of the par allele, such as a promoter sequence that results in a change in the expression of the encoded protein, by modifying the par allele.

[0066] Both the par allele and the par allele include a gene having a coding sequence encoding a protein referred to herein as the "PAR protein", which protein includes 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, although this protein can also be annotated as CXXCXXXXXXX[K / R]AXXGHX[R / N]XH (SEQ ID NO: 37) (wherein 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 the zinc finger C2H2-type domain, preferably the zinc finger K2-2-like domain as defined herein, the protein includes an EAR motif having the consensus amino acid sequence DLNXXP (SEQ ID NO: 58) or DLNXP (SEQ ID NO: 59) (wherein X can be any naturally occurring amino acid) (see Kagale et al., 2010). Preferably, the protein is at most 400 amino acids, where the protein includes one or two EAR motifs as indicated herein and the zinc finger K2-2-like domain as defined herein. Preferably, the protein is at most 400 amino acids, where the protein includes only one or two EAR motifs as indicated herein and only one zinc finger K2-2-like domain as defined herein, i.e., does not include additional EAR motifs as defined herein and additional zinc finger K2-2-like domains as defined herein.In addition to a maximum size of 400 amino acids, only one or two EAR motifs as indicated herein, and the characteristics of a defined single zinc finger K2-2-like domain, the PAR protein can include only one additional zinc finger domain having the zinc finger consensus sequence C.{2}C.{12}H.{3}H, which can be noted as CXXCXXXXXXXXXXXXHXXXH (SEQ ID NO: 38), but more preferably does not include an additional zinc finger domain having the zinc finger consensus sequence C.{2}C.{12}H.{3}H (SEQ ID NO: 38).

[0067] Accordingly, the present invention provides a nucleic acid associated with apomixis in plants, wherein said nucleic acid comprises a nucleotide sequence encoding a PAR protein as defined herein. The present invention also provides a promoter sequence and 3' UTR operably linked to the nucleotide sequence encoding said PAR protein. Taraxacum officinale includes one dominant Par allele capable of inducing apomixis and two sexual counterparts encoding PAR proteins having the amino acid sequences of SEQ ID NOs: 1, 6, or 11, namely, par allele-1 and par allele-2. The Par allele includes a gene having the nucleotide sequence of SEQ ID NO: 5, par allele-1 includes a par gene having the nucleotide sequence of SEQ ID NO: 10, and par allele-2 includes a par gene having the nucleotide sequence of SEQ ID NO: 15. The par gene includes 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. par gene-1 includes 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. par gene-2 includes 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. Accordingly, 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) A variant of any one of a) to e); and g) A fragment of any one of a) to f) to provide a nucleic acid related to apomixis in plants, comprising at least one of the above.

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

[0069] Preferably, the nucleic acid is functional in apomixis. In one embodiment, the nucleic acid of the present 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) A variant of any one of a) to e); and g) A fragment of any one of a) to f) comprises or consists of at least one of the above.

[0070] Preferably, the nucleic acid of the present embodiment and / or a product derived therefrom, such as its RNA transcript or the encoded protein, directs apomixis. For example, a plant containing the nucleic acid indicates that the plant exhibits apomixis, which means that the plant has the ability to develop embryos from meiotic or non-meiotic egg cells. Preferably, the nucleic acid and / or a product derived therefrom, such as its RNA transcript or the encoded protein, is preferably functional in apomixis when present in a plant or plant cell, and more preferably, it is capable of inducing or being able to induce apomixis.

[0071] In another embodiment, the nucleic acid of the present 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) a variant of any one of a) to e); and g) a fragment of any one of a) to f) comprises at least one of or consists of them.

[0072] Preferably, when the nucleic acid of the present embodiment and / or a product derived therefrom, such as its RNA transcript or the encoded protein, is preferably present in a homozygous state in a plant or plant cell, it does not induce or is unable to induce apomixis. In other words, the presence of the nucleic acid of the present embodiment may indicate a non-apomictic phenotype or a sexual phenotype. For example, a plant containing the nucleic acid indicates that the plant has a sexual phenotype, that is, it is impossible to develop embryos from egg cells.

[0073] The Par allele may be a dominant allele. When the Par allele is dominant, in order to confirm that the plant has an apomictic phenotype, all alleles at the Par locus of the plant need to be evaluated as par alleles, and the presence of a single Par allele is sufficient to indicate that the plant is capable of apomixis.

[0074] The nucleic acids of the present invention can be used for screening and / or genotyping. Optionally, the functionality of the putative nucleic acid or gene and / or its derivatives in apomixis, or the ability of the putative nucleic acid and / or its derivatives to induce apomixis, can be evaluated by reducing the expression, by silencing or knocking out the nucleic acid or gene in the apomictic plant, for example by introducing a premature stop into the coding sequence of the gene. The subsequent loss of the apomictic phenotype implies that the putative nucleic acid and / or its derivatives are capable of inducing apomixis. The ability to induce apomixis can also be evaluated 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 may be the sunflower isolate A68 modified to lose the apomictic phenotype by reducing the expression of the functional Par allele (e.g., by deletion or knockout). Such a loss-of-function apomictic plant may be the sunflower isolate A68 containing the Par allele, wherein SEQ ID NO: 23 is modified to any one of SEQ ID NOs: 24-27 (see Table 2). Such a loss of the functional apomictic plant of the sunflower isolate A68 may be obtained by targeted genome editing using a CRISPR-Cas9 / guide RNA complex, wherein, as exemplified herein, the guide RNA (also referred to herein as gRNA) contains the target-specific sequence of SEQ ID NO: 19. Deletion of the Par allele in the sunflower isolate A68 results in a loss of apomixis, and thus a loss of apomixis. If the putative nucleic acid or its derivatives have the ability to induce apomixis, for example, by transfecting the isolate with a vector containing the nucleic acid and / or encoding the derivative, introducing the nucleic acid or derivative into the isolate will result in the restoration (or rescue) of the apomictic phenotype. Such a vector preferably contains sequences suitable for driving the expression of the encoded derivative in the isolate.By way of example, the putative nucleic acid that presumably encodes the PAR protein of the present invention may be operably linked in the vector to the promoter defined herein by SEQ ID NO: 2 and optionally to the 3’UTR defined herein by SEQ ID NO: 4. In the case of the dandelion isolate A68, high seed set in the absence of cross-pollination is a clear indicator of apomixis. Alternative explanations for self-pollination in this isolate can be excluded because, due to unbalanced triploid male and female meiosis, the sexually produced egg cells and pollen grains would have extremely low fertility.

[0075] Preferably, the variant nucleic acid as defined herein is a homolog or ortholog of the Par allele or par allele gene, promoter, coding sequence, and / or 3' UTR of the Lactuca sativa isolate A68 as defined herein. Preferably, the variant nucleic acid and / or the product derived therefrom, such as its RNA transcript or the encoded protein, when preferably present in a plant or plant cell, is related to apomixis as defined herein and can optionally induce or be capable of inducing apomixis. The above variant preferably encodes a PAR protein as defined herein or is operably linked to a sequence encoding a PAR protein. Orthologs of the Par gene and par gene identified in Lactuca sativa isolate A68 in other plant species can be identified based on the characteristics of the PAR protein as defined herein. Such genes can encode any one of the PAR proteins selected from the group consisting of, but not limited to: PAR protein from pineapple (Ananas comosus) (e.g., UniProtKB:A0A199URK4), PAR protein from Apostasia shenzhenica (e.g., UniProtKB:A0A2I0AZW3), PAR protein from Arabidopsis thaliana (e.g., UniProtKB:Q8GXP9, A0A178V2S4, O81793, A0A178V1Q3, A0MFC1, O81801), PAR protein from Arabidopsis lyrata subsp. Lyrata (e.g., UniProtKB:D7MC52 or D7MCE8), PAR protein from Arachis ipaensis (e.g., SEQ ID NO:45 or SEQ ID NO:49), PAR protein from Brachypodium distachyon (e.g., UniProtKB:I1J0D9), Brassica oleracea var.PAR proteins derived from Brassica oleracea (e.g., UniProtKB: A0A0D3A1Q6 or A0A0D3A1Q3), PAR proteins derived from Brassica campestris (e.g., UniProtKB: A0A398AHT1), PAR proteins derived from Brassica rapa (e.g., SEQ ID NO: 47), PAR proteins derived from Brassica rapa subsp. Pekinensis (e.g., UniProtKB: M4D574 or M4D571), PAR proteins derived from Brassica oleracea (e.g., UniProtKB: A0A3P6ESB1 or A0A3P6F726), PAR proteins derived from Brassica campestris (e.g., UniProtKB: A0A3P5ZMM3 or A0A3P5Z1M1), PAR proteins derived from Cajanus cajan (e.g., SEQ ID NO: 46), PAR proteins derived from Capsella rubella (e.g., UniProtKB: R0H2J1 or R0H0C2), PAR proteins derived from Cephalotus follicularis (e.g., UniProtKB: A0A1Q3CSK1), PAR proteins derived from Cicer arietinum (e.g., UniProtKB: A0A3Q7YBZ1, A0A1S2YZL9, A0A3Q7Y0Z6 or A0A1S2YZM6; or SEQ ID NO: 55, 56 or 57), PAR proteins of Cichorium endivia (e.g., SEQ ID NO: 39), PAR proteins derived from Cucumis sativus (e.g., UniProtKB: A0A0A0KGW4 or A0A0A0L0X7), PAR proteins derived from Cucumis melo (e.g., UniProtKB: A0A1S3BLF2 or A0A1S3B298), PAR proteins derived from Cucumis sativus (e.g., UniProtKB: A0A0A0KAW8), PAR proteins derived from Cucurbita moschata (e.g., SEQ ID NO: 43), Cuscuta americanaPAR proteins derived from Brassica campestris (e.g., UniProtKB: A0A484MGR1), PAR proteins derived from Dendrobium catenatum (e.g., UniProtKB: A0A2I0V7N9, A0A2I0X2T2, or A0A2I0W0Q8), PAR proteins derived from Dorcoceras hygrometricum (e.g., UniProtKB: A0A2Z7D3Y1), PAR proteins derived from Eutrema salsugineum (e.g., UniProtKB: V4LSH0; or SEQ ID NO: 44), PAR proteins derived from Fagus sylvatica (e.g., UniProtKB: A0A2N9E5Y5, A0A2N9HAB9, or A0A2N9H993), PAR proteins derived from Genlisea aurea (e.g., UniProtKB: S8E1M6), PAR proteins derived from Glycine max (e.g., SEQ ID NO: 51, 52, 53, or 54), PAR proteins derived from Gossypium hirsutum (e.g., UniProtKB: A0A1U8LDU9), PAR proteins derived from Helianthus annuus (e.g., SEQ ID NO: 21), PAR proteins derived from Hevea brasiliensis (e.g., SEQ ID NO: 42), PAR proteins of Hieracium aurantiacum (e.g., SEQ ID NO: 40), PAR proteins derived from Juglans regia (e.g., UniProtKB: A0A2I4E6B1), PAR proteins derived from Lactuca sativa (e.g., UniProtKB: A0A2J6KZF7; or SEQ ID NO: 22), PAR proteins derived from Lagenaria siceraria (e.g., SEQ ID NO: 48), PAR proteins derived from Medicago truncatula (e.g., UniProtKB: G7K024), PAR proteins derived from MorusPAR proteins from Notabilis (e.g., UniProtKB: W9SMY3 or W9SMQ7), PAR proteins from Velvet bean (Mucuna pruriens) (e.g., UniProtKB: A0A371ELJ8), PAR proteins from Nicotiana attenuata (e.g., UniProtKB: A0A1J6IQI6), PAR proteins from Nicotiana sylvestris (e.g., UniProtKB: A0A1U7VXJ0), PAR proteins from Tobacco (Nicotiana tabacum) (e.g., UniProtKB: A0A1S4A651 or A0A1S3YHQ2), PAR proteins from Rice subspecies Japonica (Oryza sativa subsp. Japonica) (e.g., UniProtKB: B9FGH8), PAR proteins from Oryza barthii (e.g., UniProtKB: A0A0D3FWX3), PAR proteins from Proso millet (Panicum miliaceum) (e.g., UniProtKB: A0A3L6Q010 or A0A3L6T1D6), PAR proteins from Parasponia andersonii (e.g., UniProtKB: A0A2P5BMI5), PAR proteins from White poplar (Populus alba) (e.g., UniProtKB: A0A4U5PSY9), PAR proteins from Black cottonwood (Populus trichocarpa) (e.g., UniProtKB: B9H661), PAR proteins from Pomegranate (Punica granatum) (e.g., UniProtKB: A0A2I0IBB9, A0A218XB85 or A0A218W102), PAR proteins from Senecio cambrensis (e.g., SEQ ID NO: 41), PAR proteins from Peach (Prunus persica) (e.g., SEQ ID NO: 50), PAR proteins from Trema orientale (e.g., UniProtKB: A0A2P5EB04), PAR proteins from TrifoliumPAR proteins derived from Medicago pratense (e.g., UniProtKB:A0A2K3N851), PAR proteins derived from Trifolium subterraneum (e.g., UniProtKB:A0A2Z6MYD3 or A0A2Z6MDR7), PAR proteins derived from Trifolium pratense (e.g., UniProtKB:A0A2K3PR44), PAR proteins derived from Vitis vinifera (e.g., UniProtKB:A0A438C778, A0A438ESC4 or A0A438DBR4) and PAR proteins derived from Zea mays (e.g., UniProtKB:A0A1D6HF46, B6UAC5, A0A3L6F4S1, A0A3L6EMC6, A0A3L6EMC6, K7UHQ6 or A0A1D6KHZ4). Such genes can also encode PAR proteins selected from the group consisting of: PAR proteins derived from Actinidia chinensis (UniProtKB:A0A2R6S2S9), PAR proteins derived from Beta vulgaris (UniProtKB:XP_010690656.1), PAR proteins derived from Solanum tuberosum (UniProtKB:XP_015159151.1), PAR proteins derived from Solanum lycopersicum (UniProtKB:A0A3Q7GXB3), PAR proteins derived from Capsicum baccatum (UniProtKB:A0A2G2WJR7), PAR proteins derived from Solanum melongena (UniProtKB:AVC18974.1), PAR proteins derived from Glycine soja (GenBank accession: XP_028201014.1, XP_006596577.1 or UniprotKB:A0A445M3M6), PAR proteins derived from Arachis hypogaea (UniProtKB:A0A444WUX5), PAR proteins derived from Phaseolus vulgaris (UniProtKB:V7CIF6), PAR proteins derived from DaucusPAR proteins derived from carrot (GenBank accession: XP_017245413.1), PAR proteins derived from common wheat (Triticum aestivum) (UniProtKB: A0A3B6RP64), PAR proteins derived from indica rice (Oryza sativa subsp. indica) (UniProtKB: A2YH63), PAR proteins derived from japonica rice (UniProtKB: Q5Z7P5), and PAR proteins derived from cacao (Theobroma cacao) (UniProtKB: A0A061DL63). The present invention encompasses these orthologous genes, their promoter -ter sequences, coding sequences (including cDNA and mRNA sequences), and 3' UTRs.

