American Journal of Plant Sciences, 2011, 2, 111-119
doi:10.4236/ajps.2011.22013 Published Online June 2011 (
Copyright © 2011 SciRes. AJPS
Difference of Curcumin Content in Curcuma longa
L. (Zingiberaceae) Caused by Hybridization with
Other Curcuma Species
Hiroshi Hayakawa1,2, Yukio Minaniya1, Katsura Ito1, Yoshinori Yamamoto1, Tatsuya Fukuda1
1Faculty of Agriculture, Kochi University, Nankoku, Japan; 2United Graduate School of Agricultural Sciences, Ehime University,
Nankoku, Japan.
Received November 29th, 2010; revised January 24th, 2011; accepted January 30th, 2011.
Curcumin, which is traditionally known to have effects on various types of diseases in humans, is found in Curcuma
longa L. Previous reports have indica ted that the curcumin co ntent varies b etween the different lines of this sp ecies. To
clarify the differences in the amounts of curcumin between the lines, we investigated the outcomes of cultivation ex-
periments with the hybridization or introgression between C. longa and other Curcuma species using the matK gene of
chloroplast DNA (cpDNA) and the external transcribed spacer (ETS) of nuclear DNA (nrDNA). The results show that
there is heterogeneity of the ETS and incongruence between the matK and the ETS phylogenetic trees, suggesting that
hybridization and introgression had taken place in the diversification of the various lines of C. longa. Moreover, al-
though all of the lines had the same cpDNA haplotype of C. longa, the lines of homogeneous C. longa had a high con-
tent of curcumin, whereas the lines created by hybridization and introgression with other Curcuma species had a me-
dium or low level. These results suggest that the difference of cu rcumin conten t among the various lines of C. longa was
caused by hybridization and introgression with other Curcuma species.
Keywords: Curcuma longa, Curcumin, Hybridization, Introgression, Molecular Analysis, Nuclear DNA
1. Introduction
Many chemicals in plants are potential drugs for humans
and natural products from plants are found in many
therapeutic formulations. Moreover, conscious efforts to
search for desirable traits in plants have been underway
for the past century, and in recent decades species with
desirable traits have come to be regarded as important
biological resources in need of conservation [1].
Curcuma longa L., which belongs to the ginger family,
Zingiberaceae, is a perennial widely used as a spice, a
colorant and also as a major ingredient of curry powder
[2]. This species has a long history of use as a traditional
medicine in China and India [3], reflecting it’s diverse
and beneficial health effects. In addition, the curcuma
species contains phenolic compounds found in the plant’s
rhizomes. Traditionally, curcumin is well-known to have
therapeutic effects on a variety of human diseases, and
the cancer preventive activity of curcumin is being inten-
sively studied all over the world. Experiments in animal
models indicate that it is a preventive agent against vari-
ous types of cancer [4]. Specifically, curcumin inhibits
the cell growth of various cancer cell lines, induces
apoptosis of cancer cells [5-7], and was effective on the
cell-cycle regulation of cancer cells [8].
According to previous study there is a difference in the
curcumin content among individuals of C. longa [9,10],
however, it remains unclear why the curcumin content is
different. To identify the lines with high curcumin con-
tent, Hayakawa et al. [10,11] developed a molecular
marker. Recent molecular phylogenetic study using
chloroplast DNA (cpDNA) sequences indicated that C.
longa has some closely related species. Also, a large
number of previous studies of species within the genus
Drosophila had illuminated the relationship between
genetic distance and reproductive isolation [12,13]. One
possibility is that the difference in curcumin content
within C. longa was caused by including hybrids be-
tween C. longa and other Curcuma species. It is very
difficult to detect polymorphisms of morphology of the
rhizomes and cpDNA sequences within this species. Al-
Difference of Curcumin Content in Curcuma longa L. (Zingiberaceae) Caused by
Hybridization with Other Curcuma Species
though this hypothesis could be clarified by comparing
nuclear DNA (nrDNA) sequences, unfortunately, such
study has not been done so far.
