Larval phenotype of the wild type Dazao, bd and bdf.

The epidermis of bd is dark black gray and the epidermis of bdf is light black gray at 5th instar day 3 of silkworm. The bar indicates 1 centimeter.

Positional cloning of the bd locus

(A) Using 532 BC1 individuals to map the bd locus between the PCR marker C and I. The numbers above DNA markers indicate recombination events. (B) Using 1162 BC1individulas to narrow the bd locus to ∼400 kb region. (C) The partial enlarged view of responsible region of bd. In this region contains 4 predicted genes, BGIBMGA012517, BGIBMGA012518, BGIBMGA012519 and BGIBMGA014589. (D) The analysis of nucleotide differences of bd responsible region. The green block indicated the deletion of the genome in bd mutants. The grey lines indicated the SNP and the red lines indicated the Indel of bdfmutants.

Spatiotemporal expression pattern of Bm-mamo.

(A) Temporal expression of Bm-mamo. The molting stage and adult stage this gene is (B) M: moulting, W: wandering stage, P: Pupal stage, A: adult stage. The 1th to 5 th denote the first instar of larvae to fifth instar of larvae, respectively. (B) Tissues expression of the 4th instar molting larvae. The Bm-mamo have relatively high expression level in midgut, head and epidermis. (C) Detailed analysis of Bm-mamo at the 4th larval stage in epidermis of Dazao strain. The Bm-mamo is up-regulation expression during molting stage. HCS indicates head capsule stage.

Effects of Bm-mamo siRNA injection on pigmentation formation.

(A) The siRNA was introduced by microinjection followed by electroporation. The “+” and “-” indicate the positive and negative poles of the electrical current. Scale bars, 1 cm (B) Partial magnification of siRNA experimental group and negative control group. Scale bars, 1 cm (C) and (D) Relative expression levels of Bm-mamo in a negative control and RNAi group as determined by quantitative RT–PCR analysis. The means ± s.d. Numbers of samples are shown at the upper right in each graph. **P<0.01, paired Student’s t-test. (NS, not significant).

Disruption of Bm-mamo using CRISPR/CAS9.

(A) Larval phenotype in G3 forth instar larva of Dazao targeted for Bm-mamo. Scale bars, 1 cm (B) The after 36 hours of laying eggs, the pigmentation of the eggs indicates that the eggs produced by knockout homozygous females cannot undergo normal pigmentation and development. (C) Genomic structure of Bm-mamo. Open reading frame (blue), untranslated region (black). The gRNA 1 and gRNA 2 sequences are shown. (D) Sequences of the Bm-mamo knock out individuals. Line 1 and line 2 deletion 15 and 71 bp, respectively.

The DNA recognize sequence logo of Bm-mamo protein and the analysis of downstream target genes.

(A) The DNA recognize sequence logo of Bm-mamo-s protein. (B) The DNA recognize sequence logo of Bm-mamo-L protein. (C) According to MEME’s FIMO program package, potential downstream target genes of Bm-mamo protein in the silkworm genome were identified.

The melanin metabolism pathway and Fluorescence quantitative PCR of relative genes.

(A) The melanin metabolism pathway. The blue indicates amino acid and catecholamine, the red indicates the name of genes, the black indicates the name of enzymes. (B) Relative expression levels of eight genes in heterozygous type of Bm-mamo knockout group (blue) and homozygous Bm-mamo knockout group (red) as determined by quantitative RT–PCR analysis. The means ± s.d. **P<0.01, paired Student’s t-test.

The expression level of cuticular protein genes were detected by qRT-PCR. The means ± s.d. **P<0.01, paired Student’s t-test.