A Near Telomere‐To‐Telomere Genome Assembly of <i>Coffea arabica</i> (Mundo Novo) Provides Insights Into Its Secondary Metabolism

Arabica coffee (Coffea arabica) dominates global coffee production, accounting for over 60% of the world's coffee trade. The Mundo Novo cultivar, predominantly grown in Yunnan, China, represents a significant germplasm resource. However, the absence of a high-quality reference genome has hindered comprehensive genetic research and in-depth investigation of secondary metabolic pathways in Arabica. In this study, we present the first near telomere-to-telomere (T2T) genome assembly of Arabica, achieved through the integration of PacBio HiFi, Oxford Nanopore ultra-long, and Hi-C sequencing technologies, representing the highest-quality Arabica genome to date. Phylogenetic analysis of N-methyltransferases (NMTs), the key enzymes responsible for caffeine biosynthesis, revealed their independent evolution across caffeine-producing clades including coffee, cacao, and tea. Furthermore, GO enrichment analysis of expanded gene families at the Arabica ancestral node, combined with fruit-specific transcriptomic profiling, revealed that glycosyltransferases likely play a critical role in the secondary metabolism of Arabica. Notably, functional characterisation demonstrated that a UGT (uridine diphosphate glycosyltransferase, UGT) from the UGT29 subfamily, which exhibited increased gene copy number in the Arabica subgenome C than its ancestor, can directly convert Rebaudioside A (Reb A) into Rebaudioside M (Reb M) through a single-step enzymatic glycosylation. This direct pathway represents a crucial advancement over conventional multi-UGTs biosynthetic routes of Reb M, which is a highly desirable sweetener whereas with limited natural abundance. Taken together, this study not only provides a valuable genomic resource for studying the unique secondary metabolic processes in C. arabica but also accelerates innovative research frontiers for the synthetic biological production of the valuable sweetener Reb M.