Domesticated species often exhibit convergent phenotypic evolution, termed the domestication syndrome, of which loss of seed dormancy is a component. To date, dormancy genes that contribute to parallel domestication across different families have not been reported. Here, we cloned the classical stay-green G gene from soybean and found that it controls seed dormancy and showed evidence of selection during soybean domestication. Moreover, orthologs in rice and tomato also showed evidence of selection during domestication. Analysis of transgenic plants confirmed that orthologs of G had conserved functions in controlling seed dormancy in soybean, rice, and Arabidopsis. Functional investigation demonstrated that G affected seed dormancy through interactions with NCED3 and PSY and in turn modulated abscisic acid synthesis. Therefore, we identified a gene responsible for seed dormancy that has been subject to parallel selection in multiple crop families. This may help facilitate the domestication of new crops.
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The sequencing data for rice accessions from this study have been deposited into the Sequence Read Archive under accession PRJNA407820.
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We thank Q. Xie from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, for providing access to the split ubiquitin-based yeast two-hybrid library. This work was supported by the Chinese Academy of Sciences (grant nos. XDA08000000 and QYZDJ-SSW-SMC014) and the National Natural Science Foundation of China (grant nos. 31525018, 91531304, and 31788103).
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 Comparison of the CDS sequences between G (from Kuaiqingpi) and g (from DN50) in soybean.
The bottom line refers to the protein sequence. Transmembrane motifs in G are marked with a box. Gray boxes indicate reserved motifs; the red box indicates the missing transmembrane motif in the g protein.
a, Subcellular localization of the G protein. The fluorescence of G-GFP and g-GFP specifically matched that of the chlorophyll autofluorescence signal, confirming exclusive chloroplast targeting of G. G-GFP, G-GFP fusion; g-GFP, g-GFP fusion; Vec-GFP, control with empty vector. Scale bar, 5 μm. A representative result of three independent experiments is shown. b, Chlorophyll accumulation in the seed coats of DN50 and transgenic lines. DN50 and the T3 transgenic lines are used. TC-1 and TC-2 indicate the two independent transformants that are generated by transformation of an 8,261-bp G genomic sequence from Kuaiqingpi into DN50, and OE-1 and OE-2 indicate the two independent overexpression transformants that were generated by transformation of the G coding sequence from Kuaiqingpi, which was driven by the CaMV 35S promoter, into DN50. Seed coats were dissected from mature seeds for chlorophyll analysis. Values represent the means ± standard error of three independent experiments. ****P ˂ 0.0001; adjusted P values were calculated by one-way ANOVA with Dunnett’s multiple-comparisons test. c, Germination rates of DN50, OE1, and OE2. Freshly harvested mature seeds are used. d, Germination of DN50, TC-1 and TC-2 after 6 months of storage. The seeds were incubated under dark conditions at 28 ºC. Values represent the means ± standard error of three independent experiments.
a, FST values for all SNP sites between G. soja and landrace. The horizontal dashed line indicates the genome-wide threshold (top 5% of the genome) of selection signals. The red line denotes the G gene, i.e., Glyma.01G198500. b, π values for all SNP sites between G. soja and landrace. The horizontal dashed line indicates the genome-wide threshold (top 5% of the genome) of selection signals. c, Whole-genome screening of selection sweeps using XP-CLR values. The horizontal dashed line indicates the genome-wide threshold (top 5% of the genome) of selection signals.
Colored boxes denote families: blue, Poaceae; green, Nelumbonaceae; orange, Solanaceae; red, Brassicaceae; purple, Rosaceae; yellow, Fabaceae.
AT, Arabidopsis thaliana; Atrl, Amborella trichopoda; Brara, Brassica rapa; Fve, Fragaria vesca; Glyma, Glycine max; Medtr, Medicago truncatula; MDP, Malus domestica; Nta, Nicotiana tabacum; Nnu, Nelumbo nucifera; Os, Oryza sativa; Prupe, Prunus persica; Seita, Setaria italic; Sobic, Sorghum bicolor; Soly, Solanum tuberosum; Zm, Zea mays.
a, FST values for all SNP sites between Oryza rufipogon and Oryza sativa. The horizontal dashed line indicates the genome-wide threshold (top 5% of the genome) of selection signals. The red line denotes the OsG gene, i.e., LOC_Os03g01014. b, π values for all SNP sites between Oryza rufipogon and O. sativa. The horizontal dashed line indicates the genome-wide threshold (top 5% of the genome) of selection signals. c, Whole-genome screening of selection sweeps using XP-CLR values. The horizontal dashed line indicates the genome-wide threshold (top 5% of the genome) of selection signals.
a, EHHS values across G region for Oryza rufipogon and O. sativa. The gray box denotes the G gene. b, EHHS values across the G region for S. pimpinellifolium (PIM), S. lycopersicum var. cerasiforme (CER), and S. lycopersicum (BIG). The gray box denotes the G gene.
