A chromosome-level <i>Mitragyna parvifolia</i> genome unveils spirooxindole alkaloid diversification and mitraphylline biosynthesis

Monoterpene indole alkaloids (MIAs) found in the Rubiaceae have varied pharmaceutical uses. Spirooxindole alkaloids are a structural subtype of MIAs with a unique spiro[pyrrolidine-3,3'-oxindole] ring system. Despite their intriguing structures and potent bioactivities, the evolution and diversification of spirooxindole alkaloids remain poorly understood. We report a high-quality chromosome-scale genome assembly of Mitragyna parvifolia, a tree species of the Rubiaceae family that predominantly produces the spirooxindole alkaloid mitraphylline. Comparative genomics, including comprehensive synteny and phylogeny analyses across the MIA-producing order Gentianales revealed a whole-genome duplication event underlying the divergence of the Cinchonoideae alliance from the Coffeeae alliance, leading to diversification of MIA biosynthesis. Transcriptome analyses of young and mature leaves, stems, stipules, and roots integrated with MIA profiling and genome analyses revealed several candidates in the MIA biosynthetic pathway. Functional characterization of selected candidates led to the elucidation of the biosynthesis of the antiproliferative spirooxindole mitraphylline in M. parvifolia. These genomic and transcriptomic resources are invaluable to identify the evolutionary origins and diversification of MIAs and spirooxindole alkaloids.