Molecular cytogenetic Molecular cytogenetic analysis of sex analysis of sex

chromosomes in the chromosomes in the lepidopteran pestslepidopteran pests

Frantisek Marec

Laboratory of Molecular Cytogenetics Biology Centre ASCR, Institute of Entomology,

Ceske Budejovice, Czech Republic

Codling moth Codling moth ((Cydia Cydia pomonellapomonella))

Lepidoptera: Tortricidae

Eurasian origin, but during last 200 years dispersed around the world

Key pest in orchards:

… but only bisexual releasesMale-only releases not possible- genetic sexing strains not available

SIT against codling mothSIT against codling mothOkanagan-Kootenay Sterile Insect Release Program (OK-SIR) in Canada

Genetic sexing using transgenic Genetic sexing using transgenic femalesfemales

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

SIT with transgenic female strainSIT with transgenic female strain

Cold treatment

Only malesNOT GMO

IrradiatedRelease of sterile males

XX

Wild-type males

Females expressing transgene

Transgenic line

Tools for codling moth transgenesis

by Lisa G. NevenYakima Agricultural Research Laboratory, Wapato, USA

Codling moth cytogenetics

by Frantisek’s lab

Mitotic chromosomes2n = 56

Fukova I, Nguyen P, Marec F (2005) Genome 48: 1083-1092.

Tools of molecular cytogeneticsfor W-chromosome identification

Laser microdissection of W-chromatinLaser microdissection of W-chromatinfrom polyploid nuclei of female larvaefrom polyploid nuclei of female larvae

DOP-PCRamplification

Fukova I, Traut W, Vitkova M, Nguyen P, Kubickova S, Marec F (2007) Chromosoma 116: 135-145.

WZ

Codling mothCodling mothW-chromosome W-chromosome painting probepainting probe

Fukova et al. (2007) Chromosoma 116: 135-145.

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)

Sex identification by multiplex PCR

F M

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)

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

Type of sequenceNumber of selected

BAC clones

W-specific 3

Z-linked genes

Per 2

Ket 2

Ldh 4

Tpi 4

Z-enriched 39

Total: 54

BAC clones selected from CM BAC-BAC clones selected from CM BAC-librarylibrary

by means of sex-chromosome by means of sex-chromosome sequences sequences

Magda Martina

W-chromosome mapping by BAC-FISH

Probes: BAC clones selected using sequences isolated by laser microdissection of W-body

CpW2, CpW5, and CpW27

Martina

W-BAC-FISH

W27-1-BAC

W-BAC-FISH

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

Z-chromosome mapping by BAC-Z-chromosome mapping by BAC-FISHFISH

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

CreationCreation of Z-chromosome of Z-chromosome DNA DNA librarylibrary and development of Z- and development of Z-

painting probepainting 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

A low specificity of FISH with the Z-chromosome painting probe

Merged image Z-probe image

Ongoing work …

Thank you …