# Barcoding theDendrobium (Orchidaceae) Species and Analysis of the Intragenomic Variation Based on the Internal Transcribed Spacer 2

Authors: Xiaoyue Wang; Xiaochen Chen; Pei Yang; Lili Wang; Jianping Han
Journal: BioMed Research International
 (2017)

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## Abstract

Many species belonging to the genusDendrobium are of great commercial value. However, their difficult growth conditions and high demand have caused many of these species to become endangered. Indeed, counterfeitDendrobiumproducts are common, especially in medicinal markets. This study aims to assess the suitability of the internal transcribed spacer 2 (ITS2) region as a marker for identifyingDendrobium and to evaluate its intragenomic variation inDendrobiumspecies. In total, 29,624 ITS2 copies from 18 species were obtained using 454 pyrosequencing to evaluate intragenomic variation. In addition, 513 ITS2 sequences from 26Dendrobium species were used to assess its identification suitability. The highest intragenomic genetic distance was observed inDendrobium chrysotoxum (0.081). The average intraspecific genetic distances of each species ranged from 0 to 0.032. Phylogenetic trees based on ITS2 sequences showed that mostDendrobium species are monophyletic. The intragenomic and intraspecies divergence analysis showed that greater intragenomic divergence is mostly correlated with larger intraspecific variation. As a major ITS2 variant becomes more common in genome, there are fewer intraspecific variable sites in ITS2 sequences at the species level. The results demonstrated that the intragenomic multiple copies of ITS2 did not affect species identification.


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## Body

## 1. Introduction

Dendrobium is one of the three largest genera of the Orchidaceae family and comprises more than 1,000 species distributed throughout the Asian tropical and subtropical regions as well as Oceania, with 78 species of this genus recorded in China alone [1]. The flowers ofDendrobium come in a rich variety of colors and shapes, and in recent years they have increased significantly in commercial value as ornamental flowers. In addition,Dendrobium is also well known for its medical value. In fact, one of the earliest records of Orchidaceae plants in ancient Chinese literature is Shen Nong’s classic herbal text written approximately 1,500 years ago. Approximately 33 species ofDendrobium are used as clinical medications [2], includingDendrobium officinale, also known as “Tie Pi Feng Dou,” andDendrobium nobile, also known as “Jin chai shi hu,” as described in the Chinese Pharmacopoeia. Each year, large numbers ofDendrobium species are needed for both the flower and medicinal markets. Adulterants and substitutes have become popular in the markets, especially for medicinal purposes. Thus, an effective method of species identification is very necessary.In eukaryotic genomes, rDNA arrays are often present in hundreds of copies, with copy number varying among different species [3–5]. As a tool to study evolution, the rDNA copy number per genome and sequence variation between species can be used to study phylogenetic relationships and biodiversity [4]. The internal transcribed spacer (ITS) is part of a multicopy gene that encodes ribosomal RNA subunits in all eukaryotic genomes. ITS regions have been used to study biodiversity in bacteria [6], insects [7], marine organisms [8–10], and plants [11], as well as many others. Due to their powerful discriminatory ability and stability amongDendrobium species, rDNA sequences have been used for identification and classification purposes [12, 13]. Among the numerousDendrobium species,D. officinale has received the greatest amount of attention due to its high medicinal value in China. Ding et al. established a database that included 21Dendrobium species labelled “Feng Dou” herbs on the market and proposed that rDNA ITS sequences could be used to identifyDendrobium species with high accuracy [14]. Indeed, Zhang et al. accurately identifiedD. officinale from its adulterants using full-length ITS regions [15]. Furthermore, Li et al. performed phylogenetic analyses and identifiedDendrobium species using rDNA ITS sequences, and their classification based on ITS sequences was identical to traditional classifications for most species [16].ITS2 is commonly used to infer phylogenetic relationships and has been employed as a DNA barcode for identification purposes. The genes in this region are thought to have evolved in concert, leading to a homogenization of all copies of this gene across the genome [17, 18]. To date, the ITS2 region has been used to identify plants [19–21], fungi [22–24], and insects [25]. Although ITS/ITS2 is extremely useful for both species identification and phylogenetic analyses, it does have drawbacks. One significant problem is the fact that it is present in multiple copies in the genome. Phylogenetic studies typically use consensus sequences that average over all copies in a genome, thereby concealing most intragenomic variation. Indeed, the intragenomic variation and intraspecies divergence in ITS2 present significant challenges for genetic diversity analyses and species identification. In contrast, the evaluation of ITS2 sequences for identification and phylogenetic purposes might prove useful for deep research into intragenomic and intraspecific diversity. While intraspecific divergence inDendrobium has been studied, the issue of intragenomic diversity revealed by multicopy has received increased attention due to the development of next-generation sequencing technology. Here we used pyrosequencing to sequence 18 selected species ofDendrobium to perform ITS2 intragenomic diversity analysis. Intra- and interspecific variations among different species were also evaluated using ITS2 sequences in 26 species ofDendrobium. Our results indicate that the ITS2 region is a valuable tool for identifying species and analyzing phylogenetic relationships.


