American Journal of Plant Sciences, 2011, 2, 15-26
doi:10.4236/ajps.2011.21002 Published Online March 2011 (http://www.SciRP.org/journal/ajps)
15
Development of a Molecular Marker to Identify a
Candidate Line of Turmeric (Curcuma longa L.)
with a High Curcumin Content
—Development of Molecular Marker of Turmeric
Hiroshi Hayakawa1,2, Tetsuya Kobayashi2, Yukio Minaniya2,3, Katsu ra Ito4, Akira Miyazaki2,
Tatsuya Fukuda2*, Yoshinori Yamamoto2
1United Graduate School of Agricultural Sciences, Ehime University, Monobe, Nankoku, Japan; 2Faculty of Agriculture, Kochi Uni-
versity, Monobe, Nankoku, Japan; 3Graduate School of Integrated Arts and Sciences, Kochi University, Monobe, Nankoku, Japan;
4JST Innovation Satellite Kochi, Laboratory of Applied Entomology, Faculty of Agriculture, Kochi University, Monobe, Nankoku,
Japan.
Received October 13th, 2010; revised November 15th, 2010; accepted November 29th, 2010.
ABSTRACT
Dried and fresh rh izomes of the spice turmeric (Curcuma longa L.) are well kno wn in traditional medicine, and curcu-
min is widely used in various geograph ic regions. Although there are differences in th e amount of curcumin within this
species, identification of the ca ndidate lin e by rhizome is difficult beca use of the relative simplicity of its morp hologica l
characteristics. To accura tely identify lines of C. longa with a high content of cu rcumin, we analysed several sequences
of chloroplast DNA. First, to determine the appropriate outgroup taxa in which to conduct infra specific analyses of C.
longa, we reconstructed the molecular phylogenetic tree of C. longa and its allied species. The results showed that C.
aromatica and C. zedoa ria are closely related to C. longa. Next, to develop a molecular marker for identifying lines of
C. longa with a high content of curcumin, a network analysis using chloroplast microsatellite regions was performed.
Results showed that a unique haplotype within C. longa corresponds to the high curcumin content line. Therefore, the
chloroplast microsatellite regions used for the analysis allowed us to determine the lines of this species with high cu r-
cumin content.
Keywords: Chloroplast DNA, Curcuma, Curcuma longa, Curcumin, Molecular Analysis
1. Introduction
Traditional medicine is known to be fertile ground for the
source of modern medicines [1]. One medicine in that
category is curcumin, a yellow coloring agent present in
the spice turmeric (Curcuma longa L.) that belongs to the
ginger family (Zingiberaceae). Besides its use in cooking
to add color and as a preservative, turmeric is used in
Indian traditional medicine to treat various common ail-
ments including stomach upset, flatulence, dysentery,
ulcers, jaundice, arthritis, sprains, wound s, acne, and skin
and eye infections [2]. In this way, curcumin is widely
used in various geographic regions in which the mor-
phologies of the dried or fresh rhizomes of Curcuma
plants are highly similar, and it is therefore difficult to
correctly identify individual Cur cu ma species.
Recent molecular phylogenetic studies have revealed
new aspects of the relationships among Curcuma species.
For example, to identify the rhizomes of C. longa, Sasaki
et al. [3] developed a molecular identification method fo r
Curcuma species using an amplification-refractory muta-
tion system analysis of 18S ribosomal ribonucleic acid
(rRNA) and the trnK gene. Although this method can
identify species in a line of samples with high accuracy,
the process is complicated. This marker is sensitive to
experimental conditions of the polymerase chain reaction
(PCR) because judgment is based on the presence or ab-
sence of PCR products. In addition, Minami et al. [4]
reported that Curcuma species can b e identified using an
intergenic spacer between trnS and trnfM of chloroplast

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
16
deoxyribonucleic acid (cpDNA). Although this method is
useful for identifying C. longa from various Curcuma
rhizomes, the study by Minami et al. [4] may require
re-evaluation since the number of sample species used
were few, and it did not employ appropriate outgroup
taxa. The same can be said of the study by Sasaki et al.
