Identification of Genetic Elements Associated with <i>EPSPS</i> Gene Amplification

<div><p>Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (<i>EPSPS</i>) gene evolved in the weed species <i>Amaranthus palmeri</i> to confer resistance to glyphosate, the world’s most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the <i>EPSPS</i> gene and genomic regions flanking <i>EPSPS</i> loci in <i>A. palmeri</i>, and searched for mobile genetic elements or repetitive sequences. The <i>EPSPS</i> gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two <i>EPSPS</i> loci in susceptible (S) <i>A. palmeri</i>, and that only one of these was amplified in glyphosate-resistant (R) <i>A. palmeri</i>. The <i>EPSPS</i> gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified <i>EPSPS</i> copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs) were identified next to <i>EPSPS</i> gene copies only in R individuals. Additionally, a putative <i>Activator</i> (<i>Ac</i>) transposase and a repetitive sequence region were associated with amplified <i>EPSPS</i> genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the <i>EPSPS</i> gene.</p>