Spatial and single-cell expression analyses reveal complex expression domains in early wheat spike development

Wheat is an important crop for global food security and a better understanding of the molecular mechanisms driving spike and spikelet development can inform the engineering of more productive varieties. In this study, we integrated single-molecule fluorescence in situ hybridization (smFISH) and single-cell RNA sequencing (scRNA-seq) to generate an atlas of cell types and expression domains during the early stages of wheat spike development. We characterized spatiotemporal expression of 99 genes by smFISH in 48,225 cells at the early transition (W1.5), late double ridge (W2.5) and lemma primordia (W3.25) stages. These cells were clustered into 21 different expression domains, including four beneath the developing spikelets and three different meristematic regions, which were consistent across spikelets and sections. We also identified genes differentially expressed during the transition of the inflorescence meristem into a terminal spikelet. The smFISH study was complemented by the scRNA-seq profiling of 26,009 cells from W2.5 and W3.25 that were clustered into 23 distinct cell types. We annotated the single-cell clusters as meristem, boundary domains, cortex, central spike, epidermis, and vasculature by using known markers, co-expression analysis of genes selected from the smFISH study, and correlation matrices of clusters identified in both studies. The scRNA-seq data uncovered genes associated with boundary genes TCP24 and FZP , as well as the meristematic genes AGL6 and ULT1. The smFISH and scRNA-seq studies provided complementary tools for characterizing the gene networks that regulate spike development, supporting the functional characterization of key regulatory genes, and informing the engineering of more productive wheat spikes.