AU2015323980B2 - Plants with engineered endogenous genes - Google Patents

Abstract

Genetically engineered plants expressing altered Glucan Water Dikinase and having elevated levels of starch are provided. Methods of genetically engineering plants to express altered Glucan Water Dikinase, and genetic constructs are provided. Methods of breeding genetically engineered plants homozygous for a mutated gene encoding an altered Glucan Water Dikinase are described. Methods of agricultural processing and animal feed using the genetically engineered plants are also provided.

Description

PLANTS WITH ENGINEERED ENDOGENOUS GENES

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62/056,852, which was filed September 29, 2014, and is incorporated herein by reference as if fully set forth.

[0003] The sequence listing electronically filed with this application titled "Sequence Listing," which was created on September 29, 2015 and had a size of 159,500 bytes is incorporated by reference herein as if fully set forth.

[0004] GOVERNMENT SUPPORT STATEMENT

[0005] This invention was made with government support under award number DE-AR0000042 awarded by the Advanced Research Projects Agency Energy, ARPA-E. The government has certain rights in the invention.

[0006] FIELD

[0007] The disclosure herein relates to genetically improved plants having optimized endogenous nucleic acid sequences encoding altered glucan water dikinase, and having elevated levels of starch. The disclosure also relates to optimized nucleic acids encoding altered glucan water dikinase, methods of optimizing endogenous nucleic acids, methods of increasing starch levels in plants, and methods of making and propagating the genetically improved plants.

[0008] BACKGROUND

[0009] Plants synthesize starch in vegetative tissues during the daytime and degrade the starch at night to mobilize the resulting sugar in order to support the energy needs of the plant. Vegetative plant cells express a series of enzymes to initiate mobilization of transitory starch during the nighttime. Glucan Water Dikinase ("GWD"), which phosphorylates starch is one of these enzymes. GWD transcript levels were shown to undergo diel fluctuation

(Smith et al. Plant Phys. Preview, April 29, 2014). Increasing the starch content of biomass can increase the energy content (calories) in animal feed or improve glucose extraction from biomass for the production of ethanol or other biochemicals.

[0010] Different molecular methods exist for manipulating plant characteristics. Almost all of these methods rely on inserting new, synthetic or recombinant nucleic acids into a plant through the process of transformation. The nucleic acids thus inserted may encode a ribonucleic acid (RNA) or protein, which is expressed by the transformed plant and thereby changes the plant phenotype. In many cases, the nucleic acid may encode a heterologous protein or produce more of an endogenous protein. Similarly, the transformed nucleic acids may produce RNA that through a variety of mechanisms (such as RNA interference, antisense RNA, etc.) reduce expression of an endogenous gene thereby "silencing" the gene and production of its product. In all cases the nucleic acid inserted into the plant is expressed in a dominant manner; that is, its presence has a direct effect on the plant's characteristics. More recently, it has been demonstrated that by expressing nucleic acids that encode deoxyribonucleic acid (DNA) altering proteins (such as nucleases) in an organism, the organism's genome can be permanently altered, even after the inserted nucleic acids have been removed, and endogenous genes optimized. In this way it is possible to not only generate beneficial dominant traits, but also generate very specific, targeted mutations as the basis to create beneficial recessive traits, which would have been otherwise extremely difficult to find and develop for commercial applications. Currently there are no recessive traits created using nucleases in commercial use in row crops. Recessive traits generated using nucleases have been previously demonstrated in plants and plant cells, but never in fully developed, multicellular corn and sorghum plants, including hybrid corn and sorghum. Like dominant traits, recessive traits may have commercial value and may have specific commercial advantages (security and regulatory benefits in particular) over dominant traits. Such recessive traits will require new methods of propagating, tracking, and delivering the trait, particularly in hybrid crops.

[0011] One problem with dominant traits, particularly in hybrid and cross-pollinating crops, such as corn, is that they can be readily transferred to other lines of the same species. In regions of the world where farmers generate at least part of their own seed for planting, this affords the opportunity to breed a dominant trait into a farmer's existing lines, without paying the technology owner. The established trait business model currently requires seed and trait purchasers to pay the trait provider a royalty and licenses commonly limit use of the trait to a single planting and prohibit breeding. For many traits, monitoring unlicensed breeding is nearly impossible, and substantial unlicensed trait transfer (pirating) of traits occurs in some parts of the world. Depending on the trait, pirating or transferring the trait into a useable line without paying the technology owner can be an easy task and difficult for technology providers to detect. For example, pest resistance or agronomic traits, that do not require any other materials for their use, such as an herbicide resistance or specific fertilizer, are nearly impossible to detect if they have been transferred into a different line. Subsequent generations can be generated and tracked by a breeder using commercially available test strips or phenotypically if the trait confers an easily scorable phenotype. Because the trait is dominant, it may not need to be homozygous in the progeny for farmers to use it, and thereby enables easy continued breeding and use outside of the technology licensor's awareness.

[0012] In contrast to dominant traits, a recessive trait needs to be homozygous in the crop in order to phenotypically observed or easily scored. Simple test strips may not be available to track the molecular basis of the trait, and accurate breeding of a recessive trait made through the use of a nuclease may require at least polymerase chain reaction (PCR) to detect. In this case, none of the progeny resulting from an outcross of the homozygous parent carrying the trait will display the trait and extended breeding, tracking, and in some cases hybrid crosses will be required to use such a trait.

This makes pirating of the technology considerably more expensive and difficult than with dominant traits. The process of making, maintaining, and providing a recessive trait requires additional steps not necessary in the production of dominant traits, and therefore requires the use of novel processes in seed and trait production.

[0013] Recessive traits that are based on optimized genes containing a specific genetic mutation may also have regulatory advantages over dominant traits made using transgenic technologies. Because such a recessive trait may not contain any newly introduced heterologous DNA, in many parts of the world it may not be regulated as a transgenic crop.

[0014] SUMMARY

[0015] In as aspect, the invention relates to a genetically engineered plant comprising an engineered nucleic acid encoding an altered Glucan Water Dikinase, wherein the plant has an elevated level of starch in comparison to a plant of the same genetic background comprising a wild type Glucan Water Dikinase.

[0016] In an aspect the invention relates to a method for genetically engineering a plant to comprise an altered Glucan Water Dikinase. The method comprises contacting at least one plant cell comprising a target sequence in an endogenous gene encoding a Glucan Water Dikinase with a vector comprising a first nucleic acid encoding a nuclease capable of inducing a double-strand break at the target sequence. The method also comprises selecting a plant cell that includes an alteration in the target sequence. The method also comprises regenerating a genetically engineered plant including the alteration from the plant cell.

[0017] In an aspect, the invention relates to a method of increasing a starch level in a plant. The method comprises expressing a nucleic acid encoding a nuclease capable of inducing a double-strand break at a target sequence, where the target sequence is a sequence in an endogenous gene encoding a Glucan Water Dikinase. The method also comprises selecting a homozygous plant that comprises an alteration in the target sequence and has an elevated level of starch in comparison to a plant of the same genetic background comprising a wild type Glucan Water Dikinase.

[0018] In an aspect, the invention relates to a method of agricultural processing. The method comprises expressing a nucleic acid encoding a nuclease capable of inducing a double-strand break at the target sequence, where the target sequence is a sequence in an endogenous gene encoding a Glucan Water Dikinase. The method may comprise selecting a homozygous plant that includes an alteration in the target sequence and has an elevated level of starch in comparison to a plant of the same genetic background comprising a wild type Glucan Water Dikinase. The method may also comprise processing the homozygous plant.

[0019] In an aspect, the invention relates to a method of preparing animal feed. The method comprises expressing a nucleic acid encoding a nuclease capable of inducing a double-strand break at the target sequence, where the target sequence is a sequence in an endogenous gene encoding a Glucan Water Dikinase. The method may comprise selecting a homozygous plant that includes an alteration in the target sequence and has an elevated level of starch in comparison to a plant of the same genetic background comprising a wild type Glucan Water Dikinase. The method may also comprise performing at least one procedure selected from the group consisting of: harvesting, bailing, shredding, drying, ensiling, pelletizing, combining with a source of edible fiber fiber, and combining with plant biomass.

[0020] In an aspect, the invention relates to a method for producing a genetically engineered plant homozygous for an engineered nucleic acid that encodes an altered Glucan Water Dikinase comprising performing any one of the method for genetically engineering a plant comprising an altered Glucan Water Dikinase described herein.

[0021] In an aspect, the invention relates to a synthetic nucleic acid promoter having a sequence with at least with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of: SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), SEQ ID NO: 82 (ZmU3P1), SEQ ID NO: 84 (ZmU3P2) and SEQ ID NO: 86 (MzU3.8P).

[0022] In an aspect, the invention relates to a genetic construct comprising a first engineered nucleic acid sequence encoding a Cas9 nuclease. The Cas9 nuclease is capable of cleaving a target sequence included in an endogenous nucleic acid encoding Glucan Water Dikinase in a plant.

[0023] In an aspect, the invention relates to a kit for identifying a modified sequence of an endogenous gene encoding Glucan Water Dikinase in a sample. The kit comprises first primer and a second primer. The first primer and the second primer are capable of amplifying a target sequence included in the endogenous gene encoding Glucan Water Dikinase. The target sequence comprises a nucleic acid sequence with at least 90% identity to a reference sequence selected from SEQ ID NOS: 1 - 4, 75, 170 - 184 186, 187, 189 - 193. The kit may also comprise one or more component for detecting at a modification in the amplified region of the target sequence. The modification may be a modified sequence of an endogenous gene encoding Glucan Water Dikinase in any of genetically engineered plants described herein.

[0024] In an aspect, the invention relates to a method of identifying a modified sequence of an endogenous gene encoding Glucan Water Dikinase in a sample. The method comprises contacting a sample with a first primer and a second primer. The method comprises amplifying a target sequence included in the endogenous gene encoding Glucan Water Dikinase. The target sequence comprises a nucleic acid sequence with at least 90% identity to a reference sequence selected from SEQ ID NOS: 1 - 4, 75, 170 - 184 186, 187, 189 - 193. The method also comprises detecting modification of the target sequence. The modification may be a modified sequence of an endogenous gene encoding Glucan Water Dikinase in any of genetically engineered plants described herein.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustration, there are shown in the drawings particular embodiments. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0027] FIG. 1 illustrates the vector pAG4715 for expressing meganuclease.

[0028] FIG. 2 illustrates the vector pAG4716 for expressing meganuclease.

[0029] FIG. 3 illustrates PCR detection of mutants.

[0030] FIG. 4 illustrates a chart depicting starch content (mg starch per gram dry weight) across populations of mutant homozygous, heterozygous and hemizygous corn plants produced by using vectors pAG4715 and pAG4716. Lines 195, 20, 19, 18, 9 and 6 are control plants.

[0031] FIG. 5 illustrates starch content in green tissue of gwd knock-out corn mutants: bar 1 is wild type (WT) plant, bar 2 is M17, bar 3 is M18, bar 4 is M1, bar 5 is M20, bar 6 is M13/M12, bar 7 is M9, bar 8 is M7/M11, bar 9 is M4/M14, bar 10 is M11/M12, bar 11 is M15, bar 12 is M14, bar 13 is M11, bar 14 is M11/M10 and bar 15 is M13.

[0032] FIG. 6 illustrates starch staining in gwd knock-out (GWDko) meganuclease cobs.

[0033] FIG. 7 illustrates the vector pAG4800 for expressing ZmCas9.

[0034] FIG. 8 illustrates the vector pAG4804 for expressing sgRNA scaffold and ZmCas9.

[0035] FIG. 9 illustrates starch accumulation in the pAG4804 maize events.

[0036] FIG. 10 illustrates starch accumulation in the pAG4806 maize events.

[0037] FIG. 11 illustrates a schematic drawing of selfing and outcrossing of a targeted mutation M20 derived from the maize event 4716_164.

[0038] FIG. 12 illustrates genotyping of T1 progeny from the selfed TO 4716_164 M20 plant.

[0039] FIG. 13 illustrates genotyping of T1 progeny from the outcrossed TO 4716_164 M20 plant.

[0040] FIG. 14 illustrates genotyping of T1 progeny from the outcrossed 4716_164 M20 plant.

[0041] DETAILED DESCRIPTION

[0042] Certain terminology is used in the following description for convenience only and is not limiting.

[0043] "Engineered nucleic acid sequence," "engineered polynucleotide," "engineered oligonucleotide," "engineered DNA," or "engineered RNA" as used herein refers to a nucleic acid, polynucleotide, oligonucleotide, DNA, or RNA that differs from one found in nature by having a different sequence than one found in nature or a chemical modification not found in nature. The engineered nucleic acid sequence," "engineered polynucleotide," "engineered oligonucleotide," "engineered DNA," or "engineered RNA" may be a synthetic nucleic acid sequence, synthetic polynucleotide, synthetic oligonucleotide, synthetic DNA, or synthetic RNA. The definition of engineered nucleic acid includes but is not limited to a DNA sequence created using biotechnology tools. Such tools include but are not limited to recombinant DNA technology, chemical synthesis, or directed use of nucleases (so called "genome editing" or "gene optimizing" technologies).

[0044] "Endogenous nucleic acid" as used herein refers to a nucleic acid, polynucleotide, oligonucleotide, DNA, or RNA naturally occurring in the organism or the genome. An endogenous nucleic acid may be an endogenous gene.

[0045] "Altered protein" as used herein refers to a protein, polypeptide, oligopeptide or peptide that contains at least one amino acid change, or deletion compared to the amino acid sequence contained in a naturally occurring organism, e.g., a parent organism. An altered protein may retain or lack the biological activity of the original sequence.

[0046] As used herein, "operably linked" refers to the association of two or more biomolecules in a configuration relative to one another such that the function of the biomolecules can be performed. In relation to two or more nucleotide sequences, "operably linked" refers to the association of the nucleic acid sequences in a configuration relative to one another such that the function of the sequences can be performed. For example, a nucleotide sequence encoding a presequence or secretory leader is operably linked to a nucleotide sequence for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence; and a nucleic acid ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate binding of the ribosome to the nucleic acid.

[0047] As used herein, genetic background is defined as the sum of all genes, or a collection of specific genes (e.g., all genes but for an engineered genetic modification) in a plant. Plants of the same species may be referred to as plants having the same genes or the same genetic background. A genetically engineered plant may include an engineered nucleic acid or polynucleotide described herein but otherwise have the same genes as non genetically engineered plant of the same genetic background.

[0048] The words "a" and "one," as used in the claims and in the corresponding portions of the specification, are defined as including one or more of the referenced item unless specifically stated otherwise. This terminology includes the words above specifically mentioned, derivatives thereof, and words of similar import. The phrase "at least one" followed by a list of two or more items, such as "A, B, or C," means any individual one of A, B or C as well as any combination thereof.

[0049] An embodiment comprises a genetically engineered plant comprising an engineered nucleic acid encoding an altered Glucan Water Dikinase. The genetically engineered plant may have an elevated level of starch in comparison to a plant of the same genetic background but comprising a wild type (wt) GWD. The activity of the altered GWD may be reduced in comparison to wild type (wt) GWD included in a plant of the same genetic background. The level of reduction may be 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% based on the level of wt GWD. Activity of GWD may be tested by monitoring starch content in plants, for example, by using Fourier Transform Near-infrared (FT-NIR) Technique as described in Example 3 herein. The altered GWD may be inactive. Increased levels of starch indicate reduced GWD activity.

[0050] In an embodiment, the engineered nucleic acid in the genetically engineered plant may comprise an endogenous nucleic acid that includes at least one allele of a gwd gene encoding a GWD protein but having one or more modifications in comparison to the wild type plant. The modifications may be made made by genetic engineering of the plant or its ancestors. The endogenous nucleic acid may be one or more allele of the gwd gene in the engineered plant. The modifications may be in the gwd coding sequences. The endogenous nucleic acid may comprise, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from SEQ ID NO: 1 (Zm GWD coding sequence) or SEQ ID NO: 2 (Sb GWD coding sequence). The engineered nucleic acid may include at least one mutation relative to the endogenous nucleic acid. A mutation may include an insertion of one or more nucleotides in comparison to the endogenous nucleic acid. A mutation may include a deletion of nucleotides in comparison to the native nucleic acid. A mutation may include a substitution of one or more nucleotides in comparison to the endogenous nucleic acid. A mutation may be a combination of several mutations. The at least one mutation may be within a target sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from SEQ ID NO: 1 (Zm GWD coding sequence), SEQ ID NO: 2 (Sb GWD coding sequence), SEQ ID NO: 3 (Zm GWD Exon 24

+ introns), SEQ ID NO; 4 (SbGWD Exon 24 + introns), SEQ ID NO: 91 (GWDela), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), and SEQ ID NO: 94 (GWDe25a), SEQ ID NO: 182 (ZmGWD Exon 24), SEQ ID NO: 183 (Sb GWD Exon 24), SEQ ID NO: 184 (SbGWD Exon 7) and SEQ ID NO: 189 (Zm GWD Exon 25).

[0051] In an embodiment, the engineered nucleic acid in the genetically engineered plant may be an endogenous nucleic acid that includes at least one allele of a gwd gene encoding a GWD protein but having one or more modifications made by genetic engineering of the plant or its ancestors. The endogenous nucleic acid may be one or more allele of the gwd gene in the engineered plant. The modifications may be in the gwd coding sequences. The endogenous nucleic acid may comprise, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from SEQ ID NO: 1 (Zm GWD coding sequence) or SEQ ID NO: 2 (Sb GWD coding sequence). The engineered nucleic acid may include at least one mutation relative to the endogenous nucleic acid. A mutation may include an insertion of one or more nucleotides in comparison to the endogenous nucleic acid. A mutation may include a deletion of nucleotides in comparison to the native nucleic acid. A mutation may include a substitution of one or more nucleotides in comparison to the endogenous nucleic acid. A mutation may be a combination of several mutations. The at least one mutation may be within a target sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from SEQ ID NO: 1 (Zm GWD coding sequence), SEQ ID NO: 2 (Sb GWD coding sequence), SEQ ID NO: 3 (Zm GWD Exon 24 +

introns), SEQ ID NO; 4 (SbGWD Exon 24 + introns), SEQ ID NO: 91 (GWDela), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), and SEQ ID NO: 94 (GWDe25a), SEQ ID NO: 182 (ZmGWD Exon 24), SEQ ID NO: 183

(Sb GWD Exon 24), SEQ ID NO: 184 (SbGWD Exon 7) and SEQ ID NO: 189 (Zm GWD Exon 25).

[0052] In an embodiment, the engineered nucleic acid in the genetically engineered plant comprises a modified sequence of Exon 24 of the maize gwd gene. The engineered nucleic acid may comprise, consist essentially of, or consist of a polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from SEQ ID NOS: 12 - 40, 114 - 118, 119 - 120 and 131 - 146, which comprise mutations in the maize gwd gene.

[0053] In an embodiment, the engineered nucleic acid in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Zm GWD gene (SEQ ID NO: 1) in the position from 3030 nucleotide (nt) to 3243 nt. In an embodiment, the engineered nucleic acid in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Zm GWD gene (SEQ ID NO: 1) in the position from 3157 nt to 3213 nt. In an embodiment, the engineered nucleic acid in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Zm GWD gene Exon 24 (SEQ ID NO: 3) in the position from 81 nt to 160 nt.

[0054] In an embodiment, the Zm GWD gene in the genetically engineered plant may comprise a modified sequence with changes in the sequence relative to wild type Zm GWD (SEQ ID NO: 1) in one of SEQ ID NOS: 12 - 40, 114 - 118, 119 - 120 and 131 - 146. The sequence of Zm GWD with the changes outside of the positions where the changes are located may have at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the corresponding regions of SEQ ID NO: 1. The changes may be the same or different from one of SEQ ID NOS: 12 - 40, 114 - 118, 119 - 120 and 131 - 146.

[0055] In an embodiment, the engineered nucleic acid in the genetically engineered plant may comprise a modified sequence of Exon 24 of the sorghum gwd gene. The engineered nucleic acid may comprise, consist essentially of, or consist of a polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from SEQ ID NO: 106 (Sb47151 (WT + ins)_Exon 24), and SEQ ID NO: 107 (Sb47152 (WT + del)_Exon 24), which are mutations in Exon 24 in the sorghum gwd gene. In an embodiment, the engineered nucleic acid in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Sb GWD gene (SEQ ID NO: 2) in the position from 3030 nt to 3243 nt. The engineered nucleic acid in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Sb GWD gene (SEQ ID NO: 2) in the position from 736 nt to 969 nt. The altered GWD may be encoded by any one of the engineered nucleic acids herein.

[0056] In an embodiment, a genetically engineered plant may comprise an altered Zea mays GWD (Zm GWD). The altered ZmGWD may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from SEQ ID NOS: 45 - 73 (Zm GWD mutant proteins M1 M29), 121 -125 (Zm GWD mutant proteins M32 - M36), 126 - 127 (Zm GWD mutant proteins M38 - M39) and 147 - 162 (Zm GWD mutant proteins M40 M55).

[0057] In an embodiment, the altered ZmGWD protein in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Zm GWD protein (SEQ ID NO: 43) in the positions from 1040 amino acid (aa) to 1120 aa. The altered ZmGWD protein in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Zm GWD protein (SEQ ID NO: 43) in the positions from 1054 aa to 1081 aa. The altered ZmGWD protein in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Zm GWD protein (SEQ ID NO: 43) in the positions from 1011 aa to 1057 aa. The altered ZmGWD protein in the genetically engineered plant may comprise a modified sequence having one or more modifications within a region of the wild type Zm GWD protein (SEQ ID NO: 43) in the positions from 1082 aa to 1116 aa.

[0058] In an embodiment, the Zm GWD protein in the genetically engineered plant may comprise a modified sequence with changes in the sequence relative to wild type Zm GWD (SEQ ID NO: 43) in one of SEQ ID NOS: 45 - 73 (Zm GWD mutant proteins M1 - M29), 121 -125 (Zm GWD mutant proteins M32 - M36), 126 - 127 (Zm GWD mutant proteins M38 - M39) and 147 - 162 (Zm GWD mutant proteins M40 - M55). The sequence of Zm GWD with the changes outside of the positions where the changes are located may have at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the corresponding regions of SEQ ID NO: 43. The changes may be the same or different from one of SEQ ID NOS: 45 - 73 (Zm GWD mutant proteins M1 - M29), 121 -125 (Zm GWD mutant proteins M32 - M36), 126 - 127 (Zm GWD mutant proteins M38 - M39) and 147 - 162 (Zm GWD mutant proteins M40 - M55).

[0059] In an embodiment, a genetically engineered plant may comprise an altered Sorghum bicolor GWD (Sb GWD). The altered Sb GWD may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from SEQ ID NO: 194(Sb GWD mutant protein Sb4715_1WT + ins) and SEQ ID NO: 195 (Sb GWD mutant protein Sb4715_2 WT + del). Nucleic acids, nucleotide sequences proteins or amino acid sequences herein can be isolated, purified, synthesized chemically, or produced through recombinant DNA technology. All of these methods are well known in the art.

[0060] In an embodiment, the genetically engineered plant may be any type of plant. The genetically engineered plant may be but is not limited to a monocotyledonous plant, a dicotyledonous plant, a C4 plant, a C3 plant, corn, soybean, rice, sugar cane, sugar beet, sorghum, switchgrass, miscanthus, eucalyptus, wheat, alfalfa, willow, or poplar. The genetically engineered plant may be derived from an energy crop plant, a forage crop plant, or a food crop plant. The energy crop plant may be a corn plant, a switchgrass plant, a sorghum plant, a poplar plant, or a miscanthus plant. The forage crop plant may be a corn plant, an alfalfa plant, a sorghum plant or a soybean plant. The food crop plant may be a corn plant, a wheat plant, a soybean plant, a rice plant, or a tomato plant.

[0061] The genetically engineered plant may be a transgenic plant or a mutant plant. The genetically engineered plant may be a progeny of a transgenic plant or a mutant plant, or a descendant of a transgenic plant or a mutant plant.

[0062] The genetically engineered plant may be a conventional mutant having one or more mutations in a nucleic acid sequence of a gene encoding GWD that result in inhibited expression of the GWD or reduced activity of GWD. The mutations may be deletions, insertions or substitutions of nucleic acids in a sequence of the GWD encoding gene. The conventional mutant may have an altered level of vegetative starch compared to a non-mutant plant of the same genetic background but expressing wild type GWD.

[0063] As used herein, the genetically engineered plant may refer to a whole transgenic plant or mutant plant or a part thereof. The part may be but is not limited to one or more of leaves, stems, flowers, buds, petals, ovaries, fruits, or seeds. The part may be callus from a transgenic plant or a mutant plant. A genetically engineered plant may be regenerated from parts of a transgenic plant or a mutant plant or plants. A genetically engineered plant may be a product of sexual crossing of a first transgenic plant and a second transgenic plant or a non-transgenic plant where the product plant retains an engineered nucleic acid introduced to the first transgenic plant. A genetically engineered plant may be a product of sexual crossing of a first mutant plant and a second non-mutant plant where the product plant retains a mutation introduced to the first mutant plant. The transgenic plant or the mutant plant may be any one of the transgenic plants or mutant plants described herein.

[0064] In an embodiment, a method for genetically engineering a plant that includes an altered Glucan Water Dikinase is provided. The method may include contacting at least one plant cell that comprises a target sequence in an endogenous gene encoding a Glucan Water Dikinase with a vector. The vector may include a first nucleic acid encoding a nuclease capable of inducing a single-strand break or a double-strand break at the target sequence. The vector may be introduced by transforming or otherwise genetically engineering a plant. Transforming may be Agrobacteriurm-mediated transformation using a vector that includes a first nucleic acid encoding a nuclease. The nuclease may cleave the target sequence as described previously (Puchta et al. 1993; Wright et al. 2005; Wehrkamp-Richter et al. 2009; Cong et al., 2013; Belhaj et al., 2013, all of which are incorporated herein by reference as if fully set forth). The nuclease may be but is not limited to a meganuclease, Cas9 nuclease, a zinc finger nuclease, or a transcription activator-like effector nuclease.

[0065] As stated, the nuclease may be a meganuclease. Meganucleases may introduce single stranded or double stranded DNA breaks and have recognition sites ranging between 14 to 40 nucleotides in length providing good specificity. For use of meganucleases for targeted modification, see Rosen et al., 2006; Wehrkamp-Richter et al. 2009; Djukanovic et al., 2013, all of which are incorporated herein by reference as if fully set forth. The meganuclease may be a LAGLIDADG homing endonuclease (LHE). LAGLIDADG homing endonucleases (LHEs) are native gene-targeting proteins with their coding sequences found in introns or inteins. See Arnould et al., 2011, which is incorporated herein by reference as if fully set forth. The meganuclease may be a I-Crel homing endonuclease. As used herein, the I Crel homing endonuclease is a meganuclease naturally occurring in chloroplasts of Chlamydomonas reinhardtii, and is a well characterized protein containing a single sequence motif important for nuclease enzymatic activity. See Heath et al., 1997, which is incorporated herein by reference as if fully set forth. The I-Crel endonuclease is suitable for protein engineering and was used for targeted genome modifications in several species including plants. See Rosen et al., 2006; Arnould et al., 2007; Djukanovic et al., 2013, all of which are incorporated herein by reference as if fully set forth. The meganuclease may be I-DmoI, I-Scel, E-Dmel or DmoCre. Other meganucleases may be used.

[0066] The meganuclease may be encoded by a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of SEQ ID NO: 108 (4715_meganuclease) and SEQ ID NO: 109 (4716_meganuclease).

[0067] In an embodiment, the nuclease may be a Cas9 nuclease. Cas9 nuclease is the nuclease used in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems (Cong et al., 2013; Belhaj et al., 2013, both of which are incorporated herein by reference as if fully set forth. The CRISPR/Cas9 is the genome editing technology that due to its low cost, high efficiency and relative simplicity to engineer has a potential of becoming a technology of choice for genome editing in various species, but has not been demonstrated to work in multi-cellular plants by using stable transformation. The CRISPR/Cas9 system may include a Cas9 nuclease and a single guide RNA (sg RNA). The Cas9 nuclease herein may be encoded with a nucleic acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 74 (Cas9 nuclease) or SEQ ID NO: 75 (ZmCas9). The nuclease may have affinity for a sequence that enables the nuclease to cleave the target sequence, or it may be guided to the target sequence by using an sgRNA. The vector herein may further include a second nucleic acid sequence encoding an sgRNA. The targeted modification of the endogenous gene may be made by expressing the Cas9 and sgRNA in a plant cell. The sgRNA chimera molecule may contain an untranslated CRISPR RNA (crRNA), a 20 bp spacer sequence complementary to the target genomic DNA sequence with a 3 bp protospacer adjacent motif (PAM) sequence (Jinek et al., 2012, which is incorporated herein by reference as if fully set forth). The Cas9 nuclease may be expressed from PPDK, CaMV 35S, Actin, or Ubiquitin promoters in plants, such as Arabidopsis, corn, tobacco, rice, wheat, and sorghum. The sgRNAs may be expressed from primarily RNA Polymerase III promoters U6 or U3 and from RNA polymerase II promoter CaMV E35S (Belhaj et al., 2013; Upadhyay et al., 2014, both of which are incorporated by reference herein as if fully set forth). The sgRNAs may be expressed from SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), ZmU3P1 (SEQ ID NO: 82), ZmU3P2 (SEQ ID NO: 84), or ZmU3.8 promoter (SEQ ID NO: 86) described herein. The promoter may have 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to one of SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), ZmU3P1 (SEQ ID NO: 82), ZmU3P2 (SEQ ID NO: 84), or ZmU3.8 promoter (SEQ ID NO: 86). The promoter may have a length equal to 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the length in nucleotides of one of SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), ZmU3P1 (SEQ ID NO: 82), ZmU3P2 (SEQ ID NO: 84), or ZmU3.8 promoter (SEQ ID NO: 86). The percent identity of promoters shorter than SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), ZmU3P1 (SEQ ID NO: 82), ZmU3P2 (SEQ ID NO: 84), or ZmU3.8 promoter (SEQ ID NO: 86 may be as set forth above along the length of the shorter promoter. Cas9 nuclease may introduce a single stranded break or double stranded DNA break into an endogenous nucleic acid included in genomic DNA. Subsequently, breaks introduced by Cas9 in genomic DNA may be repaired via two distinct mechanisms NHEJ (non homologous ends joining) and HR (homologous recombination) (Symington and Gautier, 2011, which is incorporated herein by reference as if fully set forth).

[0068] In an embodiment, the nuclease may be a transcription activator like effector nucleases (TALEN). As used herein, TALENs refer to proteins derived from Xanthomonas. TALENs are customizable fusion proteins comprising an engineered DNA-binding domain of TAL effectors fused to DNA cleavage domains of FokI endonuclease (Boch and Bonas, 2010; Christian et al., 2010; Joung and Sander, 2013; Li et al., 2011, all of which are incorporated herein by reference as if fully set forth). These chimeric proteins may work in pairs of two monomers for targeting FokI endonuclease to a specific DNA sequence within a genome for DNA cleavage. The TAL DNA-binding domain may be modified to recognize different sequences (Cermak et al., 2011, which is incorporated herein by reference as if fully set forth).

[0069] In an embodiment, the nuclease may be a zinc-finger nuclease. (Wright et al. 2005; Shukla et al., 2009, both of which are incorporated herein by reference as if fully set forth).

[0070] In an embodiment, the nuclease may be any other nuclease suitable for targeted modification of the target sequence.

[0071] The target sequence may be a target gene. The target gene may be an endogenous gene that is native to the plant. The target sequence may be a gwd gene of a plant The target sequence may be contained within SEQ ID NO: 1 (Zm GWD coding sequence) or SEQ ID NO: 2 (Sb GWD coding sequence). The target sequence may be any nucleic acid sequence included in an exon of an endogenous nucleic acid encoding GWD. The target sequence may be included in an exon of an endogenous nucleic acid encoding a maize GWD. The target sequence may be included in an exon of an endogenous nucleic acid encoding a sorghum GWD. The target sequence may be included in Exon, 1, Exon 7, Exon 24, or Exon 25 of an endogenous nucleic acid encoding GWD. The target sequence may be a target sequence for the meganuclease. The target sequence may comprise, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 41 (Meganuclease GWD-9/10x.272 target sequence (pAG4715)) or SEQ ID NO: 42 (Meganuclease GWD-7/8x target sequence (pAG4716)). The target sequence may be the target sequence for Cas9 nuclease. The target sequence may comprise, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 91 (GWDela), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), or SEQ ID NO: 94 (GWDe25a). The sgRNA may be capable of binding a target sequence selected from the group consisting of SEQ ID NO: 91 (GWDela), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), and SEQ ID NO: 94 (GWDe25a). The target sequence may be any sequence that hybridizes with the sgRNA. The nuclease may have affinity for a sequence that enables the nuclease to cleave the target sequence, or it may be guided to the target sequence by using an sgRNA.

[0072] Once expressed, the nuclease will introduce one stranded or double stranded DNA breaks in the target sequence. For example, nuclease may delete a short segment that then may be partially repaired by the cell's DNA repair mechanisms, but leaving a lesion within the target sequence. The repaired target sequence may include an alteration. The alteration may include a mutation. The mutation may be at least one of an insertion, a deletion, or a substitution of one or more nucleotides in the target sequence. The mutation may be a null mutation. As used herein, the term "null mutation" refers to a mutation in a gene that leads to its not being transcribed into RNA or translated into a functional protein. Because of the mutation in the target sequence, the native nucleic acid sequence may encode an altered GWD. The activity of the altered GWD may be reduced. The level of reduction may be 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the activity level of wild type GWD and may be tested by monitoring starch content in plants by using Fourier Transform Near-infrared (FT-NIR) Technique as described as described in Example 3 herein. The altered GWD may be inactive. The genetically engineered plant having the alteration or progeny thereof may have an elevated level of starch in comparison to a non genetically engineered plant of the same genetic background.

[0073] The method may include selecting a plant cell that includes an alteration of the target sequences. The method may include regenerating the plant including the alteration from the plant cell. The genetically engineered plant may be homozygous for the alteration.

[0074] The genetically engineered plant may be heterozygous for the alteration. The genetically engineered plant herein may be heterozygous for the gene that includes the mutation. The gene may include the engineered nucleic acid encoding the altered GWD. The heterozygous plants may include alleles of the endogenous gene that encode a wild type, unaltered, GWD. The heterozygous plant may also include hemizygous plants when at least one allele of the gene encoding GWD is missing. A heterozygous plant may be phenotypically indistinguishable from the wild type plants and may not have elevated levels of starch. To produce homozygous plants with elevated levels of starch, a heterozygous genetically engineered plant may be self-crossed. Progeny may be obtained from such crosses. The progeny may include homozygous, heterozygous and wild type plants. A heterozygous plant may be phenotypically indistinguishable from the wild type plants. The method may include analyzing the progeny for the presence of the alteration and selecting a progeny plant that includes the alteration.

[0075] In an embodiment, the method may further include crossing a heterozygous genetically engineered plant to another genetically engineered plant heterozygous for the same alteration. The method may include selecting a first progeny plant that is homozygous for the alteration. The method may further include crossing the genetically engineered plant to a wild type plant of the same genetic background. Progeny may be obtained from such crosses. The progeny may include heterozygous and wild type plants. The method may include selecting a first progeny plant that is heterozygous for the alteration. The method may further include selfing the first heterozygous progeny plant and selecting a second progeny plant that is homozygous for the alteration.

[0076] A genetically engineered plant herein may be homozygous or heterozygous for the gene that includes the mutation and may include a transgene encoding a nuclease. The transgene encoding the nuclease may be segregated away during the above-described crosses.

[0077] An embodiment comprises a method for producing a genetically engineered plant homozygous for an engineered nucleic acid encoding a protein. The engineered nucleic acid may encode a recessive trait. The recessive trait may include a cleaved endogenous target sequence of a gene.

The recessive trait may only be observed in plants that do not contain an unaltered, wild-type, allele of the gene. The method may comprise making an engineered nucleic acid by modifying a sequence of an endogenous nucleic acid. The method may also comprise breeding the recessive trait into other crop lines. The method may comprise maintaining the trait in the crop lines. The method may comprise generating homozygous progeny. The method may include making hybrid seed with a recessive trait.

[0078] An embodiment comprises a plant genetically engineered by any one of methods described herein is provided.

[0079] An embodiment comprises a method of increasing a starch level in a plant. The method may comprise expressing a nucleic acid encoding a meganuclease in a plant. The method may comprise expressing a nucleic acid that encodes a TALEN in a plant. The method may comprise expressing a first nucleic acid that encodes a Cas9 nuclease and a second nucleic acid that encodes a desired guide RNA that target a specific sequence. Expression of the nucleic acid(s) in the plant may alter the function or coding of an endogenous DNA sequence. Expression of the nucleic acid(s) in the plant may alter the activity of GWD and starch metabolism in the plant. The plant may be any transgenic or mutant plant herein. The plant may be a progeny of the transgenic or mutant plant. The nucleic acid(s) may be included in a genetic construct(s). The method may comprise making any genetically engineered plant herein. The genetically engineered plant or its progeny may be the plant, in which starch levels may be increased by the method herein.

[0080] A genetic construct having a nucleic acid encoding a meganuclease that inactivates or inhibits expression of the GWD protein involved in mobilization of starch in a plant in may be expressed at any point in the methods. The nucleic acid may be expressed prior to the step of processing the plant. The nucleic acid may be expressed during the step of processing the plant. The expression may be induced. Upon the expression of the nucleic acid(s), the genetically engineered plant may have an altered level of vegetative starch compared to the level of starch in a non-genetically engineered plant of the same genetic background but lacking the one or more genetic construct.

[0081] Any genetically engineered plant herein may be provided in a method of agricultural processing, a method of preparing animal feed, or a method of feeding an animal. A step of providing the genetically engineered plant may include obtaining it from another party that produced it. A step of providing may include making the genetically engineered plant. The genetically engineered plant may be a transgenic plant or mutant plant. The step of providing may include transforming the plant by contacting the plant with any one of the genetic constructs herein. The step of providing may include stable transformation of the plant by any of the methods described herein, or known methods. The step of providing may include genetically engineering the plant by cleaving a gene encoding a protein involved in starch metabolism at a cleavage site recognized by a nuclease transiently expressed in the plant after contacting the plant with a genetic construct comprising a polynucleotide encoding the nuclease. The step of providing may also include regenerating the plant from a tissue of the genetically engineered plant having an altered level of vegetative starch. The step of providing may include obtaining a progeny of the genetically engineered plant resulted from self pollination or cross-pollination between the genetically engineered plant and non-genetically engineered plant. The step of providing may include obtaining homozygous progeny. The homozygous progeny may be inbred plants. The homozygous progeny may be hybrid plants. The genetically engineered plant may be used in a variety of subsequent methods or uses. The step of providing may include procuring the genetically engineered plant. The step of providing may include making the genetically engineered plant available for further processing steps. The step of providing may include making the genetically engineered plant available as part of an animal diet.

[0082] In the method of agricultural processing, the genetically engineered plant may be a feedstock engineered with elevated levels of starch and/or expressing one or more polysaccharide degrading enzyme. The feedstock may include any genetically engineered plant herein alone or in combination with other components. The other components may include other plant material. Agricultural processing may include manipulating or converting any agricultural feedstock including the genetically engineered plant for a particular product or use. Agricultural processing may comprise drying the genetically engineered plant. Agricultural processing may comprise fermenting the genetically engineered plant. Agricultural processing may comprise hydrolyzing the genetically engineered plant with one or more an exogenous enzymes to obtain a biochemical product. The exogenous enzymes may be lignin degrading enzymes, cellulose degrading enzymes, or hemicellulose degrading enzymes. The exogenous enzymes may be glycosidases, xylanases, cellulases, endoglucanases, exoglucanases, cellobiohydrolases, p-xylosidases, feruloyl esterases, P-glucosidases, and amylases. The exogenous enzymes may be purchased from a vendor and may comprise Accellerase© 1000, Accellerase© 1500, Accelerase© TRIOTM, and Accellerase © XY available from Genencor International (Rochester, NY). Exogenous enzymes may comprise Cellic, CTEC, HTEC available from Novozymes (Denmark). The exogenous enzymes may comprise starch degrading enzymes. The exogenous enzymes may comprise an amylase or an invertase. The method of agricultural processing may include simultaneous saccharification and fermentation of soluble sugars to produce ethanol.

[0083] A method of agricultural processing herein may comprise harvesting the genetically engineered plants having elevated levels of starch for use as a feedstock in agricultural processing. The method may include combining the genetically engineered plant with plant biomass. The plant biomass may include non-genetically engineered plants. The plant biomass may be genetically engineered plant biomass. The genetically engineered plant biomass may express polysaccharide degrading enzymes. By combining the genetically engineered plant with the plant biomass that express polysaccharide degrading enzyme, the method herein may not require harsh pretreatments to improve cellulose cell wall accessibility to exogenous enzymes. The methods herein may utilize any methods and compositions for consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes described in U.S. Patent Application No. 13/414,627, filed March 7, 2012; and International Patent Application No. PCT/US2012/028132, filed March 7, 2012, which are incorporated herein by reference as if fully set forth. Plants with altered levels of elevated starch were described International Patent Application No. PCT/US2011/041991, filed June 27, 2011; and U.S. Patent Application No. 13/806,654, filed March 19, 2013; and U.S. Patent Application No. 13/793,078, filed March 11, 2013, which are incorporated herein by reference as if fully set forth.

[0084] The genetically engineered plant may be provided in a method of preparing animal feed. Preparing animal feed may comprise combining the genetically engineered plant with animal feed stuffs, including but not limited to corn, grain, soybeans, and/or other forage. Preparing animal feed may comprise ensiling the genetically engineered plant to make silage. Preparing animal feed may comprise combining the genetically engineered plant with distillers' grains. Preparing animal feed may comprise pelletizing the genetically engineered plant into feed pellets. Preparing animal feed may comprise combining the genetically engineered plant with a source of edible fiber. Preparing animal feed may comprise combining the genetically engineered plant with a source of protein. Preparing animal feed may comprise combining the genetically engineered plant with one or more carbohydrates as a source of energy. Preparing animal feed may comprise combining the genetically engineered plant with one or more exogenous enzymes described herein.

