Under the guidance of

1. INTRODUCTION

2. OBJECTIVES

3. REVIEW OF LITERATURE

4. MATERIALS AND METHODS

5. RESULTS AND DISCUSSION

6. SUMMARY AND CONCLUSION

7. REFERENCES

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

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.

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.

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.

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

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

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

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.

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 142F GAGCGCTTAAGTGGTCATAGG

R CTACAACGCCCCAAAACAAT

1) CAMS 153F TGCACAAATATGAATCCCAAGA

R AAGTCAGCAAACACATCTGACAA

1) CAMS 163F TCCATATAGCCCGTGTGTGA

R GCGTGGGAATACAATGCTAGA

1) CAMS 647F CGGATTCGGTTGAGTCGATA

R GTGCTTTGGTTCGGTCTTTC

1) CAMS 405F 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

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 - - -

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)

• 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.

P1 P2 P3 P1 P2 P3

CAMS 142 CAMS 153

P1 P2 P3

CAMS 163

P1 P2 P3 P1 P2 P3

CAMS 405Bands formed

for SSR

primers:

5)4)

6)

• 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.

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Electronic Journal of Plant Breeding (Mar 2011), 2(1), pp:67-76

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chilli samples (Capsicum annum) using ISSR-PCR and FISSR-PCR marker

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