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Applying DNA barcodes for identification of plant species in the family Araliaceae
Gene 499 (2012) 76–80
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Applying DNA barcodes for identification of plant species in the family Araliaceae Zhihua Liu ⁎, Xu Zeng, Dan Yang, Guiyan Chu, Zhengrong Yuan, Shilin Chen Center for Computational Biology and Bioinformatics, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
a r t i c l e
i n f o
Article history: Accepted 12 February 2012 Available online 2 March 2012 Keywords: Araliaceae DNA barcoding ITS2
a b s t r a c t An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification. © 2012 Elsevier B.V. All rights reserved.
1. Introduction There are about 50 genera and 1350 species in Araliaceae around the world and 23 genera (two endemic, one introduced) and 180 species (82 endemic, seven introduced) in China. Over one hundred species have been used for the medical purposes in virtue of biological constituents such as triterpenoid saponins (dama and oleanane), diterpene, flavones, coumarins, and phenols. The Chinese Pharmacopeia 2010 has admitted fifteen botanical origins, e.g. Panax ginseng (Ginseng Radix et Rhizome), P. notoginseng (Notoginseng Radix et Rhizome), Acanthopanax gracilistylus (Acanthopanacis cortex) and so on, all of which are rare and famous traditional herbs. P. ginseng is nowadays mainly used to promote resistance to physical, chemical, and biological press and boost general vitality (Shi et al., 2007). P. notoginseng has hemostatic and cardiovascular effects on arresting internal and external bleeding, alleviating swelling and pain, dispersing blood clots, eliminating blood stasis and promoting blood circulation (Lau et al., 2009). A. gracilistylus has been used for a thousand years in the treatment of rheumatic or rheumatoid arthralgia with flaccidity of extremities, retarded walking and lack of strength in children, edema. In addition, a lot of species are generally used in Chinese folk such as Aralias spp., Tupidanthus spp., Schefflera spp., etc. Since the roots or barks are used in clinical practice, adulterations are commonly found in the market. For instance, A. gracilistylus, the
Abbreviations: TCM, Traditional Chinese Medicine; ITS, Internal Transcribed Spacer; IMPLAD, Institute of Medicinal Plant Development; HMM, Hidden Markov Model; K2P, Kimura 2-Parameter; NJ, Neighbor-joining; BS, Bootstrap Support; CBOL, Consortium for the Barcode of Life. ⁎ Corresponding author. Tel.: + 86 10 57833112; fax: + 86 10 57833112. E-mail address: [email protected] (Z. Liu). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.02.016
botanical origin of the well-known Traditional Chinese Medicine (TCM), Wujiapi, has several congeners and adulterations, A. henryi (Oliv.) Harms, A. giraldii Harms, A. simonii Schneid., A. trifoliatus (Linn.) Merr., A. sessiliflorus (Rupr. et Maxim.) Seem, Periploca sepium Bge (Asclepiadaceae), Lycium chinense Mill (Solanaceae), L. barbarum L., Hedyotis hedyotidea DC. (Rubiaccac), and Rubus cochinchinensis Tratt. (Rosaceae). Roots of Raphanus sativus are mistaken for Ginseng Radix et Rhizome. Authentication has been usually attempted by chemical and microscopic methods. The chemicals are variable upon environmental conditions, and microscopic analysis is time-consuming and laborious which depends largely on the experience of observers. Accordingly, an accurate, sensitive and simple method is urgently needed. DNA barcoding is a new biological tool for accurate, rapid and automated species identification using a short fragment of the genomic DNA, which has been widely used especially in authentication of Chinese medicinal plants (Hebert et al., 2003). At present, rbcL, matK, psbA-trnH, rpoC1, and ITS2 have been popularly used as DNA barcodes in plant worldwide. In recent years, some researches employed the psbA-trnH barcode to identify species of medicinal pteridophytes and within the genus of Dendrobium (Ma et al., 2010; Yao et al., 2009). Chen et al. (2010) have proved that ITS2 is a universal barcode in the identification of plants and allied species, as ITS2 correctly identified 92.7% cases of over 6600 samples in seven phyla (Angiosperms, Gymnosperms, Ferns, Mosses, Liverworts, Algae, and Fungi). Since then, the ITS2 region has been shown to be applicable in discrimination among a wide range of plants within families of Asteraceae, Rutaceae, Rosaceae and so on (Gao et al., 2010; Liu et al., 2012; Luo et al., 2010; Pang et al., 2011; Yao et al., 2010). Here, we discriminated species in Araliaceae by DNA barcoding technique, which proved that ITS2 served as the best marker for the identification of Araliaceae species.
