The genetic architecture of water-soluble protein content and its genetic relationship to total protein content in soybean

Abstract

Water-soluble protein content (WSPC) is a critical factor in both soybean protein quality and functionality. However, the underlying genetic determinants are unclear. Here, we used 219 soybean accessions and 152 recombinant inbred lines genotyped with high-density markers and phenotyped in multi-environments to dissect the genetic architectures of WSPC and protein content (PC) using single- and multi-locus genome-wide association studies. In the result, a total of 32 significant loci, including 10 novel loci, significantly associated with WSPC and PC across multi-environments were identified, which were subsequently validated by linkage mapping. Among these loci, only four exhibited pleiotropic effects for PC and WSPC, explaining the low correlation coefficient between the two traits. The largest-effect WSPC-specific loci, GqWSPC8, was stably identified across all six environments and tagged to a linkage disequilibrium block comprising two promising candidate genes AAP8 and 2 S albumin, which might contribute to the high level of WSPC in some soybean varieties. In addition, two genes, Glyma.13G123500 and Glyma.13G194400 with relatively high expression levels at seed development stage compared with other tissues were regarded as promising candidates associated with the PC and WSPC, respectively. Our results provide new insights into the genetic basis of WSPC affecting soybean protein quality and yield.

Figure 1

Phenotypic analysis of protein content (PC) and water-soluble protein content (WSPC) in the 219 soybean accessions. The histograms on the diagonal show the phenotypic distribution of each trait across six environments. The values above the diagonal are pairwise correlation coefficients between traits, and the plots below the diagonal are scatter plots of compared traits. PCE1-E6, denote the protein content in six different environments; WSPCE1-E6, denote the water-soluble protein content in the corresponding environments.

Figure 2

Genetic architecture of soybean protein content (PC) and water-soluble protein content (WSPC). (a) and (b) Manhattan plot for the BLUP of soybean PC and WSPC across six environments by genome-wide association mapping. Red horizontal lines depict the Bonferroni-adjusted significance threshold (P < 4.95 × 10⁻⁶). The x axis shows the 20 soybean chromosomes, and the y axis shows the significance expressed as −log₁₀ P value. (c) Associations between 25 loci aligned on the upper boundary and 14 phenotype values (contain two traits across six environments and their BLUP) aligned on the lower boundary. Positions of loci correspond to the above panel. Deep red, red, pink, and gray lines represent significant associations between SNPs and phenotype value with threshold levels of P < 1.0 × 10⁻¹¹, P < 1.0 × 10⁻⁹, P < 1.0 × 10⁻⁷, P < 1.0 × 10⁻⁵, respectively.

Figure 3

Associations, genomic locations and the pattern of pairwise LD of SNPs associated with water-soluble protein content (WSPC) on chromosome 8. (a) A 2.5-Mb region of the major-effect quantitative trait loci (GqWSPC8) harboring the peak SNP, AX-94048210 on chromosome 8. The most significantly associated SNP is shown with a big blue dot. Red horizontal lines depict the Bonferroni-adjusted significance threshold (P < 4.95 × 10⁻⁶). The x axis shows the genomic position, and the y axis shows the significance expressed as −log₁₀ P value. (b) Soybean genome region around the SNP marker, AX-94048210 on chromosome 8, whose position is indicated by a vertical gray dashed line (0.25-Mb) on the top panel. (c) The extent of linkage disequilibrium (LD) in the regions based on pairwise r ² values. The r ² values are indicated using the color intensity index. Heatmap showing LD between each pair of markers that passed the Bonferonni threshold in GWAS.

Figure 4

Epistatic interaction between AX-93822697_T_A and AX-93952504 _G_T associated with PC, and candidate gene for each SNP locus. (a) Box plot of PC based on different genotypes in soybean accessions. (b) Phenotypic differences between genotype combinations of the two SNP. (c) and (d) Candidate genes for AX-93822697_T_A and AX-93952504 _G_T loci, respectively. The proposed causal genes are indicated in red. The bottom panel depicts the extent of linkage disequilibrium in the regions based on pairwise r ² values. The r ² values are indicated using the color intensity index shown.

Figure 5

QTLs for soybean protein content (PC) and water-soluble protein content (WSPC) on soybean chromosomes by linkage mapping in RIL population. The lines link denotes epistatic associations between QTL and QTL. Blue line denotes two QTLs in different chromosomes, while red line denotes two QTLs in the same chromosome. The outside/inside wheat-colored circle indicates the LOD/PVE value curve for investigated traits across environments. The outermost circle indicates the 20 soybean chromosomes, QTLs for PC/WSPC, the position and linked markers of these QTLs on the chromosomes.

Figure 6

The genetic overlap between protein content (PC) and water-soluble protein content (WSPC) in the GWAS population. (a) Quantitative trait locus (QTL) categories and their number. (b) Associations between genotypes of SNP AX-93995056 and PC. Box plot of PC in 14 A-type and 195 G-type soybean accessions. The vertical axis indicates the PC. The PC of A-type accessions was significantly higher than that of G-type accessions (t test, P = 3.17 × 10⁻⁸). (c) Associations between genotypes of SNP AX-94048210 and WSPC. Box plot of PC in 50 A-type and 169 G-type soybean accessions. The vertical axis indicates the WSPC. The WSPC of G-type accessions was significantly higher than that of A-type accessions (t test, P = 1.01 × 10⁻²⁹).