Integrating growth physiology and calcium status to reveal the genetic basis of lettuce tipburn via GWAS

Tipburn (TB) is a devastating physiological disorder in lettuce (Lactuca sativa) linked to localized calcium (Ca2+) deficiency. While biparental population studies have identified tipburn-linked QTLs, the core genetic mechanisms across diverse germplasm remain poorly understood. We utilized an integrative physiological-genetic framework to dissect TB genetic architecture in a diversity panel of 498 lettuce accessions evaluated over six years. Structural equation and response surface modeling established that TB risk is driven by a supply-demand imbalance regulated by plant biomass (PB) and developmental rate (DS). Although Ca2+ content significantly influenced TB incidence, it did not account for all phenotypic variance, suggesting the existence of Ca2+-independent resistance pathways. To isolate these, we performed GWAS on residual TB that accounted for PB, DS, and bulk Ca content, effectively decoupling plant physiology from resistance. We identified a stable cellular integrity hub on chromosomes 2 (BIN 2.21) and 8 (BIN 8.04), providing resistance regardless of biomass, developmental rate, or bulk Ca2+ status. Conversely, a major locus on chromosome 7 (BIN 7.17) acted as a developmental hub, where resistance is entirely mediated by bolting time and mineral partitioning. Candidate gene analysis at BIN 2.21 identified phosphoinositide phospholipase C (PLC) as a putative primary regulator; sequence analysis revealed a highly divergent PLC protein variant strongly associated with the lowest TB incidence. Other candidates include catalases (BIN 8.04) involved in oxidative stress mitigation and myosin-11 motor proteins. Our findings move beyond simple trait associations to identify stable, vigor-independent genetic targets, providing a roadmap for breeding high-yielding, tipburn-resilient lettuce cultivars.