Allele-specific SNP markers for the new low linolenic mutant genotype of winter oilseed rape

Abstract

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.

A Multiplex Fluorescent PCR Assay in Molecular Breeding of Oilseed Rape

“…The FAD3 genes encode for encoplasmic delta-15 linoleate desaturase responsible for desaturation of linoleic acid (C18:2) into linolenic acid. As a result of cloning and sequencing of FAD3 genes from wildtype and LL mutant B. napus plants, we reported two point mutations (BnaA.FAD3 and BnaC.FAD3) responsible for disruption of the FAD3 genes expression and function (Mikolajczyk et al, 2010b) in the new LL mutant rapeseed line (Spasibionek, 2006). One point mutation comprised a C to T transition in the mutant bnaA.fad3 gene leading to a possible Arg to Cys substitution.…”

“…previously by Mikolajczyk et al (2010b) for the analysis of splicing variant (see Table 3 for primer details). The PCR was carried out in a 96-well plate (Brandt, Wertheim, Germany) sealed with silicone compression mat (Axygen, Union City, CA, USA) in a reaction volume of 6 μl containing 2.5 µl of Type-it Microsatellite PCR Kit (Qiagen, Hilden, Germany), 0.2 mM of each primer, and 1 µl of DNA template (50-100 ng).…”

“…We developed genetic markers for monitoring FAD3 alleles in breeding programs. The detection of wild-type and mutant FAD3 alleles comprises two steps: independent PCR amplification of short SNP fragments and a detection of the SNPs based on microsequencing method (SNaPshot) with the use of allele-specific primers (Mikolajczyk et al, 2010b). The SNaPshot assay enabled precise and unambiguous detection of this allelic variability.…”

“…Both polymorphic sites were analysed independently by single-base primer extension reaction (microsequencing) with primers varying in length as described previously by Mikolajczyk et al (2010b). The first oligonucleotide, mutA-1f (35 nt), was used for detection of alleles in the locus BnaA.FAD3, the second, mutC-45F (45 nt), was used for the locus BnaC.FAD3 (see Table 3 for primer details).…”

“…The low linolenic mutant genotypes were monitored with the use of the developed SNaPshot assay (Mikolajczyk et al, 2010b), detecting wild-type and mutant alleles of the FAD3 desaturase genes in the AC allotetraploid genome of B. napus. The FAD3 genes encode for encoplasmic delta-15 linoleate desaturase responsible for desaturation of linoleic acid (C18:2) into linolenic acid.…”