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 CENH3 (centromere-specific histone 3 variant) 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 CENH3 genes.

Description

197072001240 HAPLOID INDUCER STRAWBERRY LINES AND METHODS OF PRODUCING AND USING THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 657,605, filed on June 7, 2024, which is incorporated by reference herein in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (197072001240seqlisting.xml; Size: 184,457 bytes; and Date of Creation: June 6, 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 homozygous octoploid strawberry lines for producing true breeding F1 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 modern 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 F1 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. 1sf-6744554197072001240

[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 F1 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 F1 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 F1 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 within each of the four subgenomes simultaneously. 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) F1 hybrid strawberry seed. To our knowledge, the methods described herein provide the first such system enabled through use of a haploid inducer line in strawberry.

[0008] In some aspects, provided herein is a strawberry plant, such as a haploid-inducing strawberry plant, or a plant part thereof, comprising one or more genetic modifications resulting in decreased expression of one or more CENH3 genes. In some embodiments, one or more of the CENH3 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: 8-14 and 53-56. In some embodiments, one or more of the CENH3 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: 1-7 and 58-61. In some embodiments, the one or more genetic modifications comprise a modification of an enhancer of one or more of the CENH3 2sf-6744554197072001240 genes, a modification of a promoter of one or more of the CENH3 genes, a modification of a coding region of one or more of the CENH3 genes, a modification of an intron of one or more of the CENH3 genes, a modification of methylation status of one or more of the CENH3 genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the CENH3 genes, expression of an RNA interference construct that targets an mRNA of one or more of the CENH3 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 CENH3 genes, a modification of a promoter of one or more of the CENH3 genes, a modification of a coding region of one or more of the CENH3 genes, modification of an intron of one or more of the CENH3 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 one or more genetic modifications comprise a deletion, insertion, or one or more nucleotide changes in a coding region of one or more of the CENH3 genes. In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) or plant part has decreased expression of CENH3 proteins relative to a strawberry plant of the same species lacking the one or more genetic modifications. In certain embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) t or plant part lacks detectable expression of CENH3 proteins. In some embodiments, the strawberry plant (e.g., haploid- inducing strawberry plant) is diploid, triploid, tetraploid, pentaploid, hexaploid, septaploid, octoploid, or nonaploid, or has a ploidy of 10x, 11x, 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 strawberry plant (e.g., haploid-inducing strawberry plant) is diploid. In certain embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a member of the genus Fragaria. 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 daltoniana, Fragaria emeiensis, Fragaria hayatae, Fragaria iinumae, Fragaria mandshurica, Fragaria nilgerrensis, Fragaria nubicola, or Fragaria pentaphylla. In certain embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a plant of the species Fragaria vesca. 3sf-6744554197072001240

[0010] In certain embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a Fragaria vesca plant of the subspecies Fragaria vesca ssp. vesca, Fragaria vesca ssp. americana, or Fragaria vesca ssp. bracteata. In some embodiments the one or more Fragaria vesca CENH3 (herein referred to as “FvCENH3”) 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: 8. In some embodiments, the one or more FvCENH3 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: 1. In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a plant of the species Fragaria vesca and wherein the one or more CENH3 genes comprise FvCENH3. In certain embodiments, the one or more genetic modifications comprise a modification of an enhancer of FvCENH3, a modification of a promoter of FvCENH3, a modification of a coding region of FvCENH3, modification of an intron of FvCENH3, or any combination thereof relative to a wild-type Fragaria vesca plant.

[0011] In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is octoploid. In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a plant of the species Fragaria x ananassa, Fragaria chiloensis, Fragaria virginiana, or Fragaria iturupensis. In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is Fragaria chiloensis plant of the subspecies Fragaria chiloensis ssp. chiloensis, Fragaria chiloensis ssp. lucida, Fragaria chiloensis ssp. pacifica, or Fragaria chiloensis ssp. sandwicensis.

[0012] In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a plant of the species Fragaria x ananassa. In some embodiments, one or more of the Fragaria x ananassa CENH3 (herein referred to as “FaCENH3”) 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: 9-14 and 53-56. In some embodiments, one or more of the FaCENH3 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: 2-7 and 58-61. In certain embodiments, the one or more FaCENH3 genes comprise: a 4sf-6744554197072001240 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: 9 (Fragaria x ananassa CENH3-1a protein, hereinafter “FaCENH3-1a”); 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: 10 (Fragaria x ananassa CENH3-1b protein, hereinafter “FaCENH3-1b”); 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: 11 (Fragaria x ananassa CENH3- 2a protein, hereinafter “FaCENH3-2a”); 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: 12 (Fragaria x ananassa CENH3-2b protein, hereinafter “FaCENH3-2b”); 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: 13 (Fragaria x ananassa CENH3-3 protein, hereinafter “FaCENH3-3”); 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: 14 (Fragaria x ananassa CENH3-4 protein, hereinafter “FaCENH3-4”). In certain embodiments, the one or more FaCENH3 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: 53 (Fragaria x ananassa CENH3-1a protein, hereinafter “FaCENH3-7a”); 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: 54 (Fragaria x ananassa CENH3-1b protein, hereinafter “FaCENH3-7b”); 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: 55 (Fragaria x ananassa CENH3-2a protein, hereinafter “FaCENH3-7c”); 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: 56 (Fragaria x ananassa CENH3-2b protein, hereinafter “FaCENH3-7d”). 5sf-6744554197072001240

[0013] In certain embodiments, the one or more FaCENH3 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: 2 (Fragaria x ananassa CENH3-1a gene, hereinafter “FaCENH3-1a”); 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: 3 (Fragaria x ananassa CENH3-1b gene, hereinafter “FaCENH3-1b”); 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: 4 (Fragaria x ananassa CENH3-2a gene, hereinafter “FaCENH3-2a”); 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: 5 (Fragaria x ananassa CENH3-2b gene, hereinafter “FaCENH3-2b”); 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: 6 (Fragaria x ananassa CENH3-3 gene, hereinafter “FaCENH3- 3”); 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: 7 (Fragaria x ananassa CENH3-4 gene, hereinafter “FaCENH3-4”). In certain embodiments, the one or more FaCENH3 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: 58 (Fragaria x ananassa CENH3-1a gene, hereinafter “FaCENH3-7a”); 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: 59 (Fragaria x ananassa CENH3-1b gene, hereinafter “FaCENH3-7b”); 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: 60 (Fragaria x ananassa CENH3-2a gene, hereinafter “FaCENH3-7c”); 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: 61 (Fragaria x ananassa CENH3-2b gene, hereinafter “FaCENH3-7d”)

[0014] In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a plant of the species Fragaria x ananassa and the one or more FaCENH3 genes 6sf-6744554197072001240 comprise one, two, three, four, five, or all six of FaCENH3-1a, FaCENH3-1b, FaCENH3-2a, FaCENH3-2b, FaCENH3-3, and FaCENH3-4. In certain embodiments, the one or more FaCENH3 genes comprise FaCENH3-1a, FaCENH3-1b, FaCENH3-2a, FaCENH3-2b, FaCENH3-3, and FaCENH3-4. In some embodiments, the one or more genetic modifications comprise: a modification of an enhancer of FaCENH3-1a, a modification of a promoter of FaCENH3-1a, a modification of a coding region of FaCENH3-1a, a modification of an intron of FaCENH3-1a, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a modification of an enhancer of FaCENH3-1b, a modification of a promoter of FaCENH3-1b, a modification of a coding region of FaCENH3-1b, a modification of an intron of FaCENH3-1b, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a modification of an enhancer of FaCENH3-2a, a modification of a promoter of FaCENH3-2a, a modification of a coding region of FaCENH3-2a, a modification of an intron of FaCENH3-2a, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a modification of an enhancer of FaCENH3-2b, a modification of a promoter of FaCENH3-2b, a modification of a coding region of FaCENH3-2b, a modification of an intron of FaCENH3-2b, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a modification of an enhancer of FaCENH3-3, a modification of a promoter of FaCENH3-3, a modification of a coding region of FaCENH3-3, a modification of an intron of FaCENH3-3, or any combination thereof relative to an unmodified Fragaria x ananassa plant; and / or a modification of an enhancer of FaCENH3-4, a modification of a promoter of FaCENH3-4, a modification of a coding region of FaCENH3-4, a modification of an intron of FaCENH3-4, or any combination thereof relative to an unmodified Fragaria x ananassa plant.

[0015] In some embodiments, the strawberry plant (e.g., haploid-inducing strawberry plant) is a plant of the species Fragaria x ananassa and the one or more FaCENH3 genes comprise one, two, three, or all four of FaCENH3-7a, FaCENH3-7b, FaCENH3-7c, and FaCENH3-7d. In some embodiments, the one or more genetic modifications comprise: a modification of an enhancer of FaCENH3-7a, a modification of a promoter of FaCENH3-7a, a modification of a coding region of FaCENH3-7a, a modification of an intron of FaCENH3- 7a, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a modification of an enhancer of FaCENH3-7b, a modification of a promoter of FaCENH3-7b, a modification of a coding region of FaCENH3-7b, a modification of an intron of FaCENH3- 7b, or any combination thereof relative to an unmodified Fragaria x ananassa plant; a 7sf-6744554197072001240 modification of an enhancer of FaCENH3-7c, a modification of a promoter of FaCENH3-7c, a modification of a coding region of FaCENH3-7c, a modification of an intron of FaCENH3- 7c, or any combination thereof relative to an unmodified Fragaria x ananassa plant; and / or a modification of an enhancer of FaCENH3-7d, a modification of a promoter of FaCENH3-7d, a modification of a coding region of FaCENH3-7d, a modification of an intron of FaCENH3- 7d, or any combination thereof relative to an unmodified Fragaria x ananassa plant.

[0016] In some aspects, provided is a method of producing the strawberry plants (e.g., haploid-inducing strawberry plants) described herein. In some embodiments, the decreased expression of the one or more CENH3 genes is achieved by gene disruption, gene knockout, gene knockdown, disruption of an endogenous amino acid sequence (including, for example, conserved amino acids or amino acid motifs), introduction of amino acids or amino acid motifs, 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.

[0017] In some embodiments, the method comprises contacting a plurality of cells of a parent strawberry plant with one or more Cas nucleases complexed with an RNA molecule comprising a polynucleotide sequence 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 a protospacer sequence within one or more of the FvCENH3 and / or FaCENH3 genes. In some embodiments, the Cas nuclease is complexed with an RNA molecule comprising 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 a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 15-34. 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 8sf-6744554197072001240 one or more CENH3 genes. In some embodiments, the method comprises 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 CENH3 genes.

[0018] 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 a sperm cell 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 contracting a plurality of tetrahaploid reduced egg cells from the donor octoploid strawberry plant with a plurality of sperm cells of the haploid inducing strawberry plant. 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 sperm cell to the reduced egg cell, thereby contacting the reduced egg cell with the sperm cell. 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 tetraploid embryo and an endosperm having a ploidy of greater than 8x.

[0019] In some aspects, provided herein is method of producing true homozygous octoploid strawberry seed, the method comprising (a) contacting an egg cell from a haploid- inducing strawberry plant described herein with a tetrahaploid reduced sperm cell from a donor octoploid strawberry plant, and (b) producing a doubled tetrahaploid cell from the egg cell, wherein the doubled tetrahaploid cell comprises the nuclear genome of the donor octoploid strawberry plant and the cytoplasmic genome of the haploid-inducing strawberry plant. In some embodiments, step (a) comprises contacting a plurality of egg cells from the haploid-inducing strawberry plants with a plurality of sperm cells from a donor octoploid strawberry plant. In some embodiments, the haploid-inducing strawberry plant and the donor octoploid strawberry plants are from different species of Fragaria. In some embodiments, the 9sf-6744554197072001240 haploid-inducing strawberry plant is diploid. In certain embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca. In some embodiments, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, step (a) comprises contacting the stigma of a pistil of the haploid-inducing strawberry plant with the pollen from the donor octoploid strawberry plant and allowing formation of a pollen tube and migration of the tetrahaploid reduced sperm cell to the egg cell, thereby contacting the egg cell with the tetrahaploid reduced sperm cell. In some embodiments, the method further comprises allowing the haploid-inducing strawberry plant to form a plurality of seeds.

[0020] In some aspects, provided herein is a method of producing true homozygous octoploid strawberry plants, the method comprising (a) contacting a plurality of tetrahaploid reduced egg cells from a maternal octoploid strawberry plant (e.g., an unedited maternal octoploid strawberry plant) with a plurality of sperm cells of an edited haploid-inducing octoploid strawberry plant described herein, (b) allowing the plurality of reduced egg cells to form a plurality of tetrahaploid seeds comprising the maternal genome of the octoploid strawberry plant (e.g., the unedited maternal genome) and lacking the edited, haploid- inducing paternal genome, (c) collecting the plurality of seeds and allowing them to germinate to form a plurality of plants, (d) determining the presence of one or more tetrahaploid plants in the plurality of plants, and (e) producing one or more doubled tetrahaploid plants (also referred to herein as a “true homozygous octoploid”) from the one or more tetrahaploid plants. Additionally, step (d) may include determining the presence of one or more spontaneously doubled tetrahaploids.

[0021] In some aspects, provided herein is a method of producing true homozygous octoploid strawberry plants, the method comprising (a) contacting a plurality of tetrahaploid reduced egg cells from an edited maternal haploid-inducing octoploid strawberry plant described herein with a plurality of sperm cells of an a paternal octoploid strawberry plant (e.g., an unedited octoploid strawberry plant), (b) allowing the plurality of reduced egg cells to form a plurality of tetrahaploid seeds comprising the paternal genome from the paternal octoploid strawberry plant (e.g., the unedited paternal genome) and lacking the edited, haploid-inducing maternal genome, (c) collecting the plurality of seeds and allowing them to germinate to form a plurality of plants, (d) determining the presence of one or more tetrahaploid plants in the plurality of plants, and (e) producing one or more doubled 10sf-6744554197072001240 tetrahaploid plants (also referred to herein as a “true homozygous octoploid”) from the one or more tetrahaploid plants. Additionally, step (d) may include determining the presence of one or more spontaneously doubled tetrahaploids.

[0022] In some aspects, provided herein is a method of producing true homozygous octoploid strawberry plants, the method comprising (a) contacting a plurality of tetrahaploid reduced egg cell from an edited haploid-inducing diploid strawberry plant described herein with a plurality of sperm cells of an octoploid strawberry plant (e.g., an unedited octoploid strawberry plant), (b) allowing the plurality of reduced egg cells to form a plurality of tetrahaploid seeds comprising the paternal genome of the octoploid strawberry plant and lacking the edited, haploid-inducing maternal genome of the diploid strawberry plant, (c) collecting the plurality of seeds and allowing them to germinate to form a plurality of plants, (d) determining the presence of one or more tetrahaploid plants in the plurality of plants, and (e) producing one or more doubled tetrahaploid plants (also referred to herein as a “true homozygous octoploid”) from the one or more tetrahaploid plants. Additionally, step (d) may include determining the presence of one or more spontaneously doubled tetrahaploids. In certain such embodiments, the tetrahaploid seedling will comprise the unedited cytoplasmic genome of the diploid mother and the unedited octoploid genome of the father.

[0023] In some embodiments of the method of producing a true homozygous octoploid strawberry plant, 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 tube to the reduced egg cell, thereby contacting the reduced egg cell with the sperm cell. In certain embodiments, step (d) comprises determining the presence of the maternal genome and the paternal genome in the one or more plants, wherein tetrahaploid plants within the plurality of plants are determined based on the absence of the maternal genome and the presence of the paternal genome.

