Bigdye version 3.1 terminator mix

Genomic DNA of 19 cultivars was extracted from 1-week-old seedlings by using the cetyltrimethyl ammonium bromide (CTAB) method51 . Primers to amplify the full-length gene were designed based on the type-b ALP coding sequence from NCBI database (Accession No.FJ529695) (Table 2). The PCR conditions were set to 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 60 °C for 45 s and 72 °C for 50 s, and a final extension at 72 °C for 10 min. 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 at the Western Australia State Agricultural Biotechnology Centre. PCR and DNA sequencing were repeated three times to ensure the accuracy.

The chromosome-specific primers were used to amplify the genomic DNA of 19 cultivars. The PCR products were ligated into pGEM-T Easy vector (Promega, Madison, WI, USA) following the manufacturer’s protocol and then the hybrid vector was transformed into competent cells of E. coli strain DH-5α. Plasmids were extracted using the Magic Mini Plasmid Prep kit (Promega, Madison, WI, USA) and the extracted DNA was amplified using BigDye^@version 3.1 terminator mix (Applied Biosystems) for Sanger sequencing. The program Bioedit 7.0 was used for sequence analysis. Geneious^® software (R7) was used for multiple alignment of the translated amino acid sequences and phylogenetic analysis.

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).