# Patent

## Title
Brassica napus plants comprising mutated FAD2 alleles

## Abstract

The present invention relates to a mutated nucleic acid sequence of a delta-12 oleate desaturase enzyme (FAD2)—encoding nucleic acid from a Cruciferae plant, wherein said mutated nucleic acid sequence comprises the non-conservative mutation of at least one nucleotide of at least one codon representing an amino acid selected from the group consisting of: —the amino acid at position 259 of the amino acid sequence of said FAD2, —the amino acid at position 216 of the amino acid sequence of said FAD2, —the amino acid at position 90 of the amino acid sequence of said FAD2, —the amino acid at position 116 of the amino acid sequence of said FAD2, —the amino acid at position 276 of the amino acid sequence of said FAD2.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage entry of International Application Serial No. PCT/IB2007/001384, filed May. 25, 2007, which claims the benefit of European Application Ser. No. 06290855.3 filed May. 29, 2006.
The present invention relates to new genetic and protein markers useful for selecting plants having a high oleic acid content.
High oleic acid content of vegetable oils is a desirable trait both because of the health benefits and of the stability to oxidation and heat of oleic acid. In particular, it has been shown that oleic acid is effective in lowering plasma cholesterol levels (Bonanome et al., 1988; Liu et al., 2002). Furthermore, its single insaturated bond makes oleic acid (C18:1) a much less vulnerable fatty acid than its multiply insaturated counterparts. For instance, the rate of oxidation of linolenic acid (C18:3) is 100 times that of oleic acid (Debruyne, 2004).
One of the most promising paths towards such plants is to select plants essentially deprived of FAD2 activity. Indeed, FAD2 (delta12 oleate desaturase) catalyses the transformation of oleic acid (C18:1) to linoleic acid (C18:2); plants having lowered FAD2 activity thus enjoy higher oleic acid content thanks to limited catabolism of the latter.
As such, WO 2004/072259 provides a Brassica napus plant having high oleic acid content, wherein its fad2 gene presents a STOP codon instead of a Glutamine (Q) codon at position 185 of FAD2.
Furthermore, US 2005/0039233 provides a Brassica juncea plant having high oleic acid content, wherein its fad2 gene presents a STOP codon instead of a Tryptophan (W) codon at position 101 of FAD2.
Brassica napus (rapeseed) is an amphidiploid which contains the genomes of two diploid ancestors, Brassica rapa (the A genome) and Brassica oleracea (the C genome) (Pires et al., 2004). The oleic acid content is 61% for the traditional oil of rapeseed (Stan Skrypetz, 2005).
The present invention notably arises from the finding, by the Inventors, of five previously unrecognised mutations in the fad2 genes of Brassica napus plants:

    
    
        G to A substitution at position 269 of the fad2A gene (fad2 gene from B. rapa genome), corresponding to a W to STOP codon substitution at position 90 of the FAD2A protein,
        C to T substitution at position 346 of the fad2A gene (fad2 gene from B. rapa genome), corresponding to a Q to STOP codon substitution at position 116 of the FAD2A protein,
        C to T substitution at position 646 of the fad2C gene (fad2 gene from B. oleracea genome), corresponding to a P to S substitution at position 216 of the FAD2C protein,
        C to T substitution at position 775 of the fad2A gene (fad2 gene from B. rapa genome), corresponding to a P to S substitution at position 259 of the FAD2A protein,
        C to T substitution at position 827 of the fad2A gene (fad2 gene from B. rapa genome), corresponding to a T to M substitution at position 276 of the FAD2A protein,

which correlate with high oleic acid content of plants harbouring them. In particular, the oleic acid content of these mutated rapeseeds varies from 66% to 82% according to environmental culture conditions.

    
    


Thus, the present invention relates to a mutated nucleic acid sequence of a delta-12 oleate desaturase enzyme (FAD2)-encoding nucleic acid from a plant, in particular a Cruciferae plant, wherein said mutated nucleic acid sequence comprises the non-conservative mutation of at least one nucleotide of at least one codon representing an amino acid selected from the group consisting of:

    
    
        the amino acid at position 90 of the amino acid sequence of said FAD2,
        the amino acid at position 116 of the amino acid sequence of said FAD2,
        the amino acid at position 216 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 215 when said FAD2 sequence is constituted of 383 amino acids,
        the amino acid at position 259 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 258 when said FAD2 sequence is constituted of 383 amino acids,
        the amino acid at position 276 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 275 when said FAD2 sequence is constituted of 383 amino acids,
    
    
    or a homologous nucleic acid sequence derived from said mutated nucleic acid sequence by substitution, insertion, or deletion of at least one nucleotide, and presenting at least 90% homology with said mutated nucleic acid sequence, provided that said homologous nucleic acid sequence presents said mutation and does not encode a functional FAD2,
    or a hybridizing nucleic acid sequence which hybridizes under stringent conditions to said mutated nucleic acid sequence, provided that the complementary sequence of said hybridizing nucleic acid sequence presents said mutation and does not encode a functional FAD2,
    or the complementary sequences of said mutated, homologous, or hybridizing nucleic acid sequences.


The homology percentage of a given nucleic acid sequence with respect to the mutated nucleic acid sequence is calculated by counting the number of identical nucleotides between said given nucleic acid sequence and the mutated nucleic acid sequence for all positions of an optimal sequence alignment of said given nucleic acid sequence and the mutated nucleic acid sequence and by dividing this number by the total number of nucleotides of said given sequence.
The optimal sequence alignment can be produced manually, optionally by introducing gaps between nucleotides, or by using computer-based alignment tools, such as BLAST (Altschul et al. (1997) Nucleic Acids Res 25: 3389-402).
“Stringent conditions” for a given nucleic acid sequence can be easily determined by the man skilled in the art, for instance as described in Sambrook et al. (2001). Particular stringent conditions notably encompass 15 mM NaCl/1.5 mM sodium citrate (0.1×SSC) and 0.1% sodium lauryl sulphate (SDS) at 50-65° C.
As intended herein, FAD2 from Cruciferae are usually constituted of 383 or 384 amino acids. However, it is within the ordinary skills of the man skilled in the art to adapt the positions given for the above defined mutations to FAD2 having more or less than 383 or 384 amino acids.
An exemplary alignment of FAD2 sequences from various Cruciferae species is given in FIG. 14.
As intended herein, a nucleic acid sequence which “does not encode a functional FAD2” relates to a nucleic acid sequence which encodes a FAD2 having an activity lower than that of the corresponding natural FAD2 measured in the same conditions, in particular the encoded FAD2 has no activity.
Advantageously, mutant plants carrying nucleic acids with the above-defined mutations have an increased oleic acid content as compared to otherwise genetically identical plants (i.e. plants which are genetically identical except for said mutations) and to the corresponding wild-type plants. Preferably, mutant plants carrying nucleic acids with the above-defined mutations have an oleic acid content of at least 65%, more preferably of at least 70%.
In an embodiment, the invention particularly relates to a mutated nucleic acid sequence as defined above, wherein said mutated nucleic acid sequence comprises at least one mutation selected from:

    
    
        a substitution of the codon representing the amino acid at position 90 of the amino acid sequence of said FAD2 by a STOP codon,
        a substitution of the codon representing the amino acid at position 116 of the amino acid sequence of said FAD2 by a STOP codon,
        a substitution of the codon representing the amino acid at position 216 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 215 when said FAD2 sequence is constituted of 383 amino acids, by a codon representing any amino acid different from P,
        a substitution of the codon representing the amino acid at position 259 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 258 when said FAD2 sequence is constituted of 383 amino acids, by a codon representing any amino acid different from P,
        a substitution of the codon representing the amino acid at position 276 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 275 when said FAD2 sequence is constituted of 383 amino acids, by any amino acid different from T.
    
