Classifications

• • 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)
• • 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

Definitions

• This invention relates to fatty acid desaturases and nucleic acids encoding desaturase proteins . More particularly, the invention relates to nucleic acids encoding delta-12 and delta-15 fatty acid desaturase proteins that affect fatty acid composition in plants, polypeptides produced from such nucleic acids and plants expressing such nucleic acids.
• Alterations in fatty acid composition of vegetable oils is desirable for meeting specific food and industrial uses.
• Brassica varieties with increased monounsaturate levels (oleic acid) in the seed oil, and products derived from such oil, would improve lipid nutrition.
• Canola lines which are low in polyunsaturated fatty acids and high in oleic acid tend to have higher oxidative stability, which is a useful trait for the retail food industry.
• Delta-12 fatty acid desaturase also known as oleic desaturase is involved in the enzymatic conversion of oleic acid to linoleic acid.
• Delta-15 fatty acid desaturase (also known as linoleic acid desaturase) is involved in the enzymatic conversion of linoleic acid to ⁇ -linolenic acid.
• a microsomal delta-12 desaturase has been cloned and characterized using T-DNA tagging. Okuley, et al . , Plant Cell 6:147-158 (1994).
• the nucleotide sequences of higher plant genes encoding microsomal delta-12 fatty acid desaturase are described in Lightner et al . , W094/11516. Sequences of higher plant genes encoding microsomal and plastid delta-15 fatty acid desaturases are disclosed in Yadav, N.
• the invention comprises Brassicaceae or Helianthus seeds, plants and plant lines having at least one mutation that controls the levels of unsaturated fatty acids in plants.
• One embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a mutation from a mutant delta-12 fatty acid desaturase conferring altered fatty composition in seeds when the fragment is present in a plant.
• a preferred sequence comprises a mutant sequence as shown in Fig. 2.
• Another embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a mutation from a mutant delta-15 fatty acid desaturase.
• a plant in this embodiment may be soybean, oilseed Brassica species, sunflower, castor bean or corn.
• the mutant sequence may be derived from, for example, a Brassica napus, Brassica rapa, Brassica juncea or Helianthus delta-12 or delta-15 desaturase gene.
• Another embodiment of the invention involves a method of producing a Brassicaceae or Helianthus plant line comprising the steps of: (a) inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species; (b) obtaining progeny plants from the mutagenized cells; (c) identifying progeny plants that contain a mutation in a delta-12 or delta-15 fatty acid desaturase gene; and (d) producing a plant line by selfing or crossing. The resulting plant line may be subjected to mutagenesis in order to obtain a line having both a delta-12 desaturase mutation and a delta-15 desaturase mutation.
• Yet another embodiment of the invention involves a method of producing plant lines containing altered fatty acid composition
• a method of producing plant lines containing altered fatty acid composition comprising: (a) crossing a first plant with a second plant having a mutant delta-12 or delta-15 fatty acid desaturase; (b) obtaining seeds from the cross of step (a) ; (c) growing fertile plants from such seeds; (d) obtaining progeny seed from the plants of step (c) ; and (e) identifying those seeds among the progeny that have altered fatty acid composition.
• Suitable plants are soybean, rapeseed, sunflower, safflower, castor bean and corn.
• Preferred plants are rapeseed and sunflower.
• the invention is also embodied in vegetable oil obtained from plants disclosed herein, which vegetable oil has an altered fatty acid composition.
• SEQ ID NO:l shows a hypothetical DNA sequence of a Brassica Fad2 gene.
• SEQ ID NO : 2 is the deduced amino acid sequence of SEQ ID NO:l.
• SEQ ID NO: 3 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 316.
• SEQ ID NO: is the deduced amino acid sequence of SEQ ID NO: 3.
• SEQ ID NO: 5 shows a hypothetical DNA sequence of a Brassica Fad2 gene.
• SEQ ID NO: 6 is the deduced amino acid sequence of SEQ ID NO : 5.
• SEQ ID NO: 7 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 515.
• SEQ ID NO : 8 is the deduced amino acid sequence of SEQ ID NO: 7.
• SEQ ID NO : 9 shows the DNA sequence for the coding region of a wild type Brassica Fad2-D gene.
• SEQ ID NO: 10 is the deduced amino acid sequence for SEQ ID NO: 9.
• SEQ ID NO: 11 shows the DNA sequence for the coding region of the IMC 129 mutant Brassica Fad2-D gene.
• SEQ ID NO: 12 is the deduced amino acid sequence for SEQ ID NO: 11.
• SEQ ID NO: 13 shows the DNA sequence for the coding region of a wild type Brassica Fad2-F gene.
• SEQ ID NO: 14 is the deduced amino acid sequence for SEQ ID NO: 13.
• SEQ ID NO: 15 shows the DNA sequence for the coding region of the Q508 mutant Brassica Fad2-F gene.
• SEQ ID NO: 16 is the deduced amino acid sequence for SEQ ID NO: 15.
• SEQ ID NO: 17 shows the DNA sequence for the coding region of the Q4275 mutant Brassica Fad2-F gene.
• SEQ ID NO: 18 is the deduced amino acid sequence for SEQ ID NO:17.
• SEQ ID NOS: 19-27 show oligonucleotide sequences.
• SEQ ID NO: 28 shows the genomic DNA sequence for the Fad2-U gene from Brassica .
• SEQ ID NOS: 30-31 show genomic sequences located upstream from the start codon of Brassica Fad2-D genes.
• Figure 1 is a histogram showing the frequency distribution of seed oil oleic acid (C 18:1 ) content in a segregating population of a Q508 X Westar cross.
• the bar labeled WSGA 1A represents the C 18;1 content of the Westar parent.
• the bar labeled Q508 represents the C 18:1 content of the Q508 parent.
