https://orcid.org/0000-0001-5381-7880
ABSTRACT
Rapid identification of herbicide-resistant weeds plays a crucial role in their management. Glyphosate resistance in Lolium perenne is a growing issue in New Zealand and can be conferred by a single nucleotide polymorphism (SNP) causing amino acid substitutions at codon 106 in the 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) gene. High-throughput molecular (HTM) methodologies that can detect such SNPs include high-resolution melting and real-time quantitative PCR probe assays. Here, we compare the effectiveness of both assays with a more labour-intense derived cleaved amplified polymorphic sequence (dCAPS) test for detecting SNPs at codon 106. The results showed that both HTM assays detected mutations at this codon, and their results were consistent with those of the dCAPS. Unlike dCAPS, however, the HTM assays over-estimated homozygosity for the glyphosate resistance allele. Nevertheless, cost-effective high-throughput detection of resistance is more important than the zygosity status when initiating an on-farm management response. In summary, both HTM assays successfully identified glyphosate resistance alleles at codon 106 in the EPSPS gene. Given that both assays are more cost-effective and can screen a larger number of samples in a timely manner compared to the dCAPS method, they can be used as quick tests for detecting glyphosate resistance in L. perenne.
Introduction
Weeds pose a significant threat to agriculture and food safety worldwide (Baucom Citation2019). Weed management practices include chemical (i.e. herbicide application) and non-chemical (e.g. cultivation) approaches (Monaco et al. Citation2001). However, herbicide application is the most common method for weed management. Nevertheless, continuous applications of herbicides with the same mode of action lead to the selection of individuals carrying mutations that enable them to survive herbicide application (Neve Citation2007). Globally, over 500 unique cases of herbicide-resistant weeds have been documented (Heap Citation2023), with over 25 cases having been documented in New Zealand (Buddenhagen et al. Citation2021; Ghanizadeh and Harrington Citation2021). While the number of herbicide resistance cases recorded in New Zealand is smaller than those in other countries such as Australia (Harrington and Ghanizadeh Citation2023), recent surveys indicate that the rate of herbicide resistance is greater than previously believed (Buddenhagen et al. Citation2021; Buddenhagen et al. Citation2022).
Glyphosate is one of the most effective non-selective herbicides being used to control annual and perennial weeds primarily because it is cost-effective and has low mammalian toxicity compared to other herbicides (Nandula Citation2010). It was once speculated that weeds would not develop resistance to glyphosate (Bradshaw et al. Citation1997). However, over 50 weed species have evolved resistance to this herbicide to date (Heap Citation2023). In New Zealand, glyphosate resistance was first reported for Lolium perenne and Lolium multiflorum in some vineyards in Marlborough (Ghanizadeh and Harrington Citation2021). To the best of our knowledge, glyphosate resistance has not been documented in other weed species in New Zealand, but a recent survey showed that glyphosate resistance in L. perenne is common in vineyards in New Zealand (Buddenhagen et al. Citation2022). Given glyphosate has been applied widely in many habitats over the last two decades, it is expected that there are unidentified cases of resistance to this herbicide likely existing in New Zealand (Harrington et al. Citation2016).
The first step to managing herbicide-resistant weeds is to identify them correctly and rapidly (Burgos et al. Citation2013). The conventional method for confirming herbicide-resistant weeds is whole-plant dose–response assays (Burgos et al. Citation2013). The primary advantage of this method is that it does not require sophisticated equipment. Nevertheless, this approach is labour-intensive and time-consuming, especially when a large number of putatively herbicide-resistant samples need to be assessed (Burgos Citation2015). An alternative approach is to use ‘quick tests’ to confirm herbicide resistance in weeds (Burgos et al. Citation2013). As indicated by their name, quick tests can provide a rapid indication of the herbicide resistance status of weed species; however, such tests should be accurate, inexpensive, and able to reliably process a large number of samples in a short time (Burgos et al. Citation2013). Various quick tests, including seed-bioassays (Tal and Rubin Citation2004), DNA-based assays (Délye et al. Citation2004; Délye et al. Citation2011), and enzymatic assays (Dayan et al. Citation2015) have been developed for screening herbicide-resistant weeds. Most of these alternative tests yield results quickly, typically in 1–14 days.
