Immomix

In this study, six microsatellites (Table II) were selected as previously described by Breuillin et al. [10] and Kubisiak et al. [39] . Amplifi cations were carried out in 50 μl reactions, which contained 25 μl 2X PCR Master Mix (Immo-Mix, Bioline, 25020), 40-40 pmol each primer, 20-40 ng of genomic DNA and nuclease free water were run out. Microsatellites were amplifi ed in a Primus (MWG Biotech) thermocycler using the following thermal profi le: 3 min initial

Barley genomic DNA from the above lines was extracted using the method described by Edwards et al. (1991) (link). PCR was performed to confirm the presence of the AtVHA-C transgene in the transformed plants using a forward (5 0 -AGA GAC TCG TAA ACA AGA G-3 0 ) and reverse (5 0 -CAG CCA TGG CTC CTG CA-3 0 ) primer, which amplified a fragment of 299 bp in size. The Hv-VRT2 vernalisation gene (GenBank DQ201168) was used as a control gene, and this gene was amplified using Hv-VRT2 specific forward (5 0 -CCG AAT GTA CTG CCG TCA TCA CAG-3 0 ) and reverse (5 0 -TGG CAG AGG AAA ATA TGC GCT TGA-3 0 ) primers, which amplifies a fragment of 280 bp in size. PCR reactions included 2 Â reaction ImmoMix (Bioline), 10 mM of forward and reverse primers, 1 mg mL -1 BSA, MilliQ water and 0.5 mL of template DNA. The PCR conditions used were similar for both the VRT2 and the VHA-C (95 C for 10 min, followed by 1 min at 94 C, 1 min at 55 C and 1 min at 72 C, repeated for 35 cycles, with a final extension of 72 C for 10 min). PCR products were visualised by gel electrophoresis using a 2% agarose gel containing Gel Red Nucleic acid stain (Biotium) (5 ml per 100 ml of gel) and a photograph of the gel was obtained (Molecular imager, Gel Doc imaging system, Bio-Rad).

Amplifi cations of 50 μl PCR reaction containing 25 μl 2 × PCR Master Mix (ImmoMix, Bioline, 25020), 40-40 pmol of each primer, 20-40 ng of genomic DNA and nuclease free water were run. SR6R and LR1 primer pair [42] (Integrated DNA Technologies, Inc.) were used to amplify the full length of ITS region, with the following amplifi cation protocol: 3 min initial denaturing at 95 °C, followed by 5 cycles of 1 min at 95 °C, 1 min annealing at 50 °C, 1 min at 72 °C and 25 cycles of 1 min at 90 °C, 1 min annealing at 50 °C, 1 min at 72 °C and 15 min fi nal extension at 72 °C. The large intron of the tef1 gene was amplifi ed by the EF1-728F and EF1-986R primer pair [19] according to the previously described protocol with a temperature of 56 °C rather than 50 °C. PCR was performed in a Primus (MWG Biotech) thermocycler. Amplifi cation products were subjected to electrophoresis in a 0.7% agarose gel containing EtBr and visualized by UV illumination. The PCR products were purifi ed by using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, 740609). Purifi ed amplifi cation products were sequenced by MWG Biotech Company in Germany.