• Figure 2 shows the nucleotide sequences for a Brassica Fad2-D wild type gene (Fad2-D wt) , IMC129 mutant gene (Fad2-D GA316 IMC129) , Fad2-F wild type gene (Fad2-F wt) , Q508 mutant gene (Fad2-F TA515 Q508) and Q4275 mutant gene (Fad2-F GA908 Q4275) .
• Figure 3 shows the deduced amino acid sequences for the polynucleotides of Figure 2.
• fatty acids herein are percent by weight of the oil of which the fatty acid is a component.
• 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.
• mutagenesis refers to the use of a mutagenic agent to induce random genetic mutations within a population of individuals. The treated population, or a subsequent generation of that population, is then screened for usable trait (s) that result from the mutations.
• a “population” is any group of individuals that share a common gene pool.
• M 0 is untreated seed.
• M- is the seed (and resulting plants) exposed to a mutagenic agent
• M 2 is the progeny (seeds and plants) of self-pollinated M x plants
• M 3 is the progeny of self-pollinated M 2 plants
• M 4 is the progeny of self-pollinated M 3 plants.
• M 5 is the progeny of self-pollinated M 4 plants.
• M 6 is the progeny of self-pollinated plants of the previous generation.
• selfed as used herein means self -pollinated.
• Stability or “stable” as used herein means that with respect to a given fatty acid component, the component is maintained from generation to generation for at least two generations and preferably at least three generations at substantially the same level, e.g., preferably + . 5%.
• the method of invention is capable of creating lines with improved fatty acid compositions stable up to + . 5% from generation to generation.
• the above stability may be affected by temperature, location, stress and time of planting. Thus, comparison of fatty acid profiles should be made from seeds produced under similar growing conditions. Stability may be measured based on knowledge of prior generation.
• Brassica plants whose seed oil contains less than 2% erucic acid. The same varieties have also been bred so that the defatted meal contains less than 30 ⁇ mol glucosinolates/gram.
• Canola refers to plant variety seed or oil which contains less than 2% erucic acid (C 22:1 ) , and meal with less than 30 ⁇ mol glucosinolates/gram.
• Applicants have discovered plants with mutations in a delta-12 fatty acid desaturase gene. Such plants have useful alterations in the fatty acid compositions of the seed oil. Such mutations confer, for example, an elevated oleic acid content, a decreased, stabilized linoleic acid content, or both elevated oleic acid and decreased, stabilized linoleic acid content. Applicants have further discovered plants with mutations in a delta-15 fatty acid desaturase gene. Such plants have useful alterations in the fatty acid composition of the seed oil, e.g., a decreased, stabilized level of c_-linolenic acid.
• nucleic acid fragments comprising sequences that carry mutations within the coding sequence of delta-12 or delta-15 fatty acid desaturases .
• the mutations confer desirable alterations in fatty acid levels in the seed oil of plants carrying such mutations.
• Delta-12 fatty acid desaturase is also known as omega-6 fatty acid desaturase and is sometimes referred to herein as Fad2 or 12-DES.
• Delta-15 fatty acid desaturase is also known on omega-3 fatty acid desaturase and is sometimes referred to herein as Fad3 or 15-DES.
• a nucleic acid fragment of the invention may be in the form of RNA or in the form of DNA, including cDNA, synthetic DNA or genomic DNA.
• the DNA may be double- stranded or single-stranded, and if single-stranded, can be either the coding strand or non-coding strand.
• An RNA analog may be, for example, mRNA or a combination of ribo- and deoxyribonucleotides .
• Illustrative examples of a nucleic acid fragment of the invention are the mutant sequences shown in Fig. 3.
• a nucleic acid fragment of the invention contains a mutation in a microsomal delta-12 fatty acid desaturase coding sequence or a mutation in a microsomal delta-15 fatty acid desaturase coding sequence.
• Such a mutation renders the resulting desaturase gene product nonfunctional in plants, relative to the function of the gene product encoded by the wild-type sequence.
• the non- functionality of the delta-12 desaturase gene product can be inferred from the decreased level of reaction product (linoleic acid) and increased level of substrate (oleic acid) in plant tissues expressing the mutant sequence, compared to the corresponding levels in plant tissues expressing the wild-type sequence.
• a nucleic acid fragment of the invention may comprise a portion of the coding sequence, e.g., at least about 10 nucleotides, provided that the fragment contains at least one mutation in the coding sequence.
• the length of a desired fragment depends upon the purpose for which the fragment will be used, e.g., PCR primer, site- directed mutagenesis and the like.
• a nucleic acid fragment of the invention comprises the full length coding sequence of a mutant delta-12 or mutant delta-15 fatty acid desaturase, e.g., the mutant sequences of Fig. 3.
• a nucleic acid fragment is about 20 to about 50 nucleotides (or base pairs, bp) , or about 50 to about 500 nucleotides, or about 500 to about 1200 nucleotides in length.
• the invention relates to an isolated nucleic acid fragment of at least 50 nucleotides in length that has at least 70% sequence identity to the nucleotide sequences of SEQ ID NO: 30 or SEQ ID NO -.31. In some embodiments, such nucleic acid fragments have at least 80% or 90% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 31. Sequence identity for these and other nucleic acids disclosed herein can be determined, for example, using Blast 2.0.4 (Feb. 24, 1998) to search the nr database (non-redundant GenBank, EMBL, DDBT and PDB) .
• BLAST 2.0.4 is provided by the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov). Altschul, S.F. et al., Nucleic Acids Res., 25:3389-3402 (1997).
