Patent Description:
Soybean (Glycine max) is an important legume crop worldwide due to its ability to fix atmospheric nitrogen. Soybean also serves as a major source of animal feed protein, and its oil has uses ranging from cooking/frying to the production of biodiesel. Typically, a hydrogenation process is used to increase heat stability and improve the shelf life and taste of soybean oil. However, hydrogenation increases the cost of production and results in the formation of trans fatty acids, which have been linked to cardiovascular disease in humans.

Provided herein are materials and methods for modifying soybean oil composition by reducing or eliminating expression of the delta-fifteen fatty acid desaturase <NUM> (FAD3) A gene without the use of transgenesis.

The methods described herein utilize sequence-specific, TAL effector endonucleases to introduce mutations in the FAD3A coding sequence, thereby knocking out gene function. These methods can mediate FAD3A silencing without insertion of a transgene. Plants containing transgenes are highly regulated in certain jurisdictions, including Europe, and the costs to obtain regulatory approval can be very high. The methods described herein can expedite the production of new varieties, and can be more cost-effective than transgenic or traditional breeding approaches.

The present invention provides a method for making a soybean plant containing a mutation in: one or more FAD2-1A alleles, one or more FAD2-1B alleles, and one or more FAD3A alleles; the method comprising: (a) providing soybean plant parts or plant cells, wherein the plant parts or plant cells comprise a mutation in one or more FAD2-1A alleles and a mutation in one or more FAD2-1B alleles, wherein each FAD2-1A allele has a sequence as set forth in SEQ ID NO: <NUM>, or has a sequence with at least <NUM>% identity to SEQ ID NO: <NUM>; wherein each FAD2-1B allele has a sequence as set forth in SEQ ID NO:<NUM>, or has a sequence with at least <NUM>% identity to SEQ ID NO:<NUM>; and wherein the plant parts or plant cells comprise at least a functional FAD3A allele, (b) contacting the plant parts or plant cells with one or more TAL effector endonucleases targeted to an endogenous FAD3A sequence, wherein each of the TAL effector endonucleases is targeted to a pair of sequences as set forth in SEQ ID NOS <NUM> and <NUM>, (c) regenerating the plant parts or plant cells into whole soybean plants, and (d) selecting from the whole soybean plants a soybean plant comprising a mutation in one or more FAD3A alleles, wherein each mutated FAD3A allele comprises a deletion of the cytosine nucleotide at position <NUM> of SEQ ID NO:<NUM>. The soybean plant parts or plant cells can be selected from the group consisting of cotyledon cells, seeds, embryos, embryogenic calli cells, and pollen cells. The contacting can include transforming the soybean plant cells with one or more vectors encoding the one or more rare-cutting endonucleases. The one or more rare-cutting endonucleases can be TAL effector endonucleases. Each mutated FAD3A allele can have a sequence as set forth in any of SEQ ID NOS:<NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>, or a sequence with at least <NUM>% identity to any of SEQ ID NOS:<NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>. The method can include introducing into the plant cells one or more TAL effector endonuclease proteins, culturing the plant cells to generate plant lines, and/or isolating, from the plant cells, genomic DNA containing at least a portion of the FAD3A locus.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

Commodity soybean oil is made up principally of five fatty acids: palmitic acid (<NUM>%), stearic acid (<NUM>%), oleic acid (<NUM>%), linoleic acid (<NUM>%), and linolenic acid (<NUM>%). Plant oils with low linolenic acid content may increase the oxidative and frying stability of soybean oil. Such oils also may be healthier, particularly since hydrogenation (a chemically-induced reduction reaction that saturates fatty acids) will reduce the levels of trans fatty acids, which are known to increase risk of heart disease. Traditional breeding, mutagenesis, and siRNA strategies have been used to generate soybean varieties containing reduced levels of linolenic acid. See, e.g., <NPL>. Transgenes expressing RNAi constructs are subject to variation in transgene expression, however, and conventional breeding efforts can be a lengthy and costly process.

Enzymes responsible for the biosynthetic progression from palmitic acid to linolenic acid have been identified. For example, the FAD3 enzymes are responsible for converting linoleic acid precursors to linolenic acid precursors during oil accumulation in developing soybean seeds. Due to ancient polyploidization and multiple duplication events, three copies of FAD3 (FAD3A, FAD3B, and FAD3C) exist in the soybean genome. FAD3A and FAD3B have <NUM>% sequence identity at the DNA level, and the encoded proteins have <NUM>% sequence identity at the amino acid level. FAD3A and FAD3C have <NUM>% sequence identity at the DNA level, and the encoded proteins have <NUM>% sequence identity at the amino acid level. FAD3B and FAD3C have <NUM>% sequence identity at the DNA level, and the encoded proteins have <NUM>% sequence identity at the amino acid level. Plants homozygous for naturally occurring FAD3A mutant alleles can have a modest decrease in linolenic acid composition (to about <NUM>% of the total fatty acid content), as described elsewhere (<NPL>). FAD3 double mutants have shown decreased linolenic acid (<NUM>%) in total seed oil, while mutations in FAD3A, FAD3B, and FAD3C resulted in a further decrease (<NUM>%) in linolenic acid content (<NPL>).

The present invention provides a method for generating soybean plants that have reduced (e.g., decreased or completely abolished) FAD3A activity. In some embodiments, the methods described herein can be used to generate soybean varieties having oil with a decreased linolenic acid component that is <NUM>% or less (e.g., <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, or <NUM>% or less) of the total fatty acid content. In some embodiments, this modification of soybean oil composition can be achieved by completely knocking out expression of the FAD3A genes. According to some of the methods provided herein, both alleles of FAD3A genes are inactivated using non-transgenic techniques. Removing the gene products of FAD3A can severely reduce the conversion of linoleic acid precursors to linolenic acid precursors in soybean seeds.

As used herein, the terms "fatty acid content" and "fatty acid composition" refer to levels of specific fatty acids relative to total fatty acids. "Fatty acid content" and "fatty acid composition" can be represented as a percentage. For example, the overall fatty acid content/fatty acid composition of oil from wild type soybean plants is about <NUM>% palmitic acid, <NUM>% stearic acid, <NUM>% oleic acid, <NUM>% linoleic acid, and <NUM>% linolenic acid. The content of particular fatty acids also can be described related to total fatty acids. For example, the "linolenic acid content" is the level of linolenic acid relative to total fatty acids, such that the "linolenic acid content" within oil from wild type soybean plants is about <NUM>%. Similarly, the "oleic acid content" within oil from wild type soybean plants is about <NUM>%, and the "linoleic acid content" within oil from wild type soybean plants is about <NUM>%.

To accomplish reduced or even complete elimination of FAD3A, FAD3B, and/or FAD3C expression, for example, an engineered, rare-cutting nuclease (e.g., a transcription activator-like (TAL) effector endonuclease) can be designed to recognize a conserved region of all FAD3 genes and create a double-strand break. Improper repair due to Non-Homologous End Joining (NHEJ) at each DNA break site can generate missense and/or nonsense mutations in the FAD3A, FAD3B, and/or FAD3C coding regions, rendering the FAD3A, FAD3B, and/or FAD3C RNA transcripts unstable and targeted for degradation prior to translation. The method of the present invention comprises contacting the plant parts or plant cells with one or more TAL effector endonucleases targeted to an endogenous FAD3A sequence, wherein each of the TAL effector endonucleases is targeted to a pair of sequences as set forth in SEQ ID NOS <NUM> and <NUM>.

In soybean, there are at least three members (A, B, and C) of the FAD3 gene family. Representative examples of naturally occurring soybean FAD3A, FAD3B, and FAD3C nucleotide sequences are shown in TABLE <NUM> (SEQ ID NOS:<NUM>, <NUM>, and <NUM>). The soybean plants, cells, plant parts, seeds, and progeny thereof described herein can have a mutation in each of the endogenous FAD3A alleles, such that expression of each gene is reduced or completely abolished. Alternatively, the soybean plants, cells, plant parts, seeds, and progeny thereof described herein may have a mutation in at least one FAD3A allele, such that expression of each gene is reduced or completely abolished. The soybean plants, cells, parts, seeds, and progeny can have decreased levels of linolenic acid as compared to wild type soybean plants, cells, parts, seeds, and progeny.

