Increased saturated fat in soybeans

Abstract

Materials and methods are provided for producing soybean varieties with altered oil composition as a result of one or more mutations that regulate the expression of the SACPD-C gene, the FATB-1A gene, or both the SACPD-C and FATB-1A genes. For example, soybean plants, plant parts, or plant cells are provided that produce oil with an increased saturated fatty acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking one or more mutations.

Description

Background Art

[0001] Soybean (Glycine max) is an important leguminous 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 applications ranging from cooking / oil cuisine to industrial uses and biodiesel. Soybean oil contains 11% palmitic acid (C16:0), 4% stearic acid (C18:0), 23% oleic acid (C18:1), 54% linoleic acid (C18:2), and 7.6% linolenic acid (C18:3). The total saturated fatty acids (palmitic acid and stearic acid) account for approximately 15% of the total fatty acid composition.

[0002] The above fatty acid composition of soybean oil may not be optimal for use in certain food and cosmetic manufacturing applications. Some limitations can be overcome by chemical hydrogenation, but the trans fatty acids produced as a result of partial hydrogenation are associated with unfavorable health effects. Palm oil or palm kernel oil (both of which are derived from the African oil palm tree (Elaeis guineensis)) is solid at room temperature, and its fractions provide a wide range of functional melting profiles, acting as a substitute for partially hydrogenated oils in food applications. This oil has a high melting point and a high saturated fat content, which is ideal for creating a desirable skin feel for creams and cosmetics and an appealing mouthfeel for confections. The unique chemistry of palm oil can also withstand the high temperatures associated with cooking, and the oil's resistance to spoilage gives products containing this oil a long shelf life.

[0003] Palm oil cultivation is often associated with environmental sustainability issues, which makes palm oil an unpopular choice. There is pressure to find alternatives to palm oil for food and cosmetic applications. One of the main drawbacks of non-palm fats based on imported fats such as shea, cocoa butter, and others is the high cost of the raw materials. Furthermore, these fats may also raise sustainability concerns. Liquid oils such as rapeseed oil, sunflower oil, or soybean oil cannot replace all palm fractions in all applications. It is highly desirable to be able to provide soybean varieties with sufficiently elevated palmitic and stearic acid content to produce solid fats that can be used to replicate the properties of the palm oil fraction.

[0004] Improving the nutritional and commercial quality of soybean oil can add further value to oil-based products. To provide products with higher nutrient content and greater stability, changes in the content and composition of soybean oil are needed to increase the saturated fatty acid content. Furthermore, soybean oil with increased saturated fatty acid content may reduce the need for industrial hydrogenation of polyunsaturated oils for food applications, thereby mitigating the negative health effects associated with trans fats. [Overview of the project]

[0005] This disclosure features soybean plants, plant parts, and plant cells that produce oil having an elevated saturated fatty acid content. This disclosure provides materials and methods for producing soybean varieties that produce soybeans having a saturated fatty acid content of more than approximately 15% by weight of the total fatty acid content. This disclosure is at least in part based on the finding that mutations modulating the expression of the SACPD-C gene, the FATB-1A gene, or both the SACPD-C and FATB-1A genes in soybean plants, plant parts, or plant cells can enhance the accumulation of saturated fatty acids such as stearic acid and palmitic acid for the production of solid fats or fractions of palm oil, cocoa butter, or other foreign fats with a functional melting profile, without hydrogenation. Furthermore, this disclosure is based on targeted mutations modulating the expression of the SACPD-C gene, the FATB-1A gene, or both the SACPD-C and FATB-1A genes to avoid the pleiotropic deletions associated with random mutagenesis, such as adverse effects on development seen in complete knockout mutants or nonspecific overexpression mutants.

[0006] Accordingly, one aspect of the present disclosure is a soybean plant, plant part, or plant cell comprising one or more mutations that modulate the expression of the SACPD-C gene, the FATB-1A gene, or both the SACPD-C and FATB-1A genes, wherein the plant, plant part, or plant cell produces an oil having an increased saturated fatty acid content compared to the oil produced from a corresponding soybean plant, plant part, or plant cell lacking one or more mutations, and the one or more mutations that modulate the expression of the SACPD-C gene comprises targeted mutations induced by low-frequency cleavage endonucleases. Soybean plants, plant parts, or plant cells may contain mutations that result in reduced expression of the SACPD-C gene. Mutations resulting in reduced expression of the SACPD-C gene may be mutations in one or more alleles of the SACPD-C gene or in their operatively linked promoters. Mutations resulting in reduced expression of the SACPD-C gene may be knockout mutations. Knockout mutations may be seed-specific knockout mutations. Seed-specific knockout mutations may involve the substitution of a seed-specific promoter at the native genomic locus of the SACPD-C gene with a promoter that is low in activity or undetectable in developing soybean seeds. Mutations resulting in reduced expression of the SACPD-C gene may be in the sequences shown in SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, or 47. Mutations resulting in reduced expression of the SACPD-C gene may be knock-in mutations of a functional SACPD-C gene operatively linked to a promoter that is low in activity or undetectable in developing soybean seeds. In one or more of the embodiments described above, a promoter that has low activity or no detectable activity in developing soybean seeds may be a nodule-specific gene promoter. In one or more of the embodiments described above, a soybean plant, plant part, or plant cell contains a mutation that results in increased expression of the FATB-1A gene. The mutation that increases the expression of the FATB-1A gene may be a targeted substitution of the endogenous promoter of the FATB-1A gene with an overexpression promoter. The overexpression promoter may be a strongly seed-specific promoter, optionally, the FAD2A promoter, or the FAD2B promoter.

[0007] In another embodiment, the present disclosure is a method for generating soybean plants containing mutations that regulate the expression of the SACPD-C gene, the FATB-1A gene, or both the SACPD-C and FATB-1A genes, (a) A population of soybean plant cells from a soybean plant that produces oil having a saturated fatty acid content of approximately 15% of the total fatty acid composition, (i) A mutation resulting in decreased expression of the SACPD-C gene, wherein the mutation is a targeted mutation induced by a low-frequency cleavage endonuclease, (ii) A mutation that results in increased expression of the FATB-1A gene, or (iii) Contacting with one or more nucleic acid sequences including a combination thereof, (b) Select cells from the population that have decreased expression of the SACPC-C gene, increased expression of the FATB-1A gene, or decreased expression of the SACPC-C gene and increased expression of the FATB-1A gene. (c) A method comprising regenerating selected plant cells into soybean plants. Decreasing the expression of the SACPD-C gene may include inducing mutations in one or more alleles of the SACPD-C gene or their operatively linked promoters. The induced mutations may be knockout mutations or seed-specific knockout mutations. Decreasing the expression of the SACPD-C gene may include replacing the seed-specific promoter at the native genomic locus of the SACPD-C gene with a promoter that is inactive or undetectable in developing soybean seeds. Decreasing the expression of the SACPD-C gene may further include delivering an expression cassette to a population of soybean plant cells containing a functional SACPD-C gene operatively linked to a promoter that is inactive or undetectable in developing soybean seeds. The promoter that is inactive or undetectable in developing soybean seeds may be a nodule-specific promoter. In one or more of the above embodiments, the method may include increasing the expression of the FATB-1A gene by replacing the endogenous promoter of the FATB-1A gene with an overexpression promoter. Increasing the expression of the FATB-1A gene may involve delivering an expression cassette containing one or more copies of the FATB-1A gene to a population of soybean plant cells. One or more copies of the FATB-1A gene may be operably linked to a strong seed-specific promoter.

[0008] In another embodiment, the disclosure features a soybean oil composition comprising soybean oil produced by a soybean plant, plant part, or plant cell containing one or more mutations that modulate the expression of the SACPD-C gene, the FATB-1A gene, or both the SACPD-C and FATB-1A genes, wherein the soybean oil has an increased saturated fatty acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking one or more mutations, and the one or more mutations that modulate the expression of the SACPD-C gene include targeted mutations induced by low-frequency cleavage endonucleases. The soybean oil composition may have a stearic acid content of more than 10%, a palmitic acid content of more than 10%, or a saturated fatty acid content of more than 20%, all percentages being on a weight basis of the total fatty acids in the oil.

[0009] Details of one or more examples are provided in the following description. Other features, purposes, and advantages are evident from the description and claims. [Brief explanation of the drawing]

[0010] This disclosure describes exemplary embodiments that are not limiting and do not exhaustive. In the drawings (which are not necessarily drawn to scale), similar numbers describe substantially similar components throughout several drawings. Similar numbers with different suffixes represent different examples of substantially similar components. The drawings generally illustrate various embodiments considered herein as examples, not as limitations.

[0011] The following refers to exemplary embodiments shown in the figures.

[0012] [Figure 1] This shows the expression profiles of the nodule-specific genes Glyma05g01360, Glyma13g44970, and Glyma14g27990 (SACPD-C) in different tissues. [Figure 2]This shows the expression profiles of seed-specific genes FAD2A (Glyma10g42470), FAD2B (Glyma20g24530), and FATB-1A (Glyma05g08060) in different tissues. [Figure 3A] Table 1 shows the normalized candidate gene expression profiles in different tissues. (A) includes young leaves, flowers, 1 cm sheaths, sheaths (10 and 14 days after full bloom (DAF)), roots, and nodule tissue. [Figure 3B] This is a continuation of Figure 3A. (B) includes seed tissue (10, 14, 21, 25, 28, 35, and 42DAF). [Figure 4] Table 2 lists TALENs (SEQ ID NOs: 15-47) for regulating GmSACPD-C gene expression according to one or more embodiments of this disclosure (e.g., approaches 1A and 1B). [Figure 5] Table 3 lists TALENs (SEQ ID NOs: 48-62) for regulating GmFATB-1A gene expression according to one or more embodiments of this disclosure (e.g., Approach 1C). [Figure 6A] Representative GmSACPD-C DNA sequences from several confirmed mutant profiles of regenerated T0 plants are shown. Underlined sequences indicate target sites for TAL effector endonucleases. (A) The wild-type GmSACPD-C sequence is shown in SEQ ID NO: 63, and mutant sequences are shown in SEQ ID NOs: 64-66. [Figure 6B] Representative GmSACPD-C DNA sequences from several confirmed mutant profiles of regenerated T0 plants are shown. Underlined sequences indicate target sites for TAL effector endonucleases. (B) Additional mutant sequences are shown in SEQ ID NOs. 67-70. [Figure 7] This shows representative GmSACPD-C DNA sequence alignments of several confirmed mutant profiles from regenerated T0 plants. Underlined sequences indicate target sites for TAL effector endonucleases. The wild-type GmSACPD-C sequence is shown in SEQ ID NO: 71, and the mutant sequences are shown in SEQ ID NOs: 72-76. [Figure 8] Approach 1B for seed-specific silencing of SACPD-C is described. (A) Exemplary geminiviral binary vector components for targeting upstream of the SACPD-C coding sequence, including a TALEN pair and a donor template, are shown. The nodule-specific promoter and a 2kb sequence of the 5'UTR (Glyma13g44970) are adjacent to the left homology arm (LHA) and right homology arm (RHA). (B) Gene targeting events during nuclease cleavage and homologous recombination with the donor template replicon are shown. According to one embodiment of this disclosure, the targeted promoter-substituted donor template sequence is shown in SEQ ID NO: 79, and the expected edited RHA sequence is shown in SEQ ID NO: 80. [Figure 9] A representative targeted promoter substitution donor template sequence (SEQ ID NO: 79) for Approach 1B is shown. [Figure 10] The expected edited RHA sequence (SEQ ID NO: 80) using the template in Figure 9 for replacing the endogenous SACPD-C gene promoter is shown. [Figure 11] Approach 1C for seed-specific overexpression (OE) of FATB-1A is described. (A) Exemplary geminiviral binary vector components for targeting upstream of the FATB-1A coding sequence, including TALEN pairs and a donor template adjacent to the LHA and RHA, are shown. (B) Gene targeting events during nuclease cleavage and homologous recombination with the donor template replicon are shown. [Figure 12] A representative targeted promoter substitution donor template sequence (SEQ ID NO: 81) for Approach 1C is shown. [Figure 13]Depiction of a DNA construct for Approach 2A for producing a plant, plant part or plant seed having tissue-specific expression of SACPD-C and FATB-1A is shown, the construct shown has two expression cassettes, cassette 1 having the coding sequence of SAPCD-C and the promoter (nodule-specific) and terminator sequences of Glyma13g44970, and cassette 2 having the coding sequence of FATB-A1 and the promoter and terminator sequences of FAD2A. The sequence of a representative construct for Approach 2A is shown in SEQ ID NO: 82.

