Fastdna extraction kit

Seed samples were collected from 39 wild populations in Europe, South America and the U.S. (Fig. 1) as described in Hierro et al. [21] . Heads were collected from up to 30 different individuals randomly chosen from across each site. A single seed (technically an achene) from at least 10 different individuals was planted in small 2 cm2 pots and grown in the greenhouse at the University of Massachusetts Boston, U.S.A. Leaf tissue from rosettes of individuals that germinated and survived was harvested after about 4 weeks. We extracted DNA from the 520 individuals using one of three methods: the Qiagen DNA extraction kit (Qiagen, Valencia, California, USA), the FastDNA extraction kit (MP Biomedicals, Solon, Ohio, U.S.), or standard CTAB methods.

Fecal DNA was extracted from samples using the FastDNA Extraction Kit (MP Biomedical, Irvine, CA, USA) and used for qPCR and preparation of 16s sequencing libraries. Prior to amplification all sample DNA was diluted to ~10 ng/µL and 1 µL of DNA was added to each PCR reaction. Reactions for each sample were run in duplicate. Quantitative PCR was conducted with B. subtilis group-specific primers ([40 (link)]; Forward: 5′-AAGTCGAGCGGACAGATGG-3′; Reverse: 5′-CCAGTTTCCAATGACCCTCCCC-3′) on a CFX96 Touch Thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following amplification program: 95 °C for 3 min followed by 30 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s with a final extension 72 °C for 5 min. Bacillus subtilis levels in samples was quantified by generation of standard curves using purified B. subtilis DNA (dilutions: 2–2 × 10⁻⁵ ng DNA/rxn). Sequencing libraries were prepared following the Earth Microbiome Project protocols, as previously described [16 (link)]. Amplicon libraries were sequenced on an Illumina Miseq at the Next Generation Sequencing Core at Colorado State University. All sequence data were processed using the DADA2 pipeline in QIIME 2 ver. 2020.8.0. Alpha diversity parameters were calculated in QIIME 2 and MyPhyloDB ver. 1.2.1 was used for calculating Bray–Curtis distances and PCoA visualizations.

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 ).