Biol. Pharm. Bull. 30(8) 1497—1502 (2007)
August 2007
1497
Species Identification of Licorice Using nrDNA and cpDNA Genetic
Markers
Kenji KONDO,*, a Mao SHIBA,a Hiroki YAMAJI,a Takashi MOROTA,a Cheng ZHENGMIN,b Pan HUIXIA,b and
Yukihiro SHOYAMAc
a
Botanical Raw Materials Research Department, Tsumura & Co.; Ibaraki 300–1192, Japan: b Xinjiang Research Institute
of Ecology and Geography, Chinese Academy of Science; Xinjiang 830011, China: and c Kyushu University, Graduate
School of Pharmaceutical Sciences; Fukuoka 812–8582, Japan. Received January 6, 2007; accepted May 2, 2007
For the accurate identification of medicinal licorice species, nucleotide sequences of four types of DNA regions were researched for 205 specimens, including three species used as licorice: Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata. The four DNA regions were the internal transcribed spacer (ITS) on nuclear ribosomal DNA, the rbcL gene, the matK gene, and the trnH–psbA intergenic region on chloroplast DNA
(cpDNA). Ten genotypes were consequently recognized as combinations of the sequence data obtained from the
four DNA regions. Species-specific genotypes were defined from the frequency of the appearance of species in
each genotype and from the phylogenetic relationships of the 10 genotypes. This revealed the possibility of identifying licorice species based on the 10 genotypes. Next, comparison of species identifications by each DNA region
suggested that efficient identification of licorice species is possible using the genetic information obtained from
the ITS and trnH–psbA intergenic region. Additionally, concerning the phylogenetic relationships of the Glycyrrhiza species used as licorice, it is suggested from the genetic information of the four types of DNA regions
that G. glabra is more closely related to G. inflata than to G. uralensis. In the G. uralensis examined, four genotypes were recognized as intra specific variations. The appearance frequency of each genotype in G. uralensis differed according to the area in China. G. uralensis may have expanded its distribution areas from western to eastern China because many licorices with the phylogenetic ancestral genotype were observed in western areas, while
many with the derivative genotype were observed in eastern areas.
Key words
licorice; identification; internal transcribed spacer; rbcL; matK; trnH–psbA intergenic region
Licorice is one of the most useful herbs in traditional Chinese medicine and Japanese Kampo medicine. In the Chinese
Pharmacopoeia, three species are described as licorice: Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata.1) The Japanese Pharmacopoeia includes two licorice
species: G. uralensis and G. glabra.2) Although these Glycyrrhiza species are identified based on the morphologic features of their aerial part, especially by their leaf and fruit
morphologies,3) it is difficult to identify species accurately
based on their root morphology, even though that is the medicinal part.
Licorice species have been identified by root morphology4,5) and by component properties.6—11) Additionally, species identification using genetic markers has been evaluated as a higher-accuracy method in recent years.12) Species
identification using RAPD and rbcL sequences on cpDNA
has been reported.13—16) Meanwhile, various hybrids have
been reported among Glycyrrhiza species.17) The species
identification using only genetic markers on cpDNA would
lead to a misidentification because cpDNA is generally inherited uniparentally.18) Additionally, since genetic markers vary
in their rates of nucleotide substitution and since there are
intra specific variations in their nucleotide sequences,12) a selection of genetic markers with a unique nucleotide sequence
for each species is needed for highly accurate species identification.
Therefore nucleotide sequences of four types of DNA regions were researched for 205 Glycyrrhiza specimens for
more accurate species identification of licorice. One of the
four DNA regions was the internal transcribed spacer (ITS)
on nuclear ribosomal DNA. Since ITS is inherited from both
parents, ITS sequences can detect genetic information on
∗ To whom correspondence should be addressed.
both parents and hybrids.19) Three of the four DNA regions
were the rbcL gene, the matK gene, and the trnH–psbA intergenic region on cpDNA. It is generally considered that they
have different rates of nucleotide substitution, known as evolution rates. The evolution rate of the rbcL gene is slower
than that of the matK gene, or intergenic regions like the
trnH–psbA.12) Suitable genetic markers for the species identification of licorice could be selected from the genetic information of these four kinds of DNA regions.
