William T. Molin

Identification of genetic elements associated with EPSPs gene amplification.

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.

Evaluation of the Interaction between Glyphosate and Glufosinate

Abstract Crops transformed to provide resistance to herbicides with two different mechanisms of action provide new opportunities for control of herbicide-resistant weeds. However, unexpected interactions may develop, especially for herbicides not generally used in tank-mixtures. The objectives of this study were to evaluate weed control and determine herbicide interactions and fluorescence responses with combinations of glyphosate and glufosinate on selected weeds prevalent in Michigan cropping systems. Field studies to determine herbicide interactions resulted in synergism only at 0.84 kg ae ha−1 of glyphosate and 0.47 kg ai ha−1 glufosinate in 2008. Early synergism (7 d after treatment [DAT]) was observed in the field at several combined rates for common lambsquarters and velvetleaf in 2009, and in the greenhouse for giant foxtail. Differences between years were perhaps due to the effect of environmental conditions on herbicide absorption and translocation. Antagonism was observed in the field in 2009 for velvetleaf, common lambsquarters, and giant foxtail especially at 840 g ae ha−1 glyphosate and 118 g ai ha−1 glufosinate, 28 DAT. Antagonism was also observed in the greenhouse for giant foxtail and Canada thistle, 28 DAT. Fluorescence measurements on Canada thistle in the greenhouse showed that glufosinate and glufosinate plus glyphosate acted rapidly to quench electron transport of photosystem II (PS II) system of photosynthesis, and the fluorescence characteristics of the glyphosate and glufosinate combinations were indistinguishable from glufosinate alone. Nomenclature: Glufosinate; glyphosate; Canada thistle, Cirsium arvense (L.) Scop.; common lambsquarters, Chenopodium album L.; giant foxtail, Setaria faberi Herrm.; velvetleaf, Abutilon theophrasti Medik.

The unique genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri : a repetitive path to resistance

BackgroundThe expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity. Amongst the most competitive weeds, Amaranthus palmeri S. Wats has rapidly evolved resistance to glyphosate primarily through massive amplification and insertion of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene across the genome. Increased EPSPS gene copy numbers results in higher titers of the EPSPS enzyme, the target of glyphosate, and confers resistance to glyphosate treatment. To understand the genomic unit and mechanism of EPSPS gene copy number proliferation, we developed and used a bacterial artificial chromosome (BAC) library from a highly resistant biotype to sequence the local genomic landscape flanking the EPSPS gene.ResultsBy sequencing overlapping BACs, a 297xa0kb sequence was generated, hereafter referred to as the “EPSPS cassette.” This region included several putative genes, dense clusters of tandem and inverted repeats, putative helitron and autonomous replication sequences, and regulatory elements. Whole genome shotgun sequencing (WGS) of two biotypes exhibiting high and no resistance to glyphosate was performed to compare genomic representation across the EPSPS cassette. Mapping of sequences for both biotypes to the reference EPSPS cassette revealed significant differences in upstream and downstream sequences relative to EPSPS with regard to both repetitive units and coding content between these biotypes. The differences in sequence may have resulted from a compounded-building mechanism such as repetitive transpositional events. The association of putative helitron sequences with the cassette suggests a possible amplification and distribution mechanism. Flow cytometry revealed that the EPSPS cassette added measurable genomic content.ConclusionsThe adoption of glyphosate resistant cropping systems in major crops such as corn, soybean, cotton and canola coupled with excessive use of glyphosate herbicide has led to evolved glyphosate resistance in several important weeds. In Amaranthus palmeri, the amplification of the EPSPS cassette, characterized by a complex array of repetitive elements and putative helitron sequences, suggests an adaptive structural genomic mechanism that drives amplification and distribution around the genome. The added genomic content not found in glyphosate sensitive plants may be driving evolution through genome expansion.

Spurred anoda (Anoda cristata) interference in wide row and ultra narrow row cotton

