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.