[0076] The nucleic acids of the present invention may be, but are not limited to, DNA such as genomic DNA, cDNA, or RNA such as mRNA. Preferably, the nucleic acids of the present invention are isolated nucleic acids. Preferably, variant nucleic acids as defined herein preferably contain 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 with any one of the sequences encoding SEQ ID NOs: 1, 6, and 11, or their complements, when pairwise alignment is performed 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 contains at least 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more nucleotide sequence identity with SEQ ID NO: 3; a variant of the coding sequence of SEQ ID NO: 5 preferably contains at least about 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more nucleotide sequence identity with SEQ ID NO: 5, etc.

[0077] Preferably, the variant differs from any one of the sequences of SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14 and 15 and the sequences encoding SEQ ID NOs: 1, 6 and 11, or their complements, by one or more nucleotide deletions, insertions, and / or substitutions, and the variant includes natural and / or synthetic / artificial variants. A "natural variant" is a variant that occurs naturally, for example, in other species of the genus Taraxacum or in other plants. Preferably, the variant is a nucleotide sequence (gene, promoter sequence or coding sequence) from different plant species, for example, a species of the genus Taraxacum different from the common dandelion in a broad sense, for example, from different cultivars, landraces or breeding lines. The variant can also be found in and / or isolated from plants other than those belonging to the genus Taraxacum.

[0078] When indicated herein, the nucleic acids of the invention also include fragments of the Par allele or genes defined by the par allele, promoters or coding sequences as defined herein, or any variants thereof. A "fragment" is at least about 10, 12, 15, 18, 20, 30, 50, 100, 150, 200, 250, 300, 500, 1000, 2000 or more contiguous nucleotides, such as 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 contiguous nucleotide sequences of the sequences encoding SEQ ID NOs: 1, 6, and 11, or variants thereof, or its complement which is preferably capable of hybridizing to said sequences. In one embodiment, such a fragment can be considered to be functional in apomixis as defined herein (preferably capable of inducing apomixis). In another embodiment, such a fragment may not be considered to be functional in apomixis, but can be considered to be associated with apomixis, for example because such a fragment can hybridize to a sequence that is functional in apomixis and thus can direct apomixis. Such fragments may be useful, for example, as PCR primers or hybridization probes, and thereby can be used as genetic markers for use in mapping assays or molecular assays and / or for identifying and / or isolating Par alleles or par alleles from other plants.

[0079] Preferably, the nucleic acid of the present 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 the promoter sequence, comprises a nucleic acid insert, preferably a double-stranded DNA insert, wherein said insert has a length 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 the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype. Preferably, the distance between the 3' end of said 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, such that preferably, said insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding the PAR protein. Preferably, said insert is located such that the 3' terminal nucleotide of the insert is homologous to the position of nucleotide 1798 of SEQ ID NO: 2 and / or nucleotide 1798 of SEQ ID NO: 5. Preferably, said insert has no open reading frame. Even more preferably, said insert is a miniature inverted-repeat transposable element (MITE) or a MITE-like sequence, wherein said MITE or MITE-like sequence contains an internal sequence having no open reading frame, and the internal sequence is flanked by terminal inverted repeats (TIRs) which are in turn flanked by short tandem repeats (target site duplications), and is a non-autonomous element. For details of MITE, TIR, and sequences, see Guo et al., Scientific Reports. June 1, 2017; 7(1):2634, which is incorporated herein by reference.The insert, preferably the MITE or MITE-like sequence, can have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, the insert is related to the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype. In a further preferred embodiment, the nucleic acid of the invention comprises or consists of regulatory sequences, preferably promoter sequences, and encompasses the insert at the position defined above herein. Preferably, the nucleic acid of the invention 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 immediately upstream of the sequence encoding the PAR protein. Optionally, the nucleic acid of the invention can comprise one or more additional transcriptional regulatory sequences.

[0080] In one embodiment, the nucleic acid of the invention can be derived from a Taraxacum line (e.g., Dandelion in a broad sense) or other species.

[0081] In one embodiment, the nucleic acid of the invention is derived from an origin different from Taraxacum or Dandelion in a broad sense.

[0082] In one embodiment, the present invention encompasses homologous or orthologous Par alleles that are derived from plants with apomixis, such as wild or cultivated plants, and / or other plants. Such homologs or orthologs can be readily isolated by using the provided nucleotide sequence or a portion thereof as a primer or a probe. For example, a fragment of the nucleotide sequence as defined herein, or its complement, can be used, for instance, to perform moderate or stringent nucleic acid hybridization methods. 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, any one variant of SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, and 15, and / or of the sequences encoding SEQ ID NOs: 1, 6, and 11, also includes nucleic acids that are naturally (or in nature) found in other plants of the genus Taraxacum, lines, or cultivars, and / or in other plants.

[0083] For optimal expression in a host or host cell, the coding sequences taught herein can be codon-optimized by using available codon usage tables to make them most preferred in plant genes, particularly in native genes for the genus or species of interest of the plant (Bennetzen and Hall, 1982, J. Biol. Chem. 257, 3026-3031; Itakura et al., 1977 Science 198, 1056-1063), by adapting the codon usage (e.g., further adapting for expression in the plant of interest). Codon usage tables for various plant species are published, for example, by Ikemura (1993, in "Plant Molecular Biology Labfax", ed. Croy, Bios Scientific Publishers Ltd.) and Nakamura et al. (2000, Nucl. Acids Res. 28, 292.), as well as in major DNA sequence databases (e.g., EMBL in Heidelberg, Germany). Thus, synthetic DNA sequences can be constructed such that the same or substantially the same protein can be made using the synthetic DNA sequences. Several techniques for modifying codon usage to that preferred by the host cell can be found in patents and the scientific literature. The exact method of modifying codon usage is not of critical importance 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, for example, by random or targeted mutagenesis (e.g., by chemical mutagenesis or CRISPR-endonuclease-mediated mutagenesis). More substantial modifications to the sequences taught herein can be routinely made by de novo DNA synthesis of the desired sequences using available techniques.

[0085] In one embodiment, the nucleic acid of the present invention can be modified so that the N-terminus of the protein of the present invention encoded by the 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, the protein of the present invention expressed in plant cells preferably starts with the dipeptide Met-Asp or Met-Ala for optimal translation initiation. Therefore, an Asp or Ala codon may be inserted following the existing Met, or the second codon Val can be replaced with a codon for Asp (GAT or GAC) or Ala (GOT, GCC, GCA, or GCG). The nucleotide sequence can also be modified to remove non-canonical splice sites.

[0086] In one embodiment, the nucleic acid of the present invention can have a (genetically) dominant function, 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, for example, an ortholog or a fragment thereof found in another plant (i.e., other than the genus Taraxacum or the broad sense dandelion).

[0087] Preferably, when the nucleic acid of the present invention is produced in a plant, it is functional and encodes a protein or a functional fragment(s) thereof that induces and / or enhances apomixis. For example, when a nucleic acid containing SEQ ID NO: 3 or 5, or a variant or fragment thereof, is expressed (transcribed and translated) and an appropriate amount of the protein of the present invention is produced in an appropriate plant tissue, the apomixis effect is significantly enhanced as compared to a plant that is different only in that it lacks the said nucleic acid. With regard to functionality, it can be easily tested by (over)expressing the nucleic acid of the present invention in an appropriate host plant such as an apomictic dandelion genus line, and analyzing the apomixis effect of the transformant in a bioassay as described in Example 2, for example. The functionality of the said nucleic acid is preferably evaluated by comparing a test plant in which one or more of these nucleic acids are (over)expressed with a control plant that is different from the test plant only in that it lacks (over)expression of the said nucleic acid. Alternatively, loss-of-function types, i.e., reduction of apomixis, can be caused by silencing or disrupting the nucleic acid of the present invention related to apomixis.

