Molecular analysis of genetic variability and relationship among Tulsi (Ocimum sanctum) varieties using RAPD markers.

Molecular analysis of genetic variability and relationship among Tulsi (Ocimum sanctum) varieties using RAPD markers.

1. WELCOME… ‹#›

2. Molecular analysis of genetic variability and relationship among Tulsi ( Ocimum sanctum ) varieties using RAPD markers. Presented By Mr. Wani Aditya Balasaheb 16-05-2023 Wani Aditya Balasaheb ‹#›

3. Introduction Need of study Objective Materials and Methods Results of the Programme Outcome of the Programme Summary of the Programme References CONTENT 16-05-2023 Wani Aditya Balasaheb ‹#›

4. Ocimum sanctum also known as Tulsi or Holy basil is an aromatic plant and it belongs to the family Lamiaceae. the chromosome number is observed to be 2n = 36. These plants are known to produce essential oils comprising of a number of aromatic compounds and Tulsi is rightly known as the “Queen of Herbs” for this reason . Krishna(Black) Tulsi is described in the Vedas and Puranas and has a long history of cultivation, of roughly 3000 years, and is therefore assumed to be of Indian origin. The metabolites (essential oils) of genus Ocimum have been reported to possess antioxidant and antifungal properties and to cure many diseases including bronchitis in Ayurveda, an Indian system of medicine. 16-05-2023 Wani Aditya Balasaheb ‹#› INTRODUCTION

5. NEED OF STUDY Tulsi plant has a great deal of essentialness for humankind, because of the complex restorative advantages it gives. The essential oils of genus Ocimum have been reported to possess antioxidant and antifungal properties. These metabolites are of immense value in the pharmaceutical, perfume and cosmetic industries (e.g. linalool, linalyl, geraniol, citral, camphor, eugenol). RAPD markers have proven to be powerful tools in the assessment of genetic variation and in the elucidation of genetic relationships within and among species. 16-05-2023 Wani Aditya Balasaheb ‹#›

6. OBJECTIVES To study genetic diversity among different Tulsi varieties using RAPD marker 16-05-2023 Wani Aditya Balasaheb ‹#›

7. MATERIALS AND METHODS MATERIAL The present study was conducted at the Department of Plant Biotechnology, K. K. Wagh College of Agricultural Biotechnology, Nashik, Maharashtra. PLANT MATERIAL The present study of molecular characterization of Tulsi genotype as shown in table no.1.These genotypes were collected from Nagarjuna Medicinal and Aromatic Plant park (Dr. P. D. K. V.), Akola Sr. No. Name of Genotype 1. Black Tulsi 2. Green Tulsi 3. Lavangi Tulsi 4. Pandharpuri Tulsi 5. Kapoori Tulsi Table no.1 List of Tulsi genotypes 16-05-2023 Wani Aditya Balasaheb ‹#›

8. Table No.2 Instruments Table No.3 Glassware's List of Equipments, Glasswares & Instruments 16-05-2023 Wani Aditya Balasaheb ‹#› Sr. No. Instruments/Equipment's 1 Autoclave 2 Hot air oven 3 Magnetic Stirrer 4 Water bath 5 Centrifuge 6 Gel electrophoresis assembly 7 Gel documentation unit 8 Deep freezer 9 Bio Spectrophotometer 10 Thermal Cycler (PCR) 11 Micropipette 12 Weighing balance 13 Microwave oven Sr. No. Glassware/ Plasticwares 1 Conical flasks 2 Beakers 3 Measuring cylinder 4 Stock Bottles 5 Microtips 6 PCR tubes 7 Eppendorf tube (1.5ml/2ml)

9. Table no. 4: List of chemicals 16-05-2023 Wani Aditya Balasaheb ‹#› Sr. no. Chemicals Sr. no. Chemicals 1 CTAB (Cetyl trimethyl ammonium bromide) buffer 10 Agarose powder 2 1 M Tris-HCl 11 Taq polymerase 3 5 M Sodium Chloride (NaCl) 12 Ethidium bromide (EtBr) 4 β- mercaptoethanol 13 6X Loading dye 5 0.5 M Ethylene diamine tetra acetic acid (EDTA) 14 Isopropanol 6 Polyvinyl pyrrolidone (PVP) 15 Chloroform: isoamyl alcohol (24:1) 7 TE buffer 16 DNA Ladder (100bp/1kbp) 8 1 X Tris: Acetate: EDTA (TAE) buffer 17 dNTPs 9 Ethanol 70% 18 PCR Buffer with Mgcl 2

