Evaluation of high-resolution melting and RT-qPCR probe assays for high-throughput detection of target-site mutations conferring glyphosate resistance in Lolium perenne

Abstract

ABSTRACT Rapid identification of herbicide-resistant weeds plays a crucial role in their management. Glyphosate resistance in Lolium perenne is a growing issue in New Zealand and can be conferred by a single nucleotide polymorphism (SNP) causing amino acid substitutions at codon 106 in the 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) gene. High-throughput molecular (HTM) methodologies that can detect such SNPs include high-resolution melting and real-time quantitative PCR probe assays. Here, we compare the effectiveness of both assays with a more labour-intense derived cleaved amplified polymorphic sequence (dCAPS) test for detecting SNPs at codon 106. The results showed that both HTM assays detected mutations at this codon, and their results were consistent with those of the dCAPS. Unlike dCAPS, however, the HTM assays over-estimated homozygosity for the glyphosate resistance allele. Nevertheless, cost-effective high-throughput detection of resistance is more important than the zygosity status when initiating an on-farm management response. In summary, both HTM assays successfully identified glyphosate resistance alleles at codon 106 in the EPSPS gene. Given that both assays are more cost-effective and can screen a larger number of samples in a timely manner compared to the dCAPS method, they can be used as quick tests for detecting glyphosate resistance in L. perenne.