Genomic DNA Extraction and Sequencing

Genomic DNA (gDNA) was isolated from whole BPH body or forewings to determine the heterozygous or homozygous genotypes of the BPHs. gDNA was extracted from one individual adult, as reported previously [63 (link)]. Briefly, one adult was homogenized in a 0.2-ml Eppendorf tube, followed by the addition of 50 μl of extraction buffer (10 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 mM NaCl, 0.2 mg/ml proteinase K). The tubes were then incubated for 30 min at 37°C, followed by 2 min at 95°C to inactivate the proteinase K, and the supernatant solution was used directly as a template for PCR.
gDNA was isolated from forewings as reported previously [64 (link)], with slight modifications. Briefly, forewings were digested in 0.5 ml extraction buffer (0.01 M Tris-HCl, 0.01 M EDTA, 0.1 M NaCl, 0.039 M dithiothreitol, 2% sodium dodecyl sulfate, 20 μg/ml, pH 8.0) for 12h at 37°C, followed by 2 min at 95°C to inactivate proteinase K. The supernatant solution was then used directly as a template for PCR. PCR products spanning NlInR2^E4 and NlInR2^E5 sgRNA target sites were amplified from the extracted gDNA using primer pairs for E4-iF/E4-iR and E5-iF/E5-iR (S20 Table), respectively. The PCR products were then used for Sanger sequencing or subcloned into pEasy-T3 cloning vector (TransGen Biotech), and then single clones were picked for Sanger sequencing.