MATERIALS AND METHODS
Materials The vouchers and localities of the 205 specimens are listed in Table 1. The vouchers are deposited in the
herbaria of Osaka University of Pharmaceutical Sciences
(OY), Tsumura & Co. (THS), University of Tokyo (TI), and
Tohoku University (TUS) in Japan.
Variable Nucleotide Sites Observed To identify G.
uralensis, G. glabra, and G. inflata accurately, nucleotides at
the following sites on the DNA regions were observed (Table
2). At the sites, some nucleotide substitutions were recognized among the three species in preliminary experiments.
Two variable sites were observed at the 187th and the 411—
413th nucleotides in the ITS sequence (accession number
AB280738 registered with GenBank). Two variable sites
were observed at the 706th and the 736th in the rbcL sequence (AB012126). One variable site was observed at the
568th—573rd in the matK sequence (AB28074). Three variable sites were observed at the 72nd, the 125th, and the 171st
in the sequence or trnH–psbA intergenic region (AB280745).
DNA Sequencing Total DNA was extracted using the
DNAeasy Plant Mini Kit (Qiagen) from the dried leaf of a
e-mail: [email protected]
© 2007 Pharmaceutical Society of Japan
1498
Vol. 30, No. 8
Table 1.
Species Identified Based on the Morphologic Features of the Aerial Part, Vouchers, Locations, and Total Genotypes (TG-1—TG-9 and ADD)
Out group (n8)
Vouchera)
OY-7
OY
OY-25
OY-18
OY-2
OY-3
OY-19
OY-20
Species
G. echinata
G. echinata
G. lepidota
G. macedonika
G. pallidiflora
G. pallidiflora
G. pallidiflora
G. pallidiflora
Geno
type
Locality
Cultivated in OY
Cultivated in OY
Canada
Italy
Cultivated in OY
Cultivated in OY
Hungary
Russia
TG-1
TG-1
TG-1
TG-1
TG-1
TG-1
TG-1
TG-1
G. glabra (n51)
Voucher
THS66496
THS66495
OY-24
OY-10
OY
TI
TUS134993
THS758-1
THS758-2
THS758-3
THS758-4
THS758-5
THS491
THS493-1
THS493-2
THS493-3
THS493-4
THS494-1
THS494-2
THS494-3
THS494-4
THS494-5
THS494-6
THS494-7
OY-22
THS41794
THS31678
THS496-1
THS496-2
OY-9
THS72275
THS72276
THS40866
THS72277
THS72278
THS72279
OY-4
OY-5
OY-11
OY
OY
OY
THS35184
THS40627
THS497-2
THS31689
THS497-1
THS498-1
THS498-2
THS498-3
THS498-4
Locality
Spain
France
Egypt
Russia
Russia
Russia
Russia
Czechoslovakia
Czechoslovakia
Czechoslovakia
Czechoslovakia
Czechoslovakia
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Italy
Turkey
Turkey
Turkey
Turkey
Turkey
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
Cultivated in OY
Cultivated in OY
Cultivated in OY
Cultivated in OY
Cultivated in OY
Cultivated in OY
—
China, Xinjiang
China, Xinjiang
Turkey
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
Genotype
TG-2
TG-2
TG-2
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-3
TG-7
TG-9
ADD
ADD
ADD
ADD
ADD
ADD
G. inflata (n18)
Voucher
THS72280
Locality
China, Xinjiang
Genotype
TG-2
THS71515
THS66047-2
THS72281
THS72283
THS72284
THS40630
THS40620
THS502-1
THS502-2
THS503-3
THS43490
THS43491
THS43492
THS43493
THS43494
THS71518
