Combining Genome-Wide Association Study and Gene-Based Haplotype Analysis to Identify Candidate Genes for Alkali Tolerance at the Germination Stage in Rice

Abstract

Salinity-alkalinity stress is one of the main abiotic factors limiting rice production worldwide. With the widespread use of rice direct seeding technology, it has become increasingly important to improve the tolerance to salinity-alkalinity of rice varieties at the germination stage. Although we have a more comprehensive understanding of salt tolerance in rice, the genetic basis of alkali tolerance in rice is still poorly understood. In this study, we measured seven germination-related traits under alkali stress and control conditions using 428 diverse rice accessions. The alkali tolerance levels of rice germplasms varied considerably during germination. Xian/indica accessions had generally higher tolerance to alkali stress than Geng/japonica accessions at the germination stage. Using genome-wide association analysis, 90 loci were identified as significantly associated with alkali tolerance. Eight genes (LOC_Os01g12000, LOC_Os03g60240, LOC_Os03g08960, LOC_Os04g41410, LOC_Os09g25060, LOC_Os11g35350, LOC_Os12g09350, and LOC_Os12g13300) were selected as important candidate genes for alkali tolerance based on the gene functional annotation and gene-CDS-haplotype analysis. According to the expression levels of LOC_Os09g25060 (OsWRKY76), it is likely to play a negative regulatory role in alkali tolerance during rice germination. An effective strategy for improving rice alkali tolerance may be to pyramid alkali-tolerant haplotypes of multiple candidate genes to obtain the optimal haplotype combination. Our findings may provide valuable genetic information and expand the use of alkali tolerance germplasm resources in rice molecular breeding to improve the alkali tolerance at the germination stage.

Keywords: alkali tolerance; genome-wide association study; germination stage; haplotype analysis; rice.

Figure 1

Phenotypic variations of germination-related traits under alkali stress and control conditions. (A) Distribution of mean germination time (MGT), germination rate (GR), germination index (GI), vigor index (VI), root length (RL), shoot length (SL), and germination energy (GE) under control and alkali stress (0.15% Na₂CO₃). Distribution of relative alkali damage of measured traits in the whole population (B), Xian and Geng subpopulations (C). (D) Correlation coefficient of alkali tolerance-related traits in the whole population. * and *** indicate significant difference at p < 0.05 and p < 0.001 (two-tailed Student’s t-test) in (A,C), respectively.

Figure 2

Genome-wide association study of five alkali tolerance-related traits. (A) RSL in the Geng subpopulation. (B) RRL in the whole population. (C) SLS in the whole population. (D) MGTS in the Xian subpopulation. (E) GES in the Geng subpopulation. (F) Number of associated traits for each of all detected loci (Supplementary Table 3). Manhattan plot (left) and Q–Q plot (right) for each panel in (A–E). Horizontal lines indicate in the Manhattan plots indicate the genome-wide suggestive thresholds. The names of candidate genes or lead SNPs are shown above the corresponding association signals.

Figure 3

Relative expression levels of candidate genes under alkali stress for 24 h. (A) In two representative Xian germplasms. (B) In two representative Geng accessions. Gene expression was normalized to that of the OsActin gene control. The relative expression levels were represented by fold change relative to the expression levels of the candidate genes. ^*p < 0.05 and ^**p < 0.01 (two-tailed Student’ t-test). Data represent means ± SD (n = 3).

Figure 4

Candidate gene analysis of locus 71 on chromosome 9. (A) Local Manhattan plot (top) and LD heat map (bottom) of locus 71 for RRL in the whole population. The red dot indicates the lead SNP rs9_14985169 and the position of candidate gene LOC_Os09g25060. (B) CDS-haplotypes of LOC_Os09g25060. (C) The distribution of RRL in the whole population for the three haplotypes of LOC_Os09g25060. Different letters above each boxplot indicate significant differences among haplotypes according to Duncan’s multiple range post-hoc test ( p < 0.05). (D) Frequency of three haplotypes of LOC_Os09g25060 in subpopulations.

Figure 5

Candidate gene analysis of locus 32 on chromosome 3. (A) Local Manhattan plot (top) and LD heat map (bottom) of locus 32 for MGTS in the Xian subpopulation. The red dot indicates the lead SNP rs3_34257992 and the position of candidate gene LOC_Os03g60240. (B) CDS-haplotypes of LOC_Os03g60240. (C) The distribution of MGTS in the whole population for the six haplotypes of LOC_Os03g60240. Different letters above each boxplot indicate significant differences among haplotypes according to Duncan’s multiple range post-hoc test ( p < 0.05). (D) Frequency of six haplotypes of LOC_Os03g60240 in subpopulations.

Figure 6

Candidate gene analysis of locus 7 on chromosome 1. (A) Local Manhattan plot (top) and LD heat map (bottom) of locus 7 for RGR in the whole population. The red dot indicates the lead SNP rs1_6533274 and the position of candidate gene LOC_Os01g12000. (B) CDS-haplotypes of LOC_Os01g12000. (C) Frequency of four haplotypes of LOC_Os01g12000 in subpopulations. (D) The distribution of GRS, RGR, and RVI in the whole population for the four haplotypes of LOC_Os01g12000. Different letters above each boxplot indicate significant differences among haplotypes according to Duncan’s multiple range post-hoc test ( p < 0.05).

Figure 7

Candidate gene analysis of locus 82 on chromosome 11. (A) Local Manhattan plot (top) and LD heat map (bottom) of locus 82 for RRL in the Xian subpopulation. The red dot indicates the lead SNP rs11_20724735 and the position of candidate gene LOC_Os11g35350. (B) CDS-haplotypes of LOC_Os11g35350. (C) The distribution of RRL in the whole population for the four haplotypes of LOC_Os11g35350. Different letters above each boxplot indicate significant differences among haplotypes according to Duncan’s multiple range post-hoc test ( p < 0.05). (D) Frequency of four haplotypes of LOC_Os11g35350 in subpopulations.

Figure 8

Optimal alkali-tolerant haplotype combination of candidate genes. (A) Combined haplotypes of LOC_Os03g60240, LOC_Os09g25060, and LOC_Os03g08960. “+” and “−” represent favorable and inferior haplotypes, respectively. (B) Comparison of the RGE among different haplotype combinations. Different letters above each histogram indicate significant differences at p < 0.05 (Least Significant Difference test).