The CYP81E gene confers herbicide resistance.

Abstract

The present disclosure relates to plants or plant parts comprising a polynucleotide encoding a CYP81E polypeptide, wherein expression of the polynucleotide confers tolerance to a synthetic auxin herbicide, such as 2,4-D, on the plant or plant part. The present disclosure further provides kits for identifying herbicide-resistant plants and methods for determining whether a plant is herbicide-resistant.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Application No. 63 / 073,276, filed on 1 September 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on 26 August 2021, is named P13673WOOO_ST25.txt and is 69,987 bytes in size.

[0003] This disclosure, as a whole, relates to compositions and methods for conferring resistance to herbicides to plants. [Background technology]

[0004] Uncontrolled and unchecked weeds can reduce the yields of several major crops by more than 50% in the current North American agricultural system. While many growers in the United States now rely heavily on chemical means (i.e., herbicides) to control weed populations, the effectiveness of this approach is gradually declining due to the increasing number of herbicide-resistant weeds. Herbicide resistance has existed in the United States since the late 1950s, but the widespread adoption of herbicide-tolerant crop varieties in the mid-1990s, along with over-reliance on one or two modes of herbicide action, has led to an exponential increase in the number of resistant weed species over the past two decades. Currently, there are 164 weed species in the United States, with documented resistance to one or more herbicides.

[0005] Understanding how weeds cope with herbicide compounds and avoid damage is a primary goal of weed science, both for developing countermeasures to combat herbicide resistance and for gaining insights into plant evolution. Studies on herbicide resistance mechanisms over the past few decades have largely focused on mutations occurring within genes encoding target enzymes directly inhibited by herbicides (target-site resistance). More recently, significant progress has been made in non-target-site resistance (NTSR) mechanisms, primarily due to the increased availability of high-throughput whole-genome / transcriptome analyses. This research strongly identifies enhanced herbicide metabolism as a major NTSR pathway, but resistance mechanisms including translocation reduction and vacuolar sequestration have also been reported. The broad use of herbicides for weed control provides an excellent platform for studying the rapid adaptation of plants to aggressive selection and for addressing evolutionary problems that are becoming increasingly manageable with advances in genomics.

[0006] Amaranthus tuberculatus is a highly problematic weed for growers throughout the Midwestern United States, due to both its high reproductive rate and its ability to readily evolve resistance to herbicides. Since the 1993 report on ALS (acetolactate synthase) inhibitor resistance in Amaranthus tuberculatus, the species has accumulated resistance to herbicides across six additional sites of action. A population discovered in Illinois in 2016 carried five modes of resistance, including resistance to photosystem II inhibitors, PPO (protoporphyrinogen oxidase) inhibitors, HPPD (4-hydroxyphenylpyruvate dioxygenase) inhibitors, and synthetic auxins. Two of the resistance traits (ALS and PPO) were found to be caused by mutations in the target sites, but the resistance mechanisms for both HPPD inhibitors and synthetic auxins remained unclear. In 2012, the population reported from Nebraska was subsequently determined to be highly resistant to 2,4-D and also tolerant to HPPD inhibitor herbicides.

[0007] Herbicide-resistant plants are useful in systems where multiple such plants are planted and crops can be produced by them, either before or after planting, when herbicides that would normally kill or harm the plants are applied due to the plants' resistance to the herbicide. Undesirable plants die or are damaged, while resistant plants survive. It is necessary to create such plants. [Overview of the project]

[0008] Compositions and methods for conferring herbicide resistance to plants, plant parts, and plant cells are provided. A modified plant having resistance to herbicides is provided, the modified plant comprising improved expression of a polynucleotide encoding the cytochrome P450 81E (CYP81E) polypeptide compared to an unmodified plant. In certain embodiments, the modified plant comprises a heterologous polynucleotide encoding the CYP81E polypeptide. Offspring of the modified plant, plant parts, and plant cells are also provided.

[0009] (a) A nucleotide sequence encoding a CYP81E polypeptide, wherein the nucleotide sequence has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1; or (b) A nucleotide sequence encoding a CYP81E polypeptide, wherein the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2, A nucleic acid molecule is provided that can confer herbicide resistance, comprising a nucleotide sequence selected from the above.

[0010] Expression cassettes, vectors, biological samples, plants, plant parts, and plant cells containing the aforementioned nucleic acid molecules are also provided.

[0011] A CYP81E polypeptide is provided that contains an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0012] A method is provided for producing plants with herbicide resistance, comprising increasing the expression of a polynucleotide encoding the CYP81E polypeptide in a plant, wherein the herbicide resistance of the plant is improved compared to a plant lacking the improved expression. In a particular embodiment, the method comprises introducing a polynucleotide encoding the CYP81E polypeptide into a plant cell, wherein the polynucleotide is operably linked to a functional heterologous promoter in the plant cell; and regenerating a plant from the plant cell.

[0013] A method for controlling undesirable vegetation in plant cultivation sites, To provide plants containing polynucleotides encoding the CYP81E polypeptide at plant cultivation sites, wherein the expression of the polynucleotide confers herbicide resistance to the plants; A method is provided which includes applying an effective amount of herbicide to a plant cultivation site.

[0014] A method for controlling the growth of herbicide-resistant weeds in plant cultivation sites, Contacting weeds with a composition containing a polynucleotide that reduces the expression or activity of the CYP81E polypeptide; Applying an effective amount of herbicide to the plant cultivation site, A method including this is provided.

[0015] Products prepared from the aforementioned plants, plant parts, and plant cells are provided, each containing a polynucleotide encoding a CYP81E polypeptide. A method for producing a plant product is also provided, comprising processing the aforementioned plant or plant part to obtain the plant product, wherein the plant product comprises a polynucleotide encoding a CYP81E polypeptide.

[0016] A method for identifying herbicide-resistant plants, Prepare biological samples derived from plants that are suspected to have herbicide resistance; The purpose is to quantify the expression of the CYP81E gene in biological samples, specifically by quantifying the differential expression of the CYP81E gene in herbicide-resistant plants compared to herbicide-sensitive plants of the same species; A method is provided that includes determining whether a plant is herbicide resistant based on quantification.

[0017] A kit for identifying herbicide-resistant plants, A kit is also provided which includes at least two primers, wherein at least two primers recognize the CYP81E gene that is differentially expressed in herbicide-resistant plants compared to herbicide-sensitive plants of the same species.

[0018] Although several embodiments are disclosed, further embodiments of the present invention will become apparent to those skilled in the art from the following modes for carrying out the invention, which illustrate and describe exemplary embodiments of the present invention. Therefore, the drawings and modes for carrying out the invention should be considered as illustrative and not limiting in nature.

[0019] The following drawings form a part of this specification and are included to further demonstrate certain specific embodiments or various aspects of the present invention. In some cases, embodiments of the present invention can be best understood by referring to the attached drawings in combination with the forms for carrying out the invention presented in this specification. The forms for carrying out the invention and the attached drawings may emphasize certain specific examples or certain aspects of the present invention. However, those skilled in the art will understand that some parts of those examples or aspects may also be used in combination with other examples or aspects of the present invention.

Brief Description of the Drawings

[0020] [Figure 1] Figure 1 is a schematic diagram of the experimental design. Within each F2 population, plants were cloned and sprayed with high and low amounts of tembotrione or 2,4-D. Based on their responses, each plant was grouped into one of four categories: RR, resistant to both 2,4-D and tembotrione; RS, resistant to 2,4-D and sensitive to tembotrione; SR, sensitive to 2,4-D and resistant to tembotrione; SS, sensitive to both 2,4-D and tembotrione. For RNA-seq analysis, the four most tolerant / sensitive plants were selected from each category. This enabled N = 8 comparisons between resistant and sensitive plants for each herbicide using only 16 plants per population. [Figure 2A] Figures 2A - B show sliding window graphs of significantly differentially expressed genes and significant SNPs. Figure 2A shows genes (DEGs) significantly differentially expressed between 2,4-D resistant and sensitive plants in CHR and NEB mapped on the Arabidopsis genome. Only genes with an FDR of 0.05 or less were considered significant. Figure 2B shows single nucleotide polymorphisms (SNPs) statistically different between 2,4-D resistant and sensitive plants in CHR and NEB mapped on the Arabidopsis genome. SNPs were called statistically significant if the PLINK analysis returned a corrected p-value of 0.05 or less. [Figure 2B]Figures 2A and 2B show sliding window graphs of significantly differentially expressed genes and significant SNPs. Figure 2A shows significantly differentially expressed genes (DEGs) between 2,4-D resistant and susceptible plants in the CHR and NEB mapped on the Aucuba genome. Only genes with an FDR of 0.05 or less were considered significant. Figure 2B shows statistically different single nucleotide polymorphisms (SNPs) between 2,4-D resistant and susceptible plants in the CHR and NEB mapped on the Aucuba genome. SNPs were considered statistically significant if the PLINK analysis returned a corrected p-value of 0.05 or less. [Figure 3A] Figures 3A and 3B show allele-specific expression of all SNPs in the scaffold 4 hotspot region in the NEB population (Figure 3A) and the CHR population (Figure 3B). The location of each SNP is given on the x-axis, and the results of the differential expression t-test (Benjamini-Hochberg adjusted p-value) between the R and S alleles are given above the bar for each locus. [Figure 3B] Figures 3A and 3B show allele-specific expression of all SNPs in the scaffold 4 hotspot region in the NEB population (Figure 3A) and the CHR population (Figure 3B). The location of each SNP is given on the x-axis, and the results of the differential expression t-test (Benjamini-Hochberg adjusted p-value) between the R and S alleles are given above the bar for each locus. [Figure 4] Figure 4 shows the phylogenetic tree of cytochrome P450 81E8 in arbitrary subsets of Amaranth populations from Illinois, Nebraska, Missouri, and Canada. Samples from this study are indicated by their population name ("CHR" or "NEB") and their 2,4-D phenotypic response. Samples beginning with a number or "N3" originated from Ontario, while samples beginning with "B", "F", "J", or "K" originated from Illinois and Missouri. [Modes for carrying out the invention]

[0021] Ayu (European aeruginosa) has evolved resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors in multiple states across the Midwestern United States. Two populations resistant to both mechanism groups—one from Nebraska (NEB) and the other from Illinois (CHR)—were studied using RNA-seq approaches to F2 mapping populations, and the genes responsible for resistance were identified.

[0022] To facilitate understanding of the present invention, certain terms are defined first. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which embodiments of the present invention belong. Many methods and materials similar, modified, or equivalent to those described herein can be used in carrying out embodiments of the present invention without excessive experimentation, and preferred materials and methods are described herein. In describing embodiments of the present invention and in claiming the invention, the following technical terms are used according to their definitions below.

[0023] It should be understood that all technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limited to any form or scope. For example, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” may refer to multiple subjects unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The word “or” means any one member of each individual list, and also includes any combination of members of such list. Furthermore, all units, prefixes, and symbols may be represented in their SI-certified form.

[0024] Numerical ranges described herein include the number defining the range and each integer within the defined range. Throughout this disclosure, various aspects of the invention are presented in range form. It should be understood that the use of range form is solely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, range descriptions should be considered to specifically disclose all possible subranges, fractions, and individual numbers within that range. For example, a range description such as 1 to 6 discloses subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, and decimals and fractions, such as 1.2, 3.8, 1. 1 / 2, and 4 3 / 4 should be considered as a specific disclosure. This applies regardless of the scope.

[0025] As used herein, the term “approximately” refers to any quantifiable variable, including but not limited to mass, volume, time, and temperature, that may occur through, for example, conventional measurement techniques and equipment. Furthermore, considering the solid and liquid processing procedures actually used, there are certain accidental errors and variations that may exist through differences in the manufacture, source, or purity of the components used to prepare the composition or to carry out the method. The term “approximately” also encompasses such variations. Whether modified by the term “approximately” or not, the claims include equivalents to quantities.

[0026] As used herein, the term “confer” means to give a plant a feature or trait, such as herbicide tolerance or resistance and / or other desirable traits.

[0027] The term “control of unwanted vegetation or weeds” should be understood to mean killing weeds and / or otherwise delaying or inhibiting their normal growth. Weeds, in their broadest sense, should be understood to mean all plants that grow in undesirable locations. Weeds in this disclosure include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the following genera: Sinapis, Lepidium, Gallium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, and Sesbania. ), Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum.Monocotyledonous weeds include, but are not limited to, weeds of the following genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, The genera include Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera. In addition, the weeds of this disclosure may include, for example, crops growing in undesirable locations. For example, native maize plants present in a field predominantly containing soybean plants can be considered weeds if maize plants are undesirable in a soybean field.

[0028] As used herein, the terms “DNA” or “DNA molecule” refer to a double-stranded DNA molecule of genomic or synthetic origin, read from the 5' (upstream) end to the 3' (downstream) end, i.e., a polymer or polynucleotide molecule of deoxyribonucleotide bases. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that in accordance with U.S. Patent Law Enforcement Rules §1.822 and as set forth in WIPO Standard ST.25 (1998), Annex 2, Tables 1 and 3.

[0029] As used herein, “endogenous gene” or “natural copy” of a gene means a gene that originates within a given organism, cell, tissue, genome, or chromosome. “Endogenous gene” or “natural copy” of a gene means a gene that has not been previously modified by human activity. Similarly, “endogenous protein” means a protein encoded by an endogenous gene.

[0030] As a whole, the term “herbicide” is used herein to mean an active ingredient that kills, controls, or otherwise adversely alters the growth of plants. A preferred amount or concentration of herbicide is an “effective amount” or “effective concentration.” “Effective amount” and “effective concentration” are intended to be amounts and concentrations sufficient to kill or inhibit the growth of similar wild-type plants, plant tissues, plant cells, or host cells, respectively, but such amounts will not kill or severely inhibit the growth of herbicide-resistant plants, plant tissues, plant cells, and host cells of the Disclosure. Typically, an effective amount of herbicide is the amount routinely used in an agricultural production system to kill the weed of interest. Such amounts are known to those skilled in the art. Herbicidal activity is what is exhibited by a herbicide useful in the Disclosure when applied directly to or to plants at any growth stage, or to the site of such plants, before planting or before germination. The observed effects depend on the plant species being controlled, the plant's growth stage, application parameters such as dilution and spray droplet size, particle size of the solid components, environmental conditions during use, the specific compound used, the specific adjuvants and carriers used, the soil type, and the amount of chemical applied. These and other factors can be adjusted, as is known in the art, to enhance non-selective or selective herbicidal activity. Generally, herbicidal treatments can be applied as PPI (pre-planting application), PPSA (post-planting surface application), or before or after germination. Post-germination treatment is typically applied to relatively immature, undesirable vegetation to achieve maximum weed control.

