EPSPS gene duplication in Palmer amaranth: relative fitness, inheritance, and duplication mechanism of the glyphosate resistance trait

Includes bibliographical references.2015 Summer.Glyphosate resistant (GR) Palmer amaranth (Amaranthus palmeri S. Wats.) is a weedy plant species that has invaded agricultural fields in at least 25 states, raising the cost of weed control to more than 4x the original cost. In most areas, the resistance is conferred through a gene amplification mechanism in which the target gene of glyphosate, 5-enolpyruvylshikimate-3-phosphate (EPSPS) is duplicated in the genome 100+ times, resulting in an overproduction of the EPSPS protein. With so much EPSPS enzyme available in each cell, glyphosate only inhibits a fraction of the proteins, leaving the rest to function as normal and ensuring plant survival. Understanding how this increase in EPSPS gene copy number and EPSPS protein production impacts relative fitness of the resistant plants was one objective of this research. Through greenhouse studies comparing high EPSPS copy GR plants with single copy sensitive plants, no difference was observed for any of the fitness characteristics measured. Both biotypes yielded similar numbers of offspring with no significant differences in germination or growth rate, revealing a complete lack of a fitness cost associated with the resistance trait. The second objective of this research was to quantify the stability of this resistance trait via multigenerational inheritance studies and within-plant EPSPS copy number variance measurements in the absence of glyphosate selection. The inheritance work found a complex pattern of EPSPS copy number transmission through the generations, a result that could be explained at least partially by the mosaic of EPSPS gene copy numbers patterns observed in both male and female Palmer amaranth plants. Copy numbers were inherited in a non-Mendelian pattern with transgressive segregation of the trait seen in both directions (more and fewer EPSPS copies found in the offspring than expected). This retention of high EPSPS copy number in the absence of a glyphosate selection pressure and no evidence of a fitness cost associated with the resistance trait possibly indicates a long-term loss of glyphosate as a control option in fields infested with GR Palmer amaranth. The last objective of this project was to better understand the mechanism of EPSPS gene duplication through sequence assembly of the EPSPS amplicon and chromosomal localization of this duplicated region. The amplicon was extended out to a little over 110kb and was found to contain mostly repetitive sequence including long direct repeats, microsatellites, and multiple transposable elements. A fluorescent in situ hybridization (FISH) assay found a single chromosomal location for the EPSPS genes, suggesting a tandem gene arrangement. These results further suggest that EPSPS duplication is achieved in Palmer amaranth via unequal recombination of the repeats surrounding the gene during mitosis and/or meiosis

Resistance to acetolactate synthase inhibitors is due to a W 574 to L amino acid substitution in the ALS gene of redroot pigweed and tall waterhemp.

Several Amaranthus spp. around the world have evolved resistance (and cross resistance) to various herbicide mechanisms of action. Populations of redroot pigweed (RRPW-R) and tall waterhemp (TW-R) in Mississippi, USA have been suspected to be resistant to one or more acetolactate synthase (ALS) inhibiting herbicides. Whole plant dose-response experiments with multiple ALS inhibitors, ALS enzyme assays with pyrithiobac, and molecular sequence analysis of ALS gene constructs were conducted to confirm and characterize the resistance profile and nature of the mechanism in the RRPW-R and TW-R populations. Two susceptible populations, RRPW-S and TW-S were included for comparison with RRPW-R and TW-R, correspondingly. The resistance index (R/S; the herbicide dose required to reduce plant growth by 50% of resistant population compared to the respective susceptible population) values of the RRPW-R population were 1476, 3500, and 900 for pyrithiobac, imazaquin, and trifloxysulfuron, respectively. The R/S values of the TW-R population for pyrithiobac, imazaquin, and trifloxysulfuron were 51, 950, and 2600, respectively. I50 values of RRPW-S and RRPW-R populations for pyrithiobac were 0.062 and 208.33 μM, indicating that the ALS enzyme of the RRPW-R population is 3360-fold more resistant to pyrithiobac than the RRPW-S population under our experimental conditions. The ALS enzyme of the TW-R population was 1214-fold resistant to pyrithiobac compared to the TW-S population, with the I50 values for pyrithiobac of ALS from TW-R and TW-S populations being 87.4 and 0.072 μM, correspondingly. Sequencing of the ALS gene identified a point mutation at position 574 of the ALS gene leading to substitution of tryptophan (W) residue with a leucine (L) residue in both RRPW-R and TW-R populations. Thus, the RRPW-R and TW-R populations are resistant to several ALS-inhibiting herbicides belonging to different chemical classes due to an altered target site, i.e., ALS. Resistance in Amaranthus spp. to commonly used ALS-inhibiting herbicides warrants an integrated weed management scheme incorporating chemical, mechanical, and cultural strategies by growers

Population Genetic Structure in Glyphosate-Resistant and -Susceptible Palmer Amaranth (Amaranthus palmeri) Populations Using Genotyping-by-sequencing (GBS)

Palmer amaranth (Amaranthus palmeri) is a major weed in United States cotton and soybean production systems. Originally native to the Southwest, the species has spread throughout the country. In 2004 a population of A. palmeri was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant (GR) crop systems. This project aims to determine the degree of genetic relatedness among eight different populations of GR and glyphosate-susceptible (GS) A. palmeri from various geographic regions in the United States by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimic acid accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. No isolation by distance was detected and A. palmeri was revealed to be a species with high genetic diversity. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri.USDA National Institute of Food and Agriculture, Hatch project [COL00719]; Colorado State University Libraries Open Access Research and Scholarship FundOpen access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Data from: Gene amplification of 5-enol-pyruvylshikimate-3-phosphate synthase in glyphosate-resistant Kochia scoparia

The widely used herbicide glyphosate inhibits the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Globally, the intensive use of glyphosate for weed control has selected for glyphosate resistance in 31 weed species. Populations of suspected glyphosate-resistant Kochia scoparia were collected from fields located in the US central Great Plains. Glyphosate dose response verified glyphosate resistance in nine populations. The mechanism of resistance to glyphosate was investigated using targeted sequencing, quantitative PCR, immunoblotting, and whole transcriptome de novo sequencing to characterize the sequence and expression of EPSPS. Sequence analysis showed no mutation of the EPSPS Pro106 codon in glyphosate-resistant K. scoparia, whereas EPSPS genomic copy number and transcript abundance were elevated three- to ten-fold in resistant individuals relative to susceptible individuals. Glyphosate-resistant individuals with increased relative EPSPS copy numbers had consistently lower shikimate accumulation in leaf disks treated with 100 μM glyphosate and EPSPS protein levels were higher in glyphosate-resistant individuals with increased gene copy number compared to glyphosate-susceptible individuals. RNA sequence analysis revealed seven nucleotide positions with two different expressed alleles in glyphosate-susceptible reads. However, one nucleotide at the seven positions was predominant in glyphosate-resistant sequences, suggesting that only one of two EPSPS alleles was amplified in glyphosate-resistant individuals. No alternatively spliced EPSPS transcripts were detected. Expression of five other genes in the chorismate pathway was unaffected in glyphosate-resistant individuals with increased EPSPS expression. These results indicate increased EPSPS expression is a mechanism for glyphosate resistance in these K. scoparia populations

pseudomolecule

Fasta file containing a pseudomolecule for the Kochia scoparia de novo transcriptome. The single longest transcript for each locus was concantenated into a single pseudomolecule to be used for RNA-Seq alignments