[0088] Vectors or plasmids can be created using the nucleic acids of the present invention for expressing the proteins of the present invention in appropriate host cells or for silencing one or more endogenous apomixis genes or gene families. Accordingly, constructs, vectors and / or plasmids containing the nucleic acids of the present invention, and / or silencing constructs are also encompassed by the present invention.

[0089] Amino acid sequence according to the present invention The present invention provides PAR proteins as defined herein. The present invention also provides proteins related to apomixis in plants, wherein the said proteins are: a) encoded by the nucleic acids of the present invention; b) have the amino acid sequences of SEQ ID NO: 1, 6 or 11; c) are variants of a) and / or b); and / or d) are fragments of any one of a) to c), Here, preferably, the protein is functional in parthenogenesis. In one embodiment, the protein of the present invention is: 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), wherein, preferably, the protein of the present invention is suitable for inducing parthenogenesis. In one embodiment, the protein of the present invention is: a) encoded by the 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), Here, preferably, the protein of the present invention is suitable for inducing parthenogenesis. The variant is preferably a PAR protein as defined herein. Preferably, the protein or protein fragment is encoded by the nucleic acid of SEQ ID NO: 3 or 5, or variants and / or fragments thereof, or such a protein comprises SEQ ID NO: 1, or variants and / or fragments thereof. Preferably, the variant preferably has at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 1, 6, or 11, respectively, when pairwise alignment is performed using the Needleman and Wunsch algorithm (global sequence alignment) with default parameters. The variant differs from the provided sequence by deletion, insertion, and / or substitution of one or more amino acid residues, and such variants include natural and / or synthetic / artificial variants. Variants of the protein having the amino acids encoded by the nucleic acid of the present invention are preferably variants of the protein encoded by any one of SEQ ID NO: 3, 5, 8, 10, 13, 15, or variants of the protein having the amino acid sequence of any one of SEQ ID NO: 1, 6, or 11, and may be homologs or orthologs. Such orthologous proteins encompassed by the present invention may be any one of the PAR proteins selected from the group consisting of, but not limited to: PAR protein from pineapple (e.g., UniProtKB:A0A199URK4), PAR protein from Apostasia shenzhenica (e.g., UniProtKB:A0A2I0AZW3), PAR protein from Arabidopsis thaliana (e.g., UniProtKB:Q8GXP9, A0A178V2S4, O81793, A0A178V1Q3, A0MFC1, O81801), PAR protein from Paralichthys olivaceus subsp. lilat (e.g., UniProtKB:D7MC52 or D7MCE8), PAR protein from Arachis hypogaea (e.g., SEQ ID NO: 45 or SEQ ID NO: 49),PAR proteins derived from Minato kamojigusa (e.g., UniProtKB: I1J0D9), PAR proteins derived from Brassica rapa var. oleifera (e.g., UniProtKB: A0A0D3A1Q6 or A0A0D3A1Q3), PAR proteins derived from Brassica campestris (e.g., UniProtKB: A0A398AHT1), PAR proteins derived from Brassica rapa (e.g., SEQ ID NO: 47), PAR proteins derived from Brassica rapa subsp. pekinensis (e.g., UniProtKB: M4D574 or M4D571), PAR proteins derived from Brassica rapa (e.g., UniProtKB: A0A3P6ESB1 or A0A3P6F726), PAR proteins derived from Brassica campestris (e.g., UniProtKB: A0A3P5ZMM3 or A0A3P5Z1M1), PAR proteins derived from Cicer arietinum (e.g., SEQ ID NO: 46), PAR proteins derived from Arabidopsis lyrata (e.g., UniProtKB: R0H2J1 or R0H0C2), PAR proteins derived from Saxifraga stolonifera (e.g., UniProtKB: A0A1Q3CSK1), PAR proteins derived from Glycine max (e.g., UniProtKB: A0A3Q7YBZ1, A0A1S2YZL9, A0A3Q7Y0Z6 or A0A1S2YZM6; or SEQ ID NO: 55, 56 or 57), PAR proteins derived from Cichorium endivia (e.g., SEQ ID NO: 39), PAR proteins derived from Cucumis sativus (e.g., UniProtKB: A0A0A0KGW4 or A0A0A0L0X7), PAR proteins derived from Cucumis melo (e.g., UniProtKB: A0A1S3BLF2 or A0A1S3B298), PAR proteins derived from Cucumis sativus (e.g., UniProtKB: A0A0A0KAW8), PAR proteins derived from Cucurbita moschata (e.g., SEQ ID NO: 43), PAR proteins derived from Celastrus orbiculatus (e.g., UniProtKB: A0A484MGR1), PAR proteins derived from Notholaena hirsuta (e.g., UniProtKB: A0A2I0V7N9, A0A2I0X2T2 or A0A2I0W0Q8), PAR proteins derived from Doroceras hygrometricum (e.g., UniProtKB: A0A2Z7D3Y1), PAR proteins derived from Eutrema salsugineum (e.g., UniProtKB: V4LSH0; or SEQ ID NO: 44),PAR proteins derived from European beech (e.g., UniProtKB:A0A2N9E5Y5, A0A2N9HAB9, or A0A2N9H993), PAR proteins derived from Gengyilia aurea (e.g., UniProtKB:S8E1M6), PAR proteins derived from soybean (e.g., SEQ ID NO: 51, 52, 53, or 54), PAR proteins derived from upland cotton (e.g., UniProtKB:A0A1U8LDU9), PAR proteins derived from sunflower (e.g., SEQ ID NO: 21), PAR proteins derived from Para rubber tree (e.g., SEQ ID NO: 42), PAR proteins of dandelion (e.g., SEQ ID NO: 40), PAR proteins derived from Persian lime (e.g., UniProtKB:A0A2I4E6B1), PAR proteins derived from lettuce (e.g., UniProtKB:A0A2J6KZF7; or SEQ ID NO: 22), PAR proteins derived from Japanese bindweed (e.g., SEQ ID NO: 48), PAR proteins derived from oil palm (e.g., UniProtKB:G7K024), PAR proteins derived from white mulberry (e.g., UniProtKB:W9SMY3 or W9SMQ7), PAR proteins derived from velvet bean (e.g., UniProtKB:A0A371ELJ8), PAR proteins derived from Nicotiana attenuata (e.g., UniProtKB:A0A1J6IQI6), PAR proteins derived from Nicotiana sylvestris (e.g., UniProtKB:A0A1U7VXJ0), PAR proteins derived from tobacco (e.g., UniProtKB:A0A1S4A651 or A0A1S3YHQ2), PAR proteins derived from Oryza sativa subsp. japonica (e.g., UniProtKB:B9FGH8), PAR proteins derived from Oryza barthii (e.g., UniProtKB:A0A0D3FWX3), PAR proteins derived from foxtail millet (e.g., UniProtKB:A0A3L6Q010 or A0A3L6T1D6), PAR proteins derived from Parasponia andersonii (e.g., UniProtKB:A0A2P5BMI5), PAR proteins derived from bitter gourd (e.g., UniProtKB:A0A4U5PSY9), PAR proteins derived from black cottonwood (e.g., UniProtKB:B9H661), PAR proteins derived from pomegranate (e.g., UniProtKB:A0A2I0IBB9,A0A218XB85 or A0A218W102), a PAR protein from Senecio cambricus (e.g., SEQ ID NO: 41), a PAR protein from peach (e.g., SEQ ID NO: 50), a PAR protein from Flammulina velutipes (e.g., UniProtKB:A0A2P5EB04), a PAR protein from Viola philippica (e.g., UniProtKB:A0A2K3N851), a PAR protein from Dimocarpus longan (e.g., UniProtKB:A0A2Z6MYD3 or A0A2Z6MDR7), a PAR protein from Viola philippica (e.g., UniProtKB:A0A2K3PR44), a PAR protein from Vitis vinifera (e.g., UniProtKB:A0A438C778, A0A438ESC4 or A0A438DBR4) and a PAR protein from Zea mays (e.g., UniProtKB:A0A1D6HF46, B6UAC5, A0A3L6F4S1, A0A3L6EMC6, A0A3L6EMC6, K7UHQ6 or A0A1D6KHZ4). Such orthologous proteins can also be PAR proteins selected from the group consisting of: a PAR protein from Actinidia arguta (UniProtKB:A0A2R6S2S9), a PAR protein from Beta vulgaris (UniProtKB:XP_010690656.1), a PAR protein from Solanum tuberosum (UniProtKB:XP_015159151.1), a PAR protein from Solanum lycopersicum (UniProtKB:A0A3Q7GXB3), a PAR protein from Capsicum annuum (UniProtKB:A0A2G2WJR7), a PAR protein from Solanum melongena (UniProtKB:AVC18974.1), a PAR protein from Phaseolus vulgaris (GenBank accession: XP_028201014.1, XP_006596577.1 or UniprotKB:A0A445M3M6), a PAR protein from Vigna radiata (UniProtKB:A0A444WUX5), a PAR protein from Phaseolus coccineus (UniProtKB:V7CIF6), a PAR protein from Daucus carota (GenBank accession: XP_017245413.1), a PAR protein from Triticum aestivum (UniProtKB:A0A3B6RP64),PAR proteins derived from Oryza sativa subsp. indica (UniProtKB: A2YH63), PAR proteins derived from Oryza sativa subsp. japonica (UniProtKB: Q5Z7P5), and PAR proteins derived from Theobroma 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 ortholog PAR proteins defined herein.

[0091] The PAR protein of the present invention, and / or a variant of the protein having SEQ ID NO: 1, 6, or 11 may be capable of inducing apomixis when present in a plant or a plant cell. The variant of the protein may be an endogenous protein or a non-endogenous protein of the plant or plant cell. Optionally, the PAR protein of the present invention and / or a variant of the protein having SEQ ID NO: 1, 6, or 11 may be capable of inducing apomixis when the expression of the protein is changed, preferably increased. Preferably, such a changed expression, preferably an increased expression, is within the egg cell. The change or increase in expression may be de novo expression of the protein in the plant or plant cell, or may be an increased expression of an endogenous protein in the plant or plant cell. Those skilled in the art know methods for increasing the expression of a protein. De novo expression of the protein in a plant or a plant cell may be induced, for example, by transfecting a construct or vector encoding the protein into the plant or plant cell, by introducing the gene encoding the protein into the progeny of the plant or plant cell, and / or by modifying an endogenous sequence to result in a sequence encoding the protein, for example, by genetic modification. Optionally, such a construct or vector may contain a sequence encoding a PAR protein operably linked to an egg cell promoter. Those skilled in the art know about egg cell promoters. Exemplary egg cell promoters capable of driving expression in plant egg cells include, but are not limited to, the promoters of egg cell-specific genes ECl.1, ECl.2, ECl.3, ECl.4, or ECl.5 (see, for example, Sprunck et al. Science, 338:1093-1097 (2012); AT2G21740; Steffen et al., Plant Journal 51:281-292 (2007)), and the Arabidopsis thaliana 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 the insert is 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 in length. Even more preferably, the insert has a length of about 1300 bp. Preferably, the insert is related to the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype. Preferably, 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, such that preferably, the insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding the PAR protein. Preferably, the insert is located such that the 3' terminal nucleotide of the insert 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 has no open reading frame. Even more preferably, the insert is a miniature inverted-repeat transposable element (MITE) or a MITE-like sequence, wherein the MITE or MITE-like sequence contains an internal sequence having no open reading frame, and the internal sequence is flanked by terminal inverted repeats (TIRs) which are in turn flanked by short tandem repeats (target site duplication), and is a non-autonomous element. For details of MITE, TIR, and sequences, see Guo et al., Scientific Reports. June 1, 2017; 7(1):2634, which is incorporated herein by reference.The insert, preferably the MITE or MITE-like sequence, can have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, the insert is related to the apomixis phenotype as defined herein and is optionally functional in the apomixis phenotype. In a further preferred embodiment, the construct or vector of the invention comprises or consists of regulatory sequences, preferably promoter sequences, and encompasses the insert at the position defined above herein. Preferably, the construct or 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 immediately upstream of the sequence encoding the PAR protein. Optionally, the construct or vector of the invention can comprise one or more additional transcriptional regulatory sequences.

