The hybrid variety and parental varieties among the 3 chilly varieties were identified by finding out the genetic polymorphism between them. It helps to identify different plant varieties, disputed plant varieties, genetic polymorphism between intraspecific crosses of plants and also to protect Plant Breeder’s Rights (PBR). Based on banding pattern on gel, identification of KA, KS and HK chilly varieties using SSR & ISSR markers was successfully carried out.

1. Under the guidance of

2. 1. INTRODUCTION
2. OBJECTIVES
3. REVIEW OF LITERATURE
4. MATERIALS AND METHODS
5. RESULTS AND DISCUSSION
6. SUMMARYAND CONCLUSION
7. REFERENCES

3.  To get a superior quality plant, the two different varieties of a plant having some novel
characters are hybridized.
 It is impossible to select hybrid plant from it’s parental plants. Because, they show
same outer appearance.
But, we can find out polymorphism between them genotypically by using molecular
markers.
DNA markers seem to be the best candidates for efficient evaluation and plant
selection.
 Molecular marker is a DNA fragment, that is associated with a certain location within
the genome and is used to identify a particular sequence of DNA in a pool of unknown
DNA.
• Highly polymorphic nature
• Co-dominant inheritance
• Frequent occurrence in genome
• Selective neutral behaviours
• Easy access
• Easy and fast assay
• High reproducibility

4.  ISSR markers (Inter Simple Sequence Repeats): used without knowing the
sequence information for genomic DNA.
 SSR markers (Simple Sequence Repeats): dominant, multi-allelic and more
informative.
 An overview of the relevant characteristics of ISSR and SSR markers
Genomic abundance Medium-high High
Polymorphism level Medium High
Locus specificity No Yes
Co dominance of alleles No Yes
Reproducibility Medium-high High
Labor intensity Low Low
Technical demands Low –medium Low -medium
Operational cost Low –medium Low
Development costs Low High
Required DNA Quantity Low Low
Amenability to automation Yes Yes
 Plant Breeder’s Rights (PBR) can be protected.

5. OBJECTIVES
 To distinguish the hybrid plant variety from the parental plants.
 To differentiate the three different varieties of Capsicum
annum.
 To identify the genetic polymorphism between the
intraspecific crosses of the plants.
 To identify the disputed plant varieties. So that, plant breeder’s
intellectual property rights can be protected.

6. Molecular markers:
 Versatile tools in various fields like taxonomy, physiology, embryology,
genetic engineering etc..
• Discovery of PCR was a landmark.
ISSR markers(Inter Simple Sequence Repeats)
 It is a semi arbitrary marker amplified by Polymerase Chain
Reaction(PCR) in the presence of one primer complementary to a target
microsatellite.
 Highly polymorphic patterns.
 Have several benefits over other markers.
 Genetic relation in Capsicum annum cultivars was analysed through ISSR
markers(5 primers used and gave total 204 clear bands).
 DNA profiling of disputed plant samples had also been done.

7. SSR markers( Simple Sequence Repeats):
 short nucleotide sequence repeats, equal to or less than 6 bases in length that vary
in number.
 Also called as microsatellites.
 Polymorphism of SSR, due to polymerase slippage during DNA replication.
 They have several advantages over other molecular markers.
 Also used for characterization of black poplars, analysis of seed dispersal and
parentage of saplings in bur oak.
Capsicum annum:
 Grown worldwide for vegetable, spice, etc..
 More than 400 varieties
 Chilli has different names.
 We used three varieties KA, KS and
HK(hybrid).
Kingdom: Plantae
Class: Equisetopsida
Order: Solanales
Family: Solanaceae
Genus: Capsicum
Species: Capsicum annum

9. Biological Material:
• Chilli varieties - KA(P1), KS(P2) and HK(Hybrid-P3)
• 5 SSR primers - CAMS 142, CAMS 153, CAMS 163, CAMS 405 & CAMS 647
• 7 ISSR PRIMERS – UBC 811, UBC 825, UBC 834, UBC 835, UBC 836, UBC 841
& UBC 844
DNA extraction:
• done by “one step extraction method”.
Materials and Reagents:
a) Edwards Solution: 200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, and
0.5% SDS
b) TE Buffer
c) 1mM EDTA
d) Extraction Buffer:Dilute Edwards Solution by 10-fold with TE Buffer to obtain
Extraction Buffer.
e) Capsicum annum seeds(3-5mg)
f) 1.5 mL plastic tube Plastic rods that fit the tube

10. Protocol:
Capsicum annum seeds+ 200μL extraction buffer,
crushed the seeds .
Centrifuged at 14,000 rpm for 5 minutes
Supernatant+ RNase
1μL of if for 20μL PCR mixture.
electrophoresed
PCR amplification:
REAGENTS QUANTITY
1) 10X reaction buffer 2 μL
1) Taq DNA polymerase 0.2 μL
1) Primer (ISSR/SSR) 2 μL
1) DNTP’s 1.6 μL
1) Template 1 μL
1) Autoclaved water 13.2 μL
Total reaction mixture 20 μL
• Applied Biosystems-2720 thermal cycler from QB

11. Following temperatures were maintained to carry out the PCR reaction,
STEPS TEMPERATURE TIME
i. Initial Denaturation 950 C 5 min
ii. Denaturation 950 C 1 min
iii. Primer annealing 550 C 2 min
iv. Elongation 720 C 1 min
v. Final elongation 720 C 10 min
vi. STORE Hold at 40 C
Reagents For 7 wells gel (8ml) For 5 wells gel (5ml)
1) Acryl amide 2.09ml 1.30ml
1) Distilled water 4.1ml 2.56ml
1) 5X TBE 1.57ml 0.98ml
1) 10% APS 131µl 81.87µl
1) TEMED 6.5µl 4.06µl
Gel was stained with Ethidium Bromide for 10 minutes and
Observed under UV Trans-lluminator.

