Villwock, S. S. C.; Gomez, K. M.; Fish, T.; Lee, J.; Doherty, A.; White, A.; Thannhauser, T. W.; Gore, M. A.; Jannink, J.-L.

Provitamin A biofortification of cassava (Manihot esculenta) is constrained by a negative genetic correlation between total carotenoid (TC) and dry matter (DM) contents, but its underlying biological mechanism remains unclear. We examined this relationship in 24 African, Latin American, and hybrid cassava genotypes by measuring gene expression and metabolites in inner and outer storage root layers that varied spatially in carotenoid accumulation, enabling comparisons across and within genotypes. TC, and particularly upstream cis-carotenes, were negatively associated with DM after accounting for genotype, and this relationship strengthened across developmental timepoints, consistent with a physiological component of the trade-off. Gene-metabolite co-expression networks constructed from genetic, non-genetic, and overall phenotypic trait components showed no significant topological overlap between carotenoid- and starch-related gene sets, indicating that the TC/DM relationship is not driven by transcriptional co-regulation of the main pathways, although individual associations between carotenoids and carbohydrate-related genes suggested possible indirect metabolic links. Instead, TC accumulation was most strongly associated with regulatory, stress-, redox-, and plastid-related genes, enriched for heme-binding and oxidoreductase functions. These findings suggest that the TC/DM trade-off is not a fixed constraint, and identify candidate regulators of carotenoid content for future functional validation to support cassava provitamin A biofortification.