Indian Journal of Biotechnology Vol 10, July 2011, pp 285-293

Genetic diversity among three Morinda species using RAPD and ISSR markers D R Singh*, Abhay K Srivastava, Amit Srivastava and R C Srivastava Division of Horticulture and Forestry, Central Agricultural Research Institute Port Blair 744 101, Andaman and Nicobar Island, India Received 8 January 2010: revised 27 August 2010; accepted 2 November 2010 A total of 22 accessions of 3 Morinda spp. (Family: Rubiaceae), namely, M. citrifolia, M. tinctoria and M. pubescens were collected from three geographical locations of India, viz., Andaman and Nicobar Islands, Tamil Nadu and Karnataka. RAPD (52) and ISSR (60) primers were employed as genetic marker to study the genotypic variation within and between Morinda spp. In RAPD, only 26 primers amplified and gave reproducible fragments, of which 11 primers were polymorphic. Among 1767 amplified DNA fragments obtained, 953 (54.33%) were polymorphic. The accessions AHD and SHE-1 were the most closely related cultivars with the highest similarity index (0.943) and BBD and MAA-1 were the most distantly related cultivar with lowest similarity index (0.387). In ISSR, of 60 primers tested, 22 gave clear and reproducible fragments. In total, 1892 fragments of different lengths were amplified, of which 1052 bands (56.02%) were polymorphic. According to ISSR results, the two most closely related cultivars were MTC and JGH with the highest similarity index (0.944) and the most distantly related cultivars were BMN with MEM and BGL-2 with lowest similarity index (0.248). Polymorphism and similarity index values for both RAPD and ISSR systems showed that both marker systems are equally effective for diversity analysis in Morinda species. Keywords: Diversity analysis, ISSR, Morinda spp., RAPD

Introduction The genus Morinda (Family: Rubiaceae) is originated in India and has 80 different species1. Later, it has been naturalized in many parts of Asia, South America, Caribbean and Polynesian Islands. The adaptation features of its seed have enhanced its natural dispersion in land through streams and rivers, using ocean currents2, by fruit-eating birds and other animals, or migrating human beings colonized the Pacific Islands for its medicinal uses3-4. It has a broad range of therapeutic and nutritional values. Interestingly, it has been found that Morinda spp. are tolerant to saline soils and can have a good sustainability for cultivation in the saline affected waste lands5. M. citrifolia (Fig. 1a), known as Noni, grows predominantly along the tropical coasts. It is an economically important species being used as a folk medicine by the tribal and aboriginals in Andaman and Nicobar Islands6. Its fruit is used worldwide for making different products. More than 200 commercial entities sell Noni products, which are distributed across the globe and enjoy an enormous market share7. It has been reported that Morinda extract has _____________ *Author for correspondence: Tel: 91-3192-250236; Fax: 91-3192-251068/233281 E-mail: [email protected]

antibacterial, antiviral, antifungal, antitumour, antihelminthic, analgesic, hypotensive, antiinflammatory and immune enhancing effects8-11. Reviews on its medicinal uses9-12 summarized that its popularity seems to hinge on a combination of its traditional uses, development and distribution of modern products and a mixture of factual and fanciful information provided directly by manufacturers and indirectly by academic researchers13. Another related species, M. pubescens (Fig. 1b) is a small tree grown in tropical regions of vacant agricultural land and comparable to M. citrifolia in its medicinal and other properties14. Decoction of stem bark of this plant used in rheumatic diseases15 and root is given in dysentery16. In the traditional system of medicine, leaves and roots of M. tinctoria (Fig. 1c) are used as astringent, deobstrent, emmengogue and to relive pain in the gout17. In recent years, a number of PCR-based DNA markers, such as RAPD, ISSR, AFLP (amplified fragments length polymorphism) and SSR (simple sequence repeat) have been widely used to investigate the genetic structure of a population18-19. The selections of RAPD and ISSR were based on their relative technical simplicity, level of polymorphism they detect, cost effectiveness, easily applicable to

