WO2014110274A2 - Generation of haploid plants - Google Patents

Abstract

The present invention provides methods and compositions for generating haploid plants. The present invention also provides plants having an inactivated endogenous CENH3 gene and expressing a heterologous CENH3 polypeptide from another species.

Description

GENERATION OF HAPLOID PLANTS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims benefit of priority to US Provisional Patent

Applictaion No. 61/750,472, filed on January 9, 2013, which is incorporated by reference for all purposes.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

[0002] The Sequence Listing written in file "Sequence Listing for 81906-895370 (212910PC)", created on December 13, 2013, 70,397 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION [0003] Typical breeding of diploid plants relies on screening numerous plants to identify novel, desirable characteristics. Large numbers of progeny from crosses often must be grown and evaluated over several years in order to select one or a few plants with a desired combination of traits.

[0004] The plant breeding process can be accelerated by producing haploid plants, the chromosomes of which can be doubled using colchicine or other means. Such doubled haploids produce instant homozygous lines in one generation, which is significantly shorter than the approximately 8-10 generations of inbreeding that is typically required for diploid breeding. Thus, methods of producing haploid plants that can be doubled to generate fertile doubled haploids can dramatically improve the efficiency and effectiveness of plant breeding by producing true-breeding

(homozygous) lines in only one generation. BRIEF SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides for methods of generating haploid plants. In some embodiments, the method comprises: crossing a first plant expressing an endogenous centromeric histone H3 (CENH3) gene to a second plant (also referred to herein as a "haploid inducer plant"), the haploid inducer plant having a genome from at least two species, wherein a majority of the genome is from a first species and the genome comprises a heterologous genomic region from a second species, wherein the heterologous genomic region encodes a CENH3 polypeptide different from the CENH3 of the first species; and selecting Fl haploid progeny generated from the crossing step.

[0006] In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide comprising a histone fold domain that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical) to the histone fold domain of the CENH3 of the first species.

[0007] In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide comprising an N-terminal tail domain having no more than 50, 45, 40, 35, 30, 25, 20, or 15 amino differences from the N-terminal tail domain of the CENH3 of the first species. [0008] In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide from the same taxonomic family as the CENH3 of the first species. In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide from the same genus as the CENH3 of the first species.

[0009] In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide comprising an N-terminal tail region that is substantially identical to the N-terminal domain of the CENH3 of the first species. In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide comprising a histone fold domain that is identical to the histone fold domain of the CENH3 of the first species and an N-terminal tail region that is different from the N-terminal tail region of the CENH3 of the first species. [0010] In some embodiments, the haploid inducer plant is a recombinant inbred line. In some embodiments, the haploid inducer plant is an introgression line.

[0011] In some embodiments, the haploid inducer plant is a diploid plant. In some embodiments, the haploid inducer plant is a allopolyploid plant. In some

embodiments, the haploid inducer plant is a tetraploid or hexaploid plant.

[0012] In some embodiments, the haploid inducer plant comprises a chromosome from the second species and said chromosome comprises the heterologous genomic region encoding the CENH3 polypeptide.

[0013] In some embodiments, the first plant is a plant of the first species. In some embodiments, the first plant is not a plant of the first species or the second species.

[0014] In some embodiments, the first plant is the pollen parent for the crossing step. In some embodiments, the first plant is the ovule parent for the crossing step.

[0015] In some embodiments, the method further comprises converting at least one selected haploid progeny into a doubled haploid plant. [0016] In some embodiments, the method of generating haploid plants comprises: crossing a first plant of a first species expressing an endogenous centromeric histone H3 (CENH3) gene to a second plant (also referred to herein as a "haploid inducer plant") comprising an inactivated endogenous CENH3 gene and further comprising an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a heterologous CENH3 polypeptide from a species different from the first species; and selecting Fl haploid progeny generated from the crossing step.

[0017] In some embodiments, the heterologous CENH3 polypeptide is from a plant of the same taxonomic family as the first species. In some embodiments, the heterologous CENH3 polypeptide is from a plant of the same genus as the first species.

[0018] In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%o, at least 90%>, or at least 95% identical) to the endogenous CENH3 polypeptide histone fold domain [0019] In some embodiments, the heterologous CENH3 polypeptide comprises an N-terminal tail region that is substantially identical (e.g., at least 70%, least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical) to the N-terminal domain of the endogenous CENH3 polypeptide. In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is identical to the endogenous CENH3 polypeptide histone fold domain and an N-terminal tail region that is different from the endogenous CENH3 polypeptide N-terminal tail region.

[0020] In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is the native CENH3 promoter of the plant comprising the expression cassette.

[0021] In some embodiments, the first plant is the pollen parent for the crossing step. In some embodiments, the first plant is the ovule parent for the crossing step.

[0022] In some embodiments, the method further comprises converting at least one selected haploid progeny into a doubled haploid plant.

[0023] In another aspect, the present invention provides for plants (or a plant cell, seed, flower, leaf, fruit, or other plant part from such plants or processed food or food ingredient from such plants) comprising an inactivated endogenous centromeric histone H3 (CENH3) gene and further comprising an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a heterologous CENH3 polypeptide from another plant species.

[0024] In some embodiments, the heterologous CENH3 polypeptide is from a plant of the same taxonomic family as the plant comprising the expression cassette. In some embodiments, the heterologous CENH3 polypeptide is from a plant of the same genus as the CENH3 of the plant comprising the expression cassette.

[0025] In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%o, at least 90%>, or at least 95% identical) to the endogenous CENH3 polypeptide histone fold domain. [0026] In some embodiments, the heterologous CENH3 polypeptide comprises an N-terminal tail region that is substantially identical (e.g., at least 70%, least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical) to the N-terminal domain of the endogenous CENH3 polypeptide. In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is identical to the endogenous CENH3 polypeptide histone fold domain and an N-terminal tail region that is different from the endogenous CENH3 polypeptide N-terminal tail region.

[0027] In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is the native CENH3 promoter of the plant comprising the expression cassette. In some embodiments, the plant, when crossed with another plant (e.g., a wild-type plant or a double haploid mutant plant), generates at least 0.1 % haploid progeny.

[0028] In another aspect, the present invention relates to methods of making a plant comprising an inactivated endogenous centromeric histone H3 (CENH3) gene and further comprising an expression cassette comprising a promoter operably linked to a polynucleotide encoding a heterologous CENH3 polypeptide from another plant species as described herein. In some embodiments, the method comprises:

transforming plant cells with a nucleic acid comprising the expression cassette; and selecting transformants comprising the nucleic acid; thereby making the plant.

[0029] Also provided are methods of generating haploid progeny from an inter- species plant cross. In some embodiments, the method comprises crossing a first plant of a first species expressing an endogenous centromeric histone H3 (CENH3) gene to a haploid inducer plant of a second species different from the first species, the haploid inducer plant lacking endogenous CENH3 expression and expressing a heterologous polypeptide comprising a heterologous amino acid sequence of at least 5 amino acids linked to an amino terminus of a protein comprising a CENH3 histone-fold domain, wherein the heterologous amino acid sequence is heterologous to the CENH3 histone- fold domain; and selecting Fl haploid progeny generated from the crossing step.

DEFINITIONS [0030] "Centromeric histone H3" or "CENH3" refers to the centromere-specific histone H3 variant protein (also known as CENP-A). CENH3 is characterized by the presence of a variable N-terminal tail domain, which does not form a rigid secondary structure, and a conserved histone fold domain made up of three a-helical regions connected by loop sections. CENH3 is a member of the kinetochore complex, the protein structure on chromosomes where spindle fibers attach during cell division, and is required for kinetochore formation and for chromosome segregation. [0031] An "endogenous" gene or protein sequence, as used with reference to an organism, refers to a gene or protein sequence that is naturally occurring in the genome of the organism.

[0032] A polynucleotide or polypeptide sequence is "heterologous" to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g. , is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).

[0033] The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters can include czs-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cz^'s-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cz^'s-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is one that is capable of initiating transcription in nearly all tissue types, whereas a "tissue- specific promoter" initiates transcription only in one or a few particular tissue types. [0034] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0035] The term "plant" includes whole plants, shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous.

[0036] "Recombinant," as used with reference to a polynucleotide sequence, refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a protein may include a promoter that is heterologous to the polynucleotide encoding the protein as the result of human manipulation (e.g. , by methods described in

Sambrook et ah, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in

Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the example above. [0037] A "transgene" is used as the term is understood in the art and refers to a heterologous nucleic acid introduced into a cell by human molecular manipulation of the cell's genome (e.g., by molecular transformation). Thus, a "transgenic plant" is a plant comprising a transgene, i.e., is a genetically-modified plant. The transgenic plant can be the initial plant into which the transgene was introduced as well as progeny thereof whose genomes contain the transgene. In some embodiments, a transgenic plant is transgenic with respect to the CENH3 gene. In some embodiments, a transgenic plant is transgenic with respect to one or more genes other than the CENH3 gene.

