BR112012003884B1 - METHODS OF DETECTING DNA FROM A CORN EVENT IN A SAMPLE COMPRISING CORN DNA, DNA DETECTION KITS, AND POLYNUCLEOTIDE - Google Patents

Abstract

AAD-1 EVENT DAS-40278-9, RELATED TRANSGENIC CORN LINES AND EVENT-SPECIFIC IDENTIFICATION THEREOF. The present invention relates, in part, to the production of a herbicide-tolerant plant and plants. The present invention includes a novel aad-1 transformation event in corn plants comprising a polynucleotide sequence, as described herein, inserted into a specific location within the genome of a corn cell. In some embodiments, said event/polynucleotide sequence can be "stacked" with other traits including, for example, other herbicide tolerance gene(s) and/or insect inhibitory proteins. Additionally, the present invention provides assays for detecting the presence of the present event in a sample (of corn grain, for example). The assays can be based on the DNA sequence of the recombinant construct inserted into the corn genome and the genomic sequences flanking the insertion site. Kits and conditions useful in conducting the tests are also provided.

Description

BACKGROUND OF THE INVENTION

The aad-1 gene (originally from Sphingobium herbicidovorans) encodes the aryloxyalkanoate dioxygenase (AAD-1) protein. The trait confers tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate herbicides (commonly referred to as "fop" herbicides, such as quizalofop) and can be used as a selective marker during plant transformation and in greenhouse production. The aad-1 gene itself, with respect to herbicide tolerance in plants, was first disclosed in WO 2005/107437 (see also US 2009-0093366).

The expression of heterologous or foreign genes in plants is influenced by where the foreign gene is inserted on the chromosome. This could be due to the chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulatory elements (e.g., enhancers) near the integration site (Weising et al., Ann. Rev. Genet 22:421-477, 1988), for example. The same gene in the same type of transgenic plant (or other organism) may exhibit a wide variation in expression level among different events. There may also be differences in spatial or temporal patterns of expression. For example, differences in the relative expression of a transgene in various plant tissues may not correspond to the patterns expected from the transcriptional regulatory elements present in the introduced gene construct. Thus, large numbers of events are often created and screened in order to identify an event that expresses an introduced gene of interest at a level satisfactory for a given purpose. For commercial purposes, it is common to produce hundreds to thousands of different events and screen from those events for a single event that has the desired levels and patterns of transgene expression. An event that has desirable levels and/or patterns of transgene expression is useful for introgression of the transgene into other genetic backgrounds through sexual crossing using conventional breeding methods. The progeny of such crosses retain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a range of varieties that are well adapted to local growing conditions.

U.S. Patent Applications 20020120964 A1 and 20040009504 A1 relate to cotton event PV-GHGT07(1445) and compositions and methods for detecting the same. WO 02/100163 relates to cotton event MONI5985 and compositions and methods for detecting the same. WO 2004/011601 relates to corn event MON863, plants and compositions and methods for detecting the same. WO 2004/072235 relates to cotton event MON88913 and compositions and methods for detecting the same.

WO 2006/098952 refers to maize event 15 3272. WO 2007/142840 refers to maize event MIR162.

U.S. Patent No. 7,179,965 relates to cotton having a cry1F event and a crylAc event.

AAD-1 corn having the specific event disclosed here has not been previously disclosed.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to corn event AAD-1 designated DAS-40278-9 having seed deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-10244 and to progeny derived therefrom. Other aspects of the invention comprise the progeny plants, seeds and grain or regenerable parts of the plants and seeds and progeny of corn event DAS-40278-9, as well as food or feed products made from any of the same. The invention also includes plant parts of corn event DAS-40278-9 which include, but are not limited to, pollen, ovule, flowers, buds, roots and leaves and plant cell nuclei, pollen cells and egg cells. The invention further relates to corn plants having tolerance to auxinic and/or aryloxyalkanoate phenoxy herbicides, novel genetic compositions of corn event DAS-40278-9 and agronomic performance aspects of corn plants comprising corn event DAS-40278-9.

The present invention relates, in part, to the production of herbicide-tolerant plants and plants. The present invention includes a novel aad-1 transformation event in corn plants comprising a polynucleotide sequence as described herein inserted into a specific location within the genome of a corn cell.

In some embodiments, said event/polynucleotide sequence may be "stacked" with other traits including, for example, other herbicide tolerance gene(s) and/or insect inhibitory proteins. However, the present invention includes plants having the single event as described herein.

Additional traits can be stacked into the plant genome via plant breeding, retransformation of the transgenic plant containing the DAS-40278-9 corn event, or addition of new traits through targeted integration via homologous recombination.

Other embodiments include excising polynucleotide sequences which comprise the corn event DAS-40278-9 including, for example, the pat gene expression cassette. Upon excision of a polynucleotide sequence, the modified event can be retargeted to a specific chromosomal location, where additional polynucleotide sequences are stacked with the corn event DAS-40278-9.

In one embodiment, the present invention encompasses a corn chromosomal target locus located on chromosome 2 approximately 20 cM between the SSR markers UMC1265 (see SEQ ID NO: 30 and SEQ ID NO: 31) and MMC0111 (see SEQ ID NO: 32 and SEQ ID NO: 33) approximately 20 cM on the 2008 DAS corn pedigree map, wherein the target locus comprises a heterologous nucleic acid. In another embodiment, the present invention encompasses a corn chromosomal target locus comprising a location defined in or by SEQ ID NO: 29 and residues thereof as described herein, as will be recognized by those of skill in the art.

In one embodiment, the present invention encompasses a method of producing a transgenic corn plant comprising inserting a heterologous nucleic acid at a position on chromosome 2 approximately 20 cM between the SSR markers UMC1265 (see SEQ ID NO: 30 and SEQ ID NO: 31) and MMC0111 (see SEQ ID NO: 32 and SEQ ID NO: 33) approximately 20 cM on the 2008 DAS corn pedigree map. In yet another embodiment, the inserted heterologous nucleic acid is flanked 5' by all or part of the 5' flanking sequence as defined herein with reference to SEQ ID NO: 29 and flanked 3' by all or part of the 5' flanking sequence as defined herein with reference to SEQ ID NO: 29.

Additionally, the present invention provides assays for detecting the presence of the event in question in a sample (of corn grain, for example). The assays can be based on the DNA sequence of the recombinant construct inserted into the corn genome and on the genomic sequences flanking the insertion site. Kits and conditions useful in conducting the assays are also provided. Thus, the present invention relates, in part, to the cloning and analysis of the DNA sequences of an integral AAD-1 insert and the border regions thereof (in transgenic corn lines). These sequences are unique. Based on these insert and border sequences, event-specific primers were generated. PCR analysis demonstrated that these events can be identified by analyzing the PCR amplicons generated with these sets of event-specific primers. Thus, these and other related procedures can be used to uniquely identify corn lines comprising the event of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a plasmid map of pDAS1740. Figure 2 shows insert components for DAS-40278-9 (pDAS1740). Figure 3 shows a restriction map and insert components for DAS-40278-9 (pDAS1740). Figure 4 shows amplicons, primers, and a cloning strategy for the DNA insert and borders for DAS-40278-9. Figure 5 illustrates primer locations relative to the insert and borders for DAS-40278-9. Figure 6 illustrates the junction and insertion regions for DAS-40278-9. Figure 7 is a production diagram mentioned in Example 7.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NOs: 1-28 are primers as described herein. SEQ ID NO: 29 provides flanking and insert sequences for the DAS-40278-9 event in question. SEQ ID NOs: 30-33 are primers for flanking markers as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in part, to the production of herbicide-tolerant plants and plants. The invention includes novel corn (maize) plant transformation events comprising subject aad-1 polynucleotide sequences, as described herein, inserted into a specific location within the genome of a corn cell. In some embodiments, said polynucleotide sequence can be "stacked" with other traits (such as other herbicide tolerance gene(s) and/or gene(s) encoding insect inhibitory proteins, for example). In some embodiments, said polynucleotide sequences can be excised and subsequently re-targeted with additional polynucleotide sequences. However, the present invention includes plants having a single event as described herein.

Additionally, the present invention provides assays for detecting the presence of the present event in a sample. Aspects of the present invention include methods of designing and/or producing any diagnostic nucleic acid molecules exemplified or suggested herein, particularly those based, in whole or in part, on the flanking sequences in question.

More specifically, the present invention relates, in part, to the transgenic corn event DAS-40278-9 (also known as pDAS1740-278), plant lines comprising this event, and the cloning and analysis of the DNA sequences of this insert and/or the border regions thereof. The plant lines of the present invention can be detected using sequences disclosed and suggested herein.

In some embodiments, the present invention relates to herbicide-tolerant corn lines and the identification thereof. The present invention relates, in part, to detecting the presence of the present event in order to determine whether the progeny of a sexual cross contain the event of interest. In addition, a method for detecting the event is included and is useful, for example, for complying with regulations requiring pre-market approval and label identification of foods derived from recombinant crop plants, for example. The presence of the present event can be detected by any well-known nucleic acid detection method, such as polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. An event-specific PCR assay is discussed, for example, by Windels et al. (Med. Fac. Landbouww, Univ. Gent 64/5b: 459462, 1999). This relates to the identification of the glyphosate-tolerant soybean event 40-3-2 by PCR using a primer set spanning the junction between the insert and flanking DNA. Specifically, one primer included a sequence from the insert and a second primer included a sequence from the flanking DNA.

Maize was modified by insertion of the aad-1 gene from S. herbicidovorans, which encodes the aryloxyalkanoate dioxygenase (AAD-1) protein. The trait confers tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate herbicides (commonly referred to as "fop" herbicides, such as quizalofop) and can be used as a selectable marker during plant transformation and in greenhouse production. Transformation of maize with a DNA fragment from plasmid pDAS1740 was performed "forward" by crossing to produce event DAS-40278-9. Genomic DNA samples extracted from twenty individual maize plants derived from five generations and four plants per generation of the DAS-40278-9 event were selected for molecular characterization of the AAD-1 maize event, DAS-40278-9. AAD-1 protein expression was tested using an AAD-1-specific rapid test strip kit. Only plants that tested positive for AAD-1 protein expression were selected for subsequent molecular characterization. Southern hybridization confirmed that the aad-1 gene is present in maize plants that tested positive for AAD-1 protein expression and the aad-1 gene was inserted as a single intact copy in these plants when hybridized with an aad-1 gene probe.

Molecular characterization of the inserted DNA in the AAD-1 corn event DAS-40278-9 is also described here. The event was produced via Whiskers transformation with the Fsp I fragment of plasmid pDAS1740. Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and determine the insert/copy number of the aad-1 gene in event DAS-40278-9. Data were generated to demonstrate the integration and integrity of the inserted aad-1 transgene into the corn genome. Characterization of the integration of noncoding regions (designed to regulate coding regions), such as promoters and terminators, of the RB7 Mar v3 and RB7 Mar v4 matrix attachment regions, as well as the stability of the transgenic insert across generations, were evaluated. The stability of the inserted DNA was demonstrated across five distinct generations of plants. Furthermore, the absence of a core sequence of the transformation plasmid, including the ampicillin resistance gene (Apr) region, was demonstrated by probes spanning almost the entire core region flanking the restriction sites (Fsp I) of plasmid pDAS1740. A detailed physical map of the insert was made based on these Southern blot analyses of event DAS-40278-9. AAD-1 protein levels were determined in corn tissues. In addition, compositional analysis was performed on corn forage and grain to investigate the equivalence between the isogenic untransformed corn line and the transgenic corn line DAS-40278-9 (unsprayed, sprayed with 2,4-D, sprayed with quizalofop, and sprayed with 2,4-D and quizalofop). The agronomic characteristics of the isogenic untransformed corn line were also compared with those of DAS-40278-9 corn.

Field expression, nutrient composition, and agronomic experiments of a nontransgenic control and a hybrid corn line containing Aryloxyalkanoate Dioxygenase-1 (AAD-1) were conducted in the same year at six sites in Iowa, Illinois (2 sites), Indiana, Nebraska, and Ontario, Canada. Expression levels are summarized here for AAD-1 protein in leaf, pollen, root, forage, whole plant, and grain, the results of agronomic determinations, and compositional analysis of forage and grain samples from control and DAS-40278-9 AAD-1 corn.

Extractable, soluble AAD-1 protein was measured using a quantitative enzyme-linked immunosorbent assay (ELISA) method in corn leaf, pollen, root, forage, whole plant, and grain. Good mean expression values were observed in root and pollen tissue, as discussed in greater detail here. Expression values were similar for all sprayed treatments, as well as for pots sprayed and unsprayed with the herbicides 2,4-D and quizalofop. Compositional analyses, including proximate composition, minerals, amino acids, fatty acids, vitamins, antinutrients, and secondary metabolites, were conducted to investigate the equivalence of DAS-40278-9 AAD-1 corn (with or without herbicide treatments) to the control. Results for DAS-40278-9 AAD-1 composite samples were all as good or better (biologically or agronomically), based on control lines and/or conventional corn, than analysis of agronomic data collected from pots with control corn and DAS-40278-9 AAD-1.

As mentioned above in the Background section, the introduction and integration of a transgene into a plant genome involves some random events (hence the name “event” for a given insertion that is expressed). That is, with many transformation techniques, such as Agrobacterium transformation, the “gene gun,” and WHISKERS, one cannot predict where in the genome a transgene will become inserted. Thus, identification of the flanking plant genomic DNA on both sides of the insert can be important for identifying a plant that has a given insertion event. For example, PCR primers can be designed that generate a PCR amplicon across the junction region of the insert and the host genome. This PCR amplicon can be used to identify a unique or distinct type of insertion event.

Since "events" are originally random events, as part of the present disclosure, at least 2500 seeds of a corn line comprising the event have been deposited and made publicly available without restriction (but subject to patent rights) with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110. The deposit has been designated as ATCC Deposit No. PTA-10244 (Yellow Dent (Zea Mays L.) hybrid corn seed: DAS-40278-9; Deposited on behalf of Dow AgroSciences LLC; Date of receipt of seed/strain(s) by ATTC: July 10, 2009; viability confirmed August 17, 2009). This deposit has been made and will be maintained in accordance with and under the terms of the Budapest Treaty with respect to deposits of seed for purposes of patent proceedings. The deposit will be held without restriction in the ATCC depository, which is a public depository, for a period of 30 years or five years after the most recent application or for the effective life of the patent, whichever is longer, and will be replaced if it becomes unviable during that period. The seeds deposited are part of the present invention. Of course, corn plants can be grown from such seeds and such plants are part of the present invention. The present invention also relates to DNA sequences contained in such corn plants that are useful for detecting such plants and their progeny. Detection methods of kits of the present invention may be directed to the identification of any one, two or even all three of these events, depending on the ultimate purpose of the test.

Definitions and examples are provided herein to help describe the present invention and to guide those skilled in the art in the practice of the invention. Unless otherwise noted, the terms should be understood in accordance with conventional usage by those skilled in the relevant field. The nomenclature for DNA bases as set forth in 37 CFR §1.822 is used.

As used herein, the term "offspring" denotes the offspring of any generation of a parent plant which comprises corn event AAD-1 DAS-40278-9.

A transgenic "event" is produced by transforming plant cells with heterologous DNA, i.e., a nucleic acid construct that includes a transgene of interest, regenerating a population of plants resulting from insertion of the transgene into the plant genome, and selecting a particular plant characterized by insertion at a particular genomic location. The term "event" refers to the original transformant and the progeny of the transformant that include the heterologous DNA. The term "event" also refers to the progeny produced by sexual cross between the transformant and another variety that includes the genomic DNA/transgene. Even after repeated backcrossing with a recurrent parent, the inserted transgenic DNA and the flanking genomic DNA (genomic DNA/transgene) of the transformed parent are present in the progeny of the cross at the same chromosomal location. The term "event" also refers to the DNA of the original transformant and progeny thereof comprising the flanking genomic sequence and inserted DNA immediately adjacent to the inserted DNA that is expected to be transferred to progeny receiving the inserted DNA, including the transgene of interest, as the result of a sexual cross of a parent line that includes the inserted DNA (e.g., the original transformant and progeny resulting from self-pollination) and a parent line that does not contain the inserted DNA.

A "junction sequence" encompasses the point at which the DNA inserted into the genome is joined to the DNA of the native corn genome flanking the insertion point, the identification or detection of one or the other of the junction sequences in a plant genetic material being sufficient to be diagnostic of the event. DNA sequences that encompass the insertions in corn events described herein and similar stretches of flanking DNA are included. Specific examples of such diagnostic sequences are provided herein; however, other sequences that overlap the junctions of the insertions or the junctions of the insertions and the genomic sequence are also diagnostic and could be used in accordance with the present invention.

The present invention relates to the identification of such flanking, junction and insert sequences. PCR primers and related amplicons are included in the invention. In accordance with the present invention, PCR analysis methods using amplicons encompassing the insert DNA and its borders can be used to identify or detect commercially available transgenic corn varieties or lines derived from the patented transgenic corn lines in question.

The entire sequences of each of these inserts, along with portions of the respective flanking sequences, are provided here as SEQ ID NO: 29. The coordinates of the flanking and insert sequences for this event with respect to SEQ ID NO: 29 (8557 base pairs in total) are printed below. This is discussed in greater detail in Example 3.8, for example.