10. ‹#› DNA Isolation Protocol Agarose gel electrophoresis Quantification of genomic DNA Purification of DNA PCR amplification Data analysis Work Plan METHODS 16-05-2023 Wani Aditya Balasaheb

11. Table no. 5 - Composition of CTAB Buffer Total volume was made up to 100 ml using autoclave water . 16-05-2023 Wani Aditya Balasaheb ‹#› Sr. No. Chemical Stock Concentration Working Concentration (100ml) Volume 1. Tris HCl 1M 100mM 10ml 2. EDTA 0.5M 20mM 4ml 3. NaCl 5M 1.4M 28ml 4. CTAB - 2% 2g 5. PVP - 1% 1g 6. β- mercaptoethanol - 0.2% 200µl

12. Fresh leaves (1g) was collect and grind with mortar- pestle. Fine crush sample was transfer to centrifuge tube containing pre-warm extraction (CTAB) buffer and mix. The extracts was incubated in water bath for 1hr at 65 o C. After incubation, equal amount of Chloroform: Isoamyl alcohol (24: 1) Was added to each sample tube and mix gently. The sample was centrifuge at 10,000 rpm for 10 min.   The aqueous phase was collected in new tubes.   Continue … 1 . DNA ISOLATION PROTOCOL 16-05-2023 Wani Aditya Balasaheb ‹#›

13. Then equal amount of ice-cold isopropanol was added and tubes was kept at -20 o C for one hour. The precipated DNA was centrifuge at 10,000 rpm for 5 min and the pellets was wash with chilled 70% ethanol and centrifuge again.   The pellet was kept for drying for about 1 hour and then the extract DNA were suspended in molecular grade water.. 16-05-2023 Wani Aditya Balasaheb ‹#›

14. Preparation & pouring of gel- 1x TAE buffer was prepared from the 50 X TAE stock solution. Agarose (0.8%) was weighed and dissolved in TAE buffer by boiling Ethidium bromide was added at a concentration 3μl/100ml TAE and mix well. Loading of samples & running the gel- The gel was allowed to set for 30 minutes after which the comb was removed carefully. DNA sample (3 μL) along with loading dye (2 μL) was loaded into the wells using a micropipette. The gel was run at a constant voltage (65V) and current (50mA) Gel Documentation- Run the gel upto 2/3rd length of the casting tray. The image was documented and saved in the gel documentation system 2. AGAROSE GEL ELECTROPHORESIS 16-05-2023 Wani Aditya Balasaheb ‹#›

15. Take 1ml TE buffer in a cuvette and calibrate the spectrometer at 260nm and 280nm. Add 1μl of each DNA sample to 99μl TE buffer and mix well. Use the TE buffer as a blank in the other cuvette of spectrometer. Note the OD260 & OD280 values on spectrometer. Calculate the OD260 /OD280 ratio. Formula :- dsDNA (µg/ml) = 50 µg x OD 260 x Dilution factor. 1000 Pure DNA has an A 260 /A 280  ratio of ~ 1.8 3. C heck Purity of DNA 16-05-2023 Wani Aditya Balasaheb ‹#›

16. RNase was add (2 μl) in 8 μl DNA sample and incubate at 37 °C for 40 min. Remove extra salts by two washings with 70% ethanol. Dry under vaccum. Minimum volume of molecular grade water was add dissolve at room temperature. Store at 4 °C. 4. PURIFICATION OF DNA 16-05-2023 Wani Aditya Balasaheb ‹#›

17. Sr. no. Primer name Sequence (5’ to 3’) 1 OPA-01 CAGGCCCTTC 2 OPA-05 GAAACGGGTG 3 OPA-06 CAATCGCCGT 4 OPA-15 TGGCGTCCTT 5. PCR (POYMERASE CHAIN REACTION) Table no. 6 List of RAPD Primers 16-05-2023 Wani Aditya Balasaheb ‹#›

18. Table no.7- PCR components for RAPD marker 16-05-2023 Wani Aditya Balasaheb ‹#› Sr. No Components Volume for each sample 1 10X PCR buffer with 17.5mM MgCl 2 1.5µl 2 dNTPs (10 mM) 1.0µl 3 Taq polymerase (3U/µl) 0.3µl 4 DNA (50 ng/µl) 1.0µl 5 RAPD Primer 1.0µl 6 Nuclease Free water 10.2µl Total 15 µl Steps Temp Duration Cycles Initial denaturation 94°C 10 min 1 Denaturation 94°C 1 min 35 cycles Annealing 30-37°C 1 min Extension 72°C 1 min Final extension 72°C 7 min 1 Hold 4°C ∞ Cycle 35 Table no.8 –PCR Program for RAPD Marker Analysis of PCR product Amplified PCR products was separated on 1.2% of agarose gel