[3]. In our view, out-groups are important for assessing
plesiomorphic or apomorphic states for each characteris-
tic and for interpreting the phylogenetic relationships
within C. longa. Alternatively, Aoi et al. [5] reported a
difference in the amount of curcumin found among indi-
viduals of C. longa. Therefore, it is necessary to use in-
fraspecific genetic markers to determine the amount of
curcumin in individuals of this species in order to acquire
it efficiently.
Large-scale sequencing of a predefined region of ap-
proximately 1500 base pairs (bp) of the cpDNA matK
has one main goal in monocotyledonous plants including
C. longa: to clarify the phylogen etic position and closely
related species in order to determine the outgroup taxa of
unknown individuals [6]. Although the sequence of the
matK gene plays an important role in identifying the out-
group taxa of C. longa, it cannot be us ed to identify lin es
in this species containing high amounts of curcumin be-
cause of its low substitution rate [7]. Thus, determining
more reliable genetic markers within C. longa requires
more informative DNA regions. In recent years, cpDNA
sequence data have been used frequently to reconstruct
the phylogenetic relationships of a wide range of land
plants [8]. Some regions were reported to have nucleo-
tide substitution rates that are sufficiently high to facili-
tate the elucidation of relationsh ips among closely related
species [7,9]. Moreover, the utility of non-coding cpDNA
regions within species-level phylogeny has been demon-
strated in many taxonomic groups of land plants [10].
However, phylogenetic analysis using regions with high
substitution rates, such as microsatellites, is often biased
by multiple substitutions at a single site. In addition, be-
cause algorithms of phylogenetic analysis can cause
problems when data do not represen t a tree-like structure
[11], we also analysed the cpDNA sequences with the
statistical parsimony network approach. This approach
reflects the genealogical relationships of the sequences
used; that is, single-mutation steps separate adjacent
haplotypes in the network, and older haplotypes are
placed at internal branching points, whereas younger
haplotypes occur towards the tip position. In the current
study, we show that this analysis of th e infraspecific rela-
tionships within C. longa can result in identification of
clear relationships not only among young and rapidly
speciating groups but also among old lineages reaching
deep into the diverg ence of this group.
Thus, the aim of our study was to develop infraspecific
genetic primers, using a phylogenetic approach, for iden-
tifying lines of C. longa with high curcumin content.
2. Materials and Methods
2.1. Cultivation and Measurements
The experiments were carried out in the fields (sandy soil)
of the Faculty of Agriculture, Kochi University, Japan
for four years (2006 -2009). Ta b le 1 lists th e sample lines
used for the cultivation experiments in this study: 2 C.
aromatica Salisb., 12 C. longa L., and 1 C. zedoaria
Rosc. Rows were constructed with a width of 70 cm and
height of 20 cm. Rhizomes were transplanted 8 cm below
the soil surface in one-row ridges, on hills separated by a
distance of 30 cm, in late May for four years. For fertile-
izer 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 four years. In addi-
tion, 200 kg/a of compost fertilizer, 15 kg/a of magnesia
lime, and 30 kg/a of chicken droppings were applied
(Chicken droppings were not used in 2006.). The ex-
perimental 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 without replicate or two rows. Rows were
covered by silver mulch. Irrigation was performed using
a sprinkler, with observation of the amount of precipita-
tion and the condition of th e plants. Fo ur or six h ills con-
taining average-sized shoots per line were sampled in
early December each year for four years.
For curcumin analysis, the curcuminoid content of cur-
cumin 1, curcumin 2, and curcumin 3 (curcumin, deme-
thoxycurcumin, and bis-deme thoxy curcumin, respectively)
of the primary branch rhizome was measured by high
performance liquid chromatography (HPLC, HITACHI,
L-2420), according to the method described by Sato et al.
[12]. We extracted curcumin by 80% of ethanol from 0.3
g of the dried and gro und samples. H PLC was perfo rmed
on a column of YMC-Pack Pro C18. Mixed acetonitrile
with 0.1% of phosphoric acid (50:50) was used for the
mobile phase. Current flowed at a rate of 1 mL per min.
The amount of injection was 10 µL.
