# Patent

## Title
Altered FAD2 and FAD3 genes in Brassica and the molecular marker-assisted detection thereof

## 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, 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 one aspect of the embodiment, two single nucleotide polymorphism (SNP) markers are provided that 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 a high oleic/low linolenic phenotype.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/US2004/003852, filed Feb. 11, 2004, published in English as International Patent Publication No. WO 2004/072259 on Aug. 26, 2004, which claims priority to U.S. Patent Application No. 60/446,429 filed Feb. 11, 2003, the entire contents of which is hereby incorporated herein by this reference.


TECHNICAL FIELD
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.
BACKGROUND
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.
Canola is a polyploid species considered to have arisen from the hybridization of Brassica oleracea, 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 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 Brassica napus, while Brassica oleracea contributed nine chromosomes from its CC genome as the maternal donor (Song et al., 1992).
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 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.
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 two times that of linoleic acid, which has two double bonds. Linoleic and linolenic acids also have the most impact on flavor and odor because they readily form hydroperoxides. High oleic oil (≧70% oleic) is less susceptible to oxidation during storage, frying and refining, and can be heated to a higher temperature without smoking, making it more suitable as cooking oil.
Two strategies are generally used to increase the oxidative stability of canola oil. In one approach, partial hydrogenation is used to lower linolenic acid content. Unfortunately, partial hydrogenation leads to the formation of trans-fatty 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 oleic 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, Ind.), which varieties produce NatreonTM oil. One such line, AG019 (a NexeraTM variety) contains 71% to 78% oleic (C18:1) and <3% linolenic (C18:3) acid. AG019 was originally created by ethyl methanesulphonate (EMS) mutagenesis and is described in U.S. Pat. No. 6,169,190 B1 to Sernyk, assigned to the assignee of the present invention.
Current methods for producing F1 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 C18:3 and increased C18:1. Similar results were reported in other studies (Yermanos and Goodin, 1965; Canvin, 1965).
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% to 10%)) indicated that the low C18:3 trait was controlled by two major loci with additive effects designated L1 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 genome (originating from Brassica oleracea) (Jourdren et al., 1996; Barret et al., 1999).
Traits that 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 linolenic content, while Somers et al. (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). Rücker and Röbbelen (1996) indicated that several minor genes are most likely involved in the desaturation step.
Heritability for C18:3 content was estimated to be 26% to 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 C18:2 or the elongation of C16:3 (Thompson, 1983).
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 linoleic acid (C18:2) (Tanhuanpaa et al., 1998; Schierholt 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 al., 1997; Schierholt et al., 2000). Chen and Beversdorf (1990) reported that the accumulation of oleic acid was controlled by at least two segregation genetic systems, one acting on chain elongation and the other involving desaturation. Heritability for C18:1 content was estimated to be 53% to 78% (Kondra and Thomas 1975) and 94% (Schierholt and Becker, 1999), respectively. Due to the higher heritability, the expression of C18:1 content is environmentally less affected and relatively stable (Schierholt and Becker, 1999).
In NexeraTM canola germplasm, one to two genes are found to control C18:1 content and at least three 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 C18:3.
Molecular maker selection is based on genotypes and is, therefore, independent from environmental effects. Molecular markers would alleviate 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.
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.
Different types of molecular markers such as RAPD (random-amplified polymorphic DNA) markers (Tanhuanpaa et al., 1995; Hu et al., 1995; Rajcan et al., 1999; Jourdren et al., 1996), RFLP (restriction fragment length polymorphism) markers (Thormann et al., 1996) and SCAR (sequence-characterized 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 identified for high C18:1 content. A RAPD marker was identified to be linked to the QTL affecting oleic acid concentration in spring turnip rape (B. rapa ssp. oleifera) and was later converted into a SCAR marker (Tanhuanpaa et al., 1996). Schierholt et al. (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.
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.
SUMMARY OF THE INVENTION
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.
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 N1 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 Brassica is also provided. The marker maps to a linkage group selected from the group consisting of N14 and N4 in the Brassica genome, and has the sequence of SEQ ID NO. 6 or a derivative thereof.
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 genes of the present invention.
In an additional embodiment, molecular markers corresponding to the 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 one aspect of the embodiment, two single nucleotide polymorphism (SNP) markers are provided that 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. In a related embodiment, methods of marker-assisted selection for high oleic and/or low linolenic oil seed plants are disclosed. In one aspect of the embodiment, methods for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm are provided. The methods include: (a) identifying one 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 N14, N4, N5 or NI and wherein the nucleic acid markers are selected from among any of SEQ. ID. NOs. 5 and 6, and (b) introgressing the traits for high oleic and/or low linolenic acid content into 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 Brassica, and comprises exposing Brassica nucleic acid to a molecular marker selected from the group consisting of SEQ. ID. Nos. 5 and/or 6.