• MEGALIGN ® DNASTAR, Madison, WI sequence alignment software can be used to determine sequence identity by the Clustal algorithm. In this method, sequences are grouped into clusters by examining the distance between all pairs. Clusters are aligned pairwise, then as groups. The Jotun Hem algorithm is also available in MEGALIGN ® . The nucleotide sequences of SEQ ID NO:30 and NO:31 are about 85% identical using the Clustal algorithm with default parameters.
• the nucleotide sequences of SEQ ID NO: 30 and SEQ ID NO: 31 are located upstream of the ATG start codon for the fad2-D gene and can be isolated from Bridger and Westar canola plants, respectively. These upstream elements contain intron-like features.
• the invention also relates to an isolated nucleic acid fragment that includes a sequence of at least 200 nucleotides.
• the fragment has at least 70% identity to nucleotides 1 to about 1012 of SEQ ID NO: 28.
• the fragment has 80% or at least 90% sequence identity to nucleotides 1 to about 1012 of SEQ ID NO: 28.
• This portion of SEQ ID NO: 28 is located upstream of the ATG start codon and has intron-like features .
• a mutation in a nucleic acid fragment of the invention may be in any portion of the coding sequence that renders the resulting gene product non-functional.
• Suitable types of mutations include, without limitation, insertions of nucleotides, deletions of nucleotides, or transitions and transversions in the wild-type coding sequence. Such mutations result in insertions of one or more amino acids, deletions of one or more amino acids, and non-conservative amino acid substitutions in the corresponding gene product.
• the sequence of a nucleic acid fragment may comprise more than one mutation or more than one type of mutation.
• Insertion or deletion 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 or deletions may also disrupt binding or 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 non- functional , 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 alanyl residue for a isoleucyl 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. Because there are only 20 amino acids encoded in a gene, substitutions that result in a non- functional gene product may be determined by routine experimentation, incorporating amino acids of a different class in the region of the gene product targeted for mutation.
• Preferred mutations are in a region of the nucleic acid encoding an amino acid sequence motif that is conserved among delta-12 fatty acid desaturases or delta- 15 fatty acid desaturases, such as a His-Xaa-Xaa-Xaa-His motif (Tables 1-3) .
• An example of a suitable region has a conserved HECGH motif that is found, for example, in nucleotides corresponding to amino acids 105 to 109 of the Arabidopsis and Brassica delta-12 desaturase sequences, in nucleotides corresponding to amino acids 101 to 105 of the soybean delta-12 desaturase sequence and in nucleotides corresponding to amino acids 111 to 115 of the maize delta-12 desaturase sequence.
• An illustrative embodiment of a mutation in a nucleic acid fragment of the invention is a Glu to Lys substitution in the HECGH motif of a Brassica microsomal delta-12 desaturase sequence, either the D form or the F form.
• This mutation results in the sequence HECGH being changed to HKCGH as seen by comparing SEQ ID NO: 10 (wild- type D form) to SEQ ID NO: 12 (mutant D form) .
• a similar mutation in other Fad-2 sequences is contemplated to result in a non-functional gene product. (Compare SEQ ID NO: 2 to SEQ ID NO : 4 ) .
• a similar motif may be found at amino acids 101 to 105 of the Arabidopsis microsomal delta-15 fatty acid desaturase, as well as in the corresponding rape and soybean desaturases (Table 5). See, e.g., WO 93/11245; Arondel, V. et al . , Science, 258:1153-1155 (1992); Yadav, N. et al., Plant Physiol . , 103:467-476 (1993). Plastid delta-15 fatty acids have a similar motif (Table 5) .
• non-conservative substitutions is substitution of a glycine residue for either the first or second histidine. Such a substitution replaces a charged residue
• glycine with a non-polar residue (glycine)
• Another type of mutation that renders the resulting gene product non- functional is an insertion mutation, e.g., insertion of a glycine between the cysteine and glutamic acid residues in the HECGH motif.
• Other regions having suitable conserved amino acid motifs include the HRRHH motif shown in Table 2, the HRTHH motif shown in Table 6 and the HVAHH motif shown in Table 3. See, e.g., WO 94/115116; Hitz, W. et al . , Plant Physiol., 105:635-641 (1994); Okuley, J., et al . , supra; and Yadav, N. et al .
• An illustrative example of a mutation in the region shown in Table 3 is a mutation at nucleotides corresponding to the codon for glycine (amino acid 303 of B . napus) .
• a non-conservative Gly to Glu substitution results in the amino acid sequence
• DRDYGILNKV being changed to sequence DRDYEILNKV (compare wild-type F form SEQ ID NO: 14 to mutant Q4275 SEQ ID NO: 18, Fig. 3) .
• Another region suitable for a mutation in a delta- 12 desaturase sequence contains the motif KYLNNP at nucleotides corresponding to amino acids 171 to 175 of the Brassica desaturase sequence.
• An illustrative example of a mutation is this region is a Leu to His substitution, resulting in the amino acid sequence (Table 4) KYHNN (compare wild-type Fad2-F SEQ ID NO: 14 to mutant SEQ ID NO: 16) .
• a similar mutation in other Fad-2 amino acid sequences is contemplated to result in a nonfunctional gene product. (Compare SEQ ID NO : 6 to SEQ ID NO: 8) .
• Brassica napus D 100-128 VWVIAHECGH HAFSDYQWLD DTVGLIFHS
• Brassica napus F 100-128 VWVIAHECGH HAFSDYQWLD DTVGLIFHS from plasmid pRF2-lC
• nucleic acid fragment containing a mutant sequence can be generated by techniques known to the skilled artisan. Such techniques include, without limitation, site-directed mutagenesis of wild-type sequences and direct synthesis using automated DNA synthesizers.
• a nucleic acid fragment containing a mutant sequence can also be generated by mutagenesis of plant seeds or regenerable plant tissue by, e.g., ethyl methane sulfonate, X-rays or other mutagens.
