Samia cynthia versus Bombyx mori: Comparative gene mapping between a species with a low-number karyotype and the model species of Lepidoptera

https://doi.org/10.1016/j.ibmb.2011.02.005Get rights and content

Abstract

We performed gene-based comparative FISH mapping between a wild silkmoth, Samia cynthia ssp. with a low number of chromosomes (2n = 25–28) and the model species, Bombyx mori (2n = 56), in order to identify the genomic components that make up the chromosomes in a low-number karyotype. Mapping of 64 fosmid probes containing orthologs of B. mori genes revealed that the homologues of either two or four B. mori chromosomes constitute the S. c. ricini (Vietnam population, 2n = 27♀/28♂, Z0/ZZ) autosomes. Where tested, even the gene order was conserved between S. c. ricini and B. mori. This was also true for the originally autosomal parts of the neo-sex chromosomes in S. c. walkeri (Sapporo population, 2n = 26♀/26♂, neo-Wneo-Z/neo-Zneo-Z) and S. cynthia subsp. indet. (Nagano population, 2n = 25♀/26♂, neo-WZ1Z2/Z1Z1Z2Z2). The results are evidence for an internal stability of lepidopteran chromosomes even when all autosomes had undergone fusion processes to form a low-number karyotype.

Graphical abstract

Highlights

► Fosmid-FISH has been applied for the first time to insect chromosomes identification and gene mapping. ► The gene-based comparative FISH mapping revealed the relationship between genomic components of Samia cynthia subspecies with reduced chromosome numbers (2n = 25–28) and Bombyx mori (2n = 56). ► Homologues of either two or four different B. mori chromosomes constitute each chromosome of the S. c. ricini low-number karyotype. ► Our results add another evidence for the internal stability of genomic regions of lepidopteran chromosomes even in species with a low-number karyotype.

Introduction

Genome rearrangements among species have been recently revealed by comparative genomic analyses. These showed that mammalian genomes have changed by reshuffling chromosomal segments from the common ancestral karyotype (Ferguson-Smith and Trifonov, 2007), while birds show a high degree of collinearity among species (Ellegren, 2010). In insects, comparative genome analysis among genome-sequenced 12 Drosophila species revealed that many orthologous genes mapped to the corresponding chromosomal arms but gene orders were scrambled between species (Ranz et al., 2001, Bhutkar et al., 2008). The availability of fully sequenced genomes provides an opportunity to study chromosomal rearrangements and evolutionary relationship among related species in detail.

Lepidoptera, moths and butterflies, consist of more than 150,000 species. They are the second largest order of animals (Kristensen and Skalski, 1999, Grimaldi and Engel, 2005). Lepidoptera have holokinetic chromosomes like aphids and bugs, and share the sex chromosome system of female heterogamety with caddis flies (Trichoptera) (Traut et al., 2007). The most common chromosome numbers of Lepidoptera range from n = 28 to n = 32 among the more than 1000 species investigated (Robinson, 1971). There are, however, also species with lower or higher number karyotypes in Lepidoptera. Species with low or high-number karyotypes are thought to have evolved by chromosomal fusion and fission from the putative ancestor with n = 31 chromosome number (Lukhtanov, 2000, Marec et al., 2010).

Since the first lepidopteran genome, that of Bombyx mori, has been accessible in public databases, comparative mapping of genes was carried out against B. mori (n = 28) by either linkage analysis (Beldade et al., 2009) or BAC-FISH (Yasukochi et al., 2009). These authors detected a high degree of conserved synteny and gene order between B. mori and two other species with the same chromosome number, Bicyclus anynana and Manduca sexta. Pringle et al. (2007) detected conserved gene order in Heliconius melpomene and suggested simple fusion events to account for the reduced chromosome number (n = 21) of this species. These studies suggested an internal stability of lepidopteran chromosomes. DNA sequencing of each 15 selected BACs from Helicoverpa armigera and Spodoptera frugiperda also revealed a high degree of conserved synteny with only a few rearrangements between B. mori and the two noctuid moths, both of them having the supposed ancestral chromosome number (n = 31) of Lepidoptera (d’Alençon et al., 2010).