[0024] In other embodiments of the method of producing a true homozygous octoploid strawberry plant, step (a) comprises contacting pollen of the donor octoploid strawberry plant with the stigma of a pistil of the haploid-inducing strawberry plant comprising the reduced egg cell and allowing formation of a pollen tube and migration of the pollen tube to the reduced egg cell, thereby contacting the reduced egg cell with the sperm cell. In certain embodiments, step (d) comprises determining the presence of the maternal genome and the 11sf-6744554197072001240 paternal genome in the one or more plants, wherein tetrahaploid plants within the plurality of plants are determined based on the presence of the maternal genome and the absence of the paternal genome.

[0025] In certain embodiments of the method of producing a true homozygous octoploid strawberry plant, step (d) further comprises determining the ploidy of the embryo from each of the one or more tetrahaploid plants and discarding any plants that (i) lacks a tetraploid embryo and / or (ii) comprises an embryo ploidy of greater than 8x. In certain embodiments, the presence (or absence) of the maternal genome and / or the paternal genome, respectively, in the one or more plants, is determined by the detection of one or more genetic markers which are present in the paternal genome and which are absent in the maternal genome. In certain embodiments, the presence (or absence) of the maternal genome and / or the paternal genome, respectively, in the one or more plants, is determined by the detection of one or more genetic markers which are present in the maternal genome and which are absent in the paternal genome.

[0026] In some embodiments of the method of producing a true homozygous octoploid strawberry plant, step (d) comprises determining the presence of the maternal genome and the paternal genome in the one or more plants, wherein tetrahaploid plants within the plurality of plants are determined based on the absence of the maternal genome and the presence of the paternal genome. In other embodiments, step (d) comprises determining the presence of the maternal genome and the paternal genome in the one or more plants, wherein tetrahaploid plants within the plurality of plants are determined based on the presence of the maternal genome and the absence of the paternal genome. In certain embodiments, step (d) further comprises determining the ploidy of the embryo from each of the one or more tetrahaploid plants and discarding any plants that (i) lacks a tetraploid embryo and / or (ii) comprises an embryo ploidy of greater than 8x. In certain embodiments, the presence (or absence) of the maternal genome and / or the paternal genome, respectively, in the one or more plants, is determined by the detection of one or more genetic markers which are present in the paternal genome and which are absent in the maternal genome. In certain embodiments, the presence (or absence) of the maternal genome and / or the paternal genome, respectively, in the one or more plants, is determined by the detection of one or more genetic markers which are present in the maternal genome and which are absent in the paternal genome. 12sf-6744554197072001240

[0027] In some embodiments of the method of producing a true homozygous octoploid strawberry plant, step (e) comprises subjecting the selected tetrahaploid plant to a tetrahaploid doubling treatment. In certain embodiments, the tetrahaploid doubling treatment comprises contacting a cell from the tetrahaploid plant with an anti-microtubule agent to form a doubled tetrahaploid cell (i.e., a true homozygous octoploid cell). In certain embodiments, the anti-microtubule agent is an anti-mitotic herbicide. In certain embodiments, the anti- microtubule agent comprises colchicine, oryzalin, nitrous oxide, trifluralin, or any combination thereof. In some embodiments of the method of producing a true homozygous octoploid strawberry plant, the doubled tetrahaploid cell is allowed to grow into a doubled tetrahaploid plant. In some embodiments, the doubled tetrahaploid plant (i.e., the true homozygous octoploid plant) is contacted with self-pollen and allowed to form seed, producing a plurality of true homozygous octoploid strawberry seed. In some embodiments, the doubled tetrahaploid plant (i.e., the true homozygous octoploid plant) is contacted with pollen from a different doubled tetrahaploid plant and allowed to form seed, producing a plurality of true breeding F1 hybrid octoploid strawberry seed.

[0028] 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 iturupensis, or a hybrid of any combination thereof. In some embodiments, the donor octoploid strawberry plant is Fragaria chiloensis plant of the subspecies Fragaria chiloensis ssp. chiloensis, Fragaria chiloensis ssp. lucida, Fragaria chiloensis ssp. pacifica, or Fragaria chiloensis ssp. 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 named “Camarosa.” In certain embodiments, the donor octoploid strawberry plant is a Fragaria x ananassa plant of the variety named “Chandler.” In certain embodiments, the donor octoploid strawberry plant is a Fragaria x ananassa plant of the variety named “Albion.”.

[0029] 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. 13sf-6744554197072001240

[0030] 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 the sperm cell 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 ga20ox gene into the true homozygous octoploid strawberry plant, wherein the runnerless true homozygous octoploid strawberry plant does not produce runners.

[0031] 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 ga20ox gene.

[0032] 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 ga20ox gene. In certain embodiments, the true homozygous octoploid strawberry plant does not produce runners. In some embodiments, the true homozygous octoploid strawberry plant, or a progenitor thereof, was subject to introduction of one or more of the genetic modifications by mutagenesis, gene editing, transgenesis, or a combination thereof followed by selection for a genetic modification that results in decreased expression of the one or more CENH3 genes. 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.

[0033] In some aspects, provided herein is a true homozygous octoploid strawberry seed produced according to a method described herein, comprising an embryo having the nuclear genome of the donor octoploid strawberry plant and the cytoplasmic genome of the haploid- inducing strawberry plant, wherein the nuclear genome and the cytoplasmic genome are from plants of different species of Fragaria. In some embodiments, the cytoplasmic genome is from a plant of the species Fragaria vesca. In some embodiments, the nuclear genome is from a plant of the species Fragaria x ananassa. In some embodiments, the true homozygous octoploid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a ga20ox gene. 14sf-6744554197072001240

[0034] In some aspects, provided herein is a true homozygous octoploid strawberry plant or a plant part thereof produced according to the method described herein, comprising at least one somatic cell having the nuclear genome of the donor octoploid strawberry plant and the cytoplasmic genome of the haploid-inducing strawberry plant, wherein the nuclear genome and the cytoplasmic genome are from plants of different species of Fragaria. In some embodiments, the cytoplasmic genome is from a plant of the species Fragaria vesca. In some embodiments, the nuclear genome is from a plant of the species Fragaria x ananassa. In some embodiments, the true homozygous octoploid strawberry plant comprises one or more genetic modifications resulting in decreased expression of a ga20ox gene. In certain embodiments, the true homozygous octoploid strawberry plant does not produce runners. In some embodiments, the true homozygous octoploid strawberry plant, or a progenitor thereof, was subject to introduction of one or more of the genetic modifications by mutagenesis, gene editing, transgenesis, or a combination thereof followed by selection for a genetic modification that results in decreased expression of the one or more CENH3 genes. 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.

[0035] In some aspects, provided herein is a true homozygous octoploid strawberry seed comprising an embryo having the nuclear genome of a first species of Fragaria and the cytoplasmic genome of a second plant species of Fragaria. In some embodiments, the first species of Fragaria is Fragaria x ananassa. In some embodiments, the second species of Fragaria is a diploid species of Fragaria. In certain embodiments, the second species of Fragaria is Fragaria vesca. In some embodiments, the true homozygous octoploid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a gibberellin ga20ox gene.

[0036] In some aspects, provided herein is a true homozygous octoploid strawberry plant or plant part thereof comprising an embryo having the nuclear genome of a first species of Fragaria and the cytoplasmic genome of a second plant species of Fragaria. In some embodiments, the first species of Fragaria is Fragaria x ananassa. In some embodiments, the second species of Fragaria is a diploid species of Fragaria. In certain embodiments, the second species of Fragaria is Fragaria vesca. In some embodiments, the true homozygous octoploid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a gibberellin ga20ox gene. In certain embodiments, the true homozygous 15sf-6744554197072001240 octoploid strawberry plant does not produce runners. In some embodiments, the true homozygous octoploid strawberry plant, or a progenitor thereof, was subject to introduction of one or more of the genetic modifications by mutagenesis, gene editing, transgenesis, or a combination thereof followed by selection for a genetic modification that results in decreased expression of the one or more CENH3 genes. 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.

[0037] In some aspects, provided herein is a method of producing uniform octoploid F1 hybrid strawberry seed comprising crossing two of the true homozygous octoploid strawberry plants described herein. In some aspects, provided herein is a uniform octoploid F1 hybrid strawberry seed produced according to said method. In certain embodiments, the uniform octoploid F1 hybrid strawberry seed comprises one or more genetic modifications resulting in decreased expression of a ga20ox gene. In some aspects, provided herein is a uniform octoploid F1 hybrid strawberry plant or a plant part thereof produced according to said method. In certain embodiments, the uniform octoploid F1 hybrid strawberry plant comprises one or more genetic modifications resulting in decreased expression of a ga20ox gene. In certain embodiments, the uniform octoploid F1 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.

[0038] In some aspects, an expression vector or isolated DNA molecule for making a strawberry plant (e.g., haploid-inducing strawberry plant), or a plant part thereof, is 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: 8-14 and 53-56. 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 a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7 and 58-61. 16sf-6744554197072001240

[0039] 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: 8-14 and 53-56. 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 a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7 and 58-61. In some embodiments, the non-coding RNA is a crRNA, a gRNA, a pegRNA, a siRNA, a miRNA, or a dsRNA.

[0040] 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 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: 8-14 and 53-56. 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 a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7 and 58-61.

[0041] 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. In some aspects, provided herein is a kit comprising an expression 17sf-6744554197072001240 vector or isolated DNA molecule or a bacterial cell described herein. 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.

[0042] In some aspects, provided herein is a genetically modified non-regenerable plant cell of a strawberry plant (e.g., haploid-inducing strawberry plant) described herein.

[0043] In some aspects, provided herein is a genetically modified plant genome of the strawberry plant (e.g., haploid-inducing strawberry plant) described herein. DESCRIPTION OF THE FIGURES

[0044] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0045] FIG. 1 depicts a schematic illustrating the production of homozygous octoploid strawberry lines by tetrahaploid induction of the maternal genome and then doubling the resulting tetrahaploid. Step (A) exemplifies the starting plant material where chromosomes are indicated by gray and white patterned vertical bars, and the oval lines surrounding the vertical bars indicate exemplary nuclei. Ploidy is indicated at the top of the nuclei. An edited cenh3 gene is indicated on the chromosomes by a white “X” mark. Pollen is indicated by a male symbol, and egg cells and megaspore mother cells are indicated by a femalesymbol. In step (B), the top row exemplifies reduced, recombined 1x cenh3-edited pollen derived from a cenh3-edited diploid Fragaria vesca strawberry plant; the middle row illustrates a reduced, recombined 4x unedited tetrahaploid egg cell derived from an unedited octoploid Fragaria x ananassa strawberry plant; and the bottom row exemplifies reduced, recombined 4x cenh3-edited pollen from a cenh3-edited octoploid Fragaria x ananassa strawberry plant. The reduced, recombined 4x unedited tetrahaploid egg cell (middle row) is contacted by either (i) the reduced, recombined 1x cenh3-edited pollen (or sperm cell) (top row), (ii) the reduced, recombined 4x cenh3-edited pollen (or sperm cell), or (iii) both. In step (C), some amount of fertilizations cause the maternal tetrahaploid egg cell to undergo embryogenesis without retention of genetic material from the cenh3-edited pollen, as indicated by the straight-rightwards arrow. The remaining eggs result in fertilizations by the haploid inducer, illustrated by: (i) the up-rightwards arrow pointing to an illustration of an exemplary 5x embryo with one set of paternal chromosomes from the cenh3-edited diploid male parent, and (ii) the down-rightwards arrow pointing to an illustration of an exemplary 18sf-6744554197072001240 8x embryo with 4 sets of paternal chromosomes from the cenh3-edited octoploid parent. Embryos are screened for presence of the maternal and / or paternal genome and have their ploidy confirmed before subjecting the tetrahaploid embryos to a doubling treatment, thereby producing true homozygous octoploid strawberry plants in step (D).

[0046] FIG. 2A depicts a schematic illustrating the production of homozygous octoploid strawberry lines by tetrahaploid induction of the paternal genome and then doubling the resulting tetrahaploid. Step (A) exemplifies the starting plant material where chromosomes are indicated by gray and white patterned vertical bars, and the oval lines surrounding the vertical bars indicate exemplary nuclei. Ploidy is indicated at the top of the nuclei. An edited cenh3 gene is indicated on the chromosomes by a white “X” mark. Pollen is indicated by a male symbol, and egg cells and megaspore mother cells are indicated by a femalesymbol. In step (B), the bottom row exemplifies reduced, recombined 4x pollen derived from an unedited octoploid Fragaria x ananassa strawberry plant, and the top row illustrates a reduced, recombined 4x tetrahaploid egg cell derived from a cenh3-edited octoploid Fragaria x ananassa strawberry plant. In other embodiments, the egg cell is a reduced, recombined 1x haploid egg cell derived from a cenh3-edited diploid Fragaria vesca strawberry plant (not illustrated). The reduced, recombined 4x cenh3-edited tetrahaploid egg cell (illustrated in the top row) or the reduced, recombined 1x cenh3-edited haploid egg cell (not illustrated), is contacted by the reduced, recombined 4x unedited pollen (bottom row). In step (C), some of the fertilizations cause the maternal tetrahaploid egg cell (illustrated in the top row) or the reduced, recombined 1x cenh3-edited haploid egg cell (not illustrated), to undergo embryogenesis without retention of nuclear genetic material from the cenh3-edited egg cell, as indicated by the straight-rightwards arrow. The remaining eggs result in fertilizations by the haploid inducer, illustrated by the down-rightwards arrow pointing to an illustration of an exemplary 8x embryo with four sets of both maternal and paternal chromosomes from octoploid parents. In embodiments where the reduced, recombined egg cells is a 1x cenh3- edited haploid egg cell, fertilization by the haploid inducer results in 5x embryos (not illustrated). Embryos are screened for presence of the maternal and / or paternal genome and have their ploidy confirmed before subjecting the tetrahaploid embryos to a doubling treatment, thereby producing true homozygous octoploid strawberry plants in step (D).

[0047] FIG. 2B depicts a schematic illustrating the production of homozygous octoploid strawberry lines by tetrahaploid induction of the paternal genome, with a cytoplasmic swap 19sf-6744554197072001240 resulting in a seed having the cytoplasmic genome of the maternal plant and the nuclear genome of the paternal plant. Pollen of octoploid (8x) F. x ananassa is used to pollinate a haploid-inducing line of diploid (2x) F. vesca, resulting in tetrahaploid embryogenesis of an embryo retaining the nuclear tetrahaploid (4x) genome of F. x ananassa and the cytoplasmic genome of F. vesca. Doubling of the resulting tetrahaploid plant produces the homozygous octoploid strawberry line, which is homozygous across the four subgenomes for the unedited 8x F. x ananassa paternal nuclear genome, and also has the cytoplasmic genome of the F. vesca maternal plant.

[0048] FIGS. 3A-3B show the configuration of CENH3 loci in octoploid strawberry. FIG. 3A shows the configuration of six homoeologous FaCENH3 loci in octoploid strawberry Fragaria x ananassa according to reference genome “Fragaria x ananassa Camarosa Genome v1.0.a2”. The six homeologs are shown, with black bars indicating the locations of the CENH3 loci. As described further herein, “FaCENH3-1a” is illustrated as the left-most black bar on chromosome 7, subgenome 1; “FaCENH3-1b” is illustrated as the right-most on chromosome 7, subgenome 1; “FaCENH3-2a” is illustrated as the left-most black bar on chromosome 7, subgenome 2; “FaCENH3-2b” is illustrated as the right-most black bar on chromosome 7, subgenome 2; “FaCENH3-3” is illustrated as the black bar on chromosome 7, subgenome 3; “FaCENH3-4” is illustrated as the black bar on chromosome 7, subgenome 4. FIG. 3B shows the configuration of six homoeologous FaCENH3 loci in octoploid strawberry Fragaria x ananassa according to reference genome “Fragaria x ananassa Royal Royce Genome v1.0.” The four homeologs are shown, with black bars indicating the locations of the CENH3 loci. As described further herein, “FaCENH3-7a” is illustrated as a black bar on chromosome 7, subgenome A; “FaCENH3-7b” is illustrated as a black bar on chromosome 7, subgenome B; “FaCENH3-7c” is illustrated as a black bar on chromosome 7, subgenome C; and “FaCENH3-7d” is illustrated as a black bar on chromosome 7, subgenome D.

