# Methods Established for EPSPS Gene Mutation Detection in Glyphosate-Resistant Rice (Oryza sativa L.)

## TL;DR

This paper introduces five methods to detect EPSPS gene mutations in glyphosate-resistant rice, enabling accurate mutation identification and large-scale screening.

## Contribution

The study establishes a comprehensive set of methods for detecting EPSPS gene mutations with varying sensitivities and applications.

## Key findings

- Five methods (Sanger, NGS, AS-PCR, qPCR, BDA) effectively detect EPSPS mutations with sensitivities ranging from 0.01% to 10%.
- Sanger, NGS, and BDA are suitable for precise mutation site characterization, while AS-PCR and qPCR are better for large-scale screening.
- The methods provide technical support for safety regulations and intellectual property protection in glyphosate-resistant rice.

## Abstract

“Rundao118” is a glyphosate-resistant rice; it contains both endogenous wild and mutated 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes. Conventional qualitative and quantitative detection methods face significant challenges for direct analysis. Here, we describe five detection methods for identifying EPSPS mutations in this rice line: (1) polymerase chain reaction (PCR) amplification-based Sanger sequencing, (2) next-generation sequencing (NGS) based on PCR amplification, (3) allele-specific PCR (AS-PCR), (4) real-time fluorescent quantitative PCR (qPCR), and (5) blocker displacement amplification (BDA). All five methods effectively identified EPSPS mutations, with the following detection sensitivities: Sanger, 10%; NGS, 1%; AS-PCR, 0.05%; qPCR, 0.01%; and BDA, 0.1%. Among these, the Sanger, NGS, and BDA methods excelled at the rapid identification of single-nucleotide mutations, making them suitable for precise mutation site characterization and identification. In contrast, the AS-PCR and qPCR methods were more appropriate for large-scale rapid screening of known mutation sites. The detection systems established in this study provide a comprehensive technical solution for rapid identification of EPSPS mutations in glyphosate-resistant rice. These methods not only enable accurate determination of mutation sequences but also effectively trace mutation origins, offering crucial technical support for both safety regulations and intellectual property protection.