The number of phylogenetic studies based on molecu-
lar data has grown enormously in recent years, and most
of the recent studies are concerned with closely related
species or variation within species. In particular, the use
of molecular markers has considerably improved our
knowledge about how past events shape the genetic di-
versity within a species [14-16]. Recently, various mo-
lecular markers have been widely analyzed to assess the
genetic variability of wild plants [17]. Among them, nu-
clear markers are mostly neutral with relatively high mu-
tation rates, and, in association with the history, provide
information to estimate the putative parents involved in
hybridization and introgression [18,19]. The polymor-
phisms of the external transcribed spacer (ETS) region in
nuclear DNA (nrDNA) are good tools for clarifying the
relationship of closely related taxa in many plant groups
[20-24]. Here, to test our hypothesis of the hybridization
of C. longa, we describe the DNA polymorphisms of the
ETS region in C. longa and its allied species and discuss
the possible reasons for the differences in curcumin con-
tent of this species.
2. Materials and Methods
2.1. Plant Materials
For the plant materials, we used 1 Curcuma alismatifo lia
Gagnep. cultivar ‘Sawang Chiang Mai’, 3 C. aromatica
Salib., 12 C. longa L. and 1 C. zedoaria Rosc. for mo-
lecular analysis in this study (Table 1). Of these, 2 C.
aromatica, 5 to 12 C. longa and 1 C. zedoaria were cul-
tivated in the field of the Faculty of Agriculture, Kochi
University, Japan for 2006-2009 (See detail Hayakawa et
al. [10]). Rhizomes were transplanted in late May for
four years. For fertilizer dressing, a total of 1.5 kg/a of N,
0.6 kg/a of P2O5, and 1.4 kg/a of K2O was applied over
Table 1. List of sample of Curcuma species used in this study.
Curcumin content1
No. Sepcies Locality
2006 2007 2008 2009
1 Curcuma alismatifolia ‘Sawang Chiang
Mai’ Chiang Mai. Thailand 2
2 C. aromatica (Kochi) Kochi Perfecture. Japan 38bcd 71b 47b 126c
3 C. aromatica (Tanegashima) Tanegashima Island. Kagoshima Perfecture.
Japan 41abcd 78bc 36b 122c
4 C. aromatica (Okinawa) Okinawa Perfecture. Japan
5 C. longa (Kochi) Kochi Perfecture. Japan 327ab 398ab 358ab 392c Hybrid
6 C. longa (Tanegashima) Tanegashima Island. Kagoshima Perfecture.
Japan 301abc 382ab 392ab 361c Hybrid
7 C. longa (Wakayama A) Wakayama Perfecture. Japan 179abcd 403ab 388ab 374c Hybrid
8 C. longa (Wakayama B) Wakayama Perfecture. Japan 1d 2
c 1
b 1
c Introgression
9 C. longa (Wakayama C) Wakayama Perfecture. Japan 404abc 396ab 390c Hybrid
10 C. longa (Okinawa A) Okinawa Perfecture. Japan 364ab 347c Hybrid
11 C. longa (Okinawa B) Okinawa Perfecture. Japan 373ab 305c Hybrid
12 C. longa (Indonesia A) Bogol. West Java. Indonesia 2849a 2777a2678a 3059a Pure line
13 C. longa (Indonesia B) Bogol. West Java. Indonesia 229abc 337ab 310c Hybrid
14 C. longa (Indonesia C) Bogol. West Java. Indonesia 2315b Pure line
15 C. longa (Vietnam A) Vietnam 2977a Pure line
16 C. longa (Vietnam B) Vietnam 3198a Pure line
17 C. zedoaria Unkonwn 1cd 2
c 1
b 1
1Curcumin content in primaly branch rhizomes (mg/100g).2Not examind. 3Type of nrDNA in Curcuna longa. Values followed by the same latter in a column of
ach year are not signigicantly at 5% level by one-way ANOVA. e
Copyright © 2011 SciRes. AJPS
Difference of Curcumin Content in Curcuma longa L. (Zingiberaceae) Caused by 113
Hybridization with Other Curcuma Species
four years. In addition, 200 kg/a of compost fertilizer, 15
kg/a of magnesia lime, and 30 kg/a of chicken droppings
were applied. The experimental plots were arranged in a
randomized complete design with two replicates, which
formed three rows. Due to a lack of seed rhizomes, some
lines of C. longa were examined with/without replicate
using two or three rows. Samples were harvested in early
December for four years. The total curcumin content
(mg/100g) of primary branch rhizomes was measured by
using High Performance Liquid Chromatography (HPLC),
according to the method described by Sato et al. [25].
