This document describes a study that aimed to identify polymorphism in different chilli varieties using DNA fingerprinting with RAPD. Seven chilli varieties were collected and genomic DNA was isolated from them using a CTAB method. Quality PCR was performed and DNA was amplified using RAPD. The PCR products were separated using urea polyacrylamide gel electrophoresis and visualized using silver staining. The study examined techniques for detecting genetic variation in chilli varieties at the DNA level.

1. IDENTIFICATION OF POLYMORPHISM
BY DNA FINGERPRINTING USING
RAPD IN CHILLI
Under the Guidance
Dr. Deepak.R (HOD)
External Guidance
Govind Rao
Submitted by
Abhinav Varma (1CR08BT001)
Sachin Subba (1CR09BT400)
Syed Mubasir (1CR05BT026)
Department of Biotechnology
CMRIT Bangalore.

2. CONTENTS
 OBJECTIVE
 REVIEW OF LITERATURE
 INTRODUCTION
 MORPHOLOGY
 CLASSIFICATION
 CLIMATIC CONDITION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION

3. OBJECTIVE
 The objective of this project is to examine the
technology of DNA fingerprinting.
 Identification of polymorphism by DNA
fingerprinting using RAPD in different variety of
chilli considered for the present study.
 7 varieties of chillies were selected for the study.
This work was carried out under the SBL
bangalore.
 Isolation of genomic DNA.
 Agarose gel electrophoresis of PCR product.
 Spectrophotometric quantification.
 Techniques to detect genetic variation of DNA.

4. REVIEW OF LITERATURE
Sources of genetic variation
Genetic variation and its
importance
 Genetic variation is also  Mutation( changes in one
called as genetic or more letters of a DNA
diversity. sequence)
 Genetic diversity is a  Mutation come in the form
necessary for survival in of single base pair (point
mutation), deletion,
a world full of changing insertion, translocation or
environmental stresses. inverse of genes.
 Genetic diversity  Recombination is the
provides the means for shuffling of DNA segments
species to adopt to  Transgenic, a tool of
changing environmental modern biotechnology.
conditions in future.

5. TECHNIQUES TO DETECT GENETIC
VARIATION
TRADATIONAL:
CLONE/SEQUENCE BASED
 Studying the character tics of MARKER :
plants & animals that are easy  Single base pair polymorphic
to observe & measure. restriction site seq & measurable
insertion or deletion
DNA BASED :
 It can be detected by
 Techniques to analyze DNA, electrophoretic techniques
developed within the last 20 FINGER PRINT MARKER :
years, enabling to locate  Most commonly in use are the
specific DNA chromosome. minisatellites.
 Minisatellites is the
GENE FLOW : comparatively low upfront cost
 A fair degree of genetic of detecting.
modification occurs through FUTURE BASED MARKER
TECHNOLOGY :
gene flow during sexual
 The generation of large insert
reproduction, followed by clone libraries for agriculture
natural selection. animal species is already
underway.

6. INTRODUCTION
ORIGIN: DISTRIBUTION:
 The orgin of chillies is believed to  Capsicum was first introduced
be as old as 7000 B.C. into Spain by Columbus in 1943.
 In 1888, experiments began for  Its cultivation spread from
cross breeding of chili plant. Mediterranean to England by
1948.
 It was originated from mexico.
 Chilli is actually reported to
 Later new breed of chilli plants nature of south america.
were evolved by crossbreeding.
 Its cultivation was known to be
 New variety of chilli i.e. Anaheim native of Peru.
was grown on 1906.
 This crop was introduced to
 There are more than 400 India by Portuguese towards the
varieties of chilli grown all over end of 15th century.
the world
 It became popular in 17th century.
 The hottest chilli is “Naga
Jolokia” which is cultivated in
hilly terrain of Assam.

7. MORPHOLOGY
BRANCHING: FRUIT:
 Chilli pant is highly branched  Fruit is berry.
herbaceous plant.  Seeds are hot & embedded on
 Its height ranges from 50 – pericarp.
100cm  Pericarp is leathery which
 Leaves are simple, alternative turns from green to purple,
with unequal margin. purple to red.
 Fruit attain full maturity in
FLOWER: around 35 days.
 Flower sometimes occur in
pairs. SEED:
 It is bell shaped, slender &  Seed start developing from 15
terminal. days of anthesis.
 Diameter of seed varies from
3-4 mm.

