*Corresponding author: jxiao@genetics.ac.cn

†These authors contributed equally to this work

Bread wheat is an essential crop worldwide, supplying over 20% of the world's daily caloric and protein needs. The grain yield of wheat largely hinges on the architecture of the inflorescence and developmental process of endosperm. To unveil regulatory mechanisms governing the spike and grain development, we conducted transcriptome and epigenome profiling across key developmental stages of spike and endosperm. Our findings unveil that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate flowering transition, spikelet development, starch and seed storage protein (SSP) genes with varying impact.

Leveraging time-series RNA-seq and ATAC-seq data, we constructed a hierarchical transcriptional regulatory network (TRN) governing spike formation, starch biosynthesis, and SSP accumulation. Integration of the TRN with genome-wide association studies (GWAS), population transcriptome and phenotypic analyses of TILLING mutants led to the identification of 36 and 42 high-confidence novel regulators for spike and grain development, respectively.

Among these regulators, TaMYB30-A1 emerged as a key player downstream of WFZP, regulating fertile spikelet number. Notably, the superior haplotype of TaMYB30-A1, characterized by a C allele at the WFZP binding site, has been enriched in wheat breeding programs in China, contributing to enhanced agronomic traits. Additionally, TaABI3-A1 was found to modulate SSP accumulation while suppressing starch biosynthesis through transcriptional regulation.

Importantly, we established a free and open access Wheat Spike Multi-Omic Database (http://39.98.48.156:8800/#/). Our study not only identifies novel regulators but also offers an effective strategy for dissecting the genetic underpinnings of wheat yield traits, with practical value for wheat breeding.