## 2. Material and Methods

### 2.1. Sampling, DNA Extraction, PCR Amplification, and Sequencing

Fresh leaves and stems of plants of the genusDendrobium were obtained from different locations (see Appendix S1, in Supplementary Material available online at https://doi.org/10.1155/2017/2734960). Samples were dried at a temperature of 45°C prior to genomic DNA extraction. DNA extraction, PCR amplification, and sequencing were performed as described in previous studies [26, 27]. Approximately 15 mg of dried leaves or 20 mg of dried stems was ground for two minutes (30 revolutions/second) in a FastPrep bead mill (MM400, Retsch, Haan, Germany). DNA was extracted using the Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). Universal primers for the ITS2 region (ITS2F/3R) were used for amplification [27]. Sequencing of the PCR products was performed bidirectionally with the same primers used for the PCR amplification using a 3730XL sequencer (Applied Biosystems, Foster, California, USA). The intragenomic data used were from a previous study by our group [28]. Other sequences were obtained from GenBank (see Appendix S2). Twenty-sixDendrobium species with more than ten sequences each were selected for identification analysis.


### 2.2. Data Analysis

ITS2 sequences in this study were subjected to hidden Markov model (HMM) [29] analysis to remove the conserved 5.8S and 26S rRNA genes. Intragenomic and intraspecific Kimura 2-parameter distances were computed using the MEGA 5.2.2 software [30] (default parameters: variance estimation method: bootstrap method; number of bootstrap replications: 1000; model: Kimura 2-parameter model; substitutions to include transition + transversions; rates among sites: uniform rates; and gaps/missing data treatment: complete deletion). Mega 5.2.2 was used to construct a neighbor-joining tree (default parameters: test of phylogeny: bootstrap method; number of bootstrap replications: 1000; model: Kimura 2-parameter model; substitutions to include: transition + transversions; rates among sites: uniform rates; and gaps/missing data treatment: complete deletion).


## 2.1. Sampling, DNA Extraction, PCR Amplification, and Sequencing

Fresh leaves and stems of plants of the genusDendrobium were obtained from different locations (see Appendix S1, in Supplementary Material available online at https://doi.org/10.1155/2017/2734960). Samples were dried at a temperature of 45°C prior to genomic DNA extraction. DNA extraction, PCR amplification, and sequencing were performed as described in previous studies [26, 27]. Approximately 15 mg of dried leaves or 20 mg of dried stems was ground for two minutes (30 revolutions/second) in a FastPrep bead mill (MM400, Retsch, Haan, Germany). DNA was extracted using the Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). Universal primers for the ITS2 region (ITS2F/3R) were used for amplification [27]. Sequencing of the PCR products was performed bidirectionally with the same primers used for the PCR amplification using a 3730XL sequencer (Applied Biosystems, Foster, California, USA). The intragenomic data used were from a previous study by our group [28]. Other sequences were obtained from GenBank (see Appendix S2). Twenty-sixDendrobium species with more than ten sequences each were selected for identification analysis.


## 2.2. Data Analysis

ITS2 sequences in this study were subjected to hidden Markov model (HMM) [29] analysis to remove the conserved 5.8S and 26S rRNA genes. Intragenomic and intraspecific Kimura 2-parameter distances were computed using the MEGA 5.2.2 software [30] (default parameters: variance estimation method: bootstrap method; number of bootstrap replications: 1000; model: Kimura 2-parameter model; substitutions to include transition + transversions; rates among sites: uniform rates; and gaps/missing data treatment: complete deletion). Mega 5.2.2 was used to construct a neighbor-joining tree (default parameters: test of phylogeny: bootstrap method; number of bootstrap replications: 1000; model: Kimura 2-parameter model; substitutions to include: transition + transversions; rates among sites: uniform rates; and gaps/missing data treatment: complete deletion).