This insertion event and other components of it are further illustrated in Figures 1 and 2. These sequences (particularly the flanking sequences) are unique. Based on these border and insert sequences, event-specific primers were generated. PCR analysis demonstrated that these maize lines can be identified in different maize genotypes by analyzing the PCR amplicons generated with these event-specific primer sets. Thus, these and related procedures can be used to uniquely identify these maize lines. The sequences identified here are unique. For example, BLAST searches against the GENBANK database did not reveal any significant homology between the cloned border sequences and sequences in the database. Detection techniques of the present invention are especially useful in conjunction with plant breeding to determine which progeny plants comprise a given event after a parent plant comprising an event of interest is crossed with another plant line in an effort to confer one or more additional traits of interest in the progeny. These PCR analysis methods benefit corn breeding programs as well as quality control, especially commercialized transgenic corn seeds. PCR detection kits for these transgenic corn lines can also now be used and made. This may also benefit product registration and product certification.

Furthermore, flanking/genomic sequences from maize can be used to specifically identify the genomic location of each insert. This information can be used to produce event-specific molecular marker systems. These can be used for accelerated breeding strategies and to establish linkage data.

Furthermore, the flanking sequence information can be used to study and characterize transgene integration processes, genomic integration site characteristics, event selection, stability of transgenes and their flanking sequences, and gene expression (especially with respect to gene silencing, solvent methylation patterns, position effects, and potential expression-related elements such as MARS [Matrix Attachment Regions] and the like). In light of the entire disclosure herein, it will be apparent that the present invention includes seeds available under ATCC Deposit No. PTA-10244. The present invention also includes a herbicide-resistant corn plant grown from a seed deposited with the ATCC under accession number PTA-10244. The present invention further includes parts of said plant, such as leaves, tissue samples, seeds produced by said plant, pollen, and the like. Further, the present invention includes progeny and/or offspring plants of plants grown from the deposited seed, preferably a herbicide-resistant corn plant, wherein said plant has a genome comprising a detectable wild-type genomic DNA/insert DNA junction sequence as described herein. As used herein, the term "corn" means sweet corn (Zea mays) and includes all varieties thereof that can be crossed with corn. The present invention further includes processes for producing crosses using a plant of the present invention as at least one parent. For example, the present invention includes an F-i hybrid plant as one or both parents of any of the plants exemplified herein. Also within the present invention is seed produced by such F-i hybrids of the present invention. The present invention includes a method of producing a F-i hybrid seed by crossing an exemplified plant with a different plant (e.g., of inbred origin) and harvesting the resulting hybrid seed. The present invention includes an exemplified plant that is either a female parent or a male parent. Characteristics of the resulting plants can be improved by carefully considering the parent plants.

A herbicide-tolerant corn plant may be improved by first sexually crossing a first parent corn plant consisting of a corn plant grown from seed of any of the lines mentioned herein and a second parent corn plant, thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that is resistant to a herbicide (or that has at least one of the events of the present invention); and self-pollinating the first progeny plant, thereby producing a plurality of second progeny plants; and then selecting from the second progeny plants a plant that is resistant to a herbicide (or that has at least one of the events of the present invention). These steps may further include backcrossing the first progeny plant or the second progeny plant with the second parent corn plant or a third parent corn plant. A corn crop comprising corn seeds of the present invention or progeny thereof may then be planted. It should also be understood that two different transgenic plants can be crossed to produce a scion that contains two independently added exogenous segregating genes. Self-pollination of appropriate progeny can produce plants that are homozygous for both of the added endogenous genes. Backcrossing to a parent plant and crossing to a non-transgenic plant are also considered, as is vegetative propagation. Other breeding methods commonly used for different traits and crops are known in the art. Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous inbred cultivar or line, which is the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait is transferred from the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting parent is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait is transferred from the donor parent.

The DNA molecules of the present invention can be used as molecular markers in a marker-assisted breeding (MAB) method. DNA molecules of the present invention can be used in methods (such as AFLP markers, RFLP markers, RAPD markers, SNPs and SSRs) that identify related agronomically useful traits, as is known in the art.

The herbicide resistance trait can be screened for in the progeny of a cross with a corn plant of the present invention (or progeny thereof and any other corn cultivar or variety) using MAB methods. DNA molecules are markers for this trait and MAB methods that are well known in the art can be used to screen for the herbicide resistance trait(s) in corn plants where at least one corn line of the present invention or progeny thereof was a parent or ancestor. The methods of the present invention can be used to identify any corn variety having the present event.

Methods of the present invention include a method of producing a herbicide tolerant corn plant wherein said method comprises breeding a plant of the present invention. More specifically, said methods may comprise crossing two plants of the present invention or a plant of the present invention and any other plant. Preferred methods further comprise selecting the progeny of said cross by analyzing said progeny for a detectable event in accordance with the present invention. For example, the present invention may be used to screen for the present event through breeding cycles with plants comprising other desirable traits, such as agronomic traits, such as those tested herein in various Examples. Plants comprising the present event and the desired trait may be detected, identified, selected and rapidly used in other breeding cycles, for example. The present event/trait may also be combined through breeding and screening in accordance with the present invention with (an) insect resistance trait(s) and/or with other herbicide tolerance traits. A preferred embodiment of the latter is a plant comprising the present event combined with a gene encoding resistance to the herbicide dicamba.

Thus, the present invention may be combined, for example, with traits encoding glyphosate resistance (e.g., resistant plant or EPSPS, GOX, bacterial GAT), glufosinate resistance (e.g., Pat, bar), acetolactate synthase (ALS)-inhibiting herbicide resistance (e.g., imidazolinones [such as imazethapyr], sulfonylureas, triazolopyrimidine sulfonanilide, pyrimidinyl thiobenzoates, and other chemicals [Csr1, SurA, et al.]), bromoxynil resistance (e.g., Bxn), resistance to HPPD (4-hydroxyphenylpyruvatedioxygenase) enzyme inhibitors, resistance to phytoene desaturase (PDS) inhibitors, resistance to herbicides that inhibit photosystem II (e.g., psbA), resistance to herbicides that inhibit photosystem I ... II (e.g., psbA), resistance to herbicides that inhibit photosystem II (e.g., psbA), resistance that inhibit protoporphyrinogen (PPO) oxidase IX (e.g., PPO-1), resistance to phenylurea herbicides (e.g., CYP76B1), dicamba-degrading enzymes (see, e.g., US 20030135879), and others could be stacked singly or in multiple combinations to confer the ability to effectively control or prevent weed drift and/or resistance to any herbicide in the aforementioned classes.

With respect to additional herbicides, some preferred ALS (also known as AHAs) inhibitors include the triazolopyrimidine sulfonanilides (such as chloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, and penoxsulam), pyrimidinyl thiobenzoates (such as bispyribac and pyrithiobac), and flucarbazone. Some preferred HPPD inhibitors include mesotrione, isoxaflutole, and sulcotrione. Some preferred PPO inhibitors include flumiclorac, flumioxazin, flufenpyr, pyraflufen, fluthiacet, butaphenacyl, carfentrazone, sulfentrazone, and the diphenylethers (such as acifluorfen, fomesafen, lactofen, and oxyfluorfen).

Additionally, AAD-1 alone or stacked with one or more additional HTC traits can be stacked with one or more additional input traits (e.g., insect resistance, fungal resistance, or stress tolerance, etc.) or output traits (e.g., increased yield, improved oil profile, improved fiber quality, etc.). Thus, the present invention can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.

Methods for integrating a polynucleotide sequence into a specific chromosomal location of a plant cell via homologous recombination have been described in the art. For example, site-specific integration, as described in U.S. Patent Application Publication No. 2009/0111188 A1, describes the use of recombinases or integrases to mediate the introduction of a donor polynucleotide sequence into a chromosomal target. Furthermore, International Patent Application No. WO 2008/021207 describes zinc finger-mediated homologous recombination for integrating one or more donor polynucleotide sequences into specific locations of the genome. The use of recombinases, such as FLP/FRT, as described in US Patent No. 6,720,475 or CRE/LOX, as described in US Patent No. 5,658,772, can be used to integrate a polynucleotide sequence into a specific chromosomal location. Finally, the use of meganucleases for targeting donor polynucleotides to a chromosomal location has been described in Puchta et al., PNAS USA 93 (1996) pages 5055-5060.

Several other methods for site-specific integration into plant cells are generally known and applicable (Kumar et al., Trends in Plant Sci. 6(4) (2001) pp. 155-159). In addition, site-specific recombination systems which have been identified in several prokaryotic and lower eukaryotic organisms can be applied in plants. Examples of such systems include, but are not limited to: the R/RS recombinase system of the pSR1 plasmid of the yeast Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol. Biol. 182: 191-203) and the Gin/gix system of the phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 170-176).

In some embodiments of the present invention, it may be desirable to integrate or stack (a) novel transgene(s) in proximity to an existing transgenic event. The transgenic event may be considered a preferred genomic locus which has been selected based on unique characteristics, such as a unique insertion site, normal Mendelian segregation and stable expression, and a superior combination of efficacy, including herbicide tolerance, and agronomic performance in and across multiple environmental locations. The newly integrated transgenes will maintain the transgene expression characteristics of the existing transformants. Furthermore, the development of assays for the detection and confirmation of the newly integrated event will be overcome, since the genomic flanking sequences and chromosomal location of the newly integrated event have already been identified. Finally, the integration of a novel transgene into a specific chromosomal location which is related to an existing transgene will facilitate introgression of the transgenes into other genetic backgrounds through sexual crossing using conventional breeding methods.

In some embodiments of the present invention, it may be desirable to excise polynucleotide sequences from a transgenic event. For example, transgenic excision, as described in U.S. Provisional Patent Application No. 61/297,628, describes the use of zinc finger nucleases to remove a polynucleotide sequence consisting of a gene expression cassette from a chromosomally integrated transgenic event. The polynucleotide sequence which is removed may be a selectable marker. Upon excision and removal of a polynucleotide sequence, the modified transgenic event may be re-targeted by insertion of a polynucleotide sequence. Excision of a polynucleotide sequence and subsequent re-targeting of the modified transgenic event confers advantages such as reuse of a selectable marker or the ability to overcome unintended changes in the plant transcriptome which result from the expression of specific genes.

The present invention discloses herein a specific site on chromosome 2 in the corn genome that is optimal for insertion of heterologous nucleic acids. Also disclosed is a 5' molecular marker, a 3' molecular marker, a 5' flanking sequence, and a 3' flanking sequence useful in identifying the location of a targeting site on chromosome 2. Thus, the present invention provides methods for introducing heterologous nucleic acids of interest into or in the general vicinity of such a target site. The present invention also encompasses a corn seed and/or a corn plant comprising any heterologous nucleotide sequence inserted into or in the general vicinity of such a target site. One option for accomplishing such targeted integration is to excise and/or substitute a different insert in place of the pat expression cassette exemplified herein. In this regard, in general, targeted homologous recombination, for example, and without limitation, may be used in accordance with the present invention.

As used herein, gene, event, or trait “stacking” is the combination of desired traits in a transgenic line. Plant breeders stack transgenic traits by crossbreeding parents so that each has a desired trait and then identifying offspring that have both of those desired traits. Another way to stack genes is by transferring two or more genes into the cell nucleus of a plant at the same time during transformation. Another way to stack genes is by retransforming a transgenic plant with another gene of interest. For example, gene stacking can be used to combine two or more different traits including, for example, two or more different insert resistance traits, insect resistance trait(s) and disease resistance trait(s), two or more herbicide resistance traits, and/or insect resistance trait(s) and herbicide resistance trait(s). The use of a selectable marker in addition to a gene of interest can also be considered gene stacking. "Homologous recombination" refers to a reaction between any pair of nucleotide sequences having corresponding sites containing a similar nucleotide sequence through which the two nucleotide sequences can interact (recombine) to form a new recombinant DNA sequence. The sites of similar nucleotide sequence are each referred to herein as a "homologous sequence". In general, the frequency of homologous recombination increases as the length of the sequence of homology increases. Thus, although homologous recombination can occur between two nucleotide sequences that are less than identical, the frequency of recombination (or efficiency) declines as the divergence between the two sequences increases. Recombination can be accomplished by placing one homologous sequence on each of the donor and target molecules, thereby generating a "single crossover" recombination product. Alternatively, two homologous sequences can be placed on each of the donor and target nucleotide sequences. Recombination between two homologous sequences on the donor with two homologous sequences on the target generates a "double crossover" recombination product. If the homologous sequences on the donor molecule flank a sequence that needs to be manipulated (e.g., a sequence of interest), double crossover recombination with the target molecule will result in a recombination product in which the sequence of interest replaces a DNA sequence that was originally among the homologous sequences on the target molecule. The exchange of DNA sequence between the target and donor through a double crossover recombination event is termed "sequence substitution."

The present AAD-1 enzyme allows transgene expression that results in tolerance to herbicide combinations that would control nearly all broadleaf and grass weeds. AAD-1 may serve as an excellent herbicide tolerant crop (HTC) trait to stack with other HTC traits (e.g., glyphosate resistance, glufosinate resistance, imidazolinone resistance, bromoxynil resistance, etc.) and insect resistance traits (Cry1F, CrylAb, Cry 34/45, etc.), for example. Additionally, AAD-1 may serve as a selectable marker to aid in the selection of primary transformants of plants genetically engineered with a second gene or group of genes.

HTC traits of the present invention can be used in novel combinations with other HTC traits (including, but not limited to, glyphosate tolerance). Such trait combinations give rise to novel methods of controlling weed species (and the like) by virtue of newly acquired resistance or inherent tolerance to herbicides (e.g., glyphosate). Thus, in addition to HTC traits, novel methods for controlling weeds using herbicides for which herbicide tolerance has been created by said enzyme in transgenic crops are within the scope of the invention. Additionally, glyphosate-tolerant crops grown worldwide are prevalent. Often, in rotation with other glyphosate-tolerant crops, control of glyphosate-resistant volunteers can be difficult in rotational crops. Thus, the use of the present transgenic traits, stacked or individually transformed into crops, constitutes a tool for controlling other HTC volunteer crops.

A preferred plant or seed of the present invention comprises, in its genome, the insert sequences as identified herein, together with at least 20-500 or more contiguous flanking nucleotides on both sides of the insert as identified herein. Unless otherwise indicated, reference to flanking sequences refers to those identified with respect to SEQ ID NO: 29 (see Table above). Again, SEQ ID NO: 29 includes the heterologous DNA inserted into the original transformant and illustrative flanking genomic sequences immediately adjacent to the inserted DNA. All or part of these flanking sequences could be expected to be transferred to progeny receiving the inserted DNA as a result of a sexual cross of a parent line that includes the event.

The present invention includes tissue cultures of regenerable cells of a plant of the present invention. Also included is a regenerated plant from such tissue culture, particularly one wherein said plant is capable of expressing all of the morphological and physiological properties of an exemplified variety. Preferred plants of the present invention have all of the physiological and morphological characteristics of a plant grown from deposited seed. The present invention further encompasses progeny of such seed and seeds possessing the quality traits of interest.

Manipulations (such as mutation, further transfection and further breeding) of plants or seeds or parts thereof may lead to the creation of what are termed “essentially derived” varieties. The International Union for the Protection of New Varieties of Plants (UPOV) has provided the following guideline for determining whether a variety has been essentially derived from a protected variety: [A] variety shall be considered to be essentially derived from another variety (“the initial variety”) when: (i) it is predominantly derived from the initial variety or from a variety which is itself predominantly derived from the initial variety, while retaining the expression of the essential characteristics which result from the genotype or combination of genotypes of the initial variety; (ii) it is clearly distinguishable from the initial variety; and (iii) except for differences which result from the act of derivation, it conforms to the initial variety in the expression of the essential characteristics which result from the genotype or combination of genotypes of the initial variety. UPOV, Sixth Meeting with International Organizations, Geneva, 30 October 1992; document prepared by the Office of the Union.

As used herein, a "lineage" is a group of plants that show little or no genetic variation among individuals for at least one trait. Such lines may be created by multiple generations of self-pollination and selection or vegetative propagation from a single parent using cell or tissue culture techniques.

As used herein, the terms "cultivar" and "variety" are synonymous and refer to a lineage which is used for commercial production. "Stability" or "stable" means, with respect to a given component, that the component is maintained from generation to generation and preferably for at least three generations at substantially the same level, e.g., preferably ±15%, more preferably ±10%, still more preferably ±5%. Stability may be affected by temperature, location, stress and timing of planting. Comparison of subsequent generations under field conditions will produce the component in a similar manner. "Commercial utility" is defined as having good plant vigor and high fertility, so that the crop can be produced by farmers using conventional production equipment and oil with the described components can be extracted from the seed using conventional crushing and extraction equipment. To be commercially useful, the yield, as measured by seed weight, oil content, and total oil produced per acre, is within 15% of the average yield of an otherwise comparable commercial canola variety without the "premium" value traits grown in the same region. "Agronomic elite" means a line having desirable agronomic traits such as yield, maturity, disease resistance, and the like, in addition to insect resistance by virtue of the present event(s). Agronomic traits, taken individually or in any combination, as set forth in the Examples below, in a plant comprising an event of the present invention, are within the scope of the present invention. Any and all of these agronomic traits and data points may be used to identify such plants, as a point at either end or both ends of a range of traits used to define such plants.

Those skilled in the art will recognize, in light of the present disclosure, preferred embodiments of the detection kits, for example, may include probes and/or primers directed to and/or comprising "junction sequences" or "transition sequences" (where the genomic flanking sequence abuts the insert sequence). For example, this includes polynucleotide probes, primers, and/or amplicons designed to identify one or both of the junction sequences (where the insert abuts the flanking sequence) as indicated in Table 1. A common design is to have a primer that hybridizes to the flanking region and a primer that hybridizes to the insert. Such primers are often each at least ~15 residues in length. With this configuration, the primers can be used to generate/amplify a detectable amplicon that indicates the presence of an event of the present invention. Such primers can be used to generate an amplicon that spans (and includes) a junction sequence as indicated above. The primer(s) "touching" the flanking sequence are typically not designed to hybridize beyond about 200 bases or beyond the junction. Thus, typical flanking primers will be designed to comprise at least 15 residues of any strand 200 bases in the flanking sequences from the start of the insert. That is, primers comprising a sequence of an appropriate size at residues ~1674-1873 and/or ~6690-6890 of SEQ ID NO: 29 are within the scope of the present invention. Insert primers can likewise be designed anywhere over the insert, but residues ~1874-2074 and ~6489-6689 can be used, for example, non-exclusively for such primer design.