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Fig. 1—Showing fruits of M. citrifolia (A), M. pubescence (B) and M. tinctoria (C)

any plant species and target those sequences which are abundant throughout the eukaryotic genome and are rapidly evolved20-21. RAPD has been the most employed technique because it is simple and fast. Despite questions about its reproducibility, its utility in diversity analysis, mapping and genotype identification has been exploited in many plant species22-23. A series of studies have indicated that ISSR could be able to produce more reliable and reproducible bands because of the higher annealing temperature and longer sequence of ISSR primers considered superior to RAPD24-25. ISSR has proved to be useful to the study of population genetic studies18,26-28, gene mapping29, germplasm identification and characterize gene bank accessions30 as well as to identify closely related cultivars31. Since, no attempt have been made to characterize the genetic diversity of Morinda cultivars through molecular markers, the present study was under taken to analyze DNA marker based genetic diversity analysis using the RAPD and ISSR primers within and between Morinda spp. for identifying elite genotype for its improvement Material and Methods Collection of Plant material

A total of 22 accessions of Morinda spp. shown in Table 1 were colleted. M. citrifolia samples contain 8 different accessions of Andaman and Nicobar Islands (A & N) and 1 accession each from Chennai (Tamil Nadu) and Bangalore (Karnataka); M. pubescens samples contain 10 accessions from Andaman and Nicobar Islands and one from Chennai; and M. tinctoria samples contain only 1 accession from Chennai. All these plant samples were planted in Horticulture Field and maintained in Germplasm Collection Block of Central Agricultural Research Institute, Port Blair, Andaman and Nicobar Islands, India.

Table 1—The collection sites of Morinda spp. Morinda spp. 1

M. citrifolia

2 3 4

“ “ “

5 6 7 8

Locations

Accessions

State

name Memeo

MEM MNJ ABH-1 SEH-1

“ “ “ M. tinctoria

Manjeri Bahai House Seamen Hostel Hadoo Jungli Ghat Sippighat Chennai

AHD JGH SGH MTC

9

M. citrifolia

Bambooflat

BFT

10

‘’

Chennai

MAA-1

11 12

Bangalore Brich Ganj

BGL-2 BCG

13

‘’ M. pubescens “

SEH-2

14

“

15 16 17 18 19 20

“ “ “ “ “ “

Seamen Hostel Hut Bay Island Bahai House Barma Nala Phoneix Bay Calicut Bidnabad Chennai

21

“

22

“

Campel Bay Island Nicobar Island

HBY ABH-2 BMN PBY CCT BBD MAA-2 KBY NCB

A&N Islands “ “ “ “ “ “ Tamil Nadu A&N Islands Tamil Nadu Karnataka A&N Islands “ “ “ “ “ “ “ Tamil Nadu A&N Islands “

DNA Isolation and PCR Amplification

Genomic DNA of 22 genotypes was extracted from 3 g fresh leaflets following the CTAB method with slight modifications32. After purification, DNA was quantified by both spectrophotometricaly and visualized under UV light after electrophoresis on 0.8% (w/v) agarose gel. The resuspended DNA was

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diluted in autoclaved ddH2O. A total of 52 RAPD (Bangalore Genei Pvt. Ltd., Bangalore, India) and 60 ISSR primers (Sigma, St. Louis, Mo.) were screened. PCR amplification were carried out in a thermal cycler in a final volume of 25 µL, containing 25 ng template DNA, 100 µM of each of the four deoxynucleotide triphosphate, 20 ng of decanucleotide primer, 1.5 mM MgCl2, 10× Taq buffer (10 mM Tris HCl pH 9.0, 50 mM KCl ) and 0.5 U Taq DNA polymerase (Bangalore Genei Pvt. Ltd., Bangalore, India). The samples were subjected to initial denaturation for 5 min at 94°C, followed by 39 cycles of 1 min at 94°C, 1 min at 36°C for RAPD and 42-64°C for ISSR, and extension for 1 min at 72°C with a final extension of 7 min at 72°C. 10 µL of amplified PCR product was separated through gel electrophoresis on 2% agarose gel stained with ethidium bromide and photographed with gel documentation system (Vilber Loubmet, France, Cat.No. Bio ID++ver.99.04)33. Analysis of RAPD and ISSR Profiles

