WO2013015782A1 - Plantes brassica produisant des huiles à faible teneur en acide alpha-linolénique - Google Patents

Plantes brassica produisant des huiles à faible teneur en acide alpha-linolénique Download PDF

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WO2013015782A1
WO2013015782A1 PCT/US2011/045235 US2011045235W WO2013015782A1 WO 2013015782 A1 WO2013015782 A1 WO 2013015782A1 US 2011045235 W US2011045235 W US 2011045235W WO 2013015782 A1 WO2013015782 A1 WO 2013015782A1
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Prior art keywords
plant
modified
locus
modified allele
allele
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PCT/US2011/045235
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English (en)
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Daren Coonrod
Kevin Brandt
Honggang Zheng
Zhizheng Chen
Richard Fletcher
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Cargill, Incorporated
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Priority to CA2836992A priority Critical patent/CA2836992A1/fr
Priority to PCT/US2011/045235 priority patent/WO2013015782A1/fr
Priority to AU2011373683A priority patent/AU2011373683B2/en
Priority to US13/685,588 priority patent/US9695434B2/en
Publication of WO2013015782A1 publication Critical patent/WO2013015782A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)

Definitions

  • This invention relates to Brassica plants, and more particularly, Brassica plants having a modified allele at a fatty acid desaturase 3D locus and/or a fatty acid desaturase 3E locus and yielding an oil with a low alpha linolenic acid content in combination with a typical, mid, or high oleic acid content.
  • Canola oil contains a relatively high (8%-10%) alpha-linolenic acid (ALA) content.
  • ALA alpha-linolenic acid
  • This trienoic fatty acid is unstable and easily oxidized during cooking, which in turn creates off-flavors of the oil. It also develops off odors and rancid flavors during storage (Hawrysh, 1990, Stability of canola oil, Chapter 7, pp. 99-122 In: F. Shahidi, ed. Canola and Rapeseed: Production, Chemistry, Nutrition, and Processing Technology, Van Nostrand Reinhold, N.Y.).
  • Reducing the ALA content level by hydrogenation increases oxidative stability of the oil. However, hydrogenation results in the production of trans fatty acids, which increases the risk for coronary heart disease when consumed.
  • an oil's oxidative stability is not determined solely by fatty acid profile, a decrease in the ALA content of canola oils generally improves the stability of the oils.
  • Brassica plants containing such a modified a iZ ) allele and modified fad3E allele can produce oils with a low ALA content (i.e. 1.5% or less ALA).
  • Such Brassica plants also can include other modified fatty acid desaturase alleles (e.g., fad2 or fad3), fatty acyl-acyl carrier protein thioesterase A2 (fatA2), and/or fatty acyl-acyl carrier protein thioesterase B (fatB) alleles to tailor the oleic acid and total saturated fatty acid content to the desired end use of the oil.
  • Brassica plants described herein are particularly useful for producing canola oils for certain food applications as the plants are not genetically modified.
  • this document features a Brassica plant (e.g., Brassica napus, Brassica juncea, or Brassica rapa plant), progeny, and seeds of the plant that include a modified allele at a fatty acid desaturase 3D (fad3D) locus and/or a fatty acid desaturase 3E (fad3E) locus, wherein the modified allele results in the production of a FAD3D and/or FAD3E polypeptide having reduced desaturase activity relative to a corresponding wild-type polypeptide.
  • fad3D fatty acid desaturase 3D
  • fad3E fatty acid desaturase 3E
  • the fad3E modified allele can include a nucleic acid encoding a truncated FAD3E polypeptide, a nucleic acid encoding a FAD3E polypeptide having a non-conservative substitution of a residue affecting substrate specificity, or a nucleic acid encoding a FAD3E polypeptide having a non-conservative substitution of a residue affecting catalytic activity.
  • thQfad3E modified allele includes a mutation in a splice donor site.
  • a modifwdfad3E allele can include a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1.
  • the fad3D modified allele can include a nucleic acid encoding a truncated FAD3D polypeptide, a nucleic acid having a deletion of an exon or a portion thereof (e.g., a deletion within exon 1 of the nucleic acid).
  • the fad3D modified allele includes a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:32.
  • a plant can include fad3E and fad3D modified alleles.
  • the fad3E and fad3D modified alleles can be mutant alleles.
  • a plant can be an Fi hybrid.
  • any of the plants described herein further can include a modified allele at a fad3A locus and/or a modified allele at a fad3B locus.
  • the fad3A and/or fad3B modified alleles can be mutant alleles.
  • afad3A modified allele can be selected from the group consisting of a) a nucleic acid encoding a FAD3 A polypeptide having a cysteine substituted for arginine at position 275 and b) a nucleic acid encoding a truncated FAD3A polypeptide.
  • a fad3B modified allele can be selected from the group consisting of a) a nucleic acid having a mutation in an exon-intron splice site recognition sequence and b) a nucleic acid encoding a truncated FAD3B polypeptide.
  • Such plants can produce seeds yielding an oil having an ALA content of 0.6 to 1.5%.
  • Plants described herein can produce seeds yielding an oil having a stearic acid content of 2.5 to 6%.
  • any of the plants described herein further can include a modified allele at a delta- 12 fatty acid desaturase (fad2) locus.
  • the fad2 modified allele can include a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glutamic acid in a His-Glu- Cys-Gly-His motif (SEQ ID NO:26).
  • the fad2 modified allele comprising a nucleic acid encoding a FAD2 polypeptide having a glutamic acid substituted for glycine in the DRDYGILNKV motif (SEQ ID NO:28) or a histidine substituted for leucine in a KYLNNP motif (SEQ ID NO:27).
  • Any of the plants described herein further can include a modified allele at two different fad2 loci.
  • One fadl modified allele can include a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glutamic acid in a His-Glu-Cys-Gly-His motif (SEQ ID NO:26).
  • Omfad2 modified allele can include a nucleic acid encoding a FAD2 polypeptide having a glutamic acid substituted for glycine in the DRDYGILNKV motif (SEQ ID NO :28) or a histidine substituted for leucine in a KYLNNP motif (SEQ ID NO:27).
  • any of the plants described herein further can include a modified allele at a fatty acyl-acyl-ACP thioesterase A2 (fatA2) locus and/or a fatty acyl-acyl-ACP thioesterase B (fatB) locus.
  • the fatA2 and/or fatB modified alleles can be mutant alleles.
  • a fatA2 modified allele results in the production of a FATA2 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2 polypeptide.
  • the fatA2 modified allele can include a nucleic acid encoding a FATA2 polypeptide having a mutation in a region (SEQ ID NO: 11) corresponding to amino acids 242 to 277 of the FATA2 polypeptide.
  • the FATA2 polypeptide can include a substitution of a leucine residue for proline at position 255.
  • a fatB modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild- type FATB polypeptide.
  • a plant can include modified alleles at four different fatB loci. At least one of the fatB modified alleles can include a nucleic acid encoding a truncated FATB polypeptide.
  • a truncated FATB polypeptide can include a nucleotide sequence selected from the group consisting of: SEQ ID NO:22 SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
  • this document features a method of producing an oil. The method includes crushing seeds produced from at least one Brassica plant described herein; and extracting oil from the crushed seeds, wherein the oil has, after refining, bleaching, and deodorizing, an ALA content of 0.6 to 1.5%. The oil further can have a stearic acid content of 2.5 to 6.0%.
  • This document also features a method for making a Brassica progeny plant.
  • the method includes crossing one or more first Brassica parent plants comprising a modified allele at a fad3E locus and/or a fad3D locus and one or more second Brassica parent plants comprising a modified allele at a different fad3 locus, wherein each modified allele results in the production of a FAD3 polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; and selecting, for one to five generations, for progeny plants having a modified allele at the fad3E locus and/or fad3D locus, and the modified allele at the different fad3 locus, thereby obtaining the Brassica plant.
  • this document features a method for making a Brassica plant.
  • the method includes obtaining one or more first Brassica parent plants comprising a modified allele at a fad3E locus and/or modified allele at a fad3D locus, wherein the fad3E oxfad3D modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; obtaining one or more second Brassica parent plants comprising a modified allele at a fad2 locus, the fad2 modified allele comprising a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glycine in a His-Glu-Cys- Gly-His motif (SEQ ID NO:26); crossing the one or more first Brassica parent plants and the one or more second Brassica parent plants; and selecting, for one to five generations, for
  • this document features a method for making a Brassica plant.
