BR112018006884B1 - METHODS AND SYSTEMS FOR PREDICTING THE RISK OF TRANSGENE S-LEARNING - Google Patents

Abstract

METHODS AND SYSTEMS FOR PREDICTING THE RISK OF TRANSGENE SILENCE. The present invention is based on the use of H3K9me2 methylation levels in plant genomes to predict transgene silencing, transgene stability and/or transgene expression level. Methods and/or systems for generating whole-genome H3K9me2 maps and their use with a designated threshold value to predict gene silencing are provided. The methods and/or systems provided herein can be used in high-throughput settings for screening large numbers of transformed events in a relatively short period of time compared to existing technologies.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of Provisional Patent Application Serial No. US 62/253,213, filed on November 10, 2015.

BASICS OF THE INVENTION

[0002] The genome of plants, for example soybean or corn plants, was successfully transformed with transgenes in the 1990s. In the last twenty years, numerous methodologies have been developed to transform the genome of plants, in which a transgene is integrated stably in the plant genome. The evolution of plant transformation methodologies has resulted in the ability to successfully introduce a transgene comprising an agronomic trait into the plant genome. The introduction of insect resistance and herbicide tolerant traits in plants provided growers with a new and convenient technological innovation for controlling insects and a broad spectrum of weeds, which was unparalleled in cultivation methods.

[0003] Current transformation methodologies rely on the random insertion of transgenes into the plant genome. As transgenic events can integrate randomly within the transcriptional sequences of genes, such events can disrupt the expression of endogenous traits and alter plant growth and development. Furthermore, transgenic events can indiscriminately integrate into locations in the genome that are susceptible to gene silencing, culminating in reduced or complete inhibition of transgene expression in the first or subsequent generations of transgenic plants. Finally, the random integration of transgenes into the plant genome requires considerable effort and cost in identifying the location of the transgenic event and in selecting transgenic events that function as designed without agronomic impact to the plant.

[0004] Therefore, there is a need for inventions that are useful for evaluating the effectiveness of transgene integration sites for gene expression and/or for predicting the risk of transgene silencing.

SUMMARY OF THE INVENTION

[0005] This invention relates to methods and systems for predicting the risk of transgene silencing and/or predicting transgene expression levels and/or predicting the stability and/or effectiveness of transgene integration sites. In one aspect, a method for predicting the risk of transgene silencing is provided. The method comprises: (a) generating histone methylation/acetylation profile data on a plant genome; (b) assembling the histone methylation/acetylation profile data from step (a) into a histone methylation/acetylation database that shows the peak numbers of the histone methylation/acetylation profiles; (c) analyzing sequences of at least one transgene insertion site; and (d) comparing the transgene insertion site sequences from step (c) with the histone methylation/acetylation database from step (b); wherein (i) if the transgene insertion site falls into peak zero or peak one of the methylation/acetylation profiles, there is no risk of transgene silencing, or (11) if the transgene insertion site falls into peak two or more of histone methylation/acetylation profiles, there is no significant risk of transgene silencing.

[0006] In one embodiment, histone methylation/acetylation profile data is generated using a chromatin immunoprecipitation sequencing (ChIP-seq) assay. In another embodiment, histone methylation/acetylation profile data is associated with methylation/acetylation selected from the group consisting of H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3, and H4K20me3 and combinations thereof. In another embodiment, the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3 and combinations thereof. In another embodiment, histone methylation/acetylation profile data is associated with H3K9me2 methylation. In another embodiment, the plant is selected from soybeans, corn, canola, cotton, wheat and rice. In another embodiment, transgene insertion site sequences are obtained by direct sequencing.

[0007] In another aspect, a computerized system is provided for predicting the risk of transgenic silencing. The system comprises: (a) a histone methylation/acetylation database showing the peak numbers of histone methylation/acetylation profiles in a plant genome; (b) an input module into which a user inputs sequences from at least one transgene insertion site; and (c) an output module shows the prediction of transgene silencing risk, wherein the prediction is based on the comparison of the introduced sequences from Step (b) and the histone methylation/acetylation profiles from Step (a).

[0008] In one embodiment, histone methylation/acetylation profile data is generated using a chromatin immunoprecipitation sequencing (ChIP-seq) assay. In another embodiment, histone methylation/acetylation profile data is associated with methylation/acetylation selected from the group consisting of H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3, H3K27me1, H3K27me3, and H4K20me3 and combinations thereof. In another embodiment, the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K9me2, H3K9me3, H3K27me1, H3K27me3, and H4K20me3 and combinations thereof. In another embodiment, histone methylation/acetylation profile data is associated with H3K9me2 methylation. In another embodiment, the plant is selected from soybeans, corn, canola, cotton, wheat and rice. In another embodiment, transgene insertion site sequences are obtained by direct sequencing.

[0009] In another aspect, a process is provided for use in a computerized system to predict the risk of transgene silencing. The process comprises: (a) introducing sequences of at least one transgene insertion site into the system provided herein by a user; and (b) receiving output from the system provided herein for predicting the risk of transgene silencing, wherein the prediction is based on comparison of introduced transgene insertion site sequences and histone methylation/acetylation profiles within the system .

BRIEF DESCRIPTION OF THE DRAWINGS

[00010] Figure 1 shows the abundance of H3K9me2 within the soybean gene traits (gene body, 2kb upstream, 1kb downstream) as a function of different expression categories (high, medium high, medium, medium low, low).

DETAILED DESCRIPTION OF THE INVENTION

[00011] The present invention is based on the use of H3K9me2 methylation levels in plant genomes to predict transgene silencing, transgene stability and/or transgene expression level. Methods and/or systems for generating whole-genome H3K9me2 maps and their use with a designated threshold value to predict gene silencing are provided. The methods and/or systems provided herein can be used in high-throughput settings for screening large numbers of transformed events in a relatively short period of time compared to existing technologies.

