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PCR-based assay to detect the EPSPS TAP-IVS substitution in Amaranthus hybridus

Abstract

Background

Amaranthus spp. are problematic weeds and competitors for nutrients in several crops, especially in soybean and corn. Resistance to glyphosate has been detected in several weed species, and a triple mutation in its EPSPS target gene was detected recently in Amaranthus hybridus.

Objective

The aim of this work was to develop a simple polymerase chain reaction (PCR) method to detect the EPSPS triple mutation in A. hybridus.

Methods

Two pairs of primers were designed for PCR-based detection of the EPSPS TAP-IVS triple mutation, which confers resistance to glyphosate, in A. hybridus.

Results

These sets of allele-specific primers were tested on five Amaranthus species and in 65 different field accessions. The PCR reaction using one set of the primers amplifies the wildtype (TAP) allele while the PCR reaction using the other pair of primers amplifies the triple mutation (IVS) allele. The presence of PCR products in both sets of primers identifies the heterozygous resistant individuals, and PCR product amplified only with the triple mutation set of primers identifies the homozygous resistant individuals. A DNA concentration test was performed and the recommend DNA amount to be used is 100 ng.

Conclusions

We developed and tested two sets of primers to detect the EPSPS TAP-IVS triple mutation and the results showed a 100% genotypic to phenotypic association. The triple mutation detection assay is easy to use and can be applied in a molecular laboratory with basic equipments. Early detection of resistance helps to better manage and control its spreading.

smooth pigweed; glyphosate; herbicide; resistance; weed control; 5-enolpyruvylshikimate-3-phosphate synthase; Amaranthaceae

1.Introduction

Weed resistance to the herbicide glyphosate is currently a problem in several crop producing regions worldwide and 53 weed species (26 dicots and 27 monocots) were identified with resistance (Heap, 2021Heap I. The international herbicide-resistant weed database. Weedscience. 2021[access April 11,2021]. Available from: www.weedscience.org
www.weedscience.org...
). Glyphosate resistance in plants can be a result of either target-site (TSR) or non-target-site resistance (NTSR), or even the occurrence of both TSR and NTSR mechanisms in the same individual. The NTSR mechanisms reported for glyphosate are reduced uptake, reduced translocation, vacuolar sequestration, rapid necrosis followed by regeneration (the Phoenix phenomenon), and enhanced degradation of aminomethylphosphonic acid (AMPA) and glyoxylate by elevated aldo-keto reductase (AKR) activity (reviewed by Duke, 2019Duke SO. Enhanced metabolic degradation: the last evolved glyphosate resistance mechanism of weeds? Plant Phys. 2019;181(4):1401-3. Available from: https://doi.org/10.1104/pp.19.01245
https://doi.org/10.1104/pp.19.01245...
). A combination of both, TSR and NTSR, was observed in several species, for instance, Amaranthus tuberculatus and Lolium rigidum, in which there was one-codon change in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and reduced translocation of glyphosate (Duke, 2011Duke SO. Glyphosate degradation in glyphosate-resistant and -susceptible crops and weeds. J Agric Food Chem. 2011;59(11):5835-41. Available from: https://doi.org/10.1021/jf102704x
https://doi.org/10.1021/jf102704x...
; Bostamam et al., 2012Bostamam Y, Malone JM, Dolman FC, Boutsalis P. Rigid ryegrass (Lolium rigidum) populations containing a target site mutation in EPSPS and reduced glyphosate translocation are more resistant to glyphosate. Weed Sci. 2012;60(3):474-9. Available from: https://doi.org/10.1614/WS-D-11-00154.1
https://doi.org/10.1614/WS-D-11-00154.1...
; Nandula et al., 2013Nandula VK, Ray JD, Ribeiro DN, Pan Z, Reddy KN. Glyphosate resistance in tall waterhemp (Amaranthus tuberculatus) from Mississippi is due to both altered target-site and nontarget-site mechanisms. Weed Sci. 2013; 61(3):374-83. Available from: https://doi:10.1614/WS-D-12-00155.1
https://doi:10.1614/WS-D-12-00155.1...
).

