Genome-wide association study reveals that the cupin domain protein OsCDP3.10 regulates seed vigour in rice

Abstract

Seed vigour is an imperative trait for the direct seeding of rice. In this study, we examined the genetic regulation of seedling percentage at the early germination using a genome-wide association study in rice. One major quantitative trait loci qSP3 for seedling percentage was identified, and the candidate gene was validated as qSP3, encoding a cupin domain protein OsCDP3.10 for the synthesis of 52 kDa globulin. Disruption of this gene in Oscdp3.10 mutants reduced the seed vigour, including the germination potential and seedling percentage, at the early germination in rice. The lacking accumulation of 52 kDa globulin was observed in the mature grains of the Oscdp3.10 mutants. The significantly lower amino acid contents were observed in the mature grains and the early germinating seeds of the Oscdp3.10 mutants compared with those of wild-type. Rice OsCDP3.10 regulated seed vigour mainly via modulating the amino acids e.g. Met, Glu, His, and Tyr that contribute to hydrogen peroxide (H₂ O₂ ) accumulation in the germinating seeds. These results provide important insights into the application of seed priming with the amino acids and the selection of OsCDP3.10 to improve seed vigour in rice.

Keywords: Oryza sativa; amino acids; genome-wide association study; rice; seed priming; seed vigour.

Figure 1

Phenotypic diversity of seedling percentage and identification of QTLs by GWAS in rice. (a) Variation and of (b) box‐plots of seedling percentage among the 203 accessions of the rice population studied. (c) Manhattan plots and (d) quantile–quantile (Q‐Q) plots for the whole population of rice accessions. The green dots indicate the qSP3 identified. (e) Identification of haplotype block of qSP3 using the 22 SNPs most significantly associated with seedling percentage. Seven tag SNPs are highlighted in red lines. Pairwise LD was determined by calculation of r ² (the square of the correlation coefficient between SNP states). (f) Seven haplotypes formed from tag SNPs where phenotypic ranges for seedling percentage are explained by haplotypes. (g) Identification of candidate genes with differential expression during seed development and germination in the leading SNP (tag SNP 33020245) region of qSP3. The leading SNP located in the coding region of candidate gene LOC_Os03g57960. Different letters above the column indicate significant difference at the 1% level according to an analysis of variance (ANOVA) test.

Figure 2

Seed vigour in the Nipponbare wild‐type (WT) and Oscdp3.10 mutants under normal and salt (150 mm NaCl) conditions. (a,b) The mutants were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system in rice. (c) Images of the seedling establishment of the WT and the mutant lines in direct seeding in soils. Scale bars are 10 mm. (d) Representative images of the seed germination of the WT and the mutant lines under normal and salt conditions in Petri dishes. Scale bars are 10 mm. Comparison of germination potential (e,g) and seedling percentage (f,h) between WT and mutant lines. Data are means (±SD), n = 30. Significant differences compared with the WT were determined using Student’s t‐test: *P < 0.05; **P < 0.01.

Figure 3

Grain traits in the Nipponbare wild‐type (WT) and Oscdp3.10 mutants. Images of grain length (a) and grain width (b). The grain length (c), width (d), 1,000‐grain weight (e), and the contents of soluble sugar (f), starch (g), and protein (h). Data are means (±SD), n = 3. Significant differences compared with the WT were determined using Student’s t‐test: *P < 0.05; **P < 0.01; n.s., not significant. (i) Comparison of 52 kDa globulin between WT and Oscdp3.10 mutants.

Figure 4

Amino acids level in the mature grains and germinating seeds of Nipponbare wild‐type (WT) and Oscdp3.10 mutants. Data are means (±SD), n = 3. Significant differences compared with the Oscdp3.10 mutants were determined using Student’s t‐test: *P < 0.05; **P < 0.01.

Figure 5

Effects of amino acids on seed vigour in Nipponbare wild‐type (WT) and Oscdp3.10 mutants under normal condition. (a) Representative images are shown of the effects of exogenous applications of Met, Glu, His, and Tyr. Scale bars are 10 mm. Comparison of the germination potential (b), and seedling percentage (c) between the WT and Oscdp3.10 mutants under the H₂O and amino acids treatments conditions. The numbers above the box‐plots indicate the relative value of the mutant compared with that of the WT. Data are means (±SD), n = 3. Significant differences compared with the WT were determined using Student’s t‐test: *P < 0.05; **P < 0.01; n.s., not significant.

Figure 6

Association between OsCDP3.10 and H₂O₂ contents during seed germination. (a) Comparison of the H₂O₂ contents between the WT and Oscdp3.10 mutants during seed germination. (b,c) Comparison of the H₂O₂ contents between the H₂O and amino acids Met, Glu, His, and Tyr treatments among WT and Oscdp3.10 mutants at 36 h and 72 h imbibition stages. Data are means (±SD), n = 3. Significant differences compared with the WT or with H₂O treatment were determined using Student’s t‐test: *P < 0.05; **P < 0.01.

Figure 7

Seed priming with amino acids improves seed vigour of salt sensitive variety Huahang 31 under salt stress (150 mm NaCl) in rice. (a) Images are shown of the effects of seed priming with amino acids Met, Glu, His, and Tyr on seedling establishment in direct seeding in soils and on seed germination in Petri dishes. Scale bars are 10 mm. Comparison of the germination potential (b), and seedling percentage (c) in various priming treatments. Data are means (±SD), n = 3. Different letters above the column indicate significant difference at the 5% level according to an analysis of variance (ANOVA) test.

Figure 8

Haplotypes of OsCDP3.10 associated with seed vigour in rice. (a) Haplotypes of OsCDP3.10 identified in the coding sequence (CDS) and the region 2 kb upstream of the gene. (b,c) Box‐plots of seedling percentage of accessions containing the different haplotypes and SNP 4. (d) The variations in SNP 4 of OsCDP3.10 as determinated using RiceVarMap. (e) Images of the seedling establishment from randomly selected accessions containing either Hap 1 or Hap 2 in direct seeding in soils. Scale bars are 10 mm. (f) Images of seed germination from randomly selected accessions containing either Hap 1 or Hap 2. Scale bars are 10 mm. (g) Comparison of 52 kDa globulin between two haplotypes groups.