Parthenogenetic methods and compositions

Abstract

Provided herein are methods and compositions for inducing parthenogenesis by expression in female gametophytes of a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell. Also included herein are promoters expressed in egg cells of plants and methods of using these promoters to express gene products in egg cells of plants.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 363,180, filed on April 19, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the fields of plant molecular biology and plant breeding.

[0004] References to sequence listings submitted electronically

[0005] An official copy of the sequence listing is submitted electronically via EFS-Web as an XML-formatted sequence listing, with the file name 8893-WO PCT_SEQ_LIST_ST26.XML, created on April 11, 2023, and having a size of 287 kilobytes, and is submitted simultaneously with this specification. The sequence listing contained in this XML-formatted file is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0006] In plants, germline cells (germline) provide transgenerational inheritance of genetic information in each progeny by producing spore mother cells during sporogenesis. For example, during sporogenesis, megaspore mother cells develop into female gametes, with egg cells and central cells forming embryos and endosperm, respectively; or microspore mother cells develop into male gametes, producing four haploid microspores, each of which further develops into mature pollen grains. A key aspect of the unique role of germline cells is to provide the genetic information that future progeny receive, with half of the genetic contribution coming from the female gametes and half from the male gametes. The egg cell is fertilized with a sperm cell to form a diploid zygote, while a second sperm cell fuses with the two polar nuclei of the central cell to form a triploid endosperm. The endosperm is the terminal vegetative tissue of the embryo, but does not contribute to the germline. After fertilization, the zygote gives rise to the embryo, a process known as zygotic embryogenesis, which is a characteristic of sexual reproduction. The newly formed embryo undergoes an embryogenic developmental program that includes underlying regulatory programs influenced by genetic determinants and epigenetic reprogramming, leading from the embryogenic cell state to the acquisition of one or more differentiated cell fates, ultimately giving rise to the plant with all its differentiated tissues.

[0007] Parthenogenesis is a form of asexual reproduction in which the growth and development of female gametes (embryos) occurs without fertilization by sperm. Female gametes produced by parthenogenesis can be haploid or diploid. Summary of the invention

[0008] Described herein is a method for expressing a gene product in an egg cell of a plant, the method comprising introducing into the plant a polynucleotide comprising (a) a polynucleotide sequence of SEQ ID NO: 37, 38 or 39, (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, 38 or 39; (c) a fragment of the polynucleotide of (a) or (b), wherein the fragment retains its ability to drive expression of a heterologous polynucleotide sequence in an egg cell, wherein the polynucleotide sequence drives expression of an operably linked heterologous polynucleotide sequence. In some aspects, the operably linked heterologous polynucleotide sequence is a Babyboom (BBM) polynucleotide encoding a BBM polypeptide that induces haploid induction when expressed in a female gametophyte. Also included herein are plants comprising a promoter operably linked to a heterologous polynucleotide sequence, wherein the promoter is selected from (a) a polynucleotide sequence of SEQ ID NO: 37, 38 or 39, (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, 38 or 39, (c) a fragment of the polynucleotide of (a) or (b), wherein the fragment retains its ability to drive expression of the heterologous polynucleotide sequence in the egg cell of the plant.

[0009] Also described herein are methods of inducing parthenogenesis, wherein the method comprises expressing in a female gametophyte a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in a plant egg cell, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof, wherein the female gametophyte is caused to parthenogenetically reproduce and form haploid embryos in the absence of pollination, and wherein the Babyboom (BBM) polynucleotide is not naturally expressed in the female gametophyte. In some aspects, the Babyboom (BBM) polynucleotide or polypeptide or a fragment thereof is a BBM1, BBM2, BMN2, BMN3, ODP2, and BBML polynucleotide or polypeptide or a fragment thereof. In some aspects, the promoter expressed in the plant egg cell is an egg cell-specific promoter or an egg cell-preferred promoter.

[0010] In some aspects, before or during the expression of a parthenogenetic factor (e.g., a BBM polynucleotide), the female gamete is not fertilized. In some aspects, BBM is expressed in an egg cell before fertilization to induce parthenogenesis, that is, the development of a zygote / embryo is performed in the absence of fertilization. In some aspects, these methods include modifying the regulatory region of an endogenous Babyboom (BBM) polynucleotide so that the Babyboom (BBM) polynucleotide is expressed in a female gamete. In some aspects, these methods include expressing a Babyboom (BBM) polynucleotide from a modified endogenous genomic BBM locus, wherein the modified endogenous genomic BBM locus comprises a modified regulatory region of an endogenous polynucleotide encoding a BBM polypeptide, wherein one or more nucleotides in the regulatory region are modified so that the BBM polypeptide is expressed in a female gamete. In some aspects, these methods include expressing Babyboom (BBM) polynucleotides from modified endogenous genomic egg cell locus, wherein the egg cell coding sequence or genomic sequence in the endogenous genomic egg cell locus is modified so that it encodes BBM polypeptides, wherein the BBM polypeptides are expressed in female gametophytes. In some aspects, female gametophytes are monocotyledonous plants or dicotyledonous plants female gametophytes. In some aspects, female gametophytes are wheat, corn, rice, oats, barley, triticale, sorghum, canola rape, Arabidopsis thaliana (Arabidopsis), cotton, sunflower, safflower, tobacco, hemp, sugarcane, soybean, lawn grass, rye, millet or flax female gametophytes. In some aspects, these methods include (a) contacting haploid embryos with a chromosome doubling agent, continuing a time period sufficient to produce double haploid embryos, (b) separating double haploid embryos, and (c) regenerating double haploid plants from the double haploid embryos of step (b). In some aspects, the methods include (a) regenerating a parthenogenetic plant from a haploid embryo comprising a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell; (b) pollinating the parthenogenetic plant of (a) with pollen from a non-haploid inducer; and (c) rescuing the haploid embryo from the parthenogenetic plant of (b).

[0011] Also included herein are methods for obtaining wheat plants that produce clonal, non-reduced, non-recombinant gametes. In some aspects, the method comprises (a) suppressing the activity of: (1) all endogenous Spo11 or Prd1, Prd2 or Prd3 polynucleotides or polypeptides in a wheat plant cell; (2) all endogenous Rec8 polynucleotides or polypeptides; (3) all endogenous Osd1 polynucleotides or polypeptides; (b) expressing a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell in a female gametophyte derived from the wheat plant cell, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof, wherein the female gametophyte is caused to parthenogenetically reproduce and form an embryo; and (c) obtaining a plant from the embryo, wherein the embryo comprises the Babyboom (BBM) polynucleotide expressed by the egg cell and the suppressed Spo11, Rec8, Osd1, Prd1, Prd2 or Prd3 polynucleotides or polypeptides, thereby producing a wheat plant that produces clonal, non-reduced, non-recombinant gametes.

[0012] Also described herein are methods for obtaining clonal apomixis plants from one or more gametophyte cells in a plant in the absence of egg cell fertilization. In some aspects, these methods include (a) expressing a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell in one or more gametophyte cells, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof that retains haploid induction activity, wherein the Babyboom (BBM) polynucleotide is not naturally expressed in female gametophytes, (b) developing embryos from the gametophyte cells in the absence of egg cell fertilization; and (c) obtaining progeny plants from one or more gametophyte cells, wherein the progeny plants contain chromosomes from the gametophyte cells of (a), thereby achieving reproduction of flowering plants in the absence of egg cell fertilization. In some aspects, in the methods and compositions described herein, the plant is a monocot or a dicot. In some aspects, in the methods and compositions described herein, the plant is wheat, cotton, sunflower, safflower, tobacco, Arabidopsis, soybean, barley, oats, rice, corn, triticale, sorghum, canola, hemp, sugarcane, rye, millet, turf grass, or flax plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1An alignment of BBM peptide sequences is shown. (Consensus sequence = SEQ ID NO: 112, TA-BBM6A = SEQ ID NO. 8, OS-BBM2 = SEQ ID NO: 25, SV-BBM2 = SEQ ID NO: 29; ZM-BBM2 = SEQ ID NO: 21; TA-BBM3A = SEQ ID NO: 2; OS-ODP2 = SEQ ID NO: 23; SV-BBM = SEQ ID NO: 29; ZM-ODP2 = SEQ ID NO: 14). Figure 1 Some A motifs in the sequences are shown in SEQ ID NOs: 107, 109, 116, 117 and 118. Figure 1 Some B motifs in the sequences are shown in SEQ ID NOs: 108, 110, 119, 120 and 121. Figure 1 Some AP2 DNA binding domains in the sequence are shown in SEQ ID NOs:111 and 122.

[0014] Figure 2 An alignment of the wheat TA-BBM native peptide sequence and the modified peptide sequence is shown. (Consensus sequence = SEQ ID NO: 113; TA-BBM3A = SEQ ID NO. 2; TA-BBM3A (ALT1) = SEQ ID NO: 42; TA-BBM3A (ALT2) = SEQ ID NO: 44; TA-BBM6A = SEQ ID NO: 8; TA-BBM6A (ALT1) = SEQ ID NO: 46; BBM6A (ALT2) = SEQ ID NO: 48). Figure 2 The A motif in the sequence is shown in SEQ ID NOs: 107 and 109. Figure 2 The B motif in the sequence is shown in SEQ ID NOs: 108 and 110. Figure 2 The conserved AP2 DNA binding domain in the sequence is shown in SEQ ID NO:111.

[0015] Brief description of sequence listing

[0016] The present disclosure can be more fully understood from the following detailed description and the accompanying sequence listing which form a part of this application.

[0017] The SEQ ID NOs for various polynucleotide and polypeptide sequences are presented in Table A. It is to be understood (as will be appreciated by one skilled in the art) that the present disclosure encompasses more than just these specific exemplary sequences.

[0018] Table A

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027] DETAILED DESCRIPTION

[0028] The disclosure herein will be described more fully hereinafter with reference to the accompanying drawings, in which some but not all possible aspects are shown. Indeed, the disclosure can be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure can satisfy applicable legal requirements.

[0029] It is to be understood that the disclosure is not to be limited to the specific examples disclosed and that modifications and other aspects are intended to be included within the scope of the appended claims.

[0030] It should also be understood that the terminology used herein is for the purpose of describing specific aspects only and is not intended to be limiting. As used in the specification and claims, the term "comprising" may include aspects of "consisting of". Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. In this specification and the claims that follow, reference will be made to several terms defined herein.

[0031] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a protein" includes one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs unless expressly stated otherwise.

[0032] All patents, publications, and patent applications mentioned in this specification are indicative of the levels of those skilled in the art to which the disclosure pertains. All patents, publications, and patent applications are incorporated herein by reference in their entirety, to the same extent as if each individual patent, publication, or patent application was expressly and individually indicated to be incorporated by reference in its entirety.

[0033] The present disclosure provides methods and compositions for inducing parthenogenesis, which include expressing in female gametophytes a BBM polypeptide or fragment thereof encoded by a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell. As shown in Example 1 herein, expression of wheat, corn, rice and Setaria officinalis BBM polypeptides in female gametophytes of wheat plants can be used to induce maternal haploid induction, thereby resulting in the production of haploid embryos without the use of pollination.

[0034] Any suitable polynucleotide encoding a BBM polypeptide or a fragment thereof that can induce maternal haploid induction in female gametophytes when expressed by a promoter expressed in an egg cell can be used in the methods and compositions of the present disclosure. In some examples, the BBM polynucleotide comprises a nucleotide sequence encoding a BBM1, BBM2, BBM3, BBM6, BMN2, BMN3, ODP2 or BBML polypeptide. In some embodiments, the BBM polynucleotide and BBM polypeptide include any of those disclosed in Publication No. US20210180077, and the document is incorporated herein by reference in its entirety. In some aspects, the BBM polynucleotide encoding the BBM polypeptide comprises a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88. In other aspects, the BBM polynucleotide comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88 when aligned over the entire length of the nucleotide sequence.

[0035] Fragments of polynucleotide sequences encoding BBM polypeptides are also encompassed in the embodiments. As used herein, a "fragment" with respect to BBM refers to a portion of a nucleotide sequence encoding a BBM polypeptide. The fragment may encode a biologically active portion of a BBM polypeptide. A nucleic acid molecule that is a fragment of a nucleotide sequence encoding a BBM polypeptide comprises at least about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, , 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 3100, 3200, 3300, 34 00、3500、3600、3700、3800、3900、4000、4100、4200、4300、4400、4500、4600、4700、4800、4900、5000、5100、5200、5300、5400、5500、5600、5700、5800、5900、6000、6100、6200、 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100 or 8200 consecutive nucleotides or up to the number of nucleotides present in the full-length nucleic acid sequence encoding a BBM polypeptide disclosed herein, such as those in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88. "Contiguous nucleotides" is used herein to refer to nucleotide residues that are immediately adjacent to each other. In some examples, the BBM polypeptide or polypeptide fragment comprises one or more of the following: an A motif, a B motif, and / or an AP2 DNA binding domain. Non-limiting examples of the A motif are shown in Figure 1 and Figure 2 , including SEQ ID NOs: 107, 109, 116, 117 and 118. Non-limiting examples of B motifs are shown in Figure 1 and Figure 2 , including SEQ ID NOs: 108, 110, 119, 120 and 121. Non-limiting examples of AP2 DNA binding domains are shown in Figure 1 and Figure 2 including SEQ ID NOs:111 and 122.

[0036] In some embodiments, the BBM polynucleotide is changed to produce a nucleotide sequence encoding a variant BBM polypeptide. In some aspects, the BBM polynucleotide encoding the variant BBM polypeptide is derived from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88 by changing one or more nucleotides by deletion, substitution, addition insertion or a combination thereof. In some examples, the nucleotide or amino acid sequence is maintained in one or more conserved regions of the BBM polypeptide (such as the A motif, the B motif or the AP2 DNA binding domain) and is changed in a non-conserved region. See, e.g. Figure 1 and Figure 2 107, 108, 109, 110, 111, 116, 117, 118, 119, 120, 121 and 122. In some examples, the nucleotide or amino acid sequence is maintained in one or more common regions of the BBM polypeptide (including the A motif, the B motif or the AP2 DNA binding domain) and is changed in the non-conserved region. Variants of the BBM polypeptides that can be used in the methods and compositions described herein retain their ability to induce haploid induction in female gametophytes when expressed by promoters expressed in egg cells. Fragments and variants can be obtained via methods including, but not limited to, site-directed mutagenesis and synthetic construction.

[0037] In some aspects, the BBM polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48. In other aspects, the BBM polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acids set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46, or 48 when aligned over the entire length of the sequence.

[0038] Fragments of BBM polypeptides are also included in the embodiments. As used herein, a "fragment" with respect to BBM refers to a portion of the amino acid sequence of a BBM polypeptide. The fragment may encode a biologically active portion of a BBM polypeptide. A polypeptide that is a fragment of a BBM polypeptide comprises at least about 150, 180, 200, 210, 225, 240, 250, 270, 290, 300, 315, 325, 330, 350, 360, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 consecutive amino acids or up to the number of amino acids present in the full-length amino acid sequence of a BBM polypeptide disclosed herein, such as those in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46, or 48. "Contiguous amino acids" is used herein to refer to amino acid residues that are immediately adjacent to each other. Polypeptide fragments that retain the haploid induction activity of BBM polypeptides and thus retain their ability to induce maternal haploid induction in female gametophytes when expressed by a promoter expressed in an egg cell. In some examples, the BBM polypeptide fragments comprise one or more of the following: an A motif, a B motif, or an AP2 DNA binding domain. Non-limiting examples of BBM B motifs are shown in Figure 1 and Figure 2 , including SEQ ID NOs: 108 and 110. Non-limiting examples of BBM AP2 DNA binding domains are shown in Figure 1 and Figure 2 including SEQ ID NO:111.

[0039] In some embodiments, the BBM polynucleotide is changed to produce a nucleotide sequence encoding a variant BBM polypeptide. In some aspects, the variant BBM polypeptide is derived from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48, for example, by changing one or more amino acid residues by deletion, substitution, addition, insertion of one or more nucleotides. In some examples, the amino acid sequence is maintained in one or more conserved regions of the BBM polypeptide (such as the A motif, the B motif or the AP2 DNA binding domain) and changed in a non-conserved region. See, e.g. Figure 1 and Figure 2 A non-limiting example of a motif is shown in Figure 1 and Figure 2 , including SEQ ID NOs: 107 and 109. Non-limiting examples of B motifs are shown in Figure 1 and Figure 2, including SEQ ID NOs: 108 and 110. Non-limiting examples of AP2 DNA binding domains are shown in Figure 1 and Figure 2 In, including SEQ ID NO: 111. Variants of BBM polypeptides useful in the methods and compositions described herein retain their ability to induce maternal haploid induction in female gametes when expressed by a promoter expressed in oocytes. Fragments and variants can be obtained via methods including, but not limited to, site-directed mutagenesis and synthetic construction.

[0040] Any BBM polynucleotide described herein can be introduced into the female gamete (including the genome of the gamete) using any suitable technique (including but not limited to gene editing or transformation). In some instances, the BBM polynucleotide is part of a recombinant nucleic acid (such as a heterologous polynucleotide and a DNA construct). As used herein, "heterologous" refers to a nucleic acid derived from an alien species, or, if derived from the same species, a nucleic acid obtained by deliberate human intervention to substantially modify its natural form in terms of composition and / or genomic locus. For example, a promoter operably linked to a heterologous structural gene, the heterologous structural gene is from a species different from the species from which the structural gene is derived, or, if from the same species, one or both of which are substantially modified from their original form and / or genomic position.

[0041] Because BBM is not naturally expressed in female gametophytes, expression of BBM is modified by operably linking the polynucleotide to a promoter that drives expression in egg cells. As used herein, "egg cell" means a female gamete that produces an embryo.

[0042] As used herein, "promoter" is an exemplary regulatory element, and generally refers to a nucleotide sequence that can control the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' of the promoter sequence. The promoter sequence comprises a proximal element and a more distal upstream element, the latter element being generally referred to as an enhancer. An "enhancer" is a nucleotide sequence that can stimulate promoter activity, and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of the promoter. An example of an enhancer is the EME described elsewhere herein.

