CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Not applicable

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA PATENT CENTER [0002] The content of the Sequence Listing XML file of the sequence listing named “PT-1109- US-PSP.xml” which is 11.7 kb in size created on July 22, 2022 and electronically submitted via Patent Center herewith the application is incorporated by reference in its entirety.

FIELD

[0003] This disclosure relates generally to Brassica plants, and more particularly, Brassica plants having modified alleles at fatty acyl-acyl carrier protein thioesterase A2-14 (FATA2-14) loci and yielding an oil with a low total saturated fatty acid content.

BACKGROUND

[0004] In recent years, diets high in saturated fats have been associated with increased levels of cholesterol and increased risk of coronary heart disease. As such, current dietary guidelines indicate that saturated fat intake should be no more than 10 percent of total calories. Based on a 2,000-calorie-a-day diet, this is about 20 grams of saturated fat a day. While canola oil typically contains only about 7% to 8% saturated fatty acids, a decrease in its saturated fatty acid content would improve the nutritional profile of the oil.

SUMMARY

[0005] This disclosure relates generally to Brassica plants, plant progeny thereof, seeds thereof, edible oils produced from the seeds of the plants and methods involving the production of the same. Generally, the plants of the present disclosure have one or more modified (e g., mutant) alleles that lead to the production of seeds yielding an edible oil having a low total saturated fatty acid content. [0006] For example, the disclosed Brassica plant, the plant progeny thereof, or seeds thereof can include a modified allele at a fatty acyl-acyl carrier protein thioesterase A2-14 (FATA2-14) locus. The modified allele can result in the production of a FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 polypeptide (SEQ ID NO: 1). [0007] The modified allele can include a nucleic acid encoding a FATA2-14 polypeptide having a mutation in a position corresponding to amino acid 139 relative to SEQ ID NO: 1. The FATA2- 14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2- 14 polypeptide can be a truncated FATA2-14 polypeptide. The truncated FATA2-14 polypeptide can be truncated at a position corresponding to amino acid 139 relative to SEQ ID NO:1.

[0008] The nucleic acid encoding the FATA2-14 polypeptide having a mutation in a position corresponding to amino acid 139 relative to SEQ ID NO:1 can have at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NON. The FATA2-14 polypeptide having a mutation in a position corresponding to amino acid 139 relative to SEQ ID NO:1, or the truncated FATA2-14 polypeptide, can have an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:3. The truncated FATA2- 14 polypeptide may be truncated by at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 225 amino acids relative to the full-length wild type FATA2-14 polypeptide of SEQ ID NO: 1.

[0009] Any of the plants, progeny thereof, and or seeds thereof described herein further can include a modified allele at one or more different fatty acyl-acyl carrier protein thioesterase B (FATB) loci (e.g., two, three or four different loci), wherein each modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide. The FATB loci can be selected from the group consisting of FATB isoform 1, FATB isoform 2, FATB isoform 3, FATB isoform 4, FATB isoform 5, FATB isoform 6, and combinations thereof

[0010] Any of the plants, progeny thereof, or seeds thereof described herein further can include a modified allele at a fatty acyl-acyl carrier protein thioesterase A2-4 (FATA2-4) locus, wherein the modified allele results in the production of a FATA2-4 polypeptide (e.g., FATA2-4 polypeptide) having reduced thioesterase activity relative to a corresponding wild-type FATA2-4 polypeptide. [0011] Any of the plants, progeny thereof, or seeds thereof described herein further can include a modified allele having a mutation at chromosome N01 quantitative trait locus (QTL1) allele. [0012] Any of the plants, progeny thereof, or seeds thereof described herein further can include a modified allele having a mutation at chromosome N19 quantitative trait locus (QTL2) allele.

[0013] Any of the plants, the plant progeny thereof, or seeds thereof described herein can include a mutant allele at a FATA2-14 locus and at least two, at least three or all four of (a)a mutant allele at a FATA2-4 locus; (b) a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus.

[0014] Any of the plants, progeny thereof, or seeds thereof described herein can be a Brassica napus, Brassica juncea, or Brassica rapa plant.

[0015] Any of the plants, progeny thereof, or seeds thereof described herein can produce a seed that yields an oil having a total saturated fatty acid content of less than 10.5%, less than 10%, less than 9%, less than 8%, less than 7%, or less than 6%.

[0016] Any of the plants, progeny thereof, or seeds thereof described herein can produce a seed that yields an oil having an oleic acid content of at or about 67% to at or about 73%, a linoleic acid content of at or about 14% to at or about 21%, and an alpha-linolenic acid content of at or about 2% to at or about 5%. The oil can further include a stearic acid having a content of at or about 1% to at or about 5%. The oil can further include a palmitic acid content of at or about 3% to at or about 6%. The oil further includes an arachidic acid content of at or about 0.4% to at or about 1.0%.