[00012] Transgene performance and hereditary stability of the transgene are important to the success of genetic improvement product concepts. Various approaches have been used in the past to evaluate transgene expression and performance, including quantitative expression analysis techniques, including, for example, RT-PCR, DNA methylation, and transgenerational stability testing. These approaches are time and resource intensive during the genetic improvement development process and require multiple generations of tracking transgenic events and could potentially lead to the cherry-picking of poor transgenic events to promote the development of downstream products.

[00013] Epigenetic marks refer to enzyme-mediated chemical modifications of DNA and its associated chromatin proteins. Typically, epigenetic marks do not alter the primary sequence of DNA, but are important in regulating genome function. Modifications to DNA (including cytosine methylation, post-translational modifications of the histone tail and histone core, and nucleosome positioning), can influence the transcriptional state and other functional aspects of chromatin. Histone modifications are a cellular system that involves transcriptional regulatory mechanisms. Both activation-type (e.g., H3K4me3 and H3K9/14ac) and repressive-type (e.g., H3K27me3, H3K9me3, H3K9me2, and H4K20me3) histone marks have been previously identified. In particular, methylation of a specific lysine (K9) on histone H3, commonly known as H3K9me2, has been characterized as an epigenetic mark indicating closed chromatin or genome-scale transcriptional repression. Methods and systems are provided for using H3K9me2 as a diagnostic tool to understand the chromatin state of the genomic location around an integrated transgene and predict the potential risk of gene silencing in transgenic events. The applicability of the methods and systems provided includes, for example, ordering or selection of events in the process of developing genetic improvements. Specifically, data for genome-wide H3K9me2 profiling in soybean roots and shoots are provided as an example. In some embodiments, the level of H3K9me2 methylation at the locus of a transgene insertion site can be used to identify/predict events with a greater probability of stable expression.

[00014] In some embodiments, whole genome H3K9me2 maps are generated for each crop plant. In some embodiment, a database is generated according to genome-wide H3K9me2 maps. In some embodiments, this database can be used to predict the chromatin structure within the locus of interest according to the methods and/or systems provided.

[00015] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as they would have for one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al. Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel FM et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1993, for definitions and terms of the art. It should be understood that this invention is not limited to the specific methodology, protocols and reagents described, as these may vary.

[00016] As used herein, the expression "about" refers to greater or less than the value or range of values indicated by 10 percent, but is not intended to designate any value or range of values for just this definition further. broad. Each value or range of values preceded by the term "about" is also intended to encompass the modality of the declared absolute value or range of values

[00017] As used herein, the term "vector" refers to a piece of DNA, typically double-stranded, which may have a piece of foreign DNA inserted into it. The vector can be, for example, of plasmid or viral origin, which normally encodes a selectable or screenable marker or transgenes. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently or match the host's chromosomal DNA. Alternatively, the vector may direct the insertion of foreign or heterologous DNA into a host chromosome.

[00018] As used herein, the term "transgene vector" refers to a vector that contains an inserted segment of DNA, the "transgene" that is transcribed into mRNA or replicated as an RNA within a host cell. The term "transgene" refers not only to the portion of the inserted DNA that is converted to RNA, but also to those portions of the vector that are necessary for RNA transcription or replication. A transgene typically comprises a gene of interest, but need not necessarily comprise a polynucleotide sequence that contains an open reading frame capable of producing a protein.

[00019] As used here, the term "transformed" or "transformation" refers to the introduction of DNA into a cell. The terms "transformant" or "transgenic" refer to plant cells, plants and the like that have been transformed or have undergone a transformation process. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA.

[00020] As used here, the expression "transgenic plant" refers to a plant whose genome has been altered by the stable integration of recombinant DNA. A transgenic plant includes a plant regenerated from an originally transformed plant cell and plants from transgenic progeny of later generations or crosses of a transformed plant.

[00021] As used herein, the term "recombinant DNA" refers to DNA that has been genetically modified and constructed outside a cell, including DNA containing naturally occurring DNA or cDNA or synthetic DNA.

[00022] As used herein, the phrase "selectable marker" or "selectable marker gene" refers to a gene that is optionally used in plant transformation to, for example, protect plant cells from a selective agent or provide resistance/tolerance to a selective agent. Only those cells or plants that receive a functional selectable marker are capable of dividing or growing under conditions with a selective agent. Examples of selective agents may include, for example, antibiotics, including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin and hygromycin. These selectable markers include the gene for neomycin phosphotransferase (npt II), which expresses an enzyme conferring resistance to the antibiotic kanamycin, and genes for the related antibiotics neomycin, paromomycin, gentamicin, and G418, or the gene for hygromycin phosphotransferase (hpt), which expresses an enzyme that confers resistance to hygromycin. Other selectable marker genes may include genes encoding herbicide resistance, including Bar (resistance against BASTA® (glufosinate ammonium), or phosphinothricin (PPT)), acetolactate synthase (ALS, resistance against inhibitors such as sulfonylureas (SUs), imidazolinones ( IMIs), triazolopyrimidines (TPs), pyrimidinyl oxybenzoates (POBs) and sulfonylamino carbonyl triazolinones that impede the first step in the synthesis of branched-chain amino acids), glyphosate, 2,4-D and metal resistance or sensitivity. The term "marker positive" refers to plants that have been transformed to include a selectable marker gene.

[00023] Various selectable or detectable markers can be incorporated into the chosen expression vector to allow the identification and selection of transformed plants, or transformants. Many methods are available to confirm the expression of selection markers in transformed plants, including, for example, DNA sequencing and PCR (polymerase chain reaction), Southern blotting, RNA blotting, immunological methods for detecting a protein expressed from of the vector, for example, precipitated protein that mediates resistance to phosphinothricin, or other proteins, such as reporter genes, ß-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), DsRed, ß-galactosidase, chloramphenicol acetyltransferase (CAT ), alkaline phosphatase and the like (See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y., 2001, the contents of which are incorporated herein by reference in their entirety).