## 3. Results

### 3.1. Intragenomic Variations in 18 Species ofDendrobium

We first investigated the levels of intragenomic variation in the ITS2 regions of 18Dendrobium medicinal materials. A summary of the intragenomic variation is provided in Table 1. In total, 29624 ITS2 copies from 18 species were obtained using 454 pyrosequencing in a previous study by our group. The numbers of ITS2 variant copies are 4 to 55, withD. crepidatum having the least andD. chrysotoxum having the most. Here, we refer to variants with an emergence frequency above 1% as “major variant(s).” Major variants representing more than 90% of ITS2 sequences were found inD. crepidatum (p1-1),D. aphyllum (P2-1),D. devonianum (p3-1),D. officinale (p4-1),D. trigonopus (p16-1),D. gratiosissimum (p15-1),D. capillipes (p13-1), andD. denneanum (p5-1), which were present at 99.27%, 95.87%, 95.43%, 93.89%, 93.34%, 93.23%, 93.03%, and 91.50% of total sequences, respectively. The major variants of each species were used to calculate the intragenomic genetic distance (IG-GD). The highest IG-GD was found inD. chrysotoxum (0.081). In contrast, the genetic distance in five species (D. officinale,D. crepidatum,D. aphyllum,D. wardianum, andD. trigonopus) was zero, indicating minimal intragenomic diversity.D. williamsonii showed the most intragenomic variant patterns of the 18 study species, showing 52 distinct major variant patterns that ranged in prevalence from 25.82% to 1.1%. The GC content of the variants from these 18 species ranged from 47.35% to 56.00%. No obvious differences were observed in the length of major variants.Table 1
Intragenomic diversity analysis of ITS2 major variants from the 18Dendrobiumspecies.
Taxon
Number of total 454 sequences
Number of total variants
Variant name (>1% emergence frequency)
Percentage of each variant (>1%)
Length (bp)
G + C content (%)
Intragenomic sequence distances (>1% emergence frequency)
D. crepidatum
1919
4
p1-1
99.27
248
50.81
0
D. aphyllum
1888
19
p2-1
95.87
248
52.42
0
D. devonianum
722
8
p3-1
95.43
248
49.20
0.004
p3-2
1.80
247
48.58
D. officinale
2554
10
p4-1
93.89
246
50.81
0∗
p4-2
4.78
245
51.02
D. denneanum
1270
15
p5-1
91.50
248
51.21
0.016
p5-2
2.36
248
50.81
p5-3
1.42
248
50.00
p5-4
1.18
248
50.81
D. nobile
578
7
p6-1
63.15
246
53.66
0.017
p6-2
14.36
246
54.07
p6-3
10.73
246
54.07
p6-4
9.69
246
52.85
D. chrysanthum
860
30
p7-1
76.74
245
52.24
0.051
p7-2
3.14
245
51.84
p7-3
3.02
244
52.05
p7-4
2.56
245
47.35
p7-5
1.74
245
48.16
p7-6
1.63
245
47.76
p7-7
1.28
245
48.57
p7-8
1.28
245
50.20
p7-9
1.28
244
52.46
D. primulinum
3690
36
p8-1
75.93
248
53.63
0.005
p8-2
9.51
248
53.23
p8-3
9.11
248
53.23
D. chrysotoxum
2366
55
p9-1
63.82
249
55.42
0.081
p9-2
19.95
249
55.82
p9-3
2.79
249
55.02
p9-4
2.20
247
52.63
p9-5
2.20
249
55.42
p9-6
1.65
247
53.04
D. loddigesii
1593
47
p10-1
39.55
248
52.02
0.009
p10-2
30.70
248
51.21
p10-3
14.44
248
52.42
p10-4
5.34
248
51.21
D. acinaciforme
1112
30
p11-1
70.05
248
52.42
0.045
p11-2
16.55
248
52.42
p11-3
6.56
248
53.23
D. williamsonii
182
52
p12-1
25.82
247
52.63
0.05
p12-2
3.85
247
52.23
p12-3
2.75
247
50.20
p12-4
3.75
247
51.01
p12-5
2.20
247
51.01
p12-6
2.20
247
51.82
p12-7
2.20
247
50.20
p12-8
2.20
247
48.99
p12-9
2.20
247
50.61
p12-10~p12-21
19.80
247–250
48.99–51.42
p12-22~p12-52
34.10
244–247
48.37–52.63
D. capillipes
1610
33
p13-1
93.03
251
50.20
0.004
p13-2
1.43
251
49.80
D. crystallinum
1197
14
p14-1
51.96
250
55.60
0.005
p14-2
39.52
250
55.60
p14-3
5.93
250
55.20
D. gratiosissimum
1167
8
p15-1
93.23
246
52.85
0.004
p15-2
5.14
246
52.44
D. trigonopus
1906
27
p16-1
93.34
251
51.00
0
D. wardianum
2778
21
p17-1
84.38
250
56.00
0∗
p17-2
12.35
251
55.78
D. pendulum
2232
19
p18-1
49.73
250
54.00
0.005
p18-2
24.82
250
53.20
p18-3
20.79
249
53.41
p18-4
1.43
250
53.60
p18-5
1.25
250
53.60
T
h
e
∗ variations were caused by insert/deletion, which was treated as complete deletion when calculating the K2P distances. Thus, the K2P distances turned out to be zero.In this study, theD. officinale sample received a total of 2554 reads of 454 pyrosequencing representing ten different variant patterns. The most common major variant represented 93.89% of the ITS2 sequences in the entire genome. After alignment, the consensus sequence of the ten variants from theD. officinale genome was 246 bp in length, with 13 variable sites, including two INDELS. The dominant sequence patterns inD. officinale were consistent with the sequences obtained via direct PCR sequencing.


