Studies On Single-Cell Marker Genes And Their Applications On Annotation Of Glandular Trichome Cells In Plants

Single-cell omics technology is the latest research frontier in the field of biology,especially the single-cell transcriptome sequencing technology(sc RNA-seq),which has been widely used in recent years.It is mainly used to reveal information such as gene expression at the overall level in a single cell,accurately reflect the heterogeneity between cells,and deeply understand the relationship between gene expression and phenotype.Plant single-cell transcriptomic studies have emerged in recent years.In this study,an analysis of marker genes for cell type annotation,a key issue of plant single-cell transcriptomic data analysis,was carried out.The database of plant cell type marker genes was established by this study,and the enzymatic hydrolysis and cell cycle-related genes affecting cell type annotation were analyzed and determined by single-cell assay using plant cell line(tobacco BY2 cell line).As an example,the marker genes obtained from this study were used for annotation of cell types(specifically glandular hair cells)of tobacco leaves based on their single-cell transcriptomic data.The main findings are as follows:1.Analysis of plant cell type marker genes: Public available plant single cell marker genes obtained from traditional laboratory validation,bulk RNA sequencing(bulk RNA-seq)and single-cell transcriptome data(sc RNA-seq)analysis were collected;the available plant sc RNA-seq data were re-analyzed by this study and marker genes for each cell type were achieved according to a unified mainstream analysis process.This analysis resulted in 24,573 cell marker genes from four plants(Arabidopsis,rice,tomato and maize).These marker genes can be used to annotate273 different cell types in 15 tissues of the plants mentioned above.2.Construction and update of a database of plant cell type marker genes(Plantsc RNAdb): Based on the plant cell marker genes obtained above,the plant cell type marker gene database named "Plantsc RNAdb" was constructed.The database provides marker gene retrieval,visualization of single-cell expression,genome browsing,and homologous gene BLAST search,etc..i.e.important basic data and tools for plant single-cell transcriptome analysis.On this basis,the database was updated to Release 2.0 up to now by this study.The current database(Release 2.0)contains 69,628 cell type marker genes from 8 species,which can be used for annotation of 364 cell types in 31 tissues of plants.3.Identification and application of plant cell cycle-related genes: Many factors can affect the accuracy of plant cell annotation,such as enzymatic hydrolysis and cell cycle changes in plant single-cell omics research.In this study,by sequencing and analyzing the tobacco BY2 cell line using RNA-seq/sc RNA-seq approaches,896 cell cycle phase-related genes were finally obtained by excluding genes affected by enzymatic hydrolysis(based on RNA-seq).Through conservation analysis with other known plant cell cycle-related genes,21 functional domain-related genes were obtained.This data will significantly improve the quality of the above-mentioned marker genes and cellular annotation.4.An application case of plant single cell marker genes---annotation of tobacco glandular trichome cells: Using the Plantsc RNAdb marker genes established in this study and the results on genes involving such as cell cycle and enzymatic hydrolysis,as a case study,we carried out a tobacco leaf single-cell(nucleus)sequencing and its cellular annotation.Using the Plantsc RNAdb marker genes,we could clearly annotate cell types in tobacco leaf cells,including three major cell groups(epidermal,vasculature,and mesophyll).Most importantly,we annotated an important cell type in tobacco leaves---glandular trichome cells.Accurate cell type annotation is the foundation and first step in plant single-cell omics analysis.The database of plant single-cell marker genes established in this study and the results on genes potentially affecting cell annotation(such as cell cycle and enzymatic hydrolysis)will contribute to plant single-cell omics research,especially the accurate annotation of cell types.