Allelic data from neutral markers (microsatellites) to assess genetic connectivity and diversity of four wetland plant species occurring in kettle holes.

Sampling of fresh plant material: Tip of the leaves of the four species was cut using garden scissors and store in tea bags with silica gels to dry. The sampling was carried out between May and August 2016. Testing of microsatellites and building of allelic database: Dried leaf samples were taken to the laboratory for genetic assessement. 12-20 mg of dried plant material was used for DNA isolation following standard protocols of NucleoSpin 96 plant II kit (MACHEREY-NAGEL, Duren, Germany). Final DNA concentration was measured with a NanoDrop instrument (NanoDrop 1000 spectrophotometer, Peqlab). For microsatellite amplification, we used published species-specific primers. We tested different primers until achieving a minimum of 10 polymorphic markers per species in a subset of 10–15 samples. All the primer pairs that worked in the test were selected and one the forward primer of each pair was fluorescent labelled (M13-FAM). Selected marked pair of primers combined with 1 µl of DNA were used for the Polymerase Chain Reactions (PCR) using GoTaq polymerase. PCR reactions included an initial denaturation temperature of 95°C for 15 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 90 seconds and 72°C for 60 seconds and a final extension at 60°C for 30 minutes. Presence of microsatellites (repetitive motives) was confirmed by Sanger sequencing, and PCR products were electrophoresed using an "ABI Prism 3130xl Genetic Analyser". Afterwards, PCR products were diluted 1:20 or 1:40 according concentrations of PCR product in the agarose gel, 0.25 μl dye-labeled size standard LIZ® was added and sequenced with 3130xl Genetic Analyser (Applied Biosystems® GeneticAnalyzers). Finally, allele size scoring was performed using GeneMapper® Software with the corresponding library of “bins” and double-checked by hand. Each txt file correspond to one marker as a final result of the GeneMapper analyses. A summary data is presented in the Excel file. Analysis in the laboratory was carried out between October 2016 and December 2017.