2.2. Molecular Analyses
For the molecular analyses, total DNA was isolated from
fresh root using a Plant Genomic DNA Mini Kit (VIO-
GENE, Sunnyvale, USA), according to the manufac-
turer’s protocol. We amplified the maturase K (matK)
gene from cpDNA and the external transcribed spacer
(ETS) region of 18S-26S rDNA from nrDNA with prim-
ers designed by Johnson and Soltis [26] and Starr et al.
[27], respectively. The isolated DNA was amplified by
PCR in a 50 µl reaction solution containing approxi-
mately 50 ng total DNA, 10 mM Tris-HCl (pH 8.3), 50
mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 1.25
units Taq DNA polymerase (TaKaRa) and 0.5 µM of
each primer pair. We used the following thermal cycle
profile for amplification by the PCR Thermal Cycler
Dice (TaKaRa): 1 min at 94˚C, 2 min at 48˚C, and 2 min
at 72˚C for 45 cycles, followed by 15 min of final exten-
sion at 72˚C. After amplification, the PCR products of
the matK and ETS region were subjected to electropho-
resis in 1% low-melting-temperature agarose gels to re-
move by-products and purify amplified products. We
sequenced the purified PCR products using a BigDye
Terminator ver. 3.1 (Applied BioSystems) and ABI Prism
3100 Genetic Analyzer (Applied BioSystems) according
to the manufacturer’s instructions. For sequencing, we
used the same primers as those used for amplification.
To construct a phylogenetic tree based on the matK
sequences of Curcuma and its allied species and the ETS
sequences of Curcuma species, the amplified regions
were aligned using ClustalW [28] and were improved
manually using MEGA 4 [29]. Phylogenetic relationships
were analyzed using the neighbor-joining (NJ) method
with PAUP* 4.08b [30]. The NJ analyses were per-
formed using MEGA 4 with Kimura’s two-parameter
model. For the NJ analyses, bootstrapping with 1000
pseudo-replicates was chosen to examine the robustness
of the clades and their phylogenetic relationships. The
matK sequences were collected from DDBJ/EMBL/
GenBank International DNA databases (Table 2).
For the ETS region, because C. longa with a medium
curcumin content could not determine its sequence
caused by putative heterozygosity, we carried out PCR-
RFLP analysis after checking the sequencing results and
alignments. The result of the alignments indicated that an
autapomorphic character of the nrDNA was the restric-
tion site Hinf I. After designating the restriction sites, the
amplified products were digested by Hinf I at 37˚C for
more than an hour. The digested DNAs were separated
on 1.0% agarose gel and the size of each band was de-
3. Results
3.1. Curcumin Content
The gradient of curcumin content between species de-
creased as follows; South Asian C. longa > domestic C.
longa and C. longa (Indonesia B) > C. aromatica > C.
zedoaria and C. longa (Wakayama B) (Table 1) [10] and
the level of curcumin content was divided into three
groups; high, medium and low.
3.2. Molecular Analyses
To construct the molecular phylogenetic tree of Curcuma
and its allied species, we determined the sequences of the
matK gene of Curcuma cpDNA and seven outgroup taxa
(Table 2). The lengths of the matK gene of the Curcuma
species varied from 1539 bp (C. alismatifolia ‘Sawang
Chiang Mai’ and C. long a (Wakayama B)) to 1554 bp (C.
thorelii). The result of the phylogenetic analysis of matK
indicated that C. longa had a conserved sequence in this
species and was closely related to C. aromatica and C.
zedoaria, whereas C. alismatifolia ‘Sawang Chiang Mai’
was located in the basal position of the phylogenetic tree
and the sister to C. thorelii with a high boot strap value
(Figure 1).