[0085] A method of agricultural processing or or a method of preparing animal feed may also comprise at least one of the operations of harvesting, baling, grinding, milling, chopping, size reducing, crushing, extracting a component from the feedstock, purifying a component or portion of the feedstock, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, chemical conversion, or chemical catalysis of the feedstock.

[0086] In an embodiment, animal feed formulations comprising increased levels of starch in vegetative tissues are provided. Animal feed formulations may be used for increasing milk and beef production by feeding animals plant material with increased levels of starch. Easily-fermentable sugars available in a fermentation process may be provided by embodiments herein. Production of biofuels may be enhanced by providing easily fermentable sugars. Methods of providing easily fermentable sugars and methods of enhancing production of biofuels are provided as embodiments herein. The animal feed formulations may comprise any one or more of the genetically engineered plants herein. The animal feed formulations may comprise the products of a method of preparing animal feed herein.

[0087] Crops with elevated levels of vegetative starch may have a variety of uses and utilities. In an embodiment, biomass from plants that accumulate elevated levels of vegetative starch relative to wild type plants are provided. The biomass may be from any genetically engineered plant herein or its progeny. These plants may have added value as feedstocks for fermentation processes or animal feed applications. For example, in a typical cellulosic process, polysaccharides, such as cellulose and hemicelluloses that are present in the biomass, are hydrolyzed to simple sugars, which may then be fermented to ethanol, butanol, isobutanol, fatty acids, or other hydrocarbons by microorganisms. Because of the recalcitrance of the biomass, the release of the simple sugars from polymers, such as cellulose and hemicelluloses, often requires the use of harsh pretreatment conditions and hydrolysis with relatively expensive mixtures of enzymes. A similar situation occurs in ruminant animals that eat forage, including corn silage, as a nutrient and an energy source. In ruminant animal, the forage is masticated and moves into the rumen, where the fiber polysaccharides, such as cellulose and hemicellulose, are hydrolyzed and fermented by the microorganisms in the rumen flora. These organisms create fatty acids that are absorbed by the animal and metabolized, providing nutrition to the animal. In either ruminant digestion or biofuels processing, any starch that is present in the biomass represents an additional source of readily fermentable sugars (namely, glucose), which are less recalcitrant to hydrolysis and can be released very easily by amylases or mild chemical treatments. As a result, any increase in the amount of starch present in the biomass will simultaneously increase the amount of fermentable sugar that can be recovered. Biomass that contains elevated levels of starch may have greater value in forage applications, where the plant material is fed to livestock or dairy animals. Again, the excess starch present in this material is more easily digested by most animals than is the cellulosic material, providing more energy per unit biomass than biomass with ordinary levels of starch. Embodiments include utilizing a plant as set forth herein for any of these methods.

[0088] Methods herein, including those in the previous paragraph, may include at least one of modifying plants to create genetically engineered plants, growing the genetically engineered plants, harvesting the genetically engineered plants, processing (for example reducing the size of the forage, ensiling, treating with an inoculant, combining with other feed components, or pelleting) them for animal feed applications as one would other forage crops, or fermenting the genetically engineered plants in a manner similar to treatments that are used in cellulosic processing. Cellulosic processing steps used may comprise pretreating and hydrolyzing the polysaccharides into their component sugars by enzymatic or chemical hydrolysis or digestion. Any one step, set of steps, or all the steps set forth in this paragraph may be provided in a method herein.

[0089] An embodiment comprises a genetic construct designed to implement a strategy for modifying levels of vegetative starch in plants. The genetic construct may comprise a first engineered nucleic acid sequence that encodes a nuclease capable of cleaving a target sequence in an endogenous nucleic acid encoding GWD. The first engineered nucleic acid may encode any one of the nucleases described herein. The genetic construct may also include a second engineered nucleic acid sequence encoding an sgRNA. The second engineered nucleic acid may encode any one the sgRNAs described herein. The genetic construct may include a promoter operably linked to the first engineered nucleic acid sequence or the second engineered nucleic acid. The operably linked promoter may allow transcription of the first engineered nucleic acid sequence encoding a nuclease, or the second engineered nucleic acid sequence encoding the sgRNA. Transcription and translation of the first engineered nucleic acid sequence may be referred to as expression of the nuclease. Upon expression, the nuclease may cut the target sequence of the endogenous nucleic acid. The endogenous nucleic acid may encode GWD. Transcription of the second nucleic acid sequence may result in production of an sgRNA that recognizes a target sequence within an endogenous nucleic acid and guides Cas9 nuclease to the target for making a break.

[0090] The genetic construct may include regions encoding nuclear localization signals. As used herein, nuclear localization signals (NLS) refers the short motifs of basic amino acid sequences within nuclear proteins. Transport of certain proteins from cytoplasm into the nucleolus to perform their specific functions occurs through the nuclear envelope and involves nuclear pore complex (NPC) (Wagner et al., 1990, which is incorporated herein by reference as if fully set forth). In this process, nuclear localization signals (NLS) play an important role as they are thought to be recognized by NPC receptors to subsequently translocate proteins through the nuclear pore complex. The NLSs fall into one of several defined categories (Garcia-Bustos et al., 1991, which is incorporated herein by reference as if fully set forth). The NLS may be the SV40 NLS from simian virus 40 large T antigen, which has been used intensively in experiments for targeted genome modifications due to its activity in various organisms, including plants (Kalderon et al., 1984; Raikhel, 1992, both of which are incorporated herein by reference as if fully set forth). The SV40 NLS may be encoded by a nucleic acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 163. The SV NLS may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 196. The NLS may be plant specific NLS sequences. Plant specific NLS sequences were also described, for example, in maize regulatory proteins opaque-2 and R (Varagona et al, 1992; Shieh et al, 1993, both of which are incorporated herein by reference as if fully set forth). The plant specific NLS may be encoded by a nucleic acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group of SEQ ID NOS: 164 (NLS1), 165 (NLS3), 166 (NLS4), 167 (NLS5), and 168 (NLS6). The plant specific NLS sequence may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group of SEQ ID NOS: 128 (NLS1), 129 (NLS3), 130 (NLS4), 169 (NLS5), and 170 (NLS6). The NLS sequence may be a derivative NLS sequence. The NLS sequences or their derivatives may be used to target meganucleases, ZFNs, TALENs, or Cas9 proteins into plant nucleus for targeted genome modification. One or more NLS sequences may be fused with an amino acid sequence of the nuclease.

[0091] The genetic construct may further include one or more regulatory sequences (also referred to as a regulatory element) operably connected to the nucleic acid encoding the nuclease. The promoter may be any kind of promoter. The promoter may be an inducible promoter. The promoter may be a constitutive promoter. The promoter may be an inducible promoter, which initiates transcription of the nucleic acid encoding the nuclease only when exposed to a particular chemical or environmental stimulus. Examples of inducible promoters include but are not limited to alcohol inducible promoters, tetracycline inducible promoters, steroid inducible promoters, or hormone inducible promoters. The promoter may be a constitutive promoter, which provides transcription of the nucleic acids or polynucleotide sequences throughout the plant in most cells, tissues, and organs, and during many but not necessarily all stages of development. The promoter may be specific to a particular developmental stage, organ, or tissue. A tissue specific promoter may be capable of initiating transcription in a particular plant tissue. Plant tissue that may be targeted by a tissue specific promoter may be but is not limited to a stem, leaves, trichomes, anthers, or seed. A constitutive promoter herein may be the rice Ubiquitin 3 promoter (OsUbi3P) or the maize ubiquitin promoter (ZmUbil). Other known constitutive promoters may be part of the genetic construct herein, and include but are not limited to Cauliflower Mosaic Virus (CAMV) 35S promoter, the Cestrum Yellow Leaf Curling Virus promoter (CMP) or the CMP short version (CMPS), the Rubisco small subunit promoter, the rice actin promoter (OsAct1P), and the maize phosphoenolpyruvate carboxylase promoter (ZmPepCP). The promoter may be a synthetic nucleic acid promoter from maize Zea mays. The synthetic nucleic acid promoter from maize may comprise, consist essentially of, or consist of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), ZmU3P1 (SEQ ID NO: 82), ZmU3P2 (SEQ ID NO: 84), or ZmU3.8 promoter (SEQ ID NO: 86). The synthetic nucleic acid promoter may have a length equal to 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the length in nucleotides of one of SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), ZmU3P1 (SEQ ID NO: 82), ZmU3P2 (SEQ ID NO: 84), or ZmU3.8 promoter (SEQ ID NO: 86). The percent identity of promoters shorter than SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), ZmU3P1 (SEQ ID NO: 82), ZmU3P2 (SEQ ID NO: 84), or ZmU3.8 promoter (SEQ ID NO: 86 may be as set forth above along the length of the shorter promoter. An embodiment comprises any one of the synthetic nucleic acid promoters described herein. The synthetic nucleic acid promoter may be operably connected with the first engineered nucleic acid or the second engineered nucleic acid molecule and may transcriptionally activate the first or the second engineered nucleic acid. As a result of transcriptional activation, the first or the second engineered nucleic acid may be expressed constitutively in a plant.

[0092] A regulatory element in a genetic construct herein may be a terminator. A terminator is capable of terminating transcription. A terminator sequence may be included at the 3' end of a transcriptional unit of the expression cassette. The transcriptional unit may encode the nuclease. The terminator may be derived from a terminator found in a variety of plant genes. The terminator may be a terminator sequence from the nopaline synthase (NOS) or octopine synthase (OCS) genes of Agrobacterium tumefaciens. The terminator may be the S. pyogenes Cas9 terminator (SEQ ID NO: 88). The terminator may be the ZmU3T terminator (SEQ ID NO: 89). The terminator sequence may be the CaMV 35S terminator from CaMV, or any of the 3'UTR sequences shown to terminate the transgene transcription in plants. For example, the terminator may be the maize PepC terminator (3'UTR). The genetic construct may be included in a vector. The genetic construct may be integrated into a genome of the genetically engineered plant. The genetic construct may be transiently expressed in the genetically engineered plant.

[0093] The genetic construct may be used for transformation of a plant. The genetic construct may be used for Agrobacterium-mediated transformation of a plant. The genetic construct may be used for transforming a plant by any known methods, for example, particle bombardment or direct DNA uptake. The genetic construct may be cloned and included into a vector.

[0094] An embodiment includes a vector comprising a genetic construct herein and appropriate for genetically engineering a plant. The vector may be an intermediate vector. The vector may be a transformation vector. Vectors incorporating a genetic construct herein may also include additional genetic elements such as multiple cloning sites to facilitate molecular cloning and one or more selectable markers to facilitate selection. A selectable marker that may be included in a vector may be a phosphomannose isomerase (PMI) gene from Escherichia coli, which confers to the transformed cell the ability to utilize mannose for growth. Selectable markers that may be included in a vector include but are not limited to a neomycin phosphotransferase (npt) gene, conferring resistance to kanamycin, a hygromycin phosphotransferase (hpt) gene, conferring resistance to hygromycin, or an enolpyruvylshikimate-3 phosphate synthase gene, conferring resistance to glyphosate. The vector may be any vector described in US application No. 13/793,078, filed March 11, 2013, which is incorporated herein by reference as if fully set forth. The vector may include a genetic construct encoding any one of the nucleases described herein. The vector may comprise a nucleic acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 108 (meganuclease 4715) or SEQ ID NO: 109 (meganuclease 4716). The vector may comprise a nucleic acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 75 (Zm Cas9). The vector may comprise a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 95 (ZmU3P1:sgRNAGWDe24b), SEQ ID NO: 96 (ZmU3P2:sgRNAGWDe24b), SEQ ID NO: 97 (ZmU3.8P:sgRNAGWDe24b), SEQ ID NO: 98 (ZmU3P2:sgRNAGWDe24c), SEQ ID NO: 99 (ZmU3P2:sgRNAGWDe25a) and SEQ ID NO: 100 (ZmU3P2:sgRNAGWDela). The vector or the genetic construct described herein may include an engineered nucleic acid. The vector may be pAG4715 (FIG.1), pAG4716 (FIG. 2), or a modification thereof replacing any one of the annotated landmarks with a counterpart otherwise described herein. Routine vector elements annotated in FIGS. 1 or 2 may be replaced by counterparts described herein or known in the art.

[0095] An embodiment includes an engineered nucleic acid comprising, consisting essentially of, or consisting of a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 41 (Meganuclease GWD-9/10x.272 target sequence (pAG4715)) or SEQ ID NO: 42 (Meganuclease GWD-7/8x target sequence (pAG4716)).

[0096] An embodiment includes an engineered nucleic acid sequence comprising, consisting essentially of, or consisting of a sequence with at least

70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from the group consisting of SEQ ID NO: 91 (GWDela), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), and SEQ ID NO: 94 (GWDe25a).

[0097] An embodiment comprises an engineered nucleic acid having a sequence as set forth in any one of the engineered nucleic acids listed herein or the complement thereof. In an embodiment, an engineered nucleic acid having a sequence that hybridizes to a nucleic acid having the sequence of any nucleic acid listed herein or the complement thereof is provided. In an embodiment, the hybridization conditions are low stringency conditions. In an embodiment, the hybridization conditions are moderate stringency conditions. In an embodiment, the hybridization conditions are high stringency conditions. Examples of hybridization protocols and methods for optimization of hybridization protocols are described in the following books: Molecular Cloning, T. Maniatis, E.F. Fritsch, and J. Sambrook, Cold Spring Harbor Laboratory, 1982; and, Current Protocols in Molecular Biology, F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Volume 1, John Wiley & Sons, 2000, which are incorporated by reference in their entirety as if fully set forth. Moderate conditions include the following: filters loaded with DNA samples are pretreated for 2 - 4 hours at 68°C in a solution containing 6 x citrate buffered saline (SSC; Amresco, Inc., Solon, OH), 0.5% sodium dodecyl sulfate (SDS; Amresco, Inc., Solon, OH), 5xDenhardt's solution (Amresco, Inc., Solon, OH), and denatured salmon sperm DNA (Invitrogen Life Technologies, Inc. Carlsbad, CA). Hybridization is carried in the same solution with the following modifications: 0.01 M EDTA (Amresco, Inc., Solon, OH), 100 [g/ml salmon sperm DNA, and 5 - 20 x 106 cpm 32p. labeled or fluorescently labeled probes. Filters are incubated in hybridization mixture for 16-20 hours and then washed for 15 minutes in a solution containing 2xSSC and 0.1% SDS. The wash solution is replaced for a second wash with a solution containing 0.1xSSC and 0.5% SDS and incubated an additional 2 hours at 200C to 29°C below Tm (melting temperature inOC). Tm

= 81.5 +16.61Logio([Na+]/(1.0+0.7[Na+]))+0.41(%o[G+C])-(500/n)-P-F. [Na+] = Molar concentration of sodium ions. %[G+C]= percent of G+C bases in DNA sequence. N = length of DNA sequence in bases. P = a temperature correction for % mismatched base pairs (~1oC per 1% mismatch). F = correction for formamide concentration (=0.63°C per 1% formamide). Filters are exposed for development in an imager or by autoradiography. Low stringency conditions refers to hybridization conditions at low temperatures between 37°C and 600C, and the second wash with higher [Na+] (up to 0.825M) and at a temperature 40oC to 48oC below Tm. High stringency refers to hybridization conditions at high temperatures over 68oC, and the second wash with [Na+] = 0.0165 to 0.0330M at a temperature 5°C to 10°C below Tm.

[0098] An embodiment comprises an engineered nucleic acid having a sequence thathas atleast 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity along its length to a contiguous portion of a nucleic acid having any one of the sequences set forth herein or the complements thereof. The contiguous portion may be any length up to the entire length of a sequence set forth herein or the complement thereof.

[0099] Determining percent identity of two amino acid sequences or two nucleic acid sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity is measured by the Smith Waterman algorithm (Smith TF, Waterman MS 1981 "Identification of Common Molecular Subsequences," J Mol Biol 147: 195 -197, which is incorporated herein by reference as if fully set forth).

[0100] An embodiment comprises engineered nucleic acids, engineered polynucleotides, or engineered oligonucleotides having a portion of the sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. Theseengineered nucleic acids, engineered polynucleotides, or engineered oligonucleotides may have a length in the range from 10 to full length, 10 to 5000, 10 to 4900, 10 to 4800, 10 to 4700, 10 to

4600,10 to 4500,10 to 4400,10 to 4300,10 to 4200,10 to 4100,10 to 4000,10 to 3900,10 to 3800,10 to 3700,10 to 3600,10 to 3500,10 to 3400,10 to 3300, 10 to 3200, 10 to 3100, 10 to 3000, 10 to 2900, 10 to 2800, 10 to 2700, 10 to 2600,10 to2500,10 to 2400,10 to2300,10 to2200,10 to2100,10 to2000,10 to1900,10to1800,10 to1700,10to1600,10to1500,10 to 1400,10to1300, 10to 1200,10to1100,10to1000,10to900,10to800,10to700,10to 600,10 to500,10to400,10to300,10to200,10to100,10to90,10to 80,10to70,10 to60,10to50,10to40,10to35,10to30,10to25,10to20,10to15,or20to 30 nucleotides, or 10, 15, 20 or 25 nucleotides. An engineered nucleic acid, engineered polynucleotide, or engineered oligonucleotide having a length within one of the above ranges may have any specific length within the range recited, endpoints inclusive. The recited length of nucleotides may start at any single position within a reference sequence (i.e., any one of the nucleic acids herein) where enough nucleotides follow the single position to accommodate the recited length. In an embodiment, a hybridization probe or primer is 85 to 100%, 90 to 100%, 91 to 100%, 92 to 100%, 93 to 100%, 94 to 100%, 95 to 100%, 96 to100%, 97 to 100%, 98 to100%, 99 to 100%, or 100% complementary to a nucleic acid with the same length as the probe or primer and having a sequence chosen from a length of nucleotides corresponding to the probe or primer length within a portion of a sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, a hybridization probe or primer hybridizes along its length to a corresponding length of a nucleic acid having the sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, the hybridization conditions are low stringency. In an embodiment, the hybridization conditions are moderate stringency. In an embodiment, the hybridization conditions are high stringency.

[0101] An embodiment comprises a kit for identifying a modified sequence of an endogenous gene encoding Glucan Water Dikinase in a sample. The kit may comprise a first primer and a second primer. The first primer and the second primer may be capable of amplifying a target sequence included in an endogenous gene encoding Glucan Water Dikinase. The target sequence may include a nucleic acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from SEQ ID NOS: 1 - 4, 75, 170 - 184 186, 187, 189 - 193. The kit may further comprise one or more component for detecting modifications in the amplified region of the target sequence. The kit may comprise the first primer comprising a nucleic acid sequence selected from SEQ ID NOS: 6, 7, 9, 11, 101, 103, 105, 110, and 111. The kit may comprise the second primer comprising a nucleic acid sequence selected from SEQ ID NOS: 5, 8, 10, 102, and 104. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 6 and the second primer comprising the nucleic sequence of SEQ ID NO: 5. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 7 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 8. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 9 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 10. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 11 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 13. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 110 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 13. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 111 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 112. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 105 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 13. The first primer and the second primer may be capable of amplifying the target sequence to produce an amplified product. The amplified product may comprise a modified target sequence. The modified target sequence may be capable of hybridizing to the sequence of the nucleic acid comprising a sequence selected of SEQ ID NO: 12 - 40, 114 - 116, 188 - 189, 19 - 120, and 131 - 162 under conditions of high stringency. The modified target sequence may be used as a probe for diagnosing the genetically engineered plants having mutations in an endogenous gene encoding the Glucan Water Dikinase. A sample may include any sample in which nucleic acids from plant matter are present. The sample may include any plant matter. The plant matter may derive from a plant or part thereof. The plant material may derive from an animal feed or food.

[0102] An embodiment provides a method of identifying a modified sequence of an endogenous gene encoding a Glucan Water Dikinase in a sample is provided. The method may include contacting a sample with a first primer and a second primer. The method may include amplifying a synthetic polynucleotide comprising a target sequence included in an endogenous gene encoding a Glucan Water Dikinase. The target sequence may be any target sequence included in the endogenous gene encoding the Glucan Water Dikinase described herein. The first primer and the second primer may be capable of amplifying the target sequence to produce an amplified product. The amplified product may be used to determine whether a plant resulted from a sexual crossing or selfing contains one or more modifications in the target sequence and diagnose specific mutants. The length of the amplified product from the sample of the mutant plant may differ from the length of the amplified product from the sample of wild type plant of the same genetic background. The amplified product from the mutant sample may be further used as probe that hybridizes to a synthetic polynucleotide comprising a specific region encoding a mutant protein under conditions of high stringency. The method may include further detecting hybridization of the at least one probe to the specific region of the target sequence.

[0103] Methods of making a genetically engineered plant, methods of increasing starch levels in plants, methods of agricultural processing, methods of preparing animal feed and methods for producing genetically engineered plants homozygous for an engineered nucleic acid that encodes an altered Glucan Water Dikinase may comprise a method of detection herein as part of making genetically engineered plants and/or identifying plants or plant biomass that comprise a genetically engineered nucleic acid herein.

[0104] The following list includes particular embodiments of the present invention. But the list is not limiting and does not exclude alternate embodiments, or embodiments otherwise described herein. Percent identity described in the following embodiments list refers to the identity of the recited sequence along the entire length of the reference sequence.

[0105] EMBODIMENTS 1. A synthetic nucleic acid promoter having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of: SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), SEQ ID NO: 82 (ZmU3P1), SEQ ID NO: 84 (ZmU3P2) and SEQ ID NO: 86 (MzU3.8P). 2. A genetic construct comprising a first engineered nucleic acid sequence encoding a Cas9 nuclease, wherein the Cas9 nuclease is capable of cleaving a target sequence in an endogenous nucleic acid encoding Glucan Water Dikinase in a plant. 3. The genetic construct of embodiment 2, wherein the first synthetic nucleic acid sequence has at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO: 74 (Cas9 nuclease) or SEQ ID NO: 75 (ZmCas9). 4. The genetic construct of one or both embodiments 2 and 3, wherein the first nucleic acid is fused to a polynucleotide sequence encoding at least one nuclear localization signal (NLS). 5. The genetic construct of any one or more of embodiments 2 - 4, wherein the polynucleotide sequence encoding the nuclear localization signal is selected from SEQ ID NOS: 163 - 168. 6. The genetic construct of any one or more of embodiments 2 - 5 further comprising a second engineered nucleic acid sequence encoding an sgRNA, and the sgRNA is capable of binding the target sequence.

7. The genetic construct of embodiments 6, wherein the second engineered nucleic acid comprises a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a sequence selected from SEQ ID NO: 135 (ZmU3P1:sgRNAGWDe24b), SEQ ID NO: 136 (ZmU3P2:sgRNAGWDe24b), SEQ ID NO: 137 (ZmU3.8P:sgRNAGWDe24b), SEQ ID NO: 138 (ZmU3P2:sgRNAGWDe24c), SEQ ID NO: 139 (ZmU3P2:sgRNAGWDe25a) and SEQ ID NO: 40 (ZmU3P2:sgRNAGWDela). 8. The genetic construct of any one or more of embodiments 2 - 7, wherein the target sequence has at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of SEQ ID NO: 91 (GWDe1a), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), and SEQ ID NO: 94 (GWDe25a). 9. The genetic construct of any one or more of embodiments 2 - 8 further comprising a first promoter operably linked to the first engineered nucleic acid and a second promoter operably linked to the second engineered nucleic acid. 10. The genetic construct of embodiment 9, wherein the first promoter or the second promoter is a synthetic nucleic acid promoter of embodiment 1. 11. The genetic construct of any one or more of embodiments 2 - 10 further comprising a terminator. 12. The genetic construct of embodiment 11, wherein the terminator comprises a nucleic acid sequence with at least 90% identity to SEQ ID NO: 88. 13. A genetic construct comprising an engineered nucleic acid sequence encoding a nuclease, wherein the nuclease is capable of cleaving a target sequence included in an endogenous nucleic acid encoding Glucan Water Dikinase. 14. The genetic construct of embodiment 13, wherein the nuclease is a meganuclease encoded by a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of SEQ ID N): 164 (4715_meganuclease) and SEQ ID NO: 165 (4716_meganuclease). 15. The genetic construct of any one or more of embodiments 13 - 14, wherein the target sequence includes a polynucleotide of SEQ ID NO: 41 (Meganuclease GWD-9/lOx.272) or SEQ ID NO: 42 (Meganucleas3e GWD 7/8x). 16. The genetic construct of any one or more of embodiments 13 - 15 comprising at least one regulatory element, wherein the regulatory element is selected from a promoter, a terminator, and an enhancer. 17. A vector comprising a genetic construct of any one or more of embodiments 2 - 16. 18. A genetically engineered plant comprising an engineered nucleic acid encoding an altered Glucan Water Dikinase and having an elevated level of starch in comparison to a non-genetically engineered plant of the same genetic background. 19. The genetically engineered plant of embodiment 18, wherein the activity of the altered Glucan Water Dikinase is reduced compared to the activity of the wild type Glucan Water Dikinase in a non-genetically engineered plant of the same genetic background. 20. The genetically engineered plant of embodiment 18, wherein the altered Glucan Water Dikinase is inactive. 21. The genetically engineered plant of any one or more of embodiments 18 - 20, wherein the engineered nucleic acid is a modified sequence of an endogenous nucleic that is one allele of a gene encoding a Glucan Water Dikinase. 22. The genetically engineered plant of any one or embodiments 18 - 21, wherein all alleles of a gene encoding Glucan Water Dikinase in the plant have the sequence of the engineered nucleic acid. 23. The genetically engineered plant of any one or more of embodiments 18 - 22, wherein the endogenous nucleic acid includes a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence of SEQ ID NO: SEQ ID NO: 1 (Zm GWD coding sequence) or SEQ ID NO: 2 (Sb GWD coding sequence). 24. The genetically engineered plant of any one or more of embodiments 18 - 23, wherein the engineered nucleic acid comprises a mutation selected from at least one of an insertion, a deletion, or substitution of one or more nucleotides in the sequence of the endogenous nucleic acid encoding a wild type GWD. 25. The genetically engineered plant embodiment 24, wherein the mutation is within a target sequence in the endogenous nucleic acid having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of SEQ ID NO: 3 (Zm GWD Exon 24 + introns), SEQ ID NO; 4 (SbGWD Exon 24 + introns), SEQ ID NO: 182 (ZmGWD Exon 24 no introns), SEQ ID NO: 183 (Sb GWD Exon 24), SEQ ID NO: 184 (SbGWD Exon 7) and SEQ ID NO: 189 (Zm GWD Exon 25). 26. The genetically engineered plant of any one or more of embodiments 18 - 25, wherein the mutation is within a target sequence in the endogenous nucleic acid having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of SEQ ID NO: 91 (GWDela), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), and SEQ ID NO: 94 (GWDe25a). 27. The genetically engineered plant of any one or more of embodiments 18 - 26, wherein the engineered nucleic acid comprises a polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group of sequences consisting of SEQ ID NOS: 12 - 40 (Zm GWD mutations - Exon 24). 28. The genetically engineered plant of any one or more of embodiments 18 26, wherein the engineered nucleic acid comprises a polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group of sequences consisting of SEQ ID NOS: 114 - 118, 188, 131 - 146 (Zm GWD mutations Exon 24), and 119 - 120 (Zm GWD mutations - Exon 25).

29. The genetically engineered plant of any one or more of embodiments 18 - 25, wherein the engineered nucleic acid comprises a polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group of sequences consisting of SEQ ID NO: 106 (Sb47151 (WT + ins)_Exon 24, and SEQ ID NO: 107 (Sb47152 (WT + ins)_Exon 24). 30. The genetically engineered plant of any one or more of embodiments 16 - 28, wherein the altered Glucan Water Dikinase comprises an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of SEQ ID NOS: 45 - 73 (Zm GWD mutant proteins M1 - M29). 31. The genetically engineered plant of any one or more of embodiments 16 - 25, wherein the altered Glucan Water Dikinase comprises an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from the group consisting of SEQ ID NOS: 121 - 125 (Zm GWD mutant proteins M32 - M36), 126 -127 (Zm GWD mutant proteins M38 - M39) and 147 - 162 (Zm GWD mutant proteins M40- M55). 32. The genetically engineered plant of any one or more of embodiments 18 - 26, wherein the altered Glucan Water Dikinase comprises an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence selected from SEQ ID NO: 82 (Sb GWD mutant protein Sb4715_1WT + ins) or SEQ ID NO: 83 (Sb GWD mutant protein Sb4715_2 WT + del). 33. The genetically engineered plant of any one or more of embodiments 18 - 33, wherein the plant is selected from the group consisting of: a monocotyledonous plant, a dicotyledonous plant, a C4 plant, a C3 plant, tomato, sugar beet, sugar cane, eucalyptus, willow, poplar, corn, sorghum, wheat, alfalfa, soybean, rice, miscanthus, and switchgrass. 34. A genetically engineered plant comprising a genetic construct of any one or more of embodiments 2 - 16.

35. A method for producing a genetically engineered plant comprising: transforming a plant cell with a vector of embodiment 17; selecting a transformed plant cell that expresses nuclease and comprises an engineered nucleic acid encoding an altered Glucan Water Dikinase; and regenerating the genetically engineered plant from the transformed plant cell, wherein the genetically engineered plant or progeny thereof has an elevated level of starch in comparison to a non-genetically engineered plant of the same genetic background. 36. The method of embodiment 35, wherein the nuclease is a meganuclease. 37. The method of embodiment 35, wherein the nuclease is a Cas9 nuclease. 38. A method for genetically engineering a plant comprising an altered Glucan Water Dikinase comprising: contacting at least one plant cell comprising a target sequence in an endogenous gene encoding a Glucan Water Dikinase with a vector comprising a first nucleic acid encoding a nuclease capable of inducing a single-strand or double-strand break at the target sequence; selecting a plant cell that includes an alteration in the target sequence; regenerating a genetically engineered plant including the alteration from the plant cell. 39. The method of embodiment 38, wherein the genetically engineered plant is homozygous for the alteration. 40. The method of embodiment 38, wherein the genetically engineered plant is heterozygous for the alteration. 41. The method of embodiment 40 further comprising selfing the heterozygous genetically engineered plant, or crossing to another genetically engineered plant heterozygous for the same alteration, and selecting a first progeny plant that is homozygous for the alteration. 42. The method of embodiment 40 further comprising crossing the genetically engineered plant to a wild type plant of the same genetic background and selecting a first progeny plant that is heterozygous for the alteration. 43. The method of embodiment 42 further comprising selfing the first heterozygous progeny plant and selecting a second progeny plant that is homozygous for the alteration. 44. The method of any one or more of embodiment 38 - 43, wherein the alteration is a mutation selected from at least one of an insertion, a deletion, or a substitution of at least one nucleotide in the target sequence. 45. The method of embodiment 44, wherein the mutation is a null mutation. 46. The method of any one or more of embodiments 38 - 44, wherein the genetically engineered plant or progeny thereof has an elevated level of starch in comparison to a non-genetically engineered plant of the same genetic background. 47. The method of any one or more embodiments 38 - 46, wherein the nuclease is selected from the group consisting of a meganuclease, Cas9 nuclease, a zinc finger nuclease, and a transcription activator-like effector nuclease. 48. The method of embodiment 47, wherein the nuclease is the meganuclease and is encoded by a sequence with at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NO: 108 (4715_meganuclease) and SEQ ID NO: 109 (4716_meganuclease). 49. The method of embodiment 48, wherein the meganuclease is capable of cutting the target sequence that comprises a polynucleotide of SEQ ID NO: 41 (target for 4715 GWD-9/lOx.272) or SEQ ID NO: 42 (target for 4716_3e GWD-7/8x276). 50. The method of embodiment 47, wherein the nuclease is the Cas9 nuclease. 51. The method of embodiment 50, wherein the Cas9 nuclease is encoded by a nucleic acid with at least 90% identity to SEQ ID NO: 74 (Cas9 nuclease) or SEQ ID NO: 75 (ZmCas9).

52. The method of embodiment 51, wherein the nucleic acid encoding the Cas9 nuclease is fused to at least one nuclear localization signal (NLS), and the NLS has a polynucleotide sequence selected from SEQ ID NOS: 163 168. 53. The method of any one of embodiments 38 - 47 and 50 - 52, wherein the vector further comprises a second nucleic acid sequence encoding an sgRNA. 54. The method of embodiment 53, wherein the sgRNA is capable of binding the target sequence, and the target sequence is selected from the group consisting of SEQ ID NO: 91 (GWDela), SEQ ID NO: 92 (GWDe24b), SEQ ID NO: 93 (GWDe24c), and SEQ ID NO: 94 (GWDe25a). 55. The method of any one or more of embodiments 53 - 54, wherein the second nucleic acid comprises a sequence with at least 90% identity to SEQ ID NOS: 95 (ZmU3P1:sgRNAGWDe24b), SEQ ID NO: 96 (ZmU3P2:sgRNAGWDe24b), SEQ ID NO: 97 (ZmU3.8P:sgRNAGWDe24b), SEQ ID NO: 98 (ZmU3P2:sgRNAGWDe24c), SEQ ID NO: 99 (ZmU3P2:sgRNAGWDe25a) and SEQ ID NO: 100 (ZmU3P2:sgRNAGWDela). 56. The method of any one or more of embodiments 38 - 55, wherein the vector further comprises a nucleic acid promoter operably linked to the first nucleic acid or the second nucleic acid. 57. The method of embodiment 56, wherein the nucleic acid promoter comprises a sequence with at least 90% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 78 (MzU3.8), SEQ ID NO: 79 (ZmU3), SEQ ID NO: 82 (ZmU3P1), SEQ ID NO: 84 (ZmU3P2) and SEQ ID NO: 86 (MzU3.8). 58. A genetically engineered plant produced by the method of any one of one of embodiments 38 - 57, or a progeny or descendant thereof, wherein the plant, progeny or descendant thereof comprises the alteration. 59. The genetically engineered plant of embodiment 58 having an elevated level of starch in comparison to a plant of the same genetic background comprising wild type Glucan Water Dikinase.

60. A method of increasing a starch level in a plant comprising expressing a nucleic acid in the plant that encodes a nuclease capable of inducing a double strand break at a target sequence and selecting a homozygous plant that includes an alteration in the target sequence and has an elevated level of starch, wherein the target sequence is included in an endogenous gene encoding a Glucan Water Dikinase only. 61. A method of agricultural processing comprising: expressing in a plant a nucleic acid encoding a nuclease capable of inducing a double-strand break at a target sequence, wherein the target sequence is included in an endogenous gene encoding a Glucan Water Dikinase; selecting a homozygous plant that includes an alteration in the target sequence and has an elevated level of starch; and processing the homozygous plant, wherein the processing comprises one or more procedures selected from harvesting, bailing, shredding, drying, fermenting, hydrolyzing with chemicals, hydrolyzing with exogenous enzymes and combining with plant biomass. The method may also comprise the method for producing a genetically engineered plant of any one or more of embodiments 63 - 71. 62. A method of preparing animal feed comprising: expressing in a plant a nucleic acid encoding a nuclease capable of inducing a double-strand break at the target sequence, wherein the target sequence is included in an endogenous gene encoding a Glucan Water Dikinase; selecting a homozygous plant that includes an alteration in the target sequence and has an elevated level of starch; and performing at least one procedure selected from the group consisting of: harvesting, bailing, shredding, drying, ensiling, pelletizing, combining with a source of edible fiber, and combining with plant biomass. The method may also comprise the method for producing a genetically engineered plant of any one or more of embodiments 63 - 71.

63. A method for producing a genetically engineered plant comprising an engineered nucleic acid that encodes an altered Glucan Water Dikinase comprising modifying a sequence of an endogenous nucleic acid of at least one allele of a gene that encodes a Glucan Water Dikinase in a plant, wherein the modified engineered nucleic acid is an engineered nucleic acid and the modified plant is the genetically engineered plant. 64. The method of embodiment 63, wherein the genetically engineered plant is homozygous for the gene that includes the mutation and all alleles include the sequence of the engineered nucleic acid. 65. The method of embodiment 63, wherein the genetically engineered plant is heterozygous for the gene that includes the mutation. 66. The method of any one or more of embodiments 63 or 65 further comprising self-crossing the genetically engineered plant and obtaining progeny. 67. The method of any one or more of embodiments 63 - 65 further comprising crossing the genetically engineered plant and a non-genetically engineered plant of the same genetic background and obtaining progeny. 68. The method of any one or more of embodiments 66 - 67 comprising analyzing the progeny for the presence of the altered Glucan Water Dikinase and selecting a progeny plant that includes the mutation. 69. The method of embodiment 63 comprising the genetically engineered plant of any one or more of embodiments 18 - 34 and 58 - 59. 70. The method of embodiment 63, wherein the step of modifying is performed by a method of any one of embodiments 35 - 36. 71. The method of embodiment 63, wherein the step of modifying is performed by using a genetic construct of any one of embodiments 2 - 16. 72. A kit for identifying a modified sequence of an endogenous gene encoding Glucan Water Dikinase in a sample, wherein the kit comprises a first primer and a second primer, wherein the first primer and the second primer are capable of amplifying a target sequence in the endogenous gene encoding Glucan Water Dikinase and the target sequence comprises a nucleic acid sequence with at least 90% identity to a reference sequence selected from SEQ ID NOS: 1 - 4, 75, 171 - 187, 189 - 193. 73. The kit of embodiment 72 urther comprising one or more component for detecting at a modification in the amplified region of the target sequence. 74. The kit of any one or more of embodiments 72 - 73 wherein, the first primer comprises a nucleic acid sequence selected from SEQ ID NOS: 6, 7, 9, 11,101,103,105,110, and 111. 75. The kit of any one or more of embodiments 72 - 74, wherein the second primer comprises a nucleic acid sequence selected from SEQ ID NOS: 5, 8, 10, 102, and 104. 76. The kit of any one or more of embodiments 72 - 75, wherein the first primer and the second primer are capable of amplifying the target sequence to produce an amplified product comprising a modified target sequence. 77. The kit of any one or more of embodiments 73 - 76, wherein the amplified target sequence comprises a sequence selected from SEQ ID NOS: 12 - 40, 106 - 107, 114 - 120, 131- 146, and 188. 78. The kit of any one or more of embodiments 72 - 76, wherein the modified target sequence is capable of hybridizing to the sequence of the nucleic acid comprising a sequence selected from SEQ ID NOS: 12 - 40, 106 107, 114 - 120, 131 - 146, and 188 under conditions of high stringency. 79. The kit of any one or more of embodiments 72 - 78, wherein the sample comprises plant matter derived from a genetically engineered plant having at least one mutation in an endogenous gene encoding the Glucan Water Dikinase. 80. A method of identifying a modified sequence of an endogenous gene encoding Glucan Water Dikinase in a sample comprising: contacting a sample with a first primer and a second primer; amplifying a target sequence included in the endogenous gene encoding Glucan Water Dikinase and the target sequence comprises a nucleic acid sequence with at least 90% identity to a reference sequence selected from SEQ ID NOS: 1 - 4, 75, 170 - 184 186, 187, 189 - 193; and detecting a modification in the target sequence. 81. The method of embodiment 79, wherein the modification in the target sequence comprises a sequence selected from SEQ ID NOS: 12 - 40, 106 - 107, 114 - 120, 131 - 146, and 188. The method of identifying may be added to any one or more of embodiments 60 - 71.

[0106] Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein.

[0107] EXAMPLES

[0108] The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and/or one or more element from an embodiment may be substituted with one or more detail from one or more example below.

[0109] Example 1. Meganuclease-based modification of GWD gene in maize and sorghum genomes

[0110] Meganuclease constructs were designed that target GWD exon 24, which is near the predicted encoded active site of the enzyme, with the intent of introducing a mutation that inactivates GWD (null mutation). Meganuclease-induced GWD DNA mutants were identified and characterized in maize and sorghum.

[0111] In order to engineer I-Crel homing endonucleases with specificities against GWD genes in maize and sorghum genomes, two nucleotide sequences were selected from the previously annotated full length GWD genes of maize and sorghum. The sequence selection was based on the existence of the high nucleotide sequence identity between maize and sorghum sequences ( 9 5 % nucleotide sequence identity) and the presence of a sequence motif in exons #24 of both crops that is required for GWD protein activity. The goal was to develop two meganuclease constructs in such a way that each of them would be specific for GWD modification in both maize and sorghum. Targeted genome modifications at the selected GWD sequences using the meganuclease approach would lead to expression of GWD protein variants lacking the active site (truncated proteins or modified proteins expressed from the frame shift containing coding sequences) and therefore being catalytically inactive. The selected sequences of ZmGWD (maize) and SbGWD (sorghum) shown below were supplied to Precision Biosciences, Inc. for designing meganucleases GWD9-10x.272 and GWD7-8x.226.

[0112] The target sequence for the meganuclease GWD-9/10x.272 (pAG4715) is: ATCCTTGTGGCAAAGAGTGTCA (SEQ ID NO: 41).

[0113] The target sequence for the meganuclease GWD-7/8x.226 target sequence (pAG4716) is: GTAGTTGGTGTAATTACACCTG (SEQ ID NO: 42).

[0114] The DNA sequences within exon 24 that are recognized by the designed meganucleases are underlined. The sequences in the uppercase letters show exon 24, while the sequences in lowercase letters represent flanking introns. The "CAT" codon that is double-underlined encodes a Histidine residue that is critical for GWD protein activity.