8. CLASSIFICATION
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Subclass : Asteridae
Order : Solanales
Family : Solanaceae
Genus : Capsicum
Species : frutescens

9. CLIMATIC CONDITON
 It can be grown in both warm & cold climatic
condition.
 The ideal temperature range is 20-25°C.
 The crop is killed in freezing temp and frost.
 Heavy continues rain during flowering results in
poor fruit.
 High temp. & dry winds are injurious to plant.

10. MATERIALS AND METHODS

11. SAMPLE COLLECTION
SAMPLE COLLECTION
7 verities of chili plants were collected from I.I.H.R agri
university, Bangalore. The samples are stored at 4⁰c.
 Mahabharath
 Sarca aroka
 Samruthi
 indam 5
 f1 hybrid
 Indam jwala
 SBL-C

12. PLASMID ISOLATION  Add 0.3ml of solution 3 and mix it
gently & incubate in ice cold
condition for 8-10mins.
 100μl of B. glycerol stock was added into
50ml of lb broth containing antibiotics
The centrifugation at 12000rpm for
12 mins
 The conical flasks were incubated
overnight at 37 C on the orbital shaker. collect supernatant and transfer to
2ml eppendorf tubes & 10-12μ was
added
 The cultures were transferred into sterile
centrifuged tubes & chilled in an ice bath collect the upper layer & transfer
for 10mins at 4 C. to 2ml eppendorf tubes.
Add equal vol of
 The cells were harvested by centrifuge at chloroform:isoamyl alcohol (24:1)
6,000rpm for 6mins at 4 C.
vial was centrifuged at
10,000rpmfor 12mins
 Collect the pellet & then add 0.2ml of ice-
cold solutin1 by using cyclo mixer.
pellet was washed with ice cold
70% ethanol
 Add 0.4ml of solution 2, mix it proper (RT)
the pellect was dried in speed
vacuum desiccators.

13. ISOLATION OF GENOMIC DNA
(CTAB METHOD)

14. DAY1
0.15g - 0.3g in 700- 900μl of CTAB buffer
& crush with the help of Motor & pestle
Transfer to 2ml of vials & incubate T 50°C for 15mins in water bath
After incubation add equal vol of chloroform: isoamyl alcohol (24:1)
Incubate at 37°C for 30mins in shaker
Centrifuge at 12000rpm for 12mins at R.T
Collect the upper layer & transfer t 2ml vials.
Add 0.5v 5M Nacl & mix it well & then add full vol of isopropanol
Storage for -20°C overnight

15. DAY2
Centrifuge at 12000rpm for 12mins at 4 C After centrifuge we get three layer
Collect pellet & allow to air dry for Collect the upper layer & transfer to 2ml
10mins vials
After air dry add 800of TE buffer Add equal vol of chloroform : isoamyl
alcohol & mix it gently
Add 6 of Rnase & incubate at 37 C for
30mins in the bath Centrifuge at 12000rpm for 12mins at
R.T
After incubation add equal vol of phenol:
chloroform: isoamly alcohol (25:24:1) Collect upper layer & transfer to 2ml
vials
Centrifuge at 12000rpm for 12mins at R.T
Add 0.1v 3m sodium acetate & mix it
well
Add full vol of absolute ethanol & mix it
well
Store at -20 C overnight

16. DAY3
Mix & centrifuge at Collect pellet & air dry for
12000rpm for 12mins at 10 – 15 mins
4°C
Add TE buffer
Collect the pellet & then add
1.5ml 70% ethanol
Dissolve the pellet gently
Dissolve pellet gently
Prepare 0.8% of agarose
gel
Centrifuge at 12000rpm for
12mins at 4°C
Load the 10μl genomic
DNA

17. SPECTROPHOTOMETRIC
QUANTIFICATION OF DNA
REQUIREMENTS :
 UV spectrophotometer
 TE buffer
 DNA sample
 Micropipette
 Absolute Ethanol

18. PROCEDURE:
Prepare a known dilution of DNA sample in the TE
buffer, which is used to dissolve the DNA sample.
Calibrate the spectrophotometer for blank using TE
buffer.
Record the OD of the sample at 260nm and 280nm.
Calculate the concentration of DNA in the sample
using the Relation

19. QUALITY PCR
REQUIREMENTS :
 Thermo stable Taq DNA polymerase
 dNTP mix (10mM)
 Chili genomic DNA
 Sterile distilled water
 PCR buffer (10x)
 Forward primers and reverse primer specific to
positive control
 Micropipettes of different ranges