[0038] The phrase "nucleic acid" or "polynucleotide sequence" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase, and/or formation of double-stranded duplexes, and do not significantly alter expression of a polypeptide encoded by that nucleic acid.

[0039] The phrase "nucleic acid sequence encoding" refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. It should be further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

[0040] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or

subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum

correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).

[0041] The phrase "substantially identical," used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below.

[0042] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0043] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection. [0044] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix {see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0045] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences {see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10^"5, and most preferably less than about 10^"20.

[0046] An "expression cassette" refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an R A or polypeptide, respectively. Antisense or sense constructs that are not or cannot be translated are expressly included by this definition. In the case of both expression of transgenes and suppression of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially identical to a sequence of the gene from which it was derived. [0047] The phrase "host cell" refers to a cell from any organism. Exemplary host cells are derived from plants, bacteria, yeast, fungi, insects or other animals. Methods for introducing polynucleotide sequences into various types of host cells are known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure. 1 Centromere competition assay. When chromosomes with a wild type centromere are mixed with chromosomes with a orthologous CENH3 mis- segregation of the mutant chromosomes occurs in early zygotic mitosis. The Fl progeny expected are diploids, aneuploids with extra chromosomes from the parent with heterologous CENH3 (star) and haploids.

[0049] Figure 2. Alignment of L. oleraceum and B. rapa CENH3 relative to A. thaliana CENH3. Positions with a 100% identity between all three alleles are shaded in black, while functionally similar substitutions are shaded in grey. Colored positions are divergent. The black line underlines the Histone Fold Domain (HFD). [0050] Figure 3. Induction of haploid A. suecica. Left, Crossing haploid inducer A. thaliana with modified CENH3 to wild-type A. suecica causes loss of the inducer's genome in the zygote. Haploids, are confirmed by flow cytometry (right). Gl and G2 nuclear peaks indicate that the allo-haploid (bottom) has half the nuclear content of the wild type (top).

[0051] Figure 4. Schematic of crosses for generating haploid plants. "Normal cross" demonstrates the production of diploid progeny in crosses of parent plants expressing native endogenous CENH3 protein. "Cross A," "Cross B", and "Cross C" demonstrate the production of haploid progeny using different types of introgression lines, in which the maternal plant expresses a CENH3 from a species other than the species that makes up the majority of the maternal plant's genome. "Cross D" demonstrates the production of haploid progeny using a transgenic maternal plant that lacks endogenous CENH3 and expresses a heterologous CENH3. The direction of these crosses is exemplary.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[0052] The inventors have discovered a method of generating haploid plants.

Specifically, the inventors have recombinantly expressed a Lepidium oleraceum CENH3 polypeptide in an Arabidopsis plant lacking a native (Arabidopsis) CENH3 polypeptide. When this recombinant plant is crossed as a maternal parent to a wild- type Arabidopsis plant, the resulting progeny have a certain percentage of haploid progeny. Accordingly, the inventors have surprisingly discovered that native CENH3 polypeptides from one species, when expressed in a different plant species, allow for subsequent generation of haploid plants. In some embodiments, this invention can be exploited by reducing or eliminating the native CENH3 polypeptide expression in a plant species and recombinantly inserting a heterologous CENH3 that is native to another species.

[0053] Notably, however, in view of the inventors' discovery, it is also believed that non-recombinant plants can be used to generate haploid plants. Specifically, in situations in which sufficiently divergent species can be crossed, plants can be generated, based from a cross from two species, such that resulting plants comprise a majority of its genome from a first species but comprise at least the CENH3 gene from a second species, thereby generating a plant in which the CENH3 genetic region from the second species is introgressed into the genetic background of the first species. As shown in the Examples, CENH3 from a first species functions

appropriately during mitosis in the background of a second species (thereby allowing for normal development of the plant), but subtly interferes with mitosis in the resulting hybrid embryos such that crossing from the "introgressed" plant to a wild- type plant results in some percentage of haploid progeny that can be selected and subsequently used as desired. This latter introgression method does not necessarily require generation of genetically modified plants. A variety of exemplary scenarios are displayed in Figure 4. II. CENH3

[0054] Centromeric histone H3 (CENH3) proteins are a well characterized class of proteins that are variants of histone H3 proteins. These specialized proteins, which are specifically associated with the centromere, are essential for proper formation and function of the kinetochore, a multiprotein complex that assembles at centromeres and links the chromosome to spindle microtubules during mitosis and meiosis. Cells that are deficient in CENH3 fail to localize kinetochore proteins and show strong chromosome segregation defects.

[0055] CENH3 proteins are characterized by a N-terminal variable tail domain and a C-terminal conserved histone fold domain made up of three a-helical regions connected by loop sections. The CENH3 histone fold domain is conserved between CENH3 proteins from different species. See, e.g., Torras-Llort et al, EMBO J.

28:2337-48 (2009). In contrast, the N-terminal tail domains of CENH3 are highly variable even between closely related species. Histone tail domains (including CENH3 tail domains) are flexible and unstructured, as shown by their lack of strong electron density in the structure of the nucleosome determined by X-ray

crystallography (Luger et al, Nature 389(6648):251-60 (1997)). Additional structural and functional features of CENH3 proteins can be found in, e.g., Cooper et al., Mol Biol Evol. 21(9): 1712-8 (2004); Malik et al, Nat Struct Biol. 10(11):882-91 (2003); Black et al, Curr Opin Cell Biol. 20(1):91-100 (2008); and Torras-Llort et al, EMBO J. 28:2337-48 (2009).

[0056] CENH3 proteins are widely found throughout eukaryotes, and a large number of CENH3 proteins have been identified. See, e.g., SEQ ID NOs: l-48. It will be appreciated that the above list is not intended to be exhaustive and that additional CENH3 sequences are available from genomic studies or can be identified from genomic databases or by well-known laboratory techniques. For example, where a particular plant or other organism species CENH3 is not readily available from a database, one can identify and clone the organism's CENH3 gene sequence using primers, which are optionally degenerate, based on conserved regions of other known CENH3 proteins.

III. Methods of Generating Haploid Plants Using Plants Having a

Heterologous Genomic Region for CENH3 [0057] In one aspect, the disclosure provides methods of generating haploid plants using plants having a genome from two or more species, such as but not limited to recombinant inbred lines or introgression lines. In some embodiments, the method comprises: crossing a first plant expressing an endogenous CENH3 gene to a second plant (also referred to herein as a "haploid inducer plant"), the haploid inducer plant having a genome from at least two species, wherein a majority of the genome is from a first species and wherein the genome comprises a heterologous genomic region from a second species, wherein the heterologous genomic region encodes a CENH3 polypeptide different from the CENH3 of the first species; and selecting Fl haploid progeny generated from the crossing step. [0058] It is believed the above method can be used for combinations of any two plant species that can be successfully crossed to generate viable, fertile progeny. Selection of the two plant species will depend upon the needs of the user, with greater divergence of species possibly reducing the resulting efficiency of the interspecies cross, but possibly improving the resulting haploid generation efficiency when the plant comprising the introgressed CENH3 is ultimately used to generate haploids due to the wider divergence between the first species CENH3 and the introgressed CENH3.

[0059] In some embodiments, the first plant and/or the second plant is a species of plant of the genus Allium, Amaranthus, Arachis, Arabidopsis, Asparagus, Atropa, Avena, Beta, Brassica, Cannabis, Capsella, Cica, Citrus, Citrullus, Capsicum,

Carthamus, Cocos, Cqffea, Cucumis, Cucurbita, Daucus, Dioscorea, Elais, Eruca, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Ipomea, Lactuca, Lepidium, Linum, Lolium, Luzula, Lycopersicon, Mains, Manihot, Majorana, Medicago, Musa, Nicotiana, Olea, Oryza, Panicum, Pennisetum, Persea, Phaseolus, Pinus, Pisum, Populus, Pyrus, Prunus, Raphanus, Saccharum, Secale, Senecio, Sesamum, Sinapis, Solarium, Sorghum, Theobroma, Trigonella, Triticum, Turritis, Vitis, Vigna, or Zea. In some embodiments, the first plant expresses an endogenous CENH3 of any species of the genus Allium, Amaranthus, Arachis, Arabidopsis, Asparagus, Atropa, Avena, Beta, Brassica, Cannabis, Capsella, Cica, Citrus, CitruUus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Dioscorea, Elais, Eruca, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Ipomea, Lactuca, Lepidium, Linum, Lolium, Luzula,

Lycopersicon, Malus, Manihot, Majorana, Medicago, Musa, Nicotiana, Olea, Oryza, Panicum, Pennisetum, Persea, Phaseolus, Pinus, Pisum, Populus, Pyrus, Prunus, Raphanus, Saccharum, Secale, Senecio, Sesamum, Sinapis, Solanum, Sorghum, Theobroma, Trigonella, Triticum, Turritis, Vitis, Vigna, or Zea. In some

embodiments, the first plant expresses an endogenous CENH3 of any of SEQ ID NOs: l-48.