Best Mode for Carrying Out the Invention

[0013] The present disclosure features soybean plants, plant parts and plant cells that can be used to produce oils having an increased saturated fatty acid content as a result of one or more mutations that regulate the expression of genes involved in fatty acid synthesis within the plant cell, as well as methods for generating such plants and oils derived from such plants. Using the methods described herein, soybean varieties having oils with a stearic acid content of at least 10% and / or a palmitic acid content of at least 10% can be generated. In some embodiments, the change in the oil composition is achieved by altering the expression of the soybean SACPD-C gene, either completely or in a seed-specific manner, or by overexpressing the soybean FATB-1A gene. According to some of the methods provided herein, the modification is achieved using non-transgenic techniques. The targeted mutations described herein minimize or avoid pleiotropic effects that can lead to deleterious phenotypes in soybean crops.

[0014] Definitions For the purposes of the present disclosure, terms are defined as follows.

[0015] The term "cis-genic" refers to the genetic modification of a plant with a natural gene encoding a trait from the plant itself or a sexually compatible donor plant. Cis-genic modification is distinguishable from transgenic modification, in which the plant is genetically modified with a gene from a non-crossable species or a synthetic gene.

[0016] "Endogenous gene" refers to a nucleic acid molecule comprising a sequence of a wild-type sequence that occurs in a wild-type plant, or a sequence having a percent identity that allows retention of the function of the encoded product (e.g., a sequence having at least 90% identity), which can be obtained from a plant or a plant part of a cell, or can be generated synthetically. Further embodiments define that the sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. In the embodiments described herein, the endogenous gene nucleotide sequence can be inserted at a locus different from that of the wild-type gene and can be operably linked to a promoter different from the wild-type gene.

[0017] "SACPD" refers to Δ9-stearoyl-acyl carrier protein desaturase. Thus, "SACPD gene" refers to a gene encoding a Δ9-stearoyl-acyl carrier protein desaturase protein. In soybean, three isoforms of the SACPD gene have been identified. Two of the isoforms of this gene, SACPD-A and SACPD-B, are expressed in both vegetative and reproductive tissues, while the third, SACPD-C (Glyma14g27990), is expressed mainly in developing seeds and root nodules.

[0018] "FATB" refers to a gene encoding palmitoyl-acyl carrier protein thioesterase.

[0019] The terms “soybean plant” or “plant part” are used broadly to include soybean plants at any developmental stage, or parts of soybean plants including plant cuttings, plant cells, plant cell cultures, plant organs, plant seeds, and phytostem plants. Plant cells are the structural and physiological units of a plant, including protoplasts and cell walls. Plant cells can be isolated single cells or aggregates of cells, e.g., brittle callus, or cultured cells, or they can be part of a more highly organized unit, e.g., plant tissue, plant organ, or plant. Thus, plant cells can be protoplasts, gamete-producing cells, or cells or aggregates of cells that can regenerate into an entire plant. For this reason, seeds, which contain multiple plant cells and can regenerate into an entire plant, are considered plant cells for the purposes of this disclosure. Plant tissue or plant organs can be seeds, protoplasts, callus, or any other group of plant cells organized into structural or functional units. Particularly useful parts of a plant include harvestable parts and parts useful for the reproduction of offspring plants. The harvestable parts of a plant may be any useful part of the plant, such as flowers, pollen, seedlings, leaves, stems, sheaths, seeds, roots, and root nodules. Parts of a plant useful for reproduction include, for example, seeds, sheaths, cuttings, seedlings, and rhizomes. "Seed" refers to any plant structure formed by the continuous differentiation of the plant's ovule following its normal maturation point at flowering, regardless of whether it is formed in the presence or absence of fertilization, and regardless of whether the seed structure is fertile or sterile.

[0020] An "expression cassette" means a DNA sequence that can direct the expression of a specific nucleotide sequence in a suitable host cell, and includes a promoter operably ligated to the nucleotide sequence of interest, which is optionally operably ligated to a termination signal and / or other regulatory element. An expression cassette may also include sequences required for the proper translation of the nucleotide sequence. The coding region typically codes for the protein of interest, but the functional RNA of interest, e.g., antisense RNA or uncoding RNA, may also code in sense or antisense direction. An expression cassette containing the nucleotide sequence of interest may be a chimeric, meaning that at least one of its components is heterogeneous with respect to at least one of the other components. An expression cassette may also be naturally occurring but obtained in a recombinant form useful for heterogeneous expression. An expression cassette may be assembled entirely extracellularly (e.g., by recombinant cloning techniques). However, an expression cassette may also be assembled using partially endogenous components. For example, an expression cassette can be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence (the endogenous sequence is then functionally linked to and controlled by the promoter sequence).

[0021] A. SACPD-C gene expression mutant Embodiments of this disclosure feature a soybean plant, plant part, or plant cell containing one or more mutations that modulate the expression of the SACPD-C gene. The one or more mutations may be located in the coding or non-coding sequence of the SACPD-C gene. The one or more mutations may be located within the SACPD-C gene, i.e., within the open reading frame of the gene, or in a region that modulates the expression of the SACPD-C gene, i.e., within the regulatory region of the SACPD-C gene, or a combination thereof. For example, a promoter-targeted mutation that disrupts the binding sequence of the SACPD-C promoter may reduce the expression of the SACPD-C gene. In some cases, one or more mutations that alter the expression of the SACPD-C gene may be located in an intron region, exon region, enhancer region, promoter region, untranslated region (UTR5' or 3'), or a combination of two or more of these regions. The genomic sequence of the Glycine max SACPD-C gene is publicly available. For example, the native genome sequence of the SACPD-C gene, Glyma.14g27990, can be downloaded from the Soybase Database (www.soybase.org). Mutations that regulate the expression of the SACPD-C gene may be in one or more alleles of the gene. A representative example of the coding sequence of the naturally occurring Glycine max SACPD-C nucleotide sequence is shown in (SEQ ID NO: 1). The coding CDS does not contain native introns and codes for the same polypeptide as the native genome sequence. In some embodiments, the soybean plants, cells, plant parts, seeds, and their offspring provided herein may have mutations in each endogenous SACPD-C allele or its promoter (e.g., seed-specific promoter), resulting in reduced or complete inhibition of gene expression in the plant or in certain tissues. Therefore, in some cases, the plants, cells, plant parts, seeds, and offspring do not exhibit detectable levels of Δ9-stearoyl-acyl carrier protein desaturase expressed from the endogenous SACPD-C gene.

[0022] In certain embodiments, expression is reduced by mutations in the SACPD-C gene or its promoter. Reducing gene expression in a plant, plant part, or plant cell involves inhibiting, interfering with, knocking out, or knocking down the gene, resulting in reduced transcription and / or translation of the gene's encoded polypeptide compared to a corresponding control plant, plant cell, or plant or population of plant cells in which the gene or polypeptide expression is not inhibited, interfering with, knocked out, or knocked down. For example, gene knockdown using RNAi technology may be used. The reduction includes any decrease in expression level (e.g., a decrease of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or even 100%) compared to a corresponding control plant, plant cell, or plant or population of plant cells. In some embodiments, reducing expression by 50% or more may be particularly useful. Expression levels can be measured using methods such as 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.

[0023] In some cases, the plants, plant cells, plant parts, seeds, and offspring provided herein may be generated using a low-frequency cleavage endonuclease (e.g., a transcription activator-like effector nuclease (TALE nuclease)) system to perform targeted knockout in one or more alleles of the SACPD-C gene. The gene targeted for knockout may have the coding sequence shown in SEQ ID NO: 1, or a SACPD-C gene having at least 75% sequence identity to SEQ ID NO: 1.

[0024] The percentage sequence identity between a specific nucleic acid and the sequence referenced by a specific sequence number is determined as follows: First, the nucleic acid is compared to the sequence indicated by the specific sequence number using the BLAST 2 Sequences (Bl2seq) program from the standalone version of BLASTZ, including BLASTN version 2.0.14. If the two compared sequences share homology, the specified output file presents the homologous regions as aligned sequences. If the two compared sequences do not share homology, the specified output file does not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where identical nucleotide residues are shown in both sequences. The percentage sequence identity is determined by dividing the number of matches by either the length of the sequence shown in the identified sequence (e.g., sequence number 1) or the segmented length (e.g., 100 consecutive nucleotide or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. The percentage sequence identity value is rounded to the nearest tenth. For example, a knockout gene may have a coding sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1.

[0025] This disclosure provides materials and methods for using low-frequency cleavage endonucleases (e.g., TALE nucleases) to produce soybean plants and related products (e.g., seeds and plant parts) that are particularly suitable for providing highly saturated fatty acid oils resulting from targeted knockout of the SACPD-C gene. Other sequence-specific nucleases, including manipulated meganucleases / homing endonucleases (e.g., I-SceI or I-CreI), zinc finger nucleases (ZFNs), and clustered and regularly arranged short palindromic repeats (CRISPR) / CRISPR-related protein 9 (Cas9), may also be used to produce desired plant materials.

[0026] Low-frequency cleavage endonucleases can be native or engineered proteins that possess endonuclease activity against nucleic acid sequences having recognition sequences (target sequences) approximately 12–40 bp in length (e.g., 14–40, 15–36, or 16–32 bp in length; see, e.g., Baker, Nature Methods 9:23–26, 2012). Typical low-frequency cleavage endonucleases induce cleavage within their recognition site, leaving alternating cleavages of 4 nucleotides (nt) with 3'OH or 5'OH overhangs. In some embodiments, the low-frequency cleavage endonuclease may be a meganuclease, for example, a wild-type or variant homing endonuclease (e.g., a homing endonuclease belonging to the dodecapeptide family (see WO2004 / 06773336)). Another type of low-frequency cleavage endonuclease is referred herein to as the "Cas9 / CRISPR system," which comprises an endonuclease from the bacterial Cas9 family and a single strand that guides the endonuclease to a DNA target sequence, generally of 20 base pairs. It is characterized by its use in combination with a guide RNA. This DNA target is generally selected to be located in the genome upstream of a so-called PAM (protospacer adjacent motif) sequence motif (NGG or NAG) recognized by Cas9. The guide RNA molecule (gRNA) (which is generally a single-stranded RNA) is introduced into living cells to confer cleavage and specificity to Cas9. This is a synthetic RNA designed to match a desired 20 bp sequence in the genome upstream of the PAM. The use of Cas9 / CRISPR in plants is outlined by Belhaj et al. (2013) (incorporated by reference). In some embodiments, low-frequency cleavage endonucleases may be fusion proteins containing a DNA-binding domain and a catalytic domain having cleavage activity. TALE nucleases and ZFNs are examples of fusions of a DNA-binding domain with the catalytic domain of the endonuclease FokI. Custom-ordered TALE nucleases are marketed under the trade name TALEN® (Cellectis, Paris, France).The specificity of activator-like (TAL) effectors depends on effector variable repeats. Polymorphisms are mainly located at repeat positions 12 and 13, which are referred to herein as repeat variable duodecimal (RVD) residues.

