Vol.6 No.1 January-June 2013, pp.21-29 @ International...

IJBR Vol.6 No.1 January-June 2013, pp.21-29 @ International Science Press, (India)

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ASSESSMENT OF GENETIC DIVERSITY IN CHAKKARAKOLLI (GYMNEMA SYLVESTRE R. BR.)

USING RANDOMLY AMPLIFIED POLYMORPHIC DNA (RAPD) MARKER FROM DIFFERENT LOCATIONS OF

GUJARAT, INDIA

Sanjay Lal, Kinnari N. Mistry* Nisha Daxini and Swati Patel Ashok & Rita Patel Institute of Integrated Studies in Biotechnology &

Allied Sciences (ARIBAS), New Vallabh Vidhya Nagar – 388121 (Gujarat) India.

Email:- [email protected]

Abstract:-Gymnema, belonging to the family Asclepiadaceae is an important medicinal plant due to its biological activity to cure diabetes, fat accumulation & extinct succulent plant in India. Twelve distinct plants of Gymnema accessions representing different geographical regions of Gujarat were characterized using RAPD primers. Five RAPD primers were used, which gave reproducible results with all the plants showing 100% polymorphism. Cluster analysis of RAPD data using UPGMA produced two major clusters & further sub clusters. Overall, molecular fingerprinting revealed the existence of considerable genetic variation in Gymnema sylvestre plants collected from various ecogeographical regions of Gujarat. Keywords: Gymnema sylvestre, Genetic diversity, RAPD, Gujarat

INTRODUCTION More than 80% of the world’s population uses natural medicines and depends on medicinal plants for health care. At present, 90% collection of herbal raw drugs used in the manufacture of Ayurveda, Siddha, Unani, and Homeopathy systems of medicine is largely from the wild out of which 70% collection involves destructive harvesting (Pandey et al. 2010). Leaves and roots are the useful parts of the plant. This climber is extensively used in almost all the Indian system of medicine as a remedy for rheumatism, cough, ulcer, and pain in eyes. It is also useful in inflammations, dyspepsia, constipation, jaundice, etc. Roots have been reported as a remedy for snakebite. The plant is popularly known as “Gudmar” for its distinctive property of temporarily destroying the taste of sweetness. Many medicinal plants are highly sensitive to the level of harvest and fragility of the ecosystem; one of them is Gudmar Madhunashini (Gymnema sylvestre) commonly known as “Gudmar” in Hindi is an important medicinal climber belonging to the family Asclepiadaceae. In India, it is found in the forests of Gujarat, Andhra Pradesh, Bihar, Chhattisgarh, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Orissa, Tamil Nadu, Uttar Pradesh, and West Bengal. Due to its heavy demand in South East Asian countries, the plant is becoming endangered and under cultivation in southern states of India, particularly in Tamil Nadu. Leaves and roots are the useful parts of the plant. The ethanolic extract of leaves demonstrated antimicrobial activity against Bacillus Spp, Pseudomonas Spp, Proteus spp & E.coli (Satdevi, et al. 2003). Recently morphological characters are supported with various kinds of biochemical and molecular data. Mostly (RAPD) Random Amplified Polymorphic DNA markers (Williams et al. 1990), (RFLP)


