A PCR plus restriction enzyme-based technique for detecting target-enzyme mutations at position Pro-106 in glyphosate-resistant Lolium perenne.

Hossein Ghanizadeh,Craig B Anderson,Andrew G Griffiths,Christopher E Buddenhagen,Kerry C Harrington,David D Fang

doi:10.1371/journal.pone.0246028

Abstract

The first step in managing herbicide-resistant weeds is to confirm their resistance status. It is, therefore, crucial to have a rapid, reliable and cost-effective technique to assess samples for herbicide resistance. We designed and evaluated three derived cleaved amplified polymorphic sequence (dCAPS) markers for detecting glyphosate resistance in Lolium perenne. conferred by non-synonymous mutations at codon-106 in the enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. The dCAPS markers involve amplification of the target region, digestion of the amplified products with restriction enzymes and gel-based visualisation of the digested products. The results showed that all three dCAPS markers could successfully detect mutations at codon-106 in the target enzyme. The dCAPS markers can also inform us of the zygosity state of the resistance allele and was confirmed by sequencing the target region of the EPSPS gene. The markers described here are effective quick tests for the monitoring and evaluation of the target-enzyme mechanism of glyphosate resistance in Lolium perenne.

Highlights

Herbicides are valuable tools for weed control and crop production; their usefulness is being undermined by the development of herbicide resistance [1, 2]
A polymerase chain reaction (PCR)-based technique for detecting target-enzyme mutations in glyphosate-resistant perennial ryegrass digestion with the corresponding restriction enzymes, the amplicons generated from the glyphosate-susceptible population SP were cleaved to yield a ~180 bp fragment, as predicted, indicating that this individual was homozygous for the wild-type glyphosate-sensitive allele (Fig 1)
Sequence confirmation of the derived cleaved amplified polymorphic sequence (dCAPS) results from a resistant individual of population O was provided by partial sequencing of the enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene using Glyf1/Glyr1 primers

Summary

Introduction

Herbicides are valuable tools for weed control and crop production; their usefulness is being undermined by the development of herbicide resistance [1, 2]. There are over 500 unique cases of herbicide-resistant weeds globally [3], and this is a growing issue compounded by the lack of new herbicide modes of action being deployed in recent years [4, 5]. Recent studies have characterized chemical classes with the potential to be developed into herbicides with novel modes of action [5]. A sustainable weed management approach, needs to be implemented to maximize the efficacy of the herbicides currently available on the market [6, 7].

Objectives

Methods

Results

Discussion

Conclusion