2.2. DNA Analysis
Total DNA was isolated from fresh root with a Plant
Genomic DNA Mini Kit (Viogene, Sunnyvale, CA, USA),
according to the manufacturer’s protocol. The PCR mix
contained approximately 100 to 200 ng of total DNA, 1
µM of each primer, 200 mM of each deoxynucleotide, 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM of MgCl2,
and 1.25 units o f Taq po lymerase. Doub le-stranded D NA
was amplified, after incubation at 94˚C for 2 min, by 45

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
17
Table 1. List of sample of curcuma species used for cultivation experiments in this study.
Nos. analysed hill1)
Species Locality
2006 2007 20082009
Curcuma aromatica (Kochi) Kochi Prefecture, Japan 6 6 4 4
C. aromatica (Tanegashima) Tanegashima Island, Kagoshima Prefecture, Japan 6 6 6 4
C. longa (Kochi) Kochi Prefecture, Japan 6 6 6 4
C. longa (Tanegashima) Tanegashima Island , Kagoshima P r efecture, Japan 6 6 6 4
C. longa (Wakayama A) Wa k ayama Prefecture, Japan 6 6 4 4
C. longa (Wakayama B) Wakayama Prefecture, Japan 6 4 4 4
C. longa (Wakayama C) Wakayama Prefectur e, Japan ---2) 6 4 4
C. longa (Okinawa A) Okinawa Prefecture, Japan --- --- 4 4
C. longa (Okinawa B) Okinawa Prefecture, Japan --- --- 4 4
C. longa (Indonesia A) Bogol, West Java, Indone sia 4 4 4 4
C. longa (Indonesia B) Bogol, West Java, Indonesia --- 4 4 4
C. longa (Indonesia C) Bogol, West Java, Indonesia --- --- --- 4
C. longa (Vietnam A) Vietnam --- --- --- 4
C. longa (Vietnam B) Vietnam --- --- --- 4
C. zedoaria Unknown 4 4 4 4
1) Four or s i x hills conta ining average-sized shoots per line were harvested; 2) Not cultivated.
cycles of incubation at 94˚C for 1.5 min, 48˚C for 2 min,
and 72˚C for 3 min, with final extension at 72˚C for 15
min. We amplified four regions from cpDNA, namely
matK, rp l16 intron 2, petB intron 1, and petB intron 2,
with primers designed by Johnson and Soltis [13] and
Nishizawa and Watano [9]. After amplification, reaction
mixtures were subjected to electrophoresis in 1% low
melting-temperature agarose gels for separation of spe-
cific amplified products. We sequenced the purified PCR
products using a Big Dye Terminator Cycle Sequencing
Kit (ABI PRISM, Perkin Elmer Applied Biosystems,
Foster City, CA, USA) and an ABI PRISM 3100-Avant
Genetic Analyzer according to the manufacturer’s in-
structions. The primers used for sequencing were the
same as those used for amplification.
To construct a phylogenetic tree based on matK se-
quences of Curcuma and its allied species, the amplified
regions were aligned using ClustalW [14] and were im-
proved manually using MEGA 4 [15]. Phylogenetic rela-
tionships were analysed using the neighbour-joining (NJ)
method with PAUP* 4.08b [16]. The NJ analyses were
performed 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. In ad-
dition, a network tree of microsatellite reg ions of cpDNA
was constructed with Network 1.4.1.2 (Fluxus Technol-
ogy Ltd., Suffolk, UK) using the median-joining method
[17].
3. Results
3.1. Curcumin Content
We mainly describe results of the cu ltivation experiments
for 2009, since data for the previous years of the study
(2006, 2007, and 2008) showed similar trends.
Table 4 shows the curcumin content in the maturity
period for the years 2006 through 2009. With respect to
the curcumin content of primary branch rhizomes in the
maturity period, total curcuminoid content, in order from
highest to lowest, was as follows: South Asian C. longa
(3198.4-2315.1 mg/100 g), with the exception of Indone-
sia B > domestic C. longa (305.0-392.2 mg/100 g), with
the exception of Wakayama B > Indonesia B (309.5
mg/100 g) > C. aromatica (121.9-126.9 mg/100 g). In-
donesia A and B and Vietnam A and B showed a high
content of accumulated curcuminoid in the rhizome.