In addition, we sequenced the ETS region of nrDNA
to detect polymorphisms among Curcuma species. The
lengths of the ETSs of Curc uma species were 514 bp (C.
alismatifolia) to 517 bp (C. aromatica). The sequences
have been deposited into the DDBJ/EMBL/GenBank
International DNA databases (C. alismatifo lia , AB588183;
C. aromatica: AB588181, C. longa: AB588182,
AB588185 and AB588186, C. zedoaria: AB588184). The
results of the phylogenetic analyses of the ETSs indi-
cated that C. longa and closely related species were di-
vided into two monophyletic groups: clade 1 and clade 2
(Figure 2). Clade 1 consisted of all individuals of C.
longa and clade 2 consisted of C. longa, C. aromatica
and C. zedoaria. Although all homogeneous C. longa
with its high curcumin content appeared in clade 1, C.
longa (Wakayama B) with its low curcumin content was
loc ted in clade 2 (Figure 2, Table 1). This suggested a
Copyright © 2011 SciRes. AJPS
Difference of Curcumin Content in Curcuma longa L. (Zingiberaceae) Caused by
Hybridization with Other Curcuma Species
Table 2. Accession numbers of matK using phylogenetic analysis of Curcuma and outgroup taxa.
Species Accession No. Reference
Curcuma aeruginosa AF478840 Kress et al. (2002)
