Molecular Biotechnology

, Volume 49, Issue 1, pp 65–76

Genetic Relationship of Curcuma Species from Northeast India Using PCR-Based Markers

Authors

  • Archana Das
    • Department of BiotechnologyIndian Institute of Technology Guwahati
  • Vigya Kesari
    • Department of BiotechnologyIndian Institute of Technology Guwahati
  • Vinod M. Satyanarayana
    • M S Swaminathan Research Foundation
  • Ajay Parida
    • M S Swaminathan Research Foundation
    • Department of BiotechnologyIndian Institute of Technology Guwahati
Research

DOI: 10.1007/s12033-011-9379-5

Cite this article as:
Das, A., Kesari, V., Satyanarayana, V.M. et al. Mol Biotechnol (2011) 49: 65. doi:10.1007/s12033-011-9379-5

Abstract

Molecular genetic fingerprints of nine Curcuma species from Northeast India were developed using PCR-based markers. The aim involves elucidating there intra- and inter-specific genetic diversity important for utilization, management, and conservation. Twelve random amplified polymorphic DNA (RAPD), 19 Inter simple sequence repeats (ISSRs), and four amplified fragment length polymorphism (AFLP) primers produced 266 polymorphic fragments. ISSR confirmed maximum polymorphism of 98.55% whereas RAPD and AFLP showed 93.22 and 97.27%, respectively. Marker index and polymorphic information content varied in the range of 8.64–48.1, 19.75–48.14, and 25–28 and 0.17–0.48, 0.19–0.48, and 0.25–0.29 for RAPD, ISSR, and AFLP markers, respectively. The average value of number of observed alleles, number of effective alleles, mean Nei’s gene diversity, and Shannon’s information index were 1.93–1.98, 1.37–1.62, 0.23–0.36, and 0.38–0.50, respectively, for three DNA markers used. Dendrograms based on three molecular data using unweighted pair group method with arithmetic mean (UPGMA) was congruent and classified the Curcuma species into two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r = 0.96), ISSR (r = 0.94), and AFLP (r = 0.97). Clustering was further supported by principle coordinate analysis. High genetic polymorphism documented is significant for conservation and further improvement of Curcuma species.

Keywords

AFLPCurcumaGenetic diversityISSRNortheastRAPD

Abbreviations

AFLP

Amplified fragment length polymorphism

EDTA

Ethylenediaminetetraacetic acid

EtBr

Ethidium bromide

ISSR

Inter simple sequence repeat

PCA

Principle coordinate analysis

PIC

Polymorphic information content

PCR

Polymerase chain reaction

RAPD

Random amplified polymorphic DNA

TE

Tris–EDTA buffer

UPGMA

Unweighted pair group method with arithmetic mean

Introduction

Curcuma belonging to the family Zingiberaceae has immense medicinal value and finds extensive use in the indigenous system of medicine [14]. Northeast (NE) India houses the vast majority of Curcuma species, many of which are endemic to the region [4]. However, the species are gradually facing the fear of being endangered in NE region as a result of ruthless uprooting of the rhizomes by the tribal people from their wild habitats for its extreme medicinal value and use in spices. Some of the species descriptions are without Latin diagnosis or type specimen, therefore the legitimate status of many species is suspicious and remains unclear (Per. Comm., Dr. Sharma, GU). Sustainable management has so far not succeeded further aggravating the extinction. For any future analysis using Curcuma, it may be useful to have information regarding its genetic diversity and relationship between cultivated and wild species. The knowledge of genetic variability is a pre requisite to study the evolutionary history of a species, as well as for other studies like intraspecific variations, genetic resources conservation, etc [5]. Thus, molecular markers based characterization of Curcuma species may contribute to analyze taxonomic relationships and intraspecific diversity of the genus. Further, the study will be useful for planning strategies for their conservation and optimal utilization.

