Academic literature on the topic 'EPSPS gene amplification'

Waterhemp is an increasingly problematic weed in the U.S. Midwest, having now evolved resistances to herbicides from six different site-of-action groups. Glyphosate-resistant waterhemp in the Midwest is especially concerning given the economic importance of glyphosate in corn and soybean production. Amplification of the target-site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was found to be the mechanism of glyphosate resistance in Palmer amaranth, a species closely related to waterhemp. Here, the relationship between glyphosate resistance and EPSPS gene amplification in waterhemp was investigated. Glyphosate dose response studies were performed at field sites with glyphosate-resistant waterhemp in Illinois, Kansas, Kentucky, Missouri, and Nebraska, and relative EPSPS copy number of survivors was determined via quantitative real-time polymerase chain reaction (qPCR). Waterhemp control increased with increasing glyphosate rate at all locations, but no population was completely controlled even at the highest rate (3,360 g ae ha−1). EPSPS gene amplification was present in plants from four of five locations (Illinois, Kansas, Missouri, and Nebraska) and the proportion of plants with elevated copy number was generally higher in survivors from glyphosate-treated plots than in plants from the untreated control plots. Copy number magnitude varied by site, but an overall trend of increasing copy number with increasing rate was observed in populations with gene amplification, suggesting that waterhemp plants with more EPSPS copies are more resistant. Survivors from the Kentucky population did not have elevated EPSPS copy number. Instead, resistance in this population was attributed to the EPSPS Pro106Ser mutation. Results herein show a quantitative relationship between glyphosate resistance and EPSPS gene amplification in some waterhemp populations, while highlighting that other mechanisms also confer glyphosate resistance in waterhemp.

With the frequency of glyphosate-resistant waterhemp increasing throughout the Midwest, the identification of resistant populations has become important for managing this species. However, high-throughput screening for glyphosate resistance in the greenhouse is tedious and inefficient. Research was conducted to document the occurrence of glyphosate-resistant waterhemp throughout the state of Illinois, and to determine whether a molecular assay for 5-enolypyruvyl-shikimate-3-phosphate synthase (EPSPS) gene amplification can be used as an alternative means to detect resistant populations. Populations throughout the state of Illinois were collected in 2010 and screened for glyphosate resistance using a whole-plant assay in a greenhouse, and survivors were examined for EPSPS gene amplification. Of 80 populations investigated, 22 were glyphosate resistant based on the greenhouse screen, and gene amplification was identified in 20 (91%) of the resistant populations. Although there are multiple mechanisms for glyphosate resistance in waterhemp, a molecular test for EPSPS gene amplification provides a rapid alternative for identification of glyphosate resistance in most populations.

In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in <i>Amaranthus palmeri</i> (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which <i>5-enolpyruvylshikimate-3-phosphate synthase</i> (<i>EPSPS</i>) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to &#x3e;160-fold increase in copies of the <i>EPSPS</i> gene than in a glyphosate-susceptible (GS) population. This increased copy number of the <i>EPSPS</i> gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb <i>EPSPS</i> cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified <i>EPSPS</i> copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The <i>EPSPS</i> gene-containing eccDNA having a size of ∼400 kb is termed <i>EPSPS</i>-eccDNA and showed somatic mosacism in size and copy number. <i>EPSPS</i>-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the <i>EPSPS</i> locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of <i>EPSPS</i>-eccDNA sheds light on various characteristics of <i>EPSPS</i>-eccDNA that favor GR in AP.

