Genotyping DNA Markers in Plant Samples

DNA was extracted by the potassium acetate method (Dellaporta et al. 1983 ). MFT was genotyped by the methods of Chono et al. (2015) (link) and Kato et al. (2017) (link), using a cleaved amplified polymorphic sequence (CAPS) marker (Chono et al. 2015 (link)). DNA templates were amplified with ExTaq DNA polymerase (TaKaRa, Shiga, Japan) and an MFT primer set (Chono et al. 2015 (link)) under conditions described in Kato et al. (2017) (link). The PCR fragments were digested by ClaI at 37°C for 1 h, separated by agarose gel electrophoresis, and visualized with Gel Red stain (Biotium, Fremont, CA, USA). ABA8ʹOH1-A was genotyped by the method of Chono et al. (2013) (link) with slight modifications. DNA templates were amplified with the S2A2 primer set (Chono et al. 2013 (link)) in a total volume of 10 μL containing 100 ng of genomic DNA, 200 μM each dNTP, 0.2 μM each primer, 0.25 U of ExTaqHS DNA polymerase (TaKaRa), and ExTaq buffer (TaKaRa). The amplification conditions used an initial denaturation at 94°C for 1 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. The PCR fragments were separated by polyacrylamide gel electrophoresis and visualized with Gel Red stain (Biotium).