THS71521
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Gansu
China, Gansu
TG-2
TG-4
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
TG-5
ADD
G. uralensis (n128)
Voucher
THS43478
THS41797
THS500-4
THS500-5
THS43478
TUS231103
TUS231099
TUS231101
THS72314-02
THS72314-03
THS72314-07
THS72314-08
THS72316-06
THS72317-05
THS72319
THS770-2
THS72270
THS72273
THS72274
THS72272
THS40621
THS500-6
THS501-2
THS43475
THS43480
THS43481
THS43483
THS44496
THS44497
THS44498
THS44499
THS44500
THS44501
THS44502
THS44503
THS44504
THS44505
THS44506
THS44507
THS44508
THS44509
THS44510
THS44511
THS44512
THS44513
THS44514
THS44515
THS44516
THS44517
THS44518
THS44519
THS44520
THS44521
THS44522
Locality
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Qinghai
China, Qinghai
China, Qinghai
China, Ningxia
China, Ningxia
China, Ningxia
China, Ningxia
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Liaoning
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
Genotype
TG-4
TG-6
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-7
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
THS44523
THS44524
THS44525
THS40629
THS42506
THS42508
THS72271
THS71543
THS42164
TUS133887
THS72316-04
THS72316-05
THS31538
THS41989
OY-1
OY-8
OY-12
OY-14
OY-15
OY-17
OY
THS43132
THS43133
THS43134
THS43135
THS43136-1
THS43136-2
THS43137
THS43483
THS71524
THS71528
THS71531
THS71534
THS71537
THS71540
THS72314-04
THS72314-05
THS72314-06
THS72315-01
THS72315-05
THS41885
TUS118100
TUS95951
TI
THS72317-01
THS72317-02
THS72317-03
THS72317-04
THS72317-06
KUN0615911
THS42163
THS32115
THS42161
THS42162
THS40393
THS40392
THS15811
THS41990
THS770-1
KUN43222
KUN43223
OY-16
OY
THS38110
THS40619
THS40622
THS40624
THS43476
THS43479
THS500-1
THS500-2
THS500-3
THS501-1
OY-21
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Gansu
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Hebei
China, Liaoning
Clutivated in OY
Clutivated in OY
Clutivated in OY
Clutivated in OY
Clutivated in OY
Clutivated in OY
Clutivated in OY
Mongolia
Mongolia
Mongolia
Mongolia
Mongolia
Mongolia
Mongolia
China, Xinjiang
China, Gansu
China, Gansu
China, Gansu
China, Gansu
China, Gansu
China, Gansu
China, Ningxia
China, Ningxia
China, Ningxia
China, Ningxia
China, Ningxia
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Nei Monggol
China, Shaanxi
China, Shanxi
China, Hebei
China, Hebei
China, Hebei
China, Heilongjiang
China, Heilongjiang
China, Heilongjiang
China, Liaoning
China, Liaoning
China, Liaoning
China, Liaoning
Cultivated in OY
Cultivated in OY
—
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
Cultivated in OY
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-8
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
TG-9
ADD
ADD
ADD
ADD
ADD
ADD
ADD
ADD
ADD
ADD
a) Herbarium abbreviations of the vouchers: OY, Osaka University of Pharmaceutical Sciences; THS, Tsumura & Co.; TI, University of Tokyo; and TUS, Tohoku University.
August 2007
Table 2.