As used herein, the terms "plants" and "plant parts" refer to cells, tissues, organs, seeds, and severed parts (e.g., roots, leaves, and flowers) that retain the distinguishing characteristics of the parent plant. "Seed" refers to any plant structure that is formed by continued differentiation of the ovule of the plant, following its normal maturation point at flower opening, irrespective of whether it is formed in the presence or absence of fertilization and irrespective of whether or not the seed structure is fertile or infertile.

The term "gene" as used herein refers to a nucleic acid sequence that includes a promoter region associated with expression of a gene product. "Gene" also encompasses intron and exon regions associated with expression of the gene product, as well as <NUM>' or <NUM>' untranslated regions associated with expression of the gene product.

The term "allele(s)" means any of one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploid) cell of an organism, alleles of a given gene are located at a specific location or locus on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes. "Heterozygous" alleles are two different alleles residing at a specific locus, positioned individually on corresponding pairs of homologous chromosomes. "Homozygous" alleles are two identical alleles residing at a specific locus, positioned individually on corresponding pairs of homologous chromosomes in the cell.

A "wild type" allele or gene refers to an allele or gene that most commonly occurs in nature, and typically is associated with the wild type phenotype. For example, a "wild type FAD3A allele" is a naturally occurring FAD3A allele (e.g., as found within naturally occurring soybean plants) that encodes a functional FAD3A protein. The terms "mutant" and "mutation" are used in connection with alleles, genes, and plant phenotypes that are different from the conventional or wild type alleles, genes, and plant phenotypes that most commonly occur in nature. A mutant plant or allele can occur in the natural population or can be produced by human intervention (e.g., by mutagenesis), and a "mutant allele" refers to an allele having one or more changes in its nucleic acid sequence when compared to the wild type allele, such that it can result in a mutant phenotype either alone or in combination with another mutant allele. In some embodiments, for example, a "mutant FAD3A allele" can be a FAD3A allele that does not encode a functional FAD3A protein; a "mutant FAD3A allele" may include one or more mutations in its nucleic acid sequence that result in no detectable amount of functional FAD3A protein in the plant or plant cell in vivo.

"Mutagenesis" as used herein refers to processes in which mutations are introduced into a selected DNA sequence. Mutations can include one or more deletions, insertions, and/or substitutions. Mutations induced by endonucleases generally are the result of a double strand break, which can yield insertions or deletions ("indels") that are detectable by deep-sequencing analysis. Such mutations typically are deletions of at least several base pairs, and can have the effect of inactivating the mutated allele. A deletion that results in a frameshift, for example, can inactivate an allele. In some cases, mutations can include large deletions that remove all or a portion of a gene of interest. Deletions can range in size from one bp to <NUM> bp or more (e.g., one to ten bp, <NUM> to <NUM> bp, <NUM> to <NUM> bp, <NUM> to <NUM> bp, <NUM> to <NUM> bp, <NUM> to <NUM> bp, <NUM> to <NUM> bp, <NUM> to <NUM> bp, or more than <NUM> bp). Mutations also can be introduced into a promoter region to decrease or inactivate expression of the corresponding gene. In the methods described herein, for example, mutagenesis can occur via double stranded DNA breaks made by TAL effector endonucleases targeted to selected DNA sequences in a plant cell. Such mutagenesis results in "TAL effector endonuclease-induced mutations" (e.g., TAL effector endonuclease-induced knockouts) and reduced expression of the targeted gene. Following mutagenesis, plants can be regenerated from the treated cells using known techniques (e.g., planting seeds in accordance with conventional growing procedures, followed by self-pollination).

As used herein, the phrase "mutation in one or more FAD alleles" refers to one or more FAD (e.g., FAD2 or FAD3) alleles with sequences that are altered such that their expression is reduced, or such that the activity of the encoded FAD protein is reduced, as compared to the corresponding wild type allele or protein. This can occur either by the mutant FAD allele encoding a non-functional FAD protein (e.g., a FAD protein having no biological activity, or a FAD protein with significantly modified or reduced biological activity, as compared to the corresponding wild type FAD protein), or by the mutant FAD allele expressing a significantly reduced amount of functional FAD protein, or no FAD protein at all. As used herein, "significantly" indicates that a result is reproducibly different, typically with a P value < <NUM>.

A mutation in a FAD nucleic acid sequence can be, for example, a missense mutation, a nonsense mutation, an insertion mutation, a deletion mutation, a frameshift mutation or a splice site mutation, or a combination of the aforementioned mutations.

The term "expression" as used herein refers to the transcription of a particular nucleic acid sequence to produce sense or antisense mRNA, and/or the translation of a sense mRNA molecule to produce a polypeptide, with or without subsequent post-translational events.

The term "reduced" as used herein with regard to expression of a FAD allele or activity of an encoded FAD polypeptide refers to any decrease in expression or activity as compared to that of a corresponding wild type FAD allele or polypeptide. For example, expression or activity can be decreased by at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%, or by <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, or <NUM> to <NUM>%. "Reducing the expression" of a gene or polypeptide in a plant or a plant cell can be achieved by inhibiting, interrupting, knocking-out, or knocking-down the gene or polypeptide, such that transcription of the gene and/or translation of the encoded polypeptide are reduced as compared to a corresponding wild type plant or plant cell. Expression levels can be assessed using methods such as, for example, reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, dot-blot hybridization, in situ hybridization, nuclear run-on and/or nuclear run-off, RNase protection, or immunological and enzymatic methods such as ELISA, radioimmunoassay, and western blotting.

The term "reduced" with regard to fatty acid content refers to a fatty acid level that is less than that in oil from a wild type soybean plant. For example, "reduced linolenic acid content" refers to a linolenic acid content that is less than the linolenic acid content found in oil from a wild type soybean plant, and "reduced linoleic acid content" is a linoleic acid content that is less than the linoleic acid content found in oil from a wild type soybean plant. The content of a particular fatty acid can be decreased by at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%, or by <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, or <NUM> to <NUM>% as compared to the content of that fatty acid in oil from a wild type soybean plant. Thus, the level of linoleic acid in oil from a soybean plant, plant part, or plant cell as provided herein may be <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, or <NUM>% or less of the total fatty acid content. The level of linolenic acid in oil from a soybean plant, plant part, or plant cell as provided herein may be <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, or <NUM>% or less of the total fatty acid content.

As used herein, the term "increased" with regard to fatty acid content refers to a fatty acid level that is more than that in oil from a wild type soybean plant. For example, "increase oleic acid content" refers to an oleic acid content that is more than the oleic acid content found in oil from a wild type soybean plant. The content of a particular fatty acid can be increased by at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%, or by <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM> to <NUM>%, or more than <NUM>% as compared to the content of that fatty acid in oil from a wild type soybean plant. Thus, the level of oleic acid in oil from a soybean plant, plant part, or plant cell as provided herein may be <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more <NUM>% or more, <NUM>% or more, or <NUM>% or more of the total fatty acid content.

As used herein, a "knockout" or "inactive" allele refers to a mutant allele that is not expressed, or to a mutant allele that encodes a protein without its normal functional activity. Knockout FAD alleles include, for example, deletion mutations of the entire coding region or a substantial part of the coding region, or frameshift or stop-codon mutations that lead to a substantial deletion or entire deletion of the protein. Knockout FAD alleles also encompass alleles having splice site mutations, deletions or insertions in the promoter region, and, with respect to FAD3, alleles with a mutation that removes or replaces any of the eight conserved histidine residues that are present within the FAD3 proteins.