[0049] FIG. 4 shows the CENH3 gene in the diploid strawberry Fragaria vesca according to the reference genome “Fragaria vesca v_4.0”. The gene (referred to herein as (“FvCENH3”) is shown with a black bar indicating its location of chromosome 7.

[0050] FIGS. 5A-C show a protein sequence alignment of CENH3 orthologs from Fragaria vesca (FvCENH3) (SEQ ID NO: 8), Fragaria x ananassa Camarosa (FaCENH3- 1a, FaCENH3-1b, FaCENH3-2,a FaCENH3-2b, FaCENH3-3, FaCENH3-4) (SEQ ID NOs: 20sf-6744554197072001240 9-14) Fragaria chiloensis (FcCENH3-7-AV, FcCENH3-7-Bi, FcCENH3-7-B1, and FcCENH3-7-B2) (SEQ ID NOs: 36-39), Fragaria iinumae (FiCENH3) (SEQ ID NO: 40), Fragaria viridis (FvirCENH3) (SEQ ID NO: 41), Potentilla micrantha (PmCENH3), (SEQ ID NO: 42), Arabidopsis thaliana (AtCENH3) (SEQ ID NO: 35), Solanum lycopersicum (SlCENH3) (SEQ ID NO: 43), Zea mays (ZmCENH3) (SEQ ID NO: 44), and Consensus (SEQ ID NO: 48). FIG. 5A shows positions 1-42 of the sequence alignment. FIG. 5B shows positions 43-108 of the sequence alignment. FIG. 5C shows positions 109-178 of the sequence alignment.

[0051] FIGS. 5D-G show a protein sequence alignment of CENH3 orthologs from Fragaria vesca (FvCENH3) (SEQ ID NO: 8), Fragaria x ananassa Royal Royce (FaCENH3-7a, FaCENH3-7b, FaCENH3-7c, and FaCENH3-7d) (SEQ ID NOs: 53-56) Fragaria chiloensis (FcCENH3-7-AV, FcCENH3-7-Bi, FcCENH3-7-B1, and FcCENH3-7- B2) (SEQ ID NOs: 36-39), Fragaria iinumae (FiCENH3) (SEQ ID NO: 40), Fragaria viridis (FvirCENH3) (SEQ ID NO: 41), Potentilla micrantha (PmCENH3), (SEQ ID NO: 42), Arabidopsis thaliana (AtCENH3) (SEQ ID NO: 35), Solanum lycopersicum (SlCENH3) (SEQ ID NO: 43), Zea mays (ZmCENH3) (SEQ ID NO: 44), and Consensus (SEQ ID NO: 57). FIG. 5D shows positions 1-60 of the sequence alignment. FIG. 5E shows positions 61- 120 of the sequence alignment. FIG. 5F shows positions 121-180 of the sequence alignment. FIG. 5G shows positions 181-189 of the sequence alignment.

[0052] FIG. 6A shows a phylogenetic tree of identified putative and characterized CENH3 orthologs as in FIGS. 5A-5C. The phylogenetic tree is a UPGMA consensus tree. The scale bar represents phylogenetic distance of 0.05 nucleotide substitutions per site. The tree is generated using software Geneious Prime 2023.2.1, with Genetic Distance Model: Jukes-Cantor.

[0053] FIG. 6B shows a phylogenetic tree of identified putative and characterized CENH3 orthologs as in FIGS. 5D-5G. The phylogenetic tree is a UPGMA consensus tree. The scale bar represents phylogenetic distance of 0.05 nucleotide substitutions per site. The tree is generated using software Geneious Prime 2025.0.2, with Genetic Distance Model: Jukes-Cantor.

[0054] FIG. 7 shows a bar graph of editing efficiencies of guide RNAs targeting different protospacers in FaCENH3 in four different varieties of Fragaria x ananassa. The x-axis 21sf-6744554197072001240 indicates the different target protospacer sites which were tested; the y-axis indicates the percent of protoplasts with successful editing. Different shades indicate different varieties of Fragaria x ananassa strawberry varieties tested. The sample size ranged from 3 replicates up to 9 replicates, with a median of 6 replicates across all data sets. The error bars indicate standard error of the mean.

[0055] FIG. 8A shows a schematic illustrating the editing windows associated with PRS878 for FvCENH3 (SEQ ID NO: 1), FaCENH3-1a (SEQ ID NO: 2), FaCENH3-1b (SEQ ID NO: 3), FaCENH3-2a (SEQ ID NO: 4), FaCENH3-2b (SEQ ID NO: 5), FaCENH3-3 (SEQ ID NO: 6), and FaCENH3-4 (SEQ ID NO: 7). The protospacer sequence is shown in bold, underlined text. UTRs are denoted in light gray; coding sequences are denoted in dark gray; and introns are denoted as a line. The nucleotides in bolded, underlined font show the (+) strand reverse complement of the PRS878 sequence (SEQ ID NO: 21). At the top of the figure, its general location is illustrated by a downwards-pointing black triangle over the third exon.

[0056] FIG. 8B shows a schematic illustrating the editing windows associated with PRS878 for FvCENH3 (SEQ ID NO: 1), FaCENH3-7a (SEQ ID NO: 58), FaCENH3-7b (SEQ ID NO: 59), FaCENH3-7c (SEQ ID NO: 60), and FaCENH3-7d (SEQ ID NO: 61). The protospacer sequence is shown in bold, underlined text. UTRs are denoted in light gray; coding sequences are denoted in dark gray; and introns are denoted as a line. The nucleotides in bolded, underlined font show the (+) strand reverse complement of the PRS878 sequence (SEQ ID NO: 21). At the top of the figure, its general location is illustrated by a downwards- pointing black triangle over the third exon.

[0057] FIG. 9A shows a schematic illustrating the editing windows associated with PRS874 for FvCENH3 (SEQ ID NO: 1), FaCENH3-1a (SEQ ID NO: 2), FaCENH3-1b (SEQ ID NO: 3), FaCENH3-2a (SEQ ID NO: 4), FaCENH3-2b (SEQ ID NO: 5), FaCENH3-3 (SEQ ID NO: 6), and FaCENH3-4 (SEQ ID NO: 7). The protospacer sequence is shown in bold, underlined text. UTRs are denoted in light gray; coding sequences are denoted in dark gray; and introns are denoted as a line. The general location of the sequence of PRS874 (SEQ ID NO: 17) is denoted by a downwards-pointing black triangle over the third exon. 22sf-6744554197072001240

[0058] FIG. 9B shows a schematic illustrating the editing windows associated with PRS874 for FvCENH3 (SEQ ID NO: 1), FaCENH3-7a (SEQ ID NO: 58), FaCENH3-7b (SEQ ID NO: 59), FaCENH3-7c (SEQ ID NO: 60), and FaCENH3-7d (SEQ ID NO: 61). The protospacer sequence is shown in bold, underlined text. UTRs are denoted in light gray; coding sequences are denoted in dark gray; and introns are denoted as a line. The general location of the sequence of PRS874 (SEQ ID NO: 17) is denoted by a downwards-pointing black triangle over the third exon.

[0059] FIG. 10A shows a schematic illustrating the editing windows associated with PRS876 for FvCENH3 (SEQ ID NO: 1), FaCENH3-1a (SEQ ID NO: 2), FaCENH3-1b (SEQ ID NO: 3), FaCENH3-2a (SEQ ID NO: 4), FaCENH3-2b (SEQ ID NO: 5), FaCENH3-3 (SEQ ID NO: 6), and FaCENH3-4 (SEQ ID NO: 7). The protospacer sequence is shown in bold, underlined text. UTRs are denoted in light gray; coding sequences are denoted in dark gray; and introns are denoted as a line. Target site PRS876 (SEQ ID NO: 19) is illustrated by a downwards-pointing black triangle over the third exon.

[0060] FIG. 10B shows a schematic illustrating the editing windows associated with PRS876 for FvCENH3 (SEQ ID NO: 1), FaCENH3-7a (SEQ ID NO: 58), FaCENH3-7b (SEQ ID NO: 59), FaCENH3-7c (SEQ ID NO: 60), and FaCENH3-7d (SEQ ID NO: 61). The protospacer sequence is shown in bold, underlined text. UTRs are denoted in light gray; coding sequences are denoted in dark gray; and introns are denoted as a line. Target site PRS876 (SEQ ID NO: 19) is illustrated by a downwards-pointing black triangle over the third exon.

[0061] FIGS. 11A-11B depict the size and growth of an exemplary sample of protoplasts derived from a cenh3 mutant strawberry line. FIG. 11A shows the cell size of the protoplasts in the exemplary sample, with bounding boxes around the protoplasts for comparison of protoplast size. FIG. 11B depicts microcalli grown from protoplasts in the exemplary sample, 0.1 – 0.3 mm in size 14 days after encapsulation of the protoplasts.

[0062] FIGS. 12A-12F depict a sequence alignment of FaCENH3-7a alleles in wild-type (N; SEQ ID NO: 77) and edited (E; SEQ ID NOs: 78-88) F. x ananassa Royal Royce plants with the translated protein sequence (SEQ ID NO: 89). FIG. 12A shows the alignment at positions 1-108 of the wild-type sequence. FIG. 12B shows the alignment at positions 109- 23sf-6744554197072001240 216 of the wild-type sequence. FIG. 12C shows the alignment at positions 217-270 of the wild-type sequence.

[0063] FIGS. 13A-13C depict a sequence alignment of FaCENH3-7b alleles in wild-type (N; SEQ ID NO: 90) and edited (E; SEQ ID NOs: 91-101) F. x ananassa Royal Royce plants with the translated protein sequence (SEQ ID NO: 102). FIG. 13A shows the alignment at positions 1-108 of the wild-type sequence. FIG. 13B shows the alignment at positions 109- 216 of the wild-type sequence. FIG. 13C shows the alignment at positions 217-270 of the wild-type sequence.

[0064] FIGS. 14A-14F depict a sequence alignment of FaCENH3-7c alleles in wild-type (N; SEQ ID NO: 103 and 104) and edited (E; SEQ ID NOs: 105-113) F. x ananassa Royal Royce plants with the translated protein sequence (SEQ ID NO: 114). FIG. 14A shows the alignment at positions 1-108 of the wild-type sequence. FIG. 14B shows the alignment at positions 109-216 of the wild-type sequence. FIG. 14C shows the alignment at positions 217-270 of the wild-type sequence.

[0065] FIGS. 15A-15C depict a sequence alignment of FaCENH3-7d alleles in wild-type (N; SEQ ID NO: 115) and edited (E; SEQ ID NOs: 116-126) F. x ananassa Royal Royce plants with the translated protein sequence (SEQ ID NO: 127). FIG. 15A shows the alignment at positions 1-108 of the wild-type sequence. FIG. 15B shows the alignment at positions 109-216 of the wild-type sequence. FIG. 15C shows the alignment at positions 217-270 of the wild-type sequence.

[0066] FIG. 16A-16F depict the results of microscopy to evaluate microspore formation in wild-type and cenh3 mutant strawberry plants. FIG. 16A shows a bar graph of the average size at which microspores developed. FIGS. 16B shows a micrograph of an exemplary smashed anther with microspores emerging. FIG. 16C shows a micrograph of the same exemplary anther at a higher magnification. FIG. 16D shows a micrograph of the same exemplary anther at yet higher magnification. FIG. 16E shows an exemplary micrograph from cenh3 mutant line E-PED524-4145 (cenh3-006), showing tetrads. FIG. 16F shows a second exemplary micrograph from cenh3 mutant line E-PED524-4145 (cenh3-006), showing tetrads.

[0067] FIGS. 17A-17B depict photographs of wild-type and cenh3 mutant strawberry lines. FIG. 17A depicts cenh3-001 (E-PED524-4004; left) and wild-type (right) plants. FIG. 24sf-6744554197072001240 17B depict two flowers and three stamens each from the wild-type (top) and cenh3-001 (E- PED524-4004; bottom) plants.

[0068] FIGS. 18A-18B depict photographs of wild-type and cenh3 mutant strawberry lines. FIG. 18A depicts cenh3-002 (E-PED524-3916; left) and wild-type (right) plants. FIG. 18B depict two flowers and three stamens each from the wild-type (top) and cenh3-002 (E- PED524-3916; bottom) plants.

[0069] FIGS. 19A-19B depict photographs of wild-type and cenh3 mutant strawberry lines. FIG. 19A depicts cenh3-003 (E-PED524-4113; left) and wild-type (right) plants. FIG. 19B depict two flowers and three stamens each from the wild-type (top) and cenh3-003 (E- PED524-4113; bottom) plants.

[0070] FIGS. 20A-20B depict photographs of wild-type and cenh3 mutant strawberry lines. FIG. 20A depicts cenh3-004 (E-PED524-4115; left) and wild-type (right) plants. FIG. 20B depict two flowers and three stamens each from the wild-type (top) and cenh3-004 (E- PED524-4115; bottom) plants.

[0071] FIGS. 21A-21B depict photographs of wild-type and cenh3 mutant strawberry lines. FIG. 21A depicts cenh3-005 (E-PED524-4135; left) and wild-type (right) plants. FIG. 21B depict two flowers and three stamens each from the wild-type (top) and cenh3-005 (E- PED524-4135; bottom) plants.

[0072] FIGS. 22A-22B depict photographs of wild-type and cenh3 mutant strawberry lines. FIG. 22A depicts cenh3-006 (E-PED524-4145; left) and wild-type (right) plants. FIG. 22B depict two flowers and three stamens each from the wild-type (top) and cenh3-006 (E- PED524-4145; bottom) plants.

[0073] FIGS. 23A-23B depict photographs of wild-type and cenh3 mutant strawberry lines. FIG. 23A depicts cenh3-007 (E-PED524-4091; left) and wild-type (right) plants. FIG. 23B depict two flowers and three stamens each from the wild-type (top) and cenh3-007 (E- PED524-4091; bottom) plants. DETAILED DESCRIPTION

[0074] 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, 25sf-6744554197072001240 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 various embodiments 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

[0075] 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 egg 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. In some embodiments, pollen from the haploid inducer lines induce the reduced egg cells to undergo embryogenesis without transfer or retention of genetic material from the paternal genome. In some embodiments, pollen from the haploid inducer lines fuses with the reduced egg cell to form a zygote wherein the maternal genome is thereafter eliminated from the zygote, generating a tetrahaploid (4x) embryo without transfer or retention of genetic material from the maternal genome. 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) F1 hybrid seed. An exemplary such method is depicted in FIG. 1.

[0076] Another advantage of the methods described herein is that the resulting F1 hybrid seed retains no foreign DNA and no targeted genetic alterations. First, de novo creation of strawberry haploid inducer lines is achieved by disrupting expression of CENH3 (centromere-specific histone 3 variant) ortholog genes in strawberry. In some embodiments, this involves the identification and targeting of CENH3 ortholog genes via nuclease-guided deletions. In some embodiments, this is done in a diploid line such as Fragaria vesca (FIG. 1, top row). In other embodiments, this is done in an octoploid Fragaria x ananassa (FIG. 1, bottom row). In the latter embodiment, edits are made to CENH3 orthologs from subgenome 1 (“sg1”), sg2 and sg3, and sg4. Next, pollen is collected from cenh3-edited strawberry lines (referred to herein as “cenh3-edited pollen” or just “cenh3-pollen”) and used to induce embryogenesis in reduced tetrahaploid egg cells (4x) from elite octoploid strawberry varieties 26sf-6744554197072001240 (8x) (FIG. 1, middle row). Embryogenesis without retention of the genomic material from the pollen occurs in some of the egg cells (FIG. 1, steps B). The resulting tetrahaploid lines (4x) are doubled with colchicine (FIG. 1, step C) to obtain fully homozygous octoploid strawberry derived from the maternal genome (8x) (FIG. 1, step D). 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 genetically uniform F1 hybrid seed.

