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InDel markers in the candidate area were used for QTL validation. The principle of InDel selection was first homozygous and different between two parents, second the sequence difference was between 5 to 100 bp and finally the sequencing depth must be greater than 10. Once the sites were determined, the 250 bp of its upstream and downstream flanking sequence was used to design primer on NCBI Primer-BLAST tools (https://www.ncbi.nlm.nih.gov/tools/primer-blast). PCR reactions contained 2 μl of genomic DNA template (50 ng/μl), 5 μl of 3G Taq Master Mix for PAGE (Vazyme, Nanjing, China), 0.25 μl primer (100 μM/μl) × 2 and 2.5 μl water. PCR steps are followed as: 95 °C for 5 min; 35 cycles of 95 °C for 15 s, 50–65 °C for 15 s, 72 °C for 30 s; and a final extension at 72 °C for 5 min. The PCR amplification products were separated on 8% polyacrylamide gel by electrophoresis of 200 V for 2.0 h. The gel was stained in 0.1% AgNO3 solution (Sinopharm Chemical Reagent, Shanghai, China), and revealed the DNA bands in 1.5% sodium hydroxide and 0.4% formaldehyde solutions (Sinopharm Chemical Reagent, Shanghai, China) [84 (link)]. According to the genotype consistent with the parents or F1, the plant height data of 1241 populations were divided into three groups. ANOVA-test and Chi-square were performed at (p < 0.01).

Total DNA of the 165 RILs and the two parental lines was extracted from the first fresh leaf of each one-mature-leaf stage seedling using the CTAB method of Doyle and Doyle [31 ]. The DNA concentration and quality were examined by electrophoresis on a 1% (w/v) agarose gel and NanoDrop One (Thermo Scientific, Waltham, MA, USA). A total of 1228 SSR primers from Shi, et al. [16 (link)] were used to screen for polymorphisms between the two parental lines. The Polymerase Chain Reaction (PCR) amplification and polyacrylamide gel electrophoresis (PAGE) refer to Song [2 (link)]: the 10 μL mixture system of PCR contains 3 μL of total DNA (15 ng·μL⁻¹), 1 μL each of forward and reverse primers (10 μmol·L⁻¹), and 5 μL of 3G Taq Master Mix for PAGE (Vazyme, Nanjing, China); the PCR reaction program in the S1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) is 95 °C for 3 min, 32 cycles (95 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s), and a final extension at 72 °C for 5 min; amplified products are separated by 6% PAGE at 150 V for 1 h, and the bands are visualized and photographed after silver staining. Amplified DNA fragments with polymorphisms were used for the analysis of the RILs and linkage mapping.