Haploid inducer strawberry lines and methods of producing and using thereof

Abstract

The present disclosure relates to systems and methods for improved breeding of elite octoploid strawberry varieties. Described herein are haploid-inducing strawberry plants, plant parts, and plant lines comprising one or more genetic modifications resulting in decreased expression of one or more DMP (Domain of Unknown Function 679 Membrane Protein) genes. Also described herein are methods of using thereof to generate true homozygous octoploid strawberry lines, as well as true breeding Fl hybrid octoploid strawberry seeds produced by crossing two true homozygous octoploid strawberry plants. Further described herein are methods and compositions for producing haploid-inducing strawberry plants, including methods for introducing genetic modifications resulting in decreased expression of one or more DMP genes.

Description

HAPLOID INDUCER STRAWBERRY LINES AND METHODS OF PRODUCING AND USING THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 552,890, filed on February 13, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (197072000640SEQLIST.xml; Size:45,916 bytes; and Date of Creation: February 3, 2025) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates generally to strawberry breeding, and more specifically to generation of fully inbred octopl oid strawberry lines and true breeding Fl hybrid octoploid strawberry seed.BACKGROUND

[0004] Strawberries are a major agronomic fruit crop worldwide, with China, Mexico, and the U.S. being among the primary producers and consumers. According to recent data, 94% of U.S. households consume strawberries, with an annual per capita consumption of 4.85 pounds. California in particular produces 75% of the entire U.S. strawberry crop, with Florida being the next closest U.S. state in terms of production.

[0005] Due to the complex genetics of the modem strawberry (e.g., having four distinct subgenomes, a tendency to outcross, and suffering from inbreeding depression ), achieving fully homozygous inbred strawberry lines to cross for the production of true-breeding F 1 hybrid strawberry seed has remained an elusive goal. Aside from inbreeding, anther culture has also been attempted in strawberry, but remains low efficiency with very few examples of limited success. E.g., Xuan et al. 2012. Haploid plant production through anther culture in day-neutral strawberry (Fragaria X ananassa Duch) CV. Albion. J. ISSAAS Vol. 18, No. 1 : 173-184. Rather, current breeding practice involves recurrent selection and the clonal, vegetative propagation of strawberry plants through a resource intensive process, during which the plants are particularly susceptible to biotic and abiotic stressors.

[0006] Therefore, there is a need in the art for improved methods of producing fully homozygous strawberry lines that can be used for generating true-breeding Fl hybrid strawberry seed. Previous attempts, however, have failed to succeed in generating true- breeding populations that also exhibit commercially acceptable vigor and quality. E.g., Dale et al. (2017). Breeding Fi hybrid day-neutral strawberries in eastern North America. ACTA HORTIC. 1156, 47-52. Another main issue with conversion of octoploid strawberry from a vegetatively propagated plant to a true seed plant is that breeding progress using current methodologies continues to provide consistent yield improvement, and allocation of resources toward inbreeding plants with traditional breeding would require many years and significant resources to develop populations and parents of sufficient homozygosity to make commercially relevant Fl hybrids. Therefore most breeders have continued to use recurrent selection and vegetative propagation. Those results may also indicate limitations on the extent of homozygosity that can be achieved by selfing octoploid strawberry. Instead, it would be desirable to have a haploid inducing strawberry variety that could generate haploid progeny, which could then be doubled to achieve true homozygosity across all four subgenomes simultaneously and uniformly.BRIEF SUMMARY

[0007] For a variety of reasons, it would be desirable to have a system for producing fully homozygous strawberry lines to cross for the production of uniform (true-breeding) Fl hybrid strawberry seed. To our knowledge, the methods described herein provide the first such system enabled through use of a haploid inducer line.

[0008] In some aspects, provided herein is a haploid-inducing strawberry plant, or a plant part thereof, comprising one or more genetic modifications resulting in decreased expression of one or more DMP genes. In some embodiments, one or more of the DMP genes comprise a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In some embodiments, one or more of the DMP genes comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In some embodiments, the one or more genetic modifications comprise a modification of an enhancer of one or more of the DMP genes, a modification of a promoter of one or more of the DMPgenes, a modification of a coding region of one or more of the DMP genes, a modification of an intron of one or more of the DMP genes, a modification of methylation status of one or more of the DMP genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the DMP genes, expression of an RNA interference construct that targets an mRNA of one or more of the DMP genes, or any combination thereof. In certain embodiments, the one or more genetic modifications comprise a modification of an enhancer of one or more of the DMP genes, a modification of a promoter of one or more of the DMP genes, a modification of a coding region of one or more of the DMP genes, modification of an intron of one or more of the DMP genes, or any combination thereof relative to an unmodified strawberry plant of the same species or an unmodified control strawberry plant. In some embodiments, the haploid-inducing strawberry plant or plant part has decreased expression of DMP proteins relative to a strawberry plant of the same species lacking the one or more genetic modifications. In certain embodiments, the haploid-inducing strawberry plant or plant part lacks detectable expression of DMP proteins. In some embodiments, the haploid-inducing strawberry plant is diploid, triploid, tetrapioid, pentapioid, hexapioid, septapioid, octoploid, or nonaploid, or has a ploidy of lOx, l lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x. In some embodiments, the plant part is selected from the group consisting of pollen, an anther, a seed, an achene, a leaf, a flower, a fruit, and a stolon. In certain embodiments, the plant part is pollen. In certain embodiments, the plant part is a seed.

[0009] In some embodiments, the haploid-inducing strawberry plant is diploid. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca, Fragaria iinumae. Fragaria nipponica, Fragaria viridis. Fragaria * bifera. Fragaria bucharica, Fragaria chinensis. Fragaria dalloniana. Fragaria emeiensis. Fragaria hayalae. Fragaria iinumae, Fragaria mandshurica. Fragaria viridis, Fragaria nilgerrensis, Fragaria nipponica, Fragaria nubicola, or Fragaria pentaphylla. In certain embodiments, the haploid- inducing strawberry plant is a plant of the species Fragaria vesca. In certain embodiments, the haploid-inducing strawberry plant is a Fragaria vesca plant of the subspecies Fragaria vesca ssp. vesca, Fragaria vesca ssp. americana, o Fragaria vesca ssp. bracteate. In some embodiments the one or more DMP genes comprise a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more DMP genes comprise a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100%identity to the polynucleotide sequence of SEQ ID NO: 16. In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca and wherein the one or more DMP genes comprise FvDMPl. In certain embodiments, the one or more genetic modifications comprise a modification of an enhancer of FvDMPl, a modification of a promoter of FvDMPl, a modification of a coding region of FvDMPl, modification of an intron of FvDMPl, or any combination thereof relative to a wild-type Fragaria vesca plant.

[0010] In some embodiments, the haploid-inducing strawberry plant is octoploid. In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa, Fragaria chiloensis, Fragaria virginiana. or Fragaria iturupensis. In some embodiments, the haploid-inducing strawberry plant is Fragaria chiloensis plant of the subspecies Fragaria chiloensis subsp. chiloensis, Fragaria chiloensis subsp. hicida. Fragaria chiloensis subsp. pacifica, o Fragaria chiloensis subsp. sandwicensis . In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, one or more of the DMP genes comprises a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5. In some embodiments, one or more of the DMP genes comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 17-20. In certain embodiments, the one or more DMP genes comprise: a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 2; a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 3; a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 4; and / or a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the one or more DMP genes comprise: a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 17; a polynucleotide sequence having at least 90%,at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 18; a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 19; and / or a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 20. In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa and the one or more DMP genes comprise one, two, three or all four of FaDMPl-1, FaDMP-l-3a, FaDMPl-3b, and FaDMPl-4. In certain embodiments, the one or more DMP genes comprise FaDMPl-1, FaDMP-l-3a, FaDMPl-3b, and FaDMPl-4. In some embodiments, the one or more genetic modifications comprise: a modification of an enhancer of FaDMPl-1, a modification of a promoter of FaDMPl-1, a modification of a coding region of FaDMPl-1, a modification of an intron of FaDMPl-1, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a modification of an enhancer of FaDMPl-2, a modification of a promoter of FaDMPl-2, a modification of a coding region of FaDMPl-2, a modification of an intron of FaDMPl-2, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a modification of an enhancer of FaDMPl-3, a modification of a promoter of FaDMPl-3, a modification of a coding region of FaDMPl-3, a modification of an intron of FaDMPl-3, or any combination thereof relative to an unmodified Fragaria x ananassa plant; and / or a modification of an enhancer of FaDMPl-4, a modification of a promoter of FaDMPl-4, a modification of a coding region of FaDMPl-4, a modification of an intron of FaDMPl-4, or any combination thereof relative to an unmodified Fragaria x ananassa plant.

[0011] In some aspects, provided herein is a method of producing the haploid-inducing strawberry plants described herein. In some embodiments, the decreased expression of the one or more DMP genes is achieved by gene disruption, gene knockout, gene knockdown, gene silencing, RNA interference, induction of methylation, or any combination thereof. In some embodiments, the method comprises introducing one or more of the genetic modifications by mutagenesis, gene editing, transgenesis, or a combination thereof. In some embodiments, the method comprises introducing one or more of the genetic modifications by gene editing using a site-directed nuclease. In certain embodiments, the site-directed nuclease is a CRISPR-associated (Cas) nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a megaTAL. In some embodiments, the method comprises contacting a plurality of cells of a parent strawberry plant with one or moreexpression vectors together comprising an expression cassette for a Cas nuclease and an expression cassette for an RNA molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of one or more of the DMP genes. In some embodiments, the method comprises contacting a plurality of cells of a parent strawberry plant with one or more Cas nucleases, wherein each Cas nuclease is complexed with a RNA molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of one or more of the DMP genes. In certain embodiments, the RNA molecule is a crRNA, a gRNA, or a pegRNA. In certain embodiments, the plurality of cells is a plurality of protoplasts. In some embodiments, the method further comprises, subsequent to the contacting step, allowing the plurality of cells of the parent strawberry plant to form calli, plants, or a combination thereof, and identifying one or more calli or plants having the genetic modifications resulting in decreased expression of one or more DMP genes.

[0012] In some aspects, provided herein is a method of producing true homozygous octoploid strawberry seed, the method comprising (a) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with pollen of a haploid-inducing strawberry plant described herein, and (b) producing a doubled tetrahaploid cell from the tetrahaploid reduced egg cell. In some embodiments, step (a) comprises contacting pollen of the haploid- inducing strawberry plant with the stigma of a pistil of the donor octoploid strawberry plant comprising the reduced egg cell and allowing formation of a pollen tube and migration of the pollen to the reduced egg cell, thereby contacting the reduced egg cell with the pollen. In some embodiments, the method further comprises allowing the donor octoploid strawberry plant to form a plurality of seeds. In certain embodiments, the method further comprises selecting one or more tetrahaploid seeds by 1) determining the ploidy of the embryo and endosperm of one or more seeds of the plurality of seeds, and 2) selecting one or more seeds having tetrapioid embryo and an endosperm having a ploidy of greater than 8x. In some embodiments, the method further comprises collecting the plurality of seeds and allowing them to germinate and form a plurality of seedlings. In some embodiments, the method further comprises collecting a plurality of embryos from the plurality of seeds, contacting the plurality of embryos with a nutrient medium suitable for inducing seedling growth, and allowing them to form a plurality of seedlings. In some embodiments, the method further comprises selecting one or more tetrahaploid seedlings by 1) determining the ploidy of thecells of one or more seedlings of the plurality of seedlings, and 2) selecting one or more tetrahaploid seedlings from the plurality of seedling. In certain embodiments, determining the ploidy of the cells of the one or more seedlings comprises obtaining a tissue sample of one or more seedlings and determining the ploidy of the cells of the tissue sample.

[0013] In some embodiments of the method of producing a true homozygous octoploid strawberry plant or seed, step (b) comprises subjecting the selected tetrahaploid cell, tetrahaploid seed, or tetrahaploid seedling to a tetrahaploid doubling treatment. In certain embodiments, the tetrahaploid doubling treatment comprises contacting the tetrahaploid cell, tetrahaploid seed, or tetrahaploid seedling with an anti -microtubule agent to form a doubled tetrahaploid cell, a doubled tetrahaploid seed, or a doubled tetrahaploid seedling. In certain embodiments, the anti -microtubule agent is an anti-mitotic herbicide. In certain embodiments, the anti-microtubule agent comprises colchicine, oryzalin, oxide, trifluralin, or any combination thereof. In some embodiments of the method of producing a true homozygous octoploid strawberry plant or seed, the doubled tetrahaploid cell or the doubled tetrahaploid seed is allowed to grow into a doubled tetrahaploid seedling. In some embodiments, the doubled tetrahaploid seedling is allowed to form seed, thus producing the true homozygous octoploid strawberry seed.

[0014] In some embodiments of the method of producing a true homozygous octoploid strawberry plant or seed, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa, Fragaria chiloensis, Fragaria virginiana. or Fragaria iliirupensis. or a hybrid of any combination thereof. In some embodiments, the donor octoploid strawberry plant is Fragaria chiloensis plant of the subspecies Fragaria chiloensis subsp. chiloensis, Fragaria chiloensis subsp. hicida. Fragaria chiloensis subsp. pacifica, o Fragaria chiloensis subsp. sandwicensis. In some embodiments, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa. In certain embodiments, the donor octoploid strawberry plant is a Fragaria x ananassa plant of the variety Camarosa.

[0015] In some embodiments, the method of producing a true homozygous octoploid strawberry plant or seed further comprises introducing one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene into the true homozygous octoploid strawberry plant or seed.

[0016] In some aspects, provided herein is a method of producing a runnerless true homozygous octoploid strawberry plant, the method comprising (a) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with pollen of a haploid-inducing strawberry plant described herein, (b) producing a doubled tetrahaploid cell from the tetrahaploid reduced egg cell, and (c) introducing one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene into the true homozygous octoploid strawberry plant, wherein the runnerless true homozygous octoploid strawberry plant does not produce runners.

[0017] In some aspects, provided herein is true homozygous octoploid strawberry seed produced according to the methods described herein. In certain embodiments, the true homozygous octoploid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene.

[0018] In some aspects, provided herein is a true homozygous octoploid strawberry plant or a plant part thereof produced according to the methods described herein. In certain embodiments, the true homozygous octoploid strawberry plant comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene. In certain embodiments, the true homozygous octoploid strawberry plant does not produce runners. In some embodiments, the plant part is selected from the group consisting of pollen, an anther, a seed, an achene, a leaf, a flower, a fruit, and a stolon.

[0019] In some aspects, provided herein is a method of producing uniform octoploid Fl hybrid strawberry seed comprising crossing two of the true homozygous octoploid strawberry plants described herein. In some aspects, provided herein is a uniform octoploid Fl hybrid strawberry seed produced according to said method. In certain embodiments, the uniform octoploid Fl hybrid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene. In some aspects, provided herein is a uniform octoploid Fl hybrid strawberry plant or a plant part thereof produced according to said method. In certain embodiments, the uniform octoploid Fl hybrid strawberry plant comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene. In certain embodiments, the uniform octoploid Fl hybrid strawberry plant does not produce runners. In some embodiments, the plant part is selected from the group consisting of pollen, an anther, a seed, an achene, a leaf, a flower, a fruit, and a stolon.

[0020] In some aspects, an expression vector or isolated DNA molecule for making a haploid-inducing strawberry plant, or a plant part thereof, described herein. In some embodiments, the expression vector or isolated DNA molecule comprises a DNA sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide sequence encoding an amino acid sequence having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In some embodiments, the expression vector or isolated DNA molecule comprises a DNA sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26.

[0021] In some embodiments, the expression vector or isolated DNA molecule comprises a DNA sequence encoding a non-coding RNA. In some embodiments, the non-coding RNA comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide sequence encoding an amino acid sequence having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In some embodiments, the non-coding RNA comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In some embodiments, the non-coding RNA is a crRNA, a gRNA, a pegRNA, a siRNA, a miRNA, or a dsRNA.

[0022] In some embodiments, the expression vector or isolated DNA molecule comprises a DNA sequence encoding a site-directed nuclease. In some embodiments, the site-directed nuclease is a Cas nuclease, a TALEN, ZFN, or a mega-TAL. In some embodiments, the site- directed nuclease comprises an amino acid sequence that confers binding to a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide sequence encoding an amino acid sequence having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In some embodiments, the site- directed nuclease comprises an amino acid sequence that confers binding to a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26.

[0023] In some aspects, provided herein is a bacterial cell comprising an expression vector or isolated DNA molecule described herein. In some embodiments, the bacterial cell is an Agrobacterium cell.

[0024] In some aspects, provided herein is a genetically modified plant, plant part, plant cell, or seed comprising an expression vector or isolated DNA molecule described herein.

[0025] In some aspects, provided herein is a kit comprising an expression vector or isolated DNA molecule or a bacterial cell described herein.DESCRIPTION OF THE FIGURES

[0026] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0027] FIG. 1 depicts a schematic illustrating the production of homozygous octoploid strawberry lines. Step A is shown at the top right, in which de novo creation of strawberry haploid inducer lines is achieved by disrupting expression of DMP, for example, through nuclease-guided deletions in the DMP gene. Circles indicate exemplary nuclei, in which chromosomes are indicated by grey and white vertical lines, from (i) diploid Fragaria vesca and (ii) octoploid Fragaria x ananassa. Ploidy is indicated at the top of the nuclei. Deletions in DMP orthologs are indicated on the chromosomes by grey diamonds. Step B is shown from the top left to the middle right, in which pollen from t / ziz -edited strawberry lines is provided for fertilization or induction of a reduced tetrahaploid egg cell. Pollen is indicated by a male “ ” symbol, and egg cells and megaspore mother cells are indicated by a femalesymbol. At the top left, a 4x reduced egg cell is provided from an elite octoploid strawberry megaspore mother cell, t / zzz -edited pollen (middle right) is used to fertilize these 4x reduced tetrahaploid egg cells. Vertical arrows indicate cell lineage. Step C is shown in the middle left, in which fertilization or haploid induction, and selection are performed. Approximately 0.5% to 8% of fertilizations cause the tetrahaploid egg cell to undergo embryogenesis without retention of genetic material from the t / zzz -edited sperm, illustrated by the downward arrow from the 4x egg cell resulting in a 4x tetrahaploid. The remaining approximately 92% to 99.5% of eggs result in fertilizations by the haploid inducer, illustrated by the leftward arrows from the 4x egg cell and the grey nuclei, retain genetic material from the t / zzz -edited pollen or fail to undergo embryogenesis and are discarded. Step D is shown at the bottom, in which fully homozygous octoploid strawberry lines are obtained through the use of colchicine to double the ploidy of the 4x tetrahaploid.

[0028] FIGS. 2A-2B show the configuration of DMP loci in diploid and octoploid strawberry. FIG. 2A shows the configuration of the DMP1 locus, “FvDMPl”, in diploid strawberry Fragaria vesca according to the reference genome “Fragaria_vesca_v4.0”. The top structure illustrates chromosome 6. The black bar indicates the DMP locus, magnified in the below structure. Dark regions in the below structure indicate exons; lighter regions indicate an untranslated region (“UTR”). FIG. 2B shows the configuration of four homeologous FaDMP loci in octoploid strawberry Fragaria x ananassa according to reference genome “Fragaria x ananassa Camarosa Genome vl.0.a2”. The four chromosome 6 homeologs are shown, with black bars indicating the locations of the DMP loci.

[0029] FIGS. 3A-3I show a protein sequence alignment of DMP orthologs from Fragaria vesca (FvDMPl), Fragaria x ananassa (FaDMPl Fvb6-4, FaDMPl Fvb6-3, and FaDMPl Fvb6-1) Fragaria chiloensis (Fchiloensis DMP1 6-Av, Fchiloensis DMP1 6-B2, and Fchiloensis DMP1 6-Bi), Fragaria iinumae (FiinumaeDMPl), Fragaria viridis (Fvzrzt / ADMPl), Potentilla micrantha (Potentilla micrantha DMP1), Solanum lycopersicum (S1DMP), Arabidopis thaliana (AtDMP9 and AtDMP8), and Zea mays (ZmDMP). FIG. 3A shows positions 1-30 of the sequence alignment. FIG. 3B shows positions 31-60 of the sequence alignment. FIG. 3C shows positions 61-90 of the sequence alignment. FIG. 3D shows positions 91-120 of the sequence alignment. FIG. 3E shows positions 121-150 of the sequence alignment. FIG. 3F shows positions 151-180 of the sequence alignment. FIG. 3Gshows positions 181-210 of the sequence alignment. FIG. 3H shows positions 211-240 of the sequence alignment. FIG. 31 shows positions 241-270 of the sequence alignment.