[0031] A “herbicide-tolerant” or “herbicide-resistant” plant is intended to be a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill or inhibit the growth of normal or wild-type plants. The level of herbicide that would normally inhibit the growth of non-tolerant plants is known and easily determined to those skilled in the art. An example is the amount recommended by the manufacturer for application. The maximum amount is an example of the amount of herbicide that would normally inhibit the growth of non-tolerant plants. In this disclosure, the terms “herbicide-tolerant” and “herbicide-resistant” are intended to be used interchangeably and have equivalent meanings and equivalent scopes. Similarly, the terms “herbicide-tolerant” and “herbicide-resistant” are intended to be used interchangeably and have equivalent meanings and equivalent scopes. Similarly, the terms “tolerant” and “resistant” are intended to be used interchangeably and have equivalent meanings and equivalent scopes. As used herein, terms such as "herbicide, etc." refer to agronomically acceptable herbicidal active ingredients (AIs) recognized in the art for herbicide compositions useful in the various embodiments described herein. As used herein, "herbicide-resistant trait" refers to a genetically modified trait that confers improved herbicide resistance to a plant compared to a wild-type plant.

[0032] In the context of inserting nucleic acids into cells, the term “introduced” means “transfection,” “transformation,” or “transduction,” and includes references to the incorporation of nucleic acids into eukaryotic or prokaryotic cells, in which case the nucleic acid may be incorporated into the cell’s genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0033] As used herein, the term “isolated DNA molecule” means a DNA molecule that is at least partially isolated from other molecules that would normally be associated with the DNA molecule in its native or natural state. In one embodiment, the term “isolated” means a DNA molecule that is at least partially isolated from some nucleic acids that would normally be adjacent to the DNA molecule in its native or natural state. Thus, a DNA molecule that has been fused to regulatory or coding sequences that are not normally associated, for example, as a result of a recombination technique, is considered isolated herein. Such molecules are considered isolated if they are integrated into the chromosomes of a host cell or exist in a nucleic acid solution with other DNA molecules, in that they are not in their native state.

[0034] As used herein, “modified” in the context of plants, seeds, plant components, plant cells, and plant genomes means a state that involves a change or alteration from their natural or native state. For example, the “native transcript” of a gene means the RNA transcript produced from an unmodified gene. Typically, a native transcript is a sense transcript. Modified plants or seeds contain molecular changes in their genetic material, including either genetic or epigenetic modifications. Typically, modified plants or seeds, or their parent or ancestral lineages, have been subjected to mutagenesis, genome editing (e.g., by methods using site-specific nucleases, but not limited to these), genetic transformation (e.g., by methods using Agrobacterium transformation or particulate guns, but not limited to these), or a combination thereof. In one embodiment, the modified plants provided herein contain no non-plant genetic material or sequences at all. In yet another embodiment, the modified plants provided herein contain no interspecies genetic material or sequences at all.

[0035] As used herein, “plant” means the whole plant, any part thereof, or a cell culture or tissue culture derived from a plant, and includes any of the whole plant, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissue, seeds, plant cells, and / or their offspring. Offspring plants may be derived from any hybrid generation, e.g., F1, F2, F3, F4, F5, F6, F7, etc. Plant cells are the biological cells of a plant, either taken from a plant or derived through a culture of cells taken from a plant.

[0036] As used herein, the term “polynucleotide” refers to a nucleic acid molecule containing multiple polymerized nucleotides, for example, at least about five consecutive polymerized nucleotides. A polynucleotide can be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. Often, a polynucleotide contains a nucleotide sequence encoding a polypeptide (or protein) or its domain or fragment. Additionally, a polynucleotide may contain a promoter, introns, enhancer regions, polyadenylation sites, translation initiation sites, 5' or 3' untranslated regions, a reporter gene, a selectable marker, etc. A polynucleotide can be single-stranded or double-stranded DNA or RNA. A polynucleotide may contain modified bases or modified backlines. A polynucleotide can be, for example, genomic DNA or RNA, a transcript (e.g., mRNA), cDNA, a PCR product, cloned DNA, synthetic DNA or RNA, etc. A polynucleotide can be combined with carbohydrates, lipids, proteins, or other substances to perform specific activities such as transformation, or to form useful compositions such as peptide nucleic acids (PNAs). A polynucleotide may contain sequences in either the sense or antisense direction. The term "oligonucleotide" is substantially equivalent to the terms amplimer, amplicon, primer, oligomer, element, target, and probe, and in some embodiments is single-stranded.

[0037] As used herein, the term “primer” encompasses any nucleic acid that can stimulate the synthesis of nascent nucleic acids in template-dependent processes such as PCR. Typically, primers are oligonucleotides of 10 to 30 nucleotides in length, but longer sequences may be used. Primers can be prepared in single-stranded or double-stranded form. Probes can be used as primers, but they are designed to bind to target DNA or RNA and do not need to be used in the amplification process.

[0038] As used herein, “promoter” includes references to DNA regions upstream of the transcription start site that are involved in the recognition and binding of RNA polymerase and other proteins that initiate transcription. A “plant promoter” is a promoter that can initiate transcription in plant cells, regardless of whether its origin is a plant cell or not. Exemplary plant promoters include, but are not limited to, those derived from plants, plant viruses, and bacteria such as Agrobacterium or Rhizobium, including genes expressed in plant cells. An example of a promoter under developmental control is a promoter that preferentially initiates transcription in a particular tissue, such as a leaf, root, or seed. Such promoters are called “tissue-preferential.” A promoter that initiates transcription only in a particular tissue is called “tissue-specific.” A “cell-type” specific promoter primarily drives expression in a particular cell type in one or more organs, for example, in vascular cells of roots or leaves. An “inducible” or “repressive” promoter is a promoter under environmental control. Examples of environmental conditions that can affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferential, cell-type-specific, and inducible promoters constitute a class of "non-constitutive" promoters. "Constitutive" promoters are those that are active under most environmental conditions.

[0039] As used herein, “recombinant” refers to nucleic acids or polypeptides, indicating that such substances have been modified as a result of the human application of a recombinant technique, such as by polynucleotide restriction and ligation, polynucleotide overlap extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if its nucleotide sequence is taken from its natural context and cloned into any type of artificial nucleic acid vector. The term recombinant can also refer to an organism that contains recombinant material; for example, a plant containing recombinant nucleic acid can be considered a recombinant plant.

[0040] A “regulatory element” refers to a nucleotide sequence located upstream (5' non-coding sequence), within it, or downstream (3' non-coding sequence) of a coding sequence, and which affects transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory elements may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences. Regulatory elements present on a recombinant DNA construct introduced into a cell may be endogenous to the cell or heterogeneous with respect to the cell. The terms “regulatory element” and “regulatory sequence” are used interchangeably herein.

[0041] A “sequence” means a continuous arrangement of nucleotides or amino acids. The boundaries of a protein-coding sequence can be determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. In some embodiments, a protein-coding molecule may include a DNA sequence that codes for a protein sequence. In some embodiments, a protein-coding molecule may include an RNA sequence that codes for a protein sequence. As used herein, “transgene expression,” “expressing a transgene,” “protein expression,” and “expressing a protein” mean the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain, which is ultimately folded into a protein.

[0042] As used herein, the terms “percentage of sequence identity” or “% sequence identity” refer to the percentage of identical nucleotides or amino acids in the linear polynucleotide or polypeptide sequence of a reference ("inquiry") sequence (or its complementary strand) compared to a test ("subject") sequence (or its complementary strand), such that the two sequences are optimally aligned (i.e., appropriate nucleotide or amino acid insertions, deletions, or gaps are less than 20% of the total of the reference sequence across the comparison window). The optimal alignment of sequences for aligning comparison windows is well known to those skilled in the art and can be performed, for example, using default parameters, by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, and the similarity search method of Pearson and Lipman, as well as by computer implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA, MEGAlign (DNAStar Inc., Madison, Wis.), and MUSCLE (version 3.6) [Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5):1792-7 (2004)], which are available as part of the sequence analysis software package of GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.). The "percentage of identity" of aligned segments of a test sequence and a reference sequence is calculated by dividing the number of identical components shared by the two aligned sequences in a portion of the aligned reference sequence segment—that is, in the entire reference sequence or a defined smaller portion of the reference sequence—by the total number of components. The percentage of sequence identity is expressed as the percentage of identity multiplied by 100. The comparison of one or more sequences may be against a full-length sequence or a portion thereof, or against a longer sequence.

[0043] As used herein, “synthetic auxin herbicide” or “auxin herbicide” means any herbicide that exerts herbicidal activity by mimicking endogenous plant auxins or inhibits the movement of auxin compounds from cells. Examples of synthetic auxin herbicides include benzoic acid, phenoxycarboxylic acid, pyridinecarboxylic acid, quinolinecarboxylic acid, semicarboazone, diflufenzopyr, 2,4-D, 2,4-DB, MCPA, MCPB, mecoprop, dicamba, clopyralide, fluroxypyr, picloram, triclopyr, aminopyralide, aminocyclopyrachlor, and quinchlorac.

[0044] As used herein, “vector” includes references to nucleic acids used in transfection of host cells into which polynucleotides can be inserted. Vectors are often replicons. Expression vectors enable the transcription of nucleic acids into which they are inserted.

[0045] CYP81E polynucleotide The plant hormone auxin functions as a central regulator of genes involved in numerous pathways of plant growth, development, and response. While indole-3-acetic acid (IAA) is the naturally occurring active auxin, many other compounds have been found to mimic the function of IAA when applied to plants. This has led to the identification and commercialization of several compounds that function as effective herbicides. While maize and other monocotyledonous crops are naturally tolerant to low levels of synthetic auxin herbicides, dicotyledonous crops such as soybeans and cotton are highly susceptible. Attempts to develop auxin herbicide-resistant varieties have focused on heterologous expression of enzymes that inactivate auxin herbicides, thereby making normally susceptible plants resistant to the herbicides.

[0046] Cytochrome P450 81E (CPY81E) sequences that confer herbicide resistance are provided. Such sequences include the amino acid sequence described in SEQ ID NO: 2, and its variants. Polynucleotide sequences encoding such amino acid sequences, including SEQ ID NO: 1, are also provided.

[0047] According to some embodiments, crops are transformed with a gene encoding the CPY81E polypeptide, which can inactivate a particular auxin herbicide, and may also be other types of herbicides.

[0048] Further polynucleotide sequences encoding the CPY81E polypeptide can be identified using methods well known in the art based on their ability to confer resistance to the herbicide of interest. For example, candidate CPY81E genes are transformed into suitable yeast strains, expressed in these strains, and selected based on their ability to oxidize test herbicides in vitro [see Siminszky et al (1999) PNAS (USA) 96:1750-1755]. Suitable yeast strains include WAT11 or WAT21, which also contain suitable plant cytochrome P450 competent reductases. After induction, cells are grown for an appropriate period (e.g., with galactose, depending on the inducible promoter used in the transformation vector), harvested, lysed, and microsomal fractions are prepared by conventional means and assayed with NADPH for their ability to oxidize 14C-labeled herbicides. The assay may be performed using whole cells in culture.

[0049] Alternatively, the CPY81E candidate gene may be expressed in tobacco, Arabidopsis, or other readily transformable herbicide-sensitive plants, and the resulting transformant plants may be evaluated for their resistance to auxin herbicides or other herbicides of interest. Plants or tissue samples taken from plants may be treated with herbicides and assays may be performed to assess the metabolic conversion rate of the parent herbicide to oxidative metabolic degradation products.

[0050] Those skilled in the art can also identify further CPY81E candidate genes based on genomic synteny and sequence similarity. In one embodiment, further gene candidates can be obtained by hybridization or PCR using sequences based on the CPY81E nucleotide sequences described above.

[0051] The PCR approach allows for the design of oligonucleotide primers for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are commonly known in the art. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY). Also see Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).

[0052] In hybridization techniques, all or part of a known polynucleotide is used as a probe, which selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA or cDNA fragments (i.e., a genomic library or cDNA library) from a selected organism. The hybridization probe can be a genomic DNA fragment, cDNA fragment, RNA fragment, or other oligonucleotide. 32They may be labeled with a detectable group such as P or any other detectable marker. Methods for preparing probes for hybridization and for constructing cDNA and genomic libraries are commonly known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY).

[0053] "Hybridizing to" or "specifically hybridizing to" means that, when a particular nucleotide sequence is present in a complex mixture (e.g., whole cell) of DNA or RNA, the molecule binds, double-strands, or hybridizes only to that specific nucleotide sequence under stringent conditions. "Substantially binding" refers to complementary hybridization between the probe nucleic acid molecule and the target nucleic acid molecule, encompassing minor mismatches that may be acceptable by mitigating the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0054] In nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization conditions" and "stringent hybridization washing conditions" are sequence-dependent and vary under various environmental parameters. Longer sequences hybridize specifically at higher temperatures. Comprehensive guidelines for nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and washing conditions are those that meet the thermal melting point (T) of a particular sequence at a given ionic strength and pH. m The temperature is selected to be approximately 5°C lower than the target temperature. Typically, under "stringent conditions," the probe hybridizes to its target subsequence but not to other sequences.

[0055] T m This refers to the temperature (under specified ionic strength and pH) at which 50% of the target sequence hybridizes perfectly to the matching probe. Very stringent conditions are the T of a particular probe. mSelected to be equivalent to: For hybridization of complementary nucleic acids having more than 100 complementary residues on a Southern blot or Northern blot filter, an example of a stringent hybridization condition is 50% formamide with 1 mg of heparin at 42°C, in which case hybridization is performed overnight. An example of a highly stringent wash condition is 0.15 M NaCl at 72°C for about 15 minutes. An example of a stringent wash condition is 0.2 × SSC washing at 65°C for 15 minutes (see Sambrook below for an explanation of SSC buffers). Often, a low stringent wash is performed before a highly stringent wash to remove background probe signals. For example, an example of a moderately stringent wash for a double helix of more than 100 nucleotides is 1 × SSC at 45°C for 15 minutes. For example, a low stringent washing for a double helix of more than 100 nucleotides is 4-6 × SSC for 15 minutes at 40°C. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically include a pH of 7.0-8.3, a sodium salt concentration of less than about 1.0 M, typically about 0.01-1.0 M (or other salt), and a temperature of at least about 30°C. Stringent conditions can also be achieved by adding an stabilizer such as formamide. Generally, a signal-to-noise ratio of 2 × (or greater) than that observed for an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected. Nucleic acids that do not hybridize to each other under stringent conditions remain substantially identical if the proteins they encode are substantially identical. This can occur, for example, when nucleic acid copies are made using the maximum codon degeneracy possible by the genetic code.

