Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (3): 350-359.doi: 10.11983/CBB18205

• EXPERIMENTAL COMMUNICATIONS • Previous Articles     Next Articles

Use of DNA Barcoding in Identifying Tropical Trees from Dipterocarpaceae

Hu Jianlin1,2,Liu Zhifang1,2,Ci Xiuqin1,Li Jie1,*   

  1. 1. Laboratory of Plant Phylogenetics and Conservation, Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223, China
    2. University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2018-09-27 Accepted:2019-02-11 Online:2019-11-24 Published:2019-05-01
  • Contact: Li Jie

Abstract:

Plants from Dipterocarpaceae are vital tropical timber trees in Southeast Asia. They have high potential in forest supervision, forest resource protection and other aspects to evaluate the efficiency of DNA barcodes. We obtained 899 sequences from 244 species for 14 genera combined with sequences from the GenBank. We amplified and sequenced four DNA regions (rbcL, matK, trnL-trnF and ITS2) in samples from the family Dipterocarpaceae. The discrimination ability of DNA barcodes was evaluated by using effective sequence ratios, characteristics of sequences, divergence of intra- and inter-varieties, and identification success rates (Best Match, Best Close Match, BLAST and neighbor-joining methods). The ITS2 fragment showed the highest identification efficiency. High-quality ITS2 sequence can be obtained from the leaves of Dipterocarpaceae plants by using an optimized amplification system. The matK fragment showed obvious genetic variation and 100% amplification and sequencing efficiency, and its identification ability was higher than with other chloroplast markers. Therefore, ITS2 and matK are suitable markers for tropical trees from Dipterocarpaceae.

Key words: Dipterocarpaceae, DNA barcoding, ITS2, matK, tropical region

Table 1

Information of experimental materials"

No. Species Voucher numbers GenBank accession number
rbcL matK trnL-trnF ITS2
1 Dipterocarpus turbinatus C. F. Gaertner JL5901 MK030561 MK051080 MK051054 MK051069
2 D. turbinatus C. F. Gaertner JL5902 MK030562 MK051081 MK051055 MK051070
3 D. retusus Blume JL9601 MK030559 MK051078 MK051052 MK193032
4 D. retusus Blume JL9602 MK030560 MK051079 MK051053 MK193031
5 Hopea chinensis (Merr.) Hand.-Mazz. JL110A MK030563 MK051082 MK193024 MK193026
6 H. chinensis (Merr.) Hand.-Mazz. JL110B MK030564 MK051083 MK051059 MK193025
7 H. hainanensis Merrill & Chun JL2201 MK030565 MK051084 MK051060 MK193029
8 H. hainanensis Merrill & Chun JL2202 MK030566 MK051085 MK051061 MK193028
9 H. hainanensis Merrill & Chun JL2203 MK030567 MK051086 MK051062 MK193030
10 Parashorea chinensis H. Wang JL6101 MK030568 MK051087 MK051056 MK051071
11 P. chinensis H. Wang JL6102 MK030569 MK051088 MK051057 MK051072
12 Shorea assamica Dyer JL3901 MK030571 MK051090 MK051063 -
13 S. assamica Dyer JL3902 MK030570 MK051089 MK051058 MK051073
14 Vatica diospyroides Symington JL7601 MK030572 MK051091 MK051064 MK051074
15 V. diospyroides Symington JL7602 MK030573 MK051092 MK193023 MK051075
16 V. guangxiensis X. L. Mo JL4801 MK030574 MK051093 MK051065 MK051076
17 V. guangxiensis X. L. Mo JL4802 MK030575 MK051094 MK051066 MK051077
18 V. mangachapoi Blanco JL111A MK030576 MK051095 MK051067 -
19 V. mangachapoi Blanco JL111B MK030577 MK051096 MK051068 MK193027

Table 2

Primer pairs used for PCR amplification and amplification protocol"

