Methods and compositions for modifying plant growth and development

Abstract

Methods and compositions are provided for modifying an agronomic characteristic in corn plants are provided. Modified corn plants are disclosed comprising a mutation in a plant growth regulatory gene. The modified plants have a reduced plant height or stature, or a lower ear height, than control plants.

Description

[0001] METHODS AND COMPOSITIONS FOR MODIFYING PLANT GROWTH AND DEVELOPMENT

[0002] RELATED APPLICATION INFORMATION

[0003] This application claims priority to United States Provisional Application Serial No, 63 / 559,248 filed February 29, 2024, the contents of which are incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING ELECTRONIC SUBMISSION OF A

[0005] SEQUENCE LISTING

[0006] Sequence Listing in ST.26 format, submitted under 37 C.F.R. § 1.821, entitled PAT- 109899- WO-SEC-l fyEQLIST.xml, 2,582,000 bytes in size, generated on February 28, 2024, and filed via EFS-Web is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.

[0007] FIELD

[0008] This disclosure relates to the field of plant biotechnology. In particular, it relates to methods and compositions for modifying agronomic characteristics of a plant.

[0009] BACKGROUND

[0010] Plants with short stature have had a major impact on world agriculture, demonstrated by the adoption of dwarf varieties of rice and wheat that gave rise to the Green Revolution. In wheat, rice and sorghum, mutations in the gibberellin synthesis or signaling pathways that led to the Green Revolution have no significant effect on the reproduction systems of the plant. The same mutations have not been utilized in com because disruption of the GA synthesis and signaling pathway has repeatedly led to dramatic distortion and masculinization of the ear (“anther ear”) and sterility (disrupted anther and microspore development) in the tassel, in addition to extreme dwarfing in some cases. See, e.g., Chen, Y et al., Plant Physiology 166: 2028-2039 (2014). These GA mutant phenotypes (off-types) in com led to significant reductions in kerne! production and a reduction in yield. Furthermore, production of anthers within the ear increases the likelihood of fungal or insect infections, which reduces the quality of the grain that is produced on those mutant ears. Forward breeding to develop semi -dwarf lines of com has not been successful, and the reproductive off- types (as well as the extreme dwarfing) of GA mutants have been challenging to overcome. Thus, the same mutations in the GA pathway that, led to the Green Revolution in other grasses have not yet been successful in corn. However, agronomic interest in short plants derives largely from their ability to resist lodging caused by wind, rain, or higher densities and thus remains an area of interest in agriculture.

[0011] SUMMARY

[0012] Methods and systems are provided for providing edited plants (e.g., corn plants) having a modified stature or height, or modified ear height, or both, relative to a control plant (e.g.. from a corresponding control plant that is unedited). In embodiments, the plants have a plant height or stature that is shorter (e.g., significantly shorter or slightly shorter) than the height of a control plant, or an ear height that is lower (e.g., significantly lower or slightly lower) that the height of a control plant, or has both a reduced plant height and a reduced ear height. Compositions are also provided for nucleic acid molecules (e.g., expression cassettes or vectors) capable of introducing targeted edits in the genome of a plant cell, particularly in the locus of select plant genes involved in regulating plant height or ear height, resulting in the creation of novel mutant alleles of the genes involved in regulating plant height or ear height (referred to herein as “plant growth regulatory genes'’). In embodiments, the resulting mutant alleles comprise sequences that when included in the genome a plant, confer the plants with a modified (e.g., shorter) plant height or stature, or a lower ear height, or both, as compared to plants not comprising the mutant alleles. Such compositions, and methods of using such compositions, enable plants to be produced that can also have an increased stalk diameter, a decrease in biomass, an increased planting density, increased harvest index, increased leaf area, reduced leaf number above the ear, increase in the ratio of the plant ear height over the plant height, and / or reduced lodging, as compared to a. control plant not having the genetic modifications of the invention.

[0013] In embodiments, the reduced stature or reduced ear height plant of the invention comprises one or more genetic modifications targeting one or more distinct genomic loci in the plant growth regulatory genes. In an embodiment, one or more genetic modifications is present within (a) the same coding region; (b) non-coding region; (c) regulatory sequence; or (d) untranslated region, of an endogenous polynucleotide encoding a polypeptide that is involved in plant growth.

[0014] In certain embodiments, the modified plant growth regulatory' gene(s) comprise one or more of the nucleotide sequences set forth in SEQ ID NO: 1-51, or a variant. fragment, or complement thereof, a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 52-102, or a variant, fragment, or complement thereof; or an endogenous regulatory' element of the foregoing. In various embodiments, the modified plant growth regulatory gene is selected from a homolog of one or more of the maize genes listed in Tables lA-lB, Tables 2A-2B, Table 3 and Table 4. In embodiments, the modified com plant exhibits a reduction in plant height or ear height of about 5% to about 50% when compared to a control corn plant not comprising the plant growth reducing genetic modification.

[0015] In embodiments, the genetic modification is introduced into the target gene through genome editing (e.g., genome modification using a site directed nuclease).

[0016] Embodiments of the invention include plants or plant cells comprising genetic modifications at one or more loci comprising the nucleotide sequence set forth in SEQ

[0017] ID NO: 1-51, or a variant fragment, or complement thereof, a nucleic acid encoding the ammo acid sequence set forth in SEQ ID NO: 52-102, or a variant, fragment, or complement thereof; or an endogenous regulator}' element of the foregoing. In various embodiments, the genetic modification is introduced into a plant growth regulators' gene that is a homolog of one or more of the maize genes listed in Tables 1 A-1 B, Tables 2A-2B, Table 3 and Table 4 (identifiable by the ZmOOOOld designation).

[0018] Further methods of the invention also include the use of mutagenesis and recombination (for example directed using chimeric oligonucleotides, meganucleases, zinc finger nucleases, TALEN or CRISPR) to introduce specific strand breaks, recombination al insertions and mutations so as to engineer In situ changes in plant genomes that result in a decrease in plant height or ear height. Thus, the invention also includes plants, varieties seed and progeny thereof that are derived from the methods of the invention.

[0019] The invention further provides methods for producing com plants having reduced stature or reduced ear height. In example embodiments, the method comprises introducing, such as via genome modification using a site directed nuclease, a mutation at one or more plant growth regulatory' gene loci of a genome of a com plant; b) selfing the plant for one or more generations to generate a progeny plant that is homozygous at each of the modified plant growth regulatoiy gene loci; and growing; the plant.

[0020] In embodiments, the editing of the one or more loci comprises editing using a DNA modification enzyme (e.g., a site directed nuclease). In embodiments, the editing comprises introducing a mutation at one or more loci comprising the nucleotide sequence of SEQ ID NO: 1-51 , or a variant, fragment, or complement thereof) or a mutation in a nucleic acid encoding one or more of SEQ ID NO: 52402, or a variant, fragment, or complement thereof. In various embodiments, the modified plant growth regulatoiy gene is selected from a homolog of one or more of the maize genes listed in Tables 1A-1B, Tables 2A-2B, Table 3 and Table 4 (identifiable by the ZmOOOOld designation).

[0021] In embodiments, the editing comprises introducing into a plant cell an expression cassette comprising (i) a nucleic acid that encodes the site-directed nuclease; and (ii) a nucleic acid that encodes at least one guide RNA (gRNA) directed to a target sequence within the one or more distinct genomic loci that are involved in regulating plant growth and development. In embodiments, the at least one gRNA is directed to a target sequence comprising SEQ ID NO: 1-51, or a nucleic acid encoding SEQ ID NO: 52-102 In various embodiments, the at least one gRNA is directed to a target sequence that is a homolog of one or more of the maize genes listed in Tables I A- IB, Tables 2A-2B, Table 3 and Table 4 (identifiable by the ZmOOOOld designation).

[0022] In example embodiments, the at least one gRNA is directed to a target sequence selected from one or more of SEQ ID NO: 103, 104, 106, 107, 111, 112, 113, 116, 117, 118. 119, 120, 121, 122. 126, 129, 130, 132. 133, 134, 135, 137. 138. 140, 143, 144, 145, 146, 147, and / or 149, including the corresponding sequences in homologous maize genes. In embodiments, the nucleic acid that encodes the site-directed nuclease is operably linked to a first promoter and the nucleic acid that encodes the at least one gRNA is operably linked to a second promoter. In embodiments, the site directed nuclease is selected from the group consisting of meganucleases (MNs), zmc- finger nucleases (ZFNs), transcription- activator tike effector nucleases (TALENs), and CRISPR-associated nucleases. In embodiments, the editing includes introducing into the one or more loci a mutation selected from the group consisting of an allele replacement, one or a plurality of base insertions, or one or a plurality of base deletions.

[0023] In embodiments, the editing further comprises regenerating a transformed TO plant from the transformed plant cell, the transformed TO plant having a plurality’ of T1 seed, wherein the plurality' of T1 seed contain a plurality’ of unique edits in the one or more distinct genomic loci involved in regulating plant growth; growing a plurality of T1 plants from the T1 seed; selfing the T1 plants for one or more generations to obtain a progeny plant that is homozygous for the one or more mutated loci.

[0024] In embodiments, the mutations at the one or more distinct genomic loci involved in regulating plant growth comprises a mutation that results in a loss of function or a partial loss of function of the one or more plant growth regulatory'- genes.

[0025] In certain embodiments, the mutations at the one or more distinct genomic loci involved in regulating plant growth comprise a mutation in a regulatory element of the plant growth regulatory gene, wherein a mutation in the regulatory' element results in an increased expression of the plant growth regulatory gene.

[0026] In other embodiments, a method of producing a com plant with a reduced height or lower ear height, or both, is provided. In example embodiments, the method comprises introducing an edit into a com plant cell at one or more distinct genomic loci that are involved regulating plant growth; and regenerating an edited com plant from the edited plant cell, wherein the edited progeny plant has a plant height that is shorter, and / or an ear height that is lower, than the plant height or ear height, respectively, of a corn plant not comprising the edits

[0027] BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0028] DEFINITIONS

[0029] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary' skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques and / or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0030] Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. For example, the phrase “a cell” refers to one or more cells, and in some embodiments can refer to a tissue and / or an organ. Similarly, the phrase “at least one”, when employed herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to ail whole number values between 1 and 100 as well as whole numbers greater than 100.