20. Reaction components:
S DNA sample Positive dNTPs (µl) Buffer (10x) Forward Reverse Taq Sterile
. volume(µl) control (µl) (µl) primer (µl) primer (µl) polymerase water (µl)
N (µl)
o
1 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
2 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
3 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
4 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
5 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0
6 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

21. QUALITY PCR PROGRAM
Heated lid 110ºC
Pre- heated lid off
Pause- off
Initial denaturation- off
Loop 1 (initial denaturation)
No. of cycles 1
Segment 94ºC 3minutes
Loop 2
No. of cycles 30
Segment 94ºC 30sec
Segment - 55ºC - 30sec
Segment - 72ºC - 1minute
Final extention 72ºC - 5minutes
Final hold 10ºC

22. AMPLIFICATION OF DNA USING RAPD
REQUIREMENTS :
 Thermostable Taq DNA polymerase
 dNTP mix (10 mM)
 Template DNA
 Sterile distilled water
 PCR buffer (10x)
 Oligonucleotide primers
 Ice bucket
 Eppendorff vials
 Micropipettes of different ranges
 Thermal cycle

23. PROCEDURE:
Set up the following reaction mixture (25 l) in the same order.
Ingredients Volume to be taken
Template DNA 10.0μl
dNTPs 2.5μl
PCR buffer 2.5μl
Primers 1.0μl
Taq DNA polymerase 0.75μl
Sterile water 8.25μl
Total 25μl

24. All those mentioned ingredients are mixed and prepared for the
total no of reactns including a blank with a particular primer
excluding template
The calculated volume of masters mix ix then transferred to
labeled PCR tubes with template source and primer.
Finally 0.33µl of Taq DNA polymerase is added to each tube
The contents of the tube are mixed with a brief spin and
transferred to PTC 200 thermal cycler
The program with following conditions is selected for the
amplification
Number cycles 30
Segment 94.0ºC 1minute
Segment 35.0ºC 1minute
Segment 72.0ºC 1minute

25. UREA POLYACRYLAMIDE GEL
ELECTROPHORESIS
REQUIREMENTS :
 Vertical electrophoresis unit
 Urea 7M
 Acrylamide 40%
 10x TBE (Tris Borate EDTA) buffer
 10%Ammonium Per Sulphate (APS)
 Tetra Ethyl Methylene Diamine (TEMED)
 Gel loading dye
 Autoclaved distilled water

26. PROCEDURE:
Preparation of gel (50ml)
Weigh 9.08g of urea and dissolved by heating in about 15ml
autoclaved distilled water.
Add 6.25ml of 40% acrylamide and 5ml of 10x TBE buffer.
Make up the volume to 50ml with autoclaved distilled water.
Add 350μl of APS and 35l of TEMED and mix well.
Immediately transfer the gel into the previously arranged
vertical electrophoresis unit.

27. Electrophoresis of the DNA
Pre-run the gel for about one hour at 100V.
To the PCR sample add 4.2l of gel loading dye.(Xylene
Cyanol).
Boil the samples for 10minutes at 85-90C.
Immediately chill the sample in ice for 2minutes.
Spin the sample at 3000rpm for 2minutes and load in top the
gel.
The electrophoresis is carried out at 150V tll the dye front
reaches the bottom of the base plate, the plates are cooled
with an ice pack during the run to prevent over-heating

28. SILVER STAINING
REQUIREMENTS :
 Gel container
 Shaker incubator
 10% acetic acid
 de-ionised water
 autoclaved double distilled water
 silver nitrate solution
 2.5% sodium carbonate and 0.02% formaldehyde.

29. PROCEDURE: Incubate for 10minutes at room
Place the gel in 5 volumes of a temperatures with shaking.
mixture of 30% ethanol and Repeat this step twice.
10% acetic acid.
Remove the deionised water and
Incubate the gel for 3 hours or add 5 gel volume of 0.1%silver
overnight with shaking at room nitrate solution.
temperatures.
Incubate for 30minutes at room
Remove the ethanol / acetic acid temperatures with shaking.
solution and add 5 gel volume
of 30% ethanol. Remove the silver nitrate
solution and wash the gel for
Incubate for 30minutes at room 20seconds under a stream of
temperatures with shaking. deionised water.
Repeat this step twice.
Add 5 gel volume of a mixture of
Remove the ethanol solution and 2.5%sodium carbonate and
add 10 gel volume of 0.02% formaldehyde
deinonised water.

