MORPHOLOGICAL, GENETIC AND BIOCHEMICAL CHARACTERIZATION OF THE ANTI-MALARIAL HERB, Artemisia annua GERMPLASM COLLECTION AT WEST VIRGINIA UNIVERSITY

Abstract

Malaria is one of the deadliest diseases in human history. Nearly half of the world's population, is at the risk in 106 countries. Only in 2016, this disease killed about 445,000 people, 72% of them being children under age five. It also accounts for US $12 billion dollars of direct costs in Africa alone. Five different species of Plasmodium cause malaria but P. falciparum is the most detrimental one, causing 50% of all malaria cases and is considered as the deadliest parasite in humans. Artemisinin (ART), a 15 C sesquiterpenoid is currently the only precursor to the most effective anti-malarial drugs. The World Health Organization (WHO) recommends the use of ARTderived drugs in combination with other anti-malarial drugs (collectively called as Artemisininbased Combination Therapies, ACT), to prevent the development of ART resistance by the parasite. The natural source of ART is Artemisia annua, an Asteraceae plant native to East Asia. ART is synthesized in 10-cell glandular trichomes and accumulates extra-cellularly in the sub cuticular space. ART is synthesized in A. annua in low quantities, about 0.01-3% DW and, due to its complex molecular structure, it cannot be synthesized chemically in an economically viable way. Even though the semi-synthesis of ART has been successful in bioreactors, ART extraction from A. annua is much more economically viable. Therefore, it is important to understand the A. annua genetic contribution to ART synthesis to improve the ART yield of the plants. We characterized a collection of 12 different genotypes of Artemisia annua at West Virginia University in terms of developmental traits, biochemical profile, and gene expression to better understand the phenotypical differences of the germplasm in the collection. Plants were analyzed for height, biomass, leaf shape and leaf size, adaxial and abaxial trichome densities for morphological characterization. Freeze dried leaf material was extracted with hexane and an internal standard and the extractions were analyzed with GC-MS. Flash frozen leaf material were used to extract total RNA and analyzed for the gene expression of 15 genes with qRT-PCR. Based on the results, it was revealed that there is high phenotypical and metabolic variation among the genotypes. Monoterpenes made up for a large component of the hexane-extracted metabolome. Camphor and endo-borneol contributed significantly to differentiate the genotypes. Out of the all the genes analyzed, gene expression of three genes (FPS, CPR, and DBR2) were significantly different among the genotypes. Based on data, genotype J has the highest accumulation of ART and DHAA with anatomical characteristics supporting towards high ART and DHAA yield. Next, I and G genotypes also showed anatomical features supporting towards high ART and DHAA accumulation. The genotypes can be used to further investigate ideal genetic variations and allelic combinations for crop improvement in future.