A Single Amino Acid Substitution in STKc_GSK3 Kinase Conferring Semispherical Grains and Its Implications for the Origin of Triticum sphaerococcum

Abstract

Six subspecies of hexaploid wheat (Triticum aestivum) have been identified, but the origin of Indian dwarf wheat (Triticum sphaerococcum), the only subspecies with round grains, is currently unknown. Here, we isolated the grain-shape gene Tasg-D1 in T sphaerococcum via positional cloning. Tasg-D1 encodes a Ser/Thr protein kinase glycogen synthase kinase3 (STKc_GSK3) that negatively regulates brassinosteroid signaling. Expression of TaSG-D1 and the mutant form Tasg-D1 in Arabidopsis (Arabidopsis thaliana) suggested that a single amino acid substitution in the Thr-283-Arg-284-Glu-285-Glu-286 domain of TaSG-D1 enhances protein stability in response to brassinosteroids, likely leading to formation of round grains in wheat. This gain-of-function mutation has pleiotropic effects on plant architecture and exhibits incomplete dominance. Haplotype analysis of 898 wheat accessions indicated that the origin of T sphaerococcum in ancient India involved at least two independent mutations of TaSG-D1 Our results demonstrate that modest genetic changes in a single gene can induce dramatic phenotypic changes.

Figure 1.

Characterization and Map-Based Cloning of the Semispherical Grain Gene Tasg-D1. (A) Plant architecture of HS2 and ND4332. Bar = 10 cm. (B) Spike, spikelet, and grain morphology of HS2 and ND4332; bar = 5 cm, 5 mm, and 5 mm, respectively. (C) The Tasg-D1 locus was mapped between markers Xgdm72 and Xgwm341 on chromosome (Chr.) 3DS near the centromeric region. (D) High-resolution linkage map of Tasg-D1. (E) The Tasg-D1 gene was fine-mapped to a 1.01-Mb region between markers 3DS-68 and 3DS-94 using recombinants. The number of recombinants between the molecular marker and Tasg-D1 is indicated. (F) Analysis of genomic architecture and grain shape analysis for each recombinant type. The number of recombinants used for phenotypic analysis was indicated on the right. The black and white rectangles represent the homozygous ND4332 and HS2 regions, respectively. (G) Predicted high-confidence genes in the mapping region according to the International Wheat Genome Sequencing Consortium wheat genome. Colored arrows indicate the orientation and order of the annotated genes. (H) Gene structure and sequence analysis of Tasg-D1 between HS2 and ND4332. The SNP information is indicated in red.

Figure 2.

Functional Analysis of Tasg-D1. (A) Gross morphology of ND4332 and its three independent EMS mutants. Bar = 10 cm. (B) and (C) Spikelet (B) and grain morphology (C) of ND4332 and the mutants; bar = 5 mm and 5 mm, respectively. Twenty grains were aligned for photograph. (D) to (I) ANOVA of plant height (D), spike length (E), spikelet density (F), thousand-grain weight (G), grain length (H), and grain length/width ratio (I) between ND4332 and the mutants. Ten plants were selected for trait analysis. (J) Gross morphology of Fielder (control) and OE-Tasg-D1 and OE-TaSG-D1 transgenic plants. Bar = 10 cm. (K) and (L) Spikelet (K) and grain morphology (L) of Fielder (control), OE-Tasg-D1, and OE-TaSG-D1; bar = 5 mm and 5 mm, respectively. Twenty grains were aligned for photograph. (M) to (R) ANOVA of plant height (M), spike length (N), spikelet density (O), thousand-grain weight (P), grain length (Q), and grain length/width ratio (R) among Fielder, OE-Tasg-D1, and OE-TaSG-D1. Eight plants were selected for data analysis. Values are means, with bars showing the sd. ** and * indicate significant differences at the 1 and 5% levels, respectively.

Figure 3.

Effects of Point Mutations in TaSG-D1 on Wheat. (A) and (B) Comparison of root length (A) and dry mass (B) between NIL-Tasg-D1 and NIL-TaSG-D1 with different concentrations of BL at the seedling stage. NIL-Tasg-D1 and NIL-TaSG-D1 are recombinants with 90% background similarity, identified from the fine-mapping population. The BL concentrations were 0, 0.001, 0.01, and 0.1 μM, respectively. Three independent biological replicates were used (five plants per treatment). (C) Expression analysis of BR biosynthesis-related and signaling-related genes in Fielder (control), OE-Tasg-D1, and OE-TaSG-D1. TaD11, TaBrd2, TaD2, and TaDwarf4 are BR biosynthesis-related genes. TaBRI1, TaBZR1, TaTUD1, TaRAVL1, and TaDLT are BR signaling-related genes. Values are means, with bars showing the sd. (D) and (E) Scanning electron microscopy of grain pericarp in HS2 and ND4332 (D) and statistical analysis (E). Bar = 100 μm. A minimum of 100 cells per sample were measured using the instrument’s software. Each parent was performed with three biological replicates. ANOVA method was conducted for statistical analysis. Values are means, with bars showing the sd. Asterisks indicate significance determined by ANOVA: *P < 0.05 and **P < 0.01.

Figure 4.

Haplotype Analysis of TaSG-D1. Three types of TREE domains were detected in 898 wheat accessions with varying ploidy levels. I indicates the HS2-type allele; II indicates the ND4332-type allele; and III indicates the third type in which the TREE domain was changed to TGEE.

Figure 5.

Phenotypic Comparison of the Parents and Six RILs. (A) to (D) ANOVA of plant height (A), grain length/width ratio (B), number of fertile spikelets per spike (C), and thousand-grain weight (D) among HS2, ND4332, and six RILs. Asterisks indicate significance determined by ANOVA: *P < 0.05 and **P < 0.01.