[0056] The following is an example of a set of hybridization / washing conditions that can be used to clone a nucleotide sequence that is a homolog of a reference nucleotide sequence: The reference nucleotide sequence is hybridized to the reference nucleotide sequence, preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA at 50°C, in which case it is washed in 2×SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA at 50°C, in which case it is washed in 1×SSC, 0.1% SDS at 50°C, still more preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA at 50°C, in which case it is washed in 0.5×SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM Hybridize in EDTA at 50°C, then wash in 0.1×SSC and 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA at 50°C, then wash in 0.1×SSC and 0.1% SDS at 65°C.

[0057] Some embodiments also relate to the use of CYP81E or its variants to confer resistance to herbicides, including auxin herbicides. “Variant” is intended to mean substantially similar sequences. In the case of polynucleotides, variants include deletions and / or additions of one or more nucleotides at one or more internal sites within a natural polynucleotide, and / or substitutions of one or more nucleotides at one or more sites within a natural polynucleotide. As used herein, “natural” polynucleotide or polypeptide includes naturally occurring nucleotide sequences or amino acid sequences, respectively. In the case of polynucleotides, conserved variants include those sequences that encode the CYP81E polypeptide described above, due to the degenerate nature of the genetic code. Naturally occurring allele variants can be identified by the use of well-known molecular biological techniques, such as polymerase chain reaction (PCR) and hybridization techniques, as outlined above. Variant polynucleotides also include synthetically derived polynucleotides, such as those produced by using site-directed mutagenesis, but still encoding the CYP81E polypeptide that confers herbicide resistance. Overall, variants of a particular polynucleotide will have at least approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the particular polynucleotide in question.

[0058] This includes variants of specific polynucleotides encoding CYP81E that confer herbicide resistance, which can be assessed by comparing the percentage of sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The percentage of sequence identity between any two polypeptides can be calculated using the sequence alignment programs and algorithms described below. When any given pair of polynucleotides is assessed by comparing the percentage of sequence identity shared by the two polypeptides they encode, the percentage of sequence identity between the two encoded polypeptides is at least approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater.

[0059] Methods for sequence alignment for comparison are well known in the art and can be performed using mathematical algorithms such as: the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; and the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. Such implementations include, but are not limited to, the following: the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and the GCG Wisconsin Genetics Software Package, Version 10, GAP, BESTFIT, BLAST, FASTA, and TFASTA (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA).

[0060] Some embodiments relate to improving the expression of the CYP81E gene in plants. The terms “improved expression” or “overexpression” as used herein mean any form of expression added to the original wild-type expression level, which may be zero (absence of expression). Methods for improving the expression of a gene or gene product are well-established in the art and include, for example, overexpression driven by a suitable promoter, the use of transcriptional or translational enhancers. Isolated nucleic acids, functioning as promoter or enhancer elements, can be introduced at a suitable position (usually upstream) of a non-heterogeneous form of a polynucleotide, thereby upregulating the expression of the nucleic acid encoding the protein of interest. For example, the endogenous promoter may be modified in vivo by mutation, deletion, and / or substitution (see Kmiec, U.S. Patent No. 5,565,350; Zarling et al., WO9322443), or the isolated promoter may be introduced into plant cells at an appropriate direction and distance from the CYP81E gene to control gene expression.

[0061] Targeted modification of the plant genome through genome editing can be used to enhance the expression of the CYP81E gene through modification of plant genomic DNA. Genome editing allows for targeted insertion of one or more nucleic acids of interest into the plant genome. Exemplary methods for introducing donor polynucleotides into the plant genome or modifying plant genomic DNA include the use of sequence-specific nucleases, such as zinc finger nucleases, engineered or native meganucleases, TALE-endonucleases, or RNA-inducible endonucleases (e.g., clustered regular-arranged short palindromic repeat (CRISPR) / Cas9, CRISPR / Cpf1, CRISPR / CasX, CRISPR / CasY, CRISPR / Cascade). Genome editing methods for modifying, deleting, or inserting nucleic acid sequences into genomic DNA are known in the art.

[0062] Expression construct The polynucleotides described herein may be provided in expression constructs. Expression constructs generally include regulatory elements that are functional in the host cells in which the expression construct is intended to be expressed. Therefore, those skilled in the art can select regulatory elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term “expression construct” refers to a combination of nucleic acid sequences that provide transcription of operably linked nucleic acid sequences. As used herein, “operably linked” means two DNA molecules linked in such a manner that one can influence the function of the other. The operably linked DNA molecules may be part of a single contiguous molecule, and may be adjacent or not. For example, a promoter is operably linked to a polypeptide-encoding DNA molecule in a DNA construct in which two DNA molecules are arranged so that the promoter can influence the expression of the DNA molecule.

[0063] As used herein, the term “heterogeneous” refers to a relationship between two or more items that originate from different sources and are therefore not ordinarily related in nature. For example, a recombinant DNA molecule encoding a protein is heterogeneous to a promoter operably linked if such a combination is not found naturally or ordinarily. In addition, a particular recombinant DNA molecule may be heterogeneous to a particular cell, seed, or organism into which it is inserted if it is not expected that this DNA molecule would not naturally exist in that particular cell, seed, or organism.

[0064] An expression construct may include a promoter sequence operably ligated to a polynucleotide sequence encoding the CYP81E polypeptide described herein. The promoter can be incorporated into the polynucleotide using standard techniques known in the art. Multiple copies of the promoter or multiple promoters may be used in the expression construct described herein. In some embodiments, the promoter may be positioned at approximately the same distance from the transcription start site in the expression construct as it is at its native genetic environment. Some variation in this distance is acceptable without substantial reduction in promoter activity. The transcription start site is typically included in the expression construct.

[0065] The embodiments relate to recombinant DNA molecules encoding the CYP81E polypeptide, in which the recombinant DNA molecule is further defined as being operably linked to a heterogeneous regulatory element. In certain embodiments, the heterogeneous regulatory element is a functional promoter in plant cells. In further embodiments, the promoter is an inducible promoter.

[0066] If the expression construct is to be supplied to or introduced into plant cells, a plant virus promoter can be used, for example, the cauliflower mosaic virus (CaMV) 35S [including the enhanced CaMV35S promoter (see, for example, U.S. Patent No. 5,106,739)] or the CaMV19S promoter or cassava vein mosaic. Other promoters that can be used in expression constructs in plants include, for example, zein promoters such as the maize zein promoter, as well as the prolifera promoter, Ap3 promoter, heat shock promoter, A. tumefaciens T-DNA 1'-promoter or T-DNA 2'-promoter, polygalacturonase promoter, petunia-derived chalcone synthase A (CHS-A) promoter, tobacco PR-1a promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al., 1993), maize Wipl promoter, maize trpA gene promoter (US Patent No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (US Patent No. 5,034,322). Constitutive promoters (e.g., CaMV promoter, ubiquitin promoter, actin promoter, or NOS promoter), promoters regulated in relation to development, and inducible promoters (e.g., such promoters that can be induced by heat, light, hormones, or chemicals) are also intended for use with the polynucleotide expression constructs described herein.

[0067] The expression construct may contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and / or an enhancer element. The transcription termination region can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. The transcription termination sequence may be positioned downstream of the coding sequence to ensure efficient termination. A signal peptide sequence is a short amino acid sequence, typically located at the amino terminus of a protein, responsible for repositioning an operably linked mature polypeptide to a wide range of post-translational intracellular and extracellular environments, from specific organelle compartments to protein action sites. Targeting gene products to intended intracellular and / or extracellular destinations through the use of operably linked signal peptide sequences is intended for use with the polypeptides described herein. Classical enhancers are cis-acting elements that increase gene transcription, and these may also be included in the expression construct. Classical enhancer elements are known in the art, but are not limited to them, and include the CaMV 35S enhancer element, the cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be located within a transcription region and are orientation-dependent. An example is the maize shrunken-1 enhancer element (Clancy and Hannah, 2002).

[0068] Where appropriate, the gene encoding the CPY81E polypeptide is codon-optimized to remove traits detrimental to expression, and codon usage is optimized for expression in specific crops (see, for example, U.S. Patent No. 6,051,760; EP0359472; EP80385962; EP0431829; and Perlak et al. (1991) PNAS USA 88:3324-3328; all of which are incorporated herein by reference).

[0069] In certain embodiments, the nucleic acid molecule includes at least one nucleotide substitution, insertion, or deletion so that the nucleic acid molecule does not describe a naturally occurring nucleic acid sequence.

[0070] CYP81E polypeptide The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain, which may or may not be modified by the addition of non-amino acid groups. It should be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as cofactors. The terms “protein” and “polypeptide” as used herein also include variants, mutants, modified forms, analogs, and / or derivatives of the polypeptides of this disclosure as described herein.

[0071] It should be understood that, with respect to the defined polypeptide, a percentage of identity higher than the value provided above encompasses preferred embodiments. Therefore, where applicable, it is preferable that the CPY81E polypeptide contains an amino acid sequence that is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to Sequence ID No. 2.

[0072] A “mutant” polypeptide is intended to be a polypeptide derived from the SEQ ID NO: 2 protein by deleting (so-called truncation) or adding one or more amino acids to the N-terminus and / or C-terminus of the native protein; deleting or adding one or more amino acids at one or more sites in the native protein; or substituting one or more amino acids at one or more sites in the native protein. Such mutants may be the result of, for example, genetic polymorphism or human manipulation. Methods of such manipulation are generally known in the art.

[0073] The term "derivative" of a protein includes peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the original protein in question, and that possess similar biological and functional activity to the original protein from which they are derived. Accordingly, functional variants and fragments of the CYP81E polypeptide, as well as the nucleic acid molecules encoding them, are also within the scope of this disclosure, regardless of the origin of the polypeptide and whether they exist in nature, unless otherwise specified.

[0074] Furthermore, those skilled in the art will further understand that mutations can introduce changes to the nucleotide sequence, thereby altering the amino acid sequence of the encoded protein without altering the protein's biological activity. For example, an isolated polynucleotide molecule encoding a CYP81E polypeptide having an amino acid sequence different from that of SEQ ID NO: 2 can be produced by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence, resulting in the introduction of one or more amino acid substitutions, additions, or deletions into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Such mutant nucleotide sequences are also covered by this disclosure. For example, preferably, a conserved amino acid substitution can be made at one or more predicted, preferably non-essential, amino acid residues. “Non-essential” amino acid residues are those that can be altered from the wild-type sequence of a protein without altering its biological activity, while “essential” amino acid residues are required for biological activity.

[0075] Deletion refers to the removal of one or more amino acids from a protein. Insertion refers to the introduction of one or more amino acid residues into a specific site in a protein. Insertions can include N-terminal and / or C-terminal fusions of a single or multiple amino acids, as well as internal sequence insertions. Generally, insertions within an amino acid sequence are smaller than N-terminal or C-terminal fusions, typically ranging from about 1 to 10 residues. Examples of N-terminal or C-terminal fusion proteins or peptides include transcription activator binding domains or activation domains used in yeast two-hybrid systems, phage coat proteins, (histidine)-6-tags, glutathione S-transferase-tags, protein A, maltose-binding proteins, dihydrofolate reductase, Tag·100 epitope, c-myc epitope, FLAG® epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope, and VSV epitope.

[0076] Substitution refers to replacing an amino acid in a protein with another amino acid that has similar properties (e.g., similar properties such as hydrophobicity, hydrophilicity, antigenicity, or the ability to form or disrupt α-helix or β-sheet structures). Amino acid substitutions are typically single-residue substitutions, but can be clustered depending on the functional constraints placed on the polypeptide, ranging from 1 to 10 amino acids; insertions are usually of the order of about 1 to 10 amino acid residues. Conservative amino acid substitutions are those in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions are not made for conserved amino acid residues or amino acid residues located within conserved motifs. A table of conserved substitutions is well known in the art [see, for example, Creighton (1984) Proteins. WH Freeman and Company (Eds)].

[0077] Amino acid substitutions, deletions, and / or insertions can be readily performed using peptide synthesis techniques well known in the art, such as solid-phase peptide synthesis, or by recombinant DNA manipulation. Methods for manipulating DNA sequences to produce each variant of protein substitution, insertion, or deletion are well known in the art. For example, techniques for producing substitutional mutations at specific sites in DNA are well known to those skilled in the art, and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange site-specific mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-specific mutagenesis, or other site-specific mutagenesis protocols.

[0078] In certain embodiments, the polypeptide includes at least one amino acid substitution, insertion, or deletion such that the polypeptide does not describe a naturally occurring amino acid sequence.

[0079] In a particular embodiment, the CYP81E polypeptide comprises at least one of the following: an alanine residue at position 9 of SEQ ID NO: 2; a serine residue at position 12 of SEQ ID NO: 2; a histidine residue at position 22 of SEQ ID NO: 2; a valine residue at position 103 of SEQ ID NO: 2; a glycine residue at position 157 of SEQ ID NO: 2; a serine residue at position 258 of SEQ ID NO: 2; a threonine residue at position 276 of SEQ ID NO: 2; a methionine residue at position 379 of SEQ ID NO: 2; an alanine residue at position 449 of SEQ ID NO: 2; a serine residue at position 450 of SEQ ID NO: 2; an alanine residue at position 463 of SEQ ID NO: 2; a valine residue at position 489 of SEQ ID NO: 2; and a leucine residue at position 491 of SEQ ID NO: 2. The positions of amino acid residues in a given amino acid sequence are typically numbered herein using the numbering of the corresponding amino acid residue positions in the Ayumodoki CYP81E amino acid sequence shown in Sequence ID No. 2.

