Non-molting dwarf (nm-d) as a mutant of Bombyx mori with a defect in purine synthesis

There are several known non-molting mutations of the silkworm , Bombyx mori , including non-molting dwarf ( nm-d ) . Larvae with this mutation hatch normally and start eating leaves, but die before the completion of the first ecdysis . Genetic analysis of the nm-d mutation would contribute to the isolation of essential genes for the larval development of lepidopteran insects. To identify the causative gene of the nm-d locus, we conducted RNA-seq based rough mapping. Using two sets of RNA-seq data, one from a pooled sample of normal larvae, and one from a pooled sample of nm-d larvae, the nm-d locus was narrowed to a 500 kb region. Among the genes located in this region, a nm-d -specific exon loss was identified in the Bombyx homolog of the ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/Inosine 5′-monophosphate cyclohydrolase) ( BmATIC ) gene, which catalyzes the final two steps of the de novo purine biosynthetic pathway in mammals. PCR and subsequent sequencing analysis revealed that a region containing exon 9 of the BmATIC gene is deleted in the nm-d larvae. A knockout allele of the BmATIC gene ( BmATIC KO ), that was generated using the CRISPR/Cas9 system, revealed that first instar knockout larvae died while exhibiting the dark brown larval body that is a typical feature of mutants that lack uric acid in the integument . Lethal larvae resulted from crosses between +/ BmATIC KO moths. The uric acid content in the whole-body of the first instar was drastically reduced in the nm-d larvae compared to normal larvae. These results indicated that the BmATIC gene is responsible for the nm-d phenotype, and that nm-d larvae have a defect in purine biosynthesis , including uric acid. We also discuss the possibility that the BmATIC mRNA is maternally transmitted to eggs. Our results indicated that RNA-seq based mapping using pooled samples is a practical method for the identification of the causative genes of lethal mutations . The non-molting dwarf ( nm-d ) locus of Bombyx mori was mapped using RNA-seq. RNA-seq analysis of nm-d mutants identified an exon loss mutation. CRISPR/Cas9 genome editing revealed that BmATIC is responsible for the nm-d phenotype. nm-d is a lethal mutant with a defect in purine synthesis.