[0092] In addition, or alternatively, such a construct or vector comprises a sequence encoding a PAR protein operably linked to the promoter of SEQ ID NO: 2. A change or increase in the expression of an 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 sequence encoding the protein may be modified, for example by genetic modification. In a preferred embodiment, the insert as defined above herein is introduced into the promoter sequence, preferably at the position defined above herein. Such functionality to induce apomixis may be evaluated by using appropriate tests for the functionality of the nucleic acid encoding the variant in apomixis, as described herein. The protein of the invention may be an isolated protein.

[0093] A "natural variant" is one that is found naturally, for example, in cultivated or wild lettuce plants and / or other plants. Fragments, i.e., non-full-length peptides of the protein of the present invention, preferably functional fragments, are also included, i.e., this fragment is 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 contiguous amino acid sequences encoded by the nucleic acids of the present invention, particularly peptides comprising or consisting of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250 or more contiguous amino acids of SEQ ID NO: 1, 6, or 11, or variants thereof (as defined herein). Sequences found naturally are also referred to herein as "wild-type".

[0094] The proteins of the present 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 recombinant host cells by expressing the nucleotide sequences taught herein that encode the proteins of the present invention. The proteins of the present invention may also be produced by expression from the nucleic acids of the present 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 present invention.

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

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

[0098] Apomictic plants and methods for producing them In a further aspect, the present invention relates to a method of producing a transgenic plant that has modified, preferably induced, apomixis, optionally comparing the plant (including, for example, plant cells, organs, seeds and plant parts) with a plant showing modified apomixis to a natural or unmodified plant. 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 genetic modifications such as, for example, at least one of random mutagenesis, targeted mutagenesis, and nucleic acid insertion.

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

[0100] The nucleic acid of the present invention can be introduced into one or more plant cells by transformation, transfection, somatic hybridization, and / or protoplast fusion. Such nucleic acid may be an exogenous nucleic acid, i.e., a nucleic acid that does not naturally exist in the plant cell.

[0101] The nucleic acids of the present invention can be introduced into one or more plant cells by modifying endogenous nucleic acids to obtain the nucleic acids of the present invention. Modification of endogenous genes preferably involves random or targeted mutations of one or more nucleotides in the coding sequence and / or in regulatory and / or promoter sequences, or insertions or deletions of short or larger sequences, such as by homologous recombination, to change the expression of endogenous proteins. Such methods preferably result in the modification of one or more endogenous par alleles to the Par allele. Random mutagenesis may be, but is not limited to, chemical mutagenesis and gamma rays. Non-limiting examples of chemical mutagenesis include, but are not limited to, EMS (ethyl methanesulfonate), MMS (methyl methanesulfonate), NaN3 (sodium azide), ENU (N-ethyl-N-nitrosourea), AzaC (azacytidine), and NQO (4-nitroquinoline 1-oxide). Optionally, mutagenesis systems 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 create plant lines having the modified genes defined herein. In TILLING, conventional chemical mutagenesis (e.g., EMS mutagenesis) is 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 change specific nucleotides or nucleic acid sequences, such as, but not limited to, oligo-directed mutagenesis, RNA-guided endonucleases (e.g., CRISPR technology), TALENs, or zinc finger technology.

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

[0103] In certain preferred embodiments, the nucleic acid is a (5’UTR) promoter sequence of a gene encoding a protein related to apomixis as defined herein. Preferably, the modification is the introduction of a nucleic acid insert, preferably a double-stranded DNA insert, wherein the insert is 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 in length. Even more preferably, the insert has a length of about 1300 bp. Preferably, the insert is related to the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype. Preferably, 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, such that preferably, the insert is introduced into a promoter sequence located immediately upstream (3’) of the sequence encoding the PAR protein. Preferably, the insert is introduced such that the 3’ terminal nucleotide of the insert 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 has no open reading frame. Even more preferably, the insert is a miniature inverted-repeat transposable element (MITE) or a MITE-like sequence, wherein the MITE or MITE-like sequence contains an internal sequence without an open reading frame, and the internal sequence is flanked by terminal inverted repeats (TIRs) that are in turn flanked by short tandem repeats (target site duplication), and is a non-autonomous element. For details of MITE, TIR, and sequences, see Guo et al., Scientific Reports. June 1, 2017; 7(1):2634, which is incorporated herein by reference.The insert, preferably the MITE or MITE-like sequence, can have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, the insert is related to the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype.

[0104] Preferably, the modification of the nucleotide sequence preferably results in the introduced or increased expression of the protein in the egg cells of plants regenerated from plant cells. 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 with SEQ ID NO: 2. Furthermore, the method of the present invention comprises the following steps: a) modifying a nucleic acid that is a sequence encoding a protein related to and / or functional in apomixis, or is operably linked to such a sequence, in one or more plant cells capable of apomixis, preferably wherein the nucleic acid is within the genome of the one or more plant cells; b) optionally, selecting plant cells containing the modified or altered nucleic acid; c) regenerating a plant from the plant cells and can include wherein preferably the protein related to and / or functional in apomixis has the amino acid sequence of the protein of the present invention described hereinabove. Preferably, the nucleic acid modified in step a) is an endogenous nucleic acid, preferably a sequence encoding 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 nucleotide sequence operably linked to such a sequence, or variants or fragments thereof, or consists of or comprises them. Preferably, the nucleic acid modified in step a) is an endogenous nucleic acid.

[0105] In certain preferred embodiments, the nucleic acid is a promoter sequence of a gene encoding a protein associated with and / or functional in apomixis as defined herein. Preferably, the modification of the nucleotide sequence preferably results in an introduced or increased expression of the protein in the egg cells of the plant regenerated from the plant cell. Preferably, the modified promoter sequence is a promoter sequence operably linked to the coding sequence of the PAR protein as defined herein. Preferably, the modified promoter sequence is modified to preferably contain the insert as defined herein at the position 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 with SEQ ID NO: 2. The present invention also provides a method for producing apomictic hybrid seeds, comprising the following steps: a) cross-fertilizing a first sexually reproducing plant with the pollen of a second plant to produce F1 hybrid seeds; b) optionally, selecting seeds containing an apomictic phenotype from the F1 seeds wherein the first plant and / or the second plant is capable of apomixis, the second plant contains the nucleic acid of the present invention, and preferably, the selection step is performed by genotyping. Preferably, the second plant is any one of SEQ ID NOs: 2 to 5 or contains the nucleic acid of the present invention encoding the protein of SEQ ID NO: 1, or a variant or fragment thereof.

[0107] ​The nucleic acid of the present invention may be contained in a chimeric gene, a gene construct or a nucleic acid vector. In one embodiment of the present invention, the nucleic acid of the present invention is used to transfer the nucleic acid into a host cell and produce a functional (preferably, capable of inducing apomixis) protein encoded by the nucleic acid in the host cell. A chimeric gene containing this nucleic acid and / or a vector may be prepared. A vector for the production of such a protein (or protein fragment or variant) in a plant cell is herein referred to as an "expression vector". The host cell is preferably a plant cell.

[0108] The construction of chimeric genes, constructs and / or vectors for the optional, although preferably stable, introduction of a nucleotide sequence encoding a protein 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 sequence encoding the protein of SEQ ID NO: 1, 6 or 11, or a functional variant and / or functional fragment 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 such that the nucleotide sequence encoding the protein can simply be inserted downstream of the promoter sequence into the vector. The vector can then be used to transform a host cell, and the nucleic acid and / or chimeric gene of the invention may be inserted into the nuclear genome or into the genome of a plastid, mitochondrion or chloroplast, and may be expressed in the host cell using a suitable promoter (e.g., McBride 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. Optionally, there is a 5' UTR sequence preceding the coding sequence. The promoter, 3' UTR and / or 5' UTR may be, for example, derived from a natural parthenogenesis gene or alternatively from other sources.

[0109] The nucleic acids taught herein that encode a protein capable of inducing apomixis taught herein can be stably inserted into the nuclear genome of a single plant cell, and using the plant cell so transformed, a transformed plant having an altered phenotype can be produced by the presence of said protein in a particular cell at a particular time. In non-limiting examples, in Agrobacterium tumefaciens, plant cells can be transformed using a T-DNA vector containing the nucleic acids taught herein that encode a protein functional in the apomixis taught herein, and thereafter, the transformed plants can be regenerated from the transformed plant cells using, for example, the procedures described in European Patent No. 0116718, European Patent Application Publication No. 0270822, PCT Published International Publication No. 84 / 02913 and Published European Patent Application No. 0242246 and Gould et al. (1991). The construction of T-DNA vectors for Agrobacterium-mediated plant transformation is well known in the art. The T-DNA vector can be either a binary vector as described in European Patent No. 0120561 and European Patent No. 0120515, or a cointegrate vector that can be integrated into the Agrobacterium Ti plasmid by homologous recombination, as described in European Patent No. 0116718. Lettuce transformation protocols are described, for example, in Michelmore et al. (1987) and Chupeau et al. (1989).

[0110] Preferred T-DNA vectors contain a promoter operably linked to the nucleotide sequence encoding the protein of the invention; for example, the promoter is operably linked to the nucleotide sequence of SEQ ID NO: 3 or variants or functional fragments thereof between the T-DNA border sequences, or is at least located to the left of the right border sequence. Preferably, the promoter is a promoter containing a nucleic acid insert, preferably a double-stranded DNA insert, wherein the insert is 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 in length. Even more preferably, the insert has a length of about 1300 bp. Preferably, the insert is related to the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype. Preferably, 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, such that preferably the insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding the PAR protein. Preferably, the insert is located such that the 3' terminal nucleotide of the insert 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 has no open reading frame. Even more preferably, the insert is a miniature inverted repeat transposable element (MITE) or MITE-like sequence, wherein the MITE or MITE-like sequence contains an internal sequence without an open reading frame, and the internal sequence is flanked by terminal inverted repeat sequences (TIRs) which are in turn flanked by short tandem repeats (duplication of the target site), and is a non-autonomous element.For details of the MITE, TIR, and sequences, see Guo et al., Scientific Reports. June 1, 2017; 7(1):2634, which is incorporated herein by reference. The insert, preferably the MITE or MITE-like sequence, can have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, the insert is related to the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype. In a further preferred embodiment, the T-DNA vector comprises or consists of regulatory sequences, preferably promoter sequences, and encompasses the insert at the position defined hereinabove. 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 immediately upstream of the sequence encoding the PAR protein. Optionally, the T-DNA vector can comprise one or more additional transcriptional regulatory sequences.

[0111] The 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 European Patent Application Publication No. 0223247), pollen-mediated transformation (e.g., as described in European Patent No. 0270356 and International Publication No. 85 / 01856), protoplast transformation such as described in U.S. Patent No. 4,684,611, plant RNA virus-mediated transformation (e.g., as described in European Patent Application Publication No. 0067553 and U.S. Patent No. 4,407,956), liposome-mediated transformation (e.g., as described in U.S. Patent No. 4,536,475), and other methods.

