Fine mapping and candidate analysis of ts1.

(A) Genetic map around the ts1-linked two markers RM3340 and RM154 on the short arm of rice chromosome 2. (B) Fine mapping of the ts1 locus. The ts1 locus was narrowed down to a 108.5 kb region between ID8378 and SSR6884 with nineteen predicted ORFs. Numbers below the chromosome bar were recombinant frequency. Mutant allele of ORF4 contains a c.+733C→T point mutation when compared with its wild allele. (C) Structure and sequence analysis of the predicted ORF4 protein. At +244 position in the sixth P subfamily motif of ORF4, the mutant protein harbored a threonine to isoleucine substitution. Amino acid with grey background indicates the point substitution position, amino acids without colorful background were consensus amino acids. White rectangle represents P motif, black rectangle represents DYW motif. (D) PCR-based SNP assay of 24 representative rice materials by SNP maker cd-733C/T. M, 2000 bp DNA marker; 1, ts1; 2, LY95; 3, cv.Wushansimiao; 4–14, randomly selected ts1/Wushansimiao F2 individuals; 15–24, segregants from recessive-class population for fine mapping. Numbers below PCR amplicons were culm numbers of each F2 individual. (E) Expression of the candidate gene ORF4 and other five rice tillering regulation genes OsSPL14, OsTB1, MOC1, HTD1, D3 in the ts1 mutant. Relative expression levels in the ts1 mutant and its wild type were detected by quantitative RT-PCR, shown as mean ±SE of three separate experiments. * and ** indicate significances at p<0.05 and p<0.01 by the Student’s t-test, respectively.