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, 70 (5), 1497-1511

TaBT1, Affecting Starch Synthesis and Thousand Kernel Weight, Underwent Strong Selection During Wheat Improvement

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TaBT1, Affecting Starch Synthesis and Thousand Kernel Weight, Underwent Strong Selection During Wheat Improvement

Yamei Wang et al. J Exp Bot.

Abstract

BRITTLE1 (BT1), responsible for unidirectional transmembrane transport of ADP-glucose, plays a pivotal role in starch synthesis of cereal grain. In this study, we isolated three TaBT1 homoeologous genes located on chromosomes 6A, 6B, and 6D in common wheat. TaBT1 was mainly expressed in developing grains, and knockdown of TaBT1 in common wheat produced a decrease in grain size, thousand kernel weight (TKW), and grain total starch content. High diversity was detected at the TaBT1-6B locus, with 24 polymorphic sites forming three haplotypes (Hap1, Hap2, and Hap3). Association analysis revealed that Hap1 and Hap2 were preferred haplotypes in modern breeding, for their significant correlations with higher TKW. Furthermore, β-glucuronidase (GUS) staining and enzyme activity assays in developing grains of transgenic rice with exogenous promoters indicated that the promoters of Hap1 and Hap2 showed stronger driving activity than that of Hap3. Evolutionary analysis revealed that BT1 underwent strong selection during wheat polyploidization. In addition, the frequency distribution of TaBT1-6B haplotypes revealed that Hap1 and Hap2 were preferred in global modern wheat cultivars. Our findings suggest that TaBT1 has an important effect on starch synthesis and TKW, and provide two valuable molecular markers for marker assisted selection (MAS) in wheat high-yield breeding.

Keywords: TaBT1; Haplotype; molecular marker; selection; starch synthesis; thousand kernel weight (TKW); wheat.

Figures

Fig. 1.
Fig. 1.
Sequence alignment and gene structures of TaBT1-6A, TaBT1-6B, and TaBT1-6D. (A) Protein sequence alignments of TaBT1-6A, -6B, and -6D. (B) Gene structures of TaBT1-6A, -6B, and -6D. Black rectangles represent exons, horizontal lines between exons signify introns, horizontal lines on the left of the first exons represent promoter regions, vertical lines signify the position of an Skn1 motif, and numbers denote size (bp).
Fig. 2.
Fig. 2.
TaBT1 is mainly expressed in tender developing grains and is localized to the chloroplast of wheat protoplast. (A) Mean relative expression of TaBT1 in different tissues of Chinese Spring. SR, seedling roots; SS, seedling stems; SL, seedling leaves; JSR, roots at the jointing stage; JSS, stems at the jointing stage; JSL, leaves at the jointing stage; HR, roots at the heading stage; HS, stems at the heading stage; HFL, flag leaves at the heading stage; HPL, penultimate leaves at the heading stage; HLS, leaf sheaths at the heading stage; HN, nodes at the heading stage; YS, young spikes; S, stamens; P, pistils; 5 DPA, 10 DPA, 15 DPA, 20 DPA, 25 DPA, 30 DPA, grains at different developmental stages, namely 5, 10, 15, 20, 25, and 30 DPA. Expression of TaBT1 in grains at 25 DPA was assumed to be 1. (B) Relative expression of TaBT1 homoeologous genes in grains at different developmental stages. Expression of TaBT1-6A in grains at 25 DPA was assumed to be 1. (C) Subcellular localization of TaBT1. GFP and TaBT1GFP fusions under the control of the Cauliflower mosaic virus 35S promoter were transiently expressed in wheat proplasts. Eighteen hours after transformation, the fluorescence signal of GFP was observed under a confocal laser scanning microscope. Chlorophyll autofluorescence (red), GFP (green), bright-field images, and an overlay of the merged fluorescence (orange) are shown in each panel. Scale bar=10 μm.
Fig. 3.
Fig. 3.
RNAi of TaBT1 caused significant shrinkage of the mature grain. (A) Grain morphology; (B) kernel length, KL; (C) kernel width, KW; (D) kernel thickness, KT; (E) TKW. Data are means ±SD of 15 plants, and asterisks indicate significant differences between TaBT1-RNAi lines and wild-type plants; *P<0.05, **P<0.01, ***P<0.001 (t-test). Scale bar=2 mm.
Fig. 4.
Fig. 4.
Starch granules in grain endosperms of TaBT1 transgenic RNAi lines. (A) Expression levels of TaBT1 in grains at 10 DPA from transgenic lines and control plants; grain starch granule morphology of (B) wild-type Fielder; (C) negative control lines; and positive lines of RNAi-L1 (D), RNAi-L2 (E), and RNAi-L3 (F); **P<0.01, ***P<0.001 (t-test). Scale bar=25 μm.
Fig. 5.
Fig. 5.
RNAi of TaBT1 altered starch granule number and grain total starch content. (A) The number of starch granules; (B) starch content. **P<0.01, ***P<0.001 (t-test).
Fig. 6.
Fig. 6.
Haplotypes at TaBT1-6B and the molecular marker development based on its polymorphism. (A) Polymorphic sites at TaBT1-6B. Vertical lines indicate sites of variation; numbers represent corresponding positions (bp); black rectangles represent loci with haplotype-specific cis-elements. (B) The InDel-2029 marker was based on the InDel at base pair –2029; cultivars with different haplotypes were discriminated on 6% denaturing polyacrylamide gels. (C) The CAPS-1664 marker was designed for the SNP at base pair –1664; the black line and arrow represent recognition sites of the restriction endonuclease AclI.
Fig. 7.
Fig. 7.
Histochemical and fluorometric GUS assays in transgenic rice grains at 21 DPA indicated that the promoters of Hap1 and Hap2 at TaBT1-6B possess stronger driving ability than that of Hap3. (A) GUS staining of transgenic rice grains. (B) β-Glucuronidase activities in transgenic rice grains. *P<0.05, **P<0.01 (t-test). Scale bar=500 μm.
Fig. 8.
Fig. 8.
Hap1 and Hap2 have a higher transcription level than Hap3 at TaBT1-6B in wheat collections. Dashed lines of each haplotype represent the average expression level in materials of the corresponding haplotype. **P<0.01, ***P<0.001 (t-test).
Fig. 9.
Fig. 9.
Distributions of TaBT1-6B haplotypes in different ecological regions and its frequency changes during the Chinese wheat breeding process. (A) 157 landraces from 10 Chinese ecological zones; (B) 348 modern cultivars from 10 Chinese ecological zones; (C) frequencies of TaBT1-6B haplotypes over decades in Chinese modern cultivars from the 1940s to 2000s; (D) common wheat cultivars from six major wheat production regions; I, North America; II, CIMMYT; III, Europe; IV, former USSR; V, China; VI, Australia.
Fig. 10.
Fig. 10.
Nucleotide diversity (π) and genetic distances at BT1 between diploid, tetraploid, and hexaploid accessions at BT1-6A (A), BT1-6D (B), and BT1-6B (C). (D) Genetic distances at BT1-6B between pairs of populations (FST). DI, diploids; TE, tetraploids; LA, landraces; MC, modern cultivars; the color gradient presents FST values from dark (1.0) to pale green (0.0).

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