Crops ›› 2017, Vol. 33 ›› Issue (5): 55-60.doi: 10.16035/j.issn.1001-7283.2017.05.010

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Identification of Morinda officinalis and Its Three Close Plant Species Based on ITS2 and psbA-trnH

Wei Ying1,Zhan Ruoting2,Liang Hongling1,Wang Haohan1,Huang Haibo1   

  1. 1School of Chinese Meteria Medica,Guangzhou University of Chinese Medicine,Guangzhou 510006, Guangdong,China
    2Research Center of Chinese Herbal Resource Science and Engineering, Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China
  • Received:2017-06-05 Revised:2017-08-08 Online:2017-10-15 Published:2018-08-26
  • Contact: Haibo Huang

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Abstract:

To identify Morinda officinalis and its three close related plant species based on ITS2 and psbA-trnH, the ITS2 and psbA-trnH of Morinda officinalis and its three close related plant species were amplified by PCR and sequenced. Genetic distance analysis, DNA Barcoding Gap and NJ tree were used to identify the above species. The results showed that the length of ITS2 was 237bp, the GC content was 70.3%, the conserved rate was 86.08%, and the mutation rate was 13.92%. The interspecific variation based on ITS2 was greater than the intraspecific variation, and there was significant DNA Barcoding Gap. The NJ tree based on ITS2 can accurately identify all the experimental species. The length of psbA-trnH was 290bp, the GC content was 20.2%, the conserved rate was 53.10%, and the mutation rate was 46.55%. The interspecies variation based on psbA-trnHwas greater than the intraspecies variation, and there was no obvious DNA Barcoding Gap. PsbA-trnH can only accurately identify two species. The species identification ability of ITS2 is better than psbA-trnH, which can accurately identify Morinda officinalis and its three closely related plants. In conclusion, based on the ITS2 and psbA-trnH sequences, Morinda officinalis and its three closely related plants can be successfully identified.

Key words: Morinda officinalis How, Morinda umbellata L. subsp. obovata Y. Z. Ruan, Morinda hainanensis Merr. et How, Morinda citrifolia L., DNA barcode, Molecular identification, DNA Barcoding Gap, Phylogenetic tree

Table 1

Experimental sample information"

编号
Serial number		 植物名
Species name		 采集地点
Collecting locations		 采集日期(年/月/日)
Collecting date		 凭证标本号
Voucher number
B1