Z. Liu et al. / Gene 499 (2012) 76–80
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and Sun, 2008; Liu and Chen, 2010; Liu et al., 2005, 2008, 2012; Zeng et al., 2011; Yuan et al., 2012).
2. Materials and methods 2.1. Plant material A total of 1113 closely related sequences belonging to 276 species from 23 diverse genera of the family Araliaceae were chosen, in which the amounts of ITS2, psbA-trnH, matK, rbcL and ycf5 were 589, 228, 177, 119 and 10, respectively. In our study, 16 samples belonging to 11 species from 5 genera, were collected from 5 provinces in China and Japan (Table 1S). The samples were authenticated by Prof. Yulin Lin of IMPLAD (the Institute of Medicinal Plant Development). And the voucher samples were deposited in the herbarium of IMPLAD. In addition, the remaining sequences were downloaded from GenBank (Table 2S). The plant materials covered more than 90% of the genera of the Araliaceae family in the territory of China, including most of the important genera (Aralia, Brassaiopsis, Dendropanax, Eleutherococcus, Hedera and Panax).
3. Results 3.1. The success rate of PCR amplification ITS2, psbA-trnH, matK, rbcL and ycf5 were all successfully amplified using one pair of universal primers per locus. They were compared in the success rates of PCR amplification. As shown in Fig. 1, ITS2 and psbA-trnH displayed the highest efficiency of PCR amplification, followed by matK and rbcL, with ycf5 being the lowest. As presented in the result, ycf5 performed poorly in this test, and a less amount of sequencing data has been deposited in GenBank. Therefore, all of ycf5 were not included in the subsequent analysis.
3.2. Genetic divergence within and between species
2.2. DNA extraction, amplification, and sequencing DNA extractions were performed using the Plant Genomic DNA Kit (Tiangen Biotech Co., China). ITS2 regions were amplified and sequenced according to Chen et al. (2010).
2.3. Data analysis Automated DNA sequencing electropherograms were proofed and edited using CodonCode Aligner v. 1.6.3. Sequences were truncated to include only the ITS2 region. The DNA sequences from GenBank were annotated and trimmed using ITS2 annotation tools based on Hidden Markov Model (HMM) (Keller et al., 2009). Sequences of plant samples were aligned with those from GenBank by ClustalX software and the genetic distances were calculated using Kimura 2-Parameter (K2P) model. The distributions of intraversus inter-specific variability were compared using DNA barcoding gap with the software TAXON DNA (Slabbinck et al., 2008). Wilcoxon signed-rank tests were performed as previously described (Yao et al., 2010) (Table 1). Moreover, BLAST 1 and the nearest distance method were used to test the power of species identification as described previously (Yao et al., 2010). The phylogenetic analysis based on ITS2 regions (53 samples from 19 genera shown in Fig. 1S) was performed by MEGA 4.0 using the neighbor-joining (NJ) method (Tamura et al., 2007). Bootstrap support (BS) values for individual clades were calculated by running 1000 bootstrap replicates of the data. All positions containing gaps and missing data were eliminated from the dataset (Liu
A favorable barcode should possess a high inter-specific divergence to distinguish different species (Gao et al., 2010). Six metrics were used to characterize inter- versus intra-specific variations (Lahaye et al., 2008). ITS2 exhibited the ability of inter-specific discrimination significantly higher than psbA-trnH, matK and rbcL (Table 2). Wilcoxon signed rank test confirmed that ITS2 had the highest inter-specific divergence between congeneric species. The results of the intra-specific differences were similar, with rbcL contributing the largest and matK the smallest variations. ITS2 was found to have high inter-specific divergence and high intra-specific variation, which indicated that ITS2 was the most suitable DNA barcode to distinguish the species of Araliaceae.
3.3. Barcoding gap assessment A robust DNA barcode should have separate and non-overlapping genetic variations between intra- and inter-specific samples (Moritz and Cicero, 2004). We examined the distributions of intra- versus inter- specific divergence in the four barcodes. In Fig. 2, the results demonstrated that the intra/inter-specific variation of ITS2, psbAtrnH, matK and rbcL exhibited distinct gaps. However, when calculations were carried out in intra-specific variation between conspecific individuals and inter-specific divergence between all hetero-specifics, there was a significant overlap without gaps. The inter-specific divergences for ITS2 were significantly higher than their corresponding intra-specific variations.
