PLANTS HAVING MUTANT SEQUENCES THAT CONFER ALTERED FATTY ACID PROFILES

Abstract: Seeds, plants and oils are provided having low FDA saturates; high oleic acid; low linoleic acid; high or low palmitic acid; low stearic acid; and low linoleic acid plus linolenic acid; and advantageous functional or nutritional properties. Plants are disclosed that contain a mutation in a delta-12 or delta-15 fatty acid desaturase gene. Preferred plants are rapeseed and sunflower plants. Plants carrying such mutant genes have altered fatty acid composition in seeds. In one embodiment, a plant contains a mutation in a region having the conserved motif His-Xaa-Xaa-Xaa-His, found in delta-12 and delta-15 fatty acid desaturases. A preferred motif has the sequence His-Glu-Cys-Gly-His. A preferred mutation in this motif has the amino acid sequence His-Lys-Cys-Gly-His. Nucleic acid fragments are disclosed that comprise a mutant delta-12 or delta-15 fatty acid desaturase gene sequence.

Description: PLANTS HAVING MUTANT SEQUENCES THAT CONFER ALTERED FATTY ACID PROFILES Technical Field This invention relates to Brassica seeds and plants having mutant sequences which confer altered fatty acid profiles on the seed oil. More particularly, the invention relates to mutant delta-12 and delta-15 fatty acid desaturase sequences in such plants which confer such profiles. Background of the Invention Diets high m saturated fats increase low density lipoprotems (LDL) which mediate the deposition of cholesterol on blood vessels. High plasma levels of serum cholesterol are closely correlated with atherosclerosis and coronary heart disease (Conner et al . , Coronary Heart Disease: Prevention, Complications, and Treatment, pp. 43-64, 1985) . By producing oilseed Brassica varieties with reduced levels of individual and total saturated fats m the seed oil, oil-based food products which contain less saturated fats can be produced. Such products will benefit public health by reducing the incidence of atherosclerosis and coronary heart disease. The dietary effects of monounsaturated fats have also been shown to have dramatic effects on health. Oleic acid, the only monounsaturated fat in most edible vegetable oils, lowers LDL as effectively as linoleic acid, but does not affect high density lipoprotems (HDL) levels (Mattson, F.H., J. Am. Diet. Assoc . , 8 . 9:387-391, 1989; Mensin et al . , New England J. Med., 321 :436-441, 1989) . Oleic acid is at least as effective in lowering plasma cholesterol as a diet low in fat and high in carbohydrates (Grundy, S.M., New England J. Med., 111:745-748, 1986; Mensink et al . , New England J. Med., 321:436-441, 1989) . In fact, a high oleic acid diet is preferable to low fat, high carbohydrate diets for diabetics (Garg et al . , New England J. Med., 319 : 829-834 , 1988) . Diets high in monounsaturated fats are also correlated with reduced systolic blood pressure (Williams et al . , J. Am. Med. Assoc. , 257 =3251-3256, 1987) . Epidemiological studies have demonstrated that the "Mediterranean" diet, which is high in fat and monounsaturates, is not associated with coronary heart disease (Keys, A., Circulation, 44 (Suppl) : 1, 1970) . Many breeding studies have been conducted to improve the fatty acid profile of Brassica varieties. Plemes and Freidt, Fat Sci. Technol . , 90(5) , 167-171 (1988) describe plant lines with reduced C 18 3 levels (2.5- 5.8%) combined with high oleic content (73-79%) . Rakow and McGregor, J. Amer. Oil Chem. Soc, 50, 400-403 (Oct. 1973) discuss problems associated with selecting mutants for linoleic and linolenic acids. In. Can. J. Plant Sci., 68, 509-511 (Apr. 1988) Stellar summer rape producing seed oil with 3% linolenic acid and 28% linoleic acid is disclosed. Roy and Tarr, Z. Pflanzenzuchtg, 95(3) , 201-209 (1985) teaches transfer of genes through an interspecific cross from Brassica j uncea into Brassica napus resulting in a reconstituted line combining high linoleic with low linolenic acid content. Roy and Tarr, Plant Breeding, 98, 89-96 (1987) discuss prospects for development of B . napus L . having improved linolenic and linolenic acid content. European Patent application 323,751 published July 12, 1989 discloses seeds and oils having greater than 79% oleic acid combined with less than 3.5% linolenic acid. Canvm, Can. J. Botany, 43, 63-69 (1965) discusses the effect of temperature on the fatty acid composition of oils from several seed crops including rapeseed. Mutations typically are induced with extremely high doses of radiation and/or chemical mutagens (Gaul, H. Radiation Botany (1964) 4:155-232) . High dose levels which exceed LD50, and typically reach LD90, led to maximum achievable mutation rates. In mutation breeding of Brassica varieties high levels of chemical mutagens alone or combined with radiation have induced a limited number of fatty acid mutations (Rakow, G.Z. Pflanzenzuchtg (1973) 69:62-82) . The low α-lmolenic acid mutation derived from the Rakow mutation breeding program did not have direct commercial application because of low seed yield. The first commercial cultivar using the low α-lmolenic acid mutation derived in 1973 was released in 1988 as the variety Stellar (Scarth, R. et al., Can. J. Plant Sci. (1988) 68:509-511) . Stellar was 20% lower yielding than commercial cultivars at the time of its release. Canola-quality oilseed Brassica varieties with reduced levels of saturated fatty acids in the seed oil could be used to produce food products which promote cardiovascular health. Canola lines which are individually low in palmitic and stearic acid content or low m combination will reduce the levels of saturated fatty acids. Similarly, Brassica varieties with increased monounsaturate levels in the seed oil, and products derived from such oil, would improve lipid nutrition. Canola lines which are low m linoleic acid tend to have high oleic acid content, and can be used m the development of varieties having even higher oleic acid content . Increased palmitic acid content provides a functional improvement in food applications. Oils high in palmitic acid content are particularly useful in the formulation of margarines. Thus, there is a need for manufacturing purposes for oils high in palmitic acid content . Decreased α-linolenic acid content provides a functional improvement in food applications. Oils which are low in linolenic acid have increased stability. The rate of oxidation of lipid fatty acids increases with higher levels of linolenic acid leading to off-flavors and off-odors in foods. There is a need in the food industry for oils low in alpha linolenic acid. 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 α-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 plas id delta-15 fatty acid desaturases are disclosed in Yadav, N., et al., Plant Physiol., 103:467-476 (1993) , WO 93/11245 and Arondel, V. et al . , Science, 258:1353-1355 (1992) . However, there are no teachings that disclose mutations in delta-12 or delta-15 fatty acid desaturase coding sequences from plants. Furthermore, no methods have been described for developing plant lines that contain delta- 12 or delta-15 fatty acid desaturase gene sequence mutations effective for altering the fatty acid composition of seeds. Summary of the Invention The present invention comprises canola seeds, plant lines producing seeds, and plants producing seed, said seeds having a maximum content of FDA saturates of about 5% and a maximum erucic acid content of about 2% based upon total extractable oil and belonging to a line in which said saturates content has been stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds and canola oil having a maximum erucic acid content of about 2%, based upon total extractable oil, are additional aspects of this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having an FDA saturates content of from about 4.2% to about 5.0% based upon total extractable oil. The present invention further comprises Brassi ca seeds, plant lines producing seeds, and plants producing seeds, said seeds having a minimum oleic acid content of about 71% based upon total extractable oil and belonging to a line in which said oleic acid content has been stabilized for both the generation to which the seed belongs and its parent generation. A further aspect of this invention is such high oleic acid seeds additionally having a maximum erucic acid content of about 2% based upon total extractable oil. Progeny of said seeds; and Brassica oil having 1) a minimum oleic acid content of about 71% or 2) a minimum oleic acid content of about 71% and a maximum erucic content of about 2% are also included m this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having an oleic acid content of from about 71.2% to about 78.3% based upon total extractable oil. The present invention further comprises canola seeds, plant lines producing seeds, and plants producing seeds, said seeds having a maximum linoleic acid content of about 14% and a maximum erucic acid content of about 2% based upon total extractable oil and belonging to a line in which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds and canola oil having a maximum linoleic acid content of about 14% and a maximum erucic acid content of about 2%, are additional aspects of this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having a linoleic acid content of from about 8.4% to about 9.4% based upon total extractable oil. The present invention further comprises Brassica seeds, plant lines producing seeds, and plants producing seeds, said seeds having a maximum palmitic acid content of about 3.5% and a maximum erucic acid content of about 2% based on total extractable oil and belonging to a line m which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds and canola having a maximum palmitic acid content of about 3.5% and a maximum erucic acid content of about 2%, are additional aspects of this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having a palmitic acid content of from about 2.7% to about 3.1% based upon total extractable oil. The present invention further comprises Brassi ca seeds, plant lines producing seeds, and plants producing seeds, said seeds having a minimum palmitic acid content of about 9.0% based upon total extractable oil and belonging to a line in which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. A further aspect of this invention is such high palmitic acid seeds additionally having a maximum erucic acid content of about 2% based upon total extractable oil. Progeny of said seeds; and Brassica oil having 1) a minimum palmitic acid content of about 9.0%, or 2) a minimum palmitic acid content of about 9.0% and a maximum erucic acid content of about 2% are also included in this invention. Preferred are seeds, plant lines producing seeds, and plants producing seeds, said seeds having a palmitic acid content of from about 9.1% to about 11.7% based upon total extractable oil . The present invention further comprises Brassica seeds, plant lines producing seeds, and plants producing seeds, said seeds having a maximum stearic acid content of about 1.1% based upon total extractable oil and belonging to a line m which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds have a canola oil having a maximum stearic acid content of about 1.1% and maximum erucic acid content of about 2%. Preferred are seeds, plant lines producing seeds, and plants producing seeds having a palmitic acid content of from about 0.8% to about 1.1% based on total extractable oil. The present invention further comprises Brassica seeds, plant lines producing seeds, and plants producing seeds, said seeds having a sum of linoleic acid content and linolenic acid content of a maximum of about 14% based upon total extractable oil and belonging to a line in which said acid content is stabilized for both the generation to which the seed belongs and its parent generation. Progeny of said seeds have a canola oil having a sum of linoleic acid content and linolenic acid content of a maximum of about 14% and a maximum erucic acid content of about 2%. Preferred are seeds, plant lines producing seeds, and plants producing seeds having a sum of linoleic acid content and linolenic acid content of from about 11.8% to about 12.5% based on total extractable oil . The invention further comprises Brassi caceae 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 mutant delta-12 fatty acid desaturase conferring altered fatty composition m seeds when the fragment is present in a plant. A preferred sequence comprises