Samia cynthia, a wild silkmoth, belongs to the family Saturniidae which is closest to the family Bombycidae including B. mori. S. cynthia has a reduced chromosome number, ranging from 2n = 25 to 2n = 28 and is about half that of B. mori. The variation is due to the variable sex chromosome constitution among geographic subspecies (Yoshido et al., 2005b). Previous study proposed repeated autosome-sex chromosome fusions resulted in the variable sex chromosome constitution as found in S. cynthia subspecies (Yoshido et al., 2010). The sex chromosome constitution in S. c. walkeri (the ailanthus silkworm, Sapporo population) females is designated as neo-W and neo-Z chromosomes, which originated by fusion of the ancestral W and Z with an autosome pair (A1). Then sex chromosome constitution (designated as neo-WZ1Z2) in S. cynthia subsp. indet. (the shinju silkworm, Nagano population) females has been formed by next evolutionary step, in which neo-W chromosome fused with an autosome (A2) and, consequently, its unfused homologue became a Z2 chromosome. In S. c. ricini (the Eri silkworm, Vietnam population), no such fusion of sex chromosomes with autosomes occurred and, the sex chromosome constitution in females is Z0, which arose from ancestral WZ by a loss of the W chromosome. Hence, the chromosome number is 2n = 27/28 in S. c. ricini with a Z0/ZZ, 2n = 26/26 in S. c. walkeri with neo-Wneo-Z/neo-Zneo-Z, and 2n = 25/26 in S. cynthia subsp. indet. with neo-WZ1Z2/Z1Z1Z2Z2 sex chromosomes in female/males.

We show here by comparative gene mapping between S. cynthia subspecies and B. mori the internal stability of lepidopteran chromosomes even when low-chromosome-number karyotypes evolve by chromosome fusion. We constructed a fosmid-library of S. cynthia and carried out gene-based comparative FISH mapping between B. mori and the three S. cynthia subspecies. Sixty-four fosmid probes which contain orthologs of B. mori genes, cytogenetically identified all chromosomes of the S. c. ricini. Furthermore, fosmid-FISH mapping identified the gene order of the neo-sex chromosomes in S. c. walkeri and S. cynthia subsp. indet.

Section snippets

Insects

S. cynthia was originally collected at three different locations: S. c. walkeri in Sapporo, Japan, S. cynthia subsp. indet. in Nagano, Japan, and S. c. ricini in Vietnam (for details, see Yoshido et al., 2005b). For rearing, we released the hatched larvae on an Ailanthus altissima tree in a field of the Field Science Center for Northern Biosphere, Hokkaido University (Sapporo, Japan).

Construction of a S. cynthia fosmid library

A fosmid library was constructed from female pupae of S. c. walkeri (Sapporo population). High molecular weight

Isolation of S. cynthia fosmid clones containing orthologs of B. mori genes

We isolated a total of 71 fosmid clones by PCR-based screening using 66 STS primer sets (Table S1). The fosmid clones carrying the orthologs of B. mori genes were selected from each of the 27 B. mori autosomes and the Z chromosome (Table 1).

Identification of individual chromosomes in S. cynthia ricini

To identify individual bivalents in S. c. ricini, we carried out FISH mapping on pachytene nuclei using the S. c. walkeri fosmid probes. In total, 64 fosmid clones mapped to single locations of S. c. ricini chromosomes (Fig. 1, Table 1). Although the clones

Discussion

We show here that each chromosome of S. cynthia can be reliably identified by FISH, using fosmid probes. A selected set of 22 probes from a genomic fosmid library was sufficient to karyotype S. cynthia (Fig. 2). Fosmid clones have been used as reliable cytogenetical markers for FISH karyotyping in animals and plants (Zhang et al., 2008, Dalzell et al., 2009, Liu et al., 2010). However, this is the first application of fosmid-FISH in Lepidoptera and – as far as we know – in all insect. Because

Acknowledgements

We thank W. Traut (Lübeck, Germany) for critical reading the manuscript and valuable suggestions. Our thanks also go to Y. Yamada (Sapporo, Japan) for supporting to rear Samia cynthia, T. Fujii (Tokyo, Japan) for providing S. c. ricini, and M. Tanaka-Okuyama, H. Ohta, E. Igari, H. Hoshida (Tsukuba, Japan) for preparing fosmid library. A.Y. received a grant 19–1114 of Japan Society for the Promotion of Science (JSPS). We acknowledge the research fellowship 21-7147 given to K.S. from JSPS.