1499
Genotypes Obtained from Nucleotide Sequences of the ITS, the rbcL Gene, the matK Gene, and the trnH–psbA Intergenic Region
ITS
(AB280738I-3)
rbcL
(AB012126R-2)
Site
Site
Genotype
I-1
I-2
I-3
ADD
matK
(AB280741M-1)
Site
Genotype
187
411—413
T
T
C
Y
TGC
CAA
TGC
YRM
R-1
R-2
trnH–psbA
(AB280745T-4)
Genotype
706
736
A
G
T
A
Site
Genotype
568—573
M-1
M-2
CTTATT
Deletion
T-1
T-2
T-3
T-4
72
125
171
C
C
T
C
A
A
A
G
T
G
T
T
The variable sites were located based on the sequences with the accession numbers. Gray backgrounds indicate nucleotide substitutions or a deletion compared with I-1, R-1,
M-1, or T-1. IUPAC ambiguity symbols are adopted (YCT, RAG, MAC).
herbarium specimen. The complete sequence of the ITS, the
partial sequences of rbcL (168 bp), matK (143 bp), and
trnH–psbA (239 bp), including the genetic polymorphisms
among the Glycyrrhiza species, were amplified with PCR
under the following conditions. The amplifying primers for
the ITS were ITS5: GGA AGT AAA AGT CGT AAC AAG
G and ITS4: TCC TCC GCT TAT TGA TAT GC.20) The amplifying primers for the rbcL were r662f: GTG CCG AAG
CAA TTT ATA AAG C and r829r: TTG CAG TGA AAC
CTC CAG TT; those for the matK were m1242f: CTT CGA
CAC TGG GTG AAA GAT G and m1384r: AGG AAC
AAG AAT AAT CTT GG; and those for the trnH–psbA were
trnH-forward: ACG GGA ATT GAA CCC GCG CA21) and
Gly-trnHR1: CAT ATG ACT TCA CAA TGT AAA ATC.
The PCR reaction mixture was: 10Gene Taq Buffer (Nippon Gene) 5 m l, dNTP mix (Nippon Gene) 4 m l, forward
primer (10 pmol/ml) 2.5 m l, reverse primer (10 pmol/ml)
2.5 m l, Gene Taq (Nippon Gene), DMSO 5 m l, D.D.W.
25.75 m l, template DNA 5 m l (5 ng). For the PCR cycle, a
modified protocol of the step-down PCR was applied.22)
Electrophoresis was performed for the amplified DNA
fragments through 1.2% TAE agarose gel. The amplified
DNA fragments were cut from the gel and purified using the
GFX PCR DNA and Gel Band Purification Kit (Amersham
Biotech). The purified PCR products were sequenced using
the BigDye Terminator Cycle Sequencing Kit ver.2.0 and a
Model 3100 automated sequencer (Applied Biosystems) following the manufacturer’s instructions. For sequencing the
ITS, matK, and trnH–psbA, their amplifying primers were
used as sequencing primers. For sequencing the rbcL, r694f:
ACT GGT GAA ATC AAA GGG C and r809r: AAG TAG
TCA TGC ATT ACG AT were designed as sequencing
primers.
Phylogenetic Analyses The phylogenetic analyses were
carried out using the PAUP software package 3.1.123) based
on the nucleotide substitutions. The most-parsimonious trees
of equal length were computed after a heuristic search of
trees using the TBR and MULPARS options.
RESULTS
Morphological Identification The 205 specimens were
identified based on their morphologic features of the aerial
part, based on Zhang et al. (1998).3) Three Glycyrrhiza
species used as licorice in China (G. glabra: n51, G.
inflata: n18, and G. uralensis: n128) and four Glycyrrhiza species as an out-group (Glycyrrhiza echinata: n
2, Glycyrrhiza lepidota: n1, Glycyrrhiza macedonika:
n1, and Glycyrrhiza pallidiflora: n4) were included in the
205 specimens.
