AU2011265499B2 - Altered FAD2 and FAD3 genes in brassica and the molecular marker-assisted detection thereof - Google Patents

Abstract

The present invention provides methods of marker assisted selection for high oleic/low linolenic traits in canola and in other oil seed crop species, as well as isolated nucleic acids for use as molecular markers in such methods. In particular, 5 molecular markers and Brassica nucleic acid corresponding to fad2 and fad3 gene mutations are disclosed. The markers of the present invention are highly useful for the direct selection of desirable fad2 and fad3 alleles during marker-assisted trait introgression and breeding. In a one aspect of the embodiment, two single nucleotide polymorphism (SNP) markers are provided which correspond to the alleles. Thus, the 10 present invention advantageously permits one of skill in the art to breed for the molecular markers described herein, or derivatives thereof, rather than breeding for a high oleic/low linolenic phenotype.

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Dow AgroSciences LLC Actual Inventor(s): Mandy Lynne Sullivan-Gilbert, Xueyi Hu, Steven Arnold Thompson, Manju Gupta Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: ALTERED FAD:2 AND FAD3 GENES IN BRASSICA AND THE MOLECULAR MARKER ASSISTED DETECTION THEREOF Our Ref: 931299 POF Code: 112152/112152 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6008q Altered FAD2 and FAD 3 Genes in Brassica and the molecular marker-assisted detection thereof The present application is a divisional application from Australian patent application 5 number 2004210965, the entire disclosure of which is incorporated herein by reference. 100011 Field of the Invention The present Invention relates generally to methods and materials for use In plant breeding. More specifically, the present invention relates to the marker assisted identification of genes encoding phenotypic traits in oil seed plant species, and in Brassica species in particular. [00021 BACKGROUND OF THE INVENTION The genus Brassica includes canola, one of the world's most important oilseed crops, and the most important oilseed crop grown in temperate geographies. Canola has been traditionally characterized as Brassica napus (a species derived as a result of inter-specific crosses of Brassica rapa and Brassica oleracea) In which erucic acid and glucosinolates have been eliminated or significantly reduced through conventional breeding, The majority of canola oil is in the form of vegetable oils produced for human consumption. There is also a growing market for the use of canola oil in industrial applications, [0003] Canola Is a polyploid species considered to have arisen from the hybridization of Brassica o/eracea, having a diploid C genome, and Brassica rapa, having a diploid A genome. Cytogenetic investigation revealed the AA and CC genomes show a degree of relatedness, being partially homologous to one another and thought to have been derived from a common ancestor genome (Prakash and Hinata, 1980). Although technically classified as IaI WO 2004/072259 PCT/US2004/003852 diploids, the genomes of both progenitor species contain a high percentage of regions duplicative of one another (Song et al., 1991). Genetic analysis revealed that the AA genome of BrassIca rapa contributed ten chromosomes to Brass/ca napus, while Brassica oleracea contributed nine chromosomes from its CC genome as the maternal donor (Song et al., 1992). [0004] The quality of edible and Industrial oil derived from a particular variety of canola seed is determined by Its constituent fatty acids, as the type and amount of fatty acid unsaturation have implications for both dietary and industrial applications. Conventional canola oil contains about 60% oleic acid (C18:1), 20% linoleic acid (C18:2) and 10% linolenic acid (18:3). The levels of polyunsaturated linolenic acid typical of conventional canola are undesirable as the oil is easily oxidized, the rate of oxidation being affected by several factors, including the presence of oxygen, exposure to light and heat, and the presence of native or added antioxidants and pro-oxidants in the oil. Oxidation causes off-flavors and rancidity of as a result of repeated frying (induced oxidation) or storage for a prolonged period (auto-oxidation). Oxidation may also alter the lubricative and viscous properties of canola oil. [00051 Oils exhibiting reduced levels of polyunsaturated fatty acids and increases in the level of monounsaturated oleic acid relative to conventional canola oil are associated with higher oxidative stability. The susceptibility of individual fatty acids to oxidation is dependent on their degree of unsaturation. Thus, the rate of oxidation of linolenic acid, which possesses three carbon-carbon double bonds, is 25 times that of oleic acid, which has only one double bond, and 2 times that of linoleic acid, which has two double 2 WO 2004/072259 PCT/US2004/003852 bonds. Linoleic and linolenic acids also have the most impact on flavor and odor because they readily form hydroperoxides. High oleic oil (270% oleic) Is less susceptible to oxidation during storage, frying and refining, and can be heated tc a higher temperature without smoking, making it more suitable as cooking oil. 100061 Two strategies are generally used to Increase the oxidative stability of canola oil. In one approach, partial hydrogenation is used to lower linolenic acid coment. Unfortunately, partial hydrogenation leads to the formation of trans-faty acids, which have been linked to elevated levels of low-density lipoprotein cholesterol (LDL or "bad" cholesterol) in the blood, and consequently, to an increased risk of coronary heart disease. The second major strategy Involves breeding programs to develop canola varieties with high olelc and low linolenic acid levels relative to conventional canola oil. High oleic and low linolenic mutants have been produced through mutagenesis (Rakow, 1973; Wong et al. 1991; Auld et al. 1992) and transgenic modification (Debonte and Hitz, 1996). Examples of commercially sold canola varieties having a fatty acid profile of C18:1 above 70% and C18:3 below 3.5% are the NexeraTM varieties, marketed by Dow AgroSciences LLC (Indianapolis, IN), which varieties produce NatreonTM oil. One such line, AGO19 (a NexeraTM variety) contains 71-78% oleic (C18:1) and <3% llnolenic (C18:3) acid. AGO19 was originally created by ethyl methanesulphonate (EMS) mutagenesis and is described in US 6,169,190 B1 to Sernyk, assigned to the assignee of the present invention. 3 WO 2004/072259 PCT/US2004/003852 [00071 Current methods for producing F 1 hybrid Brassica seeds have definite limitations in terms of cost and seed purity. Generally, these methods require stable, sib-incompatible and self-IncompatIble, nearly homozygous parental breeding lines, which parental breeding lines are available only after repeated selfing to generate inbred lines. Furthermore, Inbreeding to develop and maintain the parental lines is accomplished by labor intensive techniques, such as bud pollination, since BrassIca hybrid seed production systems based on self-incompatible traits must utilize strongly self-incompatible plants. Environmental conditions during the breeding process, such as temperature and moisture, typically affect plant lipid metabolism, thus also affecting the content level of fatty acids (Harwood, 1999). Environmental variability therefore makes the phenotypic selection of plants less reliable. Deng and Scarth (1998) found that increase in post-flowering temperature significantly reduced the levels of 018:3 and Increased C18:1. Similar results were reported In other studies (Yermanos and Goodin, 1965; Canvin, 1965). 