ENU-induced point mutations in fugu Mstn gene identified by direct sequencing. The PCR products from fugu genomic DNA screened by the HRM analysis were investigated by direct sequencing. Left side (A, C, E, G) and right side (B, D, F, H) of figures, the sequence traces of wild-type fugu (WT) and the fugu mutant (F1). Lines in green, red, black and blue; adenine, thymine, guanine and cytosine, respectively. A nucleotide in the germ cell in the founder fish (black triangles) is replaced by another nucleotide due to the ENU-treatment (red triangles), resulting in an offspring that is heterozygous at the site. The numbers mean the line of the hatched fry generated by in vitro fertilization (see Table 3).