[0060] As noted above, the second plant (comprising a majority of genetic information from a first species and the introgressed CENH3 gene from a second species) has a genome from at least two species. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the genome of the second plant is from the first species. In some embodiments, the second plant comprises a segment of genome from a second species, and the segment comprises the heterologous genomic region encoding the CENH3 polypeptide that is different from the CENH3 of the first species. In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% of more of the genome of the second plant is from the genome from the second species. In some embodiments, the second plant comprises a chromosome from the second species, and the chromosome comprises the heterologous genomic region encoding the CENH3 polypeptide that is different from the CENH3 of the first species. [0061] In some embodiments, the second plant further comprises a segment of genome from one, two, three, or more additional species. In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% of more of the genome of the second plant is from the genome from each additional species.

[0062] In some embodiments, the heterologous genomic region in the second plant encodes a CENH3 polypeptide from the same taxonomic family as the CENH3 of the first species. In some embodiments, the heterologous genomic region in the second plant encodes a CENH3 polypeptide from the same genus as the CENH3 of the first species.

[0063] In some embodiments, the heterologous genomic region in the second plant encodes a CENH3 polypeptide comprising a histone fold domain that is identical or substantially identical to the histone fold domain of the CENH3 of the first species. In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide comprising a histone fold domain that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the histone fold domain of the CENH3 of the first species. In some embodiments, the heterologous genomic region in the second plant encodes a CENH3 polypeptide comprising an N-terminal tail region that is substantially identical (e.g., at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the N-terminal tail region of the CENH3 of the first species. In some embodiments, the heterologous genomic region encodes a CENH3 polypeptide comprising a histone fold domain that is identical to the histone fold domain of the CENH3 of the first species and an N-terminal tail region that is different from the N- terminal tail region of the CENH3 of the first species.

[0064] In some embodiments, the first plant (i.e., the plant to which the second plant is crossed to generate haploid progeny) is the same species of plant as the first species of the second plant. In some embodiments, the first plant is not the same species of plant as the first species or second species of the second plant (e.g., the first plant is a third species of plant), but the first plant and second plant are sufficiently related to be capable of generating progeny. [0065] In some embodiments, the second plant (or "haploid inducer plant") is a diploid plant. In some embodiments, the second plant has higher ploidy than diploid (e.g., a tetraploid, a hexaploid, an octaploid, or a decaploid). In some embodiments, the second plant is an allopolyploid (e.g., allotetraploid) plant. [0066] In some embodiments, the second plant (or "haploid inducer plant") is not genetically modified (e.g., it is not transformed). In some embodiments, the second plant is genetically modified, for example without limitation, the second plant can comprise an unrelated genetic modification conferring an improved agronomic trait (for example, drought tolerance, herbicide resistance, insect resistance, etc.). Recombinant Inbred Lines and Introgression Lines

[0067] In some embodiments, the second plant is a recombinant inbred line. A recombinant inbred line refers to a plant line in which two or more plant species, varieties, or cultivars (e.g., related species, varieties, or cultivars) have been crossed to produce progeny which are intercrossed to produce a hybrid line that can then be self- fertilized. In some embodiments, the second plant is an introgression line. An introgression line refers to a plant line in which genetic material from one or more donor species, varieties, or cultivars has been moved, either naturally or artificially, into the parent genome of another (e.g., related) species, variety, or cultivar. The plant line may be further backcrossed back into the parent line. [0068] Recombinant inbred lines and introgression lines, methods of making recombinant inbred lines and introgression lines, and methods of screening

recombinant inbred lines and introgression lines to determine whether a gene (e.g., CENH3) is endogenous or heterologous to the parent, are known in the art. See, e.g., Y. Xu, MOLECULAR PLANT BREEDING, CAB International 2010; Cooper and

Henikoff, Mol Biol Evol 21 : 1712-18 (2004); and Hirsch et al, Mol Biol Evol 26:2877- 2885 (2009), incorporated by reference herein. Methods for producing and analyzing introgression are exemplified in Chang and de Jong, Cytogenet Genome Res 109:335- 343 (2005); Fridman et al, Science 305: 1786-1789 (2004); King et al, Chromosome Res 15: 105-113 (2007); Molnar et al, Genome 52:156-165 (2009); Nassar, Genet Mol Res 2:334-347 (2003); Navabi et al, Genome 53:619-629 (2010); and Xia, J Genet Genomics 36:547-556 (2009). IV. Methods of Generating Haploid Plants from Plants Having Inactivated Endogenous CENH3 Expression and Expressing Heterologous CENH3

[0069] In another aspect, the disclosure provides methods of generating haploid plants using plants that lack endogenous CENH3 expression and express a

heterologous CENH3 polypeptide, including but not limited to a native CENH3 from a different species, e.g., from a different plant species. In some embodiments, the method comprises: crossing a first plant of a first species expressing an endogenous CENH3 gene to a second plant (also referred to herein as a "haploid inducer plant") comprising an inactivated endogenous CENH3 gene and further comprising an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a heterologous CENH3 polypeptide from a species different from the first species; and selecting Fl haploid progeny generated from the crossing step.

[0070] In some embodiments, the heterologous CENH3 polypeptide is identical or substantially identical to {e.g., at least 99% identical to) a naturally occurring CENH3 polypeptide of a species of plant other than the first plant species. In some embodiments, the heterologous CENH3 polypeptide is a naturally occurring CENH3 polypeptide of a plant of the same family as the plant of the first species. In some embodiments, the heterologous CENH3 polypeptide is a naturally occurring CENH3 polypeptide of a plant of the same genus as the plant of the first species.

[0071] In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is substantially identical {e.g., at least about 70%>, 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the endogenous CENH3 polypeptide histone fold domain. [0072] In some embodiments, the heterologous CENH3 polypeptide comprises an N-terminal tail region that is substantially identical {e.g., at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the N-terminal tail region of the endogenous CENH3 polypeptide {e.g., the CENH3 of the first species). In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is identical to the endogenous CENH3 polypeptide histone fold domain {e.g., the CENH3 histone fold domain of the first species) and an N-terminal tail region that is different from the endogenous CENH3 N-terminal tail region (e.g., the CENH3 N-terminal tail region of the first species).

[0073] As shown in the Examples, haploid progeny can also be generated from interspecies crosses. Thus in some embodiments, a haploid inducer plant from a first species can be crossed to a plant of a second (different) species. Consistent with the description herein, in some embodiments the haploid inducer plant lacks an endogenous CENH3 expression and expresses a heterologous CENH3 polypeptide, including but not limited to a native CENH3 from a different species, e.g., from a different plant species. Alternatively, the haploid inducer plant lacks an endogenous CENH3 expression and expresses a heterologous polypeptide comprising a heterologous amino acid sequence of at least 5 amino acids linked to an amino terminus of a protein comprising a CENH3 histone-fold domain, wherein the heterologous amino acid sequence is heterologous to the CENH3 histone-fold domain. In some embodiments, the heterologous amino acid sequence is linked to the CENH3 histone-fold domain via an intervening protein sequence. In some embodiments, the intervening protein sequence comprises a non-CENH3 histone H3 tail domain. In some embodiments, the intervening protein sequence comprises a CENH3 tail domain. In some embodiments, the CENH3 tail domain is heterologous to the CENH3 histone-fold domain. In some embodiments, the heterologous amino acid sequence is at least 10 amino acids long. In some embodiments, the heterologous amino acid sequence comprises a non-CENH3 histone H3 tail domain and the non- CENH3 histone H3 tail domain is linked to an amino terminus of a CENH3 histone- fold domain. These and other non-naturally-occuring CENH3 variants functional as haploid inducer contrsutcs are described in deatil in, e.g., US Patent Publication No. 2011/0083202.