[0080] The terms "ortholog" and "paralog" encompass the evolutionary concepts used to describe the ancestral relationships of genes. A paralog is a gene within the same species that arose through the replication of an ancestral gene; an ortholog is a gene that arose through speciation, originating from a different organism, but also derived from a common ancestral gene.

[0081] The orthologs and paralogs of SEQ ID NO: 2 included in this disclosure include, but are not limited to, polypeptides comprising SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

[0082] [Table 1]

[0083] Transformation method Some embodiments relate to plant cells, plant tissues, plants, and seeds containing the recombinant DNA described herein. In some embodiments, cells, tissues, plants, and seeds containing the recombinant DNA molecule exhibit resistance to auxin herbicides.

[0084] Suitable methods for transforming host plant cells include virtually any method by which DNA or RNA can be introduced into cells (for example, cells in which a recombinant DNA construct is stably incorporated into plant chromosomes, or cells in which a recombinant DNA construct or RNA is transiently supplied to plant cells), and suitable methods are well known in the art. Two effective methods for cell transformation are Agrobacterium-mediated transformation and particulate gun-mediated transformation. Particulate gun methods are exemplified, for example, in U.S. Patents 5,550,318; 5,538,880; 6,160,208; and 6,399,861. Agrobacterium-mediated transformation methods are described, for example, in U.S. Patent 5,591,616, which is incorporated herein by reference in its entirety. Transformation of plant material is carried out in tissue culture in a nutrient medium, for example, in a mixture of nutrients that allows cells to grow in vitro. Recipient cell targets include, but are not limited to, meristematic cells, shoot tips, hypocotyls, callus, immature or mature embryos, and gamete cells such as microspores and pollen. Callus can be initiated from, but are not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores, and other tissue sources. Cells containing a gene-transferred nucleus will grow into gene-transferred plants.

[0085] In transformation, DNA is typically introduced into only a small percentage of target plant cells in any single transformation experiment. A marker gene provides an efficient system for identifying cells that have been stably transformed by receiving and integrating the recombinant DNA molecule into their cellular genome. A preferred marker gene provides a selective marker that confers resistance to a selective agonist, such as an antibiotic or herbicide. Any of the herbicides to which the plants in this disclosure may be resistant are agonists for the selective marker. Potentially transformed cells are exposed to the selective agonist. The population of viable cells generally includes cells in which the resistance-conferring gene is integrated and expressed at levels sufficient to allow for cell survival. The cells can be further tested to confirm the stable integration of the exogenous DNA. Commonly used selectable marker genes include those that confer resistance to antibiotics, such as kanamycin and paromomycin (nptll), hygromycin B (aph IV), spectinomycin (aadA), and gentamicin (aac3 and aacC4), or those that confer resistance to herbicides, such as glufosinate (bar or pat), dicamba (DM0), and glyphosate (aroA or EPSPS). Examples of such selectable markers are illustrated in U.S. Patents 5,550,318; 5,633,435; 5,780,708, and 6,118,047. Markers that provide the ability to visually screen transformants, such as genes expressing colored or fluorescent proteins like luciferase or green fluorescent protein (GFP), or genes expressing β-glucuronidase or the uidA gene (GUS), for which various chromogenic substrates are known, can also be used.

[0086] Herbicide-resistant plants Some embodiments relate to plant cells, plant tissues, plants, and seeds containing a polynucleotide encoding the CYP81E polypeptide, in which case the expression of the polynucleotide confers resistance to herbicides. The plants may be monocots or dicots, and may include, for example, the plants of rice, wheat, barley, oats, rye, sorghum, maize, grapes, tomatoes, potatoes, lettuce, broccoli, cucumbers, peanuts, melons, peppers, carrots, pumpkins, onions, soybeans, alfalfa, sunflowers, cotton, canola, and sugar beets.

[0087] Plants particularly useful in the methods disclosed herein include all plants belonging to the Viridiplantae superfamily, in particular monocots and dicots, including forage or fodder legumes, ornamental plants, food crops, trees or shrubs, selected from the list including: species of the genera Acer, Actinidia, Abelmoschus, Agave sisalana, Agropyron, Agrostis stolonifera, Allium, Amaranthus, Ammophila arenaria, and pineapple (Ananas) comosus), species of the genus Annona (spp.), celery (Apium graveolens), species of the genus Arachis (peanuts), species of the genus Artocarpus (breadfruit), asparagus (Asparagus officinalis), species of the genus Avena (e.g., oat (Avena sativa), wild oat (Avena fatua), red oat (Avena byzantina), wild oat variety sativa (Avena fatua var. sativa), wild oat hybrid (Avena hybrida)), star fruit (Averrhoa carambola), species of the genus Bambusa (Bambusa sp.), winter melon (Benincasa hispida), Brazil nut (Bertholletia excelsea), sugar beet (Beta Brassica species (e.g., Brassica napus, Brassica rapa ssp. (canola, rapeseed, rapeseed)), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum species (Capsicum spp.)), Carex elata, papaya (Carica papaya), Carissa macrocarpa, species of pecan (Carya spp.), safflower (Carthamus tinctorius), species of chestnut (Castanea spp.), kapok (Ceiba pentandra), endive (Cichorium endivia), species of cinnamon (Cinnamomum spp.), watermelon (Citrullus lanatus), species of citrus (Citrus spp.), species of coconut (Cocos spp.), species of coffee plant (Coffea spp.), taro (Colocasia esculenta), species of cola (Cola spp.), species of coriander (Coriandrum) (sativum), species of the genus Corylus (Corylus spp.), species of the genus Crataegus (Crataegus spp.), saffron (Crocus sativus), species of the genus Cucurbita (Cucurbita spp.), species of the genus Cucumis (Cucumis spp.), species of the genus Cynara (Cynara spp.), wild carrot (Daucus carota), species of the genus Desmodium (Desmodium spp.), longan (Dimocarpus longan), species of the genus Dioscorea (Dioscorea spp.), species of the genus Diospyros (Diospyros spp.), species of barnyard grass (Echinochloa spp.), oil palm (Elaeis) [e.g., Guinea oil palm (Elaeis guineensis), American oil palm (Elaeis (oleifera), finger millet (Eleusine coracana), teff (Eragrostis tef), species of the genus Erianthus (Erianthus sp.), loquat (Eriobotrya japonica), species of the genus Eucalyptus (Eucalyptus sp.), pitanga (Eugenia uniflora), species of the genus Fagopyrum (Fagopyrum spp.), species of the genus Fagus (Fagus spp.)), Festuca arundinacea, fig (Ficus carica), species of Fortunella (Fortunella spp.), species of Fragaria (Fragaria spp.), Ginkgo biloba, species of Glycine (Glycine spp.) [e.g., soybean (Glycine max), Soja hispida, or Soja max], cotton (Gossypium hirsutum), species of Helianthus (Helianthus spp.) [e.g., sunflower (Helianthus annuus)], daylily (Hemerocallis fulva), species of Hibiscus (Hibiscus spp.), species of Hordeum (Barley (Hordeum Vulgare), sweet potato (Ipomoea batatas), walnut species (Juglans spp.), lettuce (Lactuca sativa), Lathyrus species (Lathyrus spp.), lentil (Lens culinaris), flax (Linum usitatissimum), lychee (Litchi chinensis), Lotus species (Lotus spp.), loofah (Luffa acutangula), lupine species (Lupinus spp.), giant spear (Luzula sylvatica), tomato species (Lycopersicon spp.) [for example, Lycopersicon esculentum, Lycopersicon lycopersicum] (Lycopersicum), Lycopersicon pyriforme, Macrotyloma spp., Malus spp., Acerola (Malpighia emarginata), Mamey apple (Mammea americana), Mango (Mangifera indica), Manihot spp.), Sapodilla (Manilkara zapota), Medicago sativa, species of the genus Melillotus (spp.), species of the genus Mentha (spp.), Miscanthus sinensis (Japanese pampas grass), species of the genus Momordica (spp.), Morus nigra (black mulberry), species of the genus Musa (spp.), species of the genus Nicotiana (tobacco), species of the genus Olea (olive), species of the genus Opuntia (Opuntia), species of the genus Ornithopus (Ornithopus), species of the genus Oryza (Oryza spp.) [e.g., rice (Oryza sativa), Oryza latifolia], Panicum (millet) (miliaceum), switchgrass (Panicum virgatum), passion fruit (Passiflora edulis), parsnip (Pastinaca sativa), species of Pennisetum sp., species of Persea spp., parsley (Petroselinum crispum), reed (Phalaris arundinacea), species of Phaseolus spp., timothy grass (Phleum pratense), species of Phoenix spp., reed (Phragmites australis), species of Physalis spp., species of Pinus spp., pistachio (Pistacia vera), species of Pisum (spp.), species of the genus Poa (strawberry tree), species of the genus Populus (poplar), species of the genus Prosopis (prosopis), species of the genus Prunus (cherry), species of Psidium (pampas grass), pomegranate (Punica granatum), European pear (Pyrus communis), species of the genus Quercus (oak), radish (Raphanus sativus), rhubarb (Rheum rhabarbarum), species of the genus Ribes (currant).), castor bean (Ricinus communis), species of blackberry (Rubus spp.), species of sugarcane (Saccharum spp.), species of willow (Salix sp.), species of Swatonia (Sambucus spp.), rye (Secale cereale), species of sesame (Sesamum spp.), species of mustard (Sinapis sp.), species of nightshade (Solanum spp.) [e.g., potato (Solanum tuberosum), flat eggplant (Solanum integrifolium) or tomato (Solanum lycopersicum)], sorghum (Sorghum bicolor), species of spinach (Spinacia spp.), species of syzygium (Syzygium spp.), species of marigold (Tagetes spp.) (spp.), tamarind (Tamarindus indica), cacao (Theobroma cacao), species of the genus Trifolium (Trifolium spp.), cattail grass (Tripsacum dactyloides), Triticosecale rimpaui, species of the genus Triticum (Triticum spp.) [e.g., bread wheat (Triticum aestivum), durum wheat (Triticum durum), rivet wheat (Triticum turgidum), Triticum hybernum, Triticum macha, wheat (Triticum sativum), einkorn wheat (Triticum monococcum) or Triticum vulgare], dwarf nasturtium (Tropaeolum minus), nasturtium (Tropaeolum) majus), species of the genus Vaccinium, species of Vicia, species of Vigna, sweet violet (Viola odorata), species of Vitis, corn (Zea mays), wild rice (Zizania palustris), species of Zizphus (Zizphus spp.)), in particular, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrots, cauliflower, celery, collard greens, flax, kale, lentils, rapeseed, okra, onions, potatoes, rice, soybeans, strawberries, sugar beets, sugarcane, sunflowers, tomatoes, pumpkins, tea, and algae. In certain embodiments, the plants are crops. Examples of crops include, in particular, soybeans, sunflowers, canola, alfalfa, rapeseed, cotton, tomatoes, potatoes, or tobacco.

[0088] Certain embodiments include the progeny or descendant of herbicide-resistant plants as described herein, as well as seeds derived from herbicide-resistant plants and cells derived from herbicide-resistant plants.

[0089] In some embodiments, the Disclosure provides a plant-derived progeny plant or descendant plant, wherein the plant cell contains a polynucleotide operably linked to a functional promoter in at least a portion of the plant's cells, the promoter is capable of expressing a CPY81E polypeptide encoded by the polynucleotide, wherein the progeny plant or descendant plant contains a recombinant polynucleotide operably linked to a promoter in at least a portion of the plant's cells, and the expression of the CYP81E polypeptide confers herbicide resistance to the progeny plant or descendant plant.

[0090] In one embodiment, the seeds of the present disclosure preferably contain herbicide-resistant features of herbicide-resistant plants. In another embodiment, the seeds can germinate into plants, containing in at least a portion of the plant cells a polynucleotide operably linked to a functional promoter in the plant cells, the promoter can express a CYP81E polypeptide encoded by the polynucleotide, the expression of the CYP81E polypeptide confers herbicide resistance to the offspring or descendant plants.

[0091] In some embodiments, the plant cells of this disclosure can regenerate a plant or a part of a plant. In other embodiments, the plant cells cannot regenerate a plant or a part of a plant. Examples of cells that cannot regenerate a plant include, but are not limited to, the endosperm, seed coat (exocoque and pericarp), and root cap.

[0092] In another embodiment, the disclosure relates to plant cells transformed with a nucleic acid encoding the CPY81E polypeptide described herein, wherein the expression of the nucleic acid in the plant cells results in improved resistance or tolerance to herbicides compared to the wild-type variety of the plant cells.

[0093] Some embodiments provide plant products prepared from herbicide-resistant plants. In some embodiments, examples of plant products include, but are not limited to, grains, oils, and meal. In one embodiment, the plant product is a plant grain (e.g., a grain suitable for use as feed or for processing), a vegetable oil (e.g., an oil suitable for use as food or biodiesel), or a plant meal (e.g., a meal suitable for use as feed). Preferred plant products are meal, seed meal, oil, or seed-treated coated seeds. Preferably, the meal and / or oil contain CYP81E nucleic acid or CYP81E protein.

[0094] In a particular embodiment, a plant product prepared from a plant or plant part is provided, wherein the plant or plant part contains in at least a portion of the plant cells a polynucleotide operably linked to a functional promoter in the plant cells, the promoter can express a CYP81E polypeptide encoded by the polynucleotide, and the expression of the CYP81E polypeptide confers resistance to herbicides to the plant or plant part.

[0095] In some cases, the product is produced at the site where the plants were grown, and in other cases, the plant and / or parts thereof are extracted from the site where the plants were grown and the product is produced from the harvestable parts of the plant, if this can be done in a repeating cycle. For example, the plant growing step may be performed only once each time the method of the present invention is carried out, while allowing the product production process to be repeated by repeatedly extracting the harvestable parts of the plant according to the present disclosure, and, if necessary, by further processing of those parts to obtain the product. It is also possible to repeat the plant growing step and to store the plant or harvestable parts until the production of the product is subsequently carried out once more with respect to the accumulated plant or plant parts. Furthermore, the plant growing step and the product production step may overlap in time, and to a considerable extent, be carried out simultaneously or consecutively. Generally, the plant is grown for a period of time, and then the product is produced.