However, C. longa (Wakayama B) and C. zedoaria

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
18
Table 2. Accession numbers of matK using phy logenetic analy sis of Curcuma and outgroup taxa.
matK
Species Accession No. Reference
Curcuma aeruginosa AF478840 Kress et al. (2002)
C. amarissima AB047751 Cao et al. (Unpubl.)
C. aromatica AB047731 Cao et al. (2001)
C. aromatica (Kochi) AB551929 this study
C. aromatica (Tanegashima) AB551929 this study
C. aromatica (Okinawa) AB551929 this study
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 this study
C. longa (Tanegashima) AB551930 this study
C. longa (Wakayama A) AB551930 this study
C. longa (Wakayama B) AB551931 this study
C. longa (Wakayama C) AB551930 this study
C. longa (Okinawa A) AB551930 this study
C. longa (Okinawa B) AB551930 this study
C. longa (Indonesia A) AB551930 this study
C. longa (Indonesia B) AB551930 this study
C. longa (Indonesia C) AB551930 this study
C. longa (Vietnam A) AB551930 this study
C. longa (Vietnam B) AB551930 this study
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. xanthorrhiz a AB047752 Cao et al. (Unpubl.)
C. yunnanensis AB047749 Cao et al. (Unpubl.)
C. zedoaria AB551932 this study
C. zedoaria A AB047734 Cao et al. (2001)
C. zedoaria B AB047743 Cao et al. (2001)
Outgroup
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.)

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
19
Table 3. Accession numbers bas ed on network analysis of Curcuma longa and allied species.
Type of chloroplast DNA
Species rps16 intron 2 petB intron 1 petB intron 2 Haplotype
Curcuma zedoaria a1) e g A
C. aromatica (Kochi) b f i B
C. aromatica (Tanegashima) b f i B
C. longa (Kochi) c e j D
C. longa (Tanegashima) c e j D
C. longa (Wakayama A) c e j D
C. longa (Wakayama B) c e j D
C. longa (Wakayama C) c e j D
C. longa (Okinawa A) c e j D
C. longa (Okinawa B) c e j D
C. longa (Indonesia A) d f h C
C. longa (Indonesia B) c e j D
C. longa (Indonesia C) d f h C
C. longa (Vietnam A) d f h C
C. longa (Vietnam B) d f h C
1) Type of chloroplast DNA. Accession numbers; a: AB557651, b: AB557648, c: AB557650, d: AB557649, e: AB557653. f: AB557652, g: AB557657, h:
AB557655, i: AB557654, j: AB5576 5 6. Haploty pe is corresponded to Figure 3.
showed little curcuminoid content. The inside color of
the rhizome in C. longa (Wakayama B) and C. zedoaria
was white and purple, respectively, whereas C. longa,
which showed an accumulation of curcuminoid, had a
yellow-colored rhizo me (Figure 1).
In order from highest to lowest of each content of
curcumin 1, 2, and 3 showed a similar trend with the or-
der of total curcuminoid con tent. With respect to th e ratio
of its curcumin 1, 2, and 3 in domestic C. longa (with the
exception of Wakayama B) was 69%-71%, 21%-23%,
and 8%-9%, respectively. However, the ratio of curcu-
minoid content in South Asian C. longa (with the excep-
tion of Indonesia B) was 46%-52% (curcumin 1),
26%-28% (curcumin 2), and 22%-26% (curcumin 3),
indicating a low percentage of curcuminoid in curcumin
1 and a high percentage of curcuminoid in curcumin 3,
compared with domestic C. longa. The ratio of curcu min
1, 2, and 3 in Indonesia B differed from that in Indonesia
A and C and more closely resembled domestic C. longa,
despite the fact that Indonesia A, B, and C originated in
the same country. In C. aromatica, the ratio of curcumin
1, 2, and 3 was 47%-53%, 47%-52%, and 0-1%, respect-
tively. C. aromatica also had a high percentage of cur-
cumin 2.
The relative order of curcuminoid content in the pri-
mary branch rhizomes was similar throughout the four
years of the study. Morishita et al. [18] reported that
year-to-year variation in curcuminoid content is more
vigorous than that of the varietal differences in turmeric.