    


In another embodiment, the invention more particularly relates to a mutated nucleic acid sequence as defined above, wherein:

    
    
        the codon representing the amino acid at position 216 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 215 when said FAD2 sequence is constituted of 383 amino acids, is substituted by a codon representing S,
        the codon representing the amino acid at position 259 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 258 when said FAD2 sequence is constituted of 383 amino acids, is substituted by a codon representing S,
        the codon representing the amino acid at position 276 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 275 when said FAD2 sequence is constituted of 383 amino acids, is substituted by a codon representing M.
    
    


In a preferred embodiment of the above defined mutated nucleic acid sequence, the plant is of the Brassica genus.
In another preferred embodiment of the invention, the mutated nucleic acid sequence is represented by SEQ ID NO: 22 and comprises at least one mutation selected from the group constituted of:

    
    
        G to A substitution at position 269 of SEQ ID NO: 22,
        C to T substitution at position 346 of SEQ ID NO: 22,
        C to T substitution at position 646 of SEQ ID NO: 22,
        C to T substitution at position 775 of SEQ ID NO: 22,
        C to T substitution at position 827 of SEQ ID NO: 22,
    
    


In yet another embodiment of the invention, the mutated nucleic acid sequence is selected from the group constituted of:

    SEQ ID NO: 24;
    SEQ ID NO: 26;
    SEQ ID NO: 28;
    SEQ ID NO: 30;
    SEQ ID NO: 32.
    SEQ ID NO: 24 represents the fad2A gene of B. napus (i.e. the fad2 gene from B. rapa genome) harboring a G to A substitution at position 269, which corresponds to a W to STOP codon substitution at position 90 of the FAD2A protein. This mutation is in particular found in the oleic acid overproducing rapeseed line denoted ‘HOR4’ in the following Example. ‘HOR4’ has been deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41403.
    SEQ ID NO: 26 represents the fad2A gene of B. napus harboring a C to T substitution at position 346, which corresponds to a Q to STOP codon substitution at position 116 of the FAD2A protein. This mutation is in particular found in the oleic acid overproducing rapeseed line denoted ‘HOR3’ in the following Example. ‘HOR3’ has been deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41402.
    SEQ ID NO: 28 represents the fad2C gene of B. napus (i.e. the fad2 gene from B. oleracea genome) harboring a C to T substitution at position 646, which corresponds to a P to S substitution at position 216 of the FAD2C protein. This mutation is in particular found in the oleic acid overproducing rapeseed line denoted ‘HOR1’ in the following Example. ‘HOR1’ has been deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41400.
    SEQ ID NO: 30 represents the fad2A gene of B. napus harboring a C to T substitution at position 775, which corresponds to a P to S substitution at position 259 of the FAD2A protein. This mutation is in particular found in the oleic acid overproducing rapeseed line denoted ‘HOR1’ in the following Example.
    SEQ ID NO: 32 represents the fad2A gene of B. napus harboring a C to T substitution at position 827, which corresponds to a T to M substitution at position 276 of the FAD2A protein. This mutation is in particular found in the oleic acid overproducing rapeseed line denoted ‘HOR2’ in the following Example. ‘HOR2’ has been deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41401.


The present invention also relates to a nucleic acid fragment of a mutated nucleic acid as defined above, said fragment comprising at least 10, preferably at least 100, more preferably at least 1000 nucleotides, and comprising at least one nucleotide contiguous to at least one codon representing an amino acid selected from the group consisting of:

    
    
        the amino acid at position 90 of the amino acid sequence of said FAD2,
        the amino acid at position 116 of the amino acid sequence of said FAD2,
        the amino acid at position 216 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 215 when said FAD2 sequence is constituted of 383 amino acids,
        the amino acid at position 259 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 258 when said FAD2 sequence is constituted of 383 amino acids,
        the amino acid at position 276 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, or at position 275 when said FAD2 sequence is constituted of 383 amino acids,
    
    
    or the complementary sequence of said nucleic acid fragment.


In a preferred embodiment of the invention, the above-defined nucleic acid fragment comprises the mutated nucleotide.
In another preferred embodiment of the invention, the above-defined nucleic acid fragment is selected from the group constituted of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
The present invention also relates to a plant, preferably a Cruciferae plant, more preferably a plant of the Brassica genus, and even more preferably a plant of the Brassica napus species, having an increased oleic acid content, or a part of said plant, wherein the plant comprises at least one mutated nucleic acid sequence as defined above or a nucleic acid fragment as defined above which comprises the mutated nucleotide.
As intended herein, a part of plant notably relates to a plant cell, a seed, a meristem, a cal, an inflorescence, a bud, or a leaf.
In a particular embodiment of the above-defined plant, or part of plant, at least one copy of the fad2 gene which is comprised in the genome of said plant is represented by a mutated nucleic acid as defined above.
In another particular embodiment of the above-defined plant, or part of plant, each copy of the fad2 gene which is comprised in the genome of said plant is represented by a mutated nucleic acid as defined above.
Another embodiment of the invention, relates to the above-defined plant, or part of plant, wherein:

    
    
        one copy of the fad2A gene is represented by SEQ ID NO: 30 and one copy of the fad2C gene is represented by SEQ ID NO: 28, or
        one copy of the fad2A gene is represented by SEQ ID NO: 32, or
        one copy of the fad2A gene is represented by SEQ ID NO: 26, or
        one copy of the fad2A gene is represented by SEQ ID NO: 24.
    
    


In a further embodiment of the invention, the above-defined plant, or part of plant, is a plant of the Brassica napus species wherein both copies of the fad2A gene are independently represented by SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30 or SEQ ID NO: 32, and/or both copies of the fad2C gene are represented by SEQ ID NO: 28.
In yet another particular embodiment, the above-defined plant, or part of plant, is selected from the group constituted of:

    
    
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41403;
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41402;
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41400;
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41401.
    
    


The present invention also relates to the use of a nucleic acid as defined above for selecting plants, or part of plants, having an increased oleic acid content.
The present invention also relates to a method for selecting plants having an increased oleic acid content by mutagenesis, comprising the following steps:

    
    
        subjecting a plant or part of a plant to a mutagenic treatment,
        regenerating plants, or part of plants, from said treated plant or part of a plant,
        selecting from the regenerated plants those comprising at least one mutated nucleic acid sequence as defined above or a nucleic acid fragment as defined above which comprises the mutated nucleotide.
    