The eight conserved histidine residues are found within region Ia, region Ib, and region II of the FAD3 proteins. The conserved amino acids including the histidine residues within region Ia of the soybean FAD3 genes are HDCGH (SEQ ID NO:<NUM>), which are encoded by the nucleotides beginning at position <NUM> of SEQ ID NO:<NUM> (FAD3A), the nucleotides beginning at position <NUM> of SEQ ID NO:<NUM> (FAD3B), and the nucleotides beginning at position <NUM> of SEQ ID NO:<NUM> (FAD3C). The conserved amino acid sequence that contains the histidines within region Ib of the soybean FAD3 proteins is HRTHH (SEQ ID NO:<NUM>), which is encoded by nucleotides <NUM>-<NUM> of SEQ ID NO:<NUM> (FAD3A), nucleotides <NUM>-<NUM> of SEQ ID NO:<NUM> (FAD3B), and nucleotides <NUM>-<NUM> of SEQ ID NO:<NUM> (FAD3C). The conserved amino acid sequence containing the histidines within region II of the soybean FAD3 proteins is HVIHH (SEQ ID NO:<NUM>), which is encoded by nucleotides <NUM>-<NUM> of SEQ ID NO:<NUM> (FAD3A), nucleotides <NUM>-<NUM> of SEQ ID NO:<NUM> (FAD3B), and nucleotides <NUM>-<NUM> of SEQ ID NO:<NUM> (FAD3C). Mutations that disrupt any of these histidines can result in a knockout FAD3 allele. For example, an in-frame deletion encompassing any of the nucleotides encoding a conserved histidine, a frameshift mutation that occurs before the last conserved histidine, and a missense mutation occurring at any of the codons encoding a conserved histidine will result in a FAD3 knockout allele.

In the present invention, each mutated FAD3A allele comprises a deletion of the cytosine at position <NUM> of SEQ ID NO:<NUM>. Such deletions can result in knockout of the FAD3 gene, such as when they are included within a frameshift mutation, or when they are included in an in-frame deletion that removes at least one conserved histidine residue or another conserved portion of the protein, for example.

The term "rare-cutting endonucleases" herein refer to natural or engineered proteins having endonuclease activity directed to nucleic acid sequences having a recognition sequence (target sequence) about <NUM>-<NUM> bp in length (e.g., <NUM>-<NUM> bp in length). Typical rare-cutting endonucleases cause cleavage inside their recognition site, leaving <NUM> nt staggered cut with <NUM>'OH or <NUM>'OH overhangs. These rare-cutting endonucleases may be the result from fusion proteins that associate a DNA binding domain and a catalytic domain with cleavage activity. TAL-effector endonucleases are examples of fusions of DNA binding domains with the catalytic domain of the endonuclease FokI. Customized TAL effector endonucleases are commercially available under the trade name TALEN™ (Cellectis, Paris, France). For a review of rare-cutting endonucleases, see <NPL>.

In the present invention, the plants, plant cells, plant parts, seeds, and plant progeny provided by the method described herein are generated using a TAL effector endonuclease system to make targeted knockouts in the FAD3A gene. For example, nucleic acids encoding TAL effector endonucleases targeted to a pair of sequences as set forth in SEQ ID NOS <NUM> and <NUM> (see <FIG> and TABLE <NUM>) can be transformed into plant cells (e.g., cells in cotyledons), where they can be expressed. Thus, this disclosure provides materials and methods for using TAL effector endonucleases to generate plants and related products (e.g., seeds and plant parts) that are particularly suitable for production of soybean oil with decreased linolenic acid content.

In the present invention, the plants, plant parts, and plant cells described herein also include a mutation in one or more FAD2-1A alleles and a mutation in one or more FAD2-1B alleles. The FAD2 genes are responsible for converting oleic acid precursors to linoleic acid precursors during oil accumulation in developing soybean seeds. Two copies of FAD2 (FAD2-1A and FAD2-1B) exist in the soybean genome. These genes have about <NUM>% sequence identity at the DNA level, and the encoded proteins have about <NUM>% sequence identity at the amino acid level. Plants homozygous for naturally occurring FAD2-1B mutant alleles can have a modest increase (<NUM>% to <NUM>%) in oleic acid composition, as described elsewhere (<NPL>). Mutations in FAD2-1A have been developed through X-ray mutagenesis and TILLING, to produce seeds containing up to <NUM>% oleic acid (<NPL>), and mutating both the FAD2-1A and FAD2-1B alleles resulted in oil with an oleic acid content of <NUM>% (Pham et al.

Thus, the method of the present invention provides soybean plants that have reduced (e.g., lack) FAD2-1A and FAD2-1B activity in addition to reduced FAD3A activity. This is achieved by knocking out FAD3A in FAD2 mutants,. A rare cutting endonuclease (e.g., a TAL effector endonuclease) system can be used to make targeted knockouts in the FAD2-1A and/or FAD2-1B genes. In some embodiments, the soybean plants, cells, plant parts, seeds, and progeny thereof described herein can have a mutation in each of the endogenous FAD2-1A and FAD2-1B alleles (in addition to a mutation in one or more of the endogenous FAD3A alleles), such that expression of each gene is reduced or completely abolished. Alternatively, the soybean plants, cells, plant parts, seeds, and progeny thereof provided herein may have a mutation in at least one FAD2-1A allele and in at least one FAD2-1B allele (in addition to a mutation in one or more of the endogenous FAD3A alleles), such that expression of each gene is reduced or completely abolished. The soybean plants, cells, parts, seeds, and progeny can have reduced levels of linolenic acid, increased levels of oleic acid, and reduced levels of linoleic acid as compared to wild type soybean plants, cells, parts, seeds, and progeny.

In some embodiments, the methods provided herein can be used to produce plant parts (e.g., seeds) or plant products (e.g., oil) having increased oleic acid content, reduced linoleic acid content, and reduced linolenic acid content, as compared corresponding plant parts or products from wild type plants. For example, the methods described herein can be used to generate soybean varieties having oil with a decreased linolenic acid component of <NUM>% or less (e.g., <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, or <NUM>% or less) of the total fatty acid content, as well as an increased oleic acid component of at least <NUM>% (e.g., at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%) of the total fatty acid content, and a reduced linoleic acid component of <NUM>% or less (e.g., <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, or <NUM>% or less) of the total fatty acid content.

As described herein, TAL effector endonucleases can be used to generate plants and related products (e.g., seeds and plant parts) that are particularly suitable for production of soybean oil with increased oleic acid content and decreased linoleic acid content, as well as decreased linolenic acid content. For example, nucleic acids encoding TAL effector endonucleases targeted to selected FAD2-1A or FAD2-1B sequences (e.g., the FAD2-1A sequences shown in <FIG> and TABLE <NUM>, or the FAD2-1B sequences shown in <FIG> and TABLE <NUM>) can be transformed into plant cells (e.g., cells in cotyledons), where they can be expressed. To accomplish the complete elimination of FAD2-1A and FAD2-1B expression, for example, an engineered, rare-cutting nuclease can be designed to recognize a conserved region of both FAD2-<NUM> genes and create a double-strand break. Improper repair due to NHEJ at the DNA break site can generate missense and/or nonsense mutations in the FAD2-1A/1B coding regions, rendering the FAD2-1A/1B RNA transcripts unstable and targeted for degradation prior to translation. Representative examples of naturally occurring soybean FAD2-1A and FAD2-1B nucleotide sequences are shown in TABLE <NUM> (SEQ ID NOS:<NUM> and <NUM>), and also in SEQ ID NOS: <NUM> and <NUM>. The resulting soybean plants, plant parts, and/or plant cells subsequently can be analyzed to determine whether mutations have been introduced at the target site(s).

The method of the present invention provides Glycine max plants, plant parts, and plant cells with mutations in the FAD2-1A, FAD2-1B, and FAD3A genes. The genotypes of these plants, plant parts, and plant cells can be described as follows: fad2-1a fad2-1b fad3a FAD3B FAD3C, fad2-1a fad2-1b fad3a fad3b FAD3C, fad2-1a fad2-1b fad3a FAD3B fad3c, and fad2-1a fad2-1b fad3a fad3b fad3c.