### 3.2. Analysis of the ITS2 Region at Intra- and Interspecific Levels

In total, 513 ITS2 sequences from 26 species ofDendrobium were analyzed for intraspecific genetic distances (IS-GDs). The average IS-GD for each species ranged from 0 to 0.032. The average IS-GD value in seven species (D. herbaceum,D. macrostachyum,D. amoenum,D. aqueum,D. bicameratum,D. barbatulum, andD. peguanum) was zero, and the highest average IS-GD value (0.032) was found inD. hancockii (Figure 1). The number of variable sites in ITS2 sequences of each species was also calculated (Table 2).D. officinale possesses a dominant variant representing 93.89% of the sequences, and out of 61 sequences there were only five intraspecific SNPs. A similar situation was observed in three other species (D. aphyllum,D. devonianum, andD. denneanum), all of which had one dominant ITS2 variant making up more than 90% of sequences. In contrast, there were more variable sites in species where the dominant ITS2 variant made up less than 80% of the sequences. For example, the dominant variant inD. loddigesiiaccounted for only 39.55% of sequences, and this species had 28 intraspecific variable sites. In general, as the dominant ITS2 variant became more common in the genome, there were fewer intraspecific variable sites in ITS2 sequences, with the exception ofD. crepidatum.Table 2
Comparison between the numbers of intraspecific variable sites with the emergency percentage of the dominant variants in genome of ITS2 sequences of each species.
Taxon
Number of total ITS2 sequences
Number of total intraspecific variable sites
The emergency percentage of the dominant variants in genome (%)
D. crepidatum
35
36
99.27
D. aphyllum
22
4
95.87
D. devonianum
11
9
95.43
D. officinale
61
5
93.89
D. denneanum
10
4
91.50
D. chrysanthum
25
22
76.74
D. primulinum
21
25
75.93
D. chrysotoxum
27
18
63.82
D. nobile
59
12
63.15
D. loddigesii
13
28
39.55Figure 1
Intraspecific genetic distances from 26Dendrobium species revealed by ITS2 region.The BLAST1 method, which is based on similarity, was used to assess the reliability of ITS2 sequences forDendrobium species identification. In total, 383 of the 513 ITS2 sequences were correctly identified. The unidentified ITS2 sequences were distributed among six species:D. officinale,D. tosaense,D. huoshanense,D. moniliforme,D. nobile, andD. hercoglossum. The ITS2 sequences ofD. officinale andD. tosaense could not be distinguished using BLAST, and similar issues arose forD. huoshanense,D. moniliforme,D. nobile, andD. hercoglossum.