>ZmGWDExon24 aagtgatactagtgaccctctccacaattttatgcgaaccacagaaattaataatatattctattactctgcacct gacatctggctcctgctatcagTTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTA TGTGGTTGTGGTTGATGAGTTACTTGCTGTCCAGAACAAATCTTATGATA AACCAACCATCCTTGTGGCAAAGAGTGTCAAGGGAGAGGAAGAAATACC AGATGGAGTAGTTGGTGTAATTACACCTGATATGCCAGATGTTCTGTCTC ATGTGTCAGTCCGAGCAAGGAATAGCAAGgtttatcttcacagctatgttgcaagatttctt gaattttttctcttgtattgatgttgacatactagctttttcctaat (SEQ ID NO: 3)

>SbGWDExon24 aagtggtactagtgacctctccacagttttatgtgaaccacagaaattaaatatgataatatattctattactctg cacctgacatctggctcctgataacagTTGGCAGGTTATAAGCCCAGTTGAAGTATCAG GTTATGTGGTTGTGGTTGATGAGTTACTTGCTGTCCAGAACAAATCTTAT GATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGGAGAGGAAGAAA TACCAGATGGAGTAGTTGGTGTAATTACACCTGATATGCCAGATGTTCTG

TCCCATGTGTCAGTCCGAGCAAGGAATAGCAAGgtttattttcacagttatgttgcaag ctttctcagattttttttcttgtatcgatgttgacataccagttttttcctaat (SEQ ID NO: 4)

[0115] Clustal software was used to align selected ZmGWD and SbGWD sequences. (Larkin MA et al., 2007; Goujon M et al., 2010, both of which are incorporated herein by reference as if fully set forth).

CLUSTAL 2.1 multiple sequence alignment

SbGWDExon24 aagtggtactagtgacctctccacagttttatgtgaaccacagaaattaaatatgataa 59 ZmGWD_Exon24 aagtgatactagtgaccctctccacaattttatgcgaaccacagaaatta------ataa 54 ***** *********** ******** ******* *************** ****

SbGWDExon24 tatattctattactctgcacctgacatctggctcctgataacagTTGGCAGGTTATAAGCl19 ZmGWD_Exon24 tatattctattactctgcacctgacatctggctcctgctatcagTTGGCAGGTTATAAGCll4 ************************************* ** *******************

SbGWDExon24 CCAGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACTTGCTGTCCAGAACAAA179 ZmGWD_Exon24 CCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACTTGCTGTCCAGAACAAA174 ** *********************************************************

SbGWDExon24 TCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGGAGAGGAAGAAATACCA239 ZmGWDExon24 TCTTATGATAAACCAACCATCCTGTGGCAAAGAGATGTCAAGGGAGAGGAAGAAATACA234

SbGWD_Exon24 GATGGAGTAGTTGGTGTAATTACACCTGATATGCCAGATGTTCTGTCCCAGTGTCAGTC299

ZmGWD Exon24 GATGGAGTAGTTGGTGTAATTACACCTGATATGCCAGATGTTCTGTCTCATGTGTCAGTC294 *********************************************** ************

SbGWDExon24 CGAGCAAGGAATAGCAAGgtttattttcacagttatgttgcaagctttctcagatttttt359 ZmGWD_Exon24 CGAGCAAGGAATAGCAAGgtttatcttcacagctatgttgcaagatttcttgaatttttt354 ************************ ******* *********** ***** ******* SbGWDExon24 ttcttgtatcgatgttgacataccagttttttcctaat 397(SEQ ID NO: 4) ZmGWDExon24 ctcttgtattgatgttgacatactagctttttcctaat 392(SEQ ID NO: 3) ******** ************* ** ***********

[0116] Development of plant transformation vectors for expressing meganucleases:

[0117] The meganuclease sequences GWD-9/10x.272 [SEQ ID NO: 108] and GWD-7/8x.226 [SEQ ID NO: 109], which were provided by Precision Biosciences, Inc., were further modified by adding BamHI restriction site at 5' and AvrII site at 3' ends using PCR approach. Subsequently, GWD-9/10x.272 and GWD-7/8x.226 nucleotide sequences were cloned into pAG4500 vector as BamHI-AvrII fragments between the maize ubiquitin 1 gene promoter and

Nos transcriptional terminator sequences to generate respectively plant transformation vectors pAG4715 and pAG4716. FIG. 1 and FIG. 2 illustrate respective maps of pAG4715 and pAG4716 vectors. Referring to FIGS. 1 and 2, pAG4715 and 4716 include a maize ubiquitin promoter (ZmUbil), a maize ubiquitin intron (ZmUbil intron), and a polyadenilation signal NosT serving as the transcription terminator. Both vectors also include a phosphomannose isomerase gene (PMI) as a selectable marker, At NLS (nuclear localization sequence), ZmKozak, mUBQmono, T-DNA right and left borders (RB and LB, respectively), a streptothricin acetyltransferase gene, and an aminoglycoside acetyltransferse (aadA) gene conferring resistance to streptomycin. pAG4715 includes a GWD9-10x.272 meganuclease sequence [SEQ ID NO: 108] and pAG4716 includes a GWD7-8x.226 meganuclease sequence [SEQ ID NO: 109].

[0118] pAG4715 and pAG4716 were used to generate transgenic events and mutants in maize and sorghum.

[0119] Sequences of the target proteins, genes, mutants and vectors used herein are listed in Table 1. Table 1 Description of Sequences SEQ Description Type IDNO 1 ZmGWD coding sequence DNA 2 SbGWD coding sequence DNA 3 ZmGWD Exon 24 (includes introns) DNA 4 SbGWD Exon 24 (includes introns) DNA 182 ZmGWD Exon 24 (no introns) DNA 183 SbGWD Exon 24 (no introns) DNA 184 SbGWD Exon 7 (no introns) DNA 5 Mega-1 (4716) PCR Primer Reverse DNA 6 Mega-1 (4716) PCR Primer Forward DNA 7 Mega-2 (4715) PCR Primer Forward DNA 8 Mega-2 (4715) PCR Primer Reverse DNA 9 ZmGWD mega-2 PCR Primer Forward DNA 10 ZmGWD mega-2 PCR Primer Reverse DNA 11 SbGWD mega-2 PCR Primer Forward DNA 13 SbGWD mega-2 PCR Primer Reverse DNA 12 M16 (Zm GWD Exon 24 - no introns) DNA 13 M17 (Zm GWD Exon 24 - no introns) DNA 14 M18 (Zm GWD Exon 24 - no introns) DNA 15 M27 (Zm GWD Exon 24 - no introns) DNA

SEQ Description Type IDNO 16 M1 (Zm GWD Exon 24 - no introns) DNA 17 Ml1 (Zm GWD Exon 24 - no introns) DNA 18 M10 (Zm GWD Exon 24 - no introns) DNA 19 M3 (Zm GWD Exon 24 - no introns) DNA 20 M8 (Zm GWD Exon 24 - no introns) DNA 21 M14 (Zm GWD Exon 24 - no introns) DNA 22 M13 (Zm GWD Exon 24 - no introns) DNA 23 M12 (Zm GWD Exon 24 - no introns) DNA 24 M22 (Zm GWD Exon 24 - no introns) DNA 25 M23 (Zm GWD Exon 24 - no introns) DNA 26 M24 (Zm GWD Exon 24 - no introns) DNA 27 M20 (Zm GWD Exon 24 - no introns) DNA 28 M21 (Zm GWD Exon 24 - no introns) DNA 29 M4 (Zm GWD Exon 24 - no introns) DNA 30 M19 (Zm GWD Exon 24 - no introns) DNA 31 M26 (Zm GWD Exon 24 - no introns) DNA 32 M25 (Zm GWD Exon 24 - no introns) DNA 33 M15 (Zm GWD Exon 24 - no introns) DNA 34 M5 (Zm GWD Exon 24 - no introns) DNA 35 M2 (Zm GWD Exon 24 - no introns) DNA 36 M28 (Zm GWD Exon 24 - no introns) DNA 37 M6 (Zm GWD Exon 24 - no introns) DNA 38 M9 (Zm GWD Exon 24 - no introns) DNA 39 M7 (Zm GWD Exon 24 - no introns) DNA 40 M29 (Zm GWD Exon 24 - no introns) DNA 106 Mutant Sb4715_1 (Wt+ ins) DNA 107 Mutant Sb4715_2 (WT +del) DNA 41 Meganuclease GWD-9/lOx.272 target DNA sequence (pAG4715) 42 Meganuclease GWD-7/8x target sequence DNA (pAG4716) 43 ZmGWD (wild type protein) Amino acid 44 SbGWD (wild type protein) Amino acid 45 ZmGWD M1 (mutant GWD protein) Amino acid 46 ZmGWD M2 (mutant GWD protein) Amino acid 47 ZmGWD M3 (mutant GWD protein) Amino acid 48 ZmGWD M4 (mutant GWD protein) Amino acid 49 ZmGWD M5 (mutant GWD protein) Amino acid 50 ZmGWD M6 (mutant GWD protein) Amino acid 51 ZmGWD M7 (mutant GWD protein) Amino acid 52 ZmGWD M8 (mutant GWD protein) Amino acid 53 ZmGWD M9 (mutant GWD protein) Amino acid 54 ZmGWD M10 (mutant GWD protein) Amino acid 55 ZmGWD M1 (mutant GWD protein) Amino acid 56 ZmGWD M12 (mutant GWD protein) Amino acid

SEQ Description Type IDNO 57 ZmGWD M13 (mutant GWD protein) Amino acid 58 ZmGWD M14 (mutant GWD protein) Amino acid 59 ZmGWD M15 (mutant GWD protein) Amino acid 60 ZmGWD M16 (mutant GWD protein) Amino acid 61 ZmGWD M17 (mutant GWD protein) Amino acid 62 ZmGWD M18 (mutant GWD protein) Amino acid 63 ZmGWD M19 (mutant GWD protein) Amino acid 64 ZmGWD M20 (mutant GWD protein) Amino acid 65 ZmGWD M21 (mutant GWD protein) Amino acid 66 ZmGWD M22 (mutant GWD protein) Amino acid 67 ZmGWD M23 (mutant GWD protein) Amino acid 68 ZmGWD M24 (mutant GWD protein) Amino acid 69 ZmGWD M25 (mutant GWD protein) Amino acid 70 ZmGWD M26 (mutant GWD protein) Amino acid 71 ZmGWD M27 (mutant GWD protein) Amino acid 72 ZmGWD M28 (mutant GWD protein) Amino acid 73 ZmGWD M29 (mutant GWD protein) Amino acid 74 Mutant protein Sb4715 1 (WT + ins) Amino acid 75 Mutant protein Sb4715_2 (WT + del) Amino acid

[0120] Example 2. Application of TALENs for targeted modification of GWD gene in sorghum genome

[0121] Two pairs of DNA sequences were selected in each of the exons 7 and 24 of the sorghum GWD gene (SbGWD) for development of four custom TAL DNA-binding domains that will be fused to a truncated FokI nuclease sequence. The sorghum exon 24 was selected because it contains the GWD active site and to compare with other endogenous DNA editing technologies in maize, such as meganuclease and CRISP/Cas9 technologies. The sorghum exon 7 was chosen in the upstream region of the GWD gene sequence for producing shorter truncated versions of the GWD protein. Selection of the sequences for DNA-binding domains was performed on Life Technologies web site using a proprietary program. The two pairs of TAL DNA binding domains fused to truncated FokI endonuclease for targeted sorghum genome modifications in exons 7 and 24 of the GWD gene are being constructed by Life Technologies. Each pair of TALENs will recognize top and bottom strands of genomic DNA sequence at the respective GWD sites to target FokI nuclease for DNA cleavage.

[0122] SbGWD nucleotide sequences selected for TALENs-based GWD modification. The SbGWDexon7 sequence is positioned within nt 736 - 969 the SbGWD coding sequence (SEQ ID NO: 2): >SbGWDexon7 GAGGAGTATGAAGCTGCACGAGCTGAGTTAATAGAGGAATTAAATAGAG GTGTTTCTTTAGAGAAGCTTCGAGCTAAATTGACAAAAACACCTGAAGCA CCTGAGTCAGATGAACGTAAATCTCCTGCATCTCGAATGCCCGTTGATAA ACTTCCAGAGGACCTTGTACAGGTGCAGGCTTATATAAGGTGGGAGAAAG CGGGCAAGCCAAATTATCCTCCTGAGAAGCAACTG (SEQ ID NO: 184)

[0123] The SbGWDexon24 sequence is positioned within nt 3030 - 3243 the SbGWD coding sequence (SEQ ID NO: 2): >SbGWDexon24 TTGGCAGGTTATAAGCCCAGTTGAAGTATCAGGTTATGTGGTTGTGGTTG ATGAGTTACTTGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTG TGGCAAAGAGTGTCAAGGGAGAGGAAGAAATACCAGATGGAGTAGTTGG TGTAATTACACCTGATATGCCAGATGTTCTGTCCCATGTGTCAGTCCGAG CAAGGAATAGCAAG (SEQ ID NO: 183)

[0124] Underlined sequences in each exon represent selected TAL DNA binding sites with the left sequence being specific for the upper DNA strand and the right sequence targeting the bottom DNA strand. A codon encoding catalytically important Histidine residue for GWD protein activity is double underlined and in bold within exon 24. The TALENs specific for exon 7 or exon 24 will be cloned as respective pairs into pAG4500-based plant transformation vector.

[0125] Example 3. Plant Transformation and Analysis

[0126] Maize and Sorghum Transformation: DNA from Agrobacterium was extracted using the protocol described in the Plasmid pSB1 operating manual. Plant DNA was extracted using Qiagen DNeasy Plant Mini kit (69140). Maize and sorghum embryos were transformed with GWD meganuclease targeting constructs pAG4715 and/or pAG4716 according to Negrotto D et al. 2000 Plant Cell Rep 19: 798; Ishida Y et al. 1996 Nat Biotech

14: 74, which is incorporated herein by reference as if fully set forth. Briefly, embryogenic callus from wild-type AxB maize was inoculated with LBA4404 Agrobacterium cells harboring the appropriate transformation plasmid. Agrobacterium-mediated transformation of immature maize embryos was performed as described on Negrotto D et al. The expression cassettes for GWD meganucleases were cloned into the KpnI-EcoRI sites of an intermediate vector capable of recombining with the pSB1 vector in triparental mating in Agrobacterium tumefaciens strain LBA4404 using procedures reported previously (Ishida Y et al. 1996 Nat Biotech 14: 745; Hiei Y et al. 1994 Plant J 6: 271; Hiei Y and Komari T 2006 Plant Cell Tissue Organ Cult. 85: 27; Komari T et al. 1996 Plant J 10: 165). Maize (Zea mays cultivars Hill, A188 or B73) stock plants were grown in a greenhouse under 16 hours of daylight at 28°C. Immature zygotic embryos were isolated from the kernels and inoculated with the Agrobacterium solution containing the genes of interest. After inoculation immature embryos were grown in a tissue culture process for 10 - 12 weeks. Well-developed seedlings with leaves and roots were sampled for PCR analysis to identify transgenic plants containing the genes of interest. PCR positive and rooted plants were rinsed with water to wash off the agar medium, and transplanted to soil and grown in the greenhouse to generate seeds and stover.

[0127] Sorghum transformation was carried according to the protocol of Gao et al., 2005. Regeneration of the transgenic plants was performed according to Elkonin and Pakhomova, 2000.

[0128] DNA from Agrobacterium was extracted using the protocol described in the Plasmid pSB1 operating manual. Plant DNA was extracted using Qiagen DNeasy Plant Mini kit (69140).

[0129] 1OXTE+Sarkosyl-PlantDNA Isolation for 96 well plates: Briefly, a COSTAR grinding block was filled with 4 leaf samples, one 5 mm steel bead was added to each well with a sample and the storage mat block was applied using a storage mat applicator to seal the block. Samples were stored at -80°C for at least 30 min before grinding or until processing time. For processing, samples were ground for 45 sec using the Klecko Pulverizer & Secure grinder at maximum speed. Sealing was removed and discarded. Three hundred microliters of 10XTE+Sarkosyl buffer (5 mL 1M Tris, 1 mL 0.5M EDTA, 0.5g sarkosyl, 46 mL ddH 2 0) was added to each sample using a multichannel pipette and a sterile solution basin. The plate was incubated on a shaker for 10 min at 300 rpm, and spun for 3 min at 4000 rpm. Supernatant was removed and discarded, and the pellets were resuspended in 1xTE buffer. One hundred fifty microliter sample aliquots were added to the 96 well PCT plate. The PCR plate was sealed with aluminum foil. For best results, DNA isolation and PCR were performed on the same date.

[0130] Transgenic DiagnosticPCR Reaction Setup: The "complete" PCR reaction mix was as follows: 15 gl 2X GoTaq MM (GoTaq Green Master Mix (PROMEGA #M712), 3l of combined forward and reverse primers specific to the gene of interest (each mixed at 10gM), 2 gl DNA prep, and water to adjust volume to 30 gl. Twenty eight microliters of the "complete" PCR reaction mix per well were aliquoted into a PCR plate (FISHER, #14230236). Two microliters plant DNA sample were aliquoted into each well of the PCR plate. Positive control and no template negative control were used in each PCR reaction. Control Agrobacterium DNA was diluted 1:100 in TE buffer to yield clear bands. The PCR plate was sealed with a sealing mat (COSTAR #6555) and roller. PCR was performed at BIORAD PTC-100 thermocycler. The

thermocycler programs were as follows: 1) 95°C-3min; 30 cycles 95°C-30sec, 550C-30sec, 72°C-45sec; 72°C-5min; 10°C (hold), and 2) 900C for 30 min and 10°C (hold). Twelve microliters of each PCR reaction was loaded onto Ready Agarose 96 Plus gel -3% (BIORAD #161-3062) and ran at approximately 100V for 20 minutes before viewing with a BIORAD gel doc system equipped with Quantity One software. Quick-Load 50bp DNA Ladder (NEB N0473S) was used to identify the size of the PCR fragments. 1oX TBE Buffer (Promega V4251) was used.

[0131] In line monitor of starch content in living corn leaf using Fourier Transform Near-infrared(FT-NIR) Technique: The starch content in corn leaf tissue is an important factor for animal feed and biofuel production. The commonly used GOPOD assay is not applicable for real-time monitoring in living tissue because the assay is invasive, requiring physical tissue samples, and labor intensive. Predictive models of starch content based on the FT-NIR spectra of dry blends of native and maize leaf starches or corn flour (wet and dry) were developed using partial least squares regression. Three key factors determine a successful application of FT-NIR techniques for fast chemical characterization: accurate and repeatable NIR spectral acquisition, reliable calibration data, and robust chemometric analysis. For analysis, the following materials were used: a spectrophotometer (Perkin Elmer Spectrum One NTS Waltham, MA), Unscrambler® (Version 10.2., Camo Software Inc., Woodbridge, NJ), an oven, Hi-maize resistant starch (Honeyville, Brigham City, UT), and Starch (Product No. S516-500, Fisher Scientific). All blank and test samples were diluted 1OX using demonized water and the unreacted starch content was determined using a glucose oxidase-peroxidase (GOPOD) colorimetric assay (Megazyme International, Wicklow, Ireland).

[0132] Sample preparation:A total of 56 dry starch blend samples with 0- 33% starch content were prepared by mixing weight proportions of HI MAIZE© resistant starch (Honeyville, Brigham City, UT) with starch (Product No. S516-500, Fisher Scientific). The Honeyville product contains HI-MAIZE 260 (Ingredion, Bridgewater, NJ) resistant starch that has been isolated from high amylose corn hybrids produced through traditional plant breeding, and contains 33% digestible, or glycemic starch.

[0133] One hundred fifty green leaf samples from different living maize plants at certain age or with different starch accumulation were collected. One hundred samples were oven-dried and 50 samples were left undried ("wet"). Leaf samples were ground to 0.5 mm and stored in plastic sample bags for moisture equilibration. Moisture content was measured using standard methods.

[0134] Starch determination: The starch content was analyzed for starch blends, wet and dry green tissue or corn flour sample using GOPOD assay.

[0135] Scanning, processing and analyses of FT-NIR spectra: Scanning Approximately a 5 g milled sample was poured in a smaller NIRA cup, leveled, and scanned 16 times with a manual rotation between each scan. This procedure was repeated five times with separate subsamples and the resulting spectral scans were averaged. A total of 56 samples of starch blends were used to create starch models. 36 samples were used in the calibration set, 14 samples were used in the validation set, and six samples were used in the test set. The dry ground green tissue or flour models were made using 100 samples of corn leaf or seeds. For calibration, validation, and testing, 72, 20, and 8 samples were used, respectively. The wet ground corn models were made using 49 samples of corn leaf or corn seeds. 36 samples were used for calibration, 10 samples were used for validation, and three samples were used for the test set.

[0136] Processing and analyses of FT-NIR spectra - Unscrambler® (Version 10.2., Camo Software Inc., Woodbridge, NJ) was used to process and analyze the spectral data, build and validate the calibration, and test the regression models. Multiplicative scatter correction (MSC) and a 2nd derivative-based smoothing technique, such as the Savitzky-Golay (SG) technique, were used for data pretreatment. Partial least squares models using a combination of MSC with SG second derivate pretreated spectral data were developed for starch blends and milled green tissue or ground corn. Examples of measured and predicted (MSC+2nd Derivative Model) starch content of calibration, validation, and test samples are shown in Table 2.

Table 2 Measured and Predicted (MSC+2nd Derivative Model) Starch Content of Calibration, Validation, and Test Samples Starch blends Dry corn flour Wet corn flour Starch Measured Predicted Measured Predicted Measured Predicted content (%) (%) (%) (%) (%) (%)

Calibration 2.50 2.52 5.0 6.5 5.0 5.2 5.00 4.89 7.5 7.4 10.0 7.9 10.0 10.1 10.0 10.2 12.5 12.0 15.0 15.6 15.0 14.5 13.2 13.5 20.0 20.0 Validation 4.75 4.7 7.5 7.6 7.5 7.5 10.0 9.8 12.5 12.4 10.0 10.0 15.7 16.0 17.5 17.5 12.0 12.5 18.9 18.9 Test 6.5 6.55 5.0 6.5 7.2 7.3 12.5 12.45 11.5 12.5 11.2 11.5 17.5 18.2 14.5 13.2 12.2 13.5

R 2 : Starch blends: Calibration: 0.98, Validation: 0.97, Prediction: 0.97 Dry corn flour: Calibration: 0.86, Validation: 0.80, Prediction: 0.80 Wet corn flour: Calibration: 0.94, Validation: 0.80, Prediction: 0.75

[0137] Example 4. Identification and characterization of meganuclease-induced DNA mutations in the Zea mays and Sorghum bicolor GWD genes

[0138] Identification of Gene of Interest (GOI) Positive Maize and Sorghum Transformants:Leaves from maize and sorghum plants transformed with pAG4715 or pAG4716 were sampled, DNA was extracted, and screened for the presence of the meganuclease transgenes included in pAG4715 or pAG4716.

[0139] Screening of Maize and Sorghum Transformants:Plants carrying pAG4715 or pAG4716 transgenes referred to as 4715 or 4716 plants, respectively, were screened for GWD mutations using sequence analysis of PCR-amplified GWD DNA sequences.

[0140] PCR Amplification of GWD: DNA sequences surrounding the meganuclease targeting region on exon 24 were amplified using the ZmGWDmega-2 and SbGWDmega-2 primers shown in Table 3.

Table 3 Primers for Genotyping 4715 and 4716 Plants

Primer Set Primer Forward Sequence Product Name or size (bp) Reverse Mega-1 Mega-1R Reverse TGATCTTCAGCACGAG 265 (4716) GTTG (SEQ ID NO: 5) Mega-1 Mega-1F Forward GGCTCCATCTATGCCTG 265 (4716) TATC (SEQ ID NO: 6) Mega-2 Mega-2F Forward GAGCTCAGTTTCGCTGT 209 (4715) CTATC (SEQ ID NO: 7) Mega-2 Mega-2R Reverse ATGATCTTCAGCACGA 209 (4715) GGTTG (SEQ ID NO: 8) ZmGWD ZmGWD Forward GGTTATAAGCCCGGTT 204 mega-2 mega-2F GAAGTA (SEQ ID NO: 9) ZmGWD ZmGWD Reverse CTATTCCTTGCTCGGAC 204 mega-2 mega-2R TGAC (SEQ ID NO: 10) SbGWD SbGWD Forward GGCAGGTTATAAGCCC 208 mega-2 mega-2f AGTT (SEQ ID NO: 11) ZmGWD SbGWD Reverse CTATTCCTTGCTCGGAC 208 mega-2 mega-2r TGAC (SEQ ID NO: 10)

[0141] Primer sets were diluted to a final concentration of 5 gM in nuclease-free water. PCR reaction was performed as described above.

[0142] PCR samples were run on an Eppendorf Mastercycler proS (Eppendorf) using our PM155 program (95°C, 2 min; 30 cycles [95°C, 30 sec; 550C, 30 sec; 72°C, 45 sec]; 72°C, 8 min)

[0143] PCR samples were separated on Bio-Rad ReadyAgarose 96 Plus Gels, TBE (#161-3062) and visualized with a Bio-Rad gel imaging system.

[0144] FIG. 3 illustrates an example of a gel showing bands of 4715 and 4716 events or successful incorporation of transgenes from pAG4715 or pAG4716, respectively. Referring to this figure, shifted GWD bands indicate potential insertions and deletions (indels) at the GWD meganuclease targeting site and are marked by asterisks.

[0145] DNA Sequence Characterizationof the GWD Indel Alleles:

[0146] Sequencing of Initial GWD PCR Products - PCR products were sequenced at Beckman Coulter Genomics (36 Cherry Hill Dr, Danvers, MA

01923) using the same primers used for amplification (ZmGWDmega-2 or SbGWDmega-2). Sequencing allowed differentiation between three different genetic outcomes for the GWD locus, wild type, homozygous mutant, and heterozygous mutant. Wild type had no mutation within the 204 or 208 bp GWD PCR fragment, homozygous carried an indel mutation, and heterozygous carried an unresolvable sequence region (indicating at least one indel) of the GWD PCR fragment.

[0147] Cloning and Sequencing of Individual GWD Alleles. To initiate cloning, GWD was amplified with PCR using the same primer sets as above (ZmGWDmega-2 or SbGWDmega-2) from DNA derived from heterozygous plants to characterize the individual GWD alleles. PCR amplification was confirmed by running 8 gl of PCR product on agarose gels as described above and the remaining 22 gl of PCR reaction was purified with a Qiagen PCR Purification Kit (28104; Qiagen, MD, USA) and eluted in 30 gl of Elution Buffer (EB).

[0148] Purified PCR products were cloned using a TOPO@ TA Cloning Kit with One Shot TOP10 Competent E. col (K4500-01; Life Technologies) according to the protocol. For cloning procedure, 4 gl of purified PCR product was used for the ligation, ligations were incubated on ice for at least 10 min, and 50 gl of each transformation reaction was plated on LB carbenicillin (50 gg/ml) X-gal plates.

[0149] Eight E. coli colonies from each reaction were picked using a sterile pipet tip and transferred into 20 gl of sterile liquid LB with carbenicillin. GWD was then amplified by PCR using the same primer sets as above (ZmGWDmega-2 or SbGWDmega-2) and 2 l of each diluted E. coli clone culture. PCR products were confirmed and sent for sequencing as described above.

[0150] Table 4 describes ZmGWD meganuclease events zygosity, mutation types and locations. In Table 4, ZmGWD mutations were numbered 1-28. The wild type (WT) plant refers to two GWD wild type alleles. Hemizygous event refers to one GWD mutant allele and one GWD wild type allele. Heterozygous event refers to two different GWD mutant alleles. Homozygous event refers to two identical GWD mutant alleles. Table 4 ZmGWD Meganuclease Event Zygosity and Mutations

Construct/ Zygosity Mutation Mutation Mutation Mutation Event Locus#(1) Type Locus# Type Locus (2) Locus# #(1) (2)

4715 5 Hemi M18 1bp del 4715_6 WT GWD wild type 4715 11 Hemi M17 10bp del 4715 13 Hemi M16 24bp del 4715 14 Hemi M17 10bp del 4715 15 Hemi M18 1bp del 4715_18 WT GWD wild type Homo ortwo 4715_20 Heeoor mutant alleles 4715 25 Hemi M17 10bp del 4715 28 Hemi M27 16 bp del 4716 1 Hemi M1 4bp sub 4716 2 Homo M15 40bp del 4716 3 Hetero M9 15bp del M6 1bp del 4716 4 Hetero M11 17bp del M12 38bp del 4716 5 Hetero M7 4bp del M11 17bp del 4716 6 Hetero M4 9bp del M14 25bp del 4716 7 Hemi M1 4bp sub 4716 8 Homo M11 17bp del 4716_9 WT GWD wild type 4716 10 Hetero M11 17bp del M10 1bp ins 4716 11 Hetero M11 17bp del M10 1bp ins 4716 12 Hetero M3 15bp del M8 6bp del 4716 13 Homo M14 25bp del 4716 14 Homo M13 36bp del 4716 15 Hetero M11 17bp del M12 38bp del 4716 18 Hemi M20 1bp del 4716_20 WT GWD wild type 4716 22 Hetero M5 211bp ins M11 17bp del 4716 23 Homo M15 40bp del

Construct/ Zygosity Mutation Mutation Mutation Mutation Event Locus#(1) Type Locus# Type Locus (2) Locus# #(1) (2)

4716 24 Hetero M2 6bp del M14 25bp del 4716 25 Hetero M10 1 bp ins M28 27bp del 4716 26 Hetero M11 17bp del M12 38bp del 4716 27 Hetero M11 17bp del M10 1bp ins 4716 151 Hemi M10 1 bp ins 8bp 4716_152 Hetero M23 del+l5bp M22 10bp del M23 ins (7bp ins) 4716_153 WT GWD _ _ wild type 8bp 4716_154 Hetero M22 1M23 delsl5bp 47614Heel 22 1bp del M3 ins (7bp ins) 4716 155 Homo M4 9bp del 4716 157 Hemi M24 1 bp del 4716 158 Hemi M20 1bp del 4716 159 Homo M20 1bp del 4716 160 Hemi M10 1 bp ins 33bp del 4716_161 Hetero M4 9bp del M21 del± 17bp ins) 4716 162 Hetero M20 1bp del M19 4bp del 4716 163 Hemi M26 211bp ins 4716 164 Hemi M20 1bp del 4716 165 Homo M4 9bp del 4716 166 Homo M4 9bp del 4716 167 Hetero M13 36bp del M25 2bp del 4716 201 Homo M29 2bp del

[0151] DNA sequences for each clone were compared to wild type (WT) GWD using Vector NTI Advance (Version 11.5; Life Technologies). DNA sequences for wild type ZmGWD and SbGWD and transgenic events were compared and shown in the following files: ZmGWD meganuclease mutant DNA sequence alignments; ZmGWD meganuclease mutant protein sequence alignments; SbGWD meganuclease mutant DNA sequence alignments; SbGWD meganuclease mutant protein sequence alignments.

[0152] As shown below, an alignment of the sequences from three PCR products demonstrates insertions and deletions that distinguish maize mutants M5 and M26 from wild type sequence ZmGWD Exon24.

[0153] CLUSTAL 0 (1.2.1) multiple sequence alignment for ZmGWD for maize mutants M5 and M26:

ZmGWDExon24 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT60 M5 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT60 M26 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT60

ZmGWDexon24

TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG120 M5 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG120 M26 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG120

ZmGWDexon24 AGAGGAAGAAATACCAGATGGAGTA-----------------------------------145 M5 AGAGGAAGAAATACCAGATGGAGTAGTTGCAGAATTATTGAATTCTTTCATAATTGAACT180 M26 AGAGGAAGAAATACCAGATGGAGCAGTGTGCTCGGGTACAGCTTCTTATTTCAATGTCTC180

ZmGWDexon24 ------------------------------------------------------------ 145 M5 CTATGATGATGCTTTACTT--GATTGTATTATATTGATGCTCAATCATATATTGATGATT238 M26 CAGTGGGCGTCTTACCTCTATGTTTGTGTTTTTTT-TTAAGTGCAGAAATAGAGAAAGTT239

ZmGWDexon24 ------------------------------------------------------------ 145 M5 GTTGGAACTTGCTCTCCGATGCAAGGTGATCCAACGGGGGTGTGTCGCAACGTAAACAGG298 M26 CTTGCAAATATCTACTCTATGAAAAGGACAGCTATTTGGAAATA------TGTGAACAGA293

ZmGWDexon24 ------------------------------------------------------------ 145 M5 GTTTTCG-CACGAGATGGCAATAGCTCTGT-T ---AACCTAGCCTCTCACGGGCACTGTG353 M26 ACTATCCCCAGTTGCTGGGAAAAACCAAGAAGAAAGTTCCTTCAAATATCTACTCCATGA353

ZmGWDexon24 --- GTTGGTGTAATTACACCTGATATGCCAGATGTTCTGTCTCATGTGTCAGTCCGAGCA202 M5 CGGGGGTATTTAATTACACCTGATATGCCAGATGTTCTGTCTCATGTGTCAGTCCGAGCA413 M26 CGACAAGTGTCTATTACACCTGATATGCCAGATGTTCTGTCTCATGTGTCAGTCCGAGCA413

ZmGWDexon24 AGGAATAGCAAG 214 (SEQ ID NO: 182) M5 AGGAATAGCAAG 425 (SEQ ID NO: 34) M26 AGGAATAGCAAG 425 (SEQ ID NO: 31)

[0154] The below alignment of the sequences from twenty eight PCR products demonstrates modifications, such as deletions and insertions, that distinguish maize mutants M1 - M4, M6 - M25 and M27 - M29 from the wild type sequence ZmGWDExon24 (nt 3030 3243 of SEQ ID NO: 1 (ZmGWD)):

CLUSTAL 0(1.2.1) multiple sequence alignment for maize mutants M1- M4, M6 - M25 and M27 - M29: ZmGWDexon24 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M1 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M2 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M3 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M4 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M6 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M7 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M8 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M9 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M10 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M11 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M12 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M13 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M14 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M15 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M16 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M17 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M18 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M19 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M20 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M21 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M22 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M23 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M24 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M25 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M27 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M28 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 M29 TTGGCAGGTTATAAGCCCGGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60

ZmGWDexon24 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M1 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M2 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M3 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M4 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M6 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M7 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M8 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M9 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M10 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M11 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M12 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M13 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M14 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M15 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTAATTACAC 120 M16 TGCTGTCCAGAACAAATCTTATGATAAACCAAGGGAGAGGA------------------- 101 M17 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAGGG---------- 110 M18 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAA-GAGTGTCAAGGG 119 M19 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M20 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M21 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAAAT 120 M22 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M23 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M24 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M25 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M27 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAGGGAGAGAT---- 116 M28 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120 M29 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGAGTGTCAAGGG 120

ZmGWDexon24 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGTA-------ATTACACCTGATATGCCAG 173 M1 AGAGGAAGAAATACCAGATGGAGTAGTTGGAAGA-------AATACACCTGATATGCCAG 173 M2 AGAGGAAGAAATACCAGATGGAGTAGTTGTT-------------ACACCTGATATGCCAG 167 M3 AGAGGAAGAAATACCAGATGGAGCACCT----------------------GATATGCCAG 158 M4 AGAGGAAGAAATACCAGATGGAGTAA----------------TTACACCTGATATGCCAG 164 M6 AGAGGAAGAAATACCAGATGGAGTAGTTGGTAA--------ATTACACCTGATATGCCAG 172 M7 AGAGGAAGAAATACCAGATGGAGTAGTTGGT-----------TTACACCTGATATGCCAG 169 M8 AGAGGAAGAAATACCAGATGGAGTAGTTGGT-------------ATGCCAGATATGCCAG 167 M9 AGAGGAAGAAATACCAGATGGAGTAGTTG----------------------GTATGCCAG 158 M10 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGTAA------ATTACACCTGATATGCCAG 174 M11 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGTCAG------------------------ 156 M12 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGTCAGTCCGAGCAAGGAATAGCAAG---- 176 M13 AGAGGAAGAAATACCAGATGTTCTGTCTCATGTGTC------------------------ 156 M14 AGAGGAAGAATTACA--------------------------------CCTGATATGCCAG 148 M15 CTGATATGC------------------------------------------------CAG 132 M16 ------ AGAAATACCAGATGGAGTAGTTGGT-------GTAATTACACCTGATATGCCAG 148 M17 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGT-A------ATTACACCTGATATGCCAG 163 M18 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGTA-------ATTACACCTGATATGCCAG 172 M19 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGT--------ATTACACCTGATATGCCAG 172 M20 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGT--------ATTACACCTGATATGCCAG 172 M21 CTTATGATAAACC----------------------------------------ATGCCAG 140 M22 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGTGA-----------------TATGCCAG 163 M23 AGAGGAAGAAATACCAGATGGAGTAGTTGGCAAAGATAAACCTTGCACCTGATATGCCAG 180 M24 AGAGGAAGAAATACCAGATGGAGTAGTTGGTGA--------ATTACACCTGATATGCCAG 172 M25 AGAGGAAGAAATACCAGATGGAGTAGTTGG---TA------ATTACACCTGATATGCCAG 171 M27 ------------ ACCAGATGGAGTAGTTGG-------TGTAATTACACCTGATATGCCAG 157

M28 AGAGGAAGAAACACCTGATA----------------------------------TGCCAG 146 M29 AGAGGAAGAAATACCAGATGGAGTAGTTGG---TA------ATTACACCTGATATGCCAG 171

ZmGWDexon24 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 214 (SEQ ID NO: 182) M1 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 214 (SEQ ID NO: 16) M2 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 208 (SEQ ID NO: 35) M3 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 199 (SEQ ID NO: 19) M4 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 205 (SEQ ID NO: 29) M6 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 213 (SEQ ID NO: 37) M7 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 210 (SEQ ID NO: 39) M8 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 208 (SEQ ID NO: 20) M9 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 199 (SEQ ID NO: 38) M10 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 215 (SEQ ID NO: 18) M11 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 197 (SEQ ID NO: 17) M12 ----------------------------------------- 176(SEQ ID NO: 23) M13 ------------------- AGTCCGAGCAAGGAATAGCAAG 178 (SEQ ID NO: 22) M14 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 189 (SEQ ID NO: 21) M15 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 173 (SEQ ID NO: 33) M16 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 189 (SEQ ID NO: 12) M17 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 204 (SEQ ID NO: 13) M18 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 213 (SEQ ID NO: 14) M19 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 213 (SEQ ID NO: 30) M20 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 213 (SEQ ID NO: 27) M21 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 181(SEQ ID NO: 28) M22 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 204 (SEQ ID NO: 24) M23 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 221(SEQ ID NO: 25) M24 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 213 (SEQ ID NO: 26) M25 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 212 (SEQ ID NO: 32) M27 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 198 (SEQ ID NO: 15) M28 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 187 (SEQ ID NO: 36) M29 ATGTTCTGTCTCATGTGTCAGTCCGAGCAAGGAATAGCAAG 212 (SEQ ID NO: 40)

[0155] The amino acid sequences of GWD from twenty nine transgenic maize events and a wild type plant were analyzed and showed deletions and insertions in the wild type ZmGW (SEQ ID NO: 185) protein in the positions of amino acids 1040 - 1120 that distinguish this protein from maize mutants ZmGWD_Ml (SEQ ID NO: 45), ZmGWDM2 (SEQ ID NO: 46), ZmGWDM3 (SEQ ID NO: 47), ZmGWDM4 (SEQ ID NO: 48), ZmGWDM5 (SEQ ID NO: 49), ZmGWDM6 (SEQ ID NO: 50), ZmGWDM7 (SEQ ID NO: 51), ZmGWDM8 (SEQ ID NO: 52), ZmGWDM9 (SEQ ID NO: 53), ZmGWDM10 (SEQ ID NO: 54), ZmGWDM11 (SEQ ID NO: 55), ZmGWDM12 (SEQ ID NO: 56), ZmGWDM13 (SEQ ID NO: 57), ZmGWDM14 (SEQ ID NO: 58), ZmGWD_M15 (SEQ ID NO: 59), ZmGWDM16 (SEQ ID NO: 60), ZmGWDM17 (SEQ ID NO: 61), ZmGWDM18 (SEQ ID NO: 62), ZmGWDM19 (SEQ ID NO: 63), ZmGWDM20 (SEQ ID NO: 4),

ZmGWDM21 (SEQ ID NO: 65), ZmGWDM22 (SEQ ID NO: 66), ZmGWDM23 (SEQ ID NO: 67), ZmGWDM24 (SEQ ID NO: 68), ZmGWDM25 (SEQ ID NO: 69), ZmGWDM26 (SEQ ID NO: 70), ZmGWDM27 (SEQ ID NO: 71), ZmGWDM28 (SEQ ID NO: 72), and ZmGWDM29 (SEQ ID NO: 73).