Glyphosate resistance can be conferred by range of mechanisms including target enzyme modifications, target enzyme overexpression, impaired herbicide translocation, and enhanced herbicide metabolism (Shaner Citation2009; Murphy and Tranel Citation2019; Pan et al. Citation2019). In New Zealand cases of glyphosate-resistant Lolium spp., both target enzyme modification and impaired herbicide translocation mechanisms have been reported (Ghanizadeh et al. Citation2015b; Ghanizadeh et al. Citation2016). Quick tests, such as seed-based, enzymatic, and leaf dip assays, have been developed to confirm glyphosate resistance in Lolium spp. (Ghanizadeh et al. Citation2015a), regardless of the mechanism of resistance. Recently, we developed a derived cleaved amplified polymorphic sequence (dCAPS) test as a DNA-based assay to detect target enzyme modification mechanism in L. perenne (Ghanizadeh et al. Citation2021). Using modified primers, this assay amplifies the portion of the 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) gene, the enzyme targeted by glyphosate, harbouring the mutation site and generates a specific endonuclease restriction enzyme cut site which is abolished in the presence of the glyphosate resistance SNP. This enables the dCAPS assay to detect mutations conferring resistance to glyphosate at codon-106 (e.g. Pro CCA to Ser TCA) in the EPSPS gene. In addition, the zygosity state of the glyphosate resistance allele can be shown by the dCAPS markers.
While effective, the dCAPS method can be labour intensive with multiple steps through the procedure. We therefore looked to other methods that could target the same mutation but enable increased throughput at the same accuracy while being available to communities interested in quick tests for glyphosate resistance. High-resolution melting (HRM) and real-time quantitative PCR (RT-qPCR) probe assays are high-throughput molecular diagnostics that have been used for screening herbicide resistance in agricultural weeds with the target enzyme modification mechanism (Warwick et al. Citation2010; Barrantes-Santamaría et al. Citation2018). Both HRM and RT-qPCR probe assays can test hundreds of samples more rapidly compared to other molecular assays, such as dCAPS (Wang et al. Citation2022). However, to the best of our knowledge, limited information is available regarding the applicability of both assays for screening the target enzyme modification mechanism in glyphosate-resistant weeds. We aligned with SlipStream Automation (www.slipstream-automation.co.nz) a company in Palmerston North, New Zealand, specialising in high-throughput analyses, to establish and optimise HRM and RT-qPCR probe assays for this mutation conferring glyphosate resistance in L. perenne. Herein, we aimed to evaluate the applicability of HRM and RT-qPCR probe assays as high-throughput molecular diagnostic assays for detecting target-site mutations at position Pro-106 in glyphosate-resistant L. perenne and compared the performance and efficacy of both assays with those of dCAPS.
Materials and methods
Plant material and DNA extraction
Leaf samples were collected from plants (n = 63) taken from populations identified as glyphosate resistant in a random survey for evaluating the extent of glyphosate resistance in New Zealand vineyards (Buddenhagen et al. Citation2022). The leaf samples were sent overnight in an insulated container to AgResearch Grasslands campus (Palmerston North), where they were freeze-dried and stored for later use at room temperature in air-tight containers with silica gel to remove moisture. The DNA extraction for the dCAPS was performed using the method outlined by Anderson et al. (Citation2018). There were three replicates of each sample in each assay. DNA extractions for both the HRM and the RT-qPCR assays was performed by SlipStream Automation (www.slipstream-automation.co.nz, Palmerston North NZ) using their standard plate-based CTAB DNA extraction method and diluted 1:10 with nuclease-free TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0). An individual (SP6) from a known glyphosate-susceptible population was used as a control in the dCAPS, HRM and RT-qPCR assays.
dCAPS PCR amplicon restriction assay
The dCAPS markers, polymerase chain reaction (PCR) reactions and PCR conditions were previously described by Ghanizadeh et al. (Citation2021). Briefly, a 216-bp PCR amplicon was generated using Gly-dCAPS-F2: 5`-TAAAGCTCTTCCTGGGGAACGCTGGAACTGCGATGGGG-3`/ Gly-dCAPS-R2: 5`-GGTCGCTCCCTCATTCTTGGTACTCCATCAAGAA-3` primer pairs. The amplicons were digested using Sau96I enzymes (NEB, UK), with amplicons carrying a wild-type (glyphosate susceptible) allele yielding 180-bp fragments after digestion. By contrast, those with a glyphosate resistance allele at codon 106 remain uncut. The digested fragments were visualised on an agarose 1 x LB (lithium borate) 2% (w v−1) gel.
