Simultaneous detection of glyphosate and glufosinate target‐site resistance in Eleusine indica via multiplex TaqMan qPCR

BACKGROUND Continuous use of glyphosate followed by glufosinate‐ammonium has selected for multiple resistance to both herbicides in Eleusine indica worldwide. Managing such resistant weeds requires fast, accurate molecular detection assay. To address this critical need, we developed a robust multiplex TaqMan quantitative (q)PCR assay that simultaneously detects five well‐characterized target‐site resistance markers in E. indica : EPSPS copy number variation; T102I in EPSPS ; P106A and P106S in EPSPS ; and S59G in GS1‐1 . RESULTS The multiplex qPCR assay showed analytical specificity when tested on genomic DNA from nine reference accessions: three susceptible, three glyphosate‐resistant (with EPSPS CNV) and three multiple‐resistant. Subsequent analysis of 56 field‐collected samples demonstrated 98.2% concordance (55 of 56) with Sanger sequencing across all five resistance‐associated markers: EPSPS CNV, T102I, P106A, P106S and GS1‐1 S59G, confirming the reliability and practical value of the multiplex qPCR assay. Only samples 7–8 showed discordance at EPSPS position 102, where Sanger chromatograms showed overlapping peaks at this position, which is likely to be a result of heterozygous mutation distribution among amplified EPSPS gene copies. This case further underscores the advantages of the multiplex qPCR assay over Sanger sequencing in detection sensitivity and accuracy. Moreover, a strong correlation ( R 2 = 0.8935) in gene copy number estimation between the two methods across all samples further supports the reliability of the qPCR assay. CONCLUSIONS In summary, this study delivers a simple, robust and high‐throughput diagnostic tool for the rapid, simultaneous identification of dual herbicide target‐site resistance in goosegrass, offering superior sensitivity, quantitative resolution and throughput compared with Sanger sequencing.