Integrative GWAS and RNA-Seq Analysis Prioritizes a Major Chromosome 4H Locus and Candidate Genes for Barley Leaf Length

Leaf length is an important component of barley plant architecture, but its genetic basis remains incompletely understood. Here, we integrated genome-wide association study (GWAS), RNA sequencing (RNA-seq), and sequence variation analyses to investigate the genetic and transcriptional basis of barley leaf-length variation. A panel of 198 barley accessions was evaluated across two growing seasons, and contrasting long- and short-leaf accessions were subjected to RNA-seq. GWAS identified a major association signal on chromosome 4H, with a core interval of approximately 941.73 kb defined by local linkage disequilibrium analysis. RNA-seq identified 3507 and 3878 differentially expressed genes (DEGs) in flag leaves and second leaves below the flag leaf, respectively, including 2648 shared DEGs. Functional enrichment and gene set enrichment analyses indicated differences in developmental, signaling, metabolic, and temperature-responsive processes. Integration of GWAS and RNA-seq data prioritized three candidate genes, LOC123447426, LOC123449930, and LOC123450878, with different levels of supporting evidence. LOC123447426 and LOC123450878 were located within the core association interval, whereas LOC123449930 was located in the extended candidate-screening region, approximately 2.2 Mb from the lead SNP. LOC123447426 showed the strongest integrated support, with evidence from GWAS and differential expression in both leaf positions. Sequence variation, and qRT-PCR analyses provided further support for these candidates. Overall, this study prioritizes a major chromosome 4H region and three candidate genes associated with barley leaf-length variation, providing a basis for further investigation of their potential roles in leaf development. However, the current evidence does not establish causal relationships between these genes and leaf-length variation, and their biological functions remain to be experimentally verified.