[0031] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” The term “about,” as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments 120%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods and / or employ the discloses compositions, nucleic acids, polypeptides, etc. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0032] As used herein, the term “allele” refers to a variant or an alternative nucleotide sequence of a gene or at a particular genetic locus. Such an allele can be considered (i) wild-type or (ii) mutant if one or more mutati ons or edits are present in the nucleic acid sequence of the mutant allele relative to the wild-type allele. In diploids, a single allele is inherited by a progeny individual separately from each parent at each locus. The two alleles of a given locus present in a diploid organism occupy corresponding places on a pair of homologous chromosomes, although one of ordinary skill in the art understands that the alleles in any particular individual do not necessarily represent ah of the alleles that are present in the species.

[0033] A mutant allele for a gene may have a reduced or eliminated activity or expression level for the gene relative to the wild-type allele. For diploid organisms such as com and soy, a first allele can occur on one chromosome, and a second allele can occur at the same locus on a second homologous chromosome. If one allele at a locus on one chromosome of a plant is a mutant allele and the other corresponding allele on the homologous chromosome of the plant is wild type, then the plant is described as being heterozygous for the mutant allele. However, if both alleles at a locus are mutant alleles, then the plant is described as being homozygous for the mutant alleles A plant homozygous for mutant alleles at a. locus may comprise the same mutant allele or different mutant alleles if heteroallelic or biallelic.

[0034] “Allelic variation” refers to the phenomenon of variation in the sequence form of an allele at a given genetic locus. Allelic variation results in the creation of two or more allelic variants. The variants may be naturally occurring and reflecti ve of genetic differences among individuals of the same species. Such natural variations can occur as a result of natural breeding patterns. Alternatively, the variants may be non- naturally occurring, and artificially created (e.g., by a breeder or a scientist), such as using mutagenesis and / or gene editing techniques. In embodiments of the invention, allelic variants of the plant growth regulatory gene are created through gene editing methods that result in the introduction of a mutation. In additional or alternative embodiments of the invention, allelic variants of the plant growth regulatory' gene may be created through chemical mutagenesis, transposon insertion or excision, or any other known mutagenesis technique.

[0035] In example embodiments, the mutation introduced into the plant growth regulatory gene is an allele replacement, one or a plurality of base pair insertions, or one or a plurality of base pair deletions. The base pair insertions or base pair deletions may include a 3n base mutation wherein a multiple of 3 base pairs are deieted or inserted (e.g., insertion or deletion of 3bp, 6bp, 9bp, 12bp, 15bp, 18bp, etc.), thereby not affecting the reading frame of the gene. Alternatively, the base pair insertion or deletion may not be a multiple of 3 base pairs (e.g., an insertion or deletion of 2bp, 4bp, 5bp, 7bp, 11 bp, etc.), thereby affecting the reading frame of the gene.

[0036] In particular embodiments, the mutation is a truncation mutation wherein the mutation can result in the introduction of a stop codon into the gene at a location earlier than intended. Transcription of the resulting mutant allele is terminated at the earlier than intended stop codon, resulting in a truncated protein that is shorter than the corresponding wild-type protein.

[0037] In other particular embodiments, the mutation is an in-frame deletion mutation wherein deletion of an integral multiple of three base pairs (that is, 3n base pairs) occurs. Since three base pairs encode a single amino acid, the result of the in-frame deletion is that the reading frame of the transcript is maintained (that is, no frameshift mutations are introduced), however, the transcript generated from the mutant allele encodes a mutated protein that is shorter than the corresponding wild-type protein. Both the in-frame deletion and truncation mutations in the gene result in a mutated allele that encodes a shortened protein having reduced function (e.g., partial loss of function or complete loss of function) compared to the protein encoded by the wildtype (i.e., unmutated) allele. Based on the resulting phenotype, the mutant alleles at a locus may be dominant or recessive. In embodiments, an allelic variant may result in partial expression of the encoded plant growth regulatory' protein, or may result in a loss of expression of the encoded plant growth regulatory protein.

[0038] As used herein, the term “and / or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C. and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, one of more of the elements to which the “and / or’ ’ refers can also individually be present in single or multiple occurrences in the combmations(s) and / or subcombination(s).

[0039] As used herein, the phrase “associated with” refers to a recognizable and / or assayable relationship between two entities. For example, the phrase “associated with plant growth regulation” refers to a trait, locus, gene, allele, marker, phenotype, etc., or the expression thereof, the presence or absence of which can influence plant growth phenotypes, such as the plant height or ear height, of a corn plant. As such, a marker is "‘associated with” a trait when it is linked to it and when the presence of the marker is an indicator of whether and / or to what extent the desired trait or trait form will occur m a plant / germplasm comprising the marker. Similarly, a marker is "‘associated with” an allele when it is linked to it and when the presence of the marker is an indicator of whether the allele is present in a plant / germplasm comprising the marker. For example, ‘"a marker associated with an allele or mutation in a plant growth regulatory’ gene” refers to a marker whose presence or absence can be used to predict whether the allele or mutation is present and responsible for the plant height or ear height.

[0040] A “dominant stature allele” is an allele that, when present either in single copy (heterozygous) or two copies (homozygous), affects the height or stature of the plant. A “recessive stature allele” is an allele that affects the height or stature of the plant only when present in two copies (homozygous), and does not affect the height or stature of a plant when present in a single copy (heterozygous).

[0041] A “dominant ear height allele” is an allele that, when present either in single copy (heterozygous) or two copies (homozygous), affects the ear height on the plant. A "‘recessive ear height allele” is an allele that affects the ear height on the plant only when present in two copies (homozygous), and does not affect the ear height when present in a single copy (heterozygous). The term “comprising,” which is synonymous with “including,” “containing,” and “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. “Comprising” is a term of art that means that the named elements and / or steps are present, but that other elements and / or steps can be added and still fall within the scope of the relevant subject matter.

[0042] As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specifically recited. When the phrase “consists of appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0043] As used herein, the phrase “consisting essentially of’ limits the scope of the related disclosure or claim to the specified materials and / or steps, plus those that do not materially affect the basic and novel characteristic(s) of the disclosed and / or claimed subject matter.

[0044] With respect to the terms “comprising,” “consisting essentially of,” and “consisting of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include in some embodiments the use of either of the other two terms. For example, if a subject matter relates in some embodiments to com plants having a reduced plant height or ear height, it is understood that the disclosed subject matter thus also encompasses corn plants with genomic modifications that in some embodiments consist essentially of the mutated plant growth regulatory gene(s) as well as corn plants with that consist of the mutated plant growth regulatory gene(s). Similarly, it is also understood that in some embodiments the methods for the disclosed subject matter comprise the steps that are disclosed herein, in some embodiments the methods for the presently disclosed subject matter consist essentially of the steps that are disclosed, and in some embodiments the methods for the presently disclosed subject matter consist of the steps that are disclosed herein. The term “corresponding to” in the context of nucleic acid sequences or ammo acid sequences means that when the nucleic acid sequences or amino acid sequences are aligned with each other, the nucleic acids that “correspond to” certain enumerated positions in the present invention are those that align with these positions in a reference sequence, but that are not necessarily in these exact numerical positions relative to a particular nucleic acid or ammo acid sequence of the invention.

[0045] As used herein, a “cultivar” is a race or variety of a plant that has been created or selected intentionally and maintained through cultivation.

[0046] As used herein, the term “gene” refers to a hereditary’ unit including a sequence of DNAthat occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristic or trait in an organism.

[0047] A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes within a given species, generally depicted in a diagrammatic or tabular form. The genetic map is distinct from a physical map which is a description of the location of a genetic element (e.g., gene, allele, chromosomal locus, marker, etc.) on a sequenced chromosome.

[0048] As used herein, the term “human-induced mutation” refers to any mutation that occurs as a result of either direct or indirect human action. This term includes, but is not limited to, mutations obtained by any method of targeted mutagenesis and gene editing.

[0049] As used herein, “’introduced” means delivered, expressed, applied, transported, transferred, permeated, or other like term to indicate the delivery, whether of nucleic acid or protein or combination thereof, of a desired object to an object. For example, nucleic acids encoding a site directed nuclease and optionally at least one guide RNA may be introduced into a plant embry o. Likewise, extant editing machinery (comprising a site directed nuclease protein and optionally at least one guide RNA) may be introduced into sterilized com seeds upon application of appropriate cellpenetrating chemicals and / or peptides.

[0050] As used herein, “line” refers to a group of individual plants from the similar parentage with similar traits. An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance. Additionally, an elite line is sufficiently homogenous and homozygous to be used for commercial production.

[0051] Elite lines may be used in the further breeding efforts to develop new elite lines. An elite plant is any plant from an elite line.

[0052] As used herein, “locus” is a chromosomal locus or region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. A “locus” can be shared by two homologous chromosomes to refer to their corresponding locus or region.

[0053] As used herein, the terms “marker probe” and “probe” refer to a nucleotide sequence or nucleic acid molecule that can be used to detect the presence or absence of a sequence within a larger sequence, e.g., a nucleic acid probe that is complementary' to all of or a portion of the marker or marker locus, through nucleic acid hybridization Marker probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more contiguous nucleotides can be used for nucleic acid hybridization.