[0043] A promoter generally includes a core promoter (also referred to as a minimal promoter) sequence, which includes a minimal regulatory region to initiate transcription (i.e., a transcription initiation site). Typically, a core promoter includes a TATA box. A core promoter is the minimum sequence required to direct transcription initiation, and typically may not include an enhancer or other UTRs. Promoters may all be derived from natural genes, or may be composed of different elements derived from different promoters found in nature, or may even include synthetic DNA segments.

[0044] A "plant promoter" is a promoter capable of initiating transcription in a plant cell. Any suitable promoter capable of initiating transcription of a polynucleotide in a plant egg cell (i.e., expressing the polynucleotide in an egg cell) can be used in the methods and compositions of the present disclosure, including but not limited to egg cell promoters, egg cell-specific promoters, or egg cell-preferred promoters, and promoters active during female gamete development.

[0045] Exemplary promoters expressed in plant egg cells include, but are not limited to, SEQ ID NOs: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and 86. Also included in the embodiments are any of the fragments or variants thereof.

[0046] Any egg cell promoter and / or egg cell-specific promoter known to those skilled in the art may be used, such as the egg cell-specific EC1.1 and EC1.2 promoters disclosed in Sprunck et al., (2012) Science, 338, 1093-1097 and Steffen et al., (2007) Plant J., 51: 281-92, and those disclosed in US2015 / 0152430 and US 2018 / 0094273, each of which is incorporated herein by reference in its entirety.

[0047] In one embodiment, the present disclosure provides a method for inducing parthenogenesis, the method comprising expressing in a female gametophyte a BBM polypeptide or a fragment thereof encoded by a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell. As shown herein, expression of the BBM polypeptide in the female gametophyte of a plant can be used to induce maternal haploid induction, thereby resulting in the production of haploid embryos without the use of pollination.

[0048] The egg cell promoter nucleotide sequences and methods disclosed herein can be used to regulate the expression of any heterologous nucleotide sequence (such as but not limited to a BBM sequence) in a plant egg cell. In some embodiments, the promoter expressed in the egg cell is an egg cell preferred promoter. In some embodiments, the promoter expressed in the egg cell is an egg cell specific promoter.

[0049] In one embodiment, the disclosure provides a method for expressing a gene product in a plant egg cell. In some aspects, the egg cell is in a plant. In other aspects, the egg cell is isolated from a plant. As used herein, a gene product refers to a polynucleotide or a polypeptide. In some aspects, the method comprises introducing a promoter comprising a polynucleotide sequence shown in SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 into an egg cell.

[0050] In some aspects, the egg cell promoter comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 86 when aligned over the entire length of the sequence.

[0051] Fragments of the egg cell promoter are also encompassed in the embodiments.As used herein, a "fragment" with respect to an egg cell promoter refers to a portion of the nucleotide sequence of the egg cell promoter. Nucleic acid molecules that are fragments of a nucleotide sequence of an egg cell promoter comprise at least about 50, 100, 150, 180, 210, 240, 270, 300, 330, 360, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid promoter sequence disclosed herein, e.g., SEQ ID NO: 1 NO: those of 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and 86. "Contiguous nucleotides" is used herein to refer to nucleotide residues that are immediately adjacent to each other. Fragments of the nucleic acid sequence of an oocyte promoter that retain the ability to initiate transcription in an oocyte and thus retain their ability to express a heterologous polynucleotide in an oocyte.

[0052] In some embodiments, the polynucleotide of the egg cell promoter is changed to generate a nucleotide sequence encoding a variant. In some aspects, the egg cell promoter variant is derived from SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 by changing one or more nucleotides by deletion, substitution, addition insertion or a combination thereof.

[0053] "Variants" of egg cell promoters can include changes in their nucleotide sequence, wherein one or more nucleotides of the original sequence are deleted, added and / or replaced while substantially maintaining promoter function in the egg cell. One or more base pairs can be inserted, deleted or replaced within the promoter. In the case of promoter fragments, variant promoters can include changes that affect transcription of the minimal promoter operably connected thereto. Variant promoters can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing variant promoters or portions thereof.

[0054] In some embodiments, an egg cell promoter can be operably linked to a heterologous polynucleotide sequence such that a polypeptide encoded by the heterologous polynucleotide sequence is expressed in female reproductive tissues or cells (eg, egg cells) of the plant.

[0055] Any egg cell promoter polynucleotide described herein can be introduced into a female gamete (including the genome of a gamete) using any suitable technique (including, but not limited to, for example, gene editing, transformation). In some embodiments, the egg cell promoter polynucleotide can be part of a chimeric polynucleotide (such as a heterologous polynucleotide and a DNA construct). Thus, provided herein is a recombinant DNA construct comprising a nucleotide sequence operably linked to at least one heterologous sequence, the nucleotide sequence comprising: any of the sequences shown in SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 86; and ... The nucleotide sequence shown in NO:15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 has a nucleotide sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; or its functional fragment or variant. Also provided herein are embryos, plants, and parts thereof comprising a nucleotide sequence operably linked to at least one heterologous sequence, the nucleotide sequence comprising: any one of the sequences shown in SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 86; and ... The nucleotide sequence shown in NO:15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 has a nucleotide sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; or its functional fragment or variant. In some examples, the operably linked heterologous polynucleotide sequence encodes a BBM polypeptide or a functional BBM fragment or BBM variant thereof. The egg cell promoter sequence and the BBM polynucleotide sequence are heterologous relative to each other because this combination does not occur naturally. In another example, the egg cell promoter or BBM polynucleotide can be homologous, native, heterologous, or foreign to the plant cell, such as a corn egg cell promoter heterologous to a wheat female gametophyte. Fragments and variants can be obtained via any suitable method including, but not limited to, site-directed mutagenesis and synthetic construction.

[0056] The promoter sequences disclosed herein can be contained in a recombinant DNA construct or in a chimeric polynucleotide, for example, in combination with a heterologous polynucleotide (eg, as part of an engineered genomic locus).

[0057] Also provided herein is a method for regulating the expression of a polynucleotide encoding a polypeptide of interest in an egg cell. In some aspects, the method includes expressing a polynucleotide by being operably connected to a promoter nucleotide sequence expressed in an egg cell, wherein the egg cell promoter includes one or more expression regulating (modulating) / regulating (modulation) elements (EME). As used herein, "expression regulating (modulating) / regulating (modulation) elements" or "EME" refers to a nucleotide sequence that up-regulates or down-regulates the expression of one or more plant genes. EME can have one or more copies of the same sequence, and these copies are arranged in any combination of head to head, tail to head or head to tail or these configurations. EME can be derived from a plant sequence, or derived from a bacterial or viral enhancer element. Exemplary EMEs for these methods and compositions include but are not limited to SEQ ID NO:30 and those described in Publication No. WO 2018 / 183878, which are incorporated herein in their entirety. In some aspects, EME is heterologous relative to an egg cell promoter.

[0058] On the other hand, provided herein are recombinant DNA constructs or chimeric polynucleotides comprising an egg cell promoter and EME, wherein EME is heterologous to the egg cell promoter. Also provided are methods for regulating the expression of a polynucleotide sequence of interest in a plant egg cell, the method comprising expressing a polynucleotide sequence, wherein the expression of the polynucleotide sequence is regulated by a promoter expressed in an egg cell, wherein the promoter comprises one or more EME, wherein EME is heterologous to the polynucleotide sequence of interest and / or the egg cell promoter. In some aspects, the polynucleotide sequence of interest is a BBM polynucleotide described elsewhere herein. The methods and compositions disclosed herein can use different promoters with or without EME sequences and / or enhancers.

[0059] In one embodiment, a method for regulating the expression of endogenous BBM polynucleotides in plant cells is provided. The method may include changing one or more nucleotides in the regulatory region of the genomic locus of the endogenous BBM polynucleotide (e.g., BBM gene) to produce a modified regulatory region. The production of the modified regulatory region causes the BBM polypeptide to be expressed in the female gametophyte. In some embodiments, the nucleotide sequence of the promoter expressed in the egg cell is introduced into the BBM genomic locus so that the promoter is operably connected to the nucleotide sequence encoding the BBM polypeptide. In some embodiments, the endogenous BBM promoter sequence can be replaced with an egg cell promoter. In some embodiments, the endogenous BBM promoter nucleotide sequence can be changed to an egg cell promoter nucleotide sequence. The resulting modified BBM genomic locus is also provided herein.

[0060] In one embodiment, a method for regulating the expression of a coding sequence or a genomic sequence in an endogenous genomic egg cell locus in a plant cell is provided. The method may include changing one or more nucleotides in a genomic region or a coding region of a genomic locus of an egg cell polynucleotide, for example, to produce a modified genomic nucleotide sequence or a coding nucleotide sequence encoding a BBM polypeptide. The production of the modification causes the BBM polypeptide to be expressed in a female gametophyte. In some embodiments, a heterologous polynucleotide encoding a BBM polypeptide is introduced into an egg cell genomic locus so that the heterologous polynucleotide encoding the BBM polypeptide is operably connected to a regulatory region or a promoter of an egg cell locus and expressed. In some embodiments, the heterologous polynucleotide encoding the BBM polypeptide replaces an endogenous genomic nucleotide sequence or a coding nucleotide sequence in a genomic egg cell locus. In some embodiments, an endogenous genomic nucleotide sequence or a coding nucleotide sequence in a genomic egg cell locus is changed to a sequence of a heterologous polynucleotide encoding a BBM polypeptide. The resulting modified genomic egg cell locus is also provided herein.

[0061] One embodiment of the present disclosure includes methods for obtaining wheat plants that produce clonal, non-reduced, non-recombinant male and / or female gametes. In certain embodiments, the wheat plant having repressed expression and / or activity relative to the wheat plant's endogenous Spo11 or Prd1, Prd2 or PRd3; Rec8 and OSD1 polynucleotides and polypeptides is heterozygous or homozygous for repression.

[0062] The terms "suppress, suppressed, suppression, suppressing" and "silencing" are used interchangeably herein and include reducing, decreasing, decreasing, lowering, inhibiting, eliminating or preventing. "Silencing" or "gene silencing" does not specify a mechanism and includes, but is not limited to, antisense, co-suppression, viral suppression, hairpin suppression, stem-loop suppression, RNAi-based methods, and small RNA-based methods, etc. As used herein, "silencing" with respect to a target gene (such as Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1) generally refers to suppressing the level of mRNA or protein expressed by the target gene and / or the level of protein functionality.

[0063] In certain embodiments, the methods comprise suppressing endogenous wheat Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotides and polypeptides or combinations thereof in a plant or plant cell. In certain embodiments, the plant cell is a microspore mother cell (which produces haploid microspores), a megaspore mother cell (which produces haploid megaspores), or any other plant cell in which genes such as Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 are expressed.

[0064] Provided are methods for suppressing the activity of endogenous Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotides or polypeptides in wheat plant cells. In some aspects, suppressing activity also includes suppressing the expression level of Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotides or polypeptides. Any suitable method or technique can be used. In certain embodiments, Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotides or polypeptides are suppressed using antisense, co-suppression, viral suppression, hairpin suppression, stem-loop suppression, RNAi-based methods, and small RNA-based methods. In some instances, a genome editing method (including but not limited to Cas endonucleases and guide RNAs) is used to introduce polynucleotides that suppress the expression and / or activity of Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 into the genome of a plant cell. In some examples, a genome editing method (including but not limited to Cas endonucleases and guide RNAs) is used to introduce nucleotide deletions or modifications (e.g., additions or substitutions) that suppress the expression and / or activity of Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 into the genome of a plant cell. In one embodiment, any combination of RNA-based methods and gene editing methods can be used to suppress the expression and / or activity of Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1.

[0065] Those of ordinary skill in the art will readily recognize appropriate controls or references for use when evaluating or measuring the expression level or activity of Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotides or polypeptides in any embodiment of the present disclosure. For example, by way of non-limiting illustration, a plant or plant cell comprising a modified Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotide or polypeptide or comprising a Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 repressing polynucleotide is typically measured relative to a plant or plant cell that does not comprise a modified Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotide or polypeptide or does not comprise a Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 repressing polynucleotide as a control or reference plant or plant cell. In some examples, the control is a wild-type plant or cell. One skilled in the art can use assays such as PCR, Northern blot, and Western blot assays to determine Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 expression levels or activity.

[0066] In certain embodiments, wheat plants with suppressed activity relative to endogenous Spo11 or Prd1, Prd2 or PRd3; Rec8; and OSD1 polynucleotides and polypeptides are heterozygous or homozygous for suppression. These plants can be crossed, intercrossed or selfed until a wheat plant is obtained, which contains suppressed activity of each and all of the following endogenous genes: (Spo11 or Prd1, Prd2 or PRd3); Rec8; and OSD1 activity. The female gametophyte derived from this wheat plant is modified so that the Babyboom (BBM) polynucleotide encoding the BBM polypeptide or a fragment thereof is expressed in the female gametophyte to produce embryos.

[0067] Any suitable method can be used for modification. In one example, the method includes introducing a BBM polynucleotide encoding a BBM polypeptide into a coding nucleotide sequence or a genomic nucleotide sequence of a genomic egg cell locus. In another example, the method includes introducing an egg cell promoter or regulatory region into a genomic BBM locus. In another example, a chimeric polynucleotide is introduced into a female gamete, the chimeric polynucleotide comprising a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell, wherein the BBM polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof. In another example, a recombinant DNA construct is introduced into a female gamete, the recombinant DNA construct comprising a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell, wherein the BBM polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof.

[0068] Use method as described herein, can use the wheat plant derived from this embryo to produce non-reduced, non-recombinant and cloned progeny relative to parent wheat plant.The plant produced by this method allows the wheat plant to carry out clonal propagation by seed.Can grow into plant and gather seed from those plants from the seed obtained from the wheat plant, thereby allow the parent plant to carry out clonal propagation by seed.In certain embodiments, the wheat plant is a hybrid.In certain embodiments, the wheat plant is an inbred line or a variant.

[0069] As used herein, the term "wheat" refers to any species of the genus Triticum, including its precursors and progeny produced by hybridization with other species. Wheat includes "hexaploid wheat" having a genome organization of AABBDD consisting of 42 chromosomes and "tetraploid wheat" having a genome organization of AABB consisting of 28 chromosomes. Hexaploid wheat includes wheat (T.aestivum), spelta (T.spelta), mocha wheat (T.mocha), compact wheat (T.compactum), Indian round-grain wheat (T.sphaerococcum), Vavilov wheat (T.vivilovii), wild einkorn wheat (Triticum boeoticum) or domesticated forms, cultivated einkorn wheat (Triticum monococcum) and interspecific hybrids thereof. Tetraploid wheat includes T. durum (also known as durum wheat or Triticum turgidum ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspecific hybrids thereof. In addition, the term "wheat" includes possible ancestors of hexaploid or tetraploid Triticum species, such as T. uartu, T. monococcum, or T. boeoticum for the A genome, Aegilops speltoides for the B genome, and T. tauschii for the D genome (also known as Aegilops squarrosa or Aegilops tauschii). Wheat cultivars used in the present disclosure may belong to, but are not limited to, any of the species listed above. Also included are plants produced by conventional techniques using Triticum species as parents in sexual crosses with non-Triticum species, such as rye (Secale cereale), including but not limited to Triticale. In some embodiments, the wheat plants are suitable for commercial production of grain, such as commercial varieties of hexaploid wheat or durum wheat, having suitable agronomic characteristics known to those skilled in the art.

[0070] The disclosure also includes plants obtained by any method or composition disclosed herein. The disclosure also includes seeds from plants obtained by any method or composition disclosed herein. As used herein, the term "plant" refers to whole plants, plant organs (such as leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos and progeny thereof. As used herein, the term "plant" refers to whole plants, plant organs (such as leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos and progeny thereof. Plant cells can be differentiated or undifferentiated (such as callus, undifferentiated callus, immature and mature embryos, immature zygotic embryos, immature cotyledons, hypocotyls, suspension culture cells, protoplasts, leaves, leaf cells, root cells, phloem cells and pollen).

[0071] Grain is intended to mean mature seeds produced by commercial growers for purposes other than cultivating or propagating the species.

[0072] Compositions and methods of the present disclosure can be applied to a wide range of plant species, including dicotyledons and monocotyledons. Representative examples of plants that can be processed according to methods disclosed herein include, but are not limited to, wheat, cotton, sunflower, safflower, tobacco, Arabidopsis, barley, oats, rice, corn, triticale, sorghum, rye, millet, flax, sugarcane, banana, cassava, kidney bean, cowpea, tomato, potato, beet, grape, Eucalyptus (Eucalyptus), wheat grass (wheat grass), lawn grass, alfalfa, clover, soybean, peanut, citrus, papaya, Setaria sp, cocoa, cucumber, apple, Capsicum, bamboo, melon, hemp, ornamental plants (including commercial gardens and bulb flower species), fruit trees, vegetable species, Brassica species and interspecific hybrids. In one embodiment, compositions and methods of the present disclosure are applied to wheat plants.

[0073] The method of the present disclosure may include introducing a polypeptide, a polynucleotide (i.e., DNA or RNA) or a nucleotide construct (i.e., DNA or RNA) into a plant. As used herein, "introduction" means presenting a polynucleotide, a polypeptide or a nucleotide construct to a plant in a manner that allows the polynucleotide, a polypeptide or a nucleotide construct to enter the interior of a plant cell. The method of the present disclosure does not depend on a specific method for introducing a polynucleotide, a polypeptide or a nucleotide construct into a plant, as long as the polynucleotide, a polypeptide or a nucleotide construct is able to enter the interior of at least one cell of the plant. Methods for introducing a polynucleotide, a polypeptide or a nucleotide construct into a plant are known in the art, and these methods include but are not limited to gene editing, stable or transient transformation methods, and virus-mediated methods.

[0074] The compositions and methods of the present disclosure optionally include producing doubled haploid plants from haploid embryos.