Those skilled in the art will also recognize that primers and probes can be designed to hybridize, under a range of standard hybridization and/or PCR conditions, to a segment of SEQ ID NO: 29 (or its complement) and complements thereof, where the primer or probe is not perfectly complementary to the exemplified sequence. That is, some degree of mismatch can be tolerated. For a primer of approximately 20 nucleotides, for example, typically one or two or more nucleotides need not anneal to the opposite strand if the mismatched base is internal or is on the end of the primer that is opposite the amplicon. Several suitable hybridization conditions are given below. Synthetic nucleotide analogs, such as inosine, can also be used in probes. Peptide nucleic acid (PNA) probes, as well as DNA and RNA probes, can also be used. What is important is that such probes and primers are diagnostic (capable of uniquely identifying and distinguishing) the presence of an event of the present invention.

It should be noted that errors in PCR amplification can occur, which could result in minor sequencing errors, for example 5%. That is, unless otherwise indicated, the sequences listed herein were determined by generating long amplicons from corn genomic DNAs and then cloning and sequencing the amplicons. It is not uncommon to find slight differences and minor discrepancies in the sequences generated and determined in this manner, given the many amplification cycles that are required to generate enough amplicon for sequencing genomic DNAs. Those skilled in the art will recognize and be aware that any adjustments necessary due to these types of common sequencing errors or discrepancies are within the scope of the present invention.

It should also be noted that it is not uncommon for a part of the genomic sequence to be deleted, for example when a sequence is inserted during the creation of an event. Thus, some differences may also appear between the present flanking sequences and genomic sequences listed in GENBANK, for example.

Some of these differences are discussed below in the Examples section. Adjustments to the probes and primers can be made accordingly.

The components of each of the "inserts" are illustrated in Figures 1 and 2 and are discussed in greater detail below in the Examples. The DNA polynucleotide sequences of these components or fragments thereof can be used as DNA primers or probes in the methods of the present invention.

In some embodiments of the invention, compositions and methods are provided for detecting the presence of the genomic/transgenic insertion region in plants and seed and the like of a corn plant. DNA sequences are provided that comprise the subject genomic/transgenic insertion region junction sequence provided herein (between residues 1873-1874 and 6689-6690 of SEQ ID NO: 29), segments thereof, and complements of the exemplified sequences and any segments thereof. The insertion region junction sequence encompasses the junction between the heterologous DNA inserted into the genome and the corn cell DNA flanking the insertion site. Such sequences may be diagnostic for the given event.

Based on these border and insert sequences, event-specific primers can be generated. PCR analysis has demonstrated that corn lines of the present invention can be identified in different corn genotypes by analyzing PCR amplicons generated with these event-specific primer sets. These and related procedures can be used to uniquely identify such corn lines. Thus, PCR amplicons derived from such primer pairs are unique and can be used to identify such corn lines.

In some embodiments, DNA sequences comprising a contiguous fragment of the novel genomic/transgenic insert region are an aspect of the present invention. Included are DNA sequences comprising a sufficient stretch of polynucleotides from a transgenic insert sequence and a sufficient stretch of polynucleotides from corn genomic sequence from one or more of the three aforementioned corn plants and/or sequences that are useful as primer sequences for the production of a diagnostic amplicon product from one or more of these corn plants.

Related embodiments refer to DNA sequences comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more contiguous nucleotides of a transgenic portion of a DNA sequence identified herein (such as SEQ ID NO: 29 and segments thereof) or complements thereof and a similar stretch of corn DNA sequence flanking such sequences or complements thereof. Such sequences are useful as DNA primers in DNA amplification methods. Amplicons produced using such primers are diagnostic of any of the corn strains mentioned herein. Therefore, the invention also includes the amplicons produced by such DNA primers and homologous primers.

The present invention also includes methods of detecting the presence of DNA, in a sample, that corresponds to the corn event mentioned herein. Such methods may comprise: (a) contacting the sample comprising DNA with a primer set that, when used in a nucleic acid amplification reaction with DNA from at least one such corn event(s), produces an amplicon that is diagnostic of said event(s); (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon.

Other detection methods of the present invention include a method of detecting the presence of a DNA, in a sample, corresponding to at least one of said events, wherein said method comprises: (a) contacting the sample comprising DNA with a probe that hybridizes, under stringent hybridization conditions, with DNA from at least one of said corn events and which does not hybridize, under stringent hybridization conditions, with a control corn plant (DNA from an event not of interest); (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to DNA.

In still other embodiments, the present invention includes methods of producing a corn plant comprising the aad-1 event of the present invention, wherein said method comprises the steps of: (a) sexually crossing a first parent corn line (comprising an expression cassette of the present invention which confers said herbicide resistance trait to plants of said line) and a second parent corn line (which lacks said herbicide tolerance trait), thereby producing a plurality of progeny plants; and (b) selecting a progeny plant using molecular markers. Such methods may optionally comprise the additional step of backcrossing the progeny plant to the second parent corn line to produce a true breeding corn plant comprising said insect tolerance trait.

According to another aspect of the invention, methods of determining the zygosity of the progeny of a cross having any one (or more) of said three events are provided. Said methods may comprise contacting a sample comprising corn DNA with a set of primers of the present invention. Said primers, when used in a nucleic acid amplification reaction with genomic DNA from at least one of said corn events, produce a first amplicon that is diagnostic of at least one of said corn events. Such methods further comprise performing a nucleic acid amplification reaction, thereby producing the first amplicon; detecting the first amplicon; and contacting the sample comprising corn DNA with said primer set (said primer set, when used in a nucleic acid amplification reaction with genomic DNA from corn plants, produces a second amplicon comprising native corn genomic DNA homologous to the corn genomic region); and performing a nucleic acid amplification reaction, thereby producing the second amplicon. The methods further comprise detection of the second amplicon and comparison of the first and second amplicons in a sample, where the presence of both amplicons indicates that the sample is heterozygous for the transgenic insertion.

DNA detection kits can be developed using the compositions disclosed herein and methods well known in the art of DNA detection. The kits are useful for identifying present corn strain DNA in a sample and can be applied to methods for breeding corn plants containing such DNA. The kits contain DNA sequences homologous or complementary to the amplicons, for example, disclosed herein or to DNA sequences homologous or complementary to the DNA contained in the transgenic genetic elements of the present strains. These DNA sequences can be used in DNA amplification reactions or as probes in a DNA hybridization method.

The kits may also contain the reagents and materials necessary to carry out the detection method.

A "probe" is an isolated nucleic acid molecule to which is attached a conventional detectable label or reporter molecule (such as a radioactive isotope, ligand, chemiluminescent agent or enzyme). Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from one of said corn events, either from a corn plant or from a sample that includes DNA from the event. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of that target DNA sequence. "Primers" are isolated/synthesized nucleic acids that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs of the present invention relate to their use for amplification of a target nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 polynucleotides or more in length. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present invention have complete sequence similarity within the target sequence, although probes differing from the target sequence and which retain the ability to hybridize to target sequences can be designed by conventional methods.

Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. PCR primer pairs can be derived from a known sequence, for example, by using computer programs designed for that purpose.

Primers and probes based on insert and flanking DNA sequences disclosed herein can be used to confirm (and, if necessary, correct) the disclosed sequences by conventional methods, for example, by recloning and sequencing such sequences.

The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample. Nucleic acid molecules or fragments thereof are capable of specific hybridization to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of specific hybridization to each other if the two molecules are capable of forming an antiparallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be a "complement" to another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit "complete complementarity" when each 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 conventional "high stringency" conditions. Conventional stringency conditions are described by Sambrook et al., 1989. Deviations from complete complementarity are therefore permissible, provided that such deviations do not completely preclude the ability of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it is only necessary that it be sufficiently complementary in sequence to be capable of forming a stable double-stranded structure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acid sequence that will hybridize to the complement of the nucleic acid sequence to which it is being compared under conditions of high stringency. The term "stringent conditions" is functionally defined with respect to the hybridization of a nucleic acid probe to a target nucleic acid (i.e., to a particular nucleic acid sequence of interest) by means of the specific hybridization procedure discussed in Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52 and 9.56-9.58. Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplexes with complementary stretches of DNA fragments.

Depending on the intended application, varying hybridization conditions can be used to obtain varying degrees of probe selectivity for the target sequence. For applications requiring high selectivity, relatively stringent conditions can typically be employed to form the hybrids, e.g., high temperature and/or relatively low salt conditions, such as from about 0.02 M to about 0.15 M NaCl at temperatures from about 50°C to about 70°C. Stringent conditions, for example, could involve washing the hybridization filter at least twice with high-stringency wash buffer (0.2X SSC, 0.1% SDS, 65°C). Appropriate stringency conditions which promote DNA hybridization, e.g., 6.0X sodium chloride/sodium citrate (SSC) at about 45°C followed by a 2.0X SSC wash at 50°C, are known to those of skill in the art, 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0X SSC at 50°C to a high stringency of about 0.2X SSC at 50°C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. The temperature and salt can be varied, or the temperature and salt concentration can be held constant while the other variable is changed. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand. Detection of DNA sequences via hybridization is well known to those skilled in the art, and the teachings of U.S. Patent Nos. 4,965,188 and 5,176,995 are exemplary of hybridization analysis methods.

In a particularly preferred embodiment, a nucleic acid of the present invention will specifically hybridize to one or more of the primers (or amplicons or other sequences) exemplified or suggested herein, including complements and fragments thereof, under high stringency conditions. In one aspect of the present invention, a marker nucleic acid molecule of the present invention has the nucleic acid sequence set forth in SEQ ID NOs: 3-14 or complements and/or fragments thereof.

In another aspect of the present invention, a marker nucleic acid molecule of the present invention shares between 80% and 100% or 90% and 100% sequence identity with such nucleic acid sequences. In another aspect of the present invention, a marker nucleic acid molecule of the present invention shares between 95% and 100% sequence identity with such sequence. Such sequences can be used as markers in plant breeding methods to identify the progeny of genetic crosses. Hybridization of the probe to the target DNA molecule can be detected by any number of methods known to those skilled in the art, which can include, but are not limited to, fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.

With respect to the amplification of a target nucleic acid sequence (e.g., by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that allow the primer pair to hybridize only to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complement) will bind, and preferably produce a unique amplification product, the amplicon. The term "specific for (a target sequence)" indicates that a probe or primer hybridizes, under stringent hybridization conditions, only to the target sequence in a sample comprising the target sequence.

As used herein, "amplified DNA" or "amplicon" refers to the 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 resulting from a sexual cross contains the genomic DNA of the transgenic corn plant event of the present invention, DNA extracted from a tissue sample of the corn plant may be subjected to a nucleic acid amplification method using a primer pair that includes a primer derived from the flanking sequence in the plant genome adjacent to the insertion site of the inserted heterologous DNA and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic of the presence of the DNA of the event. The amplicon is of a length and has a sequence that are also diagnostic of the event. The amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair and/or the combined length of the primer pair plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500, 750, 1000, 1250, 1500, 1750, 2000 or more nucleotide base pairs (plus or minus any of the increments listed above). Alternatively, a primer pair may be derived from the flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire nucleotide sequence of the insert. One member of a primer pair derived from the plant genomic sequence may be located at a distance from the inserted DNA sequence. This distance may range from one nucleotide base pair to about twenty thousand nucleotide base pairs. Use of the term "amplicon" specifically excludes primer dimers that may be formed in the thermal DNA amplification reaction.

Nucleic acid amplification can be accomplished by any of several amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Patent No. 4,683,195 and U.S. Patent No. 4,683,202. PCR amplification methods have been developed to amplify up to 22 kb of genomic DNA. These methods, as well as other methods known in the art of DNA amplification, can be used in the practice of the present invention. The sequence of the heterologous transgenic DNA insert or flanking genomic sequence of a maize event can be verified (and corrected if necessary) by amplifying such sequences from the event using primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or cloned DNA.

The amplicon produced by these methods can be detected by a variety of techniques. Agarose gel electrophoresis and ethidium bromide staining is a well-known common method of detecting DNA amplicons. Another such method is Genetic Bit Analysis, where a DNA oligonucleotide is designed that overlaps the adjacent flanking genomic DNA sequence and the insert DNA sequence. The oligonucleotide is immobilized in wells of a microtiter slide. After PCR of the region of interest (using one primer in the insert sequence and one in the adjacent flanking genomic sequence), a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serves as a template for a single-base extension reaction using a DNA polymerase and labeled ddNTPs specific for the next expected base. Readout can be ELISA- or fluorescent-based. A signal indicates the presence of the insertion/flanking sequence due to successful amplification, hybridization, and single-base extension.

Another method is the Pyrosequencing technique, as described by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method, an oligonucleotide is designed that overlaps the junction of insert DNA and adjacent genomic DNA. The oligonucleotide is hybridized to the single-stranded PCR product from the region of interest (one primer in the insert sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, 5' adenosine phosphosulfate, and luciferin. dNTPs are added individually, and incorporation results in a clear signal that is measured. A clear signal indicates the presence of the flanking sequence/transgenic insert due to successful single- or multiple-base amplification, hybridization, and extension. Fluorescence polarization is another method that can be used to detect an amplicon of the present invention. Following this method, an oligonucleotide is designed which overlaps the junction of insert and flanking genomic DNA. The oligonucleotide is hybridized to the single-stranded PCR product of the region of interest (one primer on the insert DNA and one on the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescently labeled ddNTP. Single base extension results in the incorporation of ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgenic flanking/insert sequence due to successful amplification, hybridization and single base extension.

TAQMAN (PE Applied Biosystems, Foster City, Calif.) is a method for detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe is designed that overlaps the junction of insert and flanking genomic DNA. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. During specific amplification, Taq DNA polymerase eliminates and releases the fluorescent moiety from the quenching moiety of the FRET probe. A fluorescent signal indicates the presence of the transgenic insert/flanking sequence by virtue of successful amplification and hybridization. Molecular beacons have been described for use in sequence detection. Briefly, a FRET oligonucleotide probe is designed that overlaps the junction of the flanking and insert genomic DNA. The unique structure of the FRET probe results in it containing a secondary structure that holds the fluorescent and quenching portions in close proximity. The FRET probes and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and quenching portions. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking genomic sequence/transgenic insert by virtue of successful amplification and hybridization. Having disclosed a location in the corn genome that is optimal for an insert, the present invention also encompasses a corn seed and/or a corn plant comprising at least one non-aad-1 insert in general proximity to that genomic location. One option is to substitute a different insert in place of the aad-1 insert exemplified herein. In these general considerations, targeted homologous recombination, for example, may be used in accordance with the present invention. This type of technology is the subject of, for example, WO 03/080809 A2 and the corresponding Published U.S. Application (US 20030232410). Thus, the present invention includes plants and plant cells comprising a heterologous insert (in place of or with multiple copies of aad-1) flanked by all or a recognizable portion of the flanking sequences identified herein (e.g., residues 1-1873 and 6690-8557 of SEQ ID NO: 29). All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of the present specification. The following examples are included to illustrate procedures for practicing the invention and to demonstrate certain preferred embodiments of the invention. These examples should not be construed as limiting. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent specific approaches used to illustrate preferred modes for their practice. However, those skilled in the art will appreciate, in light of the present disclosure, that many changes can be made to these specific embodiments while still obtaining similar or similar results without departing from the spirit and scope of the invention. Unless otherwise indicated, all percentages are by weight and all solvent mixing proportions are by volume, unless otherwise noted.

The following abbreviations are used unless otherwise noted. AAD-1 aryloxyalkanoate dioxygenase-1 bp base pair °C degrees Celsius DNA deoxyribonucleic acid DIG digoxigenin EDTA ethylene diamine tetra acetic acid kb kilobase pg microgram p L microliter ml_ milliliter M molar mass OLP overlap probe PCR polymerase chain reaction PTU plant transcription unit SDS sodium dodecyl sulfate SOP standard operating procedure SSC a buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0 TBE a buffer solution containing a mixture of Tris base, boric acid, and EDTA, pH 8.3 V volts

EXAMPLES Example 1. Transformation and selection of AAD1 event pDAS1740-278

The AAD1 event, pDAS1740-278, was produced by WHISKER-mediated transformation of the Hi-ll maize line. The transformation method used is described in US Patent Application # 20090093366. An Fspl fragment of plasmid pDAS1740, also referred to as pDAB3812 (Figure 1), was transformed into the maize line. This plasmid construct contains the plant expression cassette containing the Zea mays ubiquitin 1 promoter v2 // AAD1 v3 // Zea mays PER5 3’UTR :: RB 7 MARv4 plant transcription unit (PTU).

Numerous events were produced. Those events that survived and produced healthy haloxyfop-resistant stem tissue were assigned unique identification codes representing putative transformation events and continuously transferred to fresh selection medium. Plants were regenerated from tissue derived from each single event and transferred to the greenhouse.

