Gene amplification confers glyphosate resistance in Amaranthus palmeri

Abstract

The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F(2) populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.

Fig. 1.

Increase in genomic copy number of EPSPS correlates with reduced shikimate accumulation in 12 individuals each of glyphosate-resistant (filled circles) and -susceptible (open triangles) A. palmeri plants. Increase in genomic copy number of EPSPS is relative to ALS as measured using quantitative PCR on genomic DNA. Shikimate accumulation was measured after incubation in 250 μM glyphosate in an in vivo leaf disk assay.

Fig. 2.

EPSPS genomic copy number and glyphosate resistance cosegregate in pseudo-F₂ A. palmeri populations. EPSPS copy number relative to ALS and accumulation of shikimate were determined as described in Materials and Methods. Insets: Relative copy number histograms in pseudo-F₂ populations generated using (A) hand pollination (F₁ male parent 18 relative EPSPS copies and F₁ female parent 39 relative EPSPS copies) and (B) open pollination (parental relative copy number not measured).

Fig. 3.

Increase in EPSPS genomic relative copy number is positively correlated with increase in EPSPS cDNA expression levels in selected A. palmeri glyphosate-resistant (filled circles), -susceptible (open circles), and pseudo-F₂ individuals [pseudo-F₂ hand pollinated (open triangles), open pollinated (open squares)]. Genomic copy numbers and expression levels are relative to ALS and were determined by quantitative PCR as described in Materials and Methods.

Fig. 4.

EPSPS protein levels in glyphosate-susceptible (S), glyphosate-resistant (R), and pseudo-F₂ A. palmeri plants are correlated with relative EPSPS genomic copy number. Top: Regression of normalized EPSPS quantity on increase in relative EPSPS genomic copy number; open circles: S; filled circles: R; open triangles: F₂. Bottom: Samples with <20 relative EPSPS copies had 30 μg TSP loaded per lane, and samples with >20 relative EPSPS copies had 15 μg TSP loaded per lane. Increase in relative EPSPS genomic copy number is indicated above each sample lane.

Fig. 5.

Increased EPSPS enzyme activity is positively correlated with EPSPS relative genomic copy number in four pseudo-F₂ A. palmeri plants. Glyphosate inhibition assays were normalized for TSP quantity. Data points are means and standard errors of three replications. Filled circles: 54 relative EPSPS copies, IC₅₀ (glyphosate concentration that reduced enzyme activity by 50%) = 22 μM; open circles: 39 relative copies, IC₅₀ = 15 μM; filled triangles: 8 relative copies, IC₅₀ = 36 μM; open triangles: 1 relative copy, IC₅₀ = 66 μM.

Fig. 6.

FISH mapping in an A. palmeri glyphosate-resistant individual. (A) Somatic metaphase chromosomes of glyphosate-resistant A. palmeri. (B) FISH signals from the EPSPS gene probe. (C) Merged image from A and B. Dispersed signals can be observed on every chromosome. (D) An interphase nucleus of glyphosate-resistant A. palmeri. (E) FISH signals from the EPSPS gene probe. (F) Merged image from D and E. (Scale bars, 5 μm.)