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ISSR marker based DNA profiling studies in Rauwolfia species

Gulab Khan Rohela, Prasad Bylla, Sreenu Pendli, Korra Rajender, Christopher Reuben Thammidala

Abstract


In this present study, a protocol was developed for the DNA profiling of medicinally important Rauwolfia species (05 species) by ISSR-PCR method. Initially genomic DNA was isolated from the fresh leaves (1 gram) of 24 months old Rauwolfia tetraphylla, R.serpentina, R.pentaphylla, R.vomitaria and R.micrantha. The content of genomic DNA isolated from 1 gram of fresh leaves of these five species ranges from 1.6 to 3.4 µg. Out of six inter simple sequence repeats (ISSR) primers used, HY3 primer has given the good amplification with distinct bands which were reproducible. The range of DNA bands produced by the HY3 primer were between 300 bp to 1500 bp among the five species. A dendrogram was constructed on the basis of similarity matrix data by unweighted pair group method with arithmetic average (UPGMA) cluster analysis using indigenous software. Average genetic similarity generated by HY3 has revealed 38% similarity between species of Sub cluster-A and species of Sub cluster-B which is consistent with their geographical distribution i.e. Sub cluster-A species R.tetraphylla and R.serpentina inhabit peninsular India, Sub cluster- B species R.micrantha and R. vomitoria inhabit the Himalayan foot hills. The developed protocol of DNA profiling in Rauwolfia species by ISSR-PCR method could be used efficiently for the differentiation and identification of the Rauwolfia species at molecular level.


Keywords


Rauwolfia, DNA Profiling, ISSR-PCR, Dendrogram Analysis

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References


Alatar A and Faisal M. “Encapsulation of Rauvolfia tetraphylla microshoots as artificial seeds and evaluation of genetic fidelity using RAPD and ISSR markers”. Journal of Medicinal Plants Research 6(2012):1367-1374.

Anitha S and B D Kumari.“Influence of auxins combinations on accumulation of reserpine in the callus of Rauvolfia tetraphylla L”. Pakistan Journal of Biological Sciences 10(2007): 3900-3904.

Bilal M A, A Q Shamim.“Methanolic Root Extract of Rauwolfia serpentina Benth Improves the Glycemic, Antiatherogenic, and Cardioprotective Indices in Alloxan-Induced Diabetic Mice”. Advances in Pharmacological Sciences Article ID 376429(2012). http://dx.doi.org/10.1155/2012/376429.

Douglas L. “Rauwolfia in the Treatment of Hypertension”. Integr Med (Encinitas). 14(2015):40–46. PMCID: PMC4566472.

Doyle J J and J L Doyle. “A rapid DNA isolation procedure for small quantities of fresh leaf tissue”. Phytochemical Bulletin 19(1990):11-15.

Ezeigbo I I, M I Ezeja, K G Madubuike, D C Ifenkwe DC, I A Ukweni, N E Udeh and S C Akomas. “Antidiarrhoeal activity of leaf methanolic extract of Rauwolfia serpentina”. Asian Pacific Journal of Tropical Biomedicine 2(2012): 430-432. http://dx.doi.org/10.1016/S2221-1691(12)60070-7. PMID: 23569944

Faisal M, A A Alatar, N Ahmed, M Anis and K H Ahmed KH. “Assessment of Genetic fidelity in Rauwolfia serpentina plantlets grown from synthetic (encapsulated) seeds following in vitro storage at 4ºC”. Molecules 17(2012a): 5050-5061

Faisal M, A A Alatar, N Ahmad, M Anis and A K Hegazy. “An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers”. Appl Biochem Biotechnol 168(2012b):1739-17352. https://doi.org/10.1007/s12010-012-9893-3.

Goel MK, S Mehrotra, A K Kukreja, K Shanker and S P Khanuja. “In vitro propagation of Rauwolfia serpentina using liquid medium, assessment of genetic fidelity of micro propagated plants, and simultaneous quantitation of reserpine, ajmaline, and ajmalicine”. Methods Molecular Biology 547(2009):17-33.

Gurudeeban S, T Ramanathan, K Satyavani and K Dhinesh. “Standardization of DNA Isolation and PCR Protocol for RAPD Analysis of Suaeda sp”. Asian Journal of Biotechnology 3(2011): 486-492.

Heber W Y. "Malabar Rauwolfia, Rauwolfia micrantha Hook. F”. Journal of the American Pharmaceutical Association 43(2006):141–143.

Hong L and G Huachun. “Using SSR to Evaluate the Genetic Diversity of Potato Cultivars from Yunnan Province (SW China)”. Acta Biol Cracov Ser Bot 56 (2014):16–27.

Kranthi G, U Pavan, A Mahender, R Srinivasa, K Venugopal Rao, K Kiranmayee and A Sadanandam.“Micropropagation of a valuable ethno medicinal plant Streblus asper”. Journal of Phytology 3(2011):18-23.