Leaf samples were taken for molecular analysis to verify the presence of AAD-1 transgene by Southern blot, DNA border confirmation and genomic marker-assisted confirmation. Positive T0 plants were pollinated with inbred lines to obtain T1 seed. T1 plants from event pDAS1470-278-9 (DAS-40278-9) were selected, selfed and characterized for five generations. Meanwhile, T1 plants were backcrossed and introgressed into the elite germplasm (XHH13) by marker-assisted selection for several generations. This event was generated from an independent transformed isolate. The event was selected based on its unique characteristics such as single insertion site, normal Mendelian segregation and stable expression and a superior combination of efficacy including herbicide tolerance and agronomic performance on a broad genotype basis and across multiple environmental locations. The following examples contain the data which were used to characterize event pDAS-1740-278-9.

Example 2. Characterization of event pDAS1740-278-9 via Southern Blot

Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and determine the insert/copy number of the aad-1 gene in event pDAS-1740-278-9 (DAS-40278-9). Data were generated to demonstrate the integration and integrity of the inserted aad-1 transgene into the maize genome.

Southern blot data suggested that the pDAS1740 insert/Fsp I fragment in maize event DAS-40278-9 occurred as a simple integration of a single intact copy of the aad-1 PTU from plasmid pDAS1740. Detailed Southern blot analysis was conducted using probes specific for the gene, promoter, terminator, and other regulatory elements contained within the plasmid region and descriptive restriction enzymes that have cleavage sites located within the plasmid and produce hybridizing fragments internal to the plasmid or fragments that span the junction of the plasmid with maize genomic DNA (border fragments). The molecular weights indicated from Southern hybridization to the restriction enzyme and probe combination were unique to the event and established its identification standards. These analyses also showed that the plasmid fragment had been inserted into maize genomic DNA without rearrangements of the aad-1 PTU. Identical hybridizing fragments were observed in five distinct generations of the transgenic corn event DAS-40278-9, indicating stable inheritance of the aad-1 PTU insert across generations. Hybridization with a mixture of three vector core probes located outside the Fsp I restriction site on plasmid pDAS1740 did not detect any specific DNA/gene fragments, indicating the absence of the ampicillin resistance gene and the absence of other vector core regions immediately adjacent to the Fsp I restriction sites of plasmid pDAS1740 in the transgenic corn event DAS-40278-9. The illustrated map of the insert in the aad-1 corn event DAS-40278-9 is presented in Figures 2-3.

Example 2.1 Corn leaf sample collection and genome-wide DNA (gDNA) isolation

DNA was prepared from leaves of individual plants of the DAS-40278-9 corn event. gDNA was extracted from leaf tissue harvested from individual plants carrying aad-1 from the DAS-40278-9 corn event. Transgenic corn seeds from five distinct generations of the DAS-40278-9 event were used. Twenty individual corn plants, derived from four plants per generation, for the DAS-40278-9 event were selected. In addition, gDNA was isolated from a conventional corn plant, XHH13, which contains the genetic background that is representative of the background line in which the aad-1 gene is absent.

Prior to gDNA isolation, crushed leaves were taken from each plant to test for aad-1 protein expression using a rapid test strip kit (American Bionostica, Swedesboro, NJ) according to the manufacturer's recommended procedure. Each crushed leaf sample was given a score of + or − for the presence or absence of aad-1, respectively. Only positive plants from five generations of event DAS-40278-9 were subjected to further characterization.

Maize leaf samples were collected from individual plants of event DAS-40278-9 and conventional control XHH13. Leaf samples were quickly frozen in liquid nitrogen and stored at approximately −80°C until use.

Individual genomic DNA was extracted from frozen corn leaf tissue following the standard CTAB method. When necessary, some genomic DNA was further purified with Qiagen Genomic-Tip (Qiagen, Valencia, CA) following the manufacturer's recommended procedures. After extraction, DNA was quantified spectrofluorometrically using Pico Green reagent (Invitrogen, Carlsbad, CA). DNA was then visualized on an agarose gel to confirm values from Pico Green analysis and determine DNA quality.

Example 2.2 DNA digestion and separation

For molecular characterization of DNA, nine micrograms (9 μg) of genomic DNA from the corn event DNA sample DAS-40278-9 and the conventional control were digested by adding approximately five to eleven units of selected restriction enzyme per μg of DNA and the corresponding reaction buffer to each DNA sample. Each sample was incubated at approximately 37°C overnight. Restriction enzymes EcoR I, Neo I, Sac I, Fse I, and Hind III were used for digestions (New England Biolabs, Ipswich, MA). A positive hybridization control sample was prepared by combining plasmid DNA, pDAS1740 (pDAB3812), with genomic DNA from the conventional control at a ratio approximately equivalent to 1 transgene copy per corn genome and digested using the same procedures and restriction enzyme as the test samples. DNA from the conventional corn control (XHH13) was digested using the same procedures and restriction enzymes as the test samples to serve as a negative control.

Digested DNA samples were precipitated with Quick-Precip (Edge BioSystems, Gaithersburg, MD) and resuspended in 1x Blue Juice (Invitrogen, Carlsbad, CA) to obtain the desired volume for gel loading. DNA samples and molecular weight markers were then electrophoresed through 0.8% agarose gels with IxTBE buffer (Fisher Scientific, Pittsburgh, PA) at 55–65 volts for approximately 18–22 hours to obtain fragment separation. Gels were stained with ethidium bromide (Invitrogen, Carlsbad, CA), and DNA was visualized under ultraviolet (UV) light.

Example 2.3 Southern blotting and membrane treatment

Southern blot analysis was performed essentially as described by Memelink et al. (1994) Southern, Northern and Western Blot Analysis. Plant Mol. Biol. Manual F1: 1-23. Briefly, after electrophoretic separation and visualization of DNA fragments, gels were depurated with 0.25 N HCl (Fisher Scientific, Pittsburgh, PA) for approximately 15 min and then exposed to denaturing solution (AccuGENE, Sigma, St. Louis, MO) for approximately 30 min, followed by neutralizing solution (AccuGENE, Sigma, St. Louis, MO) for at least 30 min. Southern blotting was performed overnight on nylon membranes (Roche Diagnostics, Indianapolis, IN) using a 10×SSC wick system (Sigma, St. Louis, MO). After transfer, the membranes were washed in 2x SSC solution and the DNA was bound to the membrane via UV cross-linking. This process resulted in Southern blot membranes ready for hybridization.

Example 2.4 DNA probe labeling and hybridization

DNA fragments bound to the nylon membrane were detected using a labeled probe. Probes used for the study were generated by PCR-based incorporation of a digoxigenin (DIG)-labeled nucleotide, [DIG-11]-dUTP, from fragments generated by primers specific to the gene elements and other regions of the pDAS1740 plasmid. Generation of DNA probes by PCR synthesis was performed using a PCR DIG Probe Synthesis kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's recommended procedures. A list of probes used for the study is described in Table 1. Table 1. Location and length of probes used in Southern analysis. Labeled probes were analyzed by agarose gel electrophoresis to determine their quality and quantity. A desired amount of labeled probe was then hybridized to target DNA on nylon membranes to detect specific fragments using the procedures described for DIG Easy Hyb Solution (Roche Diagnostics, Indianapolis, IN). Briefly, nylon membrane blots with DNA fixed to them were quickly washed in 2xSSC and prehybridized with 20–25 ml of DIG Easy Hyb solution prewarmed in hybridization bottles at approximately 50°C for a minimum of 30 minutes in a hybridization oven. The prehybridization solution was then decanted and replaced with 20 ml of prewarmed DIG Easy Hyb solution containing a desired amount of specific probes predenatured in boiling water for 5 minutes. The hybridization step was then conducted at approximately 40-60°C overnight in the hybridization oven.

Example 2.5 Detection

At the end of probe hybridization, DIG Easy Hyb solutions containing the probes were decanted into clean tubes and stored at −20°C. These probes could be reused 2–3 times according to the manufacturer's recommended procedure. Membrane blots were quickly rinsed and washed twice in clean plastic containers with low-stringency wash buffer (2xSSC, 0.1% SDS) for approximately 5 min at room temperature, followed by washing twice with high-stringency wash buffer (0.1xSSC, 0.1% SDS) for 15 min each at approximately 65°C. The membrane blots were then transferred to clean plastic containers and briefly washed with 1x wash buffer from the DIG Wash and Block Buffer kit (Roche Diagnostics, Indianapolis, IN) for approximately 2 minutes, followed by blocking in 1x blocking buffer for a minimum of 30 minutes, followed by incubation with an anti-DIG-AP (alkaline phosphatase) antibody (1:5,000 dilution, Roche Diagnostics, Indianapolis, IN) in 1x blocking buffer for a minimum of 30 minutes. After 2–3 washes in 1x wash buffer, specific DNA probes remained bound to the membrane blots, and DIG-labeled DNA patterns were visualized using the CDP-Star Chemiluminescent Nucleic Acid Detection System (Roche Diagnostics, Indianapolis, IN) following the manufacturer's recommendation. Blots were exposed to chemiluminescent film (Roche Diagnostics, Indianapolis, IN) for one or more time points to detect hybridizing fragments and visualize molecular weight patterns. Films were then developed with an All-Pro 100 Plus film developer (Konica SRX-101) and images were scanned for reporting. The number and sizes of bands detected were documented for each probe. DIG-labeled DNA Molecular Weight Marker II (DIG MWM II), visible after DIG detection as described, was used to determine the size of the hybridizing fragment on Southern blots.

Example 2.6 Probe Extraction

DNA probes were extracted from the membrane blots after Southern hybridization data were obtained and the membrane blots could be reused for hybridization with a different DNA probe according to the manufacturer's recommended procedures (DIG Application Manual for Filter Hybridization, (2003). Roche Diagnostics). Briefly, after signal detection and film exposure, the membrane blots were thoroughly rinsed with Milli-Q water followed by washing twice in extraction buffer (0.2N NaOH, 0.1% SDS) for approximately 15 minutes at room temperature or 37°C. The membrane blots were then quickly washed in 2xSSC and were ready for prehybridization and hybridization with another DNA probe. The membrane blots were exposed to a new chemiluminescent film to ensure that all DNA probes were extracted before proceeding to the next hybridization. The re-exposed films were kept together with the previous hybridization data package in the study archive for record.

Example 2.7 Southern Blot Results

Expected and observed sizes with a particular digest and probe, based on the known restriction enzyme sites of pDAS1740/Fsp I fragment, are provided in Table 2. Two types of fragments were identified from these digests and hybridizations: internal fragments, where known enzyme sites flank the probe region and are completely contained within pDAS1740/Fsp I fragment, and border fragments, where a known enzyme site is located at one end of the probe region and a second site is expected in the maize genome. Border fragment sizes vary by event because, in most cases, DNA fragment integration sites are unique to each event. Border fragments provide a means to locate a restriction enzyme site relative to the integrated DNA and to assess the number of DNA insertions. Based on Southern blot analyses performed in this study, it was concluded that a single copy of an intact aad-1 PTU from plasmid pDAS1740/Fsp I is inserted into the genome of maize event DAS-40278-9, as detailed in the insert map (Figures 2–3). Table 2. Predicted and observed hybridization fragments in Southern blot analysis. Table 2 (continued) Table 2 (continued) Table 2 (continued) Note: * An asterisk after the observed fragment size indicates hybridization to the endogenous sequence that was detected across all samples (including negative controls) # Doublets in conventional control, BC3S1, and some BC3S2 samples. 5 1. Expected fragment sizes are based on the plasmid map of pDAS1740 (pDAB3812) as shown in Figure 1. 2. Observed fragment sizes are assumed to be approximate from these analyses and are based on the indicated sizes of DIG-labeled DNA Molecular Weight Marker II fragments. Because of the incorporation of DIG molecules for visualization, the marker fragments are typically approximately 5-10% larger than their actual indicated molecular weight.

Restriction enzymes with a unique restriction site in plasmid pDAS1740, EcoR I, Neo I, Sac I, Fs and VHind III, were selected to characterize the aad-1 gene insert in event DAS-40278-9. The border fragment of >3382 bp, >2764 bp, >4389 bp was predicted to hybridize with the aad-1 gene probe after digestion with EcoR I, Neo I, and Sac I, respectively (Table 2). Single aad-1 hybridization bands of -12000 bp, -4000 bp, and -16000 bp were observed when EcoR I, Neo I, and Sac I were used, respectively, indicating a single insertion site of the aad-1 gene in the maize genome of event DAS-40278-9. Double digestion with Fse I and Hind III was selected to release a 3361-bp fragment that contains the plant transcription unit (PTU, promoter/gene/terminator) of aad-1 (Table 2). The predicted 3361-bp fragment was observed with the aad-1 gene probe after digestion with Fse V Hind III. Results obtained for digestion with all four enzymes/enzyme combinations of the DAS-40278-9 sample followed by hybridization to the aad-1 gene probe indicated that a single copy of an intact aad-1 PTU from plasmid pDAS1740 was inserted into the corn genome of event DAS-40278-9.

The restriction enzymes Neo I, Sac I, and Fse VHind III were selected to characterize the promoter region (ZmUbil) for aad-1 in event DAS-40278-9. Digestions with Neo I and Sac I are expected to generate a border region fragment of >3472 bp and >4389 bp, respectively, when hybridized to DNA probes, specifically in the ZmUbil promoter region (Table 2). Two hybridization bands at -6300 bp and -3600 bp were detected with the ZmUbil promoter probe after Neo I digestion. The -3600 bp band, however, was present across all sample rows, including conventional controls, suggesting that the -3600 bp band is a nonspecific signal band from homologous binding of the maize-derived ubiquitin promoter (ZmUbil) hybridized to the endogenous maize ubi gene. In contrast, the −6300 bp signal band was detected in the tested DAS-40278-9 samples but not in the conventional controls, indicating that the −6300 bp band is specific for the ZmUbil promoter probe of plasmid pDAS1740 and thus is the expected Neo l/ZmUbil band indicated in Table 2. Similarly, two hybridization bands of −3800 bp and −16000 bp were detected with the ZmUbil promoter probe after Sac I digestion. The −3800 bp band appeared in all sample rows including conventional controls and thus is considered as nonspecific hybridization of the ZmUbil promoter probe to the endogenous maize ubi. The −16000 bp hybridization band that is present only in DAS-40278-9 samples is considered the expected Sac I/ZmUbil band. Double digestion with Fse1/HindIII is expected to release the 3361-bp aad-1 PTU fragment that hybridizes to the ZmUbil promoter probe (Table 2). This 3361-bp band and a nonspecific hybridizing band at −6400 bp were detected by the ZmUbil promoter probe after digestion with Fse1/HindIII. The −6400-bp band is considered nonspecific binding of the ZmUbil promoter probe to the endogenous maize ubi gene because this band is present in all sample rows, including the conventional controls. Additionally, another band very close to −6400 bp was observed in the conventional control, BC3S1, and some of the BC3S2 samples. This additional band very close to −6400 bp is also considered nonspecific because it is present in the conventional control sample XHH13 rows and is most likely associated with the genetic background of XHH13.

The same restriction enzymes/enzyme combination, Nco I, Sac I, and Fse VHind III, were selected to characterize the terminator region (ZmPerδ) for aad-1 in event DAS-40278-9. Digestion with Nco I is expected to generate a border region fragment of >2764 bp when hybridized to DNA probes, specifically the ZmPer5 terminator region (Table 2). Two hybridization bands of −4000 bp and −3900 bp were detected with the ZmPerδ terminator probe after Nco I digestion. The −3900 bp band was present across all sample rows, including conventional controls, suggesting that the −3900 bp band is a nonspecific signal band likely due to homologous binding of the corn-derived peroxidase gene terminator probe (ZmPerδ) to the endogenous maize per gene. In contrast, the −4000 bp signal band was detected in the tested samples of DAS-40278-9 but not in the conventional controls, indicating that the −4000 bp band is specific for the ZmPerδ terminator probe of plasmid pDAS1740 and thus is the expected NeoIZZmPerδ band indicated in Table 2. A border fragment of >1847 bp is expected to hybridize to the ZmPerδ terminator probe after Sac I digestion. Two hybridization bands of −1900 bp and −9000 bp were detected with the ZmPerδ terminator probe after Sac I digestion. The −9000 bp band appeared in all sample rows including conventional controls and thus is considered as nonspecific hybridization of the ZmPerδ terminator probe to the endogenous maize per gene. The −1900 bp hybridization band that was present only in DAS-40278-9 samples is considered the expected Sac I/ZmPer5 band. Double digestion with Fse I/Hind III is expected to release the 3361 bp aad-1 PTU fragment that hybridizes to the ZmPerδ terminator probe (Table 2). This 3361 bp band and an additional −2100 bp nonspecific hybridization band were detected by the ZmPerδ terminator probe after Fse I/Hind III digestion. The additional −2100 bp band is nonspecific binding of the ZmPer5 terminator probe to the endogenous maize gene, since this band is present in all sample rows, including the negative controls. Results obtained with these digestions of the DAS-40278-9 sample, followed by the ZmUbil promoter and hybridization with the ZmPerδ terminator probe further confirmed that a single copy of an intact aad-1 PTU from plasmid pDAS1740 was inserted into the maize genome of event DAS-40278-9.

Restriction enzymes, Neo I and Sac I, were selected to characterize the remaining components of pDAS1740/Fsp I fragment in the AAD-1 corn event DAS-40278-9 (Table 2). DNA sequences of the RB7 Mar v3 and RB7 Mar v4 components have greater than 99.7% identity, therefore, DNA probes specific for RB7 Mar v3 or RB7 Mar v4 are expected to hybridize to DNA fragments containing either version of RB7 Mar. Two border fragments of >2764 bp and >3472 bp are expected to hybridize to the RB7 Mar v4 and RB7 Mar v3 probes after Neo I digestion (Table 2). Two hybridization bands of -4000 bp and -6300 bp were observed with the RB7 Mar v4 or RB7 Mar v3 probe after Neo I digestion in DAS-40278-9 samples. Similarly, two border fragments of >1847 bp and >4389 bp were predicted with the RB7 Mar v4 and RB7 Mar v3 probes after Sac I digestion (Table 2). Hybridization bands at −1900 bp and −16000 bp were detected in the DAS-40278-9 samples with either the RB7 Mar v4 or RB7 Mar v3 probe after Sac I digestion. Taken together, the Southern hybridization results obtained with these elemental probes indicated that the insert DNA in the DAS-40278-9 maize event contains an intact aad-1 PTU along with the RB7 Mar v3 and RB7 Mar v4 matrix attachment regions at the 5′ and 3′ ends of the insert, respectively.

