Corn event DP-051291-2 and its detection method

Abstract

FIELD OF THE INVENTION Embodiments disclosed herein relate to the field of plant molecular biology, and in particular to DNA constructs for conferring insect resistance to plants. Embodiments disclosed herein relate to insect-resistant corn plants containing event DP-051291-2, and assays for detecting the presence of event DP-051291-2 in samples and compositions thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 384,613, filed November 22, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing A sequence listing in XML format with the file name "108313_SequenceListing.xml" and a size of 133,622 bytes created on October 11, 2023 is filed in computer-readable format simultaneously with the specification. The sequence listing contained in this XML document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates to the field of plant molecular biology, including DNA constructs for conferring insect resistance to plants. The present disclosure also includes insect-resistant corn plants containing event DP-051291-2, as well as assays and compositions for detecting the presence of event DP-051291-2 in a sample. [Background technology]

[0004] Corn is an important crop and a major food source in many parts of the world. Damage caused by pests is a major factor in global corn crop losses, despite the use of protective measures such as chemical pesticides. Therefore, genetic engineering of crops such as corn to confer insect resistance is being undertaken with the goal of controlling insect damage and reducing the need for traditional chemical pesticides. One group of genes being utilized to create transgenic insect-resistant crops is the delta-endotoxin family from Bacillus thuringiensis (Bt). Delta-endotoxins have been successfully expressed in crop plants such as cotton, potato, rice, sunflower, and corn and have proven to provide excellent control of pests in certain circumstances. (Perlak, F. J. et al. (1990) Bio / Technology 8:939-943; Perlak, F. J. et al. (1993) Plant Mol. Biol. 22:313-321; Fujimoto, H. et al. (1993) Bio / Technology 11:1151-1155; Tu et al. (2000) Nature Biotechnology 18:1101-1104; PCT Publication WO 01 / 13731; and Bing, J. W. et al. (2000) Efficacy of Cry1F Transgenic Maize, 14 th Biennial International Plant Resistance to Insects Workshop, Fort Collins, CO).

[0005] Transgene expression in plants is known to be influenced by many different factors, including the orientation and composition of the cassettes driving expression of the individual genes of interest and their location in the plant genome, possibly due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) near the integration site (Weising et al. (1988) Ann. Rev. Genet. 22:421-477).

[0006] It would be advantageous to be able to detect the presence of a particular event in order to determine whether the progeny of a sexual cross contain the transgene of interest.

[0007] The presence of a transgene can be detected by nucleic acid detection methods, for example, by polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on commonly used genetic elements, such as promoters, terminators, and marker genes, because coding regions are interchangeable among many DNA constructs. As a result, such methods may not be useful for distinguishing between different events, particularly those made using the same or very similar DNA constructs, unless the DNA sequence of the flanking DNA adjacent to the inserted heterologous DNA is known. Summary of the Invention

[0008] The present embodiments relate to insect-resistant corn (Zea mays) plant event DP-051291-2, also known as "corn line DP-051291-2," "corn event DP-051291-2," and "DP-051291-2 corn," DNA plant expression constructs of corn plant event DP-051291-2, and methods and compositions for detecting transgene construct, flanking, and insertion (target locus) regions in corn plant event DP-051291-2 and its progeny.

[0009] In one aspect, compositions and methods relate to methods for generating and selecting insect-resistant monocotyledonous crop plants. The compositions include a DNA construct that confers insect resistance when expressed in plant cells and plants. In one aspect, a DNA construct capable of being introduced into and replicating in a host cell and that confers insect resistance to the plant cells and plants when expressed in the plant cells and plants is provided. Corn event DP-051291-2 was produced by Agrobacterium-mediated transformation of plasmid PHP74638 ( FIG. 1 ). As described herein, these events contain the IPD072Aa (polynucleotide SEQ ID NO: 4 and amino acid SEQ ID NO: 5) cassette (Table 1), which confers resistance to certain Coleopteran pests. This insect control component has demonstrated efficacy against Coleopteran insect species, particularly the Western Corn Rootworm (WCR). In some embodiments, the polynucleotide encoding the IPD072Aa polypeptide comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the IPD072Aa polypeptide comprises a sequence having 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5.

[0010] In some embodiments, compositions and methods are provided for identifying a novel corn plant designated DP-051291-2 (ATCC Accession No. PTA-127358). The methods are based on primers or probes that specifically recognize the 5' and / or 3' flanking sequences of DP-051291-2. DNA molecules are provided that contain primer sequences that, when used in a PCR reaction, will produce an amplicon unique to the transgenic event DP-051291-2. In one embodiment, corn plants and seeds containing such molecules are contemplated. Additionally, kits are provided for utilizing such primer sequences to identify the DP-051291-2 event.

[0011] Some embodiments relate to specific flanking sequences of DP-051291-2 as described herein, which can be used to develop methods for identifying DP-051291-2 in biological samples. More particularly, the present disclosure relates to the 5' and / or 3' flanking regions of DP-051291-2, which can be used to develop specific primers and probes. Further embodiments relate to methods for identifying the presence of DP-051291-2 in biological samples based on the use of such specific primers or probes.

[0012] According to some embodiments, methods for detecting the presence of DNA corresponding to corn event DP-051291-2 in a sample are provided. Such methods include (a) contacting a sample containing DNA with a set of DNA primers that, when used in a nucleic acid amplification reaction with genomic DNA extracted from corn containing event DP-051291-2, each produce an amplicon diagnostic for corn event DP-051291-2, (b) performing the nucleic acid amplification reaction, thereby producing the amplicon, and (c) detecting the amplicon. In some aspects, the primer set comprises SEQ ID NOs: 6 and 7, and optionally a probe comprising SEQ ID NO: 8.

[0013] According to some embodiments, a method for detecting the presence of a DNA molecule corresponding to the DP-051291-2 event in a sample includes: (a) contacting a sample containing DNA extracted from a corn plant with a DNA probe molecule that hybridizes to DNA extracted from corn event DP-051291-2 under stringent hybridization conditions and does not hybridize to control corn plant DNA under stringent hybridization conditions; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA extracted from corn event DP-051291-2. More specifically, a method for detecting the presence of a DNA molecule corresponding to the DP-051291-2 event in a sample includes (a) contacting a sample containing DNA extracted from a corn plant with a DNA probe molecule containing a sequence unique to the event, e.g., a junction sequence, which hybridizes to DNA extracted from corn event DP-051291-2 under stringent hybridization conditions and does not hybridize to control corn plant DNA under stringent hybridization conditions; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA.

[0014] Additionally, kits and methods for identifying event DP-051291-2 in biological samples are provided that detect DP-051291-2 specific regions.

[0015] DNA molecules are provided that contain at least one junction sequence of DP-051291-2; wherein the junction sequence spans a junction located between heterologous DNA inserted into the genome and DNA of the corn cell adjacent to the insertion site, and can be diagnostic for a DP-051291-2 event.

[0016] According to some embodiments, a method for producing an insect-resistant corn plant includes (a) sexually crossing a first parent corn line containing an expression cassette disclosed herein that confers resistance to insects with a second parent corn line lacking such expression cassette, thereby producing a plurality of progeny plants; and (b) selecting the progeny plants that are insect-resistant. Such methods can optionally include the further step of backcrossing the progeny plants with the second parent corn line to produce a true-breed insect-resistant corn plant.

[0017] Some embodiments provide a method of producing insect-resistant corn plants, comprising transforming corn cells with the DNA construct PHP74638, growing the transformed corn cells into corn plants, selecting corn plants that are resistant to insects, and further growing the corn plants into fertile corn plants that can be self-pollinated or crossed with a compatible corn variety to produce insect-resistant progeny.

[0018] Some embodiments further relate to a DNA detection kit for identifying corn event DP-051291-2 in a biological sample. The kit includes a first primer that specifically recognizes the 5'- or 3'-flanking region of DP-051291-2 and a second primer that specifically recognizes a sequence within the non-native target locus DNA of DP-051291-2 or within the adjacent DNA for use in a PCR identification protocol. Further embodiments relate to a kit for identifying event DP-051291-2 in a biological sample, the kit including a specific probe having a sequence corresponding to or complementary to a sequence having about 80% to 100% sequence identity to a specific region of event DP-051291-2. The sequence of the probe corresponds to a specific region that includes a portion of the 5'- or 3'-flanking region of event DP-051291-2. In some embodiments, the first or second primer comprises any one of SEQ ID NOs: 6-7, 9-10, 12-13, 15-16, or 18-19.

[0019] The methods and kits encompassed by embodiments disclosed herein can be used for different purposes, including, but not limited to, identifying event DP-051291-2 in products, such as, but not limited to, plants, plant material, or food or feed products (fresh or processed) containing or derived from plant material; Additionally or alternatively, the methods and kits can be used to identify transgenic plant material for the purposes of separating between transgenic and non-transgenic material. Additionally or alternatively, the methods and kits can be used to determine the quality of plant material containing corn event DP-051291-2. The kits may also contain reagents and materials necessary for carrying out the detection methods.

[0020] Further embodiments relate to DP-051291-2 corn plants or parts thereof, including but not limited to, pollen, ovules, vegetative cells, pollen cell nuclei, and egg cell nuclei of corn plant DP-051291-2, and progeny derived therefrom. In another embodiment, DNA primer molecules targeting DP-051291-2 corn plants and seeds provide specific amplicon products. [Brief explanation of the drawings]

[0021] [Figure 1]

[0023] Figure 1 shows a schematic diagram of plasmid PHP74638 containing the indicated genetic elements. The plasmid size is 66,641 bp (SEQ ID NO: 1). [Figure 2]A schematic diagram of the T-DNA region of plasmid PHP74638 is shown, showing the recombinant fragment region flanked by FRT1 and FRT87 sites, containing the pmi, mo-pat, and ipd072Aa gene cassettes intended for integration into the maize genome, and the zm-wus2, zm-odp2, mo-Flp, and DsRed2 gene cassettes not intended for integration into the maize genome. The size of the T-DNA is 23,712 bp (SEQ ID NO: 2). The zm-wus2 and zm-odp2 developmental genes were present to increase transformation efficiency, and the tissue-specific red fluorescent protein DsRed2 gene was present to allow differentiation during seed selection. [Figure 3] Figure 1 shows a schematic map of the insertion in DP-051291-2 maize based on the sequencing analysis described herein. The adjacent maize genome is represented by a horizontal black bar. A single copy of the insertion from PHP74638 and PHP50742 is integrated into the maize genome (SEQ ID NO: 2 is the T-DNA sequence). Within the insertion, the landing pad sequence from PHP50742 and the trait gene from PHP74638 are shown. SEQ ID NO: 3 is the complete insert sequence and flanking genomic regions. The FRT1 and FRT87 sites, which are targets of recombination during the SSI process, are shown. [Figure 4] Schematic diagram of transformation and development of DP-051291-2. DETAILED DESCRIPTION OF THE INVENTION

[0022] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the protein" includes one or more proteins and equivalents thereof, and the like. Unless clearly indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0023] The compositions of the present disclosure include seeds deposited under ATCC Patent Deposit No. PTA-127358, as well as plants, plant cells, and seeds derived therefrom. Applicants deposited at least 625 seeds of corn event DP-051291-2 on August 19, 2022, with the American Type Culture Collection (ATCC), Manassas, VA 20110-2209 USA (Patent Deposit No. PTA-127358). These deposits will be governed under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The seeds deposited with ATCC on August 19, 2022, will be stored at Pioneer Hi-Bred International, Inc., 7250 NW 62 ndThe deposit was taken from a deposit maintained by the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209. During the pendency of this application, this deposit will be available to the Commissioner of Patents and Trademarks and to any person whom the Commissioner determines to be entitled to the application. Upon allowance of any claims in this application, applicants will make available to the public, pursuant to 37 C.F.R. § 1.808, one or more samples of the deposit of at least 625 seeds of hybrid corn at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209. This deposit of seeds of corn event DP-051291-2 will be maintained in the ATCC public depository for 30 years, or five years from the time of the last request, or for the duration of this patent, whichever is longer, and will be replaced if they become non-viable during that period. Furthermore, applicants have satisfied all requirements of 37 C.F.R. §§ 1.801-1.809, including providing viability of samples at the time of deposit. Applicants have no authority to waive any restrictions imposed by law on the movement of biological material or its commercial transportation. Applicants do not waive any infringement of their rights conferred under this patent or any rights applicable to Event DP-051291-2 under the Plant Variety Protection Act (7 U.S.C. § 2321 et seq.). Unauthorized seed propagation is prohibited. Seeds may also be subject to restrictions.

[0024] A single copy of the insert from PHP74638 and PHP50742 was integrated into the maize genome, with the insertion containing the landing pad sequence from PHP50742 and the trait gene from PHP74638, containing the pmi, mo-pat, and ipd072Aa gene cassettes.

[0025] The first gene cassette (pmi gene cassette) contains the phosphomannose isomerase (pmi) gene from Escherichia coli (Negrotto et al., 2000). Expression of the PMI protein in plants serves as a selectable marker, allowing plant tissue growth using mannose as a carbon source. The PMI protein is 391 amino acids long and has a molecular weight of approximately 43 kDa. When present in the T-DNA region of PHP74638, the pmi gene lacks a promoter; however, it is located next to the flippase recombination target site FRT1, allowing post-recombination expression with an appropriately positioned promoter. The terminator of the pmi gene is a copy of the pinII terminator. An additional Z19 terminator is present, which is intended to prevent transcriptional interference between cassettes.

[0026] The second gene cassette (mo-pat gene cassette) contains a maize-optimized version of the phosphinothricin acetyltransferase gene (mo-pat) from Streptomyces viridochromogenes (Wohlleben et al., 1988). The mo-pat gene expresses the phosphinothricin acetyltransferase (PAT) enzyme, which confers resistance to phosphinothricin. The PAT protein is 183 amino acids long and has a molecular weight of approximately 21 kDa. Expression of the mo-pat gene is controlled by the promoter and intron regions of the Oryza sativa (rice) actin (os-actin) gene (GenBank accession CP018159; GenBank accession EU155408.1), along with a third copy of the CaMV35S terminator. To prevent transcriptional interference, two additional terminators are present: one from the Sorghum bicolor ubiquitin (sb-ubi) gene (Phytozome Gene ID Sobic.004G049900.1; U.S. Patent No. 9,725,731 [Abbitt, 2017]) and one from the γ-kapharin (sb-gkaf) gene (de Freitas et al., 1994).

[0027] The third gene cassette contains the insecticidal protein gene ipd072Aa from Pseudomonas chlororaphis (Schellenberger et al., 2016). Expression of the IPD072Aa protein in plants is effective against certain Coleoptera pests. The IPD072Aa protein is 86 amino acids long and has a molecular weight of approximately 10 kDa. Expression of the ipd072Aa gene is controlled by a promoter region from the acuminata Yunnan strain of banana streak virus [BSV(AY)] (U.S. Patent No. 8,338,662), an intron region from the maize orthologue of a hypothetical rice (Oryza sativa) protein (WO 2016 / 109157 [Abbitt and Shen, 2016]; (zm-HPLV9)), and a predicted intron region from the maize calmodulin 5 gene (U.S. Patent No. 10,059,953), in conjunction with a terminator region from a putative Arabidopsis thaliana gene in the mannose-binding protein superfamily (at-T9).

