Integrated mapping and gene editing identify BmCCP as a regulator of cocoon shape and spinning behaviour in Bombyx...

The cocoon shape in Bombyx mori (silkworm) is a construction trait shaped by cocoon-spinning behaviour, but the molecular regulation pathways remain poorly understood. Here, quantitative phenotyping, bulked segregant analysis sequencing (BSA-seq), brain transcriptomics and CRISPR/Cas9 mutagenesis were combined to identify a regulator of cocoon morphogenesis and to assess potential roles in spinning behaviour. Using representative strains L6J5 and J8, which produce short oval and long peanut-shaped cocoons, respectively, showed that cocoon shape, quantified by the cocoon aspect ratio, is a quantitative trait. BSA-seq mapped the trait to a 5.04-Mb candidate interval on chromosome 4 containing 213 annotated genes. Integration of the mapping results with brain transcriptomes from individuals with extreme cocoon phenotypes identified Bombyx mori cocoon shape-correlated protein (BmCCP) as the sole overlapping candidate gene. BmCCP was more highly expressed in the brain of strain J8 than L6J5 at the wandering stage, and the locus contained multiple associated polymorphisms. CRISPR/Cas9-mediated knockout of BmCCP in strain J8 significantly increased the cocoon aspect ratio and cocoon size. In the widely used experimental strain DaZao, which has not been artificially selected for cocoon shape, BmCCP deficiency likewise increased cocoon size and significantly reduced the larval spinning rate, while crosses with J8 further supported the role of BmCCP in regulating the cocoon aspect ratio. Together, these results identify BmCCP as a regulator of cocoon morphogenesis and provide a foundation to investigate the relationship between the cocoon morphology and spinning behaviour of the silkworm.