a mutant sequence as shown in SEQ ID NO: 3. Another embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a mutant delta-15 fatty acid desaturase. A plant in this embodiment may be soybean, oilseed Brassi ca 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 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 Brassi caceae or Helianthus species; (b) obtaining progeny plants from the mutagenized cells; (c) identifying progeny plants that contain a mutation n a delta-12 or delta-15 fatty acid desaturase gene; and (d) producing a plant line by selfing. Yet another embodiment of the invention involves a method of producing plant lines containing altered levels of unsaturated fatty acids 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 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. Brief Description of the Figures Figure 1 is a histogram showing the frequency distribution of seed oil oleic acid (C 18 x ) content in a segregating population of a Q508 X Westar cross. The bar labeled WSGA IA represents the C 18 1 content of the Westar parent. The bar labeled Q508 represents the C 18 λ content of the Q508 parent. Description of the Preferred Embodiments The U.S. Food and Drug Administration defines saturated fatty acids as the sum of lauric (C 12 0 ) , myristic (C 14 0 ) , palmitic (C 160 ) and stearic (C 1B 0 ) acids. The term "FDA saturates" as used herein means this above- defined sum. Unless total saturate content is specified, the saturated fatty acid values expressed here include only "FDA saturates." All percent fatty acids herein are percent by weight of the oil of which the fatty acid is a component. As used herein, a "line" is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. As used herein, the term "variety" refers to a line which is used for commercial production. The term "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. As used herein "M 0 " is untreated seed. As used herein, "M : " is the seed (and resulting plants) exposed to a mutagenic agent, while "M 2 " is the progeny (seeds and plants) of self-pollinated M α plants, "M 3 " is the progeny of self-pollinated M 2 plants, and "M 4 " is the progeny of self-pollinated M 3 plants. "M 5 " is the progeny of self-pollinated M 4 plants. "M 6 ", "M 7 ", etc. are each the progeny of self-pollinated plants of the previous generation. The term "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. Intensive breeding has produced 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 μmol glucosmolates/gram. "Canola" as used herein refers to plant variety seed or oil which contains less than 2% erucic acid (C 22 j) , and meal with less than 30 μ ol 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 α-lιnolenιc acid. Applicants have further discovered isolated nucleic acid fragments comprising sequences that carry mutations within the coding sequence of delta-12 or delta-15 desaturases . The mutations confer desirable alterations in fatty acid levels m 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 12-DES. Delta-15 fatty acid desaturase is also known on omega-3 fatty acid desaturase and is sometimes referred to herein as 15-DES. A nucleic acid fragment of the invention contains a mutation in a microsomal delta-12 fatty acid desaturase coding sequence or m a microsomal delta-15 fatty acid desaturase coding sequence. Such a mutation renders the resulting desaturase gene product non-functional in plants, relative to the function of the gene product encoded by the wild-type sequence. The non-functionality of the 12-DES 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. The non-functionality of the 15-DES gene product can be inferred from the decreased level of reaction product (a-lmolenic acid) and the increased level of substrate (linoleic 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. In one embodiment, 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. A mutation m 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 m the wild-type coding sequence. Such mutations result in insertions of one or more ammo acids, deletions of one or more ammo acids, and non-conservative ammo acid substitutions in the correspondmg gene product. In some embodiments, the sequence of a nucleic acid fragment may comprise more than one mutation or more than one type of mutation. Insertion or deletion of ammo 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. Ammo 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 ammo acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted ammo acids. Non-conservative ammo acid substitutions may replace an ammo acid of one class with an ammo acid of a different class. Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative ammo acid substitutions may also make a substantial change m the bulk of the residue side chain, e.g., substituting an alanyl residue for a isoleucyl residue. Examples of non-conservative substitutions mclude the substitution of a basic ammo acid for a non-polar ammo acid, or a polar ammo acid for an acidic ammo acid. Because there are only 20 ammo acids encoded m a gene, substitutions that result m a non-functional gene product may be determined by routine experimentation, incorporating ammo 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 havmg an ammo 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 correspondmg to ammo acids 105 to 109 of the Arabidopsis and Brassi ca delta-12 desaturase sequences, in nucleotides correspondmg to ammo acids 101 to 105 of the soybean delta-12 desaturase sequence and in nucleotides correspondmg to ammo acids 111 to 115 of the maize delta-12 desaturase sequence. See e.g. , WO 94/115116; Okuley et al . , Plant Cell 6:147-158 (1994) . The one letter ammo acid designations used herein are described m Alberts, B. et al . , Molecular Biology of the Cell, 3rd edition, Garland Publishing, New York, 1994. Am o acids flanking this motif are also highly conserved among delta-12 and delta-15 desaturases and are also suitable candidates for mutations in fragments of the mvention. An illustrative embodiment of a mutation m a nucleic acid fragment of the invention is a Glu to Lys substitution in the HECGH motif of a Brassi ca microsomal delta-12 desaturase sequence, either the D form or the F form. This mutation results the sequence HECGH being changed to HKCGH as seen by comparing SEQ ID NO:2 (wild- type D form) to SEQ ID NO:4 (mutant D form) A similar motif may be found at ammo acids 101 to 105 of the Arabidopsis microsomal delta-15 fatty acid desaturase, as well as in the correspondmg 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) . Among the types of mutations in an HECGH motif that render the resulting gene product non-functional are non-conservative substitutions. An illustrative example of a non-conservative substitution is substitution of a glycme residue for either the first or second histidme. Such a substitution replaces a polar residue (histidme) 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 glycme between the cystine and glutamic acid residues in the HECGH motif. Other regions having suitable conserved ammo acid motifs mclude the HRRHH motif shown in Table 2, the HRTHH motif shown m 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 . , supra. 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 Brassi ca 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 SEQ ID NO: 6 to mutant SEQ ID NO:8) . TABLE 1 Alignment of Ammo Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Position Amino Acid Sequence Arabidopsi s thaliana 100-129 IWVIAHECGH HAFSDYQWLD DTVGLIFHSF Glycine wax 96-125 VWVIAHECGH HAFSKYQWVD DWG TLHΞT Zea mays 106-135 V VIAHECGH HAFSDYSLLD DWGLVLHSS Ricmus communiε 1- 29 VMAHDCGH HAFSDYQ D DWGLILHSC Brassi ca napus D 100-128 VWVIAHECGH HAFSDYQWLD DTVG IFHS Brassica napus F 100-128 VWVIAHECGH HAFSDYQWLD DTVGLIFHS from plasmid pRF2-lC TABLE 2 Alignment of Ammo Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Position Ammo Acid Sequence Arabidopsi s thaliana 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV Glycine max 126-154 LLVPYFSWKI SHRRHHSNTG SLDRDEVFV Zea mays 136-164 LMVPYFSWKY SHRRHHSNTG SLERDEVFV Ricinus communis* 30- 58 LLVPYFSWKH SHRRHHSNTG SLERDEVFV Brassica napus D 130-158 LLVPYFSWKY SHRSHHSNTG SLERDEVFV Brassica napus F 130-158 LLVPYFSWKY SHRRHHSNTG SLERDEVFV from plasmid pRF2-lC TABLE 3 Alignment of Amino Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Position Amino Acid Sequence Arabidopsis thaliana 98- 3 33 DRDYGILNKV FHNITDTHVA HH FST PHY NAMEAT Glycme max 294 - 329 DRDYGILNKV FHHITDTHVA HHLFSTMPHY HAMEAT Zea mays 305-340 DRDYGILNRV FHNITDTHVA HHLFSTMPHY HAMEAT Ri cmuε com unis' 198-2 4 DRDYGILNKV FHNITDTQVA HHLF TMP Brassica napus D 299-334 DRDYGILNKV FHNITDTHVA HHPFSTMPHY HAMEAT Brassi ca napus F 299-334 DRDYGILNKV FHNITDTHVA HHLFSTMPHY HAMEAT from plasmid pRF2-lC TABLE 4 Alignment of Conserved Amino Acids from Microsomal Delta-12 Fatty Acid Desaturases Species Position Amino Acid Sequence Arabidopsi s thaliana 165-180 IKWYGKYLNN PLGRIM Glycine max 161-176 VAWFSLYLNN PLGRAV Zea mays 172-187 PWYTPYVYNN PVGRW Ricinus communis Λ 65- 80 IRWYSKYLNN PPGRIM Brassica napus D 165-180 IKWYGKYLNN PLGRTV Brassica napus F 165-180 IKWYGKYLNN PLGRTV from plasmid pRF2-lC TABLE 5 Alignment of Conserved Amino Acids from Plastid and Microsomal Delta-15 Fatty Acid Desaturases Species Position Amino Acid Sequence Arabidopsis thaliana" 156-177 WALFVLGHD CGHGSFSNDP KLN Brassica napus 3 114-135 WALFVLGHD CGHGSFSNDP RLN Glycine max 164-185 WALFVLGHD CGHGSFSNNS KLN Arabidopsis thaliana 94-115 WAIFVLGHD CGHGSFSDIP LLN Brassica napus 87-109 WALFVLGHD CGHGSFSNDP RLN Glycine max 93-114 WALFVLGHD CGHGSFSDSP PLN Plastid sequences TABLE 6 Alignment of Conserved Amino Acids from Plastid and Microsomal Delta-15 Fatty Acid Desaturases Species Position Am o Acid Sequence A. thaliana* 188-216 ILVPYHGWRI SHRTHHQNHG HVENDESWH B. napus 146-174 ILVPYHGWRI SHRTHHQNHG HVENDESWH Glycme max 3 196-224 ILVPYHGWRI SHRTHHQHHG HAENDESWH A thaliana 126-154 ILVPYHGWRI SHRTHHQNHG HVENDESWV Brassica napus 117-145 ILVPYHGWRI SHRTHHQNHG HVENDESWV Glycine max 125-153 ILVPYHGWRI SHRTHHQNHG HIEKDESWV a Plastid sequences The conservation of ammo acid motifs and their relative positions indicates that regions of a delta-12 or delta-15 fatty acid desaturase that can be mutated m one species to generate a non- functional desaturase can be mutated in the correspondmg region from other species to generate a non- functional 12 -DES or 15 -DES gene product m that species. Mutations m any of the regions of Tables 1-6 are specifically included within the scope of the invention, provided that such mutation (or mutations) renders the resulting desaturase gene product non-functional, as discussed hereinabove. A nucleic acid fragment containing a mutant sequence can be generated by techniques known to the skilled artisan. Such techniques mclude, 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. With mutagenesis, mutant plants havmg the desired fatty acid phenotype m seeds are identified by known techniques and a nucleic acid fragment containmg 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 12-DES or 15-DES gene. Alternatively, labeled nucleic acid probes that are specific for desired mutational events can be used to rapidly screen a mutagenized population. Seeds of Westar, a Canadian (Brassica napus) spring canola variety, were subjected to chemical mutagenesis. Mutagenized seeds were planted in the greenhouse and the plants were self-pollinated. The progeny plants were individually analyzed for fatty acid composition, and regrown either in the greenhouse or in the field. After four successive generations of self- pollinations, followed by chemical analysis of the seed oil at each cycle, several lines were shown to carry stably inherited mutations in specific fatty acid components, including reduced palmitic acid (C 16 0 ) , increased palmitic acid, reduced stearic acid (C 18 . 0 ) , increased oleic acid (C 18 x ) , reduced linoleic acid (C 1B 2 ) and reduced linolenic acid (C 18 3 ) , in the seed oil. The general experimental scheme for developing lines with stable fatty acid mutations is shown in Scheme I hereinafter. SCHEME I Westar (M 0 ) EMS Treatment v Greenhouse grow out Self-pollination <• Nursery grow out < Self-pollination v M, < Chemical analysis < Select mutants based on statistical analysis of control population < Grow out select mutants in greenhouse < Self-pollination v < Chemical analysis < Select mutants based on statistical analysis of control population < Grow out select mutants in nursery < Self-pollination v < Chemical analysis < Confirm altered fatty acid < Composition in selected lines v STABLE FATTY ACID MUTANTS Westar seeds (M 0 ) were mutagenized with ethylmethanesulfonate (EMS) . Westar is a registered Canadian spring variety with canola quality. The fatty acid composition of field-grown Westar, 3.9% C 16 0 , 1.9% C ιe 0 , 67.5% C 18 1# 17.6% C ιa 2 , 7.4% C 18 3 , <2% C20:l + C 22 2 , has remained stable under commercial production, with <± 10% deviation, since 1982. 