KASP primers designing and genotyping
KASP primers were designed according to the flanking sequences of the
SNPs/InDels (Table S6). Genotyping of the
BC2F2 and other population were
performed using the KASP platform (IntelliQube, LGC, Biosearch
Technologies) according to the KASP genotyping protocol. Total 1.6 μL of
the KASP reaction mixture included 5~10 ng DNA sample,
1.74 μM common reverse primer, 0.69μM each of Fam and Hex labelled
forward primer, and 1×KASP Master Mix. The KASP protocol was performed
by touchdown PCR as follows: step 1, 94℃ for 15 min; step 2 (touchdown),
94℃ for 20 sec, 61℃ for 60 sec (decrease 0.6℃ per cycle), 10 cycles;
step 3, 94℃ for 20sec, 55℃ for 60 sec, 26 cycles. The fluorescence
signal of amplification products was detected and classified.