2.2. DNA Extraction, PCR Amplification and Sequencing

Representative isolates were selected based on the sampling substrates and Eucalyptus genotypes for DNA extraction and sequence comparisons. DNA was extracted from 10-day-old cultures and mycelia were collected using a sterilized scalpel and transferred to 2 mL Eppendorf tubes. Total genomic DNA was extracted following the CTAB protocol described by van Burik and co-authors [31]. The extracted DNA was dissolved using 30 µL TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH 8.0), and 3 µL RNase (10 mg/mL) was added at 37 °C for 1 h to degrade RNA. Finally, DNA concentration was measured with a Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

According to previous research results, sequences of partial gene regions of translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) were used to successfully identify Calonectria species [26,28,32]. These four partial gene regions were amplified using the primer pairs EF1-728F/EF2, T1/CYLTUB1R, CAL-228F/CAL-2Rd and CYLH3F/CYLH3R, respectively, the PCR procedure was conducted as described by Liu and Chen [33], Lombard and co-authors [30] (Table 1).

Primers for amplification of tef1, tub2, cmdA, his3 and mating type gene fragments.

To obtain accurate sequences for each sequenced isolates, all PCR products were sequenced in forward and reverse directions by the same primers used for PCR amplification by the Beijing Genomics Institute, Guangzhou, China. All sequences obtained in this study were edited using MEGA v. 6.0.5 software [34] and were deposited in GenBank (https://www.ncbi.nlm.nih.gov).

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