[0030] FIG. 4 shows a phylogenetic tree of identified putative and characterized DMP orthologs. Bootstrap values are displayed at branch points, indicating how many times the same branching is observed when regenerating the phylogenetic tree 100 times, with a resampled sequence set each time. The protein alignment was generated using the MUSCLE Alignment tool (Muscle 5.1) in Geneious Prime (2023.0.3) with the “algorithm” set to “PPP”.

[0031] FIG. 5 shows a bar graph of editing efficiencies of guide RNAs targeting different protospacers in FvDMPl (RefSeq ID: XP_ 011467959) and FaDMPl (gene IDs: FxaC_21g08040, FxaC_22g06170, FxaC_22g06200 and FxaC_24g57400). The x-axis indicates the different guide RNAs tested; the y-axis indicates the percent of protoplasts with successful editing. Different colors indicate different pedigrees of strawberry tested. The sample size ranged from 2 replicates up to 10 replicates, with a median of 6.25 replicates across all data sets. The error bars indicate standard deviation.

[0032] FIGS. 6A-6B show schematics of guide RNA target sites 1 and 2, showing the two selected guide RNAs targeting DMP orthologs in Fragaria vesca and all four homeologs in Fragaria x ananassa var. Camarosa. FIG. 6A shows sequences for target site 1 (FvDMPl : residues 167-222 of SEQ ID NO: 16; FxaC_21g08040: residues 167-222 of SEQ ID NO: 17; FxaC_22g06170: residues 164-216 of SEQ ID NO: 18; FxaC_22g06200: residues 162-214 of SEQ ID NO: 19; FxaC_24g57400: residues 158-213 of SEQ ID NO: 20). FIG. 6B shows sequences for target site 2 (FvDMPl : residues 665-720 of SEQ ID NO: 16; FxaC_21g08040; FxaC_21g08040: residues 665-720 of SEQ ID NO: 17; FxaC_22g06170: residues 659-714 of SEQ ID NO: 18; FxaC_22g06200: residues 656-711 of SEQ ID NO: 19; FxaC_24g57400: residues 656-711 of SEQ ID NO: 20).DETAILED DESCRIPTION

[0033] The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the variousembodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.Overview

[0034] The methods described herein first involve the de novo creation of strawberry haploid inducer lines. Pollen from the haploid inducer lines is used to fertilize reduced eggs cells (4x) from elite octoploid (8x) strawberry varieties. Pollen from the haploid inducer lines is used to fertilize or stimulate development of reduced eggs cells (4x) from elite octoploid (8x) strawberry varieties. Pollen from the haploid inducer lines induce the reduced egg cells to undergo embryogenesis without transfer or retention of genetic material from the haploid- inducing pollen. The resulting haploid strawberry lines (4x) are doubled (for example, using colchicine) to achieve fully homozygous octoploid (8x) strawberry lines. Fully homozygous octoploid (8x) strawberry lines are crossed to determine optimal crosses for the production of genetically uniform (true-breeding) Fl hybrid seed. An exemplary such method is depicted in FIG. 1. Another advantage of the methods described herein is that the resulting Fl hybrid seed retains no foreign DNA and no targeted genetic alterations, resulting in a plant that is essentially indistinguishable from a plant generated by standard single cross hybrid breeding. First, de novo creation of strawberry haploid inducer lines is achieved by disrupting expression of DMP (Domain of Unknown Function 679 Membrane Protein) ortholog genes in strawberry. In some embodiments, this involves the identification and targeting of DMP ortholog genes via nuclease-guided deletions. In some embodiments, this is done in a diploid line such as Fragaria vesca (FIG. 1, step Ai). In other embodiments, this is done in an octoploid Fragaria x ananassa (FIG. 1, step Aii). In the latter embodiment, edits are made to DMP orthologs from subgenome 1 (“sgl”), sg2 and sg3, and a truncated DAff’-pseudogene from sg3. No DMP orthologs are known to exist on sg4 as understood from present reference assemblies.

[0035] Next, pollen is collected from t / zz / -edited strawberry lines (fdmp pollen”) and used to induce embryogenesis in reduced tetrahaploid egg cells (4x) from elite octoploid strawberry varieties (8x) (FIG. 1, step B). Embryogenesis without retention of the genomic material from the pollen occurs in about 0.5% to 8% of the egg cells (FIG. 1, step C). The resulting haploid lines (4x) are doubled with colchicine to obtain fully homozygous octoploid strawberry lines (8x) (FIG. 1, step D).

[0036] Multiple fully homozygous octoploid strawberry lines are produced and then crossed according to standard methods in the art. The best crosses are chosen to produce to genetically uniform Fl hybrid seed.

[0037] In one aspect, provided herein are haploid-inducing strawberry plants, and plant parts thereof, comprising one or more genetic modifications resulting in decreased expression of one or more DMP genes. Said haploid-inducing strawberry plant and plant parts may be crossed with octoploid strawberry plants to produce haploid (4x) strawberry plants, which may then be subject to genome doubling to produce true homozygous octoploid (8x) strawberry plants. Accordingly, also provided herein are true homozygous octoploid strawberry plants, and parts thereof.

[0038] In another aspect, provided herein are methods of producing a haploid-inducing strawberry plants by decreasing the expression of one or more DMP genes in a strawberry plant. The methods may comprise introduction of one or more genetic modifications into a strawberry plant that result in decreased expression or non-expression of one or more DMP genes, thereby producing the haploid-inducing strawberry plant. Further provided herein are expression vectors, isolated DNA molecules, bacterial cells, and kits useful in performing the methods described herein.

[0039] In yet another aspect, provided herein are methods of producing true homozygous octoploid strawberry seed and plant lines, the methods comprising crossing a haploid- inducing strawberry plant with an octoploid strawberry plant to generate a haploid (4x) cell or plant, and subjecting the haploid (4x) cell or plant to genome doubling to produce an octoploid (8x) plant having two clonal sets of 4x chromosomes. The true homozygous octoploid strawberry seed and plant lines of the present disclosure have the advantage of being monoallelic at all loci across the genome while only requiring two plant generations to produce. This stands in stark contrast to existing methods of producing inbred octoploid strawberry lines, which requires a prohibitive number of generations of selfing and may not achieve true homozygosity across all loci. Also described herein are true homozygous octoploid strawberry seeds, plants, and plant parts produced according to the methods described herein. Further provided herein uniform octoploid Fi hybrid strawberry seed, plants, and plant parts thereof produced by crossing two of said true homozygous octoploid strawberry plants.Definitions

[0040] As used herein, the term “plant” includes the whole plant or any parts or derivatives thereof, such as plant organs (e.g., harvested or non-harvested flowers, leaves, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, regenerable or non-regenerable plant cells, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, ovaries, reduced egg cells, seeds, achenes, fruits (e.g., aggregate fruits), flowers, leaves, seeds, runners, clonally propagated plants, roots, stems, cotyledons, hypocotyls, root tips (e.g., harvested tissues or organs) and the like. Any developmental stage is also included, such as seedlings, immature and mature, and the like.

[0041] As used herein, the term “strawberry plant” typically refers to any plant commonly understood by one of ordinary skill in the art to be a strawberry plant, and includes plants of the genus Fragaria.

[0042] As used herein, the term “flower” or “strawberry flower” may refer to either the aggregate inflorescence of flowers or the individual flowers making up the aggregate. As used herein, the term “fruit” or “strawberry fruit” typically refers to the aggregate fruit of the strawberry, which is the fleshy structure that is typically used in food applications and considered by laypeople as the strawberry fruit. As used herein, the term “seed” or “strawberry seed” includes both true strawberry seed and strawberry achenes. Achenes are the small seed-like structures found on the outer surface of the aggregate fruits of strawberries. Each achene arises from a single ovule of the aggregate inflorescence and, if the egg is fertilized, contains an endosperm and a most commonly a single seed. Each achene is therefore a true fruit which most commonly contains a seed.

[0043] As used herein, the term “non-regenerable” generally refers to a plant part, a plant cell, a processed plant product, or a portion of any of the foregoing, that cannot be induced to form a whole plant or that cannot be induced to form a whole plant that is capable of sexual and / or asexual reproduction.

[0044] As used herein, “ploidy” refers to the number of complete sets of chromosomes in a cell or organism. Ploidy may be annotated using “n” as the unit of complete sets of chromosomes. For example, a cell or organism with a single set of chromosomes may be referred to as “lx”, or the single set of chromosomes itself may be referred to as “lx”. Adiploid cell or organism with two sets of chromosomes may be referred to as “2x”; a triploid cell or organism with three sets of chromosomes may be referred to as “3x”; and so on.

[0045] As used herein, “diploid” refers to a cell or organism with a ploidy of 2x.

[0046] As used herein, “polyploid” refers to a cell or organism with a ploidy of greater than 2x. “Polyploid” may refer to organisms which are triploid (3x), tetrapioid (4x), pentapioid (5x), hexapioid (6x), septapioid (or heptapioid, 7x), octoploid (8x), or higher ploidies (greater than 8x).

[0047] As used herein, “octoploid” (which may also be spelled “octaploid”) refers to a cell or organism with a ploidy of 8x.

[0048] As used herein, “haploid” typically refers to a cell or organism with a ploidy half that of the parent organism. As used herein, “haploid gametes” typically refers to gamete cells with a ploidy half that of the parent organism. For example, in a diploid strawberry plant (2n=2x=14), meiosis in germline cells results in In haploid gametes (e.g., pollen and egg cells) where ln=lx=7. In another example, in an octoploid strawberry plant (2n=8x=56), meiosis in germline cells results in In haploid gametes (e.g., pollen and egg cells) where ln=4x=28. In the latter example, the 4x haploid gametes may also be referred to as tetrahaploid gametes.

[0049] As used herein, “haploid-inducing strawberry plant,” also referred to as a “haploid inducer strawberry plant,” typically refers to a strawberry plant that produces pollen that triggers the development of haploid embryos and seeds when crossed with another strawberry plant. As used herein, “haploid induction” typically refers to the process of crossing pollen from a haploid-inducing plant with a non-haploid-inducing plant to generate haploid embryos and seeds. During a normal cross with two non-haploid-inducing plant plants, two haploid sperm cells migrate down the pollen tube to the ovule. One of the sperm cells fertilizes the central cell of the ovule to form the endosperm, while the other sperm cell fertilizes the haploid reduced egg cell to form an embryo having the ploidy of the parent plant. For example, if both parent plants are octoploid, the haploid (4x) sperm and egg form an octoploid (8x) embryo in a normal fertilization process. In haploid induction, the sperm cells from the haploid-inducing plant migrate down the pollen tube to the ovule, where one of the sperm cells fertilizes the central cell to form the endosperm. However, when the other sperm cell contacts the reduced egg cell, embryogenesis is initiated, but the DNA from thesperm cell is not delivered to the egg cell or is otherwise eliminated, resulting in formation of a haploid embryo. For example, in haploid induction, if both parent plants are octoploid, the haploid (4x) egg cell does not incorporate the DNA from the haploid sperm cell (4x), resulting in a haploid (4x) embryo. In a haploid induction cross, the non-haploid-inducing plant that is pollinated by the haploid-inducing plant is typically referred to as the donor plant, and the resulting haploid embryos contain only chromosomes from the donor plant and usually have half the ploidy of the donor plant although spontaneous doubling is also sometimes observed. Accordingly, as used herein, the term “donor octoploid strawberry plant” typically refers to an octoploid strawberry plant that is pollinated with pollen from a haploid-inducing strawberry plant in a haploid induction process, resulting in haploid embryos each having one set of chromosomes from the donor octoploid strawberry plant. The haploid-inducing strawberry plant and the donor strawberry plant do not need to have the same ploidy. For example, a haploid-inducing strawberry plant may be diploid (2n=2x=14) and produce haploid (ln=lx=7) pollen that can be crossed with an octoploid (2n=8x=56) strawberry plant, resulting in tetrapioid (ln=4x=28) haploid embryos, also referred to as tetrahaploid embryos.

[0050] As used herein, “allele” refers to one of two or more alternative forms of a single gene or locus within the genome. As used herein, “monoallelic” typically describes the presence of a single allele at a given locus or set of loci within a cell or organism. As used herein, “monoallelic at over n% of the loci in the genome” typically refers to the percentage of total loci in the genome which are monoallelic. As used herein, “biallelic” typically describes the presence of two different alleles at a given locus or set of loci within a cell or organism. As used herein, “multiallelic” typically describes the presence of three or more alleles at a given locus or set of loci within a cell or organism. In allopolyploid strawberry cells or plants containing two or more subgenomes, “monoallelic” and “biallelic” typically refer to the allelic makeup of set of chromosomes within the same subgenome. For example, an octoploid strawberry plant of the species Fragaria x ananassa, which has four subgenomes, is monoallelic at all loci if the two chromosomes in a subgenome are monoallelic at all loci. Accordingly, in the case of Fragaria x ananassa, “monoallelic at over n% of the loci in the genome” typically refers to the percentage of total loci in the entire genome that are monoallelic within each subgenome, but does not indicate that n% of the loci are monoallelic across all subgenomes.

[0051] As used herein, “haplotype” refers to a distinct In set of chromosomes with a unique set of alleles. For example, a tetrapioid gamete or haploid plant where In = 4x = 28 would comprise as many as 4 unique haplotypes (1 per subgenome) for each of the basic seven chromosomes of strawberry. As used herein, each haplotype is distinct from other haplotypes in that it contains a set of alleles that confers a unique set of characteristics not conferred by other haplotypes. As used herein, “monoallelic plant” typically refers to a plant line containing a single haplotype within a subgenome, and a “biallelic plant” typically refers to a plant line containing two haplotypes.

[0052] As used herein, “clonal” describes a body of DNA that is substantially identical to another body of DNA; or a set of cells or organisms that comprise such DNA. For example, genome doubling (e.g., by colchicine treatment) of a tetrahaploid strawberry plant (ln=4x=28) results in an octoploid plant having two clonal (substantially identical) sets of chromosomes for each of the four subgenomes. Due to random errors in natural DNA replication, clonal bodies of DNA, clonal cells, or clonal organisms may not be completely identical. “Clonal” may describe two sets of chromosomes that are not completely identical in sequence but that contain the same set of alleles.

[0053] As used herein, “genetically uniform” describes a set of cells or organisms whose genomes are clonal. For example, a population of strawberry Fl hybrid seed wherein at least 99% of the population is genetically uniform indicates that 99% of the seeds of the population are Fl hybrids.

[0054] As used herein, “homozygous” describes a cell or organism in which all sets of chromosomes within a genome or subgenome encode the same allele or set of alleles at a certain chromosomal locus, a set of chromosomal loci, or at all chromosomal loci. As used herein, “homozygous plant” typically refers to a monoallelic plant or plant line having allelic uniformity across all chromosomal loci. In allopolyploid strawberry cells or plants containing two or more subgenomes, “homozygous” typically refers to sets of chromosomes within the same subgenome having the same alleles or sets of alleles at one or more chromosomal loci, and “homozygous plant” typically refers to a monoallelic plant or plant line having allelic uniformity across all chromosomal loci within each subgenome, but does not indicate that they have allelic uniformity across subgenomes. For example, an octoploid strawberry cell or plant having the same allele at a specific locus in two homologouschromosomes within in a subgenome may also be considered homozygous even though one or more alternative alleles exist in one or more different subgenomes.

[0055] As used herein, “heterozygous” describes a cell or organism in which at least one set of chromosomes in one subgenome encodes an allele or set of alleles at a certain chromosomal locus or set of chromosomal loci that is distinct from those of the other sets of chromosomes within the cell or organism. For example, an strawberry cell or plant having allele ai at locus A in one set of chromosomes in a subgenome and having allele 02 at locus A in a second set of chromosomes is heterozygous for alleles ai and a . As used herein, “heterozygous plant” typically refers to a biallelic plant or plant line. In polyploid strawberry cells or plants containing two or more subgenomes, “heterozygous” typically refers to two sets of chromosomes within the same subgenome having distinct alleles or sets of alleles at one or more chromosomal loci, and “heterozygous plant” typically refers to a plant that is biallelic for one or more loci in at least one of its subgenomes.

[0056] As used herein, “crossing” refers to the act of forming an embryo from gametes of two distinct plants or plant lines. Crossing may refer to pollinating a plant or plant line using the pollen of a different plant or plant line, e.g., using the pollen of a haploid-inducing plant line to pollinate a donor plant line. In some cases “crossing” may refer to successful fertilization of an egg by a sperm cell resulting in an embryo or may refer to induction of haploid or tetrahaploid embryo development without successful fertilization of the egg.

[0057] As used herein, “true homozygous plant line” and “true homozygous seed” typically refer to a monoallelic plant line or seed, respectively, having allelic uniformity across all chromosomal loci within a subgenome. As used herein, “true homozygous octoploid strawberry seed” typically refers to octoploid strawberry seed having allelic uniformity across all chromosomal loci within a subgenome.

[0058] As used herein, “hybrid” describes a plant or a part thereof, such as a seed, comprising two haplotypes within a subgenome. As used herein, “uniform Fl hybrid” refers to the genetically uniform first filial generation of hybrid seeds or plants resulting from the cross of two fully homozygous parent plants each having a different haplotype. As used herein, “uniform octoploid Fl hybrid strawberry seed” refers to a genetically uniform population of seed generated from the cross of two true homozygous octoploids or octoploidsderived from doubled tetrahaploid strawberry plants or plant lines wherein each parent may possess a different haplotype.

[0059] As used herein, “genetic modification” refers to any sequence or portion thereof within a nucleic acid molecule that differs from the sequence of an ancestral nucleic acid molecule. For example, a seed that contains an inserted or deleted genomic sequence that is not present in one of its parent plants comprises a genetic modification. A genetic modification may be naturally occurring or introduced. A genetic modification may be introduced via, for example: plant breeding to introduce a naturally-occurring genetic modification of one plant line into another plant line; transgenic methods; gene editing; chemical mutagenesis; and the like.

[0060] As used herein, “expression” and “expression level” refer to the relative or absolute amount of a functional gene product present in a cell. As used herein, “gene products” include, but are not limited to, nucleic acids (e.g., RNA), post-transcriptionally modified nucleic acids (e.g., spliced RNA, poly-adenylated mRNA), proteins (e.g., enzymes, structural proteins, etc.), and post-translationally modified proteins (e.g., glycoproteins, lipoproteins, etc.). The function of the gene product refers to the wild-type, unmodified, uninhibited function of the gene product. As used herein, “decreased expression” refers to a relative decrease in the amount of a functional gene product present in a cell. The decreased expression may refer to a decrease in the total amount of a gene product present in a cell (e.g., a decrease in the amount of a protein) or to a decreased in the amount of functional gene products present in a cell (e.g., a decrease in the percentage of proteins with wild-type function) or to a decreased in the function of gene products present in a cell (e.g., a decrease in the activity of proteins as compared to proteins with wild-type function). The decreased expression may be of a gene product encoded at a certain genomic locus, and may be relative to a control strawberry plant (e.g., a strawberry plant of the same species). As used herein, “non-expression” refers to the absence of a functional gene product present in a cell, or to an expression level insufficient for detection of the gene product in the cell, or to an expression level insufficient to result in the function of the gene product within the cell, or to an activity level insufficient to result in the detectable activity of the gene product within the cell.

[0061] As used herein, “DMP gene” typically refers to a gene encoding Domain of Unknown Function 679 Membrane Protein (DMP), or a gene having nucleotide or amino acid homology thereto. DMP genes may include DNA sequences encoding a DMP protein orany gene product thereof, for example, nucleic acids (e.g., RNA), post-transcriptionally modified nucleic acids (e.g., spliced RNA, poly-adenylated mRNA), DMP protein, and post- translationally modified DMP protein. DMP genes include any DMP gene orthologs, paralogs, and homeologs within a strawberry plant or cell.

[0062] As used herein, “transgenesis” typically refers to the insertion of an exogenous genetic element into the genome of an organism. Any exogenous genetic element may be inserted via transgenesis, including, but not limited to, genes, protein coding sequences, nonprotein coding sequences, regulatory sequences, spacer DNA, and the like.

