Identification and Analysis of the Terpene Synthases (TPS) Gene Family in Camellia Based on Pan-Genome

Terpenes are major determinants of tea aroma, and terpene synthases (TPSs) catalyze key steps in terpenoid biosynthesis. To capture gene-family variation beyond a single reference, we performed a pan-genome-based analysis of TPS genes across nine Camellia genomes (three wild tea relatives and six cultivated Camellia sinensis accessions) and integrated pan-transcriptome profiling across eight tissues. We identified 381 TPS genes; wild species contained more TPSs than cultivated accessions (mean 58.3 vs. 34.3), suggesting a putative contraction. Phylogenetic analysis with Arabidopsis TPSs classified Camellia TPSs into five subfamilies, dominated by TPS-b (149) and TPS-a (140), whereas TPS-c was rare (8). Gene-structure and physicochemical analyses revealed marked subfamily divergence, with TPS-c showing highly conserved coding-region length. Orthology clustering assigned 355 TPSs to 19 orthogroups, including five core groups (190 genes, 53.5%) and 14 dispensable groups (165 genes, 46.5%); core/non-core status was significantly associated with subfamily composition. Tandem and proximal duplication contributed most to TPS expansion (29.4% and 29.1%), and all orthogroups exhibited copy-number variation, with pronounced lineage-specific expansions. Ka/Ks analyses indicated pervasive purifying selection (median 0.516) but heterogeneous constraints among subfamilies. Finally, cultivated tea showed higher TPS expression in most tissues, especially mature leaf and stem, and TPS-g displayed the broadest and strongest expression. Together, these results provide a pan-genome resource for Camellia TPSs and highlight how domestication, duplication, and CNV shape terpene-related genetic diversity.