Development and multiplexing of microsatellite markers using pyrosequencing in a tetraploid plant, Vaccinium uliginosum (Ericaceae)

Background and aims – We developed a new set of microsatellite primers in the tetraploid perennial shrub, Vaccinium uliginosum L., to investigate genetic diversity and population genetic structure. Methods – Using pyrosequencing, we identified and designed primers for PCR amplification of microsatellite loci in V. uliginosum . The primers were first screened for amplification and polymorphism by PCR and agarose gel electrophoresis. PCR products of selected primers were then ligated into a vector, amplified with a universal fluorescently labelled forward primer and a specific reverse primer, and electrophoresed on a capillary sequencer to check the scorability of the peaks. Finally, multiplexes were designed and tested on ninety individuals sampled in three Belgian populations. Key results – We designed and tested a total of 52 primer pairs, of which nine yielded scorable peaks, i.e. eight di- and one tri-nucleotide loci with seven to fourteen alleles per locus in three Belgian populations. The expected heterozygosity was high, ranging from 0.52 to 0.87 (mean = 0.77). Genetic diversity (Shannon's diversity, H' ) ranged from 1.27 to 1.42 and was much higher than that observed by Albert et al. (2005) using RAPD-analyses in the same populations. This could be due to the higher polymorphism retrieved with microsatellite markers. Conclusions – The microsatellite markers we developed showed enough polymorphism to investigate genetic diversity and structure even at small spatial scales, gene and pollen dispersal (through paternity inference) or outcrossing rates