[0077] In other embodiments, unmodified 4x pollen (FIG. 2A, bottom row) is used to pollinate a modified cenh3-edited female, wherein the egg cell is induced to undergo embryogenesis, resulting in a tetrahaploid (4x) comprising the paternal nuclear genome and lacking the cenh3-edited maternal genome (FIG. 2, middle row). In other words, embryogenesis without retention of the genomic material from the egg occurs in some of the egg cells (FIG. 2, steps B). The resulting haploid lines (4x) are doubled with colchicine (FIG. 2, step C) or double spontaneously to obtain fully homozygous octoploid strawberry lines derived from the paternal genome (8x) (FIG. 2, step D). 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 genetically uniform F1 hybrid seed.

[0078] In one aspect, provided herein are haploid-inducing strawberry plants, and plant parts thereof, comprising one or more genetic modifications resulting in decreased expression (including, e.g., decreased activity) of one or more CENH3 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.

[0079] In another aspect, provided herein are methods of producing a haploid-inducing strawberry plants by decreasing the expression of one or more CENH3 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 (including, e.g., decreased activity or non-expression) of one or more CENH3 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. 27sf-6744554197072001240

[0080] 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 tetrahaploid (4x) cell or plant, and subjecting the tetrahaploid (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 homozygosity within each subgenome, while only requiring two plant generations to produce. To clarify, the true homozygous octoploid strawberry seed and plant lines of the present disclosure are homozygous within the same subgenome, but can remain heterozygous across the four different subgenomes, respective to each other. 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 are uniform octoploid F1 hybrid strawberry seed, plants, and plant parts thereof produced by crossing two of said true homozygous octoploid strawberry plants. Definitions

[0081] 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, pistil, stigma, 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.

[0082] 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 genera Fragaria.

[0083] 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 28sf-6744554197072001240 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.

[0084] 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.

[0085] As used herein, the term “nuclear genome” refers to the genome present in the nucleus of a plant cell, including, e.g., an egg cell, a sperm cell, or an embryo.

[0086] As used herein, the term “cytoplasmic genome” typically refers to the genome present in the organelles present outside the nucleus of a plant cell, such as the mitochondrial genome or the chloroplast genome of a plant cell.

[0087] 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 “1x”, or the single set of chromosomes itself may be referred to as “1x”. A diploid 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.

[0088] As used herein, “diploid” refers to a cell or organism with a ploidy of 2x.

[0089] 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), tetraploid (4x), pentaploid (5x), hexaploid (6x), septaploid (or heptaploid, 7x), octoploid (8x), or higher ploidies (greater than 8x). 29sf-6744554197072001240

[0090] As used herein, “octoploid” (which may also be spelled “octaploid”) refers to a cell or organism with a ploidy of 8x.

[0091] 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 1n haploid gametes (e.g., pollen and egg cells) where 1n=1x=7. In another example, in an octoploid strawberry plant (2n=8x=56), meiosis in germline cells results in 1n haploid gametes (e.g., pollen and egg cells) where 1n=4x=28. In the latter example, the 4x haploid gametes may also be referred to as tetrahaploid gametes.

[0092] 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, a “haploid-inducing strawberry plant” or “haploid inducer strawberry plant” may also refer to a strawberry plant that produces an egg whose genome is replaced by the genome of an unmodified strawberry plant upon fertilization by the unmodified plant and results in the development of haploid embryos and seeds. 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, in some cases, 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 or is otherwise eliminated, resulting in formation of a haploid embryo. In other cases, when the other sperm cell contacts the reduced egg cell, embryogenesis is initiated, but the maternal nuclear genome is not retained or otherwise eliminated, resulting in formation of a haploid embryo comprising the paternal nuclear 30sf-6744554197072001240 genome. For example, in haploid induction, if both parent plants are octoploid, the haploid (4x) egg cell either (i) does not incorporate the DNA from the haploid sperm cell (4x), resulting in a haploid (4x) embryo comprising the maternal nuclear genome (4x), or (ii) does not retain the DNA from the haploid egg cell (4x), resulting in a haploid (4x) embryo comprising the paternal nuclear genome (4x). 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, however the resulting haploid embryos can contain chromosomes from either the donor plant or the haploid-inducing 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 wherein the nuclear genome undergoes the haploid induction process, resulting in haploid embryos each having one set of chromosomes from the donor octoploid strawberry plant, and lacking nuclear genome from the haploid-inducing strawberry plant. The haploid- inducing plant may be a female, as in the case of female genome elimination and induction of the paternal genome; or the haploid-inducing plant may be a male, as in the case of maternal genome induction. 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 (1n=1x=7) pollen that can be crossed with an octoploid (2n=8x=56) strawberry plant, resulting in tetraploid (1n=4x=28) haploid embryos, also referred to as tetrahaploid embryos.

[0093] 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 a 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 31sf-6744554197072001240 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.

[0094] As used herein, “haplotype” refers to a distinct 1n set of chromosomes with a unique set of alleles. For example, a tetraploid gamete or haploid plant where 1n = 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.

[0095] 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 (1n=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.

[0096] As used herein, “genetically uniform” describes a set of individual plants, plant parts (e.g., seeds), or plant cells whose genomes are clonal. For example, a population of strawberry F1 hybrid seed wherein at least 99% of the population is genetically uniform indicates that 99% of the seeds of the population are clonal across sub-genomes. As used herein, “clonal” may describe a body of DNA that is substantially identical to another body of DNA, however due to random errors in natural DNA replication, the clonal bodies of DNA, clonal plants, plant parts (e.g., seeds), or plant cells may not be completely identical. “Clonal” may also describe two genomes that are not completely identical in sequence but that contain the same set of alleles.

[0097] 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 32sf-6744554197072001240 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 homologous chromosomes within in a subgenome may also be considered homozygous even though one or more alternative alleles exist in one or more different subgenomes.

[0098] 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, a strawberry cell or plant having allele a1 at locus A in one set of chromosomes in a subgenome and having allele a2 at locus A in a second set of chromosomes is heterozygous for alleles a1 and a2. 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.

[0099] 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.

[0100] 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. 33sf-6744554197072001240

[0101] 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 F1 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 F1 hybrid strawberry seed” refers to a genetically uniform population of seed generated from the cross of two true homozygous octoploids or octoploids derived from doubled tetrahaploid strawberry plants or plant lines wherein each parent may possess a different haplotype.

[0102] 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.

[0103] 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 decrease 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 decrease 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 34sf-6744554197072001240 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.

[0104] As used herein, “CENH3 gene” typically refers to a gene encoding “centromere- specific histone 3 variant” (abbreviated as “CENH3”), or a gene having nucleotide or amino acid homology thereto. Genes may include DNA sequences encoding a CENH3 protein or any gene product thereof, for example, nucleic acids (e.g., RNA), post-transcriptionally modified nucleic acids (e.g., spliced RNA, poly-adenylated mRNA), CENH3 protein, and post-translationally modified CENH3 protein. Genes also include any CENH3 gene orthologs, paralogs, and homeologs within a strawberry plant or cell.

[0105] 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, non- protein coding sequences, regulatory sequences, spacer DNA, and the like.

[0106] 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 site- specific breaks, such as double-strand breaks (DSBs) at target loci in the genome. Each site- specific 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

[0107] In one aspect, described herein is a haploid-inducing strawberry plant, or a plant part thereof, 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 CENH3 genes, such as any of the genetic modifications described herein. In some embodiments, provided herein is pollen, seed, pistil, stigma or a stolon of a haploid-inducing strawberry plant. 35sf-6744554197072001240

[0108] In another aspect, described herein is a haploid-inducing plant of the genus Potentilla, or a plant part thereof, that 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 CENH3 genes, such as any of the genetic modifications described herein. In some embodiments, provided herein is pollen, seed, pistil, stigma 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

[0109] 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), tetraploid (4x), pentaploid (5x), hexaploid (6x), septaploid (or heptaploid, 7x), octoploid (8x), or of a higher ploidy (e.g., greater than 8x, e.g., 9x, 10x, 11x, 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.

[0110] 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 × 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.

[0111] 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, or Fragaria vesca ssp. bracteate. The haploid-inducing strawberry plant may be of any variety of Fragaria vesca, for example, Rügen, Alexandria, Hawaii 4, 36sf-6744554197072001240 Baron Solemacher, Weisse Solemacher, Golden Alexandria, Quarantaine de Prin, Blanc Amélioré, Illa Martin, or Gartenfreude.

[0112] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is tetraploid, for example, a tetraploid species or hybrid of the genus Fragaria. Tetraploid species of strawberries include, for example, Fragaria corymbosa, Fragaria gracilis, Fragaria × intermedia, Fragaria moupinensis, Fragaria orientalis, and Fragaria tibetica.

[0113] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, is hexaploid, for example, a hexaploid species or hybrid of the genus Fragaria. Hexaploid species of strawberries include, for example, Fragaria moschata.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] In some embodiments, the haploid-inducing strawberry plant, or plant part thereof, has a ploidy of 10x, for example, a species or hybrid of the genus Fragaria having a ploidy of 10x. Strawberry species having a ploidy of 10x include, for example, Fragaria cascadensis and Fragaria iturupensis.

[0118] 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 37sf-6744554197072001240 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. CENH3 Genes

[0119] 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 CENH3 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 CENH3 genes (e.g., a functional CENH3 protein). The gene products encoded by the CENH3 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 CENH3 gene refers to the wild-type, unmodified function of the gene product (e.g., wild-type, unmodified function of the CENH3 protein). The decreased expression of a CENH3 gene may refer to a decrease in the total amount of a gene product encoded by a CENH3 gene present in a cell (e.g., a decrease in the amount of total CENH3 protein) or to a decrease in the amount of a functional gene product encoded by a CENH3 gene present in a cell (e.g., a decrease in the percentage of CENH3 proteins with wild-type function). In some embodiments, the one or more genetic modifications resulting in decreased expression of one or more CENH3 genes may include, but are not limited to, modification of an enhancer in one or more CENH3 genes, modification of a promoter of one or more CENH3 genes, modification of a coding region in one or more CENH3 genes, modification of an intron in one or more CENH3 genes, modification of methylation status of one or more CENH3 genes, expression of a repressor protein that targets the DNA or an mRNA of one or more CENH3 genes, and expression of an RNA interference construct that targets one or more mRNAs from one or more CENH3 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 CENH3 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 CENH3 genes. In some embodiments, the haploid- inducing strawberry plant, or plant part thereof, may comprise one or more genetic 38sf-6744554197072001240 modifications resulting in decreased activity or function of one or more CENH3 proteins or one or more domains of a CENH3 protein. In some variations, the domain of the CENH3 protein comprise an N-terminal tail, a Histone Fold Domain (HSD), or a combination thereof. The structure of CENH3 proteins, including the N-terminal tail and Histone Fold Domain, have been described previously, for example, in Wang et al. 2019 (Centromere histone H3- and phospholipase-mediated haploid induction in plants. Plant Methods 15, 42).

[0120] 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 CENH3 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) CENH3 genes.

[0121] By way of example only, a CENH3 gene is exemplified by a FvCENH3 gene from diploid strawberry Fragaria vesca, and specifically by the FvCENH3 nucleotide sequences, FvCENH3 protein sequences, and percent identities described herein. FvCENH3 protein (SEQ ID NO: 8) and nucleotide (SEQ ID NO: 1) sequences are provided in Table 1. The locus of FvCENH3 on chromosome 7 in the Fragaria vesca genome is shown in FIG. 4, and a phylogenetic tree showing the relationship between FvCENH3 and CENH3 proteins from other plant species is shown in FIG. 5. FvCENH3 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 SEQ ID NO. 8, or a fragment thereof. Gene products of FvCENH3 genes include FvCENH3 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 FvCENH3 protein of SEQ ID NO. 1, or a fragment thereof.

[0122] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-1a gene from chromosome 7 (subgenome 1, Fvb7-1) of octoploid strawberry Fragaria x ananassa (e.g., variety Camarosa), and specifically by the FaCENH3-1a nucleotide sequences, FaCENH3-1a protein sequences, and percent identities described herein. FaCENH3-1a protein (SEQ ID NO: 9) and FaCENH3-1a nucleotide (SEQ ID NO: 2) sequences are provided in Table 1. The locus of FaCENH3-1a in the Fragaria x ananassa genome is shown in FIG. 3A (top chromosome, Fvb7-1), and a phylogenetic tree showing 39sf-6744554197072001240 the relationship between FaCENH3-1a and CENH3 from other Fragaria x ananassa and other plant species is shown in FIG. 5A-C. FaCENH3-1a 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 FaCENH3-1a gene nucleotide sequence of SEQ ID NO. 2, or a fragment thereof. Gene products of FaCENH3-1a genes also include FaCENH3-1a 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 FaCENH3-1a protein of SEQ ID NO. 9, or a fragment thereof.

[0123] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-1b gene from chromosome 7 (subgenome 1, Fvb7-1) of octoploid strawberry Fragaria x ananassa (e.g., variety Camarosa), and specifically by the FaCENH3-1b nucleotide sequences, FaCENH3-1b protein sequences, and percent identities described herein. FaCENH3-1b protein (SEQ ID NO: 10) and FaCENH3-1b nucleotide (SEQ ID NO: 3) sequences are provided in Table 1. The locus of FaCENH3-1b in the Fragaria x ananassa genome is shown in FIG. 3A (top chromosome, Fvb7-1), and a phylogenetic tree showing the relationship between FaCENH3-1b and CENH3 from other Fragaria x ananassa and other plant species is shown in FIG. 5A-C. FaCENH3-1b 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 FaCENH3-1b gene nucleotide sequence of SEQ ID NO. 3, or a fragment thereof. Gene products of FaCENH3-1b genes also include FaCENH3-1b 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 FaCENH3-1b protein of SEQ ID NO. 10, or a fragment thereof.

[0124] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-2a gene from chromosome 7 (subgenome 2, Fvb7-2) of octoploid strawberry Fragaria x ananassa (e.g., variety Camarosa), and specifically by the FaCENH3-2a nucleotide sequences, FaCENH3-2a protein sequences, and percent identities described herein. FaCENH3-2a protein (SEQ ID NO: 11) and FaCENH3-2a nucleotide (SEQ ID NO: 4) 40sf-6744554197072001240 sequences are provided in Table 1. The locus of FaCENH3-2a in the Fragaria x ananassa genome is shown in FIG. 3A (second chromosome from the top, Fvb7-2), and a phylogenetic tree showing the relationship between FaCENH3-2a and CENH3 from other Fragaria x ananassa and other plant species is shown in FIG. 5A-C. FaCENH3-2a 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 FaCENH3-2a gene nucleotide sequence of SEQ ID NO. 4, or a fragment thereof. Gene products of FaCENH3-2a genes also include FaCENH3-2a 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 FaCENH3-2a protein of SEQ ID NO. 11, or a fragment thereof.