High-resolution melting (HRM) assay
The PCR amplification reaction for the HRM assay contained diluted (1:10) Slipstream-extracted template perennial ryegrass genomic DNA (2 μl), 2× LightCycler® 480 High Resolution Melting Master mix (3.5 μl) (Roche Products, New Zealand), MgCl2 (3.0 mM), forward and reverse primers (each 400 nM) and nuclease-free water to bring the reaction volume to 7 μl. The LightCycler® 480 II (Roche Products, New Zealand) amplification programme comprised an initial denaturation at 95°C for 10 min followed by 45 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 30 s. This was followed by the HRM programme consisting of an initial denaturation at 95°C for 1 min (ramp rate 4.8°C s–1), cool to 40°C for 1 min (ramp rate 2.5°C s–1), heat to 65°C for 1 s (ramp rate 2.5°C s–1) followed by heating to 95°C in continuous acquisition mode (ramp rate 0.02°C s–1; 25 acquisitions C–1) and a final cool to 40°C for 30 s (ramp rate 2.5°C s–1). A set of three primers pairs was assessed, each of which had slightly different melting temperatures, as part of a PCR and HRM optimisation process. The primers were used were: HRMA-Gly-F (5`-GGATGCCAAGGAGGAAGTAAAG-3`; Tm 62.5°C) and HRMA-Gly-R (5`-TGCATTTCCGCCAGCAGCTA-3`; Tm 66.8°C); Lp_EPSPS_HRM_F (5`-GGATGCCAAGGAGGAAGTAAA-3`; Tm 62°C) and Lp_EPSPS_HRM_R (5`-CATTTCCGCCAGCAGCTA-3`; Tm 62°C); and Lp_EPSPS_HRM_HT_F (5`-AGGATGCCAAGGAGGAAGTAAAGCTC-3`; Tm 68°C) and Lp_EPSPS_HRM_HT_R (5`-CATACGTTGCATTTCCGCCAGCA-3`; Tm 68°C). Calculated amplicon sizes for the primer pairs were 94, 92 and 102 bp, respectively. The primer pairs were assessed for PCR amplification and clarity of HRM outputs for genotype calling across a range of annealing temperatures (Ta 55°C, 60°C, 65°C and 70°C). Genotypes were called using the LightCycler® 480 II system (Roche Products, New Zealand) with negatives defined as having no amplicon after cycle 38, normalisation temperatures at 75/76°C and 89/90°C, and with a shifting value of 5%.
Real-time quantitative PCR probe (RTqPCR) assay
The RT-qPCR reaction contained diluted (1:10) SlipStream-extracted template perennial ryegrass genomic DNA (2 μl), 2× LightCycler® 480 Probes Master mix (2.5 μl) (Roche Products, New Zealand), forward (Lp_EPSPS_HRM_HT_F; 5`-AGGATGCCAAGGAGGAAGTAAAGCTC-3`) and reverse (Lp_EPSPS_HRM_HT_R; 5`-CATACGTTGCATTTCCGCCAGCA-3`) primers (each 680 nM) to generate a 102 bp amplicon template for SNP detection using FAM fluorophore-labelled Probe 1 (Lp_EPSPS_CCA_FAM; 5`-TGCG + G + C + CATTG-3`) and YAK fluorophore-labelled Probe 2 (Lp_EPSPS_TCA_YAK; 5`-TGCG + G + T + C + ATTGA-3`) primers (each 227 nM) in a final volume of 5.5 μl. The ‘plus’ symbol in front of a base denotes it was an LNA (Locked Nucleic Acid) base which increases the melting temperature (Tm) hence specificity of the oligonucleotide binding. The YAK-probe was specific to the glyphosate resistance mutation identified by the T underlined. The LightCycler® 480 II (Roche Products, New Zealand) was operated using the Dual Colour Hydrolysis Probe/UPL Probe as the detection method with both the FAM and HEX filters activated. The amplification programme contained an initial denaturation at 95°C for 4 min followed by 45 cycles of 95°C for 5 s (ramp rate 4.4°C s–1) and 70°C for 45 s (ramp rate 2.2°C s–1). This was followed by a final cool down to 40°C for 30 s (ramp rate 1.5°C s–1). Genotypes were called using the LightCycler® 480 II system (Roche Products, New Zealand) analysis profile ‘Endpoint Genotyping’ with negatives defined as having no amplicon after cycle 35.
