Introduction

Salvadora oleoides Decne belongs to the family Salvadoraceae. It is a small tree with a short twisted trunk and drooping branches, found in the arid regions of Pakistan and western India. It invaded India after closing of the east end of the Mediterranean from Africa and Arabia via Baluchistan to Sind and Rajasthan (provinces of India) then penetrated as far south else Travancore. S. oleoides commonly known in India as meetha jaal, is an oil yielding medicinal and multipurpose tree. It is deep rooted mesomorphic xerophytes as well as facultative halophytes with high salt tolerance (Mebberely, 2008). In spite the wide use of this plant in India, no effort has been made regarding its conservation strategy, as human intervention has large impact in the exploitation of this plant species.

A major objective of conservation biology is to preserve the evolutionary prospective of species by maintaining natural levels of genetic diversity (Hamrick et al.,1991). There could possibly be a genetic basis for the endangered nature if a species has very low allele frequency. Survival of a species depends on the maintenance of genetic variability within and among populations that accommodate new selection pressures brought about by environmental changes. It is very important to understand the complex processes involved in the long term evolutionary history of species such as genetic drift, mutation, gene flow within the populations (Faisal et al.,2007). Restricted populations have lower genetic diversity than widespread species. Assessment of genetic variation and its partitioning within and between populations of this plant is necessary for formulating conservation management strategies.

Molecular markers, which detect variation at the DNA level overcome most of the limitations of morphological and biochemical markers. As demonstrated by their use in various plant species, molecular markers are best suited for estimation of genetic diversity and varietal identification. Besides their unlimited numbers, molecular markers are not affected by environmental and developmental stage. Various molecular marker techniques like RFLPs (Lebrun et al.,1998), RAPD (Ashburner et al.,1997), AFLP (Teulat et al.,2000), SSRs (Rivera et al.,1999) have been used for analysis of plant genetic diversity in different regions. Although newer techniques like AFLP and SSR are preferred due to their informativeness, RAPD is still method of choice for less advanced laboratories because of its simplicity, low cost and lower infrastructure requirement. The present study was undertaken to estimate the genetic diversity and genetic relationship among Salvadora oleoides accessions using RAPD markers.

Materials and Methods

Plant materials and DNA extraction

A total of 19 samples (5 from Haryana and 7 each from Rajasthan and Gujrat) were assessed (Table 1). Young leaves were harvested and dried in ethanol on the same spot and appropriately labelled. The collected leaves were used for DNA extraction, while excess leaf materials were stored at room temperature for future DNA extraction. Total genomic DNA was extracted using a modified CTAB method based on the protocol of Doyle and Doyle (1990). Quality and concentration of total DNA was verified by UV 2450 spectrophotometer (Shimadzu, Japan) at 260 and 280 nm. Further quality of DNA was tested by submerged horizontal agarose gel (0.8%) elec­trophoresis and visualized under UV light, gel documentation system (Alpha Innotech, USA).

Table 1:

Latitude and longitude of collection sites of different accessions of Salvadora oleoides from North-West India.

RAPD analysis

84 different random decamers (50 from UBC, 34 from Operon) were screened initially. RAPD reaction and procedures were carried out as described by Williams et al.,(1990) with some modifications. The PCR reaction mixture consisted of 50 ng template DNA, 1 x PCR buffer (10 mM Tris-HCL pH 8.8, 25mM KCL, 15mM MgCl2), 200 μM dNTPs (Bangalore Genei Pvt. Ltd., India), 0.2 μM 10-base random primers and 1 unit of Taq polymerase (Bangalore Genei Pvt. Ltd., India), in a total volume of 25 μl. The PCR reaction was carried out in My cycler thermal cycler (Biorad, USA). The PCR amplification conditions for RAPD consisted of initial extended step of denaturation at 940C for 4 minutes followed by 44 cycles of denaturation at 940C for 1 min, primer annealing at 370C for 1 min and elongation at 720C for 4 min with a final extension at 720C for 10 minutes. Amplification products were separated on 1.5 % agarose (SRL, India) gel in 1X TAE buffer along with a DNA ladder (Fermentas). The DNA bands were photographed using the gel documentation system (Alpha Innotech, USA).

Data analysis

The reproducible bands were scored as present (1) or absent (0) across all accessions. Each band was regarded as locus. All calculations were done using computer program NTSYS pc version 2.02 (Rohlf, 1998). Similarity matrix was constructed using the Jaccard’s similarity coefficients and subjected to UPGMA (unweighted pair-group method with arithmetic averages) analysis to generate dendrogram. Polymorphism information content (PIC) was also calculated according to Anderson and his co-workers (Anderson et al.,1993).

The 16 primers generated amplicons ranging from 5 (UBC 66) to 13 (UBC 64 & UBC 76). The reproducibility of the bands generated by these 16 primers was confirmed by replicating the amplification twice and if needed thrice. Only the clear and unambiguous bands were considered for scoring and for further analysis. The number of polymorphic bands ranged from 5 to 13 with range of polymorphisms 33.3% (UBC 89) to 100% (UBC 51, 56, 58, 64, 66, 67, 77, 78, 86). The total number of bands generated by 16 amplifying primers was 164 with an average amplification of 10.25 bands per primer (Figure 1).

Fig. 1: –

Gel photographs showing polymorphism obtained by different primers.

