Fine mapping of Ekp-1, a locus associated with silkworm (Bombyx mori) proleg development

The silkworm homeotic mutant E-kp has a pair of rudimentary abdominal legs, called prolegs, in its A2 segment. This phenotype is caused by a single dominant mutation at the E-kp-1 locus, which was previously mapped to chromosome 6. To explore the possible association of Hox genes with proleg development in the silkworm, a map-based cloning strategy was used to isolate the E-kp-1 locus. Five E-kp-1-linked simple sequence repeat markers on chromosome 6 were used to generate a low-resolution map with a total genetic distance of 39.5 cM. Four additional cleaved amplified polymorphic sequence markers were developed based on the initial map. The closest marker to E-kp-1 was at a genetic distance of 2.7 cM. A high-resolution genetic map was constructed using nine BC1 segregating populations consisting of 2396 individuals. Recombination suppression was observed in the vicinity of E-kp-1. Four molecular markers were tightly linked to E-kp-1, and three were clustered with it. These markers were used to screen a BAC library. A single bacterial artificial chromosome (BAC) clone spanning the E-kp-1 locus was identified, and E-kp-1 was delimited to a region less than 220 kb long that included the Hox gene abdominal-A and a non-coding locus, iab-4. These results provide essential information for the isolation of this locus, which may shed light on the mechanism of proleg development in the silkworm and possibly in Lepidoptera.