Glyphosate, a nonselective herbicide and also the world's most widely used herbicide, inhibits 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme in the aromatic amino acid biosynthetic pathway. Because of its broad-spectrum and potent weed control and favorable environmental characteristics, attempts to engineer glyphosate resistance have been intensive in the past few decades. The use of at least three different mechanisms has conferred glyphosate resistance in normally sensitive crop species. Early work focused on progressive adaptation of cultured plant cells to stepwise increases in glyphosate concentrations. The resulting cells were resistant to glyphosate because of EPSPS overexpression, EPSPS gene amplification, or increased enzyme stability. Further work aimed to achieve resistance by transforming plants with glyphosate metabolism genes. An enzyme from a soil microorganism, glyphosate oxidoreductase (GOX), cleaves the nitrogen– carbon bond in glyphosate yielding aminomethylphosphonic acid. Another metabolism gene, glyphosateN-acetyl transferase (gat), acetylates and deactivates glyphosate. A third mechanism, and the one found in all currently commercial glyphosate-resistant crops, is the insertion of a glyphosate-resistant form of the EPSPS enzyme. Several researchers have used site-directed mutagenesis or amino acid substitutions of EPSPS. However, the most glyphosate-resistant EPSPS enzyme to date has been isolated fromAgrobacteriumspp. strain CP4 and gives high levels of resistance in planta. Weeds resistant to glyphosate have offered further physiological mechanisms for glyphosate resistance. Resistant field bindweed had higher levels of 3-deoxy-d-arbino-heptulosonate 7-phosphate synthase, the first enzyme in the shikimate pathway, suggesting that increased carbon flow through the shikimate pathway can provide glyphosate resistance. Resistant goosegrass has reduced translocation of glyphosate out of the treated area. Although glyphosate resistance has been achieved by numerous mechanisms, currently the only independent physiological mechanism to give adequate and stable resistance to glyphosate for commercialization of glyphosate-resistant crops has been glyphosate-resistant forms of EPSPS.

The first step in managing herbicide-resistant weeds is to confirm their resistance status. It is, therefore, crucial to have a rapid, reliable and cost-effective technique to assess samples for herbicide resistance. We designed and evaluated three derived cleaved amplified polymorphic sequence (dCAPS) markers for detecting glyphosate resistance in Lolium perenne. conferred by non-synonymous mutations at codon-106 in the enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. The dCAPS markers involve amplification of the target region, digestion of the amplified products with restriction enzymes and gel-based visualisation of the digested products. The results showed that all three dCAPS markers could successfully detect mutations at codon-106 in the target enzyme. The dCAPS markers can also inform us of the zygosity state of the resistance allele and was confirmed by sequencing the target region of the EPSPS gene. The markers described here are effective quick tests for the monitoring and evaluation of the target-enzyme mechanism of glyphosate resistance in Lolium perenne.

Master of Science
Department of Agronomy
Randall S. Currie
Mithila Jugulam
Italian ryegrass (Lolium perenne L. ssp. multiflorum (Lam.) Husnot), one of the problem weeds of the US, evolved resistance to multiple herbicides including glyphosate due to selection in Arkansas (AR). Glyphosate is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor and amplification of EPSPS gene, the molecular target of glyphosate confers resistance to this herbicide in several weed species, including Italian ryegrass from AR. The objective of this study was to determine the expression of EPSPS gene and protein as well as distribution of EPSPS copies on the genome of glyphosate-resistant Italian ryegrass (ARR) using a known susceptible Italian ryegrass (ARS) from AR. EPSPS gene copies and expression of ARR and ARS were determined using quantitative PCR with appropriate endogenous controls. EPSPS protein expression was determined using Western blot analysis. Fluorescence in situ hybridization (FISH) was performed on somatic metaphase chromosomes to determine the location of EPSPS copies. Based on qPCR analysis, ARR plants showed a wide range of 12 to 118 EPSPS copies compared to a single copy in ARS. EPSPS gene expression correlated with the gene copy number in both ARR and ARS. Individuals with high EPSPS copies showed high protein expression in Western blot analysis. FISH analysis showed presence of brighter EPSPS signals, distributed randomly throughout the genome of ARR individuals compared to a faint signal in ARS plants. Random distribution of EPSPS copies was previously reported in glyphosate-resistant Palmer amaranth. Overall, the results of this study will help understand the origin and mechanism of EPSPS gene amplification in Italian ryegrass.

