Genome-Wide Association Study and Haplotype Analysis Jointly Identify New Candidate Genes for Alkaline Tolerance at Seedling Stage in Rice

doi:10.1016/j.rsci.2025.04.006

Abstract

Abstract:

Alkaline soil is characterized by high soluble salt content, elevated pH levels, and ionic imbalance, all of which collectively intensify the harmful effects of alkaline stress on plants. To gain molecular insights into alkaline tolerance (AT), we evaluated 13 AT-related traits in 508 diverse rice accessions from the 3K Rice Germplasm Project at the seedling stage. A total of 2 929 764, 2 059 114, and 1 365 868 single nucleotide polymorphisms were used to identify alkaline-tolerance QTLs via genome-wide association studies (GWAS) in the entire population as well as in the xian and geng subpopulations, respectively. Candidate genes and their superior haplotypes were further identified through gene-based association, haplotype analysis, and gene function annotation. In total, 99 QTLs were identified for AT by GWAS, and three genes (LOC_Os03g49050 for qSSD3.1, LOC_Os05g48760 for qSKC5, and LOC_Os12g01922 for qSNC12) were selected as the most promising candidate genes. Furthermore, we successfully mined superior alleles of key candidate genes from natural variants associated with AT-related traits. This study identified crucial candidate genes and their favorable alleles for AT traits, laying a foundation for further gene cloning and the development of AT rice varieties via marker-assisted selection.

Key words: rice, alkaline tolerance, genome-wide association study, haplotype analysis, candidate gene

Ratan Kumar Ganapati, Chen Kai, Zhao Xiuqin, Zheng Tianqing, Zhang Fan, Zhai Laiyuan, Xu Jianlong. Genome-Wide Association Study and Haplotype Analysis Jointly Identify New Candidate Genes for Alkaline Tolerance at Seedling Stage in Rice[J]. Rice Science, 2025, 32(4): 537-548.

Figures/Tables 5

Table 1. Statistical summary of 13 alkaline stress-related traits measured in this study.

Trait	Mean	Range	Coefficient of variation (%)
APS (%)	71.05 ± 16.54	11.76-100.00	23.2
RL (cm)	8.85 ± 1.89	3.2-14.8	21.3
RRSNK	2.65 ± 1.27	0.83-9.22	47.7
RSKC	0.58 ± 0.19	0.06-2.99	32.7
RSNC	1.39 ± 0.46	0.14-6.12	32.7
SAT	5.9 ± 1.8	1-9	30.3
SDW (mg)	18.98 ± 8.19	2.25-49.63	43.1
SKC (mmol/L)	0.46 ± 0.22	0.06-1.08	48.2
SL (cm)	16.66 ± 3.41	6.7-29.2	20.4
SNC (mmol/L)	0.58 ± 0.13	0.17-0.82	22.2
SNK	1.56 ± 0.74	0.52-5.42	47.6
SRR	1.95 ± 0.52	0.82-4.68	26.4
SSD (d)	17.63 ± 1.95	12.0-29.0	11.0

Fig. 1. Phenotypic distributions and correlations. A, Box plots of 13 alkaline stress-related traits over 5 subpopulations. Different lowercase letters above boxes indicate significant differences by the Duncan’s multiple comparison test (P < 0.05). B, Correlations among 13 traits in whole population. The number in the middle of the cell is the Pearson correlation coefficient. The lower left section shows scatter plots between each pair of traits. *, **, and *** refer to significant correlations at the 0.05, 0.01, and 0.001 levels, respectively, by the Duncan’s multiple comparison test. APS, Average percent of survival; RL, Root length; RRSNK, Relative shoot Na+/K+ ratio; RSKC, Relative shoot K+ concentration; RSNC, Relative shoot Na+ concentration; SDW, Shoot dry weight; SRR, Shoot/root length ratio; SSD, Seedling survival day; SAT, Score of alkaline tolerance; SL, Shoot length; SNK, Shoot Na+/K+ ratio; SKC, Shoot K+ concentration; SNC, Shoot Na+ concentration.

Table 2. Six important regions detected for different alkaline stress-related traits.

Region/site (bp)	Trait	QTL	Associated SNP	Allele	P-value (Population)
24 747 225-24 894 638	RSNC	qRSNC2	Chr2_24747225	A/T	5.66e-06 (X)
	SKC	qSKC2.2	Chr2_24791853 (W), Chr2_24894638(X)	T/C	4.11e-06 (W) and 5.49e-06 (X)
	SDW	qSDW2.1	Chr2_24804466	A/G	3.54e-06 (X)
16 166 423-16 368 589	SRR	qSRR3	Chr3_16166423	G/A	8.97e-07 (W)
	RRSNK	qRRSNK3.1	Chr3_16205409	T/C	4.33e-06 (G)
	RL	qRL3	Chr3_16368589	T/C	4.18e-08 (W)
27 922 862-27 970 709	SSD	qSSD3.1	Chr3_27970709(W), Chr3_27922862(X)	C/T	2.80e-07 (W) and 2.19e-06 (X)
	RRSNK	qRRSNK3.3	Chr3_27933314	C/T	3.78e-06 (X)
	SNK	qSNK3.1	Chr3_27933314	C/T	4.72e-06 (X)
27 947 287-27 964 670	SKC	qSKC5	Chr5_27947287	A/G	2.10e-06 (W)
	RRSNK	qRRSNK5	Chr5_27957906	G/A	3.48e-06 (X)
	SNK	qSNK5.3	Chr5_27958502	C/T	5.50e-06 (X)
	RSKC	qRSKC5	Chr5_27964670	T/C	5.40e-07 (G)
17 410 717-17 710 717	RRSNK	qRRSNK10	Chr10_17560717	G/A	4.61e-07 (G)
17 410 717-17 710 717	SKC	qSKC10	Chr10_17560717	G/A	2.63e-06 (G)
532 520-557 541	SSD	qSSD12.1	Chr12_532520	T/A	1.66e-06 (W)
	SAT	qSAT12.1	Chr12_541289	T/C	1.47e-06 (W)
	SDW	qSDW12	Chr12_557541	C/T	2.76e-06 (X)
	SNC	qSNC12	Chr12_557541	C/T	4.94e-07 (X)

Fig. 2. Candidate gene analysis associated with qSSD3.1 in whole population on chromosome 3. A, High-density association analysis of qSSD3.1. B-E, Structure of candidate genes [LOC_Os03g49040 (B), LOC_Os03g49050 (C), LOC_Os03g49080 (D), and LOC_Os03g49090 (E)] and their haplotype analysis for seedling survival day (SSD) in the whole population. Different lowercase letters on top of box plots indicate significant differences based on Duncan’s multiple comparison test (P < 0.05).

Fig. 3. Candidate gene analysis associated with qSNC12 in xian subpopulation on chromosome 12. A, High-density association analysis of qSNC12. B-D, Structure of candidate genes [LOC_Os12g01916 (B), LOC_Os12g01922 (C), and LOC_Os12g01930 (D)] and their haplotype analysis for shoot Na+ concentration (SNC) in xian subpopulation. Different lowercase letters on top of box plots indicate significant differences based on Duncan’s multiple comparison test (P < 0.05).

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