    


Mutagenesis can be carried out according to methods well known to the man skilled in the art, such as those described in McCallum et al. (2000). In particular, mutagenesis is carried out using ethyl methanesulphonate (EMS).
The regeneration of plants from plant parts, such as cells, can be carried out according to methods well known to the man skilled in the art, such as those described in Herman (2005).
The present invention also relates to a method for selecting plants having an increased oleic acid content by crossing, comprising the following steps:

    
    
        crossing a plant, or part of a plant, with a parent plant, or part of a parent plant, comprising at least one mutated nucleic acid sequence as defined above or a nucleic acid fragment as defined above which comprises the mutated nucleotide,
        selecting from the progeny plants, or, part of plants, obtained by crossing, those comprising at least one mutated nucleic acid sequence as defined above or a nucleic acid fragment as defined above which comprises the mutated nucleotide.
    
    


Crossing can be carried out according to methods well known to the man skilled in the art, such as those described in Gallais (1997).
As intended herein “progeny” in particular relates to the seedlings of the crossed plants.
In a preferred embodiment of the above-defined method for selecting plants having an increased oleic acid content by crossing, the parent plant is obtained according to the above-defined method for selecting plants having an increased oleic acid content by mutagenesis.
In a particularly preferred embodiment of the above-defined method for selecting plants having an increased oleic acid content by crossing, the parent plant, or part of the parent plant, is selected from the group constituted of:

    
    
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41403;
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41402;
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41400;
        the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41401.
    
    


In particular, the following techniques can be used in the selection step of the above-defined selection methods: PCR techniques, such as the TaqMan® assay (real-time PCR) or allele-specific PCR, Northern or Southern blotting, DNA micro or macro-array, Molecular Beacons, DASH (Dynamic Allele Specific Hybridization), primer extension, oligonucleotide ligation and endonuclease cleavage.
In an embodiment of the above defined methods for selecting plants, the selection step is carried by polymerase chain reaction (PCR), preferably with at least one of the following primer pairs:

    
    
        SEQ ID NO: 12 and SEQ ID NO: 13,
        SEQ ID NO: 14 and SEQ ID NO: 15,
        SEQ ID NO: 16 and SEQ ID NO: 17,
        SEQ ID NO: 34 and SEQ ID NO: 36,
        SEQ ID NO: 35 and SEQ ID NO: 36,
        SEQ ID NO: 37 and SEQ ID NO: 13,
        SEQ ID NO: 38 and SEQ ID NO: 14,
        SEQ ID NO: 39 and SEQ ID NO: 36.
    
    


The present invention also relates to a plant as obtainable according to the above-defined methods for selecting plants.
The present invention also relates to a kit for the selection of plants having a high oleic acid content, comprising:

    
    
        at least one plant, or part of plant, comprising at least one mutated nucleic acid sequence as defined above or a nucleic acid fragment as defined above which comprises the mutated nucleotide,
        at least one nucleic acid fragment as defined above.
    
    


In a preferred embodiment of the invention, the above-defined kit comprises:

    
    
        at least one plant, or part of plant, selected from the group constituted of
        
            the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41403;
            the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41402;
            the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41400;
            the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41401;
        
        
        at least one of the following primer pairs:
        
            SEQ ID NO: 12 and SEQ ID NO: 13,
            SEQ ID NO: 14 and SEQ ID NO: 15,
            SEQ ID NO: 16 and SEQ ID NO: 17.
            SEQ ID NO: 34 and SEQ ID NO: 36,
            SEQ ID NO: 35 and SEQ ID NO: 36,
            SEQ ID NO: 37 and SEQ ID NO: 13,
            SEQ ID NO: 38 and SEQ ID NO: 14,
            SEQ ID NO: 39 and SEQ ID NO: 36.
        
        
    
    