Table 1 Wilcoxon signed rank test of the inter-specific divergences among the five loci. w+
w−
ITS2 ITS2 ITS2 ITS2 psbA-trnH psbA-trnH psbA-trnH matK matK rbcL
psbA-trnH matK rbcL ycf5 matK rbcL ycf5 rbcL ycf5 ycf5
Inter relative ranks, n, P value w+
w−
n
P
45 6 227 105 3 10 18 1 1 6
0 0 4 0 0 0 10 0 5 0
9 3 21 15 4 11 7 1 3 6
b0.0075 b0.0832 b0.0001 b0.0009 b0.1797 b0.0587 b0.4927 b0.3173 b0.2851 b0.0832
Results
ITS2 > psbA-trnH ITS2 = matK ITS2 > rbcL ITS2 > ycf5 psbA-trnH = matK psbA-trnH = rbcL psbA-trnH = ycf5 matK = rbcL matK = ycf5 rbcL = ycf5
Fig. 1. PCR amplification efficiency for five regions using a single primer pair per locus.
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Table 2 Analysis of inter-specific divergence between congeneric species and intra-specific variation for the whole sample.
All intra-specific distance Coalescent depth Theta All inter-specific distance The minimum inter-specific distance Theta prime
ITS2
psbA-trnH
matK
rbcL
0.0117 ± 0.0193 0.0169 ± 0.0264 0.0213 ± 0.0124 0.0511 ± 0.0277 0.0155 ± 0.0182 0.0431 ± 0.0338
0.0041 ± 0.0086 0.0082 ± 0.0199 0.0109 ± 0.0165 0.0141 ± 0.0100 0.0045 ± 0.0057 0.0244 ± 0.0244
0.0763 ± 0.271 0.0927 ± 0.268 0.0024 ± 0.017 0.0338 ± 0.108 0.0054 ± 0.006 0.0111 ± 0.058
0.2031 ± 0.4282 0.2861 ± 0.5829 0.1202 ± 0.3906 0.1323 ± 0.2220 0.0027 ± 0.0043 0.1316 ± 0.4027
3.4. Identification efficiency of the DNA barcodes
3.5. Species identification based on phylogenetic tree
BLAST 1 and the nearest genetic distance were two methods in the assessment of correct discrimination using different barcodes. The results based on BLAST 1 method indicated that ITS2 head the list highest identification efficiency (85.23%) at the species level, followed by psbA-trnH, rbcL and matK. At the genus level, both psbAtrnH and matK had the highest success rate (100%) and ITS2 performed well with 97.29% successful identification rate. The similar results could be obtained by the nearest genetic distance method. However, identification efficiency by the nearest genetic distance method was much lower than BLAST 1 (Table 3).
According to the neighbor-joining tree (Fig. 3 and Fig. 1S), each genus represented a monophyletic unit with the exception of some species. Most of authenticate species clades were clearly monophyletic, appearing distinctly distant from other clades. For instance, a subclade containing the genus of Kalopanax was well supported, as well as a subclade containing MacroPanax and MerrillioPanax, a subclade with a strain of OploPanax, a subclade with two strains of Osmoxylon and PseudoPanax, a subclade of Panax and a subclade of Hedera. However, the NJ tree did not identify all the authenticate species. The positions of some species were within the other
Fig. 2. Relative distribution of inter-specific divergence between congeneric species and intra-specific variation.
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Table 3 Comparison of the identification efficiency of the five candidate sequences using different methods of species identification. Method
ITS2 psbA-trnH matK rbcL
BLAST 1 Distance BLAST 1 Distance BLAST 1 Distance BLAST 1 Distance
No. of samples 589 589 228 228 177 177 119 119
Successful identification
Incorrect identification
Ambiguous identification
Species
Genus
Species
Genus
Species
Genus
85.23% 60.00% 79.39% 73.79% 56.49% 42.37% 74.71% 61.79%
97.29% 88.63% 100% 100% 100% 94.92% 100% 85.64%
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
14.77% 40.00% 20.61% 26.31% 43.51% 57.63% 25.29% 38.21%
2.71% 11.37% 0 0 0 5.08% 0 14.36%
species clades. S. brenesii (DQ007387) was nested within the genus SinoPanax. The subclade of Schefflera and Tupidanthus was confused with some species. In addition, the position of two genera (Trevesia and Brassaiopsis) was not clear in one subclade. Although ITS2 showed the high identification success rate at the genus level in phylogenetic tree, some confusing species were still not able to be distinguished well.
