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Molecular cytogenetic analysis of sex chromosomes in the lepidopteran pests

Molecular cytogenetic analysis of sex chromosomes in the lepidopteran pests. Franti s ek Marec Laboratory of Molecular Cytogenetics Biology Centre A SCR , Institute of Entomology, C esk e Bud e jovice, Czech Republic. Codling moth ( Cydia pomonella ). Lepidoptera: Tortricidae.

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Molecular cytogenetic analysis of sex chromosomes in the lepidopteran pests

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  1. Molecular cytogenetic analysis of sex chromosomes in the lepidopteran pests Frantisek Marec Laboratory of Molecular Cytogenetics Biology Centre ASCR, Institute of Entomology, Ceske Budejovice, Czech Republic

  2. Codling moth (Cydia pomonella) Lepidoptera: Tortricidae Key pest in orchards: Eurasian origin, but during last 200 years dispersed around the world

  3. SIT against codling moth Okanagan-Kootenay Sterile Insect Release Program (OK-SIR) in Canada … but only bisexual releases Male-only releases not possible - genetic sexing strains not available

  4. Genetic sexing using transgenic females Marec F, Neven LG, Robinson AS, Vreysen M, Goldsmith MR, Nagaraju J, Franz G (2005) J. Econ. Entomol.98: 248-259.

  5. Transgenic line X Wild-type males Females expressing transgene Cold treatment Irradiated Release of sterile males SIT with transgenic female strain Only males NOT GMO

  6. Tools for codling moth transgenesis by Lisa G. Neven Yakima Agricultural Research Laboratory, Wapato, USA

  7. Codling moth cytogenetics by Frantisek’s lab

  8. Mitotic chromosomes2n = 56 Fukova I, Nguyen P, Marec F (2005) Genome 48: 1083-1092.

  9. Tools of molecular cytogeneticsfor W-chromosomeidentification

  10. DOP-PCR amplification Laser microdissection of W-chromatinfrom polyploid nuclei of female larvae Fukova I, Traut W, Vitkova M, Nguyen P, Kubickova S, Marec F (2007) Chromosoma 116: 135-145.

  11. Codling mothW-chromosome painting probe WZ Fukova et al. (2007) Chromosoma 116: 135-145.

  12. Sex identification in CM eggs and larvae using molecular markers obtained by cloning of microdissected W-chromatin Fukova I, Neven LG, Barcenas NM, Gund NA, Dalikova M, Marec F (2009)

  13. Sex identification by multiplex PCR CpW2-EcoRI Cpper F M A fragment of Cpper gene from CM (an ortholog of period form Antheraea pernyi)- used as positive control of PCR reactions Fukova I, Neven LG, Barcenas NM, Gund NA, Dalikova M, Marec F (2009)

  14. CM sex-chromosome mapping by BAC-FISH • GOAL:construct a cytogenetic map of codling moth sex chromosomes (W and Z) • codling moth BAC library – available in Lisa’s lab • molecular markers of W and Z chromosomes prepared in Frantisek’s lab BAC = Bacterial Artificial Chromosome

  15. Magda Martina BAC clones selected from CM BAC-libraryby means of sex-chromosome sequences

  16. Martina W-chromosome mapping by BAC-FISH Probes:BACclones selected using sequences isolated by laser microdissection of W-body CpW2,CpW5,andCpW27

  17. W-BAC-FISH W27-1-BAC

  18. W-BAC-FISH W-BAC-23N12 (selected by CpW + CpW5)

  19. Petr Z-chromosome mapping by BAC-FISH • Search for orthologs of Bombyx mori Z-linked genes in codling moth - isolated genes: • Period • Kettin • Apterous • Triosephosphate isomerase (Tpi) • Lactate dehydrogenase (Ldh) • Z-BACs selected from CM BAC-library • Z-BAC-FISH mapping Results suggest conserved synteny

  20. Magda Jindra Creation of Z-chromosome DNA library and development of Z-painting probe • laser microdissection of Z chromosomes from spermatogonial metaphase chromosomes • DOP-PCR amplified samples tested as Z-probe by FISH • samples cloned, sequenced, analyzed, and some used to screen BAC-library

  21. A low specificity of FISH with the Z-chromosome painting probe Z-probe image Merged image Ongoing work …

  22. Thank you …

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