AU2303001A - Plants having mutant sequences that confer altered fatty acid profiles - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE

SPECIFICATION

FOR A DIVISIONAL PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT S. Name of Applicant: CARGILL, INCORPORATED Actual Inventor(s): Lorin R. DeBonte, Zhegong Fan and Willie Loh *o Address for Service: CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia Invention Title: PLANTS HAVING MUTANT SEQUENCES THAT CONFER ALTERED FATTY ACID PROFILES The following statement is a full description of this invention, including the best method of performing it known to me:- 15/02/01,all 1877.cover,l 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 in saturated fats increase low density lipoproteins (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, 20 and Treatment, pp. 43-64, 1985). By producing oilseed Brassica varieties with reduced levels of individual and total saturated fats in the seed oil, oil-based food products which contain less saturated fats can be produced. Such products will benefit public health by 25 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 30 vegetable oils, lowers LDL as effectively as linoleic acid, but does not affect high density lipoproteins (HDL) levels (Mattson, J. Am. Diet. Assoc., 89:387-391, 1989; Mensink 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, New England J. Med., 314:745-748, 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, Circulation, 44(Suppl):1, 1970).

Many breeding studies have been conducted to improve the fatty acid profile of Brassica varieties.

Pleines and Freidt, Fat Sci. Technol., 90(5), 167-171 S(1988) describe plant lines with reduced C 18 :3 levels combined with high oleic content Rakow and McGregor, J. Amer. Oil Chem. Soc., 50, 400-403 (Oct.

1973) discuss problems associated with selecting mutants 20 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 o 25 genes through an interspecific cross from Brassica juncea :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 30 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. Canvin, 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 a-linolenic 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 a-linolenic 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 e time of its release.

20 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 25 low in 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 in linoleic acid S 30 tend to have high oleic acid content, and can be used in 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 a-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 0 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 a-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 20 microsomal delta-12 fatty acid desaturase are described in Lightner et al., W094/11516. Sequences of higher plant genes encoding microsomal and plastid fatty acid desaturases are disclosed in Yadav, 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- 30 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 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 Brassica [.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 20 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 25 upon total extractable oil. Progeny of said seeds; and So. *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 in this invention. Preferred are seeds, plant 30 lines producing seeds, andplants 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 eruci-c 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 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.

0 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 Q 2% based on 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 having a maximum palmitic acid content of about 3.5% and a maximum erucic acid content of about 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 Brassica 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 30 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 35 about 2% based upon total extractable oil. Progeny of 8 said seeds; and Brassica oil having 1) a minimum palmitic acid content of about 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 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 maximum stearic acid content of about 1.1% and maximum erucic acid content of about Preferred are seeds, plant lines producing seeds, and plants producing seeds having a palmitic acid content 20 of from about 0.8% to about 1.1% based on total S" 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 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 Brassicaceae or Helianthus seeds, plants and plant lines having at least one mutation that controls the levels of unsaturated fatty acids in plants. One embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a mutant delta-12 fatty acid desaturase conferring altered fatty composition in seeds when the fragment is present in a plant. A preferred sequence comprises a mutant sequence as shown in 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 Brassica 0 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.

S 20 Another embodiment of the invention involves a method of producing a Brassicaceae or Helianthus plant line comprising the steps inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species; obtaining progeny plants from the 25 mutagenized cells; identifying progeny plants that contain a mutation in a delta-12 or delta-15 fatty acid desaturase gene; and producing a plant line by selfing.

Yet another embodiment of the invention involves a 30 method of producing plant lines containing altered levels of unsaturated fatty acids comprising: crossing a first plant with a second plant having a mutant delta-12 or delta-15 fatty acid desaturase; obtaining seeds from the cross of step growing fertile plants 35 from such seeds; obtaining progeny seed the plants of step and 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 Ficures S 10 Figure 1 is a histogram showing the frequency distribution of seed oil oleic acid (C, 8 content in a segregating population of a Q508 X Westar cross. The bar labeled WSGA 1A represents the content of the Westar parent. The bar labeled Q508 represents the content 15 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, 4 palmitic and stearic (C.

80 acids.

20 The term "FDA saturates" as used herein means this abovedefined 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 30 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 "Mo 0 l is untreated seed. As used herein, is the seed (and resulting plants) exposed to a mutagenic agent, while "M 2 1 is the progeny (seeds and plants) of self-pollinated

M,

plants,

"M

3 is the progeny of self-pollinated

M

2 plants, and is the progeny of self-pollinated

M

3 plants.

"Ms" is the progeny of self-pollinated

M

4 plants.

,M

6 1 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 The method of invention is capable of creating lines with improved fatty acid compositions stable up to from generation to generation. The 25 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.

0 30 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 Amol glucosinolates/gram.

"Canola" as used herein refers to plant variety seed or oil which contains less than 2% 'rucic acid (C 22 and meal with less than 30 gmol 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, a decreased, stabilized level of a-linolenic acid.

Applicants have further discovered isolated nucleic acid fragments comprising sequences that carry mutations within the coding sequence of delta-12 or desaturases. The mutations confer desirable alterations in fatty acid levels in the seed oil of 20 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. fatty acid desaturase is also known on omega-3 fatty acid desaturase and is sometimes referred to herein as 25 A nucleic acid fragment of the invention contains a mutation in a microsomal delta-12 fatty acid desaturase coding sequence or in 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 o* 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 13 corresponding levels in plant tissues expressing the wild-type sequence. The non-functionality of the gene product can be inferred from the decreased level of reaction product (a-linolenic 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, 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, PCR primer, sitedirected 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 fatty acid desaturase.

A mutation in a nucleic acid fragment of the S 20 invention may be in any portion of the coding sequence that renders the resulting gene product non-functional.

Suitable types of mutations include, without limitation, insertions of nucleotides, deletions of nucleotides, or transitions and transversions in the wild-type coding sequence. Such mutations result in insertions of one or more amino acids, deletions of one or more amino acids, and non-conservative amino acid substitutions in the corresponding gene product. In some embodiments, the sequence of a nucleic acid fragment may comprise more 30 than one mutation or more than one type of mutation.

Insertion or deletion of amino acids in a coding sequence may, for example, disrupt the conformation of *ooessential alpha-helical or beta-pleated sheet regions of the resulting gene product. Amino acid insertions or deletions may also disrupt binding or catalytic sites 14 important for gene product activ'ty. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids.

Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions may also make a substantial change in the bulk of the residue side chain, substituting an alanyl residue for a isoleucyl residue.

Examples of non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid. Because there are only 20 amino acids encoded in a gene, substitutions that result in a non-functional gene product may be determined by routine.experimentation, 20 incorporating amino acids of a different class in the region of the gene product targeted for mutation.

Preferred mutations are in a region of the nucleic acid having an amino acid sequence motif that is conserved among delta-12 fatty acid desaturases or delta- 25 15 fatty acid desaturases, such as a His-Xaa-Xaa-Xaa-His motif (Tables An example of a suitable region has a conserved HECGH motif that is found, for example, in nucleotides corresponding to amino acids 105 to 109 of the Arabidopsis and Brassica delta-12 desaturase sequences, in nucleotides corresponding to amino acids 101 to 105 of the soybean delta-12 desaturase sequence o* and in nucleotides corresponding to amino 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 amino acid designations used herein are described in Alberts, B. et al., Molecular Biology of the Cell, 3rd edition, Garland Publishing, New York, 1994.

Amino 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 invention. An illustrative embodiment of a mutation in a nucleic acid fragment of the invention is a Glu to Lys substitution in the HECGH motif of a Brassica microsomal delta-12 desaturase sequence, either the D form or the F form. This mutation results in the sequence HECGH being changed to HKCGH as seen by comparing SEQ ID NO:2 (wildtype D form) to SEQ ID NO:4 (mutant D form).

A similar motif may be found at amino acids 101 to 105 of the Arabidopsis microsomal delta-15 fatty acid desaturase, as well as in the corresponding rape and soybean desaturases (Table See, WO 93/11245; Arondel, V. et al., Science, 258:1153-1155 (1992); Yadav, N. et al., Plant Physiol., 103:467-476 (1993). Plastid fatty acids have a similar motif (Table 20 Among the types of mutations in an HECGH motif that render the resulting gene product non-functional are non-conservative substitutions. An illustrative example Sof a non-conservative substitution is substitution of a glycine residue for either the first or second histidine.

Such a substitution replaces a polar residue (histidine) with a non-polar residue (glycine). Another type of mutation that renders the resulting gene product nonfunctional is an insertion mutation, insertion of a glycine between the cystine and glutamic acid residues in 30 the HECGH motif.

Other regions having suitable conserved amino acid motifs include the HRRHH motif shown in Table 2, the SHRTHH motif shown in Table 6 and the HVAHH motif shown in Table 3. See, WO 94/115116; Hitz, W. et al., Plant Physiol., 105:635-641 (1994); Okuley, 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 Brassica desaturase sequence. An illustrat-ive 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 Amino Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Position Arabldopsis tbaliana 100-129 Glycine max 96-125 Zea mays 106-135 Ricinus communisa 1- 29 Brassica napus D 100-128 Brassica napus F 100-128 .a from plasmid pRF2-1C Amino Acid Sequence IWVIAHECGH HAFSDYQWLD DTVGLIFHSF VWVAHEECGH HAFSKMQWVD DVVGLTLHST VWVIAHECGH HAFSDYSLLD) DVVGLVLHSS WVMAHDCGH HAFSDYQLLD DVVGLILHSC VWVIAHECGH HAFSDYQWLD) DTVGLIFHS VWVIAHECGH HAFSDYQWLD DTVGLIFHS 0: TABLE 2 Alignment of Amino Acid Sequences from Microsomal Delta-12 Fatty Acid Desaturases Species Position Arabldopsis thaliana 130-158 Glycine max 126-154 Zea maya 136-164 Ricinus comunisa 30- S8 30 Brassica napus D 130-158 Brassica napus F 130-158 a from plasmid pRF2-1C Amino Acid Sequence LLVPYFSWKY SHRRHHSNTG SLERDEVFV LLVPYFSWKI SHRRHHSNTG SLDRflEVFV IMVPYFSWKY SERRBHSZNTG SLERDEVFV LLVPYFSWKH SEIUMffS1TG SLERDEVFV LLVPYFSWXY SHRSKHSNTG SLERDEVFV LLVPYFSWKY SHRRHHSNTG SLERDEVFV TABLE 3 Alignment of Amino Acid Sequences from Microsomal Delta-12 Fatty, Acid Desaturases species Arabldopsis thaij Glycine max Zea mays Ricinus communis Brassica napus D Brassica napuS F Position Amino Acid Sequence axaa 298-333 294-329 305-340 198-224 299- 334 299-334 DRDYGIIMICV FHNITD771VA HULFS774PHY NAI4SAT DRDYGILNKV FHHITDT{VA HHLFSTMPHY

HAMEAT

DRDYQXLNRV FHN17THmVA HNLPS?PHY HAI4EAT DRDYGILNKV MHITDTNQVA WILF 74P DRDYGILNKV FHNITDT{VA HHPFSTMPHY HA14EAT DRDYGILNKV FHN17DTHVA HHLFSTMPHY

HAMEAT

from plasmid pRP2-1C TABLE 4 Alignment of Conserved Amino Acids from Microsomal Delta-12 Fatty Acid Dsaturases Species Arabidopsi s thaliana Glycine max Zea mays Ricinus communisa Brassica napus D Brassica napus F a from plasmid pRF2-1C Po-sition 165- 180 161-176 172 -187 65- 80 165 -180 165 -180 Amino Acid Seqiuence IKWGKYLNN

PLGRIM

VAWFSLYIMN pLGRAV PWYTPYVYNN pVGRVV IRWYSICYLNN

PPGRIM

IKWYGKYLNN PLGRTv IKWYGKYLNN PLGRTV no.