• mutant plants having the desired fatty acid phenotype in seeds are identified by known techniques and a nucleic acid fragment containing the desired mutation is isolated from genomic DNA or RNA of the mutant line.
• the site of the specific mutation is then determined by sequencing the coding region of the delta-12 desaturase or delta-15 desaturase gene.
• labeled nucleic acid probes that are specific for desired mutational events can be used to rapidly screen a mutagenized population.
• the disclosed method may be applied to all oilseed Brassica species, and to both Spring and Winter maturing types within each species.
• Physical mutagens including but not limited to X-rays, UV rays, and other physical treatments which cause chromosome damage, and other chemical mutagens, including but not limited to ethidium bromide, nitrosoguanidine, diepoxybutane etc. may also be used to induce mutations.
• the mutagenesis treatment may also be applied to other stages of plant development, including but not limited to cell cultures, embryos, microspores and shoot apices. "Stable mutations" as used herein are defined as
• M 5 or more advanced lines which maintain a selected altered fatty acid profile for a minimum of three generations, including a minimum of two generations under field conditions, and exceeding established statistical thresholds for a minimum of two generations, as determined by gas chromatographic analysis of a minimum of 10 randomly selected seeds bulked together.
• stability may be measured in the same way by comparing to subsequent generations. In subsequent generations, stability is defined as having similar fatty acid profiles in the seed as that of the prior or subsequent generation when grown under substantially similar conditions.
• Mutation breeding has traditionally produced plants carrying, in addition to the trait of interest, multiple, deleterious traits, e.g., reduced plant vigor and reduced fertility. Such traits may indirectly affect fatty acid composition, producing an unstable mutation; and/or reduce yield, thereby reducing the commercial utility of the invention.
• a low mutagen dose is used in the seed treatments to create an LD30 population. This allows for the rapid selection of single gene mutations for fatty acid traits in agronomic backgrounds which produce acceptable yields.
• each form may be derived from one of the parent genomes making up the species under consideration. Plants with mutations in both forms have a fatty acid profile that differs from plants with a mutation in only one form.
• An example of such a plant is Brassica napus line Q508 , a doubly-mutagenized line containing a mutant D-form of delta-12 desaturase (SEQ ID NO: 11) and a mutant F-form of delta-12 desaturase (SEQ ID NO: 15) .
• line Q4275 which contains a mutant D-form of delta-12 desaturase (SEQ ID NO: 11) and a mutant F-form of delta-12 desaturase (SEQ ID NO:17). See Figs. 2-3.
• Preferred host or recipient organisms for introduction of a nucleic acid fragment of the invention are the oil-producing species, such as soybean ⁇ Glycine max) , rapeseed (e.g., Brassica napus , B . rapa and B . j uncea) , sunflower (Helianthus annus) , castor bean ⁇ Ri cinus communi s) , corn ( Zea mays) , and safflower ( Carthamus tinctorius) .
• a nucleic acid fragment of the invention may further comprise additional nucleic acids.
• a nucleic acid encoding a secretory or leader amino acid sequence can be linked to a mutant desaturase nucleic acid fragment such that the secretory or leader sequence is fused in- frame to the amino terminal end of a mutant delta-12 or delta-15 desaturase polypeptide.
• Other nucleic acid fragments are known in the art that encode amino acid sequences useful for fusing in- frame to the mutant desaturase polypeptides disclosed herein. See, e.g., U.S. 5,629,193 incorporated herein by reference.
• a nucleic acid fragment may also have one or more regulatory elements operably linked thereto.
• the present invention also comprises nucleic acid fragments that selectively hybridize to mutant desaturase sequences.
• a nucleic acid fragment typically is at least 15 nucleotides in length.
• Hybridization typically involves Southern analysis (Southern blotting) , a method by which the presence of DNA sequences in a target nucleic acid mixture are identified by hybridization to a labeled oligonucleotide or DNA fragment probe.
• Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzyme-labeled probe as described in sections 9.37-
• a nucleic acid fragment can hybridize under moderate stringency conditions or, preferably, under high stringency conditions to a mutant desaturase sequence.
• High stringency conditions are used to identify nucleic acids that have a high degree of homology to the probe.
• High stringency conditions can include the use of low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (0.1X SSC); 0.1% sodium lauryl sulfate (SDS) at 50-65°C.
• a denaturing agent such as formamide can be employed during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl , 75 mM sodium citrate at 42°C.
• Another example is the use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42 °C in 0.2 x SSC and 0.1% SDS.
• Moderate stringency conditions refers to hybridization conditions used to identify nucleic acids that have a lower degree of identity to the probe than do nucleic acids identified under high stringency conditions.
• Moderate stringency conditions can include the use of higher ionic strength and/or lower temperatures for washing of the hybridization membrane, compared to the ionic strength and temperatures used for high stringency hybridization.
• a wash solution comprising 0.060 M NaCl/0.0060 M sodium citrate (4X SSC) and 0.1% sodium lauryl sulfate (SDS) can be used at 50°C, with a last wash in IX SSC, at 65°C.
• a hybridization wash in IX SSC at 37°C can be used.
• Hybridization can also be done by Northern analysis (Northern blotting) , a method used to identify RNAs that hybridize to a known probe such as an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment .
• the probe is labeled with a radioisotope such as 3 P, by biotinylation or with an enzyme.
• the RNA to be analyzed can be usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al . , supra .
• a polypeptide of the invention comprises an isolated polypeptide having a mutant amino acid sequence, as well as derivatives and analogs thereof. See, e.g., the mutant amino acid sequences of Fig. 3.
• isolated is meant a polypeptide that is expressed and produced in an environment other than the environment in which the polypeptide is naturally expressed and produced. For example, a plant polypeptide is isolated when expressed and produced in bacteria or fungi.
