Figure 3

The lws^a:b mutant with a single LWS. (a) Structures of the LWSa and LWSb loci in the wild type (top) and the hybrid LWSa:b locus in the lws^a:b mutant (bottom). Black and white boxes indicate the coding region and 5′/3′ UTRs, respectively. The target sites of the gRNA exist in the 2nd exons, and the genomic region in-between (~7 kb) was deleted in the lws^a:b mutant (dotted lines). Arrowheads show approximate positions of the primers used for genotyping of F₂ siblings: white, LWSa-specific forward; black, LWSb-specific forward; grey, LWSb-specific reverse (see the Methods for their sequences). (b) Genotyping of F₂ siblings (#1~9) obtained by intercrossing heterozygous F₁s. Three genotypes (i.e., wild type, heterozygote, and homozygote) could be distinguished by genomic PCR using two pairs of three primers (see the arrowheads in (a) for their positions). The white–grey pair amplifies a product (~0.6 kb) from the hybrid LWSa:b gene, whereas the black–grey pair amplifies a product from the wild-type LWSb. Thus, No. 1–5 and No. 7 are heterozygous, No. 6 and No. 8 are wild type, and No. 9 is homozygous. A full image of this gel is available as Supplementary Fig. S1. (c) The missense substitution in LWSa:b. The original sequences of LWSa and LWSb are shown at the top with translated amino acids. Black letters show the target sequence of the gRNA⁶. The sequence of LWSa:b is shown at the bottom. The substituted nucleotide (T > A) and amino acid (Y > N) are highlighted. (d) Comparison of LWSs among vertebrates. Amino-acid sequences were collected from the GenBank database and aligned. MWSs of human and mouse are paralogues of LWS. Amino acids identical to those of the medaka LWSa (top) are shown by dots. The substituted amino acid in LWSa:b is highlighted.