| Summary: | <i>Mitragyna speciosa</i> (Kratom) is a tropical narcotic plant native to Southeast Asia with unique pharmacological properties. Here, we report the first chromosome-scale assembly of the <i>M. speciosa</i> genome. We employed PacBio sequencing to obtain a preliminary assembly, which was subsequently scaffolded using the chromatin contact mapping technique (Hi-C) into 22 pseudomolecules. The final assembly was 692 Mb with a scaffold N50 of 26 Mb. We annotated a total of 39,708 protein-coding genes, and our gene predictions recovered 98.4% of the highly conserved orthologs based on the BUSCO analysis. The phylogenetic analysis revealed that <i>M. speciosa</i> diverged from the last common ancestors of <i>Coffea arabica</i> and <i>Coffea canephora</i> approximately 47.6 million years ago. Our analysis of the sequence divergence at fourfold-degenerate sites from orthologous gene pairs provided evidence supporting a genome-wide duplication in <i>M. speciosa</i>, agreeing with the report that members of the genus <i>Mitragyna</i> are tetraploid. The STRUCTURE and principal component analyses demonstrated that the 85 <i>M. speciosa</i> accessions included in this study were an admixture of two subpopulations. The availability of our high-quality chromosome-level genome assembly and the transcriptomic resources will be useful for future studies on the alkaloid biosynthesis pathway, as well as comparative phylogenetic studies in <i>Mitragyna</i> and related species.
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