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

Abstract

Objective: Background and aims - We developed a new set of microsatellite primers in the tetraploid perennialshrub, Vaccinium uliginosum L., to investigate genetic diversity and population genetic structure.Methods - Using pyrosequencing, we identifi{ligature}ed and designed primers for PCR amplification ofmicrosatellite loci in V. uliginosum. The primers were first screened for amplification and polymorphismby PCR and agarose gel electrophoresis. PCR products of selected primers were then ligated into avector, amplified with a universal fluorescently labelled forward primer and a specific reverse primer, andelectrophoresed on a capillary sequencer to check the scorability of the peaks. Finally, multiplexes weredesigned 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. Theexpected heterozygosity was high, ranging from 0.52 to 0.87 (mean = 0.77). Genetic diversity (Shannon'sdiversity, 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 retrievedwith microsatellite markers.Conclusions - The microsatellite markers we developed showed enough polymorphism to investigategenetic diversity and structure even at small spatial scales, gene and pollen dispersal (through paternityinference) or outcrossing rates. © 2014 Botanic Garden Meise and Royal Botanical Society of Belgium.