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Restriction Fragment Length Polymorphism (Kernodle et al. 1993), (AFLP) Amplified Fragment length Polymorphism multilocus fingerprinting technique developed by Vos et al.1995, (SSR) Simple Sequence Repeat & (ISSR) Inter Simple Sequence Repeat (Zietkiewicz E et al. 1994) etc., were used to study interspecific and intraspecific divergence. The most common uses of the technique for scientific and commercial purposes include the discrimination among species for post-harvest purposes, identification of crop cultivars to settle lawsuits, marker assisted selection establishment of phylogenetic position of natural populations, certification of lack of polymorphism between in vitro and conventionally propagated plant stands and determination of genetic variation in wild and cultivated populations (Sanjay lal et al., 2011) and assessment of genetic diversity in many species, including medicinal plants due to technical simplicity and speed (Padmalata K et al. 2006) The multipurpose use has attracted global attention which prompted us to conduct studies on genetic diversity of Gymnema spp. to provide genetic data and a theoretical basis for protection of the species. Hence, an attempt was made to investigate variation among the collected different species of Gymnema by using RAPD markers. RAPD markers are based on the amplification of unknown DNA sequences using single, short and random oligonucleotide primers. Moreover the contaminating RNA that precipitates along with DNA causes many problems including suppression of PCR amplification (Pikkart et al. 1993), interference with DNA amplification involving random primers. Therefore, RAPD polymorphism is the reflection of variation of the whole genomic DNA and would be a better parameter to measure the pattern of genetic diversity of the rare and endangered plants. The genetic divergence of 0.15 to 0.28 across the Gymnema accessions using RAPD markers reflected considerable variation at DNA level (Smita Nair et al. 2006). The objectives of this study was to determine; intraspecific & interspecific genetic diversity, for identification of ideal genes, e.g. gene that encodes drugs and for recommendation based on existing information for benefit and development in medicinal sectors in India. MATERIALS AND METHOD Experimental Materials Twelve plants of Gymnema Sylvestre from different locations (Udwada, Himatnagar, Baroda, Nandigam, Anand, Vasad, Bharuch, Gandhinagar, Modasa, Gundevi, Mathasar, Surat) were collected from nearby area of Anand city, Gujarat, India. Genomic DNA isolation and RAPD PCR amplifications Initially, genomic DNA was isolated from fresh young leaves by standard CTAB (Cetyl trimethyl ammonium bromide) method (Doyle JJ et al. 1987) & modified CTAB DNA extraction protocol for plants containing high polysaccharides & polyphenol components (Porebski S et al. 1997). DNA amplification was performed in a Corbett Research gradient automatic thermal cycler using RAPD markers. Genomic DNA was amplified via the PCR reaction using 25µl reaction mixture containing 10X Taq buffer (2.5µl) (Genei, Banglore), dNTPs (2.5µl)(2.5mM each) (Genei, Banglore), Taq DNA polymerase (0.5µl) (1000U/ µl) (Genei, Banglore), primer (1µl) (40µM/ µl) (Sigma Aldrich, Bangalore), template DNA (0.5µl)(40ng/ µl) and sterile nano pure water (18µl). After initial denaturation of the DNA at 94ºC for 5 min, the thermal cycling was performed with the following temperature regimes –1 min at 94ºC, 1min at 37ºC and 2 min at 72ºC with 35 cycles and final extension at 72ºC for 7 min while hold temperature is 4ºC. Control reactions without template DNA (negative control) and without enzyme (positive control) were also run in the experiments. All the


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experiments were repeated thrice to ensure reproducibility. Amplified DNA fragments were separated by electrophoresis at 150 V for 3-4 hours on 2.0%agarose gel stained with Ethidium bromide initial and photographed by gel documentation system (Alpha Innotech, Alpha Imager EP, and U.S.A). All PCR experiments were done at least twice and best gels of the replicates were used for band scoring. RAPD data analysis & scoring Unequivocally scorable and consistently reproducible amplified DNA fragments were transformed into binary character matrix (1=presence, 0=absence). The results were analyzed on the principle that a band is considered to be ‘polymorphic’ if it is present in some individuals and absent in others, and ‘monomorphic’ if present in all individuals. Using dice coefficients, a similarity matrix involving five different species was generated with NTSYS-pc (Numerical taxonomy system, applied biostatistics, Inc., New York, USA, software version 2.02e) (Sonnate G et al. 2002). A dendogram was constructed using the unweighed pair group method with arithmetical averages (UPGMA). RESULT AND DISCUSSION

Table 1: Twelve plants of G. Sylvestre collected from different locations of Gujarat, India Sr.No. Plant Locations 1.

