Abstract: Background and Objective: M. oleifera belonging to the monogeneric family Moringaceae. It is drought-tolerant and must be cut back several times to make it branch out more. It tolerates a large range of soils and rainfall conditions. The present study has been aimed to estimate genetic relatedness among the seeds of M. oleifera from 3 different states of India i.e., Himachal Pradesh, Maharashtra and Uttarakhand using RAPD markers. Materials and Methods: Estimation of genetic relatedness among the seeds done through Isolation of genomic DNA, agarose gel electrophoresis, polymerase chain reaction (PCR) amplification by using RAPD (OPA, TC series) primers and UPGMA dendrogram. Results: Initially, nine random decamer primers were used in the study and based on the higher degree of polymorphism obtained only 4 primers were selected for further study which generated reproducible RAPD patterns. The number of bands in the amplified products for different samples which resulted from the selected RAPD primers varied between 40-50, where the size range of various amplicons for different samples varied from 202-3010 bp, approximately. Clustering based on RAPD fingerprints data revealed that the samples from different district of Himachal Pradesh (HP1B and HP2M), Uttarakhand (UK1D and UK2N) and Maharashtra (MH1L and MH2P) got paired into three separate clades at similarity level of 88.5%, showing maximum homology within their respective pairs. Conclusion: The results suggest that, high level of polymorphism was observed between the samples from south-central and northern parts of the country which showed that they were distantly related to each other.
INTRODUCTION
Moringa oleifera tree is the most admired, multipurpose and underutilized tropical tree. Moringa oleifera has great economical potential due to several reasons: it’s application in multiple industries such as cosmetics, biomedical and fuel, wide range of rich and novel products, distribution throughout many undernourished regions and tolerance to many limiting factors such as environmental or stress factors1-5. It is indigenous to Northwest India, Pakistan, Asia Minor, Western and Sub-Himalayan tracts, while it has been naturalized and cultivated in Southeast Asia, Sri Lanka, Caribbean Islands, tropical Africa, Central and South America, the greater of the tropical belt and many other countries6-10. M. oleifera also known as drumstick tree, is one of the 14 species belonging to the monogeneric family Moringaceae6. Seeds can be sown either directly or in containers without any requirement of pre-treatment and sprout readily in 1-2 weeks11. In various of its preparations M. oleifera is used for exceptionally wide range of properties like antioxidant, antihypertensive, antimicrobial, antidiabetic, antiulcer, antinflammatory, diuretic, antipyretic and antihyperlipidemic12-17. The Moringa seed extracts (both dry and aqueous) have been successfully used to purify water as it contains cationic polypeptides acting as a natural coagulant6,2,18-19. Okun et al.20 suggested that the knowledge of genetic variation as well as geographical and ecological differentiations are important for breeding and sampling strategies. The genetic diversity of the plants is closely related to their geographic distribution. Species with a wide geographic area generally have more genetic diversity21. The population structure, genetic diversity and biological factors have all three been shown highly interdependent on each other22,23. Analysis of genetic diversity in Moringa using RAPD technique has been proven very successful lately and confirms its importance in management of Moringa3,24,25. Very recently the genetic diversity of commercially grown Moringa cultivars from India was investigated in a comparative evaluation of three genetic marker systems including RAPD, ISSR and cytochrome P450-based system and the cultivars have been determined to have high degree of genetic diversity26. RAPD technique has been widely used and helps to identify a large number of markers used for estimating genetic variations27,28. The present study has been aimed to estimate genetic diversity among the seeds of M. oleifera from 3 different states of India i.e., Himachal Pradesh, Maharashtra and Uttarakhand using RAPD markers.
MATERIAL AND METHODS
Study area: Samples were collected from 3 different states of India i.e., Himachal Pradesh, Maharashtra and Uttarakhand in the month of October-November, 2016 and transported to Molecular Plant Microbe Interaction lab, Shoolini University of Biotechnology and Management Sciences, Solan for further study.
Sample collection: For the present study, seed of M. oleifera were collected from three different states of India i.e., Himachal Pradesh (Bilaspur and Mandi district), Maharashtra (Latur and Pune district) and Uttarakhand (Dehradun and Nainital district) based on the geographical distribution, morphological differences in color of seed pods and availability of samples. They were authenticated from Y.S Parmar University of Horticulture and Forestry, Solan (Himachal Pradesh). The seeds were sun-dried (to reduce the moisture content) and separated from the membranes. Thereupon, seeds were broken to remove the hard shell and the kernels were used for further studies.