[00024] Selectable marker genes are used for selection of transformed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds. Herbicide resistance genes generally encode a modified herbicide-insensitive target protein or an enzyme that degrades or detoxifies the herbicide in the plant before it acts. For example, glyphosate resistance was achieved through the use of genes encoding mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The genes and mutants for EPSPS have been disclosed in US Patent Nos. 4,940,835, 5,188,642, 5,310,667, 5,633,435, 5,633,448 and 6,566,587, the contents of which are incorporated by reference in their entirety. Resistance to glufosinate ammonium, bromoxynil and 2,4-dichlorophenoxyacetate (2,4-D) was obtained using bacterial genes encoding phosphinothricin acetyltransferase, a nitrilase, or a 2,4-dichlorophenoxyacetate monooxygenase, which detoxifies the respective herbicides. Enzymes/genes for glufosinate resistance/tolerance have been disclosed in US Patent Nos. 5,273,894, 5,276,268, 5,550,318 and 5,561,236, the contents of which are incorporated by reference in their entirety. Enzymes/genes for 2,4-D resistance have previously been disclosed in U.S. Patent Nos. 6,100,446 and 6,153,401, as well as US patent applications 2009/0093366 (AAD-1) and WO 2007/053482 (AAD-12), the contents of which are incorporated herein by reference in their entirety. Enzymes/genes for nitrilase have been previously disclosed in U.S. Patent Nos. 4,810,648, the contents of which are incorporated by reference in their entirety.

[00025] Other herbicides can inhibit the growth point or meristem, including imidazolinone or sulfonylurea, and genes for acetohydroxy acid synthase (AHAS) and acetolactate synthase (ALS) resistance/tolerance to these herbicides have been described. The genes and mutants for AHAS and mutants have been disclosed in U.S. Patent Nos. 4,761,373, 5,304,732, 5,331,107, 5,853,973 and 5,928,937, the contents of which are incorporated by reference in their entirety. The genes and mutants for ALS have been disclosed in US Patent Nos. 5,013,659 and 5,141,870, the contents of which are incorporated by reference in their entirety.

[00026] Glyphosate resistance genes include mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via introduction of recombinant nucleic acids and/or various forms of in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosate acetyl transferase (GAT), respectively). Resistance genes for other phosphono compounds include glufosinate (phosphinothricin acetyl transferase (PAT) genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes) and pyridinoxy or phenoxyproprionic acids and cyclohexones (genes encoding ACCase inhibitors). Acetyl coenzyme A carboxylase (ACCase) herbicide resistance/tolerance genes have been described in US Patents 5,162,602 and 5,498,544, the contents of which are incorporated by reference in their entirety.

[00027] A DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession number 39256 and the nucleotide sequence of the mutant gene is described in Pat. US Patent No. 4,769,061 to Comai, European Patent Application No. 0,333,033 to Kumada et al. Pat. US No. 4,975,374 to Goodman et al., disclosing nucleotide sequences of glutamine synthetase genes that confer resistance to herbicides, such as L-phosphinothricin. The nucleotide sequence of a PAT gene is provided in European application No. 0 242 246 to Leemans et al. Also DeGreef et al., Bio/Technology 7:61 (1989), describes the production of transgenic plants that express chimeric slash genes encoding PAT activity. Examples of genes that confer resistance to phenoxy propionic acids and cyclohexones, including sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall et al., Theon. Appl. Genet. 83:435 (1992). GAT genes capable of conferring resistance to glyphosate are described in WO 2005012515, to Castle et al. Genes that confer resistance to 2,4-D, fop and pyridyloxy auxin herbicides are described in WO 2005107437 and US Patent Application Serial No. 11/587,893.

[00028] Other herbicides can inhibit photosynthesis, including triazine (psbA and 1s+ genes) or benzonitrile (nitrilase gene). Przibila et al., Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. The nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. US No. 4,810,648 for Stalker and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441 and 67442. Cloning and expression of DNA encoding a glutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

[00029] For purposes of the present invention, selectable marker genes include, but are not limited to, genes encoding: neomycin phosphotransferase II (Fraley et al. (1986) CRC Critical Reviews in Plant Science, 4:1-25); cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 4250-4264); aspartate kinase; dihydrodipicolinate synthase (Perl et al. (1993) Bio/Technology, 11: 715718); tripartophane decarboxylase (Goddijn et al. (1993) Plant Mol. Bi. 22:907-912); dihydrodipicolinate synthase and desensitized aspartade kinase (Perl et al. (1993) Bio/Technology, 11:715-718); bar gene (Toki et al. (1992) Plant Physiol., 100:1503-1507 and Meagher et al. (1996) and Crop Sci., 36: 1367); tripartophane decarboxylase (Goddijn et al. (1993) Plant Mol. Biol. 22:907-912); neomycin phosphotransferase (NEO) (Southern et al. (1982) J. Mol. Appl. Gen. 1: 327; hygromycin phosphotransferase (HPT or HYG) (Shimizu et al. (1986) Mol. Cell Biol., 6:1074) ; dihydrofolate reductase (DHFR) (Kwok et al. (1986) PNAS USA 4552); phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J., 6:2513); 2,2-dichloropropionic acid dehalogenase (Buchanan - Wollatron et al. (1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (Anderson et al. US Patent No. 4,761,373; Haughn et al. (1988) Mol. Genet Genet. 221:266 ); 5-enolpyruvyl-shikimate-phosphate synthase (aroA) (Comai et al. (1985) Nature 317:741); haloarylnitrilase (Stalker et al., published PCT application WO87/04181); acetyl-coenzyme A carboxylase (Parker et al. al. (1990) Plant Physiol. 92:1220); dihydropteroate synthase (sul I) (Guerineau et al. (1990) Plant Mol. Biol. 15:127); and 32 kD photosynthetic polypeptide II (psbA) ( Hirschberg et al. (1983) Science, 222: 1346).