[0017] Also provided herein is a method of producing the disclosed Brassica plant. The method can include obtaining a first Brassica parent plant and a second Brassica parent plant. The first Brassica parent plant can include a mutant allele at a FATA2-14 locus, wherein the mutant allele results in the production of a FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 polypeptide. The second Brassica parent plant can include one or more of: (a) a mutant allele at a FATA2-4 locus; (b) a mutant allele having a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutant allele having a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus. The method may further include crossing the first Brassica plant and the second Brassica plant. The method further can include selecting for one or more generations of progeny plants having one or more of the mutant alleles of the first and/or second Brassica parent plants, thereby obtaining the Brassica plant. [0018] The disclosed Brassica plant or seeds thereof can be an Fl hybrid. The Brassica seed described herein can be an F2 seed.

[0019] Also describe herein is a method of producing a seed oil from any of the Brassica plants described in the present disclosure. The method can include crushing a seed of the Brassica plant and extracting the oil from the crushed seed. The method can further include refining, bleaching and deodorizing the extracted oil. The oil can have a total saturated fatty acid content of at or about or less than 10.5%, alternately at or about or less than 10%, alternately at or about or less than 9%, alternately at or about or less than 8%, alternately at or about or less than 7%, alternately at or about or less than 6%.

[0020] The disclosure also describes an edible oil that is produced from seeds of any of the Brassica plants described in the present disclosure. The edible oil may have an oleic acid content of at or about 56.12% to at or about 78.31%, a linoleic acid content of at or about 10.05% to at or about 28.11%, and an alpha-linolenic acid content of at or about 2.02% to at or about 4.9%. The edible oil may include a stearic acid content of at or about 1.3% to at or about 4.69%. The edible oil may include a palmitic acid content of at or about 3.41% to at or about 6.53%. The edible oil may include an arachidic acid content of at or about 0.41% to at or about 1.12%.

[0021] The disclosure also describes a plant cell of a plant described herein, wherein the plant cell includes one or more of the modified (e.g., mutant) alleles.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION

[0023] Reference will now be made in detail to certain aspects of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0024] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0025] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. [0026] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the recited macromolecule its function and properties. A summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.

Table 1: Amino Acid three and one letter symbols

[0027] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the practice of the disclosed plants, plant progeny thereof, seeds thereof and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity. Generally, the variant or modified sequence may include or greater than about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the wild-type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein.

[0028] As used herein, the phrases “% sequence identity,” “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches. An alignment can be performed using BLAST (National Center for Biological Information (NCBT) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.

[0029] Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0030] The polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.” As used herein the term “wild-type” is a term of the art understood by skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. [003 ] ] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge and/or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. [0032] As used herein, terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence,” and “nucleic acid,” are used interchangeably and refer to a sequence of nucleotides or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. The DNA polynucleotides may be a cDNA or a genomic DNA sequence.

[0033] A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

[0034] Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. The polynucleotides (i.e., polynucleotides encoding a non-heme iron-binding protein polypeptide) may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell. While polypeptides encoded by polynucleotide sequences found in coral are disclosed herein any polynucleotide sequences may be used which encodes a desired form of the polypeptides described herein. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences. [0035] The polypeptides described herein may be provided as part of a construct. As used herein, the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. The construct may be a vector including a promoter operably linked to the polynucleotide encoding the thermolabile non-heme iron-binding polypeptide. As used herein, the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. The vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments may be integrated.

[0036] It is understood that throughout the disclosure, reference to "plant" or "plants" includes progeny, i.e., descendants of a particular plant or plant line, as well as cells or tissues from the plant. Progeny of an instant plant include seeds formed on Fl, F2, F3, F4 and subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants. Seeds produced by a plant can be grown and then selfed (or, for example, outcrossed and selfed, or doubled through dihaploid) to obtain seeds homozygous for a mutant allele. The term "allele" or "alleles" refers to one or more alternative forms of a gene at a particular locus. As used herein, a "line" is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. As used herein, the term "variety" refers to a line which is used for commercial production and includes hybrid varieties and open- pollinated varieties.

