EPSPSGene Amplification Primarily Confers Glyphosate Resistance among Arkansas Palmer amaranth (Amaranthus palmeri) Populations

AbstractResearch was conducted to determine whether resistance to glyphosate among Palmer amaranth (Amaranthus palmeriS. Watson) populations within the U.S. state of Arkansas was due solely to increasedEPSPSgene copy number and whether gene copy number is correlated with resistance level to glyphosate. One hundred and fifteenA. palmeriaccessions were treated with 840 g ae ha−1glyphosate. Twenty of these accessions, selected to represent a broad range of responses to glyphosate, underwent further testing. Seven of the accessions were controlled with this dose; the rest were resistant. The effective dose to cause 50% injury (ED50) for susceptible accessions ranged from 28 to 207 g ha−1. The glyphosate-resistant (GR) accessions had ED50values ranging from 494 to 1,355 g ha−1, a 3- to 48-fold resistance level compared with the susceptible standard (SS). The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene relative copy number was determined for 20 accessions, 4 plants accession−1. Resistant plants from five GR accessions (38% of resistant plants tested) did not have increasedEPSPSgene copies. Resistant plants from the remaining eight GR accessions (62% of resistant plants tested) had 19 to 224 moreEPSPSgene copies than the SS. Among the accessions tested, injury declined 4% with every additionalEPSPScopy. ED50values were directly correlated withEPSPScopy number. The highly resistant accession MIS11-B had an ED50of 1,355 g ha−1and 150 gene copies. Partial sequences ofEPSPSfrom GR accessions withoutEPSPSamplification did not contain any of the known resistance-conferring mutations. Nearly 40% of GR accessions putatively harbor non–target site resistance mechanisms. Therefore, elevatedEPSPSgene copy number is associated with glyphosate resistance amongA. palmerifrom Arkansas.

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Variable Inheritance of AmplifiedEPSPSGene Copies in Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri)

Weed Science ◽

10.1017/wsc.2018.65 ◽

2018 ◽

Vol 67 (2) ◽

pp. 176-182 ◽

Cited By ~ 6

Author(s):

Darci A. Giacomini ◽

Philip Westra ◽

Sarah M. Ward

Keyword(s):

Copy Number ◽

Single Gene ◽

Gene Copy Number ◽

Transgressive Segregation ◽

Palmer Amaranth ◽

Gene Copy ◽

Amaranthus Palmeri ◽

Gene Copies ◽

Central United States ◽

Free Environment

AbstractGlyphosate-resistant (GR) Palmer amaranth (Amaranthus palmeriS. Watson) is considered one of the most troublesome weeds in the southern and central United States, but results of previous research to determine the mode of inheritance of this trait have been conflicting and inconclusive. In this study, we examined segregation patterns ofEPSPSgene-copy numbers in F1and F2generations ofA. palmeriand found no evidence of a Mendelian single-gene pattern of inheritance. Transgressive segregation for copy number was exhibited by several F1and all of the F2families, most likely the product ofEPSPScopy-number variation within each plant. This variation was confirmed by assaying gene-copy number across clonal generations and among individual shoots on the same plant, demonstrating thatEPSPSamplification levels vary significantly within a single plant. Increases and decreases in copy number occurred in a controlled, stress-free environment in the absence of glyphosate, indicating thatEPSPSgene amplification is a random and variable process within the plant. The ability ofA. palmerito gain or loseEPSPSgene copies is a valuable adaptive trait, allowing this species to respond rapidly to selection pressures and changing environments.

