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Fig. 3 varo is allelic to mitfa. (a) Association mapping by whole-genome resequencing placed varo on Chr6 in the vicinity of mitfa (upper). Only two potentially deleterious variants were identified (red circles, lower), both perfectly associated with phenotype and both in exons of mitfa. Numbered, anonymous loci indicate Ensembl transcript identifiers (ensdart…). (b) Two missense substitutions in varo are shown in red (nucleotide coordinates reference GRCz11). Additional alleles generated in this study are shown in black (see main text), and previously identified alleles (Johnson et al. 2011; Lister et al. 2001, 1999) are shown in grey (ts, temperature sensitive). The locations of AltR CRISPR/Cas9 targets are shown below the gene body with AltR451, AltR465 used to generate a deletion allele (see main text). pr, conserved promoter element (Baranasic et al. 2022) (c) Alignment illustrating near-invariance of the basic region of the DNA-binding domain across species. Sequence translation from the second zebrafish Mitf gene, mitfb, is included. Histidine residues are boxed for species reported to have ENU-induced or natural His→Arg missense substitutions, as in varo. Arrowheads indicate sites of missense mutations with dominant negative activities (Hallsson et al. 2000; Hansdottir et al. 2004; Hemesath et al. 1994; Philipp et al. 2011; Yamamoto et al. 2024). (d) Mice that are wild-type, heterozygous or homozygous for an H209R allele (reproduced with permission from Hansdottir et al. 2004). (e) Homozygous varo and homozygous varo with premature termination codon (mitfavp70rc1varo, 7 base pair deletion leading to 11 novel amino acids followed by a stop codon). Introduction of a non-sense mutation on the varo background restored xanthophores in larvae (arrowheads) and adults (insets) when homozygous and abolished semi-dominance when heterozygous (mitfavp70rc1varo/+). Dark spots in insets are due to iridophore iridescence. Scale bars: Upper, 200 μm; lower, 500 μm.