### 3.3. The Neighbor-Joining Tree Based on ITS2 Sequences

A neighbor-joining tree (NJ tree) was built based on the intragenomic data to determine the phylogenetic relationships between theDendrobium species. Previous studies have shown that minor variants present below 1% are difficult to detect directly with PCR or clone sequencing. Thus, we first selected the major variants for analysis (Figure 2). The results showed thatD. williamsonii (PS2503),D. trigonopus (PS2506),D. acinaciforme (PS2527), andD. capillipes (PS2502) clustered into one clade, with all other species forming a separate clade. Almost all the major variants clustered together, with the exception ofD. nobile. One of the major variants ofD. nobile (PS0766_4) introgressed intoD. officinale andD. gratiosissimum, showing a very close relationship. Another NJ tree using total intragenomic data was also constructed and is shown in Appendix S3.Figure 2
The neighbor-joining tree built using the major variants of ITS2 from 18Dendrobium species.Note. Each species was marked with a unique color. The three parts of the name of each branch represent the voucher number, the variant name of the multiple copy in the genome, and the percentage of its presence in the genome. The taxa with their voucher numbers were listed for the following:D. williamsonii(PS2503),D. trigonopus(PS2506),D. acinaciforme(PS2527),D. capillipes(PS2502),D. loddigesii(PS1748),D. crystallinum(PS2519),D. denneanum(PS0758),D. chrysotoxum(PS2501),D. officinale(PS2521),D. gratiosissimum(PS2513),D. nobile(PS0766),D. wardianum(PS2509),D. pendulum(PS2511),D. aphyllum(PS2523),D. primulinum(PS2518),D. devonianum(PS2520),D. chrysanthum(PS2515),and D. crepidatum(PS2517).Total intraspecific data were also used to construct an NJ tree for phylogenetic analysis (see Appendix S4). The results showed that most species were monophyletic except for one clade including six species (D. officinale,D. tosaense,D. huoshanense,D. moniliforme,D. nobile, andD. hercoglossum). To better clarify the relationship among these species in the main clade, these six species were used to build a separate NJ tree based on their ITS2 sequences (Figure 3). We divided this NJ tree into two major clades (Clades I and II). Clade I consists of two species,D. officinale andD. tosaense, with a bootstrap support value of 100%. Clade II consists of two subclades, with a bootstrap support value of 54%. Subclades II-I contain two species,D. moniliforme andD. huoshanense, with a bootstrap support value of 72%, and subclades II-II contain three species,D. moniliforme,D. hercoglossum, andD. nobile, with a bootstrap support value of 62%.Figure 3
The neighbor-joining tree built using ITS2 sequences of the six species.Note. Different colors in Figure 3 represent different species. Blue,D. officinale; red,D. huoshanense; green,D. nobile; purple, D. moniliforme; orange,D. tosaense; yellow,D. hercoglossum.


