FastDNA extraction kit (MP Biomedicals) was used for DNA extractions of both the 84 dried herbarium specimen tissue and 144 fresh samples from the 18 extant populations according to manufacturer's protocol. We used 12 microsatellite nuclear markers (Závada et al., 2017 (link)), and one intergenic spacer trnL‐trnF chloroplast marker (Taberlet et al., 1991 (link)) to score the genotypes of the 228 individuals of this study.
The 25 μl polymerase chain reactions (PCRs) to amplify the selected markers contained: 5 μl DNA (20–100 ng), 2.5 μl of 2.5 mM MgCl2, 2.5 μl of 2 mM deoxynucleotides (dNTPs), 2.5 μl of 10× reaction buffer, 1 μl of each primer (10 mM), 0.2 μl of Taq polymerase (5 units/μl) (New England Biolabs, Ipswich, Massachusetts) and 10.3 μl H2O. The thermocycler (MJ Research PTC‐100, Waltham, Massachusetts) conditions were 95°C for 5 min, 35 cycles of 95°C for 30 s, annealing temperature 52°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 5 min. We followed a modified amplification protocol for herbaria specimens with the KAPA3G Plant PCR Kit—Mix B (Sigma Aldrich, St. Louis, Missouri), as optimized in Schori et al. (2013 ).
The 25 μl polymerase chain reactions (PCRs) to amplify the selected markers contained: 5 μl DNA (20–100 ng), 2.5 μl of 2.5 mM MgCl2, 2.5 μl of 2 mM deoxynucleotides (dNTPs), 2.5 μl of 10× reaction buffer, 1 μl of each primer (10 mM), 0.2 μl of Taq polymerase (5 units/μl) (New England Biolabs, Ipswich, Massachusetts) and 10.3 μl H2O. The thermocycler (MJ Research PTC‐100, Waltham, Massachusetts) conditions were 95°C for 5 min, 35 cycles of 95°C for 30 s, annealing temperature 52°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 5 min. We followed a modified amplification protocol for herbaria specimens with the KAPA3G Plant PCR Kit—Mix B (Sigma Aldrich, St. Louis, Missouri), as optimized in Schori et al. (2013 ).
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