Barcodes Primers sequence (5'-3') Amplification protocol References
rbcL


1F: ATGTCACCACAAACAGAGACTAAAGC
724R: TCGCATGTACCTGCAGTAGC

94°C 4 min; 94°C 30 s, 55°C 45 s, 72°C 1 min, 5 cycles; 94°C 30 s, 54°C 45 s, 72°C 10 min, 30 cycles; 72°C 10 min Fay et al., 1997 Olmstead et al., 1992
matK
3F: CGTACAGTACTTTTGTGTTTACGA
1R: ACCCAGTCCATCTGGAAATCTTGG
94°C 4 min; 94°C 30 s, 51°C 50 s, 72°C 50 s, 35 cycles; 72°C 10 min Unpublished
390F: CGATCTATTCATTCAATATTTC
1326R: TCTAGCACACGAAAGAAGT
94°C 4 min; 94°C 30 s, 50°C 50 s, 72°C 50 s, 35 cycles; 72°C 10 min Cuénoud et al., 2002
trnL-trnF
F: GGTTCAAGTCCCTCTATCCC
R: ATTTGAACTGGTGACACGAG
94°C 4 min; 94°C 30 s, 55°C 1 min,
72°C 1 min, 35 cycles; 72°C 5 min
Taberlet et al.,1991
ITS2
ITS2 2F: ATGCGATACTTGGTGTGAAT
ITS2 3R: GACGCTTCTCCAGACTACAAT
94°C 4 min; 94°C 30 s, 55°C 45 s, 72°C 45 s, 35 cycles; 72°C 10 min Chen et al., 2010
Chen et al., 2010
ITS3: GCATCGATGAAGAACGCAGC
26SE: TAGAATTCCCCGGTTCGCTCGCCGTTAC
94°C 4 min; 94°C 30 s, 57°C 45 s, 72°C 45 s, 35 cycles; 72°C 10 min White et al., 1990; Sun et al., 1994

Table 3

The success rate of PCR amplification and sequencing"

Barcodes Amplification efficiency (%) Success rate of sequencing (%) Effective sequence ratio (%)
rbcL 100.0 100.0 100.0
matK 100.0 100.0 100.0
trnL-trnF 100.0 100.0 100.0
ITS2 100.0 89.5 89.5

Table 4

Information of sequences characteristic"

Barcodes No. of sequences Aligned length (bp) GC content (%) No. of
variable sites
Intraspecific distance (mean) Interspecific distance (mean)
rbcL 259 684 46.0 23 0.0006 0.0161
matK 207 969 35.8 27 0.0032 0.0273
trnL-trnF 365 454 27.2 46 0.0041 0.0273
ITS2 68 473 72.5 51 0.0017 0.1311

Table 5

Wilcoxon signed rank tests of intraspecific divergence among chloroplast markers in the species of Dipterocarpaceae"

W+ W- Intra relative ranks N P value Result
trnL-trnF rbcL W+=1258, W-=120 96 2.20E-07 P<0.05, trnL-trnF>rbcL
trnL-trnF matK W+=298, W-=297 43 9.93E-01 P>0.05, matK=trnL-trnF
matK rbcL W+=408, W-=57 44 9.18E-04 P<0.05, matK>rbcL

Table 6

Wilcoxon signed rank tests of interspecific divergence among chloroplast markers in the species of Dipterocarpaceae"

W+ W- Inter relative ranks N P value Result
trnL-trnF rbcL W+=8136415.5, W-=1811614.5 4560 3.78E-296 P<0.05, trnL-trnF>rbcL
trnL-trnF matK W+=236579.5, W-=156361.5 903 1.41E-07 P<0.05, trnL-trnF>matK
matK rbcL W+=411099, W-=36832 946 1.00E-13 P<0.05, matK>rbcL

Figure 1

Distribution for intra and inter-specific variation of markers in the species of Dipterocarpaceae(A) rbcL sequence; (B) matK sequence; (C) trnL-trnF sequence"

Figure 2

Comparison of the identification efficiency of barcodes base on four methods"

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