## 3.1. Intragenomic Variations in 18 Species ofDendrobium

We first investigated the levels of intragenomic variation in the ITS2 regions of 18Dendrobium medicinal materials. A summary of the intragenomic variation is provided in Table 1. In total, 29624 ITS2 copies from 18 species were obtained using 454 pyrosequencing in a previous study by our group. The numbers of ITS2 variant copies are 4 to 55, withD. crepidatum having the least andD. chrysotoxum having the most. Here, we refer to variants with an emergence frequency above 1% as “major variant(s).” Major variants representing more than 90% of ITS2 sequences were found inD. crepidatum (p1-1),D. aphyllum (P2-1),D. devonianum (p3-1),D. officinale (p4-1),D. trigonopus (p16-1),D. gratiosissimum (p15-1),D. capillipes (p13-1), andD. denneanum (p5-1), which were present at 99.27%, 95.87%, 95.43%, 93.89%, 93.34%, 93.23%, 93.03%, and 91.50% of total sequences, respectively. The major variants of each species were used to calculate the intragenomic genetic distance (IG-GD). The highest IG-GD was found inD. chrysotoxum (0.081). In contrast, the genetic distance in five species (D. officinale,D. crepidatum,D. aphyllum,D. wardianum, andD. trigonopus) was zero, indicating minimal intragenomic diversity.D. williamsonii showed the most intragenomic variant patterns of the 18 study species, showing 52 distinct major variant patterns that ranged in prevalence from 25.82% to 1.1%. The GC content of the variants from these 18 species ranged from 47.35% to 56.00%. No obvious differences were observed in the length of major variants.Table 1
Intragenomic diversity analysis of ITS2 major variants from the 18Dendrobiumspecies.
Taxon
Number of total 454 sequences
Number of total variants
Variant name (>1% emergence frequency)
Percentage of each variant (>1%)
Length (bp)
G + C content (%)
Intragenomic sequence distances (>1% emergence frequency)
D. crepidatum
1919
4
p1-1
99.27
248
50.81
0
D. aphyllum
1888
19
p2-1
95.87
248
52.42
0
D. devonianum
722
8
p3-1
95.43
248
49.20
0.004
p3-2
1.80
247
48.58
D. officinale
2554
10
p4-1
93.89
246
50.81
0∗
p4-2
4.78
245
51.02
D. denneanum
1270
15
p5-1
91.50
248
51.21
0.016
p5-2
2.36
248
50.81
p5-3
1.42
248
50.00
p5-4
1.18
248
50.81
D. nobile
578
7
p6-1
63.15
246
53.66
0.017
p6-2
14.36
246
54.07
p6-3
10.73
246
54.07
p6-4
9.69
246
52.85
D. chrysanthum
860
30
p7-1
76.74
245
52.24
0.051
p7-2
3.14
245
51.84
p7-3
3.02
244
52.05
p7-4
2.56
245
47.35
p7-5
1.74
245
48.16
p7-6
1.63
245
47.76
p7-7
1.28
245
48.57
p7-8
1.28
245
50.20
p7-9
1.28
244
52.46
D. primulinum
3690
36
p8-1
75.93
248
53.63
0.005
p8-2
9.51
248
53.23
p8-3
9.11
248
53.23
D. chrysotoxum
2366
55
p9-1
63.82
249
55.42
0.081
p9-2
19.95
249
55.82
p9-3
2.79
249
55.02
p9-4
2.20
247
52.63
p9-5
2.20
249
55.42
p9-6
1.65
247
53.04
D. loddigesii
1593
47
p10-1
39.55
248
52.02
0.009
p10-2
30.70
248
51.21
p10-3
14.44
248
52.42
p10-4
5.34
248
51.21
D. acinaciforme
1112
30
p11-1
70.05
248
52.42
0.045
p11-2
16.55
248
52.42
p11-3
6.56
248
53.23
D. williamsonii
182
52
p12-1
25.82
247
52.63
0.05
p12-2
3.85
247
52.23
p12-3
2.75
247
50.20
p12-4
3.75
247
51.01
p12-5
2.20
247
51.01
p12-6
2.20
247
51.82
p12-7
2.20
247
50.20
p12-8
2.20
247
48.99
p12-9
2.20
247
50.61
p12-10~p12-21
19.80
247–250
48.99–51.42
p12-22~p12-52
34.10
244–247
48.37–52.63
D. capillipes
1610
33
p13-1
93.03
251
50.20
0.004
p13-2
1.43
251
49.80
D. crystallinum
1197
14
p14-1
51.96
250
55.60
0.005
p14-2
39.52
250
55.60
p14-3
5.93
250
55.20
D. gratiosissimum
1167
8
p15-1
93.23
246
52.85
0.004
p15-2
5.14
246
52.44
D. trigonopus
1906
27
p16-1
93.34
251
51.00
0
D. wardianum
2778
21
p17-1
84.38
250
56.00
0∗
p17-2
12.35
251
55.78
D. pendulum
2232
19
p18-1
49.73
250
54.00
0.005
p18-2
24.82
250
53.20
p18-3
20.79
249
53.41
p18-4
1.43
250
53.60
p18-5
1.25
250
53.60
T
h
e
∗ variations were caused by insert/deletion, which was treated as complete deletion when calculating the K2P distances. Thus, the K2P distances turned out to be zero.In this study, theD. officinale sample received a total of 2554 reads of 454 pyrosequencing representing ten different variant patterns. The most common major variant represented 93.89% of the ITS2 sequences in the entire genome. After alignment, the consensus sequence of the ten variants from theD. officinale genome was 246 bp in length, with 13 variable sites, including two INDELS. The dominant sequence patterns inD. officinale were consistent with the sequences obtained via direct PCR sequencing.


