Identification of the ENU-induced nonsense mutation in fugu TILLING. A) The high-resolution melting curve (left) and the substitution plot (right). Exon 3 of the Mstn gene, which contains the C-terminal region of the Mstn protein, was examined by HRM analysis using the fugu family 4 (see Table 3). Red curve, the deviated curve due to mutation. B) Direct sequencing for the selected mutations. The selected genomic DNA with the HRM analysis was investigated by direct sequencing. Lines in green, red, black and blue; adenine, thymine, guanine and cytosine, respectively. The sequence traces of the fugu parents (Founder-♂ and -♀), control (Control), and the ENU-induced mutant (F1) are illustrated in order from left to right. Cytosine in the parents (black triangles) is substituted to adenine in the mutant (red triangle), resulting in the change from tyrosine to a stop codon at amino acid 316. C, D) Confirmation of the nonsense mutation in fugu TILLING. PCR amplicons were cloned and sequenced to confirm the substitution of the nucleotide (C). The sequence traces of the cloned wild-type and the mutant DNA are illustrated in left (WT) and right (mutation), respectively. Cytosine to adenine nucleotide transition was recognized. The cleavage product was detected with CEL I assay in the mutant DNA (D, lane 6), but not in wild-type DNA (D, lanes 1–5). Black arrow, the cleavage product. E) Nonsense mutation in the C-terminal region of fugu Mstn. The ENU-induced point mutation in fugu TILLING caused the substitution to a stop codon in the C-terminal region. Bold characteristics, six cysteines which are essential for the Mstn conformation. X, a stop codon. Black and white triangles, amino-acid change from tyrosine to a stop codon in fugu Mstn; the change from cysteine to tyrosine in medaka Mstn responsible for the phenotype, respectively.