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

Carabajal Paladino LZ, Nguyen P, Síchová J, Marec F - BMC Genet. (2014)

Bottom Line: Using 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.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.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: 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). 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.

Results: Using 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.

Conclusions: We 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.

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Mapping of genes on chromosome preparations from testes of the codling moth, Cydia pomonella. Chromosomes were counterstained by DAPI (light blue). Hybridization signals (green, red, and violet) mark the physical position of genes under study (Ace-1, Nan, RpP0, and Notch). (a) Ace-1 localized on one bivalent in the pachytene spermatocyte complement by TSA-FISH; note two discrete but unbalanced (one large and one small) hybridization signals, each representing the Ace-1 locus on one homologous chromosome. (b) TSA-FISH with the Ace-1 probe revealed a nearly middle position of the Ace-1 locus on two largest elements of male mitotic metaphase corresponding to the Z chromosomes. (c) Two Z-linked genes, RpP0 and Nan, were mapped to the Z chromosome bivalent in the pachytene nucleus by BAC-FISH. Subsequent TSA-FISH localized the Ace-1 gene between these two markers. (d) The Notch gene mapped to a subterminal region of the Z chromosome bivalent in pachytene chromosomes by TSA-FISH. (e) Two single copy genes, Ace-1 and Notch, mapped to the Z chromosome bivalent in pachytene chromosomes by two-colour TSA-FISH. (Scale bar: 10 µm.)
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Figure 1: Mapping of genes on chromosome preparations from testes of the codling moth, Cydia pomonella. Chromosomes were counterstained by DAPI (light blue). Hybridization signals (green, red, and violet) mark the physical position of genes under study (Ace-1, Nan, RpP0, and Notch). (a) Ace-1 localized on one bivalent in the pachytene spermatocyte complement by TSA-FISH; note two discrete but unbalanced (one large and one small) hybridization signals, each representing the Ace-1 locus on one homologous chromosome. (b) TSA-FISH with the Ace-1 probe revealed a nearly middle position of the Ace-1 locus on two largest elements of male mitotic metaphase corresponding to the Z chromosomes. (c) Two Z-linked genes, RpP0 and Nan, were mapped to the Z chromosome bivalent in the pachytene nucleus by BAC-FISH. Subsequent TSA-FISH localized the Ace-1 gene between these two markers. (d) The Notch gene mapped to a subterminal region of the Z chromosome bivalent in pachytene chromosomes by TSA-FISH. (e) Two single copy genes, Ace-1 and Notch, mapped to the Z chromosome bivalent in pachytene chromosomes by two-colour TSA-FISH. (Scale bar: 10 µm.)

Mentions: TSA-FISH allowed the detection of unique sequences as short as ~1300 bp on both mitotic and meiotic chromosomes of the codling moth. As previously reported, this technique provided unbalanced signals, i.e. signals, which often differed in their intensity between homologous loci or signals that were observed only on one of the homologous chromosomes (Figure 1a, b, d and Figure 1e, respectively), most likely due to chromatin structure [28,42]. However, this obstacle is circumvented as pachytene bivalents formed by paired homologues are usually used for chromosome mapping in Lepidoptera [43-48]. The signal frequency, defined as a proportion of chromosome complements bearing at least one signal per bivalent, was higher than 60%. TSA-FISH thus represents a reliable means for detection of unique sequences on lepidopteran chromosomes.


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

Carabajal Paladino LZ, Nguyen P, Síchová J, Marec F - BMC Genet. (2014)

Mapping of genes on chromosome preparations from testes of the codling moth, Cydia pomonella. Chromosomes were counterstained by DAPI (light blue). Hybridization signals (green, red, and violet) mark the physical position of genes under study (Ace-1, Nan, RpP0, and Notch). (a) Ace-1 localized on one bivalent in the pachytene spermatocyte complement by TSA-FISH; note two discrete but unbalanced (one large and one small) hybridization signals, each representing the Ace-1 locus on one homologous chromosome. (b) TSA-FISH with the Ace-1 probe revealed a nearly middle position of the Ace-1 locus on two largest elements of male mitotic metaphase corresponding to the Z chromosomes. (c) Two Z-linked genes, RpP0 and Nan, were mapped to the Z chromosome bivalent in the pachytene nucleus by BAC-FISH. Subsequent TSA-FISH localized the Ace-1 gene between these two markers. (d) The Notch gene mapped to a subterminal region of the Z chromosome bivalent in pachytene chromosomes by TSA-FISH. (e) Two single copy genes, Ace-1 and Notch, mapped to the Z chromosome bivalent in pachytene chromosomes by two-colour TSA-FISH. (Scale bar: 10 µm.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255786&req=5

Figure 1: Mapping of genes on chromosome preparations from testes of the codling moth, Cydia pomonella. Chromosomes were counterstained by DAPI (light blue). Hybridization signals (green, red, and violet) mark the physical position of genes under study (Ace-1, Nan, RpP0, and Notch). (a) Ace-1 localized on one bivalent in the pachytene spermatocyte complement by TSA-FISH; note two discrete but unbalanced (one large and one small) hybridization signals, each representing the Ace-1 locus on one homologous chromosome. (b) TSA-FISH with the Ace-1 probe revealed a nearly middle position of the Ace-1 locus on two largest elements of male mitotic metaphase corresponding to the Z chromosomes. (c) Two Z-linked genes, RpP0 and Nan, were mapped to the Z chromosome bivalent in the pachytene nucleus by BAC-FISH. Subsequent TSA-FISH localized the Ace-1 gene between these two markers. (d) The Notch gene mapped to a subterminal region of the Z chromosome bivalent in pachytene chromosomes by TSA-FISH. (e) Two single copy genes, Ace-1 and Notch, mapped to the Z chromosome bivalent in pachytene chromosomes by two-colour TSA-FISH. (Scale bar: 10 µm.)
Mentions: TSA-FISH allowed the detection of unique sequences as short as ~1300 bp on both mitotic and meiotic chromosomes of the codling moth. As previously reported, this technique provided unbalanced signals, i.e. signals, which often differed in their intensity between homologous loci or signals that were observed only on one of the homologous chromosomes (Figure 1a, b, d and Figure 1e, respectively), most likely due to chromatin structure [28,42]. However, this obstacle is circumvented as pachytene bivalents formed by paired homologues are usually used for chromosome mapping in Lepidoptera [43-48]. The signal frequency, defined as a proportion of chromosome complements bearing at least one signal per bivalent, was higher than 60%. TSA-FISH thus represents a reliable means for detection of unique sequences on lepidopteran chromosomes.

Bottom Line: Using 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.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.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: 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). 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.

Results: Using 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.

Conclusions: We 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.

Show MeSH
Related in: MedlinePlus