Korra R, Bylla P, G K Rohela, S Pendli and T C Reuben. In vitro micro propagation and confirmation of genetic fidelity using RAPD marker in ethno medicinal plant Stachytarpheta jamaicensis L. Vahl. Intern Journal of Advanced Research. 5(2017), 1494-1502. http://dx.doi.org/10.21474/IJAR01/4862.

Madhusudanan K P, S Banerjee, S P Khanuja and S K Chattopadhyay. “Analysis of hairy root culture of Rauvolfia serpentina using direct analysis in real time mass spectrometric technique”. Biomed Chromatography 22(2008):596–600.

Mahesh R, N Nirmal Kumar and R Mary Sujin. “Molecular Analysis in Rauvolfia tetraphylla L. using RAPD Markers”. Ethnobotanical Leaflets 12(2008): 1129-36.

Megahed H A, S A Salem, M M Hussein, A K Muhammad, H E Ehab and A A Sulieman. “Assessment of genetic diversity among faba bean genotypes using agro-morphological and molecular markers”.Saudi Journal of Biological Sciences 22(2015):340-350. https://doi.org/10.1016/j.sjbs.2015.02.005.

Mehrotra S, M K Goel, L U Rahman and A Kukreja. “Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina”. Plant Cell Tissue & Organ Culture 114(2013):31–8.

Mehrotra S, M K Goel, V Srivastava and L U Rahman. “Hairy root biotechnology of Rauwolfia serpentina: A potent approach for the production of pharmaceutically important terpenoid indole alkaloids”. Biotechnology Letters 37(2015):253–63.

Mehrotra S, L U Rahman, J Mishra, and A K Kukreja. “Conservation and multiplication of encapsulated micro shoots of Rauvolfia vomitoria--an endangered medicinal tree: ISSR and RAPD based evaluation of genetic fidelity of converted plantlets”. Natural Products Communication 7(2012):1647-1650.

Mukherjee A J. “Alkaloids of Rauwolfia canescens”. J. Indian chem Soc 18(1961):33-35.

Nath A, A Chattopadhyay and S R Joshi. “Biological activity of endophytic fungi of Rauwolfia serpentina Benth: An ethnomedicinal plant used in folk medicines in Northeast India”. Proc National Academic Sciences, India, Sect B Biological Sciences. 85(2015):233-240. http://dx.doi.org/10.1007/s40011-013-0184-8.

Nei M and W H Li. “Mathematical model for studying genetic variation in terms of restriction endonucleases”. Proceedings of National Academic Sciences, USA. 76(1979):5269.

Niraj T, S C Devender and S Navinder. “Assessment of genetic variations among highly endangered medicinal plant Bacopa monnieri (L.) from Central India using RAPD and ISSR analysis”. 3Biotech 2(2012):327–336. doi: 10.1007/s13205-012-0059-3

Panda S K, D Debajoyti, N Bichitra and L N Tripathy. “Phyto-pharmacognostical studies and quantitative determination of reserpine in different parts of Rauwolfia (Spp) of Eastern Odisha by UV Spectrocsopy Method”. Asian Journal of Plant Sciences and Research 2(2012):151-162.

Pietrosiuk A, M Furmanowa and B Łata. “Catharanthus roseus: micropropagation and in vitro techniques”. Phytochemistry Reviews 6(2007): 459-473.

Prajapathi N D, S S Purohit, A K Sharmam, and T Kumarmm. “A handbook of medicinal plants”. (Jodhpur: Agrobios)(2013).

Prasad B, K R Gulab, T Radha, C H Ravi, P Venkataiah, K Subhash, T R Christopher. “DNA profiling of commercial chilli pepper (Capsicum annuum L.) varieties using random amplified polymorphic DNA (RAPD) markers”. African Journal of Biotechnology 12 (2013): 4730-4735.

Raina SN, V Rani and Kojima. “RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, variety identification and phylogenetic relationships in parent (Arachis hypogea) accessions & wild species”. Genome 44(2001):763-772.

Rohela G K, P Bylla, R Korra, C Reuben.“Phytochemical screening and antimicrobial activity of leaf, stem, root and their callus extracts in Rauwolfia tetraphylla”. International Journal of Agriculture & Biology 18(2016a):521‒528. doi: 10.17957/IJAB/15.0120

Rohela G K, B Prasad, C H Ravi, K Rajender, R T Christopher. “In vitro clonal propagation of Rauwolfia tetraphylla, a relative of indian snake root plant”. Research Journal of Biotechnology 11(2015): 23-31.

Rohela G K, B Prasad, K Srinivas, A Sadanandam, C H Ravi, R T Christopher. “In vitro Plantlet Regeneration from Leaf and Stem Calluses of Rauwolfia tetraphylla (R. canescens) and Confirmation of Genetic Fidelity of Plantlets Using the ISSR-PCR Method”. Journal of Herbs, Spices & Medicinal Plants 19(2013):66-75.

Sanjay L, N M Kinnari, T Riddhi, D S Smit and B V Parth. “ Genetic Diversity assessment in six medicinally important species of Ocimum from central Gujarat (India) utilizing RAPD, ISSR and SSR Markers”. International Journal of Advanced Biological Research 2(2012): 279-288.