[0096] Auxin herbicide Synthetic auxin herbicides are also called auxin-based, growth-regulating herbicides, or Group O or Group 4 herbicides, based on their mode of action. The mode of action of synthetic auxin herbicides is that they appear to affect cell wall plasticity and nucleic acid metabolism, which can lead to uncontrolled cell division and proliferation. The group of synthetic auxin herbicides includes four chemical families: phenoxy, carboxylic acids (or pyridines), benzoic acid, and the latest family, quinoline carboxylic acids.

[0097] Phenoxy herbicides are the most common and have been used as herbicides since the discovery of (2,4-dichlorophenoxy)acetic acid (2,4-D) in the 1940s. Other examples include 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP), (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T), 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP), 2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (Cromeprop), (4-chloro-2-methylphenoxy)acetic acid (MCPA), 4-(4-chloro-o-tolyloxy)butyric acid (MCPB), and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP).

[0098] The next largest chemical family is carboxylic acid herbicides, also known as pyridine herbicides. Examples include 3,6-dichloro-2-pyridinecarboxylic acid (clopyralide), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), (2,4,5-trichlorophenoxy)acetic acid (triclopyr), and 4-amino-3,5-dichloro-6-fluoro-2-pyridyloxyacetic acid (fluroxypyr). The third chemical family is benzoic acid, examples of which include 3,6-dichloro-o-anisinic acid (dicamba) and 3-amino-2,5-dichlorobenzoic acid (colamben). The fourth and newest chemical family of auxin herbicides is the quinalocarboxylic acid family, which includes 7-chloro-3-methyl-8-quinolinecarboxylic acid (kinmelac) and 3,7-dichloro-8-quinolinecarboxylic acid (quinchlorac). This latter is unique in that, unlike other auxin-like herbicides that essentially control only broad-leaved trees or dicotyledonous plants, it also controls some grass weeds.

[0099] Synthetic auxin herbicides can be applied to plant cultivation areas containing plants and seeds provided by the compositions and methods described herein as a method for controlling weeds. The plants and seeds provided by the compositions and methods described herein contain synthetic auxin herbicide resistance traits and are therefore resistant to the application of one or more auxin herbicides. The application of the herbicide may be the recommended commercial amount (1×) or any fraction or multiple thereof, for example, twice the recommended commercial amount (2×). The amount of auxin herbicide can be expressed as acid equivalents / pound / acre (lb ae / acre) or acid equivalents / gram / hectare (g ae / ha), or as pounds / acre of the active ingredient (lb ai / acre) or grams / hectare of the active ingredient (g ai / ha), depending on the herbicide and formulation. The plant cultivation area may or may not contain weed plants at the time of herbicide application.

[0100] Herbicide application may be continuous or mixed in a tank using one, two, or a combination thereof of several auxin herbicides or any other suitable herbicides. To control a wide range of dicotyledonous weeds, monocotyledonous weeds, or both, multiple applications of one or more herbicides in combination or individually may be used over the growing season in areas containing plants expressing the CYP81E protein described herein, for example, two applications (e.g., pre-planting and post-germination application, or pre-germination and post-germination application) or three applications (e.g., pre-planting, pre-germination, and post-germination application, or pre-germination and two post-germination applications).

[0101] Control of herbicide-resistant weeds Some embodiments provide compositions and methods for controlling the growth of herbicide-resistant weeds in plant cultivation sites by contacting weeds with a composition containing a polynucleotide that reduces the expression or activity of a CYP81E polypeptide.

[0102] The regulation of the osmotic translocation of a target CYP81E gene in plants (e.g., suppression or silencing of osmotic translocation) can be achieved by locally applying a polynucleotide molecule to the plant that has a segment in a nucleotide sequence that is essentially identical or essentially complementary to the sequence of 18 or more consecutive nucleotides in either the target CYP81E gene or the RNA transcribed from the target CYP81E gene. Thereafter, the composition penetrates into the plant and induces the regulation of the osmotic translocation of the target CYP81E gene through the action of single-stranded RNA that hybridizes with the transcribed RNA, such as messenger RNA.

[0103] The polynucleotides are designed to induce regulation or repression of the ostomy of endogenous genes in plants and to have a sequence that is essentially identical or essentially complementary to the sequence of the endogenous CYP81E gene in resistant plants (which may be a coding or non-coding sequence) or the sequence of RNA transcribed from the endogenous CYP81E gene in resistant plants. "Essentially identical" or "essentially complementary" means that the polynucleotide (or at least one strand of a double-stranded polynucleotide) is designed to hybridize to the endogenous gene or RNA transcribed from the endogenous gene under physiological conditions in plant cells in order to achieve regulation or repression of the endogenous gene.

[0104] In certain embodiments, compositions and methods may include permeability enhancers and permeability enhancing treatments for conditioning the surface of plant tissues, such as leaves, stems, roots, flowers, or fruits, for permeability to plant cells by polynucleotides. The transport of polynucleotides into plant cells can be facilitated by prior or simultaneous application of polynucleotides to plant tissues. In some embodiments, the permeability enhancer is applied following the application of the polynucleotide composition. The permeability enhancer enables a pathway for polynucleotides into plant cells through the cuticle wax barrier, stomata, and / or cell wall or membrane barriers. Suitable agents for facilitating the transport of compositions into plant cells include agents that increase the permeability of the plant's outer surface, or agents that increase the permeability of plant cells to oligonucleotides or polynucleotides. Such agents for facilitating transport into plant cells may include chemical agents, physical agents, or combinations thereof.

[0105] Chemical agents for conditioning include (a) surfactants, (b) organic solvents or aqueous solutions or aqueous mixtures of organic solvents, (c) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzymes, or combinations thereof. Embodiments of the method may optionally include incubation steps, neutralization steps (e.g., neutralizing acids, bases, or oxidizing agents or inactivating enzymes), rinsing steps, or combinations thereof. Such agents for conditioning plants against penetration by polynucleotides are applied to plants by any convenient method, e.g., by spraying or coating with powder, emulsion, suspension, or solution; similarly, polynucleotide molecules are applied to plants by any convenient method, e.g., by spraying or wiping with solution, emulsion, or suspension.

[0106] Detection tool Several embodiments provide methods for identifying herbicide-resistant plants, or their cells or tissues. In some embodiments, the method involves using primers or probes that specifically recognize a portion of a gene sequence. In one embodiment, the method is based on identifying the expression level of the CPY81E gene in plants. In some embodiments, a PCR-based technique is used to quantify the differentially expressed CPY81E gene in resistant plants compared to susceptible plants before treatment. In other words, the basal expression level is higher in resistant plants compared to susceptible plants before herbicide treatment.

[0107] In some embodiments, identification is performed using polymerase chain reaction. The method may also include preparing a detectable marker specific to the CYP81E gene. In embodiments, detection is performed using enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), or RNA hybridization techniques.

[0108] In one embodiment, the method is based on the presence of a SNP between S and R plants. This can be based on the fluorescence detection of a SNP-specific hybridization probe in the PCR product, such as Taqman or Molecular Beacon. Other strategies, such as Sequenom homogeneous Mass Extend (hME) and the iPLEX genotyping system, include MALDI-TOF mass spectrometry of the SNP-specific PCR primer extension product.

[0109] Other methods include the use of KASP®, or Kompetitive Allele Specific PCR. This is based on competing allele-specific PCR, enabling scoring of single nucleotide polymorphisms (SNPs) as well as deletions and insertions at specific loci. Two allele-specific forward primers with target SNPs at their 3' ends are used, and a common reverse primer is used for both. The primers have a unique "tail" sequence (reporter nucleotide sequence) that is compatible with different fluorescent reporters (reporter molecules). The primers are brought into contact with the sample along with a mixture containing a universal fluorescence resonance energy transfer (FRET) cassette and Taq polymerase. During the rounds of the PCR cycle, the tail sequence allows the FRET cassette to bind to the DNA and emit fluorescence. For example, see Yan et al. “Introduction of high throughput and cost effective SNP genotyping platforms in soybean” Plant Genetics, Genomic and Biotechnology 2(1): 90 - 94 (2014); Semagn et al. “Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement” Molecular Breeding 33(1): 1 - 14 (2013). In this method, the emission of one fluorescent signal (reporter molecule) or the emission of the other indicates that the plant is one of two species, where the presence of both signals indicates a hybrid. The examples herein show the use of 6-carboxyfluorescein (FAM) and 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX) fluorophores, however, any convenient means of generating a measurable signal can be used. Examples, though not intended to be limiting, include tetrachlorofluorescein (TET), cyan fluorescent protein, yellow fluorescent protein, luciferase, SyBR Green I; ViC; CAL Fluor Gold 540, ROX Texas Red; CAL Fluor Red 610; CY5; Quasar 670; Quasar 705; and Fret.

[0110] In summary, a first primer is generated that recognizes a first target nucleotide sequence in the genome of the first species, a second primer is generated that recognizes a second target nucleotide sequence in the second species, and amplification is possible by a third common reverse primer that is universal to all genotypes. The "tail" reporter sequence is equipped with primers. The expression cassette contains a sequence complementary to the reporter sequence. With multiple rounds of PCR, the cassette is no longer quenched and a measurable signal is generated.

[0111] Two sets of KASP primers designed for the CPY81E position are described in SEQ ID NOs. 27-29 and 30-31. The primers for the R allele were tagged with HEX fluorophores, and the S allele was tagged with FAM.

[0112] [Table 2]

[0113] Several embodiments provide kits for identifying herbicide-resistant plants, each comprising at least two primers or probes that specifically recognize the CYP81E gene. For example, primers have been developed to amplify and / or quantify the expression of the CYP81E gene associated with SEQ ID NO: 1. By evaluating the gene expression level, those skilled in the art can determine whether a plant sample originates from a herbicide-resistant plant. In certain embodiments, the primers include SEQ ID NOs: 5 and 6. Kits for detecting the presence of SNPs between S and R plants are also provided. In certain embodiments, the primers include SEQ ID NOs: 27-29 or 30-32. In one embodiment, the kit comprises two or more primer pairs. The kit may also include one or more positive or negative controls.

[0114] In some embodiments, the kit includes a specific probe having a sequence that corresponds to or is complementary to a sequence having 80% to 100% sequence identity with a specific region of the CYP81E gene. In some embodiments, the kit includes a specific probe having a sequence that corresponds to or is complementary to a sequence having 90% to 100% sequence identity with a specific region of the CPY81E gene.

[0115] The methods, kits, and primers can be used for a variety of purposes, including, but are not limited to, identifying the presence or absence of herbicide resistance in plant materials such as plants, seeds, or cuttings; determining the presence of herbicide-resistant weeds in crop fields; and adapting herbicide regimens for the effective and economical control of weeds affecting agricultural crops.

[0116] Use in breeding methods The plants of this disclosure can be used in plant breeding programs. The objective of plant breeding is to combine various desirable traits in a single variety or hybrid. In the case of crops, these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to cold and frost, reduced time to crop maturity, higher yields, and better agrochemical quality. Uniformity of plant characteristics such as established germination and seedling establishment, growth rate, maturity, and plant and ear height is desirable in relation to the mechanical harvesting of many crops. Traditional plant breeding is an important tool in developing novel and improved commercial crops. This disclosure encompasses a method for producing a plant by crossing a first parent plant with a second parent plant, wherein one or both parent plants exhibit the phenotype described herein.

[0117] Plant breeding techniques known in this field and used in plant breeding programs include, but are not limited to, cyclic selection, bulk selection, mass selection, backcrossing, line breeding, natural pollination breeding, restriction fragment length polymorphism enhancement selection, genetic marker enhancement selection, doubling haploids, and transformation. Often, combinations of these techniques are used.

[0118] Hybrid development in plant breeding programs generally requires the development of isozygous inbred lines, crossing of these lines, and evaluation of the crosses. Many analytical methods are available to evaluate the results of these crosses. The oldest and most traditional method is the observation of phenotypic traits. Alternatively, the plant's genotype may be examined.

[0119] Genetic traits manipulated into a particular plant using transformation techniques can be transferred to another line using traditional breeding techniques well known in the field of plant breeding. For example, the backcross approach is often used to transfer introduced genes from a transformed plant to a select inbred line, the resulting offspring then containing the introduced genes. Alternatively, if an inbred line is used for transformation, the transformed plant can be crossed with a different inbred line to produce a genetically modified hybrid plant. As used herein, "crossing" may refer to a simple X×Y cross or to the process of backcrossing, depending on the context.

[0120] The development of hybrids in plant breeding programs involves three steps: (1) selection of plants from various germplasm pools for the initial breeding cross; (2) self-pollination of the selected plants from the breeding cross over several generations to create a series of inbred lines (these inbred lines are different from each other, but are both pure and highly homozygous); and (3) crossing the selected inbred lines with different inbred lines to produce hybrids. During the inbreeding process, the growth vigor of the lines decreases. When two different inbred lines are crossed to produce hybrids, the growth vigor is restored. An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrids produced by crossing a specific pair of inbred lines are always the same. Once an inbred line that produces superior hybrids is identified, the seeds of that hybrid can be reproduced indefinitely, as long as the homogeneity of the inbred parent lines is maintained.

[0121] The plants of this disclosure can be used, for example, to produce single-hybrid hybrids, ternary hybrids, or multi-hybrid hybrids. A single-hybrid hybrid is produced by crossing two inbred lines to produce Fl offspring. A multi-hybrid hybrid is produced from four inbred lines crossed in pairs (A×B and C×D), and then two F1 hybrids are crossed again as ((A×B) × (C×D)). A ternary hybrid is produced from three inbred lines, in which case two of the inbred lines are crossed (A×B), and then the resulting Fl hybrid is crossed with a third inbred line (A×B)×C. Much of the vigor and uniformity of the hybrid exhibited in the F1 hybrid is lost in the next generation (F2). As a result, the seeds produced by the hybrid are consumed rather than sown.

[0122] Embodiment The following numbered embodiments also form part of this disclosure: 1. Modified plants, or their offspring, plant parts, or plant cells that are resistant to herbicides, wherein the modified plants contain improved expression of polynucleotides encoding cytochrome P450 81E (CYP81E) polypeptides compared to unmodified plants.

[0123] 2. The modified plant is the modified plant of Embodiment 1, which contains a heterogeneous polynucleotide encoding a CYP81E polypeptide.

[0124] 3. A modified plant of Embodiment 1 or Embodiment 2, wherein the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0125] 4. A modified plant according to any one of Embodiments 1 to 3, wherein the polynucleotide encoding the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1.

[0126] 5. A modified plant according to any one of Embodiments 1 to 4, wherein the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 33 to 44.

