A Novel Genomic Rearrangement in the Amaranthus palmeri Extrachromosomal Circular DNA Provides Dual Herbicide Resistance to Glyphosate and Glufosinate

Amplification of chloroplastic glutamine synthetase ( GS2 ) has been characterized as one of the resistance mechanisms in glufosinate-resistant Amaranthus palmeri accession (MSR2). Previously, the overamplification of the glyphosate-resistance gene, 5-enolpyruvylshikimate-3-phosphate ( EPSPS ), in A. palmeri was determined to be driven by an extrachromosomal circular DNA (eccDNA). Here, a novel eccDNA is described that carries both glyphosate and glufosinate-ammonium target site due to co-duplication of their chromosomic native region, conferring resistance. Besides EPSPS , the novel replicon has a region replaced by a fragment carrying the GS2 isoforms ( GS2.1 and GS2.2 ) and other genes. The co-existence of eccDNA carrying only EPSPS was confirmed in MSR2 samples harboring dual targeting eccDNA. The genomic structure of GS2 and EPSPS amplification was also assessed in a different glufosinate-resistant A. palmeri accession (MSR1) along with MSR2. The accessions showed distinct GS2.1 and GS2.2 amplification patterns suggesting the existence of diverse replicons that were not assembled here. The EPSPS was amplified in both accessions, and a correlation was observed with the GS2 isoforms in MSR2, further supporting the co-existence 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.