[0125] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-2b gene from chromosome 7 (subgenome 2, Fvb7-2) of octoploid strawberry Fragaria x ananassa (e.g., variety Camarosa), and specifically by the FaCENH3-2b nucleotide sequences, FaCENH3-2b protein sequences, and percent identities described herein. FaCENH3-2b protein (SEQ ID NO: 12) and FaCENH3-2b nucleotide (SEQ ID NO: 5) sequences are provided in Table 1. The locus of FaCENH3-2b in the Fragaria x ananassa genome is shown in FIG. 3A (second chromosome from the top, Fvb7-2), and a phylogenetic tree showing the relationship between FaCENH3-2b and CENH3 from other Fragaria x ananassa and other plant species is shown in FIG. 5A-C. FaCENH3-2b 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 FaCENH3-2b gene nucleotide sequence of SEQ ID NO. 5, or a fragment thereof. Gene products of FaCENH3-2b genes also include FaCENH3-2b 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 FaCENH3-2b protein of SEQ ID NO. 12, or a fragment thereof.

[0126] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-3 gene from chromosome 7 (subgenome 3, Fvb7-3) of octoploid strawberry Fragaria x ananassa (e.g., variety Camarosa), and specifically by the FaCENH3-3 nucleotide sequences, 41sf-6744554197072001240 FaCENH3-3 protein sequences, and percent identities described herein. FaCENH3-3 protein (SEQ ID NO: 13) and FaCENH3-3 nucleotide (SEQ ID NO: 6) sequences are provided in Table 1. The locus of FaCENH3-3 in the Fragaria x ananassa genome is shown in FIG. 3A (third chromosome from the top, Fvb7-3), and a phylogenetic tree showing the relationship between FaCENH3-3 and CENH3 from other Fragaria x ananassa and other plant species is shown in FIG. 5A-C. FaCENH3-3 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 FaCENH3-3 gene nucleotide sequence of SEQ ID NO. 6, or a fragment thereof. Gene products of FaCENH3-3 genes also include FaCENH3-3 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 FaCENH3-3 protein of SEQ ID NO. 13, or a fragment thereof.

[0127] By way of example only, a CENH3-4 gene is further exemplified by a FaCENH3- 4 gene from chromosome 7 (subgenome 2, Fvb7-4) of octoploid strawberry Fragaria x ananassa (e.g., variety Camarosa), and specifically by the FaCENH3-4 nucleotide sequences, FaCENH3-4 protein sequences, and percent identities described herein. FaCENH3-4 protein (SEQ ID NO: 14) and FaCENH3-4 nucleotide (SEQ ID NO: 7) sequences are provided in Table 1. The locus of FaCENH3-4 in the Fragaria x ananassa genome is shown in FIG. 3A (bottom chromosome, Fvb7-4), and a phylogenetic tree showing the relationship between FaCENH3-4 and CENH3 from other Fragaria x ananassa and other plant species is shown in FIG. 5A-C. FaCENH3-4 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 FaCENH3-4 gene nucleotide sequence of SEQ ID NO. 7, or a fragment thereof. Gene products of FaCENH3-4 genes also include FaCENH3-4 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 FaCENH3-4 protein of SEQ ID NO. 14, or a fragment thereof. 42sf-6744554197072001240

[0128] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-7a gene from chromosome 7 (subgenome A) of octoploid strawberry Fragaria x ananassa (e.g., variety Royal Royce), and specifically by the FaCENH3-7a nucleotide sequences, FaCENH3-7a protein sequences, and percent identities described herein. FaCENH3-7a protein (SEQ ID NO: 53) and FaCENH3-7a nucleotide (SEQ ID NOs: 49 and 58) sequences are provided in Table 1. The locus of FaCENH3-7a in the Fragaria x ananassa genome is shown in FIG. 3B (top chromosome, chr_7A), and a phylogenetic tree showing the relationship between FaCENH3-7a and CENH3 proteins from other Fragaria x ananassa and other plant species is shown in FIG. 5D-G. FaCENH3-7a 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 FaCENH3-7a gene nucleotide sequence of SEQ ID NO. 49 or 58, or a fragment thereof. Gene products of FaCENH3-7a genes also include FaCENH3-7a 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 FaCENH3-7a protein of SEQ ID NO. 53, or a fragment thereof.

[0129] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-7b gene from chromosome 7 (subgenome B) of octoploid strawberry Fragaria x ananassa (e.g., variety Royal Royce), and specifically by the FaCENH3-7b nucleotide sequences, FaCENH3-7b protein sequences, and percent identities described herein. FaCENH3-7b protein (SEQ ID NO: 54) and FaCENH3-7b nucleotide (SEQ ID NOs: 50 and 59) sequences are provided in Table 1. The locus of FaCENH3-7b in the Fragaria x ananassa genome is shown in FIG. 3B (top chromosome, chr_7B), and a phylogenetic tree showing the relationship between FaCENH3-7b and CENH3 proteins from other Fragaria x ananassa and other plant species is shown in FIG. 5D-G. FaCENH3-7b 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 FaCENH3-7b gene nucleotide sequence of SEQ ID NO. 50 or 59, or a fragment thereof. Gene products of FaCENH3-7b genes also include FaCENH3-7b 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, 43sf-6744554197072001240 or at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the FaCENH3-7b protein of SEQ ID NO. 54, or a fragment thereof.

[0130] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-7c gene from chromosome 7 (subgenome C) of octoploid strawberry Fragaria x ananassa (e.g., variety Royal Royce), and specifically by the FaCENH3-7c nucleotide sequences, FaCENH3-7c protein sequences, and percent identities described herein. FaCENH3-7c protein (SEQ ID NO: 55) and FaCENH3-7c nucleotide (SEQ ID NOs: 51 and 60) sequences are provided in Table 1. The locus of FaCENH3-7c in the Fragaria x ananassa genome is shown in FIG. 3B (top chromosome, chr_7C), and a phylogenetic tree showing the relationship between FaCENH3-7c and CENH3 proteins from other Fragaria x ananassa and other plant species is shown in FIG. 5D-G. FaCENH3-7c 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 FaCENH3-7c gene nucleotide sequence of SEQ ID NO. 51 or 60, or a fragment thereof. Gene products of FaCENH3-7c genes also include FaCENH3-7c 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 FaCENH3-7c protein of SEQ ID NO. 55, or a fragment thereof.

[0131] By way of example only, a CENH3 gene is further exemplified by a FaCENH3-7d gene from chromosome 7 (subgenome D) of octoploid strawberry Fragaria x ananassa (e.g., variety Royal Royce), and specifically by the FaCENH3-7d nucleotide sequences, FaCENH3-7d protein sequences, and percent identities described herein. FaCENH3-7d protein (SEQ ID NO: 56) and FaCENH3-7d nucleotide (SEQ ID NOs: 52 and 61) sequences are provided in Table 1. The locus of FaCENH3-7d in the Fragaria x ananassa genome is shown in FIG. 3B (top chromosome, chr_7D), and a phylogenetic tree showing the relationship between FaCENH3-7d and CENH3 proteins from other Fragaria x ananassa and other plant species is shown in FIG. 5D-G. FaCENH3-7d 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 FaCENH3-7d gene nucleotide sequence of SEQ ID NO. 52 or 61, or a fragment thereof. Gene products of FaCENH3-7d genes also include 44sf-6744554197072001240 FaCENH3-7d 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 FaCENH3-7d protein of SEQ ID NO. 56, or a fragment thereof.

[0132] The CENH3 gene and protein sequences described herein are reference sequences and may be used by one of ordinary skill in the art to identify CENH3 in any species or variety of strawberry. For example, using methods that are routine in the art, one of ordinary skill in the art could utilize any of the CENH3 gene or protein sequences described herein to query a nucleic acid database or a genome sequence to identify CENH3 genes in the database or genome sequence. Genetic Modifications

[0133] 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.

[0134] 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 45sf-6744554197072001240 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 (including, e.g., decreased activity 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 missense or nonsense 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.

[0135] In certain embodiments, the haploid-inducing strawberry plant, or plant part thereof, comprises one or more genetic modifications resulting in decreased expression decreased expression (including, e.g., decreased activity or non-expression) of a gene product of a genomic locus. In some embodiments, genetic modifications resulting in decreased expression (including, e.g., decreased activity or non-expression) of a gene product or locus may include, but are not limited to, modification of an enhancer, modification of a promoter, 46sf-6744554197072001240 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 (including, e.g., decreased activity 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 (e.g., dCas9), modification of methylation status of gene loci, modification of splicing sites, introducing alternate splicing sites, or any combination thereof.

[0136] In some embodiments, the haploid-inducing strawberry plant comprises one or more genetic modifications resulting in decreased expression (e.g., decreased activity) of one or more CENH3 proteins described herein. In some embodiments, the haploid-inducing strawberry plant comprises one or more genetic modifications of one or more CENH3 genes. In some embodiments, the haploid-inducing strawberry comprises one or more genetic modifications resulting in decreased expression of one or more CENH3 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: 8-14 and 53-56, or fragments thereof. In some embodiments, the haploid-inducing strawberry plant comprises one or more genetic modifications of one or more CENH3 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: 1-7 and 58-61, 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 CENH3 genes, a modification of a promoter of one or more of the CENH3 genes, a modification of a coding region of one or more of the CENH3 genes, a modification of an intron of one or more of the CENH3 genes, a 47sf-6744554197072001240 modification of methylation status of one or more of the CENH3 genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the CENH3 genes, expression of an RNA interference construct that targets an mRNA of one or more of the CENH3 genes, or any combination thereof. In some embodiments, the haploid-inducing strawberry plant has decreased expression of one or more CENH3 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 CENH3 gene products 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 CENH3 gene products. In certain embodiments, the haploid-inducing strawberry plant or plant part further comprises one or more naturally-occurring inactive alleles of one or more CENH3 genes.

[0137] In some embodiments, the decreased expression (e.g., decreased activity) of one or more CENH3 proteins is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , or about 100% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications. In some embodiments, the decreased expression (e.g., decreased activity) of one or more CENH3 proteins is decreased by no more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications. In some embodiments, the decreased expression (e.g., decreased activity) of one or more CENH3 proteins is decreased is decreased by between about 10% and about 100%, between about 20% and about 100%, between about 30% and about 100%, between about 40% and about 100%, between about 50% and about 100%, between about 60% and about 100%, between about 70% and about 100%, between about 80% and about 100%, between about 90% and about 100%, between about 10% and about 90%, between about 20% and about 90%, between about 30% and about 90%, between about 40% and about 90%, between about 50% and about 90%, between about 60% and about 90%, between about 70% and about 90%, between about 80% and about 90%, between about 10% and about 80%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 60% and 48sf-6744554197072001240 about 80%, between about 70% and about 80%, 10% and about 70%, between about 20% and about 70%, between about 30% and about 70%, between about 40% and about 70%, between about 50% and about 70%, between about 60% and about 70%, between about 10% and about 60%, between about 20% and about 60%, between about 30% and about 60%, between about 40% and about 60%, between about 50% and about 60%, between about 10% and about 50%, between about 20% and about 50%, between about 30% and about 50%, between about 40% and about 50%, between about 10% and about 40%, between about 20% and about 40%, between about 30% and about 40%, between about 10% and about 30%, between about 20% and about 30%, or about 10% and 20% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications.

[0138] 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 CENH3 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 FvCENH3 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 FvCENH3 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 CENH3 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 FvCENH3 protein of SEQ ID NO. 8, or a fragment thereof.

[0139] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria vesca comprising one or more genetic modifications of an FvCENH3 gene or gene 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 CENH3 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 FvCENH3 gene nucleotide sequence of SEQ ID NO. 1, 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 49sf-6744554197072001240 FvCENH3 gene. In some variations, the one or more genetic modifications comprise a modification of an enhancer of FvCENH3, a modification of a promoter of FvCENH3, a modification of a coding region of FvCENH3, modification of an intron of FvCENH3, 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 FvCENH3 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 FvCENH3 protein.

[0140] 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 FaCENH3 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 FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, an FaCENH3-4 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 FaCENH3-1a gene, an FaCENH3- 1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and an FaCENH3-4 gene.

[0141] 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 FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3-2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, an FaCENH3-4 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 FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3- 2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, and an FaCENH3-4 protein. 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 one or more CENH3 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 50sf-6744554197072001240 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: 9-14, 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 CENH3 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: 9; 2) a CENH3 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: 10; 3) a CENH3 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: 11; 4) a CENH3 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: 12; 5) a CENH3 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: 13; and / or 5) a CENH3 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: 14.

[0142] 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 FaCENH3-7a protein, an FaCENH3-7b protein, an FaCENH3-7c protein, an FaCENH3-7d 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 one or more CENH3 51sf-6744554197072001240 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: 53-56, 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 CENH3 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: 53; 2) a CENH3 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: 54; 3) a CENH3 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: 55; and / or 4) a CENH3 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: 56.

[0143] 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 FaCENH3- 1a gene or gene product, an FaCENH3-1b gene or gene product, an FaCENH3-2a gene or gene product, an FaCENH3-2b gene or gene product, an FaCENH3-3 gene or gene product, an FaCENH3-4 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 FaCENH3-1a gene or gene product, an FaCENH3- 1b gene or gene product, an FaCENH3-2a gene or gene product, an FaCENH3-2b gene or gene product, an FaCENH3-3 gene or gene product, and an FaCENH3-4 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 CENH3 gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 52sf-6744554197072001240 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: 2-71, 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 CENH3 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: 2, or a fragment thereof; 2) one or more genetic modifications in a CENH3 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: 3, or a fragment thereof; 3) one or more genetic modifications in a CENH3 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: 4, or a fragment thereof; 4) one or more genetic modifications in a CENH3 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: 5, or a fragment thereof; 5) one or more genetic modifications in a CENH3 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: 6, or a fragment thereof; and / or 6) one or more genetic modifications in a CENH3 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: 7, or a fragment thereof. In some variations, the one or more genetic modifications comprise a modification of an enhancer of an FaCENH3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 gene), a modification of a promoter of an FaCENH3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 gene), a modification of a coding region of an FaCENH3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 gene), modification of an intron of an 53sf-6744554197072001240 FaCENH3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 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).

[0144] 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 FaCENH3- 7a gene or gene product, an FaCENH3-7b gene or gene product, an FaCENH3-7c gene or gene product, an FaCENH3-7d gene or gene product, 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 in a CENH3 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: 58-61, 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 CENH3 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: 58, or a fragment thereof; 2) one or more genetic modifications in a CENH3 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: 59, or a fragment thereof; 3) one or more genetic modifications in a CENH3 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: 60, or a fragment thereof; and / or 4) one or more genetic modifications in a CENH3 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: 61, or a fragment thereof. In some variations, the one or more genetic modifications comprise a modification of an enhancer of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an 54sf-6744554197072001240 FaCENH3-7c gene, and / or an FaCENH3-7d gene), a modification of a promoter of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d gene), a modification of a coding region of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3- 7d gene), modification of an intron of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d 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). In some embodiments, the one or more genetic modifications comprise one or more deletions, insertions, or nucleotide substitutions in a coding region of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d gene).

