Genomic characterisation and dissection of the onset of resistance to acetyl CoA carboxylase-inhibiting herbicides in a large collection of Digitaria insularis from Brazil

An in-depth genotypic characterisation of a diverse collection of Digitaria insularis was undertaken to explore the neutral genetic variation across the natural expansion range of this weed species in Brazil. With the exception of Minas Gerais, populations from all other states showed high estimates of expected heterozygosity (HE > 0.60) and genetic diversity. There was a lack of population structure based on geographic origin and a low population differentiation between populations across the landscape as evidenced by average Fst value of 0.02. On combining haloxyfop [acetyl CoA carboxylase (ACCase)-inhibiting herbicide] efficacy data with neutral genetic variation, we found evidence of presence of two scenarios of resistance evolution in this weed species. Whilst populations originating from north-eastern region demonstrated an active role of gene flow, populations from the mid-western region displayed multiple, independent resistance evolution as the major evolutionary mechanism. A target-site mutation (Trp2027Cys) in the ACCase gene, observed in less than 1% of resistant populations, could not explain the reduced sensitivity of 15% of the populations to haloxyfop. The genetic architecture of resistance to ACCase-inhibiting herbicides was dissected using a genome wide association study (GWAS) approach. GWAS revealed association of three SNPs with reduced sensitivity to haloxyfop and clethodim. In silico analysis of these SNPs revealed important non-target site genes belonging to families involved in herbicide detoxification, including UDPGT91C1 and GT2, and genes involved in vacuolar sequestration-based degradation pathway. Exploration of five genomic prediction models revealed that the highest prediction power (≥0.80) was achieved with the models Bayes A and RKHS, incorporating SNPs with additive effects and epistatic interactions, respectively

Broad Resistance to ACCase Inhibiting Herbicides in a Ryegrass Population Is Due Only to a Cysteine to Arginine Mutation in the Target Enzyme

BACKGROUND: The design of sustainable weed management strategies requires a good understanding of the mechanisms by which weeds evolve resistance to herbicides. Here we have conducted a study on the mechanism of resistance to ACCase inhibiting herbicides in a Lolium multiflorum population (RG3) from the UK. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of plant phenotypes and genotypes showed that all the RG3 plants (72%) that contained the cysteine to arginine mutation at ACCase codon position 2088 were resistant to ACCase inhibiting herbicides. Whole plant dose response tests on predetermined wild and mutant 2088 genotypes from RG3 and a standard sensitive population indicated that the C2088R mutation is the only factor conferring resistance to all ten ACCase herbicides tested. The associated resistance indices ranged from 13 for clethodim to over 358 for diclofop-methyl. Clethodim, the most potent herbicide was significantly affected even when applied on small mutant plants at the peri-emergence and one leaf stages. CONCLUSION/SIGNIFICANCE: This study establishes the clear and unambiguous importance of the C2088R target site mutation in conferring broad resistance to ten commonly used ACCase inhibiting herbicides. It also demonstrates that low levels "creeping", multigenic, non target site resistance, is not always selected before single gene target site resistance appears in grass weed populations subjected to herbicide selection pressure

A novel W1999S mutation and non-target site resistance impact on acetyl-CoA carboxylase inhibiting herbicides to varying degrees in a UK Lolium multiflorum population.

Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21).The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here.This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance

dCAPS procedures for the detection of the wild type isoleucine and mutant threonine amino acid residues at ACCase codon position 1781: (4a) <i>Nsi</i>I restricted (134 bp) correspond to the I1781 allele and unrestricted fragments (165 bp) correspond to either T1781 or L1781 alleles.

<p>(4b) <i>Nde</i>I restricted fragments (132 bp) correspond to the mutant threonine allele while the undigested band (169 bp) corresponds to either the wild type isoleucine or leucine alleles. Heterozygous plants show one each of the restricted and unrestricted PCR fragments in the two assays.</p

CAPS procedure for the detection of a thymine to cysteine change (C2088R mutation) at nucleotide position 6262 in <i>Lolium</i> spp.

<p>The <i>Hha</i> I digested fragment (126 bp) correspond to the resistant R2088 allele and the <i>Hha</i> 1 undigested fragment (161 bp) correspond to the C2088 allele. Heterozygous plants display both the 126 bp and 161 resistant and sensitive alleles respectively. Lanes 1 and 10: DNA ladder, lanes 2, 3 and 4: homozygous mutant RR2088, lanes 4, 5, 6: heterozygous CR2088 plants, lanes 7, 8, 9: homozygous wild CC2088 plants.</p

Efficacy of clethodim on different 2088 ACCase genotypes and plant growth stages. Genotypes compared using Wilcoxon rank sum test.

<p>Efficacy of clethodim on different 2088 ACCase genotypes and plant growth stages. Genotypes compared using Wilcoxon rank sum test.</p

Cycloxydim whole plant dose response tests on four plant groups.

<p>Homozygous wild type STD1-II1781 from the standard sensitive population; homozygous wild type BG2-II1781; heterozygous mutant type BG2-IT1781 and heterozygous mutant type BG2-IL1781 from population BG2.</p

Estimated GR₅₀ values for 10 ACCase inhibiting herbicides and different 2088 ACCase genotypes.

<p>Estimated GR₅₀ values for 10 ACCase inhibiting herbicides and different 2088 ACCase genotypes.</p

Herbicide rates used in whole plant dose response assays.

<p>CC2088-STD1: wild type homozygous 2088 genotype from standard sensitive population.</p><p>CC2088-RG3: wild type homozygous 2088 genotype from population RG3.</p><p>RR2088-RG3: mutant homozygous 2088 genotype from population RG3.</p

Pinoxaden whole plant dose response tests on four plant groups.

<p>Homozygous wild type STD1-II1781 from the standard sensitive population; homozygous wild type BG2-II1781; heterozygous mutant type BG2-IT1781 and heterozygous mutant type BG2-IL1781 from population BG2.</p