EPSPS Gene Copy Number Research Articles

Inheritance and mechanism of glyphosate resistance in annual bluegrass (Poa annua L.).

In initial screening, glyphosate was ineffective in controlling five Poa annua populations. These populations were tested for resistance, and studies undertaken to determine resistance mechanisms and inheritance pattern. Dose-response studies conducted at 16/12°C and 27/20°C on the five putative resistant populations showed low-level resistance (1.4- to 2.5-fold) to glyphosate. Shikimic acid accumulation in response to glyphosate confirmed differences among the populations, with greater shikimic acid accumulation in the susceptible population. The EPSPS gene copy number was 0.5- to 5.2-fold greater in one resistant population (HT) than in the susceptible (S) population, but not in the others. EPSPS gene expression was five- to tenfold higher in HT compared with the susceptible population. Target site mutations, differences in glyphosate absorption or translocation or altered expression of aldo-keto reductase (AKR) were not identified in any of the resistant populations. Crosses were successful between one resistant population and the susceptible population (P262-16♂ ✕ S♀) and inheritance of glyphosate resistance appears to be controlled by a single, nuclear dominant gene in this population. Our study identified EPSPS gene amplification in a South Australian glyphosate-resistant P. annua population (HT). This mechanism of resistance was not identified in the other four glyphosate-resistant populations, and other common mechanisms were excluded. Although the resistance mechanism in some P. annua populations remains unknown, inheritance studies with one population suggest the involvement of a single dominant gene. © 2021 Society of Chemical Industry.

Effect of EPSPS gene copy number and glyphosate selection on fitness of glyphosate-resistant Bassia scoparia in the field

The widespread evolution of glyphosate-resistant (GR) Bassia scoparia in the U.S. Great Plains poses a serious threat to the long-term sustainability of GR sugar beet. Glyphosate resistance in B. scoparia is due to an increase in the EPSPS (5-enolpyruvyl-shikimate-3-phosphate) gene copy number. The variation in EPSPS gene copies among individuals from within a single GR B. scoparia population indicated a differential response to glyphosate selection. With the continued use of glyphosate in GR sugar beet, the effect of increasing glyphosate rates (applied as single or sequential applications) on the fitness of GR B. scoparia individuals with variable EPSPS gene copies was tested under field conditions. The variation in EPSPS gene copy number and total glyphosate rate (single or sequential applications) did not influence any of the reproductive traits of GR B. scoparia, except seed production. Sequential applications of glyphosate with a total rate of 2214 g ae ha−1 or higher prevented seed production in B. scoparia plants with 2–4 (low levels of resistance) and 5–6 (moderate levels of resistance) EPSPS gene copies. Timely sequential applications of glyphosate (full recommended rates) can potentially slow down the evolution of GR B. scoparia with low to moderate levels of resistance (2–6 EPSPS gene copies), but any survivors (highly-resistant individuals with ≥ 8 EPSPS gene copies) need to be mechanically removed before flowering from GR sugar beet fields. This research warrants the need to adopt ecologically based, multi-tactic strategies to reduce exposure of B. scoparia to glyphosate in GR sugar beet.

Stability of EPSPS gene copy number in Hordeum glaucum Steud (barley grass) in the presence and absence of glyphosate selection.

Gene amplification has been shown to provide resistance to glyphosate in several weed species, including Hordeum glaucum populations in South Australia. The stability of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copies in resistant populations in the presence or absence of glyphosate selection has not been determined. Applying glyphosate to a cloned plant resulted in an increase in resistance and EPSPS copy number in the progeny of that plant compared to the untreated clone. The LD50 (herbicide concentration required for 50% mortality) increased by 75% to 79% in the progeny of the treated clones compared to the untreated in both populations (YP-17 and YP-16). EPSPS copy number estimates were higher in treated individuals compared to untreated individuals with an average of seven copies compared to six in YP-16 and 11 compared to six in YP-17. There was a positive correlation (R2 =0.78) between EPSPS copy number and LD50 of all populations. EPSPS gene copy number and resistance to glyphosate increased in H. glaucum populations under glyphosate selection, suggesting the number of EPSPS gene copies present is dependent on glyphosate selection. © 2021 Society of Chemical Industry.

No Impact of Increased EPSPS Gene Copy Number on Growth and Fecundity of Glyphosate-Resistant Kochia (Bassia scoparia)

AbstractThe level of glyphosate resistance in kochia [Bassia scoparia(L.) A. J. Scott] was reported to be due to an increase in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy number. A field study was conducted near Manhattan, KS, in 2014 and 2015 to evaluate the relationship between EPSPS gene copy number and growth and fecundity variables ofB. scopariaindividuals within suspected glyphosate-resistant (GR) populations from western Kansas. Initial assays of EPSPS gene copy and in vivo shikimate accumulation showed thatB. scopariapopulations from Finney (FN-R), Scott (SC-R), and Thomas (TH-R) counties were segregating for glyphosate resistance, with some individuals still being glyphosate susceptible (GS). A target-neighborhood competition approach was used to evaluate the competitive response of individual target plants with relatively low (classified as GS) and high (classified as GR) EPSPS gene copy number within the populations. There was no relationship observed between EPSPS gene copy number and vegetative or fecundity variables. There was no differential competitive response of target plant biomass to increasing neighbor density between individuals with low and high EPSPS gene copy number within each population. Lack of associated vegetative growth and fecundity cost to the increased EPSPS gene copy in the GRB. scopariaplants suggests that the plants are likely to persist in field populations, except when effective weed management strategies are adopted that would prevent their growth and seed production.