The usefulness of molecular markers in genetic diversity studies has been convincingly established [612]. Random amplified polymorphic DNA (RAPD) analysis is the simplest and least laborious method and has been used to estimate the genetic distances and diversity in a wide range of plant species especially at the sub-species and cultivar level [1318]. New technological developments have expanded the range of DNA polymorphisms assays for genetic mapping, marker assisted breeding, genome finger printing, and investigating genetic relatedness [19]. Amplified fragment length polymorphism (AFLP) is considered more powerful than RAPD in discriminating genetic diversity [20]. On the other hand, inter simple sequence repeats (ISSRs) markers are abundant throughout the genome and show a higher level of polymorphism than any other genetic markers [21]. The sequences flanking specific microsatellite loci in a genome are considered to be conserved within species, across species in a genus and perhaps even across the related genera [22]. Recently, microsatellite markers have been used in genetic diversity studies in certain members of Zingiberaceae [23]. Studies are available on the genetic diversity analysis using RAPD, SSR, and ISSR markers on the members of genus Curcuma [2325], but there has been no effort to analyze the endemic Curcuma species of NE India. The main goal of the present study was to access the degree of genetic diversity and to analyze the genetic proximity among the nine species of Curcuma from NE region including endemic ones using three different sets of molecular markers, viz., RAPD, ISSR, and AFLP, respectively.

Materials and Methods

Plant Material

The materials for the present study consists of nine species of Curcuma, both wild and cultivated collected from various regions of NE India (Fig. 1; Table 1). The species were collected and maintained in the departmental green house of IIT Guwahati and Botanical Garden, Gauhati University. Fresh, healthy green leaves of these samples were used for DNA extraction and subsequent fingerprinting analysis.
https://static-content.springer.com/image/art%3A10.1007%2Fs12033-011-9379-5/MediaObjects/12033_2011_9379_Fig1_HTML.gif
Fig. 1

Map locations of collection site of different Curcuma species used in the current study

Table 1

Plant materials used in the study

Species

Place of collection

Latitude and altitude

Rhizome character

Habitat

C. amada; SK 130

Amingaon

26°11′N, 56 m

Light yellow, creeping, soft, strong smell like raw mango.

Hilly slopes and moist grasslands

C. angustifolia; SK 261

Shillong

25°34′N, 1540 m

White, tuber is hard with characteristic odor.

Hilly moist slopes

C. caesia; SK270

Goalpara

26°10′N, 63.3 m

Whitish, soft tuber with mild smell, bluish inside

Moist, shady lands

C. zedoaria; SK160

Darrang

20°9′N, 104 m

Cone shaped tuber towards the end, Yellowish, with strong smell

Shady, humid places

C. aromatica; SK 335

Nagaon

26°11′N, 55 m

Hard, whitish in color with strong smell

Both shady and hot-humid places

C. longa; SK145

Barpeta

26°10′N, 56 m

Yellowish, smaller tuber, with characteristic smell

Shady, moist places

C. domestica; GS 205

Kahikuchi

26°9′N, 55.5 m

Reddish in color, larger tubers with strong smell

Shady, hot and humid places

C. domestica; GS 466

Nagaland (Tizu)

27°4′N, 900 m

Dark yellow in color, tuber has strong odor

Shady, moist, hilly places

C. spp. (wild); SK435

Kokrajhar

26°24′N, 65 m

White and hard tuber with mild smell

Hilly moist slopes

DNA Extraction

Total genomic DNA was extracted from fresh tender leaves using modified SDS method as described by Kesari et al. [26]. The quality and quantity of the extracted DNA was confirmed to be consistent both spectro-photometrically and by running the extracted DNA on 1.0% agarose gels stained with ethidium bromide.

RAPD Analysis

PCR amplification of the genomic DNA was carried out using 20 arbitrary decamer oligonucleotide primers (Operon Tech, USA) (Table 2). The reaction mixture of 20 μl contained 50 ng/μl of template DNA, 1× assay buffer (100 mM Tris sulfonic acid, pH 8.8, 15 mM MgCl2, 500 mM KCl, and 0.1% gelatin), 0.2 mM each dNTPs (B’Genei, India), 5 pmol of each primer, and 0.5 U of Taq polymerase (B’LGenei, India). The reaction was performed in 0.2 ml microfuge tubes (Dialabs). PCR amplification was carried out in a Mini Thermal Cycler (Applied Biosystems 9700). Thermal cycling conditions were as follows: pre-denaturing step of 5 min at 94°C, followed by 35 cycles each of 45 s at 94°C, annealing for 1 min at 32°C, extension for 1 min at 72°C, and followed by one final extension cycle of 5 min at 72°C. The amplification products were electrophoresed in 1.5% agarose gels in 0.5× TBE (10× stock contained 0.8 M Tris, 0.8 M boric acid, 0.5 M EDTA). The gels were photographed under a UV transilluminator.
Table 2