The clusters constructed through NTSys (2.02 PC) presented in the form of dendrogram has been shown in Figure 2. The average polymorphism generated by these bands was 90.09%. The size of the amplicons generated varied from 290 bp to 4400 bp. In the present study, the PIC ranges (Table 2) from 0.246 (UBC 89) to 0.436 (UBC 77) were also calculated. The Jaccard’s pairwise similarity coefficient values ranged from 0.21 (Panipat and Hanuman Garh) to 0.94 (Sirohi & H. Garh, Kuchh & Amreli) (Table 3). The dendrogram has put all the genotypes in two clusters (CL1 and CL2). CL2 comprises of two accessions (Jalore & Jhunjhunu) while CL1 is further divided into two groups (1 & 2). The group 1 comprises 4 accessions (Rohtak, Hisar, Rewari, M. Garh) while group 2 is further divided in two sub groups (SG1 & SG2) out of which SG1 comprises 6 accessions while SG2 comprises 7 accessions.

Fig. 2: –

UPGMA dendrogram of 19 accessions of S. oleoides based on 16 random RAPD primers.

Table 2:

List of single arbitrary primers showing total and polymorphic amplicons generated along with PIC of each pattern for 19 genotypes of Salvadora oleoides.

Table 3:

UPGMA dendrogram of 19 accessions of Salvadora oleoides based on 16 random RAPD primers. (1-Rohtak, 2-Hisar, 3-Panipat, 4-Rewari, 5-M.Garh, 6-Jodhpur, 7-Bikaner, 8-Sirohi, 9-Jalore, 10-H.Garh, 11-Jhunjhunu, 12-Sikar, 13-Jamnagar, 14-Bhavnagar, 15-G.Nagar, 16-Kuchh, 17-Junagarh, 18-Porbandar, 19-Amreli).

Discussion

The technical simplicity of the RAPD technique has facilitated its use in the analysis of genetic relationship in several genera (Wilikie et al.,1993). The major concern regarding RAPD-generated phylogenies, include homology of bands showing the same rate of migration, causes of variation in fragment mobility and origin of sequence in the genome (Stammers et al.,1995). In spite of this limitation, RAPD markers has the greatest advantage of its capability to scan across all regions of the genome hence highly suited for phylogeny studies at species level (Demeke et al.,1992).

In the present study, the medicinal plant S. oleoides showed a high percentage of genetic polymorphism of 90.09% which was near to the percentage for Cannabis sativa (79.77%) (Pinarkara et al.,2009) but higher than that of Dacydium pierrei (33.3%) (Su et al.,1999) and Cathaya argrophylla (32%) (Wang et al.,1996). Similarly, the genetic diversity index was also highly variable from 0.21 to 0.94 in case of S. oleoides accessions. These studies indicate that RAPD is sufficiently informative and powerful to access genetic variability of natural populations of S. oleoides. Thus, RAPD markers will provide a useful tool in the future design of collection strategies for germplasm conservation.

The genetic diversity of the plants is closely related to their geographic distribution. Species with a wide geographic area generally have more genetic diversity (Wilikie et al.,1993). High diversity is the reflection of adaptation to environment, which is beneficial to its propagation, resources conservation the domestication of wild species and the screen of specified locus (Hepsibha et al.,2010). This RAPD analysis showed high genetic diversity in S. oleoides accessions growing in different environments and low diversity in S. oleoides accessions in the same or adjacent regions. High similarity indices in accessions growing in adjacent regions suggest that the individuals in the population have close genetic relation among them. This situation can rise in natural populations when there is a possibility of free/random pollen flow and fertilization. The genetic similarity of the samples slightly correlated with their close geographic locations (Sayed et al.,2009).

Similarly distantly located accessions may also show high similarity indices which may be due to that these accessions may disperse out from same location or may be that these were not acclimatized to their local environment. In the present study it was found that accessions Jalore/Jhunjhunu and Panipat/Hanuman Garh/Sikar were almost molecularly identical and grouped together even though they belong to geographically different locations.

During intraspecific genetic variability study, the divisions of 19 samples of S. oleoides into two clusters as shown in Figure 2 allowed very little chances for gene flow among accessions that were geographically distant, but the probability of naturally occurring genetic cross and gene flow should be high among accessions growing near each other. So, this study concluded that the high genetic diversity among accessions in adjacent regions was mostly attributable to artificial introduction, not natural genetic differentiation.

The level and distribution of genetic diversity detected by RAPD are in overall agreement with recent studies in India (Chaurasia et al.,2009). RAPD, being a multi-locus marker with the simplest and fastest technology, have been successfully employed for determination of intra-species genetic diversity in several plant species (Gupta et al.,2010).

The calculated PIC based on the probability that two unrelated genotypes amplified from the test population will be placed into different typing groups. PIC estimates the degree of polymorphism of marker, which essentially is the proportion of individuals that are heterozygous for a marker. PIC is a good measure of the heterozygosity. It is an index of how many alleles a certain marker has and how those alleles divide. High PIC value indicates rich heterozygosity which in turn is associated with a high degree of polymorphism (Zimmer and Roalson, 2005). In case of S. oleoides, good range of PIC value was observed, which showed significant genetic diversity among S. oleoides accessions.

Conclusion

In conclusion, the result of this study indicates that the efficiency and ease of using RAPD markers for investigating genetic relationship and identification of wild medicinal plants is good tool. To the best of our knowledge, this is the first report on the characterization of S. oleoides genotypes based on commercially available primers from North-West India. The powerful capability of molecular technique to distinguish closely related genotypes based on their RAPD patterns has been brought out by this study.