orawan theanphong1, witchuda …...2002/06/10 · curcuma plants have long been known for their...

BHST. 2016, 14 (1) : 45-56 Theanphong et al.

45

Bulletin of Health, Science and Technology

BHST ISSN 0858-7531

Volume 14, Number 1, 2016 : 45-56

RAPD MARKER FOR DETERMINATION OF PHYLOGENETIC RELATIONSHIPS

OF 15 CURCUMA SPECIES FROM THAILAND

Orawan Theanphong1, Witchuda Thanakijcharoenpath

1, Chanida Palanuvej

2,

Nijsiri Ruangrungsi2, 3

and Kanchana Rungsihirunrat2*

1 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,

Chulalongkorn University, Bangkok 10330, Thailand

2 College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand

3 Faculty of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand

*Corresponding author : E-mail : [email protected]

Abstract: Curcuma, a rhizomatous herb belonging to the family Zingiberaceae, has been used

as a natural food additive, cosmetic and folk medicine in Thailand. According to the similar

morphology of Curcuma species, makes it difficult for species identification. In this study,

Random Amplified Polymorphic DNA (RAPD) was employed for determination of the

phylogenetic relationships among 15 Curcuma species from Thailand. Out of thirty random

deca-arbitrary primers, only four produced clear and reproducible polymorphic bands.

Twenty-two to twenty-eight products were amplified, with an average of 24.5 bands by each

primer. A total of 98 bands ranging from 208 to 4136 base pairs in size were amplified, among

which 39 products were found to be polymorphic. The similarity index (SI) ranged from

0.0909-0.9222. The dendrograms were constructed based on unweighted pair group method

with arithmetic averages (UPGMA). The results from the cluster diagram could be divided into

three major groups and the phylogenetic relationships were correlated with the morphological

characteristics. In conclusion, RAPD marker was successfully for differentiating among

15 Curcuma species from Thailand and providing a simple and rapid tool for differentiation.

Keywords: Curcuma, Random Amplified Polymorphic DNA, phylogenetic relationships

บทคดยอ: ประเทศไทยมการน าพชสกล Curcuma วงศ Zingiberaceae มาใชเปนสารเตมแตงอาหาร, เครองส าอาง และยารกษาโรคอยางแพรหลาย แตการจ าแนกพชสกลนท าไดยากเนองจากพชแตละชนดมลกษณะทางพฤกษศาสตรทคลายคลงกนมาก ในการศกษาน Random Amplified Polymorphic DNA (RAPD) ถกน ามาใชในการศกษาความสมพนธทางวงศวานววฒนาการ ของพชในสกล Curcuma จ านวน 15 ชนด จากประเทศไทย โดยใชไพรเมอรแบบสมทมความยาว 10 เบส จ านวน 30 ไพรเมอร พบวา มเพยง 4 ไพรเมอรทปรากฏแถบดเอนเอ และ แถบดเอนเอทมแตกตาง ของพชตวอยางทกชนด จ านวนแถบ DNA พบอยระหวาง 22-28 แถบ เฉลยไพรเมอรละ 24.5 แถบ จ านวนแถบดเอนเอทปรากฏทงหมด 98 แถบ มขนาด 208 - 4136 คเบส และพบวาในจ านวนน 39 แถบเปนแถบดเอนเอทมความแตกตางกน โดยมคาดชนความคลายคลงอยระหวาง 0.0909-0.9222 พงศาวลสรางโดยวธ unweighted pair group method with arithmetic averages (UPGMA) พบวาพชในสกล Curcuma ถกแบงออกเปน 3 กลมหลก ซงผลทไดสอดคลองกบลกษณะทางพฤกษศาสตร โดยสรปเครองหมายดเอนเอชนด RAPD เปนวธทสะดวกและรวดเรวในการจ าแนกความแตกตางในของพชสกล Curcuma จ านวน 15 ชนด จากประเทศไทย ค ำส ำคญ: Curcuma, Amplified Polymorphic DNA, ความสมพนธทางวงศวานววฒนาการ


46

INTRODUCTION

Curcuma is one of the well-known genera of the family Zingiberaceae. The genus

consists of about 110 species widely distributed in tropical Asia and the Asia-Pacific region.