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, mcludmg but not limited to ethidium bromide, nitrosoguanidme, diepoxybutane etc. may also be used to induce mutations. The mutagenesis treatment may also be applied to other stages of plant development, mcludmg but not limited to cell cultures, embryos, microspores and shoot apices. The M- L seeds were planted in the greenhouse and M : plants were individually self-pollinated. M 2 seed was harvested from the greenhouse and planted in the field m a plant-to-row design. Each plot contained six rows, and five M 2 lines were planted m each plot. Every other plot contained a row of non- mutagenized Westar as a control. Based on gas chromatographic analysis of M 2 seed, those lines which had altered fatty acid composition were self-pollinated and individually harvested. M 3 seeds were evaluated for mutations on the basis of a Z-distribution. An extremely stringent 1 m 10,000 rejection rate was employed to establish statistical thresholds to distinguish mutation events from existing variation. Mean and standard deviation values were determined from the non-mutagenized Westar control population in the field. The upper and lower statistical thresholds for each fatty acid were determined from the mean value of the population _+ the standard deviation, multiplied by the Z-distribution. Based on a population size of 10,000, the confidence interval is 99.99%. Seeds (M 3 ) from those M 2 lines which exceeded either the upper or lower statistical thresholds were replanted m the greenhouse and self-pollinated. This planting also mcluded Westar controls. The M 4 seed was re-analyzed using new statistical thresholds established with a new control population. Those M 4 lines which exceeded the new statistical thresholds for selected fatty acid compositions were advanced to the nursery. Followmg self-pollination, M 5 seed from the field were re-analyzed once again for fatty acid composition. Those lmes which remained stable for the selected fatty acids were considered stable mutations. "Stable mutations" as used herein are defmed as M 5 or more advanced lines which maintain a selected altered fatty acid profile for a minimum of three generations, mcludmg 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. Alternatively, stability may be measured in the same way by comparing to subsequent generations. In subsequent generations, stability is defmed as having similar fatty acid profiles in the seed as that of the prior or subsequent generation when grown under substantially similar conditions. The amount of variability for fatty acid content m a seed population is quite significant when smgle seeds are analyzed. Randomly selected smgle seeds and a ten seed bulk sample of a commercial variety were compared. Significant variation among the smgle seeds was detected (Table A) . The half-seed technique (Downey, R.K. and B.L. Harvey, Can. J. Plant Sci., 43:271 [1963]) m which one cotyledon of the germinating seed is analyzed for fatty acid composition and the remaining embryo grown mto a plant has been very useful to plant breeding work to select individuals in a population for further generation analysis. The large variation seen m the smgle seed analysis (Table A) is reflected in the half-seed technique. TABLE A Single Seed Analysis for Fatty Acid Composition 1 SAMPLE 16 0 16 1 18 0 18 1 18 2 18 3 20 0 20 1 22 0 22 1 Bulk 3 2 0 4 1 8 20 7 13 7 9 8 0 8 11 2 0 4 32 2 1 2 8 0 2 1 1 14 6 14 6 11 1 0 8 9 8 0 7 38 2 2 3 3 0 2 1 3 13 1 14 4 11 7 0 9 10 5 0 7 37 0 3 3 0 1 2 12 7 15 3 10 6 0 8 7 3 0 7 43 2 4 2 8 0 2 1 1 16 7 13 2 9 1 0 8 11 2 0 4 38 9 5 3 0 1 8 15 2 13 3 8 4 1 3 8 7 0 9 42 3 6 3 1 1 3 14 4 14 6 10 3 1 0 10 9 0 8 39 3 7 2 6 1 2 15 7 13 8 9 9 0 9 12 2 0 5 37 0 S 3 1 1 1 16 2 13 4 10 6 0 6 9 2 0 8 41 4 9 2 7 0 1 1 0 13 5 11 2 11 3 0 8 6 o 0 7 46 9 10 3 4 0 2 1 4 13 9 17 5 10 8 1 1 10 0 0 9 36 2 11 2 8 0 2 1 2 12 7 12 9 10 3 1 0 7 9 0 9 43 3 12 2 3 0 1 1 6 20 7 14 8 6 5 1 1 12 5 0 8 34 5 13 2 6 0 2 1 3 21 0 11 4 7 6 1 0 11 6 0 6 36 7 14 2 6 0 1 1 2 14 7 13 2 9 4 0 9 10 1 0 8 40 8 15 2 9 0 2 1 4 16 6 15 1 11 2 0 7 9 1 0 3 36 1 16 3 0 0 2 1 1 12 4 13 7 10 4 0 9 8 7 0 8 42 7 17 2 9 0 1 1 1 21 1 12 3 7 1 0 8 12 4 0 5 36 8 18 3 1 0 1 1 2 13 7 13 1 10 4 1 0 8 8 0 7 41 6 19 2 7 0 1 1 0 11 1 13 4 11 7 0 8 7 9 0 8 43 5 20 2 3 0 2 0 2 18 2 13 9 8 2 0 9 10 3 0 8 38 2 Average 2 8 0 2 1 2 15 4 13 8 9 8 0 9 9 8 0 7 39 7 Minimum 2 3 0 1 0 2 11 1 11 2 6 5 0 6 6 2 0 3 34 5 Maximum 3 4 0 2 1 8 21 1 17 5 11 7 1 3 12 5 0 9 46 9 Ranqe 1 1 0 1 1 6 9 9 6 3 5 3 0 7 6 4 0 6 12 4 Values expressed as percent of total oil Plant breeders using the half-seed technique have found it unreliable in selecting stable genetically controlled fatty acid mutations (Stefanson, B.R., In; High and Low Erucic Acid Rapeseed Oils, Ed. N.T. Kenthies, Academic Press, Inc., Canada (1983) pp. 145- 159) . Although valuable in selecting individuals from a population, the selected traits are not always transmitted to subsequent generations (Rakow, G. and McGregor, D.I., J. Amer. Oil Chem. Soc. (1973) 50:400- 403. To determine the genetic stability of the selected plants several self-pollinated generations are required (Robelen, G. In: Biotechnology for the Oils and Fats Industry, Ed. C. Ratledge, P. Dawson and J. Rattray, American Oil Chemists Society (1984) pp. 97-105) with chemical analysis of a bulk seed sample. 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. To eliminate the occurrence of deleterious mutations and reduce the load of mutations carried by the plant a low mutagen dose was used m the seed treatments to create an LD30 population. This allowed for the rapid selection of smgle gene mutations for fatty acid traits m agronomic backgrounds which produce acceptable yields. Other than changes m the fatty acid composition of the seed oil, the mutant lmes described here have normal plant phenotype when grown under field conditions, and are commercially useful. "Commercial utility" is defmed as having a yield, as measured by total pounds of seed or oil produced per acre, withm 15% of the average yield of the starting (M 0 ) canola variety grown in the same region. To be commercially useful, plant vigor and high fertility are such that the crop can be produced m this yield by farmers using conventional farming equipment, and the oil with altered fatty acid composition can be extracted using conventional crushing and extraction equipment. The seeds of several different fatty acid lines have been deposited with the American Type Culture Collection and have the followmg accession numbers. Lme Accession No. Deposit Date A129.5 40811 May 25, 1990 A133.1 40812 May 25, 1990 A144.1 40813 May 25, 1990 A200.7 40816 May 31, 1990 M3032.1 75021 June 7, 1991 M3094.4 75023 June 7, 1991 M3052.6 75024 June 7, 1991 M3007.4 75022 June 7, 1991 MM33006622..88 7755002255 June 7, 1991 M3028.10 75026 June 7, 1991 IMC130 75446 April 16, 1993 In some plant species or varieties more than one form of endogenous microsomal delta-12 desaturase may be found. In amphidiploids, 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 m only one form. An example of such a plant is Brassi ca napus lme Q508 , a doubly-mutagenized lme containing a mutant D-form of delta-12 desaturase (SEQ ID NO:l) and a mutant F-form of delta-12 desaturase (SEQ ID NO: 5) . 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., Brassi ca napus , B . rapa and B . juncea) , sunflower (Helianthus annus) , castor bean (Ri cinus communis) , corn ( Zea ways) , and safflower ( Carthamus tinctorius) . Plants accordmg to the invention preferably contain an altered fatty ac d profile. For example, oil obtamed 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. In some embodiments, oil obtamed from seeds produced by plants of the mvention may have from about 2.0% to about 5.0% saturated fatty acids, based on total fatty acid composition of the seeds. In some embodiments, 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% ff-lmolenic acid. In one embodiment of the claimed invention, a plant contams both a 12-DES mutation and a 15-DES mutation. Such plants can have a fatty acid composition comprising very high oleic acid and very low alpha- linolemc acid levels. Mutations m 12-DES and 15-DES may be combmed m a plant by making a genetic cross between 12-DES and 15-DES smgle mutant lines. A plant having a mutation m 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 m order to obtain progeny seeds . These progeny seeds are then screened m order to identify those seeds carrying both mutant genes. Alternatively, a lme possessing either a 12-DES or a 15-DES mutation can be subjected to mutagenesis to generate a plant or plant lme having mutations in both 12-DES and 15-DES. For example, the IMC 129 lme has a mutation m 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 lme can be mutagenized to induce a mutation in a 15-DES gene, resulting in a plant or plant lme carrying a mutation in a delta-12 fatty acid desaturase gene and a mutation a delta-15 fatty acid desaturase gene. Progeny includes descendants of a particular plant or plant lme, e.g., seeds developed on an instant plant. Progeny of an instant plant mclude seeds formed on F x F 2 , F 3 , and subsequent generation plants, or seeds formed on BC 1; BC 2 , BC 3 and subsequent generation plants. 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 smgle half- seed. 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. However, half-seed analysis is also known to be an inaccurate representation of genotype of the seed being analyzed. Bulk seed analysis typically yields a more accurate representation of the fatty acid profile of a given genotype. The nucleic acid fragments of the invention can be used as markers m plant genetic mappmg and plant breeding programs. Such markers may mclude 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. Marker-assisted breeding techniques may be used m 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 12- DES or 15-DES. Methods accordmg to the mvention are useful m 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, mcreased resistance to seedling fungal diseases (damping off, root rot and the like) , mcreased yield, and improved standability. While the invention is susceptible to various modifications and alternative forms, certain specific embodiments thereof are described m the general methods and examples set forth below. For example the invention may be applied to all Brassica species, including B . rapa , B . j uncea, and B . hirta , to produce substantially similar results. It should be understood, however, that these examples are not intended to limit the invention to the particular forms disclosed but, instead the invention is to cover all modifications, equivalents and alternatives falling withm the scope of the invention. This mcludes the use of somaclonal variation; physical or chemical mutagenesis of plant parts; anther, microspore or ovary culture followed by chromosome doubling; or self- or cross-pollmation to transmit the fatty acid trait, alone or m combination with other traits, to develop new Brassica lines. EXAMPLE 1 Selection of Low FDA Saturates Prior to mutagenesis, 30,000 seeds of B . napus cv. Westar seeds were preimbibed m 300-seed lots for two hours on wet filter paper to soften the seed coa . The preimbibed seeds were placed in 80 mM ethylmethanesulfonate (EMS) for four hours. Followmg mutagenesis, the seeds were rinsed three times in distilled water. The seeds were sown in 48-well flats containing Pro-Mix. Sixty-eight percent of the mutagenized seed germinated. The plants were maintained at 25°C/15°C, 14/10 hr day/night conditions m the greenhouse. At flowering, each plant was individually self-pollinated. 