The present invention also relates to a mutated FAD2 from plant encoded by a mutated nucleic acid sequence as defined above.
The present invention also relate to a nucleic acid selected from the group constituted of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39.
The present invention also relates to a specific antibody to a mutated delta-12 oleate desaturase enzyme (FAD2) from plant as defined above.
As intended herein, “antibody” also encompasses antibody fragments such as Fab, F(ab)′2, or scFv fragments. Such an antibody can be readily prepared by the man skilled in the art from mutated delta-12 oleate desaturase enzymes according to the invention by following standard procedures. In a preferred embodiment of the invention the antibody is a monoclonal antibody. Antibodies according to the invention are notably useful for detecting mutated delta-12 oleate desaturase enzymes according to the invention, in particular in plant cellular extracts.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a and 1b show partial genomic nucleotide sequences of the fad2A gene (fad2 gene from B. rapa genome) cloned from ‘LLR1’ lines (both LLR1#S007’ and ‘LLR1#PR-2601’ lines) and ‘HOR1’ lines (both ‘HOR1#S005’, ‘HOR1#B005’ and ‘HOR1#NPZ-12’ lines). The top is ‘LLR1’ sequence (SEQ ID NO: 1) and bottom is ‘HOR1’ sequence (SEQ ID NO: 3). Arrowhead indicates a single nucleotide mutation of C to T, which results in an amino acid modification. The forward and reverse primers for PCR-based mutant allele-specific marker are bolded and underlined.
FIGS. 2a and 2b show partial genomic nucleotide sequences of the fad2C gene (fad2 gene from B. oleracea genome) cloned from ‘LLR1’ lines (both LLR1#S007’and ‘LLR1#PR-2601’ lines) and ‘HOR1’ lines (both ‘HOR1#S005’, ‘HOR1#B005’ and ‘HOR1#NPZ-12’ lines). The top is ‘LLR1’ sequence (SEQ ID NO: 2) and bottom is ‘HOR1’ sequence (SEQ ID NO: 4). Arrowhead indicates a single nucleotide mutation of C to T, which results in an amino acid modification. The forward and reverse primers for PCR-based mutant allele-specific marker are bolded and underlined.
FIGS. 3a and 3b show partial genomic nucleotide sequences of the fad2A gene (fad2 gene from B. rapa genome) cloned from ‘LLR1’ lines (both LLR1#S007’ and ‘LLR1#PR-2601’ lines) and ‘HOR2’ lines. The top is ‘LLR1’ sequence (SEQ ID NO: 1) and bottom is ‘HOR2’ sequence (SEQ ID NO: 5). Arrowhead indicates a single nucleotide mutation of C to T, which results in an amino acid modification. The forward and reverse primers for PCR-based mutant allele-specific marker are bolded and underlined.
FIG. 4a and 4b show partial genomic nucleotide sequences of the fad2A gene (fad2 gene from B. rapa genome) cloned from ‘LLR1’ lines (both LLR1#S007’and ‘LLR1#PR-2601’ lines) and ‘HOR3’ lines. The top is ‘LLR1’ sequence (SEQ ID NO: 1) and bottom is ‘HOR3’ sequence (SEQ ID NO: 6). Arrowhead indicates a single nucleotide mutation of C to T, which results in a stop codon (TAG) shaded in grey. The forward and reverse primers for PCR-based mutant allele-specific marker are bolded and underlined.
FIGS. 5a and 5b show partial genomic nucleotide sequences of the fad2A gene (fad2 gene from B. rapa genome) cloned from ‘LLR1’ lines (both LLR1#S007’and ‘LLR1#PR-2601’ lines) and ‘HOR4’ lines. The top is ‘LLR1’ sequence (SEQ ID NO: 1) and bottom is ‘HOR4’ sequence (SEQ ID NO: 7). Arrowhead indicates a single nucleotide mutation of G to A, which results in a stop codon (TAG) shaded in grey. The forward and reverse primers for PCR-based mutant allele-specific marker are bolded and underlined.
FIG. 6 provides amino acid sequences of the fad2A genes, degenerated from the genomic nucleotide sequence cloned from ‘LLR1’ lines, ‘HOR1’, a published B. rapa fad2 gene and a published B. napus fad2 gene. Arrowhead indicates the position of the amino acid substitution (P to S) resulting from a single nucleotide mutation (C to T) in ‘HOR1’ line.
FIG. 7 provides amino acid sequences of the fad2C genes, degenerated from the genomic nucleotide sequence cloned from ‘LLR1’ lines, ‘HOR1’ lines, a published B. oleracea fad2 gene and a published B. napus fad2 gene. Arrowhead indicates the position of the amino acid substitution (P to S) resulting from a single nucleotide mutation (C to T) in ‘HOR1’ line.
FIG. 8 provides amino acid sequences of the fad2A genes, degenerated from the genomic nucleotide sequence cloned from ‘LLR1’ lines, ‘HOR2’ lines, a published B. rapa fad2 gene and a published B. napus fad2 gene. Arrowhead indicates the position of the amino acid substitution (T to M) resulting from a single nucleotide mutation (C to T) in ‘HOR2’ line.
FIG. 9 provides amino acid sequences of the fad2A genes, degenerated from the genomic nucleotide sequence cloned from ‘LLR1’ lines, ‘HOR3’ lines, a published B. rapa fad2 gene and a published B. napus fad2 gene. Arrowhead indicates the position of the stop codon resulting from a single nucleotide mutation (C to T) in ‘HOR3’ line.
FIG. 10 provides amino acid sequences of the fad2A genes, degenerated from the genomic nucleotide sequence cloned from ‘LLR1’ lines, ‘HOR4’ lines, a published B. rapa fad2 gene and a published B. napus fad2 gene. Arrowhead indicates the position of the stop codon resulting from a single nucleotide mutation (G to A) in ‘HOR4’ line.
FIG. 11 provides electrophoresis results of PCR products amplified from the mutant allele-specific markers for the fad2A gene (194 bp) and from the fad3 gene (internal PCR control, 57 bp) of one of the 6 DH populations. M, 25 bp DNA ladder; lane 1, ‘LLR1#S007’; lane 2, ‘HOR1#S005’; lane 3-23, DH lines from the cross of ‘LLR1#S007’ and ‘HOR1#S005’; lane 24: water control. The PCR products were separated by electrophoresis in an 2.5% agarose gel and stained with ethidium bromide.
FIG. 12 provides electrophoresis results of PCR products amplified from the mutant allele-specific markers for the fad2C gene (123 bp) and from the fad3 gene (internal PCR control, 57 bp) of one of the 6 DH populations. M, 25 bp DNA ladder; Lane 1, ‘LLR1#S007’; lane 2, ‘HOR1#S005’; lane 3-23, DH lines from the cross of ‘LLR1#S007’and ‘HOR1#S005’; lane 24: water control. The PCR products were separated by electrophoresis in an 2.5% agarose gel and stained with ethidium bromide.
FIG. 13 provides histogram showing the correlation of the mutant allele-specific markers and oleic acid content in field and greenhouse trials.
FIGS. 14a and 14b provides a sequence alignment of FAD2 from different Brassica species and from Arabidopsis thaliana. Species, accession numbers and common names are listed hereafter:















