Taq polymerase

Allele-specific PCR assays for the candidate gene markers Pain1-8c (soluble acid invertase, chromosome III), Stp23-8b(PHO1a-H_A) (L-type starch phosphorylase a, chromosome III), StpL(PHO1b)-3b, StpL(PHO1b)-3e (both L-type starch phosphorylase b, chromosome V), GP171-a (chromosome VIII), InvGE-6f (apoplastic invertase, chromosome IX), and Rca-1a (ribulose bisphosphate carboxylase/oxidase activase, chromosome X) were performed as described (Li et al. 2005 (link), 2008, 2010; Schreiber et al. 2014 (link)). An allele-specific assay was newly developed for the InvCD141-Sa allele (Draffehn et al. 2010 (link); Schreiber et al. 2014 (link)). InvCD141-Sa was amplified from 50 ng genomic DNA template in 15 µl total volume containing 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.1 % Triton X-100, 0.2 mM of each dNTP, 1 μM of each primer (Table 1) and 1.5U Taq Polymerase (Ampliqon, Odense M, Denmark). PCR conditions were: Initial denaturation for 3 min at 94 °C; 5 cycles of touch down PCR: 60 s denaturation at 94 °C, 60 s annealing at 65 °C, decreasing T_a by 1 °C per cycle, 60 s at 72 °C; then 30 cycles with 45 s denaturation, 45 s annealing at 60 °C and 45 s at 72 °C, followed by a 5 min final extension step at 72 °C. Allele-specific markers were scored as absent (0) or present (1).

Plasmids, strains, primers, and sequences used in this study are listed in Additional file 2: Tables S2, S3, S4 and S5. All oligonucleotides and double-stranded DNA fragments (gBlocks) were ordered from Integrated DNA Technologies (IDT). yEGFP was assembled into p2909 by User cloning [41 (link)], further assemblies were performed with Gibson Assembly [42 (link)] and both transformed into One Shot^® TOP10 Escherichia coli (Thermo Fisher Scientific).
S. boulardii with a uracil auxotrophy was used for as a base for all strains and obtained from previous work [37 (link)]. S. boulardii was transformed according to the protocol in Durmusoglu et al. [6 (link)]. Genomic integration cassettes were digested with restriction enzyme NotI (FastDgiest Enzyme, Thermo Scientific^™) prior to transformation. Markerless plasmids where co-transformed with pCfB6920, into strains previously transformed with pCfB2312. Genomic integration was confirmed using colony-PCR with Taq polymerase (Ampliqon). Primers flanking the integration were used to confirm the integration. Genomic DNA was extracted by boiling cells at 95 °C for 20 min in 20 mM NaOH. One single amplification band, ~ 4000 bp, on gel electrophoresis indicated a successful integration into both chromosomes. Where necessary, strains were cured for pCfB2312 and pCfB6920 after genome integration.

Genomic DNA extraction and purification from the isolates was performed using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, Mo., United States) following the manufacturer’s instructions. Purified DNA was used as a template for amplifying the 16S rRNA genes using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Frank et al., 2008 (link)) and Taq polymerase (Ampliqon, Odense, Denmark). The PCR conditions were as follows; one cycle of 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 45 s, and extension at 72°C for 2 min. A final extension step at 72°C for 10 min was then performed. Amplified products were checked by electrophoresis using 1% agarose gels, visualized after 90 min, and photographed under UV light using a G Box equipment (SynGene, Cambridge, United Kingdm). The amplified products were purified using a GenElute PCR Clean-Up Kit (Sigma-Aldrich) column, and subjected to standard Sanger DNA sequencing at Macrogen (Madrid, Spain). Strains were identified at the species level by comparing their sequences to those in the NCBI database using the BLAST tool (Altschul et al., 1997 (link)). Sequences sharing 97% identity or higher were deemed to belong to the same species.