Schematic representation of the procedure to obtain an F2 family obtained by mating F1 fish harboring the Δ31 mutation with each other (A). Table indicates the obtained proportion of heterozygous and homozygous of F2 individuals analyzed by heteroduplex mobility assay. (B). Images of heteroduplex mobility assay (HMA) for the F2 family. Fish are distinguished by HMA using genomic DNA extracted from fin clip of each F2 fish (1st HMA) where heterozygous mutants (+/-: a, b, g, and i) were identified because they showed multiple banding patterns (C). To clearly discriminate homozygous mutants from wild-type fish (individuals that show single band in 1st HMA: c, d, e, f, h, j, and k), each PCR product from the 1st HMA was mixed with a wild-type template, then reannealed to perform the 2nd HMA. The PCR products show homozygous wild-type (c′, d′, e′, f′, h′, j′, and k′), but no homozygous mutant (expected multiple banding pattern similar to heterozygotes) (D).