[0054] As used herein, the term “molecular marker” can be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying the presence / absence of a gene or allele (such as an allele at a plant growth regulatory-associated locus, such as at a locus comprising a gene corresponding to one or more of SEQ ID NO: 1-51 or a nucleic acid encoding one or more of SEQ ID NO: 52- 102, including variants, fragments, and complements thereof, such as the variants and fragments set forth in SEQ ID NO: 156-328 or a nucleotide sequence encoding the variant polypeptides set forth in SEQ ID NO:329- 501). A molecular marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from an RNA, a cDNA, etc.). The term also refers to nucleotide sequences complementary to or flanking the marker sequences, such as nucleotide sequences used as probes and / or primers capable of amplifying the marker sequence. Nucleotide sequences are “complementary” when they specifically hybridize in solution (e.g., according to Watson-Crick base pairing rules). This term also refers to the genetic markers that indicate a trait by the absence of the nucleotide sequences complementary to or flanking the marker sequences, such as nucleotide sequences used as probes and / or primers capable of amplifying the marker sequence.

[0055] As used herein, the terms “nucleotide sequence,” “polynucleotide,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid fragment” refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, nonnatural, and / or altered nucleotide bases. A “nucleotide” is a monomeric unit from which DNA or RNA polymers are constructed and consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group. Nucleotides (usually found in their 5'- monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxy cytidylate, “G” for guanylate or deoxy guanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleoti de.

[0056] As used herein, “modified” in the context of a plant, plant seed, plant part, plant cell, and / or plant genome, refers to a plant, plant seed, plant part, plant cell, and / or plant genome comprising an engineered change in the expression level and / or coding sequence of one or more of a plant growth regulatory’ gene relative to a wild-type or control plant, plant seed, plant part, plant cell, and / or plant genome, such as via a genome editing event or mutation affecting (e.g., reducing, eliminating, or increasing) the expressi on level or activity of one or more endogenous plant growth regulatory genes. The term “modified” may further refer to a plant, plant seed, plant part, plant cell, and / or plant genome having one or more mutations affecting expression of one or more endogenous plant growth regulator)' genes introduced through chemical mutagenesis, transposon insertion or excision, or any other known mutagenesis technique, or introduced through genome editing. In various embodiments, the modification can comprise fin insertion, deletion, or substitution of one or more nucleotides, or a combination of a substitution, deletion, and / or insertion of multiple nucleotides, including an insertion, substitution, or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 26-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, or more than 100 nucleotides. For clarity, therefore, a modified plant, plant seed, plant part, plant cell, and / or plant includes a mutated and / or edited plant, plant seed, plant part, plant cell, and / or plant genome having a modified expression level, expression pattern, and / or coding sequence of one or more of (a) plant growth regulatory’ gene(s) relative to a wild-type or control plant, plant seed, plant part, plant cell, and / or plant genome. Modified plants may be homozy gous or heterozygous for any given mutation or edit, and / or may be bi-allelic at the plant growth regulatory- gene locus. A modified plant is bi-allelic for a modified gene of the invention if each copy of the gene is modified by a different allele (i.e., different mutation(s) and / or edit(s)), wherein each allele lowers the expression level and / or activity of the gene. Modified plants or seeds may contain various molecular changes that affect expression of the modified genets) of the invention, including genetic and / or epigenetic modifications. Modified plants, plant parts, seeds, etc., may have been subjected to mutagenesis, genome editing or site-directed integration (e.g., without being limiting, via methods using site-specific nucleases), genetic transformation (e.g., without being limiting, via methods of Agrobacterium transformation or microprojectile bombardment), or a combination thereof. Such “modified” plants, plant seeds, plant parts, and plant cells include plants, plant seeds, plant parts, and plant cells that are offspring or derived from “modified” plants, plant seeds, plant parts, and plant cells that retain the molecular change (e.g., change in expression level and / or activity) to the plant growth regulatory gene. A modified seed provided herein may give rise to a modified plant provided herein. A modified plant, plant seed, plant part, plant cell, or plant genome provided herein may comprise a recombinant DNA construct or vector or genome edit as provided herein. A “modified plant product” may be any product made from a modified plant, plant part, plant cell, or plant chromosome provided herein, or any portion or component thereof.

[0057] As used herein, the term “nucleotide sequence identity” refers to the presence of identical nucleotides at corresponding positions of two polynucleotides, and may encompass variants, fragments or complements of the reference nucleotide sequence. Polynucleotides have “identical” sequences if the sequence of nucleotides in the two polynucleotides is the same when aligned for maximum correspondence (e.g., in a comparison window). Sequence comparison between two or more polynucleotides is generally performed by comparing porti on s of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally from about 20 to 200 contiguous nucleotides. The “percentage of sequence identity” for polynucleotides, such as about 50. 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100 percent sequence identity, can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. In some embodiments, the percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences: (b) dividing the number of matched positions by the total number of positions in the window- of comparison; and (c) multiplying the result by 100. Optimal alignment of sequences for comparison can also be conducted by computerized implementations of know algorithms, or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) and ClustalW / ClustalW2 / Clustal Omega programs available on the Internet (e.g., the website of the EMBL-EBI). Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG Package available from Accelrys, Inc. of San Diego, Calif, United States of America. See also Smith & Waterman, 1981 ; Needleman & Wunsch, 1970; Pearson & Lipman, 1988; Ausubel et al, 1988; and Sambrook & Russell, 2001. Two sequences are "optimally aligned" when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Ammo acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978) "A model of evolutionary change in proteins." in "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. M. 0. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. and Hemkoff et al. (1992) Proc. Natl. Acad. Sei, USA 89: 10915-10919. The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols, The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. Die alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website (wvw.ncbi.nlm.nih.gov). Optimal alignments, including multiple alignments, can be prepared using, e.g., PSI- BLAST, available through www.ncbi.nlm.nih.gov and described by Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

[0058] Unless otherwise stated, optimal alignment and calculation of sequence identity and similarity will be calculated by the Needleman-Wunsch global alignment and scoring algorithms (Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453) as implemented by the "needle" program, distributed as part of the EMBOSS software package (Rice, P., Longden, I., and Bleasby, A., EMBOSS: The European Molecular Biology Open Software Suite, 2000, Trends in Genetics 16, (6) pp276-277 and Madeira et al. (2022) Nucleic Acids Research Vol. 50: Web Server issue, W276- W279. which is available from EMBL-EBI on the worldwide web at ebi.ac.uk / Tools / psa / , among other sources) using default gap penalties and scoring matrices (BLOSUM62 for protein and DNAFULL for DNA).

[0059] The term “open reading frame” (ORF) refers to a nucleic acid sequence that encodes a polypeptide. In some embodiments, an ORF comprises a translation initiation codon (i.e., start codon), a translation termination (i.e., stop codon), and the nucleic acid sequence there between that encodes the ammo acids present in the polypeptide. The terms ‘‘initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides (i.e.. a codon) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

[0060] As used herein, the terms “phenotype,” “phenotypic trait” or “trait” refer to one or more traits of a plant or plant cell. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus (i.e., corresponds to a “single gene trait”). In other cases, a phenotype is the result of interactions among several genes, which in some embodiments also results from an interaction of the plant and / or plant cell with its environment,

[0061] In the case of the plant growth regulator)' com genes of the invention, phenotypic trait includes one or both of plant height and ear height. In particular embodiments, the phenotypic trait measured is a plant height and is a measure of the vertical distance from the soil surface to the first branch of the tassel. In another embodiment, the phenotypic trait measured is ear height and is a measure of the vertical distance from the soil surface to the node with the uppermost ear. It is understood that other methods for measuring plant height can be used, for example, by measuring the vertical distance from the soil surface to the highest point of the arch of the uppermost leaf whose tip is arched over, or by measuring the vertical distance from the base of the stem (at the soil surface) to the top of the canopy, or the highest part of the plant.

[0062] Other methods involve the use of computerized equipment for measuring plant height and ear height. A plant having a reduced plant height or ear height, or both, of the invention is one that has a reduction in one or both characteristics when compared to a control plant using the same methods of measurement for each.

[0063] As used herein, the term “plant” can refer to a whole plant, any part thereof, or a cell or tissue culture denved from a plant. Thus, the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, sterns, roots, etc.), plant tissues, seeds and / or plant cells.

[0064] A plant cell is a cell of a plant, taken from a plant, or denved through culture from a cell taken from a plant. Thus, the term “plant cell” includes without limitation cells within seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores. The phrase “plant part” refers to a part of a plant, including single cells and cell tissues such as plant ceils that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as scions, rootstocks, protoplasts, calli, and the like.

[0065] As used herein, the term “primer” refers to an oligonucleotide which is capable of annealing to a nucleic acid target (in some embodiments, annealing specifically to a. nucleic acid target) allowing a DNA polymerase and / or reverse transcriptase to attach thereto, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH). In some embodiments, one or more pluralities of primers are employed to amplify plant nucleic acids (e.g., using the polymerase chain reaction; PCR).

[0066] As used herein, the term “probe” refers to a nucleic acid (e.g., a single stranded nucleic acid or a strand of a double stranded or higher order nucleic acid, or a subsequence thereof) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, a probe is of sufficient length to form a stable and sequence-specific duplex molecule with its complement, and as such can be employed in some embodiments to detect a sequence of interest present in a plurality of nucleic acids.

[0067] As used herein, the terms “progeny” and “progeny plant” refer to a plant generated from vegetative or sexual reproduction from one or more parent plants. A progenyplant can be obtained by cloning or selfmg a single parent plant, or by crossing two or more parental plants. For instance, a progeny plant can be obtained by cloning or selfing of a parent plant or by crossing two parental plants and include selfings as well as the Fl or F2 or still further generations. Art Fl is a first-generation progeny produced from parents at least one of which is used for the first time as donor of a trait, while progeny of second generation (F2) or subsequent generations (F3, F4, and the like) are specimens produced from selfmgs. intercrosses, backcrosses, and / or other crosses of FIs, F2s, and the like. An Fl can thus be (and in some embodiments is) a hybrid resulting from a cross between two true breeding parents (i.e., parents that are true-breeding are each homozygous for a trait of interest or an allele thereof), while an F2 can be (and in some embodiments is) a progeny resulting from self-pollination of the Fl hybrids.

