[논문]DNA barcoding of Euphorbiaceae in Korea

Kim, Kyeonghee; Park, Ki-Ryong; Lim, Chae Eun

doi:10.12651/jsr.2020.9.4.413

DNA barcoding of Euphorbiaceae in Korea 원문보기

Journal of species research, v.9 no.4, 2020년, pp.413 - 426

Kim, Kyeonghee (National Institute of Biological Resources) , Park, Ki-Ryong (Department of Environmental and Energy Engineering, Kyungnam University) , Lim, Chae Eun (National Institute of Biological Resources)

Abstract ▼ AI-Helper

The Euphorbiaceae family features some of the most economically important plants that are sources of foods, oils, waxes, and medicines. The accurate identification of Euphorbiaceae species is critical in sustainable utilization of plant resources. We examined 234 sequences of nrDNA ITS, cpDNA rbcL and matK loci from 20 species in Euphorbiaceae in Korea and three outgroup taxa to develop efficient DNA barcodes. The three barcode loci were successfully amplified and sequenced for all Euphorbiaceae species. nrDNA ITS locus showed the highest mean interspecific K2P distance (0.3034), followed by cpDNA matK (0.0830), and rbcL (0.0352) locus. The degree of species resolution for individual barcode loci ranged from 75% (rbcL and matK) to 80% (ITS). The degree of species resolution was not enhanced with the different combinations of three barcode loci. The combined data set of the three loci(ITS+rbcL+matK) provided 80% of species resolution. These results confirm that ITS locus, as a single barcode, is the best option for barcoding of the Euphorbiaceae in Korea.

주제어

표/그림 (6)

표 Table 1. PCR/sequencing primers and PCR cycling conditions for the three barcode regions examined in this study. Primer names follow the original publications.
표 Table 2. Statistics for three DNA barcode regions used in this study.
그림 Fig. 1. Relative distributions of Kimura-2-parameter distances across three barcode loci.
표 Table 3. Species resolving ability of individual barcode regions and their combinations.
그림 Fig. 2. Summary of contrasting molecular topologies for Euphorbiaceae in Korea.
그림 Fig. 3. Neighbour-joining (NJ) tree from the combined analysis of ITS, rbcL, and matK loci from Euphorbiaceae in Korea. Numbers above the branches indicate bootstrap values ≥50% in the NJ/ Maximum Parsimony (MP) analysis, respectively.

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문제 정의

This paper was carried out with the support of the National Institute of Biological Resources (NIBR201301014, NIBR202022101), and authors thank to SeA Ryu and SuMin Han for preparing voucher specimens and laboratory assistance throughout the project.

제안 방법

In the MP analysis, all characters were equally weighed. Heuristic searches were employed with 1,000 random sequence addition replicates and tree bisection-reconnection (TBR) branch swapping. The MP bootstrap analysis (Felsenstein, 1985) were performed using 1,000 replicates with TBR branch swapping and a random addition sequence.
Table 1. PCR/sequencing primers and PCR cycling conditions for the three barcode regions examined in this study. Primer names follow the original publications.

대상 데이터

The bidirectional sequences with direct Sanger sequencing were successfully generated using the same primer sets used in PCR. All of the 234 sequence data which was newly obtained in this study was submitted to GenBank and WIGIS (Wildlife Genetic Information System, http://species.nibr.go.kr) (Appendix 1).
8%) after we aligned the dataset. In this study, matK had presented one 12 bp-long indel and two 6 bp-long indels within 798 bp of aligned sequence dataset. Another cpDNA locus, rbcL sequence was 702 bp for all accessions with 113 variable sites (16.

데이터처리

To estimate species resolution abilities for each barcode locus and the possible combinations, we conducted a tree-based identification method with the NJ tree. For each set of data, the degree of species resolution was calculated as a percentage of the total number of species correctly identified in the NJ tree divided by the total number of species examined in this study.

이론/모형

, 2018). Phylogenetic analyses were performed using neighbor-joining (NJ) and maximum parsimony (MP) methods. The NJ and MP analysis were implemented in PAUP* version 4.
Polymerase Chain Reaction (PCR) was conducted using the following thermocycler program: initial denaturation at 94℃ for 3-5 min, 35 cycles of 94℃ for 1 min, 56℃ for 1 min, 72℃ for 1 min, and final extension at 72℃ for 7 min (Table 1). The PCR products were purified with the enzymatic purification method followed by Werle et al. (1994). The purified PCR products were sequenced with the ABI Prism BigDye terminator Cycle sequencing kit (Applied Biosystems, USA) and were run on an ABI Prism 3730xl genetic analyzer (Applied Biosystems, USA).

성능/효과

A total of 78 samples representing 20 species in eight genera of the family Euphorbiaceae were included in this study (Appendix 1). Most of the species were collected from the natural populations representing 61 populations in all parts of South Korea during the research period.
Therefore, the combined analysis of three barcode loci was performed with the single-locus analysis. For individual barcode locus, the highest discrimination success rate was 80% in nrDNA ITS, followed by matK and rbcL (75% of species discrimination success rate for each locus) (Table 3).
In conclusion, this study finds that the ITS locus provides the highest degree of species identification, as well as the combination of the three barcode loci (ITS+rbcL+matK) in Euphorbiaceae in Korea. In consideration of efficiency, ITS locus is the most suitable for barcoding of the Korean Euphorbiaceae as single locus barcode.
The highest species discrimination success rate was 80% in combination of ITS+matK+rbcL, followed by combination of rbcL+matK (70%) (Table 3). In the combination of three loci which showed the highest species discrimination success rate, all species except for four species (E. esula and E. maackii, E. pekinensis and E. fauriei) were resolved (Fig. 2).
In this study, the combinations of two or more barcode loci showed no significant increase in species discrimination rate (Table 3). The combination of three barcode loci (ITS+rbcL+matK) showed 80% of species resolution (Table 3).
In total, 234 amplified fragments from three barcode loci were obtained from 78 accessions representing 20 species of Euphorbiaceae and three outgroup taxa (Table 2; Appendix 1). In regard of universality of primers, the success rate of PCR was 100% for each of the three loci for 20 Euphorbiaceae species using single primer set (Table 1).
The NJ and MP trees of combined three locus data set showed that family Euphorbiaceae in Korea was strongly resolved to be monophyletic (BS of NJ/MP=100/100) (Fig. 3). In Korean Euphorbiaceae, three subfamilies formed strongly supported monophyletic groups; these are subfm.
The multi-locus combinations of the three barcode loci did not enhance the species discrimination success rate. The highest species discrimination success rate was 80% in combination of ITS+matK+rbcL, followed by combination of rbcL+matK (70%) (Table 3). In the combination of three loci which showed the highest species discrimination success rate, all species except for four species (E.

후속연구

In addition, a DNA barcode sequence database from three barcode loci is successfully established on the basis of the correctly identified samples. The DNA barcode, as well as the sequence database of Euphorbiaceae in Korea developed in this study, will contribute to the conservation and sustainable utilization of the Euphorbiaceae (spurges) as plant resources.

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표제어: PCR

동의어: Packet Collision Rate

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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