DNA concentration, sequence analysis and species identification. We anticipated extracting DNA directly from crushed benzoin (resin) samples after sampling in this investigation, but the extracted DNA concentration was insufficient for subsequent tests (results not shown). Thus, we investigated scraping bark-like residues off the benzoin resin with a knife and treating each residue as a separate sample with DNA barcode identification, which has the advantage of preventing sequencing failure in subsequent trials due to sample mixing (Fig. 1).
In total, there were 27 batches of benzoin samples containing 40 individual bark-like residues in this study. The DNA concentrations obtained from these residues ranged from 8.0-203.8 ng/μL, and the DNA purity ranged from 1.70-2.08. Although the AX151 sample had the lowest DNA concentration of 8.0 ng/μL, it had a purity of 1.89 and was able to be successfully amplified by PCR (Table 1). Therefore, treating the samples with 95% ethanol avoided the effect of resin on sample DNA extraction and amplification. In addition, considering that the samples originated from bark-like residues in benzoin resin, whose DNA was degraded despite the DNA concentration and purity satisfying the subsequent experiments, we chose the nuclear gene ITS2 universal barcode as primers for amplification, and all the sample DNA was successfully amplified and sequenced. The complete ITS2 was obtained by removing the 5.8S and 26S regions from the sequences obtained by sequencing, which had a length of 217-230 bp.
Table 1. DNA concentration and purity of samples, and ITS2 length and GC content after sequencing.
Sample No.
|
DNA concentration (ng/μL)
|
A260
|
A280
|
DNA purity
(A260/A280)
|
ITS2 Length (bp)
|
GC content (%)
|
AX011
|
16.4
|
0.329
|
0.166
|
1.98
|
224
|
55.80
|
AX012
|
25.6
|
0.514
|
0.260
|
1.98
|
224
|
55.80
|
AX013
|
101.7
|
2.034
|
1.038
|
1.96
|
224
|
55.61
|
AX014
|
73.5
|
1.470
|
0.764
|
1.92
|
225
|
56.00
|
AX021
|
39.7
|
0.796
|
0.393
|
2.03
|
224
|
60.71
|
AX031
|
52.4
|
1.047
|
0.531
|
1.97
|
224
|
60.27
|
AX041
|
62.8
|
1.256
|
0.664
|
1.89
|
224
|
60.27
|
AX051
|
135.2
|
2.703
|
1.377
|
1.96
|
226
|
59.73
|
AX061
|
140.8
|
2.816
|
1.407
|
2.00
|
225
|
60.71
|
AX062
|
51.7
|
1.034
|
0.530
|
1.95
|
224
|
60.71
|
AX071
|
41.4
|
0.829
|
0.459
|
1.81
|
230
|
57.39
|
AX072
|
51.3
|
1.027
|
0.526
|
1.95
|
224
|
55.80
|
AX081
|
53.1
|
1.061
|
0.550
|
1.93
|
224
|
55.80
|
AX091
|
52.0
|
1.042
|
0.500
|
2.08
|
224
|
55.36
|
AX092
|
61.5
|
1.230
|
0.597
|
2.06
|
224
|
55.80
|
AX093
|
33.6
|
0.672
|
0.365
|
1.84
|
224
|
55.80
|
AX101
|
15.4
|
0.308
|
0.154
|
2.00
|
224
|
55.80
|
AX102
|
18.3
|
0.037
|
0.020
|
1.85
|
217
|
64.98
|
AX111
|
75.4
|
1.509
|
0.770
|
1.96
|
224
|
60.71
|
AX121
|
68.9
|
1.380
|
0.723
|
1.91
|
224
|
60.71
|
AX122
|
48.3
|
0.965
|
0.472
|
2.04
|
230
|
51.74
|
AX131
|
13.1
|
0.261
|
0.142
|
1.84
|
224
|
55.36
|
AX132
|
87.6
|
1.752
|
0.927
|
1.89
|
224
|
55.80
|
AX141
|
99.0
|
1.981
|
1.075
|
1.84
|
224
|
55.80
|
AX151
|
8.0
|
0.160
|
0.082
|
1.95
|
230
|
57.39
|
AX161
|
53.1
|
1.062
|
0.593
|
1.79
|
224
|
60.71
|
AX171
|
31.5
|
0.631
|
0.317
|
1.99
|
217
|
56.22
|
AX172
|
12.2
|
0.246
|
0.133
|
1.85
|
222
|
58.56
|
AX173
|
19.6