[0156] CLUSTAL 0(1.2.1) multiple sequence alignment of ZmGWD (SEQ ID NO: 43) amino acids 1040 -1120:

ZmGWD PTILVAKSVKGEEEIPDGVVGVITPDMPD----------VLS---HV---------SVR ZmGWD_M1 PTILVAKSVKGEEEIPDGVVGRNTPDMPD----------VLS---HV---------SVR ZmGWDM2 PTILVAKSVKGEEEIPDGV--VVTPDMPD----------VLS---HV---------SVR ZmGWDM3 PTILVAKSVKGEEEIPDGA-----PDMPD----------VLS---HV---------SVR ZmGWDM4 PTILVAKSVKGEEEIPDG---VITPDMPD----------VLS---HV---------SVR ZmGWDM5 PTILVAKSVKGEEEIPDGVVAELLNSFIIELYDDALLDCIILMLNHILMIVGTCSPMQGD ZmGWD_M6 PTILVAKSVKGEEEIPDGVVGKLHLICQM----------FCL---MCQSEQGIARYCLRP ZmGWDM7 PTILVAKSVKGEEEIPDGVVG-LHLICQM----------FCL---MCQSEQGIARYCLRP ZmGWDM8 PTILVAKSVKGEEEIPDGV--VGMPDMPD----------VLS---HV---------SVR ZmGWDM9 PTILVAKSVKGEEEIPDGV-----VGMPD----------VLS---HV---------SVR ZmGWD_M10 PTILVAKSVKGEEEIPDGVVGVNYT*--------------------------------- ZmGWD_Mll PTILVAKSVKGEEEIPDGVVGVRCSVSCVSPSKE*------------------------ ZmGWD_M12 PTILVAKSVKGEEEIPDGVVGVSPSK-------E*------------------------ ZmGWD_M13 PTILVAKSVKGEEEI------------PD----------VLS---HV---------SVR ZmGWD_Ml4 PTILVAKSVKGEEE--------LHLICQM----------FCL---MCQSEQGIARYCLRP ZmGWD_M15 PTILVAKSNYT*----------------------------------------------- ZmGWD_M16 P--------RERKKYQME*---------------------------------------- ZmGWD_M17 PTIL---VARERKKYQME*---------------------------------------- ZmGWD_M18 PTILVARVSRERKKYQME*---------------------------------------- ZmGWD_M19 PTILVAKSVKGEEEIPDGVVG-VHLICQM----------FCL---MCQSEQGIARYCLRP ZmGWDM20 PTILVAKSVKGEEEIPDGVVGVLHLICQM----------FCL---MCQSEQGIARYCLRP ZmGWD M21 PTILVAKSVKIL*---------------------------------------------- ZmGWDM22 PTILVAKSVKGEEEIPDGVVG---VICQM----------FCL---MCQSEQGIARYCLRP ZmGWDM23 PTILVAKSVKGEEEIPDGVVGKDKPCT*------------------------------- ZmGWDM24 PTILVAKSVKGEEEIPDGVVGELHLICQM----------FCL---MCQSEQGIARYCLRP ZmGWDM25 PTILVAKSVKGEEEIPDGVVGNYT*---------------------------------- ZmGWD_M26 PTILVAKSVKGEEEIPDGAVCSGTASYFNVSS-GRLTSMFVFFFK-CRNRESSCKYLLYE ZmGWDM27 PTILVA-----RERYQME*---------------------------------------- ZmGWDM28 PTILVAKSVKGEEE---------TPDMPD----------VLS---HV---------SVR ZmGWDM29 PTILVAKSVKGEEEIPDGVVGNYT*----------------------------------

ZmGWD ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE(SEQ ID NO: 185) ZmGWD_M1 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 45) ZmGWDM2 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 46) ZmGWDM3 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 47) ZmGWDM4 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 48) ZmGWDM5 --- PTGVCRNVNRVFARDGNSSVNLASHGHCAGVFNYT*----------- (SEQ ID NO: 49) ZmGWD_M6 VL-TTPLYLNLKDMIRNCFPSSLLLQI*------------------------- (SEQ ID NO: 50) ZmGWDM7 VL-TTPLYLNLKDMIRNCFPSSLLLQI*------------------------- (SEQ ID NO: 51) ZmGWDM8 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 52) ZmGWDM9 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 53) ZmGWD_M10 --------------------------------------------------- (SEQ ID NO: 54) ZmGWD_Mll --------------------------------------------------- (SEQ ID NO: 55) ZmGWD_M12 --------------------------------------------------- (SEQ ID NO: 56) ZmGWD_M13 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 57) ZmGWD_M14 VL-TTPLYLNLKDMIRNCFPSSLLLQI*------------------------- (SEQ ID NO: 58) ZmGWD_M15 --------------------------------------------------- (SEQ ID NO: 59) ZmGWD_M16 --------------------------------------------------- (SEQ ID NO: 60) ZmGWDMl7 --------------------------------------------------- (SEQ ID NO: 61)

ZmGWD_M18 ------------------------------------------------------ (SEQ ID NO: 62) ZmGWDM19 VL-TTPLYLNLKDMIRNCFPSSLLLQI*------------------------ (SEQ ID NO: 63) ZmGWDM20 VL-TTPLYLNLKDMIRNCFPSSLLLQI*------------------------ (SEQ ID NO: 64) ZmGWD M21 ------------------------------------------------------ (SEQ ID NO: 65) ZmGWDM22 VL-TTPLYLNLKDMIRNCFPSSLLLQI*------------------------ (SEQ ID NO: 66) ZmGWDM23 ------------------------------------------------------ (SEQ ID NO: 67) ZmGWDM24 VL-TTPLYLNLKDMIRNCFPSSLLLQI*------------------------ (SEQ ID NO: 68) ZmGWDM25 ------------------------------------------------------ (SEQ ID NO: 69) ZmGWDM26 KDSYLEICEQNYPQLLGKTKK--K-----VPSNIYSMTTSV---YYT*--- (SEQ ID NO: 70) ZmGWDM27 ------------------------------------------------------ (SEQ ID NO: 71) ZmGWDM28 ------- ARNSKVLFATCFDHTTLSELEGYDQKLFSFKPTSADITYREITE (SEQ ID NO: 72) ZmGWDM29 ------------------------------------------------------ (SEQ ID NO: 73)

[0157] For Sorghum bicolor, two meganuclease constructs were used to create GWD mutations, 4715 and 4716. First generation (TO) transformed plants could result in homozygous GWD mutants, hemizygous (WT

+ mutation) GWD mutants, or heterozygous (2 different mutations; e.g., allele 1 + allele 2) GWD mutants. The following abbreviations were used: del=deletion; ins=insertion; sub=substitution; SbGWD CDS is wild type sequence. For example, the sequence name "Sb47151 (WT+ins)" has the following meaning: Sb4715 is the construct in Sorghum bicolor; 1 is the transgenic event: WT+ins indicates that TO event 4715_1 was hemizygous for a GWD mutation, carrying a WT GWD allele and an insertion (ins) GWD allele. The same construct was used for transformation of Zea mays (Zm).

[0158] CLUSTAL nucleic acid alignment between Sorghum bicolor (Sb) GWD sequence and Sorghum bicolor GWD mutants Sb475_1 (WT+ins) and Sb47152 (WT+del) showed alterations in the sequences of the mutants compared to wild type SbGWD sequence. The SbGWDexon24 sequence is positioned within nt 3030 - 3243 the SbGWD coding sequence (SEQ ID NO: 2). The sequence of Sb4751 (WT+ins) includes a 13 nucleotide insertion in the position 3139-3149 of SbGWD and a nucleotide substitution in the position 3133-3136 of SbGWD.

[0159] As shown below, an alignment of the sequences from the three PCR products demonstrates insertions and deletions that differentiate Sb47151 (WT+ins) and Sb47152 (WT+del) and SbGWD exon 24 regions.

CLUSTAL 0(1.2.1) multiple sequence alignment: SbGWDExon24 TTGGCAGGTTATAAGCCCAGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60

Sb4715_1 (WT+ins) TTGGCAGGTTATAAGCCCAGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60 Sb4715 2 (WT+del) TTGGCAGGTTATAAGCCCAGTTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGTTACT 60

SbWD Exon24 TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGA---------- 110 b47151 (WT+ins) TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGAGTAGTTGGTGTAGTT 120 Sb4715 2 (WT+del) TGCTGTCCAGAACAAATCTTATGATAAACCAACCATCCTTGTGGCAAAGA---------- 110

SbD Exon24 --- GTGTCAAGGGAGAGGAAAAATACCAGATGGAGTAGTTGGTGTAATTACACCTGATA 167 Sb47151 (WT+ins) GGTGTATCAAGGGAGAGGAAGAAATACCAGATGGAGTAGTTGGTGTAATTACACCTGATA 180 Sb4715 2 (WT+del) --- GTGTCAAAAATACCAGATGGAG ------------ TTACACCTGATA 155

SbGWD Exon24 TGCCAATGTTCT ATGTGTCAGTCCAG AAGGAATAGCAAG214(SEQ ID NO: 183) Sb47151 (WT+ins) TCATGTTCTG T ATGTGTCA GTCAGCAAGGAATAGCAAG227(SEQ ID NO: 106) Sb4715 2 (WT+del) TGCCAATGTTCTGT ATGTGTCAGT AGCAAGGAATAGCAAG202(SEQ ID NO: 107)

[0160] The prediction of the meganuclease mutant protein amino acid sequences were made using the sequence wild type SbGWD (SEQ ID NO: 44).

[0161] Example 5. Mutant plants accumulate elevated levels of green tissue starch

[0162] Starch was assayed in the first generation (TO) transformed maize and sorghum GWD meganuclease plants. Tissues were collected, dried and milled to a fine powder. The starch content was determined by standard methods (Smith AM and Zeeman SC, Quantification of starch in plant tissues (2006) Nat Protocols 1: 11342-1345, which is incorporated herein by reference as if fully set forth). A total starch content was assayed by adapting the protocol from Megazyme International Ireland Ltd. (Megazyme kit and reagents; cat. #K-TSTA). Briefly, 85C and 50 0C heat blocks were set up. From 5 to 15 mg of dry milled tissue were placed into the 1.5 ml boil-proof microcentrifuge tubes. One milliliter of 70% ethanol was added to each tube and samples were vortexed and pelleted. Four hundred microliters of solution 1 was added to each sample. Solution 1 included 1ml of the thermostable a amylase and 29 ml of 100 mM sodium acetate buffer, pH5.0. The samples were re-suspended and vortexed. Samples were incubated for 12 minutes at 850 C for 12 minutes, and cooled for 5 minutes at room temperature. Three hundred microliters of the GOPOD reagent (Megazyme kit, cat. #K-TST) was preloaded into each well of the flat bottom 96 well assay plate. Ten microliters of samples were added to each well and compared to 1 gL, 5 gL, 10 gL, and 20 gL of glucose standard (1mg/ml), which were also added to their respective wells.

The plate was incubated at 50°C for 20 min. Absorbance was assessed at 510 nm. Referring to FIG. 4, elevated starch is shown for mutants 4715_20 (two mutant alleles), 47167 (Ml), 4716_1 (Ml), 471618 (M20), 471612 (M3/M8), 471623 (M15), 471628 (not characterized), 47163 (M9/M6), 4716_22

(M5/M11), 4716_24 (M2/M14), 2471625 (M1O/M28), 4716_15 (M11/M12), 47165 (M7/M11), 47166 (M4/M14), 4716_27 (M11/M10), 47164 (M11/M12), 471626 (M11/M12), 4716_2 (M15), 4716_13 (M14), 471613 (M14), 4716_11 M11/M10), 47168 (M11), 471610 (M11/M10), and 471614 (M13) compared to wild type plants WT_195, WT_18 and WT_6. Many of the homozygous and heterozygous events exhibited greater than 20% starch increase by weight in leaves. Based on a weighted average of starch accumulation in different tissues, we estimated total plant starch (not including grain) to be approximately 10% (weight/weight).

[0163] The levels were significantly higher was observed previously in maize using RNA interference technology, despite the low transcript abundance measured in those experiments. This was a surprising result as it was anticipated that RNAi based silencing would be dominant in the plant and have the same effect as a gene deletion or knock-out strategy.

[0164] FIG. 5 illustrates green tissue starch for selected hemizygous, homozygous, and heterozygous events. FIG. 5 shows mutation type and zygosity of transgenic corn events M17 (471514), M18 (471515), M1 (47161), M20 (471618), M3/M12 (471612), M9 (47163), M7/M11 (47165), M4/M14 (47166), M11/M12 (47164), M15 (47162), M14 (471613), M11 (47168), M11/M1O (471610) and M13 (471614) have elevated levels of starch compared to non-transgenic control WT (47169). It was observed that several events (471613 (M14), 471515 (M11/M12) and 47166 (M4/M14) have greater than average biomass.

[0165] Example 6. GWD knock-out (GWDko) cobs have increased starch levels

[0166] TO GWDko and wild type (wt) maize mutant lines were selfed, developed to maturity, dried, and cross-sectioned for staining. Cob sections were stained with Lugol's solution (5% KI) for 4 min and destained with H 2 0 overnight.

[0167] Mutant events 4716_13, 4716_26, 4716_167, 4716_164, 4716_9, and 4716-153 were analyzed for starch content. The results are shown in Table 5.

Table 5 Starch Content in Mutant Lines ConstructEvent Zygosity Green Tissue Stover Starch Starch

4716_13 Homo 229.7 38.5

4716_26 Hetero 220.7 35.3

4716_167 Hetero 112.0 31.7

4716_164 Hemi 30.5 4.0

4716_9 WT 18.8 8.4

4716_153 WT 5.7 1.2

Referring to Table 5 and FIG. 6, it was shown that homozygous (471613) and heterozygous (4716_26 and 4716_167) cobs had increased starch staining compared with hemizygous (4716_164) and wild type (47169 and 4716153) cobs.

[0168] Example 7. Construction of CRISPRICas maize transformation vectors

[0169] For constructing Cas9 expression cassette, the S. pyogenes Cas9 protein sequence containing N- and C-terminal At nuclear localization sequences (NLS) as well the 3xFLAG sequence positioned immediately after the first ATG codon (Jiang et al., 2013) (SEQ ID NO: 74) was chosen for expression in maize. The sequence of S. pyogenes Cas9 containing two SV40 nuclear localization sequences (shown in bold letters) and 3xFLAG sequence at N-terminal part (underlined sequence) for expression in maize is as follows: MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFK VLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF

LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWM TRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKP AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDT TIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDRPKKKRKVGG (SEQ ID NO: 74)

[0170] The sequence was back translated and maize codon optimized to produce ZmCas9 (SEQ ID NO: 75). The optimized ZmCas9 nucleotide sequence was synthesized by Genscript. The ZmCas9 was cloned as BamH AvrII fragment between maize ubiquitin 1 promoter (ZmUbi1P) and nopaline synthase transcriptional terminator (NosT) sequences into pAG4500 to produce pAG4800.

[0171] The work on construction of sgRNA cassettes involved: 1) identification and isolation of a maize RNA Polymerase III promoter to drive expression of sgRNA; 2) design and synthesis of sgRNA scaffold; and 3) selection of a target gene and 20 bp specific sequences within this gene for guiding Cas9 endonuclease to its target sites.

[0172] The first description of a maize sequence encoding U3 small nuclear RNA (U3snRNA) was reported by Leader et al. in 1994, who isolated MzU3.8 gene (Genebank Accession No. Z29641) (SEQ ID NO: 76) from a maize genomic DNA library and demonstrated that the MzU3.8 U3snRNA is expressed in maize protoplasts. Using BLASTN algorithm and Z29641 sequence to search the Maize Genetics and Genomics Database (http://www.maizegdb.org/)we identified a homologous sequence of maize U3 that was labeled as ZmU3 (SEQ ID NO: 77).

[0173] The ZmU3 is localized on the maize chromosome 8 and is contained within a sequence with nucleotide coordinates 163620300

163621800. The CLUSTAL 2.1 multiple nucleotide sequence alignment of putative promoter regions of MzU3.8 (SEQ ID NO: 78) and ZmU3 (SEQ ID NO: 79) demonstrated 93.8% identity between the two sequences. CLUSTAL 2.1 multiple sequence alignment: MzU3.8 GAATTCCATCTAAGTATCTTGGTAAAGCATGGATTAATTTGGATGCTCACTTCAGGTCTA 60 ZmU3 GAATTCCATCTAAGTATGTTGGTAAAGCATGGATTAATTTGGATGCCCACTTCAGGTCTA 60 ***************** **************************** *************

MzU3.8 TGCAGCTCCGGTGCCTTGTGATTGTGAGTTGTGACCGATGCTCATGCTATTTTGCATTTC 120 ZmU3 TGCAGCTCCGGTGCCTTGTGATTGTGAGTTGTGACCGATGCTCATGCTATTCTGCATTTC 120 *************************************************** ********

MzU3.8 TGCGATGTATGATGCTAGTAGATCTTCAAAACTAACAGCGCATGCCATCATCATCCACTG 180 ZmU3 TGCGATGTATGTAGCTAGTAGATCTTCAAAACTAACACCGCATGCCATCATCATCCACTG 180 *********** ************************ **********************

MzU3.8 CTTGATTTTAGTCTCACCGCTGGCCAAAAATGTGATGATGCCAGAAACCTCAACTACCTT 240 ZmU3 CTTGATTTTAGTCTCACCGCTGGCCAAAAATGTGATGATGCCAGAAACCTCAACTACCTT 240

MzU3.8 GAATCAACACGGGCCCAGCAGTGTGATGACGACAGAAACCAAAAAAAAATGAGCCAATAG 300 ZmU3 GAATCAACACGGGCCCAACAGTGTGATGACGACAGAAAC-AAAAAAAAATGAGCCAATAG 299 ***************** ********************* ********************

MzU3.8 TTCAGAAGGAGGCACTATGCAGAAACTACATTTCTGAAGGTGACTAAAAGGTGAGCGTAG 360 ZmU3 TTCAGAAGGAGGCACTATGCAGAAACTACATTTCTGAAGGTGACTAAAAGGTGAGCGTAG 359

MzU3.8 AGTGTACTTACTAGTAGTTTAGCCACCATTACCCAAATGCTTTCGAGCTTGTATTAAGAC 420 ZmU3 AGTGTAATTACTAGTAGTTTAGCCACCATTACCCAAATGCTTTCGAGCTTGTATTAAGAT 419 ***** ****************************************************

MzU3.8 TTCCTAAGCTGAGCATCATCACTGATCTGCAGG--AGGGTCGCTTCGCTGCCAAGATCAA 478 ZmU3 TTCCTAAGCTGAGCATCATCACTGATCTGCAGGCCACCCTCGCTTCGCTGCCAAGATCAA 479 ********************************* * *********************

MzU3.8 CAGCAACCATGTGGCGGCAACATCCAGCATTGCACATGGGCTAAAGATTGAGCTCTGTGC 538 ZmU3 CAGCAACCATGTGGCGGCAACATCCAGCATTGCACATGGGCTAAAGATTGAGCTTTGTGC 539 ****************************************************** *****

MzU3.8 CAAGTGTGAGCTGCAACCATCTAGGGATCAGCTGAGTTTATCAGTCTTTCCTTTTTTTCA 598 ZmU3 C---------------TCGTCTAGGGATCAGCTGAGGTTATCAGTCTTTCCTTTTTTTCA 584 * * ***************** ***********************

MzU3.8 TTCTGGTGAGGCATCAAGCTACTACTGCCTCGATCGGTTGGTGGAGGACCTGAAGCCCAC 658 ZmU3 TCCAGGTGAGGCATCAAGCTACTACTGCCTCGATTGGCTGGA------CCCGAAGCCCAC 638 * * ****************************** ** **** ** *********

MzU3.8 ATGTAGGATACCAGAATGGACCGACCCAGGACG-------------------------TA 693 ZmU3 ATGTAGGATACCAGAATGGGCCGACCCAGGACGCAGTATGTTGGCCAGTCCCACCGGTTA 698 ******************* ************* **

MzU3.8 GTGCCACCTCGGTTG-TCACACTGCGTAGAAGCCAGCTTAAAAATTTAGCTTTGGTGACT 752 ZmU3 GTGCCATCTCGGTTGCTCACA-TGCGTAGAAGCCAGCTTAAAAATTTAGCTTTGGTAACT 757 ****** ******** ***** ********************************** ***

MzU3.8 CACAGCA 759 (SEQ ID NO: 78) ZmU3 CACAGCA 764 (SEQ ID NO: 79)

[0174] Using a PCR approach with the forward primer ob2297 (SEQ ID NO: 80) and reverse primer ob2299 (SEQ ID NO: 81), the 758 bp ZmU3 promoter (ZmU3P1) (SEQ ID NO: 82) was subsequently isolated from maize genomic DNA of the maize line AxB. The forward primer ob2297 included AsiSI restriction site at its 5' end to facilitate cloning an sgRNA cassette into a pAG4500-based vector. Similarly, using a forward primer ob2343 (SEQ ID NO: 83), which contained AsiSIrestriction site at its 5' end, a shorter 398 bp version of the maize U3 promoter (ZmU3P2) (SEQ ID NO: 84) was isolated for testing efficiency of a truncated maize U3 promoter. An additional variant of the ZmU3P2 was amplified with the forward primer ob2351 (SEQ ID NO: 85) that has the Swal restriction enzyme site at its 5' end. Furthermore, a 308 bp control promoter fragment ZmU3.8P (SEQ ID NO: 86) was PCR synthesized using long primers that were designed on a MzU3.8 sequence published by Leader et al. (1994), which was shown to be expressed in maize protoplasts. The ZmU3.8P sequence also included AsiSIrestriction site at the 5' end. All amplified promoter variants were cloned into pCR-BluntII-TOPO vector (Life Technologies) and their integrity was confirmed by complete sequencing.

[0175] The sgRNA scaffold design herein is based on the published organization of an sgRNA chimera (Larson et al., 2013) and includes a 42 bp Cas9 handle hairpin (SEQ ID NO: 87) followed by a 41 bp S. pyogenes terminator (SEQ ID NO: 88). In order to improve efficiency of transcriptional termination in maize, a 37 bp putative transcription terminator sequence ZmU3T (SEQ ID NO: 89) was isolated from ZmU3 snRNA (SEQ ID NO: 77) and fused downstream of the S. pyogenes terminator (SEQ ID NO: 88). The 120 bp sgRNA scaffold (SEQ ID NO: 90) was synthesized by PCR using long primers and KOD Xtreme DNA Polymerase with the proof reading activity. The SnaBI or AscI restriction sites were added at the 3' end of the two PCR amplified sgRNA backbone DNA fragments to facilitate further cloning. The sgRNA scaffold DNA fragments synthesized in this way were cloned into pCR BluntIl-TOPO vector and sequence validated.

[0176] For testing efficiency of the CRISPR/Cas system in maize, a maize gene encoding GWD was selected for the initial targeted modifications.

[0177] The maize GWD gene has been annotated earlier and was screened for the presence of AN1 9 NGG target sequences on both sense and antisense DNA strands. The 5' end "A" in AN1 9 NGG sequence represents a conserved "Adenine" nucleotide at the transcription start of the U3 RNA Polymerase III promoter, the 3' end positioned "NGG" sequence corresponds to the required for CRISPR/Cas system activity protospacer-adjacent motif (PAM) sequence. The candidate target sequences identified in exons 1, 24, and 25 as well as in their flanking introns were further screened against Maize GDB in order to eliminate sequences that have multiple identity hits within the maize genome. This work has been done to minimize chances for off-target activity of the CRISPR/Cas system. In this analysis, only the seed sequence (12 bp) of the target sequence plus two adjacent PAM nucleotides were used in BLASTN program as it was proposed by Larson et al. (2013). Exon 1 was selected for producing an almost complete GWD knockout, while exons 24 and 25 were chosen to generate GWD variants lacking an active site that is encoded by exon 24. A list of the final 19 bp GWD target sequences (SEQ ID NOS: 131-134), which were identified for sgRNA development, is compiled in Table 6.

Table 6 GWD Gene Target Sequences With Their Corresponding SEQ ID NOS

SEQ ID Sequence Sequence GWD strand NO name 91 GWDela GGCATGAGGTGCTTACGTC antisense 92 GWDe24b CATAACCTGATACTTCAAC antisense 93 GWDe24c TCTGGCTCCTGCTATCAGT sense 94 GWDe25a TCTGCAGAAGTAGGCTTGA antisense

[0178] Each of the three variants of the maize U3 promoter, selected GWD target sequences and sgRNA backbone were assembled together by the means of fusion PCR using KOD Xtreme DNA Polymerase to construct six sgRNA expression cassettes (SEQ ID NOS: 135-140). The PCR-amplified fragments were cloned into pCR-BluntII-TOPO vector and the integrity of the synthesized sgRNA expression cassettes was verified through sequencing. The list of the PCR-synthesized sgRNA cassettes is presented in Table 7.

Table 7 The Synthesized sgRNA Expression Cassettes with Their Corresponding SEQ ID NOS

SEQ ID sgRNA cassette Flanking restriction sites NO 95 ZmU3P1:sgRNAGWDe24b AsiSI-SnaBI 96 ZmU3P2:sgRNAGWDe24b AsiSI-SnaBI 97 ZmU3.8P:sgRNAGWDe24b AsiSI-SnaBI 98 ZmU3P2:sgRNAGWDe24c AsiSI-SnaBI 99 ZmU3P2:sgRNAGWDe25a Swal-AscI 100 ZmU3P2:sgRNAGWDela AsiSI-SnaBI

[0179] Assembled sgRNA cassettes were subsequently cloned as AsiSI SnaBI fragments into pAG4800 to develop vectors pAG4804-4809 (Table 8).

Table 8 Vectors Developed for Maize CRISPR/Case System with SEQ ID NOS

Plasmid Genetic elements pAG4800 Ubi1P:Cas9 pAG4804 U3P1:sgRNAGWDe24b + UbilP:ZmCas9 pAG4805 U3P2:sgRNA GWDe24b + UbilP:ZmCas9 pAG4806 U3.8P:sgRNA GWDe24b + UbilP:ZmCas9 pAG4807 U3P2:sgRNAGWDe24c + UbilP:ZmCas9 pAG4808 U3P2:sgRNA GWDe1a + UbilP:ZmCas9 pAG4809 U3P2:sgRNA GWDe25a + UbilP:ZmCas9 pAG4817 U3P2:sgRNAGWDe25a + U3P2:sgRNAGWDe24c +

Ubi1P:ZmCas9

[0180] One additional vector pAG4817 containing two sgRNA expression cassettes was constructed by cloning ZmU3P2:sgRNAGWDe25a cassette as Swal-AscI fragment into pAG4807. This vector was developed for complete removal of the GWD exon 24 by targeting Cas9 endonuclease to two different sites that are located 364 bp apart within the maize GWD gene and flank exon 24.

[0181] The maps of the plant transformation vectors pAG4800 and pAG4804, which were constructed for development of CRISP/Cas system for maize are shown on FIGS. 7 - 8. Referring to FIG. 7, the pAG4800 vector includes the Cas9 expression cassette and the PMI expression cassette. The Cas9 expression cassette comprises a nucleotide sequence of ZmCas9 (SEQ ID NO: 75). ZmCas9 is a maize codon optimized sequence of the S. pyogenes gene encoding Cas9 fused to two At NLS at 5' and 3' ends and 3xFLAG sequence immediately after the first ATG codon. Zm Cas9 encodes the S. pyogenes Cas9 protein (SEQ ID NO: 74) containing two At nuclear localization sequences and 3xFLAG sequence at N-terminal part for expression in maize. The Cas9 cassette also includes the Zm Ubil promoter, Zm Ubil intron, mUBQMono leader, and the NosT terminator. The PMI cassette includes the PMI gene, ZmUbil promoter, mUBQMono, ZmKozak leaders and the NosT terminator. Referring to FIG. 8, the pAG4804 vector includes the GWDe24b-sgRNA scaffold cassette, the Cas9 expression cassette, and the PMI expression cassette. The GWDe24b-sgRNA scaffold cassette includes ZmU3P1 promoter, GWDed24 sequence, the sgRNA scaffold and ZmU3T terminator. The Cas9 expression cassette comprises ZmCas9 fused to two At NLS at 5' and 3' ends and 3xFLAG sequence immediately after the first ATG codon. The Cas9 cassette also includes the Zm Ubil promoter, Zm Ubil intron, mUBQMono leader, and the NosT terminator. The PMI cassette includes the PMI gene, ZmUbil promoter, mUBQMono, ZmKozak leaders and the NosT terminator.

[0182] Example 8. Generation of CRISPRICas-induced mutant plants

[0183] Identification and Characterization of CRISPRICas and Maize NLS Meganuclease-inducedMutations in the Maize GWD Gene

[0184] Maize plant transformation was performed according to the protocol described in Example 3. Screening of CRISPR/Cas-induced mutations was similar to screening of meganuclease-induced mutations methods that has been described in Example 3 with the exception of primers for genotyping and identifying mutations.

[0185] Table 9 describes primers for genotyping CRISPR/Cas plants that include 4804-4806, 4804-6 primer set; 4809, 4817, and 4804-6 primer set substituting GWDe24a-F for GWD24b-F and primers for amplifying DNA sequences surrounding the GWD meganuclease targeting region that include 4804-4807, 4804-7mut primers; 4817, 2856/2858 primers; 4837-4839, 371/429 primers.

Table 9 Primers for genotyping 4804, 4805, 4806, 4817, 4837, 4838, and 4839 plants and amplifying DNA sequences surrounding the GWD exon 24 targeting region Primer Forward or SEQ ID Product Primer Set Name Reverse Sequence NO size (bp)

4804-6 GWDe24b-F Forward CTCACAGCACATAA CCTGATACT 101 100

4804-6 sgRNA-R Reverse CGACTCGGTGCCAC TTT 102 100

4804-6 ZmCas9-F Forward AGAATCAGACCACG CAGAAG 103 186

4804-6 ZmCas9-R Reverse GCTCCTGGTCCACA TACATATC 104 186

4809/4817 GWDe24a-F Forward TGCAGAAGTAGGCT TGAGTTT 110 89

4804-7mut GWDex23-F Forward TGCTCTTCTGAACC 105 560 GATTTGA

4804-7mut ZmGWD Reverse CTATTCCTTGCTCG 13 560 mega-2R GACTGAC

4817 2856 Forward GAAGGGGATTGGAG AGGAAG 111613

4817 2858 Reverse CATGACGTTCAAAT AGCCTCA 112 613

4837-4839 371 Forward GGTTATAAGCCCGG TTGAAGTA 12 381

Primer Forward or SEQ ID Product Primer Set Name Reverse Sequence NO size (bp)

4837-4839 429 Reverse GCAGAAGTAGGCTT GAAGGAA38 113 381

[0186] Similar to previous analyses, DNA sequences for each mutant were compared to WT GWD using Vector NTI Advance (Version 11.5; Life Technologies). Mutant DNA sequences are described in Table 10.

[0187] Transgenic maize plants carrying gene editing constructs that target regions of the GWD gene have been produced and are being analyzed for mutations in the target regions of the GWD gene. GWD mutations and predicted proteins are listed in Tables 11-14. In these tables, events carrying two different GWD mutant alleles (heterozygotes) are labeled with -1 or -2, to indicate the individual alleles. Intronic sequences are presented in lowercase letters and exons 24 or 25 are shown in uppercase letters. Since pAG4817 is targeting two different locations within ZmGWD, two mutations are provided for 4817_2 and 4817_52. The first target sequence is located just upstream of the 5'end of the exon 24 and an extra "T" that is inserted into this target location is shown as a capital letter "T" within lowercase letters specific to intron 23 (see M37 sequence). M37 is an identical modification in 4817_2 and 4817_52.

[0188] All modifications introduced by CRISPR/Cas9 are highlighted (bold black = insertion; gray = deletion) and missing nucleotides are shown by dots. Corresponding numbers of deleted or inserted nucleotides are presented in the last columns of Tables 11 and 12.

[0189] Similarly, all changes to deduced protein sequences for M32-M39 are highlighted. In the cases of translation reading frame shifts and early termination of translation, all amino acids differing from wild type GWD are also highlighted and the end of protein is indicated by an asterisk(*).

Table 10 CRISPR/Cas9 induced mutations in individual transgenic 4804, 4806, and 4817 events Mutation Events and alleles M32 48042, 48043-2, 4804_4-1, 48045-2, 4806_1 M33 4804_3-1, 48045-1, 4804_7-1 M34 4804_4-2 M35 4804_6 M36 4804_7-2 M37, 4817_2 M38 M37, 4817_52 M39

Table 11 Nucleotide sequences of CRISPR/Cas9 induced mutations in individual 4804 and 4806 events Sequence DNA sequence SEQ ID Del/Ins Description NO number WT gctcctgctatcagTTGGCAGGTTATAAGCCCGGT 186 None ZmGWD TGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGT TACTTG Exon 24* M32 gctcctgctatcagTTGGCAGGTTATAAGCCCGGT 114 ±1 TTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAG TTACTTG M33 gctcctgctatcagTTGGCAGGTTATAAGCCCGGT 115 -2 T7 AGTATCAGGTTATGTGGTTGTGGTTGATGAGT TACTTG M34 gctcctgctatcagTTGGCAGGTTATAAGCCCGGT 116 -3 TP GTATCAGGTTATGTGGTTGTGGTTGATGAGT TACTTG M35 gctcctgctatcagTTGGCAGGTTATAAGCCCGGT 117 -1 GAAGTATCAGGTTATGTGGTTGTGGTTGATGAGT TACTTG M36 gctcctgctatcagTT................... 118 -37 .. ................ GGTTGTGGTTGATGAGT TACTTG *WT ZmGWD is a region of nt 81-160 of Exon 24 (SEQ ID NO: 3)

Table 12 Nucleotide sequences of CRISPR/Cas9 induced mutations in individual 4817 events Sequence DNA sequence SEQ Del/Ins Description ID number NO Wt Zm gctcctgctatcagTTGGCAGGTTATAAGCCCGGT 187 None GWD Exon TGAAGTATCAGGTTATGTGGTTGTGGTTGATGAGT TACTTG 24* M37 gctcctgctatc.iiagTTGGCAGGTTATAAGCCCGG 188 +1 TTGAAGTATCAGGTTATGTGGTTGTGGTTGATGAG TTACTTG Wt CACTCTATCTGAACTTGAAGGATATGATCAGAAAC 189 None ZmGWD TGTTTTCCTTCAAGCCTACTTCTGCAGATATA Exon 25 M38 CACTCT ATCTG.. 119 -48 ..... G................ATATA M39 CACTCTATCTGAACTTGAAGGATATGATCAGAAAC 120 +1 TGTTTTCCTTCACAGCCTACTTCTGCAGATATA

*WT ZmGWD is a region of nt 81-160 of Exon 24 (SEQ ID NO: 3)

Table 13 Partial deduced protein sequences of exon 24 in CRISPR/Cas9 mutants 4804 and 4806 Sequence Protein sequence SEQ Descripition ID NO WT WQVISPVEVSGYVVVVDELLAVQNKSYDKPTILVAKSVKGEEEIPDG 190 ZmGWD Exon 24* M32 WQVISPV*-.|. 121 M33 WQVI SPVSIRNCGNCG* 122 M34 WQVISPV||i|VSGYVVVVDELLAVQNKSYDKPTILVAKSVKGEEEIPDG 123 M35 124 1Q24PVKYQVWLWLMSYLLSRTNLMINQPSLWQRVSRERKKYQME* M36 WLWSYLLRTNLMINQPSLWQRVRKYQME* 125

*WT ZmGWD Exon 24 is a region of aa 1011-1057 of WT ZmGWD (SEQ ID NO: 43)

Table 14 Partial deduced protein sequences of exon 25 in CRISPR/Cas9 mutants 4817

Sequence Protein sequence SEQ ID Description NO WTZmGWD VLFATCFDHTTLSELEGYDQKLFSFKPTSADIT 191 Exon 25* YR M38 VLFATCFDHTTLS||||||||||||||||||||||||||||||||ADITYR 126 M39 VLFATCFDHTTLSELEGYDQKLFSFETAYF|CRY 127 _____ NL __I___A

**WT ZmGWD Exon 25 is a region of aa 1082-1116 of WT ZmGWD (SEQ ID NO: 43)

[0190] Characterizationof maize NLS meganuclease-induced mutations

[0191] To develop maize NLS meganuclease constructs pAG4837-4839, the viral SV40 NLS sequence in pAG4716 was replaced with the maize NLS sequences derived from Opaque2 (Hicks et al., PNAS,1995) (Table 15). A large variation of the induced mutations in exon 24 of the maize GWD gene was observed. These mutations included substitutions, deletions, and insertions from 1 to 114 nucleotides (Tables 16-17). Indirectly assessed efficiencies of the NLS variants were estimated as the number of events containing any modifications in the target region of the GWD gene divided by the total number of analyzed events (Table 15). Each evaluated NLS sequence supported production of the induced mutations with the NLS3 and NLS4 being the most efficient.

Table 15 Meganuclease constructs containing plant-derived NLS sequences Construct Expression NLS Protein sequence SEQ ID Relative cassette number NO efficiency (%) pAG4837 ZmUbi1P:NLS1: NLS1 MPTEERVRKRKES 128 59.1 GWD7-8x.226 NRESARRSRYRKA AHLKEL pAG4838 ZmUbilP:NLS3: NLS3 MARKRKESNRESA 129 75.0 GWD7-8x.226 RRSRYRKAAHLKE L pAG4839 ZmUbilP:NLS4: NLS4 MARKRKESNRESA 130 71.4 GWD7-8x.226 RRSRRSRYRKV

Table 16 List of representative mutations induced by maize NLS meganucleases in exon 24 of the ZmGWD gene in 4837, 4838, and 4839 events

Sequence Mutation DNA sequence SEQ ID Del/Ins Description NO WT None GAAATACCAGATGGAGTAGTTGGTGTAATTA 192 None ZmGWD* CACCTGATATGCCAGATGTTCTGTCT 4837_12 M40 GAAATACCAGATGGAGTAGTTG. TAATTA 131 -3 CACCTGATATGCCAGATGTTCTGTCT 4837_12 M41 GAAATACCAGATGGAGTAGTTGGTATAAATT 132 -2/+3 ACACCTGATATGCCAGATGTTCTGTCT 4837_16 M42 GAAATACCAGATGGAGTAGTTGGTGTAl.||TTA 133 -1 CACCTGATATGCCAGATGTTCTGTCT 4837_19 M43 GAAATACCAGATGGAGTAGTTGGTGTAANGT 134 -3/+8 AATAACACCTGATATGCCAGATGTTCTGTCT 4837_53 M44 GAAATACCAGATGGAGTAGTTGGTGT... 135 -24 ........ ... .... .... .. T.... 4838_1 M.45 GAAATACCAGATG...................136 -36 .............. .. TTCTGTCT 4838_51 M46 GAAATACCAGATGGAGTAGTTGGTGTATGAA 137 +10 CAiGTAATTACACCTGATATGCCAGATGTTC TGTCT 4838 53 M47 GAAATACCAGATGGAGTAGTTGGTGT 138 -29 CT .. .. GATATGCCAGATGTTCTGTCT........... 4839_1 M48 GAAATACCAGATGGAGTAGTTGGTG~ TTA 139 -3 CACCTGATATGCCAGATGTTCTGTCT 4839_3 M49 GAAATACCAGATGGAGTAGTTGGTGTAAATT 140 +1 ACACCTGATATGCCAGATGTTCTGTCT 4839_54 GAAATACCAGATGG........ .50 ... 141 -22

4839_57 M51 GAAATACCAGATGGAGTAGTTGGTGTCTCAT 142 +19 GCCAGATGTGAAGAAATTACACCTGATATGC CAGATGTTCTGTCT 4839_58 M52 GAAATACCAGATGGAGTAGTTGGTG 143 -21 ATGTTCTGTCT 4839 58 M53 GAAATACCAGATGGAGTAGTTGGTGT 144 -9/+2 NCGATATGCCAGATGTTCTGTCT 4839_61 M54 GAAATACCAGATGGAGTAGTTGGTGCATTTA 145 -4/+114 CTCATATTTTCTGTGATTGAATATTCTTTTC CAGATGGAGTGTCAAGGGAGAGGAAGAAATA CCAGATGGAGTGTCAAGGGAGAGGAAGAAAT ACCAGATGAAGGAAATCACC CC GATGTTCTGTCT 4839_61 M55 GAAATACCAGATGGAGT TA 146 -12 CACCTGATATGCCAGATGT

*.WT.ZmGWD is a.region of nt.3157-3213 of SEQ ID.NO:1.

Table 17 Partial deduced protein sequences of exon 24 in CRISPR/Cas9 mutants 4837, 4838, and 4839 Sequence Protein sequence SEQ ID NO Decsription WTZmGWD IPDGVVGVITPDMPDVLSHVSVRARNSK 193 Exon 24* M40 IPDGVV:|||VITPDMPDVLSHVSVRARNSK 147 M41 IPDGVVGINYT* 148 M42 IPDGVVGVLHL ICQMFCLMCQSEQGIARYCL 149 RPVLTTPLYLNLKDMIRNCFPSSLLL* M43 IPDGVVGVE* 150 M44 IPDGVVGV ,i LSHVSVRARNSK 151 M45 IPD. VLSHVSVRARNSK 152 M46 IPDGVVGV* 153 M47 IPDGVVGVSCVSPSKE* 154 M48 IPDGVVGVTPDMPDVLSHVSVRARNSK 155 M49 IPDGVVGVNYT* 156 M50 IPG 7 5GIARYCRPVLTTP

M51 IPDGVVGV C 158 M52 IPDGVVG DVLSHVSVRARNSK 159 M53 IPDGVVGVYACQ CP. 160 M54 IPDGVVGAFTHiFOD* 161 M55 IPDGV TPDMPDVLSHVSVRARNSK 162

* WT ZmGWD Exon 24 is aregion ofaa 1054-1081 of SEQ ID NO: 43.

[0192] Green Tissue StarchAssays

[0193] CRISPR/Cas and maize NLS meganuclease lines were assayed for starch in green leaf tissue. Leaf tissue harvested from 40day old events was assayed for starch according to the protocol described in Example 3. These data confirm the efficacy of our CRISPR/Cas targeting system and maize NLS meganuclease gene editing constructs.

