Gene amplification confers glyphosate resistance in Amaranthus palmeri

doi:10.1073/pnas.0906649107

Gene amplification confers glyphosate resistance in Amaranthus palmeri

^aDepartment of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523
^bDepartment of Horticulture, University of Wisconsin, Madison, WI 53706
^cMonsanto Company, St. Louis, MO 63167
^dWater Management Research Unit, US Department of Agriculture Agricultural Research Service (USDA-ARS), Fort Collins, CO 80526
^eDepartment of Crop Sciences, University of Illinois, Urbana, IL 61801
^fCrop and Soil Science Department, University of Georgia, Tifton, GA 31794
^gCrop Protection and Management Research Unit, USDA-ARS, Tifton, GA 31794
^hCrop and Soil Science Department, University of Georgia, Athens, GA 30602
ⁱSchool of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia

↵1 Present address: Western Australian Herbicide Resistance Initiative, School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia.

Edited by Charles J. Arntzen, Arizona State University, Tempe, AZ, and approved October 29, 2009 (received for review June 16, 2009)

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-F₂ 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.

Footnotes

²To whom correspondence should be addressed at: Department of Bioagricultural Sciences and Pest Management, 1177 Campus Delivery, Colorado State University, Fort Collins, CO 80523. E-mail: philip.westra{at}colostate.edu.
Author contributions: T.A.G., S.T.C., S.J.N., J.J., C.P., J.E.L., and P.W. designed research; T.A.G., W.Z., D.W., and B.B. performed research; D.L.S., W.L.P., P.J.T., A.S.C., T.L.G., T.M.W., W.K.V., and R.D.S. contributed new reagents/analytic tools; T.A.G. analyzed data; and T.A.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0910081107/DCSupplemental.