The TSR mechanisms observed are point mutations in the active site of the EPSPS gene or EPSPS gene duplication (on an extrachromosomal circular DNA (eccDNA) or in a tandem duplication at a single locus or in multiple loci throughout the genome (Gaines et al., 2010Gaines TA, Zhang W, Wang D, Bukun B, Chisholm ST, Shaner DL et al. Gene amplification confers glyphosate resistance in Amaranthus palmeri. PNAS. 2010;107(3):1029-34. Available from: https://doi.org/10.1073/pnas.0906649107
https://doi.org/10.1073/pnas.0906649107...
; Jugulam et al., 2014Jugulam M, Niehues K, Godar AS, Koo DH, Danilova T, Friebe B et al. Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia. Plant Physiol. 2014;166(3):1200-7. Available from: https://doi.org/10.1104/pp.114.242826
https://doi.org/10.1104/pp.114.242826...
; Lorentz et al., 2014Lorentz L, Gaines TA, Nissen SJ, Westra P, Strek H, Dehne HW et al. Characterization of glyphosate resistance in Amaranthus tuberculatus populations. J Agric Food Chem. 2014;62(32):8134-42. Available from: https://doi.org/10.1021/jf501040x
https://doi.org/10.1021/jf501040x...
; Nandula et al., 2014Nandula VK, Wright AA, Bond JA, Ray JD, Eubank TW, Molin WT. EPSPS amplification in glyphosate-resistant spiny amaranth (Amaranthus spinosus): a case of gene transfer via interspecific hybridization from glyphosate-resistant Palmer amaranth (Amaranthus palmeri). Pest Manag Sci. 2014;70(12):1902-9. Available from: https://doi.org/10.1002/ps.3754
https://doi.org/10.1002/ps.3754...
; Koo et al., 2018Koo DH, Molin WT, Saski CA, Jiang J, Karthik P, Jugulam M et al. Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri. PNAS. 2018;115(13):3332-7. Available from https://doi.org/10.1073/pnas.1719354115
https://doi.org/10.1073/pnas.1719354115...
; Patterson et al., 2018Patterson EL, Pettinga DJ, Ravet K, Neve P, Gaines TA. Glyphosate resistance and EPSPS gene Duplication: Convergent Evolution in Multiple Plant Species. J Hered. 2018;109(2):117-25. Available from: https://doi.org/10.1093/jhered/esx087
https://doi.org/10.1093/jhered/esx087...
). The mutations can result either in one-, two- or three-codon changes and were reported in various weed species (Sammons, Gaines, 2014; Yu et al., 2015Yu Q, Jalaludin A, Han H, Chen M, Sammons RD, Powles SB. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance. Plant Physiol. 2015;167(4):1440-7. Available from: https://doi.org/10.1104/pp.15.00146
https://doi.org/10.1104/pp.15.00146...
; Perotti et al., 2019Perotti VE, Larran AS, Palmieri VE, Martinatto AK, Alvarez CE, Tuesca D, Permingeat HR. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate resistant Amaranthus hybridus population from Argentina. Pest Manag Sci. 2019;75(5):1242-51. Available from: https://doi.org/10.1002/ps.5303
https://doi.org/10.1002/ps.5303...
). Species were observed with a single mutation at position 106 leading to substitutions from a proline to serine for Eleusine indica (Baerson et al., 2002Baerson SR, Rodriguez DJ, Tran M, Feng YM, Biest NA, Dill GM. Glyphosate-resistant goosegrass: identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol. 2002;129(3):1265-75. Available from: https://doi.org/10.1104/pp.001560
https://doi.org/10.1104/pp.001560...
), a proline to leucine on Lolium rigidum (Kaundun et al., 2011Kaundun SS, Dale RP, Zelaya IA, Dinelli G, Marotti I, McIndoe E et al. A novel P106L mutation in EPSPS and an unknown mechanism(s) act additively to confer resistance to glyphosate in a south African Lolium rigidum population. J Agric Food Chem. 2011;59(7):3227-33. Available from: https://doi.org/10.1021/jf104934j
https://doi.org/10.1021/jf104934j...
), L. multiflorum (Gonzalez-Torralva et al., 2012Gonzalez-Torralva F, Gil-Humanes J, Barro F, Brants I, Prado R. Target site mutation and reduced translocation are present in a glyphosate-resistant Lolium multiflorum Lam. biotype from Spain. Plant Physiol Biochem. 2012;58:16-22. Available from: https://doi.org/10.1016/j.plaphy.2012.06.001
https://doi.org/10.1016/j.plaphy.2012.06...
