PREMISE OF THE STUDY: Polymorphic microsatellite loci were developed and used to genotype individuals of Gentianella praecox subsp. bohemica (Gentianaceae), a highly protected taxon in Europe, to study the genetic structure of the remaining populations. METHODS AND RESULTS: Thirty-eight primer pairs were successfully amplified; of these, 12 polymorphic microsatellite loci were developed using a 454 sequencing approach and used to genotype 180 individuals of G. praecox subsp. bohemica from six populations. Allelic richness ranged between one and nine alleles per locus. We detected a high frequency of polyploid individuals (77.8%). The highest average percentage of heterozygous genotypes was identified for samples from the Hroby population (75.5%). All loci can also be amplified in the congeneric species G. praecox subsp. praecox, G. amarella subsp. amarella, and G. obtusifolia subsp. sturmiana. CONCLUSIONS: These markers will provide knowledge on patterns of gene flow and population genetic structure, which is necessary for current protection actions and for effective conservation of this species in the future.

Institute of Botany of the Czech Academy of Sciences Zámek 1 252 43 Průhonice Czech Republic; Department of Botany Faculty of Science Charles University Prague Benátská 2 128 01 Prague Czech Republic

Muzeum Cheb Krále Jiřího z Poděbrad 493 4 350 11 Cheb Czech Republic

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Brabec J. 2010. Monitoring hořečku mnohotvarého českého (Gentianella praecox subsp. bohemica) v ČR (sezóna 2010). [Monitoring of Gentianella praecox subsp. bohemica in the Czech Republic (vegetation season 2007)]. Deposit in: Central Office of Agentura Ochrany Přírody a Krajiny České Republiky, Prague, Czech Republic.

Council of the European Community. 1992. Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora. Official Journal of the European Communities Series L 206: 7–49.

Greimler J., Jang C. G. 2007. Gentianella stiriaca, a case of reticulate evolution in the northeastern and eastern Central Alps. Taxon 56: 857–870.

Hardy O. J., Vekemans X. 2002. SPAGeDi: A versatile computer program to analyse spatial genetic structure at the individual or population levels. Molecular Ecology Notes 2: 618–620.

Katoh K., Misawa K., Kuma K., Miyata T. 2002. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: 3059–3066. PubMed PMC

Kearse M., Moir R., Wilson A., Stones-Havas S., Cheung M., Sturrock S., Buxton S., et al. 2012. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics (Oxford, England) 28: 1647–1649. PubMed PMC

Königer J., Rebernig C. A., Brabec J., Kiehl K., Greimler J. 2012. Spatial and temporal determinants of genetic structure in Gentianella bohemica. Ecology and Evolution 2: 636–648. PubMed PMC

Lodhi M. A., Ye G. N., Weeden N. F., Reisch B. I. 1994. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Molecular Biology Reporter 12: 6–13.

Malausa T., Gilles A., Meglécz E., Blanquart H., Duthoy S., Costedoat C., Dubut V., et al. 2011. High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries. Molecular Ecology Resources 11: 638–644. PubMed

Meglécz E., Costedoat C., Dubut V., Gilles A., Malausa T., Pech N., Martin J. F. 2009. QDD: A user friendly program to choose microsatellite markers and design primers from large sequencing projects. Bioinformatics (Oxford, England) 26: 403–404. PubMed

Nagy S., Poczai P., Cernák I., Gorji A. M., Hegedus G., Taller J. 2012. PICcalc: An online program to calculate polymorphic information content for molecular genetic studies. Biochemical Genetics 50: 670–672. PubMed

Oberdorfer E. 1983. Pflanzensoziologische Exkursionsflora. Eugen Ulmer Verlag, Stuttgart, Germany.

Plenk K., Göd F., Kriechbaum M., Kropf M. 2016. Genetic and reproductive characterisation of seasonal flowering morphs of Gentianella bohemica revealed strong reproductive isolation and possible single origin. Plant Biology 18: 111–123. PubMed

Schuelke M. 2000. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18: 233–234. PubMed

Winfield M. O., Wilson P. J., Labra M., Parker J. S. 2003. A brief evolutionary excursion comes to an end: The genetic relationship of British species of Gentianella sect. Gentianella (Gentianaceae). Plant Systematics and Evolution 237: 137–151.

Strong fluctuations in aboveground population size do not limit genetic diversity in populations of an endangered biennial species