Probing the W chromosome of the codling moth, Cydia pomonella, with sequences from microdissected sex chromatin.
The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.
Fuková
Iva
I
Institute of Entomology, Biology Centre, ASCR, Branisovská 31, 370 05 Ceské Budejovice, Czech Republic.
Traut
Walther
W
Vítková
Magda
M
Nguyen
Petr
P
Kubícková
Svatava
S
Marec
Frantisek
F
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D020223:Chromosome Painting / Q000379:methods; D017931:DNA Primers; D017404:In Situ Hybridization, Fluorescence; D042282:Microdissection; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics*; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012728:Sex Chromatin / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012730:Sex Chromosomes / Q000235:genetics*; D013045:Species Specificity
D017931:DNA Primers; D015335:Molecular Probes; D009841:Oligonucleotides