Biochemical and Rapid Molecular Analyses to Identify Glyphosate Resistance in Lolium spp.

Lolium spp. are troublesome weeds mainly found in winter cereal crops worldwide, including Europe. In recent years resistant mechanisms have been evolved to several important herbicides. In this study we investigated the mechanisms responsible for conferring glyphosate resistance in some Lolium spp. populations. A holistic approach was used, based on dose-response experiments, determination of shikimic acid concentration in plant leaf tissue, as well as molecular analyses. More specifically, in three Lolium spp. populations the existence of a mutation in the Pro-106 codon of the 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) gene was investigated as well as the relative transcript levels of four ABC-transporter genes were monitored at three time points after glyphosate application. The results demonstrated that glyphosate resistance is a multifactor phenomenon. Relative transcript levels of the ABC-transporter genes were abundant at very early time points after glyphosate treatments. Dose-response experiments and shikimate analyses were in accordance with the findings of the quantitative PCR (qPCR) analyses. We suggest that relative expression ratio of ABC-transporter genes can be a useful tool to rapidly identify Lolium spp. populations resistant to glyphosate.

Sequence characterization of eccDNA content in glyphosate sensitive and resistant Palmer amaranth from geographically distant populations

The discovery of non-chromosomal circular DNA offers new directions in linking genome structure with function in plant biology. Glyphosate resistance through EPSPS gene copy amplification in Palmer amaranth was due to an autonomously replicating extra-chromosomal circular DNA mechanism (eccDNA). CIDER-Seq analysis of geographically distant glyphosate sensitive (GS) and resistant (GR) Palmer Amaranth ( Amaranthus palmeri ) revealed the presence of numerous small extra-chromosomal circular DNAs varying in size and with degrees of repetitive content, coding sequence, and motifs associated with autonomous replication. In GS biotypes, only a small portion of these aligned to the 399 kb eccDNA replicon, the vehicle underlying gene amplification and genetic resistance to the herbicide glyphosate. The aligned eccDNAs from GS were separated from one another by large gaps in sequence. In GR biotypes, the eccDNAs were present in both abundance and diversity to assemble into a nearly complete eccDNA replicon. Mean sizes of eccDNAs were similar in both biotypes and were around 5kb with larger eccDNAs near 25kb. Gene content for eccDNAs ranged from 0 to 3 with functions that include ribosomal proteins, transport, metabolism, and general stress response genetic elements. Repeat content among smaller eccDNAs indicate a potential for recombination into larger structures. Genomic hotspots were also identified in the Palmer amaranth genome with a disposition for gene focal amplifications as eccDNA. The presence of eccDNA may serve as a reservoir of genetic heterogeneity in this species and may be functionally important for survival.

Development of non-transgenic glyphosate tolerant wheat by TILLING

Glyphosate (N-phosphonomethyl-glycine) is the world’s most widely used broad spectrum, post-emergence herbicide. It inhibits the chloroplast-targeted enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), a component of the plant and microorganism-specific shikimate pathway and a key catalyst in the production of aromatic amino acids. Variants of EPSPS that are not inhibited by glyphosate due to particular amino acid alterations in the active site of the enzyme are known. Some of these variants have been identified in weed species that have developed resistance to glyphosate because of the strong selective pressure of continuous, heavy glyphosate use. We have used TILLING (Targeting Induced Local Lesions in Genomes), a non-transgenic, target-selected, reverse genetics, mutation breeding technique, and conventional genetic crosses, to identify and combine, through two rounds of mutagenesis, wheat lines having both T102I and P106S (so-called TIPS enzyme) mutations in both the A and the D sub-genome homoeologous copies of the wheat EPSPS gene. The combined effects of the T102I and P106S mutations are known from previous work in multiple species to minimize the binding of the herbicide while maintaining the affinity of the catalytic site for its native substrates. These novel wheat lines exhibit substantial tolerance to commercially relevant levels of glyphosate.

The First Report of Target-Site Resistance to Glyphosate in Sweet Summer Grass (Moorochloa eruciformis)

Sweet summer grass is a problematic weed in the central Queensland region of Australia. This study found glyphosate resistance in two biotypes (R1 and R2) of sweet summer grass. The level of resistance in these biotypes was greater than 8-fold. The glyphosate dose required to reduce dry matter by 50% (GR50) for the resistant populations varied from 1993 to 2100 g ha−1. A novel glyphosate resistance double point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was identified for the first time in sweet summer grass. Multiple mutations, including multiple amino acid changes at the glyphosate target site, as well as mutations involving two nucleotide changes at a single amino acid codon, were observed. Both resistant biotypes exhibited a nucleotide change of CAA to ACA in codon 106, which predicts an amino acid change of proline to a threonine (Pro-106-Thr). In addition, the R1 biotype also possessed a mutation at codon 100, where a nucleotide substitution of T for G occurred (GCT to TCT), resulting in a substitution of serine for alanine (Ala-100-Ser). Understanding the molecular mechanism of glyphosate resistance will help to design effective management strategies to control invasive weeds.

