Simultaneous changes in seed size, oil content and protein content driven by selection of SWEET homologues during soybean domestication

Abstract

Soybean accounts for more than half of the global production of oilseed and more than a quarter of the protein used globally for human food and animal feed. Soybean domestication involved parallel increases in seed size and oil content, and a concomitant decrease in protein content. However, science has not yet discovered whether these effects were due to selective pressure on a single gene or multiple genes. Here, re-sequencing data from >800 genotypes revealed a strong selection during soybean domestication on GmSWEET10a. The selection of GmSWEET10a conferred simultaneous increases in soybean-seed size and oil content as well as a reduction in the protein content. The result was validated using both near-isogenic lines carrying substitution of haplotype chromosomal segments and transgenic soybeans. Moreover, GmSWEET10b was found to be functionally redundant with its homologue GmSWEET10a and to be undergoing selection in current breeding, leading the the elite allele GmSWEET10b, a potential target for present-day soybean breeding. Both GmSWEET10a and GmSWEET10b were shown to transport sucrose and hexose, contributing to sugar allocation from seed coat to embryo, which consequently determines oil and protein contents and seed size in soybean. We conclude that past selection of optimal GmSWEET10a alleles drove the initial domestication of multiple soybean-seed traits and that targeted selection of the elite allele GmSWEET10b may further improve the yield and seed quality of modern soybean cultivars.

Keywords: SWEET; domestication; seed quality; soybean; yield.

Figure 1.

GmSWEET10a was identified as a candidate pleiotropic gene that influences seed size, fatty-acid content and protein content. (a) Genetic variations (π, F_ST and XP-EHH values) were calculated between G. soja (S) and the cultivar (C) across the 1.2-Mb genomic region of the GmSWEET10a locus. The dashed horizontal lines indicate the genome-wide thresholds (top 5% of the genome) of the selection signals. The solid lines above the plot represent genomic locations of QTLs retrieved from SoyBase (https://soybase.org/; Supplementary Table 1). The red, orange and purple lines are QTLs for the seed size, seed oil and protein contents, respectively. The black dashed lines above the x-axis are annotated genes in this region. The red dots denote the GmSWEET10a gene, i.e. Glyma.15G049200. (b) Expression pattern of GmSWEET10a in different organs in Glycine max (Gm). Expression values were obtained from Phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html#). F, flower; L, leaf; R, root; ST, stem; N, nodule; RH, root hair; SAM, shoot apical meristem; P, pod; S, seed; FPKM, fragments per kilobase of exon per million mapped. (c) Transcript abundance of GmSWEET10a in seed coats at different stages. The expression was detected by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Transcript levels were calculated relative to soybean cyclophilin 2 (GmCYP2). (d) and (e) RNA in situ hybridization of GmSWEET10a showing specific expression in the seed coats. Cross-sections of developing seeds at S2–S3 hybridized with antisense (d) or sense (e) probes for GmSWEET10a. sc, seed coat; e, embryo; p, palisade layer; hg, hourglass; tnp, thin-walled parenchyma; tkp, thick-walled parenchyma; al, aleurone layer. Scale bars, 200 μm.

Figure 2.

GmSWEET10a is a domestication gene that contributes to soybean seed size, fatty-acid content and protein content. (a) Haplotypes detected in the genomic region of GmSWEET10a. The SNP information of 871 re-sequenced accessions is derived from Zhou et al.'s data [21] and Fang et al.'s data [22]. The S/L/C indicates the accession number of soja/landrace/cultivar. (b) Median-joining network representing the relatedness of 12 GmSWEET10a haplotypes, each represented by a circle. Gray, green and blue circles represent wild soybeans, landraces and improved cultivars, respectively. (c) Frequency distribution of three haplotypes: H_I, orange; H_II, blue; H_III, green. (d) 100-seed weight, fatty-acid content and protein content of mature seeds in three haplotype populations (colors are the same as that in panel (c)). Box edges depict the interquartile range. The median is marked by a black line within the box. The number of samples in each haplotype (n) is shown under the haplotype label. The letters a, b and c indicate significant differences. P < 0.05 (Student's t-test). DW, dry weight. (e) and (f) Effect of two alleles of GmSWEET10a on seed traits. 100-seed weight, fatty-acid content and protein content of mature seeds from near-isogenic lines of GmSWEET10a with H_ I and H_ III haplotypes (e) or with H_II and H_ III haplotypes (f). NILs^A derived from the hybrid combination between HJ117 (H_I) and JY101 (H_ III). NILs^B derived from the hybrid combination between Suinong 14 (H_ III) and Enrei (H_II). Data are means ± s.d. ((e) NIL^A (H_I), n = 12; NIL^A (H_ III), n = 9; (f) n = 5). ^**P < 0.01 (Student's t-test).

Figure 3.

Effect of GmSWEET10a on seed size, fatty-acid content and protein content. (a) Genotype of the sw10a mutant edited by the CRISPR/Cas9 system. The arrow indicates the target site of the CRISPR/Cas9 editing in the region of exon 3 of GmSWEET10a. Changes in the DNA sequence in the targeted region and the amino-acid sequence of the sw10a mutant are highlighted in red. Numbers inside the brackets indicate the number of amino acids coded by the sequence. (b) Increased expression of GmSWEET10a was achieved in transgenic soybean lines OE-10a-1 and OE-10a-2 by introducing additional copies of the GmSWEET10a genomic sequence into the Williams 82 cultivar. (c) Seed appearance of the sw10a mutant, OE-10a-1 and OE-10a-2. Scale bars, 1 cm. (d)–(f) 100-seed weight (d), fatty-acid content (e) and protein content (f) of mature seeds from wild-type (WT), sw10a mutant, OE-10a-1 and OE-10a-2. DW, dry weight. Data are means ± s.d. ((d) n = 10; (e) and (f) n = 5). ^*P < 0.05; ^**P < 0.01 (Student's t-test).

