Amplification and Labeling of Repeat Families

The rye 120-bp repeat family (Bedbrook et al., 1980 (link)) was amplified by PCR from DNA clone pSc119.2 using M13 universal primers (Said et al., 2018 (link)), whereas Afa family repeat was amplified from genomic DNA of bread wheat CS using primers AS-A and AS-B (Nagaki et al., 1995 (link)). The probes pSc119.2 and Afa family repeat were labeled by PCR with digoxigenin-dUTP and biotin-dUTP (Roche, Mannheim, Germany), respectively (Said et al., 2018 (link); Said et al., 2019b (link)). Plasmid pTa71 (45S rDNA) containing a 9-kb fragment from T. aestivum with 18S-5.8S-26S rDNA and intergenic spacers (Gerlach and Bedbrook, 1979 (link)) was labeled by nick translation with either biotin-dUTP or digoxigenin-dUTP (Roche) using standard Nick Translation Mix kits (Roche) following the instructions of the manufacturer.