[0145] In some embodiments, the one or more genetic modifications in a CENH3 gene each independently comprise an insertion, a deletion, one or more nucleotide changes, or an inversion that results in decreased expression (e.g., decreased activity) of the CENH3 gene. In certain embodiments, the insertion, the deletion, the one or more nucleotide changes, or the inversion eliminates expression (e.g., eliminates activity) of the CENH3 gene. In certain embodiments, the insertion, the deletion, the one or more nucleotide changes, or the inversion decreases, but does not eliminate, expression of the CENH3 gene. In some variations, the insertion, the deletion, the one or more nucleotide changes, or the inversion is positioned in the first 70%, the first 60%, the first 50%, the first 40%, the first 30%, the first 20%, or the first 10% of the nucleotides of the CENH3 gene (e.g., of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7). In certain variations, the insertion, the deletion, the one or more nucleotide changes, or the inversion is positioned in the first 100, the first 200, the first 300, the first 400, the first 500, the first 600, the first 700, the first 800, the first 900, the first 1000, the first 1250, the first 1500, the first 1750, the first 2000, the first 2500, or the first 3000 nucleotides of the CENH3 gene (e.g., of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7). In some embodiments, the insertion, the deletion, the one or more nucleotide changes, or the inversion eliminates expression (e.g., eliminates activity) of the CENH3 gene. In some variations, the insertion, the deletion, the one or more nucleotide changes, or the inversion results in a premature stop codon present in the first 70%, the first 60%, the first 50%, the first 40%, the first 30%, the first 20%, or the first 10% of the nucleotides of a polynucleotide sequence selected from the 55sf-6744554197072001240 group consisting of SEQ ID NOs: 1-7 or of the nucleotides of the coding sequence of the CENH3 gene following the start codon in the 3’ direction, thereby eliminating expression (e.g., activity) of the CENH3 gene. In some variations, the insertion, the deletion, the one or more nucleotide changes, or the inversion results in a premature stop codon present in the first 100, the first 200, the first 300, the first 400, the first 500, the first 600, the first 700, the first 800, the first 900, the first 1000, the first 1250, the first 1500, the first 1750, the first 2000, the first 2500, or the first 3000 nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7 or of the coding sequence of the CENH3 gene following the start codon in the 3’ direction, thereby eliminating expression (e.g., activity) of the CENH3 gene.

[0146] In some embodiments, the haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa and has decreased expression of an FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3-2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, an FaCENH3-4 protein, or any combination (e.g., two, three, four, five or all six) thereof relative to 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 haploid- inducing strawberry plant is a plant of the species Fragaria x ananassa and lacks detectable expression of an FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3-2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, an FaCENH3-4 protein, or any combination (e.g., two, three, four, five or all six) thereof.

[0147] The haploid-inducing strawberry plant, or plant part thereof, may comprise one or more genetic modifications resulting in decreased expression of any combination of CENH3 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 CENH3 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, non- expression, or a combination thereof of any combination of CENH3 genes described here or known in the art. In certain embodiments, the haploid-inducing strawberry plant or plant part lacks detectable expression of CENH3 proteins.

[0148] In some embodiments, the haploid-inducing strawberry plant or plant part thereof has decreased expression (including decreased activity) of any of the CENH3 genes and / or 56sf-6744554197072001240 CENH3 proteins described herein relative to a control strawberry plant. For example, in some embodiments, the haploid-inducing strawberry plant or plant part thereof has decreased expression (e.g., decreased activity) of one or more CENH3 genes and / or one or more CENH3 proteins, wherein the decreased expression (e.g., decreased activity) of one or more CENH3 proteins CENH3 genes and / or is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% , or about 100% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications. In some embodiments, the haploid-inducing strawberry plant or plant part thereof has decreased expression (e.g., decreased activity) of one or more CENH3 genes and / or one or more CENH3 proteins, wherein the decreased expression (e.g., decreased activity) of CENH3 genes and / or one or more CENH3 proteins is decreased by no more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications. In some embodiments, the haploid- inducing strawberry plant or plant part thereof has decreased expression (e.g., decreased activity) of one or more CENH3 genes and / or one or more CENH3 proteins, wherein the decreased expression (e.g., decreased activity) of the one or more CENH3 genes and / or one or more CENH3 proteins is decreased is decreased by between about 10% and about 100%, between about 20% and about 100%, between about 30% and about 100%, between about 40% and about 100%, between about 50% and about 100%, between about 60% and about 100%, between about 70% and about 100%, between about 80% and about 100%, between about 90% and about 100%, between about 10% and about 90%, between about 20% and about 90%, between about 30% and about 90%, between about 40% and about 90%, between about 50% and about 90%, between about 60% and about 90%, between about 70% and about 90%, between about 80% and about 90%, between about 10% and about 80%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 60% and about 80%, between about 70% and about 80%, 10% and about 70%, between about 20% and about 70%, between about 30% and about 70%, between about 40% and about 70%, between about 50% and about 70%, between about 60% and about 70%, between about 10% and about 60%, between about 20% and about 60%, between about 30% and about 60%, between about 40% and about 60%, between about 50% and about 60%, between about 10% and about 50%, between about 20% and about 50%, between about 30% and about 50%, between about 40% and about 50%, between about 10% and about 40%, between about 20% and about 40%, between 57sf-6744554197072001240 about 30% and about 40%, between about 10% and about 30%, between about 20% and about 30%, or about 10% and 20% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications. 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 CENH3 proteins relative to a strawberry plant of the same species lacking the one or more genetic modifications. Characteristics of Strawberry Plants

[0149] The strawberry plants (e.g., haploid-inducing strawberry plants) described herein having one or more genetic modifications resulting in decreased expression of one or more CENH3 genes may have characteristics that differ from those of a strawberry plant lacking the one or more genetic modifications. Without wishing to be bound by theory, it is believed that decreased expression of one or more CENH3 genes may result in certain changes in mitotic and meiotic activity in plants.

[0150] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have decreased pollen germination (e.g., a decreased pollen germination rate) than a control strawberry plant lacking the one or more genetic modifications. In some embodiments, the strawberry plant has a pollen germination rate of less than 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.

[0151] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have decreased flower diameter as compared to a control strawberry plant lacking the one or more genetic modifications. In some embodiments, the strawberry plant has a flower diameter of less than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 mm. In some embodiments, the strawberry plant has a flower diameter of between 5 mm and 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 mm. 58sf-6744554197072001240

[0152] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have decreased anther width as compared to a control strawberry plant lacking the one or more genetic modifications. In some embodiments, the strawberry plant has a anther width of less than 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 mm. In some embodiments, the strawberry plant has a anther width of between 0.1 mm and 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 mm.

[0153] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have decreased filament length as compared to a control strawberry plant lacking the one or more genetic modifications. In some embodiments, the strawberry plant has a filament length of less than 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 mm. In some embodiments, the strawberry plant has a filament length of less between 0.1 mm and 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 mm.

[0154] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have delayed microspore formation as compared to a control strawberry plant lacking the one or more genetic modifications. In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have larger bud size at the time of microspore formation as compared to a control strawberry plant lacking the one or more genetic modifications. In some embodiments, the strawberry plant has an average bud size at the time of microspore formation of greater than 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.35.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 at the time of microspore formation. In certain embodiments, the strawberry plant has an average bud size at the time of microspore formation of greater than 4.7 at the time of microspore formation. In certain embodiments, the strawberry plant has an average bud size at the time of microspore formation of greater than 5.0 at the time of microspore formation.

[0155] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have buds with observable tetrads, e.g., as shown in FIGS. 16E and 16F. In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have observable tetrads in at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of buds. 59sf-6744554197072001240

[0156] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have fewer seeds per berry as compared to a control strawberry plant lacking the one or more genetic modifications. In some embodiments, the strawberry plant has fewer than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 seeds per berry. In some embodiments, the strawberry plant has between 1 and 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 seeds per berry.

[0157] In some embodiments, the strawberry plants (e.g., haploid-inducing strawberry plants) described herein have reduced cell growth as compared to a control strawberry plant lacking the one or more genetic modifications, for example, as measured by the number of microcalli regenerated from protoplasts per area of growth medium, or per lens as measured using microscopy. Plant Parts of Haploid-inducing Strawberry Plants

[0158] 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, an anther, 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.

[0159] 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 CENH3 genes described herein. In certain embodiments, the pollen comprises one or more genetic modifications in one or more CENH3 genes described herein.

[0160] 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 CENH3 genes described herein. In certain embodiments, the stolon comprises one or more genetic modifications in one or more CENH3 genes described herein.

[0161] 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 60sf-6744554197072001240 expression of one or more CENH3 genes described herein. In certain embodiments, the seed comprises one or more genetic modifications in one or more CENH3 genes described herein.

[0162] 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 CENH3 genes described herein. In certain embodiments, the achene comprises one or more genetic modifications in one or more CENH3 genes described herein.

[0163] 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 CENH3 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 CENH3 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 CENH3 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: 8-14 and 53-56, 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 CENH3 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: 1-7 and 58-61, 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 CENH3 genes, a modification of a promoter of one or more of the CENH3 genes, a modification of a coding region of one or more of the CENH3 genes, a modification of an intron of one or more of the CENH3 genes, a modification of methylation status of one or more of the CENH3 genes, expression of a repressor protein that targets the DNA or an mRNA of one or more of the CENH3 genes, expression of an RNA interference construct that targets an mRNA of one or more of the CENH3 genes, or any combination thereof. In some embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry 61sf-6744554197072001240 plant has decreased expression of one or more CENH3 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 CENH3 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 CENH3 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 CENH3 genes.

[0164] 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 CENH3 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 FvCENH3 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 FvCENH3 protein. 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 a CENH3 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 FvCENH3 protein of SEQ ID NO. 8, or a fragment thereof.

[0165] 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 FvCENH3 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 FvCENH3 gene comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 62sf-6744554197072001240 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the FvCENH3 gene nucleotide sequence of SEQ ID NO. 1, 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 FvCENH3 gene. In some variations, the one or more genetic modifications comprise a modification of an enhancer of FvCENH3, a modification of a promoter of FvCENH3, a modification of a coding region of FvCENH3, modification of an intron of FvCENH3, 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 FvCENH3 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 FvCENH3 protein.

[0166] 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 CENH3 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 FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, an FaCENH3-4 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 FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and an FaCENH3-4 gene.

[0167] 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 FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3-2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, an FaCENH3-4 protein, or any combination thereof. In certain embodiments, the 63sf-6744554197072001240 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 FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3-2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, and an FaCENH3-4 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 CENH3 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: 9-14, 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 CENH3 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: 9; 2) a CENH3 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: 10; 3) a CENH3 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: 11; and / or 4) a CENH3 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: 12; 5) a CENH3 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: 13; and / or 6) a CENH3 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 64sf-6744554197072001240 least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 14.

[0168] 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 FaCENH3-7a protein, an FaCENH3-7b protein, an FaCENH3-7c protein, an FaCENH3-7d 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 one or more CENH3 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: 53-56, 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 CENH3 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: 53; 2) a CENH3 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: 54; 3) a CENH3 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: 55; and / or 4) a CENH3 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: 56.

[0169] 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 65sf-6744554197072001240 genetic modifications of an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, an FaCENH3-4 gene, 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 FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and an FaCENH3-4 gene. 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 CENH3 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: 2-7, 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 CENH3 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: 2, or a fragment thereof; 2) one or more genetic modifications in a CENH3 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: 3, or a fragment thereof; 3) one or more genetic modifications in a CENH3 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: 4, or a fragment thereof; 4) one or more genetic modifications in a CENH3 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: 5, or a fragment thereof; 5) one or more genetic modifications in a CENH3 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: 6, or a fragment thereof; and / or 6) one or more genetic modifications in a CENH3 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%, 66sf-6744554197072001240 98%, 99%, or 100% sequence identity or complementarity to the nucleotide sequence of SEQ ID NO: 7, or a fragment thereof. In some variations, the one or more genetic modifications comprise a modification of an enhancer of an FaCENH3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 gene), a modification of a promoter of an Facenh3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 gene), a modification of a coding region of an Facenh3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 gene), modification of an intron of an Facenh3 gene (e.g., an FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, and / or an FaCENH3-4 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).

[0170] 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 FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, an FaCENH3-7d 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 in a CENH3 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: 58-61, 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 CENH3 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: 58, or a fragment thereof; 2) one or more genetic modifications in a CENH3 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: 59, or a fragment thereof; 3) one or more genetic 67sf-6744554197072001240 modifications in a CENH3 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: 60, or a fragment thereof; and / or 4) one or more genetic modifications in a CENH3 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: 61, or a fragment thereof. In some variations, the one or more genetic modifications comprise a modification of an enhancer of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d gene), a modification of a promoter of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d gene), a modification of a coding region of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d gene), modification of an intron of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d 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). In some embodiments, the one or more genetic modifications comprise one or more deletions, insertions, or nucleotide substitutions in a coding region of an FaCENH3 gene (e.g., an FaCENH3-7a gene, an FaCENH3-7b gene, an FaCENH3-7c gene, and / or an FaCENH3-7d gene).

[0171] 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 FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3-2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, an FaCENH3-4 protein, or any combination (e.g., two, three, four, five or all six) 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 an FaCENH3-1a protein, an FaCENH3-1b protein, an FaCENH3-2a protein, an FaCENH3-2b protein, an FaCENH3-3 protein, an FaCENH3-4 protein, or any combination (e.g., two, three, four, five or all six) thereof. 68sf-6744554197072001240

[0172] 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 CENH3 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 CENH3 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 CENH3 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 CENH3 proteins.

[0173] In some embodiments, the pollen, stolon, seed, or achene of the haploid-inducing strawberry plant has decreased expression of CENH3 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 CENH3 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

[0174] 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 CENH3 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 CENH3 genes into a strawberry plant by crossing with a plant having the one or more naturally occurring inactive alleles of one or more CENH3 genes. 69sf-6744554197072001240 Methods of Introducing Genetic Modifications

[0175] 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 CENH3 genes. In certain embodiments, the decreased expression is achieved by gene disruption (e.g., disruption of one or more CENH3 genes), gene knockout (e.g., knockout of one or more CENH3 genes), gene knockdown (e.g., knockdown of one or more CENH3 genes), gene silencing (e.g., silencing of one or more CENH3 genes), RNA interference (e.g., RNA interference of one or more CENH3 genes), induction of methylation (e.g., induction of methylation of one or more CENH3 genes), or any combination thereof. In some embodiments, the method comprises introducing one or more of the genetic modifications by gene editing using a site-directed nuclease.

[0176] 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 CENH3 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.

[0177] 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 Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, 70sf-6744554197072001240 Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, 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.

[0178] 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.

[0179] 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 CENH3 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.

[0180] 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 71sf-6744554197072001240 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.

[0181] 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 least 14, 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.

[0182] 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.

[0183] 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 72sf-6744554197072001240 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.).

[0184] 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 to target 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.

[0185] 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 Tnp1 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. 73sf-6744554197072001240

[0186] 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, which contribute 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.

[0187] 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. 74sf-6744554197072001240

[0188] 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.

[0189] 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, AlwI, MlyI, SbfI, SdaI, 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 double- stranded 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.

[0190] 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. 75sf-6744554197072001240

[0191] 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).

[0192] 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.

[0193] A site-specific nuclease may be a meganuclease. Meganucleases, which are commonly identified in microbes, such as the LAGLIDADG 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-CreI, I-CeuI, I-MsoI, I-SceI, I-AniI, and I-DmoI. 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 76sf-6744554197072001240 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.

[0194] 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 CENH3 gene) at or near a recognition site (e.g., a recognition site within a CENH3 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 CENH3 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 CENH3 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 CENH3 gene) at or near a recognition site (e.g., a recognition site within a CENH3 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 CENH3 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 CENH3 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.

[0195] As used herein, a repair nucleic acid molecule is a single-stranded or double- stranded DNA molecule or RNA molecule that is used as a template for modification of the 77sf-6744554197072001240 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 end- joining (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.

[0196] In some embodiments, the genetic modifications introduced by gene editing result in the decreased expression (including, e.g., decreased activity or non-expression) of one or more CENH3 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 CENH3 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 (including, e.g., decreased activity 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 CENH3 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 CENH3 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., CENH3 protein). For example, the site of the DSB or nick within the endogenous locus may be in the upstream or 5' region of the CENH3 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 CENH3 gene may be generated with a donor template molecule to direct a particular or desired mutation at 78sf-6744554197072001240 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 CENH3 gene may be achieved by substituting, inserting, deleting or inverting at least a portion of the CENH3 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 CENH3 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.

[0197] 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 of interest, 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 79sf-6744554197072001240 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 CENH3 gene. In other variations, the recognition site is within a neutral (e.g., non-coding) site within the genome.

[0198] 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 the plant 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 calcium / polyethylene 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.

