Development of Molecular Markers for the authentication of Zanthoxyli Pericarpium by the analysis of rDNA-ITS DNA barcode regions

Abstract

Objectives : Due to the morphological similarity of the pericarp and description of multi-species in National Pharmacopoeia of Korea and China, the Zanthoxylum Pericarpium is difficult to authenticate adulterant in species levels. Therefore, we introduced the sequence analysis of DNA barcode and identification of single nucleotide polymorphism(SNP) to establish a reliable tool for the distinction of Zanthoxylum Pericarpium from its adulterants. Methods : To analyze DNA barcode region, genomic DNA was extracted from twenty-four specimens of authentic Zanthoxylum species and inauthentic adulterant and the individual internal transcribed spacer regions (rDNA-ITS and ITS2) of nuclear ribosomal RNA gene were amplified using ITS1, ITS2-S2F, and ITS4 primer. For identification of species-specific sequences, a comparative analysis was performed using entire DNA barcode sequences.Results : In comparison of four Zanthoxylum ITS2 sequences, we identified 16, 4, 6, and 4 distinct species-specific nucleotides enough to distinguish Z. schinifolium, Z. bungeanum, Z. piperitum, and Z. simulans, respectively. The sequence differences were available genetic marker to discriminate four species. Futhermore, phylogenetic relationship revealed a clear classification between different Zanthoxylum species showing 4 different clusters.These results indicated that comparative analysis of ITS2 DNA barcode was an useful genetic marker to authenticate Zanthoxylum Pericarpium in species levels.Conclusions : The marker nucleotides, enough to distinguish Z. schinifolium, Z. piperitum, Z. bungeanum, and Z. simulans, were obtained at 30 SNP marker nucleotides from ITS2 sequences. These differences could be used to authenticate official Zanthoxylum Pericarpium from its adulterants as well as discriminating each four species.

Study of Some Zanthoxylum Species by Chemical and DNA Analysis Approaches

“…[ ][ ] Different gene regions are identified as targets for the molecular identification, such as ITS1 , ITS2‐S2F , and ITS4 or trnL‐F , matK , and rbcL . [ ][ ] Currently, a new method including Barcoding coupled with the Next‐Generation Sequencing (NGS) has been developed to work on damaged DNA in processed product. This technology, called Metabarcoding, uses a smaller DNA marker and short amplifying fragments about 150 bp …”

Development and Application of InDel Markers for Authentication of the Korean Herbs Zanthoxylum schinifolium and Zanthoxylum piperitum

“…Zanthoxylum formed a phylogenetic group in previous molecular phylogenetic studies [5,15,16]; however, these studies did not sufficiently resolve the relationships among some of its taxa. These studies were based on the ITS sequences of nuclear ribosomal DNA and the trnL-trnF, matK-trnK, atpB, atp-rbcL, and rbcL sequences of the cp genome [23][24][25].…”

“…Several studies have focused on developing molecular markers that can discriminate between various Zanthoxylum species used as traditional medicines in different countries. These molecular markers include amplified fragment length polymorphism (AFLP) markers for distinguishing between Zanthoxylum acanthopodium and Zanthoxylum oxyphyllum [14], sequence-related amplified polymorphism (SRAP) markers [15], internal transcribed spacer (ITS) rDNA-specific markers [16], and ISSR markers [17]. However, the use of these markers is limited by population dynamics and their reproducibility as diagnostic markers in the food market.…”

Inhibitory Effects of Essential Oil from Zanthoxylum coreanum Nakai on Melanin Production: Protection Against Re-Pigmentation after Laser Treatment

“…Grown in Korea, ZCN is morphologically similar to Z. schinifolium Siebold et Zuccarini and Z. piperitum De Candolle. 20 Although they are morphological similar, their chemical profiles and physiological functions are differ- ent. Based on genetic analysis Z. piperitum and Z. coreanum can be classified to a single group: Z. schinifolium and Z. piperitum belong to Zanthoxylum Pericarpium.…”

“…Based on genetic analysis Z. piperitum and Z. coreanum can be classified to a single group: Z. schinifolium and Z. piperitum belong to Zanthoxylum Pericarpium. 20 Recently, marker nucleotides for ZCN were detected upon analysis of rDNA-ITS DNA barcode regions in three Zanthoxylum species grown in Korea. 20 In comparison of three Zanthoxylum ITS2 sequences, 16, six, and four species-specific nucleotides were identified to distinguish among Z. schinifolium, Z. piperitum, and Z. coreanum, respectively.…”