Supplementary Figure 8 Germination variation of the wild-type and transgenic lines with different genotypes.
a, Germination of the cultivated rice HJ19 and transgenic lines. Transgenic lines were generated by introducing a genomic sequence from O. rufipogon (IRGC 105491) into HJ19. Means ± s.e.m. are shown, n = 3 independent experiments. Scale bar, 5.0 mm. b, Sequencing of PCR products containing targeted sites in OsgCR T2 plants. The red arrow indicates the deleted sequence. c, Relative expression of OsG in ZH11, CRISPR-Cas9 genome-edited (OsgCR), and overexpression (OE) lines. d–f, Germination of ZH11, OsgCR, and overexpression (OE) lines. Osg-OE and OsG-OE lines were generated by introducing DNA coding sequences from ZH11 and O. rufipogon (IRGC 105491), respectively, into ZH11. Freshly harvested spikes (d,e) and spikes stored for 6 months (f) were used in the experiments. Means ± s.e.m. are shown, n = 3 independent experiments. Scale bar, 5.0 mm.
a, FST values for all SNP sites during tomato domestication. The dashed horizontal line indicates the genome-wide threshold (top 5% of the genome) of the selection signals. The red line denotes the SolycG gene, i.e., Soly08g005010. b, π values for all SNP sites between PIM and CER. c, Whole-genome screening of selection sweeps using XP-CLR values. The horizontal dashed line indicates the genome-wide threshold (top 5% of the genome) of selection signals.
a, Gene structure and haplotype of SolycG. Four SNPs in the exons are identified in the resequenced population. Eight haplotypes are divided into two groups according to the SNP at the 14927 site. b, SNP variation in the neighbor-joining tree of the tomato population. 360 accessions were used for the tree construction. The dots indicate genotypes: the red dots indicate the G genotype, the blue dots indicate the C genotype, and the gray dots indicate an undetected genotype due to missing data at this site. The colored lines represent the classification of the accessions: purple lines represent wild relatives of tomato (Wild), green lines represent S. pimpinellifolium (PIM), orange lines represent S. lycopersicum var. cerasiforme (CER), and blue lines represent big-fruited S. lycopersicum (BIG).
a, Characterization of the AtG gene and its mutants. The genomic region of AtG is shown as a line. Exons are shown to scale as black boxes, and introns are shown as white boxes. The position of the T-DNA insertion is indicated by the triangle. b, Expression of AtG in the Col-0 and T-DNA insertional mutants. Primers for RT–PCR were designed to amplify the whole length of the coding DNA sequence of the AtG gene. A representative result of three replicate experiments is shown. c, Germination phenotype of freshly harvested seeds of Col-0, atg, atg-2, and dog1-2 without stratification. d, Germination phenotype of freshly harvested seeds of Col-0, atg, atg-2, and dog1-2 after 3 d of stratification at 4 ºC. Means ± s.e.m. are shown for n = 5 independent experiments. Each experiment consisted of about 50 seeds.
a, Coimmunoprecipitation assay of AtG with NCED3 and PSY. Proteins were expressed in Arabidopsis protoplasts. GFP and HA beads were used for immunoprecipitation. Samples of input and precipitated products were analyzed by immunoblot using anti-GFP, anti-FLAG and anti-HA. The experiment was repeated twice with similar results. Uncropped gels are available in Supplementary Fig. 14. b, BIFC assays showing the protein interaction of AtG with NCED3 and PSY in vivo. YN, BIFC-2; YC, BIFC-1. The experiment was repeated three times with similar results. Scale bar, 10 μm. c, ABA accumulation in the seeds of DN50 and transgenic lines. Newly harvested mature seeds of DN50 and T3 transgenic lines were used. A representative result of two independent experiments is shown. Means ± s.e.m. for n = 3 technical independent replicates are shown. Adjusted P values were calculated by one-way ANOVA with Dunnett’s multiple-comparisons test.
a, atg mutant plants showing a pale green phenotype as compared with Col-0. The experiment was repeated six times with similar results. Scale bar, 3 cm. b, OsgCR mutant generated by CRISPR–Cas9 showing a pale green phenotype as compared with the wild-type ZH11. The experiment was repeated three times with similar results. Scale bar, 3 cm. c,d, Chlorophyll accumulation of Col-0, atg and transgenic lines. GmG-OE and Gmg-OE were generated by introducing DNA coding sequences from Kuaiqingpi and DN50, respectively, into atg. Scale bar, 3 cm. Leaf chlorophyll of 4-week seedlings was measured and calculated. Means ± s.e.m. for n = 3 technical independent replicates are shown. The significance was calculated by one-way ANOVA with Tukey’s multiple-comparisons test, α < 0.05.
Uncropped scans of western blotting images.
Supplementary Figures 1–14
Seed coat color phenotype of accessions for GWAS
Information of the 176 resequenced rice accessions
Accessions for genetic diversity analysis of OsG in rice
Primers used in this study
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Wang, M., Li, W., Fang, C. et al. Parallel selection on a dormancy gene during domestication of crops from multiple families. Nat Genet 50, 1435–1441 (2018). https://doi.org/10.1038/s41588-018-0229-2
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