[0199] 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 80sf-6744554197072001240 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 CENH3 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.

[0200] 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 of cells 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.

[0201] 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 CENH3 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 CENH3 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 81sf-6744554197072001240 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 CENH3 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: 8-14 and 53-56. 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: 1-7 and 58-61. 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 one or more CENH3 genes described herein, a promoter of one or more CENH3 genes described herein, a coding region of one or more CENH3 genes, or an intron of one or more of the CENH3 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: 25-34. 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, SEQ ID NO: 29, or SEQ ID NO: 31.

[0202] 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 FvCENH3 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 82sf-6744554197072001240 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 FvCENH3 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 CENH3 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: 8. 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: 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 an enhancer of FvCENH3, a promoter of FvCENH3, a coding region of FvCENH3, or an intron of FvCENH3.

[0203] 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, SEQ ID NO: 29, SEQ ID NO: 31, 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, wherein a second portion of 83sf-6744554197072001240 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: 29; and wherein a third 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: 31.

[0204] 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 FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, an FaCENH3-4 gene, or any combination (e.g., two, three, four, five or all six) 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 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 FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, an FaCENH3-4 gene, or any combination (e.g., two, three, four, five or all six) 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 CENH3 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: 9-14 and 53-56. 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, 84sf-6744554197072001240 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: 2-7 and 58-61. 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 FaCENH3-1a gene, an FaCENH3-1b gene, an FaCENH3-2a gene, an FaCENH3-2b gene, an FaCENH3-3 gene, an FaCENH3-4 gene, or any combination (e.g., two, three, four, five or all six) thereof.

[0205] 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, SEQ ID NO: 29, or SEQ ID NO: 31, 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 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: 29, and wherein a third 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: 31.

[0206] In some embodiments, the decreased expression of each of the one or more of the CENH3 genes is independently achieved by introducing an insertion, a deletion, one or more nucleotide changes, or an inversion into the CENH3 gene that that results in decreased expression of the CENH3 gene. In some variations, the insertion, the deletion, the one or 85sf-6744554197072001240 more nucleotide changes, or the inversion is introduced into the first 70%, the first 60%, the first 50%, the first 40%, the first 30%, the first 20%, or the first 10% of the nucleotides of the CENH3 gene (e.g., a sequence selected from the group consisting of SEQ ID NOs: 1-7). In certain variations, the insertion, the deletion, the one or more nucleotide changes, or the inversion is introduced into the first 100, the first 200, the first 300, the first 400, the first 500, the first 600, the first 700, the first 800, the first 900, the first 1000, the first 1250, the first 1500, the first 1750, the first 2000, the first 2500, or the first 3000 nucleotides of the CENH3 gene (e.g., a sequence selected from the group consisting of SEQ ID NOs: 1-7). In some variations, the insertion, the deletion, the one or more nucleotide changes, or the inversion decreases, but does not eliminate, expression of the CENH3 gene. In some variations, the insertion, the deletion, the one or more nucleotide changes, or the inversion eliminates expression (e.g., eliminates activity) of the CENH3 gene. In some variations, the activity of the CENH3 gene is eliminated by a premature stop codon introduced into the first 70%, the first 60%, the first 50%, the first 40%, the first 30%, the first 20%, or the first 10% of a sequence selected from the group consisting of SEQ ID NOs: 1-7 or of the nucleotides of the coding sequence of the CENH3 gene following the start codon in the 3’ direction. In certain variations, the activity of the CENH3 gene is eliminated by a premature stop codon introduced into the first 100, the first 200, the first 300, the first 400, the first 500, the first 600, the first 700, the first 800, the first 900, the first 1000, the first 1250, the first 1500, the first 1750, the first 2000, the first 2500, or the first 3000 nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 1-7 or of the coding sequence of the CENH3 gene following the start codon in the 3’ direction. Compositions for Producing Haploid-inducing Strawberry Plants

[0207] 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.

[0208] 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) 86sf-6744554197072001240 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 CENH3 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: 8-14 and 53-56. 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 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: 1-7 and 58-61. 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.

[0209] 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 CENH3 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, 87sf-6744554197072001240 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: 8- 14 and 53-56. 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: 1-7 and 58-61. 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 CENH3 genes described herein, a promoter of one or more CENH3 genes described herein, a coding region of one or more CENH3 genes, or an intron of one or more of the CENH3 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: 17, SEQ ID NO: 19, SEQ ID NO: 21, or any combination thereof. 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: 17, SEQ ID NO: 19, SEQ ID NO: 21, or any combination thereof. 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: 17, 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: 19, and a third 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: 21. In some embodiments, the expression vector further comprises a DNA sequence encoding a Cas nuclease operably linked to a promoter. 88sf-6744554197072001240

[0210] 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 CENH3 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: 8-14 and 53-56. 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: 1-7 and 58-61.

[0211] 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 CENH3 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: 8-14 and 53-56. 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 89sf-6744554197072001240 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: 1-7 and 58-61. In some embodiments, the isolated DNA molecule is a synthetic oligo nucleotide.

[0212] 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 CENH3 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: 8-14 and 53-56. 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: 25-34. 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-24. 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: 17, SEQ ID NO: 19, or SEQ ID NO: 21, or a combination thereof. 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 90sf-6744554197072001240 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, SEQ ID NO: 29, or SEQ ID NO: 31, or a combination thereof. In certain embodiments, the isolated nucleic acid molecule further comprises a nucleic acid sequence for association with a CRISPR-associated (Cas) enzyme.

[0213] 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.

[0214] 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.

[0215] 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. In some embodiments, the kit further comprises instructions for using the kit components to produce a haploid-inducing strawberry plant.

[0216] 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

[0217] In some aspects, described herein are methods of producing true homozygous octoploid strawberry plant, comprising (a) generating a tetrahaploid strawberry cell or plant 91sf-6744554197072001240 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 (a1) 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 (a1) contacting a tetraploid 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 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 strawberry plant from the doubled tetrahaploid. Haploid Induction

[0218] 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 gametes 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. During a haploid induction cross beteen a non-haploid inducing plant 92sf-6744554197072001240 and a haploid-inducing plants with reduced expression of CENH3, embryogenesis is initiated in the egg cell with only the haploid genome of the male or female gamete, resulting in a haploid embryo. When pollen from a haploid-inducing plant is used to pollenate a non- haploid-inducing plant, 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 or retained in the egg cell, resulting in formation of a haploid embryo having a nuclear genome derived from only the maternal plant, and not the paternal plant. For example, in haploid induction using pollen from the haploid-inducing plant, 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 tetrahaploid. By contrast, when pollen from a non-haploid inducing plant is used to pollenate a haploid-inducing plant having decreased expression of CENH3, the sperm cells from the non-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 and the other sperm cell fertilizes the egg. Fertilization of the haploid-inducing egg cell with the non-haploid-inducing sperm cell results in retention of the paternal nuclear DNA present in the sperm cell, but elimination of the maternal nuclear DNA present in the egg cell. This results in resulting in formation of a haploid embryo having a nuclear genome derived from only the paternal plant, and a cytoplasmic genome (e.g., mitochondrial and / or chloroplast genome) derived from the maternal plant. In this scenario, if both the maternal haploid-inducing parent and the paternal parent are octoploid, haploid induction results in a tetrahaploid (4x) embryo.

[0219] 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 93sf-6744554197072001240 cell, thereby contacting the reduced egg cell with the sperm cells. 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. 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 tetraploid embryo and an endosperm having a ploidy of greater than 8x (e.g., a ploidy of 9x, 10x, 11x, or 12x).

[0220] 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 an egg cell from a haploid-inducing strawberry plant described herein. In certain embodiments, the tetrahaploid cell is a reduced sperm cell. In some embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises contacting the stigma of a pistil of the haploid-inducing strawberry plant with the pollen 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) reduced sperm cells of the pollen to the egg cell, thereby contacting the egg cell with the reduced sperm cells. In some embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises allowing the haploid-inducing strawberry plant to form a plurality 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 tetraploid embryo and an endosperm having a ploidy of greater than 8x (e.g., a ploidy of 9x, 10x, 11x, or 12x). In some embodiments, the haploid-inducing strawberry plant and the donor octoploid strawberry plants are from different species of Fragaria. In some embodiments, the haploid- inducing strawberry plant is diploid. In certain embodiments, the haploid-inducing strawberry 94sf-6744554197072001240 plant is a plant of the species Fragaria vesca. In some embodiments, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, the embryo of the true homozygous octoploid strawberry seed has a nuclear genome derived from only the donor octoploid strawberry plant and a cytoplasmic genome (e.g., mitochondrial and / or chloroplast genome) derived from the haploid-inducing strawberry plant.

[0221] 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 (1x) pollen or egg cells that can be crossed with an octoploid (8x) strawberry donor plant, resulting in tetraploid (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, tetraploid, pentaploid, hexaploid, septaploid, 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 (a1) contacting a reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a diploid, triploid, tetraploid, pentaploid, hexaploid, septaploid, 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, tetraploid, pentaploid, hexaploid, septaploid, 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 (a1) contacting a tetrahaploid reduced egg cell from a donor octoploid strawberry plant with a sperm cell from a diploid, triploid, tetraploid, pentaploid, hexaploid, septaploid, 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 an octoploid doubled tetrahaploid strawberry plant. 95sf-6744554197072001240

[0222] 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 an egg cell from a diploid, triploid, tetraploid, pentaploid, hexaploid, septaploid, 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 (a1) contacting a reduced sperm cell from a donor octoploid strawberry plant with an egg cell from a diploid, triploid, tetraploid, pentaploid, hexaploid, septaploid, 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 an egg cell from a diploid, triploid, tetraploid, pentaploid, hexaploid, septaploid, 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 (a1) contacting a tetrahaploid reduced sperm cell from a donor octoploid strawberry plant with an egg cell from a diploid, triploid, tetraploid, pentaploid, hexaploid, septaploid, 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 an octoploid doubled tetrahaploid strawberry plant. In some embodiments, the haploid-inducing strawberry plant and the donor octoploid strawberry plants are from different species of Fragaria. 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. In some embodiments, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, the embryo of the true homozygous octoploid strawberry seed has a nuclear genome derived from only the donor octoploid strawberry plant and a cytoplasmic genome (e.g., mitochondrial and / or chloroplast genome) derived from the haploid-inducing strawberry plant.

[0223] 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 96sf-6744554197072001240 from the haploid cell. In certain embodiments, the method comprises (a1) 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 (a1) 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 × 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.

[0224] 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 an egg cell 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 (a1) contacting a reduced sperm cell from a donor octoploid strawberry plant with an egg 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 an egg cell 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 (a1) contacting a 97sf-6744554197072001240 tetrahaploid reduced sperm cell from a donor octoploid strawberry plant with an egg 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 × 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. In some embodiments, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, the embryo of the true homozygous octoploid strawberry seed has a nuclear genome derived from only the donor octoploid strawberry plant (e.g., Fragaria x ananassa) and a cytoplasmic genome (e.g., mitochondrial and / or chloroplast genome) derived from the haploid-inducing strawberry plant (e.g., Fragaria vesca).

[0225] 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 (a1) 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 (a1) 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 98sf-6744554197072001240 embodiments, the octoploid haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa or Fragaria chiloensis. In certain embodiments, the octoploid haploid- inducing strawberry plant is a plant of the species Fragaria x ananassa.

[0226] 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 an egg cell 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 (a1) contacting a reduced sperm cell from a donor octoploid strawberry plant with an egg 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 an egg cell 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 (a1) contacting a tetrahaploid reduced sperm cell from a donor octoploid strawberry plant with an egg 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 species Fragaria x ananassa or Fragaria chiloensis. In certain embodiments, the octoploid haploid-inducing strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, the haploid-inducing strawberry plant and the donor octoploid strawberry plants are from different species of Fragaria. In some embodiments, the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa. In some embodiments, the embryo of the true homozygous octoploid strawberry seed has a nuclear genome derived from only the donor octoploid strawberry plant (e.g., Fragaria x ananassa) and a cytoplasmic genome (e.g., mitochondrial and / or chloroplast genome) derived from the haploid-inducing strawberry plant.

[0227] 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 99sf-6744554197072001240 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 (a1) 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 octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (a1) 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.

[0228] 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 an egg 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; and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (a1) contacting a reduced sperm cell from a donor octoploid strawberry plant with 100sf-6744554197072001240 an egg 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 an egg 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; and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (a1) contacting a tetrahaploid reduced sperm cell from a donor octoploid strawberry plant with an egg 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. In some embodiments, the embryo of the true homozygous octoploid strawberry seed has a nuclear genome derived from Fragaria x ananassa and a cytoplasmic genome (e.g., mitochondrial and / or chloroplast genome) derived from Fragaria vesca.

[0229] 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 (a1) 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 101sf-6744554197072001240 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 (a1) 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.

[0230] 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 an egg 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; and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (a1) contacting a reduced sperm cell from a donor octoploid strawberry plant with an egg 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 an egg 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; and (b) producing an octoploid doubled tetrahaploid cell or plant from the tetrahaploid cell. In certain embodiments, the method comprises (a1) contacting a tetrahaploid reduced sperm cell from a donor octoploid strawberry plant with an egg 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 102sf-6744554197072001240 certain embodiments, both the donor octoploid strawberry plant and the haploid-inducing strawberry plant are plants of the species Fragaria x ananassa.

[0231] 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 plants. 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 plants. 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 plant growth, and allowing the one or more embryos to form one or more plants. 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 plant growth, and allowing the plurality of embryos to form a plurality of plants. The method may further comprise a step of selecting one or more tetrahaploid plants by 1) determining the ploidy of one or more cells of one or more plants of the plurality of plants, and 2) selecting one or more plants having one or more tetrahaploid cells from the plurality of plants. 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 plants of the plurality of plants, and 2) selecting one or more plants having one or more tetraploid cells from the plurality of plants. In certain embodiments, determining the ploidy of one or more cells of the one or more plants comprises obtaining a tissue sample of each of the one or more plants and determining the ploidy of one or more cells of each tissue sample.

[0232] In some embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises allowing the haploid-inducing 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 plants. In certain embodiments, the step of generating a tetrahaploid strawberry plant through haploid induction comprises allowing the haploid-inducing 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 plants. In other embodiments, the step of 103sf-6744554197072001240 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 plant growth, and allowing the one or more embryos to form one or more plants. 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 plant growth, and allowing the plurality of embryos to form a plurality of plants. The method may further comprise a step of selecting one or more tetrahaploid plants by 1) determining the ploidy of one or more cells of one or more plants of the plurality of plants, and 2) selecting one or more plants having one or more tetrahaploid cells from the plurality of plants. 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 plants of the plurality of plants, and 2) selecting one or more plants having one or more tetraploid cells from the plurality of plants. In certain embodiments, determining the ploidy of one or more cells of the one or more plants comprises obtaining a tissue sample of each of the one or more plants and determining the ploidy of one or more cells of each tissue sample.

[0233] 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

[0234] 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 cells 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 plants 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 104sf-6744554197072001240 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.

[0235] 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 plants to a haploid doubling treatment. The tetrahaploid cells, tetrahaploid seeds, or tetrahaploid plants may have been selected from a plurality of cells, seeds, or plants 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 plants to a haploid doubling treatment comprises contacting the one or more tetrahaploid cells, tetrahaploid seeds, or tetrahaploid plants 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 plant with an anti-microtubule agent to form a doubled tetrahaploid cell, a doubled tetrahaploid seed, or a doubled tetrahaploid plant.

[0236] In some embodiments, the doubled tetrahaploid cell or the doubled tetrahaploid seed is allowed to grow into a doubled tetrahaploid plant. In certain embodiments, the doubled tetrahaploid plant is allowed to form seed, thus producing the true homozygous octoploid strawberry seed.