[0075] The present disclosure also provides methods of contacting haploid cells with an amount of a chromosome doubling agent before, during, after, or overlapping with any part of the separation and embryogenesis induction methods for generating a doubled haploid population of paternal gametes (producing male characteristics) or maternal gametes (producing female characteristics).

[0076] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0077] In some embodiments, parthenogenetic induction methods can be used to improve maternal haploid embryo regeneration productivity and enable gene editing to provide regenerated gene-edited maternal haploids.

[0078] On the one hand, haploid cell can be contacted with a certain amount of chromosome doubling agent to promote chromosome doubling, then regenerate homozygous diploid plant from treated haploid cell.Before, during or after microspore embryogenesis or embryo maturation, haploid microspore cell can be contacted with doubling agent.After chromosome doubling, double haploid embryo will contain 2 copies of chromosome derived by father.The efficiency of the method for obtaining double haploid plant from haploid embryo can be greater than 10%, 20%, 30%, 50%, 60%, 70%, 80% or 90%.The duration of contact between haploid cell and chromosome doubling agent can change.Contact time can be from less than 24 hours (for example 4-12 hours) to about one week.The duration of contact is generally from about 8 hours to 2 days.

[0079] Chromosome doubling methods are disclosed in: Antoine-Michard, S. et al., Plant cell, tissue organ cult. [Plant cell, tissue organ culture], Cordrecht, The Netherlands, Kluwer Academic Publishers [Ruwell Academic Publishers], 1997, 48 (3): 203-207; Kato, A., Maize Genetics Cooperation Newsletter [Maize Genetics Cooperation Newsletter] 1997, 36-37; and Wan, Y. et al., TAG [Theoretical and Applied Genetics], 1989, 77: 889-892. Wan, Y. et al., TAG [Theoretical and Applied Genetics], 1991, 81: 205-211. The disclosures thereof are incorporated herein by reference. A typical doubling method involves contacting cells with colchicine, an anti-microtubule agent or anti-microtubule herbicide, natol, nitrous oxide or any mitotic inhibitor to produce homozygous doubled haploid cells. The amount of colchicine used in the culture medium is typically 0.01%-0.2%, or about 0.05% methylamine phosphine (APM) (5-225 μM) can be used. The amount of colchicine can be in the range of about 400-600 mg / L or about 500 mg / L. The amount of natrium in the culture medium is about 0.5-20 μM. Examples of mitotic inhibitors are included in Table B. Other agents can be used together with mitotic inhibitors to improve doubling efficiency. Such agents include dimethyl sulfoxide (DMSO), adjuvants, surfactants, etc.

[0080] Table B. Chemical Chromosome Doubling Agents

[0081]

[0082]

[0083]

[0084]

[0085] As an alternative to using chemical chromosome doubling agents, by stimulating the cell cycle (and cell division) or by stimulating nuclear replication to regulate the expression of genes known to affect the plant cell cycle (genetic chromosome doubling protein), it can be used to double the chromosome set in the embryo. By regulating the expression of genes that stimulate key control points in cell cycle cells, the increase of ploidy levels in plant cells can be achieved. It is expected that other plant genes known to stimulate the plant cell cycle (or cell division) can be used to produce similar chromosome number doubling in forming maternal haploid embryos. Examples of plant genes that stimulate the cell cycle when overexpressed include cyclin A in tobacco (Yu et al., 2003), cyclin D in tobacco (Cockcroft et al., 2000, Nature 405:575-79; Schnittger et al., 2002, PNAS [Proceedings of the National Academy of Sciences of the United States of America] 99:6410-6415; Dewitte et al., 2003, Plant Cell [Plant Cell] 15:79-92), E2FA in Arabidopsis (DeVeylder et al., 2002, EMBO J [Journal of the European Molecular Biology Association] 21:1360-1368), E2FB in Arabidopsis (Magyar et al., 2005, Plant Cell [Plant Cell] 17:2527-2541). Similarly, overexpression of viral genes known to regulate plant cell cycle mechanisms can be used, such as when overexpression of the wheat dwarf virus RepA gene stimulates cell cycle progression (G1 / S transition) and cell division in corn (Gordon-Kamm et al., 2002, PNAS [Proceedings of the National Academy of Sciences of the United States] 99: 11975-11980). In contrast, when genes such as cyclin-dependent kinase inhibitor (ICK1 / KRP) in Arabidopsis are downregulated, plant genes (whose encoded products are known to inhibit the cell cycle) have been shown to cause increased cell division (Cheng et al. 2013, Plant J [Plant Journal] 75: 642-655). Therefore, downregulation of the KRP gene driven by expression with an egg cell-specific promoter can have a similar effect to overexpression of DZ470, resulting in chromosome doubling. Methods for downregulating genes such as KRP are known in the art and include expressing artificial microRNAs targeting KRP mRNA, or expressing dCas9-repressor fusions targeting the KRP promoter sequence by gRNAs directed against the sequence. Finally, plant genes are known to exist that specifically affect the endoreduplication process.When using such genes (such as, for example, the ccs52 gene or the Del1 gene), overexpression of ccs52 can lead to increased ploidy levels, as observed in Medicago sativa (Cebolla et al., 1999, EMBO J [Journal of the European Molecular Biology Organization] 18:4476 - 4484), and downregulation of Del1 can lead to increased ploidy levels, as observed in Arabidopsis thaliana (Vlieghe et al., 2005, Current Biol [Current Biology] 15:59 - 63). It is expected that other genes known to stimulate the cell cycle, G1 / S transition, or endoreduplication can be used in the methods disclosed herein to increase ploidy levels.

[0086] Repression motifs are known in the art, see, for example, Kagale and Rozwadowski (Epigenetics. 2011. 6:141 - 146). Transcriptional repression mediated by the ethylene response element binding factor - related amphiphilic repression (EAR) motif is known in plants, including the EAR motif defined by the consensus sequence patterns of LxLxL and DLNxxP (see Hiratsu et al., 2003. Plant J. [The Plant Journal] 35:177 - 192). Of interest in the present disclosure are peptides that include the amphiphilic repression motifs disclosed in WO 2013 / 109754 A1 and all references cited therein, as well as the Dr1 / DRAP1 global repressor complex (see US 7,288,695 B2 and all references cited therein), the Dr1 / DRAP1 global repressor complex including a Dr1 motif similar to the motif found in Arabidopsis thaliana MYBL2 (see Matsui K, Umemura Y, Ohme - Takagi M. 2008. Plant J. [The Plant Journal] 55:954 - 967).

[0087] Methods for inserting polynucleotides at the specific position of plant genome targeting are known in the art. Site-specific recombination systems can be utilized to realize the insertion of polynucleotides at the desired genomic position. See, for example, WO 99 / 25821, WO 99 / 25854, WO 99 / 25840, WO 99 / 25855 and WO 99 / 25853, which are incorporated herein by reference in their entirety. In short, the target polynucleotide flanked by two different recombination sites can be included in the T-DNA transfer box. The T-DNA transfer box can be introduced into a plant, which has stably incorporated the target site into its genome, and the target site flanks two different recombination sites corresponding to the site of the transfer box. Suitable recombinase is provided, and the transfer box is integrated into the target site. Thus, the target polynucleotide is integrated into the specific chromosome position in the plant genome.

[0088] Any suitable technique can be used to introduce polynucleotides into any plant cell, which can be used to target specific sites modified in the genome of a plant derived from a plant cell. Site-specific modifications that can be introduced include modifications produced using any method for introducing site-specific modifications, including but not limited to using gene repair oligonucleotides (e.g., U.S. Publication 2013 / 0019349), or by using double-strand break technology, such as TALEN, meganuclease, zinc finger nuclease, CRISPR-Cas, other endonucleases, etc. For example, the disclosed method can be used to introduce the CRISPR-Cas system into plant cells or plants for the following purposes: genome modification of target sequences in the genome of plants or plant cells, selection of plants, deletion of bases or sequences, gene editing, and insertion of target polynucleotides into the genome of plants or plant cells. Therefore, the disclosed method can be used together with the CRISPR-Cas system to provide an effective system for modifying or changing target sites and target nucleotides in the genome of plants, plant cells or seeds. Any suitable Cas endonuclease can be used in the methods and to produce the compositions described herein, such as any suitable Cas endonuclease that is capable of binding to a genomic target sequence of a plant genome and generating a double-stranded break therein.

[0089] In some embodiments, genome editing techniques can be used to introduce polynucleotides (such as egg cells or BBM polynucleotides) into the genome of a plant, or genome editing techniques can be used to edit polynucleotides (such as egg cells or BBM polynucleotides) in the genome of a plant. For example, polynucleotides can be introduced into a desired position in the genome of a plant by using a genome editing system (such as TALEN, meganuclease, zinc finger nuclease, CRISPR-Cas, etc.). For example, for the purpose of site-specific insertion, the disclosed polynucleotides can be introduced into a desired position in the genome using a CRISPR-Cas system. The desired position in the plant genome can be any desired target site for insertion, such as a genomic region.

[0090] In some embodiments, where the polynucleotide has been previously introduced into the genome, genome editing or genome engineering techniques can be used to alter or modify the introduced polynucleotide sequence, including flanking chromosomal genomic sequences. Site-specific modifications can be introduced into the disclosed compositions, including modifications produced using any method for introducing site-specific modifications, including but not limited to by using gene repair oligonucleotides, or by using site-directed genome modification tools such as TALEN, meganuclease, zinc finger nuclease, CRISPR-Cas, etc. Site-specific modifications to the disclosed polynucleotides (including genomic flanking and junction sequences) can include but are not limited to changes in codon usage, changes in regulatory elements, such as promoters, introns, terminators, enhancers, 5' or 3' untranslated regions (UTRs) or other non-coding sequences and other regions of the polynucleotides, wherein the modifications do not adversely affect the phenotypic characteristics of the resulting corn plant.

[0091] Cas polypeptides suitable for introducing site-specific modifications include, for example, Cas9, Cas12f (Cas-α, Cas14), Cas121 (Cas-β), Cas12a (Cpf1), Cas12b (C2c1 protein), Cas13 (C2c2 protein), Cas12c (C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10, or a combination or complex of these. In some aspects, transposon-associated TnpB, a programmable RNA-guided DNA endonuclease, can be used.

[0092] In some aspects, the genome editing system comprises a Cas-α (e.g., Cas12f) endonuclease and one or more guide polynucleotides that introduce one or more site-specific modifications into a target polynucleotide sequence to produce a modified target sequence. As used herein, "changed target site," "changed target sequence," "modified target site," and "modified target sequence" are used interchangeably and refer to a target sequence disclosed herein comprising at least one change or modification compared to an unchanged target sequence. Such changes or modifications include, for example: (i) replacement or substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0093] In some aspects, the genome editing system comprises a Cas-α endonuclease, one or more guide polynucleotides, and optionally a donor DNA. For example, some exemplary Cas-α endonucleases are described in WO 2020123887.

[0094] In some aspects, the genome editing system comprises a Cas polypeptide, one or more guide polynucleotides and optionally a donor DNA, and editing the target polynucleotide sequence includes non-homologous end joining (NHEJ) or homologous recombination (HR) after a double-strand break mediated by the Cas polypeptide. Once a double-strand break is induced in DNA, the DNA repair mechanism of the cell is activated to repair the break. The most common repair mechanism that combines the ends of the break is the non-homologous end joining pathway (Bleuyard et al., (2006) DNA Repair [DNA repair] 5: 1-12). The structural integrity of the chromosome is typically preserved by repair, but deletions, insertions or other rearrangements are possible (Siebert and Puchta, (2002) Plant Cell [plant cells] 14: 1121-31; Pacher et al., (2007) Genetics [genetics] 175: 21-9). Alternatively, the double-strand break can be repaired by homologous recombination between homologous DNA sequences. Once the sequence around the double-strand break is altered, for example by mature exonuclease activity involved in double-strand breaks, the gene conversion pathway can restore the original structure if there are homologous sequences, such as homologous chromosomes in non-dividing somatic cells, or sister chromatids after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA repair templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0095] In some aspects, the genome editing system comprises a Cas polypeptide, one or more guide polynucleotides and a donor DNA. As used herein, "donor DNA" is a DNA construct comprising a target polynucleotide to be inserted into the target site of the Cas polypeptide. Once a double-strand break is introduced at the target site by an endonuclease, the first and second homologous regions of the donor DNA can undergo homologous recombination with their corresponding homologous genomic regions, resulting in an exchange of DNA between the donor and target genomes. Therefore, the provided method causes the target polynucleotide of the donor DNA to be integrated into the double-strand break of the target site in the plant genome, thereby changing the original target site and generating a changed genomic target site.

[0096] In some aspects, the genome editing system comprises a base editor and a plurality of guide polynucleotides, and editing the target polynucleotide sequence comprises introducing a plurality of nucleobase edits into the target polynucleotide sequence, thereby generating variant nucleotide sequences.

[0097] One or more core bases of target polynucleotide can change chemically, and base is changed to another type from a type in some cases, for example, is changed to thymine or is changed to guanine from cytosine.In some respects, a plurality of bases, for example 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more or even more than 100, 200 or more, up to thousands of bases can modify or change, to produce the plant with a plurality of modified bases.

[0098] Any base editing complex, such as a base editing agent associated with an RNA guide protein, can be used to target and bind to a desired locus in the genome of an organism and chemically modify one or more components of the target polynucleotide.

[0099] Site-specific base conversions can be achieved to engineer one or more nucleotide changes to create one or more edits in the genome. These include, for example, site-specific base editing mediated by C·G to T·A or A·T to G·C base editing deaminases (Gaudelli et al., Programmable base editing of A·T toG·C in genomic DNA without DNA cleavage. "Nature" (2017); Nishida et al., "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353 (6305) (2016); Komor et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA." "Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage." Nature 533(7603)(2016):420-4. A catalytically "dead" or inactive Cas polypeptide (e.g., an inactive Cas9 (dCas9), Cas12f (dCas12f), or another Cas polypeptide disclosed herein) fused to a cytidine deaminase or adenine deaminase protein becomes a specific base editor that can change DNA bases without inducing DNA breaks. Base editors convert C->T (or on the opposite strand, G->A) or adenine base editors convert adenine to inosine, thereby modifying the editing window specified by the gRNA. The dCas forms a functional complex with a guide polynucleotide that shares homology with the polynucleotide sequence at the target site, and further complexes with a deaminase molecule. The guided Cas polypeptide recognizes and binds to the double-stranded target sequence, thereby opening the double strand to expose individual bases. In the case of cytidine deaminase, the deaminase deaminates the cytosine base and produces uracil. A uracil glycosylase inhibitor (UGI) is provided to prevent U from converting back to C. The DNA replication or repair mechanism then converts uracil to thymine (U to T), and the opposite base (previously G in the original GC pair) is subsequently repaired to adenine, resulting in a TA pair.

[0100] Cas endonucleases are guided by guiding nucleotides to identify double-strand breaks at specific target sites and optionally introduce the double-strand breaks into the genome of the cell. The CRISPR-Cas system provides an effective system for modifying the target site in the genome of a plant, a plant cell or a seed. Further provided is a method for using a guiding polynucleotide / Cas endonuclease system to provide an effective system for modifying the target site in the cell genome and editing the nucleotide sequence in the cell genome. Once the genomic target site is identified, a variety of methods can be used to further modify the target site. The disclosed method can be used to introduce the CRISPR-Cas system, which is used to edit the nucleotide sequence in the cell genome. The nucleotide sequence to be edited (target nucleotide sequence) can be located inside or outside the target site identified by the Cas endonuclease.

[0101] The term "Cas polypeptide" or "Cas endonuclease" refers to a polypeptide encoded by a Cas (CRISPR-associated) gene.

[0102] Cas polypeptides include but are not limited to: Cas9, Cas12f (Cas-α, Cas14), Cas12l (Cas-β), Cas12a (Cpf1), Cas12b (C2c1 protein), Cas13 (C2c2 protein), Cas12c (C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10 or a combination or complex of these.

[0103] Endonucleases are enzymes that cut the phosphodiester bonds in polynucleotide chains, and include restriction endonucleases that cut DNA at specific sites without damaging bases. Restriction endonucleases include type I, type II, type III, and type IV endonucleases, which further include subtypes. In type I and type III systems, both methylase and restriction enzyme activities are contained in a single complex. Endonucleases also include meganucleases, also referred to as homing endonucleases (HE enzymes), which are similar to restriction endonucleases, bind and cut at specific recognition sites, but for meganucleases, these recognition sites are typically longer, about 18bp or longer (patent application PCT / US12 / 30061 filed on March 22, 2012). Based on conserved sequence motifs, meganucleases have been divided into four families. These motifs participate in the coordination of metal ions and the hydrolysis of phosphodiester bonds. The notable feature of meganucleases is their long recognition sites, and also in tolerance to some sequence polymorphisms in their DNA substrates. The naming convention for meganucleases is similar to that for other restriction endonucleases. Meganucleases are also characterized by the prefixes F-, I-, or PI- for enzymes encoded by independent ORFs, introns, and inteins, respectively. One step of the recombination method involves cleavage of a polynucleotide at or near a recognition site. The cleavage activity can be used to produce a double-strand break. For a review of site-specific recombinases and their recognition sites, see Sauer (1994) Curr Op Biotechnol [New Views in Biotechnology] 5:521-7; and Sadowski (1993) FASEB [Journal of the Federation of American Societies for Experimental Biology] 7:760-7. In some instances, the recombinase is from the Integrase or Resolvase family. TAL effector nucleases are a new class of sequence-specific nucleases that can be used to cause double-strand breaks at specific target sequences in the genome of plants or other organisms. (Miller et al. (2011) Nature Biotechnology 29: 143-148). Zinc finger nucleases (ZFNs) are engineered double-strand break-inducing agents consisting of a zinc finger DNA binding domain and a double-strand break-inducing agent domain. Recognition site specificity is conferred by a zinc finger domain, which typically comprises two, three, or four zinc fingers, for example having a C2H2 structure, however other zinc finger structures are known and have been engineered. The zinc finger domain is suitable for designing polypeptides that specifically bind to a selected polynucleotide recognition sequence. ZFNs include an engineered DNA binding zinc finger domain connected to a non-specific endonuclease domain (e.g., a nuclease domain from an Ms-type endonuclease, such as Fok1).

