Rice (Oryza sativa L.) serves as a primary staple food for over half of the global population, underpinning food security and socio-economic stability (Godfray et al., 2010). Enhancing rice productivity is critical to meeting the demands of a growing population, with yield potential heavily influenced by photosynthetic efficiency and source-sink dynamics (Li et al., 2018). Leaf morphology, particularly leaf thickness, is a key factor influencing photosynthetic performance (Peng, 2000). Thicker leaves exhibit higher chlorophyll content per unit area, enhanced Rubisco activity, and greater structural robustness, improving light absorption, photosynthetic capacity, and stress resilience (Garnier et al., 1999). Furthermore, thick leaf cultivars demonstrate superior canopy architecture, exhibiting erect leaves that optimize light penetration and facilitate high-density planting, key factors for yield maximization (Khush, 2013; Qian et al., 2016). Accordingly, the International Rice Research Institute’s ideal plant type model prioritizes leaf thickness as a critical breeding trait (Peng et al., 2008).

Given it serves as a pivotal agronomic trait that directly influences photosynthetic efficiency, hydraulic regulation, and stress resilience—key determinants of crop productivity, leaf thickness has been intensively studied in different species (Jinwen et al., 2009; Coneva et al., 2017; Coneva and Chitwood, 2018), significantly advancing our understanding of the mechanisms controlling this trait. At cellular level, studies have demonstrated that basic cellular processes, cell division and cell expansion, play a critical role in mediating leaf thickness (Weston et al., 2000). For example, it was shown that cell divisions parallel to the leaf surface, drive mesophyll layer expansion, thereby enhancing leaf thickness, particularly under high-light conditions, as demonstrated in Arabidopsis (Hoshino et al., 2019).

Genetic dissection of leaf thickness has also made progress in certain species. For example, Zheng et al. (2022) identified four quantitative trait loci (QTLs) regulating leaf thickness in barley, with associated candidate genes characterized. In rice, while substantial advances have been made in understanding genes controlling leaf width, curling, and angle (e.g., Nal1, SRL1, and LC2) (Qi et al., 2008; Zhao et al., 2010; Xiang et al., 2012), only one gene, DLT (LOC_Os06g03710), has been functionally validated for its role in mesophyll cell layer thickening, identified through mutant screening (Xie et al., 2019), with no natural variation modulating leaf thickness in rice having been identified. This critical research gap, largely attributed to difficulties in high-precision phenotypic measurement in rice (Laza et al., 2006; Kanbe et al., 2008; Zhao et al., 2008).

To address these challenges, this study leverages non-destructive, high-throughput phenotyping system (Liu et al., 2014; Chen et al., 2022) and an integrated pangenome analysis framework (Wang et al., 2023) to systematically dissect the genetic architecture of leaf thickness in rice. We identify qLT9, a major-effect QTL, its causal gene OsLT9, and a putative functional variant, providing insights into leaf thickness regulation. These findings establish OsLT9 as a key target for precision breeding, while the developed molecular toolkit opens avenues for optimized canopy architecture engineering to enhance yield potential.