Generuler 1 kb ladder

A 681bp fragment of the D. gallinae mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using the primers COI1Fyuw114 (5′-AGATCTTTAATTGAAGGGGG-3′) and COI1Ryuw114 (5′- AAGATCAAAGAATCGGTGG-3′) corresponding to nucleotide positions 61 to 742 [GenBank accesion number AM921853; (19 (link))].
PCR was perfomed in a volume of 25 μl containing 12.5 μl 2x MyTaq^TM (Bioline, London, UK), 400 pM of each primer (Sigma-Aldrich, Darmstadt, Germany) and 2 μl of DNA template. PCR cycling conditions were initial denaturing at 95°C for 5 min, followed by 35 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s. Final elongation was performed at 72°C for 5 min. A T Gradient thermocycler was used (Biometra, Jena, Germany). After amplifcation, PCR products were resolved by electrophoresis in 1.0% (w/v) agarose gels, using 5X DNA loading buffer (Bioline, London, UK), Safeview Nucleic acid stain (NBS Biologicals, Cambridgeshire, UK). The GeneRuler 1 kb ladder (Thermo Fisher Scientific, Waltham, Massachusetts) was used to assess product size. Each PCR amplicon was purified using a Qiagen QIAquick PCR purifiction kit as recommended by the manufacturer (Qiagen GmbH, Hilden, Germany) and eluted in 30 μL dH₂O.

DNA was extracted from 100 mg of fresh leaves using the E.Z.N.A. ^®Plant DNA DS Kit (Omega bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. The quality of the isolated DNA was confirmed by electrophoresis using a 1.0% agarose gel, final concentration was adjusted to 40 ng μL⁻¹ for genetic analysis and stored at −20 °C for further PCR assays. Twelve iPBS (internal primer binding sequence) primers (Table S2) were used for genotyping [25 (link),34 ]. PCR assays were performed in 12.5 µL reaction mixtures containing 1 unit of Platinum™ Taq DNA polymerase (ThermoFisher Scientific, Waltham, MA, USA), 2.0 mM MgCl₂, 0.2 mM dNTPs (Invitrogen™, Carlsbad, CA, USA), 1 mM of 12–13-nt or 0.6 mM of 18-nt primers, and 40 ng genomic DNA. PCR were performed as follows: 1 denaturation cycle at 94 °C for 3 min followed by 35 cycles of 30 s denaturation at 94 °C, 45 s annealing at 49–60.5 °C, and 2 min 30 s extension at 72 °C. Final extension was for 5 min at 72 °C with a final step at 4 °C. PCR products were separated by electrophoresis at 100 V for 2.5 h in agarose gels 1.7% using 1X TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) using GeneRuler 1 Kb ladder (ThermoFisher Scientific, Waltham, MA, USA) to estimate fragment lengths. Gels were stained with GelRed (Biotium, Fremont, CA, USA) and recorded in a BIO-RAD Gel Doc™ XR equipped with an Image Lab™ Software.