Identification Of Cotton Gene Resources And DNA Methylation Mechanism Of Flooding Resistance

In recent years,with the obvious increase of flood disasters,the field of cotton flooding tolerance has become a research hotspot.Cotton is a kind of crop sensitive to flood disaster.Most of the previous studies focused on the measurement of physiological and biochemical indexes and yield loss,but lack of systematic analysis and research on the molecular mechanism and identification of flooding tolerance in cotton.In this study,a method for identifying the flooding tolerance of cotton genetic resources was established and verified.We then mapped the genome-wide single base resolution cytosine methylation profile of submergence tolerant cotton under submergence stress.By further integrating RNA-seq data,we analyzed the potential regulatory relationship between DNA methylation and gene transcriptional expression,and initially analyzed the potential epigenetic regulatory mechanism of cotton response to submergence stress.Finally,the genes related to submergence tolerance were mined and the preliminary functional analysis was carried out.The main findings are as follows:1.Carried out cotton submergence stress experiments and established a method to identify cotton submergence tolerance.This method was used to identify the submergence tolerance of 100 cotton germplasm resources.The results showed that there was less submergence resistant and more submergence tolerant germplasm,and most of them were submergence sensitive germplasm.2.The phenotypic changes,transcriptome and epitranscriptome of cotton under submergence stress were analyzed,with emphasis on the dynamic changes of methylation in submergence tolerant germplasm ZNL2067 under submergence stress.There were significant differences in stem and leaf structure,biological and economic yield,and net photosynthetic rate between submergence tolerant germplasm ZNL2067 and submergence sensitive germplasm ZL100 under flooding stress.A total of6893 DEGs were identified under submergence stress.The data volume of Raw data obtained by ZNL2067 under submergence stress was 80.42 Gb,and the methylation levels of C locus,CG,CHG and CHH sequences in the whole genome were 22.21%,54.98%,50.02% and 12.97%.For the functional regions of the genome,the methylation level of the distal promoter region and the downstream region of the gene was high,the methylation level of the gene body was low,and the methylation level of the transcription start site(TSS)and transcription end site(TES)was the lowest.Compared with normal growth treatment,the methylation levels of promoter region,exon region,intron region and upstream and downstream 2K of gene were decreased under submergence stress.A total of 3705 hypermethylated DMR and 7273 hypomethylated DMR were identified,among which 366 hypermethylated DMR and 3594 hypomethylated DMR were identified in the CG sequence of exons.The DMR length is mainly concentrated between 100 and 200 bp.KEGG enrichment indicated that DMGs were involved in the regulation of metabolic pathways,biosynthesis of secondary metabolites,and plant hormone signal transduction.3.Through the combined analysis of transcriptome and epitranscriptome,the potential regulatory mechanism between methylation dynamics and transcriptional expression changes in cotton in response to submergence stress was preliminarily analyzed.Overall,there was a negative correlation between gene expression level and methylation level.The methylation level of the promoter region of GHUGT74-2 was lower under submergence stress and increased during reoxygenation.We speculate that DNA methylation may regulate GHUGT74-2 transcription and IAA synthesis by mediating glucosinolate biosynthesis pathway,which in turn promotes adventitious roots formation and ultimately improves submergence tolerance of cotton.4.The submergence tolerance genes were excavated and the preliminary functional analysis was carried out.First,752 UGTs were identified from G.arboreum,G.barbadense,Gossypium raimondii,G.hirsutum,and these genes were systematically analyzed by bioinformatics.Then a preliminary functional analysis of GHUGT74-2 was performed.Subcellular localization indicated that GHUGT74-2protein was localized to the nucleus and cell membrane.VIGS et al.showed that GHUGT74-2 gene was involved in the response to submergence stress.Finally,the GHUGT74-2 gene was introduced into cotton and transgenic plants were obtained.