[0037] In general, this disclosure relates to Brassica plants, including B. napus, B.juncea, and A. rapa species of Brassica, that yield seeds producing oils having a low total saturated fatty acid content (e.g., at or about 10.5% or less). As used herein, total saturated fatty acid content refers to the total of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0). [0038] Brassica plants can be made so as to yield seed oils having a low total saturated fatty acid content in combination with an oleic acid content of at least 60%. Such Brassica plants can produce seed oils having a fatty acid content tailored to the desired end use of the oil (e.g., frying or food applications). For example, Brassica plants can be produced that yield seeds having a low total saturated fatty acid content, an oleic acid content of at least 60% and an a-linolenic acid content of at least 2%. Canola oils having low fatty acid contents can be useful for frying applications. [0039] Brassica plants described herein have low levels of total saturated fatty acids in the seed oil as a result of reduced activity of fatty acyl-acyl carrier protein (ACP) thioesterase A2-14 (FATA2-14). Fatty acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast to release the newly synthesized fatty acid from ACP, effectively removing it from further chain elongation in the plastid. The free fatty acid can then leave the plastid, become bound to Coenzyme-A (CoA) and enter the Kennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol (TAG) biosynthesis. Members of the FATA family prefer oleoyl (C18: l) ACP substrates with minor activity towards C18:0 and C16:0-ACPs, while members of the FATB family hydrolyze primarily saturated acyl-ACPs between 8 and 18 carbons in length. See Jones et al, Plant Cell 7:359-371 (1995); Ginalski and Rhchlewski, Nucleic Acids Res 31 :3291-3292 (2003); and Voelker T. in Genetic Engineering (Setlow, JK, ed) Vol 18, 1 1 1-133, Plenum Publishing Corp., New York (2003).

[0040] Reduced activity, including absence of detectable activity, of FATA2-14 can be achieved by modifying an endogenous fatA-2-14 allele. An endogenous fatA2-14 allele can be modified by, for example, mutagenesis, or by using homologous recombination to replace an endogenous plant gene with a variant containing one or more mutations (e.g., produced using site-directed mutagenesis). See, e.g., Townsend et al, Nature 459:442-445 (2009); Tovkach et al, Plant J., 57:747-757 (2009); and Lloyd et al, Proc. Natl. Acad. Sci. USA , 102:2232-2237 (2005). Similarly, for other genes discussed herein, the endogenous allele can be modified by mutagenesis or by using homologous recombination to replace an endogenous gene with a variant. Modified alleles obtained through mutagenesis are referred to as mutant alleles herein.

[0041] Reduced activity, including absence of detectable activity, can be inferred from the decreased level of saturated fatty acids in the seed oil compared with seed oil from a corresponding control plant. Reduced activity also can be assessed in plant extracts using assays for fatty acyl- ACP hydrolysis. See, e.g., Bonaventure et al., Plant Cell 15:1020-1033 (2003); and Eccleston and Ohlrogge, Plant Cell 10:613-622 (1998).

[0042] Genetic mutations can be introduced within a population of seeds or regenerable plant tissue using one or more mutagenic agents. Suitable mutagenic agents include, for example, ethyl methane sulfonate (EMS), methyl N-nitrosoguanidine (MNNG), ethidium bromide, diepoxybutane, ionizing radiation, x-rays, UV rays, and other mutagens known in the art. In some examples, a combination of mutagens, such as EMS and MNNG, can be used to induce mutagenesis. The treated population, or a subsequent generation of that population, can be screened for reduced thioesterase activity that results from the mutation, e.g., by determining the fatty acid profde of the population and comparing it to a corresponding non-mutagenized population. Mutations can be in any portion of a gene, including a coding sequence, an intron sequence, and regulatory elements, that render the resulting gene product non-functional or with reduced activity. Suitable types of mutations include, for example, insertions or deletions of nucleotides, and transitions or transversions in the wild-type coding sequence. Such mutations can lead to deletion or insertion of amino acids, and conservative or non-conservative amino acid substitutions in the corresponding gene product. In some aspects, the mutation is a nonsense mutation, which results in the introduction of a stop codon (TGA, TAA, or TAG) and production of a truncated polypeptide. In some aspects, the mutation is a splice site mutation which alters or abolishes the correct splicing of the pre-mRNA sequence, resulting in a protein of different amino acid sequence than the wild type. For example, one or more exons may be skipped during RNA splicing, resulting in a protein lacking the amino acids encoded by the skipped exons. Alternatively, the reading frame may be altered by incorrect splicing, one or more introns may be retained, alternate splice donors or acceptors may be generated, or splicing may be initiated at an alternate position, or alternative polyadenylation signals may be generated. In some examples, more than one mutation or more than one type of mutation is introduced.

[0043] Insertions, deletions, or substitutions of amino acids in a coding sequence may, for example, disrupt the conformation of essential alpha-helical or beta-pleated sheet regions of the resulting gene product. Amino acid insertions, deletions, or substitutions also can disrupt binding, alter substrate specificity, or disrupt catalytic sites important for gene product activity. Non- conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions may also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue. Examples of non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.

