microsatellite sequencing: leaf
Genomic DNA was isolated from ca. 15 mg dried leaf tissue of one individual using the E.Z.N.A. HP Plant DNA kit (Omega Bio-Tek, Norcross, GA, USA) with RNase treatment. The puried genomic DNA was subjected to shotgun library preparation and pyrosequencing at Macrogen (Seoul, Korea). No enrichment step was realized prior to pyrosequencing to reduce costs and to avoid bias towards particular microsatellite repeat motifs. We used MsatCommander to screen the pyrosequencing reads for microsatellite motifs (di-, tri and tetra-nucleotide loci with at least 5 repeats) and to design primers. To avoid designing primers for any locus twice, we compared all sequences of the same SSR repeat (including also the reverse complement repeats, e.g. (CTT)n and (AAG)n, in BioEdit ver. 7.0.5. We designed both forward and reverse primers for the amplication of microsatellite loci. For more details, see Van Rossum et al. Genetic diversity assessment of Helichrysum arenarium (Asteraceae) for the genetic restoration of declining populations.