Extensive conserved synteny of genes between the karyotypes of Manduca sexta and Bombyx mori revealed by BAC-FISH mapping

Abstract

Background: Genome sequencing projects have been completed for several species representing four highly diverged holometabolous insect orders, Diptera, Hymenoptera, Coleoptera, and Lepidoptera. The striking evolutionary diversity of insects argues a need for efficient methods to apply genome information from such models to genetically uncharacterized species. Constructing conserved synteny maps plays a crucial role in this task. Here, we demonstrate the use of fluorescence in situ hybridization with bacterial artificial chromosome probes as a powerful tool for physical mapping of genes and comparative genome analysis in Lepidoptera, which have numerous and morphologically uniform holokinetic chromosomes.

Methodology/principal findings: We isolated 214 clones containing 159 orthologs of well conserved single-copy genes of a sequenced lepidopteran model, the silkworm, Bombyx mori, from a BAC library of a sphingid with an unexplored genome, the tobacco hornworm, Manduca sexta. We then constructed a BAC-FISH karyotype identifying all 28 chromosomes of M. sexta by mapping 124 loci using the corresponding BAC clones. BAC probes from three M. sexta chromosomes also generated clear signals on the corresponding chromosomes of the convolvulus hawk moth, Agrius convolvuli, which belongs to the same subfamily, Sphinginae, as M. sexta.

Conclusions/significance: Comparison of the M. sexta BAC physical map with the linkage map and genome sequence of B. mori pointed to extensive conserved synteny including conserved gene order in most chromosomes. Only a few rearrangements, including three inversions, three translocations, and two fission/fusion events were estimated to have occurred after the divergence of Bombycidae and Sphingidae. These results add to accumulating evidence for the stability of lepidopteran genomes. Generating signals on A. convolvuli chromosomes using heterologous M. sexta probes demonstrated that BAC-FISH with orthologous sequences can be used for karyotyping a wide range of related and genetically uncharacterized species, significantly extending the ability to develop synteny maps for comparative and functional genomics.

Figure 1. BAC-FISH pachytene karyotype of Manduca sexta.

(a) A spermatocyte pachytene complement of n = 28 bivalents, each showing 1–3 pseudocolored hybridization signals of individual BAC probes. (b) Pachytene bivalents of the same complement as in (a) arranged according to corresponding B. mori chromosome numbers (black italic numbers). (c) Identification of the sex chromosome bivalent WZ in a pachytene oocyte by genomic in situ hybridization (GISH) combined with BAC-FISH. With GISH, the female genomic probe (green signals) highlighted the whole W-chromosome thread of the WZ bivalent, while hybridization signals of the 42G01 (red) and 22D05 (cyan) BAC clones marked the Z-chromosome thread. Chromosomes were stained with DAPI fluorochrome; BAC probes were labeled with Green-dUTP (green), Orange-dUTP (yellow), Red-dUTP (red), and Cy5-dUTP (cyan). LG, linkage group of B. mori; N, nucleolus; Z, sex chromosome bivalent (ZZ). For details, see Table S5. Bar  = 10 µm.

Figure 2. Genome wide comparison of orthologous genes between individual chromosomes of Manduca sexta and Bombyx mori.

DAPI-stained images of individual pachytene bivalents of M. sexta (blue numbers) show hybridization signals of BAC probes. Vertical bars represent corresponding parts of B. mori chromosomes (black italic numbers) drawn to relative scale in Mb taken from Kaikobase. Individual images of chromosome bivalents were obtained from different pachytene complements and/or from different preparations; therefore, their lengths differed depending on the stage and do not reflect relative sizes of the bivalents (for relative sizes, see Fig. S1). BAC probe codes are shown on the right of each chromosome image (for details, see Table S5).

Figure 3. Cross-hybridization of Manduca sexta BAC probes to male pachytene bivalents of Agrius convolvuli.

(a) Z-chromosome bivalents. (b) Chromosome 5 bivalents. (c) Chromosome 15 bivalents. Each image shows an M. sexta bivalent (left) and an A. convolvuli bivalent (right) using the same set of M. sexta BAC probes. Note that in each image the A. convolvuli bivalent shows hybridization signals in the same sequence as the corresponding M. sexta bivalent, thus indicating well conserved synteny including the gene order between the two species. BAC probe codes are shown between the bivalents (for details, see Table S5). Bar  = 5 µm.