Sequence information of RAPD and ISSR oligonucleotide primers used for amplification and polymorphism study amongst nine Curcuma species from Northeast India

S. No.

RAPD primer

Sequence (5′–3′)

ISSR primer

Sequence (5′–3′)

1

OPC 07

GTCCCGACGA

HB 12

CACCACCACGC

2

OPL 11

ACGATGAGCC

HB 13

GAGGAGGAGGC

3

OPO 08

GCTCCAGTGT

HB 14

CTCCTCCTCGC

4

OPAH 15

CTACAGCGAG

HB 15

GTGGTGGTGGC

5

OPAM 20

ACCAACCAGG

P 3

AGAGAGAGAGAGAGAGTG

6

OPAN 01

ACTCCAGGTC

P 6

CCACCACCACCACCA

7

OPAO 01

AAGACGACGG

P 8

CACCACCACCACCAC

8

OPAP 20

CCCGGATACA

807

AGAGAGAGAGAGAGAGT

9

OPAN 05

GGGTGCAGTT

809

AGAGAGAGAGAGAGAGG

10

OPAP 10

TGGGTGATCC

811

GAGAGAGAGAGAGAGAC

11

OPAA 01

AGACGGCTCC

816

CACACACACACACACAT

12

OPAB 01

CCGTCGGTAG

817

CACACACACACACACAA

13

OPAB 05

CCCGAAGCGA

818

CACACACACACACACAG

14

OPAB 14

AAGTGCGACC

824

TCTCTCTCTCTCTCTCG

15

OPAH 13

TGAGTCCGCA

825

ACACACACACACACACT

16

OPAF 02

CAGCCGAGAA

826

ACACACACACACACACC

17

OPAJ 19

ACAGTGGCCT

844

CTCTCTCTCTCTCTCTAC

18

OPX 20

CCCAGCTAGA

872

GATAGATAGATAGATA

19

OPA 08

GTGACGTAGG

17898A

CACACACACACAAC

20

OPA 12

TCGGCGATAG

17898B

CACACACACACAGT

ISSR Analysis

PCR amplification was carried out for 20 ISSR primers (Table 2). The PCR composition was same as that used for RAPD analysis with a final volume of the reaction mixture being 20 μl. The steps of temperature cycling were as follows: 94°C for 4 min, followed by 35 cycles of 45 s at 94°C, 1 min with varied temperatures as per the melting temperature of the ISSR primers used, 1 min 30 s at 72°C, and 10 min final extension step at 72°C. The amplified products were visualized in a 1.5% agarose gel containing ethidium bromide and photographed for further analysis.

AFLP Analysis

AFLP analysis was performed as described by Vos and his coworkers [27] with minor modifications. DNA (500 ng) was double digested with EcoR1 and MseI, and then ligated with adapters using T4 DNA ligase (New England Biolabs). The pre-selective-PCR product was diluted in a ratio of 1:10 with TE buffer and then used as a template for the selective amplification. The pre-selective amplification was performed at 72°C for 2 min followed by 20 cycles of denaturation (at 94°C for 20 s), primer annealing (at 56°C for 30 s) and primer extension (at 72°C for 2 min) and finally maintained at 60°C for 30 min. An aliquot of the pre-selective PCR product was electrophoresed on 1.5% agarose gel and checked for amplification. It was then diluted (1:20) with DNase free water and used as a template for the selective amplification.

The selective amplification was performed using primers from the AFLP selective primer kit. The selective amplification involved the following thermal cycling conditions: denaturation at 94°C for 2 min followed by 11 cycles of 94°C for 20 s, 66°C for 30 s, and 72°C for 2 min. The annealing temperature was reduced by 1°C every cycle till it reached 56°C. This was followed by another 20 cycles of amplification at 94°C for 20 s, 56°C for 30 s, and 72°C for 2 min, with a final extension at 60°C for 30 min. The PCR product of selective amplification (1 μl) was mixed with 0.5 μl of the GeneScan 500 ROX internal size standard (Applied Biosystems P/N 402985) and 8.5 μl of Hi-Di Formamide (Applied Biosystems P/N 4311320). The mixture was then denatured prior to separation by capillary gel electrophoresis on an automated DNA sequencer (ABI 3130, Applied Biosystems). The electropherograms generated by the sequencer were interpreted with Gene scan software. Fragments sized from 50 to 500 base pairs (bp) with a peak height >50 in the electropherogram were retained for subsequent analysis.

