Seeds of the winter oilseed rape (Brassica napus L.) line PN 3756/93 were treated with ethyl methanesulphonate to induce mutations in the fatty acid biosynthetic pathway. The seed mutagenic treatment was repeated in the M 2 generation. After treatments, individual seed and plant selections were made for changes in fatty acid composition during several generations of inbreeding. Self-pollinated plants with changed fatty acid compositions were inbred to obtain genetically homozygous and stable mutant lines. Two mutants, M-10453 and M-10464, with increased levels of oleic acid (approximately 76%) and reduced linoleic and linolenic acid contents (8.5% and 7.5%, respectively) were selected. Gene or genes controlling desaturation of oleic acid were probably mutated in these plants. The third mutant, M-681 had a very low linolenic acid content (approximately 2.6%) and increased linoleic acid content (approximately 26%). This would suggest the occurrence of mutations in genes controlling linoleic acid desaturation. The results of selection work during several generations showed that the environment had substantial influence on the composition of seed oil. This made the search for mutants with modify fatty acid compositions difficult. The induced mutants are not directly usable as new varieties, but can be used as parents in crosses for the development of high quality rapeseed varieties.Key words: Brassica napus -chemical mutagenesis -fatty acids -linoleic acid -linolenic acid -oleic acid -seed oil Seed oil of rapeseed (Brassica napus) contains approximately 45-50% erucic acid. In the 1970s, plant breeders identified and selected seeds and plants that were basically free of erucic acid (approximately 0.1%). High-yielding ÔzeroÕ erucic acid varieties were developed through crossbreeding and selection.

One of the goals in oilseed rape breeding is the identification of genotypes with low linolenic acid content in seed oil. Here, we present new genetic markers for mutant alleles of a chemically induced low linolenic rapeseed line DH219/05. Genomic clones comprising fatty acids desaturase 3 (FAD3) genes from the mutant and wild-type rapeseed lines were sequenced. Two statistically important single nucleotide polymorphisms were detected: (1) a C to T substitution in the third position of the sixth codon of the seventh exon in the BnaA.FAD3 gene and (2) a G to A transition in the 5¢ splice donor site of the sixth intron in the BnaC.FAD3 gene. Allele-specific SNP markers were designed involving detection of the wild-type and mutant alleles by SNaPshot analysis and locus-specific PCR primers. Strong negative correlation between the presence of mutant alleles in the A and C genomes and linolenic acid content was revealed by analysis of variance. Sequence analysis of transcript variants confirmed predictions on possible negative effects of mutations on FAD3 gene expression.

Development of oilseed rape (Brassica napus L.) breeding lines producing oil characterized by high oleic and low linolenic acid content is an important goal of rapeseed breeding programs worldwide. Such kind of oil is ideal for deep frying and can also be used as a raw material for biodiesel production. By performing chemical mutagenesis using ethyl methanesulfonate, we obtained mutant winter rapeseed breeding lines that can produce oil with a high content of oleic acid (C18:1, more than 75%) and a low content of linolenic acid (C18:3, less than 3%). However, the mutant lines revealed low agricultural value as they were characterized by low seed yield, low wintering, and high content of glucosinolates in seed meal. The aim of this work was to improve the mutant lines and develop high-oleic and low-linolenic recombinants exhibiting both good oil quality and high agronomic value. The plant materials used in this study included high-oleic and low-linolenic mutant breeding lines and highyielding domestic canola-type breeding lines of good agricultural value with high oleic acid content and extremely low glucosinolates content. Field trials were conducted in four environments, in a randomized complete block design. Phenotyping was performed for wintering, yield of seed and oil, and seed quality traits. Genotype × environment interaction was investigated with respect to the content of C18:1 and C18:3 acids in seed oil. Genotyping was done for the selection of homozygous high oleic and low linolenic lines using allele-specific CAPS markers and SNaPshot assay, respectively. Finally, new high oleic and low linolenic winter rapeseed recombinant lines were obtained for use as a starting material for the development of new varieties that may be of high value on the oil crop market.

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low (“00”) cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new “00” varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5′-C↓TAG-3′ sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

Flax (Linum usitatissimum L.) is an important source of oil rich in omega–3 fatty acids (especially α-linolenic acid accounting for > 50%), which is proven to have health benefits and utilized as an industrial raw material. α-Linolenic acid is a polyunsaturated fatty acid that readily undergoes oxidative transformation. Autoxidation of α-linolenic acid is the principal process contributing to the development of off-flavors, loss of color, and alteration in the nutritional value of linseed oil. However, there is huge a demand on the market for oils having different compositions of fatty acids, including the linseed oil characterized by improved stability. For this purpose, a complete diallel cross was performed in this study using six flax genotypes varying in the fatty acid content to estimate the genetic parameters. The analysis of variances carried out for the studied traits (content of oleic, linoleic and α-linolenic acid) indicated large differences among the genotypes. Variances due to GCA were much higher in magnitude than those related to SCA for the content of linoleic and α-linolenic acid, which indicated the superiority of additive gene effects in determining the inheritance of these traits. The nonadditive gene action played an important role for oleic acid content, since the magnitude of SCA effect was almost two times higher than GCA effect. The parental lines of linola (Linola KLA and Linola KLB) exhibited the highest concentration of favorable alleles for the two traits (high content of linoleic acid and low content of α-linolenic acid) and were thus found suitable for a continuous improvement program. On the basis of the SCA effect, five cross combinations, were found to be promising F1 hybrids for use as a source population for further selection, in order to achieve fatty acid changes in linseed. These combinations allow selecting varieties with 1:1 and 2:1 ratio of omega–6:omega–3 fatty acids for producing oil with an extended shelf life for food products.