4 .e or 0: 00 0O 0 0 *0 0 0* TABLE Alignment of Conserved Amino Acids from Plastid and Microsomal 25 Delta-iS atty Acid Desaturases Species Arabidopsi s thaliana& Bra saica napusa Glycine max& Arabidopsis thaliana Bra ssica napus Glycine max aPlastid sequences Positon Amino Acid Seuence 156-177 114 -135 164 -185 94 -115 87- 109 93 -114 WALFVLGHD CGHGSFSNDP

KIM

WALFVLGHD CGHGSFSNDp

RIM

WALFVLGHD CGHGSFSNNS

KIM

WAIFVLGHD CGHGSFSDIP

LIM

WALFVLGHD CGHGSFSNDP

RIM

WALFVLGHD CGHGSFSDSP PIM 0 0e to *0e* 0 *0 0 0 0e0*@O 0 18

TABLE'S

O Alignment of Conserved Amino Acids from Plastid and Microsomal Fatty Acid Desaturases Species Position Amino Acid Sequence A. thaliana" 188-216 ILVPYHGWRI SHRTHHQNHG HVENDESWH B. napus' 146-174 ILVPYHGWRI SHRTHHQNHG HVENDESWH Glycine maxa 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 amino acid motifs and their relative positions indicates that regions of a delta-12 or delta-15 fatty acid desaturase that can be mutated in one species to generate a non-functional desaturase can be mutated in the corresponding region from other species to generate a non-functional 12-DES or 15-DES gene product in that species.

Mutations in 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 include, without limitation, site-directed mutagenesis of wild-type sequences and direct synthesis using automated DNA synthesizers.

30 A nucleic acid fragment containing a mutant S sequence can also be generated by mutagenesis of plant seeds or regenerable plant tissue by, ethyl methane sulfonate, X-rays or other mutagens. With mutagenesis, mutant plants having the desired fatty acid phenotype in seeds are identified by known techniques and a nucleic acid fragment containing the desired mutation is isolated from genomic DNA or RNA of the mutant line. The site of 19 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 selfpollinations, 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 160 increased palmitic acid, reduced stearic acid (C 18 0 increased oleic acid (C, 18 reduced linoleic acid (C.

8 2 and reduced linolenic acid (C 1 3 in the seed oil.

20 The general experimental scheme for developing lines with stable fatty acid mutations is shown in Scheme I hereinafter.

*ooo* *ooo SCHEME I Westar (Mo) S| EMS Treatment v V

M,

Greenhouse grow out Self-pollination

V

4) M2 S 10 Nursery grow out Self-pollination v

M,

Chemical analysis Select mutants based on statistical analysis of control population Grow out select mutants in greenhouse I Self-pollination

V

M4 i Chemical analysis Select mutants based on statistical analysis of control population Grow out select mutants in nursery I Self-pollination

M

Chemical analysis Confirm altered fatty acid Composition in selected lines v STABLE FATTY ACID MUTANTS Westar seeds (Mo) were mutagenized with ethylmethanesulfonate (EMS). Westar is a registered Canadian spring variety with canola quality. The fatty 40 acid composition of field-grown Westar, 3.9% C 60 1.9%

C

18 0 67.5% C,: 17.6% C18, 2 7.4% C 18 3 C20:1 C 22 1 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, including but not limited to ethidium bromide, nitrosoguanidine, diepoxybutane etc. may also be used to induce mutations.

The mutagenesis treatment may also be applied to other stages of plant development, including but not limited to cell cultures, embryos, microspores and shoot apices.

The M, seeds were planted in the greenhouse and M, plants were individually self-pollinated.

M, seed was harvested from the greenhouse and planted in the field in a plant-to-row design. Each plot contained six rows, and five M 2 lines were planted in each plot. Every other plot contained a row of nonmutagenized Westar as a control. Based on gas chromatographic analysis of M, 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 in 10,000 20 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 Smean 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 30 either the upper or lower statistical thresholds were replanted in the greenhouse and self-pollinated. This planting also included Westar controls. The M, 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.

Following self-pollination, M, seed from the field were re-analyzed once again for fatty acid composition. Those lines which remained stable for the selected fatty acids were considered stable mutations.

"Stable mutations" as used herein are defined as 9 M. or more advanced lines which maintain a selected altered fatty acid profile for a minimum of three generations, including a minimum of two generations under field conditions, and exceeding established statistical thresholds for a minimum of two generations, as determined by gas chromatographic analysis of a minimum of 10 randomly selected seeds bulked together.

Alternatively, stability may be measured in the same way by comparing to subsequent generations. In'subsequent generations, stability is defined as having similar fatty acid profiles in the seed as that of the prior or subsequent generation when grown under substantially similar conditions.

20 The amount of variability for fatty acid content in a seed population is quite significant when single seeds are analyzed. Randomly selected single seeds and a ten seed bulk sample of a commercial variety were compared. Significant variation among the single seeds 25 was detected (Table The half-seed technique (Downey, R.K. and B.L. Harvey, Can. J. Plant Sci., 43:271 [1963]) in which one cotyledon of the germinating seed is analyzed for fatty acid composition and the remaining embryo grown into a plant has been very useful to plant breeding work to select individuals in a population for further generation analysis. The large variation seen in the single seed analysis (Table A) is reflected in the half-seed technique.

23 TABLE

A

Single Seed Analysis for Fattv Acid Com osition' 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..o 3 3.0 1.2 12.7 15.3 10.6 0.8 7.3 0.i 43.2 4 2.8 0.2 1.1 16.7 13.2 9.1 0.8 11.2 0.4 38.9 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 8 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 6.2 0.7 46.9 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 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 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 Range 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 o 30 Plant breeders using the half-seed technique have found it unreliable in selecting stable genetically controlled fatty acid mutations (Stefanson, 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, J. Amer. Oil Chem. Soc. (1973) 50:400- 403. To determine the genetic stability of the selected S" 40 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, 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 in the seed treatments to create an LD30 population. This allowed for the rapid selection of single gene mutations for fatty acid traits in agronomic backgrounds which produce acceptable yields.

Other than changes in the fatty acid composition of the seed oil, the mutant lines described here have normal plant phenotype when grown under field conditions, and are commercially useful. "Commercial utility" is 20 defined as having a yield, as measured by total pounds of seed or oil produced per acre, within 15% of the average yield of the starting canola variety grown in the same region. To be commercially useful, plant vigor and high fertility are such that the crop can be produced in 25 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 following accession numbers.

aoo Line Accession No. Deosit Date A129.5 40811 May 25, 1990 A133.1 40812 May 25, 1990 A144.1 40813 May 25, 1990 A200.7 40816 May 25 1990 M3032.1 75021 June May 31 199 0 M3094.4 75023 June 7, 1991 M3052.6 75024 June 7, 1991 M3007.4 75022 June 7, 1991 M3062.8 75025 June 7, 1991 *M3028.10 75026 June 7, 1991 IMC130 75446 June 7, 1991 446 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 0 mutation in only one form. An example of such a plant is Brassica napus line Q508, a doubly-mutagenized line containing a mutant D-form of delta-12 desaturase (SEQ ID NO:1) and a mutant F-form of delta-12 desaturase (SEQ ID Preferred host or recipient organisms for 25 introduction of a nucleic acid fragment of the invention are the oil-producing species, such as soybean (Glycine max), rapeseed Brassica napus, B. rapa and B.

juncea), sunflower (Helianthus annus), castor bean (Ricinus communis), corn (Zea mays), and safflower (Carthamus tinctorius).

Plants according to the invention preferably contain an altered fatty acid profile. For example, oil obtained from seeds of such plants may have from about 69 to about 90% oleic acid, based on the total fatty acid 35 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 obtained from seeds produced by plants of the invention may have from about 2t0% to about 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% a-linolenic acid.

In one embodiment of the claimed invention, a plant contains both a 12-DES mutation and a mutation. Such plants can have a fatty acid composition comprising very high oleic acid and very low alphalinolenic acid levels. Mutations in 12-DES and may be combined in a plant by making a genetic cross between 12-DES and 15-DES single mutant lines. A plant having a mutation in delta-12 fatty acid desaturase is crossed or mated with a second plant having a mutation in delta-15 fatty acid desaturase. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds are then screened in order to identify those seeds carrying both mutant genes.

Alternatively, a line possessing either a 12-DES or a 15-DES mutation can be subjected to mutagenesis to generate a plant or plant line having mutations in both 12-DES and 15-DES. For example, the IMC 129 line has a 25 mutation in the coding region (Glue 06 to Lyss 0 of the D form of the microsomal delta-12 desaturase structural gene. Cells seeds) of this line can be mutagenized to induce a mutation in a 15-DES gene, resulting in a plant or plant line carrying a mutation in a delta-12 fatty acid desaturase gene and a mutation in a delta-15 fatty acid desaturase gene.

ooe Progeny includes descendants of a particular plant or plant line, seeds developed on an instant plant.

Progeny of an instant plant include seeds formed on F 1

F

2

F

3 and subsequent generatioi plants, or seeds formed on BC,, 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, gas-liquid chromatography

(GLC)

analysis of a bulked seed sample or of a single halfseed. 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 in plant genetic mapping and plant breeding programs. Such markers may include restriction fragment length polymorphism (RFLP), random amplification 20 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 in addition to, or as an alternative to, other sorts of identification techniques. An example of marker-assisted breeding is the use of PCR primers that specifically amplify a sequence containing a desired mutation in 12- 30 DES or Methods according to the invention are useful in that the resulting plants and plant lines have desirable :oo: seed fatty acid compositions as well as superior agronomic properties compared to known lines having 35 altered seed fatty acid composition. Superior agronomic characteristics include, for extaple, increased seed germination percentage, increased seedling vigor, increased resistance to seedling fungal diseases (damping off, root rot and the like), increased yield, and improved standability.

While the invention is susceptible to various modifications and alternative forms, certain specific embodiments thereof are described in the general methods and examples set forth below. For example the invention may be applied to all Brassica species, including B.

rapa, B. juncea, 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 within the scope of the invention.

This includes 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-pollination to transmit the fatty acid trait, alone or in combination with other traits, to develop new Brassica lines.

S4 EXAMPLE 1 Selection of Low FDA Saturates 25 Prior to mutagenesis, 30,000 seeds of B. napus cv.

Westar seeds were preimbibed in 300-seed lots for two hours on wet filter paper to soften the seed coat. The preimbibed seeds were placed in 80 mM ethylmethanesulfonate (EMS) for four hours. Following 30 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 25OC/150C, 14/10 hr day/night conditions in the greenhouse. At flowering, each: plant was individually self-pollinated.