100081 Breeding for low linolenic varieties Is particularly challenging since C18:3 content is a multi-gene trait and Inherited In a recessive manner with a relatively low heritability. Genetic analysis of a population derived from the cross between "Stellar" (having a low C18:3 content (3%)) and "Drakkar" (having a "conventional" C18:3 level (9-10%)) indicated that the low C18:3 trait was controlled by two major loci with additive effects designated Li and L2 (Jourdren et al.,1996b). These two major loci controlling C18:3 content were found to correspond to two fad3 (fatty acid desaturase 3) genes; one located on A genome (originating from Brassica rapa) and the other on the C 4 WO 2004/072259 PCT/US2004/003852 genome (originating from Brassica olecera) (Jourdren et al.,1 996; Barret et aL., 1999). [00091 Traits which are continuously varying due to genetic (additive, dominance, and epistatic) and environmental influences are commonly referred to as "quantitative traits." Quantitative traits may be distinguished from "qualitative" or "discrete" traits on the basis of two factors: environmental influences on gene expression that produce a continuous distribution of phenotypes; and the complex segregation pattern produced by multigenic Inheritance. The identification of one or more regions of the genome linked to the expression of a quantitative trait led to the discovery of Quantitative Trait Loci ("QTL"). Thormann et al. (1996) mapped two QTL that explained 60% of the variance for the ilnolenic content, while Somers et a/. (1998) identified three QTL that collectively explained 51 % of the phenotypic variation of C18:3 content. A three-locus additive model was also reported by Chen and Beversdorf (1990). ROcker and Rbbelen (1996) indicated that several minor genes are most likely involved in the desaturation step. [00101 Heritability for C18:3 content was estimated to be 26 - 59% (Kondra and Thomas, 1975) (where the variability of heritability is a function of genetics as opposed to environmental factors). Complexity of the Inheritance of linolenic acid may be due to the fact that linolenic acid can be synthesized either from the desaturation of 01 8:2 or the elongation of 01 6:3 (Thompson, 1983). 5 WO 2004/072259 PCT/US2004/)03852 [0011] In contrast to linolenic acid, Inheritance of oleic acid is less complex, and the heritability of oleic acid is relatively high. It Is reported that high oleic acid content Is controlled by a major locus called fad2 (fatty acid desaturase 2) gene which encodes the enzyme responsible for the desaturation of oleic acid to Uinolelc acid (C18:2) (Tanhuanpaa et al., 1998; Schlerholt et al., 2001). All of the functional gene copies of the fad2 gene that have been reported and mapped to date are located on the A-genome-originated linkage group N5 (Scheffler et a., 1997; Schierholt et al., 2000). Chen and Beversdorf (1990) reported that the accumulation of oleic acid was controlled by at two segregation genetic systems, one acting on chain elongation and the other involving desaturatlon. Heritability for C18:1 content was estimated to be 53% to 78% (Kondra and Thomas 1975) and 94% (Schlerholt and Becker, 1999), :espectively. Due to the higher heritability, the expression of C18:1 content Is environmentally less affected and relatively stable (Schierholt and Becker, 1999). (00121 In NexeraTM canola germplasm, 1 to 2 genes are found to control C18:1 content and at least 3 genes are Involved in C18:3 expression. In segregating progenies, the distribution of seed C18:3 content is continuous, thereby making it difficult to identify genotypic classes with desirable C18:3 levels. In addition, there Is a low correlation In fatty acid content between greenhouse (GH) and field grown plants, further making it challenging to reliably select GH plants with desirable levels of 018:3. [0013] Molecular maker selection Is based on genotypes and is therefore independent from environment effects. Molecular markers would alleviate 6 WO 2004/072259 PCT/US2004/003852 the problem of the unreliable selection of plants in the greenhouse attributable.to the low correlation in fatty acid content between greenhouse grown plants and field grown plants. Significantly, molecular markers tightly linked to the genes controlling C18:1 and C18:3 content would allow early selection of plants carrying genes for high C18:1 and low C18:3. Marker assisted selection at early stage will significantly save greenhouse space, therefore, Improve the efficiency of greenhouse use, and reduce the breeding workload in the field. [0014] More generally, molecular markers have advantages over morphological markers in that: molecular markers can be highly polymorphic while morphological markers are strictly phenotype dependent; morphological markers may Interfere in the scoring of certain quantitative phenotypes while molecular markers exhibit a 1:1 relationship between genotype and phenotype (thus allowing the unambiguous scoring of all possible genotypes for a given locus); and epistatic interactions tend to limit the number of morphological markers useful In a population, while molecular markers do not interact epistatically. [00151 Different types of molecular markers such as RAPD (random amplified polymorphic DNA) markers (Tanhuanpaa et a., 1995; Hu et al, 1995; Rajcan et al., 1999;Jourdren etal., 1996), RFLP (restriction fragment length polymorphism) markers (Thormann et aL., 1996) and SCAR (sequence-characterlzed amplified region) markers (Hu et al, 1999) have been identified to be associated with low C18:3 levels in Brassica. napus. Molecular markers have also been Identifled for high C18:1 content. A RAPD 7 WO 2004/072259 PCT/US2004/l003852 marker was Identified to be linked to the QTL affecting olelc acid concentration in spring turnip rape (B. rapa ssp. o/eifera) and was later converted into a SCAR marker (Tanhuanpaa 6t al., 1996). Schlerholt et a/. (2000) identified three AFLP markers linked to a high oleic acid mutation In winter oilseed rape (B. napus L.). Tanhuanpaa et al. (1998) developed an allele-specific PCR marker for oleic acid by comparing the wild-type and high oleic allele of the fad2 gene locus in spring turnip rape (B. rapa ssp. oleifera). However, most of these markers are low-throughput markers such as RAPD, AFLP and RFLP and are not suitable for large scale screening through automation. 10016] Therefore, what is needed In the art are molecular markers suitable for identifying canola plants producing a seed oil with desired levels of high oleic and low linolenic acid which render the oil sufficiently stable for uses in various dietary and industrial applications. It would be further advantageous to map genes responsible for oleic and linolenic acid concentration and to develop high throughput PCR markers linked to high oleic and low linolenic acid content In order to facilitate the selection of these traits in oil seed crop trait Introgression and breeding. [00171 SUMMARY OF THE INVENTION 100181 The present invention provides methods of marker assisted selection In canola and other oil seed crop species, as well as Isolated nucleic acids for use as molecular markers in such methods.

