DNA条形码在热带龙脑香科树种鉴定中的应用

doi:10.11983/CBB18205

摘要/Abstract

摘要： 龙脑香科植物是东南亚地区重要的热带木材来源树种, 对其开展DNA条形码评估在林业监管及森林资源保护等方面具有非常重要的实际应用价值。通过对龙脑香科植物样品进行rbcL、matK、trnL-trnF和ITS2四个片段的扩增和测序, 结合GenBank下载的数据, 共获得龙脑香科树种14属244种共计899条序列。通过比较4个片段的通用性、序列特征、种内和种间的遗传变异, 基于Best Match (BM)、Best Close Match (BCM)、相似性搜索算法(BLAST)和邻接树(NJ) 4种方法评估DNA条形码对于龙脑香科树种的鉴定能力。结果表明, ITS2在龙脑香科树种中鉴定效率最高, 通过优化的扩增体系能够从该科植物叶片中获得较高质量的ITS2片段; 叶绿体matK片段扩增和测序效率为100%, 且种内及种间遗传变异明显, 鉴定成功率高于其它叶绿体片段, 并据此提出ITS2和matK适合作为龙脑香科树种的DNA条形码。

关键词: 龙脑香科, DNA条形码, ITS2, matK, 热带地区

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

胡建霖,刘志芳,慈秀芹,李捷. DNA条形码在热带龙脑香科树种鉴定中的应用. 植物学报, 2019, 54(3): 350-359.

Jianlin Hu,Zhifang Liu,Xiuqin Ci,Jie Li. Use of DNA Barcoding in Identifying Tropical Trees from Dipterocarpaceae. Chinese Bulletin of Botany, 2019, 54(3): 350-359.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18205

https://www.chinbullbotany.com/CN/Y2019/V54/I3/350

图/表 8

参考文献 55

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No.	Species	Voucher numbers	GenBank accession number
No.	Species	Voucher numbers	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

No.	Species	Voucher numbers	GenBank accession number
No.	Species	Voucher numbers	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

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