**3.2 MSH2 gene**

The study to check possible characters hybrids between the strains analyzed was made based on an analysis of the MSH2 gene polymorphisms with the digestion of the 875bp amplification product of a region of this gene with the restriction enzyme *Hha*I [26]. The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq DNA polymerase (Thermo Fisher Scientific), 0.2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.8, 50mM KCl, 0.8% Nonidet P40), 10 pmol of each primer (tmuts30 and tmuts41). PCR reaction was carried out under the following conditions: initial denaturation at 94°C/5 min and 30 cycles (94°C/30 sec, 55°C/1 min and 72°C/2 min). The fragment obtained by PCR was then digested with the *Hha*I restriction enzyme for 16 h at 37°C. The digestion products were analyzed by polyacrylamide gel electrophoresis in 7.5%. The gels were revealed by silver impregnation (DNA Silver Staining Kit /Amersham Biosciences).