• a polypeptide of the invention also comprises variants of the mutant desaturase polypeptides disclosed herein, as discussed above .
• a plant contains both a delta-12 desaturase mutation and a delta-15 desaturase mutation.
• Such plants can have a fatty acid composition comprising very high oleic acid and very low alpha-linolenic acid levels.
• Mutations in delta-12 desaturase and delta-15 desaturase may be combined in a plant by making a genetic cross between delta-12 desaturase and delta-15 desaturase single mutant lines.
• a plant having a mutation in delta-12 fatty acid desaturase is crossed or mated with a second plant having a mutation in delta-15 fatty acid desaturase. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds are then screened in order to identify those seeds carrying both mutant genes.
• a line possessing either a delta-12 desaturase or a delta-15 desaturase mutation can be subjected to mutagenesis to generate a plant or plant line having mutations in both delta-12 desaturase and delta-15 desaturase.
• the IMC 129 line has a mutation in the coding region (Glu 106 to Lys 106 ) of the D form of the microsomal delta-12 desaturase structural gene.
• Cells (e.g., seeds) of this line can be mutagenized to induce a mutation in a delta-15 desaturase gene, resulting in a plant or plant line carrying a mutation in a delta-12 fatty acid desaturase gene and a mutation in a delta-15 fatty acid desaturase gene.
• Progeny includes descendants of a particular plant or plant line, e.g., seeds developed on an instant plant are descendants.
• Progeny of an instant plant include seeds formed on F 1# F 2 , F 3 , and subsequent generation plants, or seeds formed on BC 1# BC 2 , BC 3 and subsequent generation plants.
• Plants according to the invention preferably contain an altered fatty acid composition.
• oil obtained from seeds of such plants may have from about 69 to about 90% oleic acid, based on the total fatty acid composition of the seed. Such oil preferably has from about 74 to about 90% oleic acid, more preferably from about 80 to about 90% oleic acid.
• oil obtained from seeds produced by plants of the invention may have from about 2.0% to about 5.0% saturated fatty acids, based on total fatty acid composition of the seeds.
• oil obtained from seeds of the invention may have from about 1.0% to about 14.0% linoleic acid, or from about 0.5% to about 10.0% c.-linolenic acid.
• Oil composition typically is analyzed by crushing and extracting fatty acids from bulk seed samples (e.g., 10 seeds) .
• Fatty acid triglycerides in the seed are hydrolyzed and converted to fatty acid methyl esters .
• Those seeds having an altered fatty acid composition may be identified by techniques known to the skilled artisan, e.g., gas-liquid chromatography (GLC) analysis of a bulked seed sample or of a single half-seed.
• GLC gas-liquid chromatography
• Half-seed analysis is well known in the art to be useful because the viability of the embryo is maintained and thus those seeds having a desired fatty acid profile may be planted to from the next generation.
• half-seed analysis is also known to be an inaccurate representation of genotype of the seed being analyzed.
• Fatty acid composition can also be determined on larger samples, e.g., oil obtained by pilot plant or commercial scale refining, bleaching and deodorizing of endogenous oil in the seeds .
• the nucleic acid fragments of the invention can be used as markers in plant genetic mapping and plant breeding programs. Such markers may include restriction fragment length polymorphism (RFLP) , random amplification polymorphism detection (RAPD) , polymerase chain reaction (PCR) or self-sustained sequence replication (3SR) markers, for example. Marker-assisted breeding techniques may be used to identify and follow a desired fatty acid composition during the breeding process.
• RFLP restriction fragment length polymorphism
• RAPD random amplification polymorphism detection
• PCR polymerase chain reaction
• 3SR self-sustained sequence replication
• 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 delta-12 desaturase or delta-15 desaturase.
• Methods according to the invention are useful in that the resulting plants and plant lines have desirable seed fatty acid compositions as well as superior agronomic properties compared to known lines having altered seed fatty acid composition.
• Superior agronomic characteristics include, for example, increased seed germination percentage, increased seedling vigor, increased resistance to seedling fungal diseases (damping off, root rot and the like) , increased yield, and improved standabilit .
• M 2 seed from individual plants were individually catalogued and stored, approximately 15,000 M 2 lines was planted in a summer nursery in Carman, Manitoba.
• the seed from each selfed plant were planted in 3 -meter rows with 6 -inch row spacing.
• Westar was planted as the check variety. Selected lines in the field were selfed by bagging the main raceme of each plant. At maturity, the selfed plants were individually harvested and seeds were catalogued and stored to ensure that the source of the seed was known.
• the selected M 3 seeds were planted in the greenhouse along with Westar controls. The seed was sown in 4-inch pots containing Pro-Mix soil and the plants were maintained at 25°C/15°C, 14/10 hr day/night cycle in the greenhouse. At flowering, the terminal raceme was self-pollinated by bagging. At maturity, selfed M 4 seed was individually harvested from each plant, labelled, and stored to ensure that the source of the seed was known. The M 4 seed was analyzed in 10-seed bulk samples. Statistical thresholds for each fatty acid component were established from 259 control samples using a Z- distribution of 1 in 800. Selected M 4 lines were planted in a field trial in Carman, Manitoba in 3 -meter rows with 6-inch spacing.
• dihaploid populations were made from the microspores of the F- L hybrids. Self-pollinated seed from dihaploid plants were analyzed for fatty acid analysis using methods described previously.
• 10-seed bulk samples were hand ground with a glass rod in a 15-mL polypropylene tube and extracted in 1.2 mL 0.25 N KOH in 1:1 ether/methanol .
• the sample was vortexed for 30 sec. and heated for 60 sec. in a 60°C water bath.
• Four mL of saturated NaCl and 2.4 mL of iso-octane were added, and the mixture was vortexed again.
• 600 ⁇ L of the upper organic phase were pipetted into individual vials and stored under nitrogen at -5°C.