Data Analysis

For all the three types of marker systems, duplicate samples from each individual were tested and only clear, unambiguous, and reproducible bands amplified in both cases were considered for the scoring and data. The numbers of polymorphic and monomorphic amplification products were determined for each primer for nine Curcuma species. Scoring was carried out as 1/0 for the presence or absence of each fragment in each sample. To avoid taxonomic ambiguities, the intensity of bands was not taken into considerations, only the presence of band was taken as indicative. To compare the efficiency of primers polymorphic information content (PIC); as a marker discrimination power, was computed using the formula PIC = 1 − ∑pi2, where pi is the frequency of ith allele at a given locus [28] and also marker index (MI) was calculated as given by Powell et al. [20]. The basic parameters for genetic diversity were calculated in the POPGENE application [29]. The polymorphism of amplification products (P), the number of observed alleles (na), the mean number of effective alleles (ne), the mean Nei’s gene diversity index (h), and the Shannon index (I) were calculated using the POPGENE software.

Level of similarity among species was established as percentage of polymorphic bands and a matrix of genetic similarity compiled using the Dice’s coefficient [30]. Applying the UPGMA [31] method using the SHAN subroutine through the NTSYS-pc (Numerical taxonomy system, 2.2 version) (Numerical taxonomy system, Applied Biostatistics, NY) [32] a dendrogram was generated representing the genetic relationship among nine Curcuma species. The correlation between the original similarity indices and cophenetic values were calculated, and the Mantel’s test [33] was performed using 250 permutations to check the goodness of fit for nine different species of Curcuma to a specific cluster in the UPGMA similarity matrix. Further, principal coordinate analysis (PCA) was undertaken for the families with modules STAND, CORR, and EIGEN of NTSYS-pc [32] using the Euclidean distances derived from the standardized values using the NTSYS-pc-2.2 software.

Results

Efficiency of Polymorphism Detection

RAPD Analysis

The genetic diversity among the nine different Curcuma species was evaluated by 12 selected primers which yielded species specific DNA profiles and proved to be informative. A total of 55 mappable RAPD markers were generated by 12 primers. The amplicons ranged between 0.3 and 1.8 kb in size. Amplicon number per primer ranged from 2 (OPAM 20) to 8 (OPC 07) with an average of 4.92. Polymorphism also varied in different species of Curcuma with a maximum of eight bands for the primer OPC 07 and a minimum of one band in the primer OPAM 20 with a mean of 4.58 (Table 3). RAPD profile of nine different Curcuma species analyzed showed the polymorphic index value of 93.22% across all the species examined in the current study. The details of amplification products, polymorphic fragments generated, PIC, and MI values for each primer were showed in Table 3. The RAPD profile generated by OPAN 01 for the nine different Curcuma species is shown in the Fig. 2a. PIC values for RAPD primers varied from 0.17 (OPAM 20) to 0.48 (OPAN 05) whereas marker indices ranged between 8.64 (OPAM 20) to 48.14 (OPAN 05).
Table 3

Degree of polymorphism and polymorphic information content for RAPD and ISSR primers in nine species of Curcuma

Markers

Primer code

Total no. of bands

Total no. of polymorphic bands

POL (%)