[0104] Catalytically active and / or inactivated Cas endonucleases can be fused to heterologous sequences (US20140068797, published on March 6, 2014). As used herein, "inactive CAS9" (dCAS9) is used to provide a transcriptional repression domain. dCAS9 has mutated and can no longer cut DNA. dCAS0 can still bind when guided to the sequence by gRNA, or it can be fused to a repressor element. As described herein, dCAS9 fused to a repressor element is abbreviated as dCAS9-REP, where the repressor element (REP) can be any known repressor motif that has been characterized in plants. The expressed guide RNA (gRNA) binds to the dCAS9-REP protein and targets the binding of the dCAS9-REP fusion protein to a specific predetermined nucleotide sequence within the promoter (promoter within T-DNA). For example, if it is expressed outside the border using a ZM-UBIPRO::dCAS9-REP::PINII TERM cassette and a U6-POL PRO::gRNA::U6 TERM cassette, and the gRNA is designed to direct the dCAS9-REP protein to bind to the SB-UBI promoter in the expression cassette SB-UBIPRO::moPAT::PINII TERM within the T-DNA, any event that integrates sequences outside the border will be bialaphos sensitive. Transgenic events that only integrate the T-DNA will express moPAT and be resistant to bialaphos. The advantage of using a dCAS9 protein fused to a repressor (as opposed to TETR or ESR) is the ability to target these repressors to any promoter within the T-DNA. TETR and ESR are limited to cognate operator binding sequences. Alternatively, a synthetic zinc finger nuclease fused to a repressor domain can be used instead of gRNA and dCAS9-REP (Urritia et al., 2003, Genome Biol. 4:231), as described above.

[0105] The type II CRISPR / Cas system from bacteria uses crRNA and tracrRNA to guide the Cas endonuclease to its DNA target. The crRNA (CRISPR RNA) contains a strand complementary to a double-stranded DNA target, and base pairs with tracrRNA (trans-activating CRISPR RNA) to form a region of RNA duplexes, which guide the Cas endonuclease to cut the DNA target. As used herein, the term "guide nucleotide" refers to a synthetic fusion of two RNA molecules - crRNA (CRISPR RNA) and tracrRNA comprising a variable targeting domain. On the one hand, the guide nucleotide comprises a variable targeting domain of 12 to 30 nucleotide sequences and an RNA fragment that can interact with the Cas endonuclease.

[0106] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize and optionally cleave a DNA target site. The guide polynucleotide can be a unimolecular or bimolecular. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence).

[0107] On the one hand, guide nucleotide and Cas endonuclease can form a complex that enables Cas endonuclease to introduce double-strand breaks at DNA target sites. Cas endonuclease (as a single effector protein or an effector complex with other components) unwinds the DNA duplex at the target sequence and optionally cuts at least one DNA chain, such as by recognizing the target sequence mediated by a polynucleotide (such as but not limited to crRNA or guide RNA) compounded with the Cas effector protein. If the correct protoplast adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence, such recognition and cutting of the target sequence by Cas endonuclease typically occurs. Alternatively, the Cas endonuclease herein may lack DNA cutting or nicking activity, but when compounded with a suitable RNA component, it can still specifically bind to the DNA target sequence. (See also U.S. Patent Application US20150082478 published on March 19, 2015 and US20150059010 published on February 26, 2015).

[0108] Cas endonucleases can occur as single effectors (class 2 CRISPR systems) or as part of larger effector complexes (class I CRISPR systems).

[0109] Cas endonucleases that have been described include, but are not limited to, for example: Cas3 (characteristic of class 1 type I systems), Cas9 (characteristic of class 2 type II systems), and Cas12 (Cpf1) (characteristic of class 2 type V systems).

[0110] Cas endonucleases and effector proteins can be used for targeted genome editing (via single and multiple double-strand breaks and gaps) and targeted genome regulation (via tethering epigenetic effector domains to Cas proteins or sgRNAs). Cas endonucleases can also be engineered to function as RNA-guided recombinases and can serve as scaffolds for assembling polyprotein and nucleic acid complexes via RNA tethers (Mali et al., 2013 Nature Methods [Natural Methods] Vol. 10: 957-963).

[0111] In some examples, the Cas endonuclease, when complexed with a cognate guide RNA, recognizes, binds, and optionally nicks or cleaves the target polynucleotide.

[0112] In one aspect of the disclosure, the variable target domain is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0113] Instruction polynucleotide enables Cas endonuclease to target recognition, binding and optionally cutting, and can be unimolecular or bimolecular.Instruction polynucleotide sequence can be RNA sequence, DNA sequence or its combination (RNA-DNA combination sequence). Optionally, instructing polynucleotide can include at least one nucleotide, phosphodiester bond or connection modification, such as but not limited to locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, thiophosphate bond, connection with cholesterol molecule, connection with polyethylene glycol molecule, connection with spacer 18 (hexaethylene glycol chain) molecule or cause cyclization 5' to 3' covalent connection. Instruction polynucleotides comprising only ribonucleic acid are also referred to as "guidance RNA" or "gRNA" (US20150082478 published on March 19, 2015 and US20150059010 published on February 26, 2015). Instruction polynucleotide can be engineered or synthetic.

[0114] The guide polynucleotide includes a chimeric non-naturally occurring guide RNA that includes regions that are not found together in nature (i.e., they are heterologous to each other). For example, the chimeric non-naturally occurring guide RNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA, and the first nucleotide sequence domain is connected to a second nucleotide sequence that can recognize a Cas endonuclease, such that the first and second nucleotide sequences are not found connected together in nature.

[0115] The guiding polynucleotide can be a double molecule (also referred to as a duplex guiding polynucleotide) comprising a cr nucleotide sequence (e.g., crRNA) and a tracr nucleotide (e.g., tracrRNA) sequence. In some cases, there is a linker polynucleotide, such as an sgRNA, connecting crRNA and tracrRNA to form a single guiding object.

[0116] In one aspect of the present disclosure, the guide nucleotide comprises a cRNA (or cRNA fragment) and a tracrRNA (or tracrRNA fragment) that can form a complex with a type II Cas endonuclease in a type II CRISPR / Cas system, wherein the guide nucleotide / Cas endonuclease complex can guide the Cas endonuclease to a plant genome target site, such that the Cas endonuclease can introduce a double-strand break into the genome target site. The guide nucleotide can be directly introduced into a plant or plant cell using any method known in the art, such as, but not limited to, particle bombardment or topical application.

[0117] In one aspect, the guide nucleotide can also be introduced indirectly by introducing a recombinant DNA molecule comprising a corresponding guide DNA sequence operably linked to a plant-specific promoter capable of transcribing the guide nucleotide in a plant cell. The term "corresponding guide DNA" includes a DNA molecule that is identical to an RNA molecule, but in which a "T" replaces each "U" of the RNA molecule.

[0118] In one aspect, the guide nucleotides are introduced via particle bombardment or using the disclosed methods for Agrobacterium transformation of a recombinant DNA construct comprising the corresponding guide DNA operably linked to a plant U6 polymerase III promoter.

[0119] On the one hand, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. An advantage of using a guide nucleotide versus a duplex crRNA-tracrRNA is that only one expression cassette needs to be made in order to express the fused guide nucleotide.

[0120] The terms "target site", "target sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", and "genomic target locus" are used interchangeably herein and refer to a polynucleotide sequence in the genome of a plant cell (including chloroplast DNA and mitochondrial DNA) at which a double-strand break is induced in the genome of the plant cell by a Cas endonuclease. The target site can be an endogenous site in the plant genome, or alternatively, the target site can be heterologous to the plant so as not to occur naturally in the genome, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.

[0121] As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome of a plant and is at the endogenous or native location of the target sequence in the genome of a plant.

[0122] "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that has been introduced into a plant genome. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the genome of the plant, but located at a different location in the genome of the plant (i.e., a non-endogenous or non-native location).

[0123] On the one hand, the disclosed method can be used to introduce into plants polynucleotides for gene suppression of target genes in plants. For several aspects of plant genetic engineering, it is desirable to reduce the activity of specific genes (also referred to as gene silencing or gene suppression). Many techniques for gene silencing are well known to those skilled in the art, including but not limited to antisense technology, siRNA, microRNA, etc. Other methods can also be used, for example, using dCas fused to transcriptional repressors and / or chromatin modification domains to induce targeted reduction of transcripts. See, for example, WO 2022087616, which is incorporated herein by reference in its entirety.

[0124] The cr nucleotide includes a first nucleotide sequence region (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA and a second nucleotide sequence (also referred to as a tracr pairing sequence) that is a part of a Cas endonuclease recognition (CER) domain. The tracr pairing sequence can hybridize with the tracr nucleotide along the complementary region and form a Cas endonuclease recognition domain or a CER domain together. The CER domain can interact with the Cas endonuclease polypeptide. The cr nucleotides and tracr nucleotides of the duplex guidance polynucleotide can be RNA, DNA and / or RNA-DNA combination sequences. In certain embodiments, the cr nucleotide molecules of the duplex guidance polynucleotide are referred to as "crDNA" (when composed of a continuous extension of DNA nucleotides) or "crRNA" (when composed of a continuous extension of RNA nucleotides) or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). The cr nucleotides may be included in fragments of crRNA naturally occurring in bacteria and archaea. The size of the crRNA fragments naturally occurring in bacteria and archaea that may be present in the cr nucleotides disclosed herein may be, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.

[0125] In some embodiments, the tracr nucleotides are referred to as "tracrRNA" (when composed of a continuous extension of RNA nucleotides) or "tracrDNA" (when composed of a continuous extension of DNA nucleotides) or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). In one embodiment, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. In the 5'- to -3' direction, tracrRNA (trans-activating CRISPR RNA) comprises (i) a sequence annealing to the repeat region of CRISPR II type crRNA and (ii) a portion containing a stem loop (Deltcheva et al., Nature [Nature] 471: 602-607). The duplex guide polynucleotide can form a complex with the Cas endonuclease, wherein the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize and bind to the target site, and optionally nick or cut the target site (introducing a single-strand or double-strand break). (US20150082478 published on March 19, 2015 and US20150059010 published on February 26, 2015).

[0126] The guide polynucleotide can also be a single molecule comprising a cr nucleotide sequence linked to a tracr nucleotide sequence (also referred to as a single guide polynucleotide). The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as a variable targeting ( V ariable T argeting) domain or VT domain) and a Cas endonuclease recognition ( C as e ndonuclease r ecognition) domain (CER domain).

[0127] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to a (targeted) target sequence (pre-spacer) that can be recognized by a guide polynucleotide / Cas endonuclease system. If the target DNA sequence is not behind the PAM sequence, the Cas endonuclease may not be able to successfully recognize the target DNA sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but typically is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.

[0128] "Random PAM" and "random protospacer adjacent motif" are used interchangeably herein and refer to a random DNA sequence adjacent to a target sequence (protospacer) recognized (targeted) by a guide polynucleotide / Cas endonuclease system. The random PAM sequence can be of any length, but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. Random nucleotides include any of the nucleotides A, C, G, or T.

[0129] The guide polynucleotide / Cas endonuclease complexes described herein are capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or a portion of a target sequence.

[0130] Proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of one or more proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein. Guidance for amino acid substitutions that are unlikely to affect the biological activity of a protein is found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, DC [National Biomedical Research Foundation, Washington, DC, USA]. Conservative substitutions, such as exchanging one amino acid for another with similar properties, would be preferred. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in protein characteristics, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by conventional screening assays. Assays for double-strand-break-inducing activity are known and typically measure the overall activity and specificity of an agent for a DNA substrate comprising a target site.

[0131] The nucleotide to be edited can be located inside or outside the target site recognized and cut by the Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification on the target site recognized and cut by the Cas endonuclease. In another embodiment, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides between the at least one nucleotide to be edited and the genomic target site.

[0132] Knockouts can be produced by indels (insertion or deletion of nucleotide bases in the target DNA sequence by NHEJ), or by specific removal of sequences that reduce or completely abolish sequence function at or near the targeted site.

[0133] Guide polynucleotide / Cas endonuclease-induced targeted mutations can occur in nucleotide sequences located within or outside the genomic target site recognized and cleaved by the Cas endonuclease.

[0134] The method for editing a nucleotide sequence in the genome of a cell may be a method by restoring the function of a non-functional gene product without using an exogenous selectable marker.

[0135] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein and at least one donor DNA into the cell, wherein the donor DNA comprises a polynucleotide of interest, and optionally, the method further comprises identifying at least one cell that has integrated the polynucleotide of interest into or near the target site.

[0136] In one aspect, the methods disclosed herein can employ homologous recombination (HR) to provide for integration of a polynucleotide of interest at a target site.

[0137] A variety of methods and compositions can be used to produce cells or organisms with a target polynucleotide inserted into a target site via the activity of a CRISPR-Cas system component as described herein. In a method described herein, via a donor DNA construct, the target polynucleotide is introduced into an organism cell. The donor DNA construct further comprises a first region and a second region of homology flanking the target polynucleotide. The first region and the second region of homology of the donor DNA have homology to the first and second genomic regions present in the target site of a cell or organism genome or flanking the target site, respectively.

[0138] Donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, can be used for genome editing, gene insertion and targeted genome regulation, and can also be used to target late mitotic cells, in which the function of endogenous HR machinery is expected to be greatly reduced (Mali et al., 2013 Nature Methods [Natural Methods] Vol. 10: 957-963).

[0139] In one embodiment, the present disclosure comprises a method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing at least one PGEN described herein and a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification of the nucleotide sequence, and the method optionally further comprises selecting at least one cell comprising the edited nucleotide sequence.

[0140] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow editing (modification) of the target genomic nucleotide sequence. (See also US20150082478 published on March 19, 2015 and WO 2015026886 published on February 26, 2015).

[0141] Further uses of the guide RNA / Cas endonuclease system have been described (see, e.g., US20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, US20150059010 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and PCT application WO2016025131 published on February 18, 2016) and include, but are not limited to, modification or replacement of a nucleotide sequence of interest (such as a regulatory element), insertion of a polynucleotide of interest, gene knockout, gene knockin, modification of splice sites and / or introduction of alternative splice sites, modification of a nucleotide sequence encoding a protein of interest, amino acid and / or protein fusions, and gene silencing caused by expression of an inverted repeat sequence in a gene of interest.

[0142] The methods described herein do not depend on a specific method for introducing a sequence into an organism or cell, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the organism. Introducing includes reference to incorporating a nucleic acid into a eukaryotic or prokaryotic cell, wherein the nucleic acid can be incorporated into the genome of the cell, and includes reference to nucleic acids, proteins, or polynucleotide-protein complexes (PGEN, RGEN) being provided transiently (directly) into the cell.

[0143] Methods for introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated introduction, transfection, transduction, cell penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical application, sexual hybridization, sexual breeding, and any combination thereof.

[0144] A variety of transient transformation methods can be used to provide or introduce polynucleotides or recombinant DNA constructs into prokaryotic and eukaryotic cells or organisms. This transient transformation method includes but is not limited to directly introducing polynucleotide constructs into plants. Nucleic acid and protein can be provided to cells by any method, including using molecules to promote the intake of any or all components (such as cell penetrating peptides and nanocarriers) of the Cas system (protein and / or nucleic acid) guided. See also US20110035836 published on February 10, 2011 and EP 2821486 A1 published on January 7, 2015.

[0145] Other methods for introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts can be used, including plastid transformation methods, and methods for introducing polynucleotides into tissues from seedlings or mature seeds."Stable transformation" is intended to mean that the nucleotide construct introduced into the organism is integrated into the genome of the organism, and can be inherited by its progeny."Transient transformation" is intended to mean that polynucleotides are introduced into the organism and are not integrated into the genome of the organism, or polypeptides are introduced into the organism.Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

[0146] A variety of methods can be used to identify those cells with an altered genome at or near the target site without using a selectable marker phenotype. Such methods can be considered to be direct analysis of the target sequence to detect any changes in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blotting, and any combination thereof.

[0147] In one aspect, the disclosed methods can be used to introduce into a plant a polynucleotide for targeted integration of a nucleotide sequence into a plant. For example, the disclosed methods can be used to introduce a T-DNA expression cassette to transform a plant comprising a target site, the transfer cassette comprising a target nucleotide sequence flanked by non-identical recombination sites. See, for example, WO 2022087616, which is incorporated herein by reference in its entirety.

[0148] On the one hand, the genome target site contains at least one group of non-identical recombination sites corresponding to those on the T-DNA expression cassette. The recombinase mediates the exchange of the nucleotide sequence flanked by the recombination site. Therefore, the disclosed method can be used to introduce the T-DNA expression cassette with targeted integration nucleotide sequence, wherein the T-DNA expression cassette flanked by non-identical recombination sites is recognized by the recombinase, and the recombinase realizes the reorganization at the non-identical recombination site. Therefore, the disclosed method and composition can be used to improve the development efficiency and the development speed of the plant containing non-identical recombination sites.

[0149] Thus, the disclosed methods can further include methods for directed, targeted integration of exogenous nucleotides into plant cells. In one aspect, the disclosed methods use recombination sites in a gene targeting system that facilitates directed targeting of desired genes and nucleotide sequences to corresponding recombination sites previously introduced into the target plant genome.

[0150] By suitable non-identical recombination sites is meant that, in the presence of an active recombinase, excision of the sequence between the two non-identical recombination sites (if any) occurs at a significantly lower efficiency than the targeted arrangement of the nucleotide sequence recombination-mediated exchange into the plant genome. Thus, suitable non-identical sites for use in the present disclosure include those sites where the recombination efficiency between the sites is low; for example, where the efficiency is less than about 30% to about 50%, preferably less than about 10% to about 30%, more preferably less than about 5% to about 10%.