• One ⁇ L samples were injected into a Supelco SP-2330 fused silica capillary column (0.25 mm ID, 30 M length, 0.20 ⁇ m df) .
• the gas chromatograph was set at 180°C for 5.5 minutes, then programmed for a 2°C/minute increase to 212 °C, and held at this temperature for 1.5 minutes. Total run time was 23 minutes. Chromatography settings were: Column head pressure - 15 psi, Column flow (He) - 0.7 mL/min., Auxiliary and Column flow - 33 mL/min. , Hydrogen flow - 33 mL/min., Air flow - 400 mL/min., Injector temperature - 250°C, Detector temperature - 300°C, Split vent - 1/15.
• Table 8 describes the upper and lower statistical thresholds for each fatty acid of interest .
• EXAMPLE 2 High Oleic Acid Canola Lines In the studies of Example 1, at the M 3 generation, 31 lines exceeded the upper statistical threshold for oleic acid (> 71.0%) . Line W7608.3 had 71.2% oleic acid. At the M 4 generation, its selfed progeny (W7608.3.5, since designated A129.5) continued to exceed the upper statistical threshold for C 18:1 with 78.8% oleic acid. M 5 seed of five self -pollinated plants of line A129.5 (ATCC 40811) averaged 75.0% oleic acid. A single plant selection, A129.5.3 had 75.6% oleic acid.
• the fatty acid composition of this high oleic acid mutant which was stable under both field and greenhouse conditions to the M 7 generation, is summarized in Table 9. This line also stably maintained its mutant fatty acid composition to the M 7 generation in field trials in multiple locations. Over all locations the self-pollinated plants (A129) averaged 78.3% oleic acid. The fatty acid composition of the A129 for each Idaho trial location are summarized in Table 10. In multiple location replicated yield trials, A129 was not significantly different in yield from the parent cultivar Westar.
• the canola oil of A129 after commercial processing, was found to have superior oxidative stability compared to Westar when measured by the Accelerated Oxygen Method (AOM) , American Oil Chemists' Society Official Method Cd 12-57 for fat stability; Active Oxygen Method (revised 1989) .
• AOM Accelerated Oxygen Method
• Westar was 18 AOM hours and for A129 was 30 AOM hours.
• Oleic Acid Canola Line Produced by Seed Mutagenesis
• Genotype C 16 0 C 18 : 0 C 18 : 1 C 18 : 2 C 18 : 3 Sat s
• An additional high oleic acid line designated A128.3, was also produced by the disclosed method.
• a 50- seed bulk analysis of this line showed the following fatty acid composition: C 16 . 3.5-6 , C ⁇ - 1.8-s , C 18:1 - 77.3%, C 18:2 - 9.0%, C 18:3 - 5.6%, FDA Sats - 5.3%, Total Sats - 6.4%.
• This line also stably maintained its mutant fatty acid composition to the M 7 generation. In multiple locations replicated yield trials, A128 was not significantly different in yield from the parent cultivar Westar.
• A129 was crossed to A128.3 for allelism studies.
• Fatty acid composition of the F 2 seed showed the two lines to be allelic.
• the mutational events in A129 and A128.3 although different in origin were in the same gene.
• An additional high oleic acid line, designated M3028.-10 was also produced by the disclosed method in Example 1.
• a 10-seed bulk analysis of this line showed the following fatty acid composition: -> C 16:0 - 3.5-s, 18:0 - 1.8 -s , C 18;1 - 77.3-s, C 18:2 - 9.0-s, C 18 . 3 - 5.6%, FDA Saturates - 5.3%, Total Saturates - 6.4%.
• M3028.10 was not significantly different in yield from the parent cultivar Westar.
• An additional low 5 linoleic acid line designated M3062.8 (ATCC 75025), was also produced by the disclosed method.
• a 10-seed bulk analysis of this line showed the following fatty acid composition: C 16:0 - 3.8%, C 18:0 - 2.3%, C 18;1 - 77.1%, C 18:2 - 8.9%, C 18:3 - 4.3%, FDA Sats-6.1%.
• This line has also 0 stably maintained its mutant fatty acid composition in the field and greenhouse. TABLE 13
• Linoleic Acid Canol a Line Produced by Seed Mutagenesis
• Linolenic Acid Canola Line Produced by Seed Mutagenesis
• Seeds of the B . napus line IMC-129 were mutagenized with methyl N-nitrosoguanidine (MNNG) .
• MNNG methyl N-nitrosoguanidine
• the MNNG treatment consisted of three parts: pre-soak, mutagen application, and wash.
• a 0.05M Sorenson's phosphate buffer was used to maintain pre-soak and mutagen treatment pH at 6.1.
• Two hundred seeds were treated at one time on filter paper (Whatman #3M) in a petri dish (100mm x 15mm) .
• the seeds were pre-soaked in 15 mis of 0.05M Sorenson's buffer, pH 6.1, under continued agitation for two hours. At the end of the pre-soak period, the buffer was removed from the plate.
• the seeds were washed with three changes of distilled water at 10 minute intervals. The fourth wash was for thirty minutes. This treatment regime produced an LD60 population.
• Treated seeds were planted in standard greenhouse potting soil and placed into an environmentally controlled greenhouse. The plants were grown under sixteen hours of light. At flowering, the racemes were bagged to produce selfed seed. At maturity, the M2 seed was harvested. Each M2 line was given an identifying number. The entire MNNG-treated seed population was designated as the Q series.
• Harvested M2 seeds was planted in the greenhouse. The growth conditions were maintained as previously described. The racemes were bagged at flowering for selfing. At maturity, the selfed M3 seed was harvested and analyzed for fatty acid composition. For each M3 seed line, approximately 10-15 seeds were analyzed in bulk as described in Example 1.