[0027] The RVD of a TAL effector corresponds to nucleotides at its target site in a direct and linear manner, with one RVD per nucleotide, exhibiting some degeneracy and seemingly context-independent. This mechanism for protein-DNA recognition enables target site prediction for novel target-specific TAL effectors, as well as target site selection and manipulation of novel TAL effectors with binding specificity to selected sites.

[0028] The TAL effector DNA-binding domain can be fused to other sequences, such as endonuclease sequences, resulting in chimeric endonucleases that target specific selected DNA sequences, leading to subsequent cleavage of DNA at or near the targeted sequence. Such cleavage in DNA (double-strand breaks) can induce mutations in wild-type DNA sequences, for example, via NHEJ or homologous recombination. In some cases, TALE nucleases can be used to facilitate site-directed mutagenesis in complex genomes, knocking out or otherwise altering gene function with high precision and efficiency. As described herein, a TALE nuclease targeting the G.max SACPD-C gene can be used to mutagenerate the endogenous gene, resulting in a plant or plant tissue lacking detectable expression of SACPD-C. The fact that some endonucleases (e.g., FokI) function as dimers can be used to enhance the target specificity of TALE nucleases. For example, in some cases, pairs of TALE nuclease monomers targeting different DNA sequences (e.g., target sequences shown in Figure 4, SEQ ID NOs: 16 and 17, 19 and 20, 22 and 23, 25 and 26, 28 and 29, 31 and 32, 34 and 35, 37 and 38, 40 and 41, 43 and 44, and 46 and 47) may be used. Relevant sequences useful in the process include “functional variants” of the disclosed sequences. Functional variants include sequences having, for example, one or more nucleotide substitutions, deletions, or insertions, where the variant retains the desired activity. Functional variants can be produced by any of several methods available to those skilled in the art, such as site-directed mutagenesis, induced mutation, identification as an allele variant, or cleavage through the use of restriction enzymes.

[0029] When two TALE nuclease recognition sites are in close proximity, inactive monomers can assemble to form a functional enzyme that cleaves DNA. Highly site-specific restriction enzymes can be constructed by requiring DNA binding to activate the nuclease. Methods for selecting endogenous target sequences and generating TALE nucleases that target such sequences can be carried out as described elsewhere. See, for example, USPat.App.Pub.No.US2011 / 0145940A1 (June 2011) (incorporated by reference). In some embodiments, software for specifically identifying TALE nuclease recognition sites is used.

[0030] The method of this disclosure includes generating soybean plants, plant cells, or plant parts having mutations in one or more endogenous genes using low-frequency cleavage endonucleases (e.g., TALE nucleases). For example, one or more nucleic acids encoding TALE nucleases targeting selected SACPD-C sequences (e.g., SACPD-C sequences shown in Table 2, "hitSeq" (Figure 4, i.e., SEQ ID NOs. 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, or 45, or sequences having at least 95% identity to sequences in Table 2) may be transformed into plant cells (e.g., protoplasts) where they can be expressed. In some cases, one or more TALE nuclease proteins may be introduced into plant cells (e.g., protoplasts). Subsequently, the cells, or plant cell lines or plant parts generated from the cells, may be subjected to nucleic acid-based assays or protein-based assays to detect the expression levels as described above. The mutation can be analyzed through a assay or using nucleic acid-based assays to detect mutations at genomic loci (e.g., PCR and DNA sequencing, or PCR and subsequent T7E1 assay) to determine whether the mutation has been introduced to the target site(s). In the T7E1 assay, genomic DNA can be isolated from pooled callus, and sequences adjacent to the TALE nuclease recognition site for SACPD-C can be PCR-amplified. The amplified product can then be denatured and re-annealed. If the re-annealed fragment forms a heteroduplex, T7 endonuclease I cleaves at the mismatch site. The digested product can be visualized by gel electrophoresis to quantify the mutagenic activity of the TALE nuclease. In some embodiments, the methods provided herein may include contacting a population of soybean plant cells (e.g., protoplasts) having a functional SACPD-C allele with a low-frequency cleavage endonuclease targeting an endogenous SACPD-C sequence, selecting cells from the population in which at least one SACPD-C allele is inactivated, and growing the selected cells into soybean plants. The plants may produce oil with elevated saturated fatty acid levels compared to control soybean plants that do not contain the inactivated SACPD-C allele. The low-frequency cleavage endonuclease can be introduced into a population of cells via a nucleic acid encoding the low-frequency cleavage endonuclease (e.g., a vector or mRNA) or as a protein. In some cases, the methods provided herein may include culturing plant cells containing the inactivated SACPD-C allele(s) to generate one or more plant lines. In addition, or or otherwise, the methods provided herein may include isolating genomic DNA containing at least a portion of the SACPD-C locus from the plant cells.

[0031] In some embodiments, methods for delivering sequence-specific nucleases to soybean plants may include Agrobacterium-mediated transformation of plant parts or plant cells (e.g., leaves, stems, petioles, internode explants, callus, or protoplasts) using T-DNA encoding the sequence-specific nuclease; microparticle gun transformation of plant parts or plant cells using one or more nucleic acids encoding the sequence-specific nuclease; and / or cell-permeable peptide-mediated transformation of plant parts or plant cells using purified sequence-specific nuclease or nucleic acids (RNA or DNA) encoding the sequence-specific nuclease. Microparticle gun transformation utilizes a microparticle gun. This method has been used to deliver genome editing reagents in various crop plants, including soybeans. The microparticle gun is based on the direct delivery of nucleic acid sequences into plant cells using metal particles (e.g., gold or tungsten particles). The system can be adapted to deliver proteins. The nucleic acid sequences to be delivered may be DNA (including large DNA fragments) and RNA (such as mRNA). Using DNA as an example, the method involves coating particles with DNA and firing the coated particles at high speed into plant tissue for the purpose of penetrating plant tissue and cell walls, thereby allowing some particles to remain inside plant cells. Once inside the cell, the DNA elutes from the particles and becomes transiently expressed or stably integrated into the host genome. Physical delivery of nucleic acid sequences to cells avoids the host range limitations sometimes encountered with Agrobacterium and eliminates the need for binary vectors. Various tissues and cell types can be transformed by the particle gun. Multiple plasmids can be delivered in high-frequency simultaneous transformations. Further delivery methods include insect vectors, transplantation, or DNA abrasion, following methods that are standard in the art.

[0032] In some embodiments, soybean lines having mutations in one or more SACPD-C alleles can be produced by polyethylene glycol (PEG)-mediated transformation. For example, protoplasts can be isolated from surface-sterilized leaves and transformed in the presence of PEG using plasmids encoding one or more sequence-specific nucleases. Transformation efficiency can be monitored by delivery of a detectable marker, such as a YFP plasmid, which can be visualized using fluorescence microscopy or flow cytometry. After PEG-mediated transformation, protoplasts can be cultured using methods and media known to those skilled in the art of protoplast culture. After a suitable length of time in the culture, callus derived from the protoplasts identified as mutants can be grown, transferred to shoot-inducing medium, and then (once roots have formed) transferred to soil and grown until mature for seed production.

[0033] In some embodiments, delivery of one or more sequence-specific nucleases to soybean plants can be achieved through transient delivery or stable integration into the host genome. To transiently deliver sequence-specific nucleases, transformed soybean plant portions or plant cells (using the method described above) can be placed on a regeneration medium without the selective agent to regenerate soybean plants. The regenerated plants can then be screened to identify those containing nuclease-induced mutations. To stably integrate the genomic manipulation reagent into the host genome, nucleic acids encoding sequence-specific nucleases can be delivered co-delivered with nucleic acids encoding plant selection markers. The selection markers can be vested on the same vector as the sequence-specific nuclease(s) or delivered as separate vectors. After transformation, soybean plant portions or plant cells can be placed on a regeneration medium containing the appropriate selective agent to regenerate transgenic soybean plants. In preferred embodiments, the soybean plants do not contain the transgene.

[0034] In some embodiments, to increase the efficiency of sequence-specific nuclease-induced mutagenesis, nucleases may be co-delivered to plant cells together with plasmids encoding one or more exonuclease proteins using a delivery method described herein (e.g., a microparticle gun). Such exonucleases include, but are not limited to, members of the TREX (Therapeutic Erythrocyte Exchange Exonuclease) family of exonucleases, such as TREX2. Other exonucleases may also be used in the methods provided herein.

[0035] Another genome manipulation tool that may be used in the methods provided herein is based on RNA-guided Cas9 nuclease from the type II prokaryotic CRISPR (clustered, regularly arranged short palindromic repeat) adaptive immune system. This system allows for the cleavage of DNA sequences adjacent to short sequence motifs called protospacer-adjacent motifs (PAMs). The cleavage is achieved by manipulating a specific crRNA that is complementary to the target sequence. The crRNA associates with heterologously expressed Cas9 endonuclease in living cells. In the crRNA / Cas9 complex, a double tracrRNA:crRNA structure acts as a guide RNA that directs the Cas9 endonuclease to a congeneral target sequence. The PAM motif present in the soybean SACPD-C gene allows for the design of SACPD-C gene-specific crRNAs for introducing mutations or inactivating one or more SACPD-C alleles in soybean plant cells, into which Cas9 endonuclease and crRNA are transfected and subsequently expressed. Therefore, in some embodiments, this approach can be used to obtain SACPD-C mutant plants as described herein.

[0036] The expression of a plant gene can be altered by inserting a copy of the nucleic acid sequence, including the genomic sequence or coding sequence of the plant gene, into a genomic locus different from the gene's locus in the plant. The copy of the genomic sequence or coding sequence is operably ligated to a promoter, where the different genomic locus has transcriptional activity. The inserted sequence may be cisgenic or endogenous and may be obtained from the plant or synthesized. In some cases, the methods provided herein may involve targeted knockout of the original endogenous SACPD-C gene and insertion of a SACPD-C expression cassette containing the coding sequence of a cisgenic SACPD-C gene operably ligated to a promoter that provides the desired expression profile at a different genomic locus. "Operatably ligated" means that each coding sequence is fused in-frame to the promoter, and as a result, the coding sequence is faithfully transcribed, spliced, and translated. The genomic locus into which the cassette is inserted may be a different location in the genome from that of the original endogenous SACPD-C gene. The locus may be on a different chromosome from the original endogenous SACPD-C gene, or on the same chromosome. Preferably, if the insertion is on the same chromosome as the endogenous gene, the genomic locus of the insertion does not capture the transcriptional activity of the promoter from the original endogenous SACPD-C gene.

[0037] A promoter is typically an expression regulatory sequence consisting of a region of a DNA molecule upstream of the point where transcription begins (generally near the start site for RNA polymerase II). Promoters are involved in the recognition and binding of RNA polymerase and other proteins to initiate and regulate transcription. Promoters typically include at least a core (basic) promoter. A promoter may also include at least one regulatory element, such as an upstream element. Such elements include an upstream activation region (UAR) and, optionally, other DNA sequences that affect the transcription of polynucleotides, such as a synthetic upstream element. The selection of a useful promoter in a method depends on the desired type of expression to be achieved, including, but not limited to, efficiency, selectivity, inducibility, desired expression level, and cell or tissue specificity. For example, tissue, organ, and cell-preferential promoters may be used that confer transcription only in or predominantly in specific tissues, organs, and cell types, respectively. In some embodiments, promoters specific to vegetative tissues such as the stem, parenchyma, basic meristem, vascular bundles, cambium, phloem, cortex, apical meristem, lateral branch meristem, apical meristem, lateral root meristem, leaf primordia, mesophyll, or leaf epidermis may be preferred regulatory regions. In some embodiments, promoters that are inactive in seeds may be useful. Other classes of promoters include, but are not limited to, inductive promoters, which confer transcription in response to inducing substances, such as external stimuli, including chemical agents, developmental stimuli, or environmental stimuli. Promoters may be promoters that preferentially express a particular tissue, organ, or other part of the plant, or that may be expressed during a particular developmental stage or under specific conditions. When referred to preferential expression, it means expression in a particular plant tissue at a higher level than in other plant tissues.

