Abstract
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.
Highlights
Rapeseed (Brassica napus L. var. oleifera) is an important oilseed crop and is the source of high-quality oil and highprotein animal feed
The results of the present study provide some information regarding the performance of the cleaved amplified polymorphic sequences (CAPS) marker and allow us to choose the best option for performing routine analyses
It is well known that the rapeseed genome is very complex [37], which is a common characteristic of genomes of all Brassica species [38]
Summary
Rapeseed (Brassica napus L. var. oleifera) is an important oilseed crop and is the source of high-quality oil and highprotein animal feed. The overall performance of the mutated plants was much lower than that of wild-type cultivars (compare the mutants with the ‘Monolit’ cultivar in Table S1 in Online Resource 12) To eliminate this disadvantage, some enhancement is needed to obtain new double-low (“00”) varieties of rapeseed having high oleic acid content in seeds. Using plants with high oleic acid content, which come from two different sources, may increase the stability of the oleic acid level in the resulting offspring and add some elite germplasm to the new segregating lines In both cases, after each crossing, there are many segregating plants for which the presence or absence of the mutated genes need to be precisely determined. The method must be optimized to obtain precise, repeatable, and explicit results
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