The Genomic Landscape of the 297 Kilobase EPSPS Amplicon in Palmer Amaranth

Amaranthus palmeri, has evolved resistance to glyphosate by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, which results in higher titers of EPSPS protein, the target site of glyphosate. A BAC library was generated using genomic DNA from a glyphosate resistant biotype. By sequencing overlapping BACs, a single consensus sequence of 297 kilobases was generated which included a single EPSPS gene of 8 exons and 7 introns in a 10,229 bp sequence. Approximately 140kb of genomic sequence was captured both 5’ and 3’ of the EPSPS locus. The genomic structure flanking the EPSPS locus was a complex configuration of tandem and inverted repeats. Also present were a ricesleeper homolog, a NAC domain containing protein, a heat shock cognate 70kD protein and a reverse transcriptase, all of which were transcribed. Whole genome shotgun sequencing (wgs) of two contrasting biotypes, sensitive and resistant, was performed on an Illumina platform and the reads were mapped to the reference amplicon, revealing significant differences in both repetitive and coding content between the biotypes. Alignment of wgs sequences from sensitive plants to the amplicon showed numerous gaps in the sequence, indicating that the amplified region was not contiguous in sensitive plants.  The origin and ends of the amplicon and the mechanism of amplification remain to be identified.  We propose that this amplicon of unprecedented length presents a unique adaption to herbicide stress and represents a mechanism for rapid evolution of herbicide resistance.