Establishment Of Single Cell Expression Analysis Method And Construction Of Cell-type-specific Regulatory Networks In Wheat Root Tips

As one type of organ having function of plant supporting and nutrients absorption,root involves in crop yield extremely.Understanding of gene transcription regulatory networks and mining of key regulatory genes during wheat root development will provide new insights to efficient use of nutrients and yield increase in wheat.Wheat root consists of different types of cells,showing high heterogeneity.However,rules of gene expression in different cell types are still unknown.In this study,we obtained high quality data of Bai Nong Ai Kang 58(AK58)root tips through single nucleus RNA-seq and single nucleus ATAC-seq.Based on this data,we construct a cell-type-specific landscape of wheat root tips.The main research results are as follows:1.An experiment method to analysis gene expression and regulatory by single nuclei in wheat root tips was established.Via the comparison of nuclei quality extracted in different buffer,we find that D Buffer can help to isolate nuclei with high quality efficiently.Then we designed a method which can extract nuclei easily for single cell experiments of wheat root tips.After applying our method in single nuclei RNA sequence experiments at BD Rhapsody and 10 x Genomics Single cell platform,we find that 10 x Genomics Single cell analysis platform works better with our method and captured more nuclei.Therefore,experiments of SnRNA-seq and SnATAC-seq at 10 x Genomics platform were applied in AK58 root tips research.After data filtering,an expression matrix containing 6,875 nuclei and chromatin accessibility profiles of 7,235 nuclei are produced.2.22 types of cells are identified in wheat root tips totally.Both t-SNE and UMAP clustered 22 types of cells in wheat root tips,according to the SnRNA-seq data.The marker genes expression pattern analysis by qPCR and RNA in situ hybridization validated the results of cell type cluster and annotation for root border cell,companion cell,protoxylem,provascular cells and proximal meristem.3.Unbalanced gene expression patterns of subgenome in wheat root at single cell resolution was observed in our research.The comparison of unbalanced subgenomes expression pattern between bulk cells and different types of cells indicates that subgenomes unbalanced gene expression patterns vary from cell types.The ratio of unbalanced homoeolog triads varies from to cell types.4.Mapping of cell clusters between SnRNA-seq data and SnATAC-seq data helps to reveal cell types.We identified 10 cell cluster based on SnATAC-seq.It is observed that some genes with cell-type-specific expression pattern also have specific chromatin accessibility in the same type of cell.5.The cell-specific regulatory networks based on TF-centre was constructed.Totally,185 TF regulon were identified and some of them such as PLT1 and MYB3R4 show a same specifity in regulate activities and expression patterns.In summary,we established a functional single cell expression analysis method.By applying our method in single cell research of AK58 root tips,we obtain transcription landscape as well chromatin accessibility maps at single cell resolution.Our study provided a new perspective to understand wheat species and the cell type specific in wheat root organ.