Sequence




Genus and species
accession number
Common name


















1
Brassica rapa
CAG26981
Wild turnip


2
Brassica rapa
CAD30827
Wild turnip


3
Brassica napus
AAS92240
Rapeseed


4
Brassica rapa
AAC99622
Wild turnip


5
Brassica napus
AAT02411
Rapeseed


6
Brassica napus
AAF78778
Rapeseed


7
Brassica oleracea
AAF14564
Cabbage


8
Brassica carinata
AAD19742
Ethiopian mustard


9
Arabidopsis thaliana
NP_187819
Wall-cress












EXAMPLE
Material and Methods
1. Plant Material
Winter rapeseed lines ‘LLR1#S007’ (wild type), ‘LLR1#PR-2601’ (wild type), ‘HOR1#S005’ (mutant type), ‘HOR1#B005’ (mutant type), ‘HOR1#NPZ-12’ (mutant type), ‘HOR2’ (mutant type), ‘HOR3’ (mutant type) and ‘HOR4’ (mutant type) were used in this study for cloning of fad2 (fatty acid desaturase-2) alleles.
‘HOR1#S005’, ‘HOR1#B005’ and ‘HOR1#NPZ-12’ have been obtained by crossing an ethyl methanesulphonate (EMS) mutant line ‘HOR1 ’ with three classical rapeseed lines (S005, B005 and NPZ-12); these three mutant lines have an oleic acid content at about 80 to 90%.
‘LLR1#S007’ and ‘LLR1#PR-2601’ are rapeseed lines with oleic acid content at about 60%.
‘HOR2’, ‘HOR3’ and ‘HOR4’ are three more independent EMS mutant lines with oleic acid content at about 75 to 85%.
Six double haploid (DH) populations were developed by microspore culture from F1 plants of crosses between (Coventry et al. 1988):
(1) ‘LLR1#S007’ and ‘HOR1#S005’ rapeseed lines,
(2) ‘LLR1#PR-2601’ and ‘HOR1#NPZ-12’ rapeseed lines,
(3) ‘LLR1#S007’ and ‘HOR1#B005’ rapeseed lines,
(4) ‘LLR1#S007’ and ‘HOR1#NPZ-12’ rapeseed lines,
(5) ‘LLR1#PR-2601’ and ‘HOR1#B005’ rapeseed lines and
(6) ‘LLR1#PR-2601’ and ‘HOR1#S005’ rapeseed lines.
Each DH population comprised of 64, 32, 115, 72, 63 and 53 lines respectively. A complete fatty acid analysis of the seeds of the DH lines and their respective parents was implemented by using gas chromatography. All the 399 DH lines were used for marker and statistical analysis. No DH population has been produced with ‘HOR2’, ‘HOR3‘and ’ HOR4’ mutant lines.
2. Genomic DNA Extraction and Quantification
DNA of both parental lines and 399 DH lines was extracted from leaves of 2-week-old field grown plants using a CTAB-based method modified from Dellaporta et al. (1983). Absorbance at 260 nm was used for DNA quantification. In a UV-microtiter plate, 195 μl of ultrapure sterile water were added into each well and then 5 μl of each DNA sample were added. The plate was then briefly agitated and read using the Spectra Max M2 microplate spectrophotometer from Molecular Devices.
3. PCR Amplification
PCR amplification reactions used for fad2 alleles cloning contained 3-4 ng/μl of genomic DNA, 1.25 μM of each primer, 2.5 mM MgCl2, 0.3 mM of each dNTP, 1×PCR buffer and 0.12 U/μl of Taq DNA polymerase. Amplifications were performed in a PTC-225 MJResearch PCR system programmed for 30 cycles of 30 sec at 94° C., 30 sec at 60° C., 30 sec at 72° C. and ending with 5 min at 72° C.
For genotyping the 399 DH, an internal PCR marker has been developed to control the PCR efficiency. A couple of primers (SEQ ID NO: 10 and SEQ ID NO: 11) has been designed on the fad3 gene to amplify a fragment at 57 bp. PCR amplification reactions used for genotyping the 399 DH lines contained 3-4 ng/μl of genomic DNA, 0.625 μM of each primer (SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10 and SEQ ID NO: 11), 1.5 mM MgCl2, 0.3 mM of each dNTP, 1×PCR buffer and 0.06 U/μl of Taq DNA polymerase. Amplifications were performed in a PTC-225 MJResearch PCR system programmed for 30 cycles of 30 sec at 94° C., 30 sec at 60° C., 10 sec at 72° C. and ending with 5 min at 72° C.
4. Cloning of Fad2 Alleles
The fad2 fragments of parental lines ‘LLR1#S007’, ‘LLR1#PR-2601’, ‘HOR1S#005’, ‘HOR1#B005’, ‘HOR1#NPZ-12’, ‘HOR2’, ‘HOR3’ and ‘HOR4’ were amplified by using primers designed with Primer 3 software on fad2 gene sequence from GenBank Accession AY577313. The fad2 fragments amplified from each of the parents by the primers FAD2BnF1 and FAD2BnR1 were ligated to pGEM®-T Easy cloning vector using a PGEM®-T Easy Vector System kit (Promega Corp., Madison, USA) per manufacturer's instructions. The ligated products were transformed into competent cells and the cells plated on LB-agar plates containing ampicillin, 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 PCR using universal M13 Forward and Reverse primers flanking the insert fragment. PCR revealed the insert fragment of the expected size. The positive clones containing the insert were sequenced by Genome Express (Meylan, France).
5. Identification of Mutations in fad2 Genes
Referring to FIGS. 1-5, primers designed with primer 3 software on fad2 gene sequence from GenBank Accession AY577313 were used to amplify genomic DNA fragments of the fad2 gene from B. napus lines ‘LLR1#S007’, ‘LLR1#PR-2601’, ‘HOR1#S005’, ‘HOR1#B005’, ‘HOR1#NPZ-12’, ‘HOR2’, ‘HOR3’ and ‘HOR4’. The primer pair FAD2BnF1 AGTGTCTCCTCCCTCCAAAAA (SEQ ID NO: 8) and FAD2BnR1: TCTTCTCACCTTGCCTGTCC (SEQ ID NO: 9) amplified a fad2 fragment of the same length (1100 bp) from each of the six parents. The amplified fragments were then cloned and sequenced to investigate the sequence differences of fad2 gene between the five parents.
6. Sequence and Data Analyses
The sequences were analysed and aligned by using Clustal W (Kyoto University Bioinformatics Center) and Genedoc Software (Nicholas et al., 1997). Linkage association between the markers and high oleic trait was determined by t-test analysis.
Results
In this study, 7 ‘LLR1’ clones (both ‘LLR1#S007’ and ‘LLR1#PR-2601’), 12 ‘HOR1’ clones (both ‘HOR1#S005’, ‘HOR1#B005’ and ‘HOR1#NPZ-12’), 12 ‘HOR2’, 20 ‘HOR3’ and 20 ‘HOR4’ clones were sequenced. The sequence alignment of these clones with B. rapa and B. oleracea fad2 sequences found in public databases identified:

    
    
        (1) two fad2 genes originating from B. rapa (fad2A) and B. oleracea (fad2C) for ‘HOR1’ lines,
        (2) one fad2 gene originating from B. rapa (fad2A) for ‘HOR2’ line,
        (3) one fad2 gene originating from B. rapa (fad2A) for ‘HOR3’ line and
        (4) one fad2 gene originating from B. rapa (fad2A) for ‘HOR4’ line.
    
    