  • the method includes obtaining one or more first Brassica parent plants comprising a modified allele at a fad3E locus and/or fad3D locus, wherein the fad3E or said fad3D modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; obtaining one or more second Brassica parent plants comprising a modified allele at a fatA2 locus, the fatA2 modified allele comprising a nucleic acid encoding a FATA2 polypeptide having a mutation in a region (SEQ ID NO:l 1) corresponding to amino acids 242 to 277 of the FADA2 polypeptide; crossing the one or more first Brassica parent plants and the one or more second Brassica parent plants; and selecting, for one to five generations, for progeny plants having the modified allele at the fa
  • the first Brassica parent plant further can include a modified allele at a fad2 locus, a modified allele at a fad3A locus, and a modified allele at a fad3B locus, wherein the fad2 modified allele comprising a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glutamic acid in a His-Glu-Cys-Gly-His motif (SEQ ID N:26), the fa d3A modified allele comprising a nucleic acid encoding a FAD3 A polypeptide having a cysteine substituted for arginine at position 275, and the fad3B modified allele comprising a fad3B nucleic acid sequence having a mutation in an exon-intron splice site recognition sequence.
  • the fad2 modified allele comprising a nucleic acid encoding a FAD2 polypeptide having
  • This document also features a method for making a Brassica plant.
  • the method includes obtaining one or more first Brassica parent plants comprising a modified allele at a fad3E locus or a fad3D locus, wherein the fad3E or fad3D modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; obtaining one or more second Brassica parent plants comprising at least one modified allele at a fatB locus, wherein the fatB modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide; crossing the one or more first Brassica parent plants and the one or more second Brassica parent plants; and selecting, for one to five generations, for progeny plants having the modified allele at the fad3E locus and/or fad3D locus, and the
  • the one or more second Brassica plants can include modified alleles at four different fatB loci. At least one of the fatB modified alleles can include a nucleic acid encoding a truncated FATB polypeptide.
  • this document features seeds of a Brassica plant comprising a modified allele at a fad3E locus and/or a modified allele at a fad3D locus.
  • the fad3E modified allele can include a nucleic acid having a mutation in a splice donor site.
  • the fad3D modified allele can include a nucleic acid having a deletion of a portion of exon 1.
  • the seeds can yield an oil having an ALA content of 0.6% to 1.5%.
  • the seeds can be F 2 seeds.
  • the Brassica plant further can include modified alleles at fad3A and/or fad3B loci.
  • the Brassica plant further can include a modified allele at a fad2 locus.
  • the Brassica plant further can include a modified allele at a fatB locus.
  • the Brassica plant further can include a modified allele at a fatA2 locus.
  • the Brassica plant further can include modified alleles at fad3A,fad3B,fad2,fatB, and fatA2 loci.
  • this document features a plant cell of a plant described herein, wherein the plant cell includes one or more of the modified alleles.
  • nucleic acid that includes a nucleic acid sequence selected from the group consisting of i) the nucleic acid sequence set forth in SEQ ID NO: 1; ii) the nucleic acid sequence set forth in SEQ ID NO:32; iii) the complement of the nucleic acid sequence set forth in i) or ii); and iv) a nucleic acid fragment of i), ii), or iii) that is at least 50 nucleotides in length and distinguishes a mutant fad3D or fad3E allele from a wild-type fad3D or fad3E allele.
  • this document features a method of making a plant line.
  • the method includes providing a population of plants; identifying one or more plants in the population containing a modified allele at a fad3E locus and/or a fad3D locus, wherein the modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; crossing one or more of the identified plants with itself or a different plant to produce seed; crossing at least one progeny plant grown from the seed with itself or a different plant; and repeating the crossing steps for an additional 0-5 generations to make the plant line, wherein the modified allele at the fad3E locus and/or the fad3D locus is present in the plant line.
  • This document also features Brassica napus seed designated 1904 and represented by American Type Culture Collection (ATCC) Accession No. PTA-11273, as well as progeny of the seed designated 1904 and represented by ATCC Accession No. PTA- 11273.
  • ATCC American Type Culture Collection
  • This document also features Brassica napus seed designated 2558 and represented by American Type Culture Collection (ATCC) Accession No. PTA-11274, as well as progeny of the seed designated 2558 and represented by ATCC Accession No. PTA- 11274.
  • ATCC American Type Culture Collection
  • FIG. 1 is an alignment of the BnFad3E sequences from 1904, IMC201, and BrFad3E (SEQ ID NOs: 1, 2, and 3, respectively).
  • the BnFad3E-2 SNP that correlates with the low ALA (CI 8:3) content in the 1904 mutant line is highlighted with a black box at position 1851 ofthis alignment.
  • a single nucleotide mutation G to A is shown, which is located in a splice donor site (see Figure 2).
  • FIG. 2 is an alignment of the nucleotide sequence of the exon 3, intron 3 border of the BnFad3A, BnFad3B, BnFad3E genes from IMC201, IMC02, Westar, 1904, 2558, and 95CB504, and the BrFad3E gene from Brassica rapa (world wide web at brassica- rapa.org) showing the single nucleotide mutation (G to A) in BnFad3E-2 from the 1904 line. See SEQ ID NOs:4-8. This mutation (G to A) is located in the last nucleotide of exon 3 of BnFad3E. Intron 3 of the BnFad3E starts from the sequence GT (see SEQ ID NO:8).
  • FIG. 3 is an alignment of the amino acid sequences of FAD3E polypeptides from B. napus and B. rapa, and FAD3 from Arabidopsis thaliana. See SEQ ID NOs:29, 30, and 31.
  • FIG. 4 is an alignment of the BnFad3D sequences from 1904 (SEQ ID NO:32) and IMC201 and 95CB504 (SEQ ID NO:33) showing a DNA deletion in the 1904 BnFad3D starting at position 575 in this alignment, which includes a portion of exon 1 and intron 1.
  • the start codon (ATG) is at position 441.
  • FIG. 5 is the amino acid sequence of the FAD3D polypeptide (SEQ ID NO:34).
  • the FAD3D polypeptide is truncated after amino acid 64.
  • this document provides Brassica plants, including B. napus, B.juncea, and B. rapa species of Brassica, that yield seeds producing oils having a low ALA content (i.e., 1.6% or less).
  • Canola oil produced from seeds having a low ALA content tends to exhibit increased stability (e.g., oxidative stability and/or flavor stability) and a useful nutritional profile, and can be used for many food applications including as a frying oil.
  • plants described herein yield seeds producing oils having a low ALA content in combination with low total saturated fatty acids (i.e., 6% or less) or very low total saturated fatty acids (i.e., having 3.6% or less).
  • 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).
  • Brassica plants described herein can produce oils having a low ALA content and a total saturated fatty acid content of 2.5 to 6.0%, 3 to 5%, 3 to 4.5%, 3.25 to 3.75%, 3.0 to 3.5%, 3.4 to 3.7%, 3.6 to 5%, 4 to 5.5%, 4 to 5%, or 4.25 to 5.25%.
  • Oils having a low ALA content and a low or very low total saturated fatty acid content have improved oxidative stability and nutritional quality and can help consumers reduce their intake of saturated fatty acids.
  • Brassica plants yield seed oils having a low ALA content in combination with a typical (60%-70%), mid (70. l%-80%), or high (>80%) oleic acid content.
  • the total saturated fatty acid content of such seed oils can be less than 6%.
  • 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 ALA content (e.g., 1.5%) or less), an oleic acid content of 60 to 70%, and a linoleic acid content of 17 to 24%).
  • Canola oils having such a fatty acid profile are particularly useful for frying applications due to the polyunsaturated fatty acid content, which is low enough to have improved oxidative stability for frying yet high enough to impart the desired fried flavor to the food being fried, and are an improvement over commodity type canola oils.
  • the fatty acid content of commodity type canola oils may be on the order of is 6 to 8%) total saturated fatty acids, 55 to 65 % oleic acid, 20 to 30%o linoleic acid, and 7 to 10%o a- linolenic acid. See, e.g., Bailey's Industrial Oil and Fat Products, Section 2.2, "Canola Oil” on pages 61-121 of Volume 2 (6th Edition, 2005).
  • Brassica plants can be produced that yield seeds having a low ALA content, mid-oleic acid content (e.g., 70.1 to 80%) oleic acid) and a low total saturated fatty acid content (e.g., ⁇ 6.0%>).
  • Canola oils having such a fatty acid profile have an oxidative stability that is higher than oils with higher ALA and lower oleic acid contents or commodity type canola oils, and are useful for coating applications (e.g., spray-coatings), formulating food products, or other applications where shelf-life stability is desired.
  • Brassica plants can be produced that yield seeds having a low ALA content, high oleic acid content (e.g., 80.1 to 90%> oleic acid) and a low total saturated fatty acid content ( ⁇ 6.0%>).
  • Canola oils having a low ALA, high oleic acid, and low total saturated fatty acid content are particularly useful for food applications requiring high oxidative stability and a reduced saturated fatty acid content.
  • Brassica plants described herein can have reduced levels of ALA (e.g., 8.0% or less) in the seed oil as a result of reduced activity of fatty acid desaturase (FAD) 3E (also known as delta- 15 desaturase). Brassica plants described herein also can have reduced levels of ALA (e.g., 3.0% or less, 2.8% or less, 2.6% or less) in the seed oil as a result of reduced activity of FAD3D. FAD3 proteins are involved in the enzymatic conversion of linoleic acid to a-linolenic acid.