In some embodiments, the plants described herein also can contain one or more transgenes. A transgene can be integrated into the soybean genome using standard transformation protocols (see, for example, <NPL>; <NPL>; and <NPL>). In some cases, a transgene can encode a protein that confers tolerance or resistance to one or more herbicides (e.g., glufonsinate, mesotrione, imidazolinone, isoxaflutole, glyphosate, <NUM>,<NUM>-D, hydroxyphenylpyruvate dioxygenase-inhibiting herbicides, or dicamba). In some cases, a transgene can encode a plant <NUM>-enolpyruvylshikimate-<NUM>-phosphate synthase (EPSPS) protein, or a modified plant EPSPS protein, where the modified plant EPSPS contains an amino acid substitution within the conserved TAMRP (SEQ ID NO:<NUM>) sequence. The substitution can be, for example, a threonine to isoleucine substitution, a proline to serine substitution, or a proline to adenine substitution. In some embodiments, a transgene can encode a bacterial EPSPS protein, an agrobacterium CP4 EPSPS protein, an aryloxyalkanoate dioxygenase (AAD) protein, a phosphinothricin N-acetyltransferase (PAT) protein, an acetohydroxyacid synthase large subunit protein, a p-hydroxyphenylpyruvate dioxygenase (hppd) protein, or a dicamba monooxygenase (DMO) protein.

In some cases, a transgene can encode a product that enhances resistance to insects (e.g., lepidopteran insects). For example, a transgene can encode a protein from Bacillus thuringiensis, such as a Cry protein (e.g., a Cry1Ac delta-endotoxin protein, a Cry1F delta-endotoxin protein, a Cry2Ab delta-endotoxin protein, or a Cry1Ac delta-endotoxin protein). In some embodiments, a transgene can enhance virus resistance. For example, a transgene can contain a sequence from a viral genome, such as an antisense sequence from a virus genome.

In some embodiments, a transgene can cause male sterility. For example, a transgene can include a pollen killer gene (e.g., an alpha amylase gene, S24 gene, or S35 gene). A transgene can further include a screenable marker, such as a fluorescent protein (e.g., GFP, YFP, RFP, BFP, or luciferase) or a gene involved in regulating seed size. In some cases, a transgene can further include a restoring factor (e.g., a functional male-sterile (MS) gene).

Also described herein are FAD2 and FAD3 nucleic acid molecules. As described herein, a nucleic acid can have a nucleotide sequence with at least about <NUM> percent sequence identity to a representative FAD3A, FAD3B, FAD3C, FAD2-1A, or FAD2-1B nucleotide sequence. For example, a nucleotide sequence can at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> percent sequence identity to a representative, naturally occurring FAD3A, FAD3B, or FAD3C nucleotide sequence as set forth in TABLE <NUM>, or to a representative, naturally occurring FAD2-1A or FAD2-1B nucleotide sequence as set forth in TABLE <NUM> (SEQ ID NOS:<NUM> and <NUM>). As described herein, at least about <NUM>% (e.g., at least <NUM>%, <NUM>%, <NUM>%, or more than <NUM>%) of the nucleotide sequence of a targeted gene can be deleted when generating the mutant.

The percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST <NUM> Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN version <NUM>. <NUM> and BLASTP version <NUM>. This stand-alone version of BLASTZ can be obtained online at fr. com/blast or at ncbi. Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1. txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2. txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output. txt); -q is set to -<NUM>; -r is set to <NUM>; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq -i c:\seq1. txt -j c:\seq2. txt -p blastn -o c:\output. txt -q -<NUM> -r <NUM>. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1. txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2. txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output. txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seql. txt -j c:\seq2. txt -p blastp -o c:\output. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:<NUM>), or by an articulated length (e.g., <NUM> consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by <NUM>. For example, a nucleic acid sequence that has <NUM> matches when aligned with the sequence set forth in SEQ ID NO:<NUM> is <NUM> percent identical to the sequence set forth in SEQ ID NO:<NUM> (i.e., <NUM> ÷ <NUM> x <NUM> = <NUM>). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, <NUM>, <NUM>, <NUM>, and <NUM> is rounded down to <NUM>, while <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> is rounded up to <NUM>. It also is noted that the length value will always be an integer.

The FAD nucleic acid molecules described herein can include target sequences for rare cutting endonucleases (e.g., TAL effector endonucleases). Methods for selecting endogenous target sequences and generating TAL effector endonucleases targeted to such sequences can be performed as described elsewhere. See, for example, <CIT>. TAL effectors are found in plant pathogenic bacteria in the genus Xanthomonas. These proteins play important roles in disease, or trigger defense, by binding host DNA and activating effector-specific host genes (see, e.g., <NPL>; <NPL>; <NPL>; <NPL>; and <NPL>). Specificity depends on an effector-variable number of imperfect, typically <NUM> amino acid repeats (<NPL>; and <CIT>). Polymorphisms are present primarily at repeat positions <NUM> and <NUM>, which are referred to herein as the repeat variable-diresidue (RVD).

The RVDs of TAL effectors correspond to the nucleotides in their target sites in a direct, linear fashion, one RVD to one nucleotide, with some degeneracy and no apparent context dependence. This mechanism for protein-DNA recognition enables target site prediction for new target specific TAL effectors, as well as target site selection and engineering of new TAL effectors with binding specificity for the selected sites.

TAL effector DNA binding domains can be fused to other sequences, such as endonuclease sequences, resulting in chimeric endonucleases targeted to specific, selected DNA sequences, and leading to subsequent cutting of the DNA at or near the targeted sequences. Such cuts (i.e., double-stranded breaks) in DNA can induce mutations into the wild type DNA sequence via NHEJ or homologous recombination, for example. In some cases, TAL effector endonucleases can be used to facilitate site directed mutagenesis in complex genomes, knocking out or otherwise altering gene function with great precision and high efficiency. As described in the Examples below, TAL effector endonucleases targeted to the soybean FAD3A, FAD3B, and FAD3C alleles can be used to mutagenize the endogenous genes, resulting in plants with reduced expression (e.g., without detectable expression) of these genes. The fact that some endonucleases (e.g., FokI) function as dimers can be used to enhance the target specificity of the TAL effector endonuclease. For example, in some cases a pair of TAL effector endonuclease monomers targeted to different DNA sequences (e.g., the underlined target sequences shown in <FIG>, <FIG>, and <FIG>) can be used. When the two TAL effector endonuclease recognition sites are in close proximity, as depicted in <FIG>, the inactive monomers can come together to create a functional enzyme that cleaves the DNA. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created.

Methods for using TAL effector endonucleases to generate plants, plant cells, or plant parts having mutations in endogenous genes include, for example, those described in the Examples herein. For example, nucleic acids encoding TAL effector endonucleases targeted to selected FAD3A, FAD3B, or FAD3C sequences (e.g., the FAD3A sequences shown in <FIG> and TABLE <NUM>, the FAD3B sequences shown in <FIG> and TABLE <NUM>, and/or the FAD3C sequences shown in <FIG> and TABLE <NUM>) can be transformed into plant cells (e.g., cells in cotyledons), where they can be expressed. The cells subsequently can be analyzed to determine whether mutations have been introduced at the target site(s), through nucleic acid-based assays or protein-based assays to detect expression levels as described above, for example, or using nucleic acid-based assays (e.g., PCR and DNA sequencing, or PCR followed by a T7E1 assay; <NPL>) to detect mutations at the genomic loci.