BRIEF DESCRIPTION OF THE FIGURES
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.
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 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 DMS100.
FIG. 3 shows genomic nucleotide sequences of the fad3c gene cloned from DMS100 and Quantum. The top is the DMS100 sequence (SEQ. ID. NO. 12) and the bottom is the Quantum sequence (SEQ. ID. NO. 13). Exons 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 Arabidopsis. The arrowhead indicates a single nucleotide mutation of G to A. The forward and reverse primers for PCR-based mutant allele-specific markers are bolded and underlined.
FIG. 4 provides a table that 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.
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.
FIG. 6 provides Invader Assay results for mutant and wild-type alleles of fad2 and fad3 genes.
FIGS. 7 and 8 provide a list of the various PCR markers developed and tested for high oleic and/or low linolenic trait identification.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates generally to methods and materials for use in plant breeding. In a preferred embodiment, the present invention relates to methods and compositions of matter for marker-assisted identification of genes encoding high oleic, low linolenic traits in canola.
By “genetic locus” is meant a location on a chromosome.
By “genomic locus” is meant a location within the entire set of chromosomes of an organism.
As used herein, “linkage disequilibrium” refers to a statistical association between two loci or between a trait and a marker.
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.
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 linolenic 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 nucleic acid sequences having substantial sequence homology with the disclosed marker sequences herein such that they are able to have the disclosed functionalities for use in marker-assisted breeding.
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 not 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 that 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.
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 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, A. M. Lesk, ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, D. W. Smith, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, A. M. Griffin and H. G. Griffin, eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, G. von Heinje, Academic Press (1987); and Sequence Analysis Primer, M. Gribskov and J. Devereux, eds., Stockton Press, New York (1991)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to those disclosed in H. Carillo and D. Lipman, 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 (J. Devereux et al., Nucleic Acids Research 12 (1):387 (1984)), BLASTP, BLASTN, FASTA and TFASTA (S. F. Atschul et al., J. Mol. Biol. 215:403 (1990)).
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.
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 predetermined 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, G. W. Snedecor and W. G. Cochran, 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.
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 nucleic 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% to 100% identity, preferably about 70% to 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% to 70% identity). Thus, conditions of “weak” or “low” 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.
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.
A “substitution,” as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
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 DMS100 was derived from an F4 bulk of a single F3 plant selection originating from the cross of Global×AG019 sister line. DMS100 is a HOLL (High Oleic and Low Linolenic) line with oleic acid content at about 77% and linolenic acid content at about 3%. Quantum is a 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 (Tanhuanpää 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.
C18:1 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, nine DMS100 clones and ten 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 DMS100 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 linoleic acid, thus leading to the accumulation of 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%).
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 DMS100 clones and six Quantum clones of fad31 and six DMS100 clones and six Quantum clones of fad32 were sequenced. Sequence analysis and alignment revealed no sequence difference between DMS100 and Quantum clones for fad31 (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 (Tanhuanpää, 2000) and Arabidopsis (Nishiuchi et al., 1994). The fad3 gene of B. rapa and Arabidopsis contains eight exons and seven introns, while the sequence examined covers exons 4 (partial), 5, 6 and 7 (partial) and introns 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. 
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 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 +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 site 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 three possible reading frames and hence, exons 7 and 8 will 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.
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 DMS100. 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 DMS100, its progeny or derivatives, or transgenic versions of its mutated fad2 and fad3 genes (SEQ. ID. NO. 7 (see FIG. 1) and SEQ. ID. NO. 12 (see FIG. 3), respectively), for purposes of developing HOLL canola.
Identification of Mutations in fad2 and fad3 Genes