WO 2004/072259 PCT/US2004/003852 [00191 In a first embodiment, an isolated and purified genetic marker associated with high oleic oil content in Brassica, is provided. The marker maps to a linkage group selected from the group consisting of N5 and NI in the Brassica genome, and has the sequence of SEQ ID NO. 5 or a derivative thereof. An Isolated and purified genetic marker associated with low linolenic oil content in Brass/ca Is also provided. The marker maps to a linkage group selected from the group consisting of N14 and N4 In the Brass/ca genome, and has the sequence of SEQ ID NO. 6 or a derivative thereof. [00201 In another embodiment, the present invention provides nucleic acid sequences corresponding to mutated fad2 and/or fad3 genes. These genes may be introduced into canola or other oil seed plants by any of a number of known methods in the art. Additionally, wild-type fad2 and/or fad3 may be altered by known In vivo or in vitro methods to correspond to the fad2 and/or fad3 genies of the present invention. 100211 In an additional embodiment, molecular markers corresponding to the fad2 anc" fad3 gene mutations are disclosed. The markers of the present invention are highly useful for the direct selection of desirable fad2 and fad3 alleles during marker-assisted trait introgression and breeding. In a one aspect of the embodiment, two single nucleotide polymorphism (SNP) markers are provided which correspond to the alleles. Thus, the present invention advantageously permits one of skill in the art to breed for the molecular markers described herein, or derivatives thereof, rather than breeding for the phenotype. 9 WO 2004/0722.59 PCT/US2004/003852 In a related embodiment, methods of marker-assisted selection for high oleic and/or 1o1N linolenic oil seed plants are disclosed. In one aspect of the embodiment, methods for reliably and predictably introgressing traits for high oleic andor low linolenic acid content into Brass/ca germplasm are provided. The methods include: (a) identifying one or more nuclelc acid markers for marker ai3sisted selection among Brassica lines to be used In a Brassica breeding program, wherein the nucleic acid markers map to at least one of linkage groups N14, N4, N5 or N1 and wherein the nucleic acid markers are selected from among any of SEQ. ID. NOs. 5 and 6, and (b) Introgressing said traits for high olelc and/or low linolenic acid content into said Brassica by performing marker-assisted selection. A further method is drawn to identifying nucleic acid as a modulator of high oleic and/or low linolenic acid content in Brass/ca, and comprises exposing Brassica nucleic acid to a molecular marker selected from the group consisting of SEQ. ID. Nos. 5 and/or 6. 100221 BRIEF DESCRIPTION OF THE FIGURES [00231 FIG. 1 shows partial genomic nucleotide sequences of the fad2 gene cloned from DMS100 and Quantum. The top is DMS100 sequence (SEQ. ID. NO. 7) and bottom Is Quantum sequence (SEQ. ID. NO. 9). The arrowhead Indicates a single nucleotide mutation of C to T, which resulted in a stop codon (TAG) (shaded). The forward and reverse primers for PCR-based mutant allele-specific marker are bolded and underlined. [00241 FIG. 2 provides amino acid sequences of the fad2 gene, degenerated from the genomic nucleotide sequence cloned from DMS100 (SEQ. ID. NO. 8), Quantum (SEQ. ID. NO. 10) and from a published Brassica napus fad2 10 WO 24)04/072259 PCT/US2004/003852 gene (BNfad2) (SEQ. ID. NO. 11). The arrowhead indicates the position of the stop codon resulting from a single nucleotide mutation (C to T) In DMS1 00. 100251 FIG. 3 shows genomic nucleotide sequences of the fad3c gene cloned from DMS100 and Quantum. The top is the DMS100 sequence (SEQ. 11D. NO. 12) and the bottom is the Quantum sequence (SEQ. ID. NO. 13). Excns are boxed, introns are unboxed, which correspond to exons 4, 5, 6 and 7 and introns 4, 5 and 6 of the fad3 gene in Brassica rapa and Arabldopsls. The arrowhead indicates a single nucleotide mutation of G to A. The forward and reverse primers for PCR-based mutant allele-specific marker are bolded and underlined. [0026] FIG. 4 provides a table which correlates the mutant allele-specific markers and fatty acid content of 184 DH lines derived from the cross of Quantum and DMS100, as well as electrophoresis results of PCR products amplified from the mutant allele-specific marker for the fad2 gene. 100271 FIG. 5 Is a QTL map of showing one major (N5) and one minor (N1) QTL region for high C18:1, and three QTL regions (N4 and N14) for low C18:3 detected by markers of the present Invention. 100281 FIG. 6 Is provides Invader Assay results for mutant and wild-type alleles of fad2 and fad3 genes. 100291 FIGs, 7 and 8 provide list of the various PCR markers developed and tested for high oleic and/or low linolenic trait identification. 10030] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present Invention relates generally methods and materials for use in plant breeding. In a preferred embodiment, the present Invention relates to 11 WO 2004/072259 PCT/US2004/003852 methods and compositions of matter for marker-assisted identification of genes encoding high oleic, low linolenic traits In canola. [0031] By "genetic locus" is meant a location on a chromosome. [00321 By "genomic locus" is meant a location within the entire set of chromosomes of an organism. 100331 As used herein, "linkage disequilibrium" refers to a statistical association between two loci or between a trait and a marker. [00341 As used herein, "marker" includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A genotype may be defined by use of one or a plurality of markers. 100351 The term "derivative", as used herein, refers to a modification of a sequence disclosed in the present invention. Illustrative of such modifications with regard to molecular markers would be the substitution, insertion, and/or deletion of one or more bases relating to a nucleic acid sequence of a marker disclosed herein that preserve, slightly alter, or increase the function of the molecular marker in identifying one or more high oleic and/or low linolenlc traits In Brassica or other oil seed crop species. Such derivatives can be readily determined by one skilled in the art, for example, using computer modeling techniques for predicting and optimizing sequence structure. The term "derivative" thus also includes nuclelc acid sequences having substantial sequence homology with the disclosed marker 12 WO 2004/07225.9 PCT/US20(14/003852 sequences herein such that they are able to have the disclosed functionalities for use In marker-assisted breeding. (00361 The term "homology", as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., Identity). A partially complementary sequence is one that at least partially inhibits an Identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term "substantially homologous." The Inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (I.