Figure 4.

Effect of GmSWEET10b on seed size, fatty-acid content and protein content. (a) Expression pattern of SWEET10b in different organs in Glycine max (Gm). Expression values were obtained from Phytozome 12 (https://phytozome.jgi.doe.gov/pz/-portal.html#). F, flower; L, leaf; R, root; ST, stem; N, nodule; RH, root hair; SAM, shoot apical meristem; P, pod; S, seed; FPKM, fragments per kilobase of exon per million mapped. (b) Transcript abundance of GmSWEET10b in seed coats at different stages. The expression was detected by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Transcript levels were calculated relative to soybean cyclophilin 2 (GmCYP2). (c) and (d) RNA in situ hybridization of GmSWEET10b showing specific expression in the seed coats. Cross-sections of developing seeds at S2–S3 hybridized with antisense (c) or sense probes (d) for GmSWEET10b. sc, seed coat; e, embryo; p, palisade layer; hg, hourglass; tnp, thin-walled parenchyma; tkp, thick-walled parenchyma; al, aleurone layer. Scale bars, 200 μm. (e) Genotypes of the sw10b mutant edited by CRISPR/Cas9 system. The arrow indicates the target site in the region of exon 3 of GmSWEET10b. Changes in DNA sequence in the targeted region and amino-acid sequence of the sw10b mutant are highlighted in red. Numbers inside brackets indicate the number of amino acids coded by the sequence. (f) Increased expression of GmSWEET10b was achieved in transgenic soybean lines OE-10b-1 and OE-10b-2 by introducing additional copies of the genomic sequence into the Williams 82 cultivar. (g) Seed appearance of sw10b mutant and overexpression lines. Scale bars, 1 cm. (h)–(j), 100-seed weight (h), fatty-acid content (i) and protein content (j) of mature seeds from wild-type (WT), sw10b mutant, OE-10b-1 and OE-10b-2. DW, dry weight. Data are means ± s.d. ((h) n = 10; (i) and (j), n = 5). ^*P < 0.05; ^**P < 0.01 (Student's t-test).

Figure 5.

GmSWEET10b is a potential domestication gene that contributes to soybean seed size, fatty-acid content and protein content. (a) Haplotypes detected in the genomic region of GmSWEET10b. The SNP information of 871 re-sequenced accessions is derived from Zhou et al.'s data [21] and Fang et al.'s data [22]. (b) Frequency distribution of three haplotypes of GmSWEET10b. (c) Genetic variations (π, F_ST and XP-EHH values) were calculated between G. soja (S) and the cultivar (C) across the 2.0-Mb genomic region of the GmSWEET10b locus. The dashed horizontal lines indicate the genome-wide thresholds (top 5% of the genome) of the selection signals. The black dashed lines above the x-axis are annotated genes in this region. The red dots denote the GmSWEET10b gene—Glyma.08G183500. (d)–(f) 100-seed weight (d), fatty-acid content (e) and protein content (f) of mature seeds in three haplotype populations. Box edges depict the interquartile range. The median is marked by a black line within the box. The number of samples in each haplotype (n) is shown under the haplotype label. The letters a, b and c indicate significant differences. P < 0.05 (Student's t-test).

Figure 6.

Sugar-transporter activities of GmSWEET10a and GmSWEET10b. (a) Characterization of GmSWEET10a and GmSWEET10b sucrose-transport activity using FLIPsuc-2-10μ in HEK293T. Sensor only (black) and AtSWEET11 (green) were used as negative and positive controls, respectively. Data are means ± s.d. (n ≥ 8). (b) Sugar-uptake transport activities of GmSWEET10a and GmSWEET10b were tested in Xenopus oocytes. Oocytes were injected with water (negative control), GmSWEET10a or GmSWEET10b cRNA. Data are means ± s.d. (n = 3). ^*P < 0.05; ^**P < 0.01 (Student's t-test). (c) and (d) Sugar content in the developing seeds at S2 (14-16 DAF) (c) and S3 (20-22 DAF) (d) stages. sw10a;10b, double mutants at GmSWEET10a and GmSWEET10b. Data are means ± s.d. (n = 3). ^*P < 0.05; ^**P < 0.01 (Student's t-test). (e) A working model for the involvement of GmSWEET10a and GmSWEET10b in seed size, oil content and protein content during soybean domestication. The expression level of GmSWEET10a is significantly increased in cultivars at the seed-filling stage, which promotes more hexose accumulation in the embryo, resulting in a larger seed size, higher oil content and lower protein content due to the increased carbohydrate state. Selection of GmSWEET10b is ongoing and might use a mechanism similar to that of GmSWEET10a. Dark-blue arrows indicate the translocation of sugars from the seed coat to the embryo. Orange arrows indicate the breakdown of Suc into Hex by invertase or sucrose synthase. The red and green arrows represent ‘increase’ and ‘decrease’, respectively. Hex, hexose; Suc, sucrose.