Development of Specific SNP Molecular Marker from Thistle in the DNA Sequences of Chloroplast TrnL-F and Matk Region Using HRM Analysis

엉겅퀴의 엽록체 TrnL-F와 Matk 영역 염기서열의 HRM 분석을 통한 특이적 SNP 분자마커의 개발

Abstract

Medicinal plants resources are becoming important assets since their usages have been expanded to the development of functional foods for human health, cosmetics and pharmaceutical industries. However, their phylogenetic origins and names are different from each country and quite often they are mixed each other resulting in the confusion for consumers. Particularly when they are very similar based on their morphological characteristics and distributed, it is extremely difficult to differentiate their origins even by specialists. Therefore, identification of each plant species is important for standardizing herbal medicine. Thistle is a medicinal and perennial plant. Obtaining information about the genetic diversity of plant populations is highly important for conservation and germplasm utilization. Although thistle is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish from other similar species from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from chloroplast genomic sequences to identify distinct Korean-specific thistle species via high resolution melting (HRM) curve analyses. We performed molecular authentication of four different kinds of thistle species from different regions using DNA sequences in the trnL-F and matK chloroplast intergenic region. The SNP markers developed in this study are useful for rapidly identifying specific thistle species from different country.

엉겅퀴는 대표적인 다년생의 약용식물이다. 최근 국제적 추세에 따라 자국의 유전자원의 발굴, 보존 등이 강화 됨에 따라 인접국가와 국내 자생 엉겅퀴 계통을 판별 할 수 있는 기준 설정에 관한 연구의 필요성이 대두되고 있지만, 분자생물학적 판별 기술의 개발은 아직 미흡한 실정이다. 본 연구에서는 국내 토종과 해외 유래 엉겅퀴종의 기원을 판별하기 위해 엽록체에 존재하는 trnL-trnF와 MatK 유전자단편에서 SNP를 이용한 판별 프라이머를 확보하였으며 이를 보완하여 보다 신속하게 판별하기 위하여 HRM 분석 기술을 이용한 판별 마커와 그 조건을 확립하였다. 그러므로, 본 연구에서 개발된 SNP 마커는 다양한 지역 또는 국가에서 서식하는 엉겅퀴 종들의 신속한 확인을 위해 매우 유용하게 이용될 것으로 생각된다.

Fig. 3. Primer sets and HRM curve analysis using the plastid intragenic regions of MatK and TrnL-F in the 4 kinds of thistle species. (A) MatK melting curves of sample from each thistle species. (B) TrnL-F and melting curves of sample from each thistle species.

Fig. 1. Sequence alignment of the MatK and TrnL-F chloroplast intergenic region in 4 kinds of thistle species. Korean species (C. japonicum and C. nipponicum), Canadian species (C. arvense) and European species S. marianum).

Fig. 2. Phylogenetic tree showing the genetic diversity of 8 plants of 4 kinds of thistle species. (A) MatK plastid intergenic regions, (B) TrnL-F plastid intergenic regions. The tree was produced using the neighbor-joining method based on intergenic sequences of the plastid intragenic regions of MatK and TrnL-F.

Table 1. List of plant materials used in this study

Table 2. Primer sequences for amplification of the MatK and TrnL-F fragments

Table 3. Primer sequences for HRM analysis of the MatK and TrnL-F fragments