Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella.

Leonela Z Carabajal Paladino,Jindra Šíchová,Petr Nguyen,František Marec

doi:10.1186/1471-2156-15-s2-s15

Abstract

BackgroundWe work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth.ResultsUsing a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome.ConclusionsWe showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.

Highlights

We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT)
fluorescence in situ hybridization (FISH) with bacterial artificial chromosomes (BAC-FISH) BAC clones 40B18 and 12O03 containing the genes Nanchung (Nan) and Ribosomal protein P0 (RpP0), respectively, were obtained from the codling moth BAC library constructed by GENEfinder Genomic Resource Laboratory (Texas A&M University, College Station, TX, USA)
We adapted the protocol of TSA-FISH, i.e. fluorescence in situ hybridization coupled with tyramide signal amplification, originally customized for the localization of genes on large mitotic metaphase chromosomes of the frog Xenopus tropicalis [30], for lepidopteran chromosomes

Summary

Introduction

We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). The codling moth SIT relies on bisexual releases, but there are reasons to believe that male-only releases would bring significant improvement to this technology as it has been demonstrated in the medfly, Ceratitis capitata, where the release of irradiated males only is severalfold more efficient in inducing sterility in wild insect populations than the release of both sexes [16,17,18,19]. A new approach for genetic sexing in the codling moth has been proposed It is based on the development of a transgenic C. pomonella strain with females carrying a dominant conditional lethal mutation (DCLM) in the female-specific W chromosome. A dominant cold sensitive mutant allele of the Notch gene, originally isolated in Drosophila melanogaster, has been chosen as a suitable DCLM [21], and the codling moth transgenesis is currently under way [22]

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Conclusion