[0151] As noted above, in one embodiment, the recombination site in the targeting cassette corresponds to the recombination site in the target site of the transformed plant. That is, if the target site of the transformed plant contains the different recombination sites of the flanks of FRT and sudden change FRT, the targeting cassette will contain the different recombination sites of the same FRT and sudden change FRT. If the FRT site is used, the FLP recombinase is needed. In the same way, when using the lox site, the Cre recombinase is needed. If the different recombination sites comprise both FRT and lox sites, both FLP and Cre recombinase will be needed in the plant cell.

[0152] Where appropriate, the nucleotide sequence to be inserted into the plant genome may be optimized to increase expression. Where mammalian, yeast or bacterial genes are used in the present disclosure, they may be synthesized using plant preferred codons to improve expression. It will be appreciated that dicot genes may also be synthesized using monocot preferred codons for expression in monocots. It will be further appreciated that all or any portion of the gene sequence may be optimized or synthetic. That is, fully optimized or partially optimized sequences may also be used.

[0153] Additional sequence modifications are known to enhance gene expression in cellular hosts and can be used in the present disclosure. These include: elimination of sequences encoding spurious polyadenylation signals and exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to an average level for a given cellular host calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid the presence of foreseeable hairpin secondary RNA structures.

[0154] The present disclosure also encompasses FLP recombination target sites (FRTs). FRTs have been identified as the minimum sequence containing two 13 base pair repeats separated by eight (8) base spacers. As long as the two 13 base repeats are separated by eight nucleotides, the nucleotides in the spacer can be replaced by a combination of nucleotides.

[0155] FRT mutation sites can be used to practice the disclosed method. Although mutation FRT sites are known (see SEQ ID NO:2,3,4 and 5 of WO1999 / 025821), it is recognized that other mutation FRT sites can be used to practice the disclosure. The disclosure is not limited to the use of specific FRT or recombination sites, but can use different recombination sites or FRT sites to target insertion and expression of nucleotide sequences in plant genomes. Therefore, other mutation FRT sites can be constructed and used based on the disclosure.

[0156] As discussed above, in the presence of a recombinase, a genomic DNA containing a target site with non-identical recombination sites causes recombination together with a carrier containing a T-DNA expression cassette with corresponding non-identical recombination sites. The nucleotide sequence of the T-DNA expression cassette between the flanking recombination sites is exchanged with the nucleotide sequence of the target site between the flanking recombination sites. In this way, the target nucleotide sequence can be accurately incorporated into the host's genome.

[0157] It is recognized that many variations of the present disclosure can be put into practice. For example, a target site with a plurality of different recombination sites can be constructed. Therefore, a plurality of genes or nucleotide sequences can be stacked or sorted at the precise position in the plant genome. Similarly, once a target site has been established in the genome, other recombination sites can be introduced by incorporating such a site into the nucleotide sequence of the T-DNA expression cassette and transferring the site to the target sequence. Therefore, once a target site has been established, it is possible to subsequently add a site or change the site by restructuring.

[0158] Another modification includes providing a promoter or transcription initiation region operably connected to the target site in the organism. Preferably, the promoter will be located 5' of the first recombination site. By transforming the organism with a T-DNA expression cassette comprising the coding region, expression of the coding region will occur when the T-DNA expression cassette is integrated into the target site. This aspect provides a method for selecting transformed cells (particularly plant cells) by providing a selective marker sequence as the coding sequence.

[0159] The present disclosure is further illustrated in the following examples. It should be understood that these examples are given by way of illustration only and not by way of limitation.

[0160] Embodiment 1. A method for inducing parthenogenesis, the method comprising:

[0161] Expressing in a female gametophyte a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell of a plant, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof, wherein the female gametophyte is made parthenogenetic and forms haploid embryos in the absence of pollination, and wherein the Babyboom (BBM) polynucleotide is not naturally expressed in the female gametophyte.

[0162] Embodiment 2. The method of embodiment 1, wherein the nucleotide sequence encoding the Babyboom (BBM) polypeptide or a fragment thereof is selected from the group consisting of: BBM1, BBM2, BMN2, BMN3, ODP2, and BBML.

[0163] Embodiment 3. The method of embodiment 1, wherein the Babyboom (BBM) polynucleotide is selected from the group consisting of:

[0164] (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0165] (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0166] (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0167] (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity;

[0168] (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

[0169] Embodiment 4. The method of embodiment 1, wherein the Babyboom (BBM) polypeptide is selected from the group consisting of:

[0170] (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0171] (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0172] (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0173] (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and

[0174] (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

[0175] Embodiment 5. The method of embodiment 1, wherein the promoter expressed in the oocyte is an oocyte-specific promoter or an oocyte-preferred promoter.

[0176] Embodiment 6. The method of embodiment 5, wherein the promoter expressed in the oocyte comprises:

[0177] (a) the polynucleotide sequence of SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 or a variant thereof;

[0178] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86, or a variant thereof;

[0179] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the operably linked Babyboom (BBM) polynucleotide encoding the Babyboom (BBM) polypeptide or the fragment thereof.

[0180] Embodiment 7. The method of embodiment 1, wherein the promoter expressed in the oocyte further comprises an expression regulatory element.

[0181] Embodiment 8. A method as described in Embodiment 1, wherein the expression of the Babyboom (BBM) polynucleotide driven by an egg cell promoter comprising an expression regulatory element increases the haploid induction frequency of the female gametophyte compared to the haploid induction frequency caused by the expression of the Babyboom (BBM) polynucleotide driven by an egg cell promoter without an expression regulatory element.

[0182] Embodiment 9. The method of embodiment 1, wherein before or during expression of the parthenogenetic factor (eg, the BBM polynucleotide) in the egg cell, the female gametophyte has not been fertilized.

[0183] Embodiment 10. The method of embodiment 1, further comprising: modifying the regulatory region of the endogenous Babyboom (BBM) polynucleotide so that the Babyboom (BBM) polynucleotide is expressed in female gametophytes.

[0184] Embodiment 11. The method according to embodiment 1, comprising:

[0185] The Babyboom (BBM) polynucleotide is expressed from a modified endogenous genomic BBM locus, wherein the genomic BBM locus comprises a modified regulatory region of an endogenous polynucleotide encoding a BBM polypeptide, wherein one or more nucleotides in the regulatory region are modified such that the BBM polypeptide is expressed in female gametophytes.

[0186] Embodiment 12. The method according to embodiment 1, comprising:

[0187] The Babyboom (BBM) polynucleotide is expressed from a modified endogenous genomic egg cell locus, wherein the coding sequence or genomic sequence of the egg cell is modified such that it encodes a BBM polypeptide, wherein the BBM polypeptide is expressed in female gametophytes.

[0188] Embodiment 13. The method of embodiment 1, wherein the Babyboom (BBM) polynucleotide has been modified from its native form using gene editing technology.

[0189] Embodiment 14. A method as described in embodiment 13, wherein the gene editing technology uses a DNA modifying enzyme, and the DNA modifying enzyme is a site-directed nuclease selected from the group comprising: mega nuclease (MN), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), Cas polypeptides such as dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9 adenine deaminase, chimeric FEN1-Fok1, Mega-TAL, nickase Cas9 (nCas9), Chimeric dCas9 non-FokI nuclease, dCpf1-non-FokI nuclease, Cas9, Cas12f (Cas-α, Cas14), Cas12l (Cas-β), Cas12a (Cpf1), Cas12b (C2c1 protein), Cas13 (C2c2 protein), Cas12c (C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10 or a combination or complex of these.

[0190] Embodiment 15. The method of embodiment 1, wherein the female gametophyte is a monocot or dicot female gametophyte.

[0191] Embodiment 16. The method of embodiment 1, wherein the female gametophyte is a wheat, cotton, sunflower, safflower, tobacco, Arabidopsis, hemp, sugarcane, soybean, barley, oats, rice, corn, triticale, sorghum, rye, millet, or flax female gametophyte.

[0192] Embodiment 17. The method according to embodiment 1, further comprising:

[0193] (a) contacting the haploid embryo with a chromosome doubling agent for a period of time sufficient to produce a doubled haploid embryo;

[0194] (b) isolating the doubled haploid embryos; and

[0195] (c) regenerating a doubled haploid plant from the doubled haploid embryo of step (b).

[0196] Embodiment 18. The method of embodiment 1, further comprising:

[0197] (a) regenerating a parthenogenetic plant from a haploid embryo comprising the Babyboom (BBM) polynucleotide operably linked to the promoter expressed in an egg cell;

[0198] (b) pollinating the parthenogenetic plant of (a) with pollen from a non-haploid inducer; and

[0199] (c) rescuing haploid embryos from the parthenogenetic plant of (b).

[0200] Example 19. A method for obtaining a wheat plant that produces clonal, non-reduced, non-recombinant gametes, the method comprising:

[0201] (a) inhibiting the following activities in wheat plant cells:

[0202] (1) All endogenous Spo11 or Prd1, Prd2 or Prd3 polynucleotides or polypeptides;

[0203] (2) all endogenous Rec8 polynucleotides or polypeptides;

[0204] (3) all endogenous Osd1 polynucleotides or polypeptides;

[0205] (b) expressing a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell in a female gametophyte derived from the wheat plant cell, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof, wherein the female gametophyte is caused to parthenogenetically reproduce and form embryos;

[0206] (c) obtaining a plant from the embryo, wherein the embryo comprises a Babyboom (BBM) polynucleotide expressed by the egg cell and a repressed Spo11, Rec8, Osd1, Prd1, Prd2 or Prd3 polynucleotide or polypeptide, thereby producing a wheat plant that produces clonal, non-reduced, non-recombinant gametes.

[0207] Embodiment 20. The method of embodiment 19, wherein the plant is a progeny hybrid plant.

[0208] Embodiment 21. A method as described in Embodiment 19, wherein the activity of the endogenous Spo11 polynucleotide or polypeptide, Rec8 polynucleotide or polypeptide, Osd1 polynucleotide or polypeptide, Prd1, Prd2 or Prd3 polynucleotide or polypeptide or a combination thereof is suppressed by introducing nucleotide modifications into its polynucleotide sequence or introducing amino acid modifications into its polypeptide sequence.

[0209] Embodiment 22. A method as described in embodiment 21, wherein the nucleotide modification is a deletion, addition or substitution of one or more nucleotides, and wherein the amino acid modification is a deletion, addition or substitution of one or more amino acids.

[0210] Embodiment 23. The method of embodiment 21, wherein the nucleotide modification is introduced by a nuclease selected from the group consisting of: TALEN, mega-nuclease, zinc finger nuclease and CRISPR-associated nuclease.

[0211] Embodiment 24. A method as described in Embodiment 21, wherein the nucleotide modification is introduced by a Cas nuclease guided by at least one guide RNA, and the Cas nuclease is, for example: Cas9, Cas12f (Cas-α, Cas14), Cas12l (Cas-β), Cas12a (Cpf1), Cas12b (C2c1 protein), Cas13 (C2c2 protein), Cas12c (C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10 or a combination or complex of these.

[0212] Embodiment 25. The method of embodiment 21, wherein the promoter expressed in the oocyte comprises:

[0213] (a) the polynucleotide sequence of SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 or a variant thereof;

[0214] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86, or a variant thereof;

[0215] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the operably linked Babyboom (BBM) polynucleotide encoding the Babyboom (BBM) polypeptide or the fragment thereof.

[0216] Embodiment 26. The method of embodiment 19, wherein the nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof is selected from the group consisting of BBM1, BBM2, BMN2, BMN3, ODP2 and BBML.

[0217] Embodiment 27. The method of embodiment 19, wherein the Babyboom (BBM) polynucleotide is selected from the group consisting of:

[0218] (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0219] (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0220] (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0221] (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity;

[0222] (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

[0223] Embodiment 28. The method of embodiment 19, wherein the Babyboom (BBM) is selected from the group consisting of:

[0224] (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0225] (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0226] (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0227] (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and

[0228] (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

[0229] Embodiment 29. A method for obtaining a clonal apomictic plant from one or more gametophytic cells in a plant in the absence of fertilization of an egg cell, the method comprising:

[0230] (a) expressing in one or more gamete cells a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof that retains haploid inducing activity, wherein the Babyboom (BBM) polynucleotide is not naturally expressed in female gametophytes;

[0231] (b) developing an embryo from said gamete cell in the absence of fertilization of an egg cell; and

[0232] (c) obtaining a progeny plant from the one or more gametophyte cells, wherein the progeny plant contains chromosomes from the gametophyte cells of (a), thereby achieving reproduction of a flowering plant in the absence of egg cell fertilization.

[0233] Embodiment 30. The method of embodiment 29, wherein the embryo is formed from an unreduced plant cell.

[0234] Embodiment 31. The method of embodiment 30, wherein the unreduced plant cell is an egg cell or is formed from a somatic cell.

[0235] Embodiment 32. The method of embodiment 29, wherein the promoter expressed in the oocyte comprises:

[0236] (a) the polynucleotide sequence of SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 or a variant thereof;

[0237] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86, or a variant thereof;

[0238] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the operably linked Babyboom (BBM) polynucleotide encoding the Babyboom (BBM) polypeptide or the fragment thereof.

[0239] Embodiment 33. The method of embodiment 29, wherein the nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof is selected from the group consisting of BBM1, BBM2, BMN2, BMN3, ODP2 and BBML.

[0240] Embodiment 34. The method of embodiment 29, wherein the Babyboom (BBM) polynucleotide is selected from the group consisting of:

[0241] (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0242] (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0243] (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0244] (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity;

[0245] (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

[0246] Embodiment 35. The method of embodiment 29, wherein the Babyboom (BBM) is selected from the group consisting of:

[0247] (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0248] (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0249] (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0250] (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and

[0251] (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

[0252] Embodiment 36. A method of regulating expression of an endogenous polynucleotide in a genomic locus of a plant cell, the method comprising:

[0253] One or more nucleotides in a regulatory region of a genomic locus comprising an endogenous polynucleotide encoding a BBM polypeptide are modified to generate a modified regulatory region, wherein generation of the modified regulatory region results in expression of the BBM polypeptide in female gametophytes.

[0254] Embodiment 37. A method as described in Embodiment 36, wherein the modification of one or more nucleotides is performed by introducing one or more nucleotides, changing one or more nucleotides, or deleting one or more nucleotides, or a combination thereof.

[0255] Embodiment 38. The method of embodiment 36, wherein the BBM polypeptide is selected from the group consisting of:

[0256] (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0257] (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0258] (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0259] (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and

[0260] (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

[0261] Embodiment 39. The method of embodiment 36, wherein the regulatory region has been modified to comprise an egg cell-specific promoter or an egg cell-specific sequence motif.

[0262] Embodiment 40. The method of embodiment 36, wherein the regulatory region has been modified to comprise a promoter expressed in the egg cell, wherein the promoter comprises:

[0263] (a) the polynucleotide sequence of SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 or a variant thereof;

[0264] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86, or a variant thereof;

[0265] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the operably linked Babyboom (BBM) polynucleotide encoding the Babyboom (BBM) polypeptide or the fragment thereof.

[0266] Embodiment 41. The method of embodiment 36, wherein the regulatory region has been modified to comprise an expression regulatory element.

[0267] Embodiment 42. A modified genomic locus of a plant cell, wherein the genomic locus comprises a modified regulatory region of an endogenous polynucleotide encoding a BBM polypeptide, wherein one or more nucleotides in the regulatory region are modified such that the BBM polypeptide is expressed in a female gametophyte.

[0268] Embodiment 43. The modified genomic locus of embodiment 42, wherein the BBM polypeptide is selected from the group consisting of:

[0269] (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0270] (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0271] (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0272] (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and

[0273] (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

[0274] Embodiment 44. The modified genomic locus of Embodiment 42, wherein the regulatory region has been modified to comprise an egg cell-specific promoter or an egg cell-specific sequence motif.

[0275] Embodiment 45. The modified genomic locus of embodiment 42, wherein the regulatory region has been modified to comprise a promoter expressed in the egg cell, wherein the promoter comprises:

[0276] (a) the polynucleotide sequence of SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 or a variant thereof;

[0277] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86, or a variant thereof;

[0278] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the operably linked Babyboom (BBM) polynucleotide encoding the Babyboom (BBM) polypeptide or the fragment thereof.

[0279] Embodiment 46. The modified genomic locus of Embodiment 42, wherein the regulatory region has been modified to comprise an enhancer expression element.

[0280] Embodiment 47. A method of regulating a genomic sequence or coding sequence of an endogenous polynucleotide in a genomic locus of a plant cell, the method comprising:

[0281] A genomic sequence or coding sequence of an endogenous genomic oocyte locus is modified to produce a modified genomic sequence or coding sequence comprising a heterologous polynucleotide encoding a BBM polypeptide, wherein the modification results in expression of the BBM polypeptide in female gametophytes.

[0282] Embodiment 48. A method as described in embodiment 47, wherein the modification of the endogenous genomic sequence or coding sequence is performed by:

[0283] (a) replacing one or more nucleotides of the endogenous coding sequence;

[0284] (b) introducing one or more nucleotides into the endogenous coding sequence;

[0285] (c) changing one or more nucleotides of the endogenous coding sequence; or

[0286] (d) deleting one or more nucleotides of the endogenous coding sequence;

[0287] or a combination thereof.

[0288] Embodiment 49. The method of embodiment 47, wherein the heterologous polynucleotide is selected from the group consisting of:

[0289] (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0290] (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0291] (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0292] (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity;

[0293] (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

[0294] Embodiment 50. The method of embodiment 47, wherein the BBM polypeptide is selected from the group consisting of:

[0295] (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0296] (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0297] (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0298] (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and

[0299] (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

[0300] Embodiment 51. The method of embodiment 47, wherein the genomic egg cell locus has been modified to comprise an enhancer expression element such that the heterologous polynucleotide is expressed in the female gametophyte, and / or the genomic egg cell locus comprises an endogenous promoter expressed in the egg cell, wherein the endogenous promoter comprises:

[0301] (a) the polynucleotide sequence of SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86 or a variant thereof;

[0302] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86, or a variant thereof;

[0303] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the operably linked Babyboom (BBM) polynucleotide encoding the Babyboom (BBM) polypeptide or the fragment thereof.