Genotypes Recognized in the Sequences of the ITS,
rbcL Gene, matK Gene, and trnH–psbA Intergenic Region
In the sequencing results for the 205 Glycyrrhiza specimens,
four genotypes (I-1, I-2, I-3, and ADD) were recognized as
combinations of nucleotides at the variable sites in the ITS
sequences (Table 2). Within the ADD genotype of the ITS,
two types of nucleotides were observed at every variable site:
Y (CT) at the 187th and YRM (CT, AG, and
AC) at the 411—413th nucleotides in the ITS sequence
(AB280738). Therefore it was considered that the ADD
genotype is a combination of the I-2 (Y, CAA) and I-3 (C,
TGC) genotypes (Table 2). This has been called “nucleotide
additivities” that are often observed in hybrids between two
species having different ITS nucleotide sequences.24) In the
rbcL sequences, two genotypes (R-1 and R-2) were recognized as combinations of nucleotides at two sites of the 706th
and the 736th in the rbcL sequence (AB012126). In the matK
sequences, two genotypes (M-1 and M-2) were recognized as
an insertion and deletion, respectively, of the six nucleotides
CTTATT at a site of the 568th—573rd in the matK sequence
(AB280741). In the trnH–psbA intergenic region, four genotypes (T-1, T-2, T-3, and T-4) were recognized as combinations of nucleotides at each of three sites of the 72nd, 125th,
and 171st in the sequence of the trnH–psbA intergenic region
(AB280745).
Total Genotypes and Frequency of Appearance of
Species by Total Genotype Ten total genotypes (TG-1—
TG-9 and ADD) were recognized as combinations of the
genotypes (I-1—I-3, ADD, R-1—R-2, M-1—M-2, and T-1—
T-4) obtained from the ITS, the rbcL gene, the matK gene,
and the trnH–psbA intergenic region (Fig. 1).
The frequency of appearance of species for each total
genotype was examined (Fig. 1). In the 205 Glycyrrhiza
specimens examined, all of the out-group (n8) had the TG1 genotype. In G. glabra identified based on the morphologic
features, the TG-2, TG-3, TG-7, TG-9, and ADD genotypes
were recognized. Most G. glabra (78%40/51) had the TG-3
genotype. In G. inflata identified based on morphologic features, the TG-2, TG-4, TG-5, and ADD genotypes were observed. Most G. glabra (78%14/18) had the TG-5 genotype. In G. uralensis identified based on the morphologic features, the TG-4, TG-6, TG-7, TG-8, TG-9, and ADD genotypes were recognized. G. uralensis with the TG-7, TG-8,
and TG-9 genotypes made up 91% (116/128).
1500
Vol. 30, No. 8
Fig. 1. Total Genotypes (TG-1—TG-9 and ADD) Consisted of the Combinations of the Genotypes (I-1—I-3, ADD, R-1—R-2, M-1—M-2, and T-1—T-4)
Obtained from the ITS, rbcL, matK, and trnH–psbA
Under the genotypes, the frequency of appearance of each species identified by aerial morphologic features is shown by each total genotype. Bold typeface indicates speciesspecific genotypes and major frequencies in each species. Phylogenetic relationships among the total genotypes were described as the only most-parsimonious tree of eight steps
long. The tree consistency index was 0.875.
Phylogenetic Relationship among Total Genotypes To
clarify phylogenetic relationships among the total genotypes,
phylogenetic analysis was performed. Only one most-parsimonious phylogenetic tree of eight steps in length was calculated from the sequence data except for the ADD genotype.
Since two types of nucleotide sequences were included in the
ADD genotype, the genetic information on it was not used in
the phylogenetic analysis. In the phylogenetic tree, two
clades, consisting of TG-2—TG-5 and TG-6—TG-9, are recognized (Fig. 1). In the former, TG-4 and TG-5 form a subclade, while in the latter, TG-8 and TG-9 form a subclade.