Inactivation of Endogenous CENH3 Expression

[0074] A number of methods can be used to inactivate or inhibit expression of endogenous CENH3 in plants. For instance, antisense technology can be

conveniently used to inactivate gene expression. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been suggested that antisense RNA inhibits gene expression by preventing the accumulation of mR A which encodes the polypeptide of interest, see, e.g., Sheehy et al, Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988); Pnueli et al, The Plant Cell 6: 175-186 (1994); and Hiatt et al, U.S. Patent No. 4,801,340.

[0075] The antisense nucleic acid sequence transformed into plants will be substantially identical to at least a portion of the endogenous gene to be repressed. The sequence, however, does not have to be perfectly identical to inhibit expression. Thus, an antisense or sense nucleic acid molecule encoding only a portion of CENH3, or a portion of the CENH3 mRNA (including but not limited to untranslated portions of the mRNA) can be useful for producing a plant in which CENH3 protein expression is suppressed. The antisense sequences of the present invention are optionally designed such that the inhibitory effect applies only to CENH3 and does not affect expression of other genes. In situations where endogenous CENH3 is to be inactivated, one method for achieving this goal is to target the antisense sequence to CENH3 sequences {e.g., untranslated mRNA sequences) not found in other proteins within a family of genes exhibiting homology or substantial homology to the CENH3 gene.

[0076] For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. For example, a sequence of between about 30 or 40 nucleotides can be used, and in some embodiments, about full length nucleotides should be used, though a sequence of at least about 20, 50, 100, 200, or 500 nucleotides can be used. [0077] Catalytic RNA molecules or ribozymes can also be used to inhibit expression of a CENH3 gene. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. [0078] A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs that are capable of self- cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA- specific ribozymes is described in Haseloff et al. Nature, 334:585-591 (1988).

[0079] Another method of suppression is sense suppression (also known as co- suppression). Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al, The Plant Cell 2:279-289 (1990); Flavell, Proc. Natl. Acad. Sci., USA 91 :3490-3496 (1994); Kooter and Mol, Current Opin. Biol. 4: 166-171 (1993); and U.S. Patents Nos. 5,034,323, 5,231,020, and 5,283,184.

[0080] Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65% to the target CENH3 sequence, but a higher identity can exert a more effective repression of expression of the endogenous sequences. In some embodiments, sequences with substantially greater identity are used, e.g., at least about 80%, at least about 95%, or 100%) identity are used. As with antisense regulation, the effect can be designed and tested so as to not significantly affect expression of other proteins within a similar family of genes exhibiting homology or substantial homology. [0081] For sense suppression, the introduced sequence in the expression cassette, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants that are overexpressers. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. In some embodiments, a sequence of the size ranges noted above for antisense regulation is used, i.e., 30-40, or at least about 20, 50, 100, 200, 500 or more nucleotides.

[0082] Endogenous gene expression may also be suppressed by means of R A interference (R Ai) (and indeed co-suppression can be considered a type of R Ai), which uses a double-stranded RNA having a sequence identical or similar to the sequence of the target gene. RNAi is the phenomenon in which when a double- stranded RNA having a sequence identical or similar to that of the target gene is introduced into a cell, the expressions of both the inserted exogenous gene and target endogenous gene are suppressed. The double-stranded RNA may be formed from two separate complementary RNAs or may be a single RNA with internally complementary sequences that form a double-stranded RNA. Although complete details of the mechanism of RNAi are still unknown, it is considered that the introduced double-stranded RNA is initially cleaved into small fragments, which then serve as indexes of the target gene in some manner, thereby degrading the target gene. RNAi is also known to be effective in plants {see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc. Natl. Acad. Sci. USA 97: 4985 (2000); Waterhouse et al, Proc. Natl. Acad. Sci. USA 95: 13959-13964 (1998); Tabara et al. Science 282:430-431 (1998); Matthew, Comp Funct. Genom. 5: 240-244 (2004); Lu, et al., Nucleic Acids Research 32(21):el71 (2004)). For example, to achieve suppression of CENH3 expression using RNAi, a double-stranded RNA having the sequence of an mRNA encoding the CENH3 protein, or a substantially similar sequence thereof (including those engineered not to translate the protein) or fragment thereof, is introduced into a plant or other organism of interest. The resulting plants/organisms can then be screened for a phenotype associated with the target protein (optionally in the presence of expression of a tailswap protein to avoid lethality) and/or by monitoring steady-state RNA levels for transcripts encoding the protein. Although the genes used for RNAi need not be completely identical to the target gene, they may be at least 70%, 80%, 90%), 95%o or more identical to the target gene sequence. See, e.g., U.S., Patent Publication No. 2004/0029283 for an example of a non-identical siRNA sequence used to suppress gene expression. The constructs encoding an R A molecule with a stem-loop structure that is unrelated to the target gene and that is positioned distally to a sequence specific for the gene of interest may also be used to inhibit target gene expression. See, e.g., U.S. Patent Publication No. 2003/0221211. [0083] The RNAi polynucleotides can encompass the full-length target RNA or may correspond to a fragment of the target RNA. In some cases, the fragment will have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000 nucleotides corresponding to the target sequence. In addition, in some embodiments, these fragments are at least, e.g., 10, 15, 20, 50, 100, 150, 200, or more nucleotides in length. In some cases, fragments for use in RNAi will be at least substantially similar to regions of a target protein that do not occur in other proteins in the organism or may be selected to have as little similarity to other organism transcripts as possible, e.g., selected by comparison to sequences in analyzing publicly-available sequence databases. [0084] Expression vectors that continually express siRNA in transiently- and stably-transfected cells have been engineered to express small hairpin RNAs, which get processed in vivo into siRNAs molecules capable of carrying out gene-specific silencing (Brummelkamp et ah, Science 296:550-553 (2002), and Paddison, et ah, Genes & Dev. 16:948-958 (2002)). Post-transcriptional gene silencing by double- stranded RNA is discussed in further detail by Hammond et ah, Nature Rev Gen 2: 110-119 (2001), Fire et al, Nature 391 : 806-811 (1998) and Timmons and Fire, Nature 395: 854 (1998).

[0085] One of skill in the art will recognize that sense (including but not limited to siRNA) or antisense transcript should be targeted to sequences with the most variance between family members where the goal is to specifically target only one histone family member, e.g., CENH3.

[0086] Yet another way to suppress expression of an endogenous CENH3 gene in a plant is by recombinant expression of a microRNA that suppresses the target gene. Artificial microRNAs are single-stranded RNAs (e.g., between 18-25 mers, generally 21 mers), that are not normally found in plants and that are processed from

endogenous miRNA precursors. Their sequences are designed according to the determinants of plant miRNA target selection, such that the artificial microRNA specifically silences its intended target gene(s) and are generally described in Schwab et al, The Plant Cell 18: 1121-1133 (2006) as well as the internet-based methods of designing such microR As as described therein. See also, US Patent Publication No. 2008/0313773. [0087] Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known and can be used to introduce mutations into or to knock out the CENH3 gene. For instance, seeds or other plant material can be treated with a mutagenic insertional polynucleotide (e.g., transposon, T-DNA, etc.) or chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, ethyl methanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from sources such as, X-rays or gamma rays can be used. Plants having a mutated or knocked-out CENH3 gene can then be identified, for example, by phenotype or by molecular techniques. [0088] Alternatively, homologous recombination can be used to induce targeted gene modifications or knockouts by specifically targeting the CENH3 gene in vivo {see, generally, Grewal and Klar, Genetics, 146: 1221-1238 (1997) and Xu et al, Genes Dev., 10:2411-2422 (1996)). Homologous recombination has been

demonstrated in plants (Puchta et al, Experientia, 50:277-284 (1994); Swoboda et al, EMBO J., 13:484-489 (1994); Offringa et al, Proc. Natl Acad. Sci. USA, 90:7346- 7350 (1993); and Kempin et al, Nature, 389:802-803 (1997)).

[0089] In applying homologous recombination technology to the genes of the invention, mutations in selected portions of an kinetochore complex protein gene sequences (including 5' upstream, 3' downstream, and intragenic regions) such as those disclosed here are made in vitro and then introduced into the desired plant using standard techniques. Since the efficiency of homologous recombination is known to be dependent on the vectors used, dicistronic gene targeting vectors as described by Mountford et al, Proc. Natl. Acad. Sci. USA, 91 :4303-4307 (1994); and Vaulont et al, Transgenic Res., 4:247-255 (1995) are conveniently used to increase the efficiency of selecting for inactivated CENH3 gene expression in transgenic plants. The mutated gene will interact with the target wild-type gene in such a way that homologous recombination and targeted replacement of the wild-type gene will occur in transgenic plant cells, resulting in suppression of CENH3 protein activity.