[0044] A Brassica plant as described herein may contain a mutant allele at a FATA2-14 locus, wherein the mutant allele results in the production of a FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 polypeptide.

[0045] The mutant allele at the FATA2-14 locus can include a nucleic acid encoding a FATA2- 14 polypeptide having a mutation in a position corresponding to amino acid 139 relative to SEQ ID NO: 1. The mutation may be a nonsense mutation in the position corresponding to amino acid 139 relative to SEQ ID NO: 1. The nonsense mutation results in the introduction of a stop codon (TGA) and the production of a truncated FATA2-14 polypeptide. The term “truncated FATA2-14 polypeptide” means that the polypeptide contains fewer amino acids than would be found in the amino acid sequence of the wild-type FATA2-14 (SEQ ID NO: 1; 366 amino acids). For example, the truncated FATA2-14 polypeptide may contain at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or at least 225 fewer amino acids than the full-length wildtype FATA2-14 polypeptide of SEQ ID NO: 1. The missing amino acids may be missing from the N-terminus, C- terminus, or a combination thereof relative to SEQ ID NO:1.

[0046] The mutant allele at a FATA2-14 locus may include a nucleic acid having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4. The FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14, or the truncated FATA2-14 polypeptide, may have an amino acid sequence at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3.

[0047] It will be readily appreciated by one skilled in the art that more than one nucleic acid molecule may encode the same polypeptide due to the degeneracy of the genetic code. Degeneracy results because a triplet code designates 20 amino acids and a stop codon. Because four bases exist which are utilized to encode genetic information, triplet codons are required to produce at least 21 different codes. The possible 4 ³ possibilities for bases in triplets give 64 possible codons, meaning that some degeneracy must exist. As a result, some amino acids are encoded by more than one triplet, i.e. by up to six. The degeneracy mostly arises from alterations in the third position in a triplet. This means that nucleic acid molecules having different sequences, but still encoding the same polypeptide are envisaged and can be employed in accordance with the plants, plant progeny thereof, seeds thereof, and the methods of the present disclosure.

[0048] The Brassica plant further can include a mutant allele at one or more different fatty acylacyl carrier protein thioesterase B (FATB) loci (e.g., two, three or four different loci), wherein each modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide. In some examples, the Brassica plant has 6 different FATB isoforms (i.e., different forms of the FATB polypeptide at different loci), which are called isoforms 1-6 herein.

[0049] The Brassica plant can have a mutation in a nucleotide sequence encoding FATB isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, or isoform 6. The mutant allele may be at one or more FATB loci, where the FATB loci is one or more combinations of FATB isoform 1, FATB isoform 2, FATB isoform 3, FATB isoform 4, FATB isoform 5, and FATB isoform 6. In some aspects, the mutant allele(s) at the FATB loci can be as described in US 9,334,483 and US2019/001479, incorporated herein by reference.

[0050] The Brassica plant further can include a mutant allele at a fatty acyl-acyl carrier protein thioesterase A2-4 (FATA2-4) locus, wherein the modified allele results in the production of a FATA2-4 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-4 polypeptide. The genomic FATA2-4 sequence is provided as SEQ ID NO:5 and the corresponding amino acid sequence as SEQ ID NO:6. The mutant allele can include a nucleic acid that encodes a FATA2-4 polypeptide having a non-conservative substitution within a helix/4- stranded sheet (4HBT) domain (also referred to as a hot-dog domain) or non-conservative substitution of a residue affecting catalytic activity or substrate specificity. The modified allele at the FATA2-4 loci can be as described in US 9,334,483 and US2019/001479, incorporated herein by reference.

[0051] The Brassica plant further can include a mutant allele having a mutation at chromosome N01 quantitative trait locus (QTL1) allele. In some aspects, the modified allele having a mutation at the chromosome N01 QTL1 allele can be as described in WO 2015/077661, incorporated herein by reference.

[0052] The Brassica plant further can include a mutant allele having a mutation at chromosome N19 quantitative trait locus (QTL2) allele. In some aspects, the modified allele having a mutation at the chromosome Nl 9 QTL2 allele can be as described in WO 2015/077661 , incorporated herein by reference.

[0053] Generally, QTL1 (Nl) and QTL2 (N19) are modified alleles that have been found to correlate to reduced saturated fatty acid content. These modified alleles and methods of identifying them are described in WO 2015/077661. In some examples, the Brassica plants may have mutations in both the QTL1 and QTL2 loci as well as in FATA2-4 and/or FATB loci.

[0054] In some examples, breeding lines of the Brassica plants that can be used in the present invention are as described in US 2019/0014791, herein incorporated by reference.