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 m 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 mam 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. Self-pollinated M 3 seed and Westar controls were analyzed in 10-seed bulk samples for fatty acid composition via gas chromatography. Statistical thresholds for each fatty acid component were established usmg a Z-distribution with a stringency level of 1 in 10,000. The selected M 3 seeds were planted in the greenhouse along with Westar controls . The seed was sown m 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. Ten M 4 plants m each row were bagged for self-pollination. At maturity, the selfed plants were individually harvested and the open pollinated plants in the row were bulk harvested. The M 5 seed from smgle plant selections was analyzed in 10-seed bulk samples and the bulk row harvest in 50-seed bulk samples. Selected M 5 lines were planted in the greenhouse along with Westar controls. The seed was grown as previously described. At flowering the terminal raceme was self-pollinated by bagging. At maturity, selfed M B seed was individually harvested from each plant and analyzed m 10-seed bulk samples for fatty acid composition. Selected M 6 lines were entered mto field trials m Eastern Idaho. The four trial locations were selected for the wide variability in growing conditions. The locations mcluded Burley, Tetonia, Lamont and Shelley (Table I) . The lines were planted m four 3-meter rows with an 8-inch spacing, each plot was replicated four times. The planting design was determined using a Randomized Complete Block Designed. The commercial cultivar Westar was used as a check cultivar. At maturity the plots were harvested to determine yield. Yield of the entries m the trial was determined by taking the statistical average of the four replications. The Least Significant Difference Test was used to rank the entries the randomized complete block design. TABLE I Trial Locations for Selected Fatty Acid Mutants LOCATION SITE CHARACTERIZATIONS BURLEY Irrigated. Long season. High temperatures during flowering. TETONIA Dryland. Short season. Cool temperatures. LAMONT Dryland. Short season. Cool temperatures. SHELLEY Irrigated. Medium season. High temperatures during flowering. To determine the fatty acid profile of entries, plants m each plot were bagged for self-pollination. The M 7 seed from smgle plants was analyzed for fatty acids in ten-seed bulk samples. To determine the genetic relationships of the selected fatty acid mutants crosses were made. Flowers of M 6 or later generation mutations were used in crossing. F 1 seed was harvested and analyzed for fatty acid composition to determine the mode of gene action. The F 2 progeny were planted in the greenhouse. The resulting plants were self-pollinated, the F 2 seed harvested and analyzed for fatty acid composition for allelism studies. The F 2 seed and parent lme seed was planted m the greenhouse, individual plants were self-pollinated. The F 3 seed of individual plants was tested for fatty acid composition using 10-seed bulk samples as described previously. In the analysis of some genetic relationships dihaploid populations were made from the microspores of the F x hybrids. Self-pollinated seed from dihaploid plants were analyzed for fatty acid analysis using methods described previously. For chemical analysis, 10-seed bulk samples were hand ground with a glass rod m 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. m 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. After phase separation, 600 μL of the upper organic phase were pipetted mto individual vials and stored under nitrogen at -5°C One μL samples were injected into a Supelco SP-2330 fused silica capillary column (0.25 mm ID, 30 M length, 0.20 μm 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/mm., Injector temperature - 250°C, Detector temperature - 300°C, Split vent - 1/15. Table II describes the upper and lower statistical thresholds for each fatty acid of interest. TABLE II Statistical Thresholds for Specific Fatty Acids Derived from Control Westar Plantings Percent Fatty Acids Genotype C 16 0 C 18 0 C 18 x C 1B 2 C 18 3 Sats * M 3 Generation(1 in 10,000 rejection rate) Lower 3.3 1.4 -- 13.2 5.3 6.0 Upper 4.3 2.5 71.0 21.6 9.9 8.3 M 4 Generation(1 in 800 rejection rate) Lower 3.6 0.8 -- 12.2 3.2 5.3 Upper 6.3 3.1 76.0 32.4 9.9 11.2 M 5 Generation (1 in 755 rejection rate) Lower 2.7 0.9 -- 9.6 2.6 4.5 Upper 5 ^ 7 2_ : _7 80.3 26.7 9.6 10.0 * Sats=Total Saturate Content At the M 3 generation, twelve lines exceeded the lower statistical threshold for palmitic acid (<_3.3%) . Line W13097.4 had 3.1% palmitic acid and an FDA saturate content of 4.5%. After a cycle in the greenhouse, M 4 seed from line W13097.4 (designated line A144) was analyzed. Line W13097.4.1 (A144.1) had 3.1% C 16 0 , exceeding the lower statistical threshold of 3.6%. The FDA saturate content for A144.1 was 4.5%. The fatty acid compositions for the M 3 , M 4 and M 5 generations of this family are summarized in Table III. TABLE III Fatty Acid Composition of a Low Palmitic Acid/Low FDA Saturate Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype 1 -16 0 '-IB 0 ^18 1 ^-18 2 ^-18 3 SatS Ot S3t Westar 3.9 1.9 67.5 17.6 7.4 5.9 7.0 W13097.4 (M 3 ) 3.1 1.4 63.9 18.6 9.5 4.5 5.6 W13097.4 (M 4 ) 3.1 1.4 66.2 19.9 6.0 4.5 5.5 A144.1.9 (M s ) 2.9 1.4 64.3 20.7 7.3 4.4 5.3 a Letter and numbers up to second decimal point indicate the plant line. Number after second decimal point indicates an individual plant. Sat=FDA Saturates c Tot Sat=Total Saturate Content The M 5 seed of ten self-pollinated A144.1 (ATCC 40813) plants averaged 3.1% palmitic acid and 4.7% FDA saturates. One selfed plant (A144.1.9) contained 2.9% palmitic acid and FDA saturates of 4.4%. Bulk seed analysis from open-pollinated (A144.1) plants at the M s generation averaged 3.1% palmitic acid and 4.7% FDA saturates. The fatty acid composition of the bulked and individual A144.1 lines are summarized in Table IV. TABLE IV Fatty Acid Composition of A144 Low Palmitic Acid/Low FDA Saturate Line Percent Fatty Acids Genotype 3 C 16 0 C 18 0 C 18 : C 1B 2 C lβ 3 Sats Tot Sat c Individually Self-Pollinated Plants A144. .1. .1 3.2 1.6 64. .4 20. .5 7 .0 4 , .8 5. .9 A144. .1. .2 3.0 1.5 67, .4 18. .6 6 .3 4 , .5 5, .7 A144. .1. .3 3.6 1.8 61. .4 22, .4 7, .5 5, .2 6, .6 A144 , .1. .4 3.2 1.5 64. .6 20. .9 6, .7 4. .7 5. .8 A144. .1. .5 3.3 1.7 60. .0 23. .9 7, .9 5. .0 6. .1 A144. .1. .6 3.1 1.4 67. .3 17, .8 6 .5 4. .6 5 .2 A144. .1. .7 3.1 1.6 67. .7 17 , .4 6, .5 4. .8 5 , .4 A144. .1. .8 3.1 1.8 66. .9 18, .7 6. .1 4. .9 5, .4 A144 .1. .9 2.9 1.4 64. .3 20 .7 7, .3 4. .4 5. .3 A144 .1 .10 3.1 1.5 62, .5 20, .4 7 .7 4. .6 5 .6 Average of Individually Self-Pollinated Plants A144.1.1-10 3.1 1.6 64.8 20.1 6.9 4.7 5.7 Bulk Analysis of Open-Pollinated Plants A144.1B 3.1 1.6 64.8 19.4 7.8 4.7 5.7 a Letter and numbers up to second decimal point indicate the plant line. Number after second decimal point indicates an individual plant. b Sat=FDA Saturates c Tot Sat=Total Saturate Content These reduced levels have remamed stable to the M 7 generations in both greenhouse and field conditions. These reduced levels have remained stable to the M 7 generation in multiple location field trails. Over all locations, the self-pollinated plants (A144) averaged 2.9% palmitic acid and FDA saturates of 4.6%. The fatty acid composition of the A144 lines for each Idaho location are summarized in Table V. In the multiple location replicated trial the yield of A144 was not significantly different in yield from the parent cultivar Westar. By means of seed mutagenesis, the level of saturated fatty acids of canola (B . napus) was reduced from 5.9% to 4.6%. The palmitic acid content was reduced from 3.9% to 2.9%. TABLE V Fatty Acid Composition of a Mutant Low Palmitic Acid/Low FDA Saturate Canola Line at Different Field Locations in Idaho Percent Fatty Acids Trial Location Cl6 0 C 18 0 c, . 8 1 1B 2 ^-lβ 3 Sats Tot Sats Burley 2.9 1.3 62.3 20.6 10.3 4.2 5.0 Tetonia 2.9 1.7 59.7 21.0 11.2 4.6 5.7 Lamont 3.1 1.8 63.2 19.5 9.0 4.9 5.9 Shelley 2.8 1.9 64.5 18.8 8.8 4.7 5.9 To determine the genetic relationship of the palmitic acid mutation in A144 (C 16 0 - 3.0%, C 18 0 - 1.5%, C 18 j - 67.4%, C 18 2 - 18.6%, C 183 - 6.3%) to other fatty acid mutations it was crossed to A129 a mutant high oleic aci (C^ Q - 3.8Ό , 18 Q - 2.3*6, C 18 1 - 75.6*0, C 18 2 - 9.5*6, C 18 3 - 4.9%) . Over 570 dihaploid progeny produced from the F hybrid were harvested and analyzed for fatty acid composition. The results of the progeny analysis are summarized in Table VB. Independent segregation of the palmitic traits was observed which demonstrates that the genetic control of palmitic acid in A144 is different from the high oleic acid mutation A129. TABLE VB Genetic Stud:ιes of Dihaploid Proαenv of A144 X A129 Freσuencv C *-16 0 Genotype Content (%) Observed Expected p-p-p2-p2- 3.0% 162 143 p+p+p2-p2- 3.4% 236 286 p+p+p2+p2+ 3.8% 175 143 EXAMPLE 2 An additional low FDA saturate lme, designated A149.3 (ATCC 40814) , was also produced by the method of Example 1. A 50-seed bulk analysis of this lme showed the following fatty acid composition: C 16 0 - 3.6%, C 18 0 - 1.4%, C 18 l - 65.5%, C 182 - 18.3%, C 183 - 8.2%, FDA Sats - 5.0%, Total Sats - 5.9%. This lme has also stably maintained its mutant fatty acid composition to the M 5 generation. In a multiple location replicated trial the yield of A149 was not significantly different in yield from the parent cultivar Westar. EXAMPLE 3 An additional low palmitic acid and low FDA saturate line, designated M3094.4 (ATCC 75023) , was also produced by the method of Example 1. A 10-seed bulk analysis of this lme showed the followmg fatty acid composition: C 16 0 - 2.7%, C 1B 0 - 1.6%, C 18 : - 66.6%, C 18 2 - 20.0%, C 18 3 - 6.1%, C 20 1 - 1.4%, C 22 ! - 0.0%, FDA Saturate 4.3%, Total Saturates - 5.2%. This lme has stably maintained its mutant fatty acid composition to the M 5 generation. In a smgle replicated trial the yield of M3094 was not significantly different in yield from the parent cultivar. M3094.4 was crossed to A144, a low palmitic acid mutation (Example 1) for allelism studies. Fatty acid composition of the F 2 seed showed the two lmes to be allelic. The mutational events in A144 and M3094, although different in origin, are in the same gene. EXAMPLE 4 In the studies of Example 1, at the M, generation, 470 lines exceed the upper statistical threshold for palmitic acid (^4.3%) . One M 3 line, W14538.6, contained 9.2% palmitic acid. Selfed progenies of this line, since designated M3007.4 (ATCC 75022) , continued to exceed to the upper statistical threshold for high palmitic acid at both the M 4 and M 5 generations with palmitic acid levels of 11.7% and 9.1%, respectively. The fatty acid composition of this high palmitic acid mutant, which was stable to the M 7 generation under both field and greenhouse conditions, is summarized in Table VI. TABLE VI Fatty Acid Composition of a High Palmitic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype Cj.6 0 C-ile 0- c c - . lB !. Sats * Westar 3.9 1.9 67.5 17.6 7.4 7.0 W114538.6 8.6 1.6 56.4 20.3 9.5 10.2 (M 3 ) M3007.2 11.7 2.1 57.2 18.2 5.1 13.9 (M 4 ) M3007.4 9.1 1.4 63.3 13.7 5.5 12.7 (M 5 ) * Sats=Total Saturate Content To determine the genetic relationship of the high palmitic mutation in M3007.4 to the low palmitic mutation in A144 (Example 1) crosses were made. The F 2 progeny were analyzed for fatty acid composition. The data presented in Table VIB shows the high palmitic group (C 16 0 > 7.0%) makes up one-quarter of the total population analyzed. The high palmitic acid mutation was controlled by one single gene mutation. TABLE VIB Genetic Studies of M3007 X A144 Frequency Genotype Content (%) Observed Expected p-p-/p-hp- <7.0 151 142 hp-hp- >7.0 39 47 An additional M 3 lme, W4773.7, contained 4.5% palmitic acid. Selfed progenies of this line, since designated A200.7 (ATCC 40816) , continued to exceed the upper statistical threshold for high palmitic acid in both the M 4 and M 5 generations with palmitic acid levels of 6.3% and 6.0%, respectively. The fatty acid composition of this high palmitic acid mutant, which was stable to the M 7 generation under both field and greenhouse conditions, is summarized in Table VII. TABLE VII Fatty Acid Composition of a High Palmitic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype ^-16 0 ^-18 0 c *—18 1 c 2 c-1B 3 Sats * Westar 3.9 1.9 67.5 17. ,6 7.4 7.0 W4773.7 4.5 2.9 63.5 19. ,9 7.1 9.3 (M 3 ) M4773.7.7 6.3 2.6 59.3 20. .5 5.6 10.8 (M 4 ) A200.7.7 6.0 1.9 60.2 20. .4 7.3 9.4 (M s ) " Sats=Total Saturate Content EXAMPLE 5 Selection of Low Stearic Acid Canola Lines In the