|
0.393
|
0.205
|
1.92
|
224
|
55.36
|
AX174
|
17.1
|
0.342
|
0.187
|
1.83
|
217
|
65.90
|
AX181
|
38.4
|
0.768
|
0.411
|
1.87
|
224
|
60.71
|
AX191
|
95.6
|
1.912
|
0.998
|
1.92
|
224
|
60.71
|
AX201
|
31.6
|
0.633
|
0.348
|
1.82
|
222
|
58.56
|
AX211
|
53.5
|
0.654
|
0.347
|
1.88
|
224
|
60.71
|
AX221
|
52.8
|
1.056
|
0.557
|
1.90
|
224
|
61.16
|
AX231
|
82.7
|
1.655
|
0.877
|
1.89
|
218
|
68.35
|
AX241
|
80.3
|
1.607
|
0.828
|
1.94
|
224
|
60.27
|
AX251
|
43.0
|
0.859
|
0.506
|
1.70
|
217
|
64.98
|
AX261
|
57.8
|
1.156
|
0.630
|
1.83
|
224
|
55.8
|
AX271
|
203.8
|
4.075
|
2.157
|
1.89
|
224
|
60.71
|
Forty individual samples were found by BLAST comparison with GenBank data from species of the genera Styrax Linn., Dimocarpus Lour., Aquilaria Lam., Ageratum L., Glycine Willd., Acronychia J. R. Forst. & G. Forst., Trema Lour., and Musa L., with a BLAST species identification rate of over 92% (Table 2). The BLAST and secondary structure analysis showed that 30 samples were derived from the genus Styrax Linn., of which 16 were identified as S. tonkinensis and 14 were identified as S. japonicus. In addition, 10 samples were derived from other genera. The BLAST and secondary analysis results of sample AX174 were different, but both indicated that the sample belongs to species of Trema Lour. Samples AX071 and AX151 were identified as Aquilaria sinensis (Lour.) Spreng., AX102 and AX215 as Dimocarpus longan Lour., AX172 and AX201 as Acronychia pedunculata (L.) Miq., and AX171 as Glycine soja Siebold & Zucc. and Glycine max (Lour.) Merr. AX122 and AX231 were identified as Ageratum conyzoides L. and Musa acuminata Colla, respectively. These species are mainly found in tropical and subtropical regions, consistent with benzoin production areas. Benzoin is resinous, and it is inferred that the tissue of these plants may have been mixed with the harvested benzoin.
Table 2. Results of BLAST comparison and secondary structure analysis of ITS2 sequences of samples.
Sample No.
|
BLAST results
|
Secondary structures results
|
Styrax Linn.
|
Other species
|
Per. Ident (%)
|
E value
|
Styrax Linn.
|
Other species
|
AX011
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX012
|
S. japonicus
S. tonkinensis
|
|
99.55%
|
1e-107
|
S. tonkinensis
|
|
AX013
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX014
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX021
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX031
|
S. tonkinensis
|
|
94.20%
|
1e-93
|
S. tonkinensis
|
|
AX041
|
S. tonkinensis
|
|
96.88%
|
3e-100
|
S. tonkinensis
|
|
AX051
|
S. tonkinensis
|
|
92.40%
|
6e-82
|
S. tonkinensis
|
|
AX061
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX062
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX071
|
|
Aquilaria sinensis (Lour.) Spreng.
(Aquilaria Lam.)
|
99.13%
|
2e-112
|
|
A. sinensis
|
AX072
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX081
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX091
|
S. japonicus
|
|
99.11%
|
4e-108
|
S. japonicus
|
|
AX092
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX093
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX101
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX102
|
|
Dimocarpus longan Lour.