  • FAD fatty acid desaturase
  • Seeds produced by a plant can be grown and then selfed (or outcrossed and selfed, or doubled through dihaploid) to obtain seeds homozygous for a modified allele.
  • allele or “alleles” refers to one or more alternative forms of a gene at a particular locus.
  • 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.
  • the term “variety” refers to a line which is used for commercial production, and includes hybrid varieties and open-pollinated varieties.
  • Reduced activity, including absence of detectable desaturase activity, of FAD3E and/or FAD3D can be achieved by modifying an endogenous fadSE or fad3D allele.
  • An endogenous fad3E or fad3D 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.
  • 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 encompassed by the term "mutant alleles" as that term is used herein.
  • Reduced desaturase activity can be inferred from the decreased level of linolenic acid (product) and in some cases, increased level of linoleic acid (the substrate) in the plant compared with a corresponding control plant. Reduced activity also can be assessed by in vitro translation of the desaturase and assaying for desaturase activity. See, for example, Goren and Fox, Protein Expr Purif. 62(2): 171-178 (2008).
  • mutagenic agents include, for example, ethyl methane sulfonate (EMS), methyl N-nitrosoguanidine (MN G), ethidium bromide, diepoxybutane, ionizing radiation, x-rays, UV rays and other mutagens known in the art.
  • EMS ethyl methane sulfonate
  • MN G methyl N-nitrosoguanidine
  • ionizing radiation x-rays
  • UV rays UV rays
  • 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 desaturase activity that results from the mutation, e.g., by determining the fatty acid profile of the population and comparing it to that of a corresponding non-mutagenized population.
  • Mutations can be in any portion of a gene, including coding sequence, exon sequence, 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.
  • the mutation is a deletion of an exon or a portion thereof, resulting in the production of a truncated polypeptide from either lack of or incorrect RNA splicing.
  • 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.
  • the gene product of an allele having a stop codon mutation typically lacks detectable desaturase activity.
  • 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.
  • one or more exons may be skipped during RNA splicing, resulting in a protein lacking the amino acids encoded by the skipped exons.
  • 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.
  • more than one mutation or more than one type of mutation is introduced.
  • PCR can be used to amplify modified alleles in genomic DNA from the plant or plant tissue, and the resulting amplification product can be isolated and sequenced to characterize the polypeptide encoded by the modified allele.
  • RT-PCR can be used to detect particular RNA transcripts.
  • Insertions, deletions, or substitutions of amino acids in a protein 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. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product nonfunctional, compared to a smaller number of inserted or deleted amino acids.
  • 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.
  • 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. Because there are only 20 amino acids encoded in a gene, substitutions that result in reduced activity may be determined by routine experimentation, incorporating amino acids of a different class in the region of the gene product targeted for mutation.
  • a plant described herein contains a modified allele at a fad3E locus.
  • a fad3E locus can include a nucleotide sequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1.
  • the nucleotide sequence set forth in SEQ ID NO: 1 is a representative nucleotide sequence of the fad3E gene from B. napus line 1904, which contains a single nucleotide mutation (G to A) in a splice donor site.
  • sequence identity refers to the degree of similarity between any given nucleic acid sequence and a target nucleic acid sequence. The degree of similarity is represented as percent sequence identity. Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. Percent sequence identity also can be determined for any amino acid sequence.
  • Percent sequence identity can be determined using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14.
  • This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (World Wide Web at "fr” dot “com” slash “blast") or the U.S. government's National Center for Biotechnology Information web site (World Wide Web at "ncbi” dot “nlm” dot "nih” dot “gov”). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ.
  • B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C: ⁇ seql .txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C: ⁇ output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting.
  • the following command will generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql .txt -j c: ⁇ seq2.txt -p blastn -o c: ⁇ output.txt -q -1 -r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences.
  • a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with sequence from the identified sequence starting with any matched position and ending with any other matched position.
  • a matched position is any position where an identical nucleotide is presented in both the target and identified sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence.
  • a plant described herein contains a modified allele at a fad3D locus.
  • a fad3D locus can include a nucleotide sequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) sequence identity to the nucleotide sequence set forth in SEQ ID NO:32.
  • the nucleotide sequence set forth in SEQ ID NO: 32 is a representative nucleotide sequence of the fad3D gene from B. napus line 1904, which contains a deletion of 164 nucleotides from exon 1.
  • B. napus line 1904 which contains a deletion of 164 nucleotides from exon 1.
  • a Brassica plant contains a modified fad3E allele and a modified fad3D allele.
  • a modified/ai/lE and a modified fad3D allele may be combined in a plant by making a genetic cross between modified lines. For example, a plant having a modified allele at a fad3E locus can be crossed or mated with a second plant having a modified allele at a fad3D locus. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds.
  • progeny seeds can be screened in order to identify those seeds carrying both modified alleles.
  • progeny are selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having modified fad3E and fad3D alleles.
  • a line having both fad3E and fad3D modified alleles is used to introgress an individual modified allele into a different line or to introgress both modified alleles into a different line.
  • a Brassica plant contains a modified fad3E allele or a modified fad3D allele, and optionally one or more modified alleles at fad3 (e.g.fad3A and/or fad3B),fatA2,fatB, and fad2 loci.
  • a Brassica plant contains a modified fad3E allele and a modified fad3D allele, and optionally one or more modified alleles at fad3 (c.g.,fad3A and/or fad3B),fatA2,fatB, and fad2 loci.
  • a Brassica plant can contain a modified fad3E allele, a modified fad3D allele, and one or more other modified fad3 alleles.
  • Brassica plants can contain the mutation from the APOLLO or STELLAR B. napus variety that confers low linolenic acid. The STELLAR and
  • the disclosed plants contain the fad3A and/or fad3B mutation from IMC02 that confer a low linolenic acid phenotype.
  • IMC02 contains a mutation in both the fad3A and fad3B genes.
  • the fad3A gene contains a C to T mutation at position 2565, numbered from the ATG in genomic DNA, resulting in the substitution of a cysteine for arginine at position 275 of the encoded FAD3A polypeptide.
  • the fad3B gene contains a G to A mutation at position 3053 from ATG in genomic DNA, located in the exon-intron splice site recognition sequence.
  • IMC02 was obtained from a cross of IMCOl x Westar. See Example 3 of U.S. Patent No. 5,750,827. IMCOl was deposited with the American Type Culture Collection (ATCC) under Accession No. 40579. IMC02 was deposited with the ATCC under Accession No. PTA-6221.
  • Other examples of fad3 mutations include nonsense mutations in fad3A and fad3B sequences. See, Example 4.
  • the mutant fad3A sequence set forth in SEQ ID NO: 9 contains a mutation at position 102, resulting in a codon change from TGG to TGA and production of a truncated FAD3A polypeptide.
  • the mutant fad3B sequence set forth in SEQ ID NO: 10 contains a mutation at position 206, resulting in a codon change from TGG to TAG and production of a truncated FAD3B polypeptide.
  • Two or more different modified fad3 alleles may be combined in a plant by making a genetic cross between modified lines. For example, a plant having a modified allele at a fad3E locus and/or a fad3D locus can be crossed or mated with a second plant having a modified allele at a fad3A or fad3B locus. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying both modified alleles. In some embodiments, progeny are selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having modified alleles at two different fad3 loci.
  • Brassica plants having a modified allele at a fad3E locus or fad3D locus also can include modified alleles controlling fatty acyl-ACP thioesterase A2 (fatA2) and/or fatty acyl-ACP thioesterase B (fatB) to tailor the total saturated fatty acid content to the end use of the oil.
  • 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 CoenzymeA (CoA) and enter the Kennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol (TAG) biosynthesis.
  • CoA CoenzymeA
  • ER endoplasmic reticulum
  • TAG triacylglycerol
  • Members of the FATA family prefer oleoyl (C18:l) ACP substrates with minor activity towards 18:0 and 16:0-ACPs, while members of the FATB family hydrolyze primarily saturated acyl-ACPs between 8 and 18 carbons in length.
  • Reduced activity of FATA2 and/or FATB 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, for example, Bon Rush et al, Plant Cell 15: 1020-1033 (2003); and Eccleston and Ohlrogge, Plant Cell 10:613-622 (1998).
  • a Brassica plant in addition to a modified allele at a fad3E locus and/or a fad3D locus, and optionally one or more other modified fad3 loci, contains a modified allele at a fatA2 locus, wherein the modified allele results in the production of a FATA2 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2 polypeptide.
  • the modified fatA2 allele can include a nucleic acid that encodes a FATA2 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.
  • 4HBT helix/4-stranded sheet
  • a Brassica plant can contain a modified allele that includes a nucleic acid encoding a FATA2b polypeptide having a substitution in a region (SEQ ID NO:l 1) of the polypeptide corresponding to residues 242 to 277 of the FATA2 polypeptide (as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. M 93041.1, protein (SEQ ID NO: 12); GenBank Accession No. NM_117374, mRNA).
  • This region of FATA2 is highly conserved in Arabidopsis and Brassica.