The mutagenized population, or a subsequent generation of that population, can be screened for one or more desired traits (e.g., altered oil composition) that result from the mutations. Alternatively, the mutagenized population, or a subsequent generation of that population, can be screened for a mutation in a FAD3A, FAD3B, or FAD3C gene. For example, the progeny M<NUM> generation of M<NUM> plants may be evaluated for the presence of a mutation in a FAD3A, FAD3B, or FAD3C gene. A "population" is any group of individuals that share a common gene pool. As used herein, "M<NUM>" refers to plant cells (and plants grown therefrom) exposed to a TAL effector nuclease, while "M<NUM>" refers to seeds produced by self-pollinated M<NUM> plants, and plants grown from such seeds. "M<NUM>" is the progeny (seeds and plants) of self-pollinated M<NUM> plants, "M<NUM>" is the progeny of self-pollinated M<NUM> plants, and "M<NUM>" is the progeny of self-pollinated M<NUM> plants. "M<NUM>" is the progeny of self-pollinated M<NUM> plants. "M<NUM>", "M<NUM>", etc. are each the progeny of self-pollinated plants of the previous generation. The term "selfed" as used herein means self-pollinated.

One or more M<NUM> soybean plants can be obtained from individual, mutagenized M<NUM> plant cells (and plants grown therefrom), and at least one of the plants can be identified as containing a mutation in a FAD3A, FAD3B, or FAD3C gene. An M<NUM> soybean plant may be heterozygous for a mutant allele at a FAD3A, FAD3B, and/or FAD3C locus and, due to the presence of the wild type allele, exhibit delta-fifteen fatty acid desaturase activity. Alternatively, an M<NUM> soybean plant may have a mutant allele at a FAD3A, FAD3B, or FAD3C locus and exhibit the phenotype of lacking delta-fifteen fatty acid desaturase activity. Such plants may be heterozygous and lack delta-fifteen fatty acid desaturase activity due to phenomena such a dominant negative suppression, despite the presence of the wild type allele, or may be homozygous due to independently induced mutations in both alleles at the FAD3A, FAD3B, or FAD3C locus.

A soybean plant carrying mutant FAD3A, FAD3B, and FAD3C alleles can be used in a plant breeding program to create novel and useful lines and varieties. Thus, in some embodiments, an M<NUM>, M<NUM>, M<NUM>, or later generation soybean plant containing at least one mutation in a FAD3A gene, at least one mutation in a FAD3B gene, and at least one mutation in a FAD3C gene can be crossed with a second soybean plant, and progeny of the cross can be identified in which the FAD3A, FAD3B, and FAD3C gene mutations are present. It is to be appreciated that the second soybean plant can contain the same FAD3A, FAD3B, and FAD3C mutations as the plant to which it is crossed, different FAD3A, FAD3B, and FAD3C mutations, or can be wild type at the FAD3A, FAD3B, and/or FAD3C loci.

It also should be appreciated that the mutagenized soybean population can be combined with other previously identified mutations, to yield increased agronomically valuable traits. For example, a FAD3A, FAD3B, and/or FAD3C mutant can be combined with a FAD2 mutant soybean plant. Such a combinatorial mutant may further increase the value of the oil profile. This combination can be obtained either by utilizing mutant plants as the material for FAD3A, FAD3B, and/or FAD3C mutagenesis, or through breeding programs.

Breeding can be carried out via known procedures. DNA fingerprinting, SNP or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant FAD3A, FAD3B, and FAD3C alleles into other soybean plants. For example, a breeder can create segregating populations from hybridizations of a genotype containing a mutant allele with an agronomically desirable genotype. Plants in the F<NUM> or backcross generations can be screened using markers developed from FAD3A, FAD3B, and FAD3C sequences or fragments thereof. Plants identified as possessing the mutant allele can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern or the MAS technology used, it may be necessary to self-pollinate the selected plants before each cycle of backcrossing to aid identification of the desired individual plants. Backcrossing or other breeding procedure can be repeated until the desired phenotype of the recurrent parent is recovered.

Successful crosses yield F<NUM> plants that are fertile and that can be backcrossed with one of the parents if desired. In some embodiments, a plant population in the F<NUM> generation can be screened for FAD3A, FAD3B, and FAD3C gene expression. For example, a plant can be identified that fails to express FAD3A, FAD3B, and FAD3C due to the presence of mutations within the FAD3A, FAD3B, and FAD3C genes, using standard methods such as, for example, a PCR method with primers based on the nucleotide sequence information for FAD3A, FAD3B, and FAD3C described herein. Selected plants then can be crossed with one of the parents and the first backcross (BC<NUM>) generation plants are self-pollinated to produce a BC<NUM>F<NUM> population that is again screened for variant FAD3A, FAD3B, and FAD3C gene expression (e.g., null versions of the FAD3A, FAD3B, and FAD3C genes). The process of backcrossing, self-pollination, and screening is repeated, for example, at least four times until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent, such that it has acceptable agronomic performance. This plant, if desired, can be self-pollinated, and the progeny subsequently can be screened again to confirm that the plant lacks FAD3A, FAD3B, and FAD3C gene expression. Cytogenetic analysis of the selected plants optionally can be performed to confirm the chromosome complement and chromosome pairing relationships. Breeder's seed of the selected plant can be produced using standard methods including, for example, field testing, confirmation of the null condition for FAD3A, FAD3B, and FAD3C, and/or analyses of oil to determine the level of linolenic acid.

In situations where the original F<NUM> hybrid resulting from the cross between a first, mutant soybean parent and a second, wild type soybean parent, is hybridized or backcrossed to the mutant soybean parent, the progeny of the backcross can be self-pollinated to create a BC<NUM>F<NUM> generation that is screened for the mutant FAD3A, FAD3B, and FAD3C alleles.

A plant breeding program using the mutant soybean plants described herein can result in novel and useful lines and varieties. As used herein, the term "variety" refers to a population of plants that share constant characteristics that separate them from other plants of the same species. A variety is often, although not always, sold commercially. While possessing one or more distinctive traits, a variety can be further characterized by a very small overall variation between individuals within that variety. A "pure line" variety may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. A variety can be essentially derived from another line or variety. As defined by the International Convention for the Protection of New Varieties of Plants (December <NUM>, <NUM>, as revised on November <NUM>, <NUM>, on October <NUM>, <NUM>, and on March <NUM>, <NUM>), a variety is "essentially derived" from an initial variety if (a) it is predominantly derived from the initial variety, or from a variety that is predominantly derived from the initial variety, while retaining expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety, (b) it is clearly distinguishable from the initial variety, and (c) except for the differences that result from the act of derivation, it conforms to the initial variety in the expression of essential characteristics that result from the genotype or combination of genotypes of the initial variety. Essentially derived varieties can be obtained, for example, by selection of a natural or induced mutant, a somaclonal variant, a variant individual from plants of the initial variety, backcrossing, or transformation. A "line" as distinguished from a variety most often denotes a group of plants used non-commercially, for example in plant research. A line typically displays little overall variation between individuals for one or more traits of interest, although there may be some variation between individuals for other traits.

The methods provided herein can be used to produce plant parts (e.g., seeds) or plant products (e.g., oil) having decreased linolenic acid content, as compared to corresponding plant parts or products from wild type plants. The fatty acid content of a plant part or a plant product can be evaluated using standard methods, such as those described in Example <NUM> herein, for example.

To completely inactivate or knock out the alleles of FAD3A, FAD3B, and FAD3C genes in G. max, sequence-specific nucleases were designed that target the nucleic acid sequence encoding conserved histidine cluster motifs required for catalytic activity. Nine TAL effector endonuclease pairs were designed to target the FAD3 gene family (i.e., three TAL effector endonuclease pairs for each FAD3 gene), using software that specifically identifies TAL effector endonuclease recognition sites, such as TALE-NT <NUM> (<NPL>). TAL effector endonuclease recognition sites for the FAD3A gene are underlined in <FIG>, TAL effector endonuclease recognition sites for the FAD3B gene are underlined in <FIG>, and TAL effector endonuclease recognition sites for the FAD3C gene are underlined in <FIG>; these also are listed in TABLE <NUM>. TAL effector endonucleases were synthesized using methods similar to those described elsewhere (<NPL>; <NPL>; and <NPL>). The TAL effector endonuclease pairs targeting FAD3 sequence are referred to as GmFAD3_T01, GmFAD3_T02, and GmFAD3_T03, wherein GmFAD3A_T01 and GmFAD3A_T03 are comparative examples and GmFAD3B_T02 is according to the present invention. GmFAD3_T01 may also referred to as GmFAD3_T01. GmFAD3_T02 may also referred to as GmFAD3_T02. GmFAD3_T03 may also referred to as GmFAD3_T03.