PIC

MI

RAPD

OPAN 01

6

5

83.33

0.33

27.43

OPAO 01

5

5

100

0.39

39.50

OPC 07

8

8

100

0.44

44.44

OPL 11

3

3

100

0.41

41.15

OPAM 20

2

1

50

0.17

8.64

OPAN 05

4

4

100

0.48

48.14

OPAB 10

5

4

80

0.38

30.61

OPO 08

6

6

100

0.36

36.21

OPA 8

3

3

100

0.42

42.79

OPA 12

7

7

100

0.45

45.14

OPAH 15

5

4

80

0.34

26.86

OPAP 20

5

5

100

0.40

40.43

Total

59

55

93.22

Mean

4.92

4.58

Range

2–8

1–8

50–100

0.17–0.48

8.64–48.1

ISSR

HB 12

4

3

75

0.33

24.99

HB 13

4

4

100

0.40

40.74

HB 14

5

5

100

0.45

45.43

HB 15

2

2

100

0.41

41.97

P3

5

5

100

0.43

43.45

P6

3

3

100

0.32

32.92

P8

4

4

100

0.46

46.91

807

1

1

100

0.44

44.44

809

3

3

100

0.34

34.56

811

4

4

100

0.25

25.92

816

4

4

100

0.37

37.04

817

4

4

100

0.37

37.04

818

1

1

100

0.19

19.75

824

5

5

100

0.35

35.55

825

4

4

100

0.48

48.14

826

4

4

100

0.32

32.09

844

4

4

100

0.38

38.27

17898 A

2

2

100

0.46

46.91

17898 B

6

6

100

0.38

38.68

Total

69

68

98.55

Mean

3.63

3.58

Range

1–6

1–6

75–100

0.197–0.481

19.75–48.14

POL polymorphism, PIC average polymorphic information content for polymorphic bands, MI marker index = POL(%) × PIC

https://static-content.springer.com/image/art%3A10.1007%2Fs12033-011-9379-5/MediaObjects/12033_2011_9379_Fig2_HTML.gif
Fig. 2

RAPD and ISSR polymorphic profiles for nine species of Curcuma from Northeast India. M, 1 kb DNA ladder; 1, C. amada; 2, C. angustifolia; 3, C. caesia; 4, C. zedoaria; 5, C. aromatica; 6, C. longa, 7, C. domestica I; 8, C. domestica II; 9, C. spp. a RAPD (OPAN 01), b ISSR (HB 12)

ISSR Analysis

Twenty ISSR primers were used to characterize the genetic diversity present among the nine species of Curcuma. Nineteen of these primers showed a total of 69 reproducible fragments that ranged from 0.2 to 0.85 kb in size. High percentage of polymorphism with all the 19 primers (98.55%) was displayed among the nine species of Curcuma with 68 polymorphic bands. It was observed that the number of visible bands ranged from 1 (807, 818) to 6 (17898 B) with an average of 3.63 whereas the average number of polymorphic bands per primer obtained was 3.58 (Table 3). The percentage of ISSR polymorphism for nine different Curcuma species studied ranged from 75 to 100%. Figure 2b displays the ISSR fingerprints using the primer HB 12. The PIC values for ISSR primers ranged from 0.19 (818) to 0.48 (825) with an average of 0.38, whereas MI ranged between 19.75 (818) to 48.14 (825).

AFLP Analysis

A total of 147 AFLP bands were recorded with four primer pair combinations and 143 of these bands were polymorphic (Table 4). The size of amplified products ranged from 50 to 500 bp. Total number of bands per primer ranged from 3 (Msel-CAA/EcoRI-ACT) to 81 (Msel-CAT/EcoRI-ACC) with an average of 36.75 whereas polymorphic bands per primer ranged from 3 (Msel-CAA/EcoRI-ACT) to 78 (Msel-CAT/EcoRI-ACC) with an average of 35.75 (Table 4). The PIC of each primer was evaluated which was found highest (0.61) for the primer combination Msel-CAG/EcoRI-AAC and the lowest (0.25) for the primer combination Msel-CAC/EcoRI-ACA (Table 4) with an average of 0.35 per primer combination. The highest MI of 59.8 obtained for the primer pair Msel-CAG/EcoRI-AAC and the primer combination Msel-CAC/EcoRI-ACA showed the lowest MI of 25 with an average of 35.18.
Table 4

Degree of polymorphism and polymorphic information content for AFLP primers applied to nine species of Curcuma

Primer combinations

Total number of bands

POL

PIC

MI

Number

%

MseI-CAA/EcoRI-ACT

3

3

100

0.28

28

MseI-CAC/EcoRI-ACA

12

12

100

0.25

25

MseI-CAG/EcoRI-AAC

51

50

98.0

0.61

59.8

MseI-CAT/EcoRI-ACC

81

78

96.3

0.29

27.9

Total

147

143

97.27

1.43

140.7

Average

36.75

35.75

0.36

35.18

POL polymorphism, PIC average polymorphic information content for polymorphic bands, MI marker index = POL(%) × PIC