Example 2.8 Absence of main part sequences

An equal molar ratio combination of three DNA fragments (Table 1) spanning nearly the entire Fsp I backbone region (4867–7143 bp in plasmid pDAS1740) of plasmid pDAS1740 was used as the backbone probe to characterize the AAD-1 maize event DAS-40278-9. Plasmid pDAS1740/Fsp I fragment was used to generate event DAS-40278-9, therefore, no specific hybridization signal was expected with the backbone probe combination (Table 2) after any restriction enzyme digestion. It was confirmed that no specific hybridization signal was detected with the backbone probe after Neo I or Sac I digestion in all DAS-40278-9 samples. Positive control lanes contained the expected hybridization bands, demonstrating that the probes were capable of hybridizing to any homologous DNA fragments if present in the samples. The data suggested that the insert in maize event DAS-40278-9 did not include any vector head sequence outside the Fsp I region of plasmid pDAS1740.

Leaf samples from five distinct generations of event DAS-40278-9 were used to conduct Southern blot analysis for molecular characterization. The integration pattern was investigated using selected combinations of restriction enzyme digestion and probe to characterize the inserted gene, aad-1, as well as the noncoding regions, including the promoter, gene expression terminator, and matrix attachment regions.

Southern blot characterization of the insert DNA in event DAS-40278-9 indicates that a single intact copy of aad-1 PTU was integrated into event DAS-40278-9. The molecular weights indicated by Southern hybridization for the restriction enzyme and probe combination were unique to the event and established its identification pattern. The hybridization pattern is identical through all five generations, indicating that the insert is stable in the maize genome. Hybridization with probes spanning the headspace region beyond the pDAS1740/Fsp I transformation fragment of plasmid pDAS1740 confirms that no vector headspace sequences had been incorporated into event DAS-40278-9.

Example 3. Cloning and Characterization of DNA Sequence in the Insert and Flanking Border Regions of Corn Event DAS-40278-9

To characterize the inserted DNA and describe the genomic insertion site, DNA sequences from the insert and border regions of event DAS-40278-9 were determined. In total, 8557 bp of genomic sequence from event DAS-40278-9 were confirmed, comprising 1873 bp of 5′-flanking border sequence, 1868 bp of 3′-flanking border sequence, and 4816 bp of insert DNA. The 4816 bp DNA insert contains an intact aad-1 expression cassette, a 259 bp partial MAR v3 over the 5′ terminus, and a 1096 bp partial MAR v4 over the 3′ terminus. Sequence analysis revealed a 21 bp insertion at the 5′-integration junction and a two base pair deletion of the insertion locus in the maize genome. A single base pair insertion was found at the 3′-integration junction between the maize genome and the DAS-40278-9 insert. Also, a single base change (T to C) was found in the insert at position 5212 in the noncoding region of the 3′ UTR. Neither of these changes affects the open reading frame composition of the aad-1 expression cassette. PCR amplification based on the insert and border sequences of event DAS-40278-9 confirmed that the border regions were of maize origin and that the junction regions could be used for event-specific identification of DAS-40278-9. Sequence analysis spanning the junction regions indicated that no new open reading frames (ORF >= 200 codons) resulted from the DNA insertion in event DAS-40278-9, and no genomic open reading frames were disrupted by the integration of DAS-40278-9 into the native maize genome. Overall, characterization of the insert and border sequences of the maize AAD-1 event DAS-40278-9 indicated that a single intact copy of the aad-1 expression cassette was integrated into the native maize genome.

Example 3.1. Extraction and Quantification of Genomic DNA

Genomic DNA was extracted from freeze-dried or freshly ground leaf tissues using a modified CTAB method. DNA samples were dissolved in 1x TE (10 mM Tris, pH 8.0, 1 mM EDTA) (Fluka, Sigma, St. Louis, MO) and quantified with the Pico Green method according to the manufacturer's instructions (Molecular Probes, Eugene, OR). For PCR analysis, DNA samples were diluted with molecular biology grade water (5 PRIME, Gaithersburg, MD) to result in a concentration of 10–100 ng/μL.

Example 3.2. PCR primers

Table 3 lists the primer sequences that were used to clone the DNA insert and flanking border regions of event DAS-40278-9, with positions and descriptions marked in Figure 4. Table 4 lists the primer sequences that were used to confirm the insert and border sequences. Primer positions are marked in Figures 4 and 5, respectively. All primers were synthesized by DNA Technologies, Inc. (Coralville, IA). Primers were dissolved in water (5 PRIME, Gaithersburg, MD) to a concentration of 100 μM for the stock solution and diluted with water to a concentration of 10 μM for the working solution. Table 3. List of primer sequences used in cloning the maize event DAS-40278-9 insert and flanking border sequence Table 3 (continued) (+): Direct sequence; (-): Complementary sequence; Table 4. List of primer sequences used in confirmation of maize genomic DNA Table 4 (continued) (+): Direct sequence; (-): Complementary sequence;

Example 3.3. Genome Walking

The GenomeWalker ® Universal kit (Clontech Laboratories, Inc., Mountain View, CA) was used to clone the 5′ and 3′ flanking border sequences of maize event DAS-40278-9. According to the manufacturer’s instruction, approximately 2.5 μg of genomic DNA from the AAD-1 maize event DAS-40278-9 was digested overnight with EcoR V, Stu I (both provided with the kit), or Sea I (New England Biolabs, Ipswich, MA). Digested DNA was purified using the DNA Clean & Concentrator®-25 (ZYMO Research, Orange, CA), followed by ligation to GenomeWalker® adapters to construct GenomeWalker® libraries. Each GenomeWalker® library was used as a DNA template for primary PCR amplification with the kit-provided adapter primer AP1 and each construct-specific 5End3812_A and 3End3812_C primers. One microliter of a 1:25 dilution of the primary PCR reaction was then used as a template for secondary PCR amplification with the pooled adapter primer AP2 and each construct-specific 5End3812_B and 3End3812_D primers. TaKaRa LA Taq® HS (Takara Bio Inc., Shiga, Japan) was used for PCR amplification. In a 50 μL PCR reaction, 1 μL of DNA template, 8 μL of 2.5 mM dNTP mix, 0.2 μM of each primer, 2.5 units of TaKaRa LA Taq™ HS DNA Polymerase, 5 μL of 10 x LA II PCR buffer (Mg2+ plus), and 1.5 μL of 25 mM MgCfe were used. Specific PCR conditions are listed in Table 5. Table 5. Genome Walking Conditions of the Maize AAD-1 Event DAS-40278-9 to Amplify the Flanking Border Regions

Example 3.4. Conventional PCR

Standard PCR was used to clone and confirm the sequence of the DNA insert and border in corn event DAS-40278-9. TaKaRa LA Taq® (Takara Bio Inc., Shiga, Japan), HotStarTaq DNA Polymerase (Qiagen, Valencia, CA), Expand High Fidelity PCR System (Roche Diagnostics, Inc., Indianapolis, IN), or Easy-A® High-Fidelity PCR Cloning Enzyme & Master Mix (Stratagene, LaJolla, CA) was used for conventional PCR amplification according to the manufacturer's recommended procedures. Specific PCR conditions and amplicon descriptions are listed in Table 6. Table 6. Conditions for Standard PCR Amplification of Border Regions from Corn Event DAS-40278-9 Table 6 (continued)

Example 3.5 PCR product detection, purification, subcloning of PCR products, and sequencing

PCR products were inspected by electrophoresis using 1.2% or 2% E-gel (Invitrogen, Carlsbad, CA) according to the product instructions. Fragment size was estimated by comparison with DNA markers. If necessary, PCR fragments were purified by excising the fragments from a 1% agarose gel in 1x TBE stained with ethidium bromide using the QIAquick Gel Extraction kit (Qiagen, Carlsbad, CA).

PCR fragments were subcloned into pCR®4-TOPO® using the TOPO TA Cloning® Kit for Sequencing (Invitrogen, Carlsbad, CA) according to the product instructions. Specifically, two to five microliters of TOPO® cloning reaction were transformed into TOP10 One Shot chemically competent cells following the manufacturer's instructions. Cloned fragments were verified by plasmid DNA miniprep (QIAprep Spin Miniprep Kit, Qiagen, Carlsbad, CA) followed by restriction digestion with EcoR I or by direct colony PCR using T3 and T7 primers provided in the kit. Plasmid DNA or glycerol stocks from selected colonies were then sent for sequencing.

After subcloning, putative target PCR products were initially sequenced to confirm that DNA fragments of the expected size had been cloned. Colonies containing appropriate DNA sequences were selected for primer walking to determine the complete DNA sequences. Sequencing was performed by Cogenics (Houston, TX).

Final assembly of the insert and border sequences was completed using Sequencher software (Version 4.8 Gene Codes Corporation, Ann Arbor, Ml). Annotation of the insert and border sequences of maize event DAS-40278-9 was performed using Vector NTI (Version 10 and 11, Invitrogen, Carlsbad, CA).

Homology searching was performed using the BLAST program against the GenBank database. Open Reading Frame (ORF) analysis using Vector NTI (Version 11, Invitrogen) was performed to identify ORFs (>= 200 codons) in the flanking border and full-length insert sequences.

Example 3.6. 5’ End Edge Sequence

A DNA fragment was amplified from each DAS-40278-9 GenomeWalker® corn event library using the 5′-end-specific pooled primer set of the transgene. A PCR product of approximately 800 bp was observed from the DAS-40278-9 EcoR V and Stu I GenomeWalker™ event libraries. The Sea I GenomeWalker® library generated a product of approximately 2 kb. The fragments were cloned into pCR®4-TOPO® and six colonies from each library were randomly selected for end sequencing to confirm that the insert contained the expected sequences. Complete primer walking sequencing of the inserts revealed that the amplified fragments from the DAS-40278-9 corn event Stu I, EcoR V, and Sea I GenomeWalker® libraries were 793, 822, and 2132 bp, respectively. The DNA fragments generated from the Stu I and EcoR V GenomeWalker® libraries were 100% matches to the DNA fragment generated from the Sea I GenomeWalker® library, suggesting that these DNA fragments were amplified from the 5′ region of the transgenic insert. BLAST searching of the resulting 1873 bp corn genomic sequence indicated high similarity to the sequence of a corn BAC clone. Furthermore, sequence analysis of the insertion junction indicated that 917 bp of MAR v3, at its 5′ end region, was truncated compared with plasmid pDAS1740/Fsp I fragment, leaving a partial MAR v3 of 259 bp at the 5′ region of the aad-1 expression cassette.

Example 3.7. End-edge sequence 3’

A DNA fragment of approximately 3 kb in size was amplified from the DAS-40278-9 Stu I GenomeWalker® library of the corn event using the pooled primer set specific for the 3′ end of the transgene. The DNA fragment was cloned into pCR®4-TOPO® and ten colonies were randomly selected for end sequencing to confirm the insertion of the expected sequences. Three colonies with the expected inserts were completely sequenced, generating a 2997 bp DNA fragment. Sequence analysis of this DNA fragment revealed a partial MAR v4 element (missing 70 bp of its 5′ region) and an 1867 bp corn genomic sequence. BLAST search showed that the 1867 bp genomic DNA sequence was a 100% match to the sequence in the same corn BAC clone that was identified with the 5′ border sequence.

Example 3.8. DNA Insertion and Junction Sequence

The DNA insert and junction regions were cloned from the maize endothelial DAS-40278-9 using PCR-based methods as previously described. Five primer pairs were designed based on the 5′ and 3′ flanking border sequences of the expected transgenic sequence. In total, five overlapping DNA fragments (1767 bp amplicon 1, 1703 bp amplicon 2, 1700 bp amplicon 3, 1984 bp amplicon 4, and 1484 bp amplicon 5) were cloned and sequenced (Figure 4). The entire insert and flanking border sequences were assembled based on the overlapping sequence between the five fragments. The end sequence confirms the presence of the 4816 bp DNA insert derived from pDAS1740/Fsp I, 1873 bp of 5′ flanking border sequence and 1868 bp of 3′ flanking border sequence. The 4816 bp DNA insert contains an intact aad-1 expression cassette, a 259 bp partial MAR v3 over the 5′ terminus and a 1096 bp partial MAR v4 over the 3′ terminus (SEQ ID NO: 29). At least two clones for each primer pair were used for primer walking to obtain complete sequence information about the DNA insert and its border sequences. Sequence analysis indicated a 21-bp insertion at the 5′-integration junction between maize genomic DNA and the integrated partial MAR v3 of pDAS1740/Fsp I. Results of BLAST search and Vector NTI analysis indicated that the 21-bp insert DNA demonstrates no homology to DNA from any plant species or to the pDAS1740 plasmid DNA. A single base pair insertion was found at the 3′-integration junction between maize genomic DNA and the partial MAR v4 of pDAS1740/Fsp I. DNA integration also resulted in a two-base pair deletion at the insertion locus of the maize genome (Figure 6). In addition, a nucleotide difference (T to C) at position 5212 bp was observed in the 3′ untranslated UTR of the DNA insert (SEQ ID NO: 29). However, none of these changes appear to be critical for aad-1 expression or create any new ORFs (>= 200 codons) across the DAS-40278-9 insert junctions.

Example 3.9. Confirmation of Maize Genomic Sequences

To confirm the insertion site of the transgene of event DAS-40278-9 in the maize genome, PCR amplification was performed with different primer pairs (Figure 4). Genomic DNA from event DAS-40278-9 and other transgenic and non-transgenic maize lines was used as a template. Two primers specific for aad-1, AI5EndO1 and AI5EndO2, and two primers designed according to the 5′ end border sequence, 1F5EndO1 and 1F5EndO2, were used to amplify DNA fragments spanning the aad-1 gene up to the 5′ end border sequence. Similarly, to amplify a DNA fragment spanning aad-1 up to the 3′ end border sequence, primer 1F3EndO5 derived from the 3′ end border sequence and the aad-1-specific primer AI3EndO1 were used. DNA fragments of expected sizes were amplified only from genomic DNA of the AAD-1 corn event DAS-40278-9, with each primer pair consisting of a primer located on the flanking border of the AAD-1 corn event DAS-40278-9 and an aad-1-specific primer. Control DNA samples did not yield PCR products with the same primer pairs, indicating that the cloned 5′ and 3′ end border sequences are in fact the upstream and downstream sequences of the inserted aad-1 gene construct, respectively. Note that a faint band of about 8 kb in size was observed in all maize samples, including the AAD-1 corn event DAS-40278-9, the AAD-1 corn event DAS-40474, and the non-transgenic maize line XHH13 when the primer pair 1F5EndO1 and AI5EndO1 was used for PCR amplification. A faint band observed (on a prepared gel) could be the result of nonspecific amplification in the maize genome with this primer pair. To further confirm the DNA insertion in the maize genome, two primers located in the 5′ end border sequence, 1F5EndO3 and 1F5End04, and two primers located in the 3′ end border sequence, 1F3EndO3 and 1F3EndO4, were used to amplify DNA fragments spanning the insertion locus. PCR amplification with the primer pair 1F5EndO3/1F3EndO3 or the primer pair 1F5EndO4/1F3EndO4 resulted in a fragment with an expected size of approximately 8 kb from the genomic DNA of corn event AAD-1 DAS-40278-9. In contrast, no PCR product resulted from the genomic DNA of corn event AAD-1 DAS-40278-9 or the non-transgenic corn line XHH13. Given that corn event AAD-1 DAS-40278-9 and event DAS-40474-7 were generated by Hill transformation followed by backcrossing of the original transgenic events to corn line XHH13, the majority of the genome in each of these two events is theoretically from corn line XHH13. It is very likely that only the flanking border sequences close to the aad-1 transgene were brought over from the original genomic DNA and preserved during the introgression process of the AAD-1 event, whereas other regions of genomic sequences could have been replaced by the genomic sequences of XHH13. Therefore, it is not surprising that no fragments were amplified from the genomic DNA of the maize AAD-1 event DAS-40474-7 and XHH13 with the primer pair 1F5EndO3/1F3EndO3 or the primer pair 1F5EndO4/1F3EndO4. Fragments of approximately 3.1 and 3.3 kb were amplified with the primer pair 1F5EndO3Z 1F3EndO3 and 1F5End04/1F3End04, respectively, in genomic DNA from corn lines Hill and B73, but not from corn line A188. The results indicate that the border sequences originated from corn line B73.

Additional cloning of B73/HIII maize genomic DNA was performed to ensure the validity of the flanking border sequences. The PCR-amplified fragments were sequenced to demonstrate that the insert DNA region was integrated into the specific location of the B73/HIII genomic DNA. Primers were designed based on the obtained sequence. The Amp 1 F/Amp 5R primer set was used to amplify a 2212 bp fragment spanning the 5′ to 3′ junctions of the native B73/HIII genome without the insert DNA. Sequence analysis revealed that there was a two base pair deletion of the native B73 genome at the transgenic insertion locus. Analysis of the DNA sequences of the cloned native B73 genomic fragment identified an ORF (>= 200 codons) located downstream of the 3′-integration junction region. Additionally, there were no other ORFs across the original locus where the maize AAD-1 event DAS-40278-9 was integrated. BLAST search also confirmed that both the 5′ and 3′ end border sequences of event DAS-40278-9 are located side by side on the same maize BAC clone.