4. Discussion 4.1. Screening of DNA barcodes in Araliaceae Screening for single or multiple regions appropriate for DNA barcoding studies has been an important research focus of species identification. Robust DNA barcodes feature inter-specific divergence greater than intra-specific divergence in a PCR recovery sequence using a single primer pair. Cytochrome c oxidase subunit 1 (CO1 gene) has been proved distinctive advantages in animal species discrimination. However, it is not favorable in plant species due to the low amount of variation in the genes and the variable structure of mitochondrial genome (Luo et al., 2010). Recent candidate barcodes are rbcL, rpoC1, ycf5, matK and psbAtrnH in the chloroplast genome, as well as ITS and ITS2 of nuclear ribosomal DNA (Kress et al., 2005). ITS2 has been regarded as a universal barcode in discrimination of more than 6600 plant samples (Chen et al., 2010). Here, a large number of ITS2 DNA sequence data of Araliaceae plants are available from GenBank, which show high efficiency
of PCR amplification and high sequence quality. In this study, ITS2 had the highest discriminatory ability among the five markers and the greatest inter-specific divergence which was significantly higher than the intra-specific variation determined by the “DNA barcoding gap” assessment and Wilcoxon signed rank tests. Furthermore, the ITS2 locus had the highest identification efficiency among all the tested markers. Recently, a two-locus combination of rbcL + matK is recommended as a plant barcode by the plant Working Group of the Consortium for the Barcode of Life (CBOL) (Group, 2009). Although rbcL offered high universality, good PCR recovery and convenient alignment, it demonstrated insufficient sequence variation to distinguish among closely related species (Kress and Erickson, 2007; Lahaye et al., 2008). It showed the rate of species identification of 74.71%, which was not as high as ITS2. The matK gene is one of the most rapidly evolving plastid coding regions; however, it needs to be improved in primer universality and efficiency of PCR amplification (Chase et al., 2007). The CBOL Plant Working Group proposed rbcL + matK as the standard barcode for land plant as successful discrimination in 72% of cases was achieved after screening rpoB, rpoC1, rbcL, matK, trnH-psbA, atpF-atpH and psbK-psbI in 550 samples belonging to angiosperm, gymnosperm and alga. Single loci, 2-locus and 3-locus barcodes could achieve correct discrimination of 69%, 75% and 76% at most, respectively. However, the results were far from the CBOL Group expected (Group, 2009). The psbA-trnH has good priming sites because it is one of the most rapidly evolving spacers in chloroplast DNA with 75 bp conserved fragments at the ends. In addition, it demonstrated good
Fig. 3. Phylogenetic analysis of ITS2 regions from 53 samples in 19 genera.
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universality and high amplification success (Shaw et al., 2005; Yao et al., 2009). Here, we proposed psbA-trnH as another candidate barcode for Araliaceae plants with correct discrimination of 100% and 79.39% at genus and species, respectively. However, psbA-trnH has a great inter-specific variation with a long sequence and frequent poly A structures in angiosperm leading to a low sequencing quality (Thomas, 2009). In addition, alignment is hard to perform because of great variations in sequence length. Ycf5 needs a further study, for the number of samples (b10 sequences) we obtained was not enough for correct discrimination.
4.2. Efficacy of ITS2 for authentication Comparison of the five barcode markers in discrimination of Araliaceae plants showed that ITS2 was the best option in the following respects. ITS2 has good primer sites due to conserved 5.8S and 26S at the ends. ITS2 is as short as about 200 bp in Araliaceae plants, which is easy for DNA extraction, amplification and sequencing. On primary ITS2 sequences analyses, three discrimination methods (phylogenetic tree, the nearest distance and BLAST 1) were performed for identifying species. Furthermore, the secondary structure of ITS2 is considered as a molecular morphological characteristic (Selig et al., 2008). Schultz et al. established a database of ITS2 secondary structures, and 216,374 ITS2 of that are predicted (Schultz et al., 2006). ITS2 is a powerful barcode in species discrimination due to rapid evolvement and great variation at genus or species level, which has been proved by CBCs method. Most recently, 99.5% species of the diatom genus have been successfully discriminated by 5.8S + ITS2 (Moniz and Kaczmarska, 2010). However, 87 out of 589 samples failed in discrimination by ITS2 barcode, e.g., E._divaricatus f. albeofructus, E. divaricatus f. tristigmatis, E._divaricatus var. chiisanensis, E. divaricatus var. flavi-flos, and so on. It is hard for DNA barcode to identify those subspecies or varieties still debated in taxonomy, indicating that some closely related species might not be correctly discriminated by DNA barcoding. It may be suitable to identify them using other molecular markers (e.g., SNP, SSR, etc.), which are assistant tools for DNA barcoding (Liu et al., 2005, 2008, 2012; Liu and Sun, 2008; Luo et al., 2010; Zeng et al., 2011; Yuan et al., 2012). Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2012.02.016.
Acknowledgments This work was supported by the National Natural Science Foundation of China (81102746), Beijing Natural Science Foundation (5113033), Scientific Research Foundation of the State Human Resource Ministry and the Education Ministry for Returned Chinese Scholars, New Star Project of Peking Union Medical College, Youth Foundation of Peking Union Medical College, the Research Fund for the Doctoral Program of Higher Education (20111106120028), “Major Drug Discovery” major science and technology research “12nd Five-Year Plan” (2012ZX09301-002-001), and China Medical Board of New York (A2009001) granted to Zhihua Liu.