  • SEQ ID NO: 13 sets forth the predicted amino acid sequence of the Brassica FATA2b polypeptide encoded by exons 2-6, and corresponding to residues 121 to 343 of the A. thaliana sequence set forth in SEQ ID NO: 12.
  • the FATA2 polypeptide can have a substitution of a leucine residue for proline at the position corresponding to position 255 of the Arabidopsis FATA2 polypeptide (i.e., position 14 of SEQ ID NO:l 1 or position
  • the proline in the B. napus sequence corresponding to position 255 m Arabidopsis is conserved among B. napus, B. rapa, B.juncea, Zea mays, Sorghum bicolor, Oryza sativa Indica (rice), Triticum aestivum, Glycine max, Jatropha (tree species), Carthamus tinctorius, Cuphea hookeriana, Iris tectorum, Perilla frutescens, Helianthus annuus, Garcinia mangostana, Picea sitchensis, Physcomitrella patens subsp.
  • Patens Elaeis guineensis, Vitis vinifera, Elaeis oleifera, Camellia oleifera, Arachis hypogaea, Capsicum annuum, Cuphea hookeriana, Populus irichocarpa, and
  • Diploknema butyracea The mutation at position 255 is associated with a low total saturated fatty acid phenotype, low stearic acid phenotype, low arachidic acid phenotype, and an increased eicosenoic acid phenotype.
  • the stearic acid content phenotype is negatively correlated with the eicosenoic acid phenotype.
  • the modified allele at a fatA2 locus includes a nucleotide sequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) sequence identity to the nucleotide sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.
  • the nucleotide sequences set forth in SEQ ID NOs: 14 and 15 are representative nucleotide sequences from the m tantfatA2b gene from B. napus line 15.24. See, U.S. Provisional Application Nos. 61/287,985 and 61/295,049.
  • a Brassica plant contains a modified allele at a fatB locus, wherein the modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide.
  • a Brassica plant contains modified alleles at two or more different fatB loci.
  • a Brassica plant contains modified alleles at three different fatB loci or contains modified alleles at four different fatB loci.
  • Brassica napus contains 6 different FATB isoforms (i.e., different forms of the FATB polypeptide at different loci), which are called isoforms 1-6 herein.
  • SEQ ID NOs: 16-21 set forth the nucleotide sequences encoding FATB isoforms 1-6, respectively, of Brassica napus.
  • the nucleotide sequences set forth in SEQ ID NOs: 16-21 have 82% to 95 %> sequence identity as measured by the ClustalW algorithm (version 1.83, default parameters). See Chenna et al, Nucleic Acids Res.. 31(13):3497-500 (2003)..
  • a modified allele at afad3E locus and a fad3D locus for example, in addition to a modified allele at afad3E locus and a fad3D locus, a
  • 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.
  • a plant can have a mutation in a nucleotide sequence encoding FAD3E and can have mutation in a nucleotide sequence encoding 2 or more FATB isoforms, e.g., FATB isoforms 1 and 2; 1 and 3; 1 and 4; 1 and 5; 1 and 6; 2 and 3; 2 and 4; 2 and 5; 2 and 6; 3 and 4; 3 and 5; 3 and 6; 4 and 5; 4 and 6; 5 and 6; 1, 2, and 3; 1, 2, and 4; 1, 2, and 5; 1, 2, and 6; 1, 3, and 4; 1, 3, and 5; 1, 3, and 6; 1, 4, and 5; 1, 4, and 6; 1, 5, and 6; 2, 3, and 4; 2, 3, and 5; 2, 3, and 6; 2, 4, and 5; 2, 4, and 6; 1, 5, and 6; 2,
  • a Brassica plant can have a mutation in a nucleotide sequence encoding a FAD3E polypeptide and can have a mutation in nucleotide sequences encoding FATB isoforms 1, 2, and 3; 1, 2, and 4; 1, 3, and 4; 2, 3, and 4; or 1, 2, 3, and 4.
  • a mutation in a FATB isoform results in deletion of a 4HBT domain or a portion thereof of a FATB polypeptide.
  • FATB polypeptides typically contain a tandem repeat of the 4HBT domain, where the N- terminal 4HBT domain contains residues affecting substrate specificity (e.g., two conserved methionines, a conserved lysine, a conserved valine, and a conserved serine) and the C-terminal 4HBT domain contains residues affecting catalytic activity (e.g., a catalytic triad of a conserved asparagine, a conserved histidine, and a conserved cysteine) and substrate specificity (e.g., a conserved tryptophan).
  • substrate specificity e.g., two conserved methionines, a conserved lysine, a conserved valine, and a conserved serine
  • catalytic activity e.g., a catalytic triad of a conserved asparagine, a conserved histidine, and a conserved cysteine
  • substrate specificity e.g., a
  • the mutation in a nucleotide sequence encoding FATB results in a non-conservative substitution of a residue in a 4HBT domain or a residue affecting substrate specificity.
  • the mutation in a nucleotide sequence encoding FATB is a splice site mutation.
  • the mutation in a nucleotide sequence encoding FATB is a nonsense mutation in which a premature stop codon (TGA, TAA, or TAG) is introduced, resulting in the production of a truncated polypeptide.
  • SEQ ID NOs:22-25 set forth the nucleotide sequences encoding fatB isoforms 1-
  • SEQ ID NO:22 is the nucleotide sequence of isoform 1 having a mutation at position 154, which changes the codon from CAG to TAG.
  • SEQ ID NO:23 is the nucleotide sequence of isoform 2 having a mutation at position 695, which changes the codon from CAG to TAG.
  • SEQ ID NO:24 is the nucleotide sequence of isoform 3 having a mutation at position 276, which changes the codon from TGG to TGA.
  • SEQ ID NO:25 is the nucleotide sequence of isoform 4 having a mutation at position 336, which changes the codon from TGG to TGA. See also U.S. Provisional Application Nos.
  • Two or more different modified FATB alleles may be combined in a plant by making a genetic cross between modified lines.
  • a plant having a modified allele at a FATB locus encoding isoform 1 can be crossed or mated with a second plant having a modified allele at a FATB locus encoding isoform 2.
  • Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying both modified alleles.
  • progeny are selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having modified alleles at two different FATB loci.
  • a plant having modified alleles at two or more different FATB isoforms can be crossed with a second plant having modified alleles at two or more different FATB alleles, and progeny seeds can be screened to identify those seeds carrying modified alleles at four or more different FATB loci. Again, progeny can be selected for multiple generations to obtain the desired plant.
  • a modified allele at a fad3E locus, a fad3D locus, a fatA2 locus and modified alleles at two or more (e.g., three or four) different fatB loci can be combined in a plant.
  • a plant having a modified allele at a fad3E locus and a fad3D locus can be crossed or mated with a second plant having a modified allele at a fatA2 locus. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds.
  • progeny seeds can be screened in order to identify those seeds carrying modified fad3E,fad3D, and fatA2 alleles.
  • Progeny can be selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having a modified allele at a fad3E locus, a modified allele at a fad3D locus, and a modified allele at a fatA2 locus.
  • progeny identified as having a modified allele at a fad3E locus, a modified allele at a fad3D locus, and a modified allele at a fatA2 locus can be crossed or mated with a second plant having modified alleles at two or more different fatB loci. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying modified fad3E,fad3D,fatA2, and fatB alleles.
  • Progeny can be selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having a modified allele at a fad3E locus, a modified allele at a fatA2 locus, and two or more different fatB loci.
  • Plants having a modified allele at a fad3E locus, a fad3D locus, a fatA2b locus, and modified alleles at three or four different fatB loci have a low ALA content, high oleic acid content, and a low total saturated fatty acid content.
  • Brassica plants described herein also can have decreased activity of a delta- 12 desaturase, which is involved in the enzymatic conversion of oleic acid to linoleic acid, to confer a mid or high oleic acid content in the seed oil.
  • Brassica plants can exhibit reduced activity of delta- 12 desaturase (also known as FAD2) in combination with reduced activity of FAD3E and optionally one or more of FAD3A, FAD3B, FATA2, and FATB.
  • delta- 12 desaturase also known as FAD2
  • FAD3E reduced activity of FAD3E
  • FATA2 FATA2
  • FATB FATB.
  • the sequences for the wild-type fad2 genes from B. napus (termed the D form and the F form) are disclosed in WO 98/56239.
  • a reduction in delta- 12 desaturase activity, including absence of detectable activity, can be achieved by mutagenesis.
  • Decreased delta- 12 desaturase activity can be inferred from the decrease level of linoleic acid (product) and increased level of oleic acid (substrate) in the plant compared with a corresponding control plant.
  • suitable fad2 mutations include the G to A mutation at nucleotide 316 within the fad2-D gene, which results in the substitution of a lysine residue for glutamic acid in a HECGH (SEQ ID NO:26) motif. Such a mutation is found within the line IMC 129, which has been deposited with the ATCC under Accession No. 40811.