[0090] Another way to suppress expression of an endogenous CENH3 gene in a plant is by use of CRISPR/Cas technology. The CRISPR/Cas system has been modified for use in prokaryotic and eukaryotic systems for genome editing and transcriptional regulation. The "CRISPR/Cas" system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR/Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR/Cas systems include type I, II, and III sub-types. Wild-type type II CRISPR/Cas systems utilize the RNA-mediated nuclease, Cas9 in complex with guide and activating RNA to recognize and cleave foreign nucleic acid. Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae- Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria,

Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al, RNA Biol. 2013 May 1; 10(5): 726-737 ; Nat. Rev.

Microbiol. 2011 June; 9(6): 467-477; Hou, et al, Proc Natl Acad Sci U S A. 2013 Sep 24;110(39): 15644-9; Sampson et al, Nature. 2013 May 9;497(7448):254-7; and Jinek, et al, Science. 2012 Aug 17;337(6096):816-21.

[0091] Yet another way to suppress expression of an endogenous CENH3 gene in a plant is by targeted mutagenesis of CENH3 via non-homologous end joining (NHEJ) repair of DNA lesions induced through sequence-specific meganucleases such as TAL effector nucleases (TALENs). NHEJ-mediated targeted genome modification has been demonstrated in plants (Qi et al, Genome Res (2013, in press); Zhang and

Voytas, Methods Mol Biol 701 : 167-77 (2011)); see also Mahfouz and Li, GM Crops 2:99-103 (2011).

Expression of Heterologous CENH3 Protein

[0092] For the plants as described herein that lack endogenous CENH3 expression, a heterologous CENH3 protein from another plant species can be introduced into and expressed in the plant using an expression cassette comprising a polynucleotide encoding the heterologous CENH3 polypeptide, directed by a promoter. [0093] Any of a number of means well known in the art can be used to drive heterologous CENH3 activity or expression in plants. Any organ can be targeted, such as shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit. Alternatively, the heterologous CENH3 polynucleotide can be expressed

constitutively (e.g., using the CaMV 35S promoter). In some embodiments, a native promoter from the CENH3 gene can be used. For example, in some embodiments the native promoter from the inactivated endogenous CENH3 (e.g., the CENH3 of the first species) can be used.

[0094] To express a heterologous CENH3 sequence as described herein in a plant, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature, e.g., Weising et al., Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the desired polypeptide is combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.

[0095] For example, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the Γ- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill.

[0096] Alternatively, the plant promoter may direct expression of the

polynucleotide of the invention in a specific tissue (tissue-specific promoters) or organ (organ-specific promoters) or may be otherwise under more precise

environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves or guard cells (including but not limited to those described in WO/2005/085449; U.S. Patent No. 6,653,535; Li et al. , Sci China C Life Sci. 2005 Apr;48(2): 181-6; Husebye, et al, Plant Physiol, April 2002, Vol. 128, pp. 1180-1188; and Plesch, et al, Gene, Volume 249, Number 1, 16 May 2000, pp. 83- 89(7)), fruit, seeds, flowers, pistils, or anthers. Suitable promoters include those from genes encoding storage proteins or the lipid body membrane protein, oleosin.

Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light.

[0097] If proper polypeptide expression is desired, a polyadenylation region at the 3 '-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.

[0098] The vector comprising the sequences {e.g., promoters or coding regions) from genes of the invention can also comprise, for example, a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.

[0099] Embodiments of the present invention also provide for a heterologous CENH3 nucleic acid operably linked to a promoter which, in some embodiments, is capable of driving the transcription of the CENH3 coding sequence in plants. The promoter can be, e.g., derived from plant or viral sources. The promoter can be, e.g., constitutively active, inducible, or tissue specific. In construction of the recombinant expression cassettes, vectors, and transgenics of the invention, different promoters can be chosen and employed to differentially direct gene expression, e.g., in some or all tissues of a plant or animal.

Constitutive Promoters

[0100] A promoter, or an active fragment thereof, can be employed which will direct expression of a nucleic acid encoding a heterologous CENH3 protein as described herein in all transformed cells or tissues, e.g., as those of a regenerated plant. The term "constitutive regulatory element" means a regulatory element that confers a level of expression upon an operatively linked nucleic molecule that is relatively independent of the cell or tissue type in which the constitutive regulatory element is expressed. A constitutive regulatory element that is expressed in a plant generally is widely expressed in a large number of cell and tissue types. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. [0101] A variety of constitutive regulatory elements useful for ectopic expression in a transgenic plant are well known in the art. The cauliflower mosaic virus 35 S (CaMV 35S) promoter, for example, is a well-characterized constitutive regulatory element that produces a high level of expression in all plant tissues (Odell et al, Nature 313:810-812 (1985)). The CaMV 35S promoter can be particularly useful due to its activity in numerous diverse plant species (Benfey and Chua, Science 250:959- 966 (1990); Futterer et al, Physiol. Plant 79: 154 (1990); Odell et al, supra, 1985). A tandem 35 S promoter, in which the intrinsic promoter element has been duplicated, confers higher expression levels in comparison to the unmodified 35 S promoter (Kay et al., Science 236: 1299 (1987)). Other useful constitutive regulatory elements include, for example, the cauliflower mosaic virus 19S promoter; the Figwort mosaic virus promoter; and the nopaline synthase (nos) gene promoter (Singer et al. , Plant Mol. Biol. 14:433 (1990); An, Plant Physiol. 81 :86 (1986)).

[0102] Additional constitutive regulatory elements including those for efficient expression in monocots also are known in the art, for example, the pEmu promoter and promoters based on the rice Actin-1 5' region (Last et al, Theor. Appl. Genet.

81 :581 (1991); Mcelroy et al, Mol. Gen. Genet. 231 : 150 (1991); Mcelroy et al, Plant Cell 2: 163 (1990)). Chimeric regulatory elements, which combine elements from different genes, also can be useful for ectopically expressing a nucleic acid molecule encoding a protein of interest (Comai et al, Plant Mol. Biol. 15:373 (1990)). [0103] Examples of constitutive promoters include those from viruses which infect plants, such as the cauliflower mosaic virus (CaMV) 35S transcription initiation region (see, e.g., Dagless, Arch. Virol. 142: 183-191 (1997)); the Γ- or 2'- promoter derived from T-DNA of Agrobacterium tumefaciens (see, e.g., Mengiste supra (1997); O'Grady, Plant Mol. Biol. 29:99-108) (1995)); the promoter of the tobacco mosaic virus; the promoter of Figwort mosaic virus (see, e.g., Maiti, Transgenic Res. 6: 143-156) (1997)); actin promoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang, Plant Mol. Biol. 33: 125-139 (1997)); alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar, Plant Mol. Biol. 31 :897-904 (1996)); ACT11 from Arabidopsis (Huang et al., Plant Mol. Biol. 33:125-139 (1996)), Cat3 from

Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251 : 196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al., Plant Physiol. 104: 1167-1176 (1994)), GPcl from maize (GenBank No. X15596, Martinez et al, J. Mol. Biol. 208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunath et al, Plant Mol. Biol. 33:97-112 (1997)), other transcription initiation regions from various plant genes known to those of skill. See also Holtorf, "Comparison of different constitutive and inducible promoters for the overexpression of transgenes in

Arabidopsis thaliana," Plant Mol. Biol. 29:637-646 (1995). Additional constitutive promoters include, e.g., the polyubiquitin gene promoters from Arabidopsis thaliana, UBQ3 and UBQ10 (Norris et al., Plant Mol. Biol. 21 :895 (1993)), are also useful for directing gene expression. Inducible Promoters

[0104] One can optionally use an inducible promoter to control (1) expression of an artificial microRNA, siRNA, or other silencing polynucleotide, (2) and

simultaneously turn on expression of the heterologous CENH3 protein, or (3) both (1) and (2). This would have the advantage of having a normal plant (e.g., one that might have higher fertility) until induction, which would then create gametes ready for inducing haploids.

[0105] Alternatively, a plant promoter may direct expression of the heterologous CENH3 polynucleotide under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as "inducible" promoters. For example, the invention can incorporate a drought- specific promoter such as a drought-inducible promoter of maize (e.g., the maize rabl7 drought-inducible promoter (Vilardell et al. (1991) Plant Mol. Biol. 17:985- 993; Vilardell et al. (1994) Plant Mol. Biol. 24:561-569)); or alternatively a cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant Mol. Biol. 33:897-909). [0106] Alternatively, plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the heterologous CENH3

polynucleotide. For example, the invention can use the auxin-response elements El promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10:

955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996)

37:906-913); a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274: 1900-1902).