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency. This Is riot to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) Interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% Identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence. [0037] The terms "identity" and "similarity", as used herein, and as known In the art, are relationships between two polypeptide sequences or two polynucleotide sequences, as determined by comparing the sequences. In the art, Identity also means the degree of sequence relatedness between two 13 WO 2004/072:L99 PCT/US2004/003852 polypeptide or two polynucleotide sequences as determined by the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press. New York (1988); Blocomputing: Informatics and Genome Projecto-, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York (1991)). Methods commonly employed to determine identity or similarity between two sequer.ces include, but are not limited to those disclosed In Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Methods to determine identity and similarity are codified In computer programs. Typical computer program methods to determine identity and similarity between two sequences include: GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)),' BLASTP, BLASTN, FASTA and TFASTA (Atschul, S. F. et al., J. Mol. Blol. 215: 403 (1990)). [00381 An "insertion" or "addition", as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule. 14 WO 2004/07229 PCT/US2004/003852 100391 The term "statistically associated" refers to the tendency of two events to occur together at a frequency greater than that attributable to chance, where the frequency attributable to chance is represented by a pre determined level of significance. Statistical association can be determined by any one of a number of significance tests well known to those in the art, for example, ANOVA or t-tests. See, e.g. Statistical Methods, Snedecor, G. W. and Cochran, W. G., Iowa State University Press, Ames, Iowa (1985). Significance levels for alpha are preferably less than 0.01. For example, levels of significance for this invention could range between 0 and about 0.250, e.g., less than about 0,0001, 0.00050, 0.0010, 0.0050, 0.010, 0.025, 0.050, 0.100, or 0.250. [00401 The term "stringency" is used herein to describe the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that "stringency" conditions may be altered by varying the parameters just described either individually or in concert. With "high stringency" conditions, nucleic acid base pairing will occur only between nuclelc acid fragments that have a high frequency of complementary base sequences (for example, hybridization under "high stringency" conditions may occur between homologs with about 85-100% identity, preferably about 70-100% Identity). With medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with an Intermediate frequency of complementary base sequences (for example, hybridization under "medium stringency" conditions may occur between homologs with about 50-70% Identity). Thus, conditions of "weak" or "low" 15 WO 2004/072259 PCT/US2004/003852 stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. [0041] As used in the present application, the term "substantial sequence homology" is used to indicate that a nucleotide sequence (in the case of DNA or RNA) or an amino acid sequence (in the case of a protein or polypeptide) exhibits substantial, functional or structural equivalence with another nucleotide or amino acid sequence. Any functional or structural differences between sequences having substantial sequence homology will be de minimis; that is they will not affect the ability of the sequence to function as indicated in the present application. Sequences that have substantial sequence homology with the sequences disclosed herein are usually variants of the disclosed sequence, such as mutations, but may also be synthetic sequences. [0042] A "substitution", as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively. 10043] Canola varieties DMS100 (mutant type) and Quantum (wild type) were used in the cloning of fad2 (fatty acid desaturase-2) and fad3 (fatty acid desaturase-3) alleles. The variety DMS1 00 was derived from an F 4 bulk of a single F 3 plant selection originating from the cross of Global X AGO1 9 sister line. DMS1 00 is a HOLL (kjigh Qlelc and Low Linolenic) line with oleic acid content at about 77% and linolenic acid content at about 3%. Quantum Is a 16 WO 2004/)72259 PCT/US2004/003852 commercial variety and contains low oleic acid (-66%) and high linolenic acid (-7%) content. As discussed in detail herein, sequencing of DMS100 genomic clones of fad2 and fad3 desaturase enzymes involved in the fatty acid synthesis pathway revealed single nucleotide mutations in each of the genes. Further sequence analyses show the mutations to be the cause of altered fatty acid contents in DMS100. These two mutations are distinct from previously published mutations (Tanhuanp&& et al. 1998; Jourdren 1996), and the use of these sequences as isolated nucleic acid conferring HOLL traits Is an aspect of the present invention. [0044] C 18:1 content in canola is influenced by a fad2 gene that encodes an enzyme (endoplasmic delta 12 oleate desaturase) responsible for the desaturation of oleic acid (C18;1) to linoleic acid (C18:2). In the Examples that follow, 9 DMS1 00 clones and 10 Quantum clones were sequenced. The sequence analysis and alignment of these clones identified a single nucleotide mutation, C to T, at position 411 that consistently occurred in the fad2 gene sequence of all the DMS1 00 clones (SEQ, ID. NO. 7), but not the Quantum clones (SEQ. ID. NO. 9) (see FIG. 1). Further analysis Indicated that this single nucleotide mutation occurred in the coding sequence (exon) of the fad2 gene (see FIG. 2). As further shown in FIG. 1, the mutation of C to T created a stop codon (TAG) that causes early termination of the polypeptide chain during translation. The stop codon results in the incorporation of only 185 amino acids into the polypeptide instead of all 384 amino acids of the full-length polypeptide (FIG. 2). The truncated polypeptide appears to have little, if any, function as an active desaturase for the desaturation of oleic acid to linolelc acid, thus leading to the accumulation of 17 WO 2004/072239 PCT/US2004/003852 oleic acids In the seeds of the mutant line. The lack of functionality of the mutant fad2 gene explains the significantly higher C18:1 content (77%) of DMS100 relative to the wild-type line Quantum (66%). [00451 The fad3 gene encodes for endoplasmic delta-15 linoleic desaturase, an enzyme responsible for the desaturation of linoleic acid (C18:2) to linolenic acid (C18:3). Two fad3 genes (fad31 and fad32) in particular have been reported to control linolenic content. Seven DMS1 00 clones and 6 Quantum clones of fad31 and 6 DMS100 clones and 6 Quantum clones of fa32 wore sequenced. Sequence analysis and alignment revealed no sequence difference between DMS100 and Quantum clones for fad3l (data not shown). However, sequence alignment revealed a single nucleotide mutation, G to A, at the first base of 5' splice site of the third intron in fad32 gene (se FIGu 3). This intron corresponds to the Intron 6 of the fad3 gene In B. rapa (Tanhuanpad, 2000) andt Arabldopsis (Nishluchi etal 1994). The fad3 gerie of B. rapa and Arabidopsis contains 8 exons and 7 introns, while the sequence examined covers exon 4 (partial), 5, 6 and 7 (partial) and intron 4, 5 and 6. This interpretation of exons/introns in the fad3 gene is supported the fact that the fad3 gene sequence is highly conserved among sequenced Brassice species and Arabidopsis. [00461 Plant introns contain highly conserved 5' splice sites (exon / Intron junction- AG/GTAAG) and 3' splice sites (intron / exon junction -TGCAG/G. The first two nucleotides in the 5' splice site intron junction sequence, +1G and +2T, have shown 100% and 99% conservation respectively among over 1000 Arabidopsls introns studied (Lorkovic, 2000 and Brown, 1996). The 18 WO 2004/072,:59 PCT/US2004/003852 accuracy of splicing depends on the mechanisms of intron signal recognition and the correct selection of 5' and 3' splice sites. Referring again to FIG. 3, the mutation of +1G to +1A at the 5' splice site (position 530) identified herein can abolish splicing or lead to exon skipping, i.e., the affected exon (exon 6) and both flanking introns are removed in a single splicing event (Lorkovic et al., 2000; Simpson et al., 1998). Such exon skipping could lead to synthesis of a polypeptide missing the amino acids encoded by the exon 6 of the fad3 gene. The mutation could also block splicing at the normal 5' splice sito and activate cryptic splice sites at different positions, which can cause cryptic splicing of the affected exon together with the downstream Intron (McCullough et al., 1993). Such cryptic splicing could lead early termination of translation and synthesis of a shorter polypeptide for delta-15 linoleate desaturase encoded by fad3. This will occur because the intron contains stop codons in all 3 possible reading frames and hence, exon 7 and 8 wili remain untranslated. The Incomplete translation of the fad3 can inactivate the enzyme and block the desaturation of linoleic acid (C18:2) to linolenic acid (C18:3), resulting in the decrease of C18:3 accumulation in canola seeds. 