[0304] Embodiment 52. A modified genomic locus of a plant cell, wherein the genomic locus comprises a modified coding sequence of an endogenous genomic egg cell locus, wherein one or more nucleotides in the coding sequence of the endogenous egg cell have been modified to express a BBM polypeptide, wherein the BBM polypeptide is expressed in a female gametophyte. See, e.g., SEQ ID NOs: 114 and 115.

[0305] Embodiment 53. The modified genomic locus of embodiment 52, wherein the heterologous polynucleotide is selected from the group consisting of:

[0306] (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0307] (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0308] (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88;

[0309] (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity;

[0310] (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

[0311] Embodiment 54. The modified genomic locus of embodiment 52, wherein the BBM polypeptide is selected from the group consisting of:

[0312] (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0313] (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0314] (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48;

[0315] (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and

[0316] (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

[0317] Embodiment 55. The modified genomic locus of embodiment 52, wherein the endogenous genomic oocyte locus has been modified to comprise an enhancer expression element.

[0318] Embodiment 56. A plant comprising a promoter operably linked to a heterologous polynucleotide sequence, wherein the promoter is selected from the group consisting of:

[0319] (a) the polynucleotide sequence of SEQ ID NO: 37, 38 or 39;

[0320] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, 38 or 39;

[0321] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the heterologous polynucleotide sequence in the egg cell of a plant.

[0322] Embodiment 57. A recombinant DNA construct comprising a promoter operably linked to a heterologous polynucleotide sequence, wherein the promoter is selected from the group consisting of:

[0323] (a) the polynucleotide sequence of SEQ ID NO: 37, 38 or 39;

[0324] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, 38 or 39;

[0325] (c) A fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of the heterologous polynucleotide sequence in the egg cell of a plant.

[0326] Embodiment 58. A method for expressing a gene product in an egg cell of a plant, the method comprising:

[0327] Introducing into the plant or plant cell or the egg cell of the plant a polynucleotide comprising a polynucleotide sequence selected from the group consisting of:

[0328] (a) the polynucleotide sequence of SEQ ID NO: 37, 38 or 39;

[0329] (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, 38 or 39;

[0330] (c) a fragment of the polynucleotide of (a) or (b), such as a fragment of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 consecutive nucleotides of (a) or (b), wherein the fragment retains its ability to drive expression of a heterologous polynucleotide sequence in an oocyte;

[0331] Wherein the polynucleotide sequence drives the expression of an operably linked heterologous polynucleotide sequence.

[0332] Embodiment 59. The method of embodiment 56, 57 or 58, wherein the operably linked sequence is a Babyboom (BBM) polynucleotide encoding a polypeptide that induces haploid induction when expressed in female gametophytes.

[0333] Embodiment 60. The method as described in Embodiment 60, wherein the operably linked sequence encodes a BBM polypeptide or a functional fragment thereof.

[0334] Embodiment 61. The method of embodiment 56, 57 or 58, wherein the plant is a monocot or a dicot.

[0335] Embodiment 62. The method of embodiment 56, 57 or 58, wherein the plant is wheat, cotton, sunflower, safflower, tobacco, Arabidopsis, soybean, barley, oats, rice, corn, triticale, sorghum, hemp, sugarcane, rye, millet, or flax.

[0336] The following examples are offered by way of illustration and not by way of limitation.

[0337] Examples

[0338] Aspects of the present disclosure are further defined in the following examples, wherein parts and percentages are by weight, and degrees are in degrees Celsius, unless otherwise stated. Although these examples illustrate aspects of the present disclosure, they are provided by way of illustration only. From the above discussion and these examples, those skilled in the art can determine the essential characteristics of aspects of the present disclosure, and, without departing from the spirit and scope of the present disclosure, various changes and modifications thereof may be made to adapt them to various uses and conditions. Therefore, from the above description, in addition to those shown and described herein, various modifications will be clear to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0339] Example 1: Methods for inducing parthenogenesis in wheat.

[0340] Polynucleotides encoding BBM peptides linked to regulatory elements active in maternal tissues or haploid cells were evaluated for their ability to induce parthenogenesis in wheat oocytes.

[0341] A. Maternal haploid induction in response to wheat BBM peptide

[0342] Polynucleotides encoding BBM-type transcription factor peptides were identified in the wheat genome on homeologous chromosome 3 (chromosomes 3A (SEQ ID NOs: 1, 2, 74, 75), 3B (SEQ ID NOs: 3, 4, 76, 77), and 3D (SEQ ID NOs: 5, 6, 78, 79)) and homeologous chromosome 6 (chromosomes 6A (SEQ ID NOs: 7, 8, 80, 81), 6B (SEQ ID NOs: 9, 10, 82, 83), and 6D (SEQ ID NOs: 11, 12, 84, 85)). An alignment of wheat BBM peptides with BBM peptides from other monocot species is shown in Figure 1 The DNA polynucleotide sequence encoding the TA-BBM peptide is operably linked to various promoters active during female gamete development.

[0343] Expression cassettes were prepared to test parthenogenesis using the wheat TA-BBM3A sequence, an ortholog of ZM-ODP2 (SEQ ID NOs: 13, 14). In one example, the ZM-DD45PRO:TA-BBM3A expression cassette contained a polynucleotide encoding the TA-BBM3A peptide (SEQ ID NO: 1) operably linked to a regulatory element, ZM-DD45 PRO (SEQ ID NO: 15). In another example, the PvEC1 PRO:TA-BBM3A expression cassette contains a polynucleotide encoding the TA-BBM3A peptide (SEQ ID NO: 1), which is operably linked to regulatory elements comprising PV-EGG CELL PRO (TR1) (SEQ ID NO: 16), EGGMIN PRO (SEQ ID NO: 17), and PV-PRO31696.1 5UTR (SEQ ID NO: 18), and this combination of regulatory elements SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 is referred to as the "PvEC1 promoter" (SEQ ID NO: 19).

[0344] A similar expression cassette was prepared to evaluate parthenogenesis using the wheat TA-BBM6A sequence, an ortholog of the ZM-BBM2 (SEQ ID NOs: 20, 21) sequence in wheat. In one example, the ZM-DD45 PRO PRO:TA-BBM6A expression cassette contained a polynucleotide encoding the TA-BBM6A peptide (SEQ ID NO: 7) operably linked to the regulatory element ZM-DD45 PRO (SEQ ID NO: 15). In another example, the PvEC1PRO:TA-BBM6A expression cassette contains a polynucleotide encoding the TA-BBM6A peptide (SEQ ID NO: 7), which is operably linked to regulatory elements comprising PV-EGG CELL PRO (TR1) (SEQ ID NO: 16), EGG MIN PRO (SEQ ID NO: 17) and PV-PRO31696.15UTR (SEQ ID NO: 18), and this combination of regulatory elements SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 is referred to as the "PvEC1 promoter" (SEQ ID NO: 19).

[0345] These expression cassettes are designed and used as parthenogenesis factors to induce egg cells to differentiate into embryonic cells and start embryogenesis. These expression cassettes are included in the construct for the transformation of wheat line SBC0456D mediated by Agrobacterium, thereby causing T-DNA to be randomly integrated into the wheat genome. Leaf samples are collected from T0 seedlings to determine the copy number and transgenic integrity of multiple different elements in the left and right borders of the T-DNA integrated into the plant genome. Only further characterization is determined as the plant of single copy for multiple different elements. After transformation, each regeneration hemizygous T0 plant containing a single stable inserted T-DNA is considered as a unique event, and the plant growth is made to maturity, allowing it to self-pollinate, and harvest mature T1 seeds. In the plant containing hemizygous BBM expression cassette, it is expected that transgenic is separated with a 1:1 ratio (transgenic positive: transgenic negative) in female gametes, and if therefore the parthenogenesis phenotype is fully penetrant and expressed, 50% is the expected maximum frequency of haploid progeny. If parthenogenesis occurs, pollination may only require fertilization of the central cell and initiate normal development of the endosperm, but does not require fertilization of the egg cell that has already initiated embryogenesis. During female gametogenesis, expression of wheat BBM polynucleotides driven by an egg cell-specific promoter in or near the embryo sac cell (particularly the egg cell) stimulates embryogenesis and parthenogenesis of the egg cell. In bread wheat, the progeny derived from the parthenogenetic egg cell are haploid (1n=3x, ABD) rather than diploid (2n=6x, AABBDD), and contain half the number of chromosomes and relative DNA content compared to the parent plant. Flow cytometric analysis of nuclei isolated from leaf tissue was used to determine the ploidy of individual plants in the T1 progeny.

[0346] The evaluation of haploid progeny demonstrated the ability of wheat BBM polynucleotide sequences to induce parthenogenesis. The percentage (%) of haploid progeny for each unique event was assessed using the number of haploid progeny divided by the total number of progeny examined to determine the haploid induction (HI) frequency. Progeny from transgenic events containing expression cassettes ZM-DD45 PRO::TA-BBM3A, Pv-EC1 PRO:TA-BBM3A were evaluated by flow cytometry to determine the HI frequency. For the ZM-DD45 PRO::TA-BBM3A expression cassette, individual events showed a range of 0% to 10% HI frequency, and showed an average ratio of 1.94% HI frequency, with a standard deviation of 4%, as shown in Table 1. For the Pv-EC1 PRO:TA-BBM3A expression cassette, individual events showed a range of 31.03% to 48.57% HI frequency, and showed an average ratio of 41.56% HI frequency, with a standard deviation of 6.97%, as shown in Table 1. Similarly, progeny from transgenic events containing the expression cassettes ZM-DD45 PRO::TA-BBM6A, Pv-EC1 PRO:TA-BBM6A were evaluated by flow cytometry to determine the frequency of haploid progeny. For the ZM-DD45 PRO::TA-BBM6A expression cassette, individual events showed a range of 0% to 6.5% HI frequency, and an average rate of 0.89% HI frequency with a standard deviation of 2.22%, as shown in Table 1. For the Pv-EC1 PRO:TA-BBM6A expression cassette, individual events showed a range of 16.7% to 56.3% HI frequency, and an average rate of 38.1% HI frequency with a standard deviation of 12.91%, as shown in Table 1.

[0347] Table 1. Haploid induction (HI) in the T1 progeny of transgenic events with wheat BBM peptides

[0348]

[0349]

[0350] As described above, the regulatory elements expressed in plant egg cells can be used to regulate the activity of the TA-BBM peptide to induce maternal haploid induction, resulting in a certain percentage of the progeny produced being haploid (having half the number of chromosomes compared to the parent). In addition, alternative regulatory elements can be used to further optimize the level of maternal haploid induction in parthenogenetic reproduction. For example, the following regulatory elements can be used in the methods disclosed herein: those disclosed in US2015 / 0152430 (promoters include but are not limited to AT-DD5 promoter, AT-DD31 promoter and AT-DD65 promoter) and those disclosed in US2018 / 0094273 (maize egg cell promoter) (see, for example, US2015 / 0152430 and US2018 / 0094273, which are incorporated herein by reference in their entirety).

[0351] In conclusion, wheat BBM polynucleotides were shown to induce parthenogenesis and produce viable haploid progeny when expressed in the egg cells of wheat female gametophytes. In addition, unexpectedly, the egg cell specific regulatory element PvEC1 promoter improved the parthenogenetic haploid frequency compared to the egg cell specific regulatory element ZM-DD45 PRO (Table 1).

[0352] B. Maternal haploid induction in response to non-wheat BBM peptides

[0353] In this example, BBM-type transcription factors (such as those disclosed in publication number US20210180077, which is incorporated herein by reference in its entirety) and polynucleotides encoding BBM-type transcription factors identified in other species were used. Haploid progeny assays were performed to evaluate whether BBM genes from different species initiate parthenogenesis in wheat. Expression cassettes encoding DNA polynucleotide sequences (SEQ ID NOs: 13-14, 20-29) encoding additional BBM peptides (described in Table 2) were operably linked to the PvEC1 promoter (SEQ ID NO: 19) active during female gamete development.

[0354] Table 2. Summary of BBM polynucleotides and peptides.

[0355]

[0356]

[0357] Progeny from transgenic events containing maize BBM-type sequences (with expression cassettes PvEC1 PRO::ZM-ODP2 and Pv-EC1 PRO:ZM-BBM2) were evaluated by flow cytometry to determine the frequency of haploid progeny in wheat. The PvEC1 PRO::ZM-ODP2 expression cassette showed an average rate of 43.1% HI frequency with a standard deviation of 12.2%, and the Pv-EC1 PRO:ZM-BBM2 expression cassette showed an average rate of 42.8% HI frequency with a standard deviation of 10.4%, as shown in Table 3. In addition, progeny from transgenic events containing rice and Setaria BBM-type sequences (with expression cassettes PvEC1 PRO::OS-ODP2, Pv-EC1 PRO:OS-BBM2, PvEC1 PRO::SV-BBM1 and Pv-EC1 PRO:SV-BBM2) were evaluated by flow cytometry to determine the frequency of haploid progeny in wheat. The PvEC1PRO::OS-ODP2 expression cassette showed an average ratio of 46.8% HI frequency with a standard deviation of 8.5%, and the Pv-EC1 PRO:OS-BBM2 expression cassette showed an average ratio of 49.7% HI frequency with a standard deviation of 18.6%, as shown in Table 3. The PvEC1 PRO::SV-BBM1 expression cassette showed an average ratio of 36.5% HI frequency with a standard deviation of 11.0%, and the Pv-EC1PRO:SV-BBM2 expression cassette showed an average ratio of 44.1% HI frequency with a standard deviation of 10.3%, as shown in Table 3.

[0358]

[0359]

[0360] In conclusion, BBM polynucleotides from maize, rice, and Setaria viridis were shown to induce parthenogenesis and produce viable haploid progeny when expressed in the egg cells of wheat female gametophytes.

[0361] Example 2: Method for inducing parthenogenesis in wheat using a modified enhancing promoter

[0362] As shown in Example 1, the wheat TA-BBM3A polynucleotide sequence is operably linked to the ZM-DD45 PRO regulatory element and exhibits low frequency of parthenogenesis as measured by the frequency of haploid progeny. In addition, alternative regulatory elements are used to further optimize the haploid induction level of the parthenogenetic maternal parent. For example, the following regulatory elements are used in the methods disclosed herein, such as those disclosed in US2015 / 0152430 (promoters include but are not limited to AT-DD5 promoter, AT-DD31 promoter and AT-DD65 promoter) and those disclosed in US2018 / 0094273 (maize egg cell promoter) (US2015 / 0152430 and US 2018 / 0094273, which are incorporated herein by reference in their entirety). In addition, further modifications that make the regulatory element include expression regulatory elements (EME) are used to further optimize the haploid induction level of the parthenogenetic maternal parent. For example, expression regulatory elements such as those disclosed in Publication No. WO 2018 / 183878, which is incorporated herein by reference in its entirety, can be used in the methods of the present disclosure.

[0363] An expression cassette encoding TA-BBM3A operably linked to a regulatory element containing at least one EME can be used in the methods of the present disclosure. The modified ZM-DD45 PRO comprising at least one EME is used to alter the level of mRNA transcription during female gametogenesis, thereby further improving and / or optimizing parthenogenetic maternal haploid induction compared to the ZM-DD45 PRO:TA-BBM3A expression cassette as shown in Example 1.

[0364] For example, exemplary promoters are shown in Table 4. As shown in Table 4, ZM-DD45PRO (SEQ ID NO: 15) was modified at different positions using one, two or three copies of ZM-AS2 EME (SEQ ID NO: 30) (shown as 1X ZM-AS2, 2X ZM-AS2 or 3X ZM-AS2, respectively). The EME position indicates the number of DNA base pairs upstream of the TATA box inserted into each corresponding EME sequence. The TATA sequence for each promoter is shown in the TATA box column.

[0365] Table 4. Exemplary promoters comprising EME modifications

[0366]

[0367] The modified regulatory elements shown in Table 4 were used to alter the mRNA transcription levels during female gametogenesis, thereby further improving and / or optimizing the induction of parthenogenetic maternal haploids compared to the TA-BBM3A polynucleotide (SEQ ID NO: 1) operably linked to the regulatory elements comprising a DNA fragment designated ZM-DD45 PRO (SEQ ID NO: 15) as described in Example 1. Expression cassettes were prepared to evaluate parthenogenesis using wheat TA-BBM3A polynucleotide sequences with modified ZM-DD45PRO regulatory sequences. In one example, the ZM-DD45 PRO: (1X ZM-AS2-20)::TA-BBM3A expression cassette contains a polynucleotide encoding a TA-BBM3A peptide (SEQ ID NO: 1) operably linked to the regulatory element ZM-DD45 PRO: (1X ZM-AS2-20) (SEQ ID NO: 31). In another example, the ZM-DD45 PRO:(2X ZM-AS2-100)::TA-BBM3A expression cassette contains a polynucleotide encoding the TA-BBM3A peptide (SEQ ID NO: 1) operably linked to a regulatory element ZM-DD45 PRO:2X ZM-AS2-100) (SEQ ID NO:36).

[0368] The evaluation of haploid progeny demonstrated the ability of modified regulatory elements operably linked to the wheat TA-BBM3A polynucleotide sequence to induce parthenogenesis. The percentage (%) of haploid progeny for each unique event was assessed using the number of haploid progeny divided by the total number of progeny examined to determine the haploid induction (HI) frequency. Progeny from transgenic events containing expression cassettes ZM-DD45 PRO::TA-BBM3A, ZM-DD45PRO:(1X ZM-AS2-20)::TA-BBM3A and ZM-DD45 PRO:(2X ZM-AS2-100)::TA-BBM3A were evaluated by flow cytometry to determine the frequency of haploid progeny. As shown in Table 5, the transgenic events showed an average rate of 28.5% HI frequency for the ZM-DD45PRO:(1X ZM-AS2-20)::TA-BBM3A expression cassette, and an average rate of 4.2% HI frequency for the ZM-DD45PRO(2X ZM-AS2-100)PRO::TA-BBM3A expression cassette, relative to an average rate of 1.9% HI frequency for the unmodified ZM-DD45PRO::TA-BBM3A (as shown in Example 1). Thus, modification of the regulatory elements operably linked to the BBM polynucleotide sequence can improve the rate of parthenogenesis and haploid progeny.

