Genome-wide association study presents insights into the genetic architecture of drought tolerance in maize seedlings under field water-deficit conditions

Abstract

Introduction: Drought stress is one of the most serious abiotic stresses leading to crop yield reduction. Due to the wide range of planting areas, the production of maize is particularly affected by global drought stress. The cultivation of drought-resistant maize varieties can achieve relatively high, stable yield in arid and semi-arid zones and in the erratic rainfall or occasional drought areas. Therefore, to a great degree, the adverse impact of drought on maize yield can be mitigated by developing drought-resistant or -tolerant varieties. However, the efficacy of traditional breeding solely relying on phenotypic selection is not adequate for the need of maize drought-resistant varieties. Revealing the genetic basis enables to guide the genetic improvement of maize drought tolerance.

Methods: We utilized a maize association panel of 379 inbred lines with tropical, subtropical and temperate backgrounds to analyze the genetic structure of maize drought tolerance at seedling stage. We obtained the high quality 7837 SNPs from DArT's and 91,003 SNPs from GBS, and a resultant combination of 97,862 SNPs of GBS with DArT's. The maize population presented the lower her-itabilities of the seedling emergence rate (ER), seedling plant height (SPH) and grain yield (GY) under field drought conditions.

Results: GWAS analysis by MLM and BLINK models with the phenotypic data and 97862 SNPs revealed 15 variants that were significantly independent related to drought-resistant traits at the seedling stage above the threshold of P < 1.02 × 10-5. We found 15 candidate genes for drought resistance at the seedling stage that may involve in (1) metabolism (Zm00001d012176, Zm00001d012101, Zm00001d009488); (2) programmed cell death (Zm00001d053952); (3) transcriptional regulation (Zm00001d037771, Zm00001d053859, Zm00001d031861, Zm00001d038930, Zm00001d049400, Zm00001d045128 and Zm00001d043036); (4) autophagy (Zm00001d028417); and (5) cell growth and development (Zm00001d017495). The most of them in B73 maize line were shown to change the expression pattern in response to drought stress. These results provide useful information for understanding the genetic basis of drought stress tolerance of maize at seedling stage.

Keywords: SNPs; field drought tolerance; genome-wide association study; maize (Zea mays L.); seedling stage.

Figure 1

Violin plots of distributions of drought resistance related traits in maize seedlings. The horizontal axis represents different traits. In a violin plot, the inner black box represents the interquartile range. The central white dot represents the median value. The outer white shape on each side represents all measured data points and the thickness represents the probability density of the data. Analysis of variance (ANOVA) was applied to examine the difference of phenotypes among different environments. Different letters indicate statistically significant differences at P ≤ 0.05. (A) 19ER, seedling emergence rate measured in Fuxin in 2019; 20ER, seedling emergence rate measured in Fuxin in 2020; ER, seedling emergence rate BLUE value calculated from two-year data; (B) 19SPH, seedling plant height measured in Fuxin in 2019; 20SPH, seedling plant height measured in Fuxin in 2020; SPH, BLUE value of seedling plant height calculated from two-year data; (C) 19GY, grain yield measured in Fuxin in 2019; 20GY, grain yield measured in Fuxin in 2020; GY, grain yield BLUE value calculated from two-year data.

Figure 2

The broad-sense heritability (H²) of drought resistance related traits in maize seedlings. 19ER, seedling emergence rate measured in Fuxin in 2019; 20ER, seedling emergence rate measured in Fuxin in 2020; ER, seedling emergence rate BLUE value calculated from two-year data; 19SPH, seedling plant height measured in Fuxin in 2019; 20SPH, seedling plant height measured in Fuxin in 2020; SPH, BLUE value of seedling plant height calculated from two-year data; 19GY, grain yield measured in Fuxin in 2019; 20GY, grain yield measured in Fuxin in 2020; GY, grain yield BLUE value calculated from two-year data.

