Studies On Gene Expression Regulation And Abortion Mechanism For Ovules Of High-frequency Female-sterile Rice

Rice is an important food crop and monocotyledonous plant,which has a small genome and relatively complete genomic information.Rice female gametophyte fertility affects the yield directly.However,we know very little about the mechanism of female sterility in rice.Therefore,exploring the mechanism of female sterility in rice has great theoretical significance and application value.In this study,high-throughput sequencing was used to compare the DNA methylation,alternative splicing patterns and lncRNA expression characteristics of a wild-type rice Gui99 and a high-frequency female sterile rice fsv1 ovules at different developmental stages in a genome-wide level.We expect to provide clues for clarifying the mechanism of rice female gametophyte sterility.DNA methylation is an essential epigenetic modification that dynamically regulates gene expression during plant development processes.Female gametophyte fertility as a major field of rice sexual reproduction research,it is of great significance to explore the methylation characteristics of ovule development in female-sterile rice.In this study,ovules were continuously acquired from the beginning of megaspore mother cell meiosis until the mature female gametophyte formation period,and global DNA methylation patterns were compared in the ovules of a high-frequency female-sterile line(fsv1)and a wild-type rice line(Gui99)using whole-genome bisulfite sequencing(WGBS).Profiling of the global DNA methylation revealed hypo-methylation.We divided the genome into different elements and calculated the methylation ratio of each genome element in the ovules of two rice lines.A total of 2348 differential methylated genes(DMGs)and 3,471 differentially methylated regions(DMRs)were screened with a threshold of methylation ratio change?2 and a FDR?0.001.Based on KEGG analysis of differentially methylated genes(DMGs),we observed more DMGs enriched in cellular component,reproduction regulation,metabolic pathway,and other pathways.In particular,many ovule development genes and plant hormone-related genes such as OsAPC6,CYP97A4,CHR721,OsSPL1 and OsPIN8 showed significantly different methylation patterns in the two rice lines,and these differences may provide important clues for revealing the mechanism of female gametophyte abortion.The alternative splicing of pre-mRNA is a crucial step in the process of gene expression.On the one hand,it cuts the introns in the mRNA precursor and joins the exons to form mature mRNA.On the other hand,it can produce abundant splice isoforms and increase protein diversity.In this study,high-throughput sequencing was used to investigate the ovule pre-mRNA alternative splicing events in different developmental stages of two rice line ovules.The results indicate that the alternative splicing events are widely distributed,about 33% of the genes whose pre-mRNA have alternative splicing events.Intron retention is the main alternative splicing form,genes having multiple transcripts and multiple exons are more susceptible to alternative splicing,but gene expression is not significantly correlated with the occurrence of alternative splicing events.We have screened 223,761 and 345 differentially splicing genes(DSG)in three different ovule developmental stages of two rice lines.we found that these differentially spliced genes were mainly enriched in metabolic pathways such as cellular components,signal transduction,and reproduction by GO functional annotation.In addition,we also found some important auxin-related genes and transcription factor genes such as OsARF5,OsARF8,OsMYB3R-2 and OsbZIP13 have significant differential splicing patterns,which may be related to female gametophytic abortion and deserve further study.Long noncoding RNAs(lncRNAs)play important and wide-ranging roles in the growth and development of plants and have become a major research focus in recent years.Therefore,we explored the profile of lncRNAs during ovule development and female gametophytic abortion.In our study,whole-transcriptome strand-specific RNA sequencing(ssRNA-seq)was performed in the ovules of a high-frequency female-sterile rice line(fsv1)and a wild-type rice line(Gui99)at the megaspore mother cell meiosis stage(stage 1),functional megaspore mitosis stage(stage 2)and female gametophyte mature stage(stage 3).By comparing two rice lines,we identified 152,233,and 197 differentially expressed lncRNAs at the three ovule developmental stages.Functional analysis of target genes of these lncRNAs indicated that many lncRNAs participate in multiple pathways such as hormone and cellular metabolism and signal transduction.Moreover,there were many differentially expressed lncRNAs acting as the precursors of some miRNAs that are involved in the development of ovules and female gametophytes.In addition,we have found that lncRNAs can act as decoys,competing with mRNAs for binding to miRNAs to maintain the normal expression of genes related to ovule and female gametophyte development.These results provide important clues for elucidating the female gametophyte abortion mechanism in rice.This study also expands our understanding about the biological functions of lncRNAs and the annotation of the rice genome.In summary,we analyzed the DNA methylation,alternative splicing and lncRNA patterns of in fsv1 and Gui99 ovules at different stages on the whole genome level,and found that these gene regulators mainly affect female gametophyte abortion by regulating ovule and female gametophyte development-related genes,transcription factor genes and plant hormone-related genes.In addition,we also constructed gene expression regulatory networks based on gene and miRNA data,and found ovule development and female gametophyte abortion is a complex and exquisite process involving multiple gene expression and regulation.The various regulatory factors interact with each other and synergistic effect at different levels of gene expression.