[0369] Table 5. Haploid induction (HI) in the T1 progeny of transgenic events with modified regulatory element ZM-DD45 PRO

[0370]

[0371]

[0372] The methods of the present disclosure may also use different promoters with or without EME sequences to improve haploid parthenogenesis as described herein.

[0373] Example 3: Identification of wheat egg cell preferred promoters

[0374] Genes that are specifically or preferentially expressed in oocytes were identified in wheat (Triticum aestivum) genomic DNA references. Polynucleotides encoding regulatory element sequences were identified on homeologous chromosome 4 in the wheat genome (SEQ ID NOs: 37, 38, 39). The spatiotemporal pattern of gene expression can be visualized by operably linking a DNA polynucleotide sequence encoding the TA-EGG regulatory sequence to a fluorescent protein and observing the fluorescent signal during female gamete development.

[0375] Example 4: Method for inducing parthenogenesis using wheat egg cell promoter

[0376] As shown in Example 1, the wheat TA-BBM3A polynucleotide sequence is operably linked to the PvEC1 PRO regulatory element and exhibits a high frequency of parthenogenesis as measured by the frequency of haploid progeny. In addition, alternative regulatory elements are used to further optimize the level of parthenogenetic maternal haploid induction. The DNA polynucleotide sequences encoding the TA-BBM3A and TA-BBM6A peptides are operably linked to the natural wheat promoters TA-EGG PRO and TA-ECPRO that are active during female gamete development.

[0377] Expression cassettes were prepared to evaluate parthenogenesis using wheat TA-EGG PRO (SEQ ID NO: 37) and TA-EC PRO (SEQ ID NO: 40) regulatory element sequences. In one example, the TA-EGG PRO:TA-BBM3A and TA-EGG PRO:TA-BBM6A expression cassettes contain polynucleotides encoding TA-BBM3A or TA-BBM6A peptides (SEQ ID NO: 1, 7) operably linked to the regulatory element TA-EGG PRO (SEQ ID NO: 37). In another example, the TA-EC PRO:TA-BBM3A and TA-EC PRO:TA-BBM6A expression cassettes contain polynucleotides encoding TA-BBM3A or TA-BBM6A peptides (SEQ ID NO: 1, 7) operably linked to the regulatory element TA-EC PRO (SEQ ID NO: 40).

[0378] The expression cassettes of these designs are used as parthenogenetic factors to induce egg cell differentiation into embryonic cells and to initiate embryogenesis. These expression cassettes are included in the construct for wheat line SBC0456D to be transformed by Agrobacterium-mediated transformation, thereby causing T-DNA to be randomly integrated into the wheat genome. Leaf samples are collected from the T0 seedlings to determine the copy number and transgenic integrity of a plurality of different elements in the left and right borders of the T-DNA that is integrated into the plant genome.

[0379] Only plants determined as single copies (hemizygous) for multiple different elements are further characterized. After transformation, each regenerated plant as hemizygous T0 containing a single stable inserted T-DNA is considered a unique event, the plant is grown to maturity, allowed to self-pollinate, and mature T1 seeds are harvested. If parthenogenesis occurs, pollination may only require fertilization of the central cell and initiate normal development of the endosperm, but does not require fertilization of the egg cell that has initiated embryogenesis. During female gametogenesis, the expression of wheat BBM polynucleotides driven by an egg cell-specific promoter in or near the embryo sac cell (especially the egg cell) stimulates embryogenesis and parthenogenesis of the egg cell. In bread wheat, the progeny derived from the parthenogenetic egg cell is haploid (1n=3x, ABD) rather than diploid (2n=6x, AABBDD), and contains half the number of chromosomes and relative DNA content compared to the parent plant. The ploidy of individual plants in the T1 progeny is determined using flow cytometric analysis of nuclei isolated from leaf tissue.

[0380] The evaluation of haploid progeny demonstrated the ability of the natural wheat egg cell promoter in combination with the natural wheat BBM polynucleotide sequence to induce parthenogenesis. The HI frequency was determined. The evaluation of transgenic events containing the expression cassette TA-EGG PRO::TA-BBM6A had an average value of 45.3% HI with a standard deviation of 12.4%, and the transgenic events containing the expression cassette TA-EGG PRO::TA-BBM3A had an average value of 35.1% HI with a standard deviation of 9.5% (Table 6). The evaluation of transgenic events containing the expression cassette TA-ECPRO::TA-BBM6A had an average value of 46.6% HI with a standard deviation of 10.0%, and the transgenic events containing the expression cassette TA-EC PRO::TA-BBM3A had an average value of 40.6% HI with a standard deviation of 18.7% (Table 6). In addition, multiple hemizygous T1 plants from four individual events containing the expression cassette TA-EGG PRO::TA-BBM6A were grown to produce progeny seeds of the T2 generation and evaluated by flow cytometry. The stability of the parthenogenetic phenotype was observed as each individual event maintained comparable HI frequencies in the T1 and T2 generations.

[0381] Table 6. Haploid induction (HI) in the T1 progeny of transgenic events with wheat egg promoters

[0382]

[0383] Thus, expression cassettes containing native wheat promoters and BBM-related polynucleotide sequences are sufficient to drive high HI ratios in wheat systems. Furthermore, the penetrance of the parthenogenetic phenotype showed that the wheat TA-EGG and TA-EC promoter sequences contain regulatory elements capable of driving expression in egg cells. Although TA-BBM3A and TA-BBM6A have only 34.9% sequence homology, both are capable of driving high HI ratios in wheat systems.

[0384] Example 5: Method for inducing parthenogenesis in wheat using modified BBM peptides

[0385] The wheat polynucleotide encoding the BBM peptide was linked to a regulatory element active in haploid cells or tissues (e.g., a promoter active during female gamete development). To increase the activity of BBM, the polynucleotide and peptide sequences of TA-BBM3A (SEQ ID NO: 1, 2) and TA-BBM6A (SEQ ID NO: 7, 8) were modified. An alignment of the native and modified wheat BBM peptides is shown in Figure 2 The PvEC1 promoter was operably linked to these modified wheat TA-BBM3A and TA-BBM6A sequences and screened for inducing parthenogenesis.

[0386] TA-BBM3A (ALT1) (SEQ ID NO: 41, 42) is produced by truncating the N-terminal domain and C-terminal domain of TA-BBM3A (SEQ ID NO: 1, 2) to contain only the start codon, base pairs 154 to 2010, and the stop codon. It retains the start codon and the conserved motifs B, A, and AP2 DNA binding domain. In another variation, TA-BBM3A (ALT2) (SEQ ID NO: 43, 44) is produced by truncating TA-BBM3A (SEQ ID NO: 1, 2) to leave the start codon and base pairs 154-231, 442-504, and 684-2010, and the stop codon. TA-BBM6A (ALT1) (SEQ ID NOs: 45, 46) was generated by truncating the N-domain and C-domain of TA-BBM6A (SEQ ID NOs: 7, 8) to contain only the start codon and base pairs 328 to 1980 and the stop codon. In another variation, TA-BBM6A (ALT2) (SEQ ID NOs: 47, 48) was generated by truncating TA-BBM6A (SEQ ID NOs: 7, 8) to leave the start codon, base pairs 328-1908 and the stop codon.

[0387] Table 7. Summary of wheat TA-BBM3A and TA-BBM6A polynucleotide and peptide truncations

[0388]

[0389]

[0390] Expression cassettes were prepared to evaluate parthenogenesis using modified wheat TA-BBM3A sequences. In one example, the PvEC1 PRO:TA-BBM3A (ALT1) expression cassette contained a polynucleotide encoding the TA-BBM3A (ALT1) peptide (SEQ ID NO: 41), which was operably linked to a regulatory element PvEC1 PRO (SEQ ID NO: 19). In another example, the PvEC1 PRO:TA-BBM3A (ALT2) expression cassette contained a polynucleotide encoding the TA-BBM3A (ALT2) peptide (SEQ ID NO: 43), which was operably linked to a regulatory element PvEC1 PRO (SEQ ID NO: 19). Similarly, expression cassettes were prepared to evaluate parthenogenesis using modified wheat TA-BBM6A sequences. In one example, the PvEC1 PRO:TA-BBM6A (ALT1) expression cassette contains a polynucleotide encoding a TA-BBM6A (ALT1) peptide (SEQ ID NO: 45), which is operably linked to a regulatory element PvEC1 PRO (SEQ ID NO: 19). In another example, the PvEC1 PRO:TA-BBM6A (ALT2) expression cassette contains a polynucleotide encoding a TA-BBM6A (ALT2) peptide (SEQ ID NO: 47), which is operably linked to a regulatory element PvEC1 PRO (SEQ ID NO: 19).

[0391] Evaluation of haploid progeny demonstrated the ability of the modified BBM peptide to induce parthenogenesis when operably linked to the PvEC1 PRO regulatory element. HI frequencies were determined as previously described. Evaluations of transgenic events containing the expression cassette PvEC1 PRO::TA-BBM3A (ALT1) had a mean of 27.2% HI with a standard deviation of 9.8%, and transgenic events containing the expression cassette PvEC1 PRO::TA-BBM3A (ALT2) had a mean of 0.0% HI with a standard deviation of 0.0% (Table 8). In addition, evaluations of transgenic events containing the expression cassette PvEC1 PRO::TA-BBM6A (ALT1) had a mean of 2.6% HI with a standard deviation of 3.1%, and transgenic events containing the expression cassette PvEC1 PRO::TA-BBM6A (ALT2) had a mean of 0.0% HI with a standard deviation of 0.0% (Table 8).

[0392] Table 8. Haploid induction (HI) in T1 progeny of transgenic events with wheat BBM polynucleotides and peptide truncations

[0393]

[0394] In summary, when expressed in the oocyte of wheat female gametophyte, the minimal N-terminal truncation and C-terminal truncation of wheat BBM polypeptide (ALT1 sequence) are sufficient to induce parthenogenesis and produce live haploid offspring, but not as effective as the full-length BBM polynucleotide sequence. However, between motifs B and A, further truncation of the N-terminus or C-terminus of wheat BBM polypeptide is not sufficient to induce parthenogenesis or haploid offspring.

[0395] Example 6: Methods for engineering natural parthenogenetic loci in wheat

[0396] To obtain an optimal parthenogenetic phenotype, as described in Example 4, promoters operably linked to TA-BBM polynucleotides were evaluated for driving egg cell-specific or preferential expression of TA-BBM in transgenic wheat plants.

[0397] A. Modification of the endogenous wheat BBM locus

[0398] Here, a gene editing method is presented to modify the promoter region of the endogenous BBM locus to obtain the expression pattern of the desired parthenogenetic phenotype. In wheat, it is expected that the promoter of a single homoeologous TA-BBM locus is modified to include an alternative egg cell specific / preferred promoter or egg cell specific / preferred sequence motif (also including a native promoter sequence or an alternative native promoter sequence) to modify the expression pattern. This modified egg cell expression pattern of a single TA-BBM homolog is expected to induce parthenogenesis in wheat.

[0399] The DNA reagents used during modification of the native locus (including but not limited to guide RNA, Cas polypeptides such as Cas9, Cas12f (Cas-α, Cas14), Cas121 (Cas-β), Cas12a (Cpf1), Cas12b (C2c1 protein), Cas13 (C2c2 protein), Cas12c (C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10, or a combination or complex of these) and transformation selectable markers are not essential for the function of the newly generated BBM alleles and can be eliminated from the genome by segregation through breeding methods known to those skilled in the art. Because the promoter TA-EGG (SEQ ID NO: 37) with the same sequence as the endogenous TA-EGG locus is inserted into the TA-BBM locus by homologous recombination, such as TraesCS3A02G395500 or TraesCS6A02G229500 as described in the IWGSCv1.0 Chinese Spring Wheat Reference Sequence Assembly and Annotation, this TA-BBM allele is difficult to distinguish from the natural mutant allele.

[0400] In order to replace the natural promoter of TA-BBM with TA-EGG promoter and enter the 5'UTR of wheat TA-BBM gene, two kinds of guide RNA (gRNA) can be prepared using corn U6 promoter and terminator. One of gRNA can include the 5' end of gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 5' upstream region of TA-BBM promoter; and one of gRNA can include the 5' end of gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 3' upstream region of TA-BBM promoter (preferably near the ATG start codon). For example, the potential gRNA targeting domain sequence for TA-BBM3A promoter and 5'UTR sequence describes but is not limited to the sequence in Table 7 (SEQ ID NO: 49-56). The flanks of the polynucleotide modification template containing TA-EGG PRO are two genomic DNA fragments (HR1 and HR2), which are derived from the upstream and downstream regions of the genomic target sequence and are homologous to the upstream and downstream regions of the genomic target sequence. Transformation can be initiated with a construct comprising gRNA, a polynucleotide modification template, a Cas box (such as a Cas9 box) and a transformation selective marker. Transgenic plants produced by this method can be evaluated by PCR analysis to confirm promoter exchange by identifying PCR amplification products, which include both TA-EGG promoter sequences and TA-BBM genome coding sequences, but do not include TA-BBM promoters and TA-BBM genome coding sequences. Successful promoter exchange events can be further characterized by sequencing the above-mentioned amplification products. It is expected that successful TA-EGG PRO:TA-BBM promoter exchange events induce parthenogenesis and result in haploid progeny as described in Example 4.

[0401] Table 9. Examples of gRNA targeting domain sequences in the TA-BBM3A promoter region

[0402]

[0403]

[0404] B. Modification of the endogenous wheat EGG CELL locus

[0405] Here, a gene editing method is presented to modify the coding region of the endogenous EGG CELL locus to obtain the expression pattern of the desired parthenogenetic phenotype. In wheat, it is expected that modification of the coding sequence of a single partly homologous TA-EGG locus to include an alternative BBM coding sequence to replace the native coding sequence can modify the expression pattern of the BBM polynucleotide. It is expected that the introduction of the BBM coding sequence downstream of the egg cell specific / preferred promoter will induce parthenogenesis in wheat.

[0406] In order to replace the natural coding sequence of TA-EGG with the TA-BBM coding sequence and enter the 5'UTR of the wheat TA-BBM gene, two kinds of guide RNA (gRNA) can be prepared using corn U6 promoter and terminator. One of the gRNAs can include the 5' end of the gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 3' upstream region of the TA-EGG promoter (preferably near the ATG start codon); and one of the gRNAs can include the 5' end of the gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 3' region of the TA-EGG locus (preferably near the stop codon). The flanks of the polynucleotide modified template containing the TA-BBM coding sequence are two genomic DNA fragments (HR1 and HR2), which are derived from the upstream and downstream regions of the genomic target sequence and are homologous to the upstream and downstream regions of the genomic target sequence. Conversion can be initiated with a construct comprising gRNA, polynucleotide modified templates, Cas boxes (such as Cas9 boxes) and conversion selectable markers. Transgenic plants produced by this method can be evaluated by PCR analysis to confirm the coding sequence exchange by identifying PCR amplification products, which contain both the TA-EGG promoter sequence and the TA-BBM genomic coding sequence, but do not contain the amplification product of the TA-EGG promoter and the TA-EGG genomic coding sequence. Successful promoter exchange events can be further characterized by sequencing the above-mentioned amplification products. It is expected that successful TA-EGG PRO:TA-BBM coding sequence exchange events induce parthenogenesis and lead to haploid progeny as described in Example 4.

[0407] Example 7: Method for producing apomictic plants

[0408] Apomixis is asexual reproduction resulting in offspring that are genetically identical to the parent. The methods disclosed herein are used to obtain apomictic plants having apomictic components having suppressed or mutated gene products that induce mitosis rather than meiosis (MiMe phenotype) and containing BBM parthenogenetic components as described in Examples 1, 2, 4-6.

[0409] In hexaploid wheat, it is expected that the MiMe phenotype may require suppression or mutation in each of the three homeologous loci. The genomic sequences of the coding regions of the SPO11-1 homeologous loci were identified from the IWGSCv1.0 Chinese Spring reference sequence and assembly (SEQ ID NOs: 57, 58, 59). The genomic sequences of the coding regions of the REC8 homeologous loci were identified from the IWGSCv1.0 Chinese Spring reference sequence and assembly (SEQ ID NOs: 60, 61, 62). The genomic sequences of the coding regions of the OSD homeologous loci were identified from the IWGSCv1.0 Chinese Spring reference sequence and assembly (SEQ ID NOs: 63, 64, 65).

[0410] Natural mutations or mutations induced by chemicals or radiation in individual homoeologous loci of MiMe genes can be identified and then hybridized together to produce individual MiMe knockout lines. Individual MiMe knockout lines of Spo11, Rec8, Prd1, Prd2, Prd3 are expected to be both male and female sterile, and individual MiMe knockout lines of Osd1 are expected to increase the ploidy of gametes. However, a combination of mutations for producing MiMe phenotypes can be produced by hybridizing plants that are heterozygous for the alleles of the MiMe locus. In this example, mutations with knockout or knockdown alleles of the MiMe locus can be combined with BBM parthenogenetic components to obtain apomixis plants (e.g., flowering plants) with cloned gametes and progeny in the absence of egg fertilization.

[0411] Another example of improving the process of knocking out or knocking down alleles in MiMe loci is to use gene editing components. The method disclosed herein uses gene editing traits, which include a first expression cassette encoding CRISPR-Cas9 gene editing polynucleotides (or other Cas polypeptides) and a second expression cassette encoding a gRNA molecule with sequence homology to MiMe genes. For example, the gRNA targeting domain sequences (SEQ ID NO: 66-73) of TA-SPO11-1, TA-REC8 and TA-OSD genomic coding sequences are described, but are not limited to the sequences shown in Table 8. It is expected that mutations at MiMe gene target sites provide methods for obtaining non-recombination and non-reduction. Alternatively, the method disclosed herein uses gene editing traits, which include a first expression cassette encoding Casα gene editing polynucleotides and a second expression cassette encoding a gRNA molecule with sequence homology to MiMe genes. It is expected that mutations at MiMe gene target sites provide methods for obtaining non-recombination and non-reduction gametes.