Given the uniqueness of the 5′-integration junction of the AAD-1 DAS-40278-9 corn event, two specific PCR primer pairs, 1F5EndT1F/1F5EndT1R and Corn278-F/Corn278-R, were designed to amplify this insert-to-plant genome junction. As expected, the desired DNA fragment was generated only in the genomic DNA of the AAD-1 DAS-40278-9 corn event, but not any other transgenic or non-transgenic corn lines. Therefore, these two primer pairs can be used as identifiers specific to the AAD-1 DAS-40278-9 corn event.

Example 4. Genomic Characterization via Flanking SSR Markers of DAS-40278-9

To characterize and describe the genomic insertion site, marker sequences located in close proximity to the insert were determined. A panel of polymorphic SRR markers was used to identify and map the location of the transgene. Event pDAS1740-278 is located on chromosome 2 approximately 20 cM between SSR markers UMC1265 and MMC0111 approximately 20 cM on the 2008 DAS corn line map. Table 6 summarizes the primer information for these two markers that were found to be in close proximity to the pDAS 1740-278 transgene. Table 6. Primer names, dye labels, locus positions, forward and reverse primer sequences, and significant scores for flanking markers associated with event pDAS1740-278.

Example 4.1. Isolation of gDNA

DNA was extracted from crushed leaves using the DNEasy 96 Plant Test kit (Qiagen, Valencia, CA). Modifications were made to the protocol to accommodate automation. Isolated gDNA was quantified using PicoGreen® dye from Molecular Probes, Inc. (Eugene, OR). The gDNA concentration was diluted to 5 ng/μL for all samples using sterile deionized water.

Example 4.2. Screening of gDNA with markers

Diluted gDNA was genotyped with a subset of simple sequence repeat (SSR) markers. SSR markers were synthesized by Applied Biosystems (Foster City, CA) with forward primers labeled with 6-FAM, HEX/VIC, or NED fluorescent tags (blue, green, and yellow, respectively). Markers were divided into groups or panels based on their fluorescent tag and amplicon size to facilitate post-PCR multiplexing and analysis. PCR was performed in 384-well assay slides with each reaction containing 5 ng of genomic DNA, 1.25X PCR buffer (Qiagen, Valencia, CA), 0.20 μM each forward and reverse primer, 1.25 mM MgCl2, 0.015 mM each dNTP, and 0.3 units of HotStart Taq DNA polymerase (Qiagen, Valencia, CA). Amplification was performed on a GeneAmp PCR System 9700 with a 384-Dual Head module (Applied Biosystems, Foster City, Calif.). The amplification program was as follows: (1) initial Taq activation at 95°C for 12 min; (2) 30 s at 94°C; (3) 30 s at 55°C; (4) 30 s at 72°C; (5) repetition of steps 2–4 for 40 cycles; and (6) final extension for 30 min at 72°C. The PCR products for each SSR marker panel underwent a multiplex reaction along with the addition of 2 μl of each PCR product from the same plant to sterilizing deionized water for a total volume of 60 μl. Of the multiplexed PCR products, 0.5 μl was placed in 384-well loading slides containing 5 μl of loading buffer comprised of a 1:100 ratio of GeneScan 500-base pair LIZ size standard and ABI HiDi Formamide (Applied Biosystems, Foster City, California). Samples were then loaded onto an ABI Prism 3730x1 DNA analyzer (Applied Biosystems, Foster City, California) for capillary electrophoresis using the manufacturer's recommendations with a total run time of 36 min. Marker data were collected by ABI Prism 3730x1 Automated Sequencer Data Collection software, Version 4.0, and extracted via GeneMapper 4.0 software (Applied Biosystems) for allele characterization and fragment size labeling.

Example 4.3 SSR Marker Results

Primer data for the flanking markers which were identified in close proximity to the transgene are listed in Table 6. The two closest associated markers, UMC1265 and MMC0111, are located approximately 20 cM away from the transgenic insert on chromosome 2.

Example 5. Characterization of the aad-1 protein in event DAS-40278-9

The biochemical properties of recombinant aad-1 protein derived from transgenic corn event DAS-40278-9 were characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Coomassie blue stained and glycoprotein detection methods), Western blot, immunodiagnostic test strip assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and protein sequencing analysis by random MS were used to characterize the biochemical properties of the protein.

Example 5.1. Immunodiagnostic strip assay

The presence of aad-1 protein in leaf tissue of DAS-40278-9 was confirmed using commercially prepared immunodiagnostic test strips from American Bionostica. The strips were able to discriminate between transgenic and nontransgenic plants by testing crude leaf extracts (data not shown). Nontransgenic extracts (XHH13) did not contain detectable amounts of immunoreactive protein. This result was also confirmed by Western blot analysis.

To test for aad-1 protein expression, an immunodiagnostic strip assay was performed. Four crushed leaves were collected from each plant for XHH13 (control plant) and event DAS-40278-9 by placing the tissue between the snap-on caps of individually labeled 1.5 mL microcentrifuge tubes. Upon receipt at the laboratory, 0.5 mL of aad-1 extraction buffer (American Bionostica, Swedesboro, NJ) was added to each tube, and the tissue was homogenized using a disposable pestle, followed by shaking the sample for ∼10 s. After homogenization, the test strip was placed in the tube and allowed to develop for ∼5 min. The presence or absence of aad-1 protein in the plant extract was confirmed based on the appearance (or lack thereof) of a test line on the immunodiagnostic strip. Once aad-1 protein expression was confirmed for the transgenic event, corn stalk tissue was harvested and lyophilized and stored at approximately −80°C until use.

Example 5.2. Purification of aad-1 protein from corn

Immunorpurified maize-derived aad-1 protein (molecular weight: ~33 kDa) or crude aqueous extracts of maize stem tissue were prepared. All leaf and stem tissues were harvested and transported to the laboratory as follows: leaves were cut from the plant with scissors and placed in cloth bags and stored at approximately −20°C for future use. Separately, stems were cut just above the soil line, placed in cloth bags, and immediately frozen at approximately −80°C for ~6 h. The stems were then placed in a freeze dryer for 5 days to remove water. Once the tissues were completely dry, they were ground into a fine powder with dry ice and stored at approximately −80°C until needed. Corn-derived aad-1 protein was extracted from lyophilized stem tissue in a phosphate-based buffer (see Table 7 for buffer components) by weighing ~30 grams of lyophilized tissue into a chilled 1000 mL glass blender and adding 500 mL of extraction buffer. The tissue was blended at high speed for 60 seconds, and soluble proteins were collected by centrifuging the sample for 20 minutes at 30,000 x g. The pellet was re-extracted as described, and the supernatants were combined and filtered through a 0.45 μm filter. Filtered supernatants were loaded at approximately +4°C onto an anti-aad-1 immunoaffinity column that was conjugated with a monoclonal antibody prepared by Strategic Biosolution Inc. (MAb 473F1 85.1; Purified Protein A; Lot #: 609.03C-2-4; 6.5 mg/mL (∼35.2 mg total)) (Windham, ME); conjugated to CNBr-activated Sepharose 4B (GE Healthcare, Piscataway, NJ). Unbound proteins were collected, and the column was washed extensively with pre-chilled 20 5 mM ammonium bicarbonate buffer, pH 8.0. Bound proteins were eluted with 3.5 M NaSCN (Sigma, St. Louis, MO), 50 mM Tris (Sigma, St. Louis, MO) buffer, pH 8.0. Seven 5 mL fractions were collected and fractions number 2 -> 7 were dialyzed overnight at approximately +4°C against 10 mM Tris buffer, pH 8.0. The fractions were examined by SDS-PAGE and Western blot and the remaining samples were stored at approximately +4°C until used for subsequent analysis. Commercially available reference substances used in this study are listed in the following Table. Table 7.

The protein that bound to the immunoaffinity column was examined by SDS-PAGE, and the results showed that the eluted fractions contained the aad-1 protein at an approximate molecular weight of 33 kDa. In addition, a Western blot was also performed and was positive for the aad-1 protein. The corn-derived aad-1 protein was isolated from ~30 g of lyophilized stalk material.

Example 5.3. SDS-PAGE and Western Blot

Freeze-dried tissue from event DAS-40278-9 and stem XHH13 (~100 mg) was weighed into 2 mL microcentrifuge tubes and extracted with ~1 mL of PBST (Sigma, St. Louis, MO) containing 10% plant protease inhibitor cocktail (Sigma, St. Louis, MO). Extraction was facilitated by the addition of 4 small beads and subjecting the sample to Geno-Grinding for 1 min. After grinding, samples were centrifuged for 5 min at 20,000xg and supernatants were mixed 4:1 with 5x Laemmli sample buffer (2% SDS, 50 mM Tris, pH 6.8, 0.2 mg/mL bromophenol blue, 50% (w/w) glycerol containing freshly added 10% 2-mercaptoethanol) and heated for 5 min at ~100°C. After a brief centrifugation, 45 μL of the supernatant was loaded directly onto a BioRad Criterion SDS-PAGE gel (Bio-Rad, Hercules, CA) fitted to a Criterion Cell gel module. A microbially derived aad-1 positive reference standard was resuspended at 1 mg/mL in PBST, pH 7.4, and further diluted with PBST. The sample was then fixed with Bio-Rad Laemmli buffer with 5% 2-mercaptoethanol and processed as previously described. Electrophoresis was performed with Tris/glycine/SDS buffer (Bio-Rad, Hercules, CA) at voltages of 150–200 V until the dye front approached the end of the gel. After separation, the gel was cut in half and one half was stained with Pierce GelCode Blue protein dye and the other half was electroblotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA) with a Mini Trans-blot electrophoretic transfer cell (Bio-Rad, Hercules, CA) for 60 min at a constant voltage of 100 volts. The transfer buffer contained 20% methanol and Bio-Rad Tris/glycine buffer. For immunodetection, the membrane was hybridized with a rabbit polyclonal antibody specific for aad-1 (Strategic Biosolution Inc., Newark, DE, Protein A Purified Rabbit Polyclonal Antibody Lot #: DAS F1 197-15 1, 1.6 mg/mL). A goat anti-rabbit IgG (H+L) and alkaline phosphatase conjugate (Pierce Chemical, Rockford, IL) was used as the secondary antibody. SigmaFast BCIP/NBT substrate was used for development and visualization of immunoreactive protein bands. The membrane was washed extensively with water to stop the reaction, and a record of the results was captured with a digital scanner (Hewlett Packard, Palo Alto, CA).

In aad-1 produced by P. fluorescens, the major protein band, as visualized on Co-omassie-stained SDS-PAGE gels, was approximately 33 kDa. As expected, the corresponding maize-derived aad-1 protein (event DAS-40278-9) was identical in size to the microbially expressed proteins. Predictably, the purified plant fractions contained a minor amount of immunoreactive impurities in addition to the aad-1 protein. The copurified proteins were probably retained on the column by weak interactions with the column matrix or by leaching of the monoclonal antibody from the column under stringent elution conditions. Other researchers have also reported the nonspecific adsorption of peptides and amino acids onto Sepharose 4B immunosorbents activated with cyanogen bromide (Kennedy and Barnes, 1983; Holroyde et al., 1976; Podlaski and Stern, 2008).

The Pseudomonas-derived aad-1 protein showed a positive signal of the expected size by Western blot analysis of the polyclonal antibody. This was also observed in the stem extract of transgenic corn DAS-40278-9. In Western blot analysis of aad-1, no immunoreactive proteins were observed in the control XHH13 extract and no alternatively sized proteins (aggregates or degradation products) were observed in the transgenic samples.

Example 5.4. Detection of post-translational glycosylation

Immunoaffinity chromatography-purified corn-derived aad-1 protein (Fraction #3) was mixed 4:1 with 5x Laemmli buffer. Microbial-derived aad-1, soybean trypsin inhibitor, bovine serum albumin, and horseradish peroxidase were diluted with Milli-Q water to approximately the concentration of plant-derived aad-1 and mixed with Bio-Rad Laemmli buffer. The proteins were then heated to ~95°C for 5 min and centrifuged at 20,000xg for 2 min to obtain a clarified supernatant. The resulting supernatants were applied directly to a Bio-RadCriterion Gel and electrophoresed with XT MES running buffer (Bio-Rad, Hercules, CA) essentially as described above, except that electrophoresis was performed at 170 V for ∼60 min. After electrophoresis, the gel was cut in half and one half was stained with GelCode Blue dye for total protein according to the manufacturer's protocol. After staining was complete, the gel was scanned with a Molecular Dynamics densitometer to obtain a permanent visual record of the gel. The other half of the gel was stained with a GelCode Glycoprotein Staining kit (Pierce Chemical, Rockford, IL) according to the manufacturer's protocol to visualize glycoproteins. Glycoproteins (with a limit of detection as low as 0.625 ng per band) were visualized as magenta bands on a light pink base. After glycoprotein staining was complete, the gel was scanned with a Hewlett Packard digital scanner to obtain a permanent visual record of the gel. After the glycosylation staining image was captured, the gel was stained with GelCode Blue to verify the presence of nonglycosylated proteins. The results showed that the maize and microbe-derived aad-1 proteins had no covalently attached carbohydrates. This result was also confirmed by peptide mass profiling.

Example 5.5. Peptide mass profile characterization by mass spectrometry and sequencing of maize-derived aad-1 and Pseudomonas

Mass spectrometry analysis of aad-1 derived from Pseudomonas and corn was conducted. The aad-1 protein derived from transgenic corn stem (event DAS-40278-9) was subjected to in-solution trypsin digestion, followed by MALDI-TOF MS and ESI-LC/MS. The masses of the detected peptides were compared with those deduced based on potential protease cleavage sites in the corn-derived aad-1 protein sequence. The theoretical cleavage was generated in silico using the freeware Protein Analysis Worksheet (PAWS) software from Proteometrics LLC. The aad-1 protein, once denatured, is readily digested by proteases and will generate numerous peptide peaks. In trypsin digestion of the aad-1 protein derived from transgenic maize (event DAS-40278-9), the detected peptide fragments spanned almost the entire protein sequence lacking only a small tryptic fragment at the C-terminal end of the protein, F248 to R253, and a short peptide fragment (2 amino acids). This analysis confirmed that the amino acid sequence of the maize-derived protein matched that of the microbial-derived aad-1 protein. Results of these analyses indicate that the amino acid sequence of the maize-derived aad-1 protein was equivalent to the protein expressed in P. fluorescens.

Example 5.5.1. Tryptic peptide fragment sequencing

In addition to peptide mass profiling, amino acid residues at the N- and C-termini of the maize-derived aad-1 protein (immunoaffinity purified from maize event DAS-40278-9) were sequenced and compared with the microbial-derived protein sequence. Protein sequences were obtained by random mass spectrometry for the first 11 residues of the microbial- and maize-derived proteins (Table 8). The amino acid sequences for both proteins were Á HAALSPLSQR (SEQ ID NO: 30) showing that the N-terminal methionine had been removed by an aminopeptidase (Table 8). The N-terminal aad-1 protein sequence was expected to be MAH AALSPLSQ R2. (SEQ ID NO: 31). These results suggest that during or after translation in maize and P. fluorescens, the N-terminal methionine is cleaved by a methionine aminopeptidase (MAP). MAPs cleave methionyl residues rapidly when the second residue on the protein is small, such as Gly, Ala, Ser, Cys, Thr, Pro, and Val (Walsh, 2006). In addition to the methionine being removed, it was shown that a small portion of the N-terminal peptide of the aad-1 protein was acetylated after the N-terminal methionine was cleaved (Table 8). This result is frequently found with proteins expressed in eukaryotes (plants), since approximately 80–90% of the N-terminal residues are modified (Polevoda and Sherman, 2003). Also, it was shown that proteins with serine and alanine at the N-termini are the most frequently acetylated (Polevoda and Sherman, 2002). The two co-translational processes, cleavage of the N-terminal methionine residue and N-terminal acetylation, are the most common modifications and occur in the vast majority (∼85%) of eukaryotic proteins (Polevoda and Sherman, 2002). However, examples demonstrating biological significance associated with N-terminal acetylation are rare (Polevoda and Sherman, 2000). Table 8. Summary of N-terminal sequence data of maize- and microbe-derived AAD-1 proteins 1Expected N-terminal sequence of the first 12 amino acid residues of AAD-1 derived from P. fluorescens and maize. 2Detected N-terminal sequences of AAD-1 derived from P. fluorescens and maize. 3Tandem MS data for the N-terminal peptides revealed a mixture of AHAALSPLSQR (acetylated) and /V-AceW-AHAALSPLSQR (acetylated). N-terminal ragged-ends were also detected (peptides corresponding to the amino acid sequences HAALSPLSQR, AALSPLSQR, and ALSPLSQR). Relative abundance, an estimate of the relative amount of peptide fragments, was made based on the corresponding LC peak areas measured at 214 nm. Notes: Superscript numbers (Rx) indicate amino acid residue numbers in the sequence. Amino acid residue abbreviations: In addition to N-acetylation, there was also a slight N-terminal truncation that appeared during purification of the maize-derived aad-1 protein (Table 8). These ragged ends resulted in the loss of amino acids A2, H3, and A4 (in varying forms and amounts) from the maize-derived protein. This truncation is believed to have occurred during purification of the aad-1 protein, since the Western blot probe of crude leaf extracts contained a single clear band at the same MW as the microbial-derived aad-1 protein. The extraction buffer for the samples subjected to Western blotting contained an excess of protease inhibitor cocktail, which contains a mixture of protease inhibitors with broad specificity for inhibiting serine, cysteine, aspartic acid, and metalloproteases and aminopeptidases.