[0028] As used herein, the term "corn" means Zea mays or maize and includes all plant varieties that can be bred with corn, including wild corn species.

[0029] As used herein, the terms "insect resistance" and "impact pest" refer to causing changes in the feeding, growth, and / or behavior of insects at any stage of development, including, but not limited to, killing the insects; retarding growth; reducing reproductive capacity; preventing feeding; and the like.

[0030] As used herein, the terms "insecticidal activity" and "insecticidal activity" are used synonymously to refer to the activity of an organism or substance (e.g., a protein) that can be measured by a number of parameters, including, but not limited to, pest mortality, pest weight loss, pest attraction, pest repellency, and other behavioral and physical changes in pests, after feeding and / or exposure of the organism or substance for an appropriate length of time. For example, an "insecticidal protein" is a protein that exhibits insecticidal activity, alone or in combination with other proteins.

[0031] As used herein, "insert DNA" refers to heterologous DNA present in an expression cassette used to transform plant material, while "flanking DNA" can be either genomic DNA naturally present in an organism such as a plant or foreign (heterologous) DNA introduced by a transformation method that is foreign to the original insert DNA molecule, e.g., a fragment associated with the transformation event. "Flanking region" or "flanking sequence," as used herein, refers to a sequence of at least 10 bp (in some narrower embodiments, at least 20 bp, at least 50 bp, and up to at least 5,000 bp) located either directly upstream and adjacent to the original, non-native insert DNA molecule and / or directly downstream and adjacent to it. Transformation procedures for exogenous DNA can result in transformants containing various flanking regions that are characteristic and unique to each transformant. When recombinant DNA is introduced into plants through conventional breeding, the flanking regions will generally remain unchanged. Single-base changes in flanking regions may occur through several generations of plant breeding and conventional breeding. A transformant will also contain a unique junction between a heterologous insert DNA piece and genomic DNA, or between two pieces of genomic DNA, or between two pieces of heterologous DNA. A "junction" is a point where two specific DNA pieces join. For example, a junction exists where an insert DNA joins adjacent DNA. Junctions are also present in transformed organisms where two DNA pieces are joined together in a modified form compared to that found in the native organism. "Junction DNA" refers to DNA that contains a junction.The junction sequences provided herein include those located between the 5' end of the insert and the corn genomic DNA, extending from the junction by at least -5 to +5 nucleotides (SEQ ID NO: 26), by at least -10 to +10 nucleotides (SEQ ID NO: 27), and by at least -25 to +25 nucleotides (SEQ ID NO: 28); and those located between the 3' end of the insert and the corn genomic DNA, extending from the junction by at least -5 to +5 nucleotides (SEQ ID NO: 29), by at least -10 to +10 nucleotides (SEQ ID NO: 30), and by at least -25 to +25 nucleotides (SEQ ID NO: 31). The junction sequences provided herein also include those located between the target locus and the 5' end of the insert. In some embodiments, SEQ ID NO: 8 or 21 of DP-051291-2 represents the junction between the target locus and the 3' end of the insert. The complete insert with flanking regions is represented by SEQ ID NO: 3. In some embodiments, the insert region and flanking regions comprise polynucleotides having at least 95%, 96%, 97%, 98%, or 99% sequence identity compared to SEQ ID NO:3.

[0032] In one embodiment, seeds, plants, and plant parts comprising corn event DP-051291-2 are provided, wherein the seeds, plants, and plant parts comprise a DNA sequence selected from SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, or a sequence having at least 95% sequence identity to SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, and a representative sample of corn event DP-051291-2 seeds has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358. In another embodiment, seeds, plants, and plant parts are provided comprising corn event DP-051291-2, wherein the seeds, plants, and plant parts comprise SEQ ID NO:3 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:3, and a representative sample of corn event DP-051291-2 seeds has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

[0033] As used herein, "heterologous" in reference to a nucleic acid sequence refers to a nucleic acid sequence that originates from a different, incompatible species, or, if originating from the same species, has been substantially altered in composition and / or genomic locus relative to its native form by deliberate human intervention. For example, a promoter operably linked to a heterologous nucleotide sequence may be from a species different from the species from which the nucleotide sequence was derived, or, if from the same species, the promoter is not found operably linked to the nucleotide sequence in nature. A heterologous protein may originate from a foreign species, or, if originating from the same species, has been substantially altered relative to its native form by deliberate human intervention.

[0034] The term "regulatory element" refers to a nucleic acid molecule that has gene-regulatory activity, i.e., the ability to affect the transcriptional and / or translational expression pattern of an operably linked transcribable polynucleotide. Thus, the term "gene-regulatory activity" refers to the ability to affect the expression of an operably linked transcribable polynucleotide molecule by affecting the transcription and / or translation of the operably linked transcribable polynucleotide molecule. Gene-regulatory activity can be positive and / or negative, and its effects can be characterized by its temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemical response qualities, as well as by quantitative or qualitative indicators.

[0035] A "promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' from the promoter sequence. A promoter sequence includes proximal and more distal upstream elements, the latter of which are often referred to as enhancers. Accordingly, an "enhancer" is a nucleotide sequence capable of stimulating promoter activity and may be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. A promoter may be derived entirely from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even contain synthetic nucleotide segments. Those skilled in the art will understand that different regulatory elements may direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. A promoter that causes expression of a nucleic acid fragment in most cell types at most all times is generally referred to as a "constitutive promoter." It is further recognized that because the exact boundaries of regulatory sequences in most cases have not been fully defined, nucleic acid fragments of different lengths may have identical or similar promoter activity.

[0036] "Translation leader sequence" refers to a nucleotide sequence located between the promoter sequence and coding sequence of a gene. The translation leader sequence is present upstream of the translation start sequence in the fully processed mRNA. The translation leader sequence can influence many parameters, including processing of the primary transcript into mRNA, mRNA stability, and / or translation efficiency.

[0037] "3' non-coding sequences" refer to nucleotide sequences located downstream of the coding sequence, including sequences encoding polyadenylation recognition sequences and other regulatory signals capable of affecting mRNA processing or gene expression. Polyadenylation signals are usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of a pre-mRNA.

[0038] A DNA construct is a collection of linked DNA molecules that provide one or more expression cassettes. The DNA construct can be a plasmid that allows autonomous replication in bacterial cells and contains various endonuclease enzyme restriction sites useful for introducing functional genetic elements, i.e., DNA molecules that provide, among other things, promoters, introns, leaders, coding sequences, and 3' termination regions; or the DNA construct can be a linear collection of DNA molecules, such as an expression cassette. The expression cassette contained within the DNA construct contains the genetic elements necessary to effect transcription of messenger RNA. The expression cassette can be designed for expression in prokaryotic or eukaryotic cells. The expression cassette of this embodiment is designed for expression in plant cells.

[0039] The DNA molecules disclosed herein are provided in an expression cassette for expression in an organism of interest. The cassette includes 5' and 3' regulatory sequences operably linked to the coding sequence. "Operably linked" means that the linked nucleic acid sequences are contiguous, and, where necessary to join two protein-coding regions, contiguous and in the same reading frame. Operably linked is intended to refer to a functional linkage between a promoter and a second sequence, where the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. The cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, such one or more additional genes may be provided on multiple expression cassettes or multiple DNA constructs.

[0040] An expression cassette may include, from the 5' to 3' direction of transcription: a transcriptional and translational initiation region, a coding region, and a transcriptional and translational termination region functional in the host organism. The transcriptional initiation region (e.g., promoter) may be native or analogous to the host organism, or may be foreign or heterologous. In addition, the promoter may be a natural sequence, or alternatively, may be a synthetic sequence. The expression cassette may additionally contain a 5' leader sequence in the expression cassette construct. Such a leader sequence may serve to enhance translation.

[0041] As used herein, the term "transgenic" should be understood to generally include any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of heterologous nucleic acid (including those originally so altered as well as those produced by sexual crossing or asexual propagation from an initial transgenic) and which harbors such heterologous nucleic acid.

[0042] Transgenic "events" are produced by transforming plant cells with one or more heterologous DNA constructs, including a nucleic acid expression cassette containing a transgene of interest, regenerating a plant population resulting from the insertion of the transgene into the plant's genome, and selecting plants characterized by the insertion at a specific genomic location. Events are phenotypically characterized by the expression of the transgene. At the genetic level, events are part of the plant's genetic makeup. The term "event" also refers to progeny produced by sexual outcrossing between a transformant and another variety, which contain heterologous DNA. After backcrossing with the recurrent parent, the progeny of that cross contain the inserted DNA and linked flanking genomic DNA from the transformed parent at the same chromosomal location. Progeny plants may contain sequence variations in the insert resulting from conventional breeding techniques. The term "event" also refers to DNA from an original transformant, including the inserted DNA and flanking sequences immediately adjacent to the inserted DNA, that would be expected to be inherited by progeny receiving the inserted DNA, including the transgene of interest, as a result of sexual mating of one parent line containing the inserted DNA (e.g., the original transformant and its progeny resulting from selfing) with a parent line that does not contain the inserted DNA.

[0043] Breeding of insect-resistant DP-051291-2 corn plants can be achieved by first sexually crossing a first parent corn plant having a transgenic DP-051291-2 event plant and its progeny derived from transformation with an expression cassette of the present embodiments that confers insect resistance with a second parent corn plant lacking the expression cassette, thereby producing a plurality of first progeny plants; and then selecting first progeny plants that are resistant to insects; and selfing the first progeny plants, thereby producing a plurality of second progeny plants; and then selecting insect-resistant plants from the second progeny plants. These steps can further include backcrossing the first insect-resistant progeny plant or the second insect-resistant progeny plant with the second parent corn plant or the third parent corn plant, thereby producing an insect-resistant corn plant. The term "selfing" refers to self-pollination, including the union of gametes and / or nuclei from the same organism.

[0044] As used herein, the term "plant" includes reference to whole plants, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny. In some embodiments, transgenic plant parts include, for example, plant cells, protoplasts, tissues, callus, embryos, and flowers, stems, fruits, leaves, and roots that have been previously transformed with a DNA molecule disclosed herein and thus originate from a transgenic plant or its progeny that are at least partially composed of transgenic cells.

[0045] As used herein, the term "plant cell" includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The plant classes that can be used are generally as broad as the higher plant classes that are amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.

[0046] "Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in stable genetic inheritance. Host plants containing the transformed nucleic acid fragment are referred to as "transgenic" plants.

[0047] As used herein, the term "progeny," in the context of event DP-051291-2, refers to the descendants of any generation of a parent plant that includes corn event DP-051291-2.

[0048] The isolated polynucleotides disclosed herein can be incorporated into recombinant constructs, typically DNA constructs, capable of being introduced into and replicated in a host cell. Such constructs can be vectors containing a replication system and sequences capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Some vectors suitable for stable transfection of plant cells or establishment of transgenic plants are described, for example, in Pouwels et al. (1985; Supp. 1987) Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, (Academic Press, New York); and Flevin et al. (1990) Plant Molecular Biology Manual, (Kluwer Academic Publishers). Typically, plant expression vectors contain, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors can also contain a promoter regulatory region (e.g., a regulatory region that controls inducible or constitutive, environmentally or developmentally regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

[0049] During the process of introducing an insert into the genome of a plant cell, it is not uncommon for some deletions or other alterations to occur in the insert and / or genomic flanking sequences. Therefore, relevant segments of the plasmid sequences provided herein may contain some minor variations, including truncations. The same is possible for the flanking and linking sequences provided herein. Thus, plants containing polynucleotides having any range of identity to the subject flanking and / or insert sequences are within the scope of the subject disclosure. Identity to the disclosed sequences can be polynucleotide sequences having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% identity, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences exemplified or described herein. Hybridization and hybridization conditions as provided herein can also be used to define such plant and polynucleotide sequences of the subject disclosure. Sequences containing flanking sequences plus the complete insert sequence can be confirmed against the deposited seeds.

[0050] In some embodiments, two different transgenic plants can be crossed to produce offspring containing two independently segregating added exogenous genes. Self-breeding of the appropriate progeny can produce plants homozygous for the added exogenous gene. Backcrossing with the parent plant and outcrossing with a non-transgenic plant are also contemplated, as is vegetative propagation.

[0051] A "probe" is an isolated nucleic acid to which a conventional synthetic detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme, has been attached. Such a probe is complementary to a strand of target nucleic acid, such as a strand of DNA isolated from corn event DP-051291-2, whether from a corn plant or a sample containing DNA from the event. Probes include not only deoxyribonucleic acid or ribonucleic acid, but also polyamide and other modified nucleotides that can specifically bind to and be used to detect the presence of a target DNA sequence.

[0052] A "primer" is an isolated nucleic acid that anneals to a complementary target DNA strand by nucleic acid hybridization, thereby forming a hybrid between the primer and the target DNA strand, which is then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. A primer pair refers to its use in amplifying a target nucleic acid sequence, for example, by PCR or other conventional nucleic acid amplification methods. "PCR" or "polymerase chain reaction" is a technique used to amplify specific DNA segments (see U.S. Pat. Nos. 4,683,195 and 4,800,159; incorporated herein by reference).

[0053] Probes and primers are of sufficient nucleotide length to specifically bind to the target DNA sequence under hybridization or reaction conditions determined by the operator. This length can be any length sufficient to be useful in the detection method of choice. Generally, lengths of 11 nucleotides or more, 18 nucleotides or more, and 22 nucleotides or more are used. Such probes and primers specifically hybridize to the target sequence under high stringency hybridization conditions. Probes and primers according to embodiments can have consecutive nucleotides of perfect DNA sequence similarity to the target sequence, but probes that differ from the target DNA sequence and retain the ability to hybridize to the target DNA sequence can be designed by conventional methods. Probes can also be used as primers, but are generally designed to bind to target DNA or RNA and are not used in the amplification process.

[0054] Specific primers can be used to amplify integration fragments to generate amplicons that can be used as "specific probes" to identify event DP-051291-2 in biological samples. When the probe hybridizes to nucleic acids in a biological sample under conditions that allow binding of the probe to the sample, the binding can be detected, thereby indicating the presence of event DP-051291-2 in the biological sample. In certain embodiments of the present disclosure, the specific probe is a sequence that specifically hybridizes to a region within the 5' or 3' flanking region of the event under appropriate conditions and also contains a portion of the exogenous DNA adjacent to it. The specific probe can comprise a sequence at least 80%, 80 and 85%, 85 and 90%, 90 and 95%, 95 and 100% identical (or complementary) to the specific region of the event.

[0055] Methods for preparing and using probes and primers are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2002. nded., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989 (hereinafter "Sambrook et al., 1989"); Ausubel et al. eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, 1995 (including periodic updates) (hereinafter "Ausubel et al., 1995"); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from known sequences using computer programs designed for this purpose, such as the PCR primer analysis tools in Vector NTI, Version 6 (Informax Inc., Bethesda, MD); PrimerSelect (DNASTAR Inc., Madison, WI); and Primer (Version 0.5®, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, primers can be manually identified by visual inspection of the sequence using guidance known to those of skill in the art.

[0056] "Kit," as used herein, refers to a set of reagents and optionally instructions for practicing embodiments of the disclosed methods, and more particularly, for identifying event DP-051291-2 in biological samples. Kits can be used whose components are specifically tailored for quality control (e.g., seed lot purity), detection of event DP-051291-2 in plant material, or in materials that contain or are derived from plant material, such as, but not limited to, food or feed products. "Plant material," as used herein, refers to material obtained from or derived from a plant.