  • Another suitable fad2 mutation can be the T to A mutation at nucleotide 515 of the fad2-F gene, which results in the substitution of a histidine residue for leucine in a KYLNNP (SEQ ID NO:27) motif (amino acid 172 of the Fad2 F polypeptide).
  • a mutation is found within the variety Q508. See U.S. Patent No. 6,342,658.
  • fad2 mutation is the G to A mutation at nucleotide 908 of the fad2-F gene, which results in the substitution of a glutamic acid for glycine in the DRDYGILNKV (SEQ ID NO:28) motif (amino acid 303 of the Fad2 F polypeptide).
  • DRDYGILNKV SEQ ID NO:28
  • amino acid 303 of the Fad2 F polypeptide amino acid 303 of the Fad2 F polypeptide
  • fad2 mutation can be the C to T mutation at nucleotide 1001 of the fad2-F gene (as numbered from the ATG), which results in the substitution of an isoleucine for threonine (amino acid 334 of the Fad2 F polypeptide). Such a mutation is found within the high oleic acid line Q7415.
  • the presence of one of the fad2-D or fad2-F mutations confers a mid- oleic acid phenotype (e.g., 70-80% oleic acid) to the seed oil, while the presence of both fad2-D and fad2-F mutations confers a higher oleic acid phenotype (e.g., >80% oleic acid).
  • Q4275 contains the fad2-D mutation from IMC 129 and afad2-F mutation at amino acid 303.
  • Q508 contains fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 172.
  • Q7415 contains the fad2-D mutation from IMC 129 and a fad2-F mutation at amino acid 334.
  • the presence of both fad2 mutations in Q4275, Q508, and Q7415 confers a high oleic acid phenotype of greater than 80% oleic acid.
  • a Brassica plant contains a modified allele at a fad3E locus and a modified allele at a fad2 locus.
  • a Brassica plant can contain a modified allele at a fad3E locus and a modified allele at a fad2 locus described above.
  • a Brassica plant also can contain a modified allele at a fad3E locus, a modified allele at a fad2 locus, and a modified allele at &fatA2 locus.
  • a Brassica plant can contain a modified allele at a fad3E locus, modified alleles at two or more different fatB loci (three or four different loci), and a fad2 locus described above.
  • a Brassica plant also can contain a modified allele at a fad3E locus, fatA2 locus, modified alleles at two or more different fatB loci (three or four different loci) and a modified allele at a fad2 locus described above.
  • a Brassica plant contains a modified allele at a fad3E locus, at least one modified allele at a different fad3 locus, a modified allele at a fatA2 locus, a modified allele at one or more different fatB loci (e.g., two or more), and a modified allele at one or more fad2 loci.
  • a Brassica plant also can contain modified alleles at a fad3E locus and a fad3D locus, modified alleles at two or more different fatB loci (three or four different loci), modified alleles at fad2 loci, and modified alleles at fad3A and/or fad3B loci described above.
  • a Brassica plant also contain a modified allele at a fad3E locus, a modified allele at a fad3D locus, a modified allele at a fatA2 locus, modified alleles at two or more different FATB loci (three or four different loci), modified alleles at fad2 loci, and modified alleles at fad3A and fad3B loci described above.
  • B. napus One commercially important Brassica crop is B. napus.
  • Commercial B. napus lines may be classified as either spring lines or winter lines. Winter lines are commonly planted in the autumn and flower in the spring after a period of vernalization over the winter. Spring lines do not require vernalization to flower and are commonly planted and harvested in the same growing season. Winter lines are common in Europe, but most winter lines fare poorly in the colder winters of Canada and the northern United States. As a consequence, most B. napus grown commercially in North America are spring lines.
  • One useful embodiment provides a Brassica plant that is a B. napus plant. Though the B. napus plant may have a winter flowering habit, one preferred implementation has a spring growing habit, i.e., it does not require vernalization to flower.
  • Hybrid Brassica varieties can be produced by preventing self-pollination of female parent plants (i.e., seed parents), permitting pollen from male parent plants to fertilize such female parent plants, and allowing Fi hybrid seeds to form on the female plants.
  • Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development.
  • pollen formation can be prevented on the female parent plants using a form of male sterility.
  • male sterility can be cytoplasmic male sterility (CMS), nuclear male sterility, molecular male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or be produced by self-incompatibility.
  • CMS cytoplasmic male sterility
  • nuclear male sterility nuclear male sterility
  • molecular male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or be produced by self-incompatibility.
  • Female parent plants containing CMS are particularly useful.
  • CMS can be, for example of the ogu (Ogura), nap, pol, tour, or mur type.
  • Ogura ogu
  • Monetan a compound that has a high degree of ogu
  • Monetan a compound that has a high degree of ogu
  • Monetan a compound that has a high degree of ogura
  • p. 199-203 a description of Ogura type CMS.
  • Genome 30:234-238 for a description of Ogura type CMS.
  • Riungu and McVetty, 2003, Can. J. Plant Sci., 83:261-269 a description of nap, pol, tour, and mur type CMS.
  • the male parent plants typically contain a fertility restorer gene to ensure that the Fi hybrids are fertile.
  • a fertility restorer gene that can overcome the Ogura type CMS.
  • Non- limiting examples of such fertility restorer genes include the Kosena type fertility restorer gene (U.S. Patent No. 5,644,066) and Ogura fertility restorer genes (U.S. Patent Nos. 6,229,072 and 6,392,127).
  • male parents can be used that do not contain a fertility restorer. Fi hybrids produced from such parents are male sterile. Male sterile hybrid seed can be inter-planted with male fertile seed to provide pollen for seed-set on the resulting male sterile plants.
  • the methods described herein can be used to form single-cross Brassica Fi hybrids.
  • the parent plants can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants.
  • the Fi seed formed on the female parent plants is selectively harvested by conventional means.
  • One also can grow the two parent plants in bulk and harvest a blend of Fi hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination.
  • three-way crosses can be carried out wherein a single-cross Fi hybrid is used as a female parent and is crossed with a different male parent that satisfies the fatty acid parameters for the female parent of the first cross.
  • the overall oleic acid content of the vegetable oil may be reduced over that of a single-cross hybrid; however, the seed yield will be further enhanced in view of the good agronomic performance of both parents when making the second cross.
  • double-cross hybrids can be created wherein the Fi progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self- pollination of female parents when forming a double-cross hybrid.
  • Hybrids described herein have good agronomic properties and exhibit hybrid vigor, which results in seed yields that exceed that of either parent used in the formation of the Fi hybrid.
  • yield can be at least 10% (e.g., 10% to 20%, 10% to 15%, 15% to 20%, or 25% to 35%) above that of either one or both parents.
  • the yield exceeds that of open-pollinated spring canola varieties such as 46A65 (Pioneer) or Q2 (University of Alberta), when grown under similar growing conditions.
  • yield can be at least 10% (e.g., 10% to 15% or 15% to 20%) above that of an open-pollinated variety.
  • Hybrids described herein typically produce seeds having very low levels of glucosinolates ( ⁇ 30 ⁇ /gram of de-fatted meal at a moisture content of 8.5%).
  • hybrids can produce seeds having ⁇ 20 ⁇ of glucosinolates/gram of defatted meal.
  • hybrids can incorporate mutations that confer low glucosinolate levels. See, for example, U.S. Patent No. 5,866,762.
  • Glucosinolate levels can be determined in accordance with known techniques, including high performance liquid chromatography (HPLC), as described in ISO 9167-1 : 1992(E), for quantification of total, intact glucosinolates, and gas-liquid chromatography for quantification of trimethylsilyl (TMS) derivatives of extracted and purified desulfoglucosinolates. Both the HPLC and TMS methods for determining glucosinolate levels analyze de-fatted or oil-free meal.
  • HPLC high performance liquid chromatography
  • TMS trimethylsilyl
  • Brassica plants disclosed herein are useful for producing canola oils with low ALA content.
  • oil obtained from seeds of Brassica plants described herein may have an ALA content of 0.5% to 1.6% (e.g., 0.5 to 1.5%, 0.5 to 1.0%, 0.5 to 0.8%, 0.6 to 1.4%, 0.6 to 1.3%, 0.6 to 1.2%, 0.6 to 1.1%, 0.6 to 1.0%, 0.6 to 0.8%, 0.7 to 1.2%, 0.7 to 1.1%), 0.8 to 1.2%), or 0.8 to 1.0%).
  • Brassica plants described herein produce canola oils with low ALA content (e.g., 0.5 to 1.6%>) and low or no total saturated fatty acids.
  • oil obtained from seeds of Brassica plants described herein may have an ALA content of 0.5 to 1.5% and a total saturated fatty acid content of 2.5 to 6%, 3 to 5%, 3 to 4.5%, 3.25 to 3.75%, 3.0 to 3.5%, 3.4 to 3.7%, 3.6 to 5%, 4 to 5.5%, 4 to 5%, or 4.25 to 5.25%».
  • the palmitic acid content of such oils can be 2.4 to 3.5%) (e.g., 2.5 to 3%> or 2.7 to 3.3%>).
