Open Access

Molecular identification of Uncaria (Gouteng) through DNA barcoding

  • Yin-lin Tang1, 2, 3,
  • Yao-sheng Wu1, 2Email author,
  • Rui-song Huang4,
  • Nai-xia Chao1, 2,
  • Yong Liu1, 5,
  • Peng Xu1, 6,
  • Ke-zhi Li1, 7,
  • Dan-zhao Cai1, 2 and
  • Yu Luo1, 2
Chinese Medicine201611:3

https://doi.org/10.1186/s13020-015-0072-7

Received: 14 January 2015

Accepted: 7 December 2015

Published: 3 February 2016

Abstract

Background

While DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng).

Methods

Genomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke’s Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbAtrnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0.

Results

The accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbAtrnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbAtrnH was higher than the corresponding intraspecific distance. Additionally, psbAtrnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method.

Conclusion

While psbAtrnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbAtrnH was a more suitable barcode for authentication of Uncaria macrophylla.

Background

Uncaria rhynchophylla (Miq.) Jacks is used to treat convulsion, hypertension, epilepsy, eclampsia, migraine, and cerebral diseases [13]. Rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine are the major components of U. rhynchophylla [4]. Oleanane and ursane-type triterpenes, (including uncarinic acids, ursolic acid, 3-hydroxyurs-12-en-27,28-dioic acid, hyperin, and catechin) were found in Uncaria [1, 5]. Uncaria comprises 34 species [6], 10 of which are found in the Guangxi Zhuang Autonomous Region. Among the 10 species of Uncaria in Guangxi, U. rhynchophylla and Uncaria macrophylla are the most widely and abundantly distributed [7]. Stems with hooks from several species of Uncaria, including U. rhynchophylla, U. macrophylla, Uncaria hirsuta, Uncaria sinensis, and Uncaria sessilifructus, have been used in Chinese medicine (CM) preparations, Gouteng in Chinese. Only the above five species plants of the genus Uncaria can serve as the botanical origins of Gouteng according to the Chinese Pharmacopeia (10th edition) [8]. Adulterants of Gouteng include Uncaria laevigata, Uncaria lancifolia, Uncaria scandens, Uncaria rhynchophylloides, and Uncaria homomalla [7, 9], due to similar organoleptic characteristics to those of U. rhynchophylla. But their chemical constituents and therapeutic effects are distinct from those of U. rhynchophylla [2, 10, 11].

DNA barcoding can accurately identify species on the basis of short standardized genes or DNA regions [12, 13], without confounding factors such as environmental influence, growth phase, and morphological diversity within species [1416]. The mitochondrial gene encoding cytochrome c oxidase subunit 1 (co1) is a potential DNA barcode in most animal species as well as some fungal species. However, the co1 gene and other mitochondrial genes from plants have limited use in identifying plant species across a wide range of taxa, due to their low genetic variations and variable mitochondrial genomes [17]. Several DNA regions, such as ITS2, psbAtrnH, matK, rbcL, ITS, ycf5, and rpoC1 [14, 1821] have been evaluated as potential DNA barcodes in medicinal plants. Among these candidate barcoding loci, the ITS2 locus not only had the highest identification efficiency among all tested regions, but also discriminated a wide range of plant taxa [14, 22]. By contrast, ITS1 was a useful barcode for identifying Salvia species [23]. The psbA–trnH intergenic region was a suitable DNA marker for identification of flowering plants [17, 18], pteridophytes [24], Lonicera japonica Thunb from Caprifoliaceae [21], and aquatic plant species [25].

The authentication of the botanical origins of Gouteng is based on the morphological characteristics, microscopic structures, or chemical components of specimens [26]. The accuracy is often affected by environmental and subjective factors, especially for dry medicinal materials from different origins [26]. Chemical analysis methods, such as high-performance liquid chromatography (HPLC) and HPLC coupled with quadrupole time-of-flight mass spectrometry, have also been studied [27]. Multiple genetic molecular markers have been used to screen Uncaria, such as random amplified polymorphic DNA (RAPD) and rDNAs (including 5.8S rDNA, ITS1, and ITS2) [28].

This study aims to determine suitable markers for DNA barcoding of the genus Uncaria. In this study, five candidate loci (ITS2, rbcL, psbAtrnH, ITS, and matK) were tested for their potential as DNA barcodes for Uncaria.

