植物学报 ›› 2019, Vol. 54 ›› Issue (3): 350-359.doi: 10.11983/CBB18205

• 研究报告 • 上一篇    下一篇

DNA条形码在热带龙脑香科树种鉴定中的应用

胡建霖1,2,刘志芳1,2,慈秀芹1,李捷1,*   

  1. 1. 中国科学院西双版纳热带植物园综合保护中心, 植物系统发育与保护生物学实验室, 昆明 650223
    2. 中国科学院大学, 北京 100049
  • 收稿日期:2018-09-27 接受日期:2019-02-11 出版日期:2019-05-01 发布日期:2019-11-24
  • 通讯作者: 李捷
  • 基金资助:
    中国科学院战略生物资源服务网络计划生物多样性保护策略(ZSSD-013和中国科学院东南亚生物多样性中心资助No.Y4ZK111B01)

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:2019-05-01 Published:2019-11-24
  • Contact: Li Jie

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rbcLmatKtrnL-trnFITS2四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ITS2matK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

表1

实验材料"

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

表2

PCR扩增引物及程序"

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

表3

条形码扩增和测序成功率"

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

表4

序列特征信息"

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

表5

叶绿体条形码片段在龙脑香科物种种内变异Wilcoxon检验"

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

表6

叶绿体条形码片段在龙脑香科物种种间变异Wilcoxon检验"

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

图1

条形码片段在龙脑香科物种种内和种间遗传变异分布(A) rbcL序列; (B) matK序列; (C) trnL-trnF序列"

图2

基于4种方法的条形码鉴定效率比较"

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