A Genome-Wide Association Study Reveals Candidate Genes Related to Salt Tolerance in Rice (Oryza sativa) at the Germination Stage

Abstract

Salt toxicity is the major factor limiting crop productivity in saline soils. In this paper, 295 accessions including a heuristic core set (137 accessions) and 158 bred varieties were re-sequenced and ~1.65 million SNPs/indels were used to perform a genome-wide association study (GWAS) of salt-tolerance-related phenotypes in rice during the germination stage. A total of 12 associated peaks distributed on seven chromosomes using a compressed mixed linear model were detected. Determined by linkage disequilibrium (LD) blocks analysis, we finally obtained a total of 79 candidate genes. By detecting the highly associated variations located inside the genic region that overlapped with the results of LD block analysis, we characterized 17 genes that may contribute to salt tolerance during the seed germination stage. At the same time, we conducted a haplotype analysis of the genes with functional variations together with phenotypic correlation and orthologous sequence analyses. Among these genes, OsMADS31, which is a MADS-box family transcription factor, had a down-regulated expression under the salt condition and it was predicted to be involved in the salt tolerance at the rice germination stage. Our study revealed some novel candidate genes and their substantial natural variations in the rice genome at the germination stage. The GWAS in rice at the germination stage would provide important resources for molecular breeding and functional analysis of the salt tolerance during rice germination.

Keywords: genome-wide association study; germination; natural variation; rice; salt stress.

Figure 1

Determination of the optimum NaCl concentration and main phenotypes under salt stress and control conditions. (a) Individual value plot of germination percentage in the presence of 0, 200, and 300 mM NaCl (each dot represents an individual). (b–d) Box plots for phenotypic values in the presence of 0 and 200 mM NaCl (the asterisks are extreme outliers). GP: germination percentage, GE: germination energy, GI: Germination index, SL: shoot length, RL: root length.

Figure 2

Principal components analysis (PCA) using genotype and phenotype data. (a). For genotype data, 295 accessions were divided into ind (indica) and jap (japonica) based on PC1 and PC2 along with an admixture group. (b). For phenotype data, no clear grouping was observed.

Figure 3

Genome-wide association mapping and LD block analysis for root length (RL) under salt stress (200 mM) conditions. (a) Manhattan plot from association mapping using the CMLM. (b) QQ plot of expected and observed P values. (c) The peak region on chromosome 4 along with the LD blocks. (d) The peak region on chromosome 12 along with the LD blocks. In (c,d) pair-wise LD between SNPs is indicated as D’ values: red indicates a value of 1 and yellow indicates 0. The LD region was 200 kb upstream and downstream of the top −log (p) value in the peak range.

Figure 4

Haplotyping and sequence analysis of Os04g0614100, which was correlated with a phenotypic difference. (a) One functional SNP of the candidate gene in the CDS region. Type 1 is the reference and type 2 is the SNP. (b) The phenotypic difference based of the functional SNP. (c) Amino acid sequence alignment using several orthologues in various rice subgroups and species. Red box indicates the target amino acid change caused by the functional SNP. (d) RNA expression levels in rice accessions with type 1 and type 2. (e) Haplotype network analysis. Circle size is proportional to the number of samples within a given haplotype. Lines between haplotypes represent mutational steps between alleles. Colors denote rice designation.