M, seed from individual plants were individually catalogued and stored, approximately 15,000

M

2 lines was planted in a summer nursery in Carman, Manitoba. The seed from each selfed plant were planted in 3 -meter rows with 6-inch row spacing. Westar was planted as the check variety. Selected lines in the field were selfed by bagging the main raceme of each plant. At maturity, the selfed plants were individually harvested and seeds were catalogued and stored to ensure that the source of the seed was known.

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 using 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 in 4-inch pots containing Pro-Mix soil and the plants were maintained at 25oC/15oC, 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 Zdistribution of 1 in 800. Selected

M

4 lines were planted 30 in a field trial in Carman, Manitoba in 3-meter rows with 6-inch spacing. Ten M 4 plants in 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 single plant selections was analyzed in- 10-seed bulk samples and the bulk row harvest in 50-seed bulk samples.

Selected M, 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

6 seed was individually harvested from each plant and analyzed in 10-seed bulk samples for fatty acid composition.

Selected

M

6 lines were entered into field trials in Eastern Idaho. The four trial locations were selected for the wide variability in growing conditions. The locations included Burley, Tetonia, Lamont and Shelley (Table The lines were planted in 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 S. maturity the plots were harvested to determine yield.

20 Yield of the entries in the trial was determined by taking the statistical average of the four replications.

The Least Significant Difference Test was used to rank the entries in the randomized complete block design.

TABLE I 25 Trial Locations for Selected Fatty Acid Mutants LOCATION SITE CHARACTERIZATIONS BURLEY Irrigated. Long season. High temperatures during flowering.

TETONIA Dryland. Short season. Cool temperatures.

S 30 LAMONT Dryland. Short season. Cool temperatures.

SHELLEY Irrigated. Medium season. High S. temperatures during flowering.

To determine the fatty acid profile of entries, plants in each plot were bagged for self-pollination.

The M, seed from single 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, or later generation mutations were used in crossing.

F, seed was harvested and analyzed for fatty acid composition to determine the mode of gene action. The F 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 line seed was planted in the greenhouse, individual plants were self-pollinated. The F, 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 S 20 the F, 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 in a 15-mL polypropylene tube and extracted in 1.2 mL 0.25 N KOH in 1:1 ether/methanol. The sample was vortexed for 30 sec. and heated for 60 sec. in a 60 0 C water bath. Four mL of saturated NaC1 and 2.4 mL of iso-octane were added, and the mixture was vortexed again. After phase separation, 600 pL of the upper organic phase were pipetted into individual vials and stored under nitrogen at -5 0 C. One At samples were injected into a Supelco SP-2330 fused silica capillary column (0.25 mm ID, 30 M length, 0.20 Am df).

The gas chromatograph was. set at 180 0 C for minutes, then programmed for a 2 0 C/minute increase to 212 0 C, and held at this temperature for 1.5 minutes.

Total run time was 23 minutes. Chromatography settings were: Column head pressure 15 psi, Column flow (He) 0.7 mL/min., Auxiliary and Column flow 33 mL/min., Hydrogen flow 33 mL/min., Air flow 400 mL/min., Injector temperature 250 0 C, Detector temperature 300 0 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 15 Percent Fatty Acids Genotype

C

16 0 Cz 1 0 Cz, 8

C

18 2

C

18 3 Sats' M3 Generation(1 in 10,000 rejection rate) S: Lower 3.3 1.4 13.2 5.3 20 Upper 4.3 2.5 71.0 21.6 9.9 8.3 M Generation(1 in 800 rejection rate) Lower 3.6 0.8 12.2 3.2 5.3 $i Upper 6.3 3.1 76.0 32.4 9.9. 11.2 Ms Generation (1 in 755 rejection rate) 25 Lower 2.7 0.9 9.6 2.6 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 Line W13097.4 had 3.1% palmitic acid and an FDA saturate content of After a cycle in the greenhouse,

M

4 seed from line W13097.4 (designated .line A144) was analyzed.

Line W13097.4.1(Al44.1) had 3.1t C, 6 0 exceeding the lower statistical threshold of The FDA saturate content for A144.1 was The fatty acid compositions for the

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 b Seed Mutagenesis i0 Percent Fatty Acids Genotypea C 16 0

C

1

C

18 :1 C 1 8 2

C

18 3 Satsb Tot Satc Westar 3.9 1.9 67.5 17.6 7.4 5.9 W13097.4

(M

3 3.1 1.4 63.9 18.6 9.5 4.5 5.6 0 W13097.4

(M

4 3.1 1.4 66.2 1'9.9 6.0 4.5 A144.1.9 (Ms) 2.9 1.4 64.3 20.7 7.3 4.4 5.3 aLetter and numbers up to second decimal point indicate .the plant line. Number after second decimal point indicates an individual plant.

bSat=FDA Saturates i CTot Sat=Total Saturate Content The M s 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 Bulk seed analysis from open-pollinated (A144.1) plants at the M, 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.

34 TABLE IV Fatty Acid Composition of A144 Low Palmitic Acid/Low FDA Saturate Line Percent Fatty Acids Genotypea

C

16 0 CS: o C 18 :I C 1 8 2

C

18 3 Satsb Tot Satc Individually Self-Pollinated Plants A144.1.1 A144.1.2 A144.1.3 A144.1.4 A144.1.5 A144.1.6 A144.1.7 A144.1.8 A144.1.9 A144.1.10 Average of 3.2 3.0 3.6 3.2 3.3 3.1 3.1 3.1 2.9 3.1 Individu 1.6 64.4 20.5 7.0 4.8 1.5 67.4 18.6 6.3 4.5 1.8 61.4 22.4 7.5 5.2 1.5 64.6 20.9 6.7 4.7 1.7 60.0 23.9 7.9 5.0 1.4 67.3 17.8 6.5 4.6 1.6 67.7 17.4 6.5 4.8 1.8 66.9 18.7 6.1 4.9 1.4 64.3 20.7 7.3 4.4 1.5 62.5 20.4 7.7 4.6 ally Self-Pollinated Plants 1.6 64.8 20.1 6.9 4.7 5.9 5.7 6.6 5.8 6.1 5.2 5.4 5.4 5.3 5.6 5.7 5.7 r r A144.1.1-10 3.1 Bulk Analysis of Open-Pollinated Plants A144.1B 3.1 1.6 64.8 19.4 7.8 4.7

S..

aLetter and numbers up to second decimal point indicate the plant line. Number after second decimal point indicates an individual plant.

bSat=FDA Saturates CTot Sat=Total Saturate Content These reduced levels have remained stable to the My 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 The fatty acid composition of the A144 liies 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 napus) was-reduced from 5.9% to 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 Trial -Percent Fatty Acids Trial Location

C

16

C

8 0

C

18 1

C

18 :2 C 18 :3 Sats Tot Sats Burley 2.9 1.3 62.3 20.6 10.3 4.2 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 20 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 :o C 18 :o

C

18 :e 67.4%, C 1 8 2 18.6%, C 18 :3 to other fatty acid mutations it was crossed to A129 a mutant high oleic acid (C 1

C

18 e: C 1 8 1 75.6%, C 18 2 Ce: 3 Over 570 dihaploid progeny produced from the F, hybrid were harvested and analyzed for fatty acid composition. The results of the progeny analysis are 30 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 in A129.

36 TABLE VB Genetic Studies of Dihaploid Progeny of A144 X A129 Frequency

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 line, designated A149.3 (ATCC 40814), was also produced by the method of Example 1. A 50-seed bulk analysis of this line showed the following fatty acid composition: Co 16

C

18 :0 C,1: 1 65.5%, C 18 :2 18.3%, C1e:3 FDA Sats 15 Total Sats This line has also stably maintained its mutant fatty acid composition to the M generation. In a multiple'location replicated trial the yield of A149 was not significantly different in yield from the parent cultivar Westar.

20 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 line showed the following fatty acid composition: C 16 0

C

18

C

18 1 66.6%, Ce8: 2 20.0%, C 18 3

C

2 0 1

C

22 1 FDA Saturate Total Saturates This line has stably maintained its mutant fatty acid composition to the M generation. In a single replicated trial the yield of 30 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, seed showed the two lines 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 i, at the M 3 generation, 470 lines exceed the upper statistical threshold for palmitic acid 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 1 he upper statistical threshold for high palmitic acid at both the M 4 and M s generations with palmitic acid levels of 11.7% and respectively. The fatty acid composition of this high palmitic acid mutant, which was stable to the My 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 Genote Percent Fatty Acids Genotype 6 18: 1 8 2 1 8: Sats 20 Westar 3.9 1.9 67.5 17.6 7.4 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 4 M3007.4 9.1 1.4 63.3 13.7 5.5 12.7

(M)

*Sats=Total Saturate Content To determine the genetic relationship of the high 30 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 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

C

1 6 :0 Genotype Content(%) Observed Expected p-p-/p-hp- <7.0 151 142 10 hp-hp- >7.0 39 47 An additional

M

3 line, W4773.7, contained 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, and Ms generations with palmitic acid levels of 6.3% and 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.

S 20 TABLE VII Fatty Acid Composition of a High Palmitic g"'I Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids 25 Genotype

C

16 :0 C 18 0

C,

1 e: C 1 8 2 C18:3 Sats' Westar 3.9 1.9 67.5 17.6 7.4 W4773.7 4.5 2.9 63.5 19.9 7.1 9.3 (M3) 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 (Ms) "Sats=Total Saturate Content 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 Line W14859.6 had 1.3% stearic acid. At the M 5 generation, its selfed progeny-(M3052.1) continued to fall within the lower statistical threshold for C 1 ,eo 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 single location replicated yield trial M3052.1 was not significantly different in yield from the parent cultivar Westar.

TABLE VIII Fatty Acid Composition of a Low Stearic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype

C,

1 0 Ce: Ce: 1 Ce18:2 C18:3 Sats Westar 3.9 1.9 67.5 17.6 7.4 5.9 20 W14859.6 5.3 1.3 56.1 23.7 9.6

(M

3 M3052.1 4.9 0.9 58.9 22.7 9.3 5.8 S 25 M3052.6 4.4 0.8 62.1 21.2 7.9 5.2

(MS)

To determine the genetic relationship of the low stearic acid mutation of M3052.1 to other fatty acid S.mutations it was crossed to the low palmitic acid mutation A144 (Example 1) Seed from over 300 dihaploid 30 progeny were harvested and analyzed for fatty acid composition. The results are summarized in 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 C18:0 Genotype Content(%) Observed Expected p-p-s-s- 87 77 152 154 p+p+s+s+ 70 77 An additional M s 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

160 C18: 0

C

181 61.7%, C 18 2 23.0%, C 18 3

FDA

saturates Total Saturates In a single location replicated yield trial M3051.10 was not significantly different in yield from the parent cultivar 20 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 S. 1.3% stearic acid in the greenhouse and field respectively. A ten-seed analysis of this line showed the following fatty acid composition:

C

1 6 0

C

1 8 0

C

18 1 66.5%, C 1 8 :2 18:4%, C 1 3 saturates 30 Total Saturates 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 F2 seed showed the two lines to be allelic. The mutational events in M3054.7, M3051.10 and M3052.1 although different in origin were in the same gene.