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Confirmation of Glyphosate-Resistant Kochia (Kochia scoparia) from Sugar Beet Fields in Idaho and Oregon

Weed Technology ◽

10.1017/wet.2017.80 ◽

2017 ◽

Vol 32 (1) ◽

pp. 27-33 ◽

Cited By ~ 3

Author(s):

Vipan Kumar ◽

Joel Felix ◽

Don Morishita ◽

Prashant Jha

Keyword(s):

Sugar Beet ◽

Copy Number ◽

Cropping Systems ◽

Gene Copy Number ◽

Glyphosate Resistance ◽

Gene Copy ◽

Resistance Level ◽

Control Programs ◽

Gene Copies

AbstractGlyphosate-resistant (GR) kochia is an increasing management concern in major cropping systems of the northwestern US. In 2014, we investigated four putative GR kochia accessions (designated as ALA, VAL, WIL, DB) collected from sugar beet fields in eastern Oregon and southwestern Idaho to characterize the level of evolved glyphosate resistance and determine the relationship between the 5-enol-pyruvylshikimate-3-phospate synthase (EPSPS) gene copy number and level of glyphosate resistance. TheEPSPSgene copy number was used as a molecular marker to detect GR kochia in subsequent surveys in 2015 and 2016. Based on LD50values from a whole-plant dose-response study, the four putative GR kochia populations were 2.0- to 9.6-fold more resistant to glyphosate than the glyphosate-susceptible (GS) accession. In anin vivoleaf-disk shikimate assay, leaf disks of GS kochia plants treated with 100-μM glyphosate accumulated 2.4- to 4.0-fold higher amounts of shikimate than the GR plants. The four GR accessions had 2.7 to 9.1 relativeEPSPSgene copies compared with the GS accession (<1EPSPSgene copies), and there was a linear relationship betweenEPSPSgene copy number and glyphosate resistance level (LD50values). The 2015 and 2016 GR kochia survey results indicated that about half of the collected populations from sugar beet fields in eastern Oregon had developed resistance to glyphosate whereas only one population from the Idaho collection was confirmed glyphosate resistant. This is the first confirmation of GR kochia in sugar beet fields in eastern Oregon and southwestern Idaho. Diversified weed control programs will be required to prevent further development and spread of GR kochia in sugar beet-based rotations in this region.

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Defense by duplication: The relation between phenotypic glyphosate resistance and EPSPS gene copy number variation in Amaranthus palmeri

10.1101/2021.04.13.439524 ◽

2021 ◽

Author(s):

Sarah B Yakimowski ◽

Zachary Teitel ◽

Christina M. Caruso

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Gene Copy Number ◽

Glyphosate Resistance ◽

Gene Copy ◽

Quantitative Relation ◽

Stabilizing Selection ◽

Amaranthus Palmeri ◽

Gene Copy Number Variation ◽

Number Variation

Gene copy number variation (CNV) has been increasingly associated with organismal responses to environmental stress, but we know little about the quantitative relation between CNV and phenotypic variation. In this study we quantify variation in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) copy number using digital drop PCR and variation in phenotypic glyphosate resistance in 22 populations of Amaranthus palmeri (Palmer Amaranth), a range-expanding agricultural weed. Overall, we detected a significant positive relation between population mean copy number and mean resistance. The majority of populations exhibited high glyphosate resistance, yet maintained low-resistance individuals resulting in bimodality in many populations. We investigated linear and threshold models for the relation between copy number and resistance, and found evidence for a threshold of ~15 EPSPS copies: there was a steep increase in resistance before the threshold, followed by a much shallower slope. Moreover, as copy number increases, the range of variation in resistance decreases. This suggests a working hypothesis that as EPSPS copies and dosage increases, negative epistatic interactions may be compensated. We detected a quadratic relation between mean resistance and variation (s.d.) in resistance, consistent with the prediction that as phenotypic resistance increases in populations, stabilizing selection decreases variation in the trait. Finally, patterns of variation across the landscape are consistent with less variation among populations in mean copy number / resistance in Georgia where glyphosate resistance was first detected, and wider variation among populations in resistance and copy number in a more northern state where resistance evolution may be at a younger evolutionary state.

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Prediction of glyphosate resistance level based on EPSPS gene copy number in Kochia scoparia

10.1101/047878 ◽

2016 ◽

Author(s):

Todd A. Gaines ◽

Abigail L. Barker ◽

Eric L. Patterson ◽

Philip Westra ◽

Eric P. Westra ◽

...