The C-terminal sequence of the maize- and microbe-derived aad-1 proteins were determined as above and compared with the expected amino acid sequences (Table 9). The results indicated that the measured sequences were identical to the expected sequences and the maize- and microbe-derived aad-1 proteins were identical and unchanged at the C-terminus. Table 9. Summary of C-terminal sequence data of maize- and microbe-derived AAD-1 proteins 1Expected C-terminal sequence of the last 10 amino acid residues of AAD-1 derived from P. fluorescens and maize. 2Detected C-terminal sequences of AAD-1 from P. fluorescens and maize. Notes: Superscript numbers (Rx) indicate amino acid residue numbers in the sequence. Amino acid residue abbreviations:

Example 6. Field expression, nutrient composition analysis and agronomic traits of a hybrid corn line containing event DAS-40278-9

The purpose of this study was to determine the levels of AAD-1 protein found in corn tissues. In addition, compositional analysis was performed on corn forage and grain to investigate the equivalence between the isogenic untransformed corn line and the transgenic corn line DAS-40278-9 (unsprayed, sprayed with 2,4-D, sprayed with quizalofop, and sprayed with 2,4-D and quizalofop). The agronomic characteristics of the isogenic untransformed corn line were also compared with DAS-40278-9 corn. Field expression, composition, and agronomic experiments were conducted at six test sites within major corn production regions of the U.S. and Canada. These sites represent regions of diverse agronomic practices and environmental conditions. The experiments were located in Iowa, Illinois (2 sites), Indiana, Nebraska, and Ontario, Canada. All site mean values for the control inputs, unsprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D, and AAD-1 + both were within literature ranges for corn. A limited number of significant differences between unsprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D, or AAD-1 + both corn and the control were observed, but the differences were not considered to be biologically significant because they were small and the results were within the ranges found for commercial corn. Plots of the composition results did not indicate any biologically significant treatment-related compositional differences between the corn lines A- unsprayed AD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D, or AAD-1 + both corn and the control. In conclusion, the compositional results for unsprayed AAD-1, AAD-1 + quizalofop, AAD-1 + 2,4-D, and AAD-1 + both corns confirm the equivalence of AAD-1 corn (Event DAS 40278-9) to conventional corn lines.

Example 6.1. Tested corn lines

Hybrid seed containing event DAS-40278-9 and control plants, which are conventional hybrid seeds of the same genetic background as the test line but do not contain event DAS-40278-9, are listed in Table 10. Table 10.

The plants described above were grown at locations within major growing regions of the U.S. and Canada. The six field test facilities, Richland, IA; Carlyle, IL; Wyoming, IL; Rockville, IN; York, NE; and Branchton, Ontario, Canada (referred to as IA, IL1, IL2, IN, NE, and ON) represent regions of diverse agronomic practices and environmental conditions for corn.

Control and test corn seed were planted at a planting rate of approximately 24 seeds per row, with seed spacing within each row of approximately 10 inches (25 cm). At each site, 4 replicate plots of each treatment were established, with each plot consisting of rows 2–25 feet apart. Plots were arranged in a randomized complete block (RCB) design, with a single randomization at each site. Each corn plot was bordered by 2 rows of a non-GM corn hybrid of similar maturity. The entire experimental site was surrounded by a minimum of 12 rows (or 30 feet) of a non-GM corn hybrid of similar relative maturity. Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable yield. Average monthly maximum and minimum temperatures, along with rainfall and irrigation, were calculated for the site. These ranges are typically found in corn production.

Example 6.2. Herbicide applications

Herbicide treatments were applied at a spray volume of approximately 20 gallons per acre (187 L/ha). These applications were designed to replicate commercial maximum application rate practices. Table 11 lists the herbicides that were used. Table 11. ae = acid equivalent. bai = active ingredient. 2,4-D (Weedar 64) was applied as 3 overhead spray applications to Test Entries 4 and 5 (seasonal total of 3 lb ae/A). Individual applications were pre-emergence and at approximately the V4 and V8 -V8.5 growth stages. Target individual application rates were 1.0 lb ae/A for Weedar 64 or 1120 g ae/ha. Actual application rates ranged from 1096 - 1231 g ae/A. Quizalofop (Assure II) was applied as a single overhead spray application to Test Entries 3 and 5. Application timing 10 was approximately the V6 growth stage. Target application rate was 0.0825 lb ae/A for Assure II or 92 g ae/ha. Actual application rates ranged from 90.8 - 103 g ai/ha.

Example 6.3. Agronomic data collection and results

Agronomic traits were recorded for all trial entries within Blocks 2, 3, and 4 at each location. Table 12 lists the traits that were measured. Table 12. Note: Heat Unit = ((MAX temp + MIN temp) / 2) - 50°F

An analysis of agronomic data collected from the control, unsprayed aad-1, aad-1 + 2,4-D, aad-1 + quizalofop, and aad-1 + both inputs was conducted. For the across-site analysis, no statistically significant differences were observed in the values for early population (V1 and V4), vigor, final population, crop damage, time to corn silk, time to pollen release, stem lodging, root lodging, disease incidence, insect damage, days to maturity, plant height, and pollen viability (shape and color) in the across-site summary analysis (Table 13). For ear height and stay green, significant paired t-tests were observed between the control and aad-1 + quizalofop inputs, but were not accompanied by significant overall treatment effects or adjusted p-values for False Discovery Rates (FDR) (Table 13). Table 13. Summary of Agronomic Trait Analysis Results Across Locations for DAS-40278-9 aad-1 (Sprayed and Unsprayed) and Control Corn a Overall treatment effect estimated using an F-test. b Comparison of sprayed and unsprayed treatments with the control using a t-test. c P-values adjusted using a False Discovery Rate (FDR) procedure. d Visual estimate on a scale of 1–9; 9 = tall plants with large, robust leaves. e Scale of 0–100%; with 0 = no damage and 100 = dead plant. f Number of heat units accumulated since the time of planting. 9 Scale of 0–100%; with % of pollen grains with ruptured walls. h Scale of 0–100%; with % of pollen grains with intense yellow color. 1 Visual estimate on a scale of 1–9, with 1 = no visible green tissue. J Visual estimate on a scale of 1–9, with 1 being poor disease resistance. k Visual estimate on a scale of 1-9, with 1 being poor insect resistance. 1 NA = statistical analysis not performed, since there is no variability across replicates or treatment. m Statistical difference indicated by P-Value <0.05.

Example 6.4. Sample collection

Samples for expression and composition analysis were collected as listed in Table 14. Table 14

Example 6.5. Determination of aad-1 protein in corn samples

Corn samples were analyzed for the amount of aad-1 protein. Soluble extractable aad-1 protein was quantified using an enzyme-linked immunosorbent assay (ELISA) kit purchased from Beacon Analytical Systems, Inc. (Portland, ME). Corn tissue samples were isolated from test plants and prepared for expression analysis by coarse grinding, freeze-drying, and fine grinding (if necessary) with a Geno/Grinder (Certi-prep, Metuchen, New Jersey). No additional preparation was required for pollen. aad-1 protein was extracted from corn tissues with a phosphate-buffered saline solution containing the detergent Tween-20 (PBST) containing 0.5% bovine serum albumin (BSA). For pollen, the protein was extracted with a PBST/0.5% BSA buffer containing 1 mg/mL sodium ascorbate and 2% protease inhibitor cocktail. Plant tissue and pollen extracts were centrifuged; the aqueous supernatant was collected, diluted with appropriate buffer if necessary, and analyzed using an aad-1 ELISA kit in a sandwich format. The kit used the following steps. An aliquot of the diluted sample and a biotinylated anti-aad-1 monoclonal antibody were incubated in the wells of a microtiter slide coated with an immobilized anti-aad-7 monoclonal antibody. These antibodies bind to the aad-1 protein in the wells and form a “sandwich” with the aad-1 protein bound between the soluble and immobilized antibodies. Unbound samples and conjugate were then removed from the slide by washing with PBST. An excess amount of streptavidin enzyme conjugate (alkaline phosphatase) was added to the wells for incubation. At the end of the incubation period, unbound reagents were removed from the slide by washing. Subsequent addition of the enzyme substrate generated a colored product. Since aad-1 was bound in the antibody sandwich, the level of color development was related to the concentration of aad-1 in the sample (i.e., lower residual concentrations result in lower color development). Absorbance at 405 nm was measured using a Molecular Devices V-max or Spectra Max 190 slide reader. A calibration curve was generated, and the concentration of aad-1 in unknown samples was calculated from the polynomial regression equation using Soft-MAX Pro® software, which was compatible with the slide reader. Samples were analyzed in duplicate wells, with the measured concentration from the duplicate wells being reported. A summary of aad-1 protein concentrations (averaged across sites) in the various corn matrices is shown in Table 15. The average aad-1 protein concentration ranged from 2.87 ng/mg dry weight in R1 stage roots to 127 ng/mg in pollen. Expression results for 10 unsprayed and sprayed lots were similar. No aad-1 protein was detected in any control samples except for one control root sample from the Indiana site. Table 15. Summary of average aad-1 protein concentration levels measured in unsprayed aad-1, aad-1 + Quizalofop, aad-1 + 2,4-D, and aad-1 + Quizalofop and 2,4-D in corn tissues a ND = value less than the Limit of Detection (LOD) of the method. b Values in parentheses are between the LOD and the Limit of Quantitation (LOQ) of the method.

Example 6.6. Compositional Analysis

Forage and corn grain samples were analyzed for nutrient content using a variety of tests. Forage analyses included ash, total fat, moisture, protein, carbohydrate, crude fiber, acid detergent fiber, neutral detergent fiber, calcium, and phosphorus. Grain analyses included proximate compositions (ash, total fat, moisture, protein, carbohydrate, crude fiber, acid detergent fiber), neutral detergent fiber (NDF), minerals, amino acids, fatty acids, vitamins, secondary metabolites, and antinutrients. Nutritional analysis results for forage and corn grain were compared with values reported in the literature (see Watson, 1982 (4); Watson, 1984 (5); I LSI Crop Composition Database, 2006 (6); OECD Consensus Document on Compositional Considerations for Maize, 2002 (7); and Codex Alimentarius Commission 2001 (8)).

Example 6.6.1. Analysis of centesimal composition, fiber and mineral content of forage

An analysis of protein, fat, ash, moisture, carbohydrate, ADF, NDF, calcium, and phosphorus in corn forage samples from the control, unsprayed aad-1, aad-1 + quizalofop, aad-1 + 2,4-D, and aad-1 + both inputs was performed. A summary of the results across all sites is shown in Table 16. For the across-site and individual-site analysis, all mean values for proximate composition, fiber, and mineral were within literature ranges. No statistical differences were observed in the across-site analysis between the control and transgenic inputs for moisture, ADF, NDF, calcium, and phosphorus. For protein and ash, significant paired t-tests were observed for unsprayed AAD-1 (protein), aad-1 + quizalofop (protein), and aad-1 + both (ash), but were not accompanied by significant overall treatment effects or FDR-adjusted p-values. For fat, significant paired t-tests and adjusted p-values were observed for aad-1 + quizalofop compared with the control, but a significant overall treatment effect was not observed. For carbohydrates, a statistically significant overall treatment effect, paired t-test, and FDR-adjusted p-value were observed between aad-1 + quizalofop and the control. Also, for carbohydrates, a significant paired t-test for the unsprayed aad-1 input was observed, but without a significant FDR-adjusted p-value. These differences are not biologically meaningful since all results across the site for these analytes were within the ranges reported in the literature for corn and the differences from the control were small (<23%). Table 16. Summary of proximate composition, fiber, and mineral analysis of corn forage from all sites. Table 16 (continued) a Combined range. b Overall treatment effect estimated using an F-test. c Comparison of transgenic treatments with control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure. e Statistical difference indicated by a P-value <0.05.

Example 6.6.2. Analysis of centesimal composition and fiber of the grain

A summary of the results for proximate compositions (protein, fat, ash, moisture, cholesterol, and carbohydrate) and fiber (ADF, NDF, and total dietary fiber) in corn grain across all sites is shown in Table 17. All results for proximate compositions and fiber were within literature ranges, and no significant differences in the across-site analysis were observed between the control and transgenic inputs for fat, ash, NDF, and total dietary fiber. For moisture, a significant overall treatment effect was observed but was not accompanied by significant paired t-tests or FDR-adjusted p-values. For ADF, a significant paired t-test was observed for a-ad-1 + both, but no overall treatment effect or significant FDR-adjusted p-value was observed. For protein and carbohydrates, significant pairwise tests, adjusted p-values, and overall treatment effects were found for unsprayed aad-1, aad-1 + quizalofop, and aad-1 + both. Since these differences were small (<12%) and all values were within literature ranges, the differences were not considered biologically significant. Table 17. Summary of proximate composition and fiber analysis of corn grain from all sites. Table 17 (continued) a Combined range. b Overall treatment effect estimated using an F-test. c Comparison of transgenic treatments with control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure. e Statistical difference indicated by a P-value <0.05. f NR = not reported g NA = statistical analysis was not performed since most of the data were <LOQ.

Example 6.6.3 Mineral Analysis of Grain

An analysis of corn grain samples for the minerals calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium, and zinc was performed. A summary of the results across all sites is shown in Table 18. All results were within the ranges reported in the literature. For the across-site analysis, no significant differences were observed for calcium, copper, iodine, and potassium. Mean results for chromium, iodine, selenium, and sodium were below the method limit of quantification. For magnesium and phosphorus, significant paired t-tests were observed for the aad-1 unsprayed and aad-1 + quizalofop inputs, but were not accompanied by significant overall treatment effects or FDR-adjusted p-values. For manganese and molybdenum, a significant paired t-test was observed for aad-1 unsprayed, but a significant FDR-adjusted p-value and overall treatment effect were not found. For the aad-1 + both entry, a significant pairwise t-test was observed for zinc, but a significant FDR-adjusted p-value or overall treatment effect was not present. Additionally, these differences from the control were small (<13%) and all values were within literature ranges where available. Table 18. Summary of corn grain mineral analysis from all sites. Table 18 (continued) a Combined range. b Overall treatment effect estimated using an F-test. c Comparison of transgenic treatments with control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure. e NA = statistical analysis was not performed since most data were < LOQ. f Statistical difference indicated by a P-value <0.05.

Example 6.6.4 Amino acid analysis of grain

Corn samples were analyzed for amino acid content in control, unsprayed aad-1, aad-1 + quizalofop, aad-1 + 2,4-D, and aad-1 + both corn and a summary of the results over all sites and by individual field site is shown in Table 19. Levels of all amino acids were within the ranges reported in the literature and no significant differences in the analysis across sites were observed for arginine, lysine, and tyrosine. Significant differences were observed for several of the amino acids in the analysis across sites. In these cases, the amino acid content of the control was lower than that of the aad-1 transgenic lines, which may be related to the lower overall protein content in the control kernel compared with the aad-1 lines. For the unsprayed aad-1 entry, significant overall treatment effects along with significant paired t-tests and FDR-adjusted p-values were found for all amino acids except arginine, glycine, lysine, tryptophan, and tyrosine. For the aad-1 + quizalofop entry, significant overall treatment effects along with significant paired t-tests and FDR-adjusted p-values were found for all amino acids except arginine, cysteine, glycine, lysine, tryptophan, and tyrosine. For the aad-1 + 2,4-D entry, significant overall treatment effects along with significant paired t-tests (with significant FDR-adjusted p-values) were found for all amino acids except arginine, aspartic acid, glycine, histidine, lysine, tyrosine, and valine. For the aad- 1 + both entry, significant overall treatment effects along with significant paired t-tests and FDR-adjusted p-values were found for all amino acids except arginine, glycine, lysine, serine, tryptophan, and tyrosine. Although there were many differences observed for amino acids, the differences were small (<15%), not observed across all sites, and all mean values were within the ranges reported in the literature. Table 19. Summary of corn kernel amino acid analysis from all sites. Table 19 (continued) Table 19 (continued) a Combined range. b Overall treatment effect estimated using an F-test. c Comparison of transgenic treatments with control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure. e Statistical difference indicated by a P-value <0.05.

Example 6.6.5. Fatty acid analysis of grain

An analysis of corn grain samples for fatty acids was performed. A summary of the results across all sites is shown in Table 20. All results for the control, unsprayed aad-1, aad-1 + quizalofop, aad-1 + 2,4-D, and aad-1 + both corn grain samples analyzed for these fatty acids were within the ranges published in the literature. Results for caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), myristoleic (14:1), pentadecanoic (15:0), pentadecenoic (15:1), heptadecanoic (17:0), heptadecenoic (17:1), gamma linolenic (18:3), eicosadienoic (20:2), eicosatrienoic (20:3), and arachidonic (20:4) acids were below the method Limit of Quantitation (LOQ). In the through-site analysis, no significant differences were observed for 16:0 palmitic, 16:1 pamitoleic, 18:0 stearic, 18:2 linoleic, 18:3 linolenic, and 20:0 arachidic acids. For 18:1 oleic and 20:1 eicosenoic, significant paired t-tests were observed for the entries aad-1 unsprayed (18:1) and aad-1 + 2,4-D (18:1 and 20:1), but were not accompanied by significant overall treatment effects or FDR-adjusted p-values. For 22:0 behenic, a significant overall treatment effect and significant paired t-tests for aad-1 + 2,4-D and aad-1 + both were found, but significant FDR-adjusted p-values were not present. Table 20. Summary of corn grain fatty acid analysis from all sites. Table 20 (continued) Table 20 (continued) a Results converted from % dry weight units to % fatty acids. b Pooled range. c Overall treatment effect estimated using an F-test. d Comparison of transgenic treatments with control using t-tests. e P-values adjusted using a False Discovery Rate (FDR) procedure. f NA = statistical analysis was not performed since most of the data were < LOQ. 9 Statistical difference indicated by P value < 0.05.

