Bulk RNA-seq Of Leafy Head And Single-cell Transcriptome Analysis Of Leaf In Brassica Rapa

Leafy head is a special product organ of Chinese cabbage.Analyzing the molecular mechanism of leafy head formation is of great value to the genetic improvement of Chinese cabbage quality.The formation of leafy head is a very complicated process,which requires three developmental stages:seedling stage,rosette stage and heading stage.There are obvious changes occur in leaf morphology during the development process.At the heading stage,the leaves of different layers also showed continuous morphological changes from the outside to the inside,including the size,color and degree of curling of the leaves;even the degree of curling of different parts of the same leaves was different.This difference in leaf morphology is usually caused by the heterogeneity of gene expression between leaf tissues and between single cells.Transcriptome sequencing has been widely used for Chinese cabbage heading.However,previous transcriptome analyses to date have focused on only the inner leaves between different stages or simply separated the blade and midrib of internal and external leaves of leafy heads.At present,there is still a lack of detailed analysis of a series of different layers of leaves from outside to inside of the whole plant at the heading stage,as well as a detailed analysis of the transcriptome of different parts of the same leaf.There is no any report on the single-cell transcriptome of Chines cabbage leaves.This study reported the transcriptome profiling of Chinese cabbage plant at the heading stage using24 spatially dissected tissues representing different regions of the inner to outer leaves.Genome-wide transcriptome analysis clearly distinguished inner leaf tissues from outer leaf tissues as well as leaf blades and leaf ribs.In particular,we analyzed the expression patterns of the genes that regulate leaf development(adaxial and abaxial polarity genes and the genes involved in cell proliferation/cell expansion)and key genes for sugar metabolism,found significant changes in gene expression at leaf layer L7,which thus were confirmed as the key transition leaves.We also observed that the key transition leaves were the first inwardly curved ones during the heading process.Surprisingly,most of the heading candidate genes identified by domestication selection analysis also showed significant expression pattern changes in the key transition leaves,supporting that the key transition leaves were closely related to the heading.Through weighted gene coexpression network analysis,we found that the key transformation leaves were controlled by a complex signal network,including not only internal hormones and protein kinases,but also external light and other stimuli.The findings of this study provide new insights to unravel the genetic control of heading traits.In order to understand the developmental process of Chinese cabbage leaf at the single-cell level,we performed single-cell transcriptome sequencing using young rosette leaves.By unsupervised clustering,16,055 high-quality cells were divided into 17 cell clusters.Using Chinese cabbage orthologous genes that have been reported for Arabidopsis cell type marker genes,eight cell types were identified,including mesophyll cells,epidermal cells,vascular cells,vascular bundle sheath cells,guard cells,proliferating cells,phloem cells and xylem cells.Through pseudo-time analysis,the differentiation and development trajectories of epidermis and guard cells,xylem and phloem,sieve element and companion cells were constructed respectively.A number of important genes that were dynamically expressed along the differentiation or development trajectories of various cell types were identified,including many key genes involved in cell fate decisions and cell function formation in Arabidopsis.In addition,the glucosinolate synthesis pathway genes were preferentially expressed in vascular bundles.This study laid the foundation for the study of Chinese cabbage leaf development and provide a very valuable data resource.To sum up,this study found the key transition leaves through establishment of transcriptomic landscape of leafy head,and drew the cell atlas of rosette leaf by single-cell RNA sequencing,which provided new insights or ways to study leafy head formation at the tissue and single cell level.