Population-scale gene expression analysis reveals the contribution of expression diversity to the modern wheat improvement.

Gene expression diversity is crucial for crop breeding, yet population genomics has focused primarily on sequence polymorphisms. A single reference genome for RNA-seq cannot handle introgression bias. Here, we conduct RNA-seq for 328 wheat lines, including landraces and elite cultivars from China and the United States, to investigate the expression variation underlying agronomic traits. Leveraging pan-genome resources, we identify 23,296 more transcripts than using the Chinese Spring reference. We construct a pan-gene regulatory atlas through eQTL analysis, revealing the tight regulation of introgressed genes. We identify 299 high-confidence candidate genes for 34 agronomic traits and resistance to 8 Blumeria graminis f. sp. tritici isolates, more than one-fifth of which were absent from the Chinese Spring. Utilizing the Kenong 9204 mutant library, 73.7% of the candidates show significant phenotypic effects. Our work mitigates the reference bias and highlights the impact of breeding-driven directional expression changes on wheat adaptation and improvement.