100471 These data strongly suggest that the single nucleotide mutations identified in the fad2 and fad3 genes are factors that account for the increase in oleic acid and decrease in linolenic acid contents in the canola line DMSI100. Using the molecular markers of the present invention or markers with substantial homology thereto, these two mutations may serve to allow marker assisted introgression Into canola lines making use of DMS1 00, its progeny or derivatives, or transgenic versions of its mutated 19 WO 2004(072259 PCT/US20041003852 fad2 and 0ad3 genes (SEQ. ID. NO. 7 (see FIG. 1) and SEO. ID. NO. 12 (see FIG. 3) respectively), for purposes of developing HOLL canola. [00481 klentification of mutations In fad2 and fad3 genes Referring to FIG. 1, primers homologous to the Arabldopsis fad2 gene sequence were used to amplify genomic DNA fragments of the fad2 gene from B. napus lines DMS1 00 and Quantum. The primer pair FAD2-2F: CAATCCCTCGCTCTTTCTCCTACC (SEQ. ID. NO. 1) and FAD2-6R: CCTTTCTTGTCACCTTCCCTGTCC (SEQ. ID. NO. 2) amplified a fad2 fragment of the same length (986 bp) from each of the two parents. The amplified fragments were then cloned and sequenced to investigate the sequence differences of fad2 gene between the two parents. (00491 Genomic DNA fragments corresponding to the fad3l and fad32 genes were amplified from DMS100 and Quantum lines using PCR. The primers f or amplification were designed from the published B. napus fad3l and fad32 gene sequences (Brunel et al. 1999, GenBank Accession AF056569 and AF056570, respectively). The fad3l fragments amplified by the primer pairs BNFD31 -CF (GAGGCTTGGACGACCACTTG)(SEQ. ID. NO. 3) aid BNFD31-CR (GACTGGACCAACGAGGAATG)(SEQ. ID. NO. 4) and fad32 fragments amplified by the primer pairs BNFD32-CF (CAAGAATTTGTCCCACAGTACAC)(SEQ. ID. NO. 14) and BNFD32-CR (CAACTGTTGTTAATCCTCCACG)(SEQ. ID. NO. 15) were cloned because these fragments covered more sequences of each gene. Seven DMS 100 clones and 6 Quantum clones of fad3l and 6 DMS100 clones and 6 Quantum clones of fad32 were sequenced. Sequence analysis and 20 WO 2004/072259 PCT/US2004/003852 alignment revealed no sequence difference between DMS1 00 and Quantum for fad3l (data not shown). However, sequence alignment revealed a single nucleotide mutation, G to A, at the first base of 5' splice site of the third Intron In fad32 gene (see FIG. 3). This intron corresponds to the Intron 6 of the fad3 gene in B. rapa (Tanhuanpda, 2000) and Arabidopsis (Nishiuchi et al. 1994). The fad3 gene of B. rapa and Arabidopsis contains 8 exons and 7 introns, while the sequence examined covers exon 4 (partial), 5, 6 and 7 (partial) and intron 4, 5 and 6. This interpretation of exons/introns in the fad3 gene is supported by the fact that the fad3 gene sequence is highly conserved among sequenced Brassica species and ArabidopsIs. [00501 Plant introns contain highly conserved 5' splice sites (exon / Intron junction- AG/GTAAG) and 3' splice sites (intron I exon junction - TGCAG/G. The firsi: two nucleotides in the 5' splice site Intron junction sequence, +1 G and +2T, have shown 100% and 99% conservation respectively among over 1000 Arabidopsis introns studied (Lorkovic, 2000 and Brown, 1996). The accuracy of splicing depends on the mechanisms of intron signal recognition and the correct selection of 5' and 3' splice sites. Referring again to FIG. 3, the mutation of +1G to +1 A at the 5' splice site (position 530) identified herein can abolish splicing or lead to exon skipping, I.e., the affected exon (exon 6) and both flanking introns are removed in a single splicing event (Lorkovic et al., 2000; Simpson et al., 1998). Such exon skipping could lead to synthesis of a polypeptide missing the amino acids encoded by the exon 6 of tho fad3 gene. The mutation could also block splicing at the normal 5' splice site and activate cryptic splice sites at different positions, which can cause cryptic splicing of the affected exon together with the downstream 21 intron (NiCuilough et al., 1993). Such cryptic splicing could lead to early termination of translation arid synthesis of a shorter polypepTiade for delta-I 5 linoleate desaturase encoded by fad3. This will occur because the intron contains stop codons in all 3 possible reading frames and hence, exon 7 and 8 will remain untrans[ated. The 5 incornpleta translation of the fd3 can inactivate the enzyme and block the desaturation of linoleic acid (C18:2) to linoienic acid (C18:3), resulting in the decrease of C 18:3 accumulation in- canola seeds. [0051] This data strongly suggests that the single nucleotide mutations identified in 0 the fad2 and fad3 genes are factors that account for the increase in oleic acid and decrease in linoleric eacid contents in the canola line DMSI00. As shown in FIGs. 1 and 3 respectively, mutant-specific primers FAD2GM (CGCACCGTGATGi-AACGGTTT) (SEQ. ID. NO, 5) and FAD3cGM (ATAAATAATGTTGATCTACTTAT) (SEQ. ID. NO. 6) were designed for purposes of 5 detecting the mutant HOLL alleles of fad2 and fad32 using PCR amplification. Using the molecular markers of the present invention, these two mutations may serve to allow marker assisted introgression into Brassica lines using the HOLL alleges of DMS100, its progeny or derivatives, or transgenic versions of its mutated fad2 and fWd3 genes (SEQ. ID. NO. 7 (FIG. 1) and SEQ. ID, NO. 12 (FIG. 3), respectively) for purposes of developing HOLL canola. [0052] Qa1eep'ent 4f -hwt tneopeefjc. SNP .- arkers for ftd2 Ad fo1$ genes 22 n a presently preferred embodiment, the single nucleotide mutations present in the fadi2 arid fad3 genes are used as SNP markers to tag the fad2 ard fad3 genes for selection of high C18:1 aid low C18:3 In canola breeding. Mutant-specific primers (FAD2GM: CGOACCGTGATGTAACGGTTT (SEQ. ID, NO. 5); and FAD3cGM: 5 ATAAATAATGTTGATCTACTTAT (SEQ. I0. NO. 6)) were designed in order to detect mutant alleiss of fad2 and fad32 using PCR amplification. The primers were designed such that the mutated base (SNP) was at the 3' end of one of the primers for allele specific PCR amplification (FIGs. 1 and 3). A list of thesvarious PCR markers developedd and tested for HO/LL trait identification are provided in FIGs. 7 and 8. The primers D specific to fed2 amplified a polymorphic band that was present in DMS1OO and DNA bulks for high oleic acid (Cl8:1), but were absent in Quantum and the DNA bulks for low oleic acid (FIG. 4). [0053] This gene-specific marker wa- tested on a doubled haploid (DH) population derived from the cross of Quantum and DMS100, where it was found that the .allele distribution was highly correlated to high C18:1 (see FIG. 4, Table 1). The fad3 allele-specific primers also amplified a polymorphic fragment that was present in DMS100 but absent in Quaritum. The analysis with the DH population indicated that this allele-specific marker was statistically associated with low C18i3 (see FIG. 4, Table 1). Thus, two gene-specific PCR-based rriarkers that directly tag fad2 and fad3 gene mutations were sucoessfully developed. Given the disclosure, variations or derivatives of the markers