For ‘HOR1’ lines, the sequence analysis identified a single nucleotide mutation in each fad2 gene.
The single nucleotide mutation in fad2A gene, C to T, at position 752 of the amplified fragment (i.e. position 775 in the full length fad2A gene) occurred in the fad2A gene sequence of all the ‘HOR1’ clones (both ‘HOR1#S005’, ‘HOR1#B005’ and ‘HOR1#NPZ-12’) (SEQ ID NO: 3) but not the ‘LLR1’ clones (both ‘LLR1#S007’ and ‘LLR1#PR-2601’) (SEQ ID. NO: 1) (see FIG. 1).
The single nucleotide mutation in fad2C gene, C to T, at position 623 of the amplified fragment (i.e. position 646 in the full length fad2C gene) occurred in the fad2C gene sequence of all the ‘HOR1’ clones (both ‘HOR1#S005’, ‘HOR1#B005’ and ‘HOR1#NPZ-12’) (SEQ ID NO: 4) but not the ‘LLR1’ clones (both ‘LLR1#S007’ and ‘LLR1#PR-2601’) (SEQ ID. NO: 2) (see FIG. 2).
Further analysis indicated that these single nucleotide mutations occurred in the coding sequence of both fad2A and fad2C genes (see FIGS. 6-7). As further shown in FIGS. 1-2 and FIGS. 6-7, the mutations of C to T in fad2A and fad2C genes occurred at the first nucleotide of a codon resulting in a Proline (P) to Serine (S) substitution at amino acid 259 and 216 respectively. After protein annotation using TMHMM Server v. 2.0 (prediction of transmembrane domains, extracellular and intracellular domains), the Proline (P) to serine (S) substitution in FAD2A protein has been localized in an extracellular domain whereas the proline (P) to serine (S) substitution in FAD2C protein has been found in a transmembrane domain. Proline is a non-polar amino acid which tends to reside within the center of the protein whereas Serine is a polar and relatively hydrophobic amino acid which tends to be present on the surface of a protein. These different properties could lead to a structural modification of the FAD2A and FAD2C proteins resulting in a non active delta 12 oleate desaturase.
For ‘HOR2’ line, the sequence analysis identified a single nucleotide mutation in the fad2A gene. The single nucleotide mutation in fad2A gene, C to T, at position 804 of the amplified fragment (i.e. position 827 in the full length fad2A gene) occurred in the fad2A gene sequence of all the ‘HOR2’ clones (SEQ ID NO: 5) but not the ‘LLR1’ clones (both ‘LLR1#S007’ and ‘LLR1#PR-2601’) (SEQ ID. NO: 1) (see FIG. 3). Further analysis indicated that this single nucleotide mutation occurred in the coding sequence of fad2A gene (see FIG. 8). As further shown in FIG. 3 and FIG. 8, the mutation of C to T in fad2A gene occurred at the second nucleotide of a codon resulting in a Threonine (T) to Methionine (M) substitution at amino acid 276. After protein annotation using TMHMM Server v. 2.0 (prediction of transmembrane domains, extracellular and intracellular domains), the Threonine (T) to Methionine (M) substitution in FAD2A protein has been localized in an extracellular domain. Methionine (M) is a non-polar amino acid which tends to reside within the center of the protein whereas Threonine (T) is a polar and relatively hydrophobic amino acid which tends to be present on the surface of a protein. These different properties could lead to a structural modification of the FAD2A protein resulting in a non active delta 12 oleate desaturase.
For ‘HOR3’ line, the sequence analysis identified a single nucleotide mutation in the fad2A gene. The single nucleotide mutation in fad2A gene, C to T, at position 323 of the amplified fragment (i.e. position 346 in the full length fad2A gene) occurred in the fad2A gene sequence of all the ‘HOR3’ clones (SEQ ID NO: 6) but not the ‘LLR1’ clones (both ‘LLR1#S007’ and ‘LLR1#PR-2601’) (SEQ ID NO: 1) (see FIG. 4). Further analysis indicated that this single nucleotide mutation occurred in the coding sequence of fad2A gene (see FIG. 9). As further shown in FIG. 4 and FIG. 9, the mutation of C to T in fad2A gene occurred at the first nucleotide of a codon creating a stop codon (TAG) that causes early termination of the polypeptide chain during translation. The stop codon results in the incorporation of only 115 amino acids into the polypeptide instead of all 384 amino acids of the full-length polypeptide.
For ‘HOR4’ line, the sequence analysis identified a single nucleotide mutation in the fad2A gene. The single nucleotide mutation in fad2A gene, G to A, at position 246 of the amplified fragment (i.e. position 269 in the full length fad2A gene) occurred in the fad2A gene sequence of all the ‘HOR4’ clones (SEQ ID NO: 7) but not the ‘LLR1’ clones (both ‘LLR1S#007’ and ‘LLR1#PR-2601’) (SEQ ID. NO: 1) (see FIG. 5). Further analysis indicated that this single nucleotide mutation occurred in the coding sequence of fad2A gene (see FIG. 10). As further shown in FIG. 5 and FIG. 10, the mutation of G to A in fad2A gene occurred at the first nucleotide of a codon creating a stop codon (TAG) that causes early termination of the polypeptide chain during translation. The stop codon results in the incorporation of only 89 amino acids into the polypeptide instead of all 384 amino acids of the full-length polypeptide.
Molecular tests were developed for genotyping the 6 double haploids populations. For dominant tests, an internal PCR marker has been developed to control the PCR efficiency. A couple of primers (SEQ ID NO: 10 and SEQ ID NO: 11) has been designed on the fad3 gene to amplify a fragment at 57 bp. Forward and reverse PCR primers (SEQ ID NO: 14 and SEQ ID NO: 15 respectively) for detecting the C752T mutation have been used for genotyping DH lines from the cross of ‘LLR1#S007’ and ‘HOR1#S005’ (see FIG. 11), the cross of ‘LLR1#PR-2601’ and ‘HOR1#NPZ-12’ (data not shown), the cross of ‘LLR1#S007’ and ‘HOR1#B005’ (data not shown), the cross of ‘LLR1#S007’ and ‘HOR1#NPZ-12’ (data not shown), the cross of ‘LLR1#PR-2601’ and ‘HOR1#B005’ (data not shown) and the cross of ‘LLR1#PR-2601’ and ‘HOR1#S005’ (data not shown). Forward and reverse PCR primers (SEQ ID NO: 12 and SEQ ID NO: 13 respectively) for detecting the C623T mutation have been used for genotyping DH lines from the cross of ‘LLR1#S007’ and ‘HOR1#S005’ (see FIG. 12), the cross of ‘LLR1#PR-2601’ and ‘HOR1#NPZ-12’ (data not shown), the cross of ‘LLR1#S007’ and ‘HOR1#B005’ (data not shown), the cross of ‘LLR1#S007’ and ‘HOR1#NPZ-12’ (data not shown), the cross of ‘LLR1#PR-2601’ and ‘HOR1#B005’ (data not shown) and the cross of ‘LLR1#PR-2601’ and ‘HOR1#SO05’ (data not shown). Forward and reverse PCR primers (SEQ ID NO: 16 and SEQ ID NO: 17 respectively) for detecting the C804T mutation have been used for testing ‘HOR2’ and wild type lines (data not shown).
Furthermore, forward and reverse PCR primers have been used for testing ‘HOR3’ (SEQ ID NO: 34 and SEQ ID NO: 36), ‘HOR4’ (SEQ ID NO: 35 and SEQ ID NO: 36) and wild type lines.
After genotyping all the 399 DH lines, it was found that the alleles distribution was highly correlated to high C18:1 (see FIG. 13, Table 1).







TABLE 1







Correlation of the mutant allele-specific markers and oleic acid content


in field and greenhouse trials of different DH lines categories.










Number of line tested
Average oleic acid content (%)











Greenhouse/Field
Greenhouse
Field





Wild type
119/133
63.1 ± 5.8
62.6 ± 3.0


With marker FAD2 1C ‘HOR1’ only
 83/102
66.2 ± 5.5
66.3 ± 3.0


With marker FAD2 2A ‘HOR1’ only
66/79
71.5 ± 4.2
  71 ± 3.3


With marker FAD2 1C ‘HOR1’ + FAD2 2A ‘HOR1’
57/67
77.1 ± 3.1
77.7 ± 2.4









Besides, PCR primers have been designed in order to provide codominant markers for determining if the mutations are found on only one (heterozygote) or on the two alleles (homozygote) of a fad2 gene.
For the C646 mutation (found in ‘HOR1’) the following PCR primers are used:







(SEQ ID NO: 37)









forward primer 5′ ATT TCC ACC CCA ACC GCT 3′



 







(SEQ ID NO: 13)









reverse primer 5′ AGG CCA CTC CCT GCG 3′






Taking advantage of the introduction of a BsrBl restriction site by the forward primer in the amplified fragment corresponding to the fad2C wild-type gene, the PCR amplification product is cut by the BsrBl enzyme, which yields the following differentiated restriction profile:

    
    
        wild-type homozygote: 16 bp+108 bp
        mutated homozygote: 124 bp
        heterozygote: 16 bp+108 bp+124 bp
    
    


For the C775T mutation (found in ‘HOR1’) the following PCR primers are used:







(SEQ ID NO: 38)







forward primer 5′ TCT ACC GCT ACG CTG CTG TC 3′


 







(SEQ ID NO: 15)







reverse primer 5′ GTG CGT GTC CGT GAT ATT GT 3′






Taking advantage of the suppression of a MnlI restriction site in the mutated fad2A gene harbouring the C775T mutation, the PCR amplification product is cut by the MnlI enzyme, which yields the following differentiated restriction profile:

    
    
        wild-type homozygote: 14 bp+26 bp+41 bp+66 bp+83 bp
        mutated homozygote: 14 bp+41 bp+83 bp+92 bp
        heterozygote: 14 bp+26 bp+41 bp+66 bp+83 bp+92 bp
    
    


For the C346T and G269A mutations (respectively found in ‘HOR3’ and ‘HOR4’) the following PCR primers are used:







(SEQ ID NO: 39)







forward primer 5′ AGT GTC TCC TCC CTC CAA AAA 3′


 







(SEQ ID NO: 36)







reverse primer 5′ ATC GAG GCA ACT CCT TGG A 3′






The PCR amplification product is cut by the Bfal enzyme, which yields the following differentiated restriction profile:

    
    
        C346T
        wild-type homozygote: 732 bp
        mutated homozygote: 410 bp+322 bp
        heterozygote: 732 bp+410 bp+322 bp
        
            G269A
        
        
        wild-type homozygote: 732 bp
        mutated homozygote: 244 bp+488 bp
        heterozygote: 732 bp+244 bp+488 bp
    
    













SEQ ID NO:
Description
















1
Partial nucleotide sequence of ‘LLR1’ fad2A


2
Partial nucleotide sequence of ‘LLR1’ fad2C


3
Partial nucleotide sequence of ‘HOR1’ fad2A


4
Partial nucleotide sequence of ‘HOR1’ fad2C


5
Partial nucleotide sequence of ‘HOR2’ fad2A


6
Partial nucleotide sequence of ‘HOR3’ fad2A


7
Partial nucleotide sequence of ‘HOR4’ fad2A


8
PCR primer for amplifying a 1100 bp fad2 fragment


9
PCR primer for amplifying a 1100 bp fad2 fragment


10
PCR primer for amplifying a 57 bp fad3 fragment


11
PCR primer for amplifying a 57 bp fad3 fragment


12
Forward PCR primer for detecting the C646T



mutation (‘HOR1’)


13
Reverse PCR primer for detecting the C646T



mutation (‘HOR1)


14
Forward PCR primer for detecting the C775T



mutation (‘HOR1)


15
Reverse PCR primer for detecting the C775T



mutation (‘HOR1’)


16
Forward PCR primer for detecting the C827T



mutation (‘HOR2’)


17
Reverse PCR primer for detecting the C827T



mutation (‘HOR2’)


18
Exemplary wild type B. napus fad2A



nucleotide sequence


19
Exemplary wild type B. napus FAD2A



protein sequence


20
Exemplary wild type B. napus fad2C



nucleotide sequence


21
Exemplary wild type B. napus FAD2C



protein sequence


22
Consensus sequence for mutated fad2


23
Consensus sequence for mutated FAD2


24
Nucleotide sequence for G269A mutated fad2


25
Protein sequence for W90STOP mutated FAD2


26
Nucleotide sequence for C346T mutated fad2


27
Protein sequence for Q116STOP mutated FAD2


28
Nucleotide sequence for C646T mutated fad2


29
Protein sequence for P216S mutated FAD2


30
Nucleotide sequence for C775T mutated fad2


31
Protein sequence for P259S mutated FAD2


32
Nucleotide sequence for C827T mutated fad2


33
Protein sequence for T276M mutated FAD2


34
Forward PCR primer for detecting the C346T



mutation (‘HOR3’)


35
Forward PCR primer for detecting the G269A



mutation (‘HOR4’)


36
Reverse PCR primer for detecting the C346T or G269A



mutation (‘HOR3/4’)


37
Forward PCR primer for detecting the C646T mutation



(codominant)


38
Forward PCR primer for detecting the C775T mutation



(codominant)


39
Forward PCR primer for detecting the C346T or G269A



mutation (codominant)









References


    Bonanome A, Grundy S M. Effect of dietary stearic acid on plasma cholesterol and lipoprotein levels. N Engl J. Med. 1988 May 12; 318(19):1244-8.
    Liu, Q., Singh, S., Green, A. (2002). High-Oleic and High-Stearic Cottonseed Oils: Nutritionally Improved Cooking Oils Developed Using Gene Silencing. J Am Coll Nutr 21: 205S-211.
    Debruyne I. Soybean Oil Processing: Quality Criteria and Flavor Reversion. Oil Mill Gazetteer. Volume 110, July 2004.
    McCallum, C. M., Comai, L., Greene, E. A., and Henikoff, S. (2000). Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol. 123, 439-442.
    Herman E. B. (2005). Media and Techniques for Growth, Regeneration and Storage 2002-2005. Volume 9 of Recent Advances in Plant Tissue Culture. Agricell Report.
    Gallais A. (1997). Théorie de la sélection en amélioration des plantes. Editions Masson.
    Coventry, J; L Kott and W Beversdorf: 1988. Manual for microspore culture of Brassica napus. University of Guelph Technical Bulletin, OAC Publication 0489
    Dellaporta S L, Wood J, and Hicks J B (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19-21.
    Nicholas K. B., Nicholas Jr H. B. & Deerfield II D. W. (1997) GeneDoc:Analysis and Visualization of Genetic Variation. http://www.psc.edu/biomed/genedoc [accessed on 6 Apr. 2005].
    Sambrook J and Russell D W (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.
    J. C. Pires, J. Zhao, M. E. Schranz, E. J. Leon, P. A. Quijada, L. N. Lukens and T. C. Osborn (2004). Flowering time divergence and genomic rearrangements in resynthesized Brassica polyploids (Brassicaceae). Volume 82 Page 675 Issue 4. Biological Journal of the Linnean Society.
    Stan Skrypetz, 2005. Le Bulletin bimensuel. Agriculture et Agroalimentaire Canada. Volume 18 Numéro 17.
    Konieczny A, Ausubel F M (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based marker. Plant J 4: 403-410

## Claims

1. A nucleic acid sequence encoding a mutated delta-12 oleate desaturase enzyme (FAD2) from a plant of Brassica napus species, wherein said nucleic acid sequence comprises :
a) nucleic acid sequence SEQ ID NO:22 in which at least one nucleotide of a codon representing the proline at position 259 of the polypeptide encoded by nucleic acid sequence SEQ ID NO:22 is mutated and produces a non-conservative substitution of said proline at position 259 of the polypeptide encoded by nucleic acid sequence SEQ ID NO:22, or
b) a nucleic acid sequence having at least 90% identity with the sequence defined in a) and which comprises a mutation producing a non-conservative substitution at an amino acid position corresponding i) to proline at position 259 of the polypeptide encoded by nucleic acid sequence SEQ ID NO:22, and which does not encode a functional FAD2; or
c) the complementary sequences of the sequence defined in a) or b).

2. A mutated nucleic acid sequence according to claim 1, wherein said mutated nucleic acid sequence is SEQ ID NO: 30.

3. A nucleic acid fragment of a mutated nucleic acid according to claim 1, said fragment comprising at least 10 nucleotides and comprising at least one nucleotide contiguous to at least one codon representing the amino acid at position 259 of the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 22 or the complementary sequence of said nucleic acid fragment.