Methods

Plant materials

Fifty-four sequences from our laboratory (all submitted to GenBank), among which 12 samples of six species of Uncaria (U. rhynchophylla, U. macrophylla, U. sessilifructus, U. hirsuta, U. lancifolia and U. homomalla) are used as Gouteng in CM markets, were collected from areas in Guangxi Province, including Rongshui, Sanjiang, Shanglin, Ningming, and Jinxi county, Nanning Sitang town, and Guangxi Medicinal Botanical Garden, in 2009 and 2010 by Professor Ruisong Huang. The plant species were identified by Shouyang Liu, Yiling Zhu, and Kejian Yan through morphological characteristics and analysis of microscopic structures [7, 10]. All of the voucher specimens (all the voucher numbers can be seen in Table 1) were deposited in the Key Laboratory of Biological Molecular Medicine Research of Guangxi Higher Education, Guangxi Medical University.
Table 1

Uncaria information used in this study

Voucher no

Species

Habitat site (county, province, country)

GenBank accession no.

ITS2

rbcL

psbAtrnH

ITS

matK

PS1001MT01

U. rhynchophylla_01

Rongshui, Guangxi, China

KM057008

KM057019

KM057031

KM057043

KM057054

PS1001MT02

U. rhynchophylla_02

Sanjiang, Guangxi, China

KM057009

KM057020

KM057032

KM057044

 

U. rhynchophylla_03

Chinaa

AJ346900

  

AJ346900

 

PS1040MT01

U. rhynchophylla_04

Chinaa

JF421552

    

URH-1

U. rhynchophylla_05

Chinaa

KF881222

 

KF881177

KF881265

 

URH-2

U. rhynchophylla_06

Chinaa

KF881223

 

KF881178

  

PS1002MT01

U. macrophylla_01

Nanning, Guangxi, China

KM057010

KM057021

KM057033

KM057045

KM057055

PS1002MT02

U. macrophylla_02

Nanning, Guangxi, China

KM057011

KM057022

KM057034

KM057046

KM057056

PS1002MT03

U. macrophylla_03

Ningming, Guangxi, China

KM057012

KM057023

KM057035

KM057047

KM057057

PS1038MT03

U. macrophylla_04

Chinaa

GQ434637

GQ436558

GQ435234

  

PS1038MT04

U. macrophylla_05

Chinaa

GQ434638

GQ436559

GQ435235

  

PS1038MT01

U. macrophylla_06

Chinaa

GQ434636

    

UMA-1

U. macrophylla_07

Chinaa

KF881209

KF881134

KF881170

  

UMA-2

U. macrophylla_08

Chinaa

KF881210

KF881135

KF881171

  

UMA-3

U. macrophylla_09

Chinaa

KF881211

KF881136

KF881172

KF881257

 

UMA-4

U. macrophylla_10

Chinaa

KF881212

KF881137

KF881173

KF881258

 

UMA-5

U. macrophylla_11

Chinaa

KF881213

  

KF881259

 

UMA-6

U. macrophylla_12

Chinaa

KF881214

KF881138

KF881174

  

UMA-7

U. macrophylla_13

Chinaa

KF881215

    

UMA-8

U. macrophylla_14

Chinaa

KF881216

KF881139

KF881175

KF881260

 

UMA-9

U. macrophylla_15

Chinaa

   

KF881261

 

PS1003MT01

U. sessilifructus_01

Nanning, Guangxi, China

KM057013

KM057024

KM057036

KM057048

KM057058

PS1003MT02

U. sessilifructus_02

Shangsi, Guangxi, China

  

KM057037

  

U. sessilifructus_03

Chinaa

GU937111

  

GU937111

 

PS1041MT02

U. sessilifructus_04

Chinaa

GQ434640

    

USE-1

U. sessilifructus_05

Chinaa

KF881195

KF881122

   

USE-2

U. sessilifructus_06

Chinaa

KF881196

KF881123

KF881160

  

USE-3

U. sessilifructus_07

Chinaa

KF881197

KF881124

KF881161

  

USE-4

U. sessilifructus_08

Chinaa

KF881198

KF881125

KF881162

  

USE-5

U. sessilifructus_09

Chinaa

KF881199

KF881126

   

USE-6

U. sessilifructus_10

Chinaa

KF881200

KF881127

   

USE-7

U. sessilifructus_11

Chinaa

KF881201

KF881128

 

KF881249

 

PS1004MT01

U. hirsuta_01

Nanning, Guangxi, China

KM057014

KM057026

KM057038

KM057049

KM057059

PS1004MT02

U. hirsuta_02

Nanning, Guangxi, China

KM057015

KM057027

KM057039

KM057050

KM057060

PS1004MT03

U. hirsuta_03

Rongshui, Guangxi, China

KM057016

KM057028

KM057040

KM057051

 

U. hirsuta_04

Chinaa

GU937110

  