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Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Applying DNA barcodes for identification of plant species in the family Araliaceae Zhihua Liu ⁎, Xu Zeng, Dan Yang, Guiyan Chu, Zhengrong Yuan, Shilin Chen Center for Computational Biology and Bioinformatics, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
a r t i c l e
i n f o
Article history: Accepted 12 February 2012 Available online 2 March 2012 Keywords: Araliaceae DNA barcoding ITS2
a b s t r a c t An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification. © 2012 Elsevier B.V. All rights reserved.
1. Introduction There are about 50 genera and 1350 species in Araliaceae around the world and 23 genera (two endemic, one introduced) and 180 species (82 endemic, seven introduced) in China. Over one hundred species have been used for the medical purposes in virtue of biological constituents such as triterpenoid saponins (dama and oleanane), diterpene, flavones, coumarins, and phenols. The Chinese Pharmacopeia 2010 has admitted fifteen botanical origins, e.g. Panax ginseng (Ginseng Radix et Rhizome), P. notoginseng (Notoginseng Radix et Rhizome), Acanthopanax gracilistylus (Acanthopanacis cortex) and so on, all of which are rare and famous traditional herbs. P. ginseng is nowadays mainly used to promote resistance to physical, chemical, and biological press and boost general vitality (Shi et al., 2007). P. notoginseng has hemostatic and cardiovascular effects on arresting internal and external bleeding, alleviating swelling and pain, dispersing blood clots, eliminating blood stasis and promoting blood circulation (Lau et al., 2009). A. gracilistylus has been used for a thousand years in the treatment of rheumatic or rheumatoid arthralgia with flaccidity of extremities, retarded walking and lack of strength in children, edema. In addition, a lot of species are generally used in Chinese folk such as Aralias spp., Tupidanthus spp., Schefflera spp., etc. Since the roots or barks are used in clinical practice, adulterations are commonly found in the market. For instance, A. gracilistylus, the
Abbreviations: TCM, Traditional Chinese Medicine; ITS, Internal Transcribed Spacer; IMPLAD, Institute of Medicinal Plant Development; HMM, Hidden Markov Model; K2P, Kimura 2-Parameter; NJ, Neighbor-joining; BS, Bootstrap Support; CBOL, Consortium for the Barcode of Life. ⁎ Corresponding author. Tel.: + 86 10 57833112; fax: + 86 10 57833112. E-mail address: [email protected] (Z. Liu). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.02.016
botanical origin of the well-known Traditional Chinese Medicine (TCM), Wujiapi, has several congeners and adulterations, A. henryi (Oliv.) Harms, A. giraldii Harms, A. simonii Schneid., A. trifoliatus (Linn.) Merr., A. sessiliflorus (Rupr. et Maxim.) Seem, Periploca sepium Bge (Asclepiadaceae), Lycium chinense Mill (Solanaceae), L. barbarum L., Hedyotis hedyotidea DC. (Rubiaccac), and Rubus cochinchinensis Tratt. (Rosaceae). Roots of Raphanus sativus are mistaken for Ginseng Radix et Rhizome. Authentication has been usually attempted by chemical and microscopic methods. The chemicals are variable upon environmental conditions, and microscopic analysis is time-consuming and laborious which depends largely on the experience of observers. Accordingly, an accurate, sensitive and simple method is urgently needed. DNA barcoding is a new biological tool for accurate, rapid and automated species identification using a short fragment of the genomic DNA, which has been widely used especially in authentication of Chinese medicinal plants (Hebert et al., 2003). At present, rbcL, matK, psbA-trnH, rpoC1, and ITS2 have been popularly used as DNA barcodes in plant worldwide. In recent years, some researches employed the psbA-trnH barcode to identify species of medicinal pteridophytes and within the genus of Dendrobium (Ma et al., 2010; Yao et al., 2009). Chen et al. (2010) have proved that ITS2 is a universal barcode in the identification of plants and allied species, as ITS2 correctly identified 92.7% cases of over 6600 samples in seven phyla (Angiosperms, Gymnosperms, Ferns, Mosses, Liverworts, Algae, and Fungi). Since then, the ITS2 region has been shown to be applicable in discrimination among a wide range of plants within families of Asteraceae, Rutaceae, Rosaceae and so on (Gao et al., 2010; Liu et al., 2012; Luo et al., 2010; Pang et al., 2011; Yao et al., 2010). Here, we discriminated species in Araliaceae by DNA barcoding technique, which proved that ITS2 served as the best marker for the identification of Araliaceae species.
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and Sun, 2008; Liu and Chen, 2010; Liu et al., 2005, 2008, 2012; Zeng et al., 2011; Yuan et al., 2012).