), and Digitaria insularis (Carvalho et al., 2012Carvalho LB, Alves PLCA, Gonzalez-Torralva F, Cruz-Hipolito HE, Rojano-Delgado AM, Prado R et al. Pool of resistance mechanisms to glyphosate in Digitaria insularis. J Agric Food Chem. 2012;60(2):615-22. Available from: https://doi.org/10.1021/jf204089d
https://doi.org/10.1021/jf204089d...
). A complete list of publications with species and their identified mutations in the EPSPS gene is available in weedscience.org (Gaines, Heap, 2021Heap I. The international herbicide-resistant weed database. Weedscience. 2021[access April 11,2021]. Available from: www.weedscience.org
www.weedscience.org...
). The Amaranthus species with reported resistance to glyphosate are A. palmeri, A. tuberculatus (syn. A. rudis), A. spinosus, and A. hybridus (syn. A. quitensis) (Heap, 2021Heap I. The international herbicide-resistant weed database. Weedscience. 2021[access April 11,2021]. Available from: www.weedscience.org
www.weedscience.org...
). In A. tuberculatus and A. palmeri a single codon change at the protein position 106 was reported and the resistance levels were usually low (≤ 10 fold) (reviewed by Sammons, Gaines, 2014). The double amino acid substitution in the EPSPS gene, known as TIPS, was reported for instance in E. indica (Yu et al., 2015Yu Q, Jalaludin A, Han H, Chen M, Sammons RD, Powles SB. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance. Plant Physiol. 2015;167(4):1440-7. Available from: https://doi.org/10.1104/pp.15.00146
https://doi.org/10.1104/pp.15.00146...
) and Bidens pilosa (Alcántara-de la Cruz et al., 2016Alcántara-de la Cruz R, Fernández-Moreno PT, Ozuna CV, Rojano-Delgado AM, Cruz-Hipolito HE, Domínguez-Valenzuela JA et al. Target and non-target site mechanisms developed by glyphosate-resistant hairy beggarticks (Bidens pilosa L.) populations from Mexico. Front Plant Sci. 2016;7:1-12. Available from: https://doi.org/10.3389/fpls.2016.01492
https://doi.org/10.3389/fpls.2016.01492...
), and the double mutation known as TIPT was detected in B. subalternans (Takano et al. 2020Takano HK, Fernandes VNA, Adegas FS, Oliveira Jr RS, Westra P, Gaines TA et al. A novel TIPT double mutation in EPSPS conferring glyphosate resistance in tetraploid Bidens subalternans. Pest Manag Sci. 2020;76(1):95-102. Available from: https://doi.org/10.1002/ps.5535
https://doi.org/10.1002/ps.5535...
). Thus far, double mutation in the EPSPS gene was not observed in Amaranthus spp. The only known triple mutation in the EPSPS gene conferring high resistance levels to glyphosate was reported for A. hybridus in Cordoba, Argentina (Perotti et al., 2019Perotti VE, Larran AS, Palmieri VE, Martinatto AK, Alvarez CE, Tuesca D, Permingeat HR. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate resistant Amaranthus hybridus population from Argentina. Pest Manag Sci. 2019;75(5):1242-51. Available from: https://doi.org/10.1002/ps.5303
https://doi.org/10.1002/ps.5303...
; Garcia et al., 2019Garcia MJ, Palma-Bautista C, Rojano-Delgado AM, Bracamonte E, Portugal J, Alcantara-de la Cruz R et al. The triple amino acid substitution TAP-IVS in the EPSPS gene confers high glyphosate resistance to the superweed Amaranthus hybridus. Int J Mol Sci. 2019;20(10):1-15. Available from: https://doi.org/10.3390/ijms20102396
https://doi.org/10.3390/ijms20102396...
). The triple amino acid substitution occurs at position 102 (ACA to ATA, Thr to Ile), at position 103 (GCC to GTG, Ala to Val), and at position 106 (CCA to TCA, Pro to Ser). The TAP-IVS triple amino acid substitution (T102I, A130V, and P106S), based on in silico conformational studies, is reported to generate an EPSPS enzyme with a functional active site and with increased restriction to glyphosate binding, due to a likely smaller active site (Perotti et al., 2019Perotti VE, Larran AS, Palmieri VE, Martinatto AK, Alvarez CE, Tuesca D, Permingeat HR. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate resistant Amaranthus hybridus population from Argentina. Pest Manag Sci. 2019;75(5):1242-51. Available from: https://doi.org/10.1002/ps.5303
https://doi.org/10.1002/ps.5303...
).