The greatest diversity occurs in India, Myanmar and Thailand and the distribution extends to

Korea, China, Australia and the South Pacific (Ravindran, Babu and Shiva, 2007). Several

Curcuma plants have long been known for their uses as food, spices and medicinal plants.

However, the botanical identity of many species is confusing owing to their similar

appearance and, probably, their natural hybridization (Skornickova, 2006). In addition,

a comprehensive taxonomic revision of the whole genus has not yet been accomplished as

there are some major problems in the taxonomic studies such as lack of type specimens and

illustrations of old species, lack of protologues with finer details in the earlier literature,

absence of important floral parts in the herbarium specimens, incomplete description of the

rhizome features in the herbarium sheets, fleshy and perishable aerial portions, etc.

(Sasikumar, 2005). As the taxonomic identification of plants in the genus Curcuma cannot

be accomplished effectively through the classical method based on plant morphology, more

information on other characters of the plants is required.

Recently, there are several DNA based molecular technique which are reliable and

powerful tools for identification of taxa at various infrageneric levels as they provide

consistent results irrespective of age, tissue origin, physiological conditions, environmental

factors, harvest, storage and processing of samples (Heubl, 2010). Several DNA marker

systems are now commonly used in genetic diversity analysis of plants. The random

amplified polymorphic DNA (RAPD) technique (Williams et al., 1990) is popularly used in

genetic studies. RAPD marker is a rapid, inexpensive and effective tool for studying genetic

relationships in various plants due to their advantages i.e. no need of prior knowledge of the

DNA sequence, the small amount of DNA used in the study and the ability to assay for many

loci simultaneously (Semagn, Bjornstad and Ndjiondjop, 2006; Zou, et al., 2011). The

RAPD technique has been reported of its application in the differentiation of several plants in

Zingiberaceae such as those of the genera Boesenbergia (Vanijajiva, Sirirugsa and

Suvachittanont, 2005), Kaempferia (Pojanagaroon et al., 2004; Vanijajiva, Sirirugsa and

Suvachittanont, 2005) and Curcuma (Sasikumar, 2005; Zou, et al., 2011; Kitamura, et al.,

2007; Syamkumar and Sasikumar, 2007).

Thus, the aim of this study was to evaluate the phylogenetic relationships of

15 Curcuma plants existing in Thailand using RAPD fingerprints. The results might provide

some useful information for taxonomic study of the genus Curcuma.

MATERIALS AND METHODS

Plant materials

Fresh rhizomes of 15 Curcuma species; compose of 12 identified Curcuma species,

3 unidentified Curcuma species and Zingiber montanum (outgroup plant) were collected in

June 2009 from different locations in Thailand (Table 1). The shape and colour of the

rhizomes were recorded. The rhizomes of all plant samples were employed for cultivation at

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical

Sciences, Chulalongkorn University, Bangkok, Thailand, for 1-2 months. Morphological

characters of the cultivated plants e.g. leaf shape, leaf base, leaf apex, colour of midrib,

colour of coma bract were recorded.


47

Table 1. Details of the plant samples used in the study

Plant samples Code Locality

C. aeruginosa Roxb. AE Prachin Buri

C. albicoma S.Q. Tong AL Chiang Mai

C. amada Roscoe AM Chiang Mai

C. angustifolia Roxb. AN Prachin Buri

C. aromatica Salisb. AR Chiang Mai

C. comosa Roxb. CO Ratchaburi

C. longa L. LO Chiang Mai

C. mangga Valeton & Zijp MA Chiang Mai

C. parviflora Wall. PA Chiang Mai

C. petiolata Roxb. PE Prachin Buri

C. rubrobracteata Skornickova RU Chiang Mai

C. sessilis Gage SE Chiang Mai

Curcuma sp. 1 CS1 Phetchabun

Curcuma sp. 2 CS2 Chiang Mai

Curcuma sp. 3 CS3 Chiang Mai

Zingiber montanum (J.Koenig)

Link ex. A,Dietr.

