Single-cell transcriptome sequencing reveals alfalfa root tip developmental landscape

Abstract Background: Root development relies on establishing stem cells and meristems, which differentiate into different heterogeneous cell types with varying temporal and spatial gradients. Constructing the developmental trajectories and dissecting the transcriptional regulatory networks of these cells, therefore, improves the functional understanding of the stem cells and meristems of dicot root tips. Single-cell RNA sequencing (scRNA-seq) has more advantages in obtaining the dynamic expression of genes in time and space than traditional RNA sequencing (RNA-seq). Pseudo-time trajectory analysis enables a re-study of the developmental process of root tips, stem cells, and meristems. Results: This study used high-throughput single-cell RNA sequencing to analyze 11,344 cells of the 5-day-old alfalfa root tips and identified 17 cell clusters and cluster-specific marker genes. Pseudo-time analysis of the single-cell transcriptome data using an unsupervised algorithm enabled the reconstruction of developmental trajectories between cells in different developmental states. The gene expression status during the differentiation of meristem cells was also revealed. Conclusions: Our study constructed a single-cell transcriptomic map of alfalfa roots and revealed heterogeneity among the different root cells. The study also conducted a single-cell resolution of the root cells during differentiation and intermediate transitions. The gene expression characteristics of the living cells provide valuable information and a resource library for studying the developmental biology of alfalfa.