C. amarissima AB047751 Cao et al. (Unpubl.)
C. alismatifolia 'Sawang Chiang Mai' AB588187 this study
C. aromatica AB047731 Cao et al. (2001)
C. aromatica (Kochi) AB551929 Hayakawa et al. (2010)
C. aromatica (Tanegashima) AB551929 Hayakawa et al. (2010)
C. aromatica (Okinawa) AB551929 Hayakawa et al. (2010)
C. attenuata GQ248110 Hollingsworth et al. (Unpubl.)
C. bicolor AF478837 Kress et al. (2002)
C. chuanezhu AB047736 Cao et al. (2001)
C. chuanhuangjiang AB047732 Cao et al. (2001)
C. chuanyujin AB047733 Cao et al. (2001)
C. comosa AF478838 Kress et al. (2002)
C. elata AB047747 Cao et al. (Unpubl.)
C. exigua AB047750 Cao et al. (Unpubl.)
C. kwangsiensis A AB047744 Cao et al. (2001)
C. kwangsiensis B AB047745 Cao et al. (Unpubl.)
C. longa (Kochi) AB551930 Hayakawa et al. (2010)
C. longa (Tanegashima) AB551930 Hayakawa et al. (2010)
C. longa (Wakayama A) AB551930 Hayakawa et al. (2010)
C. longa (Wakayama B) AB551931 Hayakawa et al. (2010)
C. longa (Wakayama C) AB551930 Hayakawa et al. (2010)
C. longa (Okinawa A) AB551930 Hayakawa et al. (2010)
C. longa (Okinawa B) AB551930 Hayakawa et al. (2010)
C. longa (Indonesia A) AB551930 Hayakawa et al. (2010)
C. longa (Indonesia B) AB551930 Hayakawa et al. (2010)
C. longa (Indonesia C) AB551930 Hayakawa et al. (2010)
C. longa (Vietnam A) AB551930 Hayakawa et al. (2010)
C. longa (Vietnam B) AB551930 Hayakawa et al. (2010)
C. longa AB047738 Cao et al. (2001)
C. phaeocaulis AB047735 Cao et al. (2001)
C. roscoeana A AB047741 Cao et al. (Unpubl.)
C. roscoeana B AF478839 Kress et al. (2002)
C. sichuanensis A AB047739 Cao et al. (Unpubl.)
C. sichuanensis B AB047740 Cao et al. (Unpubl.)
C. thorelii AF478841 Kress et al. (2002)
C. wenyujin AB047746 Cao et al. (2001)
C. xanthorrhiza AB047752 Cao et al. (Unpubl.)
C. yunnanensis AB047749 Cao et al. (Unpubl.)
C. zedoaria AB551932 Hayakawa et al. (2010)
C. zedoaria A AB047734 Cao et al. (2001)
C. zedoaria B AB047743 Cao et al. (2001)
Boesenbergia rotunda AF478827 Kress et al. (2002)
Cautleya spicata AF478834 Kress et al. (2002)
Cornukaempferia auran tiflora AF478835 Kress et al. (2002)
Curcumorpha longiflora AF478842 Kress et al. (2002)
Kaempferia marginata AB232054 Sitthithaworn and Komatsu (Unpubl.)
Scaphochlamys biloba AF478889 Kress et al. (2002)
Zingiber mioga AB047755 Cao et al. (Unpubl.)
Copyright © 2011 SciRes. AJPS
Difference of Curcumin Content in Curcuma longa L. (Zingiberaceae) Caused by 115
Hybridization with Other Curcuma Species
Figure 1. Phylogenetic tree of Curcuma and its allied species in the matK gene of cpDNA using the neighbor-joining (NJ)
method. The numbers below the branc he s indic a te the bootstrap value .
Figure 2. Phylogenetic tree of Curcuma species in the ETS region of nrDNA using the NJ method. The numbers below the
branches indicate the bootstrap value.
Copyright © 2011 SciRes. AJPS
Difference of Curcumin Content in Curcuma longa L. (Zingiberaceae) Caused by
Hybridization with Other Curcuma Species
Copyright © 2011 SciRes. AJPS
4. Discussion that C. longa (Wakayama B) was introgressive with
clade 2 because it had the cpDNA haplotype of C. longa.
However, some individuals could not be sequenced in the
ETS region because of the putative heterogeneity of C.
longa and other Curcuma species. To detect their het-
erogeneity, we conducted a PCR-RFLP analysis because
restriction of the site of Hinf I to distinguish C. longa
with other Curcuma species was in the ETS region (Fig-
ure 3). The result was that the digestion pattern of all
samples of homogeneous C. longa and C. aromatica
showed the expected patterns, and heterogeneous C.
longa showed the combined patterns of homogeneous C.
longa and C. aromatica (Figure 4). Moreover, C. longa
(Wakayama B) showed same band pattern as C. aro-
matica. We therefore confirmed that the medium and low
curcumin contents of C. longa were hybrid and intro-
gressive between C. longa and other Curcuma species on
clade 2 (Figure 4, Table 1).
In general, hybrids typically display a mosaic of parental
and intermediate morphological characters, although
extreme and novel characters appear quite often. A spe-
cies with morphological characteristics intermediate be-
tween two recognized species has always been consid-
ered to be a hybrid [31]. However, morphological char-
acteristics alone, such as the rhizomes of the Curcuma
species, are of limited value when identifying natural
hybrids, but molecular studies have greatly enhanced our
knowledge in this field [32]. Interspecific hybrids are
most commonly identified by the heterogeneity of nrDNA
and the incongruences between cpDNA and nrDNA phy-
logenies that may indicate different parental contributions
to the hybrid genome [33,34]. In particular, incongruence
between cpDNA and nrDNA phylogenies is very likely
the result of interspecific gene flow and subsequent
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
C. longa
C. aromatica
Figure 3. Expected restriction sites of Hinf I for molecular characteristics of ETS regions by PCR-RFLP. H: restriction site.