Master of Science
Agronomy
Mithila Jugulam
Common waterhemp (Amaranthus rudis) is a problematic weed species of cropping systems throughout the Midwestern states, including Kansas. Recently, waterhemp populations from Kansas were found to have evolved resistance to the widely used herbicide glyphosate as a result of amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzyme target of glyphosate. The objectives of this research were to 1) perform glyphosate dose-response study and determine the relationship between relative EPSPS genomic copies and EPSPS gene expression in glyphosate-resistant waterhemp, and 2) characterize the genomic configuration and distribution of EPSPS copies using florescence in situ hybridization (FISH) in three glyphosate-resistant waterhemp populations. Waterhemp populations from eastern Kansas were screened with 868 g ae haˉ¹ (field used rate) of glyphosate, and genomic DNA and total RNA was isolated from the survivors to determine the EPSPS genomic copies and EPSPS gene expression relative to the acetolactate synthase (ALS) gene using qPCR. Furthermore, waterhemp specific EPSPS probes were synthesized to perform florescence in situ hybridization (FISH) on these glyphosate-resistant plants. Results of these experiments indicate a positive correlation between level of glyphosate resistance, EPSPS copies, and their expression. As expected, a negative correlation was found between shikimate accumulation and EPSPS copies. Sequencing of the EPSPS gene showed no presence of the proline 106 mutation, which is known to be associated with glyphosate resistance suggesting that an insensitive EPSPS enzyme was not involved in the mechanism of glyphosate resistance. FISH analysis of resistant plants illustrated presence of amplified EPSPS copies on two homologous chromosomes, likely near the centromeric region. . This is the first report demonstrating a positive relationship between EPSPS copies and expressions, as well as chromosome configuration of EPSPS copies in glyphosate- resistant waterhemp from Kansas.

A planta daninha Amaranthus palmeri é nativa dos Estados Unidos, porém foi pela primeira vez relatada no Brasil no ano de 2015. Embora comprovadamente com resistência múltipla aos herbicidas inibidores da ALS e da EPSPS, até o momento não foram investigadas as bases moleculares da resistência. Além disso, por causa da recente introdução da planta daninha no país, alternativas de manejo com culturas tolerantes a herbicidas necessitam ser estudadas. Sendo assim, os objetivos desse trabalho são de caracterizar a espécie de planta daninha introduzida no país, identificar os mecanismos de resistência aos herbicidas inibidores da ALS e da EPSPS presentes no biótipo, e propor abordagens de manejo em ambientes dos novos eventos transgênicos resistentes a herbicidas. Um bioensaio utilizando marcadores genéticos foi desenvolvido para confirmar que a população coletada no estado do Mato Grosso (BR-R) é A. palmeri, e não A. tuberculatus, outra espécie dióica do gênero Amaranthus. Os resultados de experimentos de curvas de dose-resposta e acúmulo de chiquimato indicaram que a BR-R possui alto nível de resistência, com DL50 de 4.426 e 3.400 g glyphosate ha-1 no primeiro e segundo experimento, respectivamente, mais que o dobro da dose típicamente recomendada para o controle da espécie e, adicionalmente, observou se acúmulo mínimo de chiquimato a concentração de 1 mM nos tecidos das plantas tratadas com o herbicida. BR-R também foi resistente a herbicidas dos grupos químicos das sulfoniluréias e imidazolinonas. O mecanismo de resistência ao glyphosate encontrado nesta população foi a super expressão gência, através do aumento no número de cópias do gene da EPSPS no genoma da planta BR-R, entre 50 e 179 cópias adicionais. Além disso, duas substituições de aminoácidos foram observadas na sequência da ALS, W574L e S653N, conferindo resistência tanto a sulfoniluréias quanto a imidazolinonas. No experimento utilizandos os herbicidas correspondentes às culturas geneticamente modificadas com novos traits de tolerância a herbicidas observou se, de uma forma geral, que as associações de herbicidas apresentaram níveis de controle mais satisfatórios. Assim, esta pesquisa confirma a introdução de da espécie A. palmeri no Brasil, assim como a resistência múltipla aos herbicidas inibidores da EPSPS e da ALS. Seu manejo é mais eficaz através da associação de herbicidas, garantindo assim o uso racional das novas tecnologias de culturas geneticamente modificadas com tolerância a herbicidas.
Palmer Amaranth (Amaranthus palmeri) is a weed species native to the United States, but it was reported in Brazil for the first time in 2015. Despite this population being resistant to EPSPS and ALS inhibitors, the molecular basis of its multiple resistance is unknown up to date. Because of this species introduction to Brazil, alternatives of management with the new herbicide-tolerant crops technologies need to be studied. The objectives of this research are to characterize the weed species introduced to Brazil, identify the mechanisms conferring resistance to ALS and EPSPS inhibitors herbicides, and to propose management approaches in environments with the new genetically modified herbicide-tolerant crops. A genotyping bioassay using genetic markers was developed to confirm that the species collected in the state of Mato Grosso (BR-R) is indeed A. palmeri and not A. tuberculatus, another dioceous species in the Amaranthus genus. Dose-response experiments and shikimate accumulation bioassay data indicate high level of resistance, with LD50 of 4,426 and 3,400 g glyphosate ha-1 in the first and second experiments, respectively, higher than the double rate tipically recommended to control it, and minimal accumulation in BR-R with 1 mM of glyphosate in treated plants in the leaf disks assay. BR-R also was resistanto to sulfonilurea and imidazolinone herbicides. The mechanism conferring resistance to glyphosate identified in this population was gene amplification, with increased EPSPS copy number - between 50 and 179 more copies in BR-R. Besides, two target-site mutations were identified in the ALS gene sequencing, W574L and S653N, conferring resistance to sulfonilureas and imidazolinones. The weed control experiment, overal, herbicide tank mixtures achieved higher levels of control. Therefore, this research confirms the introduction of A. palmeri to Brazil, as well as its multiple resistance to EPSPS and ALS inhibitor herbicides. Its control is more efficient with herbicide mixtures, which guarantees more susteinable use of the new herbicide-tolerant crop technologies.

