Unique phenotype of the metamorphosis-defective mutant Ishigameyoh (gap): Establishment of a PCR-based marker for efficacious mutant maintenance in Bombyx mori

Abstract

Ishigameyoh (gap) was identified as a spontaneous mutant of Bombyx mori with an apterous and sterile phenotype. Phenotypic observations revealed that the gap mutation affected the development of imaginal discs and the primordia of adult organs, including the wings, legs, compound eyes, and reproductive organs. Therefore, Ishigameyoh (gap) is a metamorphosis-defective mutant. We found that oogenesis depends on the gap gene, whereas spermatogenesis is independent. Ovary transplantation experiments suggest a cell-autonomous requirement for the gap gene in the process of oogenesis. The gap mutation has been maintained by crossing +^gap/gap moths as the u90 strain. However, the u90 strain is difficult to maintain because +^gap/+^gap and +^gap/gap moths cannot be distinguished phenotypically. For efficacious maintenance, we established a molecular marker that is closely linked to the gap locus. First, we localized the gap locus to chromosome 5 using phenotypic markers. Second, the gap locus was narrowed down to within a 1-Mb region using PCR-based markers. Within this 1-Mb region, we finally established a PCR-based marker that amplified different-sized products from +^gap/+^gap and gap/gap offspring of the u90 strain. This marker allows the distinction of +^gap/+^gap and +^gap/gap moths with an error rate of <1% and therefore reduces the labor needed to maintain the u90 strain. Moreover, this marker helps to identify +^gap and gap individuals before pupation, which is necessary for larvae-based genetic and physiological analyses of the gap gene.