4. A nucleic acid fragment, of a mutated nucleic acid according to claim 1, said fragment comprising at least 10 nucleotides and comprising at least one nucleotide contiguous to at least one codon representing the amino acid at position 259 of the amino acid sequence encoded by nucleic acid sequence SEQ ID NO: 22, or the complementary sequence of said nucleic acid fragment;
wherein said fragment comprises the mutated nucleotide.

5. A mutated nucleic acid sequence according to claim 1, wherein the codon representing the proline at position 259 of the amino acid sequence encoded by nucleic acid SEQ ID NO: 22 is substituted by a codon representing S.

6. A plant of Brassica napus species having an increased oleic acid content, or a part of said plant, wherein the plant comprises at least one nucleic acid according to claim 1.

7. A method for selecting plants of Brassica napus species having an increased oleic acid content by mutagenesis, comprising the following steps:
subjecting a plant of Brassica napus species or part of a plant of Brassica napus species to a mutagenic treatment,
regenerating a plant, or part of plants, from said treated plant or part of a plant,
selecting from the regenerated plants those comprising at least one nucleic acid according to claim 1.

8. A method for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species comprising at least one nucleic acid according to claim 1,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 1.

9. A method for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species, comprising at least one nucleic acid according to claim 1,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 1,
wherein the parent plant is obtained according to a method for selecting plants of Brassica napus species having an increased oleic acid content by mutagenesis, comprising the following steps:
subjecting a plant of Brassica napus species or part of a plant of Brassica napus species to a mutagenic treatment,
regenerating plants from said treated plant or part of a plant,
selecting from the regenerated plants those comprising at least one nucleic acid according to claim 1.

10. A method for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species comprising at least one nucleic acid according to claim 1,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 1, wherein the parent plant is
the plant deposited at the NCIMB (Aberdeen, Scotland) on May 17, 2006 under accession number 41400.

11. A kit for the selection of plants having a high oleic acid content, comprising:
at least one plant of Brassica napus species, or part of plant of Brassica napus species, comprising a mutated nucleic acid of a delta-12 oleate desaturase enzyme (FAD2), wherein said mutated nucleic acid sequence comprises:
a) nucleic acid sequence SEQ ID NO:22 in which at least one nucleotide of a codon representing the proline at position 259 of the polypeptide encoded by nucleic acid sequence SEQ ID NO:22 is mutated and produces a non-conservative substitution of said proline at position 259 of the polypeptide encoded by nucleic acid sequence SEQ ID NO:22, or
b) a nucleic acid sequence having at least 90% identity with the sequence defined in a) or b) and which comprises a mutation producing a non-conservative substitution at an amino acid position corresponding i) to proline at position 259 of the polypeptide encoded by nucleic acid sequence SEQ ID NO:22; or
c) the complementary sequences of the sequence defined in a) or, b), and
at least one nucleic acid according to claim 3.

12. A method for selecting plants of Brassica napus species having an increased oleic acid content by mutagenesis, comprising the following steps:
subjecting a plant of Brassica napus species or part of a plant of Brassica napus species to a mutagenic treatment,
regenerating plants, from said treated plant or part of a plant,
selecting from the regenerated plants those comprising at least one nucleic acid according to claim 1;
or for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species comprising at least one nucleic acid according to claim 1,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 1;
wherein the selection step is carried by polymerase chain reaction (PCR).

13. A plant of Brassica napus species as obtainable according to a method for selecting plants of Brassica napus species having an increased oleic acid content by mutagenesis, comprising the following steps:
subjecting a plant of Brassica napus species or part of a plant of Brassica napus species to a mutagenic treatment,
regenerating plants from said treated plant or part of a plant,
selecting from the regenerated plants those comprising at least one nucleic acid according to claim 1;
or for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species comprising at least one nucleic acid according to claim 1,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 1.

14. The mutated nucleic acid sequence according to claim 1, wherein said mutated nucleic acid sequence further comprises the non-conservative mutation of at least one nucleotide of a codon representing the amino acid at position 216 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids,
or a homologous nucleic acid sequence derived from said mutated nucleic acid sequence by substitution, insertion, or deletion of at least one nucleotide, and presenting at least 90% homology with said mutated nucleic acid sequence, provided that said homologous nucleic acid sequence presents said mutation and does not encode a functional FAD2,
or a hybridizing nucleic acid sequence which hybridizes under stringent conditions to said mutated nucleic acid sequence, provided that the complementary sequence of said hybridizing nucleic acid sequence presents said mutation and does not encode a functional FAD2,
or the complementary sequences of said mutated, homologous, or hybridizing nucleic acid sequences.

15. The mutated nucleic acid sequence according to claim 14, wherein said mutated nucleic acid sequence comprises at least one substitution of the codon representing the amino acid at position 216 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, by a codon representing any amino acid different from P.

16. The mutated nucleic acid sequence according to claim 15, wherein said codon representing the amino acid at position 216 of the amino acid sequence of said FAD2 when said FAD2 sequence is constituted of 384 amino acids, is substituted by a codon representing S.

17. The mutated nucleic acid sequence according to claim 15, wherein said mutated nucleic acid sequence is represented by SEQ ID NO: 22 and comprises at least:
C to T substitution at position 646 of SEQ 10 NO: 22, and
C to T substitution at position 775 of SEQ 10 NO: 22.

18. A plant of Brassica napus species having an increased oleic acid content, or a part of said plant, wherein the plant comprises at least one nucleic acid according to claim 14.

19. A method for selecting plants of Brassica napus species having an increased oleic acid content by mutagenesis, comprising the following steps:
subjecting a plant of Brassica napus species or part of a plant of Brassica napus species to a mutagenic treatment,
regenerating plants from said treated plant or part of a plant,
selecting from the regenerated plants those comprising at least one nucleic acid according to claim 14.

20. A method for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species comprising at least one nucleic acid according to claim 14,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 14.

21. A method for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species comprising at least one nucleic acid according to claim 14,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 14,
wherein the parent plant is obtained according to a method for selecting plants of Brassica napus species having an increased oleic acid content by mutagenesis, comprising the following steps:
subjecting a plant of Brassica napus species or part of a plant of Brassica napus species to a mutagenic treatment,
regenerating plants from said treated plant or part of a plant,
selecting from the regenerated plants those comprising at least one nucleic acid according to claim 14.

22. A plant of Brassica napus species as obtainable according to a method for selecting plants of Brassica napus species having an increased oleic acid content by mutagenesis, comprising the following steps:
subjecting a plant of Brassica napus species or part of a plant of Brassica napus species to a mutagenic treatment,
regenerating plants from said treated plant or part of a plant,
selecting from the regenerated plants those comprising at least one nucleic acid according to claim 14;
or for selecting plants of Brassica napus species having an increased oleic acid content by crossing, comprising the following steps:
crossing a plant of Brassica napus species with a parent plant of Brassica napus species comprising at least one nucleic acid according to claim 14,
selecting from the progeny plants obtained by crossing, those comprising at least one nucleic acid according to claim 14.