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.