ZM Chiang Mai

DNA isolation and random amplified polymorphic DNA (RAPD) fingerprinting Fresh leaf of each plant was ground in liquid nitrogen with mortar and pestle to obtain a

fine powder. Genomic DNA was isolated from the fine powder using the DNeasy Plant Mini

Kit (Qiagen, Germany) according to the manufacturer’s protocol.

The RAPD reaction was carried out in 20 l containing 2 l of genomic DNA, 1X

amplification buffer, 3.5 mM MgCl2, 0.4 mM of each dNTP, 1.25 U of Taq DNA polymerase

(Fermentas, Canada) and 0.4 M random deca-arbitrary primers (Eurofins MWG Operon,

Germany). The amplification was performed using a DNA thermal cycler (Applied

Biosystems, USA) with an initial pre-denaturation at 95°C for 2 min, denaturation at 95°C for

45 sec, annealing at 37°C for 1 min, extension at 72°C for 2 min with 45 cycles and final

extension at 72°C for 5 min. The RAPD products were separated on 1.5% agarose gel in

TBE buffer and stained with ethidium bromide. The RAPD fragments were photographed

using a UV transilluminator and analyzed with a gel documentation system (Syngene, USA).

The PCR amplifications were repeated at least three times.


48

RAPD data analysis

The RAPD bands were scored as 0 or 1 for the absence or presence of bands,

respectively. Only clear and reproducible bands were scored as 1. The standard DNA

marker (1 kb and 100bp GeneRuler, Fermentas, Canada) was used to assign the size of each

RAPD fragment. The similarity index was calculated from the data that was generated using

Dice similarity index coefficient (Nei and Li, 1979). The dendrogram was constructed based

on the similarity matrix data using the unweighted pair group method with arithmetic

averages (UPGMA), clustering by GeneTool and GeneDirectory software.

RESULTS AND DISCUSSION

In this study, we describe a simple process which based on the amplification of

genomic DNA with single primers of arbitrary nucleotide sequence. Thirty random deca-

arbitrary primers were screened; only four primers (OPJ-20, OPS-01, OPS-19 and OPV-12)

produced clear and reproducible polymorphic bands in all plant samples (Figure 1-4).

Twenty-two to twenty-eight PCR products were amplified, with an average of 24.5 bands by

each primer. The highest number of RAPD bands (28 bands) was generated from OPS-19

while the lowest (22 bands) was from OPJ-20. A total of 98 amplified bands ranging from

208 to 4136 bp in size were amplified, with 39 polymorphic bands being observed. Primer

OPS-01 produced the highest percentage of polymorphism (44.00%) while OPJ-20 produced

the lowest (27.27%) (Table 2).

Table 2. The sequence of the oligonucleotide primers used for the RAPD analysis and the

number of PCR products obtained from Curcuma species and outgroup plant

Primer

Nucleotide

sequence

(5´ to 3´)

No. of

bands

Size of bands No. of

polymorphic

bands

Polymorphism

(%)

OPJ-20 AAGCGGCCTC 22 233-1536 6 27.27

OPS-01 CTACTGCGCT 25 375-4136 11 44.00

OPS-19 GAGTCAGCAG 28 273-2755 12 42.86

OPV-12 ACCCCCCACT 23 208-2269 10 43.48


49

Figure 1. RAPD fingerprint of 15 Curcuma and outgroup plant obtained from the

OPJ-20 primer.

Abbreviations of the plant samples are according to codes used in Table 1.

M: GeneRuler 1 kb (size shown in bp).

The polymorphic bands of each plant sample are indicated with arrows.


OPS-01 primer.




M ZE LO AR MA CO AM AE AN SE PE RU PA CS1 CS2 CS3 ZM M(100 bp)

500

250

750

1000

1500

2000

3000


500

250

750

1000

1500

2000

3000


50


OPS-19 primer.





OPV-12 primer.





