A Chromosome-Scale Genome Assembly of Mitragyna speciosa (Kratom) and the Assessment of Its Genetic Diversity in Thailand.

Mitragyna speciosa (Kratom) is a tropical narcotic plant native to Southeast Asia with unique pharmacological properties. Here, we report the first chromosome-scale assembly of the M. speciosa 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 M. speciosa diverged from the last common ancestors of Coffea arabica and Coffea canephora 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 M. speciosa, agreeing with the report that members of the genus Mitragyna are tetraploid. The STRUCTURE and principal component analyses demonstrated that the 85 M. speciosa 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 Mitragyna and related species.

Seasonal abundance and distribution of Anopheles larvae in a riparian malaria endemic area of western Thailand.

Three taxonomic groups of Anopheles larvae were morphologically identified within the Funestus Group (Minimus Subgroup and Aconitus Subgroup) (75.63%), Maculatus Group (20.47%), and Barbirostris Group (0.57%) during a two-year period in conjunction with active malaria transmission in a village near the Thai-Myanmar border in Kanchanaburi Province, western Thailand. The remaining 3.33% of anophelines collected were Anopheles culicifacies (3.07%), Anopheles philippinensis (0.17%), and Anopheles vagus (0.09%). Using an allele-specific multiplex molecular identification assay, the Minimus Subgroup consisted of Anopheles minimus (69.83%), and Anopheles harrisoni (0.06%) and 2 genetically-related species belonging to the Aconitus Subgroup, Anopheles aconitus (0.63%) and Anopheles varuna (5.12%). The Minimus and Aconitus Subgroup species were more abundant during the dry season (52.58%) than during the hot (24.95%) and wet (22.46%) seasons. The number of Anopheles larvae collected from the stream habitat was significantly higher during the second year than the first year, believed to be due to human environmental changes in the stream habitat from the building of a small check dam, which provided a more suitable and stable habitat for mosquito larval development. This study illustrates the importance of conducting site-specific studies to accurately determine vector bionomics (eg, larval habitats) and adult activity patterns and linking observations with malaria transmission dynamics in a given area.

Cloning and characterization of the bovine testicular PH-20 hyaluronidase core domain.

The core nucleotide sequence of bovine (Bos taurus) testicular PH-20 hyaluronidase was cloned using one step RT-PCR. The 5' and 3' regions were cloned separately and a sequence overlap of 124 bp facilitated the fusion of these two fragments by overlapping PCR, resulting in a concatenated sequence of 1422 bp. This nucleotide sequence and its deduced amino acid sequence were compared to homologous sequences from eight other mammal species. The bovine sequences were most similar to those of the pig, Sus scrofa (swine Spam1: 79.1% nucleotide and 70.1% amino acid similarity) and least similar to sequences from the Norway rat, Rattus norvegicus (murine Spam1: 61% nucleotide and 53.3% amino acid similarity). A phylogenetic analysis joined the red fox (Vulpes vulpes) sequence as sister to the bull-pig pair. Twelve cysteine residues were conserved among all nine aligned amino acid sequences and five proposed glycosylation sites have been identified. The feasibility of developing an effective, low-cost bovine PH-20 expression system is discussed in light of these new data.