19. 7. Data Analysis Amplification profiles of Tulsi genotypes were compared with each other and bands of DNA fragments scored manually as (1) or (0) depending on the presence or absence of a particular band respectively. The data was analyzed using 1) Paleontological Statistics (PAST 4.0) software package and dendrogram was also constructed by it. Principle Component Analysis (PCA) was computed based on Jaccard’s similarity coefficient using PAST 4.0 for comparison among Tulsi genotypes. The data obtained from the RAPD morphological characterization of Tulsi genotypes was pooled together to generate a combined dendrogram so as to get the over all picture of variation in the Tulsi genotypes. 16-05-2023 Wani Aditya Balasaheb ‹#›

20. 16-05-2023 Wani Aditya Balasaheb ‹#› Work Plan Sr. No. Month Work plan 1. November 2022     1 st week Registration for VIII semester and module selection. Reading guidelines of READY- 482. 2 nd week Crop selection and searching its varieties. Searching for literature, research papers, and review articles. 3 rd week Preparing the Outline of Research Work (ORW). 4 th week Collection of different types of Tulsi varieties. Collection of material requirements and Sterilization of types of equipment. 2. December 2022     1 st week Preparation of chemicals for isolation of genomic DNA. 2 nd week Standardization of protocol for genomic DNA isolation from Tulsi. 3 rd week Isolation of genomic DNA from Tulsi. Qualitative and quantitative analysis of genomic DNA using agarose electrophoresis. 4 th week Purification of DNA from Tulsi varieties. Quantification to determine optimum quantity of DNA for PCR analysis. 3. January 2022     1 st week PCR amplification using RAPD markers. 2 nd week Genotyping analysis through electrophoresis of PCR products. 3 rd week Data interpretation, Tabulation of results. Thesis writing, compilation of all data. 4 th week Correction and final report presentation. Final submission of thesis.

21. RESULTS OF THE PROGRAM DNA isolation Quantification of DNA Purification of DNA PCR Amplification Data Analysis 16-05-2023 Wani Aditya Balasaheb ‹#›

22. Plate 1:- Isolated DNA of Tulsi genotypes run on 0.8% agarose gel. Lane (L)- L1-Black Tulsi L2-Lavangi L3- Green Tulsi L4-Pandharpuri L5-Kapoori   1. DNA Isolation 16-05-2023 Wani Aditya Balasaheb ‹#› L1 L2 L3 L4 L5

23. ‹#› 2. DNA Quantification Sr. No. 260 nm/280 nm Black Tulsi 1.18 Lavangi 1.30 Green Tulsi 1.26 Pandharpuri 1.24 Kapoori 1.25 Table No 12. Results of Quantification of Genomic DNA 16-05-2023 Wani Aditya Balasaheb

24. 3. Purification of DNA Plate 2 :- Purification of Tulsi genotypes by RNase enzyme. Lane (L)- L1-Black Tulsi L2-Lavangi L3- Green Tulsi L4-Pandharpuri L5-Kapoori 16-05-2023 Wani Aditya Balasaheb ‹#› L1 L2 L3 L4 L5

25. ‹#› Sr. No. 260 nm/280 nm Black Tulsi 1.96 Lavangi 1.92 Green Tulsi 1.98 Pandharpuri 1.81 Kapoori 1.86 Table No 13. Results of Quantification of Purified Genomic DNA 16-05-2023 Wani Aditya Balasaheb

26. 4. PCR AMPLIFICATION ‹#› Plate 3:- Amplification profile of Tulsi genotypes by OPA 01 Primer Amplifications using RAPD Primer 16-05-2023 Wani Aditya Balasaheb Plate 4:- Amplification profile of Tulsi genotypes by OPA 05 Primer Note :- L- 1kb Ladder 1- Black Tulsi 2- Lavangi 3- Green Tulsi 4- Pandharpuri 5- Kapoori L 1 2 3 4 5 10000 9000 4000 2000 1500 1000 750 500 250 L 1 2 3 4 5 10000 9000 7000 5000 4000 3000 2000 1500 1000 750   500     250

27. Plate 5:- Amplification profile of Tulsi genotypes by OPA 06 Primer Plate 6:- Amplification profile of Tulsi genotypes by OPA 15 Primer 16-05-2023 Wani Aditya Balasaheb ‹#› Note :- L- 1kb Ladder 1- Black Tulsi 2- Lavangi 3- Green Tulsi 4- Pandharpuri 5- Kapoori L 1 2 3 4 5 10000 9000 8000 7000 5000 3000 2000 1500 1000 750 500 250 10000 9000 4000 2000 1500 1000 750 500 250 L 1 2 3 4 5

