Geneamp 2720 thermal cycler

The selective PCR mix was prepared consisting of 1.2 µl 10× PCR buffer II, 0.72 µl 25 mM MgCl₂, 0.24 µl(10 mM) deoxynucleotide triphosphate mix, 0.07 µl Amplitaq 360 DNA polymerase (Applied Biosystems), 0.5 µl of Msel primer (5.0 µM), 0.3 µl EcoRI (1.0 µM) IRD-700 labeled primer (Integrated DNA Technologies, Coralville, IA), 6.97 µl nanopure^® water, and 1.5 µl of the preamplification template DNA. This step was performed in the dark due to light sensitivity of the labeled primers. Selective amplification was performed on a GeneAmp 2720 thermal cycler (Applied Biosystems) with one pre-PCR cycle (30 s at 94 °C, 30 s at 65 °C, 1 min at 72 °C), 12 cycles of 30 s at 94 °C, 30 s at 65 °C → 56 °C, 60 s at 72 °C, and 23 cycles of 30 s at 94 °C, 30 s at 65 °C → 56 °C and 60 s at 72 °C. Blue stop solution (LI-COR Biosciences, Lincoln, NE) (2.5 µl) was used to end the reaction. The product was then denatured for 3 min at 94 °C and stored at −20 °C.

Genomic DNA was isolated from 238 P. linearis individuals and 152 P. dioica individuals using the LiCl extraction protocol described by Hong et al. (1992) (link) as modified by van Oppen et al. (1995) (link). Polymerase chain reaction (PCR) for 10 selected microsatellite markers (Varela-Álvarez et al., 2017 (link), 2018a (link); Supplementary Table 1) were performed separately for each locus in a 20 μl reaction volume containing 5–50 ng genomic DNA. Amplifications were conducted following the PCR programs and conditions as described in Varela-Álvarez et al. (2017 (link), 2018a) (link) using a GeneAmp 2720 thermal cycler (Applied Biosystems). Amplified fragments were separated electrophoretically using an ABI PRISM 3130xl (Applied Biosystems) automated capillary sequencer at CCMAR, Portugal, and sized with GeneScan-350ROX size standard (Applied Biosystems). In addition, genotype data from samples from two locations previously used in Varela-Álvarez et al. (2018b) (link) was added to the data set: 22 genotypes from P. linearis from Belém and 30 genotypes from P. dioica from Buarcos, both locations in Portugal. Alleles were scored in GENEMAPPER v.4.1 (Applied Biosystems). Binning and allele rounding were checked with GENEMAPPER v.4.1 and TANDEM (Matschiner and Salzburger, 2009 (link)) for the full data set containing 442 multilocus genotypes (Supplementary Appendix 1).