To assess the activity of the TAL effector endonucleases targeting the FAD3A gene, NHEJ frequencies were assessed via deep sequencing of transformed soybean protoplasts. TAL effector endonuclease activity at endogenous target sites in G. max was measured by expressing the TAL effector endonucleases in protoplasts and surveying the TAL effector endonuclease target sites for mutations introduced by NHEJ. Briefly, G. max immature seeds were taken from <NUM> to <NUM> day old plants under high light conditions. Protoplasts were isolated according to the aforementioned methods described by <NPL>.

TAL effector endonuclease-encoding plasmids, together with a YFP-encoding plasmid, were introduced into G. max protoplasts by PEG-mediated transformation as described elsewhere (<NPL>). Twenty-four hours after treatment, transformation efficiency was measured by evaluating an aliquot of the transformed protoplasts using a fluorescent microscope to monitor YFP fluorescence. Transformation efficiency was determined to be <NUM>%. The remainder of the transformed protoplasts were harvested, and genomic DNA was prepared by a salt-extraction based method (<NPL>). Using the genomic DNA prepared from the protoplasts as a template, a fragment encompassing the TAL effector endonuclease recognition site was amplified by PCR. The PCR product was then subjected to <NUM> pyro-sequencing. Sequencing reads with insertion/deletion (indel) mutations in the spacer region were considered as having been derived from imprecise repair of a cleaved TAL effector endonuclease recognition site by NHEJ. Mutagenesis frequency was calculated as the number of sequencing reads with NHEJ mutations out of the total sequencing reads. The values were then normalized by the transformation efficiency.

The raw activity of the GmFAD3 TAL effector endonuclease pairs, GmFAD3A_T01, GmFAD3A _T02, and GmFAD3A_T03, against FAD3A, FAD3B, and FAD3C are summarized in TABLE <NUM>. TAL effector endonucleases GmFAD3A_T02 had the highest activity at the intended target FAD3A (<NUM>%) and decreased activity at FAD3B and FAD3C (<NUM>% and <NUM>%, respectively).

A correlation was observed between the number of SNPs within the TAL effector endonuclease binding sites and the relative mutation frequencies (<FIG>). Raw mutation frequencies at FAD3A target sites (containing <NUM> SNPs) for TAL effector endonuclease pairs GmFAD3_T01, GmFAD3_T02, GmFAD3_T03 were <NUM>%, <NUM>% and <NUM>%, respectively. After normalizing TAL effector endonuclease mutation frequencies at FAD3A, the relative mutation frequencies at FAD3B and FAD3C were determined. Target sites with one or two SNPs decreased mutation frequencies to ~<NUM> or <NUM>%, respectively, relative to the activity of the corresponding TAL effector endonuclease at FAD3A; target sites with four SNPs decreased mutation frequencies to <NUM>%; target sites with five SNPs decreased mutation frequencies to <NUM>%, and target sites with more than five SNPs decreased mutation frequencies to undetectable levels. Whereas these data do not account for relative position of the SNPs, they provide evidence for TAL effector endonuclease target site specificity, indicating that target sites with five or more SNPs are unlikely to be recognized and cleaved.

FAD3 mutations within soybean protoplasts, which were introduced using the FAD3A TALE nuclease pairs, were further analyzed. Both insertions and deletions were observed, with the majority of the mutations being deletions. A list of the TAL effector endonuclease-induced FAD3 mutations identified in soybean cells is provided by SEQ ID NOS:<NUM>-<NUM>. Specific mutations introduced into FAD3A by GmFAD3_T01. <NUM> are set forth in the sequences of SEQ ID NOS:<NUM>-<NUM>. Specific mutations introduced into FAD3B by GmFAD3_T01. <NUM> are set forth in the sequences of SEQ ID NOS:<NUM>-<NUM>. Specific mutations introduced into FAD3A by GmFAD3_T02. <NUM> are set forth in the sequences of SEQ ID NOS:<NUM>-<NUM>. Specific mutations introduced into FAD3B by GmFAD3_T02. <NUM> are set forth in the sequences of SEQ ID NOS:<NUM>-<NUM>. Specific mutations introduced into FAD3A by GmFAD3_T03. <NUM> are set forth in the sequences of SEQ ID NOS: <NUM>-<NUM>. Specific mutations introduced into FAD3B by GmFAD3_T03. <NUM> are set forth in the sequences of SEQ ID NOS:<NUM>-<NUM>. Specific mutations introduced into FAD3C by GmFAD3_T03. <NUM> are set forth in the sequences of SEQ ID NOS: <NUM>-<NUM>. It was observed that the majority of mutations included a deletion of the nucleotide at position <NUM> of the <NUM> nucleotide spacer. Specifically, using TAL endonuclease pair GmFAD3_T01. <NUM>, a deletion of the adenine nucleotide at position <NUM> of SEQ ID NO:<NUM> (FAD3A) was observed. Using TAL endonuclease pair GmFAD3_T02. <NUM>, a deletion of the cytosine nucleotide at position <NUM> of SEQ ID NO:<NUM> (FAD3A) was observed. Using TAL endonuclease pair GmFAD3_T03. <NUM>, a deletion of the adenine nucleotide at position <NUM> of SEQ ID NO:<NUM> (FAD3A) was observed. Using TAL endonuclease pair GmFAD3_T01. <NUM>, a deletion of the adenine nucleotide at position <NUM> of SEQ ID NO:<NUM> (FAD3B) was observed. Using TAL endonuclease pair GmFAD3_T02. <NUM>, a deletion of the cytosine nucleotide at position <NUM> of SEQ ID NO:<NUM> (FAD3B) was observed. Using TAL endonuclease pair GmFAD3_T03. <NUM>, a deletion of the adenine nucleotide at position <NUM> of SEQ ID NO:<NUM> (FAD3B) was observed. Using TAL endonuclease pair GmFAD3_T03. <NUM>, a deletion of the adenine nucleotide at position <NUM> of SEQ ID NO:<NUM> (FAD3C) was observed.

Following verification that FAD3 TAL effector endonuclease created targeted modifications at endogenous target sites, experiments were conducted to create soybean plants with mutations in FAD3A, FAD3B, and FAD3C. To accomplish this, each of the FAD3 TAL effector endonuclease pairs was cloned into a bacterial vector, with TAL effector endonuclease expression driven by the cauliflower mosaic virus <NUM> promoter. Such vectors can be delivered to plant cells by Agrobacterium-mediated transformation or by using biolistics.

Transgenic soybean plants expressing the TAL effector endonucleases were generated using standard transformation protocols (<NPL>; <NPL>; and <NPL>). Following transformation of soybean half cotyledons (variety Bert) with sequences encoding the GmFAD3_T02 TAL effector endonuclease, putatively transgenic plants were regenerated. Both WT and FAD2-1A, FAD2-1B mutant soybean lines were transformed. The plants were transferred to soil, and after approximately <NUM> weeks of growth, a small leaf was harvested from each plant for DNA extraction and genotyping. From four independent transformations (designated as experiments Gm183, Gm184, Gm205, and Gm206), a total of <NUM> events were generated. All T0 transgene-positive plants were then subjected to a T7 Endonuclease <NUM> (T7E1) assay to identify plants with mutations at the FAD3A, FAD3B, and/or FAD3C TAL effector endonuclease recognition sites (<NPL>). Briefly, a PCR product spanning the TAL effector endonuclease recognition site was generated, denatured, and allowed to reanneal. T7E1 was added to the annealed products to cleave heteroduplexes generated when a wild type DNA fragment annealed with a fragment carrying a TAL effector endonuclease-induced mutation, and cleavage products were visualized by agarose gel electrophoresis. Alternatively, the PCR amplicons could be directly sequenced to assess whether mutations have occurred at the FAD3A, FAD3B and/or FAD3C target sites.

