PCR multiplexing for maximising genetic analyses with limited DNA samples: an example in the collared flycatcher, Ficedula albicollis

In population and evolutionary genetics it is commonly recognised that more reliable results are often obtained when the number of loci analysed is increased, however lack of DNA is one of several factors which can limit the possibility to increase the number of loci assessed in a given study. A promising way to overcome the problem is the simultaneous amplification of several loci within the same reaction i.e. multiplex PCR. The purpose of the present work was to develop a series of microsatellite multiplexes using a recently released commercial multiplex buffer in order to demonstrate the potential usefulness of PCR multiplexing in a non-model organism. We developed ten multiplex sets of primers for the amplification of 77 microsatellite markers in collared flycatchers, each reaction requiring 30 ng of genomic DNA. The multiplexed microsatellite markers provide an easy and cost effective method for collection of genotype data thereby reducing the quantity of reagents and importantly reducing the quantity of DNA required for obtaining successful amplification to less than 4 ng per locus.