[0107] Plant promoters inducible upon exposure to chemical reagents that may be applied to the plant, such as herbicides, antibiotics, or a foreign hormone such as estradiol, are also useful for expressing the heterologous CENH3 polynucleotide. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. A CENH3 coding sequence can also be under the control of, e.g., a tetracycline -inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11 :465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11 : 1315-1324; Uknes et al., Plant Cell 5: 159-169 (1993); Bi et al, Plant J. 8:235-245 (1995)).

[0108] Examples of useful inducible regulatory elements include copper-inducible regulatory elements (Mett et al, Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717 (1988)); tetracycline and chlor-tetracycline -inducible regulatory elements (Gatz et al., Plant J. 2:397-404 (1992); Roder et al., Mol. Gen. Genet. 243:32-38 (1994); Gatz, Meth. Cell Biol. 50:411-424 (1995)); ecdysone inducible regulatory elements (Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318 (1992); Kreutzweiser et al., Ecotoxicol. Environ. Safety 28: 14-24 (1994)); heat shock inducible regulatory elements (Takahashi et al., Plant Physiol.

99:383-390 (1992); Yabe et al, Plant Cell Physiol. 35: 1207-1219 (1994); Ueda et al, Mol. Gen. Genet. 250:533-539 (1996)); and lac operon elements, which are used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression (Wilde et al, EMBO J. 11 : 1251-1259 (1992)). An inducible regulatory element useful in generating the plants of the invention also can be, for example, a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al, Plant Mol. Biol. 17:9 (1991)) or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et αΙ., ΜοΙ. Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471 (1990)).

Tissue-Specific Promoters

[0109] Another alternative is to downregulate the endogenous CENH3 protein (e.g., by gene silencing) in a specific tissue {e.g., at least in the mature gametophytes (either pollen or embryo sac)) and to replace it only in this specific tissue with a specific promoter that drives expression of the heterologous CENH3 protein. Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. In some embodiments, the same tissue-specific promoter is used to drive an artificial microRNA, siRNA, or other silencing polynucleotide and the heterologous CENH3 polynucleotide.

[0110] Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue, or epidermis or mesophyll. Reproductive tissue-specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof. In some embodiments, the promoter is cell-type specific, e.g., guard cell-specific.

[0111] Other tissue-specific promoters include seed promoters. Suitable seed- specific promoters are derived from the following genes: MACl from maize (Sheridan (1996) Genetics 142:1009-1020); Cat3 from maize (GenBank No. L05934, Abler (1993) Plant Mol. Biol. 22:10131-1038); vivparous-1 from Arabidopsis (Genbank No. U93215); atmycl from Arabidopsis (Urao (1996) Plant Mol. Biol. 32:571-57;

Conceicao (1994) Plant 5:493-505); napA from Brassica napus (GenBank No. J02798, Josefsson (1987) JBL 26: 12196-1301); and the napin gene family from Brassica napus (Sjodahl (1995) Planta 197:264-271).

[0112] A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can also be used to express polynucleotides encoding heterologous CENH3 polypeptides. For example, promoters controlling patatin, the major storage protein of the potato tuber, can be used, see, e.g., Kim (1994) Plant Mol. Biol. 26:603-615; Martin (1997) Plant J. 11 :53-62. The ORF13 promoter from Agrobacterium rhizogenes that exhibits high activity in roots can also be used (Hansen (1997) Mol. Gen. Genet. 254:337-343. Other useful vegetative tissue- specific promoters include: the tarin promoter of the gene encoding a globulin from a major taro {Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra (1995) Plant Mol. Biol. 28: 137-144); the curculin promoter active during taro corm development (de Castro (1992) Plant Cell 4:1549-1559) and the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto (1991) Plant Cell 3:371-382).

[0113] Leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCS1 and RBCS2 are expressed in developing tomato fruits (Meier (1997) FEBS Lett.

415:91-95). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka (1994) Plant J. 6:311-319, can be used. Another leaf-specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter, see, e.g., Shiina (1997) Plant Physiol. 115:477-483; Casal (1998) Plant Physiol. 116: 1533-1538. The Arabidopsis thaliana myb-related gene promoter (Atmyb5) described by Li (1996) FEBS Lett. 379: 117-121, is leaf- specific. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage. A leaf promoter identified in maize by Busk (1997) Plant J.

11 : 1285-1295, can also be used. [0114] Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters. For example, the "SHOOTMERISTEMLESS" and "SCARECROW" promoters, which are active in the developing shoot or root apical meristems, described by Di Laurenzio (1996) Cell 86:423-433; and, Long (1996) Nature 379:66-69; can be used. Another useful promoter is that which controls the expression of 3-hydroxy-3- methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell. 7:517-527). Also useful are knl-related genes from maize and other species which show meristem-specific expression {see, e.g., Granger (1996) Plant Mol. Biol. 31 :373-378; Kerstetter (1994) Plant Cell

6: 1877-1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51) and the Arabidopsis thaliana K AT1 promoter {see, e.g., Lincoln (1994) Plant Cell 6: 1859-1876). [0115] One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.

[0116] In another embodiment, the heterologous CENH3 polynucleotide is expressed through a transposable element. This allows for constitutive, yet periodic and infrequent expression of the constitutively active polypeptide. The invention also provides for use of tissue-specific promoters derived from viruses including, e.g. , the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92: 1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong

phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer {1996) Plant Mol. Biol. 31 : 1129-1139).

V. Plant Breeding [0117] Parent plants as described herein {e.g., plants having a genome from at least two species, wherein a majority of the genome is from a first species and the genome comprises a heterologous genomic region from a second species, wherein the heterologous genomic region encodes a CENH3 polypeptide different from the CENH3 of the first species; or plants comprising an inactivated endogenous CENH3 gene of a first plant species and further comprising an expression cassette comprising a polynucleotide encoding a heterologous CENH3 polypeptide from a species different from the first plant species) can be crossed either as a pollen parent or an ovule parent to another plant that expresses an endogenous CENH3 gene. This crossing step will result in at least some progeny (e.g., at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or more) that are haploid and comprise only chromosomes from the plant that expresses the endogenous CENH3 gene. Thus, the present disclosure provides for the generation of haploid plants having all of their chromosomes from a plant of interest by crossing the plant of interest with a plant that expresses a divergent CENH3 (e.g., a plant expressing a CENH3 transgene or a plant having CENH3 introgressed into the genome) and collecting the resulting haploid seed. [0118] Once generated, haploid plants can be used for a variety of useful endeavors, including but not limited to the generation of doubled haploid plants, which comprise an exact duplicate copy of chromosomes. Such doubled haploid plants are of particular use to speed plant breeding, for example. A wide variety of methods are known for generating doubled haploid organisms from haploid organisms. [0119] Somatic haploid cells, haploid embryos, haploid seeds, or haploid plants produced from haploid seeds can be treated with a chromosome doubling agent.

Homozygous double haploid plants can be regenerated from haploid cells by contacting the haploid cells, including but not limited to haploid callus, with chromosome doubling agents, such as colchicine, anti-microtubule herbicides, or nitrous oxide to create homozygous doubled haploid cells.

[0120] Methods of chromosome doubling are disclosed in, for example, U.S. Patent No. 5,770,788; 7,135,615, and US Patent Publication No. 2004/0210959 and

2005/0289673; Antoine-Michard, S. et al, Plant Cell, Tissue Organ Cult., Cordrecht, the Netherlands, Kluwer Academic Publishers 48(3):203-207 (1997); Kato, A., Maize Genetics Cooperation Newsletter 1997, 36-37; and Wan, Y. et al, Trends Genetics 77: 889-892 (1989). Wan, Y. et al, Trends Genetics 81 : 205-211 (1991), the disclosures of which are incorporated herein by reference in their entirety for all purposes. Methods can involve, for example, contacting the haploid cell with nitrous oxide, anti-microtubule herbicides, or colchicine. Optionally, the haploids can be transformed with a heterologous gene of interest, if desired.

[0121] Double haploid plants can be further crossed to other plants to generate Fl , F2, or subsequent generations of plants with desired traits.

VI. Plants Expressing Heterologous CENH3 Protein

[0122] In yet another aspect, the present disclosure provides plants comprising an inactivated endogenous CENH3 gene and further comprising a heterologous CENH3 gene from another species. In some embodiments, the plant (e.g., a plant of a first plant species) comprises an inactivated endogenous CENH3 gene and further comprises an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a heterologous CENH3 polypeptide from another plant species (e.g., a second plant species).

