Chinese Bulletin of Botany ›› 2019, Vol. 54 ›› Issue (3): 350-359.DOI: 10.11983/CBB18205
• EXPERIMENTAL COMMUNICATIONS • Previous Articles Next Articles
Jianlin Hu1,2,Zhifang Liu1,2,Xiuqin Ci1,Jie Li1,*
Received:
2018-09-27
Accepted:
2019-02-11
Online:
2019-07-01
Published:
2019-11-24
Contact:
Jie Li
Jianlin Hu,Zhifang Liu,Xiuqin Ci,Jie Li. Use of DNA Barcoding in Identifying Tropical Trees from Dipterocarpaceae[J]. Chinese Bulletin of Botany, 2019, 54(3): 350-359.
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 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 |
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 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 |
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 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 |
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 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 |
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 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 |
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 |
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
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