Diagnostic prediction of acaricide resistance gene frequency using quantitative real-time PCR with resistance allele-specific primers in the two-spotted spider mite Tetranychusurticae population (Acari: Tetranychidae)

Abstract

Due to the development of acaricide resistance in Tetranychus urticae, a rapid and accurate monitoring method for acaricide resistance gene frequency is required for resistance management.In this study, we developed a new diagnostic gene frequency prediction method, using quantitative real-time PCR with allele-specific primer sets, for mutation of I1017F in chitin synthase I, G126S in mitochondrial cytochrome b, and H110R in the PSST subunit of mitochondrial electron transport complex I, which are involved in etoxazole, bifenazate, and pyridaben resistance in T. urticae, respectively. Resistance allele frequencies computed using the 2 −ΔΔCq method, in mixtures of resistance and wild-type (susceptible) alleles, were strongly correlated with the actual frequencies of the resistance alleles in DNA samples for all three acaricides (regression slopes of 0.945-0.996; R 2 > 0.99). This result strongly indicates that resistance allele frequency in a population can be accurately predicted using a diagnostic quantitative real-time PCR method with a resistance allele-specific primer set. Finally, we applied this method to nine field populations and successfully determined resistance allele frequencies for the three acaricides. Overall, this diagnostic prediction method may contribute to the development of acaricide resistance management strategies, via monitoring resistance allele frequencies for a wide range of acaricides.

Identification and characterization of striking multiple‐insecticide resistance in a Tetranychus urticae field population from Greece

“…Moderate to high RRs to METI‐I acaricides have been reported previously 13,28,68 . A point mutation in psst/NDUFS7 of complex I has previously been associated with resistance to METI‐Is in both Tetranychus and Panonychus species, 9,69,70 and this mutation was also identified in the Trz population, although not fixed. In addition, an A55T substitution was also uncovered, but this mutation was not in a conserved region or near the binding site of METI‐Is.…”

freqpcr: estimation of population allele frequency using qPCR ΔΔCq measures from bulk samples

“…Maeoka et al . (2020) demonstrated that a general ΔΔCq method without restriction enzyme treatment could be used for allele-frequency measurement if a specific primer set were to be designed to amplify only the R allele at the target locus.…”

“…Although Eq. 11 is defined for the RED-ΔΔCq method, it is also applicable to the ΔΔCq method by Maeoka et al . (2020) by substituting and to and , respectively.…”

“…A longer digestion period (3 h) was also required to quantify etoxazole resistance in the protocol by Osakabe et al (2017). Maeoka et al (2020) demonstrated that a general ΔΔCq method without restriction enzyme treatment could be used for allele-frequency measurement if a specific primer set was designed to amplify only the R allele at the target locus. Similar to the RED-ΔΔCq method, DNA samples with unknown mixing ratios were dispensed and amplified using primer sets corresponding to TG and HK loci, respectively.…”

“…Quantitative PCR (qPCR), based on real-time PCR, is also used for the point estimation of allele frequency (Germer et al 2000). Osakabe et al (2017) and Maeoka et al (2020) developed diagnostic methods for acaricide resistance in the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), where they used a bulk sample to measure the frequency of the resistant point mutation in field mite populations. To calculate the point estimate, these studies compared the relative quantity of the resistance allele with an internal reference (housekeeping gene) in the sample, which is known as the ΔΔCq method (Livak and Schmittgen 2001).…”