500

250

750

1000

1500

2000

3000


500

250

750

1000

1500

2000

3000


51

According to the four primers that produced clear and reproducible polymorphic bands,

the OPJ-20 primer produced the polymorphic bands of 1045 bp in C. longa, 404 bp in

C. petiolata, 233 and 515 bp in Curucuma sp. 1 and 268 and 530 bp in Z. montanum

(Figure 1). The polymorphic bands of 1070 and 4000 bp in C. longa, 2321 bp in

C. aromatica, 527 bp in C. amada, 2923 bp in C. aeruginosa, 556 bp in C. sessilis, 788 bp in

C. rubrobracteata, 1563 and 4136 in Curcuma sp. 1, 375 and 1393 bp in Z. montanum were

generated by the OPS-01 primer (Figure 2). This primer generated the approximately 780 bp

characteristic band of all Curcuma species, which was not observed in outgroup plant

(Figure 2). The polymorphic bands of 1302 bp in C. zedoaria, 1107 bp in C. longa, 883 bp in

C. aromatica, 706 bp in C. comosa, 1529 bp in C. angustifolia, 1664 bp in C. parviflora,

1099 and 1355 bp in Curcuma sp. 2, 1132 bp in Curcuma sp. 3, 273, 1440 and 2755 bp in

Z. montanum were generated by the OPS-19 primer (Figure 3). The OPV-12 primer

produced the polymorphic bands of 208, 1449 and 2269 in C. mangga, 2161 bp in

C. aeruginosa, 574 and 702 in C. angustifolia, 464 and 834 bp in C. rubrobracteata, 559 bp

in Curcuma sp. 2 and 646 bp in Curcuma sp. 3 (Figure 4). RAPD method can be used to

reproducibly amplify segment of genomic DNA form closely related species and

polymorphisms among the amplification products can be detected through examination of an

ethidium bromide stained agarose gel.

The pair-wise comparisons of the RAPD profiles based on both of the shared and

unique amplification bands were used to generate a similarity index. Among 15 Curcuma

species including outgroup plant, Dice similarity index ranged from 0.0909 to 0.9222

(Table 3). The highest genetic similarity index (0.9222) was found between C. longa and

C. zedoaria, whereas the lowest (0.0909) was found between C. comosa and Z. montanum.

A dendrogram was constructed according to the UPGMA cluster analysis using Dice

similarity coefficient. The UPGMA dendrogram showed the division of 15 Curcuma species

into tree main clusters (Figure 5). Cluster I was divided into four subgroups Ia, Ib, Ic and Id,

respectively. Subgroup Ia, consists of C. longa and C. zedoaria, with 0.9222 similarity index.

Subgroup Ib includes C. angustifolia and C. sessilis, with 0.6372 similarity index. Subgroup

Ic consists of C. comosa, C. amada and C. mangga, with 0.5737-0.7995 similarity index.

Subgroup Id includes 5 species, C. aromatica, C. aeruginisa, Curcuma sp. 1, Curcuma sp. 2

and Curcuma sp. 3, with 0.4333-0.8518 similarity index. C. parviflora was clustered in

cluster II while C. rubrobracteata and C. petiolata were clustered in cluster III, with 0.8553

similarity index. Outgroup plant, Z. montanum, was completely separated from the Curcuma

species.

The result was similar to those previously reported by Angel et al. (2008) and

Syamkumar and Sasikumar (2007). Based on RAPD and ISSR marker, C. longa and

C. zedoaria were clustered in the same group whereas C. aeruginosa was classified in

different subgroup from C. comosa and C. amada. In addition, based on ITS, trnK and

chloroplast DNA sequences, C. rubrobracteata was grouped with C. petiolata and C. longa

with C. zedoaria (Cao et al., 2001; Zaveska et al., 2012).

The genetic relationships through RAPD marker were also correlated with the

morphological characteristic. The important morphological characters of each cluster were

summarized in Table 4. The results were similar to those previously reported by Sirirugsa et

al. (2007). Based on morphological characters including the presence or absence and shape

of stylodial glands and the shape of bract apex, Sirirugsa et al. (2007) divided Curcuma

species in Thailand into 5 groups i.e. Alismatifolia, Cochinchinensis, Ecomata, Longa and

Petiolata.