DNA fragment sizes on agarose gel were estimated by comparing with 500 bp to 1 kb DNA size markers. The bands were scored ‘1’ for presence and ‘0’ for absence in DNA samples amplified to create a binary data matrix. The data obtained by scoring the RAPD and ISSR profiles with different primers individually as well as collectively were subjected to the construction of similarity matrix using Jaccard’s coefficients34. The similarity values were used for cluster analysis. Sequential hierarchical agglomerative non-overlapping (SHAN) clustering was done using unweighted pair group method with arithmetic averages (UPGMA) method. Data analysis was done using NTSYS-PC, version 2.0235. The similarity matrix was obtained after multivariate analysis using the Dice coefficient similarity36. Results RAPD Analysis

Amplified products were observed for each accession of M. citrifolia, M. tinctoria and M. pubescens using RAPD-PCR analysis with 52 arbitrary random primers (Figs 2a & b). A total of 26 RAPD primers were amplified and 11 showed polymorphic banding pattern. The codes and sequences of the amplified primers are shown in (Table 2). A total number of 1767 DNA fragments were amplified from 26 primers, among them 953 (54.33%) were polymorphic. The results of the

Fig. 2 (a & b)—Amplification with RAPD primers OPH 11 (a) and OPH-31 (b): Mx & My, 500 bp ladder (a); Mx, 1 kb & My, 500 bp ladder (b); lane 1, MEM; 2, MNJ; 3, ABH-1; 4, SEH-1; 5, AHD; 6, JGH; 7, SGH; 8, MTC; 9, BFT; 10, MAA-1; 11, BGL-2; 12, BCG; 13, SEH-2; 14, HBY; 15, ABH-2; 16, BMN; 17, PBY; 18, CCT; 19, BBD; 20, MAA-2; 21, KBY; & 22, NCB (a & b).

consensus tree indicated that tree was divided into two major clusters, each having 11 accessions showing 45% similarity (Fig. 3). The cluster I sub-divided in two sub-clusters with 82% similarity. The first sub-cluster had 8 collections including 1 accession of M. tinctoria and 7 accessions of M. citrifolia from A & N, and second subcluster had 3 collections each from A & N, Bangalore and Chennai. The second major cluster divided in two sub-sub-clusters with 76% similarity. The first sub-cluster had all 5 accessions of M. pubescens from South Andaman and second had 6 accessions, 3 from Andaman and 1 each from Chennai, Kambel Bay and Nicobar. The similarity coefficient based on the 1767 DNA products ranged from 0.387 to 0.943. In RAPD analysis two most closely related genotypes were AHD and SHE-1 with the highest similarity index

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Table 2—Amplified primers and sequences for RAPD profiling No.

Primer code

Sequance 5'-3'

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

OPH-1 OPH-2 OPH-3 OPH-4 OPH-5 OPH-6 OPH-7 OPH-8 OPH-9 OPH-10 OPH-11 OPH-13 OPH-14 OPH-15 OPH-17 OPH-19 OPH-20 OPH-21 OPH-31 OPH-33 OPH-34 OPH-36 OPH-39 OPH-41 OPH-42 OPH-43 OPH-44 OPH-45

AATCGGGCTG CAATCGCCGT TCTGTGCTGG GACCGCTTGT GTTGCGATCC TCGCCGCAAA AGCGTCACTC GTCCGTACTG GGTGCTCCGT GACCGACCCA CGGTTCCCCC GTCTGACGGT CAGCTCAAGT CGATCGAGGA AAGCAGCAAG AGCATTCGGT CACCGTTCTG ACTCCGCAGT TAGACAGTCG AGGCCGTATC ATGAGTCCAC TCAAACTCGG TAGCCGTCAA ATTTGATCGC ACGCTGATCA AACCGACGGG TGCCCTGCCT CTTGCCTCCC

Fig. 3—Dendrogram showing similarity coefficient of Morinda accessions based on RAPD analysis