[00030] Also included are genes that encode resistance to: chloramphenicol (Herrera-Estrella et al. (1983) EMBO J., 2:987-992); methotrexate (Herrera-Estrella et al. (1983) Nature, 303:209213; Meijer et al. (1991) Plant Mol Bio, 16:807-820 (1991); hygromycin (Waldron et al. (1985) Plant Mol. Biol 5:103-108; Zhijian et al. (1995) Plant Science, 108: 219-227 and Meijer et al. (1991) Plant Mol. Bio 16:807820); streptomycin (Jones et al. (1987) Mol. Gen. Genet., 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res., 5:131-137); bleomycin (Hille et al. (1986) Plant Mol. Biol. 7: 171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127-136); bromoxynil (Stalker et al. (1988) Science, 242:419-423); 2,4-D ( Streber et al. (1989) Bio/Technology, 7: 811-816); glyphosate (Shaw et al. (1986) Science, 233:478-481); and phosphinothricin (DeBlock et al. (1987) EMBO J., 6:2513-2518). All references cited in the disclosure are incorporated herein by reference in their entirety, unless otherwise indicated.

[00031] The above list of selectable marker and reporter genes is not intended to be limiting. Any reporter or selectable marker gene is encompassed by the present invention. If necessary, these genes can be sequenced by methods known in the art.

[00032] Selectable marker and reporter genes are synthesized for optimal expression in the plant. That is, the gene's coding sequence was modified to increase expression in plants. The synthetic marker gene is designed to be expressed in plants at a higher level, resulting in greater transformation efficiency. Methods for synthetic optimization of genes are available in the art. In fact, several genes have been optimized to increase gene product expression in plants.

[00033] The marker gene sequence can be optimized for expression in a particular plant species or, alternatively, it can be modified for optimal expression in plant families. Preferred plant codons can be determined from the highest frequency codons in the proteins expressed in greatest quantities in the specific plant species of interest. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. academic Sci. USA, 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research, 17: 477-498; Pat. US No. 5,380,831; and Pat. No. 5,436,391, incorporated herein by reference. In this way, nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence can be optimized or synthetic. That is, fully optimized or partially optimized sequences can also be used.

[00034] Furthermore, several transformation strategies using the Agrobacterium-mediated transformation system have been developed. For example, the binary vector strategy is based on a two-plasmid system where the T-DNA is on a different plasmid than the rest of the Ti plasmid. In a cointegration strategy, a small portion of the T-DNA is placed on the same vector than the foreign gene, whose vector subsequently recombines with the Ti plasmid.

[00035] As used herein, the expression "plant" includes dicotyledonous plants and monocotyledonous plants. Examples of dicotyledonous plants include tobacco, Arabidopsis, soybean, tomato, papaya, canola, sunflower, cotton, alfalfa, potato, vine, pigeon pea, pea, Brassica, chickpea, beet, rapeseed, watermelon, melon, pepper, peanut, pumpkin , radish, spinach, zucchini, broccoli, cabbage, carrots, cauliflower, celery, bok choy, cucumber, eggplant and lettuce. Examples of monocot plants include corn, rice, wheat, sugar cane, barley, rye, sorghum, orchids, bamboo, banana, cattail, lilies, oats, onion, millet, and triticale.

[00036] As used herein, the term "plant" includes an entire plant and any descendant, cell, tissue or part of a plant. The term "plant parts" includes any part(s) of a plant, including, for example, and without limitation: seed (including mature seed and immature seed), a plant cutting: a plant cell; a plant cell culture; a plant organ (e.g. pollen, embryo, flowers, fruits, twigs, leaves, roots, stems and explants). A plant tissue or plant organ may be a seed, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant containing the physiological and morphological characteristics of the plant from which the cell or tissue was obtained and of regenerating a plant with significantly the same plant genotype. In contrast, some plant cells are not capable of being regenerated to produce plants. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, root tips, hairs, flowers, seeds, cobs, cobs, barks or stalks.

[00037] Parts of plants include parts that can be harvested and parts useful for propagating progeny plants. Plant parts useful for propagation include, for example, and without limitation: seed; fruit; a cut; a seedling; a bulb and a rhizome. A harvestable part of a plant may be any useful part of a plant, including, for example, and without limitation: flower; pollen; changes; bulb; sheet; stalk; fruit; seed and root.

[00038] As used herein, the expression "described plant cell" or "transformed plant cell" refers to a plant cell that is transformed with stably integrated, non-natural, recombinant DNA, for example, by Agrobacterium-mediated transformation or by bombardment using microparticles coated with recombinant DNA or other means. A plant cell of this invention may be an originally transformed plant cell that exists as a microorganism or as a progenic plant cell that is regenerated into differentiated tissue, for example, in a transgenic plant with stably integrated non-natural recombinant DNA or seed. or pollen derived from a progenic transgenic plant.

[00039] As used herein, the term "consensus sequence" refers to an artificial amino acid sequence in a conserved region of an alignment of amino acid sequences of homologous proteins, for example, as determined by a CLUSTALW alignment of the amino acid sequence of homologous (functional) proteins.