28. DATA ANALYSIS FOR RAPD 16-05-2023 Wani Aditya Balasaheb ‹#› Fig no. 1. Dendrogram of RAPD Marker

29. The dendrogram of RAPD shows three cluster in which cluster I includes Lavngi and Pandharpuri tusli, second cluster II include cluster I and Kapoori tulsi and cluster III includes cluster II and Green tulsi. The Black tulsi showing dissimilarity with other genotypes. 16-05-2023 Wani Aditya Balasaheb ‹#›

30. OUTCOME OF THE PROGRAMME 16-05-2023 Wani Aditya Balasaheb ‹#› The present study entitled “ Molecular analysis of genetic variability and relationship among Tulsi (Ocimum sanctum) varieties using RAPD marker”. CTAB Protocol was found to be the best for isolation of genomic DNA of Tulsi. The RNA contamination was completely removed through RNase A treatment, which resulted in DNA with no impurities and suitable for RAPD analysis. The protocol for RAPD assay in Tulsi was standardized. The results showed that the 0.3 µl Tag polymerase, 1 µl of dNTPs, 1 µl primer, 1.5 µl PCR buffer with MgCl 2 , 1 µl template DNA, 35 total cycles and 1.2 percent agarose gel gave optimum amplification. 7 RAPD primers were screened and in which 4 RAPD primers of them showed amplifications respectively were selected for RAPD profiling of five Tulsi varieties.

31. SUMMARY OF THE PROJECT The project “ Molecular analysis of genetic variability and relationship among Tulsi ( Ocimum sanctum ) varieties using RAPD marker ” was successfully carries out by following summary was obtained. CTAB protocol was found to be the best for isolation of genomic DNA of Tulsi. The agarose gel electrophoresis was shown that there was sufficient amount of DNA with RNA contamination. The purification of isolated DNA was carried out by RNase A enzyme A total 4 RAPD primers were used for PCR amplification and all primers were amplified and analysis of varieties revealed good level of genetic polymorphism which allowed unique banding pattern. ‹#› 16-05-2023 Wani Aditya Balasaheb

32. The dendrogram of RAPD shows three cluster in which cluster I includes Lavngi and Pandharpuri tusli, second cluster II include cluster I and Kapoori tulsi and cluster III includes cluster II and Green tulsi. The Black tulsi showing dissimilarity with other genotypes. 16-05-2023 Wani Aditya Balasaheb ‹#›

33. Bhadra P. and Sethi L. ( 2020 ). A Review Paper on Tulsi Plant. Ind. J of Natural Sci ., 20854-20860. Bilal A. ( 2020 ). Molecular Characterization of Ocimum Species Using Random Amplified Polymorphic DNA Method and Gene Identification Using Sanger Sequencing Method . J Mol Biomark Diagn 9: 423. Borah R., Biswas S. P. ( 2018 ). Tulsi excellent source of phytochemicals . Int. j. agric. environ. biotechnol. (IJEAB) 3: 5, 1732-1738. Doyle J. J. and Doyle J. L. ( 1990 ). Isolation of plant DNA from fresh tissue. Opoola J. , Oziegbe M. ( 2019 ). Characterization of Ocimum tenuiflorum (linn.) Morpho-types Using RAPD Markers . Notulae Scientia Biologicae ,11(4):417-420. REFERENCES 16-05-2023 Wani Aditya Balasaheb ‹#›

34. Patel H., Fougat R., Kumar S., Mistry J. and Kumar M. ( 2014 ). Detection of genetic variation in Ocimum species using RAPD and ISSR markers. 3 Biotech., 1-11. Sairkar P. , Vijay N. , Silawat N. , Garg R. K. , Chouhan S., Batav N., Sharma R. , Mehrotra NN. (2012 ). Inter-species Association of Ocimum Genus as Revealed through Random Amplified Polymorphic DNA Fingerprinting. Sci Secure J Biotech 1(1): 1-8 Taleyzzaman M,Jain P, Verma R, Iqbal Z, Mirza M. ( 2021 ). Eugenol as a Potential Drug Candidate: a Review. Bentham Sci. Pub. , 1804-1815. 16-05-2023 Wani Aditya Balasaheb ‹#›

35. THANK YOU… 16-05-2023 Wani Aditya Balasaheb ‹#›