Ultrapure nuclease free water

Two primer pairs were used to amplify the ACCase gene sequence in D. insularis: FE35332 Forward (5′-ATG​TCC​ACT​CCT​GAA​TTC​CCA-3′), FE35333 Reverse (5′-CAT​TCT​GAG​GGA​AGT​ATC​AT-3′). PCR was performed in 25 μL reaction volume containing 5.0 µL of GoTaq Buffer, 0.5 µL of 10 mM dNTPs, 1.5 µL of 25 mM MgCl₂, 0.5 µL of 10 µM of each forward and reverse primers, 0.2 µL of GoTaq G2 Hot Start Polymerase (Promega), and 14.8 µL of ultrapure nuclease-free water (Sigma). PCR cycling conditions were: one cycle at 95°C for 2 min, 35 cycles at 94°C for 30 s, 58°C for 30 s, 72°C for 90 s, and final extension at 72°C for 10 min. PCR product was run on 1.0% agarose gel to verify the amplicon size of 1.5 kb. The amplified samples were purified and sequenced in a Genetic Analyzer 3,500 instrument (Applied Biosystems, Thermo Fisher) following manufacturer’s instructions. Four individuals per population were sequenced using the original amplification primers and the following three internal sequencing forward primers: FE35334 Sequencing Forward 1 (5′- TGG​GAG​AGC​AAA​GCT​TGG​GGT-3′), FE35407 Sequencing Forward 2 (5′- GAA​GTG​CTG​CTA​TTG​CCA​GTG​C -3′) and FE35408 Sequencing Forward 3 (5′- GAC​CCA​CCA​GAC​AGA​CCT​GTT​A -3′). The chromatograms were manually read using Bioedit version 7.2.5 software (Hall, 1999 ) to screen the seven known target-site mutations.

To test the specificity of in silico selected primers, a generic quantitative PCR was carried out with a panel of reference DNAs. Primer and probes were obtained from Metabion (Planegg, Germany) (Table 5). The SYBR^® Green qPCR was performed in a total volume of 20 µL. For the PCR reaction mixture, 10 µL SsoAdvanced universal SYBR^® Green supermix (2×) (Biorad Laboratories GmbH, Munich, Germany), 10 pmol of each primer (forward and reverse), ultrapure nuclease-free water (Sigma-Aldrich, St. Louis, MO, USA) and 5 µL of template DNA were used. The cycling conditions in the SYBR^® Green real-time PCR were 98.0 °C (3 min, activating of Taq polymerase), followed by 40 cycles at 95.0 °C for 15 s and at 60.0 °C for 30 s. After each cycle, the light emitted by the fluorophore was measured. The melting curve was constructed from 65 to 95 °C at 0.5-°C increments with a dwell time of 5 s at each temperature. Real-time PCR results were analysed using the CFX Maestro software suite (Version: 3.1.15; Biorad Laboratories GmbH, Munich, Germany).