Mapping and recombination analysis of two moth colour mutations, Black moth and Wild wing spot, in the silkworm Bombyx mori

Abstract

Many lepidopteran insects exhibit body colour variations, where the high phenotypic diversity observed in the wings and bodies of adults provides opportunities for studying adaptive morphological evolution. In the silkworm Bombyx mori, two genes responsible for moth colour mutation, Bm and Ws, have been mapped to 0.0 and 14.7 cM of the B. mori genetic linkage group 17; however, these genes have not been identified at the molecular level. We performed positional cloning of both genes to elucidate the molecular mechanisms that underlie the moth wing- and body-colour patterns in B. mori. We successfully narrowed down Bm and Ws to ~2-Mb-long and 100-kb-long regions on the same scaffold Bm_scaf33. Gene prediction analysis of this region identified 77 candidate genes in the Bm region, whereas there were no candidate genes in the Ws region. Fluorescence in-situ hybridisation analysis in Bm mutant detected chromosome inversion, which explains why there are no recombination in the corresponding region. The comparative genomic analysis demonstrated that the candidate regions of both genes shared synteny with a region associated with wing- and body-colour variations in other lepidopteran species including Biston betularia and Heliconius butterflies. These results suggest that the genes responsible for wing and body colour in B. mori may be associated with similar genes in other Lepidoptera.

Figure 1

Phenotypes and linkage maps of the Bm and Ws mutations. (a) Phenotypes of B. mori wild type (p50T), Bm mutant (No. 908), Ws mutant (u42) and B. manderina. Arrowheads indicate the spot at the apex of the wing. (b) Linkage map of group 17. The loci are labelled based on their position in centimorgan units (left) and the locus name (right). Abbreviations: Bm, Black moth; Ws, Wild wing spot; ow, waxy translucent; bts, brown head and tail spot; nm-g, non-molting glossy; nsd-2, non-susceptibility to DNV-2; Suc-1, sucrase-1 (Banno et al., 2010).

Figure 2

Mapping of the Bm and Ws mutations in linkage group 17. (a) Physical map and scaffold of linkage group 17. Black and grey lines indicate the physical map and the scaffold, respectively. The upper and lower figures indicate the whole and upstream regions of linkage group 17, respectively. The upper numbers indicate the positions that correspond to each gene (Ito et al., 2008, 2009, 2010; Niwa et al., 2010). nsd-2 could not be mapped onto the physical map of group 17, because it was located on a non-mapped scaffold (Bm_scaf131). ow was mapped to the Bm_scaf154 (Ito et al., 2009). (b) The narrowed down candidate region on the Bm_scaf33. The dotted arrows indicate the results of the detailed linkage analysis to narrow down the region linked to the Bm (upper) and Ws (lower) mutations (Table 2). The black boxes are candidate regions of each mutations.

Figure 3

Inversion in chromosome 17 of B. mori No. 908 strain carrying the Black moth loci. The inverted order of FISH signals between 4D3C (yellow) and 1G10A (red) is apparent compared with the p50 strain. BAC codes are shown in the same colours as the signals. Marker sequence (see Yasukochi et al. 2006) or GenBank accession numbers for the BACs are shown on the left of the black bar, which represents B. mori chromosome 17 drawn to a relative scale in Mb taken from KAIKObase. White and black scale bars represent 5 μm and 5 Mb for the bivalents and chromosome 17, respectively. See Table 1 for details of the BAC probe information.

Figure 4

Localisation of Bm and Ws regions narrowed down to the Bm_scaf33 and their positional relationships with candidate regions of carbonaria, HmSb and HmYb. The brown, purple, black, blue and green bars indicate the Bm, Ws, carbonaria, HmSb and Hmyb regions, respectively. The red bar indicates the overlapping region for all genes. The carbonaria gene determines the phenotype of industrial melanism in the British peppered moth, B. betularia (van't Hof et al., 2011). The HmSb and HmYb genes exhibit phenotypes with a hindwing margin and a yellow hindwing bar in H. melpomene, respectively (Ferguson et al., 2010). Lower bars indicate 25 predicted genes. A, BGIBMGA005665; B, 005664; C, 005663; D, 005548; E, 005662; F, 005661; G, 005549; H, 005660; I, 005550; J, 005659; K, 005551; L, 005658; M, 005552; N, 005553 and 005554; O, 005555; P, 005556; Q, 005657; R, 005656; S, 005557; T, 005655; U, 005558; V, 005654; W, 005653; and X, 005652. The letters correspond to Supplementary Figure S3 and Suppementary Table S3.

Figure 5

RT-PCR analysis of the candidate genes of Bm and Ws. Stage-specific expression profiles of three candidate genes, BGIBMGA005658 (L), 005657 (Q) and 005655 (T), were investigated with p50T (wild type), No. 908 (Bm mutant) and u42 (Ws mutant) strains. P and A indicate pupa and adult, respectively. The numbers under the P and A bars show the day for each stage. 18S ribosomal RNA was used as an internal control.