[0112] In a further embodiment, the nucleic acids of the invention may be introduced by somatic hybridization. Somatic hybridization may be performed by protoplast fusion (see, e.g., Holmes, 2018).

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

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

[0115] Similarly, the selection and regeneration of transgenic plants from transgenic cells are well known in the art. Clearly, protocols are specifically configured to regenerate transformants with high frequency for various species and even for various varieties or cultivars of a single species. The invention also encompasses progeny of transgenic plants that exhibit apomixis and contain the nucleic acids and / or proteins of the 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 plastid genome transformation is that it can reduce the risk of spread of the introduced gene(s). Plastid genome transformation can be carried out as known in the art, see, for example, Sidorov et al. (1999) or Lutz et al. (2004).

[0117] The resulting transgenic plants can be used in conventional plant breeding schemes to produce more transgenic plants containing the introduced gene. Single-copy transformants can be selected, for example, using Southern blot analysis or PCR-based methods or Invader® technology assays (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. The sequence of the plant DNA adjacent to the insertion site of the introduced gene can also be determined, thereby enabling the development of "event-specific" detection methods for routine use. See, for example, WO 01 / 41558, which describes elite event detection kits (such as PCR detection kits) based on the integrated and adjacent (genomic) sequences.

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

[0119] A promoter that can be operably linked to SEQ ID NO: 3, or variants or fragments thereof, may be, for example, a constitutively active promoter, such as the following: the strong constitutive 35S promoter or enhanced 35S promoter of cauliflower mosaic virus (CaMV) of each isolate CM1841 (Gardner et al., 1981), CabbB-2 (Franck et al., 1980), and CabbB-JI (Hull and Howell, 1987) (the "35S promoter"); the 35S promoter described by Odell et al. (1985) or in U.S. Patent No. 5,164,316, promoters derived from the ubiquitin family (e.g., Christensen et al., 1992; the maize ubiquitin promoter of European Patent No. 0342926; see also Cornejo et al., 1993), the gos2 promoter (de Pater et al., 1992), the emu promoter (Last et al., 1990), the Arabidopsis actin promoter such as the promoter described by An et al. (1996), the promoter described by Zhang et al. (1991), and the promoter described in U.S. Patent No. 5,641,876 or the rice actin 2 promoter described in International Publication No. WO 07 / 0067; the promoter of cassava vein mosaic virus (International Publication No. WO 97 / 48819, Verdaguer et al., 1998), the pPLEX series of promoters derived from Subterranean Clover Stunt virus (International Publication No. WO 96 / 06932, particularly the S7 promoter), the alcohol dehydrogenase promoter, e.g., pAdh1S (GenBank accession numbers X04049, X00581), and the TR1' promoter and TR2' promoter that drive the expression of the 1' and 2' genes of T-DNA, respectively (the "TR1' promoter" and "TR2' promoter", respectively) (Velten et al., 1984), the Figwort Mosaic Virus promoter described in U.S. Patent No. 6,051,753 and EP426641, the histone gene promoter, e.g., the Ph4a748 promoter derived from Arabidopsis (PMB 8:179-191), or others.

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

[0121] Constitutive production of the protein of the present invention can be costly to the fitness of the plant. Thus, in one embodiment, it is preferable to use a promoter whose activity is inducible. Examples of inducible promoters are wound-inducible promoters, such as the MPI promoter described by Cordera et al. (1994) that is induced by wounding (e.g., caused by insects or physical wounding), or the COMPTII promoter (International Publication No. 0056897) or the RP1 promoter described in U.S. Patent No. 6,031,151. Alternatively, the promoter may be inducible by chemicals such as dexamethasone as described by Aoyama and Chua (1997) and in U.S. Patent No. 6,063,985, or by tetracycline (see TOPFREE or TOP10 promoters, Gatz, 1997 and Love et al., 2000).

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

[0123] In one embodiment, a promoter of a natural parthenogenesis gene is used. For example, the promoter of the Par allele or the par allele of the genus Taraxacum may be isolated and operably linked to the coding region encoding the protein according to the present invention. In one embodiment, the promoter (upstream transcriptional regulatory region, for example, 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 natural parthenogenesis 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, the promoter is a promoter containing a nucleic acid insert, preferably a double-stranded DNA insert, where the insert is 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 in length. Even more preferably, the insert has a length of about 1300 bp. Preferably, the insert is related to the parthenogenesis phenotype as defined herein and is optionally functional in the parthenogenesis phenotype. Preferably, 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, such that preferably, the insert is located within a promoter sequence located immediately upstream (3') of the sequence encoding the PAR protein. Preferably, the insert is located such that the 3' terminal nucleotide of the insert 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 has no open reading frame. Even more preferably, the insert is a miniature inverted-repeat transposable element (MITE) or a MITE-like sequence, where the MITE or MITE-like sequence contains an internal sequence without an open reading frame, and the internal sequence is flanked by terminal inverted repeats (TIRs) that are in turn flanked by short tandem repeats (target site duplication), and is a non-autonomous element. For details of MITE, TIR, and sequences, see Guo et al., Scientific Reports. June 1, 2017; 7(1):2634, which is incorporated herein by reference.The insert, preferably the MITE or MITE-like sequence, can have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. Preferably, the insert is related to the apomictic phenotype as defined herein and is optionally functional in the apomictic phenotype. The promoter can have the nucleotide sequence of SEQ ID NO: 2. Also, sequences longer than the sequences described herein may be used. The region up to about 2000 bp upstream of the translation start codon of the coding region can contain transcriptional regulatory elements (i.e., promoters). Thus, in one embodiment, the nucleotide sequence at 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 present invention can be isolated and tested for promoter activity, and if functional, the above sequence can be operably linked to the sequence encoding the protein of the present invention taught herein. The promoter activity of the entire sequence and fragments thereof can be tested, for example, by deletion analysis, whereby the 5' portion and / or 3' portion 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 the coding sequence is upstream (i.e., 5') of a suitable 3' untranslated region ("3' end" or 3' UTR). Suitable 3' ends include the 3' ends 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 the natural parthenogenesis gene, or a 3' UTR derived therefrom, is used. For example, any 3' UTR derived from SEQ ID NO: 4, or variants or fragments thereof, may be used. The 3' UTR may have the nucleotide sequence of SEQ ID NO: 4.

[0126] In one embodiment, a promoter having the nucleotide sequence of SEQ ID NO: 2, or a variant and / or fragment herein, may be operably linked to a nucleic acid encoding the protein of the invention. Preferably, the nucleotide sequence encoding the protein is capable of inducing parthenogenesis as taught herein, and more preferably has the amino acid sequence of SEQ ID NO: 1, or a variant and / or fragment thereof. Preferably, the promoter and the coding sequence are further operably linked to the 3' UTR of SEQ ID NO: 4, or a variant and / or fragment 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 the plant genome as hybrid gene sequences, whereby the coding sequences are in-frame linked to a gene encoding a selectable or scorable marker (U.S. Patent No. 5,254,799; Vaeck et al., 1987), for example, the neo (or nptII) gene encoding kanamycin resistance (European Patent No. 0242236), etc., such that the plant expresses a fusion protein that is readily detectable.

[0129] All or part of the sequence encoding the protein of the present 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 produce recombinant viruses. This is particularly suitable for the production and subsequent purification of the protein, preferably the isolated protein. Transformation of bacteria with all or part of the coding sequences taught herein incorporated into a suitable cloning vehicle can be carried out by conventional methods, preferably using the conventional electroporation techniques described by Maillon et al. (1989) and International Publication No. 90 / 06999. With respect to expression in prokaryotic host cells, the codon usage frequency of the nucleic acid sequence may be optimized accordingly (as described for the plants herein). Intron sequences need to be removed, and other adaptations for optimal expression may be made as known. Such prokaryotic host cells containing and / or expressing the nucleic acids of the present invention are encompassed by the present invention. Such host cells can be used to produce the protein and / or nucleic acids of the present invention.

[0130] The DNA sequence of the nucleic acid of the present invention can be further varied in a translationally neutral manner to introduce changes in the DNA sequences that may be inhibitory present in the gene portion and / or in the codon usage frequency, for example by adapting the codon usage frequency to the most preferred codon usage frequency in plants, for example in the host plants described herein, preferably in certain related plant genera.

[0131] According to one embodiment of the present invention, the protein of the present invention targets intracellular organelles such as plastids, preferably chloroplasts, mitochondria, etc. or is secreted from cells, as a result, potentially optimizing the stability and / or expression of the protein. Similarly, the protein may target vacuoles. For this purpose, in one embodiment of the present invention, the chimeric gene of the present invention includes a coding region encoding a signal or target peptide linked to the region encoding the protein of the present invention. Particularly preferred peptides included in the protein of the present invention are transit peptides for chloroplasts or other plastids targeting the overlapping transit peptide region from plant genes where the gene product is targeted to plastids, the optimized transit peptide of Capellades et al. (U.S. Patent No. 5,635,618), the transit peptide of ferredoxin-NADP+ oxidoreductase from spinach (Oelmuller et al., 1993), the transit peptides described by 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 such peptides extracellularly, such as the secretion signal of potato proteinase inhibitor II (Keil et al., 1986), the secretion signal of rice alpha-amylase 3 gene (Sutliff et al., 1991) and the secretion signal of tobacco PR1 protein (Cornelissen et al., 1986). Particularly useful signal peptides according to the present invention include chloroplast transit peptides (e.g., Van Den Broeck et al., 1985), or the optimized chloroplast transit peptides of U.S. Patent No. 5,510,471 and U.S. Patent No. 5,635,618 that cause the transport of proteins to chloroplasts, secretion signal peptides, or peptides that direct proteins to other plastids, mitochondria, ER, or another organelle that is the target.Signal sequences for targeting to intracellular organelles or for secretion outside of or to the cell wall of a plant cell are found in proteins that are naturally targeted or secreted, 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 protein of the invention taught herein is co-expressed, optionally under the control of various promoters, with other proteins that control, preferably enhance or induce, apomixis or apomixis in a single host. Such other genes may be, for example, genes for conferring apomixis such as polysporous reproduction described in International Publication No. WO 2017 / 039452A1, which is incorporated herein by reference.

[0133] In another embodiment, the protein of the invention is transgenically introduced into a germplasm preferably containing other interesting genes such as genes for conferring apomixis (e.g., genes for polysporous reproduction). Through mating and selection, hybrids are created that can stack several interesting genes.

[0134] Co-expression host plants can be readily obtained by transforming plants that already express the proteins of the present invention or by crossing plants transformed with various nucleic acids of the present invention. It is understood that various proteins can be expressed in the same plant or each 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 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 both selection purposes and weed control options, the transgenic plants of the present invention are also transformed with DNA encoding a protein conferring resistance to a herbicide, for example, a broad-spectrum herbicide such as, for example, glufosinate ammonium as the active ingredient (e.g., Liberty® or Basta; resistance is conferred by the PAT or bar gene; see European Patent No. 0242236 and European Patent No. 0242246) or glyphosate (e.g., RoundUp®; resistance is conferred by the EPSPS gene; see, for example, European Patent No. 0508909 and European Patent No. 0507698), which are herbicides based on. Using a herbicide resistance gene (or other gene conferring a desired phenotype) as a selectable marker further has the advantage of avoiding the introduction of an antibiotic resistance gene.

