A Two-in-One Strategy: Target and Nontarget Site Mechanisms Both Play Important Role in IMI-Resistant Weedy Rice

Abstract

The introduction of Clearfield technology allows the use of imidazolinone (IMI) herbicides to control weedy rice. Imidazolinone herbicides stop the acetolactate synthase (ALS) enzyme from synthesizing branched-chain amino acids, resulting in the death of the plant. Since the launch of Clearfield technology in Malaysia in 2010, many farmers have replaced traditional cultivars with Clearfield (CL) rice lines (MR220-CL1 and MR220-CL2). This technology was initially effective; however, in recent years, local farmers have reported the reduced efficacy of IMI herbicides in controlling the spread of weedy rice. Under IMI herbicide treatment, in previous weedy rice studies, the target-site resistance (TSR) mechanism of the ALS gene has been suggested as a key factor conferring herbicide resistance. In our study, a combination of ALS gene sequencing, enzyme colorimetric assay, and a genome-wide association study (GWAS) highlighted that a non-target-site resistance (NTSR) can be an alternative molecular mechanism in IMI-resistant weedy rice. This is supported by a series of evidence, including a weak correlation between single nucleotide polymorphisms (SNPs) within the ALS exonic region and ALS enzyme activity. Our findings suggest that the adaptability of weedy rice in Clearfield rice fields can be more complicated than previously found in other rice strains.

Keywords: clearfield rice; herbicide-resistant weedy rice; imidazolinone; non-target-site resistance; target-site resistance.

Figure 1

Positions of the three nonsynonymous mutations within the ALS gene and the corresponding amino acid listed below the SNPs. The SNPs are underlined and bolded.

Figure 2

Haplotype analysis of the ALS gene. All weedy rice samples are grouped into nine haplotype groups. Sus: IMI-susceptible weedy rice; Res: IMI-resistant weedy rice; Amb: IMI-R ambiguous weedy rice.

Figure 3

ALS enzyme colorimetric assay, with bar plots showing the absorbance reading of weedy rice samples at 520 nm. The letter “n” represents the number of samples in each haplotype group. IMI-R, IMI-S, and IMI-Ra represent the average absorbance reading of resistant, susceptible, and ambiguous samples, respectively.

Figure 4

Manhattan plot showing a peak on chromosome 2 (blue arrow) (with –log10 (p-value) > 5.8) where the ALS gene is located. A lower peak on chromosome 3 (green arrow) was identified as a potential gene contributing toward IMI resistance in weedy rice.