Udwada, South Gujarat 2. Himatnagar, North Gujarat 3. Baroda, East Gujarat 4. Nandigam, North Gujarat 5. Anand , South Gujarat 6. Vasad, South Gujarat 7. Bharuch, South Gujarat 8. Gandhinagar, North Gujarat 9. Modasa, North Gujarat 10. Gundevi, South Gujarat 11. Mathasar, North Gujarat 12. Surat, South Gujarat

In RAPD total 5 random primers were used to check the genetic variation among twelve plants of G.sylvestre collected from different location within zones of Gujarat. The RAPD banding pattern of RAPD analysis for five primers is shown in figure 2, 3, 4, 5 for primer RPI-3, RPI-4, RPI-5, and OPA-03 respectively. The details of banding are shown in table 2.All five primers gave amplification in all plants of different locations. Total 195 bands were observed and all were polymorphic. This resulted in polymorphism of 100%. Primer RPI-4 gave maximum number (53) of bands while minimum numbers of (17) bands were given by OPD-02 primer. The Amplicon were observed ranging from 100bp-1900bp where

Gymnema sylvestre


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the largest (1900bp) amplicon was amplified by primer OPD-02 & shortest (100bp) was amplified by RPI-4. . Table 2: Sequence of RAPD primers & Analysis of amplified bands in twelve ecotypes of G. Sylvestre

plants

Primer Sequence Range of Amplicon (bp)

Total No. of bands

Total No. of polymorphic bands

PIC value

RPI-3 ACGCGACAGT 150-1500bp 53 53 0.89498 RPI-4 AATCGCGTGC 100-1800bp 40 40 0.86 RPI-5 AATCGGGGCT 150-1800bp 47 47 0.898144 OPA-03 GGACCCACAC 230-1800bp 17 17 0.726644 OPD-02 AGTCAGCATC 250-1900bp 38 38 0.857341 Total 195 195 Ave- 0.84721

Figure 1: RAPD banding pattern for RPI-3 primer




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Figure 4: RAPD banding pattern for OPA-03

Table 3: Similarity Index based on Jaccard’s coefficient

From above similarity index, lowest similarity index value is 0.083333 & highest is 0.875000 giving 0.3360 as an average similarity index value which shows that they have very low similarities between each plant of G.sylvestre. In our study, RAPD markers were successfully used to differentiate all twelve plants of G.sylvestre from each other. Thus, on the basis of RAPD the findings of this study are similar to the observations of Smita Nair et al. (2006) The RAPD analysis detected high degree of genetic variations. The highest similarity index (0.875000) was observed between sample of Baroda and Himatnagar. The least similarity index (0.083333) was observed between sample of Udwada and Mathasur. The mean similarity was observed reasonably high (0.3360).This indicates high genetic variation among twelve G.sylvestre plants. Similar variation were observed in dendogram constructed using UPGMA method (Figure 3) as well as in 2-D & 3-D plots (Figure-5 & 6) In dendogram, two clear clusters were observed (A & B), Plants from Udwada, Himatnagar, Baroda, Vasad, Bharuch, Modasa, Gundevi, Gandhinagar, Nandigam, Mathasur, Surat were in cluster A, while plant of Anand was in cluster B. The cluster A was subdivided into A1 & A2 which is further bifurcated into A1a & A1b followed upto A1aaa1 & A1aaa2 while on other side upto A1aab.Plant from Udwada, Himatnagar, Baroda belongs to sub-sub cluster A1aaa1, while cluster A1aaa2 includes plant from Vasad. The cluster


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A1aab involves plant from Bharuch & Modasa, then the plant from Gundevi & Gandhinagar belongs to cluster A1ab while plant from Nandigam belongs to cluster A1b. The cluster A2 includes plant from Mathasur and Surat.