Abstract A field experiment was conducted in 2000, 2001, and 2002 at Stoneville, MS, to determine the effect of spurred anoda interference on yield loss of two cotton cultivars, ‘Delta Pine 5415’ and ‘Pima S-6’, grown under wide (1 m) (WR) and ultra narrow (0.25 m) row (UNR) spacings. The relationship between spurred anoda density and dry weight per plot was linear each year. At a spurred anoda density of 8 m−2, spurred anoda dry weight per plot was 507, 322, and 777 g m−2 in 2000, 2001, and 2002, respectively. However, spurred anoda did not interfere with seed cotton yield in 2001, which was probably attributable to the low branch development in that year. Yield losses exceeded 55% at a spurred anoda density of 8 m−2 compared with controls in both WR and UNR. The effect of spurred anoda density on boll numbers was nearly identical in 2000 and 2002, regardless of cotton cultivar and row spacing. Boll weights decreased in response to spurred anoda interference. Spurred anoda interference resulted in a decrease in cotton branch dry weight in WR but not in UNR. The yield decrease as a result of spurred anoda interference in WR was due to reduction in boll retention or fruiting sites (predicated on a decrease in branch weight). However, in UNR, the yield decrease was due to plant mortality; the plant density of both cotton cultivars decreased by one plant for each additional spurred anoda, but the yield per plant for surviving plants remained constant. Neither WR nor UNR cotton had significant advantage in response to spurred anoda interference. The decreased boll weight observed in UNR, and the failure to increase boll numbers m−2 to compensate for decreased boll weight in UNR compared with WR, may limit its appeal to cotton producers. Nomenclature:u2003Spurred anoda, Anoda cristata (L.) Schlecht. ANVCR; cotton, Gossypium hirsutum L. ‘DP 5415’; Gossypium barbadense L. ‘Pima S-6’.

Survey of the genomic landscape surrounding the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant Amaranthus palmeri from geographically distant populations in the USA

BACKGROUNDnGlyphosate 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.nnnRESULTSnPolymerase 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.nnnCONCLUSIONnEPSPS 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.

Survey of the genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri from geographically distant populations in the United States

BACKGROUNDnGlyphosate 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.nnnRESULTSnPolymerase 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.nnnCONCLUSIONnEPSPS 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.

Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri

Significance Glyphosate is a nonselective herbicide used around the globe for weed control in glyphosate-resistant (GR) and noncrop situations. The extensive and exclusive use of glyphosate has led to the evolution of herbicide resistance in many crop weeds. The molecular target of glyphosate, the 5-enolpyruvlyshikimate-3-phosphate synthase (EPSPS) gene, confers resistance upon amplification and was first documented in GR Amaranthus palmeri. We now report that amplified EPSPS copies in GR A. palmeri are present in the form of extrachromosomal circular DNA molecules (eccDNAs) with various conformations. We discovered that eccDNAs are transmitted to the next generation by tethering to mitotic and meiotic chromosomes. These results represent a report of extrachromosomal structures that drive rapid adaptive evolution in higher organisms. Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs). Amaranthus palmeri, a crop weed, can develop herbicide resistance to glyphosate [N-(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplified EPSPS, the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplified EPSPS copies in glyphosate-resistant (GR) A. palmeri are present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock’s postulated innate systems [McClintock B (1978) Stadler Genetics Symposium] that can rapidly produce soma variation, amplify EPSPS genes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution.

Detection of the onset of glyphosate-induced soybean plant injury through chlorophyll fluorescence signal extraction and measurement

Abstract In this study, chlorophyll fluorescence (ChlF) was used to detect the onset of soybean plant injury from treatment of glyphosate, the most widely used herbicide. Thirty-six pots of nonglyphosate-resistant soybean were randomly divided into three groups and treated with different doses of glyphosate solutions. The three treatment groups were control (CTRL) group (with no glyphosate treatment), 0.25X group (treated with 0.217 u2009 u2009 kg · ae / ha solution of glyphosate), and 0.5X group (treated with 0.433 u2009 kg · ae / ha solution of glyphosate). Three kinds of fluorescence measurements, steady-state fluorescence spectra, Kautsky effect parameters, and ChlF-related spectral indices were extracted and generated from the measurements in the glyphosate treatment experiment. The mean values of these fluorescence measurements for each of the CTRL group, the 0.25X group, and the 0.5X group were calculated. Glyphosate-induced leaf injury was then analyzed by examining the separability of these mean values at 6, 24, 48, and 72 hours after the treatment (HAT). Results indicate that the peak position of far-red ChlF shows an obvious blue shift for glyphosate-treated soybean, and peak values of steady-state fluorescence spectra for the three groups can be significantly distinguished from each other at 48 HAT and later. Four Kautsky effect parameters, Fv, Fv/Fm, Area, and PI, are parameters sensitive to glyphosate treatment, showing some differences between the CTRL group and treated groups at 24 HAT, and significant differences among the three groups at and beyond 48 HAT. Moreover, ChlF-related spectral indices, R 683 2 / ( R 675 · R 690 ) and R 690 / R 655 , are also shown to be useful in detection of the glyphosate injury, though they are less effective than the steady-state fluorescence spectra and the Kautsky effect parameters. Based on the presented results, it can be concluded that glyphosate-induced soybean injury can be detected in a timely manner by the ChlF measurements, and this method has the potential to be further developed into practical use.