[0057] Using primers and probes based on the flanking DNA and insert sequences disclosed herein, the disclosed sequences can be confirmed (and modified, if necessary) by conventional methods, for example, by recloning and sequencing such sequences. The nucleic acid probes and primers hybridize to the target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from the transgenic event in a sample.

[0058] A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if it exhibits complete complementarity or minimal complementarity. As used herein, molecules are said to exhibit "complete complementarity" when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they can hybridize to each other with sufficient stability to allow them to remain annealed to each other under at least conventional "low stringency" conditions. Similarly, molecules are said to be "complementary" if they can hybridize to each other with sufficient stability to allow them to remain annealed to each other under conventional "high stringency" conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and Haymes et al., "Nucleic Acid Hybridization, a Practical Approach," IRL Press, Washington, DC (1985), and thus deviations from perfect complementarity are acceptable as long as such deviations do not completely abolish the ability of the molecules to form double-stranded structures. For a nucleic acid molecule to serve as a primer or probe, there need only be sufficient complementarity to enable the sequences to form a stable double-stranded structure under the specific solvent and salt concentrations employed.

[0059] In hybridization reactions, specificity is typically a function of post-hybridization washes, with the determining factors being the ionic strength and temperature of the final wash solution. m ) is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. For DNA-DNA hybrids, T m can be approximated from the formula of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: T m T = 81.5°C + 16.6(logM) + 0.41(%GC) - 0.61(%form) - 500 / L, where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the hybrid length in base pairs. m decreases by approximately 1°C for every 1% mismatch; thus, to hybridize sequences of desired identity, T m For example, if sequences with greater than 90% identity are desired, the hybridization and / or washing conditions can be adjusted. m Generally, stringent conditions are those that achieve a T for a specific sequence and its complementary sequence at a defined ionic strength and pH. m However, in some embodiments, other stringency conditions may be applied, with highly stringent conditions being selected to be about 5°C lower than T m Moderately stringent conditions may utilize hybridization and / or washing at temperatures 1, 2, 3, or 4° C. lower than T; m Hybridization and / or washing at temperatures 6, 7, 8, 9, or 10° C. lower than T may be utilized; low stringency conditions may be utilized at temperatures 6, 7, 8, 9, or 10° C. lower than T m and the like. Hybridization and / or washing at temperatures 11, 12, 13, 14, 15, or 20° C. lower than the standard temperature may be utilized.

[0060] This formula, hybridization and washing compositions, and the desired T m Using the above, one of skill in the art will understand that variations in the stringency of hybridization and / or wash solutions are essentially described. If the degree of desired mismatch is below T 45°C (aqueous solution) or 32°C (formamide solution), m If this results in a high SSC concentration, the user may choose to increase the SSC concentration so that a higher temperature can be used. Extensive guides to nucleic acid hybridization can be found in Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel, et al., eds. (1995) and Sambrook et al. (1989).

[0061] In some embodiments, a complementary sequence has the same length as the nucleic acid molecule to which it hybridizes. In some embodiments, a complementary sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, a complementary sequence is 1%, 2%, 3%, 4%, or 5% longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, a complementary sequence is complementary on a nucleotide-by-nucleotide basis, meaning that there are no mismatched nucleotides (every A pairs with a T and every G pairs with a C). In some embodiments, a complementary sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or fewer mismatches. In some embodiments, a complementary sequence contains 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or fewer mismatches.

[0062] "Percent (%) sequence identity" to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical to each amino acid residue or nucleotide in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative amino acid substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST and BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including which algorithms are required to achieve maximum alignment across the full length of the sequences under comparison. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions in the query sequence × 100).

[0063] With respect to the amplification of a target nucleic acid sequence using a particular amplification primer pair (e.g., by PCR), stringent conditions are those that allow the primer pair to hybridize only to the target nucleic acid sequence that, if the primers had the corresponding wild-type sequence (or its complement), would bind in a DNA thermal amplification reaction and, optionally, produce a unique amplification product, an amplicon.

[0064] As used herein, "amplified DNA" or "amplicon" refers to a nucleic acid amplification product of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a corn plant obtained from a sexual cross contains transgenic event genomic DNA from a corn plant disclosed herein, DNA extracted from a corn plant tissue sample is subjected to a nucleic acid amplification method using a DNA primer pair comprising a first primer derived from a flanking sequence adjacent to the insertion site of the inserted heterologous DNA and a second primer derived from the inserted heterologous DNA, producing an amplicon that is diagnostic of the presence of the event DNA. Alternatively, the second primer can be derived from the flanking sequence. The amplicon has a length and sequence that is also diagnostic of the event. The amplicon can range in length from the total length of the primer pair plus one nucleotide base pair to any length amplicon producible by the DNA amplification protocol. Alternatively, the primer pair can be derived from flanking sequences on either side of the inserted DNA, resulting in the production of an amplicon that contains the entire insert nucleotide sequence of the PHP74638 expression construct as well as a portion of the sequence flanking the transgenic insert. The members of the primer pair derived from the flanking sequence can be located at a distance from the inserted DNA sequence, which can range from one nucleotide base pair up to the limit of the amplification reaction. The use of the term "amplicon" specifically excludes primer dimers that may form in a DNA thermal amplification reaction.

[0065] Nucleic acid amplification can be achieved by any of a variety of nucleic acid amplification methods known in the art, including PCR. Various amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202, and Innis et al. (1990) (supra). PCR amplification methods have been developed to amplify up to 22 Kb of genomic DNA and up to 42 Kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods of DNA amplification, as well as other methods known in the art, can be used in practicing embodiments of the present disclosure. It is understood that several parameters of specific PCR protocols may need to be adjusted to suit specific laboratory conditions and may be slightly modified and still achieve similar results. These adjustments will be apparent to those skilled in the art.

[0066] Amplicons produced by these methods can be detected by several techniques, including, but not limited to, Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994), in which DNA oligonucleotides are designed that overlap both the adjacent flanking DNA sequence and the inserted DNA sequence. These oligonucleotides are immobilized in the wells of a microwell plate. Following PCR of the region of interest (e.g., using one primer in the inserted sequence and one primer in the adjacent flanking sequence), the single-stranded PCR product is hybridized to the immobilized oligonucleotide and can serve as a template for a single-base extension reaction using DNA polymerase and labeled ddNTPs specific for the expected next base. Readouts can be fluorescent or ELISA-based. A signal indicates the presence of the insert / flanking sequence due to successful amplification, hybridization, and single-base extension.

[0067] Another detection method is the pyrosequencing technique as described by Winge (2000) Innov. Pharma. Tech. 00:18-24. In this method, oligonucleotides are designed to overlap the junctions between the flanking DNA and the insert DNA. The oligonucleotides are hybridized to single-stranded PCR products from the region of interest (e.g., one primer for the inserted sequence and one for the flanking sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate, and luciferin. dNTPs are added separately, and a light signal is measured upon incorporation. The light signal indicates the presence of the transgene insert / flanking sequence due to successful amplification, hybridization, and single- or multi-base extension.

[0068] Fluorescence polarization, described by Chen et al. (1999) Genome Res. 9:492-498, is another method that can be used to detect amplicons. Using this method, oligonucleotides are designed to overlap the junction between the flanking and inserted DNA. These oligonucleotides are hybridized to single-stranded PCR products of the region of interest (e.g., one primer for the inserted DNA and one for the flanking DNA sequence) and incubated in the presence of DNA polymerase and fluorescently labeled ddNTPs. Single-base extension results in the incorporation of ddNTPs. Incorporation can be measured using a fluorometer as a change in polarization. The change in polarization indicates the presence of the transgene insert / flanking sequence due to successful amplification, hybridization, and single-base extension.

[0069] Quantitative PCR (qPCR) has been described as a method for detecting and quantifying the presence of DNA sequences and is well understood in the instructions provided by commercial product manufacturers. Briefly, in one such qPCR method, a FRET oligonucleotide probe is designed to overlap the junction of the flanking and insert DNA. The FRET probe and PCR primers (one primer for the insert DNA sequence and one for the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety, which separates from the quenching moiety of the FRET probe. The fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization.

[0070] Molecular beacons for use in sequence detection have been described as described by Tyangi et al. (1996) Nature Biotech. 14:303-308. Briefly, a FRET oligonucleotide probe is designed to overlap the junction of the flanking and insert DNA. The unique structure of the FRET probe results in it having a secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (e.g., one primer for the insert DNA sequence and one for the flanking sequence) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, the FRET probe hybridizes to the target sequence, resulting in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. The resulting fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization.

[0071] Hybridization reactions using probes specific for sequences found within the amplicon are yet another method used to detect amplicons produced by PCR reactions.

[0072] Examples of pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, and the like, particularly Coleoptera.

[0073] Of interest are larvae and adults of the order Coleoptera, including weevils of the families Anthribidae, Bruchidae, and Curculionidae, such as, but not limited to, Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspessus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (Citrus root weevil); Hypera punctata Fabricius (clover leaf weevil); wevil); Lissorhoptrus oryzophilus Kuschel (grain soup weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian sugarcane weevil); M. hemipterus sericeus Olivier (white cane weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (sunflower seed weevil); S. sordidus LeConte LeConte) (gray sunflower seed weevil); Sphenophorus maidis Chittenden (corn weevil); S. livis vaurier (S.livis Vaurie (sugarcane weevil); Rhabdoscelus obscurus Boisduval (New Guinea Pig sugarcane weevil); fleas, cucumber beetles, rootworms, leaf beetles, potato beetles, and foliar pests of the family Chrysomelidae, including but not limited to: Chaetocnema ectypa Horn (dessert corn flea beetle); C. pulicaria Melsheimer (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberi Smith & Lawrence Smith & Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn beetle); D. virgifera virgifera Leconte (D.virgifera virgifera LeConte (Western corn rootworm); Leptinotarsa ​​decemlineata Say (Colorado potato beetle); Oulema melanopus Linnaeus (cereal leaf beetle); Phylloterteta cruciferae Goeze (corn leaf beetle); Zygogramma exclamationis Fabricius; Coccinellidae beetles, including but not limited to: Epilachna varivestis marsanto Mulsant (Mexican bean beetle); chafers and other beetles of the Scarabaeidae family, including but not limited to: Antitrogus parvulus Britton (tildus sugarcane grub); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Dermolepida albohirtum Waterhouse (grayback sugarcane beetle); Euetheola humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi Blackburn Blackburn (French sugarcane grub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub); P. latifrons Leconte (P.latifrons LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); dermestidae beetles, Elateridae wireworms, Eleodes and Melanotus genera, e.g. M. communis Gylenhal Gyllenhal (wireworms); Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles of the Scolytidae family; beetles of the Tenebrionidae family; beetles of the Cerambycidae family, including but not limited to Migdolus fryanus Westwood (longhorn beetle); and beetles of the Buprestidae family, including but not limited to Aphanisticus cochinchinae seminulum Obenberger (leaf-mining jewel beetle).

[0074] In some embodiments, the DP-051291-2 corn event can further comprise a stack of additional traits. Plants comprising a stack of polynucleotide sequences can be obtained by either or both traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming transgenic plants comprising the genes disclosed herein with subsequent genes, and co-transforming genes into a single plant cell. As used herein, the term "stacked" includes having multiple traits present in the same plant (i.e., both traits integrated into the nuclear genome, one trait integrated into the nuclear genome and one trait integrated into the plastid genome, or both traits integrated into the plastid genome).

[0075] In some embodiments, the DP-051291-2 corn event disclosed herein, alone or stacked with one or more additional insect resistance traits, can be stacked with one or more additional input traits (e.g., herbicide resistance, fungus resistance, virus resistance, stress resistance, disease resistance, male sterility, stalk strength, etc.) or output traits (e.g., increased yield, modified starch, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought tolerance, etc.). In this manner, the present embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost-effectively control several agronomic pests.

[0076] In further embodiments, the DP-051291-2 corn event is a toxin that can be used to treat a variety of diseases, including but not limited to, Cry3B toxins as disclosed in U.S. Pat. Nos. 8,101,826, 6,551,962, 6,586,365, 6,593,273, and WO 2000 / 011185; mCry3B toxins as disclosed in U.S. Pat. Nos. 8,269,069, and 8,513,492; or the Cry34 / 35 toxins disclosed in U.S. Patent Nos. 7,309,785, 7,524,810, 7,985,893, 7,939,651, and 6,548,291. In further embodiments, the DP-051291-2 corn event is transgenic with one or more additional transgenic events containing these Bt insecticidal toxins and other Coleopteran active Bt insecticidal traits, such as event MON863 disclosed in U.S. Pat. No. 7,705,216; event MIR604 disclosed in U.S. Pat. No. 8,884,102; event 5307 disclosed in U.S. Pat. No. 9,133,474; event DAS-59122 disclosed in U.S. Pat. No. 7,875,429; event DP-4114 disclosed in U.S. Pat. No. 8,575,434; event MON disclosed in U.S. Pat. No. 9,441,240; 87411; event DP-23211 disclosed in WO 2019 / 209700; and event MON88017 disclosed in U.S. Pat. No. 8,686,230, all of which are incorporated herein by reference.In some embodiments, the DP-051291-2 corn event can be stacked with MON-87429-9 (MON87429 event); MON87403; MON95379; MON95275; MON87427; MON87419; MON-00603-6 (NK603); MON-87460-4; LY038; DAS-06275-8; BT176; BT11; MIR162; GA21; MZDT09Y; SYN-05307-1; DP-915635-2; DP-23211; DP-910521-2; DAS-01131-3; and DAS-40278-9.

[0077] In further embodiments, the DP-051291-2 corn event can be stacked with one or more of the following additional herbicide-resistance traits provided: Glyphosate herbicides act by inhibiting the EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase). This enzyme is involved in the biosynthesis of aromatic amino acids essential for plant growth and development. Various enzymatic mechanisms are known in the art that can be utilized to inhibit this enzyme. Genes encoding such enzymes can be operably linked to the genetic regulatory elements of the present disclosure. In one embodiment, selectable marker genes include, but are not limited to, genes encoding glyphosate resistance genes: mutant EPSPS genes, such as the 2mEPSPS gene, the cp4 EPSPS gene, the mEPSPS gene, and the aroA gene; and glyphosate degradation genes, such as the glyphosate acetyltransferase gene (gat) and the glyphosate oxidase gene (gox). These traits are currently commercially available as Gly-Tol™, Optimum® GAT®, Agrisure® GT, and Roundup Ready®. Resistance genes for glufosinate and / or bialaphos compounds include the dsm-2, bar, and pat genes. The bar and pat traits are currently commercially available as LibertyLink®. Also included are resistance genes that provide resistance to 2,4-D, such as the aad-1 gene (note that the aad-1 gene has additional activity against alloxyphenoxypropionic acid herbicides) and the aad-12 gene (note that the aad-12 gene has additional activity against pyridyloxyacetic acid synthetic auxins). These traits are commercially available as Enlist® Crop Protection Technology. Resistance genes to ALS inhibitors (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) are known in the art.

