Identification of Single Nucleotide Polymorphic Loci and Candidate Genes for Seed Germination Percentage in Okra under Salt and No-Salt Stresses by Genome-Wide Association Study

Abstract

Excessive soil salinity is a major stressor inhibiting crops' growth, development, and yield. Seed germination is a critical stage of crop growth and development, as well as one of the most salt-sensitive stages. Salt stress has a significant inhibitory effect on seed germination. Okra is a nutritious vegetable, but its seed germination percentage (GP) is low, whether under salt stress conditions or suitable conditions. In this study, we used 180 okra accessions and conducted a genome-wide association study (GWAS) on the germination percentage using 20,133,859 single nucleotide polymorphic (SNP) markers under 0 (CK, diluted water), 70 (treatment 1, T1), and 140 mmol/L (treatment 2, T2) NaCl conditions. Using the mixed linear model (MLM) in Efficient Mixed-model Association eXpedated (EMMAX) and Genome-wide Efficient Mixed Model Association (GEMMA) software, 511 SNP loci were significantly associated during germination, of which 167 SNP loci were detected simultaneously by both programs. Among the 167 SNPs, SNP2619493 on chromosome 59 and SNP2692266 on chromosome 44 were detected simultaneously under the CK, T1, and T2 conditions, and were key SNP loci regulating the GP of okra seeds. Linkage disequilibrium block analysis revealed that nsSNP2626294 (C/T) in Ae59G004900 was near SNP2619493, and the amino acid changes caused by nsSNP2626294 led to an increase in the phenotypic values in some okra accessions. There was an nsSNP2688406 (A/G) in Ae44G005470 near SNP2692266, and the amino acid change caused by nsSNP2688406 led to a decrease in phenotypic values in some okra accessions. These results indicate that Ae59G004900 and Ae44G005470 regulate the GP of okra seeds under salt and no-salt stresses. The gene expression analysis further demonstrated these results. The SNP markers and genes that were identified in this study will provide reference for further research on the GP of okra, as well as new genetic markers and candidate genes for cultivating new okra varieties with high GPs under salt and no-salt stress conditions.

Keywords: GWAS; candidate gene; germination; okra; salt tolerance.

Figure 1

Frequency distribution of GP in 180 okra accessions.

Figure 2

Population structure of 180 okra cultivars and 10 wild species using SNP genotyping data. a: population structure based on K = 4 using the ADMIXTURE program. The x-axis represents the okra accessions, and the y-axis represents the probability that an individual belongs to a subgroup. Yellow, green, blue, and red represent Pop I, Pop II, Pop III, and Pop IV, respectively.

Figure 3

PCA of 180 okra cultivars and 10 wild species. PC1 and PC2 refer to the first and second principal components, respectively. The colors of dots correspond to the structure grouping.

Figure 4

LD block analysis of SNP2619493 on chromosome 59. Regional Manhattan plot (top) and LD heatmap (bottom) surrounding the SNP2619493. Red dotted line represents the significance threshold of −log10 (p) = 4. Red circle indicates SNP2619493, and blue circles indicate the SNP loci that are not significantly associated. Triangles indicate individual haplotype blocks.

Figure 5

LD block analysis of SNP2692266 on chromosome 44. Regional Manhattan plot (top) and LD heatmap (bottom) surrounding the SNP2692266. Red dotted line represents the significance threshold of −log10 (p) = 4. Red circles indicate SNP2692266, and blue circles indicate the SNP loci that are not significantly associated. Triangles indicate individual haplotype blocks.

Figure 6

The position of the nsSNPs in the candidate genes. (a) The position of nsSNP2626294 in the Ae59G004900 gene; (b) the position of nsSNP2688406 in the Ae44G005470 gene. Orange squares represent the exon regions, and black lines represent the noncoding regions.

Figure 7

Phenotypes of the accessions with different nsSNP2626294 in Ae59G004900 alleles. (a,c,e) represent the GP-CK-3 d, GP-T1-3 d, and GP-T2-3 d of the 180 accessions, respectively; CC represents the homozygous reference allele, CT represents the heterozygous allele, TT represents the homozygous mutant allele; n represents the number of accessions; * and ** represent significant differences at p < 0.05 and p < 0.01 by the t-test, respectively. (b,d,f) represent the gene expression levels of Ae59G004900 under the CK, T1, and T2 conditions, respectively. W1, W2, and W3 represent the CC-type accessions 18, 34, and 70, respectively; M1, M2, and M3 represent the TT-type accessions 32, 99, and 157, respectively.

Figure 8

Phenotypes of the accessions with different nsSNP2688406 in Ae44G005470 alleles. (a,c,e) represent the GP-CK-7 d, GP-T1-7 d, and GP-T2-7 d of the 180 accessions, respectively; AA represents the homozygous reference allele, and GG represents the homozygous mutant allele; n represents the number of accessions; and * and ** represent significant differences at p < 0.05 and p < 0.01 by the t-test, respectively. (b,d,f) represent the gene expression levels of Ae44G005470 under the CK, T1, and T2 conditions, respectively. W1, W2, and W3 represent the AA-type accessions 18, 34, and 70, respectively; M4, M5, and M6 represent the GG-type accessions 66, 82, and 141, respectively.