[0063] As used herein, “gene editing” typically refers to a type of genetic modification in which DNA is inserted, deleted or substituted in the genome of an organism using one or more natural or engineered nucleases. Gene editing may be carried out using site-specific nucleases, guided nucleases, or a combination thereof. The nuclease creates one or more sitespecific breaks, such as double-strand breaks (DSBs) at target loci in the genome. Each sitespecific break may be repaired, for example via non-homologous end joining (NHEJ), resulting in a genetic modification in the genome at the target locus; or via homologous recombination of the target locus with a provided repair nucleic acid molecule comprising homology to the target genomic sequence and the desired genetic modification.Haploid-inducing Strawberry Plants and Parts Thereof

[0064] In one aspect, described herein is a haploid-inducing strawberry plant, or a plant part thereof, that produces pollen that triggers the development of haploid embryos and seeds when crossed with another strawberry plant. In some embodiments, the haploid-inducing strawberry plant or part thereof comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes, such as any of the genetic modifications described herein. In some embodiments, provided herein is pollen, seed, or a stolon of a haploid-inducing strawberry plant.

[0065] In another aspect, described herein is a haploid-inducing plant of the genus Potentilla, or a plant part thereof, that produces pollen that triggers the development of haploid embryos and seeds when crossed with another strawberry plant. In some embodiments, the haploid-inducing plant of the genus Potentilla or part thereof comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes, such as any of the genetic modifications described herein. In some embodiments,provided herein is pollen, seed, or a stolon of a haploid-inducing plant of the genus Potentilla. In some embodiments, the haploid-inducing strawberry plant is a plant of the genus Potentilla. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Potentilla micrantha.Ploidy and Species of Strawberry Plant

[0066] In one aspect, described herein is a haploid-inducing strawberry plants. The haploid-inducing strawberry plants may be of any ploidy, for example, diploid (2x), triploid (3x), tetrapioid (4x), pentapioid (5x), hexapioid (6x), septapioid (or heptapioid, 7x), octaploid (8x), or of a higher ploidy (e.g., greater than 8x, e.g., 9x, lOx, l lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x). The haploid-inducing strawberry plants may be of any taxonomic distinction known to those of skill in the art as strawberry plants. In some embodiments, the haploid-inducing strawberry plant is a plant of the family Rosaceae. In some embodiments, the haploid-inducing strawberry plant is a plant of the genus Fragaria.

[0067] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is diploid, for example, a diploid species of the genus Fragaria. Diploid species of strawberry plants include, for example, Fragaria vesca, Fragaria iinumae, Fragaria nipponica, Fragaria viridis, Fragaria x bifera, Fragaria bucharica, Fragaria chinensis, Fragaria daltoniana, Fragaria emeiensis, Fragaria hayatae, Fragaria iinumae, Fragaria mandshurica, Fragaria viridis, Fragaria nilgerrensis, Fragaria nipponica, Fragaria nubicola, and Fragaria pentaphylla.

[0068] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is a plant of the species Fragaria vesca. The haploid-inducing strawberry plant may be of any subspecies of Fragaria vesca, for example, Fragaria vesca ssp. vesca, Fragaria vesca ssp. americana, o Fragaria vesca ssp. bracteate. The haploid-inducing strawberry plant may be of any variety of Fragaria vesca, for example, Riigen, Alexandria, Baron Solemacher, Weisse Solemacher, Golden Alexandria, Quarantaine de Prin, Blanc Ameliore, Illa Martin, or Gartenfreude.

[0069] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is tetrapioid, for example, a tetrapioid species or hybrid of the genus Fragaria. Tetrapioid species of strawberries include, for example, Fragaria corymbosa, Fragariagracilis, Fragaria x intermedia, Fragaria moupinensis, Fragaria orientalis, and Fragaria tibetica.

[0070] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is hexapioid, for example, a hexapioid species or hybrid of the genus Fragaria. Hexapioid species of strawberries include, for example, Fragaria moschata.

[0071] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is octoploid, for example, an octoploid species or hybrid of the genus Fragaria. Octoploid species of strawberries include, for example, Fragaria x ananassa, Fragaria chiloensis, Fragaria virginiana, and Fragaria iturupensis.

[0072] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is a plant of the species Fragaria x ananassa. The haploid-inducing strawberry plant may be of any of Fragaria x ananassa, for example, Camarosa, Sweet Charlie, Chandler or Seascape.

[0073] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is a plant of the species Fragaria chiloensis. The haploid-inducing strawberry plant may be of any subspecies of Fragaria chiloensis, for example, Fragaria chiloensis subsp. chiloensis, Fragaria chiloensis subsp. lucida, Fragaria chiloensis subsp. pacifica, and Fragaria chiloensis subsp. sandwicensis.

[0074] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, has a ploidy of lOx, for example, a species or hybrid of the genus Fragaria having a ploidy of lOx. Strawberry species having a ploidy of lOx include, for example, Fragaria cascadensis and Fragaria iturupensis.

[0075] In some embodiments the haploid-inducing strawberry plant, or plant part thereof, is a hybrid between any of the strawberry species described herein, or is a synthetic polyploid derived from a diploid species. In some embodiments, the haploid inducing strawberry plant is a synthetic polyploid derived from any diploid species, hybrid or synthetic polyploid.DMP Genes

[0076] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, comprises one or more genetic modifications resulting in decreased expression ofone or more DMP genes. In some variations, the haploid-inducing strawberry plant, or plant part thereof, comprises one or more genetic modifications resulting in a decreased amount of a functional gene product encoded by one or more DMP genes (e.g., a functional DMP protein). The gene products encoded by the DMP genes may include, but are not limited to, nucleic acids (e.g., RNA), post-transcriptionally modified nucleic acids (e.g., spliced RNA, poly-adenylated mRNA), proteins (e.g., enzymes, structural proteins, etc.), and post- translationally modified proteins (e.g., glycoproteins, lipoproteins, etc.). The function of the gene product at the DMP gene refers to the wild-type, unmodified function of the gene product (e.g., wild-type, unmodified function of the DMP protein). The decreased expression of a DMP gene may refer to a decrease in the total amount of a gene product encoded by a DMP gene present in a cell (e.g., a decrease in the amount of total DMP protein) or to a decrease in the amount of a functional gene product encoded by a DMP gene present in a cell (e.g., a decrease in the percentage of DMP proteins with wild-type function). In some embodiments, the one or more genetic modifications resulting in decreased expression of one or more DMP genes may include, but are not limited to, modification of an enhancer in one or more DMP genes, modification of a promoter of one or more DMP genes, modification of a coding region in one or more DMP genes, modification of an intron in one or more DMP genes, modification of methylation status of one or more DMP genes, expression of a repressor protein that targets the DNA or an mRNA of one or more DMP genes, and expression of an RNA interference construct that targets one or more mRNAs from one or more DMP genes. In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, may comprise one or more genetic modifications resulting in non-expression of one or more DMP genes. In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, may comprise one or more genetic modifications resulting in decreased expression, non-expression, or a combination thereof of a plurality of DMP genes.

[0077] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes. In other embodiments, the haploid-inducing strawberry plant, or plant part thereof, comprises one or more genetic modifications resulting in decreased expression of two or more (e.g., two, three, four, or more) DMP genes.

[0078] By way of example only, a DMP gene is exemplified by a FvDMPl gene from diploid strawberry Fragaria vesca, and specifically by the FvDMPl nucleotide sequences,protein sequences, and percent identities described herein. FvDMPl protein (SEQ ID NO: 1) and nucleotide (SEQ ID NO: 16) sequences are provided in Table 1. The locus of FvDMPl on chromosome 6 in the Fragaria vesca genome is shown in FIG. 2B, and a phylogenetic tree showing the relationship between FvDMPl and DMP genes of other plant species is shown in FIG. 4. DMP genes, and gene products thereof, include nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FvDMPl gene nucleotide sequence of SEQ ID NO. 16, or a fragment thereof. Gene products of DMP genes include DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FvDMPl protein of SEQ ID NO. 1, or a fragment thereof.

[0079] By way of example only, a DMP gene is exemplified by a FaDMPl-1 gene from chromosome Fvb6-1 of octoploid strawberry Fragaria x ananassa, and specifically by the FaDMPl-1 nucleotide sequences, protein sequences, and percent identities described herein. FaDMPl-1 protein (SEQ ID NO: 2) and nucleotide (SEQ ID NO: 17) sequences are provided in Table 1. The locus of FaDMPl-1 in the Fragaria x ananassa genome is shown in FIG. 2B, and a phylogenetic tree showing the relationship between FaDMPl-1 and DMP genes of other Fragaria x ananassa and other plant species is shown in FIG. 4. DMP genes, and gene products thereof, include nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FaDMPl-1 gene nucleotide sequence of SEQ ID NO. 17, or a fragment thereof. Gene products of DMP genes also include DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FaDMPl-1 protein of SEQ ID NO. 2, or a fragment thereof.

[0080] By way of example only, a DMP gene is exemplified by a FaDMPl-2 gene from chromosome Fvb6-3 of octoploid strawberry Fragaria x ananassa, and specifically by the FaDMPl-2 nucleotide sequences, protein sequences, and percent identities described herein. FaDMPl-2 protein (SEQ ID NO: 3) and nucleotide (SEQ ID NO: 18) sequences are provided in Table 1. The locus of FaDMPl-2 in the Fragaria x ananassa genome is shownin FIG. 2B (FaDMPl-3a), and a phylogenetic tree showing the relationship between FaDMPl-2 and DMP genes of other Fragaria x ananassa and other plant species is shown in FIG. 4. DMP genes, and gene products thereof, include nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FaDMPl-2 gene nucleotide sequence of SEQ ID NO. 18, or a fragment thereof. Gene products of DMP genes also include DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FaDMPl-2 protein of SEQ ID NO. 3, or a fragment thereof.

[0081] By way of example only, a DMP gene is exemplified by a FaDMPl-3 gene from chromosome Fvb6-3 of octoploid strawberry Fragaria x ananassa, and specifically by the FaDMPl-3 nucleotide sequences, protein sequences, and percent identities described herein. FaDMPl-3 protein (SEQ ID NO: 4) and nucleotide (SEQ ID NO: 19) sequences are provided in Table 1. The locus of FaDMPl-3 in the Fragaria x ananassa genome is shown in FIG. 2B (FaDMPl-3b), and a phylogenetic tree showing the relationship between FaDMPl-3 and DMP genes of other Fragaria x ananassa and other plant species is shown in FIG. 4. DMP genes, and gene products thereof, include nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FaDMPl-3 gene nucleotide sequence of SEQ ID NO. 19, or a fragment thereof. Gene products of DMP genes also include DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FaDMPl-3 protein of SEQ ID NO. 4, or a fragment thereof.

[0082] By way of example only, a DMP gene is exemplified by a FaDMPl-4 gene from chromosome Fvb6-4 of octoploid strawberry Fragaria x ananassa, and specifically by the FaDMPl-4 nucleotide sequences, protein sequences, and percent identities described herein. FaDMPl-4 protein (SEQ ID NO: 5) and nucleotide (SEQ ID NO: 20 sequences are provided in Table 1. The locus of FaDMPl-4 in the Fragaria x ananassa genome is shown in FIG. 2B, and a phylogenetic tree showing the relationship between FaDMPl-4 and DMP genes of other Fragaria x ananassa and other plant species is shown in FIG. 4. DMP genes,and gene products thereof, include nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FaDMPl-4 gene nucleotide sequence of SEQ ID NO. 20, or a fragment thereof. Gene products of DMP genes also include DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FaDMPl-4 protein of SEQ ID NO. 5, or a fragment thereof.Table 1. Sequences of DMP genes from Strawberry species. “NT” refers to nucleotide sequences, and “AA” refers to amino acid sequences.Genetic Modifications

[0083] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, comprises one or more genetic modifications. Genetic modifications may be generated by modification of any nucleic acid sequence or genetic element by insertion, deletion, or substitution of one or more nucleotides in a nucleic acid molecule. This can occur by a replacement of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide, a chemical alteration of at least one nucleotide, or a combination thereof as long as the result is a detectable (e.g., by PCR, DNA sequencing, chromatography, etc.) change of nucleotide sequence compared to the sequence of the nucleic acid molecule prior to modification. Such modifications can be achieved by any of several well-known methods known in the art including, but not limited to, random mutagenesis, gene editing, insertion of a recombinant nucleic acid, crossing of an unmodified plant (e.g., strawberry plant) with a modified plant (e.g., strawberry plant) to introduce the modification of the modified plant into the unmodified plant, and the like. A genetic modification may be naturally occurring or non-naturally occurring.

[0084] The genetic modifications described herein may be present in any known genetic element including, but not limited to, protein-coding sequences, non-protein-coding sequences, promoter regions, 5' untranslated leaders, genes, exons, introns, poly-A signal sequences, 3' untranslated regions, regions encoding small RNAs (such as microRNAs and small -interfering RNAs), and any other sequences that affect transcription or translation of one or more nucleic acid sequences. In some embodiments, genetic modifications may include, but are not limited to, modifying or replacing nucleotide sequences of interest (such as a regulatory elements), gene disruption, gene knockout, gene knockdown, gene knock-in, gene silencing (including, e.g., by expressing an inverted repeat into a gene of interest), RNA interference (including, e.g., by insertion and / or expression of an RNA interference construct), modification of methylation status, modification of splicing sites, introducing alternate splicing sites, or any combination thereof. As used herein, gene disruption refers to the alteration or insertion of a sequence into a gene or locus that results in decreased expression or non-expression of a functional gene product (e.g., a functional protein gene product) of the gene. A gene disruption may be achieved by introduction of a genetic modification in a protein-coding sequence, including, but not limited to, as a mis-sense or non-sense mutation, or an insertion, deletion, or substitution. As used herein, a knockout is a genetic modification wherein a gene or gene product has been rendered completely inoperative. A knockout of a gene product may be achieved by introduction of a genetic modification in a protein-coding sequence of a gene or any non-protein-coding or regulatory sequence described herein. As used herein, a knockdown is a genetic modification wherein a gene or gene product has been rendered partially inoperative. A knockdown of a gene product may be achieved by introduction of a genetic modification in a protein-coding sequence of a gene or in a non-protein-coding or regulatory sequence, or insertion of a trans-acting element, such as a construct that expresses an inverted repeat of the gene product or a construct that expresses a DNA- or RNA-binding protein such as a transcriptional repressor which may include, for example, a deactivated targeted nuclease such as deactivated CRISPR-associated (Cas) nuclease e.g., dCas9). As used herein, knock-in represents the replacement or insertion of a DNA sequence at a specific DNA locus in a cell. Knock-ins may include, but are not limited to, specific insertion of a heterologous amino acid coding sequence in a coding region of a gene, an insertion of a transcriptional regulatory element in a genetic locus, or any of several methods of inserting a DNA sequence into a call that are known to one of ordinary skill in the art.

[0085] In certain embodiments, the haploid-inducing strawberry plant, or plant part thereof, comprises one or more genetic modifications resulting in decreased expression or non-expression of a gene product of a genomic locus. In some embodiments, genetic modifications resulting in decreased expression or non-expression of a gene product or locus may include, but are not limited to, modification of an enhancer, modification of a promoter, modification of a 5’ untranslated leader, modification of a coding region, modification of a non-coding region, insertion and / or expression of an RNA interference construct that targets an mRNA, modification of a region encoding a small RNA, modification of methylation status of a genomic locus, expression of a repressor protein that targets a DNA or mRNA sequence, and any other sequences that affect transcription or translation of one or more nucleic acid sequences. In some embodiments, genetic modifications resulting in decreased expression or non-expression of a gene product or locus may include, but are not limited to, modifying or replacing nucleotide sequences of interest (such as a regulatory elements), gene disruption, gene knockout, gene knockdown, gene knock-in, gene silencing (including, e.g., by inserting and / or expressing an inverted repeat into a gene of interest), RNA interference (including, e.g., by insertion and / or expression of an RNA interference construct), expression of a repressor protein (c.g, dCas9), modification of methylation status of gene loci, modification of splicing sites, introducing alternate splicing sites, or any combination thereof.

[0086] In some embodiments, the haploid-inducing strawberry plant comprises one or more genetic modifications resulting in decreased expression of one or more DMP proteins described herein. In some embodiments, the haploid-inducing strawberry plant comprises one or more genetic modifications of one or more DMP genes. In some embodiments, the haploid-inducing strawberry comprises one or more genetic modifications resulting in decreased expression of one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the list consisting of SEQ ID NOs: 1-15, or fragments thereof. In some embodiments, the haploid-inducing strawberry plant comprises one or more genetic modifications of one or more DMP genes comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting SEQ ID NOs: 16-26, or a fragment thereof. In certain embodiments, the one ormore genetic modifications comprise a modification of an enhancer of one or more of the DMP genes, a modification of a promoter of one or more of the DMP genes, a modification of a coding region of one or more of the DMP genes, a modification of an intron of one or more of the DMP genes, a modification of methylation status of one or more of the DMP genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the DMP genes, expression of an RNA interference construct that targets an mRNA of one or more of the DMP genes, or any combination thereof. In some embodiments, the haploid- inducing strawberry plant has decreased expression of one or more DMP genes described herein relative to a strawberry plant (e.g., a control strawberry plant, e.g., a strawberry plant of the same species) lacking the one or more genetic modifications. In certain embodiments, the haploid-inducing strawberry lacks detectable expression of one or more DMP genes described herein relative to a strawberry plant (e.g., a control strawberry plant, e.g, a strawberry plant of the same species) lacking the one or more genetic modifications. In certain embodiments, the haploid-inducing strawberry lacks detectable expression of any DMP genes. In certain embodiments, the haploid-inducing strawberry plant or plant part further comprises one or more naturally-occurring inactive alleles of one or more DMP genes.

[0087] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising one or more genetic modifications resulting in decreased expression of one or more DMP genes. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising one or more genetic modifications resulting in decreased expression of an FvDMPl gene. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising one or more genetic modifications resulting in decreased expression of an FvDMPl protein. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising one or more genetic modifications resulting in decreased expression of a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FvDMPl protein of SEQ ID NO. 1, or a fragment thereof.

[0088] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising one or more genetic modifications of an FvDMPl gene orgene product. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FvDMPl gene nucleotide sequence of SEQ ID NO. 16, or a fragment thereof. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising genetic modifications in both alleles of an FvDMPl gene. In some variations, the one or more genetic modifications comprise a modification of an enhancer of FvDMPl, a modification of a promoter of FvDMPl, a modification of a coding region of FvDMPl, modification of an intron of FvDMPl, or any combination thereof relative to a control Fragaria vesca plant (e.g., a Fragaria vesca plant lacking one or more of the genetic modifications). In some embodiments, the haploid- inducing strawberry plant is a plant of the species Fragaria vesca and has decreased expression of FvDMPl protein relative to a Fragaria vesca plant lacking the one or more genetic modifications. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca and lacks detectable expression of FvDMPl protein.

[0089] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications resulting in decreased expression of one or more DMP genes. In certain embodiments, the haploid- inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications resulting in decreased expression of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3 gene, an FaDMPl-4 gene, or any combination thereof. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications resulting in decreased expression of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3 gene, and an Fa-DMPl-4 gene.

[0090] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications resulting in decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, an FaDMPl-4 protein, or any combination thereof. In certain embodiments, the haploid- inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications resulting in decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, and an Fa-DMPl-4 protein. In certainembodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications resulting in decreased expression of one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the list consisting of SEQ ID NOs: 2-5, or fragments thereof. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications resulting in decreased expression of 1) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 2; 2) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 3; 3) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 4; and / or 4) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 5.

[0091] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications of an FaDMPl-1 gene or gene product, an FaDMPl-2 gene or gene product, an FaDMPl-3 gene or gene product, an FaDMPl-4 gene or gene product, or any combination thereof. In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications of an FaDMPl-1 gene or gene product, an FaDMPl-2 gene or gene product, an FaDMPl-3 gene or gene product, and an FaDMPl-4 gene or gene product. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17-20, or a fragment thereof. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa comprising: 1) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 17, or a fragment thereof; 2) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 18, or a fragment thereof; 3) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 19, or a fragment thereof; and / or 4) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 20, or a fragment thereof. In some variations, the one or more genetic modifications comprise a modification of an enhancer of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), a modification of a promoter of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), a modification of a coding region of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), modification of an intron of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), or any combination thereof relative to a control Fragaria x ananassa plant (e.g., a wild-type or unmodified Fragaria x ananassa plant lacking one or more or all of the genetic modifications).

[0092] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa and has decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, an FaDMPl-4 protein, or any combination (e.g., two, three, or all four) thereof relative to a control Fragaria x ananassa plant (e.g., aFragaria ananassa plant lacking one or more or all of the genetic modifications). In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa and lacks detectable expression of FaDMPl-1 protein, FaDMPl-2 protein, FaDMPl-3 protein, FaDMPl-4 protein, or any combination (e.g., two, three, or all four) thereof.

