Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in the domesticated silkworm, Bombyx mori

The domesticated silkworm, Bombyx mori , and its wild progenitor, B. mandarina , are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori . Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori . To demonstrate the efficacy of targeted knockouts in B. mandarina , we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y . In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like ( apt-like ) in B. mori × B. mandarina F 1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F 1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F 1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori . Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication. Brief abstract Bombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina , we confirmed this gene”s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like ( apt-like ) in B. mori × B. mandarina F 1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication. Graphical TOC/Abstract