[0078] These resistance genes most commonly result from point mutations in the ALS-encoding gene sequence. Other ALS inhibitor resistance genes include the hra gene, csr1-2 gene, Sr-HrA gene, and surB gene. Some of these traits are sold under the trade name Clearfield®. Herbicides that inhibit HPPD include pyrazolones, such as pyrazoxyfen, benzofenap, and topramezone. Triketones, such as mesotrione, sulcotrione, tembotrione, and benzobicyclon; and diketonitriles, such as isoxaflutole. These exemplary HPPD herbicides can be tolerated by known traits. Examples of HPPD inhibitors include the hppdPF_W336 gene (resistance to isoxaflutole) and the avhppd-03 gene (resistance to meostrian). An example of an oxynil herbicide resistance trait is the bxn gene, which has been shown to confer resistance to the herbicide / antibiotic bromoxynil. Dicamba resistance genes include the dicamba monooxygenase gene (dmo) as disclosed in WO 2008 / 105890. Resistance genes to PPO or PROTOX inhibitor herbicides (e.g., acifluorfen, butafenacil, flupropadil, pentoxazone, carfentrazone, furazolate, pyraflufen, aclonifen, azafenidin, flumioxazin, flumiclorac, bifenox, oxyfluorfen, lactofen, fomesafen, fluoroglycofen, and sulfentrazone) are known in the art.Exemplary genes that confer resistance to PPO include the wild-type Arabidopsis thaliana PPO enzyme (Lermontova I and Grimm B, (2000) Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol 122:75-83.), the B. subtilis PPO gene (Li, X. and Nicholl D. 2005. Development of PPO inhibitor-resistant cultures and crops. Pest Manag. Sci. 61:277-285 and Choi KW, Han O, Lee HJ, Yun YC, Moon YH, Kim MK, Kuk YI, Han SU and Guh JO, (1998) Generation of resistance to the diphenyl ether herbicide, oxyfluorfen, via expression of Examples of herbicides that confer resistance to transgenic tobacco plants include the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants. Biosci Biotechnol Biochem 62:558-560. Pyridinoxy- or phenoxypropionic acid and cyclohexone resistance genes include genes encoding ACCase inhibitors (e.g., Acc1-S1, Acc1-S2, and Acc1-S3). Exemplary genes conferring resistance to cyclohexanedione and / or aryloxyphenoxypropanoic acids include haloxyfop, diclofop, fenoxyprop, fluazifop, and quizalofop. Finally, herbicides can inhibit photosynthesis, including psbA gene-mediated resistance (resistance to triazines), 1s+ gene-mediated resistance (resistance to triazines), and nitrilase gene-mediated resistance (resistance to benzonitriles).The selectable markers listed above are not intended to be limiting; any herbicide resistance gene is encompassed by the present disclosure.

[0079] In some embodiments, the disclosed compositions can be introduced into a plant's genome using genome editing technology, or a polynucleotide already introduced into a plant's genome can be edited using genome editing technology. For example, the disclosed polynucleotides can be introduced into a desired location in a plant's genome by using a genome editing system such as TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas. For example, the disclosed polynucleotides can be introduced into a desired location in the genome using a CRISPR-Cas system for site-specific insertion. The desired location in the plant genome can be any target site where insertion is desired, such as a genomic region suitable for breeding, or a target site located in a genomic window containing an existing trait of interest. The existing trait of interest can be either an endogenous trait or a previously introduced trait.

[0080] In some embodiments, if the disclosed polynucleotides have previously been introduced into a genome, genome editing or genome engineering techniques can be used to alter or modify the introduced polynucleotide sequence, including adjacent chromosomal genomic sequences. Site-specific modifications that can be introduced into the disclosed compositions include, but are not limited to, those generated using any method for introducing site-specific modifications, such as through the use of sequence repair oligonucleotides alone, or through the use of site-specific genome modification tools, such as TALENs, meganucleases, zinc finger nucleases, and CRISPR-Cas, with or without donor DNA. Site-specific modifications to the disclosed polynucleotides (including genomic flanking and junction sequences) can include, but are not limited to, changes in codon usage, regulatory elements such as promoters, introns, terminators, enhancers, 5' or 3' untranslated regions (UTRs), or other non-coding sequences, and other regions of the polynucleotide, where the modifications do not adversely affect the phenotypic characteristics of the resulting corn plant. DP-051291-2 event plants containing modified polynucleotide sequences are also contemplated herein.

[0081] Suitable Cas polypeptides for introducing site-specific modifications include, for example, Cas9, Cas12f (Cas-α, Cas14), Cas12l (Cas-beta), Cas12a (Cpf1), Cas12b (C2c1 protein), Cas13 (C2c2 protein), Cas12c (C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas5, Cas6, Cas7, Cas8, Cas10, or combinations or complexes thereof. In some embodiments, transposon-associated TnpB, a programmable RNA-guided DNA endonuclease, can be used.

[0082] In some embodiments, the genome editing system comprises a Cas-alpha (e.g., Cas12f) endonuclease and one or more guide polynucleotides that introduce one or more site-specific modifications into a target polynucleotide sequence, resulting in a modified target sequence. As used herein, the terms "altered target site," "altered target sequence," "altered target site," and "altered target sequence" are used interchangeably and refer to a target sequence disclosed herein that contains at least one modification or alteration when compared to the unaltered target sequence. Such modifications or alterations include, for example, (i) a substitution or substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0083] In some embodiments, the genome editing system comprises a Cas-alpha endonuclease, one or more guide polynucleotides, and optionally donor DNA. Some exemplary Cas-alpha endonucleases are described, for example, in WO2020123887.

[0084] In some embodiments, the genome editing system comprises a Cas polypeptide, one or more guide polynucleotides, and optionally donor DNA, and editing the target polynucleotide sequence comprises non-homologous end joining (NHEJ) or homologous recombination (HR) following a Cas polypeptide-mediated double-strand break. When a double-strand break is introduced into DNA, the cell's DNA repair machinery is activated to repair the break. The most common repair mechanism for joining broken ends together is the non-homologous end joining pathway (Bleuyard et al., (2006) DNA Repair 5:1-12). As a result, deletions, insertions, or other rearrangements are possible (Siebert and Puchta, (2002) Plant Cell 14:1121-31; Pacher et al., (2007) Genetics 175:21-9). Alternatively, the double-strand break can be repaired by homologous recombination between homologous DNA sequences. If the sequence near the double-strand break is altered, for example, by exonuclease activity involved in double-strand break maturation, gene conversion pathways can repair the original structure if homologous sequences are available, such as homologous chromosomes in non-dividing somatic cells or sister chromatids after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA repair templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0085] In some embodiments, the genome editing system includes a Cas polypeptide, one or more guide polynucleotides, and donor DNA. As used herein, "donor DNA" refers to a DNA construct containing a polynucleotide of interest to be inserted into the genomic target site of the Cas polypeptide. When a double-strand break is introduced into the target site by an endonuclease, the first and second regions of homology in the donor DNA can undergo homologous recombination with their corresponding genomic regions of homology, resulting in DNA exchange between the donor DNA and the target genomic region. Thus, the provided method incorporates a polynucleotide of interest in the donor DNA into the double-strand break in the target site in a plant genome, thereby modifying the original target site and generating a modified genomic target site.

[0086] In some embodiments, the genome editing system comprises a base editor and a plurality of guide polynucleotides, and editing the target polynucleotide sequence comprises introducing a plurality of nucleic acid base edits into the target polynucleotide sequence resulting in a variant nucleotide sequence. Other embodiments include modified DP-051291-2 event plants produced using the genome editing system.

[0087] One or more nucleic acid bases of the target genome sequence can, in some cases, be chemically altered to change the base from one type to another, for example, from cytosine to thymine, or from adenine to guanine. In some embodiments, multiple bases can be altered or modified, for example, 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, or even 100, 200 or more, up to several thousand bases, to generate plants with multiple altered bases.

[0088] Any base editing complex, such as a base editor associated with an RNA-guided polypeptide (e.g., dCas associated with a deaminase), can be used to target and bind to a desired locus within the genome of an organism and chemically modify one or more nucleotides of the target genomic sequence.

[0089] Site-specific nucleotide base conversion can be achieved to engineer one or more nucleotide changes to create one or more edits in the genome. These include, for example, site-specific base editing mediated by C·G to T·A or A·T to G·C base editing deaminase enzymes (Gaudelli et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage,” Nature (2017); Nishida et al., “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems,” Science 353(6305) (2016); Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature 533(7603) (2016):420-4). A catalytically "dead" or inactive Cas (dCas) polypeptide fused to a cytidine deaminase or adenine deaminase protein, such as inactive Cas9 (dCas9), Cas12f (dCas12f), or another Cas polypeptide disclosed herein, becomes a specific base editor that can modify DNA bases without inducing DNA cleavage. A base editor converts C to T (or G to A on the opposite strand), or an adenine base editor converts adenine to inosine, resulting in an A to G change within the editing window specified by the guide polynucleotide. Any molecule that effects a change in a nucleic acid base is a "base editor." dCas forms a functional complex with a guide polynucleotide that shares homology with the genomic sequence of the target site and further complexes with a deaminase molecule. The induced Cas polypeptide recognizes and binds to the target sequence, opening the duplex to expose individual bases. In the case of cytidine deaminase, the deaminase deaminates the cytosine base to produce uracil.A uracil glycosylase inhibitor (UGI) is provided to prevent the conversion of U back to C. DNA replication or repair machinery then converts the uracil to thymine (U to T) and then repairs the opposite base (the former G of the original GC pair) to an adenine, creating a TA pair.

[0090] One or more nucleotides of the inserted event and / or the adjacent genomic DNA can be modified using prime editing techniques. See, for example, Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019). The prime editing complex includes a prime editing protein containing an RNA-guided DNA nicking domain, such as a Cas nickase (e.g., Cas9 nickase, Casd12f1 nickase), fused to a reverse transcriptase domain and complexed with a PEG-RNA. The PEG-RNA complex can introduce a targeted DNA edit at the desired location in the genome by binding to the target DNA and nicking the PAM-containing strand. The resulting 3' end hybridizes to a selected primer binding site, which then primes reverse transcription of a new DNA sequence containing the desired edit using the PEG-RNA as a reverse transcriptase template.

[0091] The resulting regulatory expression elements of the disclosed recombinant expression cassettes may be truncated or may comprise a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% identity, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the regulatory element sequences exemplified or described herein.

[0092] Other modifications may include modifications to other portions of the DNA of the DP-051291-2 event. In some embodiments, genome engineering techniques can be used to relocate one or more expression cassettes described herein to one or more different locations on the same chromosome or on different chromosomes of maize or a different crop. In such embodiments, a polynucleotide comprising one or more of the junction sequences described herein (SEQ ID NOs: 26 and / or 29) may be partially or completely retained or removed by the expression cassette. Additionally, the genomic flanking sequences described herein may also be partially or completely retained or removed with the expression cassette.

[0093] In another embodiment, genome engineering techniques can be used to co-locate one or more transgenes or expression cassettes physically adjacent to the 5' or 3' junction sequences described herein. In terms of physical location on a chromosome, the juxtaposed transgenes and / or expression cassettes can be separated from the 5' or 3' junction sequences by, for example, about 1 megabase (MB; 1 million nucleotides), about 500 kilobases (Kb; 1000 nucleotides), about 400 Kb, about 300 Kb, about 200 Kb, about 100 Kb, about 50 Kb, about 25 Kb, about 10 Kb, about 5 Kb, about 4 Kb, about 3 Kb, about 2 Kb, about 1 Kb, about 500 nucleotides, about 250 nucleotides, or less. In terms of genetic distance on a chromosome, co-located transgenes and / or expression cassettes can be separated from the 5' or 3' junction sequences by, for example, about 10 cM, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, 0.1 cM. For example, one or more expression cassettes resulting from one or more additional transgenic events described above can be co-located in physical proximity to the 5' or 3' junction sequences described herein.

[0094] In another embodiment, a polynucleotide containing one of the junction sequences (SEQ ID NO: 26 or 29) can be introduced at one or both ends of the inserted heterologous DNA. For example, the polynucleotide containing the 5' junction sequence can be deleted and replaced with a polynucleotide containing the 3' junction sequence, or vice versa.

[0095] In another embodiment, genome editing techniques can be used to modify previously introduced polynucleotides by inverting at least one of the polynucleotides of the inserted DNA of the DP-051291-2 event.

[0096] Using such genome editing techniques, previously introduced polynucleotides can be modified by inserting, deleting, and / or substituting one or more nucleotides within the introduced polynucleotide. Alternatively, double-strand break techniques can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that can be added include, but are not limited to, additional expression elements, such as enhancer and promoter sequences. Sequences that can be deleted include, but are not limited to, regulatory elements or portions thereof that do not adversely affect function when deleted. Modifications to adjust expression patterns (e.g., reducing the expression level of an insecticidal polypeptide in a specific tissue) are also contemplated by site-specific modifications to the introduced expression cassette.

[0097] In another embodiment, genome modification techniques can be used to delete or modify all or a portion of one or more expression cassettes of the DP-051291-2 event, deposited with the ATCC on August 19, 2022, and having accession number PTA-127358. In this embodiment, the resulting corn plants derived from the DP-051291-2 event, deposited with the ATCC on August 19, 2022, and having accession number PTA-127358, can contain a portion of the expression cassettes described herein, no expression cassettes described herein, or modifications of the expression cassettes described herein.

[0098] In another embodiment, using targeted DSB technology, additional insecticidally active proteins can be placed within the genome of a plant in close proximity to the compositions of the present disclosure disclosed herein to create a molecular stack of insecticidally active proteins.

[0099] In another embodiment, the polynucleotide sequences disclosed herein are used in a method comprising designing a guide polynucleotide, e.g., a guide RNA (gRNA), that recognizes the polynucleotide sequence, synthesizing or obtaining the guide polynucleotide, and introducing the guide polynucleotide as part of a genome modification composition to modify DNA of the DP-051291-2 event, deposited with the ATCC on August 19, 2022, and having accession number PTA-127358. Such resulting modifications may include polynucleotide sequences having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% identity, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence exemplified or described herein.

[0100] Embodiments include modified DP-051291-2 event plants generated using the genome modification techniques described herein.

[0101] One embodiment includes a corn plant comprising the genotype of corn event DP-051291-2, wherein the genotype comprises a nucleotide sequence as set forth in SEQ ID NO:26 and SEQ ID NO:29, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:26 and SEQ ID NO:29.

[0102] Another embodiment includes a corn plant comprising the genotype of corn event DP-051291-2 of any previous embodiment, wherein the genotype comprises a nucleotide sequence set forth in SEQ ID NO:27 and SEQ ID NO:30, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27 and SEQ ID NO:30.

[0103] Another embodiment includes a corn plant comprising the genotype of corn event DP-051291-2 of any previous embodiment, wherein the genotype comprises the nucleotide sequence set forth in SEQ ID NO:28 and SEQ ID NO:31, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:28 and SEQ ID NO:31.

[0104] One embodiment includes a DNA construct comprising operably linked expression cassettes, one of the expression cassettes comprising: 1) BSV(AY) promoter; 2) zm-HPLV9 intron; 3) ipd072Aa; and 4) AT-T9 Terminator.

[0105] Another embodiment includes a plant comprising a DNA construct comprising two operably linked expression cassettes of any of the previous embodiments.

