PMC

Graphical genotypes of recombinant plants. Blue and yellow bars indicate chromosome of Epi-d1 and Kasalath respectively. (A) Graphical genotype of Epi-d1. (B) Graphical genotype of Kasalath. (C–F) Graphical genotypes of recombinant lines 184, 186, 198, and 211 are shown. The phenotypes of these lines are shown on the right of the chromosome.

ChIP assay of D1 gene. (A, 1–4) The regions amplified to detect the chromatin state are indicated as arrows. (B) Nucleosomes were precipitated by anti-acetylated histone H3K9 (α-H3K9-Ac) and anti-dimethylated histone H3K9 (α-H3K9-me²) Input and no antibody were also amplified as positive and negative controls, respectively. The precipitated nucleosomes of Nipponbare (Nip), Epi-normal (Epi-N), and Epi-dwarf (Epi-d) were amplified by using primers of heterochromatic gene, euchromatic gene, and D1 gene.

DNA methylation analysis of Epi-d1. (A) The appearance of chimeric plants from Epi-dwarf seeds treated with inhibitor of DNA methylation, 5-aza-2′ deoxycytidine. Values in the graph indicate the ratio of chimeric plants from Epi-dwarf (percentage). (B) Restriction map of the candidate region of Epi-d1 gene. HindIII sites are described as H. (C and D) The restriction map of first and second 7.5-kb region digested by 2 HindIII sites. (E) The restriction map of 10.6-kb region contains the D1 gene. (F and G) The probes used are indicated as broken line and simple line, respectively. (C–E) Arrows indicate the sites of DNA methylation sensitive restriction enzyme, AccII (CGCG). (F–H) The genomic DNA of Nipponbare (Nip), Epi-normal (Epi-N), and Epi-dwarf (Epi-d) were digested by HindIII or double digested by HindIII and AccII. Digested DNAs were hybridized with repeat probe (F) and D1 probe (G). 5s rRNA probe was also hybridized as a control (H).

Mapping analysis of Epi-d1. (A) Gross morphology of Epi-d1 mutant (Center) and magnified view of its dwarf panicle and seed in dwarf tillers (Left) and normal panicle and seed in normal tillers (Right). Arrowheads indicate tillers (vegetative branch shoots), asterisks indicate panicles (inflorescences), and white circles indicate grains. (B) Epi-d1 shows varying phenotypes from completely dwarf to completely normal. (C) Chimeric phenotype in panicle. A panicle showing 2 separate rachises with either small round or normal grains (Left). A panicle showing small round and normal grains arranged alternately on the rachis (Center). A panicle showing a random distribution of the 2 grain phenotypes (Right). (D) Epi-d1 was mapped into 59 cM of chromosome 5, then narrowed down to a 33.5-kb region between 2 molecular markers, D1-1002 and D1-1005. In this region, there are 3 8.6-kb tandem repeats (red arrows) and 4 ORFs are predicted by RiceGAAS. There are 11 polymorphisms in this 33.5-kb region between Nipponbare and Epi-d1 mutant. The numbers indicate the positions of polymorphisms in this region. (E) Comparison of sequence of D1 gene between Epi-normal, Epi-dwarf, and Nipponbare. (F–H) The phenotypes of d1 loss-of-function mutant are shown. (I–K) The phenotypes of Epi-dwarf plant are shown. (L–N) The phenotypes of F₁ plant derived from cross between Epi-dwarf and d1 are shown. The gross morphologies are shown in F, I, and L; panicles in G, J, and M, and seeds in H, K, and N. (O) Total RNAs were extracted from 4 independent chimeric plants by separating tissues with different phenotypes. RNA blots were hybridized with D1 probe. N and d indicate RNA extracted from normal and dwarf tillers, respectively. (P) Total RNAs were extracted from Epi-normal and Epi-dwarf plants and hybridized with D1 probe. (Q) Total RNAs were extracted from Epi-normal and Epi-dwarf plants and hybridized with probes of D1 and its neighbor genes. Hypothetical and DnaJ gene are located 7 kb downstream and 4 kb upstream of the D1 gene, respectively. N and d indicate RNA extracted from Epi-normal and Epi-dwarf, respectively. Actin1 is hybridized as a control (O–Q).