Keyword(s):

Gene Duplication ◽

Empirical Model ◽

Copy Number ◽

Gene Copy Number ◽

Glyphosate Resistance ◽

Gene Copy ◽

Kochia Scoparia ◽

Resistance Level ◽

Epsps Gene ◽

Fallow Systems

AbstractGlyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Sugarbeet fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska. The GR samples all had increased EPSPS gene copy number, with median population values up to 11. An empirical model was developed to estimate the level of glyphosate-resistance in K. scoparia based on EPSPS gene copy number. The results suggested that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number, and further increases in EPSPS gene copy number could increase resistance levels up to 8-fold relative to susceptible K. scoparia. These trends suggest that continued glyphosate selection pressure is selecting for higher EPSPS copy number and higher resistance levels in K. scoparia. By including multiple K. scoparia samples lacking EPSPS gene duplication, our empirical model provides a more realistic estimate of fold-resistance due to EPSPS gene copy number compared to methods that do not account for normal variation of herbicide response in susceptible biotypes.

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EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia

PLoS ONE ◽

10.1371/journal.pone.0168295 ◽

2016 ◽

Vol 11 (12) ◽

pp. e0168295 ◽

Cited By ~ 18

Author(s):

Todd A. Gaines ◽

Abigail L. Barker ◽

Eric L. Patterson ◽

Philip Westra ◽

Eric P. Westra ◽

...

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Glyphosate Resistance ◽

Gene Copy ◽

Kochia Scoparia ◽

Resistance Level ◽

Epsps Gene ◽

Whole Plant

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Nosocomial Outbreak of Carbapenemase-Producing Proteus mirabilis With Two Novel Salmonella Genomic Island 1 Variants Carrying Different blaNDM–1 Gene Copies in China

Frontiers in Microbiology ◽

10.3389/fmicb.2021.800938 ◽

2022 ◽

Vol 12 ◽

Author(s):

Lang Yang ◽

Hong He ◽

Qichao Chen ◽

Kaiying Wang ◽

Yanfeng Lin ◽

...

Keyword(s):

Proteus Mirabilis ◽

Copy Number ◽

Genomic Island ◽

Gene Copy Number ◽

Carbapenem Resistance ◽

Multidrug Resistant ◽

Gene Copy ◽

Nosocomial Outbreak ◽

Gene Copy Number Variation ◽

Gene Copies

NDM-1-producing multidrug-resistant Proteus mirabilis brings formidable clinical challenges. We report a nosocomial outbreak of carbapenem-resistant P. mirabilis in China. Six P. mirabilis strains collected in the same ward showed close phylogenetic relatedness, indicating clonal expansion. Illumina and MinION sequencing revealed that three isolates harbored a novel Salmonella genomic island 1 carrying a blaNDM–1 gene (SGI1-1NDM), while three other isolates showed elevated carbapenem resistance and carried a similar SGI1 but with two blaNDM–1 gene copies (SGI1-2NDM). Four new single nucleotide mutations were present in the genomes of the two-blaNDM–1-harboring isolates, indicating later emergence of the SGI1-2NDM structure. Passage experiments indicated that both SGI variants were stably persistent in this clone without blaNDM–1 copy number changes. This study characterizes two novel blaNDM–1-harboring SGI1 variants in P. mirabilis and provides a new insight into resistance gene copy number variation in bacteria.

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Deciphering the Mechanism of Glyphosate Resistance in Amaranthus palmeri by Cytogenomics

Cytogenetic and Genome Research ◽

10.1159/000521409 ◽

2022 ◽

pp. 1-7

Author(s):

Dal-Hoe Koo ◽

Rajendran Sathishraj ◽

Bernd Friebe ◽

Bikram S. Gill

Keyword(s):

Genome Size ◽

Copy Number ◽

Fold Increase ◽

Glyphosate Resistance ◽

Resistance Mechanisms ◽

Fish Analysis ◽

Amaranthus Palmeri ◽

Artificial Chromosomes ◽

Genetic Elements ◽

Meiotic Chromosomes

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 Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS 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 EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EPSPS-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 EPSPS 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 EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.

