Pangenome identification and functional characterization of AHL genes in wheat (Triticum aestivum L.) reveal the role of TaAHL67 in grain weight regulation

AT-hook motif nuclear localized (AHL) proteins function as plant-specific transcription factors that regulate gene expression and chromatin remodeling, thereby involving various aspects of plant growth, development, and responses to abiotic stresses. However, the AHL gene family in wheat (Triticum aestivum) remains incompletely characterized. In this study, we identified 1835 TaAHL genes based on a pan-genome of 21 high-quality wheat genomes, which can be classified into 133 group. These group were further classified into 43 core TaAHL (present in all 21 varieties) and 91 dispensable TaAHL (absent in at least one varieties), including 17 soft-core (retained in 90% varieties), 30 outer shells (> 10%), and 43 line-specific TaAHL (≤ 10%). Phylogenetic analysis categorized these genes into two distinct clades. Expression pattern analysis revealed that TaAHL genes displayed tissue-specific expression profiles across all growth stages of wheat and showed responses to a variety of abiotic stresses. Drought survival rate association analysis suggests that the TaAHL24 may be involved in drought stress response. Among 29 candidate genes highly expressed in developing spikes and grains, TaAHL67 was strongly associated with thousand-grain weight (TGW). A gene-based Kompetitive Allele-Specific PCR (KASP) marker for TaAHL67 was developed and validated using a panel of 343 wheat varieties, revealing that the favorable haplotype (TaAHL67-HAP II) significantly increased TGW by 11.5–21.0%. Evolutionary analysis further indicated that TaAHL67-HAP II had undergone positive selection in modern cultivars, highlighting its important breeding value. This study conducted a comprehensive characterization and functional analysis of the AHL gene family in wheat based 21 pangenome, identifying a candidate gene, TaAHL67, associated with TGW, and developing and validating a KASP marker. These findings provide valuable gene resources for improving grain yield in wheat molecular breeding programs.