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.

Fitness Cost Associated With Enhanced EPSPS Gene Copy Number and Glyphosate Resistance in an Amaranthus tuberculatus Population

The evolution of resistance to pesticides in agricultural systems provides an opportunity to study the fitness costs and benefits of novel adaptive traits. Here, we studied a population of Amaranthus tuberculatus (common waterhemp), which has evolved resistance to glyphosate. The growth and fitness of seed families with contrasting levels of glyphosate resistance was assessed in the absence of glyphosate to determine their ability to compete for resources under intra- and interspecific competition. We identified a positive correlation between the level of glyphosate resistance and gene copy number for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) glyphosate target, thus identifying gene amplification as the mechanism of resistance within the population. Resistant A. tuberculatus plants were found to have a lower competitive response when compared to the susceptible phenotypes with 2.76 glyphosate resistant plants being required to have an equal competitive effect as a single susceptible plant. A growth trade-off was associated with the gene amplification mechanism under intra-phenotypic competition where 20 extra gene copies were associated with a 26.5 % reduction in dry biomass. Interestingly, this growth trade-off was mitigated when assessed under interspecific competition from maize.

Gene Flow Risks From Transgenic Herbicide-Tolerant Crops to Their Wild Relatives Can Be Mitigated by Utilizing Alien Chromosomes

Integration of a transgene into chromosomes of the C-genomes of oilseed rape (AACC, 2n = 38) may affect their gene flow to wild relatives, particularly Brassica juncea (AABB, 2n = 36). However, no empiric evidence exists in favor of the C-genome as a safer candidate for transformation. In the presence of herbicide selections, the first- to fourth-generation progenies of a B. juncea × glyphosate-tolerant oilseed rape cross [EPSPS gene insertion in the A-genome (Roundup Ready, event RT73)] showed more fitness than a B. juncea × glufosinate-tolerant oilseed rape cross [PAT gene insertion in the C-genome (Liberty Link, event HCN28)]. Karyotyping and fluorescence in situ hybridization–bacterial artificial chromosome (BAC-FISH) analyses showed that crossed progenies from the cultivars with transgenes located on either A- or C- chromosome were mixoploids, and their genomes converged over four generations to 2n = 36 (AABB) and 2n = 37 (AABB + C), respectively. Chromosome pairing of pollen mother cells was more irregular in the progenies from cultivar whose transgene located on C- than on A-chromosome, and the latter lost their C-genome-specific markers faster. Thus, transgene insertion into the different genomes of B. napus affects introgression under herbicide selection. This suggests that gene flow from transgenic crops to wild relatives could be mitigated by breeding transgenic allopolyploid crops, where the transgene is inserted into an alien chromosome.

Mechanism(s) of Glyphosate Resistance in a Selected Plantago lanceolata (L.) R Biotype

In 2003, a glyphosate-resistant plantago (Plantago lanceolata L.) population located in the Robertson district of South Africa was subjected to different glyphosate dosages and the highest dosage (7200 g a.e. ha−1) gave no acceptable levels of control. Here we reconfirm resistance and investigate the mechanism of glyphosate resistance. Dose-response curves indicated that the glyphosate dosage rate causing 50% survival (LD50) for the resistant (R) biotype is 43 times greater than for the susceptible (S) biotype, i.e., 43-fold resistant to glyphosate. Investigation into the molecular mechanism of plantago showed shikimate accumulation of the R biotype was lower than that of the S biotype. The reported 31P and 13C nuclear magnetic resonance (NMR) spectra show rapid glyphosate translocation into the young untreated leaves of the S biotype. No glyphosate translocation was observed in the R biotype. A point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, resulting in an amino acid substitution was also observed, indicating two unique glyphosate resistance mechanisms within the R biotype. The rapid evolution of glyphosate-resistant weeds threatens the usage of the world’s most important herbicide (glyphosate), which is essential in world food production and further limits grower options for weed control. New weed management strategies will be necessary to combat plantago R biotypes.