[0055] Also provided herein are methods for producing a Brassica plant. Methods for producing a Brassica plant may including crossing a first Brassica parent plant and a second Brassica parent plant. The first and second Brassica praent plants may have one or more genotypic or phenotypic trains that are desirable for breeding into one or more generations of progeny plants. For example, the first Brassica parent plant may include a mutant allele at a FATA2-14 locus, wherein the mutate allele results in production of a FATA2- 14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 polypeptide. The second Brassica parent plant may include at least one mutation selected from the group consisting of (a) a mutant allele at a FATA2-4 locus; (b) a mutant allele having a mutation at chromosome N01 quantitative trait locus

1 (QTL1) allele; (c) a mutant allele having a mutation at chromosome N19 quantitative trait locus

2 (QTL2) allele; and (d) a mutant allele at a FATB locus. Additionally or alternatively, the second Brassica parent plant may include at least two mutations selected from the group consisting of (a) a mutant allele at a FATA2-4 locus; (b) a mutant allele having a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutant allele having a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus. Additionally or alternatively, the second Brassica parent plant may include at least three mutations selected from the group consisting of (a) a mutant allele at a FATA2-4 locus; (b) a mutant allele having a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutant allele having a mutation at chromosome Nl 9 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus. Alternatively or additionally, the second Brassica parent plant may include (a) a mutant allele at a FATA2-4 locus; (b) a mutant allele having a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutant allele having a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus. [0056] Following crossing of the first and second Brassica parent plants, one or more generations of progeny plants are then selected that have one or more of the mutant alleles of the first and/or second Brassica parent plants. For example, the progeny plants may be selected for a mutant FATA2-14 allele and at least one of, at least two of, at least three of, or all four of (a) a mutant allele at a FATA2-4 locus; (b) a mutant allele having a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutant allele having a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus. The progeny plants may be an Fl, F3, F3, F4, or subsequent generation of progeny produced from crossing the first and second Brassica parent plants.

[0057] Brassica plants disclosed herein are useful for producing edible oils. As used herein, the terms “edible solid fat”, “edible liquid fat”, or “edible oil” refer to a fat or oil that is suitable for human consumption. Edible solid fats, edible liquid fats, and edible oils are typically compositions including triacylglycerols (“TAG”). The edible solid fats, edible liquid fats, and edible oils may be obtained from plant, animal, or microbial sources.

[0058] For example, oil obtained from seeds of Brassica plants described herein may have a total saturated fatty acid content of less than 10.5%, less than 10%, less than 9%, less than 8.5%, less than 8%, less than 7.%%, or less than 7%. The oil may have a total saturated fatty acid content between 2% and 10.5%, between 3% and 10%, between 4% and 9%, or between 5% and 8%.

[0059] The oil obtained from the seeds of the Brassica plants described herein may have an oleic acid content of at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. For example, the oil may have an oleic acid content between 60% and 85%, between 65% and 80%, or between 70% and 80%. The oil may include about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% oleic acid.

[0060] The oil obtained from the seeds of the Brassica plants described herein may have a linoleic acid content of at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20% or at least 21%. For example, the oil may have a linoleic acid content between 14% and 21%, between 15% and 21%, between 16% and 21%, between 17% and 21%, or between 18% and 21%. The oil may include about 14%, 15%, 16%, 17%, 18%, 19%, 20%, or 21% linoleic acid. [0061] The oil obtained from the seeds of the Brassica plants described herein may have an a- linoleic acid content of at least 2%, at least 3%, at least 4%, or at least 5%. For example, the oil may have an a-linoleic acid content between 2% and 5%, between 3% and 5%, or between 4% and 5%. The oil may include about 2%, 3%, 4%, or 5% a-linoleic acid.

[0062] The oil obtained from the seeds of the Brassica plants described herein may have a palmitic acid content of at least at least 3%, at least 4%, at least 5% or at least 6%. For example, the oil may have a palmitic acid content between 3% and 6%, between 4% and 6%, or between 5% and 6%. The oil may include about 3%, 4%, 5%, or 6% palmitic acid.

[0063] The oil obtained from the seeds of the Brassica plants described herein may have a palmitic acid content of at least at least 3%, at least 4%, at least 5% or at least 6%. For example, the oil may have a palmitic acid content between 3% and 6%, between 4% and 6%, or between 5% and 6%. The oil may include about 3%, 4%, 5%, or 6% palmitic acid.

[0064] The oil obtained from the seeds of the Brassica plants described herein may have a stearic acid content of at least 1%, at least 2%, at least 3%, at least 4%, or at least 5%. For example, the oil may have an a-linoleic acid content between 1% and 5%, 2% and 5%, between 3% and 5%, or between 4% and 5%. The oil may include about 1%, 2%, 3%, 4%, or 5% a-linoleic acid.