disclosed herein (including markers of various 2~3 WO 2004/072259 PCT/US2004/003852 types) based for example, on substantial homology over a sufficient number of base pairs, will be readily apparent to one of skill in the art. [00541 Through genetic and QTL mapping using the DH population derived from the cross of Quantum x DMS1 00, one major (N5) and one minor (N1) QTL region for high 018:1, and three QTL regions (N4 and N14) for low C18:3 have been found (FIG. 5). This QTL mapping result is consistent with the genetic analysis that high C18:1 Is controlled by one major gene and low C18:3 is controlled by multiple genes. The ad2 gene-based marker was located exactly at the mapped location of the major QTL locus for C18:1, supporting the fact that this QTL corresponds to the functional fad2 gene that is affected by the mutation in DMS100. This is also consistent with the previous studies that fad2 gene is located on the linkage group N5 (Schlerholt, 2000). The location of the fad3 gene-based marker matches exactly with the mapped location of one of the major QTL loci for C18:3 on the linkage group 14 (C genome), supporting the conclusion that this QTL is the fad3c; (fad3 in the C genome, previously called fad32) gene and it is also affected by the second mutation In DMS1 00. [00551 For molecular marker methods, see generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in: Genome Mapping in Plants (ed. Andrew 'H. Paterson) by Academic Press/R. G. Landis Company, Austin, Tex., pp.. 7-21. [00561 All publications, patents, and patent applications cited herein are hereby incorporated by reference. Unless otherwise noted herein, standard 24 WO 2004/072259 PCT/US2004/003852 methods of DNA purification, restriction enzyme digestion, agarose gel analysis, DNA fragment isolation, ligation and transformation may be used for purposes of the present invention. Such methods are described In, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989), and Ausubel et al., Current Protocols In Molecular Biology (New York: John Wiley and Sons) (1987), both of which are also incorporated by reference herein. [0057] The present Invention has of necessity been discussed herein by reference to certain specific methods and materials. The enumeration of these methods and materials was merely Illustrative, and in no way constitutes any limitation on the scope of the present invention, it is to be expected! that those skilled in the art may discern and practice variations of or alternatives to the specific teachings provided herein, without departing from the scops of the present invention. 10058] EXAMPLES Example 1: Plant Material Canola varieties DMS100 (mutant type) and Quantum (wild type) were used in this study for cloning of fad2 (fatty acid desaturase-2) and fad3 (fatty acid desaturase-3) alleles. DMS100 Is a HOLL (High Qleic and Low Lnolenic) line with oleic acid content at about 77% and linolenic acid content at about 3%. It is derived from an F4 bulk of a single F3 plant selection originating from the cross of Global x AGO1 9 sister line. Quantum is a commercial variety and contains low oleic acid (-66%) and high linolenic acid (~7%) content. A double haploid (DH) population was developed by microspore culture from F1 plants of the cross between canola line Quantum and DMS1 00. The DH 25 WO 2004/072259 PCI/US2004/003852 population comprised of 604 lines. A complete fatty acid analysis of the seeds of the DH lines and their parents was implemented by using gas chromatography. Of the 604 DH lines, 183 were randomly selected for marker analysis and mapping. [00591 Example 2: Genomic DNA extraction and quantification DNA of both parental lines and 183 DH lines was extracted from the leaves of 2-week-old greenhouse grown plants using Qlagen DNeasy 96 Plant Test Kit. The details of DNA extraction procedures are described In the DNeasyTm 96 Plant Test Kit Handbook. This kit allowed DNA to be extracted in a 96-well format for a high throughput extraction. [00601 For DNA quantification, PicoGreen dye was diluted 200 fold Into IX TE buffer. In a microtiter plate, 100 pl of the diluted PicoGreen dye solution were added into each well and then 5 pl of each DNA sample or DNA standards (5 pg/ml, 10 pg/mI and 20 pg/ml) were added. The plate was then agitated on a plate shaker briefly and read using the Spectra Max GEMINIS XS microplate fluorometer from Molecular Devices. 100611 Example 3: PCR amplification PCR amplification reactions contained 20-30 ng of genomic DNA, 0.25 pM 1 0-mer primer, 2.5 mM MgCl2, 0.2mM of each dNTP, 1 X PCR buffer and 0.6 units of Tag DNA polymerase. Amplifications were performed In a GeneAmp PCR System 9700 programmed for 35 cycles of 45 sec at 94 0C, 30 sec at 55-60 "C, 1 min at 72 OC and ending with 7 min at 72 0C. 26 WO 2004/072259 PCT/US2004/003852 [00621 Example 4: Cloning of fad2 and fad3 alleles The fad2 fragments of parental lines DMS1 00 and wild-type line Quantum were amplified by using the primers homologous to Arab/dopsis or B. rapa fad2 gene sequences (Tanhuanpda et al. 1998). The ad2 fragments amplified from each of the parents by the primers FAD2-2F and FAD2-6R were cloned and sequenced. The primers FAD2-2F and FAD2-6R correspond to the primers 2 and 6 of Tanhuanpdd et al. (1998), respectively. The sequences of these two primers are: FAD2-2F: CAATCCCTCGCTCTTTCTCCTACC FAD2-6R: CCTTTOTTGTCACCTTCCCTGTCC [00631 The DNA sequences of the fad3l, and fad32 loci for C1 8:3 of B. napus were searched and retrieved from GenBank. The GenBank accession number for fad3l and fad32 are AF056569 and AF066570, respectively. Three pairs of primers for each fad3l and fad32 locus were designed from fad3l and fad32 gene sequences by using Primer Express primer designing software (PE Applied Biosystems, Foster City, California). The fac3l fragments amplified by the primers BNFD31 -CF and BNFD31--CR and the fad32 fragments amplified by the primers BNFD32-CF and BNFD32-CR from each of the parents were cloned and sequenced. 100641 The PCR amplification products of interest were resolved by agarose gel electrophoresis, and the bands of Interest were excised from the gel. The excised bands were placed in a microfuge tube containing sterilized water and heated for 5 min in boiling water, The dissolved DNA was amplified by PCR with the corresponding primer pairs. The amplified products were ligated to PCR2.1 -TOPO cloning vector using a TA-cloning kit (Invitrogen 27 WO 2004/072259 PCT/US2004/003852 Corp., San Diego, Calif) per manufacturer's instructions. The ligated products were then transformed Into competent cells and plated on LB-agar plates containing ampicillin or kanamycin, X-GAL and IPTG to enable white/blue selection. White colonies in the transformation plates were picked and identification of the cloned PCR products were verified by a digest with the restriction enzyme EcoR 1, which revealed the vector DNA fragment and the insert fragment of the expected size. The positive clones containing the insert were sequenced by Sequetech Corporation (Mountain View, Calif.). [00651 Example 5: invader assay Invader Assay kits specific to fad2 and fad3 gene mutations were developed through Third Wave Technologies (Madison, Wisconsin). The concentration of DNA samples for Invader Assay was normalized to 15 ng/pl using QiaGen Bio-Robot 3000 (Valencia, California). Invader Assay was performed in 96 well plates per manufacturer's Instruction. In brief, DNA samples were denatured at 95 "C for 10 minutes. 