EXAMPLE 6 High Olei Acid Canola Lines In the studies of Example 1, at the M 3 generation 31 lines exceeded the upper statistical threshold for oleic acid Line W7608.

3 had 71.2% oleic acid.

At the M 4 generation, its selfed progeny (W7608.3.

5 since S designated A129.5) continued to exceed the upper statistical threshold for with 78.8% oleic acid.

M

seed of five self-pollinated plants of line A129.5

(ATCC

40811) averaged 75.0% oleic acid. A single plant selection, A129.5.3 had 75.6% oleic acid. The fatty acid composition of this high oleic acid mutant, which was stable under both field and greenhouse conditions to the M, generation, is summarized in Table IX. This line also 2 stably maintained its mutant fatty acid composition to the M generation in field trials in multiple locations.

Over all locations the self-pollinated plants (A129) taveraged 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, A29 was not significantly different in yield from the parent cultivar Westar.

The canola oil of A129, after commercial S. processing, was found to have superior oxidative 0 stability compared to Westar when measured by the S. 30 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.

42 TABLE IX Fatty Acid Composition of a High Oleic Acid Canola Line Produced by Seed Mutagenesis Percent Fatty Acids Genotype

C

16 0 Ce 8 0

C

18

C

18 2

C

1 :3 Sats Westar 3.9 1.9 67.5 17.6 7.4 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 (Ms) Sats=Total Saturate Content TABLE X Fatty Acid Composition of a Mutant High Oleic Acid Line at Different Field Locations in Idaho Percent Fatty A~i~n 0 *0 Percent Fatty Acids Location -Burley 20 Tetonia Lamont Shelley Sats=Total

C

16 0 C18: 0 3.3 2.1 3.5 3.4 3.4 1.9 3.3 2.6 Saturate Content

C

1 8 1 77.5 77.8 77.8 30.0

C

1 8 :2 8.1 6.5 7.4 5.7

C

1 8 :3 6.0 4.7 6.5 4.5 Sats 6.3 7.7 5.7 4.5 7.7 0 00 *00* 0 The genetic relationship of the high oleic acid 25 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 C 18 C 1 8 :1 62.9%,C 18 :2 20.0%, C 18 :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 43 controlled the inheritance of the high oleic acid phenotype.

TABLE XB Genetic Studies of A129 X Global Frequency C18: Genot 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 C 8 0 C 18 66.4%, 18.1%, C 1 8 3 was made to determine the inheritance of the oleic acid desaturase and linoleic acid desaturase. In the F, 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 20 1:2:1:2:4:2:1:2:1 segregation of two independent, codominant genes (Table XC). A line was selected from the cross of A129 and IMC01 and designated as IMC130 (ATCC deposit no. 75446) as described in U.S. Patent Application No. 08/425,108, incorporated herein by 25 reference.

TABLE XC Genetic Studies of A129 X IMC 01 Frequency Genotype Ratio bservedte 30 od-od-ld-ld---- 0 Observed Expected 30 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+d+ 1 8 12 An additional high oleic acid line, designated A128.3, was also produced by the disclosed method. A seed bulk analysis of this line showed the following fatty acid composition:

C

16 0 C,: 0

C

18 1 77.3%, C 18 :2 C 18 3 FDA Sats Total Sats This line also stably maintained its mutant fatty acid composition to the M, 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:

C

16 0

C

1 e:o C 1 e: 1 77.3%, C18 C 1 :3 FDA Saturates Total Saturates In a single location replicated yield trial M3028.10 was not significantly different in yield from the parent cultivar Westar.

SEXAMPLE 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 (S Line W12638.8 had 9.4% linoleic acid. At the M 4 and M s generations, its selfed progenies S 30 [W12638.8, since designated A133.1 (ATCC 40812)] continued to exceed the statistical threshold for low C 1 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, 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 line showed the following fatty acid composition:

C,

16 C 18 0 C 18 1 77.1%, c,:2

C

18 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 Mutagenesis Percent Fatty Acids 15 Genotype

C

16 0

C

182 e C 18

C

18 2

C

18 Satsb Westar 3.9 1.9 67.5 17.6 7.4 W12638.8 3.9 2.3 75.0 9.4 6.1 20 W12638.8.

1 4.1 1.7 74.6 10.2 5.9 7.1 20

(M

4 A133.1.8 3.8 2.0 77.7 8.4 5.0 (Ms) t aLetter and numbers up to second decimal point indicate the plant line. Number after second decimal point 25 .indicates an individual plant.

bSats=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 Line W14749.8 had 5.3% linolenic acid and 15.0% linoleic acid. At the M, and Ms generations, its selfed progenies [W14749.8, since designated M3032 (ATCC 75021)] continued to exceed the statistical threshold for low 3 with linolenic acid levels of 2.7% and 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 s 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 Mutaenesis Percent Fatty Acids 20 Genotype

C

1 6: 0 Ce:0 C 18 :1 Ce: 2 C18.

3 Sats Westar 3.9 1.9 67.5 17.6 7.4 W14749.8 4.0 2.5 69.4 15.0 5.3 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 (M)Sats=

S

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.o 0

C

18 :0

C

1 8 :1 63.1%, C 18 2 17.8%, C 1 9.3%.

The cross and progeny resulting, from were coded as 89B60303.

The F, 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.

Ind. -idual plants were selected in 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 self ing rows and in plots for preliminary yield and agronomic evaluations. At flowering the F 3 plants in the selfing rows were selfpollinated. 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 line were self-pollinated. At maturity the F 25 seed was harvested from each and analyzed for fatty acid composition.

The F 5 line 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 O9 30 for planting the yield plots in the field. At flowering, the F 5 plants in each selfing-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 deteriined 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 trialing. At flowering-the

F

plants were self-pollinated. At maturity the F, seed was harvested and analyzed for fatty acid composition. Three F, seed lines which had fatty acid profiles most similar to IMC 129 (Table XIII) were selected and planted in the field as self ing rows, the remaining seed was bulked together for yield trialing. The high oleic fatty acid profile of IMC 129 was maintained through seven generations of selection for fatty acid and agronomic traits in 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 20 Fatty Acid Composition of Advanced Breeding Generation with High Oleic Acid Trait (IMC 129 X Legend) Fatty Acid Composition(%) F, Selections of 89860303 Co C C C of 89B60303

C

16 0

C

18 0

C

18 1

C

18 2

C

18 :3 25 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 30 introduced into a different genetic background by combining crossing and selection methods with the generation of dihaploid populations from the microspores of the F, hybrids. IMC 129 was crossed to Hyola 41, a commercial spring Brassica napus variety. Hyola 41 has the following fatty acid composition:

C

16 :0 C 18 :0

C

18 :1 64.9%,

C,,

8 2 Ce:3 The cross and progeny resulting from the cross were labeled 90DU.146.

The F, seed was planted from the cross and a dihaploid population was made from the F, microspores using standard procedures for Brassica napus.

Each DH, plant was self-pollinated at flowering to produce DH, seed. At maturity the DH, seed was harvested and analyzed for fatty acid composition. DH, individuals which expressed the high oleic fatty acid profit of IMC 129 were advanced to the next generation in the C greenhouse. For each individual selected five DH, seeds were planted. At flowering the DH 2 plants were selfpollinated. At maturity the DH 2 seed was harvested and analyzed for fatty acid composition. The DH 2 seed which g was similar in fatty acid composition to the IMC 129 parent was advanced to the field as a selfing row. The remaining DH, seed of that group was bulked and planted in plots to determine yield and agronomic characteristics of 20 the line. At flowering individual DH, plants in the selfing row were self-pollinated. At maturity the DH, seed was harvested from the individual plants to determine fatty acid composition. Yield of the selections was determined from the harvested plots.

25 Based on fatty acid composition, yield and agronomic characteristics selections were advanced to the next generation in the greenhouse. The DH, seed produced in the greenhouse by self-pollination was analyzed for fatty acid composition. Individuals which were similar to the 30 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 Hyola41) Fatty Acid Composition(%) of 90DU.146 at Multiple Locations Cso 6 Ce: 0

C

8 1

C

18 2

-C

1 :3 Aberdeen 3.7 2.6 75.4 8.1 7.2 Blackfoot 3.3 2.4 75.5 8.8 Idaho Falls 3.7 3.1 75.0 7.5 8.1 Rexberg 3.9 3.7 75.3 7.0 Swan Valley 3.5 3.4 74.5 7.0 7.3 Lamont 3.9 2.8 72.0 10.1 8.4 EXAMPLE Canola Lines 0508 and 04275 Seeds of the B. napus line IMC-129 were mutagenized with methyl N-nitrosoguanidine (MNNG). The MNNG treatment consisted of three parts: pre-soak, 20 mutagen application, and wash. A 0.05M Sorenson's phosphate buffer was used to maintain pre-soak and mutagen treatment pH at 6.1. Two hundred seeds were treated at one time on filter paper (Whatman #3M) in a petri dish (100mm x 15mm). The seeds were pre-soaked in 25 15 mls 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 10mM concentration of MNNG in 0.05M Sorenson's buffer, pH 6.1, was prepared prior to use. Fifteen ml of 30 10m MNNG was added to the seeds in each plate. The seeds were incubated at 22 0 C+3 0 C in the dark under constant agitation for four 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 into an environmentally controlled greenhouse. The plants were grown under sixteen hours of light. At flowering, the racemes were bagged to produce selfed seed. At maturity, the M2 seed was harvested. Each M2 line was given an identifying number. The entire MNNG-treated seed population was designated as the Q series.

Harvested M2 seeds was planted in the greenhouse.

The growth conditions were maintained as previously described. The racemes were bagged at flowering for selfing. At maturity, the selfed M3 seed was harvested and analyzed for fatty acid composition. For each M3 g seed line, approximately 10-15 seeds were analyzed in bulk as described in Example 1.

High oleic-low linoleic M3 lines were selected from the M3 population using a cutoff of >82% oleic acid 20 and 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 25 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 30 Line 16:0 18:0 18:1 18:2 18:3 SA129* 4.0 2.4 77.7 7.8 4.2 Q508 3.9 2.1 84.9 2.4 2.9 SFatty acid composition of A129 is the average of 35 50 self-pollinated plants grown with the M3 population

M

4 generation Q508 plants had poor agronomic qualities in the field compared to Westar. Typical plants were slow growing relative to Westar, lacked early vegetative vigor, were short in stature, tended to be chlorotic and had short pods. The yield of Q508 was very low compared to Westar.

The M 4 generation Q508 plants in the greenhouse tended to be reduced in vigor compared to Westar.

However, Q508 yields in the.greenhouse were greater than Q508 yields in the field.

TABLE XVI Fatty Acid Composition of Seed Oil from Greenhouse-Grown 0508, IMC 129 and Westar.

C

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 Westarb 3.9 1.9 67.5 17.6 7.4 >5.8 Q508c 3.9 2.1 84.9 2.4 2.9 'Average or 50 self-pollinated plants bData from Example 1 cAverage of 50 self-pollinated plants Nine other M4 high-oleic low-linoleic lines were also identified: Q3603, Q3733, Q4249, Q6284, Q6601, Q6761, Q7415, Q4275, and Q6676. Some of these lines had good agronomic characteristics and an elevated oleic acid level in seeds of about 80% to about 84%.