[0038] A promoter of interest may have strong or weak transcriptional activity. Those skilled in the art will understand that promoter sequences can be modified to provide a range of expression levels for operably linked heterologous nucleic acid molecules. Generally, a “weak promoter” is intended to drive the expression of a coding sequence at low levels. “Low levels” are intended to be around 1 / 10,000 transcript to 1 / 100,000 transcript to 1 / 500,000 transcript. Conversely, a strong promoter drives the expression of a coding sequence at high levels, or at around 1 / 10 transcript to 1 / 100 transcript to 1 / 1,000 transcript. It is recognized that enhancers may be used in combination with the promoter region to increase transcription levels.

[0039] Altering expression using a knockout mutation in the original endogenous SACPD-C gene can result in all SACPD-C gene expression being controlled by an operablely linked promoter. For the SACPD-C to be knocked in, a nodule-specific promoter may be desired. In some cases, the method involves identifying an endogenous gene that matches the desired expression profile and cloning the regulatory elements of the endogenous gene. For example, suitable promoters to include in an expression cassette for nodule-specific SACPD-C expression may include, for instance, the promoters of the nodule-specific genes Glyma05g01360 and Glyma13g44970. Sequence IDs 2-5 show the promoter and terminator sequences for Glyma05g01360 and Glyma13g44970. In other situations, tissue-specific, step-specific, or inducible expression may be desired.

[0040] Any method that provides efficient transformation may be used. For example, methods for plant cell transformation include the use of Ti or Ri plasmids, microinjection, electroporation, DNA microparticle guns, and liposome fusion. In many cases, it is desirable to have constructs bounded by T-DNA on one or both sides, particularly left and right boundaries, and more specifically, a right boundary. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as the mode of transformation, but T-DNA boundaries may find use for other modes of transformation.

[0041] In one embodiment, the method provided herein may include targeted knockout of the original endogenous SACPD-C gene and targeted insertion of SACPD-C (genomic or CDS) into a locus having a gene sequence near the promoter of interest. Knocking out the original endogenous SACPD-C gene may result in all SACPD-C gene expression being controlled by the promoter of interest. Preferably, the promoter is inactive in the developing seed and is, for example, a nodule or root-specific promoter. In other situations, stage-specific or inducible expression may be desired. In some cases, the method includes identifying an endogenous gene that matches the desired expression profile. Several methods and software programs are available for identifying genes with desired expression characteristics. These include, but are not limited to, RNA sequencing (whole transcriptome shotgun sequencing). Once a gene with the desired expression profile is identified, it is understood that the promoter sequence (usually upstream of or near the gene of interest), as opposed to the actual gene expressed by the promoter, is a key component used in the method. The final step is to determine the specific type of genome editing required to capture the transcriptional activity of the identified promoter.

[0042] In some cases, the methods provided herein may involve seed-specific knockout of the SACPD-C gene. For example, a geminiviral sequence may be used as a gene targeting vector to target the endogenous promoter of the SACPD-C gene and replace it with an inactive promoter in developing seeds. Geminiviruses are a large family of plant viruses containing a circular single-stranded DNA genome, and their sequences may be used as gene targeting vectors. For example, a geminiviral genome may be manipulated to contain a desired modification adjacent to a sequence homologous to a target locus. In some cases, this may be achieved by replacing a non-essential geminiviral nucleotide sequence (e.g., a CP sequence) with a desired repair template. Examples of geminiviruses include cabbage leaf curl virus, tomato golden mosaic virus, bean yellow wilt virus, African cassava mosaic virus, wheat streak virus, miscanthus streak mastrevirus, tobacco yellow wilt virus, tomato yellow leaf curl virus, bean golden mosaic virus, beet curly top virus, corn streak virus, and tomato pseudo-curly top virus.

[0043] Therefore, the repair template contains homology to the promoter sequence of the endogenous SACPD-C gene. Typically, the repair template contains nucleic acids that replace the endogenous target sequence in the plant, flanked by sequences homologous to the endogenous sequences on both sides of the target. The flanking homologous sequences may be referred to as “homologous arms.” In this case, the endogenous sequence is replaced by one of the promoter sequences described above. Within the repair template, the flanking homologous sequences may have any preferred length. The preferred length of the flanking homologous sequence is related to the length of the desired substitution. Thus, the length may be at least about 25 nt and may include sequences of 750 nt or more. In some cases, the flanking homologous sequences may be longer than 800 nt, 900 nt, or 1,000 nt. The repair template and DNA viral plasmid may be prepared using techniques that are standard in the art. Constructions containing the repair template may be delivered to plant cells, for example, using a particulate gun. Alternatively, the repair template may be delivered using Agrobacterium-mediated transformation, insect vectors, transplantation, or DNA polishing, according to methods that are standard in the art.

[0044] In addition to repair templates, this method includes endonucleases that can be specially ordered to target specific nucleotide sequences and generate double-strand breaks in or near those sequences. Examples of such specially ordered endonucleases include ZFNs, meganucleases, and TALE nucleases, as well as the CRISPR / Cas system described above. Similar to TALE nucleases, components of the CRISPR / Cas system (Cas9 endonucleases and crRNA and tracrRNA, or cr / tracrRNA hybrids) can be delivered to cells in a geminiviral construct.

[0045] After infecting or transfecting plants with repair templates and associated endonucleases, it may be determined using any preferred method whether seed-specific knockout of the endogenous SACPD-C gene has occurred. For example, PCR-based methods may be used to confirm whether the genomic target site contains the repair template sequence and / or whether precise recombination has occurred at the 5' and 3' ends of the repair template.

[0046] The disclosed strategies may be combined with conventional breeding methods and the targeted approaches described above. Targeted gene editing of SACPD-C gene expression may be performed on any line, species, or cultivar of the soybean of interest, but is not limited to these. In some embodiments, targeted gene editing or other genetic modifications may be performed in the germplasm or other plant tissues that already possess features (e.g., genetic predisposition) for producing oil with an increased saturated oil content.

[0047] B.FATB-1A gene expression mutant Embodiments of this disclosure feature a soybean plant, plant portion, or plant cell containing one or more mutations that regulate the expression of the FATB-1A gene. The one or more mutations may be located within the regulatory regions of the FATB-1A gene, such as the enhancer region, promoter region, UTR region (5' or 3'), silencer region, or combinations of regions. Genomic sequences associated with the Glycine max FATB-1A locus are publicly available. For example, the sequence of the native soybean FATB-1A gene, Glyma05g08060, can be downloaded from the Soybase Database (www.soybase.org). Mutations may be located at genomic loci different from those of the endogenous FATB-1A gene. For example, the coding sequence of a naturally occurring G.max FATB-1A nucleotide sequence (e.g., a representative sequence is shown in (SEQ ID NO: 6)) may be inserted into or into any locus of the genome, thereby providing at least two functional FATB-1A genes. The encoding CDS does not contain native introns and encodes the same polypeptide as the native genome sequence, resulting in increased or elevated gene expression in plants or in certain tissues (e.g., in developing seeds). Thus, in some cases, plants, cells, plant parts, seeds, and offspring exhibit elevated levels of acyl-ACP thioesterase expressed from one or more soybean FATB-1A genes.

[0048] The gene editing techniques described above for regulating the expression of the SACPD-C gene may be modified to enhance the expression of FATB-1A. In one or more embodiments, the methods provided herein may involve targeted substitution of the FATB-1A promoter in an overexpression promoter. The promoter may be a native soybean promoter, which may be a seed-specific promoter, such as the promoters of genes encoding β-conglycinin and lectins. A suitable promoter may be selected based on the expression profile of the seed-specific gene. In some cases, the method involves identifying an endogenous gene that matches the desired expression profile. Several methods and software programs are available for identifying genes with desired expression characteristics. These include, but are not limited to, RNA sequencing (whole transcriptome shotgun sequencing). Once a gene with the desired expression profile has been identified, it is understood that the promoter sequence (usually upstream of or near the gene of interest), as opposed to the actual gene expressed by the promoter, is a key component used in the method. The final step is to determine the specific type of genome editing required to capture the transcriptional activity of the identified promoter. A suitable seed-specific promoter may be one that drives expression at a specific developmental stage. Preferably, the promoter provides high expression in developing seeds. More preferably, high expression in seeds, e.g., developing seeds, is combined with no expression or very low levels of expression in other tissues. In some cases, the promoter is from a gene encoding a fatty acid desaturase enzyme. For example, as described in the examples, the expression profiles of GmFAD2A (Glyma10g42470) and GmFAD2B (Glyma20g24530) have been identified by the inventors as suitable candidates for driving FATB-1A overexpression. Thus, a geminivirus may be designed to target the endogenous FATB-1A promoter and replace it with a promoter of the endogenous FAD2A or FAD2B gene.TALENs targeting the FATB-1A 5'-UTR region can be designed based on the 5'-UTR sequence. Exemplary TALENs are shown in Figure 5 (Table 3), including SEQ ID NOs. 49 and 50, 52 and 53, 55 and 56, 58 and 59, and 61 and 62.

[0049] In some cases, mutations that enhance soybean FATB-1A expression can be untargeted. For example, an expression cassette containing the coding sequence of the soybean FATB-1A gene operably ligated to a strong promoter or seed promoter can be inserted into any genomic locus (e.g., by a microparticle gun method). A suitable cisgenic promoter can be selected based on the desired expression profile. For example, the promoter can be selected based on high expression in developing seeds and no or low levels of expression in other tissues. In some cases, the operably ligated promoter may be the sequence shown in SEQ ID NOs: 7 or 8. The cassette may contain the termination sequence of GmFAD2A (Glyma10g42470) or GmFAD2B (Glyma20g24530) (SEQ ID NOs: 9 and 10, respectively).

[0050] Increased expression encompasses any degree of increase in total expression level compared to the corresponding control plant, plant cell, or population of plants or plant cells (e.g., increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%). For example, expression may increase by approximately 2-fold, 5-fold, or 10-fold compared to the control plant, plant cell, or population thereof. Expression levels may be measured using methods such as 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.

[0051] The disclosed strategies may be combined with conventional breeding methods and the targeted approaches described above. Targeted gene editing of FATB-1A gene expression may be performed on any line, species, or cultivar of the soybean of interest, but is not limited to these. In some embodiments, targeted gene editing or other genetic modifications may be performed in the germplasm or other plant tissues that already possess features (e.g., genetic predisposition) for producing oil with an increased saturated oil content.

[0052] C. Accumulated traits Embodiments characterized by soybean plants, plant parts, or plant cells having mutations that modulate the expression of both the SACPD-C and FATB-1A genes, wherein the plants, plant parts, or plant cells produce oil having an increased saturated fatty acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking one or more mutations, are within the scope of the disclosure. In particular, the disclosure features plant lines having one or more mutations directed toward increasing the saturated fatty acid content of the oil produced. In some cases, the plant line may, in conjunction with tissue-specific expression of soybean stearoyl-ACP desaturase and / or overexpression of soybean stearoyl-ACP thioesterase, provide transcription or transcription and translation of one or more other sequences of interest.