(0.943) and the two most distantly related cultivar were BBD and MAA-1 with the lowest similarity index (0.387). Within species diversity, the two most closely related accessions were AHD and SHE-1 (0.943) and the two distant accessions were MEM and MAA-1 (0.750) for M. citrifolia; while for M. pubescens, the two most closely related accessions were BCG and ABH-2 (0.906) and the distant accessions were BBD and BCG (0.700) (Table 3). ISSR Analysis

A total of 60 ISSR primers based on dinucleotide, tetranucleotide or pentanucleotide repeats were used to amplify each genotype of M. citrifolia, M. tinctoria and M. pubescens (Figs 4a & b). Among the primers tested, 38 primers amplified but only 22 primers (Table 4) those produced clear bands and had reproducibility were selected for further analysis. A total number of 1892 fragments with different lengths were clearly amplified from 22 accessions, among which 1052 bands (56.02%) were polymorphic. The results showed that 10 amplified primers were monomorphic and 12 were polymorphic. Among the

Fig. 4 (a & b)—Amplification with ISSR primer UBC 7 (a) and UBC 34 (b): Mx, 1 kb & My, 500 bp ladder (a); Mx, 500 bp & My, 1 kb ladder (b); lane 1, MEM; 2, MNJ; 3, ABH-1; 4, SEH-1; 5, AHD; 6, JGH; 7, SGH; 8, MTC; 9, BFT; 10, MAA-1; 11, BGL-2; 12, BCG; 13, SEH-2; 14, HBY; 15, ABH-2; 16, BMN; 17, PBY; 18, CCT; 19, BBD; 20, MAA-2; 21, KBY; & 22, NCB (a & b).

primers studied, the highest number of bands were generated with primer UBC-10 (132 bands), while the lowest number of 34 bands were generated with primer UBC-54. The results of the consensus tree from ISSR data indicated that tree was divided into

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Table 3—Similarity index for RAPD

two major clusters each having 11 accessions with 38% similarity (Fig. 5). The first major cluster divided into 2 sub-clusters with 85% similarity; first sub-cluster had 8 collections including M. tinctoria and second had 3 collections, 1 each from Andaman, Chennai and Bangalore. The second major cluster again divided into 2 sub-clusters, first having 5 accessions and second having 6 accessions with 67% similarity. According to the ISSR results, the similarity coefficient based on the 1892 DNA products ranged from 0.248 to 0.944. The most closely related genotypes were MTC and JGH with the highest similarity index (0.944) and the most distantly related genotypes were BMN, MEM and BGL-2 with lowest similarity index (0.248) (Table 5). The ISSR results showed the same pattern of clustering as in RAPD. Within species diversity studies by ISSR, the two most close accessions were AHD and SHE-1 (0.95) and the most distant accessions were BGL-2 and MEM (0.77) for M. citrifolia; while for M. pubescens, the two most close accessions were NCB and KBY (0.86) and the most distant accessions were BCG and CCT (0.58).

Table 4—Amplified primers and sequences for ISSR profiling No.

Primer code

Sequance 5'-3'

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

UBC-1 UBC-2 UBC-3 UBC-7 UBC-8 UBC-9 UBC-10 UBC-14 UBC-15 UBC-18 UBC-19 UBC-20 UBC-34 UBC-35 UBC-36 UBC-45 UBC-46 UBC-47 UBC-54 UBC-55 UBC-56 UBC-57

ATATATATATATATATT ATATATATATATATATG ATATATATATATATATC AGAGAGAGAGAGAGAGT AGAGAGAGAGAGAGAGC AGAGAGAGAGAGAGAGG GAGAGAGAGAGAGAGAT CTCTCTCTCTCTCTCTA CTCTCTCTCTCTCTCTG CACACACACACACACAG GTGTGTGTGTGTGTGTA GTGTGTGTGTGTGTGTC AGAGAGAGAGAGAGAGYT AGAGAGAGAGAGAGAGYC AGAGAGAGAGAGAGAGYA CTCTCTCTCTCTCTCTRG CACACACACACACACART CACACACACACACACARC TCTCTCTCTCTCTCTCRG ACACACACACACACACYT ACACACACACACACACYA ACACACACACACACACYG

R=Purines Y=Pyrimidine

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Combined RAPD and ISSR analysis