studies of Example 1, at the M 3 generation, 42 lines exceeded the lower statistical threshold for stearic acid (<1.4%) . Lme W14859.6 had 1.3% stearic acid. At the M 5 generation, its selfed progeny (M3052.1) continued to fall withm the lower statistical threshold for C 1B o with 0.8% stearic acid. The fatty acid composition of this low stearic acid mutant, which was stable under both field and greenhouse conditions is summarized in Table VIII. In a smgle location replicated yield trial M3052.1 was not significantly different yield from the parent cultivar Westar. TABLE VIII Fatty Acid Compcisition of a Low Stearic Acid Canola Lme Produced by Sieed Mutagenesis Percent Fatty Acids Genotype c ""16 0 18 o c '-18 1 C ^18 2 lβ 3 Sats Westar 3.9 1.9 67.5 17.6 7.4 5.9 W14859.6 5.3 1.3 56.1 23.7 9.6 7.5 (M 3 ) M3052.1 4.9 0.9 58.9 22.7 9.3 5.8 (M 4 ) M3052.6 4.4 0.8 62.1 21.2 7.9 5.2 (M 5^ ) To determine the genetic relationship of the low stearic acid mutation of M3052.1 to other fatty acid mutations it was crossed to the low palmitic acid mutation A144 (Example 1) . Seed from over 300 dihaploid progeny were harvested and analyzed for fatty acid composition. The results are summarized m Table VIIIB. Independent segregation of the palmitic acid and stearic acid traits was observed. The low stearic acid mutation was genetically different from the low palmitic acid mutations found in A144 and M3094. TABLE VIIIB Genetic Studies of M3052 X A144 Frequency c *-16 0 + ^ c '- 18 Genotype C Coonntteenntt ((%%)) Observed Expected p-p-s-s- <<44..99%% 87 77 p-p-s-s-/p+p+s-s- 44..00%%<<XX<<55..6% 152 154 p+p+s+s- >>55..66%% 70 77 An additional M 5 line, M3051.10, contained 0.9% and 1.1% stearic acid in the greenhouse and field respectively. A ten-seed analysis of this line showed the following fatty acid composition: C 16 0 - 3.9%, C 18 0 - l.l*ε, C 1B 1 - 61.7-s, C 18 2 ~ 23.0*s, C 18 3 - 7.6-s, FDA saturates - 5.0%, Total Saturates - 5.8%. In a single location replicated yield trial M3051.10 was not significantly different in yield from the parent cultivar Westar. M3051.10 was crossed to M3052.1 for allelism studies. Fatty acid composition of the F 2 seed showed the two lines to be allelic. The mutational events in M3051.10 and M3052.1 although different in origin were in the same gene. An additional M s line, M3054.7, contained 1.0% and 1.3% stearic acid in the greenhouse and field respectively. A ten-seed analysis of this line showed the following fatty acid composition: C 160 - 4.0%, C 18 0 - 1.0%, C 18 j - 66.5%, C 182 - 18:4%, C 18 3 - 7.2%, saturates - 5.0%, Total Saturates - 6.1%. In a single location replicated yield trial M3054.7 was not significantly different in yield from the parent cultivar Westar. M3054.7 was crossed to M3052.1 for allelism studies. Fatty acid composition of the F 2 seed showed the two lines to be allelic. The mutational events in M3054.7, M3051.10 and M3052.1 although different m origin were m the same gene . EXAMPLE 6 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. seed of five self-pollinated plants of lme 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 IX. This line also stably maintained its mutant fatty acid composition to the M 7 generation m 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 X. 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 processmg, 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) . The AOM of Westar was 18 AOM hours and for A129 was 30 AOM hours. TABLE IX Fatty Acid ι Composition of a High Oleic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Westar 3.9 1.9 67.5 17.6 7.4 7.0 W7608.3 3.9 2.4 71.2 12.7 6.1 7.6 (M 3 ) W7608.3. 5 3.9 2.0 78.8 7.7 3.9 7.3 (M 4 ) A129.5.3 3.8 2.3 75.6 9.5 4.9 7.6 (M 5 ) Sats=Total Saturate Content TABLE X Fatty Ac: Ld Composition of a Mutant High Oleic Acid Line at Different Fieild Locations in Idaho Percent Fatty Acids Location (-16.0 -18 0 (-18 1 c '-18 2 iB 3 Sats Burley 3.3 2.1 77.5 8.1 6.0 6.5 Tetonia 3.5 3.4 77.8 6.5 4.7 8.5 Lamont 3.4 1.9 77.8 7.4 6.5 6.3 Shelley 3.3 2.6 80.0 5.7 4.5 7.7 Sats=Total Saturate Content The genetic relationship of the high oleic acid mutation A129 to other oleic desaturases was demonstrated in crosses made to commercial canola cultivars and a low linolenic acid mutation. A129 was crossed to the commercial cultivar Global (C 16 0 - 4.5%, C 18 0 - 1.5%, C 18 x - 62.9%,C 18 2 - 20.0%, C 1B 3 - 7.3%) . Approximately 200 F 2 individuals were analyzed for fatty acid composition. The results are summarized in Table XB. The segregation fit 1:2:1 ratio suggesting a single co-dominant gene controlled the inheritance of the high oleic acid phenotype. TABLE XB Genetic Studies of A129 X Global Frequency (-18 0 Genotype Content (%) Observed Expected od-od- 77.3 43 47 od-od+ 71.7 106 94 od+od+ 66.1 49 47 A cross between A129 and IMC 01, a low linolenic acid variety (C 16 0 - 4.1%, C 1B 0 - 1.9%, C 1B x - 66.4%, C 182 - 18.1%, C 1B 3 - 5.7%) , was made to determine the inheritance of the oleic acid desaturase and linoleic acid desaturase. In the F l hybrids both the oleic acid and linoleic acid desaturase genes approached the mid-parent values indicating a co-dominant gene actions. Fatty acid analysis of the F 2 individuals confirmed a 1:2:1:2:4:2:1:2:1 segregation of two independent, co- dominant genes (Table XC) . A line was selected from the cross of A129 and IMCOl and designated as IMC130 (ATCC deposit no. 75446) as described in U.S. Patent Application No. 08/425,108, incorporated herein by reference. TABLE XC Genetic Studies of A129 X IMC 01 Frequency Genotype Ratio Observed Expected od-od-ld-ld- 1 11 12 od-od-ld-ld+ 2 30 24 od-od-ld+ld+ 1 10 12 od-od+ld-ld- 2 25 24 od-od+ld-ld+ 4 54 47 od-od+ld+ld+ 2 18 24 od+od+ld-ld- 1 7 12 od+od+ld-ld+ 2 25 24 od+od+ld+ld+ 1 12 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 0 - 3.5%, C 1B 0 - 1.8%, C 18 x - 77.3%, C 182 - 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 (ATCC 75026) , was also produced by the disclosed method in Example 1. A 10-seed bulk analysis of this line showed the following fatty acid composition: - 16 o ~ 3.5-ά, C 18 0 - 1.8-s, C 18 1 - 77.3-ε, C 18 2 - 9.0-s, C 18 3 5.6%, FDA Saturates - 5.3%, Total Saturates - 6.4%. In a single location replicated yield trial M3028.10 was not significantly different in yield from the parent cultivar Westar. EXAMPLE 7 Low Linoleic Acid Canola In the studies of Example 1, at the M 3 generation, 80 lines exceeded the lower statistical threshold for linoleic acid ( <_ 13.2%) . Line W12638.8 had 9.4% linoleic acid. At the M 4 and M 5 generations, its selfed progenies [W12638.8, since designated A133.1 (ATCC 40812)] continued to exceed the statistical threshold for low C 18 2 with linoleic acid levels of 10.2% and 8.4%, respectively. The fatty acid composition of this low linoleic acid mutant, which was stable to the M 7 generation under both field and greenhouse conditions, is summarized in Table XI. In multiple location replicated yield trials, A133 was not significantly different in yield from the parent cultivar Westar. An additional low linoleic acid line, designated M3062.8 (ATCC 75025) , was also produced by the disclosed method. A 10-seed bulk analysis of this lme showed the following fatty acid composition: C 16 0 - 3.8%, C 18 0 - 2.3%, C 1B λ - 77.1%, C 18 2 - 8.9%, C 18 3 - 4.3%, FDA Sats-6.1%. This line has also stably maintained its mutant fatty acid composition in the field and greenhouse. TABLE XI Fatty Acid Composition of a Low Linoleic Acid Canola Line Produced by Seed Mutagsnesis Percent Fatty Acids Genotype C. 16 0 ^18 0 C lB 1 (-18 2 (-18 3 Sats b Westar 3.9 1.9 67.5 17.6 7.4 7.0 W12638.8 3.9 2.3 75.0 9.4 6.1 7.5 (M 3 ) W12638.8.1 4.1 1.7 74.6 10.2 5.9 7.1 (M 4 ) A133.1.8 3.8 2.0 77.7 8.4 5.0 7.0 (M 5 ) "Letter and numbers up to second decimal point indicate the plant line. Number after second decimal point indicates an individual plant. b Sats=Total Saturate Content EXAMPLE 8 Low Linolenic and Linoleic Acid Canola In the studies of Example 1, at the M 3 generation, 57 lines exceeded the lower statistical threshold for linolenic acid (<. 5.3%) . Line W14749.8 had 5.3% linolenic acid and 15.0% linoleic acid. At the M 4 and M 5 generations, its selfed progenies [W14749.8, since designated M3032 (ATCC 75021)] continued to exceed the statistical threshold for low C 18 3 with linolenic acid levels of 2.7% and 2.3%, respectively, and for a low sum of linolenic and linoleic acids with totals of 11.8% and 12.5% respectively. The fatty acid composition of this low linolenic acid plus linoleic acid mutant, which was stable to the M 5 generation under both field and greenhouse conditions, is summarized in Table XII. In a single location replicated yield trial M3032 was not significantly different in yield from the parent cultivar (Westar) . TABLE XII Fatty Acid Composition of a Low Linolenic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype C 16 0 C 1B 0 C 18 x C 1B 2 C 1B 3 Sats Westar 3. .9 1.9 67.5 17.6 7.4 7.0 W14749.8 4. .0 2.5 69.4 15.0 5.3 6.5 (M 3 ) M3032.8 3 , .9 2.4 77.9 9.1 2.7 6.4 (M 4 ) M3032.1 3. .5 2.8 80.0 10.2 2.3 6.5 (M 5 ) Sats=Total Saturate Content EXAMPLE 9 The high oleic acid mutation of A129 was introduced into different genetic backgrounds by crossing and selecting for fatty acid and agronomic characteristics. A129 (now renamed IMC 129) was crossed to Legend, a commercial spring Brassica napus variety. Legend has the following fatty acid composition: C 16 0 - 3.8-ϊ, C 1B 0 - 2.1-s, C 18 1 - 63.1-s, C 18 2 - 17.8-s, C 18 3 - 9.3-s. The cross and progeny resulting from were coded as 89B60303. The F x seed resulting from the cross was planted in the greenhouse and self-pollinated to produce F 2 seed. The F 2 seed was planted in the field for evaluation. Individual plants were selected m the field for agronomic characteristics. At maturity, the F 3 seed was harvested from each selected plant and analyzed for fatty acid composition. Individuals which had fatty acid profiles similar to the high oleic acid parent (IMC 129) were advanced back to the field. Seeds (F 3 ) of selected individuals were planted in the field as selfing rows and m plots for preliminary yield and agronomic evaluations. At flowering the F 3 plants in the selfing rows were self- pollinated. At maturity the F 4 seed was harvested from individual plants to determine fatty acid composition. Yield of the individual selections was determined from the harvested plots. Based on fatty acid composition of the individual plants and yield and agronomic characteristics of the plots F 4 lines were selected and advanced to the next generation in the greenhouse. Five plants from each selected lme were self-pollinated. At maturity the F 5 seed was harvested from each and analyzed for fatty acid composition. The F s lme with the highest oleic fatty profile was advanced to the field as a selfing row. The remaining F 5 seed from the five plants was bulked together for planting the yield plots m the field. At flowering, the F s plants in each selfmg-row were self-pollinated. At maturity the F 6 self-pollinated seed was harvest from the selfing row to determine fatty acid composition and select for the high oleic acid trait . Yield of the individual selections was determined from the harvested plots . Fifteen F 6 lines having the high oleic fatty profile of IMC 129 and the desired agronomic characteristics were advanced to the greenhouse to increase seed for field trialmg. At flowering the F 6 plants were self-pollinated. At maturity the F 7 seed was harvested and analyzed for fatty acid composition. Three F 7 seed lines which had fatty acid profiles most similar to IMC 129 (Table XIII) were selected and planted m the field as selfing rows, the remaining seed was bulked together for yield trialmg. The high oleic fatty acid profile of IMC 129 was maintained through seven generations of selection for fatty acid and agronomic traits m an agronomic background of Brassica napus which was different from the parental lines. Thus, the genetic trait from IMC 129 for high oleic acid can be used in the development of new high oleic Brassica napus varieties. TABLE XIII Fatty Acid Composition of Advanced Breeding Generation with High Oleic Acid Trait (IMC 129 X Legend) Fatty Acid Composition (%) F 7 Selections of 89B60303 *-16 0 (-18 0 -18 1 (-18 2 ^-18 3 93.06194 3.8 1.6 78.3 7.7 4.4 93.06196 4.0 2.8 77.3 6.8 3.4 93.06198 3.7 2.2 78.0 7.4 4.2 The high oleic acid trait of IMC 129 was also introduced into a different genetic background by combining