[0053] Providing plants transformed for a synergistic effect may involve the use of multiple distinct nucleic acid constructs or transformation events. For example, multiple constructs as described above may be introduced into plant cells by the same or different methods, including introducing such traits by including two transcription cassettes in a single transformation vector, simultaneous transformation of two expression constructs, retransformation using plant tissue expressing one construct with an expression construct for a second gene, or by crossing transgenic plants via traditional plant breeding methods, as long as the resulting product is a plant having both features incorporated into its genome. In some cases, soybean plants are transformed and regenerated using the constructs described above. The regenerated plants with the desired sequence are self-pollinated to remove the gene-editing plasmid and retain the targeted mutation. Lines of the resulting null isolates with the specific mutation may then be crossed to provide plant seeds or plants exhibiting a synergistic effect. For example, a null isolate from a seed-specific SACPD-C knockout line can be crossed with a null isolate from a seed-specific FATB-1A overexpression line, and a null isolate from a ubiquitous knockout SACPD-C line can be crossed with a null isolate that overexpresses both SACPD-C and FATB-1A in a seed-specific manner.

[0054] Any combination of approaches may be used to achieve combinational regulation of the GmSACPD-C and GmFATB-1A genes. For example, combinational regulation may include TALEN-mediated knockout of one or more SACPD-C alleles, insertion of a first linear cisgenic cassette containing a nodule promoter operably linked to the coding sequence of GmSACPD-C, and insertion of a second linear cisgenic cassette containing a seed promoter operably linked to the coding sequence of GmFATB-1A. In some cases, the first linear cisgenic cassette has the sequence shown in SEQ ID NO: 13, containing nodule Glyma13g44970 promoter-GmSACPD-C-Glyma13g44970 terminator, and the second cisgenic cassette has the sequence shown in SEQ ID NO: 14, containing seed FAD2A promoter-GmFATB1A-FAD2A terminator.

[0055] One or more soybean plants may be obtained from individual mutagenic plant cells (and plants grown therefrom), and at least one of the plants may be identified as containing a mutation that modulates the expression of the SACPD-C gene or the FATB-1A gene. A population of soybean plants sharing a common gene pool is provided. For example, "M0" may be used to refer to plant cells (and plants grown therefrom) exposed to TAL effector nuclease, while "M1" refers to seeds produced by self-pollinated M0 plants and plants grown from such seeds. "M2" is the offspring (seeds and plants) of self-pollinated Mi plants, "M3" is the offspring of self-pollinated M2 plants, and "M4," "M5," "M6," etc., are, respectively, offspring of self-pollinated plants of the previous generation. As used herein, the term "selfed" means self-pollinated.

[0056] In some cases, at least one of the plants may be identified as containing a mutation in the SACPD-C gene, and at least one of the plants may be identified as containing a knock-in SACPD-C gene. Soybean plants carrying mutant alleles can be used in plant breeding programs to create novel and useful lines and varieties. Thus, in some embodiments, soybean plants containing a mutation in the endogenous SACPD-C gene are crossed with a second soybean plant containing at least one insertion of the SACPD-C gene operably linked to a promoter that does not drive expression in developing seeds, and offspring of the hybrid in which the gene mutation is present are identified. In other embodiments, soybean plants containing at least one mutation that modulates the expression of the SACPD-C gene and at least one mutation that modulates the expression of the FATB-1A gene are crossed with a second soybean plant, and offspring of the hybrid in which the gene mutation is present are identified. It is understood that the second soybean plant may contain the same or different mutations as the plant it is crossed with, or it may be wild-type in terms of SACPD-C or FATB-1A gene expression.

[0057] Breeding can be carried out through known procedures. DNA fingerprinting, SNPs, or similar techniques can be used in marker-assisted selection (MAS) breeding programs to transfer or breed mutations that regulate the expression of the SACPD-C or FATB-1A allele to other soybean plants. For example, breeders can create segregated populations from genotype crosses containing mutant alleles with agronomically desirable genotypes. Plants of the F2 generation or backcross generation can be screened using markers or fragments developed from the mutant sequences. Plants identified as having the mutation can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected genetic pattern or the MAS technique used, it may be necessary to self-pollinate the selected plants before each backcross cycle to aid in the identification of individual plants of interest. Backcrossing or other breeding procedures may be repeated until the desired phenotype of the repeating parent is restored.

[0058] A successful cross yields F1 plants that are fertile and, if desired, can be backcrossed with one of the parents. In some embodiments, the F2 generation plant population is screened for SACPD-C and FATB-1A gene expression, for example, according to standard methods, to identify plants that fail to express SACPD-C in developing seeds and overexpress FATB-1A due to a mutation. The selected plants are then crossed with one of the parents, and the first backcross (BC1) generation plants are self-pollinated to produce a BC1F2 population, which is again screened for variant gene expression. The process of backcrossing, self-pollination, and screening is repeated at least four times, for example, until the final screening produces plants that are fertile and reasonably similar to the repeating parent. These plants can, if desired, be self-pollinated, and the offspring can then be screened again to confirm that the plants lack SACPD-C expression in developing seeds and overexpress FATB-1A. Selected plants can be randomly subjected to cytogenetic analysis to confirm chromosome complementation and chromosome pairing relationships. Seeds from the breeders of the selected plants can be produced using standard methods, including, for example, oil analysis to determine the levels of saturated fatty acids, including stearic acid and palmitic acid.

[0059] In situations where the original F1 hybrid obtained from a cross between a first mutant soybean parent and a second wild-type soybean parent is crossed with or backcrossed with the mutant soybean parent, the offspring of the backcross can be self-pollinated to produce a BC1F2 generation, which can then be screened for mutations.

[0060] The results of plant breeding programs using the mutant soybean plants described herein may be novel and useful lines and varieties. As used herein, the term “variety” refers to a group of plants that share invariant characteristics that distinguish them from other plants of the same species. Varieties are often (but not always) commercially available. While possessing one or more distinctive traits, a variety may be further characterized by very small overall variability among individuals within that variety. A “purebred” variety may be produced by several generations of self-pollination and selection, or by vegetative propagation from a single parent using tissue or cell culture techniques. A variety may, in essence, originate from another line or variety. A variety is “essentially derived” from an initial variety if a) it is predominantly derived from the initial variety, or from a variety predominantly derived from the initial variety, while retaining the expression of essential characteristics resulting from the genotype or combination of genotypes of the initial variety; b) it is clearly distinguishable from the initial variety; and c) it matches the initial variety in the expression of essential characteristics resulting from the genotype or combination of genotypes of the initial variety, except for differences resulting from the act of induction. Essentially derived varieties can be obtained, for example, by selecting natural or induced mutants, somatic cell breeding system variants, variant individuals from early variety plants, backcrossing, or transformation. "Lineages," as distinguished from varieties, almost always refer to groups of plants used non-commercially, for example, in plant research. Lineages typically show little overall variability between individuals for one or more traits of interest, although some variability may exist between individuals for other traits.

[0061] The methods provided herein may be used to produce plant parts (e.g., seeds) or plant products (e.g., oils) having an increased saturated fatty acid content compared to the corresponding plant parts or products from wild-type plants. The fatty acid content of the plant parts or plant products may be evaluated using standard methods.

[0062] D. High-saturated fatty acid soybeans and their uses The mutations described herein provide soybean plants, plant parts, or plant cells that can produce oil having an increased saturated fatty acid content compared to oil produced from corresponding soybean plants, plant parts, or plant cells lacking one or more mutations.

[0063] In one or more embodiments of this disclosure, the mutation results in a soybean plant, plant part, or plant cell capable of producing an oil containing a total saturated fatty acid content of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, and up to about 80%. The total saturated fatty acid content is preferably in the range of about 20% to about 50%. The percentages of fatty acids expressed herein are on a weight basis unless otherwise specified. Oil extracted from soybean seeds produced by soybean plants has increased stability and superior cooking properties compared to oil extracted from standard soybean seeds having a lower saturated fatty acid content. Furthermore, the oil has a higher level of solids than commercial soybean oil, making it a more preferable material for the preparation of food products such as margarine, soy flour, soy milk, and shortening. Transesterification of the oil can further increase the solids content and the usefulness of the oil in the preparation of food products. A higher saturated fatty acid content may provide, for example, a substitute for palm oil fraction or cocoa butter.

[0064] A higher saturated fatty acid content may result from one or more of the following: an increase in the level of stearic acid and / or an increase in the level of palmitic acid. For example, embodiments of the present disclosure include soybean oil having a stearic acid content of at least about 10% of the total fatty acid composition, e.g., about 10-24%, soybean oil having a palmitic acid content of at least about 10% of the total fatty acid composition, e.g., about 10-24%, and combinations thereof. For example, the resulting palmitic acid concentration may be at least about 14% of the total fatty acid composition, while the resulting stearic acid concentration may be at least about 10% of the total fatty acid composition, or the palmitic acid content of the soybean lineage of the present invention may be at least about 10%, while the stearic acid content is at least about 20% or more. A specific application generally determines the desired total saturated fatty acid content. For example, the relative levels of palmitic acid and stearic acid content can be varied to suit the specific needs of the application.

[0065] Embodiments of this disclosure include soybeans having desired palmitic acid and stearic acid content, and varying content of oleic acid, linoleic acid, and linolenic acid. The levels of these fatty acids and others can be adjusted for specific applications. For example, it is within the scope of the invention to modify the soybean plants, plant parts, and plant cells described herein by incorporating other fatty acids that may be desired (by genetic modification or other means).

[0066] Soybeans and their extracted oils can be used in a variety of applications. For example, soybean oil as described herein can be used, in part or in whole, to replace palm oil, cocoa butter, or other foreign oils. Soybean oil can act as a raw material for cost-effective mixtures with other saturated fats and / or triacylglycerols through transesterification. Food applications include, for example, margarine and shortening, as well as products containing these ingredients (e.g., baked goods and confectionery). The high saturated fatty acid content is also advantageous for skincare compositions. For example, palmitic acid promotes the regeneration of natural oils and helps the skin maintain its protective barrier. Stearic acid acts primarily as a lubricant; it allows the skin to maintain a proper moisture balance. Therefore, extracted soybean oil can be included in topical compositions, including creams, lotions, and spray oils, which can be easily applied to hair, skin, and nails.

[0067] High-saturated fatty acid soybeans can be used in the production of soy-based food products such as tofu and soy milk. Furthermore, soybeans can be ground into whole-fat soy flour, which can be used in candies, gravy, sauces, frozen desserts, pasta, meat products, and baked goods. Soy flour can be used to increase the protein content of baked goods without affecting texture. [Examples]

[0068] The following embodiments are intended to illustrate the above invention and should not be construed as limiting its scope. Those skilled in the art will readily recognize that the examiner may suggest many other ways in which the invention can be carried out. Numerous variations and modifications can be made while remaining within the scope of the invention.

[0069] I. Expression profile of the SACPD gene The majority of the identified variations in stearic acid content in soybean oil are attributed to mutations in the Δ9-stearoyl-acyl carrier protein desaturase (SACPD) gene. These enzymes desaturate stearoyl-ACP to oleoil-ACP in plastids. Three isoforms of SACPD have been identified in soybeans. Two of the isoforms, SACPD-A and SACPD-B, are active in both vegetative and reproductive tissues, while the third, SACPD-C (Glyma14g27990), is expressed primarily in developing seeds and root nodules. In soybeans, chemically mutagenic SACPD-C mutant lines have been reported to produce oil with stearic acid content ranging from 6 to 14%, which is 1.5 to 3 times the level found in wild-type seeds of the Williams 82 cultivar. Sequencing of candidate genes revealed that all of these lines possess an amino acid substitution in the gene encoding SACPD-C, which is required for the conversion of stearic acid to oleic acid. Further studies also revealed that SACPD-C mutants have several defects in nitrogen-fixing root nodules and leaf structure.

[0070] II. Expression Profiles of FatA and FatB Genes As the final step in the fatty acid biosynthesis pathway, acyl-acyl carrier protein (ACP) thioesterases determine the chain length of the acyl group that leaves the plastid for further metabolism in the cytosol. Higher plant acyl-ACP thioesterases can be divided into two distinct classes based on their amino acid sequence: FatA and FatB. FatA thioesterases primarily hydrolyze unsaturated 16:1 and 18:1-ACPs, while FatB is for C8-C16 saturated acyl-ACPs.