Gene Diversity Between Species

In this study, relatively higher level of polymorphism and genetic diversity among the nine Curcuma species was revealed by RAPD, ISSR, and AFLP markers. Screening genetic diversity at the interspecific level, the average values of na, ne, and h were ranged from 1.93–1.99, 1.37–1.62, and 0.24–0.36, respectively (Table 5). The mean Shannon’s indexes (I) for Curcuma species were 0.53, 0.50, and 0.38 based on RAPD, ISSR, and AFLP, respectively (Table 5).
Table 5

Genetic diversity parameters in nine species of Curcuma

Parameter

Value

RAPD

ISSR

AFLP

The number of observed alleles, na

1.93 ± 0.24

1.99 ± 0.12

1.97 ± 0.16

The mean number of effective alleles, ne

1.62 ± 0.31

1.57 ± 0.30

1.37 ± 0.30

The mean Nei’s gene diversity index, h

0.36 ± 0.14

0.34 ± 0.13

0.24 ± 0.15

Shannon index, I

0.53 ± 0.19

0.51 ± 0.17

0.38 ± 0.19

Each value = mean ± SD

Genetic Diversity Analysis as Revealed by RAPD, ISSR, and AFLP

Based on the RAPD, ISSR, and AFLP analyses, the similarity coefficient among all the species of Curcuma were calculated. The genetic similarity value derived from the RAPD data ranged from 0.098 between C.longa and C.zedoaria to 0.806 between C. domestica I and C. domestica II. The genetic similarity coefficients for all the nine species of Curcuma depicted with ISSR markers showed a wide range from 0.278 (between C. angustifolia and C. caesia) to 0.756 (between C. aromatica and C. longa). Similarly, for AFLP, the lowest genetic similarity coefficient was for the pair of C. angustifolia and C. longa (0.04) while the highest value (0.648) was calculated for the pair of Curcuma species C. amada and C. zedoaria, indicating a broad genetic basis.

In the present study, the genetic relationships among nine species of Curcuma were analyzed by 12 RAPD, 19 ISSR, and 4 AFLP primer combinations on the basis of Dice genetic distance [30]. Resulting clusters were expressed as UPGMA dendrograms constructed using SHAN neighbor-joining tree separately for each molecular marker used. The coefficients on the x-axis represent the similarity indices (DICE) of the different species chosen for the study. Based on UPGMA clustering algorithm from RAPD, the genotypes were grouped into two major clusters at a similarity index value of 0.20 (Fig. 3a). C. amada and C. zedoria were the two extremes in the dendrogram. Cluster I consisted of only one group having C. caesia and C. zedoria. Cluster II consists of individuals belonging to domestic species along with C. angustifolia and C. aromatica. Within cluster II, three subgroups were evident, one containing C. amada and C. spp., while subgroup 2 included C. angustifolia and C. longa. In subgroup 3, C. aromatica was placed with the domestic varieties C. domestica I and II. Similarly, the dendrogram obtained from ISSR profiles showed two distinct groups for nine species of Curcuma studied at a similarity index value of 0.34 (Fig. 3b), placing C. caesia in first and the rest in the second cluster. Cluster II again formed three distinct subgroups where C. amada and C. zedoaria are found to form subgroup 1 separately with C. angustifolia. The cultivated species are grouped with C. aromatica and C. spp. in subgroups 2 and 3, respectively. The dendrogram prepared based on AFLP analysis for studied Curcuma species where again formed two clusters showing higher level of diversity. The AFLP discriminated most genotypes and grouped individuals together though belonging to two different species (such as C. amada and C. caesia) (Fig. 3c). C. angustifolia alone formed cluster I. In cluster II, cultivated species are grouped together along with other closer species. C. amada, C. zedoaria, and C. spp. were placed in subgroup 1, where as C. longa and C. domestica I was placed in subgroup 2. In subgroup 3, C. domestica II, C. aromatica, and C. caesia grouped separately.
https://static-content.springer.com/image/art%3A10.1007%2Fs12033-011-9379-5/MediaObjects/12033_2011_9379_Fig3_HTML.gif
Fig. 3

Dendrograms representing the genetic variability among nine species of Curcuma from Northeast India as revealed by UPGMA cluster analysis. The genetic distances were from Dice similarity coefficient. a RAPD, b ISSR, c AFLP