• High oleic-low linoleic M3 lines were selected from the M3 population using a cutoff of >82% oleic acid and ⁇ 5.0% linoleic. From the first 1600 M3 lines screened for fatty acid composition, Q508 was identified. The Q508 M3 generation was advanced to the M4 generation in the greenhouse. Table 15 shows the fatty acid composition of Q508 and IMC 129. The M4 selfed seed maintained the selected high oleic-low linoleic acid phenotype (Table 16) .
• Fatty acid composition of A129 is the average of 50 self -pollinated plants grown with the M3 population M 4 generation Q508 plants had poor agronomic qualities in the field compared to Westar. Typical plants were slow growing relative to Westar, lacked early vegetative vigor, were short in stature, tended to be chlorotic and had short pods. The yield of Q508 was very low compared to Westar.
• the M 4 generation Q508 plants in the greenhouse tended to be reduced in vigor compared to Westar. However, Q508 yields in the greenhouse were greater than Q508 yields in the field.
• M4 high-oleic low-linoleic lines were also identified: Q3603, Q3733, Q4249, Q6284, Q6601, Q6761, Q7415, Q4275, and Q6676. Some of these lines had good agronomic characteristics and an elevated oleic acid level in seeds of about 80% to about 84%.
• Q4275 was crossed to the variety Cyclone. After selfing for seven generations, mature seed was harvested from 93GS34-179, a progeny line of the Q4275 Cyclone cross. Referring to Table 17, fatty acid composition of a bulk seed sample shows that 93GS34 retained the seed fatty acid composition of Q4275. 93GS34-179 also maintained agronomically desirable characteristics.
• F 2 seed of the 92EF population was planted in the greenhouse to analyze the genetics of the Q508 line.
• F 3 seed was analyzed from 380 F2 individuals.
• the C 18:1 levels of F 3 seed from the greenhouse experiment is depicted in Figure 1.
• the data were tested against the hypothesis that Q508 contains two mutant genes that are semi -dominant and additive: the original IMC 129 mutation as well as one additional mutation.
• the hypothesis also assumes that homozygous Q508 has greater than 85% oleic acid and homozygous Westar
• the fatty acid composition of representative F3 individuals having greater than 85% oleic acid in seed oil is shown in Table 18.
• the levels of saturated fatty acids are seen to be decreased in such plants, compared to Westar. TABLE 18
• Plants of Q508, IMC 129 and Westar were grown in the greenhouse. Mature leaves, primary expanding leaves, petioles and roots were harvested at the 6-8 leaf stage, frozen in liquid nitrogen and stored at -70°C. Lipid extracts were analyzed by GLC as described in Example 1. The fatty acid profile data are shown in Table 19.
• RNA from seeds of IMC 129, Q508 and Westar plants was isolated by standard methods and was used as template.
• the RT-amplified fragments were used for nucleotide sequence determination.
• the DNA sequence of each gene from each line was determined from both strands by standard dideoxy sequencing methods .
• Q508 line was derived from IMC 129. The same base change was also detected in Q508 and IMC 129 when RNA from leaf tissue was used as template.
• the G to A mutation at nucleotide 316 was confirmed by sequencing several independent clones containing fragments amplified directly from genomic DNA of IMC 129 and Westar. These results eliminated the possibility of a rare mutation introduced during reverse transcription and PCR in the RT-PCR protocol. It was concluded that the IMC 129 mutant is due to a single base transversion at nucleotide 316 in the coding region of the D gene of rapeseed microsomal delta 12 -desaturase .
• the mutation in the D gene of IMC 129 and Q508 mapped to a region having a conserved amino acid motif (His-Xaa-Xaa-Xaa-His) found in cloned delta-12 and delta- 15 membrane bound-desaturases (Table 20) .
• the primers used to specifically amplify delta-12 desaturase F gene RNA from the indicated tissues were sense primer 5 ' -GGATATGATGATGGTGAAAGA-3 ' and antisense primer 5 ' -TCTTTCACCATCATCATATCC-3 ' .
• the primers used to specifically amplify delta-12 desaturase D gene RNA from the indicated tissues were sense primer 5' -GTTATGAAGCAAAGAAGAAAC-3' and antisense primer 5'- GTTTCTTCTTTGCTTCATAAC-3' .
• RNA of both the D and F gene was expressed in seed and leaf tissues of IMC 129, Q508 and wild type Westar plants .
• vi tro transcription and translation analysis showed that a peptide of about 46 kD was made. This is the expected size of both the D gene product and the F gene product, based on sum of the deduced amino acid sequence of each gene and the cotranslational addition of a microsomal membrane peptide.
• PCR primers Based on the single base change in the mutant D gene of IMC 129 described in above, two 5' PCR primers were designed. The nucleotide sequence of the primers differed only in the base (G for Westar and A for IMC 129) at the 3' end. The primers allow one to distinguish between mutant fad2-D and wild-type Fad2-D alleles in a DNA-based PCR assay. Since there is only a single base difference in the 5' PCR primers, the PCR assay is very sensitive to the PCR conditions such as annealing temperature, cycle number, amount, and purity of DNA templates used. Assay conditions have been established that distinguish between the mutant gene and the wild type gene using genomic DNA from IMC 129 and wild type plants as templates.
• Conditions may be further optimized by varying PCR parameters, particularly with variable crude DNA samples.
• a PCR assay distinguishing the single base mutation in IMC 129 from the wild type gene along with fatty acid composition analysis provides a means to simplify segregation and selection analysis of genetic crosses involving plants having a delta-12 fatty acid desaturase mutation.
