Dissecting The Genetic Basis Of Chromatin Accessibility In Rice Pollen At Single-cell Resolution

Pollen is an essential component of sexual reproduction in plants.In rice,microspore mother cells undergo meiosis to form microspores,which then undergo two rounds of mitosis to ultimately form mature tricellular pollen containing one vegetative cell and two sperm cells.Although this process has been well-studied at the cytological level,little is known about cell fate determination and the epigenetic regulatory mechanisms involved.This study uses a single-cell ATAC-seq experimental procedure developed by the research team to obtain high-quality single-cell ATAC-seq data of the rice hybrid Shanyou 63 pollen and analyzes the genetic basis of rice pollen chromatin accessibility variation at the singlecell level.The main findings of this study are as follows:1.Combine clustering results and confirmed genes to identify all cell types and obtain a list of differentially expressed genes.After single-cell clustering analysis,we confirmed the presence of three cell types in our data: microspore cell,sperm cell,and vegetative cell,with sperm cell further divided into two developmental stages,bicellular and tricellular pollen.There were 1,466 microspore cells,524 bicellular sperm cells,2,330 tricellular sperm cell,and 478 vegetative cells.In addition,we obtained a list of differentially expressed genes for each cell type and constructed a pseudotime differentiation trajectory for different cell types.2.Identify similarities and differences in transcription factor activity among different cell types.We performed an analysis of transcription factor activity and found that there were some common transcription factor families with high activity across different cell types,such as the MYB and NAC families.However,we also found some specific transcription factors with high activity in different cell types,such as the B3 and WRKY transcription factors that were uniquely active in vegetative cells compared to other cell types.These differences may be related to the developmental stage of different cell types.3.Obtain research results on differences in chromatin accessibility,CG methylation,and microspore activity among different cell types.Our study found that differences in chromatin accessibility between sperm cells and vegetative cells were mainly attributed to the combined effect of the upstream 1 kb region of the promoter and the distal intergenic region of genes.In addition,we analyzed the CG methylation levels of different cell types and found that the CG methylation level was lowest in vegetative cells,while the tricellular sperm cells had the highest level of CG methylation.Regarding transposon activity,Class I transposon activity was significantly higher in microspores than in vegetative cells and sperm cells,while Class II transposon activity was higher in vegetative cells than in microspores and sperm cells.This suggests that the activity of different types of transposons exhibits a dynamic trend during pollen development.4.A genetic recombination map of Shanyou 63 pollen was constructed,and eight potential biased segregation loci were detected and analyzed for the timing of biased segregation occurrence.Since Shanyou 63 is a hybrid,the genotype of each pollen grain is different.This study constructed a large-scale genetic recombination map of Shanyou 63 pollen using genotype information contained in single-cell sequencing data and detected eight potential biased segregation loci.Among them,the genotype at the locus on chromosome 4 tended to be from the paternal parent,Minghui 63,while the genotypes at the other seven loci tended to be from the maternal parent,Zhenshan 97.Combined with cell type annotation results,biased segregation on chromosome 4 was found to have occurred during the microspore stage.5.A pseudo–Bulked Segregant Analysis(pseudo-BSA)method was proposed for quantitative trait locus(QTL)mapping of molecular traits in single-cell data,and its reliability was demonstrated by using an introgression line as validation material.PseudoBSA can overcome the disadvantage of low coverage in single-cell sequencing technology,accurately identify the association between target traits and variant loci,and thus resolve the genetic basis of molecular traits at the single-cell level.6.For the first time,the genetic basis of chromatin accessibility differences in rice pollen nuclei at the single-cell level was analyzed.Using genotype and chromatin accessibility data from all cells,this study identified a total of 15,376 chromatin accessibility QTLs using the pseudo-BSA method,including 2,278 cis-QTLs and 13,098trans-QTLs.Furthermore,this study investigated the QTL loci regulating chromatin accessibility in different cell types and constructed a genetic regulatory network of chromatin accessibility in different cell types,revealing differences in regulatory patterns among different cell types.In summary,this study systematically analyzed the genetic basis of chromatin accessibility variation in rice pollen and the differences in chromatin accessibility among different cell types,providing information for understanding the pollen development process and its regulation.