Isolation and characterization of microsatellite loci in Pulsatilla patens (L.) Mill. (Ranunculaceae) a rare and endangered plant species in Europe

Abstract

Pulsatilla patens is a rare and endangered plant species in many areas of Europe and protected under the Bern Convention and it is listed in Annex II and Annex IV to the Habitats Directive. In this study we developed 12 novel microsatellite loci using via 454 sequencing. We determined 11,220 contigs with a length of 156–11,384 bp. Within this dataset, we identified 319 SSR motifs in 301 contigs. All markers were genotyped on 56 individuals from three populations located in Poland. The number of alleles and expected heterozygosity were 2–12 (mean 3.7) and 0.142–0.820 (0.541 on average) respectively. The markers described in this study will be useful for evaluating genetic diversity of P. patens populations, could be applied to investigate the biological aspects and to develop effective conservation programs for the European populations of this species.

Pulsatilla patens (L.) Mill. (Ranunculaceae) is a long-lived perennial herb. It is a lowland species with a circumpolar range, found in all three continents in the northern hemisphere (Hulten and Fries 1986). Pulsatilla patens is widespread in Central and Eastern Europe, with its western range extending to Sweden and Germany (Akeroyd 1993). In North America, the species occurs mostly in the Central and Western United States, in Central and Northwestern Canada and in Eastern Alaska. Pulsatilla patens shows a preference for dry, sun-exposed sites. In Europe, it can be found in thermophilous grasslands and coniferous forest. Within its range in Europe, P. patens is considered to be critically endangered in many areas. The species is protected under the Bern Convention, and it is listed in Annex II and Annex IV to the Habitats Directive (Council of Europe 1979; European Communities 2004) due to a small number of localities, low abundance and the gradual disappearance of populations. The species decline is related to changes in land use, especially in forestry practices where efficient wildfire prevention and termination of cattle grazing in forests have led to the formation of a continuous moss layer or strongly grass dominated vegetation, which severely hinders the regeneration of P. patens (Kalamees et al. 2005). Long-term protection and management plans aimed at preserving P. patens populations should involve habitat and environmental monitoring as well as the quantification of genetic diversity within and among populations. Microsatellite markers, also referred to as simple sequence repeat (SSR) markers, are widely used in ecological studies and can also be employed to investigate the genetic diversity of populations. In this study, we developed nuclear microsatellite markers for P. patens with the involvement of GS Junior next generation sequencing (Roche 454 Life Sciences, Branford, CT, USA).