[0237] 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, trifluralin, or nitrous oxide. 105sf-6744554197072001240

[0238] 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 plants. From a plurality of tetrahaploid cells, seeds, or plants. 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, 10x, 11x, or 12x). True Homozygous Octoploid Strawberry Seed and Plants

[0239] 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 two clonal sets of chromosomes, each clonal set comprising a haploid set of chromosomes from each of four subgenomes.

[0240] 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. 106sf-6744554197072001240

[0241] Methods of measuring homozygosity and heterozygosity within genomes and subgenomes of strawberry plants are known in the art. In one exemplary method, in order to estimate the within-subgenome rate of heterozygosity, whole genome sequencing may be conducted on parent octoploid plants, and doubled tetrahaploids derived from those parent octoploid plants, using paired end next generation sequencing (e.g., Illumina Novaseq with paired end 150nt reads in a 500nt insert library). Read pairs are mapped to a repeat masked version of a phased assembly of the parent octoploid plant (e.g., of the F. x ananassa Camarosa genome (2n=8x=56)) using Burrows-Wheller Alignment (BWA). Read pairs are retained for analysis if they uniquely map to a single chromosome within a single subgenome. Variants are called at specific loci and the rate of within-subgenome heterozygosity is calculated.

[0242] In the parental octoploid lines the average rate of within-subgenome heterozygosity is typically calculated to be approximately 1.5%, while the rate of within- subgenome heterozygosity in doubled tetrahaploids is typically less than 0.05% using the same methods, showing that the doubled tetrahaploids are approximately 30 times more homozygous than the octoploid parents after a single generation.

[0243] In some embodiments, the true homozygous octoploid strawberry plant, plant part, plant line, or seed has a rate of within-subgenome heterozygosity of less than 1.5%, less than 1.0%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%, or less than 0.005%.

[0244] In some aspects, provided herein is a homozygous octoploid strawberry plant, plant part, plant line, or seed comprising at least one cell having a nuclear genome from a first species of Fragaria and a cytoplasmic genome from a second species of Fragaria. The first and second species of Fragaria may each independently be any species of Fragaria known in the art or described herein. In some embodiments, the nuclear genome and the cytoplasmic genome are both from octoploid species of Fragaria. In some embodiments, the nuclear genome is from an octoploid species of Fragaria and the cytoplasmic genome is from a diploid species of Fragaria. In certain embodiments, the nuclear genome is from a plant of the species Fragaria x anannasa. In certain embodiments, the cytoplasmic genome is a from a plant of the species Fragaria vesca. In certain embodiments, the nuclear genome is from a plant of the species Fragaria x anannasa and the cytoplasmic genome is a from a plant of the species Fragaria vesca. 107sf-6744554197072001240

[0245] In some aspects, provided herein is a runnerless true homozygous octoploid strawberry plant. In some embodiments, the runnerless true homozygous octoploid strawberry plant comprises one or more genetic modifications resulting in decreased expression of a gibberellin 20-oxidase (ga20ox) gene. In some embodiments, the runnerless true homozygous octoploid strawberry plant does not produce runners or produces fewer runners as compared to a control plant that lacks the one or more genetic modifications resulting in decreased expression of the ga20ox gene.

[0246] 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 F1 Hybrid Strawberry Seed

[0247] In one aspect, described herein are methods of breeding and producing a uniform octoploid F1 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 F1 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 F1 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 F1 hybrid strawberry seed.

[0248] In some embodiments, the methods of breeding and producing a uniform octoploid F1hybrid strawberry seed comprise crossing two true homozygous octoploid strawberry plants. In some variations, crossing two true homozygous octoploid strawberry 108sf-6744554197072001240 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 F1 hybrid strawberry seed is produced by crossing the two true homozygous octoploid strawberry plants and allowing seeds to form.

[0249] In some embodiments, the methods of breeding and producing uniform octoploid F1 hybrid strawberry seed comprise maintaining one or more true homozygous octoploid strawberry lines, one or more F1 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 F1 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.

[0250] In further aspects, provided herein is a uniform F1 hybrid strawberry seed produced by the methods described herein. In some embodiments, provided herein is a population of uniform octoploid F1 hybrid strawberry seed. In certain embodiments, at least 50% of the uniform octoploid F1 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 F1 hybrid strawberry seed are genetically uniform. Obtaining Plant Lines

[0251] In some embodiments, the methods of breeding and producing uniform octoploid F1 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 F1 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 / or Fragaria 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 109sf-6744554197072001240 obtained from sources including, but not limited to, natural diversity, existing breeding programs, or any combination thereof.

[0252] 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

[0253] In some embodiments, the methods of breeding and producing uniform octoploid F1 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 F1 hybrid strawberry plant. In some variations, the methods of breeding and producing uniform octoploid F1 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 octo...

Claims

197072001240 CLAIMS What is claimed is:

1. A strawberry plant, or a plant part thereof, comprising one or more genetic modifications resulting in decreased expression of one or more CENH3 genes.

2. The strawberry plant, or plant part thereof, of claim 1, wherein one or more of the CENH3 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: 8-14 and 53-56.

3. The strawberry plant, or plant part thereof, of claim 1 or 2, wherein one or more of the CENH3 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: 1-7 and 58-61.

4. The 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 CENH3 genes, a modification of a promoter of one or more of the CENH3 genes, a modification of a coding region of one or more of the CENH3 genes, modification of an intron of one or more of the CENH3 genes, or any combination thereof relative to an unmodified strawberry plant of the same species or an unmodified control strawberry plant, optionally wherein the one or more genetic modifications comprise a deletion, insertion, or one or more nucleotide changes in a coding region of one or more of the CENH3 genes.

5. The strawberry plant, or plant part thereof, of any one of claims 1-4, wherein the strawberry plant is diploid.

6. The strawberry plant, or plant part thereof, of claim 5, wherein the strawberry plant is a plant of the species Fragaria vesca.

7. The strawberry plant, or plant part thereof, of claim 5 or 6, wherein the one or more CENH3 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:

8. 210sf-6744554197072001240 8. The strawberry plant, or plant part thereof, of any one of claims 5-7, wherein the one or more CENH3 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:

1.

9. The strawberry plant, or plant part thereof, of any one of claims 1-4, wherein the strawberry plant is octoploid.

10. The strawberry plant, or plant part thereof, of claim 9, wherein the strawberry plant is a plant of the species Fragaria x ananassa.

11. The strawberry plant, or plant part thereof, of claim 9 or 10, wherein one or more of the CENH3 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: 9-14 and 53-56.

12. The strawberry plant, or plant part thereof, of any one of claims 9-11, wherein one or more of the CENH3 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: 2-7 and 58-61.

13. The strawberry plant, or plant part thereof, of any one of claims 9-12, wherein the one or more CENH3 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: 53; 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: 54; 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: 55; and / or 211sf-6744554197072001240 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:

56.

14. The strawberry plant, or plant part thereof, of any one of claims 9-13, wherein the one or more CENH3 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: 58; 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: 59; 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: 60; 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:

61.

15. The strawberry plant, or plant part thereof, of any one of claims 1-14, wherein the expression or activity of one or more CENH3 genes is decreased by no more than about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications.

16. The strawberry plant, or plant part thereof, of any one of claims 1-15, wherein the expression or activity of one or more CENH3 genes is decreased by at least about 1%, 5%, 10%, 20%, 30%, 40%, or 50% as compared to a control plant, plant part, or plant cell lacking the one or more genetic modifications.

17. The strawberry plant of any one of claims 1-16, wherein the strawberry plant has: 212sf-6744554197072001240 (a) decreased pollen germination, flower diameter, anther width, filament length, fruit per berry, cell growth rate, or any combination thereof as compared to a control strawberry plant lacking the one or more genetic modifications; (b) delayed microspore formation as compared to a control strawberry plant lacking the one or more genetic modifications; and / or (c) the strawberry plant has a larger bud size at which microspores form as compared to a control strawberry plant lacking the one or more genetic modifications as compared to a control strawberry plant lacking the one or more genetic modifications.

18. The strawberry plant, or plant part thereof, of any one of claims 1-17, 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.

19. The strawberry plant, or plant part thereof, of any one of claims 1-18, wherein the strawberry plant is a haploid-inducing strawberry plant.

20. A method of producing the strawberry plant of any one of claims 1-19.

21. The method claim 20, wherein the decreased expression of the one or more CENH3 genes is achieved by gene disruption, gene knockout, gene knockdown, gene silencing, RNA interference, induction of methylation, or any combination thereof.

22. The method of claim 20 or 21, comprising introducing one or more of the genetic modifications by mutagenesis, gene editing, transgenesis, or a combination thereof into a strawberry plant, plant part, or plant cell to produce the strawberry plant.

23. The method of any one of claims 20-22, further comprising selecting a strawberry plant, plant part, or plant cell having decreased expression of the one or more CENH3 genes to produce the strawberry plant.

24. The method of claim 20-23, comprising introducing one or more of the genetic modifications by gene editing using a site-directed nuclease. 213sf-6744554197072001240 25. The method of any one of claims 20-24, 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 CENH3 genes, optionally wherein the RNA molecule is a crRNA, a gRNA, or a pegRNA.

26. The method of any one of claims 20-25, 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 CENH3 genes, optionally wherein the RNA molecule is a crRNA, a gRNA, or a pegRNA.

27. 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 a sperm cell of the haploid-inducing strawberry plant of claim 19, and (b) producing a doubled tetrahaploid cell from the tetrahaploid reduced egg cell.

28. The method of claim 27, 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 tube to the reduced egg cell, thereby contacting the reduced egg cell with the sperm cell.

29. The method of claim 28, further comprising allowing the donor octoploid strawberry plant to form a plurality of seeds.

30. A method of producing true homozygous octoploid strawberry seed, the method comprising (a) contacting an egg cell from the haploid-inducing strawberry plant of claim 19 with a tetrahaploid reduced sperm cell from a donor octoploid strawberry plant, and (b) producing a doubled tetrahaploid cell from the egg cell, wherein the doubled tetrahaploid cell comprises the nuclear genome of the donor octoploid strawberry plant and the cytoplasmic genome of the haploid-inducing strawberry plant. 214sf-6744554197072001240 31. The method of claim 30, wherein step (a) comprises contacting the stigma of a pistil of the haploid-inducing strawberry plant with the pollen from the donor octoploid strawberry plant and allowing formation of a pollen tube and migration of the pollen tube to the egg cell, thereby contacting the egg cell with the tetrahaploid reduced sperm cell.

32. The method of claim 31, further comprising allowing the haploid-inducing strawberry plant to form a plurality of seeds.

33. The method of claim 29 or 32, further comprising: (a) collecting the plurality of seeds and allowing them to germinate and form a plurality of seedlings; or (b) 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.

34. The method of claim 33, 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.

35. The method of any one of claims 27-34, wherein step (b) comprises subjecting the selected tetrahaploid cell, tetrahaploid seed, or tetrahaploid seedling to a tetrahaploid doubling treatment.

36. The method of claim 35, wherein 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, optionally wherein the anti-microtubule agent comprises colchicine, oryzalin, oxide, trifluralin, or any combination thereof.

37. The method of any one of claims 27-36, wherein the donor octoploid strawberry plant is a plant of the species Fragaria x ananassa.

38. A method of producing a runnerless true homozygous octoploid strawberry plant comprising the steps of the method of any one of claims 27-37 and further comprising introducing one or more genetic modifications resulting in decreased expression of a 215sf-6744554197072001240 gibberellin 20-oxidase (GA20ox) gene into the true homozygous octoploid strawberry plant, wherein the runnerless true homozygous octoploid strawberry plant does not produce runners.

39. A true homozygous octoploid strawberry seed produced according to the method of any one of claims 27-38.

40. A true homozygous octoploid strawberry plant or a plant part thereof produced according to the method of any one of claims 27-38.

41. A true homozygous octoploid strawberry seed produced according to the method of any one of claims 27-38, comprising an embryo having the nuclear genome of the donor octoploid strawberry plant and the cytoplasmic genome of the haploid-inducing strawberry plant, wherein the nuclear genome and the cytoplasmic genome are from plants of different varieties of strawberry.

42. A true homozygous octoploid strawberry plant or a plant part thereof produced according to the method of any one of claims 27-38, comprising at least one somatic cell having the nuclear genome of the donor octoploid strawberry plant and the cytoplasmic genome of the haploid-inducing strawberry plant, wherein the nuclear genome and the cytoplasmic genome are from plants of different varieties of strawberry.

43. The true homozygous octoploid strawberry seed of claim 41 or the true homozygous octoploid strawberry plant or plant part of claim 42, wherein the different varieties of strawberry are different species of Fragaria, optionally wherein the first species of Fragaria is Fragaria x ananassa and the second species of Fragaria is Fragaria vesca.

44. The true homozygous octoploid strawberry seed of claim 41 or the true homozygous octoploid strawberry plant or plant part of claim 42, wherein the different varieties of strawberry are different varieties of Fragaria x ananassa.

45. A true homozygous octoploid strawberry seed comprising an embryo having the nuclear genome of a first variety of strawberry and the cytoplasmic genome of a variety of strawberry.

46. A true homozygous octoploid strawberry plant or plant part thereof comprising an embryo having the nuclear genome of a first variety of strawberry and the cytoplasmic genome of a second variety of strawberry. 216sf-6744554197072001240 47. The true homozygous octoploid strawberry seed of claim 56 or the true homozygous octoploid strawberry plant or plant part of claim 46, wherein the first and second varieties of strawberry are different species of Fragaria, optionally wherein the first species of Fragaria is Fragaria x ananassa and the second species of Fragaria is Fragaria vesca.

48. The true homozygous octoploid strawberry seed of claim 56 or the true homozygous octoploid strawberry plant or plant part of claim 46, wherein the first and second varieties of strawberry are two different varieties of Fragaria x ananassa.

49. The true homozygous octoploid strawberry plant, or plant part thereof, of any one of claims 40, 42-44, and 46-48, 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.

50. A method of producing uniform octoploid F1hybrid strawberry seed comprising crossing two true homozygous octoploid strawberry plants of any one of claims 40, 42-44, and 46-48.

51. A uniform octoploid F1hybrid strawberry seed produced according to the method of claim 50.

52. A uniform octoploid F1 hybrid strawberry plant or a plant part thereof produced according to the method of claim 50.

53. The uniform octoploid F1 hybrid strawberry plant, or plant part thereof, of claim 52, 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.

54. An expression vector or isolated DNA molecule for making the strawberry plant, or plant part thereof, of any one of claims 1-19.

55. The expression vector or isolated DNA molecule of claim 54, 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: 8-14 and 53-56. 217sf-6744554197072001240 56. The expression vector or isolated DNA molecule of claim 54 or 44, 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 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: 1-7 and 58-61.

57. The expression vector or isolated DNA molecule of any one of claims 54-56, comprising a DNA sequence encoding a non-coding RNA, optionally wherein the non-coding RNA is a crRNA, a gRNA, a pegRNA, a siRNA, a miRNA, or a dsRNA.

58. The expression vector or isolated DNA molecule of claim 57, 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: 8-14 and 53-56.

59. The expression vector or isolated DNA molecule of claim 57 or 58, 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 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: 1-7 and 58-61.

60. A bacterial cell comprising the expression vector or isolated DNA molecule of any one of claims 54-59, optionally wherein the bacterial cell is an Agrobacterium cell.

61. A genetically modified plant, plant part, plant cell, or seed comprising the expression vector or isolated DNA molecule of any one of claims 54-59.

62. A kit comprising the expression vector or isolated DNA molecule of 54-59 or the bacterial cell of claim 60.

63. A genetically modified non-regenerable plant cell of the strawberry plant of any one of claims 1-19. 218sf-6744554197072001240 64. A genetically modified plant genome of the strawberry plant of any one of claims 1- 219sf-6744554

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