(Dimocarpus Lour.)
|
|
|
|
D. longan
|
AX111
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX121
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX122
|
|
Ageratum conyzoides L.
(Ageratum L.)
|
99.13%
|
2e-112
|
|
A. conyzoides
|
AX131
|
S. japonicus
|
|
98.66%
|
5e-107
|
S. japonicus
|
|
AX132
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX141
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX151
|
|
A. sinensis
|
100%
|
4e-113
|
|
A. sinensis
|
AX161
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX171
|
|
Glycine soja Siebold & Zucc.
(Glycine Willd.)
|
97.70%
|
9e-100
|
|
G. soja
|
Glycine max (L.) Merr.
(Glycine Willd.)
|
97.70%
|
9e-100
|
|
G. max
|
AX172
|
|
Acronychia pedunculata (L.) Miq.
(Glycine Willd.)
|
99.55%
|
1e-108
|
|
A. pedunculata
|
AX173
|
S. japonicus
|
|
99.11%
|
1e-108
|
S. japonicus
|
|
AX174
|
|
Trema cannabinum var. dielsianum (Hand.Mazz.) C. J. Chen
(Trema Lour.)
|
100%
|
1e-107
|
|
Trema micranthum (L.) Blume
|
Trema nitidum C. J. Chen
(Trema Lour.)
|
100%
|
1e-107
|
|
Trema orientale (L.) Blume
(Trema Lour.)
|
100%
|
1e-107
|
|
Trema tomentosa (Roxb.) H.Hara
(Trema Lour.)
|
100%
|
1e-107
|
|
AX181
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX191
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX201
|
|
Acronychia pedunculata (L.) Miq.
(Acronychia J. R. Forst. & G. Forst.)
|
99.10%
|
1e-107
|
|
A. pedunculata
|
AX211
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
AX221
|
S. tonkinensis
|
|
96.43%
|
3e-99
|
S. tonkinensis
|
|
AX231
|
|
Musa acuminata Colla
(Musa L.)
|
96.33%
|
1e-97
|
|
M. acuminata
|
AX241
|
S. tonkinensis
|
|
97.32%
|
2e-101
|
S. tonkinensis
|
|
AX251
|
|
D. longan
|
100%
|
1e-107
|
|
D. longan
|
AX261
|
S. japonicus
|
|
99.55%
|
9e-110
|
S. japonicus
|
|
AX271
|
S. tonkinensis
|
|
96.88%
|
9e-100
|
S. tonkinensis
|
|
We used the 14 samples identified by BLAST as S. japonicus as group 1 and the 16 samples identified as S. tonkinensis as group 2. Twenty base variable loci were identified by ITS2 sequence alignment with S. japonicus and S. tonkinensis species in GenBank (Table 3). Base sites 13, 24, 46, 75, 78, 102, 104, 126, and 219 can be used as specific identification sites for these two species. However, it was also found that there were different base sites in the collected samples from these two species, such as G at base site 7 and C at base site 189 in group 2, while S. japonicus and S. tonkinensis species downloaded from GenBank had A and T for base sites, respectively. In addition, there are two base sites in group 2 that are identical to those of S. japonicus (base sites 32 and 209), and there are also two base variable sites (base sites 60 and 185).
Table 3. BLAST comparison of base difference sites of ITS2 sequences from Styrax Linn. samples with S. japonicus and S. tonkinensis. 1) S. japonicus has the following accession numbers in Genbank: AB114900, LC534305, LC600960, and MF349070; 2) S. tonkinensis has the following accession numbers in Genbank: JF421547, MF096782, MF096783, MF096784, MF096785, and MF096786. Grpoup1 is the 14 samples identified as S. japonicus and Grpoup2 is the 16 samples identified as S. tonkinensis. Bolded black indicates differential loci for S. japonicus and S. tonkinensis.
