Figure 4

Identification of OsACS1 mutants. (a) Heteroduplex mobility assays identifying three of the OsACS1 mutants (acs1 152 s3, acs1 228 s1 and acs1 576 s1). Denaturating polyacrylamide gel electrophoresis showing different heteroduplex DNA band patterns formed after PCR amplification and mismatch cleavage by Fennel Crude Extract (FCE) incubation. The presence of two bands of about 600 and 850 bp in the case of acs1 228 s1 sample and 700 and 750 bp in the case of acs1 576 s1 sample, indicate heteroduplex digestions in the 1480 bp amplicon (ACS1 4). The presence of two mutations in the same 1014 bp amplicon (ACS1 1–3) of the same individual (acs1 152 s3) generates four bands of about 250, 450, 550 and 750. M: molecular weight marker (100 bp marker, Thermo Fisher Scientific Inc.). Pool: positive mismatch pool formed by mixing four individual DNA samples. Arrows indicate mismatch digested bands. Lanes 2–5: individuals DNA samples of the positive mismatch pool. Mutant samples are indicated by bold number lane. (b) Identification of nucleotide changes by sequencing. Heterozygous acs1 mutants (upper panel) and wt (lower panel) sequences are represented. Black arrows indicate the base substitution. Direct nucleotide sequencing of the acs1 152 s3 mutant revealed the heterozyogus G to A and T to G transitions at gene nucleotides position 174 and 58 respectively which resulted in substitution of GT/AG at intron 1 5′UTR and the amino acid C to G change at position 20 (C20G). Direct nucleotide sequencing of acs1 228 s1 and acs1 576 s1 mutants revealed the heterozigous G to A and C to G transitions at gene nucleotides position 1266 and 1351 which resulted in substitution of amino acids S to N and L to P at amino acid positions 314 (S314N) and 354 (L354P) respectively.