[00040] As used herein, the term "homolog" refers to a nucleic acid or a protein in a group of proteins that perform the same biological function, for example, proteins that belong to the same protein family or similar nucleic acids that provide an increased characteristic common in transgenic plants of the present invention. Homologs are expressed by homologous genes. With reference to homologous genes, homologs include orthologs, that is, genes expressed in different species that evolved from common ancestral genes by speciation and encode proteins that retain the same function, but do not include paralogs, that is, genes that are related by duplication, but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially created variants. Degeneracy of the genetic code provides the possibility of replacing at least one base of the protein coding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be altered. When optimally aligned, homologous genes have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity along the entire length of the gene identified as being associated with conferring an increased trait when expressed in plant cells. In one aspect of the invention, the homologous genes have a nucleic acid or amino acid sequence similarity that has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with a consensus sequence of proteins, nucleotides and homologues disclosed here.

[00041] Homologs can be identified by amino acid sequence comparison, for example, manually or through a computer-based tool using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA and Smith-Waterman . A local sequence alignment program, (e.g. Basic Local Alignment Search Tool (BLAST)) can be used to search a sequence database to find similar sequences and the Summary Expectation value (E value) used to measure the base similarity of the sequence. Because a protein hit with the best E-value for a particular organism may not necessarily be an ortholog, i.e., have the same function, or be the only ortholog, a reciprocal analysis is used to filter out sequences of hits with significant E-values for orthological identification. Reciprocal analysis involves searching for significant hits against a database of amino acid sequences from the base organism that are similar to the analysis protein sequence. A hit can be identified as an ortholog when the best bit of the reciprocal analysis is the analysis protein itself or a protein encoded by a gene duplicated after speciation. Another aspect of the DNA-encoded homologs useful in the transgenic plants of the invention are those proteins that differ from a disclosed protein as a result of the deletion or insertion of one or more amino acids in a native sequence.

[00042] As used herein, the expression "percent identity" or "% identity" refers to the extent to which DNA sequences or protein segments are invariant over a sequence alignment window, e.g., nucleotide sequences or amino acid sequences. An "identity fraction" for a sequence aligned with a reference sequence is the number of identical components that are shared by the sequences, divided by the length of the alignment, not including gaps introduced by the alignment algorithm. "Percent identity" is the fraction of the identity times 100. The percent identity is calculated over the aligned length, preferably using a local alignment algorithm, for example, BLASTp.

[00043] As used herein, the expression "functional activity" or "functionally active" refers to proteins/enzymes for use in accordance with the present invention that have the ability to provide stress tolerance, which can result in increased performance. Transfer of functional activity to plant or bacterial systems may involve a nucleic acid sequence, encoding the amino acid sequence for a protein of the present invention, integrated into a protein expression vector appropriate for the host in which the vector will reside. One way to obtain a nucleic acid sequence that encodes a protein with functional activity is to isolate the native genetic material of the bacterial species that produce the protein of interest, using information deduced from the amino acid sequence of the protein, as disclosed herein. Native sequences can be optimized for expression in plants. Optimized polynucleotides can also be designed based on the protein sequence.

[00044] As used herein, the terms "control sequences" and "regulatory sequences" are interchangeable and refer to nucleic acid sequences useful for gene expression/transcription in plants. "Control sequences" or "regulatory sequences" may include, but are not limited to, promoters, operators, enhancers, origins of replication, ribosome binding sites, termination signals and polyadenylation.

[00045] As used herein, the term "promoter" refers to regulatory DNA to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells, whether or not its origin is a plant cell, for example, it is well known that Agrobacterium T-DNA promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses and bacteria, such as Agrobacterium and Bradyrhizobium bacteria. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, including leaves, roots, or seeds. These promoters are referred to as “tissue preferred.” Promoters that initiate transcription only in certain tissues are referred to as "tissue specific". A "cell type" specific promoter primarily induces expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "repressible" promoter is a promoter that is under environmental or chemical control. Examples of environmental conditions that can affect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue-specific, tissue-preferential, cell type-specific, and inducible promoters belong to the class of “nonconstitutive” promoters. A "constitutive" promoter refers to a promoter that is active under most conditions and in most tissues.

[00046] Numerous promoters that are active in plant cells have been previously described. These include promoters present in plant genomes, as well as promoters from other sources, including the nopaline synthase (NOS) and octopine synthetase (OCS) promoters carried on tumor-inducing plasmids from Agrobacterium tumefaciens and the CaMV35S promoters from Agrobacterium tumefaciens virus. cauliflower mosaic, as disclosed in US Patents 5,164, 316 and 5,322,938. Useful promoters derived from plant genes are found in US Patent No. 5,641,876, which describes a rice actin promoter; US Patent No. 7,151,204, which discloses a corn chloroplast aldolase promoter and a corn aldolase promoter (FDA); and US Patent Application Publication 2003/0131377 disclosing a corn nicotianamine synthetase promoter. These and numerous other promoters that function in plant cells are known to those skilled in the art and are available for use in the recombinant polynucleotides described herein to provide expression of the desired genes in transgenic plant cells.

[00047] Additionally, promoters can be altered to contain multiple "enhancer sequences" to help elevate gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, expression of the selected protein can be improved. These enhancers are often found 5' to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted upstream (5') or downstream (3') of the coding sequence. In some cases, these 5' enhancer elements are introns. Particularly useful as enhancers are the rice actin 1 5' introns (see, e.g., U.S. Patent No. 5,641,876) and the rice actin 2 genes, the intron(s) of the dehydrogenase gene. of corn alcohol, the intron of the corn heat shock protein 70 gene, e.g., U.S. Patent No. 5,593,874) and the corn shrunken gene 1 intron. See also US patent application publication 2002/0192813A1, which describes 5', 3' and intron elements useful in designing effective plant expression vectors.

