BackgroundPalmer amaranth achieves resistance to glufosinate by overproducing the chloroplastic glutamine synthetase (GS2) protein, a result of the amplification and overexpression of its nuclear coding gene. This study examined how amplifiedGS2copies are inherited, identified their physical location in the cell, and investigated the mechanism ofGS2amplification.ResultsSegregation analysis revealed that inheritance of amplifiedGS2copies deviates from classical Mendelian patterns, with poor correlation between plant level resistance andGS2amplification. Fluorescencein situhybridization revealed chromosomal insertions ofGS2and potential extrachromosomal circular DNA (eccDNA), and variability inGS2amplification both among individual plants and within cells (not all cells in plants with highGS2copy number showed GS2 amplification). The unpredictable inheritance patterns and distribution ofGS2copies across multiple chromosomes suggest a role for eccDNA inGS2amplification. This was confirmed through eccDNA sequencing, which also identified multiple isoforms ofGS2.ConclusionThis is the second documented case of herbicide resistance conferred by eccDNA-mediated target-site gene amplification in Palmer amaranth.