Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

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.

Identification and Application of BhAPRR2 Controlling Peel Colour in Wax Gourd (Benincasa hispida)

“…Cleaved amplified polymorphic sequences (CAPS) markers have been widely used in molecular marker-assisted breeding. This technique has had profound applications in tomato (Ashish et al, 2021), rapeseed (Matuszczak et al, 2020), and maize (Pan et al, 2021). In this study, we conducted a genotypephenotype joint analysis by KASP markers, compressing the mapping interval.…”

Omics: The way forward to enhance abiotic stress tolerance inBrassica napusL

“… 47 More than 75% of detected SNPs were useful and could be exploited in modern molecular breeding schemes. 47 , 110 Likewise, SNP analysis was documented to study the heat stress on different rapeseed varieties under field conditions, and data were recorded from flowering to maturity phase for different agronomic traits. Association mapping (AM) for various agronomic traits was executed to detect heat-related QTLs.…”

Validation of CAPS marker WR003 for the leaf rust resistance gene Lr1 and the molecular evolution of Lr1 in wheat

“…However, the use of molecular markers provides a direct aid in selecting disease-resistant varieties and speeds up the breeding process (Juliana et al 2018;Rani et al 2019). CAPS markers are cost-efficient and simple tools and are extensively used by breeders (Michikawa et al 2019;Matuszczak et al 2020). In this study, the CAPS marker WR003's specificity for Lr1 was validated in a set of wheat varieties and their progenitor Ae.…”