[0093] The haploid-inducing strawberry plant, or plant part thereof, may comprise one or more genetic modifications resulting in decreased expression of any combination of DMP genes described herein or known in the art. In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, may comprise one or more genetic modifications resulting in non-expression of any combination of DMP genes described here or known in the art. In further embodiments, the haploid-inducing strawberry plant, or plant part thereof, may comprise one or more genetic modifications resulting in decreased expression, nonexpression, or a combination thereof of any combination of DMP genes described here or known in the art. In certain embodiments, the haploid-inducing strawberry plant or plant part lacks detectable expression of DMP proteins.

[0094] In some embodiments, the haploid-inducing strawberry plant or plant part thereof has decreased expression of DMP proteins relative to a control strawberry plant. Control strawberry plants include, for example, a plant of the same species as the haploid-inducing strawberry plant that lacks one or more or all of the genetic modifications comprised by the haploid-inducing strawberry plant, a wild-type plant of the same species as the haploid- inducing strawberry plant, an unmodified plant of the same species as the haploid-inducing strawberry plant, or a null segregant strawberry plant, which underwent the same genetic manipulation, but is lacking one or more or all of the genetic modifications comprised by the haploid-inducing strawberry plant. In certain embodiments, the haploid-inducing strawberry plant or plant part thereof has decreased expression of DMP proteins relative to a strawberry plant of the same species lacking the one or more genetic modifications.Plant Parts of Haploid-inducing Strawberry Plants

[0095] Also provided herein are plant parts of haploid-inducing strawberry plants. The plant part may be any plant part known in the art, including, but not limited to, a flower, a pistil, a leaf, a stem, a petiole, a cutting, a tissue, a seed coat, an ovule, pollen, a sperm cell, a root, a fruit (e.g., an aggregate fruit), a cotyledon, a hypocotyl, a protoplast, an embryo, ananther, a seed, an achene, a stolon (also known as a runner), a callus, a cell culture, or any portion thereof. Plant parts can be obtained by lifting, cutting, snapping, grinding, or otherwise disassociating the plant part from the plant.

[0096] Accordingly, provided herein is pollen of a haploid-inducing strawberry plant. In some embodiments, the pollen comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes described herein. In certain embodiments, the pollen comprises one or more genetic modifications in one or more DMP genes described herein.

[0097] Also provided herein is stolon of a haploid-inducing strawberry plant. In some embodiments, the stolon comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes described herein. In certain embodiments, the stolon comprises one or more genetic modifications in one or more DMP genes described herein.

[0098] Also provided herein is seed of a haploid-inducing strawberry plant. In some embodiments, the seed comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes described herein. In certain embodiments, the seed comprises one or more genetic modifications in one or more DMP genes described herein.

[0099] Also provided herein is an achene of a haploid-inducing strawberry plant. In some embodiments, the achene comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes described herein. In certain embodiments, the achene comprises one or more genetic modifications in one or more DMP genes described herein.

[0100] In some embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant comprises one or more genetic modifications resulting in decreased expression of one or more DMP proteins described herein. In some embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant comprises one or more genetic modifications of one or more DMP genes. In some embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant comprises one or more genetic modifications resulting in decreased expression of one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the list consisting of SEQ ID NOs: 1-15, or fragments thereof. In some embodiments,the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant comprises one or more genetic modifications of one or more DMP genes comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26, or a fragment thereof. In certain embodiments, the one or more genetic modifications comprise a modification of an enhancer of one or more of the DMP genes, a modification of a promoter of one or more of the DMP genes, a modification of a coding region of one or more of the DMP genes, a modification of an intron of one or more of the DMP genes, a modification of methylation status of one or more of the DMP genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the DMP genes, expression of an RNA interference construct that targets an mRNA of one or more of the DMP genes, or any combination thereof. In some embodiments, the pollen, stolon, seed, or achene of the haploid- inducing strawberry plant has decreased expression of one or more DMP genes described herein relative to a pollen, stolon, seed, or achene of a strawberry plant (e.g., a control strawberry plant, e.g., a strawberry plant of the same species) lacking the one or more genetic modifications. In certain embodiments, the haploid-inducing strawberry lacks detectable expression of one or more DMP genes described herein relative to a pollen, stolon, seed, or achene of a strawberry plant (e.g., a control strawberry plant, e.g., a strawberry plant of the same species) lacking the one or more genetic modifications. In certain embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant lacks detectable expression of any DMP genes. In certain embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant further comprises one or more naturally-occurring inactive alleles of one or more DMP genes.

[0101] In some embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of an FvDMPl gene. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of an FvDMPl protein. In certain embodiments, the pollen, stolon, seed, or achene is from ahaploid-inducing strawberry plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FvDMPl protein of SEQ ID NO. 1, or a fragment thereof.

[0102] In some embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria vesca and comprises one or more genetic modifications of an FvDMPl gene or gene product. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria vesca and comprises one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FvDMPl gene nucleotide sequence of SEQ ID NO. 16, or a fragment thereof. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria vesca and comprises genetic modifications in both alleles of an FvDMPl gene. In some variations, the one or more genetic modifications comprise a modification of an enhancer of FvDMPl, a modification of a promoter of FvDMPl, a modification of a coding region of FvDMPl, modification of an intron of FvDMPl, or any combination thereof relative to a wild-type or unmodified Fragaria vesca plant. In some embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria vesca and has decreased expression of FvDMPl protein relative to a pollen, stolon, seed, or achene from a control Fragaria vesca plant (e.g., a Fragaria vesca plant lacking one or more of the genetic modifications). In certain embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant is a plant of the species Fragaria vesca and lacks detectable expression of FvDMPl protein.

[0103] In some embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3gene, an FaDMPl-4 gene, or any combination thereof. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3 gene, and an Fa-DMPl-4 gene.

[0104] In some embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, an FaDMPl-4 protein, or any combination thereof. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, and an Fa-DMPl-4 protein. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the list consisting of SEQ ID NOs: 2-5, or fragments thereof. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of 1) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 2; 2) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 3; 3) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 4; and / or 4) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ IDNO: 5.

[0105] In some embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications of an FaDMPl-1 gene or gene product, an FaDMPl-2 gene or gene product, an FaDMPl-3 gene or gene product, an FaDMPl-4 gene or gene product, or any combination thereof. In some embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications of an FaDMPl-1 gene or gene product, an FaDMPl-2 gene or gene product, an FaDMPl-3 gene or gene product, and an FaDMPl-4 gene or gene product. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and comprises one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17-20, or a fragment thereof. In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and comprises: 1) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 17, or a fragment thereof; 2) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 18, or a fragment thereof; 3) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 19, or a fragment thereof; and / or 4) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQID NO: 20, or a fragment thereof. In some variations, the one or more genetic modifications comprise a modification of an enhancer of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), a modification of a promoter of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), a modification of a coding region of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), modification of an intron of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl- 1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), or any combination thereof relative to a pollen, stolon, seed, or achene of a control Fragaria x ananassa plant (e.g., a wild-type or unmodified Fragaria x ananassa plant lacking one or more or all of the genetic modifications).

[0106] In some embodiments, the pollen, stolon, seed, or achene is from a haploid- inducing strawberry plant of the species Fragaria x ananassa and has decreased expression of an FaDMPl-1 protein, an FaDMPl -2 protein, an FaDMPl -3 protein, an FaDMPl -4 protein, or any combination (e.g, two, three, or all four) thereof relative to a pollen, stolon, seed, or achene of a control Fragaria x ananassa plant (e.g, a Fragaria ananassa plant lacking one or more or all of the genetic modifications). In certain embodiments, the pollen, stolon, seed, or achene is from a haploid-inducing strawberry plant of the species Fragaria x ananassa and lacks detectable expression of FaDMPl-1 protein, FaDMPl -2 protein, FaDMPl -3 protein, FaDMPl -4 protein, or any combination (e.g., two, three, or all four) thereof.

[0107] The pollen, stolon, seed, or achene of the haploid-inducing strawberry plant may comprise one or more genetic modifications resulting in decreased expression of any combination of DMP genes described herein or known in the art. In some embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant may comprise one or more genetic modifications resulting in non-expression of any combination of DMP genes described here or known in the art. In further embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant may comprise one or more genetic modifications resulting in decreased expression, non-expression, or a combination thereof of any combination of DMP genes described here or known in the art. In certain embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant lacks detectable expression of DMP proteins.

[0108] In some embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant has decreased expression of DMP proteins relative to a control strawberry plant. Control strawberry plants include, for example, a plant of the same species as the haploid-inducing strawberry plant that lacks one or more or all of the genetic modifications comprised by the haploid-inducing strawberry plant, a wild-type plant of the same species as the haploid-inducing strawberry plant, an unmodified plant of the same species as the haploid-inducing strawberry plant, or a null segregant strawberry plant, which underwent the same genetic manipulation, but is lacking one or more or all of the genetic modifications comprised by the haploid-inducing strawberry plant. In certain embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant has decreased expression of DMP proteins relative to a pollen, stolon, seed, or achene of a strawberry plant of the same species lacking the one or more genetic modifications.Methods and Compositions for Producing Haploid-inducing Strawberry Plants

[0109] In one aspect, described herein are methods of producing haploid-inducing strawberry plants. In some embodiments, the method comprises introducing one or more genetic modifications resulting in decreased expression of one or more DMP genes described herein into a strawberry plant. In certain embodiments, the method further comprises introducing one or more naturally-occurring inactive alleles of one or more DMP genes into a strawberry plant by crossing with a plant having the one or more naturally-occurring inactive alleles of one or more DMP genes.Methods of Introducing Genetic Modifications

[0110] In some embodiments, the methods of producing haploid-inducing strawberry plants described herein comprise introducing one or more genetic modifications into a strawberry plant. In certain embodiments, the step of introducing one or more genetic modifications into a strawberry plant results in a decreased expression of one or more DMP genes. In certain embodiments, the decreased expression is achieved by gene disruption (e.g., disruption of one or more DMP genes), gene knockout (e.g., knockout of one or more DMP genes), gene knockdown (e.g., knockdown of one or more DMP genes), gene silencing (e.g., silencing of one or more DMP genes), RNA interference (e.g., RNA interference of one or more DMP genes), induction of methylation (e.g., induction of methylation of one or more DMP genes), or any combination thereof. In some embodiments, the method comprisesintroducing one or more of the genetic modifications by gene editing using a site-directed nuclease.[OHl] In certain embodiments, the genetic modifications are introduced by gene editing. Any of several gene editing methods known in the art may be used to introduce the genetic modifications of the one or more DMP genes. In some variations, gene editing is performed with one or more natural or engineered site-directed nucleases including, but not limited to, RNA-guided nucleases, meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector-based nucleases (TALENs). In further variations, gene editing is performed with RNA-guided nucleases including, but not limited to, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) nucleases. In yet additional variations, gene editing may be performed using a deactivated Cas nuclease fused to an engineered reverse transcriptase in a process known as prime editing. Methods of gene editing are numerous, well-known and routine in the art, and are described in US17 / 045747, US16 / 977020, and US16 / 961396, which are herein incorporated in their entirety.

[0112] An engineered nuclease may be a guided nuclease, which may function as a ribonucleoprotein (RNP) complex with a guide RNA. According to some embodiments, a guided nuclease may be selected from the group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, CasZ, MAD7, and homologs or modified versions thereof, Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), and homologs or modified versions thereof). The DNA construct or molecule encoding a guided nuclease may be delivered with or without a guide nucleic acid.

[0113] For guided nucleases, a guide nucleic acid molecule may be further provided to direct the guided nuclease to a target site in the genome of the plant via base-pairing or hybridization to cause a DSB or nick at or near the target site. The guide nucleic acid may be transformed or introduced into a plant cell or tissue as a guide nucleic acid molecule, or as a recombinant DNA molecule, construct or vector comprising a transcribable DNA sequence encoding the guide nucleic acid operably linked to a promoter or plant-expressible promoter.The promoter may be a constitutive promoter, a tissue-specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter.

[0114] In some embodiments, the guide nucleic acid comprises a first segment comprising a nucleotide sequence that is complementary to a sequence in a target nucleic acid (e.g., a target nucleic acid of a DMP gene described herein) and a second segment that interacts with a guided nuclease protein. In some embodiments, the first segment of a guide comprising a nucleotide sequence that is complementary to a sequence in a target nucleic acid corresponds to a CRISPR RNA (crRNA or crRNA repeat). In some embodiments, the second segment of a guide comprising a nucleic acid sequence that interacts with a guided nuclease protein corresponds to a trans-acting CRISPR RNA (tracrRNA). In some embodiments, the guide nucleic acid comprises two separate nucleic acid molecules (a polynucleotide that is complementary to a sequence in a target nucleic acid and a polynucleotide that interacts with a guided nuclease protein) that hybridize with one another. In other embodiments, the guide nucleic acid is a single polynucleotide (e.g., a gRNA). In some embodiments, the guide nucleic acid may comprise DNA, RNA or a combination of DNA and RNA.

[0115] A protospacer-adjacent motif (PAM) may be present in the genome immediately adjacent and upstream to the 5' end of the genomic target site sequence complementary to the targeting sequence of the guide RNA, immediately downstream (3') to the sense (+) strand of the genomic target site (relative to the targeting sequence of the guide RNA) as known in the art. See, e.g., Wu, X. et al. 2014. "Target specificity of the CRISPR-Cas9 system," Quant Biol. 2(2): 59-70. The genomic PAM sequence on the sense (+) strand adjacent to the target site (relative to the targeting sequence of the guide RNA) may comprise 5'-NGG-3'. However, the corresponding sequence of the guide nucleic acid (immediately downstream (3') to the targeting sequence of the guide RNA) may generally not be complementary to the genomic PAM sequence.

[0116] The guide nucleic acid may typically be a non-coding RNA molecule that does not encode a protein. The targeting sequence of the guide nucleic acid may be at least 10 nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15-30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. The targeting sequence may be at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a DNA sequence at the genomic target site.

[0117] In addition to the targeting sequence, a guide nucleic acid may further comprise one or more other structural or scaffold sequence(s), which may bind or interact with an RNA-guided endonuclease. Such scaffold or structural sequences may further interact with other RNA molecules (e.g., tracrRNA). Methods and techniques for designing targeting constructs and guide nucleic acids for genome editing and site-directed integration at a target site within the genome of a plant using a guided nuclease are known in the art.

[0118] In prime editing, a modified guided nuclease, such as a Cas nickase, is fused to an engineered reverse transcriptase (RT) and complexed with a non-coding RNA typically referred to as a pegRNA. A pegRNA comprises three components, from 5’ to 3’: 1) guide sequence complementary to the protospacer on non-PAM strand of the target site, 2) a template sequence comprising the desired nucleotide edit, and 3) a primer binding site complementary to the protospacer on the PAM strand of the target site. Guide sequence- mediated binding of the nuclease-RT fusion to the target site results in a nick in the PAM strand of the target site. The primer binding site of the pegRNA then binds to the nicked (PAM) strand of the target, which positions the template sequence 3’ of the PAM strand to serve as a template for reverse transcription. Ligation of the newly-synthesized strand and the nicked PAM strand results in successful introduction of the desired edit into the gene. Prime editing allows for precise editing of genomic sequences and is further described, for example, in Anzalone et al. ("Search-and-replace genome editing without double-strand breaks or donor DNA. " Nature 576.7785 (2019): 149-157.).

[0119] An engineered nuclease may be a site-specific nuclease. Several site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, are not nucleic acid-guided and instead rely on their protein structure to determine their target site for causing the DSB or nick, or they are fused, tethered or attached to a DNA- binding protein domain or motif. The protein structure of the site-specific nuclease (or the fused / attached / tethered DNA binding domain) may target the site-specific nuclease to the target site. According to many of these embodiments, non-nucleic acid-guided site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, may be designed, engineered and constructed according to known methods totarget and bind to a target site at or near the genomic locus of an endogenous gene of a plant to create a DSB or nick at such genomic locus to knockout or knockdown expression of the gene via repair of the DSB or nick, which may lead to the creation of a mutation or insertion of a sequence at the site of the DSB or nick, through cellular repair mechanisms, which may be guided by a donor template molecule.

[0120] In some embodiments, a site-specific nuclease is a recombinase. A recombinase may be a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif, or other recombinase enzyme known in the art. A recombinase or transposase may be a DNA transposase or recombinase attached or fused to a DNA binding domain. Non-limiting examples of recombinases include a tyrosine recombinase attached, etc., to a DNA recognition motif provided herein is selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnpl recombinase. In an aspect, a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc-finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, a serine recombinase attached to a DNA recognition motif provided herein is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase. In another aspect, a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.

[0121] A site-specific nuclease may be a zinc finger nuclease (ZFN). ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain (or a cleavage half-domain), which may be derived from a restriction endonuclease (e.g., FokI). The DNA binding domain may be canonical (C2H2) or non-canonical (e.g., C3H or C4). The DNA-binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site. Multiple zinc fingers in a DNA- binding domain may be separated by linker sequence(s). ZFNs can be designed to cleave almost any stretch of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain (e.g., derived from the FokI nuclease) fused to a DNA-binding domain comprising a zinc finger array engineered to bind a target site DNA sequence. The DNA-binding domain of a ZFN may typically be composed of 3-4 (or more) zinc-fingers. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger .alpha. -helix, whichcontribute to site-specific binding to the target site, can be changed and customized to fit specific target sequences. The other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities.

[0122] Methods and rules for designing ZFNs for targeting and binding to specific target sequences are known in the art. See, e.g., US Patent App. Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. The FokI nuclease domain may require dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFN monomer can cut the target site if the two- ZF-binding sites are palindromic. A ZFN, as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site. Without being limited by any theory, because the DNA-binding specificities of zinc finger domains can be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any target sequence (e.g., at or near a gene in a plant genome). Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly. In an aspect, a method and / or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more ZFNs. In another aspect, a ZFN provided herein is capable of generating a targeted DSB or nick.

[0123] A site-specific nuclease may be a TALEN enzyme. TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain (e.g., FokI). When each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double-stranded DNA break at the target site. Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity.

[0124] TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain. In some aspects, the nuclease is selected from a group consisting of PvuII, MutH, TevI, FokI, Alwl, Mlyl, Sbfl, Sdal, StsI, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a doublestranded DNA break at the target site. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

[0125] Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence, such as at or near the genomic locus of a gene in a plant. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

[0126] Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALEs. PvuII functions as a highly specific cleavage domain when coupled to a TALE (see Yank et al. 2013. PLoS One. 8: e82539). MutH is capable of introducing strand-specific nicks in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41 : e83). TevI introduces double-stranded breaks in DNA at targeted sites (see Beurdeley et al., 2013. Nature Communications. 4: 1762).

[0127] The relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNAWorks can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. See Doyle et al., Nucleic Acids Research (2012) 40: W117-122.; Cermak et al., Nucleic Acids Research (2011) 39:e82; and tale-nt.cac.cornelledu / about. In another aspect, a TALEN provided herein is capable of generating a targeted DSB.

[0128] A site-specific nuclease may be a meganuclease. Meganucleases, which are commonly identified in microbes, such as the LAGLID ADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (>14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp). According to some embodiments, a meganuclease may comprise a scaffold or base enzyme selected from the group consisting of I-Crel, I-Ceul, I-Msol, I-Scel, I- Anil, and I-Dmol. The engineering of meganucleases can be more challenging than ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity. Additionally, meganucleases have been combined with transcription activator-like (TAL) effectors to generate megaTALs, which fuse the DNA- binding region of the TAL to allow recognition of specific sequences to meganucleases to increase the activity and specificity of cleavage (see, e.g., Boissel et al. "megaTALs: a rare- cleaving nuclease architecture for therapeutic genome engineering." Nucleic acids research 42.4 (2014): 2591-2601). Thus, a meganuclease may be selected or engineered to bind to a genomic target sequence in a plant, such as at or near the genomic locus of a gene. In another aspect, a meganuclease provided herein is capable of generating a targeted DSB.