Referring to FIG. 1, primers homologous to the Arabidopsis fad2 gene sequence were used to amplify genomic DNA fragments of the fad2 gene from B. napus lines DMS100 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.
Genomic DNA fragments corresponding to the fad31 and fad32 genes were amplified from DMS100 and Quantum lines using PCR. The primers for amplification were designed from the published B. napus fad31 and fad32 gene sequences (Brunel et al., 1999, GenBank Accession AF056569 and AF056570, respectively). The fad31 fragments amplified by the primer pairs BNFD31-CF (GAGGCTTGGACGACCACTTG) (SEQ. ID. NO. 3) and 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 DMS100 clones and six Quantum clones of fad31 and six DMS100 clones and six Quantum clones of fad32 were sequenced. Sequence analysis and alignment revealed no sequence difference between DMS100 and Quantum for fad31 (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 (Tanhuanpää, 2000) and Arabidopsis (Nishiuchi et al., 1994). The fad3 gene of B. rapa and Arabidopsis contains eight exons and seven introns, while the sequence examined covers exons 4 (partial), 5, 6 and 7 (partial) and introns 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. 
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 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 +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 exon 6 of the 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 intron (McCullough et al., 1993). Such cryptic splicing could lead to 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 three possible reading frames and hence, exons 7 and 8 will 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.
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 DMS100. As shown in FIGS. 1 and 3, respectively, mutant-specific primers FAD2GM (CGCACCGTGATGGTTAACGGTTT) (SEQ. ID. NO. 5) and FAD3cGM (ATAAATAATGTTGATCTACTTAT) (SEQ. ID. NO. 6) were designed for purposes of 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 alleles of DMS100, its progeny or derivatives, or transgenic versions of its mutated fad2 and fad3 genes (SEQ. ID. NO. 7 (FIG. 1) and SEQ. ID. NO. 8 (FIG. 3), respectively) for purposes of developing HOLL canola.
Development of Mutant Allele-Specific SNP Markers for fad2 and fad3 Genes
In a presently preferred embodiment, the single nucleotide mutations present in the fad2 and fad3 genes are used as SNP markers to tag the fad2 and fad3 genes for selection of high C18:1 and low C18:3 in canola breeding. Mutant-specific primers (FAD2GM: CGCACCGTGATGGTTAACGGTTT (SEQ. ID. NO. 5); and FAD3cGM: ATAAATAATGTTGATCTACTTAT (SEQ. ID. NO. 6)) were designed in order to detect mutant alleles 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). Lists of the various PCR markers developed and tested for HO/LL trait identification are provided in FIGS. 7 and 8. The primers specific to fad2 amplified a polymorphic band that was present in DMS100 and DNA bulks for high oleic acid (C18:1), but were absent in Quantum and the DNA bulks for low oleic acid (FIG. 4).
This gene-specific marker was 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 Quantum. The analysis with the DH population indicated that this allele-specific marker was statistically associated with low C18:3 (see FIG. 4, Table 1). Thus, two gene-specific PCR-based markers that directly tag fad2 and fad3 gene mutations were successfully developed. Given the disclosure, variations or derivatives of the markers disclosed herein (including markers of various 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.
Through genetic and QTL mapping using the DH population derived from the cross of Quantum×DMS100, one major (N5) and one minor (N1) QTL region for high C18:1, 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 fad2 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 the fad2 gene is located on the linkage group N5 (Schierholt, 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 DMS100.
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.
All publications, patents, and patent applications cited herein are hereby incorporated by reference. Unless otherwise noted herein, standard 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.
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 scope of the present invention.
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 Oleic and Low Linolenic) 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×AG019 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 DMS100. The DH 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.
Example 2
Genomic DNA Extraction and Quantification
DNA of both parental lines and 183 DH lines was extracted from the leaves of two-week-old greenhouse-grown plants using Qiagen 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.
For DNA quantification, PicoGreen dye was diluted 200-fold into 1×TE buffer. In a microtiter plate, 100 μl of the diluted PicoGreen dye solution were added into each well and then 5 μl of each DNA sample or DNA standards (5 μg/ml, 10 μg/ml and 20 μg/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.
Example 3
PCR Amplification
PCR amplification reactions contained 20 to 30 ng of genomic DNA, 0.25 μM 10-mer primer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 1×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 seconds at 94° C., 30 seconds at 55° C. to 60° C., 1 minute at 72° C. and ending with 7 minutes at 72° C.
Example 4
Cloning of fad2 and fad3 Alleles
The fad2 fragments of parental lines DMS100 and wild-type line Quantum were amplified by using the primers homologous to Arabidopsis or B. rapa fad2 gene sequences (Tanhuanpää et al., 1998). The fad2 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 Tanhuanpää et al. (1998), respectively. The sequences of these two primers are:











FAD2-2F:
CAATCCCTCGCTCTTTCTCCTACC




 



FAD2-6R:
CCTTTCTTGTCACCTTCCCTGTCC






The DNA sequences of the fad31 and fad32 loci for C18:3 of B. napus were searched and retrieved from GenBank. The GenBank accession number for fad31 and fad32 are AF056569 and AF066570, respectively. Three pairs of primers for each fad31 and fad32 locus were designed from fad31 and fad32 gene sequences by using Primer Express primer designing software (PE Applied Biosystems, Foster City, Calif.). The fad31 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.
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 five minutes 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 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 I, 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.).
Example 5
Invader Assay
Invader Assay kits specific to fad2 and fad3 gene mutations were developed through Third Wave Technologies (Madison, Wis.). The concentration of DNA samples for Invader Assay was normalized to 15 ng/μl using QiaGen Bio-Robot 3000 (Valencia, Calif.). Invader Assay was performed in 96-well plates per manufacturer's instruction. In brief, DNA samples were denatured at 95° C. for ten minutes. Seven μl of the denatured DNA (15 ng/μl) and 8 μl of reaction mix (3 μl oligo mix and 5 μl of 24 mM MgCl2) were added into each well of 96-well Invader Assay plates. Then, each reaction was overlaid with 15 μl of mineral oil and the plates were incubated in the BioOven III from St. John Associates, Inc. (Beltsville, Md.) at 63° C. for four hours. The reaction plates were read using the Spectra Max GEMINIS XS microplate fluorometer 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.
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 (Wisconsin University). Linkage association between the markers and high oleic or low linolenic (HO/LL) traits were determined by t-test analysis. The genetic linkage map was generated with JoinMap 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.
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## Claims

1. A method for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm, said method comprising:
identifying one or more nucleic acid markers that map to at least one of linkage groups N14, N4, N5 or N1, wherein the nucleic acid markers comprise a nucleotide sequence within a Fad2 gene that is capable of hybridizing to the nucleic acid molecule of SEQ ID NO:8 under high stringency conditions, but is not capable of hybridizing to the nucleic acid molecule of SEQ ID NO:7 under high stringency conditions;
crossing a Brassica plant comprising the identified one or more nucleic acid markers with another Brassica plant that does not comprise the identified one or more markers; and
selecting progeny from the cross that comprise the identified one or more nucleic acid markers.

2. The method of claim 1, wherein Brassica is canola.

3. A method for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm, said method comprising:
identifying one or more nucleic acid markers that map to at least one of linkage groups N14, N4, N5 or N1, wherein the nucleic acid markers comprise a nucleotide sequence within a Fad3 gene that is capable of hybridizing to the nucleic acid molecule of SEQ ID NO:12 under high stringency conditions, but is not capable of hybridizing to the nucleic acid molecule of SEQ ID NO:13 under high stringency conditions;
crossing a first Brassica plant comprising the identified one or more nucleic acid markers with a second Brassica plant that does not comprise the identified one or more markers; and
selecting progeny from the cross that comprise the identified one or more nucleic acid markers.

