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 SSRlociare amplifiedusingspecificprimersthat are adjustedby5’-M13tailstoenabletheuseofuniversalfluorescentlylabeledprimer.Fourdifferentflurophores are usedfortheprimerlabeling(6-FAM,VIC,NED,PET),allowingforsubsequentmultiplexingofthereactions. Thereaction is performedinafinalvolumeof20μlcontaining10ngoftemplategenomicDNA,reactionbuffer(consistingof10mMTris-HCl(pH8),KCl50mM,0.1%Triton-X100and1.5mMMgCl2),200μMdNTPs(each),1UofTaqpolymerase,8pmoloftheM13-tailedlocusspecificforwardprimer,6pmolofthefluorescentlylabeleduniversalM13forwardprimer,10pmolofthelocusspecificreverseprimer.
Amplificationwasperformedasfollows:94°Cfor5min (1 cycle);followedby35cyclesofdenaturation(94°C/45s),annealingattemperaturecorrespondingtothelocus–specificprimer(1min)andextension(72°C/1min) and 72°C for 5min (1 cycle).