[0129] In some embodiments, gene editing comprises (a) inducing a DSB in the genome of a cell at a cleavage site (e.g., a cleavage site within a DMP gene) at or near a recognition site (e.g., a recognition site within a DMP gene) for a natural or engineered nuclease by expressing in the cell the natural or engineered nuclease recognizing said recognition site and inducing said DSB at the cleavage site; (b) introducing into the cell a repair nucleic acid molecule comprising an upstream flanking region having homology to the DNA region upstream of the preselected site (e.g., the recognition site within the DMP gene) and / or adownstream flanking DNA region having homology to the DNA region downstream of the preselected site (e.g., the recognition site within the DMP gene) for allowing homologous recombination between said flanking region or regions and said DNA region or regions flanking said preselected site; and (c) selecting a cell wherein said repair nucleic acid molecule has been used as a template for making a modification of said genome at said preselected site. In other embodiments, gene editing comprises (a) inducing a DSB in the genome of a cell at a cleavage site (e.g., a cleavage site within a DMP gene) at or near a recognition site (e.g., a recognition site within a DMP gene) for a natural or engineered nuclease by introducing into the cell the natural or engineered nuclease recognizing said recognition site and inducing said DSB at the cleavage site; (b) introducing into the cell a repair nucleic acid molecule comprising an upstream flanking region having homology to the DNA region upstream of the preselected site (e.g., the recognition site within the DMP gene) and / or a downstream flanking DNA region having homology to the DNA region downstream of the preselected site (e.g., the recognition site within the DMP gene) for allowing homologous recombination between said flanking region or regions and said DNA region or regions flanking said preselected site; and (c) selecting a cell wherein said repair nucleic acid molecule has been used as a template for making a modification of said genome at said preselected site.

[0130] As used herein, a repair nucleic acid molecule is a single-stranded or doublestranded DNA molecule or RNA molecule that is used as a template for modification of the genomic DNA at the preselected site in the vicinity of or at the cleavage site. As used herein, use as a template for modification of the genomic DNA, means that the repair nucleic acid molecule is copied or integrated at the preselected site by homologous recombination between the flanking region(s) and the corresponding homology region(s) in the target genome flanking the preselected site, optionally in combination with non-homologous endjoining (NHEJ) at one of the two ends of the repair nucleic acid molecule (e.g., in case there is only one flanking region). Integration by homologous recombination will allow precise joining of the repair nucleic acid molecule to the target genome up to the nucleotide level, while NHEJ may result in small insertions / deletions at the junction between the repair nucleic acid molecule and genomic DNA.

[0131] In some embodiments, the genetic modifications introduced by gene editing result in the decreased expression or non-expression of one or more DMP genes. In gene editing,the introduction of a DSB or nick may be used to introduce targeted genetic modifications in the genome of a plant. According to this approach, genetic modifications, such as deletions, insertions, inversions and / or substitutions may be introduced at a target site (e.g., a target site within a nucleotide of a DMP gene) via imperfect repair of the DSB or nick to produce a knock-out or knock-down of a gene. Such genetic modifications may be generated by imperfect repair of the targeted locus even without the use of a donor template molecule, and can result in decreased expression or non-expression of an endogenous gene product. For example, genetic modifications may be produced by an indel (insertion or deletion of nucleotide bases in a target DNA sequence through NHEJ), or by specific removal of sequence that reduces or completely destroys the function of sequence at or near the targeting site. A knockout of a DMP gene may be achieved by inducing a DSB or nick at or near the endogenous locus of the gene that results in non-expression of the gene product (e.g., a DMP protein), whereas a knockdown of a gene may be achieved in a similar manner by inducing a DSB or nick at or near the endogenous locus of the gene that is repaired imperfectly at a site that does not affect the coding sequence of the gene in a manner that would eliminate the function of the gene product (e.g., DMP protein). For example, the site of the DSB or nick within the endogenous locus may be in the upstream or 5' region of the DMP gene (e.g., a promoter and / or enhancer sequence) to affect or reduce its level of expression. Similarly, such targeted knockout or knockdown mutations of a DMP gene may be generated with a donor template molecule to direct a particular or desired mutation at or near the target site via repair of the DSB or nick. The donor template molecule may comprise a homologous sequence with or without an insertion sequence and comprising one or more mutations, such as one or more deletions, insertions, inversions and / or substitutions, relative to the targeted genomic sequence at or near the site of the DSB or nick. For example, targeted knockout mutations of a DMP gene may be achieved by substituting, inserting, deleting or inverting at least a portion of the DMP gene, including, but not limited to, by introducing a frame shift or premature stop codon into a protein coding sequence of the gene. A deletion of a portion of a DMP gene may also be introduced by generating DSBs or nicks at two target sites and causing a deletion of the intervening target region flanked by the target sites.

[0132] In some embodiments, the genetic modifications are introduced by transgenesis. Transgenes may include, but are not limited to, one or more protein-coding sequences operably linked to a plant-expressible promoter, one or more transcribable DNA sequences encoding an RNA molecule operably linked to a plant-expressible promoter, a gene ofinterest, a marker gene, or any combination thereof. Methods for the introduction of transgenes in plants are well-known and routine in the art. In some embodiments, transgenesis comprises (a) inducing a DSB in the genome of a cell at a cleavage site at or near a recognition site for a natural or engineered nuclease by expressing in the cell the natural or engineered nuclease recognizing said recognition site and inducing said DSB at the cleavage site; (b) introducing into the cell a repair nucleic acid molecule comprising an upstream flanking region having homology to the DNA region upstream of the preselected site, a downstream flanking DNA region having homology to the DNA region downstream of the preselected site, and a transgene region flanked by the upstream and downstream flanking DNA regions and comprising the transgene to be inserted at the preselected site; and (c) selecting a cell wherein said repair nucleic acid molecule has been used as a template for making a modification of said genome at said preselected site. In other embodiments, transgenesis comprises (a) inducing a DSB in the genome of a cell at a cleavage site at or near a recognition site for a natural or engineered nuclease by introducing into the cell the natural or engineered nuclease recognizing said recognition site and inducing said DSB at the cleavage site; (b) introducing into the cell a repair nucleic acid molecule comprising an upstream flanking region having homology to the DNA region upstream of the preselected site, a downstream flanking DNA region having homology to the DNA region downstream of the preselected site, and a transgene region flanked by the upstream and downstream flanking DNA regions and comprising the transgene to be inserted at the preselected site; and (c) selecting a cell wherein said repair nucleic acid molecule has been used as a template for making a modification of said genome at said preselected site. In some variations, the recognition site is a site within a DMP gene. In other variations, the recognition site is within a neutral (e.g., non-coding) site within the genome.

[0133] In some embodiments, the genetic modification comprises introducing proteins, nucleic acids, or a combination thereof into a plant cell. The introduction of the proteins, nucleic acids, or combination thereof into the plant cell may be achieved by any of several means known and routinely-used in the art. In some embodiments, the introduction of the proteins, nucleic acids, or combination thereof into the plant cell comprises isolating protoplasts, transfecting the protoplasts, encapsulating the protoplasts, and regenerating plants from the protoplasts. In other embodiments, the introduction of the proteins, nucleic acids, or combination thereof into the plant cell comprises biolistic transformation. In certain embodiments, the introduction of the proteins, nucleic acids, or combination thereof into theplant cell comprises isolating immature plant embryos, bombarding the embryos with particles comprising nucleic acids, and regenerating plants from the immature embryos. Numerous additional transformation methods may be used to introduce the proteins, nucleic acids, or combination thereof into a suitable plant or plant cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant (cell) such as microinjection, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the cal cium / poly ethylene glycol method for protoplasts (Krens et al. (1982) Nature 296: 72-74; Negrutiu et al. (1987) Plant. Mol. Biol. 8: 363-373); electroporation of protoplasts (Shillito et al. (1985) Bio / Technol. 3: 1099-1102); microinjection into plant material (Crossway et al. (1986) Mol. Gen. Genet. 202: 179-185); DNA or RNA-coated particle bombardment (Klein et al. (1987) Nature 327: 70) infection with (non-integrative) viruses and the like.

[0134] Accordingly, in some embodiments, the method of producing the haploid- inducing strawberry plant comprises contacting a plurality of cells of a strawberry plant with one or more nucleic acid molecules, proteins, or a combination thereof into a plurality of cells of a strawberry plant. In some embodiments, the method comprises introducing one or more nucleic acid molecules, proteins, or a combination thereof into a plurality of cells of a strawberry plant. In certain embodiments, the plurality of cells is a plurality of protoplasts. In some embodiments, the method further comprises, subsequent to the contacting step: allowing the plurality of cells (e.g., protoplasts) of the parent strawberry plant to form calli, plants, or a combination thereof; determining the presence of one or more of the genetic modifications in the calli or plants; and identifying one or more calli or plants as having the genetic modifications resulting in decreased expression of one or more DMP genes. Methods of detecting genetic modifications are known in the art and include, for example, PCR amplification, DNA sequencing, hybridization assays (e.g., in situ hybridization), chip-based assays, reporter assays, and the like. A method of detecting genetic modifications in strawberry plants is described in Example 2.

[0135] In some embodiments, the method comprises contacting a plurality of cells of a parent strawberry plant with one or more expression vectors comprising an expression cassette for a site-directed nuclease (e.g., a Cas nuclease, a meganuclease, a TALEN, a ZFN, or a megaTAL). In certain embodiments, , the method comprises contacting a plurality ofcells of a parent strawberry plant with one or more expression vectors comprising an expression cassette for a site-directed nuclease contacting a plurality of cells of a parent strawberry plant with one or more expression vectors together comprising an expression cassette for a Cas nuclease and an expression cassette for a guide RNA molecule.

[0136] In some embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant with one or more expression vectors together comprising an expression cassette for a Cas nuclease and an expression cassette for a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of one or more of the DMP genes described herein. In other embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant with one or more Cas nucleases, wherein each Cas nuclease is complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of one or more of the DMP genes described herein. The guide RNA molecule may be a crRNA, a gRNA, or a pegRNA. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence encoding one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or moreconsecutive nucleotides of an enhancer of one or more DMP genes described herein, a promoter of one or more DMP genes described herein, a coding region of one or more DMP genes, or an intron of one or more of the DMP genes described herein. In certain embodiments, the guide RNA has 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34. In certain embodiments, the guide RNA has 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27 or SEQ ID NO: 33

[0137] In some embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant of the species Fragaria vesca with one or more expression vectors together comprising an expression cassette for a Cas nuclease and an expression cassette for a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of an FvDMPl gene. In other embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant of the species Fragaria vesca with one or more Cas nucleases, wherein each Cas nuclease is complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of an FvDMPl gene. The guide RNA molecule may be a crRNA, a gRNA, or a pegRNA. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence encoding a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 16. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17,18, 19, 20, or more consecutive nucleotides of an enhancer of FvDMPl, a promoter of FvDMPl, a coding region of FvDMPl, or an intron of FvDMPl.

[0138] In other embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant of the species Fragaria vesca with one or more Cas nucleases, wherein each Cas nuclease is complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27 or SEQ ID NO: 33, or a combination thereof. In certain embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells protoplasts of a parent strawberry plant of the species Fragaria vesca with a plurality of Cas nucleases, wherein a first portion of the plurality of the Cas nucleases are each complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27, and wherein a second portion of the plurality of the Cas nucleases are each complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 33.

[0139] In some embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant of the species Fragaria x ananassa with one or more expression vectors together comprising an expression cassette for a Cas nuclease and an expression cassette for one or more guide RNA molecules having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18,19, 20, or more consecutive nucleotides of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3 gene, an FaDMPl-4 gene, or any combination (e.g., two, three, or all four) thereof. In other embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant of the species Fragaria x ananassa with one or more Cas nucleases, wherein each Casnuclease is complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3 gene, an FaDMPl-4 gene, or any combination (e.g., two, three, or all four) thereof. The guide RNA molecule may be a crRNA, a gRNA, or a pegRNA. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence encoding a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15 In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In certain embodiments, the guide RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of an enhancer, a promoter, a coding region, or an intron of an FaDMPl-2 gene, an FaDMPl-3 gene, an FaDMPl-4 gene, or any combination (e.g., two, three, or all four) thereof.

[0140] In other embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells (e.g., protoplasts) of a parent strawberry plant of the species Fragaria x ananassa with one or more Cas nucleases, wherein each Cas nuclease is complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27 or SEQ ID NO: 33, or a combination thereof. In certain embodiments, the method of producing the haploid-inducing strawberry plant comprises contacting a plurality of cells protoplasts of a parent strawberry plant of the species Fragaria x ananassa with a plurality of Cas nucleases, wherein a first portion of the plurality of the Cas nucleases are each complexed with a guide RNA molecule having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27, and wherein a second portion of the plurality of the Cas nucleases are each complexed with a guide RNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 33.Compositions for Producing Haploid-inducing Strawberry Plants

[0141] In some aspects, provided herein are expression vectors, isolated DNA molecules, bacterial cells, and kits for making a haploid-inducing strawberry plant described herein. Also provided are genetically modified plants, plant parts, plant cells, and seeds comprising said expression vectors and isolated DNA molecules.

[0142] In one aspect, provided herein are expression vectors for making a haploid- inducing strawberry plant described herein. In some embodiments, the expression vector comprises one or more expression cassettes. The expression cassettes may comprise, for example, a nucleic acid encoding site-directed nuclease operably linked to a promoter, or a nucleic acid encoding a non-coding RNA (e.g., a guide RNA, an siRNA, or the like) operably linked to a promoter. In some embodiments, the expression vector comprises at least a first expression cassette comprising a nucleic acid encoding a guide RNA operably linked to a promoter. In certain embodiments, the expression vector comprises one or more additional expression cassettes, for example, a second expression cassette comprising a nucleic acid encoding a Cas nuclease operably linked to a promoter, and / or one or more additional expression cassettes each comprising a nucleic acid encoding a guide RNA operably linked to a promoter. In certain embodiments, the expression vector comprises a DNA sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence encoding one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the group consisting SEQ ID NOs: 1-15. In certain embodiments, the expression vector comprises a DNA sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotidesequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In some embodiments, the expression vector comprises a DNA sequence encoding a site-directed nuclease. In certain embodiments, the site-directed nuclease is a Cas nuclease, a TALEN, ZFN, or a mega-TAL.

[0143] In some embodiments, provided herein is an expression vector for making a haploid-inducing strawberry plant, wherein the expression vector comprises at least a first expression cassette comprising a nucleic acid encoding a non-coding RNA molecule operably linked to a promoter. In certain embodiments, the non-coding RNA is a crRNA, a gRNA, a pegRNA, a siRNA, a miRNA, or a dsRNA. In some embodiments, the non-coding RNA molecule (e.g., guide RNA) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence encoding one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In certain embodiments, the non-coding RNA molecule (e.g., guide RNA) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In certain embodiments, the non-coding RNA molecule (e.g., guide RNA) has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of an enhancer of one or more DMP genes described herein, a promoter of one or more DMP genes described herein, a coding region of one or more DMP genes, or an intron of one or more of the DMP genes described herein. In certain embodiments, the non-coding RNA molecule is a guide RNA having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27 or SEQ ID NO: 33, or both. In certainembodiments, the expression vector comprises a first expression cassette comprising a nucleic acid encoding a guide RNA having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27 or SEQ ID NO: 33, or both. In certain embodiments, the expression vector comprises a first expression cassette comprising a nucleic acid encoding a guide RNA having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27, and a second expression cassette comprising a nucleic acid encoding a guide RNA having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 33. In some embodiments, the expression vector further comprises a DNA sequence encoding a Cas nuclease operably linked to a promoter.

[0144] In some embodiments, provided herein is an expression vector comprising a DNA sequence encoding a site-directed nuclease operably linked to a promoter. In certain embodiments, the site-directed nuclease is a Cas nuclease, a TALEN, ZFN, or a mega-TAL. In some embodiments, the expression vector comprises a DNA sequence encoding a TALEN, ZFN, or a mega-TAL operably linked to a promoter, wherein the TALEN, ZFN, or a mega- TAL comprises an amino acid sequence that confers binding to a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence encoding one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In some embodiments, the expression vector comprises a DNA sequence encoding a TALEN, ZFN, or a mega-TAL operably linked to a promoter, wherein the TALEN, ZFN, or a mega-TAL comprises an amino acid sequence that confers binding to a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26.

[0145] In some embodiments, provided herein is an isolated DNA molecule for making a haploid-inducing strawberry plant, wherein the isolated DNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to at least 15, 16, 17, 18, 19, 20, 50, 100, or more consecutive nucleotides of a nucleotide sequence encoding one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In some embodiments, provided herein is an isolated DNA molecule for making a haploid-inducing strawberry plant, wherein the isolated DNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to at least 15, 16, 17, 18, 19, 20, 50, 100, or more consecutive nucleotides of a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In some embodiments, the isolated DNA molecule is a synthetic oligo nucleotide.

[0146] In some embodiments, provided herein is an isolated nucleic acid molecule for making a haploid-inducing strawberry plant, wherein the isolated nucleic acid molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to at least 15, 16, 17, 18, 19, 20, 50, 100, or more consecutive nucleotides of a nucleotide sequence encoding one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. In some embodiments, provided herein is an isolated nucleic acid molecule for making a haploid- inducing strawberry plant, wherein the isolated RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100%identity or complementarity to at least 15, 16, 17, 18, 19, 20, 50, 100, or more consecutive nucleotides of a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26. In certain embodiments, the isolated nucleic acid molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34. In certain embodiments, the isolated nucleic acid molecule has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27 or SEQ ID NO: 33, or both. In some embodiments, the isolated nucleic acid molecule comprises DNA, RNA, or both. In some embodiments, the isolated nucleic acid molecule is an isolated RNA molecule. In certain embodiments, the isolated RNA molecule is a guide RNA. In certain embodiments, the guide RNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to SEQ ID NO: 27 or SEQ ID NO: 33, or both. In certain embodiments, the isolated nucleic acid molecule further comprises a nucleic acid sequence for association with a CRISPR-associated (Cas) enzyme.

[0147] In some aspects, provided herein are genetically modified plants, plant parts, plant cells, or seed comprising any of the expression vectors or isolated DNA molecules described herein. In some embodiments, the genetically modified plants, plant parts, plant cells, or seeds are genetically modified strawberry plants, strawberry plant parts (e.g., achenes, stolons, or any strawberry plant part described herein), strawberry plant cells, or strawberry seeds.

[0148] In other aspects, provided herein is a bacterial cell comprising any of the expression vectors or isolated DNA molecules described herein. In some embodiments, the bacterial cell is an Agrobacterium cell. In some aspects, provided herein are genetically modified plants, plant parts, plant cells, or seed comprising said bacterial cell.

[0149] In yet other aspects, provided herein is a kit for producing a haploid-inducing strawberry plant comprising one or more of the expression vectors, isolated DNA molecules, isolated RNA molecules, bacterial cells, or any combination thereof described herein. Insome embodiments, the kit further comprises instructions for using the kit components to produce a haploid-inducing strawberry plant.

[0150] In yet other aspects, provided herein is a kit for producing a true homozygous octoploid strawberry plant comprising one or more cells or plant parts of any of the haploid- inducing strawberry plants described herein. In some embodiments, the kit further comprises instructions for using the kit components to produce a true homozygous octoploid strawberry plant. In some embodiments, the kit further comprises a chromosome doubling agent described herein. In certain embodiments, the kit further comprises colchicine, oryzalin, oxide, trifluralin, or any combination thereof.Methods of Producing True Homozygous Octoploid Strawberry Lines

[0151] In some aspects, described herein are methods of producing true homozygous octoploid strawberry plant, comprising (a) generating a tetrahaploid strawberry cell or plant through haploid induction, and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce a doubled tetrahaploid strawberry cell or plant. A doubled tetrahaploid cell or plant may also be described as a homozygous octoploid cell or plant having the same haplotype for each of the chromosomes within each of the subgenomes of the plant. In some embodiments, the method comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a haploid-inducing strawberry plant described herein, and (b) producing a doubled tetrahaploid cell or plant from the tetrahaploid cell. In some embodiments, the method comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a haploid-inducing strawberry plant described herein, and (b) producing an octoploid cell or plant from the doubled tetrahaploid cell or plant. In some embodiments, the method comprises (al) contacting a reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a haploid-inducing strawberry plant described herein, (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant, and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce a doubled tetrahaploid strawberry plant. In some embodiments, the method comprises (al) contacting a tetrapioid reduced egg cell (a tetrahaploid cell) from a donor octoploid strawberry plant with a sperm cell from a haploid-inducing strawberry plant described herein, (a2) allowing a tetrahapploid embryo to form and grow into a tetrahapploid strawberry plant, and (b) subjecting the tetrahapploid strawberry plant to genome doubling to produce an octoploid strawberry plant form the doubled tetrahaploid.Haploid Induction

[0152] In some embodiments, the methods of producing true homozygous octoploid strawberry plant, or plant part thereof, comprise generating a tetrahaploid strawberry plant through haploid induction, or the process of crossing pollen from a haploid-inducing strawberry plant with a donor (non-haploid-inducing) strawberry plant to generate embryos and seeds with half the ploidy of the donor strawberry plant. During a normal cross with two non-haploid-inducing plant plants, two haploid sperm cells migrate down the pollen tube to the ovule. One of the sperm cells fertilizes the central cell of the ovule to form the endosperm, while the other sperm cell fertilizes the haploid reduced egg cell to form an embryo having the ploidy of the parent plant. For example, if both parent plants are octoploid, the haploid (4x) sperm and egg form an octoploid (8x) embryo in a normal fertilization process. In haploid induction, the sperm cells from the haploid-inducing plant migrate down the pollen tube to the ovule, where one of the sperm cells fertilizes the central cell to form the endosperm. However, when the other sperm cell contacts the reduced egg cell, embryogenesis is initiated, but the DNA from the sperm cell is not delivered to the egg cell, resulting in formation of a haploid embryo. For example, in haploid induction, if both parent plants are octoploid, the haploid (4x) egg cell does not incorporate the DNA from the haploid sperm cell (4x), resulting in a haploid (4x) embryo, which may also be referred to herein as a tetrehaploid.