Chloris truncata and C. virgata, which are major weeds in cotton and grain crops in the sub-tropical region of Australia, have recently emerged as potential weeds of the future in southern Australia. Glyphosate, an inhibitor of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS), is the most widely used non-selective post-emergence herbicide globally. As a result of over-reliance on glyphosate combined with dominance of reduced tillage systems for weed control, glyphosate-resistant populations of C. truncata have already been reported in Australia. C. virgata is also considered hard to kill with glyphosate, but resistance has not been reported so far in the literature. Studies on growth, development and seed biology of C. truncata and C. virgata were conducted to better understand the biology of these emerging weed species. Under field conditions, C. truncata and C. virgata required 748-786 degree-days (Cd) and 1200 Cd respectively to progress from emergence to mature seed production. Freshly produced seeds of C. virgata were dormant for about 2 months, whereas 16-40% of seeds of C. truncata germinated within a week after maturation. Seed dormancy of C. virgata was released by the pre-treatment with 564 mM NaClO for 30 minutes. Exposure to light significantly increased germination of C. truncata seed from 0-2% in the dark to 77-84% in the light, and of C. virgata seed from 2-35% in the dark to 72- 85% in the light. Seeds of these two species could germinate over a wide temperature range (10-40oC), with maximum germination at 20-25oC for C. truncata and 15-25oC for C. virgata. The predicted base temperature for germination was 9.2-11.2oC for C. truncata and much lower 2.1-3.0oC for C. virgata. Seedling emergence of C. virgata (76% for seeds present on soil surface) was significantly reduced by burial at 1 (57%), 2 (49%) and 5 cm (9%), whereas seedling emergence of C. truncata was completely inhibited by burial of seed even at a shallow depth (0.5 cm). Under field conditions, both C. truncata and C. virgata seeds persisted in the soil for at least 11 months and seasons with below-average spring-summer rainfall increased seed persistence. Detailed studies were undertaken to identify glyphosate-resistant populations and to understand the mechanism of glyphosate resistance in C. truncata and C. virgata. Glyphosate resistance (GR) was confirmed in five populations of C. truncata and four populations of C. virgata. GR plants were 2.4 to 8.7-fold (C. truncata) and 2 to 9.7-fold (C. virgata) more resistant and accumulated less shikimate after glyphosate treatment than susceptible (S) plants. The differences in shikimate accumulation indicated that glyphosate did reach the target site but inhibited the EPSPS enzyme of each population differently. Glyphosate absorption and translocation did not differ between GR and S plants of either C. truncata or C. virgata. Two target-site EPSPS mutations (Pro-106-Leu and Pro-106-Ser) were likely to be the primary mechanism of glyphosate resistance in C. virgata but no previously known target-site mutations were identified in C. truncata. The C. virgata population with Pro-106-Leu substitution was 2.9 to 4.9-fold more resistant than those with Pro-106-Ser substitution. The primary mechanism of resistance to glyphosate in C. truncata was a combination of target-site EPSPS mutation (Glu-91-Ala) and amplification of the EPSPS gene. There were 16 to 48-fold more copies of the EPSPS gene in GR plants compared to S plants, with the number of EPSPS copies found to be variable both between and within populations.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2017