## 3.2. Analysis of the ITS2 Region at Intra- and Interspecific Levels

In total, 513 ITS2 sequences from 26 species ofDendrobium were analyzed for intraspecific genetic distances (IS-GDs). The average IS-GD for each species ranged from 0 to 0.032. The average IS-GD value in seven species (D. herbaceum,D. macrostachyum,D. amoenum,D. aqueum,D. bicameratum,D. barbatulum, andD. peguanum) was zero, and the highest average IS-GD value (0.032) was found inD. hancockii (Figure 1). The number of variable sites in ITS2 sequences of each species was also calculated (Table 2).D. officinale possesses a dominant variant representing 93.89% of the sequences, and out of 61 sequences there were only five intraspecific SNPs. A similar situation was observed in three other species (D. aphyllum,D. devonianum, andD. denneanum), all of which had one dominant ITS2 variant making up more than 90% of sequences. In contrast, there were more variable sites in species where the dominant ITS2 variant made up less than 80% of the sequences. For example, the dominant variant inD. loddigesiiaccounted for only 39.55% of sequences, and this species had 28 intraspecific variable sites. In general, as the dominant ITS2 variant became more common in the genome, there were fewer intraspecific variable sites in ITS2 sequences, with the exception ofD. crepidatum.Table 2
Comparison between the numbers of intraspecific variable sites with the emergency percentage of the dominant variants in genome of ITS2 sequences of each species.
Taxon
Number of total ITS2 sequences
Number of total intraspecific variable sites
The emergency percentage of the dominant variants in genome (%)
D. crepidatum
35
36
99.27
D. aphyllum
22
4
95.87
D. devonianum
11
9
95.43
D. officinale
61
5
93.89
D. denneanum
10
4
91.50
D. chrysanthum
25
22
76.74
D. primulinum
21
25
75.93
D. chrysotoxum
27
18
63.82
D. nobile
59
12
63.15
D. loddigesii
13
28
39.55Figure 1
Intraspecific genetic distances from 26Dendrobium species revealed by ITS2 region.The BLAST1 method, which is based on similarity, was used to assess the reliability of ITS2 sequences forDendrobium species identification. In total, 383 of the 513 ITS2 sequences were correctly identified. The unidentified ITS2 sequences were distributed among six species:D. officinale,D. tosaense,D. huoshanense,D. moniliforme,D. nobile, andD. hercoglossum. The ITS2 sequences ofD. officinale andD. tosaense could not be distinguished using BLAST, and similar issues arose forD. huoshanense,D. moniliforme,D. nobile, andD. hercoglossum.


## 3.3. The Neighbor-Joining Tree Based on ITS2 Sequences

A neighbor-joining tree (NJ tree) was built based on the intragenomic data to determine the phylogenetic relationships between theDendrobium species. Previous studies have shown that minor variants present below 1% are difficult to detect directly with PCR or clone sequencing. Thus, we first selected the major variants for analysis (Figure 2). The results showed thatD. williamsonii (PS2503),D. trigonopus (PS2506),D. acinaciforme (PS2527), andD. capillipes (PS2502) clustered into one clade, with all other species forming a separate clade. Almost all the major variants clustered together, with the exception ofD. nobile. One of the major variants ofD. nobile (PS0766_4) introgressed intoD. officinale andD. gratiosissimum, showing a very close relationship. Another NJ tree using total intragenomic data was also constructed and is shown in Appendix S3.Figure 2
The neighbor-joining tree built using the major variants of ITS2 from 18Dendrobium species.Note. Each species was marked with a unique color. The three parts of the name of each branch represent the voucher number, the variant name of the multiple copy in the genome, and the percentage of its presence in the genome. The taxa with their voucher numbers were listed for the following:D. williamsonii(PS2503),D. trigonopus(PS2506),D. acinaciforme(PS2527),D. capillipes(PS2502),D. loddigesii(PS1748),D. crystallinum(PS2519),D. denneanum(PS0758),D. chrysotoxum(PS2501),D. officinale(PS2521),D. gratiosissimum(PS2513),D. nobile(PS0766),D. wardianum(PS2509),D. pendulum(PS2511),D. aphyllum(PS2523),D. primulinum(PS2518),D. devonianum(PS2520),D. chrysanthum(PS2515),and D. crepidatum(PS2517).Total intraspecific data were also used to construct an NJ tree for phylogenetic analysis (see Appendix S4). The results showed that most species were monophyletic except for one clade including six species (D. officinale,D. tosaense,D. huoshanense,D. moniliforme,D. nobile, andD. hercoglossum). To better clarify the relationship among these species in the main clade, these six species were used to build a separate NJ tree based on their ITS2 sequences (Figure 3). We divided this NJ tree into two major clades (Clades I and II). Clade I consists of two species,D. officinale andD. tosaense, with a bootstrap support value of 100%. Clade II consists of two subclades, with a bootstrap support value of 54%. Subclades II-I contain two species,D. moniliforme andD. huoshanense, with a bootstrap support value of 72%, and subclades II-II contain three species,D. moniliforme,D. hercoglossum, andD. nobile, with a bootstrap support value of 62%.Figure 3
The neighbor-joining tree built using ITS2 sequences of the six species.Note. Different colors in Figure 3 represent different species. Blue,D. officinale; red,D. huoshanense; green,D. nobile; purple, D. moniliforme; orange,D. tosaense; yellow,D. hercoglossum.


