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

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.

Automated summary

Rapid identification of herbicide-resistant weeds is crucial for their management. This study compared the effectiveness of two high-throughput molecular (HTM) assays with a labor-intensive method for detecting glyphosate-resistant weeds. Both HTM assays successfully detected mutations at the targeted codon and showed consistent results with the labor-intensive method. However, the HTM assays overestimated the homozygosity of the resistance allele. Despite this, cost-effective high-throughput detection of resistance is more important than accurately determining the zygosity status when initiating management actions. Overall, the HTM assays are effective and efficient tools for detecting glyphosate resistance in Lolium perenne.