[00048] In some embodiments, sufficient expression in plant seed tissues is desired to affect improvements in seed composition. Exemplary promoters for use in modifying seed composition include seed gene promoters such as napin, as disclosed in U.S. Patent No. 5,420,034, corn oleosin L3, as disclosed in U.S. Patent No. 6,433,252), zein Z27, as disclosed by Russell et al. (1997) Transgenic Res. 6(2): 157-166), globulin 1, as disclosed by Belanger et al (1991) Genetics 129:863-872), glutelin 1, as disclosed by Russell (1997, supra) and antioxidant peroxiredoxin (Perl), as disclosed by Stacy et al. (1996) Plant Mol Biol. 31(6):1205- 1216.

[00049] As used herein, the term "operably linked" refers to the association of two or more DNA fragments into a recombinant DNA construct, such that the function of one, e.g., protein-coding DNA, is controlled by the another, for example, a prosecutor.

[00050] Recombinant DNA constructs are assembled using methods well known to those skilled in the art and typically comprise a promoter operably linked to DNA, the expression of which provides the improved agronomic characteristic. Other building blocks may include additional regulatory elements, including 5' leaders and introns to enhance transcription, 3' untranslated regions (e.g., signals and polyadenylation sites), and DNA for transit or signal peptides.

[00051] The recombinant DNA constructs described here also generally include a 3' element that typically contains a signal and polyadenylation site. Well-known 3' elements include those of Agrobacterium tumefaciens genes, such as nos 3', tml 3', tmr 3', tms 3', ocs 3', tr73', for example, disclosed in US Patent No. 6,090. 627; 3' elements of plant genes such as wheat (Triticum aesevitum) heat shock protein 17 (Hspl 73), a wheat ubiquitin gene, a wheat fructose-1,6-bisphosphatase gene, a rice glutelin gene, a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all of which are disclosed in US Patent Application Publication No. 2002/0192813; and the pea (Pisum sativum) ribase bisphosphate carboxylase gene (rbs 3') and 3' elements of the genes within the host plant.

[00052] The constructs and vectors may also include a transit peptide for directing a gene to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see, for example, US Patent No. 5,188,642 and US Patent No. 5,728,925.

[00053] As used herein, the term "expressed" refers to a protein that is expressed or produced in a plant cell when its cognate DNA is transcribed into mRNA that is translated into protein. As used herein, the term "suppressed" refers to decreased expression or activity of a protein. Typically, a protein is suppressed in a plant cell when there is a decrease in the amount and/or activity of the protein in the plant cell. The presence or activity of the protein may be decreased by any amount, up to and including a total loss of expression and/or activity of the protein.

[00054] As used herein, the term "characteristic" refers to a physiological, morphological, biochemical or physical characteristic of a particular plant or plant material or cell. In some cases, this characteristic is visible to the human eye, including seed or plant size, or can be measured by biochemical techniques, including detecting the protein, starch or oil content of the seed or leaves, or by observing a metabolic process or physiological, (e.g., by measuring carbon dioxide absorption), or by observing the expression level of a gene or genes (e.g., using Northern analysis, RT-PCR, microarray gene expression assays), or reporter gene expression systems, or by agricultural observations including stress tolerance, yield or pathogen tolerance.

[00055] As used herein, the term "overexpression" refers to a greater level of expression of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any stage developmental or temporal for the gene. Overexpression can occur when, for example, genes are under the control of a strong expression signal, including one of the promoters described here or the cauliflower mosaic virus 35S transcription initiation region known in the art. Overexpression can occur throughout a plant or in specific plant tissues, depending on the promoter used.

[00056] As used herein, a "non-genic sequence" or "non-genic genomic sequence" is a native DNA sequence found in the nuclear genome of a plant, having a length of at least 1 Kb and devoid of any open reading frames , gene sequences or gene regulatory sequences. Furthermore, the non-genic sequence does not comprise any intronic sequences (i.e., introns are excluded from the definition of non-genic). The non-genic sequence cannot be transcribed or translated into protein. Many plant genomes contain nongenic regions, where up to 95% of the genome may be nongenic and these regions may be composed primarily of repetitive DNA.

[00057] As used herein, a "gene region" is defined as a polynucleotide sequence comprising an open reading frame encoding an RNA and/or polypeptide. The gene region may also encompass any identifiable adjacent 5' and 3' non-coding nucleotide sequences involved in regulating expression of the open reading frame up to approximately 2 kb upstream of the coding region and 1 kb downstream of the coding region, but possibly further. upstream or downstream. A genic region further includes any introns that may be present in the genic region. Furthermore, the genic region may comprise a single gene sequence, or multiple gene sequences interspersed with short gaps (less than 1 Kb) of non-gene sequences.

[00058] As used herein, the term "hypomethylation" or "hypomethylated", in reference to a DNA sequence, defines a reduced state of methylated DNA nucleotide residues in a given DNA sequence. Typically, decreased methylation relates to the number of methylated adenine or cytosine residues, relative to the average level of methylation found in non-genic sequences present in the genome of a plant, for example, soybean or corn.

[00059] The term "in close proximity to a genic region", when used in reference to a non-genic sequence, defines the relative location of the non-genic sequence to a genic region. Specifically, the number of genic regions within a 40 Kb neighborhood (i.e., within 40 Kb at each end of the selected optimal soybean genomic loci sequence) is analyzed. The number of gene regions can vary from a minimum of 1 gene to a maximum of 18 genes within the 40 Kb neighborhood.

[00060] The term "recombination evidence", as used herein, refers to the frequencies of meiotic recombination between any pair of genomic markers across a chromosomal region comprising the selected sequence. Recombination frequencies were calculated based on the ratio of the genetic distance between markers (in centimorgan (cM)) over the physical distance between markers (in megabases (Mb)). For a selected sequence to have evidence of recombination, the selected sequence must contain at least one recombination event between two markers flanking the selected sequence, as detected using a high-resolution marker dataset generated from multiple mapping populations.