crossing and selection methods with the generation of dihaploid populations from the microspores of the F x hybrids. IMC 129 was crossed to Hyola 41, a commercial spring Brassica napus variety. Hyola 41 has the followmg fatty acid composition: C 16 0 - 3.8%, C 18 0 - 2.7%, C lβ ! - 64.9%, C 182 - 16.2%, C 1B 3 - 9.1%. The cross and progeny resulting from the cross were labeled 90DU.146. The F j seed was planted from the cross and a dihaploid (DH X ) population was made from the F λ microspores using standard procedures for Brassica napus . Each OH 1 plant was self-pollinated at flowering to produce OH 1 seed. At maturity the D^ seed was harvested and analyzed for fatty acid composition. DH X individuals which expressed the high oleic fatty acid profit of IMC 129 were advanced to the next generation m the greenhouse. For each individual selected five DH j seeds were planted. At flowering the DH 2 plants were self- pollmated. At maturity the DH 2 seed was harvested and analyzed for fatty acid composition. The DH 2 seed which was similar in fatty acid composition to the IMC 129 parent was advanced to the field as a selfing row. The remaining DH 2 seed of that group was bulked and planted in plots to determine yield and agronomic characteristics of the lme. At flowering individual DH 3 plants m the selfing row were self-pollinated. At maturity the DH 3 seed was harvested from the individual plants to determine fatty acid composition. Yield of the selections was determined from the harvested plots. Based on fatty acid composition, yield and agronomic characteristics selections were advanced to the next generation in the greenhouse. The DH 4 seed produced in the greenhouse by self-pollination was analyzed for fatty acid composition. Individuals which were similar to the fatty acid composition of the IMC 129 parent were advanced to the field to test for fatty acid stability and yield evaluation. The harvested DH 5 seed from six locations maintained the fatty acid profile of the IMC 129 parent (Table XIV) . TABLE XIV Fatty Acid Composition of Advanced Dihaploid Breeding Generation with High Oleic Acid Trait (IMC 129 X Hvola41) Fatty Acid Composition (%) DH5 of 90DU.146 at Multiple Locations C 160 C 18 0 C 18 C 182 C 1B Aberdeen 3, .7 2.6 75.4 8.1 7.2 Blackfoot 3, .3 2.4 75.5 8.8 7.5 Idaho Falls 3 , .7 3.1 75.0 7.5 8.1 Rexberg 3 .9 3.7 75.3 7.0 6.5 Swan Valley 3 .5 3.4 74.5 7.0 7.3 Lamont 3 .9 2.8 72.0 10.1 8.4 EXAMPLE 10 Canola Lines 0508 and 04275 Seeds of the B. napus lme IMC-129 were mutagenized with methyl N-nitrosoguanidine (MNNG) . The MNNG treatment consisted of three parts: pre-soak, mutagen application, and wash. A 0.05M Sorenson' s phosphate buffer was used to mamtam 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 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. A lOmM concentration of MNNG in 0.05M Sorenson' s buffer, pH 6.1, was prepared prior to use. Fifteen ml of 10m MNNG was added to the seeds m each plate. The seeds were incubated at 22 0 C±3 0 C m the dark under constant agitation for four (4) hours. At the end of the incubation period, the mutagen solution was removed. 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 mto 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 lme 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 usmg 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 XV shows the fatty acid composition of Q508 and IMC 129. The M4 selfed seed maintained the selected high oleic-low linoleic acid phenotype (Table XVI) . TABLE XV Fatty Acid Composition of A129 and High Oleic Acid M3 Mutant Q508 Line # 16 :0 18:0 18 :1 18 :2 18:3 A129 * 4 . 0 2 . 4 77 . 7 7 . 8 4 . 2 0508 3 . 9 2 . 1 84 . 9 2 . 4 2 . 9 "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 m 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 m the greenhouse tended to be reduced in vigor compared to Westar. However, Q508 yields m the greenhouse were greater than Q508 yields m the field. TABLE XVI Fatty Acid Composition of Seed Oil from Greenhouse-Grown Q508, IMC 129 and Westar, FDA Line 16 :0 18 :0 18 :1 18:2 18:3 Sats IMC 4.0 2.4 77.7 7.8 4.2 6.4 129 a Westar" 3.9 1.9 67.5 17.6 7.4 >5.8 Q508 c 3.9 2.1 84.9 2.4 2.9 6.0 a Average of 50 self-pollinated plants b Data from Example 1 c Average of 50 self-pollinated plants Nine other M4 high-oleic low-l oleic 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 lme of the Q4275 Cyclone cross. Referring to Table XVII, 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. After more than seven generations of selfing of Q4275, plants of Q4275, IMC 129 and 93GS34 were field grown during the summer season. The selections were tested m 4 replicated plots (5 feet X 20 feet) in a randomized block design. Plants were open pollinated. No selfed seed was produced. Each plot was harvested at maturity, and a sample of the bulk harvested seed from each lme was analyzed for fatty acid composition as described above. The fatty acid compositions of the selected lmes are shown in Table XVII. Table XVII Fatty Acid Composition of Field Grown IMC 129, Q4275 and 93GS34 Seeds Lme Fatty Acid Composition (%) Ci6 0 c *-lB 0 Clβ 1 (-18 2 (-IB 3 FDA Sats IMC 129 3.3 2.4 76.7 8.7 5.2 5.7 Q4275 3.7 3.1 82.1 4.0 3.5 6.8 93GS34-179 2.6 2.7 85.0 2.8 3.3 5.3 The results shown in Table XVII show that Q4275 maintained the selected high oleic - low linoleic acid phenotype under field conditions. The agronomic characteristics of Q4275 plants were superior to those of Q508. M 4 generation Q508 plants were crossed to a dihaploid selection of Westar, with Westar serving as the female parent. The resulting Fl seed was termed the 92EF population. About 126 Fl individuals that appeared to have better agronomic characteristics than the Q508 parent were selected for selfing. A portion of the F 2 seed from such individuals was replanted in the field. Each F2 plant was selfed and a portion of the resulting F3 seed was analyzed for fatty acid composition. The content of oleic acid m F 3 seed ranged from 59 to 79%. No high oleic (>80%) individuals were recovered with good agronomic type. A portion of the F 2 seed of the 92EF population was planted in the greenhouse to analyze the genetics of the Q508 lme. F 3 seed was analyzed from 380 F2 individuals. The C 18 λ 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 has 62-67% oleic acid. The possible genotypes at each gene in a cross of Q508 by Westar may be designated as: AA = Westar Fad2 a BB = Westar Fad2 b aa = Q508 Fad2 a bb = Q508 Fad2 b Assuming independent segregation, a 1:4:6:4:1 ratio of phenotypes is expected. The phenotypes of heterozygous plants are assumed to be indistinguishable and, thus, the data were tested for fit to a 1:14:1 ratio of homozygous Westar: heterozygous plants: homozygous Q508. Phenotypic # of Ratio Weistar Alleles Genotype 1 4 AABB (Westar) 4 3 AABb,AaBB,AABb,AaBB 6 2 AaBb,AAbb,AaBb,AaBb, aaBB,AaBb 4 1 Aabb, aaBb,Aabb, aaBb 1 0 aabb (Q508) Using Chi-square analysis, the oleic acid data fit a 1:14:1 ratio. It was concluded that Q508 differs from Westar by two major genes that are semi-dominant and additive and that segregate independently. By comparison, the genotype of IMC 129 is aaBB. The fatty acid composition of representative F3 individuals having greater than 85% oleic acid in seed oil is shown in Table XVIII. The levels of saturated fatty acids are seen to be decreased in such plants, compared to Westar. TABLE XVIII 92EF F ? Individuals with >85% C 1B , in Seed Oil F3 Plant Fatty Acid Composition (%) Identifier C16:0 C18 :0 C18:l C18:2 C18:3 FDASA +38068 3.401 1.582 85.452 2.134 3.615 4.983 +38156 3.388 1.379 85.434 2.143 3.701 4.767 +38171 3.588 1.511 85.289 2.367 3.425 5.099 +38181 3.75 1.16 85.312 2.968 3.819 4.977 +38182 3.529 0.985 85.905 2.614 3.926 4.56 +38191 3.364 1.039 85.737 2.869 4.039 4.459 +38196 3.557 1.182 85.054 2.962 4.252 4.739 +38202 3.554 1.105 86.091 2.651 3.721 4.713 +38220 3.093 1.16 86.421 1.931 3.514 4.314 +38236 3.308 1.349 85.425 2.37 3.605 4.718 +38408 3.617 1.607 85.34 2.33 3.562 5.224 +38427 3.494 1.454 85.924 2.206 3.289 4.948 +38533 3.64 1.319 85.962 2.715 3.516 4.959 EXAMPLE 11 Leaf and Root Fatty Acid Profiles of Canola Lines IMC-129, Q508, and Westar 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 XIX. The data m Table XIX indicate that total leaf lipids in Q508 are higher in C 18 1 content than the C 18 2 plus C 18 3 content. The reverse is true for Westar and IMC 129. The difference in total leaf lipids between Q508 and IMC 129 is consistent with the hypothesis that a second Fad2 gene is mutated in Q508. The C 16 3 content in the total lipid fraction was about the same for all three lines, suggesting that the plastid FadC gene product was not affected by the Q508 mutations. To confirm that the FadC gene was not mutated, chloroplast lipids were separated and analyzed. No changes in chloroplast C 16:1 , C 162 or C 163 fatty acids were detected in the three lines. The similarity in plastid leaf lipids among Q508, Westar and IMC 129 is consistent with the hypothesis that the second mutation in Q508 affects a microsomal Fad2 gene and not a plastid FadC gene. TABLE XIX MATURE EXPANDING LEAF LEAF PETIOLE ROOT West 12 9 30508 West 12 9 30508 West 12 9 3Q508 West 12 9 3Q508 16 0 12 1 1 1 9 10 1 16 4 16 1 1 1 3 2 1 7 23 S 1 1 9 2 1 1 2 1 9 12 0 16 1 0 8 0 6 1 1 0 7 0 6 1 1 1 0 1 3 1 4 - 1 6 2 2 3 2 2 2 0 2 8 3 1 2 8 1 8 2 2 1 8 16 3 14 7 15 0 14 0 6 3 5 4 6 9 5 7 4 6 5 7 18 0 2 2 1 6 1 2 2 5 2 B 1 5 3 7 4 0 1 6 3 6 2 9 2 5 18 1 2 8 4 9 16 7 3 8 8 3 3 8 0 4 9 12 9 46 9 3 5 6 1 68 8 18 2 12 6 11 5 6 8 1 3 3 13 8 4 9 2 0 7 18 3 5 2 28 0 30 4 4 4 I B 3 50 6 50 3 46 0 54 2 50 0 33 5 40 4 33 2 2 5 3 43 8 3 8 7 12 3 EXAMPLE 12 Sequences of Mutant and Wild-Type Delta-12 Fatty Acid Desaturases from B . napus Primers specific for the FAD2 structural gene were used to clone the entire open reading frame (ORF) of the D and F 12-DES genes by reverse transcriptase polymerase chain reaction (RT-PCR) . 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. Sequence analysis revealed a G to A transversion at nucleotide 316 (from the translation initiation codon) of the D gene in both IMC 129 (SEQ ID NO:3) and Q508, compared to the sequence of Westar (SEQ ID NO:l) . The transversion changes the codon at this position from GAG to AAG and results in a non-conservative substitution of glutamic acid, an acidic residue, for lysine a basic residue. The presence of the same mutation in both lines was expected since the 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 m the coding region of the D gene of rapeseed microsomal delta 12-desaturase. A smgle base transition from T to A at nucleotide 515 of the F gene was detected m Q508 compared to the Westar sequence. The mutation changes the codon at this position from CTC to CAC, resulting m the non- conservative substitution of a non-polar residue, leucine, for a polar residue, histidme, m the resulting gene product. No mutations were found m the F gene sequence of IMC 129 compared to the F gene sequence of Westar. These data support the conclusion that a mutation in a delta-12 desaturase gene sequence results m alterations in the fatty acid profile of plants containing such a mutated gene. Moreover, the data show that when a plant lme or species contams two delta-12 desaturase loci, the fatty acid profile of an individual havmg two mutated loci differs from the fatty acid profile of an individual having one mutated locus. The mutation in the D gene of IMC 129 and Q508 mapped to a region having a conserved ammo acid motif (His-Xaa-Xaa-Xaa-His) found m cloned delta-12 and delta- 15 membrane bound-desaturases (Table XX) . Table XX Alignment of Ammo Acid Sequences of Cloned Canola Membrane Bound-Desaturases Desaturase Gene Sequence 3 Position Canola-fad2-D(mutant) AHKCGH 109-114 Canola-Fad2-D AHECGH 109-114 Canola-Fad2-F AHECGH 109-114 Canola-FadC GHDCAH 170-175 Canola-fad3 (mutant) GHKCGH 94-99 Canola-Fad3 GHDCGH 94-99 Canola-FadD GHDCGH 125-130 (FadD = Plastid delta 15, Fad3 = Microsomal delta-15) , (FadC = Plastid delta-12, Fad2 = Microsomal delta-12) a One letter amino acid code; conservative substitutions are underlined; non-conservative substitutions are bold. EXAMPLE 13 Transcription and Translation of Microsomal Delta-12 Fatty Acid Desaturases Transcription in vivo was analyzed by RT-PCR analysis of stage II and stage III developing seeds and leaf tissue. The primers used to specifically amplify 12-DES 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 12-DES D gene RNA from the