Multiple resistance to glyphosate, paraquat and ACCase-inhibiting herbicides in Italian ryegrass populations from California: confirmation and mechanisms of resistance.

Glyphosate, paraquat and acetyl CoA carboxylase (ACCase)-inhibiting herbicides are widely used in California annual and perennial cropping systems. Recently, glyphosate, paraquat, and ACCase- and acetolactate synthase (ALS)-inhibitor resistance was confirmed in several Italian ryegrass populations from the Central Valley of California. This research characterized the possible mechanisms of resistance. Multiple-resistant populations (MR1, MR2) are resistant to several herbicides from at least three modes of action. Dose-response experiments revealed that the MR1 population was 45.9-, 122.7- and 20.5-fold, and the MR2 population was 24.8-, 93.9- and 4.0-fold less susceptible to glyphosate, sethoxydim and paraquat, respectively, than the susceptible (Sus) population. Accumulation of shikimate in Sus plants was significantly greater than in MR plants 32 h after light pretreatments. Glyphosate resistance in MR plants was at least partially due to Pro106-to-Ala and Pro106-to-Thr substitutions at site 106 of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS gene copy number and expression level were similar in plants from the Sus and MR populations. An Ile1781-to-Leu substitution in ACCase gene of MR plants conferred a high level of resistance to sethoxydim and cross-resistance to other ACCase-inhibitors. Radiolabeled herbicide studies and phosphorimaging indicated that MR plants had restricted translocation of 14 C-paraquat to untreated leaves compared to Sus plants. This study shows that multiple herbicide resistance in Italian ryegrass populations in California, USA, is due to both target-site and non-target-site resistance mechanisms. © 2017 Society of Chemical Industry.

EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia.

Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number.

Genomic Stability of Palmer amaranth Plants Derived by Macro-Vegetative Propagation

qPCR (quantitative polymerase chain reaction) and random amplified polymorphic DNA (RAPD) were utilized to investigate genetic stability of Palmer amaranth cloned plants over 10 generations. DNA from original parent Palmer amaranth plants (grown from seeds) was re-analyzed using qPCR, and confidence levels for determining ΔΔCt (threshold crossing) values were established. ANOVA was used to determine variation (margin of error) of these ΔΔCt values. This margin of error was applied to qPCR analysis of DNA from eight individual parent plants and their descendants (10th generation) so that possible differences in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene copy number could be ascertained. This method (and the associated error) indicated a lack of agreement in ΔΔCt values of DNA from plants of these two generations. qPCR analysis showed that in five out of eight clones, EPSPS gene copy number varied more than the calculated error (P = 0.05). A second technique to monitor genetic stability, RAPD was used to determine possible changes in genomic DNA due to extended cloning of these regenerated plants. RAPD analysis showed that four out of the eight clones differed when the profiles of the two generations were compared. Results show that qPCR and RAPD analysis point to the fact that several Palmer amaranth clones experienced changes in genome structure over 10 generations. Although the glyphosate resistance trait was retained, results suggest that during cloning studies, the genetic stability of macro-vegetatively propagated lines should be monitored.

Stability and culture medium limitations of gene amplification in glyphosate resistant carrot cell lines

The stability of resistance to the herbicide glyphosate caused by amplification of the 5-enolpyruvylshiki-mate-3-phosphate synthase (EPSPS) gene was followed in six cloned Daucus carota (carrot) cell lines and other carrot lines grown in the absence of the selection agent for periods up to about four years. The lines originated from three different carrot suspension cultures with high levels of glyphosate resistance. The cloned lines lost resistance with a half-time of about two years. The resistance was correlated with decreased levels of EPSPS enzyme activity and EPSPS gene copy number. In some, but not all cases, glyphosate resistance was correlated with increased DNA content as measured by flow cytometry. The C1 wild type cell line, which had 2.3-times as much nuclear DNA as carrot plants, also had a correspondingly higher chromosome number, about 42 versus 18. The medium containing high glyphosate (70 mmol · L−1) was much less toxic if the K+ level was decreased, indicating that growth inhibition of the most resistant lines was caused in part by the high K+ concentration of the medium. These studies show that the selected glyphosate resistance was unstable and was lost slowly when grown away from the selection agent and that high K+ concentrations limited growth of resistant lines at high glyphosate concentrations.