[0136] Alternatively or in addition, other selectable marker genes such as antibiotic resistance genes may be used. Since it is generally not recognized to retain antibiotic resistance genes in transformed host plants, these genes can be removed again following the selection of transformants. There are various techniques for the removal of transgenes. One method of removal is to flank the transgene with lox sites and, following selection, cross the transformed plant with a CRE recombinase-expressing plant (see, for example, EP506763B1). Site-specific recombination results in the excision of the marker gene. Another site-specific recombination system is the FLP / FRT system described in EP686191 and U.S. Patent No. 5,527,695. Site-specific recombination systems such as CRE / LOX and FLP / FRT can also be used for the purpose of gene stacking. Furthermore, a one-component excision system has been described (see, for example, WO97 / 37012 or WO95 / 00555).

[0137] Preferably, the nucleic acids of the invention are used to create transgenic plant cells, plants, plant seeds, etc., and any derivatives / offspring thereof, with an enhanced apomictic phenotype. Transgenic plants with enhanced apomixis can be created, as described herein, by transforming plant host cells with the nucleic acids of the invention or variants and / or fragments thereof that preferably encode a protein having the amino acid sequence of SEQ ID NO: 1 under the control of a suitable promoter, and by regenerating transgenic plants from said cells. Preferably, the transgenic plants of the invention exhibit enhanced apomixis as compared to controls with non-transformed vectors or empty vectors. As a result, for example, transgenic lettuce plants with enhanced apomixis are provided. Thus, a transformed plant expressing the protein according to the invention exhibits enhanced apomixis if it shows a significant increase in apomixis as compared to controls transformed with non-transformed or empty vectors. By expressing an appropriate amount of the protein of the invention capable of inducing apomixis at appropriate times and / or positions, the enhancement of the apomictic phenotype can be fine-tuned. Such fine-tuning may be carried out by determining the most suitable promoter and / or by selecting transgenic "events" showing the desired expression level.

[0138] Transformants, hybrids or inbred lines that express the desired level of the protein of the present invention and / or contain the nucleic acid of the present invention at the desired or desired level can be selected, for example, by analyzing the copy number (Southern blot analysis), the mRNA transcription level (e.g., RT-PCR using primer pairs or adjacent primers capable of amplifying the protein of the present invention), or by analyzing the presence and level of the unitary reproductive protein in various tissues (e.g., SDS-PAGE; ELISA assay, etc.). By way of example, single-copy transformants may be selected for regulatory reasons, but the sequences adjacent to the insertion site of the transgene are analyzed, preferably sequenced, to characterize the "event". Transgenic events that result in high or medium expression of the protein of the present invention are selected for further development until high-performance elite events with stable transgenes are obtained.

[0139] Transformants that express the protein of the present invention and / or contain the nucleic acid of the present invention may also contain other transgenes, for example, other transgenes that confer disease resistance or resistance to other biotic and / or abiotic stresses, or that confer apomixis. To obtain such plants with "stacked" transgenes, other transgenes may be introduced into the transformant, or the transformant may be subsequently transformed with one or more other genes, or alternatively, several chimeric genes may be used to transform plant lines or varieties, either way. For example, several transgenes may be present on a single vector, or on various vectors that are co-transformed.

[0140] In one embodiment, the following genes are combined with the nucleic acids of the present invention: known disease resistance genes, particularly genes that confer enhanced resistance to necrotrophic pathogens, virus resistance genes, insect resistance genes, abiotic stress resistance genes (such as drought tolerance, salt tolerance, heat tolerance or cold tolerance, etc.), herbicide resistance genes, and the like. Thus, the stacked transformants can have a broader biotic and / or abiotic stress resistance against pathogen resistance, insect resistance, nematode resistance, salinity, low temperature stress, heat stress, water stress, etc. Also, the silencing approach may be combined with the expression approach in a single plant. For example, the silencing of the Par allele may be combined with the expression of the par allele, or vice versa. Optionally, the nucleic acids of the present invention may be used to suppress apomixis, for example, by silencing, knocking down, or reducing the expression of apomixis 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 (such as a promoter sequence) of the Par allele(s) present in the plant or plant cell, or by introducing RNAi targeting the transcript of the Par allele(s). Thus, the present invention also provides a method for reducing or eliminating apomixis in a plant or plant cell, comprising the following steps: a) reducing or eliminating the expression of a nucleic acid capable of inducing apomixis and / or functional in apomixis, as defined herein, in one or more plant cells; b) selecting the plant cells in which the expression has been reduced or eliminated; c) regenerating a plant from the plant cells and including.

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

[0142] All plants, plant parts (e.g., seeds, cells, tissues), and plant products (e.g., fruits) and progeny of any of the transgenic plants described herein are included herein. For the above-mentioned whole plants, plant parts, plant products and progeny, due to 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 apomictic gene, and / or without being limited thereto, by using genomic mutation analysis such as sequence-based genotyping (SBG) or KeyGene® SNPSelect analysis, they can be identified. Also, an "event-specific" PCR diagnostic method can be developed. In this case, the PCR primers are based on plant DNA adjacent to the inserted transgene. See U.S. Patent No. 6,563,026. Similarly, an event-specific AFLP fingerprint or RFLP fingerprint for identifying transgenic plants or any plants, seeds, tissues or cells derived therefrom may be developed.

[0143] The transgenic plants according to the present invention preferably do not exhibit undesirable phenotypes such as a decrease in yield, an enhanced susceptibility to diseases (especially necrotrophs), or undesirable structural changes (dwarfing, deformation), etc., and, when such phenotypes are found in the primary transformants, they can be removed by conventional methods. It is understood that any of the transgenic plants described herein may be heterozygous, homozygous or hemizygous with respect to the transgene.

[0144] The present invention also relates to plants, seeds, plant parts (e.g., plant cells) and plant products obtainable or obtainable by the methods detailed herein, preferably comprising the proteins of the invention, the nucleic acids of the invention and / or the constructs of the invention. Preferably, said proteins, nucleic acids and / or constructs are capable of inducing apomixis and / or are functional in apomixis as detailed herein. The plants of the invention are preferably of the species listed herein as suitable host plants. Such methods include the transfer of the nucleic acids of the invention from plants to progeny, and / or the transformation of plant cells with the nucleic acids of the invention as transgenes, and the subsequent regeneration of plants from said plant cells. Preferably, the plants, plant parts and / or plant products are not of the genus Lactuca in the broad sense, but contain the nucleic acids of the invention, wherein said plants or plant cells are preferably of the species listed herein as suitable host plants, preferably from a family selected from the group consisting of Brassicaceae, Cucurbitaceae, Fabaceae, Gramineae, Solanaceae and Asteraceae (Compositae).

[0145] Preferably, the plants, plant parts and / or plant products contain the nucleic acids of the invention by genetic modification or by gene transfer, wherein preferably said nucleic acids are integrated into the genomes of the plants, plant parts and / or plant products. Preferably, said plants, plant parts and / or plant products are capable of apomixis and / or are functional in apomixis. Even more preferably, said plants, plant parts and / or plant products are further capable of apomixis. The present invention provides seeds, plant parts or plant products or plant cells of the plants of the invention.

[0146] The present invention also relates to plant parts and plant products derived from the plants of the present invention, wherein the plant parts and / or plant products comprise the protein of the present invention as defined herein, the nucleic acid of the present invention as defined herein, and / or the construct of the present invention as defined herein, which may be fragments as defined herein that enable the assessment of the presence of such protein, nucleic acid or construct in the plant from which the plant part of the plant product is 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-proliferative materials.

[0147] Any plant can be a suitable host, but most preferably, the host plant species is required to be a plant species that is expected to benefit from enhanced or reduced apomixis. Suitable hosts include any plant species. In particular, cultivars or breeding lines with otherwise good agricultural characteristics are preferred. A person skilled in the art, together with appropriate control plants, knows how to create transgenic plants and evaluate apomixis to test whether the nucleic acids and / or proteins taught herein, and / or their variants or fragments, can confer the required increase or decrease in apomixis onto the host plant.

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

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

[0150] In a preferred embodiment, the plants, plant parts, plant cells or seeds taught herein are from plant species selected from the group consisting of the genus Taraxacum, the genus Actaea, the genus Artemisia, the genus Capsicum, the genus Solanum, the genus Cucumis, the genus Zea, the genus Gossypium, the genus Glycine, the genus Triticum, the genus Oryza, the genus Allium, the genus Brassica, the genus Helianthus, the genus Beta, the genus Cichorium, the genus Chrysanthemum, the genus Pennisetum, the genus Secale, the genus Hordeum, the genus Medicago, the genus Phaseolus, the genus Rosa, the genus Lilium, the genus Coffea, the genus Linum, the genus Cannabis, the genus Manihot, the genus Daucus, the genus Cucurbita, the genus Citrullus, and the genus Sorghum.

[0151] Suitable host plants include, for example, maize / corn (Zea mays species), wheat (Triticum species), barley (e.g., Hordeum vulgare), oats (e.g., Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp, e.g., Glycine max), cotton (Gossypium species, e.g., Gossypium hirsutum, G. barbadense), Brassica spp. (e.g., Brassica napus, B. juncea, B. oleracea, B. rapa, etc.), sunflower (Helianthus annuus), safflower, yam, cassava, alfalfa (Medicago sativa), rice (Oryza species, e.g., Oryza sativa indica cultivar group or japonica cultivar group), forage grasses, pearl millet (Pennisetum spp., e.g., P. glaucum, P.(glaucum)), tree species (pine (Pinus), poplar, fir, castor oil plant, etc.), camphor tree, coffee tree, oil palm, coconut, vegetable species, for example, pea, zucchini, beans (for example, Phaseolus species), pepper, cucumber, artichoke, asparagus, eggplant, broccoli, garlic, leek, lettuce, onion, daikon radish, turnip, tomato, potato, Chinese cabbage, carrot, cauliflower, chicory, celery, spinach, endive, water chestnut, beet, fruits having pulp (grape, peach, plum, strawberry, mango, apple, plum, cherry, apricot, banana, blackberry, blueberry, citrus, kiwi, fig, lemon, lime, nectarine, raspberry, watermelon, orange, grapefruit, etc.), ornamental species (for example, rose, petunia, chrysanthemum, lily, gerbera species), herbs (mint, parsley, basil, thyme, etc.), woody trees (for example, Populus species, Salix species, Quercus species, Eucalyptus species), fiber species, for example, flax (Linum usitatissimum) and hemp (Cannabis sativa) are included.

[0152] Marker-assisted selection and introgression or combination of one or more Par alleles The nucleic acid of the present invention can be used for marker-assisted selection of Par alleles or par alleles of Lactuca species and / or other plant species, and for creating intra- or inter-specific hybrids with plants in which and / or to which a Par allele or par allele (or variant) is found, for the transfer and / or combination of different or identical Par alleles or par alleles into and / or in such plants for the purpose of creating intra- or inter-specific hybrids with plants in which and / or to which a Par allele or par allele (or variant) is found, and for the transfer and / or combination of these into and / or in such plants. It can be used as a gene marker for this purpose.

