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

Wheat is important for global food security and understanding the molecular mechanisms driving spike and spikelet development can inform the development 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 clusters 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, late double ridge, and floret primordia stages. These cells were grouped into 21 different expression domains, including four in the basal region of the developing spikelets and three different meristematic regions, which were consistent across spikelets and sections. Using induced mutants, we revealed functional roles associated with the specific expression patterns of LFY in intercalary meristems, SPL14 in inflorescence meristems, and FZP in glume axillae. Complementary scRNA-seq profiling of 26,009 cells from W2.5 and W3.5 stages identified 23 distinct cell clusters. We used the scRNA-seq information to impute the expression of 74,464 genes into the spatially anchored smFISH-labelled cells and generated a public website to visualize them. We used experimental and imputed expression profiles, together with co-expression studies and correlation matrices, to annotate the scRNA-seq clusters. From co-expression analyses, we 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 dissecting gene networks that regulate spike development and identifying new co-expressed genes for functional characterization.