[0065] The oil obtained from the seeds of the Brassica plants described herein may have an arachidic acid content of at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, or at least 1.0%. For example, the oil may have an arachidic acid content between 0.4% and 1.0%, 0.5% and 1.0%, 0.6% and 1.0%, 0.7% and 1.0%, 0.8% and 1.0%, or between 0.9% and 1.0%. The oil may include about 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% arachidic acid.

[0066] The fatty acid composition of seeds can be determined by first crushing and extracting oil from seed samples (e.g., bulk seeds samples of 10 or more seeds). TAGs inthe seed are hydrolyzed to produce free fatty acids, which then can be converted to fatty acid methyl esters and analyzed using techniques known to the skilled artisan, e.g., gas liquid chromatography (GLC) according to AOCS Procedure Ce le-91. Near infrared (NIR) analysis can be performed on whole seed according to AOCS Procedure Am- 192 (revised 1999).

[0067] Seeds harvested from plants described herein can be used to make a crude canola oil or a refined, bleached, and deodorized (RBD) canola oil with a low or no total saturated fatty acid content. Harvested canola seed can be crushed by techniques known in the art. The seed can be tempered by spraying the seed with water to raise the moisture to, for example, 8.5%. The tempered seed can be flaked using smooth roller with, for example, a gap setting of 0.23 to 0.27 mm. Heat may be applied to the flakes to deactivate enzymes, facilitate further cell rupturing, coalesce the oil droplets, or agglomerate protein particles in order to ease the extraction process. Typically, oil is removed from the heated canola flakes by a screw press to press out a major fraction of the oil from the flakes. The resulting press cake contains some residual oil.

[0068] Crude oil produced from the pressing operation typically is passed through a settling tank with a slotted wire drainage top to remove the solids expressed out with the oil in the screw pressing operation. The clarified oil can be passed through a plate and frame filter to remove the remaining fine solid particles. Canola press cake produced from the screw pressing operation can be extracted with commercial n-Hexane. The canola oil recovered from the extraction process is combined with the clarified oil from the screw pressing operation, resulting in a blended crude oil. [0069] Free fatty acids and gums typically are removed from the crude oil by heating in a batch refining tank to which food grade phosphoric acid has been added. The acid serves to convert the non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present in the crude oil. The phosphatides and the metal salts are removed from the oil along with the soapstock. The oil-acid mixture is treated with sodium hydroxide solution to neutralize the free fatty acids and the phosphoric acid in the oil-acid mixture. The neutralized free fatty acids, phosphatides, etc. (soapstock) are drained off from the neutralized oil. A water wash may be done to further reduce the soap content of the oil. The oil may be bleached and deodorized before use, if desired, by techniques known in the art.

[0070] Oils obtained from plant described herein can have increased oxidative stability, which can be measured using, for example, an Oxidative Stability Index Instrument (e.g., from Omnion, Inc., Rockland, MA) according to AOCS Official Method Cd 12b-92 (revised 1993). Oxidative stability is often expressed in terms of "AOM" hours.

[0071] In the Tables described herein, the fatty acids are referred to by the length of the carbon chain and number of double bonds within the chain. For example, C14_0 refers to myristic acid; C16_0 refers to palmitic acid; Cl 8_0 refers to stearic acid; Cl 8 1 refers to oleic acid; C18_2 refers to linoleic acid; C18_3 refers to a-linolenic acid; C20_0 refers to arachidic acid; C20_l refers to eicosenoic acid, C22_0 refers to behenic acid, C22_l refers to erucic acid, C24_0 refers to lignoceric acid, and C24_l refers to nervonic acid. "Total Sats" refers to the total ofC14_0, C16_0, C18_0, C20_0, C22_0, and C24_0. Representative fatty acid profiles are provided for each of the specified samples. [0072] Unless otherwise indicated, all percentages refer to wt % based on total wt% of fatty acids in the oil.

[0073] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Examples

[0074] Plants producing an oil with low total saturated fatty acid content were produced by subjecting a population of B. Napus IMC201 seeds to chemical mutagenesis and identifying the targeted FatA genes. Prior to mutagenesis, IMC201 seeds were pre-imbibed in 700 gm seed lots by soaking for 15 min then draining for 5 min at room temperature. This was repeated four times to soften the seed coat. The pre-imbibed seeds then were treated with 4 mM methyl N- nitrosoguanidine (MNNG) for three hours. Following the treatment with MNNG, seeds were drained of the mutagen and rinsed with water for one hour. After removing the water, the seeds were treated with 52.5 mM ethyl methanesulfonate (EMS) for sixteen hours. Following the treatment with EMS, the seeds were drained of mutagen and rinsed with water for 1.5 hours. This dual mutagen treatment produced an LD50 with the seed population. Three thousand bulk M2 generation seeds were planted. Upon maturity, M3 seed (2500 individuals) was harvested from 2500 M2 plants.