7 d of the denatured DNA (15 ng/l 1) and 8 si of reaction mix (3 pl1 oligo mix and 5 p of 24 mM MgCl2) were added into each well of 96-well Invader Assay plates. Then, each reaction was overlaid with 15 sl of mineral oil and the plates were Incubated in the BloOven Il from St. John Associates, Inc. (Beltsville, Maryland) at 63 0C for 4 hours. The reaction plates were read using the Spectra Max GEMINIS XS micrcplate fluororeter from Molecular Devices for fluorescent signals. Percent signal over background for the mutant allele was divided by the percent signal for wild-type allele for each sample to calculate the ratio. The genotypes of the samples were determined based on the calculated ratio. Results are provided in FIG. 6. 28 WO 2004/072259 PCT/US2004/(103852 [0066] Example 6: Sequence and data analyses The sequences were analyzed and aligned by using SeqWeb (version 2) web-based sequence analysis software in GCG software package (Wiscondin University). Linkage association between the markers and high oleic or low linolenic (HO/LL) traits were determined by f-test analysis. The genetic linkage map was generated with JolnMap V2.0 computer software using a minimum LOD of 3.0. Map distance was converted to centiMorgans using the Kosambi function. Putative QTL regions associated with the C18:1 and C18:3 were located by interval mapping using the MapQTL V 3.0 software. A LOD score of 3.0 was used to Identify regions potentially affecting the two fatty acid traits. References 100671 Arondel, V., B. Lemieux, I. Hwang, S. Gibson, H.M. Goodman and C.R Somervilla. 1992. Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arab/dops/s. Science 258:1353-1355. [00681 Auld, D. L., M. K. Heikkinen, D. A. Erickson, J. L. Sernyk, J. E. Romero. 1992. Rapeseed mutants with reduced levels of polyunsaturated fatty acids and increased levels of oleic acid. Crop Sci. 32, 657-662. [0069] Barret, P., R. Delourme, D. Brunet, C. Jourdren, R. Horvals and M. Renard. 1999. Mutations in Li and L2 genes of Brassica napus L. Induce low linolenic acid content In the seeds, GCIRC 1999 Canberra, Australia. 100701 Brown, J.W.S. 1996. Arabidopsis Intron mutations and pre-mRNA splicing. Plant J. 10: 771-780. 100711, Brunel, D., N. Roger and G. Pelletier. 1999. Development of amplified consensus genetic markers (ACGM) In Brassica napus from Arabldopsis thallana sequences of known biological function. Genome 42: 387-402. 100721 Canvin, D.T. (1965). The effect of temperature on the oil content and fatty acid composition of the oils from several oil seed crops. Canadian Journal of Botany, 43,63-69. 29 WO 2004/0722:59 PCT/US2004/I03852 [00731 Dobonte, L.R. and W.D. Hitz. Canola oil having increased oleic acid and decreased linolenic acid content and its manufacture using transgenic plants. CODEN: JSXXAM. US 5850026 A 981215. Application: US 96-675650 960703. CAN 130:65607. 100741 Chen, J. L. and W. D, Beversdorf, 1990. A comparison of traditional and haploid-derived populations of oilseed rape (Brassica napus L.) for fatty acid composition of the seed oil. Euphytica 51: 59-65. [0075] Deng, X. and R. Scarth. 1998. Temperature effects on fatty acid composition during development of low linolenic oilseed rape (Brassica napus L.) Journal of the Americar: Oil Chemists' Society 75:759-766. [00761 J.L. Harwood, Lipid Synthesis and Manufacture (ed. FD. Gunstone) Sheffield Academic Press, Sheffield (1999). 100771 Hu J., Quiros C., Arus P., Struss D. and RObbelen G. 1995. Mapping of a gene determining linolenic acid concentration in rapeseed with DNA-based markers. Theor. Apple. Genet. 90: 258-262. [00781 Hu J., Ll G., Struss D. and Quiros C.F. 1999. SCAR and RAPD markers assoclatod with 18-carbon fatty acids in rapeseed, Brassica napus. Plant Breeding 118: 145-150. [00791 Jourdren C., P. Barret, D. Brunel, R. Delourme and M. Renard. 1996. Specific molecular marker of the genes controlling linolenic acid content in rapeseed. TAG 93:512-518. 100801 Kondra, Z.P., and P.M. Thomas. 1975. Inheritance of oleic, linoleic and linolenlc acids In seed oil of rapeseed (Brassica napus). Can. J. Plant Sol. 55:205 210. 10081] Lorkovic, Z.J., D.A.W. Kirk, M.H.L. Lambermon and W. Filipowicz. 2000. Pre mRNA splicing In higher plants. Trends in Plant Science. 5:160-167, [0082] McCullough, A.J., H. Lou and M.A. Schuler. 1993. Factors affecting authentic 5' splice site selection in plant nuclei. Mol. Cell. Biol. 13: 1323-1331. [00831 Nishiuchl, T., M. Nishimura, V. Arondel and K. Iba. 1994. Genomic nucleotide sequence of a gene encoding a microsomal W-3 fatty acid desaturase from Arabidopsis thallana. Plant Physiol. 105: 767-768. 30 WO 2004/)72239 PCT/U S20041/003852 100841 Prakash, S. and K. Hinata, 1980 Taxonomy, cytogenetics, and origin of crop Brassica, a review. Opera. Bot. 55:1-59. [00851 Rajcan I., Kasha K.J., Kott L.S. and Beversdorf W.D. 1999. Detection of molecular markers associated with linolenic and erucic acid levels in spring rapeseed (Brasslca napus). Euphytica 105: 173-181. [0086] Rakow, G. 1973. Selektion auf linol- und linolen-saureghalt in rapssamen nach mutegener behand-lung. Z. Planzen.69:205-20 9 . [0087] ROcker, B.; Rbbbelen, G. (1996): Impact of low linolenic acid content on seed yield of winter oilseed rape (Brassica napus L.). Plant Breeding 115 p. 226-230. [00881 Scheffler, J.A., A.G. Sharpe, H. Schmidt, P. Sperling, I.A.P. Parkin, W. Lohs, D.J. Lydlate and E. Heinz. 1997. Dasaturase multigene families of Brassica napus arose through genome duplication. Theor Appi Genet 94:583-591. [00891 Schlerholt A., B. ROcker and H.C. Becker Ecke. 2001. Inheritance of high oleic acid mutations in winter oilseed rape (Brassica napus L.). Crop Sci. 41: 1444 1449. [00901 Schierholt A., H.C. Becker and W. Ecke. 2000, Mapping a high oleic acid mutation In winter oilseed rape (Brassica napus L.). Theor Appi Genet 101: 897-901. 10091] Schlerholt, A. and H.C.Becker. 1999. Genetic and environmental variability of high oleic acid content in winter oilseed rape. GCiRC 1999. Canberra, Australia. [00921 Simpson CG, McQuade C, Lyon J, Brown JWS (1998). Characterization of exon skipping mutants of the COP1 gene from Arabidopsis. Plant J 17: 125-131. [0093] Somers D.J., Friesen K.R.D. and Rakow G. 1998. Identification of molecular markers associated with linoleic acid desaturation In Brassica napus. Theor. Apple. Genet. 913: 897-903. 100941 Song, KM, JY Suzuki, MK Slocum, PH Williams, TC Osborn (1991) A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci, Theor Appi Genet 82:296-304. [0095] TanhuanpidP. 2000. Mapping and cloning of Fad3 gene in Brassica rapa subsp. Olelfera. GenBank direct submission. GenBank Accession AF308975, AF308976, AF308977 and AF308978. 31 WO 2004/072259 PCT/US2004/003852 [0096] Tanhuanpaa P., J. Vilkki and M. Vihinen. 1998. Mapping and cloning of FAD2 gene to develop allele-specific PCR for oleic acid in spring turnip rape (Brasslca rapa ssp. oleifera). Molecular Breeding 4: 543-550. [00971 Tanhuanp&& P.K., Vilkki J.P. and Vilkki H.J. 1995. Association of a RAPD marker with linolenic acid concentration in the seed oil of rapeseed (Brasslca napus L.). Genome 38: 414-416. 100981 Thompson K.F. 1983. Breeding winter oilseed rape, Brassica napus. Adv. Apple. Biol 7:1-104. [00991 Thormann C.E., Romero J., Mantet J. and Osborn T.C. 1996. Mapping loci controlling the concentrations of erucic and linolenic acids in seed oil of Brassica napus L. Theor. Appi. Genet 93: [001001 282-286. [00101]Wong, R., J.D. Patel, 1, Grant, J. Parker, D. Charne, M. Elhalwagy and E. Sys. 1991. The development of high oleic canola. GCIRC 1991 Congress, Saskatoon, Canada. A16:53. [00102]Yadav, N.S., A. Wierzbicki, M. Aegerter, C.S, Caster, L. Perez-Grau, A.J. Kinney, N.D. Hitz, J. R. Booth Jr., B.Schweiger, K.L. Stecca, S.M. Allen, M. Blackwell, R.S. Reiter, T.J. Carlson, S.H. Russell, K.A. Feldmann, J. Pierce and J. Browse. 1993. Cloning of higher plant w-3 fatty acid desaturases. Plant Physiol. 103: 467-476 [00103jYermanos, D.M and J. R. Goodin. 1965. Effects of temperatures during plant development on fatty acid composition of linseed oil. Agronomy. J. 57:453-454. 32

