2.3. Identifying Target Site Mutations in the Genes of Fungicide Target Proteins
This protocol is extracted from research article:
Fungicide Resistance Evolving in Ramularia collo-cygni Population in Estonia
Microorganisms, Jul 15, 2021; DOI: 10.3390/microorganisms9071514

Genomic DNA from R. collo-cygni isolates was extracted by the thermolysis method [45]. All the PCR reactions were done in a 25 µL volume consisting of 10.9 µL MilliQ water, 5.0 µL 5 × DreamTaq PCR buffer (Thermo Fisher Scientific, Waltham, MA, USA), 100 µM of each dNTP, 10 µM of specific forward and reverse primers (Table S1), 1 unit of DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), and 1.0 µL DNA (about 10 ng µL−1).

PCR reactions to obtain the sequences of the Sdh gene were performed using primers SdhB_Rcc_Final_F and SdhB_Rcc_Final_R for SdhB subunit and SdhC_Rcc_Final_F and SdhC_Rcc_Final_R for SdhC subunit (Table S1) [34]. The amplification was performed using the following conditions: initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 15 s, annealing at 57 °C (SdhB) or 50 °C (SdhC) for 30 s, extension at 72 °C for 1 min and a final extension at 72 °C for 10 min.

The amplification of the CYP51 gene was performed using primers KES2230 and KES2231 for amplification (Table S1) [33]. The conditions for amplification were: initial denaturation at 95 °C for 60 s, 35 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 120 s and a final elongation step at 72 °C for 5 min.

The amplification of the CytB gene was performed using primers RCCcytobF and RCCcytobR for amplification (Table S1) [30]. The conditions for amplification were: initial denaturation at 95 °C for 90 s, 35 cycles at 95 °C for 60 s, 55 °C for 45 s, 72 °C for 120 s and a final elongation step at 72 °C for 5 min.

All PCR products were sequenced using the same forward and reverse primers using an Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) in the Estonian Biocentre in Tartu. The sequences obtained were analysed against non-redundant databases and DK05 reference sequence using blastn and blastx search tools publicly available at NCBI [46]. Target-site mutations in each gene were identified.

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