Example 6.6.6. Vitamin analysis of grain

The levels of vitamins A, B1, B2, B5, B6, B12, C, D, E, niacin, and folic acid in corn grain samples from the control, unsprayed aad-1, aad-1 + quizalofop, aad-1 + 2,4-D, and aad-1 + both corn inputs were determined. A summary of the results across all sites is shown in Table 21. Vitamins B12, D, and E were not quantifiable by the analytical methods used. All reported mean results for vitamins were similar to values reported in the literature, where available. The results for vitamins without ranges reported in the literature (vitamins B5 and C) were similar to the control values obtained (<22% difference from control). For the analysis across sites, no statistical differences were observed except for vitamins B1, C, and niacin. Significant paired t-tests for Vitamin B1 were observed between control and unsprayed aad-1, aad-1 + quizalofop, and aad-1 + both, but were not accompanied by significant overall treatment effects or FDR-adjusted p-values. For Vitamin C, a significant overall treatment effect was observed, along with significant paired t-tests and FDR-adjusted p-values for aad-1 + quizalofop and aad-1 + 2,4-D. Similarly for Niacin, a significant overall treatment effect was observed, along with significant paired t-tests and FDR-adjusted p-values for aad-1 + quizalofop and aad-1 + both. A significant paired t-test for aad-1 + 2,4-D was also found for Niacin for the aad-1 + 2,4-D input, but was not accompanied by a significant overall treatment effect or FDR-adjusted p-value. Since differences were not observed across sites and values were within literature ranges (where available), the differences were not considered biologically significant.

Table 21. Summary of corn grain vitamin analysis from all sites. Table 21 (continued) a Combined range. b Overall treatment effect estimated using an F-test. c Comparison of transgenic treatments with control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure. e Statistical difference indicated by a P-value <0.05. f NR = not reported 9 NA = statistical analysis was not performed since most of the data were <LOQ.

Example 6.6.7. Antinutrient and secondary metabolite analysis of grain

Secondary metabolite (coumaric acid, ferulic acid, furfural, and inositol) and antinutrient (phytic acid, raffinose, and trypsin inhibitor) levels in corn grain samples from the control, unsprayed aad-1, aad-1 + quizalofop, aad-1 + 2,4-D, and aad-1 + both corn inputs were determined. A summary of the results across all sites is shown in Tables 22 and 23. For the across-site analysis, all values were within the literature ranges. No significant differences between the results for the aad-1 inputs and control inputs were observed in the across-site analysis for inositol and trypsin inhibitor. The results for furfural and raffinose were below the limit of quantification of the method. Significant paired t-tests were observed for coumaric acid (aad-1 unsprayed, aad-1 + 2,4-D, and aad-1 + both) and ferulic acid (aad-1 + quizalofop and aad-1 + both). These differences were not accompanied by significant overall treatment effects or FDR-adjusted p-values and were similar to the control (difference < 10%). A significant overall treatment effect, paired t-test, and FDR-adjusted p-value were found for phytic acid (aad-1 unsprayed). Since all results were within literature ranges and were similar to the controls (difference < 11%), these differences were not considered to be biologically meaningful. Table 22. Summary of secondary metabolite analysis of corn grain from all sites. a Combined range. b Overall treatment effect estimated using an F-test. c Comparison of transgenic treatments with control using t-tests. 5 d P-values adjusted using a False Discovery Rate (FDR) procedure. e Statistical difference indicated by a P-value <0.05. f NA = statistical analysis was not performed since most data were <LOQ. Table 23. Summary of antinutrient analysis of corn grain from all sites. a Combined range. b Overall treatment effect estimated using an F-test. c Comparison of transgenic treatments with control using t-tests. d P-values adjusted using a False Discovery Rate (FDR) procedure. e Statistical difference indicated by a P-value <0.05. f NA = statistical analysis was not performed since most of the data were <LOQ.

Example 7 - Additional agronomic experiments

Agronomic traits of corn line 40278 compared to a near-isogenic corn line were evaluated across a variety of environments. Treatments included four genetically distinct hybrids and their appropriate near-isogenic control hybrids tested across a total of 21 locations. The four test hybrids were mid- to late-maturing hybrids ranging from 99 to 113 days relative maturity. Experiment A tested event DAS-40278-9 on the Inbred C x conversion 10 BC3S1 genetic background. This hybrid has a relative maturity of 109 days and was tested at 16 locations (Table 24). Experiment B tested the Inbred E x conversion BC3S1 hybrid background, a hybrid with a relative maturity of 113 days. This hybrid was tested at 14 locations using a slightly different set of locations than Experiment A (Table 24). Experiments C and D tested the BC2S1 x Inbred D conversion and BC2S1 x Inbred F conversion hybrid bases, respectively. Both of these hybrids have a relative maturity of 99 days and were tested at the same 10 sites. Table 24, Agronomic experiment locations For each experiment, a randomized complete block design was used with two replicates per location and two plant plots. Root length was 20 ft, and each row was grown at 34 seeds per row. Standard regional agronomic practices were used in managing the experiments. Data were collected and analyzed for eight agronomic traits: plant height, ear height, stalk lodging, root lodging, final population, grain moisture, test weight, and yield. The parameters plant height and ear height provide information about the appearance of the hybrids. The agronomic traits percent stalk lodging and root lodging determine the harvestability of a hybrid. Final population counts measure seed quality and seasonal growing conditions that affect yield. Percent grain moisture at harvest defines hybrid maturity, and yield (bushels/acre adjusted for moisture) and test weight (weight in pounds of a bushel of corn adjusted for 15.5% moisture) describe hybrid reproductive capacity. Analysis of variance was conducted across field sites using a linear model. Input and location were included in the model as fixed effects. Mixed models, including location and location by input as random effects, were explored, but location by input explained only a small portion of the variance and its variance component was often not significantly different from zero. For stock and root lodging, a logarithmic transformation was used to stabilize the variance; however, means and ranges are reported on the original scale. Significant differences were declared at the 95% confidence level. The significance of an overall treatment effect was estimated using a t-test. Results of these agronomic characterization experiments can be found in Table 2. No statistically significant differences were found for any of the four 40278 hybrids compared to the inbred controls (a p < 0.05) for the parameters of ear height, stem lodging, root lodging, grain moisture, test weight, and yield. Final population counts and plant height were statistically different in Experiments A and B, respectively, but similar differences were not observed in comparisons to the other 40278 hybrids tested. Some of the variation observed may be due to low levels of genetic variability remaining from the backcrossing of event DAS-40278-9 into the elite inbred lines. The overall range of values for the measured parameters are all within the range of values obtained for traditional corn hybrids and do not lead to a conclusion of increased weakness. In summary, agronomic characterization data indicate that 40278 corn is biologically equivalent to conventional corn. Table 25. Analysis of agronomic characteristics Experiment A Experiment B Experiment C Experiment D

Example 8 - Using the DAS-40278-9 corn event insertion location for targeted integration

Consistent agronomic performance of the DAS-40278-9 corn event transgene over multiple generations under field conditions suggests that these identified regions surrounding the DAS-40278-9 corn event insertion site constitute good genomic locations for targeted integration of other transgenic genes of interest. Such targeted integration overcomes the problems with the so-called "position effect" and the risk of creating a mutation in the genome upon integration of the transgene into the host. Other advantages of such targeted integration include, but are not limited to, reducing the large number of transformation events that must be screened and tested before obtaining a transgenic plant that exhibits the desired level of transgene expression without also exhibiting abnormalities resulting from an inadvertent insertion of the transgene at an important locus in the host genome. Furthermore, such targeted integration allows stacking of transgenes, making the production of elite plant lines with both genes more efficient. Using the disclosed teaching, those skilled in the art are able to target polynucleic acids of interest to the same insertion site on chromosome 2 as in corn event DAS-40278-9 or to a site in close proximity to the insertion site in corn event DAS-40278-9. One such method is disclosed in International Patent Application No. WO2008/021207, incorporated herein by reference in its entirety. Briefly, up to 20 Kb of genomic sequence 5' flanking the insertion site and up to 20 Kb of genomic sequence 3' flanking the insertion site (portions of which are identified by reference to SEQ ID NO: 29) are used to flank the gene or genes of interest that are intended to be inserted into a genomic location on chromosome 2 via homologous recombination. The gene or genes of interest may be placed exactly as they are in the insertion site of corn event DAS-40278-9 or may be replaced anywhere within the 20 Kb regions surrounding the insertion sites of corn event DAS-40278-9 to confer a consistent level of transgene expression without detrimental effects on the plant. DNA vectors containing the gene or genes of interest and flanking sequences may be delivered to plant cells via one of several methods known to those of skill in the art including, but not limited to, Agrobacterium-mediated transformation. Insertion of the donor DNA vector into the target site of corn event DAS-40278-9 may be further enhanced by one of several methods including, but not limited to, co-expression or up-regulation of recombination enhancement genes or down-regulation of endogenous recombination suppression genes. Furthermore, it is known in the art that double-stranded cleavage of specific sequences in the genome can be used to increase the frequency of homologous recombination, therefore, insertion at the insertion site of maize event DAS-40278-9 and its flanking regions can be enhanced by expression of naturally occurring or engineered sequence-specific endonucleases for cleaving these sequences. Thus, using the teaching provided herein, any heterologous nucleic acid can be inserted onto maize chromosome 2 at a target site located between a 5' molecular marker discussed in Example 4 and a 3' molecular marker discussed in Example 4, preferably within SEQ ID NO: 29 and/or regions thereof as discussed elsewhere herein.

Example 9 - Excision of the pat gene expression cassette from corn event DAS-40278-9

Removal of a selectable marker gene expression cassette is advantageous for targeted insertion into the genomic loci of corn event DAS-40278-9. Removal of the pat selectable marker from corn event DAS-40278-9 allows reuse of the pat selectable marker for targeted integration of polynucleic acids into chromosome 4 in subsequent generations of corn.

Using the disclosed teaching, those skilled in the art are able to excise polynucleic acids of interest from corn event DAS-40278-9. One such method is disclosed in U.S. Provisional Patent Application No. 61/297,628, incorporated herein by reference in its entirety.

Briefly, sequence-specific endonucleases, such as zinc finger nucleases, are engineered to recognize, bind to, and cleave specific DNA sequences flanking the gene expression cassette. Zinc finger nucleases are delivered to the plant cell by crossing a parent plant containing transgenic zinc finger nuclease expression cassettes with a second parent plant containing the DAS-40278-9 corn event. The resulting progeny are grown to maturity and analyzed for loss of the pat expression cassette via leaf mocking with a glufosinate-containing herbicide. Progeny plants that are not resistant to the herbicide are molecularly confirmed and advanced to self-fertilization. Excision and removal of the pat expression cassette are molecularly confirmed in progeny obtained from self-fertilization. Using the teaching provided herein, any heterologous nucleic acid may be excised from chromosome 2 at a target site located between a 5' molecular marker and a 3' molecular marker as discussed in Example 4, preferably within SEQ ID NO: 29 or the indicated regions thereof.

Example 10 - Stem breaking strength

Stalk breakage refers to the breaking of corn stalks by high winds following applications of growth-regulating herbicides, usually during periods of rapid growth. Mechanical “push” tests, which use a bar to physically push the corn to simulate damage due to high winds, were conducted on hybrid corn containing the DAS-40278-9 event and control plants not containing the DAS-40278-9 event. Treatments were completed at four different geographic locations and were replicated four times (with one exception, one experiment was replicated only three times). Plots consisted of eight rows: four rows of each of the two hybrids, with two rows containing the DAS-40278-9 event and two rows without the event. Each row was twenty feet long. Corn plants were grown to the V4 stage of development and a commercial herbicide containing 2,4-D (Weedar 64, Nufarm Inc., Burr Ridge, IL) was applied at rates of 1120 g ae/ha, 2240 g ae/ha, and 4480 g ae/ha. Seven days after herbicide application, a mechanical push test was performed. The mechanical push test for stalk breakage consisted of pulling a 4-foot bar down the two rows of corn to simulate wind damage. The bar height and travel speed were set to provide a low level of stalk breakage (10% or less) with untreated plants to ensure a test severe enough to demonstrate a difference between treatments. The direction of the stalk breakage treatment was applied against the slope of the corn.

Two of the experimental sites experienced high winds and thunderstorms 2-3 days after application of 2,4-D herbicide. On two consecutive days, a thunderstorm began in Huxley IA. Wind speeds of 2 to 17 m s'1 with high speeds of 33 m s'1 were reported on the planted field side. Wind direction was variable. On one day, a thunderstorm was reported in Lanesboro MN. High wind speeds were reported at the planted field site. In addition, both thunderstorms produced rain. The combination of rain and wind was attributed to the reported stem breakage damage.

Assessments of stem breakage damage resulting from the mechanical push test (and inclement weather conditions) were made by visually scoring the percentage of damage. Prior to the mechanical push test, lodging counts were made for hybrid corn containing event DAS-40278-9 and controls. Several days after the push test, lodging counts within the plot were re-evaluated. The percentage of leaning and reduced lodging within the plot was determined (Table 26). Data from the experiments demonstrated that hybrid corn containing event DAS-40278-9 was less prone to stem breakage compared to null plants following a 2,4-D application. Table 26: Stem breakage tolerance of DAS-40278-9 corn following a V4 application of 2,4-D amine. Percent stem rupture was calculated for hybrid corn plants containing the DAS-40278-9 event and compared with control plants which did not contain the event. 1 Thunderstorms and high winds occurred 2-3 days after application in two experiments 2 Treatments replicated four times in a randomized complete block design (one experiment was completed with only three replicates) 3 Means corrected for occurrences in untreated (untreated means 5 forced to zero)

Example 11 - Grain protein analysis

Grain with increased total protein content was produced from hybrid corn containing event DAS-40278-9 when compared with control plants not containing the event. Two consecutive multisite field experiments were conducted that included no spray and herbicide treatments with three different herbicide combinations. In 7 of the 8 statistical comparisons, event DAS-40278-9 produced grain with significantly higher total protein content (Table 27). These data are corroborated by analyses of 15 individual amino acids. Table 27: Grain protein content from multisite field experiments

Example 12 - Additional Agronomic Experiments

Agronomic characteristics of hybrid corn containing event DAS-40278-9 compared with near-isogenic corn lines were collected from multiple field experiments across diverse geographic environments during one growing season. Data were collected and analyzed for agronomic traits as described in Example 7. The results for hybrid corn lines containing event DAS-40278-9 when compared to null plants are listed in Table 28. Additionally, agronomic traits for the 10 hybrid corn lines containing event DAS-40278-9 and null plants sprayed with the herbicides quizalofop (280 g ae/ha) at the V3 growth stage and 2,4-D (2,240 g ae/ha) sprayed at the V6 growth stage are described in Table 29. Table 28: Yield, percent moisture, and final population 15 results for hybrid corn containing event DAS-40278-9 when compared to a near-isogenic control line. Table 29: Yield, percent moisture, percent stock lodging, percent root lodging, and total population for hybrid corn lines containing event DAS-40278-9 when compared to the near-isogenic line control.

Claims (5)

1. A method of detecting DNA from a corn event comprising SEQ ID NO: 29 in a sample comprising corn DNA, wherein said method comprises contacting said sample with a first and a second primer, wherein said primers are diagnostic for AAD-1 corn comprising SEQ ID NO: 29 as present in seed deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-10244, said primers are defined wherein: the first primer is selected from the group consisting of SEQ ID NOs: 5, 7, 14-16, 19-22, 24, 26, and 28 and binds to a flanking sequence selected from the group consisting of residues 1-1873 of SEQ ID NO: 29, residues 6690-8557 of SEQ ID NO: 29, and complements thereof; and said second primer is selected from the group consisting of SEQ ID NOs: 1-4, 6, 8-13, 17, 18, 23, 25, and 27 and binds to an insert sequence comprising residues 1874-6689 of SEQ ID NO: 29 or the complement thereof; subjecting said sample to polymerase chain reaction; and assaying with an amplicon generated between said primers. 2. A method of detecting DNA from a corn event comprising SEQ ID NO: 29 in a sample comprising corn DNA, wherein said method comprises contacting said sample with a polynucleotide that is diagnostic for an AAD-1 corn comprising SEQ ID NO: 29, as present in seed deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-10244, said polynucleotide comprising a sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO: 29, residues 6679 to 6700 of SEQ ID NO: 29, and complements thereof; wherein said method further comprises subjecting said sample and said polynucleotide to stringent hybridization conditions; and assaying said sample by hybridization of said polynucleotide to said DNA. 3. DNA detection kit for detecting DNA from a corn event 5 comprising SEQ ID NO: 29, comprising a first primer selected from the group consisting of SEQ ID NOs: 5, 7, 14-16, 19-22, 24, 26, and 28 and a second primer selected from the group consisting of SEQ ID NOs: 1-4, 6, 8-13, 17, 18, 23, 25, and 27. 4. DNA detection kit for detecting DNA from a corn event 10 comprising SEQ ID NO: 29, characterized in that it comprises a polynucleotide selected from the group consisting of residues 1863 to 1875 of SEQ ID NO: 29, residues 6679 to 6700 of SEQ ID NO: 29, and their complements, wherein said polynucleotide hybridizes under stringent wash conditions to a nucleotide sequence selected from the group consisting of residues 1863 to 1875 of SEQ ID NO: 29, residues 6679 to 6700 of SEQ ID NO: 29 and their complements. 5. Polynucleotide, characterized by the fact that it presents the sequence of SEQ ID NO: 29.