Sarika R D, S A Dhanashree, A P Bhausaheb. “Extraction and evaluation of indole alkaloids from Rauwolfia serpentina for their antimicrobial and anti proliferative activities”. International Journal of Pharmacy and Pharmaceutical Sciences 5(2012):329-334.

Shafie M, B Shafie, M Sayed, H Zain, M Z Abdullah and M S Ramisah. “RAPD and ISSR markers for comparative analysis of genetic diversity in wormwood capillary (Artemisia capillaris) from Negeri Sembilan, Malaysia”. Journal of Medicinal Plants Research 5(2011):4426-4437.

Shetty M R, G A Harisha, Y Jayanth, A H Kumar. “Production of secondary metabolites from in vitro cultures of Rauwolfia serpentina (L.) Benth”. International Journal of Science, Technology & Research 2(2014): 844–852.

Singh D K, B Srivastava and A Sahu. “Spectrophotometric determination of Rauwolfia alkaloids: estimation of reserpine in pharmaceuticals”. Anal Science 20(2004): 571-73.

Sitthiwong K, T Mastui, S Sukprakarn, N Okuda and Kosugi. “Classification of pepper (Capsicum annuum L.) accessions by RAPD analysis”. Biotechnology 4(2005):305.

Sonibare M A and G U Akpan GU. "In vitro Callus Induction and Antioxidant Activity of Rauwolfia vomitoria Afzel. (Apocynaceae)”. Nigerian Journal of Pharmaceutical Research 12(2016):105-115.

Spiridonova E V, D M Adnof, I O Andreev and VA Kunakh. “Dynamics of genome changes in Rauwolfia serpentina callus tissue upon the switch to conditions of submerged cultivation”. Tsitol 42(2008):35-41.

Srivastava A, A K Tripathi, R Pandey, R K Verma and M M Gupta. “Quantitative determination of reserpine, ajmaline, and ajmalicine in Rauvolfia serpentina by reversed-phase high-performance liquid chromatography”. Journal of Chromatographical Science 44(2006):557–60.

Srivastava P P, K Vijayan, A K Awasthi, B Sarat Chandra. “Genetic analysis of Morus alba through RAPD & ISSR markers”. Indian Journal of Biotechnology 3(2004): 527-532.

Sudha G G and S Seeni S. “In vitro propagation of Rauwolfia micrantha, a rare medicinal plant. Plant Cell, Tissue and Organ Culture. 44(1996): 243. https://doi.org/10.1007/BF00048530

Sujatha D, C H Ravi, L Raghuvardhan, B Prasad, K R Gulab, A Sadanandam and R T Christopher. “In vitro plantlet regeneration and genetic transformation of sponge gourd (Luffa cylindrica L.)”. African Journal of Plant Sciences 7(2013):244-252. doi: 10.5897/AJPS12.196.

Tiwari K L, S K Jadhav and A Kumar. “Random amplified polymorphic DNA (RAPD) molecular marker based genetic relationship study of medicinal plants collected from tribal region of Chhattisgarh,, India”. Research Journal of Biotechnology 6(2011):63-66.

Vakil R J. “A clinical trial of Rauwolfia serpentina in essential hypertension. Br Heart J 9(1949):350.

Wang X M, X Q Hou, Y Q Zhang, R Yang, S F Feng and Y Li. “Genetic diversity of the endemic and medicinally important plant Rheum officinale as revealed by Inter-Simpe Sequence Repeat (ISSR) Markers”. International Journal of Molecular Science 13(2012):3900–3915. doi: 10.3390/ijms13033900.

Williams J G, A R Kubelik, K J Livak, J A Rafalski and S V Tingey. “DNA polymorphisms amplified by arbitrarily primers are useful as genetic markers”. Nucleic Acids Research 18 (1990):6531-6535.

Wong K F and R M Taha. “The effect of 2,4-Dichlorophenoxyacetic acid and 6-benzylaminopurine on callus induction and plantlet regeneration from Allamanda catharitica: A valuable medicinal plant”. Research Journal of Biotechnology 7(2002):75.

Yamuna Prasad S, V B Singh, K S Praveen, K Vimlesh and P Ram. “Genetic Diversity among Promising Lines and their F1 Progeny of Sponge Gourd [Luffa cylindrica (Roem) L.]”. International Journal of Current Microbiology & Applied Sciences 6(2017): 2756-2768. https://doi.org/10.20546/ijcmas.2017.607.386.

Zhao K G, Z Ming-Qin and Q C Long. “Genetic Diversity and Discrimination of Chimonanthus praecox (L.) Link Germplasm Using ISSR and RAPD Markers”. Hortiscience 42(2007):1144–1148.

Zhou Y, Z Chune, Y Huanling, L Yanju and T Rongtao. “Application of ISSR markers in detection of genetic variation among Chinese yam (Dioscorea opposita Thunb) cultivars”. Life Science Journal 5(2008):6-12.




DOI: https://doi.org/10.21746/aps.2018.7.5.13

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