Mutations in a β-group of solute carrier gene are responsible for egg and eye coloration of thebrown egg 4(b-4) mutant in the silkworm,Bombyx mori

The brown egg 4 ( b-4 ) is a recessive mutant in the silkworm ( Bombyx mori ), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the β-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany ( mah ). Accordingly, we named this gene Bmmah . We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah . Several adult moths in generation 0 (G 0 ) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes. Highlight Responsible region for the brown egg 4 ( b-4 ) mutation was narrowed down by double digest restriction-site associated DNA sequencing (ddRAD-seq). The gene structure was disrupted in one of the candidate genes, Bombyx mori mahogany ( Bmmah ), in the b-4 mutant strains. CRISPR/Cas9-mediated gene knockout and complementation test confirmed that the Bmmah is responsible for the b-4 phenotypes. The Bmmah encoded a putative amino acid transporter that belongs to the β-group of solute carrier family. The Bmmah gene is essential for normal colorization of eggs, compound eyes, and ganglions. Graphical abstract