2. Materials and methods 2.1. Plant material A total of 1113 closely related sequences belonging to 276 species from 23 diverse genera of the family Araliaceae were chosen, in which the amounts of ITS2, psbA-trnH, matK, rbcL and ycf5 were 589, 228, 177, 119 and 10, respectively. In our study, 16 samples belonging to 11 species from 5 genera, were collected from 5 provinces in China and Japan (Table 1S). The samples were authenticated by Prof. Yulin Lin of IMPLAD (the Institute of Medicinal Plant Development). And the voucher samples were deposited in the herbarium of IMPLAD. In addition, the remaining sequences were downloaded from GenBank (Table 2S). The plant materials covered more than 90% of the genera of the Araliaceae family in the territory of China, including most of the important genera (Aralia, Brassaiopsis, Dendropanax, Eleutherococcus, Hedera and Panax).
3. Results 3.1. The success rate of PCR amplification ITS2, psbA-trnH, matK, rbcL and ycf5 were all successfully amplified using one pair of universal primers per locus. They were compared in the success rates of PCR amplification. As shown in Fig. 1, ITS2 and psbA-trnH displayed the highest efficiency of PCR amplification, followed by matK and rbcL, with ycf5 being the lowest. As presented in the result, ycf5 performed poorly in this test, and a less amount of sequencing data has been deposited in GenBank. Therefore, all of ycf5 were not included in the subsequent analysis.
3.2. Genetic divergence within and between species
2.2. DNA extraction, amplification, and sequencing DNA extractions were performed using the Plant Genomic DNA Kit (Tiangen Biotech Co., China). ITS2 regions were amplified and sequenced according to Chen et al. (2010).
2.3. Data analysis Automated DNA sequencing electropherograms were proofed and edited using CodonCode Aligner v. 1.6.3. Sequences were truncated to include only the ITS2 region. The DNA sequences from GenBank were annotated and trimmed using ITS2 annotation tools based on Hidden Markov Model (HMM) (Keller et al., 2009). Sequences of plant samples were aligned with those from GenBank by ClustalX software and the genetic distances were calculated using Kimura 2-Parameter (K2P) model. The distributions of intraversus inter-specific variability were compared using DNA barcoding gap with the software TAXON DNA (Slabbinck et al., 2008). Wilcoxon signed-rank tests were performed as previously described (Yao et al., 2010) (Table 1). Moreover, BLAST 1 and the nearest distance method were used to test the power of species identification as described previously (Yao et al., 2010). The phylogenetic analysis based on ITS2 regions (53 samples from 19 genera shown in Fig. 1S) was performed by MEGA 4.0 using the neighbor-joining (NJ) method (Tamura et al., 2007). Bootstrap support (BS) values for individual clades were calculated by running 1000 bootstrap replicates of the data. All positions containing gaps and missing data were eliminated from the dataset (Liu
A favorable barcode should possess a high inter-specific divergence to distinguish different species (Gao et al., 2010). Six metrics were used to characterize inter- versus intra-specific variations (Lahaye et al., 2008). ITS2 exhibited the ability of inter-specific discrimination significantly higher than psbA-trnH, matK and rbcL (Table 2). Wilcoxon signed rank test confirmed that ITS2 had the highest inter-specific divergence between congeneric species. The results of the intra-specific differences were similar, with rbcL contributing the largest and matK the smallest variations. ITS2 was found to have high inter-specific divergence and high intra-specific variation, which indicated that ITS2 was the most suitable DNA barcode to distinguish the species of Araliaceae.
3.3. Barcoding gap assessment A robust DNA barcode should have separate and non-overlapping genetic variations between intra- and inter-specific samples (Moritz and Cicero, 2004). We examined the distributions of intra- versus inter- specific divergence in the four barcodes. In Fig. 2, the results demonstrated that the intra/inter-specific variation of ITS2, psbAtrnH, matK and rbcL exhibited distinct gaps. However, when calculations were carried out in intra-specific variation between conspecific individuals and inter-specific divergence between all hetero-specifics, there was a significant overlap without gaps. The inter-specific divergences for ITS2 were significantly higher than their corresponding intra-specific variations.
Table 1 Wilcoxon signed rank test of the inter-specific divergences among the five loci. w+
w−
ITS2 ITS2 ITS2 ITS2 psbA-trnH psbA-trnH psbA-trnH matK matK rbcL
psbA-trnH matK rbcL ycf5 matK rbcL ycf5 rbcL ycf5 ycf5
Inter relative ranks, n, P value w+
w−
n
P
45 6 227 105 3 10 18 1 1 6
0 0 4 0 0 0 10 0 5 0
9 3 21 15 4 11 7 1 3 6
b0.0075 b0.0832 b0.0001 b0.0009 b0.1797 b0.0587 b0.4927 b0.3173 b0.2851 b0.0832
Results
ITS2 > psbA-trnH ITS2 = matK ITS2 > rbcL ITS2 > ycf5 psbA-trnH = matK psbA-trnH = rbcL psbA-trnH = ycf5 matK = rbcL matK = ycf5 rbcL = ycf5
Fig. 1. PCR amplification efficiency for five regions using a single primer pair per locus.