indicated tissues were sense primer 5' -GTTATGAAGCAAAGAAGAAAC-3 ' and antisense primer 5' -GTTTCTTCTTTGCTTCATAAC-3' . The results indicated that mRNA of both the D and F gene was expressed m seed and leaf tissues of IMC 129, Q508 and wild type Westar plants. In 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 ammo acid sequence of each gene and the cotranslational addition of a microsomal membrane peptide. These results rule out the possibility that non¬ sense or frameshift mutations, resulting in a truncated polypeptide gene product, are present in either the mutant D gene or the mutant F gene. The data, in conjunction with the data of Example 12, support the conclusion that the mutations in Q508 and IMC 129 are in delta-12 fatty acid desaturase structural genes encoding desaturase enzymes, rather than in regulatory genes. EXAMPLE 14 Development of Gene-Specific PCR Markers Based on the smgle base change in the mutant D gene of IMC 129 described m 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- ype 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 smgle base mutation m 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. EXAMPLE 15 Transformation with Mutant and Wild Type Fad3 Genes B . napus cultivar Westar was transformed with mutant and wild type Fad3 genes to demonstrate that the mutant Fad3 gene for canola cytoplasmic linoleic desaturase 15-DES is nonfunctional. Transformation and regeneration were performed using disarmed Agrobacteri um tumefaciens essentially following the procedure described m WO 94/11516. Two disarmed Agrobacterium strains were engineered, each containing a Ti plasmid having the appropriate gene linked to a seed-specific promoter and a correspondmg termination sequence. The first plasmid, pIMCHO, was prepared by inserting mto a disarmed Ti vector the full length wild type Fad3 gene m sense orientation (nucleotides 208 to 1336 of SEQ ID 6 m WO 93/11245) , flanked by a napm promoter sequence positioned 5' to the Fad3 gene and a napm termination sequence positioned 3' to the Fad3 gene. The rapeseed napm promoter is described in EP 0255378. The second plasmid, pIMC205, was prepared by inserting a mutated Fad3 gene in sense orientation mto 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 ammo acid at codon 96 of the gene product was thereby changed from aspartic acid to lysine. See Table XX. A bean ( Phaseolus vulgaris) phaseolin (7S seed storage protein) promoter fragment of 495 base pairs, starting with 5'- TGGTCTTTTGGT-3 ' , was placed 5' to the mutant Fad3 gene and a phaseolin termination sequence was placed 3' to the mutant Fad3 gene. 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 discemable stems formed from the callus, shoots were excised and transferred to elongation medium. The elongated shoots were cut, dipped in Rootone™, rooted on an agar medium and transplanted to potting soil to obtain fertile Tl plants. T2 seeds were obtained by selfing the resulting Tl plants. Fatty acid analysis of T2 seeds was carried out as described above. The results are summarized in Table XXI. Of the 40 transformants obtained using the pIMCHO plasmid, 17 plants demonstrated wild type fatty acid profiles and 16 demonstrated overexpression. A proportion of the transformants are expected to display an overexpression phenotype when a functioning gene is transformed in sense orientation into plants. Of the 307 transformed plants having the pIMC205 gene, none exhibited a fatty acid composition indicative of overexpression. This result indicates that the mutant fad3 gene product is non-functional , since some of the transformants would have exhibited an overexpression phenotype if the gene product were functional. Table XXI Overexpression and Co-suppression Events in Westar Populations Transformed with pIMC205 or pIMCHO Construct Number of α-Linolenic Overexpression Co-Suppressio Wild Type Transformants Acid Events Events Events Range <%) (>10% linolenic) (<4.0 linolenic) pIMCHO 40 2 4 20 6 16 7 17 PIMC205 307 4.6 - 10.4 0 0 307 Fatty acid compositions of representative transformed plants are presented in Table XXII. 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 . Table XXII Fatty Acid Composition of T2 Seed From Westar Transformed With pIMC205 or pIMCHO . Line Fatty Acid Composition < ) C16:0 C18:0 C18: 1 C18:2 C18:3 652-09 pIMCHO 4 7 3 3 65 6 B 1 14 8 overexpression 663-40 4 9 2 1 62 5 23 2 3 6 pIMCHO co- suppression 205-284 3 7 1 8 68 8 15 9 6 7 PIMC205 To the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various specific embodiments herein described and illustrated may be further modified to incorporate features shown in other of the specific embodiments. The foregoing detailed description has been provided for a better understanding of the invention only and no unnecessary limitation should be understood therefrom as some modifications will be apparent to those skilled in the art without deviating from the spirit and scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Cargill, Incorporated (ii) TITLE OF INVENTION: PLANTS HAVING MUTANT SEQUENCES THAT CONFER ALTERED FATTY ACID PROFILES (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Fish & Richardson, P.C., P.A. (B) STREET: 60 South Sixth Street, Suite 3300 (C) CITY: Minneapolis (D) STATE: MN (E) COUNTRY: USA (F) ZIP: 55402 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOΞ (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: PCT/US96/ (B) FILING DATE: 13-DEC-1996 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/572,027 (B) FILING DATE: 14-DEC-1995 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Ellinger, Mark S. (B) REGISTRATION NUMBER: 34,812 (C) REFERENCE/DOCKET NUMBER: 07148/049WO1 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 612/335-5070 (B) TELEFAX: 612/288-9696 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1155 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Brassica napus (ix) FEATURE: (D) OTHER INFORMATION: Wild type D form. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1. ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAG AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 GAA ACC GAC ACC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192 lie Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 TGC TTC TAC TAC NTC GCC ACC ACT TAC TTC CCT CTC CTC CCT CAC CCT 240 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAA GGG TGC GTC 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95 CTA ACC GGC GTC TGG GTC ATA GCC CAC GAA TGC GGC CAC CAC GCC TTC 336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 AGC GAC TAC CAG TGG CTT GAC GAC ACC GTC GGT CTC ATC TTC CAC TCC 384 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGC AGC CAC 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His 130 135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCG TTG 576 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 TAC TTA GCC TTC AAC GTC TCG GGA AGA CCT TAC GAC GGC GGC TTC CGT 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 TTC CGT TAC GCC GCC GGC CAG GGA GTG GCC TCG ATG GTC TGC TTC TAC 768 Phe Arg Tyr Ala Ala Gly Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 GGA GTC CCG CTT CTG ATT GTC AAT GGT TTC CTC GTG TTG ATC ACT TAC 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAC GAT TCG TCC GAG TGG 864 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 GAT TGG TTC AGG GGA GCT TTG GCT ACC GTT GAC AGA GAC TAC GGA ATC 912 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 TTG AAC AAG GTC TTC CAC AAT ATT ACC GAC ACG CAC GTG GCC CAT CAT 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 CCG TTC TCC ACG ATG CCG CAT TAT CAC GCG ATG GAA GCT ACC AAG GCG 1008 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTC GAT GGG ACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TAT GTG GAA CCG 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 GA 1155 (2) INFORMATION FOR SEQ ID NO:2: (l) SEQUENCE CHARACTERISTICS. (A) LENGTH: 384 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear i) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION. SEQ ID NO: 2 Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val lie Ala His Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 Phe Arg Tyr Ala Ala Gly Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 (2) INFORMATION FOR SEQ ID NO:3 : U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1155 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Uli) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Brassica napus (vii) IMMEDIATE SOURCE: (B) CLONE: IMC129 (ix) FEATURE: (D) OTHER INFORMATION: G to A transversion mutation at nucleotide 316 of the D form. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAG AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 GAA ACC GAC ACC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 TGC TTC TAC TAC NTC GCC ACC ACT TAC TTC CCT CTC CTC CCT CAC CCT 240 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAA GGG TGC GTC 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 * 90 95 CTA ACC GGC GTC TGG GTC ATA GCC CAC AAG TGC GGC CAC CAC GCC TTC 336 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His Ala Phe 100 105 110 AGC GAC TAC CAG TGG CTT GAC GAC ACC GTC GGT CTC ATC TTC CAC TCC 384 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGC AGC CAC 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His 130 135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCG TTG 576 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 TAC TTA GCC TTC AAC GTC TCG GGA AGA CCT TAC GAC GGC GGC TTC CGT 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 TTC CGT TAC GCC GCC GGC CAG GGA GTG GCC TCG ATG GTC TGC TTC TAC 768 Phe Arg Tyr Ala Ala Gly Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 GGA GTC CCG CTT CTG ATT GTC AAT GGT TTC CTC GTG TTG ATC ACT TAC 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAC GAT TCG TCC GAG TGG 864 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 GAT TGG TTC AGG GGA GCT TTG GCT ACC GTT GAC AGA GAC TAC GGA ATC 912 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 TTG AAC AAG GTC TTC CAC AAT ATT ACC GAC ACG CAC GTG GCC CAT CAT 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 CCG TTC TCC ACG ATG CCG CAT TAT CAC GCG ATG GAA GCT ACC AAG GCG 1008 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lvs Ala 325 330 335 ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTC GAT GGG ACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TAT GTG GAA CCG 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 GA 1155 (2) INFORMATION FOR SEQ ID NO:4 : U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 384 amino acids (B) TYPE: ammo acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 Phe Arg Tyr Ala Ala Gly Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 * 285 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr H s Val Ala His His 305 310 315 320 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 (2) INFORMATION FOR SEQ ID NO: 5. U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1155 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Brassica napus (ix) FEATURE: (D) OTHER INFORMATION: Wild type F form. (xi) SEQUENCE DESCRIPTION- SEQ ID NO.5 ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAA AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lvs Ser 1 5 10 15 GAA ACC GAC AAC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 TGC TTC TAC TAC GTC GCC ACC ACT TAC TTC CCT CTC CTC CCT CAC CCT 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAG GGC TGC GTC 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95 CTA ACC GGC GTC TGG GTC ATA GCC CAC GAG TGC GGC CAC CAC GCC TTC 336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 AGC GAC TAC CAG TGG CTG GAC GAC ACC GTC GGC CTC ATC TTC CAC TCC 384 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGA CGC CAC 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCT TTG 576 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 TAC TTA GCC TTC AAC GTC TCG GGG AGA CCT TAC GAC GGC GGC TTC GCT 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 TAC CGC TAC GCT GCT GTC CAA GGA GTT GCC TCG ATG GTC TGC TTC TAC 768 Tyr Arg Tyr Ala Ala Val Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 GGA GTT CCG CTT CTG ATT GTC AAT GGG TTC TTA GTT TTG ATC ACT TAC 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAT GAC TCG TCT GAG TGG 864 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 GAT TGG TTG AGG GGA GCT TTG GCC ACC GTT GAC AGA GAC TAC GGA ATC 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 TTG AAC AAG GTC TTC CAC AAT ATC ACG GAC ACG CAC GTG GCG CAT CAC 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 CTG TTC TCG ACC ATG CCG CAT TAT CAT GCG ATG GAA GCT ACG AAG GCG 1008 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTG CAT GGG ACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Leu His Gly Thr Pro Val 340 345 350 GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TAT GTG GAA CCG 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 GA 1155 (2) INFORMATION FOR SEQ ID NO:6 : U) SEQUENCE CHARACTERISTICS (A) LENGTH: 384 ammo acids (D) TOPOLOGY: linear Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 • Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val lie Ala His Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 " 240 Tyr Arg Tyr Ala Ala Val Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Leu His Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 (2) INFORMATION FOR SEQ ID NO: 7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1155 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Brassica napus (vii) IMMEDIATE SOURCE: (B) CLONE: Q508 Ux) FEATURE: (D) OTHER INFORMATION: T to A transversion mutation at nucleotide 515 of the F form. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAA AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 GAA ACC GAC AAC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 TGC TTC TAC TAC GTC GCC ACC ACT TAC TTC CCT CTC CTC CCT CAC CCT 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAG GGC TGC GTC 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95 CTA ACC GGC GTC TGG GTC ATA GCC CAC GAG TGC GGC CAC CAC GCC TTC 336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 AGC GAC TAC CAG TGG CTG GAC GAC ACC GTC GGC CTC ATC TTC CAC TCC 384 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGA CGC CAC 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CAC AAC AAC CCT TTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr His Asn Asn Pro Leu 165 170 175 GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCT TTG 576 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 TAC TTA GCC TTC AAC GTC TCG GGG AGA CCT TAC GAC GGC GGC TTC GCT 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672 Cys His Phe His Pro Asn Ala Pro lie Tyr Asn Asp Arg Glu Arg Leu 210 215 220 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 TAC CGC TAC GCT GCT GTC CAA GGA GTT GCC TCG ATG GTC TGC TTC TAC 768 Tyr Arg Tyr Ala Ala Val Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 GGA GTT CCG CTT CTG ATT GTC AAT GGG TTC TTA GTT TTG ATC ACT TAC 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAT GAC TCG TCT GAG TGG 864 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 GAT TGG TTG AGG GGA GCT TTG GCC ACC GTT GAC AGA GAC TAC GGA ATC 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 TTG AAC AAG GTC TTC CAC AAT ATC ACG GAC ACG CAC GTG GCG CAT CAC 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 CTG TTC TCG ACC ATG CCG CAT TAT CAT GCG ATG GAA GCT ACG AAG GCG 1008 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTG CAT GGG ACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Leu His Gly Thr Pro Val 340 345 350 GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TAT GTG GAA CCG 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 GA 1155 (2) INFORMATION FOR SEQ ID NO: 8 : U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 384 amino acids (B) TYPE: ammo acid (D) TOPOLOGY: linear Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION. SEQ ID NO: 8 : Met Gly Ala Gly Gly Arg Met Gin Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 " 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gin Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr His Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu Thr Val Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gin Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 Tyr Arg Tyr Ala Ala Val Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Leu His Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380

Claims: WHAT IS CLAIMED IS: 1. An isolated nucleic acid fragment comprising a sequence of at least about 10 nucleotides from a Brassicaceae or Helianthus delta-12 fatty acid desaturase gene having at least one mutation, wherem said gene is effective for altering fatty acid composition in Brassi caceae or Helianthus seeds and wherem said sequence mcludes said at least one mutation. 2. The nucleic acid fragment of claim 1, wherein said sequence comprises a full-length coding sequence of said gene . 3. The nucleic acid fragment of claim 1, wherein said mutant desaturase gene encodes a microsomal gene product . 4. The nucleic ac d fragment of claim 1, wherein said at least one mutation compriseε a mutation in a region of said desaturase gene encoding a His-Glu-Cys-Gly-His am o acid motif . 5. The nucleic acid fragment of claim 4, wherein said at least one mutation comprises a non-conservative am o acid substitution in said region. 6. The nucleic acid fragment of claim 5, wherein said at least one mutation comprises the sequence His- ys-Cys- 7. The nucleic acid fragment of claim 1, wherein said mutant desaturase gene is from a Brassi ca napus plant. 8. The nucleic ac d fragment of claim 1, wherein said gene is the D form of a Brassi caceae microsomal gene. 9. The nucleic acid fragment of claim 1, wherein said at least at least one mutation comprises the sequence ys-Tyr-His-Asn-Asn-Pro. 10. A plant of the Brassi caceae or Helianthus families other than Brassica napus, said plant containing a sequence of at least 10 nucleotides from a delta-12 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a His-Xaa-Xaa- Xaa-His amino acid motif and wherein said mutation confers an altered fatty acid composition in seeds of said plant . 11. The plant of claim 10, wherein said plant contains a full-length coding sequence of said mutant gene. 12. The plant of claim 10, wherein said motif comprises the sequence His-Glu-Cys-Gly-His. 13. The plant of claim 10, wherein said gene is from a Brassica napus plant . 14. The plant of claim 10, wherein said plant is a Brassica rapa plant. 15. An isolated nucleic acid fragment comprising a sequence of at least about 10 nucleotides from a Brassicaceae or Helianthus delta-15 fatty acid desaturase gene having at least one mutation, wherein said gene is effective for altering fatty acid composition in Brassicaceae or Helianthus seeds and wherein said sequence mcludes said at least one mutation. 16. The nucleic acid fragment of claim 15, wherem said sequence comprises a full-length coding sequence of said gene. 17. The nucleic acid fragment of claim 15, wherein said at least one mutation comprises a mutation in a region of said desaturase gene encoding a His-Asp-Cys- Gly-His amino acid motif. 18. The nucleic acid fragment of claim 15, wherein said mutant desaturase gene is from a Brassica napus plant. 19. A Brassi caceae or Helian thus plant containing a sequence of at least 10 nucleotides from a delta-15 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a His-Xaa-Xaa- Xaa-His ammo acid motif and wherein said mutation confers an altered fatty acid composition in seeds of said plant. 20. The plant of claim 19, wherein said plant contains a full-length coding sequence of said mutant gene. 21. The plant of claim 19, wherein said motif comprises the sequence His-Asp-Cys-Gly-His . 22. The plant of claim 19, wherein said mutant desaturase gene is from a Brassica napus plan . 23. The plant of claim 19, wherein said plant is a Brassi ca napus plant. 24. A Brassicaceae or Helianthus plant containing: a) a sequence of at least about 10 nucleotides from a delta-12 fatty acid desaturase gene having at least one mutation, said at least one delta-12 gene mutation in a region encoding a His-Xaa-Xaa- Xaa-His ammo acid mot if ; b) a sequence of at least 10 nucleotides from a delta-15 fatty acid desaturase gene having at least one mutation, said at least one delta-15 gene mutation in a region encoding a His-Xaa-Xaa- Xaa-His ammo acid motif, said delta-12 gene mutation and said delta-15 gene mutation conferring an altered fatty acid composition m seeds of said plant . 25. A Brassicaceae or Helian thus plant containing a sequence of at least about 10 nucleotides from a delta-12 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a Tyr- Leu-Asn-Asn-Pro ammo acid motif and wherein said mutation confers an altered fatty acid composition in seeds of said plant. 26. A vegetable oil extracted from seeds produced by the plant of claim 10. 27. The oil of claim 26, wherein said oil has, followmg crushing and extraction of said seeds, from about 1% to about 10% linoleic acid based on total fatty acid composition. 28. The oil of claim 26, wherein said oil has from about 69% to about 90% oleic acid based on total fatty acid composition. 29. A vegetable oil extracted from seeds produced by the plant of claim 19. 30. The oil of claim 29, wherein said oil has, followmg crushing and extraction of said seeds, from about 0.5% to about 10% αr-lιnolenιc acid based on total fatty acid composition. 31. A vegetable oil extracted from seeds produced by the plant of claim 24. 32. A vegetable oil extracted from seeds produced by the plant of claim 25. 33. A method for producing a Brassicaceae or Helianthus plant lme, comprising the steps of: a) inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species; b) obtaining one or more progeny plants from said cells; c) identifying at least one of said progeny plant that contains a delta-12 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a His- Xaa-Xaa-Xaa-His amino acid motif; and d) producing said plant line from said at least one progeny plant by self- or cross-pollmation, said plant line having said at least one delta-12 gene mutation. 34. The method of claim 33, wherein said plant lme produces seeds yielding an oil having a stabilized linoleic acid content from about 1% to about 14%. 35. The method of claim 33, further comprising the steps of : e) inducing mutagenesis m cells of said plant line; f) obtaining one or more progeny plants from said plant line cells; g) identifying at least one of said plant lme progeny plants that contains a delta-15 fatty acid desaturase gene having at least one delta-15 gene mutation, said at least one delta-15 gene mutation in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif; h) producing a second plant lme from said at least one plant line progeny plant by self- or cross-pollmation, said second plant line having said at least one delta-12 gene mutation and said at least one delta-15 gene mutation. 36. The method of claim 33, wherein said starting variety is a Brassica napus variety. 37. The method of claim 36, wherein said mutation is in a first form of delta-12 fatty acid desaturase. 38. The method of claim 37, further comprising the step of crossing a plant of said plant line to a plant having a mutation in a second form of delta-12 fatty acid desaturase. 39. The method of claim 38, wherein said second mutation is m a region other than a region encoding a His-Xaa-Xaa-Xaa-His ammo acid motif. 40. The method of claim 36, further comprising the steps of: e) inducing mutagenesis cells of said plant lme; f) obtaining one or more progeny plants from said plant lme cells; g) identifying at least one of said plant line progeny plants that contams a second delta-12 fatty acid desaturase gene having at least one mutation, said second gene mutation in a region other than a region encoding a His-Xaa-Xaa-Xaa-His ammo acid motif; and h) producing a second plant line from said at least one plant line progeny plant by self- or cross-pollination, said second plant line having said first delta-12 gene mutation and said second delta-12 gene mutation. 41. A method for producing a Brassicaceae or Helianthus plant lme, comprising the steps of: a) inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species; b) obtaining one or more progeny plants from said cells; c) identifying at least one of said progeny plants that contains a delta-15 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a His- Xaa-Xaa-Xaa-His ammo acid motif; and d) producing said plant lme from said at least one progeny plant by self- or cross-pollination, said plant lme having said delta-15 gene mutation.

Inventors: DEBONTE R LORIN, FAN ZHEGONG, LOH H-T WILLIE, DEBONTE, R., LORIN, FAN, ZHEGONG, LOH, H.-T., WILLIE
Applicants: CARGILL INC, DEBONTE R LORIN, FAN ZHEGONG, LOH H T WILLIE, CARGILL, INCORPORATED, DEBONTE, R., LORIN, FAN, ZHEGONG, LOH, H.-T., WILLIE
Application year: 1996
Main classification: A01H1
IPC codes: C07C53, C07C57, C12N9, C12N15