[0127] 6. A modified plant according to any one of embodiments 1 to 5, wherein a polynucleotide is operably linked to a functional promoter in the plant cell.

[0128] 7. A modified plant according to any one of Embodiments 1 to 6, wherein the herbicide is an auxin herbicide.

[0129] 8. A modified plant of any one of Embodiments 1 to 7, wherein the auxin herbicide is 2,4-D.

[0130] 9. A modified plant of any one of Embodiments 1 to 8, wherein the plant is a dicotyledonous plant.

[0131] 10. A modified plant of any one of Embodiments 1 to 9, wherein the plant is a crop.

[0132] 11. A modified plant of any one of Embodiments 1 to 10, wherein the plant is a soybean, cotton, canola, tobacco, tomato, potato, alfalfa, sugar beet, or sunflower.

[0133] 12. A modified plant comprising any one of Embodiments 1 to 11, further comprising a second herbicide resistance trait.

[0134] 13. (a) A nucleotide sequence encoding a CYP81E polypeptide, wherein the nucleotide sequence has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1; or (b) A nucleotide sequence encoding a CYP81E polypeptide, wherein the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2, A nucleic acid molecule containing a nucleotide sequence selected from the following.

[0135] 14. The nucleic acid molecule of Embodiment 13 is an isolated, synthetic, or recombinant nucleic acid molecule.

[0136] 15. An expression cassette comprising nucleic acid molecules of Embodiment 13 or Embodiment 14, operably linked to a functional heterologous promoter in plant cells.

[0137] 16. A vector comprising a nucleic acid molecule of Embodiment 13 or Embodiment 14; or an expression cassette of Embodiment 15.

[0138] 17. A CYP81E polypeptide comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0139] 18. A plant, plant part, or plant cell comprising a nucleic acid molecule of Embodiment 13 or Embodiment 14; an expression cassette of Embodiment 15; a vector of Embodiment 16; or a polypeptide of Embodiment 17.

[0140] 19. A biological sample comprising a nucleic acid molecule of Embodiment 13 or Embodiment 14; an expression cassette of Embodiment 15; a vector of Embodiment 16; or a polypeptide of Embodiment 17.

[0141] 20. A method for producing herbicide-resistant plants, comprising increasing the expression of a polynucleotide encoding a CYP81E polypeptide in the plants, wherein the herbicide resistance of the plants is improved compared to plants lacking the improved expression.

[0142] 21. A method of Embodiment 20 comprising introducing a polynucleotide encoding a CYP81E polypeptide into plant cells, wherein the polynucleotide is operably linked to a functional heterologous promoter in the plant cells; and regenerating a plant from the plant cells.

[0143] 22. The method of Embodiment 20 or Embodiment 21, wherein the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0144] 23. Any one of Embodiments 20 to 22, wherein the polynucleotide encoding the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1.

[0145] 24. Any one of Embodiments 20 to 23, wherein the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 33 to 44.

[0146] 25. The herbicide is an auxin herbicide, according to any one of embodiments 20 to 24.

[0147] 26. The auxin herbicide is 2,4-D, according to any one of the embodiments 20 to 25.

[0148] 27. The plant is a dicotyledonous plant, according to any one of embodiments 20 to 26.

[0149] 28. The plant is a crop, according to any one of embodiments 20 to 27.

[0150] 29. Any one of embodiments 20 to 28, wherein the plant is a soybean, cotton, canola, tobacco, tomato, potato, alfalfa, sugar beet, or sunflower plant.

[0151] 30. A method for controlling undesirable vegetation in a plant cultivation site, comprising: providing a plant containing a polynucleotide encoding a CYP81E polypeptide in a plant cultivation site, wherein the expression of the polynucleotide confers resistance to herbicides to the plant; and applying an effective amount of herbicide to the plant cultivation site.

[0152] 31. The method of Embodiment 30, wherein the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0153] 32. The method of Embodiment 30 or Embodiment 31, wherein the polynucleotide encoding the CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1.

[0154] 33. Any one of embodiments 30 to 32, wherein a polynucleotide is operably linked to a functional heterologous promoter in a plant cell.

[0155] 34. The herbicide is an auxin herbicide, one of the methods of Embodiments 30 to 33.

[0156] 35. The auxin herbicide is 2,4-D, according to any one of the embodiments 30 to 34.

[0157] 36. The plant is a dicotyledonous plant, according to any one of embodiments 30 to 35.

[0158] 37. Any one of embodiments 30 to 36, wherein the plant is a soybean, cotton, canola, tobacco, tomato, potato, alfalfa, sugar beet, or sunflower.

[0159] 38. A method for controlling the growth of herbicide-resistant weeds in a plant cultivation site, comprising: contacting the weeds with a composition containing a polynucleotide that reduces the expression or activity of CYP81E polypeptide; and applying an effective amount of herbicide to the plant cultivation site.

[0160] 39. The method of Embodiment 38, wherein the polynucleotide is a double-stranded RNA, a single-stranded RNA, or a double-stranded DNA / RNA hybrid polynucleotide.

[0161] 40. The method of Embodiment 38 or Embodiment 39, wherein the polynucleotide comprises a sequence that is essentially identical or essentially complementary to at least 18 consecutive nucleotides of SEQ ID NO: 1.

[0162] 41. A polynucleotide having a length of 26 to 60 nucleotides, any one of the embodiments 38 to 40.

[0163] 42. The CYP81E polypeptide has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2, according to any one of Embodiments 38 to 41.

[0164] 43. The herbicide is an auxin herbicide, one of the methods of Embodiments 38 to 42.

[0165] 44. The auxin herbicide is 2,4-D, according to any one of the embodiments 38 to 43.

[0166] 45. The weed is Amaranthium, according to any one of embodiments 38 to 44.

[0167] 46. ​​A composition comprising an agent that enables polynucleotides to penetrate from the surface of the weed into the weed cells, according to any one of embodiments 38 to 45.

[0168] 47. A product prepared from any one of the plants, plant parts, or plant cells of Embodiments 1 to 12, comprising a polynucleotide encoding a CYP81E polypeptide.

[0169] 48. The product of Embodiment 47, wherein the product is feed, seed coarse powder, oil, or seed-treated coated seeds.

[0170] 49. A method for producing a plant product, comprising processing one of the plants or plant parts of Embodiments 1 to 12 to obtain a plant product, wherein the plant product comprises a polynucleotide encoding a CYP81E polypeptide.

[0171] 50. The method of Embodiment 49, wherein the plant product is feed, seed coarse powder, oil, or seed-treated coated seeds.

[0172] 51. A method for identifying herbicide-resistant plants, comprising: preparing a biological sample derived from a plant suspected to be herbicide-resistant; quantifying the expression of the CYP81E gene in the biological sample, wherein the CYP81E gene is expressed differentially in herbicide-resistant plants compared to herbicide-sensitive plants of the same species; and determining, based on the quantification, that the plant is herbicide-resistant.

[0173] 52. The method of Embodiment 51, wherein the biological sample is derived from Ayu.

[0174] 53. The method of Embodiment 51 or Embodiment 52, wherein the herbicide is an auxin herbicide.

[0175] 54. A method for quantifying the expression of the CYP81E gene, comprising quantifying CYP81E mRNA, as one of embodiments 51 to 53.

[0176] 55. A method for quantifying the expression of the CYP81E gene, comprising quantifying the CYP81E polypeptide, as described in any one of Embodiments 51 to 54.

[0177] 56. The CYP81E gene has at least four times differential expression in herbicide-resistant plants compared to herbicide-sensitive plants before herbicide application, according to any one of embodiments 51 to 55.

[0178] 57. The CYP81E gene has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, according to any one of embodiments 51 to 56.

[0179] 58. Quantifying expression is a method of any one of embodiments 51 to 57, comprising amplifying nucleic acid using at least two primers.

[0180] 59. Any one of the embodiments 51 to 58, wherein at least two primers include SEQ ID NO: 5 and SEQ ID NO: 6.

[0181] 60. A kit for identifying herbicide-resistant plants, comprising at least two primers, wherein at least two primers recognize the CYP81E gene differentially expressed in herbicide-resistant plants compared to herbicide-sensitive plants of the same species.

[0182] 61. The CYP81E gene has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, in a kit according to Embodiment 60.

[0183] 62. A kit of Embodiment 60 or Embodiment 61, further comprising at least one of a positive control and a negative control.

[0184] 63. A kit comprising any one of embodiments 60 to 62, further comprising components of a qRT-PCR solution.

[0185] 64. A kit in which the plant is Amaranthus spp. and the herbicide is an auxin herbicide, any one of embodiments 60 to 63.

[0186] All publications and patent applications described herein indicate the level of skill of those skilled in the art to which the present invention belongs. Every publication and patent application is incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0187] While the aforementioned invention has been described in some detail with examples and illustrations for the sake of clear understanding, it is clear that certain modifications and alterations may be carried out within the scope of the attached claims.

[0188] The following examples are provided for illustrative purposes only and are not intended as limitations. [Examples]

[0189] [Example 1]

[0190] Resistive response Two populations of Amaranthus spp. exhibiting resistance to HPPD inhibitors and 2,4-D were identified from both Illinois (referred to as "CHR") (Evans et al. 2019) and Nebraska (referred to as "NEB") (Bernards et al. 2012). Herbicide-resistant plants from each population were crossed with a herbicide-sensitive Amaranthus spp. population (WUS; first collected in Brown County, Ohio), and F1 seeds were screened to confirm resistance to both HPPD inhibitors and 2,4-D. To screen these F1 populations, plants were grown under the previously described greenhouse conditions (Lillie et al., 2020) and treated with an initial identification dose of mesotrione (220g ai ha). -1 In addition to Callisto, a 1% v / v crop oil concentrate was sprayed, followed by a late post treatment with 2,4-D (560g ae ha). -1In addition to 2,4-D amine, 0.25% v / v non-ionic surfactant was sprayed. Application of all herbicides was carried out using a movable nozzle spray chamber as described previously (Lillie et al. 2020). Among each of the NEB-derived and CHR-derived F1 lines, pairs of full-sibling F1 survivors were mated to form several segregating pseudo-F2 populations. Since spinach is dioecious, F1 plants cannot be self-pollinated to create a true F2 population.

[0191] A single pseudo-F2 (hereinafter referred to as F2) population was selected from each of NEB and CHR, and several hundred seeds from each F2 were germinated on moist filter paper in a growth chamber set at a 12-hour day / night cycle (35 °C / 15 °C) for 48 hours. Weed Lite Mix [a 3:1:1:1 mixture of LC1 (Sun Gro Horticulture Canada):Soil:Peat:Torpedo Sand] was filled in 50 cm 3 pots, and the germinated seedlings were transplanted and grown in a greenhouse until the plants reached a height of 4 - 6 cm. Then, 100 plants from each F2 population were transplanted into 3.8 L round pots filled with Weed Lite Mix and grown until the plants reached a height of 8 - 10 cm. Then, tissues were collected from the smallest fully expanded leaves, immediately placed in liquid nitrogen, and stored at -80 °C until RNA extraction. All tissues were collected within 2 hours from 10 am to noon on the same day. Tissues were collected before herbicide application, and herbicide-treated tissues were not included in this study. Identifying potential resistance genes induced by herbicide application without using extensive (and expensive) time-course RNAseq studies is extremely difficult due to the differential effects of herbicide treatment on stress and death pathways between resistant and susceptible plants (Giacomini et al., 2018).

[0192] All F2 plants continued to grow for an additional 3 weeks until each plant produced multiple lateral buds. At the time of lateral bud formation, the lateral buds were cut off, immersed in rooting hormone, and placed on a flat filled with moist soil at 400 cm 3The plants were transplanted into inserts. This flat was covered with a 15 cm transparent plastic dome (to maintain high humidity) until the clones established a good root system (approximately 3-4 weeks). Four clones were generated from each plant, and each clone was treated with either a high or low dose of an HPPD inhibitor or 2,4-D to determine the phenotype of each F2 individual for multiple herbicide resistances. Low and high doses of the HPPD inhibitor were 27 g and 270 g ha of Tempotrione, respectively. -1 (Laudis) was used. The low and high doses of 2,4-D were 560 g and 2240 g, respectively. -1 (2,4-D amine) was used. Clones were visually assessed for herbicide damage using a scale of 1 to 10 on 14DAT and 21DAT (a score of 10 indicated no plant damage).

[0193] The cloning and spraying procedures were repeated on 70 different plants from each population to produce enough data for Fisher's exact test to assess whether the two resistance traits were independently separated from each other. Using a visual rating scale cutoff of 3, plants were scored as either susceptible or resistant, and the count data for each category was sent to R for analysis using fisher.test(alternative="two.sided").

[0194] Based on visual evaluation of clones in both quantities using 21DAT, F2 plants were ranked from lowest to highest resistance to both tenbotrione and 2,4-D. Within each F2 population, plants were then grouped into four categories: (1) RR, resistant to both 2,4-D and tenbotrione; (2) RS, resistant to 2,4-D and sensitive to tenbotrione; (3) SR, sensitive to 2,4-D and resistant to tenbotrione; and (4) SS, sensitive to both 2,4-D and tenbotrione. The four most resistant and sensitive plants in each category (16 plants in total from each population and 32 plants overall) were selected for RNA extraction using a Trizol-based method (Simms et al. 1993), followed by treatment with DNase I. After checking the quality and quantity of the samples by running them on a Qubit analyzer and on a 1% agarose gel, respectively, the samples were sent to the Roy J. Carver Biotechnology Center at the University of Illinois, Urbana-Champaign, for Illumina library construction and sequencing.

[0195] RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA-seq Sample Prep kit. Libraries were quantified by qPCR and sequenced across four lanes on a HiSeq 4000 using the HiSeq 4000 sequencing kit version 1. Fastq files were created using bcl2fastq v2.17.1.14 Conversion Software (Illumina) and demultiplexed. Adapters were trimmed from the 3' end of the reads, and any leading or trailing bases with a quality score less than 30 were trimmed via Trimmomatic-0.33, retaining only reads with a length of 30 bp or longer (Bolger et al. 2014).

[0196] Trimmed read files within each subgroup (RR, RS, SR, and SS) were concatenated and assembled using Trinity v2.1.0 (Grabherr et al. 2011). All four resulting assemblies were compared with each other and clustered into groups of transcripts using CD-HIT (Li & Godzik 2006). The longest transcript from each group was used as a representative of that group to generate the final reference transcriptome.