Figure 3

Genetic relatedness among the inbred lines visualized using the heat map, dendrogram of kinship matrix and principal component analysis (PCA) in the association panel. (A) Principal component analysis (PCA) in the association population using GBS-DArT dataset, including the first three principal components. The percentage of variation among lines explained by each principal component is displayed on both the x- (PC1), y- (PC2) and z-(PC3) axes. Points in the PCA plot denote individual line; (B) The lower right corner is the genetic distance heat map of paired lines, and the upper and left are the dendrogram of kinship matrix in the association panel using GBS-DArT dataset.

Figure 4

LD decay plots of the NCCP panel. The horizontal axe is the decay distance (Kb) of LD with different r². (A–C) are the LD decay for each chromosome; (D–F) are the average LD decay for all 10 chromosomes.

Figure 5

GWAS-derived Manhattan plots showing significant SNPs associated with maize drought resistance traits using MLM. Each dot represents a SNP. The horizontal dashed black line represents the Bonferroni-corrected significant threshold of 1.02 × 10^-5. (A) 19ER: seedling emergence rate (ER) was measured in 19FX (2019 Fuxin); (B) 20ER: seedling emergence rate (ER) was measured in 20FX (2020 Fuxin); (C) ER: BLUE of seedling emergence rate across two environments; (D) 19SPH: seedling plant height (SPH) was measured in 19FX (2019 Fuxin); (E) 20SPH: seedling plant height (SPH) was measured in 20FX (2020 Fuxin); (F) SPH: BLUE of seedling plant height across two environments; (G) 19GY: grain yield (GY) was measured in 19FX (2019 Fuxin); (H) 20GY: grain yield (GY) was measured in 20FX (2020 Fuxin); (I) GY: BLUE of grain yield across two environments.

Figure 6

GWAS-derived QQ plots using MLM. (A) 19ER: seedling emergence rate (ER) was measured in 19FX (2019 Fuxin); (B) 20ER: seedling emergence rate (ER) was measured in 20FX (2020 Fuxin); (C) ER: BLUE of seedling emergence rate across two environments; (D) 19SPH: seedling plant height (SPH) was measured in 19FX (2019 Fuxin); (E) 20SPH: seedling plant height (SPH) was measured in 20FX (2020 Fuxin); (F) SPH: BLUE of seedling plant height across two environments; (G) 19GY: grain yield (GY) was measured in 19FX (2019 Fuxin); (H) 20GY: grain yield (GY) was measured in 20FX (2020 Fuxin); (I) GY: BLUE of grain yield across two environments.

Figure 7

Heat map of expression patterns of candidate genes identified by GWAS in different tissues and different drought treatments. The values used in the figure were normalized TPM value by row. Columns and rows were ordered according to similarity (hierarchical cluster analysis at the top and left. The red and navy blue represented higher and lower expression level. (A) Expression of 15 genes in different tissues of maize. (B) Expression of 15 genes in different treatments. Drought_T0 is 10 days of drought, Drought_T7 represents 7 days of rehydration after drought and Control is the negative control.

Figure 8

B73 growth plot under normal and drought conditions and the expression of candidate genes verified by qRT-PCR. (A) The left side is B73 maize under normal conditions (60% of the maximum water holding capacity), and the right side is B73 maize under drought conditions (30% of the maximum water holding capacity); (B) The expression level in the leaves of B73 seedlings under drought stress in laboratory. The value in the figure is 2^-ΔΔCt. The statistical significance was determined via an Independent Samples t-test with **P ≤ 0.01, *P ≤ 0.05, and ns represent not statistically significant (P > 0.05).

Figure 9

Distribution of temperature and rainfall in maize planting plots of Fuxin Mongolian Autonomous County in 2019 and 2020. (A, B) represent the monthly average temperatures (°C) for 2019 and 2020, including the monthly average maximum temperature, average temperature, and minimum temperature; (C, D) represent the monthly average rainfall in mm for 2019 and 2020.