• the first plasmid, pIMCHO was prepared by inserting into a disarmed Ti vector the full length wild type Fad3 gene in sense orientation (nucleotides 208 to 1336 of SEQ ID 6 in WO 93/11245) , flanked by a napin promoter sequence positioned 5' to the Fad3 gene and a napin termination sequence positioned 3' to the Fad3 gene.
• the rapeseed napin promoter is described in EP 0255378.
• the second plasmid, pIMC205 was prepared by inserting a mutated Fad3 gene in sense orientation into a disarmed Ti vector.
• the mutant sequence contained mutations at nucleotides 411 and 413 of the microsomal Fad3 gene described in W093/11245, thus changing the sequence for codon 96 from GAC to AAG.
• the amino acid at codon 96 of the gene product was thereby changed from aspartic acid to lysine. See Table 20.
• the phaseolin sequence is described in Doyle et al . , (1986) J. Biol. Chem. 261:9228-9238) and Slightom et al . , (1983) Proc. Natl. Acad. Sci. USA 80:1897-1901.
• the appropriate plasmids were engineered and transferred separately to Agrobacterium strain LBA4404. Each engineered strain was used to infect 5 mm segments of hypocotyl explants from Westar seeds by cocultivation. Infected hypocotyls were transferred to callus medium and, subsequently, to regeneration medium. Once discernable stems formed from the callus, shoots were excised and transferred to elongation medium. The elongated shoots were cut, dipped in RootoneTM, rooted on an agar medium and transplanted to potting soil to obtain fertile TI plants. T2 seeds were obtained by selfing the resulting TI plants.
• Lines 652- 09 and 663-40 are representative of plants containing pIMCHO and exhibiting an overexpression and a co- suppression phenotype, respectively.
• Line 205-284 is representative of plants containing pIMC205 and having the mutant fad3 gene.
• Fad2-D and Fad2-F High molecular weight genomic DNA was isolated from leaves of Q4275 plants (Example 5) and from Westar and Bridger canola plants. This DNA was used as template for amplification of Fad2-D and Fad2-F genes by polymerase chain reaction (PCR) . PCR amplifications were carried out in a total volume of 100 ⁇ l and contained 0.3 ⁇ g genomic DNA, 200 ⁇ M deoxyribonucleoside triphosphates, 3 mM MgS0 4 , 1-2 Units DNA polymerase and IX Buffer (supplied by the DNA polymerase manufacturer) . Cycle conditions were: 1 cycle for 1 min at 95°C, followed by 30 cycles of 1 min at 94°C, 2 min at 55°C and 3 min at 73°C. The Fad2-D gene was amplified once using Elongase ®
• PCR primers were:
• CAUCAUCAUCAUCTTCTTCGTAGGGTTCATCG SEQ ID NO: 23
• CUACUACUACUATCATAGAAGAGAAAGGTTCAG SEQ ID NO: 24
• the Fad2-F gene was independently amplified 4 times, twice with Elongase ® and twice with Taq polymerase (Boehringer Mannheim) .
• the PCR primers used were: 5 ' CAUCAUCAUCAUCATGGGTGCACGTGGAAGAA3 ' (SEQ ID NO: 25) and 5 ' CUACUACUACUATCTTTCACCATCATCATATCC3 ' (SEQ ID NO: 26) for the 5' and 3' ends of the gene, respectively.
• Amplified DNA products were resolved on an agarose gel, purified by JetSorb ® and then annealed into pAMPl (Gibco-BRL) via the (CAU) 4 and (CUA) 4 sequences at the ends of the primers, and transformed into E. coli DH5 ⁇ .
• the Fad2-D and Fad2-F inserts were sequenced on both strands with an ABI PRISM 310 automated sequencer (Perkin-Elmer) following the manufacturer's directions, using synthetic primers, AmpliTaq ® DNA polymerase and dye terminator.
• the Fad2-D gene was found to have intron-like sequences upstream of the ATG start codon (SEQ ID NO: 30 and SEQ ID NO: 31) .
• the coding sequence of the gene derived from IMC 129 contained a G to A mutation at nucleotide 316 (Fig. 2) .
• a single base transversion from G to A at nucleotide 908 was detected in the F gene sequence of the Q4275 amplified products, compared to the wild type F gene sequence (Fig. 2) .
• This mutation changes the codon at amino acid 303 from GGA to GAA, resulting in the non- conservative substitution of a glutamic acid residue for a glycine residue (Table 3 and Fig. 3) .
• Expression of the mutant Q4275 Fad2-F delta-12 desaturase gene in plants alters the fatty acid composition, as described hereinabove .
• Fad2-U High molecular weight genomic DNA was isolated from the leaves of Bridger and Westar Brassica plants by standard methods.
• the Fad2-U gene was amplified in a 100 ⁇ l total reaction containing 1 ⁇ M of each primer, 0.3 ⁇ g genomic DNA, 200 ⁇ M dNTP, 3 mM MgS0 4 , lx Buffer (supplied by the manufacturer of the DNA polymerase) , and 1-2 units of Elongase DNA polymerase (BRL) .
• the amplification conditions included one cycle for 1 min at 95°C, 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and elongation at 72°C for 3 min. Subsequently, the reaction was incubated at 72 °C for an additional 10 min. Fad2U gene was amplified twice from Westar and twice from Bridger genomic DNAs using the following primers:
• ADDRESSEE Fish & Richardson P.C., P. A.
• MOLECULE TYPE Genomic DNA
• FEATURE :
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO: 19: GGATATGATG ATGGTGAAAG A 21
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO: 20: TCTTTCACCA TCATCATATC C 21
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO:21: GTTATGAAGC AAAGAAGAAA C 21
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO:22: GTTTCTTCTT TGCTTTGCTT CATAAC 26
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO:24: CUACUACUAC UATCATAGAA GAGAAAGGTT CAG 33
• MOLECULE TYPE Genomic DNA
• FEATURE

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