The separation approach as revealed by the Mantel test comparing the results of RAPD, ISSR, and AFLP, indicated a significant correlation within the nine different Curcuma species. The cophenetic correlation coefficient between dendrogram and the original similarity matrix were also significant for RAPD (r = 0.96), ISSR (r = 0.94), and AFLP (r = 0.97) supporting a good degree of confidence in the association obtained for the nine species of Curcuma. PCA derived on the basis of RAPD data illustrated that the first three principal coordinate components accounted for 24.99, 21.45, and 13.78% variation, respectively, among the Curcuma species. While for ISSR and AFLP marker-based PCA maps showed the three coordinates describing 25.77, 17.36, and 11.91% (for ISSR) and 32.17, 14.25, and 11.64% (for AFLP) of the total variance, respectively. Thus, the first three most informative coordinates accounted for 60.22, 55.04, and 58.06% of the genetic similarity variance based on RAPD, ISSR, and AFLP markers. PCA showed the multidimensional relationships that describe portions of the genetic variance in a data set for Curcuma species (Fig. 4a–c).
https://static-content.springer.com/image/art%3A10.1007%2Fs12033-011-9379-5/MediaObjects/12033_2011_9379_Fig4_HTML.gif
Fig. 4

Principle co-ordinate map for the first, second and third principle coordinate estimated for RAPD, ISSR, and AFLP markers for nine species of Curcuma from Northeast India

Discussion

Curcuma along with other species of Zingiberaceae display diversity in habitat, ethno botanical use, and morphology [25]. Very little is known about phylogenetic relationship among taxa and genetic diversity. Detailed knowledge about genetic relationships among wild and cultivated species of Curcuma will enhance the utilization value of wild species for any future study. A few studies based on morphological, anatomical, and biochemical characterization of Curcuma species and cultivars have been attempted earlier [3438]. Relying much on the morphological characters alone in species delimitation has its own limitations since they are not always completely representative of the genetic structure [39]. Conventional taxonomic techniques in conjunction with molecular biology tools may go a long way in providing accurate and powerful ways of analyzing genetic relationship in the genus Curcuma. However, not much has been done on molecular characterization in Curcuma [40] Molecular markers assume great significance, as these methods detect polymorphisms by assaying subsets of the total amount of DNA sequence variation in a genome. Earlier diversity studies have been reported in Curcuma species of all over India by using only RAPD and ISSR markers [25, 41].

The present work is the first attempt to assess the genetic relationship of nine economically important indigenous species of Curcuma (cultivated and wild) from NE India using three different sets of molecular markers (RAPD, ISSR, and AFLP). The percentage of polymorphism by ISSR markers was found highest (98.55) as compared to RAPD (93.22%) and AFLP (97.27%) in the studied Curcuma species. This indicates that the efficiency of ISSR markers in terms of amplification of a large number of fragments is high, compared to RAPD. This coincides with the observations found in other monocots, viz., rice bean and barley by Muthusamy and Fernandez et al., respectively [42, 43]. AFLP technology is another powerful tool for detection and evolution in germplasm collections and in the screening of biodiversity as well as for fingerprinting studies [4446]. It is also clear from the present investigation that AFLP technique was able to differentiate intraspecific relations of the Curcuma species by a higher number of unique markers compared to RAPD and ISSR techniques. Similar findings were reported among four Zingiber species from Western Ghats, South India using AFLP markers [47]. However, ISSR marker demonstrated a different polymorphic capability compared with RAPD and AFLP and was found to be most informative in characterizing closely related Curcuma species from NE India. Yang and his coworkers [48] also stated that ISSR assay can provide more informative data than other techniques.