GU937110

 

UHI-1

U. hirsuta_05

Chinaa

KF881235

    

PS1005MT01

U. lancifolia_01

Jingxi, Guangxi, China

KM057017

KM057029

KM057041

KM057052

KM057061

Razafimandimbison et al. 713 (S)

U. lancifolia_02

Unknowna

KC737634

KC737740

 

KC737634

 

ULA-1

U. lancifolia_03

Chinaa

KF881218

KF881140

KF881176

KF881262

 

ULA-2

U. lancifolia_04

Chinaa

KF881219

  

KF881263

 

ULA-3

U. lancifolia_05

Chinaa

KF881220

  

KF881264

 

ULA-4

U. lancifolia_06

Chinaa

KF881221

    

PS1006MT01

U. homomalla_01

Shanglin, Guangxi, China

KM057018

KM057030

KM057042

KM057053

KM057062

Munzinger 177

U. homomalla_02

Unkowna

KC737633

KC737739

 

KC737633

 

UHO-1

U. homomalla_03

Chinaa

KF881202

KF881129

KF881163

KF881250

 

UHO-2

U. homomalla_04

Chinaa

KF881203

KF881130

KF881164

KF881251

 

UHO-3

U. homomalla_05

Chinaa

KF881204

KF881131

KF881165

KF881252

 

UHO-4

U. homomalla_06

Chinaa

KF881205

KF881132

KF881166

KF881253

 

UHO-5

U. homomalla_07

Chinaa

KF881206

 

KF881167

KF881254

 

UHO-6

U. homomalla_08

Chinaa

KF881207

 

KF881168

KF881255

 

UHO-7

U. homomalla_09

Chinaa

KF881208

KF881133

KF881169

KF881256

 

PS1039MT01

U. sinensis_01

Chinaa

FJ980386

GQ436560

GQ435236

FJ980386

 

USI-1

U. sinensis_02

Chinaa

 

KF881146

   

USI-2

U. sinensis_03

Chinaa

 

KF881147

KF881183

KF881271

 

USI-3

U. sinensis_04

Chinaa

   

KF881272

 

USI-4

U. sinensis_05

Chinaa

KF881234

KF881148

KF881184

KF881273

 

Razafimandimbison 304 (LBR, MO, P, TAN)

U. africana_01

Gabona

AJ414545

AJ347006

 

AJ414545

 

Taylor, Chanderbali, and Bourne 12075 (MO)

U. guianensis_01

Guyanaa

AJ414546

AJ347007

 

AJ414546

 

Andersson et al. 2031 (GB)

U. tomentosa_01

Unknowna

GQ852159

  

GQ852159

 

Andersson et al. 2038 (GB)

U. tomentosa_02

Unknowna

 

GQ852363

   

BioBot06438

U. tomentosa_03

Area de Conservacion Guanacaste, Rincon Rainforest, Sendero Venado, Costa Ricaa

 

JQ593902

   

BioBot06439

U. tomentosa_04

Area de Conservacion Guanacaste, Rincon Rainforest, Sendero Venado, Costa Ricaa

 

JQ593903

   

Razafimandimbison et al. 766 (S)

U. lanosa_01

Unkowna

KC737635

KC737741

 

KC737635

 

UYU-1

U. yunnanensis_01

Chinaa

KF881243

KF881156

KF881191

KF881281

 

UYU-2

U. yunnanensis_02

Chinaa

KF881244

    

UYU-3

U. yunnanensis_03

Chinaa

KF881245

KF881157

 

KF881282

 

UYU-4

U. yunnanensis_04

Chinaa

KF881246

KF881158

KF881193

KF881283

 

UYU-5

U. yunnanensis_05

Chinaa

KF881247

 

KF881194

  

UYU-6

U. yunnanensis_06

Chinaa

KF881248

KF881159

 

KF881284

 

WP2E0309

U. appendiculata_01

Papua New Guineaa

 

JF738785

   

WP1D0176

U. appendiculata_02

Papua New Guineaa

 

JF738676

   

WP5E1207

U. appendiculata_03

Papua New Guineaa

 

JF739007

   

Razafimandimbison et al. 768 (S)

U. scandens_01

Unknowna

KC737636

KC737742

 

KC737636

 

USC-1

U. scandens_02

Chinaa

KF881236

KF881149

KF881185

KF881274

 

USC-2

U. scandens_03

Chinaa

KF881237

KF881150

KF881186

KF881275

 

USC-3

U. scandens_04

Chinaa

KF881238

KF881151

KF881187

KF881276

 

