Figure 5

Identification of homozygous mutants among OsSGR 389 s2 mutant descendants. (a) Electrophoretic analysis to discard heterozygous mutant plants obtained by self-fertilization of sgr 389 s2. Individual DNA samples were subjected to PCR amplification, denaturation, re-annealing, Fennel Crude Extract (FCE) heteroduplex mismatch cleavage and denaturing polyacrylamide gel analysis. The 926 bp band corresponding to the amplified fragment is present in all samples, while cleaved bands (380 and 506 bp) were detected in heterozygous mutant descendants (D4, D8, D10, E1, E4, E6 and E9). No cleavage bands were observed either in wild type or mutant homozygous descendants (D1, D3, D5, D6, D7, E3, E5, E8 and E10) highlighted in bold. (b) Electrophoretic analysis to discard homozygous wild type descendants. Homozygous descendants (D1, D3, D5, D6, D7, E3, E5, E8 and E10) individual DNA was mixed with wild type DNA (1:1 w/w) and subjected to PCR amplification, denaturation, re-annealing, FCE mismatch cleavage incubation and polyacrylamide gel analysis. Cleaved bands (380 and 506 bp) were detected in homozygous mutant descendants (D1, D3, D5, D7 and E10 in bold). Homozygous wild type individuals DNA could not form mismatches, and consequently no mismatch cleaved bands were visualized (D6, E3, E5 and E8). M: Molecular weight marker (100 bp marker, Thermo Fisher Scientific Inc.).