[0068] As used herein, the phrase “recombination” refers to an exchange of DNA fragments between two DNA molecules or chromatids of paired chromosomes (a “crossover”) over in a region of similar or identical nucleotide sequences. A “recombination event” is herein understood to refer in some embodiments to a meiotic crossover.

[0069] As used herein, the term “reference sequence” refers to a defined nucleotide sequence used as a basis for nucleotide sequence comparison.

[0070] As used herein, the term “regenerate,” and grammatical variants thereof, refers to the production of a plant from tissue culture.

[0071] As used herein, the phrase “stringent hybridization conditions” refers to conditions under which a polynucleotide hybridizes to its target subsequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and can be different under different circumstances. Longer sequences typically hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Sambrook & Russell, 2001. Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Exemplary stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides).

[0072] Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Additional exemplary stringent hybridization conditions include 50% formamide, 5xSSC, and 1% SDS incubating at 42° C.; or SSC, 1% SDS, incubating at 65° C.; with one or more washes in 0.2*SSC and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures can vary between about 32° C. and 48° C. (or higher) depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references (see e.g., Ausubel et al., 1999).

[0073] As used herein, the term “trait” refers to a phenotype of interest, a gene that contributes to a phenotype of interest, as well as a nucleic acid sequence associated with a gene that contributes to a phenotype of interest. For example, a “plant height trait” or an “ear height trait” refers to a phenotype as well as a gene that contributes to plant growth regulation e.g., plant height or ear height) and has a nucleic acid sequence (e.g., a plant growth regulatory gene) that is associated with a growth response, including a shorter plant height or lower ear height.

[0074] As used herein, the term “transgene” refers to a nucleic acid molecule introduced into an organism or one or more of its ancestors by some form of artificial transfer technique. The artificial transfer technique thus creates a “transgenic organism” or a “transgenic cell. ” It is understood that the artificial transfer technique can occur in an ancestor organism (or a cell therein and / or that can develop into the ancestor organism) and yet any progeny individual that has the artificially transferred nucleic acid molecule or a fragment thereof is still considered transgenic even if one or more natural and / or assisted breeding steps results in the artificially transferred nucleic acid molecule being present in the progeny individual.

[0075] As used herein, the term “targeted mutagenesis” or “mutagenesis strategy” refers to any method of mutagenesis that results in the intentional mutagenesis of a chosen gene. Targeted mutagenesis includes the methods CRISPR, TILLING, TALEN, and other methods which may be used to achieve the same outcome.

[0076] DETAILED DESCRIPTION

[0077] Provided herein are plants comprising non-naturally occurring allelic modifications (mutations) of plant growth regulatory' genes that modify the stature or ear height of the resulting plant. Various means of introducing mutations that result in the non- naturally occurring alleles or mutations into the com plant are also disclosed, which include transgenic means, gene editing, and breeding. Markers for identifying the presence of these non-naturally occurring alleles or mutations in the plant are also disclosed. As used herein, the terms “phenotype,” “phenotypic trait” or “trait” refer to a distinguishable characteristic(s) of a genetically controlled trait.

[0078] In some embodiments, the plants provided herein are a non-naturally occurring variety of corn having the desired trait. A “non-naturally occurring variety of corn” is any variety of com that does not naturally exist in nature. A non-naturally occurring variety' of com may be produced by any method known in the art, including, but not limited to, transforming a com plant or germplasm, transfecting a com plant or germplasm, and crossing a naturally occurring variety of com with a non-naturally occurring variety of com. In some embodiments, a “non-naturally occurring variety of com” may comprise one of more heterologous nucleotide sequences. In some embodiments, a “non-naturally occurring variety of com” may comprise one or more non-naturally occurring alleles of a naturally occurring gene (i.e., non-naturally occurring mutations introduced into a gene that naturally occurs in corn). In some embodiments, a non-naturally occurring variety ofcom may comprise anon-natural combination of one or more non-naturally occurring alleles of a plant growth regulatory gene.

[0079] A "subject plant or plant cell" is one in which genetic alteration, such as a mutation, has been affected as to a gene of interest to create a non-naturally occurring and novel allele, or is a plant or plant cell which is descended from a plant or cell so altered and

[0080] non-genome edited control plant, plant seed, plant part, plant cell and / or plant genome. As used herein, a “contror plant, plant seed, plant part, plant ceil and / or plant genome may also be a plant, plant seed, plant part, plant cell and / or plant genome having a similar (but not the same or identical) genetic background to a modified plant, plant seed, plant part, plant cell and / or plant genome, if deemed sufficiently similar for comparison of the characteristics or traits to be analyzed.

[0081] I. Novel, non-naturally occurring plant regulatory genes that result in a modified agronomic trait

[0082] Methods and compositions are provided for producing corn plants having a modified agronomic characteristic, including com plants having a reduced plant height or a lower ear height compared to a control plant. Such plants comprise non-naturally occurring modifications at one or more genetic loci corresponding to a plant growth regulatory' gene, These plants can be grown in environmental conditions that are not ideal for com plants not having a. reduced stature or reduced ear height, or in planting densities that are higher than what is conducive for plants not having a reduced stature or reduced ear height, thus contributing to an improvement in the overall yield of the modified com plants.

[0083] The present invention discloses methods and compositions for generating modified com plants having a reduced plant height or a lower ear height using mutagenesis and genome editing techniques. In various embodiments, the modified corn plants have a modification in one or more genetic loci of a plant growth regulatory gene. The one or more plant growth regulatory genets) comprise the SEQ ID NO: 1-51, or a nucleic acid encoding SEQ ID NO: 52-102, including variants, fragments, and complements thereof. In various embodiments, the corn plant has a modification in one or more plant growth regulatory genes that are homologous to one or more of the maize genes listed in Tables 1A-1B, Tables 2A-2B, Table 3 and Table 4 (identifiable by the ZmOOOOld designation in accordance with the Maize Genetics and Genomics Database found on the internet at maizegdb.org). In certain embodiments, a homologous sequence is an amino acid sequence (or a nucleotide sequence that encodes an amino acid sequence) that shares at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in any of SEQ ID NO:52-102, including variants, fragments, and complements thereof, such as the variant and fragment nucleotide sequences set forth in SEQ ID NO: 156- 328 or a nucleotide sequence encoding the variant polypeptides set forth in SEQ ID 0:329-501.

[0084] In embodiments, the modification results in a partial loss of function or a loss of function of a protein encoded by the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 9, 10, 11, 14. 15, 16, 17, 18, 19, 20, 24, 27, 2.8, 30, 31, 32, 33, 35, 36. 38, 41, 42, 43, 44, 45, and / or 47 or a nucleic acid encoding SEQ ID NO: 2, 53, 55, 56, 60, 61, 62, 65, 66, 67, 68, 69, 70, 71 , 75, 78, 79, 81, 82, 83, 84, 86, 87, 89, 92, 93, 94, 95, 96. and / or 98. In other embodiments, the modification results in an increased expression of one or more of the nucleotide sequences of SEQ ID NO: 3, 4, 6, 7, 8, 12, 13, 21 , 22, 23, 25, 26, 29, 34, 37, 39, 40, 46, 48, 49, 50, and / or 51, or a nucleic acid encoding SEQ ID NO: 54, 55, 57, 58, 59, 63, 64, 72, 73, 74, 76, 77, 80, 85, 88, 90, 91, 97, 99, 100, 101, and / or 102. In certain embodiments, the modification that results in an increased expression occurs in a regulatory element of the nucleotide sequence of SEQ ID NO: 3, 4, 6, 7, 8, 12. 13, 21, 22, 23, 25, 26. 29, 34, 37, 39. 40. 46, 48, 49, 50, and / or 51, or in a regulatory element of a nucleic acid encoding SEQ ID NO: 54, 55, 57, 58, 59, 63, 64, 72, 73, 74. 76, 77, 80, 85, 88, 90, 91, 97, 99, 100. 101, and / or 102. Further provided herein is a method of improving an agronomic characteristic of a population of com plants grow in a field, the method comprising: providing the population of com plants, modifying the plant height of the population of the com plants by introducing a genetic modification that results in reduced stature of the population of the com plants; and growing the population of plants in a crop growing environment, wherein an agronomic characteristic of the population of the com plants is improved compared to a control population of plants, In certain aspects, the corn plants exhibit an increase in standability when compared to the control plant. In certain aspects, the population of corn plants exhibit an increase in early season canopy thereby resulting in improved weed control and / or less applied herbicide per acre. In certain aspects, the population of com plants exhibit a decrease in early season disease. In certain aspects, the population of corn plants leave reduced net residue per bushel of grain yield after harvest.

[0085] In an embodiment, the modified corn plant comprises one or more genetic modifications targeting one or more distinct genomic loci that are involved in plant growth regulation.

[0086] According to embodiments of the present disclosure, a modified com plant(s) is / are provided that comprise (i) a plant height (at maturity) of less than 2000 mm, less than 1950 mm, less than 1900 mm, less than 1850 mm, less than 1800 mm, less than 1750 mm, less than 1700 mm, less than 1650 mm, less than 1600 mm, less than 1550 mm, less than 1500 mm, less than 1450 mm, less than 1400 mm, less than 1350 mm, less than 1300 mm, less than 1250 mm, less than 1200 mm, less than 1150 mm, less than 1100 mm, less than 1050 mm, or less than 1000 mm, and / or (ii) an ear height (at maturity) of less than 2000, less than 1900, less than 1800, less than 1700, less than 1600, less than 1500, less than 1450, less than 1400, less than 1350, less than 1300, less than 1250, less than 1200, less than 1150, less than 1100, less than 1050, or less than 1000 mm. Any such plant height trait or range that is expressed in millimeters (mm) may be converted into a different unit of measurement based on known conversions (e.g., one inch is equal to 2.54 cm or 25.4 millimeters, and millimeters (mm), centimeters (cm) and meters (m) only differ by one or more powers of ten), Thus, any measurement provided herein is further described in terms of any other comparable units of measurement according to known and established conversions. However, the exact plant height and / or ear height of a modified com plant may depend on the environment and genetic background. Thus, the change in plant height and / or ear height of a modified com plant may instead be described in terms of a minimum difference or percent change relative to a. control plant.

