A rearranged <i>Amaranthus palmeri</i> extrachromosomal circular DNA confers resistance to glyphosate and glufosinate

Some herbicide-resistant weeds become resistant by generating additional copies of specific loci. For example, amplification of the locus encoding chloroplastic glutamine synthetase (GS2) produces herbicide resistance in the glufosinate-resistant Palmer amaranth (Amaranthus palmeri) accession MSR2. Previously, overamplification of the glyphosate-resistant gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Palmer amaranth was determined to be driven by an extrachromosomal circular DNA (eccDNA). Here, we describe a rearranged eccDNA that confers resistance to both glyphosate and glufosinate ammonium due to the coduplication of the native chromosomal regions that contain the genes that encode for these herbicides target proteins. In addition to EPSPS, the replicon carries 2 GS2 isoforms (GS2.1 and GS2.2) and other genes. MSR2 samples harbored eccDNA carrying only EPSPS coexisting with eccDNAs harboring both EPSPS and GS2. A second glufosinate-resistant Palmer amaranth accession (MSR1) showed distinct GS2.1 and GS2.2 amplification patterns from MSR2, suggesting the existence of diverse replicons in Palmer amaranth. EPSPS copy number was correlated with both GS2 isoforms copy number in MSR2, further supporting the coexistence of these genes in the same replicon. These findings shed light on the complexity of eccDNA formation in plant systems, with the collection and accumulation of extra pieces of DNA.