  • the stearic acid content of such oils can be 0.7 to 2.5% (e.g., 0.8 to 1.7%, 0.9 to 1.5%, or 1.0 to 1.5%).
  • an oil has an ALA content of 0.5 to 1.5%, an oleic acid content of 60 to 70% (e.g., 62 to 68%, 63 to 67%, or 65 to 66%), and a total saturated fatty acid content of 5 to 10%>.
  • an oil has an ALA content of 0.6 to 1.5% (e.g., 0.7 to 1.4%, 0.8 to 1.3%, or 0.9 to 1.2%) and an oleic acid content of 71 to 80% (e.g., 72 to 78%, 72 to 76%, 73 to 75%, 74 to 77%, 74 to 78%, or 75 to 80%).
  • the total saturated content of such an oil can be 3 to 8%> (e.g., 4 to 6%>, 4 to 5.5%>, 4 to 5%>, 5 to 7%), 6 to 8%), or 7 to 8%).
  • a canola oil can have an ALA content of 0.5 to 1.5%, an oleic acid content of 85 to 87% (e.g., 86 to 87%), and a total saturated fatty acid content of 5 to 6%.
  • an oil has an ALA content of 0.5 to 1.5%, an oleic acid content of 81 to 90% (e.g., 82 to 88% or 83 to 87%) oleic acid and a total saturated fatty acid content of 3.5 to 4.5% (e.g., 3.75 to 4.25%, 3.9 to 4.1%, or 4.0%).
  • Oils described herein can have an eicosenoic acid content of 1.0 to 1.9%.
  • an oil can have an eicosenoic acid content of 1.0 to 1.4%, 1.1 to 1.3%, 1.1 to 1.6%, 1.2 to 1.6%, 1.4 to 1.9%, in addition to a low ALA content.
  • Oils described herein can have a linoleic acid content of 3.5 to 26%, e.g., 3.7 to 4.5%, 8 to 10%, 9 to 12%, 10 to 13%, 11 to 13%, 12 to 16%, 13 to 16%, 14 to 18%, or 14 to 22%), in addition to a low ALA content.
  • Oils described herein have an erucic acid content of less than 2% (e.g., less than 1%, 0.5%, 0.2, or 0.1%) in addition to a low ALA content.
  • 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 in the 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)
  • 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 ALA content.
  • Harvested canola seed can be crushed to extract crude oil and, if desired, refined, bleached and deodorized by techniques known in the art. See, e.g., Bailey's Industrial Oil and Fat Products, Volume 5, "Edible Fat and Oil Products: Processing Technologies” (6th Edition, 2005). Briefly, refining refers to removing most if not all free fatty acids and other impurities such as phosphatides or protein substances from a crude oil.
  • One common method of refining involves treating an oil with a strong base, followed by extensive washings with water.
  • Bleaching refers to a process that removes natural pigments (e.g., carotenoids, chlorophylls, and xanthophylls) and other impurities such as metal cations (e.g., Fe, Cu and Zn). Bleaching can be done by absorbing such pigments and/or cations on a natural bleaching earth or clay, which is usually added to an oil under vacuum and high temperature.
  • Deodorizing refers to the removal of relatively volatile trace components (e.g., ketones, aldehydes, alcohols) from an oil that contribute to flavor, odor, and color. Deodorizing is usually done by injecting steam into an oil heated to high temperatures (e.g., about 470°F to about 510°F) under high vacuum (e.g., ⁇ 5 mm Hg).
  • 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 a 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.
  • 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.
  • 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.
  • Further oil can be extracted from the press cake produced from the screw pressing operation using known solvent extraction techniques, e.g., using commercial n-hexane extraction.
  • the canola oil recovered from the solvent extraction process is combined with the clarified oil from the screw pressing operation, resulting in a blended crude oil.
  • 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 acid-oil 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.
  • Oil may be bleached and deodorized before use, if desired, by techniques known in the art.
  • Oils obtained from plants 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.
  • Oils obtained from plants described herein also can have increased flavor stability, which can be measured using, for example, trained test panels in room-odor tests according to Mounts, J. Am. Oil Chem. Soc. 56:659-663, 1979 and the AOCS Recommended Practice Cg 2-83 for the Flavor Evaluation of Vegetable Oils (Methods and Standard Practices of the AOCS, 4th Edition (1989)).
  • the technique encompasses standard sample preparation and presentation, as well as reference standards and method for scoring oils.
  • oils having a low ALA content can be used for food applications or for frying.
  • Oils having a low ALA content in combination with a low (6% or less) or very low (3.5% or less) total saturated fatty acid content can be used to replace or reduce the amount of saturated fatty acids and hydrogenated oils (e.g., partially hydrogenated oils) in various food products such that the levels of saturated fatty acids and trans fatty acids are reduced in the food products.
  • canola oils having a low ALA content in combination with a low total saturated fatty acid content and a mid or high oleic acid content can be used to replace or reduce the amount of saturated fats and partially hydrogenated oils in processed or packaged food products, including bakery products such as cookies, muffins, doughnuts, pastries (e.g., toaster pastries), pie fillings, pie crusts, pizza crusts, frostings, breads, biscuits, and cakes, breakfast cereals, breakfast bars, puddings, and crackers.
  • bakery products such as cookies, muffins, doughnuts, pastries (e.g., toaster pastries), pie fillings, pie crusts, pizza crusts, frostings, breads, biscuits, and cakes, breakfast cereals, breakfast bars, puddings, and crackers.
  • an oil described herein can be used to produce sandwich cookies that contain no or reduced levels of partially hydrogenated oils in the cookie and/or creme filling.
  • the cookies also have a reduced total saturated fatty acid content.
  • Such cookie compositions can include, for example, in addition to canola oil, flour, sweetener (e.g., sugar, molasses, honey, high fructose corn syrup, artificial sweetener such as sucralose, saccharine, aspartame, or acesulfame potassium, and combinations thereof), eggs, salt, flavorants (e.g., chocolate, vanilla, or lemon), a leavening agent (e.g., sodium bicarbonate or other baking acid such as monocalcium phosphate monohydrate, sodium aluminum sulfate, sodium acid pyrophosphate, sodium aluminum phosphate, dicalcium phosphate, glucano-deltalactone, or potassium hydrogen tartrate, or combinations thereof), and optionally, an emulsifier (e.g., mono- and digly
  • a creme filling composition can include, in addition to canola oil, sweetener (e.g., powdered sugar, granulated sugar, honey, high fructose corn syrup, artificial sweetener, or combinations thereof), flavorant (e.g., vanilla, chocolate, or lemon), salt, and, optionally, emulsifier.
  • sweetener e.g., powdered sugar, granulated sugar, honey, high fructose corn syrup, artificial sweetener, or combinations thereof
  • flavorant e.g., vanilla, chocolate, or lemon
  • salt e.g., emulsifier
  • Canola oils (e.g., with a low ALA, low total saturated fatty acid and low or high oleic acid content) also are useful for frying applications due to the polyunsaturated content, which is low enough to have improved oxidative stability for frying yet high enough to impart the desired fried flavor to the food being fried.
  • canola oils can be used to produce fried foods such as snack chips (e.g., corn or potato chips), French fries, or other quick serve foods.
  • Oils described herein also can be used to formulate spray coatings for food products (e.g., cereals or snacks such as crackers).
  • the spray coating can include other vegetable oils such as sunflower, cottonseed, corn, or soybean oils.
  • a spray coating also can include an antioxidant and/or a seasoning.
  • Oils described herein also can be use in the manufacturing of dressings, mayonnaises, and sauces to provide a reduction in the total saturated fat content of the product. Oils described herein can be used as a base oil for creating structured fat solutions such as microwave popcorn solid fats or canola butter formulations.
  • the nucleic acids described herein can be used as markers in plant genetic mapping and plant breeding programs.
  • markers may include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA detection (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) or microsatellite, for example.
  • Marker-assisted breeding techniques may be used to identify and follow a desired fatty acid composition (e.g., low linolenic acid) during the breeding process.
  • a nucleic acid described herein such as the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:32, or the complement thereof, can be used to identify one or more individual plants that possess the polymorphic allele correlated with the desired linolenic acid content. Those plants then can be used in a breeding program to combine the polymorphic allele with a plurality of other alleles at other loci that are correlated with a desired variation (e.g., in fatty acid composition).
  • a desired variation e.g., in fatty acid composition
  • a fragment of the nucleic acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:32, or the complement thereof, that is at least 50 nucleotides in length can be used to distinguish a modified fad3 allele from a wild- type Fad3 allele (e.g., by allele-specific hybridization or by PCR).
  • Techniques suitable for use in a plant breeding program include, without limitation, backcrossing, mass selection, pedigree breeding, bulk selection, crossing to another population and recurrent selection. These techniques can be used alone or in combination with one or more other techniques in a breeding program.
  • each identified plant is selfed or crossed to a different plant to produce seed, which is then germinated to form progeny plants.