[0153] In some embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen, or a part thereof, from a haploid-inducing strawberry plant described herein. In certain embodiments, the tetrahaploid cell is a reduced egg cell. In certain embodiments, the tetrahaploid cell is a reduced egg cell and the part of the pollen from the haploid-inducing strawberry plant is a sperm cell. In some embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises contacting pollen of the haploid-inducing strawberry plant with the stigma of a pistil of the donor octoploid strawberry plant comprising the reduced egg cell, allowing formation of a pollen tube and migration of one or more (e.g., two) sperm cells of the pollen to the reduced egg cell, thereby contacting the reduced egg cell with the sperm cells.

[0154] In some embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises allowing the donor octoploid strawberry plant to form aplurality of seeds. In certain embodiments, the plurality of seeds comprises one or more tetrahaploid seeds. The method may further comprise a step of selecting one or more tetrahaploid seeds of the plurality of seeds by 1) determining the ploidy of the embryo and / or endosperm of one or more seeds of the plurality of seeds, and 2) selecting one or more seeds having a tetrahaploid embryo. In certain embodiments, the step of selecting one or more tetrahaploid seeds of the plurality of seeds comprises 1) determining the ploidy of the embryo and / or endosperm of one or more seeds of the plurality of seeds, and 2) selecting one or more seeds having a tetrapioid embryo and an endosperm having a ploidy of greater than 8x (e.g., a ploidy of 9x, lOx, l lx, or 12x).

[0155] The haploid-inducing strawberry plant and the donor strawberry plant may have the same ploidy or different ploidies. For example, a haploid-inducing strawberry plant may be diploid (2x) and produce haploid (lx) pollen that can be crossed with an octoploid (8x) strawberry donor plant, resulting in tetrapioid (4x) embryos, also referred to as tetrahaploid embryos. Accordingly, in some embodiments, the method of producing true homozygous octoploid strawberry plant, or plant part thereof, comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a diploid, triploid, tetrapioid, pentapioid, hexapioid, septapioid, or octoploid haploid-inducing strawberry plant described herein, and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a diploid, triploid, tetrapioid, pentapioid, hexapioid, septapioid, or octoploid haploid-inducing strawberry plant described herein, (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant, and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce a doubled tetrahaploid strawberry plant. In certain embodiments, the method comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a diploid, triploid, tetrapioid, pentapioid, hexapioid, septapioid, or octoploid haploid-inducing strawberry plant described herein, and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a diploid, triploid, tetrapioid, pentapioid, hexapioid, septapioid, or octoploid haploid-inducing strawberry plant described herein, (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant, and (b) subjecting the tetrahaploidstrawberry plant to genome doubling to produce an octoploid doubled tetrahaploid strawberry plant.

[0156] In some embodiments, the method of producing a true homozygous octoploid strawberry plant, or plant part thereof, comprises (a) contacting a haploid cell from a donor octoploid strawberry plant with pollen from a diploid haploid-inducing strawberry plant described herein, and (b) producing an octoploid cell from a doubled tetrahaploid or plant from the haploid cell. In certain embodiments, the method comprises (al) contacting a reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a diploid haploid-inducing strawberry plant described herein, (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant, and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce a doubled tetrahaploid strawberry plant. In certain embodiments, the method comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a diploid haploid-inducing strawberry plant described herein, and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a diploid haploid-inducing strawberry plant described herein, (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant, and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce an octoploid doubled tetrahaploid strawberry plant. In some embodiments, the diploid haploid-inducing strawberry plant is a plant of the species Fragaria vesca, Fragaria iinumae, Fragaria nipponica. Fragaria viridis, Fragaria x bifera, Fragaria bucharica, Fragaria chinensis, Fragaria daltoniana, Fragaria emeiensis, Fragaria hayatae, Fragaria iinumae, Fragaria mandshurica, Fragaria viridis, Fragaria nilgerrensis, Fragaria nipponica, Fragaria nubicola, or Fragaria pentaphylla. In certain embodiments, the diploid haploid-inducing strawberry plant is a plant of the species Fragaria vesca.

[0157] In other embodiments, the method of producing true homozygous octoploid strawberry plant, or plant part thereof, comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from an octoploid haploid-inducing strawberry plant described herein, and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a reduced egg cell from a donor octoploid strawberry plant with a sperm cell from an octoploid haploid-inducing strawberry plant described herein, (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant, and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce a doubled tetrahaploid strawberry plant. In certain embodiments, the method comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from an octoploid haploid-inducing strawberry plant described herein, and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with a sperm cell from an octoploid haploid- inducing strawberry plant described herein, (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant, and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce an octoploid doubled tetrahaploid strawberry plant. In some embodiments, the octoploid haploid-inducing strawberry plant is a plant of the speices Fragaria x ananassa or Fragaria chiloensis. In certain embodiments, the octoploid haploid- inducing strawberry plant is a plant of the speices Fragaria x ananassa.

[0158] Haploid induction may comprise intraspecific haploid induction (e.g., using pollen of a haploid-inducing Fragaria x ananassa plant to fertilize a donor Fragaria x ananassa plant) or interspecific haploid induction (e.g., using pollen of a haploid-inducing Fragaria vesca plant to fertilize a donor Fragaria x ananassa plant). Accordingly, in some embodiments, the method of producing true homozygous octoploid strawberry plant, or plant part thereof, comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of different species; and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of different species; (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant; and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce a doubled tetrahaploid strawberry plant. In certain embodiments, the method comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid- inducing strawberry plant are plants of different species; and (b) producing an octoploiddoubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of different species; (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant; and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce an octoploid doubled haploid strawberry plant. In certain embodiments, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa, and the haploid-inducing strawberry plant is a plant of the species Fragaria vesca.

[0159] In other embodiments, the method of producing true homozygous octoploid strawberry plant, or plant part thereof, comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of the same species; and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of the same species; (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant; and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce a doubled tetrahaploid strawberry plant. In certain embodiments, the method comprises (a) contacting a tetrahaploid cell from a donor octoploid strawberry plant with pollen from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of the same species; and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (al) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a haploid-inducing strawberry plant described herein, wherein the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of the same species; (a2) allowing a tetrahaploid embryo to form and grow into a tetrahaploid strawberry plant; and (b) subjecting the tetrahaploid strawberry plant to genome doubling to produce an octoploid doubled tetrahaploid strawberry plant. In certain embodiments, both the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of the species Fragaria x ananassa.

[0160] In some embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises allowing the donor octoploid strawberry plant to form a plurality of seeds and subsequently allowing one or more seeds of the plurality to germinate and form one or more seedlings. In certain embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises allowing the donor octoploid strawberry plant to form a plurality of seeds and subsequently allowing two or more seeds of the plurality to germinate and form a plurality of seedlings. In other embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises collecting one or more embryos from the plurality of seeds, contacting the one or more embryos with a nutrient medium suitable for inducing seedling growth, and allowing the one or more embryos to form one or more seedlings. In certain embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises collecting a plurality of embryos from the plurality of seeds, contacting the plurality of embryos with a nutrient medium suitable for inducing seedling growth, and allowing the plurality of embryos to form a plurality of seedlings. The method may further comprise a step of selecting one or more tetrahaploid seedlings by 1) determining the ploidy of one or more cells of one or more seedlings of the plurality of seedlings, and 2) selecting one or more seedlings having one or more tetrahaploid cells from the plurality of seedling. In some embodiments, the step of selecting one or more tetrahaploid seeds of the plurality of seeds comprises 1) determining the ploidy of one or more cells of one or more seedlings of the plurality of seedlings, and 2) selecting one or more seedlings having one or more tetrapioid cells from the plurality of seedlings. In certain embodiments, determining the ploidy of one or more cells of the one or more seedlings comprises obtaining a tissue sample of each of the one or more seedlings and determining the ploidy of one or more cells of each tissue sample.

[0161] Methods of determining the ploidy of a cell are known in the art and are described, for example, in Example 3 of the present disclosure and Galbraith et al (Galbraith et al. 1983. “Rapid flow cytometric analysis of the cell cycle in intact plant tissues.” Science 220, no. 4601 : 1049-1051) and reviewed in, for example, Ochatt et al (Ochatt et al. 2011. "Ploidy level determination within the context of in vitro breeding." Plant Cell, Tissue and Organ Culture (PCTOC) 104.3 (2011): 329-341).Haploid Doubling

[0162] In some embodiments, the methods of producing true homozygous octoploid strawberry plants, or plant parts thereof, comprise subjecting a tetrahaploid strawberry cell or plant to genome doubling to produce a doubled tetrahaploid strawberry cell or plant. Genome doubling, also known as chromosome doubling, refers to the process of disrupting mitosis in a parent cell or plant in order to generate daughter cell or plants having two clonal copies of the genome of the parent cell or plant. In the case of haploid doubling, mitosis is disrupted in a haploid parent cell or plant in order to generate doubled-haploid daughter cells or plant having two clonal copies of the haploid gene of the parent cell or plant. This results in a true homozygous daughter cell or plant which is monoallelic at all loci across its two haploid sets of chromosomes. For example, if haploid induction in an octoploid (8x) strawberry donor plant to generate a tetrahaploid (4x) cell or plant, genome doubling of the tetrahaploid (4x) cell or plant would result in generating two clonal copies of the 4x haploid chromosomes, resulting in an octoploid (8x) cell or plant having two 4x clonal sets of chromosomes that are monoallelic across all loci.

[0163] In some embodiments, the methods of producing true homozygous octoploid strawberry plants, or plant parts thereof, comprise subjecting one or more tetrahaploid cells, tetrahaploid seeds, or tetrahaploid seedlings to a haploid doubling treatment. The tetrahaploid cells, tetrahaploid seeds, or tetrahaploid seedlings may have been selected from a plurality of cells, seeds, or seedlings grown from an octoploid strawberry donor plant that has been contacted with pollen or a part thereof (e.g., sperm cells) from a haploid-inducing strawberry plant, as described herein. In some embodiments, subjecting one or more tetrahaploid cells, tetrahaploid seeds, or tetrahaploid seedlings to a haploid doubling treatment comprises contacting the one or more tetrahaploid cells, tetrahaploid seeds, or tetrahaploid seedlings with an agent that causes genome doubling or chromosome doubling, such as an antimitotic agent. In certain embodiments, the antimitotic agent is an anti -microtubule agent. In some embodiments, the antimitotic agent comprises colchicine, oryzalin, oxide, trifluralin, or any combination thereof. In some embodiments, the antimitotic agent comprises colchicine. In some embodiments, the antimitotic agent is an antimitotic herbicide. Antimitotic herbicides include, for example, oryzalin, trifluralin, and amiprofos-methyl (APM). In some embodiments, the haploid doubling treatment comprises contacting the tetrahaploid cell, tetrahaploid seed, or tetrahaploid seedling with an anti -microtubule agent to form a doubled tetrahaploid cell, a doubled tetrahaploid seed, or a doubled tetrahaploid seedling.

[0164] In some embodiments, the doubled tetrahaploid cell or the doubled tetrahaploid seed is allowed to grow into a doubled tetrahaploid seedling. In certain embodiments, the doubled tetrahaploid seedling is allowed to form seed, thus producing the true homozygous octopl oid strawberry seed.

[0165] Methods of chromosome doubling in plants are known in the art and reviewed, for example, in Hooghvorst et al. (2021. "Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops." Plant Cell Reports 40.2: 255-270.). Methods of chromosome doubling include, but are not limited to, treatment with mitotic inhibitors such as colchicine, oryzalin, trifluran, or nitrous oxide.

[0166] In some embodiments, haploid doubling occurs spontaneously. Accordingly, in some embodiments, provided herein are methods of producing true homozygous octoploid strawberry plant, comprising (a) generating a tetrahaploid strawberry cell or plant through haploid induction, and (b) allowing the tetrahaploid strawberry plant to undergo spontaneous genome doubling to produce a doubled tetrahaploid strawberry cell or plant. The method may further comprise a step of selecting one or more doubled tetrahaploid cells, seeds, or seedlings. From a plurality of tetrahaploid cells, seeds, or seedlings. In some embodiments, the method comprises selecting one or more doubled tetrahaploid seeds from a plurality of seeds by 1) determining the ploidy of the embryo of one or more seeds of the plurality of seeds, and 2) selecting one or more seeds having an octoploid embryo. In certain embodiments, the step of selecting one or more tetrahaploid seeds of the plurality of seeds comprises 1) determining the ploidy of the embryo and endosperm of one or more seeds of the plurality of seeds, and 2) selecting one or more seeds having an octoploid embryo and an endosperm having a ploidy of greater than 8x (e.g., a ploidy of 9x, lOx, 1 lx, or 12x).True Homozygous Octoploid Strawberry Seed and Plants

[0167] In some aspects, provided herein are true homozygous octoploid strawberry plants, plant parts thereof, plant lines, and seeds produced by the methods described herein. In some embodiments, the true homozygous octoploid strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, the true homozygous octoploid strawberry plant, or part thereof, comprises two clonal sets of chromosomes. In certain embodiments, the true homozygous octoploid strawberry plant, or part thereof, comprises twoclonal sets of chromosomes, each clonal set comprising a haploid set of chromosomes from each of four subgenomes.

[0168] In some embodiments, the true homozygous octoploid strawberry plant, plant part, plant line, or seed is monoallelic across a majority of loci in the genome. In some embodiments, the true homozygous octoploid strawberry plant, plant part, plant line, or seed is monoallelic at more than 50%, more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.9%, more than 99.99%, or 100% of the loci in the genome.

[0169] In some embodiments, provided herein is a population of true homozygous octoploid strawberry seed. In certain embodiments, at least 50% of the true homozygous octoploid strawberry seed are genetically uniform. In some variations, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% of the true homozygous octoploid strawberry seed are genetically uniform. Genetic uniformity may be determined by any means known in the art including, for example, DNA-hybridization based methods (e.g., SNP-chip, DNA sequencing methods (e.g., whole genome sequencing or genotyping by sequencing (GBS)), and the like.Methods of Breeding and Producing Uniform Octoploid Fi Hybrid Strawberry Seed

[0170] In one aspect, described herein are methods of breeding and producing a uniform octoploid Fi hybrid strawberry seed comprising crossing two true homozygous octoploid strawberry plants. In some embodiments, the methods comprise producing one or more true homozygous octoploid strawberry plants using the methods described herein. In certain embodiments, the methods comprise performing a plurality of crosses between pairs of true homozygous octoploid strawberry lines selected from a plurality of true homozygous octoploid strawberry lines, evaluating the traits of the Fl generation of plants, and selecting one or more of the pairs of true homozygous octoploid strawberry lines that, when crossed, resulted in desirable traits in the Fl generation. In certain embodiments, the method further comprises crossing the one or more selected pairs of true homozygous octoploid strawberry lines to produce the uniform octoploid Fl hybrid strawberry seed.

[0171] In some embodiments, the methods of breeding and producing a uniform octoploid Fi hybrid strawberry seed comprise crossing two true homozygous octoploid strawberry plants. In some variations, crossing two true homozygous octoploid strawberry plants comprises contacting pollen from a first true homozygous octoploid strawberry plant with the stigma of a pistil of a second true homozygous octoploid strawberry plant. In some embodiments, the uniform octoploid Fi hybrid strawberry seed is produced by crossing the two true homozygous octoploid strawberry plants and allowing seeds to form.

[0172] In some embodiments, the methods of breeding and producing uniform octoploid Fl hybrid strawberry seed comprise maintaining one or more true homozygous octoploid strawberry lines, one or more Fl hybrid strawberry lines, or a combination thereof. In some variations, the inbred and / or hybrid octoploid strawberry lines are maintained via vegetative propagation, selfing, apomixis, or any combination thereof. Additional methods of maintaining strawberry lines are well-known in the art. In some embodiments, the methods of breeding and producing uniform octoploid Fl hybrid strawberry seed comprise maintaining an inventory of true homozygous octoploid strawberry lines from which haplotypes may be selected for rapid deterministic combination of the haplotypes.

[0173] In further aspects, provided herein is a uniform Fl hybrid strawberry seed produced by the methods described herein. In some embodiments, provided herein is a population of uniform octoploid Fl hybrid strawberry seed. In certain embodiments, at least 50% of the uniform octoploid Fl hybrid strawberry seed are genetically uniform. In some variations, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.99% of the uniform octoploid Fl hybrid strawberry seed are genetically uniform.Obtaining Plant Lines

[0174] In some embodiments, the methods of breeding and producing uniform octoploid Fl hybrid strawberry seed comprise obtaining a set of octoploid strawberry lines. In preferred embodiments, the set of octoploid strawberry lines is genetically diverse and comprises a large and diverse pool of haplotypes. In some embodiments, the methods of breeding and producing uniform octoploid Fl hybrid strawberry seed comprises obtaining a set of lines of the same or related species of octoploid strawberry plant (e.g., Fragaria x ananassa and / orFragaria chiloensis). The set of strawberry lines may be obtained from any source and by any methods known in the art. In some embodiments, the set of octoploid strawberry lines is obtained from sources including, but not limited to, natural diversity, existing breeding programs, or any combination thereof.

[0175] In some embodiments, the set of octoploid strawberry lines is a genetically diverse founder population of plants. These plants may be collected from existent germplasm sources such as wild progenitor species and landraces. In some instances, these plants may have high genetic load and may not have undergone the narrowing of genetic variability attributable to the elite selection practices imposed on modern cultivated materials. As such, many possess traits promoting their fitness within non-agrarian environments in the case of wild progenitors, or have been adapted to vastly differing cultivation practices and agricultural environments in the case of landraces. In either instance, these founding octoploid strawberry lines may contain suites of both desirable and undesirable agronomic characteristics that may be recombined, selected, and complemented to develop a commercializable product.Heterotic Groups

[0176] In some embodiments, the methods of breeding and producing uniform octoploid Fl hybrid strawberry seed comprise organizing the set of octoploid strawberry lines obtained into a plurality of heterotic groups, wherein each heterotic group comprises a haplotype, and wherein the haplotypes are grouped based on observed or predicted heterotic performance when combined in the octoploid Fl hybrid strawberry plant. In some variations, the methods of breeding and producing uniform octoploid Fl hybrid strawberry seed comprise organizing the set of octoploid strawberry lines into three or more heterotic groups, four or more heterotic groups, five or more heterotic groups, six or more heterotic groups, seven or more heterotic groups, or eight or more heterotic groups. The obtained set of octoploid strawberry lines may be organized by assigning their membership into complementary heterotic groups based upon heterotic patterns observed from preliminary estimation or prediction of their combining ability for traits and environments of interest. In this context, the combining ability is an estimation of the value of an octoploid strawberry plant as a parent as inferred by progeny testing in an established factorial or hierarchical mating design.