[0197] Dose-response data from previous studies showed that the CHR population exhibited approximately 15-fold resistance to mesotrione and approximately 9-fold resistance to 2,4-D compared to the WUS population (Evans et al. 2019). Similar levels of 2,4-D resistance have been reported in the NEB population, showing 10-fold resistance compared to the Nebraska 2,4-D susceptible population (Bernards et al. 2012), although this susceptible population was restored to susceptibility by pretreatment with the cytochrome P450 inhibitor malathion (Figueiredo et al. 2018). In the case of tenbotrione, 43-fold resistance was observed in the CHR population and 15-fold resistance in the NEB population compared to the WUS population (Murphy and Tranel). (2019). In both the CHR and NEB populations, resistance to tenbotrione and 2,4-D appeared to be independently segregated (p-values ​​= 0.2457 and 0.1457, respectively). By selecting four F2 plants with each resistance combination (RR, RS, SR, and SS), we were able to perform eight overlapping comparisons for each of the two resistance traits from just 16 plants in each population (Figure 1). [Example 2]

[0198] Analysis of differential transcription and differential gene expression Using the following parameters: -b 100--bias--single--rf-stranded-l 255-s 40, each sample was aligned to a reference transcriptome assembly using kallisto (Bray et al. 2016). These pseudo-alignments were then analyzed for differential expression using sluice (Pimentel et al. 2017) with herbicide sensitivity assessment (R vs S) as a condition. Sluice analysis was performed for all four comparisons: tenbotrione resistance vs sensitivity for the NEB population, tenbotrione resistance vs sensitivity for the CHR population, 2,4-D resistance vs sensitivity for the NEB population, and 2,4-D resistance vs sensitivity for the CHR population (n=8). The transcripts were further mapped to a gene model derived from the Amaranth reference genome assembly (Lightfoot et al. 2017; Genbank accession GCA_000753965.1) to calculate differential expression at the gene level and fix the genes to scaffolds, thereby potentially identifying arbitrary physical clusterings of differentially expressed genes (DEGs). Using GMAP (Wu & Watanabe 2005), the transcripts were aligned to the genome in a splice-aware manner (--cross-species-n 1--min-trimmed-coverage=0.80--min-identity=0.80). This gene transcript mapping table was then sent to a sluice, which was run again in gene mode to calculate differential gene expression between herbicide-resistant and susceptible cohorts. Genes with a Benjamini-Hochberg corrected p-value (Benjamini & Hochberg 1995) of 0.1 or less were considered DEGs and used in further analysis.

[0199] The transcriptome was assembled into 57,106 transcripts with a total length of 98,112,700 bp. All 32 libraries (16 for each population) were sequenced to a minimum of 40 million reads per sample (total sequenced reads ranged from 40,800,978 bp to 54,938,593 bp). Over 80% of the reads were aligned to the transcriptome for each sample, resulting in an average alignment of 81.3% across all libraries, providing approximately 40× coverage across the entire transcriptome.

[0200] In the CHRF2 population, there were 39 differentially expressed transcripts (DETs) between 2,4-D resistant and 2,4-D sensitive plants, and 121 DETs between tenbotrione resistant and sensitive plants. In the NEB F2 population, 1445 differentially expressed transcripts were found between 2,4-D resistant and sensitive plants, and 115 differentially expressed transcripts were found between tenbotrione resistant and sensitive plants.

[0201] From the differentially expressed genes that emerged from the data of all four comparisons, the most likely candidates for herbicide resistance were identified based on their relative ranking, polyploidy, and gene annotation as potential metabolic resistance genes, as supported by previous publications suggesting herbicide metabolism-based resistance mechanisms in these populations (Figueiredo et al., 2018; Evans et al., 2019).

[0202] Quantitative PCR primers were developed for each candidate gene (Table 3).

[0203] Primers were also prepared for six housekeeping genes, and PCR efficiency was calculated for all primer sets using a five-step logarithmic scale serial dilution of cDNA. Only primer sets that showed PCR efficiency close to 100% (+ / -5%) were retained and used for further analysis.

[0204] [Table 3]

[0205] To validate the results of differential analysis, subsets of F2 plants were selected from both CHR-derived and NEB-derived populations, including individuals used and not used in RNA-seq (n=14). RNA was extracted from all samples using the Trizol method (described above), and the RNA was converted to cDNA using the ProtoScript First Strand cDNA Synthesis Kit (NEB). Quantitative PCR was performed three times with each primer set for each sample, using a combination of 5 μL of iTaq Universal SYBR Green Supermix (Bio-Rad), 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 3 μL of nuclease-free water, and 1 μL of cDNA. Three housekeeping genes were run on each plate for each sample and used as endogenous controls, with assays performed 2-3 times to ensure consistent results. A susceptible parent (WUS) was used as a reference sample. -ΔΔCt Relative expression was calculated using the method (Livak & Schmittgen 2001). These expression values ​​were then regression-regressed against R phenotypic evaluations (stats v3.6.1) to test for significant linear relationships for each population.

[0206] One of the transcripts most significantly differentially expressed in the 2,4-D resistant CHR population was cytochrome P450 (CYP81E8), also identified as isoflavone 2'-hydroxylase. This same cytochrome P450 was also found to be significantly overexpressed in 2,4-D resistant plants in the NEB population, suggesting the possibility of shared resistance mechanisms between the two populations despite their different geographical origins. Quantitative PCR analysis confirmed the overexpression of CYP81E8, revealing a strong correlation between its expression and the phenotypic response to 2,4-D in both populations (Table 4).

[0207] Other putative resistance genes underwent the same qPCR validation process, which confirmed that glucosyltransferase (UDP-glucose flavonoid 3-O-glucosyltransferase) expression was higher in NEB plants resistant to HPPD inhibitors. The ABC transporter, which emerged as a DET in the CHR population of Tempotrione, was also found to correlate with resistance in both populations, not only to HPPD inhibitors but also to 2,4-D resistance. All genes were also tested for genome copy number augmentation using qPCR-based assays, but no evidence of gene duplication was found for any of these DETs.

[0208] [Table 4]

[0209] By measuring differential expression at the gene level, we (1) increased power and eliminated confounding information caused by minor transcription isoforms, and (2) enabled the later mapping of genes to the genome for spatial gene expression profiling. In the CHR population, we obtained 90 and 31 differentially expressed genes (DEGs) for 2,4-D comparison and tenbotrione comparison, respectively. Here again, the NEB population gave higher values, with 676 DEGs found for 2,4-D comparison and 268 DEGs for tenbotrione comparison. [Example 3]

[0210] Co-expression cluster analysis We tested significant clustering of DEGs using CROC (Pignatelli et al. 2009). CROC searches for clusters using a hypergeometric distribution test, calculating the probability of obtaining k DEGs (out of all n genes) in a sliding window along each scaffold. A significant cluster was defined as having an adjusted p-value (FDR) less than 0.05, using a window size of 1 Mbp and an offset size of 500 kbp. Using a sliding window approach, the resulting clustering along each of the 16 longest scaffolds was visualized using R v3.5.1 (R Core Team 2018). The number of DEGs within each window was counted with a window size of 500 kb and a step size of 500 kb, and plotted using a custom R script.

[0211] Additionally, overpresentation of DEGs at the chromosomal level was tested by totaling the number of DEGs across each chromosome and comparing this number to the predicted number of DEGs on that chromosome using Fisher's exact test in R. Adjusted p-values ​​(p.adjust,method='bonferroni') were calculated.

[0212] Genes differentially expressed between the 2,4-D resistant and susceptible biotypes of both CHR and NEB were found to be physically clustered together in a small number of chromosomal regions. CROC analysis revealed significant clustering in regions on scaffold 4 for both populations, and significant regions on scaffold 7 for the NEB population (Table 5; Figure 2A).

[0213] While no significant regional clustering for DEG was observed between HPPD-resistant and HPPD-sensitive plants, Fisher's exact test for DEG overpresentation across chromosomal scaffolds indicated a significantly higher number of DEGs for NEB on scaffolds 6 and 13 than expected. This overpresentation analysis also identified the previously found significant clustering for 2,4-D comparisons on scaffold 4 (for CHR and NEB) and scaffold 7 (for NEB), as well as clustering for NEB on scaffold 13. The small sample size (n=8) may have been insufficient for adequate resolution of co-expression clusters in the HPPD comparisons.

[0214] [Table 5] [Example 4]

[0215] Condition-specific SNP Single nucleotide polymorphisms were recalled using best practices outlined in GATK v3.7 (Van der Auwera et al. 2013). Clean reads from each RNA-seq sample were initially mapped to the Aucuba japonica genome using STAR v2.5.3 (Dobin et al. 2012) with the following parameters: --outSAMtype BAM SortedByCoordinate--quantMode TranscriptomeSAM GeneCounts--sjdbGTFtagExonParentTranscript Parent. Reading groups were assigned, PCR duplication was removed using Picard Tools v1.95 (The Broad Institute 2019), and then the sequences, extended into intron regions, were hard clipped using the GATK SplitNCigarReads tool. To correct any systematic bias in the quality of each aligned base, GATK BaseRecalibrator was run using a series of high-quality SNPs. Since a high-quality SNP dataset does not exist for Aesculus nigricans, we first created a set from the data generated herein by first running the first round of variant calls on uncalibrated data using the GATK HaplotypeCaller and GenotypeGVCFs functions, and then hard filtering SNPs using the following strict parameters: QD<2.0;FS>60.0;MQ<40.0;MQRankSum<-12.5;ReadPosRankSum<-8.0. After base recalibration, we ran variant calls again on calibrated data, in this case using HaplotypeCaller (parameters: -dontUseSoftClippedBases-stand_call_conf 20.0--variant_index_type LINEAR--variant_index_parameter 128000-ERC GVCF) and Genotype GVCF.SNPs were extracted from the final mutant file and filtered to include only biallelelic SNPs that passed the following parameters: -window 35-cluster 3-filter QD<2.0-filter FS>30.0.

[0216] From this final SNP dataset, condition-specific SNPs were identified using the case / control association analysis in PLINK v1.9 (Chang et al. 2015; Steiss et al. 2012). Due to the small sample sizes (n=8) for the comparison of herbicide resistance versus herbicide sensitivity, an adaptive Monte Carlo permutation test with 1000 replicates was also run as part of this association analysis. SNPs that differed between R plants and S plants with corrected p-values ​​of 0.05 or less were defined as condition-specific SNPs. Similar to DEG, these condition-specific SNPs were visualized using a sliding window approach with a window size of 500kb and a step size of 500kb.

[0217] To check for the presence of any resistance-specific SNPs in these populations, SNPs were called across all genes, and condition-specific SNPs (those that varied between resistant and susceptible plants) were identified using Fisher's exact test in PLINK v1.9. Using an adjusted p-value cutoff of 0.05, 10 and 192 SNPs were found to be associated with resistance in the 2,4-D resistance vs. susceptibility comparison of CHR and NEB, respectively. In both populations, SNPs were found to cluster in the same regions where clustering was found in DEG. In CHR, 9 of the 10 SNPs were found in the scaffold 4 region containing the CYP81E8 gene, while the other SNPs were found in scaffold 6. Within the scaffold 4 cluster, significant SNPs were found in both the CYP81E8 gene and the PIN3 auxin efflux carrier gene (this is interesting considering that 2,4-D is a synthetic auxin). However, 2,4-D resistance is unlikely to be attributable to any one of these SNPs, as they are in linkage disequilibrium with each other, making it difficult to pinpoint the location of the causative mutant. Fine mapping of this region is currently underway. In NEB, 182 SNPs were found in scaffold 4, six in scaffold 7 (where expression analysis also showed a DEG cluster), and four other SNPs scattered across scaffolds 1, 2, and 16. A sliding window graph illustrates the clustering of these SNPs, and when compared with a DEG sliding window graph, co-occurrence of DEG and SNP clustering is shown (Figure 2B). No significant SNPs were found between resistant and susceptible plants in HPPD comparisons. The lack of SNP clustering in HPPD comparisons may be due to the more complex nature of this resistance trait, as it has been demonstrated to be polygenic in these populations (Murphy and Tranel, 2019). [Example 5]

[0218] Analysis of allele-specific expression Considering the co-occurrence of both differential gene expression and condition-specific SNPs in several regions of the genome, the hypothesis of allele-specific expression was tested using read count data for each condition-specific SNP to identify all heterozygous individuals (those showing expression of each allele). Then, using homozygous resistant and susceptible plants at each SNP site, each SNP was classified as R or S, and then, using the read count data for each R or S-associated SNP in heterozygous individuals, the significant difference in read depth between R and S SNPs was tested using R(rstatix). SNPs and their associated adjusted p-values ​​(Benjamini-Hochberg, p=0.1) were plotted across scaffold 4 cluster regions using R(ggpubr).

[0219] Clustering of condition-specific SNPs exhibiting differential gene expression regions suggested the presence of allele-specific expression. Allele-specific expression (ASE) is defined as a form of allele imbalance in which one parent's allele is preferentially expressed over the other (Knight 2004). In the scaffold 4 cluster, nine SNPs were found to be statistically significant differentially expressed for NEB (Figure 3A). For all but one, the R allele was significantly more highly expressed than the S allele, suggesting that some cis-acting factor is involved in this region and consequently regulates its expression. In the CHR population, four SNPs were present in this scaffold 4 region in heterozygous individuals, three of which showed significantly different expression between the two alleles (Figure 3B), but here again, the R allele was more highly expressed than the S allele. While ASE may exist elsewhere along this region, only SNPs found to be present in a heterozygous state across three or more individuals were included in this analysis. [Example 6]

[0220] Cytochrome 81E8 phylogenetic analysis Both the CHR and NEB populations exhibited the same upregulated alleles for 2,4-D resistance in the CYP81E8 gene, raising the question of whether this putative resistance allele evolved independently in each population. Using previously published whole-genome datasets from Illinois and Canadian Ayu samples (Kreiner et al. 2019), we constructed phylogenetic trees to examine the evolutionary relationships of CYP81E8 from each population. Whole-genome or whole-transcriptome datasets were aligned to the CDS of CYP81E8 using bowtie2 (Langmead & Salzberg 2012) (parameters: --no-unal-tL 20). The sorted bam files were then fed into the same GATK SNP pipeline to generate filtered vcf files. The SNPRelate package in R allowed me to convert this vcf file to a gds file, and then use this gds file to create a dendogram based on the relationships (snpgdsHCluster;snpgdsCutTree, n.perm=5000).