52

Tab

le 3

. S

imil

arit

y m

atri

x o

f C

urc

um

a an

d o

utg

roup p

lants

gen

erat

ed u

sing

Dic

e si

mil

arit

y c

oef

fici

ent


53

Figure 5. Dendrogram produced by UPGMA cluster analysis of RAPD data showing the

genetic relationship among 15 Curcuma plants and outgroup plant

According to the UPGMA dendrograms based on RAPD profiles, 15 Curcuma plants

were divided into three clusters (Figure 5). Curcuma species in cluster I was considered as

the Longa group as they share the unique approximately 500 bp band in OPJ-20 primer and

approximately 1200 bp band in OPV-12 primer (Figure 1 and 4). These plants have the

curved acicular anther spurs and cylindrical stylodial gland along with coma bract with acute

bract apex. Curcuma sp. 1, Curcuma sp. 2 and Curcuma sp. 3 were clustered with

C. aeruginosa and C. aromatica. They share the unique approximately 1300 bp band in

OPS-19 primer and approximately 800 bp band in OPV-12 primer (Figure 3 and 4).

Moreover, comparison to the morphological characters, three unidentified Curcuma species

were also found to be closely related to C. aeruginosa, the Longa group, as they have

greenish-yellow or greenish-blue rhizomes together with reddish purple mid ribs and leaf

sheaths. Therefore, three unidentified Curcuma species should be classified as the Longa

group. C. parviflora (clusters II) was considered as the Alismatifolia group because this plant

has the coma bract and obtuse to rounded or acute bract apex; the anther spurs and stylodial

gland are absent whereas C. rubrobracteata and C. petiolata (clusters III) were considered as

the Petiolata group because these plans have the straight acicular anther spurs, clavate

C. angustifolia

Ic

C. rubrobracteata

C. longa

C. zedoaria

C. amada

C. mangga

C. comosa

C. sessilis

C. aromatica

C. aeruginosa

Curcuma sp. 1

Curcuma sp. 2

Curcuma sp. 3

C. parviflora

C. petiolata

Z. montanum

Ia

Ib

Id

II

II

I

10093857870635548413326


54

stylodial gland and coma bract with rounded to obtuse bract apex. RAPD marker is a rapid,

inexpensive and effective tool for studying genetic relationships in various plants due to their

advantages i.e. no need of prior knowledge of the DNA sequence, the small amount of DNA

used in the study and the ability to assay for many loci simultaneously (Semagn, Bjornstad

and Ndjiondjop, 2006; Zou, et al., 2011). The RAPD technique has been reported of its

application in the differentiation of several plants in Zingiberaceae such as those of the

genera Boesenbergia (Vanijajiva, Sirirugsa and Suvachittanont, 2005), Kaempferia

(Pojanagaroon et al., 2004; Vanijajiva, Sirirugsa and Suvachittanont, 2005) and Curcuma

(Sasikumar, 2005; Zou, et al., 2011; Kitamura, et al., 2007; Syamkumar and Sasikumar,

2007). Furthermore, sequence characterized amplified regions (SCARs) could be further

developed as an alternative tool for differentiation plants that have similar morphological

characteristics and also monitoring the quality of herbal medicines.

CONCLUSION

In conclusion, our results suggested the ability of RAPD fingerprint in differentiating

15 Curcuma species and the correlated classifications of Curcuma species by RAPD maker

and morphological characteristics. RAPD fingerprints along with morphological

characteristics remains the most reliable and useful techniques for studying the relationships

among the different Curcuma taxa.

ACKNOWLEDGEMENTS

The authors acknowledged the 90th

anniversary of Chulalongkorn University fund

(Ratchadaphiseksomphot Endowment Fund) for financial support and the authors are

thankful to Assoc. Prof. Thatree Phadungcharoen, Department of Pharmacognosy and

Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University,

Thailand and Assist. Prof. Dr. Thaya Jenjittikul, Department of Plant Science, Faculty of

Science, Mahidol University, Thailand for the sample identification and valuable

information.