Results
dCAPS
Amplification of DNA samples using the dCAPS primer pairs yielded the expected amplicon size (216 bp) for all samples. The cleavage of the amplicons with the restriction enzyme (Sau96I), however, yielded different patterns of digestion among samples (). Out of 63 samples, 17 samples revealed an uncut glyphosate resistance allele amplicon (216 bp) where a target-site resistance single nucleotide polymorphism (SNP) mutation at codon 106 prevented cleavage by the restriction enzyme, indicating that these samples were homozygous for the glyphosate resistance allele (RR genotype). The rest of the samples consistently yielded both an uncut glyphosate resistance allele amplicon and digested glyphosate-susceptible allele amplicon (180 bp), indicating that these samples were heterozygous for the glyphosate resistance allele (RS genotype). The glyphosate-susceptible individual (SP6) showed it was homozygous for the glyphosate susceptibility allele (SS genotype). These results were consistent among the three replicates of each sample. Overall, the results of the dCAPS assay confirmed that all tested samples were resistant to glyphosate, with the target-site mutation playing a role in the glyphosate resistance mechanism. This was expected as the populations from which the plants had been selected had been phenotyped previously for resistance (Buddenhagen et al. Citation2022). This dataset provided a comparator for development of the following high-throughput methods.
Figure 1. The patterns of digested amplicons observed for some of the glyphosate-resistant Lolium perenne samples in the dCAPS assay. The presence of the 216 bp amplicon indicates the presence of resistant alleles in the EPSPS gene, whereas the ∼180 bp amplicon indicates the presence of the glyphosate-sensitive allele. RS and RR represent glyphosate-resistant individuals that were heterozygous and homozygous, respectively, for the resistance allele identified at codon 106 in the EPSPS gene. Individual SP6 is homozygous for the susceptibility allele (SS) whereas individual O2 is heterozygous for the resistance/susceptible alleles (RS) at position Pro-106 (Ghanizadeh et al. Citation2015b). CK indicates dCAPS amplicons prior to digestions with Sau3a.
High-resolution melting assay
The high-resolution melting (HRM) assay was used to determine the presence of target site mutations at codon 106 in the EPSPS gene in L. perenne (). To optimise the assay, three different primer pairs were designed and tested at four different annealing temperatures (55°C, 60°C, 65°C, 70°C). The results showed that all three primer pairs could discriminate the glyphosate-resistant phenotype from the susceptible one in most tested conditions and annealing temperature had little influence, however the best results with the clearest melting curve profiles were achieved with the Lp_EPSPS_HRM_F/R primer pairs at an annealing temperature of 60°C (Supplementary Figure 1). This primer pair and amplification conditions generated a 92 bp product and was used for assessing target-enzyme mutations at codon 106 position in the EPSPS gene in all samples, a subset of which is shown in . The results showed that the HRM assay detected the presence of the mutations at codon 106 and confirmed the outcome of the dCAPS assay, although there was a considerable difference between the two assays in determining the zygosity state of the glyphosate resistance allele (). Two samples (10527, 10542) did not generate data (). In contrast to the dCAPS results, a larger number of samples assessed with the HRM assay were assessed as homozygous for the glyphosate resistance allele at codon 106. In addition, there were some inconsistencies for the zygosity state among replicates of two samples (). Overall, the HRM assay was effective in detecting variation at codon 106 and while it did not always accurately describe the zygosity state of the mutations, this method did identify the presence of SNP alleles conferring glyphosate resistance in all cases.
Figure 2. High-resolution melting (HRM) SNP genotyping analysis of the codon 106 mutation in the EPSPS gene conferring glyphosate resistance in Lolium perenne. This is a normalised and temperature-shifted difference plot using the SS genotype as the baseline.
Note: HRM: high-resolution melting analysis; RS: individuals heterozygous for the glyphosate resistance (R) SNP (T allele) and the susceptible (S) SNP (C allele); SS: individuals (SP6 control) homozygous for the susceptible (S) SNP (C allele); RR: individuals homozygous for the susceptible (R) SNP (T allele).