[0221] Phylogenetic analysis of the CYP81E8 gene revealed the evolutionary relationship between the CYP81E8 alleles from both the CHR and NEB populations, as well as from other Auricularia populations from Illinois, Missouri, and Canada. The CYP81E8 alleles from CHR and NEB were divided into three groups: (1) 2,4-D susceptible alleles from NEB, (2) 2,4-D susceptible alleles from CHR, and (3) 2,4-D resistant alleles from both CHR and NEB (Figure 4). The close clustering of 2,4-D resistant-associated CYP81E8 from CHR and NEB, along with the segregation of wild-type susceptible alleles from CHR and NEB, provides sufficient evidence that the R alleles in both populations share a common evolutionary origin.

[0222] Discussion of Examples 1-6 In these examples, strong candidate genes for metabolic-based herbicide resistance were found for 2,4-D in both CHR and NEB populations. Both cytochrome P450 (CYP81E8) and ABC transporter (ABCC10) showed a certain degree of overexpression in 2,4-D-resistant plants compared to 2,4-D-sensitive plants. These results support the earlier study (Figueiredo et al., 2018) in which the cytochrome P450 inhibitor malathion reversed the resistance phenotype, suggesting that 2,4-D resistance in the NEB population is likely mediated by cytochrome P450. The putative resistance allele of this gene was co-isolated with further resistant plants from the F2 population, but fine-mapping is currently underway.

[0223] However, our findings regarding HPPD inhibitor resistance were not very clear. One candidate gene, UDP-glucose flavonoid 3-O-glucosyltransferase, was found to be overexpressed in tenbotrion-resistant plants compared to tenbotrion-sensitive plants. Primary function annotation of this gene indicated its involvement in fruit maturation, but further studies suggested its possible involvement in xenobiotic metabolism through glycosylation of exogenous substances (Greisser et al., 2008). The lack of further candidate HPPD inhibitor resistance genes may be due to their polygenicity (Oliveira et al., 2018), which makes it difficult to identify the resistance locus. Additionally, our RNA-seq approach primarily focused on identifying genes contributing to resistance via constitutive differential expression, which could result in missing other resistance-constituting changes among plants. Recent RNA-seq studies investigating mesotrione resistance in Amarantha japonica, including treated plants, have found some evidence of cytochrome P450a induction in resistant plants compared to susceptible plants (Kohlhase et al., 2019). However, the final list of differentially expressed transcripts in this study was approximately 4800, making it difficult to identify the causative resistance gene. Studies using gene mapping approaches to identify HPPD inhibitor resistance genes in NEB and CHR populations are currently underway.

[0224] The fact that the plants were not treated with herbicides prior to RNA-seq meant that the identification of co-expression networks was not extensively pursued in this study. Without this shared treatment, it seemed unlikely that co-expression analysis would yield anything meaningful, as it measures random differences in expression across two populations. In fact, when we first started working on co-expression networks, we obtained no informative results at all.

[0225] In addition to identifying candidate herbicide resistance genes, these data also reveal several insights into the regulation of herbicide resistance. Physical clustering of DEGs observed for 2,4-D resistance provides evidence of co-expression of colocalized genes, a phenomenon observed in many other species, including yeast (Cohen et al. 2000), Arabidopsis thaliana (Williams & Bowles 2004), C. elegans (Chen & Stein 2006), and humans (Trinklein et al. 2004). While some examples of such co-expression clustering are found between adjacent gene pairs, co-expression across longer chromosomal intervals has also been reported (Lercher & Hurst 2006; Reimegard et al. 2017). The ability of herbicides to alter the genomic landscape of weed species has recently been demonstrated in morning glory (Ipomoea purpurea), where evidence of selective sweeping was found in five genomic regions within the glyphosate-resistant population (Van Etten et al., 2020). Interestingly, enrichment of herbicide detoxification genes was evident within these regions.

[0226] One of the main implications of such clustering is the possibility of shared mechanisms for gene regulation in these regions. Gene expression regulation is a complex process involving selective interactions between transcription factors and enhancers, chromatin opening and closing to enable / prevent transcription, and interactions between these two processes (Voss & Hager 2014). The inventors examined the upstream regions of all DEGs and searched for overpresentation of transcription factor binding sites (TFBSs), but found no evidence of shared enhancer elements. Previous studies investigating the regulatory mechanisms of physically clustered co-expressed genes have shown that co-expressed gene pairs are often regulated by shared transcription factors, while larger regions of co-expression spanning 10-20 genes are influenced by changes in chromatin structure (Batada et al. 2007). However, only a few examples have been studied so far, and the interdependence of regulatory mechanisms makes it difficult to pinpoint the direct cause of gene expression. In any case, more research is needed in these populations to determine the influence of chromatin state on gene expression patterns.

Claims

1. A modified plant, or its offspring, plant part, or plant cell, containing a heterologous polynucleotide encoding a cytochrome P450 81E (CYP81E) polypeptide having at least 90% sequence identity with SEQ ID NO:

2.

2. The modified plant, or its offspring, plant part, or plant cell according to claim 1, wherein the modified plant, or its offspring, plant part, or plant cell, is resistant to auxin herbicides.

3. The modified plant, or its offspring, plant part, or plant cell according to claim 1, wherein the CYP81E polypeptide has at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

2.

4. The modified plant, or its offspring, plant part, or plant cell according to claim 1, wherein the polynucleotide encoding the CYP81E polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

5. The modified plant, its offspring, plant part, or plant cell according to claim 1, wherein the polynucleotide is operably linked to a functional heterologous promoter in the plant cell.

6. The auxin herbicide is 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP), (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T), 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP), 2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (Cromeprop), 2-(4-chloro-2-methylphenoxy)acetic acid (MCPA), 4-(4-chloro-2-methylphenoxy)butanoic acid (MCPB), 2-(4-chloro-2-methylphenoxy)propane Selected from the group consisting of acids (MCPP), 3,6-dichloro-2-pyridinecarboxylic acid (clopyralide), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), [(3,5,6-trichloropyridine-2-yl)oxy]acetic acid (triclopyr), [(4-amino-3,5-dichloro-6-fluoropyridine-2-yl)oxy]acetic acid (fluroxypyr), 3,6-dichloro-2-methoxybenzoic acid (dicamba), 3-amino-2,5-dichlorobenzoic acid (chloramben), 7-chloro-3-methylquinoline-8-carboxylic acid (kinmelac), and 3,7-dichloroquinoline-8-carboxylic acid (quinchlorac), The modified plant according to claim 2, or its offspring, plant part, or plant cell.

7. The auxin herbicide is 2,4-D, as described in claim 6, for the modified plant, or its offspring, plant part, or plant cell.

8. The modified plant according to claim 1, or its offspring, plant part, or plant cell, wherein the plant is a dicotyledonous plant.

9. The modified plant according to claim 1, or its offspring, plant part, or plant cell, wherein the plant is a crop.

10. The modified plant according to claim 1, or its offspring, plant part, or plant cell, wherein the plant is a soybean, cotton, canola, tobacco, tomato, potato, alfalfa, sugar beet, or sunflower.

11. The modified plant further comprises a second herbicide-resistant trait, as described in claim 1, or its offspring, plant part, or plant cell.

12. (a) A nucleotide sequence encoding the CYP81E polypeptide, A nucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1; or (b) A nucleotide sequence encoding the CYP81E polypeptide, CYP81E polypeptide has a nucleotide sequence that is at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

2. Includes a nucleotide sequence selected from, Nucleic acid molecule.

13. The nucleic acid molecule according to claim 12, wherein the nucleic acid molecule is an isolated, synthetic, or recombinant nucleic acid molecule.

14. An expression cassette comprising the nucleic acid molecule according to claim 12, operably linked to a functional heterologous promoter in plant cells.

15. A vector comprising the nucleic acid molecule described in claim 12.

16. A biological sample containing the nucleic acid molecule described in claim 12.

17. A plant, plant part, or plant cell containing the nucleic acid molecule described in claim 12.

18. A CYP81E polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

2.

19. A method for producing a plant resistant to an auxin herbicide, comprising introducing a polynucleotide encoding a CYP81E polypeptide having at least 90% sequence identity with SEQ ID NO: 2 into a plant cell, and regenerating a plant from the plant cell.

20. The method according to claim 19, wherein the polynucleotide is operably linked to a functional heterologous promoter in a plant cell.

21. The method according to claim 19, wherein the CYP81E polypeptide has at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

2.

22. The method according to claim 19, wherein the polynucleotide encoding the CYP81E polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

23. The auxin herbicide is 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP), (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T), 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP), 2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (Cromeprop), 2-(4-chloro-2-methylphenoxy)acetic acid (MCPA), 4-(4-chloro-2-methylphenoxy)butanoic acid (MCPB), 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) The method according to claim 19, wherein a selection is made from the group consisting of ), 3,6-dichloro-2-pyridinecarboxylic acid (clopyralide), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), [(3,5,6-trichloropyridine-2-yl)oxy]acetic acid (triclopyr), [(4-amino-3,5-dichloro-6-fluoropyridine-2-yl)oxy]acetic acid (fluroxypyr), 3,6-dichloro-2-methoxybenzoic acid (dicamba), 3-amino-2,5-dichlorobenzoic acid (chloramben), 7-chloro-3-methylquinoline-8-carboxylic acid (kinmelac), and 3,7-dichloroquinoline-8-carboxylic acid (quinchlorac).

24. The method according to claim 23, wherein the auxin herbicide is 2,4-D.

25. The method according to claim 19, wherein the plant is a dicotyledonous plant.

26. The method according to claim 19, wherein the plant is a crop.

27. The method according to claim 19, wherein the plant is a soybean, cotton, canola, tobacco, tomato, potato, alfalfa, sugar beet, or sunflower.

28. A method for controlling undesirable vegetation in plant cultivation sites: To provide plants containing polynucleotides encoding the CYP81E polypeptide, which has at least approximately 90% sequence identity with Sequence ID No. 2, at plant cultivation sites; Applying an effective amount of auxin herbicide to the plant cultivation site, A method comprising the expression of the polynucleotide conferring resistance to auxin herbicides to plants.

29. The method according to claim 28, wherein the CYP81E polypeptide has at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

2.

30. The method according to claim 28, wherein the polynucleotide encoding the CYP81E polypeptide has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

31. The method according to claim 28, wherein the polynucleotide is operably linked to a functional heterologous promoter in a plant cell.

32. The herbicide is 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP), (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T), 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP), 2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (Cromeprop), 2-(4-chloro-2-methylphenoxy)acetic acid (MCPA), 4-(4-chloro-2-methylphenoxy)butanoic acid (MCPB), 2-(4-chloro-2-methylphenoxy)propanoic acid (M Selected from the group consisting of CPP), 3,6-dichloro-2-pyridinecarboxylic acid (clopyralide), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), [(3,5,6-trichloropyridine-2-yl)oxy]acetic acid (triclopyr), [(4-amino-3,5-dichloro-6-fluoropyridine-2-yl)oxy]acetic acid (fluroxypyr), 3,6-dichloro-2-methoxybenzoic acid (dicamba), 3-amino-2,5-dichlorobenzoic acid (chloramben), 7-chloro-3-methylquinoline-8-carboxylic acid (kinmelac), and 3,7-dichloroquinoline-8-carboxylic acid (quinchlorac), The method according to claim 28.

33. The method according to claim 32, wherein the auxin herbicide is 2,4-D.

34. The method according to claim 28, wherein the plant is a dicotyledonous plant.

35. The method according to claim 28, wherein the plant is a soybean, cotton, canola, tobacco, tomato, potato, alfalfa, sugar beet, or sunflower.

36. A product prepared from a plant, plant part, or plant cell as described in claim 1, comprising a polynucleotide encoding the CYP81E polypeptide as described in claim 1.

37. The product according to claim 36, wherein the product is feed, seed coarse powder, oil, or seed-treatment-coated seed.

38. A method for producing a plant product, comprising processing a plant or plant part as described in claim 1 to obtain a plant product, wherein the plant product comprises a polynucleotide encoding the CYP81E polypeptide as described in claim 1. method.

39. The method according to claim 38, wherein the plant product is feed, seed powder, oil, or seed-treatment-coated seed.

40. A method for identifying auxin herbicide-resistant plants, Prepare a biological sample derived from a plant that is suspected to have resistance to auxin herbicides; The objective is to quantify the expression of the CYP81E gene in a biological sample that has at least 90% sequence identity with SEQ ID NO: 1, To quantify the differential expression of the CYP81E gene in auxin-resistant plants compared to auxin-sensitive plants of the same species; Based on quantification, it is possible to determine whether plants are resistant to auxin herbicides, A method that includes this.

41. The method according to claim 40, wherein the biological sample is derived from Amanita japonica.

42. The method according to claim 40, wherein the auxin herbicide is 2,4-D.

43. The method according to claim 40, wherein quantifying the expression of the CYP81E gene includes quantifying CYP81E mRNA.

44. The method according to claim 40, wherein quantifying the expression of the CYP81E gene comprises quantifying the CYP81E polypeptide.

45. The method according to claim 40, wherein the CYP81E gene has at least four times differential expression in auxin herbicide-resistant plants compared to auxin herbicide-sensitive plants before application of the auxin herbicide.

46. The method according to claim 40, wherein the CYP81E gene has at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

47. The method according to claim 40, wherein quantifying expression comprises amplifying a nucleic acid using at least two primers.

48. The method according to claim 47, wherein at least two primers include SEQ ID NO: 5 and SEQ ID NO:

6.

49. A kit for identifying auxin herbicide-resistant plants, comprising at least two primers, wherein at least two primers recognize the CYP81E gene which has at least 90% sequence identity with SEQ ID NO: 1, which is differentially expressed in auxin herbicide-resistant plants compared to auxin herbicide-sensitive plants of the same species. kit.

50. The kit according to claim 49, wherein the CYP81E gene has at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

51. The kit according to claim 49, further comprising at least one of a positive control and a negative control.

52. The kit according to claim 49, further comprising the components of a qRT-PCR solution.

53. The kit according to claim 49, wherein the plant is Amaranthus spp. and the auxin herbicide is 2,4-D.

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