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.
Presented By
Mr. Wani Aditya Balasaheb
16-05-2023 Wani Aditya Balasaheb 2

• Introduction
• Need of study
• Objective
• Materials and Methods
• Results of the Programme
• Outcome of the Programme
• Summary of the Programme
• References
CONTENT

1. Ocimum sanctum also known as Tulsi or Holy basil is an aromatic plant and it belongs to the
family Lamiaceae.
2. the chromosome number is observed to be 2n = 36.
3. 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 .
4. 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.
5. 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.
INTRODUCTION

NEED OF STUDY
1. Tulsi plant has a great deal of essentialness for humankind, because of the complex
restorative advantages it gives.
2. 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).
3. 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.

OBJECTIVES
1. To study genetic diversity among different Tulsi varieties using RAPD marker

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

Table No.2 Instruments Table No.3 Glassware's
List of Equipments, Glasswares & Instruments
16-05-2023
Wani Aditya Balasaheb
8
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)

Table no. 4: List of chemicals
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 Mgcl2

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

Table no. 5 - Composition of CTAB Buffer
Total volume was made up to 100 ml using autoclave water.
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

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

Then equal amount of ice-cold isopropanol was added and tubes was kept at -20 oC 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..

⮚ 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

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 OD260 x Dilution factor.
1000
Pure DNA has an A260/A280 ratio of ~1.8
3. Check Purity of DNA

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

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

Table no.7- PCR components for RAPD marker
Sr. No Components
Volume for each
sample
1
10X PCR buffer with
17.5mM MgCl2
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

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.

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

RESULTS OF THE PROGRAM
1. DNA isolation
2. Quantification of DNA
3. Purification of DNA
4. PCR Amplification
5. Data Analysis

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
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

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
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

4. PCR AMPLIFICATION
26
Plate 3:- Amplification profile of Tulsi genotypes
by OPA 01 Primer
Amplifications using RAPD Primer
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

by OPA 06 Primer
by OPA 15 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
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

DATA ANALYSIS FOR RAPD
Fig no. 1. Dendrogram of RAPD Marker

• 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.

OUTCOME OF THE PROGRAMME
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 MgCl2, 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.

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.
31

• 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.

• 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

• 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.

THANK YOU…

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

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