[0106] A further embodiment includes a plant comprising a DNA construct comprising two operably linked expression cassettes of any preceding embodiment, wherein the plant is a corn plant.

[0107] One embodiment includes a plant comprising the sequence shown in SEQ ID NO:21 or a sequence having at least 95% sequence identity to SEQ ID NO:21.

[0108] In one embodiment, a representative sample of seeds of the corn event comprises corn event DP-051291-2, which has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

[0109] Other embodiments include plant parts of corn event DP-051291-2, a representative sample of seeds of which has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358 of any of the previous embodiments.

[0110] One embodiment includes seeds comprising corn event DP-051291-2, the seeds comprising a DNA molecule selected from SEQ ID NO:26 and SEQ ID NO:29, wherein a representative sample of said corn event DP-051291-2 seeds has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

[0111] Another embodiment includes a corn plant or part thereof grown from seed comprising corn event DP-051291-2, the corn plant or part thereof comprising a DNA molecule selected from SEQ ID NO:26 and SEQ ID NO:29, wherein a representative sample of said corn event DP-051291-2 seed comprises the corn plant or part thereof deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358 of any previous embodiment.

[0112] Further embodiments include transgenic seed produced from corn plants of corn event DP-051291-2, wherein a representative sample of seeds of said corn event has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358 of any preceding embodiment.

[0113] Other embodiments include a transgenic corn plant or portion thereof grown from seed produced from a corn plant of corn event DP-051291-2, a representative sample of seed of which has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358 of any of the previous embodiments.

[0114] One embodiment includes an isolated nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 21 and 26-31, and full-length complements thereof.

[0115] One embodiment includes an amplicon comprising a nucleic acid sequence selected from SEQ ID NOs: 21-25 and their full-length complements.

[0116] One embodiment includes a biological sample or extract derived from a corn event DP-051291-2 plant, tissue, or seed, comprising a nucleotide sequence that is, or is complementary to, a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, wherein the nucleotide sequence is detectable in the sample or extract using a nucleic acid amplification method or a nucleic acid hybridization method, and a representative sample of the corn event DP-051291-2 seed has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

[0117] Another embodiment is a biological sample or extract from a corn event DP-051291-2 plant, tissue, or seed, comprising a nucleotide sequence that is or is complementary to a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, wherein the nucleotide sequence is detectable in the sample or extract using a nucleic acid amplification method or a nucleic acid hybridization method, and a representative sample of the corn event DP-051291-2 seed has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358 of any previous embodiment, and the biological sample or extract comprises a transgenic corn event DP-051291-2 plant, plant tissue, or seed.

[0118] Another embodiment includes a biological sample or extract derived from corn event DP-051291-2 plants, tissues, or seeds, said sample or extract comprising a nucleotide sequence that is or is complementary to a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method; a sample of corn event DP-051291-2 seeds representative of any previous embodiment has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358; and said biological sample or extract is a DNA sample extracted from transgenic corn plant event DP-051291-2, said DNA sample comprising one or more nucleotide sequences selected from SEQ ID NOs:21-31 and complements thereof.

[0119] Another embodiment includes a biological sample or extract derived from corn event DP-051291-2 plants, tissues, or seeds, wherein the sample or extract comprises a nucleotide sequence that is, or is complementary to, a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, wherein the nucleotide sequence is detectable in the sample or extract using a nucleic acid amplification or nucleic acid hybridization method; a representative sample of the corn event DP-051291-2 seeds has been deposited with the American Type Culture Collection (ATCC) having accession number PTA-127358 of any of the previous embodiments; and the biological sample or extract is selected from corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals produced in whole or in part to contain corn by-products.

[0120] One embodiment is a method for producing hybrid corn seed, comprising: a) sexually crossing a first inbred corn line comprising a nucleotide sequence selected from SEQ ID NOs: 21-31 with a second inbred line having a different genotype; b) growing offspring from said mating; and and c) harvesting the hybrid seeds produced thereby.

[0121] Another embodiment includes the method of producing hybrid corn seed of any previous embodiment, wherein the first inbred corn line is the female parent or the male parent.

[0122] One embodiment is a method for producing a corn plant that is resistant to Coleopteran pests, comprising: a) sexually crossing a first parent corn plant with a second parent corn plant, thereby producing a plurality of first generation progeny plants, wherein the first or second parent corn plant comprises event DP-051291-2; b) producing a plurality of second generation progeny plants by selfing the first generation progeny plants; and c) selecting from said second generation progeny plants that contain said event DP-051291-2 and are resistant to Coleoptera pests. The method includes:

[0123] Another embodiment is a method for producing hybrid corn seed, comprising: a) sexually crossing a first inbred corn line that contains the DNA construct comprising two operably linked expression cassettes of any of the previous embodiments with a second inbred line that does not contain the DNA construct comprising two operably linked expression cassettes of any of the previous embodiments; and b) harvesting the hybrid seeds produced thereby.

[0124] Another embodiment includes the method of producing a corn plant that is resistant to Coleoptera pests of any previous embodiment, further comprising backcrossing a second generation progeny plant that includes corn event DP-051291-2 with the parent plant that lacks the corn event DP-051291-2 DNA, thereby producing a backcross progeny plant that is resistant to Coleoptera pests.

[0125] One embodiment is a method of producing a corn plant that is resistant to corn rootworm, comprising: a) crossing a first parent corn plant with a second parent corn plant, thereby producing a plurality of first generation progeny plants, wherein the first or second parent corn plant comprises event DP-051291-2; b) selecting first generation progeny plants containing said event DP-051291-2; c) backcrossing the first generation progeny plants of step (b) with a parent plant lacking corn event DP-051291-2 DNA, thereby producing a plurality of backcross progeny plants; and d) selecting from the backcross progeny plants a plant containing the event DP-051291-2; wherein the selected backcross progeny plant of step (d) comprises SEQ ID NO: 21, 26 or 29.

[0126] Another embodiment includes the method for producing a corn rootworm resistant corn plant of any previous embodiment, wherein the plant of the first parent corn line is the female or male parent.

[0127] Another embodiment includes hybrid seed produced by the method for producing corn rootworm resistant corn plants of any previous embodiment.

[0128] One embodiment is a method for determining the zygosity of a corn plant containing event DP-051291-2 in a biological sample, comprising: a) contacting the sample with a first pair of DNA molecules and a second, different pair of DNA molecules; 1) when used in a nucleic acid amplification reaction containing Corn event DP-051291-2 DNA, produces a first amplicon that is diagnostic of event DP-051291-2; and 2) contacting, which when used in a nucleic acid amplification reaction containing corn genomic DNA other than DP-051291-2 DNA, produces a second amplicon that is diagnostic of corn genomic DNA other than DP-051291-2 DNA; b) performing a nucleic acid amplification reaction; and c) detecting the amplicons so produced, wherein detection of the presence of both amplicons indicates that the sample is heterozygous for corn event DP-051291-2 DNA, and detection of only the first amplicon indicates that the sample is homozygous for corn event DP-051291-2 DNA. The method includes:

[0129] Another embodiment includes a method of determining the zygosity of a corn plant comprising event DP-051291-2 in a biological sample of any previous embodiment, wherein the first DNA molecule pair comprises primer pair SEQ ID NOs: 6 and 7.

[0130] Another embodiment includes a method of determining the zygosity of a corn plant comprising event DP-051291-2 in a biological sample of any previous embodiment, wherein the first and second pair of DNA molecules comprise a detectable label.

[0131] Further embodiments include a method of determining the zygosity of a corn plant containing event DP-051291-2 in a biological sample, wherein the first and second pair of DNA molecules contain detectable labels as in any preceding embodiment, and wherein the detectable labels are fluorescent labels.

[0132] Another embodiment includes a method of determining the zygosity of a corn plant comprising event DP-051291-2 in a biological sample, wherein the first and second pair of DNA molecules comprise a detectable label as in any preceding embodiment, and the detectable label is covalently attached to one or more of the primer molecules.

[0133] One embodiment is a method for detecting the presence of a nucleic acid molecule unique to event DP-051291-2 in a sample containing corn nucleic acid, comprising: a) contacting the sample with a pair of primers that, when used in a nucleic acid amplification reaction with genomic DNA from event DP-051291-2, generate an amplicon that is diagnostic for event DP-051291-2; b) performing a nucleic acid amplification reaction, thereby producing an amplicon diagnostic for Event DP-051291-2; and c) Detecting amplicons diagnostic for Event DP-051291-2 The method includes:

[0134] Another embodiment includes a method of detecting the presence of a nucleic acid molecule unique to event DP-051291-2 in a sample containing corn nucleic acid of any previous embodiment, wherein the nucleic acid molecule diagnostic of event DP-051291-2 is an amplicon produced by nucleic acid amplification chain reaction.

[0135] Another embodiment includes a method of detecting the presence of a nucleic acid molecule unique to event DP-051291-2 in a sample containing corn nucleic acid of any preceding embodiment, further comprising contacting the sample with a probe.

[0136] Further embodiments include methods for detecting the presence of nucleic acids specific to event DP-051291-2 in a sample containing corn nucleic acids, further comprising contacting the sample with the probe of any preceding embodiment, wherein the probe comprises a detectable label.

[0137] Further embodiments include a method of detecting the presence of a nucleic acid molecule unique to event DP-051291-2 in a sample containing corn nucleic acid, further comprising contacting the sample with a probe, wherein the probe comprises the detectable label of any preceding embodiment, and wherein the detectable label is a fluorescent label.

[0138] Further embodiments include a method of detecting the presence of a nucleic acid molecule unique to event DP-051291-2 in a sample containing corn nucleic acid, further comprising contacting the sample with a probe, wherein the probe comprises the detectable label of any preceding embodiment, and wherein the detectable label is covalently associated with the probe.

[0139] One embodiment includes a plurality of polynucleotide primers comprising one or more polynucleotides that target event DP-051291-2 DNA templates in a sample to produce, as a result of a polymerase chain reaction process, an amplicon that is diagnostic of event DP-051291-2.

[0140] Another embodiment includes a plurality of polynucleotide primers according to any previous embodiment, wherein: a) the first polynucleotide primer comprises the nucleotide sequence as set forth in SEQ ID NO:6 and its complement; b) The second polynucleotide primer comprises the nucleotide sequence as set forth in SEQ ID NO: 7 and its complement.

[0141] Another embodiment includes the primer of any previous embodiment, wherein said first primer and said second primer are at least 18 nucleotides.

[0142] One embodiment is a method for detecting the presence of DNA corresponding to event DP-051291-2 in a sample, comprising: a) contacting a sample containing corn DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions to DNA from corn event DP-051291-2 and does not hybridize under said stringent hybridization conditions to non-DP-051291-2 corn plant DNA; b) subjecting the sample and probe to stringent hybridization conditions; and c) detecting hybridization of the probe to the DNA; Here, detection of hybridization indicates the presence of event DP-051291-2.

[0143] One embodiment includes a kit for detecting nucleic acids unique to event DP-051291-2, comprising at least one nucleic acid molecule of contiguous polynucleotides of sufficient length to function as a primer or probe in a nucleic acid detection method, wherein the nucleic acid, upon amplification of or hybridization with a target nucleic acid sequence in a sample, and subsequent detection of the amplicon or hybridization with the target sequence, is a diagnostic indicator of the presence of a nucleic acid sequence unique to event DP-051291-2 in the sample.

[0144] Another embodiment includes a kit for detecting nucleic acids unique to event DP-051291-2, comprising at least one nucleic acid molecule of contiguous polynucleotides of sufficient length to function as a primer or probe in a nucleic acid detection method, wherein the nucleic acid, upon amplification of or hybridization with a target nucleic acid sequence in a sample, followed by detection of the amplicon or hybridization with the target sequence, is a diagnostic indicator of the presence of a nucleic acid sequence unique to event DP-051291-2 in the sample of any previous embodiment, wherein the nucleic acid molecule comprises a nucleotide sequence from SEQ ID NOs: 6-31.

[0145] Another embodiment includes a kit for detecting nucleic acids unique to event DP-051291-2, comprising at least one nucleic acid molecule of a contiguous polynucleotide of sufficient length to function as a primer or probe in a nucleic acid detection method, wherein the nucleic acid, upon amplification of or hybridization with a target nucleic acid sequence in a sample, followed by detection of the amplicon or hybridization with the target sequence, is a diagnostic indicator of the presence of a nucleic acid sequence unique to event DP-051291-2 in the sample of any of the previous embodiments, and wherein the nucleic acid molecule is a primer selected from SEQ ID NOs: 6-31 and complements thereof.

[0146] Another embodiment includes a corn plant comprising the genotype of corn event DP-051291-2 of any previous embodiment, wherein the genotype comprises a nucleotide sequence having 1, 2, 3, 4 or 5 nucleotide changes in one or more of SEQ ID NOs:26-28, SEQ ID NOs:29-31, or SEQ ID NO:3.

[0147] Another embodiment includes a corn plant comprising the genotype of corn event DP-051291-2 of any previous embodiment, further comprising a nucleotide sequence as set forth in SEQ ID NO:3, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:3.

[0148] One embodiment includes a method of modifying a DP-051291-2 corn event, a representative sample of seeds of the corn event has been deposited with the ATCC under accession number PTA-127358, the method comprising applying genome modification techniques to a DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event.

[0149] Another embodiment includes a method of modifying the DP-051291-2 corn event, wherein a representative sample of seeds of the corn event has been deposited with the ATCC under accession number PTA-127358, comprising applying genome modification techniques to a DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event of any previous embodiment, wherein the DNA of the DP-051291-2 corn event is modified to generate a modified DNA sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.

[0150] Another embodiment includes a method of modifying the DP-051291-2 corn event, wherein a representative sample of seeds of the corn event has been deposited with ATCC under accession number PTA-127358, comprising applying genome modification techniques to the DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event of any previous embodiment, comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence having all or a portion of SEQ ID NO:26 or SEQ ID NO:29 duplicated in the modified DNA sequence.

[0151] Another embodiment includes a method of modifying the DP-051291-2 corn event deposited with ATCC under accession number PTA-127358, wherein a representative sample of seeds of the corn event comprises applying genome modification techniques to the DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event of any previous embodiment, comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence that includes a removal from SEQ ID NO:3.

[0152] Further embodiments include a method of modifying the DP-051291-2 corn event, wherein a representative sample of seeds of the corn event has been deposited with ATCC under accession number PTA-127358, comprising applying genome modification techniques to the DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event, comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence comprising deletions from SEQ ID NO:3 of any previous embodiment, wherein the deletions comprise deletions from one or more regulatory elements of SEQ ID NO:3 that do not substantially affect activity of the one or more regulatory elements.

[0153] Another embodiment includes a method of modifying a DP-051291-2 corn event, wherein a representative sample of seeds of the corn event has been deposited with ATCC under Accession No. PTA-127358, comprising applying genome engineering techniques to the DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event; comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence comprising an excision from SEQ ID NO:3 of any previous embodiment; comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence having all or a portion of SEQ ID NO:26 or SEQ ID NO:29 excised from the modified DNA sequence.

[0154] Another embodiment includes a method of modifying the DP-051291-2 corn event, wherein a representative sample of seeds of the corn event has been deposited with ATCC under accession number PTA-127358, comprising applying genome modification techniques to the DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event; comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence comprising a removal from SEQ ID NO:3 of any previous embodiment; comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence, wherein at least 30% of SEQ ID NO:3 has been removed from the modified DNA sequence.