[0087] According to embodiments of the present disclosure, modified com plants are provided that have (i) a plant height that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% less than the height of a wild-type or control plant, and / or (ii) an ear height that is less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, or less than 95% of the ear height of the wild-type or control plant.

[0088] According to embodiments of the present disclosure, modified corn plants are provided that comprise a plant height between 5% and 75%, between 5% and 50%, between 10% and 70%, between 10% and 65%, between 10% and 60%, between 10% and 55%, between 10% and 50%, between 10% and 45%, between 10% and 40%, between 10% and 35%, between 1014 and 30%, between 10% and 25%, between 10% and 20%, between 10% and 15%, between 10% and 10%, between 10% and 75%. between 25% and 75%, between 10% and 50%, between 20% and 50%, between 25% and 50%, between 30% and 75%, between 30% and 50%, between 25% and 50%, between 15% and 50%, between 20% and 50%, between 25% and 45%, or between 30% and 45% less than the plant height of a wild-type or control plant, and / or an ear height that is between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 100%, between 10% and 75%, between 10% and 50%, between 10% and 40%, between 10% and 30%, between 10% and 20%, between 25% and 75%, between 25% and 50%, between 50% and 75%. between 8% and 20%, or between 8% and 15% less than the wild-type or control plant.

[0089] According to embodiments of the present disclosure, a modified com plant is provided that has a lodging frequency that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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%, or 100% less or lower than a wild-type or control plant. A modified com plant may have a lodging frequency that is between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 8.5%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, betw-een 5% and 10%, between 10% and 100%, between 10% and 75%, between 10% and 50%, between 10% and 40%, between 10% and 30%, between 10% and 20%, between 25% and 75%, between 25% and 50%, or between 50% and 75% less or lower than a wildtype or control plant. Further provided are populations of com plants having increased lodging resistance and a reduced lodging frequency. Populations of modified com plants are provided having a lodging frequency that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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%, or 100% less or lower than a population of wild-type or control plants. A population of modified com plants may comprise a lodging frequency that is between 5% and 100%, between 5% and 95%, between 5% and 90%, between 5% and 85%, between 5% and 80%, between 5% and 75%, between 5% and 70%, between 5% and 65%, between 5% and 60%, between 5% and 55%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 100%, between 10% and 75%, between 10% and 50%, between 10% and 40%, between 10% and 30%, between 10% and 2.0%, between 25% and 75%, between 25% and 50%, or between 50% and 75% less or lower than a population of wild-type or control plants, which may be expressed as an average over a specified number of plants or crop area of equal density.

[0090] In certain embodiments, the invention provides a modified com plant that can be planted at a higher density than a wild-type or control com plant. In embodiments, the planting density is at least about 30,000 to about 75,000 plants per acre, or least about 50,000 plants; 55,000 plants; 58,000 plants; 60,000 plants; 62,000 plants; or at least about 64,000 plants per acre. In certain aspects, the com plants are planted in a plurality of rows having a row width of about 8 inches to about 30 inches, about 10 inches to about 20 inches, or about 15 inches in width. In certain aspects, the corn plants yield an average of about 1 bushel more per acre, about 2 bushels per acre, about 3, about 5. about 5, about 10, about 15, about 20, or about 30 more bushes per acre compared to control corn plants. In certain embodiments, the short stature coni plant of the invention has a stem or stalk diameter that is at least 5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%. 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% greater than the stem diameter of the wildtype or control plant not having the genetic modifications described herein.

[0091] In an embodiment, the plant is substantially tolerant to lodging as measured at a single plant level or at an increased planting density, compared to a control plant or control population of plants. In an embodiment, the plant comprises up to about 10% less number of leaves compared to the control plant. In an embodiment, the plant growth regulation is characterized by the shortening of distance between one or more internodes that are present above or below a female reproductive part of the corn plant. Modified corn plants, whose average internode lengths are reduced compared to the wild-type plants are provided. For example, average internode length (2nd internode length and / or 4th internode length relati ve to the position of the ear) that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% less than the same or average internode length of a wildtype or control plant are provided. The 2nd internode’’ refers to the second internode below the ear of the com plant, likewise, the “4th internode” refers to the fourth internode below the ear of the com plant.

[0092] In an embodiment, in addition to reducing plant stature as a result of introducing one or more genetic modifications targeting one or more distinct genomic loci that are involved in regulating plant height, one or more of the following agronomic characteristics of the plant is also increased or reduced: harvest index of the plant is increased; leaf area is increased; leaf number above the ear i s reduced, ratio of the plant ear height over the plant height is increased; and the yield is increased at higher planting density, as compared to a wild-type or control plant not comprising the genetic modifications. In aii embodiment, such plant growth regulation does not substantially affect flowering time. In an embodiment, the flowering time does not change by more than about 5-10 comparative relative maturity (CRM) or plus or minus 10% GDU or 125- 250 growing degree units (GDU), compared to a control plant not comprising' the modifications in one or more genetic loci that are involved in plant growth regulation, wherein 25 GDU is equivalent to about 1 day and 1 CRM is about 1 day. In an embodiment, the plant growth regulation does not substantially alter root architecture of the plant or does not significantly increase root lodging, compared to a control plant not comprising the modifications.

[0093] II. Gene editing

[0094] The plant growth regulatory genes described herein can be targeted within the genome of a recipient plant cell to create the novel allele sequences. Such methods include, but are not limited to, meganucleases designed against the plant genomic sequence of interest CRISPR-Cas9, TALENs, and other technologies for precise editing of genomes (Feng, et al. Cell Research 23: 1229-12.32, 2013, WO 2013 / 026740); Cre- lox site-specific recombination; FLP-FRT recombination (Li et al . (2009) Plant Physiol 151 : 1087-1095); Bxbl -mediated integration (Yau et al. Plant J (201 1 ) 701 : 147-166); zine-finger mediated integration (Wright et al. (2005) Plant J 44:693-705); Cai et al. (2009) Plant Mol Biol 69:699-709); and homologous recombination (Lieberman-Lazarovich and Levy (201 1 ) Methods Mol Biol : 51-65); prime editing and transposases (Anzalone, A. et al., Nat Biotechnol. 2020 Jul;38(7): 824-844); translocation; and inversion.

[0095] Various embodiments of the methods described herein use gene editing. In some embodiments, gene editing is used to mutagenize the genome of a plant to produce plants having novel alleles that confer a shortened plant height or ear height in a com plant. The novel alleles may be created by targeted introduction of mutations in the genome of a plant at the plant growth regulatory’ gene loci provided herein. Editing may be achieved through the use of editing expression cassettes. The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e , a promoter), a polynucleotide of interest, and a transcriptional and translational termination region (re., termination region) functional in the organism of interest, i.e., a plant or bacteria. The promoters of the invention are capable of directing or driving transcription and expression of a coding sequence in a host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cel] or to each other. As used herein, a chimeric gene or a chimeric nucleic acid molecule comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0096] A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and correct mRNA polyadenylation. The termination region may be native with the transcriptional initiation region, may be native with the operably linked DM A sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence of interest, the plant host, or any combination thereof). Appropriate transcriptional terminators are those that are known to function in plants and include the CAMV pSOYl terminator, the trnl terminator, the nopaline synthase terminator and the pea rbcs E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator may be used. Termination regions used in the expression cassettes can be obtained from, e.g., the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991 ) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:962.7-9639.

[0097] Additional regulator}' signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U. S. Pat. Nos. 5,039,523 and 4,853,331 ; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter “Sambrook 1 1”; Davis et al, eds. (1980).

[0098] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DN A fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0099] A number of promoters can be used in the practice of the inven tion, The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, inducible, tissue-preferred, or other promoters for expression in the organism of interest See, for example, promoters set forth in WO 99 / 43838 and in US Patent Nos: 8,575,425; 7,790,846, 8, 147,856; 8,586832; 7,772,369; 7,534.939, 6,072,050, 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;

[0100] 5,399,680; 5,268,463; 5,608,142; and 6,177,611 ; herein incorporated by reference. In some embodiments, the promoter used herein comprises an exogenous promoter. The term “exogenous promoter,” refers to a promoter that is not found in plants in nature, for example, a synthetic promoter. In some embodiments, provided herein are plants transformed with and expressing gene-editing machinery as described above, which, when crossed with a target plant, result in gene editing in the target plant.

[0101] In general, gene editing may involve transient, inducible, or constitutive expression of the gene editing components or systems. Gene editing may involve genomic integration or episomal presence of the gene editing components or systems.

[0102] Gene editing generally refers to the use of a site-directed nuclease (including but not limited to CRISPR / Cas, zinc fingers, meganucleases, and the like) to cut a nucleotide sequence at a desired location. This may be to cause an insertion / deletion (“indel”) mutation, (i.e., “SDN1”), a base edit (i.e., “SDN2”), or allele insertion or replacement (i.e., “SDN3”). SDN2 or SDN3 gene editing may comprise the provision of one or more recombination templates (e.g., in a vector) comprising a gene sequence of interest that can be used for homology directed repair (HDR) within the plant (i.e., to be introduced into the plant genome). In some embodiments, the gene or allele of interest is one that is able to confer to the plant an improved trait, e.g., modified plant height or ear height. The recombination template can be introduced into the plant to be edited either through transformation or through breeding with a donor plant comprising the recombination template. Breaks in the plant genome may be introduced within, upstream, and / or downstream of a target sequence. In some embodiments, a double strand DNA break is made within or near the target sequence locus. In some embodiments, breaks are made upstream and downstream of the target sequence locus, which may lead to its excision from the genome. In some embodiments, one or more single strand DNA breaks (nicks) are made within, upstream, and / or downstream of the target sequence. Any of these DNA breaks, as well as those introduced via other methods known to one of skill in the art, may induce HDR. Through HDR, the target sequence is replaced by the sequence of the provided recombination template comprising an allele of interest, e.g., SEQ ID NO: 1- 51, or a polynucleotide encoding a polypeptide having the sequence of any one of SEQ ID NO: 52-102, may be provided on / as a template. By designing the system such that one or more single strand or double strand breaks are introduced within, upstream, and / or downstream of the corresponding region in the genome of a plant not comprising the gene sequence of interest, this region can be replaced with the template.