The process to confirm whether a population is resistant or not is composed of bioassays and molecular assays. In the bioassays, seeds from the resistant population are sowed and the herbicide of interest is applied to the seedlings at the recommended stage, and this whole process can take several weeks to a few months to be executed and the results to be available (Burgos, 2015Burgos NR. Whole-plant and seed bioassays for resistance confirmation. Weed Sci. 2015;63(SP1):152-65. Available from: https://doi.org/10.1614/WS-D-14-00019.1
https://doi.org/10.1614/WS-D-14-00019.1...
). In the other hand, molecular assays aiming to detect the resistance at the genetic level, which means finding changes in the DNA that are associated with resistance, can be faster performed from several days to a few weeks (Délye et al., 2015Délye C, Duhoux A, Pernin F, Riggins CW, Tranel PJ. Molecular mechanisms of herbicide resistance. Weed Sci. 2015;63(SP1):91-115. Available from: https://doi.org/10.1614/WS-D-13-00096.1
https://doi.org/10.1614/WS-D-13-00096.1...
). However, molecular assays can be more expensive when costly equipments are required, such as Sanger sequencing platforms. It is very important to highlight the complementarity of bioassay and molecular assay results, and the information each one brings to the understanding of weed resistance.

Thus, this study was performed with the aim of developing a simple molecular method to detect the EPSPS triple mutation in A. hybridus. We took advantage of the polymerase chain reaction (PCR) and the PCR amplification of specific alleles (PASA) (also called allele-specific PCR, ASPCR) (Corbett, Tardif, 2006) strategies to design allele-specific primers for the mutations leading to the EPSPS triple amino acid substitution which confers high levels of resistance to glyphosate.

2.Material and Methods

2.1 Sample preparation and DNA extraction

Leaf samples were collected and used for DNA isolation. Samples were either dried leaves collected in the field and brought to the laboratory or fresh leaf tissue from germinated seeds collected in the greenhouse. Genomic DNA was extracted from approximately 100 mg of leaf tissue using the Wizard Genomic DNA Purification Kit (Promega) and following the manufacturer’s instructions. DNA quality was verified on agarose gels and quantity was measured using the DeNovix instrument (Wilmington, Delaware). DNA samples were stored at -20⁰C until further use.

2.2 Primer design and PCR assays for EPSPS triple mutation detection

The EPSPS DNA sequences from A. hybridus available in Genbank under accession numbers MG595170.1 (201 bp; resistant) and MG595171.1 (201 bp; sensitive) along with internally available sequenced samples of A. hybridus, A. viridis, A. deflexus, and A. spinosus, were used for designing the primers. The sequences were aligned, and a forward primer was designed upstream of the EPSPS triple mutation region corresponding to 102, 103, and 106 amino acid positions. This forward primer is common for wildtype and mutant alleles and was named ‘Wildtype Forward’ (WT-F; Table 1). Two reverse primers were designed: the ‘Wildtype Reverse’ (WT-R) primer amplifies the wildtype (TAP) allele, and the ‘Triple Mutation Resistant Reverse’ (TMR-R) primer amplifies the triple mutation (IVS) allele. Primers designed in this study are listed in Table 1. Primers EPSF1 and EPSR8 are available in the literature (Gaines et al., 2010Gaines TA, Zhang W, Wang D, Bukun B, Chisholm ST, Shaner DL et al. Gene amplification confers glyphosate resistance in Amaranthus palmeri. PNAS. 2010;107(3):1029-34. Available from: https://doi.org/10.1073/pnas.0906649107
https://doi.org/10.1073/pnas.0906649107...
) and were used to amplify a 195 bp long fragment of EPSPS gene which contains the 102-106 positions, and this amplification was used as a PCR positive control to confirm that the DNA of all samples are of good quality for PCR purposes. All primers were synthesized by and purchased from the company Exxtend (Paulínia, São Paulo).

