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문제 정의
- The main objectives of the present study are (1) to develop a DNA barcode database for Korean Schisandraceae, (2) to assess the level of differentiation of the species, and (3) to evaluate the utility of DNA barcodes.
제안 방법
- For each PCR reaction, 1 µL of total DNA was included in a 20 µL reaction mixture with Solg EF-Taq DNA polymerase (Solgent, Daejeon, Korea). Amplification of the target regions was conducted with a Veriti thermal cycler (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: initial denaturation at 95℃ for 2 min, 35 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 1 min, followed by a final extension at 72℃ for 5 min. PCR products were examined on a 1% agarose gel in 1× TBE buffer, purified and were sent to Solgent for sequencing, which prepared the sequencing reaction using the same primers used in PCR with the BigDye Terminator v3.
- Bootstrap analyses (Felsenstein, 1985) of 500 pseudoreplicates were conducted using both MP and NJ criteria. For the bootstrap analysis using the MP method, heuristic searches were employed with a simple sequence addition in PAUP* to evaluate the support for the Korean Schisandraceae data. For the expanded data matrix, 10,000 pseudoreplicates were sampled using the “fast” stepwise-addition option in PAUP*.
- Gaps resulting from multiple alignments of indels were treated as missing. Heuristic searches were used with 100 replicates of random sequence additions with tree bisection-reconnection branch swapping, with all of the best trees saved at each step (MulTrees). Kimura’s two-parameter model was employed to calculate the distance matrix in NJ analyses.
- japonica. We obtained DNA sequences of rbcL, matK, and the ITS regions of all accessions of the three species published in Zhang et al. (2015) and analyzed these together with our data to examine whether the Korean accessions can be distinguished from the Chinese and Japanese accessions by the DNA barcodes. In addition, exemplar accessions of other species were included in a more inclusive analysis (Appendix 1).
대상 데이터
- The authors thank SeA Ryu, Wha Jung Suh, Jin-Dong Lee, EuiHo Eom, BongSeok Kim, SeongWon Lee, and YoungTae Yang for their help throughout this project. We are grateful to two anonymous reviewers for their critical reviews of the manuscript.
- In addition, exemplar accessions of other species were included in a more inclusive analysis (Appendix 1). The expanded data included 36 accessions from 13 species of Schisandra and 13 from five of Kadsura. Phylogenetic analyses were conducted using the maximum parsimony (MP) and neighbor-joining methods (NJ) in PAUP* 4.
이론/모형
- Distribution of interspecific distance (red) and intraspecific distance (blue) calculated using Kimura’s two-parameter model.
성능/효과
- chinensis. A combined analysis of the three regions showed that S. repanda is a sister to S. chinensis in both the NJ and MP methods. Our data indicate that all of the DNA barcode regions included in this study (rbcL, matK, and ITS) are useful for identifying a species of Schisandraceae in Korea.
- Thirteen samples of Schisandraceae in total were included in this study (Table 1). For the relatively common and widespread species of S. chinensis, seven plants were included to represent their distributional range. For K.
- Therefore, using three concatenated regions is the best option for DNA barcoding in Schisandraceae in Korea. The combined analyses of the expanded data showed that both Schisandra and Kadsura were not monophyletic (Fig. 2). Previous molecular phylogenetic studies of Schisandraceae also produced similar results (Hao et al.
- chinensis, in which four ribotypes were found among seven accessions. The final alignment of the concatenated data with Illicium anisatum as an outgroup resulted in 2,274 sites, of which 194 sites were variable and 44 were parsimoniously informative (Table 2).
후속연구
- , 2015). Further detailed systematic studies are needed to investigate the generic boundaries of the two genera.
- chinensis is widely distributed in northeastern China, Mongolia, far eastern Russia, Korea, and Japan. Further studies are necessary to determine the geographic structure of the species with more samples of all distributional ranges and rapidly evolving molecular markers.
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