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Since the initial report of glyphosate-resistant (GR) Amaranthus palmeri S. Watson in 2006, resistant populations have been reported in 28 states. The mechanism of resistance is amplification of a 399-kb extrachromosomal circular DNA, called the EPSPS replicon, and is unique to glyphosate-resistant plants. The replicon contains a single copy of the 10-kb 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene which causes the concomitant increased expression of EPSP synthase, the target enzyme of glyphosate. It is not known whether the resistance by this amplification mechanism evolved once and then spread across the country or evolved independently in several locations. To compare genomic representation and variation across the EPSPS replicon, whole genome shotgun sequencing (WGS) and mapping of sequences from both GR and susceptible (GS) biotypes to the replicon consensus sequence was performed. Sampling of GR biotypes from AZ, KS, GA, MD and DE and GS biotypes from AZ, KS and GA revealed complete contiguity and deep representation with sequences from GR plants, but lack of homogeneity and contiguity with breaks in coverage were observed with sequences from GS biotypes. The high sequence conservation among GR biotypes with very few polymorphisms which were widely distributed across the USA further supports the hypothesis that glyphosate resistance most likely originated from a single population. We show that the replicon from different populations was unique to GR plants and had similar levels of amplification., Competing Interests: The authors have declared that no competing interests exist.

Gene copy number variation is a predominant mechanism used by organisms to respond to selective pressures from the environment. This often results in unbalanced structural variations that perpetuate as adaptations to sustain life. However, the underlying mechanisms that give rise to gene proliferation are poorly understood. Here, we show a unique result of genomic plasticity in Amaranthus palmeri : a massive, ∼400-kb extrachromosomal circular DNA (eccDNA) that harbors the 5-ENOYLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE ( EPSPS ) gene and 58 other genes whose encoded functions traverse detoxification, replication, recombination, transposition, tethering, and transport. Gene expression analysis under glyphosate stress showed transcription of 41 of these 59 genes, with high expression of EPSPS , as well as genes coding for aminotransferases, zinc finger proteins, and several uncharacterized proteins. The genomic architecture of the eccDNA replicon is composed of a complex arrangement of repeat sequences and mobile genetic elements interspersed among arrays of clustered palindromes that may be crucial for stability, DNA duplication and tethering, and/or a means of nuclear integration of the adjacent and intervening sequences. Comparative analysis of orthologous genes in grain amaranth ( Amaranthus hypochondriacus ) and waterhemp ( Amaranthus tuberculatus ) suggests that higher order chromatin interactions contribute to the genomic origins of the A. palmeri eccDNA replicon structure., (© 2020 American Society of Plant Biologists. All rights reserved.)

Background: Glyphosate resistance in Amaranthus palmeri, one of the most prevalent herbicide-resistant weeds in the USA, is attributable to amplification and increased expression of the gene encoding the target site of glyphosate, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The EPSPS gene and the surrounding 287 kilobases (kb) of amplified sequence are unique to glyphosate-resistant plants and termed the EPSPS cassette. It has only been sequenced in one A. palmeri population from Mississippi. This research compares EPSPS cassettes in seven resistant and five sensitive populations from geographically distant locations within the USA, including Mississippi, Arizona, Kansas, Maryland, Delaware and Georgia., Results: Polymerase chain reaction (PCR) products from 40 primer pairs specific to the cassette were similar in size and sequence in resistant populations. Several primer pairs failed to generate PCR products in sensitive populations. Regions of the cassette sequenced in the resistant populations were found to be nearly identical to those from Mississippi. Gene expression analysis showed that both EPSPS and another gene in the cassette, a reverse transcriptase, were elevated in all resistant populations tested relative to the sensitive populations., Conclusion: EPSPS cassettes from distant resistant populations were nearly homologous. Considering the complexity of the cassette, and the degree of similarity among some cassette sequences, the results are consistent with the hypothesis that glyphosate resistance probably evolved once and then rapidly spread across the USA. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background: Amaranthus spinosus, a common weed of pastures, is a close relative of Amaranthus palmeri, a problematic agricultural weed with widespread glyphosate resistance. These two species have been known to hybridize, allowing for transfer of glyphosate resistance. Glyphosate-resistant A. spinosus was recently suspected in a cotton field in Mississippi., Results: Glyphosate-resistant A. spinosus biotypes exhibited a fivefold increase in resistance compared with a glyphosate-susceptible biotype. EPSPS was amplified 33-37 times and expressed 37 times more in glyphosate-resistant A. spinosus biotypes than in a susceptible biotype. The EPSPS sequence in resistant A. spinosus plants was identical to the EPSPS in glyphosate-resistant A. palmeri, but differed at 29 nucleotides from the EPSPS in susceptible A. spinosus plants. PCR analysis revealed similarities between the glyphosate-resistant A. palmeri amplicon and glyphosate-resistant A. spinosus., Conclusions: Glyphosate resistance in A. spinosus is caused by amplification of the EPSPS gene. Evidence suggests that part of the EPSPS amplicon from resistant A. palmeri is present in glyphosate-resistant A. spinosus. This is likely due to a hybridization event between A. spinosus and glyphosate-resistant A. palmeri somewhere in the lineage of the glyphosate-resistant A. spinosus plants. Published 2014. This article is a U.S. Government work and is in the public domain in the USA., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

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 (EPSPS) gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world's most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS 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 EPSPS loci in susceptible (S) A. palmeri, and that only one of these was amplified in glyphosate-resistant (R) A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs) were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac) transposase and a repetitive sequence region were associated with amplified EPSPS 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 EPSPS gene.

The efficacy of the commercial glyphosate [( N-phosphonomethyl) glycine] formulations Roundup Ultra, Touchdown and Engame were compared for the control of prickly sida ( Sida spinosa L.), morningglory ( Ipomeae hederacea var. integriuscula Gray), sicklepod ( Senna obtusifolia L.) and purple nutsedge ( Cyperus rotundus L.). Engame is a new formulation of glyphosate that contains glyphosate acid and 1-aminomethanamide dihydrogen tetraoxosulfate (AMADS), a proprietary mixture of sulfuric acid and urea, other than glyphosate salt and surfactants. Injury by Engame differed from Roundup Ultra and Touchdown in that necrotic lesions formed on leaves several hours after treatment. Leaves of very susceptible species, such as prickly sida, were rapidly, although incompletely, desiccated and then became chlorotic and died in a manner typical of other glyphosate formulations. Engame was 2–3 times more active to growth inhibition than either the Roundup Ultra or Touchdown formulations, based on GR50 comparisons expressed on an acid equivalent basis. The GR50 estimates did not change over the 3 week evaluation period for prickly sida and purple nutsedge, and after 2 weeks after treatment for morningglory. The GR50 estimates for sicklepod decreased over the 3 week evaluation period indicating a slower response to glyphosate. The application of AMADS alone caused minute necrotic lesions on sicklepod and purple nutsedge, and lesions up to 3 mm in diameter on prickly sida and morningglory. Further injury from AMADS was not noted and plants resumed growth without apparent delay. At glyphosate rates above 1120 g ha−1, greater than 80% control was achieved at 7 days after treatment. These results demonstrate that glyphosate efficacy can be further enhanced by formulations that apparently improve uptake and translocation. [ABSTRACT FROM AUTHOR]