Total DNA was extracted from young leaves of individual plants by the CTAB method, and polymorphism data of 67 SSR markers distributed among the 12 chromosomes (Supplemental Table 2) were collected. Kawasaki-Tanaka and Fukuta (2014) (link), Wunna et al. (2016) (link), Khan et al. (2017) (link) and Odjo et al. (2017) (link) also used the SSR markers, and they demonstrated that these polymorphisms could differentiate among the upland Japonica, lowland Japonica and Indica Groups. PCR was performed in a 10-μL mixture containing 1 μL sterile H2O, a total 1.5 μL of forward and reverse primers (each 2 μM), 7.5 μL of 2× Quick Taq HS Dye Mix (Toyobo Co., Ltd. Japan) and 5 μL DNA concentrated to about 5 to 10 ng/μL. PCR amplification was performed with the following profile: 94°C for 2 min, followed by 40 cycles of 30 s at 94°C, 30 s at 55°C and 1 min at 68°C. The amplified products were separated by electrophoresis in 2% agarose gel in 1× TAE buffer at 150 V for 90 or 120 min, and DNA polymorphisms were detected by ethidium bromide staining.
The banding patterns were scored based on the presence (1) or absence (0) in each SSR markers’ allele. These polymorphism data were used to classify the accessions by Ward’s hierarchical clustering method in JMP software (v. 7.0.2; SAS Institute, Inc., Cary, NC, USA).