12. PRIMER SEQUENCE
A) ISSR PRIMERS
1) UBC 834 AGAGAGAGAGAGAGAGYT
1) UBC 811 GAGAGAGAGAGAGAGAC
1) UBC 825 ACACACACACACACACT
1) UBC836 AGAGAGAGAGAGAGAGYA
1) UBC 841 GAGAGAGAGAGAGAGAYC
1) UBC 835 CTCTCTCTCTCT
1) UBC 844 CTCTCTCTCTCTCTCTRC
B) SSR PRIMERS
1) CAMS 142
F GAGCGCTTAAGTGGTCATAGG
R CTACAACGCCCCAAAACAAT
1) CAMS 153
F TGCACAAATATGAATCCCAAGA
R AAGTCAGCAAACACATCTGACAA
1) CAMS 163
F TCCATATAGCCCGTGTGTGA
R GCGTGGGAATACAATGCTAGA
1) CAMS 647
F CGGATTCGGTTGAGTCGATA
R GTGCTTTGGTTCGGTCTTTC
1) CAMS 405
F TTCTTGGGTCCCACACTTTC
R AGGTTGAAAGGAGGGCAATA
• Single letter abbreviations for mixed base positions, R = (A, G), Y = (C, T),
F= forward primer, R= reverse primer.
The markers (ISSR and SSR) were used as primers in the PCR component and are listed below

13. RESULTS AND DISCUSSIONS
DNA presence confirmed by running
on Agarose gel
(P1= KA, P2= KS and P3=HK)
P1 P2 P3
PRIMERS
Number of bands formed in Capsicum
annum varieties
KA(P1) KS(P2) HK(P3)
ISSR primers
UBC 811 2 2 2
UBC 825 4 4 1
UBC 834 2 1 1
UBC 835 - - -
UBC 836 1 - -
UBC 841 - - -
UBC 844 - - -

14. P1 P2 P3 P1 P2 P3 M
UBC 811 UBC 825
P1 P2 P3 P1 P2 P3 M
UBC 836 UBC 841
P1 P2 P3
UBC 834
1) 2)
3)

15. • ISSR primers produced a total of 20 bands.
• we can tell all the three varieties are different based on the band thickness.
• On the whole, ISSR primers gave DNA profile of the 3 varieties.
• The bands did not show polymorphism but, the three different varieties can be
differentiated.
PRIMERS
Number of bands formed in Capsicum annum
varieties
KA(P1) KS(P2) HK(P3)
SSR primers
CAMS 142 2 2 2
CAMS 153 2 2 2
CAMS 163 3 2 3
CAMS 405 4 4 4
6 5 7
• A total of 50 bands were formed for 5 SSR primers.
• Clear bands were observed.
• CAMS 647 gave polymorphic bands.
• The bands of KA and KS variety are seen in HK variety and that confirmed the hybrid
variety.

16. P1 P2 P3 P1 P2 P3
CAMS 142 CAMS 153
P1 P2 P3
CAMS 163
P1 P2 P3 P1 P2 P3
CAMS 405
Bands formed
for SSR
primers:
5)4)
6)

17. • In the recent days, many molecular markers have
been developed and are the powerful tools for
successful plant breeding.
• SSR & ISSR analysis can be used for genetic identity,
parentage, clone and strain identification and
taxonomic studies of closely related species.
• Useful to describe the genetic similarities and
dissimilarities.
• Since CAMS 647 SSR primer gave polymorphic
bands to identify the hybrid plant variety, our project
concludes that, ISSR and SSR primers are useful to
identify plant varieties, to analyze the disputed plant
samples.

18. 1. Kumar P, Gupta V K, Misra A K, Modi D R And Pandey B K, Potential of
Molecular Markers in Plant Biotechnology, Plant Omics Journal (2009), 2(4):
pp:141-162.
2. Mishra P K, Fox R T V, Culhamm A, Inter-simple sequence repeat and
aggressiveness analyses revealed high genetic diversity, recombination and
longrange dispersal in Fusarium culmorum, School of Plant Sciences, The
University of Reading, Whiteknights, Reading RG6 6AS, UK, Association of
Applied Biologists(2003) ,pp:1-7.
3. Fang D, Roose M L, Identification of closely related citrus cultivars with
intersimple sequence repeat markers, Theoritical And Applied Genetics (1997),
95,pp: 408–417.
4. Avni S Patel, Sasidharan N, Ashish G Vala and Vinay kumar, Genetic relation in
Capcicum annum [L.] cultivars through microsatellite markers: SSR and ISSR,
Electronic Journal of Plant Breeding (Mar 2011), 2(1), pp:67-76
5. Lekha D Kumar, Kathirvel M, Rao G V, Nagaraju J, DNA profiling of disputed
chilli samples (Capsicum annum) using ISSR-PCR and FISSR-PCR marker
assays, Forensic Science International (2001), 116; pp: 63-68
6. Eshbaugh WH Peppers: History and exploitation of a serendipitous new crop
discovery. In: Janick J, Simon JE, editors. New crops. New York, USA: Wiley
(1993) , pp: 132–139.