Table 1
List of primers used in this study

2.3 EPSPS gene amplification and Sanger sequencing

An EPSPS fragment of 195 bp, which contains the 102-106 positions, was amplified from all samples using primers EPSF1 and EPSR8 (Table 1) as previously described (Gaines et al., 2010Gaines TA, Zhang W, Wang D, Bukun B, Chisholm ST, Shaner DL et al. Gene amplification confers glyphosate resistance in Amaranthus palmeri. PNAS. 2010;107(3):1029-34. Available from: https://doi.org/10.1073/pnas.0906649107
https://doi.org/10.1073/pnas.0906649107...
). The PCR reaction was performed in a final volume of 25 µL containing 5.0 µL of 5X GoTaq Buffer, 0.5 µL of 10 mM dNTPs (Sinapse), 1.5 µL of 25 mM MgCl2, 0.5 µL of each primer at 10 µM, 0.2 µL of GoTaq G2 Hot Start Polymerase (Promega), and 14.8 µL of ultrapure nuclease-free water (Sigma). The PCR conditions were as follow: 95⁰C for 3 min, 32 cycles of 95⁰C for 30 s, X⁰C (see Table 1 for specific annealing temperatures) for 30 s, 72⁰C for 1 min, and a final extension step of 72⁰C for 5 min. PCR amplification was verified on 1% agarose gels and using the 100 bp molecular weight (ladder, Sinapse). PCR products were purified using the ExoSAP-IT™ Express reagent (Thermo Fisher) following manufacturer instructions. Purified PCR products were 10X diluted and 1 µL was used in the sequencing reaction. Sequencing reactions were performed with the BigDye cycle sequencing terminator kit (Thermo Fisher) and sequencing was performed in an Applied Biosystems 3500 Genetic Analyzer instrument (Thermo Fisher). All sequence electropherograms were visually checked for quality and consistency before sequences were assembled and aligned using UGENE (Unipro, 2012; Okonechnikov et al., 2012Okonechnikov K, Golosova O, Fursov M. UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012;28(8):1166-7. Available from: https://doi.org/10.1093/bioinformatics/bts091
https://doi.org/10.1093/bioinformatics/b...
).

2.4 Testing the EPSPS triple mutation detection assay

The primer pairs WT-F + WT-R and WT-F + TMR-R were tested in Amaranthus samples from the following five species: A. palmeri, A. hybridus, A. viridis, A. deflexus, and A. spinosus. The EPSF1 and EPSR8 primers (Gaines et al., 2010Gaines TA, Zhang W, Wang D, Bukun B, Chisholm ST, Shaner DL et al. Gene amplification confers glyphosate resistance in Amaranthus palmeri. PNAS. 2010;107(3):1029-34. Available from: https://doi.org/10.1073/pnas.0906649107
https://doi.org/10.1073/pnas.0906649107...
) were used to amplify a 195 bp long fragment of EPSPS gene, which contains the 102-106 positions, and the fragment was sequenced for all the samples tested for the five species listed above. Another test was performed with samples from A. hybridus known to be glyphosate resistant. All the tests were performed three times in three different PCR reactions and runs. The Amaranthus species determination was performed using the PCR assay with intron 1 sequence from the EPSPS gene as previously published (Wright et al., 2016Wright AA, Molin WT, Nandula VK. Distinguishing between weedy Amaranthus species based on intron 1 sequences from the 5-enolpyruvylshikimate-3-phosphate synthase gene. Pest Manag Sci. 2016;72(12):2347-54. Available from: https://doi.org/10.1002/ps.4280
https://doi.org/10.1002/ps.4280...
; data not shown).

2.5 DNA concentration and sensitivity of EPSPS triple mutation detection assay

This sensitivity assay was performed to test three amounts of DNA to detect the EPSPS triple mutation. The DNA samples of a wildtype TAP-allele (sensitive) and two resistant (a heterozygous TAP/IVS and a homozygous IVS) A. hybridus samples were diluted to three concentrations: 100, 10, and 1.0 ng µL-1, and PCR reactions were performed as described above. PCR amplification was verified on 1% agarose gels and used the 100 bp molecular weight (ladder, Sinapse).