[0075] DNA from the M3 seeds were used in tilling assays using the following tilling primer to identify mutants within the targeted regions of identified BoFatA genes.

[0076] A mutant line, M3B-1492-02, was identified to carry the modified allele in the gene BoFatA2-14. A DH (doubled haploid) population was developed from a cross with the pedigree 03LC.034*3/M3B-1492-03//5/03LC.034LLl*9/Salomon-005/4/03LC.0 34*5/mIMC201. DH lines that carried the modified FatA2-14 allele were selected for the data referenced in Tables 2 and 3. Note that the statistical analysis in Tables 2 and 3 are different.

[0077] Table 2 includes the total saturates of near isogenic lines with different combinations of the low saturated genes, namely, mutant alleles at the FATA2-14 locus, the FATA2 locus, QTL1 (N01) and QTL2 (N19). The statistical analysis of Table 2 is as follows. Statistical analysis was performed in R (R Core Team 2015). The Ismeans package (Length 2016) was used to calculate means for each grouping of similar low sat alleles (Table 2). R Core Team (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available on the World Wide Web at R-project.org/. Russell V Lenth (2016). Least- Squares Means: The R Package Ismeans. Journal of Statistical Software, 69(1), 1-33. Available on the World Wide Web at CRAN.R-project.org/package=lsmeans.

Table 2. Fatty Acid Means of Near Isogenic Lines with different combinations of Low Saturate Genes. [0078] Table 3 includes data comparing the total saturates with and without the FATA2- 14 allelic mutations. The statistical analysis of Table 3 is as follows. Statistical analysis was performed in R (R Core Team 2015). The Ismeans package (Length 2016) was used to calculate least square means for each grouping of similar low sat alleles. Pairwise comparisons with Tukey ’ s adjustments were made for the same groupings with and without the presence of FatA2-14 (Table 3). R Core Team (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available on the World Wide Web at R-project.org/. Russell V. Lenth (2016). Least- Squares Means: The R Package Ismeans. Journal of Statistical Software, 69(1), 1-33. Available on the World Wide Web at CRAN.R-project.org/package=lsmeans.

Table 3. Total Saturates of Near Isogenic Lines with different combinations of Low Saturate Genes.

Additional enumerated examples of the invention described herein are enumerated below. It will be understood by an ordinarily skilled artisan that these examples are not the only embodiments of the invention as described herein.

Enumerated Examples

1. A Brassica plant, or progeny thereof, comprising a mutant allele at a fatty acyl-acyl carrier protein thioesterase A2-14 (FATA2-14) locus, wherein the mutant allele results in the production of a FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 polypeptide.

2. The plant of example 1, wherein the mutant allele comprises a nucleic acid encoding a FATA2-14 polypeptide having a mutation in a position corresponding to amino acid 139 relative to SEQ ID NO: 1.

3. The plant of example 1 or example 2, wherein the FATA2-14 polypeptide having reduced thioesterase activity is a truncated FATA2-14 polypeptide.

4. The plant of any one of examples 1-3, wherein the plant further comprises a mutant allele at one or more fatty acyl-acyl carrier protein thioesterase B (FATB) loci, wherein the mutant allele results in the production of a FATB polypeptide having a reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide. 5. The plant of example 4, wherein the FATB loci is selected from the group consisting of FATB isoform 1, FATB isoform 2, FATB isoform 3, FATB isoform 4, FATB isoform

5. FATB isoform 6, and combinations thereof.

6. The plant of any one of examples 1-5, wherein the plant further comprises a mutant allele at a fatty acyl-acyl carrier protein thioesterase A2-4 (FATA2-4), wherein the mutant allele results in the production of a FATA2-4 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-4 polypeptide.

7. The plant of any one of examples 1-6, wherein the plant further comprises a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele.

8. The plant of any one of examples 1-7, wherein the plant further comprises a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele.

9. The plant of any one of examples 1-8, wherein the plant comprises at least two selected from the group consisting of: (a) a mutant allele at a FATA2-4 locus; (b) a mutation at chromosome N01 quantitative trait locus 1 (QTL 1) allele; (c) a mutation at chromosome N19 quantitative trait locus 2 (QTL 2) allele; and (d) a mutant allele at a FATB locus.

10. The plant of any one of examples 1-9, wherein the plant comprises a nucleic acid having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NO:4.

11. The plant of any one of examples 1-10, wherein the FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 has an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:3.

