 Data analysis method | STS markers :
The reaction mixture for PCR amplification consisted of 25 ng total DNA, 200 mM of each dNTP (Boehringer Mannheim), 20 pmol primers (5' and 3' primers), 2 units of Taq DNA polymerase (Perkin-Elmer), PCR buffer (10 mM Tris pH 8.3 at 25°C, 50 mM KCl, and 0.001% (w/v) gelatin), and 40 mM MgCl2 in 20.0 μL volume. Amplification was performed in GeneAmp PCR System 9600 (Perkin-Elmer) with 35 cycles of 94°C (1 min), 60°C (1 min), and 72°C (2 min), and a final cycle of 72°C for 7 min. The amplified DNA products were electrophoresed on 3.0% agarose gels in 0.5 x TBE buffer at 120 V for 2 h, and stained with ethidium bromide. The size standard marker for electrophoretic analysis was mixture of HindIII digests of λDNA and HaeIII digests of φX174DNA.
CAPS markers :
PCR amplification was performed using the GeneAmp PCR System 9600 and the following amplification condition: 30 cycles of 94°C (1 min), 60°C (2 min), and 72°C (3 min), and a final cycle of 72°C for 7 min. The amplified products of each primer pair were digested with 28 restriction enzymes (PstI, HindIII, BamHI, EcoRI, ApaI, XhoI, KpnI, HaeIII, DraI, XbaI, SalI, EcoT14I, MspI, HinfI, EcoRV, BglII, SacI, HhaI, EcoT22I, HapII, ScaI, AfaI, MluI, PshBI, MboI, MvaI, SacII, and HincII). For some markers, the amplified products were further digested with 14 more restriction enzymes (AccII, AluI, AvaII, BcnI, Cfr13I, AccI, AvaI, BanII, Cfr10I, EaeI, HaeII, MflI, Bsp1286I, and TthHB8I). The digested products were electrophoresed on 2.0% agarose gels in 0.5 x TBE buffer at 120 V for 2 h, and stained with ethidium bromide. For dCAPS marker development, we performed the genomic DNA sequencing to identify nucleotide polymorphisms between Nipponbare and Kasalath, and created a unique restriction site into the PCR product in one of varieties by combination of the nucleotide polymorphism with mismatch primer sequences. PCR amplifications with dCAPS primers were carried out at the same condition as that with CAPS primers and the resultant products were digested with the enzyme. The digested products was electrophoresed on 4.0% agarose gels in 0.5 x TBE buffer at 120 V for 2 h, and stained with ethidium bromide. |