In the present study, the difference of curcuminoid con-
tent in Curcuma species had not only varietal difference
but also year-to-year variation.
3.2. DNA Analysis
To construct the molecular phylo genetic tree o f Curcuma
and its allied species, we determined sequences of the
matK gene of Curcuma cpDNA and seven outgroups of
Boesenbergia rotunda, Cautleya spicata, Cornukaemp-
feria aurantiflora, Curcumorpha longiflora, Hedychium
greenei, Scaphochlamys biloba , and Zingiber mioga. The
lengths of the matK gene of Curcuma species varied
from 1831 bp (C. longa (Wakayama B)) to 1846 bp (C.
thorelii).
The results of the phylogenetic analysis indicated that
Curcuma species consists of a monophyletic group (Fig-
ure 2), with the genus Curcu ma primarily divided into
two monophyletic groups: clade 1 and clade 2. Clade 1,
located at the most basal monophyletic position of Cur -

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
20
Table 4. Curcumin content at maturity 2006-2009.
Year Species Curcumin 1 content1) Curcumin 2 content1) Cu rcumin 3 content1) Curcuminoids content1)
Curcuma aromatica (Kochi) 59.5efg (47)2) 65.7c (52)2) 1.0d (1)2) 126.2c (100)
C. aromatica (Tanegashima) 64.7defg (53) 56.7c (47) 0.5d (0) 121.9c (100)
C. longa (Kochi) 274.6c (70) 87.5c (22) 30.1d (8) 392.2c (100)
C. longa (Tanegashima) 254.6cde (70) 77.7c (22) 29.1d (8) 361.3c (100)
C. longa (Wakayama A) 260.3cde (69) 83.9c (22) 30.3d (8) 374.5c (100)
C. longa (Wakayama C) 268.4cd (69) 89.0c (23) 32.1d (8) 389.5c (100)
C. longa (Okinawa A) 246.6cde (71) 72.7c (21) 27.8d (8) 347.1c (100)
C. longa (Okinawa B) 213.8cde (70) 63.5c (21) 27.7d (9) 305.0c (100)
C. longa (Indonesia A) 1588.0a (52) 813.6a (26) 657.5b (22) 3059.1a (100)
C. longa (Indonesia B) 209.1cdef (68) 72.7c (23) 27.8d (9) 309.5c (100)
C. longa (Indonesia C) 1169.9b (50) 618.2b (27) 526.9c (23) 2315.1b (100)
C. longa (Vietnam A) 1427.1a (48) 840.1a (28) 709.5b (24) 2976.7a (100)
C. longa (Vietnam B) 1474.7a (46) 884.8a (28) 838.9a (26) 3198.4a (100)
C. longa (Wakayama B) 0.5g (49) 0.7c (51) 0.0d (0) 1.c (100)
2009
C. zedoaria 0.8fg (70) 0.4c (30) 0.0d (0) 1.2c (100)
C. aromatica (Kochi) 19.2b (41) 27.1b (58) 0.2b (1) 46.5b (100)
C. aromatica (Tanegashima) 15.4b (42) 20.9b (58) 0.1b (1) 36.3b (100)
C. longa (Kochi) 239.1ab (67) 86.4ab (24) 32.2ab (9) 357.6ab (100)
C. longa (Tanegashima) 273.3ab (70) 85.6ab (22) 32.8ab (9) 391.7ab (100)
C. longa (Wakayama A) 268.4ab (69) 84.2ab (22) 35.8ab (10) 388.3ab (100)
C. longa (Wakayama C) 271.6ab (69) 88.0ab (22) 35.9ab (9) 395.5ab (100)
C. longa (Okinawa A) 256.0ab (70) 78.9ab (22) 29.0ab (8) 364.0ab (100)
C. longa (Okinawa B) 265.0ab (71) 74.8ab (22) 33.3ab (9) 373.1ab (100)
C. longa (Indonesia A) 1294.7a (49) 652.5a (24) 730.6a (27) 2677.8a (100)