[0155] Further embodiments include a method of modifying the DP-051291-2 corn event deposited with ATCC under accession number PTA-127358, wherein a representative sample of seeds of the corn event comprises applying genome modification techniques to the DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event, and modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence comprising a removal from SEQ ID NO:3 of any previous embodiment, wherein at least 80% of SEQ ID NO:3 is removed from the modified DNA sequence.

[0156] Another embodiment includes a method of modifying the DP-051291-2 corn event deposited with ATCC under accession number PTA-127358, wherein a representative sample of seeds of the corn event comprises applying genome modification techniques to the DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event, comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence comprising a removal from SEQ ID NO:3 of any previous embodiment, wherein all of SEQ ID NO:3 is removed from the modified DNA sequence.

[0157] One embodiment includes a method of generating guide polynucleotides for use with the DP-051291-2 Corn Event Genome Editing System, the method comprising designing one or more guide polynucleotides that recognize at least a portion of SEQ ID NO:3, and synthesizing the guide polynucleotides.

[0158] Another embodiment includes a method of modifying DNA of the DP-051291-2 event having accession number PTA-127358, comprising introducing the one or more guide polynucleotides for use with the DP-051291-2 corn event genome editing system of any previous embodiment as part of a genome modification composition into the DNA of the DP-051291-2 event, thereby modifying the DNA of the DP-051291-2 event.

[0159] One embodiment includes a DP-051291-2 corn event genome editing system comprising a CAS polypeptide, one or more guide polynucleotides, and DP-051291-2 corn event donor DNA.

[0160] One embodiment includes a method of modifying at least one expression cassette of the DP-051291-2 event deposited with the ATCC, having accession number PTA-127358, comprising modifying the at least one expression cassette using genome editing techniques, wherein the resulting corn plant derived from the DP-051291-2 event comprises the at least one modified cassette.

[0161] Another embodiment includes a method of modifying at least one expression cassette of the DP-051291-2 event deposited with the ATCC having accession number PTA-127358, comprising modifying the at least one expression cassette using genome editing techniques, wherein the resulting corn plant derived from the DP-051291-2 event comprises at least one modified cassette of any preceding embodiment, and comprises modifying expression of IPD072Aa.

[0162] One embodiment includes a method of controlling Coleopteran insects, comprising exposing the Coleopteran insects to insect-resistant corn plants of event DP-051291-2.

[0163] Another embodiment includes a method for controlling Coleopteran insects, comprising exposing Coleopteran insects to insect-resistant corn plants of event DP-051291-2, wherein the Coleopteran insect is corn rootworm.

[0164] Another embodiment includes a method of controlling Coleopteran insects, comprising exposing Coleopteran insects to insect-resistant corn plants of event DP-051291-2, wherein damage from the Coleopteran insects is controlled on corn roots from event DP-051291-2.

[0165] Another embodiment includes a method of producing a commodity plant product comprising processing grain produced from a corn event DP-051291-2 plant comprising a nucleotide sequence that is, or is complementary to, a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, a representative sample of the corn event DP-051291-2 seed having been deposited with the American Type Culture Collection (ATCC) under Accession Number PTA-127358, wherein the grain is processed into a commodity plant product selected from corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products, and wherein the commodity plant product comprises a detectable amount of the nucleotide sequence.

[0166] In some embodiments, corn plants containing the DP-051291-2 event can be treated with a seed treatment, which can be a fungicide, insecticide, or herbicide.

[0167] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0168] The following example is included to more fully describe an embodiment of the development of corn event DP-051291-2, which resulted from the construction of 84 different construct designs tested in multiple transformation vectors to create 217 different vectors, and the production of approximately 7,112 events (both early and late testing) containing IPD072 as the single insect control gene over approximately six years. Thousands of individual plants (selfs and hybrids) were analyzed, many of which were tested over the years across diverse environments using one or more criteria, including, but not limited to, molecular testing, protein expression, efficacy testing, agronomic testing, and / or field testing, for the creation and selection of corn event DP-051291-2. Efficacy was primarily determined by analysis of corn rootworm nodal injury scores (CRWNIS), including high pressure positions.

[0169] Locus discovery and characterization included 2,711 TO plants that generated 6,285 events over approximately six years, each containing IPD072 as a single insect control gene, and 4,557 later events derived from 21 different vectors. Year 1 of field testing included 71 commercial line events. Of this pool, five were advanced to year 2 field testing, and two were further advanced to year 3 field testing. After the third year of testing, corn event DP-051291-2 was selected.

[0170] Example 1. Cassette design for transgenic plants containing constructs encoding IPD072Aa The cassette design for IPD072Aa expression used in the molecular stack to generate commercial line events was selected based on efficacy and expression in genetic testing transformation experiments. A number of different regulatory elements (promoters, introns) and other elements (terminators) were evaluated in genetic testing experiments. Using a number of different regulatory elements, expression patterns were evaluated for agronomic performance and trait efficacy.

[0171] The genetic elements contained in the selected event construct, the ipd072Aa gene cassette in the T-DNA region of plasmid PHP74638, are listed in Table 1 .

[0172] [Table 1]

[0173] Example 2. Transformation of maize by Agrobacterium transformation and regeneration of transgenic plants containing ipd072Aa, mo-pat, and pmi genes Maize event DP-051291-2 was generated using three sequential transformation steps: (1) inserting a specific integration site sequence into the maize genome to create an initial SSI "landing pad"; (2) resetting the initial landing pad by replacing the first selectable marker with a second selectable marker to create the final SSI "landing pad"; and (3) using site-specific integration (SSI) to insert the intended trait gene into the final SSI landing pad. After each transformation, the maize genome was characterized using Southern-by-sequencing (SbS) to ensure the intended insertion was present with sequence integrity and that no unintended plasmid-derived sequences were present in the genome.

[0174] The DP-051291-2 corn event was generated by Agrobacterium-mediated SSI transformation with plasmid PHP74638. Agrobacterium-mediated SSI was performed essentially as described in U.S. Patent Application Publication No. 2017 / 0240911, which is incorporated herein by reference.

[0175] A total of 1,289 immature embryos were infected with PHP74638. After 105 days of selection and regeneration, a total of 120 TO plantlets were regenerated. Samples were taken from all TO plantlets for PCR analysis to confirm the presence and copy number of the inserted ipd072Aa, pmi, and mo-pat genes. In addition to this analysis, TO plantlets were analyzed by PCR for the presence of specific Agrobacterium binary vector backbone sequences and for the developmental genes zm-odp2 and zm-wus2, as disclosed in U.S. Patent Nos. 7,579,529 and 7,256,322 (incorporated herein by reference in their entireties). Plants determined to contain a single copy of the inserted genes ipd072Aa, pmi, and mo-pat, no Agrobacterium backbone sequences, and no developmental genes, were selected for further greenhouse propagation. Samples from these PCR-selected T0 quality events were collected for further analysis using Southern Bifid sequencing to confirm that the inserted gene was located at the correct target locus (also referred to herein as the "landing pad") and was free of any gene disruption. Corn event DP-051291-2 was confirmed to contain a single copy of the T-DNA (see Examples 3 and 4). These selected T0 plants were assayed for trait efficacy and protein expression. T0 plants that met all criteria were advanced and crossed with inbred lines to generate seeds for further testing. A schematic diagram of the transformation and event development process is shown in Figure 4.

[0176] Example 3. Identification of corn event DP-051291-2 Polymerase chain reaction (PCR) amplification of the unique region of the introduced genetic element can distinguish test plants from their non-transgenic counterparts and can be used to screen for the presence of the inserted T-DNA region of plasmid PHP74638.

[0177] To detect the ipd072Aa, mo-pat, and pmi genes contained within DP-051291-2 maize and the genomic junction spanning the insertion site of DP-051291-2 maize, regions spanning 72 to 113 bp were amplified using primers and probes specific to each unique sequence. Additionally, a 79-bp region of the endogenous reference gene, high mobility group A (HMG; GenBank accession number AF171874.1), was used in duplex with each assay for qualitative and quantitative evaluation of each assay and was validated to demonstrate the presence of sufficient quality and quantity of DNA in the PCR reactions (Krech et al., 1999). Data from HMG were used in scoring calculations. Data were compared to the performance of either validated positive or copy number calibrators and negative genomic controls.

[0178] The real-time PCR reaction utilized the 5' nuclease activity of a heat-activated DNA polymerase. Two primers and one probe were annealed to the target DNA, with the probe containing a 5' fluorescent reporter dye and a 3' quencher dye. During each PCR cycle, the polymerase cleaved the reporter dye from the annealed probe, releasing a fluorescent signal that increased with each subsequent cycle. The cycle at which the emission intensity of the sample amplicon rose above the detection threshold was designated C. T If no amplification occurred, the C calculated by the instrument T Not 40 C T A value was assigned.

[0179] When determining the copy number of test samples, copy number calibrators (samples known to contain a defined copy of the gene of interest, e.g., 1 or 2 copies) were used as controls for both the endogenous gene and the gene of interest. A fold difference was used to apply a copy number to each test sample. The fold difference or fold change was calculated as 2- ΔCT As above, ΔC is calculated for the test samples and copy number calibrators using the formula: T was calculated. Copy number 1 was applied to sample populations that produced fold changes between 0 and 0.7 when compared to a two-copy calibrator, with a maximum range of 0.75. Similarly, copy number 2 was applied to sample populations that produced fold changes ranging from 1.5 to 2.2 when compared to a single-copy calibrator, with a maximum range of 0.91. Copy number 3 was applied to sample populations that produced fold changes ranging from 1.3 to 1.5 when compared to a two-copy calibrator, with a maximum range of 0.35.

[0180] Genomic DNA was isolated from DP-051291-2 maize leaf tissue for approximately 100 plants from each of the T1 and T2 generations. DNA samples were extracted using an alkaline buffer consisting of sodium hydroxide, ethylenediaminetetraacetic acid disodium salt dihydrate (Na2-EDTA), and Tris-HCl. Approximately 5 ng of template DNA was used per reaction.

[0181] Each assay supporting the targeting event and transgene was multiplexed with an HMG endogenous reference assay. Reaction mixtures containing all components for both the gene of interest and the endogenous gene for the PCR reaction were prepared. Bioline SensiFast™ Probe Lo-ROX Master Mix, a base master mix containing 30% bovine serum albumin (BSA) as an additive, was used. Individual primer and probe concentrations varied from reaction to reaction, ranging from 600 nM to 900 nM for primers and 80 nM to 120 nM for probes, depending on the optimal concentrations established during assay validation. Assay controls included water or Tris-EDTA (TE) buffer (10 mM Tris pH 8.0, 1 mM EDTA), as well as no template controls (NTCs) consisting of copy number calibrators and negative controls, all of which were validated for each assay performed. The primers and probes used for each PCR analysis are listed in Tables 2 and 3. The annealing temperatures and cycle numbers used during PCR analysis are listed in Table 4.

[0182] Genomic DNA samples isolated from collected leaf samples of 200 DP-051291-2 maize plants (100 plants from each of the T1 and T2 generations), along with copy number calibrators, negative, and non-template controls (NTCs), were subjected to quantitative real-time PCR (qPCR) amplification using primer pairs and probes specific to the genes ipd072Aa, mo-pat, and pmi, as well as event-specific junctions to the maize genome, for specific identification of the PHP74638-derived DNA insertion in DP-051291-2 maize. For assay and DNA quality monitoring, maize HMG was included in the duplex with each reaction as an endogenous control. Each qPCR reaction was set up in a total volume of 6 μL containing approximately 5 ng (1.0 μL volume) of isolated genomic DNA.

[0183] The PCR target site and expected PCR product size for each primer / probe set are shown in Table 2. Primer and probe sequence information supporting each target region is shown in Table 3. PCR reagents and reaction conditions are shown in Table 4. Approximately 5 ng of maize genomic DNA was used for all PCR reactions in this study.

[0184] [Table 2]

[0185] [Table 3]

[0186] [Table 4]

[0187] [Table 5]

[0188] The results of qPCR copy number analysis showed stable integration and segregation of a single copy of the gene of interest within the T-DNA of plasmid PHP74638, demonstrating its transmission to the next generation.

[0189] PCR products ranging in size from 72 bp to 113 bp representing the DP-051291-2 maize insertion site and genes within the T-DNA from plasmid PHP74638 were amplified and observed in DP-051291-2 maize leaf samples and eight copy number calibrator genome controls, but were absent from each of the eight negative genome controls and eight NTC controls. For all samples, each assay was performed in duplex, and the DP-051291-2 event insertion site and all genes were analyzed a total of four times, with identical results observed. All data generated were analyzed using C T value, ΔC T Values ​​and copy numbers (if applicable) were calculated.

[0190] The maize endogenous reference gene HMG was used to amplify a 79 bp PCR product and observed in leaf samples from DP-051291-2 maize as well as eight copy number calibrators and eight negative genomic controls. Amplification of the endogenous gene was observed in C T This was not observed in the eight NTC controls tested without generating values.

[0191] To evaluate the sensitivity of the construct-specific PCR assay, DP-051291-2 corn DNA was diluted with control corn genomic DNA to yield test samples containing various amounts of DP-051291-2 corn (5 ng, 1 ng, 500 pg, 250 pg, 100 pg, 50 pg, 20 pg, 10 pg, and 5 pg) totaling 5 ng of corn DNA. These various amounts of DP-051291-2 corn DNA corresponded to 100%, 20%, 10%, 5%, 2%, 1%, 0.4%, 0.2%, and 0.1% of the DP-051291-2 corn DNA in total corn genomic DNA, respectively. The various amounts of DP-051291-2 corn DNA were subjected to real-time PCR amplification for the ipd072Aa, mo-pat, and pmi genes and the insertion site. Based on these analyses, the limits of detection (LODs) for DP-051291-2 maize in 5 ng of total DNA were determined to be approximately 20 pg (0.4%) for ipd072Aa, 10 pg (0.2%) for mo-pat, and 10 pg (0.2%) and 20 pg (0.4%) for pmi, the insertion site representing event DP-051291-2. The determined sensitivity of each assay described is sufficient for many screening applications. Each concentration was tested a total of five times. When amplification of the tested target was not detected in each replicate, the preceding concentration was determined to be the limit of sensitivity.

[0192] Real-time PCR analysis of event DP-051291-2 using event-specific and construct-specific primer / probe sets confirmed stable integration and segregation of a single copy of the event's plasmid PHP74638 T-DNA in the tested leaf samples, as demonstrated by quantified detection of the event insertion site DP-051291-2 and the ipd072Aa, pmi, and mo-pat genes in DP-051291-2 maize. These results were reproducible across all replicate qPCR analyses performed. A maize endogenous reference gene assay for detecting hmg-A was amplified as expected in all test samples and negative controls, and was not detected in the NTC sample. The sensitivity of each assay under the described conditions ranged from 20 pg to 10 pg DNA (0.4% to 0.2%), both of which are sufficient for many PCR-based screening applications.

