Scheme of the experimental design:

For detailed information, please see: Presentation or Poster

Please note, that the set of 19 SSR markers does not include species/sub-species specific markers. The genotyping system is based on comparing SSR profiles of unknown samples with profiles characteristics for individual species/subspecies. For detailed information, please see: Christelová et al. 2011.  

PCR amplification:

19 SSR loci are amplified using specific primers that are adjusted by 5’-M13 tails to enable the use of universal fluorescently labeled primer. Four different flurophores are used for the primer labeling (6-FAM, VIC, NED, PET), allowing for subsequent multiplexing of the reactions.  The reaction is performed in a final volume of 20 μl containing 10 ng of template genomic DNA, reaction buffer (consisting of 10mM Tris-HCl (pH 8), KCl 50 mM, 0.1% Triton-X100 and 1.5 mM MgCl₂), 200 μM dNTPs (each), 1U of Taq polymerase, 8 pmol of the M13-tailed locus specific forward primer, 6 pmol of the fluorescently labeled universal M13 forward primer, 10 pmol of the locus specific reverse primer.

Amplification was performed as follows: 94°C for 5 min (1 cycle); followed by 35 cycles of denaturation (94°C/45 s), annealing at temperature corresponding to the locus–specific primer (1 min) and extension (72°C/1 min) and 72°C for 5 min (1 cycle).

Three independent PCR reactions were performed in order to improve the accuracy of alleles binning.