Isolation of total DNA from seeds of M. oleifera: Total DNA was extracted using standardized protocol defined by Sambrook et al.29 with slight alterations. Crushing of 100-150 mg of seed content was done in 1-2 mL of extraction buffer (100 mM TrisHCl, 50 mM EDTA, 500 mM NaCl, 0.01% β-mercaptoethanol) using autoclaved pestle-mortar. 130-150 μL of 20 % sodium dodecyl sulphate (SDS) was added to the mixture/mL of extraction buffer for denaturation of proteins and incubated at 65°C for 15 min. Then the mixture was centrifuged in 1.5 mL microfuge tube at 10,000 rpm for 10 min. The resulted supernatant was filtered over a miracloth filtration membrane and transferred to a new microfuge tube. Equal volume of phenol/chloroform (1:1) was added, shaked vigorously for 1 min and further centrifuged at 3,000 rpm for 30 min. Aqueous phase containing nucleic acid was collected and 2.5 volume of absolute ethanol were added to precipitate the DNA in a fresh microfuge tube. Mixed the solution gently and incubated on ice for 1 min. The solution was then centrifuged at 12,000 rpm for 20 min to pellet down the precipitates. The pellet was washed with 70% ethanol, air dried and resuspended in 50 μL of 10X TE buffer (10 mM TrisHCl, 50 mM EDTA). The qualitative and quantitative analysis of DNA was done by agarose gel electrophoresis (using 1% agarose) and spectrophotometry (determining the optical density at 260 and 280 nm).
Genetic diversity analysis of M. oleifera seeds using random amplified polymorphic DNA (RAPD) technique: Isolated DNA from seeds of M. oleifera was used to study the genetic variation using RAPD technique. RAPD analysis was performed by the method described by Williams et al.30, with some modifications. The standard RAPD reaction was set up by using random decamer primers. Nine decamer primers [OPA-1 (5’-CAGGCCCTTC-3’), OPA-3 (5’-AGTCAGCCAC-3’), OPA-9 (5’-GGGTAACGCC-3’) OPA-13 (5’-CAGCACCCAC-3’), OPA-14 (5’-TCTGTGCTGG-3’), TC-1 (5’-GGAGTACTGG-3’), TC-2 (5’-GGTCTAGAGG-3’), TC-3 (5’-GAGTCTCAGG-3’), TC-4 (5’-GGACTGCAGA-3’)] were used in this study. PCR was conducted in 25 μL mixture containing 1 μL (~100 ng) DNA, 2.5 μL of 2 mM dNTPs, 2 μL (10 pM) of RAPD primer and 0.3 μL (5 U μL–1) of Taq DNA polymerase (Bangalore Genei Pvt. Ltd.) with 10X PCR buffer (containing 15 mM MgCl2). Temperature conditions for PCR included initial denaturation at 94°C for 5 min, followed by 40 cycles comprised of 3 stages with denaturation at 94°C for 30 sec, annealing at 36°C for 1 min and elongation at 72°C for 1 min. The additional final elongation step was conducted at 72°C for 7 min. The final PCR products were analyzed by performing electrophoresis using 1% agarose.
RAPD product scoring and data analysis: The RAPD fragments obtained upon agarose gel electrophoresis were scored for presence (1) and absence (0) of amplification bands for each sample. The data of similarity coefficients was generated and used to construct the dendrogram indicating the genetic relatedness or differentiation among the M. oleifera seeds. Data was compiled as binary 0-1 matrix, ‘1’ represents the presence of a band and ‘0’ represents the absence of a band in specified size range. All high and low intensity bands were considered in statistical analysis. Dendrogram was produced from the distance matrix by UPGMA (Unweighted Pair-Grouped Method Arithmetic) average31, constructed using online D-UPGMA program (DendroUPGMA: A dendrogram construction utility), version-2015.
RESULTS AND DISCUSSION
DNA isolation: Total DNA was extracted from 6 samples of M. oleifera seeds i.e., a sample each from Bilaspur and Mandi districts of Himachal Pradesh, Latur and Pune district of Maharashtra and also from Dehradun and Nainital district of Uttarakhand. DNA bands of good quality were observed upon agarose gel electrophoresis (Fig. 1).
| Fig. 1: | Isolated DNA of 6 M. oleifera samples HP1B and HP2M from Himachal Pradesh (Bilaspur and Mandi districts, respectively), UK1D and UK2N from Uttarakhand (Dehradun and Nainital districts, respectively), MH1L and MH2P Maharashtra (Latur and Pune districts, respectively), M: 1kb DNA Ladder (TaKaRa) |
| Table 1: Maximum band size, minimum band size and the total number of amplified fragments obtained for all the 4 selected RAPD primers viz., TC-1, TC-2, TC-3 and TC-4 | |||
| Band size (bp) | |||
| Primers | Maximum |
Minimum |
Amplified fragments |
| TC-1 | 1500 |
295 |
46 |
| TC-2 | 961 |
221 |
40 |
| TC-3 | 3010 |
202 |
42 |
| TC-4 | 2250 |
495 |
49 |
The concentration of DNA determined at 260 nm was found to be 7.5 μg μL–1 with OD of 1.5 and dilution factor of 100. Correspondingly, the purity of DNA was confirmed with A260 nm/A280 nm ratio which was found to be of range 1.8-2.0.
