Genome-Wide Association Study of Brown Rice Weight Identifies an RNA-Binding Protein Antagonistically Regulating Grain Weight and Panicle Number

doi:10.1016/j.rsci.2025.06.002

Abstract

Abstract:

Rice grain yield is primarily determined by three key agronomic traits: panicle number, grain number per panicle, and grain weight (GW). However, the inherent tradeoffs among these yield components remain a persistent challenge in rice breeding programs. Notably, compared with GW, brown rice weight (BRW) provides a more direct metric associated with actual grain yield potential. In this study, we conducted a two-year replicated genome-wide association study to elucidate the genetic architecture of BRW and identify new loci regulating GW. Among seven consistently detected loci across experimental replicates, four were not co-localized with previously reported genes associated with BRW or GW traits. BRW1.1, one of the four newly identified loci, was found to encode a novel RNA-binding protein. Functional characterization revealed that BRW1.1 acts as a negative regulator of BRW, potentially through modulating mRNA translation processes. Intriguingly, through integrated analysis of mutant phenotypes and haplotype variations, we demonstrated that BRW1.1 mediates the physiological tradeoff between GW and panicle number. This study not only delineates the genetic determinants of BRW but also identifies BRW1.1 as a promising molecular target for breaking the yield component tradeoff in precision rice breeding.

Key words: genome-wide association study, brown rice weight, RNA-binding protein, tradeoff, grain weight, panicle number, grain number, rice yield

Zhou Lin, Jiang Hong, Huang Long, Li Ziang, Yao Zhonghao, Li Linhan, Ji Kangwei, Li Yijie, Tang Haijuan, Cheng Jinping, Bao Yongmei, Huang Ji, Zhang Hongsheng, Chen Sunlu. Genome-Wide Association Study of Brown Rice Weight Identifies an RNA-Binding Protein Antagonistically Regulating Grain Weight and Panicle Number[J]. Rice Science, 2025, 32(4): 525-536.

Figures/Tables 8

Fig. 1. Variation of brown rice weight (BRW) in the rice population. A and B, Frequency distributions of BRW of the rice population in 2019 (A) and 2020 (B). C and D, BRW comparisons among subpopulations in 2019 (C) and 2020 (D). ADM, Admixed; AUS, aus; IND, indica; TEJ, temperate japonica; TRJ, tropical japonica. **, P < 0.01 by the Student’s t-test.

Table 1. Statistical analysis of brown rice weight.

Year	Mean (mg)	SD (mg)	CV (%)	Maximum (mg)	Minimum (mg)	Ratio	Skewness	Kurtosis	h²
2019	22.29	4.15	18.62	39.18	9.87	3.97	0.53	0.94	0.93
2020	21.10	4.01	19.00	37.38	9.18	4.07	0.56	0.98	0.92

Fig. 2. Genome-wide association study (GWAS) of brown rice weight. A and B, Manhattan plots of GWAS results in 2019 (A) and 2020 (B). The x-axis shows the chromosome (Chr.) positions, and the y-axis indicates the -log10 (Transformed P-value) (-log10P). Red dashed lines denote the significance threshold. Blue-highlighted regions with arrows mark seven consistently detected loci across experimental replicates. Blue fonts denote the loci co-localized with known grain size- and grain weight-related genes, and red fonts indicate novel loci. C and D, Quantile-Quantile plots of GWAS results in 2019 (C) and 2020 (D).

Fig. 3. Candidate gene analysis of BRW1.1. A, Linkage disequilibrium (LD) block analysis of BRW1.1. Manhattan plot of genome-wide associated study result for BRW1.1 is shown at the top, with grey and red dashed lines indicating the significance threshold and the lead single nucleotide polymorphism (SNP) position, respectively. y-axis indicates the -log10(Transformed P-value) (-log10P). LD heatmap of BRW1.1 for pairwise correlations (r2) between SNPs is displayed at the middle with barcodes denoting SNP positions. Four open reading frames (ORFs) in the LD block of the lead SNP are shown at the bottom. A red arrow points the lead SNP location at the genomic region of four ORFs. B, Expression heatmap of ORFs across tissues. Inflorescences P2-P6 represent inflorescences of different lengths (P2, 3-6 cm; P3, 7-10 cm; P4, 11-15 cm; P5, 16-22 cm; P6, 23-30 cm). Seeds S1-S5 represent seeds at different days after pollination (DAP) (S1, 0-2 DAP; S2, 3-4 DAP; S3, 5-10 DAP; S4, 11-20 DAP; S5, 21-29 DAP). C, Expression profile of ORF2 examined by real-time qPCR assays. Actin was used as internal control. Endosperm 5, 10, 15, 20, and 25 repsent endosperms at 5, 10, 15, 20, and 25 DAP. D, SNPs in the ORF2 coding sequence. Missense and synonymous mutations are indicated in red and black, respectively. The corresponding amino acids are shown in parenthesis. E, Predicted protein domains of ORF2. Locations of missense mutations are marked by red arrows. CC, Coiled-coil.

Fig. 4. Brown rice phenotypes of BRW1.1 knockout mutants. A, Sequencing results of Zhonghua 11 (WT) and BRW1.1 knockout lines (brw1.1-1, brw1.1-2, and brw1.1-3). Genome editing targets are indicated by red dashed lines. Locations of premature termination codons are provided in parenthesis. PAM, Protospacer adjacent motif (AGG). B, Brown rice morphology of WT and BRW1.1 knockout lines. Brown rice length, width, and thickness are shown respectively. Scale bars, 1 cm. C, Measurements of brown rice weight, length, width, and thickness of WT and BRW1.1 knockout lines. Data are mean ± SE (n = 30). **, P < 0.01 by the Student’s t-test; ns, No significance.

Fig. 5. Grain phenotypes of BRW1.1 knockout mutants. A, Grain length, width, and thickness of Zhonghua 11 (WT) and BRW1.1 knockout lines (brw1.1-1, brw1.1-2, and brw1.1-3). Scale bars, 1 cm. B, Measurements of 1000-grain weight, and grain length, width, and thickness of BRW1.1 knockout lines. Data are mean ± SE (n = 30). *, P < 0.05; **, P < 0.01 by the Student’s t-test.

Fig. 6. Molecular function of BRW1.1. A, Subcellular localization of BRW1.1-GFP (green fluorescent protein) fusion protein in rice protoplasts. Chloroplasts autofluorescence indicates protoplast vigor. Scale bars, 5 μm. B, Gene Ontology enrichment of the top 100 co-expressed genes of BRW1.1.

Fig. 7. BRW1.1-mediated tradeoff between grain weight and panicle number. A, Plant architecture of BRW1.1 knockout lines (brw1.1-1, brw1.1-2, and brw1.1-3). Scale bars, 15 cm. B, Measurements of panicle number, grain number per panicle, and plant height of BRW1.1 knockout lines. Data are mean ± SE (n = 30). *, P < 0.05; **, P < 0.01 by the Student’s t-test; ns, No significance. C, Major BRW1.1 haplotypes (Hap. A-E) in the population of 3K Rice Genome Project. Single nucleotide polymorphisms within the coding sequence are displayed on the upper, and haplotype variation is shown at the lower. Accession numbers of different haplotypes are indicated in parenthesis. D, 1000-grain weight and panicle number of haplotypes. **, P < 0.01 by the Student’s t-test. E, Distribution of haplotypes in subpopulations. ADM, Admixed; AUS, aus; IND, indica; TEJ, temperate japonica; TRJ, tropical japonica.

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