Chromosome-level genome assembly of Tilia mandshurica reveals divergence of duplicated TPP genes associated with fructose-rich nectar formation.

Tilia mandshurica is an important woody nectar plant, but the genomic and transcriptional basis of its nectar sugar accumulation remains poorly understood. Here, we generated a chromosome-level reference genome for T. mandshurica and integrated comparative genomics, gene duplication analysis, RNA-seq across seven floral developmental stages, qRT-PCR validation, carbohydrate measurements and metabolite profiling. Gene family analysis revealed lineage-specific expansion of families involved in carbohydrate metabolism, ion transport and specialized metabolism. Ks and synteny analyses identified an ancient Eocene whole-genome duplication likely shared with Craigia yunnanensis, whereas duplication-mode analysis indicated that subsequent small-scale duplications contributed to lineage-specific duplicate retention. Among the enriched carbohydrate-related categories, trehalose-phosphatase activity provided a link between genome evolution and floral sugar accumulation. Sixteen trehalose-phosphate phosphatase (TPP) genes separated into fructose-positive C1 and fructose-negative C2 classes, with strong divergence at both the gene level (Δ = 1.060) and module level (Δ = 1.294). C1 TPP genes tracked fructose accumulation, whereas C2 genes followed starch-related developmental programs. Coexpression analysis further revealed contrasting network architectures: C1 TPP genes formed a convergent network with enriched vacuolar/tonoplast-related targets, whereas C2 genes showed a dispersed architecture with no consensus targets. C1-co-expressed candidates included genes related to vacuolar ion homeostasis, cell-wall/glycan remodeling, and isoprenoid/carotenoid metabolism. Together, these results support a model in which duplicated TPP genes are associated with distinct carbohydrate-associated transcriptional programs for starch remobilization and fructose-rich nectar formation. This study provides a genomic resource for Tilia and identifies candidate TPP-centered networks for nectar sugar accumulation in woody honey plants.