Figure 1.
Dimorphic flowers of buckwheat and schematic presentation of the intra-morph incompatibility response in buckwheat.
Short-styled flowers of buckwheat have long stamens and vice versa. A pollen grain from a long-styled plant germinates and the pollen tube successfully elongates to reach the ovary in the pistil of a short-styled plant, whereas it germinates but fails to elongate in the style of long-styled flower.
Figure 2.
Expression of genes selected by in silico subtraction as determined by RT-PCR.
The expression of genes corresponding to the 15 contigs selected by in silico subtraction was examined by RT-PCR using cDNA from long styles (LS) and short styles (SS) as templates. See Table S1 for RT-PCR primers.
Figure 3.
Analysis of a chimeric mutant plant and a short-styled-specific gene (SSG).
(A) A chimeric plant generated by ion-beam mutagenesis. Red and blue circles indicate long-styled (LS) and short-styled (SS) flowers, respectively. (B) PCR amplification of gene fragments for SSG1-SSG4 using genomic DNA isolated from the chimeric plant as template. M, molecular marker (25-bp DNA ladder, Invitrogen). (C) Southern blot analysis of SSG2 and SSG3 (S-ELF3) using genomic DNA isolated from the Kitawase cultivar (LS, SS) or Kyushu PL4 (PL4) as template. The star indicates the band corresponding to SSG2. Fragment sizes of the λHindIII marker shown at the right are in kb.
Figure 4.
PCR survey of S-ELF3 (SSG3) in 47 buckwheat landraces and modern cultivars.
The numbering of individual plants corresponds to that shown in Table S2. L, long-styled plant. S, short-styled plant. N, negative control. M, 1-kb DNA ladder (GenDireX).
Figure 5.
Expression analysis of S-ELF3 transcripts.
cDNA prepared from roots, stems, leaves, pistils, stamens and pollen was used for RT-PCR analysis. ACTIN was amplified as a loading control. DBF: one day before flowering. DF: the day of flowering.
Figure 6.
Analysis of S-ELF3 in Fagopyurm species.
(A) PCR survey of S-ELF3 in heteromorphic and self-incompatible species, F. cymosum and F. urophyllum. LS, plant with long-styled flowers. SS, plant with short-styled flowers. N, negative control. M, molecular marker (1-kb DNA Ladder, GeneDireX) (B) Southern blot analysis of S-ELF3 in F. cymosum, F. tataricum, and F. urophyllum. Fragment sizes of the λHindIII marker shown at the right are in kb. See Table S2 for accession numbers (C9142 and C9143).
Figure 7.
The gene structure and phylogeny of S-ELF3 in five Fagopyrum species, including the SC Kyushu PL4 line, which contains the Sh allele of F. homotropicum, are shown. Species in blue and red font exhibit heteromorphic SI and homomorphic SC, respectively. Dark brown boxes and lines represent 5′- and 3′-untranslated regions and introns, respectively. Coding regions are colored blue. Red boxes and line indicate large insertions (>400 bp) and nonsense mutation, respectively. The phylogenetic tree in the inset was obtained by the Neighbor-joining method. The S-ELF3 sequence from F. urophyllum was used as an outgroup. The bootstrap numbers (500 replicates) are shown next to the branches. The scale bar corresponds to 0.02 substitution per nucleotide site.