Table 18 Starch Content in CRISPR/Cas and Maize NLS Meganuclease Lines

Vector Event Starch SD Vector Event Starch SD (mg (mg glucose/100 glucose/100 mg DW) mg DW)

[% DW] [% DW] WT WT 1.0 1.4 4806 2 1.2 0.1 4804 58 1.1 0.1 4806 52 1.2 0.6 4804 60 1.2 0.1 4806 101 1.4 0.2 4804 59 1.3 0.2 4806 53 1.7 0.2 4804 61 2.4 0.2 4806 154 2.0 0.3 4804 53 9.3 1.9 4806 57 2.2 0.7 4804 6 9.6 0.3 4806 152 2.7 0.9 4804 4 11.2 1.4 4806 1 3.3 0.3 4804 54 11.9 0.5 4806 205 4.0 1.0 4804 51 12.4 0.9 4806 207 4.6 0.6 4804 57 14.1 1.8 4806 209 5.9 1.0 4804 62 16.7 0.4 4806 210 7.1 0.9 4804 56 16.7 0.7 4806 208 7.4 0.6 4804 7 17.0 0.6 4806 155 7.5 1.0 4804 63 17.9 0.4 4806 55 8.6 1.4 4804 5 18.0 0.2 4806 56 9.4 1.8 4804 52 18.2 0.8 4806 206 10.1 1.4 4804 3 18.5 0.9 4806 54 11.0 0.9 4804 64 19.0 0.7 4806 151 15.7 1.4 4804 2 19.5 1.2 4807 1 0.7 0.0 4804 55 20.5 0.9 4807 6 0.7 0.1 4804 1 27.8 1.7 4807 5 0.7 0.1 4805 1 1.2 0.3 4807 2 0.7 0.0 4805 103 11.2 0.7 4807 4 1.0 0.1 4805 53 11.5 1.0 4807 3 2.1 0.4 4805 56 11.7 0.7 4809 1 10.1 0.8 4805 55 13.8 2.3 4809 2 10.6 0.5 4805 101 14.2 0.4 4809 4 13.3 0.7 4805 104 15.0 1.2 4809 3 17.8 1.3 4805 51 15.0 1.2 4817 54 14.2 0.7 4805 54 15.6 1.0 4817 55 18.0 1.1 4805 2 16.4 0.7 4817 51 19.2 0.8 4806 204 0.9 0.1 4817 1 19.4 0.8 4806 203 0.9 0.1 4817 53 20.7 0.5 4806 202 0.9 0.1 4817 52 20.9 0.8 4806 201 1.0 0.1

Table 19 Starch Content in CRISPR/Cas and Maize NLS Meganuclease 4837, 4838 and 4839 Lines Vector Event Starch (mg glucose/100 mg DW SD

[%DW] WT 1 1.0 0.2 4837 8 1.3 0.2 4837 51 1.8 0.5 4837 7 1.8 0.3 4837 16 2.2 0.5 4837 4 2.3 0.2 4837 1 2.3 0.2 4837 15 2.5 0.1 4837 53 3.3 0.5 4837 9 3.6 0.5 4837 3 4.7 1.0 4837 12 6.2 1.4 4837 17 16.5 0.5 4837 11 17.0 0.7 4837 14 18.2 0.6 4837 2 18.6 1.1 4837 5 19.3 1.3 4837 19 20.0 1.1 4837 18 20.4 0.4 4837 10 22.7 2.1 4837 6 23.3 0.9 4837 52 24.7 1.4 4838 4 1.2 0.1 4838 3 1.7 0.4 4838 53 1.8 0.7 4838 2 3.3 0.4 4838 54 6.9 1.2 4838 51 19.3 1.4 4838 52 19.9 1.2 4838 1 20.0 1.4 4839 53 1.1 0.1 4839 63 1.1 0.1 4839 62 1.3 0.1 4839 52 1.4 0.2 4839 54 2.1 0.4 4839 56 2.3 0.2 4839 51 2.4 1.1 4839 57 2.7 0.3 4839 58 2.8 0.8 4839 55 22.8 1.7

Vector Event Starch (mg glucose/100 mg DW SD

[%DW] 4839 61 23.5 1.1 4839 60 24.5 1.1 4839 59 28.0 3.3

[0194] Referring to Tables 18 and 19, it was observed that many of the events exhibited high starch, which ranged approximately 3%-27.8%.

[0195] Example 10. Green tissue starch assays

[0196] All of the CRISPR/Cas lines were assayed for starch in green leaf tissue as well as in dried stover leaves, stalks, and cobs. Leaf tissue harvested from 40 day old CRISPR/Cas events were assayed for starch according to our protocol described in Example 3. FIG. 9 illustrates starch accumulation in the pAG4804 maize events. Referring to FIG. 9, all seven TO maize 4804 events had high starch, which ranged 9.6-27.8%, indicating that all currently unresolved GWD sequences were the result of two different GWD mutations rather than one wild type and one mutant allele (hemizygote). FIG. 10 illustrates starch accumulation in the pAG4806 maize events. Referring to FIG. 10, both TO maize 4806 events had low starch, suggesting that the one unresolved GWD sequence is a hemizygote. These data confirm the efficacy of the CRISPR/Cas targeting system herein, which includes new GWD guide RNA targeting sequences and new U3 promoters used for expression of the guide RNAs.

[0197] Example 11. Breeding recessive mutations for elite inbred introgression and testing

[0198] The advent of new methods for precision DNA engineering and mutagenesis provides a means of generating targeted recessive and dominant mutations for the development of new and beneficial plant traits. Some of these methods include targeting specific regions of genes with meganucleases, Talens, and the CRISPR/Cas system. Tracking and advancing targeted mutations present a new challenge for trait development, because, unlike traditional transgenic plant traits, they do not carry dominant T-DNA expression cassettes and selectable markers. Generation of targeted mutations in maize and sorghum using TALENS, ZFN, meganuclease and CRISPR/Cas methods described herein can lead to the creation of new methods for screening and breeding these unique plant traits.

[0199] Example 12. Tracking and breeding targeted mutations

[0200] Transformation (TO) Generation Genotypes: Gene-specific mutations identified using the described methods resulted in first generation transformed (TO) plants with one of three different genotypes: 1) one wild type gene allele and one mutant allele, 2) two different mutant alleles, or 3) two identical mutant alleles. These mutant allele combinations were designated as hemizygous, heterozygous, and homozygous, respectively.

[0201] Molecular Methods for Tracking Targeted Mutations: The specific sequence characteristics of a targeted DNA mutation (e.g., substitution, deletion, insertion, or combination) relative to the wild type sequence were, and may be, tracked using methods described herein when breeding the mutation into other lines, or expanding the existing lines through breeding. To differentiate wild type, hemizygous, heterozygous, and homozygous lines in a TO and T1+ (progeny derived from TO parental lines and beyond) segregating populations, at least five methods could be used. These methods include: 1) PCR of the mutation site with gel electrophoresis for size separation, 2) PCR with restriction enzyme digestion and gel electrophoresis to generate a mutation specific restriction pattern, 3) PCR with direct sequencing, 4) PCR with cloning and sequencing, and 5) PCR using primers that bind or do not bind to mutation sites.

[0202] Homozygous DNA sequences from either wild type or engineered, altered, or optimized endogenous nucleic acids would be easily analyzed by PCR, size determination, and, or DNA sequencing in the targeted or mutated region. In contrast, DNA sequences that possess different alleles may result in a portion of sequence that would be difficult to analyze using sequencing, PCR or size determination due to differences in the two allelic sequences (e.g., a wild type and a mutant or two different mutant sequences). PCR products from these types of targeted events would require cloning to isolate and effectively sequence each allele.

[0203] Once the sequence of the targeted mutation has been confirmed, a mutation-specific molecular strategy for tracking is established. As mentioned previously, this strategy will be dependent on the characteristics of the mutation. In tracking the gwd mutations generated herein, PCR specific reactions were developed for each of the mutations and engineered endogenous (optimized) nucleic acids described herein.

[0204] Breeding Crosses and Selfing: A main goal of developing traits with targeted mutations induced with transgenes is to isolate the mutations by separating the desired mutation from the transgene. This can be accomplished through genetic crosses and is most effective through outcrosses (higher frequency of recovered mutant plants that are transgene negative), which involves crossing TO pollen to the female component of a non-transgenic plant. This can also be accomplished at a lower frequency using selfing, which involves self-pollinating (using TO pollen to pollinate the same TO plant). Sibbing is another option and involves crosses between two genetically identical plants, which would be expected to have the same outcome as a self cross.

[0205] TO plants carrying targeted mutations can be selfed to generate homozygous plants and would result in different numbers and types of progeny depending on the TO zygosity. Homozygous TO plants would generate 100% homozygous progeny, hemizygous TO plants would segregate 1:2:1 (homozygous: hemizygous: wild type) for the mutant allele, and heterozygous TO plants would segregate 1:2:1 (homozygous targeted allele 1: heterozygous: homozygous targeted allele 2).

[0206] TO plants carrying targeted mutations can also be outcrossed into other lines and would result in different numbers and types of progeny depending on the TO zygosity. Homozygous TO plants would generate 100% hemizygous progeny, hemizygous TO plants would generate 50% hemizygous progeny and 50% wild type progeny, and heterozygous TO plants would generate hemizygous progeny, 50% with targeted allele 1 and 50% with targeted allele 2.

[0207] Because all TO plants would be carrying transgenes, the transgene insertion location is most commonly different, and the number of transgene insertions could differ, the segregation patterns for the transgenes have the potential to vary considerably between each TO transformation event/plant. To identify transgene-negative plants, PCR would be applied to the progeny from any cross (self, sib, or outcross) with TO plants. Transgene negative plants would be identified by the absence of a transgene-specific PCR product. The transgene-negative plants would then be screened for the targeted mutation using the molecular diagnostics approach defined during the initial characterization of the TO plants.

[0208] Targeted mutations isolated from the transgene can then be maintained and bred for testing and introgressions with the continued use of the trait-specific molecular diagnostics protocol.

[0209] An Example Tracking and Breeding Procedure for a Targeted Mutation: The molecular tracking and breeding procedure for a targeted mutation is described herein. Tracking of the GWD gene from maize (M20, ZmGWDM20 from event 4716_164) is described herein. Generation and initial sequence characterization of this and other meganuclease-induced targeted mutations in maize and sorghum has been described in Examples herein. TO 4716_164 plants were hemizygous for the M20 mutation initially and carried an unknown number of T-DNA insertions. The M20 mutation is a recessive single base pair (bp) deletion in exon 24 of ZmGWD, which results in a mutation at an MluCI restriction enzyme site in the wild type sequence. The small size of this deletion required use of the MluCI RFLP with gel electrophoresis because it could not be differentiated from wild type with gel electrophoresis alone.

[0210] FIG. 11 illustrates a schematic drawing of selfing and outcrossing of a targeted mutation M20 derived from the maize event 4716_164. Referring to FIG. 11, TO 4716_164 plants were selfed and outcrossed to generate progeny for efficacy testing and introgressions, respectively. To identify homozygous M20 progeny from the TO selfed parent, PCR with the meganuclease targeting gene and the target region of the GWD gene was performed. FIG. 12 illustrates genotyping of T1 progeny from the selfed TO 4716_164 M20 plant. Referring to FIG. 12, PCR products from meganuclease GOI and ZmGWD target region were digested with MluCI, separated on 5% polyacrylamide and stained with ethidium bromide. This revealed plants that did not carry the T-DNA (Meganuclease) but were homozygous for M20. These plants were maintained for testing.

[0211] To identify hemizygous M20 progeny from the TO outcrossed parent, PCR of the selectable marker gene, PMI, the meganuclease gene, and the target region was performed, followed by 3% agarose gel electrophoresis and ethidium bromide staining. This also allowed identification of T-DNA negative plants. FIG. 13 illustrates genotyping of T1 progeny from the outcrossed TO 4716_164 M20 plant. MluCI restriction digests were then performed on the same PCR products from T-DNA negative plants and separated them on a 5% polyacrylamide gel stained with ethidium bromide. FIG. 14 illustrates genotyping of T1 progeny from the outcrossed 4716_164 M20 plant. These plants were maintained for further introgressions and future testing. References An, G et al., 2005. Reverse genetic approaches for functional genomics of rice. Plant molecular biology, 59(1), pp. 1 1 1 - 2 3 . Available at: http://www.ncbi.nlm.nih.gov/pubmed/16217606 [Accessed July 25, 2012].

Arnould S, Perez C, Cabaniols JP, Smith J, Gouble A, Grizot S, Epinat JC, Duclert A, Duchateau P, Paques F. (2007) Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J. Mol. Biol. 371: 49-65.

Arnould S, Delenda C, Grizot S, Desseaux C, Paques F, Silva GH, Smith J. (2011) The I-CreI meganuclease and its engineered derivatives:

Applications from cell modifi cation to gene therapy. Protein Eng. Des. Sel. 24: 27-31.

Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V. (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9: 39-48.

Boch J. & Bonas U. (2010) Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu. Rev. Phytopathol. 48: 419-436.

Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39: e82

Chi-Ham CL et al., 2010. The intellectual property landscape for gene suppression technologies in plants. Nature Biotechnology, 28 (1):32-36.

Christian, M et al., 2010. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 186(2), pp. 7 5 7 - 6 1 . Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2942870&to ol=pmcentrez&rendertype=abstract [Accessed July 14, 2012].

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819-823.

Djukanovic V, Smith J, Lowe K, Yang M, Gao H, Jones S, Nicholson MG, West A, Lape J, Bidney D, Falco SC, Jantz D, Lyznik LA. (2013) Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-Crel homing endonuclease. The Plant Journal 76: 888-899.

Elkonin LA, Pakhaomova NV. (2000) Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum. Plant Cell Tissue and Organ Culture 61: 115-123.

Frizzi A & Huang S, 2010. Tapping RNA silencing pathways for plant biotechnology. Plant biotechnology journal, 8(6), pp. 6 5 5 - 7 7 . Available at: http://www.ncbi.nlm.nih.gov/pubmed/20331529 [Accessed July 24, 2012].

Gao Z, Xie X, Ling Y, Muthukrishnan S, Liang GH. (2005) Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection. Plant biotechnology journal, 3, pp. 591- 599.

Garcia-Bustos J, Heitman J, Hall MN (1991) Nuclear protein localization. Biochim Biophys Acta 1071: 83-101.

Gasiunas G, Barrangou R, Horvath P, Siksnys V. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 109 (39): 2579-2586.

Goujon M et al., 2010 A new bioinformatics analysis tools framework at EMBL-EBI (2010) Nucleic acids research Jul, 38 Suppl: W695-9 doi:10.1093/nar/gkq313

Heath PJ, Stephens KM, Monnat RJ Jr., Stoddard BL. (1997) The structure of I-Crel, a group I intron-encoded homing endonuclease. Nat. Struct. Biol. 4: 468-76.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821.

Joung JK & Sander JD. (2013) TALENs: a widely applicable technology for targeted genome editing. Nature Reviews (Mol Cell Biol) 14: 49-55.

Kalderon D, Richardson WD, Markham AF, Smith AE (1984) Sequence requirements for nuclear l6cation of simian virus 40 large T antigen. Nature 311: 33-38.

Larkin MA et al., 2007 ClustalW and ClustalX version 2 Bioinformatics, 23(21): 2947-2948. doi:10.1093/bioinformatics/btm4O4

Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. (2013) CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8(11): 2180-2196.

Leader DJ, Connelly S, Filipowicz W, Brown JWS. (1994) Characterisation and expression of a maize U3 snRNA gene. Biochimica et Biophysica Acta 1219: 145-147.

Liang Z, Zhang K, Chen K, Gao C. (2014) Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas System. Journal of Genetics and Genomics 41: 63-68.

Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B. (2011) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39: 359-372.

Maniatis T, Fritsch EF and J. Sambrook J, 1982. Molecular Cloning Cold Spring Harbor Laboratory.

Puchta H, Dujon B and Hohn B, 1993. Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic acids research, 21(22), pp.5034 40. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=310614&too 1=pmcentrez&rendertype=abstract.

Raikhel NV (1992) Nuclear targeting in plants. Plant Physiol 100: 1627-1632.

Rosen LE et al., (2006) Homing endonuclease I-Crel derivatives with novel DNA target specificities. Nucleic Acids Res. 34: 4791-4800.

Shieh MW et al., (1993) Nuclear targeting of the maize R protein requires two nuclear localization sequences. Plant Physiology 101: 353-361.

Shukla VK et al. (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459: 437-441.

Sikora P et al., 2011. Mutagenesis as a tool in plant genetics, functional genomics, and breeding. Internationaljournal of plant genomics, 2011, p.314829. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3270407&to ol=pmcentrez&rendertype=abstract [Accessed July 25, 2012].

Smith AM and Zeeman SC, 2006. Quantification of starch in plant tissues. Nat. Protocols 1:1342-1345.

Smith TF, Waterman MS, 1981. Identification of Common Molecular Subsequences. J Mol Biol 147: 195 -197.

Symington LS, Gautier J. (2011) Double-Strand Break End Resection and Repair Pathway Choice. Annual Review of Genetics. 45: 247-271.

Till BJ et al., 2007 Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol. 7:19.

Upadhyay SK et al., (2014) RNA-guided genome editing for target gene mutations in wheat. Genes, Genomes, Genetics 3: 2233-2238.

Vainstein A et al., 2011. Permanent genome modifications in plant cells by transient viral vectors. Trends in biotechnology, 29(8), pp. 3 6 3 - 9 .

Available at: http://www.ncbi.nlm.nih.gov/pubmed/21536337 [Accessed July 30, 2012].

Varagona MJ et al., (1992) Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2. The Plant Cell 4: 1213-1227.

Wagner P et al.,1990) Active transport of proteins into the nucleus. FEBS 275: 1-5.

Wehrkamp-Richter S et al., 2009. Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction. Plant physiology and biochemistry: PPB / Societ frangaise de physiologie vigitale, 47(4), pp. 2 4 8 - 5 5 . Available at: http://www.ncbi.nlm.nih.gov/pubmed/19136269 [Accessed July 17, 2012]

Weise, S.E. et al., (2012) Engineering starch accumulation by manipulation of phosphate metabolism of starch. P. Biotech. J. 10, 545-554.

Wright D et al., 2005. High-frequency homologous recombination in plants mediated by zinc-finger nucleases. The Plant journal: for cell and molecular biology, 44(4), pp. 6 9 3 - 7 0 5 . Available at: http://www.ncbi.nlm.nih.gov/pubmed/16262717 [Accessed July 19, 2012].Yon J, Fried M. (1989) Precise gene fusion by PCR. Nucleic Acids Res. 17 (12): 4895.

Larkin MA et al., 2007 ClustalW and ClustalX version 2 Bioinformatics,

23(21): 2947-2948. doi:10.1093/bioinformatics/btm4O4 Goujon M et al., 2010 A new bioinformatics analysis tools framework at EMBL-EBI (2010) Nucleic acids research Jul, 38 Suppl: W695-9 doi:10.1093/nar/gkq313

[0212] The references cited throughout this application, are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0213] It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.

AGR_PT024_1WO_SequenceListing_EFS SEQUENCE LISTING <110> Agrivida, Inc. <120> PLANTS EXPRESSING MODIFIED GLUCAN WATER DIKINASE

<130> AGR-PT024.1WO <150> US 62/056,852 <151> 2014-09-29 <160> 196

<170> PatentIn version 3.5 <210> 1 <211> 4416 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(4416) <223> ZmGWD coding sequence

<400> 1 atgtccggat tcagtgccgc ggccaacgca gcggcggctg agcggtgcgc gctcgcgttc 60

cgcgcacggc ccgcggcctc ctcgccagcg aagcggcagc agcagccgca gccagcgtcc 120

ctccgacgca gcgggggcca gcgccgcccc acgacgctct ccgcctctag ccgcggcccc 180

gtcgtgccgc gcgccgtcgc cacgtccgcg gaccgcgcgt cccccgacct tatcggaaag 240

ttcacgctgg attccaactc cgagctccag gtcgcagtga acccagcgcc gcagggtttg 300 gtgtcagaga ttagcctgga ggtgaccaac acaagcggtt ccctgatttt gcattgggga 360

gcccttcgcc cggacaagag agattggatc ctcccgtcca gaaaacctga tggaacgaca 420

gtgtacaaga acagggctct caggacacct tttgtaaagt caggtgataa ctccactcta 480 aggattgaga tagatgatcc tggggtgcac gccattgagt tcctcatctt tgacgagaca 540

cagaacaaat ggtttaaaaa caatggccag aattttcagg ttcagttcca gtcgagccgc 600 catcagggta ctggtgcatc tggtgcctcc tcttctgcta cttctacctt ggtgccagag 660 gatcttgtgc agatccaagc ttaccttcgg tgggaaagaa ggggaaagca gtcatacaca 720

ccagagcaag aaaaggagga gtatgaagct gcacgagctg agttaataga ggaagtaaac 780 agaggtgttt ctttagagaa gcttcgagct aaattgacaa aagcacctga agcacctgag 840 tcggatgaaa gtaaatcttc tgcatctcga atgcccatcg gtaaacttcc agaggatctt 900

gtacaggtgc aggcttatat aaggtgggag caagcgggca agccaaacta tcctcctgag 960 aagcaactgg tagaatttga ggaagcaagg aaggaactgc aggctgaggt ggacaaggga 1020

atctctattg atcagttgag gcagaaaatt ttgaaaggaa acattgagag taaagtttcc 1080 aagcagctga agaacaagaa gtacttctct gtagaaagga ttcagcgcaa aaagagagat 1140 atcacacaac ttctcagtaa acataagcat acacttgtgg aagataaagt agaggttgta 1200

ccaaaacaac caactgttct tgatctcttc accaagtctt tacatgagaa ggatggctgt 1260 Page 1

AGR_PT024_1WO_SequenceListing_EFS gaagttctaa gcagaaagct cttcaagttc ggcgataaag agatactggc aatttctacc 1320

aaggttcaaa ataaaacaga agttcacttg gcaacaaacc ataccgaccc acttattctt 1380 cactggtctt tggcaaaaaa tgctggagaa tggaaggcac cttctccaaa tatattgcca 1440

tctggttcca cattgctgga caaggcgtgt gaaactgaat ttactaaatc tgaattggat 1500 ggtttgcatt accaggttgt tgagatagag cttgatgatg gaggatacaa aggaatgcca 1560 tttgttcttc ggtctggtga aacatggata aaaaataatg gttctgattt tttcctagat 1620

ttcagcaccc atgatgtcag aaatattaag gcaattttaa agggcaatgg tgatgctggt 1680 aaaggtactg ctaaggcatt gctggagaga atagcagatc tggaggaaga tgcccagcga 1740 tctcttatgc acagattcaa tattgcagca gatctagctg accaagccag agatgctgga 1800

cttttgggta ttgttgggct ttttgtttgg attagattca tggctaccag gcaactaaca 1860 tggaataaga actataatgt gaagccacgt gagataagca aagcacagga taggtttaca 1920 gatgatcttg agaatatgta caaagcttat ccacagtaca gagagatatt aagaatgata 1980

atggctgctg ttggtcgcgg aggtgaaggt gatgttggtc aacgcattcg tgatgagata 2040 ttagtaatac agagaaataa tgactgcaaa ggtggaatga tggaagaatg gcaccagaaa 2100

ttgcacaaca atacaagccc agatgatgta gtgatatgcc aggccttaat tgattatatc 2160

aagagtgact ttgatataag cgtttactgg gacaccttga acaaaaatgg cataaccaaa 2220

gagcgtctct tgagctatga tcgtgctatt cattcagaac caaatttcag aagtgaacag 2280

aaggcgggtt tactccgtga cctgggaaat tacatgagaa gcctaaaggc tgtgcattct 2340 ggtgctgatc ttgaatctgc tatagcaagt tgtatgggat acaaatcaga gggtgaaggt 2400

ttcatggttg gtgttcagat caatccagtg aagggtttac catctggatt tccggagttg 2460

cttgaatttg tgcttgaaca tgttgaggat aaatcagcgg aaccacttct tgaggggcta 2520 ttggaagctc gagttgaact gcgccctttg cttcttgatt cgcgtgaacg catgaaagat 2580

cttatatttt tggacattgc tcttgattct accttcagga cagcaattga aaggtcatat 2640 gaggagctga atgatgcagc cccagagaaa ataatgtact tcatcagtct tgtccttgaa 2700 aatcttgcgc tttcaattga cgacaatgaa gacatcctgt attgtttaaa gggatggaac 2760

caagccttgg aaatggctaa gcaaaaagac gaccaatggg cgctctatgc taaagcattt 2820 cttgacagaa acagacttgc ccttgcgagc aagggagaac aataccataa tatgatgcag 2880 ccctctgctg agtatcttgg ctcgttactc agcatagacc aatgggcagt caatatcttc 2940

acagaagaaa ttatacgcgg tggatcagct gctactctgt ctgctcttct gaaccgattt 3000 gatcctgttt taaggaatgt tgctcacctc ggaagttggc aggttataag cccggttgaa 3060

gtatcaggtt atgtggttgt ggttgatgag ttacttgctg tccagaacaa atcttatgat 3120 aaaccaacca tccttgtggc aaagagtgtc aagggagagg aagaaatacc agatggagta 3180 gttggtgtaa ttacacctga tatgccagat gttctgtctc atgtgtcagt ccgagcaagg 3240

aatagcaagg tactgtttgc gacctgtttt gaccacacca ctctatctga acttgaagga 3300 Page 2

AGR_PT024_1WO_SequenceListing_EFS tatgatcaga aactgttttc cttcaagcct acttctgcag atataaccta tagggagatc 3360

acagagagtg aacttcagca atcaagttct ccaaatgcag aagttggcca tgcagtacca 3420 tctatttcat tggccaagaa gaaatttctt ggaaaatatg caatatcagc cgaagaattc 3480

tctgaggaaa tggttggggc caagtctcgg aatatagcat acctcaaagg aaaagtacct 3540 tcatgggtcg gtgtcccaac gtcagttgcg ataccatttg gcacttttga gaaggttttg 3600 tcagatgggc ttaataagga agtagcacag agcatagaga agcttaagat cagacttgcc 3660

caagaagatt ttagtgctct aggtgaaata agaaaagtcg tccttaatct tactgctcct 3720 atgcaattgg ttaatgagct gaaggagagg atgctaggct ctggaatgcc ctggcctggt 3780 gatgaaggag acaagcgttg ggagcaagca tggatggcta ttaaaaaggt ttgggcatca 3840

aaatggaacg aaagagcata ttttagcaca cgcaaggtga aacttgatca tgagtacctt 3900 tcgatggctg ttctcgtgca agaagttgtg aatgcagatt atgcttttgt cattcatacc 3960 acaaacccat cgtctggaga ttcttctgag atatatgctg aagtggtgaa agggcttggc 4020

gagaccctcg tgggagccta tcctggtcgt gctatgagct ttgtttgcaa aaaagatgac 4080 cttgactctc ccaagttact tggttaccca agcaagccaa ttggtctctt cataaggcaa 4140

tcaatcatct tccgttccga ctccaacggt gaggacctgg aaggttatgc tggagcagga 4200

ttatatgata gtgtaccgat ggatgaggag gatgaggttg tacttgatta tacaactgac 4260

cctcttatag tagaccgtgg attccgaagc tcaatcctct caagcatagc acgggctggc 4320

catgccatcg aggagctata tggttctcct caggacgtcg agggagtagt gaaggatgga 4380 aaaatctatg tagtccagac aagaccacag atgtag 4416

<210> 2 <211> 4410 <212> DNA <213> Sorghum bicolor

<220> <221> misc_feature <222> (1)..(4410) <223> SbGWD coding sequence

<400> 2 atgaccggat tcagtgccgc ggcctccgca gcagcggcgg cggagcggtg cgcgctcgcg 60

atccgcgcac ggcccgcggc ctcctcgcca gcgaagcggc agcagcagtc ggcgtccctc 120 agacgcagcg ggggccagcg ccgccccacc acgctcgctg cctcccgccg cagcccagtc 180

gtcgtgcccc gcgccatcgc cacgtccgcg gaccgcgcgt cccacgacct tgtcggaaag 240 ttcacgctgg attccaactc cgagctcctg gttgcagtga acccagcgcc gcagggtttg 300 gtgtcggtga tcggcctgga ggtgaccaac acaagcggtt ccctgattct gcattgggga 360

gtccttcgcc cggacaagag agattggatc ctcccatcca gacaacctga tggaacgacg 420 gtgtacaaga acagggctct taggacgcct tttgtaaagt ctggtgataa ctctactctt 480

Page 3

AGR_PT024_1WO_SequenceListing_EFS agaattgaga tagatgatcc tgcggtgcaa gctattgagt tcctcatctt tggcgagaca 540 cagaacaaat ggtttaaaaa caatggccag aattttcaga ttcagctcca gtcgagccgc 600 catcagggta atggtgcatc tggtgcctcc tcttctgcta cttctacctt ggtgccagag 660

gatcttgtgc agatccaagc ttaccttcgg tgggaaagaa agggaaagca gtcatacaca 720 ccagagcaag aaaaggagga gtatgaagct gcacgagctg agttaataga ggaattaaat 780 agaggtgttt ctttagagaa gcttcgagct aaattgacaa aaacacctga agcacctgag 840

tcagatgaac gtaaatctcc tgcatctcga atgcccgttg ataaacttcc agaggacctt 900 gtacaggtgc aggcttatat aaggtgggag aaagcgggca agccaaatta tcctcctgag 960

aagcaactgg tagaacttga ggaagcaagg aaggaactgc aggctgaggt ggacaaggga 1020 atctctattg atcaattgag gcagaaaatt ttgaaaggaa acattgagag taaagtttcc 1080

aagcagctga agaacaagaa gtacttctct gtagaaagga ttcagcgcaa aaagagagat 1140 atcatgcaac ttctcagtaa acataagcat acagttatgg aagagaaagt agaggttgca 1200 ccaaaacaac caactgttct tgatctcttc accaagtctt tacatgagaa ggatggctgt 1260

gaagttctaa gcagaaagct cttcaagttc ggtgataaag agatactggc aatttccacc 1320

aaggttcaaa ataaaacaga agttcacttg gcaacaaacc atacggagcc acttattctt 1380

cactggtctt tggcaaaaaa ggctggagaa tggaaggcac ctccttcaaa tatattgcca 1440 tctggttcca aattgctaga catggcgtgt gaaactgaat ttactagatc tgaattggat 1500

ggtttgtgtt accaggttgt tgagatagag cttgatgatg gaggatacaa aggaatgcca 1560

tttgttctta ggtctggtga aacatggata aaaaataatg gttccgattt tttcctagat 1620

ttcagcaccc gtgataccag aaatattaag ttaaaggaca atggcgatgc tggtaaaggc 1680 actgctaagg cgttgctgga gagaatagca gatctggagg aagatgccca gcgatctctt 1740

atgcataggt tcaatattgc agcagatcta gctgacgaag ccagagatgc tggactgttg 1800

ggtattgttg gactttttgt ttggattagg ttcatggcta ccaggcaact aacatggaat 1860

aagaactata atgtgaagcc acgtgagata agcaaagcac aagataggtt tacagatgat 1920 cttgagaata tgtacagaac ttatcctcag tacagagaga tactaagaat gataatggct 1980

gctgttggtc gtggaggtga aggtgacgtt ggtcaacgca ttcgtgatga gatattagta 2040 atacagagaa ataatgactg caaaggtgga atgatggaag aatggcacca gaaattgcac 2100

aacaatacaa gcccagatga tgtagtgata tgccaggcat taattgatta tataaaaaat 2160 gattttgata taagcgttta ctgggacacc ttgaacaaaa atggcataac caaagagcgt 2220

ctcttgagct atgatcgtgc tattcattca gaaccaaatt tcagaagtga acagaaggag 2280 ggtttactcc gtgacctggg aaattacatg agaagcctaa aggctgtgca ttctggtgct 2340 gatcttgaat ctgctatagc aacttgtatg ggatacaaat cagagggtga aggtttcatg 2400

gttggcgttc agatcaatcc agtgaagggt ttgccatctg gatttcctga gttgcttgaa 2460 tttgtgcttg accatgttga ggataaatca gcagaaccac ttcttgaggg gctattggaa 2520

Page 4

AGR_PT024_1WO_SequenceListing_EFS gctcgagttg atctgcgccc tttgcttctt gattcgcctg aacgcatgaa agatcttata 2580 tttttggaca ttgctcttga ttctaccttc aggacagcaa ttgaaaggtc atatgaggag 2640 ctcaatgatg cagccccaga gaaaataatg tacttcatca gtcttgtcct tgaaaatctt 2700

gcgttttcaa ttgacgacaa tgaagacatc ctgtattgct taaagggatg gaaccaagcc 2760 ttggaaatgg ctaagcaaaa agacgaccaa tgggctcttt acgctaaagc atttcttgac 2820 agaatcagac ttgcccttgc gagcaaggga gaacagtacc ataatatgat gcagccctca 2880

gctgaatatc ttggctcgtt actcagcata gacaaatggg cagtcaatat cttcacagaa 2940 gaaattatac gcggtggatc agctgctact ctgtccgctc ttctgaaccg atttgatcct 3000

gttctaagga acgttgctaa ccttggaagt tggcaggtta taagcccagt tgaagtatca 3060 ggttatgtgg ttgtggttga tgagttactt gctgtccaga acaaatctta tgataaacca 3120

accatccttg tggcaaagag tgtcaaggga gaggaagaaa taccagatgg agtagttggt 3180 gtaattacac ctgatatgcc agatgttctg tcccatgtgt cagtccgagc aaggaatagc 3240 aaggtactgt ttgcaacctg ttttgaccat accactctgt ctgaacttga aggatatgat 3300

cagaaactgc tttccttcaa gcctacttct gcagatataa cctataggga gatcacagag 3360

agtgagcttc agcaatcaag ttctccaaat gcagaagttg gccatgcagt accatctatt 3420

tcattggcca agaagaaatt tcttggaaaa tatgcaatat cagctgaaga attcaccgag 3480 gaaatggttg gggccaagtc tcggaatata gcatacctca aaggaaaagt accttcatgg 3540

gttggtgttc caacgtcagt tgcgatacca tttggcactt ttgagaaggt tttgtcagat 3600

ggtcttaata aggaagtagc acaaaccata gagaagctta agatcaggct tgctcaagaa 3660

gattttagtg ctctaggtga aataagaaaa gccgttctta atcttactgc tcctatgcaa 3720 ttggttaatg agctgaagga gaggatgcta ggctctggaa tgccctggcc tggtgatgaa 3780

ggcaacaggc gctgggagca agcatggatg gctattaaaa aggtttgggc atcaaaatgg 3840

aatgaaagag catattttag cacacgcaag gtgaaactca atcatgagta cctttcgatg 3900

gctgttcttg tgcaagaagt tgtgaatgca gattatgctt ttgtcattca tactacaaac 3960 ccatcgtctg gagattcttc tgagatatat gctgaagtcg tgaaagggct cggagagact 4020

ctcgtgggag cctatcctgg tcgtgctatg agctttgttt gcaaaaaaga tgaccttgac 4080 tctcccaagt tacttggtta cccgagcaag ccaattggtc tcttcataag gcgatcgatc 4140

atctttcgtt ctgactccaa cggcgaggat ctggaaggtt atgccggagc aggattatat 4200 gatagtgtac cgatggatga ggaggatgaa gtcgtacttg attacacaac tgaccctctt 4260

atagtagatc gtggattccg aaattcaata ctctcaagca tcgcacgggc tggccatgcc 4320 attgaagagc tatatggttc tcctcaggac gtcgagggtg tagtgaagga tggaaaaatc 4380 tatgtagtcc agacaagacc acagatgtag 4410

<210> 3 <211> 392 <212> DNA Page 5

AGR_PT024_1WO_SequenceListing_EFS <213> Zea mays

<220> <221> misc_feature <222> (1)..(392) <223> ZmGWD Exon 24 _introns <400> 3 aagtgatact agtgaccctc tccacaattt tatgcgaacc acagaaatta ataatatatt 60 ctattactct gcacctgaca tctggctcct gctatcagtt ggcaggttat aagcccggtt 120

gaagtatcag gttatgtggt tgtggttgat gagttacttg ctgtccagaa caaatcttat 180 gataaaccaa ccatccttgt ggcaaagagt gtcaagggag aggaagaaat accagatgga 240 gtagttggtg taattacacc tgatatgcca gatgttctgt ctcatgtgtc agtccgagca 300

aggaatagca aggtttatct tcacagctat gttgcaagat ttcttgaatt ttttctcttg 360 tattgatgtt gacatactag ctttttccta at 392

<210> 4 <211> 397 <212> DNA <213> Sorghum bicolor

<220> <221> misc_feature <222> (1)..(397) <223> SbGWD Exon 24-introns

<400> 4 aagtggtact agtgacctct ccacagtttt atgtgaacca cagaaattaa atatgataat 60

atattctatt actctgcacc tgacatctgg ctcctgataa cagttggcag gttataagcc 120 cagttgaagt atcaggttat gtggttgtgg ttgatgagtt acttgctgtc cagaacaaat 180

cttatgataa accaaccatc cttgtggcaa agagtgtcaa gggagaggaa gaaataccag 240

atggagtagt tggtgtaatt acacctgata tgccagatgt tctgtcccat gtgtcagtcc 300

gagcaaggaa tagcaaggtt tattttcaca gttatgttgc aagctttctc agattttttt 360 tcttgtatcg atgttgacat accagttttt tcctaat 397

<210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, Mega-1(4716) PCR primer reverse <400> 5 tgatcttcag cacgaggttg 20

<210> 6 <211> 21 <212> DNA <213> Artificial Sequence

Page 6

AGR_PT024_1WO_SequenceListing_EFS <220> <223> Synthetic construct, Mega-1 (4716) PCR primer forward

<400> 6 ggctccatct atgcctgtat c 21

<210> 7 <211> 22 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, Mega-2 (4715) PCR primer forward <400> 7 gagctcagtt tcgctgtcta tc 22

<210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, Mega-2 (4715) PCR primer reverse <400> 8 atgatcttca gcacgaggtt g 21

<210> 9 <211> 22 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, ZmGWDmega-2 PCR primer forward

<400> 9 ggttataagc ccggttgaag ta 22

<210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ZmGWDmega-2 PCR primer reverse

<400> 10 ctattccttg ctcggactga c 21

<210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, SbGWD mega-2 PCR primer forward <400> 11 ggcaggttat aagcccagtt 20

<210> 12 Page 7

AGR_PT024_1WO_SequenceListing_EFS <211> 189 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(189) <223> M16 <400> 12 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aagggagagg aagaaatacc agatggagta 120 gttggtgtaa ttacacctga tatgccagat gttctgtctc atgtgtcagt ccgagcaagg 180 aatagcaag 189

<210> 13 <211> 204 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(204) <223> M17

<400> 13 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaggg agaggaagaa 120

ataccagatg gagtagttgg tgtaattaca cctgatatgc cagatgttct gtctcatgtg 180

tcagtccgag caaggaatag caag 204

<210> 14 <211> 213 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(213) <223> M18 <400> 14 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaagag tgtcaaggga 120

gaggaagaaa taccagatgg agtagttggt gtaattacac ctgatatgcc agatgttctg 180 tctcatgtgt cagtccgagc aaggaatagc aag 213

<210> 15 <211> 198 <212> DNA <213> Zea mays

Page 8

AGR_PT024_1WO_SequenceListing_EFS <220> <221> misc_feature <222> (1)..(198) <223> M27 <400> 15 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaggg agagatacca 120 gatggagtag ttggtgtaat tacacctgat atgccagatg ttctgtctca tgtgtcagtc 180

cgagcaagga atagcaag 198

<210> 16 <211> 214 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(214) <223> M1

<400> 16 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgg aagaaataca cctgatatgc cagatgttct 180

gtctcatgtg tcagtccgag caaggaatag caag 214

<210> 17 <211> 197 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(197) <223> M11 <400> 17 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tgtcagatgt tctgtctcat gtgtcagtcc 180

gagcaaggaa tagcaag 197

<210> 18 <211> 215 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(215) <223> M10

Page 9

AGR_PT024_1WO_SequenceListing_EFS <400> 18 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tgtaaattac acctgatatg ccagatgttc 180

tgtctcatgt gtcagtccga gcaaggaata gcaag 215

<210> 19 <211> 199 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(199) <223> M3 <400> 19 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagcacctga tatgccagat gttctgtctc atgtgtcagt 180

ccgagcaagg aatagcaag 199

<210> 20 <211> 208 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(208) <223> M8

<400> 20 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tatgccagat atgccagatg ttctgtctca 180 tgtgtcagtc cgagcaagga atagcaag 208

<210> 21 <211> 189 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(189) <223> M14 <400> 21 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

Page 10

AGR_PT024_1WO_SequenceListing_EFS agaggaagaa ttacacctga tatgccagat gttctgtctc atgtgtcagt ccgagcaagg 180 aatagcaag 189

<210> 22 <211> 178 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(178) <223> M13

<400> 22 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg ttctgtctca tgtgtcagtc cgagcaagga atagcaag 178

<210> 23 <211> 176 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(176) <223> M12

<400> 23 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tgtcagtccg agcaaggaat agcaag 176

<210> 24 <211> 204 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(204) <223> M22

<400> 24 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tgtgatatgc cagatgttct gtctcatgtg 180

tcagtccgag caaggaatag caag 204

<210> 25 <211> 221 <212> DNA <213> Zea mays Page 11

AGR_PT024_1WO_SequenceListing_EFS

<220> <221> misc_feature <222> (1)..(221) <223> M23

<400> 25 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgg caaagataaa ccttgcacct gatatgccag 180 atgttctgtc tcatgtgtca gtccgagcaa ggaatagcaa g 221

<210> 26 <211> 213 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(213) <223> M24

<400> 26 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgg tgaattacac ctgatatgcc agatgttctg 180

tctcatgtgt cagtccgagc aaggaatagc aag 213

<210> 27 <211> 213 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(213) <223> M20 <400> 27 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgg tgtattacac ctgatatgcc agatgttctg 180 tctcatgtgt cagtccgagc aaggaatagc aag 213

<210> 28 <211> 181 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(181) Page 12

AGR_PT024_1WO_SequenceListing_EFS <223> M21 <400> 28 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaaat 120

cttatgataa accatgccag atgttctgtc tcatgtgtca gtccgagcaa ggaatagcaa 180 g 181

<210> 29 <211> 205 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(205) <223> M4 <400> 29 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtaattac acctgatatg ccagatgttc tgtctcatgt 180

gtcagtccga gcaaggaata gcaag 205

<210> 30 <211> 210 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(210) <223> M19

<400> 30 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tgtacacctg atatgccaga tgttctgtct 180

catgtgtcag tccgagcaag gaatagcaag 210

<210> 31 <211> 425 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(425) <223> M26 <400> 31 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

