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. 2024 Sep 24;15(10):1241.
doi: 10.3390/genes15101241.

Whole-Genome Bisulfite Sequencing (WGBS) Analysis of Gossypium hirsutum under High-Temperature Stress Conditions

Zhaolong Gong  1 , Juyun Zheng  1 , Ni Yang  1 , Xueyuan Li  1 , Shuaishuai Qian  1 , Fenglei Sun  1 , Shiwei Geng  1 , Yajun Liang  1 , Junduo Wang  1
Affiliations

Whole-Genome Bisulfite Sequencing (WGBS) Analysis of Gossypium hirsutum under High-Temperature Stress Conditions

Zhaolong Gong et al. Genes (Basel). .

Abstract

Background: DNA methylation is an important part of epigenetic regulation and plays an important role in the response of plants to adverse stress.

Methods: In this study, whole-genome bisulfite sequencing (WGBS) was performed on the high-temperature-resistant material Xinluzao 36 and the high-temperature-sensitive material Che 61-72 at 0 h and 12 h under high-temperature stress conditions.

Results: The results revealed that the Gossypium hirsutum methylation levels of CG and CHG (H = A, C, or T) decreased after the high-temperature stress treatment, and the methylation level of the A subgenome was significantly greater than that of the D subgenome. The methylation level of CHH increased, and the methylation level of CHH in the D subgenome was significantly greater than that in the A subgenome after high-temperature stress treatment. The methylation density of CG is lower than that of CHG and CHH, and the methylation density of the middle region of chromosomes is greater than that of both ends, which is opposite to the distribution density of genes. There were 124 common differentially methylated genes in the CG, CHG, and CHH groups, and 5130 common DEGs and differentially methylated genes were found via joint analysis with RNA-seq; these genes were significantly enriched in the biosynthesis of plant hormones, thiamine metabolism, glutathione metabolism, and tyrosine metabolism pathways. DNA methylation did not affect the expression of many genes (accounting for 85.68% of the differentially methylated genes), DNA methylation-promoted gene expression was located mainly in the downstream region of the gene or gene body, and the expression of inhibitory genes was located mainly in the upstream region of the gene.

Conclusions: This study provides a theoretical basis for further exploration of the gene expression and functional regulatory mechanism of G. hirsutum DNA methylation under high-temperature stress conditions.

Keywords: G. hirsutum; RNA-seq; WGBS; high temperature.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
(a) Cytosine and average methylation levels of each context at 0 h and 40 h under the high-temperature stress treatment for Xinluzao 36 and Che 61–72. (b) The proportion of methylated cytosine in Xinluzao 36 and Che 61–72 under the high-temperature stress treatment at 0 h and 40 h.
Figure 2
Figure 2
(a) CG methylation levels of each subgenome at 0 h and 40 h under the high-temperature stress treatment in Xinluzao 36 and Che 61–72; (b) CHG methylation levels in each subgenome at 0 h and 40 h under the high-temperature stress treatment in Xinluzao 36 and Che 61–72; and (c) CHH methylation levels in each subgenome at 0 h and 40 h under the high-temperature stress treatment in Xinluzao 36 and Che 61–72. (error bars represent the mean ± SE of three replicates, ** p < 0.01. ns means no statistical significance).
Figure 3
Figure 3
The methylation distribution map from outside to inside was as follows: gene density, CG (car 61–72, 0 h), CHG (car 61–72, 0 h), CHH (car 61–72, 0 h), CG (car 61–72, 12 h), CHG (car 61–72, 12 h), CHH (car 61–72, 12 h), CG (Xinluzao 36, 0 h), CHG (Xinluzao 36, 0 h), CHH (Xinluzao 36, 0 h), CG (Xinluzao 36, 12 h), CHG (Xinluzao 36, 12 h), and CHH (Xinluzao 36, 12 h) chromosomal range density distribution information. The blue-to-yellow color represents the direction in which the density increased.
Figure 4
Figure 4
(a) CG methylation levels in genetically different regions of the genome (upstream, gene body, and downstream); (b) CHG methylation levels in genetically different regions of the genome (upstream, gene body, and downstream); and (c) CHH methylation levels in genetically different regions of the genome (upstream, gene body, and downstream).
Figure 5
Figure 5
(a) Venn diagram of the differential methylation regions (DMRs) at 0 h and 12 h under high-temperature stress treatment; different colors represent different methylation types, and the numbers represent the number of DMRs. (b) Venn diagram of the differential methylation regions (DMRs) at 0 h and 12 h under high-temperature stress treatment; the different colors represent different methylation types and the numbers represent the number of DMRs. (c) Venn diagram of the differential methylation regions (DMRs) at 0 h under high-temperature stress treatment; the different colors represent different methylation types and the numbers represent the number of DMRs. (d) Venn diagram of the differential methylation regions (DMRs) at 6 h and 72 h under high-temperature stress treatment; the different colors represent different methylation types and the numbers represent the number of DMRs.
Figure 6
Figure 6
(a) Venn diagram of the differentially expressed genes identified via RNA-seq; (b) Venn diagram of the differentially methylated genes; (c) Venn diagram of the differentially methylated genes and the DEGs; (d) scatter plot of the DEGs identified via KEGG enrichment analysis; (e) scatter plot of the differentially methylated gene KEGG enrichment analysis; and (f) KEGG enrichment analysis scatter plot of the differentially methylated genes and common DEGs.
Figure 7
Figure 7
(a) WGCNA gene clustering dendrogram; (b) correlation and significance of the module with Xinluzao 36 and Che 61–72 at 0 h and 12 h; and (c) linear relationship between the expression levels of eight candidate genes and methylated water.
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