USC-4

U. scandens_05

Chinaa

KF881239

KF881152

KF881188

KF881277

 

USC-5

U. scandens_06

Chinaa

KF881240

KF881153

 

KF881278

 

USC-6

U. scandens_07

Chinaa

KF881241

KF881154

KF881189

KF881279

 

USC-7

U. scandens_08

Chinaa

KF881242

KF881155

KF881190

KF881280

 

HITBC:Liana Mengsong 107_7_4

U. laevigata_01

Mengsong, Yunnan, Chinaa

 

KF181471

  

HG004898

ULAE-1

U. laevigata_02

Chinaa

KF881224

KF881142

KF881179

KF881266

 

ULAE-2

U. laevigata_03

Chinaa

KF881225

  

KF881267

 

ULAE-3

U. laevigata_04

Chinaa

KF881226

KF881143

 

KF881268

 

ULAE-4

U. laevigata_05

Chinaa

KF881227

KF881144

KF881180

KF881269

 

ULAE-5

U. laevigata_06

Chinaa

KF881228

 

KF881181

  

ULAE-6

U. laevigata_07

Chinaa

KF881229

  

KF881270

 

ULAE-7

U. laevigata_08

Chinaa

KF881230

 

KF881182

  

Total no. of sequences

 

257

77

63

49

58

10

aFrom GenBank

In total, 257 accessions related to the five candidate DNA barcoding sites (ITS2, rbcL, psbAtrnH, ITS, and matK) from 89 samples belonging to 15 species of Uncaria were analyzed in this study. All accession data were downloaded from GenBank, except for the above 54 sequences, which were amplified and sequenced in our laboratory. All datasets of Uncaria species used in the study contained more than two samples, except for Uncaria africana, Uncaria guianensis, and Uncaria lanosa. Some accessions in which the sequences contained undetermined bases or were from sp. species (taxa of species unclear or unnamed) were not selected. In this study, the correctness of the accessions downloaded from GenBank was tested through blasting against those of congener plants. Only the sequences with both a similarity ratio and query cover ratio higher than 90 % in the same species were suitable for selection. However, some accessions containing inversion sequences were collected in this dataset because they could influence the sequence divergence and supply some important genetic characters [29]. The total data and sample information used in this study are shown in Table 1.

DNA extraction, PCR amplification, and sequencing

In this study, genomic DNA was extracted from the freshly dried leaves of Uncaria plants by the improved protocol of a new rapid Plant Genomic DNA Extraction Kit (centrifugal column type, DP3112; Bioteke Corporation, Beijing, China). The Uncaria leaves were ground in liquid nitrogen, and the cell nuclear separation solution (3 ml for 0.5 g sample) was immediately added to the samples to remove impurities from the cytoplasm before the cell nuclei were lysed [30]. PCR amplification of the five candidate DNA barcode sites was performed in a Tprofessional Gradient 96 Type (Biometra, Göttingen, Germany) with approximately 30 ng of genomic DNA as a template in a 25-µL reaction mixture. Each reaction contained 1 × PCR buffer (2.0 mM MgCl2, 0.2 mM each dNTP, 0.1 µM each primer; synthesized by Sangon Biotech, Co., Ltd., Shanghai, China), and 1.0 U Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd., Dalian, China). The primers and reaction conditions used were the same as those used by Chen et al. [14]. The PCR products were electrophoresed in a 1.5 % agarose gel in 1 × TAE buffer, then purified with a TIANGel Midi Purification Kit (Tiangen Biotech Co. Ltd, Beijing, China). The purified PCR products were bidirectionally sequenced with appropriate amplification primers (Additional file 1) in an ABI-PRISM3730 instrument (Thermo Fisher Scientific, MA, USA) by Sangon Biotech, Co., Ltd., Shanghai, China.

Sequence alignment and data analysis

Sequence assembly and consensus sequence generation were performed by CodonCode Aligner 3.7.1 (CodonCode Co., MA, USA) by trimming the low quality sequence and primer areas. The matK and rbcL regions were delimited by alignment with known sequences in databases by CodonCode Aligner. After removal of the psbA and trnH genes at the ends of psbAtrnH, the boundary of the psbAtrnH intergenic spacer was determined according to the annotations of similar sequences in GenBank. The five candidate DNA barcodes were aligned by ClustalW (EMBL-EBI, Heidelberg, German). Kimura 2-Parameter (K2P) genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0 (The Biodesign Institute, AZ, USA) [31]. All interspecific and intraspecific distances, including theta prime, minimum interspecific distance, theta, and coalescent depth for all accessions of each locus, were calculated and compared to evaluate the interspecific divergence and intraspecific variation by the K2P model. Meanwhile, statistical analysis of the distribution divergency of the genetic distance between different sequences was performed through the Wilcoxon signed-rank test to assess the barcoding gap for different candidate loci with SPSS software (SPSS 16.0: International Business Machines Corporation Statistical Product and Service Solutions, Armonk, New York, USA), which the test statistical W+ and W− were calculated for two side test, as described previously [14, 22]. The BLAST1 and nearest distance methods were used to evaluate the species identification efficiency [32, 33].

