Philip Westra

Gene amplification confers glyphosate resistance in Amaranthus palmeri

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.

Mechanism of Resistance of Evolved Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri)

Evolved glyphosate resistance in weedy species represents a challenge for the continued success and utility of glyphosate-resistant crops. Glyphosate functions by inhibiting the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The resistance mechanism was determined in a population of glyphosate-resistant Palmer amaranth from Georgia (U.S.). Within this population, glyphosate resistance correlates with increases in (a) genomic copy number of EPSPS, (b) expression of the EPSPS transcript, (c) EPSPS protein level, and (d) EPSPS enzymatic activity. Dose response results from the resistant and an F(2) population suggest that between 30 and 50 EPSPS genomic copies are necessary to survive glyphosate rates between 0.5 and 1.0 kg ha(-1). These results further confirm the role of EPSPS gene amplification in conferring glyphosate resistance in this population of Palmer amaranth. Questions remain related to how the EPSPS amplification initially occurred and the occurrence of this mechanism in other Palmer amaranth populations and other glyphosate-resistant species.

Inheritance of Resistance to The Auxinic Herbicide Dicamba in Kochia (Kochia scoparia)

Abstract The inheritance of resistance to the auxinic herbicide dicamba was examined in a kochia population from Nebraska. An inbred, resistant line was developed by selection and selfing over seven generations to ensure any resistance alleles would be homozygous in the parents. An inbred, susceptible line was similarly developed, but without selection. Dose–response experiments with dicamba determined a glyphosate-resistant concentration required to inhibit dry weight accumulation by 50% (GR50) of 45 and 1,331 g ae ha−1 for the susceptible and resistant populations, respectively. F1 crosses were made between resistant and susceptible inbred individuals by hand-pollination, and the F1 plants were selfed to produce F2 plants. The F2 population was screened with 280 g ha−1 dicamba, a rate that could discriminate between susceptible and resistant plants. A total of eight F2 families were screened twice. In the first screen, seven F2 families segregated in a 3:1 ratio, consistent with a single dominant allele controlling resistance, and in the second screen six F2 families segregated in a 3:1 ratio. F2 individuals were selfed, the F3 progeny were tested with 280 g ha−1 dicamba, and the genotype of each F2 parent was determined based on F3 progeny segregation. F3 family segregation was consistent with the F2 parents having a 1:2:1 homozygous-susceptible:heterozygote:homozygous-resistant pattern, confirming that resistance to dicamba in kochia is likely conferred by a single allele with a high degree of dominance. Nomenclature: Dicamba, kochia, Kochia scoparia (L.) Schrad. KCHSC

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.

Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Genes encoding enolpyruvylshikimate phosphate synthase are tandemly arranged on chromosomes of field-evolved glyphosate-resistant Kochia scoparia. Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

Impact of Genetic Background in Fitness Cost Studies: An Example from Glyphosate-Resistant Palmer Amaranth

Abstract Since its discovery in 2005, glyphosate-resistant Palmer amaranth has become a major problem for many farmers in the southern United States. One mechanism of resistance found in a Georgia population of glyphosate-resistant Palmer amaranth is amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene throughout the genome, with some resistant plants containing and expressing more than 100 EPSPS genes. Such high numbers of EPSPS genes and protein production could result in a fitness cost to resistant plants due to (1) metabolic cost of overproduction of this enzyme and (2) disruption of other genes after insertion of the EPSPS gene. A greenhouse experiment was set up to investigate differences in growth and reproduction between glyphosate-susceptible and -resistant Palmer amaranth plants. Measurements included growth rate, plant height/volume ratio, final biomass, photosynthetic rate, inflorescence length, pollen viability, and seed set. This study found no significant fitness costs for plants with the resistance trait. This study also provided a clear example of how controlling for genetic background is important in fitness cost studies and how potentially misleading results can be obtained if only a few fitness traits are measured. These results indicate that glyphosate-resistant Palmer amaranth plants with high EPSPS gene copy numbers are likely to persist in field populations, even in the absence of glyphosate, potentially leading to long-term loss of glyphosate as a control option for Palmer amaranth. Nomenclature: Glyphosate; Palmer amaranth, Amaranthus palmeri S. Wats AMAPA.

Characterization of glyphosate resistance in Amaranthus tuberculatus populations.