Difference of Curcumin Content in Curcuma longa L. (Zingiberaceae) Caused by 117
Hybridization with Other Curcuma Species
Figure 4. PCR-RFLP profile of various lines of C. longa and C. aromatica. Arrows indicate expected fragments of both C.
longa and C. aromatica. M: size marker. A: C. aromatica. L: C. longa. Plant number corresponds to the numbers in Table 1.
chloroplast capture. In fact, some studies indicate that
introgression and asymmetric capture of the cpDNA are
common phenomena in hybridized species [19,35]. Our
results indicated that hybrid and introgressive individuals
with other Curcuma species were included in C. longa
although hybrid and introgressive have same haplotype
of C. longa based on matK sequences of cpDNA (Figure
1). Moreover, it is very interesting that homogeneous C.
longa has a high curcumin content, and that a heteroge-
neous hybrid of the Curcuma genome has a medium
amount of curcumin. Additionally, an introgressive sam-
ple with incongruent haplotypes between cpDNA and
nrDNA has a low content of curcumin (Table 1, Figure
4). Therefore, the pattern of decreased curcumin content
was congruent with the hybridization or introgression
between C. longa and other Curcuma species, such as C.
aromatica, which have low curcumin content. These re-
sults indicated that hybridization or introgression with
other Curcuma species could affect the content of cur-
cumin of C. longa. In this study, C. longa proved to be
the seed parent of the hybrid and introgression samples
because all of the haplotypes on the matK of cpDNA
matched this species. In the future, an analysis of the
curcumin content of hybrid and introgression samples in
which C. longa is the pollen parent needs to be con-
ducted. In addition, the recent resurgence in plant devel-
opment study has been accelerated, in part, by success in
elucidating the molecular genetic basis of plant devel-
opmental processes, including the isolation and charac-
terization of genes that synthesize curcumin in C. longa
[36-38]. As a result of these studies, it is considered very
important to isolate and analyze the homologous genes of
C. longa apart from other Curcuma species. As for hy-
brids, Allard [39] claimed that interspecific hybrids are
very useful in introducing genetic divergence, and, in
fact, hybrids have been used for many crops and orna-
mental plants. The way it was stated now appears that it
is negating the well established knowledge of hybrid
vigor and our results could not support this claim because
interspecific hybrids contribute to the decrease in the
curcumin content of C. longa.
Genetic variation is one of the fundamental underpin-
nings of biological diversity. The genetic structure and
history of a given species is an important research focus,
because this knowledge is needed to plan species con-
servation and to understand the evolutionary processes
leading to diversity. Our study results suggest that re-
productive isolation mechanisms were not acting in the
case of the small phylogenetic distances among the Cur-
cuma species (Figure 1). The evolution of reproductive
isolation is one of the defining characteristics of speci-
ation [40], and reproductive isolation contributes to the
diversification of species by creating genetically inde-
pendent lineages and phylogenetic tree branches [41].
Each branching of the tree is a speciation event; however,
reproductive isolation alone does not create a new branch.
Each branch by itself cannot produce the phenotypic di-
vergence represented by the angular departure of a
branch from the ancestral form [41]. Therefore, the di-
versification in the Curcuma species may have other
factors involved rather than just reproductive isolation. In
the future, it will be necessary to consider the phyloge-
netic implications in order to understand the detailed
evolutionary history of the Curcuma species.
In summary, we have provided a hypothesis for the
differences of curcumin by analyzing cpDNA and nrDNA
Copyright © 2011 SciRes. AJPS
Difference of Curcumin Content in Curcuma longa L. (Zingiberaceae) Caused by
Hybridization with Other Curcuma Species
data. Our results, using a molecular approach, were highly
effective in revealing the histories of hybridization and
introgression of C. longa. However, our data was less
effective in definitively answering the question concern-
ing the differences of curcumin content. Further studies
will be needed to determine whether more comprehensive
samplings and additional genetic evidence support the
working hypothesis we have developed here.
5. Acknowledgements
We wish to thank T. Kobayashi, A. Miyazaki, R. Ara-
kawa and J. Yokoyama. T. Yoshida, A. Matsuzawa, A.
Hirata, Y. Muramatsu, M. Saito, R. Ueda, K. Ohga, N.
Yokoyama, M. Muroi, and K. Matsuyama for providing
much help. This study was partly supported by a grant-
in-aid for scientific research from the Ministry of Educa-
tion, Science, and Culture of Japan (T.F.).
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