Fig 1.
Phenotypes of the ts1 mutant and its wild type.
(A) Plant architecture of the ts1 mutant and its wild type at tillering stage. Bar = 20 cm. (B) Plant architecture of wild type, the ts1 mutant, ts1/wushansimiao F1 plants, and cv.Wushansimiao at maturity. Bar = 30 cm. (C) Comparison of culm numbers between the ts1 mutant and its wild type. (D) Comparison of panicle numbers among LY95, ts1, ts1/Wushansimiao F1 plant, and Wushansimiao. (E) Comparison of panicle length and internode lengths between the ts1 mutant and its wild type. P represents panicle length; I, II, III, IV, V, VI represent the first, second, third, fourth, fifth, sixth internode length, respectively. (F) Comparison of spikelets per panicle between the ts1 mutant and its wild type. (G) Morphological observation of tiller buds of the ts1 mutant and its wild type. White arrows point to the position of tiller buds. Bar = 3 mm. (H) Longitudinal sections of shoot apexes of the ts1 mutant and its wild type. Black arrows indicate active tiller buds, white arrow indicates inactive tiller buds. Bar = 200μm. WT, wild type rice line LY95. Each value represents the mean ± SE of 10 replicates. Significant differences of mean values in (C), (E), and (F) were determined by the Student’s t-test. Double asterisks indicate significant differences at P<0.01. Significant differences of mean values in (D) were determined by the Duncan test. a, b, c, and d represent 4 levels of significant differences at P<0.05.
Fig 2.
Primary mapping and QTL analysis of ts1 locus in ts1/Wushansimiao F2 population.
(A) Genotypes of SSR marker RM3340 for a pair of bulks and F2 individuals with extreme trait values. P1, ts1; P2, cv.Wushansimiao; L, L bulk; H, H bulk; Lanes 1–10 are F2 individuals with culm numbers = 1 or 2. Lane 11–20 are F2 those with culm numbers ≥12 at 40 DAS. Culm number of ts1 (P1) was 2.2 while Wushansimiao (P2) was 14.5 (40 DAS). (B) Frequency distribution of culm number at 40 DAS in ts1/Wushansimiao F2 population and these individual’s genotypes for marker RM3340. (C) LOD score plots showing locations of QTL for culm numbers at 40d, 50d and 60d, reproductive culm number, plant height, panicle length, and spikelets of main panicle within RM12298-RM154 on the short arm of chromosome 2. LOD curve indicates the strength of evidence for the presence of the QTL. Marker names and genetic distances between markers are shown below the chromosome.
Fig 3.
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.
Table 1.
Candidate genes in the genomic region between ID8378 and SSR6884.