[0412] Table 10. Examples of gRNA targeting domain sequences in the coding regions of the TA-SPO11-1, TA-REC8 and TA-OSD genomes

[0413]

[0414]

[0415] The construct used for the gene editing process can include an additional box containing the BBM parthenogenesis component to obtain apomixis plants with cloned gametes and progeny. The construct used for gene editing or parthenogenesis process can also be transformed into a previously transformed or gene-edited system to obtain apomixis plants with cloned gametes and progeny. Alternatively, gene editing with knockout or knockdown alleles of MiMe loci can be combined with BBM parthenogenesis components by hybridization to obtain apomixis plants with cloned gametes and progeny. In this example, gene editing with knockout or knockdown alleles of MiMe loci can be combined with BBM parthenogenesis components to obtain apomixis plants with cloned gametes and progeny.

[0416] Example 8: Methods for Engineering Natural Parthenogenetic Loci in Maize

[0417] To achieve an optimal parthenogenetic phenotype, promoters are evaluated in transgenic maize plants for driving egg cell-specific or preferential expression of ZM-BBM or ZM-ODP2 operably linked to a ZM-BBM2 or ZM-ODP2 polynucleotide.

[0418] A. Modification of the endogenous maize BBM locus

[0419] Here, a gene editing method is presented to modify the promoter region of the endogenous maize BBM2 locus or ODP2 locus to obtain the expression pattern of the desired parthenogenetic phenotype. In maize, it is expected that the promoter of a single homoeologous ZM-BBM2 or ZM-ODP2 locus is modified to include an alternative egg cell specific / preferred promoter or egg cell specific / preferred sequence motif (also including a native promoter sequence or an alternative native promoter sequence) to modify the expression pattern. This modified egg cell expression pattern of a single ZM-BBM2 or ZM-ODP2 homolog is expected to induce parthenogenesis in maize.

[0420] The DNA reagents used during the modification of the native locus (including but not limited to guide RNA, Cas endonucleases (such as Cas9, Cas12f (Cas-α, Cas14), Cas121 (Cas-β), Cas12a (Cpf1), Cas12b (C2c1 protein), Cas13 (C2c2 protein), Cas12c (C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10 or a combination or complex of these)) and transformation selectable markers are not essential for the function of the newly generated BBM allele and can be eliminated from the genome by segregation through standard breeding methods. For example, the promoter ZM-EGG PRO (SEQ ID NO: 86) is inserted upstream of the start codon of the endogenous ZM-BBM2 genomic locus or the endogenous ZM-ODP2 genomic locus. In another example, promoter ZM-DD45 (SEQ ID NO: 15) is inserted upstream of the start codon of the endogenous Zm-BBM2 or ZM-ODP2 genomic locus. In any of these cases, a gRNA can be used near the start codon or in the 5'UTR to insert the promoter sequence, or two gRNAs can be used to first excise the natural ZM-BBM2 or ZM-ODP2 promoter before inserting the egg cell promoter. Because the promoter ZM-EGG PRO (having the same sequence as the endogenous ZM-EGG locus) is inserted into the ZM-BBM2 or ZM-ODP2 locus, for example, by homologous recombination, this allele is difficult to distinguish from the natural mutant allele.

[0421] In order to replace the natural promoter of ZM-BBM2 with ZM-EGG promoter (SEQ ID NO: 86) and enter the 5'UTR of corn ZM-BBM2 gene, two guide RNAs (gRNAs) can be prepared using corn U6 promoter and terminator. One of the gRNAs can include the 5' end of the gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 5' upstream region of the ZM-BBM2 promoter; and one of the gRNAs can include the 5' end of the gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 3' upstream region of the ZM-BBM2 promoter (preferably near the ATG start codon). For example, the potential gRNA targeting domain sequence for ZM-BBM2 promoter and 5'UTR sequence describes but is not limited to the sequences in Table 9 (SEQ ID NO: 91, 92, 93, 94, 95, 96, 97 and 98).

[0422] Table 11. Examples of gRNA targeting domain sequences in the ZM-ODP2 and ZM-BBM2 promoter regions.

[0423]

[0424]

[0425] The flanks of the polynucleotide modified template containing ZM-EGG PRO are two genomic DNA fragments (HR1 and HR2), which are derived from the upstream and downstream regions of the genomic target sequence and are homologous to the upstream and downstream regions of the genomic target sequence. Conversion can be initiated with a construct comprising gRNA, polynucleotide modified template, Cas box and conversion selectable marker. Transgenic plants produced by this method can be evaluated by PCR analysis to confirm promoter exchange by identifying PCR amplification products, which include both ZM-EGG promoter sequence and ZM-BBM2 genomic coding sequence. Successful promoter exchange events can be further characterized by sequencing the above-mentioned amplification products. It is expected that successful ZM-EGG PRO: ZM-BBM2 promoter exchange events induce parthenogenesis and lead to haploid progeny.

[0426] In another example, a similar strategy can be applied to insert the ZM-DD45 PRO or ZM-EGG PRO into the ZM-ODP2 genomic locus.

[0427] Table 12. Examples of gRNA targeting domain sequences for the ZM-EGG and ZM-DD45 regions.

[0428]

[0429]

[0430] B. Modification of the endogenous maize EGG CELL locus

[0431] Here, a gene editing method is presented to modify the coding region of the endogenous EGG CELL locus to obtain a desired expression pattern of a parthenogenetic phenotype in maize. In maize, it is expected that modification of the coding sequence of a single homeologous ZM-EGG locus to include an alternative BBM2 or ODP2 coding sequence to replace the native coding sequence can modify the expression pattern of the BBM polynucleotide. It is expected that the introduction of the BBM2 or ODP2 coding sequence downstream of an egg cell specific / preferred promoter will induce parthenogenesis in maize.

[0432] In order to replace the natural coding sequence of ZM-EGG with the ZM-BBM coding sequence and enter the 5'UTR of the corn ZM-BBM2 gene, two kinds of guide RNA (gRNA) can be prepared using corn U6 promoter and terminator. One of the gRNAs can include the 5' end of the gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 3' upstream region of the ZM-EGG promoter (preferably near the ATG start codon); and one of the gRNAs can include the 5' end of the gRNA containing the following 20-bp variable targeting domain, which targets the genomic sequence in the 3' region of the ZM-EGG locus (preferably near the stop codon). The flanks of the polynucleotide modified template containing the ZM-BBM coding sequence are two genomic DNA fragments (HR1 and HR2), which are derived from the upstream and downstream regions of the genomic target sequence and are homologous to the upstream and downstream regions of the genomic target sequence. Conversion can be initiated with a construct comprising gRNA, polynucleotide modified templates, Cas endonuclease box and conversion selectable marker. Transgenic plants produced by this method can be evaluated by PCR analysis to confirm the coding sequence exchange by identifying PCR amplification products, which contain both the ZM-EGG promoter sequence and the ZM-BBM2 coding sequence. Successful coding sequence exchange events can be further characterized by sequencing the above-mentioned amplification products. Successful ZM-EGG PRO: ZM-BBM2 coding sequence exchange events are expected to induce parthenogenesis and result in haploid progeny.

[0433] In another example, similar to the above, but a single gRNA is used to produce DNA breaks between the promoter of an egg promoter (such as ZM-EGG or ZM-DD45) and the coding sequence of ZM-EGG or DD45. The coding sequence (genomic or CDS) of ZM-ODP2 or ZM-BBM2 and their terminators are introduced and integrated into the cleavage site. In these examples, the coding sequence of ZM-EGG or ZM-DD45 is not eliminated from the genome, but is separated from its functional promoter. Alternatively, the functional egg promoter drives the expression of ZM-ODP2 or ZM-BBM2. The transgenic plants produced by this method can be evaluated by PCR analysis to confirm the coding sequence exchange by identifying PCR amplification products, which include both ZM-EGG promoter sequence and ZM-BBM2 / ZM-ODP2 coding sequence. Successful coding sequence exchange events can be further characterized by sequencing the above-mentioned amplification products. Successful ZM-EGG PRO:ZM-BBM2, ZM-DD45:ZM-BBM2, ZM-EGG:ZM-ODP2 and ZM-EGG:ZM-ODP2 coding sequence exchange events are expected to induce parthenogenesis and result in haploid progeny.

Claims

1. A method for expressing a gene product in an egg cell of a plant, the method comprising: Introducing into the plant a polynucleotide comprising a polynucleotide sequence selected from the group consisting of: (d) the polynucleotide sequence of SEQ ID NO: 37, 38 or 39; (e) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, 38 or 39; (f) a fragment of the polynucleotide of (a) or (b), wherein the fragment retains its ability to drive expression of a heterologous polynucleotide sequence in an oocyte; Wherein the polynucleotide sequence drives the expression of an operably linked heterologous polynucleotide sequence.

2. The method of claim 1, wherein the operably linked heterologous polynucleotide sequence is a Babyboom (BBM) polynucleotide encoding a polypeptide that induces haploid induction when expressed in female gametophytes.

3. The method of claim 2, wherein the operably linked heterologous polynucleotide sequence encodes a BBM polypeptide or a functional fragment thereof.

4. The method of claim 1, wherein the plant is a monocot or a dicot.

5. The method of claim 1, wherein the plant is wheat, cotton, sunflower, safflower, tobacco, Arabidopsis, soybean, barley, oats, rice, corn, triticale, sorghum, canola, hemp, sugarcane, rye, millet, turf grass, or flax.

6. A plant comprising a promoter operably linked to a heterologous polynucleotide sequence, wherein the promoter is selected from the group consisting of: (a) the polynucleotide sequence of SEQ ID NO: 37, 38 or 39; (b) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37, 38 or 39; (c) A fragment of the polynucleotide of (a) or (b), wherein the fragment retains its ability to drive expression of the heterologous polynucleotide sequence in an egg cell of a plant.

7. A method for inducing parthenogenesis, the method comprising: Expressing in a female gametophyte a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell of a plant, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof, wherein the female gametophyte is made parthenogenetic and forms haploid embryos in the absence of pollination, and wherein the Babyboom (BBM) polynucleotide is not naturally expressed in the female gametophyte.

8. The method of claim 7, wherein the nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof is selected from the group consisting of BBM1, BBM2, BMN2, BMN3, ODP2, and BBML.

9. The method of claim 7, wherein the Babyboom (BBM) polynucleotide is selected from the group consisting of: (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity; (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

10. The method of claim 7, wherein the Babyboom (BBM) polypeptide is selected from the group consisting of: (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

11. The method of claim 7, wherein the promoter expressed in plant egg cells is an egg cell-specific promoter or an egg cell-preferred promoter.

12. The method of claim 7, wherein the promoter expressed in the oocyte comprises a polynucleotide comprising: (d) the polynucleotide sequence of SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86; (e) a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 86; (f) a fragment of the polynucleotide of (a) or (b), wherein the fragment retains its ability to drive expression of the operably linked polynucleotide sequence in an oocyte; wherein the promoter drives expression of the operably linked Babyboom (BBM) polynucleotide encoding the Babyboom (BBM) polypeptide or fragment thereof.

13. The method of claim 7, wherein prior to or during expression of the Babyboom (BBM) polynucleotide in the egg cell, the female gametophyte has not been fertilized.

14. The method of claim 7, further comprising: The regulatory region of an endogenous Babyboom (BBM) polynucleotide is modified so that the Babyboom (BBM) polynucleotide is expressed in female gametophytes.

15. The method of claim 7, comprising: The Babyboom (BBM) polynucleotide is expressed from a modified endogenous genomic BBM locus, wherein the modified endogenous genomic BBM locus comprises a modified regulatory region of an endogenous polynucleotide encoding a BBM polypeptide, wherein one or more nucleotides in the regulatory region are modified such that the BBM polypeptide is expressed in female gametophytes.

16. The method of claim 7, comprising: The Babyboom (BBM) polynucleotide is expressed from a modified endogenous genomic egg cell locus, wherein an egg cell coding sequence or a genomic sequence in the endogenous genomic egg cell locus is modified such that it encodes a BBM polypeptide, wherein the BBM polypeptide is expressed in female gametophytes.

17. The method of claim 7, wherein the Babyboom (BBM) polynucleotide has been modified from its native form using gene editing technology.

18. The method of claim 17, wherein the gene editing technology uses a DNA modifying enzyme, and the DNA modifying enzyme is a site-directed nuclease comprising: a meganuclease (MN), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a Cas polypeptide.

19. The method of claim 7, wherein the female gametophyte is a monocot or dicot female gametophyte.

20. The method of claim 7, wherein the female gametophyte is a wheat, cotton, sunflower, safflower, tobacco, Arabidopsis, hemp, canola, sugarcane, soybean, barley, oats, rice, corn, triticale, sorghum, turfgrass, rye, millet, or flax female gametophyte.

21. The method of claim 7, further comprising: (a) contacting the haploid embryo with a chromosome doubling agent for a period of time sufficient to produce a doubled haploid embryo; (b) isolating the doubled haploid embryos; as well as (c) regenerating a doubled haploid plant from the doubled haploid embryo of step (b).

22. The method of claim 7, further comprising: (a) regenerating a parthenogenetic plant from a haploid embryo comprising the Babyboom (BBM) polynucleotide operably linked to the promoter expressed in an egg cell; (b) pollinating the parthenogenetic plant of (a) with pollen from a non-haploid inducer; and (c) rescuing haploid embryos from the parthenogenetic plant of (b).

23. A method for obtaining a wheat plant producing clonal, non-reduced, non-recombinant gametes, the method comprising: (a) inhibiting the following activities in wheat plant cells: (1) All endogenous Spo11 or Prd1, Prd2 or Prd3 polynucleotides or polypeptides; (2) all endogenous Rec8 polynucleotides or polypeptides; (3) all endogenous Osd1 polynucleotides or polypeptides; (b) expressing a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell in a female gametophyte derived from the wheat plant cell, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof, wherein the female gametophyte is caused to parthenogenetically reproduce and form embryos; and (c) obtaining a plant from the embryo, wherein the embryo comprises a Babyboom (BBM) polynucleotide expressed by the egg cell and a repressed Spo11, Rec8, Osd1, Prd1, Prd2 or Prd3 polynucleotide or polypeptide, thereby producing a wheat plant that produces clonal, non-reduced, non-recombinant gametes.

24. The method of claim 23, wherein the plant is a progeny hybrid plant.

25. The method of claim 23, wherein the activity of the endogenous Spo11 polynucleotide or polypeptide, Rec8 polynucleotide or polypeptide, Osd1 polynucleotide or polypeptide, Prd1, Prd2 or Prd3 polynucleotide or polypeptide, or a combination thereof is suppressed by introducing nucleotide modifications into the polynucleotide sequence thereof or introducing amino acid modifications into the polypeptide sequence thereof.

26. The method of claim 25, wherein the nucleotide modification is a deletion, addition or substitution of one or more nucleotides.

27. The method of claim 25, wherein the amino acid modification is a deletion, addition or substitution of one or more amino acids.

28. The method of claim 25, wherein the nucleotide modification is introduced by a nuclease selected from the group consisting of: TALEN, meganuclease, zinc finger nuclease, and CRISPR-associated nuclease.

29. The method of claim 25, wherein the nucleotide modifications are introduced by a Cas polypeptide guided by at least one guide RNA.

30. The method of claim 23, wherein the nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof is selected from the group consisting of BBM1, BBM2, BMN2, BMN3, ODP2 and BBML.

31. The method of claim 23, wherein the Babyboom (BBM) polynucleotide is selected from the group consisting of: (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity; (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

32. The method of claim 23, wherein the Babyboom (BBM) polypeptide is selected from the group consisting of: (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

33. A method for obtaining a clonal apomictic plant from one or more gametophytic cells in a plant in the absence of fertilization of an egg cell, the method comprising: (a) expressing in one or more gamete cells a Babyboom (BBM) polynucleotide operably linked to a promoter expressed in an egg cell, wherein the Babyboom (BBM) polynucleotide comprises a nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof that retains haploid inducing activity, wherein the Babyboom (BBM) polynucleotide is not naturally expressed in female gametophytes; (b) developing an embryo from said gamete cell in the absence of fertilization of an egg cell; as well as (c) obtaining a progeny plant from the one or more gametophyte cells, wherein the progeny plant contains chromosomes from the gametophyte cells of (a), thereby achieving reproduction of a flowering plant in the absence of egg cell fertilization.

34. The method of claim 33, wherein the embryos are formed from unreduced plant cells.

35. The method of claim 34, wherein the unreduced plant cell is an egg cell.

36. The method of claim 34, wherein the unreduced plant cells are formed from somatic cells.

37. The method of claim 33, wherein the nucleotide sequence encoding a Babyboom (BBM) polypeptide or a fragment thereof is selected from the group consisting of BBM1, BBM2, BMN2, BMN3, ODP2 and BBML.

38. The method of claim 33, wherein the Babyboom (BBM) polynucleotide is selected from the group consisting of: (a) a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (c) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 20, 22, 24, 26, 28, 41, 43, 45, 47, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87 or 88; (d) A fragment of any one of the nucleotide sequences of (a), (b) or (c), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity; (e) A fragment of any one of the nucleotide sequences of (a), (b), (c) or (d), wherein the nucleotide sequence encodes an amino acid fragment having haploid inducing activity, wherein the amino acid fragment comprises an A motif, a B motif or an AP2 DNA binding domain or a combination thereof.

39. The method of claim 33, wherein the Babyboom (BBM) polypeptide is selected from the group consisting of: (a) an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (b) an amino acid sequence having at least 95% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (c) an amino acid sequence having at least 85% amino acid identity to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 21, 23, 25, 27, 29, 42, 44, 46 or 48; (d) a fragment of any one of the amino acid sequences of (a), (b) or (c), wherein the fragment has haploid inducing activity; and (e) A fragment of any of the amino acid sequences of (a), (b) or (c) or (d), wherein the fragment comprises the A motif, the B motif or the AP2 DNA binding domain or a combination thereof.

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