The T7E1 assay revealed that out of the <NUM> transgene-positive plants screened, <NUM> plants were positive for mutations at the target site. The FAD3A and FAD3B PCR amplicons of the <NUM> positive plants were then inserted into cloning vectors and subjected to Sanger sequencing to confirm and characterize the mutant profiles. The resulting reads were then aligned to the wild type sequences to determine allele types. The results are summarized in TABLE <NUM>, and representative sequences are shown in <FIG>. Together, these results confirmed the successful mutagenesis of FAD3 within T0 soybean plants, with TAL effector endonuclease GmFAD3_T02 mutagenesis frequencies at FAD3A of about <NUM> percent.

To confirm that TAL effector endonuclease-induced mutations can be stably transmitted to subsequent generations, candidate T1 plants derived from experiment Gm183 were screened for mutations within FAD3A by PCR amplification and sequencing of clones. From three different T0 events (Gm183-<NUM>, Gm183-<NUM> and Gm183-<NUM>), T1 plants harboring heterozygous or homozygous mutations within FAD3A were identified, indicating that mutations were stably transmitted to the next generation. No plants with combinations of FAD3A and FAD3B mutations were identified, indicating that the frequency of mutagenesis at FAD3B was <<NUM>% (i.e., less than <NUM> out of <NUM> events).

Sanger sequencing reads containing mutations within FAD3A were aligned to the wild type sequences to determine allele types. The results are summarized in TABLE <NUM>. A listing of mutations identified within FAD3A is shown within SEQ ID NOS <NUM>-<NUM>, <NUM>-<NUM> and SEQ ID NOS:<NUM>-<NUM>.

An alternative approach to knock out FAD2 and FAD3 genes is to simultaneously deliver two TAL effector endonucleases targeting sequence within the FAD2-1A, FAD2-1B (one TAL effector endonuclease), and FAD3 genes (the other TAL effector endonuclease). Following verification that FAD2 and FAD3 TAL effector endonuclease created targeted modifications at endogenous target sites, experiments are conducted to create soybean plants with mutations in FAD2-1A, FAD2-1B, FAD3A, FAD3B, and FAD3C. To accomplish this, each of the FAD2 and FAD3 TAL effector endonucleases are cloned into a T-DNA vector, and TAL effector endonuclease expression is driven by the cauliflower mosaic virus <NUM> promoter (Zuo et al. The T-DNA vector also contains a selectable marker that confers resistance to glufosinate. Such vectors are delivered to plant cells by Agrobacterium-mediated transformation or by using biolistics.

Transgenic soybean plants expressing the TAL effector endonucleases are generated using standard Agrobacterium rhizogenes transformation protocols (Curtin et al. Following cultivation of the T-DNA-containing A. rhizogenes strains with soybean half cotyledons (variety Bert), putatively transgenic plants are regenerated. The plants are transferred to soil, and after approximately <NUM> weeks of growth, a small leaf is harvested from each plant for DNA extraction and genotyping. Each DNA sample is first screened using PCR for the presence of T-DNA. All T-DNA-positive plants are then subjected to a T7 Endonuclease <NUM> (T7E1) assay to identify plants with mutations at the FAD2-1A, FAD2-1B, FAD3A, FAD3B, and/or FAD3C TAL effector endonuclease recognition sites (<NPL>). Briefly, a PCR product spanning the TAL effector endonuclease recognition site is generated, denatured, and allowed to reanneal. T7E1 is added to the annealed products to cleave heteroduplexes generated when a wild type DNA fragment annealed with a fragment carrying a TAL effector endonuclease-induced mutation, and cleavage products are visualized by agarose gel electrophoresis. Alternatively, the PCR amplicons can be directly sequenced to assess whether mutations occurred at the FAD2-1A, FAD2-1B, FAD3A, FAD3B and/or FAD3C target sites.

Following verification that soybean plants with mutations in FAD2-1A, FAD2-1B (<FIG>), FAD3A, FAD3B, and FAD3C have been generated, the mutant plants are subjected to crossing to yield combinatorial mutants of FAD2-1A, FAD2-1B, FAD3A, FAD3B, and/or FAD3C. This is accomplished by acquiring pollen from a young flower that has opened for the first time; the flower is separated and collected to transfer the desired pollen to the stigma. The resulting progeny are then genotyped to confirm successful crosses.

Soybean lines are grown to maturity and allowed to self-fertilize, giving rise to seeds that are homozygous mutant in either FAD3A (designated aaBBCC), FAD3B (designated AAbbCC), FAD3C (designated AABBcc), FAD3A and FAD3B (designated aabbCC), FAD3B and FAD3C (designated AAbbcc), or FAD3A, FAD3B, and FAD3C (designated aabbcc). These seeds are analyzed for fatty acid composition. Briefly, individual soybean seeds are pulverized, and DNA is prepared from a portion of the ground tissue and analyzed to establish the genotype of each seed. The remaining pulverized tissue from FAD3A homozygous (aaBBCC), FAD3B homozygous (AAbbCC), FAD3C homozygous (AABBcc), FAD3A and FAD3B homozygous (aabbCC), FAD3B and FAD3C homozygous (AAbbcc), or FAD3A, FAD3B, and FAD3C homozygous (aabbcc) knockout seeds is used to determine fatty acid composition using fatty acid methyl esters (FAME) gas chromatography (<NPL>). The results of the analysis likely show that the single mutant has a modest reduction in linolenic acid, whereas the double and triple mutants are likely to show a more significant reduction.

The oil profile from seed was assessed from FAD3A mutant soybean plants (designated as fad3a) and FAD2-1A FAD2-1B FAD3A homozygous mutant soybean plants (designated as fad2-1a fad2-1b fad3a) (<FIG>). Seed from T1 homozygous mutant lines were collected and assessed for oil composition by gas chromatographic analysis of fatty acid methyl esters (GC FAME Analysis). Significant changes in linolenic and linolenic acid levels were observed in oil from fad3a plants, relative to oil from WT plants: linolenic acid levels decreased from <NUM> ± <NUM>% to <NUM> ± <NUM>%, while linoleic acid levels increased from <NUM> ± <NUM>% to <NUM> ± <NUM>%. Surprisingly, significant changes in the levels of oleic and stearic acid levels also were observed: oleic acid levels decreased from <NUM> ± <NUM>% to <NUM> ± <NUM>%, and stearic acid levels decreased from <NUM> ± <NUM>% to <NUM> ± <NUM>%.

Specifically, five biological replicates of seeds from fad3a plants were found to have linolenic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%; linoleic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%; oleic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%; stearic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%; and palmitic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%.

Four biological replicates of WT seed were found to have linolenic acid levels of <NUM>%, <NUM>%, <NUM>%, and <NUM>%; linoleic acid levels of <NUM>%, <NUM>%, <NUM>%, and <NUM>%; oleic acid levels of <NUM>%, <NUM>%, <NUM>%, and <NUM>%; stearic acid levels of <NUM>%, <NUM>%, <NUM>%, and <NUM>%; and palmitic acid levels of <NUM>%, <NUM>%, <NUM>%, and <NUM>%.