[0103] In embodiments, the site directed nuclease is selected from the group consisting of meganucleases (MNs), zinc-fmger nucleases (ZFNs), transcription- activator like effector nucleases (TALENs), Cas9 nuclease, Cfpl nuclease, dCas9-Fokl, dCpfl - Fold, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FENI -Fold, and Mega- TALs, and CRISPR-associated nucleases.

[0104] In embodiments, the editing is performed by transforming a plant cell with an expression cassette comprising (i) a nucleic acid that encodes the site-directed nuclease; and (ri) a nucleic acid that encodes at least one guide RNA (gRN A) directed to a target sequence The gRNA is directed to a target sequence which comprises the plant growth regulatory gene described herein. In particular embodiments, the gRNA is directed to a target sequence comprising SEQ ID NO: 1-51, or a polynucleotide encoding a polypeptide having the sequence of any one of SEQ ID NO: 52-102. In embodiments of the editing expression cassette, the nucleic acid that encodes the site- directed nuclease is operably linked to a first promoter while the nucleic acid that encodes the at least one gRNA is operably linked to a second promoter, which may be the same or different from the first promoter. In some embodiments, the expression cassette may further comprise one or more additional regulatory elements, such as an enhancer operably linked to the first promoter or the second promoter. In some embodiments, mutations in the genes or wild-type alleles of interest described herein may be generated without the use of a recombination template via targeted introduction of DNA double strand breaks. Such breaks may be repaired through the process of non-homologous end joining (NHEJ), which can result in the generation of small insertions or deletions (indels) at the repair site. Such indels may lead to frameshift mutations causing premature stop codons or other types of loss-of- function mutations in the targeted genes. In certain embodiments of the present invention, the mutation(s) occur within, or within 1 -5, 5-25, or 25 to 50 nucleotides upstream or downstream, of a target position in the plant growth regulatory genes set forth in SEQ ID NO: 1 -51. In various embodiments, the target positions are selected from the positions identified in Table 3 (corresponding to the target regions set forth in SEQ ID NO: 103-153), and the insertion, substitution or deletion of one or more nucleotides occurs within the target position, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides upstream of the double-strand break (DSB) that typically occurs 3 nucleotides upstream of the protospacer motif (PAM) site (designated as "NGG” in the target sequences herein; See Wu et al. (2014) Quant Biol. 2(2): 59-70, specifically Figure 1).

[0105] In some embodiments, gene editing may involve transient, inducible, or constitutive expression of the gene editing components or systems in the target plant. Gene editing may also involve genomic integration or episomal presence of the gene editing components or systems m the target plant.

[0106] In certain embodiments, the nucleic acid modification or mutation is effected by a (modified) zinc-finger nuclease (ZFN) system. The ZFN system uses artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain that can be engineered to target desired DNA sequences. Exemplary methods of genome editing using ZFNs can be found for example in U.S. Patent Nos. 6,534,261 ; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6.866,997; 6,933,113; and 6,979,539.

[0107] In certain embodiments, the nucleic acid modification is effected by a (modified) meganuclease, which are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). Exemplary' method for using meganucleases can be found in US Patent Nos: 8,163,514; 8,133,697, 8,021 ,867; 8,1 19,361; 8,119,381; 8,124,369; and 8,129,134, which are specifically incorporated by reference.

[0108] In certain embodiments, the nucleic acid modification is effected by a (modified) CRISPR / Cas complex or system. In certain embodiments, the CRISPR / Cas system or complex is a class 2 CRISPR / Cas system. In certain embodiments, said CRISPR / Cas system or complex is a type II, type V, or type VI CRISPR / Cas system or complex. The CRISPR-'Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas protein can be programmed by an RNA guide (gRNA) to recognize a specific nucleic acid target, in other words the Cas enzyme protein can be recruited to a specific nucleic acid target locus (which may comprise or consist of RNA and / or DNA) of interest using said short RNA guide.

[0109] In general, the CRISPR / Cas or CRISPR system is as used herein foregoing documents refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene and one or more of, atracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a ‘‘direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9. e.g. CRISPR RNA and, where applicable, transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.

[0110] In certain embodiments, the gRNA is a chimeric guide RNA or single guide RNA (sgRNA). In certain embodiments, the gRNA comprises a guide sequence and a tracr mate sequence (or direct repeat). In certain embodiments, the gRNA comprises a guide sequence, a tracr mate sequence (or direct repeat), and a tract sequence. In certain embodiments, the CRISPR / Cas system or complex as described herein does not comprise and / or does not rely on the presence of a tracr sequence ( e.g. if the Cas protein is Cas 12a).

[0111] The Cas protein as referred to herein, such as but not limited to Cas9, Cas 12a (formerly referred to as Cpfl), Cas 12b (formerly referred to as C2cl), Cas 13a (formerly referred to as C2c2), CasI4, C2c3, Casl3b protein, may originate from any suitable source, and hence may include different orthologues, originating from a variety' of (prokaryotic) organisms, as is well documented m the art. In certain embodiments, the Cas protein is (modified) Cas9, preferably (modified) Staphylococcus aureus Cas9 (SaCas9) or (modified) Streptococcus pyogenes Cas9 (SpCas9). In certain embodiments, the Cas protein is Cas 12a, optionally from Acidaminococcus sp., such as Acidaminococcus sp. BV3L6 Cpfl (AsCasl2a) or Lachnospiraceae bacterium Cas 12a, such as Lachnospiraceae bacterium MA2020 or Lachnospiraceae bacterium MD2006 (LBCasl2a). See U.S. Pat. No. 10,669,540, incorporated herein by reference in its entirety. Alternatively, the Cas 12a protein may be from Moraxella bovoculi AAX08 00205 [Mb2Casl2a] or Moraxella bovoculi AAX11_OO2O5 [Mb3Casl2a]. See WO 2017 / 189308, incorporated herein by reference in its entirety. In certain embodiments, the Cas protein is (modified) C2c2, preferably Leptotrichia wadei C2c2 (LwC2c2) or Listeria newyorkensis FSL M6- 0635 C2c2 (LbFSLC2c2). In certain embodiments, the (modified) Cas protein is C2cl. In certain embodiments, the (modified) Cas protein is C2c3. In certain embodiments, the (modified) Cas protein is Cast 3b. Other Cas enzymes are available to a person skilled in the art.

[0112] Gene editing methods and compositions are also disclosed in US Pat. Nos 10,519,456 and 10,285,348 82, the entire content of which is herein incorporated by reference.

[0113] The gene-editing machinery (e.g., the DNA modifying enzyme) introduced into the plants can be controlled by any promoter that can drive recombinant gene expression in plants. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter, e.g., a pollen-specific promoter or a sperm cell specific promoter, a zygote specific promoter, or a promoter that is highly expressed in sperm, eggs and zygotes (e.g., prOsActinl). Suitable promoters are disclosed in U.S. Pat. No. 10,519,456, the entire content of which is herein incorporated by reference.

[0114] III. Selection of plants with novel alkies and improved traits.

[0115] In addition to the phenotypic traits, the genetic characteristic of the plant as represented by its genetic marker profile can be used to select plants of desired traits. The term “marker-based selection’’ refers to the use of genetic markers to detect one or more nucleic acids from the plant, where the nucleic acid is associated with a desired trait to identify plants that carry genes or alleles for desirable (or undesirable) traits. Markers include but are not limited to Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Poly morphic DNAs (RAPDs), Arbitrarily Primed Polymerase Cham Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats

[0116] (SSRs) which are also referred to as Microsatellites, and Single Nucleotide

[0117] Polymorphisms (SNPs). There are known sets of public markers that are being examined by ASIA and other industry groups for their applicability in standardizing determinations of what constitutes an essentially derived variety under the US Plant Variety Protection Act. However, these standard markers do not limit the type of marker and marker profile which can be employed in breeding or developing backcross conversions, or in distinguishing varieties or plant parts or plant cells or verify a progeny pedigree. Corn genome data and markers are disclosed, for example, in MaizeGBD (Woodhouse el al. (2021) BMC Plant Biol 21, 385) located at the world wide web at maizegdb.org / .

[0118] The term ‘‘associated with” as used herein refers to a recognizable and / or detectable relationship between two entities. For example, the phrase “associated with reduced plant height” or “associated with lower ear height” refers to a trait, locus, gene, allele, marker, phenotype, etc., or the expression product thereof, the presence or absence of which can influence or indicate an extent and / or degree to which a plant or its progeny exhibits a change in its plant height or ear height, respectively, as compared to a control plant. As such, a marker is “associated with” a trait when it is linked to it and when the presence of the marker is an indicator of whether and / or to what extent the desired trait or trait form will occur in a planrigermplasm comprising the marker. Similarly, a marker is “associated with” an allele when it is linked to it and when the presence (or absence) of the marker is an indicator of whether the allele is present (or absent) in a plant, germplasm, or population comprising the marker.

[0119] The term “allele(s)” refer to any of one or more alternative forms of a gene, all of which alleles relate to at least one trait or characteristic. In a diploid cell, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. The term “genotype” and variants thereof refer to the genetic composition of an organism, including, for example, whether a diploid organism is heterozygous (i.e., has two different alleles for a given gene or QTL) or homozygous (i.e., has the same allele for a given gene or QTL) for one or more genes or loci (e.g., a SNP, a haplotype, a gene mutation, an insertion, or a deletion).