[0071] III. Regulation of GmSACPD expression Sequence-specific nucleases were designed to completely inactivate or knock out the GmSACPD-C gene (also known as "eccentric knockout"). A TAL effector endonuclease pair was designed to target GmSACPD-C on the first exon (Figure 4, showing Table 2).

[0072] TAL effector endonucleases were selected for expression in soybean cells. The activity of these TAL effector endonucleases was evaluated at their endogenous target sites in soybeans. Each TAL effector endonuclease was cloned into a T-DNA vector downstream of an inducible promoter, then transformed into Agrobacterium rhizogenes strains, and subsequently used to infect soybean hemicotyledons to produce transgenic hairy roots. Three weeks after infection, the hairy roots were harvested, frozen in liquid nitrogen, and genomic DNA was prepared using standard methods.

[0073] To determine whether NHEJ-mediated mutations are generated at target sites in the soybean genome by TAL effector endonuclease, DNA from nine hairy roots is subjected to a PCR enrichment assay. Samples containing TAL effector endonuclease-induced NHEJ mutations may lack restriction enzyme sites within the spacer sequence, resulting in an undigested PCR product that appears as a full-length band on the gel. Therefore, an undigested PCR product is observed for the GmSACPD-C gene.

[0074] Undigested PCR products are cloned and sequenced to verify that they contain TAL effector endonuclease-induced mutations. PCR products are cloned using a commercially available cloning kit according to the manufacturer's instructions. Individual clones derived from a given undigested fragment are sequenced, and their DNA sequences are aligned with the wild-type GmSACPD-C gene sequence.

[0075] Plants containing the inactivated or knocked-out GmSACPD-C gene will be grown, and the nodule formation phenotype will be evaluated.

[0076] To provide a cisgenic expression cassette that can be used to rescue GmSACPD-C gene expression in root or nodule tissue containing an inactivated or knocked-out GmSACPD-C gene, we searched the soybean expression database SoyBase and identified two nodule-specific genes. Glyma05g01360 and Glyma13g44970 were selected as candidates based on their expression profiles. Both genes are highly expressed in nodules and roots, like SACPD-C, while showing no expression or very low levels of expression in developing seeds (Figures 1 and 3A-B). Sequence IDs 2-5 show the respective promoter and terminator sequences for Glyma05g01360 and Glyma13g44970. A linear cisgenic cassette containing a nodule promoter operably ligated to GmSACPD-C was cloned and introduced into soybean plants using a microparticle gun method.

[0077] To provide seed-specific knockout of GmSACPD-C, a geminivirus is designed to replace the endogenous GmSACPD-C promoter with a nodule-specific promoter. The construct is delivered to soybean plants via Agrobacterium-mediated transformation.

[0078] IV. Regulation of GmFATB-1A expression Overexpression of the GmFATB-1A gene in developing seeds is achieved using one or more of the following methods.

[0079] Two seed-specific genes, GmFAD2A (Glyma10g42470) and GmFAD2B (Glyma20g24530), were selected as candidate seed promoters based on their expression profiles (i.e., highly expressed in developing seeds and absent or very low levels of expression in other tissues (Figures 2 and 3A-B)). Sequence ID 6 shows the coding sequence for GmFATB-1A. Sequence IDs 7-10 show the promoter and terminator sequences for GmFAD2A (Glyma10g42470) and GmFAD2B (Glyma20g24530).

[0080] To enhance GmFATB-1A expression, a linear cisgenic cassette containing a seed promoter operably linked to GmFATB-1A is synthesized. The cisgenic cassette is introduced into soybean plants along with a linear or cyclic selective marker gene via co-delivery by a microparticle gun.

[0081] In parallel, to provide a seed-specific promoter knock-in for driving the overexpression of the FATB-1A gene, a geminivirus is designed to replace the endogenous GmFATB-1A promoter with a promoter sequence that drives the expression of endogenous GmFAD2A or GmFAD2B. The coding sequences for GmFAD2A and GmFAD2B are provided in SEQ ID NOs. 11 and 12, respectively. A TALEN targeting GmFAD2A (or GmFAD2B) upstream of the endogenous FATB-1A locus is designed on the first exon. The construct is delivered to soybean plants via Agrobacterium-mediated transformation.

[0082] Regulation of the expression of both V.GmSACPD-C and GmFATB-1A (Approach 2A) The technique for TALEN-mediated knockout of gmSACPD-C is combined with the introduction of a linear cisgenic cassette having a Glyma13g44970 promoter operably linked to a GmSACPD-C coding sequence having a Glyma13g44970 terminator, and a linear cisgenic cassette having a FAD2A promoter operably linked to GmFATB-1A having an FAD2A terminator.

[0083] VI. Fatty acid composition analysis The fatty acid content is analyzed from the seeds of soybean lines transformed using one or more of the above constructs. One to five seeds each of the knockout, cisgenic, and control soybean lines are ground for oil extraction. The oil is extracted from the ground soybean seeds and derivatized into methyl esters. The resulting fatty acid methyl esters are extracted in hexane and separated by gas chromatography (GC).

[0084] Analysis of the fatty acid composition of the seed oils shows that stearate (C18:0) levels and / or palmitate (C16:0) levels are significantly higher than those obtained from seed oils of non-transformed control plants. Total saturated fatty acid levels are increased to approximately 20-40%.

[0085] VII. Decreased SACPD-C expression due to targeted mutations (Approach 1A) After verifying that TAL effector endonucleases produced targeted modifications at endogenous target sites, experiments were conducted to create soybean plants with mutations in GmSACPD-C. To achieve this, TAL effector endonuclease pairs were cloned into bacterial vectors and delivered to plant cells by Agrobacterium-mediated transformation or by using a microparticle gun.

[0086] Transgenic soybean plants expressing TAL effector endonuclease were generated using a standard transformation protocol. After transformation of soybeans (cv BERT) with the sequence encoding the GmSACPD-C-T03 TAL effector endonuclease, putative transgenic plants were regenerated. The plants were transplanted to soil, and after approximately 4 weeks of growth, small leaves were collected from each plant for DNA extraction and genotyping. From independent transformations, events #1-#5 with biallelic or homozygous mutations at the target site were generated. DNA samples were analyzed by next-generation sequencing of the GmSACPD-C DNA sequence adjacent to the GmSACPD-C-T03 TAL effector endonuclease binding site. The resulting reads were then aligned relative to the wild-type sequence to determine the alleles. The results are summarized in Table 4, and representative sequences, SEQ ID NOs. 63-71, are shown in Figures 6A-B and 7. Combined, these results confirm the successful mutagenesis of GmSACPD-C in T0 soybean plants using the TAL effector endonuclease GmSACPD-C-T03. [Table 4]

[0087] VII. Seed-specific silencing of SACPD-C by targeted promoter substitution (Approach 1B) A genomic manipulation reagent to replace the endogenous SACPD-C promoter with a nodule-specific promoter was delivered to soybean protoplasts. Protoplasts were prepared using conventional methods. Briefly, soybean seeds were grown in vitro on germination medium under sterile conditions five days prior to transformation. The first true leaves and hypocotyls of the soybean seedlings were then digested overnight. Isolation and transformation were performed the day after overnight digestion. During isolation, the protoplasts were first screened to ensure a proper yield of 1 million cells. These cells were subjected to several washes in wash buffer and divided into 200,000 cells for each construct used for either validation via a yellow fluorescent protein (YFP) cassette or extraction of genomic DNA.

[0088] A geminiviral binary vector for targeted promoter substitution was constructed (schematically shown in Figure 8(A)). Protoplast cells were transformed using plasmids encoding each TALEN pair ("GmSACPD-C-T10" SEQ ID NOs. 42 and 43) along with a geminiviral donor molecule (SEQ ID NO: 79) via polyethylene glycol in a MMg buffer suspension culture. Figure 8(B) shows the targeted substitution event.

[0089] Genomic DNA was extracted from protoplasts, and PCR was performed to amplify each homology arm of the insert DNA containing the nodule promoter, thereby molecularly detecting successful gene targeting and donor molecule insertion. Two primer pairs were designed to amplify each homology arm. For each pair, one primer bound to the genomic DNA outside the homology arm, and the other bound to the newly inserted DNA, in this case the nodule promoter. The expected DNA band lengths amplified from these PCR reactions were 1144 base pairs for the left homology arm (LHA) and 1171 base pairs for the right homology arm (RHA). Targeted editing at the SACPD-C site was confirmed by gel electrophoresis. The expected sequence of the RHA sequence is shown in Figure 10 (SEQ ID NO: 80).

[0090] VIII. Seed-specific upregulation of FATB-1A by targeted promoter substitution (Approach 1C) Genome manipulation reagents for FATB-1A were delivered to soybean protoplasts. Here, soybean seeds were grown in vitro on germination medium under sterile conditions 5 days prior to transformation. The first true leaves and hypocotyls of the soybean seedlings were then digested overnight. Isolation and transformation were performed the day after overnight digestion. During isolation, the protoplasts were first screened to ensure a proper yield of 1 million cells. These cells were subjected to several washes in wash buffer, and they were divided into 200,000 cells for each construct, which was used either for validation via a yellow fluorescent protein (YFP) cassette or for extraction of its genomic DNA.

[0091] Geminivirus binary vectors for targeted promoter substitution were constructed using the TALEN pairs “GmFATB1A-T2” SEQ ID NOs. 52 and 53, “GmFATB1A-T3” SEQ ID NOs. 55 and 56, and “GmFATB1A-T4” SEQ ID NOs. 58 and 59, respectively (schematically shown in Figure 11(A)). Protoplast cells were transformed using plasmids encoding each TALEN pair, along with a geminivirus donor molecule (SEQ ID NO: 81), via polyethylene glycol in a suspension culture in MMg buffer. Figure 11(B) shows the targeted substitution event. Genomic DNA was extracted from the protoplasts, and successful gene targeting and donor molecule insertion were molecularly detected by amplifying each homology arm of the insert DNA containing the FAD2A promoter using PCR. Two primer pairs were designed to amplify each homology arm. For each pair, one primer bound to the genomic DNA outside the homology arm, and the other bound to the newly inserted DNA, in this case the FAD2A promoter. The expected DNA band lengths amplified from these PCR reactions were 1365 base pairs for LHA and 1430 base pairs for RHA. Gel electrophoresis confirmed targeted editing at the FATB-1A site. Sequence IDs 55 and 56 were selected for advancement based on the activity of the TALEN pairs.

[0092] IX. Seed-specific upregulation of FATB-1A and nodule / leaf-specific expression of SACPD-C by cisgenic cassettes (Approach 2A) We construct linear cisgenic constructs for tissue-specific expression of the SACPD-C and FATB-1A genes, having the sequence shown in Sequence ID No. 82.

[0093] Immature cotyledons are excised from immature soybean pods and grown in liquid culture on a shaker until soybean somatic cell embryogenesis callus is formed (4-8 weeks). The soybean somatic cell embryogenesis callus is co-bombarded using cassettes 1 and 2 (Figure 13) and gold particles coated with DNA constructs containing a selection marker. After a 1-week quiescence in regenerating medium, the selection agent is added. The selection medium is changed weekly for approximately 4 weeks. The transformed embryogenesis callus is then cut into 1-2 mm pieces and placed on charcoal-rich maturation medium for 4-8 weeks. After drying the transformed mature embryos, they are transferred to rooting medium.

[0094] Other embodiments of the Disclosure are possible. While the above description contains many specificities, these should be construed as merely providing examples of currently preferred embodiments of the Disclosure, and not limiting the scope of the Disclosure. Various combinations or partial combinations of specific features and aspects of the embodiments may be made and are also intended to remain within the scope of the Disclosure. Various features and aspects of the disclosed embodiments may be combined with or substituted for each other to form various embodiments. Therefore, it is intended that at least some scope of the Disclosure should not be limited by the embodiments described herein.