C. longa (Indonesia B) 229.7ab (68) 77.0ab (23) 29.9ab (9) 336.6ab (100)
C. longa (Wakayama B) 0.4b (71) 0.3b (29) 0.0b (0) 0.7b (100)
2008
C. zedoaria 0.5b (49) 0.6b (51) 0.0b (0) 1.0b (100)
C. aromatica (Kochi) 30.60abc (43) 39.35ab (56) 0.87ab (1) 70.81b (100)
C. aromatica (Tanegashima) 37.53ab (48) 38.74abc (51) 1.54ab (2) 77.81bc (100)
C. longa (Kochi) 260.38bcd (65) 96.32bcd (24) 41.01bc (10) 397.71ab (100)
C. longa (Tanegashima) 259.06bcd (68) 86.29bcd (23) 36.88bc (10) 382.24ab (100)
C. longa (Wakayama A) 270.04ab (67) 93.36ab (23) 39.65ab (10) 403.05ab (100)
C. longa (Wakayama C) 266.83abcd (67) 97.22abc (24) 40.19abc (10) 404.24abc (100)
C. longa (Indonesia A) 1276.73a (53) 687.15a (25) 813.05a (23) 2776.93a (100)
C. longa (Indonesia B) 149.72abcd (66) 58.11abcd(25) 21.17abc (9) 229.00abc (100)
C. longa (Wakayama B) 1.18d (51) 0.99cd (49) 0.10c (4) 2.27c (100)
2007
C. zedoaria 1.28cd (67) 0.38d (29) 0.08c (5) 1.74c (100)
C. aromatica (Kochi) 16.05bc (43) 20.96ab (56) 0.60abc (2) 37.61bcd (100)
C. aromatica (Tanegashima) 18.36abc (45) 22.02ab (54) 0.51bc (1) 40.89abcd (100)
C. longa (Kochi) 226.72ab (69) 68.90a (21) 31.60ab (10) 327.21ab (100)
C. longa (Tanegashima) 219.47ab (73) 56.67a (19) 25.23ab (8) 301.36abc (100)
C. longa (Wakayama A) 127.72abc (71) 37.66ab (21) 13.87abc (8) 179.25abcd (100)
C. longa (Indonesia A) 1358.64a (48) 679.14a (24) 811.43a (28) 2849.21a (100)
C. longa (Wakayama B) 0.26c (34) 0.43b (56) 0.07c (9) 0.76d (100)
2006
C. zedoaria 0.43c (57) 0.33b (43) 0.00c (0) 0.76cd (100)
1) Curcumin content in primary branch rhizomes (mg/100g). 2) Percentage of curcumin 1, 2, and 3 content to curcuminoids content. Values followed by the
same letter in a column are not significantly different at 5% level by one-way ANOVA.

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
21
(a) (b)
(c) (d)
Figure 1. Rhizomes of the genus Curcuma. (a) C. aromatica (typical type); (b) C. longa (WakayamaB); (c) C. longa (Kochi); (d)
C. zedoaria. Scale bar = 5 cm.
cuma, includes C. aeruginosa, C. bicolor, and C. thorelii.
Clade 2 includes the following 17 species: C. amarissima,
C. aromatica, C. chuanezhu, C. chuanhuangjiang, C.
chuanyujin, C. comosa, C. elata, C. exigua, C. kwangsien-
sis, C. longa, C. phaecaulis, C. roscoeana, C. sichuanen-
sis, C. wenyujin, C. xanthorriza, C. yunnanensis, and C.
zedoaria. With the exception of C. roscoeana B, clade 2
was further divided into two groups: subclade 1 and sub-
clade 2. Subclade 1 included the following 12 samples: C.
chuanezhu, C. comosa, C. elata, C. exigua, C. kwang-
siensis B, C. phaeocaulis, C. sichuanensis B, C. xantho r-
riza, C. yunnanensis B, C. zedoaria, C. zedoaria A, and
C. zedoaria B. The remaining Curcuma species were
grouped under subclade 2.
