Fig 1.
Surface features of seed coats in water-permeable cultivar Tachinagaha and its impermeable NIL.
The structures of seed coats on the dorsal side of seeds were observed with SEM. Cracks (arrows) and pits (arrowheads) were observed in Tachinagaha (A) and its impermeable NIL (TA-HS) (B), respectively. C to F, Surface features after removing the cuticle with hot (60°C) 1 M NaOH. The cracks observed in Tachinagaha show a ladder-like structure (C and E) in which palisade cells are partly connected (E). Pits in TA-HS are closed (D and F). Bars in A to D = 50 μm; E and F = 5 μm.
Fig 2.
Frequencies of cracks and pits on seed coats.
The numbers of cracks and pits per area (505 × 675 μm) in seed coats on the dorsal sides of seeds were significantly different (p < 0.001) between water-permeable cultivar Tachinagaha and its impermeable NIL (TA-HS)(n = 40). Error bars indicate the maximum errors of estimates at 95%.
Fig 3.
Fine-mapping of the QTL for hard seededness (qHS1).
A segregating family of a backcross inbred line derived from a cross between Tachinagaha and a wild soybean accession was used for fine-mapping. Graphical genotypes are presented for plants (B5F4) with recombination in the genomic region harboring the QTL (A) and their progeny (B5F6) homozygous for recombinant genotypes (B). Open, dark gray, and black bars indicate regions homozygous for the allele from Tachinagaha (TA allele), heterozygous, and homozygous for the allele from the wild soybean (AO allele), respectively. Light gray bars indicate a region in which recombination occurred. Phenotypes for permeability are presented at right. In (A), the permeability of the B5F4 plants was evaluated based on the results of a progeny test (Seg, segregating for permeability; Fixed, homozygous for permeability). In (B), HS (%) indicates percent of hard seeds. C, The delineated region of 93 kb included 10 annotated genes in the Williams 82 genome sequence database (Phytozome Glyma1.0). D, Mutation site in the endo-1,4-β-glucanase gene (Glyma02g43680) and predicted amino acid sequences. Underline, PvuII restriction site.
Fig 4.
Amino acid sequence comparison among endo-1,4-β-glucanases from soybean and other plant species.
The predicted amino acid sequences of qHS1 and its homoeologous copy Glyma14g05200 were compared with predicted endo-1,4-β-glucanases of Lotus japonicus (AK339581), Medicago truncatula (Medtr5g093090 and Medtr3g110130), and Arabidopsis thaliana (At2g32990; AtGH9B8). The catalytic residues and amino acids involved in substrate binding of previously characterized glycoside hydrolase family 9 enzymes, OsCel9A, rice (Oryza sativa L.) endo-1,4-β-glucanase (Uniprot Q0JPJ1) and TfCel9A, Thermobifida fusca endo/exo-1,4-β-glucanase, are labeled with “Cat” and subsite numbers (+2 to -4), respectively. Arrow, substitution from isoleucine in Tachinagaha (qHS1-SS) to serine in TA-HS (qHS1-HS).
Fig 5.
Calcofluor white staining in micro-sections of seed coat.
Cross-sections of seed coats from the dorsal side of seeds of permeable cultivar Tachinagaha (A) and its impermeable NIL (TA-HS) (B) were stained with 0.1% calcofluor white solution and examined under UV illumination by confocal microscopy. Fluorescence indicates accumulation of β-1,4-glucan derivatives in palisade cells (PA), hourglass cells (HG), and outer and inner layers of the aleurone layer (AL). A fluorescent line (arrow) is observed in the outer layer of palisade cells in TA-HS (B), but not in Tachinagaha (A). C, Intensity of fluorescence averaged over 30 positions per genotype at the same relative positions along the long axis of the palisade cell. Blue and red lines are averages for TA-HS and Tachinagaha, respectively, with the maximum errors of estimates at 95% (light blue or pink dashed lines). Scale bars in A and B = 50 μm.
Fig 6.
Seed coat characteristics of transgenic plants carrying the impermeable qHS1 allele.
A soybean cultivar with a permeable seed coat, Kariyutaka (KA), was transformed with the 6,273-bp genomic region containing the putative promoter and coding region of qHS1 from TA-HS. T2 plants of three transgenic T1 plants (KA-qHS1; T2-1 to T2-3; gray bars) were compared with the T2 line transformed with the pMDC123-GFP construct (KA-GFP; open bars), which possessed only the GFP cassette. A, Average percentage of hard seeds (n = 60–100); B, Average number of cracks per area (270 × 361 μm) in seed coats on the dorsal side of seeds (n = 40). Error bars indicate the maximum errors of estimates at 95%.
Fig 7.
CAPS marker to detect the SNP in qHS1.
A, A fragment of 443 bp harboring the SNP can be digested by PvuII in the impermeable TA-HS, but not in the permeable Tachinagaha. ND, not digested; D, digested. M, Ladder marker (DNA-035, TOYOBO, Tokyo, Japan). B, Variation of seed coat permeability in 194 cultivated accessions. C, Genotypic constitutions at the CAPS marker for 69 impermeable seeds produced by 28 cultivated accessions.