Figure 5: Dendogram based on UPGMA method showing genetic relationship among twelve plants of G.sylvestre by RAPD markers, Udw-Udwada, Him-Himatnagar, Bar- Baroda, Vas- Vasad, Bha-Bharuch, Mod-Modasa, Gan-Gandhinagar, Gun-Gundevi, Nan-Nandigam, Mat-Mathasur, Sur-

Surat, And-Anand

Dim-1-0.00 0.22 0.43 0.65 0.87

Dim-2

-0.70

-0.42

-0.13

0.16

0.44 udw Him

Bar

Nan

And Vas

Bha

Gan

Mod

Gun

MatSur

Figure 6a: 2-D plot


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Figure 6b: 3-D plot Figure 6: Phylogenetic relationship among twelve plants of G. Sylvestre revealed by RAPD primers: Figure 6a: 2-D plot, Figure 6b: 3-D plot. The genetic diversity obtained in this study might be useful in future strategies for evolution of desired genotypes. Our results are in agreement with previous studies reporting that the samples from various parts of state did not form well defined groups and were interspread with each other observed by Smita Nair and R. Keshavchandran 2006. The genetic variability has been assessed in G. Sylvestre plants from Western Ghats of Maharashtra using RAPD & ISSR molecular markers. The populations recorded high level of gene differentiation (GST= 0.41 based on ISSR) & gene flow (Nm=0.92 based on RAPD). The high level of gene differentiation among the populations attributed to antomophilus pollination, wind seed dispersal, out crossing and antrogenic activity (Shahnawaz et al. 2011), similarly high degree of polymorphism is observed in our study of genetic variation in G.sylvestre using RAPD markers. Many other members of Asclepiadaceae have been genetically characterized by using molecular markers such as Gymnema sylvestre R.Br, a natural potential to chemical means of blood sugar regulation. In particular report of Siddiqui et al. (2000), attempted to characterize 18 accessions of Gymnema, which were earlier identified as variants based on morphological and biochemical evaluations using isozyme and RAPD markers. Study revealed the existence of considerable variations at the molecular level in the Gymnema germplasm, which confirms the earlier results based on morphological and biochemical studies. The results could be used correlating the molecular fingerprints with desirable morphological and biochemical features. For efficient conservation and management of such threatened species as Gymnema the genetic composition of the species in different geographic location needs to be assessed. Yet there has been no previous report on the use of these methods to characterize the genetic diversity of Gymnema species. Hence, a study was undertaken to provide unambiguous identification of Gymnema germplasm and to characterize its ecotypes. A relatively simple & easy method for detection of genetic variation & subsequent decision making for hybridization program is RAPD-PCR. This technique has been previously applied on different plant

SurMat

Gun

ModGan

Bha

Vas

And

Nan

BarHimudw


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species. The RAPD technique applied to assess molecular polymorphism in Vigna (Kaga et al. 1996), mung bean (Lakhanpaul et al. 2000), Pea (Simioniuc et al. 2000), Wheat (Gorji et al. 2010). Even used for assessment of genetic diversity in different cultivars of Catharanthus roseus & resulted in high degree of genetic variation using RAPD, ISSR, SSR markers (Sanjay lal et al. 2011). Through some research & studies of genetic diversity, it has been observed that domestication, selection and breeding have caused a decreased in genetic variation (McGarth et al. 1999). Several aspects of plants responses to different environmental conditions have impact through this phenomenon such as susceptible for surviving in unfavorable conditions. Hence, in order to avoid such problem in future, it becomes necessary to study inter & intra specific genetic diversity. In the present study, we selected G.sylvestre, since it has important medicinal uses and can be grown easily in normal environmental conditions. CONCLUSION RAPD markers proved potential tools for analyzing genetic variation among twelve G. Sylvestre plants collected from 12 locations of Gujarat. So, it can be used to design a strategy to maintain or enhance the genetic diversity of future varieties. The polymorphism data generated can be used for plant breeding, crop improvement programs & also might be useful in future strategies for evaluation of desired genotypes & further development of new G. sylvestre plants. ACKNOWLEDGEMENT Authors are grateful to Charutar Vidya Mandal (CVM) Vallabh Vidyanagar, Gujarat for providing platform for this research work. We are also thankful to our director of Ashok and Rita Patel Institute of Integrated Study & Research in Biotechnology and Allied Sciences (ARIBAS) New Vallabh Vidya Nagar, for providing the facilities and their valuable suggestions during our research work. REFERENCES

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