  • At least one such progeny plant is then selfed or crossed with a different plant to form a subsequent progeny generation.
  • the breeding program can repeat the steps of selfing or outcrossing for an additional 0 to 5 generations as appropriate in order to achieve the desired uniformity and stability in the resulting plant line, which retains the polymorphic allele.
  • analysis for the particular polymorphic allele will be carried out in each generation, although analysis can be carried out in alternate generations if desired.
  • selection for other useful traits is also carried out, e.g., selection for disease resistance. Selection for such other traits can be carried out before, during or after identification of individual plants that possess the desired polymorphic allele.
  • Marker-assisted breeding techniques may be used in addition to, or as an alternative to, other sorts of identification techniques.
  • An example of marker-assisted breeding is the use of PCR primers that specifically amplify a sequence containing a desired mutation in the fad3D or fad3E sequence.
  • the fatty acids are referred to by the length of the carbon chain and number of double bonds within the chain.
  • C14:0 refers to myristic acid
  • CI 6:0 refers to palmitic acid
  • CI 8:0 refers to stearic acid
  • CI 8: 1 refers to oleic acid
  • C18:2 refers to linoleic acid
  • C18:3 refers to ALA
  • C20:0 refers to archidic 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
  • C24: l refers to nervonic acid.
  • Total Sats refers to the total of C14:0, C16:0, C18:0, C20:0, C22:0, and C24:0.
  • Representative fatty acid profiles are provided for each of the specified samples
  • Brassica plant lines 1904 and 2558 are Brassica plant lines 1904 and 2558.
  • Plants producing an oil with a low ALA content were obtained by subjecting a population of B. napus IMC201 seeds to chemical mutagenesis and selecting for low linolenic acid content ( ⁇ 1.5%).
  • the typical fatty acid composition of field grown IMC201 is 3.6% C16:0, 1.8% C18:0, 76% C18: l, 12.5% C18:2, 3% C18:3, 0.7% C20:0, 1.5% C20:l, 0.3%C22:0, 0% C22:l, with total saturates of 6.4%.
  • IMC201 seeds Prior to mutagenesis, IMC201 seeds were pre-imbibed in 700 gm seed lots by soaking for 1 min then draining for 5 min at room temperature. This was repeated four times to soften the seed coat.
  • the pre-imbibed seed then were treated with 4 mM methyl N-nitrosoguanidine (M G) 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 one and one half hours. This dual mutagen treatment produced an LD 50 with the seed population.
  • M G methyl N-nitrosoguanidine
  • Lines 1904 and 2558 were selected from the mutagenized population of IMC201 seeds as follows. Three thousand bulk M2 generation seeds were planted. Upon maturity, M3 seed (2500 individuals) was harvested from 2500 M2 plants and analyzed via a modified method for gas chromatograph determination of fatty acid profile per the American Oil Chemist's Society protocol (AOCS Ce lc-89).
  • the oil from the seeds was first treated to convert the acylglycerols to fatty acid methyl esters ("FAMEs") and vials of the FAMEs were placed in a gas chromatograph for analysis in accordance with a modified version of American Oil Chemist's Society Official Method Ce 1-62 that employed an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA) equipped with a fused silica capillary column (5 m x 0.180 mm and 0.20 ⁇ film thickness) packed with a polyethylene glycol based DB- Wax® for liquid phase separation (J&W Scientific, Folsom, CA). Hydrogen (H2) was used as the carrier gas at a flow rate of 2.5 mL/min and the column temperature was isothermal at 200°C. Seed from each plant was tested via this method in replicates of two.
  • FAMEs fatty acid methyl esters
  • Lines 1904 and 2558 were identified as having low linolenic acid content in seed oil. M3 seeds of lines 1904 and 2558 were planted (50 per line) and the resulting plants were self pollinated. M4 seeds were harvested from the plants and bulk seed samples (approximately 20 seeds) were analyzed via GC. The results are presented in Table 1 Lines 1904 and 2558 had ALA contents ranging from approximately 0.70% to 1.95%. Line 1904 was deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-11273 and line 2558 was deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-11274. TABLE 1
  • M4 individuals were self pollinated to generate M5 seeds and further evaluated in an environmentally controlled plant growth chamber.
  • Seeds from M3B- 2558-35 and M3-B 1904-35 were planted in Premier Pro-Mix BX potting soil (Premier Horticulture, Quebec, Canada) in four inch plastic pots. Planted seeds were watered and stratified at 5°C for 5 days and germinated at 20°C day temperature and 17°C night temperature (20/17) in Conviron ATC60 controlled-environment growth chambers (Controlled Environments Limited, Winnipeg, MB). Each genotype combination was randomized and replicated 10 times in each of two separate growth chambers.
  • one chamber was reduced to a diurnal temperature cycle of 15°C day temperature and 12°C night temperature (15/12) while the other remained at 20/17.
  • the temperature treatments were imposed to identify the effects of temperature on fatty acid composition. Plants were watered five times per week and fertilized bi-weekly using a 20:20:20 (NPK) liquid fertilizer at a rate of 150 ppm. Plants were bagged individually to ensure self pollination and genetic purity of the seed. Seeds from each plant were harvested individually at physiological seed maturity. The fatty acid profile of the seeds was determined using the modified GC method described above (replicates of two).
  • Fatty acid data from plants grown under the different temperature regimes was analyzed in two ways. First, data was analyzed separately as different environments and then it was pooled and analyzed across environments. Data was analyzed in SAS (SAS Institute, 2003) using proc glm to estimate differences in mean fatty acid values. Table 2 contains the population size, mean value and standard deviation of oleic, linoleic and linolenic fatty acid of seeds produced by plants carrying mutant fad3 alleles and grown in two environmental growth chambers set at different diurnal temperature regimes (20°C day/17°C night; 15°C day/12°C night) as discussed above.
  • Genotypes 1904-35 and 2558-35 are mutant allele combinations and vl030 hybrid and IMC02 are controls.
  • the 1904-35, 2558-35, and IMC02 lines each contain mutant fad3A and fad3B alleles, while line 1904-35 also contains a mutant fad3E allele and a mutant fad3D allele (see below).
  • Means with different letters are significantly different as determined by a Student- Newman-Keuls mean separation test.
  • lines 1904-35 and 2558-35 can reach an alpha-linolenic content less than vl030 and IMC02.
  • Genome mapping, map-based gene cloning, and direct-sequencing strategies were used to identify loci associated with the ⁇ 1.5% linolenic fatty acid content in the 1904-35 line described in Example 1.
  • a DH (doubled haploid) population was developed from a cross between 1904-35 and 95CB504, a B line (maintainer).
  • the two parental lines were screened with 1066 SNP (single nucleotide polymorphism) markers using the
  • MassARRAY platform (Sequenom Inc., San Diego, CA) to identify polymorphic SNP markers between the two parents; 174 polymorphic SNP markers were identified.
  • Comparative genome mapping was performed to locate the identified QTL in Brassica napus chromosomes and further identify the Brassica rap& BAC (Bacterial Artificial Chromosome) clones encompassing the identified SNP markers and the candidate genes in the identified QTL interval for the ⁇ 1.5% linolenic acid content using publicly available Brassica and Arabidosis genome sequences, genes, genetic linkage maps, and other information from the world wide web at brassica.bbsrc.ac.uk/, and ncbi.nlm.nih.gov/.
  • QTL mapping identified two QTLs for low linolenic acid content ( ⁇ 1.5% C18:3).
  • Comparative genome mapping located one QTL on the N3 chromosome in Brassica napus (A3 in Brassica rapa) and further identified a Fad3E candidate gene which is located at IcM from the SNP marker that showed significant association with CI 8:3 content.
  • the IcM interval between the SNP marker and Fad3E gene is 248 kb according to co-linearity with the Arabidopsis genome.
  • Example 3 describes the second QTL on the N5 chromosome in Brassica napus (A5 in Brassica rapa).
  • FadSE genes from chromosome N3 of the Brassica napus genome were sequenced from 1904-35, 95CB504 and IMC201. The sequences were analyzed using BLAST (the Basic Local Alignment Search Tool) and DNASTAR/Lasergene 8.0
  • FIG. 1 shows the sequence alignment of the BnFad3E gene from 1904-35 and IMC201, and the BrFad3E located in Brassica rapa BAC, KBrH013B15 from the world wide web at brassica-rapa.org.
  • the nucleotide substitution of a "A" in 1904-35 for "G” in IMC201 and 95CB504 at position 18 1 of this alignment position 1756 in SEQ
  • FIG. 3 shows the alignment of FAD3E amino acid sequences from 1904 BnFAD3E-2 and IMC201 BnFAD3E-2 (SEQ ID NO:29), BrFAD3E deduced from BrFad3E (world wide web at brassica-rapa.org) (SEQ ID NO:30), and AtFAD3
  • fad3E-2 SNP allele results in an altered consensus sequence at the "splice donor site" for RNA splicing. Therefore, the RNA splicing of fad3E-2 primary transcript (pre-mRNA) cannot be processed to create a mature RNA (mRNA).