References (47)

  • H. Ellegren

    Evolutionary stasis: the stable chromosomes of birds

    Trends Ecol. Evol.

    (2010)
  • The International Silkworm Genome Consortium

    The genome of a lepidopteran model insect, the silkworm Bombyx mori

    Insect Biochem. Mol. Biol.

    (2008)
  • K.W. Wolf

    The structure of condensed chromosomes in mitosis and meiosis of insects

    Int. J. Insect Morphol. Embryol.

    (1996)
  • K.P. Arunkumar et al.

    WildSilkbase: an EST database of wild silkmoths

    BMC Genomics

    (2008)
  • N. Backström et al.

    Genetic mapping in a natural population of collared flycatchers (Ficedula albicollis): conserved synteny but gene order rearrangements on the avian Z chromosome

    Genetics

    (2006)
  • N. Backström et al.

    A gene-based genetic linkage map of the collared flycatcher (Ficedula albicollis) reveals extensive synteny and gene-order conservation during 100 million years of avian evolution

    Genetics

    (2008)
  • P. Beldade et al.

    A gene-based linkage map for Bicyclus anynana butterflies allows for a comprehensive analysis of synteny with the lepidopteran reference genome

    PLoS Genet.

    (2009)
  • A. Bhutkar et al.

    Chromosomal rearrangement inferred from comparison of 12 Drosophila genomes

    Genetics

    (2008)
  • J. Cabrero et al.

    Location and expression of ribosomal RNA genes in grasshoppers: abundance of silent and cryptic loci

    Chromosome Res.

    (2008)
  • E. d’Alençon et al.

    Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high rates of local genome rearrangements

    Proc. Natl. Acad. Sci. U.S.A.

    (2010)
  • P. Dalzell et al.

    Standardized reference ideogram for physical mapping in the saltwater crocodile (Crocodylus porosus)

    Cytogenet. Genome Res.

    (2009)
  • P.M. Datson et al.

    Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism?

    Chromosome Res.

    (2006)
  • J. De Prins et al.

    Karyology and sex determination

  • J. Dubcovsky et al.

    Ribosomal RNA multigene loci: nomads of the Triticeae genomes

    Genetics

    (1995)
  • M.A. Ferguson-Smith et al.

    Mammalian karyotype evolution

    Nat. Rev. Genet.

    (2007)
  • H. Fujiwara et al.

    Stability and telomere structure of chromosomal fragments in two different mosaic strains of the silkworm, Bombyx mori

    Zool. Sci.

    (2000)
  • D.K. Griffin et al.

    Whole genome comparative studies between chicken and turkey and their implications for avian genome evolution

    BMC Genomics

    (2008)
  • D.A. Grimaldi et al.

    Evolution of the Insects

    (2005)
  • N.P. Kristensen et al.

    Phylogeny and palaeontology

  • C. Liu et al.

    Karyotyping in melon (Cucumis melo L.) by cross-species fosmid fluorescence in situ hybridization

    Cytogenet. Genome. Res.

    (2010)
  • V.A. Lukhtanov

    Sex chromatin and sex chromosome systems in non-ditrysian Lepidoptera (Insecta)

    J. Zool. Syst. Evol. Res.

    (2000)
  • F. Marec et al.

    Meiotic pairing of sex chromosome fragments and its relation to atypical transmission of a sex-linked marker in Ephestia kuehniella (Insecta: Lepidoptera)

    Heredity

    (2001)
  • F. Marec et al.

    Rise and fall of the W chromosome in Lepidoptera

  • Cited by (0)

    View full text