Significant changes in fatty acid levels within seed oil from fad2-1a fad2-1b fad3a soybean plants also were observed as compared to the fatty acid levels within fad2-1a fad2-1b soybean plants (<FIG>). The average linolenic acid level within oil from fad2-1a fad2-1b fad3a plants was <NUM> ± <NUM>%, significantly lower than oil from fad2-1a fad2-1b soybean plants (<NUM> ± <NUM>%). Further, and surprisingly, linoleic acid levels decreased from <NUM> ± <NUM>% in fad2-1a fad2-1b lines to <NUM> ± <NUM>% in fad2-1a fad2-1b fad3a lines, and oleic acid levels increased from <NUM> ± <NUM>% in fad2-1a fad2-1b lines to <NUM> ± <NUM>% in fad2-1a fad2-1b fad3a lines. Together, these results indicate that stacking mutations within the FAD2-<NUM> and FAD3A genes can decrease linolenic and linoleic acid levels to below <NUM>%, and increases oleic acid levels to over <NUM>%.

Specifically, <NUM> biological replicates of seeds from fad2-1a fad2-1b fad3a plants were found to have linolenic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%; linoleic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%; oleic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%; stearic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%; and palmitic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%.

Twenty biological replicates of seeds from fad2-1a fad2-1b plants were found to have linolenic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%; linoleic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%; oleic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%; stearic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%; and palmitic acid levels of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%.

To obtain soybean lines that have the FAD3 mutations but lack the FAD3 TAL effector endonuclease construct and its associated selectable marker, progeny are screened for the aabbcc genotype. Mutant, transgene-free segregants are then sought using a PCR strategy that utilized two sets of primer pairs: one spanning the TAL effector endonuclease coding sequence and the other the endogenous alcohol dehydrogenase gene of soybean (a control for the PCR reaction). These results indicate the feasibility of creating transgene-free lines with mutations in the FAD3A, FAD3B, and FAD3C genes.

Further, T1 plants containing FAD3A mutations within the FAD2 KO background were assessed by PCR for the presence of transgene sequence (TABLE <NUM>). Of the <NUM> T1 plants assayed, <NUM> were positive for transgene sequence and two were negative (i.e., null segregants for the TAL effector endonuclease transgene). Importantly, the two transgene-free T1 plants harbored mutations within FAD3A. These two plants were self-pollinated to produce homozygous-mutant, transgene-free fad2-1a fad2-1b fad3a soybean plants.

To completely inactivate or knock out the alleles of FAD2-1A and FAD2-1B genes in soybean, sequence-specific nucleases were designed that target the protein coding region in the vicinity of the start codon. Eight TAL effector endonuclease pairs were designed to target the FAD2-<NUM> gene family within the first <NUM> bp of the coding sequence using software that specifically identifies TAL effector endonuclease recognition sites, such as TALE-NT <NUM> (<NPL>). The TAL effector endonuclease recognition sites for the FAD2-<NUM> genes are listed in <FIG>. TAL effector endonucleases were synthesized using methods similar to those described elsewhere (<NPL>; <NPL>; and <NPL>). TALEN activity was verified using a yeast assay, and TALEN pair FAD2_T04 was chosen for stable transformation in soybean plants.

Candidate transgenic plants were regenerated and transferred to soil. After approximately <NUM> weeks of growth, a small leaf was harvested from each plant for DNA extraction and genotyping. Each DNA sample was first screened using PCR for the presence of transgenic DNA. All transgene-positive plants were then subjected to a T7E1 assay to identify plants with mutations at the FAD2-1A and FAD2-1B TAL effector endonuclease recognition site (<NPL>). Briefly, a PCR product spanning the TAL effector endonuclease recognition site was generated, denatured, and allowed to reanneal. T7E1 endonuclease was added to the annealed products to cleave heteroduplexes generated when a wild type DNA fragment annealed with a fragment carrying a TAL effector endonuclease-induced mutation, and cleavage products were visualized by agarose gel electrophoresis. Four plants showed evidence of TAL effector endonuclease-induced mutations (Gm026-<NUM>, Gm026-<NUM>, Gm027-<NUM> and Gm027-<NUM>). In addition, all four plants had mutations at both FAD2-1A and FAD2-1B, indicating that both genes were mutagenized simultaneously.

To determine if mutations introduced by TAL effector endonucleases in leaf tissue were transmitted to the next generation, seeds were collected from T0 plants Gm026-<NUM>, Gm026-<NUM> and Gm027-<NUM>. In each T1 population, <NUM>-<NUM> individual plants were genotyped to confirm transmission of the mutations. Both FAD2-1A and FAD2-1B mutations segregated in the T1 progeny of GM026-<NUM>. In contrast, only FAD2-1A or FAD2-1B mutations were transmitted to the T1 progeny of GM-<NUM>-<NUM> and GM027-<NUM>, respectively. The heritable mutations within GM026-<NUM> are shown in <FIG>.

Seed from field-grown GM026-<NUM> soybean plants was evaluated for protein content and fatty acid composition (<FIG>). GM026-<NUM> soybean plants were directly compared to control plants, Glycine max (L. cultivar Bert, which do not contain FAD2-1A or FAD2-1B mutations. Twenty-seven different properties were evaluated, including moisture content (by forced draft oven evaluation), as well as protein, crude fat, tryptophan, cysteine, methionine, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, threonine, total lysine, tyrosine, valine, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha linolenic acid, and total fatty acid content. Unexpectedly, an increase in protein content was observed in GM026-<NUM> lines compared to the wild type control (compare <NUM> to <NUM>; compare <NUM> to <NUM>; compare <NUM> to <NUM>).

Plants containing combinations of mutations that knock out activity of one or more FAD2 and FAD3 genes or proteins are produced, either by targeting one gene or multiple genes using TAL effector endonucleases, or by crossing plants with mutations in different FAD2 and/or FAD3 genes. Plants containing a series of mutation combinations are produced. For example, combination <NUM> is FAD2-<NUM> (WT) FAD2-1B (WT) fad3a (mutant) FAD3B (WT) FAD3C (WT). This combination of mutations and WT genes is also written as FAD2-1A FAD2-1B fad3a FAD3B FAD3C. Combination <NUM> and other combinations are set forth in TABLE <NUM>, where combinations not encompassed by the appended claims are indicated by an asterisk (*).

Plants having mutations in FAD2 and FAD3 genes were grown in field conditions in Minnesota, and phenotyped. Tested plants contained the genotype fad2-1afad2-1b fad3a FAD3B FAD3C. Several plants having different mutations in FAD3A were tested, including those with a -43bp deletion, a -4bp deletion, and a combination of the -<NUM> bp and -4bp deletions (i.e., a compound heterozygous mutant). Seed oil produced by the field-grown plants was assessed by FAME. Results from the FAME testing are shown in <FIG>.

The FAD2 KO background included the Gm026-<NUM> FAD2-1A (SEQ ID and FAD2-1B alleles (SEQ ID NO:<NUM>) alleles.

Claim 1:
A method for making a soybean plant comprising a mutation in:
one or more FAD2-1A alleles, one or more FAD2-1B alleles, and one or more FAD3A alleles;
the method comprising:
(a) providing soybean plant parts or plant cells, wherein the plant parts or plant cells comprise a mutation in one or more FAD2-1A alleles and a mutation in one or more FAD2-1B alleles,
wherein each FAD2-1A allele has a sequence as set forth in SEQ ID NO: <NUM>, or has a sequence with at least <NUM>% identity to SEQ ID NO: <NUM>;
wherein each FAD2-1B allele has a sequence as set forth in SEQ ID NO:<NUM>, or has a sequence with at least <NUM>% identity to SEQ ID NO:<NUM>;
and wherein the plant parts or plant cells comprise at least a functional FAD3A allele,
(b) contacting the plant parts or plant cells with one or more TAL effector endonucleases targeted to an endogenous FAD3A sequence, wherein each of the TAL effector endonucleases is targeted to a pair of sequences as set forth in SEQ ID NOS <NUM> and <NUM>,
(c) regenerating the plant parts or plant cells into whole soybean plants, and
(d) selecting from the whole soybean plants a soybean plant comprising a mutation in one or more FAD3A alleles,
wherein each mutated FAD3A allele comprises a deletion of the cytosine nucleotide at position <NUM> of SEQ ID NO:<NUM>.