Results

PCR amplification and base composition of the five loci of Uncaria

The sequence length and GC content of the five candidate loci (ITS2, rbcL, psbAtrnH, ITS, and matK) were obtained from the CodonCode Aligner and Clustal W alignment results (Table 2). The GC content of psbAtrnH was the lowest, while that of ITS2 was the highest. The variability of the length range of the psbAtrnH intergenic spacer was greater than that of the other candidates. The psbAtrnH region of U. macrophylla was more divergent than that of the other Uncaria plants.
Table 2

Analysis of the five candidate barcode loci of Uncaria

Items

ITS2

rbcL

psbAtrnH

ITS

matK

Species numbers

14

15

10

14

7

Accession no.

77

63

49

58

10

Length range (average) (bp)

210–221 (220)

512–656 (608)

235–315 (287)

607–621 (616)

757–814 (808)

Average of GC content (%)

66.3

43.0

24.8

62.8

33.1

No. of variable sites in all taxa

41

16

173

86

13

No. of indels in all taxa

2

0

39

14

0

BLAST method (identification efficiency [%])

87.0

42.9

95.9

91.4

80

Nearest distance method (identification efficiency [%])

92.2

76.2

95.9

84.5

80

Genetic interspecific divergence and intraspecific variation

Six parameters (Table 3) represented the genetic divergences of species in Uncaria. In a comparison of the intraspecific distances of the five candidate barcodes among Uncaria species, the intraspecific distance of psbAtrnH was higher than that of the other loci at the species level. Meanwhile, the interspecific genetic distance of the psbAtrnH intergenic spacer exhibited the highest divergence according to the interspecific distance, theta prime, and minimum interspecific distance. The interspecific distance of ITS2 was the second highest after psbAtrnH. All interspecific divergences of ITS2, psbAtrnH, and ITS were greatly higher than the corresponding intraspecific divergences. Furthermore, the overall mean distance of psbAtrnH was the highest among the five loci (Fig. 1).
Table 3

Calculation of interspecific and intraspecific divergences for Uncaria

Parameters

ITS2

rbcL

psbAtrnH

ITS

matK

Intraspecific divergence theta

0.0044 ± 0.0063

0.0010 ± 0.0013

0.0674 ± 0.0508

0.0080 ± 0.0089

0.0010 ± 0.0003

Coalescent depth

0.0171 ± 0.0292

0.0022 ± 0.0025

0.1060 ± 0.0705

0.0153 ± 0.0151

0.0012 ± 0.0000

All intraspecific distance

0.0059 ± 0.0128

0.0010 ± 0.0021

0.0480 ± 0.0401

0.0047 ± 0.0079

0.0009 ± 0.0006

Theta prime

0.0340 ± 0.0089

0.0040 ± 0.0021

0.0986 ± 0.0299

0.0253 ± 0.0050

0.0060 ± 0.0024

Minimum interspecific distance

0.0151 ± 0.0141

0.0009 ± 0.0017

0.0192 ± 0.0232

0.0104 ± 0.0092

0.0030 ± 0.0028

All interspecific distance

0.0348 ± 0.0166

0.0042 ± 0.0033

0.1068 ± 0.0468

0.0239 ± 0.0102

0.0057 ± 0.0027

Fig. 1

Distribution of overall mean distance for all sequence pairs among five loci. The number at right y axis is the estimates of average evolutionary divergence over all sequence pairs for each locus, which is the base substitutions per site from averaging over all sequence pairs. Analyses were conducted by the maximum composite likelihood method in MEGA6 [31]

The psbAtrnH intergenic spacer had the highest interspecific divergence among all the loci based on the Wilcoxon signed-rank test. The second highest interspecific divergence was shown by ITS2. The scale of the interspecific divergence of psbAtrnH was higher than ITS2, ITS, matK and rbcL, respectively (all P < 0.001), that of ITS2 was higher than ITS, matK and rbcL, respectively (all P < 0.001, Table 4). Furthermore, the intraspecific divergences between ITS and matK, rbcL and matK, ITS2 and matK, psbAtrnH and matK, and ITS and rbcL did not exhibit any significant differences (P > 0.05, Table 5).
Table 4