Six species, C. aromatica, C. longa, C. kwangsiensis,
C. roscoeana, C. sichuanensis, and C. zedoaria, were
analysed using more than one sample. Of these, C.
kwangsiensis (subclades 1 and 2), C. roscoeana (clade 2
and subclade 1), and C. sichuanensis (subclades 1 and 2)
contained paraphyletic assemblages. Although three
samples of C. zedoaria were included in the same clade
(subclade 1), each sample belonged to a different lineage
within the subclade; therefore, th is species also was con-
sidered to be paraphyletic. Four samples of C. aromatica
were included in the monophyletic group with C.
chuanyujin and C. chuanhuan gjiang in subclade 2. How-

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
22
Figure 2. Phylogenetic tree of Curcuma and its allied species using the neighbour-joining (NJ) method. The numbers below
the branches indicate the bootstrap value. Black squares indicate high curcumin cont ent. Black circles indicate medium cur-
cumin content. White circles indicate low curcumin content.
ever, because there was no synapomorphic character in
this group, we were unable to determine whether C. aromatica was monophyletic or paraph yletic. In add itio n, 13 samples of C. longa did not share a synapomorphic

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
23
character that would place them in the monophyletic
group; therefore, this species is also considered to show
polytomy. We were unable to determine whether C.
longa was monophyletic or paraphyletic.
To establish the genetic polymorphisms within C.
longa, we determined the chloroplast microsatellite re-
gions of rp l16 intron 2, petB intron 1, and petB intron 2
of C. aromatica, C. longa, and C. zedoaria. The se-
quence lengths of rpl16 intron 2, petB intron 1, and petB
intron 2 were 42-46 bp, 168-169 bp, and 47-62 bp, re-
spectively. Network relationships of each individual by
statistical parsimony showed that C. longa included two
haplotypes: C and D (Figure 3). The genetic relationship
between haplotype C and D was distant. It is notable that
haplotype C was closely related to C. aromatica and C.
zedoaria despite haplotype C being the most distinct of
the C. longa haplotypes.
4. Discussion
4.1. Phylogenetic Relationship of Curcuma and
Its Allied Species
To clarify the phylogenetic relationships of Curcuma and
its allied species, we determined the nucleotide se-
quences of the cpDNA matK region for all species of the
genus examined in this study. Only a few characters were
considered in the phylogenetic analysis. We suggest that
the degree of genetic differentiation among species of the
genus Curcuma is small, despite the fact that these spe-
cies are morphologically diverse, as evidenced by shoot/
root architecture and floral coloration. Therefore, it is
likely that the genus lineage has undergone recent, rapid
Figure 3. Parsimony network of C. longa, C. aromatica, and
C. zedoaria based on cpDNA rpl16 intron 2, petB intron 1
and petB intron 2. Haplotype letters correspond to those in
Table 3. White circles indicate the taxa in which haplo-
types were found. Black circles between haplotypes rep-
resent a mutational step.
radiation. To confirm the scenario of rapid radiation in
the genus Curcu ma, we also determined the high-sub-
stitution regions of the regions of the rps16 intron (ac-
cession number AB557658) and trnG (UCC) intron (ac-
cession numbers AB557659 and AB557660), and the
two intergenic regions of trnV (UAC)-trnM (CAU) (ac-
cession number AB55766) and petD-rpoA (accession
number AB557662). No variable characters were de-
tected even though the utility of these regions for intras-
pecific phylogeny was well determined [9]. This result
also supports the hypothesis that the evolutionary history
of the Curcum a genus underwent re c ent divers ificati o n.
Several reports of the rapid radiation of continental
plant species have been published [19,20], although most
of these examples are reported to have occurred on oce-
anic islands [21,22]. In most oceanic examples, rapid
radiation is hypothesized to have been driven by low
levels of competition in new habitats [23]. However,
none of the samples used in the present study occur on
oceanic islands. Alternatively, the concept of key inno-
vations often is employed to explain the rapid radiation
of a lineage [20,23,24]; therefore, the evolution of any
key innovations in Curcuma may lead to rapid radiation.
In the future, more studies should compare the morphol-
ogic and/or physiologic characters of Curcuma and its
allied species to determine the nature of any key innova-
tions.
