Nanopore sequencing for genotyping zzyIR10230 that rescued the male sterility of ZZY10330

Fertile GFP-expressing F20 male hybrids were mated with L4 stage C. nigoni female worms in three batches. Genomic DNA was extracted from the females of 21st generation backcrossing progeny. A total of 371 F21 adult females were picked for DNA extraction. GFP-expressing F20 male hybrids were genotyped with PCR to ensure the boundary of X-linked introgression was the same with zzyIR10330, which produced the male sterility in C. nigoni background. It was expected that 100% of the F21 female worms carried the GFP-flagged zzyIR10330 as a result of sex-linked segregation of the introgression, and 50% carried the non-GFP-flagged zzyIR10230 both as a heterozygote.