Maize (Zea mays subsp mays) was domesticated from its wild ancestor, teosinte (Zea mays subsp parviglumis). Maize’s distinct morphology and adaptation to diverse environments required coordinated changes in various metabolic pathways. However, how the metabolome was reshaped since domestication remains poorly understood. Here, we report a comprehensive assessment of divergence in the seedling metabolome between maize and teosinte. In total, 461 metabolites exhibited significant divergence due to selection. Interestingly, teosinte and tropical and temperate maize, representing major stages of maize evolution, targeted distinct sets of metabolites. Alkaloids, terpenoids, and lipids were specifically targeted in the divergence between teosinte and tropical maize, while benzoxazinoids were specifically targeted in the divergence between tropical and temperate maize. To identify genetic factors controlling metabolic divergence, we assayed the seedling metabolome of a large maize-by-teosinte cross population. We show that the recent metabolic divergence between tropical and temperate maize tended to have simpler genetic architecture than the divergence between teosinte and tropical maize. Through integrating transcriptome data, we identified candidate genes contributing to metabolic divergence, many of which were under selection at the nucleotide and transcript levels. Through overexpression or mutant analysis, we verified the roles of Flavanone 3-hydroxylase1, Purple aleurone1, and maize terpene synthase1 in the divergence of their related biosynthesis pathways. Our findings not only provide important insights into domestication-associated changes in the metabolism but also highlight the power of combining omics data for trait dissection.

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