Real-time quantitative PCR assays for quantification of L1781 ACCase inhibitor resistance allele in leaf and seed pools of Lolium populations

The I1781L amino acid substitution in the target ACCase enzyme causes broad resistance to ACCase inhibitor herbicides in several monocotyledenous weeds of agronomic importance. This mutation results from a substitution of an adenine (A) residue by either a thymine (T) or cytosine (C) at position 5341 in Alopecurus myosuroides Huds and at an equivalent position in Lolium species, Avena fatua L. and Setaria viridis (L.) Beauv. Two different procedures, the PCR-based allele-specific assay (ASA) and the derived cleaved amplified polymorphic sequence (dCAPS) method, have previously been described for detecting this mutation. These methods are, however, only amenable to low sample throughput and are used in the analysis of single plants. Here, an alternative high-throughput ARMS/Scorpion real-time quantitative PCR (Q-PCR) method for measuring levels of the I1781L mutation in pools of leaf and seed samples of Lolium populations is presented. The limit of detection for C and T mutant alleles in a background of wild-type A is 0.02 and 0.0003% respectively. In this study, DNA from batches of 24 leaf segments measuring 0.5 cm from different plants or 1000 seeds could be conveniently extracted and accurately analysed. As part of assay validation, the comparative analysis of five geographically distinct Lolium populations with dCAPS and Q-PCR procedures demonstrated the accuracy of the latter method, and the three possible II1781, IL1781 and LL1781 ACCase genotypes being distributed as predicted by the Hardy-Weinberg principle. Given the dominance of the L1781 over the I1781 allele at recommended field rates for most ACCase inhibitors, the frequency of herbicide survivors in the field owing only to the presence of the I1781L mutation is thus predicted to be 2pq + q(2), where p and q are the frequencies of the I1781 and L1781 alleles as determined by Q-PCR. The Q-PCR assay established allows detection of very low levels of the L1781 ACCase mutation before resistance would normally be discernible in the field. Therefore, it offers the opportunity to tackle resistance at its very onset, potentially avoiding implementation of complicated and often costly weed management practices.