Q4275 was crossed to the variety Cyclone. After selfing for seven generations, mature seed was harvested from 93GS34-179, a progeny line of the Q4275 Cyclone cross. Referring to Table 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 in 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 line was analyzed for fatty acid composition as described above. The fatty acid compositions of the selected lines are shown in-Table

XVII.

Table XVII Fatty Acid Composition of Field Grown IMC 129, Q4275 and 93GS34 Seeds Line Fatty Acid .Composition

C

1 C 1:0 C18: C 1 8:2 C18: 3 FDA Sats IMC 129 3.3 2.4 76.7 8.7 5.2 5.7 S' 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 S: 25 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, 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 in F 3 seed ranged from 59 to 79%.

No high oleic 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 line. F, seed was analyzed from 380 F2 individuals. The C 18 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 Fad2a BB Westar Fad2b aa Q508 Fad2 a bb Q508 Fad2 b 20 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 Westar Alleles Genotype 4 AABB(Westar) 4 3 AABb,AaBB,AABb,AaBB 6 2 AaBb,AAbb,AaBb,AaBb,aaBB,AaBb 30 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 0 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% Ci Seedil F3 Plant Fatty Acid Composition Identifier C16:0 C18:0 IC18:1 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 329 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 20 20 +38202 3.554 1.105 86.091 2.651 3.721 4.713 S* +38220 3.093 1.16 86.421 1.931 3.514 4.314 +38236 3.308 1.349 85.425 2.37 3.605 4.718 384 +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 56 EXAMPLE.11 Leaf and Root Fatty Acid Profiles of Canola Lines IMC-129, 0508. 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 0 C. Lipid extracts were analyzed by GLC as described in Example 1.

The fatty acid profile data are shown in Table XIX.

The data in Table XIX indicate that total leaf lipids in Q508 are higher in C 1 content than the 2 plus Ce: 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 20 mutated, chloroplast lipids were separated and analyzed.

No changes in chloroplast C 1 6

C

6 2 or C 6 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 25 in Q508 affects a microsomal Fad2 gene and not a plastid FadC gene.

TABLE XIX MATURE

EXPANDING

LEAF LEAF PETIOLE

ROOT

West. 129 3Q508 West. 129 3 08o We-. 1 I 0 s 0*eS .00.

6 s 0O000 *oOO*o S 0

S

*o 0 ooo 06 g.

*ooo DiDO.*

S*

5 16:0 12.1 11.9 10.1 16.4 16.1 11.3 21.7 23.5 11.9 16:1 0.8 0.6 1.1 0.7 0.6 1.1 1.0 1.3 1.4 16:2 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 2.8 1.5 3.7 4.0 1.6 18: 1 2.8 4.9 16.7 3.8 8.3 38.0 4.9 12.9 46.9 18:2 12.6 11.S 6.8 13.3 13.8 4.9 20.7 18.3 S.2 18:3 50.6 50.3 46.0 54.2 50.0 33.5 40.4 33.2 25.3 west. 129 3Q508 21.1 21.9 12.0 3.6 2.9 3.5 6.1 68.8 28.0 30.4 4.4 43.8 38.7 12.3 ±u 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 15 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 20 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:1). The transversion changes the codon at this position from GAG to AAG and results in a non-conservative substitution of glutamic acid, an dic residue, for lysine a basic residue. The pi :e 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 in the coding region of the D gene of rapeseed micrpsomal delta 12-desaturase.

A single base transition from T to A at nucleotide 515 of the F gene was detected in Q508 compared to the Westar sequence. The mutation changes the codon at this position from CTC to CAC, resulting in the nonconservative substitution of a non-polar residue, leucine, for a polar residue, histidine, in the resulting gene product. No mutations were found in the F gene sequence of IMC 129 compared to the F gene sequence of 20 Westar.

These data support the conclusion that a mutation in a delta-12 desaturase gene sequence results in alterations in the fatty acid profile of plants containing such a mutated gene. Moreover, the data show that when a plant line or species contains two delta-12 desaturase loci, the fatty acid profile of an individual having 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 amino acid motif (His-Xaa-Xaa-Xaa-His) found in cloned delta-12 and deltamembrane bound-desaturases (Table XX).

Table XX Alignment of Amino Acid Sequences of Cloned Canola Membrane Bound-Desaturases i Desaturase Gene Canola-f ad2-D (mu~tant) Canola-Fad2-D

I

Sequencea

AHKCGH

AHECGH

AHECGH

GHDQCA{

Canola-Fad2

-F

Canola- FadC Position 109-1-14 109-114 109-114 170-175 94-99 I4.

Canola-fad3 (mutant) !9HKCGH

I

Canola-Fad3

GILD

I ~H~CGH [Canola-FadD I GHCGH 1125-130 (FadD Plastid delta 15, Fad3 Microsomal (FadC Plastid delta-.2, Fad2 Microsomal delta-12) one letter amino acid code; conservative substitutions are underlined; non-conservative substitutions are in bold.

*t 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 51-GGATATGATGATGGTGAAAGA-31 and antisense primer 5' -TCTTTCACcATCATCATATCC- The primers used to specifically amplify 1*2-DES D gene RNA from the indicated tissues were sense primer 5'-GTTATGAAGCAAGAAGAAAC-3' and ant isense primer 5' -GTTTCTTCTTTGCTTCTAC-.

3 The results indicated that mRNA of both the D and F gene was expressed in seed and leaf tissues of IMC 129, 0508 and wild type Westar plants.

In vitro transcription and translation analysis showed that a peptide of about 46 kD was made. This is the expected size of both the D gene product and the F gene product, based on sum of the deduced amino acid sequence of each gene and the cotranslational addition of a microsomal membrane peptide.

These results rule out the possibility that nonsense 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.

15 EXAMPLE 14 Development of Gene-Specific PCR Markers Based on the single base change in the mutant D gene of IMC 129 described in above, two 5' PCR primers were designed. The nucleotide sequence of the primers 20 differed only in the base (G for Westar and A for IMC 129) at the 3' end. The primers allow one to distinguish between mutant fad2-D and wild-type Fad2-D alleles in a .DNA-based PCR assay. Since there is only a single base difference in the 5' PCR primers, the PCR assay is very sensitive to the PCR conditions such as annealing temperature, cycle number, amount, and purity of DNA S. 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 30 plants as templates. Conditions may be further optimized by varying PCR parameters, particularly with variable crude DNA samples. A PCR assay distinguishing the single base mutation in IMC 129 from the wild type gene along with fatty acid composition analysis provides a means to 61 simplify segregation and selection analysis of genetic crosses involving plants having a delta-12 fatty acid desaturase mutation.

EXAMPLE 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 Agrobacterium tumefaciens essentially following the procedure described in 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 Scorresponding termination sequence. The first plasmid, pIMC110, was prepared by inserting into a disarmed Ti vector the full length wild type Fad3 gene in sense orientation (nucleotides 208 to 1336 of SEQ ID 6 in WO S 20 93/11245), flanked by a napin promoter sequence Spositioned 5' to the Fad3 gene and a napin termination sequence positioned 3' to the Fad3 gene. The rapeseed napin promoter is described in EP 0255378.

The second plasmid, pIMC205, was prepared by 25 inserting a mutated Fad3 gene in sense orientation into a disarmed Ti vector. The mutant sequence contained mutations at nucleotides 411 and 413 of the microsomal Fad3 gene described in W093/112 4 5 thus changing the sequence for codon 96 from GAC to AAG. The amino acid at 30 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 TGGTCTTTTGGT-3', was placed 5' to the mutant Fad3 gene 35 and a phaseolin termination sequence was placed 3' to the 62 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 discernable 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 T1 plants. T2 seeds were obtained by selfing the resulting T1 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 pIMC110 S. 20 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.

25 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 30 phenotype if the gene product were functional.

63 Table XXI Overexpression and Co-suppression Events in Westar Populations Transformed with pIMC205 or pIMC110.

Construct er of -Lin o l enic Overexpresion Co-Suppression Wild Type (3,1 0 l i n l n i c) 0 l i o l e n i c) PIMC110 40 2.4 2-20.6 16 7 17 p C205 307 4.6 10.4 0 __>mlnlnc .g'to.,

B

Fatty acid compositions of representative transformed plants are presented in Table XXII. Lines 652-09 and 663-40 are representative of plants containing pIMC110 and exhibiting an overexpression and a cosuppression phenotype, respectively. Line 205-284 is representative of plants containing pIMC205 and having the mutant fad3 gene.

Table

XXII

15 Fatty Acid Composition of T2 Seed From Westar Transformed With pIMC205 or pIMC110.

.ine Fatty Acid Composition C16 10 C180O C18tL C18s2 C18a3 652-09 pIC110 4.7 3.3 656 overexpression 6 6 8.1 14.8 S 663-0 4.9 2.1 62.5 232 3.6 co-suppression 20-284 3.7 .8 68.8 15.9 6. pIMC205 *15 9 6.

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.

30 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 64 skilled in the art without deviating from the spirit and scope of the appended claims.

Page(s)7-S are claims pages they appear after the sequence listing SEQUENCE LISTING GENERAL INFORMATION: O 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: ADDRESSEE: Fish Richardson, P.A.

STREET: 60 South Sixth Street, Suite 3300 CITY: Minneapolis STATE: MN COUNTRY: USA ZIP: 55402 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US96/20090 FILING DATE: 13-DEC-1996

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/572,027 FILING DATE: 14-DEC-1995

CLASSIFICATION:

S (viii) ATTORNEY/AGENT INFORMATION: NAME: Ellinger, Mark S.

REGISTRATION NUMBER: 34,812 REFERENCE/DOCKET NUMBER: 07148/049WO1 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 612/335-5070 TELEFAX: 612/288-9696 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Brassica napus (ix) FEATURE: OTHER INFORMATION: Wild type F form.

66 (Xi) SEQUENCE DESCRIPTION: SEQ IV 110:1: ATG GOT GCA GGT Met Gly Ala Gly 1

OGA

Gly AGA ATG CAA GTG Arg Met Gin Val CCT CCC TCC AAG Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Giu Thr Asp GTC GGA GAA Val Gly Glu ATC AAG CGC GTA Ile Lys Arg Val TGC GAG ACA CCG Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser CTC AAG AAA GCA Leu Lys Lys Aia

ATC

Ile 40 CCA CCG CAC TOT Pro Pro His Cys.