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Table 2 Analysis of inter-specific divergence between congeneric species and intra-specific variation for the whole sample.
All intra-specific distance Coalescent depth Theta All inter-specific distance The minimum inter-specific distance Theta prime
ITS2
psbA-trnH
matK
rbcL
0.0117 ± 0.0193 0.0169 ± 0.0264 0.0213 ± 0.0124 0.0511 ± 0.0277 0.0155 ± 0.0182 0.0431 ± 0.0338
0.0041 ± 0.0086 0.0082 ± 0.0199 0.0109 ± 0.0165 0.0141 ± 0.0100 0.0045 ± 0.0057 0.0244 ± 0.0244
0.0763 ± 0.271 0.0927 ± 0.268 0.0024 ± 0.017 0.0338 ± 0.108 0.0054 ± 0.006 0.0111 ± 0.058
0.2031 ± 0.4282 0.2861 ± 0.5829 0.1202 ± 0.3906 0.1323 ± 0.2220 0.0027 ± 0.0043 0.1316 ± 0.4027
3.4. Identification efficiency of the DNA barcodes
3.5. Species identification based on phylogenetic tree
BLAST 1 and the nearest genetic distance were two methods in the assessment of correct discrimination using different barcodes. The results based on BLAST 1 method indicated that ITS2 head the list highest identification efficiency (85.23%) at the species level, followed by psbA-trnH, rbcL and matK. At the genus level, both psbAtrnH and matK had the highest success rate (100%) and ITS2 performed well with 97.29% successful identification rate. The similar results could be obtained by the nearest genetic distance method. However, identification efficiency by the nearest genetic distance method was much lower than BLAST 1 (Table 3).
According to the neighbor-joining tree (Fig. 3 and Fig. 1S), each genus represented a monophyletic unit with the exception of some species. Most of authenticate species clades were clearly monophyletic, appearing distinctly distant from other clades. For instance, a subclade containing the genus of Kalopanax was well supported, as well as a subclade containing MacroPanax and MerrillioPanax, a subclade with a strain of OploPanax, a subclade with two strains of Osmoxylon and PseudoPanax, a subclade of Panax and a subclade of Hedera. However, the NJ tree did not identify all the authenticate species. The positions of some species were within the other
Fig. 2. Relative distribution of inter-specific divergence between congeneric species and intra-specific variation.
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Table 3 Comparison of the identification efficiency of the five candidate sequences using different methods of species identification. Method
ITS2 psbA-trnH matK rbcL
BLAST 1 Distance BLAST 1 Distance BLAST 1 Distance BLAST 1 Distance
No. of samples 589 589 228 228 177 177 119 119
Successful identification
Incorrect identification
Ambiguous identification
Species
Genus
Species
Genus
Species
Genus
85.23% 60.00% 79.39% 73.79% 56.49% 42.37% 74.71% 61.79%
97.29% 88.63% 100% 100% 100% 94.92% 100% 85.64%
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
14.77% 40.00% 20.61% 26.31% 43.51% 57.63% 25.29% 38.21%
2.71% 11.37% 0 0 0 5.08% 0 14.36%
species clades. S. brenesii (DQ007387) was nested within the genus SinoPanax. The subclade of Schefflera and Tupidanthus was confused with some species. In addition, the position of two genera (Trevesia and Brassaiopsis) was not clear in one subclade. Although ITS2 showed the high identification success rate at the genus level in phylogenetic tree, some confusing species were still not able to be distinguished well.
