DNA Super-barcoding and Authentication of Species in Araliaceae Family

Abstract

The Araliaceae family includes 50 genera and around 1500 species worldwide. Overharvesting and adulteration of high-value medicinal plants from this family, particularly Panax species, have caused trouble in supply systems. The whole plastid genome (plastome) and 45S nuclear ribosomal DNA (nrDNA) sequences, referred to as super-barcodes, have emerged as a promising approach for correctly identifying plant species, a vital first step in the authentication system for combating economically motivated adulteration (EMA). This dissertation was conducted to assess the efficiency of these super-barcodes in classifying Araliaceae species and identifying specific barcoding loci for Panax species authentication. Eleven newly characterized complete plastomes and 64 previously reported complete plastomes were included, representing 56 species from 26 genera in the Araliaceae family and Centella asiatica (Apiaceae). The maximum likelihood (ML) tree was constructed from 75 plastome sequences and showed the monophyly of 25 genera with 100% bootstrap support. The genus Aralia was polyphyletic. The relaxed molecular clock estimation based on the 78 plastid genes indicated that subsequent speciation occurred within the Araliaceae genera from 8.04 to 0.67 million years ago (MYA) during the Middle Miocene and Pliocene. The collection of 74 complete plastomes revealed 43,458 variants. The 45S nrDNA sequences of 22 Araliaceae species showed 97.5 to 100% identities with 383 variants. Critical examination of plastome and 45S nrDNA super-barcoding data would provide new insights to improve knowledge of the evolutionary history of Araliaceae species. Furthermore, based on the phylogenetic tree, a total of fifteen SNPs in the plastid coding gene sequences were selected and successfully developed Kompetitive allele-specific PCR (KASP) markers, enabling clear identification among three genera: Panax, Aralia, and Eleutherococcus. Panax species native to Vietnam are designated as endangered species due to illegal harvesting and EMA from related species. Conservation of Panax species in Vietnam is hindered by a lack of genetic resources and molecular techniques. Seven P. vietnamensis individuals, representing three varieties: var vietnamensis, var. fuscidiscus, and var. langbianesis, were collected from Vietnamese habitats to generate NGS data for assemblies of whole plastome and 45S nrDNA sequences. The ML phylogenetic trees of the complete plastomes and 45S nrDNA indicated nine major Panax clades. The plastomes of P. vietnamensis var. vietnamensis and var. langbianesis were almost identical and clustered within the P. vietnamensis clade. However, P. vietnamensis var. fuscidiscus formed a distinct clade with P. zingiberensis, which was sister to the clade of P. vietnamensis. Significant intraspecific variation and phylogenetic incongruence within the P. japonicus clade and within the P. vietnamensis var. fuscidiscus and P. zingiberensis clade suggested an ambiguous species delimitation. Sixteen Panax-specific SNPs were developed as KASP markers, a high throughput SNP genotyping platform, which were highly accurate in the authentication of 114 Panax individuals collected from Vietnam. In conclusion, the two investigations demonstrated the effectiveness of the DNA super-barcoding approach through the use of whole plastome and 45S nrDNA sequences. The results of this study widened the field of phylogenetic analysis and species identification in the Araliaceae family. Furthermore, the markers developed through the analysis of Panax species entire plastome and 45S nrDNA sequences will aid in the detection of counterfeit ginseng in Vietnam's ginseng industry.