[0153] Based on these arrays, a variety of many marker assays can be developed. The development of marker assays generally involves the identification of polymorphisms between the Par allele and the par allele, and as a result, the polymorphism is a genetic marker that "marks" a specific allele. Then, the polymorphism(s) is used in the marker assay. For example, the sequences of the Par alleles taught herein are correlated with the presence or enhancement of apomixis. This can be done, for example, by screening apomictic plant material and / or non-apomictic plant material for the Par allele or a portion of the nucleotide sequence of the par allele taught herein in order to correlate a specific allele with apomixis or non-apomixis. Thus, PCR primers or probes can be created to detect such nucleotide sequences in samples (e.g., RNA, cDNA, or genomic DNA samples) obtained from (non-)apomictic plant material. The sequences or portions thereof are compared, and polymorphic markers correlated with apomixis are identified. Then, polymorphic markers such as SNP markers linked to the Par allele or the par allele can be developed into rapid molecular assays for screening plant material for the presence or absence of apomictic alleles. Thus, the presence of the Par allele or the par allele linked to the marker is indicated by the presence or absence of these "genetic markers", and the detection of the Par allele or the par allele can be replaced by the detection of the genetic marker.

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

[0155] In one aspect, PCR primers and / or probes, molecular markers, and kits for detecting the nucleic acids of the present invention, or related or derived RNA sequences (such as transcripts), are provided. Degenerate or specific PCR primer pairs for amplifying the nucleic acids of the present invention from a sample can be synthesized based on the nucleotide sequences taught herein or their variants, as is 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) may be used as primers or probes.

[0156] Similarly, DNA fragments containing the Par allele or the sequence of the par allele taught herein, or their complements, can be used as hybridization probes. The detection kits provided herein comprise either Par (allele-) specific primers and / or Par (allele-) specific probes, and related protocols, to use the above primers or probes, and as a result, detect the nucleic acids of the present invention in a sample. Such detection kits can be used, for example, to determine whether a plant has been transformed with the nucleic acids of the present invention, or to screen the germplasm of dandelions and / or other plant species for the presence of the Par allele and optionally for zygosity determination. Accordingly, in one embodiment, a method for detecting the presence or absence of the nucleotide sequence encoding the protein of the present invention in a plant tissue, such as a dandelion tissue, or its nucleic acid sample, is provided. The method of the present invention comprises: a) Obtaining a plant tissue sample or its nucleic acid sample from one or more plants; b) Analyzing a nucleic acid sample using a molecular marker assay for the presence or absence of one or more markers linked to the par allele, wherein the marker assay detects the presence of the nucleic acid of the present invention associated with apomixis, and optionally c) Selecting a plant comprising one or more of said markers for further use, can be included. Alternatively or in addition, the method of the present invention comprises: a) Obtaining a plant tissue sample or its nucleic acid sample from one or more plants; b) Analyzing a nucleic acid sample using a molecular marker assay for the presence or absence of one or more markers linked to the par allele, wherein the marker assay detects the presence of the nucleic acid of the present invention associated with non - apomixis, and optionally c) Selecting a plant comprising one or more of said markers for further use, can be included.

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

[0158] Application of apomixis The nucleic acids and / or proteins of the present invention may be used to confer apomixis to increase ploidy, for screening (e.g., one or more apomictic loci in plants or plant cells), genotyping, and / or for the production of doubled haploids. Preferably, said use is within plant biotechnology and / or breeding, i.e., within / on plants or plant cells.

[0159] Apomixis is an element of apomixis, and the genes of apomixis are used in combination with the genes of apomixis (e.g., diplospory) to create apomixis, and as a result, preferably, apomixis can be used for the applications listed herein. These genes can be introduced into sexually reproducing crops by transformation, gene transfer, or by modifying appropriate endogenous genes, thereby converting the apomixis genes into apomeiotic (or diplosporous) genes. Using knowledge of the structure and function of apomixis genes, these genes can also be modified so that endogenous sexual reproduction genes become apomixis genes. A preferred use is to place the apomixis gene under an inducible promoter so that apomixis can be switched off when sexual reproduction creates new genotypes and switched on when apomixis is required to propagate elite genotypes.

[0160] Nucleic acids or derivatives thereof can be used as components of apomixis. Functional gametophytic apomixis requires both apomixis and apomixis. Apomixis can be achieved by a combination of mutations that affect meiosis (Crismani et al., 2013), resulting in no reduction of chromosomes in the megaspore, i.e., mitotic rather than meiotic results. Somatic cells that take on the fate of the gametophyte through epigenetic changes (Grimanelli, 2012) can also give rise to non-reduced spore-like cells that have the potential to produce non-reduced gametes (egg cells). In another embodiment, apomixis is effected by transgenic or non-transgenic expression of natural apomixis genes. By any means, the appropriate temporal and spatial expression of the nucleic acids of the invention that can form non-reduced egg cells and induce apomixis can induce egg cells to behave as zygotes and divide in the absence of fertilization.

[0161] Apomictic genes can be used in a completely new way, for example, not directly as a tool for apomixis. For example, in apomixis, both apogamy and parthenogenesis are combined in a single plant. However, if apogamy is used in one generation and parthenogenesis is used in the next generation, the ploidy level increases by apogamy and decreases by parthenogenesis, so that the sexual gene pool of the crop at the diploid and polyploid levels is likely to be linked. Such a thing is very useful because polyploid populations can tolerate more mutations and can be better for mutagenesis. Polyploid plants can also be considered stronger. However, diploid populations are better for selection, and diploid mating is better for genetic mapping, construction of BAC libraries, etc. Polyploid parthenogenesis can produce haploids that can mate with diploids. Diploid diplospory can produce non-reduced 2n egg cells that are fertilized by pollen from polyploids to produce polyploid offspring. Thus, the alternation of apogamy and parthenogenesis in different breeding generations links the diploid and polyploid gene pools.

[0162] Another use of nucleic acids and their derivatives (transcripts or encoded proteins) not involving apoptosis is the production of haploid progeny, which can be used for the production of haploids and genomic doubling of doubled haploids (DH) (e.g., spontaneous genomic doubling, colchicine, sodium azide or other chemicals). Doubled haploids can be used as parents to produce sexual F1 hybrids. Doubled haploids are the fastest way to make plants homozygous. While doubled haploids can make plants homozygous, on the other hand, for the second fastest method, self-pollination, it takes 5 to 7 generations for diploid plants to reach a significantly high level of homozygosity. There are several methods for producing doubled haploids. In some plant species, haploids can be produced by microspore culture. Other methods include the production of haploid embryos (female gametophyte development) by pollination with irradiated pollen (melon) or pollination with specific pollinator stocks (maize, potato). These methods have limitations such as cost, genotype infertility, labor intensity, etc. In some crops, there is no method for producing haploids (e.g., tomato). The dominant allele of the parthenogenesis gene may significantly improve the frequency of female gametophyte development and potentially reduce the cost of haploid production.

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

[0164]

Table 1

[0165] [Table 2] [Examples]

[0166] Example 1 Materials and methods Plant material Wild-type apomictic triploid dandelion A68 and sexual diploid dandelion FCH72.

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

[0168] Plant transformation method 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). Taraxacum officinale A68 explants obtained from in vitro propagated plants of subcultured seeds grown on half - strength MS20 medium containing 0.8% agar were used as starting materials for plant transformation. 50 ml of an overnight culture of Agrobacterium tumefaciens (Rhizobium radiobacter) such as strain C58C1 with a binary vector in LB medium was used for co - culture at a 10 - fold dilution (resuspended and diluted with liquid MS20). The explants were cut into pieces approximately 0.5 cm 2 in size and co - cultured for 2 - 3 days. Subsequently, the explants were transferred to callus induction medium (CIM; MS 4.4 g l−1 containing sucrose 20 g l−1, micro - and macro - nutrients, agar 8 g l−1, BAP 1 mg l−1, IAA 0.2 mg l−1, glufosinate 3 mg l−1 for plant selection, vancomycin 100 mg l−1 and cefotaxime 100 mg l−1, pH 5.8). The explants were transferred to fresh CIM every week. When callus appeared, the callus was transferred to shoot induction medium (SIM; MS 4.4 g l−1 containing sucrose 20 g l−1, micro - and macro - nutrients, agar 8 g l−1, zeatin 2 mg l−1, IAA 0.1 mg l−1, GA3 0.05 mg l−1, glufosinate 3 mg l−1 for plant selection, vancomycin 100 mg l−1 and cefotaxime 100 mg l−1, pH 5.8). Finally, shoots with a diameter of several cm were rooted in rooting medium (RM; MS 2.2 g l−1 containing sucrose 20 g l−1, micro - and macro - nutrients, agar 8 g l−1, vancomycin 100 mg l−1 and cefotaxime 100 mg l−1, pH 5.8). The rooted shoots were transferred to potted soil in a greenhouse.

[0169] Results For the rooted plants obtained from Agrobacterium transformation, genotyping was performed by PCR for the presence of each T-DNA encoding Cas9 and guide RNA-1 or guide RNA-2 in the plant genome. The plants that were positive in this test (designated herein as transgenic plants) were grown until fruiting. Individual transgenic plants derived from individual calli containing any one of these constructs had normal viable dark black-gray seeds, and some of such plants had abnormal light gray seeds (see Table 2). These light gray seeds were found to be empty, lacking embryos, infertile, and did not germinate. Control plants (negative for T-DNA or transformed with a 35S-GUS control construct) never had similar abnormal light gray seeds, and all control plants had normal seed heads containing fertile black-gray seeds. Next, genotyping was performed for all transgenic plants by amplicon sequencing of the guide RNA-1 target genomic DNA region on the Illumina MiSeq System. All transgenic plants showing abnormal light gray seeds were found to have small deletions or small insertions in the parthenogenetic gene, more particularly, 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 DSB.

[0170] None of the transgenic plants with normal black seeds had any changes in the gene sequence. Table 2 summarizes the observed small deletions or small insertions and their effects on the translation of the protein sequence of the coding sequence, and Figure 1 shows the multiple alignment of the amplicons.

[0171] For the fruit set observed in transgenic plants having a small deletion in the gene of SEQ ID NO: 5, it was interpreted as an indicator of loss of the apomictic phenotype (referred to herein as apomixis loss or LoA), and further loss of the parthenogenetic phenotype (parthenogenesis loss or LoP). Apomictic plants always have the dominant Par allele.

[0172] The high seed set of triploid dandelions in the absence of cross-pollination by other plants is a clear indicator of apomixis. Self-pollination can be excluded as an alternative explanation because, due to unbalanced male and female meiosis in triploids, the sexually produced egg cells and pollen grains would have extremely low fertility. The deletion of the Par allele results in LoP and thus LoA. However, LoA may also be caused by disturbances in other developmental processes. Therefore, LoP plants are a subset of LoA plants, and further tests are required to identify the observed phenotype as a LoP loss phenotype.

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

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

[0175] Example 2 Using a gene essential for apomixis, the apomictic trait can be transferred to plants without apomixis or without apomixis. The transfer of such an apomictic trait can be carried out using either the gene essential for apomixis or the coding sequence of the gene having SEQ ID NO: 5 or a homologous gene. A binary vector driven by its natural promoter or a female gamete-specific promoter is prepared with a T-DNA having at least the gene of SEQ ID NO: 5 or a homologous gene. This gene construct is transformed into a plant without apomixis, such as lettuce or Arabidopsis thaliana, by Agrobacterium-mediated transformation. Plants tested positive for the presence of the transgene are evaluated for the occurrence of apomixis. Since the trait is dominant, the test is performed on primary transformed plants (T0). For apomixis, it can be detected microscopically in non-apomictic plants by Nomarski Differential Interference Microscopy (DIC) of ovules permeabilized with methyl salicylate (Van Baarlen et al. 2002). In the absence of cross-fertilization or self-fertilization, apomictic egg cells develop into embryos. In plants harboring the above-mentioned T-DNA, at least some of such embryos are found.