Species
|
Base site
|
7
|
13
|
24
|
32
|
33
|
46
|
60
|
75
|
77
|
78
|
84
|
102
|
S. japonicus1)
|
A
|
T
|
A
|
T
|
T
|
T
|
A
|
T
|
T
|
A
|
T
|
A
|
Group1
|
A
|
T
|
A
|
T
|
T
|
T
|
A
|
T
|
T
|
A
|
C
|
A
|
S. tonkinensis2)
|
A
|
C
|
C
|
C
|
C/T
|
C
|
A
|
C
|
T/A/C
|
G
|
C
|
G/A
|
Group2
|
G
|
C
|
C
|
T
|
T
|
C
|
G/A
|
C
|
C
|
G
|
C
|
G
|
Species
|
104
|
105
|
106
|
109
|
126
|
185
|
113
|
189
|
200
|
209
|
219
|
|
S. japonicus1)
|
G
|
C
|
A
|
T
|
G
|
T
|
A
|
T
|
T
|
A
|
T
|
|
Group1
|
G
|
C
|
A
|
T
|
G
|
T
|
A
|
T
|
T
|
A
|
T
|
|
S. tonkinensis2)
|
A
|
T/C
|
G/A
|
T/C
|
A
|
G
|
A/G
|
T/C
|
T/C
|
G/A
|
C
|
|
Group2
|
A
|
C
|
A
|
C
|
A
|
G/C
|
A
|
C
|
C
|
A
|
C
|
|
ITS2 secondary structure analysis
The ITS2 database was used for homology prediction analysis of the secondary structures of 48 major species of the genus Styrax Linn. The ITS2 secondary structures of all species were folded into the typical structure of a central loop with four helices (I, II, III, and IV), with helix III being the longest, followed by helices I, II, and IV. Helix II was the most conserved, with the central main loop relatively conserved and rich in purine bases46-48. The ITS2 secondary structure predictions of 40 individual samples are shown in Table 4. By homology prediction, 30 samples were derived from species of the genus Styrax Linn., 16 of which were identified as S. tonkinensis and 14 as S. japonicus. In addition, 10 samples were derived from species of other genera. The ITS2 secondary structures of the two species identified as S. tonkinensis and S. japonicus in the benzoin samples are shown in Fig. 2A. The helix positions and angles of the secondary structures of these two species are completely different, with helices I and III being more variable due to multiple unpaired bases and positional differences. In contrast, the shapes of helices II and IV, although basically the same, also have unpaired bases, leading to differences. S. tonkinensis has three slightly different secondary structures, as shown in Fig. 2A a), b), and c). The red dashed boxes are the base pairs between the base of helix III and the first unpaired loop on the helix arm of S. tonkinensis a) and S. tonkinensis b), and these differences are due to differences in the position of helix III. S. tonkinensis has three ITS2 secondary structures with different base sites, such as bases A, C, and T at position 32 of helix I (black arrows). In addition, there are hCBCs in helix III (109/179: C-G→T-G, red arrows). We compared the DNA sequences of the two sets of basal samples obtained by BLAST identification. The ITS2 secondary structure sequence of group 1 was identical to that of S. japonicus, with the presence of hCBCs (73/84: G-C→G-T) only on helix II (Table 4). The results of the predicted secondary structure of ITS2 for group 2 homologs were the same as those of S. tonkinensis, but the differences in the position of helix III were clear, and there were also variations in base sites. For example, sample AX051 differed from the other 15 samples by the secondary structure at base 60 on the main loop (A) and the presence of hCBCs on helix III (103-185: C-C→C-G), but the rest of the sites were identical. Compared with S. tonkinensis a) and S. tonkinensis b), the bases at sites 7 and 60 of the main loop of group 2 are G, the bases at site 32 of the top asymmetric loop of helix I are T, the bases at site 77 of the top asymmetric loop of helix II are C, while the base 100/188 of helix III lacks the A-T base pair and has hCBCs (103-185: C-C→C-G), and base 209 of the top asymmetric loop of helix IV is A. The samples compared to S. tonkinensis c) group 2 have G bases at sites 7 and 60 of the main loop; T-T bases at sites 32-33 of the top asymmetric loop of helix I; different base pairs at 100-106/182-188 of helix III; and the presence of hCBCs on helix IV (200-215: T-G→C-G), as shown in Table 4. Therefore, the secondary structures of ITS2 of S. tonkinensis and S. japonicus differ significantly, and the species can be further precisely identified based on this information. In addition, the ITS2 secondary structures of the remaining 46 species of the genus Styrax Linn. are shown in Figure 2B. The size, position, and angle of the four helices and the bases on the helix vary among the 38 species, except for the four species pairs of S. calvescens and S. formosanus, S. formosanus and S. dasyanthus, S. formosanus and S. confusus, and S. confusus and S. hemsleyanus. The primary sequences in these four groups of species are identical to each other. We tried to analyze the differences in their secondary structures by homologous folding, but unfortunately, the ITS2 secondary structures of the four groups of species were also identical.
