Highly polymorphic microsatellite markers in Pulsatilla vulgaris (Ranunculaceae) using next-generation sequencing.

PREMISE OF THE STUDY: We developed novel microsatellite markers for the perennial plant Pulsatilla vulgaris (Ranunculaceae) to investigate the effects of fragmentation on gene flow in this imperiled species. METHODS AND RESULTS: We identified microsatellites and developed primers based on 454 shotgun sequences. We identified 14 markers that were polymorphic and produced clean bands. Of these, eight could be analyzed as diploids. Genotyping of 97 individuals across two populations revealed these markers to be highly polymorphic with seven to 17 alleles per locus and observed heterozygosity from 0.41 to 0.83. CONCLUSIONS: The markers are highly informative and will be used to test if the reintroduction of shepherding in southern Germany improves genetic connectivity among fragmented populations of P. vulgaris. The combination of diploid and tetraploid markers presented here will be useful in resolving the polyploidization history of this and related species.