4. Discussion 4.1. Screening of DNA barcodes in Araliaceae Screening for single or multiple regions appropriate for DNA barcoding studies has been an important research focus of species identification. Robust DNA barcodes feature inter-specific divergence greater than intra-specific divergence in a PCR recovery sequence using a single primer pair. Cytochrome c oxidase subunit 1 (CO1 gene) has been proved distinctive advantages in animal species discrimination. However, it is not favorable in plant species due to the low amount of variation in the genes and the variable structure of mitochondrial genome (Luo et al., 2010). Recent candidate barcodes are rbcL, rpoC1, ycf5, matK and psbAtrnH in the chloroplast genome, as well as ITS and ITS2 of nuclear ribosomal DNA (Kress et al., 2005). ITS2 has been regarded as a universal barcode in discrimination of more than 6600 plant samples (Chen et al., 2010). Here, a large number of ITS2 DNA sequence data of Araliaceae plants are available from GenBank, which show high efficiency
of PCR amplification and high sequence quality. In this study, ITS2 had the highest discriminatory ability among the five markers and the greatest inter-specific divergence which was significantly higher than the intra-specific variation determined by the “DNA barcoding gap” assessment and Wilcoxon signed rank tests. Furthermore, the ITS2 locus had the highest identification efficiency among all the tested markers. Recently, a two-locus combination of rbcL + matK is recommended as a plant barcode by the plant Working Group of the Consortium for the Barcode of Life (CBOL) (Group, 2009). Although rbcL offered high universality, good PCR recovery and convenient alignment, it demonstrated insufficient sequence variation to distinguish among closely related species (Kress and Erickson, 2007; Lahaye et al., 2008). It showed the rate of species identification of 74.71%, which was not as high as ITS2. The matK gene is one of the most rapidly evolving plastid coding regions; however, it needs to be improved in primer universality and efficiency of PCR amplification (Chase et al., 2007). The CBOL Plant Working Group proposed rbcL + matK as the standard barcode for land plant as successful discrimination in 72% of cases was achieved after screening rpoB, rpoC1, rbcL, matK, trnH-psbA, atpF-atpH and psbK-psbI in 550 samples belonging to angiosperm, gymnosperm and alga. Single loci, 2-locus and 3-locus barcodes could achieve correct discrimination of 69%, 75% and 76% at most, respectively. However, the results were far from the CBOL Group expected (Group, 2009). The psbA-trnH has good priming sites because it is one of the most rapidly evolving spacers in chloroplast DNA with 75 bp conserved fragments at the ends. In addition, it demonstrated good
Fig. 3. Phylogenetic analysis of ITS2 regions from 53 samples in 19 genera.
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universality and high amplification success (Shaw et al., 2005; Yao et al., 2009). Here, we proposed psbA-trnH as another candidate barcode for Araliaceae plants with correct discrimination of 100% and 79.39% at genus and species, respectively. However, psbA-trnH has a great inter-specific variation with a long sequence and frequent poly A structures in angiosperm leading to a low sequencing quality (Thomas, 2009). In addition, alignment is hard to perform because of great variations in sequence length. Ycf5 needs a further study, for the number of samples (b10 sequences) we obtained was not enough for correct discrimination.
4.2. Efficacy of ITS2 for authentication Comparison of the five barcode markers in discrimination of Araliaceae plants showed that ITS2 was the best option in the following respects. ITS2 has good primer sites due to conserved 5.8S and 26S at the ends. ITS2 is as short as about 200 bp in Araliaceae plants, which is easy for DNA extraction, amplification and sequencing. On primary ITS2 sequences analyses, three discrimination methods (phylogenetic tree, the nearest distance and BLAST 1) were performed for identifying species. Furthermore, the secondary structure of ITS2 is considered as a molecular morphological characteristic (Selig et al., 2008). Schultz et al. established a database of ITS2 secondary structures, and 216,374 ITS2 of that are predicted (Schultz et al., 2006). ITS2 is a powerful barcode in species discrimination due to rapid evolvement and great variation at genus or species level, which has been proved by CBCs method. Most recently, 99.5% species of the diatom genus have been successfully discriminated by 5.8S + ITS2 (Moniz and Kaczmarska, 2010). However, 87 out of 589 samples failed in discrimination by ITS2 barcode, e.g., E._divaricatus f. albeofructus, E. divaricatus f. tristigmatis, E._divaricatus var. chiisanensis, E. divaricatus var. flavi-flos, and so on. It is hard for DNA barcode to identify those subspecies or varieties still debated in taxonomy, indicating that some closely related species might not be correctly discriminated by DNA barcoding. It may be suitable to identify them using other molecular markers (e.g., SNP, SSR, etc.), which are assistant tools for DNA barcoding (Liu et al., 2005, 2008, 2012; Liu and Sun, 2008; Luo et al., 2010; Zeng et al., 2011; Yuan et al., 2012). Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2012.02.016.
Acknowledgments This work was supported by the National Natural Science Foundation of China (81102746), Beijing Natural Science Foundation (5113033), Scientific Research Foundation of the State Human Resource Ministry and the Education Ministry for Returned Chinese Scholars, New Star Project of Peking Union Medical College, Youth Foundation of Peking Union Medical College, the Research Fund for the Doctoral Program of Higher Education (20111106120028), “Major Drug Discovery” major science and technology research “12nd Five-Year Plan” (2012ZX09301-002-001), and China Medical Board of New York (A2009001) granted to Zhihua Liu.
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