[0095] The scope of this disclosure should be determined by the appended claims and their legal equivalents. The scope of this disclosure includes other embodiments that may be apparent to those skilled in the art. Thus, the scope of this disclosure is not limited by anything other than the appended claims, and here, references to singular elements are not intended to mean “only” unless expressly stated so, but rather “one or more.” All structural, chemical, and functional equivalents to elements of the preferred embodiments described above that are known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Furthermore, no device or method needs to address all the problems that the disclosure seeks to solve in order to be encompassed by the claims. Furthermore, elements, components, or method steps in this disclosure are not intended to be open to the public, whether or not they are expressly described in the claims.

[0096] The above description of various preferred embodiments of this disclosure is presented for illustrative and explanatory purposes only. It is not intended to be exhaustive or to limit the disclosure to exact embodiments, and many modifications and variations are obviously possible in light of the above teachings. The exemplary embodiments, as described above, have been selected and described to best illustrate the principles of this disclosure and its practical applications, thereby enabling other persons skilled in the art to best utilize this disclosure in various embodiments with various modifications to suit their specific intended use. The scope of this disclosure is intended to be defined by the claims appended herein.

[0097] Various examples are provided. These and other examples are within the scope of the following claims.

[0098] [Sequence List] 1) GmSACPD-C code sequence (Sequence ID 1) 2) Nodule-specific Glyma05g01360 promoter sequence 2KB (SEQ ID NO: 2) 3) Nodule-specific Glyma13g44970 promoter sequence 2KB (SEQ ID NO: 3) 4) Nodule-specific Glyma05g01360 terminator sequence 903bp (SEQ ID NO: 4) CCTCTCCACCTAGATCTTGTATTTGGTTTGTATGGGAGTATGTTTGAAGCTATAGCGCCTGTGGTTGTATACCTGTATTTCTGTGCAGTGTTTTGTGATTTTGTTTTAGAATAAACTGCAAAATTGATCTTCCAAAGATTATGGCATCACCTCATTAATGTTTTTAAGATTTTTGTTATCAAATTAGTCCCACAAATATCTAAAATGTTACCACATTTGT TCACATAGAGGACTAAAGAAGTGGTAACACAAAATAATTTAGTATTTGATTTGTATCTCTGTGGGATTGATTTGTTGACCAAAATCTTTGGAGGACCAGTTCAAGGACTTACTCTATCGATTTATACTTTCAGTTTCAGGCAGTCAAGTAATACTGTATATTCTGTTGTCTATATTGTGGATCATGCATAACTAAACTATCAAGTATCCCTGTATATACAAGTTGT CTAAATTGTGAAAGTTACATATACAAGAAATTTCCTATATACCGTTAGAGAGTAGATAAACTATATACCGTTAGAGAGTTTTCATGCTAATTGTTAAATTAAATACTACGCTTTCTAACCTTCCATCTTTTGAACCAATAAAAAATGTTTCTACTCATCGTATTCTCCATTAAGTTGAAATATTACCAATTAAGTTATGTTACAAAATTTTCCCTCGGAATCTGAA GGTTTGCTTGTATAGCAGTGCTCATTTTTTTCTAAAAGAAAATATACAATTGCTTCCCCTTTACGAATAGTAAAGTCATTCACAACGAAGTAAAGAACAGAAGTAACACAGCAGCTACCATCTCATCCAAGAAAAAAAAAAAGTAATTCTGGAGCTATTTGTTTCAGATATTCAACTTCTAAAAGAATATTAAACTTAAAACAGGTAAAAATGATAAACAAATACC 5) Nodule-specific Glyma13g44970 terminator sequence 1KB (SEQ ID NO: 5) 。 6) GmFATB-1A code sequence (sequence number 6) 7) Seed-specific GmFAD2A promoter sequence 2KB (SEQ ID NO: 7) 8) Seed-specific GmFAD2B promoter sequence 2KB (Sequence ID 8) 9) Seed-specific GmFAD2A terminator sequence 1KB (SEQ ID NO: 9) 。 10) Seed-specific GmFAD2B terminator sequence 1KB (SEQ ID NO: 10) 。 11) GmFAD2A code sequence (sequence number 11) 12) GmFAD2B code sequence (sequence number 12) 13) Linear cisgenic cassette 1 [Root nodule Glyma13g44970 promoter-GmSACPD-C-Glyma13g44970 terminator] (Sequence ID 13) 14) Linear cisgenic cassette 2 [Seed FAD2A promoter-GmFATB1A-FAD2A terminator] (Sequence ID 14) 15) TALEN GmSACPD-C-T01(hitSeq) (SEQ ID NO: 15) that targets the GmSACPD-C gene coding sequence. TGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGA 16) TALEN GmSACPD-C-T01(leftSeq) (SEQ ID NO: 16) that targets the GmSACPD-C gene coding sequence. TGGAGGGATGGGCCTCG 17) TALEN GmSACPD-C-T01(rtSeq) (Sequence ID 17) that targets the GmSACPD-C gene coding sequence. TCCACGGGCTTCAGCAG 18) TALEN GmSACPD-C-T02(hitSeq) (Sequence ID 18) that targets the GmSACPD-C gene coding sequence. TGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAA 19) TALEN GmSACPD-C-T02(leftSeq) (Sequence ID 19) that targets the GmSACPD-C gene coding sequence. TGGGTCCTACCGCTGCT 20) TALEN GmSACPD-C-T02(rtSeq) (Sequence ID 20) that targets the GmSACPD-C gene coding sequence. TTTGTGGCTGCCAGCAT 21) TALEN GmSACPD-C-T03(hitSeq) (Sequence ID 21) that targets the GmSACPD-C gene coding sequence. TCCTCCCTGACCCCTCCTTCCGCATGAAGAGTTCAGCCATCAGGTGAA 22) TALEN GmSACPD-C-T03(leftSeq)(SEQ ID NO: 22) that targets the GmSACPD-C gene coding sequence. TCCTCCCTGACCCCTCC 23) TALEN GmSACPD-C-T03(rtSeq) (SEQ ID NO: 23) that targets the GmSACPD-C gene coding sequence. TTCACCTGATGGCTGAA 24) TALEN GmSACPD-C-T04(hitSeq) (Sequence ID 24) that targets the GmSACPD-C gene coding sequence. TCCCTGACCCCTCCCTTCCGCATGAAGAGTTCAGCCATCAGGTGAAGGA 25) TALEN GmSACPD-C-T04(leftSeq) (Sequence ID 25) that targets the GmSACPD-C gene coding sequence. TCCCTGACCCCTCCCTT 26) TALEN GmSACPD-C-T04(rtSeq) (Sequence ID 26) that targets the GmSACPD-C gene coding sequence. TCCTTCACCGEATGGCT 27) TALEN GmSACPD-C-T05(hitSeq) (Sequence ID 27) that targets the GmSACPD-C gene coding sequence. TACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGA 28) TALEN GmSACPD-C-T05(leftSeq) (Sequence ID 28) that targets the GmSACPD-C gene coding sequence. TACCTGATGAGTACTTT 29) TALEN GmSACPD-C-T05(rtSeq) (Sequence ID 29) that targets the GmSACPD-C gene coding sequence. TCCTCGGTGACCATATC 30) TALEN GmSACPD-C-T06(hitSeq) (SEQ ID NO: 30) that targets the 5'UTR of the GmSACPD-C gene. TCCGCGGCGCCGTTCAAAGCCCGGAAGGCCCACTCAATGCCTCCAGAAA 31) TALEN GmSACPD-C-T06(leftSeq)(SEQ ID NO: 31) that targets the 5'UTR of the GmSACPD-C gene. TCCGCGGCGCCGTTCAA 32) TALEN GmSACPD-C-T06(rtSeq) (SEQ ID NO: 32) that targets the 5'UTR of the GmSACPD-C gene. TTTCTGGAGGCATTGAG 33) TALEN GmSACPD-C-T07(hitSeq) (SEQ ID NO: 33) that targets the 5'UTR of the GmSACPD-C gene. TAAATTATCAACAAACCAAGGGCTAATCACTAGTCACACCCTTTACAAA 34) TALEN GmSACPD-C-T07(leftSeq)(SEQ ID NO: 34) targeting the 5'UTR of the GmSACPD-C gene TAAATTATCAACAAACC 35) TALEN GmSACPD-C-T07(rtSeq) (SEQ ID NO: 35) that targets the 5'UTR of the GmSACPD-C gene. TTTGTAAAGGGTGTGAC 36) TALEN GmSACPD-C-T08(hitSeq) (SEQ ID NO: 36) that targets the 5'UTR of the GmSACPD-C gene. TAGTCACACCCTTTACAAAATATCTCCAACCTCTCCAGTTCCACTCAA 37) TALEN GmSACPD-C-T08(leftSeq) (SEQ ID NO: 37) that targets the 5'UTR of the GmSACPD-C gene. TAGTCACACCCTTTACA 38) TALEN GmSACPD-C-T08(rtSeq) (SEQ ID NO: 38) that targets the 5'UTR of the GmSACPD-C gene. TTGAGTGGAACTGTGGA 39) TALEN GmSACPD-C-T09(hitSeq) (SEQ ID NO: 39) that targets the 5'UTR of the GmSACPD-C gene. TCAAGTACAATAGACACGTAATCAAAACCATGCAGATACGAACCTGCCA 40) TALEN GmSACPD-C-T09 (leftSeq) (Sequence ID 40) that targets the 5'UTR of the GmSACPD-C gene. TCAAGTACAATAGACAC 41) TALEN GmSACPD-C-T09(rtSeq) (SEQ ID NO: 41) that targets the 5'UTR of the GmSACPD-C gene. TGGCAGGTTCGTATCTG 42) TALEN GmSACPD-C-T10(hitSeq) (Sequence ID 42) that targets the 5'UTR of the GmSACPD-C gene. TCACCACCCAAACCCTTCCACAACTTCCGTGTTCTTCTAGAAAAGCCCA 43) TALEN GmSACPD-C-T10 (leftSeq) (SEQ ID NO: 43) that targets the 5'UTR of the GmSACPD-C gene. TCACCACCCAAACCCTT 44) TALEN GmSACPD-C-T10(rtSeq) (SEQ ID NO: 44) targeting the 5'UTR of the GmSACPD-C gene. TGGGCTTTTCTAGAAGA 45) TALEN GmSACPD-C-T11(hitSeq) (SEQ ID NO: 45) that targets the 5'UTR of the GmSACPD-C gene. TTCCGCCGTTAAACGCTGCGGTTTCCGCGGCGCCGTTCAAAGCCCGGAA 46) TALEN GmSACPD-C-T11(leftSeq) (Sequence ID 46) that targets the 5'UTR of the GmSACPD-C gene. TTCCGCCGTTAAACGCT 47) TALEN GmSACPD-C-T11(rtSeq) (SEQ ID NO: 47) that targets the 5'UTR of the GmSACPD-C gene. TTCCGGGCTTTGAACGG 48) TALEN GmFATB1A-T01(hitSeq) (SEQ ID NO: 48) targeting the 5'UTR of the GmFATB-1A gene. TTAGTCCGATTGATTTCTCGATATCATTTAAGGCTAAGGTTGACCTCTA 49) TALEN GmFATB1A-T01 (leftSeq) (Sequence ID 49) targeting the 5'UTR of the GmFATB-1A gene. TTAGTCCGATTGATTTC 50) TALEN GmFATB1A-T01(rtSeq) (SEQ ID NO: 50) that targets the 5'UTR of the GmFATB-1A gene. TAGAGGTCAACCTTAGC 51) TALEN GmFATB1A-T02(hitSeq) (SEQ ID NO: 51) that targets the 5'UTR of the GmFATB-1A gene. TCTTCTAACTTGGCGTATATTTTGCATGCAGCGACCTTAGAAATTCATTA 52) TALEN GmFATB1A-T02 (leftSeq) (Sequence ID 52) that targets the 5'UTR of the GmFATB-1A gene. TCTTCTAACTTGCGTAT 53) TALEN GmFATB1A-T02(rtSeq) (SEQ ID NO: 53) that targets the 5'UTR of the GmFATB-1A gene. TAATGAATTTCTAAGGh 54) TALEN GmFATB1A-T03 (hitSeq) (SEQ ID NO: 54) targeting the 5'UTR of the GmFATB-1A gene. TTTGCATTTCTCTTCTTTATCCCCTTTCTGTGGAAGGTGGGAGGGAAAA 55) TALEN GmFATB1A-T03 (leftSeq) (Sequence ID 55) targeting the 5'UTR of the GmFATB-1A gene. TTTGCATTTCTCTTCTT 56) TALEN GmFATB1A-T03(rtSeq) (SEQ ID NO: 56) that targets the 5'UTR of the GmFATB-1A gene. TTTTCCCTCCCACCTTC 57) TALEN GmFATB1A-T04 (hitSeq) (SEQ ID NO: 57) that targets the 5'UTR of the GmFATB-1A gene. TGTGATATAACTGATGTGCTGTGCTGTTATTATTTGTTATTTGGGGTGA 58) TALEN GmFATB1A-T04 (leftSeq) (Sequence ID 58) targeting the 5'UTR of the GmFATB-1A gene. TGTGATATAACTGATGT 59) TALEN GmFATB1A-T04(rtSeq) (SEQ ID NO: 59) targeting the 5'UTR of the GmFATB-1A gene. TCACCCCAAATAACAAA 60) TALEN GmFATB1A-T05 (hitSeq) (SEQ ID NO: 60) targeting the 5'UTR of the GmFATB-1A gene. TTATTATTTGTTATTTGGGGTGAAGTATAATTTTTTGGGTGAACTTGGA 61) TALEN GmFATB1A-T05 (leftSeq) (SEQ ID NO: 61) that targets the 5'UTR of the GmFATB-1A gene. TTATTATTTGTTATTTG 62) TALEN GmFATB1A-T05(rtSeq) (SEQ ID NO: 62) that targets the 5'UTR of the GmFATB-1A gene. TCCAAGTTCACCCAAAA 63) GmSACPD-C fragment Bert wt (SEQ ID NO: 63) CAATGCCTCCAGAAAAGAAAGAAATTTTCAAGTCCTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCCTGACCCCTCCCTTCCGCATGAAGAGTTCAGCCATCAGGTGAAGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACGCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 64) GmSACPD-C fragment T0 event #1 (-26 nt) (SEQ ID NO: 64) CAATGCCTCCAGAAAAGAAAGAAATTTTCAAGTCCTTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCCTCAGCCATCAGGTGAAGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACGCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 65) GmSACPD-C fragment T0 event #2 (-14 nt) (SEQ ID NO: 65) CAATGCCTCCAGAAAAGAAAGAAATTTTCAAGTCCTTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCCTGACCCCTCCCTTCCGCCATCAGGTGAAGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACGCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 66) GmSACPD-C fragment T0 event #3 (-52nt) (Sequence ID 66) CAATGCCTCCAGAAAAGAAAGAAATTTTCAAGTCCTTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGAAGAGTTCAGCCATCAGGTGAAGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACGCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 67) GmSACPD-C fragment T0 event #4 allele 1 (-5nt) (Sequence ID 67) CAATGCCTCCAGAAAAGAAAGAAATTTTCAAGTCCTTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCTTCGAAGAGTTCAGCCATCA GGTGAAGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACGCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 68) GmSACPD-C fragment T0 event #4 allele 2 (-7 / +3nt) (Sequence ID 68) CAATGCCTCCAGAAAAGAAAGAAATTTTCAAGTCCTTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTTCGTCAGAGTTCAGCCATCA GGTGAAGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACGCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 69) GmSACPD-C fragment T0 event #5 allele 1 (-8nt) (Sequence ID 69) CAATGCTCCAGAAAAGAAAGAAATTTTCAAGTCCTTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTAAGAGTTCAGCCATCAGG TGAAGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 70) GmSACPD-C fragment T0 event #5 allele 2 (-14nt) (Sequence ID 70) CAATGCCTCCAGAAAAGAAAGAAATTTTCAAGTCCTTGGAGGGATGGGCCTCGGAGTGGGTCCTACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTTCAGCCATCAGGTGA AGGAGCTTCGCGAACGCACTAAAGAGTTACCTGATGAGTACTTTGTGGTGCTGGTGGGTGATATGGTCACCGAGGACCGCTTCCCACTTACCAGACCATGATCAACAACCTTGATGGAGTGAAAGATGACAGCGGCACGAG 71) GmSACPD-C fragment Bert wt (SEQ ID NO: 71) ACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTTCCGCATGAAGAGTTCAGCCATCAGGTGAAGGAGCTT 72) GmSACPD-C fragment T0 event #1 (Sequence ID 72) ACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCCTCAGCCATCAGGTGAAGGAGCTT 73) GmSACPD-C fragment T0 event #2 (Sequence ID 73) ACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTTCCGCCATCAGGTGAAGGAGCTT 74) GmSACPD-C fragment T0 event #3 (SEQ ID NO: 74) ACCGCTGCTGAAGCCCGTGAAGAGTTCAGCCATCAGGTGAAGGAGCTT 75) GmSACPD-C fragment T0 event #4 allele 1 (Sequence ID 75) ACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTTCGAAGAGTTCAGCCATCAGGTGAAGGAGCTT 76) GmSACPD-C fragment T0 event #4 allele 2 (Sequence ID 76) ACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTTCGTCAGAGTTCAGCCATCAGGTGAAGGAGCTT 77) GmSACPD-C fragment T0 event #5 allele 1 (Sequence ID 77) ACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCCTGACCCCTCCCTAAGAGTTCAGCCATCAGGTGAAGGAGCTT 78) GmSACPD-C fragment T0 event #5 allele 2 (Sequence ID 78) ACCGCTGCTGAAGCCCGTGGAGCAATGCTGGCAGCCACAAAACTTCCTCCTGACCCCTCCCTTCAGCCATCAGGTGAAGGAGCTT 79) Artificial Sequence for Seed-Specific Silencing of SACPD-C (Targeted Promoter Substitution Donor Template Sequence LHA(SACPD-C)-Nodule Pro and 5'UTR(Glyma13g44970)-RHA(SACPD-C)) (SEQ ID NO: 79) 80) Expected edited RHA sequence - nodule promoter / UTR insert - RHA (SEQ ID NO: 80) 81) Artificial sequences for seed-specific upregulation of FATB-1A (targeted promoter substitution donor template sequences LHA(FATB-1A)-FAD2A Pro and 5'UTR(Glyma10g42470)-RHA(FATB-1A)) 82) Artificial sequence for tissue-specific expression of SACPD-C and FATB-1A genes (SEQ ID NO: 82)

