Development and validation of functional kompetitive allele-specific PCR markers for herbicide resistance in Brassica napus

Abstract

Effective weed control in the field is essential for maintaining favorable growing conditions and rapeseed yields. Sulfonylurea herbicides are one kind of most widely used herbicides worldwide, which control weeds by inhibiting acetolactate synthase (ALS). Molecular markers have been designed from polymorphic sites within the sequences of ALS genes, aiding marker-assisted selection in breeding herbicide-resistant rapeseed cultivars. However, most of them are not breeder friendly and have relatively limited application due to higher costs and lower throughput in the breeding projects. The aims of this study were to develop high throughput kompetitive allele-specific PCR (KASP) assays for herbicide resistance. We first cloned and sequenced BnALS1 and BnALS3 genes from susceptible cultivars and resistant 5N (als1als1/als3als3 double mutant). Sequence alignments of BnALS1 and BnALS3 genes for cultivars and 5N showed single nucleotide polymorphisms (SNPs) at positions 1676 and 1667 respectively. These two SNPs for BnALS1 and BnALS3 resulted in amino acid substitutions and were used to develop a KASP assay. These functional markers were validated in three distinct BC1F2 populations. The KASP assay developed in this study will be valuable for the high-throughput selection of elite materials with high herbicide resistance in rapeseed breeding programs.

Imidazolinone Resistance in Oilseed Rape (Brassica napus L.): Current Status, Breeding, Molecular Markers and Prospects for Application in Hybrid Seed Purity Improvement

“…Since SNPs in coding triplets are capable of making amino acid substitutions, this can lead to changes in the agronomic traits of plants. Therefore, specific single-nucleotide changes are used as an important tool for the genetic improvement of crops [24][25][26][27]. The first oilseed rape mutants resistant to imidazolinones were obtained by Swanson et al in 1989 by chemical mutagenesis of microspores using 20 µM ethyl nitrosourea (ENU) in combination with a selective medium containing 40 µg/L imazethapyr (or a herbicide under the commercial name Pursuit) [28].…”

“…Functional markers targeting polymorphic regions within genes are directly related to phenotypic variability. Such markers, due to their complete linkage to alleles of the trait locus, are preferred for selection over DNA markers not located in the gene itself, such as RAPDs, SSRs and AFLPs [27,[39][40][41].…”

“…Currently, two markers each have been published in open sources for BnAHAS1R (Ser653Asp) and BnAHAS3R (Trp574Leu) mutations, which are responsible for resistance to imidazolinones (Table 2) [27,34,35,42,43]. that can detect the presence/absence of SNPs associated with resistance or susceptibility to herbicides of the imidazolinone group [34,42].…”

“…The markers described above require fragment separation by electrophoresis after PCR amplification, making their application relatively limited due to high cost and low throughput [27]. Mass plant testing is essential for plant breeding programs, so methods are constantly being improved.…”

“…In 2023, Shi et al, investigating the 5N mutant, developed functional KASP markers for both mutations of this mutant: BnAHAS3R (Trp574Leu) and BnAHAS1-2R (Trp574Leu) [27]. Table 2 summarizes the markers for the BnAHAS3R(Trp574Leu) mutation since our review focuses on resistance to herbicides of the imidazolinone group.…”