Page 13

AGR_PT024_1WO_SequenceListing_EFS tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagcagtgtg ctcgggtaca gcttcttatt tcaatgtctc 180 cagtgggcgt cttacctcta tgtttgtgtt tttttttaag tgcagaaata gagaaagttc 240

ttgcaaatat ctactctatg aaaaggacag ctatttggaa atatgtgaac agaactatcc 300 ccagttgctg ggaaaaacca agaagaaagt tccttcaaat atctactcca tgacgacaag 360 tgtctattac acctgatatg ccagatgttc tgtctcatgt gtcagtccga gcaaggaata 420

gcaag 425

<210> 32 <211> 212 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(212) <223> M25

<400> 32 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgg taattacacc tgatatgcca gatgttctgt 180

ctcatgtgtc agtccgagca aggaatagca ag 212

<210> 33 <211> 173 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(173) <223> M15 <400> 33 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtaattacac 120 ctgatatgcc agatgttctg tctcatgtgt cagtccgagc aaggaatagc aag 173

<210> 34 <211> 425 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(425) <223> M5 <400> 34 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 Page 14

AGR_PT024_1WO_SequenceListing_EFS tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgc agaattattg aattctttca taattgaact 180 ctatgatgat gctttacttg attgtattat attgatgctc aatcatatat tgatgattgt 240

tggaacttgc tctccgatgc aaggtgatcc aacgggggtg tgtcgcaacg taaacagggt 300 tttcgcacga gatggcaata gctctgttaa cctagcctct cacgggcact gtgcgggggt 360 atttaattac acctgatatg ccagatgttc tgtctcatgt gtcagtccga gcaaggaata 420

gcaag 425

<210> 35 <211> 208 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(208) <223> M2 <400> 35 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgt tacacctgat atgccagatg ttctgtctca 180

tgtgtcagtc cgagcaagga atagcaag 208

<210> 36 <211> 187 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(187) <223> M28 <400> 36 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa acacctgata tgccagatgt tctgtctcat gtgtcagtcc gagcaaggaa 180 tagcaag 187

<210> 37 <211> 213 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(213) <223> M6 Page 15

AGR_PT024_1WO_SequenceListing_EFS <400> 37 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgg taaattacac ctgatatgcc agatgttctg 180 tctcatgtgt cagtccgagc aaggaatagc aag 213

<210> 38 <211> 199 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(199) <223> M9 <400> 38 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tatgccagat gttctgtctc atgtgtcagt 180

ccgagcaagg aatagcaag 199

<210> 39 <211> 210 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(210) <223> M7 <400> 39 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tttacacctg atatgccaga tgttctgtct 180

catgtgtcag tccgagcaag gaatagcaag 210

<210> 40 <211> 212 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(212) <223> M29

<400> 40 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 Page 16

AGR_PT024_1WO_SequenceListing_EFS agaggaagaa ataccagatg gagtagttgg taattacacc tgatatgcca gatgttctgt 180

ctcatgtgtc agtccgagca aggaatagca ag 212

<210> 41 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, Meganuclease GWD target sequence pAG4715 <400> 41 atccttgtgg caaagagtgt ca 22

<210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, Meganuclease target sequence pAG4716

<400> 42 gtagttggtg taattacacc tg 22

<210> 43 <211> 1469 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(1469) <223> ZmGWD

<400> 43 Met Ser Gly Phe Ser Ala Ala Ala Asn Ala Ala Ala Ala Glu Arg Cys 1 5 10 15

Ala Leu Ala Phe Arg Ala Arg Pro Ala Ala Ser Ser Pro Ala Lys Arg 20 25 30

Gln Gln Gln Pro Gln Pro Ala Ser Leu Arg Arg Ser Gly Gly Gln Arg 35 40 45

Arg Pro Thr Thr Leu Ser Ala Ser Ser Arg Gly Pro Val Val Pro Arg 50 55 60

Ala Val Ala Thr Ser Ala Asp Arg Ala Ser Pro Asp Leu Ile Gly Lys 70 75 80

Phe Thr Leu Asp Ser Asn Ser Glu Leu Gln Val Ala Val Asn Pro Ala 85 90 95

Pro Gln Gly Leu Val Ser Glu Ile Ser Leu Glu Val Thr Asn Thr Ser Page 17

AGR_PT024_1WO_SequenceListing_EFS 100 105 110

Gly Ser Leu Ile Leu His Trp Gly Ala Leu Arg Pro Asp Lys Arg Asp 115 120 125

Trp Ile Leu Pro Ser Arg Lys Pro Asp Gly Thr Thr Val Tyr Lys Asn 130 135 140

Arg Ala Leu Arg Thr Pro Phe Val Lys Ser Gly Asp Asn Ser Thr Leu 145 150 155 160

Arg Ile Glu Ile Asp Asp Pro Gly Val His Ala Ile Glu Phe Leu Ile 165 170 175

Phe Asp Glu Thr Gln Asn Lys Trp Phe Lys Asn Asn Gly Gln Asn Phe 180 185 190

Gln Val Gln Phe Gln Ser Ser Arg His Gln Gly Thr Gly Ala Ser Gly 195 200 205

Ala Ser Ser Ser Ala Thr Ser Thr Leu Val Pro Glu Asp Leu Val Gln 210 215 220

Ile Gln Ala Tyr Leu Arg Trp Glu Arg Arg Gly Lys Gln Ser Tyr Thr 225 230 235 240

Pro Glu Gln Glu Lys Glu Glu Tyr Glu Ala Ala Arg Ala Glu Leu Ile 245 250 255

Glu Glu Val Asn Arg Gly Val Ser Leu Glu Lys Leu Arg Ala Lys Leu 260 265 270

Thr Lys Ala Pro Glu Ala Pro Glu Ser Asp Glu Ser Lys Ser Ser Ala 275 280 285

Ser Arg Met Pro Ile Gly Lys Leu Pro Glu Asp Leu Val Gln Val Gln 290 295 300

Ala Tyr Ile Arg Trp Glu Gln Ala Gly Lys Pro Asn Tyr Pro Pro Glu 305 310 315 320

Lys Gln Leu Val Glu Phe Glu Glu Ala Arg Lys Glu Leu Gln Ala Glu 325 330 335

Val Asp Lys Gly Ile Ser Ile Asp Gln Leu Arg Gln Lys Ile Leu Lys 340 345 350

Gly Asn Ile Glu Ser Lys Val Ser Lys Gln Leu Lys Asn Lys Lys Tyr 355 360 365

Phe Ser Val Glu Arg Ile Gln Arg Lys Lys Arg Asp Ile Thr Gln Leu Page 18

AGR_PT024_1WO_SequenceListing_EFS 370 375 380

Leu Ser Lys His Lys His Thr Leu Val Glu Asp Lys Val Glu Val Val 385 390 395 400

Pro Lys Gln Pro Thr Val Leu Asp Leu Phe Thr Lys Ser Leu His Glu 405 410 415

Lys Asp Gly Cys Glu Val Leu Ser Arg Lys Leu Phe Lys Phe Gly Asp 420 425 430

Lys Glu Ile Leu Ala Ile Ser Thr Lys Val Gln Asn Lys Thr Glu Val 435 440 445

His Leu Ala Thr Asn His Thr Asp Pro Leu Ile Leu His Trp Ser Leu 450 455 460

Ala Lys Asn Ala Gly Glu Trp Lys Ala Pro Ser Pro Asn Ile Leu Pro 465 470 475 480

Ser Gly Ser Thr Leu Leu Asp Lys Ala Cys Glu Thr Glu Phe Thr Lys 485 490 495

Ser Glu Leu Asp Gly Leu His Tyr Gln Val Val Glu Ile Glu Leu Asp 500 505 510

Asp Gly Gly Tyr Lys Gly Met Pro Phe Val Leu Arg Ser Gly Glu Thr 515 520 525

Trp Ile Lys Asn Asn Gly Ser Asp Phe Phe Leu Asp Phe Ser Thr His 530 535 540

Asp Val Arg Asn Ile Lys Leu Lys Gly Asn Gly Asp Ala Gly Lys Gly 545 550 555 560

Thr Ala Lys Ala Leu Leu Glu Arg Ile Ala Asp Leu Glu Glu Asp Ala 565 570 575

Gln Arg Ser Leu Met His Arg Phe Asn Ile Ala Ala Asp Leu Ala Asp 580 585 590

Gln Ala Arg Asp Ala Gly Leu Leu Gly Ile Val Gly Leu Phe Val Trp 595 600 605

Ile Arg Phe Met Ala Thr Arg Gln Leu Thr Trp Asn Lys Asn Tyr Asn 610 615 620

Val Lys Pro Arg Glu Ile Ser Lys Ala Gln Asp Arg Phe Thr Asp Asp 625 630 635 640

Leu Glu Asn Met Tyr Lys Ala Tyr Pro Gln Tyr Arg Glu Ile Leu Arg Page 19

AGR_PT024_1WO_SequenceListing_EFS 645 650 655

Met Ile Met Ala Ala Val Gly Arg Gly Gly Glu Gly Asp Val Gly Gln 660 665 670

Arg Ile Arg Asp Glu Ile Leu Val Ile Gln Arg Asn Asn Asp Cys Lys 675 680 685

Gly Gly Met Met Glu Glu Trp His Gln Lys Leu His Asn Asn Thr Ser 690 695 700

Pro Asp Asp Val Val Ile Cys Gln Ala Leu Ile Asp Tyr Ile Lys Ser 705 710 715 720

Asp Phe Asp Ile Ser Val Tyr Trp Asp Thr Leu Asn Lys Asn Gly Ile 725 730 735

Thr Lys Glu Arg Leu Leu Ser Tyr Asp Arg Ala Ile His Ser Glu Pro 740 745 750

Asn Phe Arg Ser Glu Gln Lys Ala Gly Leu Leu Arg Asp Leu Gly Asn 755 760 765

Tyr Met Arg Ser Leu Lys Ala Val His Ser Gly Ala Asp Leu Glu Ser 770 775 780

Ala Ile Ala Ser Cys Met Gly Tyr Lys Ser Glu Gly Glu Gly Phe Met 785 790 795 800

Val Gly Val Gln Ile Asn Pro Val Lys Gly Leu Pro Ser Gly Phe Pro 805 810 815

Glu Leu Leu Glu Phe Val Leu Glu His Val Glu Asp Lys Ser Ala Glu 820 825 830

Pro Leu Leu Glu Gly Leu Leu Glu Ala Arg Val Glu Leu Arg Pro Leu 835 840 845

Leu Leu Asp Ser Arg Glu Arg Met Lys Asp Leu Ile Phe Leu Asp Ile 850 855 860

Ala Leu Asp Ser Thr Phe Arg Thr Ala Ile Glu Arg Ser Tyr Glu Glu 865 870 875 880

Leu Asn Asp Ala Ala Pro Glu Lys Ile Met Tyr Phe Ile Ser Leu Val 885 890 895

Leu Glu Asn Leu Ala Leu Ser Ile Asp Asp Asn Glu Asp Ile Leu Tyr 900 905 910

Cys Leu Lys Gly Trp Asn Gln Ala Leu Glu Met Ala Lys Gln Lys Asp Page 20

AGR_PT024_1WO_SequenceListing_EFS 915 920 925

Asp Gln Trp Ala Leu Tyr Ala Lys Ala Phe Leu Asp Arg Asn Arg Leu 930 935 940

Ala Leu Ala Ser Lys Gly Glu Gln Tyr His Asn Met Met Gln Pro Ser 945 950 955 960

Ala Glu Tyr Leu Gly Ser Leu Leu Ser Ile Asp Gln Trp Ala Val Asn 965 970 975

Ile Phe Thr Glu Glu Ile Ile Arg Gly Gly Ser Ala Ala Thr Leu Ser 980 985 990

Ala Leu Leu Asn Arg Phe Asp Pro Val Leu Arg Asn Val Ala His Leu 995 1000 1005

Gly Ser Trp Gln Val Ile Ser Pro Val Glu Val Ser Gly Tyr Val 1010 1015 1020

Val Val Val Asp Glu Leu Leu Ala Val Gln Asn Lys Ser Tyr Asp 1025 1030 1035

Lys Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu 1040 1045 1050

Ile Pro Asp Gly Val Val Gly Val Ile Thr Pro Asp Met Pro Asp 1055 1060 1065

Val Leu Ser His Val Ser Val Arg Ala Arg Asn Ser Lys Val Leu 1070 1075 1080

Phe Ala Thr Cys Phe Asp His Thr Thr Leu Ser Glu Leu Glu Gly 1085 1090 1095

Tyr Asp Gln Lys Leu Phe Ser Phe Lys Pro Thr Ser Ala Asp Ile 1100 1105 1110

Thr Tyr Arg Glu Ile Thr Glu Ser Glu Leu Gln Gln Ser Ser Ser 1115 1120 1125

Pro Asn Ala Glu Val Gly His Ala Val Pro Ser Ile Ser Leu Ala 1130 1135 1140

Lys Lys Lys Phe Leu Gly Lys Tyr Ala Ile Ser Ala Glu Glu Phe 1145 1150 1155

Ser Glu Glu Met Val Gly Ala Lys Ser Arg Asn Ile Ala Tyr Leu 1160 1165 1170

Lys Gly Lys Val Pro Ser Trp Val Gly Val Pro Thr Ser Val Ala Page 21

AGR_PT024_1WO_SequenceListing_EFS 1175 1180 1185

Ile Pro Phe Gly Thr Phe Glu Lys Val Leu Ser Asp Gly Leu Asn 1190 1195 1200

Lys Glu Val Ala Gln Ser Ile Glu Lys Leu Lys Ile Arg Leu Ala 1205 1210 1215

Gln Glu Asp Phe Ser Ala Leu Gly Glu Ile Arg Lys Val Val Leu 1220 1225 1230

Asn Leu Thr Ala Pro Met Gln Leu Val Asn Glu Leu Lys Glu Arg 1235 1240 1245

Met Leu Gly Ser Gly Met Pro Trp Pro Gly Asp Glu Gly Asp Lys 1250 1255 1260

Arg Trp Glu Gln Ala Trp Met Ala Ile Lys Lys Val Trp Ala Ser 1265 1270 1275

Lys Trp Asn Glu Arg Ala Tyr Phe Ser Thr Arg Lys Val Lys Leu 1280 1285 1290

Asp His Glu Tyr Leu Ser Met Ala Val Leu Val Gln Glu Val Val 1295 1300 1305

Asn Ala Asp Tyr Ala Phe Val Ile His Thr Thr Asn Pro Ser Ser 1310 1315 1320

Gly Asp Ser Ser Glu Ile Tyr Ala Glu Val Val Lys Gly Leu Gly 1325 1330 1335

Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Met Ser Phe Val 1340 1345 1350

Cys Lys Lys Asp Asp Leu Asp Ser Pro Lys Leu Leu Gly Tyr Pro 1355 1360 1365

Ser Lys Pro Ile Gly Leu Phe Ile Arg Gln Ser Ile Ile Phe Arg 1370 1375 1380

Ser Asp Ser Asn Gly Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly 1385 1390 1395

Leu Tyr Asp Ser Val Pro Met Asp Glu Glu Asp Glu Val Val Leu 1400 1405 1410

Asp Tyr Thr Thr Asp Pro Leu Ile Val Asp Arg Gly Phe Arg Ser 1415 1420 1425

Ser Ile Leu Ser Ser Ile Ala Arg Ala Gly His Ala Ile Glu Glu Page 22

AGR_PT024_1WO_SequenceListing_EFS 1430 1435 1440

Leu Tyr Gly Ser Pro Gln Asp Val Glu Gly Val Val Lys Asp Gly 1445 1450 1455

Lys Ile Tyr Val Val Gln Thr Arg Pro Gln Met 1460 1465

<210> 44 <211> 1469 <212> PRT <213> Sorghum bicolor

<220> <221> MISC_FEATURE <222> (1)..(1469) <223> SbGWD <400> 44 Met Thr Gly Phe Ser Ala Ala Ala Ser Ala Ala Ala Ala Ala Glu Arg 1 5 10 15

Cys Ala Leu Ala Ile Arg Ala Arg Pro Ala Ala Ser Ser Pro Ala Lys 20 25 30

Arg Gln Gln Gln Ser Ala Ser Leu Arg Arg Ser Gly Gly Gln Arg Arg 35 40 45

Pro Thr Thr Leu Ala Ala Ser Arg Arg Ser Pro Val Val Val Pro Arg 50 55 60

Ala Ile Ala Thr Ser Ala Asp Arg Ala Ser His Asp Leu Val Gly Lys 70 75 80

Phe Thr Leu Asp Ser Asn Ser Glu Leu Leu Val Ala Val Asn Pro Ala 85 90 95

Pro Gln Gly Leu Val Ser Val Ile Gly Leu Glu Val Thr Asn Thr Ser 100 105 110

Gly Ser Leu Ile Leu His Trp Gly Val Leu Arg Pro Asp Lys Arg Asp 115 120 125

Trp Ile Leu Pro Ser Arg Gln Pro Asp Gly Thr Thr Val Tyr Lys Asn 130 135 140

Arg Ala Leu Arg Thr Pro Phe Val Lys Ser Gly Asp Asn Ser Thr Leu 145 150 155 160

Arg Ile Glu Ile Asp Asp Pro Ala Val Gln Ala Ile Glu Phe Leu Ile 165 170 175

Page 23

AGR_PT024_1WO_SequenceListing_EFS Phe Gly Glu Thr Gln Asn Lys Trp Phe Lys Asn Asn Gly Gln Asn Phe 180 185 190

Gln Ile Gln Leu Gln Ser Ser Arg His Gln Gly Asn Gly Ala Ser Gly 195 200 205

Ala Ser Ser Ser Ala Thr Ser Thr Leu Val Pro Glu Asp Leu Val Gln 210 215 220

Ile Gln Ala Tyr Leu Arg Trp Glu Arg Lys Gly Lys Gln Ser Tyr Thr 225 230 235 240

Pro Glu Gln Glu Lys Glu Glu Tyr Glu Ala Ala Arg Ala Glu Leu Ile 245 250 255

Glu Glu Leu Asn Arg Gly Val Ser Leu Glu Lys Leu Arg Ala Lys Leu 260 265 270

Thr Lys Thr Pro Glu Ala Pro Glu Ser Asp Glu Arg Lys Ser Pro Ala 275 280 285

Ser Arg Met Pro Val Asp Lys Leu Pro Glu Asp Leu Val Gln Val Gln 290 295 300

Ala Tyr Ile Arg Trp Glu Lys Ala Gly Lys Pro Asn Tyr Pro Pro Glu 305 310 315 320

Lys Gln Leu Val Glu Leu Glu Glu Ala Arg Lys Glu Leu Gln Ala Glu 325 330 335

Val Asp Lys Gly Ile Ser Ile Asp Gln Leu Arg Gln Lys Ile Leu Lys 340 345 350

Gly Asn Ile Glu Ser Lys Val Ser Lys Gln Leu Lys Asn Lys Lys Tyr 355 360 365

Phe Ser Val Glu Arg Ile Gln Arg Lys Lys Arg Asp Ile Met Gln Leu 370 375 380

Leu Ser Lys His Lys His Thr Val Met Glu Glu Lys Val Glu Val Ala 385 390 395 400

Pro Lys Gln Pro Thr Val Leu Asp Leu Phe Thr Lys Ser Leu His Glu 405 410 415

Lys Asp Gly Cys Glu Val Leu Ser Arg Lys Leu Phe Lys Phe Gly Asp 420 425 430

Lys Glu Ile Leu Ala Ile Ser Thr Lys Val Gln Asn Lys Thr Glu Val 435 440 445

Page 24

AGR_PT024_1WO_SequenceListing_EFS His Leu Ala Thr Asn His Thr Glu Pro Leu Ile Leu His Trp Ser Leu 450 455 460

Ala Lys Lys Ala Gly Glu Trp Lys Ala Pro Pro Ser Asn Ile Leu Pro 465 470 475 480

Ser Gly Ser Lys Leu Leu Asp Met Ala Cys Glu Thr Glu Phe Thr Arg 485 490 495

Ser Glu Leu Asp Gly Leu Cys Tyr Gln Val Val Glu Ile Glu Leu Asp 500 505 510

Asp Gly Gly Tyr Lys Gly Met Pro Phe Val Leu Arg Ser Gly Glu Thr 515 520 525

Trp Ile Lys Asn Asn Gly Ser Asp Phe Phe Leu Asp Phe Ser Thr Arg 530 535 540

Asp Thr Arg Asn Ile Lys Leu Lys Asp Asn Gly Asp Ala Gly Lys Gly 545 550 555 560

Thr Ala Lys Ala Leu Leu Glu Arg Ile Ala Asp Leu Glu Glu Asp Ala 565 570 575

Gln Arg Ser Leu Met His Arg Phe Asn Ile Ala Ala Asp Leu Ala Asp 580 585 590

Glu Ala Arg Asp Ala Gly Leu Leu Gly Ile Val Gly Leu Phe Val Trp 595 600 605

Ile Arg Phe Met Ala Thr Arg Gln Leu Thr Trp Asn Lys Asn Tyr Asn 610 615 620

Val Lys Pro Arg Glu Ile Ser Lys Ala Gln Asp Arg Phe Thr Asp Asp 625 630 635 640

Leu Glu Asn Met Tyr Arg Thr Tyr Pro Gln Tyr Arg Glu Ile Leu Arg 645 650 655

Met Ile Met Ala Ala Val Gly Arg Gly Gly Glu Gly Asp Val Gly Gln 660 665 670

Arg Ile Arg Asp Glu Ile Leu Val Ile Gln Arg Asn Asn Asp Cys Lys 675 680 685

Gly Gly Met Met Glu Glu Trp His Gln Lys Leu His Asn Asn Thr Ser 690 695 700

Pro Asp Asp Val Val Ile Cys Gln Ala Leu Ile Asp Tyr Ile Lys Asn 705 710 715 720

Page 25

AGR_PT024_1WO_SequenceListing_EFS Asp Phe Asp Ile Ser Val Tyr Trp Asp Thr Leu Asn Lys Asn Gly Ile 725 730 735

Thr Lys Glu Arg Leu Leu Ser Tyr Asp Arg Ala Ile His Ser Glu Pro 740 745 750

Asn Phe Arg Ser Glu Gln Lys Glu Gly Leu Leu Arg Asp Leu Gly Asn 755 760 765

Tyr Met Arg Ser Leu Lys Ala Val His Ser Gly Ala Asp Leu Glu Ser 770 775 780

Ala Ile Ala Thr Cys Met Gly Tyr Lys Ser Glu Gly Glu Gly Phe Met 785 790 795 800

Val Gly Val Gln Ile Asn Pro Val Lys Gly Leu Pro Ser Gly Phe Pro 805 810 815

Glu Leu Leu Glu Phe Val Leu Asp His Val Glu Asp Lys Ser Ala Glu 820 825 830

Pro Leu Leu Glu Gly Leu Leu Glu Ala Arg Val Asp Leu Arg Pro Leu 835 840 845

Leu Leu Asp Ser Pro Glu Arg Met Lys Asp Leu Ile Phe Leu Asp Ile 850 855 860

Ala Leu Asp Ser Thr Phe Arg Thr Ala Ile Glu Arg Ser Tyr Glu Glu 865 870 875 880

Leu Asn Asp Ala Ala Pro Glu Lys Ile Met Tyr Phe Ile Ser Leu Val 885 890 895

Leu Glu Asn Leu Ala Phe Ser Ile Asp Asp Asn Glu Asp Ile Leu Tyr 900 905 910

Cys Leu Lys Gly Trp Asn Gln Ala Leu Glu Met Ala Lys Gln Lys Asp 915 920 925

Asp Gln Trp Ala Leu Tyr Ala Lys Ala Phe Leu Asp Arg Ile Arg Leu 930 935 940

Ala Leu Ala Ser Lys Gly Glu Gln Tyr His Asn Met Met Gln Pro Ser 945 950 955 960

Ala Glu Tyr Leu Gly Ser Leu Leu Ser Ile Asp Lys Trp Ala Val Asn 965 970 975

Ile Phe Thr Glu Glu Ile Ile Arg Gly Gly Ser Ala Ala Thr Leu Ser 980 985 990

Page 26

AGR_PT024_1WO_SequenceListing_EFS Ala Leu Leu Asn Arg Phe Asp Pro Val Leu Arg Asn Val Ala Asn Leu 995 1000 1005

Gly Ser Trp Gln Val Ile Ser Pro Val Glu Val Ser Gly Tyr Val 1010 1015 1020

Val Val Val Asp Glu Leu Leu Ala Val Gln Asn Lys Ser Tyr Asp 1025 1030 1035

Lys Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu 1040 1045 1050

Ile Pro Asp Gly Val Val Gly Val Ile Thr Pro Asp Met Pro Asp 1055 1060 1065

Val Leu Ser His Val Ser Val Arg Ala Arg Asn Ser Lys Val Leu 1070 1075 1080

Phe Ala Thr Cys Phe Asp His Thr Thr Leu Ser Glu Leu Glu Gly 1085 1090 1095

Tyr Asp Gln Lys Leu Leu Ser Phe Lys Pro Thr Ser Ala Asp Ile 1100 1105 1110

Thr Tyr Arg Glu Ile Thr Glu Ser Glu Leu Gln Gln Ser Ser Ser 1115 1120 1125

Pro Asn Ala Glu Val Gly His Ala Val Pro Ser Ile Ser Leu Ala 1130 1135 1140

Lys Lys Lys Phe Leu Gly Lys Tyr Ala Ile Ser Ala Glu Glu Phe 1145 1150 1155

Thr Glu Glu Met Val Gly Ala Lys Ser Arg Asn Ile Ala Tyr Leu 1160 1165 1170

Lys Gly Lys Val Pro Ser Trp Val Gly Val Pro Thr Ser Val Ala 1175 1180 1185

Ile Pro Phe Gly Thr Phe Glu Lys Val Leu Ser Asp Gly Leu Asn 1190 1195 1200

Lys Glu Val Ala Gln Thr Ile Glu Lys Leu Lys Ile Arg Leu Ala 1205 1210 1215

Gln Glu Asp Phe Ser Ala Leu Gly Glu Ile Arg Lys Ala Val Leu 1220 1225 1230

Asn Leu Thr Ala Pro Met Gln Leu Val Asn Glu Leu Lys Glu Arg 1235 1240 1245

Page 27

AGR_PT024_1WO_SequenceListing_EFS Met Leu Gly Ser Gly Met Pro Trp Pro Gly Asp Glu Gly Asn Arg 1250 1255 1260

Arg Trp Glu Gln Ala Trp Met Ala Ile Lys Lys Val Trp Ala Ser 1265 1270 1275

Lys Trp Asn Glu Arg Ala Tyr Phe Ser Thr Arg Lys Val Lys Leu 1280 1285 1290

Asn His Glu Tyr Leu Ser Met Ala Val Leu Val Gln Glu Val Val 1295 1300 1305

Asn Ala Asp Tyr Ala Phe Val Ile His Thr Thr Asn Pro Ser Ser 1310 1315 1320

Gly Asp Ser Ser Glu Ile Tyr Ala Glu Val Val Lys Gly Leu Gly 1325 1330 1335

Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Met Ser Phe Val 1340 1345 1350

Cys Lys Lys Asp Asp Leu Asp Ser Pro Lys Leu Leu Gly Tyr Pro 1355 1360 1365

Ser Lys Pro Ile Gly Leu Phe Ile Arg Arg Ser Ile Ile Phe Arg 1370 1375 1380

Ser Asp Ser Asn Gly Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly 1385 1390 1395

Leu Tyr Asp Ser Val Pro Met Asp Glu Glu Asp Glu Val Val Leu 1400 1405 1410

Asp Tyr Thr Thr Asp Pro Leu Ile Val Asp Arg Gly Phe Arg Asn 1415 1420 1425

Ser Ile Leu Ser Ser Ile Ala Arg Ala Gly His Ala Ile Glu Glu 1430 1435 1440

Leu Tyr Gly Ser Pro Gln Asp Val Glu Gly Val Val Lys Asp Gly 1445 1450 1455

Lys Ile Tyr Val Val Gln Thr Arg Pro Gln Met 1460 1465

<210> 45 <211> 74 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE Page 28

AGR_PT024_1WO_SequenceListing_EFS <222> (1)..(74) <223> ZmGWD_M1_aa_1040-1120

<400> 45 Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Arg Asn Thr Pro Asp Met Pro Asp Val Leu Ser 20 25 30

His Val Ser Val Arg Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly 35 40 45

Glu Glu Glu Ile Pro Asp Gly Val Val Gly Arg Asn Thr Pro Asp Met 50 55 60

Pro Asp Val Leu Ser His Val Ser Val Arg 70

<210> 46 <211> 79 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(79) <223> ZmGWD_M2 _1040-1120)

<400> 46

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Val Thr Pro Asp Met Pro Asp Val Leu Ser His Val 20 25 30

Ser Val Arg Ala Arg Asn Ser Lys Val Leu Phe Ala Thr Cys Phe Asp 35 40 45

His Thr Thr Leu Ser Glu Leu Glu Gly Tyr Asp Gln Lys Leu Phe Ser 50 55 60

Phe Lys Pro Thr Ser Ala Asp Ile Thr Tyr Arg Glu Ile Thr Glu 70 75

<210> 47 <211> 76 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(76) <223> ZmGWD_M3 _aa1040-1120 Page 29

AGR_PT024_1WO_SequenceListing_EFS <400> 47

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Ala Pro Asp Met Pro Asp Val Leu Ser His Val Ser Val Arg 20 25 30

Ala Arg Asn Ser Lys Val Leu Phe Ala Thr Cys Phe Asp His Thr Thr 35 40 45

Leu Ser Glu Leu Glu Gly Tyr Asp Gln Lys Leu Phe Ser Phe Lys Pro 50 55 60

Thr Ser Ala Asp Ile Thr Tyr Arg Glu Ile Thr Glu 70 75

<210> 48 <211> 78 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(78) <223> ZmGWD_M4 _aa_1040-1120

<400> 48

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Ile Thr Pro Asp Met Pro Asp Val Leu Ser His Val Ser 20 25 30

Val Arg Ala Arg Asn Ser Lys Val Leu Phe Ala Thr Cys Phe Asp His 35 40 45

Thr Thr Leu Ser Glu Leu Glu Gly Tyr Asp Gln Lys Leu Phe Ser Phe 50 55 60

Lys Pro Thr Ser Ala Asp Ile Thr Tyr Arg Glu Ile Thr Glu 70 75

<210> 49 <211> 95 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(95) <223> ZmGWD_M5_aa_1040-1120

<400> 49 Page 30

AGR_PT024_1WO_SequenceListing_EFS Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Ala Glu Leu Leu Asn Ser Phe Ile Ile Glu Leu Tyr 20 25 30

Asp Asp Ala Leu Leu Asp Cys Ile Ile Leu Met Leu Asn His Ile Leu 35 40 45

Met Ile Val Gly Thr Cys Ser Pro Met Gln Gly Asp Pro Thr Gly Val 50 55 60

Cys Arg Asn Val Asn Arg Val Phe Ala Arg Asp Gly Asn Ser Ser Val 70 75 80

Asn Leu Ala Ser His Gly His Cys Ala Gly Val Phe Asn Tyr Thr 85 90 95

<210> 50 <211> 73 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(73) <223> ZmGWD_M6 _aa_1040-1120

<400> 50

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Lys Leu His Leu Ile Cys Gln Met Phe Cys Leu 20 25 30

Met Cys Gln Ser Glu Gln Gly Ile Ala Arg Tyr Cys Leu Arg Pro Val 35 40 45

Leu Thr Thr Pro Leu Tyr Leu Asn Leu Lys Asp Met Ile Arg Asn Cys 50 55 60

Phe Pro Ser Ser Leu Leu Leu Gln Ile 70

<210> 51 <211> 72 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(72) <223> ZmGWD_M7 _aa_1040-1120 Page 31

AGR_PT024_1WO_SequenceListing_EFS <400> 51

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Leu His Leu Ile Cys Gln Met Phe Cys Leu Met 20 25 30

Cys Gln Ser Glu Gln Gly Ile Ala Arg Tyr Cys Leu Arg Pro Val Leu 35 40 45

Thr Thr Pro Leu Tyr Leu Asn Leu Lys Asp Met Ile Arg Asn Cys Phe 50 55 60

Pro Ser Ser Leu Leu Leu Gln Ile 70

<210> 52 <211> 79 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(79) <223> ZmGWD_M8 _aa_1040-1120

<400> 52

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Met Pro Asp Met Pro Asp Val Leu Ser His Val 20 25 30

Ser Val Arg Ala Arg Asn Ser Lys Val Leu Phe Ala Thr Cys Phe Asp 35 40 45

His Thr Thr Leu Ser Glu Leu Glu Gly Tyr Asp Gln Lys Leu Phe Ser 50 55 60

Phe Lys Pro Thr Ser Ala Asp Ile Thr Tyr Arg Glu Ile Thr Glu 70 75

<210> 53 <211> 76 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(76) <223> ZmGWD_M9_aa_1040-1120

<400> 53 Page 32

AGR_PT024_1WO_SequenceListing_EFS Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Met Pro Asp Val Leu Ser His Val Ser Val Arg 20 25 30

Ala Arg Asn Ser Lys Val Leu Phe Ala Thr Cys Phe Asp His Thr Thr 35 40 45

Leu Ser Glu Leu Glu Gly Tyr Asp Gln Lys Leu Phe Ser Phe Lys Pro 50 55 60

Thr Ser Ala Asp Ile Thr Tyr Arg Glu Ile Thr Glu 70 75

<210> 54 <211> 25 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <223> ZmGWD_M10 _aa_1040-1120

<400> 54

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Val Asn Tyr Thr 20 25

<210> 55 <211> 34 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(34) <223> ZmGWD_M11_aa_1040-1120 <400> 55

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Val Arg Cys Ser Val Ser Cys Val Ser Pro Ser 20 25 30

Lys Glu

<210> 56 <211> 27 Page 33

AGR_PT024_1WO_SequenceListing_EFS <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(27) <223> ZmGWD_M12 _1040-1120 <400> 56 Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Val Ser Pro Ser Lys Glu 20 25

<210> 57 <211> 60 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(60) <223> ZmGWD_M13 _aa_1040-1120

<400> 57 Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Val Leu Ser His Val Ser Val Arg Ala Arg Asn Ser Lys Val Leu 20 25 30

Phe Ala Thr Cys Phe Asp His Thr Thr Leu Ser Glu Leu Glu Gly Tyr 35 40 45

Asp Gln Lys Leu Phe Ser Phe Lys Pro Thr Ser Ala 50 55 60

<210> 58 <211> 65 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(65) <223> ZmGWD_M14_aa_1040-1120 <400> 58

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Leu His 1 5 10 15

Leu Ile Cys Gln Met Phe Cys Leu Met Cys Gln Ser Glu Gln Gly Ile 20 25 30

Page 34

AGR_PT024_1WO_SequenceListing_EFS Ala Arg Tyr Cys Leu Arg Pro Val Leu Thr Thr Pro Leu Tyr Leu Asn 35 40 45

Leu Lys Asp Met Ile Arg Asn Cys Phe Pro Ser Ser Leu Leu Leu Gln 50 55 60

Ile

<210> 59 <211> 11 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(11) <223> ZmGWD_M15_aa_1040-1120 <400> 59

Pro Thr Ile Leu Val Ala Lys Ser Asn Tyr Thr 1 5 10

<210> 60 <211> 10 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(10) <223> ZmGWD_M16_aa_1040-1120

<400> 60 Pro Arg Glu Arg Lys Lys Tyr Gln Met Glu 1 5 10

<210> 61 <211> 15 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(15) <223> ZmGWD_M17_aa_1040-1120

<400> 61 Pro Thr Ile Leu Val Ala Arg Glu Arg Lys Lys Tyr Gln Met Glu 1 5 10 15

<210> 62 <211> 18 <212> PRT <213> Zea mays Page 35

AGR_PT024_1WO_SequenceListing_EFS

<220> <221> MISC_FEATURE <222> (1)..(18) <223> ZmGWD_M18_aa_1040-1120

<400> 62 Pro Thr Ile Leu Val Ala Arg Val Ser Arg Glu Arg Lys Lys Tyr Gln 1 5 10 15

Met Glu

<210> 63 <211> 71 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(71) <223> ZmGWD_M19 _aa_1040-1120

<400> 63

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Val His Leu Ile Cys Gln Met Phe Cys Leu Met 20 25 30

Cys Gln Ser Glu Gln Gly Ile Ala Arg Tyr Cys Leu Arg Pro Val Leu 35 40 45

Thr Thr Pro Leu Tyr Leu Asn Leu Lys Asp Met Ile Arg Asn Cys Phe 50 55 60

Pro Ser Ser Leu Leu Leu Gln 70

<210> 64 <211> 73 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(73) <223> ZmGWD_M20_aa_1040-1120

<400> 64 Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Val Leu His Leu Ile Cys Gln Met Phe Cys Leu Page 36

AGR_PT024_1WO_SequenceListing_EFS 20 25 30

Met Cys Gln Ser Glu Gln Gly Ile Ala Arg Tyr Cys Leu Arg Pro Val 35 40 45

Leu Thr Thr Pro Leu Tyr Leu Asn Leu Lys Asp Met Ile Arg Asn Cys 50 55 60

Phe Pro Ser Ser Leu Leu Leu Gln Ile 70

<210> 65 <211> 12 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(12) <223> ZmGWD_M21_aa_1040-1120

<400> 65

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Ile Leu 1 5 10

<210> 66 <211> 70 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(70) <223> ZmGWD_M22_aa_1040-1120 <400> 66

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Val Ile Cys Gln Met Phe Cys Leu Met Cys Gln 20 25 30

Ser Glu Gln Gly Ile Ala Arg Tyr Cys Leu Arg Pro Val Leu Thr Thr 35 40 45

Pro Leu Tyr Leu Asn Leu Lys Asp Met Ile Arg Asn Cys Phe Pro Ser 50 55 60

Ser Leu Leu Leu Gln Ile 70

<210> 67 <211> 27 <212> PRT Page 37

AGR_PT024_1WO_SequenceListing_EFS <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(27) <223> ZmGWD_M23_aa_1040-1120 <400> 67 Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Lys Asp Lys Pro Cys Thr 20 25

<210> 68 <211> 73 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(73) <223> ZmGWD_M24_aa_1040-1120

<400> 68

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Glu Leu His Leu Ile Cys Gln Met Phe Cys Leu 20 25 30

Met Cys Gln Ser Glu Gln Gly Ile Ala Arg Tyr Cys Leu Arg Pro Val 35 40 45

Leu Thr Thr Pro Leu Tyr Leu Asn Leu Lys Asp Met Ile Arg Asn Cys 50 55 60

Phe Pro Ser Ser Leu Leu Leu Gln Ile 70

<210> 69 <211> 24 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(24) <223> ZmGWD_M25 _aa_1040-1120 <400> 69

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Page 38

AGR_PT024_1WO_SequenceListing_EFS Asp Gly Val Val Gly Asn Tyr Thr 20

<210> 70 <211> 95 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(95) <223> ZmGWD_M26_aa_1040-1120 <400> 70 Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Ala Val Cys Ser Gly Thr Ala Ser Tyr Phe Asn Val Ser Ser 20 25 30

Gly Arg Leu Thr Ser Met Phe Val Phe Phe Phe Lys Cys Arg Asn Arg 35 40 45

Glu Ser Ser Cys Lys Tyr Leu Leu Tyr Glu Lys Asp Ser Tyr Leu Glu 50 55 60

Ile Cys Glu Gln Asn Tyr Pro Gln Leu Leu Gly Lys Thr Lys Lys Lys 70 75 80

Val Pro Ser Asn Ile Tyr Ser Met Thr Thr Ser Val Tyr Tyr Thr 85 90 95

<210> 71 <211> 13 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(13) <223> ZmGWD_M27_aa_1040-1120 <400> 71

Pro Thr Ile Leu Val Ala Arg Glu Arg Tyr Gln Met Glu 1 5 10

<210> 72 <211> 72 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(72) <223> ZmGWD_M28_aa_1040-1120 Page 39

AGR_PT024_1WO_SequenceListing_EFS <400> 72

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Thr Pro 1 5 10 15

Asp Met Pro Asp Val Leu Ser His Val Ser Val Arg Ala Arg Asn Ser 20 25 30

Lys Val Leu Phe Ala Thr Cys Phe Asp His Thr Thr Leu Ser Glu Leu 35 40 45

Glu Gly Tyr Asp Gln Lys Leu Phe Ser Phe Lys Pro Thr Ser Ala Asp 50 55 60

Ile Thr Tyr Arg Glu Ile Thr Glu 70

<210> 73 <211> 24 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(24) <223> ZmGWD_M29 _aa_1040-1120

<400> 73

Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Asn Tyr Thr 20

<210> 74 <211> 1417 <212> PRT <213> Streptococcus pyogenes

<220> <221> MISC_FEATURE <222> (1)..(1147) <223> Cas9 protein <400> 74

Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp 1 5 10 15

Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val 20 25 30

Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu 35 40 45

Page 40

AGR_PT024_1WO_SequenceListing_EFS Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 50 55 60

Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His 70 75 80

Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 85 90 95

Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 100 105 110

Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 115 120 125

Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe 130 135 140

Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn 145 150 155 160

Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His 165 170 175

Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 180 185 190

Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu 195 200 205

Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 210 215 220

Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 225 230 235 240

Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 245 250 255

Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys 260 265 270

Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 275 280 285

Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 290 295 300

Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 305 310 315 320

Page 41

AGR_PT024_1WO_SequenceListing_EFS Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 325 330 335

Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 340 345 350

Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 355 360 365

Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu 370 375 380

Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly 385 390 395 400

Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys 405 410 415

Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 420 425 430

Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser 435 440 445

Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 450 455 460

Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 465 470 475 480

Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg 485 490 495

Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile 500 505 510

Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln 515 520 525

Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 530 535 540

Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 545 550 555 560

Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 565 570 575

Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 580 585 590

Page 42

AGR_PT024_1WO_SequenceListing_EFS Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 595 600 605

Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp 610 615 620

Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile 625 630 635 640

Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu 645 650 655

Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu 660 665 670

Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 675 680 685

Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 690 695 700

Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 705 710 715 720

Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile 725 730 735

His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val 740 745 750

Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly 755 760 765

Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 770 775 780

Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 785 790 795 800

Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 805 810 815

Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 820 825 830

Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 835 840 845

Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp 850 855 860

Page 43

AGR_PT024_1WO_SequenceListing_EFS Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile 865 870 875 880

Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu 885 890 895

Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu 900 905 910

Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 915 920 925

Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 930 935 940

Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu 945 950 955 960

Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser 965 970 975

Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 980 985 990

Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp 995 1000 1005

Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala 1010 1015 1020

His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys 1025 1030 1035

Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys 1040 1045 1050

Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile 1055 1060 1065

Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn 1070 1075 1080

Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1085 1090 1095

Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp 1100 1105 1110

Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met 1115 1120 1125

Page 44

AGR_PT024_1WO_SequenceListing_EFS Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1130 1135 1140

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 1145 1150 1155

Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe 1160 1165 1170

Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1175 1180 1185

Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu 1190 1195 1200

Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile 1205 1210 1215

Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu 1220 1225 1230

Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly 1235 1240 1245

Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn 1250 1255 1260

Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala 1265 1270 1275

Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln 1280 1285 1290

Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 1295 1300 1305

Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp 1310 1315 1320

Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1325 1330 1335

Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr 1340 1345 1350

Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1355 1360 1365

Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1370 1375 1380

Page 45

AGR_PT024_1WO_SequenceListing_EFS Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg 1385 1390 1395

Ile Asp Leu Ser Gln Leu Gly Gly Asp Arg Pro Lys Lys Lys Arg 1400 1405 1410

Lys Val Gly Gly 1415

<210> 75 <211> 4272 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ZmCas9 <400> 75 ggatcctaaa ccatggatta caaggaccac gacggcgatt acaaggacca cgacattgat 60 tacaaggacg acgacgataa gatggctccc aagaagaaga ggaaggttgg catccacggg 120

gtgccggctg ctgacaagaa gtactcgatc ggcctcgata ttgggactaa ctctgttggc 180

tgggccgtga tcaccgacga gtacaaggtg ccctcgaaga agttcaaggt cctgggcaac 240

accgatcggc attctatcaa gaagaatctc attggcgctc tcctgttcga ctcaggggag 300 accgctgagg ctacgaggct caagaggacc gcccgcaggc ggtacacgcg caggaagaat 360

cgcatctgct acctgcagga gattttctcc aacgagatgg cgaaggttga cgattctttc 420

ttccacaggc tggaggagtc attcctcgtg gaggaggata agaagcacga gcggcatcca 480

atcttcggca acattgtcga cgaggttgcc taccacgaga agtaccctac gatctaccat 540 ctgcggaaga agctcgtgga ctccacagat aaggcggacc tccgcctgat ctacctcgct 600

ctggcccaca tgattaagtt caggggccat ttcctgatcg agggggatct caacccggac 660

aatagcgatg ttgacaagct gttcatccag ctcgtgcaga cgtacaacca gctcttcgag 720

gagaacccca ttaatgcgtc aggcgtcgac gcgaaggcta tcctgtccgc tcgcctctcg 780 aagtctagga ggctggagaa cctgatcgcc cagctgccgg gcgagaagaa gaacggcctg 840

ttcgggaatc tcatcgctct cagcctgggg ctcacgccaa acttcaagtc gaatttcgat 900 ctcgctgagg acgccaagct gcagctctcc aaggacacat acgacgatga cctggataac 960

ctcctggccc agatcggcga tcagtacgcg gacctgttcc tcgctgccaa gaatctgtcg 1020 gacgccatcc tcctgtctga tattctcagg gtgaacaccg agattacgaa ggctccgctc 1080

tcagcctcca tgatcaagcg ctacgacgag caccatcagg atctgaccct cctgaaggcg 1140 ctggtcaggc agcagctccc cgagaagtac aaggagattt tcttcgatca gtccaagaac 1200 ggctacgctg ggtacattga cggcggggcc agccaggagg agttctacaa gttcatcaag 1260

ccgattctgg agaagatgga cggcacggag gagctcctgg tgaagctcaa tcgcgaggac 1320 ctcctgagga agcagcggac attcgataac ggcagcatcc cacaccagat tcatctcggg 1380

Page 46

AGR_PT024_1WO_SequenceListing_EFS gagctgcacg ccatcctgag gcggcaggag gacttctacc ctttcctcaa ggataaccgc 1440 gagaagatcg agaagattct gaccttccgc atcccgtact acgtcggccc actcgcccgc 1500 ggcaactccc gcttcgcttg gatgacccgc aagtcagagg agaccatcac gccgtggaac 1560

ttcgaggagg tggtcgacaa gggcgctagc gctcagtcgt tcatcgagag gatgacgaat 1620 ttcgacaaga acctgccaaa tgagaaggtg ctccctaagc actcgctcct gtacgagtac 1680 ttcacagtct acaacgagct cactaaggtg aagtatgtga ccgagggcat gaggaagccg 1740

gctttcctgt ctggggagca gaagaaggcc atcgtggacc tcctgttcaa gaccaaccgg 1800 aaggtcacgg ttaagcagct caaggaggac tacttcaaga agattgagtg cttcgattcg 1860

gtcgagatca gcggcgttga ggacaggttc aacgcctccc tggggaccta ccacgatctc 1920 ctgaagatca ttaaggataa ggacttcctg gacaacgagg agaatgagga tatcctggag 1980

gacattgtgc tgacactcac tctgttcgag gaccgggaga tgatcgagga gcgcctgaag 2040 acttacgccc atctcttcga tgacaaggtc atgaagcagc tcaagaggag gaggtacacc 2100 ggctggggga ggctgagcag gaagctcatc aacggcattc gggacaagca gtccgggaag 2160

acgatcctcg acttcctgaa gagcgatggc ttcgcgaacc gcaatttcat gcagctgatt 2220

cacgatgaca gcctcacatt caaggaggat atccagaagg ctcaggtgag cggccagggg 2280

gactcgctgc acgagcatat cgcgaacctc gctggctcgc cagctatcaa gaaggggatt 2340 ctgcagaccg tgaaggttgt ggacgagctc gtgaaggtca tgggcaggca caagcctgag 2400

aacatcgtca ttgagatggc ccgcgagaat cagaccacgc agaagggcca gaagaactca 2460

cgcgagagga tgaagaggat cgaggagggc attaaggagc tggggtccca gatcctcaag 2520

gagcacccgg tggagaacac gcagctgcag aatgagaagc tctacctgta ctacctccag 2580 aatggccgcg atatgtatgt ggaccaggag ctggatatta acaggctcag cgattacgac 2640

gtcgatcata tcgttccaca gtcattcctg aaggatgact ccattgacaa caaggtcctc 2700

accaggtcgg acaagaaccg gggcaagtct gataatgttc cttcagagga ggtcgttaag 2760

aagatgaaga actactggcg ccagctcctg aatgccaagc tgatcacgca gcggaagttc 2820 gataacctca caaaggctga gaggggcggg ctctctgagc tggacaaggc gggcttcatc 2880

aagaggcagc tggtcgagac acggcagatc actaagcacg ttgcgcagat tctcgactca 2940 cggatgaaca ctaagtacga tgagaatgac aagctgatcc gcgaggtgaa ggtcatcacc 3000

ctgaagtcaa agctcgtctc cgacttcagg aaggatttcc agttctacaa ggttcgggag 3060 atcaacaatt accaccatgc ccatgacgcg tacctgaacg cggtggtcgg cacagctctg 3120

atcaagaagt acccaaagct ggagtccgag ttcgtgtacg gggactacaa ggtttacgat 3180 gtgcgcaaga tgatcgccaa gtcggagcag gagattggca aggctaccgc caagtacttc 3240 ttctactcta acattatgaa tttcttcaag acagagatca ctctggccaa tggcgagatc 3300

cggaagcgcc ccctcattga gaccaacggc gagacggggg agatcgtgtg ggacaagggc 3360 agggatttcg cgaccgtcag gaaggttctc tccatgccac aagtgaatat cgtcaagaag 3420

Page 47

AGR_PT024_1WO_SequenceListing_EFS acagaggtcc agactggcgg gttctctaag gagtcaattc tgcctaagcg gaacagcgac 3480 aagctcatcg cccgcaagaa ggactgggac ccgaagaagt acggcgggtt cgacagcccc 3540 actgtggcct actcggtcct ggttgtggcg aaggttgaga agggcaagtc caagaagctc 3600

aagagcgtga aggagctcct ggggatcacg attatggaga ggtccagctt cgagaagaac 3660 ccgatcgatt tcctggaggc gaagggctac aaggaggtga agaaggacct gatcattaag 3720 ctccccaagt actcactctt cgagctggag aacggcagga agcggatgct ggcttccgct 3780

ggcgagctcc agaaggggaa tgagctcgct ctgccgtcca agtatgtgaa cttcctctac 3840 ctggcctccc actacgagaa gctcaagggc agccccgagg acaacgagca gaagcagctg 3900

ttcgtcgagc agcacaagca ttacctcgac gagatcattg agcagatttc cgagttcagc 3960 aagcgcgtga tcctggccga cgcgaatctg gataaggtcc tcagcgcgta caacaagcac 4020

cgcgacaagc caatcaggga gcaggctgag aatatcattc atctcttcac cctgacgaac 4080 ctcggcgccc ctgctgcttt caagtacttc gacacaacta tcgatcgcaa gaggtacaca 4140 tcgactaagg aggtcctgga cgcgaccctc atccaccagt ctattacagg cctgtacgag 4200

actcggattg atctgtcgca gctcggcggg gataggccca agaagaagag gaaggtcggc 4260

ggctgaccta gg 4272

<210> 76 <211> 1007 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(1007) <223> MzU3.8

<400> 76 gaattccatc taagtatctt ggtaaagcat ggattaattt ggatgctcac ttcaggtcta 60

tgcagctccg gtgccttgtg attgtgagtt gtgaccgatg ctcatgctat tttgcatttc 120 tgcgatgtat gatgctagta gatcttcaaa actaacagcg catgccatca tcatccactg 180 cttgatttta gtctcaccgc tggccaaaaa tgtgatgatg ccagaaacct caactacctt 240

gaatcaacac gggcccagca gtgtgatgac gacagaaacc aaaaaaaaat gagccaatag 300 ttcagaagga ggcactatgc agaaactaca tttctgaagg tgactaaaag gtgagcgtag 360 agtgtactta ctagtagttt agccaccatt acccaaatgc tttcgagctt gtattaagac 420

ttcctaagct gagcatcatc actgatctgc aggagggtcg cttcgctgcc aagatcaaca 480 gcaaccatgt ggcggcaaca tccagcattg cacatgggct aaagattgag ctctgtgcca 540

agtgtgagct gcaaccatct agggatcagc tgagtttatc agtctttcct ttttttcatt 600 ctggtgaggc atcaagctac tactgcctcg atcggttgga cttggacctg aagcccacat 660 gtaggatacc agaatggacc gacccaggac gtagtgccac ctcggttgtc acactgcgta 720

gaagccagct taaaaattta gctttggtga ctcacagcac gaccttactt gaacaggatc 780 Page 48

AGR_PT024_1WO_SequenceListing_EFS tgttctatag gatcgtactg ttgcatcttt gattaataag aaggcaagta cttaaacctg 840

gttgatgaga atttgacctg tgggccagag cgtgatttaa cggccaggac tttgccttgg 900 tgcattgtct ggagctgcag atgatcgttc ttggccaggc ttaatgtctg gctagggtgg 960

cctacaggct gtttgacagg tttctcaatt tttttgctct gctgcag 1007

<210> 77 <211> 1501 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(1501) <223> ZmU3 <400> 77 tagttgtcta ttaataaatt ttatcatgtg tagctgactt aaaagacatg taatctagtg 60 cgcatgcaat ctcagcatgc aaacatatat atttttgaac ttgtgatatt tttatacagt 120

atatcataat agataaaatt agacaacaca gaactaaaat tataatatta atactaattt 180

ggaccatacc attaccaaat atgttgaact aaatcattct tgaagtcaat atgcttttat 240

agtttgatat atccatgatt tctgaattcc atctaagtat gttggtaaag catggattaa 300 tttggatgcc cacttcaggt ctatgcagct ccggtgcctt gtgattgtga gttgtgaccg 360

atgctcatgc tattctgcat ttctgcgatg tatgtagcta gtagatcttc aaaactaaca 420

ccgcatgcca tcatcatcca ctgcttgatt ttagtctcac cgctggccaa aaatgtgatg 480

atgccagaaa cctcaactac cttgaatcaa cacgggccca acagtgtgat gacgacagaa 540 acaaaaaaaa atgagccaat agttcagaag gaggcactat gcagaaacta catttctgaa 600

ggtgactaaa aggtgagcgt agagtgtaat tactagtagt ttagccacca ttacccaaat 660

gctttcgagc ttgtattaag atttcctaag ctgagcatca tcactgatct gcaggccacc 720

ctcgcttcgc tgccaagatc aacagcaacc atgtggcggc aacatccagc attgcacatg 780 ggctaaagat tgagctttgt gcctcgtcta gggatcagct gaggttatca gtctttcctt 840

tttttcatcc aggtgaggca tcaagctact actgcctcga ttggctggac ccgaagccca 900 catgtaggat accagaatgg gccgacccag gacgcagtat gttggccagt cccaccggtt 960

agtgccatct cggttgctca catgcgtaga agccagctta aaaatttagc tttggtaact 1020 cacagcacga ccttacttga acaggatctg ttctatagga tcgtactgtt gcatctttga 1080

ttaataagaa ggcaagtact taaacctggt tgatgagaat ttgacctgtg ggccagagcg 1140 tgattaacgg ccaggactct ttgccttggt gcattgtctg gagctgcaga tgatcgttct 1200 tggccaggct taatgtctgg ctagggtggc ctacaggctg tttgacaggt ctctcaattt 1260

ttttgctctg ctgcaggtga tcatttgact caacgccatt aatgattgac tttttgatct 1320 gtgctgcgtt tgaagaaacc tactccagct agcttttcct cagcatttgc actcaaatta 1380

Page 49

AGR_PT024_1WO_SequenceListing_EFS agagggccag atatcttgct cgcttttgcc atcagtaata aagttttcct taggtgtgat 1440 gcatttgaag gggatttaag gaggttattt ctgtcaccag ctgtttttgc ttagtgttgc 1500 t 1501

<210> 78 <211> 761 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(761) <223> MzU3.8 promoter region <400> 78 gaattccatc taagtatctt ggtaaagcat ggattaattt ggatgctcac ttcaggtcta 60 mtgcagctcc ggtgccttgt gattgtgagt tgtgaccgat gctcatgcta ttttgcattt 120 cmtgcgatgt atgatgctag tagatcttca aaactaacag cgcatgccat catcatccac 180

tgcttgattt tagtctcacc gctggccaaa aatgtgatga tgccagaaac ctcaactacc 240 ttgaatcaac acgggcccag cagtgtgatg acgacagaaa ccaaaaaaaa atgagccaat 300

agttcagaag gaggcactat gcagaaacta catttctgaa ggtgactaaa aggtgagcgt 360

agagtgtact tactagtagt ttagccacca ttacccaaat gctttcgagc ttgtattaag 420

acttcctaag ctgagcatca tcactgatct gcaggagggt cgcttcgctg ccaagatcaa 480

cagcaaccat gtggcggcaa catccagcat tgcacatggg ctaaagattg agctctgtgc 540 caagtgtgag ctgcaaccat ctagggatca gctgagttta tcagtctttc ctttttttca 600

ttctggtgag gcatcaagct actactgcct cgatcggttg gacttggacc tgaagcccac 660

atgtaggata ccagaatgga ccgacccagg acgtagtgcc acctcggttg tcacactgcg 720 tagaagccag cttaaaaatt tagctttggt gactcacagc a 761

<210> 79 <211> 764 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(764) <223> ZmU3 promoter region

<400> 79 gaattccatc taagtatgtt ggtaaagcat ggattaattt ggatgcccac ttcaggtcta 60 tgcagctccg gtgccttgtg attgtgagtt gtgaccgatg ctcatgctat tctgcatttc 120 tgcgatgtat gtagctagta gatcttcaaa actaacaccg catgccatca tcatccactg 180

cttgatttta gtctcaccgc tggccaaaaa tgtgatgatg ccagaaacct caactacctt 240 gaatcaacac gggcccaaca gtgtgatgac gacagaaaca aaaaaaaatg agccaatagt 300

Page 50

AGR_PT024_1WO_SequenceListing_EFS tcagaaggag gcactatgca gaaactacat ttctgaaggt gactaaaagg tgagcgtaga 360 gtgtaattac tagtagttta gccaccatta cccaaatgct ttcgagcttg tattaagatt 420 tcctaagctg agcatcatca ctgatctgca ggccaccctc gcttcgctgc caagatcaac 480

agcaaccatg tggcggcaac atccagcatt gcacatgggc taaagattga gctttgtgcc 540 tcgtctaggg atcagctgag gttatcagtc tttccttttt ttcatccagg tgaggcatca 600 agctactact gcctcgattg gctggacccg aagcccacat gtaggatacc agaatgggcc 660

gacccaggac gcagtatgtt ggccagtccc accggttagt gccatctcgg ttgctcacat 720 gcgtagaagc cagcttaaaa atttagcttt ggtaactcac agca 764

<210> 80 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ob 2297 forward primer

<400> 80 gcgatcgcca tctaagtatg ttggtaaagc atgg 34

<210> 81 <211> 36 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, ob2299 reverse primer <400> 81 tgctgtgagt taccaaagct aaatttttaa gctggc 36

<210> 82 <211> 758 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(758) <223> ZmU3P1 <400> 82 catctaagta tgttggtaaa gcatggatta atttggatgc ccacttcagg tctatgcagc 60 tccggtgcct tgtgattgtg agttgtgacc gatgctcatg ctattctgca tttctgcgat 120

gtatgtagct agtagatctt caaaactaac accgcatgcc atcatcatcc actgcttgat 180 tttagtctca ccgctggcca aaaatgtgat gatgccagaa acctcaacta ccttgaatca 240

acacgggccc aacagtgtga tgacgacaga aacaaaaaaa aatgagccaa tagttcagaa 300 ggaggcacta tgcagaaact acatttctga aggtgactaa aaggtgagcg tagagtgtaa 360 ttactagtag tttagccacc attacccaaa tgctttcgag cttgtattaa gatttcctaa 420

gctgagcatc atcactgatc tgcaggccac cctcgcttcg ctgccaagat caacagcaac 480 Page 51

AGR_PT024_1WO_SequenceListing_EFS catgtggcgg caacatccag cattgcacat gggctaaaga ttgagctttg tgcctcgtct 540

agggatcagc tgaggttatc agtctttcct ttttttcatc caggtgaggc atcaagctac 600 tactgcctcg attggctgga cccgaagccc acatgtagga taccagaatg ggccgaccca 660

ggacgcagta tgttggccag tcccaccggt tagtgccatc tcggttgctc acatgcgtag 720 aagccagctt aaaaatttag ctttggtaac tcacagca 758

<210> 83 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ob2343 forward primer

<400> 83 gcgatcgcag tttagccacc attacccaaa tgc 33

<210> 84 <211> 398 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(398) <223> ZmU3P2

<400> 84 gcgatcgcag tttagccacc attacccaaa tgctttcgag cttgtattaa gatttcctaa 60

gctgagcatc atcactgatc tgcaggccac cctcgcttcg ctgccaagat caacagcaac 120 catgtggcgg caacatccag cattgcacat gggctaaaga ttgagctttg tgcctcgtct 180

agggatcagc tgaggttatc agtctttcct ttttttcatc caggtgaggc atcaagctac 240

tactgcctcg attggctgga cccgaagccc acatgtagga taccagaatg ggccgaccca 300

ggacgcagta tgttggccag tcccaccggt tagtgccatc tcggttgctc acatgcgtag 360 aagccagctt aaaaatttag ctttggtaac tcacagca 398

<210> 85 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ob2351 forward primer <400> 85 cgatttaaat agtttagcca ccattaccca aatgc 35

<210> 86 <211> 308 <212> DNA <213> Zea mays

Page 52

AGR_PT024_1WO_SequenceListing_EFS <220> <221> misc_feature <222> (1)..(308) <223> ZmU3.8P

<400> 86 gcgatcgcgc ttcgctgcca agatcaacag caaccatgtg gcggcaacat ccagcattgc 60 acatgggcta aagattgagc tctgtgccaa gtgtgagctg caaccatcta gggatcagct 120 gagtttatca gtctttcctt tttttcattc tggtgaggca tcaagctact actgcctcga 180

tcggttggac ttggacctga agcccacatg taggatacca gaatggaccg acccaggacg 240 tagtgccacc tcggttgtca cactgcgtag aagccagctt aaaaatttag ctttggtgac 300 tcacagca 308

<210> 87 <211> 42 <212> DNA <213> Streptococcus pyogenes

<220> <221> misc_feature <222> (1)..(42) <223> Cas9 handle hairpin

<400> 87 gttttagagc tagaaatagc aagttaaaat aaggctagtc cg 42

<210> 88 <211> 41 <212> DNA <213> Streptococcus pyogenes

<220> <221> misc_feature <222> (1)..(41) <223> S. pyogenes terminator

<400> 88 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt t 41

<210> 89 <211> 37 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(37) <223> ZmU3T

<400> 89 gctctgctgc aggtgatcat ttgactcaac gccatta 37

<210> 90 <211> 120 <212> DNA Page 53

AGR_PT024_1WO_SequenceListing_EFS <213> Artificial Sequence <220> <223> Synthetic construct, sgRNA scaffold <400> 90 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60 ggcaccgagt cggtgctttt tttgctctgc tgcaggtgat catttgactc aacgccatta 120

<210> 91 <211> 19 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, GWDe1a antisense

<400> 91 ggcatgaggt gcttacgtc 19

<210> 92 <211> 19 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, GWDe24b antisense

<400> 92 cataacctga tacttcaac 19

<210> 93 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, GWDe24c sense <400> 93 tctggctcct gctatcagt 19

<210> 94 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, GWDe25a antisense <400> 94 tctgcagaag taggcttga 19

<210> 95 <211> 911 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, ZmU3P1:sgRNA_GWDe24b

<400> 95 Page 54

AGR_PT024_1WO_SequenceListing_EFS gcgatcgcca tctaagtatg ttggtaaagc atggattaat ttggatgccc acttcaggtc 60 tatgcagctc cggtgccttg tgattgtgag ttgtgaccga tgctcatgct attctgcatt 120 tctgcgatgt atgtagctag tagatcttca aaactaacac cgcatgccat catcatccac 180

tgcttgattt tagtctcacc gctggccaaa aatgtgatga tgccagaaac ctcaactacc 240 ttgaatcaac acgggcccaa cagtgtgatg acgacagaaa caaaaaaaaa tgagccaata 300 gttcagaagg aggcactatg cagaaactac atttctgaag gtgactaaaa ggtgagcgta 360

gagtgtaatt actagtagtt tagccaccat tacccaaatg ctttcgagct tgtattaaga 420 tttcctaagc tgagcatcat cactgatctg caggccaccc tcgcttcgct gccaagatca 480

acagcaacca tgtggcggca acatccagca ttgcacatgg gctaaagatt gagctttgtg 540 cctcgtctag ggatcagctg aggttatcag tctttccttt ttttcatcca ggtgaggcat 600

caagctacta ctgcctcgat tggctggacc cgaagcccac atgtaggata ccagaatggg 660 ccgacccagg acgcagtatg ttggccagtc ccaccggtta gtgccatctc ggttgctcac 720 atgcgtagaa gccagcttaa aaatttagct ttggtaactc acagcacata acctgatact 780

tcaacgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 840

aaagtggcac cgagtcggtg ctttttttgc tctgctgcag gtgatcattt gactcaacgc 900

cattatacgt a 911

<210> 96 <211> 543 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, ZmU3P2:sgRNA_GWDe24b

<400> 96 gcgatcgcag tttagccacc attacccaaa tgctttcgag cttgtattaa gatttcctaa 60

gctgagcatc atcactgatc tgcaggccac cctcgcttcg ctgccaagat caacagcaac 120

catgtggcgg caacatccag cattgcacat gggctaaaga ttgagctttg tgcctcgtct 180 agggatcagc tgaggttatc agtctttcct ttttttcatc caggtgaggc atcaagctac 240

tactgcctcg attggctgga cccgaagccc acatgtagga taccagaatg ggccgaccca 300 ggacgcagta tgttggccag tcccaccggt tagtgccatc tcggttgctc acatgcgtag 360

aagccagctt aaaaatttag ctttggtaac tcacagcaca taacctgata cttcaacgtt 420 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 480

accgagtcgg tgcttttttt gctctgctgc aggtgatcat ttgactcaac gccattatac 540 gta 543

<210> 97 <211> 453 <212> DNA <213> Artificial Sequence

Page 55

AGR_PT024_1WO_SequenceListing_EFS <220> <223> Synthetic construct, ZmU3.8P:sgRNA_GWDe24b

<400> 97 gcgatcgcgc ttcgctgcca agatcaacag caaccatgtg gcggcaacat ccagcattgc 60

acatgggcta aagattgagc tctgtgccaa gtgtgagctg caaccatcta gggatcagct 120 gagtttatca gtctttcctt tttttcattc tggtgaggca tcaagctact actgcctcga 180 tcggttggac ttggacctga agcccacatg taggatacca gaatggaccg acccaggacg 240

tagtgccacc tcggttgtca cactgcgtag aagccagctt aaaaatttag ctttggtgac 300 tcacagcaca taacctgata cttcaacgtt ttagagctag aaatagcaag ttaaaataag 360

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gctctgctgc 420 aggtgatcat ttgactcaac gccattatac gta 453

<210> 98 <211> 543 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, ZmU3P2:sgRNA_GWDe24c

<400> 98 gcgatcgcag tttagccacc attacccaaa tgctttcgag cttgtattaa gatttcctaa 60 gctgagcatc atcactgatc tgcaggccac cctcgcttcg ctgccaagat caacagcaac 120

catgtggcgg caacatccag cattgcacat gggctaaaga ttgagctttg tgcctcgtct 180

agggatcagc tgaggttatc agtctttcct ttttttcatc caggtgaggc atcaagctac 240

tactgcctcg attggctgga cccgaagccc acatgtagga taccagaatg ggccgaccca 300 ggacgcagta tgttggccag tcccaccggt tagtgccatc tcggttgctc acatgcgtag 360

aagccagctt aaaaatttag ctttggtaac tcacagcatc tggctcctgc tatcagtgtt 420

ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 480

accgagtcgg tgcttttttt gctctgctgc aggtgatcat ttgactcaac gccattatac 540 gta 543

<210> 99 <211> 545 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ZmU3P2:sgRNA_GWDe24a <400> 99 atttaaatag tttagccacc attacccaaa tgctttcgag cttgtattaa gatttcctaa 60 gctgagcatc atcactgatc tgcaggccac cctcgcttcg ctgccaagat caacagcaac 120

catgtggcgg caacatccag cattgcacat gggctaaaga ttgagctttg tgcctcgtct 180 agggatcagc tgaggttatc agtctttcct ttttttcatc caggtgaggc atcaagctac 240

Page 56

AGR_PT024_1WO_SequenceListing_EFS tactgcctcg attggctgga cccgaagccc acatgtagga taccagaatg ggccgaccca 300 ggacgcagta tgttggccag tcccaccggt tagtgccatc tcggttgctc acatgcgtag 360 aagccagctt aaaaatttag ctttggtaac tcacagcatc tgcagaagta ggcttgagtt 420

ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 480 accgagtcgg tgcttttttt gctctgctgc aggtgatcat ttgactcaac gccattaggc 540 gcgcc 545

<210> 100 <211> 543 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ZmU3P2:sgRNA_GWDe1a <400> 100 gcgatcgcag tttagccacc attacccaaa tgctttcgag cttgtattaa gatttcctaa 60 gctgagcatc atcactgatc tgcaggccac cctcgcttcg ctgccaagat caacagcaac 120

catgtggcgg caacatccag cattgcacat gggctaaaga ttgagctttg tgcctcgtct 180

agggatcagc tgaggttatc agtctttcct ttttttcatc caggtgaggc atcaagctac 240

tactgcctcg attggctgga cccgaagccc acatgtagga taccagaatg ggccgaccca 300 ggacgcagta tgttggccag tcccaccggt tagtgccatc tcggttgctc acatgcgtag 360

aagccagctt aaaaatttag ctttggtaac tcacagcagg catgaggtgc ttacgtcgtt 420

ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 480

accgagtcgg tgcttttttt gctctgctgc aggtgatcat ttgactcaac gccattatac 540 gta 543

<210> 101 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, GWDe24b-F primer

<400> 101 ctcacagcac ataacctgat act 23

<210> 102 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Syntehtic construct, sgRNA-R primer <400> 102 cgactcggtg ccacttt 17

<210> 103 Page 57

AGR_PT024_1WO_SequenceListing_EFS <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, ZmCas9-F primer

<400> 103 agaatcagac cacgcagaag 20

<210> 104 <211> 22 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, ZmCas9-R primer

<400> 104 gctcctggtc cacatacata tc 22

<210> 105 <211> 21 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, GWDex23-F primer

<400> 105 tgctcttctg aaccgatttg a 21

<210> 106 <211> 227 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, Sb4715_1 (WT+INS)_Exon24 <400> 106 ttggcaggtt ataagcccag ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggagtagt tggtgtagtt 120 ggtgtatcaa gggagaggaa gaaataccag atggagtagt tggtgtaatt acacctgata 180

tgccagatgt tctgtcccat gtgtcagtcc gagcaaggaa tagcaag 227

<210> 107 <211> 202 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, Sb4715_2(WT+del)_Exon24

<400> 107 ttggcaggtt ataagcccag ttgaagtatc aggttatgtg gttgtggttg atgagttact 60

tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagttacacc tgatatgcca gatgttctgt cccatgtgtc 180

Page 58

AGR_PT024_1WO_SequenceListing_EFS agtccgagca aggaatagca ag 202

<210> 108 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, meganuclease 4715 <400> 108 atggcaccga agaagaagcg caaggtgcat atgaatacaa aatataataa agagttctta 60 ctctacttag cagggtttgt agacggtgac ggttccatct ttgccaggat caggccttct 120

caatctcgga agttcaagca ccagctgacg ctcgagttca aggtcactca gaagacacag 180 cgccgttggt tcctcgacaa gctggtggac gagatcggtg tgggttacgt gacggacgat 240

ggcagcgtct ccttttactc tctgtcccag atcaagcctt tgcataattt tttaacacaa 300 ctacaacctt ttctaaaact aaaacaaaaa caagcaagtt tagttttaaa aattattgag 360 caacttccgt cagcaaaaga atccccggac aaattcttag aagtttgtac atgggtggat 420

caaattgcag ctctgaatga ttcgaagacg cgtaaaacaa cttctgaaac cgttcgtgct 480

gtgctagaca gtttaccagg atccgtggga ggtctatcgc catctcaggc atccagcgcc 540

gcatcctcgg cttcctcaag cccgggttca gggatctccg aagcactcag agctggagca 600 ggttccggca ctggatacaa caaggaattc ctgctctacc tggcgggctt cgtcgacggg 660

gacggctcca tctatgccac tatcaggccg aggcagtcgg tgaagttcaa gcactttctg 720

gagctcagtt tcgctgtcta tcagaagaca cagcgccgtt ggttcctcga caagctggtg 780

gacgagatcg gtgtgggtta cgtgtatgac agtggcagta cttcccggta cctgctgtcc 840 gagatcaagc ctctgcacaa cttcctgacc cagctccagc ccttcctgaa gctcaagcag 900

aagcaggcca acctcgtgct gaagatcatc gagcagctgc cctccgctaa ggaatccccg 960

gacaagttcc tggaggtgtg cacctgggtg gaccagatcg ccgctctgaa cgactccaag 1020

acccgcaaga ccacttccga aaccgtccgc gccgttctag acagtctctc cgagaagaag 1080 aagtcgtccc cctaa 1095

<210> 109 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, meganuclease 4716 <400> 109 atggcaccga agaagaagcg caaggtgcat atgaatacaa aatataataa agagttctta 60 ctctacttag cagggtttgt agacggtgac ggttccatct atgcctcgat cacgcctagt 120

caacatctga agttcaagca ccagctgagg ctctggttcg atgtcgctca gaagacacag 180 cgccgttggc tcctcgacaa gctggtggac gagatcggtg tgggttacgt gtatgaccag 240

Page 59

AGR_PT024_1WO_SequenceListing_EFS ggcagcgtct cctattaccg tctgtccgag atcaagcctt tgcataattt tttaacacaa 300 ctacaacctt ttctaaaact aaaacaaaaa caagcaaatt tagttttaaa aattattgaa 360 caacttccgt cagcaaaaga atccccggac aaattcttag aagtttgtac atgggtggat 420

caaattgcag ctctgaatga ttcgaagacg cgtaaaacaa cttctgaaac cgttcgtgct 480 gtgctagaca gtttaccagg atccgtggga ggtctatcgc catctcaggc atccagcgcc 540 gcatcctcgg cttcctcaag cccgggttca gggatctccg aagcactcag agctggagca 600

ggttccggca ctggatacaa caaggaattc ctgctctacc tggcgggctt cgtcgacggg 660 gacggctcca tctatgcctg tatccatcct gatcaagcta ataagttcaa gcaccggctg 720

cggctctatt tcattgtcag tcagaagaca cagcgccgtt ggttcctcga caagctggtg 780 gacgagatcg gtgtgggtta cgtgtatgac aggggcggcg tctcccatta ccagctgtcc 840

cagatcaagc ctctgcacaa cttcctgacc cagctccagc ccttcctgaa gctcaagcag 900 aagcaggcca acctcgtgct gaagatcatc gagcagctgc cctccgccaa ggaatccccg 960 gacaagttcc tggaggtgtg cacctgggtg gaccagatcg ccgctctgaa cgactccaag 1020

acccgcaaga ccacttccga aaccgtccgc gccgttctag acagtctctc cgagaagaag 1080

aagtcgtccc cctaa 1095

<210> 110 <211> 21 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, GWDe24a-F

<400> 110 tgcagaagta ggcttgagtt t 21

<210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, 2856 forward primer

<400> 111 gaaggggatt ggagaggaag 20

<210> 112 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, 2858 reverse primer <400> 112 catgacgttc aaatagcctc a 21

<210> 113 Page 60

AGR_PT024_1WO_SequenceListing_EFS <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, 429 reverse primer

<400> 113 gcagaagtag gcttgaagga a 21

<210> 114 <211> 77 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M32

<400> 114 gctcctgcta tcagttggca ggttataagc ccggtttgaa gtatcaggtt atgtggttgt 60 ggttgatgag ttacttg 77

<210> 115 <211> 74 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M33

<400> 115 gctcctgcta tcagttggca ggttataagc ccggttagta tcaggttatg tggttgtggt 60

tgatgagtta cttg 74

<210> 116 <211> 73 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M34 <400> 116 gctcctgcta tcagttggca ggttataagc ccggttgtat caggttatgt ggttgtggtt 60

gatgagttac ttg 73

<210> 117 <211> 75 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M35

<400> 117 gctcctgcta tcagttggca ggttataagc ccggtgaagt atcaggttat gtggttgtgg 60

ttgatgagtt acttg 75

<210> 118 Page 61

AGR_PT024_1WO_SequenceListing_EFS <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M36

<400> 118 gctcctgcta tcagttggtt gtggttgatg agttacttg 39

<210> 119 <211> 19 <212> DNA <213> Artificial Sequence

<220> <223> Syntehtic construct, e25a - 48

<400> 119 cactctatct gcagatata 19

<210> 120 <211> 68 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, e25a+1

<400> 120 cactctatct gaacttgaag gatatgatca gaaactgttt tccttcacag cctacttctg 60

cagatata 68

<210> 121 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M32 peptide

<400> 121 Trp Gln Val Ile Ser Pro Val 1 5

<210> 122 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M33 peptide <400> 122

Trp Gln Val Ile Ser Pro Val Ser Ile Arg Leu Cys Gly Cys Gly 1 5 10 15

<210> 123 <211> 46 <212> PRT Page 62

AGR_PT024_1WO_SequenceListing_EFS <213> Artificial Sequence <220> <223> Synthetic construct, M34 peptide <400> 123

Trp Gln Val Ile Ser Pro Val Val Ser Gly Tyr Val Val Val Val Asp 1 5 10 15

Glu Leu Leu Ala Val Gln Asn Lys Ser Tyr Asp Lys Pro Thr Ile Leu 20 25 30

Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro Asp Gly 35 40 45

<210> 124 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M35 peptide <400> 124

Trp Gln Val Ile Ser Pro Val Lys Tyr Gln Val Met Trp Leu Trp Leu 1 5 10 15

Met Ser Tyr Leu Leu Ser Arg Thr Asn Leu Met Ile Asn Gln Pro Ser 20 25 30

Leu Trp Gln Arg Val Ser Arg Glu Arg Lys Lys Tyr Gln Met Glu 35 40 45

<210> 125 <211> 35 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M36 peptide <400> 125

Trp Leu Trp Leu Met Ser Tyr Leu Leu Ser Arg Thr Asn Leu Met Ile 1 5 10 15

Asn Gln Pro Ser Leu Trp Gln Arg Val Ser Arg Glu Arg Lys Lys Tyr 20 25 30

Gln Met Glu 35

<210> 126 <211> 19 <212> PRT <213> Artificial Sequence

Page 63

AGR_PT024_1WO_SequenceListing_EFS <220> <223> Synthetic construct, M38 peptide

<400> 126 Val Leu Phe Ala Thr Cys Phe Asp His Thr Thr Leu Ser Ala Asp Ile 1 5 10 15

Thr Tyr Arg

<210> 127 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M39 peptide <400> 127 Val Leu Phe Ala Thr Cys Phe Asp His Thr Thr Leu Ser Glu Leu Glu 1 5 10 15

Gly Tyr Asp Gln Lys Leu Phe Ser Phe Thr Ala Tyr Phe Cys Arg Tyr 20 25 30

Asn Leu

<210> 128 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, NLS 1 protein <400> 128

Met Pro Thr Glu Glu Arg Val Arg Lys Arg Lys Glu Ser Asn Arg Glu 1 5 10 15

Ser Ala Arg Arg Ser Arg Tyr Arg Lys Ala Ala His Leu Lys Glu Leu 20 25 30

<210> 129 <211> 27 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, NLS3 protein

<400> 129 Met Ala Arg Lys Arg Lys Glu Ser Asn Arg Glu Ser Ala Arg Arg Ser 1 5 10 15

Arg Tyr Arg Lys Ala Ala His Leu Lys Glu Leu Page 64

AGR_PT024_1WO_SequenceListing_EFS 20 25

<210> 130 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, NLS4 protein <400> 130

Met Ala Arg Lys Arg Lys Glu Ser Asn Arg Glu Ser Ala Arg Arg Ser 1 5 10 15

Arg Arg Ser Arg Tyr Arg Lys Val 20

<210> 131 <211> 54 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M40

<400> 131 gaaataccag atggagtagt tgtaattaca cctgatatgc cagatgttct gtct 54

<210> 132 <211> 58 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M41

<400> 132 gaaataccag atggagtagt tggtataaat tacacctgat atgccagatg ttctgtct 58

<210> 133 <211> 56 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M42 <400> 133 gaaataccag atggagtagt tggtgtatta cacctgatat gccagatgtt ctgtct 56

<210> 134 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M43 <400> 134 gaaataccag atggagtagt tggtgtagag taataacacc tgatatgcca gatgttctgt 60

Page 65

AGR_PT024_1WO_SequenceListing_EFS ct 62

<210> 135 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M44 <400> 135 gaaataccag atggagtagt tggtgttctg tct 33

<210> 136 <211> 21 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M45 <400> 136 gaaataccag atgttctgtc t 21

<210> 137 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M46

<400> 137 gaaataccag atggagtagt tggtgtatga acacgtaatt acacctgata tgccagatgt 60

tctgtct 67

<210> 138 <211> 28 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M47 <400> 138 gaaataccag atggagtagt tggtgtct 28

<210> 139 <211> 54 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M48

<400> 139 gaaataccag atggagtagt tggtgttaca cctgatatgc cagatgttct gtct 54

<210> 140 <211> 58 <212> DNA Page 66

AGR_PT024_1WO_SequenceListing_EFS <213> Artificial Sequence <220> <223> Synthetic construct, M49 <400> 140 gaaataccag atggagtagt tggtgtaaat tacacctgat atgccagatg ttctgtct 58

<210> 141 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M50 <400> 141 gaaataccag atgggatatg ccagatgttc tgtct 35

<210> 142 <211> 76 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M51

<400> 142 gaaataccag atggagtagt tggtgtctca tgccagatgt gaagaaatta cacctgatat 60 gccagatgtt ctgtct 76

<210> 143 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M52 <400> 143 gaaataccag atggagtagt tggtgatgtt ctgtct 36

<210> 144 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M53 <400> 144 gaaataccag atggagtagt tggtgtcaga tatgccagat gttctgtct 49

<210> 145 <211> 167 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M54

<400> 145 Page 67

AGR_PT024_1WO_SequenceListing_EFS gaaataccag atggagtagt tggtgcattt actcatattt tctgtgattg aatattcttt 60 tccagatgga gtgtcaaggg agaggaagaa ataccagatg gagtgtcaag ggagaggaag 120 aaataccaga tgaaggaaat acacctgata tgccagatgt tctgtct 167

<210> 146 <211> 38 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, M55 <400> 146 gaaataccag atggagttac acctgatatg ccagatgt 38

<210> 147 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M40 pepide <400> 147

Ile Pro Asp Gly Val Val Val Ile Thr Pro Asp Met Pro Asp Val Leu 1 5 10 15

Ser His Val Ser Val Arg Ala Arg Asn Ser Lys 20 25

<210> 148 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M41 peptide

<400> 148 Ile Pro Asp Gly Val Val Gly Ile Asn Tyr Thr 1 5 10

<210> 149 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M42 peptide <400> 149