Wilcoxon signed-rank test for interspecific divergences

W+

W−

Inter relative rank

n

P value

Result

ITS2

rbcL

W+ = 1.00, W− = 639.50

1282

2.25 × 10−210

ITS2 > rbcL

ITS2

psbAtrnH

W+ = 506.72, W− = 98.66

957

1.42 × 10−149

ITS2 < psbAtrnH

ITS2

ITS

W+ = 365.08, W− = 744.61

1358

8.42 × 10−143

ITS2 > ITS

ITS2

matK

W+ = 0.00, W− = 16.50

32

7.93 × 10−7

ITS2 > matK

rbcL

psbAtrnH

W+ = 360.00, W− = 0.00

719

2.27 × 10−119

rbcL < psbAtrnH

rbcL

ITS

W+ = 442.81, W− = 20.38

862

8.05 × 10−141

rbcL < ITS

rbcL

matK

W+ = 22.63, W− = 17.86

41

0.0193

rbcL < matK

psbA-trnH

ITS

W+ = 27.27, W− = 287.47

560

1.80 × 10−92

psbA-trnH > ITS

psbA-trnH

matK

W+ = 0.00, W− = 16.50

32

7.93 × 10−7

psbA-trnH > matK

ITS

matK

W+ = 0.00, W− = 16.50

32

7.93 × 10−7

ITS > matK

Table 5

Wilcoxon signed-rank test for intraspecific divergences

W+

W−

Intra relative rank

n

P value

Result

ITS2

rbcL

W+ = 23.12, W− = 45.44

149

7.54 × 10−6

ITS2 > rbcL

ITS2

psbAtrnH

W+ = 60.70, W− = 11.00

124

1.90 × 10−20

ITS2 < psbAtrnH

ITS2

ITS

W+ = 49.59, W− = 37.93

127

0.0166

ITS2 > ITS

ITS2

matK

W+ = 2.00, W− = 0.00

4

0.1025

ITS2 = matK

rbcL

psbAtrnH

W+ = 46.00, W− = 0.00

101

1.19 × 10−16

rbcL < psbAtrnH

rbcL

ITS

W+ = 29.17, W− = 26.60

84

0.3788

rbcL = ITS

rbcL

matK

W+ = 2.00, W− = 0.00

4

0.1025

rbcL = matK

psbAtrnH

ITS

W+ = 10.50, W− = 34.22

70

4.23 × 10−12

psbAtrnH > ITS

psbAtrnH

matK

W+ = 1.00, W− = 2.50

4

0.2763

psbAtrnH = matK

ITS

matK

W+ = 2.00, W− = 0.00

4

0.1025

ITS = matK

Analysis of barcoding gaps

As a barcode for identifying botanical species, the divergence between species should be higher than the variation within species [34]. Although the histogram of the K2P genetic distance analysis revealed a partial overlap “barcoding gap” between the intraspecific and interspecific divergence of ITS2 or psbAtrnH (Fig. 2), the intraspecific variation of psbAtrnH and ITS2 was considerably lower than the distribution of their interspecific divergence. The genetic divergence distribution of ITS was similar to that of ITS2. No clear “barcoding gap” corresponding to the rbcL or matK loci was observed, wherein the genetic distance distribution of more than 90 % of accessions was less than 0.020. However, the distribution of the interspecific divergence of ITS2 and psbAtrnH provided a better resolution than that of rbcL and matK.
Fig. 2

Distribution of divergence between interspecific and intraspecific genetic distance for five candidate barcoding loci among 257 accessions. a ITS2; b rbcL; c psbAtrnH; d ITS; e, matK. x axis, genetic distance; y axis, distribution of genetic divergence

Identification efficiency and characteristics of Clustal W alignment

The BLAST and nearest distance methods were employed to test the applicability of the five loci for species identification of Uncaria. psbAtrnH presented 95.9 % identification efficiency with both the BLAST and nearest distance methods at the species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method, whereas rbcL showed only 76.2 % by the nearest distance method and 42.9 % by the BLAST method (Table 2). Meanwhile, psbAtrnH of U. macrophylla exhibited more obvious characteristics than U. rhynchophylla and the other species tested (Figs. 3, 4, 5). Two insertion fragments existed in the psbAtrnH sequence of U. macrophylla, including a serial seven A fragment at 171–177 bp, and another double repeat “ATTAAA” at 234–247 bp. The psbAtrnH intergenic spacer can be used as a barcode for the identification of Uncaria plants. The phylogeny of Uncaria ITS2 (computed model: Maximum Composite Likelihood) [31] showed that only four accessions (4/77 accessions) were in the incorrect taxonomic category (Fig. 6), which was less than the other loci tested. Thus, ITS2 could be another suitable DNA barcode for Uncaria.

