Comparative analysis of monoterpene indole alkaloid composition and genotypic variation in Thai Mitragyna speciosa

Mitragyna speciosa (commonly known as kratom) is a tropical tree native to Southeast Asia, ethnobotanically used for pain relief and fatigue management. Kratom has gained popularity in Western countries for its dose-dependent stimulant and opioid-like effects, and its reported use in the self-treatment of opioid withdrawal. Kratom leaves accumulate over 50 monoterpene indole alkaloids (MIAs) and oxindole alkaloids that have pharmaceutical significance, to which many of these effects are attributed. In this study, we characterized eight kratom accessions originating from Central and Southern Thailand which exhibited phenotypic variation in leaf morphology, including differences in shape, margins, and vein coloration. To authenticate and evaluate genotypic variation among these accessions, we employed DNA barcoding using four loci: the nuclear ITS, and three plastid barcodes including rbcL, MatK, and trnH-psbA. No polymorphisms were detected using ITS and rbcL barcodes. However, sequence analyses revealed insertion-deletion polymorphisms in trnH-psbA, and single nucleotide polymorphisms in both MatK and trnH-psbA, resolving intraspecific variation and separating the accessions into two distinct haplotypes. Targeted metabolite profiling was conducted using UPLC-MS to quantify 17 MIAs and oxindole alkaloids from both young and mature leaves across all kratom accessions. Mitragynine was the most predominant alkaloid in mature leaves, reaching up to 1.19% w/w of leaf dry mass, whereas juvenile leaves accumulated speciociliatine as the major alkaloid, at levels up to 1.14% w/w. Notably, strictosidine, the central precursor of MIA biosynthesis, was detected exclusively in juvenile leaves, which also exhibited significantly higher levels of upstream intermediates including corynantheidine, and iso-corynantheidine compared with mature leaves. In addition, juvenile leaves were dominated by 3R MIAs, whereas mature leaves accumulated higher levels of 3S MIAs. However, the relative distribution of 3S and 3R stereoisomers remained consistent across accessions. Under the conditions examined, leaf developmental stage exerted a greater influence on alkaloid composition than accession or haplotype variation. Despite visible distinction in leaf vein coloration, alkaloid profiles at maturity remained largely consistent across all accessions. The developmental chemotype patterns presented in this study provide a valuable framework for targeted breeding, metabolic engineering, and controlled cultivation strategies aimed at optimizing specific MIA profiles, particularly those of pharmacological interest.