## 4. Discussion

Ribosomal DNA (rDNA) is present in multiple copies of tandem repeats per genome [31], and two noncoding spacers (internal transcribed spacer 1 and 2) divide each transcriptional unit into three subunits: 18S, 5.8S and 28S. Each tandem can contain variations, thus leading to intragenomic variation. Many studies have addressed genomic divergence inDendrobium, but most of these have been focused on intra- and interspecific levels of variation [32–36]. It is thought that biodiversity at the species level is generally overestimated due to intragenomic variation [37]. In this study we therefore focused on the intragenomic level, aiming to identify relationships between intragenomic diversity and intraspecific diversity. Sequence-based methods have replaced many traditional approaches such as allozyme or restriction enzyme polymorphisms, which is valid as long as appropriate marker(s) is selected [38]. Traditional approaches (e.g., RAPD, AP-PCR, and AFLP) generally require high-quality DNA for amplification, which can lead to problems with reproducibility and accuracy. Sequence-based methods should be more objective and stable, enhancing our ability to assess biodiversity and identify species [39]. In addition, experimental error and subjective factors such as scoring PCR bands on a gel are eliminated or minimized in sequence-based protocols.The ITS2 locus has already been proposed as a universal DNA barcode, particularly in plants, and it has been shown that plants can be identified at the species and genus level with more than 97% accuracy [27, 40]. Although the China plant BOL group suggested ITS as the core barcode for seed plants, ITS2 has several advantages compared with the full-length ITS region [41]. First, ITS2 is shorter than ITS, which simplifies PCR amplification. Moreover, ITS2 has secondary structure in all eukaryotes [42, 43]. This molecular morphological characteristic strengthens the power of its discriminatory ability. In addition to species identification applications, ITS2 and its secondary structure have been used as effective tools for phylogenetic analyses in insects, corals, and yeast [44–47]. As these transcribed spacers are highly divergent, they can also be used to estimate low levels of genetic diversity among related species [48]. Liu et al. evaluated the resolution of five regions (rbcL,matK, ITS, ITS2, andtrnH-psbA), ultimately suggesting anrbcL + ITS2 barcode combination as the most suitable marker for analyzing biodiversity in the Dinghushan National Nature Reserve (DNNR) in China [49].Among all theDendrobium species,D. officinale is undoubtedly the most valuable, owing to its low production but high price and clinical efficacy in the clinic. Previous studies using ISSR, RAPD, and SRAP revealed distinct genetic differences and extensive genetic diversity among different populations ofD. officinale[34, 50, 51]. However, the intraspecific genetic diversity ofD. officinale (intraspecific genetic distance, average: 0.001; Max: 0.013) as revealed by ITS2 sequences turned out to be relatively low compared with results from other approaches. From the 61 ITS2 sequences obtained fromD. officinale, only five variable sites were detected after alignment. Across the whole genome,D. officinale has a single dominant variant that represents 93.89% of ITS2 sequences. These results indicate that the ITS2 regions are relatively conserved among different populations ofD. officinale. Due to the low production and high price ofD. officinale, there are so many closely related species appearing as adulterants in the herbal market. These adulterants are species that have morphological characteristics similar to each other, making traditional taxonomic identification difficult, particularly after processing into medicinal slices. According to this result above, ITS2 can be an effective molecules tool for identifying commercialD. officinale and otherDendrobiumspecies.In the Chinese Pharmacopoeia (2015 edition),D. officinale is described as an independent species that is the source of the herbal medicine “Tie Pi Shi Hu.” However, this species has already been accepted as a synonym ofD. catenatum,D. tosaense, and several others in flora of China and the other research [52]. In our study, ITS2 sequences from these two species were grouped into a single clade with 100% bootstrap support. The NJ tree described here demonstrates that, at the very least,D. officinale andD. tosaense are extremely closely related at the genetic level, consistent with other results from China. Therefore, we agree thatD. officinale andD. tosaense should be accepted as synonyms ofD. catenatum. In a previous study, a phylogenetic tree including twelve samples ofDendrobium species was constructed [50]. The three speciesD. moniliforme,D. hercoglossum, andD. nobile were grouped in the same clade, similar to classifications based on inflorescence color and the results from this study.


## 5. Conclusion

In this study, we analyzed intragenome and intraspecies divergence to find that, in most cases, greater intragenomic divergence is correlated with larger intraspecific variation. The results of this study strongly confirm that the direct PCR sequencing data were credible because all the dominant sequences in high-throughput sequencing in each species were detected by direct PCR. Thus, the multiple copies in ITS2 did not affect the species identification inDendrobium. Therefore, we demonstrate that ITS2 is an effective tool forDendrobium species identification.



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*Source: 2734960-2017-10-17.xml*