Fig. 3

ClustalW results of psbAtrnH of Uncaria plants. Identical positions are shown as dot; indels as dash; the red box site show a seven A repeat inserted at 171–177 bp, the differences of U. macrophylla from U. rhychophylla and other Uncaria species

Fig. 4

ClustalW results of psbAtrnH of Uncaria plants. Identical positions are shown as dot; indels as dash; the red box site show a cis-repeats of ATTAAA insertion at 234–239 bp, the differences of U. macrophylla from U. rhychophylla and other Uncaria species

Fig. 5

ClustalW results of psbAtrnH of Uncaria plants. Identical positions are shown as dot; indels as dash; the red box site show a cis-repeats of ATTAAA insertion at 241-247 bp, the differences of U. macrophylla from U. rhychophylla and other Uncaria species

Fig. 6

Phylogeny tree of Uncaria ITS2. The evolutionary history was inferred using the neighbor-joining method, the evolutionary distances were computed using the maximum composite likelihood model. Only four accessions labeled by triangular, square or circular symbol were incorrectly taxonomic category

Discussion

Significance of authentication of Uncaria by DNA barcoding

Gouteng is commonly exploited as the major ingredient herb of CM prescriptions for hypertension or migraine treatment [2, 35]. The amount of stems with hooks of U. rhynchophylla (Gouteng) required in traditional clinic and pharmaceutical production, has been increased; while the natural growth of U. rhynchophylla, U. hirsuta and U. macrophylla which could serve as the botanical origins of Gouteng was limited with the rising of collection. Some other species of the genus Uncaria are often collected to adulterate Guoteng, such as U. laevigata, U. lancifolia, U. scandens [7]. Therefore, the correct genotypic identification of Uncaria plant material is essential in order to protect public health and for industrial production.

Although some methods have been developed to distinguish Uncaria plants based on morphotype, microcharacter, or physical and chemical reactions [8, 9], these are dependent on taxonomy experts. Currently, the genetic molecular markers for the genus Uncaria were related to RAPD, rDNA, and ITS, while DNA barcoding assays have not yet been reported. This study included 11 of 12 species of Uncaria in China, with U. rhynchophylloides missing in the screen for suitable DNA barcodes for Uncaria.

In the present study, psbAtrnH presented 95.9 % identification efficiency for Uncaria accessions tested with both BLAST and nearest distance methods at the species or genus level. ITS2 also exhibited high identification efficiency at 92.2 or 87 % with the nearest distance or BLAST method, respectively.

Quality and amplification efficiency of DNA from Uncaria

The DNA of Uncaria was not extracted efficiently, due to the large amounts of polysaccharides, polyphenols, and alkaloids present in the samples. A cell nuclear separation solution was used to remove the impurities from genomic DNA [30]. The quality of the DNA extracted from the Uncaria plants satisfied the requirements for PCR amplification and sequencing. The efficiency of both PCR amplification and sequencing for psbAtrnH was the highest among the five candidate loci. Specifically, PCR amplification showed 96.7 % efficiency, while sequencing showed 100 % efficiency. Because the average GC content of ITS2 was 66.3 %, which was higher than that of the other loci, the resulting DNA extract was slightly difficult to amplify.

Selection of candidate DNA barcodes

In this study, the length of psbAtrnH of Uncaria ranged from 235 to 315 bp (mean 287 bp), which was longer than that of ITS2, but shorter than that of rbcL, ITS, and matK. Additionally, psbAtrnH of Uncaria exhibited the highest interspecific divergence among the five loci tested, based on the results of six parameters of the K2P model or Wilcoxon signed-rank test of interspecific divergence. The interspecies divergence of psbAtrnH was higher than the relevant intraspecies variation. Furthermore, psbAtrnH of U. macrophylla was significantly distinct from that of U. rhynchophylla and the other species because of two insertion fragments: one was a seven A repeat inserted at 171–177 bp and the other was two cis-repeats of ATTAAA at 233–247 bp (Figs. 3, 4, 5). Although one TAAAAAA repeat was observed at 171–177 bp in psbAtrnH from Uncaria yunnanensis, no double cis-repeats of ATTAAA were observed at 233–247 bp. Meanwhile, one inversion sequence of length 73–74 bp with identity ratios of more than 98 % in psbAtrnH of Uncaria was found in this study (Additional file 2). The intragenic variation of the genus Uncaria was large because of this inversion phenomenon existing in psbAtrnH. This situation was also observed in psbAtrnH of Aconitum L. [29]. The characteristics of the insertion sequences in psbAtrnH could effectively authenticate Uncaria species.