4.2. Genetic Markers to Identify Lines with High
Curcumin Content
Simple molecular marker assays that do not rely on spe-
cialized equipment or reagents underpin routine research
activities in many laboratories worldwide. Recent re-
search has shown that the non-coding portion of the plas-
tid genome is more variable than previously anticipated
[9]. Mutational hot spots have been reported from in-
tronic or intergenic cpDNA regions of a number of or-
ganisms. Such hot spots are usually characterized by the
presence of mononucleotide repeats [25] and a high in-
cidence of duplications and other types of indels [26,27].
Short inversions [28] and minisatellites [29,30 ] have also
been identified but seem to occur less frequently. In the
present study, we were able to determine the sequencing
repeat of three regions in Curcuma species, rpl16 intron
2, petB intron 1 and petB intron 2, providing ev idence fo r
additional and previously unrecognised types of poly-
morphic regions in the cpDNA of this group.
Identifying mutational hot spots in cpDNA is of ut-
most interest for studies below the species level. These
so-called microsatellites have been used to detect intras-
pecific chloroplast polymorphisms in various species
from a number of plant families [7]. In many cases, the

Development of a Molecular Marker to Identify a Candidate Line of Turmeric (Curcuma longa L.) with
a High Curcumin Content
24
amplified fragments also contained size-variable sites in
the target species. Thus, microsatellites may provide
reasonable genetic markers to amplify the polymorphic
chloroplast regions from any grasses, crops, fruits, and
vegetables of interest. As the position of mutational hot
spots in general is usually not conserved across species,
these primer pairs will have to be tested in any new taxon
on the basis of trial and error. However, the present re-
port could extend th e application of microsatellites to the
Curcuma species. In fact, we observed three instances of
haplotype sharing within C. longa, together with a con-
siderable degree of intraspecific variation. Moreover, it is
interesting that C. longa exhibits unique chloroplast hap-
lotypes (e.g. haplotype C) that are involved in lines with
high curcumin content (Figure 3). This result indicates
that the microsatellite regions are useful for identifying C.
longa candidate lines with high curcumin content. Fur-
ther studies will determine whether more comprehensive
sampling and addition al evidence will support the results
of the present study regarding the identification of C.
longa lines with high curcumin content.
In this study, we showed that the cpDNA of C. longa
can be divided into two groups, expecting to expose a
cryptic species with the appropriate biologic characteris-
tics. Cryptic species are closely related species that differ
critically in genetic, physiologic, behavioral, and eco-
logical traits [31], and the abundance of morphologically
cryptic or unrecognized species, even in well-known taxa,
suggests that there are more species than are currently
recognized or estimated [32]. Recent studies have re-
ported that cryptic species seem to be especially common
in certain taxa [33-36], and similar evidence for cryptic
species was found in our results. Therefore, in order to
verify this hypothesis with respect to C. longa, further
reciprocal crosses among the haplotypes discussed in the
present study may lead to a conclusion of cryptic diver-
sity. In the future, additional research will be needed to
determine if the various cryptic species differ in their
ecological requirements and tolerance to potential envi-
ronmental stresses. There is also the need to re-examine
the alpha taxonomy of the four species showing cryptic
diversity. Without this basic taxonomic research, these
cryptic species will remain in taxonomic crypsis [37].
Our limited data set does not allow us to decide the
most appropriate genetic marker at the present time. To
accurately map the distribution of C. longa, and to
monitor phylogeographic patterns within this species in
more detail, we are currently applying the chloroplast
markers developed in the present study to a large number
of specimens sampled throughout the distributional range
of this species. In addition, genetic and phylogenetic
studies in Curcuma species have lagged behind those of
other crop species. The network analysis presented here,
along with the polymorphic information presented, pro-
vide valuable genetic resources for future studies of C.
longa and related species.
5. Acknowledgements
We wish to thank R. Arakawa and J. Yokoyama, T. Yo-
shida, A. Matsuzawa, A. Hirata, Y. Muramatsu, M. Saito,
R. Ueda, K. Ohga, N. Yokoyama, M. Muroi, and K. Ma-
tsuyama for providing much help. This study was partly
supported by a grant-in-aid for scientific research from
the Ministry of Education, Science, and Culture of Japan
(to TF).
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