Ile Pro Asp Gly Val Val Gly Val Leu His Leu Ile Cys Gln Met Phe 1 5 10 15

Cys Leu Met Cys Gln Ser Glu Gln Gly Ile Ala Arg Tyr Cys Leu Arg 20 25 30

Page 68

AGR_PT024_1WO_SequenceListing_EFS Pro Val Leu Thr Thr Pro Leu Tyr Leu Asn Leu Lys Asp Met Ile Arg 35 40 45

Asn Cys Phe Pro Ser Ser Leu Leu Leu 50 55

<210> 150 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M43 peptide <400> 150

Ile Pro Asp Gly Val Val Gly Val Glu 1 5

<210> 151 <211> 20 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M44 peptide

<400> 151

Ile Pro Asp Gly Val Val Gly Val Leu Ser His Val Ser Val Arg Ala 1 5 10 15

Arg Asn Ser Lys 20

<210> 152 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M45 peptide <400> 152

Ile Pro Asp Val Leu Ser His Val Ser Val Arg Ala Arg Asn Ser Lys 1 5 10 15

<210> 153 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M46 peptide <400> 153 Ile Pro Asp Gly Val Val Gly Val 1 5

Page 69

AGR_PT024_1WO_SequenceListing_EFS <210> 154 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M47 peptide <400> 154 Ile Pro Asp Gly Val Val Gly Val Ser Cys Val Ser Pro Ser Lys Glu 1 5 10 15

<210> 155 <211> 27 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M48 peptide <400> 155

Ile Pro Asp Gly Val Val Gly Val Thr Pro Asp Met Pro Asp Val Leu 1 5 10 15

Ser His Val Ser Val Arg Ala Arg Asn Ser Lys 20 25

<210> 156 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M49 peptide

<400> 156 Ile Pro Asp Gly Val Val Gly Val Asn Tyr Thr 1 5 10

<210> 157 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M50 peptide <400> 157

Ile Pro Asp Gly Ile Cys Gln Met Phe Cys Leu Met Phe Gln Ser Glu 1 5 10 15

Gln Gly Ile Ala Arg Tyr Cys Leu Arg Pro Val Leu Thr Thr Pro Leu 20 25 30

Tyr Leu Asn Leu Lys Asp Met Ile Arg Asn Cys Phe Pro Ser Ser Leu 35 40 45

Page 70

AGR_PT024_1WO_SequenceListing_EFS Leu Leu Gln Ile 50

<210> 158 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M51 peptide

<400> 158 Ile Pro Asp Gly Val Val Gly Val Ser Cys Gln Met 1 5 10

<210> 159 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M52 peptide <400> 159

Ile Pro Asp Gly Val Val Gly Asp Val Leu Ser His Val Ser Val Arg 1 5 10 15

Ala Arg Asn Ser Lys 20

<210> 160 <211> 23 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M53 peptide

<400> 160 Ile Pro Asp Gly Val Val Gly Val Arg Tyr Ala Arg Cys Ser Val Ser 1 5 10 15

Cys Val Ser Pro Ser Lys Glu 20

<210> 161 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, M54 peptide <400> 161 Ile Pro Asp Gly Val Val Gly Ala Phe Thr His Ile Phe Cys Asp 1 5 10 15

Page 71

AGR_PT024_1WO_SequenceListing_EFS <210> 162 <211> 24 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, M55 peptide <400> 162 Ile Pro Asp Gly Val Thr Pro Asp Met Pro Asp Val Leu Ser His Val 1 5 10 15

Ser Val Arg Ala Arg Asn Ser Lys 20

<210> 163 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, SV40 NLS coding sequence <400> 163 ccgaagaaga agcgcaaggt g 21

<210> 164 <211> 96 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, NLS1 coding sequence

<400> 164 atgcctaccg aggaaagagt gaggaaaaga aaggaatcca atagagaatc agccagacgc 60

tccagataca ggaaagccgc tcacctgaaa gaactg 96

<210> 165 <211> 81 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, NLS3 coding sequence <400> 165 atggccagga aaagaaagga atccaataga gaatcagcca gacgctccag atacaggaaa 60 gccgctcacc tgaaagaact g 81

<210> 166 <211> 72 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct, NLS4 coding sequence

<400> 166 Page 72

AGR_PT024_1WO_SequenceListing_EFS atggccagga aaagaaagga atccaataga gaatcagcca gacgctccag acgctccaga 60 tacaggaagg tg 72

<210> 167 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, NLS5 coding sequence

<400> 167 atgtcggagc gaaagcgacg agagaagctc 30

<210> 168 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, NLS6 coding sequence

<400> 168 atgatcagcg aggctcttcg caaagctata gggaagcgg 39

<210> 169 <211> 10 <212> PRT <213> Artificial Sequence

<220> <223> Synthetic construct, NLS5 peptide <400> 169

Met Ser Glu Arg Lys Arg Arg Glu Lys Leu 1 5 10

<210> 170 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, NLS6 peptide

<400> 170 Met Ile Ser Glu Ala Leu Arg Lys Ala Ile Gly Lys Arg 1 5 10

<210> 171 <211> 204 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(204) <223> T1_ZmGWDmega-2F-2R

Page 73

AGR_PT024_1WO_SequenceListing_EFS <400> 171 ggttataagc ccggttgaag tatcaggtta tgtggttgtg gttgatgagt tacttgctgt 60

ccagaacaaa tcttatgata aaccaaccat ccttgtggca aagagtgtca agggagagga 120 agaaatacca gatggagtag ttggtgtaat tacacctgat atgccagatg ttctgtctca 180

tgtgtcagtc cgagcaagga atag 204

<210> 172 <211> 560 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(560) <223> T2_GWDex23-F + ZmGWDmega-2R <400> 172 tgctcttctg aaccgatttg atcctgtttt aaggaatgtt gctcacctcg gaaggtaaaa 60 atgtaaaatc tatgactgct gttgaacttc ttttactttg tatccccagt atatgaacac 120

ataattctaa ggactacttt gggaactcaa atccccttcg ggattgaagg ggattggaga 180

ggaagttagt ttattttcac ctcaatcctc tcctatcccg aaggggattt gaggttccca 240

aagtagccct aaaagtgata ctagtgaccc tctccacaat tttatgcgaa ccacagaaat 300 taataatata ttctattact ctgcacctga catctggctc ctgctatcag ttggcaggtt 360

ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact tgctgtccag 420

aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg agaggaagaa 480

ataccagatg gagtagttgg tgtaattaca cctgatatgc cagatgttct gtctcatgtg 540 tcagtccgag caaggaatag 560

<210> 173 <211> 613 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(613) <223> T3_2856 + 2858

<400> 173 gaaggggatt ggagaggaag ttagtttatt ttcacctcaa tcctctccta tcccgaaggg 60

gatttgaggt tcccaaagta gccctaaaag tgatactagt gaccctctcc acaattttat 120 gcgaaccaca gaaattaata atatattcta ttactctgca cctgacatct ggctcctgct 180

atcagttggc aggttataag cccggttgaa gtatcaggtt atgtggttgt ggttgatgag 240 ttacttgctg tccagaacaa atcttatgat aaaccaacca tccttgtggc aaagagtgtc 300 aagggagagg aagaaatacc agatggagta gttggtgtaa ttacacctga tatgccagat 360

gttctgtctc atgtgtcagt ccgagcaagg aatagcaagg tttatcttca cagctatgtt 420 Page 74

AGR_PT024_1WO_SequenceListing_EFS gcaagatttc ttgaattttt tctcttgtat tgatgttgac atactagctt tttcctaatg 480

aaggtactgt ttgcgacctg ttttgaccac accactctat ctgaacttga aggatatgat 540 cagaaactgt tttccttcaa gcctacttct gcagatataa cctataggta cttgaggcta 600

tttgaacgtc atg 613

<210> 174 <211> 381 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(381) <223> T4_ZmGWDmega-2F + 429 <400> 174 ggttataagc ccggttgaag tatcaggtta tgtggttgtg gttgatgagt tacttgctgt 60 ccagaacaaa tcttatgata aaccaaccat ccttgtggca aagagtgtca agggagagga 120

agaaatacca gatggagtag ttggtgtaat tacacctgat atgccagatg ttctgtctca 180

tgtgtcagtc cgagcaagga atagcaaggt ttatcttcac agctatgttg caagatttct 240

tgaatttttt ctcttgtatt gatgttgaca tactagcttt ttcctaatga aggtactgtt 300 tgcgacctgt tttgaccaca ccactctatc tgaacttgaa ggatatgatc agaaactgtt 360

ttccttcaag cctacttctg c 381

<210> 175 <211> 737 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(737) <223> T5_GWDex23-F + 429 <400> 175 tgctcttctg aaccgatttg atcctgtttt aaggaatgtt gctcacctcg gaaggtaaaa 60

atgtaaaatc tatgactgct gttgaacttc ttttactttg tatccccagt atatgaacac 120 ataattctaa ggactacttt gggaactcaa atccccttcg ggattgaagg ggattggaga 180 ggaagttagt ttattttcac ctcaatcctc tcctatcccg aaggggattt gaggttccca 240

aagtagccct aaaagtgata ctagtgaccc tctccacaat tttatgcgaa ccacagaaat 300 taataatata ttctattact ctgcacctga catctggctc ctgctatcag ttggcaggtt 360

ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact tgctgtccag 420 aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg agaggaagaa 480 ataccagatg gagtagttgg tgtaattaca cctgatatgc cagatgttct gtctcatgtg 540

tcagtccgag caaggaatag caaggtttat cttcacagct atgttgcaag atttcttgaa 600 Page 75

AGR_PT024_1WO_SequenceListing_EFS ttttttctct tgtattgatg ttgacatact agctttttcc taatgaaggt actgtttgcg 660

acctgttttg accacaccac tctatctgaa cttgaaggat atgatcagaa actgttttcc 720 ttcaagccta cttctgc 737

<210> 176 <211> 778 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(778) <223> T6_GWDex23-F + 2858

<400> 176 tgctcttctg aaccgatttg atcctgtttt aaggaatgtt gctcacctcg gaaggtaaaa 60 atgtaaaatc tatgactgct gttgaacttc ttttactttg tatccccagt atatgaacac 120 ataattctaa ggactacttt gggaactcaa atccccttcg ggattgaagg ggattggaga 180

ggaagttagt ttattttcac ctcaatcctc tcctatcccg aaggggattt gaggttccca 240

aagtagccct aaaagtgata ctagtgaccc tctccacaat tttatgcgaa ccacagaaat 300

taataatata ttctattact ctgcacctga catctggctc ctgctatcag ttggcaggtt 360 ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact tgctgtccag 420

aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg agaggaagaa 480

ataccagatg gagtagttgg tgtaattaca cctgatatgc cagatgttct gtctcatgtg 540

tcagtccgag caaggaatag caaggtttat cttcacagct atgttgcaag atttcttgaa 600 ttttttctct tgtattgatg ttgacatact agctttttcc taatgaaggt actgtttgcg 660

acctgttttg accacaccac tctatctgaa cttgaaggat atgatcagaa actgttttcc 720

ttcaagccta cttctgcaga tataacctat aggtacttga ggctatttga acgtcatg 778

<210> 177 <211> 395 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(395) <223> T7_2856 + ZmGWDmega-2R

<400> 177 gaaggggatt ggagaggaag ttagtttatt ttcacctcaa tcctctccta tcccgaaggg 60

gatttgaggt tcccaaagta gccctaaaag tgatactagt gaccctctcc acaattttat 120 gcgaaccaca gaaattaata atatattcta ttactctgca cctgacatct ggctcctgct 180 atcagttggc aggttataag cccggttgaa gtatcaggtt atgtggttgt ggttgatgag 240

ttacttgctg tccagaacaa atcttatgat aaaccaacca tccttgtggc aaagagtgtc 300 Page 76

AGR_PT024_1WO_SequenceListing_EFS aagggagagg aagaaatacc agatggagta gttggtgtaa ttacacctga tatgccagat 360

gttctgtctc atgtgtcagt ccgagcaagg aatag 395

<210> 178 <211> 572 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(572) <223> T8_2856 + 429 <400> 178 gaaggggatt ggagaggaag ttagtttatt ttcacctcaa tcctctccta tcccgaaggg 60

gatttgaggt tcccaaagta gccctaaaag tgatactagt gaccctctcc acaattttat 120 gcgaaccaca gaaattaata atatattcta ttactctgca cctgacatct ggctcctgct 180 atcagttggc aggttataag cccggttgaa gtatcaggtt atgtggttgt ggttgatgag 240

ttacttgctg tccagaacaa atcttatgat aaaccaacca tccttgtggc aaagagtgtc 300

aagggagagg aagaaatacc agatggagta gttggtgtaa ttacacctga tatgccagat 360

gttctgtctc atgtgtcagt ccgagcaagg aatagcaagg tttatcttca cagctatgtt 420 gcaagatttc ttgaattttt tctcttgtat tgatgttgac atactagctt tttcctaatg 480

aaggtactgt ttgcgacctg ttttgaccac accactctat ctgaacttga aggatatgat 540

cagaaactgt tttccttcaa gcctacttct gc 572

<210> 179 <211> 381 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(381) <223> T9_ZmGWDmega-2F+429

<400> 179 ggttataagc ccggttgaag tatcaggtta tgtggttgtg gttgatgagt tacttgctgt 60 ccagaacaaa tcttatgata aaccaaccat ccttgtggca aagagtgtca agggagagga 120 agaaatacca gatggagtag ttggtgtaat tacacctgat atgccagatg ttctgtctca 180

tgtgtcagtc cgagcaagga atagcaaggt ttatcttcac agctatgttg caagatttct 240 tgaatttttt ctcttgtatt gatgttgaca tactagcttt ttcctaatga aggtactgtt 300

tgcgacctgt tttgaccaca ccactctatc tgaacttgaa ggatatgatc agaaactgtt 360 ttccttcaag cctacttctg c 381

<210> 180 <211> 422 Page 77

AGR_PT024_1WO_SequenceListing_EFS <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(422) <223> T10_ZmGWDmega-2F+ 2858 <400> 180 ggttataagc ccggttgaag tatcaggtta tgtggttgtg gttgatgagt tacttgctgt 60

ccagaacaaa tcttatgata aaccaaccat ccttgtggca aagagtgtca agggagagga 120 agaaatacca gatggagtag ttggtgtaat tacacctgat atgccagatg ttctgtctca 180

tgtgtcagtc cgagcaagga atagcaaggt ttatcttcac agctatgttg caagatttct 240 tgaatttttt ctcttgtatt gatgttgaca tactagcttt ttcctaatga aggtactgtt 300

tgcgacctgt tttgaccaca ccactctatc tgaacttgaa ggatatgatc agaaactgtt 360 ttccttcaag cctacttctg cagatataac ctataggtac ttgaggctat ttgaacgtca 420 tg 422

<210> 181 <211> 208 <212> DNA <213> Sorghum bicolor

<220> <221> misc_feature <222> (1)..(208) <223> T11_SbGWDmega-2F + ZmGWDmega-2R

<400> 181 ggcaggttat aagcccagtt gaagtatcag gttatgtggt tgtggttgat gagttacttg 60

ctgtccagaa caaatcttat gataaaccaa ccatccttgt ggcaaagagt gtcaagggag 120 aggaagaaat accagatgga gtagttggtg taattacacc tgatatgcca gatgttctgt 180

cccatgtgtc agtccgagca aggaatag 208

<210> 182 <211> 214 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(214) <223> Zm GWD Exon 24 <400> 182 ttggcaggtt ataagcccgg ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120

agaggaagaa ataccagatg gagtagttgg tgtaattaca cctgatatgc cagatgttct 180 gtctcatgtg tcagtccgag caaggaatag caag 214

Page 78

AGR_PT024_1WO_SequenceListing_EFS <210> 183 <211> 214 <212> DNA <213> Sorghum bicolor

<220> <221> misc_feature <222> (1)..(214) <223> SbGWD Exon 24

<400> 183 ttggcaggtt ataagcccag ttgaagtatc aggttatgtg gttgtggttg atgagttact 60 tgctgtccag aacaaatctt atgataaacc aaccatcctt gtggcaaaga gtgtcaaggg 120 agaggaagaa ataccagatg gagtagttgg tgtaattaca cctgatatgc cagatgttct 180

gtcccatgtg tcagtccgag caaggaatag caag 214

<210> 184 <211> 234 <212> DNA <213> Sorghum bicolor

<220> <221> misc_feature <222> (1)..(234) <223> SbGWD Exon 7

<400> 184 gaggagtatg aagctgcacg agctgagtta atagaggaat taaatagagg tgtttcttta 60

gagaagcttc gagctaaatt gacaaaaaca cctgaagcac ctgagtcaga tgaacgtaaa 120

tctcctgcat ctcgaatgcc cgttgataaa cttccagagg accttgtaca ggtgcaggct 180 tatataaggt gggagaaagc gggcaagcca aattatcctc ctgagaagca actg 234

<210> 185 <211> 81 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(81) <223> ZmGWD aa1040-1120

<400> 185 Pro Thr Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro 1 5 10 15

Asp Gly Val Val Gly Val Ile Thr Pro Asp Met Pro Asp Val Leu Ser 20 25 30

His Val Ser Val Arg Ala Arg Asn Ser Lys Val Leu Phe Ala Thr Cys 35 40 45

Page 79

AGR_PT024_1WO_SequenceListing_EFS Phe Asp His Thr Thr Leu Ser Glu Leu Glu Gly Tyr Asp Gln Lys Leu 50 55 60

Phe Ser Phe Lys Pro Thr Ser Ala Asp Ile Thr Tyr Arg Glu Ile Thr 70 75 80

Glu

<210> 186 <211> 76 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(76) <223> WT ZmGWD_nt 81-160 Exon 24 <400> 186 gctcctgcta tcagttggca ggttataagc ccggttgaag tatcaggtta tgtggttgtg 60

gttgatgagt tacttg 76

<210> 187 <211> 76 <212> DNA <213> Zea mays

<220> <221> misc_feature <222> (1)..(76) <223> Wt ZmGWD Exon 24 <220> <221> misc_feature <222> (1)..(76) <223> Wt ZmGWD Exon 24_nt 81-160

<400> 187 gctcctgcta tcagttggca ggttataagc ccggttgaag tatcaggtta tgtggttgtg 60 gttgatgagt tacttg 76

<210> 188 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct, M37 <400> 188 gctcctgcta tctagttggc aggttataag cccggttgaa gtatcaggtt atgtggttgt 60 ggttgatgag ttacttg 77

<210> 189 <211> 67 <212> DNA Page 80

AGR_PT024_1WO_SequenceListing_EFS <213> Zea mays

<220> <221> misc_feature <222> (1)..(67) <223> Wt ZmGWD Exon 25 <400> 189 cactctatct gaacttgaag gatatgatca gaaactgttt tccttcaagc ctacttctgc 60 agatata 67

<210> 190 <211> 47 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(47) <223> Wt ZmGWD aa 1011-1057

<400> 190 Trp Gln Val Ile Ser Pro Val Glu Val Ser Gly Tyr Val Val Val Val 1 5 10 15

Asp Glu Leu Leu Ala Val Gln Asn Lys Ser Tyr Asp Lys Pro Thr Ile 20 25 30

Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro Asp Gly 35 40 45

<210> 191 <211> 35 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(35) <223> Wt ZmGWD aa 1082-1116

<400> 191 Val Leu Phe Ala Thr Cys Phe Asp His Thr Thr Leu Ser Glu Leu Glu 1 5 10 15

Gly Tyr Asp Gln Lys Leu Phe Ser Phe Lys Pro Thr Ser Ala Asp Ile 20 25 30

Thr Tyr Arg 35

<210> 192 <211> 57 <212> DNA <213> Zea mays Page 81

AGR_PT024_1WO_SequenceListing_EFS

<220> <221> misc_feature <222> (1)..(57) <223> Wt ZmGWD_nt 3157-3213

<400> 192 gaaataccag atggagtagt tggtgtaatt acacctgata tgccagatgt tctgtct 57

<210> 193 <211> 28 <212> PRT <213> Zea mays

<220> <221> MISC_FEATURE <222> (1)..(28) <223> Wt ZmGDW_aa1054-1081 <400> 193 Ile Pro Asp Gly Val Val Gly Val Ile Thr Pro Asp Met Pro Asp Val 1 5 10 15

Leu Ser His Val Ser Val Arg Ala Arg Asn Ser Lys 20 25

<210> 194 <211> 1045 <212> PRT <213> Sorghum bicolor

<220> <221> MISC_FEATURE <222> (1)..(1045) <223> Sb4715_2 WT + del <400> 194

Met Thr Gly Phe Ser Ala Ala Ala Ser Ala Ala Ala Ala Ala Glu Arg 1 5 10 15

Cys Ala Leu Ala Ile Arg Ala Arg Pro Ala Ala Ser Ser Pro Ala Lys 20 25 30

Arg Gln Gln Gln Ser Ala Ser Leu Arg Arg Ser Gly Gly Gln Arg Arg 35 40 45

Pro Thr Thr Leu Ala Ala Ser Arg Arg Ser Pro Val Val Val Pro Arg 50 55 60

Ala Ile Ala Thr Ser Ala Asp Arg Ala Ser His Asp Leu Val Gly Lys 70 75 80

Phe Thr Leu Asp Ser Asn Ser Glu Leu Leu Val Ala Val Asn Pro Ala 85 90 95

Page 82

AGR_PT024_1WO_SequenceListing_EFS Pro Gln Gly Leu Val Ser Val Ile Gly Leu Glu Val Thr Asn Thr Ser 100 105 110

Gly Ser Leu Ile Leu His Trp Gly Val Leu Arg Pro Asp Lys Arg Asp 115 120 125

Trp Ile Leu Pro Ser Arg Gln Pro Asp Gly Thr Thr Val Tyr Lys Asn 130 135 140

Arg Ala Leu Arg Thr Pro Phe Val Lys Ser Gly Asp Asn Ser Thr Leu 145 150 155 160

Arg Ile Glu Ile Asp Asp Pro Ala Val Gln Ala Ile Glu Phe Leu Ile 165 170 175

Phe Gly Glu Thr Gln Asn Lys Trp Phe Lys Asn Asn Gly Gln Asn Phe 180 185 190

Gln Ile Gln Leu Gln Ser Ser Arg His Gln Gly Asn Gly Ala Ser Gly 195 200 205

Ala Ser Ser Ser Ala Thr Ser Thr Leu Val Pro Glu Asp Leu Val Gln 210 215 220

Ile Gln Ala Tyr Leu Arg Trp Glu Arg Lys Gly Lys Gln Ser Tyr Thr 225 230 235 240

Pro Glu Gln Glu Lys Glu Glu Tyr Glu Ala Ala Arg Ala Glu Leu Ile 245 250 255

Glu Glu Leu Asn Arg Gly Val Ser Leu Glu Lys Leu Arg Ala Lys Leu 260 265 270

Thr Lys Thr Pro Glu Ala Pro Glu Ser Asp Glu Arg Lys Ser Pro Ala 275 280 285

Ser Arg Met Pro Val Asp Lys Leu Pro Glu Asp Leu Val Gln Val Gln 290 295 300

Ala Tyr Ile Arg Trp Glu Lys Ala Gly Lys Pro Asn Tyr Pro Pro Glu 305 310 315 320

Lys Gln Leu Val Glu Leu Glu Glu Ala Arg Lys Glu Leu Gln Ala Glu 325 330 335

Val Asp Lys Gly Ile Ser Ile Asp Gln Leu Arg Gln Lys Ile Leu Lys 340 345 350

Gly Asn Ile Glu Ser Lys Val Ser Lys Gln Leu Lys Asn Lys Lys Tyr 355 360 365

Page 83

AGR_PT024_1WO_SequenceListing_EFS Phe Ser Val Glu Arg Ile Gln Arg Lys Lys Arg Asp Ile Met Gln Leu 370 375 380

Leu Ser Lys His Lys His Thr Val Met Glu Glu Lys Val Glu Val Ala 385 390 395 400

Pro Lys Gln Pro Thr Val Leu Asp Leu Phe Thr Lys Ser Leu His Glu 405 410 415

Lys Asp Gly Cys Glu Val Leu Ser Arg Lys Leu Phe Lys Phe Gly Asp 420 425 430

Lys Glu Ile Leu Ala Ile Ser Thr Lys Val Gln Asn Lys Thr Glu Val 435 440 445

His Leu Ala Thr Asn His Thr Glu Pro Leu Ile Leu His Trp Ser Leu 450 455 460

Ala Lys Lys Ala Gly Glu Trp Lys Ala Pro Pro Ser Asn Ile Leu Pro 465 470 475 480

Ser Gly Ser Lys Leu Leu Asp Met Ala Cys Glu Thr Glu Phe Thr Arg 485 490 495

Ser Glu Leu Asp Gly Leu Cys Tyr Gln Val Val Glu Ile Glu Leu Asp 500 505 510

Asp Gly Gly Tyr Lys Gly Met Pro Phe Val Leu Arg Ser Gly Glu Thr 515 520 525

Trp Ile Lys Asn Asn Gly Ser Asp Phe Phe Leu Asp Phe Ser Thr Arg 530 535 540

Asp Thr Arg Asn Ile Lys Leu Lys Asp Asn Gly Asp Ala Gly Lys Gly 545 550 555 560

Thr Ala Lys Ala Leu Leu Glu Arg Ile Ala Asp Leu Glu Glu Asp Ala 565 570 575

Gln Arg Ser Leu Met His Arg Phe Asn Ile Ala Ala Asp Leu Ala Asp 580 585 590

Glu Ala Arg Asp Ala Gly Leu Leu Gly Ile Val Gly Leu Phe Val Trp 595 600 605

Ile Arg Phe Met Ala Thr Arg Gln Leu Thr Trp Asn Lys Asn Tyr Asn 610 615 620

Val Lys Pro Arg Glu Ile Ser Lys Ala Gln Asp Arg Phe Thr Asp Asp 625 630 635 640

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AGR_PT024_1WO_SequenceListing_EFS Leu Glu Asn Met Tyr Arg Thr Tyr Pro Gln Tyr Arg Glu Ile Leu Arg 645 650 655

Met Ile Met Ala Ala Val Gly Arg Gly Gly Glu Gly Asp Val Gly Gln 660 665 670

Arg Ile Arg Asp Glu Ile Leu Val Ile Gln Arg Asn Asn Asp Cys Lys 675 680 685

Gly Gly Met Met Glu Glu Trp His Gln Lys Leu His Asn Asn Thr Ser 690 695 700

Pro Asp Asp Val Val Ile Cys Gln Ala Leu Ile Asp Tyr Ile Lys Asn 705 710 715 720

Asp Phe Asp Ile Ser Val Tyr Trp Asp Thr Leu Asn Lys Asn Gly Ile 725 730 735

Thr Lys Glu Arg Leu Leu Ser Tyr Asp Arg Ala Ile His Ser Glu Pro 740 745 750

Asn Phe Arg Ser Glu Gln Lys Glu Gly Leu Leu Arg Asp Leu Gly Asn 755 760 765

Tyr Met Arg Ser Leu Lys Ala Val His Ser Gly Ala Asp Leu Glu Ser 770 775 780

Ala Ile Ala Thr Cys Met Gly Tyr Lys Ser Glu Gly Glu Gly Phe Met 785 790 795 800

Val Gly Val Gln Ile Asn Pro Val Lys Gly Leu Pro Ser Gly Phe Pro 805 810 815

Glu Leu Leu Glu Phe Val Leu Asp His Val Glu Asp Lys Ser Ala Glu 820 825 830

Pro Leu Leu Glu Gly Leu Leu Glu Ala Arg Val Asp Leu Arg Pro Leu 835 840 845

Leu Leu Asp Ser Pro Glu Arg Met Lys Asp Leu Ile Phe Leu Asp Ile 850 855 860

Ala Leu Asp Ser Thr Phe Arg Thr Ala Ile Glu Arg Ser Tyr Glu Glu 865 870 875 880

Leu Asn Asp Ala Ala Pro Glu Lys Ile Met Tyr Phe Ile Ser Leu Val 885 890 895

Leu Glu Asn Leu Ala Phe Ser Ile Asp Asp Asn Glu Asp Ile Leu Tyr 900 905 910

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AGR_PT024_1WO_SequenceListing_EFS Cys Leu Lys Gly Trp Asn Gln Ala Leu Glu Met Ala Lys Gln Lys Asp 915 920 925

Asp Gln Trp Ala Leu Tyr Ala Lys Ala Phe Leu Asp Arg Ile Arg Leu 930 935 940

Ala Leu Ala Ser Lys Gly Glu Gln Tyr His Asn Met Met Gln Pro Ser 945 950 955 960

Ala Glu Tyr Leu Gly Ser Leu Leu Ser Ile Asp Lys Trp Ala Val Asn 965 970 975

Ile Phe Thr Glu Glu Ile Ile Arg Gly Gly Ser Ala Ala Thr Leu Ser 980 985 990

Ala Leu Leu Asn Arg Phe Asp Pro Val Leu Arg Asn Val Ala Asn Leu 995 1000 1005

Gly Ser Trp Gln Val Ile Ser Pro Val Glu Val Ser Gly Tyr Val 1010 1015 1020

Val Val Val Asp Glu Leu Leu Ala Val Gln Asn Lys Ser Tyr Asp 1025 1030 1035

Lys Pro Thr Ile Leu Val Glu 1040 1045

<210> 195 <211> 840 <212> PRT <213> Sorghum bicolor

<220> <221> MISC_FEATURE <222> (1)..(840) <223> Sb4715_2 WT + del <400> 195

Val Val Pro Arg Ala Ile Ala Thr Ser Ala Asp Arg Ala Ser His Asp 1 5 10 15

Leu Val Gly Lys Phe Thr Leu Asp Ser Asn Ser Glu Leu Leu Val Ala 20 25 30

Val Asn Pro Ala Pro Gln Gly Leu Val Ser Val Ile Gly Leu Glu Val 35 40 45

Thr Asn Thr Ser Gly Ser Leu Ile Leu His Trp Gly Val Leu Arg Pro 50 55 60

Asp Lys Arg Asp Trp Ile Leu Pro Ser Arg Gln Pro Asp Gly Thr Thr 70 75 80 Page 86

AGR_PT024_1WO_SequenceListing_EFS

Val Tyr Lys Asn Arg Ala Leu Arg Thr Pro Phe Val Lys Ser Gly Asp 85 90 95

Asn Ser Thr Leu Arg Ile Glu Ile Asp Asp Pro Ala Val Gln Ala Ile 100 105 110

Glu Phe Leu Ile Phe Gly Glu Thr Gln Asn Lys Trp Phe Lys Asn Asn 115 120 125

Gly Gln Asn Phe Gln Ile Gln Leu Gln Ser Ser Arg His Gln Gly Asn 130 135 140

Gly Ala Ser Gly Ala Ser Ser Ser Ala Thr Ser Thr Leu Val Pro Glu 145 150 155 160

Asp Leu Val Gln Ile Gln Ala Tyr Leu Arg Trp Glu Arg Lys Gly Lys 165 170 175

Gln Ser Tyr Thr Pro Glu Gln Glu Lys Glu Glu Tyr Glu Ala Ala Arg 180 185 190

Ala Glu Leu Ile Glu Glu Leu Asn Arg Gly Val Ser Leu Glu Lys Leu 195 200 205

Arg Ala Lys Leu Thr Lys Thr Pro Glu Ala Pro Glu Ser Asp Glu Arg 210 215 220

Lys Ser Pro Ala Ser Arg Met Pro Val Asp Lys Leu Pro Glu Asp Leu 225 230 235 240

Val Gln Val Gln Ala Tyr Ile Arg Trp Glu Lys Ala Gly Lys Pro Asn 245 250 255

Tyr Pro Pro Glu Lys Gln Leu Val Glu Leu Glu Glu Ala Arg Lys Glu 260 265 270

Leu Gln Ala Glu Val Asp Lys Gly Ile Ser Ile Asp Gln Leu Arg Gln 275 280 285

Lys Ile Leu Lys Gly Asn Ile Glu Ser Lys Val Ser Lys Gln Leu Lys 290 295 300

Asn Lys Lys Tyr Phe Ser Val Glu Arg Ile Gln Arg Lys Lys Arg Asp 305 310 315 320

Ile Met Gln Leu Leu Ser Lys His Lys His Thr Val Met Glu Glu Lys 325 330 335

Val Glu Val Ala Pro Lys Gln Pro Thr Val Leu Asp Leu Phe Thr Lys 340 345 350 Page 87

AGR_PT024_1WO_SequenceListing_EFS

Ser Leu His Glu Lys Asp Gly Cys Glu Val Leu Ser Arg Lys Leu Phe 355 360 365

Lys Phe Gly Asp Lys Glu Ile Leu Ala Ile Ser Thr Lys Val Gln Asn 370 375 380

Lys Thr Glu Val His Leu Ala Thr Asn His Thr Glu Pro Leu Ile Leu 385 390 395 400

His Trp Ser Leu Ala Lys Lys Ala Gly Glu Trp Lys Ala Pro Pro Ser 405 410 415

Asn Ile Leu Pro Ser Gly Ser Lys Leu Leu Asp Met Ala Cys Glu Thr 420 425 430

Glu Phe Thr Arg Ser Glu Leu Asp Gly Leu Cys Tyr Gln Val Val Glu 435 440 445

Ile Glu Leu Asp Asp Gly Gly Tyr Lys Gly Met Pro Phe Val Leu Arg 450 455 460

Ser Gly Glu Thr Trp Ile Lys Asn Asn Gly Ser Asp Phe Phe Leu Asp 465 470 475 480

Phe Ser Thr Arg Asp Thr Arg Asn Ile Lys Leu Lys Asp Asn Gly Asp 485 490 495

Ala Gly Lys Gly Thr Ala Lys Ala Leu Leu Glu Arg Ile Ala Asp Leu 500 505 510

Glu Glu Asp Ala Gln Arg Ser Leu Met His Arg Phe Asn Ile Ala Ala 515 520 525

Asp Leu Ala Asp Glu Ala Arg Asp Ala Gly Leu Leu Gly Ile Val Gly 530 535 540

Leu Phe Val Trp Ile Arg Phe Met Ala Thr Arg Gln Leu Thr Trp Asn 545 550 555 560

Lys Asn Tyr Asn Val Lys Pro Arg Glu Ile Ser Lys Ala Gln Asp Arg 565 570 575

Phe Thr Asp Asp Leu Glu Asn Met Tyr Arg Thr Tyr Pro Gln Tyr Arg 580 585 590

Glu Ile Leu Arg Met Ile Met Ala Ala Val Gly Arg Gly Gly Glu Gly 595 600 605

Asp Val Gly Gln Arg Ile Arg Asp Glu Ile Leu Val Ile Gln Arg Asn 610 615 620 Page 88

AGR_PT024_1WO_SequenceListing_EFS

Asn Asp Cys Lys Gly Gly Met Met Glu Glu Trp His Gln Lys Leu His 625 630 635 640

Asn Asn Thr Ser Pro Asp Asp Val Val Ile Cys Gln Ala Leu Ile Asp 645 650 655

Tyr Ile Lys Asn Asp Phe Asp Ile Ser Val Tyr Trp Asp Thr Leu Asn 660 665 670

Lys Asn Gly Ile Thr Lys Glu Arg Leu Leu Ser Tyr Asp Arg Ala Ile 675 680 685

His Ser Glu Pro Asn Phe Arg Ser Glu Gln Lys Glu Gly Leu Leu Arg 690 695 700

Asp Leu Gly Asn Tyr Met Arg Ser Leu Lys Ala Val His Ser Gly Ala 705 710 715 720

Asp Leu Glu Ser Ala Ile Ala Thr Cys Met Gly Tyr Lys Ser Glu Gly 725 730 735

Glu Gly Phe Met Val Gly Val Gln Ile Asn Pro Val Lys Gly Leu Pro 740 745 750

Ser Gly Phe Pro Glu Leu Leu Glu Phe Val Leu Asp His Val Glu Asp 755 760 765

Lys Ser Ala Glu Pro Leu Leu Glu Gly Leu Leu Glu Ala Arg Val Asp 770 775 780

Leu Arg Pro Leu Leu Leu Asp Ser Pro Glu Arg Met Lys Asp Leu Ile 785 790 795 800

Phe Leu Asp Ile Ala Leu Asp Ser Thr Phe Arg Thr Ala Ile Glu Arg 805 810 815

Ser Tyr Glu Glu Leu Asn Asp Ala Ala Pro Glu Lys Ile Met Tyr Phe 820 825 830

Ile Ser Leu Val Leu Glu Asn Leu 835 840

<210> 196 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct, SV NLS peptide <400> 196

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AGR_PT024_1WO_SequenceListing_EFS Pro Lys Lys Lys Arg Lys Val 1 5

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Claims (25)

CLAIMS What is claimed is:

1. A genetically engineered plant comprising an engineered nucleic acid encoding an altered Glucan Water Dikinase, wherein the engineered nucleic acid comprises a polynucleotide selected from the group consisting of a sequence as set forth in SEQ ID NO: 114, 115, 116, 117, or 118 and the plant has an elevated level of starch in comparison to a plant of the same genetic background comprising a wild type Glucan Water Dikinase.

2. The genetically engineered plant of claim 1, wherein the altered Glucan Water Dikinase is inactive.

3. The genetically engineered plant of claim 1, wherein the altered Glucan Water Dikinase comprises an amino acid sequence as set forth in SEQ ID NO: 121, 122, 123, or 124.

4. A method for genetically engineering a plant comprising an altered Glucan Water Dikinase comprising: contacting at least one plant cell comprising a target sequence in an endogenous gene encoding a Glucan Water Dikinase with a vector comprising a first nucleic acid encoding a nuclease capable of inducing a double-strand break at the target sequence and a second engineered nucleic acid sequence encoding an sgRNA, wherein the nuclease is a Cas9 nuclease encoded by a nucleic acid comprising the sequence as set forth in SEQ ID NO: 75 (ZmCas9), and the second nucleic acid is capable of binding the target sequence included in an endogenous gene encoding a Glucan Water Dikinase and comprising the sequence as set forth in SEQ ID NO: 92 (GWDe24b); selecting a plant cell that includes an alteration in the target sequence; and regenerating a genetically engineered plant comprising the alteration from the plant cell.

5. The method of claim 4, wherein the genetically engineered plant is homozygous for the alteration.

6. The method of claim 4, wherein the genetically engineered plant is heterozygous for the alteration.

7. The method of claim 6 further comprising selfing the heterozygous genetically engineered plant, or crossing to another genetically engineered plant heterozygous for the same alteration, and selecting a first progeny plant that is homozygous for the alteration.

8. The method of claim 6 further comprising crossing the genetically engineered plant to a wild type plant of the same genetic background and selecting a first progeny plant that is heterozygous for the alteration.

9. The method of claim 8 further comprising selfing the first heterozygous progeny plant and selecting a second progeny plant that is homozygous for the alteration.

10. The method of claim 4, wherein the alteration comprises the mutation in an amino acid sequence of the Glucan Water Dikinase as set forth in SEQ ID NO: 121, 122, 123, or 124.

11. The method of claim 4, wherein the genetically engineered plant comprising the alteration or a progeny thereof has an elevated level of starch in comparison to a non-genetically engineered plant of the same genetic background.

12. The method of claim 4, wherein the vector further comprises a first nucleic acid promoter operably linked to the first nucleic acid.

13. The method of claim 4, wherein the vector further comprises a second nucleic acid promoter operably linked to the second nucleic acid, wherein the second nucleic acid promoter has a sequence as set forth in SEQ ID NO: 82 (ZmU3P1).

14. The method of claim 4, wherein the second nucleic acid comprises a sequence of SEQ ID NO: 95 (ZmU3P1:sgRNAGWDe24b).

15. A genetically engineered plant produced by the method of any one of claims 4-14, or a progeny or descendant thereof, wherein the plant, progeny or descendant thereof comprises the alteration.

16. The genetically engineered plant of claim 15 having an elevated level of starch in comparison to a plant of the same genetic background comprising a wild type Glucan Water Dikinase.

17. A method of increasing a starch level in a plant comprising performing the method of any one of claims 4-14.

18. A method of agricultural processing comprising: performing the method of any one of claims 4-14; and processing the homozygous plant, wherein the processing comprises one or more procedures selected from harvesting, bailing, shredding, drying, fermenting, hydrolyzing with chemicals, hydrolyzing with exogenous enzymes and combining with plant biomass.

19. A method of preparing animal feed comprising: performing the method of any one of claims 4-14; and performing at least one procedure selected from the group consisting of: harvesting, bailing, shredding, drying, ensiling, pelletizing, combining with a source of edible fiber, and combining with plant biomass.

20. A genetic construct comprising a first engineered nucleic acid sequence encoding a Cas9 nuclease and a second engineered nucleic acid sequence encoding an sgRNA, wherein the Cas9 nuclease comprises the sequence as set forth in SEQ ID NO: 75 (ZmCas9) and is capable of cleaving a target sequence included in an endogenous nucleic acid encoding Glucan Water Dikinase in a plant, and the sgRNA is capable of binding the sequence included in the target sequence and set forth in SEQ ID NO: 92 (GWDe24b).

21. The genetic construct of claim 20, wherein the first nucleic acid is fused to a polynucleotide sequence encoding at least one nuclear localization signal (NLS).

22. The genetic construct of claim 21, wherein the polynucleotide sequence is selected from SEQ ID NOS: 163 - 168.

23. The genetic construct of claim 20, wherein the second engineered nucleic acid comprises a sequence of SEQ ID NO: 95 (ZmU3P1:sgRNAGWDe24b).

24. The genetic construct of claims 20 further comprising the first nucleic acid promoter operably linked to the first engineered nucleic acid.

25. The genetic construct of claims 20 further comprising the second nucleic acid promoter operably linked to the second engineered nucleic acid, wherein the second nucleic acid promoter has a sequence as set forth in SEQ ID NO: 82 (ZmU3P1).