[0177] In defining heterotic groups, the main goal is identifying subpopulations of the set of octoploid strawberry lines based on employment of a clustering procedure that maximizessome measure of interpopulation combining ability. Exhaustive evaluation of all possible parental combinations for the final octoploid Fl hybrid strawberry, as in the case of a factorial mating scheme, is infeasible for all but a trivial number of potential parents (i.e. even a partial diallel ignoring reciprocal crosses scales at (n+3)! / (4!(n-l)!) crosses per n parents). Yet, hierarchical mating schemes necessitate an understanding and judgment of which set of “testers” or analogous constructs should serve as a relevant and efficient basis or frame of reference for inferring combining ability. The sample of testers selected invariably biases perceptions of existing heterotic patterns. Furthermore, the relative importances of traits and environments of interest used to infer these combining abilities are dynamic and depend upon market trends. As such, predictive modeling is essential and the process of assigning and refining heterotic groups and testers to represent them is one of iterative improvement and refinement throughout repeated cycles of the breeding process. Nonetheless, once preliminary heterotic group membership is assigned, interpopulation improvement of the true homozygous octoploid strawberry lines and development of hybrid octoploid strawberry plants may proceed.Evaluating Characteristics of Octoploid Fl Hybrid Strawberry Plants

[0178] In some embodiments, the methods of breeding and producing uniform octoploid Fl hybrid strawberry seed comprise evaluating one or more characteristics of an octoploid Fl hybrid plant grown from the uniform octoploid Fl hybrid strawberry seed. Methods for evaluating strawberry plant characteristics are numerous and well-known in the art. The one or more characteristics evaluated may include, but are not limited to, plant size, plant vigor, fruit yield, fruit color, fruit flavor, fruit sweetness, fruit aroma, abiotic stress resistance, disease resistance, pest resistance, and the like. The strawberry plants to be evaluated may then be grown under different geographical, climatic, and soil conditions, and further selections can be made during, and at the end of, the growing season. Promising advanced breeding strawberry lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for three years at least. The best strawberry lines are candidates for new commercial strawberry cultivars. These processes, which lead to the final step of marketing and distribution, usually take from five to ten years from the time the first cross or selection is made.

[0179] In some embodiments, the method of breeding a uniform octoploid Fl hybrid strawberry seed comprises repeating the steps of the method using the one or morecharacteristics of one or more octoploid Fl hybrid strawberry plants grown from the uniform octoploid Fl hybrid strawberry seed evaluated to guide the breeding of candidate octoploid strawberry lines, the selecting of candidate octoploid strawberry lines, or both. In some variations, the method comprises repeating the steps of the method two, three, four, five, six, seven, eight, nine, or ten times or more. In additional variations, the repeating of the steps of the method iteratively informs a genome prediction model for improved prediction of heterotic performance. In certain variations, the improved prediction of heterotic performance allows for rapid combination of haplotypes with strong heterotic performance and acceleration of breeding programs.Genetically Modified Plants, Plant Parts, Plant Cells, and Processed Plant Products

[0180] In yet another aspect, provided herein are genetically modified plants, plant parts, and plant cells grown from haploid-inducing strawberry plants, true homozygous octoploid strawberry seed, and uniform octoploid Fl strawberry seed described herein. Also provided herein are processed plant products derived from the genetically modified plants, plant parts, or plant cells provided herein. In some embodiments, the genetically modified plant parts, genetically modified plant cells, and processed plant products provided herein are non- regenerable.

[0181] In some embodiments, genetically modified strawberry plants and genetically modified strawberry plant parts are provided herein. The genetically modified strawberry plants and strawberry plant parts may be grown from true homozygous octoploid strawberry seed or uniform octoploid Fl strawberry seed described herein. Alternatively, the genetically modified strawberry plants and strawberry plant parts may be regenerated from a genetically modified cell of a haploid-inducing strawberry plant, a true homozygous octoploid strawberry plant, or a uniform octoploid Fl strawberry plant described herein. In certain embodiments, the genetically modified strawberry plants and strawberry plant parts may be regenerated from a genetically modified cell of a haploid-inducing strawberry plant, wherein the genetically modified cell comprises a genetic modification resulting in decreased expression of one or more DMP genes described herein. Genetically modified strawberry plants can be obtained from a genetically modified strawberry seed. Genetically modified strawberry plant parts can be obtained by cutting, snapping, grinding or otherwise disassociating the part from the strawberry plant. The strawberry plant part may be any strawberry plant part known in the art, including, but not limited to, a flower, a pistil, a leaf, a stem, a petiole, a cutting, a tissue,a seed coat, an ovule, pollen, a sperm cell, a root, a fruit (e.g., an aggregate fruit), a cotyledon, a hypocotyl, a protoplast, an embryo, an anther, a seed, an achene, a stolon (also known as a runner) or any portion thereof. In certain embodiments, a genetically modified strawberry plant part provided herein is a non-regenerable portion of a genetically modified strawberry plant part. As used in this context, a “non-regenerable” portion of a genetically modified strawberry plant part refers to a portion that cannot be induced to form a whole strawberry plant or that cannot be induced to form a whole strawberry plant (e.g., through in vitro culture) that is capable of sexual and / or asexual reproduction. A non-regenerable portion of a genetically modified strawberry plant part may be a portion of a flower, a pistil, a leaf, a stem, a petiole, a cutting, a tissue, a seed coat, an ovule, pollen, a sperm cell, a root, a fruit (e.g., an aggregate fruit), a cotyledon, a hypocotyl, a protoplast, an embryo, an anther, an achene, a stolon (also known as a runner) or any portion thereof.

[0182] In some embodiments, a non-regenerable or non-propagating strawberry plant cell is provided herein. As used in this context, a “non-regenerable strawberry plant cell” is a cell from a strawberry plant which cannot be regenerated into a whole strawberry plant that is capable of sexual and / or asexual reproduction through in vitro culture. The non-regenerable strawberry plant cell may be in a strawberry plant or strawberry plant part described herein. The non-regenerable strawberry plant cell may be a cell in a stolon, an achene, the hull or pericarp of said achene, a seed, or in the seedcoat of said seed. Mature strawberry plant organs, including a mature leaf, a mature stem, a mature root, or a mature stolon contain at least one non-regenerable cell. In certain embodiments, the non-regenerable strawberry plant cell is a somatic cell.

[0183] Also provided herein is a cell culture or tissue culture of non-regenerable or regenerable cells or tissue of a genetically modified strawberry plant or genetically modified strawberry plant part described herein, wherein the non-regenerable or regenerable cells one or more genetic modifications resulting in decreased expression of one or more DMP genes described herein. Preferably, the regenerable cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, pistils, ovules, stems, petioles, roots, root tips, stolons (also known as runners), fruits (e.g., aggregate fruits), seeds, achenes, flowers, cotyledons, and / or hypocotyls of a genetically modified plant or a genetically modified plant part grown from a haploid-inducing strawberry plant embryo or seed, a true homozygous octoploid strawberry seed, or a uniform octoploid Fl strawberry seed described herein.

[0184] In some embodiments, provided herein is a processed strawberry plant product derived from a genetically modified strawberry plant, plant part, or plant cell described herein. In certain embodiments, the processed plant product contains sufficient nucleic acid (e.g., DNA or RNA) and / or protein material from the genetically modified strawberry plant, plant part, or plant cell to detect nucleic acid and / or protein sequences corresponding to the haplotypes of one or two true homozygous octoploid strawberry plants described herein, the haplotypes of a uniform octoploid Fl strawberry seed described herein, or both. In some embodiments, the processed strawberry plant product is non-regenerable, z.e., cannot be induced to form a whole strawberry plant or that cannot be induced to form a whole strawberry plant that is capable of sexual and / or asexual reproduction.

[0185] A processed strawberry plant product may be a seed, an achene, a fruit (e.g., an aggregate fruit), a root, a vegetable, or any plant part described herein, and may be blended as a commodity or other product which moves through commerce and is derived from a genetically modified strawberry plant or a genetically modified plant part. In some embodiments, the commodity or other product can be tracked through commerce by detecting nucleic acid and / or protein sequences of the genetically modified plant or plant part from which they were obtained. In certain embodiments, the processed strawberry plant product comprises a detectable amount of nucleotide and / or protein sequences corresponding to the haplotypes of one or two true homozygous octoploid strawberry plants described herein, the haplotypes of a uniform octoploid Fl strawberry seed described herein, or both. In certain embodiments, the commodity or other product is produced in or maintained in the genetically modified strawberry plant or plant part from which the commodity or other product has been obtained. Such commodities or other products of commerce include, but are not limited to, plant parts, fruit, biomass, oil, meal, food starch, syrup, sugar, animal feed, flour, flakes, bran, lint, hull, processed seed, seed, seedless fruit, puree, juice, juice concentrate, pulp, pomace, preserve, or sauce. The processed plant product may be a food product that is processed by any means known in the art, e.g., canned, steamed, boiled, fried, dried, blanched, juiced, pureed, and / or frozen etc.

[0186] In some embodiments, provided herein is a genetically modified strawberry plant, strawberry plant part, or strawberry plant cell comprising one or more genetic modifications resulting in decreased expression of one or more DMP proteins described herein. In some embodiments, genetically modified strawberry plant, strawberry plant part, or strawberryplant cell comprises one or more genetic modifications of one or more DMP genes. In some embodiments, genetically modified strawberry plant, strawberry plant part, or strawberry plant cell comprises one or more genetic modifications resulting in decreased expression of one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to an amino acid sequence selected from the list consisting of SEQ ID NOs: 1-15, or fragments thereof. In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell comprises one or more genetic modifications of one or more DMP genes comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26, or a fragment thereof. In certain embodiments, the one or more genetic modifications comprise a modification of an enhancer of one or more of the DMP genes, a modification of a promoter of one or more of the DMP genes, a modification of a coding region of one or more of the DMP genes, a modification of an intron of one or more of the DMP genes, a modification of methylation status of one or more of the DMP genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the DMP genes, expression of an RNA interference construct that targets an mRNA of one or more of the DMP genes, or any combination thereof. In some embodiments, genetically modified strawberry plant, strawberry plant part, or strawberry plant cell has decreased expression of one or more DMP genes described herein relative to a strawberry plant (e.g., a control strawberry plant, e.g., a strawberry plant of the same species) lacking the one or more genetic modifications. In certain embodiments, genetically modified strawberry plant, strawberry plant part, or strawberry plant cell lacks detectable expression of one or more DMP genes described herein relative to a strawberry plant (e.g., a control strawberry plant, e.g., a strawberry plant of the same species) lacking the one or more genetic modifications. In certain embodiments, genetically modified strawberry plant, strawberry plant part, or strawberry plant cell lacks detectable expression of any DMP genes. In certain embodiments, genetically modified strawberry plant, strawberry plant part, or strawberry plant cell further comprises one or more naturally-occurring inactive alleles of one or more DMP genes.

[0187] In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of an FvDMPl gene. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of an FvDMPl protein. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and comprises one or more genetic modifications resulting in decreased expression of a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FvDMPl protein of SEQ ID NO. 1, or a fragment thereof.

[0188] In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and comprises one or more genetic modifications of an FvDMPl gene or gene product. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and comprises one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FvDMPl gene nucleotide sequence of SEQ ID NO. 16, or a fragment thereof. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and comprises genetic modifications in both alleles of an FvDMPl gene. In some variations, the one or more genetic modifications comprise a modification of an enhancer of FvDMPl, a modification of a promoter of FvDMPl, a modification of a coding region of FvDMPl, modification of an intron of FvDMPl, or any combination thereof relative to a control Fragaria vesca plant (e.g., a Fragaria vesca plant lacking one or more of the genetic modifications). In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberryplant cell is a plant or from a plant of the species Fragaria vesca and has decreased expression of FvDMPl protein relative to a control Fragaria vesca plant (e.g., a Fragaria vesca plant lacking one or more of the genetic modifications). In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria vesca and lacks detectable expression of FvDMPl protein.

[0189] In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of one or more DMP genes. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3 gene, an Fa-DMPl-4 gene, or any combination thereof. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 gene, an FaDMPl-2 gene, an FaDMPl-3 gene, and an Fa-DMPl-4 gene.

[0190] In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, an FaDMPl-4 protein, or any combination thereof. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, and an Fa-DMPl-4 protein. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of one or more DMP proteins having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or99% sequence similarity to an amino acid sequence selected from the list consisting of SEQ ID NOs: 2-5, or fragments thereof. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications resulting in decreased expression of 1) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO:2; 2) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 3; 3) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 4; and / or 4) a DMP protein having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 5.

[0191] In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications of an FaDMPl-1 gene or gene product, an FaDMPl-2 gene or gene product, an FaDMPl-3 gene or gene product, an FaDMPl-4 gene or gene product, or any combination thereof. In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications of an FaDMPl-1 gene or gene product, an FaDMPl-2 gene or gene product, an FaDMPl-3 gene or gene product, and an FaDMPl-4 gene or gene product. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 17-20, or a fragment thereof. In certain embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and comprises: 1) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 17, or a fragment thereof; 2) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 18, or a fragment thereof; 3) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 19, or a fragment thereof; and / or 4) one or more genetic modifications in a DMP gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 20, or a fragment thereof. In some variations, the one or more genetic modifications comprise a modification of an enhancer of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), a modification of a promoter of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), a modification of a coding region of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), modification of an intron of an FaDMPl gene (e.g., an FaDMPl-1 gene, an FaDMPl-1 gene, an FaDMPl-1 gene, and / or an FaDMPl-1 gene), or any combination thereof relative to a control Fragaria x ananassa plant (e.g., a wild-type or unmodified Fragaria x ananassa plant lacking one or more or all of the genetic modifications).

[0192] In some embodiments, the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and has decreased expression of an FaDMPl-1 protein, an FaDMPl-2 protein, an FaDMPl-3 protein, an FaDMPl -4 protein, or any combination (e.g., two, three, or all four) thereof relative to a control Fragaria x ananassa plant (e.g., a Fragaria ananassa plant lacking oneor more or all of the genetic modifications). In certain embodiments the genetically modified strawberry plant, strawberry plant part, or strawberry plant cell is a plant or from a plant of the species Fragaria x ananassa and lacks detectable expression of FaDMPl-1 protein, FaDMPl-2 protein, FaDMPl-3 protein, FaDMPl-4 protein, or any combination (e.g., two, three, or all four) thereof.

[0193] In some embodiments, provided herein is a processed strawberry plant product derived from any of the foregoing embodiments of genetically modified strawberry plants, plant parts, or plant cells, wherein the processed strawberry plant product comprises a detectable amount of nucleotide and / or protein sequences corresponding to the haplotypes of one or two true homozygous octoploid strawberry plants described herein, the haplotypes of a uniform octoploid Fl strawberry seed described herein, or both of the genetically modified plant, plant part, or plant cell. In some embodiments, the product is selected from the group consisting of plant biomass, oil, meal, food starch, syrup, animal feed, flour, flakes, bran, lint, hulls, processed seed, puree, juice, juice concentrate, pulp, pomace, preserve, or sauce. In certain embodiments, the processed plant product is non-regenerable.EXAMPLES

[0194] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.Example 1: General MethodsPlants

[0195] An octoploid Fragaria x ananassa (PED504) and a diploid Fragaria vesca (PED503) were initiated in vitro by meristem extraction using hormone free 'A MS media (Murashige & Skoog (1962) “A revised medium for rapid growth and bio assays with tobacco tissue cultures” Physiologia Plantarum, 15(3): 473-497). The hormone free ’A MS media was supplemented with 30 g / L sucrose and plants were then grown under 16 hour day / 8 hour night lighting regime with cool white fluorescent lighting. After establishment, plants were micropropagated on the same medium.RNPs

[0196] RNPs were prepared by standard methods known in the art. In short, 2 pL of NEB buffer 2.1 (lOx stock) was placed into a 1.5 mL microcentrifuge tube with 10-40 pg of crRNA (CRISPR RNA) and an equal mass of Cas nuclease. The final volume was adjusted to 20 pL using nuclease free water.Protoplasts

[0197] Approximately 0.3 g of leaves from 3 to 4 week old explants were removed under aseptic conditions and sliced into thin sections 0.5mm to 1mm in width. Protoplasts were prepared from the sliced leaf tissue essentially as described in Barcelo et al. (Barcelo et al. (2019) “Isolation and culture of strawberry protoplasts and field evaluation of regenerated plants” Scientia Horticulturae 256: 108552). Protoplast cells were quantified using a Biirker hemocytometer before transfection.Transfection

[0198] Protoplasts were combined with freshly prepared RNPs and transformed using standard PEG transfection methods, essentially as described in Y. Gou et al. (Gou et al.(2020) “Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vescaf Plant Cell, Tissue, and Organ Culture 141(1): 41-53). In short, plasmid DNA were added to 100 pL of isolated protoplast solution (approximately 2 x 105protoplasts per mL) in 2 mL microfuge tubes. An equal volume of PEG solution (40% PEG 4000 (wt / vol), 0.2 M mannitol, and 100 mM CaCh) was immediately mixed with the protoplasts by shaking gently and thoroughly. The amount of plasmid DNA was determined experimentally between 5-80 pg. Transfection mixtures were incubated at room temperature for 2-40 minutes, as determined experimentally, and then diluted with 440 pL W5 solution (2 mM MES (pH 5.7), 154 mM NaCl, 5 mM glucose, 125 mM CaCh, and 5 mM KC1) at room temperature, and the transfection process terminated by gentle shaking. Protoplasts were centrifuged at 100 x g for 1 minute at room temperature. Finally, the protoplasts were gently resuspended with 200 pL WI solution (4 mM MES (pH 5.7), 0.5 M mannitol, and 20 mM KC1) and incubated in the dark for 20-25 hours at room temperature.Plant regeneration

[0199] Protoplasts were plated and maintained under sterile conditions until callus formation. Callus with confirmed edits was regenerated essentially as described in Barcelo etal. (Barcelo et al. (2019) “Isolation and culture of strawberry protoplasts and field evaluation of regenerated plants” Scientia Horticulturae 256: 108552).Example 2: Identification of target sites and guide RNA designIdentification DMP orthologs

[0200] Candidate DMP orthologs from strawberry were identified by comparing DMP protein sequences from Zea mays (ZmDMP, RefSeq ID: NP 001144876) and Arabidopsis thaliana (AtDMP8 and AtDMP9, RefSeq IDs: NP 001031010 and NP 198781) using Protein Blast, tblastn, and Clustal Omega workflows.

[0201] FvDMPl (RefSeq ID: XP 011467959) from diploid strawberry Fragaria vesca and four FaDMPl homeologs (Gene IDs: FxaC_21g08040, FxaC_22g06170, FxaC_22g06200 and FxaC_24g57400) from octoploid strawberry Fragaria x ananassa (Gene IDs based on Fragaria x ananassa Camarosa Genome vl ,0.a2: www[dot]rosaceae[dot]org / Analysis / 9642085) were identified as putative orthologs of ZmDMP (RefSeq ID: NP 001144876) and AtDMP8 and AtDMP9 (RefSeq IDs: NP_001031010 and NP_198781).

[0202] The four homeologs in octoploid strawberry were located on three subgenomes with both FxaC_22g06170 and FxaC_22g06200 on subgenome Fvb6-3. FxaC_22g06200 was likely a pseudogene as there was a premature stop codon making the predicted protein product truncated. Configuration of DMP loci in diploid and octoploid Fragaria is shown in FIG. 2

[0203] Additionally, we identified DMP orthologs from Potentilla micrantha and three closely related Fragaria species (Fragaria chiloensis. Fragaria iinumae and Fragaria viridis). The protein sequence alignment and a phylogenetic tree of DMP orthologs from Fragaria species, Zea mays, Arabidopsis thaliana and Solanum lycopercicum are shown in FIGS. 3A-3I and FIG. 4, respectively. In FIG. 4, bootstrap values are displayed at branch points, indicating how many times the same branching is observed when regenerating the phylogenetic tree 100 times, with a resampled sequence set each time. The protein alignment was generated using the MUSCLE Alignment tool (Muscle 5.1) in Geneious Prime (2023.0.3) with the “algorithm” set to “PPP”.Guide RNA design

[0204] Protospacer sequences targeting FvDMPl (RefSeq ID: XP 011467959) or FaDMPl homeologs (gene IDs: FxaC_21g08040, FxaC_22g06170, FxaC_22g06200 and FxaC_24g57400) in Table 2 were selected. Guide RNAs were synthesized based on protospacer sequence and scaffold sequence and were screened in vivo (Fig. 5).Table 2: Protospacer sequencesEditing in protoplasts

[0205] RNPs with the synthesized guide RNAs and a Cas nuclease were prepared as in Example 1. Protoplasts were generated, transfected with the RNPs, and regenerated as described in Example 1.Sequence-based confirmation of editing

[0206] Primers were designed to amplify each DMP candidate region based on the reference genome, and one or more long range direct PCRs were performed using crude lysate from transfected protoplasts. Alternatively, DNA was extracted from callus, leaf or other plant material. PCR products were pooled by transfection sample and a seqWell library preparation performed to generate an Illumina library. Samples were loaded onto an Illumina iSeq and sequenced with a Paired End 150nt sequencing kit. Sequences were analyzed by aligning fastq files to reference sequences, and mutations adjacent to target PAM sites for each targeted DMP gene were tabulated relative to a control. Editing efficiency was calculated based on the frequency of observed mutations (FIG. 5) and used to calculate howmany plants should be screened to identify the DMP gene knockouts required to confer a haploid induction phenotype. Protospacer PRS265 and PRS271 were selected based on the observed editing efficiencies in protoplasts (FIG. 5).Example 3: Haploid inducer system for strawberryCreation of haploid inducer lines

[0207] An octoploid Fragaria x ananassa (PED504) and a diploid Fragaria vesca (PED503) are edited with guides PRS265 and PRS271, selected based on their high editing efficiency as demonstrated in Example 2. These guide RNAs are specific to FvDMPl and all homeologs of FaDMPl as illustrated in FIG. 6.