4. The method of claim 3, wherein Brassica is canola.

5. A method for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm, said method comprising:
identifying nucleic acid markers that map to at least one of linkage groups N14, N4, N5 or N1, wherein the nucleic acid markers comprise
a nucleic acid marker comprising a nucleotide sequence within a Fad2 gene that is capable of hybridizing to the nucleic acid molecule of SEQ ID NO:8 under high stringency conditions, but is not capable of hybridizing to the nucleic acid molecule of SEQ ID NO:7 under high stringency conditions, and
a nucleic acid marker comprising a nucleotide sequence within a Fad3 gene that is capable of hybridizing to the nucleic acid molecule of SEQ ID NO:12 under high stringency conditions, but is not capable of hybridizing to the nucleic acid molecule of SEQ ID NO:13 under high stringency conditions;

crossing a Brassica plant comprising the identified markers with another Brassica plant that does not comprise the identified nucleic acid markers; and
selecting progeny from the cross that comprise the identified nucleic acid markers.

6. The method of claim 5, wherein Brassica is canola.

7. A method for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm, said method comprising:
identifying one or more nucleic acid markers that map to at least one of linkage groups N14, N4, N5 or N1, wherein the nucleic acid markers comprise SEQ ID NO:5;
crossing a first Brassica plant comprising the identified one or more nucleic acid markers with a second Brassica plant; and
selecting progeny from the cross that comprise the identified one or more nucleic acid markers.

8. The method according to claim 7, wherein Brassica is canola.

9. The method according to claim 7, wherein the one or more nucleic acid markers consist of a nucleic acid marker consisting of the polynucleotide of SEQ ID NO:5, wherein the presence of the nucleic acid marker consisting of the polynucleotide of SEQ ID NO:5 is determined in the first Brassica plant and the selected progeny by polymerase chain reaction, and wherein the Brassica plants are canola plants.

10. A method for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm, said method comprising:
identifying one or more nucleic acid markers that map to at least one of linkage groups N14, N4, N5 or N1, wherein the nucleic acid markers comprise SEQ ID NO:6;
crossing a first Brassica plant comprising the identified one or more nucleic acid markers with a second Brassica plant, wherein the first Brassica plant comprises a G to A mutation at the first base of the 5′ splice site of the third intron in the Fad3 gene; and
selecting progeny from the cross that comprise the identified one or more nucleic acid markers.

11. The method according to claim 10, wherein Brassica is canola.

12. The method according to claim 10, wherein the one or more nucleic acid markers consist of a nucleic acid marker consisting of the polynucleotide of SEQ ID NO:6, wherein the presence of the nucleic acid marker consisting of the polynucleotide of SEQ ID NO:6 is determined in the first Brassica plant and the selected progeny by polymerase chain reaction, and wherein the Brassica plants are canola plants.

13. A method for reliably and predictably introgressing traits for high oleic and/or low linolenic acid content into Brassica germplasm, said method comprising:
identifying one or more nucleic acid markers that map to at least one of linkage groups N14, N4, N5 or N1, wherein the nucleic acid markers comprise
a first nucleic acid marker comprising SEQ ID NO:5 and
a second nucleic acid marker comprising SEQ ID NO:6;

crossing a first Brassica plant comprising the identified one or more nucleic acid markers with a second Brassica plant; and
selecting progeny from the cross that comprise the identified one or more nucleic acid markers.

14. The method according to claim 13, wherein Brassica is canola.

15. The method according to claim 13, wherein the one or more nucleic acid markers consist of two nucleic acid markers,
the first nucleic acid marker consisting of the polynucleotide of SEQ ID NO:5, and
the second nucleic acid marker consisting of the polynucleotide of SEQ ID NO:6,

wherein the presence of the two nucleic acid markers is determined in the first Brassica plant and the selected progeny by polymerase chain reaction, and wherein the Brassica plants are canola plants.