[0120] In one embodiment, the markers used to identify the plants comprising the alleles disclosed herein are SNPs. Non-limiting examples of SNP genotyping methods include hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing and coded spheres. Such methods are well known and disclosed in e.g., Gut, LG., Hum. Mutat. 17: 475-492 (2001); Shi, Clin. Chem. 47(2): 164-172 (2001); Kwok, Pharmacogenomics 1(1): 95-100 (2000); and Bhattramakki and Rafalski, Discovery’ and application of single nucleotide polymorphism markers in plants, in PLANT GENOTYPING: THE DNA FINGERPRINTING OF PLANTS, CABI Publishing, Wallingford (2001). A wide range of commercially available technologies utilize these and other methods to interrogate SNPs, including Masscode SupTM / Sup (Qiagen, Germantown. MD, (Hologic, Madison, Wl), (Applied Biosystems, Foster City, CA), (Applied Biosystems, Foster City', CA) and Beadarrays SupTM / Sup (Illumina, San Diego, CA),

[0121] In some embodiments, an assay (e.g., generally a two-step allelic discrimination assay or similar), a KASP SupTM / Sup assay (generally a one-step allelic discrimination assay defined below or similar), or both can be employed to identify the SNPs that associate with modified plant height or ear height. In an exemplary two-step assay, a forward primer, a reverse primer, and two assay probes that recognize two different alleles at the SNP site (or hybridization oligos) are employed. The forward and reverse primers are employed to amplify genetic loci that comprise SNPs that are associated with modified plant or ear height. The particular nucleotides that are present at the SNP positions are then assayed using the probes. In some embodiments, the assay probes and the reaction conditions are designed such that an assay probe will only hybridize to the reverse complement of a 100% perfectly matched sequence, thereby permitting identification of which allele (s) that are present based upon detection of hybridizations. In some embodiments, the probes are differentially labeled with, for example, fluorophores to permit distinguishing between the two assay probes in a single reaction. Exemplary methods of amplifying include employing a polymerase chain reaction (PCR) or ligase chain reaction (LCR) using a nucleic acid isolated from a com plant or germplasm as a template in the PCR or LCR.

[0122] These SNP markers can be used in a marker assisted breeding program to move traits, such as native traits or traits conferred by transgenes or traits conferred by genome editing, into a desired plant background. As used herein, the term "‘native trait” refers to a trait already existing in germplasm, including wild relatives of crop species, or that can be produced by recombination of existing traits. For example, progeny plants from a cross between a donor com plant comprising in its genome a modified plant growth regulatory' gene of the invention, and a recipient com plant not comprising said modification can be screened to detect the presence of the markers associated with modified plant height or ear height, Plants comprising said markers can be selected and verified for modified plant height or ear height as compared to control plants.

[0123] IV. Plant Transformation

[0124] Once the gene editing cassette has been cloned into an expression system, it is transformed into a plant cell. The receptor and target expression cassettes of the present invention can be introduced into the plant cell in a number of art-recognized ways. The term “introducing” in the context of a polynucleotide, for example, a nucleotide construct of interest, is intended to mean presenting to the plant the polynucleotide in such a manner that the polynucleotide gams access to the interior of a cell of the plant. Where more than one polynucleotide is to be introduced, these polynucleotides can be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into the host cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part, of a breeding protocol. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, only that the polynucleotide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotides into plants are known in the art including, but not limited to, transient transformation methods, stable transformation methods, and virus-mediated methods.

[0125] “Transient transformation” in the context of a polynucleotide is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant.

[0126] By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a plant is intended the introduced polynucleotide is stably incorporated into the plant genome, and thus the plant is stably transformed with the polynucleotide.

[0127] “Stable transformation” or “stably transformed” is intended to mean that a polynucleotide, for example, a nucleotide construct described herein, introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983)), the pat and bar genes, which confer resistance to the herbicide glufosinate (also called phosphin othri cm; see White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990) and U.S. Pat. Nos. 5,561,236 and 5,276,268), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelniann, Mol Cell Bio], 4: 2929-2931 ), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1 104 (1983)), the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642), the glyphosate N-acetyltransferase (GAT) gene, which also confers resistance to glyphosate (Castle et al. (2004) Science, 304:1151-1154; U.S. Patent App. Pub. Nos. 20070004912, 20050246798, and 20050060767); and the mannose-6- phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

[0128] Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the deliveiy of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, mi croinj ection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.

[0129] Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). For the construction of vectors useful in Agrobacterium transformation, see, for example, US Patent Application Publication No. 2006 / 0260011, herein incorporated by reference.

[0130] Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. For the construction of such vectors, see, for example, US Application No. 2006026001 1, herein incorporated by reference.

[0131] Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well know n in the art. Transformed tissue is regenerated on selectable medium canydng the antibiotic marker present between the binary plasmid T-DNA borders.

[0132] Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert panicles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Bi ologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.

[0133] Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both of these techniques are suitable for use with this invention. Co-transformalion may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093- 1096 (1986)).

[0134] Patent Applications EP 0 292. 435, EP 0 392 225, and WO 93 / 07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology' 8: 833-839 (1990)) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93 / 07278 and Koziel et al. (Biotechnology 1 1 : 194-200 (1993)) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. Tins technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS- lOOOHe Biolistics device for bombardment. Transformation of monocotyledons using Agrobacterium has also been described.

[0135] See, WO 94 / 00977 and U.S. Pat. No. 5,591,616, both of which are incorporated herein by reference. See also, Negrotto el al., Plant Cell Reports 19: 798-803 (2000), incorporated herein by reference.

[0136] The genetic properties engineered into the genome-edited or transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting.

[0137] Use of the advantageous genetic properties of the genome-edited or transgenic plants and seeds according to the invention can further be made in plant breeding.

[0138] Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multi-line breeding, variety blend, interspecific hybridization, aneupioid techniques, etc. Thus, the genome edited or transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines that, for example, increase the geographical range of cultivation. Many suitable methods for transformation using suitable selection markers such as kanamycm, binary vectors such as from Agrobacterium and plant regeneration as, for example, from tobacco leaf discs are well known in the art.

[0139] V, Commodity Products

[0140] The present disclosure provides a commodity product that is derived from a corn plant of the invention. As used herein, a ‘‘commodity product” generally refers to any composition or material that includes material derived or processed from a plant, seed, plant cell, or plant part comprising the short stature corn plant of the invention. Commodity products may be viable (e.g., seeds) or nonviable (e.g., com meal). Nonviable commodity products include but are not limited to nonviable seeds and grains; processed seeds, seed parts, and plant parts; dehydrated plant tissue, frozen plant tissue, and processed plant tissue; seeds and plant parts processed for animal feed for terrestrial and / or aquatic animal's consumption, oil, meal, flour, flakes, bran, fiber, milk, cheese, paper, cream, wine, ethanol, and any other food for human consumption; and biomasses and fuel products. Viable commodity products include but are not limited to seeds and plant cells.

[0141] In some embodiments, the modified corn plant described herein can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). In a further embodiment, the modified com plant of the invention may be combined with one or more Bt insecticidal toxins or other non-Bt insecticidal proteins.

[0142] In some embodiments, the modified com plants of the invention can be crossed with com plants containing other desired com traits, e.g., transgenic or dsgenic traits, or a combination thereof, and the resulting properties of the progeny plan ts evaluated. For example, the modified com plant of the invention can be crossed or combined with corn plants including one or more combinations of the following elite corn events: DP-202216-6, MON810; DAS-59122-7; MIR604; MON89034; MON863;

[0143] MON87411; MON87403; MON87427; MON-00603-6 (NK.603); MON-87460-4; MON-88017-3; LY038; TCI 507; 5307; DAS-06275-8; BT176; BT11; MIR162; GA21; MZDT09Y; SYN-05307-1 ; DP-0041 14-3; and DAS-40278-9. EXAMPLES

[0144] The following examples provide illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

[0145] Example 1 - Selection of candidate plant height genes

[0146] To identify candidate genes associated with plant height, 242 genomic regions containing QTLs that were shown to be associated with plant height and / or ear height were identified. The regions were ranked according to the magnitude of the effect on one or both parameters and the top 10 regions with the largest effect were selected. Within the top 10 genomic regions, the 1143 genes within these regions were ranked using proprietary' software that associates evidence to each gene from available data to support a role for the gene on a specific phenotype or physiological pathway.

[0147] Maximum weight was assigned to mutant phenotype in maize, next in importance was mutant phenotype in Arabidopsis, and next was inclusion of the gene in published reports of QTL affecting plant height in maize. 'The phenotype and physiological pathway are determined by specifying one or more key words. The 1 143 genes were ranked according to parameters of interest for short stature, The top 74 genes were selected for targeted edits.

[0148] Example 2 - Construct Design

[0149] The top 74 genes described in Example 1 were selected for knockout via CRISPR / Cas9. Single guide RNAs (sgRNAs) targeting these genes were designed.

[0150] Plant transformation constructs were designed for targeting each of the selected genes. The constructs were as in The Plant Cell, Vol. 32: 1397-1413, 2020, comprising the SpCas9 gene driven by the maize ubiquitin- 1 promoter and a sgRNA with the sequence indicated in the tables below, driven by the maize RNA Polymerase III promoter U6 (Kor SD, Chowdhury N, Keot AK, Yogendra K, Chikkaputtaiah C, Sudhakar Reddy P. RNA Pol III promoters-key players in precisely targeted plant genome editing. Front Genet. 2023 Jan 4:13:989199. doi:

[0151] 10.3389 / fgene.2022.989199. PMID: 36685866; PMCID: PMC9845283). Each construct included the bar gene cassette conferring resistance to the herbicide BASTA® to allow for selection of transformed plants. The construct also included a cassette containing the fluorescent protein DsRed driven by the aleurone-expressed promoter of barley (Hordewn vulgar e) HvLtp2 to facilitate selection of seed free of the T-DNA as described in Xu et al.. Plant Biotechnology Journal (2021), pp. 1-3, doi: 10.1111 / pbi.13575.