[0123] In some embodiments, the heterologous CENH3 polypeptide is identical or substantially identical to (e.g., at least 99% identical to) a naturally occurring CENH3 polypeptide of another species of plant than the first plant species. In some embodiments, the heterologous CENH3 polypeptide is a naturally occurring CENH3 polypeptide of a plant of the same family as the plant comprising the expression cassette (e.g., the first plant species). In some embodiments, the heterologous CENH3 polypeptide is a naturally occurring CENH3 polypeptide of a plant of the same genus as the plant comprising the expression cassette (e.g., the first plant species).

[0124] In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is substantially identical (e.g., at least about 70%>, 75%>, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the endogenous CENH3 polypeptide histone fold domain. In some embodiments, the heterologous CENH3 polypeptide comprises an N-terminal tail region that is substantially identical (e.g., at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the N-terminal tail region of the endogenous CENH3 polypeptide (e.g., the CENH3 of the first species). In some embodiments, the heterologous CENH3 polypeptide comprises a histone fold domain that is identical to the endogenous CENH3 polypeptide histone fold domain (e.g., the CENH3 histone fold domain of the first species) and an N-terminal tail region that is different from the endogenous CENH3 N-terminal tail region (e.g., the CENH3 N- terminal tail region of the first species).

[0125] In some embodiments, the plant comprises an expression cassette as described herein, e.g., in Section IV above. In some embodiments, the expression cassette comprises a promoter that is a constitutively active promoter. In some embodiments, the expression cassette comprises a promoter that is a native CENH3 promoter. In some embodiments, the expression cassette comprises a promoter that is the native CENH3 promoter of the plant comprising the expression cassette (e.g., the native CENH3 promoter of the first plant species).

[0126] The plants expressing heterologous CENH3 proteins as described herein can be used according to the breeding methods described herein. In some embodiments, a plant as described herein, when crossed with a second plant (e.g., a wild-type plant expressing an endogenous CENH3 protein or a double haploid mutant plant), generates at least some progeny (e.g., at least 0.1 %, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%), 8%), 9%), 10%), 15%), 20%) or more) that are haploid. In some embodiments, the haploid progeny comprise only chromosomes from the second plant (e.g., wild-type or double haploid mutant plant).

VII. Production of Plants Expressing Heterologous CENH3 Protein [0127] In another aspect, the present disclosure provides methods of making plants expressing heterologous CENH3 protein as described herein. In some embodiments, the method comprises: transforming plant cells with a nucleic acid comprising an expression cassette as described herein; and selecting transformants comprising the nucleic acid; thereby making the plant.

[0128] Nucleic acid constructs as described herein may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the nucleic acid construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the nucleic acid constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment. Alternatively, the nucleic acid constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.

[0129] Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of nucleic acid constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3:2717- 2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985). Biolistic transformation techniques are described in Klein et al., Nature 327:70-73 (1987).

[0130] Agrobacterium tumefaciens-mediatGd transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803 (1983).

[0131] Transformed plant cells which are derived by any of the above

transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased disease resistance compared to a control plant that was not transformed or

transformed with an empty vector. Such regeneration techniques rely on

manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).

[0132] The nucleic acid constructs disclosed herein can be used to confer the characteristics described herein, including the ability to generate haploid progeny, as described herein, on essentially any plant. Thus, the invention has use over a broad range of plants, including dicots or monocots, including e.g., species from the genera Allium, Amaranthus, Arachis, Arabidopsis, Asparagus, Atropa, Avena, Beta,

Brassica, Cannabis, Capsella, Cica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Dioscorea, Elais, Eruca, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Ipomea, Lactuca, Lepidium, Linum, Lolium, Luzula, Lycopersicon, Malus, Manihot, Majorana,

Medicago, Musa, Nicotiana, Olea, Oryza, Panicum, Pennisetum, Persea, Phaseolus, Pinus, Pisum, Populus, Pyrus, Prunus, Raphanus, Saccharum, Secale, Senecio, Sesamum, Sinapis, Solanum, Sorghum, Theobroma, Trigonella, Triticum, Turritis, Vitis, Vigna, or Zea.

VIII. Examples

[0133] The following examples are offered to illustrate, but not to limit the claimed invention. Example 1: Mitotic and meiotic complementation of Brassicaceae CENH3 alleles

[0134] In this example, CENH3 alleles with Brassicaceae (the family comprising A. thaliana) are surveyed to determine whether CENH3 heterologs functionally complement the cenh3 mutation in mitosis and meiosis.

[0135] Constructs are generated that express untagged CENH3 heterologs under the control of native A. thaliana CENH3 regulatory sequences. The functions of the evolutionarily derived CENH3 variant is then assayed in the following ways: (1) Complementation of the mitotic functions of an A. thaliana cenh3 mutant. Mitotic complementation will be inferred if plants expressing the CENH3 heterolog are viable in the cenh3 homozygous mutant background. (2) CENH3 variant functionality during meiosis. Some alleles of CENH3 are functional in mitosis but have meiosis- specific defects (Ravi et al., Genetics 186:461-471 (2010)). Meiotic complementation of CENH3 heterologs will be inferred if the plants in (1) are fertile and produce siliques with a seed set comparable to wild-type A. thaliana. (3) Evaluation of centromere activity. A. thaliana centromeres with foreign CENH3 will be compared to centromeres with native CENH3. Plants from (2) will be crossed to a Ler gll, an A. thaliana accession with wild-type chromosomes. The recessive gll mutation allows genome elimination to be scored easily, as haploids will show the gll phenotype (absence of leaf hairs). If the two sets of parental chromosomes have centromeres of unequal strength, chromosomal mis-segregation in the zygote is expected. Defects in segregation will be inferred if aneuploids and haploids are recovered in Fl progeny.

Example 2: Functional complementation of A. thaliana cenh3 with CENH3 from other species

[0136] To assay the effect of evolutionarily-derived substitutions in CENH3 we generated constructs that express untagged CENH3 orthologs under the control of native A. thaliana CENH3 regulatory sequences. We surveyed CENH3 alleles within and beyond the family Brassicaceae (containing A. thaliana) to refine the evolutionary boundaries for mitotic and meiotic complementation. We assayed functional complementation in the following ways:

[0137] 1) Does the CENH3 variant complement the mitotic functions of an A. thaliana cenh3 mutant? Mitotic complementation will be inferred if plants expressing the CENH3 ortholog are viable in the cenh3 homozygous mutant background. [0138] 2) Is the CENH3 variant functional during meiosis? Some alleles of CENH3 are functional in mitosis but have meiosis-specific defects (M. Ravi, et al. PLoS Genet., 7(6):el002121 ( 2011)). Meiotic complementation of CENH3 heterologs is inferred if the plants in (1) are fertile and produce siliques with a seed set comparable to wild-type A. thaliana. [0139] 3) How do A. thaliana centromeres with foreign CENH3 compete against centromeres with native CENH3? Plants from (2) were be crossed to a Ler gll, a A. thaliana accession with wild-type chromosomes. The recessive gll mutation allows genome elimination to be scored easily, as haploids will show the gll phenotype (absence of leaf hairs). If the two sets of parental chromosomes have centromeres of unequal strength, chromosomal mis-segregation in the zygote is expected (Figure 1). Defects in segregation will be inferred if aneuploids and haploids are recovered in Fl progeny.

[0140] Here we show data indicating that centromeres built on heterologous CENH3 proteins are functionally weaker than native centromeres. We tested CENH3 alleles from two species Lepidium oleraceum and Brassica rapa (Figure 2; A.

thaliana CENH3 polypeptide (SEQ ID NO: 10); L. oleraceum CENH3 polypeptide (SEQ ID NO:49); B. rapa CENH3 polypeptide (SEQ ID NO:50)). Arabidopsis and L. oleraceum are sister species (estimated time of divergence 8-36 MY A), while the ancestors of B. rapa and A. thaliana ancestors split off from one another during the earliest radiation of the Brassicaceae family (estimated time of divergence 24-40 MYA) (A. Franzke, et al., Trends Plant Sci., 16(2): 108-16 (2011)). Both !.

oleraceum CENH3 and B. rapa CENH3 were able to functionally complement the A. thaliana cenh3 mutation, i.e. plants expressing only the orthologous CENH3 were viable and fertile. However, when crossed to wild-type A. thaliana these

complemented lines exhibited segregation defects. In fact, we recovered a significant fraction of haploid Fl progeny from these crosses, representing a complete loss of chromosomes that contained heterologous CENH3. For both the heterologous CENH3 proteins we tested three independent transgenic lines in crosses to wild type and recovered haploids at consistent frequencies (Table 1).