Table 4. Comparison of base differences on the helices of S. tonkinensis and S. japonicus with the samples. Grpoup1 was the 14 samples identified as S. japonicus, and Grpoup2 was the 16 samples identified as S. tonkinensis by BLAST. -: represents no differences in base sequences.
Species
|
Helix I
|
Helix II
|
Helix III
|
Helix IV
|
Main loop
|
S. japonicus
|
-
|
hCBCs (73/84: G-C→G-T)
|
-
|
-
|
-
|
Group1
|
S. tonkinensis a)
|
32: T→C
|
77: C→A;
C→T
|
Position difference;
100/188:-→A-T
hCBCs (103-185: C-C→C-G)
|
209: A→G
|
7: G→A;
60: G→A
|
S. tonkinensis b)
|
Group2
|
S. tonkinensis c)
|
32/33:T-T→C-C
|
-
|
Position difference;
100-106/182-188: Base pairs difference
|
hCBCs (200-215: T-G→C-G)
|
7: G→A;
60: G→A
|
Group2
|
The species of the 27 batches of benzoin samples collected were inferred from a comprehensive BLAST analysis and by analysis of the ITS2 secondary structures of 40 individual samples, as shown in Table 5. The AX01 sample contained two species of the genus Styrax Linn., S. japonicus and S. tonkinensis. No species of the genus Styrax Linn were detected in the four batches of samples (AX15, AX20, AX23, and AX25), and only other plant species were detected, but this does not mean that they were not benzoin. The contamination of commercially available benzoin samples was prominent, accounting for 29.6% of the samples, which is related to the harvesting and transportation processes at the site of origin.
Table 5. Comprehensive determination of species in 27 batches of benzoin samples. Y represents Styrax Linn. species, N represents other genus species, and - represents Styrax Linn. species not detected.
Batch No.
|
Included species
|
Judgment results
|
AX01
|
S. Japonicus; S. tonkinensis
|
Y
|
AX02
|
S. tonkinensis
|
Y
|
AX03
|
S. tonkinensis
|
Y
|
AX04
|
S. tonkinensis
|
Y
|
AX05
|
S. tonkinensis
|
Y
|
AX06
|
S. tonkinensis
|
Y
|
AX07
|
S. japonicus; A. sinensis
|
Y/N
|
AX08
|
S. japonicus
|
Y
|
AX09
|
S. japonicus
|
Y
|
AX10
|
S. japonicus; D. longan
|
Y/N
|
AX11
|
S. tonkinensis
|
Y
|
AX12
|
S. tonkinensis; A. conyzoides
|
Y/N
|
AX13
|
S. japonicus
|
Y
|
AX14
|
S. japonicus
|
Y
|
AX15
|
A. sinensis
|
-/N
|
AX16
|
S. tonkinensis
|
Y
|
AX17
|
S. japonicus; G. soja ; G. max; A. pedunculata; Trema Lour.
|
Y/N
|
AX18
|
S. tonkinensis
|
Y
|
AX19
|
S. tonkinensis
|
Y
|
AX20
|
A. pedunculata
|
-/N
|
AX21
|
S. tonkinensis
|
Y
|
AX22
|
S. tonkinensis
|
Y
|
AX23
|
M. acuminata
|
-/N
|
AX24
|
S. tonkinensis
|
Y
|
AX25
|
D. longan
|
-/N
|
AX26
|
S. japonicus
|
Y
|
ZX27
|
S. tonkinensis
|
Y
|