[00061] As used herein, the term "relative location value" is a calculated value that defines the distance of a genomic locus from its corresponding chromosomal centromere. For each selected sequence, the genomic distance from the native location of the selected sequence to the centromere of the chromosome on which it is located is measured (in Bp). The relative location of the selected sequence within the chromosome is represented as the ratio of its genomic distance to the centromere relative to the length of the specific chromosomal arm (measured in Bp) on which it is found. These relative location values for the ideal non-genic genomic loci can be generated for different plants, the relative location values for the data set can range from a minimum ratio of 0 to a maximum of 0.99682 genomic distance.

[00062] The term "exogenous DNA sequence", as used herein, is any nucleic acid sequence that has been removed from its native location and inserted into a new location, altering the sequences flanking the nucleic acid sequence that has been moved. For example, an exogenous DNA sequence may comprise a sequence from another species.

[00063] "Recombination" refers to a process of exchanging genetic information between two polynucleotides, including, but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination. As used herein, "homologous recombination (HR)" refers to the specialized form of such exchange that occurs, for example, during the repair of double-strand breaks in cells via homology-directed repair mechanisms. This process requires homology of nucleotide sequences, uses a "donor" molecule to repair the template of a "target" molecule (i.e., the nucleotide sequence that has undergone the double-strand break), and is variously known as "uncross-linked gene conversion ", or "short trait gene conversion", because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve correction of heteroduplex DNA mismatches that form between the broken target and the donor and/or "synthesis-dependent strand annealing", in which the donor is used to resynthesize genetic information that will become part of the target and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide. For HR-directed integration, the donor molecule contains at least 2 regions of homology to the genome ("homology arms") of at least 50-100 base pairs in length. See, for example, U.S. Patent Publication No. 20110281361, the contents of which are incorporated herein by reference.

[00064] As used here, the term "gene expression" refers to the conversion of information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as closure, polyadenylation, methylation and editing and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, myristylation and glycosylation.

[00065] As used herein, the term "substantially homologous" or "substantial homology", with respect to a contiguous nucleic acid sequence, refers to contiguous nucleotide sequences that hybridize under stringent conditions to a reference nucleic acid sequence. For example, nucleic acid sequences that are substantially homologous to a reference nucleic acid sequence are those nucleic acid sequences that hybridize under stringent conditions (e.g., the Moderate Stringency conditions defined above) to the reference nucleic acid sequence. . Substantially homologous sequences may have at least 80% sequence identity. For example, substantially homologous molecules may contain from about 80% to 100% sequence identity, such as about 81%; around 82%; around 83%; around 84%; about 85%; around 86%; around 87%; around 88%; around 89%; about 90%; around 91%; about 92%; around 93%; about 94% about 95%; about 96%; about 97%; about 98%; around 98.5%; about 99%; around 99.5%; and about 100%. The property of substantial homology is closely related to specific hybridization. For example, a nucleic acid molecule is especially hybridizable when there is a sufficient degree of complementarity to prevent non-specific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired, for example, under stringent hybridization conditions.

[00066] In some cases, "homologous" may be used to refer to the relationship of a first gene to a second gene by descent from a common ancestral DNA sequence. In these cases, the term homologous indicates a relationship between the genes separated by the speciation event (see ortholog) or the relationship between the genes separated by the gene duplication event (see paralog). In other cases, "homologous" may be used to refer to the level of sequence identity between one or more polynucleotide sequences, in such cases the one or more polynucleotide sequences do not necessarily descend from a common ancestral DNA sequence. Those skilled in the art are aware of the meaning of the term "homologue" and appreciate the proper application of the term.

[00067] Although the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes can be made without departing from the invention. All publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies that may be used in connection with the invention. All cited patents, patent applications and sequence information on cited websites and public databases are also incorporated by reference.

EXAMPLES Example 1

[00068] In accordance with the methods and/or systems provided herein, database data and/or maps can be used to evaluate or predict the performance and/or stability of transgenes through local chromatin state profiling, in particular the abundance of H3K9me2, in the vicinity of the locus inserted by transgenes. As predicted here, a threshold value can be assigned to predict the potential risk of transgene silencing for transgene integration occurring with genomic regions highly enriched in H3K9me2. To assess local chromatin structure, a genome-wide histone3 lysine (K) 9 dimethylation (H3K9me2) map in soybean (Glycine maxcv Maverick is constructed. H3K9me2 are frequently observed within the silenced heterochromatic regions, including centromere and telomere-carrying repetitive sequences This H3K9me2 genome map allows prediction of chromatin structure (open or compacted) at any locus in the soybean genome and provides an assessment of silencing potential.

[00069] Shoot and root tissues of soybean (Glycine max) cv Maverick are collected at stage v1 (~ 12 days after planting) and then cross-linked using 1% formaldehyde. The cross-linked tissues are used for the chromatin immunoprecipitation (ChIP) assay using the H3K9me2 antibody (Millipore 17-648), in which normal rabbit IgG (Millipore Cat. 12-370) is used as a negative control. Three independent ChIP assays of each tissue are performed and the ChIPated DNA is prepared for sequencing using HiSeq. Sequencing reads are listed in Table 1.

[00070] Analysis of ChIP-Seq data - The raw sequencing data from the H3K9me2 ChIP-seq experiments are aligned using Bowtie to the Glycine Max Williams82 genome. The MACS (ChIP-seq model-based analysis) peak finding algorithm can be used to identify regions of H3K9me2 enrichment in background, where a "peak" represents a genomic stretch that is predicted to be enriched for H3K9me2 compared to its genomic neighborhood. Conversely, a "ridge" represents the genomic location that has the greatest enrichment within a predicted "peak".