TTC

Phe ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser CTC ATC TOG GAC Leu Ile Trp Asp

ATC

Ile ATC ATA GCC TCC Ile Ile Ala Ser

TGC

Cys TTC TAC TAC GTC Phe Tyr Tyr Val

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe

CCT

Pro CTC CTC COT CAC Leu Leu Pro His CTC TCC TAC TTC Leu Ser Tyr Phe

GCC

Ala TOO CCT CTC TAC Trp Pro Leu Tyr 0CC TOC CAA 000 Ala Cys Gin Giy TGC GTC Cys Vai CTA ACC 000 Leu Thr Giy AGC GAC TAC Ser Asp Tyr 115

GTC

Val 100 TOO OTC-ATA 0CC Trp Val Ile Ala

CAC

His 105 GAA TGC GGC CAC Oiu Cys Gly His CAC GCC TTC His Ala Phe 110 TTC CRC TC Phe His Ser CR0 TOO CTT GAC Gin Trp Leu Asp

GAC

Asp 120 ACC GTC GGT CTC Thr Vai Oly Leu

ATC

Ile 125 TTC CTC Phe Leu 130 CTC GTC CCT TAO Leu Val' Pro Tyr

TTC

Phe 135 TCC TOO AAG TAC Ser Trp Lys Tyr

AGT

Ser 140 CAT COA COO CRC His Arg Arg His

CAT

His 145 TCC AAC ACT GOC Ser Asn Thr Oly

TCC

Ser 150 CTC GAG AGA GCC Leu Glu Arg Asp GAA OTG Glu Val 155 TTT GTC CCC Phe Vai Pro

AAG

Lys 160 AAG AAG TCA GAC Lys Lys Ser Asp

ATC

Ile 165 AAG TOO TAC 000 Lys Trp, Tyr Oly

AAG

Lys .170 TAC CTC AAC AAC Tyr Leu Asn Asn CCT TTG Pro Leu 175 384 432 480 528 576 624 672 OGA COO ACC Gly Arg Thr TAC TTA 0CC Tyr Leu Ala 195

GTG

Val 180 ATO TTA ACO OTT Met Leu Thr Val CR0 Gin 185 TTC ACT CTC GGC Phe Thr Leu Oly TOO COO TTO Trp Pro Leu 190 GGC TTC GCT Gly Phe Ala TTC AAC OTC TCG Phe Asn Val Ser

OGA

Gly 200 AGA OCT TAO GAO Arg Pro Tyr Asp

GC

Gly 205 TOO CRT Cys His 210 TTC CRC CCC AAC Phe His Pro Asn

OCT

Ala 215 CCC ATC TAC Pro Ile Tyr AAC GAC Asn Asp 220 GCC GTC Ala Val 235 CdO GAG CGT CTC Arg Giu Axg Leu CR0 Gin 225 ATA TAC ATC TCC Ile Tyr Ile Ser

GAC

Asp 230 OCT GOC ATC CTC Ala Gly Ile Leu TOO TAC GOT Cys Tlyr Oly

CTC

Leu 240 TTC COT TAC 0CC Phe Arg Tyr Ala 000 Ala 245 000 CR0 OCR OTO Ala Gin Gly Val 0CC Ala 250 TCG ATO OTC TGC Ser Met Val Cys TTC TAC Phe Tyr 255

GGA

Gly

GAT

Asp

TTG

Le 305

CTG

Leu

ATA

S le

GT'

Val

GAC

Asp

GA

GTC

Val

CAG

Gin

TGG

Trp 290

AAC

Asn

TTC

Phe

AAG

Lys

AAG

Lys

AGG

Arg 370

CCG

Pro

CAC

His 275

TTG

L~u

AAG

Lys

TCC

Ser

CCG

Pro

GCG

Ala 355

CAA

Gin

CTT

Leu 260

ACG

Thr

AGG

Arg

GTC

Val

ACG

Thr

ATA

Ile 340

ATG

Met

GGT

Giy

CTG

Leu

CAT

His

GGA

Gly

TTC

Phe

ATG

Met 325

CTG

Leu

TGG

Trp

GAG

Giu

ATT

Ile

CCT

Pro

GCT

Ala

CAC

His 310

CCG

Pro

GGA

Giy

AGG

Arg

AAG

Lys GTC AAT Val Asn TCC CTG Ser Leu 280 TTG GCT Leu Ala 295 AAT ATT Asn Ile CAT TAT His Tyr GAG TAT Glu Tyr GAG GCG Giu Ala 360 AAA GGT Lys Gly 375 67

TC

Phe

CAC

His

GTT

Val

GAC

Asp

GCG

Ala 330

CAG

Gin

GAG

Giu

TTC

Phe

GTG

Val

GAT

Asp

AGA

Arg 300

CAC

His

GAA

Glu

GAT

Asp

ATC

Ile

TAC

Tyr 380 TTG ATC Leu Ile 270 TCG TCC Ser Ser 285 GAC TAC Asp Tyr GTG GCG Val Ala GCT ACC Mla Thr GGG ACG Giy Thr 350 TAT GTG Tyr Val 365 AAC AAT Asn Asn

ACT

Thr

GAG

Giu

GGA

Gly

CAT

His

AAG

Lys 335

CCG

Pro

GAA

Glu

AAG

Lys

TAC

Tyr

TGG

Trp

ATC

Ile CAiT His 320

GCG

Ala

GTG

Val

CCG

Pro TTA T Leu 816 864 912 960 1008 1056 1104 1153 1155 0.

INFORMATION FOR SEQ ID NO:2: Soo* Val.

Met SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Gly Ala Giy Gly Arg Met Gin Val Ser Pro Pro Thr Asp Thr Ile Lys Arg Val Pro Cys Giu Thr 25 Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys 40 Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile 55 Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu 75 Ser Tyr Phe Ala Trp, Pro Leu Tyr Trp Ala Cys Thr Gly Val Trp, Val Ile Ala His Giu Cys Gly 100 105 Ser Pro Phe Ile Leu Gin His Lys Pro Lys Ile Pro Gly His 110 Ser Thr Ser Ser Pro Vai Phe 68 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 Giu 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 Giy Trp Pro Leul- *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 Giu 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 Ala Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Vai 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 Giu 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 Giu Ala Thr Lys Ala :325 330 335 Ile Lys Pro Ile Leu Giy Giu Tyr Tyr Gin Phe Asp Gly Thr Pro Val 340 345 350 Vai Lys Ala Met Trp Arg Giu Ala Lys Giu Cys Ilie Tyr Val Giu Pro 355 360 365 Asp Arg Gin Gly Giu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu .370 375 380 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: 8 LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 69 (vi) ORIGINAL SOURCE: ORGANISM: Brassica napus (vii) IMMEDIATE SOURCE: CLONE: Q508 (ix) FEATURE: OTHER INFORMATION: T to A transversion mutation at nucleotide 515 of the F form.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATG

Met 1 GGT GCA GGT Gly Ala Gly

GGA

Gly 5 AGA ATG CAA GTG Arg Met Gin Val

TCT

Ser 10 CCT CCC TCC AAG Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Glu Thr Asp GTC GGA GAA Val Gly Glu ATC AAG CGC GTA Ile Lys Arg Val TGC GAG ACA CCG Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser CTC AAG AAA GCA Leu Lys Lys Ala

ATC

Ile 40 CCA CCG CAC TGT Pro Pro His Cys

TTC

Phe 144 192 ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser CTC ATC TGG GAC Leu Ile Trp Asp

ATC

Ile ATC ATA GCC TCC Ile Ile Ala Ser

TGC

0 Cys TTC TAC TAC GTC GCC ACC ACT TAC TTC Phe Tyr Tyr Val Ala Thr Thr Tyr Phe 70

CCT

Pro CTC CTC CCT CAC Leu Leu Pro His CTC TCC! TAC TTC Leu Ser Tyr Phe TGG CCT CTC TAC Trp Pro Leu Tyr

TGG

Trp 90 GCC TGC CAA Ala Cys Gin CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115

GTC

Val 100 TGG GTC ATA CC Trp Val Ile Ala

CAC

His 105 GAG TGC GGC CAC Giu Cys Gly His GGG TGC GTC Giy Cys Val CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser CAG TGG CTT GAC Gin Trp Leu Asp

GAC

Asp 120 ACC GTC GGT CTC Thr Vai Giy Leu

ATC

Ile 125 336 384 432 480 TTC CTC Phe Leu 130 CTC GTC CCT TAC Leu Val Pro Tyr TCC TGG AAG TAC Ser Trp Lys Tyr CAT CGA CGC CAC His Arg Arg His

CAT

His 145 TCC AAC ACT GCC Ser Aen Thr Gly

TCC

Ser 150 CTC GAG AGA GAC Leu Giu Arg Asp

GAA

Giu 155 GTG TTT GTC CCC Val Phe Val Pro AAG AAG TCA GAC Lys Lye Ser Asp ATC AAG TGG TAC GGC AAG TAC CAC AAC AAC CCT TTG 528 Ile Lye Trp Tyr Gly Lys Tyr His Aen Aen Pro Leu 165 *170 175 GGA CGC ACC Gly Arg Thr

GTG

Val 180 ATG ITA ACG GTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GCC Phe Thr Leu Gly TGG CCG TTG Trp Pro Leu 190 576 624 TAC TTA GCC TTC AAC GTC TCG GGA AGA CCT TAC GAC GGC GGC TTC GCT Tyr Leu Ala Phe Asn Val Ser Giy Arg Pro Tyr Asp Gly Gly Phe Ala 195 200 205 TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Aen

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Aen

GAC

Asp 220 CGC GAG CGT CTC Arg Glu Arg Leu

CAG

Gln 225 ATA TAC ATC TCC Ile Tyr Ile Ser

GAC

Asp 230 GCT GGC ATC CTC Ala Gly Ile Leu

GCC

Ala 235 GTC TGC TAC GGT Val Cys Tyr Gly

CTC

Leu 240 TTC CGT TAC GCC Phe Arg Tyr Ala

GCC

Ala 245 GCG CAG GGA GTG Ala Gin Gly Val

GCC

Ala 250 TCG ATG GTC TGC Ser Met Val Cys TTC TAC Phe Tyr 255 GGA GTC CCG Gly Val Pro TTG CAG CAC Leu Gin His 275

CTT

Leu 260 CTG ATT GTC AAT Leu Ile Val Asn

GGT

Gly 265 TTC CTC GTG TTG Phe Leu Val Leu ATC ACT TAC Ile Thr Tyr 270 TCC GAG TGG- Ser Glu Trp ACG CAT CCT TCC Thr His Pro Ser

CTG

Leu 280 CCT CAC TAC GAT Pro His Tyr Asp

TCG

Ser 285 GAT TGG Asp Trp 290 TTG AGG GGA GCT Leu Arg Gly Ala

TTG

Leu 295 GCT ACC GTT GAC Ala Thr Val Asp

AGA

Arg 300 GAC TAC GGA ATC Asp Tyr Gly Ile

TTG

0 Leu 305 AAC AAG GTC TTC Asn Lys Val Phe

CAC

His 310 AAT ATT ACC GAC Asn Ile Thr Asp

ACG

Thr 315 CAC GTG GCG CAT His Val Ala His

CAT

His 320 720 768 816 864 912 960 1008 1056 1104 1153 1155 CTG TTC TCC ACG Leu Phe Ser Thr

ATG

Met 325 CCG CAT TAT CAC Pro His Tyr His

GCG

Ala 330 ATG GAA GCT ACC Met Glu Ala Thr AAG GCG Lys Ala 335 ATA AAG CCG Ile Lys Pro GTT AAG GCG Val Lys Ala 355