Table 1. A summary of the results from all three molecular diagnostic assays for detecting glyphosate resistance alleles at codon 106 in the EPSPS gene. RR = homozygous resistance allele, RS = heterozygous resistance/susceptible alleles, SS = homozygous susceptible allele, N/A = not available.
Real-time quantitative PCR probe assay
The real-time quantitative PCR (RT-qPCR) probe assay detected glyphosate-resistant and -susceptible individuals successfully, indicating this method can be used as a high-throughput assay for glyphosate resistance screening (, ). However, as with the HRM assay, the zygosity state of the resistance allele detected by the RT-qPCR assay was not entirely consistent with the results of the dCAPS assay. This may be due to the proximity between the heterozygous RS and homozygous RR in the endpoint fluorescence scatter plot whereas the homozygous susceptible (SS) individuals had greater separation for easier discrimination (). Nevertheless, the accuracy rate for the zygosity state in the RT-qPCR probe assay was greater than that in the HRM assay, with almost 25% of the samples correctly identified as heterozygous based on the results of the dCAPS method (). As with the HRM method, the RT-qPCR probe assay accurately identified all samples harbouring a SNP allele conferring resistance to glyphosate, resistance, except for sample 10527 which gave a false negative outcome. This same sample did not generate a consistent output using HRM suggesting that the DNA sample may been sub-optimal.
Figure 3. Real-time quantitative PCR probe assay (RT-qPCR) probe SNP genotyping analysis of the codon 106 mutation in the EPSPS gene conferring glyphosate resistance in Lolium perenne using an endpoint fluorescence scatter plot. FU: fluorescence units; green triangles: individuals homozygous for the susceptible (R) SNP allele (RR); red triangles: individuals heterozygous for the glyphosate resistance (R) and the susceptible (S) SNP alleles (RS); blue triangles: individuals homozygous for the susceptible (S) SNP allele (SS); grey circles: negative controls.
Discussion
The genus Lolium is comprised of several problematic weed species, including L. perenne. In New Zealand, L. perenne is an important forage crop, nevertheless it is a challenging weed in vineyards and arable crops (Buddenhagen et al. Citation2022; Ghanizadeh et al. Citation2022). In vineyards, glyphosate application has been the most used management practice for L. perenne for many years (Harrington et al. Citation2016), resulting in the evolution of glyphosate resistance in this species in most of the New Zealand vineyards surveyed to date (Buddenhagen et al. Citation2022).
Management and mitigation of herbicide-resistant weeds require a timely identification of resistant individuals (Burgos et al. Citation2013). Rapid identification of herbicide resistance facilitates rapid recommendation and implementation of alternative management practices to prevent economic losses (Burgos Citation2015). Molecular diagnostic assays enable rapid diagnosis of herbicide-resistant weeds; however, these assays have been primarily used for the diagnosis of target enzyme modification mechanisms (Délye and Michel Citation2005; Délye et al. Citation2011). Target enzyme modification is one of the commonly identified glyphosate-resistant mechanisms in weeds (Murphy and Tranel Citation2019). The target enzyme modification mechanism of glyphosate resistance can be conferred by amino acid substitutions at codon position 106 alone or in combination with amino acid substitutions at codons 102 and 103 (Murphy and Tranel Citation2019). Hence, codon 106 can be used as a conserved target for the development of molecular diagnostic assays since this codon has been observed in all cases of glyphosate resistance with the target enzyme modification mechanism.
