Fluorescence In Situ Hybridization Protocol

For the 42 males selected from 22 localities (including five specimens collected in 2005 and 2007 from two localities), the best preparations were used for fluorescence in situ hybridization. FISH was carried out as described previously^{21 (link)} using an 18S rDNA probe from orthopteran labelled through PCR with biotin-16-dUTP (Roche Diagnostics GmbH, Germany). A probe from the telomeric DNA sequence (TTAGG)_n was generated by PCR in the absence of a template, using TTAGG_F (5′- TAA CCT AAC CTA ACC TAA CCT AA-3′) and TTAGG_R (5′-GGT TAGGTT AGG TTA GGT TAG G-3′)^{29 (link)} as primers. The visualization of hybridized DNA labelled with biotin-16-dUTP or digoxigenin-11-dUTP (Roche, Diagnostics GmbH, Germany) was performed with avidin-FITC (Invitrogen, USA) or anti-digoxigenin rhodamine (Roche Diagnostics GmbH, Germany), respectively. Digital images were obtained using a CCD DS-U1 camera coupled to fluorescence microscope. The software NIS-Elements BR2 was used for camera control and the merging of DAPI and fluorochrome images of the paints.