2.6 Testing the EPSPS triple mutation detection assay in multiple field accessions

This EPSPS triple mutation detection assay was used in our large-scale resistance monitoring program for Amaranthus spp. with samples collected in 2020. For this study, Amaranthus spp. seeds were collected from soybean growing regions throughout Brazil. These seeds were sown in plastic containers containing substrate and plants were kept in a greenhouse under 32°C/25°C (day/night) in a 16 h photoperiod. The samples were collected from Rio Grande do Sul (RS, 33 samples), Paraná (PR, 15 samples), Minas Gerais (MG, 1 samples), and Mato Grosso (MT, 16 samples) states in Brazil. Leaf samples were harvested from individual plants and were used for DNA extraction as described above. A total of 65 samples were analyzed using this method and sequenced using the Sanger Sequencing method (described above) for confirmation purpose.

3.Results and Discussion

Two sets of allele-specific primers were developed for detection of the triple mutation in the EPSPS gene, which confers resistance to glyphosate and was first identified in A. hybridus. The primer pair WT-F + WT-R was designed to amplify the wildtype (TAP) allele and the primer pair WT-F + TMR-R was designed to amplify the triple mutation (IVS) allele. For a better understanding and helping the interpretation of results, a schematic diagram is presented in Figure 1. Both primer pairs were tested and amplified one band only (Figure 2). All the results were confirmed through Sanger sequencing the amplified fragments (Figure 3). Using this detection assay, two PCR reactions are performed to determine whether the samples are wildtype or are carrying the triple mutation, as shown in Figure 1. Thus, the presence of a PCR band when using the WT-F + WT-R primers, and absence of PCR band amplified with the WT-F + TMR-R primers, showed that the sample has the wildtype (sensitive) allele. When there was a PCR band amplified with the WT-F + WT-R primers and a PCR band amplified with the WT-F + TMR-R primers, for the same sample, it showed that the sample has the TAP-IVS (resistant) allele in a heterozygous state. When there was no PCR band amplified with the WT-F + WT-R primers, and only a PCR band amplified with the WT-F + TMR-R primers, it showed that the sample has the homozygous IVS (resistant) allele.

Figure 1
Schematic diagram of expected results using the PCR-based detection of EPSPS triple mutation assay. The plus sign (+) denotes presence of PCR band and the minus sign (-) denotes absence of PCR band in the agarose gel. Presence of PCR band when using EPSF1and EPSR8 primer pair is required to verify the quality of the DNA and as a PCR positive control

Figure 2
Electrophoresis of PCR products amplified with primers for detection of EPSPS triple mutation in five Amaranthus spp.: 1) A. hybridus, 2) A. hybridus heterozygous with triple mutation, 3) A. hybridus homozygous with triple mutation, 4) A. viridis, 5) A. spinosus, 6) A. deflexus, 7) A. palmeri, 8) PCR negative control. Primers for amplifying an EPSPS gene fragment and confirm DNA quality (EPSF1 and EPSR8; Gaines et al., 2010Gaines TA, Zhang W, Wang D, Bukun B, Chisholm ST, Shaner DL et al. Gene amplification confers glyphosate resistance in Amaranthus palmeri. PNAS. 2010;107(3):1029-34. Available from: https://doi.org/10.1073/pnas.0906649107
https://doi.org/10.1073/pnas.0906649107...
) (A); primers (WT-F + WT-R) for amplifying the wildtype allele (no triple mutation. B); primers (WT-F + TMR-R) for amplifying the allele with triple mutation (resistant IVS-allele; C). L is the ladder with 100 bp molecular weight [sizes from bottom to top: 100, 200, 300, 400, 500 (brightest), 600, 700, 800, 900, 1,000 bp]