Species-Specific Genotypes Species-specific genotypes
were defined from the frequency of appearance of species for
each total genotype and from the phylogenetic relationships
of the total genotypes (Fig. 1). The TG-1 of the total genotype is a specific genotype of the out group examined. TG-2
is of G. glabra or G. inflata. TG-3 is of G. glabra. TG-4 and
TG-5 are of G. inflata because TG-4 and TG-5 form a subclade in the phylogenetic tree and they are closely related. On
the other hand, TG-6—TG-9, forming a clade in the phylogenetic tree, are species-specific genotypes of G. uralensis. Additionally, licorices with the ADD genotype are hybrids between G. uralensis and either G. glabra or G. inflata because
the ADD genotype consisted of the I-2 and I-3 genotypes of
the ITS (Table 2). The I-2 genotype was observed in TG-2—
TG-5 of the total genotypes that are defined as the speciesspecific genotypes of G. glabra or G. inflata, and the I-3
genotype was recognized in TG-6—TG-9 of the total genotypes that are defined as the species-specific genotype of G.
uralensis.
The accuracy of licorice species identification based on the
total genotypes was estimated as about 96% (197/205) in this
study because only three samples were identified as belonging to different species based on their aerial morphologic
features, and five samples with TG-2 of the total genotype
could not be distinguished between G. glabra and G. inflata.
Maternal Species of Hybrids To determine maternal
species of the hybrids with the ADD genotype, their geno-
types obtained from the rbcL, matK, and trnH–psbA sequences on cpDNA were examined (Table 3). The samples
with the ADD genotype are hybrids between G. uralensis and
either G. glabra or G. inflata. Additionally, concerning genetic information on cpDNA, it was suggested that the R-1
genotype of the rbcL and the T-4 genotype of the trnH–psbA
were recognized in G. uralensis alone, and the M-2 genotype
of the matK and the T-3 genotype of trnH–psbA were observed in G. inflata alone. From this information, the maternal species of hybrids with the ADD genotype were assumed
(Table 3). In this result, the species-specific genotype of G.
inflata was recognized in G. uralensis identified from the
morphologic features with the ADD genotype, G. uralensis
and G. inflata were recognized in G. glabra, and G. uralensis
was recognized in G. inflata (Table 3). Therefore it is suggested that cross-hybridizations among G. uralensis, G.
glabra, and G. inflata occurred.
DISCUSSION
Selection of Efficient Genetic Markers for Species
Identification of Licorice The species identification of
licorice is possible from the total genotype obtained from the
nucleotide sequences of the ITS on nrDNA, and the rbcL
gene, the matK gene on cpDNA. Meanwhile, comparing the
species identification according to the genetic information of
each DNA region, the identification of G. uralensis, G.
glabra, and G. inflata is possible based on the genetic information from the ITS and the trnH–psbA intergenic region
alone. G. uralensis is distinguishable by the I-3 genotype of
the ITS (Fig. 1). At the same time, G. glabra and G. inflata
have the I-2 genotype and the out group examined has the I-1
genotype. Additionally, G. glabra and G. inflata are distinguishable by having the T-2 and T-3 genotypes of the
trnH–psbA intergenic region, respectively.
Phylogenetic Relationships among G. uralensis, G.
glabra, and G. inflata From the rbcL sequences, Hayashi
et al.15) reported that G. uralensis was more closely related to
August 2007
Table 3.