Claims (11)

1. A method for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm, comprising: 5 (a) identifying two or more nucleic acid markers for marker assisted selection among Brassica lines to be used in a Brassica breeding program, wherein the nucleic acid markers map to at least one of linkage groups N5 or N1 and at least one of linkage groups N14 or N4 and wherein the nucleic acid markers are selected from among any of SEQ ID NO 5 or derivatives thereof and SEQ ID NO 6 or derivatives thereof; and 10 (b) introgressing said traits for high oleic and/or low linolenic acid content into said Brassica by performing marker-assisted selection.

2. A method for identifying nucleic acid as a modulator of high oleic and/or low linolenic acid content in Brassica, the method comprising: 15 exposing Brassica nucleic acid to a molecular marker having a sequence of SEQ. ID. No. 5 and a molecular marker having a sequence of SEQ. ID. No. 6.

3. An isolated and purified genetic marker associated with high oleic oil content in Brassica, said marker mapping to a linkage group selected from the group consisting of 20 N14 and N4 in the Brassica genome, said marker having a sequence from SEQ. ID. NO. 6 or a derivative thereof, when used in the method of claim 1 or claim 2.

4. The isolated and purified genetic marker of claim 3 wherein Brassica is canola. 25

5. A Brassica plant, or parts thereof, which evidences high oleic and/or low linolenic acid content, said Bressica plant comprising a genome, homozygous with respect to genetic alleles which are native to a first parent and nonnative to a second parent of the Brassica plant, wherein said second parent evidences significantly less oleic 30 and/or higher IVnolenic acid content than said first parent and said Brassica plant comprises alleles from said first parent that evidences high oleic and/or low linolenic acid content in a hybrid or inbred combination in at least one locus selected from a locus mapping to a linkage group selected from the group consisting of N5 and N1 of a Brassica species and mapped by the marker set forth as SEQ. ID. NO. 5 and at least one 35 locus selected from a locus mapping to a linkage group selected from the group 33 consisting of N'14 and N4 of a Brassica species and mapped by the marker set forth as SEQ. ID. NO. 6.

6. The Brassica plant, or parts thereof, of claim 5 comprising the progeny of a 5 cross between first and second inbred lines, alleles conferring high oleic and/or low linolenic acid content being present in the homozygous state in the genome of one or the other or both of said first and second inbred lines such that the genome of said first and second inbreds together donate to the hybrid a complement of alleles necessary to confer the high oleic and/or low linolenic acid content. 10

7. The method of claims I or 2 wherein Brassica is canola.

8. An isolated nucleic acid from a Brassica species comprising a fad2 and a fad3 gene having a sequence mutation corresponding to position 411 of the DMS100 fad2 15 gene, and a sequence mutation corresponding to position 530 of the DMS100 fad32 gene.

9. An isolated nucleic acid of claim 8 wherein the sequence mutation of the fad3 gene is a sequence mutation of +1G to +1A at the 5' splice site corresponding to position 20 530 of the DMS100 fad32 gene.

10. A method of claim 1 or claim 2, substantially as hereinbefore described with reference to the Figures and/or Examples. 25

11. An isolated nucleic acid of claim 8 substantially as hereinbefore described with reference to the Figures and/or Examples. 34