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 Eurasian origin, but during last 200 years dispersed around the world Key pest in orchards:

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

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

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 chromosomes 2n = 56 Fukova I, Nguyen P, Marec F (2005) Genome 48: 1083-1092.

9. Tools of molecular cytogenetics for W-chromosome identification

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

11. WZ Codling moth W-chromosome painting probe 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 FM CpW2-EcoRI Cpper 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. Type of sequence Number of selected BAC clones W-specific3 Z-linked genes Per2 Ket2 Ldh4 Tpi4 Z-enriched39 Total: 54 BAC clones selected from CM BAC-library by means of sex-chromosome sequences MagdaMartina

16. W-chromosome mapping by BAC-FISH Probes:BAC clones selected using sequences isolated by laser microdissection of W-body CpW2, CpW5, and CpW27 Martina

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

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

19. Z-chromosome mapping by BAC-FISH 1.Search for orthologs of Bombyx mori Z-linked genes in codling moth - isolated genes: Period Kettin Apterous Triosephosphate isomerase (Tpi) Lactate dehydrogenase (Ldh) 2.Z-BACs selected from CM BAC-library 3.Z-BAC-FISH mapping Petr Results suggest conserved synteny

20. 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 Jindra Magda

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

22. Thank you …