[0208] A Fragaria x ananassa plant designated PED504-Fa-Inducer bearing 8 nonfunctional alleles of dmp and a Fragaria vesca plant designated PED503-Fv-Inducer bearing two nonfunctional alleles of dmp are selected from a population of plants edited with PRS265 and PRS271.

[0209] After in vitro propagation, 5 plants each of PED504-Fa-Inducer and PED503-Fv- Inducer are rooted and grown to maturity under standard greenhouse conditions (for example, heating below 55°F and cooling at 66°F). Pollen is harvested from mature flowers of PED504-Fa-Inducer and PED503-Fv-Inducer and is used to pollinate emasculated flowers of 10 unmodified octoploid Fragaria x ananassa varieties. Upon maturation of the berries all seeds (achenes) of the unmodified plants are collected, surface disinfected and placed into hormone free U MS basal salts for in vitro germination.

[0210] Germinated seeds are grown in vitro until the second true leaf forms, at which point a leaf is harvested and flow cytometry is performed essentially as described in Galbraith et al., (Galbraith et al. (1983) “Rapid flow cytometric analysis of the cell cycle in intact plant tissues” Science 220(4601): 1049-1051). In short, intact nuclei are extracted into Galbraith buffer, then filtered and stained with propidium iodide. DNA content of nuclei is determined by applying the samples to a BD Accuri™ C6 Flow Cytometer. Gating is performed and genomic DNA content is calculated by comparing the peak area for the sample to the known position of the internal control. Tetrahaploids are identified based on relative DNA content of nuclei between samples and internal controls, i.e. samples with approximately half the DNA content of unmodified octoploid Fragaria x ananassa controls are classified as tetrahaploid.

[0211] Tetrahaploid plants resulting from crosses with PED503-Fv-Inducer are further distinguished by application of molecular markers for Fragaria vesca specific genes due to the similar DNA content of true hybrids (pentapioids) versus induced tetrahaploids.

[0212] The tetrahaploid induction rate for PED 504 -Fa-Inducer varies from 0.5% to 7% among the ten populations of plants recovered, as determined by the number of haploid embryos observed divided by the total number of embryos that develop. The tetrahaploid induction rate for PED503-Fv-Inducer varies from 0.5% to 8% among the ten populations of plants recovered.Haploid doubling

[0213] Tetrahaploids are propagated in vitro on ’A MS medium with 0.5 mg / L 6- benzylaminopurine (BAP) and are subsequently doubled to produce fully homozygous octoploid plants with treatment of 0.1 to 0.3% colchicine solution in vitro for 4 to 24 hours. Successful doubling of tetrahaploids is initially identified by plant morphology (larger and thicker leaves) and is then confirmed by flow cytometry using the same methods as above. Resulting fully homozygous octoploid plants have approximately twice the DNA content of the unmodified tetrahaploid plants and are similar in DNA content to unmodified octoploid parents.Production of uniform Fl hybrid octoploid strawberry

[0214] Fully homozygous octoploid plants are crossed with one another to produce uniform true seed populations of Fl hybrid octoploid Fragaria x ananassa. Crosses are performed to identify the best breeding parents for commercial production.Example 4: Pollination with DMP-Edited Pollen

[0215] After performing pollination with t / ziz -edited pollen as described herein, two octoploid individuals were analyzed by testing markers at six loci. Individual E-PED8913- 042 is the offspring of a selfed Fragaria x ananassa Camarosa plant with an edited DMP gene. Individual E-PED8912-033 is the offspring of a cross between a Fragaria x ananassa Quinault plant and a Fragaria vesca Alexandria plant with an edited DMP gene. Each marker was on a different chromosome. The results are shown in Table 3 below.Table 3: Marker Analysis, “hom” = homozygous; “het” = heterozygous

Claims

CLAIMSWhat is claimed is:

1. A haploid-inducing strawberry plant, or a plant part thereof, comprising one or more genetic modifications resulting in decreased expression of one or more DMP genes.

2. The haploid-inducing strawberry plant, or plant part thereof, of claim 1, wherein one or more of the DMP genes comprise a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15.

3. The haploid-inducing strawberry plant, or plant part thereof, of claim 1 or 2, wherein one or more of the DMP genes comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26.

4. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1-3, wherein the one or more genetic modifications comprise a modification of an enhancer of one or more of the DMP genes, a modification of a promoter of one or more of the DMP genes, a modification of a coding region of one or more of the DMP genes, a modification of an intron of one or more of the DMP genes, a modification of methylation status of one or more of the DMP genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the DMP genes, expression of an RNA interference construct that targets an mRNA of one or more of the DMP genes, or any combination thereof.

5. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1-3, wherein the one or more genetic modifications comprise a modification of an enhancer of one or more of the DMP genes, a modification of a promoter of one or more of the DMP genes, a modification of a coding region of one or more of the DMP genes, modification of an intron of one or more of the DMP genes, or any combination thereof relative to an unmodified strawberry plant of the same species or an unmodified control strawberry plant.

6. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1-5, wherein the haploid-inducing strawberry plant or plant part has decreased expression of DMPproteins relative to a strawberry plant of the same species lacking the one or more genetic modifications.

7. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1-6, wherein the haploid-inducing strawberry plant or plant part lacks detectable expression of DMP proteins.

8. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1-7, wherein the haploid-inducing strawberry plant is diploid, triploid, tetrapioid, pentapioid, hexapioid, septapioid, octoploid, or nonaploid, or has a ploidy of lOx, 1 lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x.

9. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1-8, wherein the haploid-inducing strawberry plant is diploid.

10. The haploid-inducing strawberry plant, or plant part thereof, of claim 9, wherein the haploid-inducing strawberry plant is a plant of the species Fragaria vesca, Fragaria iinumae. Fragaria nipponica, Fragaria viridis, Fragaria x bifera, Fragaria bucharica, Fragaria chinensis, Fragaria daltoniana, Fragaria emeiensis, Fragaria hayatae, Fragaria mandshurica, Fragaria nilgerrensis, Fragaria nubicola, or Fragaria pentaphylla11. The haploid-inducing strawberry plant, or plant part thereof, of claim 9, wherein the haploid-inducing strawberry plant is a plant of the species Fragaria vesca.

12. The haploid-inducing strawberry plant, or plant part thereof, of claim 11, wherein the haploid-inducing strawberry plant is a Fragaria vesca plant of the subspecies Fragaria vesca ssp. vesca, Fragaria vesca ssp. americana, o Fragaria vesca ssp. bracteate.

13. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 9-12, wherein the one or more DMP genes comprise a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity the amino acid sequence of SEQ ID NO: 1.

14. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 9-13, wherein the one or more DMP genes comprise a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 16.

15. The haploid-inducing strawberry plant, or plant part thereof, of claim any one of claims 11-14, wherein the haploid-inducing strawberry plant is a plant of the species Fragaria vesca and wherein the one or more DMP genes comprise FvDMPl.

16. The haploid-inducing strawberry plant, or plant part thereof, of claim 15, wherein the one or more genetic modifications comprise a modification of an enhancer of FvDMPl, a modification of a promoter of FvDMPl, a modification of a coding region of FvDMPl, modification of an intron of FvDMPl, or any combination thereof relative to a wild-type Fragaria vesca plant.

17. The haploid-inducing strawberry plant, or plant part thereof, of claim any one of claims 1-6, wherein the haploid-inducing strawberry plant is octoploid.

18. The haploid-inducing strawberry plant, or plant part thereof, of claim 17, wherein the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa, Fragaria chiloensis, Fragaria virginiana. or Fragaria iturupensis.

19. The haploid-inducing strawberry plant, or plant part thereof, of claim 18, wherein the haploid-inducing strawberry plant is a Fragaria chiloensis plant of the subspecies Fragaria chiloensis subsp. chiloensis, Fragaria chiloensis subsp. hicida. Fragaria chiloensis subsp. pacifica, o Fragaria chiloensis subsp. sandwicensis.

20. The haploid-inducing strawberry plant, or plant part thereof, of claim 19, wherein the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa.

21. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 17-20, wherein one or more of the DMP genes comprises a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5.

22. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 17-21, wherein one or more of the DMP genes comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs:

23. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 17-22, wherein the one or more DMP genes comprise: a. a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 2; b. a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 3; c. a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 4; and / or d. a polynucleotide sequence encoding an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 5.

24. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 17-23, wherein the one or more DMP genes comprise: a. a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 17; b. a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 18; c. a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 19; and / or d. a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO: 20.

25. The haploid-inducing strawberry plant, or plant part thereof, of claim 17-24, wherein the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa and the one or more DMP genes comprise one, two, three or all four of FaDMPl-1, FaDMP-l-3a, FaDMPl-3b, and FaDMPl-4.

26. The haploid-inducing strawberry plant, or plant part thereof, of claim 25, wherein the one or more DMP genes comprise FaDMPl-1, FaDMP-l-3a, FaDMPl-3b, and FaDMPl-4.

27. The haploid-inducing strawberry plant, or plant part thereof, of claim 25 or 26, wherein the one or more genetic modifications comprise: a. a modification of an enhancer of FaDMPl-1, a modification of a promoter of FaDMPl-1, a modification of a coding region of FaDMPl-1, a modification of an intron of FaDMPl-1, or any combination thereof relative to an unmodified Fragaria x ananassa plant; b. a modification of an enhancer of FaDMPl-2, a modification of a promoter of FaDMPl-2, a modification of a coding region of FaDMPl-2, a modification of an intron of FaDMPl-2, or any combination thereof relative to an unmodified Fragaria x ananassa plant; c. a modification of an enhancer of FaDMPl-3, a modification of a promoter of FaDMPl-3, a modification of a coding region of FaDMPl-3, a modification of an intron of FaDMPl-3, or any combination thereof relative to an unmodified Fragaria x ananassa plant; and / or d. a modification of an enhancer of FaDMPl-4, a modification of a promoter of FaDMPl-4, a modification of a coding region of FaDMPl-4, a modification of an intron of FaDMPl-4, or any combination thereof relative to an unmodified Fragaria x ananassa plant.

28. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1- 27, wherein the plant part is selected from the group consisting of pollen, an anther, a seed, an achene, a leaf, a flower, a fruit, and a stolon.

29. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1- 27, wherein the plant part is pollen.

30. The haploid-inducing strawberry plant, or plant part thereof, of any one of claims 1- 27, wherein the plant part is a seed.

31. A method of producing the haploid-inducing strawberry plant of any one of claims 1- 30.

32. The method claim 31, wherein the decreased expression of the one or more DMP genes is achieved by gene disruption, gene knockout, gene knockdown, gene silencing, RNA interference, induction of methylation, or any combination thereof.

33. The method of claim 31 or 32, comprising introducing one or more of the genetic modifications by mutagenesis, gene editing, transgenesis, or a combination thereof.

34. The method of claim 33, comprising introducing one or more of the genetic modifications by gene editing using a site-directed nuclease.

35. The method of claim 34, wherein the site-directed nuclease is a CRISPR-associated (Cas) nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a megaTAL.

36. The method of any one of claims 31-35, comprising contacting a plurality of cells of a parent strawberry plant with one or more expression vectors together comprising an expression cassette for a Cas nuclease and an expression cassette for an RNA molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of one or more of the DMP genes.

37. The method of any one of claims 31-36, comprising contacting a plurality of cells of a parent strawberry plant with one or more Cas nucleases, wherein each Cas nuclease is complexed with a RNA molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of one or more of the DMP genes.

38. The method of claim 36 or 37, wherein the RNA molecule is a crRNA, a gRNA, or a pegRNA.

39. The method any one of claims 36-38, wherein the plurality of cells is a plurality of protoplasts.

40. The method of any one of claims 36-39, wherein the method further comprises, subsequent to the contacting step, allowing the plurality of cells of the parent strawberry plant to form calli, plants, or a combination thereof, and identifying one or more calli or plants having the genetic modifications resulting in decreased expression of one or more DMP genes.

41. A method of producing true homozygous octoploid strawberry seed, the method comprising (a) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with pollen of the haploid-inducing strawberry plant of any one of claims 1-30, and (b) producing a doubled tetrahaploid cell from the tetrahaploid reduced egg cell.

42. The method of claim 41, wherein step (a) comprises contacting pollen of the haploid- inducing strawberry plant with the stigma of a pistil of the donor octoploid strawberry plant comprising the reduced egg cell and allowing formation of a pollen tube and migration of the pollen to the reduced egg cell, thereby contacting the reduced egg cell with the pollen.

43. The method of claim 42, further comprising allowing the donor octoploid strawberry plant to form a plurality of seeds.

44. The method of claim 43, further comprising selecting one or more tetrahaploid seeds by 1) determining the ploidy of the embryo and endosperm of one or more seeds of the plurality of seeds, and 2) selecting one or more seeds having tetrapioid embryo and an endosperm having a ploidy of greater than 8x.

45. The method of claim 43 or 44, further comprising collecting the plurality of seeds and allowing them to germinate and form a plurality of seedlings.

46. The method of claim 43 or 44, further comprising collecting a plurality of embryos from the plurality of seeds, contacting the plurality of embryos with a nutrient medium suitable for inducing seedling growth, and allowing them to form a plurality of seedlings.

47. The method of claim 45 or 46, further comprising selecting one or more tetrahaploid seedlings by 1) determining the ploidy of the cells of one or more seedlings of the plurality of seedlings, and 2) selecting one or more tetrahaploid seedlings from the plurality of seedling.

48. The method of claim 47, wherein determining the ploidy of the cells of the one or more seedlings comprises obtaining a tissue sample of one or more seedlings and determining the ploidy of the cells of the tissue sample.

49. The method of any one of claims 41-48, wherein step (b) comprises subjecting the selected tetrahaploid cell, tetrahaploid seed, or tetrahaploid seedling to a tetrahaploid doubling treatment.

50. The method of claim 49, wherein the tetrahaploid doubling treatment comprises contacting the tetrahaploid cell, tetrahaploid seed, or tetrahaploid seedling with an antimicrotubule agent to form a doubled tetrahaploid cell, a doubled tetrahaploid seed, or a doubled tetrahaploid seedling.

51. The method of claim 50, wherein the anti -microtubule agent is an anti-mitotic herbicide.

52. The method of claim 50, wherein the anti -microtubule agent comprises colchicine, oryzalin, oxide, trifluralin, or any combination thereof.

53. The method of any one of claims 50-52, wherein the doubled tetrahaploid cell or the doubled tetrahaploid seed is allowed to grow into a doubled tetrahaploid seedling.

54. The method of any one of claims 50-53, wherein the doubled tetrahaploid seedling is allowed to form seed, thus producing the true homozygous octoploid strawberry seed.

55. The method of any one of claims 41-54, wherein the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa, Fragaria chiloensis, Fragaria virginiana. or Fragaria iliirupensis. or a hybrid of any combination thereof.

56. The method of any one of claims 55, wherein the donor octoploid strawberry plant is Fragaria chiloensis plant of the subspecies Fragaria chiloensis subsp. chiloensis, Fragaria chiloensis subsp. hicida. Fragaria chiloensis subsp. pacifica, o Fragaria chiloensis subsp. sandwicensis .

57. The method of any one of claims 56, wherein the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa.

58. The method of any one of claims 57, wherein the donor octoploid strawberry plant is a Fragaria x ananassa plant of the variety Camarosa.

59. The method of any one of claims 41-58, wherein the method further comprises introducing one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene into the true homozygous octoploid strawberry plant or seed.

60. A method of producing a runnerless true homozygous octoploid strawberry plant comprising the steps of the method of any one of claims 41-58 and further comprising introducing one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene into the true homozygous octoploid strawberry plant, wherein the runnerless true homozygous octoploid strawberry plant does not produce runners.

61. A true homozygous octoploid strawberry seed produced according to the method of any one of claims 41-60.

62. The true homozygous octoploid strawberry seed of claim 61, wherein the true homozygous octoploid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene.

63. A true homozygous octoploid strawberry plant or a plant part thereof produced according to the method of any one of claims 41-60.

64. The true homozygous octoploid strawberry plant of claim 63, wherein the true homozygous octoploid strawberry plant comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene.

65. The true homozygous octoploid strawberry plant of claim 64, wherein the true homozygous octoploid strawberry plant does not produce runners.

66. The true homozygous octoploid strawberry plant, or plant part thereof, of any one of claims 63-65, wherein the plant part is selected from the group consisting of pollen, an anther, a seed, an achene, a leaf, a flower, a fruit, and a stolon.

67. A method of producing uniform octoploid Fi hybrid strawberry seed comprising crossing two true homozygous octoploid strawberry plants of any one of claims 63-65.

68. A uniform octoploid Fi hybrid strawberry seed produced according to the method of any one of claims 63-65.

69. The uniform octoploid Fi hybrid strawberry seed of claim 68, wherein the uniform octoploid Fi hybrid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene.

70. A uniform octoploid Fi hybrid strawberry plant or a plant part thereof produced according to the method of any one of claims 63-65.

71. The uniform octoploid Fi hybrid strawberry plant of claim 70, wherein the uniform octoploid Fi hybrid strawberry plant comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (GA20ox) gene.

72. The uniform octoploid Fi hybrid strawberry plant of claim 71, wherein the uniform octoploid Fi hybrid strawberry plant does not produce runners.

73. The uniform octoploid Fi hybrid strawberry plant, or plant part thereof, of any one of claims 70-72, wherein the plant part is selected from the group consisting of pollen, an anther, a seed, an achene, a leaf, a flower, a fruit, and a stolon.

74. An expression vector or isolated DNA molecule for making the haploid-inducing strawberry plant, or a plant part thereof, of any one of claims 1-30.

75. The expression vector or isolated DNA molecule of claim 74, comprising a DNA sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide sequence encoding an amino acid sequence having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15.

76. The expression vector or isolated DNA molecule of claim 74 or 75, comprising a DNA sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutivenucleotides of a polynucleotide having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-2677. The expression vector or isolated DNA molecule of any one of claims 74-76, comprising a DNA sequence encoding a non-coding RNA.

78. The expression vector or isolated DNA molecule of claim 77, wherein the non-coding RNA comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide sequence encoding an amino acid sequence having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15.

79. The expression vector or isolated DNA molecule of claim 77 or 78, wherein the noncoding RNA comprises a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26.

80. The expression vector or isolated DNA molecule of any one of claims 74-79, wherein the non-coding RNA is a crRNA, a gRNA, a pegRNA, a siRNA, a miRNA, or a dsRNA.

81. The expression vector or isolated DNA molecule of any one of claims 74-80, comprising a DNA sequence encoding a site-directed nuclease.

82. The expression vector or isolated DNA molecule of claim 81 wherein the site-directed nuclease is a Cas nuclease, a TALEN, ZFN, or a mega-TAL.

83. The expression vector or isolated DNA molecule of claim 81 or 82, wherein the site- directed nuclease comprises an amino acid sequence that confers binding to a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide sequence encoding an amino acid sequence having at least96%, at least 97%, at least 95%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15.

84. The expression vector or isolated DNA molecule of any one of claims 81-83, wherein the site-directed nuclease comprises an amino acid sequence that confers binding to a polynucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity or complementarity to 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a polynucleotide having at least 96%, at least 97%, at least 95%, at least 99%, or 100% identity to an polynucleotide sequence selected from the group consisting of SEQ ID NOs: 16-26.

85. A bacterial cell comprising the expression vector or isolated DNA molecule of any one of claims 74-84.

86. The bacterial cell of claim 85, wherein the bacterial cell is an Agrobacterium cell.

87. A genetically modified plant, plant part, plant cell, or seed comprising the expression vector or isolated DNA molecule of any one of claims 74-84.

88. A kit comprising the expression vector or isolated DNA molecule of claim 74-84 or the bacterial cell of claim 85 or 86.

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