Geneelute plant genomic miniprep kit

Genomic DNA was isolated from fresh leaves using the GeneElute Plant Genomic Miniprep Kit (Sigma).
PCR amplifications were carried out using 80 ng of genomic DNA, 5 μl 10X Buffer, 1 μl 10 mM dNTPs, 2 μl 10 μM forward and reverse primers, 2U of Triple-Master Polymerase Mix (Eppendorf). After the first DNA denaturation step at 94°C for 3 min, amplifications were run for 30 cycles consisting of 1 min at 94°C, 1 min at 69°C, and 1 min at 72°C. A final elongation step was then run at 72°C for 7 min. PCR products were analyzed on 1.5% agarose gel and purified with the PCR product purification kit “GFX^TM PCR DNA and Gel Band Purification” Kit (GE Healthcare). The purified amplifications were then cloned using the pGem-T Easy vector (Invitrogen) and transformed into JM109 competent Escherichia coli cells by the heat shock method. Positive clones were selected and their plasmids were sequenced at both insert ends with SP6 and T7 primers. The full fragment sequence was obtained by primer walking using specific primers (Supplementary Table S1). Regions of ambiguous reads were re-sequenced with additional primers.

DNA of each RIL and parental lines PG2 and Grecale was extracted from fresh leaves by using the GeneElute Plant Genomic Miniprep Kit (Sigma, Waltham, MA, USA). After checking DNA concentration and quality by agarose gel-electrophoresis and NanoDrop2000 (Thermo Scientific™, Waltham, MA, USA), each DNA sample was diluted to 50 ng/µL and sent to TraitGenetics GmbH (Gatersleben, Germany) [88 ] for the genotyping assays with the wheat SNP 25K chip array developed by Illumina CSPro^R (San Diego, CA, USA), as described by [28 (link)].