2.6 PCR and qRT-PCR analysis
DNA was extracted from leaves of barley seedlings according to Qiu et al. (2011). The target DNA fragment was examined by PCR amplification and agarose gel electrophoresis. The presence of 1-kb insertion to the coding region of HvAACT1 (Fujii et al., 2012) was examined using PCR of full length coding sequences in 110 Tibetan wild barley accessions and Dayton. Gene expression of HvAACT1 in 10-mm root tips of the three barley genotypes was determined after 2 h of 5 μM Al exposure. The expression of the major glycolysis and Pi transporter genes in 10-mm root tips of XZ29 and XZ9 were also determined after 0.5, 2, 6, 12, and 24 h of 5 μM Al treatments. RNA was extracted using Tiangen RNAprep Pure Plant Kit (Tiangen, China), and cDNA was synthesized using the Reverse Transcriptase kit (Takara, Japan). Gene expression was determined using SYBR Green Supermix (Bio-Rad, USA).HvBetaTublin was used as a reference gene. The primers are listed in Table S1.