DNA isolation and polymerase chain reaction
Sorghum leaf tissues were harvested from 14 days old seedlings for DNA isolation. Total genomic DNA was extracted from three individual plants for each of the 373 genotypes (separate extractions) using a modified CTAB method according to (Mace et al . (2003). DNA was amplified in a multiplex-Polymerase Chain reaction (PCR) targeting Sulfotransferase and Ubiquitin genes. The Sulfotransferase primer pair (PDStriga l5b: forward 5’-CAAACCCATCGGACATCTTC-3’ ;PDStrigalgs 5b: reverse 5’ -CAGCATGTCCTCGTACCTGA-3’ ) as well as the Ubiquitin primers (forward 5’ -ATGTCCTGCTGCGAAGCTAT-3’ ;reverse 5’ -GCTAGAGCACCCGAGGAGTA-3’ ), have been previously described by Gobenaet al . (2017) and Mallu et al . (2021), respectively. PCR reactions were performed in 20 µl volumes using MyTaq™ DNA polymerase kit (Bioline, Meridian Biosciences). The cycling conditions were 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, 62.5°C for 30 seconds, an extension of 72°C for 1 minute and a final extension of 72°C for 7 minutes using a Bio-Rad thermal cycler. The PCR amplicons were resolved in a 2% (w/v) agarose gel stained with Safe ViewTM Fire Red (Biotium) and visualized under UV light.