Figure 3
Electropherogram alignment of EPSPS gene fragments. Alignment showing the codons for 101 to 107 amino acid. The triple amino acid substitution TAP-IVS corresponding to positions 102 (ACA to ATA, Threonine to Isoleucine), 103 (GCG to GTC, Alanine to Valine), and 106 (CCA to TCA, Proline to Serine) are highlighted in red. In position 105 there is a mutation in the resistant homozygous sample leading to a synonymous substitution (CGC to CGA, arginine). The A. hybridus EPSPS sequence (MG595171.1) was used as “Reference”

These sets of allele-specific primers were tested on five Amaranthus species and they were able to amplify the EPSPS fragments in all the tested species (Figure 2). There is a high conservation in the EPSPS gene region corresponding to the active target site (Padgette et al., 1991Padgette SR, Re DB, Gasser CS, Eichholtz DA, Frazier RB, Hironaka CM et al. Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site. J Biol Chem. 1991;266(3):22364-9. Available from: https://doi.org/10.1016/S0021-9258(18)54580-4
https://doi.org/10.1016/S0021-9258(18)54...
; Sammons, Gaines, 2014), where the triple mutation is located. The 195 bp EPSPS gene fragment amplified (as described in the methods) was sequenced for all five species and confirmed the results obtained with the allele-specific primers. Due to this high EPSPS sequence conservation the allele-specific primers can be used to genotype other Amaranthus species, although the EPSPS triple mutation was found only in A. hybridus until now (Garcia et al., 2019Garcia MJ, Palma-Bautista C, Rojano-Delgado AM, Bracamonte E, Portugal J, Alcantara-de la Cruz R et al. The triple amino acid substitution TAP-IVS in the EPSPS gene confers high glyphosate resistance to the superweed Amaranthus hybridus. Int J Mol Sci. 2019;20(10):1-15. Available from: https://doi.org/10.3390/ijms20102396
https://doi.org/10.3390/ijms20102396...
; Perotti et al., 2019Perotti VE, Larran AS, Palmieri VE, Martinatto AK, Alvarez CE, Tuesca D, Permingeat HR. A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate resistant Amaranthus hybridus population from Argentina. Pest Manag Sci. 2019;75(5):1242-51. Available from: https://doi.org/10.1002/ps.5303
https://doi.org/10.1002/ps.5303...
). Another important information is that double mutations as the TI-PS and TI-PT which are reported in E. indica (Yu et al., 2015Yu Q, Jalaludin A, Han H, Chen M, Sammons RD, Powles SB. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance. Plant Physiol. 2015;167(4):1440-7. Available from: https://doi.org/10.1104/pp.15.00146
https://doi.org/10.1104/pp.15.00146...
) and B. subalternans (Takano et al., 2020Takano HK, Fernandes VNA, Adegas FS, Oliveira Jr RS, Westra P, Gaines TA et al. A novel TIPT double mutation in EPSPS conferring glyphosate resistance in tetraploid Bidens subalternans. Pest Manag Sci. 2020;76(1):95-102. Available from: https://doi.org/10.1002/ps.5535
https://doi.org/10.1002/ps.5535...
), respectively, were not observed in any of the Amaranthus species studied so far. This information about the double mutation is important due to the fact that the reverse primer (TMR-R) might be able to amplify the double mutant allele depending on its sequence if it happens to arise in nature.

The sensitivity of the PCR for EPSPS triple mutation detection assay was tested using three DNA concentrations in the PCR, 100, 10 and 1 ng, and the assay showed consistent results for the three amounts (Figure 4). However, the 1 ng concentration resulted in a faint band and could lead to dubious interpretation of the results. Thus, the recommended DNA amount to be used in this triple mutation detection assay is 100 ng in a 25 µL reaction. PCR reactions with DNA amounts below 1 ng were not tested and thus, are not recommended for use.