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Effect of EPSPS Gene Copy Number and Glyphosate Selection on Fitness of Glyphosate-Resistant Bassia Scoparia in The Field

10.21203/rs.3.rs-155310/v1 ◽

2021 ◽

Author(s):

Charlemagne Ajoc Lim ◽

Prashant Jha ◽

Vipan Kumar ◽

Alan T. Dyer

Keyword(s):

Sugar Beet ◽

Seed Production ◽

Great Plains ◽

Copy Number ◽

Gene Copy Number ◽

Reproductive Traits ◽

Gene Copy ◽

Epsps Gene ◽

Gene Copies

Abstract 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. We tested the hypothesis of reduced GR B. scoparia fitness (reproductive traits) to increasing glyphosate rates (applied as single or sequential applications) potentially experienced within a GR sugar beet field. 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 2,214 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.

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Evolutionary Trajectories toward Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00574-21 ◽

2021 ◽

Author(s):

Alessandra Carattoli ◽

Gabriele Arcari ◽

Giulia Bibbolino ◽

Federica Sacco ◽

Dario Tomolillo ◽

...

Keyword(s):

High Risk ◽

Klebsiella Pneumoniae ◽

Copy Number ◽

Residual Activity ◽

Gene Copy Number ◽

Resistance Mechanisms ◽

University Hospital ◽

Gene Copy ◽

Carbapenemase Gene ◽

Evolutionary Trajectories

From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA) resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the ST512, ST101 and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among them, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69 and KPC-70 showed amino acid substitutions, insertions and deletions in the Ω loop of the protein. KPC-29 has the duplication, while the novel KPC-67 has the triplication of the KDD triplet in the 270-loop of the protein. Genomics performed on contemporary resistant and susceptible clones underlined that those novel mutations emerged in bla KPC-3 genes located on conserved plasmids: in ST512, all bla KPC-3 mutant genes were located in pKpQIL plasmids, while the three novel bla KPC-3 mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy number pTOPO vector, the Ω loop mutated variants showed CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA-resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.

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High variability in SSU rDNA gene copy number among planktonic foraminifera revealed by single-cell qPCR

ISME Communications ◽

10.1038/s43705-021-00067-3 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Tamara Milivojević ◽

Shirin Nurshan Rahman ◽

Débora Raposo ◽

Michael Siccha ◽

Michal Kucera ◽

...

Keyword(s):

Single Cell ◽

Community Composition ◽

Copy Number ◽

Planktonic Foraminifera ◽

Gene Copy Number ◽

Ssu Rdna ◽

Gene Copy ◽

Rdna Gene ◽

Gene Copies ◽

Average Gene

AbstractMetabarcoding has become the workhorse of community ecology. Sequencing a taxonomically informative DNA fragment from environmental samples gives fast access to community composition across taxonomic groups, but it relies on the assumption that the number of sequences for each taxon correlates with its abundance in the sampled community. However, gene copy number varies among and within taxa, and the extent of this variability must therefore be considered when interpreting community composition data derived from environmental sequencing. Here we measured with single-cell qPCR the SSU rDNA gene copy number of 139 specimens of five species of planktonic foraminifera. We found that the average gene copy number varied between of ~4000 to ~50,000 gene copies between species, and individuals of the same species can carry between ~300 to more than 350,000 gene copies. This variability cannot be explained by differences in cell size and considering all plausible sources of bias, we conclude that this variability likely reflects dynamic genomic processes acting during the life cycle. We used the observed variability to model its impact on metabarcoding and found that the application of a correcting factor at species level may correct the derived relative abundances, provided sufficiently large populations have been sampled.

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