1501
Maternal Species of the Hybrids with the ADD Genotype Assumed from the cpDNA Genotype
Species
identified based
on morphologic
features
G. uralensis
Genotype
Voucher
Locality
ITS
rbcL
matK
trnH–psbA
THS40619
THS40622
China, Xinjiang
China, Xinjiang
ADD
ADD
R-1
R-1
M-2
M-1
T-3
T-1
THS40624
THS43476
THS43479
China, Xinjiang
China, Xinjiang
China, Xinjiang
ADD
ADD
ADD
R-1
R-1
R-1
M-2
M-1
M-1
T-3
T-3
T-2
G. glabra
THS500-1
THS500-2
THS500-3
THS501-1
OY-21
THS31689
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
Cultivated in OY
Turkey
ADD
ADD
ADD
ADD
ADD
ADD
R-2
R-2
R-2
R-1
R-2
R-1
M-1
M-1
M-1
M-2
M-1
M-1
T-4
T-4
T-4
T-3
T-1
T-2
G. inflata
THS497-1
THS498-1
THS498-2
THS498-3
THS498-4
THS71521
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Xinjiang
China, Gansu
ADD
ADD
ADD
ADD
ADD
ADD
R-2
R-1
R-1
R-1
R-1
R-2
M-1
M-2
M-2
M-2
M-2
M-1
T-4
T-3
T-3
T-3
T-3
T-1
Maternal species
assumed from
cpDNA genotype
G. inflata
G. uralensis
or G. glabra
G. inflata
G. inflata
G. uralensis
or G. glabra
G. uralensis
G. uralensis
G. uralensis
G. inflata
G. uralensis
G. uralensis
or G. glabra
G. uralensis
G. inflata
G. inflata
G. inflata
G. inflata
G. uralensis
Bold typeface indicates the key genotypes of identification. Gray backgrounds indicate that maternal species is different from species identified based on the morphologic features.
Fig. 2.
Samples with the Total Genotype Mapped for Each Province in China
G. inflata than to G. glabra because G. uralensis and G. inflata had the same rbcL sequence, which is the R-2 genotype
in this study, and the rbcL sequence of G. glabra had two nucleotide substitutions from G. uralensis and G. inflata, which
is the R-1 genotype. However, our results indicate that the R1 genotype is observed in G. uralensis, G. glabra, and G. inflata although the R-2 genotypes are recognized only in G.
uralensis (Fig. 1). Therefore accurate phylogenetic relationships among species could not be estimated from only few
samples and few genetic markers. In particular, since the ex-
istence of hybrids was reported in Glycyrrhiza species,17) G.
inflata with the R-2 genotype might be a hybrid. Our phylogenetic tree calculated with the genetic information on the
ITS, rbcL, matK, and trnH–psbA sequences clearly indicates
that G. glabra is more closely related to G. inflata than to G.
uralensis (Fig. 1).
Distributions of G. uralensis, G. glabra, and G. inflata
in China The 205 samples examined with the total genotypes were mapped for each province in China (Fig. 2). G.
glabra and G. inflata with the TG-2—TG-5 genotypes were
1502
distributed in northwestern China. Meanwhile, G. uralensis
with TG-6—TG-9 genotypes was distributed from northeastern to northwestern China. The hybrids between G. uralensis
and either G. glabra or G. inflata with the ADD genotype
were distributed in northwestern China where G. uralensis,
G. glabra, and G. inflata are mixed.
In G. uralensis, the four types of the total genotypes (TG6—TG-9) were recognized as an intra specific variation. In
our result, only one sample with the TG-6 genotype was
found in Xinjiang province (Fig. 2). The TG-6 genotype has
a comparatively ancestral nucleotide sequence among TG6—TG-9 because the sequence of TG-6 has only one nucleotide substitution from the most ancestral sequence calculated based on phylogenetic analysis (Fig. 1). G. uralensis
with TG-7—TG-9 was found in wide areas from northeastern to northwestern China. The appearance frequencies of
TG-8 were higher in western China, and those of TG-9 were
higher in eastern China (Fig. 2). It is considered that TG-9 is
derived from the TG-8 phylogenetically because the TG-9
was formed by the occurrence of a nucleotide substitution
from TG-8 (Fig. 1). Therefore G. uralensis may have expanded its distribution from western to eastern China because there were many licorices with the phylogenetic ancestral genotypes (TG-6 and TG-8) in western areas and many
of the derivative genotype (TG-9) in eastern areas (Fig. 2).
Acknowledgments We express our sincere thanks to Dr.
H. Hayashi of Gifu Pharmaceutical University, Dr. G. Kusano of Osaka University of Pharmaceutical Sciences, Dr. H.
Ohashi of Tohoku University, and J. Murata of Tokyo University for providing materials.
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