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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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Diagram integrating Ace-1 into a physical map of the codling moth Z chromosome. The gene-based physical map was adopted from Nguyen et al. [26]. The mean relative position of Ace-1 was calculated from physical distances between Ace-1 and RpP0 hybridization signals related to physical distances between RpP0 and Nan in 10 ZZ bivalents. Major genes conferring insecticide resistance are underlined, Ace-1 is in red.
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Figure 2: Diagram integrating Ace-1 into a physical map of the codling moth Z chromosome. The gene-based physical map was adopted from Nguyen et al. [26]. The mean relative position of Ace-1 was calculated from physical distances between Ace-1 and RpP0 hybridization signals related to physical distances between RpP0 and Nan in 10 ZZ bivalents. Major genes conferring insecticide resistance are underlined, Ace-1 is in red.

Mentions: The probe derived from partial coding sequence of the Acetylcholinesterase 1 (Ace-1) gene provided a clear and intense hybridization signal localized nearly in the middle of a male pachytene bivalent (Figure 1a). Chromosomal position of the Ace-1 was also confirmed by TSA-FISH performed on male mitotic chromosomes, which mapped Ace-1 to an interstitial region of the two largest elements in the codling moth karyotype (Figure 1b) identified previously as the Z sex chromosomes [23,26]. Comparison of the physical map of the Cydia pomonella Z chromosome with a reference genome of the silkworm Bombyx mori [26] suggests that Ace-1 is located between two anchoring loci, Ribosomal protein P0 (RpP0) and Nanchung (Nan). In order to test this prediction we combined BAC-FISH and TSA-FISH in two subsequent steps. First, BAC-derived probes containing RpP0 and Nan were hybridized to male pachytene chromosomes of the codling moth. As expected, both markers were localized interstitially on a single pachytene bivalent (Figure 1c). Then the slides were stripped and reprobed using TSA-FISH protocol. The Ace-1 gene mapped between the two anchoring genes adjacent to RpP0 (Figure 1c). Physical distances between the genes were measured using ImageJ and relative position of Ace-1 was calculated, which allowed its integration in the physical map of the codling moth Z chromosome (Figure 2).


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)

Diagram integrating Ace-1 into a physical map of the codling moth Z chromosome. The gene-based physical map was adopted from Nguyen et al. [26]. The mean relative position of Ace-1 was calculated from physical distances between Ace-1 and RpP0 hybridization signals related to physical distances between RpP0 and Nan in 10 ZZ bivalents. Major genes conferring insecticide resistance are underlined, Ace-1 is in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Diagram integrating Ace-1 into a physical map of the codling moth Z chromosome. The gene-based physical map was adopted from Nguyen et al. [26]. The mean relative position of Ace-1 was calculated from physical distances between Ace-1 and RpP0 hybridization signals related to physical distances between RpP0 and Nan in 10 ZZ bivalents. Major genes conferring insecticide resistance are underlined, Ace-1 is in red.
Mentions: The probe derived from partial coding sequence of the Acetylcholinesterase 1 (Ace-1) gene provided a clear and intense hybridization signal localized nearly in the middle of a male pachytene bivalent (Figure 1a). Chromosomal position of the Ace-1 was also confirmed by TSA-FISH performed on male mitotic chromosomes, which mapped Ace-1 to an interstitial region of the two largest elements in the codling moth karyotype (Figure 1b) identified previously as the Z sex chromosomes [23,26]. Comparison of the physical map of the Cydia pomonella Z chromosome with a reference genome of the silkworm Bombyx mori [26] suggests that Ace-1 is located between two anchoring loci, Ribosomal protein P0 (RpP0) and Nanchung (Nan). In order to test this prediction we combined BAC-FISH and TSA-FISH in two subsequent steps. First, BAC-derived probes containing RpP0 and Nan were hybridized to male pachytene chromosomes of the codling moth. As expected, both markers were localized interstitially on a single pachytene bivalent (Figure 1c). Then the slides were stripped and reprobed using TSA-FISH protocol. The Ace-1 gene mapped between the two anchoring genes adjacent to RpP0 (Figure 1c). Physical distances between the genes were measured using ImageJ and relative position of Ace-1 was calculated, which allowed its integration in the physical map of the codling moth Z chromosome (Figure 2).

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