Preliminary Study On The Epigenetic Mechanism Of DNA Methylation In Regulating The Fertility Of Cotton CMS-D2 Restorer Line Under High-temperature Stress

In recent years,with the increasing cost of manual hybrid seed production year by year,cytoplasmic male sterility(CMS)has gradually become a research hotspot in the field of cotton heterosis utilization.However,the anther development of the restorer line of CMS conditioned by Gossypium harknessii cytoplasm(CMS-D2)is susceptible to consecutive high-temperature(HT)stress,which limits the widespread application of "three-line" cotton hybrids.Although some studies have reported that DNA methylation is involved in regulating plant anther development,the potential regulatory role of genomewide DNA methylation dynamics in the fertility of cotton CMS-D2 restorer line under HT stress is still lacking in systematic analysis and exploration.In this study,the whole-genome bisulfite sequencing(WGBS)technique was first used to compare and analyze four samples during the critical period of anther development(flower buds about 3mm in length)of the maintainer line ZB(HT-tolerant)and its nearisogenic restorer line ZBR(HT-sensitive)under HT stress and suitable temperature(ST)conditions,and the single-base resolution cytosine methylation maps of cotton anther development under HT stress were deciphered.Subsequently,through the integrated transcriptome data,we further explored the relationship between the differences in epigenomic changes and transcriptional expression changes in response to HT stress during the anther development of ZB and ZBR.Combined with the experimental results of in vitro spray treatment,the potential epigenetic mechanism of anther inferility of cotton CMS-D2 restorer line induced by HT stress was preliminarily revealed.The main findings are as follows:1.Taking the maintainer line ZB as an example,the cotton anther genome was found to display approximately 31.6%,68.7%,61.8%,and 21.8% methylation across all sequenced C sites and in the CG,CHG and CHH sequence contexts,respectively.This represents the percentage of DNA methylation levels(MLs)in the whole anther genome.In gene-rich regions on cotton chromosomes,the density of transposable elements(TEs)is low,accompanied by a relatively low ML;in contrast,TE-rich regions have a higher density of cytosine methylation distribution.The preference of cytosine methylation sites in cotton anthers is highly correlated with DNA sequence contexts and sequence regions with a high or low density of cytosine methylation,but it has not been changed under HT stress.The CG and CHG MLs in ZBR showed only a slight increase under HT stress,whereas ZB exhibited obvious CHH demethylation,especially in promoter and repeat regions.2.In the cotton anther genome,the degree of promoter methylation did not appear to be significantly correlated with gene expression levels and only promoter-unmethylated genes presented relatively higher expression levels,whereas there was an apparent positive correlation between the degree of DNA methylation in gene body regions and gene expression levels.Additionally,the majority of differentially expressed genes(DEGs)in response to HT stress were found not to be associated with corresponding DNA methylation variations.3.Through systematic analysis of the DNA methylation patterns of TEs in cotton anthers,we found that the DNA MLs of different TEs were highly correlated with their sequence lengths.More differentially methylated TEs(DMTEs)in maintainer line ZB presented obvious demethylation in the CG,CHG and CHH contexts under HT stress;conversely,its near-isogenic restorer line ZBR showed more hypermethylated TEs compared with ZB.4.Integrated methylome and transcriptome data analysis revealed that DNA methylation-mediated transcriptional changes of genes involved in the ‘oxidative phosphorylation' pathway,including GhNDUS7,GhCOX6 A,GhCX5B2 and GhATPBM,may play a vital role in anther development under HT stress.5.The flower buds and leaves of ZBR were sprayed with different concentrations of DNA methyltransferase inhibitor 5-azacytidine(5-azaC)under HT stress,indicating that DNA demethylation can contribute to the normal development of cotton anthers.However,the flower buds and leaves of ZB were sprayed with different concentrations of DNA methylation accelerator methyl trifluoromethanesulfonate(MTFMS)under HT stress,indicating that increased DNA MLs can only partially inhibit anther development.