RAPD analysis: Isolated DNA from all different samples of M. oleifera seeds was used for study of genetic variations using RAPD technique. The standard RAPD reaction was set up by using standard RAPD primers. Initially, nine random decamer primers (OPA-1, OPA-3, OPA-9, OPA-13, OPA-14, TC-1, TC-2, TC-3, TC-4) were used in the study and based on the higher degree of polymorphism obtained only 4 primers viz.,TC-1, TC-2, TC-3 and TC-4 were selected for further study which generated reproducible RAPD patterns (Fig. 2). A total of 46, 40, 42 and 49 bands were scored by using TC-1, TC-2, TC-3 and TC-4 primer, respectively (Table 1). The number of bands in the amplified products for different samples which resulted from the selected RAPD primers varied between 40-50, where the size range of various amplicons for different samples varied from 202-3010 bp, approximately.
| Fig. 2(a-d): | RAPD banding patterns of 6 samples of M. oleifera using 4 selected random primers which showed higher degree of polymorphism, (a) TC1 primer, (b) TC2 primer, (c) TC3 primer and (d) TC4 primer HP1B and HP2M samples from Himachal Pradesh, UK1D and UK2N samples from Uttarakhand and MH1L and MH2P samples from Maharashtra, M: 1 kb DNA Ladder (TaKaRa) |
Clustering based on RAPD fingerprints data revealed that the samples ‘HP1B and HP2M’ (from 2 separate districts of Himachal Pradesh), ‘UK1D and UK2N’ (from 2 separate districts of Uttarakhand) and ‘MH1L and MH2P’ (from 2 separate districts of Maharashtra) were grouped or got paired-up into 3 separate clades at similarity level of 88.5%, which revealed that they showed maximum homology within their respective pairs and it also favors the fact that the paired samples do belong to the same region or same state of the country. The results stand in support of the generalization that 2 closely related organisms, species or varieties of same species would be expected to yield more similar banding patterns than the ones those are distant in evolutionary terms32. The samples ‘MH1L and MH2P’ from Maharashtra (south-central state of the country) were paired in a very divergent independent group, which immediately separated them from all other 4 samples from the Himachal Pradesh and Uttarakhand states (viz., northern states of the country) which got clustered into a separate clade. This revealed high level of polymorphism between the samples from the south-central and northern parts of the country and showed that the samples were distantly related to each other. A high level of polymorphism detected in the present study indicates that the primers TC-1, TC-2, TC-3 and TC-4 could be employed in future for RAPD based genetic diversity studies of various varieties of M. oleifera.
Our results suggest the existence of variations among the samples of M. oleifera from different regions of the country as all the 6 samples were divided into 2 main clusters where the pair of Maharashtra samples was found to have a distant relationship with other 2 pairs of samples from Himachal Pradesh and Uttarakhand states of the country as shown in Fig. 3. For future studies, RAPD technique can be used as a more reliable and specific tool for identifying variations occurring within the species. However, RAPD markers are generally cheap, fast, simple to carry out in comparison with different other methods, such as ISSR, AFLP and SNP in detecting polymorphisms, they are nevertheless not easy to reproduce33. It will continue to be relevant particularly in studies of genetic diversity in underutilized medicinal species as long as other DNA-based methods remain unavailable in terms of upper hand in cost, time and labor27,34. Previously, assessment of genetic diversity in M. oleifera has been described using AFLP and RAPD markers2,35,36.
| Fig. 3: | Dendrogram for samples of M. oleifera derived from RAPD fingerprints generated by using 4 different primers produced from the distance matrix based upon UPGMA (Unweighted Pair-Grouped Method Arithmetic) average HP1B and HP2M samples from Himachal Pradesh, UK1D and UK2N samples from Uttarakhand, MH1L and MH2P samples from Maharashtra |
Similarly, Mgendi et al.36, investigated the genetic diversity by RAPD markers between cultivated and non-cultivated provenances of M. oleifera from coastal regions of Tanzania and obtained 89.6% polymorphism using 12 RAPD primers whereas Abubakar et al.24, obtained 75% polymorphism using 24 RAPD primers. In another study to check the genetic diversity in 16 accessions from the germplasm bank of Brazil, RAPD primers generated a total of 95 fragments of which 59 were polymorphic3. All of these reports indicate the genetic behavior of M. oleifera. The RAPD primers used in this study are effective in generating polymorphisms in M. oleifera from three different states of India.
CONCLUSION
In the present study, we have used RAPD to find out genetic diversity of M. oleifera between South-Central and Northern states of India. RAPD data revealed that the samples from different regions got paired into 3 separate clades at similarity level of 88.5%, showing maximum homology within their respective pairs and high level of polymorphism was observed between the samples from south-central and northern parts of the country which showed that they were distantly related to each other. RAPD is a fast and sensitive method and provides reproducible fingerprints of complex genomes without prior sequence information and provides a cost-effective method for the precise evaluation of variability.
SIGNIFICANCE STATEMENT
This study has discovered the existence of the considerable genetic variation between the samples of northern and south-central part of the country, which could be beneficial for the scientific community in exploring the possibility for the presence of different types of species of M. oleifera existing around the distant regions in India and even across the world. Moreover, this also encourages the researchers in a direction to uncover the critical areas of human interest which may include the exploration of some novel phytochemical, medicinal and nutritional properties which are still undiscovered in the species under study till date. Thus, a new drive in researchers may arrive that could lead to rapid strides in the field of human health care considering the historical cultural background of M. oleifera claiming it to be a miracle tree having medicinal properties.