In this research, we compared the versatility of two diagnostic molecular assays, HRM and RT-qPCR probe assay, to a previously developed dCAPS assay for detecting variations at codon 106 in the EPSPS gene in L. perenne. HRM and RT-qPCR probe assays have been used to screen target enzyme mechanisms in weeds resistant to acetyl-CoA carboxylase (ACCase) and acetolactate synthase-inhibiting herbicides (Warwick et al. Citation2010; Barrantes-Santamaría et al. Citation2018). However, to our knowledge, no attempts have been made to detect target enzyme mutations at codon 106 of the EPSPS gene in glyphosate-resistant plants using HRM or RT-qPCR assays. The results of this research showed that both assays proved efficient in distinguishing glyphosate-resistant individuals from susceptible ones, and their glyphosate resistance allele identification outcome was congruent with that of the dCAPS assay. However, the determination of the resistance allele zygosity state was inconsistent among the assays. Previously, we showed that the zygosity state determined by the dCAPS assay was always consistent with DNA sequencing results (Ghanizadeh et al. Citation2021). Based on the dCAPS assay, most of the tested resistant samples were heterozygous for the glyphosate resistance allele at codon 106 of the EPSPS gene in this research. By contrast, the outcome of the HRM and RT-qPCR probe assays revealed that most of the samples were homozygous for the glyphosate resistance allele. The zygosity state significantly impacts the level of herbicide resistance, with semi-dominant resistance alleles conferring lower levels of resistance in heterozygous individuals than in homozygous ones (Ghanizadeh et al. Citation2019). When comparing the high-throughput assays with the dCAPS results, in most cases where the upper dCAPS undigested fragment (216 bp) representing the resistance allele was brighter than the lower 180 bp band representing the susceptibility allele, the HRM in particular would identify the sample as homozygous for the resistant alleles (; ). However, as qualitative assays to rapidly identify the presence of the codon-106 resistance alleles, both HRM and RT-qPCR probes were very effective.
The HRM and RT-qPCR probe assays are rapid and sensitive methods for the determination of SNPs (Zhang et al. Citation2013), and have advantages over the dCAPS assay. For instance, unlike the dCAPS, the HRM assay is automated, making it more cost-effective and less labour-intensive. Similarly, the RT-qPCR probe assay can assess a larger number of samples, as samples can be pooled, making this assay even more cost-effective, less labour-intensive, and more rapid than the dCAPS and HRM assays. Despite the advantages outlined above, the performance of HRM and RT-qPCR probe assays depends on factors such as sample uniformity, amplicon size, DNA quality and concentration (Zhang et al. Citation2013). These factors can limit the accuracy of both assays if they are violated. For instance, the presence of natural SNPs that are not associated with resistance mechanisms can affect the melting profile of amplicons, decreasing the accuracy of the outcome for the HRM assay (Barrantes-Santamaría et al. Citation2018). Cousins et al. (Citation2013) noted that nucleotide composition greatly influences the melting profile in HRM assays, with nucleotide transversions yielding a similar melting profile becoming indistinguishable on some HRM instruments. The presence of SNPs within the region of the RT-qPCR probe can also interfere with the amplification of the alleles of interest, leading to false negative or false positive results (Alfaro et al. Citation2019). Given the genetic variability that exists among different populations of L. perenne, the presence of natural SNPs in the EPSPS gene cannot be ruled out. These variants may have interfered with the heterozygosity calls by HRM and TaqMan assays, leading to an underestimation of the number of heterozygous individuals identified by both assays compared to the dCAPS assay, which is less sensitive to the presence of SNPs around the alleles of interest. However, to reduce the likelihood of interference from flanking SNPs, the amplicons were designed to be short (∼100 bp) which also increases the sensitivity of methods such as HRM (Zhang et al. Citation2013). Sample uniformity and quality may influence the consistency of results, but the leaf material was harvested and treated in a consistent way and any discrepancies among the assays were mostly due to intrinsic features of the assays themselves.
Despite the advantages and disadvantages of the molecular diagnostic assays evaluated in this research, selecting a molecular diagnostic quick assay is primarily dependent on the objectives of the research. For instance, in large-scale survey experiments where researchers assess a large number of samples, the RT-qPCR probe would allow a preliminary assessment of the resistance mechanism conferred by the target enzyme modification in a short time since the objective of such research is to confirm if samples are glyphosate-resistant. However, if the aim of the research is to explore the genetics of the resistance allele, then the dCAPS assay would be more accurate in assessing the zygosity state of samples.
Conclusion
The molecular diagnostic assays examined in this research showed an acceptable ability to rapidly detect the target-enzyme mutations at codon 106 in the EPSPS gene in L. perenne. Although these assays can only inform the molecular basis of one of the glyphosate resistance mechanisms, they are effective for assessing a large number of samples to provide a rapid confirmation of glyphosate resistance. For instance, if any of the assays confirmed glyphosate resistance, the farmers can be advised to use alternative chemicals to prevent further build-up of resistance. By aligning with SlipStream Automation, the protocol has been scaled up and is available as a service for use by land-based industry communities.
Supplementary Figure 1 Assessment of different primer pairs for clarity of high
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