The three marker systems, RAPD, ISSR, and AFLP analyzed different segments of genome and hence revealed different genetic information [45, 49]. Results showed that RAPD, ISSR, and AFLP are very powerful methods of characterization of genetic relationship among species of Curcuma. The dendrograms based on three markers data were basically same with minor changes showing inter-specific differences were more significant compared to intra-varietal ones. In RAPD cluster analysis, C. ceasia and C. zedoaria formed cluster I. All other species were grouped into the cluster II along with cultivated species. Cluster II formed 3 subgroups where in C. spp. is placed with C. amada in subgroup 1 showing that the wild species does not make an independent cluster. The adjoined group of cultivated varieties with wild ones suggests that they have been evolved in course of time. C. domestica I, II, and C. aromatica have physiological similarity of strong aroma and were also sub-grouped together in the RAPD dendrogram having least genetic distance. Coinciding with the results of RAPD, the clusters based on ISSR analysis divides the Curcuma species at their genetic distances segregating them more precisely. ISSR analysis has placed C. caesia separately whereas other species were placed together with the cultivated species in another cluster. C. amada, C. angustifolia, and C. zedoaria were found to be genetically closer to each other and placed in a single group inferring their vegetative and topological similarity also. However, the genetic distance between C. aromatica and the domestic varieties of Curcuma was found to be the same in both RAPD and ISSR analysis. Different hierarchical positions of the nine Curcuma species in the dendrograms elucidated that genomes of each species are not exactly the same. Like ISSR, AFLP analysis was also found to provide a high resolution for the detection of genetic diversity and structures between and within species of Curcuma. AFLP markers separated the three varieties of a single species (C. longa, C. domestica I and II) in two subgroups depicting that intraspecific variations also exists (Fig. 3c). This supports the findings of Kavitha et al. [47] where intraspecific genetic diversity is studied of four Zingiber species using AFLP markers. C. angustifolia was placed in a separate cluster I inferring to its wild nature. C. domestica II, C. aromatica, and C. caesia were found to form a subgroup which shows a more precise discrimination among them. This can be explained that C. domestica II and C. aromatica are highlands species, possess geographic similarity and have a strong aroma; thus are found to be related. Dendrogram revealed that the species that are the derivatives of genetically similar type clustered more together. Similar findings were reported by Vanijajiva and his group in a genetic relationship study among Boesenbergia and related genera [50]. In all three cluster patterns C. domestica (I and II), C. longa, and C. aromatica are cohabiting having much similarity in floral, vegetative and rhizome characters. C. spp. also share many common vegetative and rhizome traits with the four species mentioned above. C. longa and C. domestica (I and II) as used in Indian spices and cultivated largely hence can be inferred as evolutionarily very active species. In all the three markers based dendrograms, C. aromatica is found to be genetically close to C. domestica II, both of which share a similar ecological niche in the highlands of NE India. It is evident from the data that genetic segregation does exist among the species of Curcuma both wild and domestic. These data were comparable with many works on other genera of Zingiberaceae [23, 51].

Based on Mantel tests [33], strong correlations observed and were statistically significant for each of the three marker systems used independently to study the diversity patterns of nine Curcuma species. Muthusamy [42] observed the same results while studying Vigna umbellata landraces for genetic variations. PCA further helped in depicting the variability among the species in the three dimensional modes. In case of RAPD, the first three coordinates accounted for 60.22% at the maximum. Whereas for ISSR and AFLP, the variability accounted for by three coordinates was 55.04 and 58.06% of the total variance, respectively. It was evident from the 3D plot that the species from hilly areas fall close to each other genetically. This analysis allowed understanding the spatial distribution of studied species and broadly classifies them. Similar result was reported by Islam and his group in intraspecific variation study of C. zedoaria [52]. From PCA analysis it is evident that both the methods, phenogram and three-dimensional plots of PCA were effective in studying genetic relationships and the groups found were comparable.

Conclusion

In conclusion, employing molecular marker-based study of genetic variations facilitates in the delineation of Curcuma species in dendrograms which are suggestive of an evolutionary pattern among Curcuma. The results provided an insight into the phylogenetic relationship between cultivated and wild relatives of Curcuma. ISSR markers are more discriminating than RAPD and AFLP to evaluate the genetic diversity/relationship among Curcuma species from the rich flora of NE India. Although RAPD markers are very quick and easy to develop but reproducibility is less than ISSR which detect at predetermined sites, such as DNA repetitive regions of the genome which are known as fast evolving sequences. ISSR fingerprinting opens new and interesting possibilities in the characterization of the Curcuma plants especially from NE region of India which still awaits proper systematic identification. Exploration and evaluation of diversity would be of great significance for in situ conservation of important Curcuma species especially for their scientific and commercial programmes. Furthermore, the scientific data presented here indicates that the application of PCR-based fingerprinting using whole DNA and arbitrary primers would provide a rapid and sensitive method for detection of genetic variations among the different species of the genus and also other genera of Zingiberaceae.

Acknowledgments

Authors are thankful to Ministry of Human Resources Development (MHRD), Government of India for fellowship. Thanks to Dr Sarma, Gauhati University for kind supply of study material. LR acknowledges funding by the Ministry of Information Technology, Government of India.

Copyright information

© Springer Science+Business Media, LLC 2011