Claims

1. A soybean plant, soybean plant part, or soybean plant cell containing one or more mutations that regulate the expression of both the SACPD-C and FATB-1A genes, The soybean plant, soybean plant part, or soybean plant cell produces an oil having an increased saturated fatty acid content compared to the oil produced from a corresponding soybean plant, soybean plant part, or soybean plant cell lacking one or more mutations. The one or more mutations that regulate the expression of the SACPD-C gene include targeted mutations induced by low-frequency cleavage endonucleases. The soybean plant, soybean plant portion, or soybean plant cell contains a mutation that results in reduced expression of the SACPD-C gene, the mutation that results in reduced expression of the SACPD-C gene includes substitution of the seed-specific promoter at the native genomic locus of the SACPD-C gene with a promoter that is less active or undetectable in developing soybean seeds. The soybean plant, soybean plant portion, or soybean plant cell contains a mutation that results in increased expression of the FATB-1A gene, and the mutation resulting in increased expression of the FATB-1A gene is a targeted substitution of the endogenous promoter of the FATB-1A gene in an overexpression promoter. Soybean plant, soybean plant part, or soybean plant cell.

2. The soybean plant, soybean plant portion, or soybean plant cell according to claim 1, wherein the mutation causing a decrease in the expression of the SACPD-C gene is a mutation in one or more alleles of the SACPD-C gene or in a promoter operably linked to them.

3. The soybean plant, soybean plant portion, or soybean plant cell according to claim 1 or 2, wherein the mutation causing a decrease in the expression of the SACPD-C gene is a knockout mutation.

4. The soybean plant, soybean plant portion, or soybean plant cell according to claim 3, wherein the knockout mutation is a seed-specific knockout mutation.

5. The soybean plant, soybean plant portion, or soybean plant cell according to claim 4, wherein the seed-specific knockout mutation comprises the substitution of a seed-specific promoter at the native genomic locus of the SACPD-C gene with a promoter that is underactive or undetectable in developing soybean seeds.

6. The soybean plant, soybean plant portion, or soybean plant cell according to claim 5, wherein the mutation is in a sequence shown in SEQ ID NOs: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, or 45, or a sequence having at least 95% identity to a sequence shown in SEQ ID NOs: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, or 45.

7. A soybean plant, soybean plant portion, or soybean plant cell according to any one of claims 1 to 6, comprising a knock-in mutation of a functional SACPD-C gene operably linked to the promoter which has low activity or undetectable activity in developing soybean seeds.

8. The soybean plant, soybean plant portion, or soybean plant cell according to claim 5 or 7, wherein the promoter having low activity or undetectable activity in the developing soybean seed is a nodule-specific gene promoter.

9. The soybean plant, soybean plant portion, or soybean plant cell according to claim 11, wherein the promoter or overexpression promoter is a strongly seed-specific promoter.

10. A method for generating soybean plants containing mutations that regulate the expression of both the SACPD-C and FATB-1A genes, (a) A population of soybean plant cells from a soybean plant that produces oil having a saturated fatty acid content of 15% of the total fatty acid composition, (i) A mutation resulting in a decrease in the expression of the SACPD-C gene, wherein the mutation is a targeted mutation induced by a low-frequency cleavage endonuclease, and the mutation resulting in a decrease in the expression of the SACPD-C gene includes the substitution of a seed-specific promoter at the native genomic locus of the SACPD-C gene with a promoter that is low in activity or undetectable in developing soybean seeds, (ii) A mutation that results in increased expression of the FATB-1A gene, wherein the mutation resulting in increased expression of the FATB-1A gene is a targeted substitution of the endogenous promoter of the FATB-1A gene in the overexpression promoter, Contacting one or more nucleic acid sequences containing a combination, (b) Selecting cells from the population in which the expression of the SACPD-C gene is decreased and the expression of the FATB-1A gene is increased, (c) A method comprising regenerating selected plant cells into a soybean plant.

11. The method according to claim 10, wherein reducing the expression of the SACPD-C gene is a mutation in one or more alleles of the SACPD-C gene or in a promoter operatively linked thereto.

12. The method according to claim 11, wherein reducing the expression of the SACPD-C gene includes replacing the seed-specific promoter at the native genomic locus of the SACPD-C gene with the promoter that has low activity or no detectable activity in developing soybean seeds.

13. The method according to claim 11, further comprising delivering to the population of soybean plant cells an expression cassette containing a functional SACPD-C gene operably linked to the promoter which has low activity or no detectable activity in developing soybean seeds.

14. The method according to any one of claims 10 to 13, wherein increasing the expression of the FATB-1A gene comprises delivering an expression cassette containing one or more copies of the FATB-1A gene to a population of soybean plant cells.

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