ITS2 was another suitable locus for distinguishing different species of Uncaria. The length range of ITS2 was 210–221 bp (mean 219.9 bp), which was the shortest among the five loci. Consequently, 95.8 % efficiency could be reached by PCR amplification. In a comparison of the interspecific genetic distances of the five candidate barcodes among Uncaria species, the mean interspecific distance of ITS2 was higher than its mean intraspecific divergence, and the values were second only to those of psbAtrnH (Table 3). Based on the phylogenetic analysis of ITS2 by the neighbor-joining method and the evolutionary distances computed by the Maximum Composite Likelihood model, more than 93 % of Uncaria at the species level in this study were divided into monophyla as recognized species. Among 77 accessions of ITS2, comprising 14 species of Uncaria, only four accessions were in an incorrect taxonomic category, according to the construction of a phylogenetic tree for ITS2 (Fig. 6). Uncaria manifested complex morphological features and genetic backgrounds, and even some specimens with obvious differences in appearance possessed similar ITS sequences [28]. This could explain the existence of some accessions that appeared in different monophyla from their original morphological taxa. Some species submitted to GenBank may have been wrongly categorized. Sequences with lengths of less than 100 bp, those with ambiguous bases containing more than one “N”, or those belonging to unnamed species (such as those with spp. and aff. in the species name) were excluded [20] from this study to guarantee the reliability of the selected sequences.

A better “barcoding gap” was observed between the interspecific divergence and intraspecific variation of ITS2 compared with the other loci. ITS, which contained three fragments (ITS1, 5.8S rDNA, ITS2), exhibited a similar identification efficiency to that of ITS2. Both rbcL and matK were unsuitable genetic loci for authentication of the botanical origins of Gouteng, because of the absence of a clear barcoding gap between the interspecific divergence and intraspecific variation by the K2P model. The overall mean distance of rbcL was only 0.002 and that for matK was 0.005, as computed by the Maximum Composite Likelihood model (Fig. 1). Moreover, we found that the combination of psbAtrnH with ITS2 would provide a better result for the authentication of Uncaria plants, and could even distinguish between incorrect and correct taxa or identify some cryptic species. Currently, a preliminary system for DNA barcoding of herbal materials has been established based on a two-locus combination of ITS2 and psbAtrnH barcodes [36]. Recently, ITS2 was successfully exploited in a survey involving commercial Rhodiola products, including decoction pieces [37].

psbAtrnH and ITS2 also exhibited high authentication power for different species of Uncaria. Both psbAtrnH and ITS2 revealed the distinct divergence of U. macrophylla from U. rhynchophylla and the other species at the species level.

Conclusion

While psbAtrnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbAtrnH was a more suitable barcode for authentication of U. macrophylla.

Abbreviations

ITS: 

internal transcribed spacer

psbAtrnH

gene spacer between psbA and trnH in chloroplast DNA

K2P: 

Kimura 2-Parameter

CM: 

Chinese medicine

HPLC: 

high-performance liquid chromatography

RAPD: 

random amplified polymorphic DNA

Declarations

Authors’ contributions

YSW conceived and designed the study. YLT, RSH, PX, KZL extracted DNA and performed PCR; YLT, DZC and YLuo checked the quality of PCR products and analyzed the sequencing results; YLT, NXC and YLiu analyzed the data; YSW wrote and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to Mr. Shou-yang Liu and Mr. Yi-ling Zhu, Mr. Ke-jian Yan for their help with the collection and identification of the Uncaria specimens. We appreciate the essential comments provided by Dr. Hui Yao and all others that much improved the manuscript. We thank the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, P.R.China, for providing the calculation and analysis platform. The work was supported by Guangxi Natural Science Foundation (2010GXNSFA013145); Scientific Research Project Foundation for Higher Education School from Guangxi Provincial Department of Education (201203YB038); National Natural Scientific Foundation of P.R. China (No.31260069).

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Biochemistry and Molecular Biology, Guangxi Medical University
(2)
Key Laboratory of Biological Molecular Medicine Research of Guangxi Higher Education
(3)
Clinical Laboratory, Maternal and Child Health Hospital of Guangxi
(4)
Guangxi Academy of Minority Nationality Medicine and Pharmacology
(5)
School of Pharmacy, Guangdong Medical College
(6)
Liuzhou People’s Hospital
(7)
Affiliated Cancer Hospital of Guangxi Medical University

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© Tang et al. 2016

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