[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 construct The binary vector was constructed with the following components encoded on the T-DNA region; the Arabidopsis thaliana EC1.1 promoter (as in Sprunk et al., 2012) driving the expression of the Par allele CDS sequence of dandelion (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 selectable neomycin phosphotransferase gene (nptII). Techniques suitable for creating such binary vectors are Gateway®, Golden Gate, or Gibson Assembly® (see, for example, Ma et al., 2015). The transgenic lines harboring this T-DNA were numbered with code pKG10824.

[0178] Plant transformation method 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, by Curtis et al. Any other method suitable for genetic transformation of lettuce, such as those described by Michelmore et al. (1987) or Chupeau et al. (1989), may be used to create plants harboring the desired T-DNA.

[0179] Results As described below under the section "DNA constructs", plants that were tested positive for the presence of the transgene were evaluated for apomictic development. Since the trait is dominant, the tests were performed on primary transformed plants (T0). In the absence of cross- or self-fertilization, apomictic egg cells develop into embryos. To prevent fertilization of any plants harboring the transgene, the plants were grown in a greenhouse and all flowers were manually emasculated prior to microscopic observation. Emasculation was performed by clipping the bracts before the corolla grew. For apomixis, it can be detected microscopically in non-apomictic plants by Nomarski Differential Interference Microscopy (DIC) of cleared ovules. Here, a clearing method using chloral hydrate was applied, which is a commonly used method for clearing plant ovules for microscopic imaging (e.g., Franks et al. 2016). 75 hours after emasculation, flower buds were harvested and the ovules were cleared with chloral hydrate. Multiple embryos were observed in the cleared ovules in all seven transgenic lines evaluated (see Table 3 showing data for five of these lines). Figure 3 shows an example of such observed embryos. In some single ovules, multiple embryos were observed (polyembryony). Figure 4 shows an example of the observed polyembryony. However, polyembryony was observed at a much lower frequency than single embryos. In non-emasculated transgenic lines, embryos could already be observed before male gametogenesis was complete and thus before fertilization. Also, in these non-emasculated transgenic plants, polyembryony was observed in some rare cases. In non-transformed control plants, which were emasculated and imaged by the same method, no embryos were observed at all.

[0180]

Table 3

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

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

[0183] The inventors 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 (SEQ ID NO: 2) of the Par allele at a distance of 102 bp upstream (3') of the start codon, and it was identified that this sequence is not present in the sexual counterparts (SEQ ID NOs: 7 and 12). This MITE or MITE-like sequence is expected to direct the parthenogenetic phenotype, for example, by being responsible for changes in the expression level of the encoded protein, and can be considered the cause of such a parthenogenetic phenotype.

[0184] These specific polymorphisms, insertions, or deletions of the parthenogenetic alleles can be introduced into non-parthenogenetic plants by chemical mutagenesis or targeted gene editing of the sexual allele homologs of the parthenogenetic genes of the present invention. For example, the promoter sequence of the PAR gene may be replaced with the promoter of the Taraxacum allele, i.e., SEQ ID NO: 2, and as shown above, a MITE sequence may be introduced into the PAR gene of the non-parthenogenetic plant at a position homologous to the MITE sequence of the Taraxacum Par allele. When such specific polymorphisms, insertions, or deletions of the parthenogenetic alleles are introduced, the plant will acquire 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-fertilization or self-fertilization, parthenogenetic egg cells develop into embryos. In plants harboring the above-described specific polymorphisms, insertions, or deletions, at least some of such embryos are found.

[0185] Example 4 Triploid and tetraploid Taraxacum apomicts were crossed as pollen donors with diploid Taraxacum koksaghyz plants. The pollen donors themselves were obtained by crossing sexual Taraxacum with apomictic Taraxacum brevicorniculatum pollen donors. Thus, the apomixis genes were derived from Taraxacum brevicorniculatum (Kirschner et al. 2012). Triploid progeny plants were tested using PCR markers for the presence of the Par allele and the diplospory (Dip) allele (see International Publication No. WO 2017 / 039452 A1) and for the production of apomictic seeds. 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 polyspore reproductive gene VPS13 to specifically amplify the Dip allele. Using these primers, the presence of the Dip allele resulted in a 829 bp PCR product, while the absence of this allele did not result in a 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 the Par allele could be distinguished by the length of the PCR product.

[0188]

Table 4

[0189] As shown in Table 5 reported in this specification below, 56 progeny plants were tested, and a 100% correlation was observed between the presence of the Par allele and apomixis. Plants that produced apomictic seeds and were negative for the DIP and PAR markers were not observed.

[0190]

Table 5

[0191] Therefore, it can be concluded that the markers developed from the Par locus of Taraxacum officinale also identified the presence of apomixis in different species of Taraxacum, and this presence is further evidence that the Par allele causes apomixis.

[0192] Example 5 Construction of a gamma-ray irradiated deletion population of apomictic A68 Approximately 3 × 2000 seeds from clone A68 were gamma-irradiated at 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 the greenhouse. After a vernalization period of two months at below 10 °C, the plants were grown again in a heated greenhouse. More than 90% of the plants flowered and produced seeds. The plants were classified according to whether they exhibited the apomixis loss phenotype (LoA). Apomictic A68 plants spontaneously produce seeds and form large white seed heads with a dark brown center, where the seeds (achenes: fruits with one seed) are attached to the receptacle. In the apomixis loss phenotype, the center of the seed head is lighter and, in many cases, the seed head has a reduced diameter. Finally, 102 plants were identified as having the apomixis loss phenotype.

[0193] Single-dose dominant markers can be mapped to autopolyploid plants using the method of Wu et al. (1992). A segregation analysis approach (Michelmore et al. 1991) was used to find AFLP markers (Vos et al. 1995) linked to the Par locus. 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 the 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). In the case of the Par pool, apomictic plants were used. One hundred and forty-seven AFLP primer combinations (Vos et al. 1995) were screened for the presence of fragments in pool A and the absence of fragments in pool B. Control fragments in both pools were verified in individuals from both pools. A genetic map of the chromosomal region of the Par locus was constructed based on the TJX3-20 × A68 cross (76 plants) using 17 AFLP markers. Fourteen out of the 17 AFLP markers co-segregated tightly with the Par phenotype. This is an indication of suppression of recombination near the Par locus.

[0194] When one of the three homologous chromosomes is partially deleted, the single-dose AFLP markers located in the deletion region will be lost. AFLP analysis of LoA plants showed that some LoA plants had lost one or more AFLP markers genetically linked to the Par locus. LoA plants lacking Par-linked AFLP markers were crossed with diploid pollen donors, and then tetraploid progeny were produced. This indicated that these LoA plants had lost the apomixis phenotype but were still of the polysporic type and produced unreduced egg cells. These LoA plants could be ranked based on the number of genetically linked AFLP markers of Par they lacked. The number of lost AFLP markers is an indicator of the size of the deletion. The AFLP marker most frequently lost in LoA plants was considered to be the closest to the Par locus. Plant i34 had lost the fewest PAR-linked AFLP markers and was thus considered to have the smallest deletion.

[0195] Example 6 Genotype and allele-specific expression of the Par gene in the megagametophyte of apomictic dandelion plants versus Par deletion and sexual plants Cells and tissues from various developmental stages of the gametophyte were isolated by laser-assisted microdissection (LAM) using an SL μCut instrument (2001, Medical Micro Instruments, Glattbrugg, Switzerland) that uses a solid UV-A laser (wavelength approximately 350 nm) to cut tissues, as described by Wuest et al. (2010) and Florez-Rueda et al. (2020). Subsequently, transcriptome analysis was performed. RNA was extracted using the PicoPure (trademark) RNA isolation kit according to the manufacturer's (Thermo Fisher Scientific) instructions. To maintain the initial differences in expression between samples, after reverse transcription into DNA, 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 of Dutch origin (abbreviated: APO), 2. the tetraploid PAR deletion progeny (abbreviated: DEL) from the 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 of French origin (abbreviated: SEX).

[0197] For each plant line, five different developmental stages / tissue types were sampled (Table 6). In the case of very young stages, a single sample was analyzed. From mature embryo sacs, the central cell and the egg apparatus (egg cell and synergids) were sampled in triplicate. Altogether, these represent nine samples for each plant line (Table 6).

[0198]

Table 6

[0199] Linear amplified DNA was sequenced on an Illumina Hiseq platform. Individual reads were mapped to the sequence of the Par gene (Figure 5). Expression of the Par gene was not detected in any of the PAR deletion or SEX plants (all stages and tissues). In the APO line, reads specific to the Par gene were found in both the egg apparatus and the central cell, in all samples of the mature gametophyte. 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 the apomict, while it is not expressed in seven samples of the deletion line or seven samples of the sexual line, which are in the same developmental state. This further emphasizes that the ectopic expression of the gene in the central cell and the egg apparatus is the cause of the loss of egg cell arrest and, as a result, the apomictic development of the embryo.

[0201] As also shown in Example 3, the expression of the Par gene in the apomict of these cells cannot be suppressed as in the sexual case, probably due to the influence of the MITE sequence in the promoter region. Since MITE is large, it may physically prevent the binding of transcription factors of the Par gene.

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Claims

1. 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) a variant or fragment of any one of a) to e). A nucleic acid associated with parthenogenesis in plants, comprising at least one of: Preferably functional in parthenogenesis Nucleic acid.

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

3. a) encoded by the nucleic acid of claim 1; b) having the amino acid sequence of SEQ ID NO: 1, 6 or 11; and / or c) a variant or fragment of a) and / or b); A protein associated with parthenogenesis in plants, Preferably functional in parthenogenesis protein.

4. 1. A plant or plant cell that is not of the species Taraxacum officinale in the broad sense, comprising a nucleic acid according to claim 1 and / or a protein according to claim 3, preferably from a family selected from the group consisting of Brassicaceae, Cucurbitaceae, Fabaceae, Gramineae, Solanaceae, Asteraceae (Composites), Rosaceae and Poaceae.

5. 5. A plant or plant cell according to claim 4, which contains a nucleic acid according to claim 1 by genetic modification or by gene transfer, preferably said nucleic acid being integrated into the genome of said plant or plant cell.

6. 6. A plant or plant cell according to claim 4 or 5, which is capable of parthenogenesis.

7. A plant or plant cell according to any one of claims 4 to 6, further capable of apomixis, preferably capable of apomixis.

8. A seed, plant part or plant product of a plant or plant cell according to any one of claims 4 to 7.

9. a) introducing into one or more plant cells a nucleic acid according to claim 1 capable of inducing parthenogenesis; b) selecting a plant cell containing said nucleic acid, preferably wherein said nucleic acid is integrated into the genome of said plant cell; c) regenerating a plant from said plant cell; A method for producing a parthenogenetic plant, comprising:

10. A method for producing an apomictic plant, comprising steps a) to c) of claim 9, wherein the one or more plant cells of step a) are capable of apomicsis.

11. 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:

12. b) selecting, preferably by genotyping, from said F1 seeds containing an apomictic phenotype. The method of claim 11 further comprising:

13. The method includes the steps of claim 11 or 12, c) growing at least one F1 plant from said F1 hybrid seed. The method of producing an apomictic hybrid plant further comprises:

14. A plant, seed, plant part or plant product obtainable by the method according to any one of claims 9 to 13.

15. Use of a nucleic acid according to claim 1 or 2 or a protein according to claim 3 for screening parthenogenesis genes in a plant or plant cell, for genotyping a plant or plant cell for parthenogenesis and / or for conferring parthenogenesis to a plant or plant cell.

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