Variation in the gene encoding acetolactate-synthase in Lolium species and proactive detection of mutant, herbicide-resistant alleles

Lolium species (ryegrasses) are genetically highly variable plants that are both forage crops and major weeds across the globe. As weeds, they rapidly evolve resistance under the selective pressure of acetolactate-synthase (ALS) inhibitors, the most resistance-prone herbicide group. Quick and accurate diagnosis is therefore of importance to prevent resistance spread in ryegrass. To develop proactive molecular tools for the detection of mutant, resistant ALS alleles, we assessed variation in the ryegrass ALS gene. Sequencing the full 1929-bp ALS coding sequence in 59 plants from six distant locations revealed a total of 208 polymorphic nucleotide positions (one every 9.3 nucleotides). The heterogeneous distribution of synonymous and non-synonymous substitutions along the ALS coding sequence suggested that nucleotide variation of ALS is shaped by purifying and background selection. Using regions of the ALS coding sequence with a low number of polymorphic nucleotide sites, five derived cleaved amplified polymorphic sequence (dCAPS) assays were developed targeting codons crucial for herbicide sensitivity. These enabled the first detection in ryegrass of a Pro-197-Thr substitution that confers herbicide resistance. Most assays could also be used to genotype Festuca and Vulpia plants. These dCAPS assays should prove powerful tools for both resistance diagnosis and population genetics studies.