30. Incubate at room temperature with shaking. Bands will start
appearing slowly.
Incubate until band appears.
Stop the reaction by washing with 1% acetic acid.
Wash several times with deionised water for 10 minutes each
The gel might now be observed over an illuminating source of
white light for better result and documented.
For preserving the gel, place it in 20ml of a 20% glycerol
solution.
Keep the gel between two layers of gelatin [aper and dry for 3
days at 37ºC.

31. RESULTS AND DISCUSSION

32. GENOMIC DNA ISOLATION AGAROSE GEL
 In the present study DNA ELECTROPHORESIS
was isolated from chilli  After isolation of G.DNA
leaves following the CTAB from chilli leaves sample
method. were loaded into 0.8%
 Method described by Doyle agarose gel.
(1987) with few  To cross check the presence
modification. or absence of G.DNA in
 About 320 g of pure G.DNA isolated sample.
could be isolated by this  In agarose gel we observed
method. respective G.DNA bands
with little streaking.
 These streaking might be
because breakage of DNA.

33. QUALITY PCR
 To cross check quality of
G.DNA in test sample
 We arrange quality PCR
using test chilli G.DNA
sample.
 +ve control along with
specific primer, loaded into
0.8% agarose gel.
 Out of 7 chilli varities only
6 varities were amplified
throughout along with +ve
control
 Indicates only 6 chilli
G.DNA quality was good.

34. RAPD PCR
 The RAPD technique was
standardize by adapting
various temp, primer conc.
 After standardize of RAPD
program routine analysis
was done with a PCR.
 Program having 2mins
initial denauration (94⁰c) .
1min denauration (94⁰C)
1min annealing (35⁰c)
1min extension (72⁰c)
For about 30 cycles, this was
followed by one final
extension 72⁰c for about
5mins.

35. SCREENING OF RAPD PRIMERS
 RAPD analysis of isolated chilli plant G.DNA was
carried out with 10 different oligonucleotide random
primer.
 Out of 10 random primers tested for chilli only 4
primers showing amplification with test sample.
 So that we use only 4 selected primers to study
polymorphism in 6 chilli varities.

36. PRIMER 1
 Produce moderate level of
polymorphic in 6 chilli
verities.
 F1 hybrid showing 4
amplified fragments with
different molecular weight.
 Samruthi, indam5 & jwala
showing 3 amplified
fragments with different
mol.wgt
 Magabharathi showing 2
amplified fragments with
different mol.wgt

37. PRIMER 2
 Produce moderate level
of polymorphic
fragments in 6 chilli
verities.
 F1 hybrid, magabharathi
showing 2 amplified
fragments with different
mol.wgt
 Samruthi showing 1
amplified fragments.
 Indam 5 & aroka suphar
no fragments.

38. PRIMER 3
 Produces moderate level
of polymorphic bands in
6 chilli verities.
 F1 hybrid showing 4
amplified fragments with
different mol.wgt
 Magabharathi, indam5,
samruthi, jwala showing
3 amplified fragments.
 Aroka suphar showing 2
amplified fragments.

39. PRIMER 4
 Produce moderate level
of polymorphic
fragments in 6 chilli
verities as depicted in fig.
 Samruthi showing 4
fragments
 Indam 5 showing 3
fragments
 Magabharathi showing 2
fragments.
 Jwala no fragments.

40. Primers used and their sequence in 5’ to 3’ direction
1 CTATAAGCCA
2 GGTGACGCAG
3 CCGGTGTGGG
4 TGCCCGTCGT
5 CCCTGTCGCA
6 TAGCCTAGGC
7 CTGAGACGGA
8 GGCAGCAGGT
9 GAATGCGACG
10 ATGACGTTGA

41. UREA ACRLY AMIDE GEL
 Well 1 – samruthi, primer 4
 Well 2 - mahabharath,
primer 3
 Well 3-samruthi, primer 2
 Well 4- f1 hybrid, primer 2
 Well 5- mahabharath,
primer 2
 Well 6- indam 5, primer 1
 Well 7- ladder dna
 Well 8- mahabharath,
primer 4
 Well 9- samruthi, primer 4
 Well 10- f1 hybrid, primer 4
 Well 11- jwala, primer 3
 Well 12- jwala, primer 4
 Well 13- mahabharath,
primer 3

42. CONCLUSION
 Polymorphism between genotypes is due to either a
nucleotide base change that alters the ability of the
primer to anneal to the DNA template within the
amplified fragment.
 All the primers cannot amplify all the verities of
chilies. Variation was seen as certain primers could
separate.
 Our study reflected the tremendous genetic diversity
available among the genotypes.
 The rich genetic diversity in which breeding efforts
depend can be utilized for current & future.

43. THANK YOU