55

Tab

el 4

. T

he

imp

ort

ant

morp

ho

logic

al c

har

acte

rs o

f ea

ch c

lust

er

1


56

REFERENCES

Angel G, Makeshkumar T, Mohan C, Vimala B and Nambinsan B. 2008. Genetic diversity analysis of starchy

Curcuma species using RAPD markers. Journal of Plant Biochemistry and Biotechnology. 17: 173-176.

Cao H, Sasaki Y, Fushimi H and Komatsu K. 2001. Molecular analysis of medicinally-Used Chinese and

Japanese Curcuma based on 18S rRNA gene and trnK Gene sequences. Biological & Pharmaceutical

Bulletin. 24:1389-1394.

Heubl G. 2010. New aspects of DNA-based authentication of Chinese medicinal plants by molecular biological

techniques. Planta Medica. 76:1963-1974.

Kitamura C, Nagoe T, Prana M, Agusta A, Ohashi K and Shibuya H. 2007. Comparison of Curcuma sp. in

Yakushima with C. aeruginosa and C. zedoaria in Java by trnK gene sequence, RAPD pattern and

essential oil component. Journal of Natural Medicines. 61:239-243.

Nei M and Li W. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases.

Proceedings of the National Academy of Sciences of the United States of American. 76:5269-5273.

Pojanagaroon S, Praphet R, Kaewrak C and Yotdi A. 2004. Characterization of Krachai-Dam (Kaempferia

parviflora) cultivars using RAPD markers, morphological traits and chemical components of essential

oil from rhizomes. Proceedings of the 42nd Kasetsart University Annual Conference, Kasetsart,

Thailand. 56-65.

Ravindran P, Babu KN and Shiva K. 2007. Botany and crop improvement of Turmeric. In: Ravindran PN,

Nirmal BK and Sivaraman K, editors. Turmeric The genus Curcuma. Boca Raton: CRC Press; pp. 15-

70.

Sasikumar, B. 2005. Genetic resources of Curcuma: diversity, characterization and utilization. Plant Genetic

Resources. 3:230-251.

Semagn K, Bjornstad A and Ndjiondjop M. 2006. An overview of molecular marker methods for plants. African

Journal of Biotechnology. 5:2540-2568.

Sirirugsa P, Larsen K and Maknoi C. 2007. The genus Curcuma L. (Zingiberaceae): distribution and

classification with reference to species diversity in Thailand. Gardens' Bulletin Singapore. 59:203-220.

Skornickova J. 2006. Curcuma - Stunning beauty, hidden treasure. GardenWise. 2-5.

Syamkumar S and Sasikumar B. 2007. Molecular marker based genetic diversity analysis of Curcuma species

from India. Scientia Horticulturae. 112:235-241.

Vanijajiva O, Sirirugsa P and Suvachittanont W. 2005. Confirmation of relationships among Boesenbergia

(Zingiberaceae) and related genera by RAPD. Biochemical Systematics and Ecology. 33:159-170.

Williams J, Kubelik A, Livak K, Rafalski J and Tingey S. 1990. DNA polymorphisms amplified by arbitrary

primers are useful as genetic markers. Nucleic Acids Research.18:6531-6535.

Zou X, Dai Z, Ding C, Zhang L, Zhou Y and Yang R. 2011. Relationships among six medicinal species of

Curcuma assessed by RAPD markers. Journal of Medicinal Plants Research. 5:1349-1354.

Zaveska E, Fer T, Sida O, Krak K, Marhold K and Leong-Skornickova J. 2012. Phylogeny of Curcuma

(Zingiberaceae) based on plastid and nuclear sequences: Proposal of the new subgenus Ecomata.

TAXON. 61:747-63.

orawan theanphong1, witchuda …...2002/06/10 · curcuma plants have long been known for their...

Documents