Fig. 4.

Cardinal is the causal gene that underlies the spontaneous re mutation. a) Illustration of the cardinal gene and the relative positions of the 5 sgRNAs. A 1-bp deletion (in red) in red-eyed G₁ male #1 and a 99-bp deletion (in red) in red-eyed G₁ male #3 are also shown. The 1-bp deletion was found in the other sequenced red-eyed G₁ individuals (male #1, 5, 6, and 7). None of the deletions was found in black-eyed G₁ individuals. The sgRNA sequences near the deletion sites are underlined. b) Design for germline knockout of cardinal (G₁) and the crossing scheme used to generate homozygous cardinal knockout (G₃). The wild-type (+) ere color allele is not shown and the re allele is indicated as a red box. c) G₁ black-eye (left) and red-eye (right) phenotypes. Four red-eyed G₁ females and 7 red-eyed G₁ males were identified among approximately 500 black-eyed G₁ progeny. Sequence confirmation of the knockout is shown in (a). d) Four phenotypes observed in the G₃ progeny shown in (b). From left to right: red-eyed male, black-eyed male, yellow-eyed female, black-eyed female. The G₁ red-eye male has the 99-bp deletion (G₁ red-eye male #3). (e) The eyes in the G₃ yellow-eyed female pupa turn red over the course of pupal development. E1–3: 1-, 2-, and 3-day-old pupae. To highlight the eye phenotype, the contrast was adjusted to 20% and the brightness was adjusted to 20% (for E1–2) and 40% (E3). F) One-day-old G₃ adult eye phenotypes of the red-eyed male (F1), yellow-turn-red-eyed female (F2), and black-eyed female (F3). To highlight the eye phenotypes, the contrast of images was adjusted to 25% and the brightness to 25% (F1–2) and 50% (F3). All unmodified photos are found in the Supplementary File.