The evolution of glyphosate-resistant weeds has recently increased dramatically. Six suspected glyphosate-resistant Amaranthus tuberculatus populations were studied to confirm resistance and determine the resistance mechanism. Resistance was confirmed in greenhouse for all six populations with glyphosate resistance factors (R/S) between 5.2 and 7.5. No difference in glyphosate absorption or translocation was observed between resistant and susceptible individuals. No mutation at amino acid positions G101, T102, or P106 was detected in the EPSPS gene coding sequence, the target enzyme of glyphosate. Analysis of EPSPS gene copy number revealed that all glyphosate-resistant populations possessed increased EPSPS gene copy number, and this correlated with increased expression at both RNA and protein levels. EPSPS Vmax and Kcat values were more than doubled in resistant plants, indicating higher levels of catalytically active expressed EPSPS protein. EPSPS gene amplification is the main mechanism contributing to glyphosate resistance in the A. tuberculatus populations analyzed.

Interspecific hybridization transfers a previously unknown glyphosate resistance mechanism in Amaranthus species

A previously unknown glyphosate resistance mechanism, amplification of the 5‐enolpyruvyl shikimate‐3‐phosphate synthase gene, was recently reported in Amaranthus palmeri. This evolved mechanism could introgress to other weedy Amaranthus species through interspecific hybridization, representing an avenue for acquisition of a novel adaptive trait. The objective of this study was to evaluate the potential for this glyphosate resistance trait to transfer via pollen from A. palmeri to five other weedy Amaranthus species (Amaranthus hybridus, Amaranthus powellii, Amaranthus retroflexus, Amaranthus spinosus, and Amaranthus tuberculatus). Field and greenhouse crosses were conducted using glyphosate‐resistant male A. palmeri as pollen donors and the other Amaranthus species as pollen recipients. Hybridization between A. palmeri and A. spinosus occurred with frequencies in the field studies ranging from <0.01% to 0.4%, and 1.4% in greenhouse crosses. A majority of the A. spinosus × A. palmeri hybrids grown to flowering were monoecious and produced viable seed. Hybridization occurred in the field study between A. palmeri and A. tuberculatus (<0.2%), and between A. palmeri and A. hybridus (<0.01%). This is the first documentation of hybridization between A. palmeri and both A. spinosus and A. hybridus.

Genetic diversity of jointed goatgrass (Aegilops cylindrica) determined with RAPD and AFLP markers.

Abstract Two DNA molecular marker techniques were used to evaluate genetic diversity in 58 accessions of jointed goatgrass and 6 accessions of the related wild species barb goatgrass. Random amplified polymorphic DNA (RAPD) assays were performed on 8 U.S. and 50 Eurasian jointed goatgrass accessions using 30 random decamer primers. The frequency of scorable polymorphic bands within jointed goatgrass was 6 out of 90 (6.7%). Cluster analysis of RAPD data showed small genetic distances (values of 0.005 or less) among jointed goatgrass accessions. To validate the effectiveness of RAPD techniques to detect genetic diversity in tetraploid Aegilops species, six accessions of barb goatgrass were assayed using a subset of 20 decamer primers (from the original 30). RAPD data for barb goatgrass were pooled with jointed goatgrass data from the same primers. A total of 63 scorable bands were generated, of which 27 (43%) were polymorphic between two or more accessions. RAPD analysis readily distinguished between the two species and detected much greater levels of genetic diversity within barb goatgrass than between the jointed goatgrass accessions. Amplified fragment length polymorphism (AFLP) assays were performed on a subset of the 58 jointed goatgrass accessions, 3 U.S. and 13 Eurasian. These accessions were selected to represent a range in geographic diversity within our collection. Ten primer combinations generated 560 scorable bands of which 28 (5%) were polymorphic. Cluster analysis of AFLP data showed a slightly smaller range in genetic distance (0.0002 to 0.0022) among accessions compared with RAPD results; however, AFLPs distinguished among all but 2 of the 16 accessions surveyed. Although AFLP produced more scorable bands than RAPD did, both methods revealed limited genetic diversity in jointed goatgrass. Nomenclature: Jointed goatgrass, Aegilops cylindrica L. AEGCY; barb goatgrass, Aegilops triuncialis L. AEGTR.

Above winter wheat

Above, a hard red winter wheat (Triticum aestivum L. em. Thell.), is adapted for dryland production in the west central Great Plains of the United States. It carries a nontransgenic source of tolerance to imidazolinone herbicides derived by mutation induction with sodium azide. Above was developed cooperatively by the Colorado and Texas Agricultural Experiment Stations and released to seed producers in September 2001. Key words: Triticum aestivum, wheat (winter), cultivar description, herbicide tolerance