[0152] Example 3 - Plant Transformation

[0153] Maize variety’ KN5585 was transformed using previously described methods (Liu, et al... 2020, Plant Cell 32:1397-1413). Transformed tissue was selected on medium containing 10 mg / ml of glufosinate-amnioniiim and putative transformants were cultivated to produce plants. These TO plants were grown to maturity and selfpollinated to produce T1 seed.

[0154] Example 4 - Molecular Cimraeterizatiim of Transformed Plants

[0155] Leaf tissue was collected from putative transformants and DNA was extracted from this leaf tissue. The extracted DNA was analyzed essentially as described previously (Liu et al 2020 Plant Cell 32: 1397-1413). Seed from those TO plants determined to comprise a desirable mutation was harvested and used to grow T1 plants. All TOs were backcrossed to the transformation line KN5585. T1 plants were grown to maturity. Tissue was collected from the TI plants to screen for the lack of the spCas9 gene, to confirm the presence of the desired mutation in the gene of interest, and to screen for homozygous mutants at the gene of interest The Tl plants sho wn to lack the spCas9 gene and to comprise a desired mutation in the gene of interest were selfpollinated (most plants) or backcrossed to KN5585 (some) to produce T2. seed.

[0156] Example 5 - Phenotypic Evaluation of Mutants in T2 trials

[0157] T2 seed from T1 plants determined to have a desired mutation in the gene of interest was grown in a randomized design in a sunhouse under ambient conditions for evaluation of plant growth characteristics. Each of the plants for a given gene mutation was derived from a single event.

[0158] Plants height and ear height were measured at the time of tassel ernergence / maturity. Plant and ear height were calculated as the distance from the surface of the soil to the first branch of the tassel and from the s urface of the soil to the node with the uppermost ear, respectively.

[0159] Populations of plants containing a mutation were compared to their corresponding null segregants and the difference in the mean of the plant height or ear height relative to the null segregants within each population was determined. Based on these evaluations, plant height and ear height was shown to be statistically different from null segregants in the list of mutations shown in Table 1A (plant height) or Table IB (ear h ei ght). The "contrast” column is a represen tation of the genotype for each of the mutations compared to the null (N) segregants. The specific mutation is indicated by one or more nucleotide residues followed by a (-) to represent a deletion, or a (+) to represent an addition. A substitution is presented by “>” where the nucleotide(s) preceding the > represent the native nucleotide(s) and the nucleotide(s) following the > represent the nucleotides that were substituted. Heterozygous mutations are indicated by WT+Mu, where one allele contains the WT nucleotide(s) and the other allele contains the mutation following ‘"Mu.” The estimate column represents the height difference in the sample means of the mutant and the null segregant. A negative value in the estimate column indicates that the mutated plant had a lower plant height or ear height relative to the null segregant. A positive value in the estimate column indicates that the mutated plant had a higher plant height or ear height relative to the null segregant. Table 1 A: List of gene mutants that had significantly different plant height compared to a control (null segregant) in the T2 trial

[0160] Table I B. List of gene mutants that had significantly different ear height compared to a control (null segregant) in the T2 trial

[0161] Based on the data shown in Table 1 A, the mutant alleles for genes d003308, J003448, d042450, and d031871 resulted in plants with statistically significant decreases in plant height relative to control plants, while the mutant alleles for genes d039691 , d040737, d042492, d042499, and d017907 (homozygous and heterozygous) resulted in plants w i th statistically significant increases in plant height relative to control plants.

[0162] Based on the data shown in Table IB, the mutant alleles for genes d003300, d003583, d041056, 4042492, and dOl 7907 resulted in plants with statistically significant decreases in ear height relative to control plants, while the mutant alleles for genes d003583, d041056, d042492, and dOl 7907 resulted in plants with statistically significant increases in ear height relative to control plants.

[0163] Example 6 - T3 Field Trial design and data

[0164] The trial for T3 plants was planted in Changchun City, Jilin province, China. The trial consisted of five completely randomized plots of 10 plants / plot. Each plot in the replicate was homozygous for a frame shift mutation in the gene of interest or a null segregant. The two plots of 10 plants each, 10 homozygous mutant plants and 10 null segregant plants, were planted side by side. All blocks were surrounded by two rows of wild type plants.

[0165] The phenotype of the plants was collected on a per plant basis. Plant and ear height were calculated as the di stance from the ground to the first branch of the tassel and from the ground to the node with the uppermost ear, respectively. The results are shown in Tables 2 A and 2B.

[0166] Table 2A. Plant height results showing genes that result in lower plant height when mutated (P-value < 0.05). The column ‘estimate’ is the difference, measured in centimeters, in height between the mutant and the control. If the estimate is a negative value the mutant was shorter than the control (null segregants). Table 2.B. Ear height results showing genes that result in lower ear height when mutated (P-value < 0.05). The column ‘estimate’ is the difference, measured in centimeters, in ear height between the mutant and the control. If the estimate is a negative value, the ear height of the mutant was lower than the control.

[0167] *T--5 means starting from the Sth base forward of the target sequence. “34bp(-) means there are 34 base deletions

[0168] Table 3 lists the genomic sequences of plant growth regulatory genes in maize reference variety7B73_v5 with exons in bold text and target positions (for sense strand) underlined.

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Claims

Claims1. A modified com plant comprising a mutation in one or more of: a. the nucleotide sequence set forth in SEQ ID NO: 1-51, or a variant, fragment, or complement thereof, b. a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 52-102, or a variant, fragment, or complement thereof; or c. an endogenous regulatory element of (a) or (b). wherein said com plant has an improved agronomic characteristic relative to the same characteristic in a control plant.

2. The modified com plant of claim 1, wherein said mutation comprises an addition, substitution or deletion of one or more nucleotides in said nucleotide sequence, and wherein the improved agronomic characteristic is a reduced plant height or reduced ear height, or both, relative to a control plant.

3. The modified corn plant of claim 1, wherein the mutation results in a loss of function or a partial loss of function of a protein encoded by the nucleotide sequence.

4. The modified corn plant of claim 3, wherein the nucleotide sequence is selected from: a. the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 5, 9, 10, 11, 14. 15, 16, 17, 18, 19, 20. 24, 27, 28, 30, 31.

32. 33, 35, 36, 38, 41. 42, 43, 44, 45, and / or 47, or a variant, fragment, or complement thereof; or b. a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 2, 53, 55, 56, 60, 61, 62, 65, 66, 67, 68, 69, 70, 71. 75, 78, 79, 81,82, 83, 84, 86, 87, 89, 92, 93, 94, 95, 96, and / or 98, or a variant, fragment, or complement thereof.

5. The modified com plant of claim 1 , wherein the mutation results in an increase in the expression level of a protein encoded by the nucleotide sequence.

6. The modified com plant of claim 5, wherein the nucleotide sequence is selected from: a. the nucleotide sequence set forth in SEQ ID NO: 3, 4, 6, 7, 8, 12, 13, 21, 22, 23, 25, 26, 29, 34, 37, 39, 40, 46, 48, 49, 50, and / or 51, or a variant, fragment, or complement thereof; b. a nucleic acid encoding the ammo acid sequence set forth in SEQ ID NO: 54, 55, 57, 58, 59, 63, 64, 72, 73, 74, 76, 77, 80, 85, 88, 90, 91, 97, 99, 100, 101, and / or 102, or a variant, fragment or complement thereof; or c. an endogenous regulatory element of (a) or (b).

7. The modified com plant of claim 5, wherein said endogenous regulatory' sequence is a promoter or an enhancer of said nucleotide sequence.

8. A plant part of the modified com plant of any of claims 1-7.

9. The plant part of claim 8, wherein said part is a cell or a seed.

10. A method of generating a modified corn plant having an improved agronomic characteristic, wherein said method comprises introducing into a population of com plant cells: a. a site-directed nuclease capable of generating a double strand break in a target site of the ceils, or a nucleic acid encoding said nuclease; andb. a guide RNA (gRNA) or a nucleic acid encoding said gRNA; wherein said gRNA and said site-directed nuclease form a complex, wherein said gRN A hybridizes to said target site, wherein said complex generates a modification at said target site, and wherein said target site is present in any one of SEQ ID NO: 1 -51 or a nucleic acid encoding any one of SEQ ID NO: 52-102.1 1. The method of claim 10, further comprising regenerating a plant from the population of com plant cells and selecting plants having said improved agronomic characteristic.

12. The method of claim 10 or 11, wherein said target site is selected from SEQ IDNO: 103, 104, 106, 107, 11 1, 112, 113, 116, 117, 118, 119, 120, 121, 122, 126, 129,130, 132, 133, 134, 135, 137, 138, 140, 143, 144, 145, 146, 147, and / or 149, or a complement thereof.

13. The method of any of claims 10-12, wherein the site-directed nuclease is a Cas nuclease.

14. The method of claim 13, wherein the site-directed nuclease is Cas9 or Cast 2.

15. The method of any of claims 10-14, wherein said agronomic characteristic is reduced plant height or reduced ear height, or both.

16. A plant generated by the method of any of claims 10-15.

17. A plant of any of claims 1-7 and 16, wherein the height of the plant is less than200 cm, less than 195 cm, less than 190 cm, less than 185 cm, less than 180 cm, less than 175 cm, less than 170 cm, less than 165 cm, less than 160 cm less than 155 cm, less than 150 cm, less than 145 cm, less than 140 cm, less than 135 cm, less than130vcm, less than 125 cm, less than 120 cm, less than 115 cm, less than 110 cm, less than 105 cm, or less than 100 cm.

18. The plant of any of claims 1 -7 and 16, wherein the ear height on the plant is less than 100 cm, less than 90 cm, less than 85 cm, less than 80 cm, less than 75 cm, less than 70 cm, less than 65 cm, less than 60 cm, less than 65 cm, less than 50 cm, less than 45 cm, less than 40 cm, less than 35 cm, or less than 30 cm.

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