 巴戟天
M. officinalis
 海南省琼海市会山镇
N：19°3′32.11″
E：110°14′55.03″		 2016/8/31

 HS201608311
B2

 巴戟天
M. officinalis
 福建省漳州市南靖县和溪镇林中村
N：24°53′53.16″
E：117°13′21.15″		 2015/11/29

 HX201511292
B3

 巴戟天
M. officinalis
 福建省漳州市南靖县和溪镇月明村
N：24°57′26.81″
E：117°15′46.77″		 2015/11/29

 HX201511293
B4

 巴戟天
M. officinalis
 福建省漳州市南靖县和溪镇林坂村
N：24°53′4.80″
E：117°14′0.59″		 2015/11/29

 HX201511294
B5

 巴戟天
M. officinalis
 福建省漳州市南靖县和溪镇迎新村
N：24°54′14.05″
E：117°17′39.63″		 2015/11/29

 HX201511295
B6

 巴戟天
M. officinalis
 广东省云浮市罗定市附城街道康任村
N：22°50′24.49″
E：111°32′38.48″		 2015/1/30

 LD20150130
HB1

 海滨木巴戟
M. citrifolia
 海南万宁兴隆热带植物所
N：18°43′56.39″
E：110°11′46.09″		 2016/8/29

 WN2016082913
HB2

 海滨木巴戟
M. citrifolia
 海南万宁兴隆热带植物所
N：18°44′0.96″
E：110°11′39.56″		 2016/8/30

 WN2016083015
HB3

 海滨木巴戟
M. citrifolia
 海南万宁兴隆热带植物所
N：18°44′2.42″
E：110°11′39.50″		 2016/8/29

 WN2016082911
HB4

 海滨木巴戟
M. citrifolia
 海南万宁兴隆热带植物所
N：18°44′1.40″
E：110°11′39.91″		 2016/8/29

 WN2016082916
HN1

 海南巴戟
M. hainanensis
 海南省乐东县尖峰镇
N：18°44′51.89″
E：108°52′34.56″		 2016/8/30

 JF2016083025
HN2

 海南巴戟
M. hainanensis
 海南省乐东县尖峰镇
N：19°3′34.89″
E：110°14′56.46″		 2016/8/31

 JF2016083121
HN3

 海南巴戟
M. hainanensis
 海南省乐东县尖峰镇
N：18°44′45.20″
E：108°52′6.57″		 2016/8/30

 JF2016083023
Y1

 羊角藤
M. umbellatasubsp.obovata
 广东省梅州市平远县仁居镇
N：24°48′40.22″
E：115°53′21.93″		 2016/7/16

 RJ201607162
Y2

 羊角藤
M. umbellatasubsp.obovata
 福建省龙岩市长汀县汀州镇
N：25°50′7.09″
E：116°21′28.28″		 2016/2/8

 TZ201602082
Y3

 羊角藤
M. umbellatasubsp.obovata		 广东省梅州市平远县上举镇相思谷
N：24°47′53.84″
E：115°56′54.88″		 2016/1/24

 SJ201601241
Y4

 羊角藤
M. umbellatasubsp.obovata
 广东省梅州市平远县石正镇南台山
N：24°32′25.84″
E：115°50′21.24″		 2016/1/23

 SZ201601231
Y5

 羊角藤
M. umbellatasubsp.obovata
 广东省广州市番禺区大学城广州中医药大学
N：23°3′36.93″
E：113°23′46.32″		 2016/4/20

 PY201604202
Y6

 羊角藤
M. umbellatasubsp.obovata
 福建省龙岩市长汀县汀州镇
N：25°50′7.09″
E：116°21′28.28″		 2016/2/8

 TZ201602083

Table 2

Primer sequence information"

条形码片段
Bar code fragment		 引物名称
Primer name		 碱基序列
Base sequence
ITS2 ITS2F 5'-ATGCGATACTTGGTGTGAAT-3'
ITS3R 5'-GACGCTTCTCCAGACTACAAT-3'
psbA-trnH Fwd 5'-GTTATGCATGAACGTAATGCTC-3'
Rev 5'-CGCGCATGGTGGATTCACAATCC-3'

Table 3

Sequence information"

ITS2 psbA-trnH
序列数Sequence No. 19 19
序列长度Sequence length (bp) 237 290
GC含量GC content (%) 70.3 20.2
保守率Conservative rate (%) 86.08 53.10
变异率Variation rate (%) 13.92 46.55
简约信息率Simple information rate (%) 13.08 46.55
单碱基突变率(%)
Single base variation rate		 0.84
 0
平均种内序列分歧度
Mean diversity of intraspecific sequence		 0.001
 0.000
平均种间序列分歧度
Mean diversity of interspecific sequence		 0.063
 0.279
总序列分歧度
Total diversity of sequence		 0.065
 0.279
遗传变异系数
Genetic coefficient of variation		 0.982
 1.000

Table 4

Intraspecific and interspecific genetic distance ITS2, psbA-trnH"

物种名
Species Name		 巴戟天
M. officinalis		 羊角藤
M. umbellatasubsp.obovata		 海南巴戟
M. hainanensis		 海滨木巴戟
M. citrifolia
巴戟天M. officinalis 0,0
羊角藤M. umbellata subsp. obovata 0.041,0.004 0.005,0
海南巴戟M. hainanensis 0.038,0 0.046,0.004 0,0
海滨木巴戟M. citrifolia 0.127,0.797 0.134,0.787 0.144,0.797 0,0

Fig.1

DNA Barconding Gap"

Fig.2

The NJ tree of Morinda officinalis and its three closely related plants based on the ITS2 sequence"

Fig.3

The NJ tree of Morinda officinalis and its three closely related plants based on the psbA-trnH sequence"

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