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, 111 (50), 17797-802

Molecular Basis of a Shattering Resistance Boosting Global Dissemination of Soybean

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Molecular Basis of a Shattering Resistance Boosting Global Dissemination of Soybean

Hideyuki Funatsuki et al. Proc Natl Acad Sci U S A.

Abstract

Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1. The gene for the shattering-resistant genotype, pdh1, was defective, having a premature stop codon. The functional gene, Pdh1, was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world's leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1, or genes with the same role, will possibly be useful for crop improvement.

Keywords: QTL; crop improvement; dirigent protein; map-based cloning; pod dehiscence.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Pod dehiscence and pod-wall torsion of near-isogenic cultivated soybean [Glycine max Merr. (L.)] lines for qPDH1 under low-humidity conditions. (Scale bars: 10 mm.) (A) Dried pods of the shattering-resistant (SR) line 85R (Left) and the shattering-susceptible (SS) line 85S (Right) at ambient humidity [∼40% relative humidity (RH)]. (B) Percentages of dehisced pods of 85R, 85H, and 85S after drying for 3 wk and 6 wk at 30% RH (mean ± SE; n = 8). The 85H indicates HC1-85H, the parental line of 85R and 85S with the heterozygous genotype at qPDH1. Different letters denote significant differences (P < 0.001). (C) Pod walls of 85R (Left) and 85S (Right) at 22% RH, dehisced after natural drying. (D) Torsion angles of dehisced pod walls of 85R, 85H, and 85S at 30% RH (mean ± SE; n = 6). Different letters denote significant differences (P < 0.001).
Fig. 2.
Fig. 2.
Map-based cloning of cultivated soybean [Glycine max Merr. (L.)] qPDH1. (A) Predicted ORFs in the previously determined candidate region of chromosome 16 (blue arrows) and predicted ORFs in the region delimited in the present study (pink boxes). BAC H88I22 indicates a BAC clone carrying the qPDH1 locus of a shattering-susceptible (SS) cultivar, Misuzudaizu. Red triangles indicate positions of single nucleotide polymorphisms (SNPs) between Hayahikari [shattering-resistant (SR)] and Toyomusume (SS). (B) Results from semiquantitative RT-PCR for transcripts of ORF1 and ORF2 in pod walls of near-isogenic lines for qPDH1. R and S indicate lines 85R and 85S, respectively. (C) cDNA structure of ORF2 of Toyomusume. (D) DNA and deduced amino acid sequences around the SNP. Red letters indicate the SNP and the resulting amino acid residue or termination signal. The SNP can be recognized by a restriction enzyme, Nhe I. (E) PCR–RFLP genotyping of SR and SS cultivars at the SNP using Nhe I. SR cultivars (leftmost lanes) and SS cultivars (rightmost lanes) are listed in SI Materials and Methods. In these cultivars, the presence of an SR or SS allele at qPDH1 on chromosome 16 was suggested in this study or previous studies (14, 15, 17, 48, 56). (F) Percentages of pod dehiscence of an SR cultivar, Jack, plants, which were nontransformed, transformed only with the vector, or transformed with ORF2 from Toyomusume, at 30% RH (mean ± SE; n = 3, 2, and 8). Different letters indicate significant differences (P < 0.001). Although nontransformed Jack plants were grown in a different growth chamber, pod dehiscence was simultaneously monitored in the same chamber. (G) Torsion angles of dehisced pod walls of two-seeded (bar 2) or three-seeded (bar 3) pods of nontransformed Jack plants, Jack plants transformed only with the vector, or Jack plants transformed with ORF2 from Toyomusume at 30% RH (mean ± SE; n = 3, 3, 2, 2, 8, and 6). Different letters indicate significant genotypic differences (P < 0.05), detected by two-way ANOVA.
Fig. 3.
Fig. 3.
Cultivated soybean [Glycine max Merr. (L.)] Pdh1 expression patterns. (Scale bars: 100 μm.) (A) Results of RT-PCR targeting Pdh1 transcripts in several tissues. CYP2 corresponds to cyclophilin 2, used as a reference. (B) In situ hybridization for Pdh1 with an RNA probe in the antisense direction (Anti-sense) and with the probe in the sense direction (Sense) to detect nonspecific binding. (C) Relative expression levels of Pdh1 in the endocarp and the remaining portion of 5-wk-old pod walls detected by quantitative RT-PCR with reference to CYP2. (D) Cross-sections of inner parenchyma of 2- to 6-wk-old pod walls stained with phloroglucinol-HCl to reveal the degree of lignification. (Scale bar: 100 μm.) (E) Changes in pod length, pod width, and pod-wall dry weight during pod growth. (F) Relative expression levels of Pdh1 in 1- to 6-wk-old pod walls (means ± SE; n = 3). Different letters indicate significant differences (P < 0.05) between tissues (C) or among stages (F). 1W–8W correspond to the number of weeks after anthesis. Mat indicates maturity.
Fig. 4.
Fig. 4.
Genotypes for the single-nucleotide polymorphism (SNP) of Pdh1 and shattering degrees of accessions in soybean germplasms. (A) Proportion of Pdh1/pdh1 genotypes in various soybean germplasm pools: landraces from East, Southeast, and South Asian countries from a mini-core collection, old Japanese cultivars, modern Japanese cultivars, old North American cultivars, and modern North American cultivars. Old and modern North American cultivars correspond, respectively, to “ancestral” and “elite” North American cultivars in a previous study by Hyten et al. (32). Gray and black bars indicate percentages of accessions featuring SS and SR genotypes, respectively, at the SNP. The accessions used are listed in Tables S1 and S2. (B) Histograms of accessions with shattering scores, grouped by the shattering-susceptible (SS) or the shattering-resistant (SR) genotype at the Pdh1locus. The score increases with shattering degree as observed 2 wk after harvest (www.ars-grin.gov/). Accessions with “SHATLATE” values deposited in the GRIN database were analyzed.

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