[0193] Example 4. Southern by Sequencing (SbS) Analysis of DP-051291-2 Maize for Integrity and Copy Number Southern by Sequencing (SbS) analysis utilizes probe-based sequence capture, next-generation sequencing (NGS) techniques, and bioinformatics procedures to capture, sequence, and identify inserted DNA within the maize genome (Zastrow-Hayes et al., 2015). By compiling a large number of maize sequencing reads and mapping them against the intended insertion sequence (including the intended expression cassette from PHP74638, including right and left border elements, and the landing pad sequence from PHP50742), the linearized transformation plasmid map, and the endogenous genome reference, unique junctions resulting from the inserted DNA are identified in bioinformatics analysis. This information is used to determine the number and organization of insertions within the plant genome and to confirm the absence of the plasmid backbone or other unintended plasmid sequences.

[0194] Genomic DNA samples isolated from 10 individual plants of the T1 generation of DP-051291-2 maize (five transgenic plants and five null segregants) were analyzed by SbS to determine the insert copy number and organization within the plant genome and to confirm the absence of plasmid backbones or other unintended sequences. SbS was also performed on control maize DNA samples from maize plants that were not genetically modified and had the same genetic background as DP-051291-2 maize but did not contain the DP-051291-2 insert, as well as on positive control samples (control maize DNA spiked with PHP74638, PHP50742, PHP16072, PHP5096, PHP46438, PHP21139, or PHP31729 plasmid DNA) to confirm that the assay could reliably detect plasmid fragments within genomic DNA.

[0195] Biotinylated capture probes for hybridization to the plasmid sequences were designed and synthesized by Roche NimbleGen, Inc. (Madison, WI). Probe sets were designed to target all sequences within the PHP74638, PHP50742, PHP16072, PHP5096, PHP46438, PHP21139, and PHP31729 plasmids.

[0196] Next-generation sequencing libraries were constructed for DNA samples from DP-051291-2 corn plants, control corn plants, and positive control samples. SbS was performed as described by Zastrow-Hayes et al. (2015) in Plant Genome. Target sequences were enriched by hybridizing the sequencing library to capture probes through two rounds of hybridization. After NGS (Illumina, NextSeq), sequencing reads were trimmed and assessed for quality assurance. Reads were aligned to the corn genome and plasmid sequences, and reads containing both genome and plasmid sequences were identified as junction reads. Alignment of junction reads to the transformation construct indicated the relative boundaries of the inserted DNA relative to the expected insertion.

[0197] To identify junctions containing endogenous maize sequences, a control maize genomic DNA library was captured and sequenced in the same manner as the DP-051291-2 maize plants, which increased the probability that endogenous junctions captured by the probes would be detected in the control sample, thereby allowing their identification and removal in the DP-051291-2 maize samples.

[0198] SbS analysis of five transgenic plants from the segregating T1 generation of DP-051291-2 maize containing the inserted DNA yielded sequencing reads aligned to the intended insertion. Each of these plants contained two unique genomic insertion junctions, one at each end of the insertion; the two unique junctions were identical across the five plants. Five null segregants from the T1 generation of DP-051291-2 maize that were negative for the DP-051291-2 insertion each yielded sequencing reads for endogenous genetic elements derived from the maize genome. There were no junctions between the plasmid sequences detected in these plants and the maize genome, indicating that these plants did not contain insertions derived from PHP74638, PHP50742, PHP16072, PHP5096, PHP46438, PHP21139, or PHP31729.

[0199] SbS analysis was performed on the T1 generation of DP-051291-2 maize to determine the insert copy number and organization within the plant genome and to confirm the absence of plasmid backbones or other unintended sequences. Genomic DNA extracted from leaf tissue of 10 individual plants from the segregating T1 generation of DP-051291-2 maize and one non-GM control maize plant, along with a plasmid-positive control sample, was analyzed by SbS using capture probes covering the entire sequence of all plasmids utilized to generate DP51291 maize: PHP74638, PHP50742, PHP16072, PHP5096, PHP46438, PHP21139, and PHP31729. Captured sequencing reads were aligned to sequences from the intended inserts and PHP74638, PHP50742, PHP16072, PHP5096, PHP46438, PHP21139, and PHP31729. SbS detected single copies of the inserted DNA from PHP74638 and PHP50742 in five of ten positive plants from the segregating T1 generation of DP-051291-2 maize, and no insertions were detected in the five null segregating plants. Furthermore, no plasmid backbone or other unintended plasmid sequences were detected in plants from the T1 generation of DP-051291-2 maize. Junctions between the plasmid and maize genome sequences were not detected in the non-GM control maize, indicating that it did not contain any insertions. SbS performed on positive control samples (control maize DNA spiked with PHP74638, PHP50742, PHP16072, PHP5096, PHP46438, PHP21139, or PHP31729 plasmid DNA) provided sequence coverage across the entire length of each plasmid, demonstrating that this assay can reliably detect plasmid fragments within the maize genome.

[0200] SbS analysis of the T1 generation of DP-051291-2 maize demonstrated that DP-051291-2 maize contained a single copy of the inserted DNA from PHP74638 and PHP50742, in the expected configuration, and no additional insertions, plasmid backbones, or other unintended sequences were present in its genome.

[0201] Example 5. Insect Efficacy of Corn Event DP-051291-2 Hybrid corn plants containing event DP-051291-2, which expresses the insect-active IPD072Aa protein for protection against certain Coleoptera pests, including corn rootworm (CRW), were evaluated in the field. Controls consisted of corn plants of the same hybrid background that did not contain event DP-051291-2 or other events active against CRW (referred to as control corn). Data were statistically analyzed using linear mixed models.

[0202] Field trials were conducted at 13 locations located in commercial corn-growing regions of North America: Brookings, SD; Champaign, IL; Johnston, IA #1; Johnston, IA #2; Mankato, MN; Lanesboro, MN; Dunkerton, IA; Readlyn, IA; Shabonna, IL; Mineral Point, WI; Windfall, IN; York, NE; and Fairmont, NE. Efficacy data were not collected at four of the 13 locations (Johnston, IA #1, Mankato, MN, Lanesboro, MN, and Windfall, IN) due to low corn rootworm nodal injury scores (CRWNIS) of less than 0.75 on negative control roots.

[0203] Planting was performed in single-row (10-foot-long) test plots in a three-replicate, complete, randomized block design. Each replicate included DP-051291-2 corn as well as negative control corn. Prior to planting, 200 kernels from each seed lot were characterized by PCR analysis to confirm the presence of the DP-051291-2 event. All locations were planted in fields with a history of natural corn rootworm infestation. Additionally, at all 13 locations, when plants reached the V2-V4 growth stage, an 8-foot section of each plot was manually infested with non-diapausing western corn rootworm eggs. Eggs were infested at a targeted infestation rate of approximately 750 eggs / plant. Eggs were injected into the soil approximately 4 inches deep, approximately 2 inches on each side of each plant.

[0204] Root-feeding larvae were assessed for injury when plants were approximately at the R2 growth stage. Five corn plants from each plot were targeted, tagged, manually excavated, and the soil washed clean with pressurized water. Each root was visually assessed for the amount of larval feeding. Only healthy, representative plants were selected for data collection. A Corn Rootworm Nodal Injury Score (CRWNIS) was recorded for each plant using the Iowa State 0-3 Nodal Injury Scale described by Oleson et al. (2005).

[0205] The mean root damage rating results for CRW nodal roots for both DP-051291-2 corn and control corn are provided in Table 5. These results indicate that the insect-active IPD072Aa protein expressed in DP-051291-2 corn provides protection from feeding against CRW compared to the negative control corn.

[0206] [Table 6]

[0207] Example 6. Agronomic and Yield Field Evaluation of Corn Event DP-051291-2 The field trial involving DP-051291-2 was to generate yield data and evaluate other agronomic traits. All inbred and hybrid material tested for the event was generated from a single TO plant.

[0208] Hybrid Exam Over the two-year trial, seven hybrid trials were planted across a total of 70 growing locations, with two replicate entry lists at each location. Grain was collected from 52 of the 70 locations. The experiment was conducted as a randomized split-plot treatment design. The main plot was the hybrid background, and the subplots were the transgene status (event DP-051291-2 or WT). The subplots were randomized within the main plot, and the main plot was randomized within each replicate. Two replicates were conducted at each field location each year. Plots were four rows spaced 76 cm apart, ranging in length from 4.4 to 5.3 m, with a 0.5 m alley between each plot. At each planting location, various observations and data were collected throughout the growing season. The following agronomic characteristics were analyzed for comparison with the wild-type entry (WT). Data generated for the hybrid agronomic trial included the following agronomic traits (Table 6): 1.) EARHT: The measurement from the ground to the point of attachment of the most developed flower spike on the plant. Flower spike height is measured in inches. 2.) Growing Degree Units Emitted (GDUSHD): The measurement is recorded as the total cumulative growing degree units when 50% of the plants in the plot have tassels shedding pollen. For this dataset, one day is approximately 1.5 growing degree days. 3.) Growing Degree Units for Silk (GDUSLK): The measurement is recorded as the total cumulative growing degree units when 50% of the plants in the plot have exposed silk. For this data set, the daily equivalent is approximately 1.5 growing degree days. 4.) Culm Length (PLTHT): Drone measurement from ground level to base of flag leaf. Culm length is measured in inches. 5.) Moisture (MST): Measurement of percent grain moisture at harvest. 6.) Yield: Recorded weight of grain harvested from each plot. Reported bushel / acre yield calculations were made by adjusting for moisture measurements for each plot.

[0209] Inbred Test The inbred trial was planted across 13 locations as an entry list with two replicates per location. Grain was collected from four locations for analysis. The experiment was conducted as a randomized split-plot treatment design. The main plot was the inbred background and the subplots were the transgene status (event DP-051291-2 or WT). Crop data and observations were collected for the inbred trial and analyzed for comparison to the wild-type entry (WT). Data generated for the inbred trial included the following agronomic traits (Table 7): 1.) EARHT: The measurement from the ground to the point of attachment of the most developed flower spike on the plant. Flower spike height is measured in inches. 2.) Growing Degree Units Emitted (GDUSHD): The measurement is recorded as the total cumulative growing degree units when 50% of the plants in the plot have tassels shedding pollen. For this dataset, one day is approximately 1.5 growing degree days. 3.) Growing Degree Units for Silk (GDUSLK): The measurement is recorded as the total cumulative growing degree units when 50% of the plants in the plot have exposed silk. For this data set, the daily equivalent is approximately 1.5 growing degree days. 4.) Culm Length (PLTHT): Measurement from ground to base of flag leaf. Culm length is measured in inches. 5.) Photometric kernels per spike (PHTKPE): The number of kernels per spike determined by spike photometry.

[0210] Test results To evaluate the hybrid data, a multi-location analysis was performed using a mixed model framework. In the multi-location analysis, the event was considered a fixed effect. The factors of location, context, examiner, examiner by event, location by context, location by examiner, location by event, location by examiner by event, and personnel within location were considered random effects. Spatial effects, including range and plot within the location, were considered random effects to remove irrelevant spatial noise. Heterogenous residuals were assumed for each location, with autoregressive correlation as AR1 × AR1. T-tests were performed to compare events with WT. Differences were considered statistically significant if their P values ​​were less than 0.05. Yield analysis was performed using ASREML (VSN International Ltd; Best Linear Unbiased Prediction; Cullis, B. Ret al (1998) Biometrics 54:1-18, Gilmour, AR et al (2009); ASReml User Guide 3.0, Gilmour, AR, et al (1995) Biometrics 51:1440-50).

[0211] To evaluate the inbred data, multi-location analyses were conducted using a mixed model framework. In multi-location analyses, events were considered fixed effects. The factors of location, context, events, location by events, and personnel within locations were considered random effects. Spatial effects, including range and plot within the location, were considered random effects to remove irrelevant spatial noise. Autoregressive correlation was assumed for each location, with AR1 × AR1, assuming heterogeneous residuals. T-tests were performed to compare events with the wild-type (WT). Differences were considered statistically significant if their P values ​​were less than 0.05. Yield analyses were performed using ASREML (VSN International Ltd; Best Linear Unbiased Prediction; Cullis, B. Ret al (1998) Biometrics 54:1-18; Gilmour, AR et al (2009); ASReml User Guide 3.0, Gilmour, AR, et al (1995) Biometrics 51:1440-50).

[0212] [Table 7]

[0213] [Table 8]

[0214] Example 7. Protein Expression and Concentration Protein extraction IPD072Aa, PAT, and PMI protein concentrations were determined using a quantitative enzyme-linked immunosorbent assay (ELISA) that had been internally validated to demonstrate method suitability. For analysis of IPD072Aa, PAT, and PMI protein concentrations, non-herbicide-treated processed tissue subsamples were weighed to the following target weights: 5 mg pollen, 10 mg leaves, 20 mg grain and roots, and 30 mg foliage. Pollen, leaf, grain, and foliage samples analyzed for IPD072Aa protein were extracted with 0.60 ml of chilled 25% StabilZyme Select in phosphate-buffered saline containing polysorbate 20 (PBST). Root samples analyzed for IPD072Aa protein were extracted with chilled H5 buffer, consisting of 90 mM HEPES, 140 mM sodium chloride, 1.0% polyethylene glycol, 1.0% PVP40, 1.0% bovine serum albumin, 0.007% thimerosal, and 0.3% polysorbate 20. Samples analyzed for PAT and PMI proteins were extracted with 0.60 ml of chilled PBST. All extracted samples were centrifuged, and the supernatant was then removed and prepared for analysis.

[0215] Determination of IPD072Aa protein concentration Prior to analysis, samples were diluted as applicable with 25% StabilZyme Select in PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated on plates pre-coated with IPD072Aa-specific antibodies. After incubation, unbound material was washed from the plate, and the bound IPD072Aa protein was incubated with a different IPD072Aa-specific antibody conjugated to the enzyme horseradish peroxidase (HRP). Unbound material was washed from the plate. Detection of bound IPD072Aa-antibody complexes was achieved by adding a substrate that generates a colored product in the presence of HRP. The reaction was stopped with an acidic solution, and the optical density (OD) of each well was determined using a plate reader.

[0216] Determination of PAT protein concentration Before analysis, samples were diluted appropriately with PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were co-incubated with PAT-specific antibodies conjugated to the enzyme HRP in a plate pre-coated with another PAT-specific antibody. After incubation, unbound material was washed off the plate. Detection of bound PAT-antibody complexes was achieved by adding a substrate that generates a colored product in the presence of HRP. The reaction was stopped with an acidic solution, and the OD of each well was determined using a plate reader.

[0217] Determination of PMI protein concentration Before analysis, samples were appropriately diluted with PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated on a plate precoated with a PMI-specific antibody. After incubation, unbound material was washed off the plate, and the bound PMI protein was incubated with another PMI-specific antibody conjugated to the enzyme HRP. Unbound material was washed off the plate. Detection of the bound PMI-antibody complex was achieved by adding a substrate that generates a colored product in the presence of HRP. The reaction was stopped with an acidic solution, and the OD of each well was determined using a plate reader.

[0218] Calculation for determining protein concentration The calculations necessary to convert the OD values ​​obtained for each set of sample wells into protein concentration values ​​were performed using SoftMax Pro GxP (Molecular Devices) microplate data software.

[0219] A standard curve was included with each ELISA plate. The equation for the standard curve was determined by software, which related the OD values ​​obtained for each set of standard curve wells to their respective standard curve concentrations (ng / ml) using a quadratic fit. Sample concentration values ​​were adjusted for the dilution factor, expressed as 1:N, by multiplying the interpolated concentration by N.

