GWAS and RNA-seq analysis uncover candidate genes associated with alkaline stress tolerance in maize (Zea mays L.) seedlings

Abstract

Soil salt-alkalization is a common yet critical environmental stress factor for plant growth and development. Discovering and exploiting genes associated with alkaline tolerance in maize (Zea mays L.) is helpful for improving alkaline resistance. Here, an association panel consisting of 200 maize lines was used to identify the genetic loci responsible for alkaline tolerance-related traits in maize seedlings. A total of nine single-nucleotide polymorphisms (SNPs) and their associated candidate genes were found to be significantly associated with alkaline tolerance using a genome-wide association study (GWAS). An additional 200 genes were identified when the screen was extended to include a linkage disequilibrium (LD) decay distance of r² ≥ 0.2 from the SNPs. RNA-sequencing (RNA-seq) analysis was then conducted to confirm the linkage between the candidate genes and alkali tolerance. From these data, a total of five differentially expressed genes (DEGs; |log2FC| ≥ 0.585, p < 0.05) were verified as the hub genes involved in alkaline tolerance. Subsequently, two candidate genes, Zm00001d038250 and Zm00001d001960, were verified to affect the alkaline tolerance of maize seedlings by qRT-PCR analysis. These genes were putatively involved protein binding and "flavonoid biosynthesis process," respectively, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses. Gene promoter region contains elements related to stress and metabolism. The results of this study will help further elucidate the mechanisms of alkaline tolerance in maize, which will provide the groundwork for future breeding projects.

Keywords: RNA-seq; alkali tolerance; candidate genes; genome-wide association study; maize.

Figure 1

Morphological change in maize under control (CK) and alkaline (AT) treatment. Comparison of roots of maize inbred line He344; inbred line k10; inbred line Mo17; and inbred line Zheng58 under control and alkaline treatment for 10 days. The alkali-sensitive inbred lines Mo17 and He344 are from the Lancaster group, and the alkali-tolerant inbred lines Zheng58 and K10 are from the PA group.

Figure 2

Distributions of and correlations between 10 relative phenotypic traits. The frequency distribution histograms of 10 traits are located on the diagonal line, the area below the diagonal line is the scatter plot of the traits, and the area above is the correlation coefficient between each pair of traits. ^*, ^**, and ^*** indicate significance at p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 3

Manhattan plots of GWAS results showing the significant SNPs associated with 10 relative phenotypic traits. The traits included the following: the relative root average diameter (RRAD), relative root fresh weight (RRFW), relative root surface area (RRSA), relative root volume (RRV), relative shoot fresh weight (RSFW), relative root dry weight (RRDW), relative root length (RRL), relative root tip number (RRTN), relative shoot dry weight (RSDW), and relative seeding length (RSL).

Figure 4

Venn diagram of DEGs distributed in maize inbred lines K10. The treatments included the normal control treatment for 3 and 4 days (CK), and the alkaline treatment for 3 and 4 days (AT). The alkaline treatment for 3 and 4 days are labeled “K3D_AT”and “K4D_AT,” respectively. The five treatment-line comparisons groups included the following: alkaline treatment for 3 days vs. normal control treatment for 3 days, upregulated expression (K3D_ATvsK3D_CK.up); alkaline treatment for 3 days vs. normal control treatment for 3 days, downregulated expression (K3D_ATvsK3D_CK.down); alkaline treatment for 4 days vs. normal control treatment for 4 days, upregulated expression (K4D_ATvsK4D_CK.up); alkaline treatment for 4 days vs. normal control treatment for 4 days, downregulated expression (K4D_ATvsK4D_CK.down); and candidate genes.

Figure 5

GO annotations and KEGG pathways of DEGs. (A) Bubble chart of GO classifications of five DEGs. (B) Chord chart of GO annotation corresponding to five DEGs. (C) The red box is the KEGG pathway map of Zm00001d02761, Zm00001d001820, and Zm00001d001960 genes.

Figure 6

qRT-PCR validation of the GWAS and RNA-seq results. Expression of five candidate genes in alkaline-tolerant inbred line K10 and the alkaline-sensitive inbred line Mo17. Expression analysis was conducted on leaf that were collected at 3 and 4 days under normal control treatment and alkaline treatment, respectively. ^** indicate significance at p < 0.01.

Figure 7

The characterization of candidate genes. (A) Evolutionary tree of Zm00001d038250 protein. (B) Evolutionary tree of Zm00001d001960 protein. (C) The promoter region of Zm00001d038250 gene. (D) The promoter region of Zm00001d001960 gene.