The Bombyx mori karyotype and the assignment of linkage groups

Abstract

Lepidopteran species have a relatively high number of small holocentric chromosomes (Bombyx mori, 2n = 56). Chromosome identification has long been hampered in this group by the high number and by the absence of suitable markers like centromere position and chromosome bands. In this study, we carried out fluorescence in situ hybridization (FISH) on meiotic chromosome complements using genetically mapped B. mori bacterial artificial chromosomes (BACs) as probes. The combination of two to four either green or red fluorescence-labeled probes per chromosome allowed us to recognize unequivocally each of the 28 bivalents of the B. mori karyotype by its labeling pattern. Each chromosome was assigned one of the already established genetic linkage groups and the correct orientation in the chromosome was defined. This facilitates physical mapping of any other sequence and bears relevance for the ongoing B. mori genome projects. Two-color BAC-FISH karyotyping overcomes the problem of chromosome recognition in organisms where conventional banding techniques are not available.

Figure 1.—

Detection of the WZ bivalent (Z is linkage group 1) using Z-BACs 9A5H, 14I7D, 5H3E, and a female-derived whole-genomic probe for the W chromosome (A). Red signals are from Z-BACs and green signals are from the whole-genomic probe. The chromosomes are counterstained with DAPI (light blue). The arrow points to the heterochromatic block of an autosomal bivalent (see text). N, nucleolus. Bar, 10 μm. (B and C) Linkage map with RAPD loci (in black) from Yasukochi (1998) and the map positions of BACs (in red) corresponding to the signals on the Z chromosome.

Figure 2.—

Identification of individual chromosomes and comparison with the linkage map. Note that the lengths of chromosome bivalents cannot be compared in this figure as they are derived from different cells. Numbering of linkage groups follows Fujii et al. (1998) and Yasukochi (1998) (for details see Yasukochi et al. 2005). Linkage maps show RAPD loci from Yasukochi (1998) (in black), including the markers used to select the BACs for FISH (red, green, or yellow text on the right). The sites of BAC-FISH signals correspond nearly always with those map positions. For the BACs used, see Table 1.

Figure 2.—

Identification of individual chromosomes and comparison with the linkage map. Note that the lengths of chromosome bivalents cannot be compared in this figure as they are derived from different cells. Numbering of linkage groups follows Fujii et al. (1998) and Yasukochi (1998) (for details see Yasukochi et al. 2005). Linkage maps show RAPD loci from Yasukochi (1998) (in black), including the markers used to select the BACs for FISH (red, green, or yellow text on the right). The sites of BAC-FISH signals correspond nearly always with those map positions. For the BACs used, see Table 1.

Figure 3.—

BAC-FISH karyotype of B. mori. (A) An oocyte pachytene nucleus. Bar, 10 μm. (B) The bivalents from this nucleus arranged according to their lengths together with their diagnostic representation.