High-quality genomic DNA of ND3331 and Zang1817 was extracted and sequenced with an average 6 × coverage of the assembled genome using the Illumina NovaSeq 6000 platform as 2 × 150 bp reads. High-quality reads were aligned to the Chinese Spring IWGSC RefSeq v1.0 using the Burrows-Wheeler Aligner 0.7.15 program with default parameters (Li and Durbin 2009 (link)). Insertion/deletion (InDel) calling was performed using the HaplotypeCaller module, and InDels between Nongda3331 and Zang1817 were used to develop molecular markers for QTL analysis (Dataset S1). Primer 3 was used to design the sequences of InDel primers.
DNA amplification was programmed for an initial 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, and finally 5 min at 72 °C. A 10 µL PCR reaction system was used, containing 5 µL of 2 × Taq PCR StarMix (GenStar, Beijing, China), 1.5 µL of DNA template (about 50–100 ng), 1.5 µL of each InDel primer, and double-distilled H2O. The PCR products were analyzed on 8% non-denaturing polyacrylamide gels with silver staining.
DNA amplification was programmed for an initial 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, and finally 5 min at 72 °C. A 10 µL PCR reaction system was used, containing 5 µL of 2 × Taq PCR StarMix (GenStar, Beijing, China), 1.5 µL of DNA template (about 50–100 ng), 1.5 µL of each InDel primer, and double-distilled H2O. The PCR products were analyzed on 8% non-denaturing polyacrylamide gels with silver staining.
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