Genetic diversity of 22 accessions was estimated using combined RAPD and ISSR data. The results indicated that consensus tree was divided into two major clusters, each having 11 accessions showing 42% similarity (Fig. 6). The first major cluster divided into 2 sub-clusters with 84% similarity, the first su-bcluster consisted of 8 collections including M. tinctoria and the second one with 3 collections. The second cluster divided into 2 sub-clusters with 76% similarity and had 5 and 6 accessions,

Fig. 5—Dendrogram showing similarity coefficient of Morinda accessions based on ISSR analysis

respectively. The combined data showed the same pattern as clustered in RAPD and ISSR. The combined data revealed that the two most closely related cultivars were SEH-1 and AHD-1 with the highest similarity index (0.948) and the most distantly related cultivars were BMN with BGL-2 with low similarity index (0.335) (Table 6). Within species diversity studies, the two most closely related accessions were AHD and SHE-1 (0.95) and the distant accessions were BGL-2 and MEM (0.76) for

Fig. 6—Dendrogram showing similarity coefficient of Morinda accessions based on combined RAPD-ISSR analysis

Table 5—Similarity index for ISSR

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Table 6—Similarity index for combined RAPD-ISSR

M. citrifolia; while for M. pubescens, the two most closely accessions were NCB and KBY (0.86) and the distant accessions were BCG and BBD (0.64). Discussion Morphological characters in plants may be affected by environmental conditions and a species grown in different environmental conditions may be different morphologically. Thus, the use of morphological characters for classification may result in discrepancy. Efficiency of a molecular marker technique depends on the amount of polymorphism it can detect among the set of accessions under investigation. Genotype distribution on the consensus tree based on the combined banding patterns of RAPD and ISSR may significantly differ because it is possible that each technique amplify different parts of the genome. It is, therefore, better to use the combination of banding patterns of the two techniques in order to use more segments sites of the genome that will increases the validity of the consensus tree. RAPD analysis has been used to study the genetic relationship in a number of grasses like switch and

forage37-40. RAPD and ISSR has been extensively used in many crops and a comparison of both concluded that ISSR would be a better tool than RAPD for phylogenetic studies41-45. Nagaoka and Ogihara (1997) have also reported that the ISSR primers produced several times more information than RAPD markers in wheat. In a study on 16 barley cultivars form different countries, higher similarity index was observed with ISSRs in comparison to RAPDs47. In the present study, RAPD results showed BBD and MAA-1 as the most divergent ones, while BMN, MEM and BGL-2 accessions were most diverse as per the ISSR results. On the other hand, the combined RAPD-ISSR results showed that BMN and BGL-2 accessions were the most divergent acccessions. A close genetic similarity was observed in some of the cultivars analyzed as shown by high values of similarity index. Based on similarity matrix using simple matching coefficient, the similarity values between all the Morinda spp. were from 38-94% for RAPD, 25-94% for ISSR and 33-95% for combined RAPD-ISSR analysis. Although, the similarities

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detected with ISSRs (0.944) between MTC and JGH were nearly same as observed with RAPDs (0.943) between AHD and SHE-1. Also for combined RAPD-ISSR, it was found 0.948 between SEH-1 and AHD-1, which is similar to RAPD and ISSR result. M. tinctoria from Chennai showed close relationship with M. citrifolia in all cases, i.e., RAPD, ISSR and combined RAPD-ISSR analysis. This may be due to either seed dispersal through sea or the seedlings transported from one place to another by migrating people for its medicinal uses. Genetic variations observed in some of the accessions are very narrow like BMN and MEM and between BMN and BGL-2, it might be because of less distance between accessions. This study provides evidence that RAPD and ISSR polymorphisms could be used as efficient tools for the detection of similarities and phylogenetic relationships between the studied genotypes. A similar observation was made by several other studies48-52.

8 9

10 11

12

13

14

15

Acknowledgment The authors express their sincere thanks to Central Instrumentation Facility (CIF), Central Agricultural Research Institute, Port Blair, Andaman and Nicobar Island, India for technical assistance. Authors are also thankful to World Noni Research Foundation, Chennai for financial assistance for the study.

16

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