Table 1:

[0141] The implication of this result is that natural alleles of CENH3, when introduced into a non-native genetic background, can act as haploid inducers because they result in "weaker" centromeres. This suggests a simple introgression-based approach for the generation of a non-transgenic haploid inducer, whereby an introgression overlapping with the CENH3 gene effectively generates a plant where the native CENH3 is replaced with a non-native allele from a sister species. Nearly all species have a single functional CENH3 gene. Moreover, in contrast to the GFP- tailswap haploid-inducer line that is male sterile, the heterologous CENH3 lines have the advantage of being fully fertile, a quality desirable in most crop species. [0142] The L. oleraceum CENH3 gene has 12 amino acid substitutions in its histone fold domain (HFD) relative to A. thaliana and >30 in its N-terminal tail region (Figure 2). To map the domain within the CENH3 protein causing segregation defects, we tested a chimeric proteins where the N-terminal tail of A. thaliana CENH3 is fused to the histone fold domain (HFD) of L. oleraceum CENH3 and vice versa. Both the chimeric transgenes functionally complemented the A. thaliana cenH3, i.e. plants expressing only the chimeric CENH3 proteins are viable and fertile. With respect to segregation defects we only have results from one of the chimeras: A. thaliana N-terminal tail fused to L. oleraceum HFD (At-tail::Lo-HFD). Strikingly, we recovered no haploids when we crossed At-tail::Lo-HFD complemented plants to wild-type individuals (Table 1). While this result does not rule out the role of the HFD in causing genome elimination, it clearly points out that the divergence in the N- terminal tail domain plays a role for orthologous CENH3 proteins to act as haploid inducers.

[0143] This result is promising with regards to the non-transgenic approach because the N-terminal tail domain is the most rapidly evolving region of CENH3 and is the region that differentiates first with evolutionary distance. Species close enough to be crossed for the purposes of generating introgression lines are likely to have

accumulated lineage-specific substitutions in their CENH3 N-terminal tails.

[0144] It is a significant advantage if a haploid inducer line could be used for genome elimination not only within its own species but also in crosses to its close relatives. This is particularly relevant in a genus such as Brassica where there are multiple commercially relevant crop species that can be crossed to one another. Additionally, the non-transgenic haploid inducer could be developed as an introgression in a genetic background different from the crop species, requiring an interspecies or interploidy cross. As a proof of concept we tested whether we could generate haploids in the allopolyploid species Arabidopsis suecica by crossing it to the A. thaliana tailswap line. A. suecica, originated from a single hybridization event that took place 5-300 thousand years ago between A. thaliana (2n =10) and A.

arenosa (2n = 16) (T. Sail, et al., J. Evol. Biol. 16(5): 1019-29 (2003)).

[0145] Since the A. thaliana GFP-tailswap haploid inducer line tends to be predominantly male sterile, we pollinated multiple GFP-tailswap plants without emasculation, with pollen from wild-type A. suecica. Seeds were germinated on MS media and of the 576 germinated seedlings that were transplanted only 241 survived. Two of the surviving seedlings were identified as haploids from their sterile phenotype and A.suecicaASks, appearance. Flow cytometry and molecular karyotyping confirmed the haploid genotype (Figure 3). Only a small fraction of the 241 plants appeared to be products of the interspecies cross with the majority being products of selfing of the tailswap line. Engineering a male sterile haploid inducer line could eliminate this background. In this case the interspecific cross as a method for genome elimination appears to be straightforward and yields haploids with a frequency of at least 1%. [0146] In conclusion, despite the pitfalls to this approach, namely interspecies barrier, differences in parental chromosome numbers, and endosperm imbalance, we have demonstrated the feasibility of this method.

[0147] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of generating a haploid plant, the method

comprising:

crossing a first plant expressing an endogenous centromeric histone H3 (CENH3) gene to a haploid inducer plant, the haploid inducer plant having a genome from at least two species, wherein a majority of the genome is from a first species and the genome comprises a heterologous genomic region from a second species, wherein the heterologous genomic region encodes a CENH3 polypeptide different from the CENH3 of the first species; and

selecting Fl haploid progeny generated from the crossing step.

2. The method of claim 1, wherein the heterologous genomic region encodes a CENH3 polypeptide comprising a histone fold domain that is at least 70% identical to the histone fold domain of the CENH3 of the first species.

3. The method of claim 1, wherein the heterologous genomic region encodes a CENH3 polypeptide comprising a histone fold domain that is at least 90% identical to the histone fold domain of the CENH3 of the first species.

4. The method of claim 1, wherein the heterologous genomic region encodes a CENH3 polypeptide from the same family as the CENH3 of the first species.

5. The method of claim 1, wherein the heterologous genomic region encodes a CENH3 polypeptide from the same genus as the CENH3 of the first species.

6. The method of claim 1, wherein the heterologous genomic region encodes a CENH3 polypeptide comprising an N-terminal tail region that is substantially identical to the N-terminal domain of the CENH3 of the first species.

7. The method of claim 1, wherein the heterologous genomic region encodes a CENH3 polypeptide comprising a histone fold domain that is identical to the histone fold domain of the CENH3 of the first species and an N- terminal tail region that is different from the N-terminal tail region of the CENH3 of the first species.

8. The method of claim 1 , wherein the haploid inducer plant is a recombinant inbred line.

9. The method of claim 1 , wherein the haploid inducer plant is an introgression line.

10. The method of claim 1, wherein the haploid inducer plant is a diploid plant.

11. The method of claim 1 , wherein the haploid inducer plant is an allopolyploid plant.

12. The method of claim 11 , wherein the haploid inducer plant is tetraploid or hexaploid.

13. The method of claim 1 , wherein the haploid inducer plant comprises a chromosome from the second species and said chromosome comprises the heterologous genomic region encoding the CENH3 polypeptide.

14. The method of claim 1 , wherein the first plant is a plant of the first species.

15. The method of claim 1 , wherein the first plant is not a plant of the first species or the second species.

16. The method of claim 1, wherein the first plant is the pollen parent for the crossing step.

17. The method of claim 1, wherein the first plant is the ovule parent for the crossing step.

18. The method of claim 1, further comprising converting at least one selected haploid progeny into a doubled haploid plant.

19. A method of generating a haploid plant, the method comprising: crossing a first plant of a first species expressing an endogenous centromeric histone H3 (CENH3) gene to a haploid inducer plant comprising an inactivated endogenous CENH3 gene and further comprising an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a heterologous CENH3 polypeptide from a species different from the first species; and

selecting Fl haploid progeny generated from the crossing step.

20. The method of claim 19, wherein the heterologous CENH3 polypeptide comprises a histone fold domain that is at least 70% identical to the endogenous CENH3 polypeptide histone fold domain.

21. The method of claim 19, wherein the first plant is the pollen parent for the crossing step.

22. The method of claim 19, wherein the first plant is the ovule parent for the crossing step.

23. The method of claim 19, further comprising converting at least one selected haploid progeny into a doubled haploid plant.

24. A plant comprising an inactivated endogenous centromeric histone H3 (CENH3) gene and further comprising an expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a heterologous CENH3 polypeptide from another plant species.

25. The plant of claim 24, wherein the heterologous CENH3 polypeptide comprises a histone fold domain that is at least 70% identical to the endogenous CENH3 polypeptide histone fold domain.

26. The plant of claim 24, wherein the heterologous CENH3 polypeptide comprises a histone fold domain that is at least 90% identical to the endogenous CENH3 histone fold domain.

27. The plant of claim 24, wherein the heterologous CENH3 polypeptide is from a plant of the same family as the plant comprising the expression cassette.

28. The plant of claim 24, wherein the heterologous CENH3 polypeptide is from a plant of the same genus as the plant comprising the expression cassette.

29. The plant of claim 24, wherein the heterologous CENH3 polypeptide comprises an N-terminal tail region that is substantially identical to the N-terminal domain of the endogenous CENH3 polypeptide.

30. The plant of claim 24, wherein the heterologous CENH3 polypeptide comprises a histone fold domain that is identical to the endogenous CENH3 polypeptide histone fold domain and an N-terminal tail region that is different from the endogenous CENH3 polypeptide N-terminal tail region.

31. The plant of claim 24, wherein the promoter is a constitutively active promoter.

32. The plant of claim 24, wherein the promoter is the native CENH3 promoter of the plant comprising the expression cassette.

33. The plant of claim 24, wherein the plant, when crossed with another plant, generates at least 0.1% haploid progeny.

34. A method of making a plant of claim 24, the method comprising:

transforming plant cells with a nucleic acid comprising the expression cassette; and

selecting transformants comprising the nucleic acid; thereby making the plant.