[00071] According to this example, ChIP-seq data is consistent between technical replicates. To reduce variation and noise resulting from ChIP-seq data, three technical replicates of ChIP-seq are performed individually. To examine whether the ChIP pull-downs from each round are consistent and reflect genuine H3K9me2 modification in the soybean genome, the pull-down from each technical representative can be compared. Among the three technical replicates, the ChIP-seq data are consistent with each other, indicating that the pull-downs are consistent across each round of ChIP experiments. Therefore, the CHIP-seq protocols used in this example are highly replicable in root and shoot technical replicate samples.

Example 2

[00072] H3K9me2 enrichment is associated with heterochromatic regions and gene silencing - H3K9me2 is associated with heterochromatin (compact chromatin structure) and one of the highly conserved histone marks responsible for transcriptional repression in eukaryotes. To determine whether H3K9me2 is associated with heterochromatic region and gene silencing in soybean, H3K9me2 enrichment within the genic region (measured as the number of predicted H3K9me2 "peaks" within a genic region - greater number of peaks represents greater extent of H3K9me2 enrichment) H3K9H3K9me2) is investigated and compared with the expression level of the corresponding gene (measured using RNAseq technology, in normalized read count units). Here, we observed that gene regions with zero or one H3K9me2 peak show a significant range of gene expression. On the other hand, gene regions with more than two H3K9me2 peaks are associated with relatively low levels of gene expression. This indicates that the higher enrichment of H3K9me2 is highly associated with the lower levels of gene expression in soybean.

[00073] In addition to understanding the content of H3K9me2 in genic regions, we identified higher levels of H3K9me2 in transposable elements that are often associated with highly repetitive and transcriptionally repressed regions within the genome. We compared the distribution of H3K9me2 enrichment between transposable elements and genes in the soybean genome. On average, the H3K9me2 enrichment of most transposon elements is much higher than that of normal genes. These data further demonstrate that H3K9me2 enrichment is highly associated with the repressive status of the soybean genome.

[00074] Based on our understanding of how H3K9me2 abundance is associated with gene expression, we categorize genes based on their expression levels into five different expression categories - High, High Medium, Medium, Low Medium and Low - and select a representative number of 500 genes from each category (see Figure 1). For each gene within each category, the abundance of H3K9me2 in the gene region was calculated. The gene region is then divided into three sections - gene body, 2kb upstream and 1kb downstream. A mean value for the abundance of H3K9me2 within each section is plotted for each expression category in Figure 1. The High and Medium High expression categories can be differentiated from the Low and Medium Low expression categories based on the abundance of H3K9me2 in the three sections. genes. This provides unique insight and a statistical model of how H3K9me2 data can be used to predict the expression level for a specific transgene of interest. Together, the above data indicated that H3K9me2 is a conserved repressive histone mark in soybean, as well as the abundance of H3K9me2 is associated with the heterochromatic region and transcriptional repression in soybean. We conclude that a threshold is linked to acceptable expression levels, where the gene region has a peak from H3K9me2 profiling data mapped to soybean genomes. According to this example, a database for the soybean genome is established based on H3K9me2 profiling data.

Example 3

[00075] Assessment of H3K9me2 enrichment using soybean key events to validate prediction of expression levels based on H3K9me2 profiling data. To evaluate the practical use of applying the H3K9me2 map as a tool to assess the potential risk of transgene silencing, nine major soybean events from the Enlist and Insect Resistance constructs are selected and listed in Table 2. insertion of these main events are already known, the enrichment profiles of H3K9me2 in the neighborhood (5kb) of the inserted site in the transgene are evaluated to predict the risk of gene silencing. As all key events are selected based on stable transgene expression and stability, the transgene is expected to be inserted into a locus with low H3K9me2 enrichment (peak no. < 1). In the 5kb window of the site inserted per transgene among nine distinct events, six events showed zero peak and three events showed an H3K9me2 peak. Low H3K9me2 enrichment corresponds to stable transgene expression. This indicates that H3K9me2 profiling provides an effective tool for predicting the risk of gene silencing.

Claims (7)

1. Method for predicting the risk of transgene silencing, characterized by the fact that it comprises: (a) generating histone methylation/acetylation profile data in the genome of a plant from a physical assay; (b) assembling the histone methylation/acetylation profile data from step (a) into a histone methylation/acetylation database that shows the peak numbers of the histone methylation/acetylation profiles; (c) analyze sequences from at least one transgene insertion site from a transgenic event; (d) compare the transgene insertion site sequences from step (c) with the histone methylation/acetylation database from step (b); whereby (i) if the transgene insertion site falls into peak zero or peak one of the histone methylation/acetylation profiles, there is no risk of transgene silencing, or (ii) if the transgene insertion site falls into peak two or more of histone methylation/acetylation profiles, there is significant risk of transgene silencing; (e) select the transgenic event with the lowest probability of transgene silencing; and (f) obtaining a plant comprising the selected transgenic event. 2. Method according to claim 1, characterized by the fact that histone methylation/acetylation profile data is generated using a chromatin immunoprecipitation sequencing (ChIP-seq) assay. 3. Method according to claim 1, characterized by the fact that the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K4me2, H3K4me3, H3K9/14ac, H3K9me2, H3K9me3 , H3K27me1, H3K27me3 and H4K20me3 and combinations thereof. 4. Method according to claim 1, characterized by the fact that the histone methylation/acetylation profile data is associated with histone methylation/acetylation selected from the group consisting of H3K9me2, H3K9me3, H3K27me1, H3K27me3 and H4K20me3 and combinations of the same. 5. Method according to claim 1, characterized by the fact that the histone methylation/acetylation profile data is associated with H3K9me2 methylation. 6. Method, according to claim 1, characterized by the fact that the plant is selected from soybeans, corn, canola, cotton, wheat and rice. 7. Method according to claim 1, characterized by the fact that the transgene insertion site sequences are obtained by direct sequencing.