For population development and QTL mapping, total DNA was extracted using 2 cm-long leaf sample following the method of Zheng et al. [34 ]. PCR amplification was performed according to Chen et al. [35 (link)]. The products were visualized on 6% non-denaturing polyacrylamide gels using silver staining or on 2% agarose gels using Gelred staining. Three DNA markers were used, including functional marker Si9337 for Hd1, functional marker Se9153 and closely linked marker RM5436 for Ghd7 [10 (link),17 (link)].
For expression analysis, penultimate leaves of rice lines in the R1-NIL population were harvested at 7:00 am in 17HZ and 9:00 am in 17LS, 2 h after sunrise. Total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN, Hilden, German). First-strand cDNA was synthesized using ReverTra AceR Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed on Applied Biosystems 7500 using SYBR qPCR Mix Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Actin1 was used as the endogenous control. The data were analyzed according to the 2-ΔCt method. Three biological replicates and three technical replicates were used. The primers were selected from previous studies [10 (link),20 (link),36 (link)].
For expression analysis, penultimate leaves of rice lines in the R1-NIL population were harvested at 7:00 am in 17HZ and 9:00 am in 17LS, 2 h after sunrise. Total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN, Hilden, German). First-strand cDNA was synthesized using ReverTra AceR Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed on Applied Biosystems 7500 using SYBR qPCR Mix Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Actin1 was used as the endogenous control. The data were analyzed according to the 2-ΔCt method. Three biological replicates and three technical replicates were used. The primers were selected from previous studies [10 (link),20 (link),36 (link)].
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