The quality of canola oil is determined by its constituent fatty acids such as oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Most canola cultivars normally produce oil with about 55-65% oleic acid and 8-12% linolenic acid. High concentrations of linolenic acid lead to oil instability and off-type flavor, while high levels of oleic acid increase oxidative stability and nutritional value of oil. Therefore, development of canola cultivars with increased oleic acid and reduced linolenic acid is highly desirable for canola oil quality. In this study, we have mapped one locus that has a major effect and one locus that has a minor effect for high oleic acid and two loci that have major effects for low linolenic acid in a doubled haploid population. The major locus for high C18:1 was proven to be the fatty acid desaturase-2 (fad2) gene and it is located on the linkage group N5; the minor locus is located on N1. One major QTL for C18:3 is the fatty acid desaturase-3 gene of the genome C (fad3c) and it is located on N14. The second major QTL resides on N4 and is the fad3a gene of the A genome. We have sequenced genomic clones of the fad2 and fad3c genes amplified from an EMS-induced mutant and a wild-type canola cultivar. A comparison of the mutant and wild-type allele sequences of the fad2 and fad3c genes revealed single nucleotide mutations in each of the genes. Detailed sequence analyses suggested mechanisms by which both the mutations can cause altered fatty acid content. Based on the sequence differences between the mutant and wild-type alleles, two single nucleotide polymorphism (SNP) markers, corresponding to the fad2 and fad3c gene mutations, were developed. These markers will be highly useful for direct selection of desirable fad2 and fad3c alleles during marker-assisted trait introgression and breeding of canola with high oleic and low linolenic acid.

No abstract

The plant fad3 (Fatty Acid Desaturase 3) gene encodes an endoplasmic delta-15 linoleate desaturase which is responsible for the desaturation of linoleic acid (C18:2) to linolenic acid (C18:3). Two fad3 genes ( fad3a and fad3c) were reported to control linolenic content in Brassica napus, with fad3a located on linkage group N4 and fad3c on N14 (Barret et al. 1999;Brunel et al. 1999;Hu et al. 2006). The high level of linolenic acid in canola oil is undesirable because linolenic acid is highly unsaturated and easily oxidized to cause off-flavor and rancidity of the oil, thus resulting in a shortened shelf life. Breeding canola varieties with low linolenic acid in oilseeds is one of major objectives for many canola breeding programs. Low C18:3 mutants have been produced through mutagenesis (Rakow 1973;Auld et al. 1992). The development of canola varieties with fatty acid profile of C18:1 above 70% and C18:3 below 3.0% in oilseeds, for example, Natreon varieties, is a major objective of the canola breeding program in Dow AgroSciences. A single nucleotide mutation of ϩ1G to ϩ1A at the 5Ј splice site of intron 6 of the B. napus fad3c gene was previously identified by comparing the wild-type and high oleic mutant allele of the locus (Hu et al. 2006). This mutant was developed through ethyl methanesulphonate (EMS) mutagenesis and contains reduced C18:3 content in oil seed. An allele-specific PCR marker diagnostic to the mutation was developed. Linkage analysis with 183 doubled haploid lines derived from the cross of Quantum and DMS100 indicated that this allele-specific marker was significantly correlated to low C18:3. A high-throughput Invader ® assay detecting the SNP generated from the fad3c gene mutation clearly differentiated the homozygous mutant, homozygous wild-type and heterozygous genotypes, thus allowing specific selection of the fad3c alleles conferring low C18:3 content (Hu et al. 2006).The molecular characterization of mutations in splicing site sequences in Arabidopsis provides valuable information on plant pre-mRNA splicing. As in other organisms, plant genes contain highly conserved 5Ј splice site (exon/intron junction-AG/GTAAG) and 3Ј splice site (intron/exon junction-TGCAG/G). The first two nucleotides in the 5Ј splice site intron junction sequence, ϩ1G and ϩ2T, have shown 100% and 99% conservation respectively among over 1000 Arabidopsis introns studied (Lorkovic et al. 2000;Brown 1996). The accuracy of splicing depends on the mechanisms of intron signal recognition and the correct selection of 5Ј and 3Ј splice sites. Mutations in splice sites can abolish splicing or lead to exon skipping, i.e., the affected exon Abstract Two genes ( fad3a and fad3c) encode for endoplasmic delta-15 linoleate desaturase, which is responsible for the desaturation of linoleic acid (C18:2) into linolenic acid (C18:3) in canola (Brassica napus). The canola mutant line DMS100 carries a G-to-A base substitution at the 5Ј splice site, located at ϩ1 G, of fad3c intron 6 and contains reduced C18:3 content in oil seeds. Reverse ...