  • a 2 nd QTL was also identified for low linolenic acid content. Comparative genomics located this 2nd QTL on the N5 chromosome of Brassica napus and further identified a Fad3D candidate gene on chromosome N5.
  • the Fad3D genes from chromosome N5 of the Brassica napus genome were sequenced from 1904-35, 95CB504, and IMC201. The sequences were analyzed using BLAST and DNASTAR/Lasergene 8.0 (DNASTAR, Inc).
  • FIG. 4 shows the sequence alignment of a portion of the BnFad3D gene from 1904-35, 95CB504 and IMC201.
  • a deletion was identified in one of the two Fad3D isoforms from the 1904-35 mutant line that was not present in 95CB504 and IMC201.
  • the mutant type BnFad3D from 1904-35 has a deletion including a portion of exon 1 (from position 575 to position 739).
  • exon 1 starts at position 441 and ends at position 739.
  • exon 1 is only 134 bp long. Therefore, it is believed the deletion mutation in 1904 BnFad3D induced a non-functional truncated
  • RNA splicing truncated protein with 64 amino acids
  • RNA splicing truncated protein with 64 amino acids
  • Fad3D deletion was 1904-35-specific.
  • 215 lines were used from multiple populations, which carry all Fad3 isoforms, for the multiple regression analysis.
  • a population of B. napus IMC201 seeds was subjected to chemical mutagenesis as set forth in Example 1. Approximately 200,000 treated seeds were planted in standard greenhouse potting soil and placed into environmentally controlled greenhouse. The plants were grown under sixteen hours of day light. At maturity, M2 seed was harvested from the plants and bulked together. The M2 generation was planted and leaf samples from the early, post-cotyledon stage of development from 8 plants were pooled and DNA was extracted from leaves of these plants. The leaf harvest, pooling and DNA extraction was repeated for approximately 32,000 plants, and resulted in approximately forty 96- well blocks containing mutagenized B. napus IMC201 DNA. This grouping of mutagenized DNA is referred to below as the original DNA mutagenesis library.
  • Example 1 approximately 200,000 treated seeds from the dual mutagen treatment described in Example 1 were planted in standard greenhouse potting soil and placed into environmentally controlled greenhouse. The plants were grown under sixteen hours of day light. At maturity, M2 seed was harvested from the plants and bulked together. This M2 generation was planted in greenhouses and, at flowering, plants were bagged in groups of four to facilitate cross-pollination that would occur in parallel with the majority self pollination events, and seed from this generation was harvested.
  • Genomic DNA from three seeds per plant of this M3 generation was isolated in 96-well blocks; a collection of mutagenized DNA from this process is referred to below as the new Tilling DNA mutagenesis library.
  • the original DNA mutagenesis library and the new Tilling DNA mutagenesis library were screened to identify stop-codon containing fad3A and fad3B mutant alleles.
  • PC reactions were performed using B. napus IMC201 genomic DNA original mutagenesis library or new Tilling DNA mutagenesis library.
  • PCR products from the original mutagenesis library were analyzed using temperature gradient capillary electrophoresis on a REVEAL® instrument (Transgenomics Inc.), which allows PCR reactions containing heterogeneous PCR products to be distinguished from reactions containing only homogeneous products, as would be the case if a SNP existed in genomic DNA from chemical mutagenesis and subsequent PCR amplification.
  • the PCR products from the new Tilling DNA mutagenesis library were sequenced directly using an Applied Biosystems (Life Technologies) 3730 DNA sequencer using the manufacturer's recommendations .
  • the mutant Fad3A sequence is shown in SEQ ID NO: 9 and contains a mutation at position 102, changing the codon from TGG to TGA.
  • the mutant Fad3B sequence is shown in SEQ ID NO: 10 and contains a mutation at position 206, resulting in a codon change from TGG to TAG.
  • a double ha loid population was generated by collecting F ⁇ microspores from the cross, treating the microspores with colchicine, and propagating them in vitro. Plantlets formed in vitro from the microspores were moved to a greenhouse and inflorescences that formed were self pollinated. Seed was harvested from the DHi plants at maturity and analyzed for fatty acid profile. Seeds from those plants exhibiting low and high linolenic acid content were grown in the greenhouse. Table 3 contains the fatty acid profile of a bulk sample of seeds produced by each of 5-10 greenhouse-grown plants of a DHi population designated Husker.
  • a cross was made between 2558-35 (Example 1) and Dumpling-314, a double haploid B-line (maintainer) developed from a cross between IMC106RR and Jetton, a known winter rapeseed variety.
  • a double haploid population was generated as described in Example 5. Seed was harvested from the DHi plants at maturity and analyzed for fatty acid profile. Seeds from those plants exhibiting low linolenic acid content were grown in the greenhouse. Table 4 contains the fatty acid profile of a bulk sample of seeds produced by each of 10 greenhouse-grown plants of a DHi population designated Vest.
  • a hybrid canola line yielding seeds with an ALA content of less than 1.5% was produced by introducing genes from line 1904-35 (Example 1) into a commercially grown hybrid variety, Victory® vl03 .
  • Hybrid vl035 has an average oleic acid content of 65% and an ALA content of 2.8%.
  • Plants of the line 1904-35, and the inbreds 1035R and 95CB504, were planted in a greenhouse.
  • Inbred 1035R is the male parent of vl035.
  • Inbred 95CB504 is the B line female parent of vl035. Plants of 1035R and 1904-35 were cross pollinated in the greenhouse, as were 95CB504 and 1904-35, as shown in Table 5.
  • BCi-B progeny which were selfed (BCiS). Plants with low total saturates were selected from the BQ-B selfed progeny, and backcrossed to 95CB504 to produce BC 2 -B progeny. Fi progeny from the cross of 1035R and 1904-35 were backcrossed to 1035R to produce BCi-R progeny, which were selfed. Plants with low linolenic were selected from the BCi-R selfed progeny, and backcrossed to 1035R to produce BC 2 -R progeny. Backcrossing, selection, and self-pollination of the BC-B and BC-R progeny were continued for multiple generations.
  • the 95CB504 male sterile A line, 00OA05 was converted to a low linolenic phenotype in parallel with the conversion of the 95CB504 B line.
  • Table 6 shows the mean CI 8:3 content of selected lines of converted BC 3 S 5 generation parent lines compared unconverted 95CB504 and 1035R.
  • Hybrid seed was generated by hand, using BC 1 S 3 generation plants of the 95CB504 B line as the female parent and BCiS 3 generation plants of the 1035R R line as the male parent.
  • the hybrid seed was grown at 5 locations x 4 replications in Western Canada. In the trial plot locations, some individual plants were bagged for self pollination (5 locations x 2 reps) and seeds harvested at maturity. The remaining plants were not bagged (5 locations x 4 reps) and seeds were harvested in bulk. As such, the bulk samples had some level of out crossing with non-low linolenic fatty acid lines in adjacent plots. Seeds from the individual and bulk samples were analyzed for fatty acid content. Seeds from control plants of line Q2, hybrid vl035 and commercial variety 46A65 were also harvested individually and in bulk..
  • Table 7 shows the fatty acid profile of the individually bagged samples and bulked samples for hybrid 1904-35 and controls. The results indicate that seed produced by Hybrid 1904-35 has a statistically significant decrease in 18:3 content relative to the controls.

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Abstract

L'invention concerne des plantes Brassica produisant des huiles à faible teneur en acide alpha-linolénique, ainsi que des procédés de production de ces plantes.
PCT/US2011/045235 2010-05-25 2011-07-25 Plantes brassica produisant des huiles à faible teneur en acide alpha-linolénique WO2013015782A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA2836992A CA2836992A1 (fr) 2011-07-25 2011-07-25 Plantes brassica produisant des huiles a faible teneur en acide alpha-linolenique
PCT/US2011/045235 WO2013015782A1 (fr) 2011-07-25 2011-07-25 Plantes brassica produisant des huiles à faible teneur en acide alpha-linolénique
AU2011373683A AU2011373683B2 (en) 2011-07-25 2011-07-25 Brassica plants yielding oils with a low alpha linolenic acid content
US13/685,588 US9695434B2 (en) 2010-05-25 2012-11-26 Brassica plants yielding oils with a low alpha linolenic acid content

Applications Claiming Priority (1)

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PCT/US2011/045235 WO2013015782A1 (fr) 2011-07-25 2011-07-25 Plantes brassica produisant des huiles à faible teneur en acide alpha-linolénique

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US9695434B2 (en) 2010-05-25 2017-07-04 Cargill, Incorporated Brassica plants yielding oils with a low alpha linolenic acid content
WO2015087058A1 (fr) * 2013-12-10 2015-06-18 The University Of York Acides nucléiques et polypeptides de désaturases
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CN104894146A (zh) * 2015-05-26 2015-09-09 昆明理工大学 一种δ12-脂肪酸脱氢酶基因的新用途

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