ATA

Ile 340 CTG GGA GAG TAT Leu Gly Glu Tyr

TAT

Tyr 345 CAG TTC GAT GGG Gin Phe Asp Gly ACG CCG GTG Thr Pro Val 350 GTG GAA CCG Val Glu Pro 4 4 4* *e ATG TGG AGG GAG Met Trp Arg Glu

GCG

Ala 360 AAG GAG TGT ATC Lys Glu Cys Ile

TAT

Tyr 365 GAC AGG Asp Arg 370 CAA GGT GAG AAG Gin Gly Glu Lys

AAA

Lys 375 GGT GTG TTC TGG Gly Val Phe Trp

TAC

Tyr 380 AAC AAT AAG TTA T Asn Asn Lys Leu 4* a 4 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid 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 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe 40 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile 55 Lys Arg Ser Ile Ala Ser Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 70 75 Leu Leu Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Phe 0 Gly Asp Leu 305 Leu 0e Val *oe Ser Thr Asp Leu 130 Ser Lys Arg Leu His 210 Ile Arg Val Gln Trp 290 Asn Phe Lys Lys Tyr Phe Ala Gly Val Trp 100 Tyr Gin Trp 115 Leu Val Pro Asn Thr Gly Ser Asp Ile 165 Thr Val Met 180 Ala Phe Asn 195 Phe His Pro Tyr Ile Ser Tyr Ala Ala 245 Pro Leu Leu 260 His Thr His 275 Leu Arg Gly Lys Val Phe Ser Thr Met 325 Pro Ile Leu 340 Ala Met Trp 355 Gln Gly Glu Trp Pro Val Ile Leu Asp Tyr Phe 135 Ser Leu 150 Lys Trp Leu Thr Val Ser Asn Ala 215 Asp Ala 230 Ala Gin Ile Val Pro Ser Ala Leu 295 His Asn 310 Pro His Gly Glu Arg Glu Lys Lys 375 Leu Ala Asp 120 Ser Glu Tyr Val Gly 200 Pro Gly Gly Asn Leu 280 Ala Ile Tyr Tyr Ala 360 Gly Tyr His 105 Thr Trp Arg Gly Gin 185 Arg Ile Ile Val Gly 265 Pro Thr Thr His Tyr 345 Lys val Trp 90 Glu Val Lys Asp Lys 170 Phe Pro Tyr Leu Ala 250 Phe His Val Asp Ala 330 Gin Glu Phe Ala Cys Gly Tyr Glu 155 Tyr Thr Tyr Asn Ala 235 Ser Leu Tyr Asp Thr 315 Met Phe Cys Trp Cys Gly Leu Ser 140 Val His Leu Asp Asp 220 Val Met Val Asp Arg 300 His Glu Asp Ile Tyr 380 Gin His Ile 125 His Phe Asn Gly Gly 205 Arg Cys Val Leu Ser 285 Asp Val Ala Gly Tyr 365 Asn Gly His 110 Phe Arg Val Asn Trp 190 Gly Glu Tyr Cys Ile 270 Ser Tyr Ala Thr Thr 350 Val Asn Cys Ala His Arg Pro Pro 175 Pro Phe Arg Gly Phe 255 Thr Glu Gly His Lys 335 Pro Glu Lys Val Phe Ser His Lys 160 Leu Leu Ala Leu Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro Leu Asp Arg 370 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Brassica napus (ix) FEATURE: OTHER INFORMATION: Wild type D form.

(xi) SEQUENCE DESCRIPTION: SEQ ID

ATG

Met 1 GGT GCA GGT Gly Ala Gly AGA ATG CAA GTG Arg Met Gln Val

TCT

Ser 10 CCT CCC TCC AAA Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC AAC Glu Thr Asp Asn ATC AAG COC GTA Ile Lys Arg Val

CCC

Pro 25 TGC GAG ACA CCG Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser GTC GGA GAA Val Gly Glu CTC AAG AAA Leu Lys Lys GCA ATC Ala Ile CCA CCG CAC TGT Pro Pro His Cys ATC CCT Ile Pro CGC TCT TTC TCC Arg Ser Phe Ser CTC ATC TGG GAC Leu Ile Trp Asp ATC ATA GCC TCC Ile Ile Ala Ser 96 144 192 240 288 336

TGC

CyB TTC TAC TAC GTC Phe Tyr Tyr Val

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe

CCT

Pro 75 CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro CTC TCC TAC TTC Leu Ser Tyr Phe TGG CCT CTC TAC Trp Pro Leu Tyr GCC TGC CAG Ala Cys Gin CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115

GTC

Val 100 TGG GTC ATA GCC Trp Val Ile Ala

CAC

His 105 GAG TGC GOC CAC Glu Cys Gly His GGC TGC GTC Gly Cys Val CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser CAG TOG CTG GAC Gin Trp Leu Asp

GAC

Asp 120 ACC GTC GGC CTC Thr Val Gly Leu

ATC

Ile 125 TTC CTC CTC GTC CYT TAC TTC TCC TG AAG TAC AGT CAT CGA CGC CAC Phe leu leu Val Xaa Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140

CAT

His 145 TCC AAC ACT GGC Ser Asn Thr Gly CTC GAG AGA GAC Leu Giu Arg Asp

GAA

Glu 155 GTG TTT GTC CCC Val Phe Val Pro 432 480 528 AAG AAG TCA GAC Lys Lys Ser Asp

ATC

Ile 165 AAG TGG TAC GGC Lys Trp Tyr Gly

AAG

Lys 170 TAC CTC AAC AAC Tyr Leu Asn Asn CCT TTG Pro Leu 175 GGA CGC ACC Gly Arg Thr

GTG

Val 180 ATG TTA ACG OTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGG CCT TTG 576 Trp Pro Leu 190 TAC TTR GCC TTC AAC GTC TCG GOG AGA CCT TAC GAC GGC GOC TTC GCT Tyr leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Giy Giy Phe Ala 195 200 205 TGC CAT TTC Cys His Phe 210 CAG, ATA TAO Gin Ile Tyr 225 TAC CGC TAC Tyr Arg Tyr GGA GTT OCT Gly Val Pro TTG CAG CAC Leu Gin His 275 GAT TGG TTG Asp Trp Leu 290 TTG AAC AAG Leu Asn Lys 305 OTG rro TOG Leu Phe Ser ATA AAG COG S le Lys Pro GTT AAG GCG Val Lys Ala V. **355 *GAC AGG CAA Asp Arg Gin 370

GA

CAC CCC AAC His Pro Asn ATC TOC GAC Ile Ser Asp 230 GOT GOT RTC Ala Ala Xaa 245 OTT CTG RTT Leu Leu Xaa 260 ACG CAT COT Thr His Pro AGG GGA GOT Arg Gly Ala GTC TTC CAC Val Phe His 310 ACC ATG COG Thr Met Pro 325 ATA OTG GGA Ile Leu Giy 340 ATG TGG AGG Met Trp Arg GGT GAG AAG Gly Glu Lys

GOT

Ala 215

GOT

Ala

CAA

Gin

GTC

Val

TC

Ser

TTG,

Leu 295

AAT

Asn

CAT

His

GAG

Giu

GAG

Giu

AAA

Lys 375 000 ATC TAO AAO GAO CGT GAG OGT OTC Pro Ile Tyr GO ATC OTC Gly Ile Leu GGA GTT GC Gly Val Ala 250 AAC GGG TTO Asn Gly Phe 265 OTG COT CAC Leu Pro His 280 GOC ACC GTT Ala Thr Val ATC ACG GAC Ile Thr Asp TAT CAT GOG Tyr His Ala 330 TAT TAY CAG Tyr Tyr Gin 345 GOG AAG GAG Ala Lys Glu 360 GGT GTG TI'C Giy Val Phe Asn Asp Arg 2? 0 GCC GTO TGC Ala Val Cys 235 TCG ATG GTC Ser Met Val TTA GTT TTG Leu Val Leu TAT GAC TCG Tyr Asp Ser 285 GAC AGA GAC Asp Arg Asp 300 ACG CAC GTG Thr His Val 315 ATG GAA GOT Met Giu Ala TTC GAT GGG Phe Asp Giy TGT ATO TAT Cys Ile Tyr 365 TOG TAO AAC Trp Tyr Asn 380 Glu Arg Leu TAO GOT OTO Tyr Giy Leu 240 TOO TTC TAO Cys Phe Tyr 255 ATO ACT TAdO Ile Thr Tyr 270 TOT GAG TG Ser Glu Trp TAC GGA ATO Tyr Gly Ile GOG OAT CAC Ala His His 320 ACG AAG GOG Thr Lye Ala 335 ACG OCG GTO Thr Pro Val 350 GTG GAA COG Val Oiu Pro AAT AAG TTA T Asn Lys Leu 672 720 768 816 864 912 960 1008 1056 1104 1.153 1155 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARATERISTICS: LENGTH: 384 amino acids TYPE: amino acid TOPOLOGY: linear 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 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 Vai Gly Giu Leu Lye Lye Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 Ile Cys 0e Leu Ser Phe His 145 Lys Gly Tyr Cys Gin 225 Tyr **Gly Leu Asp Leu 305 Leu Val Asp Pro Phe Ser Thr Asp Leu.

130 Ser Lys Arg Leu His 210 Ile Arg Val Gin Trp 290 Asn Phe Lys Lys Arg 370 Arg Tyr Tyr Gly Tyr 115 Leu Asn Ser Thr Ala 195 Phe Tyr Tyr Pro His 275 Leu Lys Ser Pro Ala 355 Gin Ser Tyr Phe Val 100 Gin Val Thr Asp Val 180 Phe His Ile Ala Leu 260 Thr Arg Val Thr Ile 340 Met Gly Phe Val Ala 85 Trp Trp Xaa Gly Ile 165 Met Asn Pro Ser Ala 245 Leu His Gly Phe Met 325 Leu Trp Glu Ser Ala 70 Trp Val Leu Tyr Ser 150 Lys Leu Val Asn Asp 230 Xaa Xaa Pro Ala His 310 Pro Gly Arg Lys Tyr 55 Thr Pro Ile Asp Phe 135 Leu Trp Thr Ser Ala 215 Ala Gin Vai Ser Leu 295 Asn Hi s Giu Giu Lys 375 Leu Ile Thr Tyr Leu Tyr Ala His 105 Asp Thr 120 Ser Trp Giu Arg Tyr Gly Val Gin 185 Gly Arg 200 Pro Ile Gly Ile Gly Vai Asn Gly 265 Leu Pro 280 Ala Thr Ile Thr Tyr His Tyr Tyr 345 Ala Lys 360 Gly Val Trp Phe Trp 90 Giu Val Lys Asp Lys 170 Phe Pro Tyr Leu Ala 250 Phe His Val Asp Ala 330 Gin Glu Asp Pro 75 Ala Cys Gly Tyr Glu 155 Tyr Thr Tyr Asn Ala 235 Ser Leu Tyr Asp Thr 315 Met Phe Cys Ile Lau Cys Gly Leu Ser 140 Val Leu Leu Asp Asp 220 Val Met Val Asp Arg 300 His Glu Asp Ile Ile Leu Gin His Ile 125 His Phe Asn Giy Gly 205 Arg Cys Val Leu Ser 285 Asp Val Ala Gly Tyr 365 Ile Pro Gly His 110 Phe Arg Vai Asn Trp 190 Gly Giu Tyr Cys Ile 270 Ser Tyr Ala Thr Thr 350 Val Ala His.

