Mutations in a β-group of solute carrier gene are responsible for egg and eye coloration of the brown 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 {beta}-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 (G0) 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. HighlightO_LIResponsible region for the brown egg 4 (b-4) mutation was narrowed down by double digest restriction-site associated DNA sequencing (ddRAD-seq). C_LIO_LIThe gene structure was disrupted in one of the candidate genes, Bombyx mori mahogany (Bmmah), in the b-4 mutant strains. C_LIO_LICRISPR/Cas9-mediated gene knockout and complementation test confirmed that the Bmmah is responsible for the b-4 phenotypes. C_LIO_LIThe Bmmah encoded a putative amino acid transporter that belongs to the {beta}-group of solute carrier family. C_LIO_LIThe Bmmah gene is essential for normal colorization of eggs, compound eyes, and ganglions. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/436376v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10d1d56org.highwire.dtl.DTLVardef@12df5org.highwire.dtl.DTLVardef@1703701org.highwire.dtl.DTLVardef@2b5bcf_HPS_FORMAT_FIGEXP M_FIG C_FIG