[0220] Adjusted concentration = interpolated sample concentration x dilution factor

[0221] The adjusted sample concentration values ​​determined by SoftMax Pro GxP Software were converted from ng / ml to ng / mg sample weight as follows:

number

[0222] The reportable assay lower limit of quantitation (LLOQ) (ng / ml) was calculated as follows:

[0223] Reportable assay LLOQ (ng / ml) = (lowest standard concentration - 10%) x minimum dilution

[0224] The LLOQ (ng / mg sample weight) was calculated as follows:

number

[0225] result Protein concentration results (mean, standard deviation, and range) of IPD072Aa, PAT, and PMI proteins were determined for roots (V6, V9, R1, and R4 growth stages), leaves (V9, R1, and R4 growth stages), pollen (R1 growth stage), stems (R4 growth stage), and grain (R6 growth stage) of DP-051291-2 maize, as shown in Table 8.

[0226] [Table 9]

[0227] The above description of various exemplary embodiments of the present disclosure is not intended to be exhaustive or to limit the scope to the precise forms disclosed. While specific embodiments and examples have been described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the art will recognize. The teachings provided herein can be applied to other purposes other than the examples described above. Many modifications and variations are possible in light of the above teachings and, accordingly, are within the scope of the appended claims.

[0228] These and other changes can be made in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the scope to the specific embodiments disclosed in the specification and the claims.

[0229] The entire disclosure of each document cited in the Background, Detailed Description, and Examples (including patents, patent applications, journal articles, abstracts, manuals, books, or other disclosures) is incorporated herein by reference in its entirety.

[0230] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, concentrations, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees Celsius; and pressure is at or near atmospheric.

Claims

1. 1. A corn plant comprising the genotype of corn event DP-051291-2, wherein the genotype comprises a nucleotide sequence as set forth in SEQ ID NO:26 and SEQ ID NO:29, or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:26 and SEQ ID NO:

29.

2. 2. The corn plant of claim 1, wherein the genotype comprises the nucleotide sequences set forth in SEQ ID NO:27 and SEQ ID NO:30, or nucleotide sequences having at least 90% sequence identity to SEQ ID NO:27 and SEQ ID NO:

30.

3. 2. The corn plant of claim 1, wherein the genotype comprises the nucleotide sequences set forth in SEQ ID NO:28 and SEQ ID NO:31, or nucleotide sequences having at least 90% sequence identity to SEQ ID NO:28 and SEQ ID NO:

31.

4. A DNA construct comprising operably linked expression cassettes, one of said expression cassettes comprising: 1) BSV(AY) promoter; 2) zm-HPLV9 intron; 3) ipd072Aa; and 4) A DNA construct containing the at-T9 terminator.

5. A plant comprising the DNA construct of claim 4.

6. The plant of claim 5 , wherein the plant is a corn plant.

7. A plant comprising the sequence shown in SEQ ID NO:21 or a sequence having at least 95% sequence identity to SEQ ID NO:

21.

8. Corn event DP-051291-2, a representative sample of seed of said corn event having been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

9. 9. A plant part of the corn event of claim 8.

10. A seed comprising corn event DP-051291-2, the seed comprising a DNA molecule selected from SEQ ID NO:26 and SEQ ID NO:29, wherein a representative sample of the corn event DP-051291-2 has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

11. A corn plant or part thereof grown from the seed of claim 10.

12. A transgenic seed produced from the corn plant of claim 8.

13. A transgenic corn plant or part thereof grown from the seed of claim 12.

14. An isolated nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 21 and 26-31, and full-length complements thereof.

15. An amplicon comprising a nucleic acid sequence selected from SEQ ID NOs: 21-25 and the full-length complement thereof.

16. 1. A biological sample derived from a corn event DP-051291-2 plant, tissue, or seed, comprising a nucleotide sequence that is, or is complementary to, a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, wherein the nucleotide sequence is detectable in the sample using a nucleic acid amplification method or a nucleic acid hybridization method, and a representative sample of the corn event DP-051291-2 seed has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

17. 17. The biological sample of claim 16, comprising a plant, plant tissue or seed of transgenic corn event DP-051291-2.

18. 18. The biological sample of claim 17, wherein the biological sample is a DNA sample extracted from the transgenic corn plant event DP-051291-2, and the DNA sample comprises one or more of the nucleotide sequences selected from SEQ ID NOs:21-31, and complements thereof.

19. 17. The biological sample of claim 16, selected from corn flour, corn meal, corn syrup, corn oil, corn starch and cereals manufactured to contain, in whole or in part, corn by-products.

20. 1. An extract derived from a corn event DP-051291-2 plant, tissue, or seed, and comprising a nucleotide sequence that is, or is complementary to, a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, wherein a representative sample of the corn event DP-051291-2 seed has been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358.

21. 21. The extract of claim 20, wherein the nucleotide sequence is detectable in the extract using nucleic acid amplification or nucleic acid hybridization techniques.

22. 22. The extract of claim 21, wherein the extract comprises a plant, plant tissue, or seed of transgenic corn plant event DP-051291-2.

23. 23. The extract of claim 22, wherein the composition is selected from corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured to contain, in whole or in part, corn by-products, said composition comprising a detectable amount of said nucleotide sequence.

24. 1. A method for producing hybrid corn seed, comprising: a. sexually crossing a first inbred corn line comprising a nucleotide sequence selected from SEQ ID NOs:21-31 with a second inbred line having a different genotype; b. Growing offspring from said mating; and c. harvesting the hybrid seed produced thereby.

25. 25. The method of claim 24, wherein the first inbred corn line is a female parent or a male parent.

26. 1. A method for producing a corn plant that is resistant to Coleoptera pests, comprising the steps of: a. sexually crossing a first parent corn plant with a second parent corn plant, thereby producing a plurality of first generation progeny plants, wherein the first or second parent corn plant comprises event DP-051291-2; b. selfing the first generation progeny plants to produce a plurality of second generation progeny plants; and c) selecting from said second generation progeny plants which comprise said event DP-051291-2 and are resistant to Coleoptera pests.

27. 1. A method for producing hybrid corn seed, comprising: a. sexually crossing a first inbred corn line containing the DNA construct of claim 4 with a second inbred line that does not contain the DNA construct of claim 4; and b. harvesting the hybrid seed produced thereby.

28. 27. The method of claim 26, further comprising backcrossing a second generation progeny plant comprising corn event DP-051291-2 to the parent plant lacking the corn event DP-051291-2 DNA, thereby producing a backcross progeny plant that is resistant to Coleoptera pests.

29. 1. A method for producing a corn plant that is resistant to corn rootworm, comprising: a. crossing a first parent corn plant with a second parent corn plant, thereby producing a plurality of first generation progeny plants, wherein the first or second parent corn plant comprises event DP-051291-2; b. Selecting a first generation progeny plant comprising said event DP-051291-2; c. backcrossing the first generation progeny plants of step (b) with a parent plant lacking the corn event DP-051291-2 DNA, thereby producing a plurality of backcross progeny plants; and d. selecting from the backcross progeny plants a plant comprising the event DP-051291-2; wherein the selected backcross progeny plant of step (d) comprises SEQ ID NO: 21, 26 or 29.

30. 30. The method of claim 29, wherein the plant of the first parent corn line is the female parent or the male parent.

31. 30. A hybrid seed produced by the method of claim 29.

32. 1. A method for determining the zygosity of a corn plant containing event DP-051291-2 in a biological sample, comprising: a. contacting the sample with a first pair of DNA molecules and a second, different pair of DNA molecules; i. when used in a nucleic acid amplification reaction containing Cohn event DP-051291-2 DNA, produces a first amplicon that is diagnostic of event DP-051291-2; and ii. contacting, which when used in a nucleic acid amplification reaction containing corn genomic DNA other than DP-051291-2 DNA, produces a second amplicon that is diagnostic of corn genomic DNA other than DP-051291-2 DNA; b. performing a nucleic acid amplification reaction; and c. detecting the amplicons so produced, wherein detection of the presence of both amplicons indicates the sample is heterozygous for corn event DP-051291-2 DNA, and detection of only the first amplicon indicates the sample is homozygous for corn event DP-051291-2 DNA.

33. 33. The method of claim 32, wherein the first pair of DNA molecules comprises the primer pair of SEQ ID NOs: 6 and 7.

34. 33. The method of claim 32, wherein the first and second DNA molecule pairs comprise detectable labels.

35. 35. The method of claim 34, wherein the detectable label is a fluorescent label.

36. 35. The method of claim 34, wherein the detectable label is covalently attached to one or more of the primer molecules.

37. 1. A method for detecting the presence of a nucleic acid molecule unique to event DP-051291-2 in a sample containing corn nucleic acids, comprising: a. contacting the sample with a pair of primers that, when used in a nucleic acid amplification reaction with genomic DNA from event DP-051291-2, produces an amplicon that is diagnostic for event DP-051291-2; b. performing a nucleic acid amplification reaction, thereby producing said amplicon diagnostic of event DP-051291-2; and c. Detecting said amplicon diagnostic of event DP-051291-2. A method comprising:

38. 38. The method of claim 37, wherein the nucleic acid molecule diagnosing a DP-051291-2 event is an amplicon produced by the nucleic acid amplification chain reaction.

39. 38. The method of claim 37, further comprising contacting the sample with a probe.

40. 40. The method of claim 39, wherein the probe comprises a detectable label.

41. 41. The method of claim 40, wherein the detectable label is covalently attached to the probe.

42. A plurality of polynucleotide primers comprising one or more polynucleotides that target the event DP-051291-2 DNA template in the sample to produce, as a result of a polymerase chain reaction process, an amplicon that is diagnostic of event DP-051291-2.

43. 43. The plurality of polynucleotide primers of claim 42, a. the first polynucleotide primer comprises the nucleotide sequence as set forth in SEQ ID NO:6 and its complement; b. A polynucleotide primer, wherein the second polynucleotide primer comprises the nucleotide sequence as set forth in SEQ ID NO:7 and its complement.

44. 44. The primer of claim 43, wherein the first primer and the second primer are at least 18 nucleotides.

45. 1. A method of detecting the presence of DNA corresponding to event DP-051291-2 in a sample, comprising: a. contacting the sample containing corn DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions to DNA from corn event DP-051291-2 and does not hybridize under said stringent hybridization conditions to non-DP-051291-2 corn plant DNA; b. subjecting the sample and probe to stringent hybridization conditions; and c. detecting hybridization of the probe to the DNA; wherein detecting hybridization indicates said presence of event DP-051291-2.

46. 1. A kit for detecting nucleic acids unique to event DP-051291-2, comprising at least one nucleic acid molecule of a contiguous polynucleotide of sufficient length to function as a primer or probe in a nucleic acid detection method, wherein the nucleic acid, upon amplification of or hybridization with a target nucleic acid sequence in a sample, and subsequent detection of the amplicon or hybridization with the target sequence, is a diagnostic indicator of the presence of a nucleic acid sequence unique to event DP-051291-2 in the sample.

47. 47. The kit of claim 46, wherein the nucleic acid molecule comprises a nucleotide sequence from SEQ ID NOs: 6-31.

48. 47. The kit of claim 46, wherein the nucleic acid molecule is a primer selected from SEQ ID NOs: 6-31 and complements thereof.

49. 4. The corn plant of claim 3, wherein the genotype comprises a nucleotide sequence having 1, 2, 3, 4, or 5 nucleotide changes in one of SEQ ID NO:28 or SEQ ID NO:

31.

50. 2. The corn plant of claim 1, further comprising the nucleotide sequence set forth in SEQ ID NO:3, or a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:

3.

51. 1. A method of modifying a DP-051291-2 corn event, a representative sample of seeds of the corn event having been deposited with the ATCC under accession number PTA-127358, the method comprising applying genome modification techniques to a DNA sequence of the DP-051291-2 corn event to modify the DNA of the corn event.

52. 52. The method of claim 51, comprising modifying the DNA of the DP-051291-2 corn event to generate a modified DNA sequence having at least 90% sequence identity to SEQ ID NO:

3.

53. 52. The method of claim 51, comprising modifying the DNA of the DP-051291-2 cone event to generate a modified DNA sequence having all or a portion of SEQ ID NO:26 or SEQ ID NO:29 duplicated in the modified DNA sequence.

54. 52. The method of claim 51, comprising modifying the DNA of the DP-051291-2 cone event to generate a modified DNA sequence that includes a deletion from SEQ ID NO:

3.

55. 55. The method of claim 54, wherein said excision comprises an excision from said one or more regulatory elements of SEQ ID NO: 3 that does not substantially affect the activity of said one or more regulatory elements.

56. 55. The method of claim 54, comprising modifying the DNA of the DP-051291-2 cone event to generate a modified DNA sequence, wherein all or a portion of SEQ ID NO:26 or SEQ ID NO:29 is removed from the modified DNA sequence.

57. 55. The method of claim 54, comprising modifying the DNA of the DP-051291-2 cone event to generate a modified DNA sequence, wherein at least 30% of SEQ ID NO:3 is removed from the modified DNA sequence.

58. 58. The method of claim 57, wherein at least 80% of SEQ ID NO: 3 is excised from the modified DNA sequence.

59. 58. The method of claim 57, wherein all of SEQ ID NO: 3 is excised from the modified DNA sequence.

60. DP-051291-2. A method for generating guide polynucleotides for use with the Corn Event Genome Editing System, comprising: designing one or more guide polynucleotides that recognize at least a portion of SEQ ID NO:3; and synthesizing the guide polynucleotides.

61. 61. A method of modifying the DNA of the DP-051291-2 event having accession number PTA-127358, comprising introducing one or more guide polynucleotides of claim 60 into the DNA of the DP-051291-2 event as part of a genome modification composition to modify the DNA of the DP-051291-2 event.

62. A DP-051291-2 corn event genome editing system comprising a CAS polypeptide, one or more guide polynucleotides, and DP-051291-2 corn event donor DNA.

63. 1. A method of modifying at least one expression cassette of the DP-051291-2 event deposited with the American Type Culture Collection (ATCC) having accession number PTA-127358, comprising modifying at least one expression cassette using genome editing techniques, wherein the resulting corn plant derived from the DP-051291-2 event comprises at least one modified cassette.

64. 64. The method of claim 63, wherein the method comprises altering expression of IPD072Aa.

65. 1. A method of making a commodity plant product, comprising processing kernels produced from a corn event DP-051291-2 plant comprising a nucleotide sequence that is, or is complementary to, a sequence selected from SEQ ID NO:26 and SEQ ID NO:29, a representative sample of the corn event DP-051291-2 seed having been deposited with the American Type Culture Collection (ATCC) under accession number PTA-127358, wherein the kernels are processed into a commodity plant product selected from corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals made to contain, in whole or in part, corn by-products, and wherein the composition / commodity plant product comprises a detectable amount of the nucleotide sequence.

66. 1. A method for controlling Coleopteran insects, comprising exposing said Coleopteran insects to insect-resistant corn plants of event DP-051291-2.

67. 67. The method of claim 66, wherein the Coleopteran insect is a corn rootworm.

68. 67. The method of claim 66, wherein damage from Coleopteran insects is controlled on corn roots from event DP-051291-2.

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