2.4 Mutation analysis and screening of homozygous mutation line
Microinjected eggs were placed in incubator to hatch, the neonates we maintained in the artificial diet and maintained until pupation. After the yellow peach moth emergence, one hind leg was removed to extract DNA. Then the adults were individually placed in a small plastic box with code until to use, and 10% honey water was provided. Genomic DNA was extracted using the GenoDirect PCR kit following the manufacturer’s instruction (Herogen Biotech, Co. Ltd, China). According to the GOBP1 and GOBP2 sgRNA locations within each gene, the forward and reverse primers were designed to detect the deletion of the gene, respectively (Figure 1). The gene deletion was ensured by using pair of primers and expected to amplify a small fragment of GOBP1 and GOBP2 (about 350 bp and 600 bp). A pair of specific primers are developed for each gene to determine if the gene deletion mutation is homozygous or heterozygous (Figure 1). Genotypes of the gene deletion mutation can be discriminated according to the banding pattern of PCR amplified products, and all PCR primers showed in (Supplementary Table S1). The screened moths with the same banding pattern, which small fragments of genomic we expected, was mixed and allowed to produce homozygous mutants.