Molecular Characterization of Wheat Cultivars

Genomic DNA was extracted from 50 mg of wheat flour for each wheat cultivar using the GeneAll Exgene Plant SV mini kit (GeneAll, Seoul, Korea) following the manufacturer’s instructions. The extracted genomic DNA was quantified and its quality assessed on a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and then it was diluted to 50 ng/μL. PCR analysis to discriminate between 1Bx7 and 1Bx7^OE was performed using the method described by [5 (link),17 (link)]. PCR was performed in a reaction volume of 20 μL using 150 ng of genomic DNA, 1.25 U of Go Taq DNA polymerase (Promega, USA), 1× Green Go Taq reaction buffer (containing 1.5 mM MgCl₂), 200 μM of dNTP mix (Bioneer, Daejeon, Korea) and 10 pmol each of forward and reverse primers. Left junction primers were: forward 5′-ACGTGTCCAAGCTTTGGTTC-3′ and reverse 5′-GATTGGTGGGTGGATACAGG-3′, and right junction primers were forward 5′-CCACTTCCAAGGTGGGACTA-3′ and reverse 5′-TGCCAACACAAAAGAAGCTG-3′ [17 (link)]. Amplification conditions for PCR were an initial cycle at 95 °C for 5 min, followed by 34 cycles of 94 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min, and then a final extension at 72 °C for 5.25 min. PCR products were resolved on 1.5–2.0% agarose gels in 0.5× Tris borate EDTA (TBE) buffer, stained with ethidium bromide and visualized under UV.