Wheat ALP Gene Fragment Amplification

The primer pairs used in this study were the same as being published by Zhang et al. [47 (link)] to amplify TaALP fragments from the genomic DNA of wheat varieties, Living Stone, Chinese Spring (CS), Spitfire, Drysdale, RAC875, Lincoln, Kauz, Excalibur, Chara, Baxter, Mace, Bonnie Rock, Gliadius, Greygory, Kukri, Westonia, Yitpi, Wyalketchem, Bethleyhem, and Eagle Rock. PCR amplification cycles consisted of 1 cycle =3 min 95 °C; 35 cycles = 30 s 95 °C, 30 s 60–62 °C, 1 min 72 °C; 1 cycle = 5 min 72 °C. The target PCR products were separated by 1.5% (w/v) agarose gel electrophoresis, and the expected fragments were purified from the gel using a Gel Extraction Kit (Promega, Madison, WI, USA). Subsequently, the purified PCR products were amplified using BigDye@version 3.1 terminator mix (Applied Biosystems) and submitted for Sanger sequencing. Alignment of ALPs was carried out using the MUSCLE add-on tool in Geneious Pro software (v10.2.2).