Figure 4
Electrophoresis of PCR products amplified with primers for detection of EPSPS triple mutation and three DNA concentrations for testing the sensitivity of the assay. Primers (EPSF1 and EPSR8; Gaines et al., 2010Gaines TA, Zhang W, Wang D, Bukun B, Chisholm ST, Shaner DL et al. Gene amplification confers glyphosate resistance in Amaranthus palmeri. PNAS. 2010;107(3):1029-34. Available from: https://doi.org/10.1073/pnas.0906649107
https://doi.org/10.1073/pnas.0906649107...
) for amplifying an EPSPS gene fragment and confirm DNA quality as a PCR positive control (A); primer pair (WT-F + WT-R) for amplifying the wildtype (TAP) allele (B); primers (WT-F + TMR-R) for amplifying the IVS allele (C). Presence of PCR band in B only denotes a wildtype (TAP) allele; presence of PCR bands in B and C denotes the heterozygous resistant (TAP/IVS) allele; presence of PCR band in C only denotes the homozygous resistant (IVS) allele. DNA dilutions: 100 ng (well 1, 5, 9), 10 ng (well 2, 6, 10), 1 ng (well 3, 7, 11), negative control (well 4, 8, 12), L is the ladder with 100 bp molecular weight [sizes from bottom to top: 100, 200, 300, 400, 500 (brightest), 600, 700, 800, 900, 1,000 bp]

A set of 65 field accessions of Amaranthus spp., collected from various agricultural regions in Brazil in a resistance monitoring program, was genotyped using the PCR detection assay for the triple mutation in the EPSPS target gene. The distribution of the number of samples per state and the results are detailed in Table 2. Sensitive and resistant (IVS-allele) samples were observed in the Paraná and Rio Grande do Sul states, whereas in the Minas Gerais and Mato Grosso states all the sampled plants were sensitive (Table 2). For this study, only one sample from Minas Gerais was tested and interestingly it was identified as A. viridis and showed the wildtype genotype.

Table 2
Genotyping results using the PCR detection assay for the triple mutation of the EPSPS target gene. A total of 65 Amaranthus accessions collected from four Brazilian states (Paraná, Rio Grande do Sul, Minas Gerais, and Mato Grosso). The species column shows the Amaranthus spp. found in each state

A few things can influence the PCR detection assay and can affect the results, giving false negatives or even false positives, and thus are important to be mentioned here. False negative results can be due to the presence of PCR inhibitors, for instance, substances originated from the sample or were introduced during sample processing or even remaining from the DNA extraction process (Schrader et al., 2012Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors: occurrence, properties, and removal. J Applied Microbiol. 2012;113(5):1014-26. Available from: https://doi.org/10.1111/j.1365-2672.2012.05384.x
https://doi.org/10.1111/j.1365-2672.2012...
). Such organic or inorganic substances, among them calcium ions, urea, phenol, polysaccharides, and proteinases, can be present in the DNA solution and interfere with different steps of a PCR and ultimately inhibit the amplification of the targeted amplicon. Troubleshooting the DNA extraction process will only be required if the PCR with primers EPSF1 and EPSR8 result in no amplification at all. Thus, there should be a minimum DNA quality where the PCR reaction can occur without interference, and most DNA purification/extraction kits commercially available result in high quality DNA.

Overall, this PCR-based assay for detecting the EPSPS triple mutation conferring resistance to glyphosate in A. hybridus will fulfil the gap of lacking a simple method for detecting glyphosate resistance, which is spreading in Brazil and South America. This assay will enable laboratories with basic equipments, such as PCR thermocycler and electrophoresis apparatus, to identify Amaranthus plants harboring the IVS triple mutation. By having the results in a few days on whether the Amaranthus plants in a crop field are resistant or not, the growers and weed management technical teams can make faster and better decisions on approaches for controlling the weed.

4.Conclusions

We developed and tested two sets of primers to detect the EPSPS triple mutation in A. hybridus and the results showed a 100% genotypic to phenotypic association. The triple mutation detection assay is easy to use and can be applied in a molecular laboratory with basic equipments and help in the assessment of Amaranthus with resistance to glyphosate. The identification and confirmation of glyphosate resistance in Amaranthus plants can improve the decision on the management strategy to be applied in the problematic fields.

Acknowledgements

We would like to acknowledge Danilo Cestari for critically reading and reviewing the manuscript before submission. We would like to acknowledge Marcelo Nicolai for providing the Amaranthus palmeri DNA sample used in this study.

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Edited by

Approved by:
Editor in Chief: Carlos Eduardo Schaedler
Associate Editor: Marcos Yanniccari

Publication Dates

  • Publication in this collection
    12 Jan 2022
  • Date of issue
    2022

History

  • Received
    9 June 2021
  • Accepted
    17 Sept 2021
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