Cys Ala His Arg Pro Pro 175 Pro Phe Arg Gly Phe 255 Thr Glu Gly His Lys 335 Pro Giu Ser Pro Vai Phe Serf Hi's Lys 160 Leu Leu.

Ala Leu, Leu 240 Tyr Tyr Trp Ile His 320 Ala Val Pro Phe Trp, Tyr 380 Asn Asn Lys Leu INFORM4ATION FOR SEQ ID NQ:7: SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: nucleic acid STR~ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Brassica napus (vii) IMMEDIATE SOURCE: CLONE: IMC129 (ix) FEATURE: OTHER INFORMATION: G to A transversion mutation at nucleotide 316 of the D form.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ATG

Met 1 GGT GCA GGT Gly Ala Gly

GGA

Gly 5 AGA ATG CAA GTG Arg Met Gin Val CCT CCC TCC AAA Pro Pro Ser Lys AAG TCT Lys Ser GAA ACC GAC Glu Thr Asp GTC GGA GAA **Val Gly Glu *..ATC CCT CGC le Pro Arg .*50 ATC AAG CGC Ile Lys Arg GTA CCC Val Pro TGC GAG ACA CCG Cys Glu Thr Pro CCC TTC ACT Pro Phe Thr AAA CGC TCG Lys Arg Ser CTC AAG AAA GCA Leu Lye Lys Ala CCA CCG CAC TGT Pro Pro His Cys

TTC

Phe TCT TTC TCC Ser Phe Ser

TAC

Tyr 55 CTC ATC TOG GAC Leu Ile Trp Asp

ATC

Ile ATC ATA GCC TCC Ile Ile Ala Ser

TGC

Cys TTC TAC TAC GTC Phe Tyr Tyr Val

GCC

Ala 70 ACC ACT TAC TTC Thr Thr Tyr Phe CTC CTC CCT CAC Leu Leu Pro His

CCT

Pro 144 192 240 288 336 CTC TCC TAC TTC Leu Ser Tyr Phe

GCC

Ala 85 TOG CCT CTC TAC Trp Pro Leu Tyr GCC TGC CAG GGC Ala Cys Gin Gly TGC GTC Cys Val CTA ACC GGC Leu Thr Gly AGC GAC TAC Ser Asp Tyr 115

GTC

Val 100 TOG GTC ATA GCC Trp Val Ile Ala

CAC

His 105 AAG TGC GGC CAC Lye Cys Gly His CAC GCC TTC His Ala Phe 110 TTC CAC TCC Phe His Ser CAG TOG CTG GAC Gin Trp Leu Asp ACC GTC GGC CTC Thr Val Gly Leu

ATC

Ile 125 Phe Leu 130 CTC GTC CYT TAC Leu Val Xaa Tyr

TTC

Phe 135 TCC TGG AAG TAC Ser Trp Lye Tyr

AGT

Ser 140 CAT CGA CGC CAC His Arg Arg His 432

CAT

His 145 TCC AAC ACT GGC Ser Asn Thr Gly

TCC

Ser 150 CTC GAG. AGA GAC Leu Giu Arg Asp

GAA

Glu 155 GTG TTT GTC CCC Val Phe Val Pro

AAG

Lys 160 AAG AAG TCA GAC Lys Lye Ser Asp

ATC

Ile 165 AAG TOG TAC GOC Lys Trp Tyr Gly

AAG

Lys 170 TAC CTC AAC AAC Tyr Leu Asn Aen CCT TTG Pro Leu 175 GGA CGC ACC Gly Arg Thr TAC TTR GCC Tyr Leu Ala 195

GTG

Val 180 ATG TTA ACG GTT Met Leu Thr Val

CAG

Gin 185 TTC ACT CTC GGC Phe Thr Leu Gly TGG CCT TTG Trp Pro Leu 190 GGC TTC GCT Gly Phe Ala TTC AAC GTC TCG Phe Asn Val Ser

GGG

Gly 200 AGA CCT TAC GAC Arg Pro Tyr Asp

GGC

Gly 205 TGC CAT Cys His 210 TTC CAC CCC AAC Phe His Pro Asn

GCT

Ala 215 CCC ATC TAC AAC Pro Ile Tyr Asn

GAC

Asp 220 CGT GAG CGT CTC Arg Glu Arg Leu

CAG

Gin 225 ATA TAC ATC TCC Ile Tyr Ile Ser

GAC

Asp 230 GCT GGC ATC CTC Ala Gly Ile Leu

GCC

Ala 235 GTC TGC TAC GGT Val Cys Tyr Gly

CTC-

Leu 240 TAC CGC TAC GCT Tyr Arg Tyr Ala

GCT

Ala 245 RTC CAA GGA GTT Xaa Gin Gly Val

GCC

Ala 250 TCG ATG GTC TGC Ser Met Val Cys TTC TAC Phe Tyr 255 GGA GTT CCT Gly Val Pro TTG CAG CAC Leu Gin His 275 GAT TGG TTG Asp Trp Leu 290

CTT

Leu 260 CTG RTT GTC AAC Leu Xaa Val Asn

GGG

Gly 265 TTC TTA GTT TTG Phe Leu Val Leu ATC ACT TAC Ile Thr Tyr 270 TCT GAG TGG Ser Glu Trp ACG CAT CCT TCC Thr His Pro Ser

CTG

Leu 280 CCT CAC TAT GAC Pro His Tyr Asp

TCG

Ser 285 576 624 672 720 768 816 864 912 960 1008 1056 1104 1153 1155 AGG GGA GCT Arg Gly Ala

TTG

Leu 295 GCC ACC GTT GAC Ala Thr Val Asp

AGA

Arg 300 GAC TAC GGA ATC Asp Tyr Gly Ile

TTG

Leu 305 AAC AAG GTC TTC Asn Lys Val Phe

CAC

His 310 AAT ATC ACG GAC Asn Ile Thr Asp CAC GTG GCG CAT His Val Ala His CTG TTC TCG ACC Leu Phe Ser Thr

ATG

Met 325 CCG CAT TAT CAT Pro His Tyr His

GCG

Ala 330 ATG GAA GCT ACG Met Glu Ala Thr AAG GCG Lys Ala 335 ATA AAG CCG Ile Lys Pro

ATA

Ile 340 CTG GGA GAG TAT Leu Gly Glu Tyr

TAY

Tyr 345 CAG TTC GAT GGG Gin Phe Asp Gly ACG CCG GTG Thr Pro Val 350 GTG GAA CCG Val Glu Pro GTT AAG GCG Val Lys Ala 355 ATG TGG AGG GAG Met Trp Arg Glu

GCG

Ala 360 AAG GAG TGT ATC Lys Glu Cys Ile

TAT

Tyr 365 GAC AGG Asp Arg 370 CAA GGT GAG AAG Gin Gly Glu Lys

AAA

Lys 375 GGT GTG TTC TGG Gly Val Phe Trp

TAC

Tyr 380 AAC AAT AAG TTA T Asn Asn Lys Leu INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid TOPOLOGY: linear (ii) 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 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr 25 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 40 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser 55 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 70 75 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 90 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 Xaa 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 Xaa Gin Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 Gly Val Pro Leu Leu Xaa 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 S"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 Gln Phe Asp Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Gu Ala Lys Gu Cys He Tyr Val Gu Pro 355 360 365 Asp Arg Gin Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

This application is a divisional application of 12932/97, the subject matter which is incorporated herein by way of reference

Claims (31)

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, wherein said gene is effective for altering fatty acid composition in Brassicaceae or Helianthus seeds and wherein said sequence includes 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 Smutant desaturase gene encodes a microsomal gene product.

4. The nucleic acid fragment of claim 1, wherein said 15 at least one mutation comprises a mutation in a region of O said desaturase gene encoding a His-Glu-Cys-Gly-His amino acid motif. The nucleic acid fragment of claim 4, wherein said at least one mutation comprises a non-conservative amino 20 acid substitution in said region.

6. The nucleic acid fragment of claim 5, wherein said at least one mutation comprises the sequence His-Lys-Cys- Gly-His.

7. The nucleic acid fragment of claim 1, wherein said mutant desaturase gene is from a Brassica napus plant.

8. The nucleic acid fragment of claim 1, wherein said gene is the D form of a Brassicaceae microsomal gene.

9. The nucleic acid fragment of claim I, wherein said at least at least one mutation comprises the sequence Lys-Tyr-His-Asn-Asn-Pro. A plant of the Brassicaceae 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. 20 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 25 Brassicaceae or Helianthus seeds and wherein said sequence includes said at least one mutation. 81

16. The nucleic acid fragment of claim 15, wherein said sequence comprises a full-length coding sequence of said gene.

17. The nucleic acid fragment of claim 15, wherein 5 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 Brassicaceae or Helianthus 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- 15 Xaa-His amino 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 0 the sequence His-Asp-Cys-Gly-His.

22. The plant of claim 19, wherein said mutant desaturase gene is from a Brassica napus plant.

23. The plant of claim 19, wherein said plant is a Brassica napus plant. o*o oooo

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 amino acid motif; b) a sequence of at least 10 nucleotides from a fatty acid desaturase gene having at least one mutation, said at least one gene mutation in a region encoding a His-Xaa-Xaa- Xaa-His amino acid motif, said delta-12 gene mutation and said delta-15 gene mutation conferring an altered fatty acid composition in seeds of said plant. A Brassicaceae or Helianthus plant containing a 15 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 amino acid motif and wherein said mutation confers an altered fatty acid composition in 20 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, following crushing and extraction of said seeds, from 25 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. The oil of claim 29, wherein said oil has, following crushing and extraction of said seeds,- from 5 about 0.5% to about 10% a-linolenic 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 0. 33. A method for producing a Brassicaceae or Helianthus plant line, comprising the steps of: a) inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species; 15 b) obtaining one or more progeny plants from G" 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* S. 20 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 4* one progeny plant by self- or cross-pollination, said plant line having said at least one delta-12 25 gene mutation.

34. The method of claim 33, wherein said plant line produces seeds yielding an oil having a stabilized linoleic acid content from about 1% to about 14%. 84 The method of claim 33, further comprising the steps of: e) inducing mutagenesis in 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 line progeny plants that contains a delta-15 fatty acid desaturase gene having at least one delta-15 gene 0 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 line from said at least one plant line progeny plant by self- or cross-pollination, 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 t variety is a Brassica napus variety. 20 37. The method of claim 36, wherein said mutation is in a first form of delta-12 fatty acid desaturase. S.

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 in a region other than a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif. The method of claim 36, further comprising the steps of: e) inducing mutagenesis in 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 line progeny plants that contains 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 amino 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 line, comprising the steps of: inducing mutagenesis in cells of.a starting variety of a Brassicaceae or Helianthus species; 20 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 amino acid motif; and... d) producing said plant line from said at least one progeny plant by self- or cross-pollination, said plant line having said delta-15 gene S 30 mutation. S C. DATED this 16 th day of February, 2001 CARGILL, INCORPORATED By their Patent Attorneys: LAWRIE CALLIAN LAWRIE *JS 0A