‘Universal’ PCR assays detecting mutations in acetyl‐coenzyme A carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds

Abstract

Délye C, Pernin F & Michel S (2011). ‘Universal’ PCR assays detecting mutations in acetyl‐coenzyme A carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds. Weed Research51, 353–362. Summary Herbicides inhibiting acetyl‐coenzyme A carboxylase (ACCase) or acetolactate synthase (ALS) are key for grass weed control. Yet, numerous cases of resistance have evolved. Using the derived cleaved amplified polymorphic sequence method, we developed molecular assays to detect amino acid replacements at the seven ACCase codons (1781, 1999, 2027, 2041, 2078, 2088 and 2096) and at two ALS codons (197 and 574) known to play a role in herbicide resistance in grass weeds. For each codon, one assay detecting all known amino acid replacements endowing herbicide resistance was developed. The nine assays were successfully used to genotype ACCase and ALS in 39 grass species. Their flexible design enables easy detection of new mutations at the targeted codons. Because they can be implemented with basic molecular biology facilities and no previous knowledge of the ACCase or ALS sequence of the grass weed of interest, these assays are tools of choice to easily detect resistance caused by alteration(s) of ACCase or ALS in such species.

Molecular Basis for Resistance to Fluazifop-P-Butyl in Itchgrass (Rottboellia cochinchinensis) from Costa Rica

“…DNA from 10 individual plants, five from putative resistant and five from susceptible R. cochinchinensis populations was extracted and successfully PCR amplified and sequenced with primers that had been designed to amplify regions A and B of the CT domain of the ACCase gene in grasses, which contain all known mutation sites that confer resistance to ACCase inhibiting herbicides (Délye & Michel, 2005;Délye et al, 2011). Nucleotide and amino acid sequences were then aligned to each other and to the chloroplastic ACCase genes of other grass weeds (Figure 1).…”

Using next‐generation sequencing to detect mutations endowing resistance to pesticides: application to acetolactate‐synthase (ALS)‐based resistance in barnyard grass, a polyploid grass weed

“…Thus, seeking resistance-endowing mutations scattered along a gene requires the development of a set of assays. 20 Yet, only sequencing captures the full nucleotide variation in a DNA region of interest. Today, the rise of next-generation sequencing (NGS) technologies (described by Metzker 29 ) has opened up the possibility of using sequencing for pesticide resistance diagnosis.…”

“…15 -18 This is also true of dCAPS assays. 19,20 Following technology evolution, allele-specific PCR has been at the basis of 'hi-tech', high-throughput assays implementing quantitative PCR. 21,22 Other genotyping techniques allowing high-throughput detection of mutations at known positions have also been used to develop pesticide resistance diagnosis, such as MALDI-TOF, 23 TaqMan, 24,25 SimpleProbe, 26 DNA microarrays 27 or SNaPshot.…”

Molecular Mechanisms of Herbicide Resistance

“…This easy DNA extraction protocol is fast and costs almost nothing. It can be performed from different plant tissues (leaf, root, green stem), either fresh or dried (Délye et al 2011); however, Brutus extraction from ripe seeds is often not suitable for PCR.…”

“…The dCAPS primer is located close to the mutation of interest, and contains one or more mismatched nucleotides so as to create a restriction enzyme recognition site encompassing the mutation of interest (Neff et al 1998). The (d)CAPS technique has been successfully used to detect mutations conferring herbicide resistance, including in the ACCase and ALS genes (e.g., Délye and Boucansaud 2008;Délye et al 2011;.…”