12. The plant of any one of examples 1-11, wherein the plant is a Brassica napus, Brassica juncea, or Brassica rapa plant. 13. The plant of any one of examples 1-12, wherein the plant is an Fl hybrid.

14. The plant of any one of examples 1-13, wherein the plant produces a seed that yields an oil having a total saturated fatty acid content of less than 10.5%, less than 10%, less than 9%, less than 8%, less than 7%, or less than 6%.

15. A seed of the plant of any one of examples 1-14, wherein the seed comprises the mutant allele at a FATA2-14 locus, wherein the mutant allele results in the production of a FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 polypeptide.

16. The seed of example 15, wherein the mutant allele comprises a nucleic acid encoding a FATA2-14 polypeptide having a mutation in a position corresponding to amino acid 139 relative to SEQ ID NO:1.

17. The seed of example 15 or 16, wherein the FATA2-14 polypeptide having reduced thioesterase activity is a truncated FATA2-14 polypeptide.

18. The seed of any one of examples 15-17, wherein the seed yields an oil having a total saturated fatty acid content of less than 10.5%, less than 10%, less than 9%, less than 8%, less than 7%, or less than 6%.

19. The seed of any one of examples 15-18, wherein the seed further comprises a mutant allele at one or more FATB loci, wherein the mutant allele results in the production of a FATB polypeptide having a reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide.

20. The seed of example 19, wherein the FATB loci is selected from the group consisting of FATB isoform 1, FATB isoform 2, FATB isoform 3, FATB isoform 4, FATB isoform 5, FATB isoform 6, and combinations thereof. 21. The seed of any one of examples 15-20, wherein the seed further comprises a mutant allele at a FATA2-4 locus, wherein the mutant allele results in the production of a FATA2-4 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-4 polypeptide.

22. The seed of any one of examples 15-21, wherein the seed further comprises a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele.

23. The seed of any one of examples 15-22, wherein the seed further comprises a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele.

24. The seed of any one of examples 15-23, wherein the seed comprises a nucleic acid having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the nucleotide sequence of SEQ ID NO:4.

25. The seed of any one of examples 15-24, wherein the FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 has an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:3.

26. The seed of any one of examples 15-25, wherein the seed comprises at least two selected from the group consisting of: (a) a mutant allele at a FATA2-4 locus; (b) a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus.

27. The seed of any one of examples 15-26, wherein the seed yields an oil having an oleic acid content of between 65% to 80%, a linoleic acid content of between 14% to 21%, and an alpha-linolenic acid content of between 2% and 5%. 28. The seed of example 27, wherein the oil further includes a stearic acid content between 1% and 5%.

29. The seed of example 27, wherein the oil further includes a palmitic acid content between 3% and 6%.

30. The seed of example 27, wherein the oil further includes an arachidic acid content between 0.4% and 1.0%.

31. The seed of any one of examples 15-30, wherein the seed is an F2 seed.

32. A method of producing a Brassica plant, comprising: crossing a first Brassica parent plant with a second Brassica parent plant, wherein the first Brassica parent plant includes a mutant allele at a FATA2-14 locus, wherein the mutant allele results in the production of a FATA2-14 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2-14 polypeptide, and wherein the second Brassica plant includes at least two selected from the group consisting of: (a) a mutant allele at a FATA2-4 locus; (b) a mutant allele having a mutation at chromosome N01 quantitative trait locus 1 (QTL1) allele; (c) a mutant allele having a mutation at chromosome N19 quantitative trait locus 2 (QTL2) allele; and (d) a mutant allele at a FATB locus, and selecting for one or more generations of progeny plants having one or more of the mutant alleles of the first and/or second Brassica parent plant, thereby obtaining the Brassica plant.

33. A method of producing an oil, comprising: crushing a seed of any one of the plants of examples 1-14; and extracting the oil from the crushed seed, the oil having a total saturated fatty acid content of less than 10.5%, less than 10%, less than 9%, less than 8%, less than 7%, or less than 6%.

34. The method of example 33, further comprising refining, bleaching and deodorizing the extracted oil.

35. An edible oil produced by the method of example 33 or 34, wherein the canola oil has an oleic acid content between 65% and 80%, a linoleic acid content between 14% and 21%, and an alpha-linolenic acid content between 2% and 5%.

36. The edible oil of example 35, further comprising a stearic acid content between 1% and 5%.

37. The edible oil of example 35 or 36, further comprising a palmitic acid content between 3% and 6%.

38. The edible oil of any one of examples 35-37, further comprising an arachidic acid content between 0.4% and 1.0%.

39. A plant cell of a plant of any of examples 1-14, wherein the plant cell comprises the mutant allele.