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.

家蚕 Bombyx mori有几种已知的非蜕皮突变，包括非蜕皮矮化 ( nm-d ) 。带有这种突变的幼虫正常孵化并开始吃叶子，但在第一次蜕皮完成之前死亡。nm-d突变的遗传分析将有助于分离鳞翅目昆虫幼虫发育的必需基因。为了鉴定nm-d基因座的致病基因，我们进行了基于 RNA-seq 的粗略作图。使用两组 RNA-seq 数据，一组来自正常幼虫的汇集样本，另一组来自nm-d幼虫的汇集样本，nm-d基因座缩小到 500 kb 区域。在位于该区域的基因中，在 ATIC（5-氨基咪唑-4-羧酰胺核糖核苷酸转化酶/肌苷 5'-单磷酸环化水解酶）（BmATIC）基因的Bombyx同源物中鉴定出nm-d特异性外显子丢失，该基因催化哺乳动物从头嘌呤生物合成途径的最后两个步骤。PCR 和随后的测序分析显示，在nm-d幼虫中，含有BmATIC基因外显子 9 的区域被删除。BmATIC基因的敲除等位基因( BmATIC ^KO )，这是使用 CRISPR/Cas9 系统生成的，揭示了第一龄敲除的幼虫死亡，同时表现出深棕色的幼虫体，这是外皮中缺乏尿酸的突变体的典型特征。致死幼虫由 +/ BmATIC ^KO蛾之间的杂交产生。与正常幼虫相比，nm-d幼虫的一龄全身尿酸含量显着降低。这些结果表明BmATIC基